| As a heavy pollutant,heavy metals have caused damage to the environment that can not be ignored.With the acceleration of the industrialization process,more and more industrial waste water and domestic sewage were discharged into the water.Silver(?)ions,lead(?)ions and mercury(?)ions have been quite common and seriously endangering human health and the environment pollution.In this paper,based on the different specific structures which can be formed by these three heavy metal ions and specific nucleic acid sequences,we developed a highly sensitive and highly selective detection method,which can detect the silver,lead,mercury heavy metal ions in the water and food.The main contents of this thesis are as follows:1.Based on the coordination of Ag(?)ions and cytosine-rich nucleic acid sequences to form cytosine-Ag(?)-cytosine,the coordination of Pb(?)ions and guanine-rich nucleic acid sequences to form G-quadruplex,the coordination of Hg(?)ions and thymidine-rich nucleic acid sequences to form thymidine-Hg(?)-thymidine,we designed a double-labeled fluorescent nucleic acid probe which can detect silver ions,lead ions and mercury ions sim?Ltaneously.In this method,the 5'-end of nucleic acid probe was rich in cytosine and labeled by a fluorescein-based dye hexchloro fluorescein(HEX),it can coordinate with Ag(?)ions to form C-Ag(?)-C specific structure.The 3'-end of nucleic acid probe was rich in thymine and labeled by a fluorescein-based dye Cy5,it can coordinate with Hg(?)ions to form T-Hg(?)-T specific structure.The middle of the nucleic acid sequence was rich in guanine,and in the presence of Pb(?)ions,it can form G-quadruplex structure which has a good quenching performance.Therefore,the probe can detect Ag(?)ions,Pb(?)ions and Hg(?)ions in the sample sim?Ltaneously.2.When Ag(?)ions were detected,firstly,the G-rich nucleic acid sequences in the middle of probe were formed into G-quadruplex after a certain concentration of Pb(?)ions added into the buffer system,and then adding different concentrations of Ag(?)ions to the system,the C-rich nucleic acid sequence at 5'-end was folded to form a hairpin structure in the presence of Ag(?)ions,so that the fluorescent group HEX and G-quadruplex closed to each other and then occurred to varying degrees of fluorescence quenching.From the experimental data,we conducted that there is a good linear relationship between quenching degree of HEX and Ag(?)ions concentration at 1-80 nmol/L,and the detection limit is 47 pmol/L.After optimizing the system,the optimum conditions are as follows:the pH is 7.0,the concentration of sodium ions and Pb(?)ions are 0.4 mol/L and 400 nmol/L,respectively.3.When Hg(?)ions were detected,firstly,the G-rich nucleic acid sequences in the middle of probe were formed into G-quadruplex after a certain concentration of Pb(?)ions added into the buffer system,and then adding different concentrations of Hg(?)ions to the system,the T-rich nucleic acid sequence at 3'-end was folded to form a hairpin structure in the presence of Hg(?)ions,so that the fluorescent group Cy5 and G-quadruplex closed to each other and then occurred to varying degrees of fluorescence quenching.From the experimental data,we conducted that there is a good linear relationship between quenching degree of Cy5 and Hg(?)ions concentration at 1-80 nmol/L,and the detection limit is 99 pmol/L.After optimizing the system,the optimum conditions are as follows:the pH is 7.0,the concentration of sodium ions and Pb(?)ions are 0.4 mol/L and 300 nmol/L,respectively.4.When Pb(?)ions were detected:Method for detecting Pb(?)ions by Ag(?)ions:firstly,the C-rich nucleic acid sequences was folded to form a hairpin structure after a certain concentration of Ag(?)ions added into the buffer system,then adding different concentrations of Pb(?)ions to the system,the G-rich nucleic acid sequence which at the middle of probe was formed into G-quadruplex in the presence of Pb(?)ions,so that the fluorescent group HEX and G-quadruplex closed to each other and then occurred to varying degrees of fluorescence quenching.From the experimental data,we conducted that there is a good linear relationship between quenching degree of HEX and Ag(?)ions concentration at 1-80 nmol/L,and the detection limit is 54 pmol/L.After optimizing the system,the optimum conditions are as follows:the pH is 7.0,the concentration of sodium ions and Ag(?)ions are 0.4mol/L and 400 nmol/L,respectively.Method for detecting Pb(?)ions by Hg(?)ions:firstly,the T-rich nucleic acid sequences was folded to form a hairpin structure after a certain concentration of Hg(?)ions added into the buffer system,then adding different concentrations of Pb(?)ions to the system,the G-rich nucleic acid sequence which at the middle of probe was formed into G-quadruplex in the presence of Pb(?)ions,so that the fluorescent group Cy5 and G-quadruplex closed to each other and then occurred to varying degrees of fluorescence quenching.From the experimental data,we conducted that there is a good linear relationship between quenching degree of Cy5 and Hg(II)ions concentration at 1-80 nmol/L,and the detection limit is 116 pmol/L.After optimizing the system,the optimum conditions are as follows:the pH is 7.0,the concentration of sodium ions and Hg(?)ions are 0.4 mol/L and 400 nmol/L,respectively.5.By examining the sensitivity,selectivity and standard curve,we concluded that,this method has high sensitivity and specificity for the detection of Ag(?)ions,Pb(?)ions and Hg(?)ions,and has strong anti-interference ability to the related anions and cations in the environment.By this method,we tested these three heavy metals content in water samples and life samples which commonly easy to be rich in heavy metal such as bean sprouts,mushrooms and so on.The res?Lts showed that low concentration of Ag(?)ions,Pb(?)ions and Hg(?)ions co?Ld be detected,and the above detection concentration was verified by spiked recovery experiment,the recovery rate is greater than 90%,indicating that the test res?Lts are accurate.Finally,comparing the sensitivity and selectivity of the two methods for detecting Pb(?)ions,it can be concluded that the method by Ag(?)ions detecting Pb(?)ions is more accurate.6.Ag(?)ions,Pb(?)ions,Hg(?)ions can mediate the interaction between fluorescent groups which at both ends of OND fluorescent probe and G-quadruplex,which res?Lting in fluorescence quenching.We studied the quenching mechanism by using ?Ltraviolet absorption spectrum and thermodynamic method.The res?Lts showed that the quenching types of the two fluorescent groups interact with the G-quadruplex are static quenching,the binding ratio of the fluorescent group bound to the G-quadruple is 1:1,and the larger binding constant proves that the combination between the G-quadruplex and fluorescent groups is very stable.Finally,the thermodynamic parameters indicated that these three metal ions-mediated reactions are spontaneous and the force types are van der waals force and hydrogen bond interaction. |
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