Argonaute-triggered and enhancement-by-mixing nano-SERS-transduced triple cascade amplification strategy: Transforming ultrasensitive molecular detection of foodborne pathogenic bacteria.

The emergence of diseases attributable to foodborne pathogens poses a significant threat to human health. Pyrococcus furiosus Argonaute (PfAgo), a novel member of programmable nucleases, is repurposed for molecular detection owing to its programmable and sequence-specific nucleic acid cleaving capabilities. In this work, a triple cascade amplification strategy termed as PASS was developed for pathogenic bacteria biosensing. The species-specific invA gene was amplified using loop-mediated isothermal amplification (LAMP) (1st signal amplification). The amplicons were recognized by PfAgo, which triggered targeted and programmable nucleic acid cleavage. Such cleavage resulted in the degradation of a linker sequence that was pre-designed to link AuNPs-based surface-enhanced Raman scattering (SERS) nanoprobes with corresponding magnetic nanoprobes, in which the nucleic acid signal can be transduced and amplified to SERS signals (2nd amplification). It was also discovered that simple physical mixtures of as-made SERS nanoprobes with AgNPs@PVA to form mixing nano-SERS probes, giving rise to a more than 30-fold enhancement of SERS signals of reporting molecule 4-Mercaptobenzoic acid (4-MBA), termed Enhancement-by-Mixing nano-SERS signals. This further amplified the signals (3rd amplification). The PASS strategy achieved single-cell sensitivity (1 CFU/mL) across a broad dynamic range (10-10 CFU/mL) within 60 min. In summary, for the first time, a conceptually novel biosensing strategy was constructed, whose advantages led to the creation of a novel paradigm of molecular diagnostics.