Glucose oxidase (GOX) or lactate oxidase (LOX) were immobilized in an osmium-based three-dimensional redox hydrogel that electrically connected the enzyme's redox centers to electrodes. The enzyme "wiring" hydrogel was formed by cross-linking poly(1-vinylimidazole) (PVI) complexed with Os-(4,4'-dimethylbpy)2Cl (termed PVI15-dmeOs) with poly(ethylene glycol) diglycidyl ether (peg). Glucose and lactate sensors exhibited typical limiting current densities of 250 and 500 microA/cm2, respectively. When the electrodes were poised at 200 mV (SCE), the currents resulting from electrooxidation of ascorbate, urate, acetaminophen, and L-cysteine were negligible. When a Nafion film was employed, the linear range was extended from 6 to 30 mM glucose and from 4 to 7 mM lactate. The redox potential of the gel-forming polymer was 95 mV (SCE). Glucose and lactate measurements performed in bovine calf serum correlated well with a substrate calibration in phosphate buffer.

Enzyme electrodes based on a redox hydrogel formed upon complexing water-soluble poly(1-vinylimidazole) (PVI) with [Os(bpy)2Cl]+ and cross-linked with water-soluble poly(ethylene glycol) diglycidyl ether (molecular weight 400, peg 400) are described. The properties of the electrodes depended on their polymers' osmium content, the extent of cross-linking, the pH, and the ionic strength in which they were used. The redox hydrogels' electron diffusion coefficients (De) increased with osmium content of their polymers. The De values were 1.5 x 10(-8), 1.3 x 10(-8), and 4.3 x 10(-9) cm2/s for PVI3-Os, PVI5-Os, and PVI10-Os, respectively, the subscripts indicating the number of monomer units per osmium redox center. De decreased with increasing ionic strength and increased upon protonation of the polymer. In glucose electrodes, made by incorporating into their films glucose oxidase (GOX) through covalent bonding in the cross-linking step, glucose was electrooxidized at > 150 mV (SCE). The characteristics of these electrodes depended on the GOX concentration, film thickness, O2 concentration, pH, NaCl concentration, and electrode potential. The steady-state glucose electrooxidation currents were independent of the polymers' osmium content in the studied (3-10 monomer units per osmium center) range. Electrodes containing 39% GOX reached steady-state glucose electrooxidation current densities of 400 microA/cm2 and, when made with thick gel films, were selective for glucose in the presence of physiological concentrations of ascorbate and acetaminophen.

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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.

Glucose oxidase (GOX) was electrically 'wired' to glassy carbon electrodes by redox hydrogels based on poly (N-vinyl imidazole) (PVI) complexed with Os(bpy) 2 Cl 2 (PVI-Os). The hydrogels were formed by crosslinking PVI-Os and glucose oxidase with poly(ethylene glycol) diglycidyl ether (PEGDGE). Glucose was electrooxidized on the PVI-Os based 'wired' enzyme electrodes at 350 mV vs SCE. The dependence of the electrooxidation current on pH, ionic strength, film thickness, weight fraction of the enzyme in the redox hydrogel and oxygen partial pressure for electrodes rotating at 1000 rpm is described.Polymeric redox mediators have been used for the transport of electrons between active sites of redox enzymes and electrodes (1-11). In complexes between high molecular weight redox polymers and redox enzymes, electrons are transferred efficiently from the substrate reduced redox active site of the enzyme to redox centers of the polymers (7,12) Our research focuses on water soluble redox polymers that are crosslinked on electrode surfaces to form 3-dimensional redox hydrogel, to the polymer skeletons of which enzymes are covalently bound and in which segments of the polymer may also form complexes with the enzymes. Redox polymers based on poly (vinyl pyiridine) complexed with Os (bpy)2Cl +/2+ (bpy = 2,2* bipyridine) have been used to 'wire' i.e. to electrochemically connect enzymes to electrodes. By quarternizing part of the free pyridine rings on the polymer with bromoethylamine we formed earlier a water soluble, PEGDGE crosslinked redox polyamine, POsEA (4,5).Electrooxidation of ascorbate, urate, acetaminophen and other readily electrooxidizable substrates often interferes with assays of analytes such as glucose. The problem of the resulting poor specificity for glucose can be alleviated by operating the electrodes at less oxidizing potentials, where the rate of electrooxidation of interferents can be slower. Alternatively, membranes can placed on electrodes to exclude anionic interferents or large interfèrent molecules. An example of such a membrane is Nafion (13). Recently we showed that interference by ascorbate, urate and aoetaminophen can be altojgether eliminated by their oxidation in a crosslinked overlayer of horseradish peroxidase by in situ generated or externally added H2O2