RCA-based assay using multivalent aptamer magnetic beads for sensitive detection of Pseudomonas aeruginosa

Pseudomonas aeruginosa (P. aeruginosa), an important opportunistic pathogen responsible for severe infections and life-threatening diseases, is an underestimated foodborne threat to food safety and public health. Rapid and accurate monitoring in food matrices is therefore crucial. Consequently, there is a pressing and urgent demand for developing highly sensitive detection methods for this pathogen. Here, we developed a fluorescent aptasensor based on multivalent aptamer-functionalized magnetic beads. The multivalent aptamers were in-situ synthesized on the surface of magnetic beads though rolling circle amplification technology. The capture ability of multivalent aptamers toward P.aeruginosa was significantly enhanced due to the synergistic effect. The carboxyfluorescein-labeled complementary sequences were hybridized with the multivalent aptamers to form a sensing platform. In the presence of P. aeruginosa, it competitively bound to the aptamers, triggering the dissociation of the complementary sequences. After magnetic separation, the fluorescence intensity of the supernatant was enhanced, enabling the quantitative detection of P. aeruginosa. By integrating multivalent aptamers and magnetic separation technology, the aptasensor exhibited a linear range of 101 – 107 CFU/mL for P. aeruginosa, with a limit of detection as low as 2 CFU/mL. Furthermore, the aptasensor was successfully applied for the detection of P. aeruginosa in milk samples, with recoveries ranging from 89.33% – 106.35%. These results demonstrate that the aptasensor possesses sufficiently good analytical performance and promising practical application potential.