A microfluidic chip suitable for active single-cell multi-omics

Abstract

The application relates to the technical field of gene therapy, and specifically discloses a microfluidic chip suitable for active single-cell multi-omics, which comprises a capture channel, a first microsphere interception channel, a second microsphere interception channel and a cell interception channel; the capture channel is used for contacting microspheres with cells; the outlet of the first microsphere interception channel is communicated with the capture channel, is used for flowing a first microsphere solution, and is used for intercepting the microspheres in the first microsphere solution in the capture channel; the outlet of the second microsphere interception channel is communicated with the capture channel, is used for flowing a second microsphere solution, and is used for intercepting the microspheres in the second microsphere solution in the capture channel; and the outlet of the cell interception channel is communicated with the capture channel, is used for flowing a cell solution, and is used for intercepting the cells in the cell solution in the capture channel. In the scheme, the above channels can realize rapid capture of single cells, can avoid waste of a large number of cells, and can realize precise pairing of a single cell and two different microspheres.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of gene therapy, and in particular to a microfluidic chip suitable for active single-cell multi-omics. BACKGROUND

[0002] Single-cell multi-omics technology is generally used to measure multiple dimensions of information of the same cell, including genome, transcriptome, epigenome, proteome, etc. Single-cell multi-omics technology can solve scientific problems such as average effect caused by population sample sequencing, low cell starting amount that cannot be sequenced, and cell heterogeneity. In addition, the high-resolution data produced by single-cell technology helps researchers discover more detailed genomic structural features and more intuitively understand gene expression regulation, early embryonic development, tumor formation and development, etc.

[0003] In the field of cell gene therapy, in addition to traditional surgical resection, radiotherapy and chemotherapy, cell therapy is a new way. Cell therapy mainly relies on the modification and reinfusion of human immune cells to kill cancer cells.

[0004] For example, CAR-T immunotherapy has achieved preliminary success in the treatment of hematological tumors in existing clinical applications; TCR-T cell therapy, which recognizes tumor-specific antigens from the cell membrane surface or intracellular sources, is expected to make breakthroughs in the treatment of solid tumors; and in the study of immunotherapy for lung cancer, TCR specificity that can target tumor cells is one of the important links to solve off-target effects.

[0005] In the process of cell therapy, single-cell capture can be used to isolate and obtain single cells for subsequent analysis and research. Among them, microfluidic technology is a commonly used single-cell capture technology.

[0006] In existing microfluidic technology, the commonly used method for capturing single cells and microspheres is to wrap single cells and microsphere reagents required for subsequent reactions into droplet microreactors, dilute the cell suspension to a certain concentration, and make it uniformly dispersed, then take multiple equal volumes of samples from it, or divide it into equal volumes to generate single dispersed droplets. The cells in the suspension are randomly dispersed into droplets, and the number of cells in the droplets conforms to the Poisson distribution. This method is fast and simple, but the single-cell wrapping probability is low, and the highest can only reach about 40%, and the multi-cell droplets (containing two or more cells in the droplet) account for a large proportion. This way a large number of cells are lost, and only single-microsphere and single-cell capture can be achieved. SUMMARY

[0007] Therefore, the purpose of the present application is to provide a microfluidic chip suitable for active single-cell multi-omics, which can solve some or all of the above problems.

[0008] To achieve the above technical purposes, the application provides a microfluidic chip suitable for active single-cell multi-omics, comprising:

[0009] a capture channel for contacting microspheres with cells;

[0010] a first microsphere interception channel, an outlet of the first microsphere interception channel being communicated with the capture channel, for flowing a first microsphere solution, and for intercepting microspheres in the first microsphere solution in the capture channel;

[0011] a second microsphere interception channel, an outlet of the second microsphere interception channel being communicated with the capture channel, for flowing a second microsphere solution, and for intercepting microspheres in the second microsphere solution in the capture channel;

[0012] a cell interception channel, an outlet of the cell interception channel being communicated with the capture channel, for flowing a cell solution, and for intercepting cells in the cell solution in the capture channel.

[0013] Further, first and second interception valves are arranged at intervals on the capture channel;

[0014] The first and second interception valves are used to control the opening and closing of the capture channel;

[0015] The outlet of the first microsphere interception channel, the outlet of the second microsphere interception channel, and the outlet of the cell interception channel are all located between the first and second interception valves.

[0016] Further, it further comprises a wrapping channel;

[0017] The outlet of the capture channel is communicated with the wrapping channel;

[0018] The capture channel is used to flow a flushing liquid to flush the microspheres and cells to the wrapping channel after the microspheres and cells are contacted;

[0019] The wrapping channel is used to flow a wrapping liquid to wrap the microspheres and cells with droplets of the wrapping liquid.

[0020] Further, the height of the wrapping channel is greater than the height of the capture channel.

[0021] Further, the height of the wrapping channel is at least twice the height of the capture channel.

[0022] Further, a first microsphere interception valve is arranged on the first microsphere interception channel;

[0023] The first microsphere interception valve is used to intercept the microspheres in the first microsphere solution in the capture channel;

[0024] The second microsphere cutoff valve is arranged on the second microsphere cutoff channel;

[0025] The second microsphere cutoff valve is arranged on the second microsphere cutoff channel;

[0026] The cell cutoff valve is arranged on the cell cutoff channel;

[0027] The cell cutoff valve is arranged on the cell cutoff channel;

[0028] Further, the capture channel and / or the cell cutoff channel is an arc channel with an arc-shaped top surface;

[0029] The first cutoff valve, the second cutoff valve and the cell cutoff valve are pneumatic valves.

[0030] Further, the waste liquid channel is further included;

[0031] The inlet of the waste liquid channel is communicated with the capture channel, for the waste liquid in the capture channel to flow out.

[0032] Further, a third cutoff valve is arranged on the capture channel;

[0033] The third cutoff valve is arranged between the inlet of the waste liquid channel and the outlet of the cell cutoff channel, for the cells in the cell liquid to be cut off between the third cutoff valve and the inlet of the waste liquid channel.

[0034] Further, the first sheath liquid cutoff channel and the second sheath liquid cutoff channel are further included;

[0035] The outlet of the first sheath liquid cutoff channel is communicated with the first microsphere cutoff channel;

[0036] The outlet of the first microsphere cutoff channel is communicated with the second microsphere cutoff channel.

[0037] As can be seen from the above technical solutions, the present application provides a microfluidic chip suitable for active single-cell multi-omics, which comprises a capture channel, a first microsphere cutoff channel, a second microsphere cutoff channel and a cell cutoff channel; the capture channel is used for contacting microspheres and cells; the outlet of the first microsphere cutoff channel is communicated with the capture channel, for the first microsphere solution to flow, and for the microspheres in the first microsphere solution to be cut off in the capture channel; the outlet of the second microsphere cutoff channel is communicated with the capture channel, for the second microsphere solution to flow, and for the microspheres in the second microsphere solution to be cut off in the capture channel; the outlet of the cell cutoff channel is communicated with the capture channel, for the cell liquid to flow, and for the cells in the cell liquid to be cut off in the capture channel.

[0038] The channel can realize rapid capture of single cells, avoid waste of a large number of cells, break through the Poisson distribution problem in high-throughput single cell sequencing technology, and quickly complete sorting analysis for target cells. Meanwhile, the scheme can realize precise pairing of a single cell and two different microspheres, increase the accuracy of detection, broaden the detection range, increase the reliability of experimental results, and facilitate data standardization and visualization. BRIEF DESCRIPTION OF DRAWINGS

[0039] In order to more clearly illustrate the technical solutions in the embodiments of the present application or the prior art, the drawings needed to be used in the embodiments or prior art description will be briefly introduced. Obviously, the drawings in the following description are only some embodiments of the present application, and other drawings can be obtained by those skilled in the art without creative labor.

[0040] Figure 1 A schematic diagram of each channel of a microfluidic chip suitable for active single cell multi-omics provided by the embodiments of the present application;

[0041] Figure 2 A process variation diagram of the main structure manufacturing process of a microfluidic chip suitable for active single cell multi-omics provided by the embodiments of the present application;

[0042] Figure 3 A process variation diagram of the main structure manufacturing process of a microfluidic chip suitable for active single cell multi-omics provided by the embodiments of the present application;

[0043] In the drawings:

[0044] 10, first microsphere interception channel; 11, first microsphere interception valve;

[0045] 20, second microsphere interception channel; 21, second microsphere interception valve;

[0046] 30, cell interception channel; 31, cell interception valve;

[0047] 40, capture channel; 41, first interception valve; 42, second interception valve; 43, third interception valve;

[0048] 50, wrapping channel;

[0049] 60, waste liquid channel; 61, waste liquid interception valve;

[0050] 70, first sheath liquid interception channel; 71, first sheath liquid interception valve;

[0051] 80, second sheath liquid interception channel; 81, second sheath liquid interception valve;

[0052] 91, positive glue layer; 92, base layer; 93, first negative glue layer; 94, second negative glue layer; 95, third negative glue layer. DETAILED DESCRIPTION

[0053] The technical solutions of the embodiments of the present application will be clearly and completely described below with reference to the drawings. Obviously, the described embodiments are only part of the embodiments of the present application, rather than all the embodiments of the present application. Based on the embodiments in the present application, all other embodiments obtained by those skilled in the art without creative work fall within the scope of the present application.

[0054] In the description of the embodiments of the present application, it should be noted that the orientations or positional relationships indicated by the terms "center", "upper", "lower", "left", "right", "vertical", "horizontal", "inner", "outer", etc. are based on the orientations or positional relationships shown in the drawings, and are only for the convenience of describing the embodiments of the present application and simplifying the description, and therefore cannot be understood as indicating or implying that the devices or elements referred to must have a particular orientation, be constructed and operated in a particular orientation, and therefore cannot be understood as limiting the embodiments of the present application. In addition, the terms "first", "second", "third" are only for the purpose of description, and cannot be understood as indicating or implying relative importance.

[0055] In the description of the embodiments of the present application, it should be noted that unless otherwise explicitly specified and limited, the terms "mounting", "connecting", "connection" should be understood in a broad sense, for example, it can be fixed connection, or replaceable connection, or integral connection, it can be mechanical connection, or electrical connection, it can be direct connection, or indirect connection through an intermediate medium, or internal communication of two elements. For those skilled in the art, the specific meaning of the above terms in the embodiments of the present application can be understood according to the specific circumstances.

[0056] Please refer to Figure 1 The microfluidic chip suitable for active single-cell multi-omics provided in the embodiments of the present application comprises a capture channel 40, a first microsphere interception channel 10, a second microsphere interception channel 20, and a cell interception channel 30.

[0057] The capture channel 40 is used for contacting the microspheres with the cells, that is, the microsphere solution and the cell solution can both flow into the capture channel 40.

[0058] The outlet of the first microsphere interception channel 10 is communicated with the capture channel 40; the first microsphere interception channel 10 is used for flowing the first microsphere solution. The microspheres in the first microsphere solution can be intercepted in the capture channel 40 through the first microsphere interception channel 10. The first microsphere solution can be used as a proteomic microsphere solution; in actual application, the first microsphere solution may, for example, be a polystyrene microsphere solution, a magnetic bead microsphere solution, etc.

[0059] The outlet of the second microsphere flow-through channel 20 is communicated with the capture channel 40; the second microsphere flow-through channel 20 is used for flowing the second microsphere solution. The microspheres in the second microsphere solution can be cut off in the capture channel 40 through the second microsphere flow-through channel 20. The second microsphere solution can be a transcriptome microsphere solution; in practical applications, the second microsphere solution can be, for example, a hydrogel microsphere solution, a gel microsphere solution, etc. It should be noted that in practical applications, the components of the first microsphere solution and the second microsphere solution can be replaced, that is, the first microsphere solution is used as a transcriptome microsphere solution, and the second microsphere solution is used as a proteome microsphere solution. The first microsphere solution and the second microsphere solution can also be other multi-omics microsphere solutions.

[0060] The outlet of the cell flow-through channel 30 is communicated with the capture channel 40; the cell flow-through channel 30 is used for flowing the cell solution; the cells in the cell solution can be cut off in the capture channel 40 through the cell flow-through channel 30.

[0061] The single cell capture performed by the microfluidic chip provided in the embodiment can break through the Poisson distribution problem in high-throughput single cell sequencing technology, and can quickly complete sorting analysis for target cells, so that the single cells can be quickly, accurately and simply captured.

[0062] Meanwhile, in the process of capturing single cells, the first microsphere flow-through channel 10 and the second microsphere flow-through channel 20 can be used simultaneously to capture and pair two kinds of microspheres and single cells; the first microsphere flow-through channel 10 and the second microsphere flow-through channel 20 can also be used only one of them to capture single microspheres and single cells. That is, the microfluidic chip in the embodiment can also widen the use scene of the microfluidic chip and increase its applicability.

[0063] Taking the simultaneous use of the first microsphere flow-through channel 10 and the second microsphere flow-through channel 20 as an example, the microfluidic chip in the embodiment can cut off the microspheres in the first microsphere solution, the microspheres in the second microsphere solution and the single cells in the capture channel 40, and at least has the following advantages:

[0064] 1. Different microspheres can carry different markers or probes to detect single cells in multiple aspects. For example, one kind of microsphere carries an antibody against a specific antigen on the surface of a cell, and another kind of microsphere carries a fluorescent probe for detecting a specific molecule in the cell, so that the cell can be analyzed from the outside and the inside of the cell at the same time, the results are verified with each other, and the accuracy of detection is improved.

[0065] 2、Capture two kinds of microspheres can detect multiple biomarkers or molecules at the same time, and obtain more comprehensive cell information. For example, in disease diagnosis, one microsphere is used to detect inflammation-related markers, and the other microsphere is used to detect tumor-related markers, which can more accurately determine the type and state of the disease.

[0066] 3、In control experiments or internal references, one of the microspheres can be used as a control and compared with the other microsphere used for actual detection. By changing the signal of the control microsphere, non-specific interference or systematic errors in the experimental process can be excluded, ensuring the reliability of the experimental results. For example, in drug screening experiments, a microsphere with a specific reaction to cells is used as a control, and another microsphere carrying the drug to be screened is used for experiments at the same time to verify the effect of the drug.

[0067] 4、The control microsphere can be used as an internal reference to standardize data under different sample or experimental conditions. Due to differences between different experimental batches or samples, the signal of the control microsphere can be used as a reference to compare the signals of other microspheres, thereby eliminating the influence of these differences on the results.

[0068] 5、Two kinds of microspheres can be combined with single cells through different interaction modes to improve the efficiency of capture. For example, one microsphere has a ligand that binds to a specific receptor on the cell surface, and the other microsphere has a functional group that interacts with the matrix around the cell, and the two work together to more stably capture single cells.

[0069] 6、For different types of single cells, two microspheres with different specificities can be used for differential capture. For example, in a complex cell population, one microsphere specifically captures a certain type of cancer cell, and the other microsphere captures normal cells, thereby achieving specific separation and analysis of different cell types.

[0070] 7、Two kinds of microspheres can carry different reagents or functional molecules, respectively, to form a multifunctional reaction system around the single cell. For example, one microsphere carries an enzyme, and the other microsphere carries a substrate, when the two microspheres are wrapped together in a system with a single cell, a specific chemical reaction can be carried out locally around the cell to study the response of the cell to the reaction.

[0071] 8、In practical applications, one microsphere can be used for preliminary processing or labeling, and then another microsphere can be introduced for subsequent operation according to experimental needs. This step-by-step operation method can more flexibly control the experimental process and achieve fine analysis of single cells.

[0072] It should be noted that the way of stopping microspheres or single cells in the channel on the microfluidic chip can use specific fluorescent dyes or fluorescently labeled antibodies to label the cells, and then control the flow and stop of the fluid in each channel by using a micropump.

[0073] For example, specific molecules in the cell, such as nucleic acids, proteins, etc., are labeled with fluorescent dyes, or specific antigens on the cell surface are recognized with fluorescently labeled antibodies. In this way, the labeled cells will emit a fluorescent signal when excited by light of a specific wavelength. At the same time, since the size of the channel on the microfluidic chip is generally tens of microns, and the diameter of most eukaryotic cells is between 10 to 30 microns, and the size of the microspheres varies slightly, for example, the size of the polyacrylamide microspheres is generally between tens of nanometers to hundreds of microns; therefore, the channel on the microfluidic chip can be set to allow only a single cell and a single microsphere to flow. The recognition point can be provided in the capture channel 40, and when the microsphere or cell is recognized to enter the capture channel 40, the microsphere solution or cell solution is stopped by closing the valve, so that the single microsphere or single cell is stopped in the capture channel 40.

[0074] As an embodiment, the capture channel 40 can be provided with a chamber; the microspheres and single cells entering the capture channel 40 can fall into the chamber and are not easily washed away by the solution.

[0075] In one embodiment, the first microsphere stopping valve 11 is provided on the first microsphere stopping channel 10; the first microsphere stopping valve 11 is used to stop the microspheres in the first microsphere solution in the capture channel 40; the second microsphere stopping valve 21 is provided on the second microsphere stopping channel 20; the second microsphere stopping valve 21 is used to stop the microspheres in the second microsphere solution in the capture channel 40; the cell stopping valve 31 is provided on the cell stopping channel 30; the cell stopping valve 31 is used to stop the cells in the cell solution in the capture channel 40.

[0076] Compared with the above-mentioned method of using a micropump to control the flow and stop of the fluid for stopping, the first microsphere stopping valve 11, the second microsphere stopping valve 21 and the cell stopping valve 31 in the present embodiment can more accurately control the flow and stop of the fluid, realize the accurate regulation of the flow rate and flow of the cell or microsphere suspension, and enable the target cells to accurately stay at the capture site. This ensures the accuracy and repeatability of the capture.

[0077] At the same time, the good sealing property of the stopping valve can effectively prevent the backflow of the fluid, and maintain the unidirectional flow of the fluid in the microfluidic chip. This helps to maintain the stable distribution of the cells or microspheres in the chip, avoids the re-entry of the captured cells or microspheres into the fluid due to backflow, and ensures the stability of the capture process.

[0078] As an implementation, the first microsphere cutoff valve 11, the second microsphere cutoff valve 21 and the cell cutoff valve 31 are all pneumatic valves, which are usually located in a specific area of the chip and cooperate with other structure layers in the channel. Specifically, the pneumatic valve can realize precise control of the flow of liquid or gas in the microfluidic chip by adjusting the gas pressure. For example, a specific channel can be opened or closed, and the flow rate and flow volume of the fluid can be adjusted. When it is necessary to prevent the fluid from flowing in a certain channel, the pneumatic valve can close the channel by applying pressure to make the film layer rise, thereby preventing the fluid from passing through. When it is necessary to let the fluid flow, the film layer will drop to open the channel when the pressure is released.

[0079] In an embodiment, the first cutoff valve 41 and the second cutoff valve 42 are arranged at intervals on the capture channel 40; the first cutoff valve 41 and the second cutoff valve 42 are used to control the on-off of the capture channel 40; the outlet of the first microsphere cutoff channel 10, the outlet of the second microsphere cutoff channel 20 and the outlet of the cell cutoff channel 30 are all located between the first cutoff valve 41 and the second cutoff valve 42.

[0080] The first cutoff valve 41 and the second cutoff valve 42 can cut off the two kinds of microspheres and single cells therebetween, which helps the contact between the microspheres and the single cells. Specifically, the movement of the microspheres and the single cells in the solution has a certain randomness. The space between the first cutoff valve 41 and the second cutoff valve 42 can give the microspheres and the single cells more opportunities to approach each other and physically or chemically combine.

[0081] In practical application, the width of the capture channel 40 can be set to be greater than the width of the first microsphere cutoff channel 10, greater than the width of the second microsphere cutoff channel 20 and greater than the width of the cell cutoff channel 30, so as to improve the fault tolerance of the particle cutoff landing point.

[0082] In another embodiment, it further includes a wrapping channel 50; the outlet of the capture channel 40 communicates with the wrapping channel 50; the capture channel 40 is used to flow the flushing liquid after the microspheres contact with the cells, so as to flush the microspheres and the cells to the wrapping channel 50; the wrapping channel 50 is used to flow the wrapping liquid, so that the droplets of the wrapping liquid wrap the microspheres and the cells.

[0083] The injection of the flushing liquid into the capture channel 40 can flush away the uncombined part, improve the purity of the target (the microspheres combined with the single cells) in the subsequent wrapping process, and reduce the interference in the subsequent experiment.

[0084] After the flushing liquid flushes the microspheres combined with the single cells into the wrapping channel 50, the wrapping liquid flows into the wrapping channel 50 to wrap the single cells and the microspheres, so as to complete the single cell multi-omics capture or the transcriptome capture of cell interaction. The wrapping liquid can be oil liquid such as fluorocarbon oil, fluorinated oil, mineral oil and silicone oil.

[0085] It should be noted that the second stop valve 42 on the capture channel 40 can be used to control the flow and stop of the flushing liquid.

[0086] In one embodiment, the height of the wrapping channel 50 is greater than the height of the capture channel 40. Specifically, increasing the height of the wrapping channel 50 can reduce the contact area of the droplet with the wrapping channel 50, thereby reducing the adhesion of certain substances in the reagent composition to the channel and causing the droplet to be pulled apart.

[0087] Optionally, the height of the wrapping channel 50 is at least twice the height of the capture channel 40. Specifically, the height of the capture channel 40 can be 40 microns, and the height of the wrapping channel 50 is greater than 80 microns. In practical applications, the size of the droplet formed after the wrapping liquid wraps the target object is generally 120 microns. In this embodiment, the height of the wrapping channel 50 is set to be at least twice the height of the capture channel 40 to ensure that the droplet can flow individually while reducing its contact area with the wrapping channel.

[0088] In one embodiment, the capture channel 40 and / or the cell interception channel 30 is an arc channel with an arc-shaped top surface; the first stop valve 41, the second stop valve 42, and the cell stop valve 31 are pneumatic valves.

[0089] In applications, the pneumatic valve closes the channel by applying pressure to make the membrane adhere to the inner wall of the channel, or opens the channel by removing the pressure to make the membrane away from the inner wall of the channel. In this embodiment, the capture channel 40 and / or the cell interception channel 30 is an arc channel, which can increase the sealing effect when the membrane contacts the wall.

[0090] Optionally, the first microsphere interception channel 10 and the second microsphere interception channel 20 are both rectangular channels, which can reduce the influence of the side edges of the channels on the flow rate of the particles.

[0091] In another embodiment, it further includes a waste liquid channel 60; the inlet of the waste liquid channel 60 is connected to the capture channel 40, for the waste liquid in the capture channel 40 to flow out.

[0092] Optionally, a waste liquid stop valve 61 is arranged on the waste liquid channel 60, for controlling the on-off of the waste liquid channel 60.

[0093] Optionally, a third stop valve 43 is arranged on the capture channel 40; the third stop valve 43 is arranged between the inlet of the waste liquid channel 60 and the outlet of the cell interception channel 30, for intercepting the cells in the cell liquid between the third stop valve 43 and the inlet of the waste liquid channel 60.

[0094] Similarly, the third shut-off valve 43 can be a pneumatic valve. When the third shut-off valve 43 detects that a cell has passed through the outlet of the cell interception channel 30 at the identification point, it closes the capture channel 40 to ensure that the cell is blocked between the third shut-off valve 43 and the outlet of the cell interception channel 30, thus preventing the cell from being carried away by the fluid.

[0095] Optionally, the microfluidic chip provided in this embodiment further includes: a first sheath fluid interception channel 70 and a second sheath fluid interception channel 80; the outlet of the first sheath fluid interception channel 70 is connected to the first microsphere interception channel 10; and the outlet of the first microsphere interception channel 10 is connected to the second microsphere interception channel 20.

[0096] A first sheath fluid cut-off valve 71 may be provided on the first sheath fluid interception channel 70; a second sheath fluid cut-off valve 81 may be provided on the second sheath fluid interception channel 8; the flow and cut-off of sheath fluid in the first sheath fluid interception channel 70 and the second sheath fluid interception channel 80 can be controlled respectively by the first sheath fluid cut-off valve 71 and the second sheath fluid cut-off valve 81.

[0097] In the above embodiments, the microfluidic chip can be fabricated through the following steps, please refer to [link / reference]. Figure 2 It includes a positive adhesive layer 91, a base layer 92, a first negative adhesive layer 93, and a second negative adhesive layer 94.

[0098] The first step is to homogenize the positive resist layer 91 set on the substrate layer 92, and then to form a rectangular channel by photolithography and development of the positive resist layer 91.

[0099] The second step involves heat-melting the rectangular channel at a temperature above 150°C for at least 3 hours to form an arc-shaped channel. This arc-shaped channel allows the PDMS film in the pneumatic valve to adhere more easily to the upper wall of the channel, thus sealing it off. The height of the arc-shaped channel can be 35 micrometers.

[0100] The third step involves overlaying a slightly higher second negative adhesive layer 94 onto the arc-shaped positive adhesive layer 91. The resulting flow channel structure is rectangular, which reduces the influence of the flow channel sides on particle velocity. The flow channel for overlaying the second negative adhesive layer 94 can be a first microsphere intercepting channel 10 and a second microsphere intercepting channel 20. The height of the rectangular flow channel formed after overlaying the second negative adhesive layer 94 can be 40 micrometers.

[0101] The fourth step is to engrave a first negative adhesive layer 93 with a thickness greater than 80 micrometers on the encapsulation channel 50. This structure can reduce the contact between the droplets and the channel wall, thereby reducing the wetting effect of the droplets.

[0102] For instructions on manufacturing the aforementioned pneumatic valve, please refer to [link / reference needed]. Figure 3The third negative adhesive layer 95 is used as the main structure of the pneumatic valve by etching a flow channel on the third negative adhesive layer 95. The height of the second positive adhesive layer 95 after etching can be 30 microns.

[0103] The above is only a preferred embodiment of the present application and is not intended to limit the present application. Although the present application has been described in detail with reference to the examples, those skilled in the art can modify the technical solutions described in the examples or replace some of the technical features with equivalent ones without departing from the spirit and principles of the present application. Any modification, equivalent replacement, improvement, etc. made within the spirit and principles of the present application shall be included in the protection scope of the present application.

Claims

1. A microfluidic chip suitable for active single-cell multi-omics, characterized in that, include: A capture channel (40) is provided for the microspheres to contact the cells; The first microsphere interception channel (10) has its outlet connected to the capture channel (40) for supplying the flow of the first microsphere solution and for trapping the microspheres in the first microsphere solution within the capture channel (40). The outlet of the second microsphere interception channel (20) is connected to the capture channel (40) for supplying the flow of the second microsphere solution and for trapping the microspheres in the second microsphere solution within the capture channel (40); A cell interception channel (30) is provided, the outlet of which is connected to the capture channel (40) for supplying cell fluid flow and for trapping cells in the cell fluid within the capture channel (40), thereby enabling the capture channel (40) to simultaneously capture the two microspheres and cells. The capture channel (40) is provided with a first shut-off valve (41) and a second shut-off valve (42) at intervals. The first shut-off valve (41) and the second shut-off valve (42) are used to control the opening and closing of the capture channel (40); The outlets of the first microsphere interception channel (10), the second microsphere interception channel (20), and the cell interception channel (30) are all located between the first shut-off valve (41) and the second shut-off valve (42).

2. The microfluidic chip suitable for active single-cell multi-omics according to claim 1, characterized in that, Also includes: Parcel access (50); The outlet of the capture channel (40) is connected to the package channel (50). The capture channel (40) is used to allow the flushing liquid to flow after the microspheres come into contact with the cells, thereby flushing the microspheres and cells into the encapsulation channel (50). The encapsulation channel (50) is used to allow the encapsulation fluid to flow so that the droplets of the encapsulation fluid encapsulate the microspheres and cells.

3. The microfluidic chip suitable for active single-cell multi-omics according to claim 2, characterized in that, The height of the package channel (50) is greater than the height of the capture channel (40).

4. The microfluidic chip suitable for active single-cell multi-omics according to claim 3, characterized in that, The height of the package channel (50) is at least twice that of the capture channel (40).

5. The microfluidic chip suitable for active single-cell multi-omics according to claim 1, characterized in that, A first microsphere shut-off valve (11) is provided on the first microsphere interception channel (10); The first microsphere shut-off valve (11) is used to shut off the microspheres in the first microsphere solution within the capture channel (40); A second microsphere shut-off valve (21) is provided on the second microsphere interception channel (20); The second microsphere shut-off valve (21) is used to shut off the microspheres in the second microsphere solution within the capture channel (40); A cell shut-off valve (31) is provided on the cell interception channel (30). The cell shut-off valve (31) is used to shut off the cells in the cell fluid within the capture channel (40).

6. The microfluidic chip suitable for active single-cell multi-omics according to claim 5, characterized in that, The capture channel (40) and / or the cell interception channel (30) are arc-shaped channels with an arc-shaped top surface; The first shut-off valve (41), the second shut-off valve (42) and the cell shut-off valve (31) are pneumatic valves.

7. The microfluidic chip suitable for active single-cell multi-omics according to any one of claims 1 to 6, characterized in that, Also includes: waste liquid channel (60); The inlet of the waste liquid channel (60) is connected to the capture channel (40) for the waste liquid in the capture channel (40) to flow out.

8. The microfluidic chip suitable for active single-cell multi-omics according to claim 7, characterized in that, A third shut-off valve (43) is provided on the capture channel (40); The third shut-off valve (43) is located between the inlet of the waste liquid channel (60) and the outlet of the cell interception channel (30) to shut off the cells in the cell fluid between the third shut-off valve (43) and the inlet of the waste liquid channel (60).

9. The microfluidic chip suitable for active single-cell multi-omics according to claim 1, characterized in that, Also includes: First sheath fluid interception channel (70) and second sheath fluid interception channel (80); The outlet of the first sheath fluid interception channel (70) is connected to the first microsphere interception channel (10). The outlet of the second sheath fluid interception channel (80) is connected to the second microsphere interception channel (20).

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