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

以蛋清中的亲和素为蛋白质，生物素为配体，对蛋白质与其配体相互作用的电化学检测进行了研究，获得了以下新的信息。通过柔红霉素、尼罗蓝和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。当这些电活性配体与亲和素结合时，其电极响应降低。电活性化合物本身不会因为加入亲和素而发生这种电极响应的变化。因此，证实了这种下降是基于亲和素与生物素的特异性结合。间隔物的长度对电极响应的影响也较小。柔红霉素和尼罗蓝标记的配体通过吸附作用聚集在电极表面，从而实现了高灵敏度的分析。然而，二茂铁标记的配体并没有显示出吸附活性。然后对离子交换聚合物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修饰电极上积累，并被灵敏地检测到。利用标记配体与生物素对亲和素有限结合部位的竞争反应，间接检测生物素。利用这些标记的配体，建立了一种基于电化学检测的均相检测方法。利用碘乙酰生物素与硫醇的反应，发展了一种新型的电化学检测方法，其化学反应受配体与蛋白质结合的控制。

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.