Microfluidic chip suitable for active single-cell multi-omics

Abstract

A microfluidic chip suitable for active single-cell multi-omics, comprising a capture channel, a first microsphere interception channel, a second microsphere interception channel, and a cell interception channel. The capture channel is configured for enabling contact between microspheres and cells. The first microsphere interception channel has an outlet in communication with the capture channel and is configured for allowing a first microsphere solution to flow and for intercepting microspheres in the first microsphere solution within the capture channel. The second microsphere interception channel has an outlet in communication with the capture channel and is configured for allowing a second microsphere solution to flow and for intercepting microspheres in the second microsphere solution within the capture channel. The cell interception channel has an outlet in communication with the capture channel and is configured for allowing a cell solution to flow and for intercepting cells in the cell solution within the capture channel. In the present solution, the above channels enable rapid capture of single cells, preventing excessive cell loss, and achieving precise pairing of individual cells with two different types of microspheres.

Description

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

[0001] This application claims priority to Chinese Patent Application No. 202411790374.8, filed on December 6, 2024, entitled “A Microfluidic Chip for Active Single-Cell Multi-Omics”, the entire contents of which are incorporated herein by reference. Technical Field

[0002] This application relates to the field of gene therapy technology, and in particular to a microfluidic chip suitable for active single-cell multi-omics. Background Technology

[0003] Single-cell multi-omics technology is typically used to measure multiple dimensions of information from the same cell, including the genome, transcriptome, epigenome, and proteome. Single-cell multi-omics technology can address scientific challenges such as the averaging effect of population-based sequencing, low initial cell counts preventing sequencing, and high cellular heterogeneity. Furthermore, the high-resolution data produced by single-cell technology helps researchers discover more refined genomic structural features and gain a more intuitive understanding of processes such as gene expression regulation, early embryonic development, and tumor formation and development.

[0004] In the field of cell and gene therapy, in addition to traditional methods such as surgical resection, radiotherapy, and chemotherapy, cell therapy is an emerging approach. Cell therapy mainly relies on modifying and reinfusing the body's own immune cells to kill cancer cells.

[0005] For example, CAR-T immunotherapy has achieved preliminary success in the treatment of hematologic malignancies in existing clinical applications; TCR-T cell therapy, by recognizing tumor-specific antigens from the cell membrane surface or intracellular sources, is expected to make breakthroughs in the treatment of solid tumors; and in immunotherapy research on diseases such as lung cancer, it has been found that TCR specificity that can target tumor cells will be one of the important links in solving off-target effects.

[0006] In cell therapy, single-cell capture can be used to isolate and obtain individual cells for subsequent analysis and research. Microfluidics is a commonly used single-cell capture technique.

[0007] In existing microfluidic technologies, the commonly used method for capturing single cells and microspheres generally involves encapsulating single cells and the microsphere reagents required for subsequent reactions within a droplet microreactor. The cell suspension is diluted to a specific concentration and uniformly dispersed. Then, equal volumes are repeatedly sampled or divided into equal volumes to generate monodisperse droplets. Cells in the suspension are randomly dispersed into the droplets, and the number of cells in each droplet follows a Poisson distribution. This method is rapid and simple; however, the single-cell encapsulation probability is low, reaching a maximum of only about 40%, and a large proportion of these droplets are multi-cell droplets (containing two or more cells). This method results in the loss of a significant number of cells and can only capture single microspheres and single cells. Summary of the Invention

[0008] In view of this, the purpose of this application is to provide a microfluidic chip suitable for active single-cell multi-omics, which can solve some or all of the above problems.

[0009] To achieve the above technical objectives, this application provides a microfluidic chip suitable for active single-cell multi-omics, comprising:

[0010] The trapping channel is used to allow the microspheres to contact the cells;

[0011] A first microsphere interception channel, the outlet of which is connected to the capture channel, is used to supply the flow of the first microsphere solution and to trap the microspheres in the first microsphere solution within the capture channel;

[0012] The second microsphere interception channel has an outlet connected to the capture channel for supplying the flow of the second microsphere solution and for trapping the microspheres in the second microsphere solution within the capture channel.

[0013] A cell interception channel, the outlet of which is connected to the capture channel, is used for the flow of cell fluid and for trapping cells in the cell fluid within the capture channel.

[0014] Furthermore, a first shut-off valve and a second shut-off valve are provided at intervals on the capture channel;

[0015] The first shut-off valve and the second shut-off valve are used to control the opening and closing of the capture channel;

[0016] The outlets of the first microsphere interception channel, the second microsphere interception channel, and the cell interception channel are all located between the first shut-off valve and the second shut-off valve.

[0017] Furthermore, it also includes: parcel access;

[0018] The exit of the capture channel is connected to the package channel;

[0019] The capture channel is used to allow the flushing fluid to flow after the microspheres come into contact with the cells, thereby flushing the microspheres and cells into the encapsulation channel;

[0020] The encapsulation channel is used to allow the encapsulation fluid to flow, so that the droplets of the encapsulation fluid encapsulate the microspheres and cells.

[0021] Furthermore, the height of the package channel is greater than the height of the capture channel.

[0022] Furthermore, the height of the package channel is at least twice that of the capture channel.

[0023] Furthermore, a first microsphere shut-off valve is provided on the first microsphere interception channel;

[0024] The first microsphere shut-off valve is used to stop the microspheres in the first microsphere solution within the capture channel;

[0025] A second microsphere shut-off valve is provided on the second microsphere interception channel;

[0026] The second microsphere shut-off valve is used to stop the microspheres in the second microsphere solution within the capture channel;

[0027] A cell shut-off valve is provided on the cell interception channel;

[0028] The cell shut-off valve is used to shut off the cells in the cell fluid within the capture channel.

[0029] Furthermore, the capture channel and / or the cell interception channel are arc-shaped channels with an arc-shaped top surface;

[0030] The first shut-off valve, the second shut-off valve, and the cell shut-off valve are pneumatic valves.

[0031] Furthermore, it also includes: waste liquid channel;

[0032] The inlet of the waste liquid channel is connected to the capture channel, and is used to allow the waste liquid in the capture channel to flow out.

[0033] Furthermore, a third shut-off valve is provided on the capture channel;

[0034] The third shut-off valve is located between the inlet of the waste liquid channel and the outlet of the cell interception channel, and is used to shut off the cells in the cell fluid between the third shut-off valve and the inlet of the waste liquid channel.

[0035] Furthermore, it also includes: a first sheath fluid interception channel and a second sheath fluid interception channel;

[0036] The outlet of the first sheath fluid interception channel is connected to the first microsphere interception channel;

[0037] The outlet of the first microsphere interception channel is connected to the second microsphere interception channel.

[0038] As can be seen from the above technical solutions, this 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 microspheres to contact cells; the outlet of the first microsphere interception channel is connected to the capture channel, used for the flow of a first microsphere solution, and used to trap microspheres in the first microsphere solution within the capture channel; the outlet of the second microsphere interception channel is connected to the capture channel, used for the flow of a second microsphere solution, and used to trap microspheres in the second microsphere solution within the capture channel; the outlet of the cell interception channel is connected to the capture channel, used for the flow of cell fluid, and used to trap cells in the cell fluid within the capture channel.

[0039] This approach enables rapid capture of single cells through the aforementioned channels, avoiding the waste of a large number of cells, overcoming the Poisson distribution problem in high-throughput single-cell sequencing technology, and allowing for rapid sorting and analysis of target cells. Furthermore, this approach allows for precise pairing of single cells with two different types of microspheres, increasing detection accuracy, broadening the detection range, enhancing the reliability of experimental results, and facilitating data standardization and visualization. Attached Figure Description

[0040] To more clearly illustrate the technical solutions in the embodiments of this application or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of this application. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0041] Figure 1 is a schematic diagram of the channels of a microfluidic chip suitable for active single-cell multi-omics provided in an embodiment of this application;

[0042] Figure 2 shows the changes in the fabrication process of the main structure of a microfluidic chip suitable for active single-cell multi-omics according to an embodiment of this application;

[0043] Figure 3 is a diagram showing the manufacturing process of a pneumatic valve body for a microfluidic chip suitable for active single-cell multi-omics, according to an embodiment of this application.

[0044] In the diagram: 10, First microsphere interception channel; 11, First microsphere shut-off valve; 20, Second microsphere interception channel; 21, Second microsphere shut-off valve; 30, Cell interception channel; 31, Cell shut-off valve; 40, Capture channel; 41, First shut-off valve; 42, Second shut-off valve; 43, Third shut-off valve; 50, Encapsulation channel; 60, Waste liquid channel; 61, Waste liquid shut-off valve; 70, First sheath fluid interception channel; 71, First sheath fluid shut-off valve; 80, Second sheath fluid interception channel; 81, Second sheath fluid shut-off valve; 91, Positive colloid layer; 92, Base layer; 93, First negative colloid layer; 94, Second negative colloid layer; 95, Third negative colloid layer. Detailed Implementation

[0045] The technical solutions of the embodiments of this application 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 this application. Based on the embodiments in this application specification, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection claimed in this application.

[0046] In the description of the embodiments of this application, it should be noted that the terms "center," "upper," "lower," "left," "right," "vertical," "horizontal," "inner," and "outer," etc., indicate the orientation or positional relationship based on the orientation or positional relationship shown in the accompanying drawings. They are only for the convenience of describing the embodiments of this application and simplifying the description, and do not indicate or imply that the device or element referred to must have a specific orientation, or be constructed and operated in a specific orientation. Therefore, they should not be construed as limitations on the embodiments of this application. In addition, the terms "first," "second," and "third" are used for descriptive purposes only and should not be construed as indicating or implying relative importance.

[0047] In the description of the embodiments of this application, it should be noted that, unless otherwise explicitly specified and limited, the terms "installation," "connection," and "linking" should be interpreted broadly. For example, they can refer to a fixed connection, a replaceable connection, or an integral connection; they can refer to a mechanical connection or an electrical connection; they can refer to a direct connection or an indirect connection through an intermediate medium; and they can refer to the internal connection of two components. Those skilled in the art can understand the specific meaning of the above terms in the embodiments of this application according to the specific circumstances.

[0048] Please refer to Figure 1. A microfluidic chip suitable for active single-cell multi-omics provided in this embodiment includes: a capture channel 40, a first microsphere interception channel 10, a second microsphere interception channel 20, and a cell interception channel 30.

[0049] The capture channel 40 is used to allow the microspheres to contact the cells, meaning that both the microsphere solution and the cell fluid can flow into the capture channel 40.

[0050] The outlet of the first microsphere interception channel 10 is connected to the capture channel 40; the first microsphere interception channel 10 is used to supply the flow of the first microsphere solution. The first microsphere interception channel 10 can trap the microspheres in the first microsphere solution within the capture channel 40. The first microsphere solution can be a proteomic microsphere solution; in practical applications, the first microsphere solution can be, for example, a polystyrene microsphere solution, a magnetic bead microsphere solution, etc.

[0051] The outlet of the second microsphere interception channel 20 is connected to the capture channel 40; the second microsphere interception channel 20 is used to supply the flow of the second microsphere solution. The second microsphere interception channel 20 can trap the microspheres in the second microsphere solution within the capture channel 40. 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 and second microsphere solutions can be interchanged, that is, the first microsphere solution can be used as a transcriptome microsphere solution, and the second microsphere solution as a proteome microsphere solution. The first and second microsphere solutions can also be other multi-omics microsphere solutions.

[0052] The outlet of the cell interception channel 30 is connected to the capture channel 40; the cell interception channel 30 is used to supply cell fluid flow; the cell interception channel 30 can trap cells in the cell fluid within the capture channel 40.

[0053] The microfluidic chip provided in this embodiment can overcome the Poisson distribution problem in high-throughput single-cell sequencing technology for single-cell capture. It can quickly complete the sorting and analysis of target cells, and achieve rapid, accurate and simple capture of single cells.

[0054] Simultaneously, during the capture of single cells, the first microsphere interception channel 10 and the second microsphere interception channel 20 can be used simultaneously to capture and pair both types of microspheres and single cells at the same time; alternatively, only one of the first microsphere interception channel 10 and the second microsphere interception channel 20 can be used to achieve the capture of a single type of microsphere and single cell. That is, the microfluidic chip in this embodiment can also broaden the application scenarios of microfluidic chips and increase their applicability.

[0055] 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 trap microspheres in the first microsphere solution, microspheres in the second microsphere solution, and single cells within the trapping channel 40, which has at least the following advantages:

[0056] 1. Because different microspheres can carry different markers or probes, multifaceted detection of single cells can be performed. For example, one type of microsphere carries an antibody against a specific antigen on the cell surface, while another carries a fluorescent probe to detect specific molecules inside the cell. This allows for simultaneous analysis of the cell from both external and internal perspectives, cross-validating results and improving detection accuracy.

[0057] 2. Capturing two types of microspheres allows for the simultaneous detection of multiple biomarkers or molecules, providing more comprehensive cellular information. For example, in disease diagnosis, one type of microsphere can be used to detect inflammation-related markers, while the other can be used to detect tumor-related markers, enabling a more accurate determination of the disease type and stage.

[0058] 3. In control experiments or internal references, one type of microsphere can serve as a control, compared with another microsphere used for actual detection. By observing the signal changes of the control microsphere, non-specific interference or systematic errors during the experiment can be eliminated, ensuring the reliability of the experimental results. For example, in drug screening experiments, a microsphere known to have a specific response to cells can be used as a control, simultaneously tested with another microsphere carrying the drug to be screened, to verify the drug's efficacy.

[0059] 4. Control microspheres can serve as an internal reference for standardizing 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 act as a benchmark, allowing comparison with the signals of other microspheres to eliminate the influence of these differences on the results.

[0060] 5. The two types of microspheres can bind to single cells through different interaction mechanisms, thereby improving capture efficiency. For example, one type of microsphere has ligands that bind to specific receptors on the cell surface, while the other type has functional groups that interact with the surrounding matrix. The combined effect of both can more stably capture single cells.

[0061] 6. For different types of single cells, two types of microspheres with different specificities can be used for differentiated capture. For example, in complex cell populations, one type of microsphere specifically captures a particular type of cancer cell, while another type captures normal cells, thereby achieving specific separation and analysis of different cell types.

[0062] 7. Two types of microspheres can each carry different reagents or functional molecules, forming a multifunctional reaction system around a single cell. For example, one type of microsphere carries an enzyme, and the other carries a substrate. When both types of microspheres are co-encapsulated with a single cell in a system, specific chemical reactions can occur locally within the cell, allowing for the study of the cell's response to these reactions.

[0063] 8. In practical applications, depending on experimental needs, one type of microsphere can be used for preliminary treatment or labeling before introducing another type of microsphere for subsequent operations. This step-by-step approach allows for more flexible control of the experimental process, enabling precise analysis of single cells.

[0064] It should be noted that the channels on the microfluidic chip can be closed by microspheres or single cells using specific fluorescent dyes or fluorescently labeled antibodies to label the cells, and then the flow and cessation of fluid in each channel can be controlled by micropumps.

[0065] For example, fluorescent dyes can be used to label specific molecules within cells, such as nucleic acids and proteins, or fluorescently labeled antibodies can be used to recognize specific antigens on the cell surface. In this way, the labeled cells will emit a fluorescent signal when excited by light of a specific wavelength. Meanwhile, since the channel width on a microfluidic chip is typically tens of micrometers, while the diameter of most eukaryotic cells is between 10 and 30 micrometers, and the size of microspheres varies slightly (e.g., polyacrylamide microspheres are generally between tens of nanometers and hundreds of micrometers), the channels on the microfluidic chip can be configured to allow only a single cell or a single microsphere to flow. A recognition point can be set within the capture channel 40. When a microsphere or cell is detected entering the capture channel 40, the flow of the microsphere solution or cell fluid is stopped by closing a valve or other means, thus trapping the single microsphere or single cell within the capture channel 40.

[0066] As one implementation, a chamber may be provided inside the capture channel 40; microspheres and single cells entering the capture channel 40 can fall into the chamber and are not easily washed away by the solution.

[0067] In one embodiment, a first microsphere cutoff valve 11 is provided on the first microsphere interception channel 10; the first microsphere cutoff valve 11 is used to cut off the microspheres in the first microsphere solution within the capture channel 40; a second microsphere cutoff valve 21 is provided on the second microsphere interception channel 20; the second microsphere cutoff valve 21 is used to cut off the microspheres in the second microsphere solution within the capture channel 40; a cell cutoff valve 31 is provided on the cell interception channel 30; the cell cutoff valve 31 is used to cut off the cells in the cell fluid within the capture channel 40.

[0068] Compared to the aforementioned method of using micropumps to control the flow and stop of fluid for cutoff, this embodiment, by setting the first microsphere cutoff valve 11, the second microsphere cutoff valve 21, and the cell cutoff valve 31, can more precisely control the flow and stop of fluid, achieving precise regulation of the flow rate and volume of the cell or microsphere suspension, allowing the target cells to accurately remain at the capture site. This ensures the accuracy and repeatability of the capture.

[0069] Meanwhile, the excellent sealing performance of the shut-off valve effectively prevents fluid backflow, maintaining unidirectional fluid flow within the microfluidic chip. This helps maintain a stable distribution of cells or microspheres within the chip, preventing captured cells or microspheres from returning to the fluid due to backflow, thus ensuring the stability of the capture process.

[0070] In one implementation, the first microsphere shut-off valve 11, the second microsphere shut-off valve 21, and the cell shut-off valve 31 described above are all pneumatic valves, typically located in specific areas of the chip and cooperating with other structural layers within the channels. Specifically, the pneumatic valves, by regulating gas pressure, can achieve precise control of the flow of liquid or gas within the channels of the microfluidic chip. For example, specific channels can be opened or closed, adjusting the fluid velocity and flow rate. When it is necessary to prevent fluid from flowing in a certain channel, the pneumatic valve can close the channel by applying pressure to raise the thin film layer, preventing fluid from passing through. Conversely, when it is necessary to allow fluid to flow, releasing the pressure causes the thin film layer to descend and open the channel.

[0071] In one embodiment, a first shut-off valve 41 and a second shut-off valve 42 are spaced apart on the capture channel 40; 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 outlet of the first microsphere interception channel 10, the outlet of the second microsphere interception channel 20 and the outlet of the cell interception channel 30 are all located between the first shut-off valve 41 and the second shut-off valve 42.

[0072] The first shut-off valve 41 and the second shut-off valve 42 can separate the two types of microspheres and single cells, facilitating contact between them. Specifically, the movement of microspheres and single cells in solution has a certain degree of randomness. The space between the first shut-off valve 41 and the second shut-off valve 42 allows microspheres and single cells more opportunities to approach each other and undergo physical or chemical bonding.

[0073] In practical applications, the width of the capture channel 40 can be set to be greater than the width of the first microsphere interception channel 10, greater than the width of the second microsphere interception channel 20, and greater than the width of the cell interception channel 30, in order to improve the fault tolerance rate of the particle cutoff landing point.

[0074] In another embodiment, it further includes: a wrapping channel 50; an outlet of a capture channel 40 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, thereby flushing the microspheres and cells to the wrapping channel 50; the wrapping channel 50 is used to allow wrapping fluid to flow, so that the droplets of wrapping fluid wrap the microspheres and cells.

[0075] Injecting flushing fluid into the capture channel 40 can wash away unbound portions, improving the purity of the target material (microspheres bound to single cells) during subsequent encapsulation, while reducing interference in subsequent experiments.

[0076] After the flushing fluid washes the microspheres containing single cells into the encapsulation channel 50, encapsulation fluid flows into the encapsulation channel 50 to encapsulate the single cells and microspheres, completing the single-cell multi-omics capture or transcriptome capture of cell interactions. The encapsulation fluid can be an oil such as fluorocarbon oil, fluorinated oil, mineral oil, or silicone oil.

[0077] It should be noted that the second shut-off valve 42 on the capture channel 40 can be used to control the flow and stop of the flushing fluid.

[0078] In one embodiment, the height of the encapsulation channel 50 is greater than the height of the capture channel 40. Specifically, increasing the height of the encapsulation channel 50 reduces the contact area between the droplet and the encapsulation channel 50, thereby reducing the likelihood of certain substances in the reagent component adhering to the channel and causing the droplet to be pulled and broken.

[0079] Optionally, the height of the encapsulation channel 50 is at least twice that of the capture channel 40. Specifically, the height of the capture channel 40 can be 40 micrometers, and the height of the encapsulation channel 50 is greater than 80 micrometers. In practical applications, the size of the droplets formed after the encapsulation liquid encapsulates the target object is generally around 120 micrometers. In this embodiment, setting the height of the encapsulation 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.

[0080] In one embodiment, 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.

[0081] In application, the pneumatic valve applies pressure to cause the membrane to adhere to the inner wall of the channel to close the channel, or releases pressure to cause the membrane to leave the inner wall of the channel to open the channel. In this embodiment, the capture channel 40 and / or the cell interception channel 30 are curved channels, which can increase the sealing effect when the membrane contacts the wall surface.

[0082] Optionally, both the first microsphere interception channel 10 and the second microsphere interception channel 20 are rectangular channels, which can reduce the influence of the channel sides on the particle velocity.

[0083] 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.

[0084] Optionally, a waste liquid shut-off valve 61 is provided on the waste liquid channel 60 to control the opening and closing of the waste liquid channel 60.

[0085] Optionally, 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, and is used 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.

[0086] 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.

[0087] 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.

[0088] 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.

[0089] In the above embodiments, the microfluidic chip can be fabricated through the following steps, as shown in Figure 2, which includes a positive adhesive layer 91, a substrate layer 92, a first negative adhesive layer 93, and a second negative adhesive layer 94.

[0090] 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.

[0091] 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.

[0092] 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.

[0093] 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.

[0094] The fabrication of the aforementioned pneumatic valve can be referred to Figure 3, which includes a base layer 92 and a third negative adhesive layer 95; the main structure of the pneumatic valve is formed by etching flow channels onto the third negative adhesive layer 95. The height of the etched second positive adhesive layer 95 can be 30 micrometers.

[0095] The above are merely preferred embodiments of this application and are not intended to limit the present invention. Although this application has been described in detail with reference to examples, those skilled in the art can still modify the technical solutions described in the foregoing examples or make equivalent substitutions for some of the technical features. However, any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of this application should be included within the protection scope of this application.

Claims

1. A microfluidic chip suitable for active single-cell multi-omics, characterized in that, include: The capture channel (40) is used to allow 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 second microsphere interception channel (20) has its outlet 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).

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 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).

3. The microfluidic chip suitable for active single-cell multi-omics according to claim 1, characterized in that, Also includes: Parcel channel (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.

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 greater than the height of the capture channel (40).

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

6. The microfluidic chip suitable for active single-cell multi-omics according to claim 2, 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).

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 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.

8. The microfluidic chip suitable for active single-cell multi-omics according to any one of claims 1 to 7, characterized in that, It 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.

9. The microfluidic chip suitable for active single-cell multi-omics according to claim 8, 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).

10. 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 first microsphere interception channel (10) is connected to the second microsphere interception channel (20).

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