Chemiluminescence flow biosensor for glucose based on gold nanoparticle-enhanced activities of glucose oxidase and horseradish peroxidase

In this work, a novel chemiluminescence (CL) flow biosensor for glucose was proposed. Glucose oxidase (GOD), horseradish peroxidase (HRP) and gold nanoparticles were immobilized with sol-gel method on the inside surface of the CL flow cell. The CL detection involved enzymatic oxidation of glucose to d-gluconic acid and H(2)O(2), and then the generated H(2)O(2) oxidizing luminol to produce CL emission in the presence of HRP. It was found that gold nanoparticles could remarkably enhance the CL respond of the glucose biosensor. The enhanced effect was closely related to the sizes of gold colloids, and the smaller the size of gold colloids had the higher CL respond. The immobilization condition and the CL condition were studied in detail. The CL emission intensity was linear with glucose concentration in the range of 1.0 x 10(-5)molL(-1) to 1.0 x 10(-3)molL(-1), and the detection limit was 5 x 10(-6)molL(-1) (3sigma). The apparent Michaelis-Menten constant of GOD in gold nanoparticles/sol-gel matrix was evaluated to be 0.3mmolL(-1), which was smaller than that of GOD immobilized in sol-gel matrix without gold nanoparticles. The proposed biosensor exhibited short response time, easy operation, low cost and simple assembly, and the proposed biosensor was successfully applied to the determination of glucose in human serum.     

Bienzymatic glucose biosensor based on co-immobilization of peroxidase and glucose oxidase on a carbon nanotubes electrode

A bienzymatic glucose biosensor was proposed for selective and sensitive detection of glucose. This mediatorless biosensor was made by simultaneous immobilization of glucose oxidase (GOD) and horseradish peroxidase (HRP) in an electropolymerized pyrrole (PPy) film on a single-wall carbon nanotubes (SWNT) coated electrode. The amperometric detection of glucose was assayed by potentiostating the bienzymatic electrode at -0.1 versus Ag/AgCl to reduce the enzymatically produced H(2)O(2) with minimal interference from the coexisting electroactive compounds. The single-wall carbon nanotubes, sandwiched between the enzyme loading polypyrrole (PPy) layer and the conducting substrate (gold electrode), could efficiently promote the direct electron transfer of HRP. Operational characteristics of the bienzymatic sensor, in terms of linear range, detection limit, sensitivity, selectivity and stability, were presented in detail.     

A novel and simple biomolecules immobilization method: electro-deposition ZrO2 doped with HRP for fabrication of hydrogen peroxide biosensor

For the first time, a very novel and simple immobilization method for fabrication of hydrogen peroxide biosensor was reported in this paper. The biocompatible composite HRP-ZrO(2) thin films were synthesized on gold electrode surface based on electro-deposition zirconia doped with horseradish peroxidase (HRP) by cyclic voltammetry scanning in KCl solution containing ZrO(2) and HRP. The fabricated process of biosensor was characterized by electrochemical impedance spectroscopy (EIS) and the surface topography of the prepared films was imaged by atomic force microscope (AFM). The HRP in HRP-ZrO(2) thin films kept its bioactivity and exhibited excellent electrocatalytical response to the reduction of H(2)O(2). Experimental conditions influencing the biosensor performance such as pH, potential were optimized. The resulting biosensor (HRP-ZrO(2)/Au electrode) showed a linear response to H(2)O(2) over a concentration range from 0.02 to 9.45mM with a detection limit of 2muM based on a signal-to-noise ratio of 3 under optimized conditions. The apparent Michaelis-Menten constant (K(M)(app)) was evaluated to be 8.01mM, which indicated the HRP in HRP-ZrO(2) thin films kept its native bioactivity and had high affinity for H(2)O(2). Moreover, the proposed biosensor showed high sensitivity, good reproducibility and long-term stability. What is more, this immobilization methodology widened biosensor application in biomolecules immobilization and could further develop for other protein and biomolecules immobilization.     

Biosensor based on polyaniline-Prussian Blue/multi-walled carbon nanotubes hybrid composites

In this work, a novel route for fabrication polyaniline (PANI)-Prussian Blue (PB) hybrid composites is proposed by the spontaneous redox reaction in the FeCl(3)-K(3)[Fe(CN)(6)] and the aniline solution. With the introduction of multi-walled carbon nanotubes (MWNTs), the PANI-PB/MWNTs system shows synergy between the PANI-PB and MWNTs which amplified the H(2)O(2) sensitivity greatly. A linear range from 8 x1 0(-8) to 1 x 10(-5)M and a high sensitivity 508.1 8 microA microM cm(2) for H(2)O(2) detection are obtained. The composites also show good stability in neutral solution. A glucose biosensor was further constructed by immobilizing glucose oxidase (GOD) with Nafion and glutaraldehyde on the electrode surface. The performance factors influencing the resulted biosensor were studied in detail. The biosensor exhibits excellent response performance to glucose with the linear range from 1 to 11 mM and a detection limit of 0.01 mM. Furthermore, the biosensor shows rapid response, high sensitivity, good reproducibility, long-term stability and freedom of interference from other co-existing electroactive species.     

Construction of an amperometric glucose biosensor based on the immobilization of glucose oxidase onto electrodeposited Pt nanoparticles-chitosan composite film

One-step construction of Pt nanoparticles-chitosan composite film (PtNPs-CS) was firstly proposed as a novel immobilization matrix for the enzymes to fabricate glucose biosensor. This novel interface embedded in situ PtNPs in CS hydrogel was developed by one-step electrochemical deposition in solution containing CS and chloroplatinic acid (H(2)PtCl(6)). Several techniques, including scanning electron microscopy (SEM), cyclic voltammetry (CV), electrochemical impedance spectroscopy (EIS) and chronoamperometry were employed to characterize the assembly process and performance of the biosensor. Under the optimized experimental conditions, the resulting biosensor exhibited excellent linear behavior in the concentration range from 1.2 μM to 4.0 mM for the quantitative analysis of glucose with a limit of detection of 0.4 μM at a signal-to-noise ratio of 3. The apparent Michaelis-Menten constant (K(M)(app)) was evaluated to be 2.4 mM, showing good affinity. The proposed biosensor offered good amperometric responses to glucose due to the nanostructured sensing film provided plenty of active sites for the immobilization of glucose oxidase (GOD).     

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.     

Glucose oxidase as label in histological immunoassays with enzyme-amplification in a two-step technique: coimmobilized horseradish peroxidase as secondary system enzyme for chromogen oxidation

A sensitive staining procedure for glucose oxidase (GOD) as marker in immunohistology is described. The cytochemical procedure involves a two-step enzyme method in which GOD and horseradish peroxidase (HRP) are coimmobilized onto the same cellular sites by immunological bridging or by the principle of avidin-biotin interaction. In this coupled enzyme technique, H2O2 generated during GOD reaction is the substrate for HRP and is utilized for the oxidation of chromogens such as 3,3'-diaminobenzidine or 3-amino-9-ethylcarbazole. Due to the immobilization of the capture enzyme HRP in close proximity to the marker enzyme (GOD), more intense and specific staining is produced than can be obtained with soluble HRP as coupling enzyme in the substrate medium. Indirect antibody labelled and antibody bridge techniques including the avidin (streptavidin)-biotin principle have proven the usefulness of this GOD labelling procedure for antigen localization in paraffin sections. Antigens such as IgA in tonsil, alpha-fetoprotein in liver and tissue polypeptide antigen in mammary gland served as models. The immobilized two-step enzyme procedures have the same order of sensitivity and specificity as comparable immunoperoxidase methods. The coupled GOD-HRP principle can be superior to conventional immunoperoxidase labelling for the localization of biomolecules in tissue preparations rich in endogenous peroxidase activities.     

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 third-generation hydrogen peroxide biosensor based on horseradish peroxidase immobilized in a tetrathiafulvalene-tetracyanoquinodimethane/multiwalled carbon nanotubes film

A new third-generation biosensor for H(2)O(2) assay was developed on the basis of the immobilization of horseradish peroxidase (HRP) in a nanocomposite film of tetrathiafulvalene-tetracyanoquinodimethane (TTF-TCNQ)/multiwalled carbon nanotubes (MWCNTs) modified gold electrode. The prepared HRP/TTF-TCNQ/MWCNTs/Au electrode was used for the bioelectrocatalytic reduction of H(2)O(2), with a linear range from 0.005 to 1.05mM and a detection limit of 0.5muM for amperometric sensing of H(2)O(2). In addition, a novel method on the basis of electrochemical quartz crystal microbalance (EQCM) measurements was proposed to determine the effective enzymatic specific activity (ESA) of the immobilized HRP for the first time, and the ESA was found to be greater at the TTF-TCNQ/MWCNTs/Au electrode than that at the MWCNTs/Au or TTF-TCNQ/Au electrode, indicating that the TTF-TCNQ/MWCNTs film is a good HRP-immobilization matrix to achieve the direct electron transfer between the enzyme and the electrode.     

Determination of serum glucose by horseradish peroxidase-catalysed imidazole chemiluminescence coupled to a micro-flow-injection system.

The reactivity of flow-injection (FI)-horseradish peroxidase (HRP)-catalysed imidazole chemiluminescence (CL) was studied for continuous determination of hydrogen peroxide (H(2)O(2)) and serum glucose with immobilized glucose oxidase. Light emission by the HRP-catalysed imidazole CL was obtained when immobilized HRP, alkaline imidazole (in Tricine solution, pH 9.3) and H(2)O(2) were reacted at room temperature. The optimal pH for the CL reaction was 9.3 and the optimal concentration of imidazole was 100 micromol/L. When no imidazole was added, the light intensity of the same H(2)O(2) specimen decreased to a level that could not be quantitatively determined. The spectrum of the light emitted by imidazole CL was in the range 400-600 nm with a peak at 500 nm. The calibration equation for determination of H(2)O(2) was y = 9860x(2) + 3830x + 11,700, where y = light intensity (RLU) and x = concentration of H(2)O(2) (micromol/L). The detection limit of H(2)O(2) was 5 pmol, and the reproducibility of the H(2)O(2) assay was 2.3% of the coefficient of variation (H(2)O(2) 48 micromol/L, n = 13). The CL method was successfully applied to assay glucose after on-line generation of H(2)O(2) with the immobilized glucose oxidase column, resulting in good reproducibility (CV = 3.3% and 1.0% for the standard glucose and the control serum, respectively).     

Development of amperometric biosensor for glucose based on a novel attractive enzyme immobilization matrix: calcium carbonate nanoparticles

Calcium carbonate nanoparticles (nano-CaCO3) may be a promising material for enzyme immobilization owing to their high biocompatibility, large specific surface area and their aggregation properties. This attractive material was exploited for the mild immobilization of glucose oxidase (GOD) in order to develop glucose amperometric biosensor. The GOD/nano-CaCO3-based sensor exhibited a marked improvement in thermal stability compared to other glucose biosensors based on inorganic host matrixes. Amperometric detection of glucose was evaluated by holding the modified electrode at 0.60 V (versus SCE) in order to oxidize the hydrogen peroxide generated by the enzymatic reaction. The biosensor exhibited a rapid response (6s), a low detection limit (0.1 microM), a wide linear range of 0.001-12 mM, a high sensitivity (58.1 mAcm-2M-1), as well as a good operational and storage stability. In addition, optimization of the biosensor construction, the effects of the applied potential as well as common interfering compounds on the amperometric response of the sensor were investigated and discussed herein.     

Amperometric hydrogen peroxide biosensor based on the immobilization of HRP on nano-Au/Thi/poly (p-aminobenzene sulfonic acid)-modified glassy carbon electrode

A novel hydrogen peroxide biosensor was fabricated for the determination of H(2)O(2). The precursor film was first electropolymerized on the glassy carbon electrode with p-aminobenzene sulfonic acid (p-ABSA) by cyclic voltammetry (CV). Then thionine (Thi) was adsorbed to the film to form a composite membrane, which yielded an interface containing amine groups to assemble gold nanoparticles (nano-Au) layer for immobilization of horseradish peroxidase (HRP). The electrochemical characteristics of the biosensor were studied by CV and chronoamperometry. The factors influencing the performance of the resulting biosensor were studied in detail. The biosensor responded to H(2)O(2) in the linear range from 2.6 x 10(-6) mol/L to 8.8 x 10(-3) mol/L with a detection limit of 6.4 x 10(-7) mol/L. Moreover, the studied biosensor exhibited good accuracy and high sensitivity. The proposed method was economical and efficient, making it potentially attractive for the application to real sample analysis.     

A porous poly(acrylonitrile-co-acrylic acid) film-based glucose biosensor constructed by electrochemical entrapment

A novel glucose biosensor was constructed by electrochemical entrapment of glucose oxidase (GOD) into porous poly(acrylonitrile-co-acrylic acid), which was synthesized via radical polymerization of acrylonitrile and acrylic acid. The obtained biosensor showed a better stability and higher sensitivity than the biosensor prepared by simple physical adsorption. Effects of some experimental variables such as immobilization time, enzyme concentration, pH, applied potential, and temperature on the amperometric response of the sensor were investigated. The biosensor exhibited a rapid response to glucose (< 30s) with a linear range of 5 x 10(-6) to 3 x 10(-3)M and a sensitivity of 6.82 mAM(-1)cm(-2). The apparent Michaelis-Menten constant (K(M)(app)) was 7.3mM.     

A novel glucose biosensor based on the nanoscaled cobalt phthalocyanine-glucose oxidase biocomposite

We report on the utilization of a novel nanoscaled cobalt phthalocyanine (NanoCoPc)-glucose oxidase (GOD) biocomposite colloid to create a highly responsive glucose biosensor. The biocomposite colloid is constructed under enzyme-friendly conditions by adsorbing GOD molecules on CoPc nanoparticles via electrostatic interactions. The glucose biosensor can be easily achieved by casting the biocomposite colloid on a pyrolytic graphite electrode (PGE) without any auxiliary matter. It has been found that GOD can be firmly immobilized on PGE surface and maintain its bioactivity after conjugating with NanoCoPc. NanoCoPc displays intrinsic electrocatalytic ability to the oxidation of the product of enzymatic reaction H2O2 and shows a higher catalytic activity than that of bulk CoPc. Under optimal conditions, the biosensor shows a wide linear response to glucose in the range of 0.02-18 mM, with a fast response (5s), high sensitivity (7.71 microA cm(-2) mM(-1)), as well as good thermostability and long-term life. The detection limit was 5 microM at 3 sigma. The general interferences coexisted in blood except ascorbic acid and L-cysteine do not affect glucose determination, and further coating Nafion film on the surface of the biosensor can effectively eliminate the interference from ascorbic acid and L-cysteine. The biosensor with Nafion film has been used for the determination of glucose in serum with an acceptable accuracy.     

The potential use of hydrazine as an alternative to peroxidase in a biosensor: comparison between hydrazine and HRP-based glucose sensors

The potential use of hydrazine sulfate was examined for the catalytic reduction of enzymatically generated H2O2 in a biosensor system. The performance of the hydrazine-based sensor was compared with an HRP-based glucose sensor as a model of a biosensor. Hydrazine and HRP were covalently immobilized onto a conducting polymer layer with glucose oxidase. The direct electron transfer reactions of the immobilized hydrazine and HRP onto the poly-5,2':5,2'-terthiophene-3'-carboxylic acid (poly-TTCA) layer were investigated by using cyclic voltammetric method and the electron transfer rate constants were determined. The glucose oxidase- and hydrazine-immobilized sensor efficiently reduced the enzymatically generated H2O2 at -0.15 V versus Ag/AgCl. The surface of this GOx/hydrazine/poly-TTCA-based glucose sensor was characterized by QCM, SEM, and ESCA. Glucose-sensing properties were studied using cyclic voltammetric and chronoamperometric techniques. Various experimental parameters were optimized according to the amount of hydrazine, pH, the temperature, and the applied potential. A linear calibration plot was obtained in the concentration range between 0.1 and 15.0 mM, and the detection limit was determined to be 40.0+/-7.0 microM. Interferences from other biological compounds were studied. The long-term stability of the GOx/hydrazine sensor was better than that of the one based on a GOx/HRP biosensor. The proposed glucose sensor was successfully applied to human whole blood and urine samples for the detection of glucose.     

A reagentless optical biosensor based on the intrinsic absorption properties of peroxidase

During the reversible reaction between peroxidase (HRP) and H(2)O(2), several peroxidase intermediate species, showing different molecular absorption spectra, are formed which can be used for H(2)O(2) determination; when H(2)O(2) is generated in a previous enzymatic reaction, the substrate involved in this reaction can also be determined. On this basis, a new family of fully reversible reagentless optical biosensors containing HRP is presented; glucose determination is used as a model. The biosensor (which can be used for at least 6 months and/or more than 750 measurements) is prepared by HRP and glucose oxidase entrapment in a polyacrylamide gel matrix. A mathematical model (in which optical, kinetic and transport aspects are considered) relating the measured absorbance with the substrate concentration is also presented together with a simple methodology for characterization of this kind of biosensor. Regarding the optical model, the Kubelka-Mulk theory of reflectance does not give good results and the biosensors are better described by the Rayleigh theory of polymer solutions. Under working conditions, linear response ranges from 1.5x10(-6) to 3.0x10(-4)M glucose and CV was about 4%. This biosensor has been applied for glucose determination in fruit juices and synthetic serum samples without sample pretreatment.     

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.     

Glucose biosensor based on the room-temperature phosphorescence of TiO2/SiO2 nanocomposite

The direct immobilization of glucose oxidase (GOD) on TiO₂/SiO₂ nanocomposite and its application as glucose biosensor were investigated. The room-temperature phosphorescence of TiO₂/SiO₂ nanocomposite can be quenched by hydrogen peroxide (H₂O₂). The detection of glucose may be accomplished by monitoring the formation of hydrogen peroxide which generated in the oxidation process of glucose with the catalysis of GOD. To our surprise, by using a 96-hole polyporous plate accessory of fluorescence spectrophotometer, the biosensor exhibits excellent linear response to glucose concentrations ranging from 1.0 × 10⁻⁹ to 1.0 × 10⁻² M with a detection limit of 1.2 × 10⁻¹⁰ M. The TiO₂/SiO₂ nanocomposite can be used as both supporting material and signal transducer. The phosphorescence intensity and color of the biosensor change obviously and even could be observed with naked eyes by continuous addition of glucose. Based on the room-temperature phosphorescence of TiO₂/SiO₂ nanocomposite, a new method of solid substrate-room-temperature phosphorimetry (SS-RTP) for glucose determination is proposed. A glucose biosensor was fabricated with wide determination concentration range, low detection limit, high sensitivity, and fast response time. And the biosensor has been successfully applied to the determination of glucose in human blood serum. The coacervation of GOD enzyme and its interaction with TiO₂/SiO₂ nanocomposite enlarge the surface area and enhance the chemical stability of GOD. The nice biocompatibility, large surface area, good chemical stability and nontoxicity of the TiO₂/SiO₂ nanocomposite have made this material suitable for functioning as biosensor.     

A novel hydrogen peroxide biosensor based on the immobilization of horseradish peroxidase onto Au-modified titanium dioxide nanotube arrays

In this study, we report on a promising H(2)O(2) biosensor based on the co-immobilization of horseradish peroxidase (HRP) and chitosan onto Au-modified TiO(2) nanotube arrays. The titania nanotube arrays were directly grown on a Ti substrate using anodic oxidation first; a gold thin film was then uniformly coated onto the TiO(2) nanotube arrays by an argon plasma technique. The morphology and composition of the fabricated Au-modified TiO(2) nanotube arrays were characterized by scanning electron microscopy (SEM) and energy-dispersive X-ray spectroscopy (EDS). Cyclic voltammetry and chronoamperometry were used to study and to optimize the performance of the resulting electrochemical biosensor. The effect of pH, applied electrode potential, the presence of the electron-mediator methylene blue, and the anodic oxidation time of the Ti substrate on the electrochemical biosensor has been systemically studied. Our electrochemical measurements show that the Au-modified TiO(2) nanotube arrays provide excellent matrices for the immobilization of HRP and that the optimized electrochemical biosensor exhibits long linearity, a low detection limit, high stability and very good reproducibility for the detection of H(2)O(2). Under the optimized conditions the linearity of the developed biosensor for the detection of H(2)O(2) is observed from 5 x 10(-6) to 4 x 10(-4) moll(-1) with a detection limit of 2 x 10(-6) moll(-1) (based on the S/N=3).     

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.