| Budget Amount *help |
¥3,600,000 (Direct Cost: ¥3,600,000)
Fiscal Year 2000: ¥1,500,000 (Direct Cost: ¥1,500,000)
Fiscal Year 1999: ¥2,100,000 (Direct Cost: ¥2,100,000)
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| Research Abstract |
Two examples of electrochemical gene sensor are described. We first introduced a ferrocene derivative with psoralen moiety. A gel-shift assay using a plasmid DNA (pBR322) showed that the compound binds irreversibly to DNA double strands upon UV radiation. This is due to photoadduct formation between the psoralen moiety and nucleases in the DNA duplex. In cyclic voltammetric measurements, the compound gave reversible redox waves, which were ascribed to the electrode reaction of the ferrocene moiety. Thus, the compound was expected to be useful for redox labeling of double-helical DNA ; the reagent which is bound covalently to DNA gives the double-strand a redox-active function. Based on these results, a possible application to electrochemical gene targeting was demonstrated in a combined use in a DNA-modified electrode system ; a gold electrode modified with a DNA probe was first treated with the target DNA to form the DNA double-strand gave redox waves upon a subsequent treatment with
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the labeling reagent while the redox activity for the DNA-modified electrode without the hybridization procedure (single-stranded DNA) was about one-half. Next, a DNA modified electrode, which can report DNA hybridization, was introduced. We have been investigating various DNA-modified electrode systems. By taking advantage of the principle of an "ion-channel sensor", some of those have been applying to electrochemical sensing of DNA-binding molecules and ions as well as DN-binding proteins. Here, the chemoreceptive surface adlayer was improved to a DNA/ferrocene bilayer format to give an analyte-dependent electrochemical signal. Electrode preparation was made as follows ; a gold electrode was first modified with 11-aminoalkanethiol and was subsequently reacted with 1, 1-ferrocendicarboxylic acid. Reaction with an oligodeoxyribonucleic acid with an amino function gave a modified electrode with a DNA/ferrocene interfacial structure. Cyclic voltammetric measurements revealed those reversible redox waves due to the electrode reaction of ferrocene suppressed to one-half upon formation of the DNA layer. If the electrode was treated with an aqueous solution of DNA having specific base sequence to the immobilized DNA, the redox waves were further suppressed to one-tenth. These results suggest that the electrode system can be applicable to the electrochemical detection of DNA hybridization without any kind of reporter molecules. Less
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