A Novel Aptamer Selection Strategy for Pseudomonas aeruginosa and Its Application as a Detecting Probe in a Hybrid Lateral Flow Assay

<i>Pseudomonas aeruginosa</i> is a clinically significant pathogen with high antibiotic resistance, necessitating rapid and reliable diagnostic methods. In this study, we developed a whole-cell aptamer selection method for <i>P. aeruginosa</i> using an Eppendorf-tube-based SELEX system, where bacterial cells were directly incubated with an ssDNA library. This configuration enhanced the recovery of bound aptamers and overcame the cell quantity limitations often encountered in microtiter-plate-based SELEX. After 10 selection rounds, six aptamer candidates were obtained and evaluated for affinity. Molecular docking analysis revealed that aptamer T1 possessed the highest target selectivity. To demonstrate diagnostic applicability, aptamer T1 was integrated into a hybrid lateral flow immunoassay (LFIA), replacing the conventional detection antibody. In this format, the AuNP-aptamer complex bound to the target bacteria and was captured by a specific antibody immobilized on the test line. The LFIA achieved a visual detection limit of 2.34 × 10² CFU/mL within 15 min, showing high specificity and suitability for point-of-care applications. This study presents the first demonstration of an aptamer-antibody hybrid LFIA for bacterial detection and highlights the potential of aptamers as low-cost, rapidly synthesized recognition elements adaptable for the detection of other infectious agents.