Oxygen-independent glucose microsensor based on glucose oxidase

A glucose oxidase-based needle-type microsensor is described that is independent of the local oxygen concentration. The sensor consists of an enzyme-coated platinum microelectrode that is inserted in a glass capillary with a tip of 5–25 μm, and connected to the environment via an agar membrane. Oxygen is supplied to the enzyme coating from the shaft of the capillary. Depending on the electrode configuration, the sensor has a response time of 1–10 s, and the measuring range extends from 0.01 to 0.4 mmol l^?1 or from 0.4 to 10 mmol l^?1. The signal is not affected by stirring.     

Biosensors for determination of glucose with glucose oxidase immobilized on an eggshell membrane

A glucose biosensor using an enzyme-immobilized eggshell membrane and oxygen electrode for glucose determination has been fabricated. Glucose oxidase was covalently immobilized on an eggshell membrane with glutaraldehyde as a cross-linking agent. The glucose biosensor was fabricated by positioning the enzyme-immobilized eggshell membrane on the surface of a dissolved oxygen sensor. The detection scheme was based on the depletion of dissolved oxygen content upon exposure to glucose solution and the decrease in the oxygen level was monitored and related to the glucose concentration. The effect of glutaraldehyde concentration, pH, phosphate buffer concentration and temperature on the response of the glucose biosensor has been studied in detail. Common matrix interferents such as ethanol, d-fructose, citric acid, sodium benzoate, sucrose and l-ascorbic acid did not give significant interference. The resulting sensor exhibited a fast response (100 s), high sensitivity (8.3409 mg L⁻¹ oxygen depletion/mmol L⁻¹ glucose) and good storage stability (85.2% of its initial sensitivity after 4 months). The linear response is 1.0×10⁻⁵ to 1.3×10⁻³ mol L⁻¹ glucose. The glucose content in real samples such as commercial glucose injection preparations and wines was determined, and the results were comparable to the values obtained from a commercial glucose assay kit based on a spectrophotometric method.     

Study on glucose oxidase fermentation coupled with membrane dialysis

The repression of glucose oxidase (GOD) biosynthesis by catabolites during fermentation can be alleviated through membrane dialysis fermentation (MDF). The results show that the volumetric enzyme productivity of MDF was two times higher than that of the control (fermentation without dialysis), and its total enzyme activity was increased by 30–50%. The operation conditions of MDF, such as pore size of the membrane, initiating time for membrane dialysis, and volume of dialysate used, were optimized. The content of amino acids and organic acids in the fermentation broth and the dialysate in the reservoir were assessed by amino acid analyzer and ionic chromatography, respectively. The relationship among the contents of pyruvic acid, gluconic acid, and enzyme activity during fermentation was analyzed quantitatively. Furthermore, the effect of membrane dialysis technology applied to the low-yield strain was found to be more effective than that applied to the high-yield strain.     

Microfabricated glucose biosensor with glucose oxidase entrapped in sol-gel matrix

A microfabricated glucose biosensor based on an amperometeric hydrogen peroxide electrode has been developed. A sol-gel layer with 5 A pore size and 2 mum thickness was used as the glucose oxidase entrapping matrix. The sol-gel matrix formed over the silicon-based sensor has good mechanical and chemical stability, and the ability to entrap a large amount of enzyme. The miniaturized electrode sensing system is composed of platinum as both working and counter electrodes and silver as a reference electrode. Nafion(R) coating was applied as the interference limiting layer. A series of technologies, such as standard photolithography, electron beam evaporation and image reverse lift-off were utilized for mass production allowing 143 electrodes to be produced at the same time. The effect of oxidable interferences was <10% of the background value of the sensor response. Calibration tests of a series of individual sensors manufactured from the same silicon wafer and dip coated in the same conditions, showed a highly reproducible response characteristics (linear range up to 500 mg dl(-1) and mean sensitivity of 0.54+/-0.14 nA mg(-1) dl(-1) (n=10)).     

Comparison of amperometric measuring principles for determinations of glucose with electrodes based on glucose oxidase

The three measuring principles for glucose determination (H₂O₂ formation, O₂ consumption, or enzymatically reduced acceptor) were studied with an apparatus that was the same for all measurements, with virtually identical electrode design. The methods were compared with regard to measurement range, sensitivity, time behaviour, operational lifetime, precision and accuracy. Essential differences were found with respect to measurement range as well as to time. Measurements under anaerobic conditions have advantages over those done aerobically.     

Amperometric determination of bonded glucose with an MnO(2) and glucose oxidase bulk-modified screen-printed electrode using flow-injection analysis

A screen-printed amperometric biosensor based on carbon ink double bulk-modified with MnO₂ as a mediator and glucose oxidase as a biocomponent was investigated for its ability to serve as a detector for bonded glucose in different compounds, such as cellobiose, saccharose, (-)-4-nitrophenyl-β-d-glucopyranoside, as well as in beer samples by flow-injection analysis (FIA). The biosensor could be operated under physiological conditions (0.1 M phosphate buffer, pH 7.5) and exhibited good reproducibility and stability. Bonded glucose was released with glucosidase in solution, and the free glucose was detected with the modified screen-printed electrode (SPE). The release of glucose by the aid of glucosidase from cellobiose, saccharose and (-)-4-nitrophenyl-β-d-glucopyranoside in solution showed that stoichiometric quantities of free glucose could be monitored in all three cases.The linear range of the amperometric response of the biosensor in the FIA-mode flow rate 0.2 mL min⁻¹, injection volume 0.25 mL, operation potential 0.48 V versus Ag/AgCl) extends from 11 to 13,900 μmol L⁻¹ glucose in free form. The limit of detection (3σ) is 1 μmol L⁻¹ glucose. A concentration of 100 μmol L⁻¹ yields a relative standard deviation of approximately 7% with five injections. These values correspond to the same concentrations of bonded glucose supposed that it is liberated quantitatively (incubation for 2 h with glucosidase).Bonded glucose could be determined in beer samples using the same assay. The results corresponded very well with the reference procedure.     

Direct electrochemistry of glucose oxidase and a biosensor for glucose based on a glass carbon electrode modified with MoS2 nanosheets decorated with gold nanoparticles

An electrochemical glucose biosensor was developed by immobilizing glucose oxidase (GOx) on a glass carbon electrode that was modified with molybdenum disulfide (MoS₂) nanosheets that were decorated with gold nanoparticles (AuNPs). The electrochemical performance of the modified electrode was investigated by cyclic voltammetry, and it is found that use of the AuNPs-decorated MoS₂ nanocomposite accelerates the electron transfer from electrode to the immobilized enzyme. This enables the direct electrochemistry of GOx without any electron mediator. The synergistic effect the MoS₂ nanosheets and the AuNPs result in excellent electrocatalytic activity. Glucose can be detected in the concentration range from 10 to 300 μM, and down to levels as low as 2.8 μM. The biosensor also displays good reproducibility and long-term stability, suggesting that it represents a promising tool for biological assays. Figure

A MoS₂-based glucose sensor has been prepared by gold nanoparticles-decorated MoS₂ nanocomposite, which exhibited excellent electrocatalytic activity, reproducibility and long-term stability. It was applied to determine glucose concentration in human serum, suggest the sensor maybe promising for practical application.     

Novel glucose biosensor based on a glassy carbon electrode modified with hollow gold nanoparticles and glucose oxidase

A novel glucose biosensor is presented as that based on a glassy carbon electrode modified with hollow gold nanoparticles (HGNs) and glucose oxidase. The sensor exhibits a better differential pulse voltammetric response towards glucose than the one based on conventional gold nanoparticles of the same size. This is attributed to the good biological conductivity and biocompatibility of HGNs. Under the optimal conditions, the sensor displays a linear range from 2.0?×?10^?6 to 4.6?×?10^?5?M of glucose, with a detection limit of 1.6?×?10^?6?M (S/N?=?3). Good reproducibility, stability and no interference make this biosensor applicable to the determination of glucose in samples such as sports drinks.

Interaction of glucose oxidase with alkyl-substituted sepharose 4B

Glucose oxidase (GOD) is often used in immobilized forms for determination of glucose. To examine the possibility of its adsorption by hydrophobic interactions, palmityl-substituted Sepharose 4B (Sepharoselipid) was employed as an adsorptive matrix. Various conditions were used in tests to improve the limited immobilization of the enzyme observed under normal (native) conditions, including use of high concentrations of denaturing agents. Of the denaturants used, only the cationic detergent dodecyl trimethyl ammonium bromide was effective in denaturing the protein and exposing its hydrophobic sites for interaction with alkyl residues on the support. This, followed by the process of renaturation, provided catalytically active immobilized preparations. The apoenzyme, prepared by treatment of the holoenzyme with acidified (NH4)2SO4 or thermal denaturation, was totally immobilized on the support. Furthermore, it was shown that either flavin adenine dinucleotide (FAD) or the alkyl residues, not both, may interact with the nucleotide site at any given time. Results are discussed in terms of high rigidity of GOD molecule and limited exposure of hydrophobic sites in its native structure. The observations are in accord with suggestions in the literature that the FAD pocket is a very narrow channel of hydrophobic properties, adapted to accept its natural coenzyme.     

Amperometric determination of glucose with a ferrocene-mediated glucose oxidase/polyacrylamide gel electrode

Enzyme electrodes were constructed by immobilization of glucose oxidase and ferrocene into cross-linked polyacrylamide gels. Electrogenerated ferrocinium ion acts as a direct electron mediator between glucose oxidase and a reticulated vitreous carbon (RVC)/graphite support bed. The electrode is easily constructed, gives a current response proportional to glucose concentrations up to 30 mM, and has good chemical stability in water and air.     

Biocomposites of covalently linked glucose oxidase on carbon nanotubes for glucose biosensor

The formation of covalently linked composites of multi–walled carbon nanotubes (MWCNT) and glucose oxidase (GOD) with high-function density for use as a biosensing interface is described. The reaction intermediates and the final product were characterized by using FT–IR spectroscopy, and the MWCNT-coated GOD nanocomposites were examined by atomic force microscopy (AFM) and transmission electron microscopy (TEM). Interestingly, it was found that the GOD–MWCNT composites are highly water soluble. Electrochemical characterization of the GOD–MWCNT composites that were modified on a glassy carbon electrode shows that the covalently linked GOD retains its bioactivity and can specifically catalyze the oxidation of glucose. The oxidation current shows a linear dependence on the glucose concentration in the solution in the range of 0.5–40 mM with a detection limit of 30 μM and a detection sensitivity of 11.3 μA/mMcm². The present method may provide a way to synthesize MWCNT related composites with other biomolecules and for the construction of enzymatic reaction-based biofuel cells and biosensors. Supported by grants from the National Natural Science Foundation of China (NSFC, No. 20125515; 90206037; 20375016) and the Natural Science Foundation of Jiangsu Province (Grant No. BK 2004210)     

Frequency response of the glucose sensor based on immobilized glucose oxidase membrane

Frequency response of the glucose sensor based on the immobilized glucose oxidase membrane was investigated experimentally by giving the sinusoidal change of glucose concentration to the glucose sensor and observing its output signal. Observed values of gains and phase lags of the frequency response of the glucose sensor followed the frequency response model of the first-order with dead time; The time constant and also the dead time were estimated and found to decrease as the amount of enzyme immobilized in the membrane increased and the thickness of the membrane decreased.

     

Thermal inactivation and conformational lock studies on glucose oxidase

In this study, the dissociative thermal inactivation and conformational lock theories are applied for the homodimeric enzyme glucose oxidase (GOD) in order to analyze its structure. For this purpose, the rate of activity reduction of glucose oxidase is studied at various temperatures using β-d-glucose as the substrate by incubation of enzyme at various temperatures in the wide range between 40 and 70 °C using UV–Vis spectrophotometry. It was observed that in the two ranges of temperatures, the enzyme has two different forms. In relatively low temperatures, the enzyme is in its dimeric state and has normal activity. In high temperatures, the activity almost disappears and it aggregates. The above achievements are confirmed by dynamic light scattering. The experimental parameter “n” as the obvious number of conformational locks at the dimer interface of glucose oxidase is obtained by kinetic data, and the value is near to two. To confirm the above results, the X-ray crystallography structure of the enzyme, GOD (pdb, 1gal), was also studied. The secondary and tertiary structures of the enzyme to track the thermal inactivation were studied by circular dichroism and fluorescence spectroscopy, respectively. We proposed a mechanism model for thermal inactivation of GOD based on the absence of the monomeric form of the enzyme by circular dichroism and fluorescence spectroscopy.     

Laccase/glucose oxidase electrode for determination of glucose

The electrode involves a layer of co-immobilized glucose oxidase and laccase in a gelatin membrane placed over a modified oxygen electrode. Hexacyanoferrate(III) is added to the samples to oxidize reductive interferents such as ascorbic acid, and the hexacyanoferrate(II) formed is re-oxidized by a laccase-catalyzed reaction. Ascorbic acid is completely eliminated up to a concentration of 20 mM in the sample.     

Development of antimicrobial packaging materials with immobilized glucose oxidase and lysozyme

Packaging based on immobilization of antimicrobial enzymes provides a promising form of active packaging systems applicable in food processing. Glucose oxidase and lysozyme were immobilized by the Ugi reaction with cyclohexyl isocyanide and glutaraldehyde on polyamide and ionomer films partially hydrolysed by hydrochloric acid. The immobilization of the enzymes on the surface of films was confirmed by FT-IR spectroscopy and the films were characterized by the specific activity of the immobilized enzymes. The enzyme migration into model solutions and the effect of pH, temperature and storage time on the activity of immobilized enzyme were also evaluated. Immobilization of lysozyme onto polyamide and ionomer films resulted in the loss of enzyme activity. The polyamide and ionomer films with immobilized glucose oxidase inhibited the growth of bacteria Escherichia coli CNCTC 6859, Pseudomonas fluorescens CNCTC 5793, Lactobacillus helveticus CH-1, Listeria ivanovii CCM 5884 and Listeria innocua CCM 4030 on agar media.      

Derivatization of glucose and 2-deoxyglucose for transition metal complexation: substitution reactions with organometallic 99mTc and Re precursors and fundamental NMR investigations

Synthetic strategies for the bifunctionalization of glucose and 2-deoxyglucose at position C-1 for transition metal coordination are reported. In particular organometallic technetium and rhenium complexes for potential use in diagnostic nuclear medicine were synthesized and investigated. Specifically, a common iminodiacetic acid (IDA) moiety was O-glycosidically connected through an ethylene spacer group to produce the pure alpha- (in case of 2-deoxyglucose) and beta-anomer (in case of glucose). Reaction of the sugar derivatives with the organometallic precursor [M(H2O)3(CO)3]+ (M = 99mTc, Re) produced single products in high yield, which are water-soluble and water-stable. The displacement of the three water molecules of the metal precursor and thus the tridentate coordination of the metal-tricarbonyl core exclusively via the amine and the two carboxylic acid functionalities of the IDA chelate was verified by means of 1D and 2D 1H NMR spectroscopy, mass spectrometry, and IR spectroscopy. The radioactive-labeled products (99mTc) proved their excellent stability in vitro in physiological phosphate buffer (pH = 7.4) and human plasma over a period of 24 h at 37 degrees C.     

Wiring of glucose oxidase with graphene nanoribbons: an electrochemical third generation glucose biosensor

A reagentless third generation electrochemical glucose biosensor was fabricated based on wiring the template enzyme glucose oxidase (GOx) with graphene nanoribbons (GN) in order to create direct electron transfer between the co-factor (flavin adenine dinucleotide, FAD) and the electrode. The strategy involved: (i) isolation of the apo-enzyme by separating it from its co-enzyme; (ii) preparation of graphene nanoribbons (GN) by oxidative unzipping of multi-walled carbon nanotubes; (iii) adsorptive immobilization of GNs on the surface of a screen printed carbon electrode (SPCE); (iv) covalent attachment of FAD to the nanoribbons; (v) recombination of the apo-enzyme with the covalently bound FAD to the holoenzyme; and (vi) stabilization of the bio-layer with a thin membrane of Nafion. The biosensor (referred to as GN/FAD/apo-GOx/Nafion/SPCE) is operated at a potential of +0.475 V vs Ag/AgCl/{3 M KCl} in flow-injection mode with an oxygen-free phosphate buffer (pH 7.5) acting as a carrier. The signals are linearly proportional to the concentration of glucose in the range from 50 to 2000 mg?L^?1 with a detection limit of 20 mg?L^?1. The repeatability (10 measurements, at 1000 mg?L^?1 glucose) is ±1.4% and the reproducibility (5 sensors, 1000 mg?L^?1 glucose) is ±1.8%. The biosensor was applied to the determination of glucose in human serum.

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Graphical abstract Wiring of the apo-enzyme of glucose oxidase (apo-GOx) with graphene nanoribbons (GN) bound to FAD at a screen-printed carbon electrode (SPCE). Cyclic voltammetric and amperometric responses to various glucose concentrations.

     

Tin disulfide nanoflakes decorated with gold nanoparticles for direct electrochemistry of glucose oxidase and glucose biosensing

Previously, we have prepared nanoflake-like tin disulfide (SnS2) and used for the immobilization of proteins and biosensing. We have now modified an electrode with a composite consisting of nanoflake-like SnS2 decorated with gold nanoparticles (Au-NPs) and have immobilized glucose oxidase (GOx) on its surface in order to study its direct electrochemistry. Scanning electron microscopy, electrochemical impedance spectroscopy, Fourier transform IR spectroscopy and cyclic voltammetry were used to examine the interaction between GOx and the AuNP-SnS2 film. It is shown that the composite film has a larger surface area and offers a microenvironment that facilitates the direct electron transfer between enzyme and electrode surface. The immobilized enzyme retains its bioactivity and undergoes a surface-controlled, reversible 2-proton and 2-electron transfer reaction, with an apparent electron transfer rate constant of 3.87 s ^-1. Compared to the nanoflake-like SnS2-based glucose sensor, the GOx-based biosensor exhibits a lower detection limit (1.0 :M), a better sensitivity (21.8 mA?M ^-1 ?cm ^-2), and a wider linear range (from 0.02 to 1.3 mM). The sensor displays excellent selectivity, good reproducibility, and acceptable stability. It was successfully applied to reagentless sensing of glucose at ?0.43 V.