Integrated enzyme-linked immunosensor with biofunctionalized ion-selective membranes by pulstrode delivery of substrate

A new principle for electrochemical enzyme immunoassays is proposed where the enzyme substrate is selectively delivered by an electrochemical excitation pulse to the location where the biomolecular interaction takes place. This is achieved by covalent attachment of the capture antibody on the surface of a polymeric ion-selective membrane, which also serves to monitor the enzyme activity over time by open circuit chronopotentiometry. The membrane biofunctionalization was accomplished by using a click chemistry protocol in addition to N-hydroxysuccinimide ester crosslinking and was characterized using confocal microscopy and electrochemical impedance spectroscopy. To demonstrate the principle, a choline-oxidase labelled antibody served as the detection reporter in a sandwich immunoassay for the determination of human lysozyme in saliva in the range of 10–100 μg mL−1 with a detection limit of 0.7 μg mL−1 (3σ). The enzyme-immunocomplex at the sensing surface catalyzes the oxidation of choline, an ion marker to which the membrane is selective and that is released from the membrane by a pulstrode protocol (galvanostatic pulse of 1 s duration and 10 μA magnitude). The enzyme turnover is interrogated by measuring the open circuit potential of the electrode over time after the release pulse. A model based on diffusion and enzyme kinetics is developed to rationalize the sensing principle and to guide the experiments. The results with this integrated immunosensor compare well to that of a conventional enzyme-linked immunoassay. © 2023 The Authors