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.