A system and method for continuous single-cell mass spectrometry analysis based on ultrasound-enabled sample injection

Abstract

The application discloses a kind of single-cell mass spectrum analysis system and method based on ultrasonic continuous sample feeding, it is related to single-cell mass spectrum analysis technical field, including: ultrasonic module, for cell suspension is carried out ultrasonic vibration;Sample feeding module is connected with ultrasonic module, for the cell sample in cell suspension is carried out continuous sample feeding, its export end is aligned with the sampling port position of mass spectrometer;Visual adjustment module, its observation port is towards the sampling port position of mass spectrometer and the export end of sample feeding module, for the sampling port position of mass spectrometer and the export end of sample feeding module are aligned.Ultrasonic module includes centrifugal tube, sample feeding bottle, gasket, piezoelectric ceramic piece connected with centrifugal tube outer wall, circuit board connected with piezoelectric ceramic piece, power switch.The application overcomes the low single-cell utilization rate and detection flux problem in existing single-cell mass spectrum analysis, guarantees the integrity of live single cell and the high signal-to-noise ratio of single-cell mass spectrum detection signal.

Description

Technical Field

[0001] This invention relates to the field of single-cell mass spectrometry analysis technology, and more specifically to a single-cell mass spectrometry analysis system and method based on continuous ultrasound sample introduction. Background Technology

[0002] Cells are the most basic structural and functional building blocks of organisms. Traditional analytical methods focus on a specific population of cells, neglecting the heterogeneity between cells. The root cause of this heterogeneity lies in the random processes that occur during cellular metabolism. Therefore, developing a simple and effective single-cell analysis method is crucial for understanding these random processes. Mass spectrometry has become the primary method for single-cell analysis due to its high sensitivity, label-free operation, simultaneous analysis of multiple substances, relative quantification, and powerful structural characterization capabilities. From the perspective of ionization technology, single-cell mass spectrometry analysis methods currently mainly include three categories: secondary ion mass spectrometry (SIMS), matrix-assisted laser desorption / ionization mass spectrometry (MALDI-MS), and electrospray ionization mass spectrometry (ESI-MS). SIMS and MALDI-MS require complex sample preparation and ionization in a high-vacuum environment, which significantly damages cell samples and reduces the cell throughput. ESI-MS, using an atmospheric pressure ion source, eliminates the need for additional sample processing, thus improving the sensitivity of single-cell mass spectrometry analysis.

[0003] Intact live single-cell electroemission mass spectrometry (ILCEI-MS) is suitable for complex multiphase cell samples with intact viable cells. It requires no sheath fluid or sheath gas assistance and significantly reduces ion inhibition and matrix effects. However, the current ILCEEI-MS method has very low single-cell utilization and detection throughput, mainly because cell sedimentation prevents the cell sample from entering the constant-diameter capillary nozzle in time, thus failing to be detected by mass spectrometry. Therefore, establishing a single-cell mass spectrometry analysis system based on continuous ultrasound injection is of great significance in ensuring the integrity of live single cells and a high signal-to-noise ratio for single-cell mass spectrometry detection signals. Summary of the Invention

[0004] The purpose of this invention is to overcome the problems of low single-cell utilization and detection throughput in existing single-cell mass spectrometry analysis, and to ensure the integrity of live single cells and the high signal-to-noise ratio of single-cell mass spectrometry detection signals.

[0005] To achieve the above objectives, the present invention provides the following technical solution: A single-cell mass spectrometry analysis system based on continuous ultrasound sample introduction includes an ultrasound module, a sample introduction module, and a visualization adjustment module; The ultrasound module is used to vibrate the cell suspension with ultrasound. The sample introduction module is connected to the ultrasound module and is used to continuously introduce cell samples from the cell suspension; the outlet end of the sample introduction module is aligned with the sampling port of the mass spectrometer. The observation port of the visualization adjustment module faces the sampling port of the mass spectrometer and the outlet of the injection module, so as to align the sampling port of the mass spectrometer with the outlet of the injection module.

[0006] Optionally, the ultrasound module includes a centrifuge tube storing a cell suspension, a sample bottle for placing the centrifuge tube, a piezoelectric ceramic plate connected to the outer wall of the centrifuge tube, a circuit board connected to the piezoelectric ceramic plate via a wire, and a power switch; the sample bottle has a cavity, the cavity is sealed with a gasket, and the centrifuge tube is placed inside the cavity; The circuit board and power switch are located outside the sample inlet bottle; The wire passes through the perforated sample vial and connects to the piezoelectric ceramic plate and circuit board on the outer wall of the centrifuge tube.

[0007] Optionally, the perforated portion of the sample vial is sealed with adhesive.

[0008] Optionally, the ultrasonic module also includes a cooling device for cooling the circuit board.

[0009] Optionally, the cooling device is a fan aligned with the circuit board.

[0010] Optionally, the injection module includes a nitrogen supply device, a highly conductive copper wire, and a capillary nozzle with the same inner diameter. The nitrogen supply device includes a nitrogen source and a nitrogen supply pipe connected to the nitrogen source. The outlet of the nitrogen supply pipe is located above the cell suspension in the centrifuge tube and does not come into contact with the cell suspension. One end of the highly conductive copper wire is inserted into the cell suspension, and the other end is connected to the positive terminal of the high-voltage power supply of the mass spectrometer. The inlet end of the capillary nozzle with constant inner diameter is inserted into the cell suspension, and the outlet end is aligned with the sampling port of the mass spectrometer.

[0011] Optionally, the visualization adjustment module is an optical microscope.

[0012] Optionally, the system is suitable for continuous ultrasonic injection of cell suspensions with different cell densities and for single-cell mass spectrometry analysis.

[0013] A single-cell mass spectrometry analysis method based on continuous ultrasound sample introduction, using the single-cell mass spectrometry analysis system based on continuous ultrasound sample introduction described above, includes the following steps: Step 1: Assemble the ultrasonic module and test its working status; Step 2: Prepare a cell suspension and add it into the centrifuge tube of the ultrasound module, then seal it with a gasket. Step 3: Assemble the injection module and check its working status; Step 4: Adjust the position of the mass spectrometer sampling port and the position of the sample injection module outlet. Step 5: Set parameters; Step 6: Connect the power supply and perform data acquisition and processing using the mass spectrometer.

[0014] As can be seen from the above technical solutions, the present invention provides a single-cell mass spectrometry analysis system and method based on continuous ultrasound sample introduction, which has the following technical advantages compared with the prior art: 1) Simple and effective: This invention uses a piezoelectric ceramic plate connected to a centrifuge tube to achieve non-destructive ultrasound. The device is lightweight and convenient, with extremely low operation difficulty. During operation, it can maintain the integrity of the cell morphology and prevent leakage of cell contents that would result in low coverage of target sample ions, which is beneficial for comprehensive analysis of the biological information of a single intact cell.

[0015] 2) High utilization rate and detection throughput: This invention greatly slows down the cell sedimentation rate by using ultrasonic vibration to vibrate the cell suspension in the sample vial, thereby further improving the utilization rate of single-cell samples and also greatly increasing the detection throughput of single-cell mass spectrometry analysis, providing an efficient strategy for existing single-cell mass spectrometry sample introduction methods. Attached Figure Description

[0016] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on the provided drawings without creative effort.

[0017] Figure 1 This is a schematic diagram of the single-cell mass spectrometry analysis system based on continuous ultrasound sample introduction of the present invention; Figure 2 This is a schematic diagram showing the density and state of A549 cells extracted from the supernatant of cell suspension at different times in the blank group and the ultrasound group, respectively, in a specific embodiment of the present invention. Figure 3 This is a schematic diagram illustrating the offline collection numbers of A549 cells in the control group and the ultrasound group, respectively, in a specific embodiment of the present invention; Figure 4 This is a visual imaging schematic diagram of the electroemission process of A549 cells in a specific embodiment of the present invention; Figure 5This is a schematic diagram of the mass spectrometry detection throughput of A549 cells at different cell densities in the blank group and the ultrasound group, respectively, in a specific embodiment of the present invention; Figure 6 In a specific embodiment of the present invention, A549 cells were subjected to mass spectrometry analysis of total ion chromatograms and extracted ion chromatograms in the blank group and the ultrasound group, respectively. Figure 7 This is a schematic diagram showing the mass spectra and ion content of a single A549 cell in a blank group and an ultrasound group, respectively, in a specific embodiment of the present invention. In this diagram, 1 represents a piezoelectric ceramic sheet; 2 represents a circuit board; 3 represents a power switch; 4 represents a gasket; 5 represents a centrifuge tube; 6 represents a sample vial; 7 represents a capillary nozzle with equal inner diameter; 8 represents a nitrogen supply pipe; 9 represents a high-conductivity copper wire; 10 represents an optical microscope; and 11 represents a mass spectrometer. Detailed Implementation

[0018] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0019] Example 1

[0020] This embodiment discloses a single-cell mass spectrometry analysis system based on continuous ultrasound sample introduction. See [link to documentation]. Figure 1 It includes an ultrasound module, a sample injection module, and a visualization adjustment module.

[0021] The ultrasonic module is used to vibrate the cell suspension with ultrasound. It includes a centrifuge tube 5 containing the cell suspension, a sample vial 6 for holding the centrifuge tube 5, a piezoelectric ceramic plate 1 connected to the lower end of the outer wall of the centrifuge tube 5, a circuit board 2 connected to the piezoelectric ceramic plate 1 via wires, and a power switch 3. The circuit board 2 and power switch 3 are located outside the sample vial 6. The circuit board 2 is used to adjust the power. The wires pass through the perforated sample vial 6 and connect to the piezoelectric ceramic plate 1 and the circuit board 2 on the outer wall of the centrifuge tube 5. The perforated portion of the sample vial 6 is sealed with adhesive. A cavity is provided inside the sample vial 6, and the centrifuge tube 5 is placed vertically within the cavity to ensure stable fixation. The cavity is sealed with a gasket 4 to guarantee the airtightness of the centrifuge tube 5.

[0022] In this embodiment, the gasket 4 is preferably a double-layer rubber gasket, and the piezoelectric ceramic sheet 1 is preferably a circular piezoelectric ceramic sheet.

[0023] The ultrasonic module may also include a cooling device, which may be a fan located below the circuit board 2, for cooling the circuit board 2.

[0024] The sample introduction module is connected to the ultrasound module and is used for continuous sample introduction of cell samples in cell suspension. It includes a nitrogen supply device, a high-conductivity copper wire 9, and a capillary nozzle 7 with equal inner diameter. The capillary nozzle 7 with equal inner diameter is used to replace the commercial nozzle of the nanoliter electrospray ion source.

[0025] The nitrogen supply device includes a nitrogen source and a nitrogen supply pipe 8 connected to the nitrogen source. The nitrogen source is high-purity nitrogen. The nitrogen supply pipe 8 passes through the sample inlet bottle 6 and the gasket 4, and its outlet is located above the cell suspension in the centrifuge tube 5, without contacting the cell suspension, thus achieving gas-liquid interface conversion inside the sample inlet bottle 6. One end of the highly conductive copper wire 9 passes through the sample inlet bottle 6 and the gasket 4 and is inserted into the bottom of the cell suspension, while the other end is connected to the positive terminal of the high-voltage power supply of the mass spectrometer 11. The inlet end of the constant inner diameter capillary nozzle 7 passes through the sample inlet bottle 6 and the gasket 4 and is inserted into the bottom of the cell suspension, while its outlet end is aligned with the sampling port of the mass spectrometer 11. The outlet end of the sample inlet module is aligned with the sampling port of the mass spectrometer 11.

[0026] Visualization adjustment module: The optical microscope 10 equipped with the ion source is used to observe and adjust the relative position of the capillary nozzle 7 with the same inner diameter and the sampling port of the mass spectrometer 11 in real time to ensure that the electroemission of single-cell samples is normal, so as to guarantee online mass spectrometry analysis.

[0027] To comprehensively consider the effects of cell integrity, cell sedimentation rate, and the heat generated by the piezoelectric effect of ultrasound on cell activity, the optimal working time is to use a cycle of 10 seconds of ultrasound followed by 50 seconds of rest.

[0028] Example 2

[0029] This embodiment discloses a single-cell mass spectrometry analysis method based on continuous ultrasound sample introduction. The method uses the aforementioned system for continuous single-cell sample introduction and single-cell mass spectrometry analysis, and includes the following steps: Step 1: Assemble the ultrasound module and test its operational status. A wire of a certain length is passed through the perforated sample bottle 6 through the piezoelectric ceramic plate 1, and the hole is sealed with glue to prevent air leakage. The outer wall of the centrifuge tube 5 is tightly connected to the piezoelectric ceramic plate 1. 50 μL of ultrapure water is added to the centrifuge tube 5. The power of the device is turned on. Continuous and uniform bubbles are observed under the microscope, indicating that the ultrasonic device is working well.

[0030] Step 2: Prepare a cell suspension and add it to the centrifuge tube 5 of the ultrasound module, sealing it with gasket 4. A549 cells in the logarithmic growth phase were used to prepare a single-cell suspension and counted, with a cell density of 1×10⁻⁶ cells. 4 ~8.5×10 5 The cells were centrifuged once to remove the culture medium and resuspended in 150 mM ammonium formate solution. The cells were then centrifuged again to remove the 150 mM ammonium formate solution and resuspended in 40 mM ammonium formate solution (unless otherwise specified, all cell samples in this invention are pretreated and resuspended in 40 mM ammonium formate solution after two centrifugations) to obtain a cell suspension.

[0031] Step 3: Assemble the injection module and check its working status: Add 50 μL of cell suspension to centrifuge tube 5 and seal it with gasket 4. Then, insert the syringe needle into gasket 4 to insert the inlet end of capillary nozzle 7 with equal inner diameter and the 2 mm diameter high-conductivity copper wire 9 into the bottom of centrifuge tube 5. The other end of the copper wire is connected to the positive terminal of the mass spectrometer high-voltage power supply. Turn on the gas cylinder switch and control the pressure at 200~300 psi. The high-purity nitrogen gas outlet with a restricted position is inserted above the cell suspension. The gas-liquid interface conversion inside sample bottle 6 causes the cell sample to be input into the inlet end of capillary nozzle 7 with equal inner diameter. The droplets continuously flow out from the outlet end of the capillary nozzle, proving that capillary nozzle 7 with equal inner diameter is not blocked.

[0032] Preparation of constant inner diameter capillary nozzle 7: Eight fused silica capillaries with an outer diameter of 360μm and an inner diameter of 16-25μm and a length of about 30-40cm were cut. The polyimide coating was ablated and removed from the etched end on one side at 4.2-5.2cm. The silica capillaries were etched using gravity-assisted etching to obtain a constant inner diameter capillary nozzle 7 with a certain taper angle at the tip and a slender front end.

[0033] Step 4: Adjust the position of the sampling port of mass spectrometer 11 and the exit position of the sample injection module: Using an optical microscope 10 equipped with an ion source, a clear 50x image of the tip of the capillary nozzle 7 with an equal inner diameter is obtained by adjusting the position of the capillary nozzle 7 and focusing with the fine adjustment knob. The optimal distance for single-cell electroemission is 1-2 mm in front of the center of the mass spectrometer sampling port, but the specific distance depends on the single-cell jet situation.

[0034] Step 5: Set parameters: Mass spectrometry signals were acquired, and the parameters were set as follows: positive ion mode, voltage controlled at 1.2–2.0 kV, and electroemission current controlled at 0.18–0.28 μA, although slight variations may occur depending on specific experimental conditions. The conditions used were as follows: ion transmission tube temperature 350 °C; resolution 30,000; injection time 50 ms; AGC 1 × 10⁻⁶. 5 Scan range, m / z 100-1000.

[0035] Step 6: Connect the power supply and perform data acquisition and processing using mass spectrometer 11. Obtain the total ion chromatogram (TIC) and extracted ion chromatogram (EICofm / z 760.5838) of the cells using ThermoXcaliburQualBrowser and mass spectrometry peak alignment Python software. Among all cell events, single-cell characteristic ion signal intensities >3×10⁻⁶ are selected. 5 This is a single-celled event.

[0036] Example 3

[0037] like Figure 2 The figure shows the density and state of A549 cells in the supernatant of cell suspensions extracted at different times in the blank group and the ultrasound group.

[0038] 1. Set up the ultrasonic module by vertically placing the centrifuge tube 5 connected to the circular piezoelectric ceramic plate inside the sample vial 6 without the need for a gasket 4 for sealing; 2. Prepare a single-cell suspension from A549 cells in the logarithmic growth phase and count the cells, setting a cell density of 4.5 × 10⁻⁶. 5 Cells / mL, after cell sample pretreatment to obtain cell suspension. A blank group: 100 μL of cell suspension from five groups was added to centrifuge tube 5 and allowed to stand for 0, 10, 20, 30, and 50 min respectively. 5 μL of supernatant was collected to observe the density and state of A549 cells (the extraction position was kept consistent each time using a 3D manipulator). An ultrasonic group: 100 μL of cell suspension from five groups was added to centrifuge tube 5 and ultrasonicated for 0, 10, 20, 30, and 50 min respectively. 5 μL of supernatant was collected to observe the density and state of A549 cells.

[0039] 3. During the 0-50 min settling period, the cell density in the supernatant of the blank group gradually decreased with increasing settling time, and after 30 min, almost no cells were visible in the supernatant. In contrast, during the 0-50 min settling period of the ultrasound group, the number of cells in the supernatant did not decrease significantly with increasing ultrasound cycle time. Therefore, ultrasound has the effect of delaying cell sedimentation, and the cell morphology remains intact.

[0040] Example 4

[0041] like Figure 3 and Figure 5 The figure shows the offline collection number of A549 cells in the blank group and the ultrasound group, and the mass spectrometry detection throughput at different cell densities. Figure 4 It is a visualization of the normal electroemission process of A549 cells.

[0042] 1. Offline Collection: The system setup process is the same as described above. It's worth noting that the electroemission process caused by energizing the high-voltage power supply for mass spectrometry is not included. Only a centrifuge tube 5 containing 50 μL McCoy's 5A medium (the medium used for A549 cell culture) is placed between the tip of the capillary nozzle 7 and the mass spectrometer sampling port to collect single-cell droplets flowing from the outlet of the capillary nozzle 7 until the cell suspension has completely flowed out and been counted. An A549 cell density of 4.25 × 10⁻⁶ cells was selected. 5 Cells / mL, 25μm inner diameter capillary nozzle 7, set up a blank group: no sonication of cell suspension, sonication group: sonication of cell suspension, each group repeated three times (Group1, Group2, Group3). Figure 3 It can be seen that, under the action of ultrasound, the number of cells collected from the outlet end of the capillary nozzle 7 with the same inner diameter in the ultrasound group is about twice as many as the number of cells collected in the control group.

[0043] 2. Mass spectrometry detection: The system setup process is the same as described above. A549 cell suspensions of different densities (1×10⁻⁶) were used. 4 cells / mL, 1×10 5 Cells / mL, 25 μm inner diameter capillary nozzle 7, set up a blank group: cell suspensions of different densities were not sonicated, and a sonicated group: cell suspensions of different densities were sonicated. Single-cell characteristic peaks (m / z 703.57-703.58, 734.57-734.58, 760.58-760.59, 813.68-813.69, 876.79-876.81, 922.78-922.79, 950.81-950.82) with signal intensity > 3 × 10⁻⁶. 5 The presence of ions with any mass-to-charge ratio constitutes a single-cell event, yielding the final single-cell mass spectrometry detection throughput. Figure 5 It can be seen that, under the action of ultrasound, the mass spectrometry detection throughput of the ultrasound group with different cell densities was higher than that of the blank group, especially at a cell density of 1×10⁻⁶. 5 At cell density of 1000 cells / mL, the mass spectrometry throughput of the ultrasonic group was more than three times that of the blank group. Furthermore, this invention is also applicable to continuous ultrasonic injection of cell suspensions with different cell densities for single-cell mass spectrometry analysis, and the final single-cell mass spectrometry throughput is significantly improved compared to the blank group.

[0044] Example 5

[0045] like Figure 6 and 7The image shows the total ion chromatogram, extracted ion chromatogram, and detection mass spectra of A549 cells under both the blank and ultrasound groups, along with the number of ions contained.

[0046] 1. The system setup process is the same as described above. A549 cells at a density of 1×10⁻⁶ were selected. 5 Cells / mL, 25μm inner diameter capillary nozzle 7, set up blank group: no sonication of cell suspension, sonication group: sonication of cell suspension. Figure 6 It can be seen that in the TIC and EIC (m / z 760.5838) of A549 cells, the single-cell peak distribution in the TIC image of the ultrasound group is more uniform, which further confirms that ultrasound has the effect of delaying cell sedimentation and achieves the goal of improving cell utilization and detection throughput in single-cell mass spectrometry analysis.

[0047] 2. By Figure 6 Extended Figure 7 The mass spectra of A549 cells and the number of ions contained therein were observed. The single-cell characteristic peak shapes of the A549-blank group and the A549-ultrasound group were almost identical, with 738 and 714 ions detected respectively. This once again proves that the single-cell mass spectrometry analysis system based on continuous ultrasonic injection does not damage the complete morphology of single cells and preserves the true biological information within the cells.

[0048] The various embodiments in this specification are described in a progressive manner, with each embodiment focusing on the differences from other embodiments. The same or similar parts between the various embodiments can be referred to each other.

[0049] The above description of the disclosed embodiments enables those skilled in the art to make or use the invention. Various modifications to these embodiments will be readily apparent to those skilled in the art, and the general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of the invention. Therefore, the invention is not to be limited to the embodiments shown herein, but is to be accorded the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. A single-cell mass spectrometry analysis system based on continuous ultrasound sample introduction, characterized in that, Includes an ultrasound module, a sample injection module, and a visualization adjustment module; The ultrasonic module is used to vibrate the cell suspension with ultrasound, and includes a centrifuge tube storing the cell suspension, a sample bottle for placing the centrifuge tube, a piezoelectric ceramic plate connected to the outer wall of the centrifuge tube, a circuit board connected to the piezoelectric ceramic plate by a wire, and a power switch; the sample bottle is provided with a cavity, the cavity is sealed with a gasket, and the centrifuge tube is placed in the cavity; The circuit board and power switch are located outside the sample inlet bottle; The wire passes through the perforated sample vial and connects to the piezoelectric ceramic plate and circuit board on the outer wall of the centrifuge tube, while the operating parameters are set. The sample introduction module is connected to the ultrasound module and is used to continuously introduce cell samples in the cell suspension. The outlet end of the sample introduction module is aligned with the sampling port of the mass spectrometer and includes a nitrogen supply device, a highly conductive copper wire, and a capillary nozzle with the same inner diameter. The nitrogen supply device includes a nitrogen source and a nitrogen supply pipe connected to the nitrogen source. The outlet of the nitrogen supply pipe is located above the cell suspension in the centrifuge tube and does not come into contact with the cell suspension. One end of the highly conductive copper wire is inserted into the cell suspension, and the other end is connected to the positive terminal of the high-voltage power supply of the mass spectrometer. The inlet end of the capillary nozzle with constant inner diameter is inserted into the cell suspension, and the outlet end is aligned with the sampling port of the mass spectrometer. The observation port of the visualization adjustment module faces the sampling port of the mass spectrometer and the outlet of the injection module, so as to align the sampling port of the mass spectrometer with the outlet of the injection module.

2. The single-cell mass spectrometry analysis system based on continuous ultrasound sample introduction according to claim 1, characterized in that, The perforated portion of the sample vial is sealed with adhesive.

3. The single-cell mass spectrometry analysis system based on continuous ultrasound sample introduction according to claim 1, characterized in that, The ultrasonic module also includes a cooling device for cooling the circuit board.

4. The single-cell mass spectrometry analysis system based on continuous ultrasound sample introduction according to claim 3, characterized in that, The cooling device is a fan aligned with the circuit board.

5. The single-cell mass spectrometry analysis system based on continuous ultrasound sample introduction according to claim 1, characterized in that, The visualization adjustment module is for optical microscopes.

6. The single-cell mass spectrometry analysis system based on continuous ultrasound sample introduction according to claim 1, characterized in that, The system is suitable for continuous ultrasonic injection of cell suspensions with different cell densities and for single-cell mass spectrometry analysis.

7. A single-cell mass spectrometry analysis method based on continuous ultrasound sample introduction, comprising performing single-cell mass spectrometry analysis using the single-cell mass spectrometry analysis system based on continuous ultrasound sample introduction as described in any one of claims 1-6, characterized in that, Includes the following steps: Step 1: Assemble the ultrasonic module and test its working status; Step 2: Prepare a cell suspension and add it into the centrifuge tube of the ultrasound module, then seal it with a gasket. Step 3: Assemble the injection module and check its working status; Step 4: Adjust the position of the mass spectrometer sampling port and the position of the sample injection module outlet. Step 5: Set parameters; Step 6: Connect the power supply and perform data acquisition and processing using the mass spectrometer.

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