Hydrogen peroxide Is efficiently electroreduced at an electrode modified with a hydrophilic, permeable film of horseradish peroxidase (HRP) covalently bound to a 3-dlmenslonal epoxy network having polyvinyl pyridine (PVP)-complexed [Os(bpy)2CI]3+/2+ redox centers. The sensitivity of the resulting H202 cathode at 0.0 V (SCE) Is 1 A cm*1 2 3456M'1.Its current increases linearly with H202 concentration In the 1 X 10"7-2 X 10"4 M range. Related NAD(P)H cathodes are based on stoichiometric homogeneous reduction of 02 to H202 by NADH or NAD(P)H. The reduction Involves two known steps. In the first step, NAD(P)H transfers two electrons and a proton to a dissolved qulnoid. The qulnolds are typically derived of phenazlnes; however phenothiazlne and phenoxazlne derivatives are also useful. In the second step, two electrons and a proton are transferred from the reduced qulnoid to 02. This reaction produces H202 and the original qulnoid. Because the two reactions are quantitative, the sensitivity and the linear range of the resulting NADH and NADPH electrodes are Identical with those of the H202 electrode, 1 A cm'2 M~1 and 1 X 10~7-2 X 10"4 M, respectively.
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 μA cm−2 at 10 mM choline concentration, while the anodic current density of the directly wired enzyme is only 5 μA 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 μA 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 μA cm−2 are observed for both enzymes. These currents are much lower than those observed in the directly wired enzyme anodes.
A thermostable soybean peroxidase-based biosensor was formed by cross-linking and electrically "wiring" the enzyme through a redox-conducting hydrogel to a glassy carbon electrode. At 65 °C the sensitivity of the sensor decayed only at a rate of only <2%/h. The sensor maintained, at 25 °C, the 1 A cm"1 2 M"1 sensitivity and the 0.1-200 µ dynamic range of the earlier "wired" horseradish peroxidase-based sensors.The life of a biosensor is often limited by that of its enzyme. YSI1 and others2 realize operational stability by designing sensors with an excess of enzyme and a membrane that reduces the flux of substrate. These devices maintain their performance specifications even after most of their enzyme has become inactive, as their residual enzyme suffices to react with the restricted substrate flux. Other stabilization efforts focused on slowing the inactivation of the enzyme. Assuming that inactivation resulted in irreversible changes in protein folding, researchers modified the protein surface or fixed its structure by cross-linking for better stability.3 4"5 Reaction of amino acid residues, known to contribute to instability, also extended the life of sensors in continuous operation.6 Superior thermostability resulted from covalent bonding of an enzyme in a phospholipid-modified surface, resembling the enzyme's natural environment in a membrane.7•8 Enhancing the thermostability of sensors by making them with enzymes isolated from thermophilic organisms has been considered,9 though these enzymes either were unavailable in the needed quantity or did not have the needed activity. Recently, a thermostable peroxidase isolated from soybean has become commercially available.10 **In tests by the manufacturer, this enzyme had a half-life of > 12 h at 80 °C.
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