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 determination of 7-hydroxycoumarin. Horseradish peroxidase (HRP) labeled anti-7-hydroxycoumarin antibody was employed in the determination, and the enzyme activity was measured as a function of current due to the reduction of hydrogen peroxide in the presence of a mediator (potassium ferrocyanide). Sensor preparation involved chemical immobilization of 7-hydroxy-coumarin-BSA conjugate on a glassy carbon electrode surface using a water soluble carbodiimide, 1-ethyl-3(3-dimethyl-aminopropyl) carbodiimide, as coupling reagent in the presence of N-hydroxysuccinimide. The competition between the free and immobilized forms of the hapten for their HRP labeled antibody in solution allows for the quantitation of free 7-hydroxycoumarin. The sensor can be regenerated by simply immersing the electrode tip in acidic media. Under these conditions the hapten-antibody complex was selectively disrupted without adversely affecting the sensor performance. The conditions for removing the non-specific adsorption of the enzyme conjugate were also studied. The results showed that the detection range obtained for the hapten was physiologically relevant.