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.     

Kinetic flow-injection determination of hydrogen peroxide by use of iron(III)-catalyzed coloration and its application to the determination of biological substances.

A kinetic flow-injection (FI) method is described for the determination of hydrogen peroxide. This method is based on an iron(III)-catalyzed oxidative coupling of 4-aminoantipyrine with N,N-dimethylaniline by hydrogen peroxide. By measuring the change in the absorbance of the dye formed at 560 nm, 1 x 10(-6) - 6 x 10(-4) M hydrogen peroxide could be determined with a sampling rate of 15 h(-1). The relative standard deviation (n = 30) was 0.8% for 5 x 10(-5) M hydrogen peroxide. There was little interference of the co-existing ions and compounds. After introducing some immobilized enzyme reactors to the FI system, the proposed method allowed the determination of glucose and uric acid ranging from 1 x 10(-6) to 6 x 10(-4) M with relative standard deviations of below 2%. The applicability of the method was demonstrated by determining these substances in serum samples.     

Amperometric sensor for hydrogen peroxide based on electric wire composed of horseradish peroxidase and toluidine blue-multiwalled carbon nanotubes nanocomposite

A kind of nanocomposites with good dispersion in water was prepared through noncovalent adsorption of toluidine blue (Tb) on multiwalled carbon nanotubes (MWCNT) for electric communication between horseradish peroxidase (HRP) and electrode. The nanocomposites could be conveniently cast on electrode surface. With the aid of chitosan, HRP was then immobilized on the nanostructure to form a reagentless amperometric sensor for hydrogen peroxide. UV-vis spectroscopy and electrochemical impedance spectroscopy were used to characterize the adsorption of Tb on MWCNT. The presence of both Tb as mediator of electron transfer and MWCNT as conductor enhanced greatly the enzymatic response to the reduction of hydrogen peroxide. The novel biosensor exhibited fast response towards hydrogen peroxide with a detection limit of 1.7x10(-6)M and the linear range extended up to 4x10(-4)M without the interference of ascorbic acid and uric acid. The Michaelis-Menten constant (K'(m)) of the immobilized HRP was evaluated to be 0.16mM.     

Chemiluminometric flow-injection method for determination of free l-malate in wine with co-immobilized malate dehydrogenase/NADH oxidase

A flow-injection system with a co-immobilized malate dehydrogenase/reduced nicotineamide adenine dinucleotide (NADH) oxidase reactor and a chemiluminometer is described for the determination of free l-malate in wine. Malate dehydrogenase and NADH oxidase were co-immobilized on poly(vinyl alcohol) beads and packed into a stainless-steel column (5 cm x 4 mm i.d.). The hydrogen peroxide produced was detected chemiluminometrically via a luminol-hexacyanoferrate(III) reaction. The calibration graph was linear from 3 x 10(-7) M to 2.5 x 10(-4) M (the linear correlation coefficient was 0.9998); the detection limit (signal-to-noise ratio, 3) was 8 x 10(-8) M. The sample throughput was 30 h(-1) without carryover. The ractor was renewed every 2 weeks.     

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.     

A novel biosensor for specific determination of hydrogen peroxide: catalase enzyme electrode based on dissolved oxygen probe

A biosensor for the specific determination of hydrogen peroxide was developed using catalase (EC 1.11.1.6) in combination with a dissolved oxygen probe. Catalase was immobilized with gelatin by means of glutaraldehyde and fixed on a pretreated teflon membrane served as enzyme electrode. The electrode response was maximum when 50 mM phosphate buffer was used at pH 7.0 and at 35 degrees C. The biosensor response depends linearly on hydrogen peroxide concentration between 1.0x10(-5) and 3.0x10(-3) M with a response time of 30 s. The sensor is stable for >3 months so in this period >400 assays can be performed.     

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%.     

Use of crude extract of lentil plant (Lens culinaris Medikus) in peroxidase-based analyses: fast kinetic determination of hydrogen peroxide and sarcosine in urine

Peroxidase-catalysed reactions are used in a wide variety of analytical applications, most of them based on the final quantification of hydrogen peroxide. Clinical tests for glucose, cholesterol, creatine, creatinine or uric acid in blood or urine and enzyme-linked immunosorbent assays for pesticides, hepatitis or acquired immune deficiency syndrome are good examples of such applications. The most widely used and commercially available peroxidase for biotechnological processes and analytical applications is horseradish peroxidase followed, although in much lower proportion, by soybean peroxidase. The high commercial interest in peroxidases has led to the search for new sources of these enzymes. This work describes the analytical use of lentil plant peroxidase (LPP), which is a new peroxidase extracted from lentil plants (Lens culinaris Medikus); an abundant post-harvest agricultural waste in the area of Castilla y León (Spain). A procedure for the quantification of hydrogen peroxide in urine is first proposed using crude extract of lentil plant instead of the purified enzyme. This procedure is then applied to the determination of sarcosine; a natural amino acid that has attracted considerable interest in clinical diagnostics since urinary sarcosine was proposed and later questioned as a biomarker for prostate cancer. Under the action of sarcosine oxidase, sarcosine is oxidized by molecular oxygen to give glycine, formaldehyde and hydrogen peroxide that is quantified according to the previously proposed procedure. The limit of detection for both hydrogen peroxide and sarcosine is around 5?×?10(-7)?M. In the determination of sarcosine, the high selectivity of the overall enzymatic reaction, the simple sample treatment and instrumentation, the high-sample throughput and the use of LPP in the plant extract instead of the purified enzyme provide a rapid and inexpensive procedure with characteristics very suitable for routine analysis in a clinical laboratory.     

Electrocatalytic Reduction of H2O2 and Oxygen on the Surface of Thionin Incorporated onto MWCNTs Modified Glassy Carbon Electrode: Application to Glucose Detection

A new H₂O₂ enzymeless sensor has been fabricated by incorporation of thionin onto multiwall carbon nanotubes (MWCNTs) modified glassy carbon electrode. First 50 μL of acetone solution containing dispersed MWCNTs was pipetted onto the surface of GC electrode, then, after solvent evaporations, the MWCNTs modified GC electrode was immersed into an aqueous solution of thionin (electroless deposition) for a short period of time <5–50 s. The adsorbed thin film of thionin was found to facilitate the reduction of hydrogen peroxide in the absence of peroxidase enzyme. Also the modified electrode shows excellent catalytic activity for oxygen reduction at reduced overpotential. The rotating modified electrode shows excellent analytical performance for amperometric determination of hydrogen peroxide, at reduced overpotentials. Typical calibration at ?0.3 V vs. reference electrode, Ag/AgCl/3 M KCl, shows a detection limit of 0.38 μM, a sensitivity of 11.5 nA/μM and a liner range from 20 μM to 3.0 mM of hydrogen peroxide. The glucose biosensor was fabricated by covering a thin film of sol–gel composite containing glucose oxides on the surface of thionin/MWCNTs modified GC electrode. The biosensor can be used successfully for selective detection of glucose based on the decreasing of cathodic peak current of oxygen. The detection limit, sensitivity and liner calibration rang were 1 μM, 18.3 μA/mM and 10 μM–6.0 mM, respectively. In addition biosensor can reach 90% of steady currents in about 3.0 s and interference effect of the electroactive existing species (ascorbic acid–uric acid and acetaminophen) is eliminated. The usefulness of biosensor for direct glucose quantification in human blood serum matrix is also discussed. This sensor can be used as an amperometric detector for monitoring oxidase based biosensors.     

Amperometric hydrogen peroxide biosensor based on horseradish peroxidase-labeled nano-Au colloids immobilized on poly(2,6-pyridinedicarboxylic acid) layer by cysteamine.

A sensitive hydrogen peroxidase (H2O2) amperometric sensor based on horseradish peroxidase (HRP)-labeled nano-Au colloids has been proposed. Nano-Au colloids were immobilized by the thiol group of cysteamine, which was associated with the carboxyl groups of poly(2,6-pyridinedicarboxylic acid) (PPDA). With the aid of the hydroquinone, the sensor displayed excellent electrocatalytical response to the reduction of H2O2. Compared with the non-Au-colloid modified electrode, i.e., PPDA/HRP, the Au-colloid modified electrode exhibited better performance characteristics, including stability, reproducibility, sensitivity and accuracy. The biosensor shows a linear response to H2O2 in the range of 3.0 x 10(-7) - 2 x 10(-3) M. The detection limit was 1.0 x 10(-7) M.     

Fabrication of herbicide biosensors based on the inhibition of enzyme activity that catalyzes the scavenging of hydrogen peroxide in a thylakoid membrane.

A novel herbicide biosensor with a thylakoid modified membrane electrode is presented. Thylakoid, isolated from spinach leaves, was entrapped in a membrane of poly (vinylalcohol) with the styrylpyridinium group (PVA-SbQ). The thylakoid membrane was fixed on the surface of a platinum electrode. It was found that the enzymes in thylakoid kept their activity for several months in the membrane. The oxidative current of hydrogen peroxide in a Tris-HCl buffer solution (pH 7.4) was detected at the modified electrode by a differential pulse voltammetric method. In the presence of herbicides, the oxidation current from the hydrogen peroxide decreased due to an inhibitor effect on the enzymes in thylakoid compared with that in the absence of the herbicides. The changes in the oxidation current at the electrode were proportional to the herbicide concentrations. The sensor could be used to detect herbicides in concentration ranges of 3 x 10(-9) - 1.5 x 10(-7) M for paraquat, 1 x 10(-8) - 3 x 10(-7) M for diuron, 4 x 10(-8) - 3 x 10(-6) M for prometryn, 5 x 10(-8) - 5 x 10(-6) M for atrazine and 1 x 10(-7) - 5 x 10(-6) M for ametryn, respectively. The enzyme activity on scavenging hydrogen peroxide in the modified PVA-SbQ membrane was examined.     

Sol-gel-derived amperometric biosensor for hydrogen peroxide based on methylene green incorporated in Nafion film

A hydrogen peroxide biosensor was fabricated by coating a sol-gel-peroxidase layer onto a Nafion-methylene green modified electrode. Immobilization of methylene green (MG) was attributed to the electrostatic force between MG(+) and the negatively charged sulfonic acid groups in Nafion polymer, whereas immobilization of horseradish peroxidase was attributed to the encapsulation function of the silica sol-gel network. Cyclic voltammetry and chronoamperometry were employed to demonstrate the feasibility of electron transfer between sol-gel-immobilized peroxidase and a glassy carbon electrode. Performance of the sensor was evaluated with respect to response time, sensitivity as well as operational stability. The enzyme electrode has a sensitivity of 13.5 muA mM(-1) with a detection limit of 1.0x10(-7) M H(2)O(2), and the sensor achieved 95% of the steady-state current within 20 s.     

Chemiluminometric method for the determination of glycerol in wine by flow-injection analysis with co-immobilized glycerol dehydrogenase/NADH oxidase

A flow-injection method for the determination of glycerol in wine is described. Glycerol dehydrogenase and NADH oxidase were co-immobilized on poly (vinyl alcohol) beads and incorporated in a flow-injection system. The hydrogen peroxide produced was detected chemiluminometrically via a luminol-hexacyanoferrate (III) reaction. Wine was diluted 1000-fold with water and sample solution (50 microl) was injected into the carrier stream. The calibration graph was linear in the range 3 x 10(-7)-3 x 10(-4) M; the detection limit was 7 x 10(-8) M and the sample throughout was 30 h(-1) without carryover.     

Flow-injection determination of L-histidine with an immobilized histidine oxidase from Brevibacillus borstelensis KAIT-B-022 and chemiluminescence detection.

A chemiluminometric flow injection analytical system for the quantitation of L-histidine is described. Histidine oxidase (EC 1.4.3.-) from Brevibacillus borstelensis KAIT-B-022 was immobilized on tresylated poly(vinyl alcohol) beads and packed into a stainless-steel column. The hydrogen peroxide produced was detected chemiluminometrically by a flowthrough sensor containing immobilized peroxidase (EC 1.1 1.1.7). The maximum sample throughput was 10 h(-1). The calibration graph was linear from 0.05 to 5 mM; the detection limit (signal to noise ratio = 3) was 0.01 mM. The activity of immobilized histidine oxidase reduced to 65% of the initial value after 350 injections. The system was applied to the determination of L-histidine in fish meat, such as salmon, tunny, bonito, and mackerel.     

An enzyme reactor electrode for determination of amino acids

L-Leucine can be determined with an enzyme reactor electrode containing L-amino acid oxidase immobilized with glutaraldehyde to glass. The reactor also contains immobilized catalase which splits the hydrogen peroxide formed. Oxygen for the reaction is also supplied by adding hydrogen peroxide to the samples. The electrode is an ammonia gas sensor. The calibration curve is strictly linear with Nernstian slope between 3·10^-5 and 10^-3 M leucine.     

Incorporation of horseradish peroxidase in a Kieselguhr membrane and the application to a mediator-free hydrogen peroxide sensor.

Horseradish peroxidase was incorporated in a kieselguhr membrane. The electron-transfer process of the enzyme was examined by cyclic voltammetry. It was observed that the electron-transfer reactivity of horseradish peroxidase was greatly enhanced, and that direct electrochemistry was accordingly feasible. Using the merits of the direct electron-transfer reactivity of horseradish peroxidase and its specific enzymatic catalysis towards hydrogen peroxide, an unmediated hydrogen peroxide biosensor was constructed. The calibration plot of this hydrogen peroxide sensor was linear in the range of 2.0 x 10(-6) mol/L - 6.5 x 10(-4) mol/L. The relative standard deviation was 4.1% for 6 successive determinations at a concentration of 1.0 x 10(-4) mol/L. The detection limit was 1.0 x 10(-6) mol/L.     

Sensitive biosensor for choline and acetylcholine involving fast immobilization of a bienzyme system on a disposable membrane

A biosensor was prepared for the determination of choline or acetylcholine by co-immobilizing choline oxidase and cholinesterase on a chemically preactivated membrane ready for use. This rapid procedure allows the coupling to be performed in a few minutes. The determination is based on the electrochemical detection of enzymatically generated hydrogen peroxide. This sensor has a detection limit of 5 × 10^?8 M. The response was obtained in 2 min and was linear up to 2 × 10^?5 M.     

Grape tissue-based electrochemical sensor for the determination of hydrogen peroxide

The use of grape tissue as a source of catalase for the determination of hydrogen peroxide is reported. A slice of grape tissue attached to the membrane of a Clark-type oxgen sensor was used to monitor the oxidation of hydrogen peroxide by catalase. At the steady state, the sensor responds linearly to hydrogen peroxide in the concentration range 1 × 10^?5–5 × 10^?4 M. The response time (T₉₀) was of the order of 1 min for this sensor. No interference was observed from ethanol, amino acids, glucose and lactic acid. The long-term stability of the grape tissue sensor was much better than previously reported immobilized enzyme and liver tissue-based hydrogen peroxide sensors.     

Flow-through chemiluminescence sensor using immobilized histamine oxidase from Arthrobacter crystallopoietes KAIT-B-007 and peroxidase for selective determination of histamine.

A flow sensor with immobilized oxidases is proposed for the determination of histamine in fish meat. Chemiluminometric measurement of histamine was based on the luminol reaction with hydrogen peroxide produced by immobilized histamine oxidase (EC 1.4.3.-.) and peroxidase (EC 1.11.1.7.) within a flow cell. Histamine oxidase was found in cells of Arthrobacter crystallopoietes KAIT-B-007 isolated from soil. The oxidase and peroxidase were coimmobilized covalently on tresylated hydrophilic vinyl polymer beads and packed into transparent PTFE; the tubing was used as the flow cell. One assay for histamine was done at intervals of 2 min without carryover. The calibration curve for histamine was linear from 0.1 microM to 50 microM. The response was reproducible within 1.25% of the relative standard deviation for 115-replicate injections of 50 microM histamine. The sensor system was applied to the determination of histamine in fish meat extracts.     

Aluminum electrode modified with manganese hexacyanoferrate as a chemical sensor for hydrogen peroxide

A chemically modified electrode was fabricated based on manganese hexacyanoferrate (MnHCF) film. The MnHCF was used as a modifier immobilized onto an aluminum electrode. Stability of the electroactive film formed on the Al electrode surface indicated that MnHCF is a suitable material for the preparation of modified electrodes. The analytical applicability of the modified electrode for the determination of hydrogen peroxide was examined. A linear response in concentration range of 6.0x10(-7)-7.4x10(-3) M (r=0.9997) was obtained with detection limit of 2.0x10(-7) M for the determination of hydrogen peroxide. The modified electrode exhibited a good selectivity for H(2)O(2) in real samples. The mentioned electrode has advantages of being highly stable, sensitive, inexpensive, ease of construction and use.