| Reactive oxygen species (ROS) produced from the metabolism of oxygen by livingorganisms are classically known as indicators for oxidative stress and potential contributors toaging and diseases ranging from cancers to neurodegeneration. Therefore, to simultaneouslydetermine multiple ROS is deemed to be important for biochemistry, molecular cell biology,drug discovery and early-stage disease diagnosis. However, it is not easy to accomplish thesimultaneous detection of these two ROS due to their low homeostasis concentrations, highreactivity, and fast interconversion.In this paper, we focus on the development of efficient analytical method for thesimultaneous detection of ROS in living cells. A versatile programmable microfluidic system fordetecting ROS at the cell level was developed by using home-synthesized fluorescent probescombined with electrokinetic manipulation. Experiments were carried out by using samples fromcell extract to single cell, which is fabricated to achieve high-throughput analysis. In addition,the results for O2ﾃつｷ-and H2O2was calibrated by dual-calibration coefficient protocol. Finally, theredox state of hepatoma cells was characterized by a reversible florescent probe. Five chaptersare included as follows:In the chapter one, the biological function and mutual transformation of reactive oxygenspecies were overviewed, then we carried out a detailed review on the application of microfluidicchips in the analysis of single cell components, the difficulties and shortcomings of single-cellROS analysis and the necessity to carry out the topics of research ideas.In the chapter two, a method for the first time to simultaneously determine O2ﾃつｷ-and H2O2inmacrophage RAW264.7 cell extracts by microchip electrophoresis with laser-inducedfluorescence detection (MCE-LIF) was developed. Using this method,O2ﾃつｷ-and H2O2in phorbolmyristate acetate (PMA)-stimulated macrophage RAW 264.7 cell extracts were determined. Themethod has paved a way for simultaneously determining two or more ROS in a biological systemwith high resolution.In the chapter three, we present a more accurate method to perform quantification of O2ﾃつｷ-andH2O2simultaneously in human HepG2 cells based on microchip electrophoresis. The rationale isthat the proposed dual-calibration coefficient protocol takes into account both the complexmatrix effect of the biological system and real time decaying ofO2ﾃつｷ-and H2O2, providing moreaccurate and quantitative analysis. In the chapter four, we present a method for the high-throughput and simultaneousmeasurement of O2ﾃつｷ-and H2O2in individual human U937 cells with laser-induced fluorescencedetection for the first time. The proposed method is simple and potent for high-throughputsingle-cell analysis of cell population of considerable size, generating statistical significanceresults.In the chapter five, we developed a method for the simultaneous detection of the binary stateof reduction-oxidation, relying on laminar flow and an in-house synthesized reversiblefluorescence probe. Normal hepatocytes and hepatoma cells were used as models to exploit thephysiological differences between normal and malignant state. The preferential reduction incancer cells compared with normal cells was observed by confocal-microscope. The results showthe existence of significant heterogeneity of redox status in cancer cells compared with normalcells, giving efficient information for tumor growth and therapy.
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