Single cell capture chip, system and use method of system

Abstract

The invention provides a single cell capture chip and system and a use method of the system.The system comprises a liquid inlet and outlet device, a detection device and a control device, and the control device controls the liquid inlet and outlet device according to the capture rate; the method specifically comprises the following steps: a control device controls a piston to move towards a first direction so as to enable cell sap in a first liquid storage tank to flow to an outlet end through a capture well area of a single cell capture chip; then, the steps S1 and S2 are executed circularly until the cell capture rate reaches a preset capture rate; the method comprises the following steps: S1, after standing, detecting the capture rate of cells by a detection device; if the preset capture rate is not reached, controlling the piston to move towards a second direction opposite to the first direction so as to push the cell sap in the capture trap area to move towards the inlet end; s2, after standing, detecting the capture rate of the cells by a detection device; and if the preset capture rate is not reached, the piston is controlled again to move towards the first direction. According to the scheme, the cell capture rate can be increased, and the capture rate of complex cell samples can also be increased.

Description

Technical Field

[0001] The present application relates to the field of cell capture technology, and in particular to a single cell capture chip, system, and method for using the system. Background Art

[0002] In the field of life science and medical research, single-cell capture technology is of vital importance. This technology can accurately separate single cells from complex cell populations, providing researchers with the possibility to deeply explore the heterogeneity between cells. Cell heterogeneity is common in various cell populations. Even cells with the same origin and similar morphology have significant differences in gene expression, functional status and phenotypic characteristics. With the help of single-cell capture technology, researchers can clearly identify these differences, laying the foundation for revealing the mechanism of cell differentiation, exploring the root causes of diseases, and formulating more accurate and effective treatment plans. For example, in tumor research, the heterogeneity of tumor cells often leads to poor treatment effects. Single-cell capture technology can accurately locate tumor cell subpopulations with special drug resistance or high invasiveness, providing a key basis for the formulation of personalized treatment plans; in stem cell research, this technology helps to analyze the molecular pathways by which stem cells differentiate into different cell types and promote the development of regenerative medicine.

[0003] Single-cell capture chips and their use of internal negative pressure to draw in cell fluid have been used to some extent in existing technologies. Their working principle is to create a negative pressure environment within the single-cell capture chip, using the pressure differential to draw cell fluid containing cells into the chip, thereby achieving cell capture.

[0004] However, this technical solution has obvious shortcomings in practical applications. The most prominent problem is the low cell capture rate. The low capture rate causes a large number of target cells to be lost during the capture process, resulting in a serious waste of sample cells. Some of these samples are collected with great difficulty from the patient's body, while others are carefully cultivated in the laboratory at the cost of a lot of manpower and material resources. The waste of sample resources not only directly increases the economic cost of the research, but also greatly increases the difficulty of subsequent data analysis, seriously restricting the widespread application and in-depth development of this technology in the field of single-cell research. At present, scientific researchers are in urgent need of developing innovative technologies to improve the capture efficiency of single-cell capture technology, break through the bottleneck of existing technologies, and give full play to the huge potential of single-cell capture technology in life science research. Summary of the Invention

[0005] The purpose of this specification is to provide a single-cell capture chip, system, and method for using the system to address the problems of low capture rate, difficulty in processing complex samples, and insufficient sample cell utilization in existing single-cell capture methods.

[0006] To solve the above technical problems, the first aspect of the present specification provides a single-cell capture system, comprising: an inlet and outlet liquid device having a cylinder, a piston, and a propulsion mechanism for controlling the reciprocating motion of the piston in the cylinder, wherein the cylinder is used to be detachably sealed and connected to the outlet end of the single-cell capture chip; wherein the inlet end of the single-cell capture chip is provided with a first liquid storage tank for containing cell fluid; a detection device for detecting the capture rate of cells; a control device for controlling the inlet and outlet liquid device according to the capture rate, wherein the control method includes: the control device controls the piston to move in a first direction so that the liquid in the first liquid storage tank is The cell fluid flows to the outlet end through the capture trap area of ​​the single-cell capture chip; then S1 and S2 are executed cyclically until the cell capture rate reaches the preset capture rate; wherein, S1: after standing still for a predetermined period of time, the detection device detects the cell capture rate; if the capture rate does not reach the preset capture rate, the control device controls the piston to move in a second direction opposite to the first direction to push the cell fluid in the capture trap area toward the inlet end; S2: after standing still for a predetermined period of time, the detection device detects the cell capture rate; if the capture rate does not reach the preset capture rate, the control device controls the piston to move in the first direction again.

[0007] In some embodiments, the propulsion mechanism includes: a motor, electrically connected to the control end of the control device, and a mechanical transmission mechanism, one side of which is connected to the motor shaft of the motor and the other side is connected to the piston handle; the mechanical transmission mechanism converts the rotational motion of the motor into linear motion of the piston in the cylinder.

[0008] In some embodiments, the detection device includes: a microscopic imaging device for imaging the capture well area; a processor for performing cell recognition on the image formed by the microscopic imaging device, determining the number of captured cells based on the recognition results, and then calculating the cell capture rate.

[0009] In some embodiments, the single cell capture system further comprises: an interactive device for acquiring an application scenario input by a user; and a control device for automatically executing operations with different parameters according to different application scenarios input by the user.

[0010] A second aspect of this specification provides a method for using a single-cell capture system, which is used for the single-cell capture system according to any one of the first aspects, the method comprising: dripping a target cell solution into a first liquid storage tank of a single-cell detection chip; setting operating parameters, wherein the operating parameters include a preset capture rate; controlling the single-cell capture system to start working; after the system reaches the preset capture rate and stops working, removing the single-cell capture chip for single-cell sequencing.

[0011] A third aspect of this specification provides a method for using a single-cell capture system, which is used for the single-cell capture system described in any one of the first aspects, the method comprising: dripping target cell fluid into the first liquid storage tank of the single-cell detection chip; setting operating parameters, wherein the operating parameters include a preset capture rate; controlling the single-cell capture system to start working; after the system reaches the preset capture rate and stops working, adjusting the piston to a predetermined state; absorbing the cell fluid in the first liquid storage tank; adding a staining solution to the first liquid storage tank; controlling the piston to move in the direction in which the cylinder becomes larger so that the staining solution is in full contact with the cells; and conducting subsequent research after the staining is completed.

[0012] A fourth aspect of this specification provides a method for using a single-cell capture system, which is used for the single-cell capture system described in any one of the first aspects, the method comprising: dripping target cell liquid into a first liquid storage tank of a single-cell detection chip; setting operating parameters, wherein the operating parameters include a preset capture rate; controlling the single-cell capture system to start working; after the system reaches the preset capture rate and stops working, imaging the capture well area of ​​the single-cell capture chip through a microscopic imaging device.

[0013] In some embodiments, the capture well area of ​​the single-cell capture chip includes a marking well; imaging the capture well area of ​​the single-cell capture chip by a microscopic imaging device includes: using a low-magnification microscopic imaging device to perform low-magnification imaging of the entire capture well area of ​​the single-cell capture chip; after finding the target cell from the low-magnification imaging results, marking the position of the target cell according to the marking well on the single-cell capture chip; and using a high-magnification microscopic imaging device to perform high-magnification imaging of the target cell at the marked position.

[0014] A fifth aspect of this specification provides a single-cell capture chip, used in the single-cell capture system described in any one of the first aspects; the single-cell capture chip has an inlet end and an outlet end, a first liquid storage tank for containing cell fluid is provided at the inlet end, and a second liquid storage tank is provided at the outlet end, one end of the second liquid storage tank is connected to the outlet end of the single-cell capture chip, and the other end is used for detachable sealed communication with the liquid inlet and outlet device; the capture well area includes capture wells that are arrayed and cover the capture well area.

[0015] In some embodiments, labeling wells are provided at multiple locations within the capture well region arranged in an array. The diameter of the labeling wells is smaller than the diameter of the cells to be captured. The labeling wells are used to mark locations within the capture well region.

[0016] In some embodiments, each position of the label capture well region is varied by combining label wells with different opening shapes.

[0017] In some embodiments, a combination of N marking wells with different opening shapes is used to represent N-base marking data, and the row and column positioning of each position is represented by the N-base marking data.

[0018] The single-cell capture chip, system, and method for using the system provided in this specification automatically detect the cell capture rate after "sucking in" the cells, and perform a "spitting out" process of the cell fluid when the capture rate does not reach the preset capture rate. The "spitting out" process is when the control device 30 controls the piston 12 to move in a second direction opposite to the first direction (that is, movement toward the opening of the first end of the cylinder 11) to push the cell fluid in the capture trap area toward the inlet end. During the "spitting out" process, cells that were not captured during the previous "sucking in" process pass through the capture trap area again and are captured during this "spitting out" process. Of course, some cells are still not captured and move toward the first liquid storage tank or enter the first liquid storage tank along with the cell fluid.

[0019] If the cell capture rate does not reach the preset capture rate, the above-mentioned "inhalation" and "spitting" processes can be repeated. Each time the process is performed, new cells will be captured, thereby improving the cell capture rate. Moreover, each time the "inhalation" or "spitting" process is performed, the captured cells will not escape from the capture trap, but may rotate in the capture trap due to the action of the fluid. It can be seen that the single-cell capture system provided in this specification can improve the cell capture rate by controlling the piston movement through the control device to repeatedly realize the "inhalation" and "spitting" process of the cell fluid.

[0020] Single-cell capture chips can capture cells by gravity or by dielectrophoresis. These two types of single-cell capture chips exist in the prior art. This solution does not improve their capture principles, so the capture trap structure and capture principle will not be described in detail.

[0021] In addition to improving cell capture rates, the single-cell capture chip, system, and method for using the system provided in this specification also have the following technical effects:

[0022] 1. Impurity cleaning

[0023] The existing technology only uses a single negative pressure suction. Once impurities enter the chip with the cell fluid, they can easily clog the capture trap and become difficult to remove. This solution can remove impurities stuck in the capture trap from the chip by repeating the "suction" and "spit" operations. Multiple "suction" and "spit" cycles can continuously clean impurities and keep the capture trap unobstructed, thereby increasing the number of effective capture traps and thus improving the cell capture rate. For example, when processing cell samples containing impurities such as tissue fragments and protein aggregates, after 3-5 cycles, the probability of impurities clogging the capture trap can be significantly reduced, creating a good environment for cell capture.

[0024] 2. Break up cell aggregates

[0025] In the single inhalation of existing technologies, cell aggregates are not only difficult to be captured themselves, but also hinder the surrounding free cells from approaching the capture trap. In the cyclic operation of this scheme, the cell fluid flows repeatedly, and the shear force generated can effectively break up the cell aggregates. Each time the cell fluid is "spitted out", the aggregated cells are subjected to a reverse impact force; when they are "inhaled" again, they experience liquid flow in a different direction. After multiple cycles, the aggregates gradually disintegrate into single cells or small cell clusters, increasing the chances of cells contacting the capture trap. When capturing tumor cell samples, tumor cells often form aggregates due to the action of intercellular adhesion molecules. After multiple cycles of operation, the breakup rate of aggregated cells can reach more than 70%, greatly improving the comprehensiveness of tumor cell capture.

[0026] 3. Adapting to cell size differences

[0027] The cell sizes in complex samples vary greatly, and existing technologies cannot take both into account in a single aspiration. During the circulation process of this solution, the movement trajectories and distribution states of cells of different sizes are constantly adjusted under repeated liquid flow. Larger cells are affected by liquid flow and have a relatively small range of movement, but multiple cycles can also make them move in different areas of the chip, increasing the possibility of contact with suitable capture traps; although smaller cells are easy to flow with the liquid, they will also be more evenly distributed in the chip due to multiple cycles. Taking a sample mixed with large-volume macrophages and small-volume lymphocytes as an example, after multiple cycles of "inhalation" and "spitting", the capture rates of both cell types have been significantly improved compared to existing technologies, increasing by 30% and 40% respectively, effectively solving the capture problem caused by cell size differences. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] In order to more clearly illustrate the implementation methods of the present application or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the implementation methods or the description of the prior art. Obviously, the drawings described below are only some implementation methods recorded in this application. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative labor.

[0029] Figure 1 Schematic diagram of the single cell capture system provided for this specification;

[0030] Figure 2 A schematic diagram of an image formed by a microscopic imaging device;

[0031] Figure 3 Schematic diagram of the control method of the single-cell capture system;

[0032] Figure 4 Schematic diagram of the method for using a single-cell capture system for single-cell sequencing;

[0033] Figure 5 Schematic diagram of the method for using a single-cell capture system for fluorescent staining;

[0034] Figure 6 Schematic diagram of the method for using a single-cell capture system for high-precision cell imaging;

[0035] Figure 7 This is a schematic diagram of a labeled well in the capture well area of ​​a single-cell capture chip provided in this specification. DETAILED DESCRIPTION

[0036] In order to enable those skilled in the art to better understand the technical solutions in this application, the following will clearly and completely describe the technical solutions in the embodiments of this application in conjunction with the drawings in the embodiments of this application. Obviously, the described embodiments are only part of the embodiments of this application, not all of the embodiments. Based on the embodiments of this application, all other embodiments obtained by ordinary technicians in this field without making creative efforts should fall within the scope of protection of this application.

[0037] To address the problems of low capture rate, difficulty in processing complex samples, and insufficient utilization of sample cells in existing single-cell capture methods, this specification provides a single-cell capture system. This system, when used in conjunction with a single-cell capture chip, can improve the capture rate of single-cell capture, increase the utilization of sample cells, and can also process complex samples.

[0038] Specifically, if Figure 1 The single cell capture system provided in this specification includes a liquid inlet and outlet device 10, a detection device 20 and a control device 30. Figure 1 In the figure, 40 indicates a single-cell capture chip.

[0039] The single-cell capture chip 40 has a solution inlet and outlet. A first liquid reservoir 41 is located at the inlet for storing cell fluid. The cell fluid to be studied can be injected into the first liquid reservoir 41. The first liquid reservoir 41 is open at both ends. One end is sealed and connected to the inlet of the single-cell capture chip, while the other end is open and used for injecting cell fluid.

[0040] The outlet end of the single cell capture chip 40 can be directly connected to the barrel 11 of the liquid inlet and outlet device 10, and the two are detachably sealed and connected.

[0041] Without the second liquid reservoir 42, liquid can enter the tube connecting the outlet of the single-cell capture chip 40 and the liquid inlet and outlet device 10. This tube can be longer and / or thicker to accommodate a larger amount of cell fluid. In some cases, the tube may contain less cell fluid, and some of the cell fluid may enter the barrel 11 of the liquid inlet and outlet device 10, thereby contaminating the barrel 11 and requiring replacement of the barrel 11 for the next cell capture experiment.

[0042] To avoid replacing the barrel 11, in some embodiments, as Figure 1 As shown, a second liquid storage tank 42 can also be provided at the outlet of the single cell capture chip 40. The second liquid storage tank 42 is open at both ends, one end of which is sealed and connected to the outlet of the single cell capture chip, and the other end is open and used for removable sealed communication with the liquid inlet and outlet device 10.

[0043] The liquid inlet and outlet device 10 comprises a cylinder 11, a piston 12, and a propulsion mechanism 13 that controls the reciprocating motion of the piston 12 within the cylinder 11. The cylinder 11 is configured to be removably and sealedly connected to the outlet of the single-cell capture chip 40. The inlet of the single-cell capture chip 40 is provided with a first liquid reservoir 41 for containing cell fluid.

[0044] The barrel 11 is open at both ends. The first end opening of the barrel 11 is designed to be removably sealed and connected to the outlet of the single-cell capture chip 40 or the opening of the second liquid storage tank 42. The piston 12 is located in the middle of the barrel 11. During reciprocating motion within the barrel 11, the piston 12 always engages the inner wall of the barrel 11. The piston handle is located on the side of the piston 12 away from the first end of the barrel 11, and the piston handle drives the piston body to reciprocate within the barrel 11.

[0045] The propulsion mechanism 13 includes a motor and a mechanical transmission mechanism. The motor is electrically connected to the control terminal of the control device 30. One side of the mechanical transmission mechanism is in driving connection with the motor shaft, and the other side is in driving connection with the piston handle. The mechanical transmission mechanism converts the rotational motion of the motor into linear motion of the piston 12 within the cylinder 11. In some embodiments, the mechanical transmission mechanism may include a lead screw. The propulsion mechanism 13 can be implemented in a variety of ways, which will not be described in detail in this specification.

[0046] Detection device 20 is used to detect the cell capture rate. In some embodiments, detection device 20 may include a microscopic imaging device and a processor. The microscopic imaging device is used to image the capture trap area, and the processor is used to identify cells in the image generated by the microscopic imaging device, determine the number of captured cells based on the identification results, and then calculate the cell capture rate. Image recognition and counting methods are prior art and will not be described in detail in this specification. Figure 2 A schematic diagram of an image formed by a microscopic imaging device.

[0047] The capture rate may be the ratio of cells captured in the capture well area to the total number of cells in the cell fluid injected into the single cell capture chip, wherein the total number of cells in the cell fluid injected into the single cell capture chip may be a value input by the user.

[0048] The control device 30 is used to control the liquid inlet and outlet device 10 according to the capture rate.

[0049] Control method such as Figure 3 As shown, the control device 30 first controls the piston 12 to move in a first direction so that the cell fluid in the first liquid storage tank 41 flows to the outlet end through the capture trap area 43 of the single cell capture chip 40; then the following S1 and S2 are cyclically executed until the cell capture rate reaches the preset capture rate.

[0050] S1: After a predetermined period of rest, the detection device 20 detects the cell capture rate. If the capture rate does not reach the preset capture rate, the control device 30 controls the piston 12 to move in a second direction opposite to the first direction to push the cell fluid in the capture trap area toward the inlet end.

[0051] S2: After a predetermined period of time, the detection device 20 detects the capture rate of cells. If the capture rate does not reach the preset capture rate, the control device 30 controls the piston 12 to move toward the first direction again.

[0052] The preset capture rate may be input by the user, or may be a default value pre-set in the control device 30 .

[0053] The control device 30 controls the piston 12 to move in a first direction (i.e., away from the opening at the first end of the cylinder 11), thereby creating a negative pressure within the single-cell capture chip 40. This causes the cell fluid in the first fluid reservoir 41 to be drawn into the single-cell capture chip 40, whereupon the cell fluid flows through the capture trap region 43 toward the outlet. This process can be referred to as the "suction" process of the cell fluid.

[0054] During this process, some cells are captured by the trap, but some remain uncaptured. These uncaptured cells may remain suspended in the liquid within the single-cell capture chip, or they may enter the secondary fluid reservoir (if provided) or the tubing between the inlet and outlet devices and the single-cell capture chip. In existing technologies, cells that are not captured after the initial "absorption" of the cell fluid are unusable and therefore wasted.

[0055] The single-cell capture system provided in this specification automatically performs a cell capture rate test after "inhaling" the cells, and performs a "spitting out" process of the cell fluid when the capture rate does not reach the preset capture rate. The "spitting out" process, that is, the control device 30 controls the piston 12 to move in a second direction opposite to the first direction (that is, movement toward the opening of the first end of the cylinder 11) to push the cell fluid in the capture trap area toward the inlet end. During the "spitting out" process, the cells that were not captured in the previous "inhalation" process pass through the capture trap area again and are captured in this "spitting out" process. Of course, some cells are still not captured and move toward the first liquid storage tank or enter the first liquid storage tank along with the cell fluid.

[0056] If the cell capture rate does not reach the preset capture rate, the above-mentioned "inhalation" and "spitting" processes can be repeated. Each time the process is performed, new cells will be captured, thereby improving the cell capture rate. Moreover, each time the "inhalation" or "spitting" process is performed, the captured cells will not escape from the capture trap, but may rotate in the capture trap due to the action of the fluid. It can be seen that the single-cell capture system provided in this specification can improve the cell capture rate by controlling the piston movement through the control device to repeatedly realize the "inhalation" and "spitting" process of the cell fluid.

[0057] Single-cell capture chips can capture cells by gravity or by dielectrophoresis. These two types of single-cell capture chips exist in the prior art. This solution does not improve their capture principles, so the capture trap structure and capture principle will not be described in detail.

[0058] In addition to improving the cell capture rate, the single cell capture system provided in this manual also has the following technical effects:

[0059] 1. Impurity cleaning

[0060] The existing technology only uses a single negative pressure suction. Once impurities enter the chip with the cell fluid, they can easily clog the capture trap and become difficult to remove. This solution can remove impurities stuck in the capture trap from the chip by repeating the "suction" and "spit" operations. Multiple "suction" and "spit" cycles can continuously clean impurities and keep the capture trap unobstructed, thereby increasing the number of effective capture traps and thus improving the cell capture rate. For example, when processing cell samples containing impurities such as tissue fragments and protein aggregates, after 3-5 cycles, the probability of impurities clogging the capture trap can be significantly reduced, creating a good environment for cell capture.

[0061] 2. Break up cell aggregates

[0062] In the single inhalation of existing technologies, cell aggregates are not only difficult to be captured themselves, but also hinder the surrounding free cells from approaching the capture trap. In the cyclic operation of this scheme, the cell fluid flows repeatedly, and the shear force generated can effectively break up the cell aggregates. Each time the cell fluid is "spitted out", the aggregated cells are subjected to a reverse impact force; when they are "inhaled" again, they experience liquid flow in a different direction. After multiple cycles, the aggregates gradually disintegrate into single cells or small cell clusters, increasing the chances of cells contacting the capture trap. When capturing tumor cell samples, tumor cells often form aggregates due to the action of intercellular adhesion molecules. After multiple cycles of operation, the breakup rate of aggregated cells can reach more than 70%, greatly improving the comprehensiveness of tumor cell capture.

[0063] 3. Adapting to cell size differences

[0064] The cell sizes in complex samples vary greatly, and existing technologies cannot take both into account in a single aspiration. During the circulation process of this solution, the movement trajectories and distribution states of cells of different sizes are constantly adjusted under repeated liquid flow. Larger cells are affected by liquid flow and have a relatively small range of movement, but multiple cycles can also make them move in different areas of the chip, increasing the possibility of contact with suitable capture traps; although smaller cells are easy to flow with the liquid, they will also be more evenly distributed in the chip due to multiple cycles. Taking a sample mixed with large-volume macrophages and small-volume lymphocytes as an example, after multiple cycles of "inhalation" and "spitting", the capture rates of both cell types have been significantly improved compared to existing technologies, increasing by 30% and 40% respectively, effectively solving the capture problem caused by cell size differences.

[0065] In some embodiments, the single-cell capture system further includes an interactive device for obtaining user input of an application scenario. An application scenario refers to the intended use or further operation after the single cell is captured. Application scenarios may include single-cell sequencing, fluorescent staining, and high-precision cell imaging.

[0066] The control device 30 is electrically connected to the interactive device. Operational parameters corresponding to each application scenario can be pre-set in the control device 30. These operating parameters may include, for example, a preset capture rate, a resting time period (each resting time period can be the same or different), an inhalation or exhalation speed (the inhalation speed and exhalation speed can be the same or different, and the inhalation speed and exhalation speed can be different each time), the number of times S1 and S2 are cycled, and whether the piston 12 returns to a predetermined state at the end of the capture process (the predetermined state refers to the state in which the piston 12 is closest to the cylinder 11, i.e., the state in which the enclosed space within the cylinder 11 is minimized).

[0067] The control device 30 automatically performs different operations using different parameters according to different application scenarios input by the user.

[0068] By setting up an interactive device, after the user injects the cell fluid into the first liquid storage tank of the single-cell capture chip, the user can select the application scenario through the interactive device, and after clicking the "Start" button of the system, the single-cell capture process is automatically executed using the default operating parameters in the control device 30; or after selecting the application scenario, the default operating parameters in the control device 30 can be adjusted through the interactive device, and then the "Start" button of the system is clicked to automatically execute the single-cell capture process using the adjusted operating parameters.

[0069] The "start" button can be set in the interactive device.

[0070] This manual provides a method for using a single-cell capture system, which corresponds to the application scenario of "single-cell sequencing". Figure 4 As shown, the method includes the following S11 to S14.

[0071] S11: Drop the target cell solution into the first liquid storage tank of the single-cell detection chip.

[0072] S12: Setting operating parameters, wherein the operating parameters include a preset capture rate.

[0073] The operating parameters may include a preset capture rate, a resting time period (each resting time period may be the same or different), and an inhalation or exhalation speed (the inhalation speed and exhalation speed may be the same or different, and the inhalation speed and exhalation speed may also be different each time). Of course, the operating parameters may also include other content.

[0074] S13: Control the single cell capture system to start working.

[0075] The single-cell capture system can be equipped with a start button, which the user can use to transmit the start command to the single-cell capture system. The single-cell capture system can also be equipped with a voice recognition device, which the user can use to transmit the start command to the single-cell capture system through voice. The user can also transmit the start command to the single-cell capture system through other means, and the single-cell capture system has corresponding settings.

[0076] S14: After the system reaches a preset capture rate and stops working, a single-cell capture chip is used for single-cell sequencing.

[0077] After receiving the user's start-up instruction, the single-cell capture system automatically performs the above-mentioned "inhalation" and "spitting" operations, and automatically stops working after the capture rate reaches the preset capture rate.

[0078] Users can remove the single-cell capture chip from the single-cell capture system and transfer it to other instruments for single-cell sequencing.

[0079] This manual provides a method for using the single cell capture system, which corresponds to the "fluorescence staining" application scenario. Figure 5 As shown, the method includes the following S21 to S28.

[0080] S21: Drop the target cell solution into the first liquid storage tank of the single cell detection chip.

[0081] S22: Setting operating parameters, wherein the operating parameters include a preset capture rate.

[0082] The operating parameters may include a preset capture rate, a resting time period (each resting time period may be the same or different), and an inhalation or exhalation speed (the inhalation speed and exhalation speed may be the same or different, and the inhalation speed and exhalation speed may also be different each time). Of course, the operating parameters may also include other content.

[0083] S23: Control the single cell capture system to start working.

[0084] The single-cell capture system can be equipped with a start button, which the user can use to transmit the start command to the single-cell capture system. The single-cell capture system can also be equipped with a voice recognition device, which the user can use to transmit the start command to the single-cell capture system through voice. The user can also transmit the start command to the single-cell capture system through other means, and the single-cell capture system has corresponding settings.

[0085] S24: After the system reaches a preset capture rate and stops working, the piston is adjusted to a predetermined state.

[0086] After receiving the user's start-up instruction, the single-cell capture system automatically performs the above-mentioned "inhalation" and "spitting" operations, and automatically stops working after the capture rate reaches the preset capture rate.

[0087] The predetermined state refers to a state in which the piston 12 is located closest to the cylinder 11 , that is, a state in which the enclosed space in the cylinder 11 is the smallest.

[0088] The piston can be adjusted to a predetermined state by manually sending an adjustment command to the control device 30. Alternatively, the control device 30 can automatically identify the application scenario and determine whether the piston needs to be adjusted to the predetermined state based on the specific application scenario. If necessary, the control device 30 identifies the state of the piston after the capture rate reaches a predetermined capture rate. If the piston is not in the predetermined state, it automatically adjusts it to the predetermined state. Specifically, the control device 30 can identify the state of the piston based on historical control records of the piston.

[0089] S25: Aspirate the cell fluid in the first fluid storage tank.

[0090] After the preset capture rate is reached, the remaining cells in the cell fluid are no longer needed, so the remaining cell fluid can be aspirated.

[0091] S26: Add dyeing solution into the first liquid storage tank.

[0092] By adding the dye solution to the first liquid storage tank, that is, reusing the first liquid storage tank, it is unnecessary to set up another liquid storage tank for the dye solution, thereby reducing the structural complexity of the single cell capture chip and reducing the chip production cost.

[0093] S27: Control the piston to move toward the first direction so that the staining solution is in full contact with the cells.

[0094] That is, S27 absorbs the staining liquid into the single-cell capture chip, and S27 absorbs the staining liquid through the reused piston, that is, reuses the outlet and piston of the single-cell capture chip, which can make the system structure of single-cell capture and staining simpler and less complex.

[0095] S27 can be executed only once, or after the staining solution is fully in contact with the cells and has been left to stand for a period of time, the control device 30 controls the piston to move in the second direction, so that the single-cell capture chip spits out the mixture of the staining solution and the cell fluid into the first liquid storage tank, and then controls the piston to move in the first direction again, that is, to absorb the mixture of the staining solution and the cell fluid.

[0096] The aforementioned process of "discharging" and "inhaling" the dye solution and cell solution mixture can be repeated multiple times. Because the cell solution is completely present in the single-cell capture chip and the dye solution is completely present in the first liquid reservoir before S27 is executed, the cyclic "discharging" and "inhaling" process of the dye solution and cell solution mixture gradually increases the dye solution concentration in the single-cell capture chip, thereby improving the dyeing effect.

[0097] The number of cycles of the "discharging" and "inhaling" processes of the dye solution and cell solution mixture can be set by the user as an operation parameter.

[0098] S28: Follow-up studies are performed after staining is completed.

[0099] Users can remove the single-cell capture chip from the single-cell capture system and transfer it to other instruments for further research.

[0100] This manual provides a method for using a single cell capture system, which corresponds to the application scenario of "high-precision cell imaging". Figure 6 As shown, the method includes the following S31 to S35.

[0101] S31: Drop the target cell solution into the first liquid storage tank of the single-cell detection chip.

[0102] S32: Setting operating parameters, wherein the operating parameters include a preset capture rate.

[0103] The operating parameters may include a preset capture rate, a resting time period (each resting time period may be the same or different), and an inhalation or exhalation speed (the inhalation speed and exhalation speed may be the same or different, and the inhalation speed and exhalation speed may also be different each time). Of course, the operating parameters may also include other content.

[0104] S33: Control the single cell capture system to start working.

[0105] The single-cell capture system can be equipped with a start button, which the user can use to transmit the start command to the single-cell capture system. The single-cell capture system can also be equipped with a voice recognition device, which the user can use to transmit the start command to the single-cell capture system through voice. The user can also transmit the start command to the single-cell capture system through other means, and the single-cell capture system has corresponding settings.

[0106] S34: After the system reaches the preset capture rate and stops working.

[0107] After receiving the user's start-up instruction, the single-cell capture system automatically performs the above-mentioned "inhalation" and "spitting" operations, and automatically stops working after the capture rate reaches the preset capture rate.

[0108] S35: Imaging the capture well area of ​​the single cell capture chip using a microscopic imaging device.

[0109] In some embodiments, the user can detach the single-cell capture chip from the single-cell capture system and transfer it to other instruments for imaging operations.

[0110] In other embodiments, the single-cell capture system itself is equipped with an imaging device. Therefore, S35 can also perform the imaging operation directly on the single-cell capture system without removing the single-cell capture chip.

[0111] In some embodiments, the capture well region of the single-cell capture chip includes a marker well. Accordingly, S35 can first use a low-magnification microscopic imaging device to perform low-magnification imaging of the entire capture well region of the single-cell capture chip; after finding the target cell from the low-magnification imaging results, the target cell's location is marked according to the marker well on the single-cell capture chip; and a high-magnification microscopic imaging device is used to perform high-magnification imaging of the target cell at the marked location. The single-cell capture system can also be provided with an automatic control system to control the high-magnification microscope to align with the location marked by the target marker well, so that the single-cell capture system can automatically find, capture, and output high-magnification imaging results of the target cell after the user marks the target cell.

[0112] Furthermore, the control device 30 can automatically adjust the lens of the high-power microscopic imaging device to align with the marked position according to the marked position of the target cell, and automatically control the high-power microscopic imaging device to perform imaging.

[0113] The above application scenarios are merely examples. In fact, the single cell capture system provided in this specification can also be used in other application scenarios. For specific usage methods, please refer to the above three usage methods provided in this specification.

[0114] This specification also provides a single-cell capture chip for use in any of the above-described single-cell capture systems to achieve single-cell capture. The single-cell capture chip has an inlet and an outlet. A first liquid reservoir for containing cell fluid is provided at the inlet, and a second liquid reservoir is provided at the outlet. One end of the second liquid reservoir is connected to the outlet of the single-cell capture chip, and the other end is configured for removable, sealed communication with a liquid inlet and outlet device. The capture trap area includes capture traps arranged in an array that covers the entire capture trap area.

[0115] In some embodiments, labeling wells are provided at multiple locations within the arrayed capture well region, wherein the diameter of the labeling wells is smaller than the diameter of the cells to be captured. The labeling wells are used to mark locations within the capture well region.

[0116] Typically, the cells to be captured are relatively small, the openings of the capture wells in the single-cell capture chip are relatively small, and the edges between the openings of each capture well are also very narrow. The existing technology does not have the process for printing position marks on the single-cell capture chip, and therefore, it is impossible to mark the position of the capture well by printing a mark.

[0117] The labeling wells provided herein are similar to the capture wells—pits within the cell flow path of a single-cell capture chip. The differences are: 1. The labeling wells have a smaller diameter; 2. The number of labeling wells is smaller. When fabricating a single-cell capture chip, the labeling wells can be fabricated simultaneously with the capture wells, using the same fabrication process. Therefore, the single-cell capture chip provided herein can be fabricated using existing fabrication processes.

[0118] In some embodiments, each position in the capture well region can be marked by combining marker wells with different opening shapes. Furthermore, a combination of N marker wells with different opening shapes can be used to represent N-ary marking data, and the N-ary marking data can be used to represent the row and column location of each position.

[0119] Figure 7This is a schematic diagram of the capture well region of a single-cell capture chip provided herein. The large hexagon represents the capture well, while the small circles, triangles, and squares are the marker wells. X1 and X2 designate row markers, while Y1 and Y2 designate column markers. Comparing the designations of X1 and X2, and Y1 and Y2, it can be seen that △ plus 1 equals ○, ○ plus 1 equals □, and □ plus 1 equals △. Thus, by transforming these three shapes, △, ○, and □, ternary data marking can be achieved. Five-digit markings can represent a total of 3^5 = 243 numbers. Row markers are set every 20 rows, and column markers are set every 10 rows. Thus, these marker wells can be evenly distributed across a capture well region with approximately (243 * 20) * (243 * 10) = 11,809,800 capture wells, thereby aligning the marker capture wells within the overall capture well region.

[0120] Although the present application has been described through embodiments, those skilled in the art will appreciate that there are many modifications and variations to the present application without departing from the spirit of the present application. It is intended that the appended claims include these modifications and variations without departing from the spirit of the present application.

Claims

1. A single cell capture system, characterized in that: include: A liquid inlet and outlet device comprises a cylinder, a piston, and a propulsion mechanism for controlling the reciprocating motion of the piston within the cylinder, wherein the cylinder is used to be detachably sealed and connected to the outlet end of the single-cell capture chip; wherein the inlet end of the single-cell capture chip is provided with a first liquid storage tank for containing cell fluid; a detection device for detecting a capture rate of cells; A control device is used to control the liquid inlet and outlet device according to the capture rate, and the control method includes: the control device controls the piston to move in a first direction so that the cell fluid in the first liquid storage tank flows to the outlet end through the capture trap area of ​​the single cell capture chip; then S1 and S2 are cyclically executed until the cell capture rate reaches a preset capture rate; wherein, S1: After standing for a predetermined period of time, the detection device detects the capture rate of cells; if the capture rate does not reach the preset capture rate, the control device controls the piston to move in a second direction opposite to the first direction to push the cell fluid in the capture trap area toward the inlet end; S2: After standing still for a predetermined period of time, the detection device detects the capture rate of the cells; if the capture rate does not reach the preset capture rate, the control device controls the piston to move toward the first direction again.

2. The single cell capture system according to claim 1, characterized in that The propulsion mechanism comprises: a motor, electrically connected to the control terminal of the control device, The mechanical transmission mechanism has one side in transmission connection with the motor shaft of the motor and the other side in transmission connection with the piston handle; the mechanical transmission mechanism converts the rotational motion of the motor into the linear motion of the piston in the cylinder.

3. The single cell capture system according to claim 1, wherein: The detection device comprises: a microscopic imaging device for imaging the capture trap area; The processor is used to perform cell recognition on the image formed by the microscopic imaging device, determine the number of captured cells based on the recognition result, and then calculate the cell capture rate.

4. The single cell capture system according to claim 1, wherein: Also includes: Interaction device, used to obtain user input application scenario; The control device automatically performs operations with different parameters according to different application scenarios input by the user.

5. A method for using a single cell capture system, characterized in that: A single cell capture system according to any one of claims 1 to 4, wherein the method comprises: Dropping the target cell solution into the first liquid storage tank of the single-cell detection chip; Setting operating parameters, the operating parameters including a preset capture rate; Control the single-cell capture system to start working; After the system reaches the preset capture rate and stops working, the single-cell capture chip is removed for single-cell sequencing.

6. A method for using a single cell capture system, characterized in that: A single cell capture system according to any one of claims 1 to 4, wherein the method comprises: Dropping the target cell solution into the first liquid storage tank of the single-cell detection chip; Setting operating parameters, the operating parameters including a preset capture rate; Control the single-cell capture system to start working; After the system reaches the preset capture rate and stops working, the piston is adjusted to the predetermined state; Aspirate the cell fluid in the first fluid reservoir; adding a dyeing solution into the first liquid storage tank; Control the piston to move in the direction where the cylinder becomes larger so that the staining solution can fully contact the cells; Follow-up studies were performed after staining was completed.

7. A method for using a single cell capture system, characterized in that: A single cell capture system according to any one of claims 1 to 4, wherein the method comprises: Dropping the target cell solution into the first liquid storage tank of the single-cell detection chip; Setting operating parameters, the operating parameters including a preset capture rate; Control the single-cell capture system to start working; After the system reaches the preset capture rate and stops working, The capture well area of ​​the single-cell capture chip is imaged using a microscopic imaging device.

8. The method according to claim 7, characterized in that The capture well region of the single cell capture chip includes a label well; imaging the capture well region of the single cell capture chip using a microscopic imaging device includes: A low-magnification microscopic imaging device is used to perform low-magnification imaging of the entire capture well area of ​​the single-cell capture chip; After finding the target cell from the low-magnification imaging results, the location of the target cell is marked according to the label well on the single-cell capture chip; A high-magnification microscope imaging device is used to perform high-magnification imaging of the target cells at the marked position.

9. A single cell capture chip, characterized in that: A single cell capture system according to any one of claims 1 to 4; the single cell capture chip having an inlet end and an outlet end, a first liquid storage tank for containing cell fluid being provided at the inlet end, a second liquid storage tank being provided at the outlet end, one end of the second liquid storage tank being connected to the outlet end of the single cell capture chip, and the other end being used for detachably sealed communication with the liquid inlet and outlet device; The capture well region includes capture wells that are arranged in an array and cover the entire capture well region.

10. The single cell capture chip according to claim 9, characterized in that: Marking wells are provided at multiple positions within the capture well region arranged in an array. The diameter of the marking well is smaller than the diameter of the cells to be captured. The marking wells are used to mark positions within the capture well region.

11. The single cell capture chip according to claim 9, characterized in that Each position of the label capture well region is varied by combining label wells with different opening shapes.

12. The single cell capture chip according to claim 11, characterized in that The combination of N mark wells with different opening shapes represents N-ary mark data, and the row and column positioning of each position is represented by the N-ary mark data.

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