Dual-mode CRISPR-Cas12a assisted aptasensor enabling quantifiable and naked-eye detection of Aflatoxin M1

Detecting Aflatoxin M1 (AFM1) in milk is vital due to its toxicity and health risks. This study presents a dual-mode (FRET + colorimetric) CRISPR-Cas12a aptasensor using fluorescent silica magnetic nanohybrids (FSMN) as fluorescence donors and gold nanoparticles (AuNPs) as quenchers. In the absence of AFM1, activated Cas12a cleaves ssDNA on FSMN, preventing their adsorption onto AuNPs, thus retaining fluorescence and inducing AuNP aggregation. Upon AFM1 binding, aptamer-target interaction reduces Cas12a activity, allowing intact FSMN to adsorb onto AuNPs, quenching fluorescence via FRET and inhibiting AuNP aggregation. The sensor demonstrated a linear response from 0.1 to 100 ng/mL, with detection limits of 0.20 ng/mL (fluorescence) and 0.31 ng/mL (colorimetric). Recovery rates in spiked milk samples ranged from 94.2 % to 104.5 %, which showed excellent agreement with results obtained using a commercial ELISA kit, confirming the method’s reliability. The entire assay completed within 1 h, offering a rapid naked-eye colorimetric response along with quantifiable fluorescence confirmation, providing a reliable and field-deployable platform for AFM1 monitoring in food safety applications.