CN115023599A

CN115023599A - Single-sheath microfluidic chip

Info

Publication number: CN115023599A
Application number: CN202080091173.2A
Authority: CN
Inventors: 郑·霞; 戈帕库马尔·卡马拉克沙库鲁普
Original assignee: Ebs International Co ltd
Current assignee: Ebs International Co ltd
Priority date: 2020-01-13
Filing date: 2020-01-13
Publication date: 2022-09-06
Also published as: BR112022012881A2; EP4090932A4; EP4090932A1; JP2023514941A; WO2021145854A1

Abstract

Microfluidic devices and methods for focusing components in a fluid sample are described herein. The microfluidic device features a microfluidic chip having a microchannel with a narrowed portion that narrows in width; and a flow focusing region downstream of the microchannel. The flow focusing region includes: a positively sloped floor that reduces the height of the flow focusing region; sidewalls that taper to reduce the width of the flow focusing region to geometrically narrow the flow focusing region. The apparatus and method may be used for sex sorting of sperm cells to improve performance and eligibility.

Description

Single-sheath microfluidic chip

Background of the present application

Technical Field

The present application relates to microfluidic chip designs, and more particularly to microfluidic chips that use laminar flow from single sheath and geometric focusing to separate particles or cellular material.

Background

Microfluidics enables the preparation and processing of samples, such as a variety of particles or cellular materials, using small volumes. In isolating samples, such as viable and motile sperm from non-viable or immotile sperm, or separation by sex, the process is often time consuming and subject to stringent volume constraints. Current separation techniques, for example, do not produce the desired yield or do not process large amounts of cellular material in a timely manner. Furthermore, existing microfluidic devices do not effectively focus or orient sperm cells.

Therefore, there is a need for a microfluidic device and a separation method using the device that is continuous, has a high throughput, saves time, and causes negligible or minimal damage to the various components being separated. In addition, such apparatus and methods may be further adapted for use in the biological and medical fields, not only in sperm sorting, but also in the separation of blood and other cellular materials, including viruses, organelles, globular tissues, colloidal suspensions, and other biological materials.

Disclosure of Invention

It is an object of the present invention to provide a microfluidic device and a method that can focus and orient particles or cellular material as described in the independent claims. Embodiments of the invention are given in the dependent claims. Embodiments of the present invention can be freely combined with each other if they are not mutually exclusive.

In some aspects, the invention features a microfluidic device for sperm cell sexing and feature enrichment. The microfluidic device may include at least one flow focusing region where the components are focused or redirected by the geometry of the region. At least a portion of the flow focusing region decreases in height and at least a portion narrows in width from an upstream end to a downstream end of the flow focusing region, thereby geometrically narrowing the flow focusing region.

According to some embodiments, the invention features a microfluidic chip that includes a microchannel having a narrowed portion that narrows in width; a flow focusing region downstream of the microchannel, the flow focusing region including a positively sloped floor that reduces a height of the flow focusing region; and a sidewall that tapers to reduce a width of the flow focusing region to geometrically narrow the flow focusing region.

In another embodiment, the microfluidic chip may include: a sample microchannel; two sheath fluid microchannels intersecting the sample microchannel to form an intersection region; a downstream microchannel fluidly connected to the crossover region; and a downstream flow focusing region fluidly connected to the downstream microchannel. The downstream microchannel may have a narrowed portion with a narrowed width. The flow focusing region may include: a positively sloped floor that reduces the height of the flow focusing region; sidewalls that taper to reduce a width of the flow focusing region to geometrically narrow the flow focusing region. The sample microchannel is configured to flow a sample fluid mixture, the two sheath fluid microchannels each being configured to flow a sheath fluid into the intersection region to induce laminar flow, and to compress the sample fluid mixture flowing from the sample microchannel from at least two sides at least horizontally such that the sample fluid mixture is surrounded by a sheath flow and compressed into a trickle. The crossover region and the downstream flow focusing region are configured to focus material in the sample fluid mixture. Compression of the sample fluid mixture causes the material in the sample fluid mixture to aggregate, thereby focusing the material at or near the center of the downstream microchannel.

In some embodiments, the narrowing portion of the microchannel comprises a tapered sidewall. In other embodiments, the positively sloped floor and the tapered sidewall occur simultaneously from an upstream end to a downstream end of the flow focusing region. The positively sloped floor and the tapered sidewalls begin at a plane that is perpendicular across the flow focusing region. In other embodiments, the sample microchannel includes a constriction downstream of the inlet of the sample microchannel. The narrowing region may include a positively sloped floor that reduces the height of the narrowing region; and sidewalls that taper to reduce the width of the narrowing region. The positively sloped floor and the tapered sidewalls can geometrically narrow the narrowing region.

In one embodiment, the outlet of the sample microchannel may be at or near the mid-height of the outlet of each of the two sheath fluid microchannels. The inlet of the downstream microchannel may be at or near the mid-height of the outlet of each of the two sheath fluid microchannels. In other embodiments, the outlet of the sample microchannel is at or near the mid-height of the intersection region. The inlet of the downstream microchannel is located at or near the mid-height of the crossover region. In yet another embodiment, the outlet of the sample microchannel and the inlet of the downstream microchannel may be aligned or not aligned.

In some embodiments, the microfluidic chip may further comprise an interrogation zone downstream of the flow focusing region. The microfluidic chip may further comprise an expansion region downstream of the interrogation region. The expansion region may include a negatively sloped floor that increases the height of the narrowing region; and an extension having sidewalls that widen to increase a width of the extension. In other embodiments, the microfluidic chip may further comprise a plurality of output microchannels downstream of and fluidically coupled to the expansion regions.

According to other embodiments, the present invention provides a method of using the microfluidic chip. In some embodiments, the invention features a method of focusing particles in a fluid flow, the method including providing a microfluidic chip; flowing a fluid mixture comprising the particles into a sample microchannel and into the intersection region; flowing a sheath fluid through the two sheath fluid microchannels and into the intersection region such that the sheath fluid induces a laminar flow and compresses the fluid mixture at least horizontally from at least two sides, wherein the fluid mixture is surrounded by sheath fluid and compressed into a thin stream, the particles being constricted to the thin stream surrounded by the sheath fluid; flowing the fluid mixture and a sheath fluid into the downstream microchannel, wherein the constriction of the downstream microchannel horizontally compresses a thin stream of the fluid mixture; flowing a fluid mixture and a sheath fluid into the focusing region, wherein the positively sloped floor and the tapered sidewalls further constrict the fluid mixture flow and redirect particles in the fluid, thereby focusing the particles.

In other embodiments, the invention features a method of producing a sperm cell fluid having a gender bias. The method may include providing a microfluidic chip; flowing a semen fluid including sperm cells into the sample microchannel and into the intersection region; flowing a sheath fluid through the two sheath fluid microchannels and into the crossover region such that the sheath fluid induces laminar flow and compresses the seminal fluid at least horizontally from at least two sides, wherein the seminal fluid is surrounded by sheath fluid and compressed into a thin stream; flowing the semen fluid and sheath fluid into the downstream microchannel, wherein the constriction horizontally compresses the thin stream of semen fluid; flowing the semen fluid and sheath fluid into the focusing region, wherein the positively sloped floor and the gradually narrowing sidewall further constrict the semen fluid to focus the sperm cells at or near the center of the semen flow; determining a chromosome type of the sperm cells in the sperm stream, wherein each sperm cell is a Y chromosome bearing sperm cell or an X chromosome bearing sperm cell; and sorting the Y chromosome bearing sperm cells from the X chromosome bearing sperm cells to produce a sex-skewed sperm cell fluid comprising predominantly Y-chromosome bearing sperm cells.

One of the unique and inventive features of the present invention is the physical limitation of the channel geometry of the flow focusing region. Without wishing to limit the invention to any theory or mechanism, it is believed that the technical features of the invention facilitate the elimination of secondary sheath fluid structures from microfluidic devices, such that the use of secondary sheath fluid is not required to focus/orient sperm cells, thus reducing the volume of sheath fluid used compared to prior devices having two focusing regions using sheath fluid for flow compression. This provides the additional benefit of reducing the operating costs of the equipment and supplies, further simplifying the complexity of the system. None of the existing references or techniques known at present possess the unique inventive features of the present invention.

The inventive features of the present invention result in Y-skewed sperm cells of equal purity, better performance and better function than prior devices using sheath fluids with two focusing regions, with surprising results. For example, the microfluidic device of the present invention unexpectedly improves sperm cell orientation, which is believed to improve eligibility, i.e., a greater number of cells are detected, sorted, and ablated. In addition, the device of the invention can improve the resolution between the sperm cell carrying the Y chromosome and the sperm cell carrying the X chromosome, thereby effectively distinguishing the sperm cell carrying the Y chromosome.

In addition, the prior references give reverse teaching to the present invention. For example, in contrast to the present invention, U.S. patent No. 7311476 teaches the use of sheath fluid to focus fluid flow in the microfluidic chip disclosed therein having at least two regions, where each region introduces sheath fluid to focus the sheath fluid around the surrounding particle, and the second (downstream) region requires the introduction of additional sheath fluid to achieve the necessary focusing.

In some embodiments, the microfluidic chip comprises a plurality of layers having a plurality of channels disposed therein, including: a sample input channel into which a sample fluid mixture of components to be separated is input; and two focusing regions including a primary focusing region for focusing the particles in the sample fluid and a secondary focusing region for focusing the particles in the sample fluid, wherein one of the focusing regions includes introducing sheath fluid through one or more sheath fluid channels and the other focusing region includes geometric compression without introducing additional sheath fluid. Geometric compression refers to physical constriction resulting from the narrowing of the sample channel dimension in both the vertical and horizontal axes (i.e., from above and below and from the left and right, relative to the direction of travel of the sample channel). In some aspects, the primary focusing region may incorporate a combination of geometry and sheath fluid introduction. However, the second focusing region does not utilize additional sheath fluid for flow focusing or particle orientation. In other aspects, the microfluidic chip can be loaded into a microfluidic chip cartridge that is mounted on a microfluidic chip mount.

In some embodiments, the sample input channel and the one or more sheath fluid channels are disposed at one or more planes of the microfluidic chip. For example, the sheath fluid channel may be provided in a different plane to that in which the sample input channel is provided. In other embodiments, the sample input channel and the sheath fluid channel are disposed in one or more structural layers, or between structural layers of the microfluidic chip. For example, the one or more sheath fluid channels may be provided in a different structural layer to that in which the sample input channel is provided.

In one embodiment, the sample input channel may be tapered at the entry point into the intersection region with the sheath fluid channel. In another embodiment, the sheath fluid channel may be tapered at the entry point into the intersection region with the sample input channel. In some embodiments, the microfluidic device may include one or more output channels fluidically coupled to the sample channel. The one or more output channels may each have an output disposed at an end thereof. In other embodiments, the microfluidic chip may further include one or more notches disposed at a bottom edge of the microfluidic chip to separate outputs of the output channels.

In some embodiments, the microfluidic chip system includes an interrogation device that interrogates and identifies a component of the sample fluid mixture in the sample input channel in an interrogation chamber located downstream of the flow focusing region. In one embodiment, the interrogation device includes a radiation source configured to emit a light beam to illuminate and excite the constituent in the sample fluid mixture. The emitted light generated by the light beam is received by the objective lens. In another embodiment, the interrogation device may include a detector, such as a photomultiplier tube (PMT), Avalanche Photodiode (APD), or silicon photomultiplier tube (SiPM).

In some embodiments, the microfluidic chip comprises a sorting mechanism that sorts the components in the sample fluid mixture by selectively acting on individual components in the sample fluid mixture. In one embodiment, the sorting mechanism may include laser killing/ablation. Other examples of sorting mechanisms that may be used in accordance with the present invention include, but are not limited to, particle deflection/electrostatic manipulation, droplet sorting/deflection, mechanical sorting, fluidic switching, piezoelectric actuation, optical manipulation (optical trapping, holographic steering, and photonic/radiation pressure), Surface Acoustic Wave (SAW) deflection, electrophoresis/electrical disruption, micro-voiding (laser induced, electrically induced). In some embodiments, the separated components are moved into one of the output channels and the unselected components flow out through the other output channel.

In other embodiments, the microfluidic chip may be operatively coupled to a computer that controls pumping of a sample fluid mixture or sheath fluid to the microfluidic chip. In another embodiment, the computer is capable of displaying the components in a field of view acquired by a CCD camera disposed above an interrogation window in the microfluidic chip.

In some embodiments, the cells being isolated may include at least one of: viable and motile sperm separated from non-viable or non-motile sperm; sorting the separated sperm according to sex and other sex; stem cells isolated from cells in the population; separating one or more labeled cells, including sperm cells, from unlabeled cells by an ideal/undesirable trait; genes isolated in nuclear DNA according to specific characteristics; cells isolated based on surface labeling; cells isolated based on membrane integrity or activity; cells isolated based on a potential or predicted reproductive state; cells isolated based on frozen viability; cells separated from contaminants or debris; isolating healthy cells from the damaged cells; red blood cells separated from white blood cells and platelets in a plasma mixture; or any cell separated into the corresponding fractions from any other cellular components.

Any feature or combination of features described herein is included within the scope of the present invention provided that the features included in any such combination are not mutually inconsistent as will be apparent from the context, this specification, and the knowledge of one of ordinary skill in the art. Other advantages and aspects of the invention will become apparent from the following detailed description and claims.

Drawings

The features and advantages of the present invention will become apparent with reference to the following detailed description when taken in conjunction with the accompanying drawings.

Fig. 1A is a bottom view of a top layer of a microfluidic device according to an embodiment of the present invention.

Fig. 1B is a top view of the bottom layer of the microfluidic device.

Fig. 1C is a side view of a top layer stacked on a bottom layer of the microfluidic device.

Figure 2A is a close-up view and a cross-sectional side view of the intersection region of the top layer shown in figure 1A.

FIG. 2B is a close-up view and a cross-sectional side view of the intersection region of the bottom layer shown in FIG. 1B.

FIG. 2C is a close-up view and a cross-sectional side view of the intersection region of the stacked layers shown in FIG. 1C.

FIG. 3A is a close-up view and a cross-sectional side view of the flow focusing region of the top layer shown in FIG. 1A.

FIG. 3B is a close-up view and a cross-sectional side view of the flow focusing region of the bottom layer shown in FIG. 1B.

FIG. 3C is a close-up view and a cross-sectional side view of the flow focusing region of the stack of layers shown in FIG. 1C.

FIG. 4 is a close-up view of the flow focusing region shown in FIG. 1B.

FIG. 5 is a non-limiting embodiment of top and side views of a downstream microchannel and flow focusing region. This embodiment shows the simultaneous geometric compression of the narrowing portion of the downstream microchannel and the floor and sidewalls of the flow focusing region.

Fig. 6 shows a close-up view and a cross-sectional side view of the output channel region of the bottom layer shown in fig. 1B.

Figure 7 is a non-limiting example of a flow chart of a semen fluid sample gender inclination method.

Detailed Description

Before turning to the figures, which illustrate the illustrative embodiments in detail, it is to be understood that the disclosure is not limited to the details or methodology set forth in the specification or illustrated in the figures. It is also to be understood that the terminology is for the purpose of description and should not be regarded as limiting. Throughout the drawings, the same or similar reference numerals have been used in an effort to designate the same or similar parts.

The following is a list of elements corresponding to the specific elements referred to herein:

100 microfluidic chip

110 sample microchannel

111 inlet of sample microchannel

112 narrowing region

113 outlet of sample microchannel

114 bottom surface of the narrowing region

115 side walls of the narrowing region

120 downstream of the microchannel

122 narrow part

124 downstream of the microchannel

125 narrow part of the side wall

130 flow focal zone

132 bottom surface of flow focusing region

135 flow focusing region sidewall

137 upstream end of flow focusing region

138 downstream end of flow focusing region

140 sheath fluid microchannel

143 sheath fluid microchannel exit

145 cross section

150 interrogation zone

160 extension area

162 bottom surface of extension region

165 sidewalls of the extension region

170 output microchannel

In one aspect, the present disclosure relates to a microfluidic chip design and method that can separate particle or cellular material, such as sperm and other particles or cells, into various components and fractions. For example, various embodiments of the invention provide for separating components in a mixture, such as separating viable and motile sperm from non-viable or immotile sperm; sorting and separating sperm according to sex and other sex; isolating stem cells from cells in the population; separating one or more labeled cells from unlabeled cells to distinguish desired/undesired traits; isolating genes in the nuclear DNA according to specific characteristics; isolating cells based on the surface markers; isolating cells based on membrane integrity (activity), potential or predicted reproductive status (fertility), frozen survival, etc.; separating the cells from the contaminants or debris; isolating healthy cells (e.g., bone marrow extraction) from damaged cells (i.e., cancer cells); red blood cells from white blood cells and platelets in the plasma mixture; separating any cells from any other cellular components into the corresponding fractions.

In another aspect, various embodiments of the present invention provide a system and method particularly useful for sorting sperm cells to produce a sexual semen product in which viable, forward-motile sperm cells are primarily Y chromosome-bearing sperm cells. In some embodiments, the systems and methods of the present invention are capable of producing a gender sorted or gender skewed semen product that includes at least 55% Y chromosome bearing sperm cells. In other embodiments, the systems and methods can produce a sexual semen product that can include about 55% to about 90% Y chromosome bearing sperm cells. In still other embodiments, the systems and methods can produce a sexual semen product that can include at least about 90% or at least about 95% or at least about 99% Y chromosome-bearing sperm cells.

Although the following focuses on separating viable and motile sperm from non-viable or non-motile sperm, or separating sperm by gender and other gender sorting variants, or separating one or more labeled cells from unlabeled cells to distinguish between desirable/undesirable characteristics, etc., the present invention may be extended to other types of particles, organisms or cellular material, capable of being interrogated in a fluid by fluorescence techniques, or capable of being manipulated to different outputs between different fluid streams.

Various embodiments of the microfluidic chip utilize one or more flow channels having a substantially laminar flow and a flow focusing region for focusing and/or directing one or more components in a fluid, allowing the one or more components to be interrogated for identification and separated into streams that recede into one or more outputs. In addition, various sorting techniques may be used, for example, particle deflection/electrostatic manipulation; droplet sorting/deflection; mechanical sorting; switching the fluid; piezoelectric driving; optical manipulation (optical trapping, holographic steering and photon/radiation pressure); laser killing/ablation; surface Acoustic Wave (SAW) deflection; electrophoresis/electrical interruption; microcavitation (laser-induced, electrical-induced); or by magnetic (i.e., using magnetic beads) the various components of the mixture are subjected to one or more sorting processes on the chip. Thus, various embodiments of the present invention thus provide for continuous focusing and separation of components without potential damage and contamination to prior art methods, particularly in sperm separation. The continuous process of the present invention also provides substantial time savings in separating the fluid components.

Microfluidic chip assembly

Referring to fig. 1A-6, the invention features a microfluidic chip (100). Non-limiting embodiments of the microfluidic chip (100) include: a sample microchannel (110); two sheath fluid microchannels (140) intersecting the sample microchannel (110) to form an intersection region (145); a downstream microchannel (120) fluidly connected to the crossover region (145), the downstream microchannel (120) having a narrowed portion (122) of narrowing width; and a downstream flow focusing region (130) fluidly connected to the downstream microchannel (120). The flow focusing region (130) may include a positively sloped bottom surface (132) that reduces a height of the flow focusing region; a sidewall (135) that tapers to reduce a width of the flow focusing region, thereby geometrically narrowing the flow focusing region (130).

Without wishing to limit the invention to a particular theory or mechanism, the sample microchannel (110) is configured to flow a sample fluid mixture, and the two sheath fluid microchannels (140) are each configured to flow a sheath fluid into the crossover region (145). The flow of sheath fluid causes a laminar flow and a compression of the sample fluid mixture flowing from the sample microchannel (110) from at least two sides, at least horizontally, such that the sample fluid mixture is surrounded by sheath fluid and compressed into a thin flow. In further iterations, additional sheath fluids may be incorporated to focus and/or adjust the position of the sample stream within the microchannel. Such that the sheath fluid may be introduced from one or more directions (e.g., top, bottom, and/or sides), and may be introduced simultaneously or sequentially.

In some embodiments, the narrowed portion (122) of the microchannel includes tapered sidewalls (125). For example, the sidewalls (125) may taper such that the width of the microchannel decreases from 150um to 125 um.

In some embodiments, the positively sloped floor (132) and the tapered sidewalls (135) occur simultaneously from an upstream end (137) to a downstream end (138) of the flow focusing region. Thereby, the positively sloped bottom surface (132) and the tapering side wall (135) have the same starting point. For example, the positively sloped floor (132) and the tapered sidewalls (135) begin from a plane that perpendicularly intersects the flow focusing region (130).

In other embodiments, the sample microchannel (110) includes a constriction region (112) downstream of the inlet (111) of the sample microchannel. The narrowing region (112) may include a positively sloped floor (114) that reduces the height of the narrowing region, and sidewalls (115) that taper to reduce the width of the narrowing region. The positively sloped floor (114) and the tapered side walls (115) can geometrically narrow the narrowing region (112).

In some embodiments, the outlet (113) of the sample microchannel may be at or near the mid-height of the outlet (143) of each of the two sheath fluid microchannels. The downstream microchannel outlet (124) may be at or near the mid-height of the outlet (143) of each of the two sheath fluid microchannels. The outlet (113) of the sample microchannel is aligned with the inlet (124) of the downstream microchannel. In other embodiments, the outlet (113) of the sample microchannel may be at or near the mid-height of the intersection region, and the inlet (124) of the downstream microchannel may be at or near the mid-height of the intersection region.

Without wishing to limit the invention to a particular theory or mechanism, the crossover region (145) and the downstream flow focusing region (130) are configured to focus material in the sample fluid mixture. For example, compression of the sample fluid mixture aggregates the material within the sample fluid mixture such that the material is focused at or near the center of the downstream microchannel.

In some embodiments, the microfluidic chip (100) may further include a plurality of output microchannels (170), the plurality of output microchannels (170) being located downstream of the expansion region (160) and fluidly coupled to the expansion region (160). The output microchannel (170) is configured to output a fluid, which may have a composition such as particles or cellular material. The output channels may each have an output disposed at an end thereof. In other embodiments, the microfluidic chip may further comprise one or more recesses disposed at a bottom edge of the microfluidic chip to separate outputs and provide an accessory for external tubing and the like. Non-limiting embodiments of the chip may include three output channels, including two side output channels and a central output channel disposed between the side channels.

In some embodiments, the microchannels and various regions of the microfluidic chip may be dimensioned to achieve a desired flow rate that meets the objectives of the present invention. In one embodiment, the microchannels may have substantially the same dimensions, however, it will be understood by those skilled in the art that the dimensions of any or all of the channels in the microfluidic chip may vary differently (i.e., between 50 and 500 microns) so long as the desired flow rate is achieved.

In some other embodiments, the microfluidic chip may further include an interrogation zone (150) downstream of the flow focusing region (130). In some other embodiments, the microfluidic chip may further include an expansion region (160) downstream of the interrogation region (150). The extension region (160) may include a negatively sloped floor (162) that increases the height of the narrowing region, and an extension portion having sidewalls (165), the sidewalls (165) widening to increase the width of the extension region.

In one embodiment, the interrogation device includes a chamber having an opening or window cut into the microfluidic chip. The opening or window may be fitted with a lid to close the interrogation chamber. The cover may be made of any material having the required transmission requirements, such as plastic, glass, and may even be a lens. In one embodiment, the window and cover allow viewing of the components of the fluid mixture flowing through the interrogation chamber and are acted upon by a suitable radiation source configured to emit a high intensity beam of light having any wavelength that matches the excitation of the components.

Although a laser may be used, it will be appreciated that other suitable radiation sources, such as Light Emitting Diodes (LEDs), arc lamps, etc., may be used to emit a beam of exciting components. In another embodiment, the light beam may be transmitted to the component through an optical fiber embedded in the microfluidic chip at the opening.

In some embodiments, a high intensity laser beam from a suitable laser of a preselected wavelength, such as a 355nm Continuous Wave (CW) (or quasi-CW) laser, is required to excite the components (i.e., sperm cells) in the liquid mixture. The laser emits a laser beam through the window to illuminate the components flowing through the interrogation zone of the chip. Since the laser beam can vary in intensity across the width of the microchannel, with the highest intensity generally being at the center of the microchannel (e.g., the middle portion of the channel width) and thus decreasing, the flow focusing region must focus the sperm cell at or near the center of the fluid flow where optimal illumination occurs at or near the center of the illumination laser spot. This may improve the accuracy of the interrogation and identification process without being bound by a particular concept.

In some embodiments, the high intensity light beam interacts with the components such that emitted light induced by the light beam is received by the objective lens. The objective lens may be arranged in any suitable position with respect to the microfluidic chip. In one embodiment, the emission light received by the objective lens is converted to an electronic signal by an optical sensor, such as a photomultiplier tube (PMT) or photodiode. The electronic signal may be digitized by an analog-to-digital converter (ADC) and sent to a Digital Signal Processor (DSP) based controller. The DSP-based controller monitors the electronic signal and may then trigger the sorting mechanism.

In other embodiments, the interrogation device may include a detector, such as a photomultiplier tube (PMT), Avalanche Photodiode (APD), or silicon photomultiplier tube (SiPM). For example, the optical sensor of the interrogation device may be an APD, which is a photodiode with substantial internal signal amplification by an avalanche process.

In some embodiments, a piezoelectric actuator assembly may be used to sort desired components in a fluid mixture as the components exit the interrogation zone after passing through the interrogation. The trigger signal sent to the piezoelectric actuator is determined by the raw sensor signal to activate the particular piezoelectric actuator assembly upon detection of the selected constituent. In some embodiments, a flexible membrane made of a suitable material, such as one of stainless steel, brass, titanium, nickel alloy, polymer, or other suitable material having a desired elastic response, is used with an actuator to push a target component in a microchannel into an output channel (170) to separate the target component from a fluid mixture. The actuator may be of the piezoelectric, magnetic, electrostatic, hydraulic or pneumatic type.

In alternative embodiments, a piezoelectric actuator assembly or suitable pumping system may be used to pump the sample fluid into the microchannel (110) towards the intersection region (145). The sample piezoelectric actuator assembly may be arranged at a sample inlet (111). By pumping the sample fluid mixture into the main microchannel, a controlled measurement of the inter-component spacing therein can be made so that a more controlled relationship can be established between the components as they enter the microchannel (110).

Other embodiments of sorting or separation mechanisms that may be used in accordance with the present invention include, but are not limited to, droplet sorting, mechanical separation, fluidic switching, acoustic focusing, holographic trapping/steering, and photonic pressure/steering. In a preferred embodiment, the sorting mechanism for sex sorting sperm cells includes laser killing/ablation of selected X chromosome bearing sperm cells.

In laser ablation, a laser is activated when an X-chromosome bearing sperm cell is detected during interrogation. The laser emits a high intensity beam of light directed at X chromosome bearing sperm cells located in the center of the fluid. The high intensity beam is configured to cause DNA and/or membrane damage to the cell, resulting in sterility or killing of X chromosome bearing sperm cells. Thus, the final product is composed primarily of viable sperm cells with the Y chromosome. In a preferred embodiment, the reduction in cross-sectional area of the flow focusing region geometrically compresses the sperm-carrying fluid. Geometric compression of the fluid concentrates the sperm cells in the fluid, thereby focusing the sperm cells at or near the center of the microchannel. Since the intensity of the laser beam varies across the width of the microchannel, with the highest intensity generally being at the center of the microchannel and thus decreasing, the flow focusing region must focus the sperm cell at or near the center of the flow where the laser beam has the highest intensity causing the greatest damage to the selected sperm cell.

Chip operation

In one embodiment, as described above, the components to be separated include, for example, separating viable and motile sperm from non-viable or non-motile sperm; sorting and separating sperm according to sex and other sex; isolating stem cells from cells in the population; separating one or more labeled cells from unlabeled cells to distinguish desired/undesired traits; isolating genes in the nuclear DNA according to specific characteristics; isolating cells based on the surface markers; isolating cells based on membrane integrity (activity), potential or predicted reproductive status (fertility), frozen viability, etc.; separating the cells from the contaminants or debris; isolating healthy cells (e.g., bone marrow extraction) from damaged cells (i.e., cancer cells); red blood cells from white blood cells and platelets in the plasma mixture; separating any cells from any other cellular components into respective fractions; damaged cells, contaminants or debris or any other biological material that needs to be separated. The component may be a cell or bead treated or coated with a crosslinker molecule, or a cell or bead embedded with a fluorescent or luminescent marker molecule. The components may have various physical or chemical properties, such as size, shape, material, texture, and the like.

In one embodiment, heterogeneous populations of components may be measured simultaneously, different numbers or similar number schemes of each component examined (e.g., multiplexed measurements), or the components may be examined and differentiated based on labels (e.g., fluorescence), images (due to size, shape, different absorption, scattering, fluorescence, luminescence characteristics, fluorescence or luminescence emission curves, fluorescence or luminescence decay lifetimes), and/or particle location, among others.

In one embodiment, a focusing method may be used in order to position the components for interrogation in the interrogation chamber. The first constriction step of the present invention is accomplished by inputting a fluid sample containing components, such as sperm cells, through a sample input (111) and inputting a sheath fluid or buffer fluid through a sheath or buffer microchannel (140). In some embodiments, the components are pre-stained with a dye (e.g., Hoechst dye) to allow fluorescence and for detection imaging. First, the components in the sample fluid mixture flow through the microchannel (110) and have random directions and locations. At the intersection region (145), the sheath or buffer fluid flowing from the sheath or buffer microchannel (140) compresses the sample mixture flowing in the microchannel (110) at least horizontally on at least two, if not all, sides of the flow. As a result, the components are focused and compressed into a thin stream, and the components (e.g., sperm cells) move toward the center of the channel width. This step has the advantage of using less sheath fluid, since the sheath fluid is only introduced at one location in the chip.

In another embodiment, the invention comprises a second constriction step, wherein the sample mixture comprising the components is at least horizontally further compressed by said constriction region (122) of said downstream microchannel. This step utilizes physical or geometric compression rather than another intersection of sheath fluids. Thus, by the second constriction step of the present invention, the sample fluid is focused in the center of the channel and the components flow along the center of the channel. In a preferred embodiment, the components flow in the form of approximately a single file. Without wishing to be bound by a particular theory or mechanism, the physical/geometric compression has the advantage of reducing the volume of the sheath fluid, since the second crossover point of the sheath fluid is eliminated.

In a preferred embodiment, the invention includes a focusing step in which the component-containing sample mixture is further compressed in a flow focusing region (130) using physical or geometric compression rather than another intersection of sheath fluids. The sample mixture is also closer to the top surface of the focal region (130) by the upwardly inclined bottom surface. Thus, by the focusing step of the present invention, the sample fluid is focused in the center of the channel and the components flow along the center of the channel in the form of an approximately single file. Without wishing to be bound by a particular theory or mechanism, the physical/geometric compression has the advantage of reducing the volume of the sheath fluid, since the second crossover point of the sheath fluid is eliminated.

Thus, the microfluidic devices described herein may be used in the focusing methods described above. In one embodiment, the present invention provides a method of focusing particles in a fluid. The method may comprise providing any of the microfluidic devices described herein, flowing a fluid mixture comprising particles into a sample microchannel (110) and into an intersection region (145), flowing a sheath fluid through the two sheath fluid microchannels (140) and into the intersection region (145), such that the sheath fluid induces laminar flow and compresses the fluid mixture at least horizontally from at least two sides, wherein the fluid mixture is surrounded by the sheath fluid and compressed into a thin stream, the particles being constricted to the thin stream surrounded by the sheath fluid; flowing the fluid mixture and a sheath fluid into the downstream microchannel (120), wherein the narrowed portion (122) of the downstream microchannel (120) horizontally compresses a thin stream of the fluid mixture; flowing a fluid mixture and a sheath fluid into the focusing region (130), wherein a positively sloped floor (132) and tapered sidewalls (135) of the focusing region further constrict the flow of the fluid mixture and redirect particles in the fluid, thereby focusing the particles.

By introducing a sheath fluid and/or physical structure at the constriction and focusing region to compress the fluid mixture, the particles of the fluid mixture are constricted into a relatively smaller, narrower fluid defined by the sheath fluid. For example, introduction of a sheath fluid into the sample microchannel (110) through two sheath fluid channels (130) can compress the fluid mixture stream from both sides into a relatively small, narrow stream while maintaining laminar flow. The flow of the fluid mixture and sheath fluid in the focusing region causes further narrowing of the flow of the fluid mixture and reorientation of the particles in the flow caused by physical structures, such as the narrowing of the raised floor (132) and sidewalls (135) of the focusing region, to focus the particles.

In some embodiments, the component of the sample is a sperm cell, and due to its pancake-like or flat tear-drop-like head, the sperm cell can be reoriented in a predetermined direction when subjected to the focusing step-i.e., its plane is perpendicular to the beam direction. Thus, the sperm cell may have a preference for its body orientation when passed through a two-step focusing process. In particular, the sperm cells tend to be more stable with their flat body perpendicular to the direction of compression. By controlling the sheath fluid or buffer fluid, a uniform orientation of the sperm cells can be achieved starting from a random orientation. The sperm cells not only form a single file in the center of the channel, but they also achieve a uniform orientation. Thus, the components introduced into the sample input (which may be other types of cells or other materials as described previously) undergo a focusing step, which allows the components to move in a single file and in a more uniform direction (depending on the type of component), which allows the components to be interrogated more easily.

In combination with the foregoing embodiments, the present invention also provides a method of producing a fluid having sex-skewed sperm cells. Referring to fig. 6, the method can include providing any of the microfluidic devices described herein, flowing a semen fluid including sperm cells into the sample microchannel (110)) and into the intersection region (145); flowing a sheath fluid through the two sheath fluid microchannels (140) and into the intersection region (145) such that the sheath fluid induces laminar flow and compresses the seminal fluid at least horizontally from at least two sides, wherein the seminal fluid is surrounded by sheath fluid and compressed into a thin stream; flowing the semen fluid and sheath fluid into the downstream microchannel (120), wherein the narrowed portion (122) of the downstream microchannel (120) horizontally compresses the thin stream of semen fluid; flowing the semen fluid and sheath fluid into a focusing region (130), wherein the positively sloped floor (132) and tapered sidewalls (135) further constrict the semen fluid to focus the sperm cells at or near the center of the semen flow; determining a chromosome type of the sperm cells in the sperm stream, wherein each sperm cell is a Y chromosome bearing sperm cell or an X chromosome bearing sperm cell; and sorting Y chromosome bearing sperm cells from the X chromosome bearing sperm cells, thereby producing a fluid comprising predominantly sex-skewed sperm cells of the Y chromosome bearing sperm cells.

In some embodiments, any of the interrogation devices described herein can be used to determine the chromosome type of a sperm cell. In one embodiment, the microfluidic chip (100) may further include an interrogation zone (150) located downstream of the flow focusing region (130). An interrogation device may be coupled to the interrogation zone (150) and used to determine a chromosome type of the sperm cell and sort the sperm cell based on the chromosome type. The interrogation device may include a radiation source that irradiates and excites the sperm cell, and the response of the sperm cell is indicative of a chromosome type in the sperm cell. The reaction of the sperm cells can be detected by an optical sensor. In other embodiments, the interrogation device may also include a laser source. The Y chromosome bearing sperm cells are sorted from the X chromosome bearing sperm cells by laser ablation, which exposes the cells to a high intensity laser source, thereby damaging or killing the cells identified as bearing the X chromosome. In one embodiment, the sex-skewed sperm cell is comprised of at least 55% of sperm cells bearing the Y chromosome. In another embodiment, the sex-tipped sperm cells are comprised of approximately 55% to 99% Y chromosome bearing sperm cells. In another embodiment, the sex-tipped sperm cells consist of at least 99% Y chromosome-bearing sperm cells.

In one embodiment, the component is detected in the interrogation chamber using a radiation source. The radiation source emits a beam (which may be through an optical fiber) that is focused at the center of the channel width. In one embodiment, a component, such as a sperm cell, is directed by a focal region such that a planar surface of the component is directed toward the beam. Furthermore, all components are preferably focused when passing through the radiation source, aligned in the form of a single file. When the component passes through the radiation source and is subjected to the beam, the component fluoresces indicating the desired component. For example, for a sperm cell, the fluorescence intensity of an X chromosome cell is different from that of a Y chromosome cell; alternatively, cells carrying one property may fluoresce at a different intensity or wavelength, while cells carrying a different set of properties fluoresce differently. In addition, the shape, size, or any other distinguishing indicator of the components may be viewed.

In one embodiment, the sample contains a component (e.g., biological material) that is interrogated by other methods. In general, methods for interrogation may include direct visual imaging, for example using a camera, and may utilize direct bright light imaging or fluorescence imaging; alternatively, more sophisticated techniques may be used, such as spectroscopy, transmission spectroscopy, spectroscopic imaging or scattering, such as dynamic light scattering or dispersive wave spectroscopy. In some cases, the optical interrogation zone may be used with additives, such as chemicals that bind to or affect components of the sample mixture or beads that are functionalized to bind and/or fluoresce in the presence of certain materials or diseases. These techniques can be used to measure cell concentration, detect disease, or detect other parameters that characterize a component.

However, in another embodiment, if fluorescence is not used, polarized light backscattering methods may also be used. The components are interrogated using spectroscopy and the spectra of components that have a positive result and fluorescence (i.e., components that react to the tag) are identified for separation. In some embodiments, the components may be identified for separation based on their reaction or binding with additives or sheath fluids or buffers, or by using the natural fluorescence of the components, or the fluorescence of substances associated with the components, as identification tags or background tags, or to meet selected size, or surface characteristics, etc. In one embodiment, the components to be discarded and the components to be collected may be selected by a computer and/or operator upon completion of the component analysis.

Continuing with the beam-induced fluorescence embodiment, the emitted beam is collected by an objective lens and subsequently converted to an electronic signal by an optical sensor. The electronic signals are then digitized by an analog-to-digital converter (ADC) and sent to an electronic controller for signal processing. The electronic controller may be any electronic processor with sufficient processing power, such as a DSP, a microcontroller unit (MCU), a Field Programmable Gate Array (FPGA), or even a Central Processing Unit (CPU). In one embodiment, a DSP-based controller monitors the electronic signal and may then trigger the sorting mechanism when the desired component is detected. In another embodiment, an FPGA-based controller monitors the electronic signals and then communicates with a DSP controller or independently triggers the sorting mechanism upon detection of the desired component. In some other embodiments, the optical sensor may be a photomultiplier tube (PMT), an Avalanche Photodiode (APD), or a silicon photomultiplier tube (SiPM). In a preferred embodiment, the optical sensor can be an APD that detects the response of the sperm cell to interrogation.

In one embodiment of the sorting mechanism, a piezoelectric actuator is used to separate selected or desired components in the interrogation chamber into desired output channels. In an exemplary embodiment, when the target or selected component reaches the cross-sectional point of the ejection channel and microchannel, an electronic signal activates a driver to trigger the actuator. This causes the actuator to contact and push the diaphragm, compressing the ejection chamber and squeezing a strong jet of buffer or sheath fluid into the microchannel, thereby pushing the selected or desired component into the desired output channel.

In some embodiments, the separated components are collected from the respective output channels (170) for storage, further separation, or processing, such as cryopreservation. In some embodiments, the output components may be electronically characterized to detect component concentrations, pH measurements, cell counts, electrolyte concentrations, and the like.

Chip box and base

In some embodiments, the microfluidic chip may be loaded into a microfluidic chip cartridge that is mounted on a chip mount. The chip mount is mounted to a translation stage to allow fine positioning of the mount. For example, the microfluidic chip mount is configured to hold the microfluidic chip in a predetermined position such that the interrogating beam intercepts the fluid component. In one embodiment, the microfluidic chip mount is made of a suitable material, such as an aluminum alloy or other suitable metal/polymer material. The body of the submount may be any suitable shape, but its configuration depends on the layout of the chip. In a further embodiment, the body of the base is configured to receive and engage with an external conduit for transporting fluid/sample to the microfluidic chip. Gaskets or O-rings of any desired shape may be provided to maintain a tight seal between the microfluidic chip and the microfluidic chip mount. The gasket may be a single piece or multiple pieces in any configuration and may be of any desired material (i.e., rubber, silicone, etc.). In one embodiment, the spacer is bonded or adhered (using epoxy) to a layer of the microfluidic chip. The gasket is configured to facilitate sealing and to stabilize or balance the microfluidic chip in the microfluidic chip mount. The details of the chip cartridge and the base and the mechanism for attaching the chip to the cartridge and the base are not described in any detail, as one of ordinary skill in the art will recognize that these devices are well known and can be of any configuration to accommodate microfluidic chips so long as the objectives of the present invention are met.

In some embodiments, the pumping mechanism includes a system with a pressurized gas that provides pressure for pumping the sample fluid mixture from the gas reservoir (i.e., sample tube) to the sample input of the chip. In other embodiments, a collapsible container with sheath fluid or buffer herein is disposed in a pressurized container, and pressurized gas pushes the fluid such that the fluid is piped to the sheath fluid or buffer input of the chip.

In one embodiment, a pressure regulator regulates the gas pressure within the gas tank, and another pressure regulator regulates the gas pressure within the vessel. The mass flow regulator controls the pumping of either sheath fluid or buffer input fluid through the tubing, respectively. Thus, the tubing is used to initially load the fluid into the chip and may be used throughout the chip to load the sample fluid into the sample input.

In accordance with the present invention, when instructions stored on a computer-readable medium are executed, for example, by a computing device or processor, any of the operations, steps, control options, etc. may be implemented by instructions stored on a computer-readable medium (e.g., memory, database, etc.) that cause the computing device or processor to perform any of the operations, steps, control options, etc. described herein. In some embodiments, the operations described in this specification may be implemented as operations performed by a data processing device or processing circuitry on data stored on one or more computer-readable storage devices or received from other sources. A computer program (also known as a program, software application, script, or code) can be written in any form of programming language, including compiled or interpreted languages, declarative or procedural languages, and it can be deployed in any form, including as a stand-alone program or as a module, component, subroutine, object, or other unit suitable for use in a computing environment. A program can be stored in a portion of a file that holds other programs or data, in a file dedicated to the program, or in multiple coordinated files. A program can be deployed to be executed on one computer or on multiple computers that are interconnected by a communication network. Processing circuitry suitable for the execution of a computer program includes, by way of example, both general and special purpose microprocessors, and any one or more processors of any kind of digital computer. Processing circuitry suitable for the execution of a computer program includes, by way of example, both general and special purpose microprocessors, and any one or more processors of any kind of digital computer.

In one embodiment, the user interface of the computer system comprises a computer screen that displays the components in a field of view acquired by a CCD camera on the microfluidic chip. In another embodiment, the computer controls any external equipment, such as a pump (if used), to pump any sample fluid, sheath fluid, or buffer fluid to the microfluidic chip, and also controls any heating equipment that sets the temperature of the fluid input to the microfluidic chip.

It should be noted that the orientation of the various elements may differ according to other illustrative embodiments, and that these variations are intended to be covered by the present disclosure. The mechanisms and arrangements of the microfluidic chip as shown in the various illustrative embodiments are illustrative only. Although only a few embodiments have been described in detail in this disclosure, many modifications are possible (e.g., sizes, dimensions, structures, shapes and proportions of the various elements, values of parameters, mounting arrangements, use of materials, colors, orientations, etc.) without materially departing from the novel teachings and advantages of the subject matter described herein. Some elements shown as integrally formed may be constructed of multiple parts or elements, the position of elements may be reversed or otherwise varied, and the nature or number of discrete elements or positions may be altered or varied. The order or sequence of any process, logical algorithm, or method steps may be varied or re-sequenced according to alternative embodiments. Other substitutions, modifications, changes and omissions may also be made in the design, operating conditions and arrangement of the various illustrative embodiments without departing from the scope of the present disclosure.

As used herein, the term "about" refers to plus or minus 10% of the reference number.

While the preferred embodiments of the invention have been shown and described, it will be obvious to those skilled in the art that changes may be made therein without departing from the scope of the appended claims. Accordingly, the scope of the invention is to be limited only by the following claims. The reference numerals referred to in the following claims are exemplary and are only for the convenience of examining the present patent application and are not intended to limit the scope of the claims in any way to the specific features having corresponding reference numerals in the drawings. In some embodiments, the drawings presented in this patent application are drawn to scale, including angles, dimensional ratios, and the like. In some embodiments, the drawings are merely representative and the claims are not limited by the size of the drawings. In some embodiments, description of the invention described herein using the phrase "comprising" includes embodiments that may be described as "consisting essentially of … …" or "consisting of … …," and thus a written description claiming one or more embodiments of the present invention requires compliance with the invention using the phrase "consisting essentially of … …" or "consisting of … ….

Claims

1. A microfluidic chip (100) comprising:

a microchannel (120) having a narrowed portion (122) that narrows in width; and

a flow focusing region (130) downstream of the microchannel (120); it includes:

a positively sloped floor (132) that reduces the height of the flow focal region;

a sidewall (135) that tapers to reduce a width of the flow focusing region, thereby geometrically narrowing the flow focusing region (130).

2. The microfluidic chip (100) of claim 1, wherein the narrowed portion (122) of the microchannel comprises a tapered sidewall (125).

3. The microfluidic chip (100) of claim 1, wherein the positively sloped floor (132) and the tapered sidewall (135) occur simultaneously from an upstream end (137) to a downstream end (138) of the flow focusing region.

4. The microfluidic chip (100) of claim 1, wherein the positively sloped bottom surface (132) and the tapered sidewalls (135) begin from a plane that is perpendicular across the flow focusing region (130).

5. A microfluidic chip (100) comprising:

a sample microchannel (110);

two sheath fluid microchannels (140) intersecting the sample microchannel (110) to form an intersection region (145);

a downstream microchannel (120) fluidly connected to the crossover region (145), the downstream microchannel (120) having a narrowed portion (122) of narrowing width; and

a downstream flow focusing region (130) fluidly connected to the downstream microchannel (120), the downstream flow focusing region (130) including a positively sloped floor (132) that reduces a height of the flow focusing region; a sidewall (135) that tapers to reduce a width of the flow focusing region, thereby geometrically narrowing the flow focusing region (130);

wherein the sample microchannel (110) is configured to flow a sample fluid mixture, wherein the two sheath fluid microchannels (140) are each configured to flow a sheath fluid into the intersection region (145) to induce laminar flow and to compress the sample fluid mixture flowing from the sample microchannel (110) at least horizontally from at least two sides such that the sample fluid mixture is surrounded by a sheath fluid and compressed into a thin stream.

6. The microfluidic chip (100) of claim 5, wherein the sample microchannel (110) comprises a constriction region (112) downstream of an inlet (111) of the sample microchannel, wherein the constriction region (112) comprises:

a positively sloped floor (114) that lowers the height of the narrowing region; and

a sidewall (115) that tapers to reduce a width of the narrowing region;

wherein the positively sloped floor (114) and the tapering sidewalls (115) geometrically narrow the narrowing region (112).

7. A microfluidic chip (100) according to claim 5, wherein the outlet (113) of the sample microchannel is located at or near the mid-height of the outlet (143) of each of the two sheath fluid microchannels, wherein the inlet (124) of the downstream microchannel is located at or near the mid-height of the outlet (143) of each of the two sheath fluid microchannels.

8. Microfluidic chip (100) according to claim 7, wherein the outlet (113) of the sample microchannel and the inlet (124) of the downstream microchannel are aligned.

9. A microfluidic chip (100) according to claim 5, wherein the outlet (113) of the sample microchannel is located at or near the middle height of the intersection region.

10. A microfluidic chip (100) according to claim 5, wherein the inlet (124) of the downstream microchannel is located at or near the middle height of the intersection region.

11. The microfluidic chip (100) of claim 5, wherein the intersection region (145) and the downstream flow focusing region (130) are configured to focus material in the sample fluid mixture.

12. The microfluidic chip (100) of claim 5, wherein compression of the sample fluid mixture concentrates the material within the sample fluid mixture such that the material is focused at or near the center of the downstream microchannel.

13. The microfluidic chip (100) of claim 5, further comprising an interrogation region (150) downstream of the flow focusing region (130).

14. The microfluidic chip (100) of claim 13, further comprising an expansion region (160) downstream of the interrogation region (150), the expansion region (160) comprising:

a negatively sloped floor (162) that increases the height of the extension region; and

an extension having sidewalls (165), the sidewalls (165) widening to increase a width of the extension.

15. The microfluidic chip (100) of claim 14, further comprising a plurality of output microchannels (170), the plurality of output microchannels (170) being located downstream of the extension regions (160) and fluidically coupled to the extension regions (160).

16. A method of focusing particles in a fluid flow, comprising:

providing a microfluidic chip (100);

the microfluidic chip (100) comprises:

a sample microchannel (110);

a downstream microchannel (120) fluidly connected to the intersection region (135), the downstream microchannel (120) having a narrowed portion (122) of narrowing width; and

flowing a fluid mixture comprising particles into the sample microchannel (110) and into an intersection region (145);

flowing a sheath fluid through the two sheath fluid microchannels (140) and into the intersection region (145) such that the sheath fluid induces laminar flow and compresses the fluid mixture at least horizontally from at least two sides, wherein the fluid mixture is surrounded by sheath fluid and compressed into a thin stream, wherein the particles are constricted to the thin stream surrounded by the sheath fluid;

flowing the fluid mixture and a sheath fluid into the downstream microchannel (120), wherein the constriction (122) of the downstream microchannel (120) horizontally compresses a thin stream of the fluid mixture; and

flowing the fluid mixture and a sheath fluid into the focusing region (130), wherein the positively-sloped floor (132) and the tapered sidewalls (135) further constrict the fluid mixture stream and redirect the particles within the fluid mixture stream, thereby focusing the particles.

17. A method of producing a fluid having sex-skewed sperm cells, the method comprising:

providing a microfluidic chip (100);

the microfluidic chip (100) comprises:

a sample microchannel (110);

flowing a semen fluid comprising sperm cells into the sample microchannel (110) and into an intersection region (145);

flowing sheath fluid through the two sheath fluid microchannels (140) and into the intersection region (145) such that the sheath fluid induces laminar flow and compresses the semen fluid at least horizontally from at least two sides, wherein the semen fluid is surrounded by sheath fluid and compressed into a thin stream;

flowing the semen fluid and sheath fluid into the downstream microchannel (120), wherein the narrowed portion (122) of the downstream microchannel (120) horizontally compresses the thin stream of semen fluid; and

flowing the semen fluid and sheath fluid into the focusing region (130), wherein the positively-sloped floor (132) and the tapering sidewall (135) further constrict the semen fluid to focus the sperm cells at or near the center of the semen fluid;

determining a chromosome type of the sperm cells in the seminal fluid, wherein each sperm cell is a Y-chromosome bearing sperm cell or an X-chromosome bearing sperm cell; and

sorting the Y chromosome-bearing sperm cells from the X chromosome-bearing sperm cells to produce a fluid comprising predominantly sex-skewed sperm cells of the Y chromosome-bearing sperm cells.

18. The method of claim 17, wherein the microfluidic chip (100) further comprises an interrogation zone (150) downstream of the flow focusing region (130), wherein an interrogation device coupled to the interrogation zone (150) is used to determine the chromosome type of the sperm cell and classify the sperm cell according to chromosome type.

19. The method of claim 18, wherein the interrogation device comprises a radiation source that illuminates and excites the sperm cell, wherein a response of the sperm cell is indicative of a chromosome type in the sperm cell, wherein the response of the sperm cell is detected by an optical sensor.

20. The method of claim 19, wherein the interrogation device further comprises a laser source, wherein Y chromosome-bearing sperm cells are sorted from the X chromosome-bearing sperm cells by laser ablation, wherein the X chromosome-bearing sperm cells are exposed to the laser source, which destroys or kills the cells.