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Single-cell mass spectrometry methods

A mass spectrometry and single-cell technology, which is applied to the analysis of materials, individual particle analysis, particle and sedimentation analysis, etc., can solve the problems of reducing the convenience of operation, waiting for research, and not conducive to popularization and application, so as to reduce costs and facilitate implementation Effect

Active Publication Date: 2021-01-08
TSINGHUA UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

As a result, the cost of sampling is greatly increased, the convenience of operation is reduced, and it is not conducive to popularization and application.
[0004] Therefore, methods for single-cell mass spectrometry analysis remain to be studied

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0054] Lipid substance analysis in single MCF7 cell of embodiment 1

[0055] Cell fixation: the cultured MCF7 cells (7-8 μm) were transferred to PBS, centrifuged at 1200 rpm, and the cells were resuspended in phosphate buffered saline (PBS) containing 2.5% glutaraldehyde. After 20 minutes, Cells were collected by centrifugation and resuspended in water to obtain a cell suspension.

[0056] Preparation of capillary glass tube: use a P1000 needle puller to draw a capillary glass tube with a tip diameter of 5 μm.

[0057] Mass Spectrometry:

[0058] (1) Add 0.5 μL of cell suspension into the capillary glass tube from the end of the capillary glass tube, select the capillary glass tube containing 1 cell under the microscope, and use -1.2kV voltage to migrate a single MCF7 cell to the front of the capillary glass tube.

[0059] (2) Wait for the liquid in the capillary glass tube to evaporate completely.

[0060] (3) A voltage of -+1.8 kV is applied while dropping auxiliary liqui...

Embodiment 2

[0062] Lipid analysis of MCF7 cells after embodiment 2 single photochemical derivation

[0063] Cell fixation: transfer the cultured cells to PBS. Using 1200 rpm centrifugation, the cells were resuspended in phosphate buffered saline (PBS) containing 2.5% glutaraldehyde, and after 20 minutes, centrifuged, the cells were collected and resuspended in water.

[0064] Photochemical derivation of cells: In the cell suspension, a solution of 2-acetylpyridine was added to a final concentration of 100 mM. In a quartz reaction box (such as figure 2 Shown) in, carry out ultraviolet photochemical reaction, used ultraviolet lamp is 254nm, and reaction time is 8 minutes.

[0065] Preparation of capillary glass tube: use a P1000 needle puller to draw a capillary glass tube with a tip diameter of 5 μm.

[0066] Mass Spectrometry:

[0067] (1) Add 0.5 μL of cell suspension (1-5 cells) into the capillary glass tube from the end of the capillary glass tube, select the capillary glass tube ...

Embodiment 3

[0072] Example 3 Structural identification of carbon-carbon double bond isomers of glycerophosphorylcholine (PC) in MCF7 cells after single photochemical derivatization

[0073] Cell fixation: transfer the cultured cells to PBS. Using 1200 rpm centrifugation, the cells were resuspended in phosphate buffered saline (PBS) containing 2.5% glutaraldehyde, and after 20 minutes, centrifuged, the cells were collected and resuspended in water.

[0074] Photochemical derivation of cells: In the cell suspension, a solution of 2-acetylpyridine was added to a final concentration of 100 mM. In a quartz reaction box (such as figure 2 Shown) in, carry out ultraviolet photochemical reaction, used ultraviolet lamp is 254nm, and reaction time is 8 minutes.

[0075] Preparation of capillary glass tube: use a P1000 needle puller to draw a capillary glass tube with a tip diameter of 5 μm.

[0076] Mass Spectrometry:

[0077] (1) Add 0.5 μL of cell suspension (1-5 cells) into the capillary gla...

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Abstract

The present invention proposes a single-cell mass spectrometry analysis method, which includes: fixing and diluting the cells to be tested to obtain a pretreated cell suspension; injecting the pretreated cell suspension into the inner cavity from the tail end of the capillary, and An electrode is inserted into the cavity; voltage is applied to the electrode to make the cells migrate to the tip of the capillary; the liquid in the capillary is completely evaporated; an auxiliary liquid is added to the tip of the capillary, so that the auxiliary liquid enters the capillary and contacts the cells to extract the target substance. A voltage is applied to the electrode for electrospray, so that the auxiliary liquid containing the target is ionized, ejected from the tip, and enters the inlet of the mass spectrometer for mass spectrometry analysis. The method of the present invention can realize the sampling and mass spectrometry analysis of a single cell without relying on a high-precision control platform, combined with the photochemical derivation process, can realize the identification of the double bond position of the unsaturated lipid in a single cell, and has a good application prospect .

Description

technical field [0001] The present invention relates to the field of biology. In particular, the present invention relates to single cell mass spectrometry methods. Background technique [0002] There will be large differences between individuals in the cell population of the same genotype, that is, cell heterogeneity. There are significant differences between cells in gene transcription, protein expression, metabolism and other aspects. Therefore, single-cell analysis is of great significance for the study of intercellular heterogeneity. A variety of technologies have been widely used in single-cell analysis, such as single-cell sequencing, fluorescence detection technology, microfluidic technology, flow cytometry, mass spectrometer, etc. Among them, mass spectrometers are widely used in single-cell analysis due to their high sensitivity, accurate structure identification, and quantitative analysis. However, the small size and volume of cells themselves pose great chall...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): G01N1/28H01J49/04G01N27/62G01N15/10
CPCG01N1/28H01J49/0445G01N27/62G01N15/10
Inventor 欧阳证李自帅马潇潇
Owner TSINGHUA UNIV
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