Study On The Preparation And Application Of Novel Electrochemical DNA Sensor

Nucleic acid films were immobilized onto indium-doped tin oxide (ITO) electrode through layer-by-layer self-assembly using avidin as the first layer and nucleic acid as the second layer, which functioned as the interface of the electrochemical DNA sensor. A DNA intercalator, Ru (bpy)₂dppz²⁺ ( bpy = 2, 2'-bipyridine, dppz = dipyrido [3, 2-a; 2', 3'-c] phenazine) was selected as electrochemical indicator to react with the DNA layer. Electrochemical measurement was performed in indicator-free electrolyte in which oxalate serving as sacrifice electron donor. A novel electrochemical DNA sensor had been set up. The main contents of this thesis include:1. Quartz crystal microbalance (QCM) and electrochemical method were used to verify the immobilization of DNA layer, and determine the amount of DNA immobilized. The amount of immobilized DNA was estimated by QCM to be 3.2 ng / mm² for both double-stranded (ds-) and single-stranded (ss-) calf-thymus DNA as well as polyguanylic acid (Poly-G) and polycytidylic acid (Poly-C), whereas 4.2 ng / mm² for damaged DNA by styrene oxide.2. To illustrate the advantages of the new system, a direct comparison was made between amplified (with oxalate) and non-amplified (without oxalate) DNA detection. When measured in an oxalate buffer, catalytic current was observed due to the oxidation of oxalate by electrochemically generated Ru (bpy)₂dppz³⁺, resulting in 120 fold increase in signal. Since oxalate itself produces very low oxidation current on ITO, the catalytic voltammetry leads to about 14 fold improvement in signal-to-blank ratio over the non-amplified determination. As a result, ds-DNA adsorbed from 20 ng / mL solution could be detected, which was estimated by QCM to be 160 pg / mm² on the surface. The catalytic current of ds-DNA was substantially higher than that of ss-DNA and poly-C, indicative of selective binding of the redox indicator to ds-DNA. The results have established basis for the catalytic voltammetric detection of DNA hybridization in the future work.3. Voltammetry of Ru (bpy)₂dppz²⁺ (Ru-dppz) bound to the DNA film showed strong dependence on the concentration of the metallo-intercalator. At low Ru-dppz concentrations, a single oxidation peak was observed, the potential of which shifted from 1.25 V to 1.1 V with increasing Ru-dppz concentration (Peak Ⅰ ). At high metal chelate concentrations, an additional oxidation peak emerged with a potential of 1.25V which was unaffected by the Ru-dppz concentration (Peak Ⅱ). The concentration dependence...