DEVELOPMENT OF A REGENERABLE AMPEROMETRIC IMMUNOSENSOR FOR 7-HYDROXYCOUMARIN

A regenerable immunosensor, based on a competitive assay principle and utilizing electrochemical detection, was developed for the determinat ion of 7-hydroxycoumarin. Horseradish peroxidase (HRP) labeled anti-7- hydroxycoumarin antibody was employed in the determination, and the en zyme activity was measured as a function of current due to the reducti on of hydrogen peroxide in the presence of a mediator (potassium ferro cyanide). Sensor preparation involved chemical immobilization of 7-hyd roxy-coumarin-BSA conjugate on a glassy carbon electrode surface using a water soluble carbodiimide, 1-ethyl-3(3-dimethyl-aminopropyl) carbo diimide, as coupling reagent in the presence of N-hydroxysuccinimide. The competition between the free and immobilized forms of the hapten f or their HRP labeled antibody in solution allows for the quantitation of free 7-hydroxycoumarin. The sensor can be regenerated by simply imm ersing the electrode tip in acidic media. Under these conditions the h apten-antibody complex was selectively disrupted without adversely aff ecting the sensor performance. The conditions for removing the non-spe cific adsorption of the enzyme conjugate were also studied. The result s showed that the detection range obtained for the hapten was physiolo gically relevant.