A coulometric biosensor to determine hydrogen peroxide using a monomolecular layer of horseradish peroxidase immobilized on a glass surface

A biosensor to detect hydrogen peroxide, by coulometry, down to submicromolar concentration using a monomolecular layer of horseradish peroxidase was developed. In this device 0.3 pmol of the enzyme were covalently immobilized on the glass surface of the biosensor and the enzyme layer was characterized by atomic force microscopy and activity measurements. The glass surface bearing the peroxidase was faced to a carbon electrode in a cell of 1 microl of active volume. The polarization of the working electrode at -100 mV versus Ag/AgCl, in the presence of 1,4-hydroquinone as mediator, allowed the fast reduction of the injected hydrogen peroxide via the hydroquinone-peroxidase system. This device permitted to measure the total number of H(2)O(2) molecules present in the cell in the concentration range of 0.3-100 microM H(2)O(2), with a sensitivity of 196 nC/microM H(2)O(2), which is close to the theoretical value (193 nC/microM).