Systems, methods, and devices for microfluidic chips with microchannel design that asymmetrically focuses particles

Abstract

The present invention provides an asymmetrically focused microfluidic chip and a method of asymmetrically focusing and processing particles using an asymmetrically focused microfluidic chip. The microfluidic chip includes a microfluidic channel structure having a sheath fluid channel structure originating upstream from a sheath fluid inlet, the sheath fluid channel structure including a first side channel structure and a second side channel structure, each channel structure having a plurality of prongs. A sheath fluid flows through the sheath fluid channel structure into the fluid channel at an intersection with a sample fluid flowing from the sample fluid channel. The sample fluid is asymmetrically focused in the intersection. The sample fluid may be analyzed and processed prior to exiting the channel structure via one or more outlets.

Description

Technical Field

[0001] The present disclosure relates generally to microfluidic chips, and more particularly to microfluidic systems and methods for optimizing the focusing and orientation of particles within microchannels of microfluidic systems.Microfluidic devices and methods for focusing particles in a fluid sample are described herein. Background Art

[0002] In the separation of various particles or cell materials, for example, the separation of sperm into active and motile sperm and inactive or non-motile sperm, or separation by sex, the process is usually a time-consuming task with strict volume limitations. Therefore, current separation technologies cannot, for example, produce the desired yield or process large amounts of cell material in a timely manner.

[0003] Photodamage laser systems use lasers to photodamage or kill unwanted cellular objects. However, prior art requires flow cytometers that use nozzles to interrogate and arrange individual objects in a droplet stream, and attempt to separate and photodamage the objects as they fall into various containers, which is difficult to achieve.

[0004] Therefore, there is a need for a method and apparatus that identifies and distinguishes target objects, is continuous, has high throughput, is time and cost effective, and causes negligible or minimal damage to various target objects. In addition, such apparatus and methods can also be applied to other biological and medical fields, not only sperm identification, but also blood and other cellular materials (including viruses, organelles, spherical structures, colloidal suspensions and other biological materials).

[0005] A variety of devices have been introduced for microfluidic sorting of cells and / or microparticles. In particular, it is often desirable to separate various particles or cells from a sample fluid mixture, such as separating active and motile sperm from inactive and non-motile sperm, or separating sperm by gender. If different markers are present, precise control of particle position within a microscale flow enables efficient sorting of particles. In particular, spatial differentiation of particles or cells can be achieved by exploiting the fluid dynamics generated by the physical structure of the microfluidic channel or the strong interactions between particles suspended in the flow.

[0006] Microfluidics enables the use of small volumes to prepare and process samples, such as various particles or cellular materials. When separating samples, such as separating sperm into active and motile sperm from inactive or non-motile sperm, or separating by sex, the process is often a time-consuming task and may have severe volume limitations. For example, current separation techniques cannot produce the desired yield or process large amounts of cellular material in a timely manner. In addition, existing microfluidic devices cannot effectively focus or orient sperm cells.

[0007] Therefore, there is a need for a microfluidic device and a separation method using the same, which is continuous, has high throughput, saves time, and causes negligible or minimal damage to the various particles being separated. In addition, such a device and method can also be applied 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, spherical structures, colloidal suspensions and other biological materials. Summary of the invention

[0008] The present invention provides a microfluidic channel structure for a microfluidic chip and a flow cytometer system, which provides asymmetric focusing of a sample fluid stream. The sample fluid stream is hydrodynamically focused in an asymmetric manner by asymmetrically introducing a sheath fluid or a buffer fluid from a group of opposite corresponding tines or "bones" in a structure similar to a herringbone pattern.

[0009] According to the principles of the present disclosure, a microfluidic chip includes an asymmetrically focused microchannel structure including a corresponding set of relative tines. The tines introduce a sheath fluid or a buffer fluid into a flow channel in which a sample fluid is already flowing or has been previously introduced. The asymmetric introduction of the sheath fluid or the buffer fluid into the flow channel gradually compresses, narrows, guides or focuses the sample fluid flow into a relatively narrow flow while maintaining a laminar flow or a sheath flow. The asymmetric action on the sample fluid flow further aligns, spaces or orients the particles in the sample fluid flow for interrogation, inspection or action.

[0010] Provided herein are systems and methods for asymmetrically focusing a sample fluid in a microfluidic chip for interrogation, analysis, and action on the sample fluid.

[0011] In one embodiment, a microfluidic channel structure is provided, which includes: a sheath fluid channel structure originating from upstream of a sheath fluid inlet, the sheath fluid channel structure including a first side channel structure and a second side channel structure; a sample fluid channel originating from an upstream sample fluid inlet; a flow channel disposed downstream of the sample fluid inlet and the sheath fluid inlet; wherein the first side channel structure and the second side channel structure each include a plurality of tines extending from the main sheath fluid channel to the flow channel; wherein the plurality of tines of both the first channel structure and the second channel structure intersect with the flow channel at an intersection.

[0012] In various embodiments, the intersection of the plurality of tines with the flow channel is subsequent to the sample introduction location.

[0013] In various embodiments, the plurality of tines are staggered at intersections with the flow channel.

[0014] In various embodiments, the intersections of the plurality of tines with the flow channels alternate between intersections of tines from the first side channel structure and intersections of tines from the second side channel structure.

[0015] In various embodiments, the plurality of tines includes a set of four tines.

[0016] In various embodiments, the plurality of tines intersect the flow channel at an angle between 1 degree and 90 degrees.

[0017] In various embodiments, the plurality of tines intersect the flow channel at a 45 degree angle.

[0018] In various embodiments, the height of the sheath fluid channel structure is greater than the height of the flow channel.

[0019] In various embodiments, the height of the tines is greater than the height of the flow channel.

[0020] In various embodiments, the intersection of each tine of the plurality of tines extends across the flow channel from a first side of the flow channel to a second side of the flow channel.

[0021] In various embodiments, a first portion of the sheath fluid channeling structure is disposed in a first substrate layer and a second portion of the sheath fluid channeling structure is disposed in a second substrate layer.

[0022] In various embodiments, a portion of each of the plurality of tines of each of the first side channel structure and the second side channel structure is disposed above and through a top surface of the flow channel.

[0023] In various embodiments, a portion of each of the plurality of tines of each of the first side channel structure and the second side channel structure is disposed below and through a bottom surface of the flow channel.

[0024] In various embodiments, the length of each tine of the plurality of tines of the second side channel structure is shorter than the length of each tine of the plurality of tines of the first side channel structure.

[0025] In various embodiments, the length of each tine of the plurality of tines of the first side channel structure is longer than the length of each tine of the plurality of tines of the second side channel structure.

[0026] In various embodiments, the length of each tine of the plurality of tines of the second side channel structure is longer than the length of each tine of the plurality of tines of the first side channel structure.

[0027] In various embodiments, the length of each tine of the plurality of tines of the first side channel structure is shorter than the length of each tine of the plurality of tines of the second side channel structure.

[0028] In various embodiments, the width of each tine of the plurality of tines of the second side channel structure is wider than the width of each tine of the plurality of tines of the first side channel structure.

[0029] In various embodiments, the width of each tine of the plurality of tines of the first side channel structure is narrower than the width of each tine of the plurality of tines of the second side channel structure.

[0030] In various embodiments, the width of each of the plurality of tines of the second side channel structure is narrower than the width of each of the plurality of tines of the first side channel structure.

[0031] In various embodiments, the width of each tine of the plurality of tines of the first side channel structure is wider than the width of each tine of the plurality of tines of the second side channel structure.

[0032] In various embodiments, the sample fluid channel intersects the flow channel upstream of an intersection of the sheath fluid channel structure and the flow channel.

[0033] In various embodiments, the height of the sample fluid channel is equal to the height of the flow channel.

[0034] In various embodiments, the flow channel includes a first geometric focusing region.

[0035] In various embodiments, the flow channel includes a second geometric focusing region.

[0036] In various embodiments, the first geometric focal region includes a cone.

[0037] In various embodiments, the first geometric focal region includes a slope.

[0038] In various embodiments, the first geometric focal region includes a slope and a cone.

[0039] In various embodiments, the second geometric focal region includes a cone.

[0040] In various embodiments, the second geometric focal region includes a slope.

[0041] In various embodiments, the second geometric focal region includes a slope and a cone.

[0042] In various embodiments, the flow channel includes a detection zone.

[0043] In various embodiments, the height of the detection zone is less than the height of the upstream portion of the flow channel.

[0044] In various embodiments, the width of the detection zone is less than the width of the upstream portion of the flow channel.

[0045] In various embodiments, the detection zone is disposed downstream of the intersection of the sheath fluid channeling structure and the flow channel.

[0046] In various embodiments, the microfluidic channel structure further includes an expansion region.

[0047] In various embodiments, the expansion zone is disposed downstream of the detection zone.

[0048] In various embodiments, the height of the expansion region is greater than the height of the upstream portion of the flow channel.

[0049] In various embodiments, the height of the expansion area is greater than the height of the detection area.

[0050] In various embodiments, the microfluidic channel structure further includes a set of outlet channels.

[0051] In various embodiments, the set of outlet channels includes a single outlet.

[0052] In various embodiments, the set of outlet channels includes three outlets.

[0053] In various embodiments, the ratio of widths of the set of outlet channels is 1:2:1.

[0054] In various embodiments, the set of outlet channels is disposed downstream of the detection zone.

[0055] In various embodiments, the set of outlet channels includes a primary outlet channel and a set of secondary outlet channels.

[0056] In various embodiments, the set of secondary outlet channels includes a set of waste channels.

[0057] In various embodiments, the intersection is configured to asymmetrically focus particles in a sample flow through the flow channel.

[0058] In various embodiments, the intersection is configured to asymmetrically focus and orient particles in a sample flow through the flow channel.

[0059] In various embodiments, the particles are cells.

[0060] In various embodiments, the cell is a sperm cell.

[0061] In various embodiments, the intersection is configured to asymmetrically focus the sample flow.

[0062] In various embodiments, asymmetric focusing includes allowing a first sheath fluid to flow into the flow channel from a first tine among a plurality of tines of a first side channel structure to direct the sample flow in the flow channel toward a first side wall opposite to the first tine, and allowing a second sheath fluid to flow into the flow channel from a second tine among a plurality of tines of a second side channel structure to direct the sample flow toward a second side wall opposite to the second tine.

[0063] In various embodiments, the plurality of tines of the first side channel structure are offset relative to the plurality of tines of the second side channel structure to provide asymmetric focusing of the sample flow.

[0064] In various embodiments, asymmetric focusing includes surrounding the sample flow with a sheath fluid from substantially three directions to move the sample flow toward a fourth direction, and then surrounding the sample flow with a sheath fluid from substantially three directions to move the sample flow away from the fourth direction.

[0065] In various embodiments, asymmetric focusing includes repeatedly directing the sample stream toward and away from a fourth direction by repeatedly surrounding the sample stream with sheath fluid from at least three directions.

[0066] In various embodiments, the sheath fluid channel structure comprises a herringbone structural configuration.

[0067] In various embodiments, each of the plurality of tines for the first side channel structure and the second side channel structure comprises bone configured in a herringbone structure.

[0068] In various embodiments, the width of the main sheath fluid channel is 50 microns to 1000 microns.

[0069] In various embodiments, the width of the main sheath fluid channel is 300 microns.

[0070] In various embodiments, the height of the main sheath fluid channel is 100 microns to 500 microns.

[0071] In various embodiments, the height of the main sheath fluid channel is 400 microns.

[0072] In various embodiments, the length of the main sheath fluid channel is 5000 microns to 35000 microns.

[0073] In various embodiments, the length of the main sheath fluid channel is 15311 microns.

[0074] In various embodiments, the length of the main sheath fluid channel is 24770 microns.

[0075] In various embodiments, the width of the tines in the plurality of tines of the first side channel structure and the second side channel structure is 50 microns to 500 microns.

[0076] In various embodiments, a width of a tine in the plurality of tines of the first side channel structure and the second side channel structure is 150 microns.

[0077] In various embodiments, the width of the tines in the plurality of tines of the first side channel structure is 150 microns and the width of the tines in the plurality of tines of the second side channel structure is 105 microns.

[0078] In various embodiments, the width of the tines in the plurality of tines of the first side channel structure is not equal to the width of the tines in the plurality of tines of the second side channel structure.

[0079] In various embodiments, the height of the tines in the plurality of tines of the first side channel structure and the second side channel structure is 100 microns to 500 microns.

[0080] In various embodiments, a height of the tines in the plurality of tines of the first side channel structure and the second side channel structure is 400 microns.

[0081] In various embodiments, a height of a tine in the plurality of tines of the first side channel structure and the second side channel structure is 465 microns.

[0082] In various embodiments, a height of a tine in the plurality of tines of the first side channel structure and the second side channel structure is 475 microns.

[0083] In various embodiments, a height of a tine in the plurality of tines of the first side channel structure and the second side channel structure is 490 microns.

[0084] In various embodiments, the height of the tines in the plurality of tines of the first side channel structure and the second side channel structure comprises 50 microns in the first substrate layer and 350 microns in the second substrate layer.

[0085] In various embodiments, the height of the tines in the plurality of tines of the first side channel structure and the second side channel structure comprises 65 microns in the first substrate layer and 400 microns in the second substrate layer.

[0086] In various embodiments, the height of the tines in the plurality of tines of the first side channel structure and the second side channel structure comprises 75 microns in the first substrate layer and 400 microns in the second substrate layer.

[0087] In various embodiments, the height of the tines in the plurality of tines of the first side channel structure and the second side channel structure comprises 100 microns in the first substrate layer and 300 microns in the second substrate layer.

[0088] In various embodiments, the height of the tines in the plurality of tines of the first side channel structure and the second side channel structure comprises 90 microns in the first substrate layer and 400 microns in the second substrate layer.

[0089] In various embodiments, the length of the tines in the plurality of tines of the first side channel structure and the second side channel structure is 1000 microns to 15000 microns.

[0090] In various embodiments, the length of the tines in the plurality of tines of the first side channel structure is not equal to the length of the tines in the plurality of tines of the second side channel structure.

[0091] In various embodiments, the length of the tines in the plurality of tines of the first side channel structure is 1407 microns and the length of the tines in the plurality of tines of the second side channel structure is 7460 microns.

[0092] In various embodiments, the length of the tines in the plurality of tines of the first side channel structure and the second side channel structure is 7460 microns.

[0093] In various embodiments, at the first sidewall of the flow channel, a distance between a tine of the plurality of tines of the first side channel structure and a tine of the plurality of tines of the second side channel structure is 600 microns to 1500 microns.

[0094] In various embodiments, at the first sidewall of the flow channel, a distance between a tine of the plurality of tines of the first side channel structure and a tine of the plurality of tines of the second side channel structure is 820 microns.

[0095] In various embodiments, the width of the flow channel is 50 microns to 500 microns.

[0096] In various embodiments, the width of the flow channel is 300 microns.

[0097] In various embodiments, the width of the flow channel is 250 microns.

[0098] In various embodiments, the width of the flow channel is 300 microns in a first region downstream of the intersection and 250 microns at a second region downstream of the intersection.

[0099] In various embodiments, the height of the flow channel is 100 microns to 500 microns.

[0100] In various embodiments, the height of the flow channel is 150 microns.

[0101] In various embodiments, the height of the flow channel is 67.5 microns.

[0102] In various embodiments, the length of the flow channel is 1001 microns to 30,000 microns.

[0103] In various embodiments, the length of the flow channel is 9200 microns.

[0104] In various embodiments, the length of the flow channel is 10912 microns.

[0105] In various embodiments, the length of the flow channel is 20112 microns.

[0106] In various embodiments, the length of the flow channel is 4912 microns.

[0107] In various embodiments, the length of the flow channel is 2319 microns.

[0108] In various embodiments, the length of the flow channel is 7231 microns.

[0109] In various embodiments, the interrogation region of the flow channel comprises a width of 50 microns to 200 microns.

[0110] In various embodiments, the interrogation region of the flow channel comprises a width of 135 microns.

[0111] In various embodiments, the interrogation region of the flow channel comprises a height of 50 microns to 150 microns.

[0112] In various embodiments, the interrogation region of the flow channel comprises a height of 67.5 microns.

[0113] In various embodiments, the interrogation region of the flow channel comprises a length of 100 microns to 5000 microns.

[0114] In various embodiments, the interrogation region of the flow channel comprises a length of 2319 microns.

[0115] In various embodiments, the sample fluid stream is positioned at a set height within the flow channel.

[0116] In various embodiments, the height of the plurality of tines of both the first microfluidic channel structure and the second microfluidic channel structure enables the sheath fluid stream to flow into the flow channel to position the sample fluid stream at a set height within the flow channel.

[0117] In various embodiments, the set height is at 2 / 3 of the height of the flow channel, + / - 1 / 8 of the height of the flow channel.

[0118] In various embodiments, the height is set at 2 / 3 of the height of the flow channel.

[0119] In various embodiments, the set height is at 2 / 3 of the height of the flow channel, + / - 1 / 8 of the height of the flow channel.

[0120] In various embodiments, the height is set at 3 / 4 of the height of the flow channel.

[0121] In various embodiments, the set height is not at the centerline of the flow channel.

[0122] In various embodiments, the set height is offset from a centerline of the flow channel.

[0123] In various embodiments, the height is set at 18 microns to 25 microns from the top surface of the flow channel.

[0124] In various embodiments, the height is set at 18 microns from the top surface of the flow channel.

[0125] In various embodiments, the height is set at 19 microns from the top surface of the flow channel.

[0126] In various embodiments, the height is set at 20 microns from the top surface of the flow channel.

[0127] In various embodiments, the height is set at 21 microns from the top surface of the flow channel.

[0128] In various embodiments, the height is set at 22 microns from the top surface of the flow channel.

[0129] In various embodiments, the height is set at 23 microns from the top surface of the flow channel.

[0130] In various embodiments, the height is set at 24 microns from the top surface of the flow channel.

[0131] In various embodiments, the height is set at 25 microns from the top surface of the flow channel.

[0132] In various embodiments, the set height is at least 18 microns from the top surface of the flow channel and is offset from the centerline of the flow channel.

[0133] In another embodiment, a microfluidic chip is provided, which includes: a first substrate layer and a second substrate layer; a sheath fluid channel structure originating from upstream of a sheath fluid inlet, the sheath fluid channel structure including a first side channel structure and a second side channel structure, wherein upper portions of the first side channel structure and the second side channel structure are arranged in the first substrate layer, and wherein lower portions of the first side channel structure and the partial side channel structure are arranged in the second substrate layer; a sample fluid channel originating from an upstream sample fluid inlet; a flow channel arranged downstream of the sample fluid inlet and the sheath fluid inlet; wherein the first side channel structure and the second side channel structure each include a plurality of tines extending from the main sheath fluid channel to the flow channel; wherein the plurality of tines of both the first channel structure and the second channel structure intersect with the flow channel at an intersection.

[0134] In one embodiment, a microfluidic chip is provided, wherein the microfluidic chip comprises the microfluidic channel structure of any of the above embodiments.

[0135] In one embodiment, a flow cytometer system is provided, which includes: an interrogation light source; a detector; and a microfluidic chip, the microfluidic chip including: a sheath fluid channel structure originating from upstream of a sheath fluid inlet, the sheath fluid channel structure including a first side channel structure and a second side channel structure; a sample fluid channel originating from an upstream sample fluid inlet; a flow channel disposed downstream of the sample fluid inlet and the sheath fluid inlet; an interrogation area disposed downstream of the flow channel; wherein the first side channel structure and the second side channel structure each include a plurality of tines extending from the main sheath fluid channel to the flow channel; wherein the plurality of tines of both the first channel structure and the second channel structure intersect with the flow channel at an intersection.

[0136] In one embodiment, provided is a flow cytometer system comprising the microfluidic chip of any of the above embodiments.

[0137] In one embodiment, provided is a flow cytometer system including a microfluidic chip, wherein the microfluidic chip includes the microfluidic channel structure of any of the above embodiments.

[0138] In one embodiment, a method for focusing a sample fluid flow comprises: introducing a sheath fluid as a sheath fluid flow into a sheath fluid channel structure at a sheath fluid inlet; splitting the sheath fluid flow into both a first side channel structure and a second side channel structure of the sheath fluid channel structure; introducing a sample fluid as a sample fluid flow into a sample fluid channel at the sample fluid inlet; causing the sheath fluid flow to flow into a plurality of tines of the first side channel structure; causing the sheath fluid flow to flow into a plurality of tines of the second side channel structure; causing the sample fluid flow to flow into a flow channel disposed downstream of the sample fluid inlet and the sheath fluid inlet; causing the sample fluid flow in the flow channel to intersect with the sheath fluid flow from the plurality of tines of the first side channel structure and the plurality of tines of the second side channel structure at an intersection; and asymmetrically focusing the sample fluid flow at the intersection by the sheath fluid flow, wherein the sample fluid flow is focused into a smaller, narrower flow by asymmetrically introducing the sheath fluid flow from the plurality of tines of the first side channel structure and from the plurality of tines of the second side channel structure.

[0139] In one embodiment, a method for focusing a sample fluid stream includes asymmetrically focusing the sample fluid stream by repeatedly introducing a sheath fluid stream so that the sample fluid stream alternately intersects in a flow channel.

[0140] In one embodiment, a method of focusing a sample fluid flow comprises asymmetrically focusing the sample fluid flow in the microfluidic channel structure of any of the above embodiments.

[0141] In one embodiment, a method of focusing a sample fluid flow comprises asymmetrically focusing the sample fluid flow in a microfluidic chip comprising the microfluidic channel structure of any embodiment.

[0142] In one embodiment, provided is a method for focusing a sample stream, the method comprising positioning the sample stream at a location offset from a centerline of a flow channel.

[0143] In various embodiments, the method further comprises positioning the sample flow using the microfluidic channel structure of any of the above embodiments.

[0144] As will be apparent from the context, this specification and the knowledge of those of ordinary skill in the art, any feature or combination of features described herein is included within the scope of the present invention as long as the features included in any such combination are not mutually contradictory. Other advantages and aspects of the present invention are apparent in the following detailed description and claims. BRIEF DESCRIPTION OF THE DRAWINGS

[0145] In order to facilitate a full understanding of the present invention, reference is now made to the accompanying drawings, in which like elements are represented by like reference numerals. These drawings should not be construed as limiting the present invention, but are intended to be illustrative and for reference purposes.

[0146] Figure 1 A top plan view of an asymmetrically focused microfluidic channel structure 100 is provided according to one embodiment.

[0147] Figure 2 A top perspective view of an intersection 600 of an asymmetrically focused microfluidic channel structure 100 is provided according to one embodiment.

[0148] Figure 3 A centerline cross-sectional view across a channel portion 314 of a tine 312 at an intersection channel 711 of an asymmetrically focused microfluidic channel structure 100 is provided according to one embodiment.

[0149] Figure 4 A top perspective view of a channel-spanning portion 314 of a tine 312 at an intersecting channel 711 of an asymmetrically focused microfluidic channel structure 100 is provided according to one embodiment.

[0150] Figure 5 A top perspective view of the lower or bottom chip layer 101 is provided according to one embodiment.

[0151] Figure 6 A detailed perspective view of an intersection 600' is provided according to one embodiment.

[0152] Figure 7A top perspective view of the upper or top chip layer 102 is provided according to one embodiment.

[0153] Figure 8 A detailed perspective view of an intersection 600" is provided according to one embodiment.

[0154] Fig. 9 A top plan view of a flow simulation of an asymmetrically focused microfluidic channel structure 100 is provided according to one embodiment.

[0155] Fig.10 A detailed plan view of a flow simulation of an intersection 600 of an asymmetrically focused microfluidic channel structure 100 is provided according to one embodiment.

[0156] Fig.11 A detailed plan view of a flow simulation of an interrogation region 800 of an asymmetrically focused microfluidic channel structure 100 is provided according to one embodiment.

[0157] Fig.12 A detailed plan view of a flow simulation of an outlet region 900 of an asymmetrically focused microfluidic channel structure 100 is provided according to one embodiment.

[0158] Fig.13 A side view of a flow simulation of an asymmetrically focused microfluidic channel structure 100 is provided according to one embodiment.

[0159] Fig.14 A detailed side view of a flow simulation of an intersection 600 of an asymmetrically focused microfluidic channel structure 100 is provided according to one embodiment.

[0160] Fig.15 A detailed side view of a flow simulation of an interrogation region 800 of an asymmetrically focused microfluidic channel structure 100 is provided according to one embodiment.

[0161] Fig.16 A velocity gradient map of an interrogation region of an asymmetrically focused microfluidic channel structure according to one embodiment is provided.

[0162] Fig.17 An illustration of sample flow locations in an interrogation region of an asymmetrically focused microfluidic channel structure according to one embodiment is provided.

[0163] Fig.18 A velocity gradient map at an interrogation region where a sample flow is positioned in an asymmetrically focused microfluidic channel structure is provided according to one embodiment.

[0164] Fig.19A representation of velocity magnitudes in opposing sets of tines at the intersection of an asymmetrically focusing microfluidic channel structure according to one embodiment is provided.

[0165] Fig. 20 A stroboscopic image of a cell (bovine sperm cell) at the interrogation region of an asymmetrically focused microfluidic channel structure according to one embodiment is provided.

[0166] Fig.21 A graphical representation of velocity gradients and sample flow positions at an interrogation region of an asymmetrically focused microfluidic channel structure according to one embodiment is provided.

[0167] Fig. 22 Microscopic images of particles in a focused sample stream at an interrogation region of an asymmetrically focusing microfluidic channel structure according to one embodiment are provided.

[0168] Fig.23 A stroboscopic image of a cell (bovine sperm cell) at the interrogation region of an asymmetrically focused microfluidic channel structure according to one embodiment is provided.

[0169] Fig.24 A graphical representation of velocity gradients and sample flow positions at an interrogation region of an asymmetrically focused microfluidic channel structure according to one embodiment is provided.

[0170] Fig.25 Microscopic images of particles in a focused sample stream at an interrogation region of an asymmetrically focusing microfluidic channel structure according to one embodiment are provided.

[0171] Fig.26 A histogram output resulting from interrogation of particles (bovine sperm cells) in a focused sample fluid stream detected in the interrogation region of an asymmetrically focused microfluidic channel structure is provided according to one embodiment.

[0172] Fig. 27 A histogram output resulting from interrogation of particles (bovine sperm cells) in a focused sample fluid stream detected in the interrogation region of an asymmetrically focused microfluidic channel structure is provided according to one embodiment.

[0173] Fig.28 and Fig.29 Respective perspective and top views of an asymmetrically focused microfluidic channel structure are provided according to one embodiment, wherein the length of one set of tines is relatively shorter than the length of another set of corresponding tines.

[0174] Fig.30 Provided in Fig.28 and Fig.29Illustration of the velocity gradient and sample flow position at the interrogation region of an asymmetrically focused microfluidic channel structure.

[0175] Fig.31 A top view of an asymmetrically focused microfluidic channel structure is provided according to one embodiment, wherein the width of one set of tines is relatively smaller than the width of another set of corresponding tines.

[0176] Fig.32 A top view of an asymmetrically focused microfluidic channel structure is provided according to one embodiment, wherein the length of one set of tines is relatively shorter than the length of another set of corresponding tines.

[0177] Fig.33 Provided in Fig.31 Illustration of the velocity gradient and sample flow position at the interrogation region of an asymmetrically focused microfluidic channel structure.

[0178] Fig.33A Provided Fig.31 Detailed plan view of a flow simulation of the intersection 3301 of an asymmetrically focused microfluidic channel structure.

[0179] Fig.34 Provided in Fig.32 Illustration of the velocity gradient and sample flow position at the interrogation region of an asymmetrically focused microfluidic channel structure.

[0180] Fig.35 A top view of an asymmetrically focused microfluidic channel structure is provided according to one embodiment, wherein the length of one set of tines is relatively shorter than the length of another set of corresponding tines.

[0181] Fig.36 Provided in Fig.35 Illustration of the velocity gradient and sample flow position at the interrogation region of an asymmetrically focused microfluidic channel structure. DETAILED DESCRIPTION

[0182] The systems and methods of this article will now be described in more detail with reference to the exemplary embodiments shown in the accompanying drawings. Although the present invention is described herein with reference to exemplary embodiments, it should be understood that the systems and methods of this article are not limited to such exemplary embodiments. Those of ordinary skill in the art and those who have access to the teachings of this article will recognize additional implementations, modifications and embodiments, as well as other applications using the present invention, which are fully considered herein to be within the scope of the systems and methods invented and claimed herein, and with respect to which the systems and methods of this article may have significant utility.

[0183] As used herein, an "action" (such as in an "action area") refers to a specific effect on an object. Such as, for example, a decision effect that enriches, sorts, or transfers, moves, destroys, or disables the object being acted upon. Without limitation, this can be laser ablation of particles in a sample stream, transfer of particles in a sample stream, or capture or suspension of particles in a sample stream.

[0184] As used herein, "asymmetric" refers to a structural feature or action that is asymmetrical or unbalanced. This can be, for example, a physical feature where corresponding elements are positioned at different or offset positions relative to a centerline, plane, or axis, or a functional feature where the functional feature is stronger or weaker, more prevalent or less prevalent, or introduced to a greater or lesser extent from one side or direction than from the opposite or corresponding direction. Without limitation, this can be fluid flowing into a channel from a first direction and a second direction, where the flow rate from the first direction is greater or less than the flow rate from the second direction, or where the fluid is introduced slightly downstream or upstream in the second direction relative to the fluid being introduced into the channel in the first direction.

[0185] As used herein, "focusing" refers to the compression of a fluid stream from at least one side by another fluid stream in a laminar fluid flow regime of a microfluidic environment, particularly where particles in or suspended in a first fluid stream are ordered, oriented, or aligned relative to a defined plane or surface, or "focusing" refers to the ordering, orientation, or alignment of particles within a fluid stream in an inertial microfluidic regime under the action of inertial forces within the fluid stream and the microfluidic channel. Focusing can be performed by one or more of geometric channel features, channel length or size, or the introduction of one or more sheath fluids or buffer fluids into the flow channel.

[0186] As used herein, "intersection", which may also be referred to as "connection", includes the connection of any two or more channels within the microfluidic channel structure of the microfluidic chip. For example, but not limited to, the intersection may include a location where a sheath fluid channel is connected to a main flow channel, a location where two or more sheath fluid channels are connected to a flow channel, a location where a sheath fluid channel and a sample channel are connected to a flow channel, a location where a sheath fluid channel is connected to a sample channel, or any combination of the above examples.

[0187] As used herein, "interrogation" refers to the process of detecting an object to reveal one or more characteristics of an object (such as identifying a characteristic of a particle). For example, this can be identifying a characteristic of a particle by exposing the particle to a light source (such as a laser beam) and detecting one or more light emissions from the particle (such as forward scatter, side scatter, or Stoke's shift emission).

[0188] As used herein, "laminar flow" refers to a non-mixing flow state in a microfluidic environment, where one or more flow streams flow through a flow channel in a non-mixing manner. Laminar flow can be defined as a flow having a particular flow or particle Reynolds number.

[0189] As used herein, "particle" refers to anything that may be present in a flowing stream. For example, a particle can be a polystyrene bead, a magnetic bead, a protein, a cell fragment, a small inorganic substance, or a cell, such as a sperm cell.

[0190] As used herein, "sample fluid" refers to a fluid that includes an analyte to be measured. The sample fluid can be, for example, a fluid mixture that includes a buffered fluid medium and a plurality of polystyrene beads, or can be a fluid mixture of sperm cells in sperm plasma, or can be a fluid mixture of sperm cells in other media with or without sperm plasma.

[0191] As used herein, "sheath fluid" or "buffer fluid" refers to a fluid that provides sheath flow and / or hydrodynamic focusing of a sample fluid in a microfluidic environment under laminar flow conditions.

[0192] As used herein, a "structure" is an element or collection of elements of a physical object. For example, a sheath fluid structure can include one or more sheath fluid channels and all elements or components of the sheath fluid channels. For example, a microfluidic channel structure can include all elements therein, such as a sample fluid channel, a flow channel, a sheath fluid channel, and an outlet channel. A structure can include one or more other structures.

[0193] As used herein, "tines" are smaller branching elements called channels and may also be referred to herein as "bones" or "branches" of a channel or microfluidic channel structure. For example, a "herringbone" structure may include one or more sets of tines or "bones."

[0194] As used herein, "group" refers to a collection or grouping of one or more objects, especially similar, associated or related things, and does not refer to an empty group or set unless explicitly stated.

[0195] Although the structures, channels, and other features herein may be referred to as “first” or “second” structures or features, this is for reference only, and these structures or features may also be considered to be “top” and “bottom,” “left” and “right,” “corresponding” or “opposite” structures unless a specific structure or spatial arrangement is explicitly indicated as required for function or operation.

[0196] In general, a microfluidic system having a microfluidic chip with a microchannel is disclosed. In one example, the microfluidic system includes at least one detection device, such as a detection site or any other detection device. The microfluidic chip can be used with at least one detection device. The microchannel is configured to receive a fluid with particles. The geometry of the cross-section of the microchannel, the flow of a buffer fluid or a sheath fluid, and the pressure and flow rate of the fluid through the microchannel optimize the focusing and orientation of the particles within the microchannel to orient the particles.

[0197] The present invention relates to a microfluidic chip having an interrogation device for detecting and interrogating an object in a sample fluid mixture, and a focused energy device for performing an action on the object or an area around the object. In one embodiment, the interrogation device interrogates the object to identify the object and determine whether the object should be targeted by the focused energy device. In one embodiment, the target object is an unwanted target object.

[0198] In one embodiment, the focused energy device is a discriminating device that discriminates target objects from non-target objects by damaging, killing, altering, disabling or destroying the target objects. The present invention is performed in a flowing, continuous fluid stream within a microfluidic network, where objects are hydrodynamically focused, positioned and oriented, non-target objects are allowed to flow through the microfluidic chip undisturbed, and target objects can be acted upon by the focused energy device, including photodamaging, killing, altering, disabling or destroying.

[0199] Various embodiments of the present disclosure provide for the selection of objects in a fluid mixture, such as: selecting active or active sperm from inactive or immobile sperm; selecting sperm via sex and other sex selection variants; selecting stem cells from cells in a population; selecting one or more labeled cells from unlabeled cells to identify desired / undesirable traits; selecting cells with desired traits; selecting genes in the nuclear DNA of cells according to specific characteristics; selecting cells based on surface markers; selecting cells based on membrane integrity (activity), potential or predicted reproductive status (fertility), cryopreservation ability, etc.; selecting cells from contaminants or debris; selecting healthy cells from damaged cells (i.e., cancer cells) (such as in bone marrow extracts); red blood cells from white blood cells and platelets in a plasma mixture; and selecting any cell from any other cell object, divided into corresponding parts; selecting damaged cells, contaminants or debris, or any other biological material desired to be identified. The object can be a cell or bead treated or coated with a linker molecule, or a cell or bead embedded with a fluorescent or (one or more) luminescent marker molecule. The object can have a variety of physical or chemical properties, such as size, shape, material, texture, etc.

[0200] In one embodiment, a heterogeneous population of objects can be measured, where each object is examined for different quantities or states of similar quantities (e.g., multiplexed measurements), or objects can be examined and distinguished based on labels (e.g., fluorescence), images (due to size, shape, different absorption, scattering, fluorescence, luminescence properties, fluorescence or luminescence emission profiles, fluorescence or luminescence decay lifetimes), and / or particle position, etc.

[0201] In addition, the subject matter of the present disclosure is also applicable to other medical applications.For example, various laminar flows discussed below can be used as the part of kidney dialysis process, wherein whole blood is cleared of waste and returned to the patient.In addition, various embodiments of the present disclosure can also be applied to other biological or medical fields, such as for selecting cells, viruses, bacteria, organelles or sub-parts, spherical structures, colloidal suspensions, lipids and lipid globules, gels, immiscible particles, spheroids, cell aggregates, microorganisms and other biomaterials.For example, according to the object selection of the present disclosure, cell "washing" can be included, wherein contaminants (such as bacteria) are removed from the cell suspension, which may be particularly useful in medical and food industry applications.In addition, the present invention has the applicability of selecting inactive cell objects from active cell objects.

[0202] The disclosed subject matter can also be used to transfer species from one solution to another where separation by filtration or centrifugation is impractical or undesirable. In addition to the applications discussed above, additional applications include, for example, selecting colloids of a given size from colloids of other sizes (for research or commercial applications), and washing particles, such as cells, eggs, etc. (effectively replacing the medium containing them and removing contaminants), or washing particles, such as nanotubes, from solutions of salts and surfactants with different salt concentrations or without surfactants.

[0203] The action of selecting species may depend on many physical properties of one or more objects, including self-motility, self-diffusion, free-fall velocity, or action under external forces, such as actuators, electromagnetic fields, or holographic optical traps. Properties that may be selected include, for example, cell motility, cell activity, object size, object mass, object density, the tendency of objects to attract or repel each other or other objects in a flow, object charge, object surface chemistry, and the tendency of certain other objects (i.e., molecules) to adhere to the object.

[0204] In one embodiment, the optimized focusing and orientation of particles by the microfluidic chip of the present disclosure provides detection by at least one detection device, and the detection includes detection of differences in DNA content in the particles. For example, the difference in DNA content may include one or more of the following: (1) a difference in DNA content of approximately 4%; or (2) the presence or absence of X / Y chromosomes. For example, in some embodiments, the at least one detection means may include one or more of the following embodiments: (1) detecting whether a cell is alive or dead, and acting on a subset of live cells based on further classification; (2) detecting the presence of an X or Y chromosome in a cell, identifying cells that include a Y chromosome, and acting on cells with a Y chromosome; (3) detecting the presence of an X or Y chromosome in a cell, identifying cells that do not include an X chromosome, and acting on non-X cells; (4) detecting the presence of an X or Y chromosome in a cell, identifying cells that include an X chromosome, and acting on cells with an X chromosome; and / or (5) detecting the presence of an X or Y chromosome in a cell, identifying cells that do not include a Y chromosome, and acting on non-Y cells. In addition, although the examples described above are specifically directed to X / Y chromosome detection, it will be understood that other DNA fragments can be used in the systems and methods of the present invention and still fall within the scope of the present disclosure. For example, other DNA fragments can be marked and sperm cells with undesirable traits can be inactivated using the same method.

[0205] Although the discussion herein may focus on identifying and selecting viable or motile sperm from non-viable or immotile sperm, or selecting sperm via sex and other sex-selective variants, or selecting one or more labeled cells from unlabeled cells to distinguish desired / undesirable traits, etc., the devices, methods and systems of the present invention can be extended to other types of particles, biological or cellular matter that can be interrogated via fluorescence techniques within a fluid flow, or can be manipulated between different fluid flows entering one or more output ports.

[0206] As described herein, various embodiments of a microfluidic chip utilize one or more flow channels having a set of substantially laminar flows that allow one or more objects to be interrogated by an interrogation device for identification and acted upon, such as via actuation or via a focused energy device, wherein the objects exit the microfluidic chip via one or more outputs. In one embodiment, objects that are not acted upon are undisturbed, and the action displaces, captures, photodamages, alters, disables, kills, or destroys the target object.

[0207] Therefore, various embodiments of the present invention provide for the selection of objects on a continuous basis, such as in a continuous closed system, without the potential damage and contamination caused by prior art methods, particularly in sperm separation. The continuous process of the present invention also provides significant time savings in selecting and identifying objects.

[0208] The sheath fluid or buffer fluid is well known in the microfluidics field, and in one embodiment, may contain nutrients well known in the art to maintain the activity of the objects (i.e., sperm cells) in the fluid mixture. Commercially available Tris, such as sold by Chata Biosystems, is an example, and the sheath fluid or buffer fluid 163 can be formulated to include the following ingredients: Water - 0.9712L; Tris - 23.88gg; Citric acid monohydrate - 11.63g; D-fructose - 8.55g. The pH is adjusted to 6.80 ± 0.05 with hydrochloric acid, and the osmotic pressure is adjusted to 270mOsm to 276mOsm with high purity fructose if necessary. The mixture is filtered using a 0.22 micron filter.

[0209] The microfluidic chip can have one or more structural layers, in which the one or more structural layers are provided with microchannels. The channel can be arranged in one or more layers, or arranged between layers. Any suitable bonding process known to those of ordinary skill in the art will provide a microfluidic chip, channel or equipment as described herein. For example, in injection molding, rather than forming two layers, two molds can be made and joined together so that injection is carried out in the cavity to obtain the chip of the present invention. Multilayers can also be etched independently in glass or another material, such as by chemical, optical or cutting process etching, and joined or bonded together with appropriate alignment to form a complete microfluidic chip.

[0210] However, one of ordinary skill in the art will appreciate that more or fewer structural layers with functional sides may be used, and with or without "blank" layers, as long as the purposes of the present invention are achieved, and that channels may be provided in any structural layer, or in different structural layers, and in any arrangement, with access to those channels through the top "blank" layer.

[0211] In conventional flow cytometer systems, such as droplet flow cytometer systems in air, since objects or cells, particularly objects or cells with asymmetric shapes, tend to be oriented when they flow close to solid surfaces, the function and improvement of object or cell orientation rely on complex nozzle designs, such as directional baffles or offset structures in nozzles. In order to avoid the complex design and high manufacturing cost of nozzle-based flow cytometer systems, the microfluidic chip and channel design described herein focus, locate and orient particles so as to optimize the analytical capabilities of the flow cytometer system or device that interrogates and acts on particles. Therefore, in one embodiment, particles (that is, cells that may have or include asymmetric or non-spherical shapes) are aligned in the channel into a restricted core volume, which may be referred to as a core flow with a width of 2 microns to 100 microns and a height of 2 microns to 100 microns, and when particles pass through the interrogation area, they are maintained in a similar and desired orientation. Therefore, more uniform scattering and detection signals are obtained, thereby contributing to increasing the sensitivity and stability of the system.

[0212] The designs of the microfluidic chip and channel structures and flow cytometer systems described herein provide core sample flow to orient flat-shaped particles, positioning the particles in the channel in a physically close to uniform arrangement, all of which improves downstream precision action by equipment such as focused energy devices or jet sorting fluid actuators to perform actions on the particles.

[0213] In order to achieve the exemplary three-dimensional hydrodynamic focusing method described herein, it is necessary to accurately deliver both the sample fluid and the sheath fluid or buffer fluid so that a constant flow can flow through the microfluidic chip. After being compressed by the sheath fluid flow or buffer fluid flow, the particles are accelerated, and the average spacing between the particles in the sample core flow is also significantly stretched thereby. The ratio of the total sheath fluid flow or buffer fluid flow rate to the sample flow rate can be adjusted between 100:1 and 1000:1. Preferably, a ratio of 200:1 to 400:1 is used in the microfluidic chip and channel structure described herein. As long as the laminar flow (non-turbulent flow) of the sample flow and the sheath fluid flow is maintained, the overall fluid flow rate in the microfluidic chip and channel structure is about 2 to 4 ml / min, 2.5 ml / min, 3 ml / min, 4 to 6 ml / min or 1 to 2 ml / min. The sheath fluid flow or buffer fluid flow introduced is constant and pulse-free to ensure a stable travel speed of the particles during interrogation and signal detection, and between the detection / interrogation position in the interrogation area and the position of the action on the particles downstream of the interrogation. This facilitates accurate signal reading and action on target particles by, for example, focused energy devices. By precisely controlling the flow of fluid through the main channel (flow channel), the total flow rate varies by less than 1% of the set flow rate, and the travel speed of the target particles for potential action varies by less than 1% from where particle interrogation and detection occurs to where the particles are acted upon.

[0214] When detecting flat shaped particles (i.e., sperm cells), one of the challenging issues is to constrain the particles to a uniform orientation when the particles pass through an interrogation beam of electromagnetic radiation, such as emitted by a laser at an interrogation position within the interrogation area. Therefore, the approximately uniform positioning of the particles in the flow channel and the corresponding orientation of the particles help to increase the sensitivity of the system. With the asymmetric hydrodynamic focusing strategy described herein, the position of the particles along the flow channel can be manipulated in a controlled manner. Therefore, by adjusting the ratio between the sheath fluid flow or the buffer fluid flow from the asymmetrically positioned or configured side channel structure, through the physical configuration of the side channel structure or the flow rate of the sheath fluid or buffer fluid passing therethrough, it is preferred for detecting flat particles to focus the sample flow to a position that is offset from the center of the channel by approximately 5 microns to 40 microns (based on a channel cross-section of, for example, 150 microns wide and 100 microns high). In general, the offset of the 0 micron to 100 micron offset position of the particle can be implemented as a fixed (e.g., from the physical channel configuration) or adjustable (e.g., from changing the flow rate) offset.

[0215] In one embodiment, pancake-shaped or paddle-shaped sperm cells are used as examples of particles. Due to their flat or flat teardrop-shaped heads, sperm cells will reorient themselves in a predetermined direction when undergoing asymmetric focusing, that is, their flat surface is perpendicular to the direction of the interrogating light source. Therefore, sperm cells have a preference for their body orientation when undergoing an asymmetric hydrodynamic focusing process. In particular, sperm cells tend to be more stable in the following situations: their flat body is perpendicular to the direction of compression, and the other orientation is achieved based on the shear force associated with the proximity of the surface, such as the top surface of the flow channel. Therefore, by designing or controlling the flow of the sheath fluid or buffer fluid, such as causing the fluid to flow asymmetrically, sperm cells that start with random orientation now achieve uniform orientation. Therefore, the sperm cells are not only arranged in a limited core volume at the center of the flow channel, but they also achieve uniform orientation.

[0216] Asymmetric focusing improves the orientation of sperm cells and the ability to distinguish the DNA content of X-sperm chromosomes from Y-sperm chromosomes (and thus X sperm from Y sperm).

[0217] Now, refer to Figure 1 , provided is a top view of a microfluidic channel structure 100 adapted or configured to hydrodynamically focus, orient and / or align particles suspended in a sample fluid as the sample fluid flows from an inlet, through a flow channel, and out of one or more outlets. The microfluidic channel structure 100 includes a sheath fluid channel structure 200, a sample fluid channel 500, a flow channel 700, an intersection 600, an interrogation region 800, which may also be referred to as and / or include an action or inspection region, and an outlet region 900.

[0218] In operation, a sheath fluid or buffer fluid, which is a fluid suitable for performing hydrodynamic focusing in a microfluidic environment, is introduced into the sheath fluid channel structure 200 through the sheath fluid inlet 202. The sheath fluid is transferred, flowed or guided into the first sheath fluid channel 230 and the second sheath fluid channel 240. The first sheath fluid channel 230 and the second sheath fluid channel 240 transfer or guide the sheath fluid into the corresponding first side channel structure 300 and the second side channel structure 400. Each of the first side channel structure 300 and the second side channel structure 400 includes a plurality of tines, respectively a first plurality of tines 310 and a second plurality of tines 410. Each plurality of tines includes a group of one or more tines, such as Figure 1 For example, first plurality of tines 310 includes at least tine 312, and second plurality of tines 410 includes at least tine 412.

[0219] The sheath fluid flows or the buffer fluid flows through the first plurality of tines 310 and the second plurality of tines 410 and enters the intersection 600 to asymmetrically focus the sample fluid. The sample fluid flows into the microfluidic channel structure 100 via the sample fluid inlet 510 and flows through the sample fluid channel 500 to the intersection. The sample fluid channel 500 may include a geometric feature 520 that includes a ramp and / or a cone to provide one or both of a horizontal or vertical reduction in channel size, and the geometric feature is used to position the sample fluid channel 500 relative to the intersection 600 and the flow channel 700, and adjust the flow characteristics of the sample fluid in the channel, such as increasing its velocity.

[0220] In the intersection 600, as Figure 2 As shown in more detail in Detail 2 of FIG. 2 , sheath fluid or buffer fluid from the first plurality of tines 310 and the second plurality of tines 410 flows into the cross channel 711 of the flow channel 700. The cross channel 711 is a portion or element of the flow channel 700 in the intersection 600 to focus and flow laminarly with the sample fluid from the sample fluid channel 500. At the first intersection 610 of the intersection 600, after the sample fluid flowing from the sample fluid channel 500 into the cross channel 711 is introduced, the sheath fluid or buffer fluid flowing out of the tines 312 and 412 flows into the cross channel 711 of the flow channel 700.

[0221] At the first intersection 610, and at any number of subsequent intersections or junctions where the tines introduce sheath fluid into the flow channel in the intersection, the flow rate or flow velocity of sheath fluid or buffer fluid entering the intersection from the sheath fluid channel structure 200 via the tines (e.g., tines 312 and 412) controls or governs the manner in which asymmetric hydrodynamic focusing of the sample fluid within the intersection occurs. Figures 9 to 15 As shown in more detail in , the sheath fluid or buffer fluid focuses the sample fluid substantially in a first direction and then in a second direction away from the first direction. The flow from each tine will control the amount of sample fluid focused in any one direction, and the flow bias of one set of tines (e.g., the first set of tines 310) to be greater or less than another set of tines (e.g., the second set of tines 410) can be used to position the sample fluid flow within the flow channel 700 and can be used to control the amount and rate of focusing of the sample fluid flow. For example, the flow rate or flow from the first set of tines 310 can be controlled to be greater or less than the flow rate or flow of the second set of tines 410 by channel size (e.g., width and height) or channel length, or by controlling the flow rate via a flow controller or pump.

[0222] In one example embodiment, by making the cross-sectional area of ​​each tine in the first group of tines smaller than the cross-sectional area of ​​each tine in the second group of tines (e.g., corresponding or opposing tines), the flow rate or flow from the first group of tines can be reduced or less than the flow rate or flow of the second group of tines. In another example embodiment, by making the length of each tine in the first group of tines longer than the length of each tine in the second group of tines (e.g., corresponding or opposing tines), the flow rate or flow from the first group of tines can be reduced or less than the flow rate or flow of the second group of tines. In either embodiment, the focusing of the sample fluid at the intersection will be biased toward the first group of tines and away from the second group of tines.

[0223] At the first intersection 610, the sheath fluid or buffer fluid from the tines 412 first intersects the sample fluid. The sheath fluid or buffer fluid from the tines 412 will surround the sample fluid on three sides (e.g., the first vertical side (which can be the left or right side), the top and the bottom), and compress, guide or focus the sample fluid toward a fourth side (e.g., the second vertical side opposite to the first vertical side in the intersection channel 711). The sheath fluid or buffer fluid from the tines 312 will then surround the sample fluid from three sides, one of which is the fourth side, and compress, guide or focus the sample fluid in the direction of the tines 412 (e.g., toward the first vertical side).

[0224] This alternating focusing is asymmetric focusing. Asymmetric focusing is when the sample fluid is focused primarily from or toward a first direction and then away from the first direction. Figure 1 As shown in , this can be achieved by alternately introducing sheath fluid or buffer fluid (e.g., from tine 412, and then from tine 312), and / or by varying the flow rate or flow rate of the sheath fluid or buffer fluid from the tine. Asymmetric focusing is relative to any position along the centerline of flow channel 700, particularly relative to the direction of flow or flow of sample fluid and / or sheath fluid or buffer fluid. Asymmetric focusing can be repeated any number of times to achieve a desired level or quality of focusing of the sample fluid flow within fluid channel 700. For example, as Figure 1 As shown in the figure, the asymmetric focusing is repeated four (4) times, but the inventors have discovered that it can be repeated 2 to 8 times to achieve different levels or qualities of focusing based on the type of sample fluid and sheath fluid or buffer fluid used and the flow rate, properties or conditions of the fluids (e.g., pressure, velocity, viscosity, concentration).

[0225] Downstream of the intersection 600 in the direction of flow, and after asymmetric focusing of the sample fluid by introduction of sheath fluid or buffer fluid from the tine sets 310 and 410, the sample fluid and sheath fluid or buffer fluid flow laminarly downward along the flow channel 700, where one or more geometric restrictions or changes in channel dimensions may further focus or condition the sample flow prior to elasticity of particles in the sample flow in the interrogation region 800. For example, restrictions 710 and 720 may be transverse cones that taper from one or both sides to horizontally reduce the channel width cross section and / or may be vertical ramps having a vertically decreasing channel height cross section. Figure 1 , the first restriction 710 is a geometric channel feature that horizontally reduces the channel width from a first width to a smaller second width, and the second restriction 720 is a geometric channel feature that horizontally reduces the channel width from a first width to a smaller second width and vertically reduces the height from a first height to a smaller second height. In another embodiment, the second restriction 720 only includes a reduction in width and does not include a reduction in height. In another embodiment, the flow channel includes only the first restriction 710. In another embodiment, the flow channel includes only the second restriction 720. In another embodiment, the flow channel does not include the first restriction 710 or the second restriction 720.

[0226] Further downstream is the interrogation region 800. In a preferred embodiment, the width of the microfluidic channel at the interrogation region 800 is less than the width of the flow channel 700, but both the width and height can be less than the width and height of the flow channel 700, or can be the same, depending on the type, nature and characteristics of the particles and sample fluid being interrogated. The purpose of the interrogation region 800 is to optimally position and adjust the flow stream, including the sample fluid stream and the sheath fluid stream or buffer fluid stream for interrogation by the interrogation device. In one embodiment, the interrogation device is a light emitter and a detector. The light emitter can be an electromagnetic radiation emitter, such as a laser, which can be a continuous wave laser or a pulsed laser, or the emitter can be another light source, such as a light emitting diode or an arc lamp. The detector can be any suitable detector, such as an avalanche photodiode or a photomultiplier tube. The emitter and detector are used to interrogate particles within or suspended in the sample fluid stream, such as dye-stained sperm cells. However, other particle types and other interrogation, labeling and identification methods are also contemplated herein.

[0227] The interrogation region 800 may also include an action region. The action region may be a downstream portion of the interrogation and detection particles, in which the particles are acted upon. For example, the particles may be acted upon by the electromagnetic radiation emitting source or another electromagnetic radiation emitting source to deactivate, cut, kill or ablate the particles. In another embodiment, an actuator is used to generate a pulse or jet of fluid to transfer particles from a sample fluid stream to a sheath fluid or a buffer fluid stream, or to another waste fluid stream. In another embodiment, a series of light sources or waveguides may be used as light traps or tweezers to transfer particles from a sample fluid stream. Regardless of which form of interrogation and action occurs within or after the interrogation region, the asymmetric focusing in the intersection 600 will be appropriately adjusted, such as by orientation, alignment and / or spacing, such as in a substantially single column, for particles in the sample fluid being interrogated.

[0228] The outlet region 900 includes an expansion region 910 and a first outlet channel 920, a second outlet channel 930, and a third outlet channel 940. The outlet channels 910, 920, and 930 each include an outlet for fluid and / or particles to exit the microfluidic channel structure 100. Downstream of the interrogation region 800, the outlet region 900 reduces the pressure by widening the channel height and / or width at the expansion region 910. In one embodiment, the sample fluid substantially exits the microfluidic channel structure 100 via the outlet 920, and the sheath fluid or buffer fluid exits via the outlets 930 and 940. In another embodiment, only the desired particles in the sample fluid exit through the outlet 920, while the waste components and the sheath fluid or buffer fluid exit through the outlets 930 and 940.

[0229] Reference now Figure 2 , a top perspective view of region 2 of the intersection 600 of the microfluidic channel structure 100 is provided. In region 2, the end of the sample fluid channel 500 is shown as leading into and connecting the intersection channel 711 of the flow channel 700. The ramp 522 and the cone 521 in the reduction region 520 reduce the width and height of the sample fluid channel 500 to increase the speed or flow rate of the sample fluid flow flowing therein. At the sample fluid outlet or introduction point 524, the height of the sample fluid channel 500 is the same as the height of the intersection channel 711 of the flow channel 700, and optionally can be less than the height of the intersection channel 711. The height of the intersection channel 711 is constant from the introduction point 524 through the intersection 600 (excluding the fork teeth).

[0230] At the first intersection 610, the tines 412 of the second set of tines 410 and the tines 312 of the first set of tines 310 connect the intersection channel 711. Figure 2 As shown in, and in more detail as Figure 3 and Figure 4As shown in region 3 of FIG. 1 , each tine (e.g., tine 312 and 412) extends completely across cross channel 711 from one side to the opposite side. In addition, a portion of the height of each tine extends above top surface 712 and below bottom surface 714 of cross channel 711. Figure 2 , the channel-spanning portion 314 of the tine 312 extends completely across the cross-channel 711 to a termination surface 316 on the opposite or opposite side of the cross-channel 711 from which the tine 312 joins the cross-channel. Each intersection or junction in the intersection 600, such as the first intersection 610, can be of the same or substantially similar configuration, or can be of a different configuration to provide desired focusing or flow conditions for the sheath fluid and the sample fluid at the terminal portion or end of the intersection 600. In an exemplary embodiment, as shown in FIG. Figure 2 As shown in FIG. 6 , the geometric configuration of each intersection is the same as that of each other intersection. Intersection 600 can be considered a focal region, or can also include other geometric channel features (e.g., Figure 1 600. In another embodiment, the focal region includes the intersection 600 and at least one of the restrictions 710 and 720.

[0231] Reference now Figure 3 , a side view of Region 3 including a channel-spanning portion 314 of a tine 312 of a microfluidic channel structure 100 is provided. As shown in Region 3, a portion of the intersection 600 is at the centerline of the intersection channel 711, and a first or lower substrate 20 and a second or upper substrate 30 are further shown. In this embodiment, an upper surface or top 712 of the intersection channel 711 is defined by the bottom of the upper substrate 30, and a bottom 714 of the intersection channel 711 is disposed within the lower substrate 20. An upper portion 320 having a top 322 and a lower portion 330 having a bottom 332 of the tine 312 (shown here as the channel-spanning portion 314) extend above the corresponding top 712 and below the bottom 714 of the intersection channel 711. The height of the tine 312 including the channel-spanning portion 314 is greater than the height of the intersection channel 711. The channel-spanning portion 314 of the tine 312 extends completely across the intersection channel 711. As shown in FIG. Figure 23, the flow of the sheath fluid or buffer fluid will be toward the observation plane and orthogonal to the flow direction shown. The sheath fluid or buffer fluid will surround the sample flow on at least three sides and will join the flow in the cross channel 711 and continue to flow downstream along the indicated flow direction. Each of the multiple sets of tines 310 and 410 will be similarly configured, with opposite, corresponding tines introduced in opposite directions and slightly offset from each other in the flow direction to asymmetrically focus the sample fluid flow.

[0232] Reference now Figure 4 , provides a side perspective view of region 3 including the termination surface 316 of the tine 312 of the microfluidic channel structure 100. The channel spanning portion 314 of the tine 312 extends from a first side 716 across the cross-channel 711 to a second side 714, includes an upper portion 320 having a top 322 extending above the top 712 of the cross-channel 711, and includes a bottom portion 330 having a bottom 332 extending below the bottom 714 of the cross-channel 711. Sheath fluid or buffer fluid flowing through the tine 312 will begin at the first side 716 where the tine 312 intersects the cross-channel 711, flow into the cross-channel 711, but will continue to flow through the cross-sectional area or volume of the channel spanning portion 314 until the sheath fluid or buffer fluid encounters the termination surface 316. In this manner, the sheath fluid or buffer fluid will surround the sample fluid on at least three sides or primarily on three sides, but may also partially encapsulate the sample fluid on a fourth side based on the fluid dynamics of the interaction of the sheath fluid or buffer fluid with the termination surface 316. In any embodiment, in the area shown in region 3, laminar flow is maintained between the sheath fluid or buffer fluid and the sample fluid. In addition, similar interactions between the sheath fluid or buffer fluid flowing from the tines into the intersection region 711 will be found at any intersection of the tines with the intersection region 711.

[0233] Table 1 below provides exemplary dimensions of various elements or structures of the microfluidic channel structure 100 .

[0234] Ranges with example values ​​are provided.

[0235]

[0236] Table 1

[0237] Reference now Figures 5 to 8, a perspective view of a first or bottom chip layer 101 and a second or upper chip layer 102 including a microfluidic channel structure 100' is provided, and the first or bottom chip layer 101 and the second or upper chip layer 102 can be connected or bonded together to form a microfluidic chip. The microfluidic channel structure 100' is substantially similar to the structure 100 described herein, and any structure identified by the figure mark and quotation marks or double quotation marks includes the same features and functions as those described for the microfluidic channel structure 100. The microfluidic channel 100' shown in the upper chip layer 102 is shown as being oriented opposite to the channel 100' shown in the lower chip layer 101, because the upper chip layer 102 will be rotated 180 degrees along its longitudinal axis to be arranged above the lower chip layer 101. One or more alignment marks can be used to align the upper chip layer 102 and the lower chip layer 101.

[0238] In an exemplary embodiment, the upper chip layer 102 includes an upper substrate 30 and the bottom chip layer 101 includes a lower substrate 20, and each of the substrates includes glass or PDMS, but in other embodiments may also include any other material suitable for use as a substrate for a microfluidic chip. However, when used with a laser, such as with a laser operating at an ultraviolet or near-ultraviolet wavelength, glass will be preferred, such as borosilicate glass. Most of the microfluidic channel structure 100' is formed in the bottom substrate 20, such as by wet etching, photolithography, electron discharge machining or other known techniques. A portion of the microfluidic channel structure 101 is also formed in the upper substrate 30. The bottom substrate 20 includes a portion of the sheath fluid inlet 202', a first sheath fluid channel 230' and a second sheath fluid channel 240', and a portion of the sheath fluid channel structure 300' and 400' including multiple tines 310' and 410'. The upper substrate 30 includes a portion of the sheath fluid inlet 202``, a first sheath fluid channel 230`` and a second sheath fluid channel 240``, and a portion of the sheath fluid channel structures 300`` and 400`` including a plurality of prongs 310`` and 410``. A sample fluid channel 500` including a sample fluid inlet 510` is formed in the bottom substrate 20.

[0239] The areas shown in regions 4 and 5 and in Figure 6 and Figure 8The intersection shown in more detail in includes a first plurality of tines (310` and 310`), a second plurality of tines (410` and 410`), and an intersection channel 711` of the flow channel 700`. The upper portion of the plurality of tines (310` and 410`) is formed in the upper substrate 30, and thus may have or provide a height or portion of the tines that is greater than the height of the intersection channel 711`, and the height or portion of the tines extends across the intersection channel 711`. The sheath fluid channel structure (230` and 240`) and the associated plurality of tines (310` and 410`) correspond to portions in the upper substrate 30 (230` and 240`, and 310` and 410`, respectively) to form or provide a complete upper surface, lower surface, and side surfaces of the tines. When attached to the lower substrate 20, the bottom surface of the upper substrate 30 forms the top of the sample channel 500', the crossover channel 711', the flow channel 700', the interrogation region 800', and the outlet region 900'.

[0240] Relatively upstream of the intersection (600` and 600``), the sample fluid channel 500` includes geometric features or restrictions 522` and 520`, which can be ramps and / or cones, and also includes a sample fluid outlet or introduction point 524`, which is used for the flow of sample fluid from the sample fluid channel 500` to the intersection channel 711`. The intersection channel 711` is a portion of the flow channel 700`. Relatively downstream of the intersection (600` and 600``), the flow channel 700` can lead to one or more of the geometric features or restrictions, such as the restriction 720`. The interrogation area 800` is used to interrogate or inspect the sample fluid or particles disposed therein, and to act on the particles. The expansion region 910' in the outlet region 900' widens to allow the sheath fluid or buffer fluid and the sample fluid to flow out through the outlet channels 920', 930' and 940' and the corresponding outlets 922', 932' and 942'.

[0241] Reference now Figures 9 to 15 , various views of flow simulation of a sample fluid 50 and a sheath fluid or a buffer fluid 60 in a microfluidic channel structure 100 are provided. Figures 9 to 15 The diagram provided in FIG. 1 represents the real-world flow of a sample fluid 50 in a laminar flow state through software simulation, wherein a sheath fluid or a buffer fluid 60 is provided in a microfluidic channel structure 100, and the software model is such as in FIG. Software simulation program.

[0242] The sample fluid 50 flows into the sample fluid inlet 510, and the sample fluid 50 flows toward the intersection portion relatively downstream, such as Fig.10 Area 6 and Fig.149. The sheath fluid is branched or diverted into two separate flows, a first flow 62 and a second flow 64, which then flow downwardly along corresponding first and second sheath fluid channels 230, 240 into corresponding first and second pluralities of tines 300, 400 before flowing into the intersection via tines such as tines 312 and 412.

[0243] In such Fig.10 The area 6 shown in FIG. Fig.14 In the region 9 shown in FIG. 1 , the sample fluid 50 flows from the region 520 through the sample fluid outlet or introduction point 524 into the cross channel 711 of the intersection 600. In the first intersection 610, the sheath fluid or buffer fluid 64 from the tines 412 flows into the cross channel 711 of the intersection 600, shifting or compressing the sample fluid 50 in the direction of the tines 312 and away from the tines 412. The sheath fluid or buffer fluid 1012 surrounds the sample fluid 50 primarily from three sides, but may also partially encapsulate the sample fluid from the fourth side. It can be seen that the sample fluid 50 is primarily directed or compressed in the first lateral direction, but is also partially directed or compressed in at least one vertical direction. After the sheath fluid or buffer fluid 64 is introduced from the tines 412, the sheath fluid or buffer fluid 62 is introduced via the tines 312. When the sheath fluid or buffer fluid 64 is introduced, it can be seen that the sheath fluid or buffer fluid 1014 in the cross channel 711 moves the sample fluid 50 away from the tines 312 and in the direction of the tines 412. In this way, the sample fluid 50 is essentially guided or compressed from three sides, but can also be partially enclosed from a fourth side. Fig.10 The area shown in 6 and Fig.14 As can be seen from the details of area 9 shown in FIG, the sample fluid 50 is asymmetrically focused. The shape of the fluid flow of the sample fluid 50 resembles an "s", a snake, or a winding river. The repeated introduction of the sheath fluid (62 and 64) from the more downstream tines (e.g., tines 1402, 1404, 1412, 1414, 1422, and 1424) causes the sample fluid 50 to be further compressed, narrowed, or focused as it advances downstream. It is the introduction of the sheath fluid 60, including repeated introduction, that provides a gradual compression, narrowing, or focusing of the sample fluid 50 stream in a manner similar to an inertial focusing state microfluidic chip having a channel design corresponding to the shape of the sample fluid 50 stream and can impart similar directional and alignment benefits thereto.

[0244] After the asymmetrical introduction of the sheath fluid (62 and 64) downstream through the intersection 600, the sample fluid 50 stream is a narrower, compressed or focused stream 52. The focused stream 52 comprises a narrower, focused stream in which the randomly oriented particles in the stream are imparted with a degree of order. As the focused stream 52 flows downstream through the length of the flow channel 700 and passes through one or more restrictions or geometric features, such as restrictions 710 and 720 comprising ramps or cones, additional orientation, conditioning, alignment and positioning of both the particles within the focused stream 52 and the focused stream 52 itself occur. Additionally, the focused stream 52, after leaving the intersection 600, has been relatively positioned toward the longitudinal centerline of the flow channel 700 and relatively positioned above the centerline 700 in the vertical direction. In particular, when the focused stream 52 reaches the interrogation area 800, as shown in FIG. Fig.11 Region 7 and Fig.15 As shown in detail in region 10 of the laser beam, the focused stream 52 will be optimally shaped and conditioned so that any particles suspended or disposed within the focused stream 52 can be interrogated or identified. For example, the stream 52 can be positioned at an optimal location within the focal plane of the laser and can have sufficiently small variations in width and particle velocity to permit individual interrogation of particles within the focused stream 52. In one embodiment, the stream position is offset from the centerline or top surface by 10 microns to 30 microns, the stream position is horizontally within 50 microns of the centerline, the stream width is 2 microns to 100 microns, and the particle velocity is approximately 5 to 15 m / s at a sheath fluid flow rate or a buffer fluid flow rate of 2 microns to 4 mL / min.

[0245] like Fig.12 As shown in region 8 of FIG. 8 , after interrogation, the focused stream 52 enters the expansion region 910 of the exit region 900 and flows out through the exit channel 920. Depending on the flow conditions, the sheath fluid or the buffer fluid 60 may flow out through one or more of the channels 920, 930, and 940.

[0246] Reference now Figures 16 to 18 , providing a velocity distribution of the fluid flow within the microfluidic channel structure 100 and a simulated position of particles within the fluid flow at the interrogation area 800 . Figures 16 to 18 The graph shown in 1660 represents the flow conditions in a channel size of 135 microns and 67.5 microns high. In the velocity magnitude graph 1660, it can be seen that the magnitude of the velocity of the fluid flowing in the channel increases inwardly toward the center of the channel, with the velocity approaching 0 m / s at the edge and being greater than 9 m / s toward the center of the channel. It can also be understood that there is a velocity gradient that increases toward the center of the channel. In the flow size graph 1700, the relative size and position of the sample flow is provided, such as Figures 9 to 151600. The position of the stream is offset from the top surface by approximately 20 microns and horizontally offset from the centerline by approximately 15 microns. The size of the stream in width and height is approximately 5 microns to 10 microns. Plot 1800 overlays the stream position shown in plot 1700 with the velocity gradient shown in plot 1600. It can be seen that the position of the sample fluid stream is at the boundary of the velocity gradient, rather than being completely positioned within a portion of the stream having a uniform velocity.

[0247] A non-uniform velocity distribution of a sample fluid stream will result in particles within the stream having varying or non-uniform velocities. For example, if one attempts to interrogate a particle at a first location and act upon a particle at a second location downstream of the first location, the velocity of the particle may vary by + / -2 m / s, and the position of the particle at the second location may vary by 1 micron to 15 microns, depending on how far downstream the second location is from the first location. Minimizing velocity variations is important for accurate measurement and action on particles. Therefore, it is desirable to obtain a more uniform velocity distribution of the sample stream and the particles therein. This can be achieved by changing the flow velocity, flow rate, or flow rate of the sheath fluid flowing from a plurality of tines into the intersection. Changing these parameters can be achieved by changing the ratio of a portion of the tines above and below the flow channel at the intersection, and by changing the relative length or cross-sectional area of ​​the tines. A flow chart showing a flow chart having a non-uniform velocity ( Figure 20 to Figure 22 ) and uniform speed ( Figure 23 to Figure 25 ) of the flow stream. Additionally, Fig.19 The graph 1900 in FIG. 1 shows the difference in surface velocity magnitude in the tines of a microfluidic channel structure between a first intersection or junction 610 of relatively high velocity and the end of an intersection region 1902 of relatively low velocity.

[0248] Reference now Figure 20 to Figure 22 and Figure 23 to Figure 25 The diagram in provides a graph with non-uniform velocity ( Figure 20 to Figure 22 ) and uniform speed ( Figure 23 to Figure 25 ) flow stream comparison.

[0249] Fig. 20 Provided is a captured stroboscopic image 2000 of approximately two thousand (2000) particles (bovine sperm cells) captured at fixed points in the interrogation region of an asymmetric microfluidic channel structure. Fig.21 The velocity gradient map 2100 and Fig. 22 As shown in the microscopic image 2200 of , the sample stream is positioned approximately 15 microns offset from the longitudinal centerline and approximately 22 microns offset from the top surface of the channel. At this location, the velocity of the fluid stream is not uniform, so the sample stream is affected by the velocity gradient.

[0250] Fig.23A captured stroboscopic image 2300 of approximately two thousand (2000) particles (bovine sperm cells) captured at a fixed point in the interrogation region of an asymmetric microfluidic channel structure is provided. Fig.24 The velocity gradient map 2400 and Fig.25 As shown in the micrograph 2500 of , the sample stream is positioned approximately 15 microns offset from the longitudinal centerline and approximately 30 microns offset from the top surface of the channel. At this location, the fluid flow is at a uniform velocity, and therefore the sample stream is subjected to a relatively constant velocity (low velocity gradient) at this location within the channel.

[0251] Stroboscopic image 2000 illustrates that the particles do not all arrive at the fixed point at the same time, and the positions of the particles can be seen to be distributed or spread out vertically down the image, rather than being substantially more concentrated at a single point as provided in stroboscopic image 2300. This is due to the particles traveling at different speeds and arriving at the fixed point at different times in image 2000 - at different distances from the fixed point compared to the relatively more constant speed shown in image 2300. Figure 20 to Figure 22 The offset shown is the height of the cross channel above the height of the fork tine below Figure 23 to Figure 25 The results of the chip shown in the figure, or it can be said that for Figure 20 to Figure 22 For the chip shown in FIG. 1 , the ratio of the portion of the tine above the cross channel to the portion of the tine below the cross channel is lower than for Figure 23 to Figure 25 The ratio of the chips shown in Figure 20 to Figure 22 As shown in FIG. 1 , the top portion is 50 microns, the total tine height is 400 microns, and Figure 23 to Figure 25 The top portion is 100 microns and the total tine height is 400 microns.

[0252] Reference now Fig.26 and Fig. 27 , provides count / intensity (2600, 2700) and area / intensity (2610, 2710) histograms of fluorescence intensity of fluorescence emitted by bovine sperm cells flowing through an interrogation area of ​​an asymmetrically focused microfluidic channel structure. Fig. 27 As can be seen from the graph 2700, the peak-to-valley ratio (resolution) is greater than that of the graph 2600. The location of the peak / area output in the graph 2710 is also relatively less shifted than the output shown in the graph 2610. This indicates that, compared to Fig.26 compared to Fig. 27 The position of the sample flow in Fig.26 In , the sample flow is more deflected and in a position within the channel such that the sample flow is subject to a greater velocity gradient and has a greater velocity deviation due to the non-uniform velocity of the flow and its associated particles. Fig. 27In the example, the sample flow has a small deflection and is located within the channel such that it is subject to relatively low velocity gradients and has low velocity deviations due to the substantially uniform velocity of the flow and its associated particles. It can therefore be seen that for interrogation and identification of particles, it is preferred to achieve uniform or substantially uniform velocity.

[0253] Reference now Figure 28 to Figure 29 , Figure 31 to Figure 32 and Fig.35 , provided are alternative embodiments of asymmetric focusing microfluidic channel structures, such as those with dimensions shown in Table 1, and Fig.30 , Figure 33 to Figure 34 and Fig.36 The velocity gradient diagram associated with . Figure 28 to Figure 29 , Figure 31 to Figure 32 and Fig.35 The asymmetric focusing microfluidic channel structure shown in Figure 1 The same functionality as the structure 100 shown in FIG. 1 is provided, however, using tine length, tine cross-sectional area, cross-channel cross-sectional area, flow channel length, flow channel cross-sectional area, the number and type of restrictions or geometric features in the flow channel (e.g., Figure 1 The position and flow characteristics of the sample fluid stream, and the velocity, position and orientation of the particles therein, are determined by changing the restrictions 710 and 720 shown in the figure and the cross-sectional area of ​​the interrogation area. Additionally, changing a portion of each tine disposed above the cross-channel is used to set the vertical position or offset of the fluid stream within the flow channel, particularly in the interrogation area.

[0254] For example, in Fig.28 and Fig.29 In one embodiment shown in the microfluidic channel structure 2800, the length of each tine in the set of tines 2840 is less than the length of each tine in the set of tines 2830. This provides a relatively higher flow rate or flow rate of the sheath fluid or buffer fluid from the set of tines 2830 than from the tines 2840, thereby positioning the sample fluid flow relatively in the center of the channel, such as Fig.30 This is shown in diagram 3000. This achieves a uniform velocity distribution of the sample fluid flow and the particles positioned therein.

[0255] In another embodiment, if Fig.31 As shown in the microfluidic channel structure 3100 of FIG. 31, the width (and cross-sectional area) of each tine in the set of tines 3130 is smaller than the width of each tine in the set of tines 3140. This provides a relatively higher flow rate or flow rate ratio of the sheath fluid or buffer fluid from the set of tines 3130 than from the tines 3140, thereby positioning the sample fluid flow relatively at the center of the channel, as shown in FIG. Fig.33This is shown in the graph 3300 in FIG. This achieves a uniform velocity distribution of the sample fluid flow and the particles positioned therein.

[0256] Reference now Fig.33A as well as Fig.31 and Fig.33 , a plan view of the flow simulation of the sample fluid 3150 and the sheath fluid or buffer fluid in the intersection 3301 of the microfluidic channel structure 3100 is provided. Fig.33A The view provided in FIG. 1 represents the real-world flow of the sample fluid 3150 under a laminar flow state through software simulation, wherein a sheath fluid or a buffer fluid (such as 3112, 3114) is provided in the microfluidic channel structure 3100, and the software simulation is such as in FIG. Software simulation program.

[0257] The sample fluid 3150 flows into the sample fluid inlet, and the sample fluid 3150 flows relatively downstream toward the intersection 3301. The sheath fluid is branched or diverted into two independent flows, a first flow and a second flow, which then flow into corresponding first and second pluralities of tines 3130, 3140 under corresponding first and second sheath fluid channels before flowing into the intersection via tines such as tines 3132 and 3142.

[0258] In the intersection 3301, the sample fluid 3150 flows from the area 3120 into the intersection channel 3171 of the intersection 3301 through the sample fluid outlet or introduction point 3124. In the first intersection 3160, the sheath fluid or buffer fluid from the fork 3142 flows into the intersection channel 3171 of the intersection 3301, shifting or compressing the sample fluid 3150 in the direction of the fork 3132 and away from the fork 3142. The sheath fluid or buffer fluid 3112 surrounds the sample fluid 3150 primarily from three sides, but may also partially encapsulate the sample fluid from a fourth side. It can be seen that the sample fluid 3150 is primarily directed or compressed in the first lateral direction, but is also partially directed or compressed in at least one vertical direction. After the sheath fluid or buffer fluid is introduced from the fork 3142, the sheath fluid or buffer fluid is introduced via the fork 3132. Upon introduction of the sheath or buffer fluid, it can be seen that the sheath or buffer fluid 3114 in the cross channel 3171 moves the sample fluid 3150 away from the tines 3132 and in the direction of the tines 3142. In this way, the sample fluid 3150 is directed or compressed from substantially three sides, but may also be partially enclosed from a fourth side. It can be seen that the sample fluid 3150 is focused asymmetrically.

[0259] The shape of the fluid flow of the sample fluid 3150 resembles an "s", a snake, or a meandering river. As the sample fluid 3150 progresses downstream, repeated introduction of sheath fluid from further downstream tines further compresses, narrows, or focuses the sample fluid 3150. It is the introduction of sheath fluid, including repeated introduction, that provides a gradual compression, narrowing, or focusing of the sample fluid 3150 stream in a manner similar to, and can impart similar directional and alignment benefits to, an inertial focusing state microfluidic chip having a channel design that corresponds to the shape of the sample fluid 3150 stream.

[0260] After the asymmetrical introduction of the sheath fluid downstream through the intersection 3301, the sample fluid 3150 stream is a narrower, compressed or focused stream 3152. The focused stream 3152 comprises a narrower, focused stream in which the randomly oriented particles in the stream are imparted with a degree of order. As the focused stream 3152 flows downstream through the length of the flow channel 3170 and passes through one or more restrictions or geometric features, both the particles within the focused stream 3152 and the focused stream 3152 itself undergo additional orientation, conditioning, alignment and positioning, which restrictions or geometric features may include ramps or cones. Additionally, the focused stream 3152, after exiting the intersection 3301, has been relatively positioned toward the longitudinal centerline of the flow channel 3170 and relatively positioned above the centerline 3170 in a vertical direction. In particular, the focused stream 3152 is relatively more centered and within the channel, as shown in FIG. Fig.33 The velocity gradient cross section 3300 is shown where the velocity gradient is small, or where the velocity is relatively uniform.

[0261] and Figures 9 to 14 Compared to the simulation of the asymmetric microfluidic channel structure 100 shown in Fig.33A The simulation representation positioning of the asymmetric microfluidic channel structure 3100 shown in the figure can be achieved by using unequal tine widths and by using different channel heights above the cross channels (711 and 3171, respectively). The tine 3131 with the asymmetric microfluidic channel structure 3100 is relatively narrow compared to the tine 3141 with the same structure (for example, compared to the tine 310 and 410 of the asymmetric microfluidic channel structure 100 with equal width). In particular, the focused stream 3152 is relatively more in the center of the channel than the focused stream 52, and a more important part or most of the focused stream 3152 is at a uniform (or low velocity gradient) flow position in the channel in the vertical direction.

[0262] In one embodiment, the stream position is offset from the centerline or top surface by 10 microns to 30 microns, the stream position is horizontally within 20 microns of the centerline, the stream width is 2 microns to 100 microns, and the particle velocity is approximately 5 to 15 m / s when the sheath fluid flow rate or the buffer fluid flow rate is 2 microns to 4 mL / min.

[0263] In another embodiment, if Fig.32 As shown in the microfluidic channel structure 3200, the length of each tine in the set of tines 3240 is less than the length of each tine in the set of tines 3230. This makes the flow rate or flow rate of the sheath fluid or buffer fluid from the set of tines 3230 relatively higher than the flow rate or flow rate of the sheath fluid or buffer fluid from the tines 3240, thereby positioning the sample fluid flow slightly toward the center of the channel. However, the height of the flow channel 3270 is also relatively smaller than, for example, Figure 1 , and thus the centering effect is reduced and the sample flow remains slightly offset, as shown in the flow channel 700. Fig.34 This is shown in the graph 3400 in FIG. This results in a non-uniform velocity distribution of the sample fluid flow and the particles positioned therein, which may be detrimental to the interrogation, identification and action of the particles.

[0264] In another embodiment, if Fig.35 As shown in the microfluidic channel structure 3500 of FIG. 35, the length of each tine in the set of tines 3540 is less than the length of each tine in the set of tines 3530. This makes the flow velocity or flow rate of the sheath fluid or buffer fluid from the set of tines 3530 relatively higher than that from the tines 3540, thereby positioning the sample fluid flow slightly toward the center of the channel. However, the height and length of the flow channel 3570 are also relatively smaller and shorter than, for example, Figure 1 The flow channel 700 shown in FIG. 1 and thus, unlike the microfluidic channel structure 3200, the centering effect is not reduced and the sample flow is as follows. Fig.36 3600. This achieves a uniform velocity distribution of the sample fluid flow and the particles positioned therein, which facilitates interrogation, identification and action on the particles.

[0265] In one embodiment, a microfluidic channel structure is provided, which includes: a sheath fluid channel structure originating from upstream of a sheath fluid inlet, the sheath fluid channel structure including a first side channel structure and a second side channel structure; a sample fluid channel originating from an upstream sample fluid inlet; a flow channel disposed downstream of the sample fluid inlet and the sheath fluid inlet; wherein the first side channel structure and the second side channel structure each include a plurality of tines extending from the main sheath fluid channel to the flow channel; wherein the plurality of tines of both the first channel structure and the second channel structure intersect with the flow channel at an intersection.

[0266] In various embodiments, the intersection of the plurality of tines with the flow channel is subsequent to the sample introduction location.

[0267] In various embodiments, the plurality of tines are staggered at intersections with the flow channel.

[0268] In various embodiments, the intersections of the plurality of tines with the flow channels alternate between intersections of tines from the first side channel structure and intersections of tines from the second side channel structure.

[0269] In various embodiments, the plurality of tines includes a set of four tines.

[0270] In various embodiments, the plurality of tines intersect the flow channel at an angle between 1 degree and 90 degrees.

[0271] In various embodiments, the plurality of tines intersect the flow channel at a 45 degree angle.

[0272] In various embodiments, the height of the sheath fluid channel structure is greater than the height of the flow channel.

[0273] In various embodiments, the height of the tines is greater than the height of the flow channel.

[0274] In various embodiments, the intersection of each tine of the plurality of tines extends across the flow channel from a first side of the flow channel to a second side of the flow channel.

[0275] In various embodiments, a first portion of the sheath fluid channeling structure is disposed in a first substrate layer and a second portion of the sheath fluid channeling structure is disposed in a second substrate layer.

[0276] In various embodiments, a portion of each of the plurality of tines of each of the first side channel structure and the second side channel structure is disposed above and through a top surface of the flow channel.

[0277] In various embodiments, a portion of each of the plurality of tines of each of the first side channel structure and the second side channel structure is disposed below and through a bottom surface of the flow channel.

[0278] In various embodiments, the length of each tine of the plurality of tines of the second side channel structure is shorter than the length of each tine of the plurality of tines of the first side channel structure.

[0279] In various embodiments, the length of each tine of the plurality of tines of the first side channel structure is longer than the length of each tine of the plurality of tines of the second side channel structure.

[0280] In various embodiments, the length of each tine of the plurality of tines of the second side channel structure is longer than the length of each tine of the plurality of tines of the first side channel structure.

[0281] In various embodiments, the length of each tine of the plurality of tines of the first side channel structure is shorter than the length of each tine of the plurality of tines of the second side channel structure.

[0282] In various embodiments, the width of each tine of the plurality of tines of the second side channel structure is wider than the width of each tine of the plurality of tines of the first side channel structure.

[0283] In various embodiments, the width of each tine of the plurality of tines of the first side channel structure is narrower than the width of each tine of the plurality of tines of the second side channel structure.

[0284] In various embodiments, the width of each of the plurality of tines of the second side channel structure is narrower than the width of each of the plurality of tines of the first side channel structure.

[0285] In various embodiments, the width of each tine of the plurality of tines of the first side channel structure is wider than the width of each tine of the plurality of tines of the second side channel structure.

[0286] In various embodiments, the sample fluid channel intersects the flow channel upstream of an intersection of the sheath fluid channel structure and the flow channel.

[0287] In various embodiments, the height of the sample fluid channel is equal to the height of the flow channel.

[0288] In various embodiments, the flow channel includes a first geometric focusing region.

[0289] In various embodiments, the flow channel includes a second geometric focusing region.

[0290] In various embodiments, the first geometric focal region includes a cone.

[0291] In various embodiments, the first geometric focal region includes a slope.

[0292] In various embodiments, the first geometric focal region includes a slope and a cone.

[0293] In various embodiments, the second geometric focal region includes a cone.

[0294] In various embodiments, the second geometric focal region includes a slope.

[0295] In various embodiments, the second geometric focal region includes a slope and a cone.

[0296] In various embodiments, the flow channel includes a detection zone.

[0297] In various embodiments, the height of the detection zone is less than the height of the upstream portion of the flow channel.

[0298] In various embodiments, the width of the detection zone is less than the width of the upstream portion of the flow channel.

[0299] In various embodiments, the detection zone is disposed downstream of the intersection of the sheath fluid channeling structure and the flow channel.

[0300] In various embodiments, the microfluidic channel structure further includes an expansion region.

[0301] In various embodiments, the expansion zone is disposed downstream of the detection zone.

[0302] In various embodiments, the height of the expansion region is greater than the height of the upstream portion of the flow channel.

[0303] In various embodiments, the height of the expansion area is greater than the height of the detection area.

[0304] In various embodiments, the microfluidic channel structure further includes a set of outlet channels.

[0305] In various embodiments, the set of outlet channels includes a single outlet.

[0306] In various embodiments, the set of outlet channels includes three outlets.

[0307] In various embodiments, the ratio of widths of the set of outlet channels is 1:2:1.

[0308] In various embodiments, the set of outlet channels is disposed downstream of the detection zone.

[0309] In various embodiments, the set of outlet channels includes a primary outlet channel and a set of secondary outlet channels.

[0310] In various embodiments, the set of secondary outlet channels includes a set of waste channels.

[0311] In various embodiments, the intersection is configured to asymmetrically focus particles in a sample flow through the flow channel.

[0312] In various embodiments, the intersection is configured to asymmetrically focus and direct particles in a sample flow through the flow channel.

[0313] In various embodiments, the particles are cells.

[0314] In various embodiments, the cell is a sperm cell.

[0315] In various embodiments, the intersection is configured to asymmetrically focus the sample flow.

[0316] In various embodiments, the asymmetric focusing includes allowing a first sheath fluid to flow into the flow channel from a first tine among a plurality of tines of a first side channel structure to direct the sample flow in the flow channel toward a first side wall opposite to the first tine, and allowing a second sheath fluid to flow into the flow channel from a second tine among a plurality of tines of a second side channel structure to direct the sample flow toward a second side wall opposite to the second tine.

[0317] In various embodiments, the plurality of tines of the first side channel structure are offset relative to the plurality of tines of the second side channel structure to provide asymmetric focusing of the sample flow.

[0318] In various embodiments, asymmetric focusing includes surrounding the sample flow with a sheath fluid from substantially three directions to move the sample flow toward a fourth direction, and then surrounding the sample flow with a sheath fluid from substantially three directions to move the sample flow away from the fourth direction.

[0319] In various embodiments, asymmetric focusing includes repeatedly directing the sample stream toward and away from a fourth direction by repeatedly surrounding the sample stream with sheath fluid from at least three directions.

[0320] In various embodiments, the sheath fluid channel structure comprises a herringbone structural configuration.

[0321] In various embodiments, each of the plurality of tines for the first side channel structure and the second side channel structure comprises bone configured in a herringbone structure.

[0322] In various embodiments, the width of the main sheath fluid channel is 50 microns to 1000 microns.

[0323] In various embodiments, the width of the main sheath fluid channel is 300 microns.

[0324] In various embodiments, the height of the main sheath fluid channel is 100 microns to 500 microns.

[0325] In various embodiments, the height of the main sheath fluid channel is 400 microns.

[0326] In various embodiments, the length of the main sheath fluid channel is 5000 microns to 35000 microns.

[0327] In various embodiments, the length of the main sheath fluid channel is 15311 microns.

[0328] In various embodiments, the length of the main sheath fluid channel is 24770 microns.

[0329] In various embodiments, the width of the tines in the plurality of tines of the first side channel structure and the second side channel structure is 50 microns to 500 microns.

[0330] In various embodiments, a width of a tine in the plurality of tines of the first side channel structure and the second side channel structure is 150 microns.

[0331] In various embodiments, the width of the tines in the plurality of tines of the first side channel structure is 150 microns and the width of the tines in the plurality of tines of the second side channel structure is 105 microns.

[0332] In various embodiments, the width of the tines in the plurality of tines of the first side channel structure is not equal to the width of the tines in the plurality of tines of the second side channel structure.

[0333] In various embodiments, the height of the tines in the plurality of tines of the first side channel structure and the second side channel structure is 100 microns to 500 microns.

[0334] In various embodiments, a height of the tines in the plurality of tines of the first side channel structure and the second side channel structure is 400 microns.

[0335] In various embodiments, a height of a tine in the plurality of tines of the first side channel structure and the second side channel structure is 465 microns.

[0336] In various embodiments, a height of a tine in the plurality of tines of the first side channel structure and the second side channel structure is 475 microns.

[0337] In various embodiments, a height of a tine in the plurality of tines of the first side channel structure and the second side channel structure is 490 microns.

[0338] In various embodiments, the height of the tines in the plurality of tines for the first side channel structure and the second side channel structure includes 50 microns in the first substrate layer and 350 microns in the second substrate layer.

[0339] In various embodiments, the height of the tines in the plurality of tines for the first side channel structure and the second side channel structure includes 65 microns in the first substrate layer and 400 microns in the second substrate layer.

[0340] In various embodiments, the height of the tines in the plurality of tines for the first side channel structure and the second side channel structure includes 75 microns in the first substrate layer and 400 microns in the second substrate layer.

[0341] In various embodiments, the height of the tines in the plurality of tines for the first side channel structure and the second side channel structure comprises 100 microns in the first substrate layer and 300 microns in the second substrate layer.

[0342] In various embodiments, the height of the tines in the plurality of tines for the first side channel structure and the second side channel structure includes 90 microns in the first substrate layer and 400 microns in the second substrate layer.

[0343] In various embodiments, the length of the tines in the plurality of tines of the first side channel structure and the second side channel structure is 1000 microns to 15000 microns.

[0344] In various embodiments, the length of the tines in the plurality of tines of the first side channel structure is not equal to the length of the tines in the plurality of tines of the second side channel structure.

[0345] In various embodiments, the length of the tines in the plurality of tines of the first side channel structure is 1407 microns and the length of the tines in the plurality of tines of the second side channel structure is 7460 microns.

[0346] In various embodiments, the length of the tines in the plurality of tines of the first side channel structure and the second side channel structure is 7460 microns.

[0347] In various embodiments, at the first sidewall of the flow channel, a distance between a tine of the plurality of tines of the first side channel structure and a tine of the plurality of tines of the second side channel structure is 600 microns to 1500 microns.

[0348] In various embodiments, at the first sidewall of the flow channel, a distance between a tine of the plurality of tines of the first side channel structure and a tine of the plurality of tines of the second side channel structure is 820 microns.

[0349] In various embodiments, the width of the flow channel is 50 microns to 500 microns.

[0350] In various embodiments, the width of the flow channel is 300 microns.

[0351] In various embodiments, the width of the flow channel is 250 microns.

[0352] In various embodiments, the width of the flow channel is 300 microns in a first region downstream of the intersection and 250 microns at a second region downstream of the intersection.

[0353] In various embodiments, the height of the flow channel is 100 microns to 500 microns.

[0354] In various embodiments, the height of the flow channel is 150 microns.

[0355] In various embodiments, the height of the flow channel is 67.5 microns.

[0356] In various embodiments, the length of the flow channel is 1001 microns to 30,000 microns.

[0357] In various embodiments, the length of the flow channel is 9200 microns.

[0358] In various embodiments, the length of the flow channel is 10912 microns.

[0359] In various embodiments, the length of the flow channel is 20112 microns.

[0360] In various embodiments, the length of the flow channel is 4912 microns.

[0361] In various embodiments, the length of the flow channel is 2319 microns.

[0362] In various embodiments, the length of the flow channel is 7231 microns.

[0363] In various embodiments, the interrogation region of the flow channel comprises a width of 50 microns to 200 microns.

[0364] In various embodiments, the interrogation region of the flow channel comprises a width of 135 microns.

[0365] In various embodiments, the interrogation region of the flow channel comprises a height of 50 microns to 150 microns.

[0366] In various embodiments, the interrogation region of the flow channel comprises a height of 67.5 microns.

[0367] In various embodiments, the interrogation region of the flow channel comprises a length of 100 microns to 5000 microns.

[0368] In various embodiments, the interrogation region of the flow channel comprises a length of 2319 microns.

[0369] In various embodiments, the sample fluid stream is positioned at a set height within the flow channel.

[0370] In various embodiments, the height of the plurality of tines of both the first microfluidic channel structure and the second microfluidic channel structure enables the sheath fluid stream to flow into the flow channel to position the sample fluid stream at a set height within the flow channel.

[0371] In various embodiments, the set height is at 2 / 3 of the height of the flow channel, + / - 1 / 8 of the height of the flow channel.

[0372] In various embodiments, the height is set at 2 / 3 of the height of the flow channel.

[0373] In various embodiments, the set height is at 2 / 3 of the height of the flow channel, + / - 1 / 8 of the height of the flow channel.

[0374] In various embodiments, the height is set at 3 / 4 of the height of the flow channel.

[0375] In various embodiments, the set height is not at the centerline of the flow channel.

[0376] In various embodiments, the set height is offset from a centerline of the flow channel.

[0377] In various embodiments, the height is set at 18 microns to 25 microns from the top surface of the flow channel.

[0378] In various embodiments, the height is set at 18 microns from the top surface of the flow channel.

[0379] In various embodiments, the height is set at 19 microns from the top surface of the flow channel.

[0380] In various embodiments, the height is set at 20 microns from the top surface of the flow channel.

[0381] In various embodiments, the height is set at 21 microns from the top surface of the flow channel.

[0382] In various embodiments, the height is set at 22 microns from the top surface of the flow channel.

[0383] In various embodiments, the height is set at 23 microns from the top surface of the flow channel.

[0384] In various embodiments, the height is set at 24 microns from the top surface of the flow channel.

[0385] In various embodiments, the height is set at 25 microns from the top surface of the flow channel.

[0386] In various embodiments, the set height is at least 18 microns from the top surface of the flow channel and is offset from the centerline of the flow channel.

[0387] In another embodiment, a microfluidic chip is provided, which includes: a first substrate layer and a second substrate layer; a sheath fluid channel structure originating from upstream of a sheath fluid inlet, the sheath fluid channel structure including a first side channel structure and a second side channel structure, wherein upper portions of the first side channel structure and the second side channel structure are arranged in the first substrate layer, and wherein lower portions of the first side channel structure and the partial side channel structure are arranged in the second substrate layer; a sample fluid channel originating from an upstream sample fluid inlet; a flow channel arranged downstream of the sample fluid inlet and the sheath fluid inlet; wherein the first side channel structure and the second side channel structure each include a plurality of tines extending from the main sheath fluid channel to the flow channel; wherein the plurality of tines of both the first channel structure and the second channel structure intersect with the flow channel at an intersection.

[0388] In one embodiment, a microfluidic chip is provided, wherein the microfluidic chip comprises the microfluidic channel structure of any of the above embodiments.

[0389] In one embodiment, a flow cytometer system is provided, which includes: an interrogation light source; a detector; and a microfluidic chip, the microfluidic chip including: a sheath fluid channel structure originating from upstream of a sheath fluid inlet, the sheath fluid channel structure including a first side channel structure and a second side channel structure; a sample fluid channel originating from an upstream sample fluid inlet; a flow channel disposed downstream of the sample fluid inlet and the sheath fluid inlet; an interrogation area disposed downstream of the flow channel; wherein the first side channel structure and the second side channel structure each include a plurality of tines extending from the main sheath fluid channel to the flow channel; wherein the plurality of tines of both the first channel structure and the second channel structure intersect with the flow channel at an intersection.

[0390] In one embodiment, provided is a flow cytometer system comprising the microfluidic chip of any of the above embodiments.

[0391] In one embodiment, provided is a flow cytometer system including a microfluidic chip, wherein the microfluidic chip includes the microfluidic channel structure of any of the above embodiments.

[0392] In one embodiment, a method for focusing a sample fluid flow comprises: introducing a sheath fluid as a sheath fluid flow into a sheath fluid channel structure at a sheath fluid inlet; transferring the sheath fluid flow into both a first side channel structure and a second side channel structure of the sheath fluid channel structure; introducing a sample fluid as a sample fluid flow into a sample fluid channel at the sample fluid inlet; causing the sheath fluid flow to flow into a plurality of tines of the first side channel structure; causing the sheath fluid flow to flow into a plurality of tines of the second side channel structure; causing the sample fluid flow to flow into a flow channel disposed downstream of the sample fluid inlet and the sheath fluid inlet; causing the sample fluid flow in the flow channel to intersect with the sheath fluid flow from the plurality of tines of the first side channel structure and the plurality of tines of the second side channel structure at an intersection; and asymmetrically focusing the sample fluid flow by the sheath fluid flow at the intersection, wherein the sample fluid flow is focused into a smaller, narrower flow by the asymmetric introduction of the sheath fluid flow from the plurality of tines of the first side channel structure and the plurality of tines of the second side channel structure.

[0393] In one embodiment, a method for focusing a sample fluid stream includes asymmetrically focusing the sample fluid stream by repeatedly introducing a sheath fluid stream to alternately cross the sample fluid stream in a flow channel.

[0394] In one embodiment, a method of focusing a sample fluid flow comprises asymmetrically focusing the sample fluid flow in the microfluidic channel structure of any of the above embodiments.

[0395] In one embodiment, a method of focusing a sample fluid flow comprises asymmetrically focusing the sample fluid flow in a microfluidic chip comprising the microfluidic channel structure of any of the above embodiments.

[0396] In one embodiment, provided is a method for focusing a sample stream, the method comprising positioning the sample stream at a location offset from a centerline of a flow channel.

[0397] In various embodiments, the method further comprises positioning the sample flow using the microfluidic channel structure of any of the above embodiments.

[0398] In one embodiment, a flow cytometer system including an interrogation device is provided. Although a laser can be used, it should be understood that other suitable radiation sources, such as light emitting diodes (LEDs), arc lamps, etc., can also be used to emit a beam of light that excites the particles. In another embodiment, the beam of light can be delivered to the particles through an optical fiber embedded in a microfluidic chip at an opening.

[0399] In some embodiments, a high intensity laser beam from a suitable laser of a preselected wavelength, such as a 355 nm continuous wave (CW) (or quasi-CW) laser or a 355 nm pulsed laser, is required to excite particles (i.e., sperm cells) in a fluid mixture. The laser emits a laser beam through a window so as to illuminate particles flowing through an interrogation area of ​​the chip. Since the intensity of the laser beam can vary along the width of the microchannel, with the highest intensity generally at the center of the flow channel (e.g., the middle of the channel width) and decreasing from there, the microfluidic channel structure containing the intersection must focus the particles at or near the center of the fluid flow, where optimal illumination occurs at or near the center of the irradiation laser spot. Without wishing to be bound by a particular belief, this can improve the accuracy of the interrogation and identification process.

[0400] In some embodiments, the high intensity light beam interacts with the particles so that the emitted light sensed by the light beam is received by the objective lens. The objective lens can be arranged at any suitable position relative to the microfluidic chip. In one embodiment, the emitted light received by the objective lens is converted into an electronic signal by an optical sensor, such as a photomultiplier tube (PMT) or a photodiode, etc. The electronic signal can be digitized by an analog-to-digital converter (ADC) and sent to a controller based on a digital signal processor (DSP). The DSP-based controller monitors the electronic signal and can then trigger a sorting mechanism.

[0401] In other embodiments, the interrogation device may include a detector such as a photomultiplier tube (PMT), an avalanche photodiode (APD), or a silicon photomultiplier tube (SiPM). For example, the optical sensor of the interrogation device may be an APD, which is a photodiode with a large amount of internal signal amplification through an avalanche process.

[0402] In some embodiments, a piezoelectric actuator assembly can be used to sort desired particles in a fluid mixture when the particles leave the interrogation zone after interrogation. A trigger signal sent to the piezoelectric actuator is determined by the sensor raw signal to activate a specific piezoelectric actuator component when the selected component is detected. In some embodiments, a flexible diaphragm made of a suitable material is used in conjunction with an actuator to push the target particles in the microchannel into an output channel, thereby isolating the target particles from the fluid mixture, such as one of stainless steel, brass, titanium, nickel alloys, polymers, or other suitable materials with a desired elastic response. The actuator can be a piezoelectric, magnetic, electrostatic, hydraulic, or pneumatic type actuator.

[0403] In an alternative embodiment, a piezoelectric actuator assembly or a suitable pumping system can be used to pump the sample fluid into the sample fluid channel toward the intersection. The sample piezoelectric actuator assembly can be arranged at the sample inlet. By pumping the sample fluid mixture into the flow channel, the inter-particle spacing therein can be controlled so that a more controlled relationship can be formed between the particles as they enter the flow channel.

[0404] Other examples of sorting or separation mechanisms that may be used in accordance with the present invention include, but are not limited to, droplet sorters, mechanical separation, fluid switching, acoustic focusing, holographic trapping / steering, and photon pressure / steering. In a preferred embodiment, the sorting mechanism for sex sorting of sperm cells includes laser killing / ablation of selected X-chromosome bearing sperm cells.

[0405] In laser ablation, when sperm cells with X chromosomes or sperm cells with Y chromosomes are detected during interrogation, the laser is activated, depending on the cell type selected or enriched by the system. The laser emits a high-intensity beam directed at the sperm cells at the center of the fluid flow. The high-intensity beam is configured to cause cell DNA and / or membrane damage, such as by cutting or ablating cells, thereby causing infertility or killing sperm cells. Therefore, depending on the cell type selected or enriched by the system, the final product is mainly composed of viable sperm cells with X chromosomes or viable sperm cells with Y chromosomes. In a preferred embodiment, the reduction in the cross-sectional area of ​​the flow focusing region vertically, horizontally, or both geometrically compresses the fluid carrying sperm cells. The geometric compression of the fluid concentrates the sperm cells in the fluid so that the sperm cells are focused at the center of the flow channel or near the center of the flow channel. In addition to asymmetric focusing at the intersection, this method can also be used. Because the intensity of the laser beam varies across the width of the flow channel, with the highest intensity generally at the center of the flow channel and decreasing from there, the microfluidic channel structure must focus the sperm cells at or near the center of the fluid stream, where the laser beam has the highest intensity, to cause maximum damage to the selected sperm cells.

[0406] In one embodiment, the particles to be separated, selected or enriched include, for example: separation of live and active sperm from non-live or inactive sperm; separation of sperm by sex, and other sex sorting variations; separation of stem cells from cells in a population; separation of one or more labeled cells from unlabeled cells to distinguish desired / undesirable traits; sperm with different desired traits; separation of genes in nuclear DNA according to specific characteristics; separation of cells based on surface markers; separation of cells based on membrane integrity (activity), potential or predicted reproductive status (fertility), cryopreservation ability, etc.; separation of cells from contaminants or debris; separation of healthy cells from damaged cells (i.e., cancer cells) (such as in bone marrow extracts); separation of red blood cells from white blood cells and platelets in a plasma mixture; and separation of any cell from any other cell component into corresponding components; damaged cells, or contaminants, or debris, or any other biological material desired to be separated. The particles can be cells or beads treated or coated with a linker molecule, or cells or beads embedded with fluorescent or (one or more) luminescent marker molecules. The particles can have a variety of physical or chemical properties, such as size, shape, material, texture, etc.

[0407] In one embodiment, a heterogeneous population of particles can be measured simultaneously, where each component is examined for a different quantity or state of similar quantities (e.g., multiplexed measurements), or particles can be examined and distinguished based on labels (e.g., fluorescence), images (due to size, shape, different absorption, scattering, fluorescence, luminescence properties, fluorescence or luminescence emission profiles, fluorescence or luminescence decay lifetimes), and / or particle position, etc.

[0408] In one embodiment, a focusing method can be used to position the particles for interrogation in the interrogation area. In some embodiments, the particles are pre-stained with a dye (e.g., Hoechst dye) to allow fluorescence and for detection imaging. Initially, the particles in the sample fluid mixture flow through the sample channel and have a random orientation and position. At the intersection, the sample mixture flowing in the flow channel is compressed at least horizontally on at least two sides of the flow by the sheath fluid or buffer fluid flowing out of the sheath channel structure, and at least vertically but in an asymmetric manner on at least two sides of the flow. For example, the sample can be compressed in an alternating manner, first mainly from three directions toward a fourth direction, and then from three directions away from the fourth direction. The sample is compressed. This can include guiding the sample flow toward the first side of the flow channel and then away from the first side toward the second side of the flow channel in an alternating manner by introducing the sheath fluid or buffer fluid asymmetrically. When the sample flow is asymmetrically focused, the flow of the sample flow through the intersection is similar to a meandering river, wherein the width and / or height of the sample flow decreases along the flow direction. Therefore, the particles are focused and compressed into a thin stream, and the particles (e.g., sperm cells) move roughly toward the center of the channel width.

[0409] In another embodiment, an additional geometric focusing or contraction step is provided, wherein the sample mixture containing the particles is further compressed at least horizontally downstream of the intersection. This includes physical or geometric compression, rather than another intersection of the sheath fluid. In preferred embodiments, the particles flow in a generally single file form, and in other embodiments, the particles flow in a generally single file form with a specified or regular spacing.

[0410] Thus, the microfluidic chips and microfluidic channel structures described herein can be used in the focusing methods described above. In one embodiment, the present invention provides a method of asymmetrically focusing particles in a fluid flow.

[0411] Through the asymmetric introduction of the sheath fluid and / or the physical structure in the contraction and focusing area, the compression of the fluid mixture contracts the particles of the fluid mixture into a relatively smaller, narrower stream defined by the sheath fluid. For example, the sheath fluid introduced asymmetrically into the intersection through the tines of the sheath fluid channel structure compresses the sample fluid flow from all sides into a relatively smaller, narrower stream while maintaining laminar flow. The flow of the sample fluid and the sheath fluid at the intersection causes further contraction of the sample fluid flow and redirection of the particles within the flow, which is caused by the asymmetric introduction of the sheath fluid from both sides of the intersection, which focuses the particles.

[0412] In some embodiments, the particles in the sample fluid are sperm cells, and due to their flat or flat teardrop-shaped heads, the sperm cells can reorient themselves in a predetermined direction when undergoing asymmetric focusing, that is, their flat surface is perpendicular to the direction of the light beam. Therefore, the sperm cells have a preference for their body orientation when undergoing a two-step focusing process. In particular, sperm cells tend to be more stable, wherein their flat body is perpendicular to the compression direction. Sperm cells starting with random orientation achieve a uniform orientation of approximately 90 to 99%, such as at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%. Sperm cells are not only arranged in a single file at the center of the channel, but they also achieve an almost uniform orientation. Therefore, the particles introduced into the sample input end experience asymmetric focusing, which can be particles of other types of cells or other materials as described above, which orients and positions the particles into a single file form and in a more uniform orientation (depending on the type of particle), which allows easier interrogation of the particles.

[0413] In addition to and / or in combination with other embodiments described herein, the present invention also provides a method for producing a fluid having sex-biased sperm cells. The method may include providing any of the asymmetrically focusing microfluidic devices described herein, causing a sperm fluid including sperm cells to flow into a sample fluid channel and into an intersection, causing a sheath fluid to flow through two sheath fluid channel structures, including flowing through corresponding multiple tines, and into the intersection, causing the sheath fluid to cause laminar flow and asymmetrically compress the sperm fluid from all sides, wherein the sperm fluid is surrounded by the sheath fluid and is compressed into a thin stream, causing the sperm fluid and the sheath fluid to flow into a downstream flow channel, determining the chromosome type of sperm cells in the sperm fluid stream, wherein each sperm cell is a sperm cell with a Y chromosome or a sperm cell with an X chromosome, and sorting sperm cells with a Y chromosome from sperm cells with an X chromosome, and vice versa, so that a fluid including sperm cells with a sex-biased sperm cells that are mainly sperm cells with a Y chromosome or sperm cells with an X chromosome is produced.

[0414] In some embodiments, any of the interrogation devices, apparatuses, or methods described herein may be used to determine the chromosome type of sperm cells. In one embodiment, the microfluidic chip may further include an interrogation region downstream of the intersection. An interrogation device coupled to the interrogation region is used to determine the chromosome type of sperm cells and to sort the sperm cells based on the chromosome type. The interrogation device may include a radiation source that irradiates and excites the sperm cells, and the response of the sperm cells indicates the chromosome type in the sperm cells. The response of the sperm cells may be detected by an optical sensor. In other embodiments, the interrogation device may further include a laser source. Sperm cells with Y chromosomes are sorted from sperm cells with X chromosomes, or sperm cells with X chromosomes are sorted from sperm cells with Y chromosomes by laser ablation, which exposes the cells to a high-intensity laser source that destroys or kills cells determined to have X chromosomes or Y chromosomes depending on the features that are enriched or selected. In one embodiment, the sex-skewed sperm cells consist of at least 55% sperm cells with Y chromosomes. In another embodiment, the sex-skewed sperm cells consist of about 55% to 99% sperm cells with a Y chromosome. In yet another embodiment, the sex-skewed sperm cells consist of at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, or at least 96% sperm cells with a Y chromosome. In another embodiment, the sex-skewed sperm cells consist of at least 99% sperm cells with a Y chromosome. In one embodiment, the sex-skewed sperm cells consist of at least 55% sperm cells with an X chromosome. In another embodiment, the sex-skewed sperm cells consist of about 55% to 99% sperm cells with an X chromosome. In yet another embodiment, the sex-skewed sperm cells consist of at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, or at least 96% sperm cells with an X chromosome. In another embodiment, the sex-skewed sperm cells consist of at least 99% X-chromosome bearing sperm cells.

[0415] In one embodiment, a radiation source is used to detect particles in an interrogation chamber. The radiation source emits a beam focused at the center of the channel width (may be via an optical fiber). In one embodiment, particles, such as sperm cells, are oriented by asymmetric focusing and / or approaching the surface so that the flat surface of the particle faces the beam. In addition, when all particles pass under the radiation source, all particles are preferably aligned into a substantially single-file form by focusing. When the particle passes under the radiation source and is acted upon by the beam, the particle emits fluorescence, which may be a Stoke shift emission indicating the desired particle. For example, for sperm cells, the fluorescence intensity emitted by X chromosome cells is different from that of Y chromosome cells; or cells carrying one trait may emit a fluorescence intensity or wavelength different from that of cells carrying a set of different traits. In addition, the shape, size, or any other distinguishing indicator of the particle can be observed.

[0416] In one embodiment, interrogation of a sample containing particles (i.e., biological material) is accomplished by other methods. In general, methods for interrogation can include direct visual imaging (such as with a camera), and can utilize direct intense light imaging or fluorescence imaging; or, more complex techniques such as spectroscopy, transmission spectroscopy, spectral imaging, or scattering, such as dynamic light scattering or diffuse wave spectroscopy, can be used. In some cases, the optical interrogation region can be used in conjunction with additives, such as chemical materials that bind to or affect the particles 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 concentrations, detect diseases, or detect other parameters that characterize particles.

[0417] However, in another embodiment, if fluorescence is not used, polarized light backscattering methods can also be used. Using spectroscopic methods, the particles are interrogated and the spectra of those particles with positive results and fluorescence (i.e., those particles that react with the label) are identified for separation. In some embodiments, particles can be identified and selected for separation based on the reaction or combination of particles with additives or sheath fluids or buffer fluids, or by using the natural fluorescence of the particles or the fluorescence of substances associated with the components as identity tags or background tags, or by conforming to selected sizes, dimensions or surface features, etc. In one embodiment, after the assay is completed, which particles to discard and which particles to collect can be selected via a computer and / or an operator.

[0418] Continuing with the embodiment of beam-induced fluorescence, the emitted light beam is then collected by an objective lens and subsequently converted into an electronic signal by an optical sensor. The electronic signal is then digitized by an analog-to-digital converter (ADC) and sent to an electronic controller for signal processing. The electronic controller can 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 then triggers a sorting mechanism when a desired component is detected. In another embodiment, an FPGA-based controller monitors the electronic signal and then communicates with the DSP controller or independently triggers a sorting mechanism when a desired component is detected. In some other embodiments, the optical sensor can 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 sperm cells to interrogation.

[0419] In one embodiment of the sorting mechanism, a piezoelectric actuator is used to isolate the selected or desired particles in the interrogation chamber into the desired output channel. In an exemplary embodiment, when the target or selected component reaches the cross-sectional point of the injection channel and the microchannel, an electronic signal activates the driver to trigger the actuator. This causes the actuator to contact the diaphragm and push it, compressing the injection chamber, and squeezing a strong jet of buffer fluid or sheath fluid into the microchannel, which pushes the selected or desired component into the desired output channel.

[0420] In some embodiments, the separated particles are collected from their respective output channels for storage, further separation, or processing, such as cryopreservation. In some embodiments, the output particles can be electronically characterized to detect particle concentration, pH measurement, cell count, electrolyte concentration, etc.

[0421] In some embodiments, the microfluidic chip can be loaded on a chip box, which is mounted on a chip holder. The chip holder is mounted to a translation stage to allow precise positioning of the holder. For example, the microfluidic chip holder is configured to hold the microfluidic chip in a predetermined position so that the interrogation beam intercepts the fluid particles. In one embodiment, the microfluidic chip holder is made of a suitable material, such as an aluminum alloy or other suitable metal / polymer material. The body of the holder can be any suitable shape, but its configuration depends on the layout of the chip. In a further embodiment, the body of the holder is configured to receive and engage an external pipeline for transferring the fluid / sample to the microfluidic chip. A gasket or O-ring of any desired shape can be provided to maintain a tight seal between the microfluidic chip and the microfluidic chip holder. Depending on the expectation, the gasket can be a single piece or multiple particles of any configuration or material (i.e., rubber, silicone, etc.). In one embodiment, the gasket is docked or bonded (using epoxy resin) to the layer of the microfluidic chip. The gasket is configured to help seal, and stabilize or balance the microfluidic chip in the microfluidic chip holder. The details of the box and holder and the mechanism for attaching the chip to the box and holder are not described in detail, as one of ordinary skill in the art will know that these devices are well known and can be any configuration that accommodates the microfluidic chip as long as the objectives of the present invention are met.

[0422] In some embodiments, the pumping mechanism comprises a system with a pressurized gas that provides the pressure for pumping the sample fluid mixture from a reservoir (i.e., a sample tube) to the sample input of the chip. In other embodiments, a collapsible container with a sheath fluid or a buffer fluid is disposed in a pressurized container, and the pressurized gas pushes the fluid so that the fluid is delivered to the sheath or buffer input of the chip via a conduit.

[0423] In one embodiment, a pressure regulator regulates the gas pressure in the reservoir, and another pressure regulator regulates the gas pressure in the container. The mass flow regulator controls the fluid pumped into the sheath or buffer input end respectively via the pipeline. Thus, the pipeline is used to initially load the fluid into the chip, and can be used throughout the chip to load the sample fluid into the sample input end.

[0424] According to the present invention, any operation, step, control option, etc. can be implemented by instructions stored on a computer-readable medium (such as a memory, a database, etc.). When, for example, a computing device or a processor executes an instruction stored on a computer-readable medium, the instruction can cause the computing device or the processor to perform any operation, step, control option, etc. described herein. In some embodiments, the operations described in this specification can be implemented as operations performed by a data processing device or a processing circuit on data stored on one or more computer-readable storage devices or received from other sources. A computer program (also referred to as a program, software, software application, script, or code) can be written in any form of programming language, including compiled or interpreted languages, declarative or procedural languages, and the computer program can be deployed in any form, including as an independent program or as a module, component, subroutine, object, or other unit suitable for use in a computing environment. The program can be stored in a portion of a file that stores other programs or data, or in a single file dedicated to the program, or in multiple collaborative files. The program can be deployed to be executed on a single computer or on multiple computers interconnected by a communication network. For example, a processing circuit suitable for executing a computer program includes both a general-purpose microprocessor and a special-purpose microprocessor, and any one or more processors of any kind of digital computer.

[0425] In one embodiment, the user interface of the computer system comprises a computer screen that displays particles in the field of view acquired by a CCD camera or other imaging sensor on the microfluidic chip. In another embodiment, the computer controls any external devices, such as pumps (if used) to pump any sample fluid, sheath fluid, or buffer fluid into the microfluidic chip, and also controls any heating devices that set the temperature of the fluid input into the microfluidic chip.

[0426] It should be noted that, according to other illustrative embodiments, the orientation of various elements can be different, and such changes are intended to be covered by the present disclosure. As shown in various exemplary embodiments, the construction and arrangement of microfluidic chip are only exemplary. Although only several embodiments are described in detail in the present disclosure, in the case of the novel teachings and advantages of this theme of this article, many modifications (for example, the size, size, structure, shape and ratio, parameter value, installation arrangement, material use, color, orientation, etc. of various elements) are possible. Some elements shown as integrally formed can be composed of multiple parts or elements, and the position of the element can be reversed or otherwise changed, and the nature or quantity or position of the discrete element can be changed or changed. According to alternative embodiments, the order or sequence of any process, logical algorithm or method step can be changed or reordered. Without departing from the scope of the present disclosure, other replacements, modifications, changes and omissions can also be carried out to the design, operating conditions and arrangement of various exemplary embodiments.

[0427] Although the present invention has been described with reference to certain preferred embodiments, it should be understood that many changes may be made within the spirit and scope of the inventive concepts described. In addition, the systems and methods herein are not limited to the scope of the specific embodiments described herein. It is fully contemplated that, in addition to those described herein, various other embodiments and modifications of the systems and methods herein will become apparent to those of ordinary skill in the art based on the foregoing description and accompanying drawings. Therefore, such other embodiments and modifications are intended to fall within the scope of the appended claims. In addition, although the systems and methods herein have been described in the context of specific embodiments, implementations, and applications, and in specific environments, it will be understood by those of ordinary skill in the art that their usefulness is not limited thereto, and that the present invention may be beneficially applied to any number of modes and environments for any purpose. Therefore, the claims set forth below should be interpreted in light of the full scope and spirit of the systems and methods disclosed herein.

Claims

1. A microfluidic channel structure, comprising: a sheath fluid channel structure originating upstream from a sheath fluid inlet, the sheath fluid channel structure comprising a first side channel structure and a second side channel structure; a sample fluid channel originating from an upstream sample fluid inlet; a flow channel disposed downstream of the sample fluid inlet and the sheath fluid inlet; wherein the first side channel structure and the second side channel structure each include a plurality of tines extending from the main sheath fluid channel to the flow channel; Wherein, a plurality of fork teeth of both the first channel structure and the second channel structure intersect with the flow channel at an intersection.

2. The microfluidic channel structure according to claim 1, wherein: The intersection of the plurality of tines with the flow channel is after a sample introduction location.

3. The microfluidic channel structure according to claim 1, wherein: The intersections of the plurality of fork teeth and the flow channel are staggered.

4. The microfluidic channel structure according to claim 1, wherein: The intersections of the plurality of tines with the flow channel alternate between intersections of tines from the first side channel structure and intersections of tines from the second side channel structure.

5. The microfluidic channel structure according to claim 1, wherein: The plurality of tines includes a set of four tines.

6. The microfluidic channel structure according to claim 1, wherein: The plurality of tines intersect the flow channel at an angle between 1 degree and 90 degrees.

7. The microfluidic channel structure according to claim 1, wherein: The plurality of tines intersect the flow channel at a 45 degree angle.

8. The microfluidic channel structure according to claim 1, wherein: The height of the sheath fluid channel structure is greater than the height of the flow channel.

9. The microfluidic channel structure according to claim 1, wherein: The height of the tines is greater than the height of the flow channel.

10. The microfluidic channel structure according to claim 1, wherein: An intersection of each tine of the plurality of tines extends across the flow channel from a first side of the flow channel to a second side of the flow channel.

11. The microfluidic channel structure according to claim 1, wherein: A first portion of the sheath fluid channel structure is disposed in a first substrate layer, and a second portion of the sheath fluid channel structure is disposed in a second substrate layer.

12. The microfluidic channel structure according to claim 1, wherein: A portion of each of the plurality of tines of each of the first and second side channel structures is disposed above and through a top surface of the flow channel.

13. The microfluidic channel structure according to claim 1, wherein: A portion of each of the plurality of tines of each of the first and second side channel structures is disposed below and through a bottom surface of the flow channel.

14. The microfluidic channel structure according to claim 1, wherein: The length of each of the plurality of tines of the second side channel structure is shorter than the length of each of the plurality of tines of the first side channel structure.

15. The microfluidic channel structure according to claim 1, wherein: The length of each of the plurality of tines of the first side channel structure is longer than the length of each of the plurality of tines of the second side channel structure.

16. The microfluidic channel structure according to claim 1, wherein: The length of each of the plurality of tines of the second side channel structure is longer than the length of each of the plurality of tines of the first side channel structure.

17. The microfluidic channel structure according to claim 1, wherein: The length of each of the plurality of tines of the first side channel structure is shorter than the length of each of the plurality of tines of the second side channel structure.

18. The microfluidic channel structure according to claim 1, wherein: The width of each of the plurality of tines of the second side channel structure is wider than the width of each of the plurality of tines of the first side channel structure.

19. The microfluidic channel structure according to claim 1, wherein: The width of each of the plurality of tines of the first side channel structure is narrower than the width of each of the plurality of tines of the second side channel structure.

20. The microfluidic channel structure according to claim 1, wherein: The width of each of the plurality of tines of the second side channel structure is narrower than the width of each of the plurality of tines of the first side channel structure.

21. The microfluidic channel structure according to claim 1, wherein: The width of each of the plurality of tines of the first side channel structure is wider than the width of each of the plurality of tines of the second side channel structure.

22. The microfluidic channel structure according to claim 1, wherein: The sample fluid channel intersects the flow channel upstream of an intersection of the sheath fluid channel structure and the flow channel.

23. The microfluidic channel structure according to claim 1, wherein: The height of the sample fluid channel is equal to the height of the flow channel.

24. The microfluidic channel structure according to claim 1, wherein: The flow channel includes a first geometric focusing region.

25. The microfluidic channel structure according to claim 24, wherein: The flow channel includes a second geometric focusing region.

26. The microfluidic channel structure according to claim 24, wherein: The first geometric focusing region includes a cone.

27. The microfluidic channel structure according to claim 24, wherein: The first geometric focusing region includes a slope.

28. The microfluidic channel structure according to claim 24, wherein: The first geometric focusing area includes a slope and a cone.

29. The microfluidic channel structure according to claim 25, wherein: The second geometric focusing area includes a cone.

30. The microfluidic channel structure according to claim 25, wherein: The second geometric focusing region includes a slope.

31. The microfluidic channel structure according to claim 25, wherein: The second geometric focusing area includes a slope and a cone.

32. The microfluidic channel structure according to claim 1, wherein: The flow channel includes a detection zone.

33. The microfluidic channel structure according to claim 32, wherein: The detection area has a height smaller than a height of an upstream portion of the flow channel.

34. The microfluidic channel structure according to claim 32, wherein: The detection area has a width smaller than a width of an upstream portion of the flow channel.

35. The microfluidic channel structure according to claim 32, wherein: The detection region is disposed downstream of an intersection of the sheath fluid channel structure and the flow channel.

36. The microfluidic channel structure according to claim 32, further comprising an expansion region.

37. The microfluidic channel structure according to claim 36, wherein: The expansion area is disposed downstream of the detection area.

38. The microfluidic channel structure according to claim 36, wherein: The height of the expansion region is greater than the height of the upstream portion of the flow channel.

39. The microfluidic channel structure according to claim 36, wherein: The height of the expansion area is greater than the height of the detection area.

40. The microfluidic channel structure of claim 32, further comprising a set of outlet channels.

41. The microfluidic channel structure according to claim 40, wherein: The set of outlet channels includes a single outlet.

42. The microfluidic channel structure according to claim 40, wherein: The set of outlet channels includes three outlets.

43. The microfluidic channel structure according to claim 40, wherein: The width ratio of the set of outlet channels is 1:2:

1.

44. The microfluidic channel structure according to claim 40, wherein: The set of outlet channels is disposed downstream of the detection region.

45. The microfluidic channel structure according to claim 1, wherein: The set of outlet channels includes a primary outlet channel and a set of secondary outlet channels.

46. ​​The microfluidic channel structure according to claim 1, wherein: The set of secondary outlet channels includes a set of waste channels.

47. The microfluidic channel structure according to claim 1, wherein: The intersection is configured to asymmetrically focus particles in a sample flow through the flow channel.

48. The microfluidic channel structure according to claim 1, wherein: The intersection is configured to asymmetrically focus and orient particles in a sample flow through the flow channel.

49. The microfluidic channel structure according to claim 48, wherein: The particles are cells.

50. The microfluidic channel structure according to claim 49, wherein: The cell is a sperm cell.

51. The microfluidic channel structure according to claim 1, wherein: The intersection is configured to asymmetrically focus the sample flow.

52. The microfluidic channel structure according to claim 51, wherein: The asymmetric focusing includes allowing a first sheath fluid to flow into the flow channel from a first tine among a plurality of tines of the first side channel structure to guide the sample flow in the flow channel toward a first side wall opposite to the first tine, and allowing a second sheath fluid to flow into the flow channel from a second tine among a plurality of tines of the second side channel structure to guide the sample flow toward a second side wall opposite to the second tine.

53. The microfluidic channel structure according to claim 51, wherein: The plurality of tines of the first side channel structure are offset relative to the plurality of tines of the second side channel structure to provide asymmetric focusing of the sample flow.

54. The microfluidic channel structure according to claim 51, wherein The asymmetric focusing includes surrounding the sample stream with a sheath fluid from substantially three directions to move the sample stream toward a fourth direction, and then surrounding the sample stream with a sheath fluid from substantially three directions to move the sample stream away from the fourth direction.

55. The microfluidic channel structure according to claim 54, wherein: The asymmetric focusing includes repeatedly directing the sample stream toward and away from the fourth direction by repeatedly surrounding the sample stream with sheath fluid from at least three directions.

56. The microfluidic channel structure according to claim 1, wherein: The sheath fluid channel structure includes a herringbone structural configuration.

57. The microfluidic channel structure according to claim 1, wherein: Each of the plurality of tines for the first side channel structure and the second side channel structure comprises bone arranged in a herringbone configuration.

58. The microfluidic channel structure according to claim 1, wherein: The width of the main sheath fluid channel is 50 microns to 1000 microns.

59. The microfluidic channel structure according to claim 1, wherein: The width of the main sheath fluid channel is 300 microns.

60. The microfluidic channel structure according to claim 1, wherein: The height of the main sheath fluid channel is 100 microns to 500 microns.

61. The microfluidic channel structure according to claim 1, wherein: The height of the main sheath fluid channel is 400 microns.

62. The microfluidic channel structure according to claim 1, wherein: The length of the main sheath fluid channel is 5000 microns to 35000 microns.

63. The microfluidic channel structure according to claim 1, wherein: The length of the main sheath fluid channel is 15311 microns.

64. The microfluidic channel structure according to claim 1, wherein: The length of the main sheath fluid channel is 24770 microns.

65. The microfluidic channel structure according to claim 1, wherein: A width of the tines in the plurality of tines of the first side channel structure and the second side channel structure is 50 microns to 500 microns.

66. The microfluidic channel structure according to claim 1, wherein: The width of the tines in the plurality of tines of the first side channel structure and the second side channel structure is 150 microns.

67. The microfluidic channel structure according to claim 1, wherein: The width of a tine in the plurality of tines of the first side channel structure is 150 microns, and the width of a tine in the plurality of tines of the second side channel structure is 105 microns.

68. The microfluidic channel structure according to claim 1, wherein: The width of the tines in the plurality of tines in the first side channel structure is not equal to the width of the tines in the plurality of tines in the second side channel structure.

69. The microfluidic channel structure according to claim 1, wherein: A height of the tines of the plurality of tines of the first side channel structure and the second side channel structure is 100 microns to 500 microns.

70. The microfluidic channel structure according to claim 1, wherein: A height of the tines in the plurality of tines of the first side channel structure and the second side channel structure is 400 microns.

71. The microfluidic channel structure according to claim 1, wherein: The height of the tines in the plurality of tines of the first side channel structure and the second side channel structure is 465 microns.

72. The microfluidic channel structure according to claim 1, wherein: The height of the tines in the plurality of tines of the first side channel structure and the second side channel structure is 475 microns.

73. The microfluidic channel structure according to claim 1, wherein: The height of the tines in the plurality of tines of the first side channel structure and the second side channel structure is 490 microns.

74. The microfluidic channel structure according to claim 1, wherein: The heights of the tines in the plurality of tines for the first side channel structure and the second side channel structure include 50 microns in the first substrate layer and 350 microns in the second substrate layer.

75. The microfluidic channel structure according to claim 1, wherein: The heights of the tines in the plurality of tines for the first side channel structure and the second side channel structure include 65 microns in the first substrate layer and 400 microns in the second substrate layer.

76. The microfluidic channel structure according to claim 1, wherein: The heights of the tines in the plurality of tines for the first side channel structure and the second side channel structure include 75 microns in the first substrate layer and 400 microns in the second substrate layer.

77. The microfluidic channel structure according to claim 1, wherein: The height of the tines in the plurality of tines for the first side channel structure and the second side channel structure includes 100 microns in the first substrate layer and 300 microns in the second substrate layer.

78. The microfluidic channel structure according to claim 1, wherein: The heights of the tines in the plurality of tines for the first side channel structure and the second side channel structure include 90 microns in the first substrate layer and 400 microns in the second substrate layer.

79. The microfluidic channel structure according to claim 1, wherein: The length of the tines in the plurality of tines of the first side channel structure and the second side channel structure is 1000 microns to 15000 microns.

80. The microfluidic channel structure according to claim 1, wherein: The length of the tines in the plurality of tines in the first side channel structure is not equal to the length of the tines in the plurality of tines in the second side channel structure.

81. The microfluidic channel structure according to claim 1, wherein: The length of the tines in the plurality of tines of the first side channel structure is 1407 microns, and the length of the tines in the plurality of tines of the second side channel structure is 7460 microns.

82. The microfluidic channel structure according to claim 1, wherein: The length of the tines in the plurality of tines of the first side channel structure and the second side channel structure is 7460 microns.

83. The microfluidic channel structure according to claim 1, wherein: At a first side wall of the flow channel, a distance between a tine of the plurality of tines of the first side channel structure and a tine of the plurality of tines of the second side channel structure is 600 micrometers to 1500 micrometers.

84. The microfluidic channel structure according to claim 1, wherein: At a first sidewall of the flow channel, a distance between a tine of the plurality of tines of the first side channel structure and a tine of the plurality of tines of the second side channel structure was 820 microns.

85. The microfluidic channel structure according to claim 1, wherein: The width of the flow channel is 50 micrometers to 500 micrometers.

86. The microfluidic channel structure according to claim 1, wherein: The width of the flow channel is 300 microns.

87. The microfluidic channel structure according to claim 1, wherein: The width of the flow channel is 250 microns.

88. The microfluidic channel structure according to claim 1, wherein: The width of the flow channel is 300 micrometers at a first region downstream of the intersection and is 250 micrometers at a second region downstream of the intersection.

89. The microfluidic channel structure according to claim 1, wherein: The height of the flow channel is 100 micrometers to 500 micrometers.

90. The microfluidic channel structure according to claim 1, wherein: The height of the flow channel is 150 microns.

91. The microfluidic channel structure according to claim 1, wherein: The height of the flow channel is 67.5 microns.

92. The microfluidic channel structure according to claim 1, wherein: The length of the flow channel is 1001 micrometers to 30000 micrometers.

93. The microfluidic channel structure according to claim 1, wherein: The length of the flow channel is 9200 microns.

94. The microfluidic channel structure according to claim 1, wherein: The length of the flow channel is 10912 microns.

95. The microfluidic channel structure according to claim 1, wherein: The length of the flow channel is 20112 microns.

96. The microfluidic channel structure according to claim 1, wherein: The length of the flow channel is 4912 microns.

97. The microfluidic channel structure according to claim 1, wherein: The length of the flow channel is 2319 microns.

98. The microfluidic channel structure according to claim 1, wherein: The length of the flow channel is 7231 microns.

99. The microfluidic channel structure according to claim 1, wherein: The interrogation region of the flow channel comprises a width of 50 microns to 200 microns.

100. The microfluidic channel structure according to claim 1, wherein: The interrogation region of the flow channel included a width of 135 microns.

101. The microfluidic channel structure according to claim 1, wherein: The interrogation region of the flow channel includes a height of 50 microns to 150 microns.

102. The microfluidic channel structure according to claim 1, wherein: The interrogation region of the flow channel included a height of 67.5 microns.

103. The microfluidic channel structure according to claim 1, wherein: The interrogation region of the flow channel includes a length of 100 microns to 5000 microns.

104. The microfluidic channel structure according to claim 1, wherein: The interrogation region of the flow channel comprised a length of 2319 microns.

105. The microfluidic channel structure according to claim 1, wherein: A sample fluid stream is positioned at a set height within the flow channel.

106. The microfluidic channel structure according to claim 105, wherein: The heights of the plurality of tines of both the first microfluidic channel structure and the second microfluidic channel structure enable the sheath fluid stream to flow into the flow channel to position the sample fluid stream at the set height within the flow channel.

107. The microfluidic channel structure according to claim 105, wherein: The set height is at 2 / 3 of the height of the flow channel and at + / - 1 / 8 of the height of the flow channel.

108. The microfluidic channel structure according to claim 105, wherein: The set height is at 2 / 3 of the height of the flow channel.

109. The microfluidic channel structure according to claim 105, wherein: The set height is 2 / 3 of the height of the flow channel and + / - 1 / 8 of the height of the flow channel.

110. The microfluidic channel structure according to claim 105, wherein: The set height is at ¾ of the height of the flow channel.

111. The microfluidic channel structure according to claim 105, wherein: The set height is not at a centerline of the flow channel.

112. The microfluidic channel structure according to claim 105, wherein: The set height is offset from a centerline of the flow channel.

113. The microfluidic channel structure according to claim 105, wherein: The set height is 18 micrometers to 25 micrometers from the top surface of the flow channel.

114. The microfluidic channel structure according to claim 105, wherein: The set height was 18 microns from the top surface of the flow channel.

115. The microfluidic channel structure according to claim 105, wherein: The set height was 19 microns from the top surface of the flow channel.

116. The microfluidic channel structure according to claim 105, wherein: The set height is 20 microns from the top surface of the flow channel.

117. The microfluidic channel structure according to claim 105, wherein: The set height was 21 microns from the top surface of the flow channel.

118. The microfluidic channel structure according to claim 105, wherein: The set height was 22 microns from the top surface of the flow channel.

119. The microfluidic channel structure according to claim 105, wherein: The set height was 23 microns from the top surface of the flow channel.

120. The microfluidic channel structure according to claim 105, wherein: The set height was 24 microns from the top surface of the flow channel.

121. The microfluidic channel structure according to claim 105, wherein: The set height is 25 microns from the top surface of the flow channel.

122. The microfluidic channel structure according to claim 105, wherein: The set height is at least 18 microns from a top surface of the flow channel and is offset from a centerline of the flow channel.

123. A microfluidic chip comprising: a first base layer and a second base layer; a sheath fluid channel structure originating upstream from a sheath fluid inlet, the sheath fluid channel structure comprising a first side channel structure and a second side channel structure, wherein upper portions of the first side channel structure and the second side channel structure are disposed in the first substrate layer, and wherein lower portions of the first side channel structure and the portion of the side channel structure are disposed in the second substrate layer; a sample fluid channel originating from an upstream sample fluid inlet; a flow channel disposed downstream of the sample fluid inlet and the sheath fluid inlet; wherein the first side channel structure and the second side channel structure each include a plurality of tines extending from the main sheath fluid channel to the flow channel; The plurality of tines of the first channel structure and the second channel structure intersect with the flow channel at the intersection.

124. A microfluidic chip comprising the microfluidic channel structure according to any one of claims 1 to 122.

125. A flow cytometer system comprising: Inquiry into the light source; Detector; as well as A microfluidic chip, comprising: a sheath fluid channel structure originating upstream from a sheath fluid inlet, the sheath fluid channel structure comprising a first side channel structure and a second side channel structure; a sample fluid channel originating from an upstream sample fluid inlet; a flow channel disposed downstream of the sample fluid inlet and the sheath fluid inlet; an interrogation region disposed downstream of the flow channel; wherein the first side channel structure and the second side channel structure each include a plurality of tines extending from the main sheath fluid channel to the flow channel; The plurality of tines of the first channel structure and the second channel structure intersect with the flow channel at the intersection.

126. A flow cytometer system comprising the microfluidic chip according to claim 123.

127. A flow cytometer system, comprising a microfluidic chip, wherein the microfluidic chip comprises the microfluidic channel structure according to any one of claims 1 to 122.

128. A method for focusing a sample fluid stream, the method comprising: introducing a sheath fluid as a sheath fluid stream into the sheath fluid channel structure at a sheath fluid inlet; transferring the sheath fluid flow into both the first side channel structure and the second side channel structure of the sheath fluid channel structure; introducing a sample fluid as a sample fluid stream into the sample fluid channel at a sample fluid inlet; allowing the sheath fluid stream to flow into a plurality of tines of the first side channel structure; allowing the sheath fluid stream to flow into the plurality of tines of the second side channel structure; allowing the sample fluid stream to flow into a flow channel disposed downstream of the sample fluid inlet and the sheath fluid inlet; intersecting the sample fluid flow in the flow channel at an intersection with sheath fluid flow from the plurality of tines of the first side channel structure and the plurality of tines of the second side channel structure; as well as The sample fluid flow is asymmetrically focused at the intersection by the sheath fluid flow, wherein the sample fluid flow is focused into a smaller, narrower flow by asymmetrically introducing the sheath fluid flow from a plurality of tines of the first side channel structure and from a plurality of tines of the second side channel structure.

129. A method for focusing a sample fluid stream, the method comprising: The sample fluid stream is asymmetrically focused by repeatedly introducing a sheath fluid stream to alternately intersect the sample fluid stream in the flow channel.

130. A method for focusing a sample fluid flow, comprising asymmetrically focusing the sample fluid flow in a microfluidic channel structure according to any one of claims 1 to 122.

131. A method for focusing a sample fluid flow, comprising asymmetrically focusing the sample fluid flow in a microfluidic chip, the microfluidic chip comprising a microfluidic channel structure according to any one of claims 1 to 122.

132. A method for focusing a sample stream, the method comprising: The sample stream is positioned at a location offset from a centerline of the flow channel.

133. The method of claim 132, wherein: The method further comprises positioning the sample flow using a microfluidic channel structure according to any one of claims 1 to 122.

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