Microfluidic System And Quantitative Detection Single Liver Cancer Research Of Hydrogen Peroxide And Nitric Oxide In The Cell

The cell is the basic unit of the organism structure and the life activities. To study andunderstand the laws of life activities, it is necessary to make cell-based research, study thestructure and function of the cell, explore the life activities of cells. There are a wide range ofspatial and temporal distribution of complex active small molecules, their level, types, contentsare closely related to the activities of the life, just as cell proliferation, cell differentiation, cellapoptosis. There is a big difference in the chemical composition, biological activity and theresponse to internal and external stimulation in different cell types, even in the same kind of cells,therefore, the cellular component analysis especially component analysis in single cell is veryimportant.Life is a complete system，the organization and the regulation of the biological molecules areinterrelated, interdependent in complex organisms，and it was also affected by kinds ofphysiological and environmental factors. The research shows that: the cell proliferation, celldifferentiation, cell apoptosis and various other activities all involves different endogenous activesmall molecules （such as reactive oxygen species, reactive nitrogen, reactive chlorine, mercaptocompounds, amino acids, glucose, H+, metal ions, anions, etc.） When the normal cells are instress conditions which is caused by the external stimuli （such as drugs, toxins, electromagnetic radiation, etc.）, the content of these active small molecules in the cell would make a significantchange with the cell response to environmental stress. The impact of the significant changewhich is caused by the levels of these active small molecule on cellular processes relatedproteins, enzymes, cytokines play a different and even opposite influence: on the one hand it caninvolve in the regulation of cell to invade, inhibit and kill the Foreign violations, make the cell torepair itself; on the other hand, it can induce apoptosis until the lesions occur. The concent levelsof the intracellular active small molecules is closely related to the cell physiological state, as wellas some major medical diseases, thus, rapid detection and quantitative analysis of active smallmolecule in the single cell level，how to use and develop varieties of instruments, accuratelyobtain the cells active small molecule quantitative information from the single-cell level, themolecular level，all these have become the precondition of the molecular mechanisms，pathogenesis and the Disease early warning，diagnosis and treatment，it has become a new fieldof inter disciplinary research in recent years.Reactive oxygen species （ROS）, such as hydrogen peroxide （H₂O₂）, nitric oxide （NO） isrepresentative radicals in biological processes, the level of reactive oxygen species in vivo isassociated with the cell Physiological and cell pathological. The interaction between thehydrogen peroxide and nitric oxide may generated a higher cytotoxic singlet oxygen （1O2）.Hydrogen peroxide and nitric oxide as a messenger molecule plays an important role in the nervous system. Therefor，qualitative, quantitative detection of H₂O₂and NO in single-cell hasimportant theoretical significance and practical value in exploring the mechanism of theirformation, transformation mechanisms, physiological role and pathogenesis.In this paper, we use a new green fluorescent probe which was design and synthesis in ourlaboratory and a commercial probe combined the new technology platform microchip to detectthe reactive oxygen species H₂O₂and NO in a single hepatoma cells at the same time. The papercontains two parts, as follows:The first chapter is the introduction, briefly expound the biological significance and thedetection methods of the intracellular reactive component H₂O₂and NO. We provide anoverview of research in the field of single-cell analysis on microfluidic system, including theapplications of single cell injection, cell lysis, electrophoretic separation and detection of isolates.The second chapter, we chose the green fluorescent probes bis （p-toluene sulfonyl）-dichloro-fluorescein which was synthesis inside the laboratory and a commercial fluorescentprobe DAF-FM DA （3-Amino4-amino methyl-2’,7’-difluorescein diacetate） as the fluorescentlabeling reagents, use microfluidic chip electrophoresis-laser induced fluorescence analysis astechnology platform to achieve the simultaneous quantitative detection of single HepG2cellsH₂O₂and NO. After pre-treatment, the probe incubation, re suspend, adjusting density, the cellsintroduce chip channel as sample, we use the electric control combined with the laser-induced fluorescence detector in simple cross chip achieve automation quantitative H₂O₂and NO insingle HepG2cell, the process mainly include: single cell injection, single cell loading, singlecell lysis, the electrophoretic separation and detection of the analyte. The method is simple, fast,easy to implement, automation,high degree of and integration.