Glucose Oxidase Concentration Research Articles

Effects of the kinematic viscosity and surface tension on the bubble take-off period in a catalase–hydrogen peroxide system

The effect of kinematic viscosity and surface tension of the solution was investigated by adding calatase, glucose oxidase, or glucose on the bubble movement in a catalase–hydrogen peroxide system. The kinematic viscosity was measured using a Cannon–Fenske kinematic viscometer. The surface tension of the solution was measured by the Wilhelmy method using a self-made apparatus. The effects of the hole diameter/cell wall thickness, catalase concentration, glucose concentration, and glucose oxidase concentration on the kinematic viscosity, surface tension, and bubble take-off period were investigated. With our system, the effects of the changes in the solution materiality on the bubble take-off period were proven to be very small in comparison to the change in the oxygen-producing rate.

An ultrasonic inactivation of Aspergillus niger glucose oxidase in aqueous solutions

The inactivation of Aspergillus niger glucose oxidase (GO) was studied in 0.02 M phosphate-citrate buffer (PCB) at various pH, temperatures of 37-59 degrees C, and sonication with low frequency (27 kHz, LF-US) and high frequency (2.64 MHz, HF-US) ultrasound. The GO inactivation was characterized by the effective first-order inactivation rate constants k(in), k(in)*, and k(in)(us), reflecting the total, thermal, and ultrasonic inactivation components. The constants strongly depended on the pH and temperature of solution, GO concentration, and the presence of acceptors of the free radicals HO* -DMF, DMSO, ethanol, butanol, octanol, and mannitol, confirming that the active radicals formed in the ultrasonic cavitation field played an important role in the GO inactivation. The activation energy in the loss of GO catalytic activity considerably decreased when the enzyme solution was treated with LF-US or HF-US. The dissociative scheme of GO inactivation is discussed. Mannitol can be used for protection of GO from inactivation with LF-US or HF-US in the food industry and immunobiotechnology.

Glutathione-capped CdTe quantum dots for the sensitive detection of glucose

A simple and sensitive assay system for glucose based on the glutathione (GSH)-capped CdTe quantum dots (QDs) was developed. GSH-capped CdTe QDs exhibit higher sensitivity to H 2O 2 produced from the glucose oxidase catalyzed oxidation of glucose, and are also more biocompatible than other thiols-capped QDs. Based on the quenching of H 2O 2 on GSH-capped QDs, glucose can be detected. The detection conditions containing reaction time, the concentration of glucose oxidase and the sizes of QDs were optimized and the detection limits for glucose was determined to be 0.1 μM; two detection ranges of glucose from 1.0 μM to 0.5 mM and from 1.0 mM to 20 mM, respectively were obtained. The detection limit was almost a 1000 times lower than other QDs-based optical glucose sensing systems. The developed glucose detection system was simple and facile with no need of complicated enzyme immobilization and modification of QDs.

Enzyme immunoassay of insulin at picomolar levels based on the coulometric determination of hydrogen peroxide

A novel electrochemical enzyme immunoassay system with a detection limit of 2 pM (10 ng L−1) was developed for the determination of insulin, an important hormone for clinical diagnosis of diabetes. An insulin-containing sample solution was added to a solution containing a certain concentration of glucose oxidase (GOx)-labeled anti-insulin antibody to undergo an immunological reaction, and unbound GOx–antibody conjugate was trapped on the insulin-immobilized glass plate. Then hydrogen peroxide generated by the enzymatic reaction with the trapped conjugate was coulometrically determined as follows. The hydrogen peroxide generated oxidized [Os(bpy)2Cl]+ to [Os(bpy)2Cl]2+ in a horseradish peroxidase (HRP)-containing polymer layer on an electrode under the open-circuit condition, and then the [Os(bpy)2Cl]2+ accumulated was reduced by applying a negative potential. The accumulation brought about a large current response and a low detection limit for hydrogen peroxide (0.5 nM): the detection limit was lowered by more two orders of magnitude than that obtained for conventional amperometric method using the same polymer-coated electrode. The enhanced sensitivity for hydrogen peroxide enabled the determination of a trace concentration of insulin with the immunoassay using the antibody modified with a hydrogen peroxide-producing enzyme. The assay time was ca. 30 min. The results suggested the possible application of the proposed method to an on-site diabetic sensing system.

Glassy Carbon Electrodes Modified with Multiwall Carbon Nanotubes Dispersed in Polylysine

AbstractWe report the analytical performance of glassy carbon electrodes (GCE) modified with a dispersion of multiwall carbon nanotubes (MWCNT) in polylysine (Plys) (GCE/MWCNT‐Plys). The resulting electrodes show an excellent electrocatalytic activity towards different bioanalytes like ascorbic acid, uric acid and hydrogen peroxide, with important decrease in their oxidation overvoltages. The dispersion of 1.0 mg/mL MWCNT in 1.0 mg/mL polylysine is highly stable, since after 2 weeks the sensitivity for hydrogen peroxide at GCE modified with this dispersion remained in a 90% of the original value. The MWCNT‐Plys layer immobilized on glassy carbon electrodes has been also used as a platform to build supramolecular architectures by self‐assembling of polyelectrolytes based on the polycationic nature of the polylysine used to disperse the nanotubes. The self‐assembling of glucose oxidase has allowed us to obtain a supramolecular multistructure for glucose biosensing. The influence of glucose oxidase concentration and adsorption time as well as the effect of using polylysine or MWCNT‐Plys as polycationic layers for further adsorption of GOx is also evaluated.

Carbon Nanotubes Based Glucose Needle-type Biosensor.

A novel needle-type biosensor based on carbon nanotubes is reported. Thebiosensor was prepared by packing a mixture of multi-wall carbon nanotubes (MWCNTs),graphite powder and glucose oxidase (Gox) freeze-dried powder into a glass capillary of 0.5mm inner diameter. The resulting amperometric biosensor was characterizedelectrochemically using amperometry in the presence of hydrogen peroxide and in thepresence of glucose. The glucose biosensor sensitivity was influenced by the glucoseoxidase concentration within the MWCNTs mixture. The optimized glucose needle-typebiosensor displayed better sensitivity and stability, and a detected range of up to 20 mM.Based on its favorable stability, the needle biosensor was first time used in real-timemonitoring system as a kind of online glucose detector. The decay of current response isless than 10% after 24-hour continuous observation.

Multiwalled Carbon Nanotube-Poly(vinyl alcohol)-Glucose Oxidase Nanobiocomposites for Amperometric Sensing

Novel nanobiocomposites multiwalled carbon nanotube-poly(vinyl alcohol)-glucose oxidase have been successfully prepared by a simple solution-evaporation method. The morphology and performance of the multiwalled carbon nanotube-poly(vinyl alcohol)-glucose oxidase film have been characterized by atomic force microscopy, cyclic voltammetry, and amperometry. The multiwalled carbon nanotube and glucose oxidase were observed to be homogeneously dispersed throughout the poly(vinyl alcohol) matrix. When compared with bare glassy carbon electrode, the sensitivity to hydrogen peroxide is greatly improved by about 115 times at multiwalled carbon nanotubepoly(vinyl alcohol) modified glassy carbon electrode. The glucose biosensor sensitivity was strongly influenced by the glucose oxidase concentration within the multiwalled carbon nanotube-poly(vinyl alcohol)-glucose oxidase composite.

Adsorption–Penetration Studies of Glucose Oxidase into Phospholipid Monolayers at the 1,2‐Dichloroethane/Water Interface

The interaction between glucose oxidase (GOx) and phospholipid monolayers is studied at the 1,2-dichloroethane/water interface by electrochemical impedance spectroscopy. Electrochemical experiments show that the presence of GOx induces changes in the capacitance curves at both negative and positive potentials, which are successfully explained by a theoretical model based on the solution of the Poisson-Boltzmann equation. These changes are ascribed to a reduced partition coefficient of GOx and an increase of the permittivity of the lipid hydrocarbon domain. Our results show that the presence of lipid molecules enhances the adsorption of GOx molecules at the liquid/liquid interface. At low lipid concentrations, the adsorption of GOx is probably the first step preceding its penetration into the lipid monolayer. The experimental results indicate that GOx penetrates better and forms more stable monolayers for lipids with longer hydrophobic tails. At high GOx concentrations, the formation of multilayers is observed. The phenomenon described here is strongly dependent on 1) the GOx and lipid concentrations, 2) the nature of the lipid, and 3) the potential drop across the interface.

Use of glucose oxidase in a membrane reactor for gluconic acid production

This article aims at the evaluation of the catalytic performance of glucose oxidase (GO) (EC.1.1.3.4) for the glucose/gluconic acid conversion in the ultrafiltration cell type membrane reactor (MB-CSTR). The reactor was coupled with a Millipore ultrafiltration-membrane (cutoff of 100 kDa) and operated for 24 h under agitation of 100 rpm, pH 5.5, and 30 degrees C. The experimental conditions varied were the glucose concentration (2.5, 5.0, 10.0, 20.0, and 40.0 mM), the feeding rate (0.5, 1.0, 3.0, and 6.0/h), dissolved oxygen (8.0 and 16.0 mg/L), GO concentration (2.5, 5.0, 10.0, and 20.0 U(GO)/mL), and the glucose oxidase/catalase activity ratio (U(GO)/U(CAT))(1:0, 1:10, 1:20, and 1:30). A conversion yield of 80% and specific reaction rate of 40 x 10(-4) mmol/h x U(GO) were attained when the process was carried out under the following conditions: D =3.0/h, dissolved oxygen =16.0 mg/L, [G] =40 mM, and (U(GO)/U(CAT)) =1:20. A simplified model for explaining the inhibition of GO activity by hydrogen peroxide, formed during the glucose/gluconic acid conversion, was presented.

Immobilization and bioactivity of glucose oxidase in hydrogel microspheres formulated by an emulsification–internal gelation–adsorption–polyelectrolyte coating method

The purpose of this study was to develop a novel microsphere formulation of glucose oxidase (GOX) with high drug loading, encapsulation efficiency and bioactivity. GOX was encapsulated in alginate/chitosan microspheres (ACMS) using an emulsification–internal gelation, followed by GOX adsorption and polyelectrolyte coating method. The factors influencing GOX loading, encapsulation efficiency and activity of the loaded GOX were investigated. The resultant ACMS in wet state were spherical with a mean diameter of about 138 μm. GOX loading was found to be pH dependent. High GOX loading and encapsulation efficiency were achieved when the pH of the adsorption medium was lower than the isoelectric point (p I) of GOX. GOX loading and encapsulation efficiency increased with increasing GOX concentration in the loading solution, but decreased with increasing chitosan concentration in the coating solution. The activity of loaded GOX increased and then decreased with increasing chitosan concentration. The activity of GOX in ACMS was maintained and showed sustained production of H 2O 2 as compared to free GOX. Around 90% of the original activity of immobilized GOX remained after lyophilization and storage at −20 °C for a month. These results suggest that the ACMS and the fabrication method are suitable for microencapsulation of proteins like GOX.

Preparation of selective micro glucose sensor without permselective membrane by electrochemical deposition of ruthenium and glucose oxidase

A carbon fiber microelectrode, surface of which ruthenium and glucose oxidase (GOx) were electrochemically codeposited, has been investigated. The Ru deposition onto the microelectrode increased current response to H 2O 2 oxidation, while decreased oxidation currents due to interfering substances, such as ascorbic acid, uric acid, p-acetamidophenol, l-cysteine and dopamine. The codeposition of Ru and GOx gave further suppression of the interfering signals with keeping the current response to H 2O 2. When amperometric glucose sensing was conducted by using the GOx and Ru modified microelectrode, an increase in GOx concentration in the deposition bath enlarged oxidation current of H 2O 2 generated from glucose oxidation by GOx. The presence of ascorbic acid in analyte gave no error in detection of glucose and errors caused by uric acid was +3% at the most for measuring 5 mM glucose, which is the normal physiological level in blood.

1,4-Benzoquinone-based electrophoretic assay for glucose oxidase

The communication demonstrates feasibility of an enzyme microassay for glucose oxidase with 1,4-benzoquinone as an acceptor of electrons. The protocol uses the plug–plug mode of electrophoretically mediated microanalysis, with nanolitre injected volumes of enzyme and reactant solutions. The reactant and product, 1,4-benzoquinone and hydroquinone, are separated during the assay by differential binding to sulfated-β-cyclodextrin used as additive to the phosphate buffer (pH 7) and monitored at selected wavelengths in their UV spectra. The assay covers glucose oxidase concentration from 0.01 to 0.1 mg ml −1. Due to the strong UV absorbance of the both reactant and product, there is no need for use of a second enzyme (peroxidase) in the present assay.

ENFET glucose biosensor produced with mesoporous silica microspheres

The biosensors based on ENFETs for direct glucose oxidase (GOD) concentration analysis have been fabricated. Mesoporous silica (MS) microspheres of different pore sizes were used as the substrates for immobilizing enzyme to construct ENFETs. The MS microspheres show significantly improved enzyme immobilization capacity. The glucose sensitive ENFET based on the modification of the gate surface of ion-sensitive field-effect transistors (ISFETs) with MS microspheres and glucose oxidase (GOD) shows obviously enhanced sensitivity and extended lifetime compared with the conventional ones. MS microspheres of different pore sizes were investigated and the sensor with remodeled MS microspheres which experienced the pore expanding treatment show better performance than untreated MS microspheres. The fabricated sensors have a linear range of 0.25–2.0 mM, and a detection limit of ca. 0.10 mM. The influence of buffer concentration, ionic strength and pH was discussed. The biosensor also has good stability and reproducibility.

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.

Development of a long-life capillary enzyme bioreactor for the determination of blood glucose

A long-life capillary enzyme bioreactor was developed that determines glucose concentrations with high sensitivity and better stability than previous systems. The bioreactor was constructed by immobilizing glucose oxidase (GOx) onto the inner surface of a 0.53 mm i.d. fused-silica capillary that was part of a continuous-flow system. In the presence of oxygen, GOx converts glucose to gluconic acid and hydrogen peroxide (H 2O 2). Hydrogen peroxide detection was accomplished using an amperometric electrochemical detector. The integration of this capillary reactor into a flow-injection (FIA) system offered a larger surface-to-volume ratio, reduced band-broadening effects, and reduced reagent consumption compared to packed column in FIA or other settings. To obtain operational (at ambient temp) and storage (at 4 °C) stability for 20 weeks, the glucose biosensing system was prepared using an optimal GOx concentration (200 mg/mL). This exhibited an FIA peak response of 7 min and a detection limit of 10 μM (S/N = 3) with excellent reproducibility (coefficient of variation, CV < 0.75%). It also had a linear working range from 10 1 to 10 4 μM. The enzyme activity in this proposed capillary enzyme reactor was well maintained for 20 weeks. Furthermore, 20 serum samples were analyzed using this system, and these correlated favorably (correlation coefficient, r 2 = 0.935) with results for the same samples obtained using a routine clinical method. The resulting biosensing system exhibited characteristics that make it suitable for in vivo application.

Real-Time Assessment of Spatial and Temporal Coupled Catalysis within Polyelectrolyte Microcapsules Containing Coimmobilized Glucose Oxidase and Peroxidase

The encapsulation of biological enzymes within polyelectrolyte microcapsules is an important step toward microscale devices for processing and analytical applications, one which could be applied to the realization of minimally invasive sensing technology. In this work, the encapsulation and functional characterization of a bienzymatic coupled catalytic system within polyelectrolyte microcapsules is described. The two components, glucose oxidase (GOx) and horseradish peroxidase (HRP), were coprecipitated with calcium carbonate microspheres, followed by layer-by-layer assembly to form ultrathin polymer film coatings that act as capsule walls after removal of the sacrificial carbonate cores. Encapsulated concentrations of GOx and HRP were determined to be 19.7 +/- 1.0 and 29.4 +/- 3.6 mg/mL, respectively. An 85% decrease in the rate of glucose consumption relative to GOx and HRP in free solution was observed, which is attributed to substrate diffusion limitations. To further understand the temporal and spatial dynamics of the two-step reaction, a technique for monitoring microscale glucose consumption was developed using confocal imaging techniques. Time-based acquisition of capsule/Amplex Red suspensions was performed, from which it was observed that the high concentration of enzyme immobilized within the capsule walls resulted in a greater rate and quantity of glucose consumption at the capsule periphery when compared to glucose consumption within the capsule interior. These findings demonstrate the function of a bienzymatic catalytic system within the controlled environment of polyelectrolyte microspheres and a novel approach to analysis of the internal reactions using confocal imaging that will allow direct comparison with reaction-diffusion modeling and further explorations to optimize the distribution and activity of the encapsulated species.

Glucose oxidase effect on dough rheology and bread quality: A study from macroscopic to molecular level

Enzymes are used in baking to improve dough handling properties and the quality of baked products. Glucose oxidase (GO) is an enzyme with oxidizing effect due to the hydrogen peroxide released from its catalytic reaction. In this study, the macroscopic effect of increasing glucose oxidase concentrations on wheat dough rheology, fresh bread characteristics and its shelf life during storage was determined. A reinforcement or strengthening of wheat dough and an improvement of bread quality can be obtained with the addition of GO, although inverse effects were obtained when excessive enzyme levels were added. The analysis of the gluten proteins at molecular level by high performance capillary electrophoresis and at supramolecular level by cryo-scanning electron microscopy revealed that the GO treatment modified gluten proteins (gliadins and glutenins) through the formation of disulfide and non-disulfide crosslinks. The high molecular weight glutenin subunits showed to be the most susceptible glutenin fraction to the oxidation action of GO. Excessive addition of GO produced an excessive crosslinking in the gluten network, responsible of the negative effect on the breadmaking properties.

Cast Thin Film Biosensor Design Based on a Nafion Backbone, a Multiwalled Carbon Nanotube Conduit, and a Glucose Oxidase Function

Novel electroanalytical sensing nanobiocomposite materials are reported. These materials are prepared by mixing multiwalled carbon nanotubes (MWNTs), a Nafion cation exchanger, and glucose oxidase (GOD) in appropriate amounts. The MWNTs are cylindrical with a diameter in the range 40-60 nm and with a length of up to several micrometers, and they provide electrical conductivity. Nafion acts as a polymer backbone to give stable and homogeneous cast thin films. Both MWNTs and Nafion provide negative functionalities to bind to positively charged redox enzymes such as glucose oxidase. The resulting biosensing composite material is inexpensive, reliable, and easy to use. The homogeneity of the MWNT-Nafion-GOD nanobiocomposite films was characterized by atomic force microscopy (AFM). Amperometric transducers fabricated with these materials were characterized electrochemically using cyclic voltammetry and amperometry in the presence of hydrogen peroxide and in the presence of glucose. Their linear response to hydrogen peroxide was demonstrated. The glucose biosensor sensitivity was strongly influenced by the glucose oxidase concentration within the nanobiocomposite film. The optimized glucose biosensor (2.5 mg/mL GOD) displayed a sensitivity of 330 nA/mM, a linear range of up to 2 mM, a detection limit of 4 microM, and a response time of <3 s.

Effects of oxidative stress on adiponectin secretion and lactate production in 3T3-L1 adipocytes

Obesity is an increasing nutritional disorder in developed countries, and oxidative stress has been identified as a key factor in numerous pathologies such as diabetes, inflammation, and atherosclerosis, which are favored by obesity. The objective of the present study was to investigate the effects of oxidative stress in 3T3-L1 adipose cells on two parameters involved in metabolic complications associated with obesity, namely adiponectin secretion and lactate production. Differentiated 3T3-L1 adipose cells were exposed to increasing concentrations of glucose oxidase. 4-Hydroxynonenal (4-HNE), a relevant lipid peroxidation by-product which may affect several metabolic processes in making covalent adducts with various molecules; adiponectin secretion; and lactate production were measured in response to glucose oxidase exposure. Results show an inhibition of adiponectin mRNA expression by glucose oxidase and a significant inverse correlation between 4-HNE formation and adiponectin secretion. Furthermore, 4-HNE alone inhibits adiponectin production by 3T3-L1. On the other hand, glucose oxidase and 4-HNE significantly stimulated lactate production by 3T3-L1 adipocytes. These results demonstrate that adipose cells are highly sensitive to oxidative stress, with subsequent decreased adiponectin secretion and increased lactate production, two events involved in the development of insulin resistance.

Covalent Immobilization of Glucose Oxidase on Well-Defined Poly(glycidyl methacrylate)−Si(111) Hybrids from Surface-Initiated Atom-Transfer Radical Polymerization

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