Microfluidic device

The present disclosure relates to microfluidic devices adapted for facilitating cytometry analysis of particles flowing therethrough. In certain embodiments, the microfluidic devices allow light collection from multiple directions. In certain other embodiments, the microfluidic devices use spatial intensity modulation. In certain other embodiments, the microfluidic devices have magnetic field separators. In certain other embodiments, the microfluidic devices have the ability to stack. In certain other embodiments, the microfluidic devices have 3-D hydrodynamic focusing to align sperm cells. In certain other embodiments, the microfluidic devices have acoustic energy couplers. In certain other embodiments, the microfluidic devices have phase variation producing lenses. In certain other embodiments, the microfluidic devices have transmissive and reflective lenses. In certain other embodiments, the microfluidic devices have integrally-formed optics. In certain other embodiments, the microfluidic devices have non-integral geographically selective reagent delivery structures. In certain other embodiments, the microfluidic devices have optical waveguides incorporated into their flow channels. In certain other embodiments, the microfluidic devices have optical waveguides with reflective surfaces incorporated into their flow channels. In certain other embodiments, the microfluidic devices have virus detecting and sorting capabilities. In certain other embodiments, the microfluidic devices display a color change to indicate use or a result.

TECHNICAL FIELD OF THE DISCLOSURE

The present disclosure relates to microfluidic cytometry systems.

BACKGROUND OF THE DISCLOSURE

Flow cytometry-based cell sorting was first introduced to the research community more than 20 years ago. It is a technology that has been widely applied in many areas of life science research, serving as a critical tool for those working in fields such as genetics, immunology, molecular biology and environmental science. Unlike bulk cell separation techniques such as immuno-panning or magnetic column separation, flow cytometry-based cell sorting instruments measure, classify and then sort individual cells or particles serially at rates of several thousand cells per second or higher. This rapid “one-by-one” processing of single cells has made flow cytometry a unique and valuable tool for extracting highly pure sub-populations of cells from otherwise heterogeneous cell suspensions.

Cells targeted for sorting are usually labeled in some manner with a fluorescent material. The fluorescent probes bound to a cell emit fluorescent light as the cell passes through a tightly focused, high intensity, light beam (typically a laser beam). A computer records emission intensities for each cell. These data are then used to classify each cell for specific sorting operations. Flow cytometry-based cell sorting has been successfully applied to hundreds of cell types, cell constituents and microorganisms, as well as many types of inorganic particles of comparable size.

Flow cytometers are also applied widely for rapidly analyzing heterogeneous cell suspensions to identify constituent sub-populations. Examples of the many applications where flow cytometry cell sorting is finding use include isolation of rare populations of immune system cells for AIDS research, isolation of genetically atypical cells for cancer research, isolation of specific chromosomes for genetic studies, and isolation of various species of microorganisms for environmental studies. For example, fluorescently labeled monoclonal antibodies are often used as “markers” to identify immune cells such as T lymphocytes and B lymphocytes, clinical laboratories routinely use this technology to count the number of “CD4 positive” T cells in HIV infected patients, and they also use this technology to identify cells associated with a variety of leukemia and lymphoma cancers.

Recently, two areas of interest are moving cell sorting towards clinical, patient care applications, rather than strictly research applications. First is the move away from chemical pharmaceutical development to the development of biopharmaceuticals. For example, the majority of new cancer therapies are bio-based. These include a class of antibody-based cancer therapeutics. Cytometry-based cell sorters can play a vital role in the identification, development, purification and, ultimately, production of these products.

Related to this is a move toward the use of cell replacement therapy for patient care. Much of the current interest in stem cells revolves around a new area of medicine often referred to as regenerative therapy or regenerative medicine. These therapies may often require that large numbers of relatively rare cells be isolated from sample patient tissue. For example, adult stem cells may be isolated from bone marrow and ultimately used as part of a re-infusion back into the patient from whom they were removed. Cytometry lends itself very well to such therapies.

There are two basic types of cell sorters in wide use today. They are the “droplet cell sorter” and the “fluid switching cell sorter.” The droplet cell sorter utilizes micro-droplets as containers to transport selected cells to a collection vessel. The micro-droplets are formed by coupling ultrasonic energy to a jetting stream. Droplets containing cells selected for sorting are then electrostatically steered to the desired location. This is a very efficient process, allowing as many as 90,000 cells per second to be sorted from a single stream, limited primarily by the frequency of droplet generation and the time required for illumination.

A detailed description of a prior art flow cytometry system is given in United States Published Patent Application No. US 2005/0112541 A1 to Durack et al.

Droplet cell sorters, however, are not particularly biosafe. Aerosols generated as part of the droplet formation process can carry biohazardous materials. Because of this, biosafe droplet cell sorters have been developed that are contained within a biosafety cabinet so that they may operate within an essentially closed environment. Unfortunately, this type of system does not lend itself to the sterility and operator protection required for routine sorting of patient samples in a clinical environment.

The second type of flow cytometry-based cell sorter is the fluid switching cell sorter. Most fluid switching cell sorters utilize a piezoelectric device to drive a mechanical system which diverts a segment of the flowing sample stream into a collection vessel. Compared to droplet cell sorters, fluid switching cell sorters have a lower maximum cell sorting rate due to the cycle time of the mechanical system used to divert the sample stream. This cycle time, the time between initial sample diversion and when stable non-sorted flow is restored, is typically significantly greater than the period of a droplet generator on a droplet cell sorter. This longer cycle time limits fluid switching cell sorters to processing rates of several hundred cells per second. For the same reason, the stream segment switched by a fluid cell sorter is usually at least ten times the volume of a single micro-drop from a droplet generator. This results in a correspondingly lower concentration of cells in the fluid switching sorter's collection vessel as compared to a droplet sorter's collection vessel.

Newer generation microfluidics technologies offer great promise for improving the efficiency of fluid switching devices and providing cell sorting capability on a chip similar in concept to an electronic integrated circuit. Many microfluidic systems have been demonstrated that can successfully sort cells from heterogeneous cell populations. They have the advantages of being completely self-contained, easy to sterilize, and can be manufactured on sufficient scales (with the resulting manufacturing efficiencies) to be considered a disposable part.

A generic microfluidic device is schematically illustrated inFIG. 1and indicated generally at10. The microfluidic device10comprises a substrate12having a fluid flow channel14formed therein by any convenient process as is known in the art. The substrate12may be formed from glass, plastic or any other convenient material, and may be substantially transparent or substantially transparent in a portion thereof. The substrate12further has three ports16,18and20coupled thereto. Port16is an inlet port for a sheath fluid. Port16has a central axial passage that is in fluid communication with a fluid flow channel22that joins fluid flow channel14such that sheath fluid entering port16from an external supply (not shown) will enter fluid flow channel22and then flow into fluid flow channel14. The sheath fluid supply may be attached to the port16by any convenient coupling mechanism as is known to those skilled in the art.

Port18also has a central axial passage that is in fluid communication with a fluid flow channel14through a sample injection tube24. Sample injection tube24is positioned to be coaxial with the longitudinal axis of the fluid flow channel14. Injection of a liquid sample of cells into port18while sheath fluid is being injected into port16will therefore result in the cells flowing through fluid flow channel14surrounded by the sheath fluid. The dimensions and configuration of the fluid flow channels14and22, as well as the sample injection tube24are chosen so that the sheath/sample fluid will exhibit laminar flow as it travels through the device10, as is known in the art. Port20is coupled to the terminal end of the fluid flow channel14so that the sheath/sample fluid may be removed from the microfluidic device10.

While the sheath/sample fluid is flowing through the fluid flow channel14, it may be analyzed using cytometry techniques by shining an illumination source through the substrate12and into the fluid flow channel14at some point between the sample injection tube24and the outlet port20. Additionally, the microfluidic device10could be modified to provide for a cell sorting operation, as is known in the art.

Although basic microfluidic devices similar to that described hereinabove have been demonstrated to work well, there is a need in the prior art for improvements to cytometry systems employing microfluidic devices. The present invention is directed to meeting this need.

SUMMARY OF THE DISCLOSURE

The present disclosure relates to microfluidic devices adapted for facilitating cytometry analysis of particles flowing therethrough. In certain embodiments, the microfluidic devices allow light collection from multiple directions. In certain other embodiments, the microfluidic devices use spatial intensity modulation. In certain other embodiments, the microfluidic devices have magnetic field separators. In certain other embodiments, the microfluidic devices have the ability to stack. In certain other embodiments, the microfluidic devices have 3-D hydrodynamic focusing to align sperm cells. In certain other embodiments, the microfluidic devices have acoustic energy couplers. In certain other embodiments, the microfluidic devices have phase variation producing lenses. In certain other embodiments, the microfluidic devices have transmissive and reflective lenses. In certain other embodiments, the microfluidic devices have integrally-formed optics. In certain other embodiments, the microfluidic devices have non-integral geographically selective reagent delivery structures. In certain other embodiments, the microfluidic devices have optical waveguides incorporated into their flow channels. In certain other embodiments, the microfluidic devices have optical waveguides with reflective surfaces incorporated into their flow channels. In certain other embodiments, the microfluidic devices have virus detecting and sorting capabilities. In certain other embodiments, the microfluidic devices display a color change to indicate use or a result.

In one embodiment, a microfluidic device system is disclosed, comprising a substrate, a microfluidic flow channel formed in said substrate, wherein said flow channel extends through a portion of said substrate adapted to facilitate cytometry analysis of cells flowing in said flow channel, a first light collection device operative to collect light emitted by said cells in a first direction, said first light collection device producing a first output, a second light collection device operative to collect light emitted by said cells in a second direction, said second light collection device producing a second output, and detection optics operative to receive said first and second outputs.

In another embodiment, a method of detecting particles in a sample is disclosed, the method comprising the steps of: a) providing a microfluidic device, said microfluidic device comprising a substrate and a microfluidic flow channel formed in said substrate, wherein said flow channel extends through a portion of said substrate adapted to facilitate cytometry analysis of cells flowing in said flow channel; b) capturing first light emitted from said cells in a first direction; c) capturing second light emitted from said cells in a second direction; d) combining said first light and said second light captured at steps (b) and (c); and e) performing cytometry analysis on said combined first and second light.

In another embodiment, a method of detecting particles in a sample is disclosed, the method comprising the steps of: a) providing a microfluidic device, said microfluidic device comprising a substrate, a first microfluidic flow channel formed in said substrate, wherein said first flow channel extends through a portion of said substrate adapted to facilitate cytometry analysis of first cells flowing in said first flow channel, and a second microfluidic flow channel formed in said substrate, wherein said second flow channel extends through a portion of said substrate adapted to facilitate cytometry analysis of second cells flowing in said second flow channel; b) producing an excitation beam aimed at said first and second flow channels; c) spatially varying said excitation beam in a first manner prior to said excitation beam reaching said first flow channel; and d) spatially varying said excitation beam in a second manner prior to said excitation beam reaching said second flow channel.

In yet another embodiment, a microfluidic device is disclosed, comprising a substrate, a microfluidic flow channel formed in said substrate, wherein said flow channel extends through a portion of said substrate adapted to facilitate cytometry analysis of cells flowing in said flow channel, a sample reception well formed onboard said substrate, said sample reception well being fluidically coupled to said flow channel, and an electromagnet disposed onboard said substrate and operative when energized to produce a magnetic field within said sample reception well.

In still another embodiment, a microfluidic device is disclosed, comprising a substrate, a microfluidic flow channel formed in said substrate, wherein said flow channel extends through a portion of said substrate adapted to facilitate cytometry analysis of cells flowing in said flow channel, and at least one leg positioned on a surface of said substrate, said at least one leg facilitating stacking said microfluidic device on another microfluidic device.

In another embodiment, a microfluidic device is disclosed, comprising a substrate, a microfluidic flow channel formed in said substrate, wherein said flow channel extends through a portion of said substrate adapted to facilitate cytometry analysis of cells flowing in said flow channel, and at least one hydrodynamic alignment structure fluidically coupled to said flow channel, said at least one hydrodynamic alignment structure operative to orient said cells such that a majority of said cells are analyzed from their largest dimension.

In yet another embodiment, a microfluidic device is disclosed, comprising a substrate, a microfluidic flow channel formed in said substrate, wherein said flow channel extends through a portion of said substrate adapted to facilitate cytometry analysis of cells flowing in said flow channel, a well disposed onboard said substrate, said well being fluidically coupled with said flow channel, and an acoustic energy coupler disposed within said well.

In still another embodiment, a microfluidic device is disclosed, comprising a substrate, a microfluidic flow channel formed in said substrate, wherein said flow channel extends through a portion of said substrate adapted to facilitate cytometry analysis of cells flowing in said flow channel, and a lens formed onboard said substrate, said lens operative to spatially modify an intensity of light passing therethrough.

In another embodiment, a microfluidic device is disclosed, comprising a substrate, a microfluidic flow channel formed in said substrate, wherein said flow channel extends through a portion of said substrate adapted to facilitate cytometry analysis of cells flowing in said flow channel, a first lens formed onboard said substrate, said first lens being disposed on a first side of said flow channel, and a second lens formed onboard said substrate, said second lens being disposed on a second side of said flow channel.

In yet another embodiment, a microfluidic device is disclosed, comprising a substrate having a first surface, a microfluidic flow channel formed in said substrate, wherein said flow channel extends through a portion of said substrate adapted to facilitate cytometry analysis of cells flowing in said flow channel, and a lens formed onboard said substrate at said first surface, said lens lying below said first surface.

In still another embodiment, a microfluidic device is disclosed, comprising a substrate having a first surface, a reagent receiving well formed onboard said substrate, a reagent structure having a reagent disposed thereon, wherein application of said reagent structure to said first surface causes said reagent to align with said reagent receiving well for transfer of reagent thereto, and a microfluidic flow channel formed in said substrate, wherein said flow channel is fluidically coupled to said reagent receiving well.

In yet another embodiment, a microfluidic device is disclosed, comprising a substrate, a microfluidic flow channel formed in said substrate, wherein said flow channel extends through a portion of said substrate adapted to facilitate cytometry analysis of cells flowing in said flow channel, and an optical waveguide formed in said flow channel.

In another embodiment, a microfluidic device is disclosed, comprising a substrate, a microfluidic flow channel formed in said substrate, wherein said flow channel extends through a portion of said substrate adapted to facilitate cytometry analysis of cells flowing in said flow channel, an optical waveguide formed in said flow channel, and a reflective surface disposed within said flow channel.

In yet another embodiment, a method of determining a pharmacological efficacy is disclosed, the method comprising the steps of: a) providing a microfluidic device, said microfluidic device comprising a substrate, a microfluidic flow channel formed in said substrate, wherein said flow channel extends through a portion of said substrate adapted to facilitate cytometry analysis of viral particles flowing in said flow channel, and a well formed onboard said substrate; b) depositing a material into said well; c) flowing said viral particles into said well; d) reacting said viral particles with said material; and e) determining a pharmacological efficacy of said material based upon said reaction.

In still another embodiment, a method of detecting particles in a sample is disclosed, the method comprising the steps of: a) providing a microfluidic device, said microfluidic device comprising a substrate, a microfluidic flow channel formed in said substrate, wherein said flow channel extends through a portion of said substrate adapted to facilitate cytometry analysis of cells flowing in said flow channel, and a dye repository formed onboard said substrate; b) depositing a dye into said well; c) performing a cytometry analysis on said cells; and d) causing said dye to exit said dye repository and enter said flow channel after said cytometry analysis is complete.

Other embodiments are also disclosed.

DESCRIPTION OF THE ILLUSTRATED EMBODIMENTS

For the purposes of promoting an understanding of the principles of the disclosure, reference will now 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 of the scope of the disclosure is thereby intended, such alterations and further modifications in the illustrated device, and such further applications of the principles of the disclosure as illustrated therein are contemplated as would normally occur to one skilled in the art to which the disclosure relates.

Microfluidic Device Having Light Collection from Multiple Directions

Certain embodiments of the present disclosure are generally directed to systems for the analysis of a sample on a microfluidic device, using cytometry (such as flow cytometry or image cytometry). In order to detect or identify cells during cytometry operations, a source (such as a laser) of electromagnetic radiation (such as visible light) is directed to a detection area. When a cell passes through the detection area, the light source causes the cell to fluoresce. This effect may be enhanced by certain dyes which are added prior to the cell reaching the detection area. In most systems, the light source is applied to only one side of the cytometry device, with the detection optics also only detecting the fluorescence from one side of the sample cell. This limits the amount of photons that are captured from the fluorescent sample cell, since the photons emitted from other directions are not captured by the detection optics. The light intensity may be increased to generate more fluorescently generated photons, however this also increases the amount of noise in the signal.

In order to capture as many of the photons generated by a fluorescing sample cell as possible, light sources may be applied to multiple sides of the detection area. In addition, the detection optics can also be placed on multiple sides the detection area. This allows the system to capture more of the emitted photons for a given excitation light intensity without increasing the noise in the received signal. Further, the method combines the photons emitted from both sides into a single collimated path that can be focused on a single photodetector. This is an improvement over using separate photodetectors for each side. The use of two photodetectors doubles the noise contributed by each photodetector. Thus, the ability to use a single photodetector improves the signal-to-noise ratio of the system.

FIG. 2schematically illustrates a system200for analyzing samples using cytometry. The system200may comprise a microfluidic device formed on substrate202(shown here from a side view) having a detection flow channel204contained therein. For simplicity and ease of illustration,FIG. 2shows a single channel within the substrate202. However, it should be appreciated that the single channel may be representative of multiple cytometry channels and a variety of possible configurations of channels as would occur to one skilled in the art. Various other cytometry components may also be contained on the cytometry chip202but are not critical to the present disclosure.

The system200may further comprise an excitation light source206, a multi-core fiberoptic cable218, focusing lenses212and220, and detection optics226. The lenses212and220may be placed outside of the substrate202as illustrated inFIG. 2, or may be mounted to or formed integrally within the substrate202depending the needs and cost considerations of the particular application. The excitation light source206may comprise a laser or other source of electromagnetic radiation known in the art. The detection optics226may comprise various light sensing means known in the art, such as photomultiplier tubes, photodiodes, or avalanche photodiodes.

In operation, excitation light source206(normally a laser) directs a light beam towards dichroic mirror208. The dichroic mirror is configured to reflect the wavelength received from the light source206to the detection area210on the chip202. Various other optical focusing components, such as lens212, may be used to ensure proper focus of the excitation light source206onto the detection area210. When a sample cell214traversing microfluidic flow channel204passes through the detection area210and fluoresces, the generated photons are emitted in many directions. Some photons will be emitted back toward the focusing lens212, pass through the dichroic mirror208(which is configured to pass the fluorescence wavelength), and enter a first end216of a multi-core fiber optic cable218. Photons that are emitted from the opposite side of chip202will be optionally focused by lens220and enter a second end222of fiber optic cable218. The combined groups of photons will then be emitted from a third end224of fiber optic cable218and received by detection optic226. The detection optic226can then collimate the combined emission from the two fiber optic cores and transmit the emission to one or more photodetectors (not shown) for measurement. In certain embodiments a series of dichroic and bandpass optical filters will be used to select specific wavelength bands and transmit them to a photodetector. In still further embodiments, separate detection optics may be used to capture the emitted photons instead using a multi core fiber optic cable.

FIG. 3schematically illustrates another embodiment of the present disclosure, shown here as system300, in which multiple light sources306are used to generate fluorescence in the sample cells314. In this embodiment, both the reflected and transmitted photons from the sample cells314will be detected from both sides of the cytometry chip302. Again, the emitted photons will enter ends316and322of the fiber optic cable318and be captured by detection optics326. In certain embodiments, the light sources306may be configured to generate different wavelengths of light. The detection optics can then detect different types of cells that have been dyed to fluoresce at the different wavelengths.

Microfluidic Device Using Spatial Intensity Modulation

Certain embodiments of the present disclosure are generally directed to systems for the analysis of a sample on a microfluidic device, using cytometry (such as flow cytometry or image cytometry). To increase overall cell throughput, multiple flow channels may be used in parallel to sort cells from a given sample. In such systems, the detection optics and associated electronic processing equipment must be able to distinguish between signals received from the individual channels. One way to accomplish this is to use individual excitation light sources on each channel, each with their own wavelength. However, this adds significant cost and complexity to the system. A single light source may also be scanned from channel to channel in a repeating fashion, with the detection optics determining which channel was scanned based on time. However this approach is also prone to errors due to the possibility of cells passing through the detection region of a given channel while the light source is exciting the detection region of a different channel.

As disclosed herein, another way to distinguish the separate channel fluorescence signals is to direct a single excitation light source on all of the channels simultaneously, but spatially vary or shape the excitation beam pattern so that a different excitation pattern is directed to the detection region of each channel. As a cell passes through the beam pattern of a particular channel, a series of time-spaced peaks will be created in the emitted fluorescence signal. Because the flow rate or speed of the cell is known, the time between the peaks, or frequency, can be used to determine which channel produced the signal. The detection optic is able to receive signals from all channels simultaneously and use varying means known in the art, such as frequency modulation/demodulation to separate and analyze the individual channel signals.

Various methods known in the art may be used to spatially vary the excitation beam within the detection region of each channel. In one embodiment, an optical element, such as a hologram or other light shifting device may be molded into the substrate of a microfluidic cytometry chip. The optical element transforms the uniform beam into spatial patterns within the detection regions of each channel. In other embodiments, the surface of the chip can be etched or coated with a non-reflective material to create the contrasting pattern elements. In still further embodiments, various mirrors, filters, or other light blocking or phase shifting means can be used to create the patterns. In still further embodiments, an active element such as a spatial light modulator or acousto-optic modulator may be employed to deliver time-varying spatial intensity patterns within the detection region of each channel. These spatial patterns may be dynamically altered at a faster rate than the progression of the cells passing through the detection region.

FIG. 4schematically illustrates one embodiment where the excitation light patterns consist of a series of bars402spaced parallel to the direction that cells404are flowing through the detection region406of parallel flow channels408,410, and412within a microfluidic device.FIG. 5illustrates sample resultant fluorescence signals508,510, and512for the channels408,410, and412, respectively. As can be seen, varying the spacing between the bars402results in a unique signature in the fluorescence signal detected for each channel, thereby allowing the detection signal for any chosen channel to be separated from the detection signals produced by the other channels.

FIG. 6shows another embodiment where the excitation pattern elements602vary perpendicular to the direction of flow in addition to varying parallel to the direction of flow. This allows the detection optics to received data about the cell604in two dimensions, as opposed to one. Using the two-dimensional fluorescence information produced as the cell604passes through the detection region, in conjunction with the particular mathematical function or shape of the pattern elements602, a two dimensional image of the cell can be created, thereby providing much more information about the cell.

Microfluidic Device Having Magnetic Field Separator

Certain embodiments of the present disclosure are generally directed to systems for the separation and analysis of a biological sample on a microfluidic device using cytometry (such as flow cytometry or image cytometry) using an electromagnetic field. In order to increase the efficiency of cell sorting operations, it is desirable to start with a sample containing only those types of cells that are desired to be studied or isolated. One method known in the art is to subject the sample to centrifugation prior to cytometry analysis. After centrifugation, the sample components will be separated into layers. The desired component can then be easily extracted. However, this introduces the possibility that the sample layers will become remixed during the transfer from the initial collection vessel to the microfluidic device. By providing the separation capabilities integral with the microfluidic device itself, the possibility of layer mixing after centrifugation is reduced. This also eliminates the need for multiple containers and reduces the possibility of outside contaminates being introduced into the sample (or potentially harmful sample components being released into the outside environment).

FIG. 7schematically illustrates a microfluidic device700operable to achieve the separation of desired biological components contained with a biological sample. In the particular illustrated embodiments, cells from a cell supply (not shown) are input to input port710are initially separated at sample well720on substrate702due to an electromagnetic force. Thereafter, the separated cells are analyzed via cytometry in analysis section712(the specific operations that occur in analysis section712are not critical to the present disclosure). According to the results of the analysis performed, the cells may be sorted into different chambers714. For simplicity and ease of illustration,FIG. 7shows single channels extending between the components, areas or sections of chip702. However, it should be appreciated that the single channels may be representative of multiple cytometry channels and a variety of possible configurations of channels as would occur to one skilled in the art.

Prior to their entry into the sample well720, the cells that the researcher or medical professional desires to analyze via cytometry may each be coupled with a magnetic particle, either in addition to or in lieu of the attachment of a fluorescent molecule to the desirable cell. The magnetic particles used in accordance with system700may be sub-micron magnetic beads or other appropriate magnetic particles as would occur to one of ordinary skill in the art. The biological sample from input port710is received in sample well720via flow channel708. Adjacent sample well720is an electromagnet722operable to create an electromagnetic field having an electromagnetic force within the sample well. In some embodiments, the electromagnet722may be temporarily, semi-permanently or permanently disposed onboard substrate702. In other embodiments, the electromagnet722is brought into proximity with sample well720without being disposed onboard, attached to, or connected with chip702. Electromagnet722may be self-contained such that the electrical energy to the electromagnet is through the use of batteries or other appropriate manners. In other embodiments, a power supply cord740is coupled to the electromagnet722to provide electrical energy thereto. In certain embodiments, the electromagnet722used to provide the magnetic field may be permanently positioned. The electromagnet may be mechanically moved into place when it is necessary to establish the magnetic field and moved out of place when it is necessary to remove the magnetic field.

The cells724to which the magnetic particles are coupled, as mentioned above, will enter the electromagnetic field, be attracted to electromagnet722and thus will be pulled toward the bottom of sample well720adjacent bottom surface720aunder the electromagnetic force. In certain embodiments, the sample well720is in flow communication with channel709leading to analysis section712at a location adjacent bottom surface720a. In such embodiments, the initially separated group of cells724(those tagged with magnetic particles) will flow into channel709and into analysis section712. To accomplish the travel of cells724into channel709, the power to electromagnet722may be temporarily, semi-permanently or permanently reduced or eliminated and thus the electromagnetic force will be correspondingly reduced or eliminated. Accordingly, with the force reduced or eliminated, the cells724will be free to travel into channel709leading to analysis section712. In some embodiments, the magnet722is strong enough to hold cells724at the bottom of well720until the magnetic force is reduced.

In another embodiment, the magnet722is not an electromagnet, but rather a permanent magnet. In this embodiment, the magnetic force due to magnet722may be overcome simply by the force of the sample fluid flow including cells724into channel709such that the cells724will exit the sample well720. In other embodiments, the physical location or magnetic strength of magnet722is altered to reduce or eliminate the magnetic force on cells724a sufficient amount so that the cells724will flow out of well720into channel709leading to analysis section712. In some embodiments, the magnet722is strong enough to hold cells724at the bottom of well720until the magnetic force is reduced.

Additionally, sample well720may optionally include input and waste ports730and732, respectively, for the introduction and removal of a wash fluid. The wash fluid may remove unwanted or undesirable material from the sample well prior to entry into the cytometry analysis. In such embodiments, as the wash fluid travels through sample well720via ports730and732, the wash fluid may force, push or direct the material in the biological sample that is not attracted to electromagnet722out through waste port732. In certain embodiments, the non-magnetized material from the biological sample will remain suspended in sample fluid and the force of the wash fluid moving through sample well720will cause the non-magnetized material to exit the well. Accordingly, the non-magnetized material is prevented from entering the cytometry analysis, thereby providing an initial step of sample purification prior to analysis and sorting. In such embodiments, the electromagnetic force pulling the cells724toward bottom surface720aof well720is greater than the force of the wash fluid moving through well720such that the cells724are not washed out through waste port732.

After the separated cells724are analyzed in section712, the cells may travel through channel713and optionally be sorted into the different wells or chambers714based on differing characteristics of the cells. In certain embodiments, the sample wells714have outlet ports (not shown) in fluid communication therewith in order to facilitate removal of the sorted sample from the wells. Sample fluid may be diverted to wells714by appropriate control of flow diverter750.

In one embodiment, the flow diverter750is a piezoelectric device that can be actuated with an electric command signal in order to mechanically divert the flow through the sorting channel713into either the well214aor the well214b, depending upon the position of the flow diverter750. In other embodiments, flow diverter750is not a piezoelectric device, but instead can be, for example, an air bubble inserted from the wall to deflect the flow, a fluid deflector moved or actuated by a magnetic field or any other flow diverter or sorting gate as would occur to one of ordinary skill in the art.

Cells may be sorted into different wells or chambers714based on the intended future use for the cells. For example, cells having the same characteristics, or phenotype, may be sorted into one well where they are fixed for viewing, and sorted into another well where they are maintained in a viable state to undergo additional functional measurements. As another example, desirable cells may be sorted into an extraction well or chamber and undesirable cells may be sorted into a waste well or chamber. Alternatively, the cells may be deposited into the wells or chambers based on volume as opposed to a sorting method. The chip may include means for physically diverting the cells into the chambers714from the analysis section712as is known in the art. Alternatively, the cells may be caused to exit the chip702after the analysis is complete.

By initiating a pre-analysis and pre-sorting separation method, system700allows the researcher or medical professional to increase the purity of the finally sorted cells as opposed to systems which provide only for analysis and sorting. This can be especially advantageous when sorting desirable stem cells through cytometry analysis because of the generally low concentration of stem cells in a biological sample and their high desirability.

The sample well720may take any convenient physical form, such as a well formed into the surface of substrate702which may be closed, may remain open and/or may include a cover. The sample well720is shown as being positioned near the top of the chip; however, it should be appreciated that the sample well may be positioned elsewhere on the substrate.

Stackable Microfluidic Devices

In certain embodiments, the present disclosure is generally directed to stackable microfluidic devices. Providing microfluidic devices in a stackable arrangement can help to protect the devices themselves from scratching, abrasion or other unwanted irritation. Stacking the microfluidic devices according to the present disclosure also helps to prevent the microfluidic devices from sticking together by separating the flat front and back surfaces of the devices. Furthermore, the embodiments disclosed herein facilitate stacking of microfluidic devices that have one or more non-flat surface. For example, one surface may have a lens attached thereto, portions of which extend out of the plane of the substrate surface. Additionally, the stackable arrangement may make it easier for a researcher, medical professional or other user to grip and manipulate the devices. It further may facilitate the use of automation and/or robotic elements to handle the microfluidic devices.

FIG. 8schematically illustrates microfluidic devices, such as cytometry chips802, in a stacked arrangement800. The cytometry chips802may be generally designed so that material from a biological sample may be analyzed thereon via cytometry (the specific operations that occur in the cytometry analysis are not critical to the present disclosure). In certain embodiments, the cytometry analysis may include image cytometry or flow cytometry, as examples. According to the results of the analysis performed, the material in the sample may optionally be sorted into one or more different wells or chambers disposed on the chips802. Alternatively, the cells may be caused to exit the chips802after the analysis is complete. It should be appreciated that the various cytometry components and sections of the chips802are not illustrated for simplicity and can vary greatly as would occur to one of ordinary skill in the art.

In certain embodiments, the cytometry analyses may occur with respect to front or top surfaces802aof the chips802and legs804may be attached to the back or bottom surfaces802bof the chips802to allow the chips to be stacked on top of each other. In the illustrated embodiment, each chip802is generally rectangular in shape and includes four legs804extending down from surface802band positioned near the four corners of the device, as best illustrated inFIG. 9. However, it should be appreciated that the legs804can number more or less than four on each chip802and/or can be positioned at other locations on the chip802as would occur to one of ordinary skill in the art. In certain embodiments, the legs804are positioned at certain locations on each chip802so that they do not interfere with any of the cytometry components or sections on surface802aof an adjacent chip802when in the stacked arrangement800.

In the illustrated embodiment, each leg804is attached to or integral with surface802bat one end and includes a widened foot portion805at the other end. Additionally, the illustrated legs804are substantially cylindrical with circular cross-sectional shapes. Each foot portion805includes a larger diameter than the remainder of leg804to provide additional stability to the stacking arrangement800. However, it should be appreciated that the chips802and/or the legs804can be shaped differently than as illustrated. As an example, foot portions805may be absent such that the legs804have a constant diameter throughout their lengths. As another example, legs804may include cross-sectional shapes that are square or rectangular. For example, in an alternative embodiment the chips1002may each include a single rectangular foot1004at opposite ends thereof, as illustrated inFIG. 10. It should be appreciated that the legs804,1004are just two non-limiting examples of numerous possible stacking elements as would occur to one of ordinary skill in the art that can be incorporated into a microfluidic device.

Microfluidic Device Having 3-D Hydrodynamic Focusing to Align Sperm Cells

In certain embodiments, the present disclosure is generally directed to a microfluidic device capable of 3-D hydrodynamic focusing to align sperm cells in a flow channel in the device. The device includes sheath fluid sub-channels joining with a sample cytometry channel having sperm cells traveling therein, the sub-channels being positioned in two different planes to three-dimensionally align the sperm cells in the sample channel in the proper orientation for a cytometry analysis.

FIG. 11schematically illustrates a microfluidic device1100formed on a substrate1102on which cells, such as sperm cells, from a biological sample (not shown) input to input port1110are aligned in section1120(as will be described in greater detail below) and analyzed via cytometry (such as, for example, flow cytometry or image cytometry) in analysis section1112(the specific operations that occur in analysis section1112are not critical to the present disclosure). According to the results of the analysis performed, the cells may optionally be sorted into one or more different wells or chambers1114. In certain embodiments, the sample wells1114have outlet ports (not shown) in fluid communication therewith in order to facilitate removal of the sorted sample from the wells. Sample fluid may be diverted to wells1114by appropriate control of flow diverter1116.

In one embodiment, the flow diverter1116is a piezoelectric device that can be actuated with an electric command signal in order to mechanically divert the flow through the flow channel into either of the wells1114, depending upon the position of the flow diverter1116. In other embodiments, flow diverter1116is not a piezoelectric device, but instead can be, for example, an air bubble inserted from the wall to deflect the flow, a fluid deflector moved or actuated by a magnetic field or any other flow diverter or sorting gate as would occur to one of ordinary skill in the art.

It should be appreciated that various components and sections shown on the chip1100are intended to show the operations of the cytometry process in a simple schematic form and the cytometry components and sections on the chip1100can vary greatly as would occur to one of ordinary skill in the art.

The cells may be sorted into the different chambers1114based on differing characteristics of the cells. Cells may be sorted into the different chambers1114based on the intended future use for the cells. For example, cells having the same characteristics, or phenotype, may be sorted into one chamber where they are fixed for viewing and sorted into another chamber where they are maintained in a viable state to undergo additional functional measurements, or properly stored for use as part of a cell-based therapeutic procedure. In a specific embodiment, sorted and isolated sperm cells may be used for artificial insemination or in-vitro fertilization for both humans and animals. Such medical procedures may be used in cases of infertility and to prevent sex-linked gene-propagated diseases from passing to a new generation. As another example, desirable cells may be sorted into an extraction well or chamber and undesirable cells may be sorted into a waste well or chamber. Alternatively, the cells may be deposited into the chambers1114based on volume as opposed to a sorting method. Alternatively, the cells may be caused to exit the chip1100after the analysis is complete.

For simplicity, the illustration ofFIG. 11shows two chambers1114; however, it should be appreciated that the microfluidic device may include more or less than two chambers as would occur to one of ordinary skill in the art. Additionally, the chambers1114are shown as being horizontally aligned near the bottom of the substrate1102. However, it should be appreciated that the chambers1114, if present, may be positioned at other locations on the substrate1102as would occur to one of ordinary skill in the art. Additionally, for simplicity and ease of illustration,FIG. 11shows single channels extending between the components, areas or sections of substrate1102. However, it should be appreciated that the single channels may be representative of multiple cytometry channels and a variety of possible configurations of channels as would occur to one skilled in the art.

FIGS. 12A and 12Bshow an example sperm cell1104which may be introduced into input port1110for cytometry analysis, withFIG. 3Ashowing a top view andFIG. 3Bshowing a side view. In certain embodiments, the sperm cell1104includes a head1105and a tail1106. As illustrated, the heads of some sperm cells are typically pancake-shaped with their width W larger than their thickness T. For the purposes of cytometry analysis, it can be beneficial to analyze the cell from the top, as it is oriented inFIG. 3A, as more information can be gained because more of the cell via its width W is available for analysis.

Accordingly, it can be desirable to orient the sperm cell1104such that the full width W is available for analysis prior to entry into the cytometry analysis section1112. This orientation provides the most uniform illumination of the cell1104as it passes through the analysis section1112of the device. For most mammalian species, this orientation is necessary to obtain the precision needed to measure the DNA difference between sperm cells that will produce females and sperm cells that will produce males. To accomplish this, chip1100can include a hydrodynamic focusing section, such as alignment section1120, to properly orient the sperm cell1104(seeFIG. 13). Section1120may be operable to align sperm cells entering sample channel1122from sample injection tube1132, the injection tube being coupled with biological sample1110. As illustrated, section1120includes sheath entry sub-channels1124and1126in the same plane, such as plane Y, and sheath entry sub-channels1128and1130in the same plane, such as plane X which is orthogonal to plane Y, to properly align and orient sperm cells flowing through sample channel1122. The sheath entry sub-channels are configured such that sheath fluids from each sub-channel into the sample channel1122work in combination to align and orient the sperm cells in the channel1122. In the particular illustrated embodiment, sub-channels1124and1126are larger in cross-sectional dimension than sub-channels1128and1130such that a greater amount of sheath fluid may enter channel1122from those locations than the amount of sheath fluid which enters from sub-channels1128and1130. Accordingly, in the particular illustrated embodiment, the sheath fluid will cause the sperm cells to be aligned with their larger width dimension generally in the X plane and their smaller thickness dimension generally in the Y plane. Additionally, the sheath flow sub-channels1124,1126,1128, and1130may also function to position the sperm cells at the center of the channel1120to better ensure that the cells pass through the focus of the optic system that may be part of the cytometry analysis.

The proper orientation is enhanced by the used of the tube1132which has a beveled end, the flat surfaces of the beveled end being aligned with or facing the incoming sheath flow entering from sub-channels1124and1126. This system will produce laminar flow between the core sample stream of sperm cells emerging from the end of the beveled tube1132and the sheath fluid passing over the end of the tube1132. The core sample stream will remain asymmetrical, having a greater width measured in the x direction than height measured in the y direction, as it passes through the analysis section1112of the chip1100. This asymmetry will enable the sperm cells to remain properly oriented as they pass through the analysis section1112.

It should be appreciated that the section1120may be an integral portion of substrate1102or may be attached to the substrate1102in an appropriate manner as would occur to one of ordinary skill in the art. In alternative embodiments, section1120may be separate from the substrate1102, with a channel connecting the output of section1120with a flow channel on the substrate1102.

Microfluidic Device Having Acoustic Energy Coupler

Certain embodiments of the present disclosure are generally directed to a microfluidic device having an acoustic energy coupler positioned within a component of the microfluidic device to disrupt the cells contained in the component. Disruption of the cells may cause the internal molecular material within the cells to be released so that the material may be observed or tested by a researcher or medical professional. In certain embodiments, the cells are sorted into a container following a cytometry analysis, such as flow or image cytometry as non-limiting examples.

FIG. 14schematically illustrates a microfluidic device1400formed on a substrate1402, on which cells or other material from a biological sample (not shown) are input to input port1410are analyzed via cytometry (such as, for example, flow cytometry or image cytometry) in analysis section1412(the specific operations that occur in analysis section212are not critical to the present disclosure). According to the results of the analysis performed, the cells may optionally be sorted into one or more different wells or chambers1414. In certain embodiments, the sample wells1414have outlet ports (not shown) in fluid communication therewith in order to facilitate removal of the sorted sample from the wells. Sample fluid may be diverted to wells1414by appropriate control of flow diverter1415.

In one embodiment, the flow diverter1415is a piezoelectric device that can be actuated with an electric command signal in order to mechanically divert the flow through the flow channel into either of the wells1414, depending upon the position of the flow diverter1415. In other embodiments, flow diverter1415is not a piezoelectric device, but instead can be, for example, an air bubble inserted from the wall to deflect the flow, a fluid deflector moved or actuated by a magnetic field or any other flow diverter or sorting gate as would occur to one of ordinary skill in the art.

It should be appreciated that the various components and sections shown on the substrate1402in the illustration ofFIG. 14are intended to show the operations of the cytometry process in a simple schematic and the cytometry components and sections on the substrate1402can vary greatly as would occur to one of ordinary skill in the art.

The cells may be sorted into the different wells or chambers1414based on differing characteristics of the cells. Cells may be sorted into the different chambers1414based on the intended future use for the cells. For example, cells having the same characteristics, or phenotype, may be sorted into one chamber where they are fixed for viewing and sorted into another chamber where they are maintained in a viable state to undergo additional functional measurements, or properly stored for use as part of a cell-based therapeutic procedure. As another example, desirable cells may be sorted into an extraction well or chamber and undesirable cells may be sorted into a waste well or chamber. Alternatively, the cells may be deposited into the chambers1414based on volume as opposed to a sorting method. The chip may include means for physically diverting the cells into the chambers1414from the analysis section1412as is known in the art. Alternatively, the cells may be caused to exit the substrate1402after the analysis is complete.

For simplicity, the illustration ofFIG. 14shows two chambers1414; however, it should be appreciated that the microfluidic device may include more or less than two chambers as would occur to one of ordinary skill in the art. Additionally, the chambers1414are shown as being horizontally aligned near the bottom of the substrate1402. However, it should be appreciated that the chambers1414, if present, may be positioned at other locations on the substrate1402as would occur to one of ordinary skill in the art. Additionally, for simplicity and ease of illustration,FIG. 14shows single channels extending between the components, areas or sections of substrate1402. However, it should be appreciated that the single channels may be representative of multiple cytometry channels and a variety of possible configurations of channels as would occur to one skilled in the art.

According to the present disclosure, at least one component on the chip1400includes an acoustic energy coupler to disrupt cells contained within the component. To illustrate an example,FIG. 15shows an acoustic energy coupler1416in the form of a probe positioned within a chamber1414. However, it should be appreciated that the acoustic energy coupler may be positioned in other components of the chip1400, including an input well or other cell collection vessel on the chip. As illustrated, acoustic energy coupler1416vibrates or oscillates in response energy received from acoustic energy source1420. It should be appreciated that coupler1416may be sized, shaped and/or otherwise configured differently and/or orientated differently in chamber1414than as illustrated as would occur to one of ordinary skill in the art.

In certain embodiments, sample fluid having cells suspended therein is collected within chamber1414. Acoustic energy coupler1416is operable to vibrate within the sample fluid in response to acoustic energy that it receives from source1420to disrupt the cells. The acoustic energy transferred to coupler1416may be in the form of sound energy (such as, for example, ultrasonic energy) so that a process of sonication may be applied to the sample fluid to agitate the cells therein. In some embodiments, coupler1416is operable to vibrate or oscillate at its resonance frequency (corresponding to its maximum amplitude) to provide optimum disruption to the cells.

In certain embodiments, the disruption to the cells is of sufficient intensity to break the cell members and release the inner molecular material of the cells into the sample fluid. The inner molecular material can then be observed or tested by a researcher or medical professional. Additionally, in the illustrated embodiment the cells are disrupted following the cytometry analysis and the sorting of the cells into the chamber1414. However, it should be appreciated that the coupler can be placed in a different chip component which houses cells prior to the cytometry analysis, such as an initial cell repository. After the cells are disrupted and the molecular material is released in the initial cell repository, the molecular material can be caused to enter section1412for cytometry analysis.

In certain embodiments, acoustic energy source1420is separate from coupler1416and may be incorporated with the external instrument system conducting the cytometry analysis with respect to the biological sample on the chip1400. In other embodiments, the acoustic energy source1420may be separately applied to the chip1400following completion of the cytometry analysis and removal of the chip1400from the external system conducting the analysis.

Microfluidic Device Having Phase Variation Producing Lens

Certain embodiments of the present disclosure are generally directed to systems for the analysis of a sample on a microfluidic device such as a cytometry chip, using cytometry (such as flow cytometry or image cytometry). In order to detect or identify cells during cytometry operations, a source (such as a laser) of electromagnetic radiation (such as visible light) is directed to a detection area. When a cell passes through the detection area within a flow channel, the light source causes the cell to fluoresce. This effect may be enhanced by certain dyes which are added prior to the cell reaching the detection area as described hereinabove.

In certain applications, it may be necessary to incorporate various optical elements into the substrate of the device or chip to vary the light-scattering nature of the various surfaces. In particular, light-shaping optics such as holographic elements or free-form lenses and mirrors may be employed to deliver intensity/phase/polarization spatial patterns or other light shifting effects on various surfaces of the chip. Because such elements can be formed or etched into the chip substrate, there is little cost to reproduce them once the initial mold or etching program is designed. More particularly, the lens used to focus an incoming excitation light beam may also be manufactured such that it will also deliver particular spatial light pattern within the channel detection region.

FIG. 16schematically shows one embodiment where a focusing lens1602is mounted to the surface of a microfluidic device1600substrate1604. As a sample cell1606passes through the detection flow channel1608, the excitation light1610is focused on the sample cell1606. The surface1612of the focusing lens1602has been formed, etched, or otherwise treated such that it will impart spatially varied phase changes on the incoming excitation light1610. The phase changes translate into an optical holographic effect on the detection region, which can be made to form a specific spatial pattern or design.

During manufacture of the focusing lens1602, the surface1612can be coated with a UV sensitive material. A pulsed laser can then be used to scan the surface1612to expose the UV material and create small variations in the surface according to a desired pattern or image. When the focusing lens1602is later mounted to the substrate1604and receives the excitation light1610, it will create a holographic effect within the detection channel1608. In certain embodiments, the focusing lens1602may be made entirely of the UV sensitive material, as opposed to merely the surface1612. In still other embodiments, the variations on the surface1612may achieved by injection molding or other means known in the art for reproducing holographic effects. In a similar fashion to holographic elements, other types of free-form lens and mirror shapes can also be combined with focusing lens1602by first constructing a master copy using micro-machining techniques known in the art such as diamond turning from a computer designed shape. This master copy can then be replicated using injection molding or other means known in the art.

The specific pattern or desired holographic effect may be selected for various uses. As one non-limiting example, the holographic lens or free-form optical element can be used to create spatially varied patterns to determine a unique “signature” for a detection channel. In order to increase overall cell throughput, multiple flow channels are often used in parallel to measure and/or sort cells in a cytometry device, such as a droplet cell sorter or a microfluidic device. In such systems, the detection optics and associated electronic processing equipment must be able to distinguish between signals received from the individual channels. One way to accomplish this is to direct a single excitation light source to all of the channels simultaneously, but spatially vary or shape the excitation beam pattern so that a different excitation pattern is directed to the detection region of each channel. As a cell passes through the beam pattern of a particular channel, a series of time-spaced peaks will be created in the emitted fluorescence signal. The phase variation lens discussed above can be used to create a holographic effect in this situation. The holographic effect transforms the uniform beam into unique spatial patterns within the detection regions of each channel.

Microfluidic Device Having Transmissive and Reflective Lenses

In prior art systems, the light source is applied to one side of the cytometry device, with the detection optics also only detecting the fluorescence from one side of the sample cell. A transmissive lens or other optic is often used to focus a portion of the emitted photons into the detection optics, however this still limits the amount of photons that are captured from the fluorescent sample cell, since the photons emitted in the opposite direction are not captured by the transmissive lens. The excitation light intensity may be increased to generate more fluorescently generated photons, however this also increases the amount of noise in the signal. Another solution is to use multiple sets of detection optics placed at different locations, although this adds significant expense.

In order to capture as many of the photons generated by a fluorescing sample cell as possible with a single detection optic, a reflective lens may be placed on the side of the sample cell opposite the detection optics. The reflective lens will capture a large portion of the photons not captured by the transmissive lens, reflect them back through the transmissive lens, and into the detection optics. This allows the system to capture more of the emitted photons for a given excitation light intensity without increasing the noise in the received signal, and without the cost of multiple detection optics.

FIG. 17schematically illustrates a system1700for analyzing samples using cytometry. The system1700may comprise a microfluidic cytometry device formed on substrate1702(shown here from a side view) having a detection flow channel1704contained therein. For simplicity and ease of illustration,FIG. 17shows a single channel within the substrate1702. However, it should be appreciated that the single channel may be representative of multiple cytometry channels and a variety of possible configurations of channels as would occur to one skilled in the art. Various other cytometry components may also be contained on the substrate1702but are not critical to the present disclosure.

The system1700may further comprise a transmissive lens1706, reflective lens1707, excitation light source1710, dichroic mirror1712and detection optics1714. The transmissive and reflective lenses1706and1707may be mounted to or formed integrally within the substrate1702as illustrated inFIG. 17, or may be placed outside of the substrate1702depending the particular application and cost considerations. The excitation light source1710may comprise a laser or other light source known in the art. The detection optics1714may comprise various light sensing means known in the art, such as photomultiplier tubes.

In operation, excitation light source1710directs a light beam towards dichroic mirror1712. The dichroic mirror is configured to reflect the wavelength received from the excitation light source1710to the detection area1716within detection channel1704. When a sample cell1718passes through the detection area1710and fluoresces, the generated photons are emitted in many directions. Some photons will be emitted back toward the focusing lens1706, pass through the dichroic mirror1712(which is configured to pass the fluorescent wavelength), and sensed by the detection optics1714. Photons that are emitted from the opposite side of chip1702will be captured by reflective lens1707, directed back to transmissive lens1706, and be focused into the detection optics1714.

The relative distance between the transmissive lens1706, the detection channel1704, and the reflective lens1707is set so that the light reflected from the reflective lens1707is also properly focused by the transmissive lens1706into the detection optics1714. For example, the reflective lens1707may need to be closer to the detection channel1704. When the transmissive and reflective lenses1706and1707are mounted directly on the substrate1702, this can be accomplished by offsetting the detection channel1704as shown inFIG. 17to ensure proper placement of the transmissive and reflective lenses relative to the detection channel1704.

Microfluidic Device Having Integrally-Formed Optics

Various optical devices, such as lenses, are often placed in the path of the incoming and/or outgoing light beams in a microfluidic device to provide the maximum photon delivery and recovery to and from the sample cell being observed. Such optical devices are also often needed to change the numerical aperture (NA) of the light emitted from the detection region so that it may be properly received by a fiberoptic cable. This is necessary because the NA of the emitted light is usually very high, while the NA of a fiberoptic cable is usually very low. If the NA of the emitted light is not reduced prior to reaching the fiberoptic cable, only a small portion of the emitted light will be successfully propagated through the fiberoptic cable.

One problem, however, is that the various incoming and outgoing light beams will be subject to refraction as they pass through various materials in the beam path. For example, when a focused excitation beam passes from the air into the substrate of a microfluidic chip (which has a normally flat surface), the beam will refract to some degree, making it more difficult to focus the beam on the intended detection region within the chip. To overcome this problem, the focusing lens or other optic can be formed into the chip substrate itself. Because the air gap between the focusing optic and the chip substrate is eliminated, and because the focusing optic and chip substrate are formed from and remain part of the same piece of material, unwanted refraction of the beam within the chip material is eliminated.

FIG. 18illustrates a system1800for analyzing samples using cytometry. The system1800may comprise a microfluidic device substrate1802(a side view section of which is shown in the figure) having a detection channel1804contained therein. For simplicity and ease of illustration,FIG. 18shows a single channel within the cytometry chip1802. However, it should be appreciated that the single channel may be representative of multiple cytometry channels and a variety of possible configurations of channels as would occur to one skilled in the art. Various other cytometry components may also be contained on the cytometry chip1802as described hereinabove but are not critical to the present disclosure.

The substrate1802further comprises an optic1806, shown here as a focusing lens. The optic1806is integrally formed into the substrate of the substrate1802. This can be accomplished by various methods known in the art. For example, the optic1806can be formed into an injection mold which is used to produce the substrate1802. In other embodiments, the optic1806can be machined into or otherwise shaped out of the chip substrate after the basic chip shape is formed.

In operation, excitation light source1810directs a light beam towards dichroic mirror1812. The dichroic mirror1812is configured to reflect the wavelength received from the excitation light source1810to the detection region1814within detection channel1804. Various other optical components known in the art may be used to direct the excitation light beam to the optic1806, the details of which are not critical to the present disclosure. When a sample cell1816passes through the detection region1814and is caused to fluoresce by the excitation light beam, the generated photons will be collected or focused by the optic1806, pass through the dichroic mirror1812(which is configured to pass the fluorescence wavelength), and be finally detected by the detection optics1816. The detection optics1816may comprise various light sensing means known in the art, such as photomultiplier tubes. In certain embodiments, other components known in the art, such as fiber optic cables, may be employed to route the emitted light beams to the detection optics1816.

It shall be understood that the optic1806may comprise any optical element for focusing, spreading, filtering, or otherwise processing the light beams received by or generated from the sample cell1816within the detection region1814. In addition, multiple optical elements may be mounted on the substrate1802. As one non-limiting example, an additional optic1818with a reflective coating may be formed into the opposite surface1820of the substrate1802to direct additional photons back to the detection optics1816. In certain applications, it may be advantageous to form the optic1818as in intrusion (as opposed to a protrusion) into the surface1820, as illustrated inFIG. 18. This allows the surface1820of the substrate1802to be placed flush against a corresponding flat surface within a mounting device (not shown).

Microfluidic Device Having a Non-Integral Geographically Selective Reagent Delivery Structure

In many cases, a reagent may need to be added to activate or otherwise prepare the sample before the cytometry analysis is executed. The required reagent may be added to the microfluidic device during manufacturing, eliminating the need for the user to add it at the time of use. However, this requires that many different types of devices will need to be kept in inventory by the manufacturer, with each device adapted for use in only a small number of applications. In order to increase manufacturing efficiency and reduce inventory cost, the reagent may be formed into a separate structure which can be mated with the microfluidic device after manufacturing. This allows the manufacturer to produce a single microfluidic device which can accept a number of different reagent delivery structures.

FIG. 19illustrates a microfluidic device1900which is configured to receive a separate reagent structure1902. The device1900may comprise a cytometry analysis portion1904that is connected to a reagent receiving portion1906by microfluidic flow channels1908. The device1900also comprises a sample receiving port1910for receiving a sample to be analyzed. Sample receiving port1910is connected to the cytometry analysis portion1904by microfluidic flow channel1912. In certain embodiments, the reagent delivery structure1902is mated to the reagent receiving portion1906before a sample is added to the device1900. This mating can be performed by the user at the time of use or by the manufacturer prior to shipping the device1900. The cytometry analysis portion1904performs cytometry analysis on the received sample and reagent mixture. For example, the cytometry analysis section1904may sort desirable cells into an extraction well1914and undesirable cells into a waste well1916. The specific operations that occur in the cytometry analysis section1904and the specific routing of the microfluidic channels are not critical to the present disclosure.

The reagent receiving portion1906is manufactured with a plurality of receiving wells1918arranged in a grid or other standardized layout. Various reagent delivery structures1902can then be manufactured which have reagent portions1920containing reagents at locations that will correspond to at least one of the receiving wells1918when the reagent delivery structure1902is mated to the reagent receiving portion1906. In certain embodiments, the location of specific reagent types within the reagent delivery structure1902will also be standardized. For example, any reagent at the location corresponding to a specific receiving well1918will always be of the same type. Because of this, the equipment used to apply reagents to the reagent delivery structure1902during manufacturing can be optimized to always apply a certain reagent type to the same location, if that reagent is being applied to the particular reagent delivery structure1902. This also allows the cytometry analysis section1904and the various microfluidic flow channels1908to be designed and optimized based on a known location of the required reagent.

The reagent delivery structure1902may be made of any material known in the art which is suitable for storing reagents. For example, the reagent structure1902may be in the form of a tape or other flexible strip, with an adhesive applied to hold the reagent structure1902in place once applied to the reagent receiving portion1906. In certain embodiments, the reagent receiving portion1906may be recessed into the device1900, with reagent delivery structure1902being made of a rigid material and sized to fit snugly within the reagent receiving portion1906, ensuring proper placement of the reagent delivery structure1902. The receiving wells1918may also comprise pins which will pierce the reagent portions1920to release or expose the reagents contained within reagent portions1920into receiving wells1918and channels1908.

FIG. 20shows another embodiment where the reagents2020contained on reagent structure2002are held within the structure2002by a soluble barrier2040. An activation material2045is included on the reagent receiving portion2006within the device2000. Once the reagent structure2002is applied to the reagent receiving portion2006, the activation material2045will cause the soluble barrier2040to dissolve, releasing the reagents2020into the reagent receiving wells2018and flow channels2008. In certain embodiments, focused laser beams or acoustic energy applied from an external source may be used to cause the soluble barrier2040to dissolve. In still further embodiments, the soluble barrier2040and/or the activation material2045can be made of a temperature sensitive material, whereby increasing the temperature after mating the reagent structure2002to the device2000will cause the soluble barrier2040to dissolve. Various materials are known in the art which will exhibit this temperature sensitive behavior. For example, the reagents can be provided in agarose, which will preserve the cells contained within the reagent until the agarose is dissolved by heating. In certain embodiments, the soluble barrier2040may incorporate micro electrical mechanical structure (MEMS) valves.

Microfluidic Device Incorporating Optical Waveguide in Flow Channel

Certain embodiments of the present disclosure are generally directed to microfluidic devices, such as cytometry chips, having cytometry channels configured as light waveguides to better direct illumination through the channels. In certain embodiments, conforming a cytometry channel into a light waveguide involves choosing materials having desirable indexes of refraction to form the channel walls. Additionally, the effectiveness of the light waveguide channel may also depend on the index of refraction of the fluid passing through the channel. While the waveguide channel according to the present disclosure is discussed herein as directing visible light for sake of brevity, light is just one non-limiting example of numerous possible types of electromagnet radiation which can be directed by the waveguide channel.

FIG. 21schematically illustrates a system2100having an example cytometry channel2102on a microfluidic device as part of a cytometry analysis (such as, for example, flow cytometry or image cytometry) occurring with respect to the device (the specific cytometry analysis operations are not critical to the present disclosure). As illustrated, channel2102has a corner2103creating two substantially perpendicular sections,2102aand2102b. System2100includes a light emitting system2106and a light collection system2107along channel section2102a, the light being used to illuminate cells for detection as part of the cytometry analysis. In the illustrated example, light collection system2107is positioned with its optical axis aligned with the axis of flow through channel section2102aand light emitting system2106is positioned with its optical axis orthogonal to the axis of flow through channel section2102. It should be appreciated that the specific configurations, positions and operations of systems2106and2107must correctly couple radiation into the system but are not critical to the present disclosure, the systems being appropriate systems as would occur to one of ordinary skill in the art. The general flow of a biological sample through channel2102is illustrated by large-head arrows2120within the channel. The light emitted from system2106is internally reflected throughout the channel2102to collection system2107and is generally represented by arrows2122. As such, channel2102may act as a light waveguide.

Channel2102receives illumination from light emitting system2106and transmits it along the length of channel segment2102ato be emitted out toward light collection system2107. To accomplish this, the channel wall2104of the channel2102may include an appropriate index of refraction along at least segment2102ato internally reflect the light as it travels through section2102a. Additionally, the channel wall2104may be masked (for example, with a reflective coating) along all or portions of its length of section2102aso the light is emitted only in desired locations. This arrangement can allow for the channel to only emit light in desired areas, which can be either a continuous or multiple point emission.

In certain embodiments, channel2102may include a wall section2108aligned with light emitting system2106and a wall section2109aligned with light collection system2107to allow for light emission into and out of the channel. Additionally, in certain embodiments, the wall sections2108and2109may be constructed of a different material having a different index of refraction from the remainder of wall2104, the different material and index of refraction being designed to allow for light emission therethrough. As another example, in embodiments in which channel wall2104is masked, the wall sections2108and2109may remain unmasked so that light may pass through.

Microfluidic Device Incorporating Optical Waveguide with Reflective Surface in Flow Channel

In certain embodiments, a reflective surface can be positioned at one end of the light waveguide channel to further assist in directing the illumination.FIG. 22schematically illustrates a system2200having an example cytometry channel2202with a channel wall2204on a microfluidic device as part of a cytometry analysis (such as, for example, flow cytometry or image cytometry) occurring with respect to the device (the specific cytometry analysis operations are not critical to the present disclosure). As illustrated, channel2202has a corner2210between substantially perpendicular channel sections2220aand2220b, and a corner2203between substantially perpendicular sections2202band2202c. Additionally, channel section2202bgenerally includes a first end2240adjacent corner2210and a second end2242adjacent corner2203. System2200includes a light emitting system2206and a light collection system2207along channel section2202b, the light being used to illuminate cells for detection as part of the cytometry analysis. In the illustrated example, light collection system2207is positioned with its optical axis aligned with the axis of flow through channel section2202band light emitting system2206is positioned with its optical axis orthogonal to the axis of flow through channel section2202b. It should be appreciated that the specific configurations, positions and operations of systems2206and2207must correctly couple radiation into the system but are not critical to the present disclosure, the systems being appropriate systems as would occur to one of ordinary skill in the art.

Additionally, a reflective surface such as mirror2230having a mirror surface2232is positioned adjacent first end2240generally opposite the light collection system2207. As illustrated, mirror2230may be integral with the channel wall2204. The mirror could be spherical in shape as shown, or it could have other shapes suitable for reflecting emissions coupled from the channel waveguide back down the channel2202. In other embodiments, the mirror2230could be a deformable mirror, generally referred to in the field as an adaptive optic. Such a mirror can have its shape adjusted rapidly under the control of a feedback system.

The general flow of a biological sample through channel2202is illustrated by large-head arrows2220within the channel. The light emitted from system2206is internally reflected throughout the channel2202to collection system2207and is generally represented by arrows2222. As such, channel2202may act as a light waveguide.

Channel2202receives illumination from light emitting system2206and transmits it along the length of channel segment2202bto be emitted out toward light collection system2207. To accomplish this, the channel wall2204of the channel2202may include an appropriate index of refraction along at least section2202bto internally reflect the light as it travels through section2202b. Additionally, the channel wall2204may be masked (for example, with a reflective coating) along all or portions of its length of section2202bso the light is emitted only in desired locations. This arrangement can allow for the channel to only emit light in desired areas, which can be either a continuous or multiple point emission. Further, spherical mirror surface2232is positioned to reflect any portion of the light which may reach mirror2230at end2240back toward light collection system2207at end2242. The use of the mirror2230improves the efficiency and effectiveness of the light waveguide channel by preventing light emitted from system2206from being transmitted out end2240opposite from system2207.

In certain embodiments, channel2202may include a wall section2208aligned with light emitting system2206and a wall section2209aligned with light collection system2207to allow for light emission into and out of the channel. Additionally, in certain embodiments, the wall sections2208and2209may be configured of a different material having a different index of refraction from the remainder of wall2204, the different material and index of refraction being designed to allow for light emission therethrough. As another example, in embodiments in which channel wall2204is masked, the wall sections2208and2209may remain unmasked so that light may pass through.

Microfluidic Device for Virus Detection and Sorting

Certain embodiments of the present disclosure is generally directed to detecting viral particles in a bodily sample by subjecting the sample to a cytometry analysis occurring on a microfluidic device, such as a cytometry chip, and sorting the viral particles into wells or chambers disposed on the chip. The cytometry analysis may be a flow cytometry analysis or an image cytometry analysis. Sorting the viral particles from the bodily sample allows the researcher or medical professional to capture the viral population for further observation or testing. As used herein, the term cytometry is used broadly and meant to include the measurement of any appropriate material, including cells and/or viral particles as two non-limiting examples

System2300, as schematically illustrated inFIG. 23, includes a microfluidic device formed on a substrate2302which allows for the detection of viral particles during the cytometry analysis and sorting of the viral particles following the analysis. As part of the system2300, a bodily sample (not shown) is input to input port2310is analyzed via cytometry on substrate2302in analysis section2312(the specific operations that occur in analysis section2312are not critical to the present disclosure). According to the results of the analysis performed, viral particles in the bodily sample input to input port2310which were detected in analysis section2312may be sorted into one or more chambers2314. In certain embodiments, the sample wells2314have outlet ports (not shown) in fluid communication therewith in order to facilitate removal of the sorted sample from the wells. Sample fluid may be diverted to wells2314by appropriate control of flow diverter2316.

In one embodiment, the flow diverter2316is a piezoelectric device that can be actuated with an electric command signal in order to mechanically divert the flow through the flow channel into any of the wells2314, depending upon the position of the flow diverter2316. In other embodiments, flow diverter2316is not a piezoelectric device, but instead can be, for example, an air bubble inserted from the wall to deflect the flow, a fluid deflector moved or actuated by a magnetic field or any other flow diverter or sorting gate as would occur to one of ordinary skill in the art.

The viral particles may further be sorted into different wells or chambers2314based on the intended future use for the particles. For example, viral particles having the same characteristics may be sorted into chamber2314awhere they are fixed for observation by electron microscopy, and sorted into chamber2314bwhere they are reacted with chemicals that are under study for their pharmacological efficacy, with the remaining sample being sorted into chambers2314cand2314daccording to other criteria. In other embodiments, all of the chambers2314may receive viral particles detected in analysis section2312. The chip2300may include means for physically diverting the viral particles into the chambers2314from the analysis section2312as is known in the art. Alternatively, the material in the bodily sample may be used to exit the chip2300after the analysis is complete.

In the illustrated embodiment there are four illustrated chambers2314; however, it should be appreciated that there may be more or less than four chambers as would generally occur to one skilled in the art. The substrate2302having a plurality of chambers2314may be designed so that a predetermined amount of viral particles is sorted into each chamber. For ease of illustration, the chambers2314are illustrated as being horizontally aligned, but it should also be appreciated that the chambers may be positioned otherwise on the chip as would generally occur to one skilled in the art. Additionally, for simplicity and ease of illustration,FIG. 23shows single channels extending between the components, areas or sections of substrate2302. However, it should be appreciated that the single channels may be representative of multiple cytometry channels and a variety of possible configurations of channels as would occur to one skilled in the art.

The bodily sample input at input port2310may be a blood sample, a urine sample, a tissue sample, a saliva sample, a cell sample, or combinations thereof, just to name a few non-limiting examples. In addition to detecting and sorting the presence of any viral particles in the bodily sample, system2300may also be configured to measure the number of viral particles in the sample. In certain embodiments, the cytometry analysis at section2312is capable of measuring the number of viral particles in the sample. In other embodiments, the viral particles sorted into the chamber(s)2314on the substrate2302and can be measured or counted after sorting.

Certain embodiments of the present disclosure are generally directed to detecting (rather than sorting) viral particles in a bodily sample by subjecting the sample to a cytometry analysis occurring on a microfluidic device, such as a cytometry chip. The cytometry analysis may be a flow cytometry analysis or an image cytometry analysis. Using microfluidic device to detect viral particles via cytometry provides increased safety and decreased risk of exposure to the researcher or medical professional due to the controlled nature of the testing on the device. Additionally, the microfluidic device allows the cytometry analysis to be run at a relatively slow rate, allowing for the small viral particles to be detected. Further, the microfluidic device also allows for simultaneous detection of cells and viral particles via one measurement device. As used herein, the term cytometry is used broadly and meant to include the measurement of any appropriate material, including cells and/or viral particles as two non-limiting examples.

In certain embodiments, the viral particles may be labeled with a specific fluorescence probe that recognizes a specific sequence of molecules in the RNA. The cytometry instrument system may detect the presence of the virus particle based on light scattering and classify the particle based on the intensity and wavelength of the fluorescence emission. In some embodiments, multiple fluorescent probes may be used and multiple viruses may be detected in the same biological sample.

In addition to detecting the presence of any viral particles in the bodily sample input at2310, system2300may also be configured to measure the number of viral particles in the sample. In certain embodiments, the cytometry analysis at section2312is capable of measuring the number of viral particles in the sample. In other embodiments, the viral particles can be sorted into wells or chambers on the substrate2302and can be measured or counted after sorting. In embodiments involving AIDS testing, the number of virus particles per unit of blood in the bodily sample can be measured via the cytometry analysis. Counting the number of virus particles, also referred to the viral load, can be an important measurement in assessing the health of an AIDS patient. If the sample remains on the substrate2302after the analysis is performed at section2312, then the safety of handling the sample is greatly improved, as the entire chip2300may be disposed of like any other contaminated medical device (e.g. like a used syringe).

Microfluidic Device Displaying Color Change to Indicate Use or a Result

Certain embodiments of the present disclosure are generally directed to a microfluidic device, such as a cytometry chip, capable of providing a visual indication following a cytometry analysis to indicate the result of the cytometry analysis. The cytometry analysis may be a flow cytometry analysis, as described above, or an image cytometry analysis. In an example embodiment, the visual indication is a color change resulting from dye flooding the components of the device after the analysis is complete if the analysis produced a positive result.

FIG. 24illustrates a system2400in which cells from a cell supply (not shown) are input to input port2410on substrate2402and are analyzed via cytometry in analysis section2412(the specific operations that occur in analysis section2412are not critical to the present disclosure). According to the results of the analysis performed, the cells may optionally be sorted into different chambers2414based on differing characteristics of the cells. In certain embodiments, the sample wells2414have outlet ports (not shown) in fluid communication therewith in order to facilitate removal of the sorted sample from the wells. Sample fluid may be diverted to wells2414by appropriate control of flow diverter2416.

In one embodiment, the flow diverter2416is a piezoelectric device that can be actuated with an electric command signal in order to mechanically divert the flow through the flow channel into either of the wells2414, depending upon the position of the flow diverter2416. In other embodiments, flow diverter2416is not a piezoelectric device, but instead can be, for example, an air bubble inserted from the wall to deflect the flow, a fluid deflector moved or actuated by a magnetic field or any other flow diverter or sorting gate as would occur to one of ordinary skill in the art.

Cells may be sorted into different wells or chambers2414based on the intended future use for the cells. For example, cells having the same characteristics, or phenotype, may be sorted into one well where they are fixed for viewing, and sorted into another well where they are maintained in a viable state to undergo additional functional measurements. Alternatively, the cells may be deposited into the wells or chambers2414based on volume as opposed to a sorting method. The chip may include means for physically diverting the cells into the chambers2414from the analysis section2412as is known in the art. Alternatively, the cells may be caused to exit the substrate2402after the analysis is complete. For simplicity and ease of illustration,FIG. 24shows single channels extending between the components, areas or sections of substrate2402. However, it should be appreciated that the single channels may be representative of multiple cytometry channels and a variety of possible configurations of channels as would occur to one skilled in the art.

In the particular embodiment illustrated inFIG. 2, a dye may be contained in a dye repository2420and stored for later use on the device. After the cells are analyzed and optionally sorted, and if the analysis produced a positive result as defined by some predetermined criteria, the dye may be allowed to flow out of repository2420and travel through the components of chip2402to provide a visual indication of the positive result via the color of the dye. In the particular illustrated embodiment, the dye may be released from repository2420, flow to and through the sample channel2409, flow through analysis section2412, and flow into optional chambers2414. In other embodiments, the dye may flow from repository2420(which may be opaque such that the dye therein is not visible) to a second non-opaque repository (not shown) in which the dye is visible external to the chip2400. However, it should be appreciated that the flow of the dye may occur in many different configurations or arrangements as would occur to one skilled in the art. To that end, the dye repository2420is shown as being positioned near the top of the chip; however, it should be appreciated that the repository may be positioned elsewhere on the chip. The dye contained within repository2420and used to provide the visual indication may be any appropriate colored dye as would occur to one skilled in the art. In other embodiments, the material contained in the repository and used to provide the visual indication is another appropriate colored material as would occur to one of ordinary skill in the art. In still other embodiments, more than one dye repository is provided, such that the color change is produced by mixing the contents of both repositories to produce a color change when mixed. The materials in the repositories may be two fluids, a fluid and a non-fluid, or two non-fluids.

Release of the dye from repository2420may be accomplished by manual operation by a user of the chip2402. Alternatively, release of the dye may be accomplished under control of the device or instrument system performing the cytometry analysis2412, such as by opening a valve maintaining the dye within the repository2420, as is known in the art. The repository2420may take any convenient physical form, such as a well formed into the surface of substrate2402, which may remain open or may include a cover that is glued in placed, snapped in place with resilient members that engage the substrate2402, slide in place under guides that extend from the substrate2402surface, or any other convenient means as would occur to one of ordinary skill in the art. The above examples are intended to be only non-limiting examples of many possible configurations.

Additionally, other manners of providing a visual indication of a result of the cytometry analysis as would occur to one of ordinary skill in the art are contemplated by the present disclosure, with the release of a dye being just one non-limiting example of numerous possible mechanisms. As an alternative example, the chip2400may include sensors incorporated therewith which are operable to provide a visual indication of a result of the cytometry analysis. As another alternative example, the visual indication can be done by providing a mechanism which immerses the chip2400in a colored or dye solution and allowing the solution to spread through the components of the chip. In another alternative example, a section of the substrate2402is coated with a light-sensitive material and may change in color due to a chemical reaction caused by electromagnetic stimulation. As another alternative example, the dye may be deposited on the chip2400in a dried form and hydrated by sheath fluid diverted to the appropriate location. The chip2400may be configured to provide a visual indication on the occurrence of a positive result of the cytometry analysis or alternatively may be configured to provide different visual indications corresponding to the different possible results of the analysis.

In embodiments in which the cytometry analysis is being run to determine if cells, tissue or other material include a dangerous or hazardous component, such as in AIDS or Hepatitis testing as examples, providing a visual indication of a positive result will provide an indication to the researcher or medical professional that the chip2400includes a dangerous or hazardous pathogen. In this way, the researcher or medical professional can sterilize, dispose of or otherwise handle the chip2400as necessary. Combined with the sterilization, the color change may function to indicate that the chip2400has been successfully decontaminated. Additionally, in many cytometry analysis environments, the researcher or medical professional may desire to run one or more additional cytometry analyses on a cell sample following a positive result to verify the accuracy of the result. The visual indication of a positive result allows the researcher or medical professional to immediately run additional testing if desired.

In other embodiments, the chip2400may be configured to simply provide a visual indication that the chip has been used. For example, providing a clear indication that the chip2400has been used may prevent accidental contamination of cell samples that could occur if a second cell sample were run through a used chip2400. Other manners of providing a visual indication that the microfluidic device has been used as would occur to one of ordinary skill in the art are contemplated by the present disclosure. As another example, the analysis section2412and/or chambers2414may include sensors coupled therewith which provide a visual indication that either the analysis in section2412is complete or the cells have been collected into the chambers2414, respectively. The sensors may themselves provide a visual indication via color change, or may direct the visual indication on the chip2400. In even a further example, a waste chamber or channel on the chip2400may be pre-coated with a chemical or dye that changes color when it comes in contact with fluid. In this way, any chip that has had fluid flow in it would be identified as used via the color change.

Additionally, in certain embodiments the chip2400may be designed to provide a visual indication when one or more steps throughout the cytometry analysis process have been completed. As an example, one or more of the cell sample input2410, analysis section2412and chambers2414may include mechanisms to provide a visual indication that the particular step associated with that component has been completed. Accordingly, a researcher or medical professional viewing the chip2400may easily determine the present status of the cytometry analysis, such as the cell sample has been loaded, the cytometry analysis is complete, or the cells have been sorted. The mechanisms to provide a visual indication at the particular stages of the cytometry analysis may be any appropriate mechanism for providing a visual indication as would occur to one skilled in the art.

With all of the embodiments disclosed herein, the use of a microfluidic device on a substrate offers many advantages, one of which is that the microfluidic device may be treated as a disposable part, allowing a new microfluidic device to be used for sorting each new sample of cells. This greatly simplifies the handling of the sorting equipment and reduces the complexity of cleaning the equipment to prevent cross contamination between sorting sessions, because much of the hardware through which the samples flow is simply disposed of. The microfluidic device also lends itself well to sterilization (such as by gamma irradiation) before being disposed of.

While the disclosure has been illustrated and described in detail in the drawings and foregoing description, the same is to be considered as illustrative and not restrictive in character, it being understood that only the preferred embodiments have been shown and described and that all changes and modifications that come within the spirit of the disclosure are desired to be protected.