A cell-bead paired capture method for single-cell omics analysis

Abstract

The present invention provides a cell-microbead pairing capture method for single-cell omics analysis, comprising the following steps: S1: providing a microfluidic capture device, comprising a separable particle capture layer and a substrate layer, wherein a plurality of capture microstructures are arranged in the particle capture layer for one-to-one pairing capture of cell-microbeads; S2: introducing a cell or microbead suspension from an inlet, and gradually completing one-to-one pairing capture of a single cell or a single microbead; S3: continuing to perfuse a controllably solidifiable solution from the inlet, and realizing the solidification of the controllably solidifiable solution according to the characteristics of the solution, so that each cell-microbead capture unit forms an independent chamber isolated from each other; S4: separating the substrate layer from the particle capture layer, forming an open cell-microbead capture unit array, and performing the next step of single-cell omics analysis. According to the present invention, a high-throughput, high-capture-efficiency, low-cost, and low-pollution cell-microbead pairing capture method is provided for single-cell omics analysis.

Description

Technical Field

[0001] The present invention relates to the field of single-cell omics analysis, and more specifically to a cell-microbead pairing capture method applied to single-cell omics analysis. Background Art

[0002] Cells are the basic structural and functional units of organisms. Multicellular organisms are composed of cells with different morphologies and functions. Cell heterogeneity is a ubiquitous biological phenomenon. Even cells that look the same may have significant heterogeneity. Therefore, it is necessary to study cell heterogeneity at the single-cell level. It is necessary to establish a research platform that can effectively analyze the chemical information of individual cells or measure the response of single cells to external stimuli, so that people can better understand certain special cell functions in cell populations and identify a small number of abnormal cells in a large number of cell populations, providing a reliable scientific basis for early diagnosis and treatment of major diseases, drug screening, and cell-to-cell interaction research. An important process for achieving single-cell analysis is to perform cell-bead pairing capture and place them in an independent environment to prevent cross-contamination between cells. Cell-bead pairing capture involves microfluidics, which is a technology that accurately controls and manipulates microscale fluids, and is mainly characterized by manipulating fluids in micro-nanoscale space. It can reduce reactions to micrometer scale, and thus can also accurately manipulate micrometer-scale cells. Single-cell analysis systems based on microfluidic technology usually have the characteristics of trace volume, high efficiency, high throughput, miniaturization, integration and portability.

[0003] At present, microfluidic chip technology has developed to a relatively mature stage. Droplet-based microfluidics and fluid mechanics-based microfluidics are widely used in the field of single-cell analysis, and commercial products based on microfluidic chip technology are also available on the market. The use of droplet microfluidics to capture single cells is to generate nanoliter to picoliter droplets in a closed microchannel network and encapsulate single cells in the droplets. Each droplet can be used as an independent single-cell microreactor, which can ultimately meet the research needs of a variety of single cells. Single-cell capture chips based on droplet microfluidics have low processing costs, consume less reagents and samples, and have a fast capture speed. However, this method has low pairing efficiency, that is, there are often cases where there is no cell encapsulation or multiple cells are encapsulated in a single droplet, and the position of the microdroplets after encapsulating a single cell is difficult to fix, which is not suitable for real-time observation of specific single cells. For example, Drop-seq and Paired-seq on the market are both based on droplets for cell-microbead pairing capture, and adopt the strategy of first pairing cells with microbeads and then forming droplets. Drop-seq has a simple structure and is easy to operate, but its cell-bead pairing efficiency is low. Paired-seq, based on the droplet strategy, improves the cell-bead pairing efficiency by applying the differential flow resistance principle and microvalve micropump structure, but its complex device structure greatly increases the cost and operation complexity, making processing and design difficult, and currently only 2000 throughput can be achieved.

[0004] The application of fluid dynamics-based microfluidics in single-cell analysis usually requires the rational design of microchannels in the chip and capture microstructures comparable to the size of cells, so as to achieve high-throughput cell-bead capture. The traditional fluid dynamics capture method has a simple operation process, does not require the use of special buffers, and the fixed capture sites are suitable for real-time observation and tracking analysis of single cells. However, the chip of this method may have cross-contamination problems, such as seq-well, where cells are often exposed to the same solutes and cells may communicate with neighboring cells, which is not a single-cell omics analysis in the full sense. Summary of the invention

[0005] The purpose of the present invention is to provide a cell-microbead pairing capture method for single-cell omics analysis, thereby solving the problems of cross-contamination, large sample consumption, complex structure and low throughput in the capture structure of the existing single-cell omics analysis platform.

[0006] In order to solve the above problems, the present invention adopts the following technical solutions:

[0007] A cell-microbead pairing capture method for single-cell omics analysis is provided, comprising the following steps: S1: providing a microfluidic capture device, the microfluidic capture device comprising a separable particle capture layer and a substrate layer, the particle capture layer being provided with an inlet, an outlet, and a plurality of capture microstructures, the capture microstructures being distributed in an array and being used for one-to-one pairing capture of cells and microbeads; S2: introducing a cell suspension or a microbead suspension from the inlet, and gradually completing one-to-one pairing capture of a single cell or a single microbead in the capture microstructure, Thus, an array consisting of a number of cell-bead capture units is formed; S3: continue to perfuse the controllable solidification solution from the inlet to replace the cell suspension or bead suspension on the particle capture layer, and solidify the controllable solidification solution in the microfluidic capture device according to the solution characteristics, so that each cell-bead capture unit forms an independent chamber isolated from each other; S4: separate the substrate layer from the particle capture layer to form an open cell-bead capture unit array, and the captured cells-beads can be subjected to the next step of single-cell omics analysis.

[0008] In step S2, the capture methods of cells and microbeads include fluid dynamics methods, external physical field capture methods, etc., wherein the external physical field includes: light, sound, magnetism, electricity, etc.

[0009] In step S3, the controllably curable solution includes photosensitive hydrogel, thermosensitive hydrogel, or various types of hydrophobic polymers.

[0010] In step S4, the next step of single-cell omics analysis includes: single-cell genome sequencing, single-cell transcriptome sequencing, single-cell whole transcriptome sequencing, and single-cell genome and transcriptome parallel sequencing.

[0011] The cell-microbead pairing capture method can be used to capture cells and microbeads of different sizes, cells and cells, and microbeads and microbeads.

[0012] The material of the substrate layer includes: glass, PDMS, organic glass, etc., preferably glass.

[0013] According to a preferred embodiment of the present invention, in step S1, a microfluidic capture device is provided, the particle capture layer comprises: an inflow zone, a capture zone, and an outflow zone, the inflow zone comprises an inlet, a branch channel, and a pre-filter connected in sequence, the capture zone comprises capture microstructures distributed in an array, each capture microstructure is composed of a plurality of microcolumns, and the plurality of microcolumns form a capture cavity internally, wherein a microstructure inlet is formed between two microcolumns, and a microstructure outlet is formed between the other two microcolumns, and one-to-one paired capture of cells and microbeads can be achieved by applying fluid dynamics methods.

[0014] Preferably, one of the microcolumns used to form the microstructure entrance adopts a deformable film structure. When the filtered cells or microbeads enter the capture area at a medium flow rate, the film structure maintains its shape, and a single-cell capture microstructure captures a cell or a microbead. Then, the flow rate is increased, the film structure is deformed, the size of the microstructure entrance becomes larger, and the captured cell or microbead is released and stored in the capture chamber.

[0015] It should be understood that the present invention takes the fluid dynamics method for one-to-one pairing capture of cells and microbeads as an example, but this is only used as an example and not as a limitation. In fact, any microfluidic capture device that can achieve one-to-one pairing capture of cells and microbeads is applicable to the present invention.

[0016] According to a preferred embodiment of the present invention, when the controllably curable solution is a photosensitive hydrogel, after the cell-microbead pairing capture is completed, the photosensitive hydrogel is introduced from the entrance of the microfluidic capture device, the microfluidic capture device is flipped over so that the substrate layer faces upward, ultraviolet light is irradiated to the particle capture layer through the mask, the photosensitive hydrogel is selectively cured to form an independent chamber, and the substrate layer is removed to form an open cell-microbead capture unit array.

[0017] According to a preferred embodiment of the present invention, the preparation method of the microfluidic capture device is as follows: 1) photolithography mask design and production: draw a microfluidic device design drawing, use high-precision printing output as multiple mask plates, each mask plate has a light-transmitting and opaque pattern, which is used to form a pattern in the exposure step; 2) photolithography mold preparation: use photoresist, mask plate and ultraviolet light for micro-manufacturing, the mold processing is completed and can be used repeatedly, which includes: cleaning silicon wafer, pre-baking, exposure, development, hardening film, and SU-8 mold production is completed; 3) inverted mold; 4) substrate bonding: use a plasma cleaning machine to plasma treat the surface of the device to be bonded and the clean glass sheet, contact the surface of the clean device with the flow channel structure with the glass sheet, and gently press to obtain a microfluidic capture device.

[0018] The key inventive point of the present invention is to adopt a strategy of first performing cell-microbead pairing and then forming an independent chamber similar to a microwell, so as to achieve higher capture efficiency, solve the problem of cross-contamination, and realize high-throughput parallel dynamic observation of the interaction between particles. It does not require complex structures such as microvalves and micropumps, greatly reducing costs, providing new ideas for cell-microbead pairing capture, and further providing new methods for single-cell sequencing and other single-cell omics analyses.

[0019] Compared with the prior art, the present invention has the following beneficial effects:

[0020] 1) The present invention adopts a strategy of first performing cell-microbead pairing and then forming independent microwells, which solves the problem of cross-contamination of capture structures in existing single-cell omics analysis platforms and is also suitable for applications where samples are relatively scarce;

[0021] 2) Compared with the method of first performing cell-bead pairing and then forming droplets, the present invention has significant advantages in pairing efficiency and real-time observation of specific single cells;

[0022] 3) The present invention uses simple fluid dynamics principles to achieve cell-microbead capture without the need for auxiliary structures such as microvalves and micropumps. The device is simple, easy to operate, and reduces costs, with commercial and industrial advantages;

[0023] 4) The present invention includes a high-density microfluidic array, which will achieve tens of thousands of sequencing throughputs in the future, enabling large-scale parallel single-cell capture and analysis;

[0024] 5) The present invention is prepared by a soft photolithography molding process, the structure is formed in one step, the process is simple and easy to implement.

[0025] In summary, the present invention provides a high-throughput, high-capture efficiency, low-cost, and low-pollution cell-microbead pairing capture method for single-cell omics analysis. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 A schematic diagram of a cell-microbead pairing capture method provided according to Example 1 of the present invention, wherein a state of using a microfluidic capture device to capture a cell-microbead pairing is shown, A is a side view, and B is a top view;

[0027] Figure 2 The process schematic diagram of step S3 of the cell-microbead pairing capture method provided in Example 1 of the present invention, wherein A is a schematic diagram of perfusing a controllably solidifiable solution to replace a cell / microbead solution in step S3, and B is a schematic diagram of selectively solidifying in a microfluidic capture device according to the characteristics of the solution in step S3;

[0028] Figure 3 A schematic diagram of the process of step S4 of the cell-microbead pairing capture method provided in Example 1 of the present invention, wherein the process of flipping the microfluidic capture device after selective solidification is achieved in step S4 to form an open cell-microbead unit array is shown;

[0029] Figure 4 A schematic diagram of the structure of a particle capture layer of a microfluidic capture device provided according to Example 2 of the present invention;

[0030] Figure 5 For Figure 4A detailed enlarged schematic diagram of a single capture microstructure in the particle capture layer shown;

[0031] Figure 6 For the use of Figure 4 Schematic diagram of the process of capturing cells by the particle capture layer when the microfluidic capture device performs cell-microbead pairing capture;

[0032] Figure 7 For the use of Figure 4 Schematic diagram of the process of further capturing microbeads by the particle capture layer after completing cell capture when the microfluidic capture device performs cell-microbead pairing capture;

[0033] Figure 8 A three-dimensional schematic diagram of forming an open cell-microbead unit array after selective solidification in steps S3-S4 of the cell-microbead pairing capture method provided in Example 2 of the present invention;

[0034] Fig. 9 Schematic diagram of the processing of the microfluidic capture device in Example 3 of the present invention.

[0035] Among them, the meanings of the figure marks are as follows: 1. inflow area, 2. capture area, 3. outflow area, 4. inlet, 5. branch channel, 6. pre-filter, 7. third channel, 8. outlet, 9. cell-microbead capture array, 10. thin film structure, 11. microstructure inlet, 12. microstructure outlet, 13. capture chamber. DETAILED DESCRIPTION

[0036] The present invention is further described below in conjunction with specific examples. It should be understood that the following examples are only used to illustrate the present invention and are not used to limit the scope of the present invention.

[0037] Example 1 A cell-microbead pairing capture method

[0038] Combination Figure 1 to Figure 3 As shown, a cell-microbead pairing capture method provided according to a preferred embodiment of the present invention comprises the following steps:

[0039] S1: First, provide a Figure 1 The microfluidic capture device shown is composed of a particle capture layer and a substrate layer, and the two layers are separable; the particle capture layer is provided with an inlet, an outlet, and a plurality of capture microstructures that can be paired one-to-one to capture cell-microbeads, and the capture microstructures are distributed in an array form.

[0040] S2: The cell suspension and microbead suspension are sequentially introduced into the particle capture layer from the inlet, and a single cell and a single microbead are captured in the capture microstructure in turn, thereby forming a large-scale cell-microbead pairing array.

[0041] S3: After the paired capture is completed, a controllable solidification solution is perfused from the inlet to replace the cell and microbead solution used in step S2, such as Figure 2 As shown in A in FIG, selective solidification is achieved in the device according to the characteristics of the solution, so that each cell-bead capture unit forms a mutually isolated chamber, forming an independent chamber, such as Figure 2 As shown in B; each independent chamber represents a microreaction unit, and each microreaction unit includes a single cell and a single microbead.

[0042] S4: First, the microfluidic capture device that has been selectively solidified in step S3 is turned upside down so that the substrate layer faces upward, and then the substrate layer is separated from the particle capture layer to form an open cell-microbead unit array, similar to a microwell structure, such as Figure 3 As shown, the captured cell-beads can be used for the next step of single-cell omics analysis.

[0043] It should be understood that the capture methods of cells and microbeads in step S2 include but are not limited to fluid dynamics methods, external physical field (light, sound, magnetism, electricity, etc.) capture methods, etc. Considering the reduction of costs and the simplification of structure, the fluid dynamics method is preferred.

[0044] It should also be understood that when the above method is used to capture a single cell and a single microbead, the microbead and the cell are captured separately and successively. Alternatively, the microbead is captured first and then the cell, or the cell is captured first and then the microbead. When capturing the microbead or the cell, the microbead or the cell is in a liquid.

[0045] In step S3, after the capture of a single cell and a single microbead is completed in each chamber, a controllable solidification solution is introduced and selective solidification occurs, thereby forming an independent chamber array of cells-microbeads to avoid cross-contamination between different cell-microbeads; each independent chamber represents a microreaction unit, and each microreaction unit includes a single cell and a single microbead.

[0046] In step S3, the controllably curable solution includes, but is not limited to, photosensitive hydrogel (which can be selectively cured by ultraviolet light); thermosensitive hydrogel (which can be selectively cured by local heating), or various types of hydrophobic polymers, etc., preferably photosensitive hydrogel.

[0047] Furthermore, if the next step of single-cell omics analysis is required after step S4, the solution can be directly perfused from the open side; if cell lysis is to be performed, the cell lysis solution can be perfused from the open side to achieve cell lysis of each pair of cells-beads in a separate chamber;

[0048] According to the present invention, the object captured by the method is not actually limited to one cell and one microbead, but may also be two microbeads or microparticles of different sizes, or two cells of different sizes.

[0049] The scope of single-cell omics analysis includes but is not limited to single-cell genome sequencing, single-cell transcriptome sequencing, single-cell whole transcriptome sequencing, single-cell genome and transcriptome parallel sequencing, etc.

[0050] The substrate layer material includes but is not limited to glass, PDMS, organic glass and the like, preferably glass.

[0051] Example 2 A microfluidic capture device and a cell-microbead pairing capture method

[0052] Combination Figure 4-5 As shown, a microfluidic capture device provided according to this preferred embodiment is shown, and the microfluidic capture device can capture cells-microbeads by a fluid dynamics method, and is described in detail as follows:

[0053] The microfluidic capture device is composed of a particle capture layer and a substrate layer, and the two layers are separable. Figure 4 As shown, the particle capture layer includes, from upstream to downstream, an inflow zone 1, a capture zone 2, and an outflow zone 3.

[0054] The inflow area 1 includes: an inlet 4, a branch channel 5, and a pre-filter 6. The inlet 4 is connected to a plurality of branch channels 5 of equal width, and the branch channels 5 are symmetrical with the third channel 7 as the symmetry axis. At the same time, a section is reserved at the rear end of the inflow area 1 to form a pre-filter 6, and the pre-filter 6 is composed of columns of the same size arranged equidistantly. The outflow area 3 includes an outlet 8.

[0055] The capture area 2 is composed of capture microstructures arranged in a high-density array. Each capture microstructure can achieve one-to-one pairing capture of cells and microbeads. A cell-microbead capture array 9 is formed by these capture microstructures.

[0056] According to the preferred embodiment, Figure 5As shown, each capture microstructure is composed of four microcolumns, and the four microcolumns form a capture cavity 13 inside, wherein a microstructure entrance 11 is formed between two microcolumns, and a microstructure exit 12 is formed between the other two microcolumns. The opening sizes of the microstructure entrance 11 and the microstructure exit 12 are both smaller than the cell particle size and the microbead particle size. One of the microcolumns used to form the microstructure entrance 11 adopts a deformable film structure 10. When the filtered cells or microbeads enter the capture area at a medium flow rate, the film structure 10 maintains its shape unchanged. A single cell capture microstructure captures a cell or a microbead through the microstructure entrance 11, and then the flow rate is increased, the film structure 10 is deformed, the size of the microstructure entrance 11 becomes larger, and the captured cell or microbead is released and stored in the capture cavity 13.

[0057] According to the preferred embodiment, a cell-microbead pairing capture method is also provided, comprising the following steps:

[0058] S1: Provide Figure 4 The microfluidic capture device shown;

[0059] S2: Introduce cell suspension and microbead suspension from the inlet in sequence to complete the capture of single cells and single microbeads in the microfluidic capture device, thereby forming a large-scale cell-microbead pairing array.

[0060] The single cell capture process is as follows:

[0061] like Figure 6 As shown, the cell suspension passes through the inlet 4 and the pre-filter 6 in sequence and enters the capture area 2. At this time, the deformation of the film structure 10 is small, and the opening size of the microstructure inlet 11 is smaller than the cell particle size, so the cells are trapped at the microstructure inlet 11; then, by applying excessive flow, the microstructure inlet 11 gradually widens as the film structure 10 deforms and becomes larger than the cell diameter, releasing the cells into the capture chamber 13. After the cells are released, the deformed film structure 10 will reduce the flow rate due to its elastic recovery, thereby returning to its original state, completing the capture of a cell.

[0062] After cell capture is completed, microbead capture is performed. The capture process of a single microbead is as follows:

[0063] like Figure 7 As shown, the microbead suspension passes through the inlet 4 and the pre-filter 6 in sequence and enters the capture area 2. At this time, the deformation of the film structure 10 is small, and the opening size of the microstructure inlet 11 is smaller than the particle size of the microbeads, so the microbeads are trapped at the microstructure inlet 11; then, by applying excessive flow, the microstructure inlet 11 gradually widens as the film structure 10 deforms and becomes larger than the diameter of the microbeads, releasing the microbeads into the capture chamber 13. After the microbeads are released, the deformed film structure 10 will reduce the flow rate due to its elastic recovery, thereby returning to its original state, completing the capture of a microbead.

[0064] It should be understood that under normal flow conditions, the filtered cells or microbeads enter the capture zone 2, one capture microstructure captures one cell or microbead, and the subsequent cells or microbeads bypass the capture microstructure through the dynamic change of fluid flow and go to the next capture microstructure, resulting in the retention of a single cell or microbead. By increasing the flow rate to deform the film structure 10 and increase the opening size of the microstructure inlet 11, the retained cells or microbeads can be successfully released and stored.

[0065] S3: If the controllable solidification solution is preferably a photosensitive hydrogel, after the cell-microbead pairing is captured, the photosensitive hydrogel is introduced from the inlet, the microfluidic capture device is flipped over so that the substrate layer faces upward, and ultraviolet light is irradiated to the microfluidic capture device through the mask. Figure 8 As shown in A in FIG, the photosensitive hydrogel undergoes selective solidification to form independent chambers, each of which contains a cell and a microbead. Figure 8 As shown in B.

[0066] S4: Separating the substrate layer from the particle capture layer to form an open cell-bead unit array, such as Figure 8 As shown in C, the captured cell-beads can be used for the next step of single-cell omics analysis.

[0067] Example 3 Preparation of a microfluidic capture device

[0068] According to the preferred embodiment, the present invention also provides a method for preparing a microfluidic capture device, the processing flow is as follows: Fig. 9 The detailed description is as follows:

[0069] (1) Photolithography mask design and production: Draw the design of the microfluidic device and use high-precision printing to output multiple masks. Each mask has a light-transmitting and opaque pattern to form a pattern in the exposure step.

[0070] (2) Photolithography mold preparation

[0071] Photolithography is a process of microfabrication using photoresist, mask and ultraviolet light. The mold can be reused after processing. The specific process is as follows:

[0072] ①Cleaning silicon wafer

[0073] ②Pre-baking (soft bake)

[0074] ③Exposure: Place the mask on top of the silicon wafer, irradiate the silicon wafer with photoresist spin-coated with ultraviolet light, and the photoresist undergoes a photochemical reaction to prepare the desired pattern on the mask;

[0075] ④ Development: Use a developer to remove the unexposed photoresist through a chemical method of development, and accurately copy the pattern on the mask to the photoresist layer;

[0076] ⑤Hard bake

[0077] The SU-8 mold is completed.

[0078] (3) Reverse molding

[0079] ① Place the SU-8 mold in an aluminum foil box of appropriate size and pour in PDMS, keeping the height at 2-3mm;

[0080] ② Heat at 120℃ for 0.5 hours. After 0.5 hours, the PDMS is solidified and the solidified PDMS is gently demolded from the mold;

[0081] (4) Substrate bonding

[0082] ① Place the surface of the device with the flow channel structure facing upwards, and drill holes from top to bottom at the entrance and exit so that the flow channel can be connected to the outside world;

[0083] ②Ultrasonic cleaning of the punched device and glass sheet, rinse with deionized water, blow dry with nitrogen, and heat to remove moisture to keep the device clean;

[0084] ③ Device overall bonding: Use a plasma cleaning machine to plasma treat the device surface to be bonded and the clean glass sheet, contact the surface of the clean device with the flow channel structure with the glass sheet, and press gently to obtain the chip.

[0085] (5) Substrate separation

[0086] After completing the cell / microbead pairing and forming an independent chamber according to the above steps, the substrate glass sheet is separated from the PDMS. The glass sheet is kept on top during the separation process, so that the chamber of the cells / microbeads captured on the PDMS is open upward after separation.

[0087] The above is only a preferred embodiment of the present invention, and is not intended to limit the scope of the present invention. The above embodiments of the present invention can also be modified in various ways. All simple, equivalent changes and modifications made according to the claims and the description of the present invention fall within the scope of protection of the claims of the present invention. The contents not described in detail in the present invention are all conventional technical contents.

Claims

1. A cell-bead pairing capture method for single-cell omics analysis, characterized in that: The following steps are involved: S1: A microfluidic capture device is provided, which includes a separable particle capture layer and a substrate layer, wherein the particle capture layer includes: an inflow area, a capture area, and an outflow area, wherein the inflow area includes an inlet, a branch channel, and a pre-filter connected in sequence, and the capture area includes capture microstructures distributed in an array, wherein each capture microstructure is composed of a plurality of microcolumns, wherein the plurality of microcolumns enclose a capture cavity inside, wherein a microstructure inlet is formed between two microcolumns, and a microstructure outlet is formed between another two microcolumns, and a one-to-one paired capture of cells and microbeads can be achieved by applying a fluid dynamics method; wherein one of the microcolumns used to form the microstructure inlet adopts a deformable film structure, and when the filtered cells or microbeads enter the capture area at a medium flow rate, the film structure maintains its shape unchanged, and a single cell capture microstructure captures a cell or microbead, and then the flow rate is increased, the film structure is deformed, the size of the microstructure inlet becomes larger, and the captured cell or microbead is released and stored in the capture cavity; S2: introducing a cell suspension or a microbead suspension from the inlet, gradually completing the one-to-one pairing capture of a single cell or a single microbead in the capture microstructure, thereby forming an array consisting of a plurality of cell-microbead capture units; S3: Continue to perfuse the controllable solidification solution from the inlet to replace the cell suspension or microbead suspension on the particle capture layer, and solidify the controllable solidification solution in the microfluidic capture device according to the solution characteristics, so that each cell-microbead capture unit forms an independent chamber isolated from each other; S4: Separating the substrate layer from the particle capture layer to form an open cell-microbead capture unit array, and the captured cell-microbeads can be subjected to the next step of single-cell omics analysis.

2. The cell-microbead pairing capture method according to claim 1, characterized in that: In step S2, the capture methods of cells and microbeads include fluid dynamics methods and external physical field capture methods, and the external physical field includes: any one of light, sound, magnetism, and electricity.

3. The cell-microbead pairing capture method according to claim 1, characterized in that: In step S3, the controllably curable solution includes a photosensitive hydrogel and a thermosensitive hydrogel.

4. The cell-microbead pairing capture method according to claim 1, characterized in that: In step S4, the next step of single-cell omics analysis includes: single-cell genome sequencing, single-cell transcriptome sequencing, single-cell whole transcriptome sequencing, and single-cell genome and transcriptome parallel sequencing.

5. The cell-microbead pairing capture method according to claim 1, characterized in that: The cell-microbead pairing capture method can be used to capture cells and microbeads of different sizes, cells and cells, and microbeads and microbeads.

6. The cell-microbead pairing capture method according to claim 1, characterized in that: The materials of the substrate layer include: glass, PDMS, and organic glass.

7. The cell-microbead pairing capture method according to claim 3, characterized in that: When the controllably curable solution is a photosensitive hydrogel, after the cell-microbead pairing capture is completed, the photosensitive hydrogel is introduced from the entrance of the microfluidic capture device, the microfluidic capture device is turned over so that the substrate layer faces upward, ultraviolet light is irradiated to the particle capture layer through the mask, the photosensitive hydrogel is selectively cured to form an independent chamber, and the substrate layer is removed to form an open cell-microbead capture unit array.

8. The cell-microbead pairing capture method according to claim 1, characterized in that: The preparation method of the microfluidic capture device is as follows: 1) Photolithography mask design and production: Draw the design of the microfluidic device and use high-precision printing to output multiple masks, each of which has a light-transmitting and light-opaque pattern to form a pattern in the exposure step; 2) Photolithography mold preparation: Use photoresist, mask and ultraviolet light for micro-manufacturing. The mold processing is completed and can be reused. It includes: cleaning silicon wafer, pre-baking, exposure, development, hardening film, and SU-8 mold production is completed; 3) Reverse mold; 4) Substrate bonding: Use a plasma cleaner to plasma treat the surface of the device to be bonded and a clean glass sheet, contact the surface of the clean device with the flow channel structure with the glass sheet, and press gently to obtain a microfluidic capture device.

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