A Novel Single-labeled Fluorescent Oligonucleotide Probe For Silver(?) Ion And Lead(?) Ion Detection

Due to the lead(?) ions and silver (?) ions pollution which caused by widely used of lead(?) ions and silver (?) ions, it has become an adverse factor in environment pollution and damage to human health. In this paper, by using the special interaction between heavy metal ions and nucleic acids, a novel single-labeled fluorescent oligonucleotide (OND) probe based on cytosine-Ag(?)-cytosine coordination and the inherent fluorescence quenching ability of G-quadruplex is designed to detect lead(?) ions and silver (?) ions. The main contents are as follows:1. Based on the inherent fluorescence quenching ability of G-quadruplex and the silver (?) ions can induce the formation of cytosine-Ag(?)-cytosine coordination, we established a detection system to measure the trace silver (?) ions. In this fluorescent OND probe, a G-rich OND was designed to follow a C-rich OND, and a fluorescein-based dye hexachloro fluorescein (HEX), was labeled at the 5'-end of the C-rich OND. Take advantage of this special probes, we can simultaneous determination the Ag (?) and Pb (?) in the solution.2. When the probe was used in the Ag (?) determination, in the presence of silver (?) ions, the C-Ag(?)-C coordination induced the labeled C-rich OND to fold into a hairpin structure which brings the fluorescent termini HEX close to the G-quadruplex, resulting in a significant fluorescence quenching of the fluorescent termini HEX. The experiment results show that silver (?) ions can be detected quantitatively, the linear response range is from 1 to 100 nmol/L with a detection limit of 21 pmol/L. After optimized the detection conditions, we selected 6.0 as the optimum pH, while choose 0.4mol/L and 50nmol/L as the optimum concentration of sodium nitrate and Pb(?) ions respectively.3. When the probe was used in the Pb(?) determination, first adding Ag(?) into the OND solution so as to the OND can form hairpin structure and brings the fluorescent termini HEX close to the C-rich sequence. In the presence of lead(?) ions, the C-rich sequence form of G-quadruplex approach to HEX and leading to a fluorescence quenching. The experiment results show that lead(?) ions can be detected quantitatively, the linear response range is from 1 to 100 nmol/L with a detection limit of 96 pmol/L. After optimized the detection conditions, we selected 6.0 as the optimum pH, while choose 0.4mol/L and 100nmol/L as the optimum concentration of sodium nitrate and Ag(?) ions respectively.4. The detection method was then tested in sensitivity, selectivity and calibration curve. It was show that this method both sensitivity in lead(?) ions and silver (?) ions detection and no an obvious interference occurs with other metal and anion ions. When this method was applied in water samples and some food, such as mushroom and tea, it can also detect the low concentration of Ag(?) and Pb(?) in those biological products. The standard addition recovery experiments verify the test results was accurate and the recoveries are in the range of 90% to 98%.5. The mechanism of the Ag(?) or Pb(?)-mediated interaction between luminophore HEX and G-quadreplex were determined by using ultraviolet absorption spectrum and thermodynamic method. We can know that both the Ag(?) and Pb(?)-mediated quenching effect of the G-quadreplex on HEX belonged to static quenching and the binding constant (Ka) and binding-site number(n) were increased with the increaseing of temperature and the HEX bound to the G-quadreplex at the ratio of 1:1, which accordance with the design of the probe. The thermodynamic parameters including enthalpy change(AH), entropy change (?S) and free energy change(AG) proofed that the binding between the HEX and G-quadreplex was a spontaneous exothermic reaction and lectrostatic interaction were the main force during the interaction.