DNAzyme-CRISPR driven dual-mode biosensor with nanozyme signal amplification for on-site Pb²⁺ detection

Lead ion (Pb²⁺) is a toxic heavy metal that poses severe threats to food safety. Traditional methods like inductively coupled plasma mass spectrometry (ICP-MS) and atomic absorption spectrometry (AAS) rely on bulky instrumentation, which are limited by high costs, complex sample preparation requirements, and inability to meet the demands for rapid on-site testing. Nanozyme-based biosensors have emerged as promising alternatives, yet single-mode sensors often suffer from matrix interference in complex food samples. Therefore, developing a rapid and reliable on-site method is critical for Pb²⁺ detection.

To address this challenge, a CRISPR/Cas12a-driven dual-mode biosensor integrating Pt/CeO₂ nanozyme-mediated peroxidase activity with GR-5 DNAzyme recognition was developed. The biosensor's design capitalizes on GR-5 DNAzyme for Pb²⁺-specific recognition, triggering CRISPR/Cas12a-mediated cleavage of electrochemical/colorimetric signal probe SH-ssDNA-Pt/CeO₂ to generate dual signals. This innovative platform synergizes electrochemical precision and colorimetric simplicity for Pb²⁺ detection in complex food matrices. The biosensor achieved an ultra-sensitive electrochemical response (linear range: 0.002-200 nM; limit of detection: 0.14 pM) alongside a robust colorimetric readout (linear range: 0.5-2000 nM; limit of detection: 0.47 nM), representing significant sensitivity improvements over conventional single-mode sensors. Crucially, the intrinsic cross-verification mechanism between orthogonal signal modalities minimized false positives while ensuring >90.5 % recovery in spiked corn, edible oil, beef and red wine samples with RSD <5 %. These results highlight the potential of the dual-mode sensor as a practical, field deployable sensing platform for the detection of Pb²⁺.

This work innovatively integrates the specificity of CRISPR with the catalytic properties of nanozymes into a self-validating electrochemical/colorimetric dual-mode system. The detection method not only establishes a robust platform for highly sensitive, reliable and visualized detection of Pb²⁺ in complex food matrices, but also pioneers a new paradigm for multi-modal biosensor design.