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 nanobiocomposite based glucose biosensor using neutral red functionalized carbon nanotubes

The performance of a new glucose biosensor based on the combination of biocatalytic activity of glucose oxidase (GOx) with the electrocatalytic properties of CNTs and neutral red (NR) for the determination of glucose is described. This sensor is comprised of a multiwalled carbon nanotubes (MWNTs) conduit functionalized with NR and Nafion (Nf) as a binder and glucose oxidase as a biocatalyst. Neutral red was covalently immobilized on carboxylic acid groups of the CNTs via carbodiimide reaction. The functionalized MWNTs were characterized by microscopic, spectroscopic and thermal methods. The MWNT-NR-GOx-Nf nanobiocomposite was prepared by mixing the GOx solution with NR functionalized CNTs followed by mixing homogeneously with Nafion. The performance of the MWNT-NR-GOx-Nf nanobiocomposite modified electrode was examined by electrochemical impedance spectroscopy and cyclic voltammetry. The catalytic reduction of hydrogen peroxide liberated from the enzymatic reaction of glucose oxidase upon glucose with NR functionalized CNTs leads to the selective detection of glucose. The excellent electrocatalytic activity and the influence of nanobiocomposite film result in good characteristics such as low potential detection of glucose with a large determination range from 1 x 10(-8) to 1 x 10(-3)M with a detection limit of 3 x 10(-9)M glucose, a short response time (with 4s), good stability and anti-interferent ability. The improved electrocatalytic activity and stability made the MWNT-NR-GOx-Nf nanobiocomposite biosensor system a potential platform to immobilize different enzymes for other bioelectrochemical applications.     

Electrodeposition of polypyrrole-multiwalled carbon nanotube-glucose oxidase nanobiocomposite film for the detection of glucose

A nanobiocomposite film consisted of polypyrrole (PPy), functionalized multiwalled carbon nanotubes (cMWNTs), and glucose oxidase (GOx) were electrochemically synthesized by electrooxidation of 0.1M pyrrole in aqueous solution containing appropriate amounts of cMWNTs and GOx. Potentiostatic growth profiles indicate that the anionic cMWNTs is incorporated within the growing PPy-cMWNTs nanocomposite for maintaining its electrical neutrality. The morphology of the PPy-cMWNTs nanocomposite was characterized by scanning electron microscopy (SEM). The PPy-cMWNTs nanocomposite was deposited homogeneously onto glassy carbon electrode. The amperometric responses vary proportionately to the concentration of hydrogen peroxide at the PPy-cMWNTs nanocomposite modified electrode at an operating potential of 0.7V versus Ag/AgCl (3M). The results indicate that the electroanalytical PPy-cMWNTs-GOx nanobiocomposite film was highly sensitive and suitable for glucose biosensor based on GOx function. The GOx concentration within the PPy-cMWNTs-GOx nanobiocomposite and the film thickness are crucial for the performance of the glucose biosensor. The amperometric responses of the optimized PPy-cMWNTs-GOx glucose biosensor (1.5 mgmL(-1) GOx, 141 mCcm(-2) total charge) displayed a sensitivity of 95 nAmM(-1), a linear range up to 4mM, and a response time of about 8s.     

Bienzyme system for the biocatalyzed deposition of polyaniline templated by multiwalled carbon nanotubes: a biosensor design

A novel method based on covalent attachment of two enzymes, glucose oxidase (GOD) and horseradish peroxide (HRP), onto carboxylic-derived multiwalled carbon nanotubes (MWNTs) for the deposition of electroactive polyaniline (PANI) under ambient conditions is described. Ultraviolet-visible spectroscopy, Fourier-transform infrared (FTIR) spectroscopy, and transmission electron microscopy were used to characterize the assembling of bienzyme and the morphology of PANI|MWNTs. Under the bienzyme biocatalytic condition, a head-to-tail structure of PANI templated by MWNTs was formed. The voltammetric characteristics of the resulting biosensor were investigated by cyclic voltammetry in the presence of glucose. The current response of PANI was linearly related to glucose concentration between 0.05 and 12.0mM with a correlation coefficient of 0.994. The synergistic performance of bienzyme, highly efficient polymerization, and templated deposition provide a general platform for the synthesis of nanowires and nanocircuits, the construction of bioelectronic devices, and the design of novel biosensors.     

Chemiluminescence flow-through biosensor for glucose with eggshell membrane as enzyme immobilization platform

In this study, a new chemiluminescence (CL) flow-through biosensor for glucose was developed by immobilizing glucose oxidase (GOD) and horseradish peroxidase (HRP) on the eggshell membrane with glutaraldehyde as a cross-linker. The CL detection involved enzymatic oxidation of glucose to D-gluconic acid and hydrogen peroxide (H2O2) and then H2O2 oxidizing luminol to produce CL emission in the presence of HRP. The immobilization condition (e.g., immobilization time, GOD/HRP ratio, glutaraldehyde concentration) was studied in detail. It showed good storage stability at 4 degrees C over a 5-month period. The proposed biosensor exhibited short response time, high sensitivity, easy operation, and simple sensor assembly, and the proposed biosensor was successfully applied to the determination of glucose in human serum.     

Amperometric biosensor for hydrogen peroxide based on ferrocene-bovine serum albumin and multiwall carbon nanotube modified ormosil composite

A novel amperometric biosensor for hydrogen peroxide (H(2)O(2)) was developed by entrapping horseradish peroxidase (HRP) in a new ormosil composite doped with ferrocene monocarboxylic acid-bovine serum albumin conjugate and multiwall carbon nanotubes (MWNTs). The ormosil was prepared using 3-(aminopropyl)triethoxysilane and 2-(3,4 epoxycyclohexyl)-ethyltrimethoxy silane as monomers. The encapsulated conjugate showed excellent electrochemistry and acted as an electron transfer mediator. The presence of MWNTs improved the conductivity of the composite film. This matrix showed a biocompatible microenvironment for retaining the native activity of the entrapped HRP and a very low mass transport barrier to the substrate, which provided a fast amperometric response to H(2)O(2). The proposed H(2)O(2) biosensor exhibited a linear range of 0.02-4.0 mM with a detection limit of 5.0 microM (S/N = 3) and a K(M)(app) value of 2.0 mM. It could be used for flow injection analysis of hydrogen peroxide with a liner range from 0.02 to 4.5 mM, sensitivity of 0.042 microA/mM and analytical time of 20 s per sample. This biosensor possessed good analytical performance and storage stability.     

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.     

The direct electron transfer of glucose oxidase and glucose biosensor based on carbon nanotubes/chitosan matrix

The glucose oxidase (GOD) is entrapped in the composite of carbon nanotubes/chitosan and direct electron transfer reaction between GOD and electrode takes place. The electron transfer rate of GOD is greatly enhanced to 7.73 s(-1) in the system, which is more than one-fold higher than that of flavin adenine dinucleotide adsorbed on the carbon nanotubes (3.1 s(-1)). This maybe results from the conformational change of GOD in the microenvironment enabling the accessibility of active site for GOD to the electrode. Additionally, the bioactivity of GOD modified in the composite on electrode surface is kept. So as-prepared electrode can be used as a glucose biosensor exhibiting higher sensitivity (0.5 microA mM(-1)) and better stability. The facile procedure of immobilizing GOD will promote the developments of electrochemical research for protein, biosensors and other bioelectrochemical devices.     

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.     

Amperometric sensor based on ferrocene-modified multiwalled carbon nanotube nanocomposites as electron mediator for the determination of glucose

A kind of nanocomposite with good dispersion in water was prepared through covalent adsorption of ferrocenecarboxaldehyde on multiwalled carbon nanotubes (MWNTs) for electrical communication between glucose oxidase (GOD) and electrode. The ferrocene-modified multiwalled carbon nanotube nanocomposites (MWNTs-Fc) could be conveniently cast on electrode surfaces. With the aid of chitosan, GOD was then immobilized on the nanostructure film to form a reagentless amperometric sensor for glucose determination. FTIR spectra and cyclic voltammetry were used to characterize the nanocomposites. The presence of both ferrocene as mediator of electron transfer and MWNTs as conductor enhanced greatly the enzymatic response to the oxidation of glucose. The novel biosensor exhibited a fast response toward glucose with a detection limit of 3.0 × 10⁻⁶ mol/L and the linear range extended up to 3.8 × 10⁻³ mol/L.     

An enzyme-chromogenic surface plasmon resonance biosensor probe for hydrogen peroxide determination using a modified Trinder's reagent

An absorption-based surface plasmon resonance (SPR(Abs)) biosensor probe has been developed for simple and reproducible measurements of hydrogen peroxide using a modified Trinder's reagent (a chromogenic reagent). The reagent enabled the determination of the hydrogen peroxide concentration by the development of deep color dyes (lambda(max)=630nm) through the oxidative coupling reaction with N-ethyl-N-(2-hydroxy-3-sulfopropyl)-3,5-dimethylaniline sodium salt monohydrate (MAOS; C(13)H(20)NNaO(4)S.H(2)O) and 4-aminoantipyrine (4-AA) in the presence of hydrogen peroxide and horseradish peroxidase (HRP). In the present study, urea as an adduct of hydrogen peroxide for color development could be omitted from the measurement solution. The measurement solution containing 5mM hydrogen peroxide was deeply colored at a high absorbance value calculated as 46.7cm(-1) and was directly applied to the SPR(Abs) biosensing without dilution. The measurement was simply performed by dropping the measurement solution onto the surface of the SPR sensor probe, and the SPR(Abs) biosensor response to hydrogen peroxide was obtained as a reflectivity change in the SPR spectrum. After investigation of the pH profiles in the SPR(Abs) biosensor probe, a linear calibration curve was obtained between 1.0 and 50mM hydrogen peroxide (r=0.991, six points, average of relative standard deviation; 0.152%, n=3) with a detection limit of 0.5mM. To examine the applicability of this SPR(Abs) biosensor probe, 20mM glucose detection using glucose oxidase was also confirmed without influence of the refractive index in the measurement solution. Thus, the SPR(Abs) biosensor probe employing the modified Trinder's reagent demonstrated applicability to other analyte biosensing tools.     

Potentiometric glucose determination in human serum samples with glucose oxidase biosensor based on iodide electrode

Glucose potentiometric biosensor was prepared by immobilizing glucose oxidase on iodide-selective electrode. The hydrogen peroxide formed after the oxidation of glucose catalysed by glucose oxidase (GOD) was oxidized by sodium molybdate (SMo) at iodide electrode in the presence of dichlorometane. The glucose concentration was calculated from the decrease of iodide concentration determined by iodide-selective sensor. The sensitivity of glucose biosensor towards iodide ions and glucose was in the concentration ranges of 1.0 × 10^?1–1.0 × 10^?6 M and 1.0 × 10^?2?1.0 × 10^?4 M, respectively. The characterization of proposed glucose biosensor and glucose assay in human serum were also investigated.     

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.     

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

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

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 0.35 V 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 0.1–10 mM glucose, with a remarkable sensitivity of 2.527 μA/mM, a detection limit of 6.7 μM estimated at a signal-to-noise ratio of 3 and fast response time (within 7 s). 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.     

A flexible and wearable glucose sensor based on functional polymers with soft-MEMS techniques

A novel biosensor for glucose measurement using functional polymers was fabricated and tested. The biosensor utilizes the physical and chemical functions of hydrophobic polydimethyl siloxane (PDMS) and hydrophilic 2-methacryloyloxyethyl phosphorylcholine (MPC) copolymerized with dodecyl methacrylate (DMA). The glucose sensor was constructed by immobilizing glucose oxidase (GOD) onto a flexible hydrogen peroxide electrode (Pt working electrode and Ag/AgCl counter/reference electrode). The electrodes were fabricated using microelectromechanical systems (MEMS) techniques onto those functional polymers. The sensor showed novel functions of flexibility and it was stretchable so that the sensor could normally work when it was released after expanding to 120% longer than that of normal length. Also, basic characteristics of the sensor were evaluated. The output current of the hydrogen peroxide electrode was linearly related to the hydrogen peroxide concentration in a range of 0.20-2.50 mmol/l, with a correlation coefficient of 0.998. GOD was then immobilized onto the surface of the sensor using MPC polymer. In this case, the current output of the glucose sensor related to the glucose level over a range of 0.06-2.00 mmol/l, with a correlation coefficient of 0.997. The calibration range includes the reported concentration of tear glucose in normal human subject (0.14 mmol/l).     

Immobilization of horseradish peroxidase on chitosan/silica sol-gel hybrid membranes for the preparation of hydrogen peroxide biosensor

A simple and effective strategy for fabrication of hydrogen peroxide (H₂O₂) biosensor has been developed by entrapping horseradish peroxidase (HRP) in chitosan/silica sol–gel hybrid membranes (CSHMs) doped with potassium ferricyanide (K₃Fe(CN)₆) and gold nanoparticles (GNPs) on platinum electrode surface. The hybrid membranes are prepared by cross-linking chitosan (CS) with 3-aminopropyltriethoxysilane (APTES), while the presence of GNPs improved the conductivity of CSHMs, and the Fe(CN)₆^3−/4− was used as a mediator to transfer electrons between the electrode and HRP due to its excellent electrochemistry activity. UV–Vis absorption spectroscopy was employed to characterize the different components in the CSHMs and their interaction. The parameters influencing the performance of the resulting biosensor were optimized and the characteristic of the resulting biosensor was characterized by cyclic voltammetry and chronoamperometry. Linear calibration for hydrogen peroxide was obtained in the range of 3.5 × 10^− 6 to 1.4 × 10^− 3 M under the optimized conditions with the detection limit (S/N = 3) of 8.0 × 10^− 7 M. The apparent Michaelis–Menten constant of the enzyme electrode was 0.93 mM. The enzyme electrode retained about 78% of its response sensitivity after 30 days. The system was applied for the determination of the samples, and the results obtained were satisfactory.     

Carbon nanotube/gold nanoparticles/polyethylenimine-functionalized ionic liquid thin film composites for glucose biosensing

A novel glucose biosensor based on immobilization of glucose oxidase (GOD) in thin films of polyethylenimine-functionalized ionic liquid (PFIL), containing a mixture of carbon nanotubes (CNT) and gold nanoparticles (AuNPs) and deposited on glassy carbon electrodes, was developed. Direct electrochemistry of glucose oxidase in the film was observed, with linear glucose response up to 12mM. The PFIL-stabilized gold nanoparticles had a diameter of 2.4+/-0.8nm and exhibited favorable stability (stored even over one month with invisible change in UV-vis spectroscopic measurements). In addition, CNT were also well dispersed in the PFIL matrix, then, the resulting CNT/AuNPs/PFIL composites film showed high electrocatalytic activity toward reduction of hydrogen peroxide and oxygen. Here, PFIL, due to its high ionic conductivity, good solubility to CNT, and stability to nanoparticles, played an important role in constructing stable CNT/AuNPs/PFIL/GOD composites. And good biocompatibility of PFIL also offered a friendly environment for the immobilization of biomolecules.     

Direct electron transfer: Electrochemical glucose biosensor based on hollow Pt nanosphere functionalized multiwall carbon nanotubes

Herein, a novel third-generation glucose biosensor based on unique hollow nanostructured Pt decorated multiwall carbon nanotubes (HPt-CNTs) composites was successfully constructed. The HPt-CNTs composites were successfully prepared and cast on the glassy carbon electrode (GCE) surface directly. With the help of electrostatic adsorption and covalent attachment, the negative l-cysteine (l-cys) and the positive poly(diallydimethylammonium) chloride (PDDA) protected gold nanoparticles (PDDA-Au) were modified on the resulting electrode surface subsequently, which provided further immobilization of glucose oxidase (GOD). Exploitation of the unique properties of HPt-CNTs composites led to the achievement of direct electron transfer between the electrode and the redox active centers of GOD, and the electrode exhibited a pair of well-defined reversible redox peaks with a fast heterogeneous electron transfer rate. In particular, the detection limit (4 × 10⁻⁷ M) of this biosensor was significantly lower and the linear range (1.2 μM–8.4 mM) was much wider than similar carbon nanotubes (CNTs) and Pt-based glucose biosensors. The resulted biosensor also showed high sensitivity and freedom of interference from other co-existing electroactive species, indicating that our facile procedure of immobilizing GOD exhibited better response and had potential application for glucose analysis.     

A bienzyme channeling glucose sensor with a wide concentration range based on co-entrapment of enzymes in SBA-15 mesopores

A novel bienzyme-channeling sensor was constructed by entrapping glucose oxidase (GOD) and horseradish peroxidase (HRP) in the mesopores of well-ordered hexagonal mesoporous silica structures (SBA-15). The SBA-15 mesoporous materials accelerated the electron transfer between the entrapped HRP and electrode. Both HRP and GOD retained their catalytic activities in the bienzyme-entrapped SBA-15 film. In presence of glucose the enzymatic reaction of GOD-glucose-dissolved oxygen system generated hydrogen peroxide in the bienzyme-entrapped mesopores, which was immediately reduced at -0.40 V by an electrocatalytic reaction with the HRP entrapped in the same mesopore to lead to a sensitive and fast amperometric response. Thus the bienzyme channeling could be used for the detection of glucose with excellent performance without the addition of any mediator. Optimization of the experimental parameters was performed with regard to pH, operating potential and temperature. The detection limit was down to 2.7 x 10(-7)M with a very wide linear range from 3.0 x 10(-6) to 3.4 x 10(-2)M. The constructed bienzyme channeling provided a strategy for amperometric detection of oxidase substrates by co-entrapping the corresponding oxidase and HRP in the mesoporous materials.