CN209906795U

CN209906795U - Micro-fluidic chip for cell separation

Info

Publication number: CN209906795U
Application number: CN201920416334.5U
Authority: CN
Inventors: 欧阳东方
Original assignee: Individual
Current assignee: Shenzhen Yawan Biomedical Co ltd
Priority date: 2019-03-29
Filing date: 2019-03-29
Publication date: 2020-01-07
Anticipated expiration: 2029-03-29

Abstract

The utility model relates to a micro-fluidic chip for cell separation. The micro-fluidic chip comprises a liquid inlet flow channel, a capturing area and a liquid outlet flow channel, wherein the liquid inlet flow channel is provided with a liquid inlet and correspondingly communicated with the liquid inlet side of the capturing area, the liquid outlet flow channel is provided with a liquid outlet and correspondingly communicated with the liquid outlet side of the capturing area; the capture zone has a plurality of capture units, each capture unit having a plurality of split-flow columns. The microfluidic chip adopts a column sorting method, the shunting column can play a role in shunting and blocking in a flow channel of a capture area, the capture flow channel and the pass flow channel are respectively formed by blocking through the shunting column, target cells to be captured can be intercepted in the capture flow channel, and other non-target cells can flow out through the pass flow channel.

Description

Micro-fluidic chip for cell separation

Technical Field

The utility model belongs to the technical field of cell detection and specifically relates to a micro-fluidic chip is used in cell separation is related to.

Background

A sample such as peripheral blood contains a plurality of cells, and how to separate target cells from the plurality of cells has been a hot point of research in the field of cell detection. For example, how to achieve efficient isolation and accurate enumeration of circulating clonal plasma cells present in the peripheral blood of a patient with a hematological cancer, such as multiple myeloma, is of great importance for early clinical diagnosis, prognosis and treatment of myeloma. The traditional methods for realizing cell separation include a density gradient centrifugation method, a physical adsorption method, a cell electrophoresis method, an immunomagnetic bead method, a flow cytometry method and the like, but the methods generally have the problem of low cell separation effect, and particularly, a simple and good separation effect separation technology for circulating cloned plasma cells in peripheral blood of patients with various myeloma cells does not exist at present.

SUMMERY OF THE UTILITY MODEL

Therefore, there is a need for a microfluidic chip for cell separation, which solves the problem of low separation effect of the conventional cell separation method.

A microfluidic chip for cell separation comprises a liquid inlet flow channel, a capture area and a liquid outlet flow channel, wherein the liquid inlet flow channel is provided with a liquid inlet and correspondingly communicated with the liquid inlet side of the capture area, the liquid outlet flow channel is provided with a liquid outlet and correspondingly communicated with the liquid outlet side of the capture area;

the capture zone has a plurality of capture units, each of the capture units having a plurality of split-flow columns; gaps among the plurality of the flow dividing columns of each capturing unit form a capturing flow channel and a passing flow channel; from the liquid inlet side to the liquid outlet side of the capturing area, one capturing flow channel is divided into a plurality of passing flow channels through at least one of the flow dividing columns.

The microfluidic chip for cell separation adopts a column sorting method, the shunting column can play a role of shunting and blocking in the flow channel of the capture area, the capture flow channel and the pass flow channel are respectively formed by the shunting column blocking, the target cells to be captured can be intercepted in the capture flow channel, and other non-target cells can flow out through the pass flow channel.

Drawings

Fig. 1 is a schematic structural view of a microfluidic chip for cell separation according to an embodiment of the present invention;

FIG. 2 is a schematic view of a portion of the pre-filtering section of FIG. 1;

FIG. 3 is a schematic view of a partial structure of the effluent filtering zone in FIG. 1;

FIG. 4 is a schematic view of a portion of the inlet channel of FIG. 1 adjacent the capture zone;

FIG. 5 is a schematic view of a portion of the trapping region of FIG. 1;

FIG. 6 is a schematic diagram of a portion of the first level of sub-trapping region of FIG. 5;

FIG. 7 is a schematic diagram of a first level sub-capture unit shown in FIG. 6;

FIG. 8 is a schematic diagram of a portion of the second level of sub-trapping region of FIG. 5;

FIG. 9 is a schematic diagram of the second-level sub-capture unit shown in FIG. 8;

FIG. 10 is a schematic diagram of a portion of the third level of sub-trapping region of FIG. 5;

FIG. 11 is a schematic structural diagram of a third stage of the sub-capture unit in FIG. 10;

FIG. 12a is a force deformation analysis of leukocytes by the capture unit of the microfluidic chip, and FIG. 12b is a force deformation analysis of circulating cloned plasma cells by the capture unit of the microfluidic chip;

FIG. 13 shows the results of various antibody staining;

FIG. 14 shows the color of different antibodies and the corresponding excitation wavelength.

Detailed Description

In order to facilitate understanding of the present invention, the present invention will be described more fully hereinafter with reference to the accompanying drawings. The preferred embodiments of the present invention are shown in the drawings. The invention may, however, be embodied in many different forms and should not be construed as limited to the embodiments set forth herein. Rather, these embodiments are provided so that this disclosure will be thorough and complete.

It will be understood that when an element is referred to as being "on" another element, it can be directly on the other element or intervening elements may also be present. When an element is referred to as being "connected" or "in communication with" another element, it can be directly connected or intervening elements may also be present.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. The terminology used in the description of the invention herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. As used herein, the term "and/or" includes any and all combinations of one or more of the associated listed items.

As shown in fig. 1, one embodiment of the present invention provides a microfluidic chip 10 for cell separation (also referred to as "microfluidic chip 10"), which includes a liquid inlet channel 11, a capture region 12, and a liquid outlet channel 13. The liquid inlet channel 11 is provided with a liquid inlet, and the liquid inlet channel 11 is correspondingly communicated with the liquid inlet side of the capture area 12. The liquid outlet flow passage 13 has a liquid outlet 132. The liquid outlet channel 13 is correspondingly communicated with the liquid outlet side of the capture area 12.

In the present embodiment, the capturing region 12 has a plurality of capturing units 120. Each capturing unit 120 has a plurality of split flow columns. Gaps between the plurality of split flow columns of each capturing unit 120 constitute a capturing flow channel for trapping target cells and a passing flow channel for passing non-target cells and the like. From the inlet side to the outlet side of the capture zone 12, a capture channel is divided into a plurality of pass-through channels by at least one splitter column.

In one specific example, the inlet channel 11 comprises a sample inlet channel 111 and a reagent inlet channel 112. The sample inlet channel 111 has a sample inlet 1111. The reagent inlet channel 112 has a reagent inlet 1121. Preferably, the sample inlet channel 111 and the reagent inlet channel 112 are merged and then correspondingly communicated with the inlet side of the capture zone 12. It is understood that in other embodiments, the sample inlet channel 111 and the reagent inlet channel 112 may be integrated into a single channel, or both channels may share a single inlet.

Further, referring to fig. 1 and fig. 2, a pre-filtering area 113 for filtering impurities is disposed in the liquid inlet channel 11. The pre-filtering section 113 has a plurality of first filtering columns 1131, and a gap between adjacent first filtering columns 1131 forms a first filtering flow path 1132. In the case where the sample inlet channel 111 and the reagent inlet channel 112 are separately provided, it is preferable that a pre-filtration region 113 is provided after the sample inlet 1111 and the reagent inlet 1121, respectively.

As shown in FIG. 2, the cross section of the first filter column 1131 is isosceles triangle at the side close to the liquid inlet and at the side close to the capturing area 12, and the angle of the top angle of the isosceles triangle at the side close to the liquid inlet is smaller than the angle of the top angle of the isosceles triangle at the side close to the capturing area 12. The first filtering columns 1131 are distributed in an array, and the first filtering columns 1131 in adjacent rows are arranged in a staggered manner in the column direction.

With reference to fig. 1 and fig. 3, the liquid outlet flow channel 13 is provided with a liquid outlet filtering area 131 for filtering impurities. The effluent liquid filtering zone 131 has a plurality of second filtering columns 1311. Gaps between adjacent second filter columns 1311 constitute second filter flow paths 1312.

As shown in fig. 3, the cross section of second filter column 1311 is isosceles triangle at the side close to the liquid outlet and at the side close to capture area 12, and the angle of the vertex of the isosceles triangle at the side close to capture area 12 is smaller than that at the side close to the liquid outlet. The plurality of second filter columns 1311 are distributed in an array, and the second filter columns 1311 in adjacent rows are arranged in a staggered manner in the column direction. It is understood that in other embodiments, the liquid outlet filtering area 131 may not be disposed in the liquid outlet flow passage 13.

As shown in fig. 4, one end of the liquid inlet channel 11 for communicating with the capturing section 12 is branched into a plurality of branched liquid inlet channels 114 through at least one stage of branching, and accordingly, the capturing section 12 is also provided with a plurality of branched channels 114 corresponding to one capturing section 12. For example, in the specific example shown in fig. 4, the inlet liquid flow path 11 is branched into 16 branched inlet liquid flow paths 114 through 4-stage branching. By branching the liquid inlet channel 11 into a plurality of branched liquid inlet channels 114, uniform and uniform sampling can be facilitated.

As shown in fig. 5, in a specific example, the capturing region 12 has a plurality of sub-capturing regions, and the capturing unit 120 includes a plurality of sub-capturing units respectively distributed in each sub-capturing region. From the liquid inlet side to the liquid outlet side of the capturing area 12, the width of the capturing flow channel of the neutron capturing unit in the next stage of sub-capturing area is smaller than that of the capturing flow channel of the neutron capturing unit in the previous stage of sub-capturing area. Different stages of sub-capture zones or different stages of sub-capture cells with different capture flow channel widths can be used to retain cells of different diameters.

More specifically, referring to fig. 5, fig. 6 and fig. 7, the capture area 12 has a first-level sub-capture area 121, and the first-level sub-capture area 121 has a plurality of first-level sub-capture units 1211. The first-stage sub-capture unit 1211 includes three first-stage stems 1212, 1213, and 1214, which are arranged in series in the lateral direction. The cross section (the section parallel to the bottom of the chip) of each of the two outer first-

stage splitter columns

1212 and 1214 is a rhombus, and the two acute ends of each first-stage splitter column are respectively towards the liquid inlet side and the liquid outlet side of the capture zone 12, while the cross section of the middle first-stage splitter column 1213 is a rhombus-like shape (i.e. a figure formed by rounding one acute end of the rhombus) with an acute end replaced by an arc, the replaced end of the arc is towards the liquid inlet side of the capture zone 12, and the rest acute end is towards the liquid outlet side of the capture zone 12. The end part of the middle first-stage shunt column 1213 facing to the liquid inlet side is replaced by an arc, so that the damage to cells when the cells are intercepted can be effectively avoided, and the cells are prevented from being punctured.

In each first-stage sub-capture unit 1211, the end of the arc substitute of the middle first-stage splitter column 1213 is closer to the liquid outlet side than the middle obtuse-angle end of each of the first-

stage splitter columns

1212 and 1214 located on both sides, the gap between the two outer first-

stage splitter columns

1212 and 1214 constitutes a first-stage capture flow channel 1215, and the gap between any one of the outer first-

stage splitter columns

1212 or 1214 and the middle first-stage splitter column 1213 constitutes a first-stage through flow channel 1216.

Further, the two first-

stage diverging columns

1212 and 1214 located on the outer side in each first-stage sub-capturing unit 1211 are identical in shape and size, the connecting line of the acute-angled ends of the two first-

stage diverging columns

1212 and 1214 located on the outer side and the connecting line of the circle center and the acute-angled end of the end replaced by the circular arc of the first-stage diverging column 1213 located in the middle are both parallel to the longitudinal direction, the connecting line of the geometric centers of the two first-

stage diverging columns

1212 and 1214 located on the outer side is parallel to the transverse direction, and the distance between the first-stage diverging column 1213 located in the middle and the two first-

stage diverging columns

1212 and 1214 located on both sides is equal. The three first-

stage branching members

1212, 1213, and 1214 are formed into a bat-like shape, and as a whole, constitute an axisymmetric pattern having a line connecting the acute-angled end and the center of a circle of the end portion replaced with the circular arc of the first-stage branching member 1213 located in the middle as the symmetry axis.

The plurality of first-stage sub-capture units 1211 in the first-stage sub-capture area 121 are distributed in an array, the first-stage sub-capture units 1211 on adjacent rows are arranged in a staggered manner in the column direction, and the distances between the first-stage sub-capture unit 1211 on one row and two adjacent first-stage sub-capture units 1211 on the other row are equal.

In one specific example, the ratio of the diagonal lengths of the first-

stage diverging columns

1212 and 1214 located at both sides in the first-stage sub-capturing unit 1211 is 0.4-0.7, preferably 0.43, and the diagonal lengths of the two obtuse-angle ends of the first-

stage diverging columns

1212 and 1214 are both 8 ± 2 μm; the ratio of the diagonal lengths of the first-stage branching pillars 1213 located in the middle is 0.2 to 0.5, and the diagonal lengths of the two blunt-angled ends of the first-stage branching pillars 1213 are 8 ± 2 μm. When the fluid passes around an obstacle, a vortex can be formed, and research shows that when a sample such as blood flows, the fluid meets the obstacle to generate a vortex, and the vortex can cause blood coagulation and blockage. The utility model discloses utilize the post to select separately the method, the reposition of redundant personnel post can play the effect that the reposition of redundant personnel blocked in the runner, and this kind of condition is applicable to the physical situation in karman vortex street. This also requires that the geometry of the partial flow column be designed to minimize the drag it creates in the flow field. Therefore, the flow dividing column with the rhombic structure is preferably adopted, the resistance coefficient can be well reduced due to the shape of the rhombus, and the flow dividing column is better streamlined due to the adoption of the proper diagonal ratio of the rhombus. Theoretically, the smaller the diagonal ratio, the least the drag coefficient of the diamond-shaped splitter, but the cross-sectional area of the splitter is required to ensure that it can trap cells, and therefore the ratio of the diagonal lengths of the

first stage splitters

1212 and 1214 is preferably 0.4-0.7, more preferably 0.43.

The closest distance (the distance between the two close obtuse-angle ends) between the two first-

stage splitter posts

1212 and 1214 located at the outer side is 28 ± 5 μm. The obtuse angle end of the middle first-stage splitter column 1213 is laterally spaced from the corresponding outer first-

stage splitter column

1212 or 1214 by 11.5 ± 2.5 μm. The center of the end of the arc substitute of the middle first-stage splitter column 1213 is longitudinally spaced from the geometric center of the outer first-

stage splitter column

1212 or 1214 by 8 ± 3 μm.

Further, the centers of the circle of the ends of the circular arc replacements of the intermediate first-stage flow-splitting columns 1213 of the two adjacent first-stage sub-capture units 1211 in the same row are spaced apart by 80 ± 5 μm as viewed in the lateral direction. Viewed in the longitudinal direction, the projection length of a line connecting the geometric center of the first-

stage diverging column

1212 or 1214 located on the outer side of the first-stage sub-capturing unit 1211 in one of the two adjacent columns and the geometric center of any one of the first-

stage diverging columns

1212 or 1214 located on the outer side in the other column in the longitudinal direction (or the projection length of a line connecting the acute angle end of the first-

stage diverging column

1212, 1213, or 1214 in one of the columns and the corresponding acute angle end of the first-

stage diverging column

1212, 1213, or 1214 in the adjacent column in the longitudinal direction) is 82 ± 8 μm.

Still further, in one particular example, the first level sub-capture area 121 is 570 ± 50 μm wide. The first level sub-capture region 121 has 10-20 rows of first level sub-capture units 1211. The overall capture area 12 includes 8-16 first level sub-capture areas 121, with the plurality of first level sub-capture areas 121 preferably arranged in a lateral sequence. Each first stage sub-capture area 121 preferably corresponds to a branch inlet channel 114.

As shown in fig. 5, 8 and 9, the capture area 12 further includes a second level sub-capture area 122. The second level sub-capture region 122 has a plurality of second level sub-capture elements 1221. The second-stage sub-capturing unit 1221 includes three second-

stage branching columns

1222, 1223 and 1224 arranged in this order in the lateral direction, in which the two second-

stage branching columns

1222 and 1224 located at the outer side are each diamond-shaped in cross section and have respective two acute-angled ends respectively facing the liquid inlet side and the liquid outlet side of the capturing zone 12, and the second-stage branching column 1223 located at the middle side is diamond-like in cross section with an acute-angled end replaced by a circular arc, and the end replaced by the circular arc faces the liquid inlet side of the capturing zone 12, and the remaining acute-angled end faces the liquid outlet side of the capturing zone 12. The end part of the middle second-stage shunt column 1223 facing to the liquid inlet side is replaced by an arc, so that the damage to cells when the cells are intercepted can be effectively avoided, and the cells are prevented from being punctured.

In each second-stage sub-trap unit 1221, the end of the arc-shaped alternate end of the middle second-stage branching column 1223 is closer to the liquid outlet side than the blunt end of the middle second-

stage branching columns

1222 and 1224 on both sides, the gap between the two outer second-

stage branching columns

1222 and 1224 constitutes a second-stage trap flow passage 1225, and the gap between either outer second-

stage branching column

1222 or 1224 and the middle second-stage branching column 1223 constitutes a second-stage passage flow passage 1226.

Further, the two second-

stage shunt columns

1222 and 1224 located on the outer side in each second-stage sub-capture unit 1221 are identical in shape and size, the line connecting the acute-angle ends of the two second-

stage shunt columns

1222 and 1224 located on the outer side and the line connecting the circle centers of the ends replaced by the circular arcs of the second-stage shunt column 1223 located in the middle and the acute-angle ends are parallel to the longitudinal direction, the line connecting the geometric centers of the two second-

stage shunt columns

1222 and 1224 located on the outer side is parallel to the transverse direction, and the distance between the second-stage shunt column 1223 located in the middle and the second-

stage shunt columns

1222 and 1224 located on the two sides is equal. The three second-

stage shunt columns

1222, 1223, and 1224 form a shape similar to a bat, and integrally constitute an axisymmetric pattern having a line connecting the circle center of the end portion replaced with the circular arc of the second-stage shunt column 1223 located in the middle and the acute-angled end as a symmetry axis.

The plurality of second-stage sub-capture units 1221 in the second-stage sub-capture area 122 are distributed in an array, the second-stage sub-capture units 1221 in adjacent rows are staggered in the column direction, and the distance between the second-stage sub-capture unit 1221 in one row and two adjacent second-stage sub-capture units 1221 in another row is equal.

In one specific example, the ratio of the diagonal lengths of the second-

stage shunt columns

1222 and 1224 in the second-stage sub-capture unit 1221 is 0.3-0.5 and the diagonal lengths of the two blunt ends of each of the second-

stage shunt columns

1222 and 1224 are 9 ± 1.5 μm; the ratio of the diagonal lengths of the second-stage splitter 1223 is 0.3 to 0.5 and the diagonal lengths of the two blunt-angled ends of the second-stage splitter 1223 are 9 ± 2 μm. The closest distance between the two second

stage shunt pillars

1222 and 1224 located at the outer side is 18 ± 2 μm. The obtuse-angled end of the middle second-stage shunt post 1223 is laterally spaced 9 ± 2 μm from the acute-angled end of the corresponding outer second-

stage shunt post

1222 or 1224. The center of the end of the arc-substituted second-stage splitter 1223 located at the middle is longitudinally spaced from the geometric center of the second-

stage splitter

1222 or 1224 located at the outer side by 12 ± 3 μm.

Further, the centers of the circle of the ends of the circular arc substitutes of the middle second-stage splitter columns 1223 of two adjacent second-stage sub-capture units 1221 in the same row are spaced apart by 73 ± 5 μm when viewed in the transverse direction. Viewed in the longitudinal direction, the projection length of the line connecting the geometric center of the outer second-

stage shunt column

1222 or 1224 of the second-stage sub-capture unit 1221 in one of the two adjacent columns and the geometric center of any one of the outer second-

stage shunt columns

1222 or 1224 in the other column in the longitudinal direction is 73 ± 5 μm.

Still further, in one particular example, the width of the second level sub-capture area 122 is 1220 ± 200 μm. The second level sub-capture regions 122 have 35-45 rows of second level sub-capture units 1221. The entire capture area 12 includes 6-10 second level sub-capture areas 122. The plurality of second level sub-capture areas 122 are preferably arranged sequentially in the lateral direction. Each second level sub-capture area 122 may correspond to one or more first level sub-capture areas 121.

As shown in fig. 5, 10 and 11, the capture area 12 further includes a third level sub-capture area 123. The third-stage sub-trapping region 123 has a plurality of third-stage sub-trapping units 1231. The third-stage sub-capturing unit 1231 includes three third-stage split streams 1232, 1233, and 1234 arranged in series in the lateral direction. Each of the third stage splitter columns 1232, 1233, 1234 has a cross-section in the shape of a rhombus with an acute end replaced by a circular arc, the end replaced by a circular arc facing the liquid inlet side of the capture zone 12, and the remaining acute end facing the liquid outlet side of the capture zone 12.

In each third-stage sub-capturing unit 1231, the end substituted by the arc of the middle third-stage splitter 1233 is closer to the liquid outlet side than the middle obtuse-angle ends of the third-stage splitters 1232 and 1234 located at the two sides, a gap between the two outer third-stage splitters 1232 and 1234 forms a third-stage capturing flow channel 1235, and a gap between any one of the outer third-stage splitters 1232 or 1234 and the middle third-stage splitter 1233 forms a third-stage passing flow channel 1236.

Further, the two third-stage split flow columns 1232 and 1234 located outside in the third-stage sub-capturing unit 1231 have the same shape and size, the connection line between the circle center of the end replaced by the circular arc of each third-stage split flow column 1232, 1233, and 1234 and the acute angle end is parallel to the longitudinal direction, the connection line between the acute angle ends of the two third-stage split flow columns 1232 and 1234 located outside is parallel to the transverse direction, and the distance between the third-stage split flow column 1233 located in the middle and the third-stage split flow columns 1232 and 1234 on both sides is equal. The three third-stage diverging columns 1232, 1233, and 1234 are formed in a shape similar to a bat, and constitute an axisymmetrical pattern having a line connecting the circle center of the end portion replaced with the circular arc of the third-stage diverging column 1233 located in the middle and the acute-angle end as a symmetry axis as a whole.

The plurality of third-stage sub-capturing units 1231 in the third-stage sub-capturing region 123 are distributed in an array, the third-stage sub-capturing units 1231 in adjacent rows are staggered in the column direction, and the distance between the third-stage sub-capturing unit 1231 in one row and the two adjacent third-stage sub-capturing units 1231 in the other row is equal.

In one specific example, the angle of the acute end of each third-stage splitter column 1232, 1233, and 1234 in the third-stage sub-capture unit 1231 is 50 ° ± 10 °, and the diagonal length of the two obtuse ends of each third-stage splitter column 1232, 1233, and 1234 is 7.65 ± 1.5 μm. The closest distance between the two outer tertiary struts 1232 and 1234 is 19 ± 3 μm, and the projection length of the line connecting the obtuse angle end of the middle tertiary strut 1233 and the acute angle end of the corresponding outer tertiary strut 1232 or 1234 in the transverse direction is 7.5 ± 2 μm. The distance between the center of the end of the arc substitute of the middle third-stage splitter column 1233 and the center of the end of the arc substitute of the outer third-stage splitter column 1232 or 1234 is 15 +/-3 μm in the longitudinal direction.

Further, the centers of the circle of the ends substituted by the circular arcs of the middle third-stage splitter column 1233 of two adjacent third-stage sub-capturing units 1231 in the same row are 58 ± 5 μm apart from each other in the transverse direction. Viewed in the longitudinal direction, the projection length of the line connecting the acute angle end of the outer tertiary splitter 1232 or 1234 of the tertiary sub-capturing unit 1231 in one of the two adjacent columns and the acute angle end of any one of the outer tertiary splitter 1232 or 1234 in the other column in the longitudinal direction is 60 ± 5 μm.

Still further, in one particular example, the width of the third level sub-capture area 123 is 1220 ± 200 μm. The third level sub-capture area 123 has 20-40 rows of third level sub-capture units. The entire capture area 12 includes 6-10 tertiary sub-capture zones 123. Preferably, one third level sub-capture area 123 corresponds to one second level sub-capture area 122.

Each third stage sub-capture area 123 exits through a branch exit flow channel 132. The plurality of branch liquid outlet flow passages 132 collectively share the same liquid outlet. Preferably, the liquid outlet filtering area 131 is disposed at an end portion close to the liquid outlet, and the plurality of branch liquid outlet flow channels 132 collectively pass through the same liquid outlet filtering area 131 to discharge the liquid from the liquid outlet.

TABLE 1

It has been found that peripheral blood of patients with multiple myeloma contains mainly three cells, namely Red Blood Cells (RBC), White Blood Cells (WBC) and circulating clonal plasma cells (clonal circulating plasma cells). There are large differences in the physical sizes and the like of these three cells, as shown in table 1. The design of the shunt columns aims to utilize the gap interval between the shunt columns of each capture unit to enable RBC and WBC to pass through smoothly, and the circulating clonal plasma cells are clamped in the shunt columns of the capture units due to the large diameter and the hard cell hardness. It is noted that although normal leukocytes and circulating clonal plasma cells overlap in size, the circulating clonal plasma cells are harder because of their difference in cell stiffness, and therefore, when leukocytes and circulating clonal plasma cells of the same size pass through the flow-distribution column, the leukocytes are crushed and deformed to a greater extent than the circulating clonal plasma cells because they are softer, and through simulation operations of fluid mechanics and contact mechanics, it is demonstrated that leukocytes of the same size (30 μm diameter, young's modulus of 200 Pa) and circulating clonal plasma cells (30 μm diameter, young's modulus of 560 Pa) are crushed and deformed, and through many different sets of experiments, the optimal flow rate and column pitch are finally found, that is, in the case of a flow rate of 6mm/s, the first primary shunt column 1213 located in the middle of the first-stage sub-capture unit 1211 is located at a distance of 11.5 μm laterally from the first-

stage shunt columns

1212 and 1214 located on both sides, the leukocytes smoothly pass through the capturing unit because they are deformed to a greater extent by compression, while circulating clonal plasma cells, which are deformed to a lesser extent by compression, are caught therein, as shown in FIGS. 12a and 12 b. When the above-mentioned distance is less than 11.5 μm, the same flow rate cannot ensure smooth passage of the white blood cells, i.e., when the distance is too small, the white blood cells are also stuck inside.

Further, an embodiment of the present invention adopts a smart signal amplification method for increasing the difference between circulating cloned plasma cells and leukocytes, by designing the height of the flow channel to be 22 ± 4 μm, which means that cells with large volume diameter will be extruded by the chip flow channel when passing through the flow channel, so that the actual diameter of the cells in the flow channel becomes larger, and the size of the circulating cloned plasma cells will increase by 1.5-2 times in the flow channel according to the poisson's ratio concept. The diameter of the white blood cells is less than 18 μm, so that the size of the white blood cells in the flow channel is not changed. Therefore, in the flow channel, the difference between the physical sizes of the normal blood cells and the circulating clone plasma cells becomes larger, and the screening of the chip is more facilitated.

The microfluidic chip for cell distribution according to an embodiment of the present invention employs a capture region design with three-level sub-capture regions, and different sub-capture regions can capture circulating cloned plasma cells with different diameters, for example, in a specific example, the first-level sub-capture region 121 can capture circulating cloned plasma cells with a diameter of 25-50 μm, the second-level sub-capture region 122 can capture circulating cloned plasma cells with a diameter of 16-25 μm, and the third-level sub-capture region 123 can be used for capturing circulating cloned plasma cells with a diameter of 14-16 μm. It is to be understood that, in other embodiments, the capture region 12 of the microfluidic chip 10 for cell separation is not limited to include three-level sub-capture regions, and may include only one-level sub-capture region, two-level sub-capture regions, four-level or more sub-capture regions, or the like.

The microfluidic chip 10 for cell separation can be applied to a process for separating cells of various diameters in various fields such as disease diagnosis and non-disease diagnosis, for example, a process for separating circulating cloned plasma cells in peripheral blood. Preferably, in the separation process, peripheral blood is sucked into the microfluidic chip for cell separation 10 at a rate of 0.5ml/h to 1 ml/h. More specifically, in one example, the microfluidic chip 10 for cell separation is used by introducing a peripheral blood sample to be detected into the sample inlet 1111 through a conduit, and the outlet 132 is connected to a peristaltic pump through the conduit, wherein the peristaltic pump provides a pulling force to suck the peripheral blood sample into the chip at a flow rate of 0.5ml to 1ml per hour, so that the peripheral blood sample passes through the whole capture area 12 and then is discharged through the outlet 132, and the circulating clonal plasma cells are trapped in the capture area 12 due to their unique physical properties.

In fact, since biocytology is a discipline that is difficult to quantify absolutely, there are many uncertainties, as well as individual variability. Some leukocytes overlap in physical size and rigidity with circulating clonal plasma cells, or have stronger adhesion to the surface and stick to the column during passage through the shunt column. Since some normal leukocytes are captured in the actual capturing process, it is necessary to perform final identification by subsequent channel cleaning and antibody staining of the microfluidic chip to identify which is the true circulating clonal plasma cell.

Accordingly, one embodiment also provides a method for cell isolation and identification, comprising the steps of:

performing cell separation on the cell sample liquid by using the microfluidic chip 10 for cell separation;

dyeing the separated microfluidic chip by using an in-situ dyeing method;

microscopic examination and identification are carried out by a fluorescence microscope.

Specifically, the in situ staining method used in the above cell isolation and identification method can be performed according to, but not limited to, the following steps:

(1) passing paraformaldehyde through the microfluidic chip 10 at a flow rate of 0.5-1ml/h under the control of an injection pump, and maintaining for 20 min;

(2) under the control of an injection pump, enabling a PBS mixed EDTA (ethylene diamine tetraacetic acid) solution to pass through the microfluidic chip 10 at a flow rate of 0.5-1ml/h, and maintaining for 10 min;

(3) under the control of an injection pump, enabling a Triton X-100 solution with the concentration of 0.1% to pass through a micro-fluidic chip 10 at the flow rate of 0.5-1ml/h and maintaining for 10 min;

(4) under the control of an injection pump, enabling the PBS mixed EDTA solution to pass through the microfluidic chip 10 at the flow rate of 0.5-4ml/h, and maintaining for 3-5 min;

(5) passing BSA solution through the microfluidic chip 10 at a flow rate of 0.5-4ml/h under the control of an injection pump, and maintaining for 20 min;

(6) under the control of a syringe pump, a mixed solution of the following antibodies is introduced: 4', 6-diamidino-2-phenylindole (4', 6-diamidino-2-phenylindole, DAPI), phycoerythrin-conjugated CD138 mouse monoclonal antibody (phycoerythrin-conjugated CD138 mouse monoclonal antibody), Alexa For 647-labeled CD45 mouse monoclonal antigen (Alexa Fluor 647-labeled CD45 mouse monoclonal antibody) and Alexa Furor 488-labeled CD19 mouse monoclonal antibody (Alexa Fluor 488-labeled CD19 mouse monoclonal antibodies) (all antibodies obtained from Thermo Fisher), were passed through a microfluidic chip at a flow rate of 0.5ml/h For 40 min;

(7) under the control of an injection pump, cells which are simultaneously positive for CD138 and DAPI and simultaneously negative for CD45 and CD19 are searched under the excitation light with the corresponding wavelength of a fluorescence microscope, and the cells are taken as circulating clone plasma cells.

As shown in fig. 13 and 14, the data of the figure verifies the success of the microfluidic chip 10, including the capture efficiency of the circulating cloned plasma cells at different flow rates, and the sampling of multiple myeloma patients at different stages, and the comparison with healthy people.

The following table is data of clinical cases

1. The capturing efficiency and specificity were preliminarily verified in the peripheral blood of a healthy person mixed with the cloned plasma cells, and U266 is a cell line of the cloned plasma cells, and the results are shown in Table 2 below.

TABLE 2

2. Data for circulating clonal plasma cells captured in peripheral blood of multiple myeloma patients and healthy humans, wherein the number of circulating clonal plasma cells in relapsed multiple myeloma patients is much higher than in remitted patients, the results are shown in table 3 below.

TABLE 3

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 represent some embodiments of the present invention, and the description thereof is specific and detailed, but not to be construed as limiting the scope of the present invention. It should be noted that, for those skilled in the art, without departing from the spirit of the present invention, several variations and modifications can be made, which are within the scope of the present invention. Therefore, the protection scope of the present invention should be subject to the appended claims.

Claims

1. The microfluidic chip for cell separation is characterized by comprising a liquid inlet flow channel, a capture area and a liquid outlet flow channel, wherein the liquid inlet flow channel is provided with a liquid inlet, the liquid inlet flow channel is correspondingly communicated with the liquid inlet side of the capture area, the liquid outlet flow channel is provided with a liquid outlet, and the liquid outlet flow channel is correspondingly communicated with the liquid outlet side of the capture area;

2. The microfluidic chip for cell separation according to claim 1, wherein the liquid inlet channel comprises a sample liquid inlet channel and a reagent liquid inlet channel, the sample liquid inlet channel has a sample liquid inlet, the reagent liquid inlet channel has a reagent liquid inlet, and the sample liquid inlet channel and the reagent liquid inlet channel are merged and then correspondingly communicated with a liquid inlet side of the capture zone;

and/or a pre-filtering area for filtering impurities is arranged in the liquid inlet flow channel, the pre-filtering area is provided with a plurality of first filtering columns, and a first filtering flow channel is formed by gaps between the adjacent first filtering columns;

and/or the liquid outlet flow channel is provided with a liquid outlet filtering area for filtering impurities, the liquid outlet filtering area is provided with a plurality of second filtering columns, and gaps between the adjacent second filtering columns form second filtering flow channels.

3. The microfluidic chip for cell separation according to claim 2, wherein the cross section of the first filter column is isosceles triangle-shaped at both the side near the liquid inlet and the side near the capture region, and the angle of the top angle of the isosceles triangle-shaped portion near the liquid inlet is smaller than the angle of the top angle of the isosceles triangle-shaped portion near the capture region; the first filter columns are distributed in an array, and the first filter columns in adjacent rows are staggered in the column direction;

and/or the cross section of the second filter column is in an isosceles triangle shape at one side close to the liquid outlet and at one side close to the capture area, and the vertex angle of the isosceles triangle part at one side close to the capture area is smaller than that of the isosceles triangle part at one side close to the liquid outlet; the second filtering columns are distributed in an array mode, and the second filtering columns in adjacent rows are arranged in a staggered mode in the column direction.

4. The microfluidic chip for cell separation according to claim 1, wherein one end of the liquid inlet channel, which is used for being in communication with the capture region, is divided into a plurality of branched liquid inlet channels through at least one stage of branch;

the capture area is provided with a plurality of capture areas, and one or a plurality of branch liquid inlet flow passages are correspondingly communicated with one capture area.

5. The microfluidic chip for cell separation according to any one of claims 1 to 4, wherein the capture region has a plurality of sub-capture regions, and the capture unit comprises a plurality of sub-capture units respectively distributed in each sub-capture region; from the liquid inlet side to the liquid outlet side of the capturing area, the width of the capturing flow channel of the neutron capturing unit in the next-stage sub-capturing area is smaller than that of the capturing flow channel of the neutron capturing unit in the previous-stage sub-capturing area.

6. The microfluidic chip for cell separation according to claim 5, wherein the first-stage sub-capture section of the capture section has a plurality of first-stage sub-capture units, and the first-stage sub-capture units comprise three first-stage flow pillars arranged in sequence in the lateral direction, wherein the cross-sections of two first-stage flow pillars located at the outer side are each diamond-shaped and have two respective acute-angle ends respectively facing the liquid inlet side and the liquid outlet side, the cross-section of the first-stage flow pillar located at the middle is diamond-like with one acute-angle end replaced by a circular arc, and the end replaced by the circular arc faces the liquid inlet side, and the remaining acute-angle end faces the liquid outlet side;

in each first-stage sub-capturing unit, the end part replaced by the circular arc of the first-stage flow dividing column in the middle is closer to the liquid outlet side than the middle obtuse-angle end of the first-stage flow dividing columns on two sides, a gap between two first-stage flow dividing columns on the outer side forms a first-stage capturing flow channel, and a gap between any one first-stage flow dividing column on the outer side and the first-stage flow dividing column in the middle forms a first-stage passing flow channel.

7. The microfluidic chip for cell separation according to claim 6, wherein the two first-stage flow distribution columns located at the outer side in each first-stage sub-capture unit have the same shape and size, a line connecting the acute-angled ends of the two first-stage flow distribution columns located at the outer side and a line connecting the circle centers of the ends replaced by the circular arcs of the first-stage flow distribution column located at the middle and the acute-angled ends are both parallel to the longitudinal direction, a line connecting the geometric centers of the two first-stage flow distribution columns located at the outer side is parallel to the transverse direction, and the distances between the first-stage flow distribution column located at the middle and the first-stage flow distribution columns located at both sides are equal;

the multiple first-stage sub-capture units in the first-stage sub-capture area are distributed in an array mode, the first-stage sub-capture units in adjacent rows are arranged in a staggered mode in the column direction, and the distances between the first-stage sub-capture units in one row and two first-stage sub-capture units close to the first-stage sub-capture units in the other row are equal.

8. The microfluidic chip for cell separation according to claim 7, wherein the ratio of the diagonal lengths of the first fractional flow pillars located at both sides in the first-stage sub-capture unit is 0.4 to 0.7 and the diagonal lengths of the respective two blunt-angle ends are 8 ± 2 μm, the ratio of the diagonal lengths of the first fractional flow pillars located in the middle is 0.2 to 0.5 and the diagonal lengths of the blunt-angle ends thereof are 8 ± 2 μm, the closest distance of the two first fractional flow pillars located at the outer sides is 28 ± 5 μm, the blunt-angle end of the first fractional flow pillar located in the middle is laterally 11.5 ± 2.5 μm from the corresponding first fractional flow pillar located at the outer side, and the center of the end substituted by the circular arc of the first fractional flow pillar located in the middle is longitudinally 8 ± 3 μm from the geometric center of the first fractional flow pillar located at the outer side;

and/or the circle centers of the ends replaced by the circular arcs of the middle first-stage flow columns of two adjacent first-stage sub-capture units in the same row are 80 +/-5 mu m apart;

and/or the projection length of the connecting line of the geometric centers of the first-stage sub-capture units positioned at the outer side in one of the two adjacent columns and the geometric centers of any first-stage sub-capture units positioned at the outer side in the other column in the longitudinal direction is 82 +/-8 μm;

and/or, the width of the first level sub-capture zone is 570 ± 50 μm;

and/or, the first level sub-capture region has 10-20 rows of first level sub-capture units;

and/or, the capture zone comprises 8-16 of the first level sub-capture zones.

9. The microfluidic chip for cell separation according to any one of claims 6 to 8, wherein the second-stage sub-capture section of the capture section has a plurality of second-stage sub-capture units, and the second-stage sub-capture units comprise three second-stage flow columns arranged in series in the lateral direction, wherein the cross-sections of the two second-stage flow columns located at the outer side are each diamond-shaped and the two acute-angle ends thereof are respectively directed to the liquid inlet side and the liquid outlet side, the cross-section of the second-stage flow column located at the middle side is diamond-like with the acute-angle end thereof being replaced by a circular arc, and the replaced end of the circular arc is directed to the liquid inlet side, and the remaining acute-angle end is directed to the liquid outlet side;

in each second-stage sub-capturing unit, the end part replaced by the circular arc of the second-stage flow dividing column in the middle is closer to the liquid outlet side than the middle obtuse-angle end of the second-stage flow dividing columns on two sides, a gap between two second-stage flow dividing columns on the outer side forms a second-stage capturing flow channel, and a gap between any one second-stage flow dividing column on the outer side and the second-stage flow dividing column in the middle forms a second-stage passing flow channel.

10. The microfluidic chip for cell separation according to claim 9, wherein the two second-stage flow distribution pillars located at the outer side in each second-stage sub-capture unit have the same shape and size, a line connecting the acute-angled ends of the two second-stage flow distribution pillars located at the outer side and a line connecting the circle centers of the ends replaced by the circular arcs of the second-stage flow distribution pillars located at the middle and the acute-angled ends are both parallel to the longitudinal direction, a line connecting the geometric centers of the two second-stage flow distribution pillars located at the outer side is parallel to the transverse direction, and the distance between the second-stage flow distribution pillar located at the middle and the second-stage flow distribution pillars located at both sides is equal;

the multiple second-stage sub-capture units in the second-stage sub-capture area are distributed in an array mode, the second-stage sub-capture units in adjacent rows are arranged in a staggered mode in the column direction, and the distances between the second-stage sub-capture units in one row and two second-stage sub-capture units close to the second-stage sub-capture units in the other row are equal.

11. The microfluidic chip for cell separation according to claim 10, wherein the second-stage particle capturing unit has a ratio of diagonal lengths of the second-stage flow pillars located at both sides of the second-stage particle capturing unit of 0.3 to 0.5 and diagonal lengths of the respective two blunt-angled ends of 9 ± 1.5 μm, a ratio of diagonal lengths of the second-stage flow pillars located in the middle of the second-stage particle capturing unit of 0.3 to 0.5 and diagonal lengths of the two blunt-angled ends of 9 ± 2 μm, a closest distance of the two second-stage flow pillars located at the outer sides of the second-stage particle capturing unit is 18 ± 2 μm, the blunt-angled end of the second-stage flow pillar located in the middle of the second-stage particle capturing unit is 9 ± 2 μm in a lateral direction from the acute-angled end of the corresponding second-stage flow pillar located at the outer side of the second-stage particle capturing unit, and a center of the arc-substituted end of the second-stage flow pillar located in the middle of the second-stage;

and/or the circle centers of the ends replaced by the circular arcs of the middle second-stage flow columns of two adjacent second-stage sub-capture units in the same row are 73 +/-5 microns away from each other;

and/or the projection length of the connecting line of the geometric centers of the first-stage sub-capture units positioned at the outer side in one of the two adjacent columns and the geometric centers of any first-stage sub-capture units positioned at the outer side in the other column in the longitudinal direction is 73 +/-5 μm;

and/or the width of the second level sub-capture zone is 1220 ± 200 μm;

and/or, the second-level sub-capture region has 35-45 rows of second-level sub-capture units;

and/or, the capture zone comprises 6-10 of the secondary sub-capture zones.

12. The microfluidic chip for cell separation according to any one of claims 6 to 8 and 10 to 11, wherein the third-stage sub-capture region of the capture region has a plurality of third-stage sub-capture units, each of the third-stage sub-capture units comprises three third-stage flow-dividing columns arranged in sequence in the transverse direction, each of the third-stage flow-dividing columns has a cross section in a rhomboid shape with an acute-angle end replaced by a circular arc, the end replaced by the circular arc faces the liquid inlet side, and the rest of the acute-angle end faces the liquid outlet side;

in each third-stage sub-capturing unit, the end part replaced by the circular arc of the middle third-stage flow dividing column is closer to the liquid outlet side than the middle obtuse-angle end of the third-stage flow dividing columns on two sides, a gap between two outer third-stage flow dividing columns forms a third-stage capturing flow channel, and a gap between any outer third-stage flow dividing column and the middle third-stage flow dividing column forms a third-stage passing flow channel.

13. The microfluidic chip for cell separation according to claim 12, wherein the two third-stage flow pillars located at the outer side in the third-stage sub-capture unit have the same shape and size, the line connecting the circle center and the acute-angle end of the end replaced by the circular arc of each third-stage flow pillar is parallel to the longitudinal direction, the line connecting the acute-angle ends of the two third-stage flow pillars located at the outer side is parallel to the transverse direction, and the distances between the third-stage flow pillar located at the middle and the third-stage flow pillars located at both sides are equal;

the multiple third-stage sub-capture units in the third-stage sub-capture area are distributed in an array mode, the third-stage sub-capture units in adjacent rows are arranged in a staggered mode in the column direction, and the distances between the third-stage sub-capture units in one row and two adjacent third-stage sub-capture units in the other row are equal.

14. The microfluidic chip for cell separation according to claim 13, wherein the angle of the acute-angled end of each third-stage flow column in the third-stage sub-capture unit is 50 ° ± 10 ° and the diagonal length of the two obtuse-angled ends is 7.65 ± 1.5 μm, the closest distance of the two outer-located third-stage flow columns is 19 ± 3 μm, the projection length of the line connecting the obtuse-angled end of the middle third-stage flow column and the acute-angled end of the corresponding outer-located third-stage flow column in the transverse direction is 7.5 ± 2 μm, and the distance between the center of the end of the circular-arc substitute of the middle third-stage flow column and the center of the end of the circular-arc substitute of the outer-located third-stage flow column in the longitudinal direction is 15 ± 3 μm;

and/or the circle centers of the ends replaced by the circular arcs of the middle third-stage flow columns of two adjacent third-stage sub-capture units in the same row are 58 +/-5 mu m away;

and/or the projection length of the connecting line of the acute angle end of the third-stage flow column positioned at the outer side of the third-stage sub-capture unit in one of the two adjacent columns and the acute angle end of any third-stage flow column positioned at the outer side in the other column in the longitudinal direction is 60 +/-5 microns;

and/or the width of the third level sub-capture zone is 1220 ± 200 μm;

and/or, the third-level sub-capture region has 20-40 rows of third-level sub-capture units;

and/or, the capture zone comprises 6-10 of the tertiary sub-capture zones.

15. The microfluidic chip for cell separation according to any one of claims 6 to 8, 10 to 11, and 13 to 14, wherein each of the flow distribution columns has a height of 22 ± 4 μm.