Application Of Novel Chemically Amplific Electrochemistry Method In DNA Detection And Environmental Analysis

The chemical amplification system developed previously in our lab wasapplied in the quantitative analysis of DNA in solution for the first time. Theassay method is based on the principle that the planar metal complexintercalates into the double helix DNA and leads to reduction in itselectrochemical signal. We also applied the same system in the quantitativeanalysis of an environmental pollution substance, phenanthrene, in acompetitive format. The main contents of our study include:1. Two DNA-intercalating planar metal complex, Fe(phen)3 andRu(bpy)₂dppz were systhesized. Electrochemical behavior of the metalcomplexes was studied using cyclic voltammetry technique before and afterthe complexes intercalated into the DNA double helix. Because the rate of themass transfer of DNA-bound metal complex is slower than the free metalcomplex in supporting electrolyte, the electrochemical signal of the formerdecreases with increasing concentration of DNA. The degree of decreasing ofoxidation current is a function of the concentration of DNA, thus founding thebasis of DNA detection. The electrochemical system is as follows:Fe(phen)₃ or Ru(bpy)₂dppz was employed as the electrochemical indicator,oxalate as the sacrificial electron donor to chemically amplify theelectrochemical signal, and tin-doped indium oxide as the working electrodeto suppress oxalate background current. Of the two metal complexes,Ru(bpy)₂dppz was superior, so it was selected as the indicator. Underoptimized conditions, the anodic current of Ru(bpy)₂dppz was amplified morethan 60 times in the presence of oxalate. In order to understand the principleof DNA detection, a positive charged electron donor, tri-n-propylamine, wasused to substitute for the negatively charged electron donor, oxalate. And theeffect of ionic strength was investigated to provide interpretation for thedecreasing of the amplification power in the presence of DNA. With anoptimized concentration of 30mmol/L oxalate and 5μmol/L Ru(bpy)₂dppz,calf thymus DNA of as low as 1×10-12mol/L was detected in solution.2. A simple and sensitive electrochemical method for quantitativedetection of phenanthrene was developed on the DNA modified electrode.Calf thymus DNA was immobilized onto ITO electrodes by alternatelayer-by-layer adsorption of avidin and double-stranded DNA. Theexperiment architecture is analogous to a protocol presently used in thecompetitive immunoassays, whereby phenanthrene analyte would competewith an electroactive intercalating complex for binding sites on DNA andmodulate the electrochemical signal. The DNA film electrode was exposed toa solution containing both the phenanthrene and Ru(bpy)2dppz. Phenanthrenewould compete with Ru(bpy)2dppz for binding sites on DNA and displacesome of the bound Ru(bpy)2dppz, resulting in a decreasing of electrochemicalsignal. The degree of decreasing of oxidation current was a function of theconcentration of phenanthrene. The detection limit of phenanthrene insolution is 1×10-7mol/L.