Full-automatic high-flux high-coverage single-cell lipidomics analysis device and method

Abstract

The invention relates to a full-automatic high-flux high-coverage single-cell lipidomics analysis device and method, and the device comprises a valve which is provided with a quantitative groove; the cell suspension injection assembly is communicated with the sample inlet of the quantitative tank through a capillary tube; the capillary chromatographic column spray needle comprises a body and a tip end, the body is communicated with the chromatographic column connector of the quantitative groove, the tip end is aligned with a sample injection conical opening of the mass spectrometer, the capillary chromatographic column spray needle is filled with filler, the capillary chromatographic column spray needle is sleeved with a metal tube, and the metal tube is connected with the capillary chromatographic column spray needle. An electric field is applied to the metal tube in time; the supercritical fluid injection part is communicated with the mobile phase inlet of the quantitative tank and is used for injecting supercritical fluid into the quantitative tank, so that a single cell sample is extracted on line. In a single experiment, the device can quickly obtain the number of analysis samples with statistical significance.

Description

Technical Field

[0001] The present invention relates to a fully automatic high-throughput and high-coverage single-cell lipidomics analysis device and method, belonging to the technical field of biological mass spectrometry analysis. Background Art

[0002] Cellular lipids play a key role in cellular life, deeply involved in important processes such as cell signaling, energy storage, and metabolic regulation. Their complex and diverse chemical structures and unique functional properties provide valuable research directions for in-depth exploration of the internal mechanisms of cellular life. Single-cell lipidome analysis, as a cutting-edge technology, can accurately reveal the types, contents, and distribution characteristics of lipids at the single-cell level, which is of irreplaceable significance for understanding cellular differentiation, developmental processes, and the occurrence and development of diseases.

[0003] Current lipidomics analysis is primarily performed using instruments such as high-performance liquid chromatography-mass spectrometry (HPLC-MS). However, due to the extremely small size of individual cell samples, single-cell analysis is not feasible. Furthermore, the analysis of each cell is equivalent to a sample in traditional metabolomics, while single-cell analysis requires analyzing a large number of cells to achieve a statistically significant number of samples within a single experiment. Consequently, single-cell analysis using instruments such as HPLC-MS suffers from drawbacks such as lengthy analysis times and severe sample dilution, making it difficult to meet the high sensitivity, high resolution, and high throughput requirements of single-cell lipidomics studies. In recent years, various direct-injection mass spectrometry methods have been developed for single-cell metabolite analysis, such as nanospray tip aspiration cytoplasmic electrospray mass spectrometry, microdroplet extraction single-cell metabolite analysis, and intact cell injection mass spectrometry. However, due to the lack of chromatographic separation, signals from high-abundance components mask those of low-abundance components, and readily ionizable components suppress the ionization of less readily ionizable components, resulting in the non-detection of low-abundance components and low lipidomics coverage. In addition, in terms of sample pre-processing, the current single-cell sorting technology requires cells to be selected one by one for extraction, which greatly affects the cell analysis throughput and also introduces errors caused by human operation. Summary of the Invention

[0004] In response to the above technical problems, the present invention provides a fully automatic high-throughput and high-coverage single-cell lipidomics analysis device and method. The present invention innovatively combines a cell suspension pressure injector with an electrically controlled four-way nanoliter quantitative injection valve to design a single-cell fully automatic injection device. By precisely controlling the pressure and tube diameter, the cell suspension can enter the system stably and orderly. At the same time, the electrically controlled four-way nanoliter quantitative injection valve can accurately control the injection volume. By optimizing the concentration of the cell suspension, a single cell is included in each injection volume, thereby achieving the required fully automatic high analysis throughput. In a single experiment, the device can quickly obtain a statistically significant number of analysis samples.

[0005] To achieve the above object, the present invention adopts the following technical solutions: A fully automated high-throughput, high-coverage single-cell lipidomics analysis device, comprising: A valve member, wherein the valve member is provided with a quantitative groove; A cell suspension injection component, the cell suspension injection component is connected to the sample injection port of the quantitative tank through a capillary; A capillary chromatographic column spray needle, comprising a body and a tip, wherein the body is connected to the chromatographic column interface of the quantitative tank, the tip is aligned with the injection cone of the mass spectrometer, the capillary chromatographic column spray needle is filled with filler, and the outer portion of the capillary chromatographic column spray needle is sheathed with a metal tube, to which an electric field is applied in a timely manner; A supercritical fluid injection component is connected to the mobile phase inlet of the quantitative tank and is used to inject supercritical fluid into the quantitative tank so that a single cell sample can be extracted online.

[0006] In the fully automatic high-throughput and high-coverage single-cell lipidomics analysis device, preferably, the valve component is a four-way nanoliter quantitative injection valve, and the capacity of the quantitative tank is 8-12nL.

[0007] In the fully automatic high-throughput and high-coverage single-cell lipidomics analysis device, preferably, the cell suspension injection component includes an air pump and a cell suspension pressure injector placed in the air pump, and the cell suspension pressure injector is connected to the sample injection port of the quantitative tank through a capillary.

[0008] The fully automatic high-throughput and high-coverage single-cell lipidomics analysis device preferably has an air pressure of 0.3-0.5 MPa applied by the air pump, and a concentration of the cell suspension in the cell suspension pressure injector of 1×10 5 -2×10 5 pieces / mL.

[0009] In the fully-automatic high-throughput and high-coverage single-cell lipidomics analysis device, preferably, the inner diameter of the capillary is 20-30 μm.

[0010] In the fully automatic high-throughput and high-coverage single-cell lipidomics analysis device, preferably, the filler is located in the body, and one end of the filler is located at the large-diameter end of the tip, and the length of the filler is 4-8 cm.

[0011] In the fully-automatic high-throughput and high-coverage single-cell lipidomics analysis device, preferably, the diameter of the tip is an inner diameter of 0.5-5 μm, and the length of the tip is 1-3 mm.

[0012] In the fully automatic high-throughput and high-coverage single-cell lipidomics analysis device, preferably, the distance between the tip and the injection cone of the mass spectrometer is 4-6 mm.

[0013] In the fully automatic high-throughput and high-coverage single-cell lipidomics analysis device, preferably, the supercritical fluid injection component is a supercritical fluid pump, the fluid flow rate is 80-120 μL / min, the fluid temperature is 50-70°C, and the fluid pressure is 8-12 MPa.

[0014] A second aspect of the present invention provides a method for operating a fully automated high-throughput, high-coverage single-cell lipidomics analysis device, comprising the following steps: preparing a cell suspension, placing the cell suspension in a cell suspension pressure injector, placing the cell suspension pressure injector in an air pump, and then inserting a capillary into the cell suspension pressure injector and connecting it to a sample injection port of a four-way nanoliter quantitative injection valve; Connecting the mobile phase inlet of the four-way nanoliter quantitative injection valve to the supercritical fluid pump, connecting the chromatographic column interface to the body of the capillary chromatographic column spray needle, and aligning the tip of the capillary chromatographic column spray needle with the injection cone of the mass spectrometer; The four-way nanoliter quantitative injection valve is adjusted to the injection mode, and pressure is applied to the air pump, so that the cell suspension is pumped into the four-way nanoliter quantitative injection valve and becomes a single cell; The four-way nanoliter quantitative injection valve is adjusted to the analysis mode, the supercritical fluid pump provides supercritical fluid to extract single cells online, and then enters the capillary chromatographic column spray needle for chromatographic separation, while an electric field is applied to the metal tube to achieve ionization, and finally enters the mass spectrometer for mass spectrometry analysis; After the analysis of a single cell is completed, the four-way nanoliter quantitative injection valve is switched to the injection mode, the air pump is turned on, and the injection analysis of the next cell is performed.

[0015] The present invention has the following advantages due to the adoption of the above technical solution: 1. The single-cell fully automated sampling device designed in this paper overcomes the throughput limitations of traditional sampling methods. Through the coordinated operation of a cell suspension pressure injector and an electrically controlled four-way nanoliter quantitative injection valve, automated, high-throughput single-cell sampling is achieved, greatly improving experimental efficiency and facilitating large-scale single-cell lipidomics studies.

[0016] 2. The application of supercritical fluid chromatography technology and the high resolution of mass spectrometry enable the device to accurately detect and analyze trace lipids in single cells, effectively avoiding the information loss caused by sample dilution in traditional methods and ensuring the high sensitivity and accuracy of the analysis results.

[0017] 3. The device and method of the present invention can be widely applied to various cell suspensions, eliminating the need for complex pretreatment procedures and is applicable to both primary cells and cell lines. Furthermore, this technology can be used for live cell analysis, preserving the physiological state of cells to the greatest extent possible, providing a powerful tool for studying the lipidomic characteristics of cells in their natural state. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 This is the operational flow chart of the fully automated high-throughput and high-coverage single-cell lipidomics analysis device; Figure 2 Schematic diagram of using a pressure injection device to disperse single cells in a cell suspension into a quantitative tank; Figure 3 Schematic diagram of online extraction of single cells using supercritical fluid and lipidome mass spectrometry analysis using a capillary column spray needle; Figure 4 Semi-quantitative standard curves established for lipid standard analysis using this device; Figure 5 Extracted ion chromatograms of four marker lipids for cellular lipidome analysis using the device of the present invention for ten consecutive injections; The reference numerals in the figures are as follows: 1-cell suspension pressure injector; 2-four-way nanoliter quantitative injection valve; 3-quantitation tank; 4-supercritical fluid pump; 5-capillary column spray needle; 6-metal tube; 7-mass spectrometer injection cone.

[0019] The markings on the four-way injection valve 2 are: W waste liquid outlet, S sample injection port, P mobile phase inlet, and C chromatographic column interface. DETAILED DESCRIPTION

[0020] To make the objectives, technical solutions, and advantages of the present invention more clear, the technical solutions of the present invention are described clearly and completely below. Obviously, the embodiments described are only some of the embodiments of the present invention, not all of them. All other embodiments derived by ordinary persons in this field based on the embodiments of the present invention without creative effort are within the scope of protection of the present invention.

[0021] Unless otherwise defined, the technical or scientific terms used in the present invention shall have the usual meanings understood by persons of ordinary skill in the field to which the present invention belongs. The words "first", "second", "third", "fourth" and similar terms used in the present invention do not indicate any order, quantity or importance, but are only used to distinguish different components. Words such as "include" or "comprise" mean that the elements or objects preceding the word include the elements or objects listed after the word and their equivalents, without excluding other elements or objects. Words such as "connect" or "connected" are not limited to physical or mechanical connections, but may include electrical connections, whether direct or indirect.

[0022] For ease of description, spatially relative terms may be used herein to describe the relationship of one element or feature relative to another element or feature as shown in the figures, such as "inside," "outside," "inner side," "outer side," "lower," "upper," etc. Such spatially relative terms are intended to encompass different orientations of the device in use or operation in addition to the orientation depicted in the figures.

[0023] In recent years, various direct-injection mass spectrometry methods have been developed for single-cell metabolite analysis, such as nanospray needle-tip aspiration cytoplasmic electrospray mass spectrometry, microdroplet extraction single-cell metabolite analysis, and intact cell injection mass spectrometry. However, due to the lack of chromatographic separation, the signals of high-abundance components mask the signals of low-abundance components, and the easily ionized components suppress the ionization of the less ionized components, resulting in the inability to detect low-abundance components and low coverage of lipidome detection. In addition, in terms of sample pretreatment, current single-cell sorting technology requires the selection of cells one by one for extraction, which greatly reduces the throughput of cell analysis and introduces errors caused by human operation.

[0024] Based on the current single-cell sorting technology, cells need to be selected one by one for extraction, which greatly affects the cell analysis throughput and also introduces the problem of human operation error. The present invention provides a fully automatic high-throughput and high-coverage single-cell lipidomics analysis device and method. The use of this device is divided into two parts. First, a cell suspension pressure injector and an electrically controlled four-way nanoliter quantitative injection valve are used for automated single-cell injection. Then, a supercritical fluid is used to perform online extraction and chromatography-mass spectrometry analysis on individual cells. The automated injection device greatly improves the efficiency of obtaining single-cell samples from a large number of cells, avoiding the errors caused by manual operation; supercritical fluid online extraction avoids dilution and provides high sensitivity; supercritical fluid chromatography-mass spectrometry has a fast analysis speed and mature technology, providing high-coverage lipidomics analysis results.

[0025] like Figure 1 、 Figure 2 As shown, the fully automatic high-throughput, high-coverage single-cell lipidomics analysis device of the present invention comprises: a cell suspension pressure injector 1, a four-way nanoliter quantitative injection valve 2, a supercritical fluid pump 4, and a capillary column spray needle 5. The cell suspension pressure injector 1 is placed in the air pump and connected to the sample injection port S of the four-way nanoliter quantitative injection valve 2 via a capillary tube. The mobile phase inlet P of the four-way nanoliter quantitative injection valve 2 is connected to the supercritical fluid pump 4. The chromatographic column interface C of the four-way nanoliter quantitative injection valve 2 is connected to the body of the capillary column spray needle 5. The tip of the capillary column spray needle 5 is aligned with the injection cone of the mass spectrometer, preferably at a distance of about 5 mm. A metal tube 6 is wrapped around the capillary column spray needle 5, and a high-voltage electric field is applied to the metal tube 6 to achieve ionization through the principle of inductive electrospray.

[0026] Further, if Figure 1 As shown, a quantitative tank 3 is provided on the four-way nanoliter quantitative injection valve 2, and its capacity is 8-12nL, preferably 10nL. The concentration of the cell suspension in the cell suspension pressure injector 1 is 1×10 5 -2×10 5 / mL, preferably 10 5 Cells / mL. Cell suspensions can be prepared using tissue samples, primary cell samples, or cultured cell lines. The inner diameter of the capillary is 20-30 μm, preferably 25 μm, which only accommodates single cells rather than cell clusters. When the cell suspension pressure injector 1 applies an air pressure of preferably 0.4 MPa through an air pump, the cells are pumped into the four-way nanoliter quantitative injection valve 2. By controlling the concentration of the suspension, an average of 1 cell is contained in 10 nL of suspension, so that a single cell sample is contained on average in the 10 nL quantitative tank. Whether the cells are pumped in or not is controlled by opening and closing the air pump.

[0027] like Figure 1As shown, the capillary chromatographic column spray needle 5 comprises a body and a tip. The body is filled with a filler, with one end of the filler located at the larger diameter end of the tip. The filler length is 4-8 cm, preferably 6 cm. The inner diameter of the tip is 0.5-5 μm, the length of the tip is 1-3 mm, and the distance between the tip and the injection cone of the mass spectrometer is 4-6 mm, preferably 5 mm. The capillary chromatographic column spray needle 5 of the present invention is homemade. The homemade method is as follows: first, a capillary with an outer diameter of preferably 360 μm and an inner diameter of 100 μm is drawn using a laser drawing instrument (such as a Shutter-P2000) to obtain a tip with an inner diameter of 0.5-5 μm and a length of 1-3 mm. Then, a suspension containing chromatographic filler particles is pumped into the capillary chromatographic column using a liquid pump to form the capillary chromatographic column. The chromatographic filler can be 3-μm Supersil ODs2 silica gel particles or other particle size fillers. The filler length is preferably about 6 cm. The filler is packed into the spray needle body, with one end adjacent to the larger diameter end of the tip.

[0028] Further, if Figure 1 As shown, the supercritical fluid pump 4 can provide supercritical fluid as an extractant and mobile phase, the fluid ratio is preferably 75% supercritical carbon dioxide + 25% methanol, the fluid pressure is stabilized at 50-70°C by a back pressure controller, the fluid pressure is 8-12MPa to maintain a supercritical state, the preferred temperature is 60°C, the preferred fluid pressure is 10MPa, and the fluid flow rate is 80-120μL / min, preferably 100μL / min.

[0029] like Figure 2 As shown, the four-way nanoliter quantitative injection valve 2 switches between two modes: Injection mode, a single-cell sample is injected into the built-in 10nL quantitative reservoir 3; in Analysis mode, the quantitative reservoir 3 is connected to the flow path of the supercritical fluid pump 4 and the capillary column needle 5, allowing the cell sample to be extracted, separated, and analyzed online. Switching between these two modes is controlled by an electric switch, eliminating the need to change the piping connection, making it simple and easy to operate.

[0030] like Figure 3As shown, in analysis mode, the four-way nanoliter quantitative injection valve 2 is switched to analysis mode (Position B) via an electric switch. A supercritical fluid pump provides 75% supercritical carbon dioxide + 25% methanol as the extractant and mobile phase. The fluid pressure is stabilized at 60°C and 10 MPa by a backpressure controller to maintain the supercritical state, and the fluid flow rate is approximately 100 μL / min. Online extraction, chromatographic separation, and mass spectrometric analysis of a single cell are performed. The chromatographic analysis lasts approximately 2.5 minutes. Electrospray ionization is induced by applying a high-voltage electric field to the metal tube 6 wrapped around the capillary column needle 5. The mass spectrometer enters data acquisition mode to record lipidomic data. After the single-cell analysis is completed, the four-way nanoliter quantitative injection valve 2 is switched to injection mode (Position A) via an electric switch, and the air pump is activated to inject the next cell.

[0031] Specifically, the operating method of the fully automatic high-throughput and high-coverage single-cell lipidomics analysis device of the present invention is as follows: Prepare a cell suspension, place the cell suspension in a cell suspension pressure injector 1, place the cell suspension pressure injector 1 in an air pump, and then insert a capillary into the cell suspension pressure injector 1 and connect it to the sample injection port S of a four-way nanoliter quantitative injection valve 2; Connect the mobile phase inlet P of the four-way nanoliter quantitative injection valve 2 to the supercritical fluid pump 4, connect the chromatographic column interface C to the body of the capillary chromatographic column spray needle 5, and align the tip of the capillary chromatographic column spray needle 5 with the injection cone of the mass spectrometer; Adjust the four-way nanoliter quantitative injection valve 2 to the injection mode, apply pressure to the air pump, and the cell suspension is pumped into the four-way nanoliter quantitative injection valve 2 and becomes a single cell; The four-way nanoliter quantitative injection valve 2 is set to the analysis mode. The supercritical fluid pump 4 provides supercritical fluid for online extraction of single cells. The supercritical fluid then enters the capillary column spray needle 5 for chromatographic separation. At the same time, an electric field is applied to the metal tube 6 to achieve ionization. Finally, the supercritical fluid enters the mass spectrometer for mass spectrometry analysis. After the single cell analysis is completed, the four-way nanoliter quantitative injection valve 2 is switched to the injection mode, the air pump is turned on, and the next cell injection analysis is performed.

[0032] The technical solution of the present invention is described in detail below with reference to specific examples.

[0033] like Figure 5As shown, using a cell suspension of a cultured cell line as an example, 10 consecutive injections were performed, recording lipidomic data for 10 cells and presenting extracted ion chromatograms of four signature lipid species. This demonstrates the robustness of the analytical method presented herein, and the comparability of single-cell lipidomic data obtained by chromatography-mass spectrometry with conventional lipidomic data. Calculations indicate that the analysis of a single cell takes less than 2.5 minutes. A statistically significant number of cell samples (>200 cells / 8 hours) can be analyzed within a single experiment.

[0034] like Figure 4 As shown, lipid standard solutions were prepared and injected into four-way nanoliter quantitative injection valve 2 at varying concentrations for analysis to establish a semi-quantitative standard curve (as shown in Table 1). This standard curve can be used for semi-quantitative analysis of single-cell lipids.

[0035] Table 1 Semi-quantitative standard curve

[0036] The above results demonstrate that the device has the capability of semi-quantitative analysis of single-cell lipidome.

[0037] Single-cell lipidomics reveals the types, content, and distribution of lipids at the single-cell level, contributing to our understanding of cellular differentiation, development, and the development and progression of diseases. Currently, the standard technical process for lipidomics analysis involves extraction of cellular lipids, chromatographic separation, and mass spectrometry detection. These manual processes limit analytical throughput and introduce human errors, making fully automated, high-throughput, and high-coverage single-cell lipidomics analysis impossible.

[0038] This invention combines a cell suspension pressure injector (1) with an electrically controlled four-way nanoliter quantitative injection valve (2) to design a fully automated device for capturing single cell samples, performing online extraction, chromatographic separation, and mass spectrometry detection. This device achieves the high throughput and coverage required for single-cell lipidomics analysis, enabling statistically significant sample analysis (>200 single-cell samples / 8 hours) within a single experiment. Supercritical carbon dioxide fluid has been demonstrated to be an excellent solvent and chromatographic mobile phase for cellular lipid analysis. By integrating supercritical fluid extraction, supercritical fluid chromatography, and mass spectrometry analysis online with the four-way nanoliter quantitative injection valve (2), the device enables rapid and detailed analysis of single-cell lipidomes. This method is universally applicable to cell suspensions prepared from various samples, eliminating the need for complex pretreatment steps and enabling live cell analysis. It holds broad application prospects in stem cell differentiation research, tumor cell typing, and clinical diagnosis. This device will become the ultimate tool for single-cell lipidomics analysis, ushering life sciences into an era of comprehensive analysis of single-cell metabolism.

[0039] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or make equivalent replacements for some of the technical features therein. However, these modifications or replacements do not deviate the essence of the corresponding technical solutions from the spirit and scope of the technical solutions of the various embodiments of the present invention.

Claims

1. A fully automatic high-throughput and high-coverage single-cell lipidomics analysis device, characterized in that: include: A valve member, wherein the valve member is provided with a quantitative groove (3); A cell suspension injection assembly, the cell suspension injection assembly being connected to the sample injection port (S) of the quantitative tank (3) via a capillary tube; A capillary chromatographic column spray needle (5), the capillary chromatographic column spray needle (5) comprising a body and a tip, the body being connected to the chromatographic column interface (C) of the quantitative tank (3), the tip being aligned with the injection cone of the mass spectrometer, the capillary chromatographic column spray needle (5) being filled with a filler, the outer portion of the capillary chromatographic column spray needle (5) being sheathed with a metal tube (6), the metal tube (6) being applied with an electric field at an appropriate time; A supercritical fluid injection component is connected to the mobile phase inlet (P) of the quantitative tank (3) and is used to inject supercritical fluid into the quantitative tank (3) so that a single cell sample can be extracted online.

2. The fully automatic high-throughput and high-coverage single-cell lipidomics analysis device according to claim 1, characterized in that The valve component is a four-way nanoliter quantitative injection valve (2), and the capacity of the quantitative tank (3) is 8-12 nL.

3. The fully automatic high-throughput and high-coverage single-cell lipidomics analysis device according to claim 1, characterized in that The cell suspension injection assembly comprises an air pump and a cell suspension pressure injector (1) placed in the air pump, and the cell suspension pressure injector (1) is connected to the sample injection port (S) of the quantitative tank (3) through a capillary tube.

4. The fully automatic high-throughput and high-coverage single-cell lipidomics analysis device according to claim 3, characterized in that The air pressure applied by the air pump is 0.3-0.5 MPa, and the concentration of the cell suspension in the cell suspension pressure injector (1) is 1×10 5 -2×10 5 pieces / mL.

5. The fully automatic high-throughput and high-coverage single-cell lipidomics analysis device according to claim 1, characterized in that: The inner diameter of the capillary is 20-30 μm.

6. The fully automatic high-throughput and high-coverage single-cell lipidomics analysis device according to claim 1, characterized in that: The filler is located in the body, and one end of the filler is located at the large-diameter end of the tip. The length of the filler is 4-8 cm.

7. The fully automatic high-throughput and high-coverage single-cell lipidomics analysis device according to claim 1, characterized in that: The inner diameter of the tip is 0.5-5 μm, and the length of the tip is 1-3 mm.

8. The fully automatic high-throughput and high-coverage single-cell lipidomics analysis device according to claim 1, characterized in that: The distance between the tip and the injection cone of the mass spectrometer is 4-6 mm.

9. The fully automatic high-throughput and high-coverage single-cell lipidomics analysis device according to claim 1, characterized in that: The supercritical fluid injection component is a supercritical fluid pump (4), the fluid flow rate is 80-120 μL / min, the fluid temperature is 50-70° C., and the fluid pressure is 8-12 MPa.

10. A method for operating a fully automated high-throughput, high-coverage single-cell lipidomics analysis device, characterized in that: The steps include: Prepare a cell suspension, place the cell suspension in a cell suspension pressure injector (1), place the cell suspension pressure injector (1) in an air pump, and then insert a capillary into the cell suspension pressure injector (1) and connect it to a sample injection port (S) of a four-way nanoliter quantitative injection valve (2); The mobile phase inlet (P) of the four-way nanoliter quantitative injection valve (2) is connected to the supercritical fluid pump (4), and the chromatographic column interface (C) is connected to the body of the capillary chromatographic column spray needle (5), and the tip of the capillary chromatographic column spray needle (5) is aligned with the injection cone of the mass spectrometer; The four-way nanoliter quantitative injection valve (2) is adjusted to the injection mode, and pressure is applied to the air pump, so that the cell suspension is pumped into the four-way nanoliter quantitative injection valve (2) and becomes a single cell; The four-way nanoliter quantitative injection valve (2) is adjusted to the analysis mode, the supercritical fluid pump (4) provides supercritical fluid to extract the single cell online, and then enters the capillary chromatographic column spray needle (5) for chromatographic separation, and at the same time, an electric field is applied to the metal tube (6) to achieve ionization, and finally enters the mass spectrometer for mass spectrometry analysis; After the analysis of a single cell is completed, the four-way nanoliter quantitative injection valve (2) is switched to the injection mode, the air pump is turned on, and the injection analysis of the next cell is performed.

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