Homogeneous amperometric ligand-binding assay amplified by a proteolytic enzyme cascade

A homogeneous electrochemically-linked ligand binding assay with enhanced sensitivity is described. A proteolytic enzyme cascade was used to amplify the signal. The avidin-biotin interaction serves to model an immunochemical reaction. Biotinylated trypsin (BT) is the trigger enzyme of the cascade, which is inhibited in the presence of avidin; this inhibition is reversed by the addition of free biotin. The liberated BT activates catalytically the second enzyme, chymotrypsin, which acts upon its immobilised electroactive substrate. The released hydrolysis product, ferrocenoyltyrosine (FcT), is then coupled to the turnover of glucose oxidase (GOx), providing a further level of amplification. The catalytic current produced is proportional to the concentration of biotin in the sample and is linear in the concentration range 0.4–5 μM.