Enzyme-Based Fiber Optic Sensor for Glucose Determination

Abstract

A new fiber-optic probe for the determination of glucose is developed. Glucose oxidase is immobilized on a preactivated Immunodyne membrane by direct application of the enzymatic solution to one side of the membrane. The membrane is then placed around the common end of a bifurcated glass fiber optic bundle, and immersed in the sample cell which contains the glucose sample, peroxidase and a colorless dye. Glucose is quantified by the color change when the reaction takes place.     

Flow Injection Analysis of Glucose by Fiber Optic Chemiluminescence Measurement

Abstract

A flow injection system was developed for the determination of glucose based on fiber optic chemiluminescence measurements. The hydrogen peroxide produced from the glucose oxidase catalyzed reaction was quantified by measuring the intensity of luminol chemiluminescence in the presence of excess ferricyanide. Glucose oxidase was immobilized on aminopropyl glass using glutaraldehyde and packed in a reactor. The chemiluminescence was transported to the detector using an optical fiber. The system responds linearly to glucose in the concentration range 0.20 mM up to 2.5 mM, with a relative precision of 5%. Several fruit juices were analyzed for their glucose content and the results were compared with a standard AOAC procedure.     

Determination of Urinary Oxalate With Disposable Oxalate Oxidase Hembrane Strips

Abstract

We describe in this paper a simple and easy method of entrapping oxalate oxidase and peroxidase in acrylamide membrane and demonstrate the use of such enzyme membrane strips in the rapid determination of urinary oxalate. A crude preparation of 45-60% acetone cut obtained from banana fruit peel (Musa paradisiaca; French plantain) homogenate serves as a source of oxalate oxidase, which decomposes oxalate into CO₂ and H₂O₂. the oxalate content of a given urine sample is determined by introducing a small enzyme membrane strip (1 × 1 cm) into an aliquot of buffered urine containing a suitable chromogen for peroxidase and then measuring the colour developed due to the interaction of peroxidase with the newly formed H₂O₂ and the chromogen. Urine samples are pretreated with sodium nitrite to eliminate the interference of ascorbic acid in the assay. the enzyme membrane assay compares well with that of wet enzyme assay of oxalate in sensitivity and reliability.     

Quantification of Glucose Using Extended Colorimetry

Abstract

The use of extended colorimetry to increase the range over which colorimetric measurments can be made without loss of sensitivity is described. Its potential for use with dehydrogenase and oxidase enzymes is demonstrated. Construction of a device for the quantification of glucose based on this technique is described. The results of assays on undiluted solutions containing glucose in the range 1 - 20mM were assesed using reflectance spectrophotometry.     

Pulse Voltammetric Biosensing System for the Rapid Determination of Glucose With Micro-Enzyme Sensor

Abstract

An electrochemically prepared micro-enzyme electrode whose diameter is 50 jim is combined with an Pt auxiliary electrode and a reference electrode to assemble a three electrode device for the rapid determination of glucose. Since the device is very small, glucose sample whose volume is only 2 μ1 can be successfully determined. Pulse voltammetry is shown to be an effective approach for making the sensing device work without any attachments such as magnetic stirrer and pump. The transient sensor output, oxidizing current for the hydrogen peroxide generated by the immobilized glucose oxidase, shows a good linearity in the glucose concentration range from 1 mM to 20 mM.     

Glucose Biosensor Based on Oxygen Electrode. Part II: Long-Term Operational Stability of the Rechargeable Glucose Biosensor

Abstract

A potentially implantable glucose biosensor for measuring blood or tissue glucose levels in diabetic patients has been developed. The glucose biosensor is based on an amperometric oxygen electrode and immobilized glucose oxidase enzyme, in which the immobilized enzyme can be replaced (the sensor recharged) without surgical removal of the sensor from the patient. Recharging of the sensor is achieved by injecting fresh immobilized enzyme into the sensor using a septum. A special technique for immobilization of the enzyme on Ultra-Low Temperature Isotropic (ULTI) carbon powder held in a liquid suspension has been developed.

In vitro studies of the sensors show stable performance during several recharge cycles over a period of 3 months of continuous operation.

Diffusion membranes which ensure linear dependence of the sensor response on glucose concentration have been developed. These membranes comprise silastic latex-rubber coatings over a microporous polycarbonate membrane. Calibration curves of the amperometric signal show linearity over a wide range of glucose concentrations (up to 16 mM), covering hypoglycemic, normoglycemic and hyperglycemic conditions.

The experimental results confirm the suitability of the sensors for in vitro measurements in undiluted human sera.     

Amperometric Determination of Picomolar Levels of Flavin Adenine Dinucleotide by Cyclic Oxidation-Reduction in Apo-Glucose Oxidase System

Abstract

Acidification of a solution of glucose oxidase in 35 percent glycerol with 10 percent sulfuric acid and a gel filtration of this acidified glucose oxidase solution yielded apo-glucose oxidase with low residual enzyme activity. Further treatment with charcoal gave apo-glucose oxidase which is devoid of enzyme activity. Using this apo-glucose oxidase, it was possible to measure flavin adenine dinucleotide amperometrically at extremely low concentrations (10_?12 M) with ease, rapidity, and convenience.     

Enzymatic Determination of Serum Adenosine-5-Triphosphate by Amperometric Measurement of the Rate of Oxygen Depletion

Abstract

ATP is determined by measuring the competition for glucose between a hexokinase-catalyzed dephospnorylation of ATP and glucose oxidase catalyzed oxidation of glucose. The ATP competition for glucose decreases the rate of oxygen consumption via the oxidation reaction, which is measured amperometrically with a Clark oxygen electrode. The decrease in the oxygen depletion is proportional to the concentration of ATP in the sample. A 10 to 50 μl sample is required and analyses are completed in 3 minutes or less.     

Ferrocene and Ferricenium Ion as Versatile Photometric Titrants of H2O2 and D-Glucose in the Presence of Peroxidase and Glucose Oxidase. A Ferrocene-Peroxidase Stairway #

Abstract

Hydrogen peroxide can routinely be determined in the presence of ferrocene (FcH) and horseradish peroxidase by monitoring at 617 nm the enzymatically produced ferricenium dye. In contrast, D-glucose can be assayed by following the fading of the ferricenium dye FcH⁺PF₆ ^? in the presence of glucose oxidase. The change in absorbance in both cases corresponds to the amount of analyte. viz. H₂O₂ or D-glucose, in solution. The routine is very simple, invariant to the concentrations of both ferrocenes/ferricenium salt and enzyme and allows numerous “one pot” measuremeats with the detection limit of 10^?4 M for both the analytes. It takes 2–4 and 5–10 min to accomplish one analysis of H₂O₂ and D-glucose in the presence of peroxidase and glucose oxidase, respectively.

     

Amperometric Multienzyme Sensor for Determination of D-Glucono-δ-Lactone

Abstract

An amperometric enzyme sensor for the determination of gluconolactone in glucose-containing samples has been developed. The interfering glucose is eliminated by an outer anti-interference layer containing hexokinase, whilst the gluconolactone reaches a glucose de-hydrogenase-glucose oxidase layer, where it is converted into glucose (by glucose dehydrogenase) and then transformed by glucose oxidase, the associated oxygen consumption can be measured at the electrode. Gluconolactone is determined over the concentration range, 0.02–1 mmo1/1, with a toleration of glucose concentration up to 2 mmo1/1.     

A Microdialysis Probe Coupled with A Miniaturized Thermal Glucose Sensor for In Vivo Monitoring

Abstract

A miniaturized thermal flow injection analysis biosensor has been coupled with a microdialysis probe for continuous subcutaneous glucose monitoring. Thermal biosensors are based on the principle of measuring the heat evolved during enzyme catalysed reactions. The system presented here consists of a miniaturized thermal biosensor with a small column containing coimmibolized glucose oxidase and catalase. The analysis buffer passes through the column at a flow rate of 60μL/min via an 1μL sample loop which is connected to a microdialysis probe.

Invitro results showed constant permeability of the probe and stability of the biosensor response during 24 hours. The response time was 85 sec giving a sample rate of 42 samples/hour.

During a load experiment, the glucose profile in a healthy volunteer was followed both in the subcutaneous tissue and blood using the microdialysis set-up proposed and comparing to blood glucose analyser.     

Biothermal Analysis Performed In Organic Solvents

Abstract

A thermal assay probe (enzyme thermistor) was used to study the performance of enzymic analyses by immobilized enzymes in organic solvents and in mixtures of organic solvents and aqueous buffer. It was found that rather small amounts of alcohols (around 5 %, v/v) in the buffer approximately doubled the registered temperature changes for glucose oxidase and catalase, mainly by increasing the total enthalpy change of the reaction. the heat production for peroxidase catalyzed reactions was as much as 45 times higher in toluene compared to buffer. the advantage of increased substrate solubility in organic solvents was demonstrated by operating a lipase column in cyclohexane. All enzymes studied showed good stability in the organic solvents used.     

Construction of a N-Acetyl-L-methionine Electrode and Determination of Acylase with an Ammonia Gas Sensor

Abstract

The N-acetyl-L-methionine electrode is based on a coupled enzymatic system consisting of acylase and L-amino acid oxidase with an ammonia gas sensor; conditions of imobilization are optimized. N-acetyl-L-methionine in the range 4×10^?5–2×10^?3M gives a linear potential vs. log(concentration) plot with a response time of 2–5 min over the range specified. This electrode combined with an L-methionine electrode, based only on L-amino acid oxidase and an ammonia gas sensor, can be used for the determination of both substrates in mixtures, thus extending the feasibility of the method. Acylase (0.1–2.00) is determined in aqueous solutions by adding N-acetyl-L-methionine to the sample, and measuring the ammonia evolved with the gas-sensing electrode.     

Detection of Laccase,Peroxidase, and Polyphenol Oxidase on a Single Polyacrylamide Gel Electrophoresis

Abstract

Laccase, polyphenol oxidase and peroxidase are widely distributed in both plants and micro-organisms. Among these enzymes, the identification of one particular activity in the presence of the others is often difficult as enzymes can often oxidise the same substrates. A method is described, that is suitable to differentiate the three activities on the same polyacrylamide gel electrophoresis. The method is based on the use of two substrates, 4-aminoN,N-diethylaniline and 4-tert-butyl-catechol, so that coloured spots appear corresponding to the different activities. Moreover, a comparison among different mushroom species belonging to the same genus is reported using the described method.     

Detection of Oxidase Generated Hydrogenperoxide by a Solid State Peroxyoxalate Chemiluminescence Detector

Abstract

The compatability of a solid state peroxyoxalate chemiluminescence detector for hydrogen peroxide with an immobilized oxidase reactor is investigated. As a model system glucose oxidase immobilized by electrostatic forces on an ion-exchanger or chemically bonded to glass beads were chosen. The former support is less suitable for immobilization of oxydases due to strong retention of hydrogenperoxide on the ion exchanger.

The relatively little flow dependence of these systems renders them suitable for low-cost manual sample injection monitors as well as in a flow injection analyses (FIA) mode with low-cost pumping systems. The system was operated with 80% acetonitrile water solutions. A detection limit of 8 × 10^?7M of glucose was achieved in directly injected samples.

Enzymes more sensitive to organic solvents can be operated with pure water and adjustment for optimal chemiluminescence condition is achieved with a make-up flow prior to detection. A detection limit of 5 × 10^?8M glucose is achieved under these conditions. The feasability of this approach to other oxidase based monitors and to detection in liquid chromatography is discussed.     

Enzyme-Induced Chemiluminescence-Determination of Blood Glucose Using Luminol

Abstract

Hydrogen peroxide, produced by the interaction of glucose with immobilized glucose oxidase, reacts with the ferricyanide-luminol system to produce chemiluminescence linearly proportional to glucose concentration. The coupled reaction is used as a sensitive precise micro method for the determination of true blood glucose. The linear range of detection is 10^?7 - 10^?4 M glucose. The method correlates quantitatively with two glucose reference techniques.     

Highly sensitive glucose sensor based on work function changes measured by an EMOSFET

In this paper, glucose is potentiometrically measured by using a specific field effect transistor, the EMOSFET. In this device, glucose oxidase is immobilized within a bovine serum albumin matrix, using glutaraldehyde. This layer is deposited on the top of an electroactive Os-polyvinylpyridine layer containing horseradish peroxidase, which is used as the gate material of the FET. The basic principle of the sensor is to measure the glucose concentration by means of measuring the change in the work function of the electroactive gate due to its redox reaction with the H2O2, generated by the reaction between glucose and glucose oxidase. The change in the work function can be detected as a change in the threshold voltage of the FET. Moreover, a measuring mode called "constant current potentiometry" has been applied to improve the sensitivity of the sensor. The sensitivity of the sensor working in this mode is found to be much higher than the Nernstian value. The experimental results show that the detection limit of the sensor can be tuned depending on the value of the applied current and the glucose oxidase concentration in the gate.     

Amperometric Determination of Galactose,Lactose and Dihydroxyacetone Using Galactose Oxidase in a Flow Injection System with Immobilized Enzyme Reactors and On-Line Dialysis

Abstract

A flow injection manifold containing a dialyzer and reactors with immobilized galactose oxidase and peroxidase was used for the determination of galactose in urine, lactose in milk and dihydroxyacetone in a biotechnological reaction medium. The hydrogen peroxide which is formed by the galactose oxidase reaction was detected by amperometric reduction of a mediator. The latter had been produced from hydrogen peroxide in a peroxidase catalyzed reaction. The hydrogen peroxide detection step was studied with several mediators and hexacyanoferrate (II) was selected. An ion exchange HPLC procedure was used to purify the galactose oxidase, in particular from catalase, and the kinetics and the selectivity of a reactor containing the immobilized enzyme was investigated. Columns for removal of certain interferents such as ascorbic acid were used in the determination of galactose in urine. The response to galactose standards was linear from the detection limit of 2 μM to 60 mM. The throughput was 45 samples per hour and the relative standard deviation 0.4%.     

Development of a Novel Silver Nanoparticles-Enhanced Screen-Printed Amperometric Glucose Biosensor

Abstract

A disposable glucose biosensor is developed by immobilizing glucose oxidase into silver nanoparticles-doped silica sol-gel and polyvinyl alcohol hybrid film on a Prussian blue-modified screen-printed electrode. The silver nanoparticles-enhanced biosensor shows a linear amperometric response to glucose from 1.25 × 10^?5 to 2.56 × 10^?3 with a sensitivity of 20.09 mA M^?1 cm^?2, which is almost double that of the biosensors without silver nanoparticles. The immobilized glucose oxidase retained 91% of its original activity after 30 days of storage in phosphate buffer (pH 6.9; 0.1 M) at 4°C. Blood glucose in a rabbit serum sample was successfully measured with the biosensor.     

Phenol oxidases production and wood degradation by a thermophilic fungusThermoascus aurantiacus

The ability of a Brazilian strain ofThermoascus aurantiacus, a thermophilic fungus, to produce extracellular phenol oxidases and to degradeEucalyptus grandis sawdust was studied.T. aurantiacus was capable of good growth in liquid culture containing 1.5% (w/v) of various lignocellulosic substrates (sugar cane bagasse, rice hulls, and chips and sawdust ofE. grandis) plus 5 mg/mL of glucose. When lignocellulosic substrates were used, enzymes involved in cellulose and hemicellulose metabolism were stimulated inT. aurantiacus. It was also found that these substrates have an inductive effect on phenol oxidase production. The most effective inducer of phenol oxidase activity wasE. grandis sawdust, which led to the production of 0.80 U/mL (o-dianisidine oxidation) on day 12. Low phenol oxidase activity was observed at cultures when only glucose was used. Cultures ofT. aurantiacus also exhibited cellobiose-quinone oxidoreductase activity when lignocellulosic materials were used as substrate. However, under our experimental conditions, lignin peroxidase activity was not detected.E. grandis sawdust supplemented with 5 mg/mL of glucose suffered a total weight loss of 6.7% accompanied by 15% lignin loss and 64.4% extractive loss after 21 d incubation withT. aurantiacus.