Peroxidase immobilized on Amberlite IRA-743 resin for on-line spectrophotometric detection of hydrogen peroxide in rainwater

The importance of atmospheric hydrogen peroxide (H₂O₂) in the oxidation of SO₂ and other compounds has been well established. A spectrophotometric method for the determination of hydrogen peroxide in rainwater is proposed. This method is based on selective oxidation of hydrogen peroxide using an on-line tubular reactor containing peroxidase immobilized on Amberlite IRA-743 resin. The hydrogen peroxide in the presence of phenol, 4-aminoantipyrine and peroxidase, produces a red compound (λ = 505 nm). Beer's law is obeyed in a concentration range of 1–100 μmol l⁻¹ hydrogen peroxide with an excellent correlation coefficient (r = 0.9991), at pH 7.0, with a relative standard deviation (R.S.D.) <2%. The detection limit of the method is 0.7 μmol l⁻¹ (4.8 ng of H₂O₂ in a 200 μl sample). Measurements of hydrogen peroxide in rain samples were carried out over the period from November 2003 to January 2005, in the central area of the Juiz de Fora city, Brazil. The concentration of H₂O₂ varied from values lower than the detection limit to 92.5 μmol l⁻¹. The effects of the presence of nonseasalt (NSS) SO₄²⁻, NO₃⁻ and H⁺ in the concentration of hydrogen peroxide in the rainwater had been evaluated. The average concentrations of H₂O₂, NO₃⁻, NSS SO₄²⁻ and SO₄²⁻ are 23.4, 18.9, 7.9 and 10.3 μmol l⁻¹, respectively. The pH values for 82% of the collected samples are greater than 5.0. The spectrophotometeric method developed in this work that uses enzyme immobilized on the resin ion-exchange compared with the amperometric method did not present any significant difference in the results.     

Flow injection amperometric determination of hydrogen peroxide by oxidation at an iridium oxide electrode

Either an iridium electrode that was anodically pretreated or a glassy carbon electrode that was coated with a film that contained iridium oxide promoted the oxidation of hydrogen peroxide. With flow injection methodology, linear calibration curves were obtained over the 2 × 10⁻⁵ −3.6 × 10−8M range with 0.1M KOH as the carrier. With solutions of pH 11, oxidation occurred at applied potentials of near 0.0 V vs. SCE, which is much lower than potentials needed at bare Pt or glassy carbon. The low overpotential suggests applications to systems where oxidation of other species limits the use of bare electrodes. Cyclic voltammetric studies show that HO₂⁻ is the electroactive species and that catalysis rather than mediation promotes the charge transfer. Dissolved oxygen does not interfere with the measurement.     

Exploiting flow injection and sequential injection anodic stripping voltammetric systems for simultaneous determination of some metals

Flow injection (FI) and sequential injection (SI) systems with anodic stripping voltammetric detection have been exploited for simultaneous determination of some metals. A pre-plated mercury film on a glassy carbon disc electrode was used as a working electrode in both systems. The same film can be repeatedly applied for at least 50 analysis cycles, thus reducing the mercury consumption and waste. A single line FI voltammetric system using an acetate buffer as a carrier and an electrolyte solution was employed. An injected standard/sample zone was mixed with the buffer in a mixing coil before entering a flow cell. Metal ions were deposited on the working electrode by applying a potential of −1.1 V vs Ag/AgCl reference electrode. The stripping was performed by anodically scanning potential of working electrode to +0.25 V, resulting a voltammogram. Effects of acetate buffer concentration, flow rate and sample volume were investigated. Under the selected condition, detection limits of 1 μg l⁻¹ for Cd(II), 18 μg l⁻¹ for Cu(II), 2 μg l⁻¹ for Pb(II) and 17 μg l⁻¹ for Zn(II) with precisions of 2–5% (n=11) were obtained. The SI voltammetric system was similar to the FI system and using an acetate buffer as a carrier solution. The SI system was operated by a PC via in-house written software and employing an autotitrator as a syringe pump. Standard/sample was aspirated and the zone was then sent to a flow cell for measurement. Detection limits for Cd(II), Cu(II), Pb(II) and Zn(II) were 6, 3, 10 and 470 μg l⁻¹, respectively. Applications to water samples were demonstrated. A homemade UV-digester was used for removing organic matters in the wastewater samples prior to analysis by the proposed voltammetric systems.     

Biosensor for phenol based on the direct electron transfer blocking of peroxidase immobilising on silica–titanium

Horseradish peroxidase (HRP) was immobilised on silica gel modified with titanium oxide. This material was employed to prepare modified carbon paste electrode. The direct electron transfer of the hydrogen peroxide reduction by HRP was blocked when immobilised on silica–titanium. This biosensor presented a very sensitive response for phenol (1 μmol l⁻¹) at an applied potential of 0 mV vs SCE. The best condition was achieved in phosphate buffer pH 6.8, ratio of hydrogen peroxide/phenol higher than 0.35. The biosensor showed a linear response range between 10 and 50 μmol l⁻¹ of phenol, adjusted by the equation j=−32.8+16.3 [phenol], for n=5 with a correlation coefficient of 0.9995. The response time of the biosensor was about 3 s.     

Detection of TMV with ODA-H(2)O(2)-HRP voltammetric enzyme-linked immunoassay system

o-Dianisidine (ODA)-H₂O₂-horseradish peroxidase (HRP) voltammetric enzyme-linked immunoassay system has firstly been used for the detection of tobacco mosaic virus (TMV). HRP catalyzes strongly the oxidation reaction of ODA by H₂O₂, the product of which produces a sensitive second order derivative linear sweep voltammetric peak at potential of −0.56 V (versus SCE) in Britton–Robinson (BR) buffer. HRP activity has been measured with this voltammetric peak and TMV detected through immunoreaction. The detection limit for HRP is 9.25×10^-7 mU l⁻¹ and the linear range is 2.5×10⁻⁶–5.0×10⁻⁴ mU l⁻¹. The detection limit for the clarified TMV is 0.25 ng ml⁻¹ and the highest dilution ratio detected for the infected leaf sap is 1:8×10⁵. The sensitivity for TMV detection with this method is higher than that with the enzyme-linked immunosorbent spectrophotometric assay (ELISA) using ODA-H₂O₂-HRP system. The processes of the enzyme-catalyzed reaction and the electro-reduction of the product of the enzyme-catalyzed reaction have been described.     

Spectrophotometric determination of maneb by ternary complex formation with PAR and CTAB

A rapid, simple, direct, and sensitive method has been developed for the determination of maneb (manganese ethylenebisdithiocarbamate) based on the formation of manganese-4-(2′-pyridylazo) resorcinol complex by a ligand displacement reaction, which is rendered water soluble by a cationic surfactant cetyltrimethylammonium bromide (CTAB) by the formation of an ion association complex. Beer's law is obeyed over the concentration range 0.08–2.4 μg/ml of the final solution at 500 nm in pH range 8–12. The molar absorptivity and Sandell's sensitivity are calculated to be 8.84 × 10⁴ l.mol⁻¹.cm⁻¹ and 0.003 μg/cm², respectively. The developed method has been applied to the determination of maneb in commercial formulations, synthetic mixture, grain samples and vegetables.     

Development of a luminol-based chemiluminescence flow-injection method for the determination of dichlorvos pesticide

A simple, fast chemiluminescence (CL) flow-injection method based on the reaction of luminol with H₂O₂ in the presence of a cationic surfactant (cyltrimethylammonium bromide, CTMAB) has been described for the direct determination of dichlorvos pesticide (DDVP). Under the optimal conditions, the CL intensity was linear to the DDVP concentration in the range of 0.02–3.1 μg ml⁻¹ (r=0.9998, n=10). The relative standard deviation was 3.4% at 0.35 μg ml⁻¹ (n=10), with a detection limit (3σ) of 0.008 μg ml⁻¹ DDVP. The possible reaction mechanism was also discussed. This method has been successfully applied to the determination of trace DDVP residue in vegetable sample and results have been compared with that of the UV method.     

Amperometric biosensors based on the immobilization of oxidases in a Prussian blue film by electrochemical codeposition

Prussian blue has been formed by cyclic voltammetry onto the basal pyrolytic graphite surface to prepare a chemically modified electrode which provides excellent electrocatalysis for both oxidation and reduction of hydrogen peroxide. It is found for the first time that glucose oxidase or -amino oxidase can be incorporated into a Prussian blue film during its electrochemical growth process. Two amperometric biosensors were fabricated by electrochemical codeposition, and the resulting sensors were protected by coverage with a thin film of Nafion. The influence of various experimental conditions was examined for optimum analytical performance. The glucose sensor responds rapidly to substrates with a detection limit of 2 × 10⁻⁶ M and a linear concentration range of 0.01–3 mM. There was no interference from 2 mM ascorbic acid or uric acid. Another ( -amino acid) sensor gave a detection limit of 3 × 10⁻⁵ M -alanine, injected with a linear concentration range of 7.0 × 10⁻⁵-1.4 × 10⁻² M. Glucose and -amino acid sensors remain relatively stable for 20 and 15 days, respectively. There is no obvious interference from anion electroactive species due to a low operating potential and excellent permselectivity of Nafion.     

A peroxidase-tetrathiafulvalene biosensor based on self-assembled monolayer modified Au electrodes for the flow-injection determination of hydrogen peroxide

The construction and performance under flow-injection conditions of an integrated amperometric biosensor for hydrogen peroxide is reported. The design of the bioelectrode is based on a mercaptopropionic acid (MPA) self-assembled monolayer (SAM) modified gold disk electrode on which horseradish peroxidase (HRP, 24.3 U) was immobilized by cross-linking with glutaraldehyde together with the mediator tetrathiafulvalene (TTF, 1 μmol), which was entrapped in the three-dimensional aggregate formed.

The amperometric biosensor allows the obtention of reproducible flow injection amperometric responses at an applied potential of 0.00 V in 0.05 mol L⁻¹ phosphate buffer, pH 7.0 (flow rate: 1.40 mL min⁻¹, injection volume: 150 μL), with a range of linearity for hydrogen peroxide within the 2.0 × 10⁻⁷–1.0 × 10⁻⁴ mol L⁻¹ concentration range (slope: (2.33 ± 0.02) × 10⁻² A mol⁻¹ L, r = 0.999). A detection limit of 6.9 × 10⁻⁸ mol L⁻¹ was obtained together with a R.S.D. (n = 50) of 2.7% for a hydrogen peroxide concentration level of 5.0 × 10⁻⁵ mol L⁻¹. The immobilization method showed a good reproducibility with a R.S.D. of 5.3% for five different electrodes. Moreover, the useful lifetime of one single biosensor was estimated in 13 days.

The SAM-based biosensor was applied for the determination of hydrogen peroxide in rainwater and in a hair dye. The results obtained were validated by comparison with those obtained with a spectrophotometric reference method. In addition, the recovery of hydrogen peroxide in sterilised milk was tested.     

Catalytic determination of cobalt at sub-nanogram levels using the oxidative coupling of 3-methyl-2-benzothiazolinone hydrazone with N-ethyl-N-(2-hydroxy-3-sulfopropyl)-3,5-dimethoxyaniline by manual and flow-injection methods

A catalytic photometric method was developed for the determination of sub-nanogram levels of cobalt. The method is based on the catalytic effect of cobalt(II) on the oxidative coupling of 3-methyl-2-benzothiazolinone hydrazone with N-ethyl-N-(2-hydroxy-3-sulfopropyl)-3,5-dimethoxyaniline (DAOS) to form a colored dye (λ_max=525 nm) in the presence of hydrogen peroxide. In this reaction system, 1,2-dihydroxybenzene-3,5-disulfonate (Tiron) acted as an effective activator for the catalysis of cobalt(II). Variation of reaction time between 5 and 10 min allows the determination range to be extended from 0.01 to 1.0 ng ml⁻¹. The reaction system can also be successfully adapted to flow-injection analysis (FIA). The dynamic range of the proposed flow-injection method was 0.01–1.0 ng ml⁻¹ and detection limit (signal/noise, S/N=3) was 5 pg ml⁻¹ at a sampling rate of 30 h⁻¹. Manual and flow-injection methods were applied to the direct determination of cobalt in pepperbush as a standard material.     

Reaction of H with H2O2 as observed by optical absorption of perhydroxyl radicals or aliphatic alcohol radicals and of OH with H2O2. A pulse radiolysis study

Two new procedures were employed for studying the reaction of hydrogen atoms with hydrogen peroxide. The absorption in the UV-range was observed either for an acidic aqueous solution containing only hydrogen peroxide or for a similar solution but also containing an aliphatic alcohol. From the increase in absorption of various alcohol radicals, a rate constant of 3.5×10⁷ dm³ mol⁻¹ s⁻¹ was determined. In addition, the rate constant for the reaction of hydroxyl radicals with hydrogen peroxide was determined to be 3.0×10⁷ dm³ mol⁻¹ s⁻¹.     

Electrochemistry and electrocatalysis with myoglobin in biomembrane-like surfactant-polymer 2C12N+PA- composite films

Biomembrane-like polyionic complex, 2C₁₂N⁺PA⁻, was prepared by reacting sodium polyacrylate (Na⁺PA⁻) with didodecyldimethylammonium bromide (2C₁₂N⁺Br⁻). Stable thin films made from 2C₁₂N⁺PA⁻, with incorporated myoglobin (Mb), on pyrolytic graphite (PG) electrodes were then characterized by electrochemistry and other techniques. Cyclic voltammetry of Mb-2C₁₂N⁺PA⁻ films showed a pair of well-defined quasi reversible peaks for MbFe(III)/Fe(II) couple at about −0.19 V versus SCE in pH 5.5 buffers. The electron transfer rate between Mb and PG electrodes was greatly facilitated in the microenvironment of 2C₁₂N⁺PA⁻ films. Square wave voltammetry data were used to estimate the apparent heterogeneous electron transfer rate constants by nonlinear regression analysis using a model featuring dispersion of formal potentials. Positions of Soret absorption bands suggested that Mb keeps its secondary structure similar to its native state in 2C₁₂N⁺PA⁻ films at the medium pH. The results of differential scanning calorimetry and X-ray diffraction suggest that synthesized 2C₁₂N⁺PA⁻ lipid films have an ordered multibilayer structure and the incorporated Mb does not disturb this structure. Oxygen was catalytically reduced by Mb-2C₁₂N⁺PA⁻ films with a significant decrease in the electrode potential. MbFe(I), a highly reduced form of Mb, was also produced in Mb-2C₁₂N⁺PA⁻ films at about –1.09 V, and could be used to catalytically reduce organohalide pollutants such as perchloroethylene (PCE) and trichloroethylene (TCE). The catalytic reduction peak currents had linear relationships with concentrations of PCE and TCE in a range of 10–100 μM. The potential applications of the film electrode as a sensor for detecting organohalides are discussed.     

Glucose oxidase/hexokinase electrode for the determination of ATP

A hydrogen peroxide based enzyme electrode for the determination of ATP has been developed by the immobilization of glucose oxidase and hexokinase. Competition between the enzymes for the substrate glucose allowed the measurement of ATP. Different immobilization procedures and different types of hexokinase have been tested. Using a BSA-glutaraldehyde procedure and hexokinase from an overproducing strain of bakers' yeast, ATP was measured in the 0.05–0.5 mmol l⁻¹ range with a detection limit of 0.01 mmol l⁻¹. ATP concentrations comparable to those reported in the literature and a good recovery were obtained when the enzyme electrode was used with human erythrocyte hemolysate.     

Third generation biosensor based on myoglobin-TiO2/MWCNTs modified glassy carbon electrode

TiO2 nanoparticles were homogeneously coated on multi-walled carbon nanotubes by hydrothermal deposition, this nanocomposite may be a promising material for myoglobin immobilization in view of its high biocompatibility and large surface. The glassy carbon electrode modified with Mb-TiO2/MWCNTs films exhibited a pair of weU defined, stable and nearly reversible cycle voltammetric peaks. The electron transfer between Mb and electrode surface, Ks of 3.08 s^-1, was greatly facilitated in the TiO2/ MWCNTs film. The electrocatalytic reductions of hydrogen peroxide were studied, the apparent Michaelis-Menten constant is calculated to be 83.10 μmol/L, which shows a large catalytic activity of Mb in the TiO2/MWCNTs film to H2O2.     

Solubilisation and binding characteristics of a recombinant beta2-adrenergic receptor expressed in the membrane of Escherichia coli for the multianalyte detection of beta-agonists and antagonists residues in food-producing animals

The on-line incorporation of cloud point extraction (CPE) to flow injection analysis (FIA) was previously based on the use of a cotton-packed column to entrap the analyte-containing surfactant aggregates after salt-induced CPE, and then the preconcentrated analyte was eluted into a separate detection cell for subsequent chemiluminescence (CL) detection (via the peroxyoxalate CL reaction). In the work, the on-line CPE/FIA technique was improved by the following: (1) sample preconcentration and CL detection were both carried out directly inside the collection column, thus avoiding the decrease in detection sensitivity due to sample dispersion and dilution, and (2) CL detection was performed through the reaction between nitrite and hydrogen peroxide, which is compatible with aqueous samples and should allow for chemical excitation to occur more efficiently inside the collection column. In addition to more effective sample preconcentration, the CL detection of the entrapped analytes directly inside the collection column, i.e., a unique heterogeneous microenvironment in which analyte-containing surfactant aggregates were embedded within the densely packed filtering material, may also contribute to the overall increase in CL intensity (e.g., a CL enhancement factor of ca. 1000). Under optimum experimental conditions, the calibration curve was found to be linear for the CL detection of bilirubin (5 to 120 μg L⁻¹), the limit of detection (S/N = 3) was 1.8 μg L⁻¹, and the R.S.D. was ca. 2.6% (n = 30) for 20 μg L⁻¹ bilirubin. Good agreements were obtained for the determination of total bilirubin in certified reference human serum samples between the present approach and an established clinical method.     

Kinetic-catalytic determination of traces of iron by the oxidative coupling reaction of 3-methyl-2-benzothiazolinone hydrazone with N,N-dimethylaniline

A new kinetic-catalytic method by the initial rate procedure for the determination of nanogram level of iron(III) is developed, which is based on its catalytic effect on the oxidative coupling of 3-methyl-2-benzothiazolinone hydrazone (MBTH) with N,N-dimethylaniline (DMA) to form an indamine dye (λ_max=590 nm) in the presence of hydrogen peroxide. Iron(II) is also determined, being oxidized to iron(III) by hydrogen peroxide. Calibration graphs obtained by the initial rate method are linear in the range 1–1000 ng ml⁻¹ Fe and as low as 10⁻⁸ M Fe(II, III) can easily be determined. The relative standard deviations are 6.6, 2.5 and 1.5% for ten determinations of 1, 20 and 60 ng ml⁻¹ of Fe(III), respectively. The method is applicable to the determination of iron in natural waters without preconcentration and separation.     

Flow-injection chemiluminescence determination of chlortetracycline using on-line electrogenerated [Cu(HIO(6))(2)](5-) as the oxidant

A novel chemiluminescence (CL) flow system for the determination of chlortetracycline is described. It is based on the direct CL reaction of chlortetracycline and [Cu(HIO₆)₂]⁵⁻ in KOH medium. The unstable [Cu(HIO₆)₂]⁵⁻ was on-line electrogenerated by constant-current electrolysis. The CL intensity was linear with chlortetracycline concentration in the range of 0.1–100 μg ml⁻¹. The determination limit was 5.3×10⁻⁸ g ml⁻¹. The whole process could be completed in 1 min. The proposed method is suitable for automatic and continuous analysis, and has been applied satisfactorily to analysis of chlortetracycline in biological fluid.     

Oxidation of thiocyanate by hydrogen peroxide - a reaction kinetic study by capillary electrophoresis

The oxidation reaction kinetics of thiocyanate by excess hydrogen peroxide has been studied by using capillary electrophoresis. The paper illustrates for the first time the use of capillary electrophoresis in studying reaction kinetics and provides a non-laborious way to determine the rate law and the rate constant for the above reaction in the pH range 6–8. Standard solutions of thiocyanate were mixed with buffer solutions of different pHs (6–8) and the reactions were initiated by adding appropriate volumes of hydrogen peroxide in capillary electrophoresis vials. Each reaction mixture was sampled at regular time intervals using an automatic injection programme to follow the progress of the reaction and identify the reaction products. The peak areas, representing the products, were integrated and their concentrations were quantified using calibration plots. The concentration profiles obtained from a series of experiments at a particular pH were then used to determine the rate law and the rate constant for the reaction. Furthermore, the rate of decomposition of hypothiocyanite formed during the reaction is determined for the first time. The rate law is zero order with respect to hypothiocyanite and first order with respect to hydrogen peroxide. The results indicate that the rate law for the oxidation reaction is zero order with respect to thiocyanate and first order with respect to hydrogen peroxide. The rate constant for the reaction at 25°C and at zero ionic strength is 3.6(±0.2)×10(⁻⁴) min⁻¹.     

Voltammetric determination of volatile free sulfide and alkylthiols as contaminants in parenteral amino acid solutions

A method for the simultaneous voltammetric determination of free sulfide and volatile alkythiols (methanethiol/ethanethiol) existing as contaminant in parenteral nutrition (PN) solutions was developed. The volatile sulfides (dihydrogensulfide and alkylthiols) formed in the formulations were distilled over 45 min at 47°C in a purpose-made Conway cell. The analytes were quantified by differential pulse cathodic stripping voltametry (DPCSV) at the hanging mercury-drop electrode and determined, simultaneously as dihydrogensulfide and alkylthiols using a 60 s preconcentration time at −300 mV (versus Ag/AgCl, Cl⁻ 3.0 mol l⁻¹). The voltammetric signals were directly linear proportional to the sulfides concentrations in the PN solutions in the range 3–20 μmol l⁻¹ and detection limits of about 2 μmol l⁻¹ were calculated. Recoveries of sulfides from PN solutions in the range 90–110% were calculated using the method and nine commercial PN formulations were analyzed. Methanethiol and ethanethiol were quantified from their composite signals, and the mutual influence of the analytes on the DPCSV measurements is discussed.     

Kinetic–catalytic–spectrophotometric determination of low concentrations of molybdenum in white wines

The paper describes the determination of the molybdenum content in white wines based on its catalytical action on the kalium iodide oxidation by hydrogen peroxide in acid medium.

The optimum reaction conditions (the catalyst, KI and H₂O₂ concentrations, the pH value, the order of the reagent additions, the temperature) have been found by studying the effect of the reaction variables.

The influence of some metallic ions (Ca²⁺, Mg²⁺, Zn²⁺, Cd²⁺, Fe²⁺ and Fe³⁺) and complexing anions (F⁻, C₂O²⁻₄, EDTA⁴⁻) on the catalyzed reaction rate was elucidated.

The molybdenum concentration was estimated by the tangent, fixed-time and fixed-absorbance method. The obtained average values for molybdenum content in white wines are within the 1.77×10⁻⁷–1.83×10⁻⁷ mol l⁻¹ range.