Electrochemical Paper-Based Nanobiosensor for Rapid and Sensitive Detection of Monkeypox Virus

Monkeypox virus (MPXV) infection was classified as a public health emergency of international concern by the World Health Organization (WHO) in 2022, being transmitted between humans by large respiratory droplets, in contact with skin lesions, fomites, and sexually. Currently, there are no available accessible and simple-to-use diagnostic tests that accurately detect MPXV antigens for decentralized and frequent testing. Here, we report an electrochemical biosensor to detect MPXV antigens in saliva and plasma samples within 15 min using accessible materials. The electrochemical system was manufactured onto a paper substrate engraved by a CO₂ laser machine, modified with gold nanostructures (AuNS) and a monoclonal antibody, enabling sensitive detection of A29 viral protein. The diagnostic test is based on the use of electrochemical impedance spectroscopy (EIS) and can be run by a miniaturized potentiostat connected to a smartphone. The impedimetric biosensing method presented excellent analytical parameters, enabling the detection of A29 glycoprotein in the concentration ranging from 1 × 10^-14 to 1 × 10^-7 g mL^-1, with a limit of detection (LOD) of 3.0 × 10^-16 g mL^-1. Furthermore, it enabled the detection of MPXV antigens in the concentration ranging from 1 × 10^-1 to 1 × 10⁴ PFU mL^-1, with an LOD of 7.8 × 10^-3 PFU mL^-1. Importantly, no cross-reactivity was observed when our device was tested in the presence of other poxvirus and nonpoxvirus strains, indicating the adequate selectivity of our nanobiosensor for MPXV detection. Collectively, the nanobiosensor presents high greenness metrics associated with the use of a reproducible and large-scale fabrication method, an accessible and sustainable paper substrate, and a low volume of sample (2.5 μL), which could facilitate frequent testing of MPXV at point-of-care (POC).