Adsorption and Electropolymerization of p-Aminophenol Reduces Reproducibility of Electrochemical Immunoassays

Abstract

Electrochemical immunoassays are a class of biosensors that achieve high specificity, low detection limits, and low cost attributed to strong antibody-antigen interactions and inexpensive electrochemical instrumentation. Electrochemical assays typically use an enzyme/substrate pair to enable electrochemical signal production. p-Aminophenyl phosphate is a commonly used enzymatic substrate which is hydrolyzed into p-aminophenol (PAP)by alkaline phosphatase. PAP exhibits low oxidation potential and electrochemical reversibility, making it an appealing candidate for assay design. The electrochemical oxidative signal from PAP is increased by its adsorption to the electrode. This is further confirmed by the prevention of PAP adsorption via self-assembled monolayers on gold electrodes reducing PAP’s oxidative current. On bare screen-printed electrodes (SPE), PAP adsorption results in oxidative current variability depending on the electroactive surface area of the working electrode. This variability could not be remedied by cleaning and reusing the same gold SPE. However, adsorption of PAP displays concentration dependence, as the decrease ofthe PAP concentration to 3.8 μM greatly improved the consistency of the measurements. PAP use is also limited in continuous monitoring applications as multiple PAP oxidations on the same electrode caused polymerization. The results from this study suggest that the use of PAP production in immunoassays with SPEs must be fine-tuned and electrodes must be cleaned or disposed of between measurements.