Paper Microfluidic Platform Using Multiplexed Isothermal Amplification and CRISPR/Cas12a for Aquatic Pathogen Detection

The global health threat posed by microbial contamination of aquatic systems demands feasible pathogen monitoring solutions. However, current detection methods are limited by expensive instrumentation and specialized personnel, which hinders their application in point-of-care testing (POCT). Here, we presented an integrated paper microfluidic platform for spatially multiplexed detection of pathogenic bacteria, including Salmonella, E. coli, C. perfringens, B. cereus, V. parahaemolyticus, S. aureus, and L. monocytogenes, selected due to their epidemiological significance and regulatory relevance in environmental and food safety monitoring. LAMP, RAA-CRISPR, and RPA-CRISPR assays were housed within physically isolated reaction chambers on two-layer chips. An engineered horseradish peroxidase (HRP) cascade-coupled crRNA modification system with DNA-conjugated labels was designed for colorimetric detection. Operation was enabled by solar-powered and portable hardware for incubation and imaging, coupled with a web application for quantitative analysis. Exceptional analytical performance was demonstrated, achieving an LOD of 1 CFU/mL, a dynamic range of 1-107 CFU/mL, high reproducibility (CV <5%), low batch-to-batch variation (<6%), low cost (£2.5 per test), and scalable integration, with a sample-to-answer time of 60 min. Successful field validation in diverse aquatic environments confirmed its practical feasibility, consistent with gold standard PCR (R2 = 0.98). This platform offers a promising POCT solution for public health protection and epidemic monitoring, particularly in resource-limited settings.