Microfluidic Chip-based Cells, Hydrogen Peroxide Detection

Various reactive oxygen species (ROS) are generated in the course of biological metabolism, such as superoxide (O2-·), hydrogen peroxide (H2O2), hydroxyl radical (HO·), nitric oxide (NO) and peroxynirte (ONOO-). They are important mediators of pathological processes in various vital processes. They can mediate much of the essential biochemistry reaction of vital processes, involved in cellular immune function, but also can create destructive modification to the chemical structure of tissues and cells and injury the normal morphology and membranous function of tissues and cells. Under normal conditions, the generation and elimination of reactive oxygen species are coexist in cells and maintain homeostasis. To maintain homeostasis, accumulation of excess hydrogen peroxide is prevented by multiple enzymatic and non-enzymatic systems such as catalase and glutathione peroxides. When they fail, either because of excess production, or because of a decrease in the capacity of the cells to eliminate them, in the presence of Fe2+ or Cu+, via the so-called Fenton reaction, H2O2 is converted to ?OH, which can result in lipid peroxidation, DNA damage and many biological effects.Many analytical techniques have been reported for determination of hydrogen peroxide, such as fluorescent spectroscopy, spectrophotometry, electrochemistry, and chemiluminescence. However, the short live and low concentration of H2O2 mean difficult to the determination, especially the determination in vivo and in situ has no breakthrough. Therefore, it is of critical importance for H2O2 detection in biological samples because of its potential to act as a diagnostic biomarker of diseases induced by oxidative stress and damage events.Micro total analysis systems (micro total analysis systems,μTAS) has become very much a'hot topic'in analytical chemistry since early 1990s due to the potential advantages of miniaturization, reduced reagent consumption, high speed and integration. Microfluidic-based research has made significant advances over the last few years, and because of their advantages including portability, low reagent and power consumption, short reaction time. Microfluidic chip-based systems for biological cell studies have attracted significant attention. Microfluidic technique has become a new platform in the field of analytical chemistry, especially in the life science, drug discovery and medicine.In this thesis, a new fluorogenic reagent synthesized by our laboratory was employed as a labeling reagent; microfluidic device coupled with Laser-induced fluorescence was employed as a technical platform for the determination of hydrogen peroxide (H2O2). Three chapters are included.In the first chapter, the progress of the application of microfluidic device in the area of cell analysis is described, including the cells culture, cells manipulate, cells lysis and cells analysis on the micrfluidic chip.In the second chapter, a new method using microchip electrophoresis with laser-induced fluorescence detection (LIF) is developed for the determination of hydrogen peroxide. Bis(p-methylbenzenesulfonyl)dichlorofluorescein (FS), a new fluorogenic reagent synthesized by our laboratory was employed as a labeling reagent. The proposed method was applied to determine hydrogen peroxide in PMA stimulated RAW264.7 macrophages. This application implies its practical value in monitoring concentration changes in hydrogen peroxide generated within heterogeneous biological samples, especially for cells.In the third chapter, based on the established analysis system in the second chapter, the loading, separation and electrophoresis determination of hydrogen peroxide in FS-derivatized RAW264.7 macrophages are presented.