A device and method for cell-microbead capture pairing for single-cell omics analysis

Abstract

The present invention provides a device and method for cell-microbead capture pairing for single-cell omics analysis, the device comprising: a microwell capture layer, which comprises: a microwell array composed of a plurality of microwells, each microwell comprising a cell capture well and a microbead capture well connected by a connecting channel; and an auxiliary capture structure, which is composed of any one of the following methods: 1) setting a physical barrier between cells and microbeads, 2) applying fluid dynamics, and 3) using an external physical field to separate cells and microbeads; through the combination of the two, a cell and a microbead fall into each cell capture well and microbead capture well, respectively, to complete a one-to-one capture pairing of cells and microbeads. The present invention overcomes the problem of low pairing efficiency due to random pairing in the prior art, the chip structure and process provided are simple, low-cost and short processing time, the operation steps are simplified, and efficient one-to-one capture pairing of cells and microbeads is achieved.

Description

Technical Field

[0001] The present invention relates to the technical field of single-cell omics analysis, and more specifically to a device and method for cell-microbead capture pairing for single-cell omics analysis. Background Art

[0002] Cells are the basic units of the structure and function of living organisms, and are also the basis for understanding and studying the laws of living organisms. As a rapidly developing emerging technology, single cell RNA sequencing (scRNA-seq) can explore cellular gene expression at single-cell resolution and reveal differences in gene expression among different cell subpopulations in tissue samples. Tang et al. first proposed single-cell transcriptome sequencing technology in 2009. Based on a large amount of data obtained from the entire transcriptome, scRNA-seq provides complete information about gene expression and its regulation in order to accurately describe the type and state of cells. Today, single-cell RNA sequencing technology can quickly determine the precise gene expression of thousands of cells and analyze the heterogeneity of expression of cells with the same genotype. It has been widely used in basic life sciences and major disease research such as development, immunity, and tumors.

[0003] Traditional single-cell analysis uses flow cytometry to measure protein expression at the single-cell level, but this method is very limited in the number of proteins it can detect; while RNA expression detection can cover all expression information, this detection is usually based on cell populations, obscuring the information of single cells. Although there have been reports of single-cell RNA expression measurements using microtiter plates or commercial microfluidic chips, these methods require sophisticated instruments, resulting in high costs for conventional transcriptome analysis of large numbers of cells.

[0004] With the development of molecular barcoding technology and sequencing library preparation technology based on microfluidic platforms, existing microfluidic technologies have achieved single-cell transcriptome sequencing of tens of thousands of cells, while reducing costs and operational difficulties. One of the most commonly used techniques is to prepare sequencing libraries by pairing each single cell with a unique barcode microbead one-to-one. In recent years, many researchers have developed a variety of technologies to improve the efficiency of pairing and separation of single cells with unique barcode microbeads. For example, in order to capture as many cells as possible from a limited sample, Zheng et al. designed a droplet-based system that uses GemCode microbeads to sequence the transcriptome of tens of thousands of single cells, with a cell capture efficiency of approximately 50%. Moon et al. used a spiral channel-based microfluidic platform to arrange high-concentration microbeads equidistantly before encapsulating cells, thereby effectively avoiding the situation where one droplet captures multiple microbeads. However, droplet-based technology still follows a random passive method to pair single cells and mRNA capture microbeads, which has fundamental limitations on the efficiency of cell-microbead pairing, can hardly handle low input samples (<500), and requires peripheral equipment support such as precise fluid control systems for droplet generation. Therefore, devices with efficient pairing and simple operation have become an urgent need for further expanding single-cell transcriptome sequencing. The large-scale pairing method of cells and microbeads based on microwell array structure has solved the problems of droplet systems to a certain extent and realized single-cell transcriptome sequencing in multiple application scenarios, but its random pairing characteristics still cause the efficiency of cell and microbead pairing to be limited by Poisson distribution.

[0005] To this end, researchers have tried to use auxiliary means to break through the bottleneck of random distribution and improve pairing efficiency. For example, RongFan et al. developed a cell-bead capture pairing method based on dielectrophoresis capture and microwell transfer, which improved the single-cell capture rate and effective transfer rate, breaking through the Poisson limit of cell capture, but this method still cannot break through the randomness of microbead capture. At the same time, the electrode design of its dielectrophoresis chip increases the difficulty of chip manufacturing and introduces additional operation steps for alignment transfer after capturing cells. Yang et al. developed a cell and microbead capture pairing microfluidic device based on fluid dynamics and valve control, which increased the pairing efficiency to 95%. However, this design requires complex processing and operation, and also limits the scalability of flux. Summary of the invention

[0006] The purpose of the present invention is to provide a device and method for cell-microbead capture pairing for single-cell omics analysis, so as to solve the problem of random pairing resulting in low pairing efficiency in the large-scale cell-microbead pairing method based on microwell array structure in the prior art.

[0007] In order to solve the above problems, the present invention adopts the following technical solutions:

[0008] According to a first aspect of the present invention, there is provided a device for cell-microbead capture pairing for single-cell omics analysis, comprising: a microwell capture layer, comprising: a microwell array composed of a plurality of microwells, each microwell comprising a cell capture well and a microbead capture well connected by a connecting channel, each cell capture well being aligned on the same side to form a cell well side for capturing cells, and each microbead capture well being aligned on the other side to form a microbead well side for capturing microbeads; and an auxiliary capture structure located above the microwell capture layer, the auxiliary capture structure being constructed by any one of the following methods: 1) setting a physical barrier between cells and microbeads, 2) applying fluid dynamics, and 3) using an external physical field to achieve separation of cells and microbeads, wherein the external physical field comprises any one of light, sound, magnetism, and electricity; through the combination of the microwell capture layer and the auxiliary capture structure, a cell falls into each cell capture well and a microbead falls into each microbead capture well, thereby completing a one-to-one cell-microbead capture pairing in each microwell.

[0009] According to one embodiment of the present invention, the auxiliary capture structure formed by setting a physical barrier between cells and microbeads to achieve separation of cells and microbeads includes: an isolation wall extending toward the connecting channel above the connecting channel of the microwell capture layer, and a cell sample flow channel and a microbead sample flow channel are respectively formed above the cell well side and the microbead well side through the isolation wall, and the bottom surfaces of the two flow channels respectively cover the cell capture well and the microbead capture well below, the front ends of the cell sample flow channel and the microbead sample flow channel are connected to the sample inlet running through the auxiliary capture structure, and the rear ends are connected to the sample outlet running through the auxiliary capture structure, which are used for the injection and outflow of cells and microbeads.

[0010] According to another embodiment of the present invention, the auxiliary capture structure constructed by applying fluid dynamics to achieve separation of cells and microbeads includes: a main channel covering the microwell array of the microwell capture layer and extending along the microwell arrangement direction, the upstream and downstream of the main channel are bifurcated to connect three branch channels, namely a sheath flow channel in the middle and a cell sample flow channel and a microbead sample flow channel on both sides, the front end of each branch channel is connected to a sample inlet running through the auxiliary capture structure, and the rear end is connected to a sample outlet running through the auxiliary capture structure, which is used for the injection and outflow of sheath flow, cells and microbeads.

[0011] According to another embodiment of the present invention, the auxiliary capture structure constructed by adopting an external physical field to achieve separation of cells and microbeads includes: a top layer and an electrode layer combined together in sequence from top to bottom, the electrode layer includes a pair of symmetrically placed upper electrode layer and lower electrode layer, which respectively serve as a capture signal electrode and a capture ground electrode for generating dielectrophoresis excitation, the upper electrode layer and the lower electrode layer respectively include: a main channel covering the microwell array of the microwell capture layer and extending along the microwell arrangement direction, the upstream and downstream of the main channel are bifurcated to connect three branch channels, which are a sheath flow channel in the middle and a cell sample flow channel and a microbead sample flow channel on both sides, the front end of each branch channel is connected to a sample inlet running through the auxiliary capture structure, and the rear end is connected to a sample outlet running through the auxiliary capture structure, which is used for the injection and outflow of sheath flow, cells and microbeads.

[0012] The microwell array of the microwell capture layer includes a single-row arrangement or a multi-row arrangement; the bottom shapes of the cell capture wells and microbead capture wells include: circular, elliptical, rectangular, and polygonal.

[0013] When the cell and microbead particle sizes are comparable, the depth of the cell capture well is 0.8-1.8 times the cell diameter, the depth of the microbead capture well is 0.8-1.8 times the microbead diameter, and the width of the connecting channel is smaller than the cell diameter; when the cell and microbead particle sizes differ greatly, the connecting channel can be omitted, and each microwell includes a directly connected cell capture well and a microbead capture well, the depth of the cell capture well is 1.2-1.8 times the cell diameter, and the depth of the microbead capture well is 1.2-1.8 times the microbead diameter.

[0014] The material of the micro-well capture layer includes but is not limited to polymethyl methacrylate, silica gel, polydimethylsiloxane, SU8, single crystal silicon, silicon oxide, etc., preferably polydimethylsiloxane (PDMS).

[0015] The auxiliary capture structure is realized by setting a physical barrier between the cells and the microbeads. The auxiliary structure material includes but is not limited to polymethyl methacrylate, silica gel, polydimethylsiloxane, SU8, single crystal silicon, silicon oxide, etc., preferably polydimethylsiloxane (PDMS).

[0016] The auxiliary capture structure is a device that uses a fluid dynamics method to separate cells and microbeads. The auxiliary structure material includes but is not limited to polymethyl methacrylate, silica gel, polydimethylsiloxane, SU8, single crystal silicon, silicon oxide, etc., preferably polydimethylsiloxane (PDMS).

[0017] The auxiliary capture device for separating cells and microbeads based on dielectrophoresis, the top layer is made of insulating transparent materials, including but not limited to polymethyl methacrylate, silica gel, SU8, polydimethylsiloxane, silicon oxide, etc., preferably polydimethylsiloxane (PDMS). The electrode layer of the double-layer electrode layer is made of conductive materials, and the conductive materials include but are not limited to conductive polymers, doped silicon, metals, etc., preferably conductive polymer silver-polydimethylsiloxane (Ag-PDMS), wherein the mass fraction of silver powder is preferably 86%. The microwell capture layer is made of insulating materials, including but not limited to polymethyl methacrylate, silica gel, polydimethylsiloxane, SU8, silicon oxide, etc., preferably polydimethylsiloxane (PDMS).

[0018] According to a second aspect of the present invention, a method for cell-bead capture pairing for single-cell omics analysis is provided, comprising the following three methods:

[0019] Method 1: Provide a device for realizing cell-microbead capture pairing by setting a physical barrier between cells and microbeads as described above. When in operation, a cell solution and a microbead solution are respectively injected from the inlets of the cell sample flow channel and the microbead sample flow channel, so that the cells and the microbeads flow in the flow channels above the cell capture well and the microbead capture well, respectively. After the cells and the microbeads fill the cell sample flow channel and the microbead sample flow channel, respectively, the cells and the microbeads are made to stand still, and the cells and the microbeads fall into the cell capture well and the microbead capture well, respectively, by gravity, thereby completing a one-to-one capture pairing of cells and microbeads in each microwell.

[0020] Method 2: Provide a device for realizing cell-microbead capture pairing by applying fluid dynamics as described above. When working, a sheath flow is injected from the inlet of the sheath flow channel, and a cell solution and a microbead solution are injected from the inlet of the cell sample flow channel and the microbead sample flow channel, respectively. The sheath flow focuses the cells and microbeads in the main flow channel above the cell well side and the microbead well side by adjusting the flow rate to avoid cross contamination. After the cells and microbeads fill the cell well side and the microbead well side, respectively, the sample injection is stopped, and the cells and microbeads fall into the cell capture well and the microbead capture well, respectively, by gravity, thereby completing the one-to-one capture pairing of cells and microbeads in each microwell.

[0021] Method three, providing a device as described above for achieving cell-microbead capture pairing by adopting an external physical field, including the following four methods: a. Adopting negative dielectrophoresis control of cells and microbeads to assist capture pairing: first, injecting sheath flow into the chip from the inlet of the sheath flow channel and the cell sample flow channel, and injecting microbead solution from the inlet of the microbead sample flow channel, using the sheath flow to restrict the microbeads to the microbead well side, using negative dielectrophoresis to make the microbeads adhere to the microbead well side, and then stopping the dielectrophoresis excitation so that the microbeads settle to the microbead capture wells of the microwell capture layer by gravity; injecting sheath flow into the chip from the inlet of the sheath flow channel and the microbead sample flow channel, and injecting microbead solution from the inlet of the cell sample flow channel; a. injecting cell solution into the chip, using sheath flow to confine cells to the side of the cell well, using negative dielectrophoresis to make cells adhere to the side of the cell well, then stopping dielectrophoresis excitation to allow cells to settle into the cell capture wells of the microwell capture layer by gravity, completing the one-to-one capture pairing of cells and microbeads in each microwell; the capture order of microbeads and cells is interchangeable; b. using negative dielectrophoresis control of cells and microbeads to assist capture pairing: first, inject sheath flow into the chip from the inlet of the sheath flow channel, and simultaneously inject microbeads and cells from the inlet of the microbead sample flow channel and the inlet of the cell sample flow channel, respectively, the sheath flow confines the microbeads and cells above the microbead well side and above the cell well side, respectively; then using The negative dielectrophoresis effect causes the microbeads and cells to adhere to the upper side of the microbead well and the upper side of the cell well, respectively. Stop injecting all fluids, then stop the dielectrophoresis excitation to allow the microbeads and cells to settle to the microbead capture wells and cell capture wells of the microwell capture layer respectively by gravity, thereby completing the one-to-one capture pairing of cells and microbeads in each microwell; c. Use positive dielectrophoresis control of cells and negative dielectrophoresis of microbeads to assist in capture pairing: first, inject sheath flow into the chip from the inlet of the sheath flow channel and the cell sample flow channel, and at the same time inject a microbead solution from the inlet of the microbead sample flow channel, use the sheath flow to confine the microbeads to the microbead well side, and use negative dielectrophoresis to cause the microbeads to adhere to the microbead well side. , stop all fluid injection, then stop dielectrophoresis excitation so that the microbeads settle into the microbead capture wells of the microwell capture layer by gravity; inject sheath flow into the chip from the inlets of the sheath flow channel and the microbead sample channel, inject cell solution from the inlet of the cell sample channel, use the sheath flow to restrict the cells near the side wall of the double-layer structure electrode on one side of the cell well, use positive dielectrophoresis to make the cells adhere to the side of the cell well, stop all fluid injection, then stop dielectrophoresis excitation so that the cells settle into the cell capture wells of the microwell capture layer by gravity, thereby completing the one-to-one capture pairing of cells and microbeads in each microwell; the capture order of microbeads and cells can be interchanged; d.Positive dielectrophoresis control of cells and negative dielectrophoresis of microbeads are used to assist in capture pairing: first, sheath flow is injected into the chip from the inlet of the sheath flow channel, and microbeads and cells are injected from the inlet of the microbead sample flow channel and the inlet of the cell sample flow channel respectively. The sheath flow restricts the microbeads and cells to the side walls of the electrode layer above the microbead well and near the side of the cell well, respectively, to avoid cross contamination; then, dielectrophoresis is used to control the cells to attach to the side walls of the upper electrode layer on the cell well side under the control of positive dielectrophoresis, and the microbeads to attach to the side walls of the lower electrode layer on the microbead well side under the control of negative dielectrophoresis, and then all fluid injection is stopped, and then dielectrophoresis excitation is stopped to allow the microbeads and cells to settle to the microbead capture well and cell capture well of the microwell capture layer respectively by gravity, thereby completing the one-to-one capture pairing of cells and microbeads in each microwell. .

[0022] The device provided by the present invention mainly includes two parts: a microwell capture layer and an auxiliary capture structure. The microwell capture layer includes three interconnected parts: a cell capture well, a microbead capture well, and a connecting channel, wherein the microbead capture well and the cell capture well located at both ends of the connecting channel respectively receive microbeads and cells falling by gravity, and the size design of each capture well and the connecting channel is used to limit the one-to-one capture pairing of microbeads and cells.

[0023] The auxiliary capture structure can be designed based on three principles: physical barrier, fluid dynamics, and external physical field. First, the auxiliary capture structure based on physical barrier can separate the microbead sample and the cell sample in space, so as to achieve their respective capture in the microbead capture well and the cell capture well. Second, the device of the auxiliary capture structure using the fluid dynamic method can use the laminar flow generated by the sheath flow and the cell solution and the microbead solution to separate the cells and microbeads, so that the cells and microbeads fall into the corresponding microwells under the action of gravity. Third, the auxiliary capture structure based on dielectrophoresis uses a new three-dimensional double-layer structure electrode. By generating efficient dielectrophoretic force in both the horizontal and vertical directions, it can control the microbeads and cells to be closely arranged on the electrode sidewalls above the microbead well and the cell well, respectively, so that after the power is turned off, the microbeads and cells fall into the corresponding microwells efficiently due to gravity. Compared with the aforementioned auxiliary capture methods based on physical barrier and fluid dynamic methods, the dielectrophoresis method enables cells and microbeads to form a regular and close arrangement under the control of electric field force, further improving the chip throughput and capture efficiency.

[0024] Compared with the existing cell-microbead capture technology, the device and method for cell-microbead capture pairing for single-cell omics analysis provided by the present invention have the following creative and beneficial effects:

[0025] The present invention proposes a cell-microbead capture pairing device for single-cell omics analysis based on a microwell structure of a special shape. The microwell includes a connected connection channel, a cell capture well and a microbead capture well. The microbead capture well and the cell capture well located at both ends of the connection channel are used to receive microbeads and cells respectively. When the particle size difference between microbeads and cells is large, the connection channel can be omitted. The microwell structure limits the one-to-one capture pairing of cells and microbeads through the size of the cell capture well and the microbead capture well, thereby allowing high-concentration sample injection, avoiding the problem of diluting the sample in the existing method to exclude the situation where multiple cells / microbeads fall into a microwell, helping to improve the capture and pairing efficiency, reduce the volume of samples to be processed, and speed up the processing speed;

[0026] 2) The auxiliary capture structure can cooperate with the micro-well capture layer to efficiently capture and pair cells and micro-beads: First, by adopting a device that sets a physical barrier between cells and micro-beads, the isolation wall is set to separate the micro-bead solution and the cell solution in space, and the micro-well capture layer is used to receive the micro-beads and cells that fall by gravity; Second, by using a fluid dynamics method to separate cells and micro-beads, the laminar flow generated by the sheath flow and the cell solution and the micro-bead solution is used to separate the cells and micro-beads by position, and the micro-well capture layer is used to receive the micro-beads and cells that fall by gravity; Third, by using dielectrophoresis to separate cells and micro-beads, a double-layer structure electrode is used as the capture electrode for cells and micro-beads. When the double-layer structure electrode is powered on, a dielectrophoretic force is generated due to the spatially non-uniform electric field generated in the vertical direction. The dielectrophoretic force is used to make the micro-beads and cells form a tight and regular arrangement at the target position. After the power is turned off, the micro-beads and cells fall into the micro-well due to gravity;

[0027] 3) Compared with the prior art, the chip structure and process of the present invention are simple, low-cost and short in processing time; the present invention does not require the support of equipment such as valve control during operation, nor does it require the distribution capture or flipping operation steps of the existing dielectrophoresis, so the operation steps are simplified; the present invention supports the simultaneous injection of cells and microbeads into the chip, further shortening the capture pairing time; the advantages of limiting the size of microwells to allow high-density samples and the tight and regular arrangement of dielectrophoresis are conducive to reducing the sample volume and improving the processing throughput;

[0028] 4) In particular, the three-dimensional double-layer electrode structure adopted by the dielectrophoresis-assisted capture structure not only breaks through the random distribution of cells and microbeads compared with the existing dielectrophoresis-based capture technology, but also can realize the synchronous capture of cells and microbeads. The electrode processing is realized through a single-step molding process, which greatly shortens the R&D and manufacturing time and reduces costs.

[0029] In summary, a device and method for cell-microbead capture pairing for single-cell omics analysis provided by the present invention overcomes the problem of low pairing efficiency caused by random pairing in the large-scale cell-microbead pairing method based on microwell array structure in the prior art. The chip structure and process provided by the present invention are simple, the cost is reduced, the processing time is short, the operation steps are simplified, and efficient one-to-one capture pairing of cells and microbeads can be achieved, which has good application prospects in the field of single-cell omics analysis. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] Figure 1 A top view of a microwell capture layer of a device for cell-microbead capture pairing applied to single-cell omics analysis according to a preferred embodiment of the present invention;

[0031] Figure 2 A top view of a micro-well capture layer without connection channels in a cell-microbead capture pairing device for single-cell omics analysis according to another preferred embodiment of the present invention;

[0032] Figure 3 This is an overall structural diagram of a cell-microbead capture pairing device applied to single-cell omics analysis according to a preferred embodiment of the present invention when an auxiliary capture structure with a separation wall is used;

[0033] Figure 4 For Figure 3 A schematic cross-sectional view of the main flow channel of the device shown;

[0034] Figure 5 For Figure 3 Schematic diagram of bead and cell capture of the device shown;

[0035] Figure 6 It is an overall structural diagram of a cell-microbead capture pairing device applied to single-cell omics analysis according to a preferred embodiment of the present invention when an auxiliary capture structure based on a fluid dynamics method is adopted;

[0036] Figure 7 For Figure 6 A schematic cross-sectional view of the main flow channel of the device shown;

[0037] Figure 8 For Figure 6 Schematic diagram of bead and cell capture of the device shown;

[0038] Fig. 9 It is an overall structural diagram of a cell-microbead capture pairing device applied to single-cell omics analysis according to a preferred embodiment of the present invention when an auxiliary capture structure based on dielectrophoresis is adopted;

[0039] Fig.10For Fig. 9 A top view of the double-layer structure electrode of the device shown;

[0040] Fig.11 For Fig. 9 A schematic cross-sectional view of the main flow channel of the device shown;

[0041] Fig.12 For Figure 6 The schematic diagram of the device shown is used to capture microbeads when using a dielectrophoresis-assisted capture structure;

[0042] Fig.13 For Figure 6 The schematic diagram of the device shown in FIG. 1 is a schematic diagram of capturing cells and forming a one-to-one pairing with microbeads when a dielectrophoresis-assisted capture structure is adopted;

[0043] Fig.14 It is an overall structural diagram of a cell-microbead capture pairing device for single-cell omics analysis according to a preferred embodiment of the present invention when a non-connected channel micro-well capture layer is used;

[0044] Fig.15 For Fig.14 A schematic cross-sectional view of the main flow channel of the device shown;

[0045] The accompanying drawings are marked as follows: 1. microwell capture layer, 2. auxiliary capture structure, 3. microwell, 4. connecting channel, 5. cell capture well, 6. microbead capture well, 7. cell well side, 8. microbead well side, 9. isolation wall, 10. cell sample flow channel, 11. microbead sample flow channel, 12. main flow channel, 13. sheath flow channel, 14. top layer, 15. double-layer structure electrode, 16. upper long thin layer, 17. lower long thin layer, 18. sample inlet, 19. sample outlet, 20. narrow channel. DETAILED DESCRIPTION

[0046] The present invention is further described below in conjunction with specific examples. It should be understood that the following examples are only used to illustrate the present invention and are not used to limit the scope of the present invention.

[0047] According to the present invention, a cell-microbead capture pairing device for single-cell omics analysis based on a microwell structure of a special shape is provided. The device mainly comprises a microwell capture layer 1 and an auxiliary capture structure 2.

[0048] like Figure 1As shown, a micro-well capture layer 1 according to a preferred embodiment of the present invention is a thin layer structure, and its upper surface has an array formed by periodically concave micro-wells 3, each micro-well 3 in the array includes three parts: a connecting channel 4, and a cell capture well 5 and a microbead capture well 6 respectively located at the two ends of the connecting channel 4, and the three parts are interconnected.

[0049] In the microwell 3 array, the connecting channels of adjacent microwells are parallel to each other and the two ends are aligned respectively, so the endpoints on the same side of the connecting channel 4 are in a common straight line, and the lines shared by the endpoints on both sides are parallel to each other. The cell capture well 5 located on one side of the connecting channel 4 is used to capture cells, and this side is defined as the cell well side 7; the microbead capture well 6 located on the other side of the connecting channel 4 is used to capture microbeads, and this side is defined as the microbead well side 8. All the cell capture wells 5 in the microwell array are exactly the same in shape and size, and all the microbead capture wells 6 are exactly the same in shape and size.

[0050] The bottom surface shape of the cell capture well 5 includes but is not limited to circular, elliptical, rectangular, polygonal and other irregular shapes, and the size is determined by the size of the captured cell to ensure that it can accommodate and can only accommodate one cell, and its length, width or diameter should preferably be 1.2-1.8 times the cell diameter. The bottom surface shape of the microbead capture well 6 includes but is not limited to circular, elliptical, rectangular, polygonal and other irregular shapes, and the size is related to the size of the captured microbeads to ensure that it can accommodate and can only accommodate one microbead, and the length, width or diameter is preferably 1.2-1.8 times the diameter of the microbead. The channel width at the intersection of the cell capture well 5 of the microwell 3 and the connecting channel 4 is smaller than the cell diameter to ensure that the captured cells will not enter the connecting channel 4 through the intersection; the channel width at the intersection of the microbead capture well 6 of the microwell 3 and the connecting channel 4 is smaller than the microbead diameter to ensure that the captured microbeads will not enter the connecting channel 4 through the intersection.

[0051] The depth of the cell capture well 5 should be guaranteed to accommodate and can only accommodate one cell, preferably 0.8-1.8 times the cell diameter; the depth of the microbead capture well 6 should be guaranteed to accommodate and can only accommodate one microbead, preferably 0.8-1.8 times the microbead diameter; there is no special requirement for the depth of the connecting channel 4, preferably the same depth as the cell capture well 5 or the same depth as the microbead capture well 6, so as to make micromachining more convenient and economical.

[0052] like Figure 2As shown, it is a micro-well capture layer 1 according to another preferred embodiment of the present invention. When the particle size difference between cells and micro-beads is large, the micro-wells 3 with periodic indentations on the upper surface of the micro-well capture layer 1 can omit the connecting groove, so that each micro-well 3 mainly includes two parts: a cell capture well 5 and a micro-bead capture well 6, and the cell capture well 5 and the micro-bead capture well 6 are directly connected. The bottom surface shape of the cell capture well 5 includes but is not limited to circular, elliptical, rectangular, polygonal and other irregular shapes, and the size is determined by the size of the captured cell to ensure that it can accommodate and can only accommodate one cell. Its length, width or diameter should preferably be 1.2-1.8 times the cell diameter, and its depth is preferably 1.2-1.8 times the cell diameter. The bottom surface shape of the micro-bead capture well 6 includes but is not limited to circular, elliptical, rectangular, polygonal and other irregular shapes, and the size is related to the size of the captured micro-beads to ensure that it can accommodate and can only accommodate one micro-bead. The length, width or diameter is preferably 1.2-1.8 times the micro-bead diameter, and the depth is preferably 1.2-1.8 times the micro-bead diameter. The micro-well 3 array can be arranged in a single row or in multiple rows.

[0053] According to the present invention, there is provided a device for cell-microbead capture pairing for single-cell omics analysis based on a microwell structure of a special shape. In addition to a microwell capture layer 1, the device also includes an auxiliary capture structure 2 located above the upper surface of the microwell capture layer 1. The auxiliary capture structure 2 has a structure that separates cells and microbeads to avoid cross-contamination, including but not limited to setting a physical barrier between cells and microbeads in time and / or space, using a fluid dynamics method to positionally separate cells and microbeads, using an external physical field (light, sound, magnetism, electricity, etc.) to positionally separate cells and microbeads, etc.

[0054] Example 1 Device formed by setting a physical barrier between cells and microbeads

[0055] In this embodiment, the device can be implemented by setting a physical barrier between the cells and the microbeads. Figure 3 As shown, the device comprises a micro-well capture layer 1 and an auxiliary capture structure 2, wherein the auxiliary capture structure 2 includes but is not limited to an isolation wall 9 arranged above the connection channel 4 and intersecting with the connection channel 4. Figure 3 3D renderings and Figure 4 As shown in the cross-sectional effect diagram, the bottom surface of the isolation wall 9 partially or completely covers the isolation channel part, and does not cover any area where the cell capture well 5 or the microbead capture well 6 is located. With the isolation wall 9 as the shared side wall, a cell sample flow channel 10 and a microbead sample flow channel 11 are formed above the cell well side 7 and the microbead well side 8, respectively, and the bottom surfaces of the two flow channels can respectively cover the cell capture well 5 and the microbead capture well 6 below, and the front end and the rear end of the sample flow channel are respectively connected to the sample inlet 18 and the sample outlet 19 that penetrate the auxiliary capture structure 2.

[0056] Combination Figure 3 to Figure 5 The working principle of the device provided in this embodiment is described as follows: when working, firstly, the cell solution and the microbead solution are injected into the cell sample flow channel 10 and the microbead sample flow channel 11 respectively from the sample inlet 18, and the setting of the isolation wall 9 can avoid cross-contamination of the cell and microbead solutions. The cells and the microbeads flow in the flow channels above the cell capture well 5 and the microbead capture well 6 respectively; after the cells and the microbeads fill the cell sample flow channel 10 and the microbead sample flow channel 11 respectively, the cells and the microbeads are made to be still, and the cells and the microbeads fall into the cell capture well 5 and the microbead capture well 6 respectively by gravity, thereby completing the one-to-one capture pairing of cells and microbeads in each microwell 3.

[0057] Example 2: Using fluid dynamics to construct a device for separating cells and microbeads

[0058] In this embodiment, the device can use fluid dynamics methods to separate cells and microbeads, such as sheath flow focusing, fluid inertial focusing, etc. When the sheath flow focusing method is used, such as Figure 6 As shown, the device includes a microwell capture layer 1 and an auxiliary capture structure 2, wherein the auxiliary capture structure 2 includes but is not limited to a main channel 12 covering the microwell 3 array and extending along the microwell repetition direction, and the upstream and downstream of the main channel 12 are respectively bifurcated to connect three branch channels, namely the sheath flow channel 13 located in the middle and the cell sample flow channel 10 and the microbead sample flow channel 11 on both sides, and each branch channel is connected to a sample inlet 18 and a sample outlet 19 connected to the outside at the beginning and end, which are used for the injection and outflow of cells, microbeads or sheath flow. The cross-sectional view of the upstream bifurcation of the main channel 13 and the cross-sectional view of the main channel part are shown in FIG. Figure 7 shown.

[0059] Combination Figure 6 to Figure 8 The working principle of the device provided in this embodiment is described as follows: when working, first, sheath flow is injected into the sheath flow channel 13 from the sample inlet 18, and at the same time, cell solution and microbead solution are injected into the cell sample channel 10 and the microbead sample channel 11 from the sample inlet 18 respectively, and the sheath flow is adjusted to focus the cells and microbeads in the main flow channel above the cell well side 7 and the microbead well side 8 respectively by adjusting the appropriate flow rate to avoid cross contamination; after the cells and microbeads are respectively covered with the cell well side 7 and the microbead well side 8, the sample injection is stopped, and the cells and microbeads fall into the cell capture well 5 and the microbead capture well 6 respectively by gravity, thereby completing the one-to-one capture pairing of cells and microbeads in each microwell 3.

[0060] Example 3: Device for separating cells and microbeads using an external physical field

[0061] In this embodiment, the device can use an external physical field (light, sound, magnetism, electricity, etc.) to separate the cells and microbeads respectively, wherein the cell-microbead separation method based on the external electric field includes but is not limited to setting up an auxiliary capture structure 2 based on dielectrophoresis.

[0062] like Fig. 9 As shown, the device includes a top layer 14, an electrode layer 15, and a micro-well capture layer 1, which are sequentially combined from top to bottom, wherein the top layer 14 and the electrode layer 15 together constitute an auxiliary capture structure. The top layer 14 is an insulating thin layer, and the electrode layer 15 has a pair of symmetrically placed double-layer electrode structures that serve as capture signal electrodes and capture ground electrodes for generating dielectrophoretic excitation, respectively. The electrode layer 15 is composed of an upper long thin layer 16 and a lower long thin layer 17 stacked up and down. Among them, the bottom surface of the lower long thin layer 17 is bonded to the upper surface of the micro-well capture layer 1, and the upper surface of the upper long thin layer 16 is bonded to the top layer 14. The gap between the double-layer electrode structure forms a flow channel as a main flow channel 12, and the bottom surface of the main flow channel 12 covers all micro-wells 3 in the extension direction. The three-dimensional effects of the upper and lower layers of the device and the schematic diagram of the decomposed structure of the electrode layer 15 are shown in the following figures. Fig. 9 and Fig.10 As shown, for the electrode layer 15, the upper elongated thin layer 16 extends into the main channel 12 relative to the lower elongated thin layer 17, that is, the main channel 12 in the upper elongated thin layer 16 is narrower, and the main channel 12 in the lower elongated thin layer 17 is wider, and the side walls of the upper elongated thin layer 16 and the lower elongated thin layer 17 facing the flow channel are both straight lines.

[0063] like Fig. 9 , Fig.10 As shown, the upstream of the main channel 12 is bifurcated to form three branch channels, namely the sheath flow channel 13 in the middle and the cell sample flow channel 10 and the microbead sample flow channel 11 on both sides. The top layer 14 and the electrode layer 15 have three sample inlets 18 that penetrate the two and are connected to the ends of the sheath flow channel 13, the cell sample flow channel 10 and the microbead sample flow channel 11. The downstream of the main channel 12 can be directly connected to the sample outlet 19, or it can be bifurcated to form multiple branch channels and outlets connected to the outside for respectively exporting cells, microbeads and sheath flow.

[0064] The electrode layer 15 has a narrow channel 20 extending from a sample inlet 18 and a sample outlet 19 that are directly connected to the main channel 12 and are parallel to the main channel 12. The narrow channel 20, the sample inlet 18, the sample outlet 19 and the flow channel form a gap that completely separates and insulates the symmetrically placed electrode layer 15.

[0065] When the dielectrophoresis capture device operates in a mode in which both cells and beads are controlled by negative dielectrophoresis, the cell capture well 5 is located within the outer flow channel range sandwiched by the side wall edge of the upper elongated thin layer 16 and the side wall edge of the lower elongated thin layer 17 on the cell well side 7; the bead capture well 6 is located within the outer flow channel range sandwiched by the side wall edge of the upper elongated thin layer 16 and the side wall edge of the lower elongated thin layer 17 on the bead well side 8.

[0066] When the dielectrophoresis capture device operates in a mode in which cells are controlled by positive dielectrophoresis and beads are controlled by negative dielectrophoresis, the cell capture well 5 is located in a flow channel range sandwiched by the downward extension of the side wall edge of the upper long thin layer 16 on the cell well side 7 and the flow channel centerline parallel to the flow channel direction; the bead capture well 6 is located in an outer flow channel range sandwiched by the downward extension of the side wall edge of the upper long thin layer 16 and the side wall edge of the lower long thin layer 17 on the bead well side 8.

[0067] Combination Figure 11 to Figure 13 For the dielectrophoresis-based assisted capture device, the working principle of the one-to-one capture pairing of cells and microbeads is described as follows:

[0068] When negative dielectrophoresis control of cells and beads is used to assist capture pairing, e.g. Fig.12 As shown, firstly, sheath flow is injected into the chip from the inlet of the sheath flow channel 13 and the cell sample channel 10, and the microbead solution is injected from the inlet of the microbead sample channel 11. The microbeads are confined near the side wall of the electrode layer 15 on the side of the microbead well 8 by the sheath flow, and the microbeads are attached to the side wall of the lower long thin layer 17 on the microbead well 8 by negative dielectrophoresis, and arranged into a single pearl chain structure parallel to the extension direction of the electrode side wall; after the pearl chain structure covers the entire side wall of the lower long thin layer 17, all fluid injections are stopped, so that the microbeads attached to the side wall of the lower long thin layer 17 stop moving; then the dielectrophoresis excitation is stopped so that the microbeads settle to the microbead capture well 6 of the microwell capture layer 1 by gravity. Next, as Fig.13 As shown, sheath flow is injected into the chip from the inlet of the sheath flow channel 13 and the microbead sample channel 11, and cell solution is injected from the inlet of the cell sample channel 10. The sheath flow is used to restrict the cells near the side wall of the electrode layer 15 on the cell well side 7. The negative dielectrophoresis effect is used to make the cells adhere to the side wall of the lower long thin layer 17 on the cell well side 7 and arrange them into a single pearl chain structure parallel to the extension direction of the electrode side wall. After the pearl chain structure covers the entire side wall of the lower long thin layer 17, all fluid injections are stopped, and the cells attached to the side wall of the lower long thin layer 17 stop moving. Then, the dielectrophoresis excitation is stopped so that the cells settle into the cell capture well 5 of the microwell capture layer 1 by gravity, and form a one-to-one pairing collection with the microbeads. The capture order of microbeads and cells can be exchanged.

[0069] When negative dielectrophoresis control of cells and microbeads is used to assist in the capture pairing working mode, in addition to the above-mentioned method of successively capturing microbeads and cells to form a pair, a method of synchronously capturing cells and microbeads can also be used to further shorten the operation time and simplify the operation process. Specifically, sheath flow is injected into the chip from the inlet of the sheath flow channel 13, and microbeads and cells are injected from the inlet of the microbead sample flow channel 11 and the inlet of the cell sample flow channel 10, respectively. The sheath flow confines the microbeads and cells to the side walls of the electrode layer 15 above the microbead well side 8 and near the side walls of the cell well side 7, respectively, to avoid cross contamination; then, negative dielectrophoresis is used to make the microbeads and cells adhere to the side walls of the lower long thin layer 17 above the microbead well side 8 and the cell well side 7, respectively, and arrange them into a single pearl chain structure parallel to the extension direction of the electrode side wall; after the pearl chain structure covers the entire side wall of the lower long thin layer 17, the injection of all fluids is stopped, so that the microbeads and cells attached to the side wall of the lower long thin layer 17 stop moving; then the dielectrophoresis excitation is stopped to allow the microbeads and cells to settle to the microbead capture well 6 and the cell capture well 5 of the microwell capture layer 1 by gravity, respectively, and complete the one-to-one pairing and collection of microbeads and cells in each microwell 3.

[0070] The dielectrophoresis-based auxiliary capture device can also use positive dielectrophoresis control of cells and negative dielectrophoresis of microbeads to assist in capture pairing during the capture process. First, sheath flow is injected into the chip from the inlets of the sheath flow channel 13 and the cell sample channel 10, and microbead solution is injected from the inlet of the microbead sample channel 11. The sheath flow is used to confine the microbeads near the side wall of the electrode layer 15 on the side of the microbead well 8. The negative dielectrophoresis effect is used to make the microbeads adhere to the side wall of the lower long thin layer 17 on the microbead well side 8, and arrange them into a single pearl chain structure parallel to the extension direction of the electrode side wall; after the pearl chain structure covers the entire side wall of the lower long thin layer 17, all fluid injections are stopped, so that the microbeads attached to the side wall of the lower long thin layer 17 stop moving; then the dielectrophoresis excitation is stopped so that the microbeads settle to the microbead capture well 6 of the microwell capture layer 1 by gravity; next , inject sheath flow into the chip from the inlet of sheath flow channel 13 and microbead sample channel 11, inject cell solution from the inlet of cell sample channel 10, use sheath flow to restrict cells near the side wall of electrode layer 15 on one side of cell well side 7, use positive dielectrophoresis to make cells adhere to the side wall of upper long thin layer 16 on cell well side 7 and arrange into a single pearl chain structure parallel to the extension direction of electrode side wall, stop all fluid injection after the pearl chain structure covers the side wall of upper long thin layer 16, stop cells attached to the side wall of upper long thin layer 16, then stop dielectrophoresis excitation to make cells settle to cell capture well 5 of microwell capture layer 1 by gravity, and form one-to-one pairing collection with microbeads. The capture order of microbeads and cells can be exchanged.

[0071] When the auxiliary capture device based on dielectrophoresis adopts the working mode of assisting the capture pairing by positive dielectrophoresis control of cells and negative dielectrophoresis of microbeads, in addition to the operation method of successively capturing microbeads and cells to form a pair as described above, a method of synchronously capturing cells and microbeads can also be adopted to further shorten the operation time and simplify the operation process. Specifically, a sheath flow is injected into the chip from the inlet of the sheath flow channel 13, and microbeads and cells are injected from the inlet of the microbead sample flow channel 11 and the inlet of the cell sample flow channel 10 respectively. The sheath flow restricts the microbeads and cells above the microbead well side 8 and near the side wall of the electrode layer 15 on the cell well side 7, respectively, to avoid cross contamination; then, a suitable frequency is selected to make the microcells attached to the side wall of the upper long thin layer 16 on the cell well side 7 under the control of positive dielectrophoresis, and at the same time, the microbeads are attached to the chip under the action of negative dielectrophoresis. On the side wall of the lower long thin layer 17 of the microbead well side 8, cells and microbeads are arranged at the side wall to form a single pearl chain structure parallel to the extension direction of the electrode side wall; after the pearl chain structure covers the side wall, all fluid injection is stopped to stop the attached microbeads and cells from moving; then the dielectrophoresis excitation is stopped to allow the microbeads and cells to settle to the microbead capture well 6 and cell capture well 5 of the microwell capture layer 1 respectively by gravity, and the microbeads and cells are collected in a one-to-one pair in the microwell 3.

[0072] In single-cell transcriptome sequencing, the particle size of the microbeads usually used is similar to or larger than the cell size. When the particle size of the microbeads and cells differs greatly, a microwell 3 in the form of a non-connected channel can be used. At this time, the auxiliary capture structure only includes a flow channel covering the microwell array, and the pairing is achieved by separating the microbeads and cells in the injection time. In operation, a microbead solution with a large particle size is first introduced, and the microbeads are adjusted to be distributed at a higher density in the flow channel, so that they fall into the microbead capture well by gravity. Next, a cell solution with a small particle size is introduced, and the cells are also distributed at a higher density in the flow channel, so that they fall into the cell capture well by gravity. When the microbeads fall, they will not fall into the cell capture well because their particle size is larger than that of the cells. When the cells fall, the microbead capture well is already occupied, so they will not fall into the microbead capture well, thereby forming a one-to-one pairing collection of cells and microbeads.

[0073] Example 4 When the particle sizes of microbeads and cells differ greatly, the auxiliary capture device based on dielectrophoresis assists the microwells without connection channels to efficiently capture and pair the cell microbeads.

[0074] like Fig.14 As shown, the microwell capture layer 1 adopts a double-row extended microwell array design, the two rows of microwells 3 are symmetrically placed, the cell capture well 5 is located on the inner side of the flow channel, and the microbead capture well 6 is located on the outer side of the flow channel. Fig.15As shown in the cross-sectional effect, the bead capture well 6 is located in the outer flow channel range sandwiched by the side wall edge of the lower long thin layer 17 and the side wall edge of the upper long thin layer 16 extending downward on the same side; and the cell capture well 5 is located in the middle flow channel part sandwiched by the side wall edge of the upper long thin layer 16 extending downward on both sides.

[0075] During the capture process, sheath flow is injected into the chip from the inlet of the sheath flow channel 13, and microbeads are injected from the inlet of the microbead sample flow channel 11 and the inlet of the cell sample flow channel 10 on both sides of the sheath flow. The sheath flow restricts the microbeads near the side walls of the electrode layer 15 on both sides of the flow channel, and the microbeads are attached to the side walls of the lower long thin layer 17 by negative dielectrophoresis and arranged into a single pearl chain structure parallel to the extension direction of the electrode side wall. After the pearl chain structure covers the entire side wall of the lower long thin layer 17, the injection of all fluids is stopped, and the microbeads attached to the side wall of the lower long thin layer 17 stop moving. Then, the dielectrophoresis excitation is stopped to allow the microbeads to settle into the microbead capture well 6 by gravity. Next, the microbeads are captured from the sheath flow channel 13. The sheath flow is injected into the chip through the inlet of the chip, and the cells are injected from the inlet of the microbead sample flow channel 11 and the inlet of the cell sample flow channel 10 on both sides of the sheath flow. The sheath flow restricts the cells near the side walls of the electrode layer 15 on both sides of the flow channel. The cells are attached to the side walls of the upper long thin layer 16 above the cell capture well by positive dielectrophoresis and arranged into a single pearl chain structure parallel to the extension direction of the electrode side wall. After the pearl chain structure covers the entire side wall of the upper long thin layer 16, all fluids are stopped from being injected, and the microbeads attached to the side walls of the upper long thin layer 16 stop moving. Then the dielectrophoretic excitation is stopped to allow the cells to settle into the cell capture well 5 by gravity, and the microbeads and cells are collected in a one-to-one pair in the microwell 3. It should be noted that the sheath flow is not necessary here, because the dielectrophoretic force has the ability to move the microbeads to the side walls of the lower long thin layer 17 and the cells to the side walls of the upper long thin layer 16. Paired capture can be achieved by injecting fluid from any one or more inlets while closing the remaining inlets. In addition, in addition to the above-mentioned method of capturing microbeads first and then capturing cells, a cell-microbead synchronous capture method can also be used, that is, a mixed sample of cell microbeads is injected into the inlet of the microbead sample flow channel 11 and the inlet of the cell sample flow channel 10 at the same time, and the microbeads are subjected to negative dielectrophoretic force and attached to the side wall of the lower long strip thin layer 17 by adjusting the appropriate frequency, while the cells are subjected to positive dielectrophoretic force and attached to the side wall of the upper long strip thin layer 16, and then by stopping the injection and releasing the dielectrophoretic excitation, the cells and microbeads fall into the corresponding microwells respectively by gravity. Sheath flow is also not necessary in this synchronous method.

[0076] The material of the micro-well capture layer 1 includes but is not limited to polymethyl methacrylate, silica gel, polydimethylsiloxane, SU8, single crystal silicon, silicon oxide, etc., preferably polydimethylsiloxane (PDMS).

[0077] The device is realized by setting a physical barrier between the cells and the microbeads. The material of the auxiliary capture structure includes but is not limited to polymethyl methacrylate, silica gel, polydimethylsiloxane, SU8, single crystal silicon, silicon oxide, etc., preferably polydimethylsiloxane (PDMS).

[0078] The device uses fluid dynamics to separate cells and microbeads, and the material of the auxiliary capture structure 2 includes but is not limited to polymethyl methacrylate, silica gel, polydimethylsiloxane, SU8, single crystal silicon, silicon oxide, etc., preferably polydimethylsiloxane (PDMS).

[0079] The auxiliary capture device based on dielectrophoresis, the top layer 14 is made of insulating transparent materials, including but not limited to polymethyl methacrylate, silica gel, SU8, polydimethylsiloxane, silicon oxide, etc., preferably polydimethylsiloxane (PDMS). The electrode layer 15 of the double-layer structure is made of conductive materials, and the conductive materials include but are not limited to conductive polymers, doped silicon, metals, etc., preferably conductive polymer silver-polydimethylsiloxane (Ag-PDMS), wherein the mass fraction of silver powder is preferably 86%. The micro-well capture layer is made of insulating materials, including but not limited to polymethyl methacrylate, silica gel, polydimethylsiloxane, SU8, silicon oxide, etc., preferably polydimethylsiloxane (PDMS).

[0080] The above is only a preferred embodiment of the present invention, and is not intended to limit the scope of the present invention. The above embodiments of the present invention can also be modified in various ways. All simple, equivalent changes and modifications made according to the claims and the description of the present invention fall within the scope of protection of the claims of the present invention. The contents not described in detail in the present invention are all conventional technical contents.

Claims

1. A cell-bead capture pairing device for single-cell omics analysis, characterized in that: include: A microwell capture layer, comprising: a microwell array composed of a plurality of microwells, each microwell comprising a cell capture well and a microbead capture well connected by a connecting channel, each cell capture well being aligned on the same side to form a cell well side for capturing cells, and each microbead capture well being aligned on the other side to form a microbead well side for capturing microbeads; and An auxiliary capture structure located above the microwell capture layer, wherein the auxiliary capture structure is formed by: 1) setting a physical barrier between the cells and the microbeads, 2) applying fluid dynamics, and 3) using an external physical field to separate the cells and the microbeads, wherein the external physical field includes any one of light, sound, magnetism, and electricity; By combining the microwell capture layer with the auxiliary capture structure, one cell falls into each cell capture well and one microbead falls into each microbead capture well, thereby completing a one-to-one capture pairing of cells and microbeads in each microwell; The auxiliary capture structure formed by setting a physical barrier between cells and microbeads to separate cells and microbeads comprises: an isolation wall extending toward the connection channel above the connection channel of the microwell capture layer, wherein a cell sample flow channel and a microbead sample flow channel are separated by the isolation wall above the cell well side and the microbead well side, and the bottom surfaces of the two flow channels respectively cover the cell capture well and the microbead capture well below, wherein the front ends of the cell sample flow channel and the microbead sample flow channel are connected to the sample inlet penetrating the auxiliary capture structure, and the rear ends are connected to the sample outlet penetrating the auxiliary capture structure, and are used for the injection and outflow of cells and microbeads; The auxiliary capture structure formed by applying fluid dynamics to achieve separation of cells and microbeads includes: a main flow channel covering the microwell array of the microwell capture layer and extending along the microwell arrangement direction, the upstream and downstream of the main flow channel bifurcating to connect three branch flow channels, namely a sheath flow channel in the middle and a cell sample flow channel and a microbead sample flow channel on both sides, the front end of each branch flow channel is connected to a sample inlet that runs through the auxiliary capture structure, and the rear end is connected to a sample outlet that runs through the auxiliary capture structure, for the injection and outflow of sheath flow, cells, and microbeads; The auxiliary capture structure, which is constructed by adopting an external physical field to achieve the separation of cells and microbeads, includes: a top layer and an electrode layer combined together in sequence from top to bottom, the electrode layer includes a pair of symmetrically placed upper electrode layer and lower electrode layer, which respectively serve as a capture signal electrode and a capture ground electrode for generating dielectrophoresis excitation, the upper electrode layer and the lower electrode layer respectively include: a main channel covering the microwell array of the microwell capture layer and extending along the microwell arrangement direction, the upstream and downstream of the main channel are bifurcated to connect three branch channels, which are a sheath flow channel in the middle and a cell sample flow channel and a microbead sample flow channel on both sides, the front end of each branch channel is connected to a sample inlet running through the auxiliary capture structure, and the rear end is connected to a sample outlet running through the auxiliary capture structure, which is used for the injection and outflow of sheath flow, cells and microbeads.

2. The cell-bead capture pairing device for single-cell omics analysis according to claim 1, characterized in that: The microwell array of the microwell capture layer includes a single-row arrangement or a multi-row arrangement; the bottom shapes of the cell capture wells and microbead capture wells include: circular, elliptical, rectangular, and polygonal.

3. The cell-bead capture pairing device for single-cell omics analysis according to claim 1, characterized in that: When the cell and microbead particle sizes are comparable, the depth of the cell capture well is 0.8-1.8 times the cell diameter, the depth of the microbead capture well is 0.8-1.8 times the microbead diameter, and the width of the connecting channel is smaller than the cell diameter; when the cell and microbead particle sizes differ greatly, the connecting channel can be omitted, and each microwell includes a directly connected cell capture well and a microbead capture well, the depth of the cell capture well is 1.2-1.8 times the cell diameter, and the depth of the microbead capture well is 1.2-1.8 times the microbead diameter.

4. The cell-bead capture pairing device for single-cell omics analysis according to claim 1, characterized in that: The material of the micro-well capture layer is selected from: polymethyl methacrylate, silica gel, polydimethylsiloxane, SU8, single crystal silicon, and silicon oxide.

5. The cell-bead capture pairing device for single-cell omics analysis according to claim 1, characterized in that: The materials of the auxiliary capture structure that uses a physical barrier between cells and microbeads or uses a fluid dynamics method to separate cells and microbeads include: polymethyl methacrylate, silica gel, polydimethylsiloxane, SU8, single crystal silicon, and silicon oxide.

6. The cell-bead capture pairing device for single-cell omics analysis according to claim 1, characterized in that: The top layer material of the auxiliary capture structure for separating cells and microbeads based on dielectrophoresis includes: polymethyl methacrylate, silica gel, SU8, polydimethylsiloxane, silicon oxide, and the electrode layer uses conductive materials including conductive polymers, doped silicon, and metals.

7. A method for cell-bead capture pairing for single-cell omics analysis, characterized in that: There are three ways: Mode 1, providing a cell-microbead capture pairing device for single-cell omics analysis according to claim 1, wherein during operation, a cell solution and a microbead solution are respectively injected from the inlets of the cell sample flow channel and the microbead sample flow channel, so that the cells and the microbeads flow in the flow channels above the cell capture well and the microbead capture well, respectively, and after the cells and the microbeads fill the cell sample flow channel and the microbead sample flow channel, respectively, the cells and the microbeads are made to stand still, and the cells and the microbeads fall into the cell capture well and the microbead capture well, respectively, by gravity, thereby completing a one-to-one capture pairing of cells and microbeads in each microwell; Mode 2, providing a cell-microbead capture pairing device for single-cell omics analysis according to claim 1, wherein when working, a sheath flow is injected from the inlet of the sheath flow channel, and a cell solution and a microbead solution are injected from the inlet of the cell sample flow channel and the microbead sample flow channel, respectively, and the sheath flow is adjusted to focus the cells and microbeads in the main flow channel above the cell well side and the microbead well side, respectively, to avoid cross contamination, and the sample injection is stopped after the cells and microbeads are respectively covered with the cell well side and the microbead well side, and the cells and microbeads fall into the cell capture well and the microbead capture well, respectively, by gravity, thereby completing the one-to-one capture pairing of cells and microbeads in each microwell; Method three, providing a cell-bead capture pairing device for single-cell omics analysis according to claim 1, comprising the following four methods: a. Adopting negative dielectrophoresis control of cells and microbeads to assist in capture pairing: first, inject sheath flow into the chip from the inlet of the sheath flow channel and the cell sample channel, and inject microbead solution from the inlet of the microbead sample channel, use the sheath flow to restrict the microbeads to the microbead well side, use negative dielectrophoresis to make the microbeads adhere to the microbead well side, and then stop dielectrophoresis excitation to allow the microbeads to settle into the microbead capture wells of the microwell capture layer by gravity; Inject sheath flow into the chip from the inlet of the sheath flow channel and the microbead sample channel, inject cell solution from the inlet of the cell sample channel, use the sheath flow to restrict the cells to the side of the cell well, use negative dielectrophoresis to make the cells adhere to the side of the cell well, then stop dielectrophoresis excitation to allow the cells to settle into the cell capture well of the microwell capture layer by gravity, and complete the one-to-one capture pairing of cells and microbeads in each microwell; the capture order of microbeads and cells can be exchanged; b. Negative dielectrophoresis control of cells and microbeads is used to assist capture pairing: first, sheath flow is injected into the chip from the inlet of the sheath flow channel, and microbeads and cells are injected from the inlet of the microbead sample flow channel and the inlet of the cell sample flow channel, respectively. The sheath flow confines the microbeads and cells above the microbead well side and above the cell well side, respectively; Then, the negative dielectrophoresis effect is used to make the microbeads and cells adhere to the upper side of the microbead well and the upper side of the cell well, respectively, and the injection of all fluids is stopped. Then, the dielectrophoresis excitation is stopped to make the microbeads and cells settle to the microbead capture well and the cell capture well of the microwell capture layer respectively by gravity, thereby completing the one-to-one capture pairing of cells and microbeads in each microwell; c. Using positive dielectrophoresis control of cells and negative dielectrophoresis of microbeads to assist in capture pairing: first, inject sheath flow into the chip from the inlets of the sheath flow channel and the cell sample channel, and inject microbead solution from the inlet of the microbead sample channel, use the sheath flow to confine the microbeads to the microbead well side, use negative dielectrophoresis to make the microbeads adhere to the microbead well side, stop all fluid injection, and then stop dielectrophoresis excitation so that the microbeads settle to the microbead capture wells of the microwell capture layer by gravity; Inject sheath flow into the chip from the inlet of the sheath flow channel and the microbead sample channel, inject cell solution from the inlet of the cell sample channel, use the sheath flow to restrict the cells near the side wall of the double-layer structure electrode on one side of the cell well, use positive dielectrophoresis to make the cells adhere to the side of the cell well, stop all fluid injections, and then stop dielectrophoresis excitation to allow the cells to settle into the cell capture wells of the microwell capture layer by gravity, thereby completing a one-to-one capture pairing of cells and microbeads in each microwell; the capture order of microbeads and cells can be interchanged; d. Using positive dielectrophoresis control of cells and negative dielectrophoresis of microbeads to assist in capture pairing: first, inject sheath flow into the chip from the inlet of the sheath flow channel, and simultaneously inject microbeads and cells from the inlet of the microbead sample flow channel and the inlet of the cell sample flow channel, respectively. The sheath flow confines the microbeads and cells to the side walls of the electrode layer above the microbead well side and near the side walls of the cell well side, respectively, to avoid cross contamination; then, using dielectrophoresis, the cells are controlled by positive dielectrophoresis to adhere to the side walls of the upper electrode layer on the cell well side, and simultaneously, the microbeads are controlled by negative dielectrophoresis to adhere to the side walls of the lower electrode layer on the microbead well side, stop injecting all fluids, and then stop dielectrophoresis excitation to allow the microbeads and cells to settle to the microbead capture wells and cell capture wells of the microwell capture layer, respectively, by gravity, thereby completing a one-to-one capture pairing of cells and microbeads in each microwell.

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