Method for injecting exogenous substances into cells based on a single-cell manipulation device

Abstract

The present application discloses a method for injecting exogenous substances into cells based on a single-cell manipulation device. The single-cell manipulation device includes a stage carrying a sample, a first probe, a first electrode disposed on the first probe, and a second electrode electrically connected to the sample. The first probe is an elongated hollow structure and includes a front end and a rear end, and the front end of the first probe acts on the cell membrane of a target cell in the sample. The method for injecting exogenous substances into cells includes: step S100, establishing a first electric field between the first electrode and the second electrode, and detecting changes in the first electric field signal during the movement of the first probe to determine that the front end of the first probe contacts the target cell; step S200, moving the front end of the first probe relative to the sample to pierce the cell membrane of the target cell; step S300, establishing a second electric field between the first electrode and the second electrode, and injecting the exogenous substance into the target cell under the action of the second electric field. The technical solution in the present application has advantages such as high precision, low damage, high throughput, and multi-functionality.

Description

Technical Field

[0001] This application relates to the field of cell manipulation technologies, and particularly to a method for injecting exogenous substances into cells based on a single-cell manipulation device. Background Art

[0002] Cell manipulation technologies play a crucial role in life science research and biomedical applications. Traditional cell manipulation methods, such as microinjection and electroporation, although effective, have limitations such as complex operation, large cell damage, and low throughput. In recent years, probe-based cell manipulation technologies have developed rapidly, providing new solutions for precise cell manipulation.

[0003] In the process of performing intracellular injection and extraction of cytoplasmic components based on a probe, it generally refers to touching the cell membrane of a target cell with the tip of a probe at the nanoscale, and performing targeted membrane rupture, introduction of target molecules, or extraction of cytoplasmic components on the target cell through mechanical force or pressure change. When the target cell is a free cell, a probe at the micron scale is also required to achieve the positioning of the target cell through mechanical force, pressure change, electromagnetic field, etc.

[0004] In actual operations, the introduction of target molecules or extraction of cytoplasmic components through pressure change places high requirements on the control accuracy of air pressure. The pressure control accuracy in the prior art often causes cell damage, and there is room for improvement. Summary of the Invention

[0005] To solve the above technical problems, this application discloses a method for injecting exogenous substances into cells based on a single-cell manipulation device, which has advantages such as high precision, low damage, high throughput, and multi-functionality, and shows great application potential in the fields of life science research and biomedical applications.

[0006] A method for injecting exogenous substances into cells based on a single-cell manipulation device is disclosed in an embodiment of this application.

[0007] The single-cell manipulation device includes a stage carrying a sample, a first probe for performing cell manipulation, a first electrode disposed on the first probe, and a second electrode electrically connected to the sample. The first probe is a slender hollow structure and includes opposite front and rear ends, and the front end of the first probe acts on the cell membrane of the target cell in the sample.

[0008] The method for injecting exogenous substances into cells includes:

[0009] Step S100, establishing a first electric field between the first electrode and the second electrode, and detecting the change of the first electric field signal during the movement of the first probe to determine the contact between the front end of the first probe and the cell membrane of the target cell.

[0010] Step S200, the front end of the first probe moves relative to the sample to pierce the cell membrane of the target cell;

[0011] Step S300, a second electric field is established between the first electrode and the second electrode, and the exogenous substance is injected into the target cell under the action of the second electric field.

[0012] The following also provides several optional methods, which are not additional limitations to the above overall solution, but only further supplements or optimizations. Without technical or logical contradictions, each optional method can be combined with the above overall solution alone, or multiple optional methods can be combined with each other.

[0013] In one embodiment, in step S100, the voltage value of the first electric field is 0.1V to 5V, and the first electric field signal is the ion current between the first electrode and the second electrode.

[0014] In one embodiment, in step S100, when establishing the first electric field, the value of the ion current is recorded as the reference value;

[0015] During the process of the first probe approaching the cell membrane of the target cell, when the ion current becomes smaller and the change amplitude compared with the reference value is greater than 10% and less than or equal to 30%, it is determined that the front end of the first probe contacts the cell membrane of the target cell.

[0016] In one embodiment, in step S300, the second electric field is a voltage pulse with a voltage value of 1V to 10V and a total duration of 0.1 second to 5 seconds, and the exogenous substance enters the cell membrane of the target cell under the action of the voltage pulse.

[0017] In one embodiment, in step S100, the front end of the first probe is positioned above the target cell and the first electric field is established. The first probe moves downward and gradually approaches the target cell, and during this process, it is determined whether the front end of the first probe contacts the cell membrane of the target cell.

[0018] In one embodiment, the single-cell manipulation device further includes a first probe assembly, and the first probe assembly includes the first probe and a first gripper for positioning the first probe; a conductive socket is fixed to the rear end of the first probe, a conductive part is provided on the first gripper, and a wire extending outside the first gripper is provided on the conductive part;

[0019] When the first probe and the first gripper cooperate with each other, the socket and the conductive part are plugged into each other.

[0020] In one embodiment, the first electrode includes a tail connected to the socket and a head near the front end of the first probe. A gap is provided between the head and the front end of the first probe to form a conduction cavity. The foreign substance is carried by an injection liquid, and the injection liquid fills the conduction cavity to conduct the first electrode and the sample.

[0021] In one embodiment, the first electrode passes through the first probe. The first electrode is strip-shaped with a maximum outer diameter ranging from 0.05 mm to 0.5 mm.

[0022] In the extending direction of the first probe, the ratio range of the length L1 of the first electrode to the length L2 of the first probe is from 0.3 to 0.9.

[0023] The material of the first electrode is one or more of platinum, gold, silver, silver chloride, carbon-based materials, silicon, indium tin oxide, and conductive polymers.

[0024] In one embodiment, the single-cell manipulation device further includes a second probe assembly and a gas pressure source assembly that provides positive or negative gas pressure for the second probe assembly. The second probe assembly includes a second probe and a second gripper.

[0025] The method for injecting cell foreign substances further includes:

[0026] Locating the target cell through the pressure change in the second probe.

[0027] In one embodiment, the single-cell manipulation device further includes a control component. The control component includes a moving platform, and the first probe assembly and the second probe assembly are fixedly connected to the moving platform; or

[0028] At least one of the first probe assembly and the second probe assembly is movably connected to the moving platform, and the two move synchronously or independently.

[0029] This application uses an electrode to realize the contact judgment between the probe and the target cell, and cooperates with voltage pulses to achieve intracellular injection. It can precisely control the processes of cell membrane rupture and cell injection, and has advantages such as high precision, low damage, high throughput, and multi-functionality.

[0030] Specific beneficial technical effects will be further explained in combination with specific structures or steps in the specific implementation manner. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] Figure 1 Schematic diagram of the first probe approaching the target cell in an embodiment of this application;

[0032] Figure 2Schematic diagram of the contact between the first probe and the cell membrane of the target cell in an embodiment;

[0033] Figure 3 Schematic diagram of the first probe piercing the cell membrane of the target cell and injecting in an embodiment;

[0034] Figure 4 Schematic flow diagram of the method for injecting cell exogenous substances in an embodiment of the present application;

[0035] Figure 5 Schematic diagram of the structure of the first probe assembly in an embodiment of the present application;

[0036] Figure 6 For Figure 5 Cooperation schematic diagram of the first probe assembly in a sectional view;

[0037] Figure 7 For Figure 5 Cooperation structure schematic diagram of the first probe and the first electrode of the first probe assembly in;

[0038] Figure 8 Cooperation schematic diagram of the first probe and the second probe in an embodiment of the present application;

[0039] Figure 9 Internal cooperation schematic diagram of the second probe assembly in an embodiment of the present application;

[0040] Figure 10 Schematic diagram of the air pressure source assembly in an embodiment of the present application;

[0041] Figure 11 For Figure 10 Working schematic diagram of the air pressure source assembly in;

[0042] Figure 12 Schematic diagram of the system framework of the cell operation device in an embodiment of the present application;

[0043] Figure 13 Schematic diagram of the process of injecting cell exogenous substances in an embodiment of the present application;

[0044] Figure 14 Schematic diagram of the process of injecting cell exogenous substances in another embodiment of the present application.

[0045] The descriptions of the reference numerals in the figure are as follows:

[0046] 10. Stage; 11. Container; 12. Sample; 21. Optical microscope objective; 22. Image detection unit; 23. Light source; 31. First probe assembly; 32. Second probe assembly; 41. First electrode; 42. Field source assembly; 43. Measurement assembly; 50. Moving platform; 60. Pneumatic source assembly; 61. Plunger pump; 61a. Inlet end; 61b. Outlet end; 61c. Plunger; 62. Solenoid valve; 62a. Common end; 62b. Normally open end; 62c. Normally closed end; 63. Three-way joint; 64. First one-way valve; 65. Second one-way valve; 66. Air filter; 67. Silencer; 68. Plug; 70. Control system;

[0047] 101. First probe; 102. First holder; 103. Socket; 104. Conductive part; 105. Wire; 106. Conducting cavity; 107. Injection liquid;

[0048] 201. Second probe; 202. Second holder; 211. Head; 212. Body; 2122. Limiting part; 2123. Pressure channel; 2124. First cylinder; 2125. Second cylinder; 2126. Third cylinder; 2127. Clamping end; 2128. Pressure end. Detailed implementation manners

[0049] Next, the technical solutions in the embodiments of the present application will be clearly and completely described in conjunction with the accompanying drawings in the embodiments of the present application. Obviously, the described embodiments are only a part of the embodiments of the present application, rather than all the embodiments. Based on the embodiments in the present application, all other embodiments obtained by those of ordinary skill in the art without creative efforts shall fall within the protection scope of the present application.

[0050] It should be noted that when a component is referred to as being "connected" to another component, it can be directly connected to the other component or there may also be an intermediate component. When a component is considered to be "disposed on" another component, it can be directly disposed on the other component or there may be an intermediate component at the same time.

[0051] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the technical field to which this application belongs. The terms used in the specification of this application herein are only for the purpose of describing specific embodiments and are not intended to limit this application. The term "and / or" used herein includes any and all combinations of one or more of the related listed items.

[0052] Refer to Att Figure 1 to Att Figure 4As shown, the present application discloses a method for injecting exogenous substances into cells based on a single-cell manipulation device. The single-cell manipulation device includes a container 11, a stage 10, a probe assembly, a control assembly, a microscopic imaging assembly, a field source assembly 42, and a measurement assembly 43. A sample 12 is carried in the container 11, and the container 11 is positioned on the stage 10. The probe assembly moves relative to the container 11 under the drive of the control assembly. During the movement, the microscopic imaging assembly provides a window for positioning the first probe 101. The microscopic imaging assembly can directly image through transmitted light during imaging by bright field to provide bright field features, or provide a fluorescence signal based on fluorescence. Attached Figure 1 to Attached Figure 5 The probe assembly in FIGS. to includes a first probe assembly. The first probe assembly includes a first probe 101 and a first gripper 102. The first probe 101 is detachably mounted on the first gripper 102. The first probe 101 is an elongated hollow structure with a front end and a rear end, and a first electrode 41 passes through the inside. When in use, the first probe 101 is filled with exogenous substances or an injection solution 107 carrying exogenous substances. The front end of the first probe 101 extends into the container 11, and the front end of the first probe 101 acts on the cell membrane of the target cell. The first electrode 41 is electrically connected to the sample 12 through the exogenous substances and / or the injection solution 107. The field source assembly 42 includes a working electrode electrically connected to the first electrode 41 and a base electrode electrically connected to a second electrode. The working electrode and the base electrode form a circuit in the sample through the first electrode 41 and the second electrode and transmit energy.

[0053] The method for injecting exogenous substances into cells includes:

[0054] Step S100: Establish a first electric field between the first electrode 41 and the second electrode. During the movement of the first probe 101, detect the change of the first electric field signal to determine that the front end of the first probe 101 contacts the cell membrane of the target cell in the sample 12;

[0055] Step S200: Move the front end of the first probe 101 relative to the sample 12 to pierce the cell membrane of the target cell;

[0056] Step S300: Establish a second electric field between the first electrode 41 and the second electrode, and inject the exogenous substances into the target cell under the action of the second electric field.

[0057] Among them, step S100 specifically further includes:

[0058] Based on the bright field features or fluorescence signals provided by the microscopic imaging assembly and through the control assembly, position the front end of the first probe 101 near the target cell (for example, as shown in Attached Figure 1 shown);

[0059] Apply an electric field between the working electrode and the base electrode through the field source assembly 42, measure the electric field signal between the working electrode and the base electrode through the measurement assembly 43 and record it as a reference value (for example, as shown in AttachedFigure 1 as shown);

[0060] The front end of the first probe 101 is gradually moved closer to the cell membrane of the target cell by the control component. The electric field signal between the working electrode and the base electrode is measured in real time by the measuring component 43 and compared with the reference value. When the change of the electric field signal meets the preset condition, it is determined that the front end of the first probe 101 contacts the cell membrane of the target cell, and the movement of the first probe 101 is stopped and this position is recorded as the reference position (for example, attached Figure 2 as shown).

[0061] Step S200 specifically further includes: moving the first probe 101 by a preset distance in a preset direction relative to the reference position by the control component to pierce the cell membrane of the target cell with the front end of the first probe 101 (for example, attached Figure 3 as shown).

[0062] Step S300 specifically further includes: applying a voltage pulse with preset conditions between the working electrode and the base electrode by the field source component 42, and the injection liquid 107 enters the cell membrane of the target cell under the action of the voltage pulse; (for example, attached Figure 3 as shown)

[0063] The control component retracts the first probe 101 to the reference position.

[0064] The first electric field signal can be in various forms. For example, a constant current condition is formed between the working electrode and the base electrode by a constant current source, and the voltage value between the working electrode and the base electrode is used as the first electric field signal. It can also be referred to as attached Figure 1 as shown. In step S100, a voltage with a preset value is applied between the working electrode and the base electrode by the field source component 42. The first electric field signal measured by the measuring component 43 is the ion current between the working electrode and the base electrode, and the value of the ion current is recorded as the reference value. That is, when establishing the first electric field, the value of the ion current is recorded as the reference value. In this embodiment, the measuring component 43 is an ammeter. In one embodiment, the field source component 42 applies a voltage of 0.1V to 5V between the working electrode and the base electrode to form the first electric field. Further, this voltage is a DC voltage, where the working electrode is the positive electrode or the negative electrode, and the corresponding base electrode is the negative electrode or the positive electrode. In other embodiments, this voltage can be various voltage values with a step of 0.5V between 0.1V and 4V, or 0.5V and 3V, or 0.5V and 1.5V, or 0.5V and 5V. In this embodiment, the range of the reference value is 10 nA (nanoampere) to 10 μA (microampere). Further, the range of the reference value is 20 nA to 950 nA.

[0065] Refer to attached Figure 2In the illustrated embodiment, in step S100, when determining whether the front end of the first probe 101 contacts the cell membrane of the target cell, the first electric field signal measured by the measurement component 43 is the ion current between the working electrode and the base electrode, and the value of the ion current is compared with a reference value. When the real-time ion current becomes smaller and the change range compared with the reference value is greater than 10% and less than or equal to 30%, it is determined that the front end of the first probe 101 contacts the cell membrane of the target cell. Further, when the change range of the real-time ion current compared with the reference value is greater than 10% and less than or equal to 20%, it is determined that the front end of the first probe 101 contacts the cell membrane of the target cell. In other embodiments, it can also be determined by the absolute value of the change in the real-time ion current. For example, when the change range of the real-time ion current is greater than or equal to 20 nA and less than or equal to 300 nA, it is determined that the front end of the first probe 101 contacts the cell membrane of the target cell. In the appendix Figure 2 When the front end of the first probe 101 is positioned near the target cell, it is positioned above the target cell and the reference value is measured. That is, in step S100, the front end of the first probe is positioned above the target cell and the first electric field is established. The first probe 101 moves downward and gradually approaches the target cell, and during this process, it is determined whether the front end of the first probe 101 contacts the cell membrane of the target cell.

[0066] In addition to being used to determine the relative position between the first probe 101 and the target cell, the first electrode 41 in the present application can also be used to achieve electric field injection. Refer to the appendix Figure 3 In the illustrated embodiment, when injecting the exogenous substance and / or the injection liquid 107, a second electric field is applied between the working electrode and the base electrode by the field source component 42, and the exogenous substance and / or the injection liquid 107 enter the cell membrane of the target cell under the action of the second electric field. Specifically, the second electric field is a voltage pulse, and the voltage value of the voltage pulse is 1 V to 10 V, and the total duration of the voltage pulse is 0.1 second to 5 seconds. The voltage values of the pulses of the voltage pulse can be the same or independently set, and the durations of the pulses can be the same or independently set. The waveforms of the pulses can be the same or independently set, such as square wave pulses, sawtooth wave pulses, or sine wave pulses. The overall pulse duty cycle of the voltage pulse is 20% to 95% or 30% to 90% or 40% to 85%.

[0067] Regarding the cooperation details between the first probe 101 and the first electrode 41, refer to the appendix Figure 5 to the appendix Figure 7In the illustrated embodiment, the body of the first probe 101 is an elongated hollow structure, made of quartz, glass, etc. Specifically, it can be formed by laser heating and drawing of a quartz capillary tube and has a geometrically rotationally symmetric structure. The interior of the body of the first probe 101 is hollow without movable parts or embedded circuits. The first probe 101 has opposite front and rear ends, both of which are open structures. The aperture of the front end is relatively reduced compared to the rear end, and it has a flat micro-operation end face. The size of the rear end is adapted to the first gripper 102. Further, a conductive socket 103 is fixed to the rear end of the first probe 101, and a conductive part 104 is provided on the first gripper 102. A wire 105 extending to the outside of the first gripper 102 and connected to the working electrode is provided on the conductive part 104;

[0068] When the first probe 101 and the first gripper 102 cooperate with each other, the socket 103 and the conductive part 104 are inserted into each other to achieve electrical connection between the first electrode 41 and the working electrode. The separable connection between the socket 103 and the conductive part 104 can facilitate the disassembly and installation of the first probe 101 and synchronously achieve the disconnection and conduction between the first electrode 41 and the working electrode during the disassembly and installation process. In Figure 6 it, the socket 103 includes a wrapping part positioned at the rear end of the first probe 101 and a plugging shaft extending from the wrapping part away from the front end of the first probe 101. The conductive part 104 is provided with a plugging cavity for receiving the plugging shaft. The plugging cavity can wrap the circumferential surface of the plugging shaft while receiving the plugging shaft to achieve stable electrical connection. In other embodiments, the structures of the socket 103 and the conductive part 104 can also be interchanged.

[0069] The first electrode 41 can extend out of the front end of the first probe 101 to be electrically connected to the sample 12 or to the environment where the sample 12 is located (such as the Figure 1 solution environment shown in the attachment), or it can also refer to the attachment Figure 7 In the illustrated embodiment, the first electrode 41 includes a tail connected to the socket 103 and a head 211 near the front end of the first probe 101. A gap is provided between the head 211 and the front end of the first probe 101 to form a conduction cavity 106. The foreign matter is carried on the injection liquid 107, and the injection liquid 107 fills the conduction cavity 106 and conducts the first electrode 41 and the sample 12. The injection liquid 107 itself is a conductive medium. This setting can avoid the first electrode 41 needing to penetrate the relatively small opening at the front end of the probe, thereby ensuring the size of the first electrode 41 and reducing the production difficulty. In terms of the details of the first electrode 41, in the attachment Figure 7In the illustrated embodiment, in the extending direction of the first probe 101, the ratio range between the length L1 of the first electrode 41 and the length L2 of the first probe 101 is from 0.3 to 0.9. Further, the first electrode 41 is strip-shaped and has a maximum outer diameter of 0.05 mm to 0.5 mm. The material of the first electrode 41 is one or more of platinum, gold, silver, silver chloride, carbon-based materials, silicon, indium tin oxide, and conductive polymers. Similarly, in Figure 1 attachment Figure 3 the base electrode realizes electrical connection with the sample 12 through a second electrode disposed in the container 11. The second electrode can be arranged in the same way as the first electrode 41 described above, or can be arranged differently. For example, the second electrode is plate-shaped or a part of the container 11 or the whole container 11 serves as the second electrode.

[0070] The cell exogenous substances themselves carry charges or obtain charges through the electric field formed by the electrodes, so as to move under the drive of the second electric field. The cell exogenous substances can be dyes, proteins, mRNAs, plasmids and other commonly injected substances in the field of cell manipulation. The exogenous substances can be a single substance or a combination of multiple substances.

[0071] Exemplarily, attachment Figure 13 and attachment Figure 14 show the injection processes of different exogenous substances. In attachment Figure 13 the exogenous substance injected in the embodiment is a dye (brand: Alexa 647), and in attachment Figure 14 the exogenous substance injected in the embodiment is a protein (such as α-synuclein) labeled with a dye (brand: Alexa 594). Figure 13 Both attachment Figure 14 and attachment

[0072] contain five stages, where:

[0073] The dotted line shown in stage I is the position of the target cell;

[0074] The highlighted part shown in stage II is the fluorescence spot at the tip of the first probe;

[0075] Stage III shows that the first probe penetrates into the target cell and injects the corresponding exogenous substance through the electric field;

[0076] Stage IV shows that the first probe withdraws from the target cell after the injection is completed;

[0077] Refer to attachment Figure 8The disclosed embodiment also discloses a technical solution for positioning a target cell by the second probe 201, which specifically includes: providing a second probe assembly including the second probe 201 and the second gripper 202, and a pneumatic source assembly 60 for providing positive or negative air pressure to the second probe 201. The microscopic imaging assembly also provides a window for positioning the second probe 201.

[0078] Before positioning the first probe 101 near the target cell, the following steps are further included:

[0079] Position the second probe 201 near the target cell through the bright-field feature or fluorescence signal provided by the microscopic imaging assembly;

[0080] The pneumatic source assembly 60 works to provide a negative pressure of a preset pressure to the second probe 201, and the front end of the second probe 201 positions the target cell through the negative pressure. The technical solution for positioning the target cell by the pressure change inside the second probe can effectively position the target system when the target cell is a free cell, facilitating the implementation of intracellular injection.

[0081] The body structure and the mating structure of the second probe 201 and the second gripper 202 can refer to those of the first probe 101 and the first gripper 102, or can also refer to the attached Figure 9 In the shown embodiment, the second gripper 202 includes a housing, and the housing includes a head 211 and a body 212 that can move relative to each other axially. The rear end of the second probe 201 passes through the head 211 and extends into the body 212; a limiting portion 2122 is provided inside the body 212. The second probe 201 extends into the body 212 and the rear end is positioned at the limiting portion 2122. A pressure channel 2123 communicating with the inside of the second probe 201 is provided on the limiting portion 2122. The limiting portion 2122 can be arranged as an annular structure inside the body 212. In terms of the specific implementation manner of the limiting portion 2122, referring to an embodiment, the body 212 is a multi-layer structure and includes:

[0082] A first cylinder 2124, located on the outer layer and open at both ends, one end is a clamping end 2127 extending into the head 211, and the other end is a pressure end 2128 connected to the peripheral device;

[0083] A second cylinder 2125, located inside the first cylinder 2124. The second cylinder 2125 includes an open end near the clamping end 2127 and a closed end located inside the first cylinder 2124. The closed end forms the limiting portion 2122;

[0084] A third cylinder 2126, located inside the first cylinder 2124 and open at both ends, one end of which abuts against the closed end of the second cylinder 2125, and the other end is close to the pressure end 2128 of the first cylinder 2124.

[0085] By fitting the second cylinder body 2125 and the third cylinder body 2126 into the first cylinder body 2124, it is possible to ensure the positioning accuracy of the limiting part 2122 relative to the overall second holder 202 while reducing the processing difficulty, thereby ensuring the spatial position of the front end of the second probe 201 relative to the overall second holder 202.

[0086] In addition to being used for positioning target cells, the second probe can also be used to achieve in-situ extraction of target cells. The air pressure source assembly 60 serves as the power source for sucking and discharging substances (cells, reagents, etc.) by the second probe. Refer to the attached Figure 10 In the illustrated embodiment, the air pressure source assembly includes a plunger pump 61, a solenoid valve 62, a three-way joint 63, a first check valve 64, a second check valve 65, an air filter 66, a silencer 67, and a plug 68. The plunger pump 61 has an inlet end 61a, an outlet end 61b, and a plunger 61c; the solenoid valve 62 has a common end 62a, a normally open end 62b, and a normally closed end 62c; the air filter 66 has an air inlet end and an air outlet end; the first check valve 64 has an "IN" port and an "OUT" port; the second check valve 65 has an "IN" port and an "OUT" port; the three-way joint 63 has three connection ports.

[0087] The plug 68 is screwed into the inlet end 61a of the plunger pump, tightened and sealed, and the air intake and exhaust of the plunger pump are both completed through the outlet end 61b. The outlet end 61b of the plunger pump is connected to the common end 62a of the solenoid valve 62 by a pipeline; the air inlet end of the air filter 66 is connected and sealed to the silencer 67 by a threaded connection, and the air outlet end of the air filter 66 is connected to the "IN" port of the first check valve 64 by a pipeline; the "OUT" port of the first check valve 64 is connected to one of the ports of the three-way joint 63 by a pipeline. Among the other two ports of the three-way joint 63, one port is connected to the normally open end 62b of the solenoid valve 62 by a pipeline, and the other port is connected to the "IN" port of the second check valve 65 by a pipeline; the normally closed end 62c of the solenoid valve 62 is connected to the pressure end 2128 of the probe assembly through a pipeline, and then communicates with the second probe, so that the entire air pressure source assembly communicates with the second probe.

[0088] The air inlet end of the air filter 66 is connected with a silencer 67 to reduce the noise of the entire gas path system for operating cells. The air outlet end of the air filter 66 is connected to the "IN" port of the first check valve 64, and the first check valve 64 prevents the gas in the entire gas path system from flowing back to the air filter 66; the second check valve 65 is used to exhaust the air in the plunger pump 61 to ensure that there is no unfiltered air in the plunger pump 61.

[0089] Before work, the common terminal 62a and the normally open terminal 62b of the solenoid valve 62 are first connected, and the normally closed terminal 62c is closed. The plunger 61c first moves upward to the uppermost position. During this process, the residual gas in the plunger pump 61 is exhausted through the three-way joint 63 and the second one-way valve 65. Then, the plunger 61c moves downward to the middle position of the plunger pump 61. During this process, air is filtered by the air filter 66 and enters the plunger pump 61 through the first one-way valve 64 and the three-way joint 63. The initial stationary position of the plunger 61c is set in the middle of the plunger pump, which can provide negative air pressure when moving downward and positive air pressure when moving upward.

[0090] In some embodiments, as Figure 11 shown, the plunger pump 61 provides positive or negative air pressure for the second probe in the form of pulses, and the plunger 61c moves intermittently within a single working stroke. When the plunger 61c moves upward intermittently, the moving distance each time is d1, and multiple positive air pressure pulses can be generated. The air pressure value of the positive air pressure pulse is P1, and the pulse period is T1. When the plunger 61c moves downward intermittently, the moving distance each time is d2, and multiple negative air pressure pulses can be generated. The air pressure value of the negative air pressure pulse is P2, and the pulse period is T2.

[0091] Figure 11 The origin O in indicates that the plunger 61c is in the initial position, and at this time, the air pressure value in the plunger is approximately equal to the atmospheric pressure. Generally, the pulse air pressure value is selected as -10Kpa - 10Kpa, and the period is less than 1s, preferably 0.5s - 1s. The air pressure value and period of the pulse are adjustable to meet different actual needs.

[0092] During the working process, the solenoid valve 62 is energized, the normally open terminal 62b is closed, and the normally closed terminal 62c is opened to connect the plunger pump 61 with the second probe, and the plunger 61c in the plunger pump 61 is controlled to move downward. At this time, the plunger pump 61 provides negative air pressure pulses for the second probe, and the second probe can suck the cells in the culture dish. If a single pulse cannot suck the cells into the interior of the second probe, multiple pulses can be continuously emitted until they are sucked in. During the process of the second probe sucking the cells, when the internal and external air pressures are balanced, the cells no longer move, so they can be kept inside the second probe.

[0093] After sucking the cells is completed, if the plunger 61c has not moved to the limit position, it can continue to move to provide pulses to suck in more cells.

[0094] When the plunger 61c moves downward to the extreme position, the normally closed end 62c of the solenoid valve 62 is closed, and the normally open end 62b is connected. The plunger 61c moves upward for reset. During the reset process, the gas in the plunger pump 61 is discharged through the three-way joint 63 and the second one-way valve 65. After the plunger 61c completes the reset, the normally open end 62b is closed and the normally closed end 62c is opened, and cells can be continuously inhaled. By repeating this operation, more cells at different sites can be inhaled into the second probe in batches.

[0095] After the cell inhalation is completed, the plunger 61c is first reset (it can also not be reset), and the plunger 61c in the plunger pump 61 is controlled to move upward. At this time, a positive air pressure pulse is provided to the second probe, and the second probe spits out cells into the container (petri dish). Since the positive air pressure is also provided in the form of a pulse, the cells can be spit out one by one until all are spit out. The working process is opposite to the cell inhalation process and will not be elaborated here.

[0096] By using positive or negative air pressure pulses, it is possible to achieve spitting out or inhaling at most one cell per operation. Providing a negative air pressure pulse may cause the cells not to enter the interior of the second probe due to insufficient suction. By continuously outputting multiple pulses, the cells can eventually enter the second probe. The process of spitting out cells is similar.

[0097] Since there is a relatively large space inside the second probe, it can temporarily store more cells. The second probe can continuously inhale multiple cells and then spit them out at one time, significantly improving the operation efficiency. In addition, by using pulses, the second probe can spit out cells one by one, so that multiple cells are distributed at different positions, eliminating the need for redistribution operations.

[0098] Combined with the attached Figure 12 The embodiment shown discloses the overall framework of the cell operation device. The probe assembly includes a first probe assembly 31 and a second probe assembly 32. Each probe assembly can work independently or cooperate with each other. The control assembly includes a moving platform 50 linked with a motor. The microscopic imaging assembly includes a light source 23, an optical microscope objective 21, and an image detection unit 22 (such as a CCD camera), etc. The light source is generally arranged directly above the stage 10. The optical microscope objective 21 and the image detection unit 22 are located below the stage. The light emitted by the light source passes through the sample, the optical microscope objective 21, and forms an image on the image detection unit 22 in the direction of the arrow shown in Figure 12 The control system 70 can be a terminal device such as a computer. The plunger pump 61, the solenoid valve 62, the motor in the moving platform 50, the motor in the stage 10, and the field source assembly in the air pressure source assembly 60 are controlled by this control system. The control system also has an imaging display unit, that is, each probe and the target cells can be displayed on the screen through the imaging system, and the operator can perform real-time operations. In Figure 12In this case, the first probe and the second probe are fixedly connected to different mobile platforms to achieve independent control of the first probe and the second probe. In other embodiments, the first probe and the second probe may also be disposed on the same mobile platform and at least one of the two is movably connected to the mobile platform, so as to achieve flexible adjustment of the two. When the mobile platform moves relative to the sample, the first probe and the second probe move synchronously or independently. In the above connection manners, each probe can be connected to the mobile platform through a corresponding gripper or directly connected to the mobile platform.

[0099] Other details of the cell manipulation device can be implemented in combination with the prior art and will not be elaborated here.

[0100] The technical features of the above-described embodiments can be combined arbitrarily. For the sake of concise description, not all possible combinations of the technical features in the above-described embodiments are described. However, as long as there is no contradiction in the combination of these technical features, it should be considered as the scope described in this specification. When the technical features in different embodiments are shown in the same drawing, it can be regarded that the drawing also discloses the combination examples of the various embodiments involved at the same time.

[0101] The above-described embodiments only represent several implementation manners of the present application, and their descriptions are relatively specific and detailed, but they should not be construed as limiting the scope of the patent application. It should be noted that for those of ordinary skill in the art, without departing from the concept of the present application, several modifications and improvements can still be made, and these all belong to the protection scope of the present application.

Claims

1. A method for injecting exogenous substances into cells based on a single cell manipulation device, characterized in that: The single-cell manipulation device comprises a stage carrying a sample, a first probe, a first electrode arranged on the first probe, and a second electrode electrically connected to the sample, wherein the first electrode is fixedly passed through the first probe, the first probe is an elongated hollow structure and comprises a front end and a rear end opposite to each other, the front end of the first probe acts on a cell membrane of a target cell in the sample, the first electrode comprises a tail and a head close to the front end of the first probe, a gap is provided between the head and the front end of the first probe to form a conducting cavity, an exogenous substance is carried in an injection liquid, the injection liquid is filled in the conducting cavity, the injection liquid is a conductive medium and conducts the first electrode and the sample; The method for injecting exogenous substances into cells comprises: Step S100, establishing a first electric field between the first electrode and the second electrode, and detecting a change in a first electric field signal during movement of the first probe to determine whether a front end of the first probe is in contact with a cell membrane of the target cell; Step S200, the front end of the first probe moves relative to the sample to penetrate the cell membrane of the target cell; Step S300, establishing a second electric field between the first electrode and the second electrode, and injecting the exogenous substance into the target cell under the action of the second electric field.

2. The method for injecting exogenous substances into cells based on a single cell manipulation device according to claim 1, characterized in that: In step S100 , the voltage value of the first electric field is 0.1V to 5V, and the first electric field signal is the ion current between the first electrode and the second electrode.

3. The method for injecting exogenous substances into cells based on a single cell manipulation device according to claim 2, characterized in that: In step S100, when the first electric field is established, the value of the ion current is recorded as a reference value; When the ion current decreases and the variation range compared with the reference value is greater than 10% and less than or equal to 30% during the process of the first probe approaching the cell membrane of the target cell, it is determined that the front end of the first probe is in contact with the cell membrane of the target cell.

4. The method for injecting exogenous substances into cells based on a single cell manipulation device according to claim 1, characterized in that: In step S300, the second electric field is a voltage pulse with a voltage value of 1V to 10V and a total duration of 0.1 seconds to 5 seconds, and the exogenous substance enters the cell membrane of the target cell under the action of the voltage pulse.

5. The method for injecting exogenous substances into cells based on a single cell manipulation device according to claim 1, characterized in that: In step S100, the front end of the first probe is positioned above the target cell and the first electric field is established. The first probe moves downward and gradually approaches the target cell and determines whether the front end of the first probe contacts the cell membrane of the target cell during the process.

6. The method for injecting exogenous substances into cells based on a single cell manipulation device according to claim 1, characterized in that: The single-cell manipulation device further includes a first probe assembly, which includes the first probe and a first clamp for positioning the first probe; a conductive socket is fixed to the rear end of the first probe, the tail of the first electrode is conductively connected to the socket, and the first clamp is provided with a conductive portion, and the conductive portion is provided with a wire extending to the outside of the first clamp; When the first probe and the first holder cooperate with each other, the socket and the conductive part are plugged into each other.

7. The method for injecting exogenous substances into cells based on a single cell manipulation device according to claim 1, characterized in that: The first electrode is in the shape of an elongated strip and has a maximum outer diameter of 0.05 mm to 0.5 mm; In the extension direction of the first probe, the ratio between the length L1 of the first electrode and the length L2 of the first probe is in a range of 0.3 to 0.9; The first electrode is made of one or more of platinum, gold, silver, silver chloride, carbon-based materials, silicon, indium tin oxide and conductive polymer.

8. The method for injecting exogenous substances into cells based on a single cell manipulation device according to claim 1, characterized in that: The single cell manipulation device further comprises a second probe assembly and an air pressure source assembly for providing positive or negative air pressure to the second probe assembly, wherein the second probe assembly comprises a second probe and a second clamp; The method for injecting exogenous substances into cells also includes: The target cell is located by pressure changes in the second probe.

9. The method for injecting exogenous substances into cells based on a single cell manipulation device according to claim 8, characterized in that: The single cell manipulation device further comprises a control component, the control component comprises a mobile platform, and the first probe and the second probe are fixedly connected to the mobile platform; or At least one of the first probe and the second probe is movably connected to the mobile platform, and the two probes move synchronously or independently.

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