TdT cascaded CRISPR/Cas12a integrated MOF-on-MOF nanozyme for ultrasensitive electrochemical detection of acetamiprid.

An electrochemical biosensor was developed for the detection of acetamiprid (ACE) by integrating a terminal deoxynucleotidyl transferase-(TdT)-cascaded CRISPR/Cas12a with a NH₂-MIL-88@PCN-224 nanozyme. ACE triggered the release of a DNA primer, initiating TdT-catalyzed generation of long poly-T strands. These strands activated the trans-cleavage activity of Cas12a, leading to the hydrolysis of phosphate-modified ssDNA on the electrode. Consequently, the adsorption of the nanozyme onto the electrode inversely correlated with the ACE concentration. The nanozyme efficiently catalyzed the oxidation of 3,3’,5,5’-tetramethylbenzidine (TMB), generating a measurable electrochemical signal. This synergistic combination of nucleic acid amplification and nanozyme catalysis achieved exceptional sensitivity, with a wide linear range from 100 fM to 10 nM and a LOD of 9.7 fM. The biosensor demonstrated high specificity, satisfactory stability and reproducibility, and was successfully applied to detect ACE in real food samples, showing excellent consistency (Pearson’s r = 0.99) with HPLC-MS results. This work provides a powerful and reliable platform for monitoring pesticide residues in food.