Simultaneous Quantitative Detection And Microfluidic Analysis Of Multiple Active Small Molecules In Single Cell

Quantitative analysis of single cells is a cutting-edge technology for life sciences.However, due to the small size, low component content and complex components ofcells, single cell manipulation and detection sensitivity, selectivity is restricted, there’sfew quantitative detection technology of single cells. Because of the contradictionbetween its great significance and unsatisfactory research status, quantitative analysisof single cells has become a challenging research. Internationally renowned academicjournal "Science" shows that quantitative analysis of single cells is principal hotspotsin2013.Currently， the main commercial single-cell analysis instruments include flowcytometry and fluorescence microscopy, the identification and classification of intactcells can be achieved with fluorescent probe, but the separation of components cannot be achieved. With microfluidic electrophoresis-based method, processing stagessuch as cell transport, single cell trapping/loading, cell lysis, electrophoreticseparation and fluorescence detection are integrated. In such cases, development of anew single cell manipulation approach which is easy to construct and implement andthat gives consideration to automation, simplicity, quantitative， throughput andprecision is advantageous.The signaling molecule nitric oxide (NO), it is involved in a number ofphysiological processes, such as blood pressure regulation in the cardiovascularsystem, neurotransmission in the central and peripheral nervous system, and immunedefense. Therefore, quantification of NO in single cell may not only help to find itsbiochemical variation within individual cells, but also help to identify its importantbiological implications, such as cell metabolism, signal transduction and diseasetreatment.In addition， interplay between reactive species within single cells has beenattracting much attention because they play a fundamental part in the regulation ofpathophysiological mechanisms. The localization of NO is greatly influenced bysuperoxide anions (O2-.) through rapid inactivation, giving rise to the formation of the potentially toxic species peroxynitrite (ONOO-). In addition to the effects mediated byNO inactivation, increasing evidence suggests additional roles for intracellularsuperoxide–nitric oxide interactions in the modulation of signaling throughredox-sensitive mechanisms. Therefore, simultaneous quantification of NO and O2-.insingle cells may not only help to find their biochemical variation within individualcells, but also help to identify the intracellular relative balance between these twospecies, and their important biological implications, such as cell metabolism, signaltransduction and disease treatment.In this work, a microchip format was developed to combine hydrostatic pressureand electrokinetic gated injection for intact cell handling and manipulation, prioring tosingle cell lysis and cellular content separation and detection. This microchip formatwas modeled on an earlier reported etched glass design used in other labs and we usea versatile programmable eight-path-electrode power supply (PEPS) andlaser-induced fluorescence detector (LIFD) microfluidic system.In the chapter two, a combination of hydrostatic pressure and electrokinetic gatedinjection was employed for intact cell manipulation. In the cell transport step,different volume of solutions were added to the five reservoirs, Cells be transportedinto the sample channel due to the hydrostatic pressure. In the single cell loading step,one cell being loaded into separation channel with the aid of electrokinetic force. Inthe lysis and electrophoresis separation step, the loaded cell was pushed byelectrokinetic flow, while one fraction of the running buffer from reservoir B entereddirectly to the reservoir SW, preventing cells from reservoir S entering the separationchannel. After that, the fluorescent product was detected with laser-inducedfluorescent detection.In the chapter three, a microchip format was developed for detection of NO insingle rat pheochromocytoma cells PC-12cells by combination of electrokinetic andhydrostatic pressure gated injection for intact cell handling and manipulation.Commercially available fluorescent probe DAR-4M AM was used for investigation ofNO intracellularly.184individual PC-12cells were detected under the sameexperimental conditions. In the chapter four, Commercially available fluorescent probe DAR-4M AM andDHE were used respectively for investigation of NO and O2-.. Under the optimizedexperimental conditions, we measured the intracellular NO and O2-.concentration inindividual PC12cells.