Ultrasensitive and rapid detection of botulinum neurotoxin serotype A via Argonaute-mediated signal cascade with a pregnancy test strip readout.

Rapid and ultrasensitive detection of botulinum neurotoxin serotype A (BoNT/A), the most lethal known toxin, is critical for food safety and clinical diagnosis. Herein, we present a novel visual detection platform integrating a programmable DNA-guided nuclease, mutant Pyrococcus furiosus Argonaute (mPfAgo), with a pregnancy test strip (PTS), collectively termed mPfAgo-PTS. In this design, BoNT/A is first captured by an immunocomplex functionalized with a DNA tag. After amplification, the resulting amplicon directs mPfAgo to cleave a customized probe conjugated to human chorionic gonadotropin (hCG), thereby releasing free hCG for visual readout on the PTS. This cascaded signal transduction and amplification strategy enables the mPfAgo-PTS platform to detect BoNT/A down to 10 fg/mL within 60 min, representing a 1000-fold improvement in sensitivity over the gold-standard mouse bioassay. High precision and sensitivity validate its robustness and versatility for practical applications in food safety and clinical screening.