Capillary Electrophoresis - Two Instances Of Electrochemiluminescence Detection For Biological Analysis

In chapter one of this paper, a simple and sensitive method for plasma prolidase activity assay was developed based on Capillary Electrophoresis combined with Electrochemiluminescence detection (CE-ECL) of the formed proline. At optimal conditions (pH 7.5, 30 mmol/L phosphate buffer, 5 mmol/L Ru(bpy)₃²⁺, potential of working electrode: 1.3V, injection: 10 kV, 10s), the detection limit of proline was 12.2 fmol (S/N = 3), corresponding tol.22×10^-8U of prodase catalyzing for Imin. In experiments, the conditions of proline detection and the enzymatic reaction system were optimized. The optimal conditions of the enzymatic reaction were: (1) preincubation: plasma diluted with 3.0×10^-3 mol/L (pH 7.6) phosphate buffer, 37℃ for 24 h with the concentration of Mn²⁺ was 1.0×10^-3 mol/L; (2) incubation: Mn²⁺, 1.0×10^-3 mol/L; glycylproline, 0.01mol/L; 37℃ for 30 min; (3) termination: 80 ℃ for 10 min. The reproducibility (RSD, n = 10) was 3.47% and the recoveries of five samples were between 97.0% and 106%. The proiidase activity of 4 plasmas samples and 2 serum samples determined by CE-ECL was in agreement with that by classic Chinard's method. The new method was also applied to detect PLD activity in serum of diabetic patients. Mean±SD was 888.18 ± 211.48 U/L for the 45 serum samples studied which obey the normal distribution. There was a significant positive correlation between serum PLD activity and fasting serum glucose concentration in the 38 subjects with normal serum triglyceride levels (r = 0.495, p< 0.002). Serum PLD activity in diabetes mellitus was higher than that in normal fasting glucose (p<0.05), which indicated that collagen degradation was accelerated in diabetes mellitus. High triglyceride concentration maybe another factor that stimulate the disorder of collagen metabolism.In chapter two, CE-ECL was applied to study the different effects of five vitamins on PLD activity. Vitamin C and vitamin B12 enhanced the PLD activity at low concentrations, while inhibit PLD at high concentrations. Vitamin B1 and vitamin H presented inhibiting effects on PLD. Vitamin B6 enhanced PLD...