Self-transducing PB@MWCNTs based immunosensor for mediator-free and clinically validated SARS-CoV-2 detection

The unprecedented impact of COVID-19 exposed the critical limitations in existing diagnostic technologies, underscoring the global demand for rapid, ultrasensitive, and reliable point-of-care (POC) detection systems. However, most current SARS-CoV-2 electrochemical sensors still rely on diffusional redox mediators or multi-step assay chemistries, which limit portability and real-world deployment. Here, we engineered a self-transducing electrochemical immunosensor by incorporating Prussian blue-decorated multi-walled carbon nanotube composite (PB@MWCNTs) integrated onto a screen-printed carbon electrode (SPCE). The PB@MWCNT hybrid intrinsically supports Fe 3+ /Fe 2+ cycling, enabling direct electron exchange without external mediators. The PB@MWCNTs/SPCE immunosensor exhibits a wide linear range (10 fg/mL–10 ng/mL) with a detection limit of 8.6 fg/mL and high specificity in PBS. The sensor delivered comparable performance in InstaView™ commercial assay buffer (LOD = 10.8 fg/mL), demonstrating reliable operation in real assay matrices within 4 min total assay workflow from sample addition to electrochemical readout. Cross-reactivity studies demonstrated that the immunosensor exhibits high specificity toward the SARS-CoV-2 nucleocapsid protein, with negligible responses to HCoV-HKU1, influenza A, RSV, and representative respiratory bacterial strains. Crucially, clinical validation using live SARS-CoV-2 (Wuhan lineage) and Omicron (BA.1) under certified BSL-3 conditions demonstrated reliable detection down to 10 3 PFU/mL—representing nearly two orders of magnitude greater sensitivity than commercial rapid antigen kits, which showed visible responses only ≥ 10 5 PFU/mL. Achieving detection at 10 3 PFU/mL highlights the platform’s ability to identify low viral burdens within clinically relevant ranges, reinforcing its practical diagnostic significance. Overall, this work establishes a clinically validated, mediator-free electrochemical sensing immunosensor that combines simple, rapid operation with high analytical sensitivity, providing a scalable and next-generation solution for decentralized respiratory virus diagnostics.