基于富含胸腺嘧啶(T)DNA适配体对Hg2+的特异性识别和核酸外切酶I(Exo Ⅰ)辅助的信号转换策略,建立了快速检测人尿液中Hg2+的荧光分析方法。固定在微孔板上的DNA适配体特异识别Hg2+后,折叠成稳定的发卡型双链结构,不能被单链特异性的Exo Ⅰ水解,核酸染料SYBR Green I(SG)插入发卡部位产生荧光信号,基于此可实现Hg2+的定量检测。优化后的最佳实验条件为:微孔板包被亲和素浓度为50 mg/L;检测DNA包被浓度为75 nmol/L;使用5×@SG以及1.2μL的Exo Ⅰ。在最佳实验条件下,体系荧光强度与Hg2+浓度的对数呈良好线性关系,线性范围为2-500 nmol/L,检出限为1.5 nmol/L(3σ)。利用本方法检测尿样中的Hg2+,加标回收率为91.2%-95.0%,相对标准偏差(n=5)为2.1%-4.6%。本方法选择性良好,操作简单,用HNO3-KMnO4溶液将尿液中其它形式的汞氧化为Hg2+后,成功实现了尿液中总汞含量的检测。
A novel fluorescence analysis method for fast detection of mercury ions in human urine was established based on DNA recognition and exonuclease I (Exo Ⅰ) assisted signal conversion strategy. The DNA immobilized on the surfaces of the microplates bound specifically with Hg2+ to fold into a stable "hairpin" double-stranded structure, thus avoided the hydrolysis by single-stranded DNA specific Exo Ⅰ. Nucleic acid dye SYBR Green I (SG) could insert into the double-stranded "hairpin" part to produce fluorescence signal emission. Based on the above facts, quantitative detection of mercury ions was achieved. The experimental results showed that, when the concentration of avidin coating solution was 50 mg/L and the concentration of the detection DNA immobilized on the microplate was 75 nmol/L, with 5×SG and 1.2 μL of Exo Ⅰ, the optimal analytical performance could be achieved. A good linear relationship between the fluorescence intensity of the system at 520 nm versus the logarithm of mercury ions concentration in the range of 2-500 nmol/L was obtained under the optimal conditions, and the detection limit was 1.5 nmol/L (3σ). The recoveries of mercury in spiked human urine samples were 96.3%-100.5% with the relative standard derivations (RSDs) of 2.1%-4.6%. The method had good selectivity with simple operation, and could be used for the detection of total mercury content after oxidizing other forms of mercury in urine into mercury ions (Ⅱ) using HNO3-KMnO4 oxidation method.
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