Fanjul-Bolado Pablo, González-García María Begoña, Costa-García Agustín
Departamento de Química Física y Analítica, Universidad de Oviedo, 33006, Oviedo, Spain.
Anal Bioanal Chem. 2006 Aug;385(7):1202-8. doi: 10.1007/s00216-006-0367-8. Epub 2006 Mar 11.
p-Nitrophenyl phosphate is one of the most widely used substrates for alkaline phosphatase in ELISAs because its yellow, water-soluble product, p-nitrophenol, absorbs strongly at 405 nm. p-Nitrophenol is also electroactive; an oxidative peak at 0.97 V (vs. an Ag pseudoreference electrode) is obtained when a bare screen-printed carbon electrode is used. When an amperometric detector was coupled to a flow-injection analysis system the detection limit achieved for p-nitrophenol was 2x10(-8) mol L(-1), almost two orders of magnitude lower than that obtained by measuring the absorbance of the compound. By use of this electrochemical detection method, measurement of 7x10(-14) mol L(-1) alkaline phosphatase was achieved after incubation for 20 min. The feasibility of coupling immunoassay to screen-printed carbon electrode amperometric detection has been demonstrated by performing an ELISA for detection of pneumolysin, a toxin produced by Streptococcus pneumoniae, which causes respiratory infections. The method is simple, reproducible, and much more sensitive than traditional spectrophotometry.
对硝基苯磷酸酯是酶联免疫吸附测定（ELISA）中碱性磷酸酶最常用的底物之一，因为其黄色的水溶性产物对硝基苯酚在405nm处有强烈吸收。对硝基苯酚也具有电活性；当使用裸丝网印刷碳电极时，在0.97V（相对于银假参比电极）处可获得一个氧化峰。当将安培检测器与流动注射分析系统耦合时，对硝基苯酚的检测限达到2×10⁻⁸ mol L⁻¹，几乎比对该化合物吸光度测量所得结果低两个数量级。通过使用这种电化学检测方法，孵育20分钟后实现了对7×10⁻¹⁴ mol L⁻¹碱性磷酸酶的测量。通过进行检测肺炎链球菌产生的毒素肺炎溶血素的ELISA，已证明将免疫测定与丝网印刷碳电极安培检测相结合的可行性，肺炎溶血素会导致呼吸道感染。该方法简单、可重复，并且比传统分光光度法灵敏得多。
