Multilabeling of ferrocenes to a glucose oxidase-digoxin conjugate for the development of a homogeneous electroenzymatic immunoassay.

A new homogeneous electroenzymatic immunoassay was developed to determine antibody concentration using glucose oxidase and ferrocene as enzymatic and electrochemical amplifier, respectively. Digoxin (Dig)-conjugated glucose oxidase (GOx) was modified with ferrocene (Fec) to form Fec-GOx-Dig conjugate. After immunocomplex formation between the Fec-GOx-Dig conjugate and the anti-digoxin antibody, the complex underwent less electrochemical reaction due to the steric hindrance of the antibody. The ferrocene multilabeled conjugate was provided for the determination of anti-digoxin antibody. Since the strategy taken here is based on a combined effect of GOx and ferrocene, i.e., enzymatic amplification by GOx and electrochemical amplification by multilabeled ferrocenes, the antibody concentration was determined in the range from 1/50 to 1/500 dilution.