Mass spectrometry injection system and method for automatic microcell analysis

Abstract

A mass spectrometry injection system and method for automatic microcell analysis, relating to the technical field of mass spectrometry. In the system, a cell suction device suctions cells in a cell suspension by using a suction needle (11) to obtain microcells; then a motion device (4) is used to clamp the suction needle (11) containing the cells from an opening into an electrical lysis cavity, and an electrical lysis device (2) is used for electrical lysis of the cells in the suction needle (11) to obtain intracellular substances; next, the motion device (4) is used to clamp the suction needle (11) to the position of a mass spectrometry injection interface. An electrospray induction device comprises the suction needle (11) and a steel tube sleeved outside the suction needle (11), and a voltage is applied to the steel tube by means of a first power supply unit, so that the cellular substances in the suction needle (11) generate electrospray, thereby implementing injection processing at the mass spectrometry injection interface. According to the system, by using a fully-automatic processing mode, the whole process from cell separation to lysis to mass spectrometry injection can be efficiently and quickly completed, the detection time is shortened, manual operation is avoided, and reliable repeatability and precision are achieved.

Description

A mass spectrometry injection system and method for automatic analysis of trace cells Technical Field

[0001] The present application relates to the technical field of mass spectrometry analysis, and in particular to a mass spectrometry injection system and method for automatic analysis of trace cells. Background Art

[0002] Mass spectrometry has the characteristics of high resolution, high sensitivity, and a wide range of substance detection, and therefore has great application prospects in trace cell detection. However, trace cells are difficult to obtain, and the entire cell detection process, from sample processing to mass spectrometry injection, is complicated, which limits the application of mass spectrometry in cell detection.

[0003] The primary prerequisite for single-cell analysis is the isolation of single cells, as cells are often in an aggregated state, closely connected to each other and difficult to distinguish. Furthermore, precise capture of target cells requires extremely precise control, so efficient single-cell acquisition remains a challenge that needs to be solved (currently, single-cell acquisition is still primarily manual, using a micromanipulation system to locate the target cell under a microscope and aspirate it with a capillary needle). Furthermore, single-cell capture requires cell lysis, and the process of releasing the contents by rupturing the cell membrane has become a key step in single-cell mass spectrometry analysis. The quality of cell lysis determines the quality of subsequent analysis. Therefore, the development of a mass spectrometry injection system that can accurately and efficiently capture and process single cells is becoming increasingly important.

[0004] Previous studies have focused more on acquiring single-cell samples, and on studying one or two parts of cell separation, processing, and mass spectrometry, and the entire system still requires a large number of manual operations.

[0005] Summary of the Invention

[0006] The embodiments of the present application provide a mass spectrometry injection system for automatic analysis of trace cells to address the problem in the prior art that previous research has focused more on obtaining single-cell samples and on one or two parts of cell separation, processing and mass spectrometry, and the entire system still requires a large number of manual operations.

[0007] Correspondingly, the embodiment of the present application also provides a mass spectrometry sampling method for automatic analysis of trace cells, which is used to ensure the implementation and application of the above method.

[0008] In order to solve the above technical problems, the present application discloses a mass spectrometry sampling system for automatic analysis of trace cells, which includes a cell aspiration device, an electrolysis device, an induced electrospray device, and a motion device;

[0009] The cell aspiration device comprises an aspiration needle for aspirating cells in a cell suspension;

[0010] The electrolysis device includes an electrolysis chamber with an opening above the electrolysis chamber;

[0011] The induced electrospray device includes an aspiration needle and a steel tube sheathed on the aspiration needle. The steel tube is connected to a first power supply unit, which is used to apply voltage to the steel tube so that the cellular material in the aspiration needle is injected into the mass spectrometer injection interface in an electrospray manner.

[0012] The motion device is used to clamp the aspiration needle so that the aspiration needle moves to a specified position; the specified position includes the position of the cell suspension, the electrolysis chamber, and the position of the mass spectrometry injection interface.

[0013] In the embodiment of the present application, a motion device is used to clamp the suction needle to the position of the cell suspension, and the cell suction device uses the suction needle to suck the cells in the cell suspension to obtain trace cells; then, the suction needle containing trace cells is clamped from the opening to the electrolysis chamber by the motion device, and the cells in the suction needle are electrolyzed by the electrolysis device to obtain intracellular substances; finally, the suction needle containing intracellular substances is clamped to the position of the mass spectrometry injection interface by the motion device, and the induction electrospray device includes a suction needle and a steel pipe sleeved outside the suction needle. Voltage is applied to the steel pipe through the first power supply unit to cause the cell substance in the suction needle to generate electrospray, thereby realizing the injection processing at the mass spectrometry injection interface. Based on trace cells, the embodiment of the present application adopts a fully automatic processing mode, which can efficiently and quickly complete the entire process from cell separation to lysis to mass spectrometry injection, shortening the entire sample detection time while avoiding manual operation. It can be used without complex training and has reliable repeatability and accuracy.

[0014] Preferably, the tip of the suction needle has a diameter of 20-50 μm.

[0015] The above-mentioned design of the needle tip caliber allows multiple cells to be sucked in at one time, and prevents the cells from touching the tip of the suction needle 11 or rupturing prematurely due to excessive pressure during the suction process.

[0016] Preferably, the electrolysis device is connected to a second power supply unit, the voltage of the second power supply unit is 3-4 kV, the duty cycle is 5%-50%, and the frequency is 250-5000 Hz.

[0017] By designing the above parameters of the second power supply unit, the electrolysis device can achieve better lysis effect.

[0018] Preferably, the electrolysis device comprises two symmetrical concave parts; and the electrolysis chamber is formed by combining the two concave parts.

[0019] Preferably, an opening is provided in the center of the concave part, and a hollow fixing part is provided in the opening; a metal sheet is provided at one end of the fixing part located on the inner side of the concave part, and a metal part is embedded in the other end; the metal part is connected to the second power supply unit, and the metal part is in contact with the metal sheet; the two metal sheets corresponding to the two concave parts form parallel plate electrodes.

[0020] Preferably, the voltage of the first power supply unit is 3-6 kV, the frequency is 50-250 Hz, and the duty cycle is 25%-50%.

[0021] By designing the above parameters of the first power supply unit, a better spray effect can be obtained, so that the time for obtaining a stable signal is longer.

[0022] Preferably, the cell aspiration device further comprises a flow control structure connected to the aspiration needle for controlling the amount of cells aspirated by the aspiration needle.

[0023] The present application also discloses a mass spectrometry sampling method for automated analysis of trace cells, based on any of the above mass spectrometry sampling systems for automated analysis of trace cells, the method comprising:

[0024] Use a pipette to aspirate the target number of cells from the cell suspension;

[0025] Using the motion device, the aspiration needle containing the cells is moved from the opening to the electrolysis chamber of the electrolysis device;

[0026] The cells in the aspiration needle are lysed by an electrolysis device to obtain intracellular substances;

[0027] The motion device is used to move the aspiration needle containing the intracellular substance to the position of the mass spectrometry injection interface;

[0028] A voltage is applied to the steel tube outside the aspiration needle by the first power supply unit, so that the cell material in the aspiration needle is injected into the mass spectrometry injection interface in an electrospray manner.

[0029] Preferably, the cells in the aspiration needle are lysed by an electrolysis device to obtain intracellular substances, comprising:

[0030] The second power supply unit is controlled to release voltage to the electrolysis device to electrolyze the cells in the aspiration needle to obtain intracellular substances; wherein the voltage of the second power supply unit is 3-4kV, the duty cycle is 5%-50%, and the frequency is 250-5000Hz.

[0031] Additional aspects and advantages of the embodiments of the present application will be given in the following description, which will become apparent from the following description or be understood through the practice of the present application. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] The above and / or additional aspects and advantages of the present application will become apparent and easily understood from the following description of the embodiments in conjunction with the accompanying drawings, in which:

[0033] FIG1 is a schematic structural diagram of a mass spectrometry injection system for automated analysis of trace cells provided in an embodiment of the present application;

[0034] FIG2 is a schematic diagram of the overall structure of the electrolysis device provided in an embodiment of the present application;

[0035] FIG3 is a schematic structural diagram of a concave member of an electrolysis device according to an embodiment of the present application;

[0036] FIG4 is a schematic structural diagram of a fixing member of an electrolysis device according to an embodiment of the present application;

[0037] FIG5 is a schematic diagram of the overall process of mass spectrometry injection provided in an embodiment of the present application;

[0038] FIG6 is a diagram showing the mass spectrometry detection effect of the control group (150 mM ammonium bicarbonate aqueous solution) provided in the examples of the present application;

[0039] FIG7 is a diagram showing the mass spectrometry detection effect of direct injection of LLC cells without lysis provided in an embodiment of the present application;

[0040] FIG8 is a diagram showing the mass spectrometry detection effect of LLC cells under ultrasonic lysis provided in an embodiment of the present application;

[0041] FIG9 is a diagram showing the mass spectrometry detection effect of LLC cells under electrolysis according to an embodiment of the present application;

[0042] FIG10 is a comparison of fluorescence-stained LLC mouse lung cancer cells under a microscope before and after electrolysis provided in an example of the present application;

[0043] FIG11 is an observation diagram of electrospray induced by laser pen illumination provided in an embodiment of the present application;

[0044] FIG12 is a schematic diagram of the process of a mass spectrometry injection system for automatic analysis of trace cells provided in an embodiment of the present application.

[0045] Among them, 11-absorption needle; 12-flow control structure; 2-electrolysis device; 21-opening; 221-electrolysis high-voltage power supply; 222-electrolysis high-voltage switch; 23-concave part; 231-opening; 232-mounting hole; 24-fixing part; 25-pin; 31-steel pipe; 321-electrospray high-voltage power supply; 322-electrospray high-voltage switch; 33-clamping part; 4-movement device; 41-mechanical claw; 5-host computer; 6-acquisition card; 7-centrifuge tube. DETAILED DESCRIPTION

[0046] The following describes embodiments of the present application in detail. Examples of the embodiments are shown in the accompanying drawings, wherein the same or similar reference numerals throughout represent the same or similar elements or elements having the same or similar functions. The embodiments described below with reference to the accompanying drawings are exemplary and are only used to explain the present application and are not to be construed as limiting the present application.

[0047] It will be understood by those skilled in the art that, unless expressly stated otherwise, the singular forms "a", "an", "said" and "the" used herein may also include the plural forms. It should be further understood that the term "comprising" used in the specification of this application refers to the presence of features, integers, steps, operations, elements and / or components, but does not exclude the presence or addition of one or more other features, integers, steps, operations, elements, components and / or combinations thereof. It should be understood that when we refer to an element as being "connected" or "coupled" to another element, it may be directly connected or coupled to the other element, or there may be intermediate elements. In addition, "connected" or "coupled" as used herein may include wireless connections or wireless couplings. The term "and / or" used herein includes all or any units and all combinations of one or more associated listed items.

[0048] It will be understood by those skilled in the art that, unless otherwise defined, all terms (including technical and scientific terms) used herein have the same meaning as commonly understood by those skilled in the art in the art to which the present invention pertains. It should also be understood that terms such as those defined in common dictionaries should be understood to have meanings consistent with their meanings in the context of the prior art and, unless specifically defined as herein, will not be interpreted in an idealized or overly formal sense.

[0049] The solution provided in the embodiment of the present application can be performed by any electronic device, such as a terminal device or a server, wherein the server can be an independent physical server, a server cluster or a distributed system composed of multiple physical servers, or a cloud server providing cloud computing services. The terminal can be a smart phone, a tablet computer, a laptop computer, a desktop computer, an intelligent speaker, a smart watch, etc., but is not limited thereto. The terminal and the server can be directly or indirectly connected via a wired or wireless communication mode, and the present application is not limited thereto. For the technical problems existing in the prior art, a mass spectrometry injection system and method for automatic analysis of micro cells provided by the present application is intended to solve at least one of the technical problems of the prior art.

[0050] The following specific embodiments describe in detail the technical solution of the present application and how the technical solution of the present application solves the above-mentioned technical problems. The following specific embodiments can be combined with each other, and the same or similar concepts or processes may not be repeated in some embodiments. The embodiments of the present application will be described below in conjunction with the accompanying drawings.

[0051] The embodiment of the present application provides a possible implementation method. FIG1 provides a schematic structural diagram of a mass spectrometry injection system for automatic analysis of trace cells.

[0052] As shown in FIG1 , the system includes a cell aspiration device, an electrolysis device 2 , an induced electrospray device, and a motion device 4 ;

[0053] The cell aspiration device includes an aspiration needle 11 for aspirating cells in a cell suspension;

[0054] The electrolysis device 2 includes an electrolysis chamber, and an opening 21 is provided above the electrolysis chamber;

[0055] The induced electrospray device includes an aspiration needle 11 and a steel tube 31 sheathed outside the aspiration needle 11. The steel tube 31 is connected to a first power supply unit, which is used to apply voltage to the steel tube 31 so that the cell material in the aspiration needle 11 is injected into the mass spectrometer injection interface in an electrospray manner.

[0056] The motion device 4 is used to clamp the aspiration needle 11 so that the aspiration needle 11 moves to a designated position; the designated position includes the position of the cell suspension, the inside of the electrolysis chamber, and the position of the mass spectrometry injection interface.

[0057] In the embodiment of the present application, the motion device 4 is used to clamp the suction needle 11 to a specified position to achieve full automation of the entire process from cell separation to lysis and then to mass spectrometry injection.

[0058] First, the moving device 4 clamps the aspirating needle 11 to the position of the cell suspension, and the aspirating needle 11 aspirates a trace amount of cells (the number is ≤ 10) from the cell suspension.

[0059] When a small amount of cells are drawn at a time, if the concentration of the cell solution is too high, it is inevitable that a large number of cells will be present and the cells will tend to clump together, making it difficult to separate (the cell concentration per unit volume is very high); if the concentration is very low, it is easy for the number of cells in the drawn cell sample solution to be very small (less than the required number) or even no cells at all (following the Poisson distribution). Therefore, in the embodiment of the present application, a cell suspension is first prepared, and the concentration per unit volume of the cell suspension is 10 4 -10 5 / ml to ensure that sufficient cells are present in each aspiration sample. When preparing the cell suspension, the cells are resuspended in an isotonic solution. By providing an ammonium bicarbonate aqueous solution to maintain cell morphology, the normal physiological environment of the cells can be simulated. This ensures that the cell morphology is not prematurely destroyed before lysis and participates in the entire detection process, avoiding the introduction of other interferences.

[0060] The cell suspension is stored in a centrifuge tube 7 , which is stored in a centrifuge box. The centrifuge box can store multiple centrifuge tubes 7 containing different samples. In the embodiment of the present application, the cell sample in any centrifuge tube 7 can be detected.

[0061] The cell aspiration device aspirates cells in the cell suspension through the aspiration needle 11 and aspirates the cells together with part of the isotonic solution outside the cells into the aspiration tube, thereby completing the cell aspiration process.

[0062] After the cell aspiration process is completed, the movement device 4 clamps the aspiration needle 11 into the electrolysis chamber, and uses the electrolysis device 2 to lyse the cells in the aspiration needle 11 to obtain intracellular substances.

[0063] In order for a cell to detect its internal substances through mass spectrometry, it must first undergo cell lysis to release the internal substances. In the embodiment of the present application, the cells are lysed by electrolysis. Electrolysis can lyse cells by applying an external electric field without directly contacting the sample without doing any treatment to the cells, and will not cause damage to the intracellular substances. There is no heat and no introduction of new substances. It can be truly efficient and harmless to the intracellular substances. The key to electrolysis is whether a high enough electric field can be generated. An electric field with a high enough field strength can achieve rapid and complete lysis of any type of cell. Different types of cells have different electric field strength requirements. For example, cells with cell walls require a stronger field strength than cells with only cell membranes. In the embodiment of the present application, the electrolysis of cells is achieved by applying a voltage to the electrolysis device 2.

[0064] After the cell lysis process is completed, the motion device 4 clamps the suction needle 11 to the position of the mass spectrometry injection interface, and uses the induced electrospray device to realize mass spectrometry injection.

[0065] The volume of trace cells is very small, and the contents inside the cells are huge. The average content of each substance is relatively low, and the content is different. It is very difficult to effectively measure the intracellular substances. Traditional mass spectrometry injection interfaces generally use electrospray injection. This soft ionization method is not only friendly to the detection substances, but also convenient and fast. Through continuous spray injection, a stable signal of the sample can be obtained all the time. However, for such trace cell samples, the traditional electrospray (ESI) injection flow rate is too fast. The flow rate of μl / min is too fast for such cell samples, even for trace cell samples, and it is impossible to obtain good detection information. Moreover, for electrospray, the slower the flow rate injection, the smaller the spray droplets produced. Such fine spray droplets are more likely to evaporate and desolvate during flight, requiring less Coulomb explosion, and the better the ionization effect, the better the detection effect of entering the mass spectrometer.

[0066] In the embodiments of this application, induced electrospray (InESI) is used. Unlike traditional electrospray methods that directly physically contact the rear electrode with the sample and then apply electricity, a steel tube 31 matching the outer diameter of the needle 11 is placed outside the needle 11. Without direct contact with the cell sample within the needle 11, the electrospray is generated by simply applying high voltage to the steel tube 31 through a first power supply unit, thereby generating an electric field. Induced electrospray can significantly extend the flow rate, thereby prolonging the signal duration and improving the signal strength.

[0067] In the embodiment of the present application, the steel tube 31 is formed with one end flush and the other closed (the end closest to the tip of the suction needle 11 is closed, and the other end is flush). This not only makes the suction needle 11 more secure, but also introduces a fixed scale, eliminating the need to control the relative distance between the suction needle 11 and the steel tube 31 each time. As long as the size of the drawn suction needle 11 is uniform, the repeatability of the entire process can be guaranteed. This method can also support more flexible and rapid testing, eliminating the need for liquid lines and controls, and further shortening the detection time by aspirating trace cells through the capillary phenomenon of the suction needle 11.

[0068] In the embodiment of the present application, the motion device 4 is used to clamp the suction needle 11 to the position of the cell suspension, and the cell suction device uses the suction needle 11 to absorb the cells in the cell suspension to obtain trace cells; then, the suction needle 11 containing trace cells is clamped from the opening 21 to the electrolysis chamber by the motion device 4, and the cells in the suction needle 11 are electrolyzed by the electrolysis device 2 to obtain intracellular substances; finally, the suction needle 11 containing intracellular substances is clamped to the position of the mass spectrometry injection interface by the motion device 4, and the induced electrospray device includes the suction needle 11 and the steel pipe 31 sleeved on the outside of the suction needle 11. A voltage is applied to the steel pipe 31 by the first power supply unit to cause the cell substance in the suction needle 11 to generate electrospray, thereby realizing the injection processing at the mass spectrometry injection interface. Based on trace cells, the embodiment of the present application adopts a fully automatic processing mode, which can efficiently and quickly complete the entire process from cell separation to lysis to mass spectrometry injection, shortening the entire sample detection time while avoiding manual operation. It can be used without complex training and has reliable repeatability and accuracy.

[0069] In an optional embodiment, as shown in FIG1 , the cell aspiration device further includes a flow control structure 12 , which is connected to the aspiration needle 11 and is used to control the amount of cells aspirated by the aspiration needle 11 .

[0070] The flow control structure 12 can be a syringe pump or an air pump. The suction needle 11 is connected to the flow control structure 12 via a highly airtight pipeline and connector. Airtightness is crucial in the entire suction process, and it plays a decisive role in the time and suction accuracy of the entire suction process. The suction accuracy requirements of the syringe pump or air pump are also very high. Generally, a tube filled with liquid (i.e., a liquid path) tends to have higher accuracy than a tube filled with air (an air path), because gas has a greater volume change than liquid and is more easily expanded and compressed. Airtightness issues will also be reflected in the suction effect.

[0071] In an optional embodiment, the tip diameter of the suction needle 11 is 20-50 μm.

[0072] The tip of the aspiration needle 11 used to aspirate cells has a larger diameter than the cell diameter. For mammalian cells, the diameter is mostly around 10 μm. At this diameter, the resistance to aspiration of cells from the needle tip is very large. If the airtightness of the pipeline is not sufficient, two situations may occur: one is that the piston of the glass syringe installed on the syringe pump (or air pump) moves a long distance without seeing any cell sample being aspirated from the needle tip, or gas directly enters the syringe during the piston's retreat; the other is that after the syringe pump (or air pump) moves a certain distance, no sufficient amount of cell sample is aspirated. Then, after the syringe pump moves a certain distance, the suction force in the tube is greater than the resistance of the needle tip, and a large amount of sample is suddenly aspirated. A syringe pump distance less than this volume will not work. Regardless of the result, it will have a negative impact on the entire aspiration process and the final extraction volume.

[0073] In this embodiment of the present application, the aspiration caliber of the aspiration needle 11 is relaxed to 20-50 μm for aspiration of trace cells (number ≤ 10). (The aspiration needle 11 can be a capillary tube with parameters of an outer diameter of 1.5 mm and an inner diameter of 0.86 mm.) This design of the needle tip caliber allows for the aspiration of multiple cells at a time, while preventing the cells from contacting the tip of the aspiration needle 11 or prematurely rupturing due to excessive pressure during the aspiration process.

[0074] In the embodiment of the present application, a capillary needle with a consistent tip diameter and neck length is obtained using a needle puller according to experimentally determined parameters as the aspiration needle 11. This batch-produced capillary needle is tightly connected to the pipeline using a two-way tube, and a syringe pump is used to control the aspiration volume of a glass syringe fixed to it, ensuring the sealing of the aspiration process and accurately controlling the aspiration volume. As long as the concentration of the cell suspension is appropriate, it can be guaranteed that the aspiration volume is sufficient at one time, and the number of cells in the tube is sufficient for detection. The operator proficiency requirement is low and automation is easy to implement.

[0075] In an optional embodiment, the electrolysis device 2 is connected to a second power supply unit, the voltage of the second power supply unit is 3-4 kV, the duty cycle is 5%-50%, and the frequency is 250-5000 Hz.

[0076] Electrolysis has high requirements for the parameters of the applied voltage. The charging time must be sufficient (the duration of high voltage). The electric field generated by a certain frequency of pulsed high voltage (high voltage, 0, high voltage, 0 switching back and forth) can obtain a better cracking effect on the basis of the same voltage size. How to balance the charging time and pulse frequency requires experimental optimization, and the final cracking time can even be at the ms level. In the embodiment of the present application, the voltage of the second power supply unit is set to 3-4kV, the duty cycle is 5%-50%, and the frequency is 250-5000Hz. On this basis, it is only necessary to control the generation and shutdown of the voltage of the second power supply unit to control the generation and disappearance of the electric field, thereby controlling the start and stop of the electrolysis device 2, which is convenient for realizing automatic control.

[0077] In an optional embodiment, as shown in FIG. 2 , the electrolysis device 2 includes two symmetrical concave members 23 ; the electrolysis chamber is composed of the two concave members 23 .

[0078] In the embodiment of the present application, a 3D-printed electrolysis device 2 is used to efficiently and contactlessly lyse the trace cells within the aspiration needle 11. As shown in Figure 2, the electrolysis device 2 is composed of two symmetrical concave members 23 combined to form an electrolysis chamber. An opening 21 is provided above the electrolysis chamber, and the shape of the opening 21 can be any shape (e.g., rectangular). During lysis, a mechanical device clamps the aspiration needle 11 through this opening 21 and inserts it into the electrolysis chamber.

[0079] In an optional embodiment, as shown in FIG3 , an opening 231 is provided at the center of the concave member 23 , and a hollow fixing member 24 is provided in the opening 231 ;

[0080] The fixing member 24 is provided with a metal sheet at one end located inside the concave member 23 and a metal member is embedded in the other end;

[0081] The metal piece is connected to the second power supply unit, and the metal piece is in contact with the metal sheet;

[0082] The two metal sheets corresponding to the two concave parts 23 form parallel plate electrodes.

[0083] Each of the two concave parts 23 has an opening 231 at its center. The fixing part 24 is located in the opening 231, passing through the inner and outer sides of the concave part 23 (the concave surface is the inner side, and the convex surface is the outer side), and the end located on the inner side extends into the electrolysis chamber. A metal sheet is attached to the end of the fixing part 24 located on the inner side of the concave part 23. After the two concave parts 23 are combined together, the two metal sheets corresponding to the two concave parts 23 can serve as parallel electrode plates in the electrolysis chamber. In order to facilitate the attachment of the metal sheet, the size of the end of the fixing part 24 located in the electrolysis chamber is larger than the size of the opening 231. At the same time, in order to facilitate assembly, the cross-sectional dimensions of the part of the fixing part 24 located in the opening 231 and the part located outside the electrolysis chamber are the same as the cross-sectional dimensions of the opening 231.

[0084] As shown in Figure 4, the fixture 24 in this embodiment of the present application is a hollow structure. One end of the metal member is connected to the second power supply unit, while the other end extends into the hollow portion of the fixture 24 and contacts the metal sheet. When the second power supply unit is controlled to apply voltage to the metal member, a uniform high-voltage electric field is formed between the two metal sheets within the electrolysis chamber, enabling the electrolysis of a small number of cells within the needle 11. Alternatively, the metal member in this embodiment of the present application may be a metal screw.

[0085] In the embodiment of the present application, referring to Figures 2 and 3 , the electrolysis apparatus 2 further includes a latch 25. Mounting holes 232 are provided on the edges of the concave member 23. When assembling the electrolysis apparatus 2, the latch 25 is inserted into two corresponding mounting holes 232 of the two concave members 23 to securely form the electrolysis chamber. Multiple latches 25 and mounting holes 232 are provided, corresponding to each other.

[0086] In an optional embodiment, the voltage of the first power supply unit is 3-6 kV, the frequency is 50-250 Hz, and the duty cycle is 25%-50%.

[0087] The inner diameter of the steel tube 31 in the embodiment of the present application is the same as the outer diameter of the aspiration needle 11, with an outer diameter of 1.7 mm and a length of 2 cm. Unlike conventional electrosprayers, which have poor resistance to isotonic saline solutions, resulting in poor spray quality and difficulty in forming a stable electrospray, the induced electrospray device in the embodiment of the present application can still produce a stable signal at the nanoliter level for over 3 minutes even with physiological saline (PBS) solutions, which are not suitable for spraying.

[0088] In an optional embodiment, the motion device 4 can be a robotic arm, which has sufficient degrees of freedom to meet the requirements of free movement between different points. The robotic arm includes a mechanical claw 41, and the suction needle 11 is provided with a steel tube 31 on the outer shell. In order to facilitate the clamping of the robotic arm, a clamping member 33 is provided on the outer shell of the steel tube 31. The robotic arm clamps the clamping member 33 through the mechanical claw 41 to indirectly clamp the suction needle 11 and move it freely between different points. The clamping member 33 is a hollow structure, and the hollow structure of the clamping member 33 is cylindrical, and the inner diameter of the cylindrical hollow structure matches the outer diameter of the steel tube 31. A hole is provided on the side of the clamping member 33, and the steel tube 31 is connected to the first power supply unit through a metal wire passing through the hole. The first power supply unit includes an electrospray high-voltage power supply 321 and an electrospray high-voltage switch 322. The external voltage of the steel tube 31 is provided by the electrospray high-voltage power supply 321 controlled by the electrospray high-voltage switch 322.

[0089] In an optional embodiment, the system also includes a host computer 5 for remotely controlling the cell aspiration volume of the cell aspiration device, the operation and shutdown of the electrolysis device 2, the operation and shutdown of the induced electrospray device, and controlling the movement device 4 to move the aspiration needle 11 to a specified position. For the application scenario of trace cells, the process from sampling and processing after system startup to obtaining 30 seconds of mass spectrometry data takes only 1 minute. No other interference (chemical reagents) are introduced during the entire online detection process. The resulting data is reliable and reflects the natural state of cells under normal physiological conditions.

[0090] In the embodiment of the present application, full automation is achieved through remote control of the host computer 5. The operation process of the system in the embodiment of the present application is shown in Figure 5. Based on the host computer 5, the motion device 4 is remotely controlled by the host computer 5. Three points (i.e., designated positions, including the position of the cell suspension, the position of the electrolysis chamber, and the position of the mass spectrometry injection interface) can be debugged and set in advance. When moving to the corresponding point, the host computer 5 starts the corresponding function of the device corresponding to the point.

[0091] First, establish a connection with the motion device 4, enable the motion device 4, and move it to the top of the cell suspension. Further enable it, move to the position of the cell suspension, immerse the suction needle 11 in the centrifuge tube 7, and at the same time program control to start the injection pump (or air pump), and start to absorb the cells in the centrifuge tube 7 according to a predetermined amount (0.2-0.5μl, the number of cells absorbed at a time ≤10). Among them, the injection pump (air pump) meets the absorption of trace cells and the flow rate requirements of electrospray, with a minimum of 0.002μl / min, and the flow rate can be controlled by the coding program of the host computer 5. After the absorption is completed, return to the top of the cell suspension, move above the cell lysis point, and then place the needle tip in the electrolysis chamber of the electrolysis device 2. Now host computer 5 sends the pulse signal that meets cracking parameter to acquisition card 6, second power supply unit comprises the electrolysis high-voltage power supply 221 and the electrolysis high-voltage switch 222 that provide voltage for electrolysis device 2, acquisition card 6 sends signal to electrolysis high-voltage switch 222, electrolysis high-voltage switch 222 starts to switch repeatedly with this frequency (electrolysis high-voltage switch 222 itself needs 24V power supply, input kV cracking high voltage needs to convey pulse signal to control, finally output the pulse high voltage of certain frequency), output the pulse high voltage used for cracking, electrolysis absorbs the cell in tube.After cracking, return to above cracking point, move to the position of mass spectrometry injection interface, now acquisition card 6 exports a signal to electrospray high-voltage switch 322 again, carries out mass spectrometry injection.Electrospray high-voltage power supply 321 can provide the DC voltage of 0-6kV, and the switching frequency of electrospray high-voltage switch 322 is between 0-250Hz, and withstand voltage 15kV, is used for controlling generation pulse waveform. The control principle of electrospray high-voltage switch 322 is similar to that of electrolysis high-voltage switch 222. The difference is that electrospray high-voltage switch 322 controls the activation and deactivation of induced electrospray and does not require the high frequency required by electrolysis high-voltage switch 222 during electrolysis. The input voltages of these two switches are controlled by two different high-voltage power supplies. The voltage parameters can be pre-set by the power supplies themselves, and the host computer 5 controls the voltage output and shutdown at the appropriate time. The entire process is completely controlled by the host computer 5, and each operation step is executed sequentially in a timed sequence, with the execution time being preset in advance.

[0092] After completing the mass spectrometry injection, it returns to the starting point, then the motion device 4 is deactivated, and finally the connection with the mechanical device is disconnected, fully automatically realizing the entire process from cell separation to lysis to mass spectrometry injection.

[0093] Among them, in the embodiment of the present application, only the devices involving voltage are controlled by the host computer 5 to control the acquisition card 6, and the acquisition card 6 then controls the output of the corresponding pulsed high voltage. The acquisition card 6 controls the high-voltage power supply (electrolysis high-voltage power supply 221, electrospray high-voltage power supply 321) by judging the digital signal sent by the host 5. The acquisition card 6 will always judge whether the signal sent by the host computer 5 is a high level or a low level. If the high voltage needs to be started at this time, the host computer 5 will send a high level to the acquisition card 6, and the acquisition card 6 will output a signal to the high-voltage switch (electrolysis high-voltage switch 222, electrospray high-voltage switch 322). At this time, the high-voltage switch will switch at a certain frequency and duty cycle.

[0094] In an embodiment of the present application, the electrolysis high-voltage power supply 221 provides a DC high voltage of 0-5kV, and the output voltage is adjusted by a 10kΩ potentiometer. The electrolysis high-voltage switch 222 has a wide range of 0-20kHz to meet the maximum requirements for optimization of the lysis voltage parameters.

[0095] The cell suction device, the electrolysis device 2 and the induced electrospray device in the embodiment of the present application do not interfere with each other. The motion device 4 combines the cell suction device, the electrolysis device 2 and the induced electrospray device to realize the whole process of cell separation (absorption), lysis and mass spectrometry injection. It has high flexibility and can be improved and replaced according to different needs. For example, for samples that do not need to be lysed, only the sample absorption and mass spectrometry injection are required; for samples pre-loaded in the syringe, only mass spectrometry injection is required for detection. The entire system can also perform rapid detection of multiple samples. After the whole process of one sample is completed, the next sample can be directly absorbed to repeat the whole process. No extra operations are required in the middle, and consumables such as the absorption needle are very convenient to disassemble and install. In addition, the cells will not be lysed immediately after absorption, but will be stored in the needle tip of the absorption needle 11. The lysis timing and lysis duration can be selected as needed.

[0096] The entire system in the embodiment of the present application has a wide range of applications. In addition to its use in mass spectrometry, it can also serve as an automatic pipetting station for simple liquid transfer. The transfer can also not use electrospray transfer, but directly use a syringe pump to precisely control the addition of samples.

[0097] In the examples of the present application, the final effect test was performed on the entire mass spectrometry injection system, and LLC mouse lung cancer cells (hereinafter referred to as LLC) were used as experimental materials, and 150mM ammonium bicarbonate was used as a cell resuspension solution. Before the experiment, the cells were resuspended in an aqueous ammonium bicarbonate solution and centrifuged three times (1200r / min, 3min, 4°C) to remove interferences other than the cells. During electrolysis, the second power supply unit was uniformly set to a voltage of 4kV, a frequency of 2kHz, a duty cycle of 10%, and lysis for 20s. During induction electrospray, the first power supply unit was uniformly set to a voltage of 5kV and a frequency of 50Hz. A control experiment was performed by setting a pure solvent group, a non-lysis group, and a lysis group. Figures 6-9 show the mass spectrometry results of LLC mouse lung cancer cells under four conditions: a control (150 mM ammonium bicarbonate aqueous solution), direct injection of LLC without lysis, sonication of LLC (without damaging the material, complete cell lysis in an ice bath until the cell suspension is homogenous and clear, with no bubbles after lysis, and the temperature is less than 21°C), and electrolysis of LLC. The sonication sample serves as a reference lysis standard. The abscissas in Figures 6-9 represent mass-to-charge ratio (m / z), and the ordinates represent intensity.

[0098] The cells used in each control group came from the same culture dish. As shown in Figures 6-9, the lipid information in these two types of cells can be obtained after cell treatment and direct sampling detection, while no effective lipid signal can be seen without treatment.

[0099] As shown in Figure 10, LLC mouse lung cancer cells after fluorescent staining with fluorescein diacetate (FDA) were compared under a microscope before and after electrolysis at a voltage of 4 kV, a frequency of 2 kHz, and a duty cycle of 10%. It can be seen that the dead cells lost fluorescence after electrolysis, proving that the electrolysis of the mass spectrometry injection system in the embodiment of the present application worked.

[0100] As shown in FIG11 , the induced electrospray plume can be clearly seen by irradiating with a laser pen, thereby verifying that the direct injection function of the mass spectrometry injection system in the embodiment of the present application is normal.

[0101] The present embodiment utilizes a multi-degree-of-freedom motion device 4 and a host computer 5 to achieve label-free, fully automated online sample injection testing for trace cells. This method is suitable for rare and difficult-to-obtain cells, and the test results can, to a certain extent, reflect the differences between cell populations. Differences between different tumor cell populations are of great significance for individualized treatment plans and the discovery of potential molecules for use as biomarkers in disease diagnosis and treatment monitoring. A more targeted approach is employed for this application scenario involving trace cells: aspiration is performed through precise fluidic control; the lysis process utilizes contactless electrolysis, which is both highly efficient and harmless to the sample being tested; and the mass spectrometry injection process utilizes induced electrospray, which not only does not damage the sample being tested but also significantly extends the duration of electrospray, making it more suitable for extremely small samples such as cells. These three processes are integrated to maximize the advantages of each component, significantly shortening the overall test run time and improving efficiency while also protecting the sample being tested. This enables online processing and injection, resulting in more reliable data, eliminating the need for manual operation, and providing high repeatability and precision.

[0102] Based on the same principle as the system provided in the embodiment of the present application, the embodiment of the present application also provides a mass spectrometry sampling method for automatic analysis of trace cells. Based on the above-mentioned mass spectrometry sampling system for automatic analysis of trace cells, as shown in FIG12 , the method includes:

[0103] Step 1201, aspirating a target number of cells from a cell suspension using an aspiration needle 11;

[0104] Step 1202 , using the movement device 4 , the aspiration needle 11 containing cells is moved from the opening 21 into the electrolysis chamber of the electrolysis device 2 ;

[0105] Step 1203: using the electrolysis device 2 to electrolyze the cells in the aspiration needle 11 to obtain intracellular substances;

[0106] Step 1204 , using the motion device 4 to move the aspiration needle 11 containing the intracellular material to the position of the mass spectrometry injection interface;

[0107] Step 1205 , applying voltage to the steel tube 31 outside the aspiration needle 11 through the first power supply unit, so as to inject the cell material in the aspiration needle 11 into the mass spectrometry injection interface in an electrospray manner.

[0108] In an embodiment of the present application, a cell aspiration device uses an aspiration needle 11 to aspirate cells from a cell suspension to obtain trace cells; then, the aspiration needle 11 containing trace cells is clamped from the opening 21 into the electrolysis chamber by the motion device 4, and the cells in the aspiration needle 11 are electrolyzed by the electrolysis device 2 to obtain intracellular substances; finally, the aspiration needle 11 containing intracellular substances is clamped to the position of the mass spectrometry injection interface by the motion device 4, and the induced electrospray device includes the aspiration needle 11 and a steel pipe 31 sleeved on the outside of the aspiration needle 11, and a voltage is applied to the steel pipe 31 by the first power supply unit to generate an electrospray of the cell substance in the aspiration needle 11, thereby realizing the injection processing at the mass spectrometry injection interface. Based on trace cells, the embodiment of the present application adopts a fully automatic processing mode, which can efficiently and quickly complete the entire process from cell separation to lysis to mass spectrometry injection, shortening the entire sample detection time while avoiding manual operation. It can be used without complex training and has reliable repeatability and accuracy.

[0109] In an optional embodiment, the electrolysis device 2 is used to electrolyze cells in the aspiration needle 11 to obtain intracellular substances, including:

[0110] Controlling the second power supply unit to release voltage to the electrolysis device 2 to electrolyze the cells in the aspiration needle 11 to obtain intracellular substances;

[0111] The voltage of the second power supply unit is 3-4 kV, the duty cycle is 5%-50%, and the frequency is 250-5000 Hz.

[0112] Each process in the mass spectrometry injection method for automatic analysis of trace cells provided in the embodiment of the present application can implement each module implemented in the system embodiments of Figures 1 to 11. To avoid repetition, they will not be described here.

[0113] The mass spectrometry injection method for automatic analysis of micro cells in the embodiments of the present application can execute the mass spectrometry injection system for automatic analysis of micro cells provided in the embodiments of the present application, and its implementation principle is similar. The actions performed in each step of the mass spectrometry injection method for automatic analysis of micro cells in each embodiment of the present application correspond to the modules and units in the mass spectrometry injection system for automatic analysis of micro cells in each embodiment of the present application. For the detailed functional description of each step of the mass spectrometry injection method for automatic analysis of micro cells, please refer to the description of the corresponding mass spectrometry injection system for automatic analysis of micro cells shown in the previous text, and will not be repeated here.

[0114] The above description is merely a preferred embodiment of the present application and an illustration of the technical principles employed. Those skilled in the art should understand that the scope of disclosure in this application is not limited to the technical solutions formed by a specific combination of the above-mentioned technical features, but also encompasses other technical solutions formed by any combination of the above-mentioned technical features or their equivalents without departing from the above-mentioned disclosed concepts. For example, a technical solution formed by replacing the above-mentioned features with (but not limited to) technical features with similar functions disclosed in this application.

Claims

1. A mass spectrometry injection system for microcellular automatic analysis, characterized in that, The system includes a cell aspiration device, an electrolysis device, an induced electrospray device, and a motion device; The cell aspiration device includes an aspiration needle for aspirating cells in a cell suspension; The electrolysis device includes an electrolysis chamber with an opening above it; The induced electrospray device includes the aspiration needle and a steel tube sleeved outside the aspiration needle. The steel tube is connected to a first power supply unit, which is used to apply a voltage to the steel tube so that the cell substances in the aspiration needle are injected in the form of electrospray at a mass spectrometry injection interface; The motion device is used to clamp the aspiration needle and move the aspiration needle to a specified position; the specified positions include the position of the cell suspension, inside the electrolysis chamber, and the position of the mass spectrometry injection interface.

2. The mass spectrometry injection system for microcellular automatic analysis according to claim 1, characterized in that, The tip diameter of the aspiration needle is 20 - 50 μm.

3. The mass spectrometry injection system for microcellular automatic analysis according to claim 1, characterized in that, The electrolysis device is connected to a second power supply unit. The voltage of the second power supply unit is 3 - 4 kV, the duty cycle is 5% - 50%, and the frequency is 250 - 5000 Hz.

4. The mass spectrometry injection system for microcellular automatic analysis according to claim 3, characterized in that, The electrolysis device includes two symmetrical concave parts; The electrolysis chamber is formed by combining the two concave parts.

5. The mass spectrometry injection system for microcellular automatic analysis according to claim 4, characterized in that, An opening is provided at the center of the concave part, and a hollow fixing part is provided in the opening; One end of the fixing part located inside the concave part is provided with a metal sheet, and the other end is embedded with a metal part; The metal part is connected to the second power supply unit, and the metal part is in contact with the metal sheet; The two metal sheets corresponding to the two concave parts form a parallel plate electrode.

6. The mass spectrometry injection system for microcellular automatic analysis according to claim 1, characterized in that, The voltage of the first power supply unit is 3 - 6 kV, the frequency is 50 - 250 Hz, and the duty cycle is 25% - 50%.

7. The mass spectrometry injection system for microcellular automatic analysis according to claim 1, characterized in that, The cell aspiration device further includes a flow control structure, which is connected to the aspiration needle and is used to control the cell aspiration amount of the aspiration needle.

8. A mass spectrometry injection method for microcellular automatic analysis, based on the mass spectrometry injection system for microcellular automatic analysis according to any one of claims 1 - 8, characterized in that, The method includes: Using the aspiration needle to aspirate a target number of cells from the cell suspension; Using the motion device to move the aspiration needle containing cells from the opening to the electrolysis chamber of the electrolysis device; Using the electrolysis device to lyse the cells in the aspiration needle by electrolysis to obtain intracellular substances; Using the motion device to move the aspiration needle containing the intracellular substances to the position of the mass spectrometry injection interface; Applying a voltage to the steel tube outside the aspiration needle through the first power supply unit to realize the injection of the cell substances in the aspiration needle in the form of electrospray at the mass spectrometry injection interface.

9. The mass spectrometry injection system for microcellular automatic analysis according to claim 8, characterized in that, The step of using the electrolysis device to lyse the cells in the aspiration needle by electrolysis to obtain intracellular substances includes: Controlling the second power supply unit to release a voltage to act on the electrolysis device to electrolyze the cells in the aspiration needle to obtain the intracellular substances; Wherein, the voltage of the second power supply unit is 3 - 4 kV, the duty cycle is 5% - 50%, and the frequency is 250 - 5000 Hz.

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