Peroxidase And Glutathione Single Cell Analysis And Dna Single Molecule Detection

In chapter one of this thesis, several main techniques for single-cell analysis including capillary electrophoresis (CE), microfluidic chip, scanning electrochemical microscopy (SECM) and high-throughput single-cell analysis were reviewed. In CE single-cell analysis, the cell injection, cytolysis, the derivatization methods of intracellular species were discussed. The detection techniques including laser-induced fluorescence (LIF) detection and electrochemical detection were introduced. In analysis-cell analysis with microfluidic chip, the approaches were focused on the detection of the intracellular species. The SECM analysis-cell analysis was also reviewed. In the chapter 142 references were cited.In chapter two, a novel high-throughput method without cytolysis for determination of enzyme activity inside single cells was developed by a combination of chemical cell perforation and an intracellular enzyme-catalyzed reaction. Peroxidase (PO) inside human neutrophils was chosen as the model system. Cells were perforated with digitonin to form micropores on the cell membrane. The perforated cells with physiological buffer saline of pH 7.4 containing hydroquinone (H₂Q) and H₂O₂ were continuously propelled by pressure through a capillary as the microsampler and microreactor. Small molecules H₂Q and H₂O₂ could diffuse into the cell interior through the micropores on the cell membrane, and the large molecule PO remained in the cell interior. Intracellular PO converted H₂Q into benzoquinone (BQ). BQ diffused out from the cell interior to the cell surface through the micropores and formed a BQ zone around the cell. The process proceeded in the capillary during cell movement. The BQ zones around every moving perforated cell were continuously delivered to the capillary outlet by hydraulic flow and detected. An average detection rate of > 1 cell/min was obtained. This paper was published in Anal. Chem. 78 (2006) 3213-3220.In chapter three, the electrochemical single-cell analysis for enzyme activity was developed using microcells on a microcell array coupled with a positionable dual...