A fluorescent biosensor using DNAzyme-mediated signal amplification for on-site ultrasensitive detection of Vibrio parahaemolyticus

Vibrio parahaemolyticus (VP), a leading cause of acute gastroenteritis in humans, is primarily transmitted via seafood consumption, necessitating ultrasensitive on-site detection. This study selected a novel VP DNAzyme to examine the mechanism behind DNAzyme and VP binding via target pull down, mass spectrometric identification, and molecular docking. This provided a feasible strategy for exploring the binding between DNAzymes and macromolecular targets. DNAzymes and rolling circle amplification (RCA) were combined to construct a novel label-free fluorescent biosensor for the sensitive and specific detection of VP in seafood. The ssDNA generated via DNAzyme self-cleavage after binding to the target resulted in the RCA-induced production of a tandem G-quadruplex (G4), which specifically bound to THT to produce a fluorescent signal proportional to the of VP quantity. This cascade amplification approach efficiently captured weak signals in complex food matrices. In optimized conditions, the biosensor demonstrated a linear detection range of 1.86 × 10⁰ to 1.86 × 10⁸ CFU mL⁻¹, while a good linear correlation was established for recognition in shrimp samples with a limit of detection (LOD) of 1.59 CFU mL⁻¹. In conclusion, this study developed a culture-free, extraction-free, highly sensitive biosensor for VP detection, exhibiting broad application potential for recognition in aquatic products.