Fabrication of polymeric ionic liquid/graphene nanocomposite for glucose oxidase immobilization and direct electrochemistry

A novel polymeric ionic liquid functionalized graphene, poly(1-vinyl-3-butylimidazolium bromide)-graphene (denoted as poly(ViBuIm(+)Br(-))-G), was synthesized. FTIR, UV-vis spectra and TEM were used to characterize the formation of as synthesized nanocomposites. Due to the modification of the polymeric ionic liquid, poly(ViBuIm(+)Br(-))-G can not only be dispersed well in aqueous solutions to form a homogeneous colloidal suspension of individual nanosheets, but also exhibit a strong positive charge. Based on self-assembly, the negatively charged glucose oxidase (GOD) was immobilized onto the poly(ViBuIm(+)Br(-))-G to form a GOD/poly(ViBuIm(+)Br(-))-G/glassy carbon (GC) electrode under mild conditions. With the advantage of both poly(ViBuIm(+)Br(-)) and graphene, poly(ViBuIm(+)Br(-))-G can provide a favorable and conductive microenvironment for the immobilized GOD and thus promote their direct electron transfer at the GC electrode. Furthermore, the GOD/poly(ViBuIm(+)Br(-))-G/GC electrode displayed an excellent sensitivity, together with a wide linear range and excellent stability for the detection of glucose. Accordingly, these unique properties of such novel nanocomposite generate a promising platform for the construction of mediator-free enzymatic biosensors.     

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.     

Enhanced direct electrochemistry of glucose oxidase and biosensing for glucose via synergy effect of graphene and CdS nanocrystals

Integrating graphene-based composites with enzyme provides a potent strategy to enhance biosensor performance due to their unique physicochemical properties. Herein we report on the utilization of graphene-CdS (G-CdS) nanocomposite as a novel immobilization matrix for the enzymes, which glucose oxidase (GOD) was chosen as model enzyme. In comparison with the graphene sheet and CdS nanocrystal, G-CdS nanocomposite exhibited excellent electron transfer properties for GOD with the rate constant (k(s)) of 5.9 s(-1) due to the synergy effect of graphene sheet and CdS nanocrystals. Further, based on the decrease of the electrocatalytic response of the reduced form of GOD to dissolved oxygen, the obtained glucose biosensor displays satisfactory analytical performance over an acceptable linear range from 2.0 to 16 mM with a detection limit of 0.7 mM, and also prevents the effects of interfering species, which is suitable for glucose determination by real samples. These results mean that this immobilization matrix not only can be used for immobilizing GOD, but also can be extended to other enzymes and bioactive molecules, thus providing a promising platform for the development of biosensors.     

Multifunctional carbon nanotubes for direct electrochemistry of glucose oxidase and glucose bioassay

Polydopamine (Pdop) has recently been shown to adsorb to a wide variety of surfaces and serves as an adhesion layer to immobilize biological molecules. In this work, the multifunctional carbon nanotube (CNT) composites were prepared though the oxidation of dopamine at room temperature and subsequent electroless silver deposition by mildly stirring. The stable immobilization and direct electron transfer of glucose oxidase were achieved on the composite film modified glassy carbon electrode. The resulting electrode gave a well-defined redox peaks with a formal potential of about −482 mV (vs. SCE) in pH 7.0 buffer. The electron transfer rate constant was estimated to be 3.6 s⁻¹, due to the combined contribution of Pdop, CNTs and Ag nanoparticles with the help of Nafion. Furthermore, the method for detecting of glucose was proposed based on the decrease of oxygen caused by the enzyme-catalyzed reaction between glucose oxidase (GOD) and glucose. The linear response to glucose ranging from 50.0 μM to 1.1 mM (R² = 0.9958), with a calculated detection limit of 17.0 μM at a signal-to-noise ratio of 3. The low calculated apparent Michaelis–Menten constant was 5.46 mM, implying the high enzymatic activity and affinity of immobilized GOD for glucose. It can reasonably be expected that this observation might hold true for other noble metal nanostructure-electroactive protein systems, providing a promising platform for the development of biosensors and biofuel cells.     

An electrochemical investigation of glucose oxidase at a CdS nanoparticles modified electrode

The direct electrochemistry of glucose oxidase (GOD) adsorbed on a CdS nanoparticles modified pyrolytic graphite electrode was investigated, where the enzyme demonstrated significantly enhanced electron-transfer reactivity. GOD adsorbed on CdS nanoparticles maintained its bioactivity and structure, and could electro-catalyze the reduction of dissolved oxygen, which resulted in a great increase of the reduction peak current. Upon the addition of glucose, the reduction peak current decreased, which could be used for glucose detection. Performance and characteristics of the fabricated glucose biosensor were assessed with respect to detection limit, sensitivity, storage stability and interference exclusion. The results showed that the fabricated biosensor was sensitive and stable in detecting glucose, indicating that CdS nanoparticle was a good candidate material for the immobilization of enzyme in glucose biosensor construction.     

Enzymatically induced formation of neodymium hexacyanoferrate nanoparticles on the glucose oxidase/chitosan modified glass carbon electrode for the detection of glucose

The formation of neodymium hexacyanoferrate (NdHCF) nanoparticles (NPs) on the surface of glucose oxidase/chitosan (GOx/CHIT) modified glass carbon electrode induced by enzymatic reaction was described and characterized. CHIT can be used not only as enzyme immobilizer, but also to provide active sites for NPs growth. Results showed that the optimized conditions of the GOx/CHIT film induced NdHCF NPs for the biosensing of glucose were 1.0mM Nd(3+) and 20.0mM Fe(CN)(6)(3-). The biocatalyzed generation of NdHCF NPs enabled the development of an electrochemical biosensor for glucose. The calculated apparent Michaelis-Menten constant was 7.5mM. The linear range for glucose detection was 0.01-10.0mM with the correlation coefficient of 0.9946, and the detection limit was 5muM (S/N=3). Furthermore, this system avoids the interferences of other species during the biosensing process and can be used for the determination of glucose in human plasma samples.     

Immobilization of creatininase, creatinase and sarcosine oxidase on iron oxide nanoparticles/chitosan-g-polyaniline modified Pt electrode for detection of creatinine

Commercial enzymes, creatininase (CA) from Pseudomonas sp, creatinase (CI) from Pseudomonas sp, sarcosine oxidase (SO) from Bacillus sp were co-immobilized onto iron oxide nanoparticles/chitosan-graft-polyaniline (Fe(3)O(4)-NPs/CHIT-g-PANI) composite film electrodeposited on surface of Pt electrode through glutaraldehyde coupling. Transmission electron microscopy (TEM) was used for characterization of Fe(3)O(4)-NPs. A creatinine biosensor was fabricated using Enzymes/Fe(3)O(4)-NPs/CHIT-g-PANI/Pt electrode as working electrode, Ag/AgCl as reference electrode and Pt wire as auxiliary electrode. The enzyme electrode was characterized by cyclic voltammetry (CV), scanning electron microscopy (SEM), Fourier transform infrared (FTIR) spectroscopic and electrochemical impedance spectroscopy (EIS). The biosensor exhibited an optimum response within 2s at pH 7.5 and 30 °C, when polarized at 0.4V vs Ag/AgCl. The electrocatalytic response showed a linear dependence on creatinine concentration ranging from 1 to 800 μM. The sensitivity of the biosensor was 3.9 μA μM(-1) cm(-2), with a detection limit of 1 μM (S/N=3). Apparent Michaelis-Menton (K(m)) value for creatinine was 0.17 mM. The biosensor showed only 10% loss in its initial response after 120 uses over 200 days, when stored at 4 °C. The biosensor measured creatinine in the serum of apparently healthy persons which correlated well with a standard colorimetric method (r=0.99).     

Immobilization of glucose oxidase on graphene oxide for highly sensitive biosensors

Glucose oxidase (GOx) was immobilized onto graphene oxide (GRO) via three different preparation methods: enzyme adsorption (EA), enzyme adsorption and crosslinking (EAC), and enzyme adsorption, precipitation and crosslinking (EAPC). EAPC formulations, prepared via enzyme precipitation with 60% ammonium sulfate, showed 1,980 and 1,630 times higher activity per weight of GRO than those of EA and EAC formulations, respectively. After 59 days at room temperature, EAPC maintained 88% of initial activity, while EA and EAC retained 42 and 45% of their initial activities, respectively. These results indicate that the steps of precipitation and crosslinking in the EAPC formulation are critical to achieve high enzyme loading and stability of EAPC. EA, EAC and EAPC were used to prepare enzyme electrodes for use as glucose biosensors. Optimized EAPC electrode showed 93- and 25-fold higher sensitivity than EA and EAC, respectively. To further increase the sensitivity of EAPC electrode, multi-walled carbon nanotubes (MWCNTs) were mixed with EAPC for the preparation of enzyme electrode. Surprisingly, the EAPC electrode with additional 99.5 wt% MWCNTs showed 7,800-fold higher sensitivity than the EAPC electrode without MWCNT addition. Immobilization and stabilization of enzymes on GRO via the EAPC approach can be used for the development of highly sensitive biosensors as well as to achieve high enzyme loading and stability.     

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.     

Layer-by-layer fabrication and direct electrochemistry of glucose oxidase on single wall carbon nanotubes

Layer-by-layer assembly of glucose oxidase (GOx) with single-wall carbon nanotubes (SWCNTs) is achieved on the electrode surface based on the electrostatic attraction between positively charged GOx in pH 3.8 buffer and negatively charged carboxylic groups of CNTs. The cyclic voltammetry and electrochemical impedance spectroscopy are used to characterize the formation of multilayer films. In deaerated buffer solutions, the cyclic voltammetry of the multilayer films of {GOx/CNT}_n shows two pairs of well-behaved redox peaks that are assigned to the redox reactions of CNTs and GOx, respectively, confirming the effective immobilization of GOx on CNTs using the layer-by-layer technique. The redox peak currents of GOx increase linearly with the increased number of layers indicating the uniform growth of GOx in multilayer films. The dependence of the cyclic voltammetric response of GOx in multilayer films on the scan rate and pH is also studied. A linear decrease of the reduction current of oxygen at the {GOx/CNT}-modified electrodes with the addition of glucose suggests that such multilayer films of GOx retain the bioactivity and can be used as reagentless glucose biosensors.     

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.     

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.     

A glucose oxidase inmobilized electrode based on modified graphite

Glucose oxidase (E. C. 1.1.3.4) was immobilized on electrochemically modified graphite to obtain an enzyme electrode. The working surface of the electrode was coated with gelatine to prevent desorption of the enzyme. In substrate (glucose) solutions the amperometric signal of the enzyme electrode was due to the electroreduction of H202 generated in the enzyme layer. The linearity of the electrode response was found up to a substrate concentration of 300 microM at a working potential of 0 mV (vs. Ag/AgCl). It was shown that the electrode did not respond to L-ascorbic and uric acid at that working potential. The response time was about 2 min. The enzyme electrode keeps about 50% of its initial activity after a one-week storage at 4 degrees C.     

Reagentless glucose biosensor based on direct electron transfer of glucose oxidase immobilized on colloidal gold modified carbon paste electrode

The direct electrochemistry of glucose oxidase (GOD) adsorbed on a colloidal gold modified carbon paste electrode was investigated. The adsorbed GOD displayed a pair of redox peaks with a formal potential of -(449+/-1) mV in 0.1 M pH 5.0 phosphate buffer solution. The response showed a surface-controlled electrode process with an electron transfer rate constant of (38.9+/-5.3)/s determined in the scan rate range from 10 to 100 mV/s. GOD adsorbed on gold colloid nanoparticles maintained its bioactivity and stability. The immobilized GOD could electrocatalyze the reduction of dissolved oxygen and resulted in a great increase of the reduction peak current. Upon the addition of glucose, the reduction peak current decreased, which could be used for glucose detection with a high sensitivity (8.4 microA/mM), a linear range from 0.04 to 0.28 mM and a detection limit of 0.01 mM at a signal-to-noise ratio of 3sigma. The sensor could exclude the interference of commonly coexisted uric and ascorbic acid.     

Amperometric l-lysine determination biosensor amplified with l-lysine oxidase nanoparticles and graphene oxide nanoparticles

We describe the amplification of amperometric l-lysine biosensor using l-lysine oxidase nanoparticles (LOxNPs) and graphene oxide nanoparticles (GrONPs) immobilized onto pencil graphite electrode (PGE). LOxNPs and GrONPs were characterized by UV spectroscopy and transmission electron microscopy (TEM). The working electrode (LOxNPs/GrONPs/PGE) was studied by scanning electron microscopy (SEM) and cyclic voltammetry at different stages of its construction. The biosensor showed optimum current at 0.7 V, pH 6.5, 35 °C, a detection limit of 0.01 μM, response time as 3.95 s and a wider linear range 0.01–1000 μM. The analytical recovery of added lysine in sera was 97 %. The within assay and between batch coefficients of variation for the biosensor were 0.068 and 0.074 % respectively. The biosensor measured l-lysine levels in sera of healthy adults and human immunodeficiency virus (HIV) patients. The biosensor exhibited good correlation with standard spectrophotometric method (R² = 0.989). The biosensor lost 35 % of its original activity after its regular uses for a period of 180 days, while being stored dry at 4 °C.     

Urchinlike MnO2 nanoparticles for the direct electrochemistry of hemoglobin with carbon ionic liquid electrode

In this paper an urchinlike MnO(2) nanoparticle was synthesized by hydrothermal method and applied to the protein electrochemistry for the first time. By using a carbon ionic liquid electrode (CILE) as the basal electrode, hemoglobin (Hb) was immobilized on the surface of CILE with chitosan (CTS) and MnO(2) nanoparticle composite materials. Spectroscopic results indicated that Hb molecules retained its native structure in the composite film. A pair of well-defined redox peaks appeared on the cyclic voltammogram with the formal peak potential as -0.180 V (vs. SCE), which indicated that direct electron transfer of Hb was realized on the modified electrode. The result can be attributed to the specific characteristic of MnO(2) nanoparticle and the advantages of CILE, which facilitated the electron transfer rate. The fabricated CTS-MnO(2)-Hb/CILE showed good electrocatalytic ability to the reduction of trichloroacetic acid (TCA). Under the optimal conditions the catalytic current was in linear to TCA concentration in the range from 0.5 to 16.0 mmol L(-1) with the detection limit calculated as 0.167 mmol L(-1) (3σ). The result indicated that urchinlike MnO(2) nanoparticle had the potential application in the third generation electrochemical biosensors.     

Ultrasensitive DNA sensor based on gold nanoparticles/reduced graphene oxide/glassy carbon electrode

We have designed a simple and novel electrochemical biosensor based on glassy carbon electrode (GCE) for DNA detection. GCE was modified with reduced graphene oxide (RGO) and gold nanoparticles (AuNPs) by the electrochemical method, which is helpful for immobilization of thiolated bioreceptors. The electrode modification processes were characterized by scanning electron microscopy (SEM) and electrochemical methods. Then a single-stranded DNA (ssDNA) probe for BRCA1 5382 insC mutation detection was immobilized on the modified electrode for a specific time. The experimental conditions, such as probe immobilization time and target DNA (complementary DNA) hybridization time and temperature with probe DNA, were optimized using electrochemical methods. The electrochemical response for DNA hybridization and synthesis was measured using electrochemical impedance spectroscopy (EIS) and cyclic voltammetry (CV) methods. The calibration graph contains two linear ranges; the first part is in the range of 3.0 × 10⁻²⁰ to 1.0 × 10⁻¹² M, and the second segment part is in the range of 1.0 × 10⁻¹² to 1.0 × 10⁻⁷ M. The biosensor showed excellent selectivity for the detection of the complementary sequences from noncomplementary sequences, so it can be used for detection of breast cancer.     

Amperometric glucose biosensor based on layer-by-layer assembly of multilayer films composed of chitosan, gold nanoparticles and glucose oxidase modified Pt electrode

A new strategy for fabricating glucose biosensor was presented by layer-by-layer assembled chitosan (CS)/gold nanoparticles (GNp)/glucose oxidase (GOD) multilayer films modified Pt electrode. First, a cleaned Pt electrode was immersed in poly(allylamine) (PAA), and then transferred to GNp, followed by the adsorption of GOD (GOD/GNp/PAA/Pt). Second, the GOD/GNp/PAA/Pt electrode was immersed in CS, and then transferred to GNp, followed by the adsorption of GOD (GOD/GNp/CS/GOD/GNp/PAA/Pt). Third, different layers of multilayer films modified Pt electrodes were assembled by repeating the second process. Film assembling and characterization were studied by quart crystal microbalance, and properties of the resulting glucose biosensors were measured by electrochemical measurements. The results confirmed that the assembling process of multilayer films was simple to operate, the immobilized GOD displayed an excellent catalytic property to glucose, and GNp in the biosensing interface efficiently improved the electron transfer between analyte and electrode surface. The amperometric response of the biosensors uniformly increased from one to six layers of multilayer films, and then reached saturation after the seven layers. Among the resulting biosensors, the biosensor based on the six layers of multilayer films was best. It showed a wide linear range of 0.5-16 mM, with a detection limit of 7.0 microM estimated at a signal-to-noise ratio of 3, fast response time (within 8s). Moreover, it exhibited good reproducibility, long-term stability and interference free. This method can be used for constructing other thin films, which is a universal immobilization method for biosensor fabrication.     

Glucose biosensor based on immobilization of glucose oxidase in poly(o-aminophenol) film on polypyrrole-Pt nanocomposite modified glassy carbon electrode

Novel Pt nanoclusters embedded polypyrrole nanowires (PPy-Pt) composite was electrosynthesized on a glassy carbon electrode, denoted as PPy-Pt/GCE. A glucose biosensor was further fabricated based on immobilization of glucose oxidase (GOD) in an electropolymerized non-conducting poly(o-aminophenol) (POAP) film that was deposited on the PPy-Pt/GCE. The morphologies of the PPy nanowires and PPy-Pt nanocomposite were characterized by field emission scanning electron microscope (FE-SEM). Effect of experimental conditions involving the cycle numbers for POAP deposition and Pt nanoclusters deposition, applied potential used in glucose determination, temperature and pH value of the detection solution were investigated for optimization. The biosensor exhibited an excellent current response to glucose over a wide linear range from 1.5 × 10⁻⁶ to 1.3 × 10⁻² M (r = 0.9982) with a detection limit of 4.5 × 10⁻⁷ M (s/n = 3). Based on the combination of permselectivity of the POAP and the PPy films, the sensor had good anti-interference ability to ascorbic acid (AA), uric acid (UA) and acetaminophen. The apparent Michaelis–Menten constant (K_m) and the maximum current density (I_m) were estimated to be 23.9 mM and 378 μA/cm², respectively. In addition, the biosensor had also good sensitivity, stability and reproducibility.     

Development of Novel Glucose oxidase Immobilization on Graphene/Gold nanoparticles/Poly Neutral red modified electrode

Glucose oxidase (GOx) was immobilized onto glassy carbon electrode (GCE) that modified by reduced graphene oxide-gold nanoparticles- poly neutral red (RGO/AuNPs/PNR) nanocomposite. The composite was analyzed by scanning electron microscope (SEM), energy dispersive x-ray (EDX) spectroscopy, atomic force microscopy (AFM), attenuated total reflectance (ATR), cyclic voltammetry (CV), chronoamperometry and electrochemical impedance spectroscopy (EIS). SEM/EDX analysis showed the morphological of the nanocomposite. AFM results showed the morphology and structure of the RGO/AuNPs and RGO surfaces. The covalent bonding between glucose oxidase and composite was confirmed by ATR technique. The electrochemical experiments were done in 100 mM phosphate buffer at pH 7 and temperature of 25 °C with three electrodes including Ag/AgCl, platinum wire and the modified GCE as the reference electrode, the auxiliary electrode and working electrode respectively. The electrochemical results confirmed the activity and direct electron transfer of immobilized enzyme. The immobilized electroactive GOx concentration was estimated 3.06 × 10⁻¹¹ mol cm⁻². The results showed the immobilized enzyme had a good stability and maintained 90% of its performance after two weeks. The nanocomposite bioanode in an air-birthing biofuel cell and 100 mM glucose concentration showed 176 μWcm⁻² Power density. This strategy could be used for GOx-based biofuel cells.