Single Neutrophil Cell Peroxidase, Hydrogen Peroxide, Active Oxygen Determination

Chapter one is the summary of methods for single-cells-analysis, the methods was classified as 3 kinds, the first kind is about detection after the separation of intracellular cintents with a micro channel, the second kind is about detection with a micro probe, the third kind is about detection with optical microscopy. The technologies used in single cells analysis, such as capillary electrophoresis, microfluidic chip, micro electrode, fiber optical sensor, scanning electric chemical microscopy, atom force microscopy, ordinary fluorescence microscopy, laser confocal scanning microscopy, total internal refraction microscopy, near field microscopy, are introduced. 190 references are reffered.In chapter two, a new method for determination of enzyme activity in single cells based on enzyme kinetics with an epi-fluorescence microscopy was developed. Peroxidase (PO) in individual neutrophils was determined with this method. PO can catalyze the reaction of ADHP with H₂O₂, and produce fluorescent resorufin. Such steps was followed to analyze single neutrophil cells: An individual cell was transferred into a capillary with electro-migration, and then the cell was transferred into a micro-liter vessel, and was lysed in the vessel by freeze-thawing and ultrasonication. The enzyme catalyzed-reaction was initiated by the addition of the buffer solution containing nonfluorescent substrates ADHP and H₂O₂ to the vessel. The fluorescent products of the reaction was excited with a 543 run laser at different reaction times and images of fluorescence were taken by an ICCD camera. The average-gray-levels of all images were measured and the corrected average-gray-levels were used for quantification. The PO activity could be determined through measuring the fluorescence of resorufm based on the slope of linear line on the plot of the fluorescence intensity versus the reaction time. The interference of intracellular metal ions was discussed. The average value of intracellular PO in single neutrophils is 3×10^-8 U/cell in our experiments. The limit of detection is 7×10^-8 U/mL.In chapter three, an epi-fluorescence microscopy for determination of intracellular...