CN115957838A

CN115957838A - Microfluidic chip and application method thereof

Info

Publication number: CN115957838A
Application number: CN202310041870.2A
Authority: CN
Inventors: 周侗; 林安丽; 崔博婧; 崔彩媚; 庞志强
Original assignee: Ruisi Life Guangdong Technology Co ltd
Current assignee: Ruisi Life Guangdong Technology Co ltd
Priority date: 2023-01-13
Filing date: 2023-01-13
Publication date: 2023-04-14
Anticipated expiration: 2043-01-13
Also published as: CN115957838B

Abstract

The invention relates to a micro-fluidic chip and a using method thereof, comprising the following steps: an inlet region formed with a sample solution channel and a buffer channel; the sorting area is connected with the inlet area, the sorting area forms a sorting channel, a first micro-column array and a second micro-column array are arranged on the sorting channel, the first micro-column array inclines towards the direction close to the central line of the sorting channel, and the second micro-column array inclines towards the direction far away from the central line of the sorting channel; the outlet area is connected with the sorting area, a first outlet channel and a second outlet channel are formed in the outlet area, and the first outlet channel and the second outlet channel are communicated with the sorting channel. The microfluidic chip can obtain the target cell collecting fluid with higher purity, improves the sorting precision, saves more purification working procedures and time for workers, and further improves the accurate purification of the target cells by using the microfluidic chip.

Description

Microfluidic chip and application method thereof

Technical Field

The invention relates to the field of cell sorting, in particular to a microfluidic chip and a using method thereof.

Background

In the field of cell separation, microfluidic chips that perform cell separation based on various physicochemical differences between different cells are widely used. Among them, label-free sorting techniques based on physical properties such as cell size and density are attracting attention because they allow for more downstream applications. Among the numerous label-free microfluidic sorting technologies, the deterministic lateral displacement technology has higher separation recovery rate and separation purity compared with other technologies such as inertia force, acoustic field force and the like due to the structure and performance similar to filter screen filtering. Meanwhile, the cell deformation condition can be controlled by adjusting the separation pressure or flow, and the method is particularly suitable for cell separation scenes needing to combine rigidity and nuclear-to-cytoplasmic ratio difference.

In the current lateral displacement microfluidic chip technology, the microfluidic chip structure characteristic size is matched with cells, so that the advantages of high precision, high flux and low cost are achieved, the sorting purity still needs to be improved, and in the current lateral displacement microfluidic chip, a fixed critical separation size (target sample size) with a fluctuation interval is set by matching fixed sorting pressure or sorting flow of a lateral displacement micro-column array used for cell sorting. When the sample difference is large or the cell sorting requirement is changed, the sorting performance of the chip is directly reduced or lost, and the application of the lateral displacement technology in the field of cell separation is greatly limited.

Disclosure of Invention

Based on the micro-fluidic chip and the use method thereof, the sorting precision of the micro-fluidic chip in the cell sorting process is improved.

A microfluidic chip, comprising: an inlet region formed with a sample solution channel and a buffer channel; the separation area is connected with the inlet area, a separation channel is formed in the separation area, the separation channel is communicated with the sample solution channel and the buffer solution channel, a first micro-column array and a second micro-column array are arranged on the separation channel, the first micro-column array and the second micro-column array are respectively formed by micro-column arrays in a distributed mode, the first micro-column array inclines towards the direction close to the central line of the separation channel, the second micro-column array inclines towards the direction far away from the central line of the separation channel, and the first micro-column array is located at the solution inlet end and the solution outlet end of the separation channel; the outlet area, the outlet area with select separately the district and connect, form first outlet channel and second outlet channel on the outlet area, first outlet channel is located the inboard of second outlet channel, first outlet channel with second outlet channel with select separately the passageway intercommunication.

The application discloses a micro-fluidic chip, a sample solution to be detected and a buffer solution enter the micro-fluidic chip through an inlet area, the sample solution channel and the buffer solution channel formed by the micro-fluidic chip respectively flow the sample solution to be detected and the buffer solution to a separation area together, the separation area is provided with a separation channel, the separation channel comprises an obliquely arranged micro-column array, the first micro-column array inclines towards the central line direction of the separation channel, after the sample solution enters the separation channel, small-sized cells pass through the first micro-column array, the flow direction of the small-sized cells is unchanged, the small-sized cells continuously move along the liquid flow direction in the whole horizontal direction, large cells laterally move along the inclination direction of the first micro-column array, namely the large cells are collected towards the central direction of the separation channel, and the small cells continuously flow along the central direction far away from the separation channel; at this time, when the sample solution enriched by the first micro-column array flows into the second micro-column array, the large cells move along the inclined direction of the second micro-column array, that is, the large cells flow in the direction away from the center of the sorting channel and mostly collect on the side wall of the sorting channel, then enter the first micro-column array, pass through the second micro-column array of the previous stage, so that the large cells enriched on the side wall of the sorting channel collect along the center of the sorting channel, the second micro-column array is located at the end of the sorting region, the large cells enriched by the sorting region flow to the large cell collecting end through the connected first outlet channel after circulating sorting and enrichment, and the rest of the solution flows out of the small cell collecting end through the second outlet channel, so that the target cell collecting solution with higher purity can be obtained, the sorting precision of the micro-fluidic chip is improved, more purification working procedures and time are saved for workers, and the precise purification of the target cells by using the chip is further improved.

In one embodiment, the number of the first micropillar array and the second micropillar array is multiple, and the second micropillar array is located between two first micropillar arrays. Through being equipped with a plurality of first microcolumn arrays and second microcolumn array, sorting channel arranges first microcolumn array and second microcolumn array through the combination, and includes the structure that a set of first microcolumn array and second microcolumn array set up crisscross at least, and further, this first microcolumn array can set up the multiunit with the crisscross structure of second microcolumn array to reach constantly further enrichment target cell to the sample solution in the last grade of sorting channel, thereby improve the target cell content of first outlet channel.

In one embodiment, the first micropillar array forms a first offset angle with the centerline direction of the sorting channel, the first offset angle being in a range of 0.5 to 12.5 °.

In one embodiment, the second micropillar array forms a second offset angle with the centerline direction of the sorting channel, the second offset angle being in a range of 0.5 to 12.5 °.

The offset angle range refers to the offset angle range between the first microcolumn array and the central line of the sorting channel, and the optimal offset angle range is set, so that the sorting channel length of the microfluidic chip and the circulating structures of the first microcolumn array and the second microcolumn array are more reasonable, and the sorting efficiency and the sorting precision are further improved.

In one embodiment, the distance between the microcolumns of the first and/or second microcolumn arrays distributed adjacently along the flow channel direction is in a range of 6 to 25 micrometers. The flow channel direction refers to the liquid flowing direction after the sample cells enter the sorting channel, the flow channel direction faces the liquid outlet end along the central line of the sorting channel, and the distance range between the microcolumns adjacently arranged in the flow channel direction is 6-25 micrometers.

In one embodiment, the distance between the micro-pillars of the first micro-pillar array and/or the second micro-pillar array which are adjacently distributed perpendicular to the flow channel direction is 10 to 60 micrometers. The flow channel direction refers to the liquid flowing direction after the sample cells enter the sorting channel, the flow channel direction faces the liquid outlet end along the central line of the sorting channel, and the distance range between the microcolumns which are adjacently distributed in the direction perpendicular to the flow channel direction is 10-60 micrometers.

In one embodiment, the height of the first micro-column array and/or the second micro-column array perpendicular to the flow channel direction is in a range of 5 to 50 micrometers. The height range of the first micro-column array and the second micro-column array in the direction vertical to the flow channel is 5-50 micrometers. The design of the size ensures that the microfluidic chip has the advantages of compact structure, high sorting precision and good reproducibility.

In one embodiment, the cross-sectional shape of the microcolumn is one of a circle, an ellipse, a triangle, a rectangle, a trapezoid, a rhombus, an L-shape, a C-shape, and a T-shape.

In one embodiment, the height of the sorting channel perpendicular to the direction of flow can be varied with different sorting pressures. The method comprises the steps that a sample solution to be detected in an inlet area and a buffer solution flow on a sorting channel after entering a sorting area, for cells capable of generating deformation, under the condition that the liquid is pressurized to enter the sorting channel, the cells deform, and therefore the height of the sorting channel, namely the height of a liquid channel formed on the sorting area, can change, the actual critical separation size of the sorting area can be adjusted by changing sorting pressure aiming at the cell samples capable of generating deformation, and the fixed critical separation size is set by matching the fixed sorting pressure or sorting flow with a micro-column array in the existing lateral displacement technology, so that when the sample difference is large or the cell sorting requirement changes, the sorting performance of a micro-fluidic chip is reduced or lost.

In one embodiment, the sorting channel has a height perpendicular to the direction of flow in the range of 15 to 60 microns. The sorting channel, namely the sorting area, is provided with a liquid channel, the height of the liquid channel, which is vertical to the fluid direction, is larger than the height of the first micro-column array and the second micro-column array, which is vertical to the fluid direction, and the height range is 15-60 micrometers.

In one embodiment, the sorting region includes a buffer solution flow channel region, a circulation sorting region, and a small cell flow channel region, the buffer solution flow channel region is formed with a buffer solution supply channel, the buffer solution supply channel is communicated with the buffer solution channel of the inlet region, the buffer solution flow channel region is located inside the circulation sorting region, the circulation sorting region is communicated with the sample solution channel and the buffer solution channel, the circulation sorting region is formed with the sorting channel, the first and second micro-column arrays are located on the circulation sorting region, the small cell flow channel region is formed with a small cell channel, and the small cell channel is communicated with the sorting channel. The buffer solution flow channel area is arranged in the center of the interior of the sorting channel, the sorting channel is formed on the circular sorting area, the small cell channel formed on the small cell flow channel area is positioned on the outer side of the circular sorting area, after fluid flows through the first micro-column array and the second micro-column array on the circular sorting area for cell sorting, most of cells collected by a target continuously flow forwards along the sorting channel, and most of small cells continuously flow forwards along the small cell flow channel area on the outer side, so that the cell sorting work is realized.

In one embodiment, the circular sorting section includes a first converging section, a diverging section and a first partition, the first converging section and the diverging section are respectively provided with the first micropillar array and the second micropillar array, the first converging section is adjacent to the inlet section, the diverging section is located at the liquid outlet end of the first converging section, the small cell flow channel section is located outside the diverging section, the first partition is arranged between the diverging section and the small cell flow channel section, and the small cell channel is communicated with the first converging section. The solution inlet end of the circular sorting area is provided with a first convergence area, the first convergence area is provided with a first microcolumn array, large cells in the sample solution are enriched along the direction of a buffer solution flow channel area facing the center, the sample solution passing through the first convergence area flows into the divergent area, the sample solution with high cell concentration enters a small cell flow channel area positioned on the outer side of the divergent area, and the enriched sample solution containing more large cells enters the divergent area, so that the sample solution is purified once. The purified sample solution flows through the second micro-column array arranged on the divergent zone and moves to the first partition part along the direction far away from the flow channel, and further, elastic collision occurs between the large cells and the side wall of the first partition part, so that the large cells are not damaged, the cell structure of the collected target cell solution is complete, the activity of the collected target cell solution is high, and the next step of use and research is facilitated. In the process, most of large cells in the sample solution obtained by the first purification are collected on the side wall of the first partition part again, and then move to the next first convergence area along the direction close to the flow channel at the outlet of the divergent area, so that the cells in the sample solution are further purified, and the content of target sorting large cells in the small cell channel is reduced.

In one embodiment, the number of the cyclic separation areas is multiple, the critical separation size among the cyclic separation areas distributed along the liquid flow direction is increased, and the critical separation size of the cyclic separation area at the next stage is 105% -200% of the critical separation size of the cyclic separation area at the previous stage. Furthermore, cells with different sizes are separated step by arranging a plurality of circulating separation areas connected in series, so that the sample solution is continuously purified to obtain large cells with higher concentration. The sizes of the adjacent circular sorting areas are different, specifically, the size of the circular sorting area at the next stage in the liquid outflow direction is 105% -200% of the size of the circular sorting area at the previous stage, the critical sorting size is increased along the flow channel direction, the sorting precision and the sorting purity of large cells are further improved, the cell separation resolution is increased, and the flow channel blocking risk is reduced.

In one embodiment, the number of the first convergent sections, the divergent sections and the first partition is plural, and the first convergent sections and the divergent sections are arranged alternately. The number of the first convergence areas and the number of the divergence areas in the circular sorting area are multiple, and the multiple first convergence areas and the multiple divergence areas are arranged in a staggered mode, so that the sorting purity is improved, and the content of large cells in the sorting channel is further improved.

In one embodiment, the buffer solution flow-path region and the small cell flow-path region are provided with a cylindrical array, and the distribution direction of the cylindrical array is the same as the liquid flowing direction. Through be equipped with the cylinder array on first convergent area and divergent district, and the cylinder in the cylinder array arranges the direction unanimous with the liquid flow direction, and this cylinder array is used for stabilizing the interior solution flow of runner.

In one embodiment, the buffer solution flow channel further comprises a flow guide part, a gap is formed between the adjacent first convergence regions and the divergence regions, the flow guide part is arranged on the outer side wall of the buffer solution flow channel region and/or the inner side wall of the first separation part, and the flow guide part is positioned at the gap. Preferably, one side of the drainage part close to the first convergence region inclines towards the direction of the central line far away from the sorting channel along the liquid flowing direction, so that the solution collected towards the direction of the central line of the sorting channel flows towards the direction of the divergence region to play a drainage effect on large cells, and one side of the drainage part close to the divergence region inclines towards the direction of the central line close to the sorting channel along the liquid flowing direction.

In one embodiment, the micro-column array comprises a circular sorting region, a buffer flow-path region, a small cell flow-path region, a circulating sorting region, a first micro-column array, a second micro-column array, a buffer flow-path region, a small cell flow-path region and a sorting channel. And further purifying the solution flowing out of the circular separation area and close to one side of the central line by the arrangement of a final separation area, and completing the final purification work. The end of the last separation zone is connected to the outlet zone, the last separation zone is located at the end of the circulating separation zone, the separation channel extends over the last separation zone, and the buffer flow channel zone extends at least partially to the end of the last separation zone, such that the buffer on the buffer flow channel zone communicates with the separation channel over the last separation zone.

In one embodiment, the final separation region includes a second convergence region on which the first micropillar array is disposed, and a second partition located outside the second convergence region. The last separation area is provided with a second convergence area, the second convergence area is provided with a first microcolumn array in a circulating mode to purify the solution purified by the circulating separation area again, the second partition part is arranged between the small cell flow channel area on the last separation area and the second convergence area to separate out small cells in the solution purified by the circulating separation area again, the last separation area is communicated with the first outlet channel of the outlet area, and the solution with high target cell concentration is obtained through the first outlet channel.

In one embodiment, the critical separation size of the last separation region is higher than that of the circulation separation region, and the critical separation size of the last separation region is 120-600% of that of the first circulation separation region. The critical separation size of the final separation area is larger than that of the first circular separation area positioned in the separation channel, so that the separation precision and the separation purity of the large cells are further improved, and the flow channel blockage risk is reduced.

In one embodiment, the buffer flow-path region extends at least partially through the end selection region, and the end of the buffer flow-path region is located above the end selection region. Because the separation channel is continuously separated and purified along the flow channel direction, the content of large cells in the tail-end separation channel is high, the content of buffer solution is reduced, and the buffer solution flow channel area at least partially extends to the end part of the tail-end separation area and is communicated with the separation channel, so that the cell activity on the tail-end separation area is favorably maintained.

In one embodiment, the first partition comprises a first partition body and a flow guide end, the flow guide end is disposed on the first partition body, the flow guide end is located at the liquid outlet end of the first convergence region, and the flow guide end is used for guiding the liquid to the small cell flow channel region. Through be equipped with the water conservancy diversion end on first partition portion, it is used for guiding the solution of last one-level sorting channel outside to flow on going into the cell runner district, and the solution that is located near sorting channel inboard then continues to get into next stage sorting channel on, the content of the big cell content in its solution, big cell then offsets along next stage array direction, according to this process of constantly carrying out the circulation purification.

In one embodiment, the flow guide end has a flow guide arc surface located outside the flow guide end, and the flow guide arc surface is offset away from the centerline of the sorting channel. The outer side of the flow guide end forms a flow guide arc surface which is deviated to the central line, so that the flow resistance between the joints of the first micro-column array and the second micro-column array is favorably reduced, and the effects of flow guide and flow stabilization are achieved. Further, the second partition has the same structure thereon as the first partition.

In one embodiment, the junction of the diversion end and the first partition body forms a bend.

In one embodiment, the buffer channel comprises a first buffer channel and a second buffer channel, the inlet zone comprising a first buffer inlet zone, a second buffer inlet zone and a sample solution inlet zone, the first buffer inlet zone forming the first buffer channel, the second buffer inlet zone forming the second buffer channel, the second buffer channel being located inside the first buffer channel. By arranging the two buffer solution channels, the sample solution channel is arranged between the first buffer solution channel and the second buffer solution channel, a sheath flow is formed when the first buffer solution enters the sorting region along the first buffer solution channel, which is beneficial to the sample cell solution to be sorted in the initial stage to flow to the middle sorting region to a greater extent, thereby reducing the loss rate of target sample cells. The flow channel design of the buffer solution and the sample solution prevents the sample cell solution to be sorted from forming turbulent flow in the microfluidic chip, is favorable for improving the sorting efficiency and sorting precision of the sample cell solution after passing through the sorting area, and improves the sample cell recovery rate of the outlet area. In one embodiment, the outlet area comprises a first outlet on which the first outlet passage is formed adjacent to the liquid outlet end of the sorting passage and a second outlet on which the second outlet passage is formed. A first outlet port of the outlet section is connected to a final sorting section of the sorting section for recovering a solution of large cells collected by a target, and a second outlet port is connected to a small cell flow channel section for recovering a solution high in small cell content.

In one embodiment, the sample solution channel, the buffer solution channel, the first outlet channel and the second outlet channel are respectively provided with a cylindrical array, and the distribution direction of the cylindrical arrays is the same as the liquid flowing direction. The cylindrical arrays are arranged on the channels of the inlet area and the outlet area, the arrangement direction of the cylinders in the cylindrical arrays is consistent with the flowing direction of liquid, and the cylindrical arrays are used for stabilizing the flow of solution in the flow channels.

Specifically, the micro-structure of the microfluidic chip can be processed and prepared by using materials such as thermosetting or thermoplastic polymers, glass, silicon and the like, and a closed structure is formed by bonding, pressing, heat sealing/welding and the like.

In a second aspect of the present application, a method for separating and purifying a microfluidic chip is disclosed, which comprises the following steps: s1: with the above microfluidic chip, the inlet region is connected to a sample solution to be processed and a buffer solution, the sample solution to be processed flows through the sample solution channel, the buffer solution flows through the buffer solution channel, the outlet region is connected to a large cell recovery liquid tube and a small cell recovery liquid tube, the first outlet channel is communicated with the large cell recovery liquid tube, and the second outlet channel is communicated with the small cell recovery liquid tube; s2: applying the same sorting pressure to a sample solution to be processed and a buffer solution, wherein the sample solution to be processed and the buffer solution pass through the sorting area, a target cell solution is recycled to the large cell recycling liquid pipe through the first outlet channel, and the rest solution is recycled to the small cell recycling liquid pipe through the second outlet channel; s3: and after the sample solution to be treated is emptied or the recovery liquid is filled, the liquid inlet of the inlet area and the liquid outlet of the outlet area are interrupted, and the sorting pressure applied by the sample solution to be treated and the buffer liquid is contacted.

In a second aspect of the present application, a separation and purification method using the above microfluidic chip is disclosed, in which a first buffer solution inlet region and a second buffer solution inlet region are connected to a buffer solution, a sample solution inlet region is connected to a sample to be treated, a first outlet region is connected to a large cell recovery liquid tube, and a second outlet region is connected to a small cell recovery liquid tube; applying equal sorting pressure to the buffer solution and the sample solution to be processed, so that the buffer solution and the sample respectively flow into the microfluidic chip from the corresponding inlets and cell sorting is completed; after the sample solution is emptied or the recovery solution is filled, the inflow and outflow of the microfluidic chip are interrupted, and the sorting pressure applied to the buffer solution and the sample solution to be processed is released, thereby completing the sorting process.

In one embodiment, the sorting pressure is determined from a sorting database, and the step of building the sorting database comprises:

s1: under the condition of different sorting pressures in the interval of 0.5-2.5 Bar, carrying out sorting test on a polymer particle sample of 2-25 micrometers, and determining the critical separation size of the microfluidic chip according to the test result;

s2: testing the sorting recovery rate of different cell samples under different sorting pressures by using the micro-fluidic chip with the confirmed critical separation size;

s3: and according to the test result, establishing a cell sorting database by combining the microfluidic chip, the critical separation size, the cell types and characteristics, the sorting pressure and the corresponding sorting recovery rate.

Wherein the critical separation size is obtained by setting sorting recovery and test data statistics, and the critical separation size comprises a positive critical separation size and a negative critical separation size. Further, the forward critical separation size is statistically derived from the minimum size data of the sample flowing into the first outlet port at 100% recovery. The negative critical separation dimension is statistically derived from the minimum dimension data of the sample flowing into the second outlet port at 100% recovery.

In one embodiment, the critical separation size ranges from 5 to 9 microns, 8 to 13 microns, 12 to 18 microns, or 16 to 24 microns, corresponding to a sorting pressure of 0.5 to 2.5Bar.

Drawings

FIG. 1 is a schematic diagram of the overall structure of a microfluidic chip;

FIG. 2 is a schematic diagram of an inlet region of the microfluidic chip;

FIG. 3 is a schematic diagram of the structure of the outlet region of the microfluidic chip;

FIG. 4 is a schematic view of the configuration of the circular sorting section;

FIG. 5 is a schematic view of the first converging and diverging regions;

FIG. 6 is a schematic diagram of the structure of the final fraction selection region;

FIG. 7 is a schematic diagram of the convergent region of the final sorting section with buffer replenishment path;

FIG. 8 is a schematic diagram of the convergent region of the final sorting section without a buffer replenishment path;

fig. 9 is a schematic structural view of the first outlet passage.

Wherein, the corresponding relation between the reference signs and the component names is as follows:

1 inlet zone, 101 sample solution channel, 102 first buffer channel, 103 second buffer channel, 11 first buffer inlet zone, 12 second buffer inlet zone, 13 sample solution inlet zone;

2 sorting zone, 201 gap, 21 buffer flow-channel zone, 22 circular sorting zone, 221 first convergent zone, 222 divergent zone, 223 first partition, 2231 first partition body, 2232 flow-directing end, 23 small cell flow-channel zone, 24 last sorting zone, 241 second convergent zone, 242 second partition;

3 outlet zone, 301 first outlet passage, 302 second outlet passage, 31 first outlet, 32 second outlet;

4 a drainage part;

a is the local structure of the first micropillar array, B is the local structure of the second micropillar array.

Detailed Description

In order that the above objects, features and advantages of the present invention can be more clearly understood, a more particular description of the invention, taken in conjunction with the accompanying drawings and detailed description, is set forth below. It should be noted that the embodiments and features of the embodiments of the present application may be combined with each other without conflict.

In the following description, numerous specific details are set forth in order to provide a thorough understanding of the present invention, however, the present invention may be practiced otherwise than as specifically described herein and, therefore, the scope of the present invention is not limited by the specific embodiments disclosed below.

A microfluidic chip according to some embodiments of the present invention is described below with reference to the accompanying drawings.

Example 1

As shown in fig. 1 to 9, the present embodiment discloses a microfluidic chip, which includes an inlet region 1, wherein the inlet region 1 is formed with a sample solution channel 101 and a buffer channel; the sorting region 2 is connected with the inlet region 1, the sorting region 2 forms a sorting channel, the sorting channel is communicated with the sample solution channel 101 and the buffer solution channel, a first micro-column array and a second micro-column array are arranged on the sorting channel, the first micro-column array and the second micro-column array are respectively formed by micro-column arrays in a distributed mode, the first micro-column array inclines towards the direction close to the central line of the sorting channel, the second micro-column array inclines towards the direction far away from the central line of the sorting channel, and the first micro-column array is located at the solution inlet end and the solution outlet end of the sorting channel; the outlet area 3, the outlet area 3 is connected with the sorting area 2, a first outlet channel 301 and a second outlet channel 302 are formed on the outlet area 3, the first outlet channel 301 is located at the inner side of the second outlet channel 302, and the first outlet channel 301 and the second outlet channel 302 are communicated with the sorting channel.

The application discloses a micro-fluidic chip, a sample solution to be detected and a buffer solution enter the micro-fluidic chip through an inlet area 1, the sample solution channel 101 and the buffer solution channel formed by the micro-fluidic chip respectively enable the sample solution to be detected and the buffer solution to flow to a sorting area 2 together, the sorting area 2 is provided with a sorting channel, the sorting channel comprises a micro-column array which is obliquely arranged, a first micro-column array inclines towards the direction of a central line of the sorting channel, after the sample solution enters the sorting channel, small-sized cells pass through the first micro-column array, the flowing direction of the small-sized cells is unchanged, the small-sized cells continue to migrate along the direction of the liquid flowing integrally in the horizontal direction, large cells laterally move along the oblique direction of the first micro-column array, namely the large cells are collected towards the central direction of the sorting channel, and the small cells continue to flow along the direction far away from the central direction of the sorting channel; at this time, when the sample solution enriched by the first microcolumn array flows into the second microcolumn array, the large cells move along the inclined direction of the second microcolumn array, that is, the large cells flow in the direction away from the center of the sorting channel and mostly collect on the side wall of the sorting channel, and then enter the first microcolumn array, the large cells enriched on the side wall of the sorting channel collect along the center of the sorting channel by the second microcolumn array of the previous stage, the first microcolumn array is located at the end of the sorting region 2, the large cells enriched by the sorting region 2 flow to the large cell collecting end through the connected first outlet channel 301 after being circularly sorted and enriched, and the rest of the solution flows out of the small cell collecting end through the second outlet channel 302, so that a target cell collecting solution with higher purity can be obtained, the sorting precision of the microfluidic chip is improved, more purification work flows and time are saved for workers, and the precise purification of the target cells by using the chip is further improved.

As shown in fig. 4 to 7, in addition to the features of the above embodiment, the present embodiment further defines: the number of the first micro-column array and the second micro-column array is multiple, and the second micro-column array is positioned between the two first micro-column arrays. Through being equipped with a plurality of first microcolumn arrays and second microcolumn array, sorting channel arranges first microcolumn array and second microcolumn array through the combination, and includes the structure that a set of first microcolumn array and second microcolumn array set up crisscross at least, and further, this first microcolumn array can set up the multiunit with the crisscross structure of second microcolumn array to reach constantly further enrichment target cell to the sample solution in the last grade of sorting channel, thereby improve the target cell content of first outlet channel 301.

In addition to the features of the above embodiment, the present embodiment further defines: the first micropillar array and the central line direction of the sorting channel form a first offset angle, and the range of the first offset angle is 0.5-12.5 degrees.

In addition to the features of the above embodiments, the present embodiment further defines: the second micropillar array and the central line direction of the sorting channel form a second offset angle, and the range of the second offset angle is 0.5-12.5 degrees.

In addition to the features of the above embodiments, the present embodiment further defines: the distance between the microcolumns of the first microcolumn array and/or the second microcolumn array which are adjacently distributed along the flow channel direction ranges from 6 microns to 25 microns. The flow channel direction refers to the liquid flowing direction after the sample cells enter the sorting channel, the flow channel direction faces the liquid outlet end along the central line of the sorting channel, and the distance range between the microcolumns adjacently arranged in the flow channel direction is 6-25 micrometers.

In addition to the features of the above embodiments, the present embodiment further defines: the flow channel direction in which the distance range between the microcolumns of the first microcolumn array and/or the second microcolumn array which are adjacently distributed in the direction perpendicular to the flow channel direction is 10-60 micrometers refers to the liquid flow direction after the sample cells enter the sorting channel, the flow channel direction faces the liquid outlet end along the center line of the sorting channel, and the distance range between the microcolumns which are adjacently distributed in the direction perpendicular to the flow channel direction is 10-60 micrometers.

In addition to the features of the above embodiments, the present embodiment further defines: the height range of the first micro-column array and/or the second micro-column array vertical to the flow channel direction is 5-50 microns. The height range of the first micro-column array and the second micro-column array in the direction vertical to the flow channel is 5-50 micrometers. The design of the size ensures that the microfluidic chip has the advantages of compact structure, high sorting precision and good reproducibility.

In addition to the features of the above embodiments, the present embodiment further defines: the cross section of the micro-column is one of circular, oval, triangular, rectangular, trapezoidal, rhombic, L-shaped, C-shaped and T-shaped.

Further, in addition to the features of the above embodiment, the present embodiment further defines: the height of the sorting channel perpendicular to the direction of flow can vary with different sorting pressures. The sample solution to be detected in the inlet area 1 and the buffer solution flow on the sorting channel after entering the sorting area 2, and for cells capable of generating deformation, under the condition that the liquid is pressurized to enter the sorting channel, the cells deform, so that the height of the sorting channel, namely the height of the liquid channel formed on the sorting area 2, can change, the actual critical separation size of the sorting area 2 can be adjusted by changing the sorting pressure aiming at the cell samples capable of generating deformation, the fixed critical separation size is set by matching the fixed sorting pressure or sorting flow with the existing lateral displacement micro-column array, and the sorting performance of the micro-fluidic chip is reduced or lost when the sample difference is large or the cell sorting requirement changes.

In addition to the features of the above embodiments, the present embodiment further defines: the height of the sorting channel perpendicular to the flow direction is in the range of 15 to 60 microns. The sorting channel, that is, the sorting area 2, is formed with a liquid channel, the height of which perpendicular to the fluid direction is larger than the height of the first micro-column array and the second micro-column array perpendicular to the fluid direction, and the height range is 15 to 60 micrometers.

In addition to the features of the above embodiments, the present embodiment further defines: the sorting area 2 comprises a buffer solution flow channel area 21, a circulating sorting area 22 and a small cell flow channel area 23, a buffer solution supply channel is formed on the buffer solution flow channel area 21 and is communicated with a buffer solution channel of the inlet area 1, the buffer solution flow channel area 21 is positioned on the inner side of the circulating sorting area 22, the circulating sorting area 22 is communicated with the sample solution channel 101 and the buffer solution channel, the circulating sorting area 22 forms a sorting channel, the first micro-column array and the second micro-column array are positioned on the circulating sorting area 22, the small cell flow channel area 23 forms a small cell channel, and the small cell channel is communicated with the sorting channel. The buffer fluid flow channel area 21 is arranged at the center of the interior of the sorting channel, the sorting channel is formed on the circular sorting area 22, the small cell channel formed on the small cell flow channel area 23 is positioned at the outer side of the circular sorting area 22, most of cells collected by a target continuously flow forwards along the sorting channel after fluid flows through the first micro-column array and the second micro-column array on the circular sorting area 22 for cell sorting, and most of small cells continuously flow forwards along the small cell flow channel area 23 at the outer side, so that the cell sorting work is realized.

As shown in fig. 4 and 5, in addition to the features of the above embodiment, the present embodiment further defines: the circular separation region 22 includes a first convergence region 221, a divergence region 222 and a first partition 223, the first convergence region 221 and the divergence region 222 are respectively provided with a first microcolumn array and a second microcolumn array, the first convergence region 221 is adjacent to the inlet region 1, the divergence region 222 is located at the liquid outlet end of the first convergence region 221, the small cell flow channel region 23 is located at the outer side of the divergence region 222, the first partition 223 is disposed between the divergence region 222 and the small cell flow channel region 23, and the small cell channel is communicated with the first convergence region 221. The solution inlet end of the circular separation area 22 is provided with the first convergence area 221, the first convergence area 221 is provided with the first microcolumn array, large cells in the sample solution are enriched along the direction of the buffer solution channel area 21 facing the center, the sample solution passing through the first convergence area 221 flows into the divergent area 222, at this time, the sample solution with high small cell concentration enters the small cell channel area 23 located on the outer side of the divergent area 222, and the enriched sample solution with more large cells enters the divergent area 222, so that the sample solution is purified once. The purified sample solution flows through the second micro-column array arranged on the divergent region 222 and moves to the first partition 223 along the direction away from the flow channel, and further, the large cells elastically collide with the side wall of the first partition 223 without damaging the large cells, so that the collected target cell solution has a complete cell structure and high activity, and is convenient for use and research in the next step. In the above process, most of the large cells in the sample solution obtained by the first purification are collected again on the sidewall of the first partition 223, and then move to the next first convergence region 221 along the direction close to the flow channel at the outlet of the divergent region 222, and the cells in the sample solution are further purified, thereby reducing the content of the target sorted large cells in the small cell channel.

In addition to the features of the above embodiments, the present embodiment further defines: the number of the circulation separation areas 22 is a plurality, the critical separation size between the circulation separation areas 22 distributed along the liquid flow direction is increased gradually, and the critical separation size of the next stage circulation separation area 22 is 105% -200% of the critical separation size of the previous stage circulation separation area 22. Further, cells of different sizes are separated step by arranging a plurality of circulating separation zones 22 connected in series, so that the sample solution is continuously purified to obtain large cells with higher concentration. The sizes of the adjacent circular separation regions 22 are different, specifically, the size of the circular separation region 22 positioned in the liquid outflow direction in the next-stage circular separation region 22 is 105-200% of the size of the previous-stage circular separation region 22, the critical separation size is increased along the flow channel direction, which is beneficial to further improving the separation precision and separation purity of large cells, increasing the resolution of cell separation and reducing the risk of flow channel blockage.

In addition to the features of the above embodiments, the present embodiment further defines: the number of the first converging sections 221, the diverging sections 222 and the first separating sections 223 is plural, and the first converging sections 221 and the diverging sections 222 are arranged alternately. The number of the first convergence regions 221 and the divergence regions 222 in the circular sorting region 22 is multiple, and the multiple first convergence regions 221 and the multiple divergence regions 222 are arranged in a staggered manner, so that the sorting purity is improved, and the large cell content in the sorting channel is further improved.

In addition to the features of the above embodiments, the present embodiment further defines: the buffer solution flow channel area 21 and the small cell flow channel area 23 are provided with cylindrical arrays, and the distribution direction of the cylindrical arrays is the same as the liquid flowing direction. By providing the first convergent section 221 and the divergent section 222 with a cylindrical array, and the arrangement direction of the cylinders in the cylindrical array is the same as the liquid flow direction, the cylindrical array is used to stabilize the solution flow in the flow channel.

In addition to the features of the above embodiments, the present embodiment further defines: and a drainage part 4 is further included, a gap 201 is formed between the adjacent first convergence region 221 and the divergence region 222, the drainage part 4 is arranged on the outer side wall of the buffer fluid channel region 21 and/or the inner side wall of the first separation part 223, and the drainage part 4 is positioned at the gap 201. Specifically, one side of the drainage part 4 close to the first convergence region 221 is inclined towards the direction far away from the central line of the sorting channel along the liquid flowing direction, so that the solution collected towards the central line of the sorting channel flows towards the direction of the divergent region 222 to perform the drainage effect on the large cells, and one side of the drainage part 4 close to the divergent region 222 is inclined towards the direction close to the central line of the sorting channel along the liquid flowing direction.

As shown in fig. 6 to 9, in addition to the features of the above embodiment, the present embodiment further defines: the device also comprises an end-stage separation area 24, wherein the end-stage separation area 24 is positioned at the liquid outlet end of the circulating separation area 22, a first micro-column array is arranged on the end-stage separation area 24, the buffer solution flow channel area 21 is positioned at the inner side of the end-stage separation area 24, the small cell flow channel area 23 is positioned at the outer side of the end-stage separation area 24, and a separation channel extends to the end-stage separation area 24. The solution flowing out of the circular separation zone 22 near the center line is further purified by the end separation zone 24 and the final purification work is completed. The end of the end separation region 24 is connected to the outlet region 3, the end separation region 24 is located at the end of the circulating separation region 22, the separation channel extends to the end separation region 24, and the buffer flow channel region 21 extends at least partially to the end of the end separation region 24, so that the buffer on the buffer flow channel region 21 communicates with the separation channel at the end separation region 24.

In addition to the features of the above embodiments, the present embodiment further defines: the final separation region 24 includes a second convergent region 241 and a second partition 242, a plurality of first micropillar arrays are disposed on the second convergent region 241, and the second partition 242 is located outside the second convergent region 241. The last separation region 24 is provided with a second convergence region 241, the second convergence region 241 is provided with a first microcolumn array in a circulating manner to purify the solution purified by the circulating separation region 22 again, a second partition part 242 is provided between the small cell flow channel region 23 on the last separation region 24 and the second convergence region 241 to separate out the small cells in the solution after passing through the circulating separation region 22 again, the last separation region 24 is communicated with the first outlet channel 301 of the outlet region 3, and the solution with high target cell concentration is obtained through the first outlet channel 301.

In addition to the features of the above embodiments, the present embodiment further defines: the critical separation size of the final stage separation region 24 is higher than that of the circular separation region 22, and the critical separation size of the final stage separation region 24 is 120-600% of that of the first circular separation region 22. The critical separation dimension of the final separation region 24 is larger than the first circular separation region 22 in the separation channel, which is beneficial to further improving the separation precision and separation purity of the large cells and reducing the risk of channel blockage.

In addition to the features of the above embodiments, the present embodiment further defines: the buffer flow-path region 21 extends at least partially across the end separation region 24, the end of the buffer flow-path region 21 being located above the end separation region 24. Since the separation and purification of the sorting channel are continuously performed along the flow channel direction, the content of large cells in the sorting channel at the tail end is high, the content of buffer solution is reduced, and the buffer solution flow channel area 21 at least partially extends to the end part of the tail-stage sorting area 24 and is communicated with the sorting channel, so that the cell activity on the tail-stage sorting area 24 is favorably maintained.

As shown in fig. 4, in addition to the features of the above embodiment, the present embodiment further defines: the first partition 223 includes a first partition body 2231 and a flow guiding end 2232, the flow guiding end 2232 is disposed on the first partition body 2231, the flow guiding end 2232 is located at the liquid outlet end of the first convergence region 221, and the flow guiding end 2232 is used for guiding the liquid to the small cell flow channel region 23. The first partition 223 is provided with a flow guide end 2232 for guiding the solution outside the previous sorting channel to flow into the small cell flow channel region 23, while the solution inside the sorting channel continues to flow into the next sorting channel, so that the content of large cells in the solution is high, and the large cells are shifted in the next array direction, thereby continuously performing the circular purification process.

In addition to the features of the above embodiments, the present embodiment further defines: the flow guide end 2232 has a flow guide curved surface that is located outside the flow guide end 2232, the flow guide curved surface being offset toward a centerline away from the sorting channel. The outer side of the flow guiding end 2232 forms a flow guiding arc surface which is offset to the center line, which is beneficial to reducing the fluid resistance between the joints of the first micro-column array and the second micro-column array, and achieves the effects of flow guiding and flow stabilizing. Further, the second partition 242 has the same structure as the first partition 223.

In addition to the features of the above embodiments, the present embodiment further defines: the junction of the diversion end 2232 and the first partition body 2231 forms a bend.

As shown in fig. 1 and 2, in addition to the features of the above embodiment, the present embodiment further defines: the buffer channel comprises a first buffer channel 102 and a second buffer channel 103, the inlet zone 1 comprises a first buffer inlet zone 11, a second buffer inlet zone 12 and a sample solution inlet zone 13, the first buffer inlet zone 11 forms the first buffer channel 102, the second buffer inlet zone 12 forms the second buffer channel 103, and the second buffer channel 103 is located inside the first buffer channel 102. By arranging two buffer solution channels, and arranging the sample solution channel 101 between the first buffer solution channel 102 and the second buffer solution channel 103, the first buffer solution forms sheath flow when entering the sorting area 2 along the first buffer solution channel 102, which is beneficial to the sample cell solution to be sorted in the initial stage to flow to the middle sorting area 2 to a greater extent, thereby reducing the loss rate of target sample cells, and by arranging the second buffer solution channel 103 on the inner side of the sample solution channel 101, after the second buffer solution enters the sorting area 2 along the second buffer solution channel 103, the sample cell solution is convenient to mix and form a middle liquid flow, thereby ensuring that the sample cell solution flows to an area with a sorting function outside the buffer solution supply channel, further, the first buffer solution channel 102, the second buffer solution channel 103 and the sample solution channel 101 in the inlet area 1 are respectively the same as the liquid flow direction in the sorting channel, which is beneficial to avoiding the sample cell solution from influencing the liquid flow direction after entering the sorting area 2 along with the buffer solution. The flow channel design of the buffer solution and the sample solution prevents the sample cell solution to be sorted from forming turbulent flow in the microfluidic chip, is favorable for improving the sorting efficiency and sorting precision of the sample cell solution after passing through the sorting area 2, and improves the sample cell recovery rate of the outlet area 3.

As shown in fig. 1 and 3, in addition to the features of the above embodiment, the present embodiment further defines: the outlet zone 3 comprises a first outlet 31, on which first outlet channel 301 is formed, and a second outlet 32, on which second outlet channel 302 is formed, the first outlet 31 being adjacent to the outlet end of the sorting channel and the second outlet 32. The first outlet port 31 of the outlet section 3 is connected to the final sorting section 24 on the sorting section 2 for recovering the large-cell solution collected by the object, and the second outlet port 32 is communicated with the small-cell flow path section 23 for recovering the solution high in small-cell content.

In addition to the features of the above embodiments, the present embodiment further defines: the sample solution channel 101, the buffer solution channel, the first outlet channel 301 and the second outlet channel 302 are respectively provided with a cylindrical array, and the distribution direction of the cylindrical arrays is the same as the liquid flowing direction. The cylindrical arrays are arranged on the channels of the inlet area 1 and the outlet area 3, the arrangement direction of the cylinders in the cylindrical arrays is consistent with the flowing direction of liquid, and the cylindrical arrays are used for stabilizing the flow of solution in the flow channels.

Example 2

The second embodiment of the application discloses a separation and purification method of a microfluidic chip. The method comprises the following steps:

s1: with the microfluidic chip, the inlet area 1 is connected with a sample solution to be processed and a buffer solution, the sample solution to be processed flows through the sample solution channel 101, the buffer solution flows through the buffer solution channel, the outlet area 3 is connected with the large cell recovery liquid tube and the small cell recovery liquid tube, the first outlet channel 301 is communicated with the large cell recovery liquid tube, and the second outlet channel 302 is communicated with the small cell recovery liquid tube;

s2: the same sorting pressure is applied to the sample solution to be processed and the buffer solution, the sample solution to be processed and the buffer solution pass through the sorting area 2, the target cell solution is recycled to the large cell recycling liquid pipe through the first outlet channel 301, and the rest solution is recycled to the small cell recycling liquid pipe through the second outlet channel 302;

s3: after the sample solution to be treated is emptied or the recovery solution is filled, the feed of the inlet region 1 and the discharge of the outlet region 3 are interrupted and the sorting pressure applied by the sample solution to be treated and the buffer solution is brought into contact.

In a second aspect of the present application, a separation and purification method using the above microfluidic chip is disclosed, wherein a first buffer solution inlet area 11 and a second buffer solution inlet area 12 are connected to a buffer solution, a sample solution inlet area 13 is connected to a sample to be processed, a first outlet 31 is connected to a large cell recovery tube, and a second outlet 32 is connected to a small cell recovery tube; applying equal sorting pressure to the buffer solution and the sample to be processed to enable the buffer solution and the sample to respectively flow into the microfluidic chip from the corresponding inlets and complete cell sorting; after the sample is emptied or the recovery liquid is filled, the inflow and outflow of the microfluidic chip are interrupted, and the sorting pressure applied to the buffer liquid and the sample to be processed is relieved, thereby completing the sorting process.

Example 3

In addition to the features of the above embodiment, the present embodiment further defines that the sorting pressure is determined from a sorting database, and the building of the sorting database includes the following steps, S1: under the condition of different sorting pressures in the interval of 0.5-2.5 Bar, carrying out sorting test on a polymer particle sample of 2-25 micrometers, and determining the critical separation size of the microfluidic chip according to the test result; s2: testing the sorting recovery rate of different cell samples under different sorting pressures by using the micro-fluidic chip with the confirmed critical separation size, and determining the optimal sorting pressure; s3: and according to the test result, establishing a cell sorting database by combining the microfluidic chip, the critical separation size, the cell types and characteristics, the sorting pressure and the corresponding sorting recovery rate.

To explain the progress, the invention adopts the microfluidic chip with the following specifications to build a sorting database: the first microcolumn array and the second microcolumn array in the microfluidic chip are both provided with isosceles triangle microcolumns, wherein the vertex angle of the triangle is 120 degrees, the offset angle of the sorting channel of the microcolumn array in the first convergence region 221 and the second convergence region 241 is 3.17 degrees, the offset angle of the sorting channel of the microcolumn array in the divergence region 222 is 5.22 degrees, the distance range between the microcolumns which are adjacently distributed in the direction perpendicular to the flow channel of the first microcolumn array and the second microcolumn array is 50 micrometers, and the critical separation sizes of the sorting regions of the first fraction and the second fraction from front to back are 4 micrometers, 5.6 micrometers and 7 micrometers. The whole critical separation size range of the chip is 8-13 microns under the sorting pressure of 0.5-2.5 Bar.

Wherein: the forward critical separation size is statistically derived from the minimum size data of the sample flowing into the primary outlet port 31 at 100% recovery. The negative critical separation dimension is statistically derived from the minimum dimension data of the sample flowing into the second outlet port 32 at 100% recovery. The first outlet port 31 is used for recovering the sorted large cell solution, and the second outlet port 32 is used for recovering the small cell solution.

(1) Particle testing:

for 5 different specifications of microfluidic chips with critical separation size design values of 2-16 micrometers, polystyrene particle samples with particle sizes of 1 micrometer, 3 micrometers, 5 micrometers, 7 micrometers, 9 micrometers, 11 micrometers, 13 micrometers, 15 micrometers, 17 micrometers and 19 micrometers are respectively used for sorting tests, and actual positive critical separation sizes and negative critical separation sizes, such as the positive critical separation size test data shown in table 1 and the negative critical separation size test data shown in table 2, are obtained.

TABLE 1

TABLE 2

(2) Cell testing: using a size 2 chip with a positive and a negative critical separation size of 5 and 3 microns, respectively, different cells with minimum sizes of 2, 4, 6, 8 and 10 microns were tested for cell recovery at sorting pressures of 0.5Bar, 0.75Bar, 1Bar, 1.25Bar, 1.5Bar, 1.75Bar, 2Bar, 2.25Bar and 2.5Bar, respectively, and the best sorting pressure data for the different sized cells shown in tables 3 and 4 was obtained.

TABLE 3

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TABLE 4

(3) Establishing a database sub-library according to the test result:

counting the data results of the particle test and the cell test to obtain a sorting database sub-library of comprehensive microfluidic chip specifications, sorting pressure and cell size characteristics shown in table 5;

TABLE 5

(4) Repeating the above operations to establish a database: and repeating the operations according to other chip specifications, sorting conditions and cell size characteristics, and finally summarizing all data to obtain a finished sorting database.

The technical features of the embodiments described above may be arbitrarily combined, and for the sake of brevity, all possible combinations of the technical features in the embodiments described above are not described, but should be considered as being within the scope of the present specification as long as there is no contradiction between the combinations of the technical features.

The above-mentioned embodiments only express several embodiments of the present invention, and the description thereof is more specific and detailed, but not construed as limiting the scope of the invention. It should be noted that, for a person skilled in the art, several variations and modifications can be made without departing from the inventive concept, which falls within the scope of the present invention. Therefore, the protection scope of the present patent shall be subject to the appended claims.

Claims

1. A microfluidic chip, comprising:

an inlet zone (1), the inlet zone (1) being formed with a sample solution channel (101) and a buffer channel;

the separation area (2), the separation area (2) is connected with the inlet area (1), the separation area (2) forms a separation channel, the separation channel is communicated with the sample solution channel (101) and the buffer solution channel, a first micro-column array and a second micro-column array are arranged on the separation channel, the first micro-column array and the second micro-column array are respectively formed by micro-column arrays in a distributed mode, the first micro-column array inclines towards the direction close to the central line of the separation channel, the second micro-column array inclines towards the direction far away from the central line of the separation channel, and the first micro-column array is located at the solution inlet end and the solution outlet end of the separation channel;

an outlet zone (3), the outlet zone (3) with the sorting zone (2) is connected, form first outlet channel (301) and second outlet channel (302) on outlet zone (3), first outlet channel (301) is located the inboard of second outlet channel (302), first outlet channel (301) and second outlet channel (302) with the sorting channel intercommunication.

2. The microfluidic chip according to claim 1,

the number of the first micro-column array and the second micro-column array is multiple, and the second micro-column array is positioned between the two first micro-column arrays; and/or

The first micropillar array and the central line direction of the sorting channel form a first offset angle, and the range of the first offset angle is 0.5-12.5 degrees; and/or

The second micropillar array and the central line direction of the sorting channel form a second offset angle, and the range of the second offset angle is 0.5-12.5 degrees.

3. The microfluidic chip according to claim 1,

the distance range between the micro-pillars adjacently distributed in the flow channel direction of the first micro-pillar array and/or the second micro-pillar array is 6-25 micrometers; and/or

The distance range between the micro-pillars which are adjacently distributed in the direction perpendicular to the flow channel direction of the first micro-pillar array and/or the second micro-pillar array is 10-60 micrometers; and/or

The height range of the first micro-column array and/or the second micro-column array vertical to the flow channel direction is 5-50 micrometers; and/or

The section of the micro-column is in one of a circle, an ellipse, a triangle, a rectangle, a trapezoid, a diamond, an L shape, a C shape and a T shape.

4. The microfluidic chip according to claim 1, wherein the height of the sorting channel perpendicular to the flow direction is variable with different sorting pressures.

5. The microfluidic chip according to claim 1 or 4, wherein the height of the sorting channel perpendicular to the flow direction is in the range of 15 to 60 μm.

6. The microfluidic chip according to claim 1, wherein the sorting region (2) comprises a buffer flow channel region (21), a circulating sorting region (22), and a small cell flow channel region (23), a buffer supply channel is formed on the buffer flow channel region (21), the buffer supply channel is communicated with a buffer channel of the inlet region (1), the buffer flow channel region (21) is located inside the circulating sorting region (22), the circulating sorting region (22) is communicated with the sample solution channel and the buffer channel, the circulating sorting region (22) forms the sorting channel, the first and second micro-column arrays are located on the circulating sorting region (22), the small cell flow channel region (23) forms a small cell channel, and the small cell channel is communicated with the sorting channel.

7. The microfluidic chip according to claim 6, wherein the circular sorting region (22) comprises a first converging region (221), a diverging region (222), and a first partition (223), the first converging region (221) and the diverging region (222) are respectively provided with the first micropillar array and the second micropillar array, the first converging region (221) is adjacent to the inlet region (1), the diverging region (222) is located at a liquid outlet end of the first converging region (221), the small cell flow channel region (23) is located outside the diverging region (222), the first partition (223) is provided between the diverging region (222) and the small cell flow channel region (23), and the small cell channel is communicated with the first converging region (221).

8. The microfluidic chip according to claim 7, wherein the number of the cyclic separation regions (22) is plural, the critical separation size between the cyclic separation regions (22) distributed along the liquid flow direction increases, and the critical separation size of the cyclic separation region (22) at the next stage is 105% to 200% of the critical separation size of the cyclic separation region (22) at the previous stage; and/or

The number of the first convergence regions (221), the number of the divergence regions (222), and the first partition (223) are plural, and the first convergence regions (221) and the divergence regions (222) are arranged alternately; and/or

The buffer solution flow channel area (21) and the small cell flow channel area (23) are provided with cylindrical arrays, and the distribution direction of the cylindrical arrays is the same as the liquid flowing direction; and/or

Still include drainage portion (4), adjacent first convergent region (221) with have clearance (201) between divergent region (222), drainage portion (4) set up buffer solution runner district (21) the lateral wall and/or on the inside wall of first partition portion (223), drainage portion (4) are located clearance (201) department.

9. The microfluidic chip according to claim 6, further comprising an end-stage separation region (24), wherein the end-stage separation region (24) is located at a liquid outlet end of the circulation separation region (22), the first microcolumn array is disposed on the end-stage separation region (24), the buffer flow-channel region (21) is located inside the end-stage separation region (24), the small-cell flow-channel region (23) is located outside the end-stage separation region (24), and the separation channel extends to the end-stage separation region (24).

10. The microfluidic chip according to claim 9, wherein the end-stage separation region (24) comprises a second convergence region (241) on which the first micropillar array is disposed and a second partition (242), the second convergence region (241) being provided with the first micropillar array, the second partition (242) being located outside the second convergence region (241); and/or

The critical separation size of the last separation area (24) is higher than that of the circulation separation area (22), and the critical separation size of the last separation area (24) is 120-600% of that of the first circulation separation area (22); and/or

The buffer flow-path region (21) extends through at least a portion of the end separation region (24), the end of the buffer flow-path region (21) being located above the end separation region (24).

11. The microfluidic chip according to claim 7, wherein the first divider (223) comprises a first divider body (2231) and a flow guide end (2232), wherein the flow guide end (2232) is disposed on the first divider body (2231), the flow guide end (2232) is located at a liquid outlet end of the first convergence region (221), and the flow guide end (2232) is configured to guide a liquid flow to the small cell flow channel region (23).

12. The microfluidic chip according to claim 11, wherein the flow-directing end (2232) has a flow-directing arc surface located outside the flow-directing end (2232), the flow-directing arc surface being offset away from a centerline of the sorting channel; and/or

The junction of the diversion end (2232) and the first partition body (2231) forms a bend angle.

13. The microfluidic chip according to claim 1, wherein the buffer channel comprises a first buffer channel (102) and a second buffer channel (103), the inlet zone (1) comprising a first buffer inlet zone (11), a second buffer inlet zone (12) and a sample solution inlet zone (13), the first buffer inlet zone (11) forming the first buffer channel (102), the second buffer inlet zone (12) forming the second buffer channel (103), the second buffer channel (103) being located inside the first buffer channel (102); and/or

Said outlet zone (3) comprising a first outlet portion (31) and a second outlet portion (32), said first outlet portion (31) having said first outlet passage (301) formed thereon, said first outlet portion (31) being adjacent to the liquid outlet end of said sorting passage, said second outlet portion (32) having said second outlet passage (302) formed thereon; and/or

The sample solution channel, the buffer solution channel, the first outlet channel and the second outlet channel are respectively provided with a cylindrical array, and the distribution direction of the cylindrical arrays is the same as the liquid flowing direction.

14. A separation and purification method of a microfluidic chip is characterized by comprising the following steps:

s1: the microfluidic chip of any one of claims 1 to 13, wherein the inlet region (1) is connected to a sample solution to be treated and a buffer solution, the sample solution to be treated flows through the sample solution channel, the buffer solution flows through the buffer solution channel, the outlet region (3) is connected to a large cell recovery liquid tube and a small cell recovery liquid tube, the first outlet channel (301) is communicated with the large cell recovery liquid tube, and the second outlet channel (302) is communicated with the small cell recovery liquid tube;

s2: applying the same sorting pressure to a sample solution to be processed and a buffer solution, wherein the sample solution to be processed and the buffer solution pass through the sorting area (2), a target cell solution is recovered to a large cell recovery liquid tube through a first outlet channel (301), and the rest solution is recovered to a small cell recovery liquid tube through a second outlet channel (302);

s3: after the sample solution to be treated is emptied or the recovery solution is filled, the feed of the inlet area (1) and the discharge of the outlet area (3) are interrupted and the sorting pressure applied by the sample solution to be treated and the buffer solution is contacted.

15. The method for separating and purifying a microfluidic chip according to claim 14, wherein the sorting pressure is determined from a sorting database, and the step of creating the sorting database comprises:

s3: and according to the test result, establishing a cell sorting database by combining the microfluidic chip, the critical separation size, the cell type and the characteristics, the sorting pressure and the corresponding sorting recovery rate.

16. The method for separating and purifying a microfluidic chip according to claim 15, wherein the critical separation size ranges from 5 to 9 micrometers, from 8 to 13 micrometers, from 12 to 18 micrometers, or from 16 to 24 micrometers, and the corresponding separation pressure is 0.5 to 2.5Bar.