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<p>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 <i>r</i> = 0.99) with HPLC-MS results. This work provides a powerful and reliable platform for monitoring pesticide residues in food.</p> Graphical Abstract <p></p>

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

  • 期刊论文

  • 发表日期：

    2026年3月28日

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  • Yue Wu,
  • Yuan Li,
  • Hua Zhang,
  • Lun Wu,
  • Yan Wei,
  • Kailang Wu,
  • Yang Yang,
  • Qinhua H. Chen

摘要

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.

Graphical Abstract