Electrochemical aptasensor based on CRISPR/Cas12a-mediated and DNAzyme-assisted cascade dual-enzyme transformation strategy for zearalenone detection

The combination of the CRISPR/Cas system and biosensing is of profound significance in small molecule diagnostics. Herein, we report a cascade dual-enzyme transformation strategy for ultrasensitive analysis of zearalenone (ZEN) by utilizing the CRISPR/Cas12a trans-cleavage activity and DNAzyme amplification. NH2-MnO2/Pd@Au NBs nanocomposites are greatly promising electrode modification material for improved electrode sensing performance. PtPd@Fe3O4 nanocomposites are synthesized as signal label materials to load the electroactive molecule methylene blue (MB). The recognition of ZEN target and aptamer is converted into DNA signal to regulate the trans-cleavage activity of the CRISPR/Cas12a on Mg2+-DNAzyme probes. After that, in the presence of DNAzyme probes and Mg2+, the MB signal changes caused by Mg2+dependent DNAzyme-assisted catalytic cleavage of signal labels on the electrode surface are evaluated. The quantitative detection of ZEN is ultimately achieved. Under optimal conditions, the as-prepared electrochemical aptasensor shows a wide linear detection range of 1 × 10-5 to 10 ng·mL−1, and the detection limit is 6.27 × 10-6 ng·mL−1. Moreover, the constructed aptasensor exhibits satisfactory selectivity, stability and repeatability, which also has a wide application prospect in the real sample analysis.