Sensitive detection of zearalenone utilizing hydrogel-based sensor with signal enhancement by RCA-assisted CRISPR/Cas12a system

Zearalenone (ZEN) is a mycotoxin that poses severe health risks to humans and livestock through contaminated cereals and feed. In this study, a hydrogel-based sensor was developed for sensitive visual detection of ZEN. The sensor integrates a DNA aptamer-crosslinked polyacrylamide hydrogel encapsulating gold nanoparticles (AuNPs) with a rolling circle amplification (RCA)-assisted CRISPR/Cas12a system for signal enhancement. Upon ZEN binding, the released complementary DNA triggers RCA, generating repetitive DNA strands that activate Cas12a. The activated Cas12a efficiently cleaves numerous single-stranded DNA crosslinkers within the hydrogel, leading to substantial dissociation and release of AuNPs for colorimetric readout. This strategy achieves high specificity and a significantly improved detection limit of 1.5 nM for ZEN, which is over 3000-fold lower than that of the aptamer-hydrogel system without amplification. The sensor enables rapid, simple, and cost-effective on-site quantification of ZEN, with the detection sensitivity and linear range well suited for food safety monitoring applications.