Single-cell capture method and micropore-coupled microchamber microfluidic chip

Abstract

A single-cell capture method, comprising the following steps: by means of a fluid, injecting single cells to be captured into a micropore-coupled microchamber microfluidic chip, wherein the chip comprises a substrate and a main channel provided thereon, the surface of the main channel is provided with a plurality of micropores and microchambers corresponding to the micropores, and the microchambers are in communication with the main channel; after the fluid fills the main channel, keeping the fluid in the main channel stationary, so that the single cells fall into the micropores under gravity; after the micropores capture the single cells, introducing the fluid again to discharge the uncaptured single cells; and after discharge, inverting and tilting the chip, so that the single cells enter the microchambers. The method solves the technical problems in the prior art of low single-cell capture efficiency, insufficient capture precision, the need for complex external devices for control, and being unable to complete single-cell capture, culture, and other operations on the same chip.

Description

Single cell capturing method and microwell coupled microcavity microfluidic chip

[0001] The present application claims priority to the Chinese patent application No. 202411643134.5, filed on November 18, 2024, and entitled "Single cell capturing method and microwell coupled microcavity microfluidic chip", the entire content of which is incorporated herein by reference. TECHNICAL FIELD

[0002] The present application belongs to the technical field of single cell capturing, and specifically relates to a single cell capturing method and a microwell coupled microcavity microfluidic chip. BACKGROUND

[0003] In the field of single cell research, microfluidic chip technology, as an important research tool, has the advantage of precise manipulation of micro-liquids. However, existing single cell capturing and analysis technologies, such as droplet microfluidics and microcavity-based capturing methods, have certain limitations. Droplet microfluidics generates small droplets by controlling the flow of two immiscible liquids, but due to its Poisson distribution law, the single cell capture efficiency is low, only about 10%-30% of the droplets contain a single cell, and multiple cell droplets are also generated, affecting the experimental precision.

[0004] Microcavity-based single cell capturing technology can improve some defects of droplet capturing technology, but it also faces the problem of low capture rate caused by Poisson distribution. To improve the capture efficiency, researchers have developed microwell chip technology, which overcomes the Poisson distribution and improves the single cell capture throughput and efficiency. However, in order to ensure the normal growth and proliferation of cells, especially adherent cells, the size of the microwell needs to be increased. However, the increase in the size of the microwell leads to a decrease in the single cell encapsulation rate, and even multiple cell capture occurs.

[0005] To further improve the precision of single cell manipulation, existing technologies use photoelectric positioning technology (such as Beacon single cell light guide platform) to transfer single cells to microcavities for culture. Although this method can achieve precise manipulation of single cells, it relies on complex micro-electro-mechanical system (MEMS) processing technology and external control circuit, increasing the cost and complexity of the equipment, making it difficult for most laboratories and companies to afford.

[0006] Therefore, the existing technology has obvious limitations in single cell capture efficiency, precision of the culture process, and equipment cost, limiting its application in extensive research. SUMMARY

[0007] The first aspect of the present application provides a single cell capturing method, comprising the following steps:

[0008] S1, injecting single cells to be captured into a chip by fluid, the chip being a microwell coupled microchamber microfluidic chip, the chip comprising a substrate and a main channel provided on the substrate, a plurality of microwells being provided on the surface of the main channel and a plurality of microchambers corresponding to the microwells, wherein the microchambers are in communication with the main channel;

[0009] S2, after the fluid fills the main channel, keeping the fluid in the main channel static, so that the single cells fall into the microwells provided on the main channel under the action of gravity;

[0010] S3, after the microwells capture single cells, again injecting fluid to discharge the single cells not captured in the main channel;

[0011] S4, after the single cells in the main channel are discharged, turning over and tilting the chip so that the microwells of the chip are above the microchambers, so that the single cells captured in the microwells enter the microchambers corresponding to the microwells from the microwells.

[0012] In some embodiments, in the step S3, the single cells not captured in the main channel are discharged by controlling the fluid flow rate of the sample inlet to ensure that the single cells in the microwells are retained in the main channel, wherein the fluid flow rate of the sample inlet is 1 uL / min to 40 uL / min.

[0013] In some embodiments, in the step S2, after the fluid fills the main channel, the fluid in the main channel is kept static, and after 1 min to 10 min, the single cells fall into the microwells provided on the main channel under the action of gravity, and then the step S3 is performed to inject fluid to discharge the single cells not captured in the main channel.

[0014] In some embodiments, the single cell capturing method further comprises a step S5 of single cell leading out method: after the single cells are led into the microchamber, the fluid in the microchamber is static, a laser is used to irradiate a position of the microchamber away from the microwell to form a bubble, the target single cell in the microchamber is led into the main channel by the method of laser-induced bubble, and fluid is injected into the main channel, and under the driving of the fluid, the target cell is led out of the chip and collected.

[0015] In some embodiments, the single cell capturing method further comprises a step S6 of single cell culture method: after the single cells are led into the microchamber, the fluid in the main channel and the microchamber is static, and a culture medium is introduced into the main channel, at this time the culture medium replaces the fluid in the main channel and the microchamber, and after the microchamber is filled with the culture medium, the single cells are cultured in the microchamber.

[0016] In some embodiments, the single cell capturing method further comprises S7 single cell labeling detection method: after the single cell is introduced into the microchamber, the fluid in the main channel and the microchamber is static, a staining agent is introduced into the main channel, at this time the staining agent replaces the fluid in the main channel and the microchamber, after the microchamber is filled with the staining agent, the single cell in the microchamber is stained and labeled by the staining agent; the state of the single cell in the microchamber is detected by combining microscopic images, and the target single cell is obtained by laser-induced bubble sorting.

[0017] In some embodiments, the single cell capturing method further comprises S8 multiple different single cell interaction method:

[0018] Firstly, the first single cell is introduced into the main channel by fluid, so that the first single cell falls into the microwell under the action of gravity, after the microwell captures the first single cell, the un-captured first single cell is discharged; the chip is turned over and tilted downward from the microwell to the microchamber direction, so that the first single cell is transferred to the corresponding microchamber by gravity.

[0019] Secondly, the second single cell is introduced into the main channel by fluid, so that the second single cell falls into the microwell under the action of gravity, after the microwell captures the second single cell, the un-captured second single cell is discharged; the chip is turned over and tilted downward from the microwell to the microchamber direction, so that the second single cell is transferred to the corresponding microchamber by gravity and is located in the same microchamber as the first single cell, completing the interaction of multiple different single cells in the microchamber.

[0020] The second aspect of the present application further provides a single cell operation method, comprising the single cell capturing method of any one of the above.

[0021] In some embodiments, the single cell operation method further comprises a single cell introduction method: after the single cell is introduced into the microchamber, the fluid in the microchamber is static, a laser is used to irradiate the position of the microchamber away from the microwell to form a bubble, the target single cell in the microchamber is introduced into the main channel by the method of laser-induced bubble, and fluid is introduced into the main channel, under the driving of the fluid, the target cell is introduced out of the chip and collected.

[0022] In some embodiments, the single cell capturing method further comprises a single cell culture method: after the single cell is introduced into the microchamber, the fluid in the main channel and the microchamber is static, a culture medium is introduced into the main channel, at this time the culture medium replaces the fluid in the main channel and the microchamber, after the microchamber is filled with the culture medium, the single cell in the microchamber is cultured.

[0023] In some embodiments, the single cell capturing method further comprises a single cell labeling detection method: after the single cells are introduced into the microchambers, the fluid in the main channel and the microchambers is static, a staining agent is introduced into the main channel, at this time the staining agent replaces the fluid in the main channel and the microchambers, after the microchambers are filled with the staining agent, the single cells in the microchambers are stained and labeled by the staining agent; the state of the single cells in the microchambers is detected by combining microscopic images, and the target single cells are obtained by laser-induced bubble sorting.

[0024] In some embodiments, the single cell capturing method further comprises a plurality of different single cell interaction methods:

[0025] First, a first kind of single cells are introduced into the main channel by fluid, so that the first kind of single cells fall into the microwells under the action of gravity, after the microwells capture the first kind of single cells, the un-captured first kind of single cells are discharged; the chip is turned upside down and tilted from the microwell to the microchamber direction, so that the first kind of single cells are transferred to the corresponding microchambers by gravity.

[0026] Again, a second kind of single cells are introduced into the main channel by fluid, so that the second kind of single cells fall into the microwells under the action of gravity, after the microwells capture the second kind of single cells, the un-captured second kind of single cells are discharged; the chip is turned upside down and tilted from the microwell to the microchamber direction, so that the second kind of single cells are transferred to the corresponding microchambers by gravity and are located in the same microchamber as the first kind of single cells, completing the interaction of the plurality of different single cells in the microchambers.

[0027] The third aspect of the present application further provides a microwell-coupled microchamber microfluidic chip, comprising: a substrate; a main channel provided on the substrate and penetrating through the substrate; a plurality of microwells uniformly provided on the main channel for capturing single cells; a plurality of microchambers corresponding to the microwells and communicating with the main channel; wherein the diameter of the microwell is d1, the depth of the microwell is t1, the diameter of the single cell is d2, d1:t1=1:2-4:5, d1:d2=1:1-4:3; the width of the microchamber is x, the length of the microchamber is y, x:y=1:1-1:3, x>d1.

[0028] In some embodiments, the spacing between adjacent two microwells and the spacing between adjacent two microchambers are both L, and 20 μm

[0029] In some embodiments, a volume above each of the microwells is V, V = πr2h, where r is a radius of the microwell and h is a height of the main channel; a single-cell concentration above each of the microwells is C, C = 1000 / V x λ, λ refers to a theoretical average number of cells in the volume above each microwell, where λ = 1, so that a number of cells above each microwell is greater than or equal to 1 cell.

[0030] In some embodiments, the microwell-coupled microchamber microfluidic chip further comprises a shunt channel, which is arranged between the microwell and the microchamber, a width of the shunt channel being less than a width of the microchamber, so that the shunt channel and the microchamber combine to form a b-shaped structure, the shunt channel and the main channel being in communication with each other, so as to introduce the single cells in the microwell into the microchamber.

[0031] Compared with the prior art, the application has the following beneficial effects:

[0032] 1. The single-cell capturing method disclosed in at least one embodiment of the application can realize efficient capturing and processing of single cells by using a microwell-coupled microchamber microfluidic chip. First, the microwell design in the microfluidic chip is used to make single cells naturally fall into the microwell under the condition of fluid staticity, realizing precise capturing of single cells; then, by introducing fluid again, the excess single cells that are not captured are removed, further improving the capturing efficiency and precision. Finally, by turning over and tilting the chip, the single cells captured in the microwell are smoothly introduced into the microchamber, ensuring that the captured single cells can be effectively transferred to the area for subsequent processing. This design greatly simplifies the operation process of single-cell capturing and improves the repeatability and efficiency of experiments, and is particularly suitable for scenarios requiring large-scale single-cell capturing, such as cell biology research and single-cell genomics analysis.

[0033] 2. The at least one embodiment of the application can also realize the exporting operation of single cells. The microchamber design allows single cells to be exported at a fixed point after entering the microchamber, and this operation is realized by laser-induced bubble technology. Specifically, a laser is used to irradiate a position of the microchamber away from the microwell, form a bubble, and through the pushing action of the bubble, the single cell in each microchamber is introduced into the main channel. This non-contact exporting method not only reduces the physical damage to single cells, but also improves the accuracy and efficiency of single-cell operation, which is beneficial to subsequent analysis and processing of single cells, such as single-cell gene sequencing or functional detection, etc.

[0034] 3. The method for culturing and staining single cells by means of the micro-pore coupled micro-chamber microfluidic chip is also provided in at least one embodiment of the present application. After the single cells are introduced into the micro-chamber, the culture medium or the staining agent is injected into the main channel to replace the fluid in the micro-chamber, and the culturing or staining marking of the single cells is completed respectively. This method effectively maintains the activity of the single cells in the micro-environment and marks the single cells through the staining technology, so that the state of the single cells can be quickly distinguished, and is suitable for subsequent cell imaging, functional testing and other research applications. The method not only improves the processing efficiency of the single cells, but also ensures the reliability of the single cell operation, and is particularly suitable for the experimental requirements of long-term culturing and analysis of the single cells.

[0035] 4. The chip disclosed in at least one embodiment of the present application also supports the interaction research of various types of single cells. Different types of single cells are introduced in sequence, and the gravity capture and chip overturning technology is used to introduce different types of single cells into the same micro-chamber, so that the interaction process between cells on the micro scale is observed. This method can realize accurate cell interaction control and is suitable for research in the fields of cell communication, cell immune response and interaction between cancer cells and immune cells, and provides strong tool support for the analysis of complex biological systems.

[0036] 5. At least one embodiment of the present application realizes the functions of single cell capture, introduction, culturing, staining and cell interaction through the innovative design of the micro-pore coupled micro-chamber microfluidic chip. The design of the chip not only improves the automation degree and accuracy of the single cell operation, but also reduces the damage to the single cells in the traditional method, and is suitable for a wide range of single cell research application scenarios, such as single cell sequencing, drug screening, cell function analysis and other fields, and has great application potential and practical value. BRIEF DESCRIPTION OF DRAWINGS

[0037] The accompanying drawings, which are included to provide a further understanding of the present application and constitute a part of the present application, illustrate the illustrative embodiments of the present application and the description thereof and do not limit the present application in any way.

[0038] FIG. 1 is a structural schematic diagram of a micro-pore coupled micro-chamber microfluidic chip provided in an embodiment of the present application;

[0039] FIG. 2 is a structural schematic diagram of a template of a micro-pore coupled micro-chamber microfluidic chip provided in an embodiment of the present application;

[0040] FIG. 3 is a COMSOL fluid dynamics simulation diagram of a micro-pore section provided in an embodiment of the present application;

[0041] FIG. 4 is a simulation diagram of fluid flow rates in a main channel and a micro-pore provided in an embodiment of the present application;

[0042] Fig. 5 is a flow chart of the single-cell operation method according to an embodiment of the present application;

[0043] Fig. 6 is a schematic diagram of the chip in a flipped state according to an embodiment of the present application.

[0044] In the figure: 10, chip; 1, substrate; 2, main channel; 21, sample inlet; 3, micropore; 4, microchamber; 5, shunt; 6, carrier; 7, first structural unit; 8, second structural unit. DETAILED DESCRIPTION

[0045] For the purpose of making the objects, technical solutions and advantages of the present application clearer, the present application is described and explained below in conjunction with the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are only used to explain the present application and should not be used to limit the present application. Based on the embodiments provided by the present application, all other embodiments obtained by those of ordinary skill in the art without creative effort belong to the scope of the present application.

[0046] Obviously, the accompanying drawings in the following description are only some examples or embodiments of the present application, and for those of ordinary skill in the art, the present application can be applied to other similar scenarios without creative effort based on these drawings. In addition, it can be understood that although the efforts made in this development process can be complex and lengthy, for those of ordinary skill in the art related to the content disclosed in the present application, some design, manufacture or production changes based on the technical content disclosed in the present application are only routine technical means and should not be understood as insufficient disclosure of the content disclosed in the present application.

[0047] In the present application, "embodiment" means that the specific features, structures or properties described in conjunction with the embodiment can be included in at least one embodiment of the present application. The appearance of this phrase in various places in the specification does not necessarily mean the same embodiment, nor is it an independent or alternative embodiment to other embodiments. It is explicitly and implicitly understood by those of ordinary skill in the art that the embodiments described in the present application can be combined with other embodiments without conflict.

[0048] In the description of the present application, "single cell" refers to a single cell. The "fluid" described in the present application is a liquid suitable for cell introduction or discharge operation, for example, various buffers for maintaining cell viability, etc. Those skilled in the art can select according to actual needs, which is common knowledge in the art and will not be described herein.

[0049] As shown in FIG. 1, the micro-pore coupled micro-chamber microfluidic chip 10 provided by the embodiments of the present application comprises a substrate 1, a main channel 2, a plurality of micro-pores 3 and a plurality of micro-chambers 4. The main channel 2 is arranged on the substrate 1 and penetrates through the substrate 1. The plurality of micro-pores 3 are arranged on the main channel 2 and are used for capturing single cells. The plurality of micro-chambers 4 correspond to the plurality of micro-pores 3 one by one and are in communication with the main channel 2.

[0050] In the above embodiments, the main channel 2 is arranged on the substrate 1 in a recessed manner and extends from one end of the substrate 1 to the other end. The plurality of micro-pores 3 are arranged on the bottom of the main channel 2 in a spaced manner along the extension direction of the main channel 2. The plurality of micro-chambers 4 are formed on the substrate 1 in a recessed manner and are in communication with the main channel 2. The plurality of micro-chambers 4 are arranged one by one corresponding to the plurality of micro-pores 3. As shown in FIG. 3, by turning over the microfluidic chip 10 to make it inclined, the single cells captured by the micro-pores 3 can enter the corresponding micro-chambers 4.

[0051] In some embodiments, the micro-pore coupled micro-chamber microfluidic chip 10 is integrally formed by using a chip template as shown in FIG. 2. The template comprises a carrier 6, a first structural unit 7 and a second structural unit 8. The first structural unit 7 is carried on the carrier 6. The shape of the first structural unit 7 corresponds to one micro-chamber 4 and the part of the main channel 2 in communication with the micro-chamber 4, and is used for forming the micro-chamber 4 and the part of the main channel 2 close to the micro-chamber 4. The second structural unit 8 is located at the position of the first structural unit 7 corresponding to the main channel 2 and has a shape corresponding to the micro-pore 3, and is used for forming the micro-pore 3 in the main channel 2. According to the number of the micro-chambers 4 in the chip 10 to be formed, a corresponding number of templates are arranged side by side to be formed. Alternatively, the microfluidic chip 10 is prepared by using photolithography and microfabrication technology.

[0052] In some embodiments, the diameter of the micro-pore 3 is d1, the depth of the micro-pore 3 is t1, the diameter of the single cell is d2, d1:t1 = 1:2-4:5, d1:d2 = 1:1-4:3; the width of the micro-chamber 4 is x, the length of the micro-chamber 4 is y, x:y = 1:1-1:3, x>d1. The width of the micro-chamber 4 refers to the dimension along the extension direction of the main channel 2, and the length of the micro-chamber 4 refers to the dimension perpendicular to the extension direction of the main channel 2.

[0053] The design basis of the microfluidic chip is to capture single cells within a specific diameter range by using the geometric characteristics of the micro-pore 3. By setting appropriate ratios of the diameter and depth of the micro-pore 3, it can be ensured that the single cells are effectively captured in the micro-pore 3 and will not be washed away or stuck in the pore. Reasonable ratio of d1 and t1 ensures that the cells can stably stay in the micro-pore 3, and at the same time ensures the smoothness when the single cells are transferred to the micro-chamber 4. The range of the ratio of d1 and d2 helps to capture single cells of different sizes.

[0054] By adjusting the size ratio of the microwell 3, the application can adapt to different sizes of single cells, improving the scope of application of the chip. This flexible size design helps to implement different types of single cell experiments and ensures stability during the capture process. The appropriate ratio setting can also reduce the physical stress on single cells during capture and transfer, reducing single cell damage.

[0055] The design of the microchamber 4 ensures sufficient space to accommodate and cultivate single cells. By setting the width x of the microchamber 4 to the length y in a ratio of 1:1 to 1:3, suitable growth space can be provided for single cells. At the same time, x is greater than the diameter d1 of the microwell 3, which ensures that single cells do not get stuck due to space limitations when falling into the microchamber 4 from the microwell 3, ensuring that single cells can smoothly enter the microchamber 4. The geometry of the microchamber 4 also helps the flow and distribution of culture medium, ensuring that single cells can uniformly contact the culture medium. This design of the size ratio of the microchamber 4 can ensure that single cells have enough space to grow and expand during cultivation, improving the effect of single cell cultivation. The design of x being greater than d1 helps single cells to be transferred smoothly, avoiding single cell damage or loss. In addition, reasonable size ratio of the microchamber 4 can also improve the flow of the culture medium in the microchamber 4, ensuring the nutrition supply of single cells, and promoting the research of single cell growth and proliferation, secretion detection, environmental stimulus response, cell interaction, genome transcriptome analysis, etc.

[0056] In some embodiments, the size of the microwell 3 can be adjusted according to specific experimental requirements. In a specific experiment, if larger or smaller single cells need to be captured, the ratio of d1 and t1 can be adjusted in a larger range. In addition, by optimizing the material, low-adhesion materials can be selected to further reduce single cell damage during capture and transfer, improving cell survival rate.

[0057] In some embodiments, when a single cell with a diameter of 15um needs to be captured, the microwell 3 has a diameter of 20um and a depth of 25um.

[0058] The COMSOL fluid dynamics simulation diagram of the microwell 3 cross-section shown in Figure 3, and Figure 4 is a fluid flow rate simulation diagram of the main channel and microwell in Figure 3. As can be seen from Figure 4, when there is fluid flow in the main channel 2, only the upper 5um or so position in the microwell 3 has flow rate, the flow rate in the microwell 3 is 0, that is, the fluid remains stationary, and the fluid in the main channel 2 has a certain flow rate, while the flow rate in the microwell 3 is 0. This means that when single cells are captured into the microwell 3, the fluid flow in the main channel 2 will not affect the single cells captured in the lower part of the microwell 3. When two single cells fall into the microwell in a stacked manner, the single cell located in the upper part of the microwell 3 will flow away with the fluid in the main channel 2. Therefore, this size ratio design greatly improves the capture rate of single cells.

[0059] In some embodiments, the size of the microchamber 4 can be adjusted as needed for the experiment. If the experimental object is a single cell of larger volume or a cell population needs to be cultured, the width and length of the microchamber 4 can be increased accordingly. In addition, the depth of the microchamber 4 can also be increased to adapt to specific experimental scenarios, ensuring that different types of single cells can grow stably. The shape of the microchamber 4 can also be changed from rectangular to circular or other geometric shapes to adapt to specific application requirements.

[0060] In some embodiments, the spacing between two adjacent microwells 3 and the spacing between two adjacent microchambers 4 are both L, where 20 pm < L < 1000 pm. In this embodiment, the spacing between two adjacent microchambers 4 is the distance between the center points of the two adjacent microchambers.

[0061] In some embodiments, the spacing between two adjacent microwells 3 is L1, where 20 pm < L1 < 1000 pm; the spacing between two adjacent microchambers 4 is L2, where 20 pm < L2 < 1000 pm, and L2 < L1. In this embodiment, the spacing between two microchambers 4 is the distance between the two adjacent sides of the two adjacent microchambers 4. It can be understood that, since the width x of the microchamber 4 is greater than the diameter d1 of the microwell 3, the spacing L2 between two adjacent microchambers 4 is smaller than the spacing L1 between two adjacent microwells 3.

[0062] The spacing design of the microwells 3 and the microchambers 4 not only ensures the efficiency of single cell capture, but also considers the overall layout and operational feasibility of the chip. The selection of the spacing ensures the independence of the microwells 3 and the microchambers 4, and provides appropriate space to avoid interference between cells during capture and transfer. Through reasonable spacing design, the mutual influence of single cells in adjacent microwells 3 or microchambers 4 is effectively prevented, thereby improving the precision of the capture and transfer process. At the same time, smaller spacing can increase the number of microwells 3 and microchambers 4 on the chip 10, improving the throughput of single cell operations and meeting the needs of high-throughput experiments. By precisely controlling the spacing, the operation of each microwell 3 and microchamber 4 is ensured to be independent, reducing the interference between cells and experimental errors. For example, when single cells are transferred from the microwells 3 to the microchambers 4, reasonable spacing avoids the cells in other microwells 3 or microchambers 4 from being disturbed by fluid, improving the precision of the operation.

[0063] In some embodiments, the micro-well coupled micro-chamber microfluidic chip 10 further comprises a diversion channel 5, which is disposed between the micro-well 3 and the micro-chamber 4, and the width of the diversion channel 5 is smaller than the width of the micro-chamber 4, so that the diversion channel 5 and the micro-chamber 4 form a "b" shape structure. The diversion channel 5 is in communication with the main channel 2, and is used to introduce the single cell in the micro-well 3 into the micro-chamber 4. The diversion channel 5 is designed as a transition channel connecting the micro-well 3 and the micro-chamber 4, and provides a path for the single cell to move from the micro-well 3 to the micro-chamber 4. The width of the diversion channel 5 is smaller than the width of the micro-chamber 4, which ensures that the single cell can smoothly pass through and fall into the micro-chamber 4, while avoiding the interference of fluid.

[0064] In some embodiments, the micro-well coupled micro-chamber microfluidic chip 10 further comprises a diversion channel 5, which is disposed between the micro-well 3 and the micro-chamber 4, and the width of the diversion channel 5 is smaller than the width of the micro-chamber 4.

[0065] In some embodiments, the diversion channel 5 is disposed between the micro-well 3 and the micro-chamber 4, and is close to one side of the micro-chamber 4 in the width direction. One side wall of the diversion channel 5 is coplanar with one side wall of the micro-chamber 4 and has a width smaller than the width of the micro-chamber 4, thereby forming the above-mentioned "b" shape structure.

[0066] Through the design of the diversion channel 5, the single cell can smoothly transition into the micro-chamber 4, reducing the capture failure caused by fluid disturbance or gravity change. At the same time, the "b" shape structure in the microfluidic chip 10 is designed to optimize the transfer process of the single cell from the micro-well 3 to the micro-chamber 4, and prevent the single cell from falling off during capture and export. This structure not only plays an important role when the single cell is transferred from the micro-well 3 to the micro-chamber 4, but also plays a protective role during the export process of the single cell from the micro-chamber 4 to the micro-well 3. Whether in the process of transferring the single cell to the micro-chamber 4 or in the export process, the inner cavity design of the "b" shape structure effectively prevents the accidental falling off of the single cell. This structure ensures that the single cell can stably move along the predetermined trajectory, reducing the loss of cells during operation.

[0067] The design of the "b" shape structure forms a "guardrail" type structure around the inner cavity of the micro-chamber 4. When the single cell is transferred from the micro-well 3 to the micro-chamber 4, the walls around the "b" shape structure prevent the single cell from deviating from the predetermined trajectory due to gravity or fluid disturbance. When the chip 10 is turned over or tilted, and the single cell moves downward from the micro-well 3 to the micro-chamber 4 under the action of gravity, the inner wall of the "b" shape structure effectively guides the single cell into the micro-chamber 4 and prevents it from moving excessively or colliding with the wall of the micro-chamber 4, thereby improving the success rate of single cell transfer. This structure can also reduce the influence of fluid disturbance on single cell transfer, ensuring that the single cell falls smoothly into the micro-chamber 4 under the action of gravity.

[0068] The "b" shape structure can also effectively prevent single cells from falling off when the single cells are inclined to be guided out of the microcavity 4 to the microwell 3. During this process, the single cells move from the microcavity 4 to the microwell 3 under the action of fluid, but due to the transition design of the "b" shape structure, the single cells can slowly transfer along the fixed path, avoiding the single cells from falling off due to too fast flow or too large inclination angle. The inner wall of the "b" shape structure can also prevent the single cells from directly falling out of the chip 10 due to fluid disturbance or excessive inclination of the chip 10 during the guiding process, ensuring that the single cells can be guided from the microcavity 4 to the microwell 3 along the expected path.

[0069] In some embodiments, the width, depth and shape of the shunt channel 5 can be appropriately adjusted according to different types of experimental requirements. For example, when larger cells are processed, the width and depth of the shunt channel 5 can be increased to ensure that the cells pass through smoothly.

[0070] In some embodiments, the depth of the shunt channel 5, the depth of the microcavity 4 and the depth of the main channel 2 are the same. By setting the depths of the shunt channel 5, the microcavity 4 and the main channel 2 to be the same, the consistency of the internal structure of the chip 10 is ensured. The same depth design can ensure that the fluid flows more stably in the chip 10, avoiding changes in fluid pressure or uneven flow rate caused by inconsistent depths. At the same time, the single cells will not be hindered during the transfer process from the microwell 3 to the microcavity 4 due to the change in depth, thereby achieving smooth transfer operation. This consistent depth design helps to simplify the manufacturing process of the chip, reduces fluid dynamic instability problems caused by depth differences, and thus improves the overall efficiency of single cell capture and culture. The same depth makes the chip structure more uniform, the fluid control more precise, reduces the need for system adjustment during operation, and improves the reliability of the experiment.

[0071] In other embodiments, the depths of the three can be appropriately adjusted according to experimental requirements. For example, the depth of the microcavity 4 can be slightly larger than the depth of the main channel 2 to provide more culture space for the single cells; or the depth of the shunt channel 5 can be optimized according to the size of the single cells to ensure that the single cells are not subjected to excessive resistance during the transfer process. For different types of single cells or experimental purposes, the depths of the various channels and the microcavity 4 can be flexibly designed.

[0072] When the depths of the shunt channel 5, the microcavity 4 and the main channel 2 are different, a smooth transition between different depths is required to avoid hindering the movement of the single cells.

[0073] In some embodiments, the volume above each microwell 3 is V, V = πr 2 h, where r is the radius of the microwell 3, h is the height of the main channel 2, and the unit of V is mm 3The concentration of single cells above each microwell 3 is C, C = 1000 / V x λ, the unit of C is cells / mL, λ represents the theoretical average number of cells in the volume above each microwell 3, and λ = 1 to ensure that the number of single cells above each microwell 3 is greater than or equal to 1. The volume above the microwell 3 is calculated based on its radius and the height of the main channel 2, and the cell concentration calculation ensures that there is at least one single cell in the volume, thereby improving the success rate of capture. By reasonably controlling the concentration of the injected single cell suspension, the concentration of single cells above each microwell 3 is ensured to meet the capture conditions.

[0074] In some embodiments, the volume above each microwell 3 is V, V = πr 2 h, where r is the radius of the microwell 3, h is the height of the main channel 2, and the unit of V is mm 3 The concentration of single cells above each microwell 3 is C, C = 1000 / V x λ, the unit of C is cells / mL, λ represents the theoretical average number of cells in the volume above each microwell 3, and λ = 1 to ensure that the number of single cells above each microwell 3 is greater than or equal to 1.

[0075] In the above embodiments, the concentration of cells above each microwell 3 is determined by theoretical calculation, ensuring the consistency and success rate of single cell capture. The concentration of single cells above each microwell 3 is precisely controlled to ensure that there is at least one single cell in each microwell 3, which greatly improves the capture efficiency in experiments. The volume V above the microwell 3 determines the capture area of the single cell, and the precise calculation of the single cell concentration C ensures that at least one single cell is captured in the area. By controlling the concentration and injection speed of the single cell suspension, it can be ensured that the single cells are uniformly distributed in the volume area above each microwell 3, and the concentration is appropriate. This design significantly improves the efficiency and consistency of single cell capture, reducing the randomness in experiments.

[0076] In the above embodiments, by reasonably controlling the concentration of the injected single cell suspension, the concentration of single cells in the area above the microwell 3 is ensured to meet the capture requirements. Through the concentration adjustment of the single cell suspension, combined with the geometric design of the microwell 3 and the main channel 2, it can be ensured that there are enough single cells in the volume area above each microwell 3, ensuring that the microwell 3 can effectively capture single cells; through precise control of the cell concentration, avoid too many or too few single cells above the microwell 3, thereby reducing the failure or interference of capture.

[0077] Theoretical calculation and concentration control are used to ensure that there is at least one single cell above each microwell 3. Such control improves the consistency and success rate of the single cell capturing process, and ensures that a single cell can be captured in each microwell 3. Concentration control ensures that the number of single cells distributed in the volume area above each microwell 3 is appropriate, avoiding too high or too low cell concentration leading to capturing failure, thereby significantly improving the efficiency of single cell capturing. By precisely controlling the concentration of single cells above the microwells 3, errors caused by uneven cell concentration in experiments can be reduced, and the repeatability and reliability of the results of each capturing experiment can be ensured.

[0078] The application also discloses a single cell capturing method, which is implemented by using the microwell-coupled microchamber microfluidic chip 10 according to any one of the above embodiments. As shown in FIG. 5, the single cell capturing method comprises the following steps:

[0079] S1, injecting single cells to be captured into the chip by fluid, wherein the chip is the microwell-coupled microchamber microfluidic chip 10, the chip 10 comprises a substrate 1 and a main channel 2 arranged on the substrate 1, a plurality of microwells 3 and microchambers 4 corresponding to the microwells 3 are arranged on the surface of the main channel 2, and the microchambers 4 are in communication with the main channel 2;

[0080] S2, after the fluid fills the main channel 2, keeping the fluid in the main channel 2 static, and allowing the single cells to fall into the microwells 3 arranged on the main channel 2 under the action of gravity;

[0081] S3, after the single cells are captured in the microwells 3, introducing fluid again to discharge the single cells not captured in the main channel 2;

[0082] S4, after the single cells in the main channel 2 are discharged, turning over and tilting the chip 10 to make the microwells 3 of the chip 10 above the microchambers 4, so that the single cells captured in the microwells 3 enter the microchambers 4 below the microwells 3 from the microwells 3.

[0083] The single cell capturing method comprises the following steps: firstly, introducing single cells into the main channel 2 by controlling fluid, and capturing the single cells in the microwells 3 by gravity. After the single cells are captured, the remaining single cells not captured are discharged from the main channel 2 by fluid. Then, the chip 10 is turned over and tilted, so that the captured single cells fall into the microchambers 4 below the microwells 3 from the microwells 3.

[0084] The method is based on the micro-pore coupled micro-chamber structure for single cell capture. First, the fluid is introduced through the main channel 2 in the microfluidic chip 10, so that the single cells are suspended therein. After the fluid fills the main channel 2, the static fluid cooperates with the gravity of the single cells to make them sink into the micro-pores 3, and the capture is completed. Then, the excess single cells that are not captured by the micro-pores 3 are discharged by injecting the fluid again, and finally the captured single cells are guided into the corresponding micro-chambers 4 by the action of gravity through the flip and tilt of the chip 10. The method uses microfluidic technology and gravity to achieve efficient capture and transfer of single cells. Through the cooperation of the micro-pores 3 and the micro-chambers 4, it is ensured that the single cells can be effectively captured and quickly transferred to the micro-chambers 4, effectively avoiding the influence of fluid disturbance on the capture, and ensuring the convenience of subsequent operation.

[0085] In some embodiments, during the single cell capture process, the fluid flow rate and introduction time can be adjusted according to experimental requirements.

[0086] In some embodiments, in S3, the single cells in the main channel 2 that are not captured are discharged by controlling the fluid flow rate of the sample inlet 21 to ensure that the single cells in the micro-pores 3 are retained in the main channel 2.

[0087] In some embodiments, in S3, the fluid flow rate of the sample inlet 21 is 1 uL / min to 40 uL / min. By precisely controlling the fluid flow rate, the effective discharge of the uncaptured single cells can be achieved, while avoiding interference with the captured single cells. The choice of flow rate helps to improve the operation stability of the system and the efficiency of cell capture. By precisely controlling the fluid flow rate, it is ensured that the uncaptured single cells in the main channel 2 are quickly discharged, while the captured single cells are stably retained in the micro-pores. This method improves the efficiency and accuracy of the capture process and reduces the influence of fluid disturbance on single cell capture.

[0088] In some embodiments, the fluid flow rate affects the suspension state of the single cells in the main channel 2 and the capture efficiency. In actual operation, the fluid flow rate can be dynamically adjusted according to different experimental requirements:

[0089] Low flow rate: suitable for discharging lighter or smaller single cells, the flow rate is controlled at 1 uL / min to 5 uL / min, which helps to prevent excessive disturbance of the fluid and ensures that the captured single cells in the micro-pores 3 are stably retained in the micro-pores.

[0090] Medium flow rate: suitable for discharging conventional single cells, the flow rate range is between 5 uL / min and 20 uL / min, which can effectively discharge the uncaptured single cells and avoid the influence of the fluid on the captured cells in the micro-pores 3.

[0091] High flow rate: suitable for quickly removing untrapped single cells, the flow rate can reach 20-40 μL / min, but the flow rate should not be too high to cause the trapped single cells to fall off the microwells 3.

[0092] In some embodiments, after the main channel 2 is filled with fluid in step S2, the fluid in the main channel 2 is kept static, and then the single cells are allowed to fall into the microwells 3 on the main channel 2 under the action of gravity after 1-10 minutes. Then step S3 is performed to remove the untrapped single cells in the main channel 2 by injecting fluid.

[0093] The fluid in the main channel 2 is kept static to allow the single cells to naturally fall into the microwells 3 under the action of gravity. According to different experimental conditions, the static time can be adjusted between 1 minute and 10 minutes to ensure that the single cells can smoothly fall into the microwells 3. Then the untrapped single cells are removed by injecting fluid to ensure that the single cells in the microwells 3 are stably retained. The static fluid and the gravity of the single cells effectively improve the trapping efficiency, and the adjustment of the static time provides a flexible operation time window for different types of single cells. This method reduces the complexity of the operation process and enhances the accuracy of the trapping.

[0094] In some embodiments, the static time can be selected according to the requirements of the experimental trapping efficiency:

[0095] Short static time (1-3 minutes): suitable for trapping lighter or smaller single cells, and the fluid static time should not be too long to avoid cell accumulation.

[0096] Long static time (4-10 minutes): suitable for trapping larger or heavier single cells to ensure that they can fully settle in the microwells 3.

[0097] In step S4, the flip angle and flip time can be accurately set according to specific needs.

[0098] If the flip angle is small, the single cells may not fall off the microwells, and if the flip angle is large, the single cells may not be transferred to the microchambers 4 and directly fall out of the microchambers 4.

[0099] In some embodiments, as shown in FIG. 6, in step S4, after the chip 10 is flipped, the angle a between the side surface where the opening of the microwell 3 is located and the horizontal plane is in the range of 30°-90°. Those skilled in the art can adjust the angle a within the above range according to the physical properties of the single cells and the viscosity of the fluid.

[0100] In the S1-S3 steps of the single-cell capturing process, the chip 10 is placed horizontally so that the main channel 2 and the microchamber 4 are on the same horizontal plane, facilitating the introduction and capture of single cells; as shown in FIG. 6, in step S4, the chip 10 is flipped by 90°-150° relative to the original horizontal position, so that the included angle a between the side surface on which the micro-well 3 is open and the horizontal plane is in the range of 30° to 90°, so that the single cells captured in the micro-well 3 slide out of the micro-well 3 under the action of gravity and enter the microchamber 4 corresponding to the micro-well 3.

[0101] Small-angle flipping (a is 60°-90°): suitable for lighter and easier to slide cells, which helps to reduce the failure of cell detachment from the micro-well 3.

[0102] Medium-angle flipping (a is 45°-60°): suitable for routine experimental operation, which can ensure the detachment of single cells and the entry of single cells into the microchamber 4.

[0103] Large-angle flipping (a is 30°-45°): suitable for heavier or difficult to slide cells.

[0104] For the flipping speed, too fast flipping speed can cause fluid disturbance, so that the captured single cells cannot be accurately transferred; too slow flipping will affect the experimental efficiency. In some embodiments, in step S4, the flipping time of the chip 10 is controlled within 1 min to 5 min, ensuring that the single cells can smoothly slide into the microchamber 4.

[0105] In some embodiments, in step S4, an automatic chip flipping device can also be introduced to accurately control the flipping angle and the flipping time, further improving the operation efficiency and accuracy, and achieving higher precision operation.

[0106] The application also provides a single-cell operation method, which is implemented by using the micro-well coupled microchamber microfluidic chip 10 according to any one of the above embodiments. The single-cell operation method includes the single-cell capturing method according to any one of the above embodiments.

[0107] In some embodiments, as shown in FIG. 5, the single-cell operation method further includes a S5 single-cell leading-out method: after the single cells are introduced into the microchamber 4, the fluid in the microchamber 4 is static, a laser is used to irradiate the position of the microchamber 4 away from the micro-well 3 to form a bubble, the target cells in the microchamber 4 are led into the main channel 2 by the method of laser-induced bubble, and the fluid is introduced into the main channel 2, under the driving of the fluid, the target cells are led out of the chip 10 and collected.

[0108] The laser-induced bubble technique uses high-energy laser to form a local bubble in the microchamber. The volume expansion and movement of the bubble can generate liquid flow, thereby pushing the single cell in the microchamber 4 to move into the main channel 2. Finally, the single cell in the main channel 2 is discharged by introducing fluid, and the cell export is completed. The application of the laser-induced bubble technique realizes the precise movement and export of the single cell, avoids the direct contact of the physical tool with the cell, reduces the damage to the cell, and maintains the integrity and activity of the single cell.

[0109] In some embodiments, as shown in FIG. 5, the single cell operation method further comprises S6 single cell culture method: after the single cell is introduced into the microchamber 4, the fluid in the main channel 2 and the microchamber 4 is static, and the culture medium is introduced into the main channel 2. At this time, the culture medium replaces the fluid in the main channel 2 and the microchamber 4, and the microchamber 4 is filled with the culture medium, and the single cell culture is carried out.

[0110] After the single cell is introduced into the microchamber 4, the static fluid helps to maintain the stability of the single cell and avoid the physical stress caused by fluid flow. Then, by introducing fresh culture medium, the original fluid is gradually replaced to ensure that the single cell can be in a suitable culture environment. The culture medium provides the nutrients required for the growth of the single cell and supports its long-term culture and observation in the microchamber 4. The static environment allows the single cell to stay in the microchamber 4 stably and avoid the influence of external fluid disturbance on the single cell. Precise operation through the microfluidic chip can effectively replace the original fluid, ensuring that the single cell is always in fresh culture medium, which helps the long-term survival and growth of the single cell. The culture environment in the microchamber 4 is precisely designed and controlled to support the long-term growth and metabolic activity of the single cell, which is suitable for further experimental operation.

[0111] In some embodiments, the culture medium can be selected according to the type of single cell and the experimental purpose, such as DMEM (Dulbecco's Modified Eagle Medium), RPMI-1640 (Roswell Park Memorial Institute 1640), FBS (Fetal Bovine Serum), etc. Different types of culture media can be prepared according to the growth needs of different cells. The introduction speed of the culture medium can be adjusted according to the experimental requirements to ensure the sufficiency and stability of the fluid replacement. In certain special experimental conditions, the fluid replacement time can be adjusted as needed to ensure that the fluid replacement operation is completed at the appropriate time.

[0112] In some embodiments, as shown in FIG. 5, the single-cell operation method further comprises S7 single-cell labeling detection method: after the single cells are introduced into the microchamber 4, the fluid in the main channel 2 and the microchamber 4 is static, and a staining agent is introduced into the main channel 2, at this time the staining agent will replace the fluid in the main channel 2 and the microchamber 4, and after the microchamber 4 is filled with the staining agent, the single cells in the microchamber 4 are stained and labeled by the staining agent; the state of the single cells in the microchamber 4 is detected by combining the microscopic image, and the target single cells are obtained by laser-induced bubble sorting.

[0113] The chemical properties of the staining agent interact with the biological markers related to the membrane integrity or activity of the single cells, and then the live cells and dead cells are distinguished by the staining reaction. For example, common dead-live staining agents include trypan blue, propidium iodide (PI) and fluorescein ester dyes. Live cells have intact cell membranes and can exclude certain staining agents, while dead cell membranes are damaged and staining agents can enter the cell interior and react with cell components, thereby producing visible staining effects. By using dead-live staining agents, live cells and dead cells in the microchamber can be quickly distinguished, which facilitates subsequent experimental operation and data analysis. The fluid is static during the staining process, reducing the physical impact of the staining agent on the single cells and ensuring the stability and consistency of the staining process. Through the microfluidic system, automatic fluid injection and replacement are achieved, simplifying the dead-live staining operation steps and reducing the error caused by manual intervention.

[0114] In some embodiments, different experiments can select different staining agents. Common staining agents such as trypan blue are suitable for quickly determining dead cells, while fluorescent dyes such as propidium iodide (PI) and calcein-AM are suitable for more accurate live-dead cell differentiation by fluorescence microscopy. Depending on the type of staining agent, the staining concentration and action time can be adjusted appropriately. For example, certain dyes may require lower concentration and longer staining time, while certain rapid dyes can complete staining in a short time. In order to further enhance the differentiation effect, two or more staining agents can be used simultaneously to label live cells and dead cells, respectively, for example, the combination of propidium iodide and calcein-AM can simultaneously show different fluorescent signals of live cells and dead cells.

[0115] In some embodiments, as shown in FIG. 5, the single-cell operation method further comprises S8 multiple different single-cell interaction methods:

[0116] First, the first single cells are introduced into the main channel 2 by fluid, so that the first single cells fall into the microwell 3 under the action of gravity, and after the microwell 3 captures the first single cells, the first single cells that are not captured are discharged; the chip 10 is turned over and tilted from the microwell 3 to the microchamber 4 direction to make the first single cells transfer to the corresponding microchamber 4 by gravity;

[0117] The second type of single cell is introduced into the main channel 2 again by fluid, and the second type of single cell falls into the microwell 3 under the action of gravity. After the second type of single cell is captured in the microwell 3, the second type of single cell that is not captured is discharged. The chip 10 is flipped and tilted downward from the microwell 3 to the microchamber 4, so that the second type of single cell is transferred to the corresponding microchamber 4 by gravity and is located in the same microchamber 4 as the first type of single cell, and the interaction of multiple different single cells in the microchamber 4 is completed.

[0118] In the above embodiment, the first type of single cell is captured into the microchamber 4 by the microfluidic chip 10 through single cell injection-microwell 3 capture-transfer to microchamber 4. At this time, there is no single cell in the microwell 3 of the chip 10. The second type of single cell is introduced into the main channel 2 again, and the second type of single cell is captured by the microwell 3. After successful capture, the single cell that is not captured in the main channel 2 is discharged by fluid. Then the chip 10 is flipped and tilted, so that the second type of single cell in the microwell 3 is transferred to the microchamber 4. At this time, two different types of single cells exist in one microchamber 4, and based on this, the interaction of two different types of single cells can be completed.

[0119] The above-mentioned multiple different single cell interaction method is based on the precise operation of the microfluidic chip 10. Through two different single cell capture and transfer, two types of single cells are introduced into the same microchamber 4. Gravity plays a core role in the whole process, and single cells rely on gravity to transfer from the microwell 3 of the main channel 2 to the microchamber 4. Each time the chip 10 is flipped, the direction of the single cell movement can be controlled to ensure that it enters the microchamber 4, thereby creating conditions for the interaction between different single cells.

[0120] In some embodiments, two or more types of single cells are sequentially captured into the microchamber 4 by the single cell capture method in the present application, and multiple single cell interaction studies are conducted.

[0121] In the present application, two or more types of single cells can be captured according to the needs, and more complex multi-cell interaction phenomena can be studied by adjusting the capture order and number. At the same time, the size and shape of the microchamber 4 can also be adjusted according to the type of single cell and the experimental goal. In addition, the system can also combine with a sensor module to monitor the chemical signals or physical changes in the cell interaction process in real time, so as to improve the accuracy and visualization effect of the experiment.

[0122] The microfluidic chip in the present application provides an efficient and accurate experimental platform for multi-cell interaction research. Through precise control of the capture, transfer and interaction of different single cells by the microfluidic chip, an efficient and repeatable platform is provided for studying the interaction between single cells. By sequentially introducing different types of single cells, the interference between different single cells in the capture process is avoided, thereby improving the accuracy and success rate of single cell interaction.

[0123] Capturing and culturing different kinds of single cells in the same microcavity 4 can simulate the real cell microenvironment, and facilitate the study of the interaction between multiple different single cells, such as communication, cooperation or competition. At the same time, through the repeated operation function of the chip, the experimenter can easily control the type and quantity of captured single cells, further improving the flexibility and controllability of the experiment.

[0124] In order to verify that the single cell capture rate of the microcavity microfluidic chip of the present application is greatly improved, a comparison test is carried out with the single cell capture method of the droplet microfluidic technology in the prior art, as follows:

[0125] Embodiments

[0126] The single cells are captured by using the microcavity microfluidic chip 10 of the present application.

[0127] (1) The single cells to be captured are injected into the microcavity microfluidic chip 10 by fluid, wherein the cell diameter is about 15 μm, the single cell suspension concentration is 1 x 10 7 cells / mL, the microcavity width is 80 μm, the microcavity length is 150 μm, and the shunt channel width is 40 μm;

[0128] (2) After the main channel 2 is filled with fluid, the fluid in the main channel 2 is kept static for 5 min, so that the single cells fall into the microcavity 4 under the action of gravity;

[0129] (3) After the single cells are captured in the microcavity 4, the fluid is introduced again to discharge the single cells not captured in the main channel 2, and the fluid flow rate is 10 μL / min;

[0130] (4) After the single cells in the main channel 2 are discharged, the chip 10 is turned over and inclined so that the microcavity 4 is located above the microcavity 4, the turning angle is 45°, and the single cells captured in the microcavity 4 are kept static for 5 min, so that the single cells captured in the microcavity 4 enter the microcavity 4 located below the microcavity 4.

[0131] Comparative examples

[0132] The single cells are captured by using the method of the droplet microfluidic technology in the prior art.

[0133] According to the above embodiments and comparative examples, the single cell capture rates are compared, as shown in Table 1.

[0134] Table 1

[0135] From the above Table 1, it can be concluded that the microwell coupled microcavity microfluidic chip of the present application is obviously superior to the droplet microfluidic method in the prior art in terms of single cell capture efficiency. Due to the precise design of the microwell and the fluid static sedimentation mechanism, the microwell coupled microcavity microfluidic chip of the present application can orderly capture and transfer single cells to the microcavity 4. Experimental data show that the single cell capture rate of this method reaches >99%, greatly improving the success rate of single cell experiments, and is particularly suitable for high-throughput and precise single cell operation scenarios. The droplet microfluidic technology has significant shortcomings in terms of capture rate and operation precision, and the single cell rate is only 10% to 30%, because the droplet microfluidic technology is highly dependent on the stability of the droplet generation process and the randomness of the fluid, which easily leads to low cell capture efficiency. At the same time, the distribution of single cells in the droplets is uneven, further reducing the success rate of capture, and there are limitations in handling a large number of single cell experiments.

[0136] Although the present application has been described in detail with reference to the foregoing embodiments, it should be understood by those skilled in the art that the technical solutions recorded in the foregoing embodiments can still be modified, or some technical features can be replaced by equivalents; and these modifications or replacements do not make the essence of the corresponding technical solutions deviate from the spirit and scope of the technical solutions of the embodiments of the present application.

Claims

1. A method of single cell capture, the method comprising, The method comprises the following steps: S1, injecting single cells to be captured into a chip by fluid, the chip being a microwell coupled microchamber microfluidic chip, the chip comprising a substrate and a main channel provided on the substrate, a plurality of microwells being provided on the surface of the main channel and a plurality of microchambers corresponding to the microwells, wherein the microchambers are in communication with the main channel; S2, after the fluid fills the main channel, keeping the fluid in the main channel static, so that the single cells fall into the microwells provided on the main channel under the action of gravity; S3, after the microwells capture the single cells, again injecting fluid to discharge the single cells not captured in the main channel; S4, after the single cells in the main channel are discharged, turning over and tilting the chip so that the microwells of the chip are above the microchambers, so that the single cells captured in the microwells enter the microchambers corresponding to the microwells from the microwells.

2. The single cell capture method of claim 1, wherein, In step S3, the single cells not captured in the main channel are discharged by controlling the fluid flow rate of the sample inlet to ensure that the single cells in the microwells are retained in the main channel, wherein the fluid flow rate of the sample inlet is 1 uL / min to 40 uL / min.

3. The single cell capture method of claim 1, wherein, In step S2, after the fluid fills the main channel, the fluid in the main channel is kept static, and after 1 min to 10 min, the single cells fall into the microwells provided on the main channel under the action of gravity, and then step S3 is performed to inject fluid to discharge the single cells not captured in the main channel.

4. The single cell capture method of claim 1, wherein, Further comprising a single cell derivation method S5: After the single cells are introduced into the microchamber, the fluid in the microchamber is static, a laser is used to irradiate a position of the microchamber away from the microwell to form a bubble, the target single cell in the microchamber is introduced into the main channel by the method of laser-induced bubble, fluid is introduced into the main channel, and under the driving of the fluid, the target cell is derived out of the chip and collected.

5. The single cell capture method of claim 1, wherein, Further comprising a single cell culture method S6: After the single cells are introduced into the microchamber, the fluid in the main channel and the microchamber is static, a culture medium is introduced into the main channel, at this time the culture medium replaces the fluid in the main channel and the microchamber, after the microchamber is filled with the culture medium, the single cells are cultured in the microchamber.

6. The single cell capture method of claim 1, wherein, Further comprising a single cell labeling and detection method S7: After the single cells are introduced into the microchamber, the fluid in the main channel and the microchamber is static, a staining agent is introduced into the main channel, at this time the staining agent replaces the fluid in the main channel and the microchamber, after the microchamber is filled with the staining agent, the single cells in the microchamber are stained and labeled by the staining agent; the state of the single cells in the microchamber is detected by combining microscopic images, and target single cells are obtained by laser-induced bubble sorting.

7. The single cell capture method of claim 1, wherein, Further comprising a method for interaction of a plurality of different single cells S8: Firstly, the first single cell is introduced into the main channel by fluid, and the first single cell falls into the microwell under the action of gravity. After the microwell captures the first single cell, the first single cell that is not captured is discharged. The chip is turned over and tilted downward from the microwell to the microchamber direction, so that the first single cell is transferred to the corresponding microchamber by gravity. Secondly, the second single cell is introduced into the main channel by fluid, and the second single cell falls into the microwell under the action of gravity. After the microwell captures the second single cell, the second single cell that is not captured is discharged. The chip is turned over and tilted downward from the microwell to the microchamber direction, so that the second single cell is transferred to the corresponding microchamber by gravity and is located in the same microchamber as the first single cell, and the interaction of multiple different single cells in the microchamber is completed.

8. A method of single cell manipulation, comprising: The single cell capture method according to any one of claims 1-3.

9. The method of single cell manipulation of claim 8, wherein, The single cell discharge method also includes: After the single cell is introduced into the microchamber, the fluid in the microchamber is static, a laser is used to irradiate a position of the microchamber away from the microwell to form a bubble, the target single cell in the microchamber is introduced into the main channel by the method of laser-induced bubble, and fluid is introduced into the main channel. Under the driving of the fluid, the target cell is discharged from the chip and collected.

10. The single cell capture method of claim 8, wherein, The single cell culture method also includes: After the single cell is introduced into the microchamber, the fluid in the main channel and the microchamber is static, and a culture medium is introduced into the main channel. At this time, the culture medium replaces the fluid in the main channel and the microchamber. After the microchamber is filled with the culture medium, the single cell is cultured in the microchamber.

11. The single cell capture method of claim 8, wherein, The single cell labeling detection method also includes: After the single cell is introduced into the microchamber, the fluid in the main channel and the microchamber is static, and a staining agent is introduced into the main channel. At this time, the staining agent replaces the fluid in the main channel and the microchamber. After the microchamber is filled with the staining agent, the single cell in the microchamber is stained and labeled by the staining agent. The state of the single cell in the microchamber is detected by combining a microscopic image, and the target single cell is obtained by laser-induced bubble sorting.

12. The single cell capture method of claim 8, wherein, The multiple different single cell interaction method also includes: Firstly, the first single cell is introduced into the main channel by fluid, and the first single cell falls into the microwell under the action of gravity. After the microwell captures the first single cell, the first single cell that is not captured is discharged. The chip is turned over and tilted downward from the microwell to the microchamber direction, so that the first single cell is transferred to the corresponding microchamber by gravity. The second single cell is introduced into the main channel by fluid again, and the second single cell falls into the microwell under the action of gravity. After the second single cell is captured in the microwell, the second single cell not captured is discharged. The chip is turned over and tilted downward from the microwell to the microchamber, so that the second single cell is transferred into the corresponding microchamber by gravity and is located in the same microchamber with the first single cell, and the interaction of multiple different single cells in the microchamber is completed.

13. A microfluidic chip with micropore coupling to a microchamber, characterized in that, Comprise: a substrate; a main channel provided on the substrate and penetrating through the substrate; a plurality of microwells uniformly provided on the main channel and used for capturing single cells; a plurality of microchambers corresponding to the microwells and in communication with the main channel; wherein the diameter of the microwell is d1, the depth of the microwell is t1, the diameter of the single cell is d2, d1:t1=1:2-4:5, and d1:d2=1:1-4:3; the width of the microchamber is x, the length of the microchamber is y, x:y=1:1-1:3, and x>d1.

14. The microfluidic chip of claim 13, wherein, The spacing between adjacent two microwells and the spacing between adjacent two microchambers are both L, and 20μm<L<1000μm.

15. The microwell coupled microchamber microfluidic chip according to claim 13, characterized in that: the volume above each microwell is V, V=πr2h, wherein r is the radius of the microwell, and h is the height of the main channel; the single cell concentration above each microwell is C, C=1000 / V×λ, and λ refers to the theoretical average number of cells in the volume above each microwell, wherein λ=1, so that the number of cells above each microwell is greater than or equal to 1 cell.

16. The microfluidic chip of claim 13, wherein, Further comprising a shunt channel provided between the microwell and the microchamber, the width of the shunt channel being less than the width of the microchamber, so that the shunt channel and the microchamber combine to form a b-shaped structure, and the shunt channel is in communication with the main channel, so as to introduce the single cell in the microwell into the microchamber.

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