Surface functionalization of polypyrrole film with glucose oxidase and viologen

A surface modification technique was developed for the functionalization of polypyrrole (PPY) film with glucose oxidase (GOD) and viologen moieties. The PPY film was first graft copolymerized with acrylic acid (AAc) and GOD was then covalently immobilized through the amide linkage formation between the amino groups of the GOD and the carboxyl groups of the grafted AAc polymer chains in the presence of a water-soluble carbodiimide. Viologen moieties could also be attached to the PPY film via graft-copolymerization of vinyl benzyl chloride with the PPY film surface followed by reaction with 4,4'-bipyridine and alpha,alpha'-dichloro-p-xylene. X-ray photoelectron spectroscopy (XPS) was used to characterize the PPY films after each surface modification step. Increasing the AAc graft concentration would allow a greater amount of GOD to be immobilized but this would decrease the electrical conductivity of the PPY film. The activity of the immobilized GOD was compared with that of free GOD and the kinetic effects were also studied. The immobilized GOD was found to be less sensitive to temperature deactivation as compared to the free GOD. The results showed that the covalent immobilization technique offers advantages over the technique involving the entrapment of GOD in PPY films during electropolymerization. The presence of viologen in the vicinity of the immobilized GOD also enabled the GOD-catalyzed oxidation of glucose to proceed under UV irradiation in the absence of O(2).     

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.     

A glucose oxidase electrode based on polypyrrole with polyanion/PEG/enzyme conjugate dopant

This study investigated a new glucose sensor prepared by electrochemical polymerization of pyrrole with polyanion/poly(ethylene glycol) (PEG)/glucose oxidase (GOD) conjugate dopants. GOD was coupled to a strong polyanion, poly(2-acrylamido-2-methylpropane sulfonic acid) (AMPS) via PEG spacer to effectively and reproducibly immobilize GOD within a polypyrrole matrix onto a Pt electrode surface. PEGs with four different chain lengths (1000, 2000, 3000, and 4000) were used as spacers to study the spacer length effect on enzyme immobilization and electrode function. After conjugation, more than 90% of the GOD bioactivity was preserved and the bioactivity of the conjugated GOD increased with longer PEG spacers. The resulting polyanion/PEG/GOD conjugate was used as a dopant for electropolymerizing pyrrole. The activity of the immobilized enzyme on the electrode ranged from 119 to 209 mU cm(-2) and the bioactivity increased with the use of longer PEG spacers. The amperometric response of the enzyme electrode was linear up to 20 mM glucose concentration with a sensitivity ranging from 180 to 270 nA mM(-1) cm(-2). The kinetic parameters Michaelis-Menten constant (K(M)(app)) and maximum current density (j(max)) depended on the amount of active enzyme, level of substrate diffusion, and PEG spacer length. An increase in the electrical charge passed during polymerization (thus, increasing polypyrrole thickness) to 255 mC cm(-2) increased the sensitivity of the enzyme electrode because of the greater amount of incorporated enzyme. However, although the amount of incorporated GOD continued to increase when the charge increased above 255 mC cm(-2), the sensitivity began to decline gradually. The condition for preparing the enzyme electrode was optimized at 800 mV potential with a dopant concentration of 1 mg ml(-1).     

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.     

Physicochemical and blood compatibility characterization of polypyrrole surface functionalized with heparin

A surface modification technique was developed for the covalent immobilization of heparin onto electrically conductive polypyrrole (PPY) film. The PPY film was first graft copolymerized with poly(ethylene glycol) methacrylate (PEGMA) and then activated with cyanuric chloride (CC). Heparin was then immobilized onto the film through the reaction between the chloride groups of CC and the amine and/or hydroxyl groups of heparin. X-ray photoelectron spectroscopy (XPS) was used to characterize the surface-modified film after each stage. The biocompatibility of the surface-modified PPY was evaluated using plasma recalcification time (PRT) and platelet adhesion. After surface modification, the film had improved wettability while retaining significant electrical conductivity. With immobilized heparin, platelet adhesion and platelet activation on PPY film was significantly suppressed, and the PRT was significantly prolonged. Electrical stimulation also plays a positive role in decreasing platelet adhesion and increasing PRT on pristine and surface-modified PPY films.     

葡萄糖氧化酶的有机相共价固定化

将葡萄糖氧化酶(GOD)在最适pH条件下冻干后,以戊二醛活化的壳聚糖为载体,分别在传统水相和1,4-二氧六环、乙醚、乙醇三种不同的有机相中进行共价固定化。通过比较水相固定化酶和有机相固定化酶的酶比活力、酶学性质及酶动力学参数,考察酶在有机相中的刚性特质对酶在共价固定化过程中保持酶活力的影响。结果表明,戊二醛浓度为0.1%、加酶量为80 mg/1 g载体、含水1.6%的1,4-二氧六环有机相固定化GOD与水相共价固定化GOD相比,酶比活力提高2.9倍,有效酶活回收率提高3倍;在连续使用7次后,1,4-二氧六环有机相固定化GOD的酶活力仍为相应水相固定化酶的3倍。在酶动力学参数方面,不论是表观米氏常数,最大反应速度还是转换数,1,4-二氧六环有机相固定化的GOD(Kmapp=5.63 mmol/L,Vmax=1.70μmol/(min.mgGOD),Kcat=0.304 s-1)都优于水相共价固定化GOD(Kmapp=7.33 mmol/L,Vmax=1.02μmol/(min.mg GOD),Kcat=0.221 s-1)。因此,相比于传统水相,GOD在合适的有机相中进行共价固定化可以获得具有更高酶活力和更优催化性质的固定化酶。该发现可能为酶蛋白在共价固定化时因构象改变而丢失生物活性的问题提供解决途径。     

Covalent immobilization of glucose oxidase on well-defined poly(glycidyl methacrylate)-Si(111) hybrids from surface-initiated atom-transfer radical polymerization

A simple one-step procedure was employed for the covalent immobilization of an atom-transfer radical polymerization (ATRP) initiator, via the robust Si-C bond, on the hydrogen-terminated Si(111) surface (Si-H surface). Well-defined poly(glycidyl methacrylate) [P(GMA)] brushes, tethered directly on the (111)-oriented single-crystal silicon surface, were prepared via surface-initiated ATRP. Kinetics study on the surface-initiated ATRP of glycidyl methacrylate revealed that the chain growth from the silicon surface was consistent with a "controlled" process. A relatively high concentration of glucose oxidase (GOD; above 0.2 mg/cm2) could be coupled directly to the well-defined P(GMA) brushes via the ring-opening reaction of the epoxide groups with the amine moieties of the enzyme. The resultant GOD-functionalized P(GMA) brushes, with the accompanying hydroxyl groups from the ring-opening reaction of the epoxide groups, serves as an effective spacer to provide the GOD with a higher degree of conformational freedom and a more hydrophilic environment. An equivalent enzyme activity above 1.6 units/cm2 [micromoles of beta-D-(+)-glucose oxidized to d-gluconolactone per minute per square centimeter] and a corresponding relative activity of about 60% could be readily achieved. The immobilized GOD also exhibited an improved stability during storage over that of the free enzyme. The GOD-functionalized silicon substrates are potentially useful to the development of silicon-based glucose biosensors.     

Assessment of in vitro bioactivity of hyaluronic acid and sulfated hyaluronic acid functionalized electroactive polymer

Electrically conductive polypyrrole (PPY) was surface functionalized with hyaluronic acid (HA) and sulfated hyaluronic acid (SHA) to improve its surface biocompatibility. The immobilization of HA on the PPY film was facilitated by the use of a cross-linker having the appropriate functional groups. The biological activity of the HA functionalized PPY film was assessed by means of an in vitro PC12 cell culture. The cell attachment on different substrates was studied and determined by bicinchoninic acid protein analysis. Cell attachment on the HA functionalized PPY film surface was significantly enhanced in the presence of nerve growth factor. The SHA functionalized PPY film was obtained by the sulfonation of the immobilized HA using pyridinesulfonate. The retention of the biological activity of the immobilized HA after sulfonation was evaluated by the in vitro assessment of the plasma recalcification time (PRT) and platelet adhesion on the substrate. The PRT observed from the SHA functionalized PPY film was significantly prolonged compared with the HA functionalized PPY. Some reduction of platelet adhesion was observed for the SHA functionalized PPY film, compared with that of the HA functionalized PPY film.     

Simultaneous co-immobilization of glucose oxidase and catalase in their substrates

Glucose oxidase (GOD) and catalase (CAT) were simultaneously co-immobilized onto magnesium silicate (Florisil®) by covalent coupling. Glucose was added in immobilization mixture and hydrogen peroxide, which is the substrate of CAT, was produced in coupling mixture during immobilization time. Therefore, co-immobilization of GOD and CAT was carried out in the presence of both their substrates: glucose and hydrogen peroxide, respectively. The effect of glucose concentration in immobilization mixture on activities of GOD and CAT of co-immobilized samples were investigated. Maximum GOD and CAT activities were determined for samples co-immobilized in the presence of 15 and 20 mM glucose, respectively. Co-immobilization of GOD and CAT in the presence of their substrates highly improved the activity and reusability of both enzymes.     

Enzyme immunosensors based on electropolymerized polytyramine modified electrodes

Highly sensitive amperometric enzyme immunosensors for human immunoglobulin G (IgG) were prepared on the basis of electrogenerated polytyramine (PTy, tyramine = p-(2-aminoethyl)-phenol) modified electrodes. Properties of PTy films changed depending on electrolysis conditions. On the basis of the found properties of the films, an effective IgG sensor was prepared: a PTy film was formed first from an acid solution on a Pt electrode, and the surface was further covered with a PTy film from an alkaline methanol solution to give a PTy doubly coated electrode on which anti-IgG was then immobilized. This electrode provided a large surface area with little non-specific adsorption of proteins. By means of the competitive enzyme immunoassay technique using glucose oxidase (GOD) labeled IgG conjugates, IgG was determined in the concentration range of c. 10 pg/ml-1 mg/ml from the oxidation current of H2O2 generated by the enzyme (GOD) reaction using the above IgG sensor. Also, an anti-IgG immobilized electrode, prepared by using a Pt electrode singly covered with a PTy film from an alkaline methanol solution, acted as an effective IgG sensor with a detection limit for IgG of c. 100 pg/ml.     

Application of an enzyme-based biofuel cell containing a bioelectrode modified with deoxyribonucleic acid-wrapped single-walled carbon nanotubes to serum

Enzyme-based biofuel cells (EFCs) are a form of biofuel cells (BFCs) that can utilize redox enzymes as biocatalysts. Applications of an EFC to an implantable system are evaluated under mild conditions, such as ambient temperature or neutral pH. In the present study, an EFC containing a bioelectrode modified with deoxyribonucleic acid (DNA)-wrapped single-walled carbon nanotubes (SWNTs) was applied to a serum system. The protection of immobilized glucose oxidase (GOD) using DNA-wrapped SWNTs was investigated in a trypsin environment, which can exist in a serum. GOD is immobilized by masking the active site onto the anode electrode. The anode/cathode system in the cell was composed of GOD/laccase as the biocatalysts and glucose/oxygen as the substrates in serum. The electrical properties of the anode in serum according to cyclic voltammetry (CV cycle) were improved using the DNA-wrapped SWNTs. Overall, an EFC that employed DNA-wrapped SWNTs and GOD immobilization in conjunction with protection of the active site increased the stability of GOD in serum, which enabled a high level of power production (ca. 190 μW/cm(2)) for up to 1 week.     

Nanoflake-like SnS₂ matrix for glucose biosensing based on direct electrochemistry of glucose oxidase

A novel biosensor is developed based on immobilization of proteins on nanoflake-like SnS? modified glass carbon electrode (GCE). With glucose oxidase (GOD) as a model, direct electrochemistry of the GOD/nanoflake-like SnS? is studied. The prepared SnS? has large surface area and can offer favorable microenvironment for facilitating the electron transfer between protein and electrode surface. The properties of GOD/SnS? are characterized by scanning electron microscopy (SEM), X-ray diffraction (XRD), UV-vis spectroscopy, Fourier transform infrared spectroscopy (FTIR) and cyclic voltammetry (CV), respectively. The immobilized enzyme on nanoflake-like SnS? retains its native structure and bioactivity and exhibits a surface-controlled, reversible two-proton and two-electron transfer reaction with the apparent electron transfer rate constant (k(s)) of 3.68 s?1. The proposed biosensor shows fast amperometric response (8s) to glucose with a wide linear range from 2.5 × 10?? M to 1.1 × 10?3 M, a low detection limit of 1.0 × 10?? M at signal-to-noise of 3 and good sensitivity (7.6 ± 0.5 mA M?1 cm?2). The resulting biosensor has acceptable operational stability, good reproducibility and excellent selectivity and can be successfully applied in the reagentless glucose sensing at -0.45 V. It should be worthwhile noting that it opens a new avenue for fabricating excellent electrochemical biosensor.     

Simultaneous co-immobilization of glucose oxidase and catalase in their substrates

Glucose oxidase (GOD) and catalase (CAT) were simultaneously co-immobilized onto magnesium silicate (florisil) by covalent coupling. Glucose was added in immobilization mixture and hydrogen peroxide which is the substrate of CAT was produced in coupling mixture during immobilization time. Therefore, co-immobilization of GOD and CAT was carried out in presence of both their substrate: glucose and hydrogen peroxide, respectively. The effect of glucose concentration in immobilization mixture on activities of GOD and CAT of co-immobilized samples were investigated. Maximum GOD and CAT activities were determined for samples co-immobilized in presence of 15 and 20 mM glucose, respectively. Co-immobilization of GOD and CAT in presence of their substrates highly improved the activity and reusability of both enzymes.     

Covalent immobilization of glucose oxidase onto poly(styrene-co-glycidyl methacrylate) monodisperse fluorescent microspheres synthesized by dispersion polymerization

In this study, micron-sized poly(styrene-co-glycidyl methacrylate) (PSt-GMA) fluorescent microspheres of 5.1microm in diameter were synthesized via dispersion polymerization of styrene and glycidyl methacrylate in the presence of 1,4-bis(5-phenyloxazol-2-yl) benzene (POPOP), which provided surface functional groups for covalent immobilization of enzymes. In an effort to study the biocompatibility of the microspheres' surface, glucose oxidase and beta-d-(+)-glucose were selected as a catalytic system for enzymatic assays. A colorimetric method was adopted in evaluating enzymatic activity by introducing horseradish peroxidase (HRP). Both the immobilization amount and the apparent activity of immobilized glucose oxidase from Aspergillus niger (GOD) were determined at different conditions. The results show that the immobilized enzymes retained approximately 28 to 34% activity, as compared with free enzymes, without pronounced alteration of the optimum pH and temperature. Kinetics studies show that the corresponding values of K(m) and V(max) are 23.2944 mM and 21.6450M/min.mg GOD for free enzymes and 35.1780 mM and 15.4799M/min.mg GOD for immobilized enzymes. The operational stability studies show that immobilized GOD could retain nearly 50% initial activity after being washed 20 times. The results suggest that the resultant PSt-GMA fluorescent microspheres provide a suitable surface for covalent immobilizing biomolecules; therefore, they have the potential of being used in fluorescence-based immunoassays in high-throughput screening or biosensors.     

Encapsulation of glucose oxidase microparticles within a nanoscale layer-by-layer film: immobilization and biosensor applications

We report on an immobilization strategy utilizing layer-by-layer encapsulated microparticles of enzymes within a nanoscale polyelectrolyte film. Encapsulation of glucose oxidase (GOD) microparticles was achieved by the sequential adsorption of oppositely charged polyelectrolytes onto the GOD biocrystal surface. The polyelectrolyte system polyallylamine/polystyrene sulfonate was used under high salt conditions to preserve the solid state of the highly water soluble GOD biocrystals during the encapsulation process. The resulting polymer multilayer capsule of about 15 nm wall thickness is permeable for small molecules (glucose), but non-permeable for macromolecules thus preventing the enzyme from leakage and at the same time shielding it from the outer environment e.g., from protease or microbial activity. Decrease of the buffer salt concentration leads to the dissolution of the enzyme under formation of μ-bioreactors. The spherical μ-bioreactors are bearing an extremely high loading of biocompound per volume. Encapsulated GOD was subsequently used to construct a biosensor by nanoengineered immobilisation of μ-bioreactor capsules onto an electrode surface. The presented approach demonstrates a general method to encapsulate highly soluble solid biomaterials and an immobilization strategy with the potential to create highly active thin and stable films of biomaterial.     

Direct electrochemistry and electrocatalysis of glucose oxidase immobilized on glassy carbon electrode modified by Nafion and ordered mesoporous silica-SBA-15

In this paper, it was found that glucose oxidase (GOD) has been stably immobilized on glassy carbon electrode modified by ordered mesoporous silica-SBA-15 and Nafion. The sorption behavior of GOD immobilized on SBA-15 matrix was characterized by transmission electron microscopy (TEM), ultraviolet–visible (UV–vis), FTIR, respectively, which demonstrated that SBA-15 can facilitate the electron exchange between the electroactive center of GOD and electrode. The direct electrochemistry and electrocatalysis behavior of GOD on modified electrode were characterized by cyclic voltammogram (CV) which indicated that GOD immobilized on Nafion and SBA-15 matrices displays direct, nearly reversible and surface-controlled redox reaction with an enhanced electron transfer rate constant of 3.89 s⁻¹ in 0.1 M phosphate buffer solution (PBS) (pH 7.12). Furthermore, it was also discovered that, in the absence of O₂, GOD immobilized on Nafion and SBA-15 matrices can produce a wide linear response to glucose in the positive potential range. Thus, Nafion/GOD-SBA-15/GC electrode is hopeful to be used in the third non-mediator's glucose biosensor. In addition, GOD immobilized on SBA-15 and Nafion matrices possesses an excellent bioelectrocatalytic activity for the reduction of O₂. The Nafion/GOD-SBA-15/GC electrode can be utilized as the cathode in biofuel cell.     

Immobilization of biocatalysts with bombyx mori silk fibroin by several kinds of physical treatment and its application to glucose sensors

Biocatalyst-immobilized Bombyx mori silk fibroin membrane was prepared. The insolubilization of the water-soluble membranes was performed by physical treatments only, i.e. stretching, compressing and standing under high humidity and methanol-immersion treatment, without any use ofcovalently binding reagent. All physical treatments performed were effective for the purpose of the immobilization of the enzymes in the membranes. The structural characterization of the glucose oxidase (GOD) immobilized membrane was performed in detail. The permeability of the substrate depends on the crystalline structure, i.e. the fraction of Silk I and Silk II of the membrane. The activity yield of the immobilized GOD was more than 80% of the value of free enzyme when 0–002% of the enzyme was entrapped in the membrane, but it decreased with increasing the concentration of the GOD in the membrane. This seems to result from diffusion limitation of the substrate. The pH and thermal stabilities of the immobilized enzyme were much improved, and were essentially independent of the methods of the immobilization. Development of the GOD or microorganism, Pseudomonas fluorescens immobilized silk fibroin membranes as glucose sensors are described.     

Glucose concentration determination by means of fluorescence emission spectra of soluble and insoluble glucose oxidase: some useful indications for optical fibre-based sensors

By using soluble and insoluble glucose oxidase, the changes in intrinsic emission fluorescence in the visible spectral region were studied as a function of glucose concentration. Insoluble glucose oxidase (GOD) was obtained by entrapment in a gelatine membrane or by covalent attachment on an agarose membrane grafted with hexamethylendiamine. The intensity of the fluorescence emission peak at 520 nm or the value of the integral fluorescence area from 480 to 580 nm were taken as physical parameters representative of the glucose concentration during the enzyme reaction. By using these parameters, linear calibration curves for glucose concentration were obtained. The extension of the calibration curve and the sensitivity of the adopted systems were found to be dependent on the enzyme state (free or immobilized) and on the immobilization method. In particular, it was found that the extent of the linear range of the calibration curves is increased of one order of magnitude when the glucose oxidase is immobilized, while the sensitivity of the measure is decreased of one order of magnitude by the immobilization process. Measures carried out by using the integral fluorescence area resulted more sensitive than those obtained with the peak size. Useful indications for the construction of optical fibre-based sensors were drawn from the reported results.     

Immobilization and direct electrochemistry of glucose oxidase on a tetragonal pyramid-shaped porous ZnO nanostructure for a glucose biosensor

A tetragonal pyramid-shaped porous ZnO (TPSP-ZnO) nanostructure is used for the immobilization, direct electrochemistry and biosensing of proteins. The prepared ZnO has a large surface area and good biocompatibility. Using glucose oxidase (GOD) as a model, this shaped ZnO is tested for immobilization of proteins and the construction of electrochemical biosensors with good electrochemical performances. The interaction between GOD and TPSP-ZnO is examined by using AFM, N(2) adsorption isotherms and electrochemical methods. The immobilized GOD at a TPSP-ZnO-modified glassy carbon electrode shows a good direct electrochemical behavior, which depends on the properties of the TPSP-ZnO. Based on a decrease of the electrocatalytic response of the reduced form of GOD to dissolved oxygen, the proposed biosensor exhibits a linear response to glucose concentrations ranging from 0.05 to 8.2mM with a detection limit of 0.01mM at an applied potential of -0.50V which has better biosensing properties than those from other morphological ZnO nanoparticles. The biosensor shows good stability, reproducibility, low interferences and can diagnose diabetes very fast and sensitively. Such the TPSP-ZnO nanostructure provides a good matrix for protein immobilization and biosensor preparation.     

Immobilization of glucose oxidase on electrodeposited nickel oxide nanoparticles: direct electron transfer and electrocatalytic activity

For the first time glucose oxidase (GOx) was successfully co-deposited on nickel-oxide (NiO) nanoparticles at a glassy carbon electrode. In this paper we present a simple fabrication method of biosensor which can be easily operated without using any specific reagents. Cyclic voltammetry was used for electrodeposition of NiO nanoparticle and GOx immobilization. The direct electron transfer of immobilized GOx displays a pair of well defined and nearly reversible redox peaks with a formal potential (E(0')) of -0.420 V in pH 7 phosphate buffer solution and the response shows a surface controlled electrode process. The surface coverage and heterogeneous electron transfer rate constant (k(s)) of GOx immobilized on NiO film glassy carbon electrode are 9.45 x 10(-13)mol cm(-2) and 25.2+/-0.5s(-1), indicating the high enzyme loading ability of the NiO nanoparticles and great facilitation of the electron transfer between GOx and NiO nanoparticles. The biosensor shows excellent electrocatalytical response to the oxidation of glucose when ferrocenmethanol was used as an artificial redox mediator. Furthermore, the apparent Michaelis-Menten constant 2.7 mM, of GOx on the nickel oxide nanoparticles exhibits excellent bioelectrocatalytic activity of immobilized enzyme toward glucose oxidation. In addition, this glucose biosensor shows fast amperometric response (3s) with the sensitivity of 446.2nA/mM, detection limit of 24 microM and wide concentration range of 30 microM to 5mM. This biosensor also exhibits good stability, reproducibility and long life time.