| Budget Amount *help |
¥3,000,000 (Direct Cost: ¥3,000,000)
Fiscal Year 2005: ¥1,300,000 (Direct Cost: ¥1,300,000)
Fiscal Year 2004: ¥1,700,000 (Direct Cost: ¥1,700,000)
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| Research Abstract |
Nucleic acid hybridization is a basic method in molecular biology and provides new possibilities in various biomedical and biochemical areas. Conventionally, fluorescence compound labeled polynucleotides were used for probes for detection of complimentary DNA by specific hybridization. However, fluorescence detection has been limited to clinical diagnosis by cost problems. In the present study, a DNA electrochemical sensor system is proposed based on the combination of sandwich hybridization of reporter probe, capture probe, and target DNA. A biotinylated DNA probe was used for capture probe for target DNA on the avidin immobilized electrode. InvA gene of Salmonella typhimurium was used for target DNA and Glucose-6-phosphate dehydrogenase was used for subsequent enzymatic electrochemical detection as reporter probe. Origonucleotide for capture probe : 5'-CACCAATATCGCCAGTACGA-biotinylated-3' and reporter probe : 5'-biotinylated-GCAGCATTAGGTATCCTG-3' were designed. Biotinylated Glucose-6
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-dehydrogenase (G6PDH, Unitika Co.) was immobilized on the 5' end of reporter probe via avidine. DNA (invA region) was prepared form cell culture of S.typhimurium by PCR. DNA sensor was constructed as follows. At first, Au electrode was modified with mercaptopropionic acid. Then PEI-Fc (ferrocene immobilized polyethylenimine)/alginic acid and diahorase (DI)/PEI layer was formed on the surface of electrode by layer-by-layer method. Finally capture probe was immobilized on the electrode via avidine. Hybridization of target DNA and the both probe on the electrode was carried at 96□. After hybridization with target DNA, electrochemical measurement was performed in the solution containing G6P as substrate and NAD+ as cofactor for enzyme reaction. As shown in Fig.2, the anodic current (at ca. 0.25 V vs. Ag/AgCl) of ferrocene moieties of PEI-Fc raised. It indicates that reporter probe was immobilized on the electrode by hybridization with target DNA and G6PDH on the probe produced NADH. The proposed method was applicable to the measurement of DNA for environmental analysis, food analysis, and etc. Less
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