Programmable Hairpin Argonaute–Nanopore Detection System for Multiplex Mycotoxin Analysis

Food samples are often simultaneously contaminated with multiple small-molecule hazardous substances. However, conventional sensing methods typically detect only a single contaminant due to the lack of accurate and multiplexed signal conversion and output mechanisms. In this work, we propose a versatile sensing platform, "programmable hairpin argonaute-nanopore detection" (PHAND), which integrates <i>Thermus thermophilus</i> Argonaute (<i>Tt</i>Ago)-mediated cleavage and a modular library of DNA hairpin probes to enable multiplex and portable detection of analytes. In the presence of specific targets, guide DNAs are released to activate <i>Tt</i>Ago proteins, which cleave the corresponding hairpin probes and generate distinct nanopore reporters. These reporters produce orthogonal, characteristic current blockade signals when blocking α-hemolysin nanopores. Using the PHAND system, we successfully achieved the simultaneous detection of three major mycotoxins─aflatoxin B₁, ochratoxin A, and zearalenone with high sensitivity and specificity. Validation in spiked real samples demonstrated satisfactory recovery and confirmed the practical applicability of our method. The PHAND system developed here supports portable and multiplexed detection of small-molecule hazardous substances, offering potential for rapid food safety screening.