Engineering of a DNAzyme-Based dimeric G-quadruplex rolling circle amplification for robust analysis of lead ion

Detecting heavy metal pollution, particularly lead ion (Pb 2 ⁺) contamination, is imperative for safeguarding public health. In this study, we introduced an innovative approach by integrating DNAzyme with rolling circle amplification (RCA) to propose an amplification sensing method termed DNAzyme-based dimeric-G-quadruplex (dimer-G4) RCA. This sensing approach allows for precise and high-fidelity Pb 2 ⁺ detection. Strategically, in the presence of Pb 2 ⁺, the DNAzyme undergoes substrate strand (S-DNA) cleavage, liberating its enzyme strand (E-DNA) to prime isothermal amplification. This initiates the RCA process, producing numerous dimer-G-Quadruplexes (dimer-G4) as the signal reporting transducers. Compared to conventional strategies using monomeric G-quadruplex (mono-G4) as the reporting transducers, these dimer-G4 structures exhibit significantly enhanced fluorescence when bound with Thioflavin T (ThT), offering superior target signaling ability for even detection of Pb 2 ⁺ at low concentration. Conversely, in the absence of Pb 2 ⁺, the DNAzyme structure remains intact so that no primers can be produced to cause the RCA initiation. This nucleic acid amplification-based Pb 2 ⁺ detection method combing with the high specificity of DNAzymes for Pb 2 ⁺ recognition ensures highly sensitive detection of Pb 2+ with a detection limit of 0.058 nM, providing a robust tool for food safety analysis and environmental monitoring.