Detection Of Soil Mercury Based On Fluorescence Spectrophotometric Method

Mercury(Hg) is a harmful metallic element to human body for its cumulative effect. Excessive intake of Hg will cause the lesions of digestive, nervous, urinary, hematopoietic and circulating system. Mercury has become one of the environmental monitoring items required to detect. Therefore, the quantitative detection of Hg is an important issue in order to understand its distribution and pollution potential. Fluorescence spectrophotometry is a powerful technique for mercury detection due to its variety of advantages such as a high sensitivity and selectivity, low detection limit and short analytical time. It has been applied in various fields, such as environmental monitoring, food safety and a variety of biomedical studies. It has provided a very effective method for rapid and accurate quantitative detection of mercury. However, it is still a major issue for quantitative detection of mercury how to further expand the scope of application of fluorescence analysis, and developed a detection method of mercury in soil samples with higher sensitivity and better accuracy. On this basis, the following four aspects were investigated in this thesis.1.A highly sensitive and selective fluorescence method of quantitative detection for mercury in soil was developed based on molecular beacon(MB), single-stranded nucleic acid(ss DNA) and nucleic acid dye Hoechst 33258. In this analytical method, the loop of MB was designed to be a sequence which is complementary to the ss DNA with T-T mismatches, the stem of MB is completely designed as C/G base pairs. In the absence of Hg2+, the interaction between Hoechst 33258 and the MBs was very weak,and the fluorescence signal of Hoechst 33258 was very low. In the presence of Hg2+, the MB and ss DNA with T-T mismatches formed double-stranded DNA(ds DNA) via T-Hg2+-T coordination structure, then Hoechst 33258 bound to hybridized ds DNA, the fluorescence intensity of Hoechst 33258 was significantly enhanced. Thus, a highly sensitive fluorescence quantitative detection method for Hg2+can be established. Under the optimum conditions, the fluorescence intensity of Hoechst 33258 exhibit good linear dependence on quantity of Hg2+ in the range of 4- 400×10-9 mol/L, and the detection limit was 3 ×10-9 mol/L(3σ). This method was applied to detect mercury in soil samples and the average recovery was from 98.3% to 103.3%. In this proposed method, the molecular beacons do not need to be modified, and it had a low detection cost, fast detection speed. However, this proposed method was relatively low sensitivity.2. A highly sensitive and selective fluorescence method of quantitative detection for mercury in soil was developed based on molecular beacon(MB), single-stranded nucleic acid(ss DNA) and nucleic acid dye SYBR Green I by synchronous fluorescence analysis. In this strategy, the fluorophore of MB was designed to be 6-carboxyfluorescein group(FAM), the loop of MB was designed to be a sequence which is complementary to the ss DNA with T-T mismatches. In the presence of Hg2+, the MBs and ss DNA with T-T mismatches formed double-stranded DNA(ds DNA) via T-Hg2+-T coordination structure, then the fluorophore FAM was separated from the quencher BHQ-1, thus the fluorophore emit fluorescence. Meanwhile, SYBR Green I binds to ds DNA, so the fluorescence intensity of SYBR Green I was significantly enhanced. When Hg2+ are detected by synchronous fluorescence analysis, the fluorescence peaks of FAM and SYBR Green I overlap completely, so the fluorescence signal of system will be significantly enhanced. Thus, highly sensitive fluorescence quantitative detection for Hg2+can be realized. Under the optimum conditions, the total fluorescence intensity of SYBR GreenⅠ and FAM exhibit good linear dependence on quantity of Hg2+ in the range of 5- 400×10-10 mol·L-1. The detection limit was 3 ×10-10 mol·L-1(3σ). This proposed method can use the total fluorescence intensity of FAM and SYBR GreenⅠ to realize the quantitative detection of Hg2+ in soil, so the detection sensitivity of the analytic method can be greatly improved, and the detection limit can be greatly reduced.3. When the quantitative analysis of Hg2+ in soil is developed by single color fluorescence method, the system are likely to produce false-positive signals once introduced into the nucleic acids that can interact with the probe. On this basis, a dual color fluorescence quantitative detection method for Hg2+ in soil was developed by synchronous fluorescence analysis based on graphene oxide(GO) and dye-labeled nucleic acids. In this strategy, two complementary dye-labeled single-stranded nucleic acids(ss DNAs) with thymine-thymine(T-T) mismatches and GO were employed. Two dyes of 6-carboxyfluorescein group(FAM) and 6-carboxy-x-rhodamine(ROX) were labeled on 3′ end of two different ss DNAs, respectively. In the absence of Hg2+, dye-labeled ss DNAs were adsorbed on GO, then the fluorescence of dyes was quenched by GO and the fluorescence signals were very weak. But in the presence of Hg2+, two complementary ss DNAs with T-T mismatches formed double-stranded DNA(ds DNA) via T-Hg2+-T coordination structure. The formation of ds DNA resulted in the release of dye-labeled ss DNA from GO, and the fluorescence effects of dyes were restored. The wavelength intervals between maximum excitation and emission of FAM and ROX are very close, so the fluorescence signals of two dyes can be obtained simultaneously using synchronous fluorescence analysis. Thus, dual color fluorescence quantitative detection for Hg2+ can be realized by simultaneously measuring fluorescence signals of FAM and ROX, respectively. Under the optimum conditions, the total fluorescence intensity of FAM and ROX exhibited a good linear dependence on Hg2+ concentration in the range from 8×10-10 ~ 8×10-8 mol·L-1, and the detection limit(3σ) of Hg2+ was estimated to be 5×10-10 mol·L-1. The method can recognize the false-positive signals that the probe interact with the nucleic acids, and the selection of quantification detection for Hg2+ in soil can be significant improved.4.A highly sensitive and selective dual color fluorescence quantitative detection method for mercury in soil was developed by synchronous scanning ?uorescence spectrometry. In this method, two complementary single-stranded nucleic acids with T-T mismatches, SYBR GreenⅠ and graphene oxide were employed. In the absence of Hg2+, two ss DNAs cannot form double-stranded DNA beause the two single-stranded nucleic acids have many T-T mismatches, and the interaction between SYBR GreenⅠand the ss DNAs were very weak,and the fluorescence signal of SYBR Green was very low. At the same time, the ROX-labeled ss DNAs were adsorbed on GO, then the fluorescence of ROX was quenched by GO and the fluorescence signals were also very weak. But in the presence of Hg2+, two complementary ss DNAs with T-T mismatches formed double-stranded DNA(ds DNA) via T-Hg2+-T coordination structure. The formation of ds DNA resulted in the release of ROX-labeled ss DNA from GO, and the fluorescence effects of ROX were restored. In addition, SYBR GreenⅠbound to hybridized ds DNA, the fluorescence intensity of SYBR GreenⅠwas significantly enhanced. Under the optimum conditions, the total fluorescence intensity of SYBR GreenⅠ and ROX exhibit good linear dependence on quantity of Hg2+ in the range of 5-500×10-10 mol·L-1, and the detection limit was 2 ×10-10 mol·L-1(3σ). This proposed method not only can recognize the false-positive signals that the probe interact with the nucleic acids, but also can use the total fluorescence intensity of FAM and SYBR GreenⅠ to realize the quantitative detection of Hg2+ in soil, so the detection sensitivity of the analytic method can be greatly improved, and the detection limit can be greatly reduced.The four methods are all successfully achieved the detection of actual soil samples. The main advantage of the first quantitative detection method is low detection cost, it can be applied to the analysis of high mercury levels in soil samples. The main advantages of the second quantitative detection method are simple operation, low detection limit and high sensitivity, it can be applied to analysis for trace mercury in the soil. The main advantage of the third quantitative detection method is that the method can recognize the false-positive signals that the probe interact with the nucleic acids, and the selection of quantification detection for Hg2+ in soil can be significant improved. It can be applied to analysis for medium mercury content in the soil. The fourth detection method not only can recognize the false-positive signals that the probe interact with the nucleic acids, but also can use the total fluorescence intensity of FAM and SYBR GreenⅠ to realize the quantitative detection of Hg2+ in soil, so the detection sensitivity of the analytic method can be greatly improved, and the detection limit can be greatly reduced. It can be applied to analysis for low mercury content in the soil.