Scanning Electrochemical Microscopy Combined With Single-cell Analysis Of Micro-reactor

This thesis consists of two parts: I. Microreactor fabrication and enzyme assay combining microreactor with Scanning Electrochemical Microscopy (SECM). Ⅱ. Quantitatively determination of peroxidase (PO) in single human neutrophils using microreactor coupled with SECM.In chapter one of this thesis, a microreactor suitable for SECM was fabricated and horseradish peroxidase (HRP) in solution was detected with the microreactor. First of all, a microccll was punched on a Plexiglas plate of ～2 mm thichness using a drill, then the HRP and H₂Q,H₂O₂ was injected with self-made micro-injector and lastly a cellulose nitrate film was covered on the microcell to fabricate the microreactor. The HRP activity was determined by detecting the product BQ diffused from the microreactor. Several factors including detection potential, concentration of H₂Q and H₂O₂, reproducibility of detection, the time of incubation and the diameter and depth of microcell were investigated and discussed. The following conditions were suitable for determination: physiological buffered saline (PBS, pH 7.4); concentration of H₂Q and H₂O₂, 2.00×10^-3 mol/L; detection potential, -0.3 V (vs. Ag/AgCl); incubation time, 20 min.; depth, 550 μm and diameter, 200 urn of the microcell. Under the optimal conditions of the experiment, the limit of detection of the method was 3.7×10^-9 U and the linear scope was between 7.50×10^-9 U - 5.63×10^-7 U. Compared with other methods, the sensitivity had been improved because HRP is free in solution and the method of incubation and flatting the cellulose nitrate solution with a glass slide was adopted.In chapter two, a method for detecting PO of single human neutrophils with microreactor by SECM was developed. In this method, firstly, a single cell perforated by digitonin was manipulated to the microcell, and lysed using a freeze-thawing technique. Then Physiological buffer saline containing 2.00×10^-3 mol/L H₂Q and...