基于分子信标及核酸染料SYBR Green Ⅰ定量检测土壤中的汞  

Highly Sensitive Fluorescence Quantitative Detection of Mercury in Soil Samples Based on Molecular Beacon and Nucleic Acid Dye SYBR Green Ⅰ

作　　者：翟琨[1,2] 向东山[2] 朱俊[1] 胡红青[1] 

机构地区：[1]华中农业大学资源与环境学院,武汉430070 [2]湖北民族学院化学与环境工程学院,恩施445000

出　　处：《分析化学》2015年第8期1125-1129,共5页Chinese Journal of Analytical Chemistry

基　　金："863"课题(No.2012AA101402);国家自然科学基金地区科学基金项目(No.21465010);国家科技支撑计划(No.2015BAD05B02)资助~~

摘　　要：利用分子信标、单链核酸（ss DNA）及核酸染料SYBR GreenⅠ,通过同步荧光分析法,建立了一种高灵敏、高选择性土壤汞（Hg2＋）的定量检测方法。在该体系中,分子信标的荧光基团设计为荧光胺（FAM）,分子信标的环设计为与ss DNA互补,但有4个T碱基错配的序列。在Hg2＋存在时,分子信标的环与ss DNA通过＂T-Hg2＋-T＂特异性结合形成双链DNA,分子信标的茎被打开,荧光基团与猝灭基团分开,荧光恢复;同时,核酸染料SYBR GreenⅠ与双链DNA作用,SYBR GreenⅠ的荧光信号显著增强。SYBR GreenⅠ与FAM的最大激发波长与最大发射波长都很接近,通过同步荧光分析法检测时,两种荧光染料的荧光峰重叠,荧光信号增强,可实现Hg2＋的高灵敏检测。体系的最优检测条件为：缓冲溶液的p H=8.0,孵育温度为50℃,孵育4 min,缓冲溶液中Na Cl的浓度为30 mmol/L,SYBR Green I工作液的体积为100 0L。在优化条件下,Hg2＋浓度在5×10 10~4.0×10 8mol/L时,SYBR GreenⅠ及FAM总的荧光强度（ΔI,为体系在存在和不存在Hg2＋时的荧光强度之差）与Hg2＋浓度（C）间有良好的线性关系,其拟合的回归方程为ΔI=1.3949C＋19.3596（R2=0.9976）,方法检出限（3σ）为3×10 10mol/L。本方法操作简单、快速、选择性好、灵敏度高、重复性好。A highly sensitive and selective fluorescence method for quantitative detection of mercury in soil was developed based on molecular beacon （ MB）, single-stranded nucleic acid （ssDNA） and nucleic acid dye SYBR Green I by synchronous fluorescence analysis. In this strategy, the fluorophore of MB was 6-carboxyfluorescein group （ FAM）, and the loop of MB was complementary to the ssDNA with four T-T mismatches. In the presence of Hg2＋, the MBs and ssDNA with T-T mismatches formed double-stranded DNA （dsDNA） via T-Hg2＋-T coordination structure, then the fluorophore FAM was separated from the quencher BHQ-1, thus emitted fluorescence. Meanwhile, SYBR Green I bound to dsDNA, so the fluorescence intensity of SYBR Green I was significantly enhanced. When subjected to synchronous fluorescence analysis, the fluorescence peaks of FAM and SYBR Green I overlapped completely, so the fluorescence signal of system could be significantly enhanced. Thus, highly sensitive fluorescence quantitative detection for Hg2＋ could be realized. In this strategy, the optimal conditions for Hg2＋ determination were as follows： buffer solution pH of 8.0, incubation temperature of 50℃, incubation time of 4 min, 30 mmol/L NaCl in buffer solution and the volume of SYBR Green I of 100μL. Under the optimum conditions, the total fluorescence intensity of SYBR Green I and FAM exhibited a good linear dependence on quantity of Hg2＋ in the range of 5.0×10^-10-400×10^-8 mol/L. The regression equation was AI= 1. 3949C＋ 19. 3596 with a correlation coefficient of 0. 9976 （R2） , where AI was the total fluorescence intensity difference in the presence and absence of Hg2＋. The detection limit was 3×10^-10 mol/L （3σ, n = 11 ）. The proposed method exhibited good precision, high selection, simple operation, fast detection speed, low detection limit and high sensitivity. This method was applied to detect mercury ion in soil and a satisfactory recovery of 97.7%- 100.6% was obtained. The detection result was consistent with

关 键 词：汞 分子信标 核酸染料SYBR GreenⅠ 定量检测 土壤 

分 类 号：X833[环境科学与工程—环境工程] O657.3[理学—分析化学]