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) 2 Cl 2 + /Os(bpy) 2 Cl 2 (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.