Microporous array chip for single cell sequencing, preparation method and application method

Abstract

The invention discloses a micropore array chip for single cell sequencing, a preparation method and an application method, and the application method comprises the following steps: respectively preparing large liquid drops and small liquid drops, adding fluorinated oil into an inlet of the micropore array chip, adding the large liquid drops into the inlet of the micropore array chip and inclining to capture the large liquid drops, and then adding the small liquid drops into the inlet of the micropore array chip and inclining to capture the small liquid drops. Discharging redundant liquid drops, placing on the ice surface, sorting the cell liquid drops through a liquid drop sorter, adding the cell liquid drops into small micropores on one side of the large micropores of the micropore array chip so as to enable the cell liquid drops and the large liquid drops to be paired one by one, performing corona treatment, heating, and placing on the ice surface again; adding the small liquid drops into an inlet of a microporous array chip, then performing corona again, sealing the microporous array chip, heating in a water bath, injecting a water-soluble buffer solution, then performing corona again to demulsify the liquid drops, and collecting a solution containing cDNA. According to the method, the large liquid drops, the cell liquid drops and the small liquid drops are paired and fused to realize single cell sequencing, so that the capturing capability of single cell mRNA is improved, and the single cell sequencing efficiency is improved.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and in particular to a micropore array chip for single-cell sequencing, a preparation method and an application method. Background Art

[0002] Single-cell sequencing technology is a revolutionary method in biomedical research in recent years. It can perform high-throughput sequencing of genomes, transcriptomes or epigenomes at the single-cell level. Traditional population sequencing technology usually measures the average signal of thousands to millions of cells, which masks the heterogeneity between individual cells. Single-cell sequencing technology can reveal the gene expression and genomic characteristics of individual cells, providing more detailed resolution and deeper biological understanding.

[0003] Single-cell sequencing technologies mainly include single-cell genome sequencing (scDNA-seq), single-cell transcriptome sequencing (scRNA-seq), and single-cell epigenome sequencing (scATAC-seq). Each method analyzes specific molecular features. For example, scRNA-seq reveals the gene expression profile of a single cell by measuring the mRNA content of the cell, and has been widely used to study cell heterogeneity, developmental processes, and disease mechanisms.

[0004] Single-cell sequencing technology has demonstrated its strong application potential in multiple research fields. In tumor research, scRNA-seq technology is used to analyze the tumor microenvironment, identify different cell subpopulations and their interactions, and reveal the heterogeneity and evolution of tumors. In immunology research, through single-cell analysis, scientists can understand in detail the gene expression profiles of different immune cell types and their role in immune responses, and explore the dynamic changes of the immune system in health and disease states. In addition, single-cell sequencing is also widely used in neuroscience research to study the gene expression of neurons and glial cells, revealing the complexity and developmental mechanisms of the nervous system.

[0005] Single-cell RNA sequencing provides scientists with the ability to gain in-depth insights into gene expression in individual cells and has been widely used in oncology, neuroscience, immunology, drug discovery, infectious diseases and other fields.

[0006] Traditional single-cell isolation methods can only detect a small number of target cells, which is time-consuming, labor-intensive and costly (using micropipette classification or laser capture microdissection to obtain single cells, and then cooperating with low-throughput single-cell sequencing technology). Currently, there are methods based on droplet co-encapsulation (the method used by 10x Genomics) and methods that capture single cells into microwells by gravity (the method used by BD) to perform single-cell separation and high-throughput single-cell sequencing. The main disadvantage of the droplet co-encapsulation method is that it will cause cell loss. When the target cells are 5,000, an excess of 8,280 cells needs to be invested. In some clinical needs, such as rare diseases or small tumors, usually only a small number of cells can be obtained, so the current sequencing methods are unacceptable for rare cell samples. Such samples require efficient medium- and low-throughput sequencing methods, and existing high-throughput technologies are not suitable for this situation.

[0007] Therefore, the single-cell sequencing methods in the prior art have the problem of being unable to efficiently capture single cells to achieve high-throughput sequencing. Summary of the invention

[0008] The embodiments of the present invention provide a microwell array chip, preparation method and application method for single-cell sequencing, aiming to solve the problem that the single-cell sequencing method in the prior art cannot efficiently capture single cells to achieve high-throughput sequencing.

[0009] In a first aspect, an embodiment of the present invention provides an application method of a microwell array chip for single-cell sequencing, wherein the application method comprises:

[0010] Large droplets are prepared by a first droplet generation chip;

[0011] Small droplets are prepared by a second droplet generation chip;

[0012] Connecting funnel-shaped connectors to the inlet and outlet of the microwell array chip respectively, adding fluorinated oil to the inlet of the microwell array chip and placing it in a vacuum environment to exhaust the air in the chip;

[0013] After adding the large droplets to the inlet of the microwell array chip, tilting the microwell array chip to allow the large droplets to drift into the large microwells in the microwell array chip by buoyancy;

[0014] adding fluorinated oil again at the inlet of the microwell array chip to remove excess large droplets;

[0015] The microwell array chip is tilted and placed on an ice surface, and the positive droplet output port of the droplet sorter is connected to the inlet port of the microwell array chip, so that the cell droplets are sorted by the droplet sorter and added to the small microwells on one side of the large microwells of the microwell array chip, so that the cell droplets are paired with the large droplets one by one;

[0016] adding fluorinated oil again at the inlet of the microwell array chip to remove excess cell droplets;

[0017] Using a corona machine, applying a corona treatment for 2-4 seconds on the microwell array chip to induce fusion of the large droplets and the cell droplets that are in contact with each other;

[0018] The microwell array chip is placed at room temperature for 20-30 minutes to allow proteinase K to fully lyse cells and release cell mRNA, then placed in a water bath at 68-75° C. for 8-12 minutes to inactivate proteinase K, and then placed on ice;

[0019] Small droplets are input through the inlet of the micropore array chip, so that they drift into the small micropores on the side of the large micropores in the micropore array chip through buoyancy to complete the one-to-one pairing between the small droplets and the droplets in the large micropores, and then excess small droplets are removed by adding fluorinated oil, and a corona treatment is applied for 2-4 seconds above the micropore array chip using a corona machine to allow the small droplets to pair and fuse with the droplets in the large micropores;

[0020] After sealing the inlet and outlet of the microwell array chip, the chip was placed in a 40-45° C. water bath for 3 hours of reverse transcription reaction;

[0021] The inlet of the microporous array chip is connected by a syringe, and a constant-current injection pump is used to push the syringe at a specific flow rate to push a water-soluble buffer solution into the microporous array chip to discharge the fluorinated oil. Air and the water-soluble buffer solution are sucked in and input into the inlet of the microporous array chip again. A corona treatment is applied for 2-4 seconds above the microporous array chip using a corona machine to induce droplet demulsification. After standing for 12-18 minutes, the water-soluble buffer solution containing cDNA is collected through the outlet of the microporous array chip for sequencing analysis.

[0022] In the second aspect, an embodiment of the present invention further provides a microwell array chip, wherein the microwell array chip is applied to the application method as described in the first aspect above, and the microwell array chip is obtained by stacking a top layer of glass-polydimethylsiloxane microwell chip, a fluid channel layer and a bottom layer of glass slide.

[0023] In a third aspect, an embodiment of the present invention further provides a method for preparing a microwell array chip, wherein the method is used to prepare the microwell array chip as described in the second aspect, and the method comprises:

[0024] Two through holes are opened on a glass slide and polydimethylsiloxane is filled in the through holes to obtain a glass substrate;

[0025] The silicon wafer mold is subjected to oxygen-plasma treatment in a vacuum chamber filled with silane vapor for more than 10 hours, the polydimethylsiloxane prepolymer from which bubbles have been removed is poured onto the silicon wafer mold for peeling, and a concave mold having a polydimethylsiloxane micropore array is obtained, and the concave mold is placed in a vacuum chamber filled with silane vapor for more than 10 hours, and the mold is turned over again to obtain a convex mold having a polydimethylsiloxane micropore array;

[0026] Pour polydimethylsiloxane prepolymer onto a convex mold with a microwell array and perform a vacuum treatment to remove air bubbles;

[0027] The glass substrate is treated with oxygen-plasma, and then placed on a convex mold with a microwell array for bonding, and then heated and cured, and then peeled off to obtain a polydimethylsiloxane microwell array-glass substrate composite;

[0028] Repeatedly spin coating polydimethylsiloxane on a silicon wafer to obtain a flat polydimethylsiloxane film, and cutting the polydimethylsiloxane film to obtain a fluid channel layer;

[0029] The fluid channel layer and the bottom glass slide are bonded and then plasma treated, a polydimethylsiloxane microwell array-glass slide substrate composite is bonded to the upper layer of the fluid channel layer, and heated at 110° C. for 10 minutes to obtain a microwell array chip;

[0030] A hydrophobic reagent was injected into the microporous array chip, and the microporous array chip was placed in a vacuum environment; after 30 seconds, the hydrophobic reagent was discharged by extraction through a syringe, and then the microporous array chip was placed at 110° C. for drying to remove the remaining hydrophobic reagent.

[0031] The embodiment of the present invention provides a micropore array chip, a preparation method and an application method, the application method comprising preparing large droplets and small droplets respectively, adding fluorinated oil to the inlet of the micropore array chip, adding large droplets to the inlet of the micropore array chip and tilting to capture large droplets, placing on an ice surface after draining excess droplets, sorting cell droplets by a droplet sorter and adding them to the large micropores of the micropore array chip so that the cell droplets and the large droplets are paired one by one, heating after corona treatment and placing on an ice surface again, adding small droplets to the inlet of the micropore array chip and corona treatment again, sealing the micropore array chip and heating in a water bath, injecting a water-soluble buffer solution and corona treatment again to break the droplets, and collecting a solution containing cDNA for sequencing analysis. The above application method realizes single-cell sequencing of cell droplets by pairing and fusing large droplets with cell droplets and small droplets; optimizing the reagent system of single-cell sequencing by step-by-step sample addition, and improving the efficiency of single-cell sequencing by improving the mRNA capture efficiency of single cells. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the accompanying drawings required for use in the description of the embodiments will be briefly introduced below. Obviously, the accompanying drawings described below are some embodiments of the present invention. For ordinary technicians in this field, other accompanying drawings can be obtained based on these accompanying drawings without paying any creative work.

[0033] Figure 1 A method flow chart of the application method of the microwell array chip provided in an embodiment of the present invention;

[0034] Figure 2 A planar structural diagram of a first droplet generation chip provided in an embodiment of the present invention;

[0035] Figure 3 A planar structural diagram of a second droplet generation chip provided in an embodiment of the present invention;

[0036] Figure 4 The overall structure diagram of the microwell array chip provided by the embodiment of the present invention;

[0037] Figure 5 A partial structural diagram of a microwell array chip provided in an embodiment of the present invention;

[0038] Figure 6 Another partial structural diagram of the microwell array chip provided by an embodiment of the present invention;

[0039] Figure 7 Another partial structural diagram of the microwell array chip provided by the embodiment of the present invention

[0040] Figure 8A flow chart of a method for preparing a microwell array chip provided in an embodiment of the present invention;

[0041] Fig. 9 A schematic diagram of the application effect of the microwell array chip provided in an embodiment of the present invention;

[0042] Fig.10 A schematic diagram of another application effect of the microwell array chip provided by an embodiment of the present invention;

[0043] Fig.11 A schematic diagram of another application effect of the microwell array chip provided in an embodiment of the present invention;

[0044] Fig.12 A schematic diagram of another application effect of the microwell array chip provided in an embodiment of the present invention;

[0045] Fig.13 A schematic diagram of the latter application effect of the microwell array chip provided by an embodiment of the present invention;

[0046] Fig.14 A schematic diagram of another application effect of the microwell array chip provided by an embodiment of the present invention;

[0047] Fig.15 A schematic diagram of another application effect of the microwell array chip provided in an embodiment of the present invention.

[0048] Figure numerals: 10, first droplet generation chip; 20, second droplet generation chip; 30, microwell array chip; 11, first reverse transcription reaction reagent inlet; 12, microsphere phase inlet; 13, first oil phase inlet; 14, first outlet; 21, second reverse transcription reaction reagent inlet; 22, second oil phase inlet; 23, second outlet; 31, glass-polydimethylsiloxane microwell chip; 32, fluid channel layer; 33, bottom glass slide; 311, large microwell; 312, small microwell. DETAILED DESCRIPTION

[0049] The following will be combined with the drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0050] It should be understood that when used in this specification and the appended claims, the terms "include" and "comprises" indicate the presence of described features, integers, steps, operations, elements and / or components, but do not exclude the presence or addition of one or more other features, integers, steps, operations, elements, components and / or combinations thereof.

[0051] It should also be understood that the terms used in the present specification are only for the purpose of describing specific embodiments and are not intended to limit the present invention. As used in the present specification and the appended claims, unless the context clearly indicates otherwise, the singular forms "a", "an" and "the" are intended to include plural forms.

[0052] It should be further understood that the term "and / or" used in the present description and the appended claims refers to and includes any and all possible combinations of one or more of the associated listed items.

[0053] See also Figure 1 As shown in the figure, the embodiment of the present application discloses an application method of a microwell array chip, which includes S101 to S112.

[0054] S101, preparing large droplets through a first droplet generation chip.

[0055] like Figure 2 As shown, the first droplet generation chip includes a first reverse transcription reaction reagent inlet, a microsphere phase inlet, a first oil phase inlet and a first outlet; the large droplets are prepared by the first droplet generation chip, including: adjusting the flow rate of the microsphere phase inlet so that each droplet contains only one microsphere; using a high-speed camera to obtain a droplet generation image, and adjusting the flow rate ratio between the first reverse transcription reaction reagent inlet and the first oil phase inlet and the microsphere phase inlet based on the diameter of the droplet in the droplet generation image, so that the microspheres and the reverse transcription reaction reagents are fused and then wrapped by the droplet generation oil to form large droplets; placing the container containing the large droplets on an ice surface for standby use.

[0056] Specifically, in order to prepare large droplets, a first droplet generation chip can be designed accordingly, and the microsphere phase inlet and the first outlet in the first droplet generation chip are connected by a pipeline; a needle-shaped pipeline changing from thick to thin is provided between the microsphere phase inlet and the confluence point of the connecting pipeline, and a needle-shaped pipeline changing from thick to thin is provided between the first outlet and the confluence point; two sections of serpentine winding pipelines connected to the first reverse transcription (RT) reaction reagent inlet are provided on both sides of the connecting pipeline at the confluence point; two sections of serpentine winding pipelines connected to the first oil phase inlet are also provided on both sides of the connecting pipeline at the confluence point. Specifically, large droplets are generated by a 70μm high large droplet generation string chip, and the chip has three inlets (RT phase, microsphere phase, oil phase) and an outlet. The first reverse transcription reaction reagent input by the first reverse transcription reaction reagent inlet includes reverse transcription buffer (RT buffer), proteinase K, DTT (dithiothreitol), RNA inhibitor and other reagents.

[0057] The droplet generation oil is loaded into a 1ml syringe, and a polyethylene tube (PE tube, outer diameter 1.22mm) is used to connect the syringe outlet to the oil phase inlet of the first droplet generation chip. Due to the small volume of RT reaction reagents and microsphere reagents, the encapsulated aqueous phase cannot be loaded into the syringe for precise flow control. To solve this problem, the microsphere reagent can be injected into the polyethylene tube and then connected to the polyethylene tube injected with fluorinated oil through a steel needle. The inlet of the polyethylene tube injected with fluorinated oil is connected to the outlet of the syringe injected with fluorinated oil, and the outlet of the polyethylene tube injected with the microsphere reagent is connected to the microsphere phase inlet of the first droplet generation chip; the syringe and the tail section PE tube are pre-installed with fluorinated oil, and the aqueous phase to be encapsulated is drawn into another section of PE tube using a pipette gun, and then the PE tubes at both ends are connected by a steel needle, so that the microsphere reagent and RT reaction reagent can be accurately controlled in flow; the first outlet is connected to the PCR tube through the PE tube.

[0058] In order to ensure the quality of single-cell sequencing, it is necessary to observe the entry speed of the microsphere phase when making large droplets, and adjust the flow rate of the microsphere reagent and the RT reaction reagent in real time so that each large droplet contains only one microsphere. Use a high-speed camera with a Nikon microscope to observe the entire process of large droplet generation, and based on this, optimize the flow rate ratio between the first reverse transcription reaction reagent inlet and the first oil phase inlet and the microsphere phase inlet to regulate the diameter of the generated large droplets. Randomly select multiple large droplet bright field images, and use Image J to measure the large droplet diameter. By replacing PCR tubes, a portion of large droplets with the highest size and microsphere encapsulation rate can be collected for subsequent experiments. The collected large droplets need to be placed on ice for later use.

[0059] S102, preparing small droplets through a second droplet generation chip.

[0060] In a specific embodiment, Figure 3 As shown, the second droplet generation chip includes a second reverse transcription reaction reagent inlet, a second oil phase inlet and a second outlet; the preparation of small droplets by the second droplet generation chip includes: using a high-speed camera to obtain a droplet generation image, and adjusting the flow rate ratio between the second reverse transcription reaction reagent inlet and the second oil phase inlet based on the diameter of the droplet in the droplet generation image, so that the reverse transcription reaction reagent is wrapped by the droplet generation oil to form small droplets; placing the container containing the small droplets on an ice surface for standby use. The second reverse transcription reaction reagent input by the second reverse transcription reaction reagent inlet includes reverse transcriptase, short oligonucleotide sequence (Template Switching Oligo), RNA inhibitor and other reagents.

[0061] The cell droplets are produced by using a 45 μm high droplet generation chip, and the specific operation process is similar to the steps used in the above step S101.

[0062] S103, connecting funnel-shaped connectors at the inlet and outlet of the microwell array chip respectively, adding fluorinated oil at the inlet of the microwell array chip and placing it in a vacuum environment to exhaust the air in the chip.

[0063] The process of droplet capture and fusion is completed in a microwell array chip (microwell array device). The overall structure of the microwell array chip is as follows: Figure 4 As shown, the arrangement structure of the large micropores on the micropore array device is as follows Figure 5 As shown. When performing droplet capture, first capture large droplets. The specific operation process is to first plug two funnel-shaped connectors (10μL tip heads) into the outlet and inlet of the microwell array chip respectively. Use a pipette to add 200μL of fluorinated oil from the inlet of the microwell array chip to wet the microwell array chip, place the microwell array chip with fluorinated oil added in a closed environment and evacuate it, that is, place the microwell array chip in a vacuum environment to empty the air in the chip.

[0064] S104, after adding the large droplets to the inlet of the microwell array chip, tilting the microwell array chip to allow the large droplets to drift into the large microwells in the microwell array chip by buoyancy.

[0065] Furthermore, a 2.5 μL large droplet is added to the inlet of the microwell array chip using a pipette, and the microwell array chip is tilted. Under the action of the buoyancy caused by the density difference between the oil phase and the water phase, the large droplet will quickly diffuse toward the outlet and drift and move with the flow of the liquid. Then, the large droplet drifts into the corresponding large microwell under the action of the buoyancy. The specific structure of the large microwell is as follows: Figure 6 As shown, the maximum depth h1 of the large micropore is 80 microns, the bottom width w1 is 90 microns, and a boss is provided on one side of the bottom of the large micropore, thereby forming a shallower concave hole on one side of the large micropore.

[0066] S105, adding fluorinated oil again to the inlet of the microwell array chip to remove excess large droplets.

[0067] Fluorinated oil was added to the inlet of the microwell array chip to flush out excess large droplets, and the capture rate of large droplets was observed using a microscope.

[0068] S106, tilt the microwell array chip and place it on an ice surface, connect the positive droplet output port of the droplet sorter to the inlet port of the microwell array chip, sort the cell droplets through the droplet sorter and add them to the small microwells on one side of the large microwells of the microwell array chip, so that the cell droplets are paired one by one with the large droplets.

[0069] Further, the cell droplets are captured. The micropore array chip can be placed on the ice surface at an angle of 30 degrees. Due to the fast sorting speed, the entrance of the micropore array chip needs to be higher than its exit. The flow rate can be slowed down a little by buoyancy, and the sorted cell droplets can also be left in the micropore array chip. After the droplet sorter starts working, the positive droplet output port of the droplet sorter is connected to the entrance of the micropore array chip, so that the cell droplets are sorted by the droplet sorter and added to the small micropores on the large micropore side of the micropore array chip. Try to avoid introducing bubbles, and the outlet of the micropore array chip is connected to a PE tube for stability.

[0070] Furthermore, in order to ensure better sorting effect and higher single-pack rate, according to the Poisson distribution formula, before the capture operation of cell droplets, a cell suspension concentration of 4500 cells / μL can be selected for the production of cell droplets. After the cell droplets are produced and before sorting, the droplets need to be placed on ice for standby. Droplet sorting is performed using the Dapu Bio Comet high-throughput screening system.

[0071] S107, adding fluorinated oil again to the inlet of the microwell array chip to remove excess cell droplets.

[0072] Fluorinated oil is added again to the inlet of the microwell array chip to remove excess cell droplets.

[0073] S108, applying a corona treatment for 2-4 seconds on the microwell array chip using a corona machine to induce fusion of the large droplets and cell droplets that are in contact with each other.

[0074] Furthermore, after the droplet capture and pairing are completed, the droplet fusion can be achieved by corona treatment. A corona machine is used to apply corona treatment for 2-4 seconds (about 3 seconds) above the microwell array chip to obtain an instantaneous AC electric field to temporarily change the stability of the droplet water-oil interface, inducing the contacting large droplets and cell droplets to fuse.

[0075] S109, placing the microwell array chip at room temperature for 20-30 minutes to allow proteinase K to fully lyse cells and release cellular mRNA, then placing it in a 68-75°C water bath for 8-12 minutes to inactivate proteinase K, and then placing the microwell array chip on ice.

[0076] After the droplets merge, the microwell array chip is placed at room temperature for 20-30 minutes to ensure the cell lysis effect, so that proteinase K can fully lyse the cells and release the cell mRNA; specifically, the microwell array chip can be placed at room temperature for 25 minutes. Then, the chip is placed in a water bath at 68-75°C for 8-12 minutes to inactivate proteinase K. Preferably, the chip can be placed in a water bath at 72°C for 10 minutes to inactivate proteinase K. After the inactivation is completed, the chip is quickly placed on ice.

[0077] S110, inputting small droplets through the inlet of the micropore array chip, allowing them to drift into the small micropores on the side of the large micropores in the micropore array chip through buoyancy to complete the one-to-one pairing between the small droplets and the droplets in the large micropores, and then removing excess small droplets by adding fluorinated oil, and applying a corona treatment for 2-4 seconds above the micropore array chip using a corona machine to allow the small droplets to pair and fuse with the droplets in the large micropores.

[0078] Since reverse transcriptase is easily inactivated when left at room temperature, the experimental progress should be fully considered in practical applications, and the sample should be prepared quickly before use and placed on ice for later use. Small droplets are input into the inlet of the microwell array chip so that the small droplets enter the small microwells on the side of the large microwells in the microwell array chip, and fluorinated oil is added to remove excess small droplets. At this time, corona treatment is required again so that the small droplets can be paired and fused with the droplets added to the large microwells. The specific steps for adding small droplets are similar to the steps for adding large droplets mentioned above.

[0079] S111, after sealing the inlet and outlet of the microwell array chip, place the chip in a 40-45°C water bath to perform reverse transcription reaction for 3 hours.

[0080] After the droplets merge, the inlet and outlet of the microwell array chip are blocked with a tip, and fluorinated oil and water are added to seal the chip; then, the chip is placed in a 40-45°C water bath for 3 hours of reverse transcription reaction. Preferably, the chip can be placed in a 42°C water bath for 3 hours of reverse transcription reaction.

[0081] S112, connecting the inlet of the microporous array chip with a syringe, pushing the syringe with a constant current injection pump at a specific flow rate to push the water-soluble buffer solution into the microporous array chip to discharge the fluorinated oil, sucking air and the water-soluble buffer solution into the inlet of the microporous array chip again, applying a corona treatment for 2-4 seconds on the microporous array chip using a corona machine to induce droplet demulsification, and after standing for 12-18 minutes, collecting the water-soluble buffer solution containing cDNA through the outlet of the microporous array chip for sequencing analysis.

[0082] Furthermore, after the droplets complete the reverse transcription reaction in the microwell array chip, the chip is taken out and the water droplets on the surface of the chip are wiped dry to prevent contamination in subsequent experiments.

[0083] In a more specific embodiment, the inlet of the microwell array chip is connected to the syringe, and the syringe is pushed by a constant flow syringe pump to push the water-soluble buffer solution into the microwell array chip at a specific flow rate to discharge the fluorinated oil, comprising:

[0084] Use a 1ml syringe to draw 1ml of water-soluble buffer solution, and connect the outlet of the syringe to the inlet of the microporous array chip through a polyethylene tube; fix the syringe on a constant current injection pump, and push the solution into the microporous array chip at a flow rate of 1500μL / h through the constant current injection pump to discharge the fluorinated oil. Use a 1mL syringe to draw 1mL of water-soluble buffer solution, connect the outlet of the syringe to the inlet of the microporous array chip through a PE tube, and connect the outlet of the microporous array chip to the PE tube. Use a constant current injection pump to push the syringe to input the water-soluble buffer solution into the microporous array chip at a flow rate of 1500μL / h to discharge the oil in the microporous array chip. About 100μL of water-soluble buffer solution is required.

[0085] At this time, the droplets are still in the micropores, but due to the replacement of the continuous phase in the above-mentioned processing steps, the surface activity of the droplets has been reduced. After the oil is drained, replace the PE tube at the outlet of the micropore array chip, and pull out the PE tube at the entrance of the micropore array chip, and absorb about 50μL of air and 105μL of water-soluble buffer solution. Push the water-soluble buffer solution into the entrance of the micropore array chip again through the syringe. After the water-soluble buffer solution is input to the appropriate position of the chip, a corona treatment of about 3 seconds is applied above the micropore array chip using a corona machine to obtain an instantaneous AC electric field to temporarily change the stability of the droplet water-oil interface and induce the droplet to break emulsion. After the droplet breaks emulsion, the chip is left to incubate for 15min to ensure the free diffusion of cDNA. After the droplet breaks emulsion, all the water-soluble buffer solution is pushed out by the air in the syringe. At this time, the water-soluble buffer solution containing cDNA (about 100μL) can be collected, and sequencing analysis can be achieved after further purification and amplification.

[0086] Based on the aqueous buffer solution containing cDNA, purification, cDNA amplification, and library construction are performed. For example, the sequencing results obtained by sequencing using a kit of Dapu Bio and an Illumina sequencer are as follows: Fig. 9 shown.

[0087] In a more specific embodiment, the water-soluble buffer solution can be an HBW solution, wherein the HBW solution includes any one or more of a Tris hydrochloric acid buffer solution, an EDTA solution, and a TWT solution. In a further embodiment, the preparation method of the water-soluble buffer solution includes: taking out a 1M, pH 7.5 Tris hydrochloric acid buffer solution, a 0.5M, pH 8.0 EDTA solution, and a TWT solution and placing them at room temperature for 20 minutes; the TWT solution is a solution obtained by dissolving Tween 20 in a 1M, pH 7.5 Tris hydrochloric acid buffer solution; adding 1mL of the above EDTA solution, 50mL of the above TWT solution, and 50mL of the above Tris hydrochloric acid buffer solution to 5L ddH2O, and shaking to obtain the water-soluble buffer solution.

[0088] The present invention also provides a microwell array chip, which is used in the application method described in the above embodiment. Figure 4 As shown, the microwell array chip is obtained by stacking a top layer of glass-polydimethylsiloxane microwell chip 31, a fluid channel layer 32 and a bottom layer of glass slide 33.

[0089] The macro-micropore 311 is located on one side of the glass-polydimethylsiloxane microporous chip 31 facing the fluid channel layer 32, and the fluid channel layer 32 is sandwiched between the glass-polydimethylsiloxane microporous chip 31 and the bottom glass slide 33. The specific structure of the macro-micropore 311 is as follows: Figure 6 and Figure 7 As shown, the maximum depth h1 of the large micropore 311 is 80 microns, and the bottom width w1 is 90 microns, that is, the diameter d1 of the bottom of the large micropore 311 is 90 microns; a boss is provided on one side of the bottom of the large micropore 311, so that a shallower small micropore 312 is formed on one side of the large micropore 311, and the depth h1 of the small micropore 312 is 50 microns, and the side of the concave hole away from the large micropore 311 is an arc shape, and the diameter d2 of the arc shape is 50 microns; the lateral distance w2 between the center of the bottom of the large micropore 311 and the center of the arc side of the concave hole is 70 microns.

[0090] The present invention also provides a method for preparing a microwell array chip. Figure 8 As shown, the preparation method is used to prepare the microwell array chip as described in the above embodiment, and the preparation method includes steps S201 to S207.

[0091] S201, opening two through holes on a glass slide and filling the through holes with polydimethylsiloxane to obtain a glass substrate.

[0092] The glass-polydimethylsiloxane microporous chip is a glass substrate with PDMS (polydimethylsiloxane) microporous attached. The glass substrate with PDMS microporous attached needs to be pre-treated to obtain the droplet inlet and outlet.

[0093] In a more specific embodiment, step S201 specifically includes: opening two through holes on the glass slide and plasma treating one side for 1 minute, and sealing the through holes on the side that has not been plasma treated with tape; using a dropper to drop uncured polydimethylsiloxane at each through hole; evacuating the glass slide and leaving it at 60°C for more than 6 hours for curing; removing the tape and cutting off excess polydimethylsiloxane; and using anhydrous alcohol to scrub and clean the side of the glass slide that has not been plasma treated.

[0094] First, drill two through holes on a 25×75mm glass slide with a hole diameter of about 3mm. Clean the drilled glass slide and treat it with plasma for 1 minute. Use tape to seal the through holes on the side of the glass slide that has not been treated with plasma, and use a dropper to add about 10 microliters of uncured PDMS to fill the two holes. Evacuate the glass slide and then place it at 60°C for more than 6 hours to cure. After curing, remove the tape, use a scalpel to cut off the excess PDMS, and use anhydrous alcohol to clean the flat side of the slide (that is, the side that has not been treated with plasma).

[0095] S202, subjecting the silicon wafer mold to oxygen-plasma treatment in a vacuum chamber filled with silane vapor for more than 10 hours, pouring the polydimethylsiloxane prepolymer from which bubbles have been removed onto the silicon wafer mold for peeling off, obtaining a concave mold having a polydimethylsiloxane micropore array, and placing the mold in a vacuum chamber filled with silane vapor for more than 10 hours, and then flipping the mold again to obtain a convex mold having a polydimethylsiloxane micropore array.

[0096] More specifically, in order to form a microwell array on a glass substrate, a mold with strong elasticity is required. The convex mold of the microwell array chip is obtained by remolding the silicon wafer mold twice with PDMS. The silicon wafer can first be treated with oxygen-plasma and treated in a vacuum chamber filled with silane vapor for more than 10 hours, and then a 10:1 PDMS prepolymer is mixed, placed in a vacuum pump to remove bubbles, poured on the silicon wafer mold, and peeled off to obtain a concave mold of the PDMS microwell array. The obtained concave mold is remolded again to obtain a convex mold of the PDMS microwell array for subsequent use; then the convex mold of the PDMS microwell array is placed in a vacuum chamber filled with silane vapor for more than 10 hours.

[0097] S203, pouring polydimethylsiloxane prepolymer onto the convex mold with the micropore array and performing a vacuum treatment to remove bubbles.

[0098] Pour PDMS prepolymer on the microwell array convex mold and place it in a vacuum pump to remove bubbles before subsequent bonding with the surface-treated glass substrate.

[0099] S204, after subjecting the glass substrate to oxygen-plasma treatment, placing it on a convex mold with a microwell array for bonding, heating and curing it, and then peeling it off to obtain a polydimethylsiloxane microwell array-glass substrate composite.

[0100] The prepared glass substrate to be attached with the microwell array was placed in an oxygen-plasma treatment machine for plasma treatment for 5 minutes, and then baked at 110°C for 30 minutes. After heating and curing, the PDMS microwell array-glass substrate composite was peeled off, and the thickness of the PDMS layer was about 150μm. The excess PDMS was cut out with a scalpel, and then the outlet and inlet of the chip were punched.

[0101] S205, repeatedly spin coating polydimethylsiloxane on the silicon wafer to obtain a flat polydimethylsiloxane film, and cutting the polydimethylsiloxane film to obtain a fluid channel layer.

[0102] In a more specific embodiment, step S205 specifically includes: spin coating polydimethylsiloxane on the silicon wafer for 130-170 seconds at a spin coating speed of 500 rpm; heating the silicon wafer at 150°C-170°C for 4-7 minutes; repeating the above spin coating and heating process 7-10 times to obtain the flat polydimethylsiloxane film. Preferably, the spin coating can be repeated 9 times at a spin coating speed of 500 rpm for 150 seconds each time to obtain a flat PDMS film with a thickness of about 720 microns. After each spin coating, the silicon wafer should be placed on a 160°C heating plate and heated for 5 minutes. Finally, the PDMS film is cut into a fluid channel layer by die cutting.

[0103] S206, bonding the fluid channel layer to the bottom glass slide and then performing plasma treatment, bonding the polydimethylsiloxane microwell array-glass slide substrate composite to the upper layer of the fluid channel layer, and heating at 110° C. for 10 minutes to obtain a microwell array chip.

[0104] First, the fluid channel layer was bonded to the bottom glass slide to form a lower chip, and plasma treatment was performed for 5 minutes; then the glass-PDMS microporous chip was bonded to the lower chip, and the glass-PDMS microporous chip was bonded to the lower chip in a 110°C heating environment for 10 minutes and then taken out.

[0105] S207, injecting a hydrophobic reagent into the microporous array chip, and placing the microporous array chip in a vacuum environment; after 30 seconds, extracting the hydrophobic reagent through a syringe, and then drying the microporous array chip at 110° C. to remove the remaining hydrophobic reagent.

[0106] Inject the hydrophobic reagent into the microporous array chip, place the microporous array chip in a confined space and evacuate it, and extract the hydrophobic reagent in the chip through a syringe after 30 seconds to discharge the hydrophobic reagent; specifically, use a 1ml syringe connected to a tube, insert the front end of the tube into the outlet of the chip, and then extract the hydrophobic reagent through a syringe. Then place the chip in a 110°C drying oven for drying to remove the remaining residual hydrophobic reagent. The chip obtained by the above preparation method can be placed in a 65°C drying oven for long-term storage.

[0107] Since the droplets are in the glass PDMS chip, it will be a challenge to reverse transcribe the droplets. You can use a flat-bed PCR instrument or a water bath for reverse transcription. Since the chip has an inlet and outlet, it was found in the experiment that the tightness of the PCR instrument's hot cover was not appropriate, which could easily introduce bubbles into the chip, which is fatal to the experiment. Therefore, a water bath was chosen to perform reverse transcription of the droplets. It has been verified that the efficiency of reverse transcription in a water bath is the same as that performed directly in a PCR instrument, the number of predicted genes remains at the same level, and the cells are clustered well together. By setting different reverse transcription conditions for testing, a cell dimensionality reduction clustering diagram is obtained. The test results are shown below. Fig.10 and Fig.11 shown.

[0108] in, Fig.10 The x1 in the figure is the cell dimension reduction cluster diagram under standard thermal cycling conditions. Fig.10 x2 in the figure is the cell dimension reduction clustering diagram under the constant temperature condition of 42℃. Fig.10 The x3 in the figure is the dimensionality reduction clustering diagram of cells under the constant temperature condition of 42℃ water bath. Fig.11 for Fig.10 The image is obtained by superimposing the three cell dimension reduction clustering maps.

[0109] The conventional method for recovering the contents of droplets is to use a demulsifier to break the emulsion droplets, and then recover them by discarding the oil phase or absorbing the water phase. However, this method is not feasible for the practical application of about 2,500 droplets in this system. Because there are 2,500 droplets with a diameter of about 100 μm and the volume of the water phase is only 1 μL, it takes 4-5 mL of oil to recover these droplets, and this trace amount of water phase is very likely to be lost during the pipetting operation. Therefore, the present application proposes a solution for recovering the contents by replacing the continuous phase in the microporous array device with a water-soluble buffer solution, and then using a corona demulsification droplet method.

[0110] Because this method is innovative, a comprehensive test plan needs to be designed to verify its rationality. The specific experimental plan is as follows: ① Make a single-stranded DNA suspension of known concentration into droplets and load the droplets into a microwell array chip. Recover the DNA in the droplets by replacing the continuous phase of the chip and demulsifying with corona, and then verify the efficiency of recovering DNA by calculation. ② Use purified mRNA of known concentration instead of cells and co-encapsulate it with RT reagents and microspheres. The experimental group loaded the droplets into the microwell array chip for on-chip reverse transcription and cDNA recovery and subsequent purification; the control group performed reverse transcription in a PCR tube and demulsified with a demulsifier. Finally, the concentrations of the purified cDNA in the two groups were compared.

[0111] In experimental scheme ②, since the number of droplets in the microwell array chip is determined, and the number of droplets in the PCR tube is estimated by volume, there may be a deviation of about 5% in the number of droplets in the experimental group and the control group. However, as long as the magnitude of cDNA in the experimental results is close, it can be shown that the scheme of using HBW for cDNA recovery in this application is reasonable.

[0112] The results obtained through testing are as follows Fig.12 and Fig.13 As shown. Among them, Fig.12 The results of the test of cDNA recovery efficiency using ssDNA to simulate cDNA. The results show that the above method can recover the cDNA content in the droplets very well. (Original) The concentration of ssDNA suspension is converted into the ssDNA content of droplets of equal volume. (Tube) Directly encapsulate ssDNA to generate droplets, take 1.5μL (about 2500 droplets) and put them into a 200μL PCR tube, break the emulsion by adding a demulsifier, and add HBW to increase the volume of the aqueous phase. (Chip) Load 2500 droplets into a microwell array chip, and recover the ssDNA in the droplets by corona demulsification.

[0113] Fig.13 The test results of cDNA recovery efficiency were tested using mRNA instead of cells. The results show that the above method can recover cDNA contents in droplets very well. (Tube RT) Directly encapsulate mRNA to generate droplets, take 1.5μL (about 2500 droplets) and put them into a 200μL PCR tube, perform constant temperature reverse transcription in a PCR instrument, break the emulsion by adding a demulsifier, and add HBW to increase the volume of the aqueous phase. (Chip RT) Directly encapsulate mRNA to generate droplets, load 2500 droplets into a microwell array chip, perform reverse transcription in a water bath, and recover cDNA in the droplets by corona demulsification.

[0114] The above results all indicate that by replacing the continuous phase in the chip to perform droplet demulsification collection, the cDNA in the droplets can be well recovered.

[0115] The present application also separately tested the demulsification rate of the droplets when using corona treatment, and the test results are shown in Table 1. The results in Table 1 show that the droplet demulsification rate is high using the above technical method, and the effect of the droplet size can be ignored in the corona demulsification operation.

[0116] Table 1

[0117]

[0118] The single-cell sequencing performance of this system was verified by unsorted cell lines and PBMCs samples. Single-cell sequencing by cell lines to verify the system performance of the single-cell sequencing system is an essential, standard, and routine experiment in the performance verification of the single-cell sequencing system. This application uses 293T cells for system verification. Cell droplets were made from a single cell suspension of 4500 cells / μL, and were manually loaded into a microwell array chip for paired sequencing experiments, which were compared with single-cell sequencing by directly encapsulating the cells. The comparison results are shown in Table 2.

[0119] Table 2

[0120] Cell number Median number of genes per cell Positive Control 539 1439 Experimental Group 669 2242

[0121] The positive control is the sequencing results of more than 500 cell lines obtained using conventional methods; the experimental control group is the sequencing results of more than 600 cell lines obtained using the technical method of this application. The number of MedianGenes measured using this method is higher, indicating that the technical method in this application has better performance indicators for single-cell sequencing.

[0122] Since the platform needs to perform single-cell sequencing on clinically sorted PBMCs samples, in order to understand the system's ability to perform single-cell sequencing on PBMCs samples and eliminate operational problems with droplet sorting, it is necessary to perform single-cell sequencing on unsorted PBMCs samples to verify the system performance. The comparison results are shown in Table 3 and Fig.14 shown.

[0123] Table 3

[0124] Sample No. Cell number Median number of genes per cell Positive Control X-4-231026 1679 1023 Experimental Group X-5-231026 1105 1685

[0125] The positive control was the sequencing result of more than 1600 PBMC cells obtained using conventional methods; the experimental control group was the sequencing result of more than 1100 PBMC cells obtained using the technical method of this application. The comparison showed that Fig.14The fitting curve of the experimental group was significantly higher than that of the positive control group, proving that the number of Median Genes measured by this method is higher.

[0126] By testing the cell clustering information of cells captured by conventional methods and the technical method of this application, the comparison results are as follows Fig.15 As shown, Fig.15 The left side is a cell cluster diagram of the positive control, and the right side is a cell cluster diagram of the experimental group. By comparison, it can be seen that there is no difference in the cell information captured by the technical method used in this application and the conventional method.

[0127] The micropore array chip, preparation method and application method provided in the embodiment of the present invention include preparing large droplets and small droplets respectively, adding fluorinated oil to the inlet of the micropore array chip, adding large droplets to the inlet of the micropore array chip and tilting to capture large droplets, placing on an ice surface after draining excess droplets, sorting cell droplets by a droplet sorter and adding them to the large micropores of the micropore array chip so that the cell droplets and large droplets are paired one by one, heating after corona treatment and placing on an ice surface again, adding small droplets to the inlet of the micropore array chip and corona treatment again, sealing the micropore array chip and heating in a water bath, injecting a water-soluble buffer solution and corona treatment again to break the droplets, and collecting a solution containing cDNA for sequencing analysis. The above application method realizes single-cell sequencing of cell droplets by pairing and fusing large droplets with cell droplets and small droplets; optimizing the reagent system of single-cell sequencing by step-by-step sample addition, and improving the efficiency of single-cell sequencing by improving the mRNA capture efficiency of single cells.

[0128] The above is only a specific embodiment of the present invention, but the protection scope of the present invention is not limited thereto. Any technician familiar with the technical field can easily think of various equivalent modifications or replacements within the technical scope disclosed by the present invention, and these modifications or replacements should be included in the protection scope of the present invention. Therefore, the protection scope of the present invention shall be based on the protection scope of the claims.

Claims

1. An application method of a microwell array chip for single-cell sequencing, characterized in that: The application method comprises: Large droplets are prepared by a first droplet generation chip; Small droplets are prepared by a second droplet generation chip; Connecting funnel-shaped connectors to the inlet and outlet of the microwell array chip respectively, adding fluorinated oil to the inlet of the microwell array chip and placing it in a vacuum environment to exhaust the air in the chip; After adding the large droplets to the inlet of the microwell array chip, tilting the microwell array chip to allow the large droplets to drift into the large microwells in the microwell array chip by buoyancy; adding fluorinated oil again at the inlet of the microwell array chip to remove excess large droplets; The microwell array chip is tilted and placed on an ice surface, and the positive droplet output port of the droplet sorter is connected to the inlet port of the microwell array chip, so that the cell droplets are sorted by the droplet sorter and added to the small microwells on one side of the large microwells of the microwell array chip, so that the cell droplets are paired with the large droplets one by one; adding fluorinated oil again at the inlet of the microwell array chip to remove excess cell droplets; Using a corona machine, applying a corona treatment for 2-4 seconds on the microwell array chip to induce fusion of the large droplets and the cell droplets that are in contact with each other; The microwell array chip is placed at room temperature for 20-30 minutes to allow proteinase K to fully lyse cells and release cell mRNA, then placed in a water bath at 68-75° C. for 8-12 minutes to inactivate proteinase K, and then placed on ice; Small droplets are input through the inlet of the micropore array chip, so that they drift into the small micropores on the side of the large micropores in the micropore array chip through buoyancy to complete the one-to-one pairing between the small droplets and the droplets in the large micropores, and then excess small droplets are removed by adding fluorinated oil, and a corona treatment is applied for 2-4 seconds above the micropore array chip using a corona machine to allow the small droplets to pair and fuse with the droplets in the large micropores; After sealing the inlet and outlet of the microwell array chip, the chip was placed in a 40-45° C. water bath for 3 hours of reverse transcription reaction; The inlet of the microporous array chip is connected by a syringe, and a constant-current injection pump is used to push the syringe at a specific flow rate to push a water-soluble buffer solution into the microporous array chip to discharge the fluorinated oil. Air and the water-soluble buffer solution are sucked in and input into the inlet of the microporous array chip again. A corona treatment is applied for 2-4 seconds above the microporous array chip using a corona machine to induce droplet demulsification. After standing for 12-18 minutes, the water-soluble buffer solution containing cDNA is collected through the outlet of the microporous array chip for sequencing analysis.

2. The application method of the microwell array chip for single-cell sequencing according to claim 1, characterized in that: The first droplet generation chip comprises a first reverse transcription reaction reagent inlet, a microsphere phase inlet, a first oil phase inlet and a first outlet; The method of preparing large droplets by using a first droplet generation chip comprises: adjusting the flow rate of the microsphere phase inlet so that each droplet contains only one microsphere; Using a high-speed camera to acquire a droplet generation image, and adjusting the flow rate ratio between the first reverse transcription reaction reagent inlet and the first oil phase inlet and the microsphere phase inlet based on the diameter of the droplets in the droplet generation image, so that the microspheres are fused with the reverse transcription reaction reagent and then wrapped by the droplet generation oil to form large droplets; Place the container of large droplets on ice until ready to use.

3. The application method of the microwell array chip for single-cell sequencing according to claim 1, characterized in that: The second droplet generation chip comprises a second reverse transcription reaction reagent inlet, a second oil phase inlet and a second outlet; The step of preparing small droplets by using the second droplet generation chip comprises: Using a high-speed camera to acquire a droplet generation image, and adjusting the flow rate ratio between the second reverse transcription reaction reagent inlet and the second oil phase inlet based on the diameter of the droplets in the droplet generation image, so that the reverse transcription reaction reagent is wrapped by the droplet generation oil to form small droplets; Place the container containing the droplets on ice until ready to use.

4. The application method of the microwell array chip for single-cell sequencing according to claim 1, characterized in that: The water-soluble buffer solution includes any one or more of Tris hydrochloric acid buffer solution, EDTA solution and TWT solution.

5. The application method of the microwell array chip for single-cell sequencing according to claim 4, characterized in that: The preparation method of the water-soluble buffer solution comprises: Take out 1M, pH 7.5 Tris hydrochloric acid buffer solution, 0.5M, pH 8.0 EDTA solution and TWT solution and place them at room temperature for 20 minutes; the TWT solution is a solution obtained by dissolving Tween 20 in 1M, pH 7.5 Tris hydrochloric acid buffer solution; Add 1 mL of the EDTA solution, 50 mL of the TWT solution and 50 mL of the Tris-HCl buffer solution to 5 L of ddH2O, and shake well to obtain the water-soluble buffer solution.

6. The application method of the microwell array chip for single-cell sequencing according to claim 1, characterized in that: The method of connecting the inlet of the microwell array chip by a syringe and driving the syringe by a constant flow syringe pump to drive the water-soluble buffer solution into the microwell array chip at a specific flow rate to discharge the fluorinated oil comprises: Use a 1 ml syringe to draw 1 ml of water-soluble buffer solution, and connect the outlet of the syringe to the inlet of the microwell array chip through a polyethylene tube; The syringe was fixed on a constant flow injection pump, and the solution was pushed into the microwell array chip at a flow rate of 1500 μL / h by the constant flow injection pump to discharge the fluorinated oil.

7. A microwell array chip, characterized in that: The microwell array chip is used in the application method as described in any one of claims 1 to 6, and the microwell array chip is obtained by laminating a top layer of glass-polydimethylsiloxane microwell chip, a fluid channel layer and a bottom layer of glass slide.

8. A method for preparing a microwell array chip, characterized in that: The preparation method is used to prepare the microwell array chip as claimed in claim 7, and the preparation method comprises: Two through holes are opened on a glass slide and polydimethylsiloxane is filled in the through holes to obtain a glass substrate; The silicon wafer mold is subjected to oxygen-plasma treatment in a vacuum chamber filled with silane vapor for more than 10 hours, the polydimethylsiloxane prepolymer from which bubbles have been removed is poured onto the silicon wafer mold for peeling, and a concave mold having a polydimethylsiloxane micropore array is obtained, and the concave mold is placed in a vacuum chamber filled with silane vapor for more than 10 hours, and the mold is turned over again to obtain a convex mold having a polydimethylsiloxane micropore array; Pour polydimethylsiloxane prepolymer onto a convex mold with a microwell array and perform a vacuum treatment to remove air bubbles; The glass substrate is treated with oxygen-plasma, and then placed on a convex mold with a microwell array for bonding, and then heated and cured, and then peeled off to obtain a polydimethylsiloxane microwell array-glass substrate composite; Repeatedly spin coating polydimethylsiloxane on a silicon wafer to obtain a flat polydimethylsiloxane film, and cutting the polydimethylsiloxane film to obtain a fluid channel layer; The fluid channel layer and the bottom glass slide are bonded and then plasma treated, a polydimethylsiloxane microwell array-glass slide substrate composite is bonded to the upper layer of the fluid channel layer, and heated at 110° C. for 10 minutes to obtain a microwell array chip; A hydrophobic reagent was injected into the microporous array chip, and the microporous array chip was placed in a vacuum environment; after 30 seconds, the hydrophobic reagent was discharged by extraction through a syringe, and then the microporous array chip was placed at 110° C. for drying to remove the remaining hydrophobic reagent.

9. The method for preparing a microwell array chip according to claim 8, characterized in that: The method of opening two through holes on the glass slide and filling the through holes with polydimethylsiloxane comprises: Two through holes were opened on the glass slide and one side was subjected to plasma treatment for 1 minute, and a tape was affixed to the through holes on the side not subjected to plasma treatment for sealing; Use a dropper to drop uncured polydimethylsiloxane into each through hole; The slide was evacuated and placed at 60°C for more than 6 hours for curing; Remove the tape and cut away any excess dimethicone; The non-plasma treated side of the slide was cleaned using an absolute alcohol wipe.

10. The method for preparing a microwell array chip according to claim 8, characterized in that: The method of repeatedly spin coating polydimethylsiloxane on a silicon wafer to obtain a flat polydimethylsiloxane film comprises: Spin-coat polydimethylsiloxane on the silicon wafer for 130-170 seconds at a spin-coating speed of 500 rpm; Heat the silicon wafer at 150°C-170°C for 4-7 minutes; The above spin coating and heating treatment process is repeated 7-10 times to obtain the flat polydimethylsiloxane film.

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