High-throughput single cell mass spectrometry method and device

Abstract

The invention relates to the technical field of single cell mass spectrometry, in particular to a high-throughput single cell mass spectrometry method and device. According to the specific technical scheme, the method comprises the following steps: (1) arranging cells in a quartz capillary tube through a single cell sorting module, and forming liquid drops containing the cells at the outlet end of the quartz capillary tube; (2) observing the number of cells in the liquid drops at an observation window away from the outlet end of the quartz capillary tube through a microscope, and removing the liquid drops by using side-spraying pulse gas if no cells exist or a plurality of cells exist; if only one cell exists, the liquid drops are transferred to the position above the side face opening of the spraying capillary tube and enter the spraying capillary tube; and (3) splitting and extracting the cells in the liquid drops by a spray solvent in the spray capillary tube, and analyzing by a mass spectrometer through an ionization module. The flow cytometry, the single cell recognition and sorting and the micro sample ionization technology are combined, so that high-flux single cell mass spectrometry is realized, and the accuracy of single cell metabolite identification is improved.

Description

Technical Field

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

[0002] Cells are the basic building blocks and functional units of organisms. Studying cells can explore the laws of life activities at a deeper level. The main challenges in analyzing cells at the cellular level are the small cell volume, extremely low substance content, and complex composition. However, nucleic acid substances have the property of being amplifiable. The polymerase chain reaction (PCR) technology can be used to amplify low-concentration DNA or RNA to a certain concentration, thus enabling the analysis of DNA and RNA in single cells. Currently, single-cell genome and transcriptome analyses have been successfully achieved. Although proteins lack effective amplification means, they have specific amino acid sequences and three-dimensional structures, showing good immunogenicity. The "immunolabeling" method can be used to analyze proteins in single cells, and common methods include microprotein blotting, fluorescence flow cytometry, flow cytometry-mass spectrometry, miniaturized immunoassays, etc. Small molecular weight, low content, complex types, non-amplifiable, and almost non-immunogenic metabolites in single cells make the analysis work difficult. Therefore, developing new, efficient, and sensitive analysis methods for single-cell chemical components is of great significance in the fields of life science and medicine.

[0003] For the mass spectrometry analysis of single cells reported currently, the commonly used analysis methods are "flow cytometry-mass spectrometry", "microscopic sampling single-cell mass spectrometry", or "surface extraction". For example, CynTOF flow cytometry-mass spectrometry ( Figure 1 a) and flow electrospray ionization mass spectrometry ( Figure 1 b) adopt the analysis form of flow cytometry. This single-cell loading mode has a relatively high throughput. Single cells will be quickly ejected and ionized, so single cells only have a short-time mass spectrometry signal. The microscopic single-cell mass spectrometry ( Figure 1 c) can obtain a long-time mass spectrometry signal compared with flow cytometry loading (the ionization methods mostly adopt enhanced technologies based on electrospray, such as NanoESI, Pulsed DC ESI, etc.). However, similar to the sampling process of "surface extraction", it is manually performed for single-cell positioning and sampling under the assistance of a microscope, relying on manual operation, with low efficiency and difficult to achieve high-throughput single-cell analysis.

[0004] Single-cell array technology disperses cells at the sampling points of the array (for example, using the micropores of the array to capture single cells). By adjusting the cell density in the cell suspension, each sampling point of the array contains only a single cell, and then combined with microdroplet extraction technology to achieve the extraction of single cells. Compared with capillary micro-sampling technology, this method can reduce the cell positioning time, thereby improving the analysis throughput. However, it still requires additional means to ensure that the number of cells at each sampling point remains consistent. In addition, when mass spectrometry imaging technology analyzes single-cell samples, it also faces problems such as short-lasting mass spectrometry signals and only being able to obtain first-order mass spectrometry data, making it difficult to carry out subsequent compound identification. Probe electrospray and ordinary nanoelectrospray often require adding a large amount of compensation solvent during the analysis process, causing the sample to be over-diluted and resulting in the loss of signals of low-concentration compounds.

[0005] Therefore, it is urgently necessary to newly develop a high-sensitivity and high-throughput device to achieve long-term and high-sample-economy analysis of ultra-trace samples. Summary of the Invention

[0006] Aiming at the deficiencies of the prior art, the present invention provides a high-throughput single-cell mass spectrometry analysis method and device, which combines flow cytometry technology, single-cell recognition and sorting, and trace sample ionization technology, thereby realizing high-throughput single-cell mass spectrometry analysis and improving the accuracy of single-cell metabolite identification.

[0007] To achieve the above object, the present invention is realized through the following technical solutions:

[0008] The present invention discloses a high-throughput single-cell mass spectrometry analysis method, including the following steps:

[0009] (1) Arrange cells in a quartz capillary through a single-cell sorting module, and form droplets containing cells at the outlet end of the quartz capillary;

[0010] (2) Observe the number of cells in the droplet through a microscope arranged beside the quartz capillary at the observation window at a distance from the outlet end of the quartz capillary. If there is no cell or there are multiple cells, remove the droplet with a side-spray pulsed gas; if only one cell exists, transfer the droplet to above the side opening of the spray capillary through a single-cell loading module and enter the spray capillary;

[0011] (3) The cells in the droplet are lysed and extracted by the spray solvent in the spray capillary, and through an ionization module, an electrospray is formed at the tip of the spray capillary and analyzed by a mass spectrometer.

[0012] Preferably, the spray solvent is a mixed solution of methanol and acetonitrile and contains 1% additive, where methanol:acetonitrile = 1:1, v / v; the additive includes but is not limited to any one of formic acid, acetic acid, and ammonia water.

[0013] Preferably, the suspension containing the cells is stirred by a magnetic stirrer.

[0014] Preferably, after one single-cell loading is completed, there is a section of blank spray solvent at the tip of the spray capillary, and the blank solvent is removed by ordinary electrospray ionization with a high flow rate.

[0015] Preferably, after one mass spectrometry data acquisition is completed, the residues of the cell analysis in the spray capillary are removed, and the spray capillary is replenished with spray solvent.

[0016] Correspondingly, a high-throughput single-cell mass spectrometry analysis device includes a single-cell sorting module and a single-cell loading module connected in sequence. The single-cell sorting module arranges single cells into a quartz capillary to form single-cell droplets at the tail end of the quartz capillary. The single-cell loading module moves the single-cell droplets into the spray capillary. The spray capillary has a side opening, and the single-cell loading module allows the droplets to enter the spray capillary through the side opening.

[0017] Preferably, the single-cell sorting module is one of flow cytometry single-cell sorting and micro-droplet nozzle single-cell sorting, and the single-cell loading module is one of a mechanical droplet moving device and an electric field controlled droplet drift device.

[0018] Preferably, the ionization module includes but is not limited to any one of Nano ESI, Pulsed DC ESI, InESI, SPI-ESI, and Chain ESI.

[0019] Preferably, the outlet end of the quartz capillary is subjected to hydrophobic treatment.

[0020] The present invention has the following beneficial effects:

[0021] 1. The present invention uses flow cytometry and image recognition technology to realize the recognition of single-cell droplets. Through bypass gas purging, droplets that do not meet the requirements are removed. At the same time, through PDMS hydrophobic treatment and the design of side openings in the spray capillary, the consistency of the micro-droplet shape and the stability during transfer are ensured.

[0022] 2. The present invention realizes parallel operation through program control, prepares the next single-cell droplet while collecting mass spectrometry data. Compared with the single-cell analysis throughput of capillary microscopy sampling < 2 cells / hour, the analysis throughput of the present invention is increased to 17 cells / hour. Through the high-voltage electromagnetic switch and the solvent replenishment mechanism, residues are removed to ensure the purity of the signal for each analysis. Description of the Drawings

[0023] Figure 1Prior art of existing single-cell mass spectrometry; (a) Flow cytometry-inductively coupled plasma mass spectrometry, (b) Flow cytometry-electrospray mass spectrometry, (c) Schematic diagram of microscopy-single-cell mass spectrometry;

[0024] Figure 2 Schematic diagram of the single-cell mass spectrometry analysis device of the present invention;

[0025] Figure 3 Physical diagram of the single-cell mass spectrometry analysis device;

[0026] Figure 4 In (a), cells flowing are observed under a microscope, and in (b), microdroplets containing cells at the outlet end of a quartz capillary;

[0027] Figure 5 Spray capillary with side holes;

[0028] Figure 6 Droplets (1 ppm, ~3 nL) of reserpine solution analyzed by CSHSCS-Chain-ESI (LTQ mass spectrometer); (a) TIC and EIC diagrams, (b) Mass spectrometry diagram corresponding to 4 minutes, where a good reserpine mass spectrometry signal (m / z 609) can be seen, (c) Mass spectrometry diagram after one droplet analysis is completed and after cleaning (only background signal, no residual reserpine);

[0029] Figure 7 Primary mass spectrometry characteristics of various cancer cells; (a) Number of mass spectrometry peaks detected from a single cell, (b) Cluster diagram of principal component analysis based on the primary mass spectrometry of cancer cells;

[0030] Figure 8 Results of CSHSCS Chain-ESI analysis of various cancer cells; (a) Typical total ion current diagram during single-cell analysis, (b)-(f) Typical mass spectrometry diagrams of different cell types, (g) Mass spectrometry diagram of blank background;

[0031] Figure 9 Lipid component analysis of various cancer cells; (a) Set of tandem mass spectrometry diagrams containing characteristic ions of m / z184 detected from cancer cells, (b) PCA analysis of multiple cancer cells and (c) Loading diagrams of PC1 and PC2. The distance between the point and the origin shows the contribution of different metabolites to cell diversity, (d) Relative intensity bar chart of metabolites that contribute greatly to the heterogeneity of different types of cancer cells, (e) Schematic diagram of the PCA-LDA analysis model, (f) Confusion matrix results identified by the PCA-LDA model of the test set;

[0032] Figure 10 Schematic diagram of the single-cell analysis platform device;

[0033] In the figure: high-voltage electric field 1, high-voltage electric field 2, quartz capillary 3, spray capillary 4, observation window 5, side opening 6, mass spectrometer 7, ion inlet 8. Detailed implementation manners

[0034] The following will clearly and completely describe the technical solutions in the embodiments of the present invention with reference to the accompanying drawings in the embodiments of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all the embodiments. All other embodiments obtained by those of ordinary skill in the art based on the embodiments of the present invention without creative efforts shall fall within the protection scope of the present invention.

[0035] Unless otherwise specified, the technical means used in the implementation examples are conventional means well known to those skilled in the art.

[0036] There are two means for mass spectrometry to identify metabolites. Usually, a single cell can only collect instantaneous mass spectrometry signals under other ionization methods, so that only the accurate mass number information of the molecule can be obtained. Due to the existence of isomers widely present in nature, it is inaccurate to identify molecules only relying on the accurate mass number information. The tandem mass spectrometry data collected by mass spectrometry contains characteristic fragment ion information of molecular structure information. However, the collection of tandem mass spectrometry data requires continuous ionization of the sample to generate stable gaseous ions. And this is exactly the content that the micro-sample ionization technology needs to solve.

[0037] Therefore, the present invention provides a high-throughput single-cell mass spectrometry analysis method and device based on the micro-sample ionization technology. A high-throughput single-cell mass spectrometry analysis method based on chain electrospray ionization (Chain ESI) is provided. In order for a single cell to be mass spectrometrically detected for a long time, sufficient tandem mass spectrometry data can be collected to improve the accuracy of component identification. The present invention adopts the electrospray mass spectrometry technology Chain ESI with a small flow rate to improve the sample economy (that is, to improve the utilization efficiency of the sample (more data can be collected with less sample)), and extend the sampling duration of a single cell during mass spectrometry analysis.

[0038] In order to improve the analysis throughput of single-cell mass spectrometry, the present invention proposes capillary side hole assisted single cell sampling (CSHSCS) to enhance the analysis ability of the ultra-low flow rate electrospray ionization technology for single cells. By integrating and assembling CSHSCS with the small flow rate electrospray mass spectrometry technology (CSHSCSESI), a high-throughput and high-capacity (a large amount of tandem mass spectrometry data can be collected from a single cell) single-cell mass spectrometry analysis platform is invented to enhance the analysis ability of the small flow rate electrospray mass spectrometry technology for single cells.

[0039] The CSHSCS ESI of the present invention uses a glass capillary as an ionization carrier, which can effectively reduce the sample consumption rate after isolating the electric field, prolong the ionization time of trace samples, and enable the mass spectrometer to calmly record the rich tandem mass spectrometry data in the sample, thereby increasing the accuracy of sample identification. However, the existing capillary sampler cannot collect a large amount of single-cell tandem mass spectrometry data.

[0040] The high-throughput single-cell mass spectrometry analysis device disclosed in the present invention comprises a single-cell sorting module and a single-cell loading module which are connected in sequence, wherein the single-cell sorting module arranges droplets into a quartz capillary, and the single-cell loading module moves the droplets into a spray capillary, wherein a hole is opened on the side of the spray capillary, and the single-cell loading module allows the droplets to enter the spray capillary through the side opening.

[0041] The single cell sorting module is one of flow cytometry single cell sorting and micro droplet nozzle single cell sorting, and the single cell loading module is one of a mechanical droplet moving device (such as a linear motor) and an electric field controlled droplet drift device (such as an electric field perpendicular to the droplet moving direction can control the droplet's running trajectory and direction). The ionization module includes but is not limited to any one of Nano ESI, Pulsed DC ESI, InESI, SPI-ESI and Chain ESI.

[0042] Based on the above device, the present invention discloses a high-throughput single-cell mass spectrometry analysis method, comprising the following steps:

[0043] (1) The cells are arranged in a quartz capillary 3 by a single cell sorting module, and a droplet containing the cells is formed at the outlet of the quartz capillary 3; the suspension containing the cells is stirred by a magnetic stirrer to ensure that the cells do not adhere to the wall or sink to the bottom, and to ensure that the cell suspension remains uniform, and then the cells are squeezed into the quartz capillary 3.

[0044] (2) The number of cells in the droplet is observed through an observation window 5 at the outlet of the quartz capillary 3 by a microscope arranged beside the quartz capillary 3. If there is no cell or multiple cells, the droplet is removed by side-spray pulse gas; if there is only one cell, the droplet is transferred to the side opening 6 of the spray capillary 4 through the single-cell loading module and enters the spray capillary 4; wherein, the observation window 5 is formed by removing the coating of the capillary 1 cm away from the outlet of the quartz capillary 3. Then, after the microscope observes a cell flowing through the observation window, a single-cell droplet is formed.

[0045] (3) The cells in the droplet are lysed and extracted by the spraying solvent in the spraying capillary 4, and through the ionization module, an electrospray is formed at the tip of the spraying capillary and analyzed by the mass spectrometer 7. The spraying solvent is a mixed solution of methanol and acetonitrile and contains 1% additive, where methanol:acetonitrile = 1:1, v / v; the additive includes but is not limited to any one of formic acid, acetic acid, and ammonia water.

[0046] Furthermore, after one single-cell sample loading is completed, there is a section of blank spraying solvent at the tip of the spraying capillary, and the blank solvent is removed by using high-flow normal electrospray ionization.

[0047] Furthermore, after one mass spectrometry data acquisition is completed, the residue of the cell analysis in the spraying capillary is removed, and the spraying solvent is replenished into the spraying capillary. As one of the implementation manners, the supply rate of the spraying solvent can be 500 nL / min.

[0048] In the present invention, the solution containing a single cell at the tip of the spraying capillary is ionized at the ion inlet 8 of the mass spectrometer 7 to form an electrospray, and then analyzed by the mass spectrometer 7. The mass spectrometer is one of a time-of-flight mass spectrometer, a quadrupole mass spectrometer, an ion trap mass spectrometer, an orbitrap mass spectrometer, a Fourier transform ion cyclotron resonance mass spectrometer, and an electromagnetic double-focusing mass spectrometer.

[0049] Furthermore, if flow cytometry is used for single-cell sorting to obtain stationary single droplets, a mechanical droplet moving device is used for single-cell sample loading; if a micro-droplet nozzle is used for single-cell sorting to obtain single droplets with an initial velocity, an electric field-controlled droplet drift device is used for single-cell sample loading.

[0050] As one of the implementation manners, Chain ESI is used as the ionization module, flow cytometry is used as the single-cell sorting module, and a mechanical micro-droplet moving device is used as the single-cell sample loading module. The specific analysis process is as follows:

[0051] (1) The cell suspension is diluted to a cell density where each single droplet contains one cell;

[0052] (2) The cell suspension is arranged in sequence after passing through a narrow capillary channel and flows out of the quartz capillary 3 in sequence, and droplets are formed at the tip of the quartz capillary 3;

[0053] (3) The quartz capillary 3 is moved towards the opening position of the spraying capillary 4 so that a single droplet is transferred into the spraying capillary;

[0054] (4) The cells in the droplet are lysed, extracted, ionized by the spraying solvent in the spraying capillary 4, an electrospray is formed at the tip, and then analyzed by the mass spectrometer.

[0055] Specifically: Refer to Figure 2 、3 As shown in Figure 10, the process of single-cell mass spectrometry analysis of the present invention is as follows:

[0056] (1) For the single-cell sorting of CSHSCS, flow cytometry is used. After the cells flow orderly through a narrow channel (a coated quartz capillary with an inner diameter of 100 μm and an outer diameter of 184 μm, and about 1 cm of the coating near the outlet end is removed to form an observation window for the microscope), a microdroplet containing cells (with a diameter of about 200 μm and a volume of about 3 nL) is formed at the outlet end of the quartz capillary. To ensure the stability of the microdroplet at the tip of the quartz capillary, the outlet end of the quartz capillary is hydrophobically treated with PDMS (polydimethylsiloxane) to keep the single-cell droplet stably spherical, facilitating its transfer into the spray capillary and preventing it from adhering to the side of the quartz capillary. The flowing cells are observed through the observation window of the quartz capillary using a microscope, and a self-developed cell recognition program based on OpenCV is used to quantify the number of cells present in the observation window. If no cell or multiple cells flow through the observation window 5, this droplet is removed by a side-spray pulsed gas; if only one cell is present, the droplet is transferred to the side opening of the spray capillary 4 by a linear motor.

[0057] Among them, the spray capillary is as Figure 5 shown. There is a small hole on the side near the tip of the spray capillary to allow the droplet to enter. The specific operation is to connect a 50 mL syringe to the tail end of the pulled capillary of the spray capillary, press the syringe to compress the air, generating a pressure of about 0.2 MPa. Then, heat the position of the pulled capillary near the tip, and finally, a small hole is extruded at the heated position through the compressed gas.

[0058] The outlet end of the quartz capillary is treated with PDMS so that the cell suspension extruded presents a spherical shape and can be well maintained at the outlet end of the quartz capillary. Subsequently, when transferred to the spray capillary, the droplet containing a single cell can be transferred relatively completely. In addition, a longer analysis time will cause the cells in the cell suspension to adhere to the wall or sink to the bottom. Specifically: the cells are dispersed in the liquid, and the liquid is an isotonic and isosmotic system, such as physiological saline or PBS. If it is pure water, the cells will rupture. In the prior art, an injection pump is used to extrude the cell suspension, but as the analysis progresses, fewer and fewer cells can reach the outlet end of the quartz capillary, or cell clusters containing multiple cells reach the outlet end of the quartz capillary simultaneously. Therefore, the homogeneity and stability of the cell suspension are crucial for single-cell analysis. Finally, the present invention abandons the cell loading method based on an injection pump and adopts a gas extrusion method to squeeze the cells into the quartz capillary, and at the same time, a small magnetic stirrer is used to maintain the homogeneity of the cell suspension.

[0059] (2) After a single cell is loaded, there is usually a blank spray solvent (methanol:acetonitrile = 1:1 (v / v), containing 1% formic acid) at the tip of the spray capillary. To remove this blank solvent, first turn on the high-voltage electric field 2 (1.5 kV) and then turn it off after 1 s. Use the high flow rate of ordinary electrospray to expel the blank solvent. Adjust the voltage and its connection time according to specific experimental conditions until the blank solvent at the tip of the spray capillary is consumed. Among them, the high-voltage electric field 1 is for ultra-low flow rate ionization and remains on. When the high-voltage electric field 2 is on, ionization is mainly based on high flow rate electrospray; when the high-voltage electric field 2 is off, the flow rate of the spray capillary is ultra-low flow rate ionization.

[0060] (3) The next single cell droplet can be prepared during the mass spectrometry data acquisition. After the mass spectrometry data acquisition for this time (the mass spectrometry data acquisition time for a single cell is 3 minutes), remove the residue of this cell analysis in the spray capillary. Turn on the high-voltage electric field 2 and keep it on for 30 s and then turn it off to eliminate the interference for the next single cell analysis. Subsequently, add fresh spray solvent from the tail end of the spray capillary, and the excess solvent will flow out from the side opening. After the solvent replenishment is completed, transfer the prepared next single cell droplet into the spray capillary to start the next round of single cell analysis.

[0061] The entire single cell analysis process is realized through program control and is monitored by a microscope and a digital camera. The microscope consists of a single-tube optical biological microscope and a CCD camera and is used to observe cells. The digital camera observes the single cell loading situation in the range of 50 - 1000 times magnification. To ensure that the cells are evenly dispersed in the suspension, a small magnetic stirrer (model M-CL, Hangzhou Qiwei Instrument Co., Ltd.) is used. Subsequently, the cell suspension is pressed into the quartz capillary by compressed nitrogen (0.2 kg·cm -2 ) (gas extrusion for 0.5 s), and droplets of about 3 nL are formed at the outlet end of the quartz capillary.

[0062] The upper computer and lower computer programs are written in Python and C++ respectively. The high-frequency electromagnetic gas valve for controlling the purge gas is used to remove unqualified droplets (i.e., droplets containing a cell number other than 1) at the outlet end of the quartz capillary. The high-voltage electromagnetic switch is used to control the on and off of the high-voltage electric field 2. The spray solvent of the spray capillary is provided by an injection pump, which is driven by a linear stepper motor. The spray solvent supply speed is 500 nL / min.

[0063] Additionally, a linear motor is used to transfer the microdroplets containing cells into the spray capillary. The activation signal for mass spectrometer data acquisition is sent by an electromagnetic relay. Once a droplet containing a single cell is identified, the automatic analysis process begins. In single-cell analysis, the mass spectrometry acquisition time is 3 minutes, and the cleaning time of the spray capillary is 0.5 minutes. While the mass spectrometry data of a single cell is being acquired, the next single-cell droplet can be prepared. Therefore, the mass spectrometry analysis of one cell can be completed within 3.5 minutes.

[0064] After the CSHSCS is set up, its ability to analyze trace samples is first tested using standards. A 3-nL droplet of a mixed standard solution (containing reserpine at a concentration of 1 ppm) is loaded into the spray capillary. As Figure 6 shown, the spectrum shows good signal-to-noise ratio and stability, successfully detecting the compound in the microdroplet, and there is no analyte residue after automated cleaning ( Figure 6 c). This indicates that CSHSCS combined with Chain-ESI has good analytical ability for trace samples.

[0065] The present invention will be further described below in conjunction with specific embodiments.

[0066] Example 1

[0067] During the actual analysis process, in order to improve the quality of cell analysis, the residual effect needs to be weakened. Therefore, the spray capillary is connected to a high-voltage power supply high-voltage electric field 2 that can control the on-off, and the residue in the spray capillary is cleaned using the high flow rate of electrospray. Specifically, when a single cell is ionized and analyzed by Chain ESI (the flow rate of Chain ESI is at the picoliter level), the high-voltage electric field 2 is turned on, so that the cell residue can be quickly flushed to prevent it from interfering with the next analysis.

[0068] After culturing A549, HCCLM3, HCK293T, MHCC97H, and SK-HEP-1 cancer cells, the culture medium is washed with PBS respectively, and then suspended with 140 mM NH4Ac aqueous solution and diluted to an appropriate cell concentration (such as 5×10 4 cells / ml). Then the cell suspension is passed through a quartz capillary, and the cells are arranged in the narrow channel and sequentially flow out of the quartz capillary, forming a droplet (~14 nL) with a diameter of ~300 μm at the front end. The measurement of the droplet size is as Figure 4 shown in b. In order to keep the droplet at the tip of the quartz capillary, the capillary is hydrophobically treated with PDMS. The droplet is observed through the observation window using a microscope, as Figure 4a. If there are no cells or multiple cells in the droplet, the droplet is purged and removed with gas. When there is only one cell in the droplet, the droplet is transferred into the orifice of the spray capillary by a linear motor, thus realizing the loading of a single cell.

[0069] Generally, after the injection of a single cell is completed, there is still a section of blank spray solvent at the tip of the spray capillary. The high-voltage electric field 2 is turned on, and the blank solvent is removed with high-flow ordinary ESI. The high-voltage value and duration of the high-voltage electric field 2 are approximately 1.5 kV and 1 s. The specific time should be adjusted according to the experimental environment as long as the purpose of consuming the blank solvent at the front end of the spray capillary can be achieved. After the blank solvent is completely removed, the high-voltage electric field 2 is disconnected, and ionization is carried out with Chain ESI. At the same time, the mass spectrometer is activated for data acquisition. During the acquisition process, the droplet of the next single cell can be prepared. After the acquisition is completed (the set acquisition duration for a single cell is 3 min), the high-voltage electric field 2 is also turned on for 30 s, and the residual single-cell residue in the spray capillary is cleaned with the high flow rate of ESI to prevent interference with the mass spectrometry analysis of the next cell. During the cleaning process, fresh spray solvent will be automatically replenished in the spray capillary. After the spray capillary is cleaned, the prepared single-cell droplet is transferred through the orifice to the spray capillary, and a new round of single-cell analysis is started.

[0070] The present invention uses Chain-ESI for single-cell metabolite analysis of various types of cancer cells, including A549, HCCLM3, HCK293T, MHCC97H, and SK-HEP-1. A total of 1100 - 1400 MS 1 characteristic peaks are detected from a single cell, as Figure 7 shown in a. Typical mass spectrometry data is as Figure 8 shown. The primary mass spectrometry data of each type of cancer cell provides a unique "fingerprint", and cancer cells can be distinguished by principal component analysis (PCA). As Figure 7 shown in b, principal component analysis of the primary mass spectrometry data of the cells well separates different types of cancer cells, proving that CSHSCS Chain-ESI has great potential in single-cell analysis.

[0071] Benefiting from the extremely low flow rate of the Chain ESI ionization method, a trace volume of a single-cell sample is well analyzed, and a large amount of secondary mass spectrometry data is collected. Lipid compounds can participate in cell development and differentiation, affect intracellular signal transduction and material transport, and play an important indicator role in the process of apoptosis. Among them, phosphatidylcholine (PC) and sphingosine (SM) in MS 2has a characteristic ion fragment with m / z 184. Using their characteristic fragment ions in the second stage and the exact molecular weight in the first stage, screening and identification studies were carried out on a variety of cancer cells, including lung cancer cell A549, renal cancer cell HCK293T, and liver cancer cells HCCLM3, MHCC97H, and SK-HEP-1. By extracting the second-stage mass spectrometry diagrams with the characteristic ion of m / z 184 in the fragment ions, a total of 1451 non-repeated second-stage mass spectrometry diagrams were obtained ( Figure 9 a). Combining the characteristic ion of m / z 184 and the exact mass number matching, a total of 485 PC and SM lipids were screened and identified (see Table 1).

[0072] To better understand the differences between these cancer cells, principal component analysis was performed using these identified lipid components. The first two principal components explained 36% and 20% of the variable differences respectively, and different types of cancer cells significantly clustered into different clusters ( Figure 9 b). To study the contribution of each lipid component to cell heterogeneity, the two-dimensional loading plot of principal component analysis shows the contribution of each lipid component to cell differences. Lipid metabolites far from the origin contribute more to the differences ( Figure 9 c). Lipid metabolites mainly gather near the origin, indicating that the compositions of most cells in a single cell are similar. In the two-dimensional loading plot of principal component analysis, the 10 components with the greatest contribution to cell differences are marked in red. Figure 9 d shows the relative distribution of these 10 lipid substances in each cancer cell, and the results indicate that they may be potential biomarkers for distinguishing these cancer cells.

[0073] In addition, principal component analysis-linear discriminant algorithm (PCA-LDA) was used to further analyze the identified lipids. After PCA dimensionality reduction, the first 10 principal components were used for LDA analysis ( Figure 9 e). The data set was divided into a training set and a test set, and the splitting ratio was 7:3. Figure 9 As shown in f, this method achieved 100% accuracy in distinguishing different cancer cell types. In summary, the above results all indicate that CSHSCS Chain-ESI has good single-cell analysis ability, can accurately distinguish and identify cancer cells, and has good application prospects for single-cell research.

[0074] Table 1 Identification results of some lipid substances

[0075]

[0076]

[0077]

[0078]

[0079] In the description of the present invention, it should be understood that the orientation or positional relationship indicated by the terms "longitudinal", "transverse", "upper", "lower", "front", "rear", "left", "right", "vertical", "horizontal", "top", "bottom", "inner", "outer", etc. is based on the orientation or positional relationship shown in the drawings, and is only for the convenience of describing the present invention, rather than indicating or implying that the device or element referred to must have a specific orientation, be constructed and operated in a specific orientation, and therefore should not be construed as a limitation to the present invention.

[0080] The embodiments described above are only descriptions of the preferred embodiments of the present invention, and do not limit the scope of the present invention. Without departing from the design spirit of the present invention, various deformations and improvements made by those of ordinary skill in the art to the technical solutions of the present invention shall fall within the protection scope determined by the claims of the present invention.

Claims

1. A high-throughput single-cell mass spectrometry analysis method, characterized in that: It includes the following steps: (1) Arrange cells in a quartz capillary through a single-cell sorting module, and form droplets containing cells at the outlet end of the quartz capillary; (2) Observe the number of cells in the droplets through a microscope arranged beside the quartz capillary at an observation window at a distance from the outlet end of the quartz capillary. If there are no cells or multiple cells, remove the droplet with a laterally sprayed pulsed gas; if only one cell exists, transfer the droplet to above the side opening of the spray capillary through a single-cell loading module and enter the spray capillary; (3) The cells in the droplet are lysed and extracted by the spray solvent in the spray capillary, and through an ionization module, an electrospray is formed at the tip of the spray capillary and analyzed by a mass spectrometer.

2. The high-throughput single-cell mass spectrometry analysis method according to claim 1, wherein: The spray solvent is a mixed solution of methanol and acetonitrile and contains 1% additive, where methanol:acetonitrile = 1:1, v / v; the additive includes but is not limited to any one of formic acid, acetic acid, and ammonia water.

3. A high-throughput single-cell mass spectrometry analysis method according to claim 1, characterized in that: The suspension containing the cells is stirred by a magnetic stirrer.

4. A high-throughput single-cell mass spectrometry analysis method according to claim 2, characterized in that: After one single-cell loading, there is a section of blank spray solvent at the tip of the spray capillary, and the blank solvent is removed by high-flow normal electrospray ionization.

5. A high-throughput single-cell mass spectrometry analysis method according to claim 2, characterized in that: After one mass spectrometry data acquisition, clear the residue of the cell analysis in the spray capillary and replenish the spray solvent into the spray capillary.

6. An apparatus for a high-throughput single-cell mass spectrometry analysis method according to any one of claims 1 to 5, characterized in that: It includes a single-cell sorting module and a single-cell loading module connected in sequence. The single-cell sorting module arranges single cells into a quartz capillary and forms single-cell droplets at the tail end of the quartz capillary. The single-cell loading module moves the single-cell droplets into the spray capillary. The spray capillary has a side opening, and the single-cell loading module allows the droplets to enter the spray capillary through the side opening.

7. The high-throughput single-cell mass spectrometry analysis device according to claim 6, wherein: The single-cell sorting module is one of flow cytometry single-cell sorting and microdroplet nozzle single-cell sorting, and the single-cell loading module is one of a mechanical droplet moving device and an electric field controlled droplet drift device.

8. A high-throughput single-cell mass spectrometry analysis device according to claim 6 or 7, characterized in that: The ionization module includes but is not limited to any one of Nano ESI, Pulsed DC ESI, InESI, SPI-ESI, and Chain ESI.

9. A high-throughput single-cell mass spectrometry analysis device according to claim 6, characterized in that: Perform hydrophobic treatment on the outlet end of the quartz capillary.

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