Electrochemical detection of the interaction between protein and its ligand using ligands labelled with electroactive compounds

使用电活性化合物标记的配体电化学检测蛋白质与其配体之间的相互作用

• 批准号: 07640795
• 负责人: TANAKA Shunitz
• 金额: $ 1.34万
• 依托单位: Hokkaido University
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (C)
• 财政年份: 1995
• 资助国家: 日本
• 起止时间: 1995 至 1996
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-07640795/
• 关键词: protein-ligand; avidin; biotin; daunomycin; electrochemical assay; Nile blue; ferrocene; enzyme assay; タンパク-リガンド相互作用; フェロセン化ビオチン; ナフィオン修飾電極; 濃縮ボルタンメトリー; 矩形波法; ニュウトラアビジン

The electrochemical detection of the interaction between protein and its ligand was investigated using avidin in egg white as protein and biotin as ligand, and the following new information was obtained.1. Some new electroactive ligands were prepared by labelling biotin with electroactive compounds such as daunomycin, Nile blue and ferrocene.2. The electrode response of these electroactive ligands decreased as these ligands bound with avidin. The electroactive compounds itself did not show such change of the electrode response by addition of avidin. Therefore, it was confirmed that the decrease was based on the specific binding between avidin and biotin. The length of spacer also affected the electrode response slightly.3. The ligands labelled with daunomycin and Nile blue were accumulated at the electrode surface by the adsorption property and it led to the high sensitive assay. However, the ligand labelled with ferrocene did not show the adsoptivity. Then the electrode modified with Nafion, one of the ion-exchange polymer, was tested and the ferrocenyl ligand was accumulated on the Nafion modified electrode when ferrocene was oxidized to ferrocenium ion and was detected sensitively.4. Using the competition reaction between the labelled ligand and biotin for the limited binding sites of avidin, biotin was also detected indirectly. The possibility of the development of a homogeneous assay based on the electrochemical detection was showed using these labelled ligand.A new type of electrochemical assay in which the chemical reaction was controlled by the binding of ligand with protein, was developed using the reaction of iodoacetyl biotin and thiol.

以蛋清中的抗生物素蛋白为蛋白质，生物素为配体，对蛋白质与其配体相互作用进行电化学检测，得到以下新信息： 1．用道诺霉素、尼罗蓝、二茂铁等电活性化合物标记生物素，制备了一些新的电活性配体。 2．当这些配体与抗生物素蛋白结合时，这些电活性配体的电极响应降低。通过添加抗生物素蛋白，电活性化合物本身并未表现出电极响应的这种变化。因此，证实该减少是基于抗生物素蛋白和生物素之间的特异性结合。垫片的长度对电极响应也有轻微影响。 3．柔红霉素和尼罗蓝标记的配体通过吸附特性聚集在电极表面，从而实现高灵敏度的测定。然而，用二茂铁标记的配体没有表现出吸附性。然后对离子交换聚合物之一的Nafion修饰的电极进行了测试，当二茂铁被氧化成二茂铁离子时，二茂铁基配体在Nafion修饰的电极上积累，并被灵敏地检测到。 4．利用标记配体与生物素之间对亲和素的有限结合位点的竞争反应，也间接检测到生物素。使用这些标记的配体显示了开发基于电化学检测的均相测定的可能性。利用碘乙酰生物素和硫醇的反应开发了一种新型电化学测定，其中化学反应是通过配体与蛋白质的结合来控制的。

项目成果

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K. Sukawara，：“使用铜 (II)-卟啉修饰的碳糊电极观察糖的电化学行为。”