Dual-trigger enhanced catalytic hairpin assembly for rapid and sensitive colorimetric detection of Pb2+ using a GR–5 DNAzyme-mediated cascade reaction

Lead ion (Pb 2+ ) is a highly toxic heavy metal with strong bioaccumulation potential, posing serious risks to environmental and food safety. In this work, we developed a dual-trigger enhanced catalytic hairpin assembly (CHA) strategy for rapid and sensitive colorimetric detection of Pb 2+ using a GR–5 DNAzyme-mediated cascade reaction. A dumbbell-shaped hairpin probe was designed to form a dimeric complex through complementary palindrome sequences of H1, with its loop region containing a ribonucleotide adenosine (rA) site. In the presence of Pb 2+ , the GR–5 DNAzyme catalyzed cleavage at the rA site, generating a new dimeric complex that efficiently initiated the CHA reaction. This dual-trigger design significantly accelerated the CHA process through the localized high-concentration trigger sequence. Consequently, an intact G-quadruplex sequence was exposed, which produced a distinct colorimetric signal. The biosensor enables accurate quantification of Pb 2+ over the linear range of 0.5–100 nM, with a detection limit as low as 51.25 pM. Spiked recovery rates in drinking water and fish samples range from 96.61 % to 109.77 %, demonstrating the biosensor's accuracy and practical applicability. These results highlight the potential of this biosensor for real-time monitoring of Pb 2+ contamination in environmental and food samples, contributing to improved public health and safety.