溶胶凝胶固定化酶流动注射化学发光法测定人体血清中的胆固醇

Determination of Cholesterol in Human Serum Based on Flow-injection Chemiluminescence Method by Sol-gel Immobilized Enzyme

采用溶胶凝胶技术分别固定了胆固醇脂酶和胆固醇氧化酶,制成固定化酶柱;人体血清中胆固醇脂在胆固醇脂酶的催化作用下生成胆固醇,胆固醇在胆固醇氧化酶的催化作用下被氧化产生H2O2,将其与鲁米诺发生耦合的化学发光反应,在模拟酶血红蛋白的催化作用下产生较强的化学发光。结合流动注射技术,建立了溶胶凝胶固定化酶流动注射化学发光法测定胆固醇的新方法。实验发现,发光强度与胆固醇的浓度在一定范围内呈良好的线性关系,总胆固醇的线性范围为1.01×10-6～2.02×10-4mol/L(r=0.9975);检出限为7.5×10-7mol/L;游离胆固醇的线性范围为5.0×10-8～2.18×10-5mol/L(r=0.9991);检出限为5.0×10-9mol/L。用生化分析仪(东芝TBA-120FR)与本方法进行对照,两种方法无显著性差异。本方法已应用于临床血清样品中胆固醇的检测。

The cholesterol esterase and its oxidase were respectively immobilized onto sol-gel（tetraethyl orthosilicate）and then an enzymatic reaction column was prepared.Hydrolysis of cholesterol ester took place in the presence of cholesterol esterase to form cholesterol under catalysis of cholesterol oxidase,and Cholesterol was oxidized to produce hydrogen peroxide which reacted with luminal in the presence of simulated enzyme hemoglobin to result in the emission of light.Thereupon,a chemiluminescence system combined with flow injection technology by sol-gel immobilized enzyme was developed for the determination of cholesterol including free and total amount in human serum.The CL intensity was well linear with the concentration of cholesterol;liner range of total and free cholesterol was 1.01×10^-6～2.02×10^-4 mol/L with a detection limit of 7.5×10^-7 mol/L and 5.0×10^-8～2.18×10^-5 mol/L with a detection limit of 5.0×10^-9 mol/L respectively.Contrasted the method with the biochemical analyzer（TBA-120FR）,both the methods had no significant difference.This method has been successfully applied for the detection of cholesterol in clinic serum samples.