Potentiometric Detection of DNA Molecules Hybridization Using Gene Field Effect Transistor and Intercalator
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Potentiometric Detection of DNA Molecules Hybridization Using Gene Field Effect Transistor and Intercalator Toshiya Sakata, Hidenori Otsuka and Yuji Miyahara Biomaterials Center, National Institute for Materials Science, 1-1 Namiki, Tsukuba, Ibaraki 305-0044, Japan ABSTRACT We propose a new concept of a gene field effect transistor (FET) for detection of allele specific oligonucleotide hybridization, which is in principle based on charge density change at the gate insulator. The electrical characteristics of the gene FET were found to shift after specific binding of biomolecules at the surface of the gate insulator. Allele specific oligonucleotide hybridization and reaction between double-stranded DNA and intercalator were successfully detected with gene FETs because they have intrinsic charges in an aqueous solution. Ability to discriminate single nucleotide polymorphism (SNP) was also examined using the gene FET. Our results show that control of hybridization temperature and utilization of intercalator lead to more precise SNP analysis using the gene FET.
INTRODUCTION Recently, various gene functional analyses have remarkably proceeded based on the completion of the decoding of the human genome [1-3]. For gene expression and genotyping analyses, several types of DNA chips and DNA microarrays have been developed and some of them are used in the field of molecular biology, pharmaceutical industry and clinical research. While fluorescent detection method is widely used for DNA chips and DNA microarrays, several new methods such as chemiluminescence and electrical current have been developed to detect specific binding of DNA molecules [4-9]. We have been investigating a new approach to realize an electrochemical detection for DNA chips. The novel concept of a gene field effect transistor (FET) is proposed in the present study for improving precision, standardization and miniaturization of a DNA chip system. The gene FET is composed of Si crystal with Si3N4/SiO2 as the gate insulator on which DNA probes are immobilized and subsequently hybridized with target DNA in sample solutions. The potentiometric detection method is based on the direct transduction of surface density change of charged biomolecules into electrical signal by the field effect and is effective for charged species such as DNA molecules[10]. One of the unique feature of our method is to utilize intercalator as charged species because they have positive charges in aqueous solutions in addition to the specific adsorption with double-stranded DNA, while intercarators are often used as fluorescent dyes in the field of molecular biology [11,12]. The introduction of intercalator after hybridization is considered to be effective to discriminate between the signal of double-stranded DNA and that of non-specifically adsorbed single-stranded DNA. In this study, we report the concept and the fundamental characteristics of gene FET, such as the direct detection of hybridization and the effect of intercalator on potentiometric measurement. Possibility for S
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