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%.     

In-line determination of metabolites and milk components with electrochemical biosensors

Electrochemical biosensors for lactate, pyruvate and β-hydroxybutyrate based on oxygen, hydrogen peroxide, and NADH sensors coupled with oxidase and dehydrogenase enzymes were developed and used in conjunction with an artificial pancreas in experiments with extracorporeal circulation. Such procedures allow the fate of these species involved in glucose metabolism to be clarified during insulin treatment of diabetic patients. Studies with a glucose oxidase electrode for in-line determination of glucose produced by hydrolysis of cellobiose in a bioreactor are reported; for the determination of glucose in the presence of high concentrations of cellobiose, the purity of glucose oxidase is important in obtaining linear calibration plots. Impurities like amylase, maltase, invertase, and galactose oxidase, which are usually present in commercial preparations of glucose oxidase, must be absent. Another application is the amperometric determination of lactose, lactate and glucose in milk samples by using a hydrogen peroxide sensor coupled with β-galactosidase, lactate oxidase and glucose oxidase. The procedures outlined are simple and the short response times enable milk to be monitored during processing.     

Fibre optic fluorometric enzyme sensors for hydrogen peroxide and lactate,based on horseradish peroxidase and lactate oxidase

An optical biosensor for the determination of hydrogen peroxide based on immobilized horseradish peroxidase is described. The fluorescence of the dimeric product of the enzyme catalysed oxidation of homovanillic acid is utilized to determine the concentration of H₂O₂. The membrane-bound enzyme is attached to a bifurcated fibre bundle permitting excitation and detection of the fluorescence by a fluorometer. The response of the sensor is linear from 1 to 130 M hydrogen peroxide; the coefficient of variation is 3%. The sensor is stable for more than 10 weeks. The operating pH for maximal sensor response is 8.15. This allows the sensor to be used in combination with oxidase reactions producing hydrogen peroxide, as is demonstrated with a co-immobilized lactate oxidase-horseradish peroxidase optode for the determination of L-lactate. The fluorescence intensity of this sensor depends linearly on the concentration of lactate between 3 and 200 M and a throughput of 10 samples per hour is possible. The precision is in the same range as that of the monoenzyme optode. The lifetime of the bienzyme sensor for lactate is considerably shorter than that of the peroxidase sensor; it is limited by the stability of the immobilized lactate oxidase enzyme. The sensor has been applied to the determination of lactate in control serum.     

Determination of Hydrogen Peroxide Concentrations By Flow Injection Analysis Based On the Enhanced Chemiluminescent Reaction Using Peroxidase

Abstract

The technique of flow injection analysis was employed in the determination of hydrogen peroxide. the method was based on the chemiluminescence reaction of luminol with H₂O₂ which is catalyzed by horseradish peroxidase and enhanced by p-iodophenol. Hydrogen peroxide was linearly detected in the range 10^?6M-10^?4M by measuring the maximum intensity of light emitted. the detection limit is about 1 · 10^?6M hydrogen peroxide. Transition metal cations at millimolar concentrations do not have any interference on the determination of hydrogen peroxide by FIA based on the enhanced chemiluminescent reaction. This technique is relatively rapid and simple, and permits measurement of up to 80 samples/hr using generally available equipment.     

Enzymatic Assay by Flow Injection Analysis with Detection by Chemiluminescence: Determination of Glucose,Creatinine, Free Cholesterol and Lactic Acid Using an Integrated Fia Microconduit

Abstract

A miniaturized flow injection system for the determination of D-glucose, L-lactic acid, creatinine and free cholesterol is described. All substrates are degraded enzymatically by means of oxidases which, along with ancillary coenzymes (creatinine assay), are immobilized on controlled porosity glass and incorporated into small PVC column reactors. The hydrogen peroxide generated by the individual oxidases is determined by chemiluminescence with an alkaline reagent containing luminol and hexacyanofer rate (III). The injection valve, flow channels, enzyme reactor and light detector are integrated into a FIA microconduit. The detection limits were 0.03 mg glucose/dl, 0.03 mg lactate/dl, 0.3 mM creatinine and 0.5 mg cholesterol/dl. The enzyme reactors all showed little change in activity over a 3 months period of operation and were found fully compatible with serum samples.     

Determination of Sulfur in Trace Amounts

Abstract

An absolute method for the determination of trace amounts of sulfur in water and organic matrices is described. The method involves decomposition of organic samples in an oxygen bomb or oxidation of water samples with hydrogen peroxide. The resultant sulfates are reduced and distilled as hydrogen sulfide by the action of stannous phosphate in anhydrous phosphoric acid. The evolved hydrogen sulfide is absorbed in zinc acetate solution, converted to methylene blue and measured spectrophotometrically at 665 mm. The range is from 2 to 40 μg of sulfur using 1 cm cells.     

MWCNT-Organoimido Polyoxomolybdate Hybrid Material: Analytical Applications for Amperometric Sensing of Hydrogen Peroxide

We report a hybrid material obtained by dispersing the polyoxometalate n-[Bu₄-N]₂[Mo₆O₁₈NC₁₃H₉] functionalized with 2-aminofluorene (POMAF) and multi-walled carbon nanotubes (MWCNTs) in 1,3-dioxolane (MWCNT- POMAF). We demonstrate the peroxidase-like activity of the hybrid material and its application for the amperometric quantification of hydrogen peroxide at −0.450 V previous drop-coating at glassy carbon electrodes, with a linear range between 1.0 μM and 6.0 μM and a detection limit of 330 nM. The reproducibility was 2.2 % using one MWCNTs-POMAF dispersion, and 3.9 % using 4 different dispersions. The sensor was successfully used for the quantification of hydrogen peroxide in enriched milk samples.     

Assay of Peracid in the Presence of Excess Hydrogen Peroxide

ABSTRACT

A simple and rapid colorimetric method for the determination of peracids in the presence of excess hydrogen peroxide is described. The method is based on the selective destruction of hydrogen peroxide by the enzyme catalase, a procedure which leaves the peracid unaffected. The residual solution, then containing only peracid, can be assayed using the stoichiometric oxidation of 2, 2′-azino-bis-(3-ethyl-benzthiazoline-6-sulfonate) diammonium salt (ABTS) in the presence of peroxidase, a reaction that can be quantified photometrically at 405 run. The double-enzyme method was extended to microtiter plate-based systems, thus providing an easy way of analyzing multiple samples for their peracid content.     

A Selective Determination of Hypoxanthine,Xanthine and Inosine by Reversed-Phase Liquid Chromatography Coupled with Enzyme Reactors

Abstract

A Selective and sensitive assay of hypoxyanthine, xanthine and inosine by reversed-phase liquid chromatography coupled with immobilized enzyme reactors is described. The flourometric detection of hydrogen peroxide using immobilized peroxidase and p-hydroxyphenylacetic acid was applied to the assay of hypoxanthine, xanthine and inosine, which were oxidized to hydrogen peroxide in the presence of the immobilized enzymes (purine nucleoside phosphorylase and/or xanthine oxidase. The enzymes were immobilized the the intermolecular cross-linking method on controlled pore-glass. The method established was applied to serum and urine samples. The detection limits of hypoxanthine, xanthine and inosine were approximately 130, 300 and 650 pg per injection, respectively.     

An Unmediated Hydrogen Peroxide Sensor Based on a Hemoglobin-sds Film Modified Electrode

ABSTRACT

An unmediated hydrogen peroxide sensor is designed in this paper by employing a hemoglobin-SDS film modified electrode. Hemoglobin exhibits direct (unmediated) electrochemistry at the modified electrode. The protein also shows elegant catalytic activity towards the electrochemical reduction of hydrogen peroxide. Consequently, a prototype hydrogen peroxide sensor is prepared. Under optimum conditions, this sensor provides a linear response over the hydrogen peroxide concentrations in the range of 1×10^-5～1×10^-4 mol/L. The detection limit was 2×10^-6 mol/L The relative standard deviation was 4.2% for 6 successive determinations of the hydrogen peroxide at 1×10^-5 mol/L. This configuration is shown to be sensitive, stable and easily fabricated. It might be useful in the biological and industrial fields.     

A Multicommuted Flow‐based System for Hydrogen Peroxide Determination by Chemiluminescence Detection Using a Photodiode

Abstract

A simple, rapid, and automated assay for hydrogen peroxide in pharmaceutical samples was developed by combining the multicommutation system with a chemiluminescence (CL) detector. The detection was performed using a spiral flow‐cell reactor made from polyethylene tubing that was positioned in front of a photodiode. It allows the rapid mixing of CL reagent and analyte and simultaneous detection of the emitted light. The chemiluminescence was based on the reaction of luminol with hydrogen peroxide catalyzed by hexacyanoferrate(III).

The feasibility of the flow system was ascertained by analyzing a set of pharmaceutical samples. A linear response within the range of 2.2–210 µmol l^?1 H₂O₂ with a LD of 1.8 µmol l^?1 H₂O₂ and coefficient of variations smaller than 0.8% for 1.0×10^?5 mol l^?1 and 6.8×10^?5 mol l^?1 hydrogen peroxide solutions (n=10) were obtained. Reagents consumption of 90 µg of luminol and 0.7 mg of hexacyanoferrate(III) per determination and sampling rate of 200 samples per hour were also achieved.     

Fluorimetric determination of hydrogen peroxide in water using acetaminophen

A fluorimetric procedure for the determination of hydrogen peroxide, based on the oxidation of acetaminophen with hydrogen peroxide in acidic medium, is described. The calibration graph was linear in the range 5.0 x 10(-8) - 2.4 x 10(-5) M hydrogen peroxide at an emission wavelength of 333 nm with excitation at 298 nm. The method has been applied to the determination of hydrogen peroxide in rain water, and the recoveries in milk samples were good.     

Determination of myo-inositol in a flow-injection system with co-immobilized enzyme reactors and amperometric detection

A selective and sensitive flow-injection system for the determination of myo-inositol (hexahydroxycyclohexane) is described. Inositol dehydrogenase, IDH, lactate dehydrogenase, LDH, and lactate oxidase, LOD, are co-immobilized on porous glass and used in a packed-bed enzyme reactor. myo-Inositol reacts to produce an equivalent amount of hydrogen peroxide, which oxidizes hexacyanoferrate(II) to hexacyanoferrate(III) in a second reactor containing immobilized peroxidase. The hexacyanoferrate(III) is then detected amperometrically at 0 mV vs. SCE in a flow-through detector. The system responds linearly to injected samples of myo-inositol (25 μl) in the concentration range 1–300 μM. The maximum throughput was 90 samples per hour. The IDH/LDH/LOD reactor was stable for at least 5 weeks.     

Heavy Atom Effects in Tellurapyrylium Dyes Useful in Photodynamic Therapy and Catalytic Generation of H2O2

Abstract

The large rate of intersystem crossing between singlet and triplet states of tellurapyrylium dyes leads to efficient generation of singlet oxygen in irradiated airsaturated aqueous solutions containing these dyes. One reaction of tellurapyrylium dyes with singlet oxygen and water is the formation of dihydroxy tellurane [tellurium(IV)] species. We have found that the photochemical generation of dihydroxy telluranes is reversible thermally. The tellurapyrylium dye is regenerated while a molecule of hydrogen peroxide is produced. The thermal generation of hydrogen peroxide coupled with a photochemical generation of singlet oxygen allows a catalytic cycle to be devised for the conversion of oxygen and water to hydrogen peroxide. The dihydroxy telluranes are efficient two-electron oxidizing agents and can be used as catalysts to accelerate reactions using hydrogen peroxide as a two-electron oxidizing agent. Examples of tellurapyrylium dye-mediated reactions of hydrogen peroxide include reactions of leucodyes normally oxidized by horseradish peroxidase and hydrogen peroxide. These processes lead to thermal and photochemical reactions that are potentially cytotoxic following the generation of singlet oxygen in photodynamic therapy. The regeneration of the original catalyst allows repeated treatment from a single dose.     

Direct determination of copper and zinc in cow milk, human milk and infant formula samples using electrothermal atomization atomic absorption spectrometry

Infant formula and powdered milk samples were suspended in a medium containing 20% ethanol, 0.65% nitric acid, 6% hydrogen peroxide and 0.1% ammonium dihydrogenphosphate and directly introduced in the electrothermal atomizer. Liquid milk samples were diluted with this suspension solution. The build-up of carbonaceous residues inside the atomizer was avoided by using a combination of hydrogen peroxide and nitric acid. Fast programs were used to simplify the heating cycles. Calibration was carried out using aqueous standards and the detection limits for copper and zinc were found to be 70 and 15 pg, respectively. The metal contents of several milk samples obtained by direct introduction of the samples agree with those obtained by means of a conventional procedure based on the total dissolution of the samples. The reliability of the methods was also confirmed by analyzing three certified reference materials. The free and protein binding fractions were measured by ultrafiltration membrane permeability of the milk samples.     

Molecularly Imprinted Electrochemical Luminescence Sensor Based on Enzymatic Amplification for Ultratrace Isoproturon Determination

A new molecularly imprinted electrochemical luminescence sensor (MIP‐ECL sensor) was developed for isoproturon (IPU) determination based on the competition reaction between IPU and glucose oxidase labeled IPU (GOD‐IPU). After competition, hydrogen peroxide produced by residual GOD‐IPU on the MIP reacted with luminol to emit electrochemiluminescence (ECL) signal. The ECL intensity decreased when the GOD‐IPU molecules were replaced by IPU molecules in the samples. IPU could be determined in the concentration range from 9×10^?11 mol/L to 5.1×10^?9 mol/L with a detection limit of 3.78×10^?12 mol/L. Water samples were assayed and recoveries ranging from 98.5 % to 102.1 % were obtained.     

Hydrogen Peroxide Sensor Based on Carbon Nanotubes/β‐Ni(OH)2 Nanocomposites

Ni(OH)₂ nanoflowers were synthesized by a simple and energy‐efficient wet chemistry method. The product was characterized by scanning electron microscopy (SEM) and X‐ray powder diffraction (XRD). Then Ni(OH)₂ nanoflowers attached multi‐walled carbon nanotubes (MWCNTs) modified glassy carbon electrodes (GCE) were proposed (MWCNTs/Ni(OH)₂/GCE) to use as electrochemical sensor to detect hydrogen peroxide. The results showed that the synergistic effect was obtained on the MWCNTs/Ni(OH)₂/GCE whose sensitivity was better than that of Ni(OH)₂/GCE. The linear range is from 0.2 to 22 mmol/L, the detection limit is 0.066 mmol/L, and the response time is <5 s. Satisfyingly, the MWCNTs/Ni(OH)₂/GCE was not only successfully employed to eliminate the interferences from uric acid (UA), acid ascorbic (AA), dopamine (DA), glucose (GO) but also NO₂^? during the detection. The MWCNTs/Ni(OH)₂/GCE allows highly sensitive, excellently selective and fast amperometric sensing of hydrogen peroxide and thus is promising for the future development of hydrogen peroxide sensors.     

Substoichiometric Isotope Dilution Analysis of Sulphur in coal

Abstract

Substoichiometric radioisotope dilution analysis has been applied to the determination of sulfur in coal. The experimental procedure involved the combustion of coal samples labelled with ³⁵S as sodium sulfate in a flowing oxýgen stream, collection of the combustion gases in hydrogen peroxide and substoíchiometric precipitation of barium sulfate. Samples of 0.2–10.0% sulfur were accurately determined with ±2% RSD by measuring the activity of the precipitates thus obtained and with comparison to standard samples carried through the same procedure. Analysis times of 15 minutes per sample were typical.     

Fluorometric Assay of Alcohols Using Alcohol Oxidase

Abstract

Fluorometric methods are described for the assay of ethanol, methanol, allyl alcohol and n-propanol using the enzyme alcohol oxidase. The enzyme catalyzes the oxidation of the alcohols to aldehydes with concomitant formation of hydrogen peroxide. The hydrogen peroxide formed is measured fluorometrically using p-hydroxy-phenylacetic acid and peroxidase. From 0.1–100 μg/ml of the alcohols and from 0.0002 to 0.050 unit/ml of alcohol oxidase can be assayed in 2 minutes with a precision of about ± 2%.     

On the Effect of Hydrogen Peroxide on the Flow‐Injection Determination of Platinum Based on Luminol Chemiluminescent Reaction

Abstract

A flow‐injection chemiluminescent (CL) method for the determination of trace amounts of Pt(IV) based on the oxidation reaction of luminol in alkaline solution is proposed. The effect of Pt(IV) on the oxidation of luminol was studied in the absence and in the presence of hydrogen peroxide. The positive effect of hydrogen peroxide as well as chloride ions on the sensitivity of measurements was observed. The developed method is characterized by a low limit of detection of Pt (LOD=0.06 ng mL^?1) and good reproducibility (RSD=2.2%). The addition of hydrogen peroxide to the reaction medium resulted in decreasing of platinum detection limit to 0.03 ng mL^?1.