BIENZYME SENSORS BASED ON ELECTRICALLY WIRED PEROXIDASE

Single-layer and bilayer bienzyme electrodes based on the combination of a three-dimensional (3-D) redox epoxy network that electrically con nects redox centers of bound horseradish peroxidase (HRP) to electrode s with a hydrogen peroxide generating enzyme, the redox centers of whi ch 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 i s electrochemically reduced at 0 mV saturated calomel electrode (SCE). When the redox centers of the H2O2 generating enzyme are also electri cally connected to the redox-epoxy network, the substrate reduced redo x 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 no t electrically connected. The single-layer bienzyme electrodes extend the range of amperometric biosensors based on directly redox-epoxy ''w ired'' enzymes to enzymes that are difficult to electrically connect t o redox polymer networks and whose preferred or only cosubstrate is ox ygen. For the difficult to wire enzyme-choline oxidase, the cathodic c urrent density in the single-layer peroxidase and choline oxide contai ning electrode is 80 muA cm-2 at 10 mM choline concentration, while th e anodic current density of the directly wired enzyme is only 5 muA cm -2. Alcohol oxidase is not electrically connected to the wiring 3-D re dox-epoxy network. The anodic current density of its redox-epoxy wired electrodes is close to nil, while the cathodic current density, obser ved in alcohol oxidase and wired peroxidase containing single-layer el ectrodes at 10 mM ethanol, is 5 muA cm-2. When well-wired enzymes, suc h as glucose oxidase or lactate oxidase, are utilized in single-layer electrodes, limiting cathodic current densities of 60 muA cm-2 are obs erved for both enzymes. These currents are much lower than those obser ved in the directly wired enzyme anodes.