Nanoarchitectonics of bacteriophage-based click fluorescent biosensing platform for detection of Pseudomonas...

Pseudomonas fluorescens (P. fluorescens) is a typical psychrotrophic bacterium responsible for raw milk spoilage and potential human infections, highlighting the need for rapid and reliable detection. In this study, we developed a clickase-based fluorescent biosensing platform for the sensitive and specific detection of P. fluorescens. Herein, a novel copper(I)/Cys-RGD nanocatalyst (namely CCRN) with excellent CuAAC reaction catalytic activity was developed, and the prepared CCRN was functionalized with a P. fluorescens-targeting bacteriophage to form a specific recognition probe (CCRN@Phage). Remarkably, integrating the CCRN@Phage probe with ConA@Fe₃O₄ enabled magnetic enrichment and bioorthogonal catalytic signal amplification for sensitive and specific detection of P. fluorescens without external reducing agents. Specifically, P. fluorescens was first enriched using ConA@Fe₃O₄ and then captured by the CCRN@Phage probe to form a sandwich complex. Upon magnetic isolation, the complex catalyzed a copper(I)-catalyzed azide-alkyne cycloaddition (CuAAC) reaction between 3-azido-7-hydroxycoumarin (azide 1) and 3-butyn-1-ol (alkyne 2), generating a fluorescent triazole product. The CCRN catalyst exhibited excellent stability and catalytic activity, functioning efficiently without dissolution or external reducing agents. The developed method for detection of P. fluorescens had a limit of detection (LOD) of 1 CFU/mL in a linear response from 10² to 10⁷ CFU/mL. This strategy was successfully applied to food samples, showing a great potential for practical applications in food safety monitoring.