DNAzyme triggered HCR/CRISPR-Cas12a cascade with dual-mode readout for ultrasensitive Pb2+ detection

Pb 2+ contamination in water and food chains poses severe public health risks due to its non-biodegradable and bioaccumulative nature. Long-term low-dose lead exposure triggers irreversible neurological injuries, especially cognitive impairment in children. Although the World Health Organization recommends a 10 μg/L limit for lead in drinking water, conventional ICP-MS requires expensive, bulky equipment and complex pretreatment, which is incompatible with rapid on-site trace lead screening. Herein, we developed a dual-mode cascade biosensor integrating the Pb 2+ -specific GR-5 DNAzyme, preassembled linear scaffold and CRISPR-Cas12a trans-cleavage for ultrasensitive quantitative and naked-eye visual Pb 2+ detection. Preassembled linear scaffolds immobilized on magnetic beads simplify the workflow and suppress background signals. Pb 2+ induces DNAzyme cleavage to release scaffold-derived DNA activators after magnetic separation and activate LbCas12a to achieve multistage signal amplification. The fluorescence channel achieves an LOD of 0.85 pM with a dynamic range of 1 pM-100 nM, while integrated, low-cost lateral flow strips support instrument-free field testing. Spiked tap-water and milk samples yielded recoveries of 97.9–107.5% (RSD < 5%), demonstrating strong anti-interference performance. Analysis of 16 real environmental water samples showed close agreement with ICP-MS (R 2 = 0.971). This facile dual-signal CRISPR-DNAzyme platform enables ultrasensitive trace Pb 2+ detection and expands CRISPR biosensing for heavy metal pollution control and food safety monitoring.