Co-immobilization Of Glucose Oxidase Research Articles

Co-immobilization of glucose oxidase and xylose dehydrogenase displayed whole cell on multiwalled carbon nanotube nanocomposite films modified electrode for simultaneous voltammetric detection of d-glucose and d-xylose

In this paper, we first report the construction of Nafion/glucose oxidase (GOD)/xylose dehydrogenase displayed bacteria (XDH-bacteria)/multiwalled carbon nanotubes (MWNTs) modified electrode for simultaneous voltammetric determination of d-glucose and d-xylose. The optimal conditions for the immobilized enzymes were established. Both enzymes retained their good stability and activities. In the mixture solution of d-glucose and d-xylose containing coenzyme NAD+ (the oxidized form of nicotinamide adenine dinucleotide), the Nafion/GOD/XDH-bacteria/MWNTs modified electrode exhibited quasi-reversible oxidation-reduction peak at −0.5V (vs. saturated calomel electrode, SCE) originating from the catalytic oxidation of d-glucose, and oxidation peak at +0.55V(vs. SCE) responding to the oxidation of NADH (the reduced form of nicotinamide adenine dinucleotide) by the carbon nanotubes, where NADH is the resultant product of coenzyme NAD+ involved in the catalysis of d-xylose by XDH-displayed bacteria. For the proposed biosensor, cathodic peak current at −0.5V was linear with the concentration of d-glucose within the range of 0.25–6mM with a low detection limit of 0.1mM d-glucose (S/N=3), and the anodic peak current at +0.55V was linear with the concentration of d-xylose in the range of 0.25∼4mM with a low detection limit of 0.1mM d-xylose (S/N=3). Further, d-xylose and d-glucose did not interfere with each other. 300-fold excess saccharides including d-maltose, d-galactose, d-mannose, d-sucrose, d-fructose, d-cellobiose, and 60-fold excess l-arabinose, and common interfering substances (100-fold excess ascorbic acid, dopamine, uric acid) as well as 300-fold excess d-xylitol did not affect the detection of d-glucose and d-xylose (both 1mM). Therefore, the proposed biosensor is stable, specific, reproducible, simple, rapid and cost-effective, which holds great potential in real applications.

Co-Immobilization of Glucose Oxidase-Catalase: Optimization of Immobilization Parameters to Improve the Immobilization Yield

Co-immobilization of glucose oxidase (EC 1.1.3.4) and catalase (EC 1. 11.1.6) on non-porus glass surfaces using ?-aminopropyltriethoxysilane and polyethyleneimine as an activator and gluteraldehyde as cross linking agent has been carried out for its potential use. Polyethyleneimine was found to be a superior immobilization activator than ?-aminopropyltriethoxysilane. In present study, the effects of rough and smooth beads and optimization of the ratio of enzyme concentrations, activator material and concentration of the cross linking agent were investigated using response surface technology. With optimized concentration of glucose oxidase to catalase ratio (0.97), polyethyleneimine (150 mg/l) and gluteraldehyde (15 ml), the effect of glass bead concentration for maximum immobilization yield was investigated. Central composite optimization strategy with 16 experiments increased immobilization yield from 82.63% to 92.74%. It was observed that 0.3 ml of beads per 120 U of glucose oxidase were necessary for higher immobilization yield.

Determination of β-glucosidase activity in soils with a bioanalytical sensor modified with multiwalled carbon nanotubes

Soil microorganisms and enzymes are the primary mediators of soil biological processes, including organic matter degradation, mineralization, and nutrient recycling. They play an important role in maintaining soil ecosystem quality and functional diversity. Moreover, enzyme activities can provide an indication of quantitative changes in soil organic matter. Beta-glucosidase (beta-Glu) activity has been found to be sensitive to soil management and has been proposed as a soil quality indicator because it provides an early indication of changes in organic matter status and its turnover. The aims of the present study were to test and use a simple and convenient procedure for the assay of beta-Glu activity in agricultural soil. The method described here is based on the enzymatic degradation of cellobiose by beta-Glu present in the soil sample and the subsequent determination of glucose produced by the enzymatic reaction using screen-printed carbon electrodes modified with multiwalled carbon nanotubes (SPCE-CNT) equipped with coimmobilized glucose oxidase and horseradish peroxidase enzymes. The potential applied to the SPCE-CNT detection was -0.15 V versus a Ag/AgCl pseudo-reference electrode. A linear calibration curve was obtained in the range 2.7-11.3 mM with a correlation coefficient. In the present study, an easy and effective SPCE-CNT-modified electrode allowed an improved amperometric response to be achieved and this is attributed to the increased surface area upon electrode modification.

Enzymatic Oxidation and Separation of Various Saccharides with Immobilized Glucose Oxidase

Glucose oxidase from Aspergillus niger, the specific enzyme for beta-D-glucose oxidation, can also oxidize other related saccharides at very slow or negligible rates. The present study aimed to compare the kinetics of D-glucose oxidation using immobilized glucose oxidase on bead cellulose for the oxidation of related saccharides using the same biocatalyst. The significant differences were observed between the reaction rates for D-glucose and other saccharides examined. As a result, k (cat)/K (M) ratio for D-glucose was determined to be 42 times higher than D-mannose, 61.6 times higher than D-galactose, 279 times higher than D-xylose, and 254 times higher than for D-fructose and D-cellobiose. On the basis of these differences, the ability of immobilized glucose oxidase to remove D-glucose from D-cellobiose, D-glucose from D-xylose, and D-xylose from D-lyxose was examined. Immobilized catalase on Eupergit and mixed with immobilized glucose oxidase on bead cellulose or co-immobilized with glucose oxidase on bead cellulose was used for elimination of hydrogen peroxide from the reaction mixture. The accelerated elimination of D-glucose and D-xylose in the presence of co-immobilized catalase was observed. The co-immobilized glucose oxidase and catalase were able to decrease D-glucose or D-xylose content to 0-0.005% of their initial concentrations, while a minimum decrease of low oxidized saccharides D-xylose, D-cellobiose, and D-lyxose, respectively, was observed.

Enhancing the longevity of microparticle-based glucose sensors towards 1 month continuous operation

Luminescent microspheres encapsulating glucose oxidase have recently been reported as potential implantable sensors, but the operational lifetime of these systems has been limited by enzyme degradation. We report here that the longevity of these enzymatic microparticle-based sensors has been extended by the coimmobilization of glucose oxidase (GOx) and catalase (CAT) into the sensor matrix. A mathematical model was used to compare the response and longevity of the sensors with and without catalase. To experimentally test the longevity, sensors were continuously operated under normoglycemic dermal substrate concentrations and physiological conditions (5.5 mM glucose and 140 μM O 2, 37 °C and pH 7.4). The sensors incorporating CAT were experimentally shown to be ∼5 times more stable than those without CAT; nevertheless, the response of sensors with CAT still changed by approximately 20%, when operated continuously for 7 days. The experimentally determined trends obtained for the variation in sensor response due to enzyme deactivation were in close agreement with modeling predictions, which also revealed a significant apparent loss in enzyme activity upon immobilization. It was further predicted via modeling that by incorporating 0.1 mM each of active GOx and CAT, the sensors will exhibit less than 2% variation in response over 1 month of continuous operation.

Immobilization of lignin peroxidase on nanoporous gold: Enzymatic properties and in situ release of H 2O 2 by co-immobilized glucose oxidase

Immobilization of enzymes on porous inorganic materials is very important for biocatalysis and biotransformation. In this paper, nanoporous gold (NPG) was used as a support for lignin peroxidase (LiP) immobilization. NPG with a pore size of 40–50 nm was prepared by dealloying Au/Ag alloy (50:50 wt%) for 17 h. By incubation with LiP aqueous solution, LiP was successfully immobilized on NPG. The optimal temperature of the immobilized LiP was ca. 40, 10 °C higher than that of free LiP. After 2 h incubation at 45 °C, 55% of the initial activity of the immobilized LiP was still retained while the free LiP was completely deactivated. In addition, a high and sustainable LiP activity was achieved via in situ release of H 2O 2 by a co-immobilized glucose oxidase. The present co-immobilization system was demonstrated to be very effective for LiP-mediated dye decolourization.

Functionalized carbon nanotube-bienzyme biocomposite for amperometric sensing

An approach to design a biocomposite bienzyme biosensor with the aim of evaluating its suitability as an amperometric sensor using functionalized multiwalled carbon nanotubes (MWCNTs) is presented. The biosensor is based on a bienzyme-channelling configuration, employing the enzymes glucose oxidase (GOx) and horseradish peroxidase (HRP), which were immobilized with toluidine blue (TB) functionalized MWCNTs. The proposed method demonstrates an easy electron transfer between the immobilized enzymes and the electrode via functionalized MWCNTs in a Nafion matrix. Co-immobilization of GOx and HRP was employed to establish the feasibility of fabricating highly effective bienzyme-based biosensors for low-level glucose determination. Bienzyme immobilized TB functionalized MWCNTs were attached to a glassy carbon electrode, and the electrochemical behavior of the sensor was studied using electrochemical impedance spectroscopy, cyclic voltammetry and chronoamperometry. The excellent electrocatalytic activity of the biocomposite film resulted in the detection of glucose under reduced over potential with a wider range of determination from 1.5 × 10 −8 M to 1.8 × 10 −3 M and with a detection limit of 3 × 10 −9 M. The sensor showed a short response time (within 2 s), good stability and anti-interferant ability. The proposed biosensor exhibits good analytical performance in terms of repeatability, reproducibility and shelf-life stability.

The interference of HEPES buffer during amperometric detection of ATP in clinical applications

HEPES-based biological buffer is subject to photooxidation upon exposure to fluorescent illumination. Thereby hydrogen peroxide is generated, which interferes with amperometric oxidoreductase-based biosensors for glucose or adenosine triphosphate (ATP). These biosensors operate at an oxidation potential above 500 mV vs. the standard calomel electrode (SCE) and involve hydrogen peroxide as the electroactive molecule detected at the electrode surface. False-positive detection of ATP was observed in HEPES buffer utilizing an amperometric microbiosensor based on the co-immobilization of glucose oxidase and hexokinase for detection of ATP in biological specimens. Electrochemical, mass spectrometric, (31)P NMR, and (1)H NMR studies indicate that complexation of ATP and HEPES induced by the presence of Ca(2+) in HEPES buffer decreases the photooxidation of HEPES. Consequently, the hydrogen peroxide background concentration is reduced, thereby leading to erroneous ATP detection at the dual-enzyme microbiosensor, which determines an increase in ATP via a reduced hydrogen peroxide signal.

A wireless magnetoelastic biosensor for rapid detection of glucose concentrations in urine samples

Abstract A wireless magnetoelastic glucose biosensor is fabricated by co-immobilizing glucose oxidase (GOx) and catalase onto a pH-sensitive-polymer-coated magnetoelastic sensor with chitosan as a supporting substrate. The GOx-catalyzed hydrolyzation of glucose produces gluconic acid, resulting in shrinking and corresponding mass decrease in the pH-responsive polymer, and consequently the resonance frequency of the magnetoelastic sensor increasing. The glucose biosensor is applied to measurement of glucose concentrations within urine samples; the shift in resonance frequency is found proportional to the glucose concentration, with a linear response obtained between 1 mM and 15 mM. The presence of acetaminophen, lactose, saccharose and galactose do not significantly interfere with glucose detection; ascorbic acid does, as expected, however its effect can be eliminated through cross-correlation with a pH-responsive magnetoelastic reference sensor or by pre-adjusting the sample to pH 7.0. A L 9 (3 4 ) orthogonal array based on the Taguchi method is used to optimize the sensor fabrication process and to determine the key factors that affect sensor performance. Assays of 15 clinical urine samples give glucose levels that are in accordance with the results by an urine analyzer, showing the proposed sensor can be applied for glucose assay in urine.

An Electron-Conducting Cross-Linked Polyaniline-Based Redox Hydrogel, Formed in One Step at pH 7.2, Wires Glucose Oxidase

A polyaniline-based, electron-conducting, glucose-permeable, redox hydrogel was formed in one step at pH 7.2 by cross-linking polymer acid-templated polyaniline with a water-soluble diepoxide, poly(ethylene glycol diglycidyl ether). Coimmobilization of glucose oxidase in the hydrogel, by co-cross-linking in the same step, led to the electrical wiring of the enzyme and to the formation of a glucose electrooxidation catalyst, allowing the electrocatalytic oxidation of glucose at a steady-state current density of 225 μA·cm-2 at 0.3 V vs Ag/AgCl.

Simultaneous co-immobilization of glucose oxidase and catalase in their substrates

Glucose oxidase (GOD) and catalase (CAT) were simultaneously co-immobilized onto magnesium silicate (florisil) by covalent coupling. Glucose was added in immobilization mixture and hydrogen peroxide which is the substrate of CAT was produced in coupling mixture during immobilization time. Therefore, co-immobilization of GOD and CAT was carried out in presence of both their substrate: glucose and hydrogen peroxide, respectively. The effect of glucose concentration in immobilization mixture on activities of GOD and CAT of co-immobilized samples were investigated. Maximum GOD and CAT activities were determined for samples co-immobilized in presence of 15 and 20 mM glucose, respectively. Co-immobilization of GOD and CAT in presence of their substrates highly improved the activity and reusability of both enzymes.

Co-immobilization of glucose oxidase and hexokinase on silicate hybrid sol–gel membrane for glucose and ATP detections

The co-immobilization of glucose oxidase (GOD) and hexokinase/glucose-6-phosphate dehydrogenase (HEX) in the silica hybrid sol-gel film for development of amperometric biosensors was investigated. The silica hybrid film fabricated by hydrolysis of the mixture of tetraethyl orthosilicate and 3-(trimethoxysiyl)propyl methacrylate possessed a three-dimension vesicle structure and good uniformity and conformability, and was ready for enzyme immobilization. The electrochemical and spectroscopic measurements showed that the silica hybrid sol-gel provided excellent matrice for the enzyme immobilization and that the immobilized enzyme retained its bioactivity effectively. The immobilized GOD could catalyze the oxidation of glucose, which could be used to determine glucose at +1.0 V without help of any mediator. The competition between GOD and HEX for the substrate glucose involving ATP as a co-substrate led to a decrease of the glucose response, which allowed us to develop an ATP sensor with a good stability. The fabricated silica hybrid sol-gel matrice offered a stage for further study of immobilization and electrochemistry of proteins.

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.

Biofuel cell based on direct bioelectrocatalysis

A biofuel cell, consisting of two 3mm diameter carbon rod electrodes and operating at ambient temperature in aqueous solution, pH 6, is described. Biofuel cell based on enzymes able to exchange directly electrons with carbon electrodes was constructed and characterized. Anode of the biofuel cell was based on immobilized Quino-hemoprotein alcohol dehydrogenase from Gluconobacter sp. 33 (QH-ADH), cathode on co-immobilized glucose oxidase from Aspergilus niger (GO(x)) and microperoxidase 8 from the horse heart (MP-8) acting in the consecutive mode. Two enzymes GO(x) and MP-8 applied in the design of biofuel cell cathode were acting in consecutive mode and by hydrogen peroxide oxidized MP-8 was directly accepting electrons from carbon rod electrode. If ethanol was applied as an energy source the maximal open circuit potential of the biofuel cell was -125 mV. If glucose was applied as energy source the open circuit potential of the cell was +145 mV. The maximal open circuit potential (270 mV) was achieved in the presence of extent concentration (over 2 mM) of both substrates (ethanol and glucose). Operational half-life period (tau(1/2)) of the biofuel cell was found to be 2.5 days.

A novel glucose ENFET based on the special reactivity of MnO 2 nanoparticles

Generally a glucose-sensitive enzyme field-effect transistor (ENFET) is based on local pH change in biomembranes resulted from the formation of gluconic acid. Here we proposed a glucose ENFET based on a new principle. The glucose ENFET was fabricated by coimmobilizing glucose oxidase (GOD) and MnO 2 nanoparticles on the gate of an ion-sensitive field-effect transistor (ISFET). The proposed glucose biosensor shows a significant local pH increase in the sensitive membrane with the increase of glucose concentration. The driving force of the pH change in our sensor is essentially different from all the other glucose ENFETs, including those prepared by bulk MnO 2. The special reaction ability of MnO 2 nanoparticles with hydrogen peroxide might be the main cause of the pH change. In addition, the influence of buffer concentration, pH and ionic strength on the glucose ENFET is investigated in detail. It is found that the ionic strength has little effect on the performance of the ENFET. Under optimal conditions, the proposed ENFET exhibits a linear response with glucose in the range of 0.025–1.90 mM, an extended dynamic upper limit of 3.5 mM glucose, and considerable good reproducibility and stability.

Glucose Amperometric Biosensor Based on the Co-immobilization of Glucose Oxidase (GOx) and Ferrocene in Poly(pyrrole) Generated from Ethanol / Water Mixtures

The co-immobilization of GOx and ferrocene in a polymeric matrix by a one-step simple method is presented. This procedure to immobilize ferrocene as mediator implies the absence of modification of the monomer or the enzyme that would lead to the loss of its activity. Ferrocene incorporation results in an increase of sensitivity compared with the sensor prepared without the redox mediator (1.5 vs 0.23 muA mmol-1 L cm-2) and the decrease of the working potential to 0.4 V. The prepared sensor shows linear response till 10 mmol L-1 and response time of 2 s, in addition to reasonable stability after one week.

A Biochemo-Mechanical System Consisting of Polyampholyte Gels with Coimmobilized Glucose Oxidase and Urease

An attempt was made to prepare a biochemo-mechanical system that is capable of converting biochemical energy created as a result of an enzyme reaction into mechanical work through the swelling and shrinking of a gel. The preparation was performed via the physical entrapment of glucose oxidase (GOD) plus urease within an amphoteric polymer network. We used N-isopropylacrylamide (NIPA) as a main constituent of the network into which acrylic acid (AAc) as an anionic monomer and 1-vinylimidazole (VI) as an cationic monomer were introduced via radical polymerization. The gelation was carried out in the presence and the absence of the enzyme(s) by cross-linking with N,N‘-methylenebisacrylamide of the monomer mixtures with molar ratios of NIPA/AAc/VI = 14:1:1 and 14:1:2. The effects of pH and temperature on the swelling degree were studied in 50 mM NaCl solution. The ampholyte gels exhibited an isoelectric point (pI) at pH 5−6 at which the swelling degree reached a minimum value. Therefore, two pH regions bringing about gel swelling lie on either side of pI, that is, pH < 5 and pH > 6. This characteristic allowed the gel to undergo a shrinking−swelling oscillation passing through pI while an increase and a decrease in pH took place via the immobilized enzyme reactions within the gel phase. When the hydrolysis of urea with the immobilized urease raises the pH in the gel, it continues shrinking at pH < pI but swelling at pH > pI after attainment of an extremely collapsed state at pI. Such volume changes take place during a decrease in the pH because of the oxidation of glucose with the immobilized GOD. As a result, the shrinking−swelling oscillation was observed by alternative addition of the two substrates into the outer medium surrounding the gel.

A stable glucose biosensor prepared by co-immobilizing glucose oxidase into poly(p-chlorophenol) at a platinum electrode.

An amperometric glucose biosensor was successfully developed by electrochemical polymerization of p-chlorophenol (4-CP) at a Pt electrode in the presence of glucose oxidase. The amperometric response of this biosensor to hydrogen peroxide, formed as the product of enzymatic reaction, was measured at a potential of 0.6 V (vs. SCE) in phosphate buffer solution. The performances of sensors, prepared at different monomer concentrations and polymerization potentials, were investigated in detail. The biosensor prepared under optimal conditions had a linear response to glucose ranging from 2.5 x 10(-4) to 1.5 x 10(-2) mol L(-1) with a correlation coefficient of 0.997 and a response time of less than 2 s. Substrate selectivity of the polymer-based enzyme electrode was tested for coexisting interferents such as uric acid and ascorbic acid, and no discernible response was observed. After 90 days, the response of the biosensor remained almost unchanged, indicating very good stability.

Flow injection analytical system for glucose with screen-printed enzyme biosensor incorporating Os-complex mediator

A novel flow injection analytical system with a screen-printed enzyme sensor incorporating Os-complex mediator has been developed. A newly created glucose biosensor was fabricated using thick-film technique and co-immobilization of glucose oxidase and Os-complex on a permeable membrane. The composition of the enzyme and mediator in the membrane and the analytical conditions were optimized. The flow injection electrochemical system showed that glucose was detected linearly in the concentration range from 0.1 to 10 mM with a detection limit of 0.03 mM by 50 μl of sample injection, giving a throughout of about 40 samples per hour. The glucose biosensors retained their constant response after more than 100 injections and storage over a month. The designed electrochemical flow system with a disposable biosensor is suitable for automatic and rapid determination of glucose.

Linear Dependence of the Potential of “Wired” Glucose Oxidase Electrodes on the Concentration of Glucose

Potentiometric assays, unlike amperometric or coulometric ones, do not require accurate definition and knowledge of the area of the measuring electrode or microcell volume. Their potential scales, however, with the logarithm of the concentration of the analyte, while in amperometric, chronoamperometric, or chronocoulometric assays the charge or current scales linearly with the concentration. Here we show that in a potentiometric glucose assay the potential can scale linearly with concentration. Such scaling is realized when the electrode is coated with a resistive, but nevertheless electron-conducting, film where glucose is electrooxidized. The film consists of a redox polymer film that “wires” reaction centers of covalently co-immobilized glucose oxidase to the electrode. After a potential pulse is applied to the “wired” enzyme electrode so that the electrode-bound redox centers are electrooxidized, the floating electrode potential decays to a value that varies linearly with the concentration of glucose.