RPA-CRISPR/Cas12a and Aptamer-Based Electrochemical Biosensing Duplex Platform for On-Site Monitoring of Antimicrobial Resistance in Water

Abstract Monitoring antimicrobial resistance (AMR) in aquatic systems is essential for tracking resistance mechanisms and identifying microbial sources. Here, we present a dual-module electrochemical sensing platform that integrates a gold-electrode-based RPA–CRISPR/Cas12a biosensor for detecting antibiotic resistance genes (ARGs) with a paper-based electrochemical aptasensor for antibiotic quantification. The system employs thiol-modified CRISPR/Cas12a probes alongside amino-functionalized graphene/thionine/gold nanoparticles (NH2-G/THI/AuNPs) nanocomposites, enabling highly sensitive and specific detection (Limits of detection: 1 copy μL–1 for ARGs and 1 nM for antibiotics). Validation was performed in drinking water, wastewater, and river water samples, demonstrating a rapid sample-to-result time of ∼60 min. Coupled with a portable electrochemical workstation, the platform allows users to introduce samples and acquire electrochemical signals with a smartphone application, giving quantitative outputs displayed through a custom web interface. Field validation showed high stability and recovery rates (95–110%) for antibiotic detection. We believe that this platform addresses critical gaps in aquatic AMR monitoring by providing a rapid, affordable, and field-deployable solution, with the potential for offering on-site surveillance in resource-limited settings.