Micro-fluidic chip suitable for active single-cell multi-omics

Abstract

The invention relates to the technical field of gene therapy, and particularly discloses a micro-fluidic 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 allowing the microspheres to be in contact with cells; an outlet of the first microsphere intercepting channel is communicated with the capturing channel and is used for allowing a first microsphere solution to flow and stopping microspheres in the first microsphere solution in the capturing channel; an outlet of the second microsphere intercepting channel is communicated with the capturing channel and is used for allowing a second microsphere solution to flow and stopping microspheres in the second microsphere solution in the capturing channel; an outlet of the cell intercepting channel is communicated with the capturing channel and is used for allowing cell sap to flow and stopping cells in the cell sap in the capturing channel. According to the scheme, rapid capture of single cells can be realized through the channels, waste of a large number of cells can be avoided, and accurate pairing of a single cell and two different microspheres can be realized.

Description

Technical Field

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

[0002] Single-cell multi-omics technology is usually 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 the average effect of population sample sequencing, low cell starting amount that cannot be sequenced, and high cell heterogeneity. In addition, the high-resolution data produced by single-cell technology can help researchers discover more detailed genomic structural features and more intuitively understand processes such as gene expression regulation, early embryonic development, tumor formation and development.

[0003] In the field of cell gene therapy, in addition to traditional surgical resection, radiotherapy and chemotherapy, cell therapy is an emerging method. Cell therapy mainly relies on the transformation and reinfusion of the body's own immune cells to kill cancer cells.

[0004] For example, CAR-T immunotherapy has achieved initial success in the treatment of hematological tumors in existing clinical applications; TCR-T cell therapy is expected to achieve breakthroughs in the treatment of solid tumors by identifying tumor-specific antigens on the cell membrane surface or intracellular sources; and in immunotherapy studies on lung cancer, it was found that the TCR specificity that can target tumor cells will be one of the important links in resolving off-target effects.

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

[0006] In the existing microfluidic technology, the commonly used method for capturing single cells and microspheres is generally to encapsulate single cells and microsphere reagents required for subsequent reactions into a droplet microreactor, dilute the cell suspension to a certain concentration, and disperse it evenly, and then take samples from it multiple times in equal volumes, or divide it in equal volumes to generate monodisperse droplets. The cells in the suspension are randomly dispersed into the droplets, and the number of cells in the droplets conforms to the Poisson distribution. This method is fast and simple, but the probability of single cell encapsulation is low, only about 40% at most, and multi-cell droplets (droplets containing two or more cells) account for a large proportion. This method causes a large number of cells to be lost and can only capture single microspheres and single cells. Summary of the invention

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

[0008] In order to achieve the above technical objectives, the present 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 intercepting channel, wherein the outlet of the first microsphere intercepting channel is connected to the capture channel, and is used for allowing the first microsphere solution to flow, and for intercepting the microspheres in the first microsphere solution in the capture channel;

[0011] a second microsphere intercepting channel, wherein the outlet of the second microsphere intercepting channel is connected to the capture channel, and is used for allowing the second microsphere solution to flow, and for intercepting the microspheres in the second microsphere solution in the capture channel;

[0012] A cell interception channel, the outlet of which is connected to the capture channel, is used for allowing the cell fluid to flow and for intercepting the cells in the cell fluid in the capture channel.

[0013] Furthermore, a first stop valve and a second stop valve are arranged at intervals on the capture channel;

[0014] The first stop valve and the second stop valve are used to control the on and off of the capture channel;

[0015] The outlet of the first microsphere intercepting channel, the outlet of the second microsphere intercepting channel and the outlet of the cell intercepting channel are all located between the first stop valve and the second stop valve.

[0016] Further, it also includes: a parcel channel;

[0017] The outlet of the capture channel is connected to the wrapping channel;

[0018] The capture channel is used for allowing flushing liquid to flow after the microspheres are in contact with the cells, so as to flush the microspheres and cells into the encapsulation channel;

[0019] The wrapping channel is used for the wrapping liquid to flow, so that the droplets of the wrapping liquid wrap the microspheres and cells.

[0020] Furthermore, the height of the wrapping channel is greater than the height of the capturing channel.

[0021] Furthermore, the height of the wrapping channel is at least twice that of the capturing channel.

[0022] Furthermore, a first microsphere stop valve is provided on the first microsphere interception channel;

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

[0024] A second microsphere stop valve is provided on the second microsphere interception channel;

[0025] The second microsphere stop valve is used to stop the microspheres in the second microsphere solution in the capture channel;

[0026] The cell interception channel is provided with a cell cut-off valve;

[0027] The cell cut-off valve is used to cut off the cells in the cell fluid in the capture channel.

[0028] Furthermore, the capture channel and / or the cell interception channel is a curved channel with a curved top surface;

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

[0030] Further, it also includes: a waste liquid channel;

[0031] The inlet of the waste liquid channel is connected to the capture channel, and is used for allowing the waste liquid in the capture channel to flow out.

[0032] Furthermore, a third stop valve is provided on the capture channel;

[0033] The third stop valve is arranged between the inlet of the waste liquid channel and the outlet of the cell interception channel, and is used to intercept the cells in the cell fluid between the third stop valve and the inlet of the waste liquid channel.

[0034] Further, it also includes: a first sheath liquid interception channel and a second sheath liquid interception channel;

[0035] The outlet of the first sheath fluid intercepting channel is connected to the first microsphere intercepting channel;

[0036] The outlet of the first microsphere intercepting channel is connected to the second microsphere intercepting channel.

[0037] It can be seen from the above technical scheme that the present application provides a microfluidic chip suitable for active single-cell multi-omics, including: a capture channel, a first microsphere interception channel, a second microsphere interception channel and a cell interception channel; the capture channel is used for contact between microspheres and cells; the outlet of the first microsphere interception channel is connected to the capture channel, for allowing the first microsphere solution to flow, and for intercepting the microspheres in the first microsphere solution in the capture channel; the outlet of the second microsphere interception channel is connected to the capture channel, for allowing the second microsphere solution to flow, and for intercepting the microspheres in the second microsphere solution in the capture channel; the outlet of the cell interception channel is connected to the capture channel, for allowing the cell fluid to flow, and for intercepting the cells in the cell fluid in the capture channel.

[0038] In this scheme, the above-mentioned channels can achieve rapid capture of single cells, avoid the waste of a large number of cells, break through the Poisson distribution problem in high-throughput single-cell sequencing technology, and quickly complete the sorting and analysis of target cells. At the same time, this scheme can achieve accurate pairing of single cells and two different microspheres, which can increase the accuracy of detection, broaden the detection range, increase the reliability of experimental results, and facilitate data standardization and visualization. BRIEF DESCRIPTION OF THE DRAWINGS

[0039] In order to more clearly illustrate the embodiments of the present application or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present application. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative labor.

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

[0041] Figure 2 A diagram showing the changes in the main structure manufacturing process of a microfluidic chip suitable for active single-cell multi-omics provided in an embodiment of the present application;

[0042] Figure 3 A diagram showing the changes in the manufacturing process of a pneumatic valve body of a microfluidic chip suitable for active single-cell multi-omics provided in an embodiment of the present application;

[0043] In the figure:

[0044] 10. First microsphere interception channel; 11. First microsphere stop valve;

[0045] 20. Second microsphere interception channel; 21. Second microsphere stop valve;

[0046] 30. Cell cut-off channel; 31. Cell cut-off valve;

[0047] 40. Capture channel; 41. First stop valve; 42. Second stop valve; 43. Third stop valve;

[0048] 50. Parcel channel;

[0049] 60. Waste liquid channel; 61. Waste liquid stop valve;

[0050] 70. First sheath liquid shutoff channel; 71. First sheath liquid stop valve;

[0051] 80. A second sheath liquid shutoff channel; 81. A second sheath liquid stop valve;

[0052] 91. Positive resist layer; 92. Base layer; 93. First negative resist layer; 94. Second negative resist layer; 95. Third negative resist layer. DETAILED DESCRIPTION

[0053] The technical solutions of the embodiments of the present application will be described clearly and completely below in conjunction with the accompanying drawings. Obviously, the described embodiments are part of the embodiments of the present application, not all of the embodiments. Based on the embodiments in the specification of the present application, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection requested by the present application.

[0054] In the description of the embodiments of the present application, it should be noted that the terms "center", "upper", "lower", "left", "right", "vertical", "horizontal", "inner", "outer", etc. indicate positions or positional relationships based on the positions or positional relationships shown in the accompanying drawings, which are only for the convenience of describing the embodiments of the present application and simplifying the description, and do not indicate or imply that the device or element referred to must have a specific orientation, be constructed and operated in a specific orientation, and therefore cannot be understood as limiting the embodiments of the present application. In addition, the terms "first", "second", and "third" are used for descriptive purposes only 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 clearly specified and limited, the terms "installed", "connected", and "connected" should be understood in a broad sense, for example, it can be a fixed connection, a replaceable connection, or an integral connection, it can be a mechanical connection, it can be an electrical connection, it can be a direct connection, it can be indirectly connected through an intermediate medium, and it can be the internal communication of two components. For ordinary technicians in this field, the specific meanings of the above terms in the embodiments of the present application can be understood according to specific circumstances.

[0056] See also Figure 1 A microfluidic chip suitable for active single-cell multi-omics provided in an embodiment of the present application includes: 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 the microspheres to contact with the cells, that is, both the microsphere solution and the cell solution can flow into the capture channel 40 .

[0058] The outlet of the first microsphere interception channel 10 is connected to the capture channel 40; the first microsphere interception channel 10 is used for the first microsphere solution to flow. 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 protein group microsphere solution; in actual application, the first microsphere solution can be, for example, a polystyrene microsphere solution, a magnetic bead microsphere solution, etc.

[0059] The outlet of the second microsphere interception channel 20 is connected to the capture channel 40; the second microsphere interception channel 20 is used for the flow of the second microsphere solution. The microspheres in the second microsphere solution can be cut off in the capture channel 40 through the second microsphere interception channel 20. The second microsphere solution can be used as a transcriptome microsphere solution; in actual application, the second microsphere solution can be, for example, a hydrogel microsphere solution, a gel microsphere solution, etc. It should be noted that in actual application, 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 interception channel 30 is connected to the capture channel 40 ; the cell interception channel 30 is used for allowing the cell fluid to flow; the cells in the cell fluid can be intercepted in the capture channel 40 through the cell interception channel 30 .

[0061] Single-cell capture using the microfluidic chip provided in this embodiment can overcome the Poisson distribution problem in high-throughput single-cell sequencing technology, quickly complete sorting and analysis of target cells, and achieve fast, accurate and easy-to-operate capture of single cells.

[0062] At the same time, in the process of capturing single cells, the first microsphere interception channel 10 and the second microsphere interception channel 20 can be used simultaneously to capture and pair two types of microspheres and single cells at the same time; the first microsphere interception channel 10 and the second microsphere interception channel 20 can also be used only one of them to achieve the capture of a single type of microsphere and a single cell. That is, the microfluidic chip in this embodiment can also broaden the use scenarios of the microfluidic chip and increase its applicability.

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

[0064] 1. Since different microspheres can carry different markers or probes, single cells can be tested in many aspects. For example, one microsphere carries antibodies against specific antigens on the cell surface, and another microsphere carries fluorescent probes for detecting specific molecules in the cell. In this way, cells can be analyzed from both the outside and inside of the cell, and the results can be verified with each other to improve the accuracy of the test.

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

[0066] 3. When conducting a control experiment or internal reference, one of the microspheres can be used as a control and compared with another microsphere used for actual detection. By comparing the signal changes of the microspheres, non-specific interference or systematic errors in the experimental process can be eliminated to ensure the reliability of the experimental results. For example, in a drug screening experiment, a microsphere known to have a specific response to cells is used as a control, and another microsphere carrying the drug to be screened is used to conduct experiments at the same time to verify the effect of the drug.

[0067] 4. The control microspheres can be used as an internal reference to standardize data from different samples or experimental conditions. Since there may be differences between different experimental batches or samples, the signal of the control microspheres can be used as a benchmark to compare the signals of other microspheres with, thereby eliminating the impact of these differences on the results.

[0068] 5. The two microspheres can bind to single cells through different interaction modes to improve the capture efficiency. 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. The two work together to capture single cells more stably.

[0069] 6. For different types of single cells, two microspheres with different specificities can be used for differentiated 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. The two microspheres can carry different reagents or functional molecules 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 and the single cell are encapsulated in a system, specific chemical reactions can be carried out locally in the cell to study the cell's response to the reaction.

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

[0072] It should be noted that the channels on the microfluidic chip can be cut off for microspheres or single cells by marking the cells with specific fluorescent dyes or fluorescently labeled antibodies, and then controlling the flow and stop of the fluid in each channel by a micropump.

[0073] For example, specific molecules in cells, such as nucleic acids and proteins, 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 fluorescent signals when excited by light of a specific wavelength. At the same time, since the channel width on the microfluidic chip is generally tens of microns, and the diameter of most eukaryotic cells is between 10 and 30 microns, the size of the microspheres is slightly different, for example, polyacrylamide microspheres are generally between tens of nanometers and hundreds of microns; therefore, the channel on the microfluidic chip can be set to only allow single cells and single microspheres to flow. An identification point can be set in the capture channel 40. When a microsphere or cell is identified to enter the capture channel 40, the microsphere solution or cell fluid stops flowing by closing the valve or the like, and the single microsphere or single cell is cut off in the capture channel 40.

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

[0075] In one embodiment, a first microsphere cut-off valve 11 is provided on the first microsphere interception channel 10; the first microsphere cut-off valve 11 is used to cut off the microspheres in the first microsphere solution in the capture channel 40; a second microsphere cut-off valve 21 is provided on the second microsphere interception channel 20; the second microsphere cut-off valve 21 is used to cut off the microspheres in the second microsphere solution in the capture channel 40; a cell cut-off valve 31 is provided on the cell interception channel 30; the cell cut-off valve 31 is used to cut off the cells in the cell fluid 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, the first microsphere stop valve 11, the second microsphere stop valve 21 and the cell stop valve 31 in this embodiment can more accurately control the flow and stop of the fluid, and achieve accurate regulation of the flow rate and flow of the cell or microsphere suspension, so that the target cells can accurately stay at the capture site, ensuring the accuracy and repeatability of the capture.

[0077] At the same time, the good sealing performance of the stop 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 cells or microspheres in the chip, prevent the captured cells or microspheres from returning to the fluid due to backflow, and ensure the stability of the capture process.

[0078] As an embodiment, the first microsphere stop valve 11, the second microsphere stop valve 21 and the cell stop valve 31 are all pneumatic valves, which are usually located in a specific area of ​​the chip and cooperate with other structural layers in the channel. Specifically, the pneumatic valve can achieve precise control of the flow of liquid or gas in the channel of the microfluidic chip by adjusting the gas pressure. For example, a specific channel can be opened or closed to adjust the flow rate and flow of the fluid. 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 raise the thin film layer to prevent the fluid from passing. When the fluid needs to flow, the thin film layer drops when the pressure is released to open the channel.

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

[0080] The first stop valve 41 and the second stop valve 42 can stop the two kinds of microspheres and single cells between them, which is conducive to 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 stop valve 41 and the second stop valve 42 can give the microspheres and the single cells more opportunities to approach each other and physically or chemically combine.

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

[0082] In another embodiment, it also includes: a wrapping channel 50; the outlet of the capture channel 40 is connected to the wrapping channel 50; the capture channel 40 is used to supply flushing liquid to flow after the microspheres and cells come into contact with each other, so as to flush the microspheres and cells to the wrapping channel 50; the wrapping channel 50 is used to supply wrapping liquid to flow, so that the droplets of the wrapping liquid wrap the microspheres and cells.

[0083] Injecting flushing fluid into the capture channel 40 can wash away the unbound portion, thereby improving the purity of the target (microspheres bound to single cells) in the subsequent encapsulation process and reducing interference in subsequent experiments.

[0084] After the flushing liquid flushes the microspheres combined with single cells into the encapsulation channel 50, the encapsulation liquid flows into the encapsulation channel 50 to encapsulate the single cells and the microspheres, thereby completing the single cell multi-omics capture or the transcriptome capture of cell interactions. The encapsulation liquid can be fluorocarbon oil, fluorinated oil, mineral oil, silicone oil or other oils.

[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 between the droplet and the wrapping channel 50, thereby reducing the adhesion of certain substances in the reagent components to the channel and causing the droplet to be pulled and broken.

[0087] Optionally, the height of the wrapping channel 50 is at least twice that of the capture channel 40. Specifically, the height of the capture channel 40 may be 40 microns, and the height of the wrapping channel 50 is greater than 80 microns. In practical applications, the size of the droplets formed after the wrapping liquid wraps the target object is generally about 120 microns. In this embodiment, setting the height of the wrapping channel 50 to be at least twice that of the capture channel 40 can reduce the contact area between the droplets and the target object while ensuring that the droplets can flow individually.

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

[0089] In application, the pneumatic valve closes the channel by applying pressure to make the film fit the inner wall of the channel or opens the channel by removing the pressure to make the film leave the inner wall of the channel. In this embodiment, the capture channel 40 and / or the cell interception channel 30 are curved channels to increase the sealing effect when the film 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 channel side on the particle flow rate.

[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, so as to allow the waste liquid in the capture channel 40 to flow out.

[0092] Optionally, a waste liquid shut-off valve 61 is provided on the waste liquid channel 60 to control the on-off of the waste liquid channel 60 .

[0093] Optionally, a third stop valve 43 is provided 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, and is used to intercept the cells in the cell fluid between the third stop valve 43 and the inlet of the waste liquid channel 60.

[0094] Similarly, the third stop valve 43 may be a pneumatic valve. When the third stop valve 43 identifies a cell passing through the outlet of the cell interception channel 30 at the identification point, the capture channel 40 is closed to ensure that the cell is intercepted between the third stop valve 43 and the outlet of the cell interception channel 30 to prevent the cell from being carried away by the fluid.

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

[0096] The first sheath liquid interception channel 70 may be provided with a first sheath liquid shut-off valve 71; the second sheath liquid interception channel 8 may be provided with a second sheath liquid shut-off valve 81; the flow and shut-off of the sheath liquid in the first sheath liquid interception channel 70 and the second sheath liquid interception channel 80 may be controlled respectively by the first sheath liquid shut-off valve 71 and the second sheath liquid shut-off valve 81.

[0097] In the above embodiment, the microfluidic chip can be manufactured by the following steps. Figure 2 , which includes a positive resist layer 91 , a base layer 92 , a first negative resist layer 93 and a second negative resist layer 94 .

[0098] In the first step, the positive photoresist layer 91 disposed on the base layer 92 is coated, and then the positive photoresist layer 91 is subjected to photolithography and development to form a rectangular channel;

[0099] In the second step, the rectangular channel is heat-melted at a temperature of more than 150° C. for more than 3 hours to form an arc channel. The arc channel can make it easier for the PDMS film in the pneumatic valve to adhere to the upper wall of the channel, thereby closing the channel. The height of the arc channel can be 35 microns.

[0100] The third step is to overlay a slightly higher second negative resist layer 94 on the positive resist layer 91 forming the arc-shaped flow channel. The formed flow channel structure is rectangular, which can reduce the influence of the flow channel side on the particle flow rate. The flow channel overlayed with the second negative resist layer 94 can be the first microsphere intercepting channel 10 and the second microsphere intercepting channel 20. The height of the rectangular flow channel formed after the second negative resist layer 94 is overlaid can be 40 microns.

[0101] In the fourth step, a first negative resist layer 93 with a thickness higher than 80 microns is overetched on the wrapped channel 50. This structure can reduce the contact between the droplets and the channel wall, thereby reducing the wetting effect of the droplets.

[0102] The production of the above-mentioned pneumatic valve can refer to Figure 3, which includes a base layer 92 and a third negative resist layer 95; a flow channel is overetched on the third negative resist layer 95 to serve as the main structure of the pneumatic valve. The height of the overetched second positive resist layer 95 can be 30 microns.

[0103] The above are only preferred embodiments of the present application and are not intended to limit the present invention. Although the present application has been described in detail with reference to the examples, those skilled in the art can still modify the technical solutions recorded in the aforementioned examples or make equivalent substitutions for some of the technical features therein. However, any modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the present application should 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) for allowing the microspheres to contact the cells; A first microsphere intercepting channel (10), the outlet of the first microsphere intercepting channel (10) being connected to the capture channel (40) and being used for allowing the first microsphere solution to flow, and for intercepting the microspheres in the first microsphere solution in the capture channel (40); A second microsphere intercepting channel (20), wherein the outlet of the second microsphere intercepting channel (20) is connected to the capture channel (40) and is used for allowing the second microsphere solution to flow, and for intercepting the microspheres in the second microsphere solution in the capture channel (40); A cell interception channel (30), wherein the outlet of the cell interception channel (30) is connected to the capture channel (40) and is used for allowing the cell fluid to flow, and for intercepting the cells in the cell fluid within the capture channel (40).

2. The microfluidic chip suitable for active single-cell multi-omics according to claim 1, characterized in that: The capture channel (40) is provided with a first stop valve (41) and a second stop valve (42) at intervals; The first stop valve (41) and the second stop valve (42) are used to control the on and off of the capture channel (40); The outlet of the first microsphere intercepting channel (10), the outlet of the second microsphere intercepting channel (20) and the outlet of the cell intercepting channel (30) are all located between the first stop valve (41) and the second stop valve (42).

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

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

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

6. The microfluidic chip suitable for active single-cell multi-omics according to claim 2, characterized in that: The first microsphere intercepting channel (10) is provided with a first microsphere stop valve (11); The first microsphere stop valve (11) is used to stop the microspheres in the first microsphere solution in the capture channel (40); The second microsphere intercepting channel (20) is provided with a second microsphere stop valve (21); The second microsphere stop valve (21) is used to stop the microspheres in the second microsphere solution in the capture channel (40); The cell interception channel (30) is provided with a cell shut-off valve (31); The cell shut-off valve (31) is used to shut off the cells in the cell fluid within the capture channel (40).

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

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

9. The microfluidic chip suitable for active single-cell multi-omics according to claim 8, characterized in that: The capture channel (40) is provided with a third stop valve (43); 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), and is used to intercept the cells in the cell fluid between the third stop valve (43) and the inlet of the waste liquid channel (60).

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

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