Dual-mode biosensor with multiple signal amplification strategy for detection of Pseudomonas aeruginosa

Pseudomonas aeruginosa (P.aeruginosa) is a prominent pathogenic bacteria in hospital-acquired infections, often leading to serious conditions such as bacteremia, sepsis, and fatal outcomes. The lengthy clinical diagnostic processes pose a challenge in quickly addressing these infections. Thus, the development of rapid and accurate detection methods is critical. In this study, we introduce a novel fluorescent-electrochemical biosensor that utilizes HCR for nucleic acid signal amplification, the high sensitivity of fluorescent nanomaterials, the efficient trans-cleaver activity of CRISPR-Cas12a, and a DNA tetrahedron (Td) containing methylene blue (MB). This sensor design involves immobilizing three aptamers on the electrode surface with Td support, improving P. aeruginosa detection. The presence of P.aeruginosa triggers the complementary chain of the aptamer (P1), which then opens the H1 hairpin, initiating a hybridization chain reaction (HCR) between H2-IRMOF3 and H3, marking the first signal amplification. The HCR product then activates the trans-cleavage activity of CRISPR-Cas12a, releasing Td-P1-MB from the electrode surface for a second signal amplification. The IRMOF3 fluorescence and MB electrochemical signals were quantified and analyzed. We optimized the assay conditions, evaluated its sensitivity, stability, and linear range, and compared it to traditional clinical methods. The biosensor exhibited a fluorescence detection range for P. aeruginosa from 4.4 × 102 to 109 CFU/mL, with a detection limit of 14 CFU/mL, and an electrochemical detection range from 4.4 × 101 to 105 CFU/mL, with a detection limit of 5 CFU/mL. This method shows high sensitivity and a wide detection range for quantifying P.aeruginosa, demonstrating significant potential for medical testing applications.