Pulsed galvanostatic control of a polymeric membrane ion-selective electrode for potentiometric immunoassays

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 (3σ). 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.