葡萄糖浓度的酶联荧光毛细分析法测定

Determination of Glucose by Enzyme Fluorescence Capillary Analysis Method via Conjugating Glucose Oxidase with Horseradish Peroxidase

该文以葡萄糖氧化酶和辣根过氧化物酶为催化剂，使含有对甲基酚的葡萄糖溶液体系通过酶偶联催化反应生成荧光物质，从而实现对葡萄糖浓度的测定。优化的实验条件为：反应时间20min；NH3-NH4Cl缓冲溶液（pH10．4）；对甲基酚浓度30．0μmol／L。分别采用荧光毛细分析法和荧光光谱分析法测定了相同浓度的系列葡萄糖溶液的荧光强度。在5．0-500．0μmol／L范围内，两种方法测得的荧光强度均与葡萄糖浓度的对数成正比。通过对比测试结果分析了两种方法的优缺点。

A fluorescence capillary analysis （FCA）method was developed for the quick determination of glucose. Glucose firstly reacted with oxygen to produce hydrogen peroxide with the catalysis of glucose oxidase（GOx）. This reaction was coupled with the oxidation reaction of p-cresol, which was catalyzed by horseradish peroxidase（HRP） in the presence of hydrogen peroxide, and produced fluo- rescent substances, the dimmer of p-cresol. Base on the fluorescence intensity of the dimmer was proportional to the logarithm of glucose concentration, the concentration of glucose was detected. The optimal experimental conditions were as the follows： reaction time： 20 min; buffer solution： pH 10. 4 NH3 - NH4Cl ; concentration of p-cresol ： 30. 0 μmol/L. Under the optimum conditions, FCA and fluorescence analysis（FA） methods were applied for the determination of glucose, respectively. The results indicated that both methods had wide concentration ranges and good sensitivity to glucose. The fluorescence intensity increased with the concentration of glucose, and the fitting curves of fluorescence intensity versus the logarithm of concentration were linear over the concentration range of 5.0 - 500. 0 μmol/L with correlation coefficients of 0. 996 （FCA） and 0. 985 （FA）. Compared with the FA method, the FCA method has the advantage of saving the reagents, but accompanied by a loss of fluorescence intensity.