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

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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.