Abstract

Vibration-enhanced mixing overcomes mass transfer limitations in paper-based RPA. • Integration of amplification, mixing, and detection in a portable μPAD system. • Rapid (20 min), sensitive detection of Vibrio parahaemolyticus for on-site use. Rapid and field-deployable detection of foodborne pathogens remains a critical challenge for ensuring food safety and public health, particularly in resource-limited settings. Herein, we firstly report a vibration-enhanced recombinase polymerase amplification (RPA) platform based on a foldable multilayer microfluidic paper-based analytical device (μPAD) for on-site detection of Vibrio parahaemolyticus ( V. parahaemolyticus ). Unlike conventional paper-based amplification systems that suffer from diffusion limitations within porous matrices, the proposed platform introduces a low-power micro-vibration module to enhance reagent mixing and improve amplification efficiency. The proposed system integrates pre-stored, air-dried RPA reagents, a portable heating unit with vibration-assisted mixing, and a smartphone-based fluorescence readout module. Isothermal amplification is initiated directly on the paper substrate by simple sample loading, enabling nucleic acid amplification and fluorescence detection within 20 min at 40 °C. The assay achieves a detection limit of 10 2  CFU/mL, comparable to that obtained using a laboratory fluorescence spectrometer. The platform exhibits high specificity and reliable performance in spiked seawater and seafood samples, with results consistent with standard culture-based methods. By addressing mass-transfer limitations in paper-based amplification and enabling system-level integration for portable detection, this platform provides a practical and scalable solution for on-site food safety monitoring. Using portable devices to perform RPA reactions on paper chips enables rapid on-site detection of pathogenic bacteria. Download: Download high-res image (103KB) Download: Download full-size ima