Disposable label-free electrochemical immunosensor using hollow porous gold sub-microcubes with surface-confined ferrocene for point-of-care detection of Mpox virus A29 protein.

Monkeypox (Mpox), a viral zoonotic disease caused by the monkeypox virus (MPXV) remains a significant global public health concern. Current laboratory diagnostics are limited by high biosafety requirements and complex workflows, preventing point-of-care (POC) use. To address these limitations, a disposable, label-free electrochemical immunosensor was developed to detect the MPXV A29 glycoprotein, a highly immunogenic and diagnostically relevant protein. Shape-controlled, reproducible, hollow porous gold sub-microcubes (HPGCs) were synthesized using AgCl as a sacrificial template. The screen-printed carbon electrode was functionalized with poly-l-cysteine and HPGCs bearing a surface-confined 6-(ferrocenyl)hexanethiol probe and antibody for selective antigen binding. Antigen binding attenuates the ferrocene electrochemistry, investigated by voltammetry and impedance spectroscopy. The high surface-to-volume ratio and electrocatalytically active centers of HPGCs enable high loading of ferrocene and antibodies. The surface-confined signaling probe eliminates the need for an external signaling probe, offering a portable, reagent-free POC testing. The sensor offers a wide linear range (0.1-3200 ng/mL), low detection limit (0.1 ng/mL), excellent selectivity against non-target proteins, high reproducibility (RSD = 3.73%), and strong recovery in spiked serum with minimal matrix effects. These findings demonstrate a robust, sensitive, and user-friendly diagnostic strategy for field-based Mpox surveillance in resource-limited settings.