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

随着人类基因组测序计划的接近完成，人们的注意力正转向蛋白质或蛋白质组的分析。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。蛋白质芯片是由固定在基质表面的各种蛋白质制成的。本计画的目的是利用我们在电化学DNA晶片研究中所获得的知识，发展一种蛋白质晶片。为了实现这一目的，我们构建了一个蛋白质芯片，通过金-硫键固定在金电极上的半胱氨酸残基的蛋白质，并试图建立一个电化学分析系统。首先，我们验证了初步的实验与肽，该系统的工作原理与设计。用电化学方法研究了固定在金电极上的带不同电荷的多肽与二茂铁羧酸的相互作用。二茂铁羧酸与阳离子肽im强结合 ...更多信息 在下一个实验中，已知可区分单链和双链DNA的Rec A蛋白及其功能结构域，L2和HPLG肽，通过具有Fmoc化学的肽合成仪合成，并测试它们与单链DNA的相互作用是否可以电化学监测。这些肽固定在各个电极上，并允许与携带电化学活性二茂铁部分的单链或双链DNA相互作用。该系统一旦建立，将可用于筛选DNA结合肽或蛋白质。电化学和石英晶体微天平实验表明，蛋白质的L2部分是重要的与DNA的核酸碱基的相互作用和HPLG部分有助于单链和双链DNA之间的区别。这些结果为电化学蛋白质芯片的发展开辟了新的前景。此外，通过使用扫描电化学显微镜，各种其他蛋白质的高通量筛选应该是可能的。少

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。

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。

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）。