CRISPR-responsive hybrid MOF assembling assay for rapid electrochemical detection of domoic acid

Domoic acid (DA), a potent neurotoxin, poses a serious threat to food safety and public health. Traditional detection methods often struggle to balance sensitivity with practical applicability. To address this, we developed an electrochemical biosensor based on a CRISPR-responsive hybrid metal-organic framework (MOF) assembling assay. This platform employs a novel negative feedback mechanism where the DA aptamer itself serves as the activator for CRISPR/Cas12a. Target binding inhibits Cas12a's trans-cleavage activity, thereby protecting the signal reporter from cleavage and enabling significant signal amplification. The system integrates dual signal amplification by using rolling circle amplification (RCA)-generated DNA nanowires as scaffolds on magnetic beads and UiO-66 MOFs loaded with abundant methylene blue (MB) molecules as efficient signal tags. This synergistic strategy effectively enhances the signal response while minimizing background noise. When coupled with portable paper-based electrodes for differential pulse voltammetry (DPV) readout, the biosensor achieved highly sensitive DA detection with a low limit of detection. Furthermore, it exhibited robust universality and specificity by successfully detecting saxitoxin (STX) in complex matrices through simple aptamer substitution, demonstrating its modular design advantage. This work not only provides a reliable and practical solution for on-site DA monitoring but also establishes a versatile and powerful biosensing platform with significant potential for point-of-care testing of a broad spectrum of hazardous substances.