A label-free and enzyme-free colorimetric method for sensitive Pb2+ detection based on a GR-5 DNAzyme-triggered CHA-HCR cascade amplification

The severe toxicity of Pb 2+ to human health, even at trace levels, the development of highly sensitive and dependable analytical methods remains of great importance. Here, we developed a label-free and enzyme-free colorimetric method for Pb 2+ assay by integrating a GR-5 DNAzyme recognition event with a catalytic hairpin assembly (CHA)-hybridization chain reaction (HCR) cascade amplification. Upon exposure to Pb 2+ , the GR-5 DNAzyme is specifically activated, catalyzing the site-specific cleavage of hairpin H1 to release the initiator strand T. The liberated T strand subsequently drives the CHA and HCR cascade reactions, generating long DNA superstructures enriched with multiple G-rich sequences. In the presence of hemin, these G-rich sequences fold into catalytically functional G-quadruplex/hemin DNAzymes, which enable a visible colorimetric signal generation. Under optimized conditions, the proposed colorimetric method delivered a linear response over the range of 50 pM to 50 nM and achieved a limit of detection of 16.71 pM. The practical applicability of the method was further demonstrated through Pb 2+ analysis in drinking water and fish samples, where satisfactory accuracy and reliability were obtained. In addition, inductively coupled plasma mass spectrometry was employed to validate the real-sample results, and the two methods showed good consistency. Owing to its simplicity, high sensitivity, and enzyme-free design, this colorimetric method shows strong potential for practical Pb 2+ monitoring in food safety and environmental analysis.