Detection, Based On The Analysis Of Microfluidic Chip Cell-africa Steady-state Material Superoxide Radicals

Cell is the basic unit of organism structure and functions. Numerous free radicals are generated in the course of biology metabolism. Superoxide anion radical (O₂^-·), hydrogen peroxide (H₂O₂), hydroxyl radicals (HO·), and peroxynirte (ONOO-) are some typical ones. They have important relation with aging and many diseases. It is reported that appropriate level of reactive oxygen species (ROS) could help to build biological immune system, while excess ROS have damages on organisms. Recent studies demonstrated that ROS can cause many diseases such as heart disease, cancer, malignancy and so on. O₂^-· serves as a precursor to other reactive oxygen species (ROS). For example, O₂^-· converts to H₂O₂ spontaneously or under action of superoxide dismutase; O₂^-· reacts with NO to form the powerful oxidant peroxynitrite. Although several methods to detect O₂^-· such as electron spin resonance (ESR), electrochemistry, fluorescence spectrometry and HPLC have been developed, there were some drawbacks for the above mentioned methods, including the large sample volume, the long analyzing time, inconvenience to operate or costliness. Therefore, rapid, sensitive detection and quantification of intracellular O₂^-· is critically important in understanding its physiological functions and pathogenesis of various diseases associated with ROS.Since 1990s, Microfluidic chip or lab on a chip provides a new technology platform for the research in chemistry, biology, microengineering and other related micro system fields, due to its shorter analyzing time and reducing sample requirements. A new method for determination of cellular non-steady substances such as superoxide free radical on the molecule level was developed, which was focused on the application of microfluidic device and free radical fluorescent probe,coupled with homely made laser-induce fluorescence (LIF). The level of cellular O₂^-· determined by this method is more clear to the real concentration. The thesis was to develop the related techniques of potentially biomedical significance. Three chapters are included:In chapter one, cellular constituent analysizing on microchip was summarized.In chapter two, a method for determination of superoxide free radical (O₂^-·) based on microchip electrophoresis with LIF was developed. Fluorescent reagent 2-chloro-1, 3-dibenzothiazolinecyclohexene (DBZTC), which was synthesized in our laboratory, was employed as the labeling reagent. Optimal determination of O₂^-· was achieved on a glass microchip, using 50mM HEPES buffer (pH7.4). Under the optimized conditions, linearity of response was obtained in the range of 4.0×10^-7–1.0×10^-5M, the detection limit (S/N = 3) was 0.15μM, the RSDs of migration time and peak area were 2.6% and 3.8%, respectively. The interference experiments was investigated and the result indicates that 1000-fold molar excess of H₂O₂ dose not interfere the determination of O₂^-· in complex system. Finally, the method has been successfully applied to determineO₂^-·in hepatocellular carcinoma (HepG2) cells as well as PMA-stimulated RAW264.7macrophages. The average recoveries were 97.3% and 98.6%, respectively.In chapter three, based on the second system, determination of O₂^-· in single HepG2 cell was studied. After incubation cells and determination microchip structure, the process in single cell analysis on microfluidic chip, including cell loading, cell separation, cellular O₂^-· determination, were explored. The further experiment was needed to optimaized.