CRISPR/Cas12a-assisted electrochemiluminescent detection of ochratoxin A based on COF@Ru coupled with a DNA tetrahedral scaffold †

Abstract

To sensitively detect ochratoxin A (OTA), a CRISPR/Cas12a-assisted electrochemiluminescence (ECL) aptasensor based on COF@Ru coupled with a DNA tetrahedral scaffold (DTS) was successfully fabricated. The covalent organic framework (COF) acted as a confined micro-reactor for the tris(2,2′-bipyridyl)ruthenium(II)/tri-n-propylamine (Ru(bpy)₃²⁺/TPrA) system and hence, evidently enhanced ECL signals. Au nanoparticles (AuNPs) coupled with DTS caused more ferrocene (Fc)-modified quenching DNA probe molecules to be attached to the surface of the sensing electrode, which could reduce the detection background. Activated CRISPR/Cas12a could effectively amplify the ECL signal since it could cut off a considerable amount of quenching DNA probe molecules and make them move away from the electrode. Owing to these strategies, the fabricated ECL aptasensor could detect OTA as low as 3.5 fg mL⁻¹ in a linear detection range of 10⁻⁵–100 ng mL⁻¹. Furthermore, the CRISPR/Cas12a and COF@Ru coupled with DTS-based ECL biosensor possessed high stability and specificity. More importantly, the ECL biosensor could effectively detect OTA in food samples, indicating that it can play a role in monitoring OTA levels in the field of food safety.