Abstract
The early and accurate detection of viral pathogens is critical for effective disease management and public health safety. This study introduces an immunosensor that integrates an electro-active evanescent-wave cavity ring-down spectroscopy (EW-CRDS) platform with a sandwich-type bioassay for label-free detection of the influenza A (H5N1) hemagglutinin (HA) protein, achieving a detection limit of 15 ng/mL. The sensor is constructed by functionalizing the EW-CRDS platform within a micro-electrochemical flow cell with a monoclonal antibody specific to the target antigen. Upon antigen binding, a secondary polyclonal antibody conjugated with a redox-active methylene blue dye is captured. This dye undergoes reversible optical signal changes during redox transitions, which are electrochemically modulated and detected with high sensitivity. Unlike conventional approaches, this sensor employs electrochemical modulation to amplify surface-specific optical signals while reducing processing time and minimizing background noise. The results demonstrate the potential of this technology for real-time monitoring and rapid, reliable diagnosis of infectious diseases, offering excellent sensitivity and ease of operation in detecting influenza viruses. This work highlights the promise of the electro-active EW-CRDS platform for antigen detection in clinical settings.