Entropy-driven DNA circuit coupled with Cas9 trans-cleavage for dual-amplified label-free electrochemical sensing of aflatoxin B1 and lead ions.

Abstract

Sensitive and reliable detection of hazardous contaminants in food and pharmaceutical products is important for public health and quality control. Herein, we developed ECG-Cas9 (Entropy-driven Circuit-assisted G-quadruplex Electrochemical Cas9 sensing) by coupling an entropy-driven DNA circuit (EDC) with Cas9 trans-cleavage and G-quadruplex-based electrochemical signal generation. Recognition of AFB1 or Pb2+ by the corresponding functional nucleic acids initiates the EDC and generates Cas9 activators. The activated Cas9 subsequently cleaves G-quadruplex-containing hairpin probes immobilized on the electrode surface, allowing the released G-quadruplex sequences to bind hemin and generate an enhanced electrochemical response. The combination of EDC amplification and Cas9 trans-cleavage provides dual signal amplification, achieving detection limits of 0.1 pg mL-1 for AFB1 and 0.5 pM for Pb2+. ECG-Cas9 also showed good selectivity toward both targets. Notably, the use of the G-quadruplex/hemin complex as the electrochemical signal reporter avoids additional electroactive labeling and simplifies probe preparation. Finally, the method was applied to the determination of AFB1 and Pb2+ in food and traditional Chinese medicine samples, with satisfactory analytical performance. These results suggest that ECG-Cas9 provides a sensitive and practical approach for contaminant analysis in complex samples.

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