DEVELOPMENT OF AN ANTIBODY-BASED BIOSENSOR FOR DETERMINATION OF 7-HYDROXYCOUMARIN (UMBELLIFERONE) USING HORSERADISH-PEROXIDASE LABELED ANTI-7-HYDROXYCOUMARIN ANTIBODY

An immunosensor based on a competitive electrochemical immunoassay was developed for the determination of 7-hydroxycoumarin. The antibody-based biosensor employed horseradish peroxidase-labelled anti-7-hydroxycoumarin, with the enzyme catalysed reaction involving the reduction of hydrogen peroxide in the presence of a mediator (hydroquinone). Sensor preparation involved immobilisation of the protein (ovalbumin/thyroglobulin)bound 7-hydroxycoumarin antigen within a Nafion film on a glassy carbon surface. The electrode was then incubated in a solution containing both the enzyme-labelled anti-7-hydroxycoumarin and increasing concentrations of the free 7-hydroxycoumarin analyte. Competition between the free and immobilised forms of the antigen allows for the quantitation of free 7-hydroxycoumarin in solution. Injections of the enzyme substrate, hydrogen peroxide, resulted in an increase in the steady-state cathodic current (-0.03 V vs. saturated calomel reference electrode). This current output was inversely proportional to the concentration of analyte in solution. Free 7-hydroxycoumarin in solution was determined between 0 and 1 mM, with a detection limit of 24 mu M.