Micro-reactor Scanning Electrochemical Microscopy For Quantitative Detection Of Biological Molecules

This thesis includes three parts: I . Scanning Electrochemical Microscopy (SECM) quantitatively determination cancer relative antigen CA15-3. II . Enzyme assay employed microreactor with SECM. III. Quantitatively determination of PO in cell abstraction by SECM.In chapter one of this thesis, we have reported a new method (SECM-ELISA) to quantitatively determine cancer relative antigen CA15-3 in the GC mode of SECM. First of all, we fabricated a microcell on the level substrate and the immunoreaction in the microcell formed the configuration of antibody(Ab)- antigen(Ag)- enzyme-labeled antibody(Ab*) on the substrate, which was called a sandwich structure. After the immunoreaction , we quantitatively determined CA15-3 by detecting BQ which was the catalyzed product of HRP. Several factors including pH, buffer concentration, electrode, detection potential and the time of detection were optimized. The following conditions were suitable for determination: buffer solution, 50 mmol/L Na2HPO₄-NaH2PO4 (pH 7.0) + 0.1mol/L KC1; detection potential, -0.4 V (vs. AgCl) ; concentration of H2Q and H2O2 , 1.0 mmol/L ; detection time, 6-8 minute after injection H2O2. Compared with other SECM-ELISA method, the substance for detection had been enriched in the microreactor. As a result, the sensitiveness had been improved .Under the conditions of the experiment, the limit of detection of the method was 2.5 U/mL and the linear scope was between 15 U/mL - 250 U/mL .The values of the CA15-3 Kit controls we detected were good agreed with the value of the Kit offered.In chapter two, we fabricated a microreactor suitable for SECM and detected HRP in solution with the microreactor. The microreactor was made by covering a thin film with plenty of hole on the cell of substrate. The diameter of the hole is smaller than the molecule diameter of HRP and bigger than the molecule diameter of H2Q, H2O2 and BQ .So HRP is sieved in the microreactor and H2Q, H2O2 and BQ are freelyin and out. As a result , HRP was determined by detection BQ diffused from the microreactor. Several factors including material of film, concentration of film solution and influence of organically solution to HRP were optimized. Under the conditions of the experiment, we have determined HRP immicroreactor triumphantly which means that this method is successful in determination some free molecule in solution by SECM.In chapter three, a method for detecting PO of cell abstraction with the microreactor by SECM was developed. The PO' S concentration of cell abstraction was 2.44 X 10^-2 U/mL and the average content of PO in single coll was found to be 1.06 X 10^-8U/cell, which was in the range of 2.5 X 10"9-4.9X 10"8U/cell detected by capillary electrophoresis.