Mass spectrum flow cytometry sample introduction system based on flow focusing

Abstract

The invention relates to a mass spectrum flow cytometry sample introduction system based on flow focusing. The system comprises a flow focusing flow channel, a single cell suspension introduction assembly and an airflow introduction part. Wherein a cavity and a microporous nozzle are arranged in the flow focusing runner, and the microporous nozzle is communicated with the cavity and a heating and solvent removing module of the mass spectrometry flow cytometer; the single-cell suspension introduction assembly comprises a capillary tube, the inner diameter of the capillary tube is far larger than the particle size of single cells in the single-cell suspension, the capillary tube is located in the cavity, and the outlet end of the capillary tube directly faces the ejection hole of the microporous nozzle; the gas flow introduction part is communicated with the atomization gas path and the flow focusing flow channel of the mass spectrometry flow cytometer and is used for introducing the atomization gas flow into the chamber, so that a pressure difference is formed between the chamber and the external environment, and the single-cell suspension is autonomously crushed into liquid drops of single cells outside the flow focusing flow channel. According to the invention, the problem of cell blockage in mass spectrum flow analysis is thoroughly solved, plug and play can be realized, and the use is convenient.

Description

Technical Field

[0001] The invention relates to a mass spectrometry flow cytometer sample introduction system based on flow focusing, belonging to the technical field of mass spectrometry detection equipment and analysis. Background Art

[0002] Cells are the basic structural and functional units of organisms, and all activities in organisms are carried out on the basis of cells. Traditional cell biology research mainly studies cells at the population level based on the origin, morphology and secretions of cells, which may mask the differences between individual cells. With the deepening of research and the continuous improvement of analytical techniques, people have gradually realized that cells have individual differences. Single-cell analysis can obtain heterogeneity and diversity information of single cells, thereby obtaining more accurate and comprehensive cell biology information. Mass cytometry is one of the most advanced cell counting platforms in the world. It can simultaneously detect more than 50 biomarkers on millions of single cells in a short time. It has opened a new door for clinical medical research and has a wide range of applications in immunology, cancer diagnosis and treatment, infectious diseases and pharmacy. Mass cytometry is considered to be the second-generation cell flow analysis technology. This technology uses metal-tagged antibodies instead of traditional fluorescent antibodies, thereby completely solving the problems of cross-talk and autofluorescence between fluorescent groups. It has the characteristics of multiple channels, low background and high data quality.

[0003] The mass spectrometry flow cytometer mainly consists of three systems, namely 1) sample introduction system, 2) inductively coupled plasma combustion system and 3) mass spectrometry detection system. The sample introduction system is crucial for the analysis of samples. The single cell suspension is introduced by an automatic sampling pump, converted into an aerosol by a pneumatic nebulizer, and dehydrated in a heating chamber before reaching the inductively coupled plasma combustion system. Among them, the nebulizer plays a vital role in the analytical performance, affecting the detection limit, sensitivity and accuracy of the instrument. In recent years, researchers have developed a sampling system specifically for inductively coupled plasma mass spectrometry analysis of single cell suspensions or single particle suspensions, using a lower flow rate (tens of microliters per minute) and a dedicated nebulizer for single cell analysis for sample extraction, which improves the sampling efficiency (more than 10 times) and also reduces the probability of multi-cell events. This nebulization strategy is also adopted in mass spectrometry flow cytometers. However, due to the use of thinner capillaries (50-100 μm), the nebulizer and capillary are prone to clogging, affecting the progress of the experiment. Summary of the invention

[0004] In view of the above technical problems, the present invention provides a mass spectrometry flow cytometer sample introduction system based on flow focusing. The system uses the flow focusing principle as a method for introducing cell samples into the mass spectrometry flow cytometer. Flow focusing is a microfluidic technology that can be used to control the generation of tiny droplets or particles with high precision. Since a thicker transmission tube and a larger aperture nozzle (100 to 500 μm) can be used, the problem of cell clogging can be completely solved.

[0005] To achieve the above object, the present invention adopts the following technical solutions:

[0006] A mass spectrometry flow cytometer sample introduction system based on flow focusing, comprising:

[0007] A flow focusing channel, wherein a chamber and a microporous nozzle are provided in the flow focusing channel, and the microporous nozzle is connected to the chamber and a heating and desolvation module of the mass spectrometry flow cytometer;

[0008] A single cell suspension introduction component, the single cell suspension introduction component comprises a capillary, the inner diameter of the capillary is much larger than the particle size of the single cell in the single cell suspension, the capillary is located in the chamber and the outlet end thereof is directly adjacent to the ejection hole of the micro-pore nozzle;

[0009] An air flow introduction component is connected to the atomization gas path of the mass spectrometer and the flow focusing channel, and is used to introduce the atomization gas into the chamber to form a pressure difference between the chamber and the external environment, so that the single-cell suspension flowing out of the outlet end of the capillary is pulled into an extremely fine liquid jet at the ejection hole and autonomously broken into droplets of single cells outside the flow focusing channel.

[0010] The mass spectrometry flow cytometer sample introduction system based on flow focusing, preferably, the flow focusing flow channel is a sleeve assembly, the sleeve assembly includes a sleeve inner tube and a sleeve outer tube nested outside the sleeve inner tube, the micropore nozzle is arranged in the sleeve outer tube, and the chamber is formed between the micropore nozzle and the sleeve outer tube.

[0011] The flow focusing-based mass cytometer sample introduction system, preferably, the single cell suspension introduction component also includes an injection tube and a tee, one end of the injection tube extends into the first inlet of the tee and is connected to the inlet end of the capillary, and the other end is connected to the automatic injection pump, the second inlet of the tee is connected to the atomization gas path of the mass cytometer, and the outlet of the tee is connected to the outer tube of the sleeve.

[0012] In the mass spectrometry flow cytometer sample introduction system based on flow focusing, preferably, one end of the sample introduction tube is detachably connected to the first inlet of the tee via a fastener.

[0013] In the flow focusing-based mass cytometer sample introduction system, preferably, the sleeve assembly is connected to the heating and desolvation module of the mass cytometer, and the airflow introduction component is connected to the atomization gas path of the mass cytometer.

[0014] In the mass spectrometry flow cytometer sample introduction system based on flow focusing, preferably, the inner diameter of the capillary is 50 to 300 μm, and the outer diameter is 300 to 500 μm.

[0015] In the mass spectrometry flow cytometer sample introduction system based on flow focusing, preferably, the micro-pore nozzle is made by 3D printing, and the aperture of the ejection hole is 100-500 μm.

[0016] In the mass spectrometry flow cytometer sample introduction system based on flow focusing, preferably, the outer diameter of the outer tube of the cannula is 4-8 mm, and the inner diameter is 2-6 mm; the outer diameter of the inner tube of the cannula is 2-6 mm, and the inner diameter is 1-3 mm.

[0017] The mass spectrometry flow cytometer sample introduction system based on flow focusing, preferably, the carrier gas flow rate of the atomizing gas is 0.1-1 L / min, and the injection speed is 10-100 μL / min.

[0018] The mass spectrometry flow cytometer sample introduction system based on flow focusing is preferably applied in the fields of mass spectrometry flow cytometer single cell analysis and inductively coupled plasma mass spectrometry single cell analysis.

[0019] The present invention adopts the above technical solution, which has the following advantages:

[0020] 1. The single cell suspension flowing out of the outlet end of the capillary in the present invention is pulled into an extremely fine liquid jet under the wrapping and squeezing action of the coaxial airflow at the ejection hole, and is autonomously broken into single cell droplets outside the flow focusing channel. This is a microfluidic technology based on flow focusing, which can be used for high-precision control of the generation of tiny droplets or particles. Since a thicker capillary and a larger aperture nozzle (100-500 μm) can be used, the problem of cell blockage can be completely solved.

[0021] 2. In the present invention, the single cell suspension can be flow-focused to generate droplets containing single cell samples under the drive of the atomizing airflow. Compared with the sample introduction method using a microfluidic atomizer, since a thicker transmission tube and a larger aperture nozzle can be used, cell clogging can be avoided; in addition, it has a simple structure, low cost, plug-and-play, and is easy to use. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1A schematic diagram of a mass spectrometry flow cytometer sample introduction system based on flow focusing provided in one embodiment of the present invention;

[0023] Figure 2 for Figure 1 A partial enlarged view of the mesocapillary and micro-pore nozzle;

[0024] Figure 3 Figures 1 and 2 are the analysis results of the four-element calibration beads in Example 1 of the present invention, wherein Figures (A), (B) and (C) are the analysis results using a commercial nebulizer, Figures (a), (b) and (c) are the analysis results using the mass spectrometry flow cytometer sample introduction system based on flow focusing of the present invention, and Figures (Aa), (Bb) and (Cc) are comparison results of the two.

[0025] Figure 4 1 is a diagram showing the analysis results of Jurket T cells in Example 1 of the present invention, wherein Figure (A) is a diagram showing the analysis results using a commercial nebulizer, Figure (a) is a diagram showing the analysis results using a mass spectrometry flow cytometer sample introduction system based on flow focusing of the present invention, and Figure (Aa) is a comparison diagram of the two results;

[0026] The following are marked in the figure:

[0027] 1-injection tube; 2-stainless steel screw; 3-metal tee; 4-plastic hose; 5-quartz sleeve outer tube; 6-capillary tube; 7-micropore nozzle; 8-quartz sleeve inner tube. DETAILED DESCRIPTION

[0028] In order to make the purpose, technical solution and advantages of the present invention clearer, the technical solution of the present invention is described clearly and completely below. Obviously, the described embodiments are part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary persons in the art without creative work are within the scope of protection of the present invention.

[0029] Unless otherwise defined, the technical or scientific terms used in the present invention shall have the usual meanings understood by persons with ordinary skills 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" and the like mean that the elements or objects appearing before 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" and the like are not limited to physical or mechanical connections, but may include electrical connections, whether direct or indirect.

[0030] 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", "below", "above", etc. Such spatially relative terms are intended to include different orientations of the device in use or operation in addition to the orientation depicted in the figures.

[0031] In recent years, researchers have developed a sampling system specifically for ICP-MS analysis of single-cell suspensions or single-particle suspensions, using a lower flow rate (tens of microliters per minute) and a dedicated nebulizer for single-cell analysis for sample extraction, which improves the sampling efficiency (more than 10 times) and also reduces the probability of multi-cell events. Mass spectrometry flow cytometers also use this nebulization strategy. However, due to the use of thinner capillaries (50-100 μm), the nebulizer and capillary are prone to clogging, affecting the progress of the experiment.

[0032] Based on the problem that atomizers and capillaries are prone to clogging during mass spectrometry flow analysis, the present invention provides a mass spectrometry flow cytometer sample introduction system and method and a mass spectrometry flow cytometer based on flow focusing. The system uses flow focusing as a cell sample introduction method for the mass spectrometry flow cytometer. Flow focusing is a microfluidic technology that can be used to control the generation of tiny droplets or particles with high precision. Since a thicker transmission tube and a nozzle with a larger aperture (100-500 μm) can be used, the problem of cell clogging can be completely solved.

[0033] Before describing the technical solution of the present invention, the flow focusing technology is first introduced.

[0034] Flow focusing (FF) is an external flow method, which can be described as the fluid flowing out of the capillary is driven by another high-speed flowing fluid, and after being focused by the small hole, a stable cone is formed at the tube mouth, and a microjet is formed at the top of the cone. The jet breaks into monodisperse droplets at a certain distance outside the small hole. In this flow, the viscous shear force in the jet is much smaller than that of similar mechanical methods for preparing microdroplets (such as direct pressure-driven jets), and no other additional forces are required (such as classic electrospraying that relies on electric field forces). Therefore, this technology is conducive to the preparation of droplets and particles of many materials. Especially in the fields of molecular biology, biochemical analysis, pharmacology, etc., since long molecular chains can only withstand limited shear forces, flow focusing can ensure that the molecular chains are not broken, which is more conducive to the preparation of monodisperse particles.

[0035] like Figure 1 , 2As shown, the mass spectrometry flow cytometer sample introduction system based on flow focusing according to the present invention comprises: a flow focusing channel, a single cell suspension introduction component and an airflow introduction component.

[0036] like Figure 1 As shown, a flow focusing channel is provided with a chamber and a microporous nozzle 7 in the flow focusing channel, and the microporous nozzle 7 connects the chamber with the central tube and plasma of the mass spectrometer; specifically, the flow focusing channel is a sleeve assembly, and the sleeve assembly includes a sleeve inner tube and a sleeve outer tube nested outside the sleeve inner tube, and the microporous nozzle 7 is arranged in the sleeve outer tube, and the chamber is formed between the microporous nozzle 7 and the sleeve outer tube. Preferably, the microporous nozzle 7 is made by 3D printing, and the aperture of the ejection hole is 100-500μm. The sleeve assembly is made of quartz, the sleeve inner tube is a quartz sleeve inner tube 8, and the sleeve outer tube is a quartz sleeve outer tube 5. The outer diameter of the sleeve outer tube is 4-8mm, and the inner diameter is 2-6mm; the outer diameter of the sleeve inner tube is 2-6mm, and the inner diameter is 1-3mm. In a preferred embodiment of the present invention, the outer diameter of the quartz sleeve outer tube 5 is 6 mm and the inner diameter is 4 mm; the outer diameter of the quartz sleeve inner tube 8 is 4 mm and the inner diameter is 2 mm.

[0037] Furthermore, the single cell suspension introduction component includes a capillary 6, the inner diameter of the capillary 6 is much larger than the particle size of the single cell in the single cell suspension, the capillary 6 is located in the chamber and its outlet end is directly adjacent to the ejection hole of the microporous nozzle 7, such as Figure 2 As shown. Figure 1 As shown, the single cell suspension introduction component also includes an injection tube 1 and a metal tee 3. One end of the injection tube 1 extends into the first inlet of the metal tee 3 and is connected to the inlet end of the capillary 6, and the other end is connected to the automatic injection pump. The second inlet of the metal tee 3 is connected to the airflow introduction component, and the outlet of the metal tee 3 is connected to the outer tube of the sleeve (that is: Figure 1 One end of the sample inlet tube 1 is detachably connected to the first inlet of the metal tee 3 through a stainless steel screw 2.

[0038] Furthermore, the airflow introduction component is connected to the atomization gas path and the flow focusing channel of the mass spectrometry flow cytometer, and is used to introduce the atomization gas into the chamber, so that a pressure difference is formed between the chamber and the external environment, so that the single cell suspension flowing out of the outlet end of the capillary 6 is pulled into an extremely fine liquid jet at the ejection hole, and is autonomously broken into droplets of single cells outside the flow focusing channel. The inner diameter of the capillary 6 is 50 to 300 μm, and the outer diameter is 300 to 500 μm. Since the inner diameter of the capillary 6 is much larger than the particle size of the single cell in the single cell suspension, the capillary 6 will not be blocked.

[0039] Specifically, Figure 2As shown, high-pressure gas is continuously and stably injected into the chamber through the airflow introduction component (i.e., the plastic hose 4), so that a pressure difference is formed between the chamber and the external environment, and then the high-pressure gas in the chamber forms a focused airflow near the ejection hole, and the airflow wraps the single-cell suspension liquid flow flowing out from the outlet end of the capillary 6. The single-cell suspension liquid flow is focused and squeezed by the airflow. Under the conditions of the pressure difference between the inside and outside of the chamber of the appropriate flow focusing channel and the liquid flow rate of the capillary 6, the diameter of the ultrafine jet can reach a size close to that of a single cell, so that the single cells can be arranged in a row to form an ultrafine jet. After the ultrafine jet is autonomously broken, a monodisperse single cell wrapped by droplets is formed. Since the frequency of the focused ultrafine jet breaking into droplets with monodisperse single cells is extremely high and the efficiency is high, a higher throughput can be achieved, that is, after the ultrafine jet is autonomously broken, a monodisperse single cell wrapped by droplets is formed, which can directly enter the mass spectrometer, and the mass spectrometer includes but is not limited to a mass spectrometer flow cytometer and an inductively coupled plasma mass spectrometer. In summary, the mass cytometer sample introduction system based on flow focusing of the present invention is not easy to be clogged and does not require complex liquid flow pipelines to form special liquid flow power to disperse single cells in the liquid flow. Instead, it uses extremely fine jets with a size close to the cell diameter and extremely fine jets to break into droplets of monodisperse single cells. The single-cell suspension has high monodisperse efficiency and high throughput and is compatible with mass cytometers and inductively coupled plasma mass spectrometers, etc.

[0040] In some preferred embodiments of the present invention, the automatic injection pump is a digital micro-injection pump, which can drive the injection pump to output the sample to be tested, and the pump speed can be set to 0-200 μL / min; the injection tube 1 is made of PEEK material, with an inner diameter of 100-500 μm; the metal tee 3 is made by precision machining (CNC), and is respectively connected to the injection tube 1, the plastic hose 4 and the quartz sleeve, which can play a role of connection and fixation; the plastic hose 4 is made of polytetrafluoroethylene, with an outer diameter of 6 mm and an inner diameter of 4 mm, and the plastic hose 4 is connected to the atomization gas path of the mass spectrometry flow cytometer, and the atomization gas flow rate is 0-1 L / min. The sleeve assembly is connected to the heating and desolvation module of the mass spectrometry flow cytometer, and the temperature of the heating and desolvation module is 190-200°C, which can play a role in desolvation of single cell droplets.

[0041] The technical solution of the present invention is described in detail below in conjunction with specific application examples.

[0042] Example 1

[0043] Taking the analysis of Jurket cells stained with four-element calibration beads and CD-45 as an example, a commercial nebulizer and the mass spectrometry flow cytometer sample introduction system based on flow focusing of the present invention were used to detect them, and the detection results were compared. The specific experimental steps are as follows:

[0044] 1) Preparation of single cell suspension

[0045] The cultured Jurket T cells were taken, centrifuged to remove the culture medium, washed once with PBS, and fixed with 4% paraformaldehyde fixative for 30 minutes.

[0046] 2) Mark and stain the cells

[0047] Take 2×10 cells fixed above 6 The cells were washed and dispersed in cell staining buffer, and the polymer probe CD45-Eu and Ir-DNA intercalator from Fluidigm, USA were used to stain the cells, and then washed thoroughly.

[0048] 3): Turn on the mass spectrometer, perform a performance self-check and instrument tuning on the mass spectrometer, and establish an analysis method:

[0049] In a preferred embodiment, the flow rate of the atomizing gas is 0.3 L / min, the auxiliary gas is 1.0 L / min, and the compensation gas is 0.76 L / min; the sample injection speed is 30 μL / min; and the heating temperature is 190° C.

[0050] Step 4): Use the above conditions to detect the four-element calibration beads and collect isotopes 140 Ce, 142 Ce, 151 Eu, 153 Eu, 165 Ho, 175 Lu and 176 Lu, record 20,000 signals; use the above conditions to detect the stained JurketT cells and collect isotope 151 Eu, 153 Eu, 191 Ir and 193 Ir, record 30000 signals.

[0051] 5): Remove the commercial nebulizer of the mass cytometer, connect the mass cytometer single cell sample introduction system based on flow focusing of the present invention to the mass cytometer, and keep the instrument parameters unchanged. Collecting isotopes 140 Ce, 142 Ce, 151 Eu, 153 Eu, 165 Ho, 175 Lu and 176 Lu, recorded 20,000 signals; tested the stained Jurket T cells and collected isotopes 151 Eu, 153 Eu, 191Ir and 193 Ir, record 30000 signals.

[0052] 6): Analyze the test results using FlowJo software and compare the results obtained using a commercial nebulizer and a mass spectrometry single-cell sample introduction system based on flow focusing, as shown in the following figure. Figure 3 , 4 shown.

[0053] like Figure 3 As shown, it is a logarithmic bivariate plot of the elements in the four-element calibration beads analyzed using a commercial nebulizer and a mass spectrometry single-cell sample introduction system based on flow focusing. Figures (A), (B) and (C) show the analysis results using a nebulizer, Figures (a), (b) and (c) show the results using a mass spectrometry single-cell sample introduction system based on flow focusing, and Figures (Aa), (Bb) and (Cc) show the comparison of the two results. From the results in the figure, it can be found that the results obtained from the analysis of the four-element calibration beads using a commercial nebulizer and a mass spectrometry single-cell sample introduction system based on flow focusing are in good agreement.

[0054] like Figure 4 As shown, it is a logarithmic bivariate plot of elements in Jurket T cells after staining using a commercial nebulizer and a mass spectrometry flow cytometry single-cell sample introduction system based on flow focusing. Figure (A) shows the analysis results using a nebulizer, Figure (a) shows the results using a mass spectrometry flow cytometry single-cell sample introduction system based on flow focusing, and Figure (Aa) shows a comparison of the two results. From the results in the figure, it can be found that the results obtained from the analysis of Jurket T cells after staining using a commercial nebulizer and a mass spectrometry flow cytometry single-cell sample introduction system based on flow focusing are in good consistency. This shows that the mass spectrometry single-cell sample introduction system based on flow focusing of the present invention can completely replace the commercial nebulizer. Since the commercial nebulizers currently in use are one-piece molded, the cost of use is extremely high and they are particularly prone to clogging. When they are clogged, they need to be replaced with a new nebulizer, which needs to be imported and is expensive. Each nebulizer costs RMB 6,000. The use of the mass spectrometry single-cell sample introduction system based on flow focusing of the present invention can not only fundamentally solve the problem of capillary clogging, but also greatly reduce the cost of use, only costing about RMB 500, and it can be plug-and-play.

[0055] The single cell suspension flowing out of the outlet end of the capillary 6 in the present invention is pulled into an extremely fine liquid jet under the wrapping and squeezing action of the coaxial airflow at the ejection hole, and is autonomously broken into single cell droplets outside the flow focusing channel. This is a microfluidic technology based on flow focusing, which can be used for high-precision control of the generation of tiny droplets or particles. Since thicker capillaries and larger aperture nozzles (100-500 μm) can be used, the problem of cell blockage can be completely solved.

[0056] 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 embodiments of the present invention.

Claims

1. A mass spectrometry flow cytometer sample introduction system based on flow focusing, characterized in that: include: A flow focusing channel, wherein a chamber and a microporous nozzle (7) are provided in the flow focusing channel, and the microporous nozzle (7) is connected to the chamber and a heating and desolvation module of the mass spectrometry flow cytometer; A single cell suspension introduction component, the single cell suspension introduction component comprising a capillary (6), the inner diameter of the capillary (6) being much larger than the particle size of the single cells in the single cell suspension, the capillary (6) being located in the chamber and the outlet end thereof being directly adjacent to the ejection hole of the microporous nozzle (7); An air flow introduction component is connected to the atomization gas path of the mass spectrometer and the flow focusing channel, and is used to introduce the atomization gas into the chamber, so that a pressure difference is formed between the chamber and the external environment, thereby causing the single-cell suspension flowing out of the outlet end of the capillary (6) to be pulled into an extremely fine liquid jet at the ejection hole and autonomously broken into droplets of single cells outside the flow focusing channel.

2. The mass spectrometry flow cytometer sample introduction system based on flow focusing according to claim 1, characterized in that: The flow focusing channel is a sleeve assembly, which comprises an inner sleeve tube and an outer sleeve tube nested outside the inner sleeve tube, the micropore nozzle (7) is arranged in the outer sleeve tube, and the chamber is formed between the micropore nozzle (7) and the outer sleeve tube.

3. The mass spectrometry flow cytometer sample introduction system based on flow focusing according to claim 2, characterized in that: The single cell suspension introduction component also includes an injection tube (1) and a tee, one end of the injection tube (1) extends into the first inlet of the tee and is connected to the inlet end of the capillary (6), and the other end is connected to an automatic injection pump, the second inlet of the tee is connected to the atomization gas path of the mass spectrometer flow cytometer, and the outlet of the tee is connected to the outer tube of the sleeve.

4. The mass spectrometry flow cytometer sample introduction system based on flow focusing according to claim 3, characterized in that: One end of the sample injection tube (1) is detachably connected to the first inlet of the tee via a fastener.

5. The mass spectrometry flow cytometer sample introduction system based on flow focusing according to claim 2, characterized in that: The sleeve assembly is connected to a heating and desolvation module of a mass spectrometry flow cytometer, and the airflow introduction component is connected to an atomization gas path of the mass spectrometry flow cytometer.

6. The mass spectrometry flow cytometer sample introduction system based on flow focusing according to claim 1, characterized in that: The inner diameter of the capillary (6) is 50 to 300 μm, and the outer diameter is 300 to 500 μm.

7. The mass spectrometry flow cytometer sample introduction system based on flow focusing according to claim 1, characterized in that: The micropore nozzle (7) is manufactured by 3D printing, and the aperture of the ejection hole is 100 to 500 μm.

8. The mass spectrometry flow cytometer sample introduction system based on flow focusing according to claim 2, characterized in that: The outer diameter of the outer tube of the sleeve is 4-8 mm, and the inner diameter is 2-6 mm; the outer diameter of the inner tube of the sleeve is 2-6 mm, and the inner diameter is 1-3 mm.

9. The mass spectrometry flow cytometer sample introduction system based on flow focusing according to any one of claims 1 to 8, characterized in that: The carrier gas flow rate of the atomizing gas is 0.1-1 L / min, and the injection speed is 10-100 μL / min.

10. The mass spectrometry flow cytometer sample introduction system based on flow focusing according to any one of claims 1 to 8, characterized in that: Its application areas are single-cell analysis by mass spectrometry flow cytometry and single-cell analysis by inductively coupled plasma mass spectrometry.

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