SPECIFIC BINDING ASSAY FOR BIOTIN BASED ON ENZYME CHANNELING WITH DIRECT ELECTRON-TRANSFER ELECTROCHEMICAL DETECTION USING HORSERADISH-PEROXIDASE

A model 'homogeneous' format enzyme channelling specific binding assay for biotin based on peroxide-sensitive horseradish peroxidase mediato rless enzyme electrodes is described. The procedure involved the immob ilisation of avidin onto the surface of printed carbon horseradish per oxidase (HRP) enzyme electrodes and the competitive binding of biotin and biotinylated glucose oxidase. Upon addition of glucose, hydrogen p eroxide was generated via the glucose oxidase label. Direct electron t ransfer between the electrodes and HRP resulted in the detection of H2 O2 by electroenzymic reduction at +50 mV vs Ag/AgCl. The cathodic curr ent response could be measured in the presence of excess biotinylated glucose oxidase by incorporation of catalase in homogeneous solution t o scavenge H2O2 generated in the bulk before it diffused to the electr ode surface. The assay showed greatest sensitivity over the range of b iotin concentrations 0.07 to 2 mu g ml(-1) in the presence of 10 mu g ml(-1) excess biotinylated glucose oxidase in the bulk solution.