Raba J, Mottola H A
Department of Chemistry, Oklahoma State University, Stillwater 74078-0447.
Anal Chem. 1994 May 1;66(9):1485-9. doi: 10.1021/ac00081a020.
Two rotating bioreactors in tandem have been incorporated into a continuous-flow/stopped-flow sample/reagent processing setup for the determination of alkaline phosphatase (EC3.1.3.1) activity in serum samples. The strategy circumvents incompatibility of buffer systems as well as that of the immobilized enzymes utilized in the bioreactors (alkaline phosphatase and alcohol oxidase, EC 1.1.3.13). The determination is indirect in nature although recorded responses are directly related to the enzyme activity in the sample. It couples the following enzyme-catalyzed reactions: (1) hydrolysis of p-nitrophenyl dihydrogen phosphate catalyzed by alkaline phosphatase, (2) enzymatic reaction between unreacted p-nitrophenyl dihydrogen phosphate with methanol, and (3) conversion of the residual methanol to the corresponding aldehyde and H2O2, catalyzed by alcohol oxidase. The H2O2 is amperometrically determined at a stationary Pt-ring electrode (applied potential + 0.600 V vs a Ag/AgCl, 3.0 M NaCl reference).
两个串联的旋转生物反应器已被整合到一个连续流动/停流的样品/试剂处理装置中，用于测定血清样品中的碱性磷酸酶（EC3.1.3.1）活性。该策略避免了缓冲系统以及生物反应器中使用的固定化酶（碱性磷酸酶和醇氧化酶，EC 1.1.3.13）之间的不相容性。尽管记录的响应与样品中的酶活性直接相关，但该测定本质上是间接的。它耦合了以下酶催化反应：（1）碱性磷酸酶催化对硝基苯基磷酸二氢酯的水解，（2）未反应的对硝基苯基磷酸二氢酯与甲醇之间的酶促反应，以及（3）醇氧化酶催化将残留甲醇转化为相应的醛和H2O2。在固定的铂环电极上通过安培法测定H2O2（相对于Ag/AgCl，3.0 M NaCl参比电极施加的电位为+ 0.600 V）。
