Co-immobilization of glucose oxidase and hexokinase on silicate hybrid sol-gel membrane for glucose and ATP detections

The co-immobilization of glucose oxidase (GOD) and hexokinase/glucose-6-phosphate dehydrogenase (HEX) in the silica hybrid sol-gel film for development of amperometric biosensors was investigated. The silica hybrid film fabricated by hydrolysis of the mixture of tetraethyl orthosilicate and 3-(trimethoxysiyl)propyl methacrylate possessed a three-dimension vesicle structure and good uniformity and conformability, and was ready for enzyme immobilization. The electrochemical and spectroscopic measurements showed that the silica hybrid sol-gel provided excellent matrice for the enzyme immobilization and that the immobilized enzyme retained its bioactivity effectively. The immobilized GOD could catalyze the oxidation of glucose, which could be used to determine glucose at +1.0 V without help of any mediator. The competition between GOD and HEX for the substrate glucose involving ATP as a co-substrate led to a decrease of the glucose response, which allowed us to develop an ATP sensor with a good stability. The fabricated silica hybrid sol-gel matrice offered a stage for further study of immobilization and electrochemistry of proteins.     

Stable enzyme biosensors based on chemically synthesized Au-polypyrrole nanocomposites

This work describes development and optimization of a generic method for the immobilization of enzymes in chemically synthesized gold polypyrrole (Au-PPy) nanocomposite and their application in amperometric biosensors. Three enzyme systems have been used as model examples: cytochrome c, glucose oxidase and polyphenol oxidase. The synthesis and deposition of the nanocomposite was first optimized onto a glassy carbon electrode (GCE) and then, the optimum procedure was used for enzyme immobilization and subsequent fabrication of glucose and phenol biosensors. The resulting nanostructured polymer strongly adheres to the surface of the GCE electrode, has uniform distribution and is very stable. The method has proved to be an effective way for stable enzyme attachment while the presence of gold nanoparticles provides enhanced electrochemical activity; it needs very small amounts of pyrrole and enzyme and the Au-PPy matrix avoids enzyme leaking. The preparation conditions, Michaelis-Menten kinetics and analytical performance characteristics of the two biosensors are discussed. Optimization of the experimental parameters was performed with regard to pyrrole concentration, enzyme amount, pH and operating potential. These biosensors resulted in rapid, simple, and accurate measurement of glucose and phenol with high sensitivities (1.089 mA/M glucose and 497.1 mA/M phenol), low detection limits (2 x 10(-6)M glucose and 3 x 10(-8)M phenol) and fast response times (less than 10s). The biosensors showed an excellent operational stability (at least 100 assays) and reproducibility (R.S.D. of 1.36%).     

Glucose biosensor prepared by glucose oxidase encapsulated sol-gel and carbon-nanotube-modified basal plane pyrolytic graphite electrode

A new glucose biosensor has been fabricated by immobilizing glucose oxidase into a sol-gel composite at the surface of a basal plane pyrolytic graphite (bppg) electrode modified with multiwall carbon nanotube. First, the bppg electrode is subjected to abrasive immobilization of carbon nanotubes by gently rubbing the electrode surface on a filter paper supporting the carbon nanotubes. Second, the electrode surface is covered with a thin film of a sol-gel composite containing encapsulated glucose oxidase. The carbon nanotubes offer excellent electrocatalytic activity toward reduction and oxidation of hydrogen peroxide liberated in the enzymatic reaction between glucose oxidase and glucose, enabling sensitive determination of glucose. The amperometric detection of glucose is carried out at 0.3 V (vs saturated calomel electrode) in 0.05 M phosphate buffer solution (pH 7.4) with linear response range of 0.2-20 mM glucose, sensitivity of 196 nA/mM, and detection limit of 50 microM (S/N=3). The response time of the electrode is < 5s when it is stored dried at 4 degrees C, the sensor showed almost no change in the analytical performance after operation for 3 weeks. The present carbon nanotube sol-gel biocomposite glucose oxidase sensor showed excellent properties for the sensitive determination of glucose with good reproducibility, remarkable stability, and rapid response and in comparison to bulk modified composite biosensors the amounts of enzyme and carbon nanotube needed for electrode fabrication are dramatically decreased.     

Imaging of ATP membrane transport with dual micro-disk electrodes and scanning electrochemical microscopy

Extracellular adenosine-5'-triphosphate (ATP) is involved in a variety of relevant regulatory mechanisms at a cellular level and has therefore been focus of extensive research. One of the major challenges associated with measuring this key regulatory analyte is the ability to detect and localize extracellular ATP with sufficient spatial and temporal resolution in physiological environments. In this study, scanning electrochemical microscopy (SECM) utilizing an amperometric micro-biosensor based on co-immobilization of the enzymes glucose oxidase and hexokinase is applied for imaging ATP transport through a porous polycarbonate membrane under physiologically relevant conditions. The enzymatic biosensor operates on competitive consumption of the substrate glucose between the immobilized enzymes glucose oxidase and hexokinase involving ATP as a co-substrate. Quantitative determination of the ATP concentration is based on a linear correlation between the glucose consumption and the ATP level. Integration of the amperometric ATP micro-biosensor into a dual micro-disk electrode configuration is achieved by immobilizing the enzymes at one of the micro-disk electrodes while the second disk serves as an unmodified amperometric probe for controlled positioning of the micro-biosensor in close proximity to the sample surface enabling quantification of the obtained current signal.     

Prussian Blue based screen printed biosensors with improved characteristics of long-term lifetime and pH stability

The promising advantages of Prussian Blue (PB) as catalyst and of the thick film screen printing technology have been combined to assemble sensors with improved characteristics for the amperometric determination of H(2)O(2). PB-modified screen printed electrodes were applied to detect H(2)O(2) at an applied potential of -0.05 V versus the internal screen printed Ag pseudoreference electrode, showing a detection limit of 10(-7) mol l(-1), a linearity range from 10(-7) to 5x10(-5) mol l(-1), a sensitivity of 234 microA mmol l(-1) cm(-2), and a high selectivity. Improved stability at alkaline pH values was also observed, which made possible their use with enzymes having an optimum basic pH. Then, the immobilisation of a single enzyme (glucose oxidase (GOD) or choline oxidase (ChOX)) or of two enzymes, acetylcholinesterase (AchE) coimmobilised with ChOX, has been performed on the surface of PB modified screen-printed electrodes (SPEs) using glutaraldehyde and Nafion. ChOX has been selected as an example of enzyme working at alkaline pH. The choline biosensors showed a detection limit of 5x10(-7) mol l(-1), a wide linearity range (5x10(-7)-10(-4) mol l(-1)), a high selectivity and a remarkable long term stability of 9 months at 4 degrees C, and at least 4 weeks at room temperature. Similar analytical characteristics and stability were observed with the acetylcholine biosensors.     

Novel biosensors for quantitative phytic acid and phytase measurement

Phytase (EC 3.1.3.26) and phytic acid (myo-inositol hexaphosphate) play an important environmental role in poultry industry and have a health aspect in food industry. Novel biosensors have been developed for simple, one step quantitative phytic acid and phytase detection. A system based on the sequentially acting enzyme phytase and pyruvate oxidase (POD) was employed for the development of phytase and phytic acid biosensors. Poly(carbamoylsulphonate) (PCS) hydrogel immobilized POD electrode was applied for the detection of phytase. It was based on the indication of phosphate ions produced by the hydrolysis of phytic acid. The phytase biosensor showed a linear response ranging from 0.5 to 6.0 units/ml. A bi-enzyme sensor based on co-immobilization of phytase and POD was developed for the detection of phytic acid on the basis of amperometric detection of the enzymatically-generated hydrogen peroxide at 0.6 V versus Ag/AgCl. It showed a linear response ranging from 0.2 to 2.0 mM with a detection limit of 0.002 mM.     

Amperometric glucose biosensor utilizing FAD-dependent glucose dehydrogenase immobilized on nanocomposite electrode

Amperometric glucose biosensors utilizing commercially available FAD-dependent glucose dehydrogenases from two strains of Aspergillus species are described. Enzymes were immobilized on nanocomposite electrode consisting of multi-walled carbon nanotubes by entrapment between chitosan layers. Unlike the common glucose oxidase based biosensor, the presented biosensors appeared to be O(2)-independent. The optimal amount of enzymes, working potential and pH value of working media of the glucose biosensors were determined. The biosensor utilizing enzyme isolated from Aspergillus sp. showed linearity over the range from 50 to 960 μM and from 70 to 620 μM for enzyme from Aspergillus oryzae. The detection limits were 4.45 μM and 4.15 μM, respectively. The time of response was found to be 60 s. The biosensors showed excellent operational stability - no loss of sensitivity after 100 consecutive measurements and after the storage for 4 weeks at 4 °C in phosphate buffer solution. When biosensors were held in a dessicator at room temperature without use, they kept the same response ability at least after 6 months. Finally, the results obtained from measurements of beverages and wine samples were compared with those obtained with the enzymatic-spectrophotometric and standard HPLC methods, respectively. Good correlation between results in case of analysis of real samples and good analytical performance of presented glucose biosensor allows to use presented concept for mass production and commercial use.     

Amperometric determination of glucose,based on the direct electron transfer between glucose oxidase and tin oxide

An amperometric glucose biosensor was designed for the detection of glucose in blood, urine, beverages, and fermentation systems. In typical glucose biosensors that employ enzymes, mediators are used for efficient electron transfer between the enzymes and the electrode. However, some of these mediators are known to be toxic to the enzymes and also must be immobilized on the surface of the electrode. We propose a mediator-free glucose biosensor that uses a glucose oxidase immobilized on a tin oxide electrode. Direct electron transfer is possible in this system because the tin oxide has redox properties similar to those of mediators. The method for immobilization of the glucose oxidase onto the tin oxide is also very simple. Tin oxide was prepared by the anodization and annealing of pure tin, and this provides a large surface area for the immobilization step because of its porosity. Glucose oxidase was immobilized onto the tin oxide using the membrane entrapment method. The proposed method provides a simple process for fabricating the enzyme electrode. Glucose oxidase immobilized onto the tin oxide, prepared in accordance with this method, has a relatively large current response when comparedto those of other glucose biosensors. The sensitivity of the biosensor was 19.55 μA/mM, and a linear response was observed between 0∼3 mM glucose. This biosensor demonstrated good reproducibility and stability.     

Mediation of glycosylated and partially-deglycosylated glucose oxidase of Aspergillus niger by a ferrocene-derivatised detergent.

A ferrocene-derivatised detergent, (11-ferrocenylundecyl) trimethylammonium bromide (FTMAB), when oxidised to the corresponding ferricinium ion, was found by electrochemical studies to be an effective electron acceptor for reduced glucose oxidase of Aspergillus niger (EC 1.13.4) and thus acts as a electron-transfer mediator between glucose oxidase and a working electrode held at a potential sufficiently positive to reoxidise reduced FTMAB. An increase in mediating activity was produced when FTMAB was present in concentrations above its critical micelle concentration. An 'enzyme electrode' was formed by adsorption of glucose oxidase and FTMAB surfactant on a graphite rod. The electrode functioned as an amperometric biosensor for glucose in phosphate-buffered saline solution. A mixed micelle of glucose oxidase and FTMAB, probably adsorbed on the electrode surface, appears to be advantageous for the amperometric determination of glucose. Additionally, glucose oxidase was treated with alpha-mannosidase. When this partially-deglycosylated glucose oxidase was incorporated in an enzyme electrode, a 100-fold increase in the second-order rate constant (k) for electron transfer between the enzyme and FTMAB was observed, together with increased current densities, with respect to the equivalent values for FTMAB and commercial glucose oxidase. The use of deglycosylated enzymes in biosensors is suggested.     

Enzyme-entrapping membranes for biosensors obtained by radiation-induced polymerization

A new method of physically immobilizing enzymes in poly(2-hydroxyethyl methacrylate) membranes was developed in order to obtain suitable biosensors. It was possible to prepare an enzyme sensor based on an oxygen Clark electrode and on glucose oxidase immobilized by low-temperature gamma radiation-induced polymerization. Temperature and pH effects on the activity of immobilized enzyme are described and the response characteristics of the resulting biosensor are summarized. The determination of glucose in standard solutions was carried out and a linear calibration curve, with an R² value of 0·9993, from the detection limit 5 × 10⁻⁵ to 1·2 × 10⁻³ was obtained. The biosensor was employed to analysis of control sera and the results were compared to those obtained by enzymatic-spectrophotometric detection.     

Fabrication of microband glucose biosensors using a screen-printing water-based carbon ink and their application in serum analysis

Microband glucose biosensors were fabricated by screen-printing a water-based carbon ink formulation containing cobalt phthalocyanine redox mediator and glucose oxidase (GOD) enzyme, then insulating and sectioning through the thick (20mum) film to expose a 3mm-long working electrode edge. The performance of these biosensors for glucose analysis was investigated at 25 degrees C. Voltammetry in glucose-containing buffer solutions established that an operating potential of +0.4V vs. Ag/AgCl was suitable for analysis under both stirring and quiescent conditions. The influence of pH on biosensor performance was established and an operational pH of 8.0 was selected. Steady-state responses were obtained under quiescent conditions, suggesting a mixed mechanism predominated by radial diffusion, indicative of microelectrode behaviour. Calibration studies obtained with these biosensors showed steady-state currents that were linearly dependent on glucose concentration from the limit of detection (0.27mM) up to 2.0mM, with a precision for replicate biosensors of 6.2-10.7%. When applied to the determination of glucose in human serum, the concentration compared favourably to that determined by a spectroscopic method. These results have demonstrated a simple means of fabricating biosensors for glucose measurement and determination in situations where low-current real-time monitoring under quiescent conditions would be desirable.     

The allosteric regulation of hexokinase C from amphibian liver.

A type C hexokinase (ATP:D-hexose-6-phosphotransferase EC 2.7.1.1) was partially purified from the liver of the frog Calyptocephalella caudiverbera. The enzyme is inhibited by glucose levels in the range of normal blood sugar concentrations. The extent of the inhibition by glucose depends on the concentration of ATP, being most marked between 1 and 5 mM ATP. Fructose, although a substrate, was not inhibitory of its own phosphorylation. The inhibitory effect of high glucose levels exhibited a strong, reversible pH dependence being most marked at pH 6.5. At pH 7.5 the inhibition by high glucose levels was a function of the enzyme concentration, the effect being stronger at high enzyme concentrations, whereas no inhibition was observed when assaying very diluted preparations. At all enzyme concentrations studied, high levels of glucose caused no inhibition at pH 8.5, whereas at pH 6.5 strong inhibition was always observed. Short times of photooxidation of hexokinase C as well as incubation with low concentrations of p-chloromercuribenzoate resulted in the loss of the inhibition by excess of glucose. Glucose-6-phosphate was found to be a strong inhibitor of hexokinase C but only at high glucose levels. The inhibitory effect of glucose-6-P follows sigmoidal kinetics at low (about 0.02 mM) glucose concentrations, the Hill coefficient being 2.3. The kinetics of the inhibition became hyperbolic at high (greater than 0.2 mM) glucose levels. These results suggest that the inhibition of hexokinase C by excess glucose is due to the interaction of glucose with a second, aldose-specific, regulatory site on the enzyme. The modification of the inhibitory effect by ATP, glucose-6-P, enzyme concentration, and pH, all of them at physiological levels, indicates a major role for hexokinase C in the regulation of glucose utilization by the liver.     

Optimization of a polypyrrole glucose oxidase biosensor

An amperometric glucose biosensor was fabricated by the electrochemical polymerization of pyrrole onto a platinum electrode in the presence of the enzyme glucose oxidase in a KCl solution at a potential of + 0·65 V versus SCE. The enzyme was entrapped into the polypyrrole film during the electropolymerization process. Glucose responses were measured by potentio-statting the enzyme electrode at a potential of + 0·7 V versus SCE in order to oxidize the hydrogen generated by the oxidation of glucose by the enzyme in the presence of oxygen. Experiments were performed to determined the optimal conditions of the polypyrrole glucose oxidase film preparation (pyrrole and glucose oxidase concentrations in the plating solution) and the response to glucose from such electrodes was evaluated as a function of film thickness, pH and temperature. It was found that a concentration of 0·3 M pyrrole in the presence of 65 U/ml of glucose oxidase in 0·01 M KCl were the optimal parameters for the fabrication of the biosensor. The optimal response was obtained for a film thickness of 0·17 μm (75 mC/cm²) at pH 6 and at a temperature of 313 K. The temperature dependence of the amperometric response indicated an activation energy of 41 kJ/mole. The linearity of the enzyme electrode response ranged from 1·0 mM to 7·5 mM glucose and kinetic parameters determined for the optimized biosensors were 33·4 mM for the K_m and 7·2 μA for the I_max. It was demonstrated that the internal diffusion of hydrogen peroxide through the polypyrrole layer to the platinum surface was the main limiting factor controlling the magnitude of the response of the biosensor to glucose. The response was directly related to the enzyme loading in the polypyrrole film. The shelf life and the operational stability of the optimized biosensor exceed 500 days and 175 assays, respectively. The substrate specificity of the entrapped glucose oxidase was not altered by the immobilization procedure.     

Amperometric glucose biosensor based on gold-deposited polyvinylferrocene film on Pt electrode

The preparations and performances of the novel amperometric biosensors for glucose based on immobilized glucose oxidase (GOD) on modified Pt electrodes are described. Two types of modified electrodes for the enzyme immobilization were used in this study, polyvinylferrocene (PVF) coated Pt electrode and gold deposited PVF coated Pt electrode. A simple method for the immobilization of GOD enzyme on the modified electrodes was described. The enzyme electrodes developed in this study were called as PVF-GOD enzyme electrode and PVF-Au-GOD enzyme electrode, respectively. The amperometric responses of the enzyme electrodes were measured at constant potential, which was due to the electrooxidation of enzymatically produced H2O2. The electrocatalytic effects of the polymer, PVF, and the gold particles towards the electrooxidation of the enzymatically generated H2O2 offers sensitive and selective monitoring of glucose. The biosensor based on PVF-Au-GOD electrode has 6.6 times larger maximum current, 3.8 times higher sensitivity and 1.6 times larger linear working portion than those of the biosensor based on PVF-GOD electrode. The effects of the applied potential, the thickness of the polymeric film, the amount of the immobilized enzyme, pH, the amount of the deposited Au, temperature and substrate concentration on the responses of the biosensors were investigated. The optimum pH was found to be pH 7.4 at 25 degrees C. Finally the effects of interferents, stability of the biosensors and applicability to serum analysis of the biosensor were also investigated.     

Direct electrochemistry of glucose oxidase and biosensing for glucose based on boron-doped carbon nanotubes modified electrode

Due to their unique physicochemical properties, doped carbon nanotubes are now extremely attractive and important nanomaterials in bioanalytical applications. In this work, selecting glucose oxidase (GOD) as a model enzyme, we investigated the direct electrochemistry of GOD based on the B-doped carbon nanotubes/glassy carbon (BCNTs/GC) electrode with cyclic voltammetry. A pair of well-defined, quasi-reversible redox peaks of the immobilized GOD was observed at the BCNTs based enzyme electrode in 0.1M phosphate buffer solution (pH 6.98) by direct electron transfer between the protein and the electrode. As a new platform in glucose analysis, the new glucose biosensor based on the BCNTs/GC electrode has a sensitivity of 111.57 microA mM(-1)cm(-2), a linear range from 0.05 to 0.3mM and a detection limit of 0.01mM (S/N=3). Furthermore, the BCNTs modified electrode exhibits good stability and excellent anti-interferent ability to the commonly co-existed uric acid and ascorbic acid. These indicate that boron-doped carbon nanotubes are the good candidate material for the direct electrochemistry of the redox-active enzyme and the construction of the related enzyme biosensors.     

AFM nanometer surface morphological study of in situ electropolymerized neutral red redox mediator oxysilane sol-gel encapsulated glucose oxidase electrochemical biosensors

Four different silica sol-gel films: methyltrimethoxysilane (MTMOS), tetraethoxysilane (TEOS), 3-aminopropyltriethoxysilane (APTOS) and 3-glycidoxypropyl-trimethoxysilane (GOPMOS) assembled onto highly oriented pyrolytic graphite (HOPG) were characterized using atomic force microscopy (AFM), due to their use in the development of glucose biosensors. The chemical structure of the oxysilane precursor and the composition of the sol-gel mixture both influenced the roughness, the size and the distribution of pores in the sol-gel films, which is relevant for enzyme encapsulation. The GOPMOS sol-gel film fulfils all the morphological characteristics required for good encapsulation of the enzyme, due to a smooth topography with very dense and uniform distribution of only small, 50nm diameter, pores at the surface. APTOS and MTMOS sol-gel films developed small pores together with large ones of 300-400nm that allow the leakage of enzymes, while the TEOS film formed a rough and incomplete network on the electrode, less suitable for enzyme immobilisation. GOPMOS sol-gel film with encapsulated glucose oxidase and poly(neutral red) redox mediator, prepared by in situ electropolymerization, were also morphologically characterized by AFM. The AFM results explain the variation of the stability in time, sensitivity and limit of detection obtained with different oxysilane sol-gel encapsulated glucose oxidase biosensors with redox mediator.     

Highly selective amperometric glucose microdevice derived from diffusion layer gap electrode

Although most of enzyme catalytic reactions are specific, the amperometric detection of the enzymatic reaction products is largely nonselective. How to improve the detection selectivity of the enzyme-based electrochemical biosensors has to be considered in the sensor fabrication procedures. Herein, a highly selective amperometric glucose biosensor based on the concept of diffusion layer gap electrode pair which we previously proposed was designed. In this biosensor, a gold tube coated with a conductive layer of glucose oxidase/Nafion/graphite was used to create an interference-free region in its diffusion layer by electrochemically oxidizing the interfering electroactive species at proper potentials. A Pt probe electrode was located in this diffusion layer of the tube electrode to selectively detect hydrogen peroxide generated from the enzyme catalytic oxidation of glucose in the presence of oxygen in the solution. In practical performance of the microdevice, parameters influencing the interference-removing efficiency, including the tip-tube opening distance, the tube electrode potential and the electrolyzing time had been investigated systematically. Results showed that glucose detection free from interferents could be achieved at the electrolyzing time of 30s, the tip-tube opening distance of 3mm and the tube electrode potential of 0.4V. The electrochemical response showed linear dependence on the concentration of glucose in the range of 1 x 10(-5) to 4 x 10(-3) M (the correlation coefficient: 0.9936, without interferents; 0.9995, with interferents). In addition, the effectiveness of this device was confirmed by numerical simulation using a model system of a solution containing interferents. The simulated results showed good agreement with the experimental data.     

Two biosensors for phenolic compounds based on mushroom (Agaricus bisporus) homogenate: comparison in terms of some important parameters of the biosensors

Interest in molecular imprinted polymer techniques has increased because they allows for the improvement of some stability characteristics of enzymes. The high stability of molecularly imprinted enzymes for a substrate can make them ideal alternatives as recognition elements for sensors. A bioimprinted mushroom tissue homogenate biosensor was constructed in a very simple way. For this purpose, sulfite was used. The enzyme, polyphenol oxidase, was first complexed by using a competitive inhibitor, sulfite, in aqueous medium and then the enzyme was immobilized on gelatin by crosslinking with glutaraldehyde on a glass electrode surface. Similarly, polyphenol oxidase uncomplexed with sulfite was also immobilized on a glass electrode in the same conditions. The aim of the study was to compare the two biosensors in terms of their repeatability and thermal, pH, and operational stability; also, the linear ranges of the two biosensors were compared with each other.     

Development of a high analytical performance amperometric glucose biosensor based on glucose oxidase immobilized in a composite matrix: layered double hydroxides/chitosan

This paper aimed at showing the interest of the composite material based on layered double hydroxides (LDHs) and chitosan (CHT) as suitable host matrix likely to immobilize enzyme onto electrode surface for amperometric biosensing application. This hybrid material combined the advantages of inorganic LDHs and organic biopolymer, CHT. Glucose oxidase (GOD) immobilized in the composite material maintained its activity well as the usage of glutaraldehyde was avoided. The process parameters for the fabrication of the enzyme electrode and various experimental variables such as pH, applied potential and temperature, were explored for optimum analytical performance of the enzyme electrode. The enzyme electrode provided a linear response to glucose over a concentration range of 1 x 10(-6) to 3 x 10(-3) M with a high sensitivity of 62.6 mA M(-1) cm(-2) and a detection limit of 0.1 muM based on the signal-to-noise ratio of 3.     

Fabrication of bienzyme nanobiocomposite electrode using functionalized carbon nanotubes for biosensing applications

Mediated biosensors consisting of an oxidase and peroxidase (POx) have attracted increasing attention because of their wider applicability. This work presents a novel approach to fabricate nanobiocomposite bienzymatic biosensor based on functionalized multiwalled carbon nanotubes (MWNTs) with the aim of evaluating their ability as sensing elements in amperometric transducers. Electrochemical behavior of the bienzymatic nanobiocomposite biosensor is investigated by Faradaic impedance spectroscopy and cyclic voltammetry. The results indicate that glucose oxidase (GOD) and horseradish peroxidase (HRP) are strongly adsorbed on the surface of the thionin (TH) functionalized MWNTs and demonstrate a facile electron transfer between immobilized GOD/HRP and the electrode via the functionalized MWNTs in a Nafion film. The functionalized carbon nanotubes act as molecular wires to allow efficient electron transfer between the underlying electrode and the redox centres of enzymes through TH. Linear ranges for these electrodes are from 10 nM to 10 mM for glucose and 17 nM to 56 mM for hydrogen peroxide with the detection limit of 3 and 6 nM, respectively. A remarkable feature of the bienzyme electrode is the possibility to determine glucose and hydrogen peroxide at a very low applied potential where the noise level and interferences from other electroactive compounds are minimal. Performance of the biosensor is evaluated with respect to response time, detection limit, selectivity, temperature and pH as well as operating and storage stability.