High-throughput Screening of Proteins by Electrochemical Protein Chip

电化学蛋白质芯片高通量筛选蛋白质

• 批准号: 12555236
• 负责人: TAKAGI Makoto
• 金额: $ 8.77万
• 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: 2000
• 资助国家: 日本
• 起止时间: 2000 至 2001
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-12555236/
• 关键词: protein chip; electrochemistry; gold electrode; electrochemical chip; ferrocene; L2-G peptide; L2 peptide; Helix G peptide; 静電的相互作用

By the near completion of the human genome sequencing project, attention is now shifting toward the analysis of proteins or proteoms. In this respect, protein chips are regarded as a high-throughput means of studying protein expression. Protein chips are made of various proteins immobilized on the surface of a substrate. The aim of this project is to develop a protein chip based on the knowledge obtained from our study of electrochemical DNA chips. To realize this purpose, we constructed a protein chip by immobilizing a protein carrying a cysteine residue on the gold electrode through the gold-sulfur linkage and tried to establish an electrochemical analysis system with it.First of all, we verified in a preliminary experiment with peptides that this system works as designed. Peptides carrying different charges immobilized on a gold electrode and their interaction with ferrocenecarboxylic acid was studied electrochemically. Ferrocenecarboxylic acid bound strongly to cationic peptides im … More mobilized on the electrode as proven by an increase in the redox current due to the ferrocene, whereas it failed to bind to anionic peptides because of electrostatic repulsion.In the next experiment, Rec A protein, known to discriminate single and double stranded DNA, and its functional domains, L2 and Helix G peptides, were synthesized by the peptide synthesizer with Fmoc chemistry and tested whether their interaction with single stranded DNA can be monitored electrochemically. These peptides were immobilized on the individual electrodes and were allowed to interact with single or double stranded DNA carrying an electrochemically active ferroncene moiety. This system, once established, should be useful for the screening of DNA binding peptides or proteins. Electrochemical as well as Quartz Crystal Microbalance experiments suggested that the L2 part of the protein is important for the interaction with nucleic bases of DNA and the Helix G part contributes to the discrimination between single and double stranded DNAs. These results opened up a new vista for the development of an electrochemical protein chip. Furthermore, high-throughput screening of various other proteins should be possible by using the scanning electrochemical microscope. Less

通过接近人类基因组测序项目的接近完成，现在注意力正在转向蛋白质或蛋白质的分析。在这方面，蛋白质芯片被认为是研究蛋白质表达的高通量手段。蛋白质芯片由固定在底物表面上的各种蛋白质制成。该项目的目的是根据我们对电化学DNA芯片的研究获得的知识开发蛋白质芯片。为了实现此目的，我们通过将蛋白质固定在金电极上的蛋白质固定在金电极上的蛋白质中，并试图与IT建立电化学分析系统。首先，我们在使用宠物的初步实验中验证了该系统以设计为宠物的实验。携带固定在金电极上的不同电荷的肽及其与铁甲基羧酸的相互作用是用电化学研究的。强烈到阳离子肽的易于……在电极上更受到动员的动员，这是由于甲茂铁的增加而证明的，而由于静电排斥，它由于静电率而无法与阴离子肽结合。在下一个实验中，蛋白质会通过鉴定单链DNA和其功能性DNA，l2和helix g2nix g2nix g2nix gynix g2nix g pletides，l2 g2通过FMOC化学，可以通过电化学测试它们是否与单链DNA相互作用。将这些胡椒化固定在单个电极上，并被允许与带有电化学活性铁烯苯基部分的单链或双链DNA相互作用。该系统一旦建立，应该对筛选DNA结合肽或蛋白质有用。电化学和石英晶体微量平衡实验表明，蛋白质的L2部分对于与DNA核碱基的相互作用很重要，而螺旋G部分则有助于歧视单链DNA和双链DNA。这些结果为开发电化学蛋白芯片开发了新的远景。此外，应使用扫描电化学显微镜对各种其他蛋白质进行高通量筛选。较少的

项目成果

S. Takenaka et al.: "Base mutation analysis by a ferrocenyl naphthalene diimide drivative"Nucleosides, Nucleotides & Nucleic Acids. 20 (4-7). 1429-1432 (2001)

S. Takenaka 等人：“二茂铁基萘二酰亚胺衍生物的碱基突变分析”核苷、核苷酸

K.Yamashita et al.: "Visualization of DNA microarrays by scanning electrochemical microscopy (SECM)"Analyst. 126. 1210-1211 (2001)

K.Yamashita 等人：“通过扫描电化学显微镜 (SECM) 实现 DNA 微阵列的可视化”分析师。

M. Takagi: "Threading intercalation to double-stranded DNA and the application to DNA sensing. Electrochemical array technique"Pure Appl. Chem.. 73 (10). 1573-1577 (2001)

M. Takagi：“双链 DNA 的螺纹插入及其在 DNA 传感中的应用。电化学阵列技术”Pure Appl。

S.Takenaka et al.: "Base mutation analysis by a ferrocenyl naphthalene diimide drivative"Nucleosides, Nucleotides & Nucleic Acids. 20・4-7. 1429-1432 (2001)

S.Takenaka等：“二茂铁基萘二酰亚胺衍生物的碱基突变分析”核苷、核苷酸和核酸20·4-7（2001）。

M.Takagi et al.: "High-throughput, high-sensitivity detection of targeted genes by ECA (Electro-Chemical Array)"Technical Digest of the 18^<th> Sensor Symposium. 387-393 (2001)

M.Takagi等人：“通过ECA（电化学阵列）对目标基因进行高通量、高灵敏度检测”第18届传感器研讨会技术文摘。