Catalytic Proximate-Hairpin Assembly (CpHA) triggered DNA-templated silver nanoclusters based assay for sensitive bacteria detection

Abstract Pseudomonas aeruginosa ( P. aeruginosa ) is a significant bacterial pathogen implicated in diverse infections and notorious for its antibiotic resistance. Rapid and reliable diagnostic methods are therefore essential for the early detection, treatment, and control of P. aeruginosa infections. This study introduces a straightforward, rapid, sensitive, and label-free fluorescence detection platform for diagnosing P. aeruginosa . The assay integrates Catalytic Proximate-Hairpin Assembly (CpHA) with DNA-templated silver nanoclusters (DNA-AgNCs) to generate signal amplification. Specifically, CpHA enhances signal amplification efficiency, thereby improving the sensitivity of the catalytic hairpin assembly (CHA) approach. The distinctive fluorescence properties of DNA-AgNCs, including stable emission, facile synthesis, and biocompatibility, enable label-free bacterial detection. The designed sensing system successfully detected P. aeruginosa within 1.5 h, comprising a 30 min target recognition phase by the F23 aptamer followed by a 60 min CpHA reaction at 37 °C. This diagnostic approach demonstrated high sensitivity (detection limit of 4.8 CFU/mL) and specificity. Furthermore, by modifying the F23 aptamer component within the detection complex, the method can be adapted for the detection of MRSA. Collectively, these findings indicate the assay provides a reliable tool for detecting P. aeruginosa , suggesting its viability for the early and rapid diagnosis of infections, thus helping prevent widespread transmission in hospital nursing.