Single-cell lipid online extraction and chromatographic separation mass spectrometry device and method based on supercritical fluid

Abstract

The invention discloses a single-cell lipid online extraction and chromatographic separation mass spectrometry device and method based on supercritical fluid. The device comprises a supercritical fluid pump, a six-way valve, a quantitative loop and a capillary chromatographic column spray needle, the capillary chromatographic column spray needle comprises a body and a tip end, the tip end is arranged to be aligned with a sample inlet of the mass spectrometer when the capillary chromatographic column spray needle supplies samples to the mass spectrometer, one port of the six-way valve is connected with the other end of the capillary chromatographic column spray needle, and the other port of the six-way valve is connected with the supercritical fluid pump; a filter with the aperture of 2.0-5.0 microns is arranged at the tail end of a quantitative ring matched with the six-way valve; and the tip of the capillary chromatographic column spray needle is filled with a chromatographic filler. The device is simple in structure and easy to operate, the chromatographic filler and the capillary tube are easy to purchase, and the cost is low; the device does not need complex pretreatment, is convenient to use and wide in application range, and can be used as an effective means for cancer diagnosis and treatment, drug screening, systematic biology, cell heterogeneity research and the like.

Description

Technical Field

[0001] The invention relates to a single-cell lipid online extraction and chromatographic separation mass spectrometry analysis device and method based on microflow supercritical fluid, belonging to the field of biological mass spectrometry analysis. Background Art

[0002] Lipids are the main components of cell membranes and play a key role in the structure and function of cells. Cell lipidome analysis can help us better understand the dynamic changes of cell membranes, signal transduction mechanisms, and the pathogenesis of diseases. There is heterogeneity in cell populations, and single-cell analysis can reveal the differentiated characteristics of a small number of cells. Single-cell lipidomics focuses on the composition and changes of intracellular membranes. Studies on single-cell lipidomics can reveal cancer subtypes (Li ZS, Cheng SM, Lin QH, et al. Single-cell lipidomics with high structural specificity by massspectrometry[J]. Nature Communications, 2021, 12(1). Saunders KDG, Von Gerichten J, Lewis HM, et al. Single-Cell Lipidomics Using Analytical Flow LC-MSCharacterizes the Response to Chemotherapy in Cultured Pancreatic Cancer Cells[J]. Analytical Chemistry, 2023, 95(39): 14727-35.) and explore cell differentiation mechanisms (Von Gerichten J, Saunders KDG, Kontiza A, et al. Single-Cell Untargeted LipidomicsUsing Liquid Chromatography and Data-Dependent Acquisition after Live Cell Selection[J]. Analytical Chemistry, 2023, 95(39): 14727-35.). Chemistry, 2024, 96(18): 6922-9.). In recent years, single-cell genome, transcriptome sequencing and proteome analysis technologies have been developed to a high degree, but the study of single-cell lipidome is still limited by analytical technology.

[0003] Traditional lipidome analysis mainly uses high performance liquid chromatography-mass spectrometry analysis technology, which requires a large sample amount (>10 6Single-cell analysis technology requires complex pretreatment and extraction processes, and a long analysis time (~30 minutes / sample) (Wang J, Wang C, Han X. Tutorial on Lipidomics [J]. Analytica Chimica Acta, 2019.). Single-cell analysis technology should avoid the dilution caused by complex pretreatment to improve analytical sensitivity; in addition, single-cell analysis technology requires high analysis throughput to meet the number of samples that reach statistical significance. In order to develop single-cell lipidome analysis technology that meets the above requirements, it is necessary to improve the methods of lipid extraction and chromatographic separation in a targeted manner.

[0004] Supercritical carbon dioxide fluid refers to a single phase of carbon dioxide with properties between liquid and gas when the temperature and pressure are both above the critical point. The diffusion coefficient of supercritical fluid is 10 to 100 times that of liquid, so it has good permeability to materials. The SFE-SFC combination method, which uses supercritical carbon dioxide fluid as both the extractant and the main component of the mobile phase, can fully dissolve and extract lipid substances from cells, and achieve efficient reversed-phase chromatography separation based on the chemical structure of different lipid substances to meet subsequent mass spectrometry analysis. However, traditional SFE-SFC-MS cannot be used for mass spectrometry analysis of single-cell samples because of its large liquid flow rate and volume, which over-dilutes single-cell samples. Summary of the invention

[0005] The purpose of the present invention is to provide a single-cell lipid online extraction and chromatography-mass spectrometry separation and analysis device and method based on microfluidic supercritical fluid, which does not require a complicated pre-treatment process. The microfluidic supercritical fluid dissolves and extracts the lipids of a single cell in a quantitative loop to avoid dilution and provide high detection sensitivity. The supercritical fluid chromatography separation speed is fast and provides a higher single-cell analysis throughput. The entire process is carried out in the flow path, which is simple and easy to operate, without sample loss and contamination, and the capillary chromatographic column spray needle used is low in cost and can be prepared by itself.

[0006] The sampling and ionization device for online extraction chromatography separation of single cell lipids provided by the present invention comprises a supercritical fluid pump, a six-way valve, a quantitative loop and a capillary chromatographic column spray needle;

[0007] Wherein, the capillary chromatographic column spray needle comprises a body and a tip, the tip is configured to align with the injection port of the mass spectrometer when supplying a sample to the mass spectrometer through the capillary chromatographic column spray needle, one port of the six-way valve is connected to the other end of the capillary chromatographic column spray needle, and the other port is connected to the supercritical fluid pump;

[0008] The quantitative ring is configured to cooperate with the six-way valve, and a filter with a pore size of 2.0 to 5.0 μm is provided at the end of the quantitative ring. The volume of the quantitative ring is 5.0 to 10.0 μL, and its inner diameter is 160 to 250 μm. The loading mode and the injection mode are realized by the cooperation of the quantitative ring and the circulation valve.

[0009] The tip of the capillary chromatographic column spray needle is filled with a chromatographic filler, such as a C18 silica gel chromatographic filler or a C30 silica gel chromatographic filler.

[0010] Preferably, the capillary chromatographic column spray needle is drawn by a capillary laser (such as a Shutter P-2000 laser drawer), which has low production cost and simple process, and can be replaced after a single use;

[0011] Preferably, the inner diameter of the capillary chromatographic column spray needle is 100 to 200 μm, and the inner diameter of the tip is 2 to 3.5 μm;

[0012] The filling length of the chromatographic packing in the tip is 5 to 8 cm.

[0013] On the basis of the above-mentioned sample introduction and ionization device, the present invention also provides a single-cell lipid online extraction and chromatography-mass spectrometry separation and analysis device based on supercritical fluid, comprising a mass spectrometer and the above-mentioned sample introduction and ionization device.

[0014] Furthermore, the present invention provides a single cell lipid online extraction and chromatography-mass spectrometry separation and analysis method based on microfluidic supercritical fluid, comprising the following steps:

[0015] S1, placing the six-way valve in the "loading" mode, and injecting a single cell to be tested into the quantitative loop of the six-way valve in the injection and ionization device;

[0016] S2, placing the six-way valve in the "injection" mode, connecting one end of the six-way valve to the supercritical fluid pump, connecting the other end to the capillary chromatographic column spray needle, turning on the supercritical fluid pump, and performing online extraction and chromatographic separation;

[0017] S3, aligning the tip of the capillary chromatographic column spray needle with the injection port of a mass spectrometer; connecting the port of the six-way valve connected to the capillary chromatographic column spray needle body to a high-voltage power supply to achieve electrospray ionization and perform mass spectrometry analysis.

[0018] After the analysis, the following washing steps are also included:

[0019] Turn off the high voltage and mass spectrometry data acquisition mode, put the six-way valve in the "load" mode, use the supercritical fluid pump to backwash the quantitative loop to avoid residue, and start the analysis of the next sample after flushing.

[0020] In step S2, the supercritical fluid used by the supercritical fluid pump is a mixed fluid of supercritical carbon dioxide and methanol, wherein the methanol ratio (volume ratio) is 10% to 25%, the flow rate is 50 to 200 μL / min, and the fluid pressure is 9 to 15 MPa.

[0021] In order to solve the problem of long separation time of liquid chromatography, the supercritical fluid chromatography (SFC) adopted in the present invention provides high-efficiency chromatographic separation in a shorter time, and provides a basis for high-throughput analysis of cell samples.

[0022] The device of the present invention has a good chromatographic retention effect on single-cell lipid substances, and provides the dimension of retention time (Retention Time) based on the mass-to-charge ratio (m / z) of the detected target substance. The separation of different substances makes it easier to identify the detected species in the database, thereby improving the credibility of the identification.

[0023] Experiments show that the single-cell lipidomics mass spectrometry data produced by the device of the present invention is consistent with the data format of traditional lipidomics, which realizes the standardization of subsequent data processing and makes it easier to compare the single-cell lipidomics data with traditional lipidomics.

[0024] Secondary mass spectrometry (MS / MS) helps confirm the structure of substances and achieve relative quantification in traditional lipidomics. The device of the present invention provides the possibility of using data-dependent acquisition (DIA) and data-independent acquisition (DDA) secondary mass spectrometry acquisition modes by applying substance separation based on the principle of chromatography, and the device of the present invention improves the quantitative analysis capability of single-cell lipidomics.

[0025] Through experimental verification, the results of single-cell analysis experiments using the device of the present invention are stable and less affected by external factors.

[0026] The device of the present invention has a simple structure and is easy to operate, and the chromatographic filler and capillary are easy to purchase and low in cost. The capillary column spray needle is convenient for rapid replacement, thereby improving the single cell analysis throughput.

[0027] The device of the present invention does not require complicated pre-treatment, is easy to use, and has a wide range of applications. It can be used as an effective means for cancer diagnosis and treatment, drug screening, systems biology, cell heterogeneity research, etc. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] Figure 1 It is a schematic diagram of the single-cell lipid online extraction chromatographic separation device of the present invention.

[0029] Figure 2 This is a schematic diagram of the loading, injection and flushing step modes when the device of the present invention is used.

[0030] Figure 3This is the standard curve used by the device of the present invention for lipid standard analysis.

[0031] Figure 4 This is a chromatographic mass spectrum of the device of the present invention when used for lipid analysis of a single oocyte.

[0032] Figure 5 The chromatographic separation of some lipid substances when the device of the present invention is used for lipid analysis of a single oocyte.

[0033] Figure 6 It is the number of lipid species that can be detected when the device of the present invention is used for lipid analysis of a single oocyte.

[0034] Figure 7 The results of analyzing different numbers of oocytes using the device of the present invention and a conventional supercritical fluid extraction-chromatographic separation device are compared.

[0035] Figure 1 Marked as:

[0036] 1. Supercritical fluid pump, 2. Quantitative loop, 3. Cell, 4. Filter (pore size 2 μm), 5. Six-way valve, 6. Capillary column spray needle, 7. High voltage power supply, 8. Mass spectrometer injection cone. DETAILED DESCRIPTION

[0037] Unless otherwise specified, the experimental methods used in the following examples are conventional methods.

[0038] Unless otherwise specified, the materials and reagents used in the following examples can be obtained from commercial sources.

[0039] like Figure 1 As shown, the sampling and ionization device for online extraction chromatography separation of single cell lipids provided by the present invention comprises a supercritical fluid pump 1, a six-way valve 5, a capillary chromatographic column spray needle 6 and a mass spectrometer. The supercritical fluid pump 1 is connected to the six-way valve 5, one end of the six-way valve 5 is connected to the capillary chromatographic column spray needle 6, and the other end of the capillary chromatographic column spray needle 6 is used to correspond to the sampling port 8 of the mass spectrometer.

[0040] In the present invention, a filter 4 with a pore size of 2.0 to 5.0 μm is arranged at the end of the quantitative ring 2 matched with the six-way valve 5, the volume of the quantitative ring 2 is 5.0 to 10.0 μL, and its inner diameter is 160 to 250 μm; the sample loading mode and the injection mode are realized by matching the quantitative ring 2 with the flow valve 6, such as Figure 2 shown.

[0041] In the present invention, the tip of the capillary chromatographic column spray needle 6 is filled with C18 silica gel chromatographic filler, and the filling length is 5 to 8 cm. The capillary chromatographic column spray needle 6 is drawn by a capillary spray needle laser (such as a Shutter P-2000 laser drawer), which has low production cost and simple process, and can be replaced after one-time use. The inner diameter of the capillary chromatographic column spray needle 4 is 100 to 200 μm, and the inner diameter of the tip is 2 to 3.5 μm.

[0042] The sample injection and ionization device of the present invention can be used to analyze single cell lipids. When analyzing single cell lipid groups, for example Figure 2 As shown, you can follow the steps below:

[0043] S1, the six-way valve 5 is placed in the "loading" mode, a single cell 3 is sucked using a capillary under a microscope, and injected into the quantitative loop 2 connected to the six-way valve 5, and the filter 4 with a pore size of 2 μm at the rear end of the quantitative loop 2 ensures that the cell is placed in the quantitative loop 2;

[0044] S2, six-way valve 5 is placed in "injection" mode, connected to supercritical fluid pump 1, and the flow rate (75% supercritical CO2 / 25% methanol, 100 μL / min) is turned on to complete online extraction and chromatographic separation;

[0045] S3, the capillary column spray needle 6 is facing the cone 8 of the mass spectrometer, 3kV high voltage is applied to the front metal part to realize electrospray ionization, and the mass spectrometry data acquisition mode is turned on, and the acquisition time is 5 minutes;

[0046] S4. Turn off the high voltage and mass spectrometry data acquisition mode, place the six-way valve 5 in the "flushing" mode, use the supercritical fluid pump 1 to reversely flush the quantitative loop 2 for 1 minute to avoid residue, and start the analysis of the next sample after flushing.

[0047] The sensitivity and analytical performance of the device of the present invention were evaluated using lipid substance standards. Figure 3 As shown, 5 μL of standard solutions containing 0.1, 1, 5, and 10 ng of a mixture of four types of lipid substances were injected respectively, and the device of the present invention was used to analyze and draw a standard curve. The detection limit was calculated based on the principle of a three-fold signal-to-noise ratio (S / N>3). The results are shown in Table 1.

[0048] Table 1 Analysis of four lipid standards

[0049] Metabolite Chinese name English name Ion mass-to-charge ratio Retention time / min Detection limit / ng Phosphatidylcholine (31:0) PC(31:0) 720.553 1.08 1.66 Phosphatidylcholine (32:0) PC(32:0) 734.569 1.19 0.33 Phosphatidylethanolamine (32:0) PE(32:0) 692.522 1.32 0.077 Sphingomyelin (d34:1) SM(d34:1) 703.571 0.92 0.37

[0050] According to the analysis results of the standard products, the method of the present invention has good analytical ability for lipid substances, high sensitivity, good linearity of the standard curve, and can be used to carry out quantitative analysis of single-cell lipid groups.

[0051] The following uses mouse oocytes as an example to illustrate the analysis method and effect of the present invention:

[0052] The present invention is used to perform online extraction chromatography mass spectrometry separation and analysis of single cell lipids. Figure 2 As shown, a single oocyte is injected into the pipeline of the six-way valve 5; the six-way valve 5 is switched to the injection mode, the capillary column needle 6 is connected, the supercritical fluid pump 1 and the mass spectrometer injection cone 8 are connected, and the high voltage is connected to the metal part in contact with the liquid, the working mode of the supercritical fluid pump 1 and the mass spectrometer is set, and the online extraction of cell metabolites and the use of chromatographic separation and ionization based on the nanoliter electrospray principle are started to collect mass spectrometry data. After the single cell is collected, the supercritical fluid pump 1 is used to backwash the quantitative loop 2, and then the next cell is collected.

[0053] After collecting cell lipidome mass spectrometry data, Compound Discoverer software was used to analyze and identify specific metabolites, and MetaboAnalyst platform was used for omics differentiation and metabolic pathway analysis.

[0054] Figure 4 This is the chromatogram of the single cell lipid as a whole in this example. It can be seen that the supercritical fluid extraction method of the present invention has a high efficiency in extracting cell lipids. From cell injection to the appearance of lipid signals, the extraction process is completed within 1 minute, and the extraction is relatively sufficient without residue. The entire analysis process can be completed within 5 minutes.

[0055] Figure 5 This is the chromatographic separation of 7 marker metabolites in the single-cell lipid mass spectrometry analysis in this example. It can be seen that the method of the present invention is applied to single-cell lipidomics analysis, with good separation effect and good peak shape.

[0056] Figure 6 This is the identification result of the single-cell lipid mass spectrometry analysis in this example. It can be seen that the method of the present invention can realize the analysis of 323 lipid substances in 12 categories, meeting the coverage requirements of traditional lipidomics analysis.

[0057] In order to verify that the microfluidic SFE-SFC method established by the method of the present invention is more suitable for the analysis of extremely small volume samples than the conventional SFE-SFC method, the results of the analysis of different numbers of oocytes by the method of the present invention and the conventional SFE-SFC method were compared.

[0058] The experimental method is: 100, 10, and 1 oocytes are taken, respectively, and the results of analysis are respectively obtained on the microfluidic SFE-SFC device of the present invention and the SFE-SFC device with a conventional flow rate (1 mL / min) and a conventional chromatographic column (250 mm×4.6 mm).

[0059] like Figure 7As shown, the microflow SFE-SFC established by the device of the present invention uses a lower flow rate and is suitable for the analysis of extremely small volume samples. It performs well with a single oocyte as a sample and has good chromatographic separation and detection effects on lipid species such as ChE, PC, PE, SM, and Cer. Conventional SFE-SFC with a flow rate of 1 mL / min has good results for lipid species extraction and chromatographic separation of 100 oocytes, but cannot give a signal when used for single cell analysis due to severe sample dilution. The experimental results show that the analytical sensitivity of the device of the present invention is greatly improved compared with the conventional SFE-SFC device, and is suitable for the analysis of extremely small volume samples such as single cells.

Claims

1. A sampling and ionization device for online extraction chromatography separation of single-cell lipids, comprising a supercritical fluid pump, a six-way valve, a quantitative loop and a capillary chromatographic column spray needle; The capillary chromatographic column spray needle comprises a body and a tip, wherein the tip is configured to be aligned with an inlet of a mass spectrometer when a sample is supplied to the mass spectrometer through the capillary chromatographic column spray needle, one port of the six-way valve is connected to the other end of the capillary chromatographic column spray needle, and the other port is connected to the supercritical fluid pump; The quantitative ring is configured to cooperate with the six-way valve, and a filter with a pore size of 2.0 to 5.0 μm is provided at the end of the quantitative ring; The tip of the capillary chromatographic column spray needle is filled with chromatographic filler.

2. The sampling and ionization device according to claim 1, characterized in that: The volume of the quantitative loop is 5.0 to 10.0 μL.

3. The sampling and ionization device according to claim 1 or 2, characterized in that: The capillary chromatographic column spray needle is obtained by capillary laser drawing.

4. The sampling and ionization device according to claim 3, characterized in that: The filling length of the chromatographic packing in the tip is 5 to 8 cm.

5. The sampling and ionization device according to claim 1, characterized in that: The inner diameter of the capillary chromatographic column spray needle is 100-200 μm, and the inner diameter of the tip is 2-3.5 μm.

6. A single-cell lipid online extraction and chromatography-mass spectrometry separation and analysis device based on microflow supercritical fluid, comprising a mass spectrometer and the sampling and ionization device according to any one of claims 1 to 5.

7. A method for single-cell lipid online extraction and chromatography-mass spectrometry separation and analysis based on microfluidic supercritical fluid, comprising the following steps: S1. Injecting a single cell to be tested into the quantitative loop in the sample injection and ionization device according to any one of claims 1 to 5; S2, connecting one end of the six-way valve to the supercritical fluid pump, and the other end to the capillary chromatographic column spray needle, turning on the supercritical fluid pump, and performing online extraction and chromatographic separation; S3, aligning the tip of the capillary chromatographic column spray needle with the injection port of a mass spectrometer; The port of the six-way valve connected to the capillary chromatographic column spray needle body is connected to a high-voltage power supply to achieve electrospray ionization and perform mass spectrometry analysis.

8. The online extraction and chromatography-mass spectrometry separation and analysis method according to claim 7, characterized in that: In step S2, the supercritical fluid used by the supercritical fluid pump is a mixed fluid of supercritical carbon dioxide and methanol, wherein the proportion of methanol is 10% to 25%.

9. The online extraction and chromatography-mass spectrometry separation and analysis method according to claim 8, characterized in that: The flow rate of the supercritical fluid is 50-200 μL / min, and the fluid pressure is 9-15 MPa.

10. A single cell lipid analysis method, comprising: The online extraction and chromatography-mass spectrometry separation and analysis device according to claim 6 is used to perform the online extraction and chromatography separation steps in the method according to any one of claims 7 to 9.

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