Electrochemical DNA Biosensors Based On Ruthenium Complex Probes And Their Applications In Environmental Toxicology

In this paper [Ru（bpy）2dppz]²⁺was chosen as the DNA probe molecules, whichhas good electrochemical properties and photoelectrochemical properties. So weconstruct an electrochemical DNA biosensor which has special response forpolycyclic organic pesticide molecules, and used to investigate the interactionbetween polycyclic organic pesticide molecules and natural double-stranded DNA（ds-DNA）. The main contents are listed as follows:1. The interaction between [Ru（bpy）2dppz]²⁺and DNA: The rutheniumcomplexes containing dppz （dipyrido[3,2-a:2′,3′-c]-phenazine） as a ligand has beenprepared. The [Ru（bpy）2dppz]²⁺（bpy=2,2-bipyridine, dppz=dipyrido [3,2-a:2′,3′-c]phenazine） complex was characterized by UV-vis,1HNMR, fluorescence spectra andelectrochemical methods and was found to exhibit the expected propeller-like strcturewith signigicant intermolecular π-stacking interactions. The binding interaction of[Ru（bpy）2dppz]²⁺with calf-thymus deoxyribonucleic acid （DNA） has been studied bysquare wave voltammetry （SWV）. The result showed that metallo-intercalators couldintercalate into the double helix of DNA, and formed a larger volume of"metallo-intercalators-DNA" complex. Because of the slow diffusion speed of thecomplex in the solution, the anodic peak current signal was decreased. The bindingparameters including of the binding constant Kaand the binding site size n, wereobtained simultaneously by the analysis of bound and free [Ru（bpy）2dppz]²⁺concentration corresponding to the limits of slow and fast binding kinetics comparedwith the experimental timescale. The binding constant （Ka） is1.7×10⁵L/mol, and thecalculated binding-site size （n） is0.84base pair, which is consistent with the reportedvalues.2. Electrochemistry of complex formation of carbaryl with ds-DNA using[Ru（bpy）2dppz]²⁺as probe: An electrochemical competition method was used to studythe interaction of carbaryl with ds-DNA. Layer-by-layer films of negatively chargedds-DNA and polycationic poly （diallyldimethylammonium chloride）（PDDA） wereassembled on the surface of a glassy carbon electrode （GCE）. The DNA intercalator[Ru（bpy）2（dppz）]²⁺was chosen as an electrochemical probe. Tripropylamine （TPA）was used as an electron donor to chemically amplify the oxidation current of theprobe. In order to examine the effects of substituting group on the binding interactionof carbaryl with DNA, the interaction of naphthalene or-naphthol with DNA werealso studied by square wave voltammetry. The values of binding constant Kbof threecompounds to DNA are determined, which were in the range of （0.2×10⁵） to （1.3×10⁵）L/mol. The correlation suggests that the functional groups may play an important role in the DNA/analyte competition binding interaction. We demonstrated that it isconducive to the combination of small molecules and DNA when the functionalgroups are hydrophobic and possess the lone-pair electrons as the electron donor.Furthermore, UV-absorption and fluorescence intensity of [Ru（bpy）2（dppz）]²⁺decreases in the presence of carbaryl. These characteristics strongly support theintercalation of carbaryl into ds-DNA.3. Electrochemiluminescence for probing interactions between heterocyclicpolycyclic aromatic hydrocarbon pesticide molecules and double-stranded DNA:Heterocyclic polycyclic aromatic hydrocarbon （heteroPAH） pesticides were widelyused in agriculture production. Very few studies have explored genotoxic ofheteroPAH pesticides, and their association with DNA damage. It was constructed thata solid-state [Ru（bpy）2（dppz）]²⁺electrochemiluminescence （ECL） sensor for studyingthe interaction of heterocyclic polycyclic aromatic hydrocarbon （heteroPAH） pesticidemolecules with ds-DNA. Layer-by-layer films of negatively charged ds-DNA andpolycationic poly（diallyldimethylammonium chloride）（PDDA） were assembled onthe surface of a glassy carbon electrode. We selected a novel transition-metalRu-complex as a true molecular―light switch‖for DNA. So the [Ru（bpy）2（dppz）]²⁺asthe probe was immobilized on the solid surface. Tripropylamine was used as anelectron donor to chemically amplify the ECL intensity of the probe. If the xenobioticmolecules compete with the probe for the same site on the DNA film, it woulddisplace the probe from the DNA binding site to decrease the ECL signal. In thispaper quinalphos, quinclorac and carbensazim were applied to study the interaction ofheteroPAH pesticide molecules with DNA. From the competition displacement curve,the values of binding constant Kbof three pesticides to DNA were determined, whichwas in the range of0.5×10⁴to2.3×10⁴L/mol.