CN110004043B - Single cell capture micro-fluidic chip - Google Patents

Abstract

The invention relates to a single cell capture micro-fluidic chip, comprising: a functional layer and a cover sheet layer; the functional layer includes: the silicon chip comprises a silicon chip body and a plurality of functional regions decorated on the silicon chip body; the plurality of functional regions includes at least: a sample feeding and pretreatment area, a capture function area and a waste liquid treatment area; the sample feeding and pre-treating area consists of cell liquid inlet, inlet liquid storage pool and inlet passage with miniature dispersing column; the capture function area consists of a micro reaction tank provided with a buffer column and a capture trap array; the waste liquid treatment area consists of an outflow channel provided with a micro dispersion column, an outlet liquid storage tank and a waste liquid outflow port; the cell liquid inlet is communicated with the inlet liquid storage tank; the inlet liquid storage tank is communicated with the micro reaction tank by means of an inflow channel; the micro reaction tank is communicated with the outlet liquid storage tank by means of an outflow channel; the outlet liquid storage tank is communicated with the outlet of the waste liquid. The microfluidic chip provided by the invention can realize uniform sample introduction of cell carrier fluid and capture of single cells in the chip.

Description

Single cell capture micro-fluidic chip

Technical Field

The invention belongs to the technical field of microfluidics, and particularly relates to a single-cell capture microfluidic chip.

Background

Since the development of microfluidic technology, it has been widely used in the fields of physical and chemical analysis, biomedical treatment, etc. The micro-fluidic chip provides a good platform for micro-scale research based on micro-nano technology, and due to the characteristics of similar scales, high analysis efficiency, high flux and the like, cell analysis on the microchip has numerous advantages, and effective research foundation and accurate analysis results can be provided for aspects such as medical analysis, drug screening, organism monitoring and the like.

In cell analysis, analysis of a single cell is important in controlling cell morphology and in the direction of accurate measurement of cells in vivo or in vitro. Unlike conventional tissue analysis of cell populations or cell spheres, the focus of research on microdevices is on achieving single cell localization and analysis, such as single cell capture in a cell carrier fluid, for subsequent functional applications.

In the prior art, micro-devices related to cell capture have been extensively studied.

The Chinese patent CN107338183A describes a cell capturing device, which is provided with a filter layer, through holes with different inlet and outlet cross-sectional areas are arranged on the filter layer to realize that cells automatically fall into along with flowing liquid and are not easy to flow out, and a single cell is reserved in each through hole through size control. However, the function realization of the device depends on a multilayer structure, so that certain technical difficulties exist in the micro-nano processing process; in addition, under the condition of a large flow rate, the flow direction is not at the same vertical height as the cells, so that the cells are easily taken out of the through holes.

The Chinese invention patent CN105441307A describes a single cell capturing chip, which realizes a high-efficiency single cell capturing function by arranging a liquid flow layer, an elastic film layer and a driving mechanism, and realizes a strong cell positioning function, so that single cells are not easy to separate from sites. However, the function of the chip is complex to realize, a driving mechanism and an elastic membrane need to be configured, and the cost is high in the micro-nano processing process.

U.S. patent No. 20150004687a1 describes a cell trapping device that achieves the function of introducing a cell dispersion and filtering CTCs by providing a housing and a filter, and disposing the filter in the housing. However, this device does not achieve single cell layer trapping, and CTCs are filtered by a filter with fine through-holes.

Disclosure of Invention

Technical problem to be solved

Aiming at the existing technical problems, the invention provides a single-cell capture microfluidic chip, which has a simple chip processing process flow, and can realize uniform sample introduction of cell carrier fluid and single-cell in-chip capture by arranging reasonable structures such as a dispersion column, a buffer column, a capture trap and the like.

(II) technical scheme

In order to achieve the purpose, the invention adopts the main technical scheme that:

a single-cell-capture microfluidic chip, comprising: a functional layer and a cover sheet layer;

the functional layer includes: the silicon chip comprises a silicon chip body and a plurality of functional regions decorated on the silicon chip body;

the plurality of functional regions includes at least: a sample feeding and pretreatment area, a capture function area and a waste liquid treatment area;

the sample feeding and pre-treating area consists of cell liquid inlet, inlet liquid storage pool and inlet passage with miniature dispersing column;

the capture function area consists of a micro reaction tank provided with a buffer column and a capture trap array;

the waste liquid treatment area consists of an outflow channel provided with a micro dispersion column, an outlet liquid storage tank and a waste liquid outflow port;

the cell liquid inlet is communicated with the inlet liquid storage tank;

the inlet liquid storage tank is communicated with the micro reaction tank by means of an inflow channel;

the micro reaction tank is communicated with the outlet liquid storage tank by means of an outflow channel;

the outlet liquid storage tank is communicated with a waste liquid outlet;

the cover sheet layer and the functional layer are combined through packaging.

Preferably, the inlet reservoir, the inflow channel, the microreactor, the outflow channel and the outlet reservoir have the same depth H1;

the micro dispersion columns, the buffer columns and the trap array have the same height H2;

the arrangement form of the capture array can be adjusted according to specific application, so that flexible combination of chips is realized, and the applicability is improved.

Preferably, the inlet reservoir and the outlet reservoir are circular or elliptical.

Preferably, the micro dispersion cylinders in the inflow channel and the outflow channel are in the shape of a cylinder or an elliptic cylinder;

the adjacent micro dispersion columns are arranged along the flow direction in a staggered manner.

Preferably, the depth dimension H1 and the height dimension H2 satisfy H1 > H2, and H1-H2 < the diameter of a single cell.

Preferably, the buffer cylinder is a cylinder, a semi-cylinder or an elliptic cylinder.

Preferably, the number of the buffer columns arranged on one side close to the communication port of the micro reaction tank and the inflow channel is increased row by row;

the number of the buffer columns arranged on one side close to the communication port of the micro reaction tank and the outflow channel is gradually reduced row by row.

Preferably, the trap well array is composed of a plurality of trap wells arranged in staggered columns;

the trapping trap is provided with a cell staying area and a carrier fluid guiding area.

Preferably, the transverse depth of the cell retention zone is H3, the longitudinal width is H4, the size is H3 ≧ single cell diameter, H4 > single cell diameter;

the carrier fluid diversion area is of a gap structure, the number N of the carrier fluid diversion areas is 0-5, and the width of the gap is H5;

2 μm < H5 < diameter of individual cells;

under the condition of a plurality of gaps, the initial included angle theta of the two outermost gaps is more than or equal to 0 degree and less than 180 degrees.

Preferably, the functional layer is made of polydimethylsiloxane;

the cover sheet layer is made of polydimethylsiloxane or glass.

(III) advantageous effects

The invention has the beneficial effects that: the single-cell capture micro-fluidic chip provided by the invention has the following beneficial effects:

(1) the chip related in the invention has only a single layer, has simple structure, greatly reduces the processing cost, can stabilize cells in the trap under the condition of fluid shearing force, is not easy to flow out, and has better function realization.

(2) The microfluidic chip provided by the invention can realize the positioning capture of single cells in the chip, the cell capture efficiency of the chip reaches more than 95% within the cell suspension inlet speed range of 0.1-0.5 mu l/s through parameter setting, and after the stable capture of the cells is achieved, the single sample consumption is effectively reduced by more than 90% compared with the cell culture dish culture with the same concentration.

Drawings

FIG. 1 is a schematic diagram of a three-dimensional structure of a single-cell capture microfluidic chip according to the present invention;

FIG. 2 is a schematic structural view of a three-dimensional structure of a single-cell-capturing microfluidic chip provided by the present invention, taken along the cross-section of FIG. 1A-A;

fig. 3 is a schematic structural diagram of a trap of a single-cell-trapping microfluidic chip functional region provided by the invention.

[ description of reference ]

11: a functional layer; 12: a cover sheet layer; 111: a cell fluid inlet; 112: an inlet liquid storage tank; 113: an inflow channel micro dispersion column; 114: a buffer column; 115: a trap well; 116: an outflow channel micro dispersion column; 117: an outlet liquid storage tank; 118: and a waste liquid outlet.

Detailed Description

For the purpose of better explaining the present invention and to facilitate understanding, the present invention will be described in detail by way of specific embodiments with reference to the accompanying drawings.

As shown in fig. 1: the embodiment discloses a single cell capture microfluidic chip, which comprises: a functional layer 11 and a cover sheet layer 12.

Wherein the functional layer 11 comprises: the silicon chip comprises a silicon chip body and a plurality of functional regions decorated on the silicon chip body;

the plurality of functional regions described herein include at least: sample introduction and pretreatment area, capture function area and waste liquid treatment area.

Specifically, the sample introduction and pretreatment area in this embodiment is composed of a cell fluid inlet 111, an inlet reservoir 112, and an inflow channel provided with a micro dispersion column.

The capture function area consists of a micro reaction tank provided with a buffer column and a capture trap array; the waste liquid treatment area consists of an outflow channel provided with a micro dispersion column, an outlet liquid storage tank 117 and a waste liquid outflow port 118; the cell fluid inlet 111 communicates with an inlet reservoir 112.

The inlet reservoir 112 communicates with the micro-reaction chamber by means of an inflow channel; the micro-reaction cell is in communication with an outlet reservoir 117 by means of an outflow channel; the outlet reservoir 117 is in communication with a waste stream outlet 118; the cover layer 12 is combined with the functional layer 11 by encapsulation.

It should be noted that: the microfluidic chip can realize the positioning capture of single cells in the chip, the cell capture efficiency of the chip reaches more than 95% within the cell suspension inlet speed range of 0.1-0.5 mu l/s through parameter setting, and the single sample consumption is effectively reduced by more than 90% compared with the cell culture dish culture with the same concentration after the steady-state capture of the cells is achieved.

The cell carrier fluid is introduced into the inlet reservoir 112 through the cell fluid inlet 111, the cells in the inlet reservoir 112 are introduced into the micro reaction cell via the inlet channel provided with the micro dispersion column, the single cells are captured by the array of capture wells 115 provided in the micro reaction cell for assay analysis, the remaining waste fluid is introduced into the outlet reservoir 117 via the outlet channel, and finally discharged out of the microfluidic chip via the waste fluid outlet 118.

What should be added is that: the capture functional region consists of a large array capture trap 115, and can realize high-throughput sample transportation and high-efficiency cell capture; the arrangement form of the capture array can be adjusted according to specific application, so that flexible combination of chips is realized, and the applicability is improved.

As shown in fig. 2: the inlet reservoir 112, the inflow channel, the micro-reaction channel, the outflow channel and the outlet reservoir 117 in this embodiment have the same depth H1; the inflow channel microdispersion posts 113, outflow channel microdispersion posts 116, buffer posts 114 and array of trap wells 115 have the same height H2.

The arrangement form of the capture array can be adjusted according to specific application, so that flexible combination of chips is realized, and the applicability is improved.

It should be noted that: the inlet reservoir 112 and the outlet reservoir 117 described in this embodiment may be provided in a circular or oval shape.

In the embodiment, the micro dispersion columns in the inflow channel and the outflow channel are cylindrical or elliptic cylinders; the adjacent micro dispersion columns are arranged along the flow direction in a staggered manner.

It should be noted here that the depth dimension H1 and the height dimension H2 in the present example satisfy H1 > H2, and H1-H2 < the diameter of a single cell.

The buffer column is a cylinder, a semi-cylinder or an elliptic cylinder.

In this embodiment, the number of the buffer columns 114 arranged on the side close to the communication port of the micro reaction tank and the inflow channel is gradually increased row by row; for example, the first column is 1, the second column is 2 or 3, the third column is 3 or 4, etc.

The number of the buffer posts 114 provided on the side close to the communication port of the micro reaction cells and the outflow passage is decreased row by row. The arrangement of the side of the communication opening of the inflow channel is just symmetrical.

The trap 115 array in this embodiment is composed of a plurality of trap traps 115 arranged in staggered columns;

the trap 115 is provided with a cell retention region and a carrier fluid diversion region.

As shown in fig. 3: in the embodiment, the transverse depth of the cell retention zone is H3, the longitudinal width is H4, the size of the cell retention zone meets the condition that H3 is not less than the diameter of a single cell, and H4 is more than the diameter of the single cell; the carrier fluid diversion area is of a gap structure, the number N of the carrier fluid diversion areas is 0-5, and the width of the gap is H5; 2 μm < H5 < diameter of individual cells.

Specifically, under the condition of a plurality of gaps, the initial included angle theta of the two outermost gaps is more than or equal to 0 degrees and less than 180 degrees.

Finally, it should be noted that: the functional layer 11 is made of polydimethylsiloxane; the cover layer 12 is made of polydimethylsiloxane or glass.

The conditions used in the examples may be further adjusted according to the conditions of the particular manufacturer, and the conditions not specified are generally the conditions in routine experiments.

In the following examples 1 to 3:

1. the preparation process of the single cell capture microfluidic chip comprises the following steps:

(1) cleaning a silicon wafer: standard cleaning of silicon wafers, placing on an electric hot plate at 200 ℃ for 15min, and drying;

(2) silicon chip modification: putting the silicon wafer into a volatilization cylinder, and dripping 1-2 drops of a modification reagent HMDS (hexamethyliselazane) into the volatilization cylinder, wherein the volatilization treatment time is more than or equal to 3 min;

(3) spin coating of the silicon wafer: pouring photoresist on the processed silicon wafer, setting the photoresist spinning speed according to the required depth or height of the pattern, and standing for 1-2 min after photoresist is homogenized;

(4) silicon wafer exposure: the pre-bake process sets the pre-bake time by determining the properties of the photoresist used; the exposure process needs to consider the power of the exposure machine and the metering needed by the material to set the exposure time; after exposure, determining the middle baking time for taking out the template, and performing middle baking treatment;

(5) and (3) developing: after being placed and cooled, the solution is placed into a developing solution; the specific time is set according to the effect, the developing is carried out for 2-3 times in the process, and the film is placed in a ventilation kitchen and dried by nitrogen;

(6) and (3) treatment of a release agent: placing the silicon wafer into a volatilization cylinder, dripping 1-2 drops of a modifying reagent tmcs (Trimethychlorosoline), volatilizing, and finishing the die processing.

(7) Preparing PDMS glue: preparing glue and homogenizing the glue;

(8) modification: placing the treated silicon wafer into a volatilization cylinder, and dripping 1-2 drops of a modifier (methyl chlorosilane) into the volatilization cylinder for a modification process for about 3 min;

(9) pouring glue: uniformly spreading the tinfoil in a vessel, placing a silicon wafer mould, slightly compacting the silicon wafer, and pouring glue to ensure that the glue on the silicon wafer has no bubbles;

(10) and (3) drying: drying in a constant-temperature drying oven at 85 ℃ for about 30 min;

(11) stripping glue: taking out after slight cooling, removing the tinfoil, carefully removing the PDMS, and separating the cured PDMS from the silicon wafer;

(12) cutting and punching: cutting along the outer frame of the chip by a cutting knife to ensure the complete structure of the chip, and punching by a puncher;

(13) cleaning and microscopic examination: cleaning the chip, and observing whether the chip channel is qualified or not by using a microscope;

(14) bonding and quality inspection: the plasma processor is used for processing the surface of the chip to be bonded, bonding the two processed surfaces and inspecting the bonding condition of the chip under a microscope;

(15) baking: baking at 65 deg.C overnight.

2. Sample solution preparation:

(1) preparing a cell liquid culture solution: RPMI-1640 culture medium, fetal bovine serum, penicillin-streptomycin double antibody, wherein the three are mixed according to the volume capacity of 100:10: 1;

(2) cell extraction: taking out cultured Hela cells from a carbon dioxide incubator, adding trypsin for cell digestion, separating adherent cells, using 2ml of single amount, centrifuging the separated mixed solution at 800r/min for 3 minutes, removing supernatant, and adding 4ml of culture medium to obtain single experimental cell suspension.

Example 1

The microfluidic chip (hereinafter, the microfluidic chip is simply referred to as a chip) is prepared in the above manner, wherein the dimensional parameters and other parameters are as follows: h1 ═ H2 ═ 20 μm, H3 ═ 16 μm, H4 ═ 16 μm, H5 ═ 5 μm, and N ═ 0. The chip was washed with deionized water and Phosphate Buffered Saline (PBS) using a syringe pump. And introducing the cell carrier fluid into the chip, controlling the injection speed to be 0.1 mu l/s, and placing the chip under a microscope for observation to finally realize the capture of the single cell in the capture well. By the embodiment 1 of the invention, the capture efficiency of the single cell in the capture trap array reaches 96%, and the single sample consumption is effectively reduced by more than 90% compared with the cell culture dish culture with the same concentration.

Example 2

The microfluidic chip is prepared in the above manner, wherein the dimensional parameters and other parameters are as follows: h1 ═ H2 ═ 20 μm, H3 ═ 16 μm, H4 ═ 16 μm, H5 ═ 5 μm, and N ═ 1. The chip was washed with deionized water and Phosphate Buffered Saline (PBS) using a syringe pump. And introducing the cell carrier fluid into the chip, controlling the injection speed to be 0.2 mu l/s, and placing the chip under a microscope for observation to finally realize the capture of the single cell in the capture well. By the embodiment 2 of the invention, the capture efficiency of the single cell in the capture trap array reaches 96%, and the single sample consumption is effectively reduced by more than 95% compared with the cell culture dish culture with the same concentration.

Example 3

The microfluidic chip is prepared in the above manner, wherein the dimensional parameters and other parameters are as follows: h1 ═ H2 ═ 20 μm, H3 ═ 16 μm, H4 ═ 16 μm, H5 ═ 5 μm, and N ═ 2. The chip was washed with deionized water and Phosphate Buffered Saline (PBS) using a syringe pump. And introducing the cell carrier fluid into the chip, controlling the injection speed to be 0.2 mu l/s, and placing the chip under a microscope for observation to finally realize the capture of the single cell in the capture well. By the embodiment 3 of the invention, the capture efficiency of the single cell in the capture trap array reaches 98%, and the single sample consumption is effectively reduced by more than 95% compared with the cell culture dish culture with the same concentration.

The technical principles of the present invention have been described above in connection with specific embodiments, which are intended to explain the principles of the present invention and should not be construed as limiting the scope of the present invention in any way. Based on the explanations herein, those skilled in the art will be able to conceive of other embodiments of the present invention without inventive efforts, which shall fall within the scope of the present invention.

Claims (7)

1. A single-cell capture micro-fluidic chip is characterized in that,

the speed range of the cell sap inlet of the chip is 0.1-0.2 mul/s;

the chip includes: a functional layer and a cover sheet layer;

the functional layer includes: the silicon chip comprises a silicon chip body and a plurality of functional regions decorated on the silicon chip body;

the plurality of functional regions includes at least: a sample feeding and pretreatment area, a capture function area and a waste liquid treatment area;

the sample feeding and pre-treating area consists of cell liquid inlet, inlet liquid storage pool and inlet passage with miniature dispersing column;

the capture function area consists of a micro reaction tank provided with a buffer column and a capture trap array;

the waste liquid treatment area consists of an outflow channel provided with a micro dispersion column, an outlet liquid storage tank and a waste liquid outflow port;

the cell liquid inlet is communicated with the inlet liquid storage tank;

the inlet liquid storage tank is communicated with the micro reaction tank by means of an inflow channel;

the micro reaction tank is communicated with the outlet liquid storage tank by means of an outflow channel;

the outlet liquid storage tank is communicated with a waste liquid outlet;

the cover plate layer and the functional layer are combined through packaging;

the inlet reservoir, the inflow channel, the micro-reaction reservoir, the outflow channel and the outlet reservoir have the same depth H1 of 20 μm;

the microdispersion columns, buffer columns and trap arrays have the same height H2 of 20 μm;

the trap well array consists of a plurality of trap wells which are arranged in staggered columns;

the trapping trap is provided with a cell retention area and a carrier fluid diversion area

The transverse depth H3 of the cell retention zone is 16 μm, and the longitudinal width H4 is 16 μm;

the carrier fluid diversion area is of a gap structure, and the width H5 of the gap is 5 mu m.

2. The microfluidic chip according to claim 1, wherein the inlet reservoir and the outlet reservoir are circular or elliptical.

3. The microfluidic chip according to claim 2,

the micro dispersion columns in the inflow channel and the outflow channel are cylindrical or elliptic cylinders;

the adjacent micro dispersion columns are arranged along the flow direction in a staggered manner.

4. The microfluidic chip according to claim 1, wherein the buffer column is a cylinder, a semi-cylinder or an elliptic cylinder.

5. The microfluidic chip according to claim 1, wherein the number of the buffer pillars disposed on the side close to the communication port of the inlet channel with the micro reaction chamber increases row by row;

the number of the buffer columns arranged on one side close to the communication port of the micro reaction tank and the outflow channel is gradually reduced row by row.

6. The microfluidic chip according to claim 5,

the number N of the gap structures in the current-carrying fluid diversion area is 0-5,

under the condition of a plurality of gaps, the initial included angle theta of the two outermost gaps is more than or equal to 0 degree and less than 180 degrees.

7. The microfluidic chip according to claim 6, wherein the functional layer is made of polydimethylsiloxane;

the cover sheet layer is made of polydimethylsiloxane or glass.

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