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<p>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<sup>3+</sup>/Fe<sup>2+</sup> cycling, enabling direct electron exchange without external mediators. The PB@MWCNTs/SPCE immunosensor exhibits a wide linear range (10&#xa0;fg/mL–10 ng/mL) with a detection limit of 8.6&#xa0;fg/mL and high specificity in PBS. The sensor delivered comparable performance in InstaView™ commercial assay buffer (LOD = 10.8&#xa0;fg/mL), demonstrating reliable operation in real assay matrices within 4&#xa0;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<sup>3</sup> PFU/mL—representing nearly two orders of magnitude greater sensitivity than commercial rapid antigen kits, which showed visible responses only ≥ 10<sup>5</sup> PFU/mL. Achieving detection at 10<sup>3</sup> 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.</p>

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

  • 期刊论文

  • 出版模式：

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  • 发表日期：

    2026年3月17日

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  • Sekar Madhu,
  • Sriramprabha Ramasamy,
  • Jung Cheol Seo,
  • Dabin Jeong,
  • Jin-A Park,
  • Sungweon Ryoo,
  • Jungil Choi

摘要

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 Fe3+/Fe2+ 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 103 PFU/mL—representing nearly two orders of magnitude greater sensitivity than commercial rapid antigen kits, which showed visible responses only ≥ 105 PFU/mL. Achieving detection at 103 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.