Signal-on fluorescent sensing strategy for Pb²⁺ detection based on 8–17 DNAzyme-mediated molecular beacon-type catalytic hairpin assembly circuit

Abstract

Based on a Pb²⁺-specific 8–17 DNAzyme-induced catalytic hairpin assembly (CHA), a simple signal-on fluorescence strategy for lead ion detection was established. 8–17 DNAzyme was used as the recognition element of Pb²⁺, which catalyzed the cleavage of the RNA base embedded in the DNA substrate strand, while releasing part of the substrate strand (S’) as CHA initiator. And two hairpin probes (H1 and H2-FQ) were designed according to the sequence of S’ for CHA, in which H2-FQ was labeled with the fluorophore FAM and quencher BHQ-1 as fluorescent “molecular switch” based on fluorescence resonance energy transfer (FRET). In the presence of Pb²⁺, the CHA reaction was triggered to form a large number of H1-H2 complexes, enabling enzyme-free isothermal amplification and a signal-on fluorescence strategy. In the concentration range of 0.5–1000 nM, the fluorescence signal increases with the increase of Pb²⁺ concentration. The quantitative detection limit of Pb²⁺ by this method is 0.5 nM, which has better detection performance compared with the FQ-labeled 8–17 DNAzyme method. The established biosensor exhibits good specificity and can be effectively used for the detection of Pb²⁺ in real samples of river water and grass carp. Through ingenious nucleic acid sequence design, DNAzyme and CHA reactions are integrated to realize the enzyme-free isothermal amplifications and sensitive detection of Pb²⁺, which holds potential versatility in food supervision and environmental monitoring.

Graphical abstract