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Development of Bioaffinity Sensor for DNA Mutation Assay

DNA突变检测生物亲和传感器的开发

基本信息

批准号：
09555266
负责人：
MAEDA Mizuo
金额：
$ 4.03万
依托单位：
KYUSHU UNIVERSITY
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (B)
财政年份：
1997
资助国家：
日本
起止时间：
1997 至 1998
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-09555266/
关键词：
DNA biosensor DNA gene assay gene mutation gene sensor cyclic voltammetry DNA binding protein gene

项目摘要

Most of DNA biosensors direct toward the detection of target DNA which has a complementary base sequence of the immobilized DNA on the electrode. On the other hand, DNA molecule can be regarded as ligands to various DNA binding proteins which recognize certain base sequences, mismatch base pair, or characteristic structure of DNA.We investigated the possibility of use of DNA binding proteins to the design of biosensor for gene assay. First, we established the system, in which DNA-protein interaction can be detected, using anti-DNA antibody and DNA modified gold electrode. In this system, ferrocyanide/ferricyanide redox couple was used as anionic redox marker to monitor the protein binding on immobilized DNA.Thus, addition of anti-DNA antibody suppressed the redox currents of marker ions dramatically in concentration dependent manner with the detection limit of 1 nM.In this case, 2-mercaptoethanol treatment of electrode surface was effective to avoid non-specific adsorption of other proteins. Then, DNA detection with the anti-DNA antibody immobilized electrode was also investigated in detail. After the study using the antibody, DNA binding protein was changed to PIT-1, which is a transcriptional factor, as sequence specific DNA binding protein. The redox current of the anionic redox marker was then augmented with the addition of PIT-i in the DNA immobilized electrode system, probably due to the cationic charges of PIT-1 which accelerates the electrochemical reaction of the marker ions. The increase of the redox current was again the concentration dependent of added PIT-1.In this way, we successfully established the detecting system of sequence specific interaction of DNA-DNA binding protein.

大多数DNA生物传感器用于检测与固定在电极上的DNA具有互补碱基序列的靶DNA。另一方面，DNA分子可以作为各种DNA结合蛋白的配体，识别特定的碱基序列、错配的碱基对或DNA的特征结构。我们探索了将DNA结合蛋白用于设计用于基因分析的生物传感器的可能性。首先，我们建立了利用抗DNA抗体和DNA修饰的金电极检测DNA-蛋白质相互作用的体系。在该体系中，亚铁氰化物/铁氰化铁还原对作为阴离子氧化还原标记物监测蛋白质与固定化DNA的结合，因此，抗DNA抗体的加入以浓度依赖的方式显著抑制了标记离子的氧化还原电流，检测下限为1 nm。在这种情况下，电极表面的2-巯基乙醇处理可以有效地避免其他蛋白质的非特异性吸附。然后，对抗DNA抗体固定电极的DNA检测进行了详细的研究。使用该抗体进行研究后，DNA结合蛋白被改变为转录因子PIT-1，作为序列特异性DNA结合蛋白。在DNA固定电极体系中，随着PIT-I的加入，阴离子氧化还原标记物的氧化还原电流增大，这可能是由于PIT-1的阳离子电荷加速了标记离子的电化学反应。氧化还原电流的增加再次与添加PIT-1的浓度有关，从而成功地建立了DNA-DNA结合蛋白序列特异性相互作用的检测体系。