Integrated chip, manufacturing method thereof and single cell capturing / culturing method

Abstract

The single cell capture / culture integrated chip comprises a silicon substrate, an upper glass substrate and a lower glass substrate, and the silicon substrate, the upper glass substrate and the lower glass substrate are bonded into a whole, the upper surface of the silicon substrate is provided with a top-layer flow channel, two ends of the top-layer flow channel are respectively provided with a top-layer flow channel inlet and a top-layer flow channel outlet which do not penetrate through the silicon substrate, and the upper glass substrate is provided with a top-layer liquid inlet, a top-layer liquid outlet, a bottom-layer liquid inlet and a bottom-layer liquid outlet; cell capturing / culturing units which penetrate through the thickness of the silicon substrate and are distributed in an array mode are formed, upper openings of the cell capturing / culturing units are vertically communicated with lower openings located in the bottom layer flow channel cavity, the outer diameter of the upper openings is larger than the outer diameter of single cells to be captured / cultured by more than two times, and the outer diameter of the lower openings is smaller than the outer diameter of the single cells to be captured / cultured. According to the chip, the top-layer flow channel cavity and the bottom-layer flow channel cavity are communicated only through the cell capturing / culturing units which are distributed in an array manner, so that a chip inner cavity is formed; the invention also provides a manufacturing method of the chip and a single cell capturing / culturing method using the chip.

Description

Technical Field

[0001] The present invention belongs to the technical field of microfluidic biochips, and specifically relates to a single cell capture / culture integrated chip and a manufacturing method thereof for realizing single cell capture and culture under high-throughput conditions using microfluidic technology, as well as a single cell capture and culture method using the integrated chip. Background Art

[0002] With the continuous improvement and development of microfluidics and micromachining technology, the advantages of micro total analysis system (μ-TAS) in the fields of biological research and clinical experimental diagnosis at the cellular level are becoming increasingly significant. Microfluidic chips have shown obvious advantages in cell analysis due to the advantages of functional unit size comparable to cell size, high precision and fast and convenient detection. This technology can integrate all current cell analysis steps and processes (such as cell manipulation, cell capture / screening, cell culture, and online real-time dynamic monitoring and analysis, etc.) on a microchip to achieve the integration of analysis operations, which can reduce damage and contamination to cell samples during operation. It is very suitable for single cell capture / culture. This type of microchip is called a microfluidic biochip. The high-throughput single cell capture / culture integrated chip of the present invention also belongs to this type of microfluidic biochip.

[0003] How to separate single cells from a certain volume of cell suspension is the premise for single-cell analysis. The traditional laboratory methods for obtaining single cells are mainly limited dilution and micromanipulation, which have disadvantages such as low efficiency, poor precision and high cost in manipulating micron-scale cell samples. The current mainstream microfluidics technology is an effective method for obtaining single cells. Compared with traditional methods, it has the advantages of high precision, small size, high throughput and high capture efficiency.

[0004] There are many types of microfluidic single-cell capture chips, which can be divided into two categories: active capture and passive capture, depending on whether external force induction is required. Among them, active capture microfluidic chips use external forces such as magnetism, electricity, and light to achieve capture; passive capture uses fluid dynamic effects to achieve capture without applying any external force or labeling.

[0005] The capture structure of microfluidic single-cell capture chips based on fluid dynamics principles can be mainly divided into:

[0006] (1) Microwell structure: The microwell structure uses the cell's own weight to settle a single cell in each independent microwell to achieve the purpose of cell capture. The single-cell capture chip using this structure has the advantages of high throughput and simple operation, but it takes a long time to complete a capture. At the same time, it may not capture a single cell or capture multiple single cells in the microwell. In addition, it is not convenient to control the exchange of solutions in the microwell, and the cell proliferation conditions are limited;

[0007] (2) Micro-column structure. Single-cell capture chips based on micro-column arrays usually design single-cell interceptors on the main fluid channel to achieve single-cell capture by controlling fluid flow. The manufacturing process of single-cell capture chips with this structure is mature and easy to operate, and the array design can achieve single-cell capture at high throughput. In addition, the design of the micro-column structure provides a certain support force for the perfusion culture of single cells, which can reduce the tensile stress on the cells and improve cell activity. However, single-cell capture chips based on micro-column arrays are usually parallel to the main fluid channel, which will cause uneven distribution of captured single cells, increase the local fluid resistance at the cell capture site, and easily cause channel blockage and multi-cell aggregation.

[0008] (3) Bypass channel structure. The single-cell capture chip based on the bypass channel is designed with a main channel with larger flow resistance and a side channel with smaller flow resistance in the microfluidic chip. When the cells flow in the main channel, they will be pushed to the side channel with smaller flow resistance. The capture structure is designed at the connection between the main channel and the side channel to achieve single-cell capture. This structure can reduce channel blockage and multi-cell aggregation, has the advantages of high throughput, reduces cell damage, and is conducive to subsequent cell culture. At the same time, its single-cell capture efficiency is also extremely high (more than 90%).

[0009] With the continuous development of single-cell analysis, higher requirements are placed on single-cell analysis microfluidic chips, requiring long-term cell culture in microfluidic devices in order to study cell differentiation, cell development and other activities.

[0010] However, the existing microfluidic biochips have the following shortcomings: 1. The functions are relatively single, some can only realize the single function of single cell capture, and some can only realize the single function of single cell culture. 2. The existing microfluidic biochips also have the problem of insufficient flux, which makes the number of single cells captured or cultured in one operation insufficient, and it is difficult to meet the needs of clinical medicine and scientific research applications. 3. The effectiveness and stability of single cell capture are not enough, and it is not possible to achieve that each single cell capture unit can capture a single cell.

[0011] Based on the above three capture structures, the present invention designs a non-planar structured high-throughput single-cell capture and culture integrated chip to meet the needs of long-term culture after single-cell capture, thereby solving the problems of low efficiency, low throughput, poor activity and difficulty in long-term culture of cells in current microfluidic chips. Summary of the invention

[0012] The technical problem to be solved by the present invention is to provide a single cell capture / culture integrated chip that uses microfluidic technology to achieve single cell capture and culture under high-throughput conditions, and a method for manufacturing the same, as well as a method for single cell capture and culture using the integrated chip. A series of problems existing in the prior art are solved: 1. The single cell capture / culture functions of single cell capture and culture are integrated into one, and the two technical problems of single cell capture and culture are solved at the same time. 2. The problem of insufficient flux that also exists in existing microfluidic biochips is solved, so that the number of single cells captured or cultured in one operation is sufficient to meet the needs of clinical medicine and scientific research applications. 3. The problem of insufficient effectiveness and stability of single cell capture that also exists in existing microfluidic biochips is solved.

[0013] In order to solve the above technical problems, the technical solution adopted by the high-throughput single-cell capture / culture integrated chip of the present invention is:

[0014] A high-throughput single-cell capture / culture integrated chip, comprising a silicon substrate, an upper glass substrate and a lower glass substrate bonded to the silicon substrate, the three being bonded together into one; characterized in that a top flow channel is provided on the upper surface of the silicon substrate, a top flow channel inlet and a top flow channel outlet that do not penetrate the silicon substrate are respectively provided at both ends of the top flow channel, and a top liquid inlet and a top liquid outlet that penetrate the upper glass substrate and are respectively connected to the top flow channel inlet and the top flow channel outlet are respectively provided on the upper glass substrate at positions corresponding to the positions of the top flow channel inlet and the top flow channel outlet; a bottom flow channel is provided on the lower surface of the silicon substrate, a bottom flow channel inlet and a bottom flow channel outlet that penetrate the silicon substrate are respectively provided at both ends of the bottom flow channel, and a bottom flow channel inlet and a bottom flow channel outlet that penetrate the upper glass substrate and are respectively connected to the bottom flow channel inlet and the bottom flow channel outlet are respectively provided on the upper glass substrate at positions corresponding to the positions of the bottom flow channel inlet and the bottom flow channel outlet The laminar flow channel inlet and the bottom flow channel outlet are connected to each other; the lower surface of the upper glass substrate is bonded to the upper surface of the silicon substrate to form a top flow channel cavity, and the upper surface of the lower glass substrate is bonded to the lower surface of the silicon substrate to form a bottom flow channel cavity. The top flow channel cavity and the bottom flow channel cavity are penetrated through the thickness of the silicon substrate to open cell capture / culture units distributed in an array. The upper opening of each cell capture / culture unit located in the top flow channel cavity is connected to the lower opening located in the bottom flow channel cavity up and down. The upper opening is more than 2 times larger than the diameter of the single cell to be captured / cultured, and the lower opening is smaller than the diameter of the single cell to be captured / cultured. The high-throughput single-cell capture / culture integrated chip connects the top flow channel cavity and the bottom flow channel cavity only through the cell capture / culture units distributed in an array, thereby forming a complete chip internal cavity structure.

[0015] The following is a further solution of the high-throughput single-cell capture / culture integrated chip of the present invention:

[0016] The top-level flow channel on the upper surface of the silicon substrate is arranged from the top-level liquid inlet to the top-level liquid outlet in a manner of being divided into multiple levels of refinement and diversion and then being coarsened and merged in multiple levels, and the top-level flow channels of the finest level are arranged parallel to each other; the bottom-level flow channel on the lower surface of the silicon substrate is also arranged from the bottom-level liquid inlet to the bottom-level liquid outlet in a manner of being divided into multiple levels of refinement and diversion and then being coarsened and merged in multiple levels, and the bottom-level flow channels of the finest level are also arranged parallel to each other.

[0017] The top flow channel on the upper surface of the silicon substrate is first differentiated into two thinner top flow channels from the top flow channel inlet to the top flow channel outlet, and the two top flow channels continue to differentiate into four thinner top flow channels, and the four top flow channels continue to differentiate into eight thinner top flow channels, and after the eight top flow channels pass through the cell capture / culture area in the middle of the top flow channels, the eight top flow channels are merged into four thicker top flow channels, the four top flow channels are merged into two thicker top flow channels, and the two top flow channels are merged into a thicker single top flow channel, and isolation is set between the differentiated top flow channels, and the eight thinnest top flow channels located in the center of the flow channel are arranged parallel to each other; The bottom flow channel on the lower surface of the silicon substrate is also differentiated into two thinner bottom flow channels from the bottom flow channel inlet to the bottom flow channel outlet, and the two bottom flow channels continue to differentiate into four thinner bottom flow channels, and the four bottom flow channels continue to differentiate into eight thinner bottom flow channels. After the eight bottom flow channels pass through the cell capture / culture area in the middle of the bottom flow channels, the eight bottom flow channels are merged into four thicker bottom flow channels, the four bottom flow channels are merged into two thicker bottom flow channels, and the two bottom flow channels are merged into a single thicker bottom flow channel. Isolations are set between the differentiated bottom flow channels, and the eight thinnest bottom flow channels located in the center of the flow channels are also arranged parallel to each other.

[0018] The high-throughput single-cell capture / culture integrated chip of the present invention comprises a silicon substrate with a thickness of 300 μm, a surface of

[100] crystal plane, and double-sided polishing. The top flow channel on the upper surface of the silicon substrate is 20 to 30 μm deep and 100 to 700 μm wide, ensuring that cells can pass smoothly under high-throughput conditions; the bottom flow channel on the lower surface of the silicon substrate is 230 to 240 μm deep and 100 to 700 μm wide, and the flow channel volume ensures the passage of cell culture fluid.

[0019] Each cell capture / culture unit is in the shape of an inverted pyramid, and its upper opening and lower opening are quadrilaterals. The upper opening is a square with a side length of 60 to 70 μm, which is much larger than the cell diameter of 10 to 20 μm and slightly narrower than the width of the single flow channel after differentiation, ensuring that the cells can pass through; the lower opening located on the bottom flow channel of the silicon substrate is a square with a side length of 5 to 10 μm, which is smaller than the cell diameter, ensuring that the cells are confined in the cell capture / culture unit.

[0020] The cell capture / culture unit provides sufficient proliferation space for the captured cells. One cell capture / culture unit can accommodate 20 to 30 cells. When in use, the flowing cell culture fluid creates a good living environment for the cells.

[0021] The top layer flow channel and the bottom layer flow channel are arranged crosswise on the upper and lower surfaces of the silicon substrate, thereby improving the area utilization of the silicon substrate. The design of the cross structure of different layers is conducive to the formation of flow velocity difference between the upper and lower layer flow channels.

[0022] In order to solve the above technical problems, the technical solution adopted by the method for manufacturing a high-throughput single-cell capture / culture integrated chip of the present invention is:

[0023] A method for manufacturing a high-throughput single-cell capture / culture integrated chip includes the manufacturing of a silicon substrate and the selection and manufacturing of an upper glass substrate and a lower glass substrate, which can be performed in any order, and bonding the three together; wherein the manufacturing steps of the silicon substrate include:

[0024] Step 1: Use a four-inch, double-sided polished, 300 μm thick silicon substrate as a substrate;

[0025] Step 2: Double-sided thermal oxidation process forms a 1 μm thick silicon oxide layer on both the front and back sides of the silicon substrate;

[0026] Step 3, spin-coating photoresist on the back side of the silicon substrate, photolithography the back side of the silicon substrate according to the design layout, and developing the bottom flow channel inlet, bottom flow channel, and bottom flow channel outlet patterns;

[0027] Step 4: Using the Samco RIE process combination, RIE etches the silicon oxide layer of the silicon substrate by 1 μm on the reverse side to form etching windows for the bottom flow channel inlet, bottom flow channel, and bottom flow channel outlet;

[0028] Step 5: Use deep reactive plasma etching to etch the back side of the silicon substrate by 240 μm to form a bottom channel, and complete the etching of the back side of the bottom channel inlet and the bottom channel outlet;

[0029] Step 6: Using a stripping process combination to remove the photoresist and impurities remaining on the silicon substrate after the previous steps;

[0030] Step 7: using a TEOS LPCVD process, i.e., a chemical vapor deposition silicon oxide process, chemically vapor depositing silicon oxide on a silicon substrate to form a silicon oxide layer with a thickness of 0.4 μm;

[0031] Step 8, spin-coating photoresist on the front side of the silicon substrate, and photolithographically developing a bottom channel inlet, a top channel, a top channel inlet, a top channel outlet, and a bottom channel outlet pattern on the front side of the silicon substrate according to the design layout, wherein the patterns of the top channel, the top channel inlet, and the top channel outlet are formed simultaneously, and the subsequent manufacturing process is the same;

[0032] Step 9: Using the Samco RIE process combination, RIE front-side etching of silicon oxide on the silicon substrate is 1.4 μm, forming etching windows for the bottom channel inlet, top channel, top channel inlet, top channel outlet, and bottom channel outlet;

[0033] Step 10: using a stripping process combination to remove the photoresist and impurities remaining on the silicon substrate after the above steps;

[0034] Step 11: Use deep reactive plasma etching to etch the silicon substrate 20 μm on the front side to form a top-layer flow channel, a top-layer flow channel inlet, and a top-layer flow channel outlet, and complete the front-side partial etching of the bottom-layer flow channel inlet and the bottom-layer flow channel outlet;

[0035] Step 12: Using TEOS LPCVD (chemical vapor deposition silicon oxide) process, chemical vapor deposition of silicon oxide 0.4 μm on the silicon substrate;

[0036] Step 13: Spin-coat photoresist on the front side of the silicon substrate, and continue to photolithographically develop the bottom channel inlet and bottom channel outlet patterns on the front side of the silicon substrate according to the design layout, and at the same time, photolithographically develop the cell capture / culture unit pattern;

[0037] Step 14: Using the Samco RIE process combination, RIE front-side etching of silicon substrate silicon oxide 0.4 μm to form bottom channel inlet, cell capture / culture unit, and bottom channel outlet etching windows;

[0038] Step 15: using a stripping process combination to remove the photoresist and impurities remaining on the silicon substrate after the above steps;

[0039] Step 16: using an anisotropic wet etching process, the anisotropic etching solution etches the silicon substrate by about 40 μm to form a cell capture / culture unit, and completes the etching of the bottom channel inlet and the bottom channel outlet;

[0040] Step 17: Use SiO2 (post-metal) etching process combined with BOE process to remove the residual silicon oxide mask on the silicon substrate, release and form the bottom flow channel inlet and bottom flow channel outlet;

[0041] Step 18: Select two 4-inch glass substrates, leave one unprocessed as the lower glass substrate, and use a laser drilling process to drill holes on the other glass substrate according to the designed layout to make an upper glass substrate.

[0042] Step 19: Align and bond the prepared silicon substrate, the upper glass substrate, and the lower glass substrate to complete the production of at least one high-throughput single-cell capture / culture integrated chip.

[0043] Further solutions of the method for making the high-throughput single-cell capture / culture integrated chip of the present invention:

[0044] As the material for making the silicon substrate, a silicon substrate plate-like assembly is used; as the material for making the upper glass substrate, an upper glass substrate plate-like assembly is used; as the material for making the lower glass substrate, a lower glass substrate plate-like assembly is used; in step nineteen, the prepared silicon substrate plate-like assembly is aligned and bonded with the upper glass substrate plate-like assembly and the lower glass substrate plate-like assembly, and an integrated chip plate-like assembly is formed by silicon-glass anodic bonding; and a silicon chip cutting machine is used to cut out individual integrated chips along the designed cutting lines, thereby completing the production of high-throughput single-cell capture / culture integrated chips in batches.

[0045] In order to solve the above technical problems, the technical solution adopted by the single cell capture / culture method of the present invention is:

[0046] A single cell capture / culture method, characterized in that, using the high-throughput single cell capture / culture integrated chip described above, first, the same cell culture solution without cells is simultaneously input into the top flow channel and the bottom flow channel of the integrated chip through the top liquid inlet and the bottom liquid inlet, and after the internal cavity of the chip is filled, the input of the solution is suspended and maintained for a set time to complete the pretreatment of the chip; then, the cell culture solution containing cells is continuously introduced into the top flow channel of the chip through the top liquid inlet, and its flow rate is flow rate a, and at the same time, the cell culture solution without cells is continuously introduced into the bottom flow channel of the chip through the bottom liquid inlet, and its flow rate is flow rate b, and the flow rate is appropriately adjusted so that the flow rate of the liquid in the top and bottom flow channels meets: flow rate a; The flow rate a is less than the flow rate b; under this condition, the cells located on the cell capture / culture unit in the top flow channel will spontaneously move to the lower opening of the cell capture / culture unit under the influence of the flow rate difference, and finally the cells will be adsorbed by this small hole under the action of the flow rate difference; when cells are captured in all cell capture / culture units, the cell culture solution without cells is continuously introduced into the top flow channel, and the flow rate does not change. The culture solution will take out the excess cells in the culture unit to ensure that each cell capture / culture unit contains a single cell. The flowing culture solution provides nutritional conditions for the cells, and finally achieves the purpose of single cell capture and culture; in the above process, after the internal cavity of the chip is filled, the excess cell culture solution overflows from the top liquid outlet and the bottom liquid outlet.

[0047] Further embodiments of the single cell capture / culture method of the present invention include:

[0048] Four hoses of appropriate size and length are used, one end of which is respectively connected to the top liquid inlet, the top liquid outlet, the bottom liquid inlet and the bottom liquid outlet; the other end of the hose connected to the top liquid inlet is connected to a cell culture solution container containing cells or not containing cells via a micro-transfer pump; the other end of the hose connected to the bottom liquid inlet is connected to a cell culture solution container not containing cells via a micro-transfer pump; the micro-transfer pump drives the delivery process of the cell culture solution to the internal cavity of the chip; the other end of the hose connected to the top liquid outlet and the bottom liquid outlet leads to a waste liquid container; after the internal cavity of the chip is filled, the excess cell culture solution overflows from the top liquid outlet and the bottom liquid outlet and flows to the waste liquid container via the hose.

[0049] The high-throughput single-cell capture / culture integrated chip of the present invention adopts the method of bonding the lower surface of an upper glass substrate with the upper surface of a silicon substrate to form a top-layer flow channel cavity, and bonding the upper surface of a lower glass substrate with the lower surface of a silicon substrate to form a bottom-layer flow channel cavity. The top-layer flow channel cavity and the bottom-layer flow channel cavity are penetrated through the thickness of the silicon substrate to open cell capture / culture units distributed in an array. The upper opening of each cell capture / culture unit located in the top-layer flow channel cavity is connected to the lower opening located in the bottom-layer flow channel cavity up and down. The upper opening is more than twice larger than the outer diameter of the single cell to be captured / cultured, and the lower opening is smaller than the outer diameter of the single cell to be captured / cultured. The chip connects the top-layer flow channel cavity and the bottom-layer flow channel cavity only through the cell capture / culture units distributed in an array, thereby forming a complete chip internal cavity structure. When in use, the same cell culture solution without cells is introduced into the top flow channel and the bottom flow channel of the integrated chip until the chip is filled with the solution; then, the culture solution containing cells is continuously introduced into the top flow channel of the chip, and at the same time, the cell culture solution without cells is continuously introduced into the bottom flow channel of the chip at a higher flow rate, and the flow rate is appropriately adjusted so that the flow rates of the liquids in the top and bottom flow channels have a sufficient flow rate difference, and the cells located on the cell capture / culture unit in the top flow channel move toward the lower opening of the cell capture / culture unit under the influence of the flow rate difference, and finally the cells are adsorbed by this small hole under the action of the flow rate difference; when cells are captured in all units, the cell culture solution without cells is continuously introduced into the top flow channel, and the flow rate does not change. The culture solution will take out the excess cells in the culture unit, which can ensure that a single cell is contained in a single unit. The flowing culture solution provides nutritional conditions for the cells, and finally the purpose of single cell capture and culture can be achieved.

[0050] The high-throughput single-cell capture / culture integrated chip of the present invention solves a series of problems existing in the prior art: 1. The single-cell capture / culture functions of single-cell capture and culture are integrated into one, solving the two technical problems of single-cell capture and culture at the same time. 2. It solves the problem of insufficient flux that also exists in the existing microfluidic biochips, so that the number of single cells captured or cultured in one operation is sufficient to fully meet the application needs in clinical medicine and scientific research applications at this stage. 3. It solves the problem of insufficient effectiveness and stability of single-cell capture that also exists in the existing microfluidic biochips. In one operation, each single-cell capture unit in the chip can capture a single cell. And the cells can be cultured and proliferated in each cell capture / culture unit.

[0051] The high-throughput single-cell capture / culture integrated chip of the present invention has the following significant advantages:

[0052] (1) Compared with the traditional microfluidic cell capture chip, the present invention not only captures cells but also provides corresponding proliferation conditions for the cells, and various activities of the cells can be observed while the cells are captured.

[0053] (2) The chip uses MEMS technology, which has good chip consistency and can be mass-produced, reducing the manufacturing cost of a single device.

[0054] (3) Compared with the microfluidic chip made on a traditional plane, the present invention innovatively designs the structure of the microfluidic chip and uses fluid mechanics to achieve cell transfer in the horizontal direction and cell capture in the vertical direction.

[0055] The present invention aims at the demand for efficient screening of high-titer hybridoma cells in monoclonal antibody preparation technology, and based on microfluidics technology and microscopic images, makes structural innovations, and develops a high-throughput single-cell capture and culture integrated chip to solve the problems of low efficiency and poor activity of captured cells under traditional technical means, and provides a technical solution for high-throughput capture and culture of hybridoma cells, which is useful for improving the capture efficiency of hybridoma cells and shortening the preparation time of monoclonal antibodies. The high-throughput single-cell capture / culture integrated chip of the present invention has the characteristics of stable and reliable single-cell capture / culture, and can be mass-produced, low-cost, and has important practical application value. BRIEF DESCRIPTION OF THE DRAWINGS

[0056] Figure 1 It is a three-dimensional schematic diagram of the appearance of the high-throughput single-cell capture / culture integrated chip of the present invention.

[0057] Figure 2 It is a three-dimensional schematic diagram of the hypothetical separation state of the silicon substrate, the upper glass substrate and the lower glass substrate of the high-throughput single-cell capture / culture integrated chip of the present invention.

[0058] Figure 3It is a three-dimensional schematic diagram of the top flow channel structure on the upper surface of the silicon substrate.

[0059] Figure 4 It is a three-dimensional schematic diagram of the bottom flow channel structure on the lower surface of the silicon substrate.

[0060] Figure 5 for Figure 3 Enlarged schematic diagram of part A in the middle.

[0061] Figure 6 for Figure 4 Enlarged schematic diagram of part B in the middle.

[0062] Figure 7 Schematic diagram of the changes in cross-sectional shape of relevant parts in each step of the silicon substrate manufacturing process.

[0063] Figure 7 It is schematic and does not correspond to the actual cross-sectional shape and length of the silicon substrate. For the convenience of representation, the imaginary cross section is a cross section that passes through the bottom channel inlet, the top channel, one of the cell capture / culture units, the bottom channel, and the bottom channel outlet. Figure 7 Contains 17 schematic diagrams of cross-sectional shape changes, including Figure 7-1 to Figure 7-1 7, respectively indicating the status after the corresponding steps are completed, divided into 2 pages.

[0064] Numbers in the figure: 1. Silicon substrate; 2. Upper glass substrate; 3. Lower glass substrate; 4. Top flow channel; 5. Top flow channel inlet; 6. Top flow channel outlet; 7. Top liquid inlet; 8. Top liquid outlet; 9. Bottom flow channel; 10. Bottom flow channel inlet; 11. Bottom flow channel outlet; 12. Bottom liquid inlet; 13. Bottom liquid outlet; 14. Cell capture / culture unit; 15. Silicon oxide layer; 16. Photoresist. DETAILED DESCRIPTION

[0065] The present invention is further described in detail below with reference to the accompanying drawings.

[0066] The high-throughput single-cell capture / culture integrated chip of the present invention is as follows: Figure 1 , Figure 2As shown, it includes a silicon substrate 1, an upper glass substrate 2 and a lower glass substrate 3 which are bonded to the silicon substrate 1, and the three are bonded to form a whole; a top flow channel 4 is provided on the upper surface of the silicon substrate 1, and a top flow channel inlet 5 and a top flow channel outlet 6 which do not penetrate the silicon substrate 1 are respectively provided at two ends of the top flow channel 4, and a top liquid inlet 7 and a top liquid outlet 8 which penetrate the upper glass substrate 2 and are respectively connected to the top flow channel inlet 5 and the top flow channel outlet 6 at positions corresponding to the positions of the top flow channel inlet 5 and the top flow channel outlet 6; a bottom flow channel 9 is provided on the lower surface of the silicon substrate 1, and a bottom flow channel inlet 10 and a bottom flow channel outlet 11 which penetrate the silicon substrate 1 are respectively provided at two ends of the bottom flow channel 9, and a top glass substrate 2 is respectively provided with positions corresponding to the positions of the bottom flow channel inlet 10 and the bottom flow channel outlet 11 which penetrate the upper glass substrate 2 and are respectively connected to the bottom flow channel inlet 10 and the bottom flow channel outlet 11 and a bottom liquid inlet 12 and a bottom liquid outlet 13 connected to the bottom flow channel outlet 11; the lower surface of the upper glass substrate 2 is bonded to the upper surface of the silicon substrate 1 to form a top flow channel cavity, and the upper surface of the lower glass substrate 3 is bonded to the lower surface of the silicon substrate 1 to form a bottom flow channel cavity. The top flow channel cavity and the bottom flow channel cavity are penetrated through the thickness of the silicon substrate 1 to open cell capture / culture units 14 distributed in an array. The upper opening of each cell capture / culture unit 14 located in the top flow channel cavity is connected to the lower opening located in the bottom flow channel cavity from top to bottom. The upper opening is more than twice larger than the outer diameter of the single cell to be captured / cultured, and the lower opening is smaller than the outer diameter of the single cell to be captured / cultured. The high-throughput single-cell capture / culture integrated chip connects the top flow channel cavity and the bottom flow channel cavity only through the cell capture / culture units 14 distributed in an array, thereby forming a complete chip internal cavity structure.

[0067] like Figure 3 As shown, the top flow channel 4 on the upper surface of the silicon substrate 1 is arranged from the top flow channel inlet 5 to the top flow channel outlet 6 in a manner of being divided into multiple levels of fine flow and then being combined into multiple levels of coarse flow, and the top flow channels 4 of the finest level are arranged parallel to each other. Figure 4 As shown, the bottom flow channel 9 on the lower surface of the silicon substrate 1 is arranged from the bottom flow channel inlet 10 to the bottom flow channel outlet 11, and is divided into multiple stages of refinement and then coarsened and merged. The bottom flow channels 9 at the finest level are also arranged parallel to each other.

[0068] Specifically, Figure 3As shown, the top flow channel 4 on the upper surface of the silicon substrate 1 first differentiates into two thinner top flow channels 4 from the top flow channel inlet 5 to the top flow channel outlet 6, and the two top flow channels 4 continue to differentiate into four thinner top flow channels 4, and the four top flow channels 4 continue to differentiate into eight thinner top flow channels 4. After the eight top flow channels 4 pass through the cell capture / culture area in the middle of the top flow channels 4, the eight top flow channels 4 merge into four thicker top flow channels 4, and the four top flow channels 4 merge into two thicker top flow channels 4, and the two top flow channels 4 merge into a thicker single top flow channel 4. The differentiated top flow channels 4 are separated, and the eight thinnest top flow channels 4 located in the center of the flow channel are arranged parallel to each other. Figure 4 As shown, the bottom flow channel 9 on the lower surface of the silicon substrate 1 is also differentiated into two thinner bottom flow channels 9 from the bottom flow channel inlet 10 to the bottom flow channel outlet 11, and the two bottom flow channels 9 are further differentiated into four thinner bottom flow channels 9, and the four bottom flow channels 9 are further differentiated into eight thinner bottom flow channels 9. After the eight bottom flow channels 9 pass through the cell capture / culture area in the middle of the bottom flow channels 9, the eight bottom flow channels 9 are merged into four thicker bottom flow channels 9, the four bottom flow channels 9 are merged into two thicker bottom flow channels 9, and the two bottom flow channels 9 are merged into a thicker single bottom flow channel 9. The bottom flow channels 9 after differentiation are separated, and the eight thinnest bottom flow channels 9 located in the center of the flow channel are also arranged in parallel with each other. The eight top flow channels 4 are arranged in parallel with each other, and the eight bottom flow channels 9 are also arranged in parallel with each other, which can improve the area utilization rate of the silicon substrate and facilitate processing and manufacturing. More importantly, since the cell capture / culture unit 14 is the key technical feature of the technical solution of the present invention, it is easy to evenly arrange the cell capture / culture unit 14 on the silicon substrate so that it has an equal chance to capture cells, thereby ensuring that each single-cell capture unit in the chip can capture a single cell in one operation.

[0069] The high-throughput single-cell capture / culture integrated chip of the present invention is as follows: Figure 3 , Figure 4 As shown, the silicon substrate 1 is a silicon substrate 1 with a thickness of 300 μm, a surface of

[100] crystal plane, and double-sided polishing. The top flow channel 4 on the upper surface of the silicon substrate 1 is 20 to 30 μm deep and 100 to 700 μm wide, ensuring that cells can pass smoothly under high-throughput conditions; the bottom flow channel 9 on the lower surface of the silicon substrate 1 is 230 to 240 μm deep and 100 to 700 μm wide, and the flow channel volume ensures the passage of cell culture fluid.

[0070] like Figure 5 As shown, each cell capture / culture unit is in the shape of an inverted pyramid, with its upper and lower openings forming a quadrilateral. The upper opening is a square with a side length of 60 to 70 μm, which is much larger than the cell diameter of 10 to 20 μm and slightly narrower than the width of the single flow channel after differentiation, ensuring that the cells can pass through. Figure 6 As shown, the opening on the bottom flow channel of the silicon substrate is a square with a side length of 5 to 10 μm, which is smaller than the diameter of the cell, ensuring that the cell is confined in the cell capture / culture unit.

[0071] like Figure 5 , Figure 6 As shown, each cell capture / culture unit 14 is in the shape of an inverted pyramid, and its upper opening and lower opening are quadrilaterals. The upper opening is a square with a side length of 60 to 70 μm, which is much larger than the cell diameter of 10 to 20 μm, and slightly narrower than the width of the single flow channel after differentiation, ensuring that the cells can pass through; the opening on the bottom flow channel 9 of the silicon substrate 1 is a square with a side length of 5 to 10 μm, which is smaller than the cell diameter, ensuring that the cells are confined in the cell capture / culture unit 14. The cell capture and culture unit provides sufficient proliferation space for the captured cells. One cell capture / culture unit 14 can accommodate 20 to 30 cells. When in use, the flowing cell culture fluid creates a good living environment for the cells.

[0072] like Figure 3 , Figure 4 As shown, the top flow channel 4 and the bottom flow channel 9 are arranged crosswise on the upper and lower surfaces of the silicon substrate 1, which improves the area utilization of the silicon substrate 1. The design of different layers of cross structures is conducive to the formation of flow velocity difference between the upper and lower flow channels. It can improve the area utilization of the silicon substrate and facilitate processing and manufacturing. More importantly, because the cell capture / culture unit 14 is a key technical feature in the technical solution of the present invention, it is easy to achieve uniform arrangement of the cell capture / culture unit 14 on the silicon substrate, so that it has an equal chance to capture cells, so as to ensure that each single cell capture unit in the chip can capture a single cell in one operation.

[0073] The method for manufacturing the high-throughput single-cell capture / culture integrated chip of the present invention comprises the manufacturing of a silicon substrate 1 and the selection and manufacturing of an upper glass substrate 2 and a lower glass substrate 3, which can be performed in any order, and bonding the three together; wherein the manufacturing of the silicon substrate 1 is as follows: Figure 7 As shown, the following steps are included:

[0074] Step 1: Use a four-inch, double-sided polished, 300 μm thick silicon substrate 1 as a substrate; Figure 7-1 shown.

[0075] Step 2: Double-sided thermal oxidation process to form a 1 μm thick silicon oxide layer 15 on both the front and back sides of the silicon substrate 1; Figure 7-2 shown.

[0076] Step 3: Spin-coat the photoresist 16 on the back of the silicon substrate 1, and photolithography the back of the silicon substrate 1 according to the design layout to develop the patterns of the bottom channel inlet 10, the bottom channel 9, and the bottom channel outlet 11; Figure 7-3 shown.

[0077] Step 4: Using the Samco RIE process combination, RIE reverse-side etching of the silicon oxide layer of the silicon substrate 1 by 151 μm forms etching windows for the bottom channel inlet 10, the bottom channel 9, and the bottom channel outlet 11; Figure 7-4 shown.

[0078] Step 5: Use deep reactive plasma etching to etch the back side of the silicon substrate by 1240 μm to form the bottom channel 9, and complete the etching of the back side of the bottom channel inlet 10 and the bottom channel outlet 11; Figure 7-5 shown.

[0079] Step 6: Use a stripping process combination to remove the photoresist 16 and impurities remaining on the silicon substrate 1 after the above steps; Figure 7-6 shown.

[0080] Step 7: Using TEOS LPCVD process, that is, chemical vapor deposition silicon oxide process, chemical vapor deposition silicon oxide is formed on the silicon substrate 1 to form a silicon oxide layer 15 with a thickness of 0.4 μm; Figure 7-7 shown.

[0081] Step 8, spin-coating a photoresist 16 on the front side of the silicon substrate 1, and photolithographically developing a pattern of a bottom channel inlet 10, a top channel 4, a top channel inlet 5, a top channel outlet 6, and a bottom channel outlet 11 on the front side of the silicon substrate 1 according to the design layout, wherein the patterns of the top channel 4, the top channel inlet 5, and the top channel outlet 6 are formed simultaneously, and the subsequent manufacturing process is the same; Figure 7 -8.

[0082] Step 9, using the Samco RIE process combination, RIE front-side etching of silicon substrate 1 by 1.4 μm of silicon oxide to form etching windows for bottom channel inlet 10, top channel 4, top channel inlet 5, top channel outlet 6, and bottom channel outlet 11; Figure 7 -9.

[0083] Step 10: Use a stripping process combination to remove the photoresist 16 and impurities remaining on the silicon substrate 1 after the above steps; Figure 7-1 0 shown.

[0084] Step 11: Use deep reactive plasma etching to etch the front side of the silicon substrate by 120 μm to form a top channel 4, a top channel inlet 5, and a top channel outlet 6, and complete the front side etching of the bottom channel inlet 10 and the bottom channel outlet 11; Figure 7-1 As shown in 1.

[0085] Step 12: Using TEOS LPCVD (chemical vapor deposition silicon oxide) process, chemical vapor deposition of silicon oxide 0.4 μm on the silicon substrate 1; Figure 7-1 2.

[0086] Step 13, spin-coat the photoresist 16 on the front side of the silicon substrate 1, and continue to photolithographically develop the bottom channel inlet 10 and bottom channel outlet 11 patterns on the front side of the silicon substrate according to the design layout, and at the same time photolithographically develop the cell capture / culture unit 14 pattern; Figure 7-1 3.

[0087] Step 14: Using the Samco RIE process combination, RIE etches 0.4 μm of silicon oxide on the silicon substrate 1 from the front, and forms etching windows for the bottom channel inlet 10, the cell capture / culture unit 14, and the bottom channel outlet 11 from left to right; Figure 7-1 4.

[0088] Step 15: Use a stripping process combination to remove the photoresist 16 and impurities remaining on the silicon substrate 1 after the above steps; Figure 7-1 5.

[0089] Step 16: using anisotropic wet etching process, the anisotropic etching solution etches the silicon substrate 1 by about 40 μm to form a cell capture / culture unit 14, and completes the etching of the bottom channel inlet 10 and the bottom channel outlet 11; Figure 7-1 6.

[0090] Step 17: Use SiO2 (post-metal) etching process combined with BOE process to remove the residual silicon oxide mask on the silicon substrate 1, release and form the bottom channel inlet 10 and the bottom channel outlet 11; Figure 7-1 7.

[0091] Step 18: Select two 4-inch glass substrates, one of which is not processed as the lower glass substrate 3, and the other is punched with holes on the glass substrate according to the designed layout using a laser punching process to make an upper glass substrate 2;

[0092] Step 19: Align and bond the prepared silicon substrate 1, upper glass substrate 2, and lower glass substrate 3 to complete the production of at least one high-throughput single-cell capture / culture integrated chip.

[0093] As the material for making the silicon substrate 1, a silicon substrate 1 plate-like aggregate is used; as the material for making the upper glass substrate 2, an upper glass substrate 2 plate-like aggregate is used; as the material for making the lower glass substrate 3, a lower glass substrate 3 plate-like aggregate is used; in step nineteen, the prepared silicon substrate 1 plate-like aggregate, the upper glass substrate 2 plate-like aggregate, and the lower glass substrate 3 plate-like aggregate are aligning and bonding, and an integrated chip plate-like aggregate is formed by silicon-glass anodic bonding; and then a silicon chip cutting machine is used to cut out individual integrated chips along the designed cutting lines, thereby completing the production of high-throughput single-cell capture / culture integrated chips in batches.

[0094] The single-cell capture / culture method of the present invention uses the above-mentioned high-throughput single-cell capture / culture integrated chip. First, the same cell culture solution without cells is simultaneously input into the top flow channel 4 and the bottom flow channel 9 of the integrated chip through the top liquid inlet and the bottom liquid inlet. After the internal cavity of the chip is filled, the input of the solution is suspended and maintained for a set time to complete the pretreatment of the chip; then, the cell culture solution containing cells is continuously introduced into the top flow channel 4 of the chip through the top liquid inlet, and its flow rate is flow rate a. At the same time, the cell culture solution without cells is continuously introduced into the bottom flow channel 9 of the chip through the bottom liquid inlet, and its flow rate is flow rate b. The flow rate is appropriately adjusted so that the flow rate of the liquid in the top and bottom flow channels 9 satisfies: flow rate a is less than flow rate b; hereby Under the above conditions, the cells located on the cell capture / culture unit 14 in the top flow channel 4 will spontaneously move to the lower opening of the cell capture / culture unit 14 under the influence of the flow velocity difference, and finally the cells will be adsorbed by this small hole under the action of the flow velocity difference; when cells are captured in all cell capture / culture units 14, the cell culture solution without cells is continuously introduced into the top flow channel 4 without changing the flow velocity, and the culture solution will bring out the excess cells in the culture unit to ensure that each cell capture / culture unit 14 contains a single cell, and the flowing culture solution provides nutritional conditions for the cells, and finally achieves the purpose of single cell capture and culture; in the above process, after the internal cavity of the chip is filled, the excess cell culture solution overflows from the top liquid outlet and the bottom liquid outlet.

[0095] During the specific operation, four hoses of suitable size and length can be used, one end of the hoses are respectively connected to the top liquid inlet, the top liquid outlet, the bottom liquid inlet and the bottom liquid outlet, the other end of the hose connected to the top liquid inlet is connected to a cell culture solution container containing cells or not containing cells via a micro-transfer pump, and the other end of the hose connected to the bottom liquid inlet is connected to a cell culture solution container not containing cells via a micro-transfer pump, and the micro-transfer pump drives the delivery process of the cell culture solution to the internal cavity of the chip; the other end of the hose connected to the top liquid outlet and the bottom liquid outlet leads to a waste liquid container; after the internal cavity of the chip is filled, the excess cell culture solution overflows from the top liquid outlet and the bottom liquid outlet and flows to the waste liquid container through the hose.

Claims

1. A high-throughput single-cell capture / culture integrated chip, comprising a silicon substrate (1), an upper glass substrate (2) and a lower glass substrate (3) bonded to the silicon substrate (1), the three being bonded together into one; characterized in that: A top flow channel (4) is provided on the upper surface of the silicon substrate (1), and a top flow channel inlet (5) and a top flow channel outlet (6) are respectively provided at both ends of the top flow channel (4) and do not penetrate the silicon substrate (1); a top liquid inlet (7) and a top liquid outlet (8) are respectively provided on the upper glass substrate (2) at positions corresponding to the top flow channel inlet (5) and the top flow channel outlet (6), and penetrate the upper glass substrate (2) and are respectively connected to the top flow channel inlet (5) and the top flow channel outlet (6); a bottom flow channel (9) is provided on the lower surface of the silicon substrate (1), and a bottom flow channel inlet (10) and a bottom flow channel outlet (11) are respectively provided at both ends of the bottom flow channel (9), and a bottom flow channel inlet (10) and a bottom flow channel outlet (11) are respectively provided on the upper glass substrate (2) at positions corresponding to the bottom flow channel inlet (10) and the bottom flow channel outlet (11), and penetrate the upper glass substrate (2) and are respectively connected to the bottom flow channel inlet (10) and the bottom flow channel outlet (11). The outlet (11) is connected to a bottom liquid inlet (12) and a bottom liquid outlet (13); the lower surface of the upper glass substrate (2) is bonded to the upper surface of the silicon substrate (1) to form a top flow channel cavity, and the upper surface of the lower glass substrate (3) is bonded to the lower surface of the silicon substrate (1) to form a bottom flow channel cavity. Cell capture / culture units (14) distributed in an array are provided between the top flow channel cavity and the bottom flow channel cavity through the thickness of the silicon substrate (1). The upper opening of each cell capture / culture unit (14) located in the top flow channel cavity is connected to the lower opening located in the bottom flow channel cavity in vertical communication. The upper opening is more than twice the diameter of the single cell to be captured / cultured, and the lower opening is smaller than the diameter of the single cell to be captured / cultured. The high-throughput single cell capture / culture integrated chip connects the top flow channel cavity and the bottom flow channel cavity only through the cell capture / culture units (14) distributed in an array, thereby forming a complete chip internal cavity structure.

2. The high-throughput single-cell capture / culture integrated chip according to claim 1, characterized in that: The top flow channel (4) on the upper surface of the silicon substrate (1) is arranged in a manner that the flow is divided into multiple stages of refinement and then coarsened and merged from the top flow channel inlet (5) to the top flow channel outlet (6), and the top flow channels (4) at the finest level are arranged parallel to each other; the bottom flow channel (9) on the lower surface of the silicon substrate (1) is also arranged in a manner that the flow is divided into multiple stages of refinement and then coarsened and merged from the bottom flow channel inlet (10) to the bottom flow channel outlet (11), and the top flow channels at the finest level are also arranged parallel to each other.

3. The high-throughput single-cell capture / culture integrated chip according to claim 2, characterized in that: The top flow channel (4) on the upper surface of the silicon substrate (1) first differentiates into two thinner top flow channels (4) from the top liquid inlet (7) to the top liquid outlet (8), and the two top flow channels (4) further differentiate into four thinner top flow channels (4), and the four top flow channels (4) further differentiate into eight thinner top flow channels (4), and after the eight top flow channels (4) pass through the cell capture / culture area in the middle of the top flow channels (4), the eight top flow channels (4) merge into four thicker top flow channels (4), and the four top flow channels (4) merge into two thicker top flow channels (4), and the two top flow channels (4) merge into a thicker single top flow channel (4), and the differentiated top flow channels (4) are separated, and the eight thinnest top flow channels (4) located in the center of the flow channel are arranged parallel to each other; The bottom flow channel (9) on the lower surface of the substrate (1) is also differentiated into two thinner bottom flow channels (9) from the bottom flow channel inlet (10) to the bottom flow channel outlet (11), and the two bottom flow channels (9) are further differentiated into four thinner bottom flow channels (9), and the four bottom flow channels (9) are further differentiated into eight thinner bottom flow channels (9). After the eight bottom flow channels (9) pass through the cell capture / culture area in the middle of the bottom flow channel (9), the eight bottom flow channels (9) are merged into four thicker bottom flow channels (9), the four bottom flow channels (9) are merged into two thicker bottom flow channels (9), and the two bottom flow channels (9) are merged into a thicker single bottom flow channel (9). Isolation is provided between the differentiated bottom flow channels (9), and the eight thinnest bottom flow channels (9) located at the center of the flow channel are also arranged parallel to each other.

4. The high-throughput single-cell capture / culture integrated chip according to claim 1, characterized in that: The silicon substrate (1) is a silicon substrate (1) with a thickness of 300 μm, a surface of [100] crystal plane, and double-sided polishing. The top flow channel (4) on the upper surface of the silicon substrate (1) is 20 to 30 μm deep and 100 to 700 μm wide, ensuring that cells can pass smoothly under high-throughput conditions; the bottom flow channel (9) on the lower surface of the silicon substrate (1) is 230 to 240 μm deep and 100 to 700 μm wide, and the flow channel volume ensures that a cell culture solution can pass through.

5. The high-throughput single-cell capture / culture integrated chip according to claim 1, characterized in that: Each cell capture / culture unit (14) is in the shape of an inverted pyramid, and its upper opening and lower opening are quadrilaterals. The upper opening is a square with a side length of 60 to 70 μm; it is slightly narrower than the width of the single flow channel after differentiation, ensuring that cells can pass through; the lower opening located on the bottom flow channel (9) of the silicon substrate (1) is a square with a side length of 5 to 10 μm, which is smaller than the cell diameter, ensuring that the cells are confined in the cell capture / culture unit (14).

6. The high-throughput single-cell capture / culture integrated chip according to claim 5, characterized in that: The cell capture / culture unit (14) provides sufficient proliferation space for the captured cells, and one cell capture / culture unit (14) can accommodate 20 to 30 cells.

7. The high-throughput single-cell capture / culture integrated chip according to claim 1, characterized in that: The top flow channel (4) and the bottom flow channel (9) are arranged in a cross pattern on the upper and lower surfaces of the silicon substrate (1), thereby improving the area utilization of the silicon substrate (1). The design of the cross structure of different layers is conducive to the formation of a flow velocity difference between the upper and lower flow channels.

8. The method for manufacturing a high-throughput single-cell capture / culture integrated chip as claimed in claim 1, comprising the steps of manufacturing a silicon substrate (1), selecting and manufacturing an upper glass substrate (2) and a lower glass substrate (3), and bonding the three substrates; wherein: The steps of manufacturing the silicon substrate (1) include: Step 1: using a four-inch, double-sided polished, 300 μm thick silicon substrate (1) as a substrate; Step 2: Double-sided thermal oxidation process to form a 1 μm thick silicon oxide layer (15) on both the front and back surfaces of the silicon substrate (1); Step 3, spin-coating a photoresist (16) on the back side of the silicon substrate (1), and photolithography the back side of the silicon substrate (1) according to the design layout to develop the patterns of the bottom flow channel inlet (10), the bottom flow channel (9), and the bottom flow channel outlet (11); Step 4: Using the Samco RIE process combination, RIE reverse-side etching of the silicon oxide layer (15) of the silicon substrate (1) by 1 μm to form etching windows for the bottom flow channel inlet (10), the bottom flow channel (9), and the bottom flow channel outlet (11); Step 5: using deep reactive plasma etching, the reverse side of the silicon substrate (1) is etched by 240 μm to form a bottom flow channel (9), and the reverse side of the bottom flow channel inlet (10) and the bottom flow channel outlet (11) are etched; Step 6: using a stripping process combination to remove the photoresist (16) and impurities remaining on the silicon substrate (1) after the above steps; Step 7: using a TEOS LPCVD process, i.e. a chemical vapor deposition silicon oxide process, chemically vapor depositing silicon oxide on the silicon substrate (1) to form a silicon oxide layer (15) with a thickness of 0.4 μm; Step 8, spin-coating a photoresist (16) on the front side of the silicon substrate (1), and photolithographically developing a pattern of a bottom channel inlet (10), a top channel (4), a top channel inlet (5), a top channel outlet (6), and a bottom channel outlet (11) on the front side of the silicon substrate (1) according to the design layout, wherein the patterns of the top channel (4), the top channel inlet (5), and the top channel outlet (6) are formed simultaneously, and the subsequent manufacturing process is the same; Step 9: Using the Samco RIE process combination, RIE front-side etching of silicon substrate (1) with 1.4 μm of silicon oxide forms etching windows for bottom channel inlet (10), top channel (4), top channel inlet (5), top channel outlet (6), and bottom channel outlet (11); Step 10: using a stripping process combination to remove the photoresist (16) and impurities remaining on the silicon substrate (1) after the above steps; Step 11: using deep reactive plasma etching, etching the silicon substrate (1) by 20 μm on the front side to form a top flow channel (4), a top flow channel inlet (5), and a top flow channel outlet (6), and completing the front side etching of the bottom flow channel inlet (10) and the bottom flow channel outlet (11); Step 12: using TEOS LPCVD (chemical vapor deposition silicon oxide) process, chemical vapor deposition of silicon oxide 0.4 μm on the silicon substrate (1); Step 13: Spin-coat the photoresist (16) on the front side of the silicon substrate (1), and continue to photolithographically develop the bottom channel inlet (10) and bottom channel outlet (11) patterns on the front side of the silicon substrate according to the design layout, and at the same time, photolithographically develop the cell capture / culture unit (14) pattern; Step 14: Using the Samco RIE process combination, RIE front-side etching of silicon substrate (1) with 0.4 μm of silicon oxide forms etching windows for the bottom flow channel inlet (10), the cell capture / culture unit (14), and the bottom flow channel outlet (11); Step 15: using a stripping process combination to remove the photoresist (16) and impurities remaining on the silicon substrate (1) after the above steps; Step 16: using an anisotropic wet etching process, the anisotropic etching solution etches the silicon substrate (1) by about 40 μm to form a cell capture / culture unit (14), and completes the etching of the bottom channel inlet (10) and the bottom channel outlet (11); Step 17: Using a SiO2 (post-metal) etching process combined with a BOE process, the residual silicon oxide mask on the silicon substrate (1) is removed to release and form a bottom flow channel inlet (10) and a bottom flow channel outlet (11); Step 18: Select two 4-inch glass substrates, one of which is not processed as the lower glass substrate (3), and the other is punched with holes according to the designed pattern using a laser punching process to form an upper glass substrate (2); Step 19: Align and bond the prepared silicon substrate (1) and the upper glass substrate (2) and the lower glass substrate (3) to complete the production of at least one high-throughput single-cell capture / culture integrated chip.

9. The method for manufacturing a high-throughput single-cell capture / culture integrated chip according to claim 8, characterized in that: As the manufacturing material of the silicon substrate (1), a silicon substrate (1) plate-like aggregate is used; as the manufacturing material of the upper glass substrate (2), an upper glass substrate (2) plate-like aggregate is used; as the manufacturing material of the lower glass substrate (3), a lower glass substrate (3) plate-like aggregate is used; in step nineteen, the prepared silicon substrate (1) plate-like aggregate, the upper glass substrate (2) plate-like aggregate, and the lower glass substrate (3) plate-like aggregate are aligned and bonded, and an integrated chip plate-like aggregate is formed by silicon-glass anodic bonding; and then a silicon chip cutting machine is used to cut out single integrated chips in batches along the designed cutting line, thereby completing the production of a high-throughput single-cell capture / culture integrated chip.

10. A single cell capture / culture method, characterized in that: Using the high-throughput single-cell capture / culture integrated chip as described in any one of claims 1 to 7, first, the same cell-free cell culture solution is simultaneously input into the top flow channel (4) and the bottom flow channel (9) of the integrated chip through the top liquid inlet (7) and the bottom liquid inlet (12). After the internal cavity of the chip is filled, the input of the solution is suspended and maintained for a set time to complete the pretreatment of the chip; then, the cell-containing cell culture solution is continuously introduced into the top flow channel (4) of the chip through the top liquid inlet (7) at a flow rate of flow rate a. At the same time, the cell-free cell culture solution is continuously introduced into the bottom flow channel (9) of the chip through the bottom liquid inlet (12) at a flow rate of flow rate b. The flow rates are appropriately adjusted so that the flow rates of the liquids in the top flow channel (4) and the bottom flow channel (9) satisfy the following conditions: flow rate a is less than or equal to 1. At flow rate b; under this condition, the cells located on the cell capture / culture unit (14) in the top flow channel (4) will spontaneously move to the lower opening of the cell capture / culture unit (14) under the influence of the flow rate difference, and finally the cells will be adsorbed by this small hole under the action of the flow rate difference; when cells are captured in all cell capture / culture units (14), a cell culture solution without cells is continuously introduced into the top flow channel (4), and the flow rate does not change. The culture solution will take out the excess cells in the culture unit, ensuring that each cell capture / culture unit (14) contains a single cell. The flowing culture solution provides nutritional conditions for the cells, and finally achieves the purpose of single cell capture and culture; in the above process, after the internal cavity of the chip is filled, the excess cell culture solution overflows from the top liquid outlet (8) and the bottom liquid outlet (13).

11. The single cell capture / culture method according to claim 10, characterized in that: Four hoses of suitable size and length are used, one end of each of the hoses being connected to a top-layer liquid inlet (7), a top-layer liquid outlet (8), a bottom-layer liquid inlet (12), and a bottom-layer liquid outlet (13), respectively; the other end of the hose connected to the top-layer liquid inlet (7) is connected to a cell culture solution container containing cells or not containing cells via a micro-transfer pump; the other end of the hose connected to the bottom-layer liquid inlet (12) is connected to a cell culture solution container not containing cells via a micro-transfer pump, and the micro-transfer pump is used to drive the cell culture solution to be transported to the internal cavity of the chip; The other end of the hose connecting the top liquid outlet (8) and the bottom liquid outlet (13) leads to a waste liquid container; after the internal cavity of the chip is filled, excess cell culture solution overflows from the top liquid outlet (8) and the bottom liquid outlet (13) and flows through the hose to the waste liquid container.

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