Pulsed galvanostatic control of ion fluxes across polymeric membrane ion-selective electrodes (ISEs) is an emerging field for potentiometric sensing. Herein we report a novel potentiometric enzyme immunoassay based on current-controlled release of an enzyme substrate, which eliminates the addition of marker ions in the sample solution. In this method, the carboxylated poly(vinyl chloride) matrix at the outer layer of the ISE membrane is employed to attach a primary antibody. A sandwich immunoassay with an alkaline phosphatase labeled antibody (ALP-Ab) as the reporter is used for the determination of human IgG (as a model protein). The large difference between the lipophilicity of the substrate ion and that of the product ion allows p-nitrophenyl phosphate to be used as the enzyme substrate for potentiometric immunosensors. After the immunoreactions, the captured ALP-Ab catalyzes the hydrolysis of the substrate ions released at the sample-membrane interface by using the pulsed galvanostatic technique. This process can be potentiometrically determined by measuring the open circuit potential of the ISE. Under optimal conditions, the potential response of the proposed immunosensor is proportional to the concentration of human IgG in the range of 50-1000 ng/mL with a detection limit of 30 ng/mL (3 sigma). Owing to simplicity and independence of sample volume and sample turbidity, the proposed potentiometric immunoassay offers a viable alternative to those based on optical absorbance.
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机译：跨聚合物膜离子选择电极（ISE）的离子通量的脉冲恒电流控制是电位感测的新兴领域。在本文中，我们报告了一种基于电流控制释放酶底物的新型电位酶免疫测定方法，该方法消除了样品溶液中标记离子的添加。在这种方法中，采用ISE膜外层的羧化聚（氯乙烯）基质连接一抗。用碱性磷酸酶标记的抗体（ALP-Ab）作为报告基因的夹心免疫测定法用于测定人IgG（作为模型蛋白）。底物离子的亲脂性与产物离子的亲脂性之间的巨大差异使得对硝基苯基磷酸酯可用作电位免疫传感器的酶底物。免疫反应后，通过使用脉冲恒电流技术，捕获的ALP-Ab催化在样品-膜界面释放的底物离子的水解。可以通过测量ISE的开路电位来电位确定该过程。在最佳条件下，建议的免疫传感器的潜在反应与人类IgG浓度成正比，范围为50-1000 ng / mL，检测极限为30 ng / mL（3 sigma）。由于样品体积和样品浊度的简单性和独立性，所提出的电位免疫测定法为基于光吸收的方法提供了可行的替代方法。
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