Sapyen W.Ruecha N.Weeranoppanant N.Khositanon C.Pasomsub E.Wu H.Yakoh A.Mahidol University2026-06-012026-06-012026-01-01Small (2026)16136810https://repository.li.mahidol.ac.th/handle/123456789/117036The 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, AgNO<inf>3</inf> 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.Materials ScienceChemistryBiochemistry, Genetics and Molecular BiologyArticleSCOPUS10.1002/smll.2025133362-s2.0-10503985203816136829