Amperometric hydrogen peroxide biosensor based on a glassy carbon electrode modified with polythionine and gold nanoparticles

We report on a novel hydrogen peroxide biosensor that was fabricated by the layer-by-layer deposition method. Thionine was first deposited on a glassy carbon electrode by two-step electropolymerization to form a positively charged surface. The negatively charged gold nanoparticles and positively charged horseradish peroxidase were then immobilized onto the electrode via electrostatic adsorption. The sequential deposition process was characterized using electrochemical impedance spectroscopy by monitoring the impedance change of the electrode surface during the construction process. The electrochemical behaviour of the modified electrode and its response to hydrogen peroxide were studied by cyclic voltammetry. The effects of the experimental variables on the amperometric determination of H₂O₂ such as solution pH and applied potential were investigated for optimum analytical performance. Under the optimized conditions, the biosensor exhibited linear response to H₂O₂ in the concentration ranges from 0.20 to 1.6?mM and 1.6 to 4.0?mM, with a detection limit of 0.067?mM (at an S/N of 3). In addition, the stability and reproducibility of this biosensor was also evaluated and gave satisfactory results.

Electrodeposition of gold nanoparticles on indium/tin oxide electrode for fabrication of a disposable hydrogen peroxide biosensor

A novel disposable third-generation hydrogen peroxide (H₂O₂) biosensor based on horseradish peroxidase (HRP) immobilized on the gold nanoparticles (AuNPs) electrodeposited indium tin oxide (ITO) electrode is investigated. The AuNPs deposited on ITO electrode were characterized by UV-vis, SEM, and electrochemical methods. The AuNPs attached on the ITO electrode surface with quasi-spherical shape and the average size of diameters was about 25 nm with a quite symmetric distribution. The direct electron chemistry of HRP was realized, and the biosensor exhibited excellent performances for the reduction of H₂O₂. The amperometric response to H₂O₂ shows a linear relation in the range from 8.0 μmol L⁻¹ to 3.0 mmol L⁻¹ and a detection limit of 2 μmol L⁻¹ (S/N = 3). The value of HRP immobilized on the electrode surface was found to be 0.4 mmol L⁻¹. The biosensor indicates excellent reproducibility, high selectivity and long-term stability.     

Horseradish peroxidase-based organic-phase enzyme electrode

An organic-phase enzyme electrode (OPEE) based on horseradish peroxidase (HRP) immobilized within Nafion on spectroscopic graphite was investigated in acetonitrile. The amperometric electrode response to hydrogen peroxide and cumene hydroperoxide present was found to be the result of the reduction of oxygen, produced upon enzymatic decomposition of both hydroperoxides (i.e., by the catalase-like activity of HRP). The electrode response was found to depend linearly on the hydroperoxide concentration up to 700 M within the range of potentials from –200 to –400 mV (versus Ag|AgCl). Detection limits of approximately 45 M for H₂O₂ and 100 M for cumene hydroperoxide were determined under the selected experimental conditions. Nernstian dependence (the open circuit voltage of HRP-based electrode versus logarithm of H₂O₂ concentration) was obtained between 0.2 and 2.0 mM, with a slope of approximately 23 mV per logarithmic unit, suggesting a catalase-like, two-electron disproportionation of the substrate in acetonitrile.     

Dendritic Silver/Silicon Dioxide Nanocomposite Modified Electrodes for Electrochemical Sensing of Hydrogen Peroxide

A novel biosensor for hydrogen peroxide was prepared by immobilizing horseradish peroxidase (HPR) on newly synthesized dendritic silver/silicon dioxide nanocomposites, which were coated on a glassy carbon electrode. The modified electrode was characterized with XPS, SEM, and electrochemical methods. This biosensor showed a very fast amperometric response to hydrogen peroxide with a linear range from 0.7 to 120 μM, a limit of detection of 0.05 μM and a sensitivity of 1.02 mA mM^?1 cm^?2. The Michaelis‐Menten constant of the immobilized HRP was estimated to be 0.21 mM, indicating a high affinity of the HRP to H₂O₂ without loss of enzymatic activity. The preparation of the proposed biosensor was convenient, and it showed high sensitivity and good stability.     

Electrochemistry and electrocatalytic properties of mixed assemblies of horseradish peroxidase, poly(diallyl dimethylammonium chloride) and gold nanoparticles on a glassy carbon electrode

Poly(diallyldimethylammonium chloride), gold nanoparticles (Au-NPs; 2–5 nm) and horseradish peroxidase (HRP) were alternately self-assembled on a glassy carbon electrode. Due to the conducting wire effect of the Au-NPs, the HRP undergoes direct electrochemistry with a small peak-to-peak separation of 33 mV and a formal potential of ?0.370 V (versus SCE). The reaction involves a single electron transfer coupled to a one-proton transfer reaction. Electrochemical impedance spectroscopy and cyclic voltammetry also were applied to characterize the self-assembly process and to study the electrochemical behavior of the immobilized HRP. Its good electrocatalytic response to the reduction of hydrogen peroxide resulted in a novel third-generation biosensor for this species.     

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.     

Fluorometric determination of hydrogen peroxide with peroxidase and 1-methyl-1,2,3,4-tetrahydro-β-carboline-3-carboxylic acid

Summary The possibility of the fluorometric determination of hydrogen peroxide using various tetrahydro--carbolines as hydrogen-donating substrates for horseradish peroxidase was examined. A simple and sensitive method has been developed by using 1-methyl-1,2,3,4-tetrahydro--carboline-3-carboxylic acid as a substrate. The fluorescence intensity was proportional to the hydrogen peroxide concentration in the range of 0.05–1 mol/l (30–600 pmol/tube) in the sample solution with a relative standard deviation of 3.8% (0.1 mol/l).     

Synthesis of multi-branched gold nanoparticles by reduction of tetrachloroauric acid with Tris base, and their application to SERS and cellular imaging

A biosensor for hydrogen peroxide was constructed by immobilizing horseradish peroxidase on chitosan-wrapped NiFe₂O₄ nanoparticles on a glassy carbon electrode (GCE). The electron mediator carboxyferrocene was also immobilized on the surface of the GCE. UV?Cvis spectra, Fourier transform IR spectra, scanning electron microscopy, and electrochemical impedance spectra were acquired to characterize the biosensor. The experimental conditions were studied and optimized. The biosensor responds linearly to H₂O₂ in the range from 1.0?×?10^?5 to 2.0?×?10^?3?M and with a detection limit of 2.0?×?10^?6?M (at S/N?=?3).

Estimation of hydrogen peroxide formed and residual ascorbate in the copper catalysed oxidation reaction of ascorbate at pH 7

A fluorescent method has been developed for determination of ascorbic acid concentrations. The method involves treatment of the ascorbic acid with Cu(II) and treating the hydrogen peroxide formed with horseradish peroxidase in the presence ofp-hydroxyphenylacetic acid, to form a fluorescentp-hydroxyphenylacetic acid dimer. The reaction is suitable for analysis of concentrations in the range from 50 M to 4 mM ascorbic acid solutions and can be used for analysis of pharmaceutical preparations but is unsuitable for analysis of ascorbate in preparations derived from natural sources. By using a slight modification of the analytical technique it is possible to measure the amount of hydrogen peroxide formed and the residual concentrations of ascorbic acid in solutions treated with varying amounts of copper ion.Abbreviations CySH cysteine - EDTA ethylenediamine tetraacetic acid - GSH reduce glutathione - HPAA hydroxyphenylacetic - HPLC high performance liquid chromatography - HRP horseradish peroxidase - TCA trichloracetic acid     

Amperometric biosensor for hydrogen peroxide based on the co-electrodeposition of horseradish peroxidase and methylene blue on an ITO electrode modified with an anodic aluminum oxide template

We report on a nano-array sensor for hydrogen peroxide (H₂O₂) that is based on a nanoporous anodic aluminum oxide template. This was used as a matrix for the co-immobilization of horseradish peroxidase (HRP) and methylene blue (MB) on the surface of an indium tin oxide electrode. The immobilized HRP retained its natural activity and MB is capable of efficiently shuttle electrons between HRP and the electrode. The new electrode was characterized by SEM and electrochemical methods. It exhibits fast response, long-term stability, high sensitivity and good selectivity to H₂O₂. Under optimized conditions, it linearly responds to H₂O₂ in the concentration range from 1.0?μM to 26?mM, with a detection limit of 0.21?μM (at S/N?=?3).

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.     

Electrochemical properties of Nile Blue covalently immobilized on self-assembled thiol-monolayer modified gold electrodes.

A monolayer of Nile Blue (NB) has been covalently immobilized on the self-assembled thiol-monolayer modified gold electrode. Cyclic voltammograms indicated a stable and reverse redox process of NB bonded on the electrode surface. The mechanisms of redox process coupling with proton transfer were proposed. The NB-modified electrode showed excellent electrocatalytic activity toward Nicotinamide adenine dinucleotide (NADH) oxidation and horseradish peroxidase (HRP) reduction. A hydrogen peroxide biosensor based on NB as a mediator has been demonstrated.     

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.     

Titrimetric determination of thiourea and silver with a picrate ion selective electrode

Summary A method has been developed for the semiautomatic potentiometric titration of thiourea with silver nitrate and of silver with thiourea, in the presence of picrate ions, using a picrate ion selective electrode. Thiourea in the range 15–1500g and silver in the range 200–1800g were determined with relative errors and relative standard deviation of about 1%.

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Zusammenfassung Eine halbautomatische potentiometrische Titrationsmethode für Thioharnstoff mit Silbernitrat bzw. umgekehrt in Gegenwart von Pikrationen mit Hilfe einer selektiven Pikratelektrode wurde entwickelt. 15–1500g Thioharnstoff bzw. 200–1800g Silber wurden mit einem relativen Fehler und einer relativen Standardabweichung von etwa ±1% bestimmt.

     

基于金纳米棒-辣根过氧化物酶协同效应的过氧化苯甲酰生物传感器

将金纳米棒(AuNRs)和辣根过氧化物酶(HRP)以自组装的方式依次修饰到Au电极表面,构建了响应过氧化苯甲酰(BPO)生物传感器。采用循环伏安法和电流时间法研究了传感器的电化学性质和最佳工作条件;由于HRP/AuNRs复合膜的协同效应,电极性能得到显著改善。在最佳工作条件下(工作电压-0.02V,pH 7的磷酸盐缓冲体系),BPO浓度在5.0×10-6～1.0×10-4 mol/L范围内与电极的电流响应值呈良好的线性关系,线性回归方程为:i(μA)=12.6796C(mmol/L)+0.2406,R=0.9993。电极的检出限为8.5×10-7 mol/L。电极用于面粉中BPO的测定,平行测定6次,平均回收率为97.9%～100.1%,相对标准偏差(RSD)为0.5%～2.1%。本方法用于商品面粉中的BPO测定,取得满意结果。     

A Mediator‐Free Bienzyme Amperometric Biosensor Based on Horseradish Peroxidase and Glucose Oxidase Immobilized on Carbon Nanotube Modified Electrode

Multiwalled carbon nanotube (CNT) modified glassy carbon electrode immobilized with horseradish peroxidase (HRP) in Nafion coating showed direct electron transfer between HRP enzyme and the CNT‐modified electrode. A mediator‐free bienzyme glucose biosensor based on horseradish peroxidase and glucose oxidase was constructed. The bienzyme biosensor exhibited a high sensitivity for glucose detection at zero applied potential.     

Immobilized and solid-state reagent systems for luminol chemiluminescence in flow systems

To facilitate the application of luminol chemiluminescence in analysis, several approaches are investigated to provide the reagents in immobilized or solid-state format. The approaches are demonstrated with flow injection systems. Luminol is covalently bound or adsorbed to the surface of small support particles and packed into flow-through reactor/detector cells. The catalyst can be either covalently immobilized heme-containing species or a positively-biased electrode in an electrochemical cell. Peroxide can be obtained electrochemically at a negatively-biased electrode. These immobilized reagent systems can be combined to yield single-channel flow systems for determination of hydrogen peroxide (0.15M detection limit) or luminol (0.1 nM detection limit).     

Enzyme biosensor based on the immobilization of HRP on SiO<Subscript>2</Subscript>/BSA/Au composite nanoparticles

In this work, an enzyme biosensor based on the immobilization of horseradish peroxidase (HRP) on SiO₂/BSA/Au/thionine/nafion-modified gold electrode was fabricated successfully. Firstly, nafion was dropped on the surface of the gold electrode to form a nafion film followed by chemisorption of thionine (Thi) as an electron mediator via the ion-exchange interaction between the Thi and nafion. Subsequently, the SiO₂/BSA/Au composite nanoparticles were assembled onto Thi film through the covalent bounding with the amino groups of Thi. Finally, HRP was immobilized on the SiO₂/BSA/Au composite nanoparticles due to the covalent conjugation to construct an enzyme biosensor. The surface topographies of the SiO₂/BSA/Au composite nanoparticles were investigated by using scanning electronic microscopy. The stepwise self-assemble procedure of the biosensor was further characterized by means of cyclic voltammetry and chronoamperometry. The enzyme biosensor showed high sensitivity, good stability and selectivity, a wide linear response to hydrogen peroxide (H₂O₂) in the range of 8.0 × 10^-6 ∼ 3.72 × 10^-3 mol/L, with a detection limit of 2.0 × 10^-6 mol/L. The Michaelies-Menten constant K_M^app K_M^{app} value was estimated to be 2.3 mM.     

An ultrasensitive hydrogen peroxide biosensor based on electrocatalytic synergy of graphene–gold nanocomposite, CdTe–CdS core–shell quantum dots and gold nanoparticles

We first reported an ultrasensitive hydrogen peroxide biosensor in this work. The biosensor was fabricated by coating graphene–gold nanocomposite (G–AuNP), CdTe–CdS core–shell quantum dots (CdTe–CdS), gold nanoparticles (AuNPs) and horseradish peroxidase (HRP) in sequence on the surface of gold electrode (GE). Cyclic voltammetry and differential pulse voltammetry were used to investigate electrochemical performances of the biosensor. Since promising electrocatalytic synergy of G–AuNP, CdTe–CdS and AuNPs towards hydrogen peroxide was achieved, the biosensor displayed a high sensitivity, low detection limit (S/N = 3) (3.2 × 10⁻¹¹ M), wide calibration range (from 1 × 10⁻¹⁰ M to 1.2 × 10⁻⁸ M) and good long-term stability (20 weeks). Moreover, the effects of omitting G–AuNP, CdTe–CdS and AuNP were also examined. It was found that sensitivity of the biosensor is more 11-fold better if G–AuNP, CdTe–CdS and AuNPs are used. This could be ascribed to improvement of the conductivity between graphene nanosheets in the G–AuNP due to introduction of the AuNPs, ultrafast charge transfer from CdTe–CdS to the graphene sheets and AuNP due to unique electrochemical properties of the CdTe–CdS, and good biocompatibility of the AuNPs for horseradish peroxidase. The biosensor is of best sensitivity in all hydrogen peroxide biosensors based on graphene and its composites up to now.     

Determination of tetraethyllead and tetramethyllead using high performance liquid chromatography and electrochemical detection

Summary A new method for the separation of tetramethyllead (TML) and tetraethyllead (TEL) was developed using high-performance liquid chromatography. The electrochemical detection was examined with different electrodes. Amperometric and pulse-amperometric techniques were investigated and the optimal working potential for each electrode was determined. Linearity for the glassy carbon electrode was observed between 350 ng and 30 g; the detection limit is 310 ng (TML) resp. 340 ng (TEL). In case of the mercury gold electrode the linearity range was 300 g–3 g and the detection limit 1.5 m (TML) resp. 1.7 g (TEL).