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1.
2.
With the aim of immobilizing glucose oxidase (GO) for routine determination of glucose, a covalent bond immobilization method on titanium (IV) chloride activated silica supports was used (1). Several parameters were studied in order to optimize the residual activity upon immobilization and during operation. The immobilized enzyme can be reutilized at 25°C for several h a day alternating with storage (4°C) for at least 3,300 h.  相似文献   

3.
Glucose oxidase (GOD) was immobilized by using glutaraldehyde crosslinking and various stabilizing agents such as BSA, gelatin, lysozyme, and polyethylenimine (PEI). Studies on the denaturation of the soluble as well as immobilized GOD were carried out for 1 h at various concentrations of guanidine hydrochloride (GdmCl) in 50 mM phosphate buffer, pH 6.0 at 25±1°C. The soluble enzyme required a GdmCl concentration of 5M for total activity loss, whereas for GOD immobilized with BSA, gelatin, lysozyme, and heat-inactivated lysozyme, the corresponding GdmCl concentration required was 8 M. GOD immobilized with PEI, however, was more stable and retained 25% activity when denatured for 1 h using 8 M GdmCl. However, after undergoing denaturation for 1 h, GOD immobilized with lysozyme regained 72% original activity within 20 min of renaturation, while GOD immobilized with BSA, PEI, gelatin, and heat-inactivated lysozyme regained only 39, 21, 20, and 25% of activity, respectively. After five cycles of repeated denaturation and renaturation with 8 M GdmCl, GOD immobilized with lysozyme retained 70% of the original activity. Refolding ability of lysozyme, glutaraldehyde crosslinkages between lysozyme and GOD, together with ionic interactions between them, appear to play an important role in the denaturation-renaturation behavior of the immobilized enzyme.  相似文献   

4.
Wu B  Zhang G  Shuang S  Choi MM 《Talanta》2004,64(2):546-553
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.  相似文献   

5.

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.

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6.
Doretti L  Ferrara D  Gattolin P  Lora S 《Talanta》1997,44(5):859-866
A new method of physically immobilizing a biomolecule of analytical interest in poly(vinyl alcohol) cryogels was developed to obtain suitable biosensors. An amperometric glucose sensor was constructed using glucose oxidase immobilized on membranes obtained by a freezing-thawing cyclic process. No chemical cross-linking agent was used. Sensor behaviour was evaluated electrochemically with a hydrogen peroxide electrode. The glucose content in standard solutions was determined and linear calibration curves in the 5 x 10(-5)-3 x 10(-3) mol 1(-1) range were obtained. Temperature and pH effects on the electrochemical response were described and kinetic parameters in the immobilized system were evaluated.  相似文献   

7.
A method for immobilizing proteins in a carbon mesoporous material (CMM) containing platinum nanoparticles (Pt-NPs) is demonstrated. Compared to pure CMM or carbon nanotubes, CMM containing Pt-NPs enhances the electron transfer and redox properties of redox enzymes, such as glucose oxidase (GOx), due to a cooperative effect of Pt-NPs and CMM. The quasi-reversible electron transfer of GOx in this system is probed, and the apparent heterogeneous electron transfer rate constants are found to be 66% larger than in pure CMM. The GOx/Pt-CMM based glucose biosensor enables the determination of glucose at a potential of 600 mV (vs. SCE). Its detection limit is 10 times lower, and the sensitivity is 16 times higher than that of the respective biosensor without Pt-NPs.  相似文献   

8.
Alexandra Sixto 《Talanta》2009,77(4):1534-1538
A new automated method for the determination of glucose in honey is proposed. The method is based on multicommutated flow analysis (MCFA) and employs an immobilized glucose oxidase reactor and spectrophotometric detection at 505 nm of the red quinoneimine formed (Trinder's method).The calibration curve obeyed a second order equation in the range 0-0.14 g L−1 (h = −2.2199 C2 + 1.3741C + 0.0077, r2 = 0.9991, where h is the peak height (absorbance) and C the concentration in g L−1). The method was validated analyzing eight commercial samples, both by the AOAC 954.11 and 977.20 official methods. According to Student's t-test of mean values, at the confidence level of 95% the results obtained with the proposed method were in agreement with those obtained by the official methods. Precision (sr(%), n = 10) was 3% and the sampling frequency of the system was 20 samples h−1.  相似文献   

9.
Absract  Rheological properties of 3% carrageenan gels formed in 0.4 M sodium chloride solution in the presence of lysozyme are studied in detail. It is shown that the addition of protein results in an increase in the gel-sol transition temperature by 2°C (transition temperature of 3% gel is 48°C, lysozyme concentration is 0.5 mg/ml). Based on the frequency dependences of dynamic moduli at various temperatures, it is revealed that systems possess viscoelastic properties at low frequencies. Within a wide frequency range up to gel-sol transition temperature, systems become elastoviscous and, at higher frequencies, they demonstrate forced transition to glassy state. It is shown that carrageenan inhibits enzyme activity of lysozyme. The interaction between enzyme and carrageenan leads to changes in lysozyme conformations, i.e., the content of α-helices increases and that of turns decreases. It is demonstrated for the first time that, in the presence of a so-called nonspecific (for the gelation of carrageenan) sodium ion, it is possible to prepare gels with the necessary structure and rheological properties and gel-sol transition temperature. These gels can release a lysozyme under the conditions of transmucosal prolonged delivery. Original Russian Text ? G.P. Yampol’skaya, A.A. Elenskii, N.V. Pan’kina, B.N. Tarasevich, V.G. Kulichikhin, 2009, published in Kolloidnyi Zhurnal, 2009, Vol. 71, No. 2, pp. 275–284.  相似文献   

10.
A flow-injection system for glucose determination is described. Glucose oxidase is immobilized on controlled porosity glass (CPG) and used in a glass column (2.5 mm diameter × 2.5 cm). The hydrogen peroxide produced by the enzymatic reaction (? 1 × 10?6 M) is detected by the current produced in a flow-through cell, with two platinum electrodes having a potential difference of 0.6 V. Glucose (0–20 mmol l?1) can be determined in blood plasma either with a dialyser in the system or, better, by incorporating a column of copper(II) diethyldithiocarbamate on CPG before the enzyme column. The results compared well with those obtained by a conventional analyser system. The glucose oxidase column showed little change in activity over a 10-month period.  相似文献   

11.
Kiba N  Ishida Y  Tsuchiya M  Furusawa M 《Talanta》1983,30(3):187-189
A thermal flow system for glucose determination is described, that utilizes a column of glucose oxidase immobilized on a cation-exchange resin (Amberlite CG50). The response is linear for glucose concentrations in the range 0.01-0.4mM. Stability and the factors influencing the response have been examined.  相似文献   

12.
A simple, one-step process, using 0.25Mp-benzoquinone dissolved in 20% dioxane at 50°C for 24 h was applied to the activation of polyacrylamide beads. The activated beads were reacted with glucose oxidase isolated fromAspergillus niger. The coupling reaction was performed in 0.1M potassium phosphate at pH 8.5 and 0–4°C for 24 h. The protein concentration was 50 mg/mL. In such conditions, the highest activity achieved was about 100 U/g solid. The optimum pH for the catalytic activity was shifted by about 1 pH unit in the acidic direction to pH 5.5. Between 35 and 50°C, the activity of the immobilized form depends on the temperature to a smaller extent than that of the soluble form. Above 50°C, the activity of immobilized glucose oxidase shows a sharper heat dependence. The enzyme-substrate interaction was not profoundly altered by the immobilization of the enzyme. The heat resistance of the immobilized enzyme was enhanced. The immobilized glucose oxidase is most stable at pH 5.5. The practical use of the immobilized glucose oxidase was tested in preliminary experiments for determination of the glucose concentration in blood sera.  相似文献   

13.
A magnetic mesoporous carbon material (i.e., mesoporous iron oxide/C, mesoFe/C) is synthesized for protein immobilization, using glucose oxidase (GOx) as model. Transmission electron microscopy images show that mesoFe/C has highly ordered porous structure with uniform pore size, and iron oxide nanoparticles are dispersed along the wall of carbon. After adsorption of GOx, the GOx-mesoFe/C composite is separated with magnet. The immobilized GOx remains its natural structure according to the reflection–absorption infrared spectra. When the GOx-mesoFe/C composite is coated on a Pt electrode surface, the GOx gives a couple of quasireversible voltammetric peaks at −0.5 V (vs. saturated calomel electrode) due to the redox of FAD/FADH2. The electron-transfer rate constant (k s) is ca. 0.49 s−1. The modified electrode presents remarkably amperometric response to glucose at 0.6 V. The response time (t 95%) is less than 6 s; the response current is linear to glucose concentration in the range of 0.2–10 mM with a sensitivity of 27 μA mM−1 cm−2. The detection limit is 0.08 mM (S/N = 3). The apparent Michaelis–Menten constant (K mapp) of the enzyme reaction is ca. 6.6 mM, indicating that the GOx immobilized with mesoFe/C has high affinity to the substrate.  相似文献   

14.
《Mendeleev Communications》2023,33(4):559-561
Electrochemical responses of glucose oxidase loaded (via electrostatic immobilization) into a surface-attached pH- and temperature-sensitive copolymer microgel were examined. The observed temperature behavior of the immobilized enzyme provides evidence that such systems enable pH-dependent regulation of activity of glucose oxidase by a (repeated) temperature cycling, which reversibly transforms the polymeric (microgel) matrix from the swollen state to the collapsed one.  相似文献   

15.
Glucose oxidase was immobilized on a Millipore (MP) filter by coating with plasma-polymerized propargyl alcohol. The resulting immobilized enzyme membrane was used as a glucose sensor. The properties as a glucose electrode system were evaluated by amperometric response with either the steady-state method or the reaction rate method. The response was proportional to concentrations of the glucose solution up to 2 mM and the sensitivity was dependent on the amount of GOD impregnated into the MP filter.  相似文献   

16.
An enzyme electrode which senses oxygen consumption for the assay of phosphate ion (10-3-10-4M), was constructed by using two enzymes together:
The competitive inhibition by phosphate ion added caused a smaller and slower oxygen consumption which could be detected by a platinum disc electrode at -0.6 V vs. SCE amperometrically. This dual enzyme electrode was also found useful for the assay of oxyacids other than phosphate, such as arsenate, tungstate, molybdate and borate.  相似文献   

17.
An optical glucose biosensor using a swim bladder membrane as an enzyme immobilization platform and an oxygen-sensitive membrane as an optical oxygen transducer has been developed. During the enzymatic reaction, glucose is oxidized by glucose oxidase with a concomitant consumption of dissolved oxygen resulting in an increase in the fluorescence intensity of the optical oxygen transducer. The fluorescence intensity is directly related to the glucose concentration. The effects of pH, temperature, buffer concentration, and selectivity have been studied in detail. The immobilized enzyme retained 80% of its initial activity after being kept for more than 10 months at 4°C. The glucose biosensor has been successfully applied to the determination of glucose content in human blood serum and urine samples. Martin M.F. Choi was on sabbatical leave at The University of North Carolina at Chapel Hill from July 2004 to July 2005.  相似文献   

18.
Glucose sensor based on glucose oxidase immobilized by zirconium phosphate.   总被引:1,自引:0,他引:1  
Amperometric glucose sensors were fabricated using glucose oxidase (GOx) entrapped in zirconium hydrogenphosphate (ZrP), and their performance was evaluated. Reportedly, alpha-ZrP is one of the candidates that are expected to improve the stability of enzymes immobilized on solid surfaces. We intercalated GOxs into ZrP (GOx/ZrP), cast the GOx/ZrP suspension in polyvinylalcohol on a platinum electrode, and dried it in a vacuum oven. The morphological layered structure was investigated by scanning electron microscopy. The enzymatic activities, which were determined by open-circuit potentiometric technique, reached the highest when GOxs were immobilized in ZrP at ca. pH 5. In vitro tests showed good linear responses in the 0-25 mM range and the sensitivity of 0.14 nA mM(-1) at 0.4 V vs. Ag/AgCl. The sensors, as made, were stable for more than 3 days within a limited deterioration.  相似文献   

19.
The authors describe enzyme based nanobiosensors for continuous monitoring of glucose, with the long term goal of using them as smart diagnostic tattoos. The method is founded on two main features: (1) The fluorescence intensity and decay times of glucose oxidase (GOx) and of GOx labeled with fluorescein (FS) or a ruthenium chelate (Ru) reversibly change during interaction with glucose; (2) The (labeled) enzyme is linked to magnetite magnetic nanoparticles (MNPs) which permits the MNPs to be physically manipulated. It is found that a stable link between MNPs and GOx is only accomplished if the number of amino groups on the GOX is artificially enlarged (to form GOxsam). Fluorescence decay data are best acquired with 8-nm MNPs where scattering is marginal; The activity of GOx is found not to be affected by immobilization on the MNPs. The various immobilized enzymes (GOxsam, GOxsam-FS and GOxsam-Ru; all on MNPs) differ only slightly in terms of linear response to glucose which ranged from 0.5 mM to at least 3.5 mM. The RSDs are about 5% (for n = 5), the detection limits are at ~50 μM, and the sensor lifetimes are >1 week.
Graphical abstract Nanobiosensors consisting of Fe3O4 magnetic nanoparticles linked to glucose oxidase, previously enriched with amino groups (GOxsam) and containing fluorescein (FS) or a ruthenium derivative (Ru), are presented as a new kind of smart tattoos for glucose monitoring.
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20.
2-Amino-4-chloro-s-triazine, a derivative of DEAE-cellulose, and acrolein/styrene copolymer were used as supports for the immobilization of glucose oxidase and catalase after being modified with diaminohexane followed by glutaraldehyde. Immobilization was carried out with optimum glucose oxidase-catalase ratios. The activity variations of the immobilized dual-enzyme systems were investigated in relation to pH and temperature. Time-dependent gluconic acid production resulting from the oxidation of glucose was monitored in a recycling fluid-bed reactor. The deactivation rates of glucose oxidase and catalase were investigated according to the first-order reaction kinetics depending on the presence of the intermediate product H2O2.  相似文献   

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