Single cell / particle precise pairing method and device based on control probe

Abstract

The invention discloses a single cell / particle precise pairing method and device based on a control probe, and the method comprises the steps: determining the size of the inner diameter of the tip end of a to-be-controlled probe according to the sizes of different types of cells / particles to be paired; preparing a cell / particle suspension; respectively and sequentially sucking the single cells / particles into a control probe channel by using a liquid driving device; the tip of the control probe extends into the receiving container until the tip of the control probe abuts against the bottom of the receiving container; driving and controlling the cells / particles sucked in the probe to flow into the tip of the probe to be in contact with the bottom of the receiving container; the control probe is lifted away, and pairing operation of two or more single cells / particles at the bottom of the receiving container is completed. Precise capture and pairing of single cells / particles are directly realized by controlling space limitation provided by the tip of the probe. According to the invention, the limitation of Poisson distribution is overcome, and the accuracy and reliability of single cell / particle pairing are improved. The device is easy to construct, convenient to operate and good in reliability.

Description

Technical Field

[0001] The present invention belongs to the field of single-cell analysis, and particularly relates to a method and device for precise pairing of single cells / particles based on a manipulation probe. Background Art

[0002] Cell pairing at the single-cell level usually allows several cells to move in a single chamber and provides high-resolution imaging and monitoring, which is crucial for biological research, such as understanding basic cell functions, developing drugs, and cancer treatment technologies. The microfluidic technology has been widely used for capturing, fixing, and analyzing single cells due to its high efficiency, high throughput, and good biocompatibility, and is applied to cell fusion, cell immunity, cell co-culture, etc. In particular, the single-cell pairing method based on microfluidic technology can help achieve the spatio-temporal study of cell interactions and provide a highly controllable method for cell heterogeneity research.

[0003] Currently, a variety of single-cell pairing methods based on microfluidic technology have been developed. The most common cell pairing method is to capture paired single cells through spatial confinement, such as in droplets, micropores, and passive traps. Although the traditional droplet method has a high throughput, it is limited by the Poisson distribution, and the cell pairing efficiency is low. Therefore, a series of methods have been developed to further improve the pairing efficiency by means of other force fields (such as electricity, magnetism, light, and sound, etc.). However, the introduction of external forces will inevitably have some difficult-to-evaluate negative impacts on the activity of the captured cells. The microfluidic chip of the passive trap type can achieve a high pairing efficiency, but there are certain difficulties in the design, manufacture, and operation of the chip, and it is also inconvenient to take out specific cells from the tightly packaged chip for subsequent research. In contrast, the micropore chip has an open-top structure, allowing for more convenient integration of cell sedimentation, manipulation, recovery, and off-chip monitoring. However, the existing technologies generally can only achieve random pairing and cannot accurately pick single cells of interest for pairing. Summary of the Invention

[0004] The present invention provides a device for precise pairing of single cells, which relies on the spatial confinement provided by the tip of the manipulation probe to bring two cells into close contact, providing a pairing technology with high pairing efficiency and small cell damage for studying cell interactions at the single-cell level.

[0005] The present invention also provides a device for precise pairing of single particles, which relies on the manipulation probe to achieve the pairing between two microparticles, etc.

[0006] The present invention adopts a more proactive method, directly relying on the spatial confinement provided by the tip of the manipulation probe to pair single cells / particles, and develops a single-cell pairing device with a simple design, easy operation, and cell-friendly, realizing the precise and efficient capture and pairing of specified single cells.

[0007] The present invention provides a method for precisely pairing single cells / particles based on a manipulation probe, which is simple to operate and has high pairing efficiency and accuracy.

[0008] A method for precisely pairing single cells / particles based on a manipulation probe includes the following steps:

[0009] (1) Determine the inner diameter size of the required manipulation probe tip according to the sizes of different types of cells to be paired, and select the required manipulation probe;

[0010] (2) Prepare a cell / particle suspension of the cells / particles to be paired;

[0011] (3) Use a liquid driving device to sequentially aspirate two or more different single cells / particles into the manipulation probe channel;

[0012] (4) Insert the tip of the manipulation probe into the receiving container until its tip abuts against the bottom of the receiving container; ensure that there is a liquid flow gap between the tip of the manipulation probe and the receiving container;

[0013] (5) Drive the cells / particles aspirated in the manipulation probe to flow into the tip of the probe through the liquid driving device, contact the bottom of the receiving container, and at the same time the solution in the channel flows out through the liquid flow gap;

[0014] (6) Lift the manipulation probe to complete the pairing operation of two or more single cells / particles at the bottom of the receiving container.

[0015] The present invention can pair two or more different cells / particles. Preferably, the present invention can achieve the pairing of two different cells / particles.

[0016] Optionally, repeating steps (1) to (6) can complete a single cell / particle pairing array without mutual interference.

[0017] In step (2), the suspension of different cells / particles can be prepared separately, or the suspension of cells / particles can be prepared in the same system. For example, for the study of cell-cell interactions at the single cell level, suspensions of different types of cells are usually prepared separately to avoid some cell-cell interactions before cell pairing. If two different microspheres or other particle systems that do not interfere with subsequent experiments are to be paired, different particles can be placed in one suspension.

[0018] Preferably, when preparing the suspension, a concentration (cell / particle density) of about or lower than 1×10 6A cell / particle suspension of [[number of cells / particles]] cells / particles / mL can be adjusted for receiving containers with different bottom areas. In principle, the lower the cell concentration, the greater the distance between cells, and the easier it is to achieve single-cell sampling with a high success rate. Of course, when the cell concentration is high, single-cell sampling can be achieved when the cells can form a single layer on the bottom of the receiving container.

[0019] Preferably, in step (5), the liquid driving device is used to drive the cells / particles aspirated in the manipulation probe to flow into the tip of the probe, and at the same time, two or more cells / particles come into contact with each other. By adopting this technical solution, the pairing of two cells in close contact with each other can be achieved, that is, the direct interaction between cells can be studied. For example, in a research system where immune cells and tumor cells need to interact through the recognition of cell membrane surface receptors and ligands, this method has unique advantages.

[0020] As a preferred embodiment, the present invention also provides a method for precise pairing of single cells / particles, comprising the following steps:

[0021] (1) Perform corresponding probe processing. The inner wall of the probe can be subjected to various surface treatments that can reduce the adsorption or adhesion of cells / particles, or the tip of the probe can be processed with a small notch structure.

[0022] (2) Add an appropriate amount of cell / particle suspension to be paired subsequently into the receiving container.

[0023] (3) Use the liquid driving device to sequentially aspirate two different single cells / particles into the probe channel.

[0024] (4) Insert the tip of the manipulation probe into the receiving container until its tip touches the bottom of the receiving container.

[0025] (5) Use the liquid driving device to pump the liquid in the channel outwards from the tip of the probe. The aspirated cells / particles are carried by the fluid and flow into the tip of the probe, coming into contact with the bottom of the receiving container, while the solution in the channel flows out through the gap between the tip of the probe and the bottom of the receiving container.

[0026] (6) Control the flow rate and volume of the liquid ejected by the manipulation probe until the two cells / particles are sequentially ejected to stay at the tip of the probe.

[0027] (7) Lift the manipulation probe away from the receiving container to complete the pairing operation of two (or more) single cells / particles on the bottom of the receiving container.

[0028] Furthermore, in order to be able to observe the pairing situation of single cells / particles in real time so as to accurately judge the pairing process, in steps (3), (4), (5), and (6), the receiving device is placed on a microscope, and the image captured by the microscope is reflected onto the computer screen in real time by a camera.

[0029] Optionally, repeating steps (1) to (6) can complete a non-interfering single cell / particle pairing array.

[0030] As another embodiment, a receiving container with a porous membrane structure is used for precise pairing of single cells / particles, including the following steps:

[0031] (a) According to the sizes of different types of cells / particles to be paired, determine the inner diameter of the tip of the manipulation probe and perform corresponding probe processing. Various surface treatments that can reduce the adsorption or adhesion of cells / particles can be carried out on the probe tube wall, and a small notch structure can also be designed and processed on the tip of the probe;

[0032] (b) Add an appropriate amount of cell / particle suspension to be paired subsequently into the receiving container;

[0033] (c) Use a liquid driving device to sequentially aspirate two different single cells / particles into the probe channel;

[0034] (d) Insert the tip of the manipulation probe into the receiving container with a porous membrane structure until its tip abuts against the porous membrane, ensuring that there is at least one micropore within the range of the tip area;

[0035] (e) Pump out the liquid in the channel from the tip of the probe through the liquid driving device. The aspirated cells / particles are carried by the fluid and flow into contact with the porous membrane and are retained, while the solution in the channel flows out through the micropores;

[0036] (f) Control the flow rate and volume of the liquid ejected by the manipulation probe until the two cells / particles are sequentially ejected and stay at the tip of the probe;

[0037] (g) Lift the manipulation probe away from the porous membrane to complete the pairing operation of two (or more) single cells / particles on the porous membrane.

[0038] In step (d), preferably, the porous membrane can be a Transwell chamber with a filter membrane pore size of 3 - 8 μm.

[0039] In the aspiration step, it can also be considered to place a part of the bottom of the manipulation probe against the receiving container, which further avoids the interference of other cells / particles to the target cells / particles and realizes rapid and accurate aspiration of cells / particles.

[0040] Preferably, the manipulation probe is a capillary probe.

[0041] Preferably, the inner diameter of the tip of the manipulation probe should be larger than the sum of the diameters of the two single cells / particles or multiple cells / particles to be paired.

[0042] As a further preference, when performing single-cell / particle pairing of different cell / particle types, the inner diameter of the manipulation probe tip should be greater than the sum of the diameters of the cell / particle pair by 5 - 10 μm. That is, the inner diameter of the manipulation probe tip should exceed the sum of the diameters of two cells / particles or multiple cells / particles by approximately 5 - 10 μm.

[0043] Preferably, the liquid driving device consists of an injection pump and a syringe.

[0044] As a further preference, the liquid driving device consists of a precision injection pump for metering liquids with picoliter-level precision and a micro syringe with a capacity less than or equal to 10 μL.

[0045] Preferably, when performing cell pairing, a certain amount of culture medium can be added in advance to the receiving container for subsequent long-term co-culture of cell pairs and monitoring of cell behavior.

[0046] Furthermore, when performing single-cell / particle pairing using the method of the present invention, it is necessary to control the injection flow rate. An excessively large pairing flow rate is likely to squeeze and deform the cells / particles, and the cells / particles will fly out after being forcibly extruded through the gap between the probe tip and the bottom of the receiving device, which not only results in a low pairing success rate but also causes damage to the cells / particles. However, if the flow rate is too low, the fluid shear force is not sufficient to make the two cells / particles stick together, which will also reduce the pairing success rate. Preferably, the injection flow rate is generally in the range of 1 - 3 nL / s. Further, in step (5), the injection flow rate in the liquid driving device is generally controlled in the range of 1 - 3 nL / s.

[0047] Furthermore, when performing single-cell / particle pairing using the method of the present invention, it is necessary to control the aspiration volume of single cells / particles and the injection volume of cell / particle pairs, that is, to operate the cells / particles with the smallest possible aspiration and injection volumes to reduce the impact on cell viability or particle shape or performance. Preferably, the aspiration volume of single cells / particles is generally 3 - 5 nL, and the injection volume of cell / particle pairs is generally within 10 nL. Further, in step (3), the liquid driving device controls the aspiration volume of single cells / particles to be generally 3 - 5 nL; in step (5), the injection volume of cell / particle pairs is generally within 10 nL.

[0048] Furthermore, when pairing single cells using the method of the present invention, the two cells fall into and stay in the probe tip region in sequence under the drive of the fluid and present a state of sticking closely together, that is, the cell membranes of the two cells are in direct contact. After the pairing is completed, the cell pair will not be significantly damaged and will always maintain the sticking state.

[0049] The method of the present invention can also achieve the pairing of two (or more) cells / particles without direct contact. By touching the bottom with the tip of the probe to fix the position of the cells / particles, it has stronger positioning compared to other methods. After determining the distance between the two cells / particles through a microscope, cells / particles pairs with appropriate distances can be selected for the next experiment.

[0050] The device and operation method of the present invention can be applied not only to the capture and pairing experiments of single cells for studying the interaction between two cells, but also to the capture and pairing experiments of single cells and single particles for single cell sequencing or studying other single cell behaviors, and can also perform multi-cell capture combinations for studying the interaction between multi-cells, etc.

[0051] Preferably, the liquid flow gap is provided by the gap left between the tip of the manipulation probe and the receiving container, or the liquid flow gap is provided by the void (such as slit or pore) of the receiving container, or the liquid flow gap is provided by the notch provided at the tip of the manipulation probe, or the liquid flow gap is provided by the concave-convex structure provided at the bottom of the receiving container, or a combination of one or more of the above methods.

[0052] The present invention also provides a single cell / particle precise pairing device for implementing the single cell / particle precise pairing method described in any one of the above technical solutions, including:

[0053] A manipulation probe, the inner diameter of the tip of which matches the size of different types of cells / particles to be paired;

[0054] A liquid driving device for driving the manipulation probe to perform sequential aspiration and pairing operations on different single cells / particles;

[0055] A receiving container for containing the cell / particle suspension of the cells / particles to be paired or receiving the paired cells / particles pairs.

[0056] As a preferred solution, it may further include one or more combinations of the following components:

[0057] A fixed probe fixing device for the manipulation probe;

[0058] An elastic connecting pipeline for connecting the liquid driving device and the manipulation probe;

[0059] A moving stage for realizing the relative movement between the receiving container and the tip of the manipulation probe;

[0060] A microscope for displaying the state of cells / particles, single cell / particle aspiration and pairing operations.

[0061] Preferably, the liquid driving device may be composed of an injection pump and a syringe. More preferably, a precision injection pump capable of metering liquid with picoliter precision is used for the injection pump, and a micro syringe with a capacity less than or equal to 10 μL is used for the syringe.

[0062] The size of the inner diameter of the tip of the manipulation probe can be determined according to the specific capture object to achieve single-cell / particle pairing of different types of cells. In theory, the inner diameter of the probe tip should be greater than the sum of the diameters of the two single cells / particles to be paired. Preferably, the inner diameter of the probe tip should exceed the sum of the diameters of the two cells / particles by about 5-10 μm. More preferably, a self-made quartz capillary probe sharpened by a pulling machine and manually ground is used. Preferably, the capillary probe can be surface-treated, including but not limited to various surface treatments on the tube wall that can reduce cell / particle adsorption or adhesion and various other structural designs.

[0063] The structural mode of the probe fixing device is a freely movable type. Preferably, a limiting device can be used to keep the probe in a vertical state. This structural mode can avoid the wear of the probe tip to a certain extent. More importantly, this structural mode can form a certain gap between the probe tip and the bottom of the receiving container, providing an outflow channel for the liquid, and the size of this gap can be adjusted by the liquid flow, so as to achieve the effect of reducing the damage of liquid shear force to cell activity. As a specific solution, the probe fixing device includes a probe fixing table and a probe fixing member for fixing the manipulation probe on the probe fixing table.

[0064] The elastic connecting pipeline is used to connect the liquid driving device and the manipulation probe, and the thickness of the pipeline can be adjusted according to the respective diameters of the liquid driving device and the probe. It can be any flexible hose with elasticity to ensure the free movement of the probe.

[0065] The receiving container is mainly used to receive the initial cell / particle suspension and the cell / particle pair. For cell pairing, various biocompatible materials can be used for the receiving container, including but not limited to various culture dishes, well plates, Transwell chambers, etc. Preferably, a chip with a microstructure is more suitable for a single-cell system. The shape of the receiving container includes but not limited to micro-columns, micro-pits, micro-planes, etc. The bottom surface of the receiving container can be designed with various different structures, including but not limited to an uneven structure, but the unevenness does not exceed half of the cell diameter, or a porous membrane structure can be added. The micropores can have various shapes, such as triangular, trapezoidal, elliptical, polygonal or other shapes, but it is necessary to ensure that its maximum exclusion size is less than the single-cell diameter.

[0066] The moving stage refers to the moving stage used to control the relative position of the receiving container and the probe, and can achieve three-dimensional movement in the x, y, and z directions.

[0067] The main advantages of the present invention are:

[0068] Compared with the droplet method and the micro-well method, the present invention directly aspirates single cells / particles by manipulating a probe, and directly realizes single-cell / particle capture and pairing relying on the spatial confinement at the tip, greatly overcoming the limitation of Poisson distribution, effectively improving the accuracy and reliability of single-cell / particle pairing, and without the need for a large number of cell / particle samples, being more suitable for the research of rare cells or other particles. At the same time, compared with the method of improving the pairing efficiency of the droplet method by means of external forces (light, sound, electricity, etc.), the present invention causes relatively less damage to cell viability.

[0069] Compared with the passive trap microfluidic chip method, the present invention does not require the design of a complex microfluidic chip, has simpler equipment requirements, is easier to construct the device, and is more convenient to operate. Moreover, it effectively solves the problem that it is difficult to carry out subsequent further operations on cell / particle pairs in a closed chip, and can realize cell / particle pairing, monitoring and subsequent processing in an open environment, improving the flexibility and accessibility of cell / particle pair analysis.

[0070] Compared with the existing methods, the present method can perform pairing under the condition of determining the respective activity states of single cells, effectively avoiding the influence of abnormal cell states on the monitoring results of cell pair behaviors.

[0071] Based on the above advantages, the present invention has broad application prospects in the fields of single-cell analysis, single-cell interaction research, etc.

[0072] In summary, the single-cell / particle precise pairing device based on a manipulated probe and its application of the present invention directly realize the precise capture and pairing of single cells / particles relying on the spatial confinement provided by the tip of the manipulated probe. The main advantages of the present invention include: the present invention greatly overcomes the limitation of Poisson distribution, effectively improves the accuracy and reliability of single-cell / particle pairing, and without the need for a large number of cell / particle samples, being more suitable for the research of rare cells, and causing relatively less damage to cell viability; the present invention can realize precise picking of single cells of interest for research; the present invention effectively solves the problem that it is difficult to carry out subsequent further operations on cell pairs in a closed chip, and can realize pairing, monitoring and subsequent processing in an open environment, improving the flexibility and accessibility of cell pair analysis; the device of the present invention is easy to construct, convenient to operate, and has good reliability. Therefore, the present invention has broad application prospects in the fields of single-cell analysis, single-cell interaction research, etc. Description of the Drawings

[0073] Figure 1 It is a schematic diagram of the single-cell precise pairing device based on a manipulated probe used in Example 1.

[0074] In the figure, 1 is a liquid driving device, 2 is an elastic connecting pipeline, 3 is a probe fixing table, 4 is a probe fixing part, 5 is a manipulation probe, 6 is a receiving container, 7 is a culture medium, and 8 is a moving table.

[0075] Figure 2 is a specific flowchart for pairing two single cells using the device of Example 1. In the figure, 9 is single cell A and 10 is single cell B.

[0076] Figure 3 is a schematic diagram for pairing two single cells using the device of Example 1.

[0077] Figure 4 is an effect diagram for pairing two single cells using the device of Example 1.

[0078] Figure 5 is a schematic diagram for pairing two single cells using a receiving container with a porous membrane structure. 11 is a porous membrane.

[0079] Figure 6 is a schematic diagram for pairing two single cells using a receiving container with a concave-convex surface structure.

[0080] Figure 7 is a schematic diagram for pairing two single cells using a probe with a small opening tip structure. Specific Embodiments

[0081] The technical solutions of the present invention will be further described below in conjunction with specific embodiments, but the protection scope of the present invention is not limited thereto.

[0082] Referring to the accompanying drawings, the preferred embodiments of the present invention will be described in detail below. The present invention is described by taking single cell pairing as an example. For the pairing of other cell-level particles, etc., the cell pairing method can be referred to.

[0083] See Figure 1 , which is a structural schematic diagram of a single cell precise pairing device based on a manipulation probe according to the present invention, including a liquid driving device 1, an elastic connecting pipeline 2, a probe fixing table 3, a probe fixing part 4, a manipulation probe 5, a receiving container 6 for containing a culture medium 7, and a moving table 8.

[0084] In this embodiment, the liquid driving device 1 is composed of a precision injection pump (measured with picoliter accuracy) and a micro-injection syringe (capacity is less than or equal to 10μL); the elastic connecting pipeline 2 can be any elastic hose; the probe fixing platform 3 can be a component of other devices or a separate fixed structure, mainly used to achieve relative fixation of the control probe 5, and the structure is not strictly limited; the probe fixing part 4 can adopt a common fixing frame, fixing clamp, or other elastic fixing parts, etc., mainly to achieve the positioning and installation of the control probe 5 on the probe fixing platform 3; the control probe 5 adopts a capillary probe, for example, a homemade quartz capillary probe that is pulled by a drawing machine and manually ground can be used; the receiving container 6 can use various biocompatible materials, including but not limited to various types of culture dishes, well plates, Transwell chambers, etc.; the moving platform 8 can select a common two-dimensional or three-dimensional motion platform.

[0085] During actual installation, the micro-injector of the liquid driving device 1 is sealedly connected to the tail end of the control probe 5 through the elastic connecting pipe 2, and the control probe 5 is fixed on the probe fixing platform 3 by the probe fixing member 4. The receiving container 6 containing the culture medium 7 is arranged on the moving platform 8, and the position of the receiving container 6 is controlled by the moving platform 8, thereby changing the position of the control probe 5 relative to the receiving container 6.

[0086] Example 1

[0087] See also Figure 1 , Figure 2 , Figure 3 and Figure 4 The device is used to pair heterotypic single cell A (K562 cell) 9 with single cell B (NK95MI cell) 10 in a conventional 384-well plate. The specific process is as follows: (1) The cell diameters of K562 cells and NK95MI cells are measured respectively, and the average diameter of the NK92MI-K562 cell pair is about 30 μm; (2) The capillary probe 5 (i.e., the manipulation probe 5) is made of a quartz capillary and then ground, and the inner diameter of the tip is about 35 μm; (3) Appropriate amounts of K562 cells / NK95MI cells are added to two 384-well plates respectively. suspension and corresponding culture medium 7, etc.; (4) a liquid driving device 1, i.e. a precision injection pump controls a micro-injector, connected to a capillary probe 5, to aspirate single cell A9 and single cell B10 at a flow rate of 1 nL / s, respectively, with an aspirated volume of 4 nL each; (5) the capillary probe is extended into another 384-well plate, i.e. the bottom of the receiving container 6, so that the tip is against the bottom of the 384-well plate, while maintaining a gap for liquid flow between the tip and the well plate, and 10 nL of liquid is ejected at a flow rate of 1 nL / s; (6) the capillary probe 5 is lifted off the 384-well plate 6 to complete the single-cell pairing. Repeating steps (1) to (6) can complete a single-cell pairing array without crosstalk, such as Figure 4 shown.

[0088] In this embodiment, the micro - liquid driving system is fixed on an automatic three - dimensional translation stage, enabling the capillary probe 5 to accurately position and move among different positions of the 384 - well plate 6.

[0089] Embodiment 2

[0090] As an explanation of Embodiment 2 of the present invention, only the differences from Embodiment 1 above will be described below.

[0091] As Figure 5 shown, the receiving container in this embodiment is a Transwell chamber, and single - cell pairing is carried out with the help of a porous membrane 11 having a micropore diameter of 8.0 μm. Similar to Embodiment 1, the precision syringe pump controls the micro - syringe 1. After connecting the capillary probe 5 to respectively aspirate 4 nL of two different single - cells A9 and single - cell B10 at a flow rate of 1 nL / s, the capillary probe is inserted into the Transwell chamber, and the tip is pressed against the porous membrane 10 to ensure that there is at least one micropore in the tip region. Then, 10 nL of liquid is ejected at a flow rate of 1 nL / s, and the liquid flows out through the small holes while the cell pairs are retained on the porous membrane 11. The capillary probe 5 is lifted away from the Transwell chamber to complete single - cell pairing.

[0092] Embodiment 3

[0093] As an explanation of Embodiment 3 of the present invention, only the differences from Embodiment 1 above will be described below.

[0094] As Figure 6 shown, the bottom of the receiving container in this embodiment has a concavo - convex structure. Similar to Embodiment 1, the precision syringe pump controls the micro - syringe 1. After connecting the capillary probe 5 to respectively aspirate 4 nL of two different single - cells A9 and single - cell B10 at a flow rate of 1 nL / s, the capillary probe is inserted into the receiving container, and the tip is pressed against the concavo - convex structure at the bottom of the receiving container. Then, 10 nL of liquid is ejected at a flow rate of 1 nL / s, and the liquid flows out through the gap formed between the probe tip and the concavo - convex structure while the cell pairs are retained at the bottom. The capillary probe 5 is lifted away from the receiving container to complete single - cell pairing.

[0095] Embodiment 4

[0096] As an explanation of Embodiment 4 of the present invention, only the differences from Embodiment 1 above will be described below.

[0097] As Figure 7As shown, the tip of the capillary probe in this embodiment has a special structural design, and a small opening is processed at the tip. This structure can form a gap with a width of about 3 μm between the tip of the capillary probe and the receiving container after the tip touches the bottom, forming a liquid flow gap. Similar to Embodiment 1, the precision syringe pump controls the micro syringe 1. After connecting the capillary probe 5 to aspirate 4 nL of two different single cells A9 and single cell B10 at a flow rate of 1 nL / s respectively, the capillary probe is inserted into the receiving container, the tip is pressed against the bottom of the receiving container, and then 10 nL of liquid is ejected at a flow rate of 1 nL / s. The liquid flows out through the gap formed between the small opening at the tip of the probe and the bottom of the receiving container, while the cell pair is retained at the bottom. The capillary probe 5 is lifted away from the receiving container to complete single cell pairing.

Claims

1. A method for precise pairing of single cells / particles based on a manipulation probe, characterized in that, It includes the following steps: (1) Determine the inner diameter size of the required manipulation probe tip according to the sizes of different types of cells / particles to be paired, and select the required manipulation probe; (2) Prepare a cell / particle suspension of the cells / particles to be paired; (3) Use a liquid driving device to sequentially aspirate two or more different single cells / particles into the manipulation probe channel respectively; (4) Insert the manipulation probe tip into the receiving container until its tip abuts against the bottom of the receiving container; Ensure that there is a liquid flow gap between the manipulation probe tip and the receiving container; (5) Drive the cells / particles aspirated in the manipulation probe to flow into the probe tip through the liquid driving device and contact the bottom of the receiving container; (6) Lift the manipulation probe to complete the pairing operation of two or more single cells / particles at the bottom of the receiving container.

2. The method for precise pairing of single cells / particles based on a manipulation probe according to claim 1, wherein When pairing cells, in step (5), multiple cells contact the bottom of the receiving container and contact each other at the same time.

3. The single cell / particle precise pairing method based on a manipulation probe according to claim 1, wherein The manipulation probe is a capillary probe; the inner diameter of the manipulation probe tip should be larger than the sum of the diameters of the two single cells / particles or multiple cells / particles to be paired; the liquid driving device is composed of an injection pump and a syringe.

4. The method for precise pairing of single cells / particles based on a manipulation probe according to claim 3, wherein The inner diameter of the manipulation probe tip should exceed the sum of the diameters of two cells / particles or multiple cells / particles by about 5 - 10 μm; the liquid driving device is composed of a precision injection pump for metering liquids with picoliter precision and a micro syringe with a capacity less than or equal to 10 μL.

5. The method for precise pairing of single cells / particles based on a manipulation probe according to claim 1, wherein In step (5), the injection flow rate is typically controlled within the range of 1 - 3 nL / s in the liquid driving device; in step (2), the concentration of the cell / particle suspension is about or lower than 1×10 6 cells / particles / mL of the cell / particle suspension.

6. The method for precise pairing of single cells / particles based on a manipulation probe according to claim 1, characterized in that In step (3), the liquid driving device controls the aspiration volume of single cells / particles to be usually 3 - 5 nL; in step (5), the injection volume of the cell / particle pair is usually within 10 nL.

7. The method for precise pairing of single cells / particles based on a manipulation probe according to claim 1, wherein The liquid flow gap is provided by the gap left between the manipulation probe tip and the receiving container, or the liquid flow gap is provided by the voids provided on the receiving container, or the liquid flow gap is provided by the notch provided on the manipulation probe tip, or the liquid flow gap is provided by the concave-convex structure provided on the bottom of the receiving container, or the liquid flow gap is provided by the receiving container with a porous membrane structure at the bottom, or a combination of one or more of the above methods.

8. The method for precise pairing of single cells / particles based on a manipulation probe according to any one of claims 1 to 7, characterized in that Step (3) is replaced with: Use a liquid driving device to sequentially aspirate two or more different single cells / particles into their respective manipulation probe channels respectively.

9. A single-cell / particle precise pairing device based on a manipulation probe for implementing the precise pairing method according to any one of claims 1 to 7, characterized in that, It includes: A manipulation probe, the inner diameter size of its needle tip matches the sizes of different types of cells / particles to be paired; A liquid driving device, used to drive the manipulation probe to perform sequential aspiration and pairing operations of different single cells / particles; A receiving container, used to hold the cell / particle suspension of the cells / particles to be paired or receive the paired cell / particle pairs.

10. The single-cell / particle precise pairing device based on a manipulation probe according to claim 9, wherein It includes one or more combinations of the following components: A fixed probe fixing device for the manipulation probe; An elastic connecting pipeline for connecting the liquid driving device and the manipulation probe; A moving stage for realizing the relative movement between the receiving container and the manipulation probe tip; A microscope for displaying the states of cells / particles, single cell / particle aspiration and pairing operations.

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