CN-116286590-B - Artificial cell for single-cell mass spectrometry and preparation method thereof

CN116286590BCN 116286590 BCN116286590 BCN 116286590BCN-116286590-B

Abstract

The invention discloses an artificial cell for single-cell mass spectrometry and a preparation method thereof, wherein the artificial cell comprises an inner water phase, an intermediate oil phase and an outer water phase, the inner water phase comprises polyethylene glycol and a polyvinyl alcohol aqueous solution, the intermediate oil phase comprises a chloroform and hexane mixture of L-alpha-phosphatidylcholine, and the outer water phase comprises PVA and an F-68 aqueous solution. Compared with a natural cell sample, the novel artificial single cell based on microfluidic self-assembly has better uniformity, stability and controllability, effectively avoids the obvious measurement difference between single cell individual samples, and effectively solves the problem that biological samples are difficult to store stably. The preparation method of the artificial cells solves the problem that standard reference substances are absent in the single-cell mass spectrometry field to a great extent, so that the research result of the single-cell mass spectrometry methodology is more accurate and reliable and the mutual acceptance ratio is improved.

Inventors

TAN SIYUAN
GONG XIAOYUN
YIN XINCHI
XUE ZHICHAO
DAI XINHUA
FANG XIANG

Assignees

中国计量科学研究院

Dates

Publication Date: 20260512
Application Date: 20230320

Claims (4)

1. An artificial cell for single cell mass spectrometry, characterized in that the artificial cell comprises an inner aqueous phase, an intermediate oil phase and an outer aqueous phase, wherein the inner aqueous phase comprises polyethylene glycol and an aqueous solution of polyvinyl alcohol, the intermediate oil phase comprises a chloroform and hexane mixture of L-alpha-phosphatidylcholine, and the outer aqueous phase comprises PVA and an aqueous solution of F-68.
2. A method of preparing an artificial cell according to claim 1, comprising the steps of: s1, constructing a microfluidic platform for preparing artificial cells: The microfluidic platform is a three-phase channel and comprises an internal water phase channel, an oil phase channel and an external water phase channel, wherein the three-phase liquid is respectively led in by five microfluidic channels, the water phase channel is a middle channel, the oil phase channel is a middle two-side channel, and the external water phase channel is an outer two-side channel; s2, preparing double-emulsion liposome artificial cells based on microfluidic self-assembly: Adding substances into the three-phase channel, extruding emulsion droplets through aqueous solution to complete a vesicle primary structure of oil-water two phases; Polyethylene glycol and polyvinyl alcohol aqueous solution are added in the inner water phase channel, a chloroform and hexane mixture of L-alpha-phosphatidylcholine is added in the oil phase channel, and PVA and F-68 aqueous solution are added in the outer water phase channel.
3. The method according to claim 2, wherein in step S2, an organic dye or a fluorescent dye is added to the internal aqueous phase channel in order to clearly observe the artificial cells of interest.
4. The method of claim 2, wherein in step S2, different amounts of the target compound are added to the internal aqueous phase channel in order to accurately simulate the histology of the internal single cells.

Description

Artificial cell for single-cell mass spectrometry and preparation method thereof Technical Field The invention belongs to the technical field of cell biology, and particularly relates to an artificial cell for single-cell mass spectrometry and a preparation method thereof. Background In recent years, single-cell mass spectrometry (SCMS) has been increasingly used in Single-cell metabolomics and proteomics as a non-labeled detection technique with high sensitivity and high specificity. Although there are other commonly used single cell metering methods (e.g., electrochemical methods, fluorescence methods, etc.), the need for specific physicochemical properties (redox) of the molecule to be tested or modification of its structure (fluorescent labeling) is often required, which greatly limits the scope of application of these methods. Compared with the methods, the single-cell mass spectrometry technology can realize high-sensitivity simultaneous detection of various biomolecules in cells without amplification or labeling. Secondly, the mass spectrum can also realize the structural analysis of unknown compounds in single cells by utilizing the characteristic fragment information of molecules in a multistage fragmentation mode. In addition, by means of isotope labeling, single-cell mass spectrometry can track the metabolic process of a specific compound on the premise of hardly influencing the metabolic behavior of cells, and meanwhile, the isotope dilution method is also a quantitative analysis means with the highest stoichiometric level at present, and the SI unit can be directly traced. The excellent characteristics enable the single-cell mass spectrometry technology to have good application prospect in the precise metering of single-cell chemical components. Although single cell mass spectrometry techniques have now achieved qualitative and quantitative analysis of partial compounds within single cells, significant inaccuracy and unreliability remain in single cell mass spectrometry studies due to the significant individual variability and physiological state instability of natural single cells. First, the natural cell types are abnormally diverse, and they are classified into plant cells, animal cells, nerve cells, white blood cells, red blood cells, platelets, phagocytes, epithelial cells, cardiac muscle cells, stem cells, cancer cells, and the like. Also, cells of the same species may exhibit significant size, morphology, and internal histology differences in organisms due to being in different stages of growth. This can lead to significant sampling and measurement differences when single cell mass spectrometry methodology studies or analytical measurements are performed, resulting in end metering results that are generally subject to poor reproducibility, poor stability, inability to trace the measurement results, etc. In order to better study the physical and chemical properties and biological functions of cells, such as microdissection, signal network, histology spectrum library and gene regulation, the concept of "artificial cells" is widely proposed by scientists, and various ideal artificial cell models, including liposome (Liposomes), polymer vesicle (Polymersomes) and multilamellar lipid vesicle (Vesosomes), have been developed in the current field. According to the structure and physiological characteristics of the cells, the artificial cells realize the simulation of cell compartment structures, internal compound types, cell membrane surface biochemical functions and the like through the structure regulation and control of vesicle materials, the encapsulation of target molecules, the modification of functional molecules and the like, and are finally successfully applied to the research of physicochemical properties and biological functions of various cells. Among these artificial cell models, liposomes are the most widely focused and reported. Liposomes are usually composed of naturally synthesized phospholipid bilayer or vesicles prepared from artificial self-assembled materials, and have attracted wide interest in the fields of drug targeted delivery, membrane protein research, bioreactors, biosensors, and the like. The liposome can realize the simulation of the size of a single living cell through the regulation and control of an amphiphilic molecular chain segment, and the characteristics of wrapping biological molecules and carrying specific molecular physiological functions make the liposome a potential substitute for single cell mass spectrum research. Through development in recent years, research on the preparation method of the artificial cells has achieved a certain result, and preliminary simulation and construction of the basic structure and biochemical characteristics of the natural cells are realized. However, liposome artificial cell development for single cell mass spectrometry is still in the vacant stage, and the main reasons are as follows: First, it is difficult to achieve u