Metallo-Toehold-Activated Catalytic Hairpin Assembly Formation of Three-Way DNAzyme Junctions for Amplified Fluorescent Detection of Hg2+

Because of their irreversible toxicological impacts on the environment and human body, the development of reliable and sensitive Hg²⁺ detection methods with high selectivity is of great significance. On the basis of the substantial signal amplification by metallo-toehold-triggered, catalytic hairpin assembly (CHA) formation of three-way DNAzyme junctions, we have constructed a highly selective and sensitive fluorescent sensing system for the determination of Hg²⁺ in different environmental water samples. The presence of the target Hg²⁺ ions can lead to the generation of T-Hg²⁺-T base mismatched metallo-toeholds, which trigger the catalytic assembly of three split-DNAzyme containing hairpins to form many Mg²⁺-dependent DNAzyme junction structures upon binding to the fluorescently quenched substrate sequences. The Mg²⁺ ions then cyclically cleave the fluorescently quenched substrate sequences of the Mg²⁺-dependent DNAzymes to generate drastically enhanced fluorescent signals for sensitively detecting Hg²⁺ at the low 4.5 pM level. The developed sensing method offers high selectivity toward the target Hg²⁺ over other possible competing metal ions due to the specific T-Hg²⁺-T bridge structure chemistry in the metallo-toehold domain, and reliable detection of spiked Hg²⁺ in environmentally relevant water samples with this method is also verified. Considering the nucleic acid nature of the trigger and assembly sequences, the developed approach thus holds great potentials for designing new enzyme-free signal amplification strategies to achieve highly sensitive determination of different DNA and RNA targets.