Redox Cycling-Based Signal Amplification in Electrochemical Immunosensors: A Personal Account

Electrochemical immunosensors are powerful tools for sensitive biomolecular detection but are often limited by low signal intensity at ultralow analyte concentrations. This personal account presents our systematic development of redox cycling-based amplification strategies to overcome this challenge. Starting with enzyme-amplified electrochemical-chemical cycling, we progressed to chemical-chemical and EC-chemical systems, enabling repeated utilization of enzymatically generated electroactive species. Mechanistic insights into electron-transfer pathways (i.e. inner sphere-philic and outer sphere- philic nature) guided the rational selection of enzyme substrates, redox mediators, and oxidant-reductant pairs. Integrating alkaline phosphatase-catalyzed reactions with multistep redox cycling substantially enhanced signal-to-background ratios and detection limits without complex electrode modification. Hydroquinone diphosphate-based systems offered superior performance due to favorable redox properties and stability. Besides, with enzyme mediated electrochemical-enzymatic cycling, we progressed to EC-enzymtic systems, enzyme itself is redox active. These approaches enabled ultrasensitive detection of protein biomarkers at femtogram-per-milliliter levels and reliable quantification of pathogenic bacteria. The account also addresses challenges in background suppression, reagent stability, and assay reproducibility, providing general design principles for robust, scalable, and sustainable electrochemical biosensors.