Intended Mismatch DNA Nanocomplexes for Ultrasensitive, Enzymatic Colorimetric Detection of MPOX Virus in Minutes Using a Paper‐Based Fast‐Flow Micromixing Device

Mpox has re-emerged as a global health threat, necessitating rapid and accessible diagnostics. This study introduces a fast-flow micromixer paper-based analytical device (fmPAD) that detects mpox virus (MPXV) with high sensitivity using a mismatch DNA probe and Ag⁺- mediated enzymatic reactivation. The fmPAD enables real-time, amplification-free detection down to 5 copies mL⁻¹ in urine and saliva using a portable colorimeter, offering a promising tool for point-of-care diagnostics.

Abstract

In 2024, mpox (formerly “monkeypox”) re-emerged as a global health threat due to the more severe Clade I strain, underscoring the need for rapid, accessible diagnostics. Traditional polymerase chain reaction (PCR) methods, while effective, demand complex laboratory infrastructure and extended processing times. To address these limitations, this study presents a fast-flow micromixer paper-based analytical device (fmPAD) designed for ultrasensitive, rapid mpox virus (MPXV) detection. The device employs a specially engineered sensing probe with a cytosine-cytosine (C─C) mismatch sequence that hybridizes with the MPXV target, reactivating urease previously inhibited by Ag⁺ ions and generating a color change in a pH-sensitive indicator. Enhanced fluid mixing and flow within the fmPAD allow efficient enzymatic reactivation, achieving a linear detection range from 1 to 1000 pm, with a limit of detection (LOD) down to 65 fM. Real viral particles in urine and saliva are also successfully detected at concentrations as low as 5 copies mL⁻¹ without DNA amplification, using a portable colorimeter—representing the lowest LOD among current MPXV DNA sensors. The fmPAD, combined with portable readout technology, enables remote, real-time analysis, offering a practical tool for monitoring and containing the spread of Clade I mpox.