Self-Powered Microfluidic Device with Laser-Converted Graphene Electrodes for Immobilization-Free Electrochemical Detection of MPOX Virus DNA via Mismatch-Driven Nanocomplexes.

The re-emergence of monkeypox virus (MPXV) underscores the urgent need for rapid and decentralized diagnostic tools. Herein, we present an innovative, immobilization-free, and label-free paper-based electrochemical fast-flow microfluidic device (eFMD) integrated with near-field communication (NFC) technology for point-of-care detection of MPXV. The platform utilizes a C─C mismatch-mediated Ag+ intercalation mechanism, generating distinct current shifts detectable via differential pulse voltammetry (DPV). A microfluidic device with optimized serpentine mixing channels ensures efficient Ag+ intercalation without external power sources. The biosensor incorporates laser-converted graphene (LCG) electrodes, offering eco-friendly, scalable, and solvent-free fabrication with rapid prototyping and high design flexibility, ideal for disposable diagnostics. Key assay parameters including buffer type, mismatch probe concentration, AgNO3 levels, and detection time were systematically optimized to enhance sensitivity. The biosensor demonstrated excellent analytical performance with a low detection limit down to 1.4 pм for MPXV DNA and significant discrimination against single-base mismatched sequences. Robust performance was further demonstrated in complex matrices, including spiked biological samples and clinically relevant lesion swab specimens, with detection sensitivity down to 1.3 copies mL- 1 and complete concordance with PCR classification. Importantly, the system allows for wireless, on-site readout via smartphones within 35 min, making it suitable for rapid MPXV screening.