Garland Rolling Circle Amplification Mediated Self-Priming Extension Strategy for Sensitive and Label-Free...

Early and accurate detection of Pseudomonas aeruginosa (P. aeruginosa) is critically important in perioperative care to prevent severe healthcare-associated infections and guide timely antimicrobial intervention. Herein, we report a novel biosensing strategy for sensitive and label-free detection of P. aeruginosa by integrating F23 aptamer-mediated target recognition, garland rolling circle amplification (RCA)-triggered self-priming extension, and SYBR Green I (SG-I)-based fluorescence readout. The capture probe, comprising the F23 aptamer and a primer strand immobilized on magnetic nanoparticles, specifically recognizes P. aeruginosa and releases the primer to initiate dumbbell probe circularization and subsequent RCA. The resulting RCA products are cleaved by a nicking endonuclease to generate fragmented DNA, which then hybridizes with a hairpin probe to prime cyclic self-extension reactions, producing abundant double-stranded DNA for SG-I intercalation and fluorescence enhancement. Under optimized conditions, the proposed method achieves a detection limit as low as 2.3 CFU/mL with a wide linear range from 10 to 10⁶ CFU/mL. The assay exhibits excellent specificity against non-target bacteria, robust stability during storage, and satisfactory anti-interference capability in complex clinical matrices. Validation using clinical samples demonstrates excellent agreement with the gold-standard colony counting method while reducing the assay time to less than 2.5 h without requiring nucleic acid extraction or thermal cycling. With its label-free design, isothermal amplification, and operational simplicity, this strategy holds great promise for point-of-care testing in perioperative settings and can be readily adapted for detecting other pathogens by substituting the corresponding aptamer.