Patent Description:
To overcome the limitations of conventional macroscale separation methods, a number of microfluidic separation techniques have been developed with many advantages of precise target control, minimized sample and reagent requirement, and capability of integration with different functional devices without the labelling process. <NUM>-4B,9B,10B Among those techniques, spiral microfluidic devices have been extensively utilized in sample preparation due to their inherent advantages including high throughput (order of <NUM>/min per a single device), simple and robust operation without any need of additional force fields like magnetic, electric, and acoustic fields, and spatially compact device configuration compared to other inertial microfluidic devices. 7B,<NUM>-36B.

In spiral microfluidic devices, lateral particle motion (in the cross-sectional view) is affected by inertial focusing by lift forces and circulating motion by additional hydrodynamic drag force caused by Dean flow. 1A,2A When a fluid flows through a curved channel, fluid elements near the channel centerline have a higher flow rate as compared to the fluid near the channel wall, and move outwards to the outer channel wall due to centrifugal effects and pressure gradient caused by the longer travel length along the outer wall compared to the inner wall, resulting in a secondary flow, the Dean flow. 1A,2A,13A,21A Depending on the size of the particle, the magnitude of the applied net lift force and the Dean drag force are changed, determining whether particles keep moving along the Dean flow or become focused on a certain equilibrium location in the channel's cross-sectional view.

The confinement ratio (CR=a/Dh, where a is the particle diameter and Dh is the hydraulic diameter of microchannel), is the key parameter with respect to the particle motion. 1A,22A-25A Generally (for moderate flow rate condition with a constraint of the Dean number, De = Rc(Dh/2r)<NUM>/<NUM> < <NUM>, where δ = Dhl<NUM>r and r represent the curvature ratio and the average radius of curvature of the channel, respectively),<NUM> in the case of a small CR (<<NUM>), the net lift force applied to particles is negligible compared to Dean drag force, resulting in the circulating motion of particles without focusing (the non-focusing mode). 24A,25A In the case of large CR(≥<NUM>), the lift force becomes stronger and comparable with Dean drag force, resulting in particle focusing on an equilibrium location determined by the competition between the net lift force and the Dean drag force (the focusing mode). In the intermediate CR (<NUM>≤CR<<NUM>), particle motion is described as the rough focusing mode. As particle size increases, both the lift force and Dean drag force increase, but with a different power; in the case of the inertial lift force (F L), F L∝ a<NUM>, and in case of the Dean drag force (F D), F D∝ a. Therefore, generally in the spiral device, as particle size increases, the equilibrium location gradually moves toward the inner wall due to the highly increased lift force, and, using this principle, particles can be separated depending on their sizes. 3A-16A,21A,22A,26A,27A.

Spiral microfluidic devices have been widely utilized for the separation of particles, especially for large CR particles,13A,14A,16A but there are some critical drawbacks which reduce their applicability. These drawbacks include narrow target size ranges (due to the difficulty in focusing particles with the small and intermediate CR conditions) and the relatively low-efficiency and somewhat unreliable separation (due to the small separation distance between focused bands of large CR particles which exist only around the inner wall side). For effective separation in such spiral devices, various approaches have been studied; including, for example, use of a two-inlets spiral device with an additional sheath flow,4A,8A,11A,12A a trapezoidal spiral device,<NUM>,<NUM>,<NUM>,<NUM> and a double-spiral device.

With respect to the spiral device with an additional sheath flow,4A,8A,11A,12A all particles (with the large and even intermediate CR conditions) are injected into the spiral channel, are focused on the outer wall side by the additional sheath flow, and start moving away from the focused flow stream to their equilibrium locations which results in their separation. The initial focusing effectively reduces the particle interaction while the particles travel to their equilibrium locations, which significantly increases separation resolution and efficiency. In addition, due to the initial focusing on the outer wall side, particles in the intermediate CR range can reach their equilibrium locations near the outer wall in a focused band, despite low applied lift force. As a result, in the spiral device with an additional sheath flow, particles can be separated with high separation performance and wide target size ranges (even particles in the intermediate CR range). In the case of separating two different sizes of particles, design channel dimensions can be designed or configured so as to have different CR regimes so that the large CR particles and the intermediate CR particles can be focused near the inner wall and the outer wall, respectively, resulting in their separation with large separation distance and high separation efficiency.

However, the use of two inlets makes the flow control complex and limits the operating flexibility such as closed-loop operation,3A,27A which reduces the applicability of such devices. Recently, a novel spiral microfluidic device with a trapezoidal cross-section was described which generates stronger Dean vortices at the outer half of the channel, resulting in significantly increased separation distance between larger and smaller particles even in a one-inlet configuration. 3A,9A,10A,13A,22A However, even in the trapezoidal spiral device, because of the low magnitude of lift force driving particle focusing, small particles with the intermediate CR may still not form a focused band, and this in turn limits the applicability of the trapezoidal spiral device. In the double spiral device,5A-7A,17A the sequential pinch effect acts to compact both sides of the focusing band resulting in a sharper and narrower band compared to single spiral device, which improves separation performance. However, the double spiral device also has the difficulty in focusing and separating particles within the intermediate CR range, and the separation performance is less than that of the two-inlet spiral device with an additional sheath flow.

In the prior art, <CIT> discloses a microfluidic chip comprising a spiral microfluidic that uses inertial forces to filter fluid components by size connected to a magnetically active channel. <CIT>discloses a fluidic device to detect, capture and/or remove disease material in a biological fluid. The device may comprises a multidirectional channel, which may be a spiral. <CIT> discloses a system comprising an inlet for receiving at least a portion of a fluid containing neutrally buoyant particles, a spiral channel within which the fluid flows, and two outlets for the fluid. <CIT> relates to separating suspended particles based on their size and mass. The particles are made to flow in a spiral channel and an array of outlets collect the separated particles. <CIT> relates to the field of rare cell and particle enriched screening and discloses using a tandem and parallel type spiral microchannel inertial separation structure.

Therefore, although significant progress has been made with respect to spiral microfluidic devices, drawbacks still exist; such as requiring precise flow control (in case of <NUM>-inlets system) and low separation performance for particles with the intermediate CR condition. There remains a need in the art for a microfluidic device and method of use, wherein the separation can be achieved with higher reliability and simpler operation, and/or separation of target samples having various size ranges can be achieved, including not only particles in the large CR range but also particles in the intermediate CR range.

Also, to extend applicability of the spiral microfluidic devices from "laboratory research level" to "real clinical application level", a fully automated and portable operating platform is desirable, and it would be advantageous for such a platform to be operated without any large imaging instrument like a microscope for high accessibility.

In a first aspect, the present invention provides a microfluidic device comprising a multidimensional double spiral (MDDS), wherein the MDDS comprises:.

The present invention is directed to a microfluidic device comprising a multi-dimensional double spiral (MDDS), which may be incorporated into a device further comprising a fully automated recirculation platform. The MDDS comprises a first spiral microchannel and a second microchannel. The first spiral microchannel and the second spiral microchannel of the MDDS may be connected sequentially or in series, such that output from the first spiral microchannel is directed into the second spiral microchannel. In a second aspect, the invention provides a method of separating particles from a sample fluid comprising a mixture of particles, the method comprising the steps of:.

Preferred features of the invention are set out in the dependent claims herein. The first spiral microchannel may be configured to concentrate the particles into a concentrated particle stream and the second spiral microchannel is configured to separate particles from the concentrated particle stream based on their sizes. The first spiral microchannel may be configured to form the concentrated particle stream on the inner wall side of the first spiral microchannel and optionally to direct the concentrated particle stream to enter the outer wall side of the second spiral microchannel. The second spiral microchannel may be configured to direct a first particle stream to the inner wall outlet and to direct a second particle stream to the outer wall outlet, wherein the first particle stream comprises particles having a larger average diameter than that of the particles in the second particle stream. Particles having more than two sizes can be separated into each outlet (see, for example, <FIG> which shows an inner wall outlet, an outer wall outlet, and three middle outlets between them). Thus, the second spiral microchannel may have one or more middle outlets to which additional streams comprising particles are directed. The device may be configured to concentrate and/or separate the particles without additional sheath flow. The first inlet of the first spiral microchannel may be the only inlet of the first spiral microchannel.

The invention also encompasses a device comprising the MDDS described herein, wherein the first spiral microchannel of the MDDS device is configured to concentrate the particles into a concentrated particle stream and the second spiral microchannel is configured to separate particles from the concentrated particle stream based on their sizes, and wherein the device further comprises a system for closed loop recirculation; wherein the inner wall outlet of the MDDS is in fluid communication with a first output reservoir and the outer wall outlet is in fluid communication with a second output reservoir, wherein the system for closed loop recirculation recirculates the fluid from the first output reservoir into the inlet of the first microchannel, and comprises a syringe in fluid communication with the first output reservoir and the inlet of the first spiral microchannel; a first check valve positioned between and in fluid communication with the first output reservoir and the syringe; and a second check valve positioned between and in fluid communication with the syringe and the inlet of the first spiral channel. The two check valves may be combined in the form of a dual-check valve.

The invention also encompasses a device comprising the MDDS described herein, wherein the first spiral microchannel of the MDDS device is configured to concentrate the particles into a concentrated particle stream and the second spiral microchannel is configured to separate particles from the concentrated particle stream based on their sizes, and wherein the device further comprises a system for closed loop recirculation; wherein the inner wall outlet of the MDDS is in fluid communication with a first output reservoir and the outer wall outlet is in fluid communication with a second output reservoir, wherein the system for closed loop recirculation recirculates the fluid from the second output reservoir into the inlet of the first microchannel, and comprises a syringe in fluid communication with the second output reservoir and the inlet of the first spiral microchannel; a first check valve positioned between and in fluid communication with the second output reservoir and the syringe; and a second check valve positioned between and in fluid communication with the syringe and the inlet of the first spiral channel. The syringe may be part of a syringe pump and/or withdrawal of the fluid from the second output reservoir and infusion into the inlet of the first spiral microchannel by the syringe may be automated. Withdrawal of the fluid from the second output reservoir and injection to the inlet reservoir by the syringe may be hand powered. The device may comprise at least two multi-dimensional double spirals (e.g., with combined inlet and outlets for simpler operation), wherein the inlet of each double spiral or the inlet of the double spirals is in fluid communication with the sample fluid and/or the second output reservoir from which fluid is recirculated.

The syringe may be part of a syringe pump and/or withdrawal of the fluid from the first output reservoir and infusion into the inlet of the first spiral microchannel by the syringe may be automated. Withdrawal of the fluid from the first output reservoir and infusion into the inlet reservoir by the syringe may be hand powered.

The device may comprise at least two multi-dimensional double spirals, wherein the first inlet of each double spiral (the inlet of the first spiral microchannel of the MDDS) is in fluid communication with the sample fluid and/or the first output reservoir from which the fluid is recirculated. Where the device comprises at least two multi-dimensional double spirals, the inlet(s) and outlet(s) for the double spiral can be combined or shared for simpler operation. Such devices comprising at least two multi-dimensional double spirals can further comprise a system for closed loop recirculation as described herein.

The second aspect of the present invention provides a method of separating particles from a sample fluid comprising a mixture of particles, the method comprising the steps of introducing the sample fluid into the inlet of the first spiral microchannel of a device of the first aspect; directing the sample fluid through the first spiral microchannel to the transition region of the device and into and through the second spiral microchannel, and directing a first particle stream to the inner wall outlet and directing a second particle stream to the outer wall outlet, and optionally wherein the first particle stream comprises particles having a larger average diameter than that of the particles in the second particle stream. The first spiral microchannel may concentrate the particles into a concentrated particle stream and the second spiral microchannel may separate particles from the concentrated particle stream based on their sizes. The method may comprise the use of a device comprises a system for closed loop recirculation as described herein. The invention may be directed to separating white blood cells from a blood sample comprising the use of a device comprises a system for closed loop recirculation as described herein.

The invention also encompasses a microfluidic device comprising a spiral microchannel wherein the device is configured for closed loop recirculation, and further wherein the device comprises a check valve that permits flow in the direction from an output reservoir to an inlet of the spiral microchannel and blocks flow in the direction from the inlet to the output reservoir.

The foregoing and other objects, features and advantages of the invention will be apparent from the following more particular description of preferred embodiments of the invention, as illustrated in the accompanying drawings in which like reference characters refer to the same parts throughout the different views. The drawings are not necessarily to scale emphasis instead being placed upon illustrating the principles of the invention.

A description of preferred embodiments of the invention follows.

As used herein, the words "a" and "an" are meant to include one or more unless otherwise specified. For example, the term "a cell" encompasses both a single cell and a combination of two or more cells and, the term "a multi-dimensional double spiral" refers to both a single multidimensional double spiral (MDDS) as well as a plurality of multidimensional double spirals.

The term "particle" and "particles" includes, but is not limited to, cells, beads, viruses, organelles, nanoparticles, and molecular complexes. The term "particle" or "particles" can include a single cell and a plurality of cells. Cells can include, but are not limited to, bacterial cells, blood cells, sperm cells, cancer cells, tumor cells, mammalian cells, protists, plant cells, and fungal cells.

A "patient" is an animal to be treated or diagnosed or in need of treatment or diagnosis, and/or from whom a biofluid is obtained. The term "patient" includes humans.

A device comprising a multi-dimensional double spiral (MDDS) can be referred to herein as an "MDDS device.

The first inlet of the first spiral microchannel of an MDDS can also be referred to herein as "the first inlet of the MDDS device," "the inlet of the MDDS device," or as "the inlet. " In embodiments where the device comprises multiple multidimensional double spirals (e.g., the quad-version described herein), the inlet of each first spiral microchannel of the multidimensional double spiral can be referred to as the "first inlet" or simply as the "inlet. " Where the device comprises multiple multidimensional double spirals, the "first inlet" of a MDDS can be shared by two or more multidimensional double spirals as discussed below.

Spiral microchannels, devices comprising such channels, and methods for the use of thereof have been described, for example, in <CIT>; <CIT>, <CIT>, <CIT>; <CIT>; <CIT>; <CIT>; <CIT>; <CIT>; and <CIT>. In microfluidic devices, particles flowing in curvilinear (such as spiral) channels are influenced by both inertial migration and secondary Dean flows. The combination of Dean flow and inertial lift results in focusing and positioning of particles at distinct positions for concentration and separation applications.

Spiral microfluidic devices have been widely utilized for sample preparation mainly as a concentrator or a separator. In such spiral devices, the particle focusing position is predominantly determined by the ratio of particle size and channel dimension; the smaller the channel dimensions, the smaller the particles that can be focused on the inner wall side. The present invention is directed to a multi-dimensional double spiral (MDDS) device, for example, in which a mixture of particles are concentrated during their passage through a first smaller-dimensional spiral channel and then separated according to their sizes during passage through the second larger-dimensional spiral channel. The devices described herein can integrate two different functions, sample concentration and separation, into a single device with one inlet configuration, and without the need of additional sheath flow. Thus, the first inlet of the first spiral microchannel may be the only inlet (e.g., for each multidimensional double spiral). In addition to possessing the advantages of conventional spiral devices (such as high throughput and simple operation), the devices described herein can provide better separation performance (e.g., separation resolution, separation efficiency, separation distance, shaper and narrow particles bands or streams) and/or can be utilized to separate particles having a wide target size range (including intermediate CR ranges) as compared to the conventional spiral devices.

As discussed above, the first aspect of the invention encompasses a spiral microfluidic device comprising a multidimensional double spiral (MDDS) device, wherein the MDDS device comprises:.

wherein the first spiral microchannel has a smaller cross-sectional area than the second spiral microchannel; wherein the cross-sectional area of the first spiral microchannel remains constant along its length (e.g., from the inlet to the transition region) and wherein the cross-sectional area of the second spiral microchannel remain constant along its length (e.g., from the transition region to the outlet); and wherein the device is configured to separate particles from a sample fluid comprising a mixture of particles. The first spiral microchannel and the second spiral microchannel may be connected sequentially by the transition region such that output from the first spiral microchannel flows into the transition region and then, from the transition region, directly into the second spiral microchannel. The first spiral microchannel may be configured to concentrate particles into a concentrate particle stream (for example, on the inner wall side of the first spiral microchannel) and the second spiral microchannel may be configured to separate the particles in the concentrated particle stream based on the particle sizes (for example, depending on the dimensions of the second spiral microchannel, particles having the larger particle sizes are directed to the inner wall side of the second spiral microchannel). It is to be understood that the transition region is a region that connects the first and second microchannels; in some examples, the transition region can be considered part of the first spiral microchannel and/or part of the second spiral microchannel.

The invention also includes, in a second aspect, a method of separating particles from a sample fluid comprising a mixture of particles, the method comprising the steps of:.

The first particle stream (directed to the inner wall outlet) can comprise particles having a larger average diameter than that of the particles in the second particle stream.

The sample fluid may be introduced into the first spiral microchannel via the first inlet; optionally, the sample fluid is placed in an inlet/input reservoir and the first inlet is in fluid communication with the inlet/input reservoir. The inlet/input reservoir may be a syringe and the sample fluid may be infused into the first spiral microchannel by actuating the syringe. The first spiral microchannel may be connected sequentially or in series to the second spiral microchannel by a microchannel transition region. The dimensions or cross-sectional area of the second spiral microchannel are larger than that of the first spiral microchannel. The particles can be concentrated into a concentrated particle stream as they pass through the first spiral microchannel and can be separated based on their sizes as they pass through the second spiral microchannel. For example, <FIG> shows an example of the configuration of the first and second spiral microchannels (where the first spiral microchannel has smaller dimensions than the second spiral microchannel) and the movement of particles/particle streams as they pass through the microchannels. When a sample fluid containing various sizes of particles is introduced into the first spiral microchannel via the inlet, the particles can have a relatively large confinement ratio (CR=a/Dh, where a is the particle diameter and Dh is the hydraulic diameter of microchannel) because the dimensions or cross-sectional area of the first spiral microchannel are small. In the first spiral microchannel, the particles become concentrated close to the inner wall side of the channel and have almost same or similar equilibrium locations. By passing through the S-shaped transition region, the concentrated particles on the inner wall side of the first spiral microchannel enter the outer wall side of the second spiral channel. As a result, the particle stream enters the second spiral microchannel in a concentrated band near the outer wall side, as if focusing the sample by the use of additional sheath flow. In the second spiral microchannel which has larger dimensions/cross-sectional area than the first spiral microchannel, the particle's CR value decreases due to the increased channel size, resulting in the equilibrium location's shift toward the outer wall side of the channel. As a result, particles form concentrated bands at different equilibrium locations depending on their sizes, which is a similar mechanism with the two-inlets spiral device with an additional sheath flow. <NUM>,<NUM>,<NUM>,<NUM>.

The inner wall is the side wall of the channel that is on the side of the microchannel that is closer to the center of the spiral (e.g., the radially inner side) whereas the outer wall is the side wall of the channel that is on the side of the microchannel that is closer to the outside or periphery of the spiral (e.g., the radially outer side). An inner wall outlet is an outlet situated or configured such that a stream on the inner wall side of the channel is directed to the inner wall outlet. An outer wall outlet is an outlet situated or configured such that a stream on the outer wall side of the channel (or a stream other than that on the inner wall side) is directed to the outer wall outlet. Where the device comprises more than two outlets, the term "inner wall outlet" refers to the outlet closest to the inner wall. Similarly, when the device comprises more than two outlets, the term "outer wall outlet" refers to the outlet closest to the outer wall. The outlet(s) situated between the inner wall outlet and the outer wall outlet are referred to herein as the middle outlet(s). In devices configured like that of <FIG>, the largest particles (e.g., the particles having the largest average diameter) of the mixture are focused on the inner wall side of the second spiral microchannel and can be collected from the inner wall outlet, and the smallest particles (e.g., particles having the smallest average diameter) of the mixture are focused on the outer wall side and can be collected from the outer wall outlet. Particles of intermediate sizes (e.g., particles having average diameters between those of the largest and smallest particles of the mixture) are focused in stream(s) between the inner wall side and the outer wall side and can be collected in one or more middle outlets (situated between the inner wall outlet and the outer wall outlet) depending on their sizes. For example, if there is more than one particle stream of intermediate sized particles and two middle outlets, then the stream with the larger sized particles of the intermediate sized particles is directed to the middle outlet closer to the inner wall and the stream with the smaller sized particles is directed to the middle outlet closer to the outer wall. <FIG> shows a configuration with three middle outlets.

The first spiral microchannel and the second spiral microchannel may be nested together. The first spiral microchannel and the second spiral microchannel may be nested together (for example, a Fermat spiral) and optionally, the transition region is S-shaped. The first spiral microchannel can, for example, spiral in the counter-clockwise direction, change direction at the transition region (for example, in the S-shaped transition region), and then the second spiral microchannel spirals in the clockwise direction (e.g., see <FIG>). Alternatively, the first spiral microchannel can spiral in the clockwise direction, change direction at the transition region, and then second spiral microchannel spirals in the counter-clockwise direction.

The second spiral microchannel may be parallel to the first microchannel. The second spiral microchannel may be positioned over or under the first spiral microchannel. The first spiral microchannel can spiral in a clockwise or counter-clockwise direction and the second spiral microchannel can spiral in the same or in the opposite direction to that of the first spiral microchannel.

Depending on the configurations of the spiral microchannels, the inlet of the first spiral microchannel can be on the circumference or periphery (outside of the spiral) of the first spiral microchannel or on the inside or center of the spiral microchannel. In addition, depending on the configuration of the spiral microchannel, the outlets can be on the circumference (outside of the spiral) of the second spiral microchannel or on the inside of the second spiral microchannel. The first spiral microchannel and the second spiral microchannel may be nested together and optionally, the transition region is S-shaped, and the inlet and the outlets are on the circumference of the channel.

The first and second spiral microchannels can each independently have a rectangular cross-section or a non-rectangular cross-section. For example, the first and second microchannels can both have a rectangular cross-section. In another example, the first and second microchannels can both have a non-rectangular cross-section, for example, both microchannels can have a trapezoidal cross-section. In yet another example, the first microchannel has a rectangular cross-section and the second microchannel has a non-rectangular cross-section. Microfluidic systems with non-rectangular cross-sections are described, for example, in <CIT>. By designing appropriate channel parameters, small particles/cells are trapped in the vortex at the outside of the microchannel wall (the outer wall) and larger particles focus along the inner microchannel wall.

An example of a non-rectangular cross-section is a trapezoidal cross-section. An additional example of a non-rectangular cross-section is a triangular cross-section. The first spiral microchannel may have a rectangular cross-section and the second spiral microchannel may have a trapezoidal cross-section. The first spiral microchannel may have a trapezoidal cross-section and the second spiral microchannel may have a trapezoidal cross-section. Microfluidic systems with trapezoidal cross-sections are described, for example, in <CIT>. In some examples, the trapezoidal cross section can be defined by a radially inner side, a radially outer side, a bottom side, and a top side, the cross section having a) the radially inner side and the radially outer side unequal in height, or b) the radially inner side equal in height to the radially outer side, and wherein the top side has at least two continuous straight sections, each unequal in width to the bottom side. The cross-section of the curvilinear microchannel may have (a) the height of the radially inner side larger than the height of the radially outer side, or (b) the height of the radially inner side is smaller than the height of the radially outer side, or (c) the top side may include at least one step forming a stepped profile, or (d) the top side may include at least one shallow region in between the radially inner side and the radially outer side. The trapezoidal cross-section may be a right trapezoidal cross section.

As described above, the dimensions and/or cross-sectional area of the first spiral microchannel is less than that of the second spiral microchannel. For example, when both spiral microchannels have a rectangular cross-section, the width and/or height (also referred to as the depth) of the first spiral microchannel is less than that of the second spiral microchannel. In another example, where the first spiral microchannel has a rectangular cross-section and the second spiral microchannel has a trapezoidal cross-section, the cross-sectional area of the first spiral microchannel is less than that of the second spiral microchannel. This is illustrated in the channel configuration described in the Examples where the first spiral channel has a rectangular cross-section with a width of <NUM> width and a height of <NUM> and the second spiral channel has a trapezoidal cross-section with a width of <NUM>, and heights of <NUM> and <NUM> for the inner wall side and the outer wall side, respectively.

The devices and methods can be used to separate particles having large, intermediate, and/or small confinement ratios. Focused particle streams comprising particles of different sizes and/or different confinement ratios can be separated from each other and directed to one or more different outlets. The confinement ratio is the ratio of the particle diameter and Dh, wherein Dh is the hydraulic diameter of the microchannel. A large CR is, for example, greater than or equal to about <NUM>. A small CR is, for example, less than <NUM>. An intermediate CR is, for example, less than about <NUM> and greater than or equal to <NUM>. The device may be configured such that at least one of the particle streams directed to an outlet (for example, the outer wall outlet or a middle outlet) comprises or consists of particles having a small CR and such that another particle stream directed to a different outlet (for example, the inner wall outlet) comprises or consists of particles having a large CR. The device may be configured such that at least one of the particle streams directed to an outlet (for example, the outer wall outlet or a middle outlet) comprises or consists of particles having an intermediate CR and such that another particle stream directed to a different outlet (for example, the inner wall outlet) comprises or consists of particles having a large CR. The outlets to which different particle streams will be directed depends on the equilibrium positions of the particles. The device may be used or configured such that particles having a small CR can be separated from other particles in the mixture (for example, from large CR particles). The device may be s used or configured such that particles having large CR can be separated from other particles in the mixture (for example, from particles having a small CR or an intermediate CR). The device may be sed or configured such that particles having intermediate CR can be separated from other particles in the mixture (for example, from particles having a large CR).

The MDDS device can comprise a single multidimensional double spiral or a plurality of multidimensional double spirals. The device may comprise, one, two, three, four, five, six, seven, eight, ten, twelve, or sixteen multidimensional double spirals. A device comprising a plurality of multidimensional spirals can be used, for example, to increase throughput and/or reduce operation time. Each multidimensional double spiral can have its own first inlet or can share a first inlet with one or more multidimensional double spirals. Similarly, each multidimensional double spiral can have its own inner wall outlet and/or outer wall outlet or can share the same inner wall outlet and/or the same outer wall outlet with one or more multidimensional double spirals. Thus, multiple different configurations are possible. The device may comprise at least two multi-dimensional double spirals, wherein each first inlet is in fluid communication with the sample fluid, for example, the sample fluid in an input reservoir. The device may comprise four multi-dimensional spirals wherein each first inlet is in fluid communication with the sample fluid. The sample fluid can, for example, be introduced into each inlet by placing the sample fluid in an input reservoir that is in fluid communication with the inlet. A set of four multi-dimensional spirals is referred herein as a "quad-version" of the MDDS device. The device may comprise eight multi-dimensional spirals; the eight multi-dimensional spirals can, for example, be made up from two quad-version of the MDDS devices. As discussed above, where the device comprises at least two multi-dimensional double spirals, the inlet(s) and/or outlet(s) for the double spiral can be combined or shared for simpler operation. For example, all or a subset of the double spirals can share an inlet and/or share an outlet (e.g., the inner wall outlet and/or the outer wall outlet). The device may comprise four multi-dimensional double spirals wherein the inlet(s) of the device is in fluid communication with the sample fluid and/or the output reservoir from which fluid is recirculated. A non-limiting example of a device comprising four multi-dimensional double spiral (referred to herein as the quad-version) is shown in <FIG>. This figure shows an exemplary quad-version in which two of the four double spirals share an inlet (Inlet <NUM>) and an inner wall (IW) outlet (IW outlet <NUM>). The other two of the four double spirals share an inlet (Inlet <NUM>) and an inner wall (IW) outlet (IW outlet <NUM>). In this configuration, the four double spirals share the same outer wall (OW) outlet. As discussed above, each double spiral may have its own inlet and/or each double spiral may have its own inner wall outlet and/or outer wall outlet.

The closed loop recirculation may be provided by a recirculation system that comprises a check-valve where only one direction of flow is allowed while the opposite direction of flow is blocked by the internal membrane. In the examples below, a dual-check-valve was used and included two different check-valves so that, once separated, output in the output reservoir can be extracted back into the input syringe at the withdrawal motion of a syringe pump and processed again through the MDDS device at the infusion motion of a syringe pump, resulting in higher purity and concentration.

As discussed above, the invention includes a microfluidic device comprising a multidimensional double spiral (MDDS) device as described herein, wherein the first spiral microchannel of the MDDS device is configured to concentrate the particles into a concentrated particle stream and the second spiral microchannel is configured to separate particles from the concentrated particle stream based on their sizes and wherein the device further comprises a system for closed loop recirculation,.

A check valve permits only one direction of flow while the opposite direction of flow is blocked, for example, by an internal membrane. The first check valve permits flow in the direction from the first output reservoir to the syringe and blocks flow in the direction from the syringe to the first output reservoir. The first check valve can comprise an inner membrane that blocks flow in the direction from the syringe to the first output reservoir when the syringe is actuated to infuse the fluid into the inlet of the first spiral channel. The second check valve permits flow in the direction from the syringe to the inlet of the first spiral microchannel and blocks flow in the direction from the inlet of the first spiral channel to the syringe. The second check valve can comprise an inner membrane that blocks flow in the direction from the inlet of the first spiral channel to the syringe when the syringe is actuated to withdraw the fluid from the first output reservoir into the syringe.

As will be understood, the device can also be configured such that the system for closed loop recirculation recirculates fluid from the second output reservoir (comprising particle have a smaller average diameter than the particles in the first output reservoir) into the MDDS device. Thus, the invention also encompasses a microfluidic device comprising a multidimensional double spiral (MDDS) device as described herein, wherein the first spiral microchannel of the MDDS device is configured to concentrate the particles into a concentrated particle stream and the second spiral microchannel is configured to separate particles from the concentrated particle stream based on their sizes and wherein the device further comprises a system for closed loop recirculation,.

The invention also includes a method of separating particles from a sample fluid comprising a mixture of particles, the method comprising the steps of:.

As referred to herein, actuation of the syringe can refer to withdrawal motion (e.g., withdrawing fluid from one of the output reservoirs) and/or infusion motion (e.g., infusion into the inlet of the first spiral microchannel). Back-and-forth motions (in other words, withdrawal and infusion motions) of the syringe and/or syringe pumps result in recirculation of fluid from the first output reservoir or the second output reservoir into the MDDS device by withdrawing fluid from the first output reservoir or the second output reservoir into the syringe and then infusing that fluid into the inlet of the first microchannel. Each time all or substantially all of the fluid in the first output reservoir or second output reservoir is recirculated in the MDDS device, a cycle of recirculation is completed. The fluid collected after being directed through the MDDS device (either after first passage through the device or after one or more cycles of recirculation) can be referred to herein as the "final output" or "final output fluid. " The methods described herein can comprise no cycle of recirculation or one or more cycles of recirculation. The method may entail one, two, three, four, five, six, seven, or eight cycles of recirculation. The number of cycles of recirculation can depend on a number of factors including, but not limited to, the desired particle separation in the final output, the desired particle purity in the final output, the desired particle concentration in the final output, the desired particle recovery in the final output, time of operation, the number of MDDS devices, etc..

The first and second check valves allow fluid from the output reservoir (either the first output reservoir or the second output reservoir) to be extracted into the syringe at the withdrawal motion of the syringe (or the syringe pump) and processed again through the MDDS device at the infusion motion of the syringe (or the syringe pump) while blocking flowing in the opposite directions, for example, toward the output reservoir from the syringe (in the case of the first check valve) and toward the syringe from the inlet of the MDDS device (in the case of the second check valve). The first check valve and second check valve can be part of the same check valve assembly or unit, for example, like the dual check valve described in the Examples section below.

The device can include one or more additional check valves. For example, for the device that comprises the system for closed loop recirculation that recirculates fluid from the first output reservoir, the additional check valve can be positioned between and in fluid communication with the inner wall outlet and the second output reservoir; this additional check valve can block flow from the second output reservoir in the direction of the first output reservoir while permitting flow from the outlet to the second output reservoir. Alternatively, for the device comprising the system for closed loop recirculation that recirculates fluid from the second output reservoir, the additional check valve can be positioned between and in fluid communication with the inner wall outlet and the first output reservoir; this additional check valve can block flow from the first output reservoir in the direction of the second output reservoir while permitting flow from the outlet to the first output reservoir.

The device comprising the system for closed loop recirculation can comprise a single multidimensional double spiral or a plurality of multidimensional double spirals. The device may comprise, one, two, three, four, five, six, seven, eight, ten, twelve, or sixteen multidimensional double spirals. Thus, multiple different configurations are possible. The device may comprise four multi-dimensional double spirals wherein the inlet(s) of the device is in fluid communication with the sample fluid and/or the output reservoir from which fluid is recirculated. Each multidimensional double spiral can have its own first inlet or can share a first inlet with one or more multidimensional double spirals of the device. Similarly, each multidimensional double spiral can have its own inner wall outlet and/or outer wall outlet or can share the same inner wall outlet and/or the same outer wall outlet with one or more multidimensional double spirals. The device comprising a plurality of multidimensional spirals as described herein can be configured to provide closed loop recirculation of the sample fluid through the first spiral microchannel of each multidimensional double spiral as described herein. For example, each inner wall outlet of the device may be in fluid communication with a first output reservoir and each outer wall outlet of the device is in fluid communication with a second output reservoir, and the system for closed loop recirculation may recirculate the fluid from the first output reservoir or the second output reservoir into the first inlet(s) of device.

The sample fluid can, for example, be introduced into the inlet by placing the sample fluid in an input reservoir that is in fluid communication with the first inlet(s). Such an input reservoir can, for example, be a syringe and the infusion motion of the syringe can introduce the sample fluid into the inlet of the first spiral microchannel. In the Examples, a set of four multi-dimensional spirals is referred to a quad-version of the MDDS device. The device may comprise eight multi-dimensional spirals; the eight multi-dimensional spirals can, for example, be made up from two quad-version of the MDDS devices.

The syringe of the recirculation system may be part of a syringe pump and/or withdrawal of the fluid from the first output reservoir and infusion into the inlet of the first spiral microchannel by the syringe may be automated. Withdrawal of the fluid from the first output reservoir and infusion to the inlet by the syringe of the recirculation system may be hand powered; optionally, a hand powered recirculation system can further comprise a pressure meter, for example, a pressure meter which monitors pressure applied at the inlet region.

The device may comprise a support that connects the MDDS device, the syringe(s), and the check valves. Where the device comprises a plurality of multidimensional spirals, such as the quad-version of the MDDS device, the support can connect the plurality of MDDS devices, the syringe(s), and the check valves. The support can, for example, be made by 3D printing. Non-limiting examples of such supports (also referred to as "connectors") are shown in <FIG> and described in the Examples below.

The MDDS device including a device comprising the MDDS device and the system for recirculation may be a portable device. Such a portable device can provide point-of-care convenience and can be particularly useful in resource-limited environments including rural areas and/or developing countries where access to health care and medical diagnostics is limited.

Various fluids comprising mixtures of particles can be used in the systems and methods described herein. Examples of mixtures include biological fluids or biofluids (e.g., a biological sample such as blood, lymph, serum, urine, mucus, sputum, cervical fluid, placental fluid, semen, spinal fluid, and fluid biopsy), liquids (e.g., water), culture media, emulsions, sewage, etc. When the biofluid is whole blood, the blood can be introduced unadulterated or adulterated (e.g., lysed, diluted). Other biological fluids or biofluids can also be used unadulterated or adulterated (e.g., the biofluid can be pre-treated in some way or diluted). For example, methods of lysing blood are known in the art. The blood sample may be diluted prior to introducing it into the inlet of the first microchannel.

The devices and methods can be used, for example, in the detection of biomarkers, microorganisms (e.g., bacterial cells, fungi, or viruses), and cells in biofluids including, but not limited to, blood, urine, saliva, and sputum. The devices and methods can be used, for example, for chemical process and fermentation filtration, water purification/wastewater treatment, sorting and filtering components of blood and other bio-fluids, concentrating colloid solutions, and purifying and concentrating environmental samples. The method can be used for separation of white blood cells from blood samples, detection of nucleated cells, detection of rare cells (e.g., circulating tumor cells) within blood samples, depletion of erythrocytes and recovery of leukocytes from G-CSF mobilized peripheral blood (PBSC), bone marrow (BM), and/or umbilical cord blood (UCB) prior to cryopreservation, removal of colloidal and supracolloidal residues from wastewater effluents, and filtration of pathogenic bacteria strains, such as E. coli O157:H7, from water.

The biological fluid may be semen. In specific methods, the device or method described herein can be used to separate sperm cells from other cells, such as immune cells, in the sample. Sperm cells can, for example, be separated based on their size and/or motility.

The biological sample may be a sputum sample. The device and/or method described herein may separate and concentrate immune cells from the other cells in the sputum sample.

The invention may be directed to a method of separating leukocytes from a blood sample using an MDDS device as described herein. The invention may include a method of separating white blood cells from a blood sample using a microfluidic device comprising a MDDS and system for closed loop recirculation, wherein the inner wall outlet of the MDDS is in fluid communication with a first output reservoir and the outer wall outlet is in fluid communication with a second output reservoir,
wherein the system for closed loop recirculation recirculates the fluid from the first output reservoir into the inlet of the first microchannel, and comprises:.

At least about <NUM>%, at least about <NUM>%, at least about <NUM>%, at least about <NUM>%, or at least about <NUM>% of the white blood cells in the blood sample may be recovered in the final output and/or the purity of the white blood cells in the final output may be at least about <NUM>%, at least about <NUM>%, at least about <NUM>%, at least about <NUM>%, or at least about <NUM>%. As described herein, combining the separation performances of the MDDS device and the advantages of a check-valve-based recirculation method, the developed separation platform shows remarkable results on the isolation of leukocytes (WBCs) in the peripheral blood from the abundant erythrocytes (RBCs). Because the platform can be operated in the fully-automated and reliable manner without any human intervention using microliter quantities of human peripheral blood (<NUM>µL), this is readily applicable to bedside or field use while allowing rapid isolation of intact, functional leukocytes amenable for functional assays. Moreover, the result of its hand-powered operation demonstrates its high applicability as a portable point-of-care (POC) device, especially for sample preparation in resource-limited environments. Also, by altering channel dimensions of the MDDS devices, separation cut-off size can be controlled so that the developed platform could be adaptable for various sample preparation applications using not only blood but also other bio-fluids including saliva, sputum, and semen. Therefore, it is believed that the developed separation platform could be used as an innovative tool to replace conventional sample preparation methodologies.

Exemplary flow rates for the MDDS devices can be in a range of between about <NUM>/min and about <NUM>/min, such as between about <NUM>/min and about <NUM>/min, or between about <NUM>/min and about <NUM>/min.

As discussed above, multiple multi-dimensional double spirals (including the first spiral microchannel and the second spiral microchannel) can be combined into a microfluidic device. Multiple sets of channels can be combined into a multiplexed microfluidic device. For example, the first and second spiral microchannels can be located on a support thereby creating a first layer and a plurality of such layers comprising a first and a second spiral microchannels is stacked and optionally, the inlets of each first spiral microchannel of each layer are in fluid communication with the sample fluid. In another example, multi-layered MDDS devices can be made by stacking single-layered MDDS devices (such single-layered MDDS devices can be a single MDDS device or can be multiple MDDS devices configured in a single layer). For example, a plasma bonding method can be used for attachment of silicon devices, and double-sided film can be used for attachment of plastic devices to one another and optionally to a support.

As described above, the second spiral microchannel may have a non-rectangular or trapezoidal cross-section thereby resulting in the alteration of the shapes and positions of the Dean vortices which generates new focusing positions for particles. For example, as described herein, a curved microchannel with a deeper inner side (along the curvature center) and a shallow outer side generates two strong Dean vortex cores near the inner wall, trapping all particles irrespective of size within the vortex. A spiral microchannel with a shallow inner side and a deeper outer side skews the vortex centers near the outer wall at the outer side and can entrain particles and cells within the vortex. However, larger particles with dominant inertial force are focused near the inner channel walls, similar to rectangular cross-section channels. Thus, by designing appropriate channel parameters, small particles/cells are trapped in the vortex at the outside wall, while relatively large particles focus along the inner microchannel wall. The threshold diameter determining whether a particle/cell is trapped within the Dean vortex or focused towards the inner channel wall is dependent on the flow rate. This enables a device to achieve good separation resolution between mixtures having a wide range of particle sizes. A trapezoidal cross-section facilitates higher particle/cell concentrations.

The separation resolution obtained using the MDDS device described herein may be greater than that of a device comprising a first spiral microchannel and a second spiral microchannel having the same cross-sectional areas (for example, a device having two spiral microchannels of the same dimensions or cross-sectional area as the second spiral microchannel of the MDDS device) but that is otherwise identical to the multi-dimensional double spiral microfluidic device. The separation resolution obtained using the MDDS device described herein may be greater than that of a device comprising only the second spiral microchannel of the MDDS and that is otherwise identical to the MDDS device. A MDDS device as described herein may have greater separation resolution as compared to a device having single spiral microchannel, wherein the single spiral microchannel has the same dimensions as the second spiral microchannel of the MDDS device. For example, as shown in <FIG>, red blood cells (RBCs) can be more effectively extracted into the outer wall side of the channel in the MDDS device as compared to the single spiral device with a low percentage (by volume) of RBCs in the inner wall side outlet.

Fluid flowing through a channel with a laminar profile has a maximum velocity component near the centroid of the cross section of the channel, decreasing to zero near the wall surface. In a curved channel, the fluid experiences centrifugal acceleration directed radially outward. Since the magnitude of the acceleration is proportional to quadratic velocity, the centrifugal force in the centroid of the channel cross section is higher than at the channel walls. The non-uniform centrifugal force leads to the formation of two counterrotating vortices known as Dean vortices in the top and bottom halves of the channel. Thus, particles flowing in a spiral channel experience a drag force due to the presence of these transverse Dean flows. Under Stokes' law, the drag force will be proportional to the Dean velocity at that point and proportional to the diameter of the particle. In the absence of other dominating forces, the Dean drag force will drive particles along the direction of flow within the vortex and finally entrain them within the core. In high aspect ratio rectangular cross section channels, this motion can be observed by observing particles moving back and forth along the channel width between the inner and outer walls with increasing downstream distance when visualized from the top or bottom.

Apart from the Dean drag force, larger particles or cells with diameters comparable to the micro-channel dimensions also experience appreciable inertial lift forces resulting in their focusing and equilibration along the channel walls. In microchannels with curvilinear geometry, the interplay between the inertial lift force and the Dean drag force reduces the equilibrium positions to just two near the inner channel wall at low flow rate, and move outward with an increase in flow rate, each within the top and bottom Dean vortex. The two equilibrium positions overlay each other along the micro-channel height and are located at the same distance from the micro-channel inner wall for a given cell size, i.e. viewed as a single position across the micro-channel width.

Spiral microchannels with trapezoidal cross sections are different from rectangular cross section microchannels, in that the maximum velocity is asymmetric along the channel cross-section resulting in the formation of stronger Dean vortex cores skewed towards the deeper channel side. These vortex cores have high probability to entrain particles within them. In spiral channels with trapezoidal cross-section, the particle focusing behavior is different from that in a rectangular channel. In a trapezoidal channel, as shown in <CIT>, particles focus near the inner channel wall at low flow rate (similar to channels with rectangular cross-section), while beyond a certain threshold flow rate, they switch to an equilibrium position located at the outer half.

Along the depth direction, according to experimental measurements, particles are focused between about <NUM> to about <NUM>% of the channel depth at flow rates of about <NUM> to about <NUM>/min. This result indicates that the distance between the focused particle and the channel wall in a trapezoidal channel in the depth direction is larger than that in the rectangular channel.

If the inner wall of the channel is deeper, strong Dean vortices will appear at the inner side, i.e., particles will be trapped near the inner side, even at high flow rates. Curved channels with this cross section can be used to collect a larger size range of particles at the inner side of the outlet and filtered particle free liquid at the outer side of the outlet, finding numerous applications in water filtration, for example. On the other hand, if the outer wall of the channel is deeper, Dean vortices are skewed towards the outer side. At the inner side, the Dean flow field is much like that in a rectangular channel. At certain flow rates, the larger particle can focus along the inner wall influenced by both Dean flow and inertial lift, while the smaller particles tend to get trapped in the vortex center at the outer side.

Two typical regimes of focusing are based on particle size, the inertial dominant and Dean dominant regimes. For small particles (e.g., <NUM> particles), the large channel dimension prevented them from focusing and these particles got trapped in the Dean vortex even at low flow rate. The larger particles (e.g., about <NUM> particles) also could not focus at the inner wall and were trapped within the Dean vortices at flow rates greater than or equal to about <NUM>/min. For example, <NUM> particles focused at the inner wall at low flow rates, about <NUM>/min, but transitioned from the inertial dominant regime to Dean dominant regime at about <NUM>/min. For the same microchannel, the <NUM> particles transitioned from the inertial regime to Dean regime at flow rates about <NUM>/min. From these results, at a flow rate of about <NUM>/min, particles >about <NUM> can be separated from smaller ones by collecting from the inner and outer outlets separately. Similarly, at a flow rate of about <NUM>/min, about <NUM> particles can be separated from a mixture of about <NUM> and about <NUM> particles. A low flow rate can be in a range of between about <NUM>/min and about <NUM>/min. Thus, a low flow rate can be a flow rate of about <NUM>/min, about <NUM>/min, about <NUM>/min, about <NUM>/min, about <NUM>/min, about <NUM>/min, about <NUM>/min, about <NUM>/min, about <NUM>/min, about <NUM>/min, about <NUM>/min, about <NUM>/min, about <NUM>/min, about <NUM>/min, about <NUM>/min, or about <NUM>/min.

The principles of the MDDS device (e.g., the difference in cross-sectional area for the first and second microchannel) can be applied to channels of various different dimensions.

In certain examples, the spiral microchannels can each independently have a radius of curvature in a range of between about <NUM> and about <NUM>. For example, the spiral microchannel can have a radius of curvature of about <NUM>, about <NUM>, about <NUM>, about <NUM>, about <NUM>, about <NUM>, about <NUM>, about <NUM>, about <NUM>, about <NUM>, about <NUM>, about <NUM>, about <NUM>, about <NUM>, about <NUM>, about <NUM>, about <NUM>, about <NUM>, about <NUM>, about <NUM>, about <NUM>, about <NUM>, or about <NUM>. The spiral microchannel can also have a length in a range of between about <NUM> and about <NUM>. For example, the curvilinear microchannel can have a length of about <NUM>, about <NUM>, about <NUM>, about <NUM>, about <NUM>, about <NUM>, about <NUM>, about <NUM>, about <NUM>, about <NUM>, about <NUM>, about <NUM>, about <NUM>, about <NUM>, about <NUM>, about <NUM>, about <NUM>, about <NUM>, about <NUM>, or about <NUM>.

For a trapezoidal cross-section spiral microchannel, there are several factors that affect the focusing position and separation efficiency, such as the width of the microchannel, inner and outer depth of the microchannel cross-section, the radius of the spiral curvature, and the slant angle. In some examples, the width can be in a range of between about <NUM> and about <NUM>, such as a width of about <NUM>, about <NUM>, about <NUM>, about <NUM>, about <NUM>, about <NUM>, about <NUM>, about <NUM>, about <NUM>, about <NUM>, about <NUM>, about <NUM>, about <NUM>, about <NUM>, about <NUM>, about <NUM>, about <NUM>, or about <NUM>.

In some examples, the outer depth can be in a range of between about <NUM> and about <NUM>, such as an outer depth of about <NUM>, about <NUM>, about <NUM>, about <NUM>, about <NUM>, about <NUM>, about <NUM>, or about <NUM>. The inner depth can be in a range of between about <NUM> and about <NUM>, such as an inner depth of about <NUM>, about <NUM>, about <NUM>, about <NUM>, about <NUM>, about <NUM>, about <NUM>, or about <NUM>. The radius of curvature can be in a range of between about <NUM> and about <NUM>, such as a radius of about <NUM>, about <NUM>, about <NUM>, about <NUM>, about <NUM>, about <NUM>, about <NUM>, or about <NUM>.

The slant angle is the angle between the top of the channel and the bottom of the channel. The slant angle can be in a range of between about <NUM> degrees and about <NUM> degrees. Thus, the slant angle can be about <NUM> degrees, about <NUM> degrees, about <NUM> degrees, about <NUM> degrees, about <NUM> degrees, about <NUM> degrees, about <NUM> degrees, about <NUM> degrees, about <NUM> degrees, about <NUM> degrees, about <NUM> degrees, about <NUM> degrees, about <NUM> degrees, about <NUM> degrees, about <NUM> degrees, about <NUM> degrees, about <NUM> degrees, about <NUM> degrees, about <NUM> degrees, about <NUM> degrees, about <NUM> degrees, about <NUM> degrees, about <NUM> degrees, about <NUM> degrees, about <NUM> degrees, about <NUM> degrees, about <NUM> degrees, about <NUM> degrees, about <NUM> degrees, or about <NUM> degrees. The slant angle of the channel affects the focusing behavior in two ways: (i) the threshold flow rate required to trap particles in the Dean vortex as a function of particle size and (ii) the location of the Dean vortex core. A large slant angle (i.e., in a range of between about <NUM> degrees and about <NUM> degrees) will lead to strong Dean at the outer side and increase the particle trapping capability. A large slant angle can also decrease the threshold flow rate required to trap particles of a given size within the Dean vortex.

The cross section of the channel can be characterized by a height of the radially inner side that is larger than a height of the radially outer side, or vice versa. The profile of the cross section can be stepped, curved, convex, or concave.

The radially inner side and the radially outer side of the trapezoidal cross section can have a height in a range of between about <NUM> microns (µm) and about <NUM>. Thus, the height of the radially inner side <NUM> can be about <NUM>, about <NUM>, about <NUM>, about <NUM>, about <NUM>, about <NUM>, about <NUM>, about <NUM>, about <NUM>, or about <NUM>, and the height of the radially outer side <NUM> can be about <NUM>, about <NUM>, about <NUM>, about <NUM>, about <NUM>, about <NUM>, about <NUM>, about <NUM>, about <NUM>, or about <NUM>. The height of the radially inner side <NUM> can be about <NUM>, or about <NUM>, or about <NUM>, and the height of the radially outer side <NUM> can be about <NUM>, or about <NUM>, or about <NUM>, or about <NUM>.

The top side and the bottom side of the trapezoidal cross section can have a width in a range of between about <NUM> and about <NUM>, such as a width of about <NUM>, about <NUM>, about <NUM>, about <NUM>, about <NUM>, about <NUM>, about <NUM>, about <NUM>, about <NUM>, about <NUM>, about <NUM>, about <NUM>, about <NUM>, about <NUM>, about <NUM>, about <NUM>, about <NUM>, or a width of about <NUM>.

For microchannels having a rectangular cross-section, an exemplary aspect ratio is between about <NUM> and about <NUM>; or between about <NUM> and about <NUM>. Exemplary average heights can be about <NUM> to about <NUM>, or about <NUM> to about <NUM>. Exemplary average widths can be about <NUM> to about <NUM>, for example, about <NUM>. In certain examples, the average height of the rectangular microchannel is about <NUM> and the average width is about <NUM>, or the average height is about <NUM> and the average width is about <NUM>. Other aspect ratios, heights and widths can also be employed for a rectangular microchannel.

Spiral microchannels can comprise one or more loops. Each of the spiral microchannel can independently be a <NUM> loop microchannel, a <NUM> loop microchannel, a <NUM> loop microchannel a <NUM> loop microchannel, a <NUM> loop microchannel, a <NUM> loop microchannel, an <NUM> loop microchannel, a <NUM> loop microchannel, a <NUM> loop microchannel, etc. The device can, for example, comprise <NUM>-loop or <NUM>-loop spiral microchannels with one inlet and two or more outlets with a radius of curvature decreasing from about <NUM> at the inlet to about <NUM> at the two outlets for efficient cell migration and focusing. The width of the channel cross-section can be about <NUM> and the inner/outer heights can be about <NUM> and about <NUM>, respectively, for the trapezoid cross-section.

A variety of particles can be separated using the microfluidic devices described herein. Larger particles can be separated from smaller particles (e.g. particles have a large CR can be separated from particles having an intermediate or small CR). Larger particles can have a diameter from about <NUM> to about <NUM>. For example, larger particles can have a diameter of about <NUM>, about <NUM>, about <NUM>, about <NUM>, about <NUM>, about <NUM>, about <NUM>, about <NUM>, about <NUM>, about <NUM>, about <NUM>, about <NUM>, about <NUM>, about <NUM>, about <NUM>, about <NUM>, about <NUM>, about <NUM>, about <NUM>, about <NUM>, about <NUM>, about <NUM>, about <NUM>, about <NUM>, about <NUM>, about <NUM>, about <NUM>, about <NUM>, about <NUM>, about <NUM>, about <NUM>, or about <NUM>. Smaller particles can have a diameter from about <NUM> to about <NUM>. For example, smaller particles can have a diameter of about <NUM>, about <NUM>, about <NUM>, about <NUM>, about <NUM>, about <NUM>, about <NUM>, about <NUM>, about <NUM>, about <NUM>, about <NUM>, about <NUM>, or about <NUM>. The flow rate can be about <NUM>/min, the larger particles can have a diameter in a range of between about <NUM> and about <NUM>, and the smaller particles can have a diameter in a range of between about <NUM> and about <NUM>. The flow rate can be about <NUM>/min, the larger particles can have a diameter in a range of between about <NUM> and about <NUM>, and the smaller particles can have a diameter in a range of between about <NUM> and about <NUM>. The flow rate can be in a range of between about <NUM>/min and about <NUM>/min, the larger particles can have a diameter in a range of between about <NUM> and about <NUM>, and the smaller particles can have a diameter in a range of between about <NUM> and about <NUM>.

The particles can be cells, such as stem cells or rare cells or blood cells (such as white blood cells and/or red blood cells). The cells can be present in a biological fluid (e.g., blood, urine, lymph, cerebrospinal fluid, and the like). The method thus encompasses methods of separating cells (for example, of different types) based on size. For example, the cells are present in a blood sample, wherein the larger cells are circulating tumor cells (CTCs), and the smaller cells are hematologic cells. The CTCs may be cancer cells (e.g., metastatic cancer cells) from a (one or more) breast cancer, colorectal cancer, kidney cancer, lung cancer, gastric cancer, prostate cancer, ovarian cancer, squamous cell cancer, hepatocellular cancer, nasopharyngeal cancer and other types of cancer cells. Because this approach does not require initial cell surface biomarker selection, it is suitable for use in different cancers of both epithelial and non-epithelial origin.

In another example, white blood cells (WBCs) can be separated from red blood cells. For example, WBCs and RBCs from a blood sample can be separated using the methods described herein.

The methods described herein can further comprise collecting and isolating the separated particles, including cells, nucleic acids and proteins. The method can further comprise downstream analysis such as immunostaining, qRT-PCR, FISH and sequencing. The method can further comprise conducting a heterogeneity study.

As will also be appreciated by those of skill in the art, the microfluidic device can further comprise other components upstream, downstream, or within a device. For example, one or more microfluidic devices can further comprise one or more collection devices (e.g., a reservoir), flow devices (e.g., a syringe, pump, pressure gauge, temperature gauge), analysis devices (e.g., a <NUM>-well microtiter plate, a microscope), filtration devices (e.g., a membrane), e.g., for upstream or downstream analysis (e.g., immunostaining, polymerase chain reaction (PCR) such as reverse PCR, quantitative PCR), fluorescence (e.g., fluorescence in situ hybridization (FISH)), sequencing, and the like. An imaging system may be connected to the device, to capture images from the device, and/or may receive light from the device, in order to permit real time visualization of the isolation process and/or to permit real time enumeration of isolated cells. In one example, the imaging system may view and/or digitize the image obtained through a microscope when the device is mounted on a microscope slide. For instance, the imaging system may include a digitizer and/or camera coupled to the microscope and to a viewing monitor and computer processor. The device may comprise a pump such as a syringe pump, a pressure pump, a peristaltic pump, or a combination of any of thereof. The device may be portable.

Spiral microchannels can be made from glass, silicone, and/or plastic. Microfluidic channels can be cast from a polymethylmethacrylate (PMMA) mold made by a precision milling process (Whits Technologies, Singapore). The patterns can be cast with Sylgard <NUM> Silicone Elastomer (PDMS) prepolymer mixed in a <NUM>:<NUM> ratio with the curing agent and cured under 80C for <NUM> hours. After curing, the PDMS mold with patterns can be peeled and plasma bonded to another <NUM> thick PDMS layer. Input and output ports can be punched prior to bonding. For the observation of particle position from the side, the device can be cut along the output section of the channel with about <NUM> distance and then a second cast can be made by keeping the device vertical to a flat bottle container. Tubings can be connected to the ports before the second cast to prevent PDMS mixer flow into the channel. The spiral microchannel may be made from plastic. A plastic device can, for example, be made by an injection molding method for its mass-production and/or disposable usage.

The invention is illustrated by the following examples which are not meant to be limiting in any way.

Inertial spiral microfluidic devices were fabricated in poly-dimethylsiloxane (PDMS) using standard micro-fabrication soft-lithographic techniques described previously. The master mold with specific channel dimensions was designed using SolidWorks® software and then fabricated by micro-milling machine (Whits Technologies, Singapore) on aluminum for PDMS casting. The PDMS replica was fabricated by molding degassed PDMS (mixed in a <NUM>:<NUM> ratio of base and curing agent, Sylgard <NUM>, Dow Corning Inc. ) on the mold and baking in the oven for <NUM> hour at <NUM>. The fluidic access holes were punched inside the device using Uni-Core™ Puncher (Sigma-Aldrich Co. SG) and the device was irreversibly bonded to a thick layer of plain PDMS using a plasma machine (Harrick Plasma, USA). The assembled device was finally placed inside an oven at <NUM> for <NUM> minutes to further enhancement of bonding strength. To efficiently and evenly deliver fluid from the sample tube to four spiral channels, 3D-printed (ProtoLab, USA) guide layer with internal fluidic channel was made, which can be inserted into PDMS device. For injection of sample fluid, a peristaltic pump (Cole-Parmer, USA) or a syringe pump (Harvard Apparatus, USA) was connected to microfluidics and the sample tube through silicone tubings (Cole-Parmer, USA).

A device can also be a plastic device fabricated by injection molding. Such a method of fabrication may offer an advantage over a PDMS device in that fabrication may be simpler and more reproducible. For example, the master mold can be designed using the same process as the PDMS device and then the plastic devices can be fabricated through injection molding. The fluidic access holes are already fabricated in the plastic device and the plastic device can be bonded to a film, such as the <NUM>™ 9795R Advanced Polyolefin Diagnostic Microfluidic Medical Tape, by pushing to seal the channels of plastic device.

<FIG> shows the channel configuration of a developed multi-dimensional double spiral (MDDS) device and its operation schematics. As shown in <FIG>, the MDDS device is composed of two spiral channels having two different dimensions. Samples containing various sizes of particles are injected into the device. In the first spiral channel, because the channel has relatively smaller dimension, particles can have larger confinement ratio (CR=a/Dh, where a is the particle diameter and Dh is the hydraulic diameter of microchannel) so that all particles become concentrated quite close to the inner wall side with having almost same equilibrium locations. While passing through the S-shaped transition region, the concentrated particles near the inner wall side of the first spiral channel enter the outer wall side of the second spiral channel. As a result, a sample enters the second spiral channel in a concentrated band near the outer wall side, as if focusing the sample by the use of additional sheath flow. In the second spiral channel which has relatively larger dimension, the particle's CR value decreases due to the increased channel size, resulting in the equilibrium location's shift toward the outer wall side. As a result, particles form a concentrated band at different equilibrium locations depending on their sizes, which is same mechanism with the two-inlets spiral device with an additional sheath flow. 4A,8A,11A,12A.

<FIG> shows size-based particle separation based on the MDDS device (<FIG>) having two-outlets configuration, compared to the single spiral channel (<FIG>) which has the same dimensions with the second spiral channel of the MDDS device. The first spiral channel has a rectangular cross-section with a width of <NUM> width and a height of <NUM>. In contrast to the first spiral channel, the second spiral channel is designed with larger dimensions and has a trapezoidal cross-section for the effective particle separation: the width is <NUM>, and heights are <NUM> and <NUM> for the inner wall side and the outer wall side, respectively. As we expected, under the optimized flow rate condition (<NUM>/min), both <NUM> and <NUM> particles were highly concentrated on the inner wall side during passing through the first spiral channel with the smaller dimension due to their high CR conditions (<NUM> particle: ~<NUM>, <NUM> particle: -<NUM>) (<FIG>). The concentrated bands enter the outer wall side of the second spiral channel (having larger dimension than the first spiral channel) and the particles become separated with two different equilibrium locations as shown in <FIG>; the changed CR values for <NUM> and <NUM> particles are -<NUM> and -<NUM>, respectively. Due to the initial focusing from the first spiral channel, particles can be separated with higher separation resolution and separation efficiency, compared to the single spiral channel, just like using an additional sheath flow. 4A,8A,11A,12A Furthermore, due to the sequential pinch effect of the double spiral channel,5A-7A,7A the focusing band becomes narrower and sharper as shown for the stream of <NUM> particles as compared to the single spiral channel.

The multi-dimensional double spiral (MDDS) device proposed here was designed as a new type of the spiral device to overcome the limitation of the spiral device with an additional sheath flow; the initial focusing of target particles can be made in the MDDS device without an additional sheath flow. <FIG> shows the channel configuration of the developed multi-dimensional double spiral (MDDS) device and its operation schematics. As shown in <FIG>, the MDDS device is composed of sequentially connected two spiral channels having two different dimensions; the first spiral channel has rectangular cross-section with <NUM> in width and <NUM> in height, and the second spiral channel was designed having larger dimension and trapezoidal cross-section for the effective particle separation with <NUM> in width and <NUM> and <NUM> in height for the inner wall side and the outer wall side, respectively. 7B <FIG> shows the trajectory of particles at the optimized flow rate condition (<NUM>/min) in the MDDS device; particles having diameters of <NUM> (green) and <NUM> (red) were used to mimic the movement of RBCs and WBCs, respectively. In the first spiral channel, all the target particles (here, which are RBCs and WBCs) are under the large confinement ratio condition (CR=a/Dh≥<NUM>, where a is the particle diameter and Dh is the hydraulic diameter of microchannel) so that RBCs as well as WBCs can be focused into the inner wall side (<FIG>); CR values of <NUM> and <NUM> particles are -<NUM> and -<NUM>, respectively. During passing through the S-shaped transition region, the concentrated stream near the inner wall side of the first spiral channel enters to the outer wall side of the second spiral channel having relatively larger dimension. In the second spiral channel, due to the increased channel dimension, RBCs no longer meet the large CR condition so that only WBCs can be focused into the inner wall side of the second spiral channel while RBCs move with being extracted into the outer wall side (<FIG>); CR values of <NUM> and <NUM> particles are -<NUM> and -<NUM>, respectively, and spiral channel with trapezoidal cross-section was used as the second spiral channel for better extraction of smaller particles, RBCs. 7B In the MDDS device, because sample fluid can be infused into the second spiral channel with a concentrated band formed near the outer wall side, as if focusing the sample by using the additional sheath flow, particle dispersion can be significantly decreased, and smaller particles can be effectively extracted into the outer-wall side of the second channel, resulting in increase of separation resolution compared to the single spiral device (<FIG> vs <FIG>); the single spiral device has the same dimension with the second spiral channel of the MDDS device.

<FIG> shows the results of blood separation in the MDDS device compared with the single spiral device. As we expected from the separation of <NUM> and <NUM> particles, although the performance varied depending on the blood dilution condition, we found that RBCs can be quite more effectively extracted into the outer wall side of the channel in the MDDS device compared to the single spiral device (<FIG> vs. <FIG>), resulting in low recovery of RBCs in the inner wall side outlet (<<NUM>% and <<NUM>% for 500x and 1000x dilution conditions, respectively, as shown in <FIG>), while both devices similarly showed great performance in the recovery of WBCs (><NUM>% in the MDDS device for all the dilution conditions, as shown in <FIG>); as the dilution rate decreases, the distribution of RBCs across channel width is broadened due to the increase of solid fraction of (mainly contributed by RBC population), which leads to decrease in RBC removal (<FIG>).

To obtain more purified and concentrated WBCs, we developed a recirculation platform using a check-valve where only one direction of flow is allowed while the opposite direction of flow is blocked by the internal membrane. The dual-check-valve we used in the platform involves two different check-valves so that once separated WBCs output can be extracted back into the input syringe at the withdrawal motion of a syringe pump and processed again through the MDDS device at the infusion motion of a syringe pump, resulting in higher purity and concentration (<FIG>). In our experiments, 500x diluted blood sample (<NUM>µL of human peripheral blood in <NUM> PBS) was determined as the initial input sample considering the hematocrit-dependent separation performance (<FIG>), the required sample volume (<NUM>µL of blood which can be drawn via finger stick), and operation time. A connector was fabricated by 3D printing to directly connect the MDDS device, syringe(s) (e.g., syringes that can be used for input and output reservoirs), and the check-valves for easier device assembly, higher portability, and minimized dead volume <FIG> and <FIG>). Through the programmed back-and-forth motions of syringe pumps (three cycles of recirculation), about <NUM> volume of highly purified and concentrated WBCs sample can be obtained within <NUM> minutes in a fully-automated manner (><NUM>% of RBC removal, ><NUM>% of WBC recovery, ><NUM>% of WBC purity at the optimized flow rate condition, <NUM>/min); for each cycle, we obtained an output having half volume of input sample where about <NUM>% of RBCs was removed while about <NUM>% of WBCs is recovered (<FIG>). To increase throughput and reduce operation time, we developed the quad-version of MDDS device (<FIG>) with a new 3D printed connector which can directly connect two quad-version of MDDS devices (involving <NUM> MDDS devices) and syringes (for input and output reservoirs) (<FIG>, <FIG>); a small pressure meter mounted connector was designed for the hand-held operation of the platform (see the section <NUM>), but the simplified version of the connector without the pressure meter was used for the general syringe-pump operation. From the three cycles of recirculation using the platform of two quad-version of MDDS devices, we can obtain about <NUM> volume of highly purified and concentrated WBCs sample within only <NUM> minutes in a fully-automated manner (><NUM>% of RBC removal, ~<NUM>% of WBC recovery, ><NUM>% of WBC purity at the optimized flow rate condition, <NUM>*<NUM>=<NUM>/min) (<FIG>).

To validate its reliability, we also tested its parallel operation using three different platforms and three different blood samples (<FIG>). The results showed that the device-dependent variation was quite small for all the blood samples and all the blood cell types as the recoveries and purity of WBCs have coefficient of variation (CV) less than <NUM>%; error bars of <FIG> represent standard deviation of the three different platforms). In the case of the sample-dependency, we found that the overall separation performance was good enough for all the blood samples (-<NUM>% of RBC removal, <NUM>-<NUM>% of WBC recovery, <NUM>-<NUM>% of WBC purity), but the recovery and purity rates of blood cells significantly changed depending on which blood sample was used. Cell type frequencies and their size distributions vary from donor to donor, which in turn leads to the different solid fraction and focusing behaviors of cells, resulting in the variation of the separation performance. Also, we found that for all the blood samples, the PMN recovery was better than the MNL recovery because generally size of PMN population (<NUM>-<NUM>) is bigger than MNL one (<NUM>-<NUM>), which corresponds with the result from the previous research using the spiral device.

Although WBCs can be efficiently separated and concentrated from the three-cycles of recirculation scheme using two-quad-version devices with very short operation time (within <NUM> minutes), the WBC purity could be still not enough for some WBC analyses; because the initial population of RBCs are about <NUM> times more than WBCs, even the output with -<NUM>% RBCs removed contains a similar number of RBCs with WBCs. For certain applications requiring higher WBC purity and concentration rather than fast operation, we designed another version of recirculation platform using one quad-version of MDDS device (<FIG>, <FIG>). The platform requires more operation time compared to the platform using two quad-version of MDDS devices, but the reduced dead volume makes it capable to process one more recirculation cycle; the four cycles of recirculation can be processed within <NUM> minutes. As shown in <FIG>, for each step, over <NUM>% of RBCs was removed while over <NUM>% of WBCs was recovered, which is slightly better performance compared to the platform using two quad-version of MDDS devices due to the reduced dead volume, and about <NUM> volume of highly purified and concentrated WBC sample was obtained from four cycles of recirculation (><NUM>% of RBC removal, -<NUM>% of WBC recovery, > <NUM>% of WBC purity). Similar to the platform using two quad-version of MDDS devices, we found the separation performance varied depending on which blood sample was used, and the overall separation performance became much better for all the blood samples (><NUM>% of RBC removal, <NUM>-<NUM>% of WBC recovery, <NUM>-<NUM>% of WBC purity) (<FIG>).

Human power could be considered as an ideal power source for driving the sample flow to operate the device in resource-poor environments. Because the MDDS device can be operated only by a sample flow without an additional sheath flow, and the recirculation method requires a simple back-and-forth motion of the input syringe, the developed platform can be operated by hand-powered syringe pushing and pulling. To find how much force is required for operating the device, we measured the applied force to the input syringe of the platform having two quad-version of MDDS devices by using a load cell which was placed between the syringe and the pusher block of the syringe pump; the output voltage from the load cell varies depending on the applied force, which is measured by a voltage-meter and transferred to an actual force value in real-time (<FIG>). From the results, the required force for the optimum flow rate (<NUM>/min) was measured about <NUM> N, which is reasonable force for hand-powered operation considering the average maximum pushing forces of male and female are over <NUM> and <NUM> N, respectively. 41B To apply proper force to the syringe on the hand-powered operation, a small pressure-meter was mounted on the 3D-printed connector; the pressure-meter is directly connected with the inlet channel of the 3D-printed connector and shows the pressure value at the inlet region. First, the pressure value was measured on the syringe pump operation under various flow-rate conditions. From the results, as we expected, we found that the load and pressure increased with a similar profile as the applied flow rate increased, and the pressure value corresponding to the optimum flow rate condition (<NUM>/min) was about <NUM> psi (<FIG>). Based on the pressure measurement from the syringe pump operation, the developed platform can be operated by simple hand-pushing and pulling motions; in the infusion step, the input syringe should be pushed while keeping pressure at the optimum pressure value (<NUM> psi) for optimum flow rate condition (<FIG>). <FIG> show the separation performance on the hand-powered operation with five different trials of three cycles of recirculation using the platform having two quad-version of MDDS devices. From the results, similar to the syringe-pump-based operation, we can obtain about <NUM> volume of highly purified and concentrated WBCs sample within only <NUM> minutes (-<NUM>% of RBC removal, -<NUM>% of WBC recovery, <NUM>-<NUM>% of WBC purity at the optimized flow rate condition, <NUM>*<NUM>=<NUM>/min) (<FIG>). Although the overall separation performance became degraded a little compared to the syringe-pump-based operation due to the inevitable flow fluctuation on the hand-powered operation, the hand-operable platform could be a very useful tool for blood preparation in resource-poor environments considering its simple and fast operating process with high reliability (less than <NUM>% of CV on the WBC recovery from the <NUM> different trials); for certain applications requiring higher WBC purity and concentration, the platform having one quad version of MDDS device could be used under hand-powered operation as well even though it requires more operation time.

The multi-dimensional double spiral (MDDS) device was fabricated in polydimethysiloxane (PDMS) following standard soft-lithographic techniques. 12B,36B The aluminum master mold with specific channel dimensions was designed using a 3D CAD software (SolidWorks <NUM>) and then fabricated by a micromilling company (Whits Technologies, Singapore) for PDMS casting. The PDMS replica was made by casting degassed PDMS (<NUM>: <NUM> mixture of base and curing agent of Sylgard <NUM>, Dow Corning Inc. ) onto the aluminum mold, followed by curing on the hot plate for <NUM> at <NUM>°. After making holes for fluidic access by disposable biopsy punches (Integra Miltex), the PDMS replica was irreversibly bonded to a glass slide using a plasma machine (Femto Science, Korea). The assembled device was placed in a <NUM>° oven for at least <NUM> to stabilize the bonding further.

Check-valve-based recirculation platform was designed to obtain more purified and concentrated WBCs. A connector of the platform was designed a 3D CAD software (SolidWorks <NUM>) and then fabricated by a 3D printer (Form <NUM>, formlabs, USA) with a specific resin (RS-F2-GPCL-<NUM>, formlabs, USA). Three different connectors were made for three different recirculation platforms having a single-version of MDDS device (<FIG> and <FIG>), two quad-version of MDDS device (<FIG> and <FIG>), and one quad-version of MDDS devices (<FIG> and <FIG>), respectively. Two kinds of check-valves were used; one is a dual-check-valve (<NUM>, QOSINA, USA) for regulating the flow direction on injection and extraction of sample, and the other is a check-valve (<NUM>, QOSINA, USA) for preventing the output in the RBC reservoir from flowing to the WBC reservoir. Using the 3D-printed connectors, we can directly connect the MDDS device, syringes (for input and output reservoirs), and the check-valves through simple and easy assembly process, resulting in the recirculation platforms having high portability and minimized dead volume. To prevent cross-contamination caused by the trapped cells on the internal membrane inside the check-valves, we used a new check-valve for each experiment; the check-valves we used are very cheap (about $<NUM>) to be used in the disposable manner.

For bead experiments, fluorescent polystyrene particles with diameter of <NUM> (<NUM>-<NUM>, Polysciences, Inc. , USA) and <NUM> (F8834, Invitrogen™, USA) were used after dilution in deionized water. For blood separation tests, we used fresh human whole blood samples purchased from Research Blood Components, LLC (Boston, MA, U. ) with dilution in 1x phosphate-buffered saline without calcium and magnesium (PBS, Corning®). For the operation of the recirculation platform, considering the hematocrit-dependent separation performance (<FIG>), the required sample volume (<NUM>µL of blood which can be drawn via finger stick), and operation time, 500x dilution condition (<NUM>µL of human peripheral blood in <NUM> 1x PBS) was chosen.

Samples were loaded to the device with the regulated flow rate by a syringe pump (Fusion <NUM>, Chemyx Inc. An inverted fluorescent microscope (IXS <NUM>, Olympus Inc. , USA) and a CCD camera (Sensicam QE, PCO, Germany) were used to observe the trajectories of the fluorescent particles and collect images from the device. Due to the absence of fluorescence, the trajectories of blood cells were observed by using a high-speed camera (Phantom v9. <NUM>, Vision Research Inc. , USA) with a certain sample rate, <NUM> pictures per second (pps).

To determine the separation efficiency, input and output samples were collected and analyzed by a flow cytometer (Accuri C6, BD Biosciences, USA) with staining the samples with the following antibodies: fluorescein isothiocyanate (FITC)-conjugated CD45 monoclonal antibody (positive for all leukocytes) and Allophycocyanin (APC)-conjugated CD66b monoclonal antibody (positive for polymorphonuclear leukocytes, PMNs); all the antibodies were purchased from eBioscience™. Considering that mononuclear leukocytes (MNLs) are composed of various cell types, and there is no efficient surface marker available to determine the total amount of MNLs, the number of MNLs was calculated as CD45-positive but CD66b-negative cells.

Claim 1:
A microfluidic device comprising a multidimensional double spiral (MDDS), wherein the MDDS comprises:
a. a first spiral microchannel comprising a first inlet;
b. a second spiral microchannel in fluid communication with the first spiral microchannel and comprising an inner wall outlet and an outer wall outlet, wherein the inner wall outlet is located on the inner wall side of the microchannel and the outer wall outlet is located on the outer wall side of the microchannel; and
c. a transition region, wherein the transition region is a microchannel that connects the first and second spiral microchannels, wherein the output from the first spiral microchannel is directed into the second spiral microchannel in the transition region;
characterized in that the first spiral microchannel has a smaller cross-sectional area than the second spiral microchannel;
wherein the cross-sectional area of the first spiral microchannel remains constant along its length and wherein the cross-sectional area of the second spiral microchannel remain constant along its length; and
wherein the MDDS is configured to separate particles from a sample fluid comprising a mixture of particles.