Glucose oxidase bienzyme electrodes for ATP, NAD+, starch and disaccharides

Based on a glucose oxidase sensor for determination of glucose several glucoseoxidase bioenzyme electrodes have been developed. Enzymes producing glucose by hydrolysis of saccharides (glucamylase, invertase, cellulase) as well as glucose consuming systems (hexo-kinase, glucose dehydrogenase) have been coupled to glucose oxidase. The function of the bienzyme systems was demonstrated by concentration measurements (blood glucose, maltose, ATP, NAD+, starch) and enzyme activity measurements (alpha-amylase, ATPase, lactate dehydrogenase).     

Development of biosensors for the simultaneous determination of sucrose and glucose, lactose and glucose, and starch and glucose

Sensors for the simultaneous determinations of sucrose and glucose, lactose and glucose, and starch and glucose were prepared by a combination of the enzyme system shown below and an oxygen electrode: The mechanism for separating the substrates with the proposed sensors is based on the time lag arising from reaction and diffusion. Invertase, beta-galactosidase, amyloglucosidase, mutarotase, and glucose oxidase were covalently immobilized on triacetyl cellulose membranes containing 1,8-diamino-4-aminomethyloctane. A glucose oxidase membrane, mutarotase membrane, three sheets of triacetyl cellulose membranes, and invertase, or beta-galactosidase or amyloglucosidase membrane were placed in that order on the tip of the oxygen electrode. Calibration curves for sucrose, lactose, and starch were linear up to 40 mM, 60-180 mM, and 10%, respectively. The simultaneous determination of sucrose and glucose, lactose and glucose, and starch and glucose was possible when the amount of glucose coexised was in the range of 2-16% sucrose, 2.8-8.3% lactose, or 0.1-1% starch. The relative errors were +/-4% for sucrose and +/-3% for lactose in 100 assays. The starch sensor was reused only five times. Each enzyme membrane was fairly stable for more than 10 days.     

Improved selectivity of microbial biosensor using membrane coating. Application to the analysis of ethanol during fermentation

A ferricyanide mediated microbial biosensor for ethanol detection was prepared by surface modification of a glassy carbon electrode. The selectivity of the whole Gluconobacter oxydans cell biosensor for ethanol determination was greatly enhanced by the size exclusion effect of a cellulose acetate (CA) membrane. The use of a CA membrane increased the ethanol to glucose sensitivity ratio by a factor of 58.2 and even the ethanol to glycerol sensitivity ratio by a factor of 7.5 compared with the use of a dialysis membrane. The biosensor provides rapid and sensitive detection of ethanol with a limit of detection of 0.85 microM (S/N=3). The selectivity of the biosensor toward alcohols was better compared to previously published enzyme biosensors based on alcohol oxidase or alcohol dehydrogenases. The biosensor was successfully used in an off-line monitoring of ethanol during batch fermentation by immobilized Saccharomyces cerevisiae cells with an initial glucose concentration of 200 g l(-1).     

A fluorescent glucose biosensor based on immobilized glucose oxidase on bamboo inner shell membrane

A fluorescent glucose biosensor was constructed by immobilizing glucose oxidase on a bamboo inner shell membrane with glutaraldehyde as a cross-linker. The detection scheme was based on the depletion of dissolved oxygen content upon exposure to glucose solution with a concomitant increase in the fluorescence intensity of an oxygen transducer, tris(4,7-diphenyl-1,10-phenanthroline)ruthenium(Pi) ditetrakis(4-chlorophenyl)borate. The enzyme immobilization, effect of pH, temperature and ionic strength have been studied in detail. The biosensor exhibited repeatable response to a 2.0 mM glucose solution with a relative standard deviation of 3.0% (n = 10). It showed good storage stability and maintained 95% of its initial response after it had been kept at 4 degrees C for 8 months. The biosensor has a linear response range of 0.0-0.6 mM glucose with a detection limit of 58 microM (S/N = 3). Common potential interferants in samples do not pose any significant interference on the response of the glucose biosensor. It was successfully applied to the determination of glucose content in some commercial wines and medical glucose injections.     

Glucose biosensor based on nanohybrid material of gold nanoparticles and glucose oxidase on a bioplatform

A simple and relatively cheap glucose biosensor based on a combination of gold nanoparticles (Au NPs) and glucose oxidase (GO(x) ) immobilized on a bioplatform eggshell membrane was established. Scanning electron microscopy showed successful immobilization of Au NPs/GO(x) on the eggshell membrane. The effects of pH, phosphate buffer concentration, and temperature on the glucose biosensor were studied in detail. The biosensor shows a linear response at a glucose concentration range of 5-525 μM. The detection limit of the biosensor is 2.5 μM (S/N = 3). The biosensor exhibits good repeatability with RSD = 3.6% (n = 6), good operational stability with over 300 measurements and long-term storage stability with a shelf life of at least 6 months. The response time is less than 60 s. The glucose level in commercial food samples has been successfully determined. The proposed work shows potential to develop cost-effective biosensors for biotechnological, biomedical and industrial use.     

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.     

Analysis of various sugars by means of immobilized enzyme coupled flow injection analysis

Glucose, maltose, sucrose, lactose, xylose, sorbose, galactose, fructose and gluconolactone were analyzed by means of immobilized pyranose oxidase as well as by the combination of immobilized glucose oxidase with immobilized glycoamylase, invertase, mutarotase, maltase (α-glucosidase) and glucose isomerase by flow injection analysis (FIA). For the simultaneous analysis of glucose and other sugars three different flow-injection configurations were applied and compared. The average error of prediction of the analyses were better than 3% in model media and better than 6% in yeast extract containing media.     

Optimization of the multienzyme system for sucrose biosensor by response surface methodology

An immobilized multienzyme- and cathodic amperometry-based biosensor for sucrose was constructed for the analysis of food and fermentation samples. The multienzyme system, comprising invertase, mutarotase and glucose oxidase (GOD), was immobilized by using glutaraldehyde as cross-linking agent. Operating parameters of the biosensor for the estimation of sucrose in the range 1–10% were standardized. Response surface methodology (RSM) based on three-factor, three-variable design was used to evaluate the effect of important variables (concentration of enzymes, (varied in the range invertase (10–50 IU), mutarotase (5–105 IU) and GOD (1–9 IU)) on the response of biosensor. In the range of parameters studied, response time decreased with decrease in the invertase and with increase in mutarotase and GOD. Mutarotase concentration above 75 IU was found to result in an increased response time due to inhibition of mutarotase by its product -D-glucose. The optimal conditions achieved for the analysis of sucrose were: invertase 10 IU, mutarotase 40 IU, and GOD 9 IU. With these conditions, the predicted and actual experimental response time values were 2.26 and 2.35 min respectively, showing good agreement.     

Purification, biochemical characterization, and gene cloning of a new extracellular thermotolerant and glucose tolerant maltooligosaccharide-forming alpha-amylase from an endophytic ascomycete Fusicoccum sp. BCC4124

An endophytic fungus, Fusicoccum sp. BCC4124, showed strong amylolytic activity when cultivated on multi-enzyme induction enriched medium and agro-industry substrates. alpha-Amylase and alpha-glucosidase activities were highly induced in the presence of maltose and starch. The purified target alpha-amylase, Amy-FC1, showed strong hydrolytic activity on soluble starch (kcat/Km=6.47 x 10(3) min(-1)(ml/mg)) and selective activity on gamma- and beta-cyclodextrins, but not on alpha-cyclodextrin. The enzyme worked optimally at 70 degrees C in a neutral pH range with t(1/2) of 240 min in the presence of Ca(2+) and starch. Maltose, matotriose, and maltotetraose were the major products from starch hydrolysis but prolonged reaction led to the production of glucose, maltose, and maltotriose from starch, cyclodextrins, and maltooligosaccharides (G3-G7). The amylase showed remarkable glucose tolerance up to 1 M, but was more sensitive to inhibition by maltose. The deduced protein primary structure from the putative gene revealed that the enzyme shared moderate homology between alpha-amylases from Aspergilli and Lipomyces sp. This thermotolerant, glucose tolerant maltooligosaccharide-forming alpha-amylase is potent for biotechnological application.     

Beta-galactosidase monitoring by a biosensor based on Clark electrode: its optimization, characterization and application

beta-Galactosidase is an hydrolase enzyme that catalyzes the hydrolysis of beta-galactosides into monosaccharides. Substrates of different beta-galactosidases include ganglioside GM1, lactosylceramides, lactose, and various glycoproteins. A novel aspect of the activity determination of beta-galactosidase was presented. A glucose oxidase biosensor based on Clark electrode was utilized in order to monitor beta-galactosidase. Immobilization of glucose oxidase was made by gelatin and glutaraldehyde as cross-linker. Several parameters such as glucose oxidase activity, gelatin amount, and glutaraldehyde percentage for cross-linking were optimized. The most important parameter, lactose concentration in working buffer was studied in detail. Optimum temperature, thermal stability, optimum pH, buffer system and its concentration effect on the biosensor system, repeatability, reproducibility, and storage and operational stabilities of the biosensor were identified. A linear detection range for beta-galactosidase was observed between 9.4 x 10(-5) and 3.2 x 10(-2)U/ml. Finally, beta-galactosidase activity in artificial intestinal juice was investigated by the biosensor and the results obtained were compared with a reference spectrophotometric method.     

Tuning the redox and enzymatic activity of glucose oxidase in layered organic films and its application in glucose biosensors

Glucose oxidase was embedded in organic films through a layer-by-layer approach, where the enzyme demonstrated significantly enhanced electron-transfer reactivity and finely tuned enzymatic activity. An unmediated, reagentless glucose biosensor was accordingly prepared with two polyethylenimine/glucose oxidase bilayers-modified pyrolytic graphite electrode. A calibration linear range of glucose was 0.5-8.9 mM with a detection limit of 50 microM and sensitivity of 0.76 microA mM(-1).     

Statistical optimization of a high maltose-forming,hyperthermostable and Ca2+-independent alpha-amylase production by an extreme thermophile Geobacillus thermoleovorans using response surface methodology

AIM: Statistical optimization for maximum production of a hyperthermostable, Ca2+-independent and high maltose-forming alpha-amylase by Geobacillus thermoleovorans. METHODS AND RESULTS: G. thermoleovorans was cultivated in 250 ml flasks containing 50 ml of chemically defined glucose-arginine medium (g l(-1): glucose 20; arginine 1.2; riboflavin 150 microg ml(-1); MgSO4. 7H2O 0.2; NaCl 1.0; pH 7.0). The medium was inoculated with 5 h-old bacterial inoculum (1.8x10(8) CFU ml(-1)), and incubated in an incubator shaker at 70 degrees C for 12 h at 200 rev min(-1). The fermentation variables optimized by 'one variable at a time' approach were further optimized by response surface methodology (RSM). The statistical model was obtained using central composite design (CCD) with three variables: glucose, riboflavin and inoculum density. An over all 24 and 70% increase in enzyme production was attained in shake flasks and fermenter because of optimization by RSM, respectively. A good coverage of interactions could also be explained by RSM. The end products of the action of alpha-amylase on starch were maltose (62%), maltotriose (31%) and malto-oligosaccharides (7%). CONCLUSIONS: RSM allowed optimization of medium components and cultural parameters for attaining high yields of alpha-amylase, and further, a good coverage of interactions could be explained. The yield of maltose was higher than maltotriose and malto-oligosaccharides in the starch hydrolysate. SIGNIFICANCE AND IMPACT OF THE STUDY: By applying RSM, critical fermentation variables were optimized rapidly. The starch hydrolysate contained a high proportion of maltose, and therefore, the enzyme can find application in starch saccharification process for the manufacture of high maltose syrups. The use of this enzyme in starch saccharification eliminates the addition of Ca2+.     

Enhancement of operational stability of an enzyme biosensor for glucose and sucrose using protein based stabilizing agents

With the incorporation of lysozyme during the immobilization step, considerable enhancement of the operational stability of a biosensor has been demonstrated in the case of an immobilized single enzyme (glucose oxidase) system for glucose and multienzyme (invertase, mutarotase and glucose oxidase) system for sucrose. Thus an increased number of repeated analyses of 750 samples during 230 days for glucose and 400 samples during 40 days of operation for sucrose have been achieved. The increased operational stability of immobilized single and multienzyme system, will improve the operating cost effectiveness of the biosensor.     

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

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

Electrochemical biosensing utilizing synergic action of carbon nanotubes and platinum nanowires prepared by template synthesis

Platinum nanowires (PtNWs) prepared by electrodeposition method with the help of porous anodic aluminum oxide (AAO) templates have been solubilized in chitosan (CHIT) together with carbon nantubes (CNTs) to form a PtNW-CNT-CHIT organic-inorganic system. The resulting PtNW-CNT-CHIT material brings capabilities for utilizing synergic action of PtNWs and CNTs to facilitate electron-transfer process in electrochemical sensor design. The PtNW-CNT-CHIT film modified electrode offered a significant decrease in the overvoltage for the hydrogen peroxide and showed to be excellent amperometric sensors for hydrogen peroxide at -0.1 V over a wide range of concentrations, and the sensitivity is 260 microAmM-1cm-2. As an application example, by linking glucose oxidase (GOx), an amplified biosensor toward glucose was prepared. The glucose biosensor exhibits a selective determination of glucose at -0.1 V with a linear response range of 5 x 10(-6) to 1.5 x 10(-2)M with a correlation coefficient of 0.997, and response time <10s. The high sensitivity of the glucose biosensor is up to 30 microAmM-1cm-2 and the detection limit was 3 microM. The biosensor displays rapid response and expanded linear response range, and excellent repeatability and stability.     

Supramolecular architecture based on the self-assembling of multiwall carbon nanotubes dispersed in polyhistidine and glucose oxidase: Characterization and analytical applications for glucose biosensing

We report for the first time the development of a sensitive and selective glucose biosensor based on the self-assembling of multiwall carbon nanotubes (MWCNTs) dispersed in polyhistidine (Polyhis) and glucose oxidase (GOx) on glassy carbon electrodes (GCE). The supramolecular architecture was characterized by SEM, FT-IR and electrochemical techniques. The optimum multistructure was obtained with five (MWCNT-Polyhis/GOx) bilayers and one layer of Nafion as anti-interferent barrier. The sensitivity at 0.700V was (1.94±0.03) mAM(-1) (r=0.9991), with a linear range between 0.25 and 5.00mM, a detection limit of 2.2μM and a quantification limit of 6.7μM with minimum interference from lactose (1.5%), maltose (5.7%), galactose (1.2%), ascorbic acid (1.0%), and uric acid (3.3%). The biocatalytic layer demonstrated to be highly reproducible since the R.S.D. for 10 successive amperometric calibrations using the same surface was 3.6%. The sensitivity of the biosensor after 15 day storage at 4°C remained at 90% of its original value. The combination of the excellent dispersing properties and polycationic nature of polyhistidine, the stability of the MWCNT-Polyhis dispersion, the electrocatalytic properties of MWCNTs, the biocatalytic specificity of GOx, and the permselective properties of Nafion have allowed building up a sensitive, selective, robust, reproducible and stable glucose amperometric biosensor for the quantification of glucose in milk samples.     

Fluorescence imaging of the activity of glucose oxidase using a hydrogen-peroxide-sensitive europium probe

A method for optical imaging of the activity of glucose oxidase (GOx) using a fluorescent europium(III) tetracycline probe for hydrogen peroxide is presented. A decay time in the microsecond range and the large Stokes shift of 210 nm of the probe facilitate intensity-based, time-resolved, and decay-time-based imaging of glucose oxidase. Four methods for imaging the activity of GOx were compared, and rapid lifetime determination imaging was found to be the best in giving a linear range from 0.32 to 2.7 m Unit/mL. The detection limit is 0.32 m Unit/mL (1.7 ng mL(-1)) which is similar to that of the time-resolved (gated) imaging using a microtiterplate reader. Fluorescent imaging of the activity of GOx is considered to be a useful tool for GOx-based immunoassays with potential for high-throughput screening, immobilization studies, and biosensor array technologies.     

Chemiluminescence flow biosensor for glucose using Mg-Al carbonate layered double hydroxides as catalysts and buffer solutions

In this work, serving as supports in immobilizing luminol reagent, catalysts of luminol chemiluminescence (CL), and buffer solutions for the CL reaction, Mg-Al-CO(3) layered double hydroxides (LDHs) were found to trigger luminol CL in weak acid solutions (pH 5.8). The silica sol-gel with glucose oxidase and horseradish peroxidase was immobilized in the first half of the inside surface of a clear quartz tube, and luminol-hybrid Mg-Al-CO(3) LDHs were packed in the second half. Therefore, a novel CL flow-through biosensor for glucose was constructed in weak acid solutions. The CL intensity was linear with glucose concentration in the range of 0.005-1.0mM, and the detection limit for glucose (S/N=3) was 0.1μM. The proposed biosensor exhibited excellent stability, high reproducibility and high selectivity for the determination of glucose and has been successfully applied to determine glucose in human plasma samples with satisfactory results. The success of this work has broken the bottleneck of the pH incompatibility between luminol CL and enzyme activity.     

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

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

Detection and substrate selectivity of new microbial D-amino acid oxidases

In order to screen for new microbial D-amino acid oxidase activities a selective and sensitive peroxidase/o-dianisidine assay, detecting the formation of hydrogen peroxide was developed. Catalase, which coexists with oxidases in the peroxisomes or the microsomes and, which competes with peroxidase for hydrogen peroxide, was completely inhibited by o-dianisidine up to a catalase activity of 500 nkat ml(-)(1). Thus, using the peroxidase/o-dianisidine assay and employing crude extracts of microorganisms in a microplate reader, a detection sensitivity for oxidase activity of 0.6 nkat ml(-)(1) was obtained.Wild type colonies which were grown on a selective medium containing D-alanine as carbon, energy and nitrogen source were examined for D-amino acid oxidase activity by the peroxidase/o-dianisidine assay. The oxidase positive colonies possessing an apparent oxidase activity > 2 nkat g dry biomass(-)(1) were isolated. Among them three new D-amino acid oxidase-producers were found and identified as Fusarium oxysporum, Verticilium lutealbum and Candida parapsilosis. The best new D-amino oxidase producer was the fungus F. oxysporum with a D-amino acid oxidase activity of about 900 nkat g dry biomass(-)(1) or 21 nkat mg protein(-)(1). With regard to the use as a biocatalytic tool in biotechnology the substrate specificities of the three new D-amino acid oxidases were compared with those of the known D-amino acid oxidases from Trigonopsis variabilis, Rhodotorula gracilis and pig kidney under the same conditions. All six D-amino acid oxidases accepted the D-enantiomers of alanine, valine, leucine, proline, phenylalanine, serine and glutamine as substrates and, except for the D-amino acid oxidase from V. luteoalbum, D-tryptophane, D-tyrosine, D-arginine and D-histidine were accepted as well. The relative highest activities (>95%) were measured versus D-alanine (C. parapsilosis, F. oxysporum, T. variabilis), D-methionine (V. luteoalbum, R. gracilis), D-valine (T. variabilis, R. gracilis) and D-proline (pig kidney). The D-amino oxidases from F. oxysporum and V. luteoalbum were able to react with the industrially important substrate cephalosporin C although the D-amino acid oxidase from T. variabilis was at least about 20-fold more active with this substrate.As the results of our studies, a reliable oxidase assay was developed, allowing high throughput screening in a microplate reader. Furthermore, three new microbial D-amino acid oxidase-producers with interesting broad substrate specificities were introduced in the field of biotechnology.