Reference Electrode-Free Electrochemical Detection Using a Bare Interdigitated Array Electrode and Initial Counterbalancing O2 Reduction

In this study, we introduce a reference electrode-free two-electrode system that utilizes the electrochemical reduction of naturally dissolved O2 in aqueous solutions to counterbalance the oxidation of 4-aminophenol (AP) at an Au interdigitated array electrode. This system minimizes the potential variation at the working electrode and eliminates the need for a large potential difference between the two electrodes. The oxidation of AP produces 4-quinoneimine, inducing electrochemical-electrochemical (EE) redox cycling. Operating in phosphate-buffered saline (pH 7.4) ensures a low anodic background current within a potential range where AP (formal potential = 0.07 V vs Ag/AgCl) undergoes oxidation. Concurrently, the reduction of O2 occurs effectively near 0.0 V. This setup enables the application of a small potential difference (0.3 V) to simultaneously achieve AP oxidation and O₂ reduction, followed by efficient EE redox cycling. Unwanted side reactions involving O₂ and H₂O₂ are minimal and do not interfere with the redox cycling. We applied this system to detect proteases (3CL protease from coronavirus SARS-CoV-2 and aminopeptidase N) that catalyze the release of AP from electro-inactive AP-conjugated peptides. Our approach demonstrates the potential for efficient EE re-dox cycling without requiring a reference electrode and added redox species.