Bioelectrochemical Characterization of Horseradish and Soybean Peroxidases

Heme peroxidase are ubiquitous enzymes catalyzing the oxidation of a broad range of substrates by hydrogen peroxide. In this paper the bioelectrochemical characterization of horseradish peroxidase (HRP) and soybean peroxidase (SBP), belonging to class III of the plant peroxidase superfamily, was studied. The homogeneous reactions between peroxidases and some common redox mediators in the presence of hydrogen peroxide have been carried out by cyclic voltammetry. The electrochemical characterization of the reactions involving enzyme, substrate and mediators concentrations allowed us to calculate the kinetic parameters for the substrate–enzyme reaction (K_MS) and for the redox mediator–enzyme reaction (K_MM). A full characterization of the direct electron transfer kinetic parameters between the electrode and enzyme active site was also performed by opportunely modeling data obtained from cyclic voltammetry and square wave voltammetry experiments. The experimental data obtained with immobilized peroxidases show enhanced direct electron transfer and excellent electrocatalytical performance for H₂O₂. Despite the structural similarities and common catalytic cycle, HRP and SBP exhibit differences in their substrate affinity and catalytic efficiency. Basing on our results, it can be concluded that peroxidase from soybean represents an interesting alternative to the classical and largely employed one obtained from horseradish as biorecognition element of electrochemical mediated biosensors.     

Tobacco Peroxidase as a New Reagent for Amperometric Biosensors

The results of testing a new enzyme, anionic tobacco peroxidase (TOP), in various amperometric biosensors are summarized. The biochemical and electrochemical properties of the enzyme are briefly characterized. As compared to the commonly used cationic peroxidase from horseradish roots, TOP exhibits a wider optimum stability pH range, higher stability to inactivation with hydrogen peroxide, and higher efficiency in direct electron-transfer processes. The enzyme immobilized by adsorption on graphite is effective in determining aminophenols and aromatic diamines under flow conditions with a detection limit of 10 nM. Upon immobilization on graphite by incorporation into a gel of a redox-active polymer (crosslinked polyvinylimidazole with osmium 4,4′-dimethylbipyridinium chloride), TOP exhibited sensitivity and stability comparable to those of horseradish peroxidase and a wider linearity range. Upon immobilization on a self-assembled thiol monolayer at a gold electrode, TOP was much superior to horseradish peroxidase in the sensitivity of determining hydrogen peroxide, regardless of the charge of the monolayer. Prospects for the further use of the native enzyme and its genetically engineered unglycosylated form are considered.__________Translated from Zhurnal Analiticheskoi Khimii, Vol. 60, No. 6, 2005, pp. 629–638.Original Russian Text Copyright © 2005 by Gazaryan, Gorton, Ruzgas, Csoregi, Schuhmann, Lagrimini, Khushpul’yan, Tishkov.Presented at the VI All-Russia Conference (with international participation) on Electrochemical Methods of Analysis (EMA-2004, Ufa, May 23–27, 2004).     

Hybrid bioelectrocatalyst for hydrogen peroxide reduction: immobilization of enzyme within organic-inorganic film of structured Prussian Blue and PEDOT

Fabrication of structured film (on glassy carbon substrate) composed of compact Prussian Blue (that has been prepared by alternate immersions and through assembling within ultra-thin layers of 4(pyrrole-1-yl)-benzoic acid, PPyBA) and poly(3,4-ethylendioxythiophene), PEDOT, is described. This functionalized film has been characterized by fast charge propagation, and it has served as a redox conducting template for permanent attachment of a model enzyme, horseradish peroxidase, HRP. The resulting organic-inorganic system acts as an effective hybrid bioelectrocatalyst for electroreduction of hydrogen peroxide, a model reactant for biosensors and biofuel cells. Among important issues are rigidity, permanence of enzyme attachment, morphology, hydrophilicity, and attractive mediating capabilities of the PEDOT-stabilized Prussian Blue based structured film.     

Highly Sensitive Determination of DAO Activity by Oxidation of a Luminescence Reagent

A highly sensitive method for measuring the activity of the enzyme diamine oxidase (DAO) independent of the type of substrate is described. The principle of the assay is to determine the amount hydrogen peroxide generated as a reaction product during oxidation of diamines by DAO. PSatto™, a highly sensitive luminescence reagent, was used to generate a signal depending on the hydrogen peroxide concentration based on the action of horseradish peroxidase. DAO is specifically captured from a sample by an antibody immobilized to microwell plates, and the substrate is added to the bound enzyme. Various diamines were used as substrates; the peroxide produced is directly proportional to the amount of DAO bound to the specific antibodies. With this very sensitive method, it is possible to detect pmol amounts of generated hydrogen peroxide in plasma matrix corresponding to the biological activity of DAO.     

辣根过氧化物酶在分析化学中的应用

辣根过氧化物酶是一种重要的分析化学试剂,对过氧化氢底物特异性强。广泛用于临床化学、环境化学和食品工业等领域。本文评述了近十年来辣根过氧化物酶在分析化学中的应用进展。包括光度法、荧光法、化学发光法、电化学传感器和光导纤维传感器法测定过氛氢及相关物质,及辣根过氧化物酶在其他无机、有机及生物物质分析中的应用。引用参考文献６０篇。     

Biosensor for H2O2 Response Based on Horseradish Peroxidase: Effect of Different Mediators Adsorbed on Silica Gel Modified with Niobium Oxide

Reagentless biosensors sensitive to hydrogen peroxide have been developed and compared. These biosensors are comprised of a carbon paste electrode modified with horseradish peroxidase (HRP) and one phenothiazine (methylene blue), one phenoxazine (meldola's blue) or one phenazine (phenazine methosulfate) dye adsorbed on silica gel modified with niobium oxide (SN). The enzyme was immobilized onto the graphite powder by cross‐linking with glutaraldehyde and mixing with one of the electron transfer mediators (dyes) adsorbed on SN. The amperometric response was based on the electrocatalytic properties of the dye to mediate electrons, which were generated in the enzymatic reaction of hydrogen peroxide under catalysis of HRP. The dependence on the biosensor response in terms of pH, buffer, HRP amounts and applied potential was investigated. The best results were found with a biosensor containing methylene blue dye showing an excellent operational stability (around 92% of the activity was maintained after 300 determinations). The proposed biosensor also presented good sensitivity (32.87 nA cm^{?2 μ}mol^?1 L) allowing hydrogen peroxide quantification at levels down to 0.52×10^?6 mol L^?1 an optimum response at pH 6.8 and at a potential of ?50 mV (vs. SCE) and showing a wide linear response range (from 1 to 700 μmol L^?1 for hydrogen peroxide).     

Oxidation of ferrocene and some substituted ferrocenes in the presence of horseradish peroxidase

The enzyme catalysed oxidation of ferrocene and some substituted ferrocenes to the corresponding ferricinium ions by hydrogen peroxide in the presence of native or immobilized horseradish peroxidase has been studied. Initial and maximum rates of oxidation have been determined. It was found that the oxidation was independent of the hydrogen peroxide concentration. The oxidation of ferrocene was effected also by horseradish peroxidase in a coupled system with glucose oxidase in the absence of any added hydrogen peroxide.     

Gas-sensing internal enzyme fiber optic biosensor for hydrogen peroxide

Feasibility is demonstrated for a novel gas-sensing, internal enzyme biosensing scheme for the selective measurement of hydrogen peroxide. Two horseradish peroxidase catalysed reactions are evaluated for the detection of hydrogen peroxide as it crosses a microporous Teflon membrane at 37 degrees C. The rate at which hydrogen peroxide crosses the membrane is determined by either a fluorescence or chemiluminescence measurement and this rate is related to the concentration of hydrogen peroxide in the sample solution. Detection limits of 0.7 mM and 10 muM are estimated for the fluorescence and chemiluminescence methods, respectively. Selectivity is demonstrated for hydrogen peroxide over ascorbic acid, uric acid and tyrosine.     

Reagentless enzyme electrode based on phenothiazine mediation of horseradish peroxidase for subnanomolar hydrogen peroxide determination

The development and characterization of a highly sensitive enzyme immobilized carbon based electrode for the determination of subnanomolar concentrations of hydrogen peroxide in aqueous samples is described. The biosensor consists of horseradish peroxidase (HRP) immobilized in solid carbon paste along with a suitable redox mediator. The latter allows the acceleration of the electroreduction of HRP in the presence of hydrogen peroxide. Several phenothiazines as mediators are investigated in a comparative manner and with respect to dimethylferrocene using cyclic voltammetry and amperometry. Insolubilization of the HRP in the solid carbon paste is achieved by cross-linking the enzyme with glutaraldehyde and bovine serum albumin. Several experimental parameters such as pH, mediator and enzyme content are considered. The hydrogen peroxide determination is better carried out in 0.1 M acetate buffer, pH 4.5, by amperometry at an applied potential of 0.0 V versus Ag/AgCl, 3 M NaCl concentration and by using the phenothiazine base as redox mediator. The biosensor response is linear over the concentration range 2 nM-10 microM with a detection limit of 1 nM. The linear range of the hydrogen peroxide response without a mediator in the biosensor is found between 2 and 40 microM. The biosensor can be used for more than 180 measurements. Additional modification of the electrode by incorporation of Nafion SAC-13 microparticles in the solid carbon paste allows detection of concentrations of hydrogen peroxide as low as 0.1 nM.     

Microcantilevers modified by horseradish peroxidase intercalated nano-assembly for hydrogen peroxide detection.

Detection of hydrogen peroxide is of industry interest and of biological importance. Here we report a new approach to hydrogen peroxide measurement using multilayer modified microcantilevers. Through a layer-by-layer nanoassembly technique, horseradish peroxidase was intercalated into a nanoscale multilayer assembly on one surface of microcantilevers. These enzyme-functionalized microcantilevers deflected in response to hydrogen peroxide concentrations in the nanomolar level. The magnitudes of bending were proportional to the concentrations of hydrogen peroxide. Furthermore, our study also showed that microcantilever technique may be used as a novel, more sensitive tool for the study of the conformational or structural changes of enzymes or proteins on surfaces.     

N,N-diethylaniline as hydrogen donor for determination of hydrogen peroxide catalyzed by metalloporphyrins as enzyme mimetic of peroxidase

Various metalloporphyrins have been used as a catalyst instead of the peroxidase for the determination of hydrogen peroxide by formation of a dye from N,N-diethylaniline (DBA) and 4-aminoantipyrine. The difference of relative catalytic activity was investigated between enzyme and enzyme mimetics. FeT(4-TAP)P [5, 10, 15, 20-tetrakis (4-trimethyl-ammoniumphenyl)-21H, 23H-porphine] was shown as the best enzyme mimetic for horseradish peroxidase (HRP) among metalloporphyrins tested. 0 to 7.0 × 10^–5 mol/L hydrogen peroxide was determined with good accuracy and reproducibility, and giving recovery of 99.7–100.7%. DEA was certified as a sensitive color reagent in enzyme mimetic assay of hydrogen peroxide, with the apparent molar absorptivity for hydrogen peroxide was 1.37 × 10⁴ L/mol·cm.     

Direct electrochemistry and electrocatalysis of horseradish peroxidase immobilized on L-glutathione self-assembled monolayers

A novel hydrogen peroxide biosensor has been fabricated based on covalently linked horseradish peroxidase （HRP） onto L- glutathione self-assembled monolayers （SAMs）. The SAMs-based electrode was characterized by electrochemical methods, and direct electrochemistry of HRP can be achieved with formal potential of-0.242 V （vs. saturated Ag/AgCl） in pH 7 phosphate buffer solution （PBS）, the redox peak current is linear to scan rate and rate constant can be calculated to be 0.042 s^-1. The HRP-SAMs- based biosensors show its better electrocatalysis to hydrogen peroxide in the concentration range of 1 × 10^-6 mol/L to 1.2 × 10^-3 mol/L with a detection limit of 4 × 10^-7 mol/L. The apparent Michealis-Menten constant is 3.12 mmol/L. The biosensor can effectively eliminate the interferences of dopamine, ascorbic acid, uric acid, catechol and p-acetaminophen.     

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.     

A Novel Amperometric Biosensor for Determination of Hydrogen Peroxide Based on Nafion and Polythionine as Well as Gold Nanoparticles and Gelatin as Matrixes

Abstract

A new biosensor for the amperometric detection of hydrogen peroxide was developed by means of immobilized horseradish peroxidase (HRP) on a platinum disk based on gold nanoparticles, nafion, polythionine (PTn), and gelatin as matrixes. The mediator (PTn) was embedded in nafion film effectively without leaching even after long periods of operation, the immobilization of the enzyme comes from the cooperative binding by the Au nanoparticles and gelatin. The fabrication procedure of the biosensor was characterized by using cyclic voltammetry (CV) and electrochemical impedance spectroscopy (EIS). The electrochemical characteristics of the enzyme electrode with respect to the effect of pH, temperature, and the operational and storage stabilities were studied. The test demonstrated that the biosensors show high stability, fast response (<20 s), and a working range 0.05 to 30.6 mM (correlation coefficient: 0.9986), a detection limit of 0.02 mM to hydrogen peroxide (H₂O₂). The analytical results by this approach were in satisfactory agreement with those by conventional methods of titration.     

Horseradish peroxidase catalyzed nitric oxide formation from hydroxyurea

Hydroxyurea represents an approved treatment for sickle cell anemia and a number of cancers. Chemiluminescence and electron paramagnetic resonance spectroscopic studies show horseradish peroxidase catalyzes the formation of nitric oxide from hydroxyurea in the presence of hydrogen peroxide. Gas chromatographic headspace analysis and infrared spectroscopy also reveal the production of nitrous oxide in this reaction, which provides evidence for nitroxyl, the one-electron reduced form of nitric oxide. These reactions also generate carbon dioxide, ammonia, nitrite, and nitrate. None of these products form within 1 h in the absence of hydrogen peroxide or horseradish peroxidase. Electron paramagnetic resonance spectroscopy and trapping studies show the intermediacy of a nitroxide radical and a C-nitroso species during this reaction. Absorption spectroscopy indicates that both compounds I and II of horseradish peroxidase act as one-electron oxidants of hydroxyurea. Nitroxyl, generated from Angeli's salt, reacts with ferric horseradish peroxidase to produce a ferrous horseradish peroxidase-nitric oxide complex. Electron paramagnetic resonance experiments with a nitric oxide specific trap reveal that horseradish peroxidase is capable of oxidizing nitroxyl to nitric oxide. A mechanistic model that includes the observed nitroxide radical and C-nitroso compound intermediates has been forwarded to explain the observed product distribution. These studies suggest that direct nitric oxide producing reactions of hydroxyurea and peroxidases may contribute to the overall pharmacological properties of this drug.     

Employing peroxidase from Thai indigenous plants for the application of hydrogen peroxide assay

Thai local plants known as banana stalk, banana blossom, banana, sugar-cane, oroxylum indicum fruit, sesbania grandiflora fruit, and pigeon pea fruit were utilized for screening peroxidase enzyme to replace costly horseradish peroxidase in the hydrogen peroxide assay. The highest peroxidase activity was found in banana stalk extracted solution. The kinetic parameters, i.e., Michaelis–Menten constant (Km) and maximum velocity (Vmax) of banana stalk peroxidase were carried out. The optimum pH and thermal stability of this enzyme were also studied. Furthermore, crude banana stalk peroxidase was applied for the determination of hydrogen peroxide in a disinfection solution without any purification. The influent parameters affecting the developed method were cautiously studied and optimized. The calibration curve of standard hydrogen peroxide was achieved between 2.0 and 10.0 μmol L^?1 with correlation coefficient (r ²) 0.995. The method validations of detection limit (LOD), limit of quantification (LOQ) and precision were investigated. The concentrations of hydrogen peroxide achieved by the developed method were correlated with the enzymatic method using commercial available horseradish peroxidase.     

Bioelectrocatalytic Reduction of Peroxide Compounds on the Peroxidase–Nafion Composite

Electrochemical reactions of peroxide compounds (hydrogen peroxide and peracetic and perbenzoic acids) on an electrode of pyrocarbon with immobilized horseradish peroxidase (HRP) are studied. The immobilization of HRP is performed in the composition of a composite with Nafion whose structure is studied by a method of scanning tunneling microscopy. The proposed composite material provides for a high catalytic activity and stability of enzyme in the reaction of reduction of peroxide compounds. It is shown that the electrocatalytic reduction of the studied compounds on the electrode with the peroxidase–Nafion composite proceeds in conditions of direct bioelectrocatalysis. The effect of the solution pH and the concentration of substrates on the electrocatalytic activity of HRP in the composition of the composite is studied. On the basis of the obtained results a possible mechanism of the electrocatalytic reduction of peroxide compounds in the presence of HRP is suggested. The rate of a bioelectrocatalytic process is defined by the nature and concentration of the substrate as well as by the electrode potential and the solution pH.     

动力学分析方法检测酶反应活性中间体的研究

提出了一种检测酶反应活性中间体的动力学新方法，利用中间体的反应活性，加入合适的竞争剂使中间体在转化产物的同时能与竞争剂反应，监测中间体与竞争剂反应的动力学过程的以获得中间体信息，该法既能研究自由基中间体，也能研究非自由基中间体，便于研究快反应和跟踪动态过程，用该法对辣根过氧化酶催经过氧化氢氧化去甲肾上腺素的中间过程进行了研究。     

Direct electrochemistry-based hydrogen peroxide biosensor formed from single-layer graphene nanoplatelet-enzyme composite film

A novel electrochemical sensing system for direct electrochemistry-based hydrogen peroxide biosensor was developed that relied on the virtues of excellent biocompatibility, conductivity and high sensitivity to the local perturbations of single-layer graphene nanoplatelet (SLGnP). To demonstrate the concept, the horseradish peroxidase (HRP) enzyme was selected as a model to form the SLGnP-TPA (tetrasodium 1,3,6,8-pyrenetetrasulfonic acid)-HRP composite film. The single-layer graphene composite film displayed a pair of well-defined and good reversible cyclic voltammetric peak for Fe(III)/Fe(II) redox couple of HRP, reflecting the enhancement for the direct electron transfer between the enzyme and the electrode surface. Analysis using electrochemical impedance spectroscopy (EIS) revealed that electrostatic attractions existed between graphene monolayers and enzyme molecules. The intimate graphene and enzyme interaction was also observed using scanning electron microscopy (SEM), which resulted in the special properties of the composite film. Ultraviolet visible spectroscopy (UV-vis) indicated the enzyme in the composite film retained its secondary structure similar to the native state. The composite film demonstrated excellent electrochemical responses for the electrocatalytic reduction of hydrogen peroxide (H₂O₂), thus suggesting its great potential applications in direct electrochemistry-based biosensors.     

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.