Triple-amplification electrochemiluminescence aptasensor integrating single-atom nanozyme catalysis with CRISPR-Cas12a/HCR cascade for zearalenone detection.

Single-atom nanozymes (SANs) are emerging as interfacial catalysts that can modulate surface-confined reactive oxygen species (ROS) generation at the electrode/solution boundary. Herein, an interface-driven electrochemiluminescence (ECL) aptasensor was constructed for the ultrasensitive detection of zearalenone (ZEN), a mycotoxin of significant concern. Oxidase-mimetic Co-N/C SANs (Co-SAC@NC) immobilized on a glassy carbon electrode catalyze the reduction of dissolved O2 to ·OH and O2·⁻, followed by in-situ generation of ROS, producing a 13-fold enhancement of luminol ECL without the addition of external H2O2. A ferrocene-labelled reporter DNA (Fc-DNA) tethered to the Co-SAC@NC surface quenches luminol ECL by trapping holes at the electrode interface; upon target binding, a magnetic-bead-supported HCR-CRISPR-Cas12a cascade is triggered, trans-cleaving the Fc-DNA and thus restoring the native ECL intensity. The concentration-dependent interfacial cleavage affords a linear range of 0.3-200 ng/mL and a LOD of 0.087 ng/mL (S/N = 3) for the determination of ZEN. This work establishes a modular interfacial amplification platform with potential for generalization by marrying SANs catalysis with a CRISPR-Cas12a/HCR nucleic acid cascade for advanced ECL bioanalysis.