Analytical Sciences

Highly Sensitive Fluorescence Quantitative Detection of Mercury in Soil Based on Non-labeled Molecular Beacon and Fluorescent Dye Hoechst 33258

Dongshan XIANG,*,** Kun ZHAI,** Qiuzhang SANG,** Boan SHI,** and Xiaohong YANG**

*Key Laboratory of Biologic Resources Protection and Utilization of Hubei Province, Hubei University for Nationalities, Enshi 445000, China
**School of Chemical and Environmental Engineering, Hubei University for Nationalities, Enshi 445000, China

A highly sensitive and selective fluorescence method of quantitative detection for mercury in soil was developed using non-labeled molecular beacon (MB), single-stranded nucleic acid (ssDNA) and fluorescent dye Hoechst 33258. In this analytical method, the loop of MB was designed to be a sequence that was complementary to the ssDNA with multiple T-T mismatches, the stem of MB was completely designed as C-G base pairs, and both ends of the MB are not modified by any fluorophore and quencher. In the absence of Hg²⁺, the interaction between Hoechst 33258 and the MB was very weak, and the fluorescence signal of Hoechst 33258 was very low. In the presence of Hg²⁺, the MB and ssDNA with multiple T-T mismatches formed a double-stranded nucleic acid (dsDNA) via the T-Hg²⁺-T coordination structure which provided binding sites for Hoechst 33258. Then Hoechst 33258 binded to A-T base pairs of dsDNA, and the fluorescence intensity of Hoechst 33258 was significantly enhanced. Thus, a highly sensitive fluorescence quantitative detection method for Hg²⁺ can be realized. In this strategy, the optimal determination conditions for Hg²⁺ were a buffer solution pH of 8.2, an incubated temperature of 50°C, an incubated time of 5 min and NaCl of 60 mmol L⁻¹. Under the optimum conditions, the fluorescence intensity of Hoechst 33258 exhibited a good linear dependence on the concentration of Hg²⁺ in the range of 5 × 10⁻⁹ – 400 × 10⁻⁹ mol L⁻¹. The fitted regression equation was ΔI = 2.1084C – 8.9587 with a correlation coefficient of 0.9943 (R²), and the detection limit of this method was 3 × 10⁻⁹ mol L⁻¹ (3σ). The proposed method had a high selection; the common substances in soil such as Ca²⁺, Mg²⁺, Mn²⁺, Fe³⁺, Cu²⁺, Pb²⁺, Al³⁺, K⁺, Na⁺, Ni²⁺, Cd²⁺, Cr³⁺, SiO₃²⁻, Cl⁻, PO₄³⁻, NH₄⁺ and S²⁻ had no interference to the detection of mercury. The proposed method had a high accuracy, and it was applied to detect mercury of ten different types of soil; the recoveries were 97.65 – 103.22%. In addition, the proposed method had a low background emission, fast detection speed and low detection cost.

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