Biological entity separation device and method of use

The current invention generally relates to apparatus and method to analyze and separate biological entities, including cells, bacteria and molecules from human blood, body tissue, body fluid and other human related biological samples. The claimed apparatus and method analyze, or detect, biological entities based on optical signals received from said entities by using optical detectors. The claimed apparatus and method further separate biological entities with using micro-actuator activated sorting devices.

BACKGROUND

The current invention generally relates to device to analyze and separate biological entities, including cells, bacteria and molecules from human blood, body tissue, body fluid and other human related biological samples, and methods to achieve the same. The disclosed devices and methods may also be utilized to separate and analyze biological entities from animal and plant samples. More particularly, the current invention relates to the methods and apparatus for achieving separation of biological entities with using one or more magnetic devices, one or more micro-fluidic devices, one or more fluorescent detectors, one or more micro-actuator activated sorting devices, individually or in combination. For description purpose, “cells” will be used predominantly hereafter as a typical representation of biological entities in general. However, it is understood that the methods and apparatus as disclosed in this invention may be readily applied to other biological entities without limitation.

Separation of biological entities from a fluid base solution, for example separating a specific type of white blood cells from human blood, typically involves a first step of identifying the target biological entities with specificity, and followed by a second step of physical extraction of the identified target biological entities from the fluid base solution. In human blood, different types of biological cells may have various types of surface antigens or surface receptors, which are also referred to as surface markers in this invention. Certain surface markers on a given type of cells may be unique to said type of cells and may be used to identify said type of cells from blood sample with specificity.

FIG.1AthroughFIG.1Cshow examples of identifying or labeling target cells1, with using superparamagnetic labels2(“SPL”) as inFIG.1A, using optical fluorescent labels3(“OFL”) as inFIG.1B, and using both the SPL2and OFL3together as inFIG.1C.

InFIG.1A, cell1has surface markers11. SPLs2are conjugated with surface antibodies or ligands, also referred to as “probe”21, which specifically bind to the surface markers11of cell1. Large quantity of SPL2having probes21are put into the solution where cell1resides. After incubation processes9, a plurality of SPLs2are bound to cell1surface with probes21selectively bound to surface markers11with specificity. Thus, cells1is magnetically identified or labeled by SPLs2, i.e. magnetically labeled cell10. A magnetic field with sufficient field gradient may be applied to cell10to produce a physical force on the SPLs2attached to the cells10surface. With sufficient strength, the physical force working through the SPLs2on cell10may be used to separate and physically remove cell10from its liquid solution.

InFIG.1B, cell1has surface markers12. OFLs3are conjugated with probes22, which specifically bind to the surface markers12of cell1. Large quantity of OFLs3having probes23are put into the solution where cell1resides. After incubation processes9, a plurality of OFLs3are bound to cell1surface with probes22selectively bound to surface markers12with specificity. Thus, cells1is optically identified or labeled by OFLs3, i.e. optically labeled cell20. By using an optical based cell separation system, cell1may be separated from its liquid solution based on the optical signal that OFL3produces under an excitation light. One type of such optical based cell separation system is a flow cytometer, wherein said liquid solution is streamed through a conduit within said flow cytometer as a continuous flow. At least one excitation light source produces a light spot upon said liquid flow through said conduit at a first optical wavelength. In presence of OFL3in the light spot, OFL3is excited by first wavelength and radiates optical light at a second wavelength. When cell1with bound OFLs3passes said light spot within said flow, OFLs3bound to cell1radiate optical signal in second wavelength, whereas strength of said optical signal as well as duration while cell1passes the light spot may be used to identify presence of cell1by the flow cytometer, which then diverts cell1into a second liquid flow path or mechanically remove cell1from the liquid flow, thus separating cell1from fluid base. In practice, OFL3bound to cell1may be in various types of fluorescent dyes or quantum dots, producing exited optical light at multiple wavelengths. A plurality of excitation light sources may also be used in same flow cytometer system to produce excitation light spots at different locations of the liquid flow with different excitation light wavelength. Combination of various wavelength produced by OFL3on same cell1may be used to increase specificity of separation of cell1, especially when a combination of various types of surface markers12is needed to specifically identify a sub-category target cell1population from a major category of same type of cells, for example CD4-T cells from other white blood cells.

InFIG.1C, cell1has both surface markers11and12. SPLs2conjugated with probes21and OFLs3conjugated with probes22are both bound to cell1surface after incubation processes9to form magnetically and optically labeled cell30. Cell30allows for separation of cell30with a combination of magnetic separation and an optical based cell separation system, where a magnetic separation through SPLs2may provide a fast first stage separation of cell category including cell30, while the optical separation through OFLs3may provide a second stage separation of cell30after magnetic separation with more specificity. Alternatively, cell30may be separated via OFLs3in a first stage and via SPLs2in a second stage. In either case, SPLs2and OFLs3together may help increase speed, efficiency and specificity in separation of cell1compared withFIG.1AandFIG.1B.

FIG.2Ashows an example of conventional magnetic separation through SPL2. In a container5, liquid solution6contains cells10ofFIG.1Aor cells30ofFIG.1Cthat are bound with plurality of SPLs2on cell surface. Magnet4, preferably a permanent magnet, is positioned in proximity to wall of container5. Magnet4has a magnetization represented by arrow41indicating a north pole (“N”) and a south pole (“S”) on top and bottom surfaces of the magnet4. Magnetic field produced by the magnetization41in the solution6is higher at the container5wall directly opposing the N surface of the magnet4, and lower at locations within solution6further away from the magnet4, thus creating a magnetic field gradient pointing towards the magnet4within the solution6. SPLs2bound to cell10/30are superparamagnetic, which are effectively non-magnetic in absence of magnetic field, but will gain magnetic moment in presence of the magnetic field produced by the magnet4. With the magnetic moment of SPLs2and the magnetic field gradient from magnet4, cells10/30will be pulled by the force produced by the magnetic field from magnet4towards magnet4. After sufficient time7, cells10/30may be depleted from solution6and form conglomerate at inside surface of the container5wall opposing magnet4. In conventional practice, solution6may be removed from container5, while maintaining magnet4position relative to container5thus cells10/30are retained as conglomerate against container5inside surface. Afterwards, magnet4may be removed from container5. With absence of magnetic field, conglomerate of cells10/30, together with any non-bound free SPLs in the conglomerate, shall self-demagnetize over extensive time to be non-magnetic and cells10/30may be removed from container5as individual cells10/30.

Conventional method as shown inFIG.2Ahas limitations in actual applications. For the SPL2to be superparamagnetic, the size of the fundamental superparamagnetic particles (“SPN”) contained in SPL2, for example iron oxide particles, shall be in the range of 10 nm (nanometer) to 30 nm, where a smaller particle size makes the particles more effectively superparamagnetic but harder to gain magnetic moment in presence of magnetic field, while a larger particle size makes the particles more difficult to become non-magnetic when magnetic field is removed. SPL2is typically composed of SPNs dispersed in a non-magnetic matrix. For example, certain SPL2is a solid sphere formed by SPNs evenly mixed within a polymer base, typically in the size of larger than 1 um (um). In another case, SPL2is solid bead formed by SPNs mixed within an oxide or nitride base, for example iron oxide nanoparticles mixed in silicon oxide base, which can be in the size of a few hundred nanometers or tens of nanometers. For the cells10/30ofFIG.2Ato be suitable for additional cellular processes, including cell culture and cell analysis, SPL2size is desirable to be smaller than the cell itself, which is usually a few ums. Thus, SPL2with sub-um size (<1 um) is desired. SPL2size less than 500 nm is more preferred. SPL2size less than 200 nm is most preferred. However, when SPL2average size is smaller, variation of SPL2size becomes larger statistically.FIG.2Bshows example schematics of single SPL2magnetic moment in the presence of an applied magnetic field. Solid curve22indicates SPL2having a population nominal size, or average size, where SPL2magnetic moment increases with higher magnetic field. With magnetic field strength increasing from 0 to Hs, nominal size SPL magnetic moment increases with field strength in a linear trend at beginning, until reaching a saturation region where magnetic moment plateaus to Ms, which is determined by the saturation moment of the SPNs material within the SPL2. For SPL2with a smaller size than nominal size, curve23indicates that at the same magnetic field strength, smaller size SPL2gains a lower moment, and thus experiencing a lower magnetic force, and requires a higher field to reach saturation magnetic moment Ms. For a larger size SPL2than nominal size, curve24indicates larger size SPL2is easier to saturate to Ms with a lower field and gains a higher moment at same magnetic field strength.

Now referring back toFIG.2A, for SPL2with sub-um size that is suitable for cell separation and cellular processes, conventional method ofFIG.2Ahas limitation of not being able to produce high magnetic field strength and strong magnetic field gradient in solution6at locations further away from the container5wall opposing magnet4N surface. Therefore, smaller size SPL2of curve23ofFIG.2Bat farther end of the container5from magnet4may be difficult to magnetize by magnet4field and experiences smaller force to move the cell10/30towards magnet4. To reach complete depletion of cells10/30in solution6within container5, it may require significant amount of time. Meanwhile, volume of container5is limited also due to magnetic field strength from magnet4may not be sufficient to magnetize the smaller SPL2of curve23ofFIG.2Bat large container5sizes. Besides overall process being slow, another drawback in conventional method ofFIG.2Ais that the operation as described inFIG.2Atypically involves air exposure of cells10/30conglomerate during the steps of solution removal and later removal of cells10/30from container5. Such air exposure poses challenge in achieving sterile separation of cells10/30for clinical purpose, as well as risk of cell10/30damage or death that negatively affects further cellular processes of cell10/30.

FIG.3Ashows another example of magnetic separation of cells10/30with SPL2in prior art. InFIG.3A, solution6containing cells10/30is passed through a column31that is filled with ferromagnetic or ferromagnetic spheres36. By applying a magnetic field across the column with magnets32and33, where dashed lines34indicates applied magnetic field direction, spheres36may be magnetized by the field and producing localized magnetic field in gaps between neighboring spheres36. Such local field and field gradient between spheres36gaps may be strong, due to the small dimensions of the gaps, to effectively magnetize SPL2of all sizes when SPL2in solution6passes through the gaps between the spheres35during a downward flow of solution6as indicated by arrow35, where SPL2may be attracted to various spheres36surface and separated from the solution6. Prior art ofFIG.3Amay effectively avoid the air exposure issue ofFIG.2A, and may possess at a higher separation speed of cells10/30thanFIG.2Aduring the flow35. However, an intrinsic issue ofFIG.3Amethod is that with the spheres36being ferromagnetic or ferromagnetic and is much larger in size than cells10/30, magnetic domains in spheres36will exist even after removal of magnets32and33from the column31. Such magnetic domains, and domain walls between the domains, will inevitably produce local magnetic field around the surface of the spheres36, which will keep the SPLs2on cells10/30magnetized and strongly attracting the cells10/30when magnets32and33are removed. Therefore, the cells10/30are inherently more difficult to be removed from the column31inFIG.3AthanFIG.2A. Cells10/30loss due to not completely removed from column31after separation is inherently high. In certain prior art method, a pressurized high speed buffer flow may be used to force wash the cells10/30from the spheres in column36. However, such forced flow will inevitably cause mechanical damage to the cells and will still leave significant percentage of cells10/30in column31due to the strong domain wall field of spheres36. Besides cells10/30loss, another intrinsic issue ofFIG.3Amethod is introducing spheres36as foreign materials in the flow of solution6, which is not desirable for sterile process needed for clinical applications.

FIG.3Bthen shows another prior similar to method ofFIG.3A, except mesh37made of ferromagnetic or ferromagnetic wires are introduced in the column31instead of spheres or blocks36. When magnetic field34is applied by the magnets32and33, wires of mesh37are magnetized and adjacent wires of mesh37produce local magnetic field around the wires. Clearances between wires of the mesh allow fluid6to flow in direction35within the column. When cells10/30is in proximity to wires of mesh37, cells10/30may be attracted to the wire surface due to the local magnetic field and field gradient produced by the wires of the mesh37. Compared toFIG.3Aprior art,FIG.3Bmay adjust size of wires and size of clearance of mesh37to tradeoff between cells10/30separation speed and cell loss in column. However, in practice, due to the gap between spheres36is much smaller than clearance size in mesh37, cells10/30separation speed inFIG.3Bis slower thanFIG.3A, whileFIG.3Bstill possesses the same cells loss issue ofFIG.3A, where domains in the wires of mesh37maintains SPL2magnetic moment after magnets32and33are removed and cells10/30are attracted to the wires by the domain and domain wall. Cells10/30loss due to the magnetic domains in wires of mesh37also exists inFIG.3B. Additionally,FIG.3Bis same asFIG.3Ain introducing mesh37as foreign materials in the flow of solution6, which is not desirable for sterile process.

FIG.3Cshows another prior art, where magnets32and33are each attached with a soft magnetic flux guide38with an apex. The flux guides38produce localized magnetic field between the apexes of the guides38with high field strength and high gradient close to the apexes.FIG.3Cshows the cross-sectional view of the conduit39, which is intrinsically a circular tubing, whereas solution6containing cells10/30flows along the tubing39length in the direction perpendicular to the cross-section view. Tubing39is positioned on one side of the gap of the apexes. Magnetic field lines34exhibit a higher density closer to the gap indicates both higher magnetic field strength and higher magnetic field gradient towards the gap. Magnetic field34produces effective force on cells10/30in solution6and pulls the cells10/30from solution6towards the tubing39inside wall that is closest to the apexes of the guides38. Prior art ofFIG.3Cwhen compared to prior art ofFIG.3AandFIG.3Bhas the advantages of: (1) not introducing foreign material in the flow path; (2) when magnets32and33are removed from tubing together with guides38, there is no ferromagnetic or ferromagnetic sphere36or mesh37in the tubing, thus avoiding the domain structures related loss of cells10/30.

However, prior art ofFIG.3Calso has intrinsic deficiencies. First deficiency is the flow speed of solution6, or flow rate, in the tubing39is limited by the prior art design ofFIG.3C. The separation speed of cells10/30of prior art as inFIG.3Cis not sufficient for many applications. Circular tubing conduit39as shown inFIG.3Cexperiences high field and high field gradient at lower end of tubing39, where cells10/30closer to the lower end of tubing39may experience a high force that pulls them to move towards the tubing39lower wall inner surface much faster. However, for the cells10/30closer to the top end of the tubing39, due to the narrow wedge gap and position of the tubing39being on one side of the gap, magnetic field and gradient is significantly lower than the lower end. Thus cells10/30closer to the top end of the tubing39experiences a much smaller force and moves to lower end of tubing39at a much slower speed. For a limited length of the tubing39in the perpendicular to cross-sectional view direction, all cells10/30within the fluid6flowing through the tubing39need to be separated from solution6to form a conglomerate on the inside surface of the tubing close to the apexes before solution6exits the tubing39. Due to slower speed of cells10/30moving from top of the tubing39, flow rate of solution6needs to be slow such that it can allow enough time for all the cells10/30near top of tubing39to be attracted into the conglomerate. If solution6flows through the tubing39at higher speed, it will cause incomplete separation of cells10/30from solution. Such limitation on flow rate due to the circular design of tubing39, where tubing top end being further away from high field and high gradient apexes cannot be cured by a smaller size tubing39. A smaller cross-sectional size circular tubing39will bring the top end of the tubing39closer to the wedge gap. However, due to the smaller cross-section size, volume of solution6flowing through the tubing39in a unit time frame, i.e. flow rate of solution6, will reduce when flow speed of solution6maintains. To maintain same flow rate as in a larger tubing39, solution6flow speed needs to increase, which then gives less time for cells10/30at top end of smaller size tubing39to move to the conglomerate site, and offsets the effect of small size tubing39.

A second deficiency ofFIG.3Cprior art is the inability to dissociate individual cell10/30from conglomerate of cells10/30and non-bound free SPL2, as the conglomerate will not self-demagnetize with ease after magnets32and33, together will guides38, are removed from tubing39in actual applications. Demagnetization of SPL2relies on the SPNs within SPL2being effectively nanoparticles. However, as the conglomerate forms an effective larger body of superparamagnetic material, the SPNs within SPL2experiences magneto-static field from a large number of closely packed SPNs from neighboring SPL2in the conglomerate, which reduces the super-paramagnetic nature of the SPNs. In one case, the SPL2of cells10/30within conglomerate requires extensive time to self-demagnetize, which is not practical for many applications. In another case, the conglomerate won't self-demagnetize due to the SPN being more ferromagnetic in conglomerate form, which is undesirable. High pressure flushing as utilized inFIG.3Ais not effective inFIG.3C, as majority of the circular tubing39inner area is occupied by empty space, while conglomerate is compacted on the lower end of tubing39, such flush will mainly flow through the top section of the tubing39without producing enough friction force on the conglomerate of cells10/30to remove the cells10/30from the tubing39lower wall. As prior art does not provide an effective method to dissociate conglomerate and remove cells10/30from tubing39, such deficiency ofFIG.3Cprior art is limiting its application.

Prior arts are limited either in causing cell loss and introducing foreign materials in the flow path, or limited in the flow rate of solution6and the ability to extract separated cells from conglomerate with an effective dissociation method.

It is desired to have a method and an apparatus that can achieve high flow rate magnetic separation of cells10/30without introducing foreign material in the flow path of the biological solution, and being able to dissociate cells10/30from conglomerate in a practically short time without damaging the cells.

SUMMARY OF THE INVENTION

This invention describes devices and methods that are able to: (1) separate biological entities based on their physical properties, including but not limited to: size, density, compressibility; (2) separate biological entities bound with SPL from biological solution; (3) analyze biological entities based on the optical signals emitted by fluorescent molecules bound to the surface receptors or antigens of said biological entities; (4) sort or separate biological entities with specificity based on the optical signals emitted by fluorescent molecules bound to the surface receptors or antigens of said biological entities with micro-actuator mechanism that are integrated into to fluidic path where said biological entities pass through within a fluidic sample.

The methods, components and apparatus as disclosed by this invention may be utilized to separate biological entities, including cells, bacteria and molecules, from human blood, human body tissue, human bones, human body fluid, human hairs, other human related biological samples, as well as biological entities from animal and plant samples alike without limitation.

For purposes of clarity and brevity, like elements and components will bear the same designations and numbering throughout the Figures, which are not necessarily drawn to scale.

DETAILED DESCRIPTION OF THE INVENTION

While the current invention may be embodied in many different forms, designs or configurations, for the purpose of promoting an understanding of the principles of the invention, reference will be made to the embodiments illustrated in the drawings and specific language will be used to describe the same. It will nevertheless be understood that no limitation or restriction of the scope of the invention is thereby intended. Any alterations and further implementations of the principles of the invention as described herein are contemplated as would normally occur to one skilled in the art to which the invention relates.

“Biological entities”, or “entities”, referred to hereafter include: cell, bacteria, virus, molecule, particles including RNA and DNA, cell cluster, bacteria cluster, molecule cluster, and particle cluster. Large entities and small entities refer to biological entities within same fluid having relatively larger physical size and smaller physical size. In one embodiment, large entities include any of: cells, bacteria, cell cluster, bacteria cluster, particle cluster, entities bound with magnetic labels, and entities bound with optical label. In another embodiment, small entities include any of: molecules, particles, virus, cellular debris, non-bound free magnetic labels, and non-bound free optical labels. In another embodiment, large entities have a physical size larger than 1 micrometer (um), and small entities have a physical size less than 1 um. In yet another embodiment, large entities have a physical size larger than 2 um, and small entities have a physical size less than 500 nanometer (nm). In yet another embodiment, large entities have a physical size larger than 5 um, and small entities have a physical size less than 2 um. Biological sample include: blood, body fluid, tissue extracted from any part of the body, bone marrow, hair, nail, bone, tooth, liquid and solid from bodily discharge, or surface swab from any part of body. Entity liquid, or fluid sample, or liquid sample, or sample solution, include: biological sample in its original liquid form, biological entities being dissolve or dispersed in a buffer liquid, or biological sample after dissociation from its original biological sample non-liquid form and dispersed in a buffer fluid. Biological entities and biological sample may be obtained from human or animal. Biological entities may also be obtained from plant and environment including air, water and soil. Entity fluid, or fluid sample, or sample may contain various types of magnetic or optical labels, or one or more chemical reagents that may be added during various steps within the embodiments of this invention. Sample flow rate is volume amount of a fluid sample flowing through a cross-section of a channel, or a fluidic part, or a fluidic path, in a unit time, where volume may be in unit of liter (l), milliliter (ml), microliter (ul), nanoliter (nl), and unit time may be in unit of minute (min), second (s), millisecond (ms), microsecond (us), nanosecond (ns). Sample flow speed is the distance of a free molecule or a free entity that travels within a liquid sample in a channel, or a fluidic part, or a fluidic path, in a unit time, where distance may be in unit of meter (m), centimeter (cm), millimeter (mm), micrometer (um). Separation efficiency is percentage of target entities within a liquid sample that are successfully separated from the liquid sample by a method designed to separate the target entities. Buffer fluid is a fluid base where biological entities may be dissolved into, or dispersed into, without introducing additional biological entities.

FIG.4shows a cross-sectional view of the first embodiment of a magnetic separation device (“MAG”) of the current invention. MAG121is composed of two magnetic field producing poles, pole102and pole103. Each of the poles102and103is composed of soft magnetic material, which may include one or more elements of iron (Fe), cobalt (Co), nickel (Ni), iridium (Ir), manganese (Mn), neodymium (Nd), boron (B), samarium (Sm), aluminum (Al). Pole102has a magnetic flux collection end1023and a tip end1021, where shape of the pole102is converging from the flux collection end1023towards the tip end1021. InFIG.4, flux collection end1023is a flat surface which is contacting, or in proximity to, the North Pole (“N”) surface of a permanent magnet104. Permanent magnet104has a magnetization as shown by arrow1041inFIG.4which points from the South Pole (“S”) surface of to the N surface of the magnet104. Magnetization1041produces magnetic field in free space, which can be described as flux lines1046emitting from N surface to returning to S surface of the magnet104. Pole102flux collection end1023being in contact with, or in close proximity to, the N surface of magnet104as shown inFIG.4, due to the soft magnetic material of pole102, magnetic flux1046from the N surface of magnet104is collected by the pole102and enters the body of the pole102through flux collection end1023. Due to the converging shape of the pole102, the collected magnetic flux is mainly channeled within the soft magnetic body of the pole102and emitted from the tip end1021of pole102. Said close proximity between flux collection end1023and N surface of magnet104may be a gap distance in between surface of1023and N surface being less than 1 mmm. Tip end1021may have a much smaller surface area than flux collection end1023, which makes flux exiting the tip end1021having a higher flux density than when flux1045is emitted by N surface of magnet104, i.e. magnetic flux1045is concentrated and thus creating a local high magnetic field and high field gradient around the tip end1021. It is preferred that tip end1021of the pole102is as small as possible, for example as a convergence point, to produce largest flux concentration for achieving highest magnetic field. However, in practice, due to manufacturing process, tip end1021may have a curved or domed shape, which shall not affect the general concept of flux concentration by the tip end1021. Pole103is similar to pole102in that pole103has a larger flux collection end1033and a smaller tip end1031, where flux collection end1033is in contact with, or in close proximity to, S surface of permanent magnet105. It is preferred that pole103and magnet105are identical to pole102and magnet104, but arranged as mirroring to pole102and magnet104around a center line1050. Magnet105magnetization1051is opposite to magnetization1041of magnet104. Magnetic flux1047collected by flux collection end1033from S surface of pole103is opposite to that of pole102, and flux emitted from tip end1031of pole103is opposite to that of tip end1021. Thus, between the gap of tip end1021and1031, emitted flux can form closed loop and further enhance the magnetic field strength and field gradient around the tip ends1021and1031. Dashed lines1045are schematic of the flux emitted from tip end1021and returns to tip end1031. Flux lines1045closer to tip end1021and1031being denser indicates stronger magnetic field and larger field gradient closer to the gap area. As shown inFIG.4, top section of pole102is tilted to the right side, while top section of pole103is tilted to the left. This tilted shape diverts the magnetic flux within poles102and103away from bottom section of the poles and helps make the tip end1021of pole102and tip end1031of pole103being the closest spaced features of the poles102and103to achieve high field in gap between tip ends1021and1031with minimizing flux leakage between lower bodies of pole102and103. InFIG.4, the tilted top sections of the poles102and103form a triangle shape, or convex shape, top surface1210of the MAG121, which will be described as “MAG wedge”1210of MAG121hereafter. Permanent magnets104and105may be composed of any of, but not limited to, Nd, Fe, B, Co, Sm, Al, Ni, Sr, Ba, O, NdFeB, AlNiCo, SmCo, strontium ferrite (SrFeO), barium ferrite (BaFeO), cobalt ferrite (CoFeO).

FIG.4embodiment includes a rigid fixed shape channel101. Channel101has a channel wall enclosing a channel space1013, where fluid sample may flow through channel101in the channel space1013along the channel101length direction that is perpendicular to the cross-section view ofFIG.4. Channel101has a top surface1012and a bottom surface1011. Bottom surface1011is formed in a shape conforming to the MAG wedge surface1210, such that when channel101is moved in direction1014to be in contact with the MAG121poles102and103, bottom surface1011of channel101is in contact with MAG wedge surface1210with no or minimal gap in between bottom surface1011and MAG wedge surface1210. Top surface1012of channel101is preferred to be conformal to bottom surface1011to produce a channel space1013with a shape that maximizes exposure of fluid sample flowing through channel101to the highest magnetic field region of the MAG wedge gap field1045.

InFIG.4embodiment, poles102and103, magnets104and105, and channel101extend in the direction perpendicular to the cross-section view ofFIG.4, which will be referred to as “length direction” hereafter. Fluid sample flows in the channel101and is contained in channel space1013along the length direction. Channel101being a rigid and fixed shape channel, the wall thickness of channel101at surface1011may be thinner than wall thickness at surface1012, such that channel101mechanical robustness is maintained by the thicker wall at surface1012, and magnetic field effect on fluid sample is enhanced by thinner wall at surface1011allowing fluid sample being closer to the MAG wedge1210and tip ends1021and1031. Channel101may be attached to a non-magnetic channel holder107at the top surface1012. Channel holder107may align channel101to MAG wedge1210, move channel101to separation position in contact with MAG121, or lift channel101away from MAG121after magnetic separation. Channel holder107may be composed of any non-magnetic material including, but not limited to, metal, non-metal element, plastic, polymer, ceramic, rubber, silicon, and glass. InFIG.4, flux collection ends1023and1033of the soft magnetic poles102and103may also be referred to as base ends1023and1033.

Permanent magnets as described in different embodiments of this invention, for example magnets104and105ofFIG.4, may each have opposite magnetization direction to that is described in the each of the figures and embodiments without affecting the designs, functions and processes of the embodiments.

FIG.5is the cross-sectional view ofFIG.4first embodiment of MAG with channel101in magnetic separation position. Channel101ofFIG.4moves along direction1014and comes into contact with MAG wedge1210surface by bottom surface1011. MAG121gap formed by tip ends1021and1031is brought into contact with, or minimal distance to, wall of channel101and the fluid sample flowing in the channel101. Channel101“C” shape matching to the MAG wedge shape helps achieve large cross-sectional area of the channel space1013to maintain a high flow rate, and at the same time confines cells10/30in the fluid sample flowing in channel101to a high field and high gradient region of the MAG121gap field as indicated by the field lines1045. Compared to prior art ofFIG.3AandFIG.3B, first embodiment MAG121does not introduce foreign material in the channel101while achieving comparable or higher magnetic field and field gradient on cells10/30flowing through the channel101. Removing of poles102and103together with magnets104and105from channel101will eliminate field generation source and avoids limitation of prior art domain related cells loss. Compared to prior art ofFIG.3C, MAG wedge ofFIG.5being in contact with the channel101wall brings highest achievable magnetic field and field gradient to the fluid sample in the channel101for a more efficient cell10/30separation. Channel101shape being conformal to MAG wedge shape allows channel101to have a large cross-sectional sample flow area, meanwhile avoids the deficiency of prior art that cells10/30at top end of a circular channel experiencing much lower magnetic field than at lower end that ultimately limits sample flow rate. Thus, sample flow rate in channel101can be higher than prior art while achieving better magnetic separation efficiency.

FIG.6is same asFIG.5, except biological entities, or cells10/30for simplicity of description, are included to describe magnetic separation by MAG121from a fluid sample6.

Fluid sample6carrying cells10/30is flown through channel101along length direction of channel101perpendicular to theFIG.6cross-sectional view. MAG121gap magnetic field magnetizes the SPL2attached to cells10/30and field gradient pulls cells10/30from the fluid6towards the MAG wedge to form conglomerate layer against the1011bottom surface of channel101. Due to MAG121design and channel101shape, cells10/30close to top surface1012experience magnetic field not significantly lower than close to bottom surface1011, and distance for cells10/30to travel from top surface1012to conglomerate on bottom surface1011is much shorter than in prior art, these characteristics allow MAG121to resolve deficiencies of prior art.

FIG.7is a side view ofFIG.6along direction61ofFIG.6. Fluid sample6carrying cells10/30flows from left to right in the channel101as indicated by arrow1010. With the MAG121gap field, cells10/30are separated from fluid6to form conglomerate on the channel wall of bottom surface1011.FIG.7shows that majority of the cells10/30are separated from liquid6at the earlier section of the channel101length, as indicated by the crowded population of cells10/30. As certain tail population cells10/30may have comparatively smaller size SPL2or fewer number of SPL2bound to it surface, time required for such tail population cells to be pulled to bottom surface1011is longer than nominal population during fluid6flowing through channel101. Thus, population of separated cells10/30will show density decrease from inlet towards outlet of channel101.

FIG.8Ais a cross-sectional view of a second embodiment of MAG of current invention. MAG122inFIG.8Ais substantially similar as MAG121, except a soft magnetic shield106is attached to the S surface of magnetic104and N surface of magnet105. Magnetic flux from S surface of magnet104and N surface of magnet105forms closure path within the soft magnetic shield106. MAG122compared to MAG121will have less magnetic flux leakage outside of the MAG122structure, where magnetic flux generated by magnets104and105are mainly confined within the soft magnetic material body of poles102and103, and shield106. MAG122is preferred in applications where magnetic interference from MAG122to other surrounding instrument or equipment is desired to be minimized.

FIG.8Bis a cross-sectional view of a third embodiment of MAG of current invention. Compared to MAG121, MAG123ofFIG.8Bincorporates only one permanent magnet108, which is attached to both poles102and103, where flux from N surface of magnet108and flux from S surface of magnetic108is conducted by poles102and103to produce MAG123gap field by tip ends1021and1031. Compared to MAG121, magnetic flux generated by magnet108is mainly confined within the soft magnetic material body of poles102and103, and MAG123is comparatively easier to assemble and produces less magnetic flux leakage.

FIG.9shows a cross-sectional view of the first embodiment MAG121being used for magnetic separation in combination with a flexible channel201.FIG.9is similar toFIG.4, except that the rigid channel101is replaced with a flexible channel201. Flexible channel201may assume any shape, including a circular shape tubing form, at its non-deformed state, but can be deformed into other shapes by external force. Wall material of channel201is deformable and may be composed of any of, but not limited to, silicone, silicone rubber, rubber, PTFE, FEP, PFA, BPT, Vinyl, Polyimide, ADCF, PVC, HDPE, PEEK, LDPE, Polypropylene, polymer, thin metal or fiber mesh coated with polymer layer, Flexible channel201is also shown inFIG.9to have a channel holder107attached to the back of channel201. Channel holder107may be composed of any non-magnetic material including, but not limited to, metal, non-metal element, plastic, polymer, ceramic, rubber, silicon, and glass. Channel201may attach to holder107through surface bonding, for example by gluing or injection molding, or via mechanical attachment through components1074ofFIG.32. Holder107has a bottom surface1070in contact with the top surface of channel201, where surface1070being preferred to be substantially conformal to the MAG121wedge shape. InFIG.9, holder107aligns attached flexible channel201to MAG121wedge gap and moves channel201towards MAG wedge gap in direction1014.

FIG.10illustrates the flexible channel201being pushed against the MAG wedge of MAG121by the channel holder107. With pressure exerted by the holder107on flexible channel201against the MAG wedge of MAG121, channel201is deformed inFIG.10with bottom surface2013of channel201becoming conformal and in surface contact to MAG wedge surface1210. Meanwhile, as holder107bottom surface1070may also be conformal to the MAG wedge shape, top surface2012of channel may also be forced into a substantially conformal shape to the MAG wedge.FIG.10depicts the “separation position” of flexible channel201relative to the MAG121during magnetic separation of cells10/30from sample fluid6. Shape of flexible channel201is substantially similar to channel101ofFIG.5andFIG.6, except such shape of channel201at separation position is result of channel201self-aligning and self-conforming to MAG wedge without the need of a manufacturing process to achieve shape of channel101. Additionally, the flow space within channel201at separation position may be adjusted to allow for larger or smaller cross-sectional area of the flow space of channel201, such that optimization of fluid sample6flow rate through channel201and cells10/30magnetic separation efficiency may be optimized. The flow space adjustment may be achieved by changing the vertical distance1071from the holder107surface1070top point in contact with channel201top surface2012, to tip ends1021and1031or to an imaginary plane where tip ends1021and1031reside. With a larger1071distance, flexible channel201is less deformed and a larger flow space is realized, which allows for a slower flow speed at the same fluid flow rate. While with a smaller1071distance, flexible channel201has a smaller flow space but top edge2012is also closer to the MAG wedge gap and tip ends1021and1031, which allows for higher magnetic field and faster separation of cells10/30. Thus optimization between flow rate and separation efficiency may be achieved with adjusting the distance1071for a given combination of MAG121design and flexible channel201. In one embodiment, distance1071is more than 0 mm and less than or equal to 1 mm. In another embodiment, distance1071is more than 1 mm and less than or equal to 3 mm. In yet another embodiment, distance1071is more than 3 mm and less than or equal to 5 mm. In yet another embodiment, distance1071is more than 5 mm and less than or equal to 10 mm. In yet another embodiment, distance1071is more than 2 times and less than or equal to 3 times of the wall thickness of flexible channel201. In yet another embodiment, distance1071is more than 3 times and less than or equal to 5 times of the wall thickness of flexible channel201. In yet another embodiment, distance1071is more than 5 times and less than or equal to 10 times of the wall thickness of flexible channel201. Flexible channel201at separation position functions similarly to channel101inFIG.6, whereFIG.10also shows that during magnetic separation, cells10/30form conglomerate along channel201wall of lower surface2013directly opposing the MAG wedge surface1210. Thickness of channel201wall at bottom surface2013may be thinner than channel201wall at top surface2012.

FIG.11illustrates after magnetic separation is completed inFIG.10, the channel holder107moves away from the MAG121in direction1015, causing the flexible channel201to separate from MAG wedge of MAG121to “lifted position” and flexible channel201may also return to its non-deformed shape, for example circular tubing as shown inFIG.11. Magnetically separated cells10/30inFIG.10may hold the conglomerate form at the bottom surface of the flexible channel201at lifted position. AfterFIG.11lifted position of flexible channels201is reached, dissociation procedures on the cells10/30within the flexible channel201to break up the conglomerate may be performed, as described inFIG.22AthroughFIG.30B. Flexible channel201returning to non-deformed shape, for example circular tubing ofFIG.11, provides a larger cross-sectional area of the channel space1013as shown inFIG.11than at separation position inFIG.10. Such larger channel space1013may be preferred for easier dissociation of cells10/30from the conglomerate form. Additional buffer fluid may be injected into the channel space1013of channel201at lifted position to assist channel201return to non-deformed shape. MAG121inFIG.9throughFIG.11may be replaced by MAG122or MAG123without limitation on described methods and processes.

FIG.12illustrates cross-sectional view of the fourth embodiment of MAG124. MAG124has three soft magnetic poles111,112and113. Center pole111is attached to N surface of permanent magnet109at a flux collection end1112, similar to flux collection end1023of pole102inFIG.4, and flux1048from magnet109N surface is conducted by pole111soft magnetic body and then emitted from a tip end1111, which is much smaller in area size than flux collection end1112, of pole111, and functions similar to tip end1021ofFIG.4to produce a local high field around tip end1111by concentrating the magnetic flux conducted from magnet109. Side poles112and113each have a flux collection end1122and1132respectively, which are attached to same top surface of a soft magnetic bottom shield114. Bottom shield114is then attached to S surface of the permanent magnet109. Thus the magnetic flux1049from the S surface of magnet109is conducted in the body of bottom shield114and divided between poles112and113and further conducted to the tip ends1121and1131of poles112and113respectively. Tip end1111is formed in proximity to tip ends1121and1131. In one embodiment, tip end1111may recess from an imaginary plane where tip ends1121and1131reside towards magnet109by an offset distance between 0 mm to 1 mm. In another embodiment, tip end1111may recess from an imaginary plane where tip ends1121and1131reside towards magnet109by an offset distance between 1 mm to 5 mm. In yet another embodiment, tip end1111may recess from an imaginary plane where tip ends1121and1131reside towards magnet109by an offset distance between 5 mm to 10 mm. Tip end1111is preferred to be spaced equally to tip ends1121and1131. Top section of pole112is tilted to the right side, while top section of pole113is tilted to the left, which is similar to pole102and pole103ofFIG.4. Such tilting is to increase gap between the main bodies of poles112and113to main body of pole111to reduce flux leakage such that flux concentration around tip ends1111,1121and1131is maximized. When flux is emitted from tip ends1111,1121and1131, since flux1048conducted by center pole111is opposite to the flux1049conducted by side poles112and113, the flux forms closure between tip ends1111to1112, and tip ends1111to1131. Thus, the magnetic flux generated by N and S surface of magnet109is conducted within bodies of poles111,112,113and shield114with minimal leakage to outside of MAG124structure. Flux density is highest around tip end1111, with tip ends1121and1131also producing high flux density, which all indicate high magnetic field and field gradient around tip ends1111,1121and1131. Compared to MAG121,122and123, MAG124has the advantage of more efficient flux closure within the MAG124soft magnetic bodies with less leakage and thus higher flux density around tip end1111to produce higher magnetic field and field gradient in channel301.

Channel301is a rigid channel similar to channel101ofFIG.4, and has a fixed shape similar to a rotated “D”. Channel301is shown to be in magnetic separation position inFIG.12, where tip ends1111,1121and1131may all be in contact with the curved bottom surface3011of the “D” shape of channel301, which provides highest possible magnetic field and field gradient that MAG124can produce in the channel space where fluid sample flows in channel301. In another embodiment, tip end1111may be in contact with the surface3011and tip ends1121and1131are not contacting surface3011. Top surface3012of channel301, in one embodiment may be on the imaginary plane where tip ends1121and1131reside, and in another embodiment top surface3012may be above the imaginary plane in between 0 mm to 1 mm, and in yet another embodiment top surface3012may be above the imaginary plane in between 1 mm to 5 mm. In one embodiment, channel301wall thickness at surface3012is thicker than wall thickness at surface3011. Channel301may be attached to a non-magnetic channel holder110at the top surface3012. Channel holder110may align channel201to MAG gap of MAG124, move channel301to separation position in contact with MAG124pole111tip end1111, or lift channel301away from MAG124after magnetic separation.

FIG.13shows a cross-sectional view of the fourth embodiment MAG124being used for magnetic separation in combination with the flexible channel201, which is same as inFIG.9. Channel holder110may be different shape than channel holder107ofFIG.9. Before magnetic separation, channel holder110is attached to channel201. Channel holder110aligns channel201to MAG gap of MAG124which is composed of tip ends1111,1121and1131as inFIG.12, and moves channel201into the MAG gap of MAG124in direction1014.

FIG.14illustrates the flexible channel201in separation position in the fourth embodiment MAG124with cells10/30being separated and form conglomerate around bottom and side walls of the channel201close to the tip ends1111,1121and1131. InFIG.14, flexible channel201is deformed similarly as inFIG.10to conform to the MAG gap boundaries, which are mainly the tip ends1111,1121and1131. Shape of channel201may be different than channel301at separation position due to flexible channel201conforming to the MAG gap boundaries under pressure from holder110. Shape of channel201inFIG.14may provide higher liquid sample flow rate with higher separation efficiency than channel301. Distance1071between the lower surface1150of holder110and tip end1111may be adjusted to optimize flow rate in channel201. Range of distance1071is same as1071described inFIG.10.

FIG.15Aillustrates cross-sectional view of the fifth embodiment MAG125. MAG125is same as MAG124, except the magnet109and bottom shield114of MAG124ofFIG.12are removed in MAG125. Permanent magnets115and116with opposing magnetizations1151and1161are placed in between poles111and112, and between poles111and113, respectively as shown inFIG.15A. Magnetizations1151and1161are horizontal inFIG.15A, which enables center pole111conducting N surface flux from both magnets115and116, while side poles112and113each conducts S surface flux from magnet115and116respectively. Compared to MAG124, MAG125may produce higher field around tip ends1111,1121and1131due to two magnets115and116are used. MAG125may also be easier to assemble than MAG124.

FIG.15Billustrates cross-sectional view of the sixth embodiment MAG126. MAG126is same as MAG124, except the side poles112and113are each attached to S surface of permanent magnets1092and1094respectively, with magnetizations1093and1095being opposite to magnetization1091of magnet109. Bottom shield114is attached to both N surface of magnet1092and1094, and S surface of magnet109, and thus forming internal flux closure in shield114between magnets109,1092and1094. Compared to MAG124, MAG126may produce higher field around tip ends1111,1121and1131due to three magnets109,1092and1092are used in MAG126.

FIG.15Cillustrates cross-sectional view of the seventh embodiment MAG127. MAG127is same as MAG126ofFIG.15B, except the bottom shield114is removed.

FIG.16illustrates two of the third embodiment MAGs123are used for magnetic separation on a pair of flexible channels201. The pair of flexible channels201are fixed on the same channel holder1020inFIG.16. The top MAG123and bottom MAG123are substantially identical, with top MAG123being upside down vertically. MAG wedges of the top and bottom MAGs123are substantially aligned with center of top and bottom channels201. The magnets108of both top and bottom MAG123may have same magnetization directions, as the arrows within magnets108inFIG.16indicate, such that the magnetic fields produced in the top and bottom channels201by the top MAG123and bottom MAG123during magnetic separation have same direction horizontal field component, which limits magnetic flux leakage between top MAG123soft magnetic poles and bottom MAG123soft magnetic poles.

FIG.17illustrates the two MAG123ofFIG.16are moved into separation position against the two flexible channels201, which is same process as inFIG.10. After reachingFIG.17separation position, fluid sample carrying cells10/30may flow through the channels201in length direction perpendicular to the cross-section view to start magnetic separation of cells10/30by top and bottom MAG123. Distance1071between the holder1021surface contacting the channel201outer edge2012and MAG123tip ends1021and1031, or the imaginary plane where tip ends1021and1031reside, may be adjusted to optimize flow rate in each of the two channels201. Range of adjustment of distance1071is same as1071described inFIG.10.

MAG123inFIG.16andFIG.17may be replaced by MAG121or MAG122, and channels201may also be replaced with channel101.

FIG.18illustrates four of the fifth embodiment MAG125being used for magnetic separation on four of flexible channels201. The four flexible channels201are fixed on the same channel holder1040as inFIG.18. The four MAG125are substantially identical. MAG gaps of the four MAG125are substantially aligned with center of each of the corresponding flexible channels201. The permanent magnets arrangement within each MAG125should be identical for example center pole of each of the four MAG125is attached to N surfaces of both magnets within each respective MAG125, and side poles of each of the four MAG125are attached to S surfaces of magnets within each MAG125, as shown inFIG.18. Thus, neighboring MAG125nearest adjacent side poles are of same magnetic polarity, and leakage from side pole to side pole between neighboring MAG125may be minimized or avoided. Additionally, four of MAGs used on four of channels201inFIG.18is only shown inFIG.18as an example of multiple channel process capability with a circular channel arrangement, where channels are positioned at center of the MAG125circular array. Fewer and more MAG125used on corresponding number of channels201may be achieved inFIG.18type circular arrangement without limitation.FIG.18multiple channel circular arrangement with MAG125is intrinsically more flexible than MAG123as inFIG.16, as two pole design ofFIG.16MAG123may lead to magnetic flux leakage through the poles of neighboring MAG123when number of MAG123is more than two.

FIG.19illustrates the four MAG125ofFIG.18are moved into separation position against the four flexible channels201, which is same process as inFIG.14. After reachingFIG.19separation position, fluid sample carrying cells10/30may flow through the channels201in length direction perpendicular to the view ofFIG.19to start magnetic separation of cells10/30by the four MAG125. Similarly as inFIG.17, distance1071between the holder1040surface contacting the channel201outer edge2012and MAG125center pole111tip end1111for each channel201and MAG125pair may be adjusted to optimize flow rate in each of the four channels201. Range of adjustment of distance1071is same as1071described inFIG.10. MAG125inFIG.18andFIG.19may be replaced by MAG124, MAG126, or MAG127, and where channels201may also be replaced with channel301.

FIG.20Aillustrates the sixth embodiment of MAG128with having a rotated “D” shape rigid channel320in separation position. MAG128is similar to MAG123, except that MAG wedge of MAG123is modified from a triangle shape to a flat top as in MAG128. MAG128pole1022is similar to pole102of MAG123, but with a flat top surface1042in pole1022instead of a tip end in pole102. Same flat top1052exists on pole1032which is similar to pole103of MAG123. Due to the flat top of the MAG wedge in MAG128, rigid channel320may have a flat bottom surface1062matching to, and being in contact with, the MAG wedge flat surface in separation position, to gain highest magnetic field and field gradient region from MAG128. Channel320may be attached to a non-magnetic channel holder1102at the top surface. Channel holder1102may align channel320to MAG wedge of MAG128, move channel320to separation position in contact with MAG128poles1022and1032tip ends, or lift channel320away from MAG128after magnetic separation.

FIG.20Billustrates the sixth embodiment MAG128being used on a flexible channel201, where channel201is attached to channel holder1102. Channel holder1102moves channel201towards MAG wedge of MAG128along direction1014.

FIG.20Cillustrates the sixth embodiment MAG128having the flexible channel201ofFIG.20Bmoved into separation position with cells10/30being separated from a liquid sample to form conglomerate at bottom surface of channel201against the top flat surface of the MAG wedge of MAG128. Channel201is forced to form into a rotated “D” shape channel by holder1102pushing channel201against the flat top of MAG wedge of MAG128, where channel201shape at separation position shows similarity to channel320ofFIG.20A. Distance1071between the holder1102bottom surface1062contacting the channel201top edge2012and MAG128pole surfaces1042and1052may be adjusted to optimize flow rate in channel201. Range of adjustment of distance1071is same as1071described inFIG.10.

Magnet108of MAG128may be replaced by placement of magnets104and105as in MAG121, and by placement of magnets104and105and bottom shield106as in MAG122.

FIG.21Aillustrates the seventh embodiment MAG129with having a “V” shape rigid channel330in separation position. MAG129is different from MAG123in pole shape, where pole1024and pole1034of MAG129have flux concentration tip ends3301and3302that forms a “V” shaped concave, instead of the triangle wedge shape of the MAG123. With the V shape MAG concave of MAG129, rigid channel330is also made into a V shape, with the lower edges3303and3304making direct contact with the surface of the tip ends3301and3302. Additionally, channel330may also preferably have a V shape notch into the channel at the top edge3305following the V shape of the3303and3304edges, which helps confine fluid sample in the V shaped channel space3306to flow closer to the pole surfaces3303and3004that provide higher field and field gradient. Channel330may be attached to a non-magnetic channel holder1103at the top surface3305. Channel holder1103may align channel330to MAG concave of MAG129, move channel323to separation position in contact with poles1024and1034tip ends surface, or lift channel330away from MAG129after magnetic separation.

FIG.21Billustrates the seventh embodiment MAG129being used with a flexible channel201, where channel201is attached to channel holder1103at the top edge of channel201. Channel holder1103moves channel201towards MAG129concave along direction1014. Channel holder1103has a triangle shape, where a convergence point of the triangle touches the channel201top edge.

FIG.21Cillustrates the seventh embodiment MAG129having the flexible channel201ofFIG.21Bmoved into separation position with cells10/30being separated from a liquid sample to form conglomerate at bottom surface of channel201against the top surfaces of the MAG concave of tip ends3301and3302of MAG129. Channel201is forced to form into a “V” shape channel by holder1103. InFIG.21C, holder1103forces channel201against the MAG concave of MAG129with the lower convergence point and deforms the top wall of the channel201downwards to move closer to the tip ends3301and3302, while the same force also causes lower wall of channel201to conform to the MAG concave of MAG129to make contact with the tip ends3301and3302top surfaces3303and3304. Thus, channel201shape inFIG.21Cat separation position shows V shape similar to channel330ofFIG.21A, which brings cells10/30in channel space3306closer to high field and high gradient tip ends3301and3302and tip surfaces3303and3304. Vertical distance1071between the holder1103bottom convergence point contacting the channel201top edge2012, and MAG129tip ends3301and3302or an imaginary plane where tip ends3301and3302reside, may be adjusted to optimize flow rate in channel201. Range of adjustment of distance1071is same as1071described inFIG.10.

Magnet108of MAG129may be replaced by placement of magnets104and105as in MAG121, and by placement of magnets104and105and bottom shield106as in MAG122.

FromFIG.22AthroughFIG.27D, various methods to demagnetize or dissociate magnetically separated cells10/30from conglomerate in MAG channel will be described. For simplicity of description, flexible channel201is used. However, channels inFIG.22AthroughFIG.27Dmay be labeled as “201/101”, indicating flexible channel201as used for description may be replaced with rigid channel101without affecting the function and results of the described method. Also for the simplicity of description, MAG123is used inFIG.22AthroughFIG.27D, while any other MAG embodiment together with corresponding channel as described in prior figures may be used under same concepts without limitation

FIG.22Ais substantially similar toFIG.10, where channel201is at separation position and cells10/30have been separated by magnetic field from MAG. InFIG.22A, MAG123is used instead of MAG121ofFIG.10. Channel holder1081may be different from channel holder107ofFIG.10by having a top surface notch that allows the cells10/30demagnetization or dissociation magnetic structure (“DMAG”), which is permanent magnet120inFIG.22A, to be able to reach closer to the channel201/101to provide sufficient field to demagnetize or dissociate cells10/30from the conglomerate in channel201/101. Such notch is preferred, but may not be required. DMAG magnet120is positioned away from MAG123ofFIG.22Awithout affecting magnetic separation of cells10/30by MAG123. DMAG magnet120magnetization is labeled as in vertical direction1201, but may also be in horizontal direction without causing functional difference. Channel201/101position relative to the MAG123and DMAG120inFIG.22Ais “Position1”.

FIG.22Bis similar toFIG.11, where channel holder1081moves channel201/101away from MAG123and come into contact with, or is in close proximity to, DMAG magnet120at the top surface of holder1081, where the magnet120may fit into the notch of holder1081to provide highest magnetic field on cells10/20conglomerate in channel201/101. Cells10/30form conglomerate after magnetic separation by MAG and do not break free from the conglomerate automatically due to SPL2on cells10/30not self-demagnetize when they are part of a conglomerate. By removing cells10/30gradually with magnetic field gradient from magnet120, for example cells10/30with higher magnetic moment SPL2that respond to weaker magnetic field from DMAG120faster, conglomerate may reach to a critical volume that remaining cells10/30in the conglomerate do not see enough magneto-static field from other cells10/30and will self-demagnetize into individual cells10/20due to the regained superparamagnetic nature of SPL2. Therefore, to dissociate cells10/30from conglomerate, removing certain amount of cells10/30, or breaking up the conglomerate from a continuous large piece into multiple smaller pieces will help cells10/30to achieve self-demagnetization. Channel201/101position relative to the MAG123and DMAG120inFIG.22Bis “Position2”. Channel201compared to channel101may have an advantage during cells10/30dissociation by DMAG magnet120, as channel201provides a larger channel space that allows farther separation between free cells10/30and from conglomerate, or between broken-up conglomerate pieces, which helps reduce magneto-static coupling and enhances self-demagnetization speed of SPL2on cells10/30. For flexible channel201, before Position2or at Position2, it is preferred to fill the channel201with additional buffer fluid to return the channel201to circular shape for larger channel space.

FIG.22Cillustrates the cells10/30in the channel201/101ofFIG.22Bbeing dissociated from conglomerate by the DMAG magnet120at Position2.

FIG.22Dillustrates the channel holder1081moves channel201/101fromFIG.22CDMAG Position2to a position, “Position3”, between MAG123and DMAG magnet120. At Position3, combined field on the cells10/30within channel201/101may be the smallest, which may help SPL2to self-demagnetize. Channel201/101may be kept at Position3for extensive time to allow SPL2and cells10/30to fully self-demagnetize and conglomerate to dissociate.

For an effective break up of conglomerate, mechanical agitations may be added to the conglomerate by the magnetic force exerted by MAG and DMAG magnets. For example, channel holder1081may alternate channel201/101between Positions1and2, or Positions2and3, or Positions1,2and3, such that alternating magnetic force by MAG and DMAG may move whole or part of the conglomerate in the channel space, thus helping break up the conglomerate into smaller pieces or cause enough cells10/30to break free from the conglomerate and conglomerate may self-dissociate. After conglomerate is sufficiently dissociated, free cells10/30may be flushed out of channel201/101at Position3or Position2.

FIG.23Aillustrates mechanical vibration may be applied to the channel holder1081by a motor130when channel201/101is at Position2or Position3ofFIG.22BandFIG.22C. Such vibration may be transferred from holder1081through wall of channel201/101and into the fluid within the channel201/101to cause localized turbulence flow at various locations within the channel201/101, which may help mechanically break up the conglomerate into small pieces to assist conglomerate dissociation.

FIG.23Billustrates ultrasound vibration by a piezoelectric transducer (“PZT”)131may be applied to the channel holder1081. Similar toFIG.23A, ultrasound vibration may be transferred into the fluid within the channel201/101to cause localized high frequency turbulence within the channel201/101, which may help mechanically break up the conglomerate into small pieces to assist conglomerate dissociation.

FIG.23Cillustrates mechanical vibration ofFIG.23Amay be applied to the channel201/101wall directly by motor130.

FIG.23Dillustrates ultrasound vibration ofFIG.23Bmay be applied to the channel201/101wall directly by PZT131.

FIG.23Eis a side view of the channel201/101along the direction61as inFIG.22D. Arrow1030represents alternating direction pulsed fluid flow may be applied to the channel liquid sample to produce a flow jittering in the liquid within the channel201/101, which may also produce local turbulence flow with fluid in channel201/101to help mechanically break up the conglomerate into small pieces to assist conglomerate self-dissociation.FIG.23Ealternating pulsed flow may be combined withFIG.23AthroughFIG.23Dvibration methods to apply to channel201/101at Position2or Position3ofFIG.22BthroughFIG.22D.

When conglomerate in channel201/101is of large size, multiple rounds of cells10/30dissociation withFIG.22BtoFIG.23Emethods, and flushing of cells10/30out of channel201/101, may be used. During each flush, a certain part of cells10/30may be washed out of channel, making dissociation of remaining cells10/30still in the conglomerate in channel201/101easier in next round.

FIG.24Ais similar toFIG.22B, where channel holder1081is in contact, or in close proximity to, DMAG magnet120after cells10/30are magnetically separated by MAG123.

Different than inFIG.22B, DMAG magnet120ofFIG.24Ais positioned on the side of and away from MAG123, and holder1081is also rotated compared toFIG.22Bto fit its top surface notch to the magnet120. Placement of magnet120inFIG.24may reduce magnetic field interference between MAG123and DMAG magnet120. Channel201/101position relative to the MAG123and DMAG120inFIG.24Ais “Position12”.

FIG.24Billustrates that after cells10/30are dissociated at Positon12ofFIG.24A, the channel201/101together with channel holder1081ofFIG.24Aare rotated away from magnet120ofFIG.24Ainto a position between MAG123and DMAG magnet120, where combined magnetic field from MAG123and DMAG magnet120on channel201/101and cells10/30therein is lowest, which is similar to Position3ofFIG.22D. Channel201/101position relative to the MAG123and DMAG120inFIG.24Bis “Position13”.

FIG.25Aillustrates DMAG structure that is same as inFIG.22B, where DMAG structure includes only permanent magnet120with magnetization1201.

FIG.25Billustrates DMAG structure that includes permanent magnet120and a soft magnetic pole1202with convergence shape towards channel201/101. Soft magnetic pole1202convergence shape helps concentrate magnetic flux from magnet120to produce higher field and high field gradient on cells10/30in channel201/101at Position2to more effectively demagnetize and dissociate the conglomerate of cells10/30.

FIG.25Cillustrates DMAG structure that includes permanent magnet120and a pair of soft magnetic poles1203and1204. Magnetization1201of magnet120is in horizontal direction, and each of poles1203and1204has an convergence shape pointing towards channel201/101, where the convergence ends of poles1203and1204form a DMAG gap sitting in, or in close proximity to, the channel holder1081top surface notch, where flux from magnet120is conducted by the poles1203and1204and concentrated in the DMAG gap to produce high field and high field gradient on cells10/30in channel201/101at Position2to more effectively demagnetize and dissociate the conglomerate of cells10/30.

FIG.25Dillustrates DMAG structure that includes an electromagnet including a soft magnetic core1205and coils1206, where electric current following in the coils1206may produce magnetization in core1205in directions of1207, and core1205functions like magnet120to product magnetic field on cells10/30in channel201/101at Position2to demagnetize or dissociate the conglomerate of cells10/30. By changing the electric current amplitude and direction in coils1206, magnetic field from core1205on cells10/30may change strength and direction. In one embodiment, DC current is applied to coils1206. In another embodiment, AC current with alternating polarities is applied to coils1206. In yet another embodiment, current applied to coils1206is programmed to vary in amplitude, or in direction, or in frequency, or in amplitude ramp up or ramp down rates, to more effectively demagnetize and dissociate the conglomerate of cells10/30.

FIG.25Eillustrates that motor130as shown inFIG.23Amay produce mechanical vibrations on DMAG structure ofFIG.25C, such vibrations may transfer from DMAG structure to holder1081through DMAG structure to holder1081contact, and finally transferred to fluid in channel201/101, where DMAG structure can be changed to any of DMAG structures described inFIG.25AthroughFIG.25D.

FIG.25Fillustrates that PAT131as shown inFIG.23Bmay produce ultrasound vibrations on DMAG structure ofFIG.25C, such vibrations may transfer from DMAG structure to holder1081through DMAG structure to holder1081contact, and finally transferred to fluid in channel201/101, where DMAG structure can be changed to any of DMAG structures described inFIG.25AthroughFIG.25D.

To achieve demagnetization and dissociation of cells10/30from conglomerate in channel201/101, an alternative method as described inFIG.26AthroughFIG.26Dmay be used without using a DMAG structure, where function of DMAG structure is achieved with same MAG.

FIG.26Ais same asFIG.22A, where channel201/101is at separation position and cells10/30are separated by magnetic field of MAG123in channel201/101, except channel holder1082may not have the top surface notch as holder1081. Channel201/101position relative to the MAG123inFIG.26Ais “Position21”.

FIG.26Billustrates channel201/101ofFIG.26Ais lifted from MAG123to a lower field position, “Position22”. At Position22channel201/101may rotate around its center as indicated by arrow210, preferable by 180 degrees. Such rotation may require channel201/101not being permanently fixed to holder1082

FIG.26Cillustrates channel201/101ofFIG.26Bafter rotation of 180 degrees at Position22, the cells10/30conglomerate formed on inner wall of channel201/101rotates together with channel wall to be at the top end of the channel201/101relative to MAG123.

FIG.26Dillustrates that channel201/101is moved from Position22closer to MAG123to a Position23in between Position21and Position22, where magnetic field from MAG123on cells12/30is stronger than Position22but weaker than Position21. Cells10/30in conglomerate at top end of channel201/101may then be pulled away by MAG123field from conglomerate and demagnetization and dissociation of conglomerate may start. The process ofFIG.26BthroughFIG.26Dmay repeat multiple times, where channel201/101may return to Position22from Position23to perform another rotation and then move back to Position23, until cells10/30are sufficiently dissociated in channel201/101. At end of demagnetization, cells10/30may be flushed out of channel201/101preferably at Position22. Mechanical vibrations and flow jittering as described inFIG.23CthroughFIG.23Emay be applied to channel201/101at Position22and Position23.

FIG.27Ais same asFIG.26A, where channel201/101is at separation position and cells10/30are separated by magnetic field of MAG123. Channel201/101position relative to the MAG123is “Position21”. Channel201/101is attached to holder1082inFIG.27A.

FIG.27Cillustrates that at Position22, dissociation of cells10/30in channel201/101may be achieved only through mechanical vibration exerted by motor130.FIG.27Cshows that motor130applies mechanical vibration to holder1082, where such vibration may be transferred from holder1082through wall of channel201/101and into the fluid within the channel201/101to cause localized turbulence flow at various locations within the channel201/101, which may help mechanically break up the conglomerate into small pieces to assist self-dissociation of cells10/30conglomerate. Motor130may also exert vibration directly on channel201/101as shown inFIG.23Cinstead of through holder1082. Alternating direction pulsed fluid flow as described inFIG.23Emay be applied to the channel liquid sample to produce a flow jittering in the liquid within the channel201/101at the same time of motor130vibration application.

FIG.27Dillustrates that at Position22, dissociation of cells10/30in channel201/101may be achieved primarily through ultrasound vibration exerted by PZT131.FIG.27Dshows that PZT131applies ultrasound vibration to holder1082, where the ultrasound vibration may be transferred into the fluid within the channel201/101to cause localized high frequency turbulence within the channel201/101, which may help mechanically break up the conglomerate into small pieces to assist self-dissociation of cells10/30conglomerate. PZT131may also exert ultrasound vibration directly on channel201/101as shown inFIG.23D. Alternating direction pulsed fluid flow as described inFIG.23Emay be applied to the channel liquid sample to produce a flow jittering in the liquid within the channel201/101at the same time of PZT131ultrasound vibration application.

FIG.28AthroughFIG.30Bdescribe embodiments of methods to assist cells10/30conglomerate dissociation by mechanical agitations, which may be applied to channel201/101as inFIG.27Bat Position22and applied to channel201/101as inFIG.22BthroughFIG.22D,FIG.23AthroughFIG.23D,FIG.24AthroughFIG.25F,FIG.26BthroughFIG.26D,FIG.27BthroughFIG.27D.

FIG.28Ashows a side view of channel201/101and holder1082along direction61ofFIG.27B, where cells10/30are magnetically separated by MAG field and form conglomerate on lower side of the channel201/101wall. Channel mounts1073may be used to attach channel201/101to channel holder1082. Channel mounts1073may fix channel201/101at sections attached to mounts1073as anchors against channel201/101deformation, compression or elongation during mechanical agitation process. Channel mounts1073may also perform a valve function that closes fluid flow into or out of flexible channel201section between two channel mounts1073before mechanical agitation process ofFIG.28A, such that fluid enclosed in channel201may more efficiently produce localized turbulence within the channel201.FIG.28Aillustrates that an externally applied force300may stretch or deform the channel201/101in a direction away from the holder1082, for example perpendicular to the channel201/101length direction. Such deformation or stretch of channel201/101builds up elastic energy in the channel201/101wall material.

FIG.28Billustrates that force300ofFIG.28Ais released, and elastic energy built up in channel201/101wall acts to spring back channel201/101towards its original non-deform and non-stretch position. Depending on channel201/101wall material property, such spring back may provide a transient turbulence flow at various locations within the channel201/101, which may help mechanically break up the cells10/30conglomerate into smaller pieces to assist self-dissociation of cells10/30conglomerate. After release of force300and channel201/101spring back, alternating flow1030may be applied similarly as inFIG.23Eto assist dissociation process of conglomerate of cells10/30, where valve function of1073may turn off to allow fluid flow within channel201/101.

TheFIG.28AandFIG.28Bchannel201/101deform/stretch and release process may be repeated as many times as needed until conglomerate of cells10/30are sufficiently dissociated, which may then be flushed out of channel201/101bybuffer fluid.

FIG.29Aillustrates an alternative method of mechanical agitation fromFIG.28A. Every aspect is same as inFIG.28A, except that a compressive force302may be applied to compress channel201in direction perpendicular to the channel201length direction, for example compressing channel201against channel holder1082as shown inFIG.29A. As liquid within channel201has limited compressibility, force302may cause channel201/101wall to expand in direction perpendicular to the view ofFIG.29A, i.e. in direction perpendicular to both channel length direction and force302direction. Such expansion of channel201/101wall will again build up elastic energy in the channel201wall material.

FIG.29Bis same asFIG.28Bin every aspect, except thatFIG.29Bis after compressive force302ofFIG.29Ais released, and elastic energy built up in channel201wall acts to spring back channel201to its original non-compressed shape. Such spring back may provide a strong transient turbulence flow at various locations within the channel201, which may help mechanically break up the cells10/30conglomerate into smaller pieces to assist self-dissociation of cells10/30conglomerate. After release of force302and channel201shape spring back, alternating flow1030may be applied similarly as inFIG.23Eto assist dissociation process of conglomerate of cells10/30, where valve function of1073may turn off

TheFIG.29AandFIG.29Bchannel201compression and release process may be repeated as many times as needed until conglomerate of cells10/30are sufficiently dissociated, which may then be flushed out of channel201.

FIG.30Aillustrates another alternative method of mechanical agitation. Every aspect is same as inFIG.28A, except that rotational twisting force303or304may be applied to channel201to twist channel201along channel length direction, as shown inFIG.30A. In one embodiment, only one of rotational force303or304is applied to one end of channel201. In another embodiment, both rotational force303and force304are applied to difference ends of the channel201in opposite rotational directions to cause the channel201to twist along channel length direction. Such twist deformation of channel201will again build up elastic energy in the channel201wall material.

FIG.30Bis same asFIG.28Bin every aspect, except thatFIG.30Bis after rotational force303and force304ofFIG.30Aare released, and elastic energy built up in channel201wall acts to spring back channel201towards its original non-twisted shape. Such spring back may provide a strong transient turbulence flow at various locations within the channel201, which may help mechanically break up the cells10/30conglomerate into smaller pieces to assist self-dissociation of cells10/30conglomerate. After release of forces303and304, and channel201shape spring back, alternating flow1030may be applied similarly as inFIG.23Eto assist dissociation process of conglomerate of cells10/30, where valve function of1073may turn off.

TheFIG.30AandFIG.30Bchannel201twist and release process may be repeated as many times as needed until conglomerate of cells10/30are sufficiently dissociated, which may then be flushed out of channel201by buffer fluid.

Mechanical forces300,302,303and304may be applied by mechanical structures that are motorized and able to apply such forces repeatedly to channel201, examples may include a flap for providing force300, a compressor for provide force302, and twisters for providing forces303and304.

FIG.31is a schematic diagram illustrating a method to use MAG to separate biological entities conjugated with magnetic labels, for example cells10/30, from a fluid solution. MAG channel ofFIG.31may be any of channels101,201,301,320, or330described in any of the figures of this specification, and MAG ofFIG.31may be any of the MAG121,122,123,124,125,126,127,128, or129described with the corresponding channel in any of the said figures. Method ofFIG.31may include the following steps in sequence. In step400, MAG channel is positioned with its outside wall contacting MAG wedge surface or pole tip ends, i.e. separation position of Position1or Position21as inFIG.5,FIG.10,FIG.12,FIG.14,FIG.17,FIG.19,FIG.20A,FIG.20C,FIG.21A,FIG.21C,FIG.22A,FIG.26A,FIG.27A. In step401, fluid sample is flown through the MAG channel in separation position. Then in step402, positive entities with magnetic labels SPL2attached, for example cells10/30, and free magnetic labels SPL2within the fluid sample are attracted by the magnetic field of MAG and agglomerate at the MAG channel wall against the MAG wedge or MAG pole tip ends. Meanwhile, in step4020, negative entities without magnetic labels SPL2attached pass through the MAG channel without being attracted. The negative entities may then be processed directly in subsequent procedures as shown by path427, where subsequent procedures may include entities analysis407, for example processes as included inFIG.79throughFIG.81, or negative entities may be passed for continued process408, for example through a UFL device as shown inFIG.46AthroughFIG.46C,FIG.50throughFIG.52, or through repeated MAG process as inFIG.54AandFIG.54B. After step402, in step403, sample may be depleted at input of the MAG channel and magnetic separation of positive entities may be completed. In step404, which is an optional step, buffer fluid may be flown through MAG channel with MAG channel still at separation position to wash off any negative entities without magnetic labels SPL2but may have resided with the conglomerate of positive entities due to non-specific bindings. Then in step405, MAG channel may be moved away from MAG to dissociation position including Position2and Position22at inFIG.11,FIG.22B,FIG.22D,FIG.24B,FIG.26B,FIG.26D,FIG.27B, and magnetic dissociation451, as shown inFIG.22AthroughFIG.26D, or mechanical dissociation452as shown inFIG.27CthroughFIG.30B, or magnetic together with mechanical dissociate453may be applied to the positive entities in MAG channel. In step406, buffer fluid may be flown through MAG channel to flush out dissociated positive entities. If positive entities are not completely dissociated,465shows that repeated dissociation process405may be applied to remaining positive entities in MAG channel after prior flush out step, until positive entities are sufficiently dissociated and flushed out of the MAG channel. In the case that fluid sample is in large volume, fluid sample may be separated into multiple sub-volumes, where after process of a sub-volume from step400to step406, a next sub-volume may be input into the MAG channel starting from step400for continued process as shown by461until completion of the fluid sample of the large volume. After positive entities are collected after step406, they may be processed in subsequent procedures as shown by path428, where subsequent procedures may include entities analysis407or continued process408.

FIG.32illustrates a method to align channel201/101to MAG gap of MAG123device. Precise alignment of channel201/101to MAG wedge or MAG pole tip ends is of importance as descried in embodiments of this invention. InFIG.32, side fixtures1074may be used to align and position channel201/101to designated locations on channel holder1081or1082, where the fixtures1074may be fit into a pre-defined slot, notch, clip or other physical features on the sides of the channel holder1081/1082. In one embodiment, channel201/101may be slightly stretched in channel length direction, thus channel201/101may have a reduced width2011in between the fixtures1074, where such stretch helps guarantee a straight channel which may be then aligned with a straight MAG wedge of MAG123. After channel201/101is attached to holder1081/1082by fixtures1074, holder1081/1082may then move channel201/101to separation position, where holder1081/1082may have pre-determined physical orientation to MAG123, for example a hinge, which aligns channel201/101to MAG wedge or MAG pole tip ends of MAG123precisely. Fixtures1074may be the same as1073as inFIG.28AthroughFIG.30B.

FIG.33AthroughFIG.37illustrate method to utilize peristaltic pumps in embodiments of this invention.

FIG.33Aillustrates a typical peristaltic pump500, which includes a rotor501, drivers502attached to the rotor501, and pump tubing504/505, where tubing504is fluid incoming section and tubing505is fluid outgoing section of the same pump tubing. When rotor501rotates in direction503, drivers502will squeeze pump tubing and force fluid to move from incoming section504to outgoing section505in directions5041and5051respectively. In the case when rotor rotates reversely to direction503, fluid moves from outgoing section505to incoming section504of the pump tubing. Connectors506and507may be optional connections to incoming fluid line and outgoing fluid line508respectively. Advantage for peristaltic pump is the tubing504/505may be included as a continuous part of an enclosed fluid line as shown inFIG.55AthroughFIG.60B, which may be made disposable and single use, as well as sterile for clinical purpose. However, due to the spaced drivers502along the circumference of the rotor501, flow rate of fluid output from section505has pulsation behavior, where flow rate increases and decreases with moving of each of the driver502. Such pulsation is not desired for MAG and UFL fluid driving.FIG.33Ashows output section505outputs fluid through connector to channel508. Channel508is preferred to be flexible tubing. Channel508may also be a section of channel201. Flow limiter parts509and510function together to effectively clamp onto the channel508to reduce the fluid flow rate passing through the limiter. With reduced flow rate through the limiter, continued fluid output from pump500section505into the channel508will build up fluid pressure within channel508. Due to the flexible nature of channel508, channel508may enlarge its width perpendicular to the channel length direction, and forms fluid reservoir within channel508with elastic stress built up in channel wall. During pulsation of output flow from pump500, when5051flow rate increases, channel508width will increase to build up stress in508channel wall and pressure within channel508and increase volume of channel508absorbs most of the instantaneous incoming flow, while flow rate520through limiter509/510into channel501shows smaller increase. When5051flow rate decreases, built-in elastic stress in channel508wall and fluid pressure in channel508continues to push fluid through the limiter509/510, and flow rate520shows smaller flow rate decrease.

FIG.33Billustrates top-down view of the inner structure of first type flow limiter509along the direction63.FIG.33Bshows that flow limiter509has a shaped trench5011, which allows fluid to flow through channel508when limiters509and510clamp onto channel508as inFIG.33A. Trench5011has entrance width511to incoming fluid and exit width512to channel201, where width511may be larger than width512. Decreased trench5011width from511to512reduces the flow rate through the limiter509/510. Flow limiter510may have same top down view and structure as limiter509when view in direction opposite to63.

FIG.33Cillustrates a second type flow limiter in same view asFIG.33A, where after flow limiters509and510clamp onto channel508, flow limiters509/510form an effective opening of514towards channel508, and opening of513towards channel201. Opening513may be smaller than opening514, which reduces flow rate through the limiter509/510.

FIG.34Ais same asFIG.33Aexcept flow limiters509/510are disengaged from the flexible channel508, where flow from pump500through channel508and channel201is continuous without limiter509/510and there is no elastic stress built up in channel508wall.

FIG.34Bis a schematic illustration of fluid flow rate520as inFIG.34Asituation, which shows large pulsation in flow rate520.FIG.34Bshows the example520flow rate value vs pump500operation time from pumping start to pumping end. Value521illustrates the high flow rate and value522illustrates low flow rate of the pulsation behavior.

FIG.35Ais same asFIG.33A, where flow limiters509/510are clamped upon flow channel508, flow rate through the flow limiters509/510is reduced, and channel508has enlarged channel width with elastic stress built up in channel508wall.

FIG.35Bis a schematic illustration of fluid flow rate520as inFIG.35Asituation, which shows pulsation reduction in flow rate520compared toFIG.34B. Value523corresponds to value521ofFIG.34B, and value524corresponds to value522ofFIG.34B.FIG.35Billustrates that limiters509/510effectively reduce520flow rate pulsation. Due to the channel508liquid pressure build up at the start of pumping, and channel508liquid pressure dissipation at end of pumping, while limiters509and510are engaged, a flow rate ramp up slope5221after pump start and flow rate ramp down slope5222after pump end may exist inFIG.35B.

FIG.36AandFIG.36Billustrate method to use flow limiter509/510to generate instantaneous high flow rate short pulse through channel201for flushing out magnetically separated entities, for example dissociated cells10/30.

FIG.36AillustratesFIG.33AandFIG.35Asituation, where flow limiters509and510are clamped onto the flexible channel508while pump500pumps fluid into channel508, where pressure is built up within the flexible channel508, and elastic stress is built up in wall of channel508. Line525represents a continuous channel201from after the limiters509/510to channel201over MAG structure. Flow rate5201represents averaged flow rate of flow rates523and524ofFIG.35Bwhen flow limiters509and510are engaged.

FIG.36Billustrates that flow limiters509and510are disengaged from the flexible channel508, similar toFIG.34Asituation, while pump500still pumps fluid into channel508, or immediately after pump500stops pumping and before pressure within channel508dissipates. At disengagement of limiters509and510, liquid pressure in channel508and elastic stress in wall of channel508produces an instant high-speed fluid pulse flow5202into channel201which may flush the magnetically separated entities out of the channel201. Such high speed short pulse flow5202may help achieve complete flush out of cells10/30with small volume of fluid that is originally contained in channel508ofFIG.36A.FIG.36Balso shows that a rigid cladding structure1075may be put into contact with channel201to help reduce deformation of flexible channel201during the cells10/30flush out to maintain the flow speed in channel201.

FIG.37is a schematic illustration of fluid flow rate pulse created by the flow limiter operation ofFIG.36AtoFIG.36B, where5201is the fluid flow rate in channel201before limiters509and510release, and5202is flow rate peak value after limiters509and510release.

FromFIG.33AthroughFIG.36B, channel508is a flexible channel, while channel201may be replaced by a rigid channel101,301,320, or330.

FIG.38AthroughFIG.43describe various embodiments of micro-fluidic chip (“UFL”) and method of use.

FIG.38Ais a top-down view of a first UFL embodiment UFL600, where micro-fluidic channels are formed as trenches into a substrate material611. UFL contains an entity fluid6020inlet602, a buffer fluid6040inlet604, a main channel601, a large entities6070outlet607, and a small entities6090outlet609. Two side channels603connect inlet602to main channel601from the two sides of the main channel601. Inlet604is directly connected to the main channel601at the center of the main channel601. Main channel601connects to outlet607at the center of the main channel601, and connects to outlet609from two sides of main channel601through two side channels608. Entities fluid6020contains both large entities6070and small entities6090. Buffer fluid6040is fluid for providing UFL function but without biological entities. Large entities6070fluid from outlet607contains mainly large entities6070and buffer fluid6040. Small entities6090fluid from out609contains mainly small entities6090and fluid of entities fluid6020and may contain certain amount of buffer fluid6040. During operation of UFL600, buffer fluid6040and entity fluid6020are simultaneously pumped into outlets604and602respectively, where buffer fluid6040flows along center line of the main channel601and entity fluid flows close to the two side of the main channel as laminar flow. Buffer fluid6040carries large entities6070to exit outlet607and entity fluid carries remaining small entities6090to exit outlet609. Channel601is substantially straight and linear along channel length direction from inlet604to outlet607.

FIG.38Bis a cross-sectional view of a portion of theFIG.38AUFL600along direction64, which includes entity fluid inlet602, buffer fluid inlet604, and part of the UFL main channel601.FIG.38Billustrates UFL600is compose of two components, substrate611and cover610. Inlets602and604, outlets607and609, channels601,603and608are formed in substrate611as trenches of same depth627and preferably formed in a single step. In one embodiment, depth627is between 100 nm to 500 nm. In another embodiment, depth627is between 500 nm to 1 um.

In yet another embodiment, depth627is between 1 lum to 10 um. In yet another embodiment, depth627is between 10 um to 100 um. In yet another embodiment, depth627is between 100 um to 1 mm. Cover610contains external access ports to inlets and outlets of UFL600to allow entities fluid6020and buffer fluid6040to enter inlets602and604, and to allow large entities6070fluid and small entities6090fluid to exit outlets607and609. Inlets602and604, outlets607and609are shown to be circular shape inFIG.38A, but may be any other shape, including ellipse, square, rectangle, triangle, polygon, as suitable for application. Access ports of cover610are clearances, i.e. holes, through cover610directly over the inlets and outlets602,604,607and609.FIG.38Bshows example of access ports621and641clearances matching to inlets602and604positions. After manufacture of the UFL600substrate611with the trenches of inlets, outlets and channels, and cover610with the access ports, cover610is positioned over the substrate611to form enclosed channels601,603and608, where cover610may bond to substrate611through any of: (1) surface to surface Van der Waals force; (2) gluing; (3) ultrasound thermal melting when one or both of substrate611and cover610being made of plastic or polymer material. Access ports clearances of cover610, for example621and641to inlets and outlets602,604,607and609are preferred to be smaller in size than the corresponding inlets and outlets, which allows for positioning error during cover610to substrate611alignments without causing function loss of UFL due to misalignment. Injectors6021and6041then show example of possible external fluid injection to inlets of UFL600through cover610access ports clearance, where the injectors6021and6041may have a larger nozzles size than the matching access ports621and641for managing positioning errors between injectors and access ports.FIG.38Bshows that entities fluid6020containing large entities612and small entities613, which may be injected by injector6021, passing through assess port621and into inlet602and passing into main channel601as side laminar flows, while buffer fluid6040may be injected by injector6041, passing through assess port641and into inlet604and passing into main channel601as center laminar flow.

Substrate611and cover610may be each composed of any of: glass, silicon, quartz, aluminum-titanium-carbon (AlTiC), SiC, SiN, silicon-oxide, alumina, plastic, PDMS, polymer, ceramic, or metal, where metal may be composed any one or any alloy of aluminum, iron, nickel, titanium, chromium, platinum, tungsten, rhenium, copper, gold, silver. In one embodiment, forming of inlets, outlets and channels in substrate611includes the steps of: (1) providing a substrate611having one substantially flat surface; (2) forming etching mask on top of said flat surface; (3) etch of substrate with a first etching method including: wet etch with fluid chemical, dry etch with chemical gas, plasma enhanced dry etch, sputter etch with ion plasma, and ion beam etch (IBE). In forming of etch mask of step (2), etch mask may be composed of photo resist (PR), which may include deposition or spin coating of PR on said flat surface; exposure by optical or ion/electron radiation with patterns of inlets, outlets and channels; development of PR after said exposure, where remaining PR with said patterns serves as etch mask. Etch mask may also be made of a hard mask material that has lower etch rate than the substrate material under the first etching method, and step (2) may include: deposition of a hard mask layer on said flat surface; deposition or spin coating PR layer on hard mask layer; exposure of said PR by optical or ion/electron radiation with patterns of inlets, outlets and channels, development of PR after said optical exposure, where remaining PR with said pattern serves as etching mask of said hard mask; hard mask is etched through with a second etch method including any of: wet etch with fluid chemical, dry etch with chemical gas, plasma enhanced dry etch, sputter etch with ion beam; removal of remaining PR layer. Second etch method and first etch method may be different in type, or different in chemistry.

In another embodiment, inlets, outlets and channels in substrate611may be formed by thermal press involving using a heated stencil with physical pattern of the inlets, outlets and channels to melt and deform part of substrate611to construct the inlets, outlets and channels, then cooling down substrate611and remove the stencil. In thermal press, substrate material is preferred to be plastic or polymer. In yet another embodiment, inlets, outlets and channels in substrate611may be formed by imprint, which involves using a stencil with physical pattern of the inlets, outlets and channels to imprint into a partially or completely melt substrate611, and then cooling the substrate611and finally removing stencil, where cooled substrate retains the pattern transferred from stencil of the inlets, outlets and channels. In imprint, substrate material is preferred to be plastic or polymer. In another embodiment, inlets, outlets and channels are formed in substrate611by injection molding, where melted substrate611materials are injected into a mold cavity where substrate611body with engraved inlets, outlet and channels are defined by the mold cavity. Cover610may compose similar to substrate611materials, and assess ports of cover610may be formed in cover610similarly as the inlets, outlet and channels formed in substrate611as described above.

FIG.38Cis a schematic diagram illustrating a single fluidic pressure node615created between two side walls of the UFL600channel601ofFIG.38Aby acoustic vibration, for example ultrasound vibration, generated by an acoustic generation device, for example a PZT614.FIG.38Cis a cross-section view along direction65ofFIG.38Afor part of the UFL600including main channel601, substrate611, cover610and PZT transducer614attached to the bottom of substrate611.FIG.38Cshows that after injection of entities fluid6020and buffer fluid6040, entities fluid6020containing large entities612and small entities613mainly flow along the edges of the channel601as laminar flow. AC voltage is applied to PZT614, where frequency (Fp) of AC voltage is preferred to be at a frequency matching to the PZT resonance frequency (Fr). PZT614produces ultrasound vibrations to the substrate611at frequency Fp. Said ultrasound vibrations transfer to the fluid contained in channel601. Channel601has channel width625defined as the normal distance between the two side walls of channel601. In one embodiment, width625is between 100 nm to 1 um. In another embodiment, width625is between 1 um to 10 um. In yet another embodiment, width625is between 10 um to 100 um. In yet another embodiment, width625is between 100 um to 500 um. In yet another embodiment, width625is between 500 um to 5 mm. When channel width625is half wavelength, or an integer multiple of half wavelength, of the ultrasound mode in the fluid within channel601at frequency Fp, standing wave may be present in between the two side walls of channel601as indicated by the dashed lines626.FIG.38Cshows when channel width625is half wavelength of fluid ultrasound mode at frequency Fp, where a single fluidic pressure node615is formed along the center line of channel601in the direction of channel length, which is perpendicular to the view ofFIG.38C. In another embodiment, channel width625is an integer times of half wavelength of fluid ultrasound mode at frequency Fp, where integer is larger than 1, and said integer number of fluidic pressure nodes may then be formed across the width635with each node being a line along the direction of channel length. Presence of standing wave626and pressure node615exert acoustic force, which is shown inFIG.38Das arrows628, on entities in the entities fluid laminar flow along the side walls of channel601and cause large size entities6070to move close to center node615during flowing through the channel601. Said acoustic force628has the characteristics of: (1) largest amplitude close to channel601side walls with force direction pointing from the side walls towards the node615; (2) smallest force, or close to zero force, around node615; (3) being linearly proportional to size of the entities; (4) is a function of the density and compressibility of both the buffer fluid6040and the entities. Due to these characteristics, with proper optimization of buffer fluid composition, buffer fluid6040laminar flow speed, and entities fluid6020laminar flow speed, large entities612may be optimized to preferably break the laminar flow barrier to enter the buffer laminar flow due to a larger acoustic force acting on large entities612, and be concentrated around the center node615.

FIG.38Dis a schematic diagram illustrating the fluid acoustic wave ofFIG.38Ccausing larger size entities612to move into buffer fluid laminar flow around center of the channel601. When fluid within the channel601exits the channel to outlets607and609, channel601center sub-channel width651ofFIG.38Ato outlet607may be much smaller than the width625of the channel601, thus only allow large entities612at center flow within channel601to exit outlet607as large entities6070fluid. While smaller entities613mainly in the close to side wall laminar flow exit channel601through side channels308to exit from outlet609as small entities6090fluid.

Frequency Fp of PZT614vibration in one embodiment is between 100 kHz to 500 kHz, between 500 kHz to 1 MHz in another embodiment, between 1 MHz to 3 MHz in yet another embodiment, between 3 MHz to 5 MHz in yet another embodiment, between 5 MHz to 10 MHz in yet another embodiment, between 10 MHz to 50 MHz in yet another embodiment, and between 50 MHz to 100 MHz in yet another embodiment. InFIG.38CandFIG.38D, PZT614may also be attached to top of cover610inFIG.38CandFIG.38D, and ultrasound vibrations from PZT614is transferred from PZT614through cover610to fluid within channel601, or through cover601to substrate611and then to the fluid within channel601.

FIG.39is a schematic diagram illustrating methods to use a UFL to separate biological entities of different sizes, where UFL may be UFL600fromFIG.38AorFIG.40A, UFL620,630and640fromFIG.41AthroughFIG.43. Sequential steps of701to705and706are substantially similar as described inFIG.38A,FIG.38B,FIG.38C, andFIG.38D, except steps703and704refer to possibility of multiple pressure nodes, as shown inFIG.41BandFIG.42B. Step707entities analysis can be performed on both the large entities6070and small entities6090, and may include processes903,904,905,906,5824,5825,5826as described inFIG.53,FIG.79,FIG.80,FIG.82andFIG.83on corresponding UFL output samples. Continued process708, for example through a MAG device as shown inFIG.44AthroughFIG.45C,FIG.47throughFIG.49, or through cascaded UFL process as inFIG.54C.

FIG.40Ais a cross-sectional view of a portion of a UFL650similar toFIG.38B. UFL650is identical to UFL600from a top-down view as inFIG.38A, except that a uniform soft magnetic layer (“SML”)616is deposited on top the substrate611of UFL650, and patterned together with the substrate611to form inlets602and604, outlets607and609, and channel601,603and608. SML616may be composed of at least one element from iron (Fe), cobalt (Co), and nickel (Ni). SML616thickness6164is between 10 nm to 100 nm in one embodiment, between 100 nm to 1 um in another embodiment, between 1 um to 10 um in yet another embodiment, between 10 um to 100 um in yet another embodiment, between 100 um to 1 mm in yet another embodiment, and between 1 mm to 3 mm in yet another embodiment. Deposition of SML layer616on substrate611may be by any of: electro-plating, vacuum plating, plasma-vapor-deposition (PVD), atomic layer deposition (ALD), chemical vapor deposition (CVD). Etching of layer616together with substrate611to form inlets602and604, outlets607and609, and channel601,603and608may be by any of: dry etch, plasma enhanced dry etch, ion plasma etch, and IBE. Layer616may be a continuous layer along the channel601length direction and forms as part of the side walls of the channel601.

FIG.40Bis similar asFIG.38Dand shows a schematic diagram illustrating the large entities612if concentrated by acoustic force628to the channel601center around the pressure node615and small entities613mainly remain around the channel601side wall. Additionally, a magnetic field617is applied in plane and induces magnetization6162in the SML layer616. For the SML layer616located as part of the side walls of the channel601, magnetization6162produces local magnetic field6163, which has strongest magnetic field strength and field gradient close to the channel601side walls. Field6163may help maintain magnetic small entities, for example free magnetic labels SPL2that is part of the entities fluid6020in positive sample after MAG separation as shown inFIG.82andFIG.83, to stay within the laminar flow close of channel601side walls and output from outlet609ofFIG.38A.

FIG.41Ais a top-down view of a second UFL embodiment UFL620, which is same asFIG.38A, except including a wider section6012of the main channel connecting between the inlet604, and the narrower channel section601ofFIG.38A. Slope6016represents a transition section6016from wider section6012to narrow section601. Channel sections6012and601are substantially straight and linear along channel length direction. Transition section6016may be a section of the main channel, where the main channel includes channel section6012connecting through the transition section6016to channel section601. Transition section6016functions to funnel fluid flow from wider section6012into the narrower section601. Channel wall of transition section6016may intersect with straight wall of wider section6012at a transition start point. Channel wall of transition section6016may intersect with straight wall of narrower section601at a transition stop point. In one embodiment, the channel shape of the transition section6016between transition start point and transition stop point may be a straight slope as shown inFIG.41A. In another embodiment, the channel shape of the transition section6016between transition start point and transition stop point may be a curvature, whereas the curvature may be tangential to one of, or both of, channel wall of wider section6012, and channel wall of narrower section601.

FIG.41Bis a cross-sectional view of UFL620along direction66inFIG.41A, which is across the wider section6012. Wider section6012has a channel width6252, which is the full wavelength of the ultrasound mode in the liquid within channel section6012at PZT614operating frequency Fp as described inFIG.38C, and effectively twice the channel width625of channel601as inFIG.38CandFIG.41C. Due channel width6252being equal to the full wavelength of ultrasound mode at Fp, two pressure nodes may exist in channel section6012, where acoustic force from the ultrasound mode may move and concentrate large entities612at each of the two nodes from the channel wall entity laminar flow.

FIG.41Cis a cross-sectional view of UFL620along direction65inFIG.41A, which is across the narrower section601, which is identical toFIG.38D. After fluid within channel section6012flows through the transition6016to channel section601, single pressure node of channel section601forces the large entities612to concentrate at channel section601center same as inFIG.41C. Wider section6012provides a first stage large entities612separation from small entities613. After transition section6016, flow rates of center buffer laminar flow and channel side wall entities laminar flow increase to about twice the speed of same flow in section6012due to the channel width reduction from6252to625. Channel section601provides a second stage large entities separation from small entities, together with the increase flow rate in channel section601, purity of large entities612in6070fluid output from outlet607, as well as purity of small entities613in6090fluid output from outlet609, may be enhanced compared to UFL600ofFIG.38A.

FIG.42Ais a top-down view of a third UFL embodiment UFL630, which is a further enhancement from the UFL620ofFIG.41A. Every aspect ofFIG.42Ais same asFIG.41A, except that when compared to UFL620ofFIG.41A, UFL630ofFIG.43Aincludes additional side channels6013that connect from around the transition section6016to side channels608, or in another embodiment directly to the outlet609, to divert side wall laminar flow of small entities613from wider section, or referred to as first stage section,6012, as shown inFIG.42B, directly to output6090without entering narrower section, or referred to as second stage section,601. Channel sections6012and601are substantially straight and linear along channel length direction. In one embodiment, side channels6013connect from first stage section6012before the transition start point of section6012intersecting section6016. In another embodiment, side channels6013connect from the transition start point of section6012intersecting section6016. In yet another embodiment, side channels6013connect from the within the transition section6016between the transition start point of section6012intersecting section6016and the transition stop point of section6012intersection section601. In yet another embodiment, side channels6013connect from the transition stop point of section6012intersecting section601. In yet another embodiment, side channels6013connect from the second stage section601after the transition stop point of section6012intersecting section601.

FIG.42Bis a cross-sectional view of UFL630along direction66inFIG.42A, which is across the wider section6012.FIG.42Bis identical toFIG.41B.

FIG.42Cis cross-sectional view of UFL630along direction65inFIG.42A, which is across the narrower section601and side channels6013. Compared toFIG.41C, side channels6013connecting from around the transition section6016ofFIG.42Acontains mainly, or purely, small entities613. While the channel601ofFIG.42Cshows large entities612separation and concentration to channel601center pressure node similar as inFIG.41C, but small entities613around section601channel walls is reduced in density when compared toFIG.41C. Due to the pre-channel section601small entities diversion by side channel6013, UFL630may have an even higher purity of large entities612in6070fluid output from outlet607, as well as higher purity of small entities613in6090fluid output from outlet609.

FIG.43is a top-down view of a fourth UFL embodiment UFL640having a multiple-stage UFL channel with sequential channel width reduction along the channel flow path.FIG.43shows a further enhancement in increasing large entities purity in6070and small entities purity in6090.FIG.43shows an additional wider width section6014is added between inlet604and channel section6012. Channel width of6014may be three times of the half wavelength of ultrasound mode of the liquid flowing through the UFL640channel at PZT frequency Fp, which is one half wavelength wider than channel width6252of section6012. Channel width of6014may also be wider than the channel width6252of next stage channel section6012by an integer times of the half wavelength, where said integer is larger than one. Channel section6014changes to reduced channel width section6012through a transition section6017. Side channels6015connect from around the transition section6017to side channels6013, or608, or directly to outlet609to divert small entities613from channel side wall laminar flow of section6014from entering section6012, thereby increasing purity of large entities concentration in section6012. Channel sections6014,6012and601are substantially straight and linear along channel length direction.

As a further extension fromFIG.43, a multiple-stage UFL640may have multiple channel sections along the UFL640channel flow path, where each earlier section of the UFL channel along the channel flow path may have a channel width that is wider than the immediate next section channel width by an integer number of a half wavelength of ultrasound mode in the fluid flow at the PZT frequency Fp, where said integer is equal to or larger than 1. Final channel section before flow exiting the outlets of the UFL640is preferred to have a channel width equal to said half wavelength in one embodiment, but may also have a channel width that equals to an integer number of said half wavelength in another embodiment where integer is larger than one. Side channels connecting to each of the transition area between adjacent channel sections divert small entities from the earlier channel in the entities laminar flow close to the earlier channel walls towards the outlet609to reduce number of small entities entry into immediate next stage channel section.

FIG.44AthroughFIG.65Billustrate various embodiments of method to utilize MAG and UFL device to separate biological entities from an entity fluid. For simplicity of description UFL600ofFIG.38Aand MAG123with channel201are used in the figures for explanation. However, UFL600may be replaced with UFL650,630,640ofFIG.40A,FIG.41A,FIG.42A,FIG.43, while MAG123may be replaced with MAG121,122,124,124,125,126,127,128,129and corresponding channel types as described in prior figures without limitation and without sacrifice of performance.

FIG.44Aillustrates first type sample processing method where biological sample is first passed through UFL600and the large entity output6070of UFL600is then passed through channel201adapted to MAG123, with a first type flow connector801connecting the UFL600large entity outlet607and MAG inlet flow as in step401ofFIG.31or step708ofFIG.39. For the in-series operation of UFL600and MAG123devices, optimal flow rate for UFL channel601and optimal flow rate for MAG channel201may be different. Optimal flow rate for UFL channel601acoustic force separation of large and small entities are determined by laminar flow condition, and separation efficiency between large and small entities. Optimal flow rate for MAG123separation is determined by the length of channel201and magnetic field force on magnetic labels attached to the entities. Direct fluidically coupled flow from UFL600outlet607to MAG channel201inlet will force the flow rate being the same through UFL600channel and MAG channel201, which may incur negative impact on separation efficiency for either one or both of UFL600and MAG123. It is necessary to decouple the fluid flows through UFL600and MAG123channel201. Flow connector801serves to decouple flow rates of the UFL600and MAG123. Output fluid6070is first injected into connector801through inlet8011, and fluid in connector801is output through outlet8012as flow as in steps401/708into inlet of channel201of MAG123. Both UFL600and MAG123channel201may operate at their respective optimal flow rate. In one embodiment where MAG123channel201optimal flow rate is larger than UFL600optimal flow rate, MAG123extracts fluid401/708from connector801faster than UFL600injects fluid6070into the connector801. A fluid level sensor100may be attached to connector801to sense fluid level remaining in connector801. If fluid level drops below a low threshold, sensor100may signal MAG123to pause flow as in steps401/708intake to wait for connector801internal liquid level to increase to another higher level before MAG123may restart extracting fluid as in steps401/708from connector801. In another embodiment where MAG123channel201optimal flow rate is smaller than UFL600optimal flow rate, MAG123extracts fluid as in steps401/708from connector801slower than UFL600injects fluid6070into the connector801. If fluid level increases above a low threshold, sensor100may signal UFL600to pause flow6070output to wait for connector801internal liquid level to drop to another lower level before UFL600may restart outputting fluid6070into connector801. Flow connector801may be in the design as shown inFIG.44A, where inlet8011is at a higher vertical location than outlet8012, where flow6070enters connector801and accumulates at outlet8012at inside of801due to gravity. Alternatively, liquid sample may be completely processed through UFL600first and stored in connector801. MAG123then extracts fluid from connector801as input into the MAG123channel201and completes processing of all liquid sample from connector801. Connector801may be made as part of an enclosed fluidic line, where during the path of flow6070from UFL600outlet607to inlet8011of connector801, to outlet8012, to flow as in steps401/708into inlet of channel201, fluid sample is not exposed to air, and being sterile.

FIG.44Billustrates first type sample processing method ofFIG.44Awith using a second type flow connector802connecting the UFL600large entity outlet607and MAG123channel201inlet. Connector802as shown inFIG.44Btakes the form similar to a vial. Flow6070enters connector802through a short length inlet tube8021of connector802and drips to bottom of the connector802due to gravity. Flow as in steps401/708is extracted from the fluid at the bottom of the connector802by a long length outlet tube8022to input of channel201. Fluid level sensor100may be attached to connector802to sense fluid level within connector802. UFL600and MAG123may both operate at their respective optimal flow rate, and fluid level sensor100may function to pause UFL600operation or MAG123operation with the same method as described inFIG.44A. Alternatively, liquid sample may be completely processed through UFL600and stored in connector802. MAG123then extracts fluid from connector801as input into the MAG123channel201and completes processing of all liquid sample from connector802. Connector802may be made as part of an enclosed fluidic line similar as connector801.

FIG.44Cillustrates first type sample processing method ofFIG.44Awith using a third type flow connector803connecting the UFL600large entity outlet607and MAG123channel201inlet. Connector803as shown inFIG.44Ctakes the form similar to a fluid bag or blood bag. Flow6070enters connector803through a bottom inlet8031fills connector803from bottom of the connector803due to gravity. Flow as in steps401/708is extracted from the fluid at the bottom of the connector803through outlet8032to input of channel201. Fluid level sensor100may be attached to connector803to sense fluid level within connector803. UFL600and MAG123may both operate at their respective optimal flow rate, and fluid level sensor100may function to pause UFL600operation or MAG123operation with the same method as described inFIG.44A. Alternatively, liquid sample may be completely processed through UFL600and stored in connector803. MAG123then extracts fluid from connector801as input into the MAG123channel201and completes processing of all liquid sample from connector803. Connector803may be made as part of an enclosed fluidic line similar as connector801.

FIG.45Aillustrates second type sample processing method where biological sample is first passed through UFL600and the small entity output6090of UFL600is then passed through MAG123, with first type flow connector801connecting the UFL small entity6090outlet609and MAG123channel201inlet.FIG.45Ais identical toFIG.44Ain every aspect except small entities flow6090from outlet609is injected into the inlet8011of connector801.

FIG.45Billustrates second type sample processing method where biological sample is first passed through UFL600and the small entity output6090of UFL600is then passed through MAG123, with second type flow connector802connecting the UFL small entity6090outlet609and MAG123channel201inlet.FIG.45Bis identical toFIG.44Bin every aspect except small entities flow6090from outlet609is injected into the inlet8021of connector802.

FIG.45Cillustrates second type sample processing method where biological sample is first passed through UFL600and the small entity output6090of UFL600is then passed through MAG123, with third type flow connector803connecting the UFL small entity6090outlet609and MAG123channel201inlet.FIG.45Cis identical toFIG.44Cin every aspect except small entities flow6090from outlet609is injected into the inlet8031of connector803.

FIG.46Aillustrates third type sample processing method where biological sample is first passed through MAG123channel201, and following procedure427or428ofFIG.31, the output of MAG123channel201is then passed through UFL600as entity fluid6020into inlet602as in step408ofFIG.31, with first type flow connector801connecting the MAG123channel201outlet and UFL600entity fluid6020inlet602. InFIG.46A, output from MAG123can be either negative entities that do not have attached SPL2, or positive entities separated by MAG123magnetic field and subsequently dissociated and flushed out of channel201as described inFIG.31. Similar as inFIG.44A, MAG123and UFL600may each operate with their respective optimal flow rate. Fluid level sensor100may be attached to connector801to sense fluid level remaining in connector801. Fluid level sensor100operates similarly as inFIG.44Ato sense fluid in connector801, and depending on the flow rate difference between MAG123and UFL600, may pause MAG123or UFL600flow to maintain fluid level in connector801above a low level or below a high level. Alternatively, liquid sample may be completely processed through MAG123first and stored in connector801. UFL600then extracts fluid from connector801as input into the inlet602and completes processing of all liquid sample from connector801. Connector801may be made as part of an enclosed fluidic line similarly as inFIG.44A.

FIG.46Bis same asFIG.46Ain every aspect, except replacing connector801with connector802, where operation of connector802and attached sensor100is same as describedFIG.44B.

FIG.46Cis same asFIG.46Ain every aspect, except replacing connector801with connector803, where operation of connector803and attached sensor100is same as describedFIG.44C.

FIG.47illustrates fourth type sample processing method where biological sample is first passed through multiple UFLs600, output fluids from the UFLs600, which can be either large entities6070or small entities6090, are then fed into inlets8011of a fourth type flow connector8010, and from connector8010outlets8012into the inlets of channels201of multiple MAGs123.FIG.47is functionally similar toFIG.44AandFIG.45A. Connector8010is also functionally same as connector801, except inlet8011of connector8010accept multiple fluid output from multiple UFLs600, and outlet8012of connector8010outputs to input of multiple channels201of multiple MAGs123.

FIG.48illustrates fifth type sample processing method where biological sample is first passed through multiple UFLs600, output fluids from the UFLs600, which can be either large entities6070or small entities6090, are then fed into inlets8021of a fifth type flow connector8020, and from connector8020outlets8022into the inlets of channels201of multiple MAGs123.FIG.48is functionally similar toFIG.44BandFIG.45B. Connector8020is also functionally same as connector802, except inlet8021of connector8020accept multiple fluid output from multiple UFLs600, and outlet8022of connector8020outputs to input of multiple channels201of multiple MAGs123.

FIG.49illustrates sixth type sample processing method where biological sample is first passed through multiple UFLs600, output fluids from the UFLs600, which can be either large entities6070or small entities6090, are then fed into inlets8031of a sixth type flow connector8030, and from connector8030outlets8032into the inlets of channels201of multiple MAGs123.FIG.49is functionally similar toFIG.44CandFIG.45C. Connector8030is also functionally same as connector803, except inlet8031of connector8030accept multiple fluid output from multiple UFLs600, and outlet8032of connector8030outputs to input of multiple channels201of multiple MAGs123.

In each ofFIG.47,FIG.48, andFIG.49, in one embodiment, same biological sample is divided and processed simultaneously through multiple UFLs600. In another embodiment, each of the UFLs600processes a different biological sample. Output from each UFL600, either large entities6070fluid from outlet607or small entities fluid from outlet609, shown as dashed lines inFIG.47,FIG.48andFIG.49, may be individually input into the inlet8011of connector8010ofFIG.47, or into inlet8021of connector8020ofFIG.48, or into inlet8031of connector8030ofFIG.49, as shown by solid lines6070/6090in each ofFIG.47,FIG.48andFIG.49. From outlet8012,8022,8032ofFIG.47,FIG.48andFIG.49respectively, following steps of401or708, each of the MAGs123ofFIG.47,FIG.48, orFIG.49may extract fluid sample from corresponding connector8010,8020, and8030into its corresponding channel201. Each of the UFL600and each of the MAG123ofFIG.47,FIG.48, orFIG.49may operate at its own respective optimal sample flow rate, which may be different between different UFLs600and different between different MAGs123within same figure. Due to the existence of the connector8010,8020, and8030, flow rate interference between the different UFLs600and MAGs123within each ofFIG.47,FIG.48andFIG.49are minimized or eliminated. Fluid level sensor100may be attached to buffers8010,8020, and8030to sense fluid level remaining in each of the flow connectors8010,8020, and8030. Fluid level sensor100operates similarly as inFIG.44AthroughFIG.44Cin sensing fluid in flow connectors8010,8020, and8030, and depending on the flow rate difference between MAGs123and UFLs600of each figure, may pause operation of one or more MAGs123, or may pause operation of one or more UFLs600of each figure to maintain fluid level in corresponding connector8010,8020, or8030to be above a low level threshold or below a high level threshold. Alternatively, liquid sample may be completely processed through all UFLs600first and stored in corresponding connector8010,8020, or8030of eachFIG.47,FIG.48andFIG.49. MAGs123then extract fluid from corresponding connector8010,8020, or8030of each figure and complete processing of all liquid sample from each corresponding connector8010,8020, or8030. Connectors8010,8020, and8030may each be made as part of a set of enclosed fluidic lines, which may include UFLs600, channels201and connections from UFLs600to each connector8010,8020,8030and from each connector8010,8020, and8030to channels201, similar as described inFIG.44AthroughFIG.44C.

FIG.50illustrates seventh type sample processing method where biological sample is first passed through multiple MAGs123, output fluids from the MAGs123channels201are then fed into inlets8011of flow connector8010ofFIG.47, and from flow connector8010outlets8012into the entity fluid inlets602of multiple UFLs600.

FIG.51illustrates eighth type sample processing method where biological sample is first passed through multiple MAGs123, output fluids from the MAGs123channels201are then fed into inlets8021of flow connector8020ofFIG.48, and from flow connector8020outlets8022into the entity fluid inlets602of multiple UFLs600.

FIG.52illustrates ninth type sample processing method where biological sample is first passed through multiple MAGs123, output fluids from the MAGs123channels201are then fed into inlets8031of flow connector8030ofFIG.49, and from flow connector8030outlets8032into the entity fluid inlets602of multiple UFLs600.

In each ofFIG.50,FIG.51, andFIG.52, in one embodiment, same biological sample is divided and processed simultaneously through multiple MAGs123. In another embodiment, each of the MAGs123processes a different biological sample. Output from each MAG123, either negative entities following procedure427, or positive entities following procedure428, may be individually input into the inlet8011of connector8010ofFIG.50, or into inlet8021of connector8020ofFIG.51, or into inlet8031of connector8030ofFIG.52, as shown by solid lines427/428in each ofFIG.50,FIG.51andFIG.52. From outlet8012,8022,8032ofFIG.50,FIG.51andFIG.52respectively, following step of408, each of the UFLs600ofFIG.50,FIG.51, orFIG.52may extract fluid sample as entity fluid6020from corresponding connector8010,8020, and8030into its corresponding entities inlet602, Each of the UFL600and each of the MAG123ofFIG.50,FIG.51, orFIG.52may operate at its own respective optimal sample flow rate, which may be different between different UFLs600and different between different MAGs123within same figure. Due to the existence of the connector8010,8020, and8030, flow rate interference between the different UFLs600and MAGs123within each ofFIG.50,FIG.51andFIG.52are minimized or eliminated. Fluid level sensor100may be attached to flow connectors8010,8020, and8030to sense fluid level remaining in each of the flow connectors. Fluid level sensor100operates similarly as inFIG.46AthroughFIG.47Cin sensing fluid in flow connectors8010,8020, and8030, and depending on the flow rate difference between MAGs123and UFLs600of each figure, may pause operation of one or more MAGs123, or may pause operation of one or more UFLs600of each figure to maintain fluid level in corresponding connector8010,8020, or8030to be above a low level threshold or below a high level threshold. Alternatively, liquid sample may be completely processed through all MAGs123first and stored in corresponding connector8010,8020, or8030of eachFIG.50,FIG.51andFIG.52. UFLs600then extract fluid from corresponding connector8010,8020, or8030of each figure and complete processing of all liquid sample from each corresponding connector8010,8020, or8030. Flow connectors8010,8020, and8030may each be made as part of a set of enclosed fluidic lines, which may include UFLs600, channels201and connections from channels201to each connector8010,8020,8030and from each connector8010,8020, and8030to UFLs600, similar as described inFIG.46AthroughFIG.46C.

FIG.53illustrates tenth type sample processing method where biological sample after being passed through one or more of UFLs600or MAGs123, output fluids from the UFLs600and MAGs123are fed into inlets of a flower connector8020or a flow connector8030, and from the flow connectors8020and8030outlets into different type of cell processing devices.FIG.53shows example of liquid sample output from MAG123channel201, including negative entities following procedure427and positive entities following procedure428, may be injected to inlet8021of connector8020or inlet8031of connector8030similar as inFIG.51andFIG.52. Alternatively, UFL600large entities output6070from outlet607or small entities output6090from outlet609may be also injected into to inlet8021of connector8020or inlet8031of connector8030similar as inFIG.48andFIG.49. After sample fluid is completely processed through UFL600or MAG123, and injected into, and stored within, connector8020or connector8030, entities analysis as in step407ofFIG.31and step707ofFIG.39may be performed by sending sample fluid containing entities from connector8020or connector8030to any of: cell counter903, cell imager904, flow cytometer or sorter905, and DNA or RNA sequencer906. Entities may be further sent to DNA or RNA sequencer906after cell counter903as in936, or after cell imager904as in946, or after flow cytometer or sorter905as in956. For sending the sample fluid from outlet8022of connector8020, or from outlet8032of connector8030, pressurized chamber800may be used to contain the connector8020or connector8030inside, and force sample fluid out of connector8020or connector8030in a steady and continuous flow stream. Chamber800may be a chamber filled with pressurized air inside. Connector8020in vial type may have an additional air port8023open to chamber800internal pressurized air to help push sample fluid out of connector8020. While connector8030may be in a flexible blood bag form, which when under pressured air of chamber800, will automatically deflate and force sample liquid out through outlet8032. To avoid back flow into UFL600or MAG123channel201, shut off valves805may be implemented on output lines from MAG123channel201and UFL600to connector8020or connector8030.

FIG.54Aillustrates eleventh type sample processing method where biological sample after being passed through a first MAG123channel201during a magnetic separation may output negative entities fluid following procedure427, or positive entities fluid following procedure428, into inlet of a second MAG123channel201input as in step408of a continued process.FIG.54Aillustrates a multi-stage MAG process.

FIG.54Billustrates twelfth type sample processing method where after biological sample passed through MAG123for magnetic separation, output fluid from MAG123channel201, containing either negative entities or position entities, may be diverted through a T-connector912into flow913. Flow913may then be re-input back into the input of the channel201of MAG123for another round of magnetic separation through T-connector911. T-connector911allows initial fluid sample input as in step401and recycled flow913input to channel201. T-connector912allows output from channel201into recycled flow913or output from MAG123as in procedures427and428. In one embodiment, recycled flow913contains negative entities, repeated magnetic separation inFIG.54Bhelps achieve complete depletion of all magnetic entities in the negative entities flow before output into427/428procedure. In another embodiment, recycled flow913contains positive entities after dissociation, repeated process as inFIG.54Bhelps increase purity in positive magnetic entities to allow wash off of non-magnetic entities that may be in the conglomerate by non-specific bindings.FIG.54Billustrates using same MAG123as a multi-cycle MAG process.

FIG.54Cillustrates thirteenth type sample processing method where biological sample after being passed through a first UFL600, output fluids from first UFL600, for example large entities6070fluid from outlet607or small entities6090fluid from outlet609, may be passed into entity fluid inlets602of one or more subsequent UFLs600as a multi-stage UFL process.

FIG.55Aillustrates first embodiment of closed and disposable fluidic lines for third type sample processing method as shown inFIG.46A, where connector801may be replaced with connector802or connector803without limitation. Input line923may connect to a sample liquid container. Input line924may connect to a MAG buffer container. Input line923and input line924are connected through a T-connector921to the inlet of the first pump tubing504/505that may be mounted into a peristaltic pump. First pump tubing504/505outlet connects to channel201which may be used as part of MAG123. Output of channel201connects to T-connector922, which connects to output line925and output line926. Output line925may connect to a MAG out sample container and output line926connects to inlet of connector801. In one embodiment, output line925may output negative entities to said MAG out sample container, and output line926may output positive entities to connector801. In another embodiment, output line925may output positive entities to said MAG out sample container, and output line926may output negative entities to connector801. Connector801outlet connects to input line9271of a second pump tubing504/505. Said second pump tubing504/505outlet then connects to UFL600sample input line6020. Input line9272may connect to UFL buffer container and connects to inlet of a third pump tubing504/505. Said third pump tubing504/505outlet then connects to UFL600buffer input line6040. UFL600large entities6070output line may connect to a large entities sample container. UFL600small entities6090output line may connect to a small entities sample container.FIG.55Aillustrates that besides the input and output lines923,924,925,9272,6070, and6090that connect to external containers, entire fluid path from sample liquid input to line923, to sample output to lines925,6070,6090, all pumps, MAG123and other fluidic line components will be externally attached to the lines ofFIG.55A. Thus, lines ofFIG.55Aare internally enclosed, suitable for single use disposable purpose and sterile applications.

FIG.55Billustrates fluidic lines ofFIG.55Abeing connected to, or attached with, various fluidic components. Input line923connects to a liquid sample container928in blood bag form. Input line924connects to buffer container929. Valves935and936are attached to lines923and924to control either sample liquid from bag928or buffer from container929is flown through T-connector921into first pump tubing504/505. First, second and third pump tubing504/505is each installed into a peristaltic pump500. Three pumps500operate to pump either sample fluid or buffer fluid into MAG123and UFL600. A flow limiter509/510may be attached to the output line from each pump500, including lines201,6020,6040to reduce flow rate pulsation from the pumps500. Channel line201is mounted into MAG123. Output line925connects to MAG out sample container934. Valve940is attached to line925and valve937is attached to line926, which control negative entities or positive entities from MAG123going into either container934or the connector801through the T-connector922. Valves940and937may both shut down the flow in lines925and926during demagnetization/dissociation process of MAG123. Input line9272may connect to UFL buffer container931. UFL output line6070connects to large entities container932and output line6090connects to small entities933. Adjustable valves939and938may be attached to the lines6070and6090to adjust the flow rate within each line of6070and6090, which in turn controls the laminar flow speed in UFL channel for channel center buffer flow and channel edge entities sample flow.

FIG.56Aillustrates second embodiment of closed and disposable fluidic lines for third type sample processing method as shown inFIG.46A.FIG.56Ais identical toFIG.55A, except the output line925is connected to a MAG sample container934, UFL output line6070is connected to a large entities container932, and UFL output line6090is attached to a small entities container933.FIG.56Aillustrates containers934,932,933are in the form of blood bags. Bags932,933,934as part of the enclose lines ofFIG.56Amay be disposable and made sterile, and may also be separated from the lines after separation process for steps407and408ofFIG.31, or steps707and708ofFIG.39.

FIG.56Bdescribes the identical process of: connecting sample container928, buffer container929, buffer container931to the lines923,924and9272respectively, same as inFIG.55B. Containers928,929,931are in blood bag form. Also same as described inFIG.55B, three pump tubing504/505are installed in the three peristaltic pumps500, valves935,936,940,937,939,938, are each attached to the corresponding lines, and flow limiters509/510may be attached at output line of each pump500same as inFIG.55B.

FIG.57Aillustrates embodiment of closed and disposable fluidic lines for first type sample processing method as shown inFIG.44A, where connector801may be replaced with connector802or connector803without limitation. Input line9271may connect to a UFL sample liquid container, and also connects to inlet of a first pump tubing504/505, which further connect to entities input line6020of UFL600. Input line9272may connect to a UFL buffer container, and also connects to inlet of a second pump tubing504/505, which further connect to buffer input line6040of UFL600. UFL600large entities output line6070connects to inlet of connector801. UFL600small entities output line6090may connect to a small entities container. Outlet of connector801connects to MAG sample input line923. MAG buffer input line924may connect to a MAG buffer container. Input lines923and924are connected through a T-connector921to the inlet of the third pump tubing504/505. Third pump tubing504/505outlet connects to channel201which may be used as part of MAG123. Output of channel201connects to T-connector922, which connects to output line925and output line926. Output lines925and926may each connect to a MAG out sample container. In one embodiment, output line925may output negative entities to a first MAG out sample container, and output line926may output positive entities to a second MAG out sample container.FIG.57Aillustrates that besides the input and output lines9271,9272,924,6090,925, and926that connect to external containers, entire fluid path from UFL sample and UFL buffer input lines9271and9272, to sample output lines6090,925and926, all pumps, MAG123and other fluidic line components will be externally attached to the lines ofFIG.57A. Thus, lines ofFIG.57Aare internally enclosed, suitable for single use disposable purpose and sterile applications.

FIG.57Billustrates fluidic lines ofFIG.57Abeing connected to, or attached with, various fluidic components. First, second and third pump tubing504/505is each installed into a peristaltic pump500. Three pumps500operate to pump either sample fluid or buffer fluid into MAG123and UFL600. A flow limiter509/510may be attached to the output line from each pump500, including lines201,6020,6040to reduce flow rate pulsation from the pumps500. Input line9271connects to a liquid sample container928in blood bag form. Input line9272connects to UFL buffer container931also in blood bag form. UFL output line6090connects to small entities container933in blood bag form. Adjustable valves939and938may be attached to the lines6070and6090to adjust the flow rate within each line of6070and6090, which in turn controls the laminar flow speed in UFL600channel for channel center buffer flow and channel edge entities sample flow. Input line924connects to MAG buffer container929. Valves935and936are attached to lines923and924to control either sample liquid from connector801or buffer fluid from container929is flown through T-connector921into third pump tubing504/505. Channel line201is mounted into MAG123. Output line925connects to first MAG out sample container934. Output line926connects to second MAG out sample container9342. Valve940is attached to line925and valve937is attached to line926, which control negative entities and positive entities from MAG123going into either container934or container9342through the T-connector922. Valves940and937may both shut down the flow in lines925and926during demagnetization/dissociation process of MAG123.

FIG.58Aillustrates embodiment of closed and disposable fluidic lines for second type sample processing method as shown inFIG.45A.FIG.58Ais identical toFIG.57Ain every aspect, except the UFL600small entities output line6090connects to the inlet of the connector801instead of the output line6070as inFIG.57A. Large entities output line6070ofFIG.58Amay connect to a large entities container.

FIG.58Billustrates fluidic lines ofFIG.58Abeing connected to, or attached with, various fluidic components.FIG.58Bis identical toFIG.57Bin every aspect, except the UFL600small entities output line6090connects to the inlet of the connector801instead of the output line6070as inFIG.57B. Large entities output line6070ofFIG.58Bconnects to a large entities container932in blood bag form.

FIG.59Aillustrates embodiment of closed and disposable fluidic lines for sample processing through a single MAG. Input line923may connect to a sample liquid container. Input line924may connect to a MAG buffer container. Input line923and input line924are connected through a T-connector921to the inlet of the pump tubing504/505that may be mounted into a peristaltic pump. Pump tubing504/505outlet connects to channel201which may be used as part of MAG123. Output of channel201connects to T-connector922, which connects to output line925and output line926. Output lines925and926may each connect to a MAG out sample container.

FIG.59Billustrates fluidic lines ofFIG.59Abeing connected to, or attached with, various fluidic components. Input line923connects to a liquid sample container928. Input line924connects to buffer container929. Valves935and936are attached to lines923and924to control either sample liquid from bag928or buffer from container929is flown through T-connector921into first tubing504/505. Pump tubing504/505is installed into a peristaltic pump500. Pump500operates to pump either sample fluid or buffer fluid into MAG123. A flow limiter509/510may be attached to the output line201from pump500to reduce flow rate pulsation from the pumps500. Channel line201is mounted into MAG123. Output line925connects to MAG out sample container934. Output line926connects to MAG out sample container9342. Valve940is attached to line925and valve937is attached to line926, which control negative entities and positive entities from MAG123going into either container934or container9342. Valves940and937may both shut down the flow in lines925and926during demagnetization/dissociation process of MAG123.FIG.59Bshows containers928,929,934and9342may be in the form of blood bags, but may also be in other physical forms of vial or bottles.

FIG.60Aillustrates embodiment of closed and disposable fluidic lines for sample processing through a single UFL600. Input line9271may connect to a UFL sample liquid container, and also connects to inlet of a first pump tubing504/505, which further connects to entities input line6020of UFL600. Input line9272may connect to a UFL buffer container, and also connects to inlet of a second pump tubing504/505, which further connects to buffer input line6040of UFL600. UFL600large entities output line6070may connect to a large entities container. UFL600small entities output line6090may connect to a small entities container.

FIG.60Billustrates fluidic lines ofFIG.60Abeing connected to, or attached with, various fluidic components. First and second pump tubing504/505is each installed into a peristaltic pump500. The two pumps500operate to pump sample fluid and buffer fluid into UFL600. A flow limiter509/510may be attached to the output line from each pump500, including lines6020and6040to reduce flow rate pulsation from the pumps500. Input line9271connects to a liquid sample container928. Input line9272connects to UFL buffer container931. UFL output line6070connects to large entities container932. UFL output line6090connects to small entities container933. Adjustable valves939and938may be attached to the lines6070and6090to adjust the flow rate within each line of6070and6090, which in turn controls the laminar flow speed in UFL600channel for channel center buffer flow and channel edge entities sample flow.FIG.60Bshows containers928,931,932and933may be in the form of blood bags, but may also be in other physical forms of vial or bottles without limitation.

FIG.61Aillustrates replacing peristaltic pumps ofFIG.56Bwith using pressurized chambers800on input sample bags to drive fluid through fluidic lines. InFIG.61A, pumps500, pump tubing504/505, and flow limiters509/510ofFIG.56Bare removed. Channel201is connected directly to T-connector921. Connector801is replaced with connector803bag. UFL entity liquid line6020is connected to connector803. Sample liquid bag928, MAG buffer bag929, connector803bag and UFL buffer bag931are each enclosed in a pressure chamber800. Pressure chamber800may operate by increasing pressure of chamber medium, for example air or other fluid, where the bags enclosed in chambers are submerged in the chamber medium. With increase of chamber medium pressure, liquid contained in the bags may be forced out of the bags and into the fluid lines.FIG.61Aoperation may need separate MAG123and UFL600operations. At first stage, valve941attached to line6020closes. Pressure in chambers800enclosing bags803and931is released. Pressure in chambers800enclosing bags928and929are increased to force sample fluid or buffer fluid into channel201to start MAG123separation. After MAG123separation and sample fluid in bag928is depleted, bag934and connector803are each filled with output samples from MAG123after MAG separation. Then, at second stage, valve937is closed and valve941is open. Chambers800around connector803and bag931increase in pressure to force connector803sample and buffer fluid in931to flow into the UFL600to start UFL separation. After sample in connector803is depleted, and UFL600separation finish, bags932and933contain large and small entities from UFL output. Connector803maybe replaced by connector8020ofFIG.52which has an air port8023.

FIG.61Billustrates replacing peristaltic pumps ofFIG.56Bwith using vacuum chambers806on output sample bags to drive fluid through fluidic lines.FIG.61Bis same asFIG.61A, except pressure chambers800are removed. Bag934,932,933, and connector803are each enclosed in a vacuum chamber806. Vacuum chamber806may operate by increasing vacuum level within each chamber806, where fluid from the fluid lines connected to the bags is forced into the bags enclosed in chambers due to fluid line pressure being larger than the vacuum pressure.FIG.61Boperation may also need separate MAG123and UFL600operations. At first stage, valve941attached to line6020closes. Vacuum in chambers806enclosing bags932and933is released. Vacuum in chambers806enclosing bags934and803are increased to force sample fluid or buffer fluid into channel201to start MAG123separation. After MAG123separation and sample fluid in bag928is depleted, bag934and connector803are each filled with output samples from MAG123after MAG separation. Then, at second stage, valve937is closed and valve941is open. Vacuum in chamber806around connector803is released. Vacuum in chambers806enclosing bags932and933are increased to force connector803sample and buffer fluid in931to flow into the UFL600to start UFL separation. After sample in connector803is depleted, and UFL600separation finish, bags932and933contain large and small entities from UFL output. Connector803maybe replaced by connector8020ofFIG.52which has an air port8023.

FIG.62Aillustrates replacing peristaltic pumps ofFIG.57Bwith using pressurized chambers800on input sample bags to drive fluid through fluidic lines. InFIG.62A, pumps500, pump tubing504/505, and flow limiters509/510ofFIG.57Bare removed. Channel201is connected directly to T-connector921. Connector801is replaced with connector803bag. MAG sample line923is connected to connector803. Sample liquid bag928, MAG buffer bag929, connector803bag and UFL buffer bag931are each enclosed in a pressure chamber800.FIG.62Amay separate UFL600and MAG123operations. At first stage, valve935attached to line923closes. Pressure in chambers800enclosing bags803is released. Pressure in chambers800enclosing bags928and931are increased to force sample fluid and UFL buffer fluid into UFL600inlets to start UFL600separation. After UFL600separation and sample fluid in bag928is depleted, bag933contains small entities fluid and connector803contains large entities fluid from UFL600separation. Then, at second stage, valve939is closed and valve935is open. Chambers800around connector803and bag929increase in pressure to force connector803large entities fluid sample or MAG buffer fluid in929to flow into channel201of MAG123to start MAG123separation. After sample in connector803is depleted, and MAG123separation finish, bags934and9342contain positive sample and negative sample from MAG123channel201output. Connector803maybe replaced by connector8020ofFIG.52.

FIG.62Billustrates replacing peristaltic pumps ofFIG.57Bwith using vacuum chambers806on output sample bags to drive fluid through fluidic lines.FIG.62Bis same as

FIG.62A, except pressure chambers800are removed. Bag934,9342,933, and connector803are each enclosed in a vacuum chamber806.FIG.62Boperation may separate MAG123and UFL600operations. At first stage, valve935attached to line923closes. Vacuum in chambers806enclosing bags933and803are increased to force sample fluid and UFL buffer fluid into inlets of UFL600to start UFL600separation. After UFL600separation and sample fluid in bag928is depleted, bag933contains small entities fluid and connector803contains large entities fluid from UFL600separation. Then, at second stage, valves938and939are closed and valve923is open. Vacuum in chamber806around connector803is released. Vacuum in chambers806enclosing bags934and9342are increased to force connector803large entities sample or MAG buffer fluid in929to flow into channel201of MAG123to start MAG123separation. After sample in connector803is depleted, and MAG123separation finish, bags934and9342contain positive sample and negative sample from MAG123channel201output. Connector803maybe replaced by connector8020ofFIG.52.

FIG.63Aillustrates replacing peristaltic pumps ofFIG.58Bwith using pressurized chambers800on input sample bags to drive fluid through fluidic lines.FIG.63Ais identical toFIG.62Ain fluid line layout and in operation of UFL600and MAG123with chambers800, except that the UFL large entities output6070connects to large entities container932in blood bag form, and small entities output6090connects to connector803.

FIG.63Billustrates replacing peristaltic pumps ofFIG.58Bwith using vacuum chambers806on output sample bags to drive fluid through fluidic lines.FIG.63Ais identical toFIG.62Bin fluid line layout and in operation of UFL600and MAG123with chambers806, except that the UFL large entities output6070connects to large entities container932in blood bag form with small entities container932enclosed in vacuum chamber806replacing container933ofFIG.62B, and small entities output6090connects to connector803.

FIG.64Aillustrates replacing peristaltic pumps ofFIG.59Bwith using pressurized chambers800on input sample bags928and929to drive fluid through channel201of MAG123. InFIG.64A, pump500, pump tubing504/505, and flow limiter509/510ofFIG.59Bare removed. Channel201is connected directly to T-connector921. Sample liquid bag928and MAG buffer bag929are each enclosed in a pressure chamber800. Pressure in chambers800enclosing bags928and929are increased to force sample fluid or buffer fluid into channel201to start MAG123separation. After MAG123separation and sample fluid in bag928is depleted, bag934and bag9342are each filled with either negative entities or positive entities from MAG123after MAG separation.

FIG.64Billustrates replacing peristaltic pumps ofFIG.59Bwith using vacuum chambers806on output sample bags934and9342to drive fluid through channel201of MAG123.FIG.64Bis same asFIG.64A, except pressure chambers800are removed. Output sample bags934and9342are each enclosed in a vacuum chamber806. Vacuum in chambers806enclosing bags934and9342are increased to force entities sample from bag928or MAG buffer fluid from bag929to flow into channel201of MAG123to start MAG123separation. After sample in bag928is depleted, and MAG123separation finish, bags934and9342contain positive sample and negative sample from MAG123channel201output.

FIG.65Aillustrates replacing peristaltic pumps ofFIG.60Bwith using pressurized chambers800on sample liquid bag928and UFL buffer bag931to drive fluid through UFL600. InFIG.65A, pump500, pump tubing504/505, and flow limiter509/510ofFIG.60Bare removed. Sample liquid bag928and UFL buffer bag931are each enclosed in a pressure chamber800. Pressure in chambers800enclosing bags928and931are increased to force sample fluid and UFL buffer fluid into UFL600inlets to start UFL600separation. After UFL600separation, sample fluid in bag928is depleted, bag932contains large entities fluid and bag933contains small entities fluid.

FIG.65Billustrates replacing peristaltic pumps ofFIG.60Bwith using vacuum chambers906on output sample bags932and933to drive fluid through UFL600.FIG.65Bis same asFIG.65A, except pressure chambers800are removed. Output sample bags932and933are each enclosed in a vacuum chamber806. Vacuum in chambers806enclosing bags932and933are increased to force sample liquid from bag928and UFL buffer fluid from bag931to flow through UFL600to start UFL separation. After sample in bag928is depleted, and UFL600separation finish, bag932contains large entities fluid and bag933contains small entities fluid.

Structures, components, and methods as described fromFIG.55AthroughFIG.65Bon enclosed fluidic lines including one UFL600and one MAG123, may be applied toFIG.47throughFIG.52without limitation, where enclosed fluidic lines including multiple MAGs123and multiple UFLs600may be achieved with replicating the components on single UFL600and single MAG123fromFIG.55AthroughFIG.65Bon each of the UFLs600and MAGs123ofFIG.47throughFIG.52.

FIG.66throughFIG.88illustrate embodiments of process flows to utilize MAG and UFL devices to separate biological entities from various biological samples. For simplicity of description, terms UFL and MAG are used in these figures for explanation. However, UFL may be any of UFL600,650,620,630,640ofFIG.40A,FIG.41A,FIG.42A,FIG.43, while MAG may be any of MAG121,122,123,124,124,125,126,127,128,129with corresponding channel types as described in prior figures without limitation and without sacrifice of performance. If a component, or a structure, inFIG.66throughFIG.88shares same name as in prior figures, it then means the same component, or same structure as in prior figures.

FIG.66illustrates embodiment of a first process flow to separate biological entities from peripheral blood using UFL and MAG. In step5801, peripheral blood sample is collected from a patient or person under test; in step5802, red blood cell lysing may be performed on said peripheral blood sample, where step5802in another embodiment may be skipped; in step5803, said blood sample from step5802, or directly from step5801, is injected in UFL entity fluid inlet602, while UFL buffer fluid is injected in outlet604; in step5804, set frequency and vibration strength of PZT attached to UFL to produce standing wave and pressure nodes in UFL fluid; in step5805, UFL outlet607outputs target sample that contains large size entities or cells; in step5806, add into target sample from step5805magnetic labels hybridized with antibodies or ligands, which specifically bind to surface antigens or receptors on target cells or entities; in step5807, target sample from step5806is incubated to form magnetic labels binding to target cells or entities; in step5808, flow target sample from step5807through MAG channel at magnetic separation positon, where during step5808, negative MAG sample may be forwarded as in5815to be collected in step5813; in step5809, target cells or entities bound with magnetic label are separated by MAG within the MAG channel; in step5810, after step5809, buffer fluid may be flown through MAG channel to wash out residue non-target entities without magnetic label, the washed out fluid may be forwarded as in5816to be collected as negative MAG sample in step5813, where step5810may be skipped in another embodiment; in step5811, after step5810or directly after step5809, separated entities conglomerate in MAG channel may be dissociated into isolated cells or entities; in step5812, buffer fluid is flown through MAG channel to washed out dissociated cells and entities in MAG channel, which, as shown by5817, may be collected as positive MAG sample in step5814.

Peripheral blood sample ofFIG.66may also be other body fluids, including but not limited to: saliva, tear, mucus, urine, secretion from various organs of body.

FIG.67illustrates an embodiment of second process flow to separate biological entities from peripheral blood using MAG. Every other aspect ofFIG.67is same asFIG.66, except step5803, step5804, and step5805ofFIG.66are removed between step5802and step5806inFIG.67. While inFIG.67, blood sample from step5802, or blood sample directly from step5801, is centrifuged in step6201to extract target sample containing white blood cells. Target sample form step6201is then sent to step5806, from step5806FIG.67flow is same as inFIG.66.

FIG.68illustrates an embodiment of third process flow to separate biological entities from peripheral blood using MAG. Every other aspect ofFIG.68is same asFIG.66, except step5803, step5804, and step5805ofFIG.66are removed between step5802and step5806inFIG.68. While inFIG.68, peripheral blood sample collected from patient or person under test as in step6301, which is same as step5801ofFIG.66, is regarded target sample. Target sample form step5802after red blood cell lysing after step6301, or directly from step6301, is then sent to step5806. From step5806,FIG.68flow is same as inFIG.66.

FIG.69illustrates an embodiment of fourth process flow to separate biological entities from peripheral blood using MAG. Every other aspect ofFIG.69is same asFIG.66, except step5801, step5802, step5803, step5804, and step5805ofFIG.66are removed before step5806inFIG.69. While inFIG.69, target sample is collected after apheresis of peripheral blood sample collected from patient or person under test. Target sample form step6401is then sent to step5806. From step5806,FIG.69flow is same as inFIG.66.

FIG.70illustrates an embodiment of fifth process flow to separate biological entities from tissue sample using UFL and MAG. Every other aspect ofFIG.70is same asFIG.66, except step5801, step5802, and step5803are removed before step5804inFIG.70. InFIG.70, tissue sample is collected in step6501. In step6502, tissue sample from step6501is dissociated in a fluid base. In step6503, dissociated tissue fluid of step6502is injected into UFL channel through inlet602and UFL buffer fluid is injected through inlet604. From step5804,FIG.70flow is same as inFIG.66. Tissue sample ofFIG.70may include any of: human body tissue aspirate, human organ tissue aspirate, bone marrow, animal body or organ tissue aspirate. Target cells or entities ofFIG.70may be rare disease cells, for example cancer cells, or micro-organisms, for example bacteria.

FIG.71illustrates an embodiment of sixth process flow to separate biological entities from tissue sample using MAG. Every other aspect ofFIG.71is same asFIG.70, except step6503, step5804, and step5805are removed before step5806inFIG.71. InFIG.71, tissue sample from step6501is dissociated in a fluid base in step6502to form target sample, and continues process in step5806. From step5806,FIG.71flow is same as inFIG.70.

FIG.72illustrates an embodiment of seventh process flow to separate biological entities from surface swab sample using UFL and MAG. Every other aspect ofFIG.72is same asFIG.66, except step5801, step5802, and step5803are removed before step5804inFIG.72. In FIG.

72, surface entities are collected in step6701by swab. In step6702, surface entities collected on swab are dissolved in a fluid base. In step6703, fluid base with dissolved surface entities from step6702is injected into UFL channel through inlet602and UFL buffer fluid is injected through inlet604. From step5804,FIG.72flow is same as inFIG.66. Surface entities ofFIG.72may be collected by swab from subjects including any of: human body, saliva, body fluid, human body discharge, animal, plant, soil, air, water, and merchandise. Target cells or entities ofFIG.72may include cells from human body, or animal body, or plant, or include micro-organisms, for example bacteria, mold, or spores.

FIG.73illustrates an embodiment of eighth process flow to separate biological entities from surface swab sample using MAG. Every other aspect ofFIG.73is same asFIG.72, except step6703, step5804, and step5805are removed before step5806inFIG.73. InFIG.73, surface entities collected on swab in step6701are dissolved in a fluid base in step6702to form target sample, and continues process in step5806. From step5806,FIG.73flow is same as inFIG.72.

FIG.74illustrates an embodiment of ninth process flow to separate biological entities from solid sample using UFL and MAG. Every other aspect ofFIG.74is same asFIG.66, except step5801, step5802, and step5803are removed before step5804inFIG.74. InFIG.74, solid sample is collected in step6901. In step6902, solid sample from step6901is dissociated in a fluid base. In step6903, dissociated solid sample fluid of step6902is injected into UFL channel through inlet602and UFL buffer fluid is injected through inlet604. From step5804,FIG.74flow is same as inFIG.66. Tissue sample ofFIG.70may include any of: solid biological products or waste generated by human, animal, or plant, powder, and soil. Target cells or entities ofFIG.74may include cells from human body, or animal body, or plant, or include micro-organisms, for example bacteria, mold, or spores.

FIG.75illustrates an embodiment of tenth process flow to separate biological entities from solid sample using MAG. Every other aspect ofFIG.75is same asFIG.74, except step6903, step5804, and step5805are removed before step5806inFIG.75. InFIG.75, solid sample from step6901is dissociated in a fluid base in step6902to form target sample, and target sample is continuously processed in step5806. From step5806,FIG.75flow is same as inFIG.74.

FIG.76Aillustrates addition of both magnetic and fluorescent labels into fluid samples for specific binding to target cells or entities.FIG.76Ashows that step5806ofFIG.66throughFIG.75may be modified to step58061, where in addition to magnetic labels, fluorescent labels hybridized with antibodies or ligands, which specifically bind to surface antigens or receptors on target cells or entities, may also be added in target sample from step5805.

FIG.76Bthen illustrates that incubation step5807ofFIG.66throughFIG.75may also be modified to step58071, which includes incubation of both magnetic and fluorescent labels at the same time to form specific binding to target cells or entities. Binding sites of magnetic labels and fluorescent labels on same target cells or entities may be different.

Steps5806and step58061may be realized in a flow connector including any one of801,802,803,8010,8020,8030of prior figures, where flow connector may contain pre-filled hybridized magnetic labels and fluorescent labels in liquid solution, or in dry powder form. Step5807and step58071may also occur in said flow connector, where said flow connector may also be located in a temperature control chamber to control incubation speed and quality. In another embodiment, said flow connector may have attached or embedded temperature control circuit to control incubation in flow connector.

FIG.77Aillustrates process of removing non-bound free magnetic labels from sample fluid by UFL before magnetic separation by MAG.FIG.77Ashows that for each ofFIG.66throughFIG.75, step5818and step5819may be added between step5807and step5808. After target sample is incubated in step5807, in step5818, target sample may be injected into second UFL through inlet602, and buffer fluid may be injected into second UFL through inlet604. In step5819, second UFL outputs target sample containing large entities from outlet607, and non-bound free magnetic labels are output from second UFL outlet609. Then in step5808, target sample containing large entities from second UFL outlet607is passed through MAG channel for magnetic separation. Target sample in step5819may contain cells10/30or entities bound with magnetic labels. Second UFL having an attached PZT that operates with a specified ultrasound vibration amplitude and frequency to create standing wave in second UFL channel fluid is assumed in step5819.

FIG.77Billustrates process of removing non-bound free magnetic labels from sample fluid by UFL after magnetic separation by MAG.FIG.77Bshows that for each ofFIG.66throughFIG.75, step5820and step5821may be added between step5812and step5814, replacing path5817. After magnetic conglomerate within MAG channel is dissociated and the positive MAG sample entities from MAG channel are flushed out as in step5812, flushed out positive MAG sample may be injected into third UFL through inlet602, and buffer fluid may be injected into third UFL through inlet604. In step5821, third UFL outputs positive MAG sample containing large entities from outlet607, and non-bound free magnetic labels are output from third UFL outlet609. Then in step5814, positive MAG sample with reduced or depleted free magnetic labels may be collected. Third UFL having an attached PZT that operates with a specified ultrasound vibration amplitude and frequency to create standing wave in third UFL channel fluid is assumed in step5821.

FIG.78Aillustrates process of removing non-bound free magnetic labels and free fluorescent labels from sample fluid by UFL before magnetic separation by MAG.FIG.78Ais similar asFIG.77A, with replacing step5807ofFIG.77Awith step58071ofFIG.76B, and replacing step5819with step58191. After adding magnetic labels and fluorescent labels into target sample as in step58061ofFIG.76A, target sample is incubated in step58071same as inFIG.76Bto form magnetic label and fluorescent label binding to target cells or entities. In step5818, target sample may be injected into second UFL through inlet602, and buffer fluid may be injected into second UFL through inlet604. In step58191, second UFL outputs target sample containing large entities from outlet607, and non-bound free magnetic labels and free fluorescent labels are output from second UFL outlet609. Then in step5808, target sample containing large entities from second UFL outlet607is flown through MAG channel for magnetic separation. Target sample in step58191may contain cells30or entities bound with magnetic and fluorescent labels. Second UFL having an attached PZT that operates with a specified ultrasound vibration amplitude and frequency to create standing wave in second UFL channel fluid is assumed in step58191.

FIG.78Billustrates process of removing non-bound free magnetic labels and free fluorescent labels from sample fluid by UFL after magnetic separation by MAG.FIG.78Bis similar asFIG.77B, with replacing step5821ofFIG.77Awith step58211. Separated entities in step5812and step5820ofFIG.78Bmay contain: cells30or entities bound with magnetic and fluorescent labels, non-bound free magnetic labels, and small amount of non-bound free fluorescent labels due to non-specific binding to conglomerate in MAG channel during magnetic separation. In step58212, third UFL outputs positive MAG sample containing large entities from outlet607, and non-bound free magnetic and free optical labels are output from third UFL outlet609. Then in step5814, positive MAG sample with reduced or depleted free magnetic labels and free fluorescent labels may be collected. Third UFL having an attached PZT that operates with a specified ultrasound vibration amplitude and frequency to create standing wave in third UFL channel fluid is assumed in step58212.

FIG.79illustrates continued process of negative MAG sample after MAG separation, as in step408ofFIG.31, through UFL to remove small entities and passing of large entities into various cell processing devices and procedures. Step5813is same as inFIG.66throughFIG.75, where negative MAG sample is collected during MAG separation of a target sample. In step5822, negative MAG sample of step5813is injected into fourth UFL inlet602, and UFL buffer is injected into inlet604of fourth UFL. In step5823, fourth UFL outputs negative MAG sample containing large entities from outlet607, and small size entities are removed from large entities and output from fourth UFL outlet609, a PZT that attaches to fourth UFL and operates with a specified ultrasound vibration amplitude and frequency to create standing wave in fourth UFL is assumed. Finally, negative MAG sample containing large entities from outlet607of fourth UFL may be sent to be analyzed by any of: cell counter903, cell imager904, flow cytometer or sorter905, DNA/RNA sequencer906. Alternatively, output from cell counter903, or output from cell imager904, or output from flow cytometer or sorter905, may be further sent to be processed by DNA/RNA sequencer906as indicated respectively by path936,946, and956. Negative MAG sample containing large entities from outlet607of fourth UFL in step5823may also be sent into the process of cell genetic modification and/or cell expansion5824. Prior to DNA/RNA sequencing in DNA/RNA sequencer906, a polymerase chain reaction (PCR) procedure on DNA/RNA sample obtained from cell lysing of large size entities from outlet607of fourth UFL from step5823may be performed, where PCR may be targeting one or more target DNA/RNA sequences and amplifies the number of target DNA/RNA sequences in the DNA/RNA sample.

FIG.80illustrates continued process of negative MAG sample after MAG separation, as in step408ofFIG.31, through UFL to retrieve small entities and passing of small entities into various molecule or small entity processing devices. After step5813ofFIG.66throughFIG.75, where negative MAG sample is collected during MAG separation of a target sample, in step5822, negative MAG sample of step5813is injected into fourth UFL inlet602, and UFL buffer is injected into inlet604of fourth UFL. In step5825, fourth UFL outputs negative MAG sample containing large entities from outlet607, and small size entities including DNA, RNA, molecules, and other small particles are output from fourth UFL outlet609, a PZT that attaches to fourth UFL and operates with a specified ultrasound vibration amplitude and frequency to create standing wave in fourth UFL is assumed. Finally, small size entities from outlet609of fourth UFL may be sent to be analyzed by any of: particle counter5835, particle imager5836, flow cytometer or sorter905, DNA/RNA sequencer906. Alternatively, output from particle counter5835, or output from particle imager5836, or output from flow cytometer or sorter905, may be further sent to be processed by DNA/RNA sequencer906as indicated respectively by path5827,5828, and956. DNA/RNA sequencer906may contain a PCR step on small size entities from outlet609of fourth UFL from step5825prior to DNA/RNA sequencing, where PCR may target one or more particular DNA/RNA sequences to amplify in quantity.

FIG.81illustrates entities analysis of negative MAG sample after MAG separation, as in step407ofFIG.31, into various analyzing devices. After step5813ofFIG.66throughFIG.75, where negative MAG sample is collected during MAG separation of a target sample, collected negative MAG sample may be sent to be analyzed by any of: cell counter903, cell imager904, flow cytometer or sorter905, particle counter5835, particle imager5836, DNA/RNA sequencer906. Alternatively, output from cell counter903, or output from cell imager904, or output from flow cytometer or sorter905, or output from particle counter5835, or output from particle imager5836, may be further sent to be processed by DNA/RNA sequencer906as indicated respectively by path936,946,956,5827, and5828. Negative MAG sample may also be sent into the process of cell genetic modification and/or cell expansion5824. DNA/RNA sequencer906may contain a PCR step on: (1) DNA/RNA obtained after cell lysing of cells contained within negative MAG sample; and (2) DNA/RNA/molecules contained within negative MAG sample. Prior to DNA/RNA sequencing, PCR may target one or more particular DNA/RNA sequences to amplify in quantity.

FIG.82illustrates continued process of positive MAG sample after MAG separation, as in step408ofFIG.31, through UFL to remove small entities and passing of large entities into various cell processing devices and procedures. Step5814is same as inFIG.66throughFIG.75, where positive MAG sample is collected after MAG separation of a target sample. In step5829, positive MAG sample of step5814is injected into fifth UFL inlet602, and UFL buffer is injected into inlet604of fifth UFL. In step5830, fifth UFL outputs positive MAG sample containing large entities from outlet607, and small size entities are removed from large entities and output from fifth UFL outlet609, a PZT that attaches to fourth UFL and operates with a specified ultrasound vibration amplitude and frequency to create standing wave in fifth UFL is assumed. Finally, positive MAG sample containing large entities from outlet607of fifth UFL may be sent to be analyzed by any of: cell counter903, cell imager904, flow cytometer or sorter905, DNA/RNA sequencer906. Alternatively, output from cell counter903, or output from cell imager904, or output from flow cytometer or sorter905, may be further sent to be processed by DNA/RNA sequencer906as indicated respectively by path936,946, and956. Positive MAG sample containing large entities from outlet607of fifth UFL in step5830may also be sent into the process of cell genetic modification and/or cell expansion5824. DNA/RNA sequencer906may contain a PCR step on DNA/RNA obtained after cell lysing of large size entities from outlet607of fifth UFL from step5830prior to DNA/RNA sequencing, where PCR may target one or more particular DNA/RNA sequences to amplify in quantity.

FIG.83illustrates continued process of positive MAG sample after MAG separation, as in step408ofFIG.31, through UFL to retrieve small entities and passing of small entities into various molecule or small entity processing devices. After step5814ofFIG.66throughFIG.75, where positive MAG sample is collected after MAG separation of a target sample, in step5829, positive MAG sample of step5814is injected into fifth UFL inlet602, and UFL buffer is injected into inlet604of fifth UFL. In step5831, fifth UFL outputs positive MAG sample containing large entities from outlet607, and small size entities including DNA, RNA, molecules, and other small particles bound by magnetic labels are output from fifth UFL outlet609, a PZT that attaches to fifth UFL and operates with a specified ultrasound vibration amplitude and frequency to create standing wave in fifth UFL is assumed. Finally, small size entities from outlet609of fifth UFL may be sent to any of: particle counter5835, particle imager5836, flow cytometer or sorter905, DNA/RNA sequencer906. Alternatively, output from particle counter5835, or output from particle imager5836, or output from flow cytometer or sorter905, may be further sent to be processed by DNA/RNA sequencer906as indicated respectively by path5827,5828, and956. DNA/RNA sequencer906may contain a PCR step on small size entities from outlet609of fifth UFL from step5831prior to DNA/RNA sequencing, where PCR may target one or more particular DNA/RNA sequences to amplify in quantity.

FIG.84illustrates entities analysis of positive MAG sample after MAG separation, as in step407ofFIG.31, into various analyzing devices. After step5814ofFIG.66throughFIG.75, where positive MAG sample is collected after MAG separation of a target sample, collected positive MAG sample may be sent to be analyzed by any of: cell counter903, cell imager904, flow cytometer or sorter905, particle counter5835, particle imager5836, DNA/RNA sequencer906. Alternatively, output from cell counter903, or output from cell imager904, or output from flow cytometer or sorter905, or output from particle counter5835, or output from particle imager5836, may be further sent to be processed by DNA/RNA sequencer906as indicated respectively by path936,946,956,5827, and5828. Positive MAG sample may also be sent into the process of cell genetic modification and/or cell expansion5824. DNA/RNA sequencer906may contain a PCR step on: (1) DNA/RNA obtained after cell lysing of cells contained within positive MAG sample; and (2) DNA/RNA/molecules contained within positive MAG sample, prior to DNA/RNA sequencing, where PCR may target one or more particular DNA/RNA sequences to amplify in quantity.

FIG.85Aillustrates adding fluorescent labels to specifically bind to target entities within negative MAG sample immediately after negative MAG sample collection.FIG.85Ashows that in step58131, immediately after step5813, where negative MAG sample is collected during MAG separation, fluorescent labels which are hybridized with antibodies or ligands, and specifically bind to surface antigens or receptors on target cells or entities are added into the negative MAG sample, and then negative MAG sample is incubated to form fluorescent labels binding to target cells or entities. Step58131may be inserted inFIG.79andFIG.80between step5813and step5822, or inserted inFIG.81immediately after step5813and before devices or processes903,904,905,906,5824,5825, and5826.

FIG.85Billustrates adding fluorescent labels to specifically bind to target entities within positive MAG sample immediately after positive MAG sample collection.FIG.85Bshows that in step58141, immediately after step5814, where positive MAG sample is collected after MAG separation, fluorescent labels which are hybridized with antibodies or ligands, and specifically bind to surface antigens or receptors on target cells or entities are added into the positive MAG sample, and then positive MAG sample is incubated to form fluorescent labels binding to target cells or entities. Step58141may be inserted inFIG.82andFIG.82between step5814and step5829, or inserted inFIG.84immediately after step5814and before devices or processes903,904,905,906,5824,5825, and5826.

FIG.86illustrates cross-sectional view of the tenth embodiment of MAG1241.FIG.86MAG1241is same design asFIG.12MAG124with variations fromFIG.12MAG123.FIG.86MAG1231is different fromFIG.12MAG123with the flux collection ends11211and11311being flat to mainly function collect flux emitted from center pole111tip end1111to form magnetic flux closure within the main pole111, side poles1120and1130, and bottom shield114. With flux collection end11211and11311being flat, highest magnetic field and highest magnetic field gradient are at the proximity to main pole111tip end1111, and thus facilitating biological entities10/30moving towards the tip end1111in the channel301. InFIG.86embodiment, flux collection ends11211and11311may also be described as soft magnetic shields of the main pole111tip end1111, where the soft shields11211and11311may help confine the magnetic flux within the gaps between tip end1111and shields11211and11311, as well as may increase effective magnetic force exerted on the biological entities10/30in channel301towards the tip end1111. InFIG.86, soft shields11211and11311flat ends being parallel to each other and form a physical space with the tip end1111, i.e. MAG gap of MAG1241, whereas the channel301is aligned by the soft shields11211and11311to be in contact with tip end1111at the bottom of the channel301. InFIG.86, the magnetic flux generated by N and S surface of magnet109is conducted within bodies of poles111,1121,1131and shield114with minimal leakage to outside of MAG1241structure. Flux density is highest around tip end1111with soft shields11211and11311producing lower flux density, enabling high magnetic field and field gradient around tip end1111. Compared to MAG121,122,123,124, MAG1241has the advantage of more efficient flux closure within the MAG1241soft magnetic bodies with least flux leakage and thus highest flux density around tip end1111to produce high magnetic force on biological entities10/30in channel301.

Channel301ofFIG.86is a rigid channel same as channel301ofFIG.12. Channel301may be attached to a non-magnetic channel holder110at the top surface3012. Channel holder110may align channel201to MAG gap of MAG1241, move channel301to separation position in contact with MAG1241pole111tip end1111, or lift channel301away from MAG1241after magnetic separation.FIG.301may be replaced with soft channel201ofFIG.13andFIG.14and operate with MAG1241ofFIG.86similarly as with MAG124inFIG.13andFIG.14.

FIG.87Aillustrates cross-sectional view of the eleventh embodiment MAG1251. MAG1251is same as MAG1241ofFIG.86, except the magnet109and bottom shield114of MAG1241ofFIG.86are removed in MAG1251. Permanent magnets115and116with opposing magnetizations1151and1161are placed in between poles111and1121, and between poles111and1131, respectively as shown inFIG.87A. Magnetizations1151and1161are horizontal inFIG.87A, which enables center pole111conducting N surface flux from both magnets115and116, while side poles1120and1130each conducts S surface flux from magnet115and116respectively. Compared to MAG1241, MAG1251may produce higher field around tip end1111due to two magnets115and116are used.

FIG.87Billustrates cross-sectional view of the twelfth embodiment MAG1261. MAG1261is same as MAG1241ofFIG.86, except the side poles1120and1130are each attached to S surface of permanent magnets1092and1094respectively, with magnetizations1093and1095being opposite to magnetization1091of magnet109. Bottom shield114is attached to both N surface of magnet1092and1094, and S surface of magnet109, and thus forming internal flux closure in shield114between magnets109,1092and1094. Compared to MAG1241, MAG1261may produce higher field around tip end1111due to three magnets109,1092and1092are used in MAG1261.

FIG.87Cillustrates cross-sectional view of the thirteenth embodiment MAG1271. MAG1271is same as MAG1261ofFIG.87B, except the bottom shield114is removed.

FIG.88Aillustrates cross-sectional view of the fourteenth embodiment MAG1242. MAG1242is same as MAG1241ofFIG.86, except that the side shield surface11212and11312are not parallel to each other, but rather side shield surface11212is substantially parallel to main pole111tip slope11112and side shield surface11213is substantially parallel to main pole111tip slope11113, where tip slopes11112and11113meet to form tip end1111. MAG gap formed by tip end1111, side shield surfaces11212and11312, ofFIG.88Amay produce higher flux concentration at tip end1111and higher effective magnetic force on biological entities10/30in channel301/201when channel301/201may be in contact with tip end1111. InFIG.88A, channel301/201may be in contact with main pole tip end1111of MAG1242, or may be in close proximity to but not in contact with main pole tip end1111.

FIG.88Billustrates cross-sectional view of the fifteenth embodiment MAG1243. MAG1243is same as MAG1242ofFIG.88A, except that the tip end1111ofFIG.88Ais replaced with a flatten tip11114inFIG.88B. MAG gap formed by flat tip end11114, side shield surfaces11212and11312, ofFIG.88Bmay avoid flat tip end11114flux saturation of main pole111to maximize the high magnetic field effective region within channel301/201when channel301/201may be in contact with tip end11114, and thus increase effective magnetic force exerted on biological entities10/30in channel301/201. The flat tip end ofFIG.88Bmay also be used to replace the tip end1111of main pole111of MAG embodiments inFIG.12throughFIG.15C,FIG.18,FIG.19, andFIG.86throughFIG.87C. InFIG.88B, channel301/201may be in contact with main pole tip end11114of MAG1243, or may be in close proximity to but not in contact with main pole tip end11114.

FIG.88Cillustrates cross-sectional view of the sixteenth embodiment MAG1244. MAG1244is same as MAG1242ofFIG.88A, except that the side shield surface11214and11314inFIG.88Care positioned above main pole tip1111, with each of side shield surface11214and11314being substantially a slope tilted in direction towards main pole tip1111to form a funnel shape that has a smaller opening between surface11214and11314when being closer to main pole tip1111. Tip ends of side shield surface11214and11314may be above main pole tip end1111. Tip ends of side shield surface11214and11314and main pole tip end1111may also be positioned within a horizontal plane.FIG.88Cside shield surface11214and11314arrangement may help produce high magnetic field and high magnetic field gradient in channel301/201and thus a higher effective magnetic force on biological entities10/30in channel301/201when channel301/201is in contact with tips ends of surface11214and11314, and may be in contact with, or in close proximity to, tip end1111of MAG1244.FIG.88Cside shield surface11214and11314arrangement may also help alignment of channel301/201to main pole tip end1111during positioning of channel301/201towards main pole tip end1111. InFIG.88C, channel301/201may be in contact with main pole tip end1111of MAG1244, or may be in close proximity to but not in contact with main pole tip end1111.

FIG.86throughFIG.88Care cross-sectional views of different embodiments of MAG designs, whereas the MAG designs extend in the direction that is into, or out of, the cross-sectional views. MAG tenth embodiment through fifteenth embodiment ofFIG.86throughFIG.88Care similar to MAG first embodiment through ninth embodiment as illustrated prior figures, whereas soft shields11211,11311,11212,11312,11213,11313, tip end1111, tip top11114, and channel101/201/301that may be in contact with, or may be in close proximity to, tip end1111or tip top11114, extend in direction of62as shown inFIG.32and being parallel to each other. Attachment of permanent magnets115,116,1092,109,1094to main pole111, side poles1120and1130as inFIG.87A,FIG.87B, orFIG.87Cmay be applied to each ofFIG.88A,FIG.88B, andFIG.88C.

FIG.89Aillustrates a cross-sectional view of channel301/201/101, similar toFIG.27B, where after magnetic separation of biological entities10/30in channel301/201/101, channel301/201/101is lifted from MAG gap of MAG embodiments inFIG.4throughFIG.21C, andFIG.86throughFIG.88B, to a lower field Position22by holder1082.

FIG.89Billustrates that at Position22ofFIG.89A, dissociation of cells10/30in channel301/201/101may be achieved by using a second channel holder1301that comes to contact channel301/201/101, where motor130is mechanically coupled to channel holder1301to produce mechanical vibration to channel holder1301, and where said mechanical vibration from motor130may then transfer through channel holder1301to channel301/201/101that is in contact with channel holder1301and may cause localized turbulence flow at various locations within the channel301/201/101, which may help mechanically break up the conglomerate into small pieces to assist self-dissociation of cells10/30conglomerate. In one embodiment, channel holder1301may push channel301/201/101and move channel301/201/101away from channel holder1082during dissociation of cells10/30. Direction of vibration exerted upon channel301/201/101through channel holder1301may be in direction61001, or in direction61002, or alternating between directions61001and61002. Channel holder1301may be in a shape that has a thinner handle connecting to motor130and a wider holder arm1302being in contact with channel301/201/101for effective vibration transfer from motor130to channel301/201/101. Channel holder1301length in the direction into or out of the cross-sectional view ofFIG.89Bmay be much shorter than channel holder1082. Channel holder1301may come into contact with channel301/201/101through existing clearances in channel holder1082without physical contact with channel holder1082.

FIG.89Cillustrates that at Position22ofFIG.89A, dissociation of cells10/30in channel301/201/101may be achieved by using a vibrator arm1303to contact channel holder1082. Motor130is mechanically coupled to vibrator arm1303to produce mechanical vibration to vibrator arm1303. Said mechanical vibration from motor130may then transfer through vibrator arm1303to channel holder1082and then to channel301/201/101that is in contact with channel holder1082and may cause localized turbulence flow at various locations within the channel301/201/101, which may help mechanically break up the conglomerate into small pieces to assist self-dissociation of cells10/30conglomerate. Direction of vibration exerted upon channel301/201/101through channel holder1082may be in direction61001, or in direction61002, or alternating between directions61001and61002. Vibrator arm1303may be in a fork shape that has a thinner handle connecting to motor130and a wider vibrator end1304being in contact with channel holder1082for effective vibration transfer from motor130to channel holder1082. Vibrator end1304may have a locking mechanism that mechanically locks onto channel holder1082to produce effective vibration transfer.

Channels101,201,301ofFIG.4thoughFIG.7,FIG.9thoughFIG.14,FIG.16thoughFIG.30B,FIG.32thoughFIG.36B,FIG.44AthoughFIG.65B,FIG.86thoughFIG.89C, may each have a channel wall thickness in the range of any of: 0.01 mm to 0.02 mm, 0.02 mm to 0.05 mm, 0.05 mm to 0.1 mm, 0.1 mm to 0.2 mm, 0.2 mm to 0.3 mm, 0.3 mm to 0.4 mm, 0.4 mm to 0.5 mm, 0.5 mm to 1 mm, 1 mm to 2 mm, and 2 mm to 5 mm.

FIG.90Ais a cross-sectional view of a portion of theFIG.38AUFL600along direction64, which includes entity fluid inlet602, buffer fluid inlet604, and part of the UFL main channel601.FIG.90Aillustrates UFL600is compose of two components, substrate611and cover610, where channels601,603and608are formed in substrate611as trenches of same depth627and preferably formed in a single step from as first surface of the substrate611. In one embodiment, depth627is between 100 nm to 500 nm. In another embodiment, depth627is between 500 nm to 1 um. In yet another embodiment, depth627is between 1 um to 10 um. In yet another embodiment, depth627is between 10 um to 100 um. In yet another embodiment, depth627is between 100 um to 1 mm. Different fromFIG.38Bembodiment, access ports for injecting fluid into the inlets602and604, and access ports for exporting fluid from the outlets607and608are formed in substrate611as single clearances as the inlets602,604and outlets607and608, enabling fluid injection or export from a second surface of the substrate611, opposing the first surface where channels601,603and608are formed.FIG.90Ashows example of access port621and inlet602are formed as a single clearance connecting from bottom second surface of substrate611to the channel603formed from top first surface of substrate611, while access port641and inlet604are formed as a single clearance connecting from bottom second surface of substrate611to the main channel601formed from top first surface of substrate611. InFIG.90A, different fromFIG.38B, cover610is a uniform cover without clearance features. Access ports in substrate611, which are also inlets and outlets of UFL600, allow entities fluid6020and buffer fluid6040to enter inlets602and604, and to allow large entities6070fluid and small entities6090fluid to exit outlets607and609. InFIG.90Aembodiment, alignment of access ports to inlets and outlets as inFIG.38Bis avoided. During manufacture of the UFL600, after substrate611are patterned on top first surface with the trenches601,603,608, clearances at locations of inlets602,604and outlets607,609may be formed through substrate611to connect from trenches to the bottom second surface. Cover610as a uniform piece may then be positioned over the substrate611top first surface to form enclosed channels601,603and608, where cover610may bond to substrate611through any of: (1) surface to surface Van der Waals force; (2) gluing; (3) ultrasound thermal melting when one or both of substrate611and cover610being made of plastic or polymer material. Injectors6021and6041then show example of possible external fluid injection to inlets of UFL600through substrate611access ports clearance, where the injectors6021and6041may have a larger nozzles size than the matching access ports621and641for managing positioning errors between injectors and access ports.FIG.90Ashows that entities fluid6020containing large entities612and small entities613, which may be injected by injector6021, passing through assess port621/602and passing into main channel601as side laminar flows, while buffer fluid6040may be injected by injector6041, passing through assess port641/604and passing into main channel601as center laminar flow.

PDMS, polymer, ceramic, or metal, where metal may be composed any one or any alloy of aluminum, iron, nickel, titanium, chromium, platinum, tungsten, rhenium, copper, gold, silver. Cover610may be composed of different material than substrate611.

In one embodiment, forming of access ports, inlets, outlets and channels in substrate611includes the steps of: (1) providing a substrate611having two substantially flat surface; (2) forming channels etching mask over first flat surface; (3) etching substrate with a first etching method including: wet etch with fluid chemical, dry etch with chemical gas, plasma enhanced dry etch, sputter etch with ion plasma, and ion beam etch (IBE), to form channels into substrate; (4) forming inlets and outlets etching mask over second flat surface of substrate opposing first flat surface; (5) etching substrate with said first etching method to form inlets and outlets in substrate connecting from second flat surface through substrate to the channels formed in step (3). In forming of etch mask of step (2) and step (4), etch mask may be composed of photo resist (PR), which may include deposition or spin coating of PR on said flat surface, then exposure by optical or ion/electron radiation with patterns of channels; development of PR after said exposure, where remaining PR with said patterns serves as etch mask. Etch mask may also be made of a hard mask material that has lower etch rate than the substrate material under the first etching method, and step (2) and step (4) may each include: deposition of a hard mask layer on said flat surface; deposition or spin coating PR layer on hard mask layer, then exposure of said PR by optical or ion/electron radiation with patterns of inlets, outlets and channels, development of PR after said optical exposure, where remaining PR with said pattern serves as etching mask of said hard mask; hard mask is etched through with a second etch method including any of: wet etch with fluid chemical, dry etch with chemical gas, plasma enhanced dry etch, sputter etch with ion beam; removal of remaining PR layer. Second etch method and first etch method may be different in type, or different in chemistry.

In another embodiment, inlets, outlets and channels in substrate611may be formed by thermal press involving using a heated stencil with physical pattern of the inlets, outlets and channels to melt and deform part of substrate611to construct the inlets, outlets and channels, then cooling down substrate611and remove the stencil. In thermal press, substrate material is preferred to be plastic or polymer. In yet another embodiment, inlets, outlets and channels in substrate611may be formed by imprint, which involves using a stencil with physical pattern of the inlets, outlets and channels to imprint into a partially or completely melt substrate611, and then cooling the substrate611and finally removing stencil, where cooled substrate retains the pattern transferred from stencil of the inlets, outlets and channels. In imprint, substrate material is preferred to be plastic or polymer. In another embodiment, inlets, outlets and channels are formed in substrate611by injection molding, where melted substrate611materials are injected into a mold cavity where substrate611body with engraved inlets, outlet and channels are defined by the mold cavity.

FIG.90Bis a cross-section view along direction65ofFIG.38Afor part of the UFL600including main channel601, substrate611, cover610.FIG.90Bembodiment functions same asFIG.38C, except PZT614is attached to the cover610and ultrasound vibration from PZT614is transferred into the ULF600channel601through cover610. InFIG.90B, cover601thickness6101is preferred to be equal or less than the thickness6111of the substrate611. In one embodiment, thickness6101is less than the thickness6111subtracting channel601depth627. Cover610thickness6101may be any one of: between 1 mm to 2 mm, between 0.5 mm to 1 mm, between 0.2 mm to 0.5 mm, between 0.1 mm to 0.2 mm.

FIG.90Cillustrates top-down viewFIG.38AUFL600with multiple PZTs attached to same UFL600device. Two or more of PZTs6141,6142,6143,6144are attached to UFL600at different locations along the main channel601and covering the main channel601. PZT6144covering at least one outlet or at least one inlet of UFL600needs to be attached to the substrate611of the UFL600inFIG.38B, while needs to be attached to the cover601inFIG.90A, opposing outlets or inlets openings in both embodiments. PZT6141,6142,6143may each be attached to the cover601or the substrate611. In one embodiment, two or more of PZTs6141,6142,6143,6144are attached to cover601ofFIG.90A. In another embodiment, two or more of PZTs6141,6142,6143,6144are attached to substrate611ofFIG.38B. In yet another embodiment, two or more of PZTs6141,6142,6143, are attached to cover601ofFIG.38B. In yet another embodiment, two or more of PZTs6141,6142,6143, are attached to substrate611of

FIG.90A. In one embodiment, at least two PZTs selected from any of6141,6142,6143,6144attached to UFL600operate at same frequency. In another embodiment, at least two PZTs selected from any of6141,6142,6143,6144attached to UFL600operate as different frequencies, with each different PZT having a different frequency causing a different standing-wave mode being generated in the channel601directly covered by each PZT, whereas channel601may have varying channel width between an inlet to an outlet of UFL600. In one embodiment, each PZT has a length61411along the channel601direction, and a width61412orthogonal to said length61411direction, and each PZT attached to UFL600has a length61411being longer than width61412. In another embodiment, each PZT attached to UFL600has a length61411being shorter than width61412. In yet another embodiment, each PZT selected from any of6141,6142,6143,6144and attached to UFL600are identical, each PZT is attached to the same cover601surface or the same substrate surface611of the UFL600, and a same alternating electrical voltage is applied simultaneously to drive each PZT attached to UFL600at the same frequency.

FIG.91Aillustrates cross-sectional view of a UFL similar toFIG.90Bbut with a flow channel having circular curvature sides walls. Channel601ofFIG.91Ais in the shape of a truncated circle, where the side walls60102and60103are part of the same circle, whereas the diameter of the circle is half wavelength, or an integer multiple of half wavelength, of the ultrasound mode in the fluid within channel601at resonance frequency, or driving frequency, Fp of PZT614, standing ultrasound wave may be present in between the two side walls60102and60103of channel601as indicated by the dashed lines626. Center of the circle that side walls60102and60103being part of is preferred to be at center of the channel as indicated by the point62601, thus the bottom edge wall60101and top edge wall60104are substantially parallel to each other and having same width. Top edge wall60104is formed by top cover610covering over substrate611. Truncated circular shape of the channel601ofFIG.91Amay be formed by etching of substrate611by isotropic, or partial isotropic and partial anisotropic etching method, including wet etch and dry etch, which etches side walls60101and60103into substantially circular curvature, whereas the bottom channel wall60101may be kept flat during said etch with having a slow etching layer, i.e. etch stop layer, at the bottom channel wall60101location that does not etch easily as the substrate611wall60102and60103. Substrate611may be a multi-layer structure that has an etch stop layer60111to form bottom wall60101during channel601etch, and etchable layer60112on top of etch stop layer60111to allow etching of the channel611intoFIG.91Achannel truncated circular shape.

FIG.91Billustrates cross-sectional view of a UFL similar toFIG.91Abut with a flow channel having a partial-circular shape formed within UFL substrate611. Channel601ofFIG.91Bis in the shape of a circle truncated only on top side, where the side walls60105is close to a full circle, whereas the diameter of the circle is half wavelength, or an integer multiple of half wavelength, of the ultrasound mode in the fluid within channel601at resonance frequency, or driving frequency, Fp of PZT614, standing ultrasound wave may be present within the circle of the channel wall60105, as indicated by the dashed lines626. Top edge wall60104is formed by top cover610covering over substrate611. Truncated circular shape of the channel601ofFIG.91Bmay be formed by etching of substrate611by isotropic, or partial isotropic and partial anisotropic etching method, including wet etch and dry etch, which etches side walls60105into substantially circular shape within substrate611.

FIG.91Cillustrates cross-sectional view of a UFL similar toFIG.91Abut with a flow channel having a circular shape formed within both UFL substrate and UFL cover. Channel601ofFIG.91Cis in the shape of a circle having a bottom channel wall60105of substantially a half circle shape formed within substrate611, and a top channel wall60104of substantially a similar half circle shape with same diameter of60105circle and formed within top cover610. Diameter of the circle formed by60104and60105is half wavelength, or an integer multiple of half wavelength, of the ultrasound mode in the fluid within channel601at resonance frequency, or driving frequency, Fp of PZT614, standing ultrasound wave may be present within the circle of the channel wall60104and60105, as indicated by the dashed lines626. The half circular shape60105of the channel601in substrate611, and half circular shape60104in cover610ofFIG.91Cmay be formed by etching of substrate611and cover by isotropic, or partial isotropic and partial anisotropic etching method, including wet etch and dry etch, which etches60104and60105into substantially circular shape within cover610and substrate611respectively. Then an alignment step of aligning channel walls60104and60105into an enclosing channel601is performed to formFIG.91Cchannel shape.

For channels601,603,608, inlets602,603, outlets607,609ofFIG.38A,FIG.38BandFIG.91A, the channels, inlets and outlets after being patterned into its shape may be coated with one layer or multiple layers of any of silicon oxide, SiN, SiC, alumina, aluminum, iron, nickel, titanium, chromium, platinum, tungsten, rhenium, copper, gold, silver, with process of PVD, CVD, PE-CVD, oxidization, ALD or PE-ALD, such coated layer is in contact with liquid sample flowing through UFL600during operation.

FIG.92Aillustrates top-down view of a UFL device600, similar as UFL600ofFIG.38A, having two inlets602and604, and two outlets607and609. Input sub-channel6042leading from inlet604to main channel601has a channel width6511. Main channel has a channel width6510. Input side-channel603leading from inlet602to main channel601has a channel width6514. Output sub-channel6072leading from main channel601to outlet607has a channel width6512. Output side-channel608leading from main channel601to outlet609has a channel width6513. In one embodiment, channel width6512is smaller than channel with6511. Channel width6512may be a percentage value of channel width6511with the percentage being within the range of any of: 10% to 20%, 20% to 40%, 40% to 60%, 60% to 80%, 80% to 100%, 100% to 150%, 150% to 200%, 200% to 500%, 500% to 1000%. In another embodiment, channel width6512is smaller than channel with6513. Channel width6512may be a percentage value of channel width6513with the percentage being within the range of any of: 10% to 20%, 20% to 40%, 40% to 60%, 60% to 80%, 80% to 100%, 100% to 150%, 150% to 200%, 200% to 500%, 500% to 1000%. Output sub-channel6072channel width6512and side channel width6513may be adjusted such that output fluid flow rate through607and609may be made different. For example,6512being equal or smaller than6513, output flow rate from outlet607is smaller than outlet609. Ratio of output fluid flow rate from outlet607to output fluid flow rate from outlet609may be estimated as (channel width6512) divided by (2 times of channel width6513). In one embodiment, channel width6511is smaller than channel width6510, channel width6512is smaller than channel width6511, channel width6512is smaller than channel width6513, channel width6513is larger than channel with6514, channel width6514is smaller than channel width6510. In one embodiment, ratio of channel width6512to channel width6513is smaller than the ratio of channel width6511to channel width6514. In another embodiment, ratio of channel width6512to channel width6513is larger than the ratio of channel width6511to channel width6514. In yet another embodiment, ratio of channel width6512to channel width6513is same as the ratio of channel width6511to channel width6514. Ultrasound generator device614, for example a PZT, may be attached to UFL600similarly as inFIG.38C, orFIG.90BthroughFIG.91C.

FIG.92Billustrates top-down view of a UFL device6000which is same asFIG.92Ain all other aspects except having only one inlet6022, and two outlets607and609. Main channel601connects directly from inlet6022to output side-channel608and output sub-channel6072. In one embodiment, channel width6512is smaller than channel width6513, channel width6514is smaller than channel width6510. Ultrasound generator device614, for example a PZT, may be attached to UFL6000similarly as inFIG.38C, orFIG.90BthroughFIG.91C.

FIG.93Aillustrates operation ofFIG.92AUFL600device. Entity fluid6020ofFIG.38Acontaining large entities10/20/30/612and smaller entities613is injected into UFL600channel through inlet602, and then passing through input side channels603with an effective volume flow rate6031into the main channel601. Buffer fluid6040ofFIG.38Ais injected into UFL600channel through inlet604, and then passing through input sub-channels6042ofFIG.92Awith an effective volume flow rate6041into the main channel601. Buffer fluid6040and entity fluid6020meet in main channel601and form a lamina flow, where buffer fluid6040flows at the center of the main channel601and entity fluid6020flows on the side of the buffer fluid6040with the channel601and along the side wall of the channel601. Due to the lamina flow, buffer fluid6040and entity fluid6020do not mix during the passage through channel601. For achieving the said lamina flow, buffer fluid6040may have a different fluid density from entity fluid6020, or buffer fluid6040may have a different viscosity than entity fluid6020, or buffer fluid6040may have a different compressibility than entity fluid6020. In one embodiment, buffer fluid6040has higher density than entity fluid. In one embodiment, buffer fluid6040has higher viscosity than entity fluid. In one embodiment, buffer fluid6040has higher compressibility than entity fluid. In main channel601, entity fluid6020may have a linear flow speed6033along the channel601edge, buffer fluid6040may have a linear flow speed6043at the center of the channel601. Linear speeds6033and6043may be different. In one embodiment, speed6033may be smaller than speed6043; in another embodiment, speed6033may be larger than speed6043; in yet another embodiment, speed6033may be substantially the same as speed6043.

InFIG.93A, ultrasonic standing wave mode created in main channel601caused by ultrasound generator device614, as shown inFIG.38CandFIG.38Dcauses the large size entities10/20/30/612to move from the entity flow6020flowing at the channel601walls at speed6033into the buffer flow6040flowing at the center of the channel601at speed6043, while all or most of the smaller entities613are maintained in entity flow6020. Small entities613may be maintained in entity flow6020by their smaller size, or larger density, or smaller compressibility than large size entities10/20/30/612. When lamina flow in channel601containing6020and6040reaches end of channel601, part of the center buffer flow6040containing the large size entities10/20/30/612flows through output sub-channel6072ofFIG.92A, exits through outlet607with an effective volume flow rate6071. While entity flow6020containing smaller entities613at side wall of channel601passes through output side channels608with an effective volume flow rate6081and exit through outlet609. In one embodiment, flow rate6071is smaller than flow rate6081with channel width6512being smaller than channel width6513inFIG.92A. In another embodiment, flow rate6071is larger than flow rate6081with channel width6512being larger than channel width6513inFIG.92A. In yet another embodiment, flow rate6071is substantially similar to flow rate6081with channel width6512being similar to6513inFIG.92A.

FIG.93Billustrates operation ofFIG.92BUFL6000device. Entity fluid6020ofFIG.38Acontaining large entities10/20/30/612and smaller entities613is injected into UFL6000channel through inlet6022with an effective volume flow rate6021into the main channel601. Ultrasonic standing wave mode, or also referred to as acoustic standing wave mode, created by ultrasound generation device, or also referred to as acoustic generation device,614in main channel601similar as shown inFIG.38CandFIG.38Dcauses the large size entities10/20/30/612flowing at the channel601edge walls to move to the center of the channel601at speed6043, i.e. a concentration of large size entities10/20/30/612at channel601center line, while all or most of the smaller entities613are maintained in entity flow6020without much concentration. Small entities613may be maintained in entity flow6020without concentration by their smaller size, or larger density, or smaller compressibility than large size entities10/20/30/612. When entity flow in channel601reaches end of channel601, center portion of the entity flow6020containing most or all large size entities10/20/30/612, and a small portion of smaller entities613, flows through output sub-channel6072ofFIG.92B, exits through outlet607with an effective volume flow rate6071. Side portion of entity flow6020containing mostly or entirely of smaller entities613passes through output side channels608with an effective volume flow rate6081and exit through outlet609. In one embodiment, flow rate6071is smaller than flow rate6081with channel width6512being smaller than channel width6513inFIG.92B. In another embodiment, flow rate6071is larger than flow rate6081with channel width6512being larger than6513inFIG.92B. In yet another embodiment, flow rate6071is substantially similar to flow rate6081with channel width6512being similar to6513inFIG.92B.

InFIG.93B, with existence of both large size entities10/20/30/612and smaller size entities613in incoming entity fluid6020, UFL6000ofFIG.93Bfunctions to output6070fluid through outlet607, with6070having a higher percentage and concentrated larger size entities10/20/30/612content and lower percentage of smaller size entities613than in the original incoming entity fluid6020, while depleting large size entities10/20/30/612population from fluid6090output from outlet609. In the case when entity fluid6020only contains large size entities10/20/30/612, UFL6000ofFIG.93Bfunctions to mainly output6070fluid with a reduced fluid volume compared to entity fluid6020through outlet607, and a higher larger size entities10/20/30/612concentration in fluid6070than in entity fluid6020.FIG.93BUFL6000functions may be effectively achieved byFIG.93AUFL600by using inlet602to inject entity flow6020and not injecting buffer flow6040through inlet604, or by using inlet604to inject entity flow6020and not injecting buffer flow6040through inlet602inFIG.93A.

InFIG.93AandFIG.93B, the flow rates6071and6081are inherent output flow rates values of the UFL600or UFL6000, meaning when outlets607and609are not connected to any external conduits and fluid6070and6090ofFIG.38Aflow out of outlets607and609freely. In the case where outlets607and609are connected to external conduits to conduct fluid6070and6090away from outlets607and609, these conduits may be used to produce additional fluid resistance on either6070or6090and causes extrinsic modification to flow rates6071and6081. In one embodiment, fluid resistance on fluid6070by conduit connected to outlet607is larger than fluid resistance on fluid6090by conduit connected to outlet609, thus causing6071being smaller than effective volume flow rate of fluid6090through outlet609, which is twice the value of6081inFIG.93AandFIG.93B. In another embodiment, fluid resistance on fluid6070by conduit connected to outlet607is smaller than fluid resistance on fluid6090by conduit connected to outlet609, thus causing6071being larger than effective volume flow rate of fluid6090through outlet609inFIG.93AandFIG.93B.

FIG.94Aillustrates embodiment of a process flow between blood or bone marrow sample collection and UFL operation. In step5801, peripheral blood sample or bone marrow sample is collected from a patient or person under test; in step5802, red blood cell lysing may be performed on sample of step5801; in step5803, said sample from step5802after lysing is injected in UFL entity fluid inlet602, while UFL buffer fluid is injected in outlet604; in step5804, UFL is operated similarly as step5804ofFIG.66. After step5804, the steps after step5804asFIG.66,FIG.70,FIG.72,FIG.74may be performed.

FIG.94Billustrates embodiment of another process flow between blood or bone marrow sample collection and UFL operation. In step5801, peripheral blood sample or bone marrow sample is collected from a patient or person under test; in step5802, red blood cell lysing may be performed on sample of step5801; in step5806, add into target sample from step5802magnetic labels, and/or fluorescent molecules, hybridized with antibodies or ligands, which specifically bind to surface antigens or receptors on target cells or entities; in step5807, target sample from step5806is incubated to form antibody-antigen or ligand-receptor binding to target cells or entities; in step5803, target sample from step5807is injected in UFL entity fluid inlet602, while UFL buffer fluid is injected in outlet604; in step5804, UFL is operated similarly as step5804ofFIG.66. After step5804, the steps after step5804asFIG.66,FIG.70,FIG.72,FIG.74may be performed.

FIG.95Aillustrates embodiment of a process flow between solid sample collection and UFL operation. In step6901, solid tissue sample is collected; in step6902, solid tissue sample from step6901is dissociated in a fluid base; in step5806, add into target sample from step6902magnetic labels, and/or fluorescent molecules, hybridized with antibodies or ligands, which specifically bind to surface antigens or receptors on target cells or entities; in step5807, target sample from step5806is incubated to form antibody-antigen or ligand-receptor binding to target cells or entities; in step5803, target sample from step5807is injected in UFL entity fluid inlet602, while UFL buffer fluid is injected in outlet604; in step5804, UFL is operated similarly as step5804ofFIG.66. After step5804, the steps after step5804asFIG.66,FIG.70,FIG.72,FIG.74may be performed.

FIG.95Billustrates embodiment of a process flow between surface sample collection and UFL operation. In step6701, surface entities are collected by swab; in step6702, surface entities collected on swab are dissolved in a fluid base; in step5806, add into target sample from step6702magnetic labels, and/or fluorescent molecules, hybridized with antibodies or ligands, which specifically bind to surface antigens or receptors on target cells or entities; in step5807, target sample from step5806is incubated to form antibody-antigen or ligand-receptor binding to target cells or entities; in step5803, target sample from step5807is injected in UFL entity fluid inlet602, while UFL buffer fluid is injected in outlet604; in step5804, UFL is operated similarly as step5804ofFIG.66. After step5804, the steps after step5804asFIG.66,FIG.70,FIG.72,FIG.74may be performed.

FIG.96illustrates embodiment of a process flow after negative MAG sample collection including UFL operation. Step5813is same as inFIG.66throughFIG.75, where negative MAG sample is collected during MAG separation of a target sample. After step5813, negative

MAG sample of step5813is concentrated by UFL without using UFL buffer including either (a) in step58222injecting negative MAG sample of step5813into inlet602or604of fourth UFL600ofFIG.92A, and no UFL buffer is injected, or (b) in step58223injecting negative MAG sample of step5813into inlet6022of fourth UFL6000ofFIG.92B. After step58222or step58223, in step58231, fourth UFL outlet607outputs sample having concentrated large size entities with small size entities being fewer than in negative MAG sample of step5813. Finally, negative MAG sample containing large entities from outlet607of fourth UFL may be sent to be analyzed by any of: cell counter903, cell imager904, flow cytometer or sorter905, DNA/RNA sequencer906. Alternatively, output from cell counter903, or output from cell imager904, or output from flow cytometer or sorter905, may be further sent to be processed by DNA/RNA sequencer906as indicated respectively by path936,946, and956. Negative MAG sample containing large entities from outlet607of fourth UFL in step5823may also be sent into the process of cell genetic modification and/or cell expansion5824. Prior to DNA/RNA sequencing in DNA/RNA sequencer906, a polymerase chain reaction (PCR) procedure on DNA/RNA sample obtained from cell lysing of large size entities from outlet607of fourth UFL from step5823may be performed, where PCR may be targeting one or more target DNA/RNA sequences and amplifies the number of target DNA/RNA sequences in the DNA/RNA sample.

FIG.97illustrates embodiment of another process flow after negative MAG sample collection including UFL operation. Step5813is same as inFIG.66throughFIG.75, where negative MAG sample is collected during MAG separation of a target sample. After step5813, negative MAG sample of step5813is concentrated by UFL without using UFL buffer including either (a) in step58222injecting negative MAG sample of step5813into inlet602or604of fourth UFL600ofFIG.92A, and no UFL buffer is injected, or (b) in step58223injecting negative MAG sample of step5813into inlet6022of fourth UFL6000ofFIG.92B. After step58222or step58223, in step58251, fourth UFL outlet609outputs sample having small size entities, including DNA/RNA/molecules/small particles, with depletion of large size entities originally included in negative MAG sample of step5813. Finally, small size entities from outlet609of fourth UFL may be sent to be analyzed by any of: particle counter5835, particle imager5836, flow cytometer or sorter905, DNA/RNA sequencer906. Alternatively, output from particle counter5835, or output from particle imager5836, or output from flow cytometer or sorter905, may be further sent to be processed by DNA/RNA sequencer906as indicated respectively by path5827,5828, and956. DNA/RNA sequencer906may contain a PCR step on small size entities from outlet609of fourth UFL from step5825prior to DNA/RNA sequencing, where PCR may target one or more particular DNA/RNA sequences to amplify in quantity.

FIG.98illustrates embodiment of a process flow after positive MAG sample collection including UFL operation. Step5814is same as inFIG.66throughFIG.75, where positive MAG sample is collected after MAG separation of a target sample. After step5814, positive MAG sample of step5814is concentrated by UFL without using UFL buffer including either (a) in step58291injecting positive MAG sample of step5814into inlet602or604of fifth UFL600ofFIG.92A, and no UFL buffer is injected, or (b) in step58292injecting positive MAG sample of step5814into inlet6022of fifth UFL6000ofFIG.92B. After step58291or step58292, in step58301, fifth UFL outlet607outputs sample having concentrated large size entities with small size entities being fewer than in positive MAG sample of step5814. Finally, positive MAG sample containing large entities from outlet607of fifth UFL may be sent to be analyzed by any of: cell counter903, cell imager904, flow cytometer or sorter905, DNA/RNA sequencer906. Alternatively, output from cell counter903, or output from cell imager904, or output from flow cytometer or sorter905, may be further sent to be processed by DNA/RNA sequencer906as indicated respectively by path936,946, and956. Positive MAG sample containing large entities from outlet607of fifth UFL in step5823may also be sent into the process of cell genetic modification and/or cell expansion5824. Prior to DNA/RNA sequencing in DNA/RNA sequencer906, a polymerase chain reaction (PCR) procedure on DNA/RNA sample obtained from cell lysing of large size entities from outlet607of fourth UFL from step5823may be performed, where PCR may be targeting one or more target DNA/RNA sequences and amplifies the number of target DNA/RNA sequences in the DNA/RNA sample.

FIG.99illustrates embodiment of another process flow after positive MAG sample collection including UFL operation. Step5814is same as inFIG.66throughFIG.75, where positive MAG sample is collected after MAG separation of a target sample. After step5814, positive MAG sample of step5814is concentrated by UFL without using UFL buffer including either (a) in step58291injecting positive MAG sample of step5814into inlet602or604of fifth UFL600ofFIG.92A, and no UFL buffer is injected, or (b) in step58292injecting positive MAG sample of step5814into inlet6022of fifth UFL6000ofFIG.92B. After step58291or step58292, in step58311, fifth UFL outlet609outputs sample having small size entities, including DNA/RNA/molecules/small particles, with depletion of large size entities originally included in positive MAG sample of step5814. Finally, small size entities from outlet609of fifth UFL may be sent to be analyzed by any of: particle counter5835, particle imager5836, flow cytometer or sorter905, DNA/RNA sequencer906. Alternatively, output from particle counter5835, or output from particle imager5836, or output from flow cytometer or sorter905, may be further sent to be processed by DNA/RNA sequencer906as indicated respectively by path5827,5828, and956. DNA/RNA sequencer906may contain a PCR step on small size entities from outlet609of fourth UFL from step5825prior to DNA/RNA sequencing, where PCR may target one or more particular DNA/RNA sequences to amplify in quantity.

FIG.100Aillustrates embodiment of a process flow including two UFLs in serial operation. In step58051, an input sample is injected into inlet602or604of UFL600ofFIG.92Aor inlet6022of UFL6000ofFIG.92B, then UFL outlet607outputs a target sample containing large size cells or entities from original input sample. After step58051, target sample from step58051is concentrated by UFL without using UFL buffer including either (a) in step58222injecting target sample from step58051into inlet602or604of fourth UFL600ofFIG.92A, and no UFL buffer is injected, or (b) in step58223injecting target sample from step58051into inlet6022of fourth UFL6000ofFIG.92B. After step58222or step58223, in step58231, fourth UFL outlet607outputs sample having concentrated large size entities with small size entities being fewer than in target sample from step58051. After step58231ofFIG.100A, processes as described inFIG.96utilizing903,904,905,906,5824after step58231may be similarly performed.

FIG.100Billustrates embodiment of another process flow including two UFLs in serial operation.FIG.100Bis same asFIG.100A, except that after step58222or step58223, in step58251, in step58251, fourth UFL outlet609outputs sample having small size entities, including DNA/RNA/molecules/small particles, with depletion of large size entities originally included in target sample from step58051. After step58251ofFIG.100B, processes as described inFIG.97utilizing5835,5836,905,906after step58251may be similarly performed.

FIG.101Aillustrates embodiment of yet another process flow including two UFLs in serial operation. In step58052, an input sample is injected into inlet602or604of UFL600ofFIG.92Aor inlet6022of UFL6000ofFIG.92B, then UFL outlet609outputs a target sample containing smaller size cells or entities from original input sample. After step58052, target sample from step58052is concentrated by UFL without using UFL buffer including either (a) in step58222injecting target sample from step58052into inlet602or604of fourth UFL600ofFIG.92A, and no UFL buffer is injected, or (b) in step58223injecting target sample from step58052into inlet6022of fourth UFL6000ofFIG.92B. After step58222or step58223, in step58226, fourth UFL outlet607outputs first sample having concentrated large size entities with small size entities being fewer than in target sample from step58052and fourth UFL outlet609outputs second sample having small size entities, including DNA/RNA/molecules/small particles, with depletion of large size entities originally included in target sample from step58052. After step58226ofFIG.101A, processes as described inFIG.96utilizing903,904,905,906,5824after step58231may be similarly performed on first sample from step58226; processes as described inFIG.97utilizing5835,5836,905,906after step58251may be similarly performed on second sample from step58226.

FIG.101Billustrates embodiment of yet another process flow including two UFLs in serial operation. In step58052, an input sample is injected into inlet602or604of UFL600ofFIG.92Aor inlet6022of UFL6000ofFIG.92B, then UFL outlet609outputs a target sample containing smaller size cells or entities from original input sample. After step58052, in step58227injecting target sample from step58052into inlet602and injecting buffer fluid into inlet604of sixth UFL600ofFIG.92A. After step58227, in step58226, sixth UFL outlet607outputs first sample having mainly large size entities with depletion of small size entities originally included in target sample from step58052; and outlet609outputs second sample having mainly smaller size entities, including DNA/RNA/molecules/small particles, with depletion of large size entities originally included in target sample from step58052. After step58228ofFIG.101B, processes as described inFIG.96utilizing903,904,905,906,5824after step58231may be similarly performed on first sample from step58228; processes as described inFIG.97utilizing5835,5836,905,906after step58251may be similarly performed on second sample from step58228.

FIG.102illustrates embodiment of a method of operating multiple UFLs in serial, or cascaded, configuration.FIG.102illustrates biological sample is passed through a first stage multiple UFLs600, output fluids from the UFLs600, which can be either large entities6070or small entities6090, are then fed into inlets8011of a fourth type flow connector8010, and from connector8010outlets8012into (a) inlet602or inlet604of one or multiple second stage UFLs600; or (b) inlet6022of one or multiple second stage UFLs6000.

FIG.103illustrates embodiment of another method of operating multiple UFLs in serial, or cascaded, configuration.FIG.102illustrates biological sample is passed through a first stage multiple UFLs600, output fluids from the UFLs600, which can be either large entities6070or small entities6090, are then fed into inlets8021of a fifth type flow connector8020, and from connector8020outlets8022into (a) inlet602or inlet604of one or multiple second stage UFLs600; or (b) inlet6022of one or multiple second stage UFLs6000.

FIG.104illustrates embodiment of yet another method of operating multiple UFLs in serial, or cascaded, configuration.FIG.104illustrates biological sample is passed through a first stage multiple UFLs600, output fluids from the UFLs600, which can be either large entities6070or small entities6090, are then fed into inlets8031of a sixth type flow connector8030, and from connector8030outlets8032into (a) inlet602or inlet604of one or multiple second stage UFLs600; or (b) inlet6022of one or multiple second stage UFLs6000.

Serial or cascade structures ofFIG.102,FIG.103, andFIG.104may be used in serial to achieve multiple stages UFL function, for example large entities6070or small entities6090from outlets607and609of second stage UFL600or UFL6000of each ofFIG.102,FIG.103, andFIG.104may be similarly injected into third stage UFL600or UFL6000of each ofFIG.102,FIG.103, andFIG.104through another intermediate8010/8020/8030connector in any combination.

FIG.105Aillustrates embodiments of another method of operating multiple UFLs in serial, or cascaded, configuration.FIG.105Aillustrates biological sample is first passed through a first UFL600, output target sample from the UFL600may be large entities6070from outlet607, as in step58051ofFIG.100AorFIG.100B, or small entities6090from outlet609, as in step58052ofFIG.101AorFIG.101B. UFL6001and UFL6002are similar to UFL600in design or operation.

In first embodiment ofFIG.105A, large entities6070target sample from UFL600may then be injected into inlet602of UFL6001, where large entities6070from UFL600may then be further separated by UFL6001into first sample containing larger size population entities of6070from UFL600and output from outlet607of UFL6001, or second sample containing smaller size population entities of6070from UFL600and output from outlet609of UFL6001. In first embodiment ofFIG.105A, ultrasound vibration generator6145attached to UFL600may be operating at any of: a higher vibration strength, a higher driving voltage, a higher resonant frequency, or having a larger area size, than ultrasound vibration generator6146attached to UFL6001; main channel601of UFL600may have any of: a narrower channel width, a deeper channel depth than main channel601of UFL6001; buffer fluid6040entering inlet604of UFL600may have any of: a smaller density, a smaller viscosity, a larger compressibility, a slower flow rate, than buffer fluid6040entering inlet604of UFL6001.

In second embodiment ofFIG.105A, smaller entities6090target sample from UFL600may then be injected into inlet602of UFL6002, where smaller entities6090from UFL600may then be further separated by UFL6001into third sample containing larger size population entities of6090from UFL600and output from outlet607of UFL6002, or fourth sample containing smaller size population entities of6090from UFL600and output from outlet609of UFL6002. In second embodiment ofFIG.105A, ultrasound vibration generator6148attached to UFL6002may be operating at any of: a higher vibration strength, a higher driving voltage, a higher resonant frequency, or having a larger area size, than ultrasound vibration generator6145attached to UFL600; main channel601of UFL6002may have any of: a narrower channel width, a deeper channel depth than main channel601of UFL600; buffer fluid6040entering inlet604of UFL6002may have any of: a same or larger density, a same or larger viscosity, a same or larger compressibility, a same or slower flow rate, than buffer fluid6040entering inlet604of UFL600.

In first embodiment ofFIG.105B, large entities6070target sample from UFL600may then be injected into inlet6022of UFL6001, where large entities6070from UFL600may then be further separated by UFL6003into fifth sample containing mostly larger size population entities of6070from UFL600and output from outlet607of UFL6003, or sixth sample containing smaller size population entities of6070from UFL600and output from outlet609of UFL6003. In first embodiment ofFIG.105B, ultrasound vibration generator6145attached to UFL600may be operating at any of: a higher vibration strength, a higher driving voltage, a higher resonant frequency, or having a larger area size, than ultrasound vibration generator6146attached to UFL6003; main channel601of UFL600may have any of: a narrower channel width, a deeper channel depth than main channel601of UFL6003.

In second embodiment ofFIG.105B, smaller entities6090target sample from UFL600may then be injected into inlet6022of UFL6004, where smaller entities6090from UFL600may then be further separated by UFL6004into seventh sample containing larger size population entities of6090from UFL600and output from outlet607of UFL6004, or eighth sample containing smaller size population entities of6090from UFL600and output from outlet609of UFL6004. In second embodiment ofFIG.105B, ultrasound vibration generator6148attached to UFL6004may be operating at any of: a higher vibration strength, a higher driving voltage, a higher resonant frequency, or having a larger area size, than ultrasound vibration generator6145attached to UFL600; main channel601of UFL6004may have any of: a narrower channel width, a deeper channel depth than main channel601of UFL600.

FIG.106Aillustrates embodiment of MAG in a module configuration. IfFIG.106A, at least one MAG unit121,122,123,124,125,126,127,128,129,1241,1242,1243,1251,1261,1272in combination with at least one channel101,201, or301, and holder107,110,1020,1040,1102,1103,1081,1082, and at least one pump500, are included in a module10801having a physical enclosure. Module10801may also have other electronic components, electronic boards, control circuit, control programs, embedded software, and peripheral structures that make module10801function as a stand-alone unit performing functions of: (1) intake of sample into channel101/201/301that is in contact with said MAG unit; (2) separation of entities10/30with said MAG unit from said sample; (3) dissociation of 10/30 from said channel; (4) output negative MAG sample and positive MAG sample as in step5813and step5814ofFIG.66throughFIG.75. Flow limiter509,510fromFIG.38AthroughFIG.36B, and valves805,935,936,937,938,939,940,941fromFIG.53throughFIG.65Bmay be included in module10801. Fluidic lines as described inFIG.59A,FIG.59B,FIG.64A,FIG.64Bmay be included as part of module10801.

FIG.106Billustrates embodiment of UFL in a module configuration. IfFIG.106B, at least one UFL600or6000, with two pumps500injecting sample into inlet602and inlet604of UFL600, or one pump500injecting sample into inlet6022of UFL6000, are included in a module10802having a physical enclosure. Module10802may also have other electronic components, electronic boards, control circuit, control programs, embedded software, and peripheral structures that make module10802function as a stand-alone unit performing functions of: (1) intake of sample into UFL600inlet602, or UFL6000inlet6022; (2) intake of buffer into UFL600inlet604; (3) separation of entities within said sample into large entities sample6070and output through outlet607of UFL600or UFL6000; (4) separation of entities within said sample into smaller entities sample6090and output through outlet609of UFL600or UFL6000. Flow limiter509,510fromFIG.38AthroughFIG.36B, and valves805,935,936,937,938,939,940,941fromFIG.53throughFIG.65Bmay be included in module10802. Fluidic lines as described inFIG.60A,FIG.60B,FIG.65A,FIG.65Bmay be included as part of module10802.

FIG.106Cillustrates embodiment of a system10800including a single module of MAG10801or a single module of UFL10802. InFIG.106C, system10800may include module10801or module10802, and may provide any of: (1) a physical enclosure for fixture of module10801or module10802; (2) electrical connections, including electrical power, electrical data communication lines, to module10801or module10802; (3) a data interface between a control unit of system10800and module10801or module10802, preferable with a standard communication protocols, including wire and wireless protocols, and including but not limited to GPIB, Bluetooth, NFC, USB, TCP/IP, serial, and parallel protocols; (4) a user interface for controlling the module10801or module10802by a user.

FIG.106Dillustrates embodiment of a system including multiple modules of MAG10801and UFL10802. InFIG.106D, system10800may include at least two modules with each module being either module10801or module10802, and provide to each said module any of: (1) a physical enclosure for fixture of all modules; (2) electrical connections, including electrical power, electrical data communication lines, to each of module10801or module10802; (3) a data interface between a control unit of system10800and each of module10801or module10802, preferable with a standard communication protocols, including wire and wireless protocols, and including but not limited to GPIB, Bluetooth, NFC, USB, TCP/IP, serial, and parallel protocols; (4) a user interface for controlling each of the module10801or module10802by a user. InFIG.106D, each module10801or module10802included in system10800may operate independently, and each said module may have fluidic lines included in each said module and without connection of said fluidic line between any of said modules.

FIG.107illustrates embodiment of a system including multiple modules of MAG10801and UFL10802with fluidic sample flowing through the modules in serial. InFIG.107, system10800may include at least two modules with each module being either module10801or module10802, and provide to each said module any of: (1) a physical enclosure for fixture of all modules; (2) electrical connections, including electrical power, electrical data communication lines, to each of module10801or module10802; (3) a data interface between a control unit of system10800and each of module10801or module10802, preferable with a standard communication protocols, including wire and wireless protocols, and including but not limited to GPIB, Bluetooth, NFC, USB, TCP/IP, serial, and parallel protocols; (4) a user interface for controlling each of the module10801or module10802by a user. InFIG.107, neighboring modules10801or module10802included in system10800may operate dependently, fluidic lines included in each said module may connect to neighbor modules, where an output sample from one module may be injected as input sample of the neighboring module.FIG.107show an example that modules from left to right function in serial, where output sample6070, or6090, or427, or428from a module10801or10802, may pass through a flow connector801,802,803,8010,8020,8030, and enters the module on the right as input sample of401, or708, or6020, or408, wherein fluidic lines between module may exist to achieve said output sample to input sample function. Fluidic lines as described inFIG.55A through58B, and inFIG.61AthroughFIG.63B, which include both MAG and UFL may be included as continuous fluidic lines with different portion of the continuous fluidic lines being part of different module10801or module10802, and said continuous fluidic lines connect across multiple modules10801or10802as shown in system10800ofFIG.107.

FIG.108AillustratesFIG.33Aflexible channel attached to the output port of a peristaltic pump having a blockage sensor in proximity to the flexible channel.FIG.108Ais substantially same asFIG.33A, except a sensor5081is included. Sensor5081may function to detect the event that the channel wall of channel508comes into physical contact with sensor5081, or to detect the event that the channel wall of channel508moves within proximity threshold to sensor5081, wherein said proximity threshold means a minimal physical distance between channel508external wall and surface of sensor5081in any range of: 0.001 mm to 0.1 mm, 0.1 mm to 1 mm, 1 mm to 2 mm, 2 mm to 10 mm, 10 mm to 20 mm. Sensor5081may connect to a control circuit5083through electrical connection5082, where control circuit5083may provide power to sensor5081and may sense the event of channel508coming into physical contact with sensor5081or the event of channel508moving within proximity threshold to sensor5081. Sensor5081may be in the form of any of: metal strip, electrode, contact surface, optical sensor set including an optical emitter and an optical sensor. Sensing by control circuit of 5083 may be through the sensing of change of parameter measured from sensor5081including any of: capacitance, inductance, thermal radiation, thermal conductivity, temperature, optical transmission or reflection, acoustic transmission or reflection, contact force or contact pressure, electrical conductivity, where said parameter may be measured for any of: between sensor5081to channel508, between sensor5081to a reference structure including but not limited to: electrical ground, temperature plate, dummy structure, where reference structure may be included in sensor5081or circuit5083, or between sensor5081to ambient environment.FIG.108Ashows that in normal operation when channel508may expand due to fluidic pressure built up in channel508caused by narrow flow path produced by clamp509and510as described inFIG.33A, but channel201after clamp509and510is not blocked, therefore channel508expansion does not cause channel508to contact sensor5081or comes below proximity threshold to sensor5081.

FIG.108Bshows that in the case of a blockage occurrence in fluidic lines after clamp509and510, channel508may further expand due to additional fluid pressure built up in channel508caused by blockage.FIG.108Bshows blockage of 5203 in channel201causes fluid flow speed in channel201significantly reduced or completely stopped, where increased fluid pressure in channel508by continued pumping of fluid sample into channel508by pump500may cause channel508to further expand and contacts sensor5081and control circuit5083may detect such event of contact and determines that a blockage event may have occurred in channel201. Pump500may then stop to avoid further fluid injection into channel508with detection of said blockage event.

FIG.109Ais identical toFIG.108Ain all other aspects except that the clamp509and510are replaced with a limiter20101. Limiter20101may take the form of a section of a tubing that connects to channel508on one end and channel201on the other end, where limiter20101may have an internal diameter20102that is much smaller than the internal diameter of either channel508or channel201, and thus function similarly as clamp509and510to reduce fluidic flow speed when fluid passes through limiter20101from channel508to channel201. The internal diameter20102may be in the range of any of: 0.01 mm to 0.1 mm, 0.1 mm to 0.2 mm, 0.2 mm to 0.3 mm, 0.3 mm to 0.4 mm, 0.4 mm to 0.5 mm, 0.5 mm to 1 mm, and 1 mm to 2 mm. The internal diameter20102relative to the internal diameter of channel201may be in the range of any of: 1% to 5%, 5% to 10%, 10% to 20%, 20% to 30%, 30% to 40%, 40% to 50%, 50% to 60%, 60% to 80%.

FIG.109Bis same asFIG.108B, with replacing clamps509and510with limiter20101similar as inFIG.109A.FIG.109Bshows that in the case of a blockage5203occurrence in channel201after limiter20101, channel508may further expand due to additional fluid pressure built up in channel508caused by blockage and may contact sensor5081and control circuit5083may detect such event of contact and determines that a blockage event may have occurred in channel201. Pump500may then stop to avoid further fluid injection into channel508with detection of said blockage event.

FIG.110Aillustrates an embodiment of a UFL600having two inlets, and an optical detector901is located around main channel601of the UFL600. UFL600ofFIG.110Ais similar to UFL600ofFIG.90CandFIG.32A, and an ultrasound generator device614is attached to UFL600. Optical detector901may include one or more optical emitters, and one or more optical detectors, where optical emitters emit light beams into the main channel601and optical detectors detect said light beams after being reflected, or scattered, by entities1/10/20/30/612/613flowing in stream6043, or stream6033, within channel601of UFL600. Said optical detectors may also detect blockage of said light beams by said entities1/10/20/30/612/613, or detect secondary light emissions by said entities1/10/20/30/612/613. Optical detector901may be used to detect, or collect, the properties of entities1/10/20/30/612/613with said properties including any of: type, size, shape, speed of moving, transparency, morphology. Optical detector901may also be used to obtain entities1/10/20/30/612/613information including any of: count of different types of entities passing through channel601within a given amount of time, or within a given volume of fluid sample; color of fluorescent molecules attached to detected entities; number of colors of fluorescent molecules attached to detected entities; fluorescence optical strength from fluorescent molecules attached to detected entities; and optical images of detected entities.

FIG.110Billustrates an embodiment of a UFL6000having one inlet6022, and an optical detector901is located around main channel601of the UFL6000. UFL6000ofFIG.110Bis similar to UFL6000ofFIG.92BandFIG.93B, and an ultrasound generator device614is attached to UFL6000. Optical detector901ofFIG.110Bis same as optical detector901ofFIG.110A. Optical detector901ofFIG.110Bfunctions to detect entities1/10/20/30/612/613and obtain information of entities1/10/20/30/612/613in stream6021flowing in channel601of UFL6000similarly as detector901ofFIG.110A.

FIG.110Cillustrates an embodiment of a UFL, which may be UFL600or UFL6000, having an optical detector901located around sample sub-channel6072of the UFL. Fluid stream6043or6021flowing in main channel601of UFL600or UFL6000carrying mainly large size entities of sample fluid becomes flow stream6071after entering sub-channel6072. Optical detector901ofFIG.110Cis same as optical detector901ofFIG.110A. Optical detector901ofFIG.110Cfunctions to detect entities1/10/20/30/612within flow stream6071and obtain information of entities1/10/20/30/612in stream6071flowing in sub-channel6072similarly as detector901ofFIG.110Adetecting and obtaining information of entities1/10/20/30/612/613in stream6033/6034flowing in channel601ofFIG.110A. InFIG.110C, an ultrasound generator device6147may optionally be attached around sub-channel6072similar to device614around channel601ofFIG.110A, to produce ultrasound standing wave similar toFIG.38CandFIG.38Dto cause entities1/10/20/30/612within flow stream6071to align to substantially center of channel6072for optical detection by detector901.

FIG.110Dillustrates another embodiment of a UFL having an optical detector901around an extended channel910in UFL.FIG.110Dshows that in UFL600or UFL6000, flow stream6021/6043/6071carrying entities1/10/20/30/612flows in channel601or channel6072and enters extended channel910and becomes flow stream9101. Extended channel910has channel width9102which may be narrower than channel width6510/6512of channel601/6072, where channel910width9102relative to channel601/6072width6510/6512may be in the range of any of: 1% to 5%, 5% to 10%, 10% to 20%, 20% to 30%, 30% to 40%, 40% to 50%, 50% to 60%, 60% to 80%, of channel601/6072width6510/6512. With effective volume flow rate being constant from flow6021/6043/6071to flow9101, linear flow speed of 9101 may be faster than linear flow speed of 6021/6043/6071, while entities1/10/20/30/612in flow9101may be spatially distributed more distant to each other than in flow6021/6043/6071, and thus enables a better spatial resolution during detection of entities1/10/20/30/612by optical detector901. Optical detector901ofFIG.110Dis same as optical detector901ofFIG.110A. Optical detector901ofFIG.110Dfunctions to detect entities1/10/20/30/612within flow stream9101and obtain information of entities1/10/20/30/612in stream9101flowing in channel910similarly as detector901ofFIG.110Adetecting and obtaining information of entities1/10/20/30/612in stream6034flowing in channel601ofFIG.110A. InFIG.110D, an ultrasound generator device6149may optionally be attached around channel910similar to device614around channel601ofFIG.110A, to produce ultrasound standing wave similar toFIG.38CandFIG.38Dto cause entities1/10/20/30/612within flow stream9101to align to substantially center of channel910for optical detection by detector901. Flow9101carrying entities1/10/20/30/612after passing detector901inFIG.110Dis designated as flow91010.

FIG.111Aillustrates an embodiment of an optical detector901having optical emitter or illuminator9011, forward scatter sensors9013and back scatter sensors9012being used to detect a biological entity1/10/20/30/612.FIG.111Adescribes more detailed structure of detector901as inFIG.110AthroughFIG.110D. InFIG.111A, flow6021/6043/6071/9101carries biological entities1/10/20/30/612through channel601/6072/910. Dashed line indicates detector901, which include components of illuminator9011, forward scatter sensors9013and back scatter sensors9012. InFIG.111A, components9011,9012are shown to be embedded within one side of the channel wall of channel601/6072/, and component9013is shown to be embedded within opposing side of the channel wall of channel601/6072/910, where components9011/9012/9013may each terminate at the channel601/6072/910side walls60112to allow light passage between channel601/6072/910and said components9011/9012/9013. When a biological entity1/10/20/30/612flows through the dashed box of 901 detector region of detection, information of biological entity1/10/20/30/612as described inFIG.110AthroughFIG.110Dmay be optically extracted by components9011,9012, and9013. Illuminator9011may include any of: light emitting diodes (LED), organic light emitting diode (OLED), laser diode, edge emitting laser. Detector9012and detector9013may each include any of: photo diode, avalanche photo diode (APD), charge-coupled device (CCD), or a complementary metal-oxide-semiconductor (CMOS) device.

InFIG.111A, illuminator9011may be driven by a first modulation signal that modulates intensity of light emitted by illuminator9011at a modulation frequency. Detected optical signal from detector9012, or from detector9013, may then be converted to second electrical signal, and a lock-in-amplification operation may then be performed with multiplying, or convolution of, the first modulation signal and second electrical signal at the modulation frequency, with necessary phase correction, and signal processing including band pass or low pass filtering, to extract the optical signal component generated from the biological entity1/10/20/30/612with higher signal to noise ratio. Extraction of said optical signal component generated from the biological entity1/10/20/30/612may be from first, second, third, or fourth harmonic of the modulation frequency during said lock-in amplification operation. Lock-in amplification operation may be achieved by feeding first modulation signal as reference signal, and second electrical signal as input signal into a lock-in amplifier control, or circuit, or component.

FIG.111Billustrates an embodiment ofFIG.111Aoptical detector901components being embedded in channel walls of channel601/6072/910, with back scatter sensor9012and the illuminator9011located in channel wall of same side, and forward scatter sensor9013located in opposing channel wall.FIG.111Bis a cross-section view ofFIG.111Aalong920cross-section line and viewing direction. InFIG.111B, illuminator9012and back scatter sensor9012are substantially around same vertical level from the bottom of the channel601/6072/910, where illuminator9012and back scatter sensor9012may be positioned with one in the front and the other in the back positions in the view ofFIG.111B. Light90112is emitted from illuminator9012toward entity1/10/20/30/612flowing in channel601/6072/910, back scatter light90122may be captured by back scatter sensor9012, and forward scatter light90132may be captured by forward scatter sensor9013. Light90122may be light90112reflected from entity1/10/20/30/612, and light90132may be light90112diffracted by, or optically scattered by, entity1/10/20/30/612, where light90122or light90132may each have same optical frequency or optical color as light90112. Light90122may be fluorescent light emitted from entity1/10/20/30/612after being excited by light90112, and light90132may be fluorescent light emitted from entity1/10/20/30/612after being excited by light90112, where light90122or light90132may each have lower optical frequency or longer optical wavelength than light90112, and where light90122or light90132may each be emitted by fluorescent molecules attached to entity1/10/20/30/612.

FIG.111Cis substantially same asFIG.111B, except thatFIG.111Cshows that back scatter sensors9012are positioned above and below the illuminator9011while embedded in the same side channel wall of channel601/6072/910.

FIG.111Dillustrates an embodiment ofFIG.111AwhereFIG.111Dis substantially same asFIG.111BorFIG.111C, except an optically transparent layer9103are coated within the channel601/6072/910internal wall of 60112 and 60113. Layer9103may be composed of single layer or a multi-layer structure, with each layer being any of: silicon nitride (SiN), silicon oxide (SiOx), silicon carbide (SiC), aluminum oxide (AlOx), aluminum nitride (AlN), zinc oxide (ZnOx), titanium nitride (TiN), titanium oxide (TiOx), magnesium oxide (MgO), diamond like carbon (DLC). Layer9103may include of any of: Si, Cu, Fe, Ti, Ta, Al, C, N, O, Tb, Sb, Ni, Cr, B, Ag, Au, Pt, Sn, Ir, Mn, Ru, W, Be, Re, Hf, Nb, Mo, Zr, Cr, V, Mg, Rh, Pd. Layer9103may coated over only the internal side walls of the channel601/6072/910, or may be coated conformally over the internal side walls and internal bottom surface of channel601/6072/910. Layer9103may be coated by thin film coating process in vacuum chamber by any of: physical vapor deposition (PVD), chemical vapor deposition (CVD), atomic layer deposition (ALD), plasma-enhanced chemical vapor deposition (PECVD), plasma-enhanced atomic layer deposition (PEALD), or molecular-beam epitaxy (MBE).

FIG.112Aillustrates an embodiment of an optical detector having illuminator array and forward scatter sensor array being used to detect a small size biological entity.FIG.112Ais same asFIG.111Ain all other aspects, except: illuminator9011ofFIG.111Aof detector901may be replaced with illuminator array ofFIG.112Athat contains five individual illuminators901101,901102,901103,901104,901105, forward scatter sensor9013ofFIG.111Aof detector901may be replaced with forward scatter sensor array ofFIG.112Athat contains five individual sensors901301,901302,901303,901304,901305, while each of said individual illuminator and individual sensor may terminate at channel wall60112. Back scatter sensor9012ofFIG.111Aof detector901may be removed inFIG.112A.FIG.112Ashows that illuminator901103emits light90112towards a small entity613in the channel, which may be carried within channel601/6072/910by flow6021, or by6033which may merge into flow6043/6071/9101. Due to the small size of the entity613, light90112may be scattered into a widely distributed light90132that may be captured by one or more of the sensors within the scatter sensor array, for example by all sensors901301,901302,901303,901304,901305.

FIG.112Bis same asFIG.112A, except that illuminator901103emits light90112towards a larger entity1/10/20/30/612, where the larger size of the entity1/10/20/30/612may block certain amount of light90112and scattered light90132from entity1/10/20/30/612may be captured by one or more of sensors of the scatter sensor array, but the number of sensors capturing light90132being fewer than inFIG.112A, for example only sensors901301and901305at the ends of the scatter sensor array as shown inFIG.112B.

ForFIG.112AandFIG.112B, each sensor of 901301, 901302, 901303, 901304, 901305, within the scatter sensor array may sense light90132: (1) at a different light color range or light wavelength range; (2) with different sensitivity; and (3) at different time during passage of entities1/10/20/30/612/613through channel601/607/910. Each illuminator of 901101, 901102, 901103, 901104, 901105 within the illuminator array may emit light90112towards center of channel601/6072/910at different strength, different optical phase, different polarization, different light emission angle relative to entity1/10/20/30/612/613, or different light emission time during passage of entities1/10/20/30/612/613through channel601/607/910. Individual sensor in sensor array or individual illuminator in illuminator array may have coordinated light sensing and light emission timing. In one embodiment, illuminators901101,901102,901103,901104,901105, are separately turned on and off to illuminate light90112towards center of channel601/6072/910, and one or more of the sensors901301,901302,901303,901304,901305, capture light90132simultaneously after each individual emission of light90112by an individual illuminator of the illuminator array. In another embodiment, illuminators901101,901102,901103,901104,901105, are separately turned on and off to illuminate light90112towards center of channel601/6072/910, and each of the sensors901301,901302,901303,901304,901305, sequentially, or separately, or individually, captures light90132after each individual emission of light90112by an individual illuminator of the illuminator array. In one embodiment, illuminator array may contain only one illuminator while scatter sensor array may contain more than one sensor. In another embodiment, illuminator array may contain more than one illuminator, while scatter sensor array may contain only one sensor. AlthoughFIG.112AandFIG.112Bshow illuminator array and scatter sensor array in one-dimensional array formation, either illuminator array or scatter sensor array may be in the form of a two-dimensional array formation, with illuminators or sensors existing in directions into or out of the viewing plane ofFIG.112AandFIG.112B. Lock-in-amplification operation as described inFIG.111A, may be performed by driving first modulation signal to modulate intensity of light90112emitted by one or more illuminators901101,901102,901103,901104,901105of illuminator array either simultaneously or individually or sequentially, at a modulation frequency. Detected optical signal from scattered light90132may be obtained from one or more of sensors901301,901302,901303,901304,901305, either simultaneously or individually or sequentially, and then may be converted to second electrical signal, and a lock-in-amplification operation may then be similarly performed as described inFIG.111A.

FIG.113Aillustrates an embodiment of an optical detector having one illuminator9011and forward scatter sensor array containing sensors901301,901302,901303,901304,901305, being used to detect a biological entity1/10/20/30/612at a first entity position11501within channel601/6072/910. InFIG.113A, illuminator9011may emit light90112towards center of channel601/6072/910. At position11501, due to size, shape, optical opacity, or optical reflectivity of entity1/10/20/30/612, scatter light90132from entity1/10/20/30/612with illumination light90112may be mainly captured by sensors901303,901304, or901305as shown inFIG.113A, while sensors901301and901302may not capture sufficient optical signal from scattered light90132due to their position relative to entity1/10/20/30/612and illuminator9011inFIG.113A. Light90132property including any of: intensity, color, light wavelength, polarization, phase, modulation, of light90132captured by each individual sensors of901303,901304, or9013, may be different. Each of components9011,901301,901302,901303,901304,901305may terminate at channel wall60112.

FIG.113BillustratesFIG.113Aentity1/10/20/30/612further moving along channel601/6072/910within flow6021/6043/6071/9101to a new position11502. InFIG.113B, illuminator9011may emit light90112towards center of channel601/6072/910. At position11502, due to size, shape, optical opacity, or optical reflectivity of entity1/10/20/30/612, scatter light90132from entity1/10/20/30/612with illumination light90112may be mainly captured by sensors901301,901302, or901305as shown inFIG.113B, while sensors901303and901304may not capture sufficient optical signal from scattered light90132due to their position relative to entity1/10/20/30/612and illuminator9011inFIG.113B. With entity1/10/20/30/612position change from11501ofFIG.113A to11502ofFIG.113B, sensors901301,901302,901303,901304,901305may capture different scatter light spatial distribution as described inFIG.113AandFIG.113Bwith sensors that capture scatter light90132being varied with position11501change to position11502.

FIG.114Aillustrates an embodiment of an optical detector having illuminator array containing illuminators901101,901102,901103,901104,901105and forward scatter sensor9013being used to detect a biological entity1/10/20/30/612at a first entity position11601in channel601/607/910with operating a first illuminator901101. InFIG.114A, illuminator901101may emit light90112towards center of channel601/6072/910, while other illuminators may not emit light90112. At position11601, due to size, shape, optical opacity, or optical reflectivity of entity1/10/20/30/612, and relative positions between entity1/10/20/30/612, illuminator901101and sensor9013, scatter light90132from entity1/10/20/30/612with illumination light90112may not reach sensor9013, or not be detected by sensor9013with sufficient optical signal inFIG.114A. Each of components9013,901101,901102,901103,901104,901105, may terminate at channel wall60112.

FIG.114Bis same asFIG.114A, except inFIG.114B, illuminator901104may emit light90112towards center of channel601/6072/910, while other illuminators may not emit light90112. At position11601, due to size, shape, optical opacity, or optical reflectivity of entity1/10/20/30/612, and relative positions between entity1/10/20/30/612, illuminator901104and sensor9013, scatter light90132from entity1/10/20/30/612with illumination light90112may be detected by sensor9013with a first optical signal intensity inFIG.114B.

FIG.114CillustratesFIG.114Aentity1/10/20/30/612further moving along channel601/6072/910within flow6021/6043/6071/9101to a new position11602. InFIG.114C, illuminator901101may emit light90112towards center of channel601/6072/910, while other illuminators may not emit light90112. At position11602, due to size, shape, optical opacity, or optical reflectivity of entity1/10/20/30/612, and relative positions between entity1/10/20/30/612, illuminator901101and sensor9013, scatter light90132from entity1/10/20/30/612with illumination light90112may reach sensor9013or be detected by sensor9013with sufficient optical signal as inFIG.114C.

FIG.114Dis same asFIG.114C, except inFIG.114D, illuminator901104may emit light90112towards center of channel601/6072/910, while other illuminators may not emit light90112. At position11602, due to size, shape, optical opacity, or optical reflectivity of entity1/10/20/30/612, and relative positions between entity1/10/20/30/612, illuminator901104and sensor9013, scatter light90132from entity1/10/20/30/612with illumination light90112may not reach or not be detected by sensor9013with sufficient optical signal inFIG.114D.

FIG.114Atogether withFIG.114B, andFIG.114Ctogether withFIG.114D, illustrate two individual illuminators901101and901104being separately operating and emitting light90112. In one embodiment, illuminators901101,901102,901103,901104,901105may be sequentially, or individually, operating and emitting light90112while other illuminators do not emit light90112, and sensor9013may be used to detect scatter light90132after each of individual illuminators901101,901102,901103,901104,901105emits light901102, and may also be used to produce a sensed light90132property including any of: intensity, color, light wavelength, polarization, phase, modulation, of light90132detected by sensor9013after individual emission of light90112by each individual illuminator of 901101, 901102, 901103, 901104, 901105 at either position11601or position11602.

FIG.115Aillustrates an example of detector signal strength at different sensor positions forFIG.113AandFIG.113Bembodiment.FIG.115Ashows that at position11501ofFIG.113A, light90112emitted from illuminator9011towards center of channel601/6072/910, may be blocked by entity1/10/20/30/612and sensors901301and901302may detect lowest strength light signal from light90132as indicated by the lowest bars, while sensors901303,901304, and901305may detect full strength light signal from light90132as indicated by the highest bars.FIG.115Aalso shows that at position11502ofFIG.113B, entity1/10/20/30/612moves with flow6021/6043/6071/9101further towards sensor901305, and thus sensors901301and901305may detect full strength light signal from light90132as indicated by the highest bar, sensors901303and901304may detect lowest strength light signal from light90132as indicated by the lowest bars, and sensor901302may detect medium strength light signal from light90132as indicated by the medium height bar. From detected light signal strength levels of sensors901301,901302,901303,901304, and901305at positions11501and11502as shown inFIG.115A, and by including information from any of: physical or designed location of illuminator9011; physical or designed location of each of said sensors; designated or designed detectable light wavelength of each of said sensors; operating timing or sequence of each of said sensors; or designated or designed detectable light polarization of each of said sensors, information related to entity1/10/20/30/612may be obtained, such information may include any of: (1) size, shape, surface scattering property, optical opacity, material composition, moving speed of entity1/10/20/30/612; (2) number of colors, or number of types, of fluorescent molecules attached to entity1/10/20/30/612, and may include numbers of molecules, or emitted optical light intensity, from each of said color, or each of said type, of said fluorescent molecules; (3) entity1/10/20/30/612being a single entity, or a cluster of multiple entities, or a conglomerate of multiple entities; (4) physical location of entity1/10/20/30/612within the channel601/6072/910; (5) distances of entity1/10/20/30/612from one or more channel walls of the channel601/6072/910.

FIG.115Billustrates an example of detector9013signal strength at different illuminator positions forFIG.114AthroughFIG.114Dembodiment.FIG.115Bshows that at position11601ofFIG.114AandFIG.114B, light90112emitted from illuminator901101towards center of channel601/6072/910, may be blocked by entity1/10/20/30/612and sensors9013may detect lowest strength light signal from light90132as indicated by the lowest bar correlating to901101, while light90112emitted from illuminator901104towards center of channel601/6072/910, may cause light90132detected by sensor9013at highest strength light signal as indicated by the highest bar correlating to901104.FIG.115Balso shows that at position11601, sensor9013detects lowest light intensity of 90132 when emitter901102emits light90112, medium light intensity of 90132 when emitter901103emits light90112, highest light intensity of 90132 when emitter901105emits light90112.FIG.115Balso shows that at position11602ofFIG.114CandFIG.114D, entity1/10/20/30/612moves further towards illuminator901105. At position11602, sensor9013detects strongest light intensity of 90132 when emitters901101,901102,901103each individually emits light90112, medium light intensity of 90132 when emitter901104emits light90112, lowest light intensity of 90132 when emitter901105emits light90112. From detected light signal strength levels by sensor9013for illuminators901101,901102,901103,901104, and901105at positions11601and11602as shown inFIG.115B, and by including information from any of: physical or designed location of sensor9013; physical or designed location of each of said illuminators; designated or designed illuminating light90112wavelength from each of said illuminators; operating timing or sequence of each of said illuminators; or designated or designed light polarization of each of said illuminators, information related to entity1/10/20/30/612may be obtained, such information may include any of: (1) size, shape, surface scattering property, optical opacity, material composition, moving speed of entity1/10/20/30/612; (2) number of colors, or number of types, of fluorescent molecules attached to entity1/10/20/30/612, and may include numbers of molecules, or emitted optical light intensity, from each of said color, or each of said type, of said fluorescent molecules; (3) entity1/10/20/30/612being a single entity, or a cluster of multiple entities, or a conglomerate of multiple entities; (4) physical location of entity1/10/20/30/612within the channel601/6072/910; (5) distances of entity1/10/20/30/612from one or more channel walls of the channel601/6072/910; (6) existence of entity1/10/20/30/612.

FIG.116Aillustrates an embodiment of an optical detector having illuminator array and forward scatter sensor array being used to detect shape of a biological entity at a first entity position with operating a first illuminator.FIG.116Ais same asFIG.112AandFIG.112B, where illuminator array containing five individual illuminators901101,901102,901103,901104,901105, and sensor array containing five individual sensors901301,901302,901303,901304,901305are used, where each individual illuminator and individual sensor may terminate at channel wall60112. InFIG.116A, illuminators901101,901102,901103,901104,901105may be individually enabled and operated to emit light90112towards center of channel601/6072/910, while sensors901301,901302,901303,901304,901305may sense scatter light90132, or in some cases emission light90112, simultaneously or individually.FIG.116Ashows an operation step11803where illuminators901101,901102,901104,901105are disabled, and only illuminator901103emits light90112, while sensors901301,901302,901303,901304,901305may sense scatter light90132, and may also sense emission light90112in one embodiment. InFIG.116A, due to the position, orientation and shape of the entity1/10/20/30/612within the channel601/6072/910, sensors901301,901302,901303may detect stronger light90132signal and sensors901304,901305may detect lower light90132signal.

FIG.116Bis same asFIG.116A, exceptFIG.116Billustrates another operation step11805where illuminators901101,901102,901103,901104are disabled, and only illuminator901105emits light90112, while sensors901301,901302,901303,901304,901305may sense scatter light90132, and may also sense emission light90112in one embodiment. InFIG.116B, due to the position, orientation and shape of the entity1/10/20/30/612within the channel601/6072/910, sensors901301,901303,901304,901305may detect stronger light90132signal and sensor901302may detect lower light90132signal.

FIG.117illustrates example of detector signal strength at different sensor positions forFIG.116AandFIG.116Bembodiments where illuminator array elements are individually operated. Light intensity detected by sensors901301,901302,901303,901304,901305is described by vertical bar for each sensor, where a higher bar indicates a stronger detected light intensity, and a lower bar indicates a lower detected light intensity. Step11803ofFIG.117corresponds to step11803ofFIG.116Awhere sensors901301,901302may detect strongest90132light signal,901303may detect medium90132light signal and sensors901304,901305may detect lowest light90132signal. Step11805ofFIG.117corresponds to step11805ofFIG.116B, where sensors901301may detect strongest90132light signal, sensor901302may detect lowest light90132signal,901303,901304,901305may detect increasingly stronger light90132signal. InFIG.117, step11801then describes the operation step where only illuminator901101emits light90112, step11802describes the operation step where only illuminator901102emits light90112, and step11804describes the operation step where only illuminator901104emits light90112, inFIG.116AorFIG.116B, while sensors901301,901302,901303,901304,901305may sense scatter light90132, and may also sense emission light90112in one embodiment. Bar heights in operation steps11801,11802, and11804describe detected light intensity by each of sensors901301,901302,901303,901304,901305in each said operation step. With the variation of detected light intensity at each of sensors901301,901302,901303,901304,901305, including light90132, and light90112in one embodiment, at different operation steps ofFIG.117where illuminators are individually operated, information of entity1/10/20/30/612may be extracted, including any of: (1) existence of entity1/10/20/30/612; (2) size, shape, surface scattering property, optical opacity, material composition, moving speed of entity1/10/20/30/612; (3) number of colors, or number of types, of fluorescent molecules attached to entity1/10/20/30/612, and may include numbers of molecules, or emitted optical light intensity, from each of said color, or each of said type, of said fluorescent molecules; (4) entity1/10/20/30/612being a single entity, or a cluster of multiple entities, or a conglomerate of multiple entities; (5) physical location of entity1/10/20/30/612within the channel601/6072/910; (6) distances of entity1/10/20/30/612from one or more channel walls of the channel601/6072/910. shape, orientation, position within channel601/6072/910,

Illuminators9011,901101,901102,901103,901104,901105ofFIG.112AthroughFIG.117may each be a multi-color LED or micro-LED unit. Illuminators901101,901102,901103,901104,901105may each also be a LED or micro-LED unit within a multi-color LED or micro-LED array or display. Each of said multi-color LED or micro-LED unit may contain multiple light emission components, with each said light emission component capable of emitting light at a different light wavelength or color, for example light emission components including LEDs or micro-LEDs emitting red, green and blue lights. Illuminators9011,901101,901102,901103,901104,901105, may be used to emit light90112at different colors or color combinations, for example alternating in emission of red, green, and blue lights from the same illuminator at different time. Sensors9013,901301,901302,901303,901304,901305ofFIG.112AthroughFIG.117may each be a monochrome or a multi-color sensing unit, for example a CMOS sensing unit. Sensors901301,901302,901303,901304,901305may each be a sensing unit within a monochrome or multi-color sensing array, for example a pixel within a monochrome or color CMOS image sensor. Sensors9011,901301,901302,901303,901304,901305may detect light90132at different colors, and may detect strength of light90132at said different colors. Illuminators9011,901101,901102,901103,901104,901105may each produce light90112in form of light pulses. Sensors9013,901301,901302,901303,901304,901305may each sense light90132in form of shuttered sensing within a designated time window.

In one embodiment, illuminators9011,901101,901102,901103,901104,901105ofFIG.112AthroughFIG.117may be placed in a spatially periodic arrangement and may produce spatially periodic illumination light90112upon entity1/10/20/30/612when entity1/10/20/30/612passes through illumination region of illuminator9011of optical detector910. Such spatially periodic illumination created by spatially periodic illuminators additionally may induce a production of temporally periodic scattered light90132when entity1/10/20/30/612passes through illumination region of illuminator9011, especially when flow speed of entity1/10/20/30/612within flow6021/6043/6071/9101is substantially constant, where said temporally periodic scattered light90132may in turn produce a detected periodic signal by sensor9013or sensors901301,901302,901303,901304,901305, at a frequency that may correlate to said spatial period of said illuminators9011,901101,901102,901103,901104,901105and may correlate to the flow speed of entity1/10/20/30/612passing through the illumination region of illuminator9011, for example said frequency may be calculated as said flow speed of said entity divided by said spatial period of said illuminators, wherein a band pass filter, or a low pass filter, or a high pass filter, which may be implemented by electronics of controller950,954, or by program of computing device955ofFIG.120AthroughFIG.122C, may be applied to said detected periodic signal by sensor9013or sensors901301,901302,901303,901304,901305to enhance signal detection from entity1/10/20/30/612and may be used to reduce noise from other entities2/3/22/613.

FIG.118Aillustrates an embodiment of an optical detector having illuminator9011, forward scatter sensors9013and back scatter sensors9012embedded in substrate611where channel601/6072/910is formed, but having optical components930positioned between wall of channel601/6072/910and each of said illuminator and sensors.FIG.118Ais substantially similar toFIG.111A, except the optical components930being used as optical intermediate path between channel601/6072/910wall and said illuminator and sensors. Optical components930may be composed of an optically transmitting material, and may be composed of single layer or a multi-layer structure, with each layer being any of: silicon nitride (SiN), silicon oxide (SiOx), silicon carbide (SiC), aluminum oxide (A10x), aluminum nitride (A1N), zinc oxide (ZnOx), titanium nitride (TiN), titanium oxide (TiOx), magnesium oxide (MgO), diamond like carbon (DLC), graphene. Optical components930may include of any of: Si, Cu, Fe, Ti, Ta, Al, C, N, O, Tb, Sb, Ni, Cr, B, Ag, Au, Pt, Sn, Ir, Mn, Ru, W, Be, Re, Hf, Nb, Mo, Zr, Cr, V, Mg, Rh, Pd. Optical components930may be deposited within substrate611by any of: PVD, CVD, ALD. PECVD, PEALD, MBE. Each of optical components930may have one end terminating at the channel601/6072/910wall60112and being in contact with flow6021/6043/6071/9101, and another end being in contact with, or aligned to, at least one of the illuminator9011, sensors9013and9012. Each of optical components930may be chemically, or physically, or biologically compatible with compositions of flow6021/6043/6071/9101, may show minimal degradation of material integrity and quality of light transmission of each of optical components930. Each of optical components930may function as an optical filter that has different optical wavelength passage property and provides optical filtering for the illuminator9011, sensors9013and9012that each of said optical components930is in contact with or aligned to. For example, optical components930in contact with sensors9012and9013may allow passage of light90132, fromFIG.111AthroughFIG.116B, that is scattered from, or emitted from, entity1/10/20/30/612to pass through while blocking light90112emitted from illuminator9011, thus enabling sensing of scattered light90132without interference from light90112. Each of optical components930may function as an optical polarizer that produces optical polarization on light90132or90112that passes through or selectively pass light90132or90112with a matching polarization. Each of optical components930may function as an optical lens that produces optical focusing, optical bending, optical diffraction, or optical divergence on light90132or90112that passes through said optical components930. For example, optical component930attached to, or aligned to, illuminator9011may focus light90112to a narrower light beam towards center of channel601/6072/910to achieve better spatial resolution during illumination by light90112. As another example, optical component930attached to, or aligned to, sensor9013may focus light90132to a narrower light beam towards sensor9013to achieve higher optical sensitivity or optical signal during detections by sensor9013.

InFIG.118A, illuminator9011may be replaced with illuminator array, for example illuminators9011,901101,901102,901103,901104,901105, and sensor9013may be replaced with sensor array, for example sensors9013,901301,901302,901303,901304,901305, as inFIG.112AthroughFIG.116B, where optical components930may exist on one or more of each of said illuminators or said sensors.

FIG.118Billustrates an embodiment of an optical detector having illuminator9011, forward scatter sensors9013and back scatter sensors9012positioned one the UFL outside surface facing the UFL channel601/6072/910walls, and having optical components930positioned between channel walls60112and each of illuminator9011and sensors9012and9013.FIG.118Bis same asFIG.118A, except that illuminator9011, sensors9012and9013are not embedded in substrate611, but rather externally attached to, or externally formed upon, the outside walls60114of the substrate611. Optical components930inFIG.118B, are same as inFIG.118A, except the optical components930have one end terminating at channel wall surface60112of channel610/6072/910, same as inFIG.118A, but another end terminates at the outside walls60114of the substrate611, connecting to, or aligned to, illuminator9011, or sensors9012and9013. InFIG.118B, illuminator9011may be replaced with illuminator array, for example illuminators901101,901102,901103,901104,901105, and sensor9013may be replaced with sensor array, for example sensors901301,901302,901303,901304,901305, as inFIG.112AthroughFIG.116B, where optical components930may exist on one or more of each of said illuminators or said sensors.

InFIG.118B, illuminator9011, and sensors9012and9013, may not be part of the substrate611but rather separated devices that may be physically aligned to each corresponding optical component930, to produce illumination light90112into the channel601/6072/910, or to detect light90122and90132from channel601/6072/910through the optical components930. The substrate611external walls60114where illuminator9011, and sensors9012and9013may be positioned may be parallel to the channel walls of channel601/6072/910, and optical components930may be substantially straight optical pathways.

FIG.119Aillustrates an embodiment of an optical detector having illuminator9011, forward scatter sensors9013and back scatter sensors9012positioned on the UFL substrate surface facing the UFL channel601/6072/910bottom surface60115, and having optical components930positioned between channel601/6072/910side walls60112and each of illuminator9011and sensors9012and9013to conduct light90112out of illuminator9011, light90122into sensor9012, and light90132into sensors9013.FIG.119Ais substantially same asFIG.118Bin every other aspect, except the illuminator9011, sensors9012and9013are either attached to, or externally positioned against the UFL substrate surface60115opposing the bottom surface60113of channel601/6072/910. InFIG.119A, optical components930are embedded in substrate611of UFL, however may have curved shapes to achieve light conduction between the side walls of channel601/6072/910and illuminator9011, sensors9012and9013. Similar as inFIG.118B, inFIG.119A, illuminator9011, and sensors9012and9013, may not be part of the substrate611but rather separated devices that may be physically aligned to each corresponding optical components930, to produce illumination light90112into the channel601/6072/910, or to detect light90122and90132from channel601/6072/910through the optical components930. InFIG.119A, illuminator9011may be replaced with illuminator array, for example illuminators901101,901102,901103,901104,901105, and sensor9013may be replaced with sensor array, for example sensors901301,901302,901303,901304,901305, as inFIG.112AthroughFIG.116B, where optical components930may exist on one or more of each of said illuminators or said sensors.

FIG.119Billustrates another embodiment of an optical detector having illuminator9011, forward scatter sensors9013and back scatter sensors9012positioned on the UFL cover surface60116facing the UFL channel601/6072/910top surface60111, and having optical components930positioned between channel601/6072/910side walls and each of illuminator9011and sensors9012and9013to conduct light90112out of illuminator9011, light90122into sensor9012, and light90132into sensors9013.FIG.119Bis identically toFIG.119Ain every other aspect except the optical components930are conducting light90112,90122and90132between601/6072/910side walls60112and top cover610of UFL. InFIG.119B, top cover610may be composed of a transparent material, or optically transmitting material that allows light of certain wavelength values to pass through with lower loss than other wavelength values. InFIG.119B, optical components930may not be in contact with illuminator9011, forward scatter sensors9013and back scatter sensors9012, but rather aligned with the positions of illuminator9011, forward scatter sensors9013and back scatter sensors9012, thus light90112,90122and90132passes through both the optical components930and top cover610. In another embodiment, top cover610may have physical clearances93011corresponding to optical components930positions and illuminator9011, forward scatter sensors9013and back scatter sensors9012positions, such that light90112,90122and90132pass through said clearances93011and said optical components930, where said clearances93011may be filled with transparent or optical filter material within said cover610. InFIG.119B, optical components930are embedded in substrate611of UFL, however may have curved shapes to achieve light conduction between the side walls of channel601/6072/910and illuminator9011, sensors9012and9013. Similar as inFIG.119A, inFIG.119B, illuminator9011, and sensors9012and9013, may not be part of the cover610but rather separated devices that may be physically aligned to each corresponding optical components930, to produce illumination light90112into the channel601/6072/910, or to detect light90122and90132from channel601/6072/910through the optical components930and cover610. InFIG.119B, illuminator9011may be replaced with illuminator array, for example illuminators901101,901102,901103,901104,901105, and sensor9013may be replaced with sensor array, for example sensors901301,901302,901303,901304,901305, as inFIG.112AthroughFIG.116B, where optical components930may exist on one or more of each of said illuminators or said sensors.

FIG.120Aillustrates an embodiment of an optical detector901having illuminator9011, forward scatter sensors9013and back scatter sensors9012with controller950of the optical detector901embedded in the UFL substrate611.FIG.120Ais same asFIG.111C, except that the illuminator9011, forward scatter sensors9013and back scatter sensors9012are connected to the embedded controller950through electrical connections951, where electrical connections951may also be embedded within substrate611. Controller950may be composed of electrical components including any of but not limited to: transistors, logic components, logic circuits, digital processor, digital signal processor, analog component, analog processor, analog circuits, analog to digital converter, digital to analog converter, digital amplifier, analog amplifier, data storage component, digital communication component, image processor, LED driver, OLED driver, photo diode driver, voltage driver, current driver, voltage sensor, current sensor, piezo driver, lock-in amplifier, phase-lock controller. Controller950connects to the optical components including: illuminator9011, forward scatter sensors9013and back scatter sensors9012, through electrical connections951to perform any of, but not limited to: providing power to said optical components, adjusting function parameters of said optical components, sensing optical signal detected by one or more of said optical components. Connections951may be conductive lines composed of any element of: copper, tungsten, tantalum, titanium, nitrogen, gold, silver, iridium, hafnium, iron, carbon. Controller950may be formed, or fabricated, within substrate611as a logic layer composed of semiconductor components prior to forming, or fabrication of, channel601/6072/910, and illuminator9011, forward scatter sensors9013and back scatter sensors9012, in substrate611, where controller950may be fabricated in a first manufacturing facility and channel601/6072/910may be fabricated in a second manufacturing facility. Substrate611may be composed of two distinctive layers, with a second layer overlapping a first layer, where controller950may be fabricated and contained within first layer, and channel601/6072/910may be fabricated within second layer, and where first and second layers of substrate611may be composed of materials including any of: glass, silicon, quartz, plastic, metal, AlTiC and quartz. An annealing process of said first layer of substrate611may be performed before second layer is formed on top of said first layer. An annealing step may be performed after forming of electrical connections951and before second layer is formed. An annealing step may be performed after second layer is formed on first layer. An annealing step may be performed after cover610is positioned, or attached to, substrate611. Said second layer may be formed on said first layer by any of: PVD or CVD film deposition, electroplating, spin coating, injection molding, or direct physical placement.

FIG.120Billustrates an embodiment of an optical detector901having illuminator9011, forward scatter sensors9013and back scatter sensors9012embedded in UFL substrate6011with controller954of the optical detector901being outside the UFL substrate611.FIG.120Bis a variation fromFIG.120A, where all other aspects are same, except controller950inFIG.120Abecomes controller954inFIG.120Bpositioned outside of ULF substrate611. InFIG.120B, electrical connections951may connect to illuminator9011, forward scatter sensors9013and back scatter sensors9012and terminate at surface electrical contacts952on the one or more of the external surfaces60114/60115of the substrate611. Electrical connections951may be embedded within substrate611. Surface electrical contacts952may be composed of any of, but not limited to: copper, silver, gold, tungsten, iridium, aluminum. Surface electrical contacts952may be in the form of surface contact pads; may be deposited on substrate611by any of: PVD or CVD film deposition, electroplating, vacuum plating, spin coating, or direct physical placement; and may be further formed into pad shape by any of: dry etch, wet etch. Electrical connections953external to substrate611then connect between surface electrical contacts952and the external controller954. Through electrical connections951and953, and surface electrical contacts952, controller954electrically connects to illuminator9011, and sensors9012and9013, and controller954functions same as controller950as inFIG.120A. Controller954may be composed of electrical components including any of but not limited to: logic components, logic circuits, digital processor, digital signal processor, analog component, analog processor, analog circuits, analog to digital converter, digital to analog converter, digital amplifier, analog amplifier, data storage component, digital communication component, image processor, LED driver, OLED driver, photo diode driver, voltage driver, current driver, voltage sensor, current sensor, piezo driver, lock-in amplifier, phase-lock controller. Controller954may perform any of, but not limited to: providing power to optical components9011,9012, or9013, adjusting function parameters of said optical components, sensing optical signal detected by one or more of said optical components. Connections951may be conductive lines composed of any element of: copper, tungsten, tantalum, titanium, nitrogen, gold, silver, iridium, hafnium, iron, carbon. Connections951may be formed, or fabricated, within substrate611as a logic layer, and may be composed of semiconductor components prior to forming, or fabrication of, channel601/6072/910, and illuminator9011, forward scatter sensors9013and back scatter sensors9012, in substrate611, where connections951may be fabricated in a first manufacturing facility and channel601/6072/910may be fabricated in a second manufacturing facility. Substrate611may be composed of two distinctive layers, with a second layer overlapping a first layer, where connections951may be fabricated and contained within first layer, and channel601/6072/910may be fabricated within second layer, and where first and second layers of substrate611may be composed of materials including any of: glass, silicon, quartz, plastic, metal, AlTiC and quartz. An annealing process of said first layer of substrate611may be performed before second layer is formed on top of said first layer. An annealing step may be performed after forming of electrical connections951and before second layer is formed. An annealing step may be performed after second layer is formed on first layer. An annealing step may be performed after cover610is positioned, or attached to, substrate611. Said second layer may be formed on said first layer by any of: PVD or CVD film deposition, electroplating, spin coating, injection molding, or direct physical placement. Connections953may be in the form of electrical probes that make electrical connection to surface contacts952through surface-to-surface contact. Connections953may be made part of controller954. Controller954may be part of, or attached to, a computing device955as described inFIG.121A.

FIG.121Aillustrates an embodiment ofFIG.120Acontroller950of the optical detector901communicating with and external computing device955through electrical connections9501,952and953. InFIG.121A, electrical connections9501may connect to embedded controller950and terminate at surface electrical contacts952on the one or more of the external surfaces60114/60115of the substrate611. Surface electrical contacts952ofFIG.121Aare same as inFIG.120B. Electrical connections953external to substrate611then connect between surface electrical contacts952and the external computing device955. Through electrical connections9501and953, and surface electrical contacts952, computing device955electrically connects to controller950, where computing device955may provide power to controller950, and may communicate with, send command to, receive information from, and exchange data with, controller950. Computing device955may contain hardware, electronics, software, algorithm, internet, data storage component, and database that may enable computing device955to control detector901through controller950. Connections9501may be conductive lines composed of any element of: copper, tungsten, tantalum, titanium, nitrogen, gold, silver, iridium, hafnium, iron, carbon. Electrical connections9501may be embedded within substrate611similar as connections951ofFIG.120A. In one embodiment, connections9501and951may be formed, or fabricated, in substrate611in same step, as described inFIG.120A. Connections953may be in the form of electrical probes that make electrical connection to surface contacts952through surface-to-surface contact. Connections953may be made part of computing device955.

FIG.121Billustrates an embodiment ofFIG.120Acontroller950of the optical detector901communicating with external computing device955through wireless means9502and9504. InFIG.121B, electrical connections9503may connect between embedded controller950and a wireless communication unit9502, where communication unit9502may be embedded in substrate611. External computing device955connects to an external wireless communication unit9504through electrical connections9505external to substrate611. Communication units9502and9504may each contain any of: an inductive coil, a wireless communication antenna, optical emitter, optical sensor, wireless communication electronics. Communication units9502and9504may transfer electrical power, analog signal or digital signal between each other through any mean of: inductive coupling, RF coupling, optical coupling. Through communication units9502and9504and electrical connections9503and9505, computing device955may wirelessly provide power to controller950, and may communicate with, send command to, receive information from, and exchange data with, controller950. Computing device955may contain hardware, electronics, software, algorithm, interne, data storage component, and database that may enable computing device955to control detector901through controller950. Connections9503may be conductive lines composed of any element of: copper, tungsten, tantalum, titanium, nitrogen, gold, silver, iridium, hafnium, iron, carbon. Electrical connections9503may be embedded within substrate611similar as connections951ofFIG.120A. In one embodiment, connections9503and951may be formed, or fabricated, in substrate611in same step, as described inFIG.120A. Connections9505and wireless communication unit9054may be made part of computing device955.

FIG.122Aillustrates an embodiment of electrically controlled optical filter9301positioned between UFL channel601/6072/910wall60112andFIG.118Ailluminator9011and scatter sensors9012and9013. InFIG.122A, controller950is same as controller950as described inFIG.120AthroughFIG.121B, and electrical connection9511is same as electrical connections951as described inFIG.120AandFIG.120B.FIG.122Ais a variation fromFIG.118A, where an optical filter9301is positioned between the optical component930and the illuminator and scatter sensors9011/9012/9013, with one end of 9301 terminating at channel601/6072/910wall60112. Optical filter9301functions to allow optical light of a first wavelength range to pass through with lower loss than a second wave length range. Optical filter9301positioned next to different optical components of the illuminator and scatter sensors9011/9012/9013may produce different optical filtering effect with allowing different first wavelength range optical light to pass with lower loss. Optical filter9301may be any of: optical low pass filter, optical high pass filter, optical band pass filter, optical band stop filter, optical shutter. Optical filter9301may provide different optical filtering effect by any of: having different material composition, having different optical coating, being a multilayer thin film structure and having different film stack configuration. In one embodiment, controller950through electrical connection9511may electrically control the optical filtering effect of optical filter9301through electrical voltage or electrical current, where an application of electrical signal from controller950may cause optical filter9301to allow certain optical light wavelength range to pass through optical filter9301with lowest loss, while a change of electrical signal from controller950may cause optical filter9301to change the optical light wavelength range that may pass through optical filter9301with lowest loss. In one embodiment, optical filter9301may be composed of liquid crystal (LC) and may be a liquid crystal tunable filter (LCTF), where LCTF may have a tunable optical wavelength between 400 nm to 2450 nm. In another embodiment, optical filter9301may be composed of electronic ink. InFIG.122A, controller950may produce an alternating electrical control signal to optical filter9301to modulate the light passage intensity through optical filter9301corresponding to said alternating control signal, for example within certain optical wavelength range, to realize a lock-in modulation function as described inFIG.111A. Optical filter9301may be embedded within substrate611similarly as illuminator9011and scatter sensors9012and9013. Optical filter9301may be externally inserted into substrate611through an opening within substrate611, where said opening may be formed within substrate611during fabrication of UFL600/6000.

FIG.122Billustrates an embodiment of electrically controlled optical lens93021/93022/93023positioned between UFL channel601/6072/910wall60112andFIG.118Ailluminator9011and scatter sensors9012and9013.FIG.122Bis same asFIG.122A, except the optical filter9301ofFIG.122Ais replaced with optical lens93021/93022/93023as inFIG.122B.FIG.122Bis a variation fromFIG.122A, where optical lens93021/93022/93023is positioned between the optical component930and the illuminator and scatter sensors9011/9012/9013. Optical lens93021may function to focus optical light from illuminator9011into the optical component930and further into channel601/6072/910, or to disperse optical light from channel601/6072/910into sensors9012and9013. Optical lens93022may function to disperse optical light from illuminator9011into the optical component930and further into channel601/6072/910, or to focus optical light from channel601/6072/910into sensors9012and9013. Optical lens93023may function similarly as lens93021or similarly as lens93022. Optical lens93023may be attached to a mechanical positioning component93024, which may move optical lens93023in direction93025towards or away from channel601/6072/910, or may move optical lens93023in direction93026along channel601/6072/910, wherein said positioning component93024may move in direction93025or93026with movement produced by a transducer comprising any of: piezo electric element, micro-electro-mechanical-system (MEMS), magnetic actuator, thermal expansion actuator, memory alloy. Optical lens93021/93022/93023may be any one of, or a combination of multiple of: convex lens, concave lens, cylindrical lens, spherical lens, or prism. Optical lens93021/93022/93023may be composed of electrically adjustable optical index material, where optical index of lens93021/93022/93023may be adjusted by an electric voltage or an electric current applied to lens93021/93022/93023, and thus optical focal length of lens93021/93022/93023may be adjusted by an electrical signal. Optical lens93021/93022/93023may be composed of an optical body containing two or more liquids, where said two or more liquids have different optical index and different dielectric constant, and wherein an applied electric voltage may alter the liquids distribution in said optical body and causing effective optical focal length change of said lens93021/93022/93023. Optical lens93021/93022/93023may be composed of a piezo-optic material, wherein an applied electric voltage may alter the optical index of said material and cause an effective optical focal length change of said lens93021/93022/93023. In one embodiment, controller950through electrical connection9511may electrically control the optical index of optical lens93021/93022/93023through electrical voltage or electrical current, where an application of electrical signal from controller950may cause optical lens93021/93022/93023to change its focal length. In one embodiment, controller950through electrical connection9511may electrically control the positioning component93024through electrical voltage or electrical current, where an application of electrical signal from controller950may cause component93024to move optical lens93023in direction93025or direction93026and thus change the optical focus or optical dispersion behavior through optical lens93023. InFIG.122B, controller950may produce an alternating electrical control signal to optical lens93021/93022/93023or to positioning component93024to modulate the light passing through the optical lens93021/93022/93023corresponding to said alternating control signal, to realize a lock-in modulation function as described inFIG.111A. Optical lens93021/93022/93023may be embedded within substrate611similarly as illuminator9011and scatter sensors9012and9013. Optical lens93021/93022/93023may be externally inserted into substrate611through an opening within substrate611, where said opening may be formed within substrate611during fabrication of UFL600/6000.

FIG.122Cillustrates an embodiment of using an optical gratings93031as optical filter and optionally using electrically positioned optical gratings93031between UFL channel601/6072/910wall andFIG.118Ailluminator9011and scatter sensors9012/9013.FIG.122Cis same asFIG.122A, except the optical filter9301ofFIG.122Ais replaced with optical gratings93031as inFIG.122B. Optical gratings93031may function similarly as optical filter9301ofFIG.122Ato allow light of certain wavelength range to pass from illuminator9011into the optical component930and further into channel601/6072/910, or to allow optical light of certain wavelength range from channel601/6072/910into sensors9012and9013. Optical filtering effect by optical gratings93031may be different from9301in the aspect that optical light of different wavelength may pass optical gratings93031and exit in different diffraction angles, similar to an optical prism, and thus orientation of optical gratings93031may be used to select which optical wavelength passes between channel601/6072/910and9011/9012/9013. Optical gratings93031may be formed as periodic straight clearances, including: slits, slots, holes, spaced across a distance that overlap illuminator9011and sensors9012/9013, wherein said clearances may be identical, oriented with an angle, and may be evenly spaced across said distance. Optical gratings93031may be fabricated directly from the UFL substrate611in area between9011/9012/9013and channel601/6072/910, as a physical structure formed within UFL substrate611material by dry etch or wet etch process to remove UFL substrate611material and form said clearances of optical gratings93031. Optical gratings93031may also be fabricated as an embedded optical element within UFL substrate611with a PVD or CVD process to provide materials forming optical gratings93031. Optical gratings93032, same as optical gratings93031, may be attached to a mechanical positioning component93024, which may move optical gratings93032in direction93025towards or away from channel601/6072/910, or may move optical gratings93032in direction93026along channel601/6072/910, or may rotate optical gratings93032in direction93027to change the orientation of said clearances, wherein said positioning component93024may move in direction93025or93026or93027with movement produced by a transducer comprising any of: piezo electric element, micro-electro-mechanical-system (MEMS), magnetic actuator, thermal expansion actuator, memory alloy. In one embodiment, controller950through electrical connection9511may electrically control the positioning component93024through electrical voltage or electrical current, where an application of electrical signal from controller950may cause component93024to move optical gratings93032in direction93025or direction93026or direction93027and thus change the optical filtering effect through optical gratings93032. InFIG.122C, controller950may produce an alternating electrical control signal to positioning component93024to modulate the light passing through the optical gratings93032corresponding to said alternating control signal, to realize a lock-in modulation function as described inFIG.111A. Optical gratings93031/93032may be externally inserted into substrate611through an opening within substrate611, where said opening may be formed within substrate611during fabrication of UFL600/6000.

Optical filter9301ofFIG.122Aand optical gratings93031ofFIG.122C, may be combined with optical lens93021/93022/93023ofFIG.122Binto an in-serial element, which may include one or more optical filter9301, or one or more optical gratings93031/93032, or one of more optical lens93021/93022/93023arranged in series, to allow optical light from illuminator9011to pass through, or to allow optical light from channel601/6072/910to pass through, all included filters, gratings, and lenses within said in-serial element.

InFIG.123A,FIG.118Aoptical detector901components, including illuminator9011, scatter sensors9012/9013, optical components930, as well as filters9301, lenses93021/93022/93023, and gratings93031/93032, may be formed on a first sub-layer61100of UFL substrate611after a first fabrication step during manufacture process of the UFL600/6000. During first fabrication step ofFIG.123A, components of optical detector901including9011/9012/9013/930/9301/93021/93022/93023/93031/93032may be fabricated on sub-layer61100with processes including any of: one or more times of deposition of single layer or multi-layer thin film stacks with any process of: PVD, CVD, ALD, MBE, plating; one or more steps of photoresist coating; one or more steps of photoresist exposure with using a photo mask; one or more steps of photoresist development and photoresist removal; one or more steps of dry etch including any of: reactive ion etch (RIE), ion beam etch (IBE), plasma etch (PE), chemical dry etch (CDE); one or more steps of wet etch. After first fabrication step ofFIG.123A, optical detector901components9011/9012/9013/930/9301/93021/93022/93023/93031/93032may be formed on sub-layer61100surface as patterned devices. Sub-layer61100may be composed any of: silicon, glass, AlTiC, ceramic, metal, polymer, plastics. After step ofFIG.123A, components of optical detector901may appear substantially patterned thin film stack islands on sub-layer61100.

In stepFIG.123B, a top layer61101may be deposited overFIG.123Asub-layer61100surface and completely covering over components of optical detector901, including9011/9012/9013/930/9301/93021/93022/93023/93031/93032. Top layer61101may exhibit uneven topography following the shape of components of optical detector901on sub-layer61100. Top layer61101may be deposited on sub-layer61100with any of: PVD, CVD, ALD, PECVD, PEALD, MBE, electro-plating, and spin coating.

In step ofFIG.123C, top layer61101surface may be planarized to be substantially a flat surface61102, or planarized with topography of step ofFIG.123Bbeing reduced, for example with surface roughness root-mean-square value reduced less than 10 nm, or less than lnm. Planarization of top layer61101may be performed by any of: IBE, PE, chemical-mechanical-polish (CMP).

In step ofFIG.123D, a trench is created within the top layer61101after step ofFIG.123C, where the trench forms bottom surface and two side walls of the channel601/6072/910of the UFL600/6000. Trench ofFIG.123Dmay be formed by: (step-1) depositing photoresist, or depositing a hard-mask layer and then a photoresist, on surface61102; (step-2) expose photoresist mask under a light source; (step-3) remove photoresist covering over the area corresponding to said trench; (step-4) etch the top layer61101, or etch the hard mask on top of surface61102and then etch the top layer61101, with etch method including any of: RIE, IBE, PE, CDE, wet etch, to remove material from said top lay61101to form said trench, where said etching of top layer61101may stop in top layer61101or stop in sub-layer61100. During etching of top layer61101to form said trench, part of one or more components of optical detector901may be exposed at the side walls of the said trench or side walls of channel601/6072/910, for example optical component930.

After step ofFIG.123D, in step ofFIG.123E, protection layer9103may be deposited and may be conformally covering overFIG.123Dtop layer61101surface, side walls and bottom surface of etched trench or channel601/6072/910. Protection layer9103may also cover over any exposed components of optical detector901after forming the trench ofFIG.123D. Protection layer9103is same as inFIG.111DandFIG.111E. InFIG.123E, protection layer9103, top layer61101and sub-layer61100may be regarded as the substrate611.

After step ofFIG.123DorFIG.123E, in step ofFIG.123F, top cover610may be positioned over the substrate611, for example contacting top layer surface61101directly if afterFIG.123Dstep, or contacting protection layer9103if after step ofFIG.123E, and forms an enclosure of the main channel601/6072/910of the UFL600/6000, where bottom surface of cover610forms top surface60111, layer9013forms side walls60112and bottom surface60113, of channel601/6072/910. An annealing step may be performed during or after the top cover610is positioned over substrate611. Top cover610may bond to substrate611through any of: (1) surface to surface Van der Waals force; (2) gluing; (3) ultrasound thermal melting; (4) metallic bond or covalent bond. Material at bottom surface of top cover610and material at top surface of substrate611contacting top cover610may exhibit element, or molecule, or material, inter-diffusion or intermixing, which may be facilitated by said annealing, such that a bonding is established.

FIG.124Aillustrates another embodiment of an optical detector901having illuminator9011, forward scatter sensors9013and back scatter sensors9012, being used to detect biological entity1/10/20/30/612. InFIG.124A, flow6021/6043/6071/9101carries biological entities1/10/20/30/612through channel601/6072/910. Dashed line indicates detector901, which include components of illuminator9011, forward scatter sensors9013and back scatter sensors9012.FIG.124Ais similar asFIG.111A, whileFIG.124Adescribes a vertical arrangement of optical components of optical detector901.

FIG.124Bshows an example ofFIG.124Aembodiment implementation, where inFIG.124B, components9011,9012are shown to be embedded within the substrate611underneath the bottom surface of channel601/6072/910, and component9013is shown to be embedded within top cover610of channel601/6072/910. When a biological entity1/10/20/30/612flows through the dashed box of 901 detector region of detection as inFIG.124A, information of biological entity1/10/20/30/612as described inFIG.110AthroughFIG.110Dmay be optically extracted by components9011,9012, and9013. Components9011and9012may terminate at bottom surface60113of channel601/6072/910. Component9013may terminate at top surface60111of channel601/6072/910. Illuminator9011may include any of: light emitting diodes (LED), organic light emitting diode (OLED), laser diode, edge emitting laser. Illuminator9011is preferred to be a vertical-cavity-surface emitting laser (VCSEL). Detector9012and detector9013may each include any of: photo diode, avalanche photo diode (APD), charge-coupled device (CCD), or a complementary metal-oxide-semiconductor (CMOS) device.

InFIG.124B, illuminator9011may be driven by a first modulation signal that modulates intensity of light emitted by illuminator9011at a modulation frequency. Detected optical signal from detector9012, or from detector9013, may then be converted to second electrical signal, and a lock-in-amplification operation may then be performed with multiplying, or convolution of, the first modulation signal and second electrical signal at the modulation frequency, with necessary phase correction, and signal processing including band pass or low pass filtering, to extract the optical signal component generated from the biological entity1/10/20/30/612with higher signal to noise ratio. Extraction of said optical signal component generated from the biological entity1/10/20/30/612may be from first, second, third, or fourth harmonic of the modulation frequency during said lock-in amplification operation. Lock-in amplification operation may be achieved by feeding first modulation signal as reference signal, and second electrical signal as input signal into a lock-in amplifier control, or circuit, or component.

Illuminator9011may be replaced by an illuminator array, for example901101/901102/901103/901104/901105, and sensor9013may each be replaced by sensor array901301/901302/901303/901304/901305, similarly as inFIG.112AthroughFIG.114D,FIG.116AandFIG.116B, where illuminator array901101/901102/901103/901104/901105and sensor array901301/901302/901303/901304/901305may function and detect entity1/10/20/30/612similarly as described inFIG.115A,FIG.115B, andFIG.117.

FIG.124Cis substantially same asFIG.124B, except that the forward scatter sensor9013may not be embedded in the top cover610but attached to the top surface60116of cover610external to channel601/6072/910. InFIG.124C, sensor9013may sense the scatter light90132with the light90132passing through top cover610, whereas light90132may pass through the top cover610where the top cover601may be substantially transparent, or light90132may pass through an optical window existing in the top cover610, where said optical window may be a patterned solid transparent material embedded within, and being part of, the top cover601.

FIG.124Dis substantially same asFIG.124B, except that the forward scatter sensor9013is not embedded in the top cover610but disposed in proximity to the top surface60116of cover610external to channel601/6072/910, where sensor9013may be positioned away from said top surface of cover610or may be in physical contact of said top surface60116of cover610. InFIG.124D, sensor9013may sense the scatter light90132with the light90132passing through top cover610, whereas light90132may pass through the top cover610where the top cover601may be substantially transparent, or light90132may pass through an optical window existing in the top cover610, where said optical window may be a patterned solid transparent material embedded within, and being part of, the top cover601.

FIG.124Eis substantially same asFIG.124B, except that the forward scatter sensor9013is not embedded in the top cover610and illuminator9011and back scatter sensor9012are not embedded in substrate611. Forward scatter sensor9013may either be disposed in proximity to the top surface60116of cover610external to channel601/6072/910, where sensor9013may be positioned away from said top surface60116of cover610or may be in physical contact of said top surface of cover610, or may be attached to the top surface60116of cover610. InFIG.124E, sensor9013may sense the scatter light90132with the light90132passing through top cover610, whereas light90132may pass through the top cover610where the top cover601may be substantially transparent, or light90132may pass through an optical window existing in the top cover610, where said optical window may be a patterned solid transparent material embedded within, and being part of, the top cover601. Illuminator9011and back scatter sensor9012may either be disposed in proximity to the bottom surface60115of substrate611external to channel601/6072/910, where illuminator9011and sensor9012may be positioned away from said bottom surface60115of substrate611or may be in physical contact of said bottom surface60115of substrate611, or may be attached to the bottom surface60115of substrate611. InFIG.124E, sensor9012may sense the scatter light90122and illuminator9011may emit light90112, with the light90112and90122passing through substrate611, whereas light90112and90122may pass through the substrate611where the substrate611may be substantially transparent, or light90112and90122may pass through an optical window existing in substrate611, where said optical window may be a patterned solid transparent material embedded within, and being part of, the substrate611.

FIG.125Ashows another example ofFIG.124Aembodiment implementation, where inFIG.125A, component9013is shown to be embedded within the substrate611underneath the bottom surface60113of channel601/6072/910, and component9011is shown to be embedded within top cover610of channel601/6072/910. Component9011may terminate at top surface60111of channel601/6072/910. Component9013may terminate at bottom surface60113of channel601/6072/910. When a biological entity1/10/20/30/612flows through the dashed box of 901 detector region of detection as inFIG.124A, information of biological entity1/10/20/30/612as described inFIG.110AthroughFIG.110Dmay be optically extracted by components9011and9013. Illuminator9011may include any of: light emitting diodes (LED), organic light emitting diode (OLED), laser diode, edge emitting laser. Illuminator9011is preferred to be a vertical-cavity-surface emitting laser (VCSEL). Detector9012and detector9013may each include any of: photo diode, avalanche photo diode (APD), charge-coupled device (CCD), or a complementary metal-oxide-semiconductor (CMOS) device.

InFIG.125A, illuminator9011may be driven by a first modulation signal that modulates intensity of light emitted by illuminator9011at a modulation frequency. Detected optical signal from detector9012, or from detector9013, may then be converted to second electrical signal, and a lock-in-amplification operation may then be performed with multiplying, or convolution of, the first modulation signal and second electrical signal at the modulation frequency, with necessary phase correction, and signal processing including band pass or low pass filtering, to extract the optical signal component generated from the biological entity1/10/20/30/612with higher signal to noise ratio. Extraction of said optical signal component generated from the biological entity1/10/20/30/612may be from first, second, third, or fourth harmonic of the modulation frequency during said lock-in amplification operation. Lock-in amplification operation may be achieved by feeding first modulation signal as reference signal, and second electrical signal as input signal into a lock-in amplifier control, or circuit, or component.

FIG.125Bis substantially same asFIG.125A, except that the illuminator9011is not embedded in the top cover610but attached to the top surface60116of cover610external to channel601/6072/910. InFIG.125B, illuminator9011may emit the light90112with the light90112passing through top cover610, whereas light90112may pass through the top cover610where the top cover601may be substantially transparent, or light90112may pass through an optical window existing in the top cover610, where said optical window may be a patterned solid transparent material embedded within, and being part of, the top cover601.

FIG.125Cis substantially same asFIG.125A, except that the illuminator9011is not embedded in the top cover610and forward scatter sensor9013is not embedded in substrate611. Illuminator9011may either be disposed in proximity to the top surface60116of cover610external to channel601/6072/910, where illuminator9011may be positioned away from said top surface60116of cover610or may be in physical contact of said top surface60116of cover610, or may be attached to the top surface of cover610. InFIG.125C, illuminator9011may emit light90112with the light90112passing through top cover610, whereas light90112may pass through the top cover610where the top cover601may be substantially transparent, or light90112may pass through an optical window existing in the top cover610, where said optical window may be a patterned solid transparent material embedded within, and being part of, the top cover601. Forward scatter sensor9013may either be disposed in proximity to the bottom surface60115of substrate611external to channel601/6072/910, where forward scatter sensor9013may be positioned away from said bottom surface60115of substrate611or may be in physical contact of said bottom surface60115of substrate611, or may be attached to the bottom surface60115of substrate611. InFIG.125C, sensor9013may sense the scatter light90132with the light90132passing through substrate611, whereas light90132may pass through the substrate611where the substrate611may be substantially transparent, or light90132may pass through an optical window existing in substrate611, where said optical window may be a patterned solid transparent material embedded within, and being part of, the substrate611.

FIG.125Dis substantially similar asFIG.124E, except the forward scatter sensor9013being disposed in proximity to the top surface60116of cover610external to channel601/6072/910, and the illuminator9011being disposed in proximity to the bottom surface60115of substrate611external to channel601/6072/910, where sensor9013may be positioned away from said top surface60116of cover610. InFIG.125D, and where illuminator9011may be positioned away from said bottom surface60115of substrate611.

Illuminator9011and sensor9012and9013inFIG.124AthroughFIG.125Dmay each be operated by controllers950and954, and by external computing device955in methods as described inFIG.120AthroughFIG.121B. Illuminator9011and sensor9012and9013inFIG.124AthroughFIG.125Dmay each be operated with components9301,93021/93022/93023/93024,93031,93032as described inFIG.122AthroughFIG.122C.

FIG.126Aillustrates an embodiment ofFIG.125Cwith said optical window930as described inFIG.125Cformed in the top cover610to allow passage of optical light90112from illuminator9011into the UFL channel601/6072/910and another optical window930as described inFIG.125Cformed in the substrate611to allow passage of optical light90132from UFL channel601/6072/910into the forward scatter sensor9013. Optical window930may be formed and function similarly as the optical component930as described inFIG.118A,FIG.118B,FIG.119A,FIG.119B,FIG.122A,FIG.122B,FIG.122C.

FIG.126Billustrates an embodiment ofFIG.125Dwith an optical window930formed in the substrate611to allow passage of optical light90112from illuminator9011into the UFL channel601/6072/910and another optical window930formed in the cover610to allow passage of optical light90132from UFL channel601/6072/910into the forward scatter sensor9013. Optical window930may be formed and function similarly as the optical component930as described inFIG.118A,FIG.118B,FIG.119A,FIG.119B,FIG.122A,FIG.122B,FIG.122C.

FIG.127Aillustrates an embodiment where an illuminator9011is embedded within the cover610or the substrate611of a UFL channel601/6072/910and light90112from the illuminator9011may be passed through an optical grating9305embedded within the cover610or substrate611of the UFL channel601/6072/910. Gratings9305may be same as gratings93031ofFIG.122C. Gratings9305may be composed of a series of isolated optical light guides, as shown inFIG.127A, with each light guide having a bottom end93052facing the light emission surface90111of illuminator9011and a top end93051facing the channel601/6072/910and entity1/10/20/30/612, where light90112emitted from illuminator9011surface90111enters bottom end93052of optical light guides, then passes through optical light guides of gratings9305, and then emitted from the top end93051of optical light guides. Top end90351may coincide with the channel wall60111/60112/60113. Each of the optical light guides of the gratings9305may have any of: different width, different length, different height. Each of the optical light guides of the gratings9305may produce a different effective optical path length for the90112light that passes through said each light guide. After light90112passes through the gratings9305, light90112may be phase modulated by gratings9305such that light90112may be optically converging or optically collimated towards center of the UFL channel601/6072/910or towards entity1/10/20/30/612.

FIG.127Billustrates an embodiment where forward scatter sensor9013is embedded within the cover610or the substrate611of a UFL channel601/6072/910and scattered light90132is passed to the forward scatter sensor9013through optical gratings9305embedded within the cover610or substrate611of the UFL channel601/6072/910. Gratings9305of FIG.127B are same as inFIG.127A, except gratings9305may be used to collect light90132from channel601/6072/910and transmit light90132to sensor9013. InFIG.127B, scattered light90132from entity1/10/20/30/612may enter top end93051of optical light guides of gratings9305, then pass through each of said optical light guides, and then exits from bottom end93052of said optical light guides and entering optical detection surface90131of sensor9013. After light90132passes through the gratings9305, light90132may be phase modulated by gratings9305such that light90132may be optically collimated or optically converged towards the optical detection surface90131of sensor9013.

FIG.127Cillustrates an embodiment where an illuminator9011is embedded within the cover610or the substrate611of a UFL channel601/6072/910and light90112from the illuminator9011may be passed through an optical phase plate9306embedded within the cover610or substrate611of the UFL channel601/6072/910. Phase plate9306may be a Fresnel lens or an optical phase array. Phase plate9306may take the form of a circularly shaped optical component having a continuously varying effective optical path along a radius direction of said circular shape for light passing through said phase plate9306. Phase plate9306may be composed of a series of isolated optical light guides that may be in the form of concentric circular rings with each light guide's circular ring having a varying effective optical path for light passing through said light guide. Phase plate9306may have a bottom end93062facing the light emission surface90111of illuminator9011and a top end93061facing the channel601/6072/910and entity1/10/20/30/612, where light90112emitted from illuminator9011surface90111enters bottom end93062of phase plate9306, then passes through phase plate9306or through optical light guides of phase plate9306, and then emitted from the top end93061of phase plate9306. Top end90361may coincide with the channel wall60111/60112/60113ofFIG.90BandFIG.91A. Each of the optical light guides of the phase plate9306may have any of: different width, different length, different height. After light90112passes through the phase plate9306, light90112may be phase modulated by phase plate9306such that light90112may be optically converged or optically collimated towards center of the UFL channel601/6072/910or towards entity1/10/20/30/612.

FIG.127Dillustrates an embodiment where forward scatter sensor9013is embedded within the cover610or the substrate611of a UFL channel601/6072/910and scattered light90132is passed to the forward scatter sensor9013through phase plate9306embedded within the cover610or substrate611of the UFL channel601/6072/910. Phase plate9306ofFIG.127Dis same as inFIG.127C, except phase plate9306may be used to collect light90132from channel601/6072/910and transmit light90132to sensor9013. InFIG.127D, scattered light90132from entity1/10/20/30/612may enter top end93061of phase plate9306or the optical light guides of phase plate9306, then pass through phase plate9306or each of said optical light guides of phase plate9306, and then exits from bottom end93062of said phase plate9306and entering optical detection surface90131of sensor9013. After light90132passes through the phase plate9306, light90132may be phase modulated by phase plate9306such that light90132may be optically collimated or optically converged towards the optical detection surface90131of sensor9013.

FIG.128Aillustrates an embodiment where an illuminator9011is located external to UFL channel601/6072/910and light90112from the illuminator9011may be passed through an optical grating9305embedded within the cover610or substrate611of the UFL channel601/6072/910.FIG.128Ais substantially similar toFIG.127A, except that gratings9305may terminate on surface60111/60112/60113facing entity1/10/20/30/612and also terminate on external surface60114/60115/60116of UFL600/600facing illuminator9011. Gratings9305may be composed of a series of isolated optical light guides, same as inFIG.127A, with each light guide having a bottom end93052facing the light emission surface90111of illuminator9011and a top end93051facing the channel601/6072/910and entity1/10/20/30/612, where light90112emitted from illuminator9011surface90111enters bottom end93052of optical light guides, then passes through optical light guides of gratings9305, and then emitted from the top end93051of optical light guides. Light emission surface90111of illuminator9011may be in contact with bottom end93052of gratings9305.

FIG.128Billustrates an embodiment where forward scatter sensor9013is located external to UFL channel601/6072/910and scattered light90132is passed to the forward scatter sensor9013through optical gratings9305embedded within the cover610or substrate611of the UFL channel601/6072/910.FIG.128Bis substantially similar toFIG.127B, except that gratings9305may terminate on surface60111/60112/60113facing entity1/10/20/30/612and also terminate on external surface60114/60115/60116of UFL600/600facing sensor9013. InFIG.128B, scattered light90132from entity1/10/20/30/612may enter top end93051of optical light guides of gratings9305, then pass through each of said optical light guides, and then exits from bottom end93052of said optical light guides and entering optical detection surface90131of sensor9013. Optical detection surface90131of detector9013may be in contact with bottom end93052of gratings9305.

FIG.128Cillustrates an embodiment where an illuminator9011is located external to UFL channel601/6072/910and light90112from the illuminator9011may be passed through an optical phase plate9306embedded within the cover610or substrate611of the UFL channel601/6072/910.FIG.128Cis substantially similar toFIG.127C, phase plate9306is also same as inFIG.127C, except that phase plate9306may terminate on surface60111/60112/60113facing entity1/10/20/30/612and also terminate on external surface60114/60115/60116of UFL600/600facing illuminator9011. Light emission surface90111of illuminator9011may be in contact with bottom end93062of phase plate9306.

FIG.128Dillustrates an embodiment where forward scatter sensor9013is located external to UFL channel601/6072/910and scattered light90132is passed to the forward scatter sensor9013through phase plate9306embedded within the cover610or substrate611of the UFL channel601/6072/910.FIG.128Dis substantially similar toFIG.127D, phase plate9306is also same as inFIG.127D, except that phase plate9306may terminate on surface60111/60112/60113facing entity1/10/20/30/612and also terminate on external surface60114/60115/60116of UFL600/600facing illuminator9011. Optical detection surface90131of detector9013may be in contact with bottom end93062of phase plate9306.

In one embodiment, spatially periodic arrangement of optical light guides of gratings9305ofFIG.127AandFIG.128Amay produce spatially periodic illumination light90112upon entity1/10/20/30/612when entity1/10/20/30/612passes through illumination region of illuminator9011of optical detector910. Such spatially periodic illumination created by periodic optical light guides may subsequently cause a production of temporally periodic scattered light90132signal when entity1/10/20/30/612passes through illumination region of illuminator9011, especially when flow speed of entity1/10/20/30/612through said illumination region is substantially constant, thereby said temporally periodic scattered light90132may in turn produce a periodic90132signal detected by sensor9013at a frequency that may correlate to said spatial period of said light guides of gratings9305and may correlate to the flow speed of entity1/10/20/30/612passing through the illumination region of illuminator9011, for example said frequency may be calculated as said flow speed of said entity divided by said spatial period of said light guides of gratings, wherein a signal filter including any of, or a combination thereof: a band pass filter, a low pass filter, a high pass filter, may be applied to said periodic signal detected by sensor9013to enhance signal detection from entity1/10/20/30/612and to reduce noise from other entities2/3/22/613. Said signal filter may be implemented by electronic components of controller950or954, or by programs within computing device955ofFIG.120AthroughFIG.122C,

FIG.129Aillustrates an embodiment of multiple optical detectors9001,9002,9003, each being same as detector901, and each having illuminator9011, forward scatter sensors9012or back scatter sensors9013positioned along the channel walls60111/60112/60113of a UFL channel601/6072/910to achieve an in-serial optical detection of biological entities1/10/20/30/612. Function of each of optical detectors9001,9002, and9003is same as detector901as inFIG.111A, except each detector9001/9002/9003may be different in any of: light90112emitted from different illuminator9011may have different wavelength or color; light90122or90132detected by different sensor9012or9013may be at different wavelength or color. Each different detector9001/9002/9003may detect existence of different type of fluorescent molecules that produce emission light of different wavelength or different color, on an entity1/10/20/30/612under illumination light90112from different illuminator9011.

FIG.129Billustrates one example of fluorescent optical signals fromFIG.129Aoptical detectors when biological entities pass through the UFL channel. Signal9021shows optical signal detected by sensor9013of each of the detectors9001,9002,9003when a first entity1/10/20/30/612passes sequentially through detector9001, detector9002, and detector9003following flow direction6021/6043/6071/9101. Illuminator9011of each of detector9001,9002,9003may emit same or different color illumination light90112. Sensor9013of detector9001may sense90132light of a first color, which may be produced by emission of a first type of fluorescent molecules that may: bind to first type of surface antigens that may exist on entity1/10/20/30/612surface, or bind to first type of DNA or RNA sections or first type of intracellular antigens inside entity1/10/20/30/612, under excitation from light90112emitted from illuminator9011of detector9001. Sensor9013of detector9002may sense90132light of a second color, which may be produced by emission of a second type of fluorescent molecules that may: bind to second type of surface antigens that may exist on entity1/10/20/30/612surface, or bind to second type of DNA or RNA sections or second type of intracellular antigens inside entity1/10/20/30/612, under excitation from light90112emitted from illuminator9011of detector9002. Sensor9013of detector9003may sense90132light of a third color, which may be produced by emission of a third type of fluorescent molecules that may: bind to third type of surface antigens that may exist on entity1/10/20/30/612surface, or bind to third type of DNA or RNA sections or third type of intracellular antigens inside entity1/10/20/30/612, under excitation from light90112emitted from illuminator9011of detector9003.

InFIG.129B, for signal9021obtained from first entity1/10/20/30/612, existence of detected signal peaks in signal9021corresponding to first entity1/10/20/30/612passing through detector9001and detector9002indicates existence of said first and second types of fluorescent molecules, and thereby existence of said first and second types of surface antigens on first entity1/10/20/30/612, or existence of first and second types of DNA or RNA sections or first and second types of intracellular antigens in first entity1/10/20/30/612. Detected signal9021peak height90012from detector9001may be used to calculate or estimate number, or abundance, of first type of surface antigen or first type of DNA or RNA sections or first type of intracellular antigens that first entity1/10/20/30/612may contain, while peak width90011, for example full-width-half-maximum, of said signal peak from detector9011may be used to calculate or estimate: physical size of first entity1/10/20/30/612; size of internal volume of first entity1/10/20/30/612where first type of DNA or RNA sections or first type of intracellular antigens are contained; or density and distribution of said first type surface antigen or first type DNA or RNA sections or first type of intracellular antigens. Similarly, peak height90022from detector9002may be used to calculate or estimate number, or abundance, of second type of surface antigen or second type of DNA or RNA sections or second type of intracellular antigens that first entity1/10/20/30/612may contain, while peak width90021may be used to calculate or estimate: physical size of first entity1/10/20/30/612; size of internal volume of first entity1/10/20/30/612where second type of DNA or RNA sections or second type of intracellular antigens are contained; or density and distribution of said second type surface antigen or second type DNA or RNA sections or second type of intracellular antigens. Non-existence of signal peak in9021from detector9003may indicate non-existence or un-detectable existence of third type of surface antigens or third type of DNA or RNA sections or third type of intracellular antigens in first entity1/10/20/30/612. By combining information retrieved, calculated from, or estimated from signal9021, including any of: number, amount, density, distribution of first and second types of surface antigens expressed by first entity1/10/20/30/612; first and second types of DNA or RNA sections contained within first entity1/10/20/30/612; first and second types of intracellular antigens contained within first entity1/10/20/30/612; size of first entity1/10/20/30/612; non-existence of third type of surface antigens or third type of DNA or RNA sections or third type of intracellular antigens, first entity1/10/20/30/612may be qualitatively, quantitatively, or categorically, described or classified or identified.

InFIG.129B, for signal9022obtained from second entity1/10/20/30/612, existence of detected signal peaks in signal9022corresponding to second entity1/10/20/30/612passing through detector9002and detector9003indicates existence of said second and third types of fluorescent molecules, and thereby existence of said second and third types of surface antigens on second entity1/10/20/30/612, or existence of second and third types of DNA or RNA section or second and third types of intracellular antigens in second entity1/10/20/30/612. Non-existence of signal peak in9022from detector9001may indicate non-existence or un-detectable existence of first type of surface antigens or first type of DNA or RNA sections or first type of intracellular antigens in second entity1/10/20/30/612. Similarly as described for signal9021, by combining information retrieved, calculated from, or estimated from signal9022, including any of: number, amount, density, distribution of second and third types of surface antigens expressed by second entity1/10/20/30/612; second and third types of DNA or RNA sections contained within second entity1/10/20/30/612; second and third types of intracellular antigens contained within second entity1/10/20/30/612; size of second entity1/10/20/30/612; non-existence of first type of surface antigens or first type of DNA or RNA sections or first type of intracellular antigens, second entity1/10/20/30/612may be qualitatively, quantitatively, or categorically, described, classified or identified, where second entity1/10/20/30/612may be categorized as being qualitatively, quantitatively, or categorically different from first entity.

InFIG.129AandFIG.129B, three detectors9001,9002,9003are shown as an example, while the number of detectors that may be used in similar manner as described inFIG.129AandFIG.129Bis not limited in detection existence of: various types of surface antigens on entities1/10/20/30/612; or various types of DNA or RNA sections or various types of intracellular antigens within entities1/10/20/30/612. In one embodiment, sensor9012or sensor9013may be composed of an imaging device, for example a CCD sensor or a CMOS image sensor, signal9021or signal9022may then be replaced with series of images captured when entity1/10/20/30/612flows through detectors9001,9002,9003, where each image may be labeled or accompanied by a time stamp of the time when said image was captured, and existence of any one type of fluorescent molecules on or within entity1/10/20/30/612as described inFIG.129AandFIG.129Bmay be observed as optical patterns, including: optical scatter pattern, optical diffraction pattern, optical interference pattern, projection image or projection shape of entity1/10/20/30/612, corresponding to passage of entity1/10/20/30/612through one or more of the detectors9001,9002,9003.

FIG.129AandFIG.129Bshow the example of each of different detectors9001,9002,9003may be used to detect a different wavelength or different color light90132, which may be emitted from different fluorescent molecules attached to, or included within, entities1/10/20/30/612. Alternatively, when sensor9013of detectors9001,9002, or9003is comprised of one or more of CCD color sensors, or one or more CMOS color sensors, sensor9013may capture light90132components of various wavelengths or various colors, during detection of light90132, wherein said different wavelength or different color components of light90132may be captured, detected, or quantized by sensor9013of a single detector of9001,9002, or9003, wherein said sensor9013may detect the amplitude90012,90022,90032of each different wavelength or color component of light90132, wherein said single sensor9013may also detect peak width90011,90021,90031of each different wavelength or color component of light90132, when entities1/10/20/30/612flow through said single detector of9001,9002, or9003containing said single sensor9013.

FIG.130Aillustrates the method to align biological entities1/10/20/30/612/613into a linear single file stream in flow channel601/6072/910, preferably at center of said flow channel601/6072/910, for optical detection by detector901as described fromFIG.110AthroughFIG.129B, with using acoustic generated fluidic pressure node as described inFIG.38C,FIG.38D,FIG.91A,FIG.91B,FIG.91C,FIG.92AthroughFIG.93B. InFIG.130A, an acoustic device, for example PZT614, may be attached to the external surface of channel601/6072/910to generate fluidic pressure node that may align entity1/10/20/30/612/613into a linear single file stream within flow of6043flowing through channel601/6072/910, which is similarly described inFIG.38D,FIG.93AandFIG.93B, where the linear single file stream of entities1/10/20/30/612/613may be aligned with the illumination region of the illuminator9011or detection region of sensors9012and9013of the detector901and thus may help enhance detection of1/10/20/30/612/613by detector901in aspect including any of: better accuracy, higher flow speed, and higher resolution.

FIG.130Billustrates the method to align biological entities1/10/20/30/612/613into a linear single file stream in flow channel601, preferably at center of said flow channel601, for optical detection detector901with using laminar flow. UFL channel600may be utilized to achieve laminar flow enabled entity1/10/20/30/612/613single file alignment for detection by detector901. InFIG.130B, sample6020containing entities1/10/20/30/612/613may be injected through inlet604, and buffer fluid or sheath fluid6040may be injected into inlet602. Buffer fluid6040after passing through side channels603as flow6031may meet sample flow6043in main channel601, where flow6031becomes sheath flow6033flowing at both sides of center sample flow6043. When flow6033and6043form a laminar flow in channel601, with fluid6020injection into inlet604may be at a higher pressure than fluid6040, entities1/10/20/30/612/613may align into a linear single file flow stream in the direction of flow6043. Said linear single file stream may be aligned with the illumination region of the illuminator9011or detection region of sensors9012and9013of the detector901and thus may help enhance detection of10/20/30/612/613by detector901in aspect including any of: better accuracy, higher flow speed, and higher resolution.

FIG.130Cillustrates the method to align biological entities1/10/20/30/612/613into a linear single file stream in flow channel601, preferably at center of said flow channel601, for optical detection detector901with using laminar flow in combination with acoustic generated fluidic pressure node.FIG.130Cis substantially same asFIG.130B, where UFL channel600may be utilized to achieve laminar flow enabled entity1/10/20/30/612/613single file alignment for detection by detector901. Same as inFIG.130B, sample6020containing entities1/10/20/30/612/613may be injected through inlet604, and buffer fluid or sheath fluid6040may be injected into inlet602. Buffer fluid6040after passing through side channels603as flow6031may meet sample flow6043in main channel601, where flow6031becomes sheath flow6033flowing at both sides of center sample flow6043. When flow6033and6043form a laminar flow in channel601, with fluid6020injection into inlet604may be at a higher pressure than fluid6040, entities1/10/20/30/612/613may align into a linear single file flow stream in the direction of flow6043. InFIG.130Can acoustic device, for example PZT614, may be attached to the external surface of channel601to generate fluidic pressure node that may further help maintain alignment of entity1/10/20/30/612/613as said linear single file stream within flow of6043flowing through channel601. Said linear single file stream of entities1/10/20/30/612/613in flow6043may be aligned with the illumination region of the illuminator9011or detection region of sensors9012and9013of the detector901and thus may help enhance detection of1/10/20/30/612/613by detector901in aspect including any of: better accuracy, higher flow speed, and higher resolution.

Said linear single file stream in flow6043may be further maintained by the aligned with the illumination region of the illuminator9011or detection region of sensors9012and9013of the detector901and thus may help enhance detection of10/20/30/612/613by detector901in aspect including any of: better accuracy, higher flow speed, and higher resolution.

FIG.131Aillustrates the method to use spatially periodic illuminators9011or optical light guides of gratings9305to detect biological entities. Illumination light90112may be emitted from illuminators9011inFIG.131A, which may be same as illuminators9011,901101,901102,901103,901104,901105ofFIG.112AthroughFIG.117. Illuminators9011inFIG.131Amay be placed in a spatially periodic arrangement and may produce spatially periodic illumination light90112upon entity1/10/20/30/612when entity1/10/20/30/612passes through illumination region of illuminator9011of optical detector910. Alternatively, illumination light90112may be emitted from optical light guides of gratings9305ofFIG.127AandFIG.128A, which may also produce spatially periodic illumination light90112upon entity1/10/20/30/612when entity1/10/20/30/612passes through illumination region of illuminator9011of optical detector910. Such spatially periodic illumination created by spatially periodic illuminators9011or light guides of gratings9305, may induce a production of temporally periodic scattered light90132when entity1/10/20/30/612passes through illumination region of illuminator9011, especially when flow speed of entity1/10/20/30/612within flow6021/6043/6071/9101is substantially constant, where said temporally periodic scattered light90132may in turn produce a detected periodic signal by sensor9013or sensors901301,901302,901303,901304,901305, at a frequency that may correlate to said spatial period of said illuminators9011or light guides of gratings9305, and may correlate to the flow speed of entity1/10/20/30/612passing through the detector region of sensor9013as inFIG.131A, for example said frequency may be calculated as said flow speed of said entity divided by said spatial period of said illuminators or light guides of gratings. InFIG.131A, where flow6021/6043/6071/9101may contain single file of entities1/10/20/30/612such that each entity1/10/20/30/612may pass sensor9013as inFIG.131Aindividually, thus producing periodic signal by sensor9013corresponding to periodic arrangement of illuminators9011or light guides of gratings9035as inFIG.131A. Flow6021/6043/6071/9101may also contain debris or small size entities2/3/22/613, which may be randomly distributed in said flow and produce effectively random noise signal from sensor9013when passing sensor9013.

FIG.131Billustrates example of detected signal by sensor9013ofFIG.131A. Signal profile90135showing periodic signal peaks represents signal induced by a single entity1/10/20/30/612that passes through sensor9013sensing region as inFIG.131A, while signal profile90136represents noise floor produced by debris or small entities2/3/22/613randomly distributed in flow6021/6043/6071/9101. Combining signals90135and90136produce effective total signal profile produced by sensor9013, where noise floor of90136may obscure signal peaks of90135from detection when population of debris or small entities2/3/22/613in flow6021/6043/6071/9101is sufficiently large.

FIG.131Cillustrates frequency domain conversion ofFIG.131Bsignal profiles90135and90136, for example after a Fourier Transformation, where signal90135ofFIG.131Bdue to its periodic nature may be converted to a signal peak90235in the frequency domain ofFIG.131C, while noise signal90136ofFIG.131Bdue to its random nature may be converted to: (1) a mainly low frequency 1/f type of noise spectrum distribution90236in frequency domain ofFIG.131C; or (2) increased side lobes around peak90235inFIG.131C. By using a band pass filter90237, or a high pass filter90238, in frequency domain ofFIG.131C, noise signal90136contribution to total signal of time domain inFIG.131Bmay be sufficiently suppressed and thus enabling a better strength of signal90135over a lowered noise level of signal90136and an effectively higher signal-to-noise-ratio (SNR) of signal90135over noise signal90136inFIG.131B, and thus a better detection of entity1/10/20/30/612may be achieved.

In one embodiment, with knowing the physical spacing between spatially periodic illuminators9011or optical light guides of gratings9305ofFIG.131A, the time spacing90335between two adjacent signal peaks in temporal signal trace90135ofFIG.131B, or the averaged time spacing90335between any two adjacent signal peaks in temporal signal trace90135ofFIG.131B, or the time spacing90336of any selected two signal peaks in temporal signal90135ofFIG.131B, may be used to calculate or estimate actual speed of movement of entity1/10/20/30/612within channel along flow6021/6043/6071/9101. Alternatively, with knowing the physical spacing between spatially periodic illuminators9011or optical light guides of gratings9305ofFIG.131A, the frequency value90337of peak90235inFIG.131Cspectrum, where90235peak frequency value09337correlates to period of signal peaks in curve90135ofFIG.131B, may also be used to calculate or estimate actual speed of movement of entity1/10/20/30/612within channel along flow6021/6043/6071/9101

FIG.132illustrates a first embodiment of a biological entity sorting device2001having a fluid path selector9701being at a first sorting position. Sorting device2001may be included within a device body9600. Device2001may be part of UFL chip600/6000and may be contained within part of substrate611of UFL chip600/6000, or may be a separate device by itself. Sorting device2001may contain a fluidic sample injection path2100for entity1/10/20/30/612injection into device2001for sorting, said path2100may be a continuation of channel601/6072/910ofFIG.110AthroughFIG.131A. In path2100, or as part of channel601/6072/910, entities1/10/20/30/612may first be detected by one or more detectors901/9001/9002/9003, where controller950through connections951may control, and sense entity1/10/20/30/612optical signal from, detectors901/9001/9002/9003. Said entity1/10/20/30/612optical signal may be processed or analyzed for category, or type, or identification, of the entities1/10/20/30/612, for example into categories of9601and9602as inFIG.132, by controller950, or by computing device955ofFIG.121AandFIG.121Bconnected to controller950. Sorting function of sorting device2001is achieved by a voice coil9708actuated rotational fluid path selector9701.FIG.132shows a substantially circular first cavity97032may be created within device body9600with a cavity wall97042. Path selector9701having a substantially circular shape may be located within said cavity97032and surrounded by cavity wall97042. Path selector circumference wall97012and cavity wall97042may be in contact, or a spacing may exist between selector wall97012and cavity wall97042. Fluid path9605in device body9600may connect from cavity wall97042to an outlet9607, and fluid path9606in device body9600may connect from cavity wall97042to another outlet9608. Fluid path9702and fluid path9703may exist within path selector9701. Path selector9701may be positioned over, or around, or onto, a central hinge9704at the center of the path selector9701, and path selector9701may rotate around hinge9704in the first cavity97032within the cavity wall97042. Actuator9707embedded with, or covered with, one or more voice coils9708may be attached to the path selector9701, where a movement of actuator9707may cause path selector9710to rotate within the cavity wall97042around hinge9704.FIG.132shows when path selector9701being at a first sorting position, where path9702within path selector9701may align with the channel2100exit at one end, and align with path9605entrance at another end, and thus establish a continuous fluid path from path2100, through path9702, and through path9605and exiting device2001through outlet9607. In the case when path selector9701rotates due to movement of actuator9707to a second sorting position, where path9703within path selector9701may align with the channel2100exit at one end, and align with path9606entrance at another end, and thus may establish a second continuous fluid path from path2100, through path9703, and through path9606and exiting through outlet9608. Actuator9707may be located within a second cavity9706in the device body9600, where second cavity9706may be created at the same step as the first cavity97032. Magnetic field9709having north and south polarities simultaneously may exist within the cavity9706, whereFIG.132shows north (N) polarity and south (S) polarity of the magnetic field9709exists side by side within cavity9706, with N polarity on the left having magnetic field direction pointing out of the plane and S polarity on the right with magnetic field direction pointing into the plane ofFIG.132, and with the magnetic field from both N and S polarities having magnetic field components perpendicular to the voice coil9708plane. Voice coil9708may be in the form of single-turn or multiple-turn coils that may be located on a surface of the actuator9707, or may be embedded within the body of the actuator9707. Voice coil9708may be created by a first thin film deposition step including: PVD, CVD, ALD, PECVD, PEALD, or by a first metal electro-plating step, and then patterned into coil form by an etching step including: RIE, IBE, wet-etch. Voice coil9708may also be formed by a single thin film deposition step including: PVD, CVD, ALD, PECVD, PEALD, or by a first metal electro-plating step, over a pre-patterned photoresist-mask, or hard-mask existing on actuator9707to create coils9708on actuator9707through the spaces within said photoresist-mask, or hard-mask, where a photoresist removal or hard-mask removal process may be used to remove said photoresist-mask, or hard-mask afterwards. Clearance97071may exist at center of the actuator9707to reduce overall mass of actuator9707, where voice coil9708may be created surrounding the clearance97071. Sensor9801may exist at one or more locations on, or embedded within, the inside boundary wall of the cavity9706, where sensor9801may sense the proximity, or distance, of the actuator9707to the boundary wall of cavity9706. Controller950may receive signal from sensor9801through electrical connection952on proximity or distance of actuator9707to the left and right boundaries ofFIG.132cavity9706, and controller950through electrical connection953may adjust or control the electric current amplitude and current direction within coil9708of actuator9707to control the movement of actuator9707and thus the rotation of path selector9701. Electric current may be applied to the voice coils9708, where the arrows on the coils9708ofFIG.132illustrate an example of a clock-wise electric current flowing within coils9708, where said current within coil9708ofFIG.132creates a magnetic field with a direction pointing into the plane ofFIG.132against N polarity and being in same direction as S polarity of magnetic field9709ofFIG.132, where a net force may be exerted by the magnetic field9709onto the voice coil9708, and cause a movement of the voice coil9708, together with the actuator9707, to move away from N polarity region and into the S polarity region of field9709as inFIG.132, and thus causing an rotation of path selector9701.

In the example as illustrated byFIG.132, entities1/10/20/30/612in a fluid sample is injected into path2100in fluid flow91010, where path2100may be an extension of channel601/6072/910. One or more detectors901/9001/9002/9003located along path2100or channel601/6072/910, similar as inFIG.118A,FIG.124A,FIG.129A, may detect optical signal from entities1/10/20/30/612, where controller950, which may be connected to detectors901/9001/9002/9003through connection951, may receive and analyze signals from detectors901/9001/9002/9003and separate entities1/10/20/30/612into type9601“solid” entity and type9602“hollow” entity. Controller950may also determine a type9602entity may be the immediate entity that will exit path2100towards path selector9701. Controller950through connection952may receive signal from sensor9801and may determine that path selector9701is at first sorting position, where type9601entities may be expected to flow through path9702and continue into path9605in the fluid flow9603and exiting the sorting device2001through outlet9607. Controller950may determine path selector9801need to rotate to a second sorting position to allow said immediate type9602entity in path2100to enter path9703and continue into path9606into flow9604and exit device2001through outlet9608. Controller950through connection953may command, or provide, or alternate, or change, electric current in voice coils9708into clock-wise direction as shown inFIG.132to cause voice coils9708to generate a magnetic field that is in the direction of S polarity, which is against the N polarity region of magnetic field9709. With voice coils9708being mostly located within N polarity region of magnetic field9709as inFIG.132, a net force may be exerted on voice coils9708by magnetic field9709with clock-wise current applied to voice coils9708, making voice coils9708, together with actuator9707, move out of N region and into S region of magnetic field9709. Movement of actuator9707that is attached to path selector9701will then cause path selector9701to rotate around hinge9704in direction9705towards said second sorting position of device2001.

During operation of sorting device2001, spacing between path selector9701wall97012and cavity97032wall97042may be minimized to form continuous flow of fluid between path2100to paths9702and9703, and further to paths9605and9606, and to avoid fluid of flow91010flowing into cavity97032or cavity9706. In one embodiment, wall97012and wall97042may be in contact during rotation of path selector9701around hinge9704, where lubricating film or anti-abrasion coating may be applied to either or both of contacting surfaces of wall97012and wall97042, wherein said lubricating film of anti-abrasion coating may be a layer composed of organic molecules, said organic molecules may be repellant to water and oil. In one embodiment, spacing between wall97012and wall97042may be any of: from 0.1 nanometer (nm) to lnm, from 1 nm to 10 nm, from 10 nm to 100 nm, from 100 nm to 200 nm, from 200 nm to 500 nm, from 500 nm to 1 micrometer (um), from 1 um to 10 um, from 10 um to 20 um. In one embodiment, cavity97032may be filled with air, or gas including but not limited to: nitrogen, helium, argon, carbon dioxide, oxygen, where spacing between wall97012and wall97042may be sufficiently small where surface tension of fluid within liquid sample flow91010maintains fluid within paths2100,9702,9703,9605,9606at first and second sorting positions and during rotation of path selector9701, where surfaces of wall97012and wall97042may be composed of, or coated with, materials that is non-wettable, or hydrophobic, for liquid within sample flow91010, thus help maintaining fluid within paths2100,9702,9703,9605,9606.

In one embodiment, cavity97032space between wall97012and wall97042, where paths9702,9703,9605and9606terminate, may be filled with cavity fluid that is biological compatible with fluid sample of flow91010,9603and9604, where switching between sorting first and second positions of path selector9701and passage of fluid between paths2100,9702,9703,9605,9606may carry certain amount of said cavity fluid into flows9603and9604, said cavity fluid may contain any of, but not limited to: water, saline, phosphate buffered saline, Ficoll. Said cavity fluid may be maintained at same, or higher, fluid pressure than flows91010,9603and9604, where fluid of sample flow91010may be maintained within paths2100,9702,9703,9605,9606. Said cavity fluid may be supplied continuously from outside device2001directly into cavity97032during operation of device2001. Said cavity fluid may be initially supplied through flow91010when entities1/10/20/30/612are not supplied to flow91010and cavity fluid may flow from path2100in between walls97012and97042into the cavity97032space, and cavity fluid may then maintain its effective volume during operation of device2001during rotation of path selector9701, where pressure within cavity fluid is maintained same as in flow91010. Said cavity fluid may be confined, or sealed within, cavity97032wall97042and not entering cavity9706, where cavity9706may be filled with air, or gas including but not limited to: nitrogen, helium, argon, carbon dioxide, oxygen, where walls of cavity9706may be composed of, or coated with, materials that is non-wettable, or hydrophobic, for cavity fluid within cavity97032, to help maintain cavity fluid within cavity97032.

InFIG.132, device body9600, path selector9701, actuator9707may each be composed of any of: glass, silicon, quartz, aluminum-titanium-carbon (AlTiC), SiC, SiN, silicon-oxide, alumina, plastic, PDMS, polymer, ceramic, or metal, where metal may be composed any one or any alloy of aluminum, iron, nickel, titanium, chromium, platinum, tungsten, rhenium, copper, gold, silver. Path selector9701and actuator9707may be created as a single piece, where voice coils9708are formed on or within the actuator9707part of said single piece. Path selector9701and actuator9707may also be created as separate pieces, and joined together afterwards through any of: soldering, welding, gluing, mechanical attachment.

FIG.133illustratesFIG.132sorting device2001at a second sorting position.FIG.133is the result of the operation as described inFIG.132example. FollowingFIG.132operation, actuator9707moves towards S region of the magnetic field9709, where controller950through sensor9801detects actuator9707proximity or distance to the right side boundary wall of cavity9706while controlling electric current amplitude and direction flowing in voice coils9708, until actuator9707rotates the path selector9701to reach second sorting position, where path9703is aligned with exit of path2100on one end and aligned with entrance of path9604on the other end. At second sorting position ofFIG.133, the immediate type9602entity exiting the path2100as inFIG.132moves into desired path9703of path selector9701following flow91010, while one or more type9602entities originally contained within path9703as shown inFIG.132may continue into path9606and ultimately exiting sorting device2001through outlet9608. From described example ofFIG.132andFIG.133, one step of sorting one type9602entity into desired flow path9703may be achieved. At second position ofFIG.133, following same operation as inFIG.132, controller950may determine the immediate exiting entity within path2100may now become type9601entity, where controller950may reverse the electric current in voice coil9708to counter-clock-wise direction, such that voice coil9708may produce a magnetic field in the N direction and the net magnetic force exerted on actuator9707from magnetic field9709may become pushing the actuator back into the N region of the magnetic field9709and cause rotation of path selector9701back to the first sorting position as shown inFIG.132to complete another sorting step of moving immediate9601entity into desired path9702of path selector9701.

FIG.134AillustratesFIG.132type sorting device2002having four sorting positions and being in first sorting position. Sorting device2002is same as sorting device2001, except four selector paths exist on path selector9701, and four exit paths exist within device body9600with each path having an outlet. At first sorting position ofFIG.134A, selector path97101may align with exit of path2100on one end and align with entrance of path96101on the other end, thus entities within path2100may continue flow through path97101, path96101and exit device2002through outlet9607.

FIG.134BillustratesFIG.134Asorting device2002in second sorting position. At second sorting position ofFIG.134B, selector path97102may align with exit of path2100on one end and align with entrance of path96102on the other end, thus entities within path2100may continue flow through path97102, path96102and exit device2002through outlet9608.

FIG.134CillustratesFIG.134Asorting device2002in third sorting position. At third sorting position ofFIG.134C, selector path97103may align with exit of path2100on one end and align with entrance of path96103on the other end, thus entities within path2100may continue flow through path97103, path96103and exit device2002through outlet9609.

FIG.134DillustratesFIG.134Asorting device2002in fourth sorting position. At fourth sorting position ofFIG.134D, selector path97104may align with exit of path2100on one end and align with entrance of path96104on the other end, thus entities within path2100may continue flow through path97104, path96104and exit device2002through outlet9610.

FIG.134AthroughFIG.134Dillustrate example of sorting entities1/10/20/30/612ofFIG.132into four different flow paths, which enables sorting of at least four different types of entities1/10/20/30/612, where controller950of sorting device2002through detectors901/9001/9002/9003of sorting device2002may be able to categorize entities1/10/20/30/612into four different types and make each type entity enter each desired selector path97101/97102/97103/97104at four sorting positions and further into outlets9607/9608/9609/9610. Similar toFIG.132andFIG.133, transition between four sorting positions ofFIG.134AtoFIG.134Dis through controller950sensing actuator9707position in cavity9706and making changes in amplitude and direction of electric current flowing in voice coil9708of actuator9707.

FIG.135AillustratesFIG.132sorting device utilizing coil lines97081of voice coil9708connecting to device body9600.FIG.135Asorting device2003serves as another example of implementation ofFIG.132. Sorting device2003, in addition to device2001as described inFIG.132, coil lines97081may connect from voice coil9708of actuator9707to device body9600, where coil lines97081connection points on device body9600may then further connect to controller950through connection953. Coil lines97081may be made as part of extension lines of voice coil9708during fabrication of voice coil9708on or within actuator9707, where coil lines97081may be part of actuator9707. Coil lines97081may be separately made and attached to both voice coil9708and device body9600at anchor points. Said anchor points may be made of metal, for example metal thin film pads, and coil lines97081may be soldered, or welded, or be in mechanical contact, to said anchor points. Coil lines97081may be made of any of: metal, conductive carbon fiber, conductive plastic line. Coil lines97081maybe composed of a metal or metal alloy including any element of: copper, gold, silver, iron, nickel, chromium, tungsten, titanium, zinc, tin. Coil lines97081may be in the form of thin wire structure and may provide spring function to actuator9707and may exert spring back force on actuator9707during movement of actuator9707within cavity9706under magnetic force generated by electric current from voice coil9708, where magnetic force from voice coil9708and spring back force from coil lines97081may work together to achieve precision in reaching desired sorting positions as inFIG.132throughFIG.134D. When current is not applied to voice coil9708, spring force by coil lines97081may return actuator9707and path selector9701to a determined position, for example as shown inFIG.135A, for repeatable initial position of sorting device2003operation. Coil lines97081may also serve as mechanical anchoring structure that may help keep the actuator9707moving within a sufficiently horizontal plane and avoid tilting of path selector9701during rotation around hinge9704.

FIG.135Billustrates an example cross-section view ofFIG.135Adevice2003along cross section direction9901ofFIG.135A. Path2100, which may also be extension of channel601/6072/910as described inFIG.132, may be formed within substrate611, which may be same as device body9600, and enclosed by top cover610.

FIG.135Cillustrates a first example view ofFIG.135Adevice2003along cross section direction9902ofFIG.135A. InFIG.135C, paths9605and9606are shown to be also formed within substrate611and enclosed by cover610. In one embodiment, paths2100,9605and9606of device2003may be formed in substrate611or device body9600in a single etching step.FIG.135Calso shows that cavity97032may be formed as a circular trench within substrate611. Hinge9704may be in the form of a solid protrusion structure from bottom surface97033of cavity97032. Path selector9701may reside within the cavity97032and may rest on top of hinge9704with a notch97041at the bottom of path selector9701. Notch97041may match to hinge9704shape and may provide rotational stability of path selector9701during operation as described inFIG.132andFIG.133. Air, various gas, or cavity fluid as described inFIG.132may reside within gap between cavity97032surface97033and bottom surface97013of path selector9701. Top surface of path selector9701may be separated from top cover610, while edge97012of path selector9701may be separated from circular wall97042of cavity97032.

FIG.135Dillustrates a second example view ofFIG.135Adevice along cross section direction9902.FIG.135Dis same asFIG.135Cin all other aspects, except the path selector9701may reside within the cavity97032and may rest on top of hinge9704directly by bottom surface97013of path selector9701without notch97041ofFIG.135C. Air, various gas, or cavity fluid as described inFIG.132may reside within gap between surfaces97013and97033, as well as between path selector9701circular side wall97012and cavity circular wall97042. Top surface of path selector9701may be separated from top cover610. Contact from hinge9704to surface97013may be a point contact to minimize fiction during operation of path selector9701as described inFIG.132andFIG.133.

FIG.135Eillustrates view ofFIG.135Adevice along cross section direction9903.FIG.135Eshows actuator9707having voice coil9708may reside within cavity9706, where coil lines97081attached to voice coil9708and substrate611may provide electrical connection between controller950and voice coil9708. Coil lines97081may function as mechanical anchors of actuator9707to substrate611and may provide spring-back function for movement of actuator9707and rotation of path selector9701as described inFIG.135A. Bottom surface97061of cavity9706may be same surface as surface97033of cavity97032when cavity97302and9706may be created in same steps during fabrication of sorting device2003. Surface97061may be above surface97033, where cavity97302and cavity9706may be created in different steps. Cavity fluid as describedFIG.132may be confined within cavity97032gap between path selector9701bottom surface97013and cavity97032surface97033, especially when surface97061is above surface97013of path selector9701, where a sufficiently narrow gap between walls97012and97042, or when walls97012and97042are in contact, may provide an effective confinement, or sealing, of cavity fluid in said cavity97032gap between surfaces97013and97033.

FIG.136Aillustrates view ofFIG.132throughFIG.135Asorting device2001,2002,2003having a first magnetic field application scheme withFIG.136Aas an example of viewing along direction9904ofFIG.135A. InFIG.136A, dashed lines illustrate boundary of actuator9707and boundary of cavity9706, both of which are hidden from view ofFIG.136A. Permanent magnet9710may be used to create neighboring N region and S region of magnetic field9709ofFIG.132throughFIG.135A, where N region may be created by a left part of magnet9710having an effective magnetization9711pointing up, and where S region may be created by a right part of magnet9710having an effective magnetization9712pointing downwards. Opposing magnetizations9711and9712of permanent magnet9710may be produced during fabrication of permanent magnet9710. Permanent magnet9710may be positioned in close proximity to but not contacting, or may be in physical contact with, bottom surface of substrate611, and permanent magnet9701may be aligned to a desired location relative to cavity9706to produce effective force on actuator9707when there is current in voice coil9708of actuator9707.

FIG.136Billustrates view ofFIG.132throughFIG.135Asorting device2001,2002,2003having a second magnetic field application scheme withFIG.136Aas an example of viewing along direction9904ofFIG.135A. To produce N region and S region of magnetic field9709similar as permanent magnet9710ofFIG.136A,FIG.136Butilizes two soft magnetic pole pieces, with left pole piece9715attached to, or placed in close proximity to, a north pole of a permanent magnet9713, and with right pole piece9716attached to, or placed in close proximity to, a south pole of same permanent magnet9713. Pole pieces9715and9716may each have a field generation surface, where left pole piece9715may conduct magnetic flux from north pole of magnet9713and emit said north magnetic flux from said field generation surface of left pole piece9715and produce N region of magnetic field9709, while right pole piece9716may conduct magnetic flux from south pole of magnet9713and emit said south magnetic flux from said field generation surface of right pole piece9716and produce S region of magnetic field9709. Said field generation surfaces of pole pieces9715and9716may be positioned in close proximity to but not contacting, or may be in physical contact with, bottom surface of substrate611, and may be aligned to a desired location relative to cavity9706to produce effective force on actuator9707when there is current in voice coil of actuator9707.

FIG.136Cillustrates view ofFIG.132throughFIG.135Asorting device2001,2002,2003having a third magnetic field application scheme withFIG.136Aas an example of viewing along direction9904ofFIG.135A.FIG.136Cis same asFIG.136B, except that soft magnetic pole piece9715ofFIG.136Bis attached to north pole of permanent magnet9717, while soft magnetic pole piece9716ofFIG.136Bis attached to south pole of another permanent magnet9718, where permanent magnets9717and9718are separate and may have magnetizations9719and9720in opposing directions.

FIG.136Dillustrates view ofFIG.132throughFIG.135Asorting device2001,2002,2003having a fourth magnetic field application scheme withFIG.136Aas an example of viewing along direction9904ofFIG.135A. To produce N region and S region of magnetic field9709similar as permanent magnet ofFIG.136A,FIG.136Dutilizes an electromagnet that has a soft magnetic pole piece9721having two field generation surfaces. Electrical coils9722conducting electric current may wrap around a section of said soft magnetic pole piece9721, where electric current in coils9722may generator magnetic flux within pole piece9721, where pole piece9721may function as an electro-magnet, with left field generation surface of pole piece9721generating N region of magnetic field9709, and right field generation surface of pole piece9721generating S region of magnetic field9709. Field generation surfaces of pole piece9721may be positioned in close proximity to but not contacting, or may be in physical contact with, bottom surface of substrate611, and may be aligned to a desired location relative to cavity9706to produce effective force on actuator9707when there is current in voice coil of actuator9707.

FIG.137illustrates sorting device2004incorporating actuator position decoders9732. Sorting device2004is similar to sorting devices2001,2002and2003. Sensor9801of sorting device2001inFIG.132may be replaced by position decoder9732of sorting device2004as inFIG.137.FIG.137shows that an encoder housing9730may be attached to actuator9707. Position encoders9731may be included within encoder housing9730. Decoders9732embedded in device body9600, may be positioned at wall edge of cavity9760to be in proximity to encoders9731. During movement of actuator9707, decoders9732may sense the change of position of encoders9731and may detect the actual position of actuator9707in cavity9706and subsequently detect rotational position of path selector9701around hinge9704.

In one embodiment, decoder9732may be composed of one or more optical sensors, and may be composed of one of more optical emitters. Encoder9731may be composed of one or more optical patterns or one or more optical reflectors. Optical sensor of decoder9732may detect optical patterns or optical reflectors of encoder9731movement and position within cavity9706, where optical emitter of decoder9732may emit probing light towards encoder9731for optical sensor of decoder9732to sense. In another embodiment, decoder9732may be composed of one or more magnetic sensors. Encoder9731may be composed of one or more magnetic field sources, for example soft magnetic poles or permanent magnets. Encoder9731movement within cavity9706may cause change of magnetic field on magnetic sensor of decoder9732, where such change of magnetic field may be caused by movement of soft magnetic poles of encoder9731in magnetic field9709, or movement of permanent magnets of encoder9731. Such change of magnetic field may be detected by magnetic sensor of decoder9732and interpreted into movement or position of actuator9707in cavity9706. In yet another embodiment, decoder9732may be composed of one or more capacitive sensors. Encoder9731may be composed of one or more conductive or insulating parts, for example metal pads or non-metal pads. Encoder9731movement within cavity9706, preferably in proximity to decoder9732, may cause change of capacitance between conductive or insulating parts of encoder9731and capacitive sensors of decoder9732, where such change of capacitance may be detected by capacitance sensor of decoder9732and interpreted into movement or position of actuator9707in cavity9706. In yet another embodiment, decoder9732may be composed of one or more acoustic sensors, and may be composed of one of more acoustic emitters. Encoder9731may be composed of one or more acoustic absorbers or one or more acoustic reflectors. Acoustic emitters of decoder9732may emit probing acoustic wave towards encoder9731, where acoustic absorbers or acoustic reflectors encoder9731may cause reflected acoustic wave in various patterns according to the movement or position of actuator9707in cavity9706, where acoustic sensor of decoder9732may detect reflected acoustic wave patterns, and position or movement of actuator9707may be interpreted from said detected reflected acoustic wave patterns.

FIG.138Aillustrates first current driving scheme of actuator voice coil9708inFIG.132throughFIG.137devices. InFIG.138A, DC type electric current may be applied in voice coil9708to move actuator9707and path selector9701between sorting positions. Slope97083of positive rotation inFIG.138Amay illustrate example of electric current in voice coil9708when path selector9701rotates from first sorting position to second sorting position inFIG.132. Initial high current value of97083in positive current direction may help start path selector9701rotation from first sorting position towards second sorting position, while lower current value of97083may indicate reaching desired second sorting position and reduction of rotational force to the value needed to maintain path selector9701to remain at second sorting position. Slope97084of negative rotation inFIG.138Athen illustrates example of electric current in voice coil9708when path selector9701rotates from second sorting position to first sorting position inFIG.132. Initial high current value of97084in negative current direction may help start path selector9701rotation from second sorting position towards first sorting position, while lower current value of97084may indicate reaching desired first sorting position and reduction of rotational force to the value needed to maintain path selector9701to remain at first sorting position.

FIG.138Billustrates second current driving scheme of actuator voice coil9708inFIG.132throughFIG.137devices. InFIG.138B, electric current is applied as modulated AC current97085. During position rotation, AC current97085shows a bias to positive current polarity at start of rotation, and gradually changes to a lower positive bias. Dashed line97083shows time averaged mean current value which is similar to the DC type current97083ofFIG.138Aduring positive rotation, whereas higher mean current value97083due to a larger AC current97085positive bias at the start of slope97083ofFIG.138B may help start path selector9701rotation from first sorting position towards second sorting position, while lower mean current value of97083due to a smaller positive bias of AC current97085may indicate reaching desired second sorting position and reduction of rotational force to the value needed to maintain path selector9701to remain at second sorting position. Similarly, dashed line97084shows time averaged mean current value which is similar to the DC type current97084ofFIG.138Aduring negative rotation, whereas higher mean current value97084due to a larger AC current97085negative bias at the start of slope97084ofFIG.138Bmay help start path selector9701rotation from second sorting position towards first sorting position, while lower mean current value of97084due to a smaller negative bias of AC current97085may indicate reaching desired first sorting position and reduction of rotational force to the value needed to maintain path selector9701to remain at first sorting position.

FIG.138BAC current frequency may be same as the resonant frequency of the combined mass of path selector9701and actuator9707during operation within cavities97032and9706.FIG.138BAC current frequency may also be at a value that correlates to said resonant frequency of the combined mass of path selector9701and actuator9707, where said AC current frequency value may be an integer multiple of said resonant frequency, said AC current frequency may be at an value that this an offset increase or an offset decrease from said resonant frequency, said AC current frequency may also be at an value that this an offset increase or an offset decrease from an integer multiple of said resonant frequency. Said AC current frequency may be in the range of any of: between 10 Hertz (Hz) to 100 Hz, between 100 Hz to 1 kilo-Hertz (kHz), between 1 kHz to 10 kHz, between 10 kHz to 100 kHz, between 100 kHz to 1 Mega-Hertz (MHz), between 1 MHz to 2 MHz, between 2 MHz to 5 MHz, between 5 MHz to 10 MHz, between 10 MHz to 100 MHz. Said resonant frequency of the combined mass may be affected by any of: mass of path selector9701, mass of actuator9707, young's modulus of path selector9701and actuator9707, spring force of coil lines97081, inductance of voice coil9708. FIG.138B AC current driving scheme may provide a faster response time thanFIG.138Ascheme in rotating path selector9701between various sorting positions, for example for sorting device2002with more than two sorting positions, which may also enable faster sorting of entities1/10/20/30/612.FIG.138BAC current driving scheme may also provide more resilience of actuator9707and path selector9701to external perturbations during sorting of entity1/10/20/30/612thanFIG.138Ascheme

FIG.139illustrates a second embodiment of a biological entity sorting device2005having a voice coil actuator9707being at a first sorting position.FIG.139is similar toFIG.132with a difference in the aspect where path selector9701and actuator9707ofFIG.132are replaced with a single actuator9707ofFIG.139, and selection of fluidic path9702and path9703, which are part of actuator9707, is achieved through linear movement of actuator9707inFIG.139, rather than rotation of path selector9701as inFIG.132. Device2005ofFIG.139may be part of UFL chip600/6000and may be contained within part of substrate611of UFL chip600/6000, or may be a separate device by itself. Sorting device2005may contain a fluidic sample injection path2100for entity1/10/20/30/612injection into device2005for sorting, said path2100may be a continuation of channel601/6072/910ofFIG.110AthroughFIG.131. In path2100, or as part of channel601/6072/910, entities1/10/20/30/612may first be detected by one or more detectors901/9001/9002/9003, where controller950through connections951may control, and sense entity1/10/20/30/612optical signal from, detectors901/9001/9002/9003. Said entity1/10/20/30/612optical signal may be processed or analyzed for category, or type, or identification, of the entities1/10/20/30/612, for example into categories of9601and9602as inFIG.139, by controller950, or by computing device955ofFIG.121AandFIG.121Bconnected to controller950.FIG.139shows a substantially rectangular cavity9706that may be created within device body9600with a cavity wall97042. Actuator9707may be located within said cavity9706. Actuator9707and cavity wall97042may be in contact, especially at the edge of actuator9707where path9702or path9703fluidically connects to path2100. Fluid path9605in device body9600may connect from cavity wall97042to an outlet9607, and fluid path9606in device body9600may connect from cavity wall97042to another outlet9608. Fluid path9702and fluid path9703may exist within actuator9707. Actuator9707may move linearly within cavity9706within the cavity wall97042. Actuator9707may be embedded with, or covered with, one or more voice coils9708. Sorting function of sorting device2005is achieved by driving electric current through voice coil9708and cause a movement of actuator9707between different sorting positions.FIG.139shows when actuator9707may be at a first sorting position, where path9703within actuator9707may align with the channel2100exit at one end, and align with path9606entrance at another end, and thus establish a continuous fluid path from path2100, through path9703, and through path9606and exiting device2005through outlet9608. In the case when actuator9707moves to a second sorting position, where path9702within actuator9707may align with the channel2100exit at one end, and align with path9605entrance at another end, and thus may establish a second continuous fluid path from path2100, through path9702, and through path9605and exiting through outlet9607. Magnetic field9709having north and south polarities simultaneously may exist within the cavity9706, whereFIG.139shows north (N) polarity and south (S) polarity of the magnetic field9709exists side by side within cavity9706, with N polarity on the left having magnetic field direction pointing out of the plane and S polarity on the right with magnetic field direction pointing into the plane ofFIG.139, and with the magnetic field from both N and S polarities having magnetic field components perpendicular to the voice coil9708plane. Voice coil9708may be in the form of single-turn or multiple-turn coils that may be located on a surface of the actuator9707, or may be embedded within the body of the actuator9707. Voice coil9708may be created by a first thin film deposition step including: PVD, CVD, ALD, PECVD, PEALD, or by a first metal electro-plating step, and then patterned into coil form by an etching step including: RIE, IBE, wet-etch. Voice coil9708may also be formed by a single thin film deposition step including: PVD, CVD, ALD, PECVD, PEALD, or by a first metal electro-plating step, over a pre-patterned photoresist-mask, or hard-mask existing on actuator9707to create coils9708on actuator9707through the spaces within said photoresist-mask, or hard-mask, where a photoresist removal or hard-mask removal process may be used to remove said photoresist-mask, or hard-mask afterwards. Clearance97071may exist at center of the actuator9707to reduce overall mass of actuator9707, where voice coil9708may be created surrounding the clearance97071. Sensor9801may exist at one or more locations on, or embedded within, the inside boundary wall97042of the cavity9706, where sensor9801may sense the proximity, or distance, of the actuator9707to the boundary wall97042of cavity9706. Controller950may receive signal from sensor9801through electrical connection952on proximity or distance of actuator9707to wall97042, and controller950through electrical connection953may adjust or control the electric current amplitude and current direction within coil9708of actuator9707to control the movement of actuator9707. Electric current may be applied to the voice coils9708, where the arrows on the coils9708ofFIG.139illustrate an example of a clock-wise electric current flowing within coils9708, where said current within coil9708ofFIG.139creates a magnetic field with a direction pointing into the plane ofFIG.139against N polarity and being in same direction as S polarity of magnetic field9709ofFIG.139, where a net force may be exerted by the magnetic field9709onto the voice coil9708, and cause a movement of the voice coil9708, together with the actuator9707, to move away from N polarity region and into the S polarity region of field9709as inFIG.139, and thus causing an movement of actuator9707to right side of the cavity9706as inFIG.139.

In the example as illustrated byFIG.139, entities1/10/20/30/612in a fluid sample is injected into path2100in fluid flow91010, where path2100may be an extension of channel601/6072/910. One or more detectors901/9001/9002/9003located along path2100or channel601/6072/910, similar as inFIG.132, may detect optical signal from entities1/10/20/30/612, where controller950, which may be connected to detectors901/9001/9002/9003through connection951, may receive and analyze signals from detectors901/9001/9002/9003and separate entities1/10/20/30/612into type9601“solid” entity and type9602“hollow” entity. Controller950may also determine a type9601entity may be the immediate entity that will exit path2100towards actuator9707. Controller950through connection952may receive signal from sensor9801and may determine that actuator9707is at first sorting position, where type9602entities may be expected to flow through path9703and continue into path9606in the fluid flow9604and exiting the sorting device2005through outlet9608. Controller950may determine actuator9707need to move to a second sorting position to allow said immediate type9601entity in path2100to enter path9702and continue into path9605into flow9603and exit device2005through outlet9607. Controller950through connection953may command, or provide, or alternate, or change, electric current in voice coils9708into clock-wise direction as shown inFIG.139to cause voice coils9708to generate a magnetic field that is in the direction of S polarity, which is against the N polarity region of magnetic field9709. With voice coils9708being mostly located within N polarity region of magnetic field9709as inFIG.139, a net force may be exerted on voice coils9708by magnetic field9709with clock-wise current applied to voice coils9708, making voice coils9708, together with actuator9707, move out of N region and into S region of magnetic field9709, resulting in movement of actuator9707towards said second sorting position of device2005.

During operation of sorting device2005ofFIG.139, spacing between actuator9707wall97022and cavity9706wall97042may be minimized to form continuous flow of fluid between path2100to paths9702and9703, and further to paths9605and9606, and to avoid fluid of flow91010flowing into cavity9706. In one embodiment, wall97022and wall97042may be in contact during movement of actuator9707, where lubricating film or anti-abrasion coating may be applied to either or both of contacting surfaces of wall97022and wall97042, wherein said lubricating film of anti-abrasion coating may be a layer composed of organic molecules, said organic molecules may be repellant to water and oil. In one embodiment, spacing between wall97022and wall97042may be any of: from 0.1 nanometer (nm) to lnm, from 1 nm to 10 nm, from 10 nm to 100 nm, from 100 nm to 200 nm, from 200 nm to 500 nm, from 500 nm to 1 micrometer (um), from 1 um to 10 um, from 10 um to 20 um. In one embodiment, cavity9706may be filled with air, or gas including but not limited to: nitrogen, helium, argon, carbon dioxide, oxygen, where spacing between wall97022and wall97042may be sufficiently small where surface tension of fluid within liquid sample flow91010maintains fluid within paths2100,9702,9703,9605,9606at first and second sorting positions and during movement of actuator9707, where surfaces of wall97022and wall97042may be composed of, or coated with, materials that is non-wettable, or hydrophobic, for liquid within sample flow91010, thus help maintaining fluid within paths2100,9702,9703,9605,9606.

In one embodiment, cavity9706, especially the space between wall97022and wall97042, where paths9702,9703,9605and9606terminate, may be filled with cavity fluid that is biological compatible with fluid sample of flow91010,9603and9604, where switching between sorting first and second positions of actuator9707and passage of fluid between paths2100,9702,9703,9605,9606may carry certain amount of said cavity fluid into flows9603and9604, said cavity fluid may contain any of, but not limited to: water, saline, phosphate buffered saline, Ficoll. Said cavity fluid may be maintained at same, or higher, fluid pressure than flows91010,9603and9604, where fluid of sample flow91010may be maintained within paths2100,9702,9703,9605,9606. Said cavity fluid may be supplied continuously from outside device2005ofFIG.139and directly into cavity9706during operation of device2005. Said cavity fluid may be initially supplied through flow91010when entities1/10/20/30/612are not supplied to flow91010and cavity fluid may flow from path2100in between walls97022and97042into the cavity9706space, and cavity fluid may then maintain its effective volume during operation of device2005and during movement of actuator9707, where pressure within cavity fluid is maintained same as in flow91010. Said cavity fluid may be confined, or sealed within, cavity9706wall97042, where walls of cavity9706may be composed of, or coated with, materials that is non-wettable, or hydrophobic.

InFIG.139, device body9600, actuator9707may each be composed of any of: glass, silicon, quartz, aluminum-titanium-carbon (AlTiC), SiC, SiN, silicon-oxide, alumina, plastic, PDMS, polymer, ceramic, or metal, where metal may be composed any one or any alloy of aluminum, iron, nickel, titanium, chromium, platinum, tungsten, rhenium, copper, gold, silver. Voice coils9708may be formed on or within the actuator9707. Spring9802may be optionally used to attach actuator9707to device body9600, or wall97042of cavity9706. Spring9802may provide spring back force in the movement direction of actuator9707to help stability and maintain actuator9707position when actuator9707reaches desired sorting positions. Spring9802may provide an enhanced resonance frequency of actuator9707and increase speed of response and movement of actuator9707during sorting operation. Spring9802may provide pressure force to maintain contact between surface97022and surface97042during movement of actuator9707. Spring9802may act as electrical connection between device body and voice coil9708, similar to coil wire97081ofFIG.137, where spring9802may connect to both voice coil9708on actuator9707and anchor points on device body9600ofFIG.139, where controller950may further connect to said anchor points through connection953to provide or control electric current in voice coil9708ofFIG.139.

Electric current in voice coil9708ofFIG.139may be applied in schemes similar as described inFIG.138AandFIG.138B. Position encoders9731ofFIG.137may be attached to actuator9707ofFIG.139similarly as inFIG.137, and position decoders9732may be embedded in device2005body9600or in cavity9706wall97042similar toFIG.137, to provide detection of actuator9707position in cavity9706ofFIG.139.

FIG.140illustrates a third embodiment of a biological entity sorting device2006having a capacitive actuator being at a first sorting position. Sorting device2006may be similar to sorting device2001ofFIG.132in every other aspect, except that actuator9707of device2006inFIG.140is moved by a capacitive driving mechanism instead of the voice coil9708of device2001inFIG.132. InFIG.140, actuator9707may include an actuator handle9804with attached actuator arms98041. A base handle9803with attached base arms98031may be included in device body9600, or within cavity9706as shown inFIG.140. Actuator arms98041and base arms98031may be parallel plates that have parallel surfaces directly facing each other, or overlapping each other, where a movement of actuator arms98041and base arms98031relative to each other may increase or decrease effective surface areas of actuator arms98041and base arms98031directly facing each other. During operation of device2006, controller950through connection953may provide electric voltage or current to actuator handle9804, and subsequently to actuator arms98041; controller950through connection953may provide electric voltage or current to base handle9803, and subsequently to base arms98031. Voltage applied to actuator arms98041and base arms98031may produce net electric charges on surfaces of actuator arms98041and base arms98031, for example negative voltage may produce negative charges, i.e. electrons, on actuator arms98041and base arms98031, while positive voltage may produce positive charges, i.e. electrons deficiencies on actuator arms98041and base arms98031. When charges on actuator arms98041and base arms98031are of same polarity, for example both having negative charges, i.e. electrons existing on actuator arms98041and base arms98031, actuator arms98041and base arms98031may repel each other and actuator arms98041may move away from the stationary base arms98031to reduce overlapping surface areas between actuator arms98041and base arms98031. Thus, actuator arms98041may push actuator move to the left side of the cavity9706and causing path selector9701to rotate around hinge9704to first sorting position as shown inFIG.140. When charges on actuator arms98041and base arms98031are of opposite polarity, for example one having negative charge and the other having negative charges, actuator arms98041and base arms98031may attract each other and actuator arms98041may move towards the stationary base arms98031to increase overlapping surface areas between actuator arms98041and base arms98031. Thus, actuator arms98041may push actuator move to the right side of the cavity9706and causing path selector9701to rotate around hinge9704to second sorting position where path9703may align with path2100of device2006. Actuator arms98041and base arms98031may comprise any of: glass, silicon, quartz, AlTiC, SiC, SiN, silicon-oxide, alumina, plastic, PDMS, polymer, ceramic, carbon, or metal, where metal may be composed any one or any alloy of aluminum, iron, nickel, titanium, tantalum, ruthenium, chromium, platinum, tungsten, rhenium, copper, gold, silver. Actuator arms98041and base arms98031may be composed of a non-metallic substrate coated with metallic layer. Actuator arms98041and base arms98031may be composed of a semiconductor material. Actuator handle9804, base handle9803, actuator arms98041and base arms98031may be part of a MEMS system included in sorting device2006. Spring9802may be optionally used to attach actuator9707or actuator handle9804to device body9600, or wall97042of cavity9706. Spring9802may provide spring back force in the movement direction of actuator9707to help stability and maintain actuator9707position when actuator9707reaches desired sorting positions. Spring9802may provide an enhanced resonance frequency of actuator9707and increase speed of response and movement of actuator9707during sorting operation. Spring9802may act as electrical connection between device body9600and actuator handle9804, where spring9802may connect to both actuator9707, or actuator handle9804, and anchor points on device body9600ofFIG.140, where controller950may further connect to said anchor points through connection953to provide or control electric voltage or current to actuator handle9804and actuator arms98041. Voltage or current applied to one of actuator arms98041or base arms98031may take the form of: (1) DC signal, which may be in form similar toFIG.138A; (2) AC signal, which may be in form similar toFIG.138B, while voltage or current applied to the other one of actuator arms98041or base arms98031may take the form of: (1) constant DC signal; (2) DC signal, which may be in form similar toFIG.138A; (3) AC signal, which may be in form similar toFIG.138B. When AC voltage or current signal is applied to at least one of actuator arms98041or base arms98031, frequency of said AC signal may be at resonant frequency of combined structure of path selector9701, actuator9707, actuator handle9804, actuator arms97041and spring9802.

Actuator sensors9801ofFIG.132may be included in cavity9706ofFIG.140. Position encoders9731ofFIG.137may be attached to actuator9707ofFIG.140similarly as inFIG.137, and position decoders9732may be embedded in device2006body9600or in cavity9706wall97042similar toFIG.137, to provide detection of actuator9707position in cavity9706ofFIG.140.

FIG.141illustrates a fourth embodiment of a biological entity sorting device2007having a capacitive actuator being at a first sorting position. Sorting device2007may be similar to sorting device2005ofFIG.139in every other aspect, except that actuator9707of device2007inFIG.141is moved by a capacitive driving mechanism instead of the voice coil9708of device2005inFIG.139. InFIG.141, actuator9707may include an actuator handle9804with attached actuator arms98041. A base handle9803with attached base arms98031may be included in device body9600, or within cavity9706as shown inFIG.141. Actuator arms98041and base arms98031may be parallel plates that have parallel surfaces directly facing each other, or overlapping each other, where a movement of actuator arms98041and base arms98031relative to each other may increase or decrease effective surface areas of actuator arms98041and base arms98031directly facing each other. During operation of device2007, controller950through connection953may provide electric voltage or current to actuator arms98041and base arms98031. Voltage or current applied to actuator arms98041and base arms98031may cause actuator arms98041to move away, or move towards, stationary base arms98031similar to operation of actuator arms98041and base arms98031inFIG.140. When charges on actuator arms98041and base arms98031are of same polarity, actuator arms98041and base arms98031may repel each other and actuator arms98041may move away from the stationary base arms98031, where actuator arms98041may push actuator9707to the left side of the cavity9706to first sorting position as shown inFIG.141. When charges on actuator arms98041and base arms98031are of opposite polarity, actuator arms98041and base arms98031may attract each other and actuator arms98041may move towards the stationary base arms98031, where actuator arms98041may push actuator9707move to the right side of the cavity9706to second sorting position where path9702may align with path2100of device2007. Actuator arms98041and base arms98031may comprise same materials and structures as inFIG.140. Actuator handle9804, base handle9803, actuator arms98041and base arms98031may be part of a MEMS system included in sorting device2007. Spring9802may be optionally used to attach actuator9707or actuator handle9804to device body9600, or wall97042of cavity9706. Spring9802may provide spring back force in the movement direction of actuator9707to help stability and maintain actuator9707position when actuator9707reaches desired sorting positions. Spring9802may provide an enhanced resonance frequency of actuator9707and increase speed of response and movement of actuator9707during sorting operation. Voltage or current application schemes to actuator arms98041and base arms98031may be same as described inFIG.140. When AC voltage or current signal is applied to at least one of actuator arms98041or base arms98031, frequency of said AC signal may be at resonant frequency of combined structure of actuator9707, actuator handle9804, actuator arms97041and spring9802.

Actuator sensors9801ofFIG.132may be included in cavity9706ofFIG.141. Position encoders9731ofFIG.137may be attached to actuator9707ofFIG.141similarly as inFIG.137, and position decoders9732may be embedded in device2007body9600or in cavity9706wall97042similar toFIG.137, to provide detection of actuator9707position in cavity9706ofFIG.141.

FIG.142illustrates a fifth embodiment of a biological entity sorting device2008having a thermal-elastic actuator being at a first sorting position. Sorting device2008may be similar to sorting device2006ofFIG.140in every other aspect, except that actuator9707of device2008inFIG.142is moved by a thermal-elastic driving mechanism instead of the capacitive driving mechanism of device2006inFIG.140. InFIG.142, actuator9707may be attached to one end of elastic arm98051through an actuator handle9804, where the other end of the elastic arm98051may attached to a base handle9805. Base handle9805may be included in device body9600, or within cavity9706as shown inFIG.142. During operation of device2008, controller950through connection953may provide electric voltage or current to actuator handle9804and base handle9805, and subsequently may cause an electric voltage across, or an electric current flowing within, elastic arm98051. Voltage or current applied to elastic arm98051, including amplitude, polarity, frequency and time of duration of applied voltage or current, may cause increase of length, i.e. elongation, or reduction of length, i.e. contraction, of elastic arm98051. When elastic arm98051elongates, elastic arm98051may push actuator9707to the left side of the cavity9706and causing path selector9701to rotate around hinge9704to first sorting position as shown inFIG.142. When elastic arm98051contracts, elastic arm98051may pull actuator9707move to the right side of the cavity9706and causing path selector9701to rotate around hinge9704to second sorting position where path9703may align with path2100of device2008. Elastic arm98051may comprise any of: glass, silicon, quartz, AlTiC, SiC, SiN, silicon-oxide, alumina, plastic, PDMS, polymer, ceramic, carbon, or metal, where metal may be composed any one or any alloy of aluminum, iron, nickel, titanium, tantalum, ruthenium, chromium, platinum, tungsten, rhenium, copper, gold, silver. Elastic arm98051may be composed of a memory metal that may change to a specific shape, or length, when elastic arm98051is heated, for example by current applied to elastic arm98051. Elastic arm98051may be composed of a metallic or non-metallic material that expands or contracts when elastic arm98051may be heated or cooled.

Actuator handle9804, base handle9805, elastic arm98051may be part of a MEMS system included in sorting device2008. Spring9802may be optionally used to attach actuator9707or actuator handle9804to device body9600, or wall97042of cavity9706. Spring9802ofFIG.142may function similarly as described inFIG.140. Voltage or current applied to elastic arm98051may take the form of: (1) constant DC signal; (2) DC signal, which may be in form similar toFIG.138A; (3) AC signal, which may be in form similar toFIG.138B. When AC voltage or current signal is applied to elastic arm98051, frequency of said AC signal may be at resonant frequency of combined structure of path selector9701, actuator9707, actuator handle9804, elastic arm97051and spring9802.

Actuator sensors9801ofFIG.132may be included in cavity9706ofFIG.142. Position encoders9731ofFIG.137may be attached to actuator9707ofFIG.142similarly as inFIG.137, and position decoders9732may be embedded in device2007body9600or in cavity9706wall97042similar toFIG.137, to provide detection of actuator9707position in cavity9706ofFIG.142.

FIG.143illustrates a sixth embodiment of a biological entity sorting device2009having a thermal-elastic actuator being at a first sorting position. Sorting device2009may be similar to sorting device2007ofFIG.141in every other aspect, except that actuator9707of device2009inFIG.143is moved by a thermal-elastic driving mechanism instead of the capacitive driving mechanism of device2007inFIG.141. InFIG.143, actuator9707may be attached to one end of elastic arm98051through an actuator handle9804, where the other end of the elastic arm98051may attached to a base handle9805. Base handle9805may be included in device body9600, or within cavity9706as shown inFIG.143. During operation of device2009, controller950through connection953may provide electric voltage or current to actuator handle9804and base handle9805, and subsequently may cause an electric voltage across, or an electric current flowing within, elastic arm98051. Voltage or current applied to elastic arm98051, including amplitude, polarity, frequency and time of duration of applied voltage or current, may cause increase of length, i.e. elongation, or reduction of length, i.e. contraction, of elastic arm98051. When elastic arm98051elongates, elastic arm98051may push actuator9707to the left side of the cavity9706to first sorting position as shown inFIG.143. When elastic arm98051contracts, elastic arm98051may pull actuator9707move to the right side of the cavity9706to second sorting position where path9702may align with path2100of device2009. Elastic arm98051may comprise same material as described inFIG.142.

Actuator handle9804, base handle9805, elastic arm98051may be part of a MEMS system included in sorting device2009. Spring9802may be optionally used to attach actuator9707or actuator handle9804to device body9600, or wall97042of cavity9706. Spring9802ofFIG.143may function similarly as described inFIG.140. Voltage or current applied to elastic arm98051may take the form of: (1) constant DC signal; (2) DC signal, which may be in form similar toFIG.138A; (3) AC signal, which may be in form similar toFIG.138B. When AC voltage or current signal is applied to elastic arm98051, frequency of said AC signal may be at resonant frequency of combined structure of actuator9707, actuator handle9804, elastic arm97051and spring9802.

Actuator sensors9801ofFIG.132may be included in cavity9706ofFIG.143. Position encoders9731ofFIG.137may be attached to actuator9707ofFIG.143similarly as inFIG.137, and position decoders9732may be embedded in device2007body9600or in cavity9706wall97042similar toFIG.137, to provide detection of actuator9707position in cavity9706ofFIG.143.

FIG.144Aillustrates example ofFIG.135Adevice having flow paths9605and9606with device body9600being covered by top covers610, and flow paths9702and9703within path selector9701being covered by separate top cover97015.FIG.144Asorting device2010serves as another example of implementation ofFIG.132. Sorting device2010, same as device2003ofFIG.135A, may have same coil lines97081connecting from voice coil9708of actuator9707to device body9600, where coil lines97081connection points on device body9600may then further connect to controller950through connection953. Coil lines97081may have same function, material and properties as inFIG.135A.FIG.144Adiffers fromFIG.135Ain that the path selector9701is capped with cover layer97015, which may cover over the paths9702and9703and share the same circular boundary wall97012as the path selector9701. Path selector9701, with the cover97015and actuator9707, may form a single rotational body around hinge9704, where said rotation body separates from cavity wall97042of third cavity97052by a gap97052, whereas cavity97032may be same as described inFIG.135A. Cover610then covers over the device body9600and cover the paths2100,9605and9606. InFIG.144A, device body9600with cover610, path selector9701with cover97015, may be fabricated as separate parts and assembled together into sorting device2010. Gap97052may be zero when walls97012and97042may be in contact and lubricating layer may exist on surfaces of walls97012and97042.

FIG.144Billustrates an example view ofFIG.144Adevice2010along cross section direction9902ofFIG.144A. InFIG.144B, paths9605and9606are shown to be also formed within substrate611and enclosed by cover610. In one embodiment, paths2100,9605and9606of device2003may be formed in substrate611or device body9600in a single etching step.FIG.144Balso shows that cavity97032may be formed as a circular trench within substrate611. Hinge9704may be in the form of a solid protrusion structure same as described inFIG.135C, from bottom surface97033of cavity97032. Path selector9701may reside within the cavity97032and may rest on top of hinge9704with a notch97041at the bottom of path selector9701. Notch97041may match to hinge9704shape and may provide rotational stability of path selector9701during operation as described inFIG.132andFIG.133. Paths9702and9703are shown to be formed within path selector9701and enclosed by cover97015. Air, various gas, or cavity fluid as described inFIG.132may reside within gap97052between walls97012and97042, and between cavity97032surface97033and bottom surface97013of path selector9701.

FIG.144Cillustrates view ofFIG.144Adevice along cross section direction9903.FIG.144Cshows actuator9707having voice coil9708may reside within cavity9706, where coil lines97081attached to voice coil9708and substrate611may provide electrical connection between controller950and voice coil9708. Coil lines97081may function same as described inFIG.135A. Bottom surface97061of cavity9706may be same surface as surface97033of cavity97032when cavity97302and9706may be created in same steps during fabrication of sorting device2010. Surface97061may be above surface97033, where cavity97302and cavity9706may be created in different steps. Cavity fluid as describedFIG.132may be confined within cavity97032, especially when surface97061is above surface97013of path selector9701, where a sufficiently narrow gap97052between walls97012and97042, or when walls97012and97042are in contact, may provide an effective confinement, or sealing, of cavity fluid in said cavity97032gap between surfaces97013and97033. InFIG.144C, actuator9707does not have cover97015ofFIG.144B.

FIG.144Dillustrates an alternative example ofFIG.144C, whereFIG.144Dmay be same asFIG.144C, except the cover97015also covers over actuator9707, and may encapsulate the voice coil9708.

FIG.144Eillustrates an alternative example ofFIG.144B, whereFIG.144Emay be same asFIG.144B, except that another cover61001may cover over the entire device body9600ofFIG.144A, forming an enclosure that encapsulates the structures including cavity97032, cavity9706, rotation entity comprising path selector9701, actuator9707and cover97015, wherein said enclosure may hermitically encapsulates said structures. Air, various gas, or cavity fluid as described inFIG.132may reside within said encapsulation by said cover61001and said device body9600. Cover601ofFIG.144Emay be part of said cover61001. Encapsulation by a cover61001over device body9600as shown inFIG.144Emay be applied to sorting devices2001,2002,2003,2004,2005,2006,2007,2008,2009,2011,2012,2013, fromFIG.132throughFIG.150similarly.

FIG.145illustrates external controller driving of sorting devices, including any of sorting devices2001,2002,2003,2004,2005,2006,2007,2008,2009,2011,2012,2013, fromFIG.132throughFIG.150.FIG.132sorting device2001is used as an example structure as sorting device2011as inFIG.145. InFIG.145, embedded controller950as inFIG.132is replaced with external controller954, similarly as described inFIG.120B, where all electronically controlled components, including detectors901/9001/9002/9003, sensors9801, voice coil9708, position decoder9732may be connected by electrical connections951to external electrical contacts952on the outside surface of device body9600, where external electrical connections953may then further connect from said contacts952to an external controller954, where controller954may realize functions of embedded controller950ofFIG.132. External controller954scheme ofFIG.145may be applied to replaced embedded controller950of sorting devices2001,2002,2003,2004,2005,2006,2007,2008,2009,2012,2013, fromFIG.132throughFIG.150similarly.

FIG.146illustrates an example of using multipleFIG.134Adevices2002in cascade arrangement to increase sorting categories. InFIG.146, output samples from outlets9607,9608,9609,9610of a first tier device2002may be sent to input path2100of four second tier sorting devices2002, where entities1/10/20/30/612within each said output sample may be further sorted by each corresponding second tier sorting device2002according to optical properties, for example types fluorescent molecules included in entities1/10/20/30/612, or physical properties, for example entity1/10/20/30/612size, with detector901/9001/9002/9003of each of said corresponding second tier sorting device2002. When one or more of said second tier sorting device2002detects same optical properties as first tier device2002, said second tier sorting device2002may function to further purify the sorted sample from outlets9607,9608,9609,9610of said first tier device2002. When one or more of said second tier sorting device2002detects different optical properties as first tier device2002, said second tier sorting device2002may function to further categorize and separate the sorted sample from outlets9607,9608,9609,9610of said first tier device2002into more categories. First tier and second tier devices2002may be isolated devices, where fluidic connections from outlets9607,9608,9609,9610of first tier device2002to input path2100of second tier sorting devices2002may be through fluidic lines or external tubing. First tier and second tier devices2002may be manufactured at different locations, or at different thickness levels, within a same device body9600, where fluidic connections from outlets9607,9608,9609,9610of first tier device2002to input path2100of second tier sorting devices2002may be through fluidic paths embedded within said device body9600. First tier and second tier devices2002ofFIG.146may each be replaced by any of sorting devices2001,2003,2004,2005,2006,2007,2008,2009,2012,2013, fromFIG.132throughFIG.150, where a second tier device may be used to further purify or to further sub-categorize output sample from a first tier device may be readily applied.

FIG.147illustrates a seventh embodiment of a biological entity sorting device2012having a voice coil actuator9707being at a first sorting position.FIG.147sorting device2012is similar to device2001ofFIG.132in operation, except the path selector9701of device2012does not have embedded flow paths9702or9703as in device2001ofFIG.132, where inFIG.147entities9601and9602are selected and passed into path9605or path9606within device body9600by a selector gate97011attached to path selector9701of device2012inFIG.147to selectively allow entities9601or9602to pass into one of path9605or path9606while blocking passage into other path.

Sorting device2012may be included within a device body9600. Device2012may be part of UFL chip600/6000and may be contained within part of substrate611of UFL chip600/6000, or may be a separate device by itself. Sorting device2012may contain a fluidic sample injection path2100for entity1/10/20/30/612injection into device2012for sorting, said path2100may be a continuation of channel601/6072/910ofFIG.110AthroughFIG.131A. In path2100, or as part of channel601/6072/910, entities1/10/20/30/612may first be detected by one or more detectors901/9001/9002/9003, where controller950through connections951may control, and sense entity1/10/20/30/612optical signal from, detectors901/9001/9002/9003. Said entity1/10/20/30/612optical signal may be processed or analyzed for category, or type, or identification, of the entities1/10/20/30/612, for example into categories of9601and9602as in

FIG.147, by controller950, or by computing device955ofFIG.121AandFIG.121Bconnected to controller950. Sorting function of sorting device2012is achieved by a voice coil9708actuated rotational fluid path selector9701.FIG.147shows a substantially circular first cavity97062may be created within device body9600with a cavity wall97042. First cavity97062ofFIG.147is different than first cavity97032ofFIG.132in that first cavity97062has opening towards flow paths2100,9605,9606to allow selector gate97011to extend into fluid flow91010and flow9603. Path selector9701having a substantially circular shape may be located within said cavity97062and surrounded by cavity wall97042. Path selector9701circumference wall97012and cavity wall97042may be in contact, or a spacing may exist between selector wall97012and cavity wall97042. Path selector9701may be positioned over, or around, or onto, a central hinge9704at the center of the path selector9701, and path selector9701may rotate around hinge9704in the first cavity97062within the cavity wall97042. Actuator9707embedded with, or covered with, one or more voice coils9708may be attached to the path selector9701, where a movement of actuator9707may cause path selector9710to rotate within the cavity wall97042around hinge9704.FIG.147shows when path selector9701being at a first sorting position, where selector gate97011attached to path selector9701is positioned at the entrance of path9606to block entities1/10/20/30/612in flow91010within path2100from entering path9606, while allowing said entities1/10/20/30/612to enter path9605as flow9603. In the case when path selector9701rotates due to movement of actuator9707to a second sorting position, where selector gate97011attached to path selector9701may be positioned at the entrance of path9605to block entities1/10/20/30/612in flow91010within path2100from entering path9605, while allowing said entities1/10/20/30/612to enter path9606. Actuator9707may be located within a second cavity9706in the device body9600, where second cavity9706may be created at the same step as the first cavity97062. Magnetic field9709having north and south polarities simultaneously may exist within the cavity9706, whereFIG.147shows north (N) polarity and south (S) polarity of the magnetic field9709exists side by side within cavity9706, with N polarity on the left having magnetic field direction pointing out of the plane and S polarity on the right with magnetic field direction pointing into the plane ofFIG.147, and with the magnetic field from both N and S polarities having magnetic field components perpendicular to the voice coil9708plane. Voice coil9708may be in the form of single-turn or multiple-turn coils that may be located on a surface of the actuator9707, or may be embedded within the body of the actuator9707. Voice coil9708may be created on top of, or within actuator9708, same as described inFIG.132. Clearance97071may exist at center of the actuator9707to reduce overall mass of actuator9707, where voice coil9708may be created surrounding the clearance97071. Sensor9801may exist at one or more locations on, or embedded within, the inside boundary wall of the cavity9706, where sensor9801may sense the proximity, or distance, of the actuator9707to the boundary wall of cavity9706. Controller950may receive signal from sensor9801through electrical connection952on proximity or distance of actuator9707to the left and right boundaries ofFIG.132cavity9706, and controller950through electrical connection953may adjust or control the electric current amplitude and current direction within coil9708of actuator9707to control the movement of actuator9707and thus the rotation of path selector9701. Electric current may be applied to the voice coils9708, where the arrows on the coils9708ofFIG.147illustrate an example of a clock-wise electric current flowing within coils9708, where said current within coil9708ofFIG.147creates a magnetic field with a direction pointing into the plane ofFIG.147against N polarity and being in same direction as S polarity of magnetic field9709ofFIG.132, where a net force may be exerted by the magnetic field9709onto the voice coil9708, and cause a movement of the voice coil9708, together with the actuator9707, to move away from N polarity region and into the S polarity region of field9709as inFIG.147, and thus causing an rotation of path selector9701.

In the example as illustrated byFIG.147, entities1/10/20/30/612in a fluid sample is injected into path2100in fluid flow91010, where path2100may be an extension of channel601/6072/910. One or more detectors901/9001/9002/9003located along path2100or channel601/6072/910, similar as inFIG.118A,FIG.124A,FIG.129A, may detect optical signal from entities1/10/20/30/612, where controller950, which may be connected to detectors901/9001/9002/9003through connection951, may receive and analyze signals from detectors901/9001/9002/9003and separate entities1/10/20/30/612into type9601“solid” entity and type9602“hollow” entity. Controller950may also determine a type9602entity may be the immediate entity that will exit path2100towards selector gate97011. Controller950through connection952may receive signal from sensor9801and may determine that path selector9701is at first sorting position, where type9601entities may be expected to flow into path9605in the fluid flow9603due to blockage of selector gate97011on path9606, and exiting the sorting device2012through outlet9607. Controller950may determine path selector9801need to rotate to a second sorting position to allow said immediate type9602entity in path2100to enter path9606into flow9604and exit device2012through outlet9608. Controller950through connection953may command, or provide, or alternate, or change, electric current in voice coils9708into clock-wise direction as shown inFIG.147to cause voice coils9708to generate a magnetic field that is in the direction of S polarity and against the N polarity region of magnetic field9709. With voice coils9708being mostly located within N polarity region of magnetic field9709as inFIG.147, a net force may be exerted on voice coils9708by magnetic field9709with clock-wise current applied to voice coils9708, making voice coils9708, together with actuator9707, move out of N region and into S region of magnetic field9709. Movement of actuator9707that is attached to path selector9701will then cause path selector9701to rotate around hinge9704in direction9705towards said second sorting position of device2012and allowing selector gate97011to block flow91010entrance into path9605and allow flow91010entrance into path9606.

Selector gate97011may comprise micro-fluidic pathways9990. Micro-fluidic pathways9990may be in the form of clearances through selector gate97011in the direction of selector gate97011movement. Micro-fluidic pathways9990may also be in the form of through trenches formed on top surface of selector gate97011facing out ofFIG.147view, or form on bottom surface of selector gate97011facing intoFIG.147view. Micro-fluidic pathways9990may have dimensions that are enough to allow passage of fluidic solution of flow91010to pass through selector gate97011during movement of selector gate97011through the fluid of flow91010, while said dimensions being small enough to block entity1/10/20/30/612from moving through selector gate97011. Micro-fluidic pathways9990may function to reduce fluidic drag on selector gate97011during movement of selector gate97011.

During operation of sorting device2012, spacing between path selector9701wall97012and cavity97062wall97042may be minimized to avoid fluid of flow91010flowing into cavity97062or cavity9706. In one embodiment, wall97012and wall97042may be in contact during rotation of path selector9701around hinge9704, where lubricating film or anti-abrasion coating may be applied to either or both of contacting surfaces of wall97012and wall97042, wherein said lubricating film of anti-abrasion coating may be a layer composed of organic molecules, said organic molecules may be repellant to water and oil. In one embodiment, spacing between wall97012and wall97042may be any of: from 0.1 nanometer (nm) to lnm, from 1 nm to 10 nm, from 10 nm to 100 nm, from 100 nm to 200 nm, from 200 nm to 500 nm, from 500 nm to 1 micrometer (um), from 1 um to 10 um, from 10 um to 20 um. In one embodiment, cavity97062may be filled with air, or gas including but not limited to: nitrogen, helium, argon, carbon dioxide, oxygen, where spacing between wall97012and wall97042may be sufficiently small where surface tension of fluid within liquid sample flow91010maintains fluid within paths2100,9605,9606at first and second sorting positions and during rotation of path selector9701, where surfaces of wall97012and wall97042may be composed of, or coated with, materials that is non-wettable, or hydrophobic, for liquid within sample flow91010, thus help maintaining fluid within paths2100,9605,9606.

In one embodiment, cavity97062space between wall97012and wall97042, may be filled with cavity fluid that is biological compatible with fluid sample of flow91010,9603and9604, where said cavity fluid may contain any of, but not limited to: water, saline, phosphate buffered saline, Ficoll. Said cavity fluid may be maintained at same, or higher, fluid pressure than flows91010,9603and9604, where fluid of sample flow91010may be maintained within paths2100,9605,9606. Said cavity fluid may be supplied continuously from outside device2012directly into cavity97062during operation of device2012. Said cavity fluid may be initially supplied through flow91010when entities1/10/20/30/612are not supplied to flow91010and cavity fluid may flow from path2100in between walls97012and97042into the cavity97062space, and cavity fluid may then maintain its effective volume during operation of device2012during rotation of path selector9701, where pressure within cavity fluid is maintained same as in flow91010. Said cavity fluid may be confined, or sealed within, cavity97062wall97042and not entering cavity9706, where cavity9706may be filled with air, or gas including but not limited to: nitrogen, helium, argon, carbon dioxide, oxygen, where walls of cavity9706may be composed of, or coated with, materials that is non-wettable, or hydrophobic, for cavity fluid within cavity97062, to help maintain cavity fluid within cavity97062.

InFIG.147, device body9600, path selector9701, actuator9707, selector gate97011may each be composed of any of: glass, silicon, quartz, aluminum-titanium-carbon (AlTiC), SiC, SiN, silicon-oxide, alumina, plastic, PDMS, polymer, ceramic, or metal, where metal may be composed any one or any alloy of aluminum, iron, nickel, titanium, chromium, platinum, tungsten, rhenium, copper, gold, silver. Path selector9701, selector gate97011, and actuator9707may be created as a single piece, where voice coils9708are formed on or within the actuator9707part of said single piece. Path selector9701, selector gate97011, and actuator9707may also be created as separate pieces, and joined together afterwards through any of: soldering, welding, gluing, mechanical attachment. Paths2100,9605,9606may be formed within device body9600in same manufacture steps simultaneously.

Encapsulation structure ofFIG.135AthroughFIG.135Emay be applied toFIG.147device2012similarly. Hinge9704form and function with path selector9701as described inFIG.135CandFIG.135Dmay be applied toFIG.147device2012similarly. Method to create field9709ofFIG.132as described inFIG.136AthroughFIG.136Dmay be applied toFIG.147device2012similarly. Position encoders9731ofFIG.137may be attached to actuator9707ofFIG.147similarly as inFIG.137, and position decoders9732may be embedded in device2012body9600or in cavity9706wall97042similar toFIG.137, to provide detection of actuator9707position in cavity9706ofFIG.147. Electric current driving methods ofFIG.138AandFIG.138Bmay be applied to device2012ofFIG.147similarly. ForFIG.138BAC current driving application to device2012ofFIG.147,FIG.138BAC current frequency may be same as the resonant frequency of the combined mass of path selector9701, selector gate91011and actuator9707.FIG.138BAC current frequency applied in device2012may also be at a value that correlates to said resonant frequency of the combined mass of path selector9701, selector gate91011and actuator9707, where said AC current frequency value may be an integer multiple of said resonant frequency, said AC current frequency may be at an value that this an offset increase or an offset decrease from said resonant frequency, said AC current frequency may also be at an value that this an offset increase or an offset decrease from an integer multiple of said resonant frequency. Said resonant frequency of the combined mass may be affected by any of: mass of path selector9701, mass of selector gate91011, mass of actuator9707; young's modulus values of path selector9701, selector gate91011and actuator9707; spring force of coil lines97081; inductance of voice coil9708. Said AC current frequency may be in the range of any of: between 10 Hertz (Hz) to 100 Hz, between 100 Hz to 1 kilo-Hertz (kHz), between 1 kHz to 10 kHz, between 10 kHz to 100 kHz, between 100 kHz to 1 Mega-Hertz (MHz), between 1 MHz to 2 MHz, between 2 MHz to 5 MHz, between 5 MHz to 10 MHz, between 10 MHz to 100 MHz. Capacitive actuator ofFIG.140, thermal-elastic actuator ofFIG.142may also be applied to device2012ofFIG.147to replace the voice coil actuator9707of device2012similarly.

FIG.148illustratesFIG.147sorting device2012at a second sorting position.FIG.148is the result of the operation as described inFIG.147example. FollowingFIG.147operation, actuator9707moves towards S region of the magnetic field9709, where controller950through sensor9801detects actuator9707proximity or distance to the right side boundary wall of cavity9706while controlling electric current amplitude and direction flowing in voice coils9708, until actuator9707rotates the path selector9701to reach second sorting position, where selector gate97011attached to path selector9701may be positioned at the entrance of path9605to block entities1/10/20/30/612in flow91010within path2100from entering path9605, while allowing said entities1/10/20/30/612to enter path9606. At second sorting position ofFIG.148, the immediate type9602entity exiting the path2100as inFIG.147moves into desired path9606and ultimately exiting sorting device2001through outlet9608. From described example ofFIG.147andFIG.148, one step of sorting one type9602entity into desired flow path9606may be achieved. At second position ofFIG.148, following same operation as inFIG.147, controller950may determine the immediate exiting entity within path2100may now become type9601entity, where controller950may reverse the electric current in voice coil9708to counter-clock-wise direction, such that voice coil9708may produce a magnetic field in the N direction and the net magnetic force exerted on actuator9707from magnetic field9709may become pushing the actuator back into the N region of the magnetic field9709and cause rotation of path selector9701back to the first sorting position as shown inFIG.147to complete another sorting step of moving immediate9601entity into desired path9605.

FIG.149illustrates an eighth embodiment of a biological entity sorting device2013having a voice coil actuator9707being at a first sorting position.FIG.149sorting device2013is similar to device2005ofFIG.139in operation, except the path selector9701of device2013does not have embedded flow paths9702or9703as in device2005ofFIG.139, where inFIG.149entities9601and9602are selected and passed into path9605or path9606within device body9600by a selector gate97011attached to path selector9701of device2013inFIG.149to selectively allow entities9601or9602to pass into one of path9605or path9606while blocking passage into other path. Device2013ofFIG.149may be part of UFL chip600/6000and may be contained within part of substrate611of UFL chip600/6000, or may be a separate device by itself. Sorting device2013may contain a fluidic sample injection path2100for entity1/10/20/30/612injection into device2013for sorting, said path2100may be a continuation of channel601/6072/910ofFIG.110AthroughFIG.131. In path2100, or as part of channel601/6072/910, entities1/10/20/30/612may first be detected by one or more detectors901/9001/9002/9003, where controller950through connections951may control, and sense entity1/10/20/30/612optical signal from, detectors901/9001/9002/9003. Said entity1/10/20/30/612optical signal may be processed or analyzed for category, or type, or identification, of the entities1/10/20/30/612, for example into categories of9601and9602as inFIG.149, by controller950, or by computing device955ofFIG.121AandFIG.121Bconnected to controller950.FIG.149shows a substantially rectangular cavity9706that may be created within device body9600with a cavity wall97042. Actuator9707may be located within said cavity9706. Actuator9707and cavity wall97042may be in contact, especially at the edge97022of actuator9707that forms part of fluid entrance into path9705or into path9706. Fluid path9605in device body9600may connect from main path2100to an outlet9607, and fluid path9606in device body9600may connect from main path to another outlet9608. Actuator9707may move linearly within cavity9706within the cavity wall97042. Actuator9707may be embedded with, or covered with, one or more voice coils9708. Sorting function of sorting device2013is achieved by driving electric current through voice coil9708and cause a movement of actuator9707between different sorting positions.FIG.149shows when actuator9707may be at a first sorting position, where selector gate97011attached to actuator9707may block entrance of path9605, while selector gate97011may form a path way together with surface97022of actuator9707to allow entities1/10/20/30/612within flow91010to flow into path9606and finally exiting device2013through outlet9608. In the case when actuator9707moves to a second sorting position, where selector gate97011attached to actuator9707may block entrance of path9606, while selector gate97011may form a path way together with surface97022of actuator9707to allow entities1/10/20/30/612within flow91010to flow into path9605and finally exiting device2013through outlet9607. Magnetic field9709having north and south polarities simultaneously may exist within the cavity9706, whereFIG.149shows north (N) polarity and south (S) polarity of the magnetic field9709exists side by side within cavity9706, with N polarity on the left having magnetic field direction pointing out of the plane and S polarity on the right with magnetic field direction pointing into the plane ofFIG.149, and with the magnetic field from both N and S polarities having magnetic field components perpendicular to the voice coil9708plane. Voice coil9708may be in the form of single-turn or multiple-turn coils that may be located on a surface of the actuator9707, or may be embedded within the body of the actuator9707. Voice coil9708may be created on top of, or within, actuator9707similarly as described inFIG.139. Clearance97071may exist at center of the actuator9707to reduce overall mass of actuator9707, where voice coil9708may be created surrounding the clearance97071. Sensor9801may exist at one or more locations on, or embedded within, the inside boundary wall97042of the cavity9706, where sensor9801may sense the proximity, or distance, of the actuator9707to the boundary wall97042of cavity9706. Controller950may receive signal from sensor9801through electrical connection952on proximity or distance of actuator9707to wall97042, and controller950through electrical connection953may adjust or control the electric current amplitude and current direction within coil9708of actuator9707to control the movement of actuator9707. Electric current may be applied to the voice coils9708, where the arrows on the coils9708ofFIG.149illustrate an example of a clock-wise electric current flowing within coils9708, where said current within coil9708ofFIG.149creates a magnetic field with a direction pointing into the plane ofFIG.149against N polarity and being in same direction as S polarity of magnetic field9709ofFIG.149, where a net force may be exerted by the magnetic field9709onto the voice coil9708, and cause a movement of the voice coil9708, together with the actuator9707, to move away from N polarity region and into the S polarity region of field9709as inFIG.149, and thus causing an movement of actuator9707to right side of the cavity9706as inFIG.149.

In the example as illustrated byFIG.149, entities1/10/20/30/612in a fluid sample is injected into path2100in fluid flow91010, where path2100may be an extension of channel601/6072/910. One or more detectors901/9001/9002/9003located along path2100or channel601/6072/910, similar as inFIG.132, may detect optical signal from entities1/10/20/30/612, where controller950, which may be connected to detectors901/9001/9002/9003through connection951, may receive and analyze signals from detectors901/9001/9002/9003and separate entities1/10/20/30/612into type9601“solid” entity and type9602“hollow” entity. Controller950may also determine a type9601entity may be the immediate entity that will exit path2100towards actuator9707. Controller950through connection952may receive signal from sensor9801and may determine that actuator9707is at first sorting position, where type9602entities may be expected to flow into path9606in the fluid flow9604and exiting the sorting device2013through outlet9608. Controller950may determine actuator9707need to move to a second sorting position to allow said immediate type9601entity in path2100to enter path9605into flow9603and exit device2013through outlet9607. Controller950through connection953may command, or provide, or alternate, or change, electric current in voice coils9708into clock-wise direction as shown inFIG.149to cause voice coils9708to generate a magnetic field that is in the direction of S polarity, which is against the N polarity region of magnetic field9709. With voice coils9708being mostly located within N polarity region of magnetic field9709as inFIG.149, a net force may be exerted on voice coils9708by magnetic field9709with clock-wise current applied to voice coils9708, making voice coils9708, together with actuator9707, move out of N region and into S region of magnetic field9709, resulting in movement of actuator9707towards said second sorting position of device2013and allowing selector gate97011to block flow91010entrance into path9606and allow flow91010entrance into path9605.

Selector gate97011may comprise micro-fluidic pathways9990similar as inFIG.147. Micro-fluidic pathways9990may be in the form of clearances through selector gate97011in the direction of selector gate97011movement. Micro-fluidic pathways9990may also be in the form of through trenches formed on top surface of selector gate97011facing out ofFIG.149view, or form on bottom surface of selector gate97011facing intoFIG.149view. Micro-fluidic pathways9990may have dimensions that are enough to allow passage of fluidic solution of flow91010to pass through selector gate97011during movement of selector gate97011through the fluid of flow91010, while said dimensions being small enough to block entity1/10/20/30/612from moving through selector gate97011. Micro-fluidic pathways9990may function to reduce fluidic drag on selector gate97011during movement of selector gate97011.

During operation of sorting device2013ofFIG.149, spacing between actuator9707wall97022and cavity9706wall97042may be minimized to form sealing of fluid between path2100to paths9605and9606, and to avoid fluid of flow91010flowing into cavity9706. In one embodiment, wall97022and wall97042may be in contact during movement of actuator9707, where lubricating film or anti-abrasion coating may be applied to either or both of contacting surfaces of wall97022and wall97042, wherein said lubricating film of anti-abrasion coating may be a layer composed of organic molecules, said organic molecules may be repellant to water and oil. In one embodiment, spacing between wall97022and wall97042may be any of: from 0.1 nanometer (nm) to 1 nm, from 1 nm to 10 nm, from 10 nm to 100 nm, from 100 nm to 200 nm, from 200 nm to 500 nm, from 500 nm to 1 micrometer (um), from 1 um to 10 um, from 10 um to 20 um. In one embodiment, cavity9706may be filled with air, or gas including but not limited to: nitrogen, helium, argon, carbon dioxide, oxygen, where spacing between wall97022and wall97042may be sufficiently small where surface tension of fluid within liquid sample flow91010maintains fluid within paths2100,9605,9606at first and second sorting positions and during movement of actuator9707, where surfaces of wall97022and wall97042may be composed of, or coated with, materials that is non-wettable, or hydrophobic, for liquid within sample flow91010, thus help maintaining fluid within paths2100,9605,9606.

In one embodiment, cavity9706, especially the space between wall97022and wall97042, may be filled with cavity fluid that is biological compatible with fluid sample of flow91010,9603and9604, where said cavity fluid may contain any of, but not limited to: water, saline, phosphate buffered saline, Ficoll. Said cavity fluid may be maintained at same, or higher, fluid pressure than flows91010,9603and9604, where fluid of sample flow91010may be maintained within paths2100,9605,9606. Said cavity fluid may be supplied continuously from outside device2013ofFIG.149and directly into cavity9706during operation of device2005. Said cavity fluid may be initially supplied through flow91010when entities1/10/20/30/612are not supplied to flow91010and cavity fluid may flow from path2100in between walls97022and97042into the cavity9706space, and cavity fluid may then maintain its effective volume during operation of device2013and during movement of actuator9707, where pressure within cavity fluid is maintained same as in flow91010. Said cavity fluid may be confined, or sealed within, cavity9706wall97042, where walls of cavity9706may be composed of, or coated with, materials that is non-wettable, or hydrophobic.

Encapsulation structure ofFIG.135AthroughFIG.135Emay be applied toFIG.149device2013similarly. Method to create field9709ofFIG.132as described inFIG.136AthroughFIG.136Dmay be applied toFIG.149device2013similarly. Position encoders9731ofFIG.137may be attached to actuator9707ofFIG.149similarly as inFIG.137, and position decoders9732may be embedded in device2013body9600or in cavity9706wall97042similar toFIG.137, to provide detection of actuator9707position in cavity9706ofFIG.149. Electric current driving methods ofFIG.138AandFIG.138Bmay be applied to device2013ofFIG.149similarly. ForFIG.138BAC current driving application to device2013ofFIG.149,FIG.138BAC current frequency may be same as the resonant frequency of the combined mass of selector gate91011and actuator9707.FIG.138BAC current frequency applied in device2013may also be at a value that correlates to said resonant frequency of the combined mass of selector gate91011and actuator9707, where said AC current frequency value may be an integer multiple of said resonant frequency, said AC current frequency may be at an value that this an offset increase or an offset decrease from said resonant frequency, said AC current frequency may also be at an value that this an offset increase or an offset decrease from an integer multiple of said resonant frequency. Said resonant frequency of the combined mass may be affected by any of: mass of selector gate91011, mass of actuator9707; young's modulus values of selector gate91011and actuator9707; spring force of coil lines97081; inductance of voice coil9708. Said AC current frequency may be in the range of any of: between 10 Hertz (Hz) to 100 Hz, between 100 Hz to 1 kilo-Hertz (kHz), between 1 kHz to 10 kHz, between 10 kHz to 100 kHz, between 100 kHz to 1 Mega-Hertz (MHz), between 1 MHz to 2 MHz, between 2 MHz to 5 MHz, between 5 MHz to 10 MHz, between 10 MHz to 100 MHz. Capacitive actuator ofFIG.141, thermal-elastic actuator ofFIG.143may also be applied to device2013ofFIG.149to replace the voice coil actuator9707of device2013similarly.

FIG.150illustratesFIG.149sorting device2013at a second sorting position.FIG.150is the result of the operation as described inFIG.149example. FollowingFIG.149operation, actuator9707moves towards S region of the magnetic field9709, where controller950through sensor9801detects actuator9707proximity or distance to the right side boundary wall of cavity9706while controlling electric current amplitude and direction flowing in voice coils9708, until actuator9707reaches second sorting position, where selector gate97011attached to actuator9707may be positioned at the entrance of path9606to block entities1/10/20/30/612in flow91010within path2100from entering path9606, while allowing said entities1/10/20/30/612to enter path9605. At second sorting position ofFIG.150, the immediate type9601entity exiting the path2100as inFIG.149moves into desired path9605and ultimately exiting sorting device2013through outlet9607. From described example ofFIG.149andFIG.150, one step of sorting one type9601entity into desired flow path9605may be achieved. At second position ofFIG.150, following same operation as inFIG.149, controller950may determine the immediate exiting entity within path2100may now become type9602entity, where controller950may reverse the electric current in voice coil9708to counter-clock-wise direction, such that voice coil9708may produce a magnetic field in the N direction and the net magnetic force exerted on actuator9707from magnetic field9709may become pushing the actuator back into the N region of the magnetic field9709and cause movement of actuator9707back to the first sorting position as shown inFIG.149to complete another sorting step of moving immediate9602entity into desired path9606.

During sorting functions as described inFIG.147throughFIG.150, selector gate97011may alternate between first and second sorting positions in between events of entity9601or entity9602exiting path2100, whereas such alternation may not achieve actual entity9601or entity9602sorting, but rather may allow sufficient amount of fluid solution flowing into both path9605and path9606to produce effective output sample flow rate through outlets9607and9608. Such alternation may be desirable in the case where one first type entities being at much smaller quantity than one second type of entities to avoid insufficiently output sample volume that contains said first type entities.

While the current invention has been shown and described with reference to certain embodiments, it is to be understood that those skilled in the art will no doubt devise certain alterations and modifications thereto which nevertheless include the true spirit and scope of the current invention. Thus the scope of the invention should be determined by the appended claims and their legal equivalents, rather than by examples given.