A novel fluorescent biosensor for sensitive detection of pathogenic bacteria based on catalytic hairpin assembly and λ-Exonuclease cleavage reaction

The present study describes the development of a fluorescent biosensor for the identification of the pathogenic bacteria Escherichia coli O157:H7. The biosensor is based on a catalytic hairpin assembly (CHA) and λ-Exonuclease cleavage reaction, which enables rapid and precise detection. This approach uses a hairpin H0 that is intended to open when it binds to the target pathogenic bacterium. When the hairpin H0 was opened, the activator domain within the hairpin H0 stem region was revealed, triggering numerous CHA cycles, generating many H1/H2 double-stranded DNAs. λ-Exonuclease cleaved the 5′-phosphorylated H2 strand in the H1/H2 double-stranded DNA to release single-stranded DNA H1, which hybridized with another hairpin H2 to start a new cycle. In both amplification reactions, a significant quantity of hairpin H2 was opened and cleaved, resulting in a fluorescent signature that could be watched in real time. By employing this innovative design philosophy, we were able to successfully identify Escherichia coli O157:H7 within a broad range of concentrations (101-108 CFU/mL), with a detection limit of 10 CFU/mL. This strategy's useful diagnostic ability was also effectively proven for identifying other target pathogenic bacteria by logically redesigning the hairpin H0.