Microporous array chips for single-cell sequencing, their fabrication methods and applications.

Abstract

This invention discloses a microporous array chip for single-cell sequencing, its preparation method, and its application method. The application method includes preparing large and small droplets separately; adding fluorinated oil to the inlet of the microporous array chip; adding the large droplets to the inlet and tilting the chip to capture them; removing excess droplets and placing the chip on ice; using a droplet sorter to sort cell droplets and adding them to the small wells on one side of the large well of the microporous array chip to ensure one-to-one pairing between cell droplets and large droplets; performing corona treatment, heating, and placing the chip on ice again; adding small droplets to the inlet of the microporous array chip and corona treatment again; sealing the microporous array chip and heating it in a water bath; injecting a water-soluble buffer solution and corona treatment again to demulsify the droplets; and collecting the solution containing cDNA. This method achieves single-cell sequencing by pairing and fusing large droplets with cell droplets and small droplets, improving the capture capacity of single-cell mRNA and thus increasing the efficiency of single-cell sequencing.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, and in particular to a microporous array chip for single-cell sequencing, its preparation method, and its application method. Background Technology

[0002] Single-cell sequencing is a revolutionary method in biomedical research in recent years, enabling high-throughput sequencing of the genome, transcriptome, or epigenome at the single-cell level. Traditional population sequencing techniques typically measure the average signal of thousands to millions of cells, which masks the heterogeneity between individual cells. Single-cell sequencing, on the other hand, can reveal gene expression and genomic characteristics of individual cells, providing more detailed resolution and a deeper understanding of biology.

[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 characteristics. For example, scRNA-seq, by measuring the mRNA content of a single cell, reveals its gene expression profile and has been widely used in studying cellular heterogeneity, developmental processes, and disease mechanisms.

[0004] Single-cell sequencing technology has demonstrated its powerful 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 tumor heterogeneity and evolutionary processes. In immunology research, through single-cell analysis, scientists can gain detailed insights into the gene expression profiles of different immune cell types and their roles in immune responses, exploring the dynamic changes of the immune system in healthy and disease states. Furthermore, single-cell sequencing is widely used in neuroscience research to study gene expression in neurons and glial cells, revealing the complexity and developmental mechanisms of the nervous system.

[0005] Single-cell RNA sequencing has provided scientists with the ability to gain in-depth understanding of gene expression in individual cells and has been widely used in fields such as oncology, neuroscience, immunology, drug discovery, and infectious diseases.

[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 sorting or laser capture microdissection to obtain single cells, followed by low-throughput single-cell sequencing technology). Currently, there are methods based on droplet co-encapsulation (used by 10x Genomics) and gravity-based capture of single cells into micropores (used by BD) for single-cell isolation and high-throughput single-cell sequencing. The main drawback of droplet co-encapsulation is cell loss; for a target of 5000 cells, an excessive amount of 8280 cells needs to be extracted. In certain clinical needs, such as rare diseases or small tumors, only a small number of cells can usually be obtained, so current sequencing methods are unacceptable for rare cell samples. These samples require efficient low-to-medium throughput sequencing methods, while existing high-throughput technologies are not suitable for this situation.

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

[0008] This invention provides a microporous array chip, its preparation method, and its application method for single-cell sequencing, aiming to solve the problem that existing single-cell sequencing methods cannot efficiently capture single cells to achieve high-throughput sequencing.

[0009] In a first aspect, embodiments of the present invention provide a method for applying a microporous array chip for single-cell sequencing, wherein the application method includes:

[0010] Large droplets were prepared using a first droplet generation chip;

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

[0012] Funnel-shaped connectors are connected to the inlet and outlet of the microporous array chip, respectively. Fluorinated oil is added to the inlet of the microporous array chip and it is placed in a vacuum environment to purge the air inside the chip.

[0013] After adding the large droplet to the inlet of the micropore array chip, the micropore array chip is tilted so that the large droplet floats into the large micropores in the micropore array chip by buoyancy.

[0014] Fluorinated oil is added again to the inlet of the micropore array chip to remove excess large droplets;

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

[0016] Fluorinated oil is added again to the inlet of the microporous array chip to remove excess cell droplets;

[0017] A corona treatment of 2-4 seconds was applied over the microporous array chip using a corona machine to induce the fusion of large droplets and cell droplets that were in contact with each other.

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

[0019] Small droplets are introduced through the inlet of the microporous array chip, allowing them to drift by buoyancy into the small micropores on the side of the large micropores in the microporous array chip, thus completing the one-to-one pairing between the small droplets and the droplets in the large micropores. Then, excess small droplets are removed by adding fluorinated oil, and a corona treatment machine is used to apply corona treatment to the microporous array chip for 2-4 seconds to make the small droplets pair and fuse with the droplets in the large micropores.

[0020] After sealing the inlet and outlet of the microporous array chip, the chip was placed in a water bath at 40-45℃ for 3 hours for reverse transcription.

[0021] Connect a syringe to the inlet of the microporous array chip. Use a constant flow syringe pump to push the syringe at a specific flow rate to push the water-soluble buffer solution into the microporous array chip to expel the fluorinated oil. Absorb air and water-soluble buffer solution and reintroduce them into the inlet of the microporous array chip. Use a corona machine to apply corona treatment to the microporous array chip for 2-4 seconds to induce droplet demulsification. After standing for 12-18 minutes, collect the water-soluble buffer solution containing cDNA through the outlet of the microporous array chip for sequencing analysis.

[0022] In a second aspect, embodiments of the present invention also provide a microporous array chip, wherein the microporous array chip is applied in the application method described in the first aspect above, and the microporous array chip is obtained by stacking a top glass-polydimethylsiloxane microporous chip, a fluid channel layer and a bottom glass slide.

[0023] Thirdly, embodiments of the present invention also provide a method for fabricating a microporous array chip, wherein the method is used to fabricate the microporous array chip as described in the second aspect, the method comprising:

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

[0025] The silicon wafer mold is subjected to oxygen-plasma treatment and treated in a vacuum chamber filled with silane vapor for more than 10 hours. The polydimethylsiloxane prepolymer with the bubbles removed is poured onto the silicon wafer mold and peeled off to obtain a concave mold with a polydimethylsiloxane micropore array. The concave mold is then placed in a vacuum chamber filled with silane vapor for more than 10 hours and then molded again to obtain a convex mold with a polydimethylsiloxane micropore array.

[0026] Polydimethylsiloxane prepolymer is poured into a convex mold with a microporous array and vacuumed to remove air bubbles;

[0027] After treating the glass substrate with oxygen-plasma, it is placed on a convex mold with a microporous array for bonding. After heating and curing, it is peeled off to obtain a polydimethylsiloxane microporous array-glass substrate composite.

[0028] Polydimethylsiloxane is repeatedly spin-coated onto a silicon wafer to obtain a flat polydimethylsiloxane film, and the polydimethylsiloxane film is cut to obtain a fluid channel layer;

[0029] After bonding the fluid channel layer to the bottom glass slide, plasma treatment is performed to bond the polydimethylsiloxane microporous array-glass slide substrate composite to the upper layer of the fluid channel layer. The microporous array chip is obtained by heating at 110°C for 10 minutes.

[0030] A hydrophobic reagent is injected into the microporous array chip, and the microporous array chip is placed in a vacuum environment. After 30 seconds, the hydrophobic reagent is extracted using a syringe and then the microporous array chip is dried at 110°C to remove the remaining hydrophobic reagent.

[0031] This invention provides a microporous array chip, its preparation method, and its application method. The application method includes preparing large and small droplets separately; adding fluorinated oil to the inlet of the microporous array chip; adding the large droplets to the inlet and tilting the chip to capture them; removing excess droplets and placing it on ice; sorting cell droplets using a droplet sorter and adding them to the large micropores of the microporous array chip to pair them one-to-one; performing corona treatment, heating, and placing it back on ice; adding small droplets to the inlet of the microporous array chip and corona treatment again; sealing the microporous array chip and heating it in a water bath; injecting a water-soluble buffer solution and corona treatment again to demulsify the droplets; and collecting the solution containing cDNA for sequencing analysis. This application method achieves single-cell sequencing by pairing and fusing large droplets with cell droplets and small droplets; it optimizes the reagent system for single-cell sequencing through stepwise sample addition and improves the efficiency of single-cell sequencing by increasing the mRNA capture efficiency of single cells. Attached Figure Description

[0032] To more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings used in the following description of the embodiments will be briefly introduced. Obviously, the drawings described below are some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0033] Figure 1 A flowchart illustrating the application method of a micro-hole array chip provided in an embodiment of the present invention;

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

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

[0036] Figure 4 This is an overall structural diagram of the micro-hole array chip provided in an embodiment of the present invention;

[0037] Figure 5 This is a partial structural diagram of the micro-hole array chip provided in an embodiment of the present invention;

[0038] Figure 6 This is another partial structural diagram of the micro-hole array chip provided in an embodiment of the present invention;

[0039] Figure 7 Another partial structural diagram of the micro-hole array chip provided in the embodiment of the present invention.

[0040] Figure 8A flowchart illustrating the method for fabricating a microporous array chip according to an embodiment of the present invention;

[0041] Figure 9 This is a schematic diagram illustrating the application effect of the micro-hole array chip provided in the embodiments of the present invention;

[0042] Figure 10 This is a schematic diagram illustrating another application effect of the micro-hole array chip provided in this embodiment of the invention;

[0043] Figure 11 This is a schematic diagram illustrating another application effect of the micro-hole array chip provided in this embodiment of the invention;

[0044] Figure 12 This is a schematic diagram illustrating another application effect of the micro-hole array chip provided in the embodiment of the present invention;

[0045] Figure 13 This is a schematic diagram illustrating the subsequent application effect of the micro-hole array chip provided in the embodiment of the present invention;

[0046] Figure 14 This is a schematic diagram illustrating another application effect of the micro-hole array chip provided in this embodiment of the invention;

[0047] Figure 15 This is a schematic diagram illustrating another application effect of the micro-hole array chip provided in the embodiments of the present invention.

[0048] Reference numerals: 10, First droplet generation chip; 20, Second droplet generation chip; 30, Microporous 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 microporous chip; 32, Fluid channel layer; 33, Bottom glass slide; 311, Large micropore; 312, Small micropore. Detailed Implementation

[0049] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some, not all, of the embodiments of the present invention. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort 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 "comprising" and "including" indicate the presence of the described features, integrals, steps, operations, elements and / or components, but do not exclude the presence or addition of one or more other features, integrals, steps, operations, elements, components and / or collections thereof.

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

[0052] It should also be further understood that the term "and / or" as used in this specification and the appended claims refers to any combination of one or more of the associated listed items and all possible combinations, and includes such combinations.

[0053] Please see Figure 1 As shown in the figure, this application discloses an application method for a micro-hole array chip, which includes steps S101 to S112.

[0054] S101. A large droplet is prepared by the first droplet generation chip.

[0055] like Figure 2 As shown, the first droplet generation chip includes a first reverse transcription reagent inlet, a microsphere phase inlet, a first oil phase inlet, and a first outlet. The process of preparing large droplets using the first droplet generation chip includes: adjusting the flow rate of the microsphere phase inlet so that each droplet contains only one microsphere; acquiring droplet generation images using a high-speed camera, and adjusting the flow rate ratio between the first reverse transcription reagent inlet and the first oil phase inlet and the microsphere phase inlet based on the droplet diameter in the droplet generation images, so that the microspheres and reverse transcription reagent are fused and then encapsulated by the droplet generation oil to form large droplets; and placing the container holding the large droplets on ice for later use.

[0056] Specifically, to prepare large droplets, a first droplet generation chip can be designed. The microsphere phase inlet and the first outlet of the first droplet generation chip are connected by a pipe. A needle-like pipe, varying in diameter from thick to thin, is provided between the microsphere phase inlet and the confluence of the connecting pipes, and a needle-like pipe, also varying in diameter from thick to thin, is provided between the first outlet and the confluence. Two serpentine winding pipes, connecting to the first reverse transcription (RT) reaction reagent inlet, are located on both sides of the connecting pipe at the confluence. Two more serpentine winding pipes, connecting to the first oil phase inlet, are also located on both sides of the connecting pipe at the confluence. Specifically, large droplets are generated using a 70μm high large droplet generation chip, which consists of three inlets (RT phase, microsphere phase, and oil phase) and an outlet. The first reverse transcription reaction reagents input through the first reverse transcription reaction reagent inlet include reverse transcription buffer (RT buffer), proteinase K, DTT (dithiothreitol), and RNA inhibitors.

[0057] The droplet-generating oil is loaded into a 1ml syringe, and the syringe outlet is connected to the oil phase inlet of the first droplet-generating chip using a polyethylene tube (PE tube, outer diameter 1.22mm). Due to the small volume of the RT reaction reagent and microsphere reagent, it is impossible to load the encapsulated aqueous phase into the syringe for precise flow control. To solve this problem, the microsphere reagent is injected into a polyethylene tube and then connected to a polyethylene tube filled with fluorinated oil via a steel needle. The inlet of the polyethylene tube filled with fluorinated oil is connected to the outlet of the syringe filled with fluorinated oil, and the outlet of the polyethylene tube filled with microsphere reagent is connected to the microsphere phase inlet of the first droplet-generating chip. The syringe and the tail section of the PE tube are pre-filled with fluorinated oil. The aqueous phase to be encapsulated is drawn into another section of the PE tube using a pipette, and then the two ends of the PE tube are connected via a steel needle, allowing for precise flow control of the microsphere reagent and RT reaction reagent. The first outlet is connected to the PCR tube via a PE tube.

[0058] To ensure the quality of single-cell sequencing, the entry velocity of the microsphere phase needs to be observed during large droplet fabrication, and the flow rates of the microsphere reagent and RT reaction reagent need to be adjusted in real time to ensure that each large droplet contains only one microsphere. The entire process of large droplet formation was observed using a Nikon microscope paired with a high-speed camera. Based on this observation, the flow rate ratio between the first reverse transcription reaction reagent inlet and the first oil phase inlet and the microsphere phase inlet was optimized to control the diameter of the generated large droplets. Multiple bright-field images of the large droplets were randomly selected, and the droplet diameter was measured using ImageJ. The largest number of large droplets with the highest size and microsphere encapsulation rate can be collected for subsequent experiments by changing the PCR tubes. The collected large droplets should be placed on ice for later use.

[0059] S102. Small droplets are prepared by the second droplet generation chip.

[0060] In specific embodiments, such as Figure 3 As shown, the second droplet generation chip includes a second reverse transcription reagent inlet, a second oil phase inlet, and a second outlet. The preparation of small droplets using the second droplet generation chip includes: acquiring droplet generation images using a high-speed camera, and adjusting the flow rate ratio between the second reverse transcription reagent inlet and the second oil phase inlet based on the droplet diameter in the droplet generation images, so that the reverse transcription reagent is coated by the droplet-generating oil to form small droplets; placing the container holding the small droplets on ice for later use. The second reverse transcription reagent input through the second reverse transcription reagent inlet includes reagents such as reverse transcriptase, template switching oligonucleotides, and RNA inhibitors.

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

[0062] S103. Connect funnel-shaped connectors to the inlet and outlet of the micro-hole array chip respectively. Add fluorinated oil to the inlet of the micro-hole array chip and place it in a vacuum environment to purge the air inside the chip.

[0063] The droplet capture and fusion process is completed in a microporous array chip (microporous array device), and the overall structure of the microporous 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 capturing droplets, large droplets are captured first. The specific procedure is as follows: First, two funnel-shaped connectors (10μL tip tips) are inserted into the inlet and outlet of the microporous array chip, respectively. Using a pipette, 200μL of fluorinated oil is added through the inlet of the microporous array chip to wet it. The microporous array chip with the added fluorinated oil is then placed in a sealed environment and a vacuum is applied, that is, the microporous array chip is placed in a vacuum environment to remove the air inside the chip.

[0064] S104. After adding the large droplet to the inlet of the micropore array chip, tilt the micropore array chip so that the large droplet drifts into the large micropores in the micropore array chip by buoyancy.

[0065] Furthermore, a 2.5 μL droplet is added to the inlet of the microporous array chip using a pipette. The chip is tilted, and due to the buoyancy caused by the density difference between the oil and water phases, the large droplet rapidly diffuses towards the outlet and drifts and moves with the liquid flow. Under the influence of buoyancy, the large droplet drifts into the corresponding micropore. The specific structure of the micropore is as follows: Figure 6 As shown. The maximum depth h1 of the large micropore is 80 micrometers, the bottom width w1 is 90 micrometers, and a boss is provided on one side of the bottom of the large micropore, thus forming a shallow concave hole on one side of the large micropore.

[0066] S105. Fluorinated oil is added again to the inlet of the micropore array chip to remove excess large droplets.

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

[0068] S106. Tilt the micropore array chip and place it on the ice surface. Connect the positive droplet output port of the droplet sorter to the inlet of the micropore array chip so that the cell droplets are sorted by the droplet sorter and added to the small micropores on the side of the large micropores of the micropore array chip so that the cell droplets are paired with the large droplets one by one.

[0069] Further, cell droplet capture is performed. The microporous array chip can be placed at a 30-degree angle on ice. Due to the high sorting speed, the inlet of the microporous array chip needs to be higher than its outlet. Buoyancy slows down the flow rate slightly, ensuring that the sorted cell droplets remain within the microporous array chip. After the droplet sorter starts working, connect the positive droplet output port of the droplet sorter to the inlet of the microporous array chip. The droplet sorter then sorts the cell droplets and adds them to the smaller pores on the side of the larger pores of the microporous array chip, minimizing the introduction of air bubbles. The outlet of the microporous array chip is stabilized using a PE tube.

[0070] Furthermore, to ensure better sorting results and a higher single-packet rate, based on the Poisson distribution formula, a cell suspension concentration of 4500 cells / μL was selected for cell droplet preparation before the cell droplet capture operation. After cell droplet preparation and before sorting, the droplets need to be placed on ice for later use. Droplet sorting was performed using the Dap BioComet high-throughput screening system.

[0071] S107. Fluorinated oil is added again to the inlet of the microporous array chip to remove excess cell droplets.

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

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

[0074] Furthermore, after droplet capture and pairing are completed, droplet fusion can be achieved through corona treatment. A corona treatment of 2-4 seconds (about 3 seconds) is applied above the microporous array chip using a corona machine to obtain a transient alternating electric field to temporarily change the stability of the droplet water-oil interface, inducing the fusion of large droplets and cellular droplets that are in contact with each other.

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

[0076] After droplet fusion, the microarray chip is placed at room temperature for 20-30 minutes to ensure cell lysis, allowing proteinase K to fully lyse cells and release cellular mRNA; specifically, the microarray 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 is placed in a water bath at 72°C for 10 minutes for proteinase K inactivation. After inactivation, the chip is immediately placed on ice.

[0077] S110. Small droplets are input through the inlet of the microporous array chip, allowing them to drift into the small micropores on the side of the large micropores in the microporous array chip by buoyancy, thus completing the one-to-one pairing between the small droplets and the droplets in the large micropores. Then, excess small droplets are removed by adding fluorinated oil. A corona treatment is applied above the microporous array chip for 2-4 seconds using a corona machine to make the small droplets pair and fuse with the droplets in the large micropores.

[0078] Since reverse transcriptase is easily inactivated at room temperature, in practical applications, the experimental schedule should be fully considered, and it should be prepared quickly before use and kept on ice for later use. Small droplets are introduced into the inlet of the microporous array chip, allowing them to enter the small wells on the side of the large wells. Fluorinated oil is added to remove excess droplets. A second corona treatment is then required to allow the small droplets to pair and fuse with the droplets already added to the large wells. The specific steps for adding small droplets are similar to those for adding large droplets.

[0079] S111. After sealing the inlet and outlet of the microporous array chip, place the chip in a water bath at 40-45℃ for 3 hours for reverse transcription.

[0080] After droplet fusion, the inlet and outlet of the microporous array chip are sealed with a tip, and fluorinated oil and water are added to seal the chip. Then, the chip is placed in a water bath at 40-45℃ for 3 hours for reverse transcription reaction. Preferably, the chip can be placed in a water bath at 42℃ for 3 hours for reverse transcription reaction.

[0081] S112. Connect the syringe to the inlet of the microporous array chip, and use a constant flow syringe pump to push the syringe at a specific flow rate to push the water-soluble buffer solution into the microporous array chip to expel the fluorinated oil. Absorb air and water-soluble buffer solution and reintroduce them into the inlet of the microporous array chip. Use a corona machine to apply corona treatment above the microporous array chip for 2-4 seconds to induce droplet demulsification. After standing for 12-18 minutes, collect 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 microporous array chip, the chip is removed and the water droplets on the chip surface are wiped dry to prevent contamination in subsequent experiments.

[0083] In a more specific embodiment, the step of connecting the syringe to the inlet of the microporous array chip and using a constant flow injection pump to push the syringe at a specific flow rate to push the water-soluble buffer solution into the microporous array chip to expel the fluorinated oil includes:

[0084] Using a 1ml syringe, draw 1ml of water-soluble buffer solution and connect the syringe outlet to the inlet of the microporous array chip via a polyethylene tube. Secure the syringe to a constant-flow syringe pump, which then propels the solution into the microporous array chip at a flow rate of 1500μL / h to expel the fluorinated oil. Alternatively, use a 1ml syringe to draw 1ml of water-soluble buffer solution and connect the syringe outlet to the inlet of the microporous array chip via a PE tube. The outlet of the microporous array chip is connected to the PE tube. Propel the solution into the microporous array chip at a flow rate of 1500μL / h using the constant-flow syringe pump, expelling the oil from the microporous array chip. Approximately 100μL of water-soluble buffer solution is required.

[0085] At this point, the droplet is still in the microwell, but due to the replacement of the continuous phase in the above steps, the surfactant on the droplet surface has decreased. After the oil is completely drained, replace the PE tube at the outlet of the microwell array chip and remove the PE tube at the inlet. Aspirate approximately 50 μL of air and 105 μL of water-soluble buffer solution. Again, use a syringe to push the water-soluble buffer solution into the inlet of the microwell array chip. After the water-soluble buffer solution is introduced to the appropriate position on the chip, apply a corona treatment for approximately 3 seconds above the microwell array chip using a corona generator. This instantaneous alternating electric field briefly alters the stability of the droplet's water-oil interface, inducing droplet demulsification. After demulsification, incubate the chip for 15 minutes to ensure free diffusion of cDNA. After demulsification, expel all the water-soluble buffer solution using air from the syringe. At this point, approximately 100 μL of water-soluble buffer solution containing cDNA can be collected. Further purification and amplification processing is then performed for sequencing analysis.

[0086] Based on the aforementioned water-soluble buffer solution containing cDNA, purification, cDNA amplification, and library construction were performed. For example, sequencing results obtained using a kit from DaP Biotech and sequencing on an Illumina sequencer are shown below. Figure 9 As shown.

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

[0088] This invention also provides a micro-hole array chip, which is applied in the application methods described in the above embodiments, such as... Figure 4 As shown, the microporous array chip is obtained by stacking a top glass-polydimethylsiloxane microporous chip 31, a fluid channel layer 32, and a bottom glass sheet 33.

[0089] The large micropore 311 is located on the side of the glass-polydimethylsiloxane microporous chip 31 facing the fluid channel layer 32, which is sandwiched between the glass-polydimethylsiloxane microporous chip 31 and the bottom glass sheet 33. The specific structure of the large micropore 311 is as follows: Figure 6 and Figure 7 As shown. The maximum depth h1 of the large micro-orifice 311 is 80 micrometers, and the bottom width w1 is 90 micrometers, meaning the diameter d1 of the bottom of the large micro-orifice 311 is 90 micrometers. A protrusion is provided on one side of the bottom of the large micro-orifice 311, thus forming a shallower micro-orifice 312 on one side of the large micro-orifice 311. The depth h1 of the micro-orifice 312 is 50 micrometers. The side of the concave hole away from the large micro-orifice 311 is arc-shaped, and the diameter d2 of this arc-shaped shape is 50 micrometers. The lateral distance w2 between the center of the bottom of the large micro-orifice 311 and the center of the arc-shaped side of the concave hole is 70 micrometers.

[0090] This invention also provides a method for fabricating a microporous array chip, such as... Figure 8 As shown, this preparation method is used to prepare the micro-hole array chip as described in the above embodiments, and the preparation method includes steps S201 to S207.

[0091] S201. Two through holes are made on the glass slide and polydimethylsiloxane is filled into the through holes to obtain a glass substrate.

[0092] Glass-polydimethylsiloxane microporous chips are glass substrates with PDMS (polydimethylsiloxane) micropores attached. The glass substrates with PDMS micropores need to be pretreated to obtain droplet inlets and outlets.

[0093] In a more specific embodiment, step S201 specifically includes: making two through holes on the glass slide and subjecting one side to plasma treatment for 1 minute; sealing the through holes on the side not subjected to plasma treatment with adhesive tape; applying uncured polydimethylsiloxane to each through hole using a dropper; evacuating the glass slide and placing it at 60°C for at least 6 hours to cure; removing the tape and cutting off any excess polydimethylsiloxane; and cleaning the side of the glass slide not subjected to plasma treatment with anhydrous alcohol.

[0094] First, drill two through holes, approximately 3mm in diameter, into a 25×75mm glass slide. Clean the slide after drilling and then subject it to plasma treatment for 1 minute. Seal the through holes on the untreated side of the slide with adhesive tape. Use a dropper to add approximately 10 microliters of uncured PDMS to the two drilled holes to fill them. Vacuum the slide and then place it at 60°C for at least 6 hours to cure. After curing, remove the tape, use a scalpel to remove excess PDMS, and clean the flat side of the slide (the untreated side) with anhydrous alcohol.

[0095] S202. 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 with the bubbles removed is poured onto the silicon wafer mold and peeled off to obtain a concave mold with a polydimethylsiloxane microporous array. The concave mold is then placed in a vacuum chamber filled with silane vapor for more than 10 hours and then molded again to obtain a convex mold with a polydimethylsiloxane microporous array.

[0096] More specifically, a highly flexible mold is needed to form a microporous array on a glass substrate. The convex mold for the microporous array chip is obtained by two PDMS molding processes using a silicon wafer mold. First, the silicon wafer is treated with oxygen-plasma and then treated in a vacuum chamber filled with silane vapor for more than 10 hours. Then, a 10:1 PDMS prepolymer is mixed, placed in a vacuum pump to remove air bubbles, poured onto the silicon wafer mold, and peeled off to obtain a concave mold for the PDMS microporous array. The concave mold is then molded again to obtain a convex mold for the PDMS microporous array for subsequent use. Subsequently, the convex mold for the PDMS microporous array is again treated in a vacuum chamber filled with silane vapor for more than 10 hours.

[0097] S203. Pour polydimethylsiloxane prepolymer into a convex mold with a microporous array and perform vacuum treatment to remove air bubbles.

[0098] Pour PDMS prepolymer onto a microporous array convex mold and place it in a vacuum pump to remove air bubbles, ready for subsequent bonding with a surface-treated glass substrate.

[0099] S204. After treating the glass substrate with oxygen-plasma, it is placed on a convex mold with a microporous array for bonding. After heating and curing, it is peeled off to obtain the polydimethylsiloxane microporous array-glass substrate composite.

[0100] The prepared glass substrate to which the micropore array is to be attached was placed in an oxygen-plasma treatment machine for 5 minutes of plasma treatment, followed by baking at 110°C for 30 minutes. After heat curing, the PDMS micropore array-glass substrate composite was obtained by peeling off, with a PDMS layer thickness of approximately 150 μm. Excess PDMS was cut off with a scalpel, and then holes were drilled at the chip's inlet and outlet.

[0101] S205. Repeated spin-coating of polydimethylsiloxane on a silicon wafer to obtain a smooth 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 onto a 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 smooth polydimethylsiloxane film. Preferably, spin-coating can be performed for 150 seconds each time at a spin-coating speed of 500 rpm, repeated 9 times to obtain a smooth PDMS film with a thickness of approximately 720 micrometers. After each spin-coating, the silicon wafer should be placed on a 160°C heating plate and heated for 5 minutes. Finally, the fluid channel layer is cut out of the PDMS film using a die-cutting tool.

[0103] S206. After bonding the fluid channel layer to the bottom glass slide, plasma treatment is performed to bond the polydimethylsiloxane microporous array-glass slide substrate composite to the upper layer of the fluid channel layer. The mixture is then heated at 110°C for 10 minutes to obtain a microporous array chip.

[0104] First, the fluid channel layer is bonded to the bottom glass slide to form the lower chip, and then subjected to plasma treatment for 5 minutes. Next, the glass-PDMS microporous chip is bonded to the lower chip, and the glass-PDMS microporous chip is heated at 110℃ for 10 minutes before being removed.

[0105] S207. Inject the hydrophobic reagent into the microporous array chip and place the microporous array chip in a vacuum environment; after 30 seconds, extract the hydrophobic reagent using a syringe, and then place the microporous array chip at 110°C to dry and remove the remaining hydrophobic reagent.

[0106] A hydrophobic reagent is injected into the microporous array chip. The microporous array chip is then placed in a sealed space and evacuated. After 30 seconds, the hydrophobic reagent is extracted from the chip using a syringe, thereby removing the hydrophobic reagent. Specifically, a 1ml syringe connected to a tube is used, with the tip of the tube inserted into the chip's outlet. The hydrophobic reagent is then extracted using the syringe. The chip is then placed in a 110°C drying oven to remove any remaining residual hydrophobic reagent. The chip obtained using the above preparation method can be stored long-term in a 65°C drying oven.

[0107] Reverse transcription of droplets within a glass PDMS chip presents a challenge. A plate PCR instrument or a water bath can be used. However, due to the chip's inlet and outlet, it was found that improper tightness of the PCR instrument's heating cap could introduce air bubbles, which is detrimental to the experiment. Therefore, a water bath was chosen for droplet reverse transcription. Verification showed that the reverse transcription efficiency in the water bath was the same as that performed directly in the PCR instrument, with the predicted gene count remaining at the same level and cells clustering well. Different reverse transcription conditions were used to obtain dimensionality-reduced cell clustering maps, and the results are shown below. Figure 10 and Figure 11 As shown.

[0108] in, Figure 10 In the diagram, x1 represents the dimensionality-reduced clustering of cells under standard thermal cycling conditions. Figure 10 x2 in the diagram represents the dimensionality reduction clustering of cells under a constant temperature of 42℃. Figure 10 x3 in the diagram represents the dimensionality reduction clustering of cells under constant temperature conditions of a 42℃ water bath. Figure 11 for Figure 10 The image is obtained by overlaying and combining the dimensionality reduction clustering diagrams of three cells.

[0109] Conventional methods for recovering droplet contents involve using a demulsifier to break up the droplets, followed by discarding the oil phase or removing the aqueous phase. However, this method is impractical for the practical application of approximately 2500 droplets in this system. Since 2500 droplets with a diameter of approximately 100 μm would have an aqueous phase volume of only 1 μL, recovering these droplets would require 4-5 mL of oil, and this trace amount of aqueous phase is highly likely to be lost during pipetting. Therefore, this application proposes a method to recover the contents by replacing the continuous phase in the microporous array device with a water-soluble buffer solution, followed by demulsification of the droplets using corona discharge.

[0110] Due to the innovative nature of this method, a comprehensive testing protocol is needed to verify its feasibility. The specific experimental protocol is as follows: ① A single-stranded DNA suspension of known concentration was prepared into droplets and loaded into a microarray chip. DNA within the droplets was recovered by changing the continuous phase of the chip and using corona demulsification. The efficiency of DNA recovery was then verified through calculation. ② Purified mRNA of known concentration was used to replace cells and co-encapsulated with RT reagents and microspheres. In the experimental group, droplets were loaded into the microarray chip for in-chip reverse transcription and cDNA recovery and subsequent purification; in the control group, reverse transcription was performed in PCR tubes, followed by demulsification with a demulsifier. Finally, the concentrations of purified cDNA in the two groups were compared.

[0111] In experimental scheme ②, since the number of droplets in the microwell array chip is fixed, while 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 between the experimental group and the control group. However, as long as the amount of cDNA in the experimental results is close in magnitude, it can be shown that the scheme for cDNA recovery using HBW in this application is reasonable.

[0112] The results obtained from the test are as follows: Figure 12 and Figure 13 As shown. Among them, Figure 12 The test results demonstrate the cDNA recovery efficiency achieved by using ssDNA to simulate cDNA. These results indicate that the above method effectively recovers the cDNA contents within droplets. (Original) The ssDNA suspension concentration was converted to the ssDNA content of an equal volume of droplets. (Tube) Droplets were generated by directly encapsulating ssDNA. 1.5 μL (approximately 2500 droplets) was placed in a 200 μL PCR tube, and demulsification was performed by adding a demulsifier. HBW was added to increase the aqueous phase volume. (Chip) 2500 droplets were loaded into a microarray chip, and the ssDNA within the droplets was recovered by corona demulsification.

[0113] Figure 13 The results of the cDNA recovery efficiency test were conducted using mRNA instead of cells. The results show that the above method can effectively recover the cDNA contents within droplets. (Tube RT) mRNA was directly encapsulated to generate droplets. 1.5 μL (approximately 2500 droplets) was placed in a 200 μL PCR tube and subjected to isothermal reverse transcription in a PCR instrument. Demulsification was achieved by adding a demulsifier, and HBW was added to increase the aqueous phase volume. (Chip RT) mRNA was directly encapsulated to generate droplets. 2500 droplets were loaded into a microarray chip and subjected to reverse transcription in a water bath. cDNA within the droplets was recovered by corona demulsification.

[0114] All the above results indicate that cDNA within droplets can be effectively recovered by replacing the continuous phase within the chip during droplet demulsification and collection.

[0115] This application also separately tested the demulsification rate of droplets 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-mentioned technique, and the influence of droplet size is negligible in corona demulsification.

[0116] Table 1

[0117]

[0118] The single-cell sequencing performance of this system was validated using unsorted cell lines and PBMCs samples. Single-cell sequencing using cell lines to validate the system performance of a single-cell sequencing system is an essential and standard routine experiment in the performance validation of single-cell sequencing systems. This application used 293T cells for system validation. Cell droplets were prepared from single-cell suspensions of 4500 cells / μL and manually loaded into microarray chips for paired sequencing experiments. The results were compared with those obtained by directly encapsulating cells for single-cell sequencing, and the comparison results are shown in Table 2.

[0119] Table 2

[0120] Cell count Median number of genes in a single cell Positive control 539 1439 experimental group 669 2242

[0121] The positive control consisted of sequencing results from over 500 cell lines obtained using conventional methods; the experimental control group consisted of sequencing results from over 600 cell lines obtained using the method described in this application. The higher number of MedianGenes obtained using this method indicates that the method described in this application provides better performance metrics for single-cell sequencing.

[0122] Since the platform requires single-cell sequencing of clinically sorted PBMCs samples, to understand the system's ability to perform single-cell sequencing on PBMCs samples and to rule out operational issues related to droplet sorting, it is necessary to perform single-cell sequencing on unsorted PBMCs samples to verify system performance. The comparison results are shown in Table 3. Figure 14 As shown.

[0123] Table 3

[0124] Sample number Cell count Median number of genes in a single cell Positive control X-4-231026 1679 1023 experimental group X-5-231026 1105 1685

[0125] The positive control consisted of sequencing results from over 1600 PBMCs obtained using conventional methods; the experimental control group consisted of sequencing results from over 1100 PBMCs obtained using the method described in this application. The comparison showed that... Figure 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 was higher.

[0126] Cell clustering information of cells captured by conventional methods and the method of this application were compared, and the results are as follows: Figure 15 As shown, Figure 15 The left side of the image shows the cell clustering diagram of the positive control group, and the right side shows the cell clustering diagram of the experimental group. The comparison shows that the cells captured using the technical method in this application have no difference in cellular information compared to conventional methods.

[0127] The microporous array chip, its preparation method, and its application method provided in this invention include the following steps: Large droplets and small droplets are prepared separately; fluorinated oil is added to the inlet of the microporous array chip; large droplets are added to the inlet of the microporous array chip and tilted to capture them; after removing excess droplets, the chip is placed on ice; cell droplets are sorted using a droplet sorter and added to the large micropores of the microporous array chip to pair them one-to-one; after corona treatment, the chip is heated and placed on ice again; small droplets are added to the inlet of the microporous array chip and corona treated again; the microporous array chip is sealed and heated in a water bath; after injecting a water-soluble buffer solution, it is corona treated again to demulsify the droplets; and the solution containing cDNA is collected for sequencing analysis. This application method achieves single-cell sequencing of cell droplets by pairing and fusing large droplets with cell droplets and small droplets; optimizes the reagent system for single-cell sequencing by stepwise sample addition; and improves the efficiency of single-cell sequencing by increasing the mRNA capture efficiency of single cells.

[0128] The above description is merely a specific embodiment of the present invention, but the scope of protection of the present invention is not limited thereto. Any person skilled in the art can easily conceive of various equivalent modifications or substitutions within the technical scope disclosed in the present invention, and such modifications or substitutions are intended to be within the scope of protection of the present invention. Therefore, the scope of protection of the present invention shall be subject to the scope of protection of the claims.

Claims

1. A method for using a microporous array chip for single-cell sequencing, characterized in that, The application method includes: Large droplets were prepared using a first droplet generation chip; Small droplets are prepared using a second droplet generation chip; Funnel-shaped connectors are connected to the inlet and outlet of the microporous array chip, respectively. Fluorinated oil is added to the inlet of the microporous array chip and it is placed in a vacuum environment to purge the air inside the chip. After adding the large droplet to the inlet of the micropore array chip, the micropore array chip is tilted so that the large droplet floats into the large micropores in the micropore array chip by buoyancy. Fluorinated oil is added again to the inlet of the micropore array chip to remove excess large droplets; The micropore array chip is tilted and placed on an ice surface. The positive droplet output port of the droplet sorter is connected to the inlet of the micropore array chip so that the cell droplets are sorted by the droplet sorter and added to the small micropores on the side of the large micropores of the micropore array chip, so that the cell droplets are paired with the large droplets one by one. Fluorinated oil is added again to the inlet of the microporous array chip to remove excess cell droplets; A corona treatment of 2-4 seconds was applied over the microporous array chip using a corona machine to induce the fusion of large droplets and cell droplets that were in contact with each other. The microwell array chip was placed at room temperature for 20-30 minutes to allow proteinase K to fully lyse the cells and release the cell mRNA. Then, it was placed in a water bath at 68-75°C for 8-12 minutes to inactivate proteinase K. After that, the microwell array chip was placed on ice. Small droplets are introduced through the inlet of the microporous array chip, allowing them to drift by buoyancy into the small micropores on the side of the large micropores in the microporous array chip, thus completing the one-to-one pairing between the small droplets and the droplets in the large micropores. Then, excess small droplets are removed by adding fluorinated oil, and a corona treatment machine is used to apply corona treatment to the microporous array chip for 2-4 seconds to make the small droplets pair and fuse with the droplets in the large micropores. After sealing the inlet and outlet of the microporous array chip, the chip was placed in a water bath at 40-45℃ for 3 hours for reverse transcription. Connect a syringe to the inlet of the microporous array chip. Use a constant flow syringe pump to push the syringe at a specific flow rate to push the water-soluble buffer solution into the microporous array chip to expel the fluorinated oil. Absorb air and water-soluble buffer solution and reintroduce them into the inlet of the microporous array chip. Use a corona machine to apply corona treatment to the microporous array chip for 2-4 seconds to induce droplet demulsification. After standing for 12-18 minutes, collect the water-soluble buffer solution containing cDNA through the outlet of the microporous array chip for sequencing analysis.

2. The application method of the microporous array chip for single-cell sequencing according to claim 1, characterized in that, 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 process of preparing large droplets using a first droplet generation chip includes: Adjust the flow rate at the microsphere inlet so that each droplet contains only one microsphere; A high-speed camera is used to acquire images of droplet generation, and the flow rate ratio between the first reverse transcription reagent inlet and the first oil phase inlet and the microsphere phase inlet is adjusted based on the diameter of the droplets in the droplet generation images, so that the microspheres are fused with the first reverse transcription reagent and then wrapped by the first oil phase to form large droplets. Place the container holding the large droplets on ice for later use.

3. The application method of the microporous array chip for single-cell sequencing according to claim 1, characterized in that, The second droplet generation chip includes a second reverse transcription reaction reagent inlet, a second oil phase inlet, and a second outlet; The process of preparing small droplets using a second droplet generating chip includes: A high-speed camera is used to acquire images of droplet generation, and the flow rate ratio between the second reverse transcription reagent inlet and the second oil phase inlet is adjusted based on the diameter of the droplets in the droplet generation images, so that the second reverse transcription reagent is wrapped by the second oil phase to form small droplets. Place the container holding the small droplets on ice for later use.

4. The application method of the microporous 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 microporous array chip for single-cell sequencing according to claim 4, characterized in that, The method for preparing the water-soluble buffer solution includes: Take out 1M Tris hydrochloric acid buffer solution, pH 7.5, 0.5M EDTA solution, pH 8.0 and TWT solution and let them stand at room temperature for 20 min; the TWT solution is the solution obtained by dissolving Tween 20 in 1M Tris hydrochloric acid buffer solution, pH 7.

5. Add 1 mL of the above EDTA solution, 50 mL of the above TWT solution, and 50 mL of the above Tris hydrochloric acid buffer solution to 5 L ddH2O, and shake well to obtain the water-soluble buffer solution.

6. The application method of the microporous array chip for single-cell sequencing according to claim 1, characterized in that, The process involves connecting a syringe to the inlet of the microporous array chip, and using a constant-flow syringe pump to push the syringe at a specific flow rate to introduce a water-soluble buffer solution into the microporous array chip to expel the fluorinated oil. This includes: Use a 1ml syringe to draw 1ml of water-soluble buffer solution, and connect the syringe outlet to the inlet of the micropore array chip through a polyethylene tube. The syringe is fixed to a constant flow injection pump, and the solution is pushed into the microporous array chip at a flow rate of 1500 μL / h by the constant flow injection pump to expel the fluorinated oil.

7. The method of using the microwell array chip for single-cell sequencing according to any one of claims 1-6, characterized in that, The microporous array chip is obtained by stacking a top glass-polydimethylsiloxane microporous chip, a fluid channel layer, and a bottom glass slide.

8. The application method of the microporous array chip for single-cell sequencing according to claim 7, characterized in that, The microporous array chip is prepared by the following method: Two through holes are made on a glass slide and polydimethylsiloxane is filled into the through holes to obtain a glass substrate; The silicon wafer mold is subjected to oxygen-plasma treatment and treated in a vacuum chamber filled with silane vapor for more than 10 hours. The polydimethylsiloxane prepolymer with the bubbles removed is poured onto the silicon wafer mold and peeled off to obtain a concave mold with a polydimethylsiloxane micropore array. The concave mold is then placed in a vacuum chamber filled with silane vapor for more than 10 hours and then molded again to obtain a convex mold with a polydimethylsiloxane micropore array. Polydimethylsiloxane prepolymer is poured into a convex mold with a microporous array and vacuumed to remove air bubbles; After the glass substrate is treated with oxygen-plasma, it is placed on a convex mold with a microporous array for bonding. After heating and curing, it is peeled off to obtain a polydimethylsiloxane microporous array-glass substrate composite as a glass-polydimethylsiloxane microporous chip. Polydimethylsiloxane is repeatedly spin-coated onto a silicon wafer to obtain a flat polydimethylsiloxane film, and the polydimethylsiloxane film is cut to obtain a fluid channel layer; After bonding the fluid channel layer to the bottom glass slide, plasma treatment is performed to bond the glass-polydimethylsiloxane microporous chip to the upper layer of the fluid channel layer. The chip is then heated at 110°C for 10 minutes to obtain a microporous array chip. A hydrophobic reagent is injected into the microporous array chip, and the microporous array chip is placed in a vacuum environment; After 30 seconds, the hydrophobic reagent is extracted using a syringe, and then the microporous array chip is dried at 110 °C to remove the remaining hydrophobic reagent.

9. The application method of the microporous array chip for single-cell sequencing according to claim 8, characterized in that, The step of creating two through holes in a glass slide and filling the through holes with polydimethylsiloxane includes: Two through holes are made on the glass slide, and one side is treated with plasma for 1 minute. The through holes on the side that has not been treated with plasma are sealed with tape. Use a dropper to apply uncured polydimethylsiloxane to each through-hole; Vacuum the glass slide and place it at 60°C for at least 6 hours to cure it. Remove the tape and cut off any excess polydimethylsiloxane; Clean the side of the glass slide that has not undergone plasma treatment by wiping it with anhydrous alcohol.

10. The application method of the microporous array chip for single-cell sequencing according to claim 8, characterized in that, The process of repeatedly spin-coating polydimethylsiloxane onto a silicon wafer to obtain a smooth polydimethylsiloxane film includes: Spin-coat polydimethylsiloxane onto a silicon wafer for 130-170 seconds at a spin speed of 500 rpm; Heat the silicon wafer at 150℃-170℃ for 4-7 minutes; Repeat the spin coating and heat treatment process 7-10 times to obtain the smooth polydimethylsiloxane film.

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