Determination of glucose in blood by flow injection analysis and an immobilized glucose oxidase column

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.     

Flow injection analysis of trace hydrogen peroxide using an immobilized enzyme reactor

Summary Flow Injection Analysis of Trace Hydrogen Peroxide Using an Immobilized Enzyme Reactor Sub-part-per-billion levels of aqueous hydrogen peroxide have been determined with a flow injection analysis system employing a single bead string reactor composed of horseradish peroxidase covalently bound to an animated macroporous polymeric adsorbent with glutaraldehyde and a passive cation exchange membrane reactor to alter pH. The chemistry relies on the peroxidase mediated oxidation of nonfluorescentp-hydroxyphenylacetate to its fluorescent dimer. The advantage of the system includes rapid throughout rates (40 samples/h), excellent detection limits (0.3 ppb H₂O₂) and large dynamic range of linear response (1 ppb-1 ppm). However, the immobilized enzyme is not useful for the analysis of organic peroxides which act as inhibitors.     

Flow-injection analysis for l-glutamate using immobilized l-glutamate oxidase: comparison of an enzyme reactor and enzyme electrode

An enzyme electrode and enzyme based on immobilized l-glutamate oxidase are used for the determination of l-glutamate in a flow-injection system. The hydrogen peroxide produced is monitored amperometrically. The enzyme reactor system surpasses the enzyme electrode system with regard to sensitivity and analytical speed. For both systems, the peak current is linearly related to the l-glutamate concentration in the range 5 × 10^?6-1 × 10^?3 M. l-Glutamate in seasoning can be determined very selectively with < 0.7% r.s.d.     

Flow enthalpimetric determination of glucose, based on oxidation by 1,4-benzoquinone and use of an immobilized glucose oxidase column

A flow enthalpimetric method for the determination of glucose is presented. The method is based on the reaction of glucose with 1,4-benzoquinone in the presence of immobilized glucose oxidase. d-Glucose concentrations ranging from 0.02 to 75mM can be determined. The method is applicable to the determination of glucose in soft drinks, wines, beers, jams and serum.     

Analytical use of immobilized glucose oxidase

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.     

Characterization of immobilized glucose oxidase—catalase and their deactivation in a fluid-bed reactor

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.     

Multicommutated flow system for the determination of glucose in honey with immobilized glucose oxidase reactor and spectrophotometric detection

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 (s_r(%), n = 10) was 3% and the sampling frequency of the system was 20 samples h⁻¹.     

A phosphate-selective electrode based on immobilized alkaline phosphatase and glucose oxidase.

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.     

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.     

Quantitative analysis of catalysis and inhibition at horseradish peroxidase monolayers immobilized on an electrode surface

Out of several tries, biotinylation of the electrode surface by means of a sacrificial biotinylated immunoglobulin, followed by the anchoring of an avidin-enzyme conjugate appears as the best procedure for depositing a horseradish peroxidase (HRP) monolayer onto an electrode surface, allowing a high-yield immobilization of the enzyme within a stable and highly catalytic coating. Cyclic voltammetry is an efficient means for analyzing the catalytic reduction of H(2)O(2) at such HRP monolayer electrodes in the presence of [Os(III)(bpy)(2)pyCl](2+) (with bpy = bipyridine and py = pyridine) as a one-electron reversible cosubstrate. The odd shapes of current-potential responses, unusual bell-shaped variation of the peak or plateau current with the substrate concentration, hysteresis and trace crossing phenomena, and dependence or lack of dependence with the scan rate, can all be explained and quantitatively analyzed in the framework of the same catalysis/inhibition mechanism as previously demonstrated for homogeneous systems, taking substrate and cosubstrate mass transport of into account. According to H(2)O(2) concentration, limiting-behavior analyses based on the dominant factors or complete numerical simulation were used in the treatment of experimental data. The kinetic characteristics derived from these quantitative treatments implemented by the determination of the amount of enzyme deposited by the newly developed droplet depletion method allowed a comparison with homogeneous characteristics to be drawn. It shows that HRP remains nearly fully active once anchored on the electrode surface through the avidin-biotin linkage. On the basis of this full mechanistic and kinetic characterization, the analytical performances in H(2)O(2) detection and amperometric immunosensor applications are finally discussed.     

Determination of cholesterol by flow injection analysis with immobilized cholesterol oxidase

The injected sample passes through a column of enzyme immobilized on controlled pore glass, at pH 7.0, and the hydrogen peroxide produced is detected amperometrically. As little as 0.2 μg of cholesterol can be detected. The method is applied to blood serum, wax-wool alcohol and an extract of butter.     

Enzyme sequence and competition electrodes based on immobilized glucose oxidase,peroxidase and catalase

The toxicologically important peroxidase substrates bilirubin and aminopyrine can be determined by combination of immobilized glucose oxidase, horseradish peroxidase and catalase, forming so-called enzyme sequence and enzyme competition electrodes. Bilirubin and aminopyrine are determined in the concentration range 5–50 μM.     

Covalent attachment of glucose oxidase to an Au electrode modified with gold nanoparticles for use as glucose biosensor

A feasible method to fabricate glucose biosensor was developed by covalent attachment of glucose oxidase (GOx) to a gold nanoparticle monolayer modified Au electrode. Cyclic voltammetry (CV) and electrochemical impedance spectroscopy (EIS) of ferrocyanide followed and confirmed the assemble process of biosensor, and indicated that the gold nanoparticles in the biosensing interface efficiently improved the electron transfer between analyte and electrode surface. CV performed in the presence of excess glucose and artificial redox mediator, ferrocenemethanol, allowed to quantify the surface concentration of electrically wired enzyme (Gamma(E)(0)) on the basis of kinetic models reported in literature. The Gamma(E)(0) on proposed electrode was high to 4.1 x 10(-12) mol.cm(-2), which was more than four times of that on electrode direct immobilization of enzyme by cystamine without intermediate layer of gold nanoparticles and 2.4 times of a saturated monolayer of GOx on electrode surface. The analytical performance of this biosensor was investigated by amperometry. The sensor provided a linear response to glucose over the concentration range of 2.0 x 10(-5)-5.7 x 10(-3) M with a sensitivity of 8.8 microA.mM(-1).cm(-2) and a detection limit of 8.2 microM. The apparent Michaelis-Menten constant (K(m)(app)) for the sensor was found to be 4.3 mM. In addition, the sensor has good reproducibility, and can remain stable over 30 days.     

Flow injection analysis for L-lactate with immobilized lactate dehydrogenase

Immobilized lactate dehydrogenase (LDH) is used for determination of L-lactate in a continuous flow system. The LDH is immobilized by reaction with glutaraldehyde onto the surface of alkylamino-bonded silica gel and packed into a column in the flow system. The reduction of NAD⁺ occurs simultaneously, and the NADH formed is detected amperometrically. The peak current is linearly related to the L-lactate concentration in the range 1–80 × 10^-6 M; 30 samples h^-1 can be analyzed with a r.s.d. of 0.5–1.5%. The immobilized LDH retains over 90% of its initial activity after repetitive use for 3 months.     

Flow injection analysis of potassium using an all-solid-state potassium-selective electrode as a detector

The concentration of potassium was determined by a combination of flow injection analysis (FIA) with an all-solid-state potassium sensor detection. The all-solid-state potassium-selective electrode possessing long-term potential stability was fabricated by coating an electroactive polypyrrole/poly(4-styrenesulfonate) film electrode with a plasticized poly(vinyl chloride) membrane containing valinomycin. The simple FIA system developed in this laboratory demonstrated sensitivity identical to that in the batch system and achieved considerably rapid assay (150 samples h⁻¹). Analyses of soy sauce and control serum samples by this FIA system yielded results in good agreement with those obtained by conventional measurements.     

The use of immobilized alcohol oxidase in the continuous flow determination of ethanol with an oxygen electrode

Immobilized alcohol oxidase was used in the determination of blood alcohol. The alcohol oxidase catalyzed the aerobic oxidation of ethanol and the oxygen concentration was monitored with an oxygen membrane electrode in a flow cell. The enzyme was immobilized either by covalent attachment via glutaraldehyde to the inside walls of nylon tubing, or by adsorption onto three separate controlled-pore glass support materials: TiO₂, SiO₂, or AL₂O₃. The supports were packed into 10 cm lengths of 3 mm i.d. glass tubing or 30 cm lengths of 5 mm i.d. nylon tubing. The five methods of immobilization were compared for stability and activity toward ethanol. Immobilization on silanized glass beads results in the highest activity and greatest stability of the reactor.     

Flow injection determination of choline in milk hydrolysates by an immobilized enzyme reactor coupled to a selective hydrogen peroxide amperometric sensor

A choline oxidase (ChO) immobilized enzyme reactor (IMER) prepared by glutaraldehyde coupling of the enzyme on aminopropyl modified controlled pore glass beads is described. The ChO-IMER was coupled, in a flow injection configuration system, to an interference free hydrogen peroxide amperometric sensor based on a Pt surface modified by an overoxidized polypyrrole film. The resulting analytical device responds selectively to choline and displays a sensitivity of 46.9 ± 0.2 μC mM⁻¹ and a limit of detection, calculated at a signal-to-noise ratio equal to 3, of 7 μM. Sensitivity remains constant for about 20 days and then starts to slowly deteriorate and after 2 months a 70% of the initial sensitivity was still retained. The application to choline determination in milk hydrolysates is demonstrated. Short- and long-term drift observed in the analytical response can be corrected by a bracketing technique.     

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.