Simultaneous Determination Of Oxidative And Antioxidant Small Molecule Species (H₂O₂, Cys And GSH) In A Single Cell Based On Multi-color Fluorescence Detection Microfluidic Device

The physiological processes of cells requires a collaborative involvement of oxidation and anti-oxidation molecules, in order to find the physiological mechanism hided in the detection of cell population, further research needed to obtain the accurate content of these small molecules in the single cell. But, due to the cell is small, many targets of small molecules in cells not only have a low concentration but have difference concentration, and some of them reactivity and can transform each other and influence each other, and these molecules can't amplification, such constraints result in the current commercial instrument is difficult to accurately quantitatively the content of these small molecules in a single cell, Lead to some analysis problem cannot be solved. For example, hydrogen peroxide?H₂O₂? is important oxidation molecules in the cell, and glutathione?GSH? and cysteine?Cys? is the important representative oxidation molecules in the cell, their physiological concentration level is closely related to the REDOX balance of the organism, and in some tumor developmentMicrofluidic chip is a emerging analysis technology which can precise manipulation and parallel processing of micro liquid, characterized by a 2D or 3D channel structure, the size scale of channel similar to the cell, fast analysis, less sample and reagent consumption, easy integration,all of these advantages make this method has become an important technology in the single cell analysis.Detector is the important component in microfluidic chip analysis system, which is used to determine the composition and content of the analytes. handled or separated by the microfluidic chip. Laser induced fluorescence detection with high sensitivity, suitable for superfine sample size determination, is widely used in microfluidic chip analysis. Combination of high sensitive laser induced fluorescence detector and the microfluidic chip electrophoresis which have characteristics of an effective chemical separation, can be used for single component analysis, the resulting method and the instrument has low consumption, fast, and multicomponent quantitative and efficient separation, can be easily automated integration advantages. however, limited to "single excitation and single emission" of the laser induced fluorescence detection technology. hence, this method in the application of multicomponent detection simultaneously in single cell is limited.Based on the above problems, this paper builded a multicolor laser induced fluorescence detector, and established a new method based on microfluidic chip electrophoresis- multicolor laser induced fluorescence to quantitative detection of H₂O₂, GSH, Cys in the single original generation mice liver cell simultaneously. And researched the content changes of H₂O₂, GSH, Cys in an acute alcoholic liver damage model. The thesis includes the following three parts:In the chapter one, we first introduced the biological significance of the molecules related to intracellular redox balance, and the usual methods to detect these molecules. Afterwards, we introduced the advances in the research of the microfluidic chip electrophoresis- laser induced fluorescence detection technology used in analysis these molecules in the single cell.In the chapter two, aims to the problem of microfluidic chip electrophoresis- laser induced fluorescence detection technology such as the excitation and emission is monotonous, we set up a double laser induced fluorescence detector,which can achieve the simultaneous and quantitative detection of fluorescent markers with different excitation and emission, and with lower detection limit, suitable for the simultaneous detection of variety molecules in the single cell, Help extend the microfluidic chip electrophoresis-laser induced fluorescence detection in single cell component analysis in the areas of application spaceIn the chapter three, Use of microfluidic multicolor fluorescence detection device, a combination of hydrostatic and electrokinetic gated injection and laboratory synthesis of fluorescent probes the FS, Cy-3-NO2, we developed a new method to quantitative detection H₂O₂, Cys and GSH in a single primary liver cell,this method can realize single cell injection, lysis, electrophoresis separation and detection in a chip. this method was used to study the different concentrations of alcohol stimulation and protection of silymarin conditions, 100 yuan in mice liver intracellular H₂O₂, Cys heterogeneity and the variation in content of GSH and cells. The method has good performance, will be for other single-celled H₂O₂, Cys and GSH in the assessment, alcohol liver injury and oxidative stress mechanism and related research provides a new way.