Electrochemical Immunosensor Employing a Potential-Guiding, Kinetically Robust, and Counterbalancing Redox Mediator in a Two-Electrode Configuration

Electrochemical immunosensors offer rapid, sensitive biomarker detection but typically rely on complex three- or two-electrode architectures with dedicated reference electrodes, complicating fabrication and miniaturization. Herein, we present an electrochemical immunosensor based on a diffusion-isolated, two-electrode system of micropatterned indium tin oxide (ITO) that eliminates the need for a separate reference electrode. A kinetically robust (fast, surface-insensitive) redox mediator [Os(bpy)₂Cl₂⁺/Os(bpy)₂Cl₂ (Os^III/Os^II)] simultaneously acts as the enzymatic substrate, the counterbalancing electroactive species, and the potential-guiding element, removing the requirement for additional electroactive species. Further, diaphorase (DI) is employed as the catalytic label in a sandwich-type immunosensor for prostate-specific antigen (PSA), rapidly reducing Os^III to Os^II in the presence of reduced nicotinamide adenine dinucleotide (NADH), enabling efficient electrochemical-enzymatic redox cycling involving the ITO electrode, Os^III, DI, and NADH. By patterning the working and auxiliary electrodes 1.0 mm apart, we achieved diffusion isolation that prevents cross-talk between electrodes. After optimizing blocking, potential difference, and incubation conditions, the sensor achieved a detection limit of approximately 20 pg/mL and a detection range of 20-10 ng/mL in spiked human serum. The results of our analysis of clinical serum samples correlated closely with those of a commercial PSA assay, demonstrating both analytical accuracy and robustness. This study establishes a streamlined, point-of-care-compatible platform that achieves stable potential control and robust kinetics without a dedicated reference electrode.