Preparation of Bienzyme Sensors with "electrically wired" peroxidase.

T. OHARA; M. VREEKE; F. BATTAGLINI; A. HELLER

ELECTROANALYSIS

Wiley

Lugar: New York; Año: 1993 vol. 5 p. 825 - 831

1040-0397

Single-layer and bilayer bienzyme electrodes based on the combination of a three-dimensional (3-D) redox epoxy network that electrically connects redox centers of bound horseradish peroxidase (HRP) to electrodes with a hydrogen peroxide generating enzyme, the redox centers of which are not connected to the redox-epoxy network, are described. In the single-layer electrodes, H2O2 generated by the first enzyme oxidizes the second enzyme HRP, which oxidizes the redox polymer network that is electrochemically reduced at 0 mV saturated calomel electrode (SCE). When the redox centers of the H2O2 generating enzyme are also electrically connected to the redox-epoxy network, the substrate reduced redox centers are oxidized by the redox polymer network, thus lowering the cathodic current. Such attenuation is avoided in bilayer electrodes, where the H2O2 producing enzyme and the redox-epoxy-HRP network are not electrically connected. The single-layer bienzyme electrodes extend the range of amperometric biosensors based on directly redox-epoxy wired enzymes to enzymes that are difficult to electrically connect to redox polymer networks and whose preferred or only cosubstrate is oxygen. For the difficult to wire enzyme-choline oxidase, the cathodic current density in the single-layer peroxidase and choline oxidase containing electrode is 80 A cm-2 at 10 mM choline concentration, while the anodic current density of the directly wired enzyme is only 5 A cm-2. Alcohol oxidase is not electrically connected to the wiring 3-D redox-epoxy network. The anodic current density of its redox-epoxy wired electrodes is close to nil, while the cathodic current density, observed in alcohol oxidase and wired peroxidase containing single-layer electrodes at 10 mM ethanol, is 5 A cm-2. When well-wired enzymes, such as glucose oxidase or lactate oxidase, are utilized in single-layer electrodes, limiting cathodic current densities of 60 A cm-2 are observed for both enzymes. These currents are much lower than those observed in the directly wired enzyme anodes.