Citation: ZHANG He, WANG Qing, YANG Mei, FU Xin. Exonuclease Ⅲ-assisted Dual-Cycle Isothermal Signal Amplification for Highly Sensitive “Turn-on” Type Detection of Mercury Ion[J]. Chinese Journal of Analytical Chemistry, ;2019, 47(6): 899-906. doi: 10.19756/j.issn.0253-3820.181635 shu

Exonuclease Ⅲ-assisted Dual-Cycle Isothermal Signal Amplification for Highly Sensitive “Turn-on” Type Detection of Mercury Ion

Hunan Province Key Laboratory of Environmental Catalysis & Waste Recycling, School of Chemistry and Chemical Engineering, Hunan Institute of Engineering, Xiangtan 411104, China

Received Date: 11 October 2018

Fund Project: This work was supported by the National Natural Science Foundation of China (No. 21005067), the Natural Science Foundation of Hunan Province, China (No.2019JJ40055) and the Scientific Research Fund of Hunan Provincial Education Department (Nos.17A044, 16B060)

An Hg²⁺-triggered exonuclease Ⅲ-assisted dual-cycle isothermal signal amplification was developed for label-free and ultrasensitive detection of Hg²⁺ using two designed hairpin probes. In the presence of Hg²⁺, the Hg²⁺ discrimination probe could hybridize with the free 3'-terminus of hairpin probe 1 by T-Hg²⁺-T coordination and a blunt 3'-terminus is formed. In this case, Exo Ⅲ could preferentially bind to the blunt 3'-terminus and catalyze the stepwise hydrolysis of mononucleotides in the 3'-to-5' direction. The released Hg²⁺ and Hg²⁺discrimination probe were recycled in a new round of reaction cycle (cycle 1). Undegraded sequence of hairpin probe 1 comprises G-quadruplex-forming sequence and I-region that was completely complementary to the free 3'-terminus of hairpin probe 2 (Ⅰ^* region) to form another blunt 3'-terminus. The new blunt 3'-terminus could trigger Exo Ⅲ-assisted cleavage again, and the I-region was released and recycled in a new round of reaction cycle (cycle 2). Cycles 1 and 2 were spontaneous cycle processes. After multiple cycles, a large number of single-stranded G-quadruplex-forming sequences could be generated. The released G-quadruplex-forming sequences could bind hemin to form stable G-quadruplex-hemin DNAzymes, which could efficiently catalyze ABTS-H₂O₂ system to produce the colored product. Under the optimal experimental conditions, the linear range for detection of Hg²⁺ was 0.3 pmol/L-50 pmol/L with a detection limit of 0.25 pmol/L, and the regression equation was ΔA_{420 nm}=0.117+0.004C_Hg²⁺(pmol/L). This Hg²⁺ sensing system had excellent selectivity for Hg²⁺ against otherpossible competing ions. The fabricated sensor was applied to detection of Hg²⁺ in environmental water samples, and the recoveries were 96.0%-106.0%, respectively. This method was simple with good selectivity, high sensitivity and strong anti-interference ability, and was suitable for the sensitive detection of Hg²⁺ in environmental water samples.
- Isothermal signal amplification,
- Exonuclease Ⅲ,
- G-quadruplex-hemin DNAzyme,
- Mercury ion,
- Colorimetric analysis
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