Label-Free Detection of Cu2+ and Hg2+ Ions Using Reconstructed Cu2+-Specific DNAzyme and G-quadruplex DNAzyme

Hui Li,Yuyan Cai ,Qiu-Fen Zhang ,Hao-Jie Xiao ,Xiaoxi Huang ,De‐Ming Kong

doi:10.1371/journal.pone.0073012

Abstract

Label-free metal ion detection methods were developed. To achieve these, a reconstructed Cu2+-specific DNA-cleaving DNAzyme (Cu2+-specific DNAzyme) with an intramolecular stem-loop structure was used. G-quadruplex-forming G-rich sequence(s), linked at the ends of double-helix stem of an intramolecular stem-loop structure, was partly caged in an intramolecular duplex or formed a split G-quadruplex. Cu2+-triggered DNA cleavage at a specific site decreased the stability of the double-helix stem, resulting in the formation or destruction of G-quadruplex DNAzyme that can effectively catalyze the 2,2′-azinobis(3-ethylbenzothiazoline)-6-sulfonic acid (ABTS)-H2O2 reaction. Based on these, two label-free, cost-effective and simple Cu2+ sensors were designed. These two sensors followed different detection modes: ‘turn-on’ and ‘turn-off’. As for the ‘turn-on’ sensor, the intramolecular stem-loop structure ensured a low background signal, and the co-amplification of detection signal by dual DNAzymes (Cu2+-specific DNAzyme and G-quadruplex DNAzyme) provided a high sensitivity. This sensor enabled the selective detection of aqueous Cu2+ with a detection limit of 3.9 nM. Visual detection was possible. Although the ‘turn-off’ sensor gave lower detection sensitivity than the ‘turn-on’ one, the characteristics of cost-effectiveness and ease of operation made it an important implement to reduce the possibility of pseudo-positive or pseudo-negative results. Combining the ability of Hg2+ ion to stabilize T-T base mismatch, above dual DNAzymes-based strategy was further used for Hg2+ sensor design. The proposed sensor allowed the specific detection of Hg2+ ion with a detection of 4.8 nM. Visual detection was also possible.

Highlights

With more and more electronic and industrial waste going to the land, rivers and air, environmental pollution has become one of the most important problems in the world
The used Cu2+-specific DNAzyme is a reconstructed version of the original one [19]. This reconstructed DNAzyme uses two oligonucleotide strands, but one strand exists as a molecular beacon-like intramolecular stem-loop structure, and the cleavage site is embedded in the double-helix stem
The other strand is a polypurine oligonucleotide (PPO), it can hybridize with the loop of MB to form a DNA triple-helix structure, providing a Cu2+ ion binding site and leading to the Cu2+-specific DNA cleavage reaction at a specific site

Summary

Introduction

With more and more electronic and industrial waste going to the land, rivers and air, environmental pollution has become one of the most important problems in the world. Methods based on DNAzymes are extremely important to meet the requirement for simple, specific and accurate detection of heavy metal ions, especially at low concentrations. Several facile fluorescent and colorimetric sensors based on heavy metal ion-specific DNAzymes have been reported [10,11,12,13,14,15,16,17]. The fluorophore/ quencher pair labeled at the two ends of the stem-loop structure can contact closely, conferring the fluorescent sensor with a low background fluorescence signal and high detection sensitivity. This fluorescent sensor needs a dual-labeled fluorescent oligonucleotide probe to produce an efficient fluorescence switch for detection. Combining the stabilizing ability of Hg2+ to T-T mismatches, this reconstructed Cu2+-specific DNAzyme was further used for label-free detection of Hg2+

Results and Discussion

Conclusion

Materials and Methods