Method for injecting exogenous substances into cells based on single cell operation device

Abstract

The present application discloses a method for injecting exogenous substances into cells based on a single-cell operation device. The single-cell operation device includes 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. The first probe is an elongated hollow structure and includes a front end and a rear end. The front end of the first probe acts on the cell membrane of the 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 the first probe detects the change of the first electric field signal during the movement to determine whether the front end of the first probe is in contact with 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 the exogenous substance is injected into the target cell under the action of the second electric field. The technical solution in the present application has the advantages of high precision, low damage, high throughput, and multifunctionality.

Description

Technical Field

[0001] The present application relates to the field of cell manipulation technology, and in particular to a method for injecting exogenous substances into cells based on a single cell manipulation device. Background Art

[0002] Cell manipulation technology plays a vital role in life science research and biomedical applications. Although traditional cell manipulation methods, such as microinjection and electroporation, are effective, they have limitations such as complex operation, great damage to cells, and low throughput. In recent years, probe-based cell manipulation technology has developed rapidly, providing new solutions for precise manipulation of cells.

[0003] In the process of probe-based intracellular injection and cytoplasmic component extraction, it generally means that the tip of the nanoscale probe touches the cell membrane of the target cell, and the target cell is membrane-perforated, the target molecule is introduced, or the cytoplasmic component is extracted through mechanical force or pressure changes. When the target cell is a free cell, a micron-scale probe is also required to locate the target cell through mechanical force, pressure changes, electromagnetic fields, etc.

[0004] In actual operations, the introduction of target molecules or the extraction of components in the cytoplasm through pressure changes places high demands on the control accuracy of gas pressure. The pressure control accuracy in existing technologies often leads to cell damage, and there is room for improvement. Summary of the invention

[0005] In order to solve the above technical problems, the present application discloses a method for injecting exogenous substances into cells based on a single-cell operating device, which has the advantages of high precision, low damage, high throughput, and multifunctionality, and shows great application potential in the fields of life science research and biomedical applications.

[0006] In one embodiment of the present application, a method for injecting exogenous substances into cells based on a single cell operation device is disclosed. The single cell manipulation device comprises 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, wherein the first probe is an elongated hollow structure and comprises a front end and a rear end opposite to each other, and the front end of the first probe acts on a cell membrane of a target cell in 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.

[0007] Several optional methods are also provided below, but they are not intended to be additional limitations on the above-mentioned overall solution, but are merely further supplements or preferences. Under the premise that there are no technical or logical contradictions, each optional method can be combined with the above-mentioned overall solution separately, and multiple optional methods can also be combined.

[0008] 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 an ion current between the first electrode and the second electrode.

[0009] In one embodiment, 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% 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.

[0010] 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 seconds to 5 seconds, and the exogenous substance enters the cell membrane of the target cell under the action of the voltage pulse.

[0011] 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 determines whether the front end of the first probe contacts the cell membrane of the target cell during the process.

[0012] In one embodiment, the single-cell manipulation device further comprises a first probe assembly, the first probe assembly comprising 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, a conductive portion is provided on the first clamp, and a wire extending to the outside of the first clamp is provided on the conductive portion; When the first probe and the first holder cooperate with each other, the socket and the conductive part are plugged into each other.

[0013] In one embodiment, the first electrode includes a tail portion connected to the socket and a head portion close to the front end of the first probe, a gap is set between the head portion and the front end of the first probe to form a conductive cavity, the foreign object is carried in an injection liquid, and the injection liquid is filled in the conductive cavity to conduct the first electrode and the sample.

[0014] In one embodiment, the first electrode passes through the first probe, and the first electrode is in the shape of a long strip with 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 material of the first electrode is at least one or more of platinum, gold, silver, silver chloride, carbon-based materials, silicon, indium tin oxide and conductive polymer.

[0015] In one embodiment, 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.

[0016] In one embodiment, the single-cell manipulation device further comprises a control component, the control component comprises a moving platform, and the first probe component and the second probe component are fixedly connected to the moving platform; or At least one of the first probe assembly and the second probe assembly is movably connected to the mobile platform, and the two move synchronously or independently.

[0017] The present application uses electrodes to determine the contact between the probe and the target cell, and cooperates with voltage pulses to achieve intracellular injection. It can accurately control the process of cell membrane rupture and cell injection, and has the advantages of high precision, low damage, high throughput, and multifunctionality.

[0018] The specific beneficial technical effects will be further explained in the specific implementation methods in combination with specific structures or steps. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 This is a schematic diagram of a first probe approaching a target cell in an embodiment of the present application; Figure 2 This is a schematic diagram of the contact between the first probe and the cell membrane of the target cell in one embodiment; Figure 3 A schematic diagram of a first probe piercing a cell membrane of a target cell and then being injected in an embodiment; Figure 4 This is a schematic diagram of the process of injecting exogenous substances into cells in one embodiment of the present application; Figure 5 This is a schematic diagram of the structure of the first probe assembly in one embodiment of the present application; Figure 6 for Figure 5 A schematic diagram of the cooperation of the first probe assembly from a cross-sectional perspective; Figure 7 for Figure 5 A schematic diagram of the matching structure of the first probe and the first electrode of the first probe assembly; Figure 8 This is a schematic diagram of the cooperation between the first probe and the second probe in one embodiment of the present application; Fig. 9 This is a schematic diagram of the internal coordination of the second probe assembly in one embodiment of the present application; Fig.10 This is a schematic diagram of an air pressure source component in one embodiment of the present application; Fig.11 for Fig.10 A schematic diagram of the working of the air pressure source assembly; Fig.12 This is a schematic diagram of the system framework of a cell manipulation device in one embodiment of the present application; Fig.13 This is a schematic diagram of the process of injecting exogenous substances into cells in one embodiment of the present application; Fig.14 This is a schematic diagram of the process of injecting exogenous substances into cells in another embodiment of the present application.

[0020] The reference numerals in the figures are described as follows: 10. stage; 11. container; 12. sample; 21. optical microscope objective lens; 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. mobile platform; 60. air pressure 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 connector; 64. first one-way valve; 65. second one-way valve; 66. air filter; 67. silencer; 68. plug; 70. control system; 101, first probe; 102, first holder; 103, socket; 104, conductive part; 105, wire; 106, conducting cavity; 107, injection liquid; 201, second probe; 202, second clamp; 211, head; 212, body; 2122, limit part; 2123, pressure channel; 2124, first cylinder; 2125, second cylinder; 2126, third cylinder; 2127, clamping end; 2128, pressure end. DETAILED DESCRIPTION

[0021] The following will be combined with the drawings in the embodiments of the present application to clearly and completely describe the technical solutions in the embodiments of the present application. Obviously, the described embodiments are only part of the embodiments of the present application, not all of the embodiments. Based on the embodiments in the present application, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of this application.

[0022] It should be noted that when a component is referred to as being "connected" to another component, it may be directly connected to the other component or there may be a central component. When a component is referred to as being "disposed on" another component, it may be directly disposed on the other component or there may be a central component at the same time.

[0023] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those generally understood by those skilled in the art to which this application belongs. The terms used herein in the specification of this application 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.

[0024] Reference Figure 1 To Attachment Figure 4 As shown, the present application discloses a method for injecting exogenous substances into cells based on a single-cell operating device, which 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. The container 11 carries a sample 12, 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 provide bright field features by directly imaging with transmitted light during the imaging process, or provide fluorescence signals based on fluorescence. Figure 1 To Attachment Figure 5The probe assembly includes a first probe assembly, which includes a first probe 101 and a first clamp 102. The first probe 101 is detachably mounted on the first clamp 102. The first probe 101 is a slender hollow structure with a front end and a rear end and a first electrode 41 passes through the interior. When in use, the first probe 101 is filled with exogenous substances or an injection liquid 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 substance and / or the injection liquid 107. The field source assembly 42 includes a working electrode electrically connected to the first electrode 41 and a base electrode electrically connected to the second electrode. The working electrode and the base electrode form a loop in the sample through the first electrode 41 and the second electrode and transmit energy.

[0025] Methods of exogenous injection include: Step S100, establishing a first electric field between the first electrode 41 and the second electrode, and detecting a change in the first electric field signal during the movement of the first probe 101 to determine whether the front end of the first probe 101 is in contact with the cell membrane of the target cell in the sample 12; Step S200, the front end of the first probe 101 moves relative to the sample 12 to penetrate the cell membrane of the target cell; Step S300: A second electric field is established between the first electrode 41 and the second electrode, and exogenous substances are injected into the target cells under the action of the second electric field.

[0026] Wherein, step S100 specifically also includes: Based on the bright field characteristics or fluorescent signals provided by the microscopic imaging component, the front end of the first probe 101 is positioned near the target cell (e.g., near the target cell) through the control component. Figure 1 shown); An electric field is applied between the working electrode and the base electrode by the field source component 42, and the electric field signal between the working electrode and the base electrode is measured by the measuring component 43 and recorded as a reference value (e.g., Figure 1 shown); The front end of the first probe 101 is gradually brought close to the cell membrane of the target cell by the control component, and 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 conditions, it is determined that the front end of the first probe 101 is in contact with the cell membrane of the target cell, the movement of the first probe 101 is stopped and the position is recorded as the reference position (for example, the position of the base electrode is close to the working electrode). Figure 2 as shown).

[0027] Step S200 specifically includes: controlling the first probe 101 of the component to move a preset distance in a preset direction compared to the reference position so that the front end of the first probe 101 pierces the cell membrane of the target cell (for example, the cell membrane of the target cell). Figure 3 as shown).

[0028] Step S300 specifically includes: applying a voltage pulse of a preset condition between the working electrode and the base electrode through 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, Figure 3 shown) The first probe 101 is withdrawn to the reference position by the control assembly.

[0029] The first electric field signal can be in various forms, for example, a constant current source is used to form a constant current condition between the working electrode and the base electrode, and the voltage value between the working electrode and the base electrode is used as the first electric field signal. Figure 1 As shown, in step S100, a voltage of a preset value is applied between the working electrode and the base electrode by the field source component 42, and 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 the first electric field is established, 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, the voltage is a DC voltage, wherein the working electrode is a positive electrode or a negative electrode, and the corresponding base electrode is a negative electrode or a positive electrode. In other embodiments, the voltage can be 0.1V to 4V or 0.5V to 3V or 0.5V to 1.5V or 0.5V to 5V, each voltage value with a step of 0.5V. In this embodiment, the range of the reference value is 10nA (nanoampere) to 10μA (microampere). Further, the range of the reference value is 20nA to 950nA.

[0030] Reference Figure 2 In the illustrated embodiment, in step S100, when judging whether the front end of the first probe 101 is in contact with the cell membrane of the target cell, 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 compared with the reference value. When the real-time ion current becomes smaller and the change amplitude compared with the reference value is greater than 10% to 30%, it is judged that the front end of the first probe 101 is in contact with the cell membrane of the target cell. Furthermore, when the real-time ion current changes by more than 10% to 20% compared with the reference value, it is judged that the front end of the first probe 101 is in contact with the cell membrane of the target cell. In other embodiments, it can also be judged by the absolute value of the real-time ion current change. For example, when the real-time ion current change amplitude is greater than or equal to 20nA to 300nA, it is judged that the front end of the first probe 101 is in contact with the cell membrane of the target cell. In the attached Figure 2In the step S100, when the front end of the first probe 101 is positioned near the target cell, it is positioned above the target cell and a reference value is measured, that is, in step S100, the front end of the first probe is positioned above the target cell and a first electric field is established, the first probe 101 moves downward and gradually approaches the target cell and determines whether the front end of the first probe 101 is in contact with the cell membrane of the target cell in the process.

[0031] In addition to determining 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 implement electric field injection. Figure 3 In the illustrated embodiment, when an exogenous substance and / or an injection liquid 107 is injected, a second electric field is applied between the working electrode and the base electrode through the field source component 42, and the exogenous substance and / or the injection liquid 107 enters 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 1V to 10V, and the total duration of the voltage pulse is 0.1 seconds to 5 seconds. The voltage values ​​of each pulse of the voltage pulse can be the same or can be set independently, and the duration of each pulse can be the same or can be set independently. The waveform of each pulse can be set the same or can be set independently, such as a square wave pulse or a sawtooth wave pulse or a sine wave pulse. The overall pulse duty cycle of the voltage pulse is 20% to 95% or 30% to 90% or 40% to 85%.

[0032] For details on the matching between the first probe 101 and the first electrode 41, refer to the attached Figure 5 To Attachment Figure 7 In the illustrated embodiment, the body of the first probe 101 is an elongated hollow structure, and the material is quartz, glass, etc. Specifically, it can be formed by drawing a quartz capillary through laser heating and has a geometric rotationally symmetrical structure. The interior of the body of the first probe 101 is hollow and has no movable parts or embedded circuits. The first probe 101 has a relative front end and a rear end, and both the front end and the rear end are open structures. The aperture of the front end is smaller than that of the rear end, and it has a flat micro-operation end face. The size of the rear end is adapted to the first clamp 102. Furthermore, a conductive socket 103 is fixed to the rear end of the first probe 101, and a conductive portion 104 is provided on the first clamp 102. The conductive portion 104 is provided with a wire 105 extending to the outside of the first clamp 102 and connected to the working electrode; When the first probe 101 cooperates with the first clamp 102, the socket 103 and the conductive part 104 are plugged into each other and realize the electrical connection between the first electrode 41 and the working electrode. The detachable connection between the socket 103 and the conductive part 104 can conveniently realize the removal and installation of the first probe 101 and simultaneously realize the disconnection and conduction between the first electrode 41 and the working electrode during the removal and installation process. Figure 6In the embodiment, the socket 103 includes a wrapping portion positioned at the rear end of the first probe 101 and a plug shaft extending from the wrapping portion away from the front end of the first probe 101, and the conductive portion 104 is provided with a plug cavity for receiving the plug shaft, and the plug cavity can wrap the circumference of the plug shaft while receiving the plug shaft to achieve a stable conductive connection. In other embodiments, the structures of the socket 103 and the conductive portion 104 can also be interchangeable.

[0033] The first electrode 41 can extend from the front end of the first probe 101 to the sample 12 or the environment where the sample 12 is located (for example, the surrounding Figure 1 The solution environment shown in the figure is used for electrical connection. Please also refer to the attached Figure 7 In the illustrated embodiment, the first electrode 41 includes a tail portion connected to the socket 103 and a head portion 211 close to the front end of the first probe 101. A gap is set between the head portion 211 and the front end of the first probe 101 to form a conductive cavity 106. The foreign matter is carried in an injection liquid 107. The injection liquid 107 is filled in the conductive 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 need for the first electrode 41 to pass through the smaller opening at the front end of the probe, thereby ensuring the size of the first electrode 41 and reducing the difficulty of production. In terms of the details of the first electrode 41, the injection liquid 107 is attached therein. Figure 7 In the illustrated embodiment, in the extension direction of the first probe 101, the ratio between the length L1 of the first electrode 41 and the length L2 of the first probe 101 is in the range of 0.3 to 0.9. Further, the first electrode 41 is in the shape of a strip and has a maximum outer diameter of 0.05 mm to 0.5 mm. The material of the first electrode 41 is at least one or more of platinum, gold, silver, silver chloride, carbon-based materials, silicon, indium tin oxide and conductive polymers. Similarly, the attached Figure 1 To Attachment Figure 3 The middle base is electrically connected to the sample 12 via a second electrode disposed in the container 11. The second electrode can be disposed similarly to the first electrode 41 described above, or can be disposed differently, for example, the second electrode is plate-shaped or a part or the entire container 11 is used as the second electrode.

[0034] The exogenous substance itself carries an electric charge or acquires an electric charge through the electric field formed by the electrode, so as to move under the driving force of the second electric field. The exogenous substance can be a common injection substance in the field of cell manipulation, such as dyes, proteins, mRNA, plasmids, etc. The exogenous substance can be a single substance or a combination of multiple substances.

[0035] For example, Fig.13 and attached Fig.14 The injection process of different exogenous substances is shown. Fig.13 The exogenous substance injected in the embodiment is a dye (brand Alexa 647), attached Fig.14The injected exogenous substance in the embodiment is a protein (e.g., α-synuclein) labeled with a dye (brand Alexa 594). Fig.13 and attached Fig.14 There are five stages in each of them, including: The dotted line shown in stage I is the location of the target cell; The highlighted part shown in stage II is the fluorescent spot at the tip of the first probe; Stage III shows that the first probe penetrates into the target cell and injects the corresponding foreign substance through the electric field; Stage IV shows that the first probe exits the target cell after injection; Stage V shows the observation of the exogenous substances in the target cells after injection.

[0036] Reference Figure 8 The illustrated embodiment also discloses a technical solution for locating target cells by means of a second probe 201, specifically comprising: providing a second probe assembly including the second probe 201 and a second holder 202 and an air pressure source assembly 60 for providing positive or negative air pressure to the second probe 201, the microscopic imaging assembly also providing a window for locating the second probe 201; Before positioning the first probe 101 near the target cell, the following steps are also included: The second probe 201 is positioned near the target cell by using the bright field feature or fluorescent signal provided by the microscopic imaging component; The air pressure source assembly 60 provides a negative pressure of a preset pressure to the second probe 201, and the front end of the second probe 201 locates the target cell through the negative pressure. The technical solution of locating the target cell through the pressure change in the second probe can effectively locate the target system when the target cell is a free cell, which is convenient for the implementation of intracellular injection.

[0037] The main structure and the matching structure of the second probe 201 and the second clamp 202 can refer to the first probe 101 and the first clamp 102, and can also refer to the attached Fig. 9 In the illustrated embodiment, the second holder 202 includes a shell, which includes a head 211 and a body 212 that are axially relatively movable. 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. The limiting portion 2122 is provided with a pressure channel 2123 that is connected to the interior of the second probe 201. The limiting portion 2122 can be an annular structure arranged inside the body 212. In the specific implementation of the limiting portion 2122, referring to an embodiment, the body 212 is a multi-layer structure and includes: The first cylinder 2124 is located at the outer layer and is 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 equipment; The second cylinder 2125 is located inside the first cylinder 2124. The second cylinder 2125 includes an open end close to the clamping end 2127 and a closed end located inside the first cylinder 2124. The closed end forms a limiting portion 2122. The third cylinder 2126 is located inside the first cylinder 2124 and is open at both ends, with one end abutting against the closed end of the second cylinder 2125 and the other end close to the pressure end 2128 of the first cylinder 2124 .

[0038] By embedding the second cylinder 2125 and the third cylinder 2126 in the first cylinder 2124, the positioning accuracy of the limit portion 2122 compared to the second clamp 202 as a whole can be guaranteed while reducing the processing difficulty, thereby ensuring the spatial position of the front end of the second probe 201 compared to the second clamp 202 as a whole.

[0039] In addition to being used to locate the target cells, the second probe can also be used to achieve in-situ extraction of the target cells. The air pressure source assembly 60 is used as a power source for the second probe to suck and spit out substances (cells, reagents, etc.). Fig.10 In the illustrated embodiment, the air pressure source assembly includes a plunger pump 61, a solenoid valve 62, a three-way connector 63, a first one-way valve 64, a second one-way valve 65, an air filter 66, a muffler 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 one-way valve 64 has an "IN" port and an "OUT" port; the second one-way valve 65 has an "IN" port and an "OUT" port; and the three-way connector 63 has three connection ports.

[0040] 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 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 muffler 67 by a threaded connection, and the air outlet end of the air filter 66 is connected to the "IN" port of the first one-way valve 64 by a pipeline; the "OUT" port of the first one-way valve 64 is connected to one of the ports of the three-way joint 63 by a pipeline, and one of the other two ports of the three-way joint 63 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 one-way 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 connected to the second probe, so that the entire air pressure source assembly is connected to the second probe.

[0041] The air filter 66 has a muffler 67 connected to the air inlet end to reduce the noise of the air path system of the entire operating cell. The air filter 66 has an exhaust end connected to the "IN" port of the first one-way valve 64, which prevents the gas in the entire air path system from flowing back to the air filter 66; the second one-way 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.

[0042] Before operation, the common end 62a and the normally open end 62b of the solenoid valve 62 are first connected, the normally closed end 62c is closed, and the plunger 61c first moves upward to the uppermost end, during which 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 which the 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 stop position of the plunger 61c is set at the middle of the plunger pump, and it can move downward to provide negative air pressure, and can also move upward to provide positive air pressure.

[0043] In some embodiments, Fig.11 As shown, the plunger pump 61 provides positive or negative air pressure to the second probe in the form of pulses, and the plunger 61c moves intermittently within a single working stroke. When the plunger 61c moves intermittently upward, each movement distance 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 intermittently downward, each movement distance 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.

[0044] Fig.11 The origin O in the figure indicates that the plunger 61c is in the initial position, and the air pressure value in the plunger is close to the atmospheric pressure. Generally speaking, the pulse air pressure value is selected to be -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 practical needs.

[0045] During operation, the solenoid valve 62 is energized, the normally open end 62b is closed, and the normally closed end 62c is opened, so that the plunger pump 61 is connected to 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 a negative air pressure pulse to 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 second probe, multiple pulses can be sent continuously until the cells are sucked in. When the second probe sucks in the cells, when the internal and external air pressures are balanced, the cells no longer move, so they can be kept in the second probe.

[0046] After the cells are sucked in, if the plunger 61c has not moved to the limit position, it can continue to move to provide pulses to suck in more cells.

[0047] When the plunger 61c moves downward to the limit position, the normally closed end 62c of the solenoid valve 62 is closed and the normally open end 62b is connected, and the plunger 61c moves upward to reset. During the reset process, the gas in the plunger pump 61 is discharged through the three-way connector 63 and the second one-way valve 65. After the plunger 61c is reset, the normally open end 62b is closed and the normally closed end 62c is opened to continue to absorb cells. The reciprocating operation can be repeated to absorb more cells at different sites into the second probe in batches.

[0048] After the cells are sucked in, the plunger 61c is reset first (or not reset), and the plunger 61c in the plunger pump 61 is controlled to move upward, and a positive air pressure pulse is provided to the second probe, and the second probe spits out cells into the container (culture dish). Because the positive air pressure is also provided in the form of pulses, the cells can be spewed out one by one until all are spewed out. The working process is opposite to the process of sucking cells, and will not be repeated here.

[0049] Using positive or negative air pressure pulses, at most one cell can be spit out or sucked in per operation. Providing a negative air pressure pulse may prevent the cell from entering the second probe due to insufficient suction. By continuously outputting multiple pulses, the cell can eventually enter the second probe. The spitting out of the cell is similar.

[0050] Since the second probe has a large space inside, it can temporarily store more cells. The second probe can continuously suck in multiple cells and then spit out the cells at once, which significantly improves the operation efficiency. In addition, the second probe can spit out cells one by one by pulsing, so that multiple cells are distributed in different positions, eliminating the redistribution operation.

[0051] Combined with Fig.12 The embodiment shown discloses the overall framework of the cell manipulation device. The probe assembly includes a first probe assembly 31 and a second probe assembly 32. Each probe assembly can work independently or in coordination with each other. The control assembly includes a mobile platform 50 linked to a motor. The microscopic imaging assembly includes a light source 23, an optical microscope objective lens 21, and an image detection unit 22 (such as a CCD camera). The light source is generally arranged directly above the stage 10, and the optical microscope objective lens 21 and the image detection unit 22 are located below the stage. The light emitted by the light source is arranged according to Fig.12The arrow direction shown in the figure passes through the sample, the optical microscope objective lens 21 and forms an image on the image detection unit 22. 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 mobile platform 50, the motor in the stage 10 and the field source component in the air pressure source component 60 are controlled by the control system. The control system also has an imaging display unit, that is, each probe and target cell can be displayed on the screen through the imaging system, and the operator can perform real-time operations. Fig.12 In the embodiment, the first probe and the second probe are fixedly connected to different mobile platforms to realize independent control of the first probe and the second probe. In other embodiments, the first probe and the second probe can also be set on the same mobile platform and at least one of them can be movably connected to the mobile platform, so as to realize flexible adjustment of the two. When the mobile platform is equivalent to the movement of the sample, the first probe and the second probe move synchronously or independently. In the above connection method, each probe can be connected to the mobile platform through a corresponding clamp or can be directly connected to the mobile platform. Other details of the cell manipulation device can be implemented in combination with existing technologies and will not be described in detail here.

[0052] The technical features of the above-mentioned embodiments can be combined arbitrarily. To make the description concise, not all possible combinations of the technical features in the above-mentioned embodiments are described. However, as long as there is no contradiction in the combination of these technical features, they should be considered to be within the scope of this specification. When the technical features in different embodiments are embodied in the same figure, it can be regarded that the figure also discloses the combination examples of the various embodiments involved.

[0053] The above-mentioned embodiments only express several implementation methods of the present application, and the descriptions thereof are relatively specific and detailed, but they cannot be understood as limiting the scope of the patent application. It should be pointed out that, for ordinary technicians in this field, several variations and improvements can be made without departing from the concept of the present application, 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 disposed on the first probe and a second electrode electrically connected to the sample, wherein the first probe is an elongated hollow structure and comprises a front end and a rear end opposite to each other, and the front end of the first probe acts on a cell membrane of a target cell in 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% 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, a conductive portion is provided on the first clamp, and a wire extending to the outside of the first clamp is provided on the conductive portion; 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 6, characterized in that: The first electrode includes a tail portion connected to the socket and a head portion close to the front end of the first probe, a gap is set between the head portion and the front end of the first probe to form a conductive cavity, the foreign object is carried by an injection liquid, and the injection liquid fills the conductive cavity and conducts the first electrode and the sample.

8. 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 passes through the first probe, and the first electrode is in a long strip shape with 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 material of the first electrode is at least one or more of platinum, gold, silver, silver chloride, carbon-based materials, silicon, indium tin oxide and conductive polymer.

9. 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.

10. The method for injecting exogenous substances into cells based on a single cell manipulation device according to claim 9, 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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