Faculty of Science, Shinshu University, 3-1-1 Asahi, Matsumoto, Nagano, Japan.
Bioelectrochemistry. 2010 Aug;79(1):147-51. doi: 10.1016/j.bioelechem.2009.12.005. Epub 2009 Dec 11.
In order to determine trace choline in human urine, a flow injection analysis (FIA) system has been developed by coupling an enzyme reactor with an electrochemiluminescence (ECL) detector. The enzyme reactor is prepared by covalently immobilizing choline oxidase (ChOx) onto the aminopropyl-controlled pore glass beads, which are then carefully packed into a micro column. The enzyme reactor catalyzes the production of hydrogen peroxide that is in direct proportion to the concentration of choline. In this study, the enzymatically produced hydrogen peroxide was detected by an ECL detector positioned at the down stream of an enzyme reactor based on the luminol/H(2)O(2) ECL system. Under the optimized condition, the enzymatic FIA/ECL provided high sensitivity for the determination of choline with the detection limit as low as 0.05 microM (absolute detection limit was at sub pmol level). The method was successfully applied in the determination of choline in the samples of human urine, and the analytical results were in good agreement with those obtained by using the microbore HPLC with an immobilized enzyme reactor-electrochemical detection system.
为了测定人尿中的痕量胆碱，我们通过将酶反应器与电化学发光（ECL）检测器相耦合，开发了一种流动注射分析（FIA）系统。酶反应器是通过将胆碱氧化酶（ChOx）共价固定在氨丙基控制孔玻璃珠上制备的，然后将其小心地填充到微柱中。酶反应器催化过氧化氢的产生，其浓度与胆碱的浓度成正比。在本研究中，基于鲁米诺/H₂O₂ ECL 体系，通过位于酶反应器下游的 ECL 检测器检测酶促产生的过氧化氢。在优化条件下，酶促 FIA/ECL 对胆碱的测定具有很高的灵敏度，检测限低至 0.05 μM（绝对检测限处于亚 pmol 水平）。该方法成功地应用于人尿样品中胆碱的测定，分析结果与使用固定化酶反应器-电化学检测系统的微柱 HPLC 获得的结果一致。
