Development of Bioaffinity Sensor for DNA Mutation Assay

大多数DNA生物传感器都是针对在电极上固定有与DNA互补碱基序列的靶DNA的检测。另一方面，DNA分子可以被看作是各种DNA结合蛋白的配体，这些DNA结合蛋白可以识别DNA的某些碱基序列、错配碱基对或特征结构，我们研究了利用DNA结合蛋白设计基因检测生物传感器的可能性。首先，我们利用抗DNA抗体和DNA修饰的金电极建立了DNA-蛋白质相互作用的检测体系。在该体系中，亚铁氰化物/铁氰化物氧化还原电对作为阴离子氧化还原标记物，用于检测固定化DNA上蛋白质的结合，抗DNA抗体的加入显著抑制了标记物离子的氧化还原电流，检测限为1 nM，并具有浓度依赖性，在这种情况下，2-巯基乙醇处理电极表面可有效避免其他蛋白质的非特异性吸附.然后，抗DNA抗体固定化电极的DNA检测也进行了详细的研究。在使用抗体的研究之后，DNA结合蛋白被改变为作为序列特异性DNA结合蛋白的转录因子PIT-1。然后，在DNA固定化电极系统中加入PIT-1，阴离子氧化还原标记物的氧化还原电流增加，这可能是由于PIT-1的阳离子电荷加速了标记物离子的电化学反应。氧化还原电流的增加也是PIT-1浓度依赖性的。这样，我们成功地建立了DNA-DNA结合蛋白序列特异性相互作用的检测体系。

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.