Scanning Electrochemical Microscopy Detection Reagent To Stimulate The Cells To Cause The Activity Of Cellular Respiration And Enzyme Activity Changes

In chapter one of this thesis, we studied how three reagents Br^-, CN^- and N₃^-inhibited the activity of respiration in a single living cell. In the detection of respiration activity, the tip was applied by a voltage at which O₂ could be deoxidized. The detected peak current originated from two parts. One was the consumption of respiration, the other was the stuck cell's salience leading to negative feedback. During detecting respiration activity, we got rid of the current from the negative feedback. By recording the respiration activity of the cell stimulated by reagents at different times, the inhibition extent of reagents to respiration activity was obtained.In chapter two, we studied how three reagents Br^-, CN^- and N₃^- inhibited the activity of myeloperoxidase in a single living cell. A method for detecting MPO activity in single living cell by scanning electrochemical microscopy (SECM) was developed. For the detection of MPO we ascertained the optimum condition of detection by the generation mode. Digitonin (Dig) in PBS could dissolve cholesterol on the cell membrane and form microhole on it. Low mass molecules could pass through the membrane while macromolecules such as enzyme were still within the cell. Consequently, H₂Q and H₂O₂ could enter the cell and react to produce BQ and H₂O under the catalysis of MPO. The produced BQ and H₂O diffused outside from the cell. When the tip was moved above the cell surface, it could determine the changes of the BQ concentration, find cells and detect the activity of intracellular MPO by SECM. When reagents was added, MPO activity decreased. By recording the MPO activity of the cell stimulated by reagents at different times, we got the inhibition extent of reagents to MPO.In chapter three, we detected MPO in single living cell by using a sub-microelectrode. We fabricated sub-microelectrode by phenol-allylphenol copolymer insulated way. After comparing the scanning curves using the tips with radius of 5 urn, 680 nm and 432 nm respectively, we found that the peak height decreased as the tip's radius decreased. When tip's radius was 432 nm, peak height was 5 pA. Smaller tip to detect MPO in single cell was impossible because the noise is 1 pA. So nanoelectrodes could not be used to detect MPO in single cell in the present method.