Resulting Glucose Biosensor Research Articles

Synthesis of perovskite-type SrTiO3 nanoparticles for sensitive electrochemical biosensing applications

In this work, SrTiO3 nanoparticles (SrTiO3NPs), a novel and effective perovskite-type material, are synthesized to modify electrode for the development of sensitive enzymatic electrochemical biosensor applications. The electrochemical behavior of glucose oxidase (GOx) immobilized on SrTiO3NPs-modified glassy carbon electrode is investigated. Scanning electron microscopy and cyclic voltammetry are used to characterize the morphology and assay property of different modified electrodes. Results illustrate that SrTiO3NPs possess large surface area and apparently enhance the direct electron transfer between GOx and modified electrode. The SrTiO3NPs-based enzyme biosensor shows a high sensitivity of 15.6mA·M−1·cm−2, wide linear range from 0.01mM to 1.2mM, and a low detection limit of 3μM (S/N=3). Furthermore, the resultant glucose biosensor has excellent selectivity, acceptable repeatability, practicality and stability. The perovskite SrTiO3NPs provide a very attractive and potential electrode material to develop electrochemical biosensors.

Mediator enhanced glucose detection using organic–inorganic hybrid supramolecular assembly on gold electrodes

In this study, a new organic–inorganic material, based on an alpha-metatungstate [H2W12O40]6− cluster, was synthesized via the hydrothermal method by using aminopyridinium (APy)+ cations as the hybrid material, and characterized by FT-IR and UV spectra analysis. The hybrid composite was immobilized on a gold electrode decorated with gold nanoparticles (AuNPs) and the modified electrode exhibited excellent electrocatalytic activities towards the reduction of H2O2 at neutral pH with a response time of <10s. The H2O2 response at the modified electrode displays a linear response ranging from 40μM to 10mM.Based on the H2O2 reduction activity, the (APy)6[H2W12O40] composite was successfully used for the fabrication of a novel enzymatic glucose biosensor. The glucose oxidase enzyme (GOx) was immobilized onto the hybrid composite on AuNPs/gold electrodes; the (APy)6[H2W12O40] composites were used as both mediation and bridging molecules. A multilayer architecture was constructed from hybrid POM alternating with (GOx), using glutaraldehyde as a covalent attachment cross-linker. Cyclic voltammetry (CV), SWV and chronoamperometry were used to determine the electrochemical properties of the mediator and characterize its reaction with (GOx). The resulting glucose biosensor exhibits a wide linear response for glucose from 40μM to 10mM with a sensitivity of 15.0μAmM−1.

Three-dimensional graphene/carbon nanotubes hybrid composites for exploring interaction between glucose oxidase and carbon based electrodes

This work presents a 3D carbon composite electrode matrix, consisting of reduced graphene oxide (RGO), carbon nanotubes, and carbon fiber felt. In this matrix, the edges of the RGO sheets are bounded to cup-stacked carbon nanotubes (CSCNTs) while the micrometer scale basal planes of the RGO sheets are suspended between the fibers. The presence of free-standing RGO sheets in which π orbitals of the basal planes do not interact with the electrode surface offers advantageous conditions to explore demanding bioelectrochemical reactions, such as the FADH2/FAD redox transition often claimed in the literature as originated from a direct electron communication between micro/nano carbon based electrodes and glucose oxidase (GOx). At least, three important questions rise from this system: (i) how the junction between the carbon nanostructures and the carbon microfibers affects the monitored electron transfer? (ii) are the monitored Faradaic peaks originated from native or denaturated GOx? (iii) is GOx still active for glucose sensing after suffering conformational changes due to the adsorption process on the surface of the electrode? It is intended here to contribute with the investigation of these questions. According to our results, the electronic interactions between CSCNTs and RGO sheets seem to modulate the performance of the aforementioned reaction, as improved kinetic parameters were observed on the 3D composite matrix rather than the obtained with 3D electrodes modified with CSCNTs or RGO sheets themselves. However, spectroscopic experiments suggested that GOx suffers significant conformational changes when adsorbed on the surface of the carbon nanostructures, leading to the denaturation of the protein. Even though the electrochemical experiments indicate that the denaturation of the enzyme does not seem to completely release the FAD moiety, the operational capability of the resulting glucose biosensor was greatly compromised.

Amperometric Glucose Biosensor Based on Effective Self-Assembly Technology for Preparation of Poly(allylamine hydrochloride)/Au Nanoparticles Multilayers.

Novel nanomaterials and nanotechnology for use in bioassay applications represent a rapidly advancing field. This study developed a novel method to fabricate the glucose biosensor with good gold nanoparticles (AuNPs) fixed efficiency based on effective self-assembly technology for preparation of multilayers composed of poly(allylamine hydrochloride) (PAH) and AuNPs. The electrochemical properties of the biosensor based on (AuNPs/PAH)n/AuNPs/glucose oxide (GOD) with different multilayers were systematically investigated. Among the resulting glucose biosensors, electrochemical properties of the biosensor with three times self-assembly processes ((AuNPs/PAH)3/AuNPs/GOD) is best. The GOD biosensor exhibited a fast amperometric response (5 s) to glucose, a good linear current-time relation over a wide range of glucose concentrations from 0.05 to 162 mM, and a low detection limit of 0.029 mM. The GOD biosensor modified with (AuNPs/PAH)n layers will have essential significance and practical application in future owing to the simple method of fabrication and good performance.

Layer-by-Layer Self-Assembling Gold Nanorods and Glucose Oxidase onto Carbon Nanotubes Functionalized Sol-Gel Matrix for an Amperometric Glucose Biosensor.

A novel amperometric glucose biosensor was fabricated by layer-by-layer self-assembly of gold nanorods (AuNRs) and glucose oxidase (GOD) onto single-walled carbon nanotubes (SWCNTs)-functionalized three-dimensional sol-gel matrix. A thiolated aqueous silica sol containing SWCNTs was first assembled on the surface of a cleaned Au electrode, and then the alternate self-assembly of AuNRs and GOD were repeated to assemble multilayer films of AuNRs-GOD onto SWCNTs-functionalized silica gel for optimizing the biosensor. Among the resulting glucose biosensors, the four layers of AuNRs-GOD-modified electrode showed the best performance. The sol-SWCNTs-(AuNRs-GOD)4/Au biosensor exhibited a good linear range of 0.01–8 mM glucose, high sensitivity of 1.08 μA/mM, and fast amperometric response within 4 s. The good performance of the proposed glucose biosensor could be mainly attributed to the advantages of the three-dimensional sol-gel matrix and stereo self-assembly films, and the natural features of one-dimensional nanostructure SWCNTs and AuNRs. This study may provide a new facile way to fabricate the enzyme-based biosensor with high performance.

Nanorod arrays composed of zinc oxide modified with gold nanoparticles and glucose oxidase for enzymatic sensing of glucose

We report on an enzymatic electrochemical glucose biosensor based on the use of zinc oxide nanorods (ZnO NRs) modified with gold nanoparticles (Au-NPs) and arranged in the form of an array. The nanorods were obtained via a hydrothermal method, and the Au-NPs were synthesized by photo-reduction. The surface of the nanoarrays was then modified with Au-NPs via electrostatic self-assembly. GOx was immobilized onto the surface of Au-NPs modified ZnO nanoarrays by electrostatic adsorption. The performance of the resulting glucose biosensors were investigated by cyclic voltammetry and amperometry. Compared to biosensors based on ZnO nanoarrays without Au-NPs, the ones described here exhibit a higher sensitivity (43.7 nA cm−2 mM−1) and a lower Kapp,M (0.78 mM). It is obvious, therefore, that the Au-NPs play an important role with respect to the performance of this biosensor.

Sn-Fe cyanogels noncovalently grafted to carbon nanotubes in a versatile biointerface design: an efficient matrix and a facile platform for glucose oxidase immobilization.

Enzyme immobilization is a powerful strategy adapted to effectively maximize the bioactivity, specificity and stability of an isolated enzyme. In this study, we demonstrate a novel and scalable procedure for facile enzyme immobilization, in which three-dimensional porous Sn-Fe hydrogels were applied to incorporate the enzyme to construct a sensing interface for an amperometric biosensor. The process was initiated from the electrodeposition of Prussian Blue (PB) on multi-walled carbon nanotube (MWCNT)-modified gold electrodes, sequentially capped with tin tetrachloride (SnCl4) solution followed by the addition of a freshly-made homogeneous mixture of enzyme and potassium ferrocyanide solution, leading to instant formation of hydrated three-dimensional (3D) porous Sn-Fe cyanogel networks, deeply set outside the produced rough layer of the MWCNT-PB complexes, providing a desirable microenvironment for the entrapped enzyme. The structural morphology and electrochemical properties of the as-prepared Sn-Fe cyanogels noncovalently grafted to MWCNTs with functionalities of electrodeposited PB were well characterized by scanning electron microscopy (SEM), ultraviolet visible spectroscopy (UV-vis) and cyclic voltammetry. The results indicate that the modified electrode with a multilayer configuration was well-organized, as proposed, and exhibited good electrical conductivity and stable catalytic activity to H2O2 electro-reduction due to the functional layer of PB. When glucose oxidase (GOx) was selected as a model enzyme, the resulting glucose biosensor exhibited a relatively low detection limit of 0.1 μM (S/N 3) with a good sensitivity of 1.68 μA mM-1 cm-2 and improved stability. The results suggest that the Sn-Fe cyanogels, with sufficient interfacial adhesion, hold promise as an attractive support material.

Controllable growth of Prussian blue nanostructures on carboxylic group-functionalized carbon nanofibers and its application for glucose biosensing

Glucose detection is very important in biological analysis, clinical diagnosis and the food industry, and especially for the routine monitoring of diabetes. This work presents an electrochemical approach to the detection of glucose based on Prussian blue (PB) nanostructures/carboxylic group-functionalized carbon nanofiber (FCNF) nanocomposites. The hybrid nanocomposites were constructed by growing PB onto the FCNFs. The obtained PB–FCNF nanocomposites were characterized by scanning electron microscopy, x-ray diffraction and x-ray photoelectron spectroscopy. The mechanism of formation of PB–FCNF nanocomposites was investigated and is discussed in detail. The PB–FCNF modified glassy carbon electrode (PB–FCNF/GCE) shows good electrocatalysis toward the reduction of H2O2, a product from the reduction of O2 followed by glucose oxidase (GOD) catalysis of the oxidation of glucose to gluconic acid. Further immobilizing GOD on the PB–FCNF/GCE, an amperometric glucose biosensor was achieved by monitoring the generated H2O2 under a relatively negative potential. The resulting glucose biosensor exhibited a rapid response of 5 s, a low detection limit of 0.5 μM, a wide linear range of 0.02–12 mM, a high sensitivity of 35.94 μA cm−2 mM−1, as well as good stability, repeatability and selectivity. The sensor might be promising for practical application.

Electrochemical glucose biosensor by electrostatic binding of PQQ-glucose dehydrogenase onto self-assembled monolayers on gold

Efficient binding of enzymes onto the electrode surface has been prerequisite for the construction of sensitive biosensors and biochips. Here, a simple and robust construction of electrochemical glucose biosensor based on pyrroloquinoline quinone-glucose dehydrogenase was demonstrated. The glucose biosensor was fabricated by binding the enzyme onto the anionic self-assembled monolayers on gold electrode via electrostatic interactions. The resulting glucose biosensor gave rise to twofold higher detection sensitivity than that by covalent conjugation under the same condition. Surface plasmon resonance and atomic force microscopy analyses revealed that electrostatic binding of the enzyme leads to much higher surface density of the enzyme. This approach will find wide applications to the development of robust enzyme-based biosensors and biochips.

Synthesis of functional SiO 2-coated graphene oxide nanosheets decorated with Ag nanoparticles for H 2O 2 and glucose detection

In this paper, we report on the first preparation of well-defined SiO 2-coated graphene oxide (GO) nanosheets (SiO 2/GO) without prior GO functionalization by combining sonication with sol–gel technique. The functional SiO 2/GO nanocomposites (F-SiO 2/GO) obtained by surface functionalization with NH 2 group were subsequently employed as a support for loading Ag nanoparticles (AgNPs) to synthesize AgNP-decorated F-SiO 2/GO nanosheets (AgNP/F-SiO 2/GO) by two different routes: (1) direct adsorption of preformed, negatively charged AgNPs; (2) in situ chemical reduction of silver salts. The morphologies of these nanocomposites were characterized by transmission electron microscopy (TEM) and scanning electron microscopy (SEM). It is found that the resultant AgNP/F-SiO 2/GO exhibits remarkable catalytic performance for H 2O 2 reduction. This H 2O 2 sensor has a fast amperometric response time of less than 2 s. The linear range is estimated to be from 1 × 10 −4 M to 0.26 M ( r = 0.998) and the detection limit is estimated to be 4 × 10 −6 M at a signal-to-noise ratio of 3, respectively. We also fabricated a glucose biosensor by immobilizing glucose oxidase (GOD) into AgNP/F-SiO 2/GO nanocomposite-modified glassy carbon electrode (GCE) for glucose detection. Our study demonstrates that the resultant glucose biosensor can be used for the glucose detection in human blood serum.

Investigation of a photoelectrochemical passivated ZnO-based glucose biosensor.

A vapor cooling condensation system was used to deposit high quality intrinsic ZnO thin films and intrinsic ZnO nanorods as the sensing membrane of extended-gate field-effect-transistor (EGFET) glucose biosensors. The sensing sensitivity of the resulting glucose biosensors operated in the linear range was 13.4 μA mM−1 cm−2. To improve the sensing sensitivity of the ZnO-based glucose biosensors, the photoelectrochemical method was utilized to passivate the sidewall surfaces of the ZnO nanorods. The sensing sensitivity of the ZnO-based glucose biosensors with passivated ZnO nanorods was significantly improved to 20.33 μA mM−1 cm−2 under the same measurement conditions. The experimental results verified that the sensing sensitivity improvement was the result of the mitigation of the Fermi level pinning effect caused by the dangling bonds and the surface states induced on the sidewall surface of the ZnO nanorods.

An amperometric glucose biosensor based on layer-by-layer GOx-SWCNT conjugate/redox polymer multilayer on a screen-printed carbon electrode

An amperometric glucose biosensor based on a multilayer made by layer-by-layer assembly of single-walled carbon nanotubes modified with glucose oxidase (GOx-SWCNT conjugates) and redox polymer (PVI-Os) on a screen-printed carbon electrode (SPCE) surface was developed. The SPCE surface was functionalized with a cationic polymer by electrodeposition of the PVI-Os, followed by alternating immersions in anionic GOx-SWCNT conjugate solutions and cationic PVI-O solutions. The purpose is to build a multilayer structure which is further stabilized through the electrodeposition of PVI-Os on the multilayer film. The electrochemistry of the layer-by-layer assembly of the GOx-SWCNT conjugate/PVI-Os bilayer was followed by cyclic voltammetry. The resultant glucose biosensor provided stable and reproducible electrocatalytic responses to glucose, and the electrocatalytic current for glucose oxidation was enhanced with an increase in the number of bilayers. The glucose biosensor displayed a wide linear range from 0.5 to 8.0 mM, a high sensitivity of 32 μA mM −1 cm −2, and a response time of less than 5 s. The glucose biosensor proved to be promising amperometric detectors for the flow injection analysis of glucose.

An electro-catalytic biosensor fabricated with Pt–Au nanoparticle-decorated titania nanotube array

A Gold–Platinum nanoparticle-decorated titania nanotubular electrode is fabricated by electrochemically depositing Au and Pt nanoparticles onto a highly-oriented titania nanotube array. The prepared electrode, characterized by SEM and EDX, shows remarkably improved catalytic activities in the oxidation of hydrogen peroxide. By modifying the electrode with glucose oxidase (GOx) the resultant glucose biosensor exhibits a high sensitivity to glucose in the range of 0 to 1.8 mM with a response time of 3 s and detection limit of 0.1 mM.

Glucose biosensors based on platinum nanoparticles-deposited carbon nanotubes in sol–gel chitosan/silica hybrid

A new strategy for fabricating a sensitivity-enhanced glucose biosensor was presented, based on multi-walled carbon nanotubes (CNT), Pt nanoparticles (PtNP) and sol-gel of chitosan (CS)/silica organic-inorganic hybrid composite. PtNP-CS solution was synthesized through the reduction of PtCl(6)(2-) by NaBH(4) at room temperature. Benefited from the amino groups of CS, a stable PtNP gel was obtained, and a CNT-PtNP-CS solution was prepared by dispersing CNT functionalized with carboxylic groups in PtNP-CS solution. The CS/silica hybrid sol-gel was produced by mixing methyltrimethoxysilane (MTOS) with the CNT-PtNP-CS solution. Then, with the immobilization of glucose oxidase (GOD) into the sol-gel, the glucose biosensor of GOD-CNT-PtNP-CS-MTOS-GCE was fabricated. The properties of resulting glucose biosensor were measured by electrochemical impedance spectroscopy (EIS) and cyclic voltammetry (CV). In phosphate buffer solutions (PBS, pH 6.8), nearly interference free determination of glucose was realized at low applied potential of 0.1V, with a wide linear range of 1.2x10(-6) to 6.0x10(-3)M, low detection limit of 3.0x10(-7)M, high sensitivity of 2.08microA mM(-1), and a fast response time (within 5s). The results showed that the biosensor provided the high synergistic electrocatalytic action, and exhibited good reproducibility, long-term stability. Subsequently, the novel biosensor was applied for the determination of glucose in human serum sample, and good recovery was obtained (in the range of 95-104%).

Enzyme-functionalized gold nanowires for the fabrication of biosensors

Gold nanowires were prepared by an electrodeposition strategy using nanopore polycarbonate (PC) membrane, with the average diameter of the nanowires about 250 nm and length about 10 μm. The nanowires prepared were dispersed into chitosan (CHIT) solution and stably immobilized onto glassy carbon electrode (GCE) surface. The electrochemical behavior of gold nanowire modified electrode and its application to the electrocatalytic reduction of hydrogen peroxide (H 2O 2) were investigated. The modified electrode allows low potential detection of hydrogen peroxide with high sensitivity and fast response time. Moreover, the good biocompatibility of nanometer-sized gold, the vast surface area of the nanowire-structure make it ideal for adsorption of enzymes for the fabrication of biosensors. Glucose oxidase was adsorbed onto the nanowire surface to fabricate glucose biosensor as an application example. The detection of glucose was performed in phosphate buffer (pH 6.98) at − 0.2 V. The resulting glucose biosensor exhibited sensitive response, with a short response time (< 8 s), a linear range of 10 − 5 –2 × 10 − 2 M and detection limit of 5 × 10 − 6 M.

Amperometric Biosensors Based on Platinum Nanowires

Platinum nanowires (PtNW) were prepared by an electrodeposition strategy using nanopore alumina template. The nanowires prepared were dispersed in chitosan (CHIT) solution and stably immobilized onto the surface of glassy carbon electrode (GCE). The electrochemical behavior of PtNW‐modified electrode and its application to the electrocatalytic reduction of hydrogen peroxide (H2O2) are investigated. The modified electrode allows low potential detection of hydrogen peroxide with high sensitivity and fast response time. As an application example, the glucose oxidase was immobilized onto the surface of PtNW‐modified electrode through cross‐linking by glutaric dialdehyde. The detection of glucose was performed in phosphate buffer at –0.2 V. The resulting glucose biosensor exhibited a short response time (<8 s), with a linear range of 10−5−10−2 M and detection limit of 5×10−6 M.

Amperometric glucose biosensor based on self-assembly hydrophobin with high efficiency of enzyme utilization

Hydrophobins are a family of natural self-assembling proteins with high biocompability, which are apt to form strong and ordered assembly onto many kinds of surfaces. These physical-chemical and biological properties make hydrophobins suitable for surface modification and biomolecule immobilization purposes. A class II hydrophobin HFBI was used as enzyme immobilization matrix on platinum electrode to construct amperometric glucose biosensor. Permeability of HFBI self-assembling film was optimized by selecting the proper HFBI concentration for electrode modification, in order to allow H2O2 permeating while prevent interfering compounds accessing. HFBI self-assembly and glucose oxidase (GOx) immobilization was monitored by quartz crystal microbalance (QCM), and characterization of the modified electrode surface was obtained by scanning electron microscope (SEM). The resulting glucose biosensors showed rapid response time within 6s, limits of detection of 0.09mM glucose (signal-to-noise ratio=3), wide linear range from 0.5 to 20mM, high sensitivity of 4.214×10−3AM−1cm−2, also well selectivity, reproducibility and lifetime. The all-protein modified biosensor exhibited especially high efficiency of enzyme utilization, producing at most 712μA responsive current for per unit activity of GOx. This work provided a promising new immobilization matrix with high biocompatibility and adequate electroactivity for further research in biosensing and other surface functionalizing.

Amperometric glucose biosensor based on multilayer films via layer-by-layer self-assembly of multi-wall carbon nanotubes, gold nanoparticles and glucose oxidase on the Pt electrode

A novel amperometric glucose biosensor based on the nine layers of multilayer films composed of multi-wall carbon nanotubes (MWCNTs), gold nanoparticles (GNp) and glucose oxidase (GOD) was developed for the specific detection of glucose. MWCNTs were chemically modified with the H2SO4–HNO3 pretreatment to introduce carboxyl groups which were used to interact with the amino groups of poly(allylamine) (PAA) and cysteamine via 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide/N-hydroxysuccinimide cross-linking reaction, respectively. A cleaned Pt electrode was immersed in PAA, MWCNTs, cysteamine and GNp, respectively, followed by the adsorption of GOD, assembling the one layer of multilayer films on the surface of Pt electrode (GOD/GNp/MWCNTs/Pt electrode). Repeating the above process could assemble different layers of multilayer films on the Pt electrode. PBS washing was applied at the end of each assembly deposition for dissociating the weak adsorption. Film assembling and characterization were studied by transmission electron microscopy and quartz crystal microbalance, and properties of the resulting glucose biosensors were measured by electrochemical measurements. The marked electrocatalytic activity of Pt electrode based on multilayer films toward H2O2 produced during GOD enzymatic reactions with glucose permitted effective low-potential amperometric measurement of glucose. Taking the sensitivity and selectivity into consideration, the applied potential of 0.35V versus Ag/AgCl was chosen for the oxidation detection of H2O2 in this work. Among the resulting glucose biosensors, the biosensor based on nine layers of multilayer films was best. It showed a wide linear range of 0.1–10mM glucose, with a remarkable sensitivity of 2.527μA/mM, a detection limit of 6.7μM estimated at a signal-to-noise ratio of 3 and fast response time (within 7s). Moreover, it exhibited good reproducibility, long-term stability and the negligible interferences of ascorbic acid, uric acid and acetaminophen. The study can provide a feasible approach on developing new kinds of oxidase-based amperometric biosensors, and can be used as an illustration for constructing various hybrid structures.

Bioelectrocatalytic application of titania nanotube array for molecule detection

A bioelectrocatalysis system based on titania nanotube electrode has been developed for the quantitative detection application. Highly ordered titania nanotube array with inner diameter of 60nm and total length of 540nm was formed by anodizing titanium foils. The functionalization modification was achieved by embedding glucose oxidases inside tubule channels and electropolymerizing pyrrole for interfacial immobilization. Morphology and microstructure characterization, electrochemical properties and bioelectrocatalytic reactivities of this composite were fully investigated. The direct detection of hydrogen peroxide by electrocatalytic reduction reaction was fulfilled on pure titania nanotube array with a detection limit up to 2.0×10−4mM. A biosensor based on the glucose oxidase–titania/titanium electrode was constructed for amperometric detection and quantitative determination of glucose in a phosphate buffer solution (pH 6.8) under a potentiostatic condition (−0.4V versus SCE). The resulting glucose biosensor showed an excellent performance with a response time below 5.6s and a detection limit of 2.0×10−3mM. The corresponding detection sensitivity was 45.5μAmM−1cm−2. A good operational reliability was also achieved with relative standard deviations below 3.0%. This novel biosensor exhibited quite high response sensitivity and low detection limit for potential applications.

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.