A method for detecting metabolites in single cells by mass spectrometry

Abstract

The application discloses a kind of single-cell metabolite in vivo in situ detection method, which comprises the following steps: establishing single-cell cytoplasm sampling volume / single-cell volume relative quantification method, obtaining the relative maximum cytoplasm extraction volume when maintaining single-cell survival;Build composite ion source, obtain the minimum sampling volume of 150 metabolites detected from single cytoplasm, obtain the sampling volume of single-cell cytoplasm in the in vivo in situ detection of metabolome;Through picoliter pump probe microscope fixed-point positioning puncture cell membrane, extract the cell cytoplasm of the above sampling volume;Metabolites in the extracted single-cell cytoplasm are ionized using composite ion source;Metabolites after ionization are detected by mass spectrometry.The application can realize multiple single-cell cytoplasm extraction of in vivo embryo during development, mass spectrometry metabolite detection, while ensuring cell survival, multiple sampling, while realizing accurate detection of trace metabolites in single cell.

Description

Technical Field

[0001] This invention belongs to the field of single-cell detection technology, specifically relating to a live-cell mass spectrometry detection method for single-cell metabolites. Background Technology

[0002] The cell is the most basic structural and physiological unit of an organism. Due to the significant heterogeneity between cells, measurements obtained from biological tissues and in vitro cell populations are insufficient to reflect the laws governing life activities at the single-cell level. To explore the fundamental laws of microscopic life activities in greater depth and obtain more precise and comprehensive information on cell biology, accurate measurement studies at the single-cell level are essential.

[0003] In situ analysis of single cells plays a crucial role in obtaining spatial information about cells, monitoring intercellular interactions, and studying the impact of the extracellular microenvironment on cell metabolism. However, current methods for analyzing single cells all require isolating individual cells and destroying their structure. Achieving in situ analysis of single cells in living organisms remains a significant international challenge.

[0004] In recent years, single-cell mass spectrometry (SCMS), as a label-free detection technique with high sensitivity and specificity, has been increasingly widely used in single-cell metabolomics and proteomics. SCMS can achieve highly sensitive simultaneous detection of multiple biomolecules within cells without amplification or labeling. Through multi-stage fragmentation, mass spectrometry can also utilize characteristic fragment information to resolve the structures of unknown compounds within single cells. However, single-cell mass spectrometry detection requires the complete destruction of cells, rendering them lifeless. To achieve in-situ, live-cell cytoplasmic mass spectrometry metabolomics quantitative detection, the following balance needs to be accurately maintained.

[0005] 1) Increasing the amount of cytoplasm extracted can improve the accuracy of mass spectrometry detection, but it will cause the cells to lose their activity.

[0006] 2) Reducing the amount of cytoplasm extracted can increase the probability of cell survival, but it will reduce the accuracy of mass spectrometry detection.

[0007] Therefore, determining the amount of cytoplasm extracted in a way that ensures both cell survival and the sensitivity of mass spectrometry detection is a key challenge that currently prevents the in vivo detection of single-cell metabolites using mass spectrometry. Summary of the Invention

[0008] To address the aforementioned shortcomings in the prior art, the present invention aims to provide a method for in vivo mass spectrometry detection of single-cell metabolites.

[0009] To achieve the above-mentioned objectives, the technical solution adopted by this invention is as follows:

[0010] A method for in vivo mass spectrometry detection of single-cell metabolites is provided, the method comprising the following steps:

[0011] Step 1: Establish a relative quantitative method for single-cell cytoplasm sampling volume / single-cell volume to obtain the relative maximum cytoplasm extraction volume when maintaining single-cell survival;

[0012] Step 2: Construct a composite ion source to obtain the minimum sampling volume for detecting 150 metabolites from a single cell cytoplasm;

[0013] Step 3: Based on the relative maximum cytoplasm extraction volume when a single cell is alive and the minimum sampling volume for detecting 150 metabolites in a single cell cytoplasm, obtain the sampling volume of single cell cytoplasm in in vivo metabolomics mass spectrometry detection.

[0014] Step 4: Using single-cell micromanipulation techniques, the cell membrane is punctured at specific points using a picoliter probe microscope to extract the cytoplasm of the sampled volume.

[0015] Step 5: Use a picoliter UV photoionization-induced electrospray composite ion source to ionize the metabolites extracted from the cytoplasm of single cells;

[0016] Step 6: Mass spectrometry is used to detect metabolites in the cytoplasm of ionized single cells.

[0017] The beneficial effects of this invention are as follows:

[0018] A live in situ single-cell metabolite sampling, detection, and analysis technique has been established, which enables multiple single-cell cytoplasmic extractions from living embryos during development and mass spectrometry metabolite detection. While ensuring cell viability and allowing for multiple samplings, it enables precise quantitative analysis of trace metabolites within single cells, ultimately obtaining spatial, temporal, and metabolite information for single cells. Attached Figure Description

[0019] Figure 1 This diagram illustrates the sampling, injection, and detection processes of the method of this invention. Detailed Implementation

[0020] The specific embodiments of the present invention are described below to enable those skilled in the art to understand the present invention. However, it should be understood that the present invention is not limited to the scope of the specific embodiments. For those skilled in the art, various changes are obvious as long as they are within the spirit and scope of the present invention as defined and determined by the appended claims. All inventions utilizing the concept of the present invention are protected.

[0021] Example

[0022] This embodiment uses mouse embryos as the research subject. Single-cell micromanipulation techniques are employed, using a picoliter-pump probe microscope to precisely puncture the cell membrane and extract the cytoplasm, achieving in-situ extraction of cytoplasm from living cells while ensuring cell viability. Simultaneously, a "picoliter-UV photoionization-induced electrospray" composite ion source was developed to improve ionization efficiency and reduce sample volume requirements. After the embryonic cells regained activity and continued to proliferate, single-cell localization sampling and mass spectrometry metabolite detection were performed again, achieving more than three cytoplasmic samplings and metabolite detections for a single cell. Specifically, the following content is included:

[0023] (1) Collection of live cytoplasm

[0024] The core of live cell cytoplasm collection lies in extracting a sufficient amount of cytoplasm to meet the conditions for mass spectrometry detection while ensuring cell viability. First, a quantitative method for the relative quantification of single-cell cytoplasm sampling volume / single-cell volume is proposed. In this embodiment, cell volume is estimated using a traditional method: the length and width of the embryonic cell are measured using an eyepiece micrometer, and half of these measurements are taken as the major and minor radii, which are then substituted into the formula to calculate the volume. Second, quantitative fluorescence labeling is used to label the cytoplasm as a whole. The fluorescence intensity of a single cell before sampling represents the cell volume, and the remaining fluorescence intensity after cytoplasm extraction represents the remaining volume. This tracking method determines the size of the cytoplasm extraction volume relative to the cell volume.

[0025] Besides biological characteristics, cellular heterogeneity leads to differences in physical properties such as size and shape among individual cells. Determining the sampling volume for each cell of varying sizes and shapes requires calculation based on the cell's inherent volume. Secondly, a method for quantitatively extracting cytoplasm from single cells using a picoliter pump is needed, tailored to the individual cell volume. Thirdly, the relative maximum cytoplasm extraction volume required to maintain cell viability needs to be determined. Finally, a technical method for quantitatively extracting single-cell cytoplasm using a microscope and a picoliter pump in combination should be established to ensure the continued survival and reproduction of embryos.

[0026] A picoliter pump uses a pressure sensor to precisely measure pressure changes in the injection needle during cell fluid extraction, thereby accurately controlling the sampling volume and ensuring that the sample is not contaminated by surrounding cells. This method enables precise control of cell fluid volume during sampling, avoiding quantitative errors caused by sampling volume. The study plans to introduce an internal standard to avoid errors in the extraction efficiency of intracellular compounds during direct sampling, ensuring that some low-concentration compounds are not difficult to detect in mass spectrometry due to matrix effects.

[0027] (2) Develop innovative ion sources, increase detection sensitivity, and establish analytical conditions.

[0028] Based on laboratory foundations, a novel combined ion source was developed to increase the sensitivity of testing trace samples and reduce the required sample volume. The photoionization mass spectrometry (APPI) ion source equipment was optimized, and a picoliter ultraviolet photoionization mass spectrometry ion source equipment was further established; simultaneously, non-contact electrospray ionization technology was developed. Subsequently, the two were combined to develop a "picoliter ultraviolet photoionization-induced electrospray" combined ion source. Through optimization of various parameters of the device, efficient ionization and high-sensitivity measurement of trace samples were achieved. By introducing an internal standard, an accurate qualitative and relative quantitative mass spectrometry analysis method for single-cell metabolites was established. After optimizing the conditions, the minimum sampling volume for detecting 150 metabolites from a single cell cytoplasm was explored.

[0029] In this embodiment, the sampling volume of single-cell cytoplasm in in vivo metabolomics mass spectrometry detection is obtained based on the relative maximum cytoplasm extraction volume when a single cell is alive and the minimum sampling volume in which 150 metabolites are detected in a single cell cytoplasm. Using single-cell micromanipulation technology, the cell membrane is punctured at specific points using a picoliter-pump probe microscope to extract the cytoplasm of the above-mentioned sampling volume. The extracted single-cell cytoplasm is ionized using a picoliter-UV photoionization-induced electrospray composite ion source. The ionized single-cell cytoplasm is then used to detect embryonic single-cell metabolites.

[0030] This application focuses on the field of life and health, aiming to establish a live in situ single-cell metabolite sampling, detection, and analysis technology. It involves multiple extractions of single-cell cytoplasm from developing live mouse embryos for mass spectrometry metabolite detection. While ensuring cell viability and allowing for multiple samplings, it achieves precise quantitative analysis of trace metabolites within single cells. Ultimately, it obtains spatial, temporal, and metabolite information for single cells.

[0031] It will be apparent to those skilled in the art that the present invention is not limited to the details of the exemplary embodiments described above, and that the invention can be implemented in other specific forms without departing from the spirit or essential characteristics of the invention. Therefore, the embodiments should be considered in all respects as exemplary and non-limiting, and the scope of the invention is defined by the appended claims rather than the foregoing description. Thus, it is intended that all variations falling within the meaning and scope of equivalents of the claims be included within the present invention.

[0032] Furthermore, it should be understood that although this specification describes embodiments, not every embodiment contains only one independent technical solution. This narrative style is merely for clarity. Those skilled in the art should consider the specification as a whole, and the technical solutions in each embodiment can also be appropriately combined to form other embodiments that can be understood by those skilled in the art.

Claims

1. A method for in vivo mass spectrometry detection of single-cell metabolites, characterized in that, The method comprises the following steps: Step 1, establishing a relative quantification method of single cell cytoplasm sampling volume / single cell volume, and obtaining a relative maximum cytoplasm extraction volume when a single cell is alive; Step 2, building a composite ion source, and obtaining a minimum sampling volume for detecting 150 metabolites from a single cytoplasm; Step 3, obtaining a sampling volume of single cell cytoplasm in metabolic group in vivo mass spectrometry detection according to the relative maximum cytoplasm extraction volume when a single cell is alive and the minimum sampling volume for detecting 150 metabolites from a single cytoplasm; Step 4, using single cell micro-operation technology, and performing fixed-point positioning and puncturing cell membrane through a picoliter pump probe microscope to extract cytoplasm of the single cell in the sampling volume; Step 5, using a picoliter ultraviolet photoionization-inducing electrospray composite ion source to ionize the extracted single cell cytoplasm; Step 6, performing mass spectrometry detection on metabolites in the ionized single cell cytoplasm.

2. The method of claim 1, wherein the single-cell metabolite live mass spectrometry method is characterized by, In step 1, an ocular micrometer is used to measure the length and width of an embryo cell, and the half of the length and the half of the width are taken as the long radius and the short radius, and then the volume is calculated by substituting the long radius and the short radius into a formula; further, a whole cell fluorescence labeling is performed on the cytoplasm by using a fluorescence quantification method, and the fluorescence intensity of a single cell before sampling represents the cell volume, and the remaining fluorescence intensity after cytoplasm extraction represents the remaining volume, so that the size of the cytoplasm extraction volume relative to the cell volume is determined.

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