Glucose biosensor based on open-source wireless microfluidic potentiostat.

Wireless potentiostats capable of cyclic voltammetry and amperometry that connect to the Internet are emerging as key attributes of future point-of-care devices. This work presents an "integrated microfluidic electrochemical detector" (iMED) three-electrode multi-potentiostat designed around operational amplifiers connected to a powerful WiFi-based microcontroller as a promising alternative to more expensive and complex strategies reported in the literature. The iMED is integrated with a microfluidic system developed to be controlled by the same microcontroller. The iMED is programmed wirelessly over a standard WiFi network and all electrochemical data is uploaded to an open-source cloud-based server. A wired desktop computer is not necessary for operation or program uploading. This method of integrated microfluidic automation is simple, uses common and inexpensive materials, and is compatible with commercial sample injectors. An integrated biosensor platform contains four screen-printed carbon arrays inside 4 separate microfluidic detection chambers with Pt counter and pseudo Ag/AgCl reference electrodes in situ. The iMED is benchmarked with K3[Fe(CN)6] against a commercial potentiostat and then as a glucose biosensor using glucose-oxidising films of [Os(2,2'-bipyridine)2(polyvinylimidazole)10Cl] prepared on screen-printed electrodes with multi walled carbon nanotubes, poly(ethylene glycol) diglycidyl ether and flavin adenine dinucleotide-dependent glucose dehydrogenase. Potential application of this cost-effective wireless potentiostat approach to modern bioelectronics and point-of-care diagnosis is demonstrated by production of glucose oxidation currents, under pseudo-physiological conditions, using mediating films with lower redox potentials.

Extracellular Electron Transfer by the Gram-Positive Bacterium Enterococcus faecalis.

Extracellular electron transfer (EET) in microbial cells is essential for certain biotechnological applications and contributes to the biogeochemical cycling of elements and syntrophic microbial metabolism in complex natural environments. The Gram-positive lactic acid bacterium Enterococcus faecalis, an opportunistic human pathogen, is shown to be able to transfer electrons generated in fermentation metabolism to electrodes directly and indirectly via mediators. By exploiting E. faecalis wild-type and mutant cells, we demonstrate that reduced demethylmenaquinone in the respiratory chain in the bacterial cytoplasmic membrane is crucial for the EET. Heme proteins are not involved, and cytochrome bd oxidase activity was found to attenuate EET. These results are significant for the mechanistic understanding of EET in bacteria and for the design of microbial electrochemical systems. The basic findings infer that in dense microbial communities, such as in biofilm and in the large intestine, metabolism in E. faecalis and similar Gram-positive lactic acid bacteria might be electrically connected to other microbes. Such a transcellular electron transfer might confer syntrophic metabolism that promotes growth and other activities of bacteria in the microbiota of humans and animals.

The In Vivo Potential-Regulated Protective Protein of Nitrogenase in Azotobacter vinelandii Supports Aerobic Bioelectrochemical Dinitrogen Reduction In Vitro.

Nitrogenase, the only enzyme known to be able to reduce dinitrogen (N2) to ammonia (NH3), is irreversibly damaged upon exposure to molecular oxygen (O2). Several microbes, however, are able to grow aerobically and diazotrophically (fixing N2 to grow) while containing functional nitrogenase. The obligate aerobic diazotroph, Azotobacter vinelandii, employs a multitude of protective mechanisms to preserve nitrogenase activity, including a "conformational switch" protein (FeSII, or "Shethna") that reversibly locks nitrogenase into a multicomponent protective complex upon exposure to low concentrations of O2. We demonstrate in vitro that nitrogenase can be oxidatively damaged under anoxic conditions and that the aforementioned conformational switch can protect nitrogenase from such damage, confirming that the conformational change in the protecting protein can be achieved solely by regulating the potential of its [2Fe-2S] cluster. We further demonstrate that this protective complex preserves nitrogenase activity upon exposure to air. Finally, this protective FeSII protein was incorporated into an O2-tolerant bioelectrosynthetic cell whereby NH3 was produced using air as a substrate, marking a significant step forward in overcoming the crippling limitation of nitrogenase's sensitivity toward O2.

Bioelectrochemical Haber-Bosch Process: An Ammonia-Producing H2 /N2 Fuel Cell.

Nitrogenases are the only enzymes known to reduce molecular nitrogen (N2 ) to ammonia (NH3 ). By using methyl viologen (N,N'-dimethyl-4,4'-bipyridinium) to shuttle electrons to nitrogenase, N2 reduction to NH3 can be mediated at an electrode surface. The coupling of this nitrogenase cathode with a bioanode that utilizes the enzyme hydrogenase to oxidize molecular hydrogen (H2 ) results in an enzymatic fuel cell (EFC) that is able to produce NH3 from H2 and N2 while simultaneously producing an electrical current. To demonstrate this, a charge of 60 mC was passed across H2 /N2 EFCs, which resulted in the formation of 286 nmol NH3  mg-1 MoFe protein, corresponding to a Faradaic efficiency of 26.4 %.

Comparative proteomics implicates a role for multiple secretion systems in electrode-respiring Geobacter sulfurreducens biofilms.

Geobacter sulfurreducens is a dissimilatory metal-reducing bacterium capable of forming thick electron-conducting biofilms on solid electrodes. Here, we employ for the first time comparative proteomics to identify key physiological changes involved in G. sulfurreducens adaptation from fumarate-respiring planktonic cells to electron-conducting biofilms. Increased levels of proteins involved in outer membrane biogenesis, cell motility and secretion are expressed in biofilms. Of particular importance to the electron-conducting biofilms are proteins associated with secretion systems of Type I, II, V and Type IV pili. Furthermore, enzymes involved in lipopolysaccharide and peptidoglycan biosynthesis show increased levels of expression in electron-conducting biofilms compared to planktonic cells. These observations point to similarities in long-range electron transfer mechanisms between G. sulfurreducens and Shewanella oneidensis, while highlighting the wider significance of secretion systems beyond that of Type IV pili identified to date in the adaptation of G. sulfurreducens to electrode respiration.

Fully Enzymatic Membraneless Glucose|Oxygen Fuel Cell That Provides 0.275 mA cm(-2) in 5 mM Glucose, Operates in Human Physiological Solutions, and Powers Transmission of Sensing Data.

Coimmobilization of pyranose dehydrogenase as an enzyme catalyst, osmium redox polymers [Os(4,4'-dimethoxy-2,2'-bipyridine)2(poly(vinylimidazole))10Cl](+) or [Os(4,4'-dimethyl-2,2'-bipyridine)2(poly(vinylimidazole))10Cl](+) as mediators, and carbon nanotube conductive scaffolds in films on graphite electrodes provides enzyme electrodes for glucose oxidation. The recombinant enzyme and a deglycosylated form, both expressed in Pichia pastoris, are investigated and compared as biocatalysts for glucose oxidation using flow injection amperometry and voltammetry. In the presence of 5 mM glucose in phosphate-buffered saline (PBS) (50 mM phosphate buffer solution, pH 7.4, with 150 mM NaCl), higher glucose oxidation current densities, 0.41 mA cm(-2), are obtained from enzyme electrodes containing the deglycosylated form of the enzyme. The optimized glucose-oxidizing anode, prepared using deglycosylated enzyme coimmobilized with [Os(4,4'-dimethyl-2,2'-bipyridine)2(poly(vinylimidazole))10Cl](+) and carbon nanotubes, was coupled with an oxygen-reducing bilirubin oxidase on gold nanoparticle dispersed on gold electrode as a biocathode to provide a membraneless fully enzymatic fuel cell. A maximum power density of 275 µW cm(-2) is obtained in 5 mM glucose in PBS, the highest to date under these conditions, providing sufficient power to enable wireless transmission of a signal to a data logger. When tested in whole human blood and unstimulated human saliva maximum power densities of 73 and 6 µW cm(-2) are obtained for the same fuel cell configuration, respectively.

Engineering of pyranose dehydrogenase for application to enzymatic anodes in biofuel cells.

In the search for improved glucose oxidising enzymes for biofuel cells, a number of Agaricus meleagris (Am) pyranose dehydrogenase mutants (mPDHs) exhibiting different degrees of glycosylation were produced using site-directed mutagenesis and electrochemically characterised. The response of electrodes modified with different mPDHs is compared in a mediated electron transfer mode, where the electrodes are modified with each of the mutants covalently attached to redox polymers based on polyvinylimidazole-bound osmium complexes using a cross-linking agent. Coating of each of the enzymes onto the graphite electrode surface is also used to screen for their capacity for direct electron transfer. The double mutant PDH exhibits the highest response to glucose at physiological pH in both direct and mediated electron transfer modes, producing a Jmax of ≈800 µA cm(-2) at room temperature and when "wired" to the Os-polymer having the highest formal potential. From the results obtained the double mPDH is proposed as the most suitable candidate for application to bioanode fabrication.

Charge transport in films of Geobacter sulfurreducens on graphite electrodes as a function of film thickness.

Harnessing, and understanding the mechanisms of growth and activity of, biofilms of electroactive bacteria (EAB) on solid electrodes is of increasing interest, for application to microbial fuel and electrolysis cells. Microbial electrochemical cell technology can be used to generate electricity, or higher value chemicals, from organic waste. The capability of biofilms of electroactive bacteria to transfer electrons to solid anodes is a key feature of this emerging technology, yet the electron transfer mechanism is not fully characterized as yet. Acetate oxidation current generated from biofilms of an EAB, Geobacter sulfurreducens, on graphite electrodes as a function of time does not correlate with film thickness. Values of film thickness, and the number and local concentration of electrically connected redox sites within Geobacter sulfurreducens biofilms as well as a charge transport diffusion co-efficient for the biofilm can be estimated from non-turnover voltammetry. The thicker biofilms, of 50 ± 9 µm, display higher charge transport diffusion co-efficient than that in thinner films, as increased film porosity of these films improves ion transport, required to maintain electro-neutrality upon electrolysis.

Photo-electrochemical communication between cyanobacteria (Leptolyngbia sp.) and osmium redox polymer modified electrodes.

Photosynthetic microbial fuel cells (PMFCs) are an emerging technology for renewable solar energy conversion. Major efforts have been made to explore the electrogenic activity of cyanobacteria, mostly using practically unsustainable reagents. Here we report on photocurrent generation (≈8.64 µA cm(-2)) from cyanobacteria immobilized on electrodes modified with an efficient electron mediator, an Os(2+/3+) redox polymer. Upon addition of ferricyanide to the electrolyte, cyanobacteria generate the maximum current density of ≈48.2 µA cm(-2).

Effect of Protection Polymer Coatings on the Performance of an Amperometric Galactose Biosensor in Human Plasma.

Galactose monitoring in individuals allows the prevention of harsh health conditions related to hereditary metabolic diseases like galactosemia. Current methods of galactose detection need development to obtain cheaper, more reliable, and more specific sensors. Enzyme-containing amperometric sensors based on galactose oxidase activity are a promising approach, which can be enhanced by means of their inclusion in a redox polymer coating. This strategy simultaneously allows the immobilization of the biocatalyst to the electroactive surface and hosts the electron shuttling units. An additional deposition of capping polymers prevents external interferences like ascorbic or uric acid as well as biofouling when measuring in physiological fuels. This work studies the protection effect of poly(2-methacryloyloxyethyl phosphorylcholine-co-glycidyl methacrylate (MPC) and polyvinylimidazole-polysulfostyrene (P(VI-SS)) when incorporated in the biosensor design for the detection of galactose in human plasma.

Further insights into the catalytical properties of deglycosylated pyranose dehydrogenase from Agaricus meleagris recombinantly expressed in Pichia pastoris.

The present study focuses on fragmented deglycosylated pyranose dehydrogenase (fdgPDH) from Agaricus meleagris recombinantly expressed in Pichia pastoris . Fragmented deglycosylated PDH is formed from the deglycosylated enzyme (dgPDH) when it spontaneously loses a C-terminal fragment when stored in a buffer solution at 4 °C. The remaining larger fragment has a molecular weight of ∼46 kDa and exhibits higher volumetric activity for glucose oxidation compared with the deglycosylated and glycosylated (gPDH) forms of PDH. Flow injection amperometry and cyclic voltammetry were used to assess and compare the catalytic activity of the three investigated forms of PDH, "wired" to graphite electrodes with two different osmium redox polymers: [Os(4,4'-dimethyl-2,2'-bipyridine)2(poly(vinylimidazole))10Cl](+) [Os(dmbpy)PVI] and [Os(4,4'-dimethoxy-2,2'-bipyridine)2(poly-(vinylimidazole))10Cl](+) [Os(dmobpy)PVI]. When "wired" with Os(dmbpy)PVI, the graphite electrodes modified with fdgPDH showed a pronounced increase in the current density with Jmax 13- and 6-fold higher than that observed for gPDH- and dgPDH-modified electrodes, making the fragmented enzyme extraordinarily attractive for further biotechnological applications. An easier access of the substrate to the active site and improved communication between the enzyme and mediator matrix are suggested as the two main reasons for the excellent performance of the fdgPDH when compared with that of gPDH and dgPDH. Three of the four glycosites in PDH: N(75), N(175), and N(252) were assigned using mass spectrometry in conjunction with endoglycosidase treatment and tryptic digestion. Determination of the asparagine residues carrying carbohydrate moieties in PDH can serve as a solid background for production of recombinant enzyme lacking glycosylation.

Membraneless glucose/oxygen enzymatic fuel cells using redox hydrogel films containing carbon nanotubes.

Co-immobilisation of three separate multiple blue copper oxygenases, a Myceliophthora thermophila laccase, a Streptomyces coelicolor laccase and a Myrothecium verrucaria bilirubin oxidase, with an [Os(2,2'-bipyridine)2 (polyvinylimidazole)10Cl](+/2+) redox polymer in the presence of multi-walled carbon nanotubes (MWCNTs) on graphite electrodes results in enzyme electrodes that produce current densities above 0.5 mA cm(-2) for oxygen reduction at an applied potential of 0 V versus Ag/AgCl. Fully enzymatic membraneless fuel cells are assembled with the oxygen-reducing enzyme electrodes connected to glucose-oxidising anodes based on co-immobilisation of glucose oxidase or a flavin adenine dinucleotide-dependent glucose dehydrogenase with an [Os(4,4'-dimethyl-2,2'-bipyridine)2(polyvinylimidazole)10Cl](+/2+) redox polymer in the presence of MWCNTs on graphite electrodes. These fuel cells can produce power densities of up to 145 µW cm(-2) on operation in pH 7.4 phosphate buffer solution at 37 °C containing 150 mM NaCl, 5 mM glucose and 0.12 mM O2. The fuel cells based on Myceliophthora thermophila laccase enzyme electrodes produce the highest power density if combined with glucose oxidase-based anodes. Although the maximum power density of a fuel cell of glucose dehydrogenase and Myceliophthora thermophila laccase enzyme electrodes decreases from 110 µW cm(-2) in buffer to 60 µW cm(-2) on testing in artificial plasma, it provides the highest power output reported to date for a fully enzymatic glucose-oxidising, oxygen-reducing fuel cell in artificial plasma.

Optimization of a membraneless glucose/oxygen enzymatic fuel cell based on a bioanode with high coulombic efficiency and current density.

After initial testing and optimization of anode biocatalysts, a membraneless glucose/oxygen enzymatic biofuel cell possessing high coulombic efficiency and power output was fabricated and characterized. Two sugar oxidizing enzymes, namely, pyranose dehydrogenase from Agaricus meleagris (AmPDH) and flavodehydrogenase domains of various cellobiose dehydrogenases (DH(CDH)) were tested during the pre-screening. The enzymes were mixed, "wired" and entrapped in a low-potential Os-complex-modified redox-polymer hydrogel immobilized on graphite. This anode was used in combination with a cathode based on bilirubin oxidase from Myrothecium verrucaria adsorbed on graphite. Optimization showed that the current density for the mixed enzyme electrode could be further improved by using a genetically engineered variant of the non-glycosylated flavodehydrogenase domain of cellobiose dehydrogenase from Corynascus thermophilus expressed in E. coli (ngDH(CtCDHC310Y)) with a high glucose-turnover rate in combination with an Os-complex-modified redox polymer with a high concentration of Os complexes as well as a low-density graphite electrode. The optimized biofuel cell with the AmPDH/ngDH(CtCDHC310Y) anode showed not only a similar maximum voltage as with the biofuel cell based only on the ngDH(CtCDHC310Y) anode (0.55 V) but also a substantially improved maximum power output (20 µW cm(-2)) at 300 mV cell voltage in air-saturated physiological buffer. Most importantly, the estimated half-life of the mixed biofuel cell can reach up to 12 h, which is apparently longer than that of a biofuel cell in which the bioanode is based on only one single enzyme.

Mediated glucose enzyme electrodes by cross-linking films of osmium redox complexes and glucose oxidase on electrodes.

Here, we report on a novel, versatile approach for the preparation of mediated enzyme electrodes, demonstrated using cross-linked films of glucose oxidase and a range of functionalised osmium complexes on graphite electrodes. Response of enzyme electrodes are optimised by evaluation of glucose response as a function of variation in ratios of [Os(2,2'-bipyridine)2(4-aminomethyl pyridine)Cl](+) redox mediator, polyallylamine support and glucose oxidase enzyme cross-linked using a di-epoxide reagent in films on graphite. Lowering of the redox potential required to mediate glucose oxidation is achieved by synthesis of complexes using (4,4'-dimethyl-2,2'-bipyridine) or (4,4'-dimethoxy-2,2'-bipyridine) as a ligand instead of (2,2'-bipyridine). Enzyme electrodes prepared using the complexes based on dimethoxy- or dimethyl-substituted bipyridines provide glucose oxidation current densities of 30 and 70 µA cm(-2) at 0.2 and 0.35 V applied potential compared to 120 µA cm(-2) at 0.45 V for the initial enzyme electrode, under pseudo-physiological conditions in 5 mM glucose, with stability of signals proving inadequate for long-term operation. Current output and stability may be improved by selection of alternate anchoring and cross-linking methodology, to provide enzyme electrodes capable for application to long-term glucose biosensors and anodes in enzymatic fuel cells.

Mediated electron transfer of cellobiose dehydrogenase and glucose oxidase at osmium polymer-modified nanoporous gold electrodes.

Nanoporous and planar gold electrodes were utilised as supports for the redox enzymes Aspergillus niger glucose oxidase (GOx) and Corynascus thermophilus cellobiose dehydrogenase (CtCDH). Electrodes modified with hydrogels containing enzyme, Os-redox polymers and the cross-linking agent poly(ethylene glycol)diglycidyl ether were used as biosensors for the determination of glucose and lactose. Limits of detection of 6.0 (±0.4), 16.0 (±0.1) and 2.0 (±0.1) µM were obtained for CtCDH-modified lactose and glucose biosensors and GOx-modified glucose biosensors, respectively, at nanoporous gold electrodes. Biofuel cells composed of GOx- and CtCDH-modified gold electrodes were utilised as anodes, together with Myrothecium verrucaria bilirubin oxidase (MvBOD) or Melanocarpus albomyces laccase as cathodes, in biofuel cells. A maximum power density of 41 µW/cm(2) was obtained for a CtCDH/MvBOD biofuel cell in 5 mM lactose and O2-saturated buffer (pH 7.4, 0.1 M phosphate, 150 mM NaCl).

Mediated electron transfer in glucose oxidising enzyme electrodes for application to biofuel cells: recent progress and perspectives.

Glucose oxidising enzyme electrodes have long been studied for their application to biosensors and, more recently, anodes in biofuel cells. At a fundamental level, insight into enzyme electron transfer and oxidation current generation at enzyme electrodes can be gained by systematic studies on integration of surfaces, biocatalysts, and artificial substrates (mediators). In this perspective, we present an overview of methods to aid the development of glucose oxidising enzyme electrodes based on mediated electron transfer for application to continuous-use anodes in a biofuel cell. Focus is placed on the rational design of mediators, based on osmium redox complexes, and screening of the activity of such complexes as mediators for glucose oxidising enzymes. An overview of the performance of enzyme electrodes, focused predominantly on crosslinked films of redox polymers and glucose oxidase, for glucose oxidation, is presented and approaches to improve both current output and stability of such enzyme electrodes are discussed.

Cross-Linkable Polymer-Based Multi-layers for Protecting Electrochemical Glucose Biosensors against Uric Acid, Ascorbic Acid, and Biofouling Interferences.

The lifetime of implantable electrochemical glucose monitoring devices is limited due to the foreign body response and detrimental effects from ascorbic acid (AA) and uric acid (UA) interferents that are components of physiological media. Polymer coatings can be used to shield biosensors from these interferences and prolong their functional lifetime. This work explored several approaches to protect redox polymer-based glucose biosensors against such interferences by designing six targeted multi-layer sensor architectures. Biological interferents, like cells and proteins, and UA and AA interferents were found to have individual effects on the current density and operational stability of glucose biosensors, requiring individual protection and treatment. Protection against biofouling can be achieved using a poly(2-methacryloyloxyethyl phosphorylcholine-co-glycidyl methacrylate) (MPC) zwitterionic polymer coating. An enzyme-scavenging approach was compared to electrostatic repulsion by negatively charged polymers for protection against AA and UA interferences. A multi-layer novel polymer design (PD) system consisting of a cross-linkable negatively charged polyvinylimidazole-polysulfostyrene co-polymer inner layer and a cross-linkable MPC zwitterionic polymer outer layer showed the best protection against AA, UA, and biological interferences. The sensor protected using the novel PD shield displayed the lowest mean absolute relative difference between the glucose reading without the interferent and the reading value with the interferent present and also displayed the lowest variability in sensor readings in complex media. For sensor measurements in artificial plasma, the novel PD extends the linear range (R2 = 0.99) of the sensor from 0-10 mM for the control to 0-20 mM, shows a smaller decrease in sensitivity, and retains high current densities. The application of PD multi-target coating improves sensor performance in complex media and shows promise for use in sensors operating in real conditions.

Tethering zwitterionic polymer coatings to mediated glucose biosensor enzyme electrodes can decrease sensor foreign body response yet retain sensor sensitivity to glucose.

Foreign body response (FBR) is a major challenge that affects implantable biosensors and medical devices, including glucose biosensors, leading to a deterioration in device response over time. Polymer shields are often used to mitigate this issue. Zwitterionic polymers (ZPs) are a promising class of materials that reduce biofouling of implanted devices. A series of ZPs each containing tetherable epoxide functional groups was synthesised for application as a polymer shield for eventual application as implantable glucose biosensors. The polymer shields were initially tested for the ability to resist fibrinogen adsorption and fibroblast adhesion. All synthesised ZPs showed comparable behaviour to a commercial Lipidure ZP in resisting fibrinogen adsorption. Nafion, a common anionic shield used against electrochemical interferents, showed higher protein adsorption and comparable cell adhesion resistance as uncoated control surfaces. However, a poly(2-methacryloyloxyethyl phosphorylcholine-co-glycidyl methacrylate) (MPC)-type ZP showed similar behaviour to Lipidure, with approximately 50% reduced fibrinogen adsorption and 80% decrease in fibroblast adhesion compared to uncoated controls. An MPC-coated amperometric glucose biosensor showed comparable current density and a 1.5-fold increase in sensitivity over an uncoated control biosensor, whereas all other polymer shields tested, including Lipidure, Nafion and a poly(ethyleneglycol) polymer, resulted in lower sensitivity and current density. Collectively, these characteristics make MPC-polymer shield coatings an appealing possibility for use in implantable glucose sensors and other implanted devices with the aim of reducing FBR while maintaining sensor performance.

Continuous ex vivo glucose sensing in human physiological fluids using an enzymatic sensor in a vein replica.

Managing blood glucose can affect important clinical outcomes during the intraoperative phase of surgery. However, currently available instruments for glucose monitoring during surgery are few and not optimized for the specific application. Here we report an attempt to exploit an enzymatic sensor in a vein replica that could continuously monitor glucose level in an authentic human bloodstream. First, detailed investigations of the superficial venous systems of volunteers were carried out using ocular and palpating examinations, as well as advanced ultrasound measurements. Second, a tubular glucose-sensitive biosensor mimicking a venous system was designed and tested. Almost ideal linear dependence of current output on glucose concentration in phosphate buffer saline was obtained in the range 2.2-22.0 mM, whereas the dependence in human plasma was less linear. Finally, the developed biosensor was investigated in whole blood under homeostatic conditions. A specific correlation was found between the current output and glucose concentration at the initial stage of the biodevice operation. However, with time, blood coagulation during measurements negatively affected the performance of the biodevice. When the experimental results were remodeled to predict the response without the influence of blood coagulation, the sensor output closely followed the blood glucose level.

Electron-transfer studies with a new flavin adenine dinucleotide dependent glucose dehydrogenase and osmium polymers of different redox potentials.

A new extracellular flavin adenine dinucleotide (FAD)-dependent glucose dehydrogenase from Glomerella cingulata (GcGDH) was electrochemically studied as a recognition element in glucose biosensors. The redox enzyme was recombinantly produced in Pichia pastoris and homogeneously purified, and its glucose-oxidizing properties on spectrographic graphite electrodes were investigated. Six different Os polymers, the redox potentials of which ranged in a broad potential window between +15 and +489 mV versus the normal hydrogen electrode (NHE), were used to immobilize and "wire" GcGDH to the spectrographic graphite electrode's surface. The GcGDH/Os polymer modified electrodes were evaluated by chronoamperometry using flow injection analysis. The current response was investigated using a stepwisely increased applied potential. It was observed that the ratio of GcGDH/Os polymer and the overall loading of the enzyme electrode significantly affect the performance of the enzyme electrode for glucose oxidation. The best-suited Os polymer [Os(4,4'-dimethyl-2,2'-bipyridine)(2)(PVI)Cl](+) had a potential of +309 mV versus NHE, and the optimum GcGDH/Os polymer ratio was 1:2 yielding a maximum current density of 493 µA·cm(-2) at a 30 mM glucose concentration.