Amperometric methylene blue-mediated sensor highly sensitive to hydrogen peroxide based on a composite membrane of regenerated silk fibroin and poly-vinyl alcohol as immobilization matrix for horseradish peroxidase

Horseradish peroxidase (HRP) was effectively entrapped in a novel composite membrane of poly-vinyl alcohol and regenerated silk fibroin, and IR was employed to provide a useful insight into the structure of the composite membrane. A methylene bluemediated sensor highly sensitive to hydrogen peroxide was constructed, which was based on the immobilization of HRP in the composite membrane. Cyclic voltammetry and amperometric measurement were utilized to demonstrate the feasibility of electron communication between immobilized HRP and a glassy carbon electrode in the bioelectrocatalytic reduction of hydrogen peroxide via methylene blue. Performance and characteristics of the sensor were evaluated with regard to response time, detection limit, selectivity, and dependence on temperature and pH as well as operating and storage stability. The sensor possesses a variety of characteristics including high sensitivity, rapid response time and a low detection limit of 0.1 μmol/L.     

Amperometric methylene blue-mediated sensor highly sensitive to hydrogen peroxide based on a composite membrane of regenerated silk fibroin and poly-vinyl alcohol as immobilization matrix for horseradish peroxidase

 Horseradish peroxidase (HRP) was effectively entrapped in a novel composite membrane of poly-vinyl alcohol and regenerated silk fibroin, and IR was employed to provide a useful insight into the structure of the composite membrane. A methylene blue-mediated sensor highly sensitive to hydrogen peroxide was constructed, which was based on the immobilization of HRP in the composite membrane. Cyclic voltammetry and amperometric measurement were utilized to demonstrate the feasibility of electron communication between immobilized HRP and a glassy carbon electrode in the bioelectrocatalytic reduction of hydrogen peroxide via methylene blue. Performance and characteristics of the sensor were evaluated with regard to response time, detection limit, selectivity, and dependence on temperature and pH as well as operating and storage stability. The sensor possesses a variety of characteristics including high sensitivity, rapid response time and a low detection limit of 0.1 μmol/L. Received: 10 January 1996/Revised: 20 March 1996/Accepted: 25 March 1996     

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.     

An amperometric biosensor based on the coimmobilization of horseradish peroxidase and methylene blue on a beta-type zeolite modified electrode

A new biosensor for the amperometric detection of hydrogen peroxide was developed based on the coimmobilization of horseradish peroxidase (HRP) and methylene blue on a beta-type zeolite modified glassy carbon electrode without the commonly used bovine serum albumin-glutaraldehyde. The intermolecular interaction between enzyme and zeolite matrix was investigated using FT-IR. The cyclic voltammetry and amperometric measurement demonstrated that methylene blue co-immobilized with HRP in this way displayed good stability and could efficiently transfer electrons between immobilized HRP and the electrode. The sensor responded rapidly to H2O2 in the linear range from 2.5 x 10(-6) to 4.0 x 10(-3) M with a detection limit of 0.3 microM. The sensor was stable in continuous operation.     

Reagentless amperometric biosensor highly sensitive to hydrogen peroxide based on the incorporation of Meldola Blue, fumed-silica and horseradish peroxidase into carbon paste

 A reagentless amperometric sensor highly sensitive to H₂O₂ has been prepared by incorporating fumed silica, horseradish peroxidase (HRP) and Meldola Blue into carbon paste. The efficient mediating ability to shift electrons between HRP and the carbon paste electrode via Meldola Blue was investigated by cyclic voltammetric and amperometric measurements. Reproducibility, response time, detection limit, selectivity and effects of applied potential, temperature and pH on the response of the sensor are reported. The high sensitivity of the sensor with a detection limit of 0.1 μmol/l arose from the high efficiency of the bioelectrocatalytic reduction of hydrogen peroxide via HRP and Meldola Blue. The dependence of the Michaelis-Menten constant on the applied potential and the mediator concentration has been investigated and the results are presented. Received: 20 December 1995/Revised: 13 March 1996/Accepted: 16 March 1996     

Poly(o-aminophenol)-modified bienzyme carbon paste electrode for the detection of uric acid

A reagentless uric acid selective biosensor constructed by immobilising uricase and horseradish peroxidase (HRP) in carbon paste without the addition of an electron transfer mediator is described. The response of the electrode is based on the enzymatic reduction of hydrogen peroxide in the presence of uric acid. Uricase and HRP were dispersed in the carbon paste and the optimum paste mixture was determined. Poly(o-aminophenol) was electropolymerised at the working surface area of the electrode acting as a conducting polymer layer. Cyclic voltammetry was used to characterise the permselective characteristics of the polymer layer. At an applied potential of 50 mV vs. Ag/AgCl, a linear response was obtained up to 1 x 10(-4) M, with a limit of detection of 3 x 10(-6) M. The sensor had a response time of 37 s. a calibration precision of 2.2% (n = 4) and an estimated sample frequency of 20 h(-1). Responses to the analyte of interest were pH dependent. The sensor was incorporated into a flow injection system for the qualification of uric acid in human serum. Results compared favourably with a standard spectrophotometric method.     

An Amperometric Biosensor Based on the Coimmobilization of Horseradish Peroxidase and Methylene Blue on a Carbon Nanotubes Modified Electrode

A novel hydrogen peroxide biosensor has been constructed based on the characteristics of the carbon nanotube. The multiwall carbon nanotube (MWNT) was used as a coimmobilization matrix to incorporate horseradish peroxidase (HRP) and electron transfer mediator methylene blue (MB) onto a glassy carbon electrode surface. Cyclic voltammetry and amperometric measurements were employed to demonstrate the feasibility of methylene blue as an electron carrier between the immobilized peroxidase and the surface of glassy carbon electrode. The amperometric response of this resulting biosensor to H₂O₂ shows a linear relation in the range from 4 μM to 2 mM. The detection limit was 1 μM when the signal to noise ratio is 3. The presence of dopamine and ascorbic acid hardly affects the sensitive determination of H₂O₂. This biosensor also possesses very good stability and reproducibility.     

基于聚乙烯醇离子液体负载HRP修饰石墨烯/纳米金复合膜的过氧化氢生物传感器

以石墨烯/纳米金修饰玻碳电极为基底, 用聚乙烯醇与离子液体复合物将辣根过氧化物酶固定于电极表面, 制备了过氧化氢生物传感器. 结果表明, 在0.1 mol/L HAc-NaAc+0.1mol/L KCl(pH=6.5)中, H₂O₂的氧化峰电流与其浓度在9.55×10^-6~6.01×10^-3 mol/L间呈良好线性关系, 检出限(3S/N)为3.3×10^-7 mol/L. 用标准加入法做回收实验, 回收率在93.4%~100.5%之间. 该传感器对H₂O₂具有较高的灵敏度和较低的检测限, 稳定性和重现性良好, 使用寿命较长, 且制作成本低, 可多次重复使用.     

An amperometric biosensor based on the coimmobilization of horseradish peroxidase and methylene blue on a β-type zeolite modified electrode

A new biosensor for the amperometric detection of hydrogen peroxide was developed based on the co-immobilization of horseradish peroxidase (HRP) and methylene blue on a β-type zeolite modified glassy carbon electrode without the commonly used bovine serum albumin-glutaraldehyde. The intermolecular interaction between enzyme and zeolite matrix was investigated using FT-IR. The cyclic voltammetry and amperometric measurement demonstrated that methylene blue co-immobilized with HRP in this way displayed good stability and could efficiently transfer electrons between immobilized HRP and the electrode. The sensor responded rapidly to H₂O₂ in the linear range from 2.5 × 10^–6 to 4.0 × 10^–3 M with a detection limit of 0.3 μM. The sensor was stable in continuous operation.     

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.     

A simplified enzyme-based fiber optic sensor for hydrogen peroxide and oxidase substrates

A simplified enzyme-based fiber optic sensor system has been developed for selective determination of hydrogen peroxide. Horseradish peroxidase (HRP) is immobilized on bovine albumin matrix with glutaraldehyde. A new fluorimetric substrate, thiamine is used to indicate the sensing process. Under optimized condition the measuring range of sensor is up to 1 x 10(-4)M hydrogen peroxide with a limit of detection of 5 x 10(-7)M in a 5 min response period. It can be easily incorporated in multienzyme sensors for biochemical substances which produce hydrogen peroxide under catalytic oxidation by their oxidase. This possibility has been tested for the determination of uric acid, D-amino acid, L-amino acid, glucose cholesterol, choline and acetylcholine, respectively, using a membrane with co-immobilized oxidase and horseradish peroxidase.     

Electrochemical characteristics of a platinum electrode modified with a matrix of polyvinyl butyral and colloidal Ag containing immobilized horseradish peroxidase

A new hydrogen peroxide biosensor was constructed, which consisted of a platinum electrode modified by a matrix of polyvinyl butyral (PVB) and nanometer-sized Ag colloid containing immobilized horseradish peroxidase (HRP), and using Co(bpy)₃³⁺ as mediator in the hydrogen peroxide solution. The electrochemical characteristics of the biosensor were studied by cyclic voltammetry and chronoamperometry. The modified process was characterized by electrochemical impedance spectroscopy and cyclic voltammetry. The HRP immobilized on colloidal Ag was stable and retained its biological activity. The sensor displays excellent electrocatalytic response to the reduction of H₂O₂. Analytical parameters such as pH and temperature were also studied. Linear calibration for H₂O₂ was obtained in the range of 1×10^–5 to 1×10^–2 M under optimized conditions. The sensor was highly sensitive to H₂O₂, with a detection limit of 2×10^–6 M, and the sensor achieved 95% of steady-state current within 10 s. The sensor exhibited high sensitivity, selectivity and stability.     

基于纳米金自组装膜固定辣根过氧化物酶的生物传感研究

将辣根过氧化物酶(HRP)通过纳米技术和自组装技术固定于电极表面,制得了酶修饰电极.纳米金与HRP形成了静电复合物并高效地保持了HRP的生物活性,以对苯二酚作为电子媒介体,差示脉冲伏安法(DPV)研究生物酶电极测定H2O2的线性范围为5.0×10-6～1.0×10-3 mol/L,检测限为2.5×10-6 mol/L,线性方程为△I=0.34765+4.05553CH2O2(mM).酶电极的表观米氏常数(K(app))为0.0675 mmol/L.实验同时证明该生物酶电极具有良好的稳定性和使用寿命.     

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).

Amperometric Biosensor for Hydrogen Peroxide Based on Electrodeposited Sub-micrometer Gold Modified Glassy Carbon Electrode

IntroductionAmperometricbiosensorofhydrogenperoxideisofpracticalimportancebecauseofitswideapplicationsinchemical,biological,clinical,environmentalandmanyotherfields.Forimprovementofsensor抯quality,vari-ouskindsofchemicalmodificationmethodshavebeendevelopedforreducingredoxoverpotentialsofH2O2atelectrodesurfaces,increasingthedetectionsensitivity,linearrange,stabilityandlivetime.Ithasbeenshownthattheuseofsub-micrometersizedmetalparticlessuchasPt-blackcansignificantlyimprovethequalityofthebiosens…     

基于二氧化锆和碳纳米管协同作用的过氧化氢生物传感器

过氧化氢作为一种重要的化工产品在纺织行业、化工行业、造纸工业、环保行业、电子行业、食品卫生行业及其他领域得到广泛的应用~([1]).     

基于等离子体聚合膜固定酶的H2O2生物传感器

以玻碳电极为基础电极,用微波等离子体技术聚合沉积聚乙二胺等离子体膜,使之形成带氨基功能团的表面,再通过戊二醛交联共价固定辣根过氧化物酶,制得H2O2生物传感器.探讨了等离子体聚合膜的形成条件(如放电功率、单体流速、单体气压和聚合时间),讨论了工作电位、介体浓度和pH值对传感器响应的影响.此外,用红外光谱对等离子体聚合膜进行了表征.该传感器在5×10-7～1.1×10-3mol/LH2O2浓度范围内有线性响应,最低检测限为0.3μmol/L.将此传感器用于实际试样回收率的测定,结果良好.     

Nanomolar hydrogen peroxide detection using horseradish peroxidase covalently linked to undoped nanocrystalline diamond surfaces

In this article, we report on the low-level detection of hydrogen peroxide, a key player in the redox signaling pathway and a toxic product in the cellular system, using a colorimetric solution assay. Amine-terminated undoped nanocrystalline diamond thin films were grown on glass using a linear-antenna microwave plasma CVD process. The diamond surface consists mainly of -NH(2) termination. The aminated diamond surface was decorated with horseradish peroxidase (HRP) enzyme using carbodiimide coupling chemistry. The success of the HRP immobilization was confirmed by X-ray photoelectron spectroscopy (XPS). The enzymatic activity of immobilized HRP was determined with a colorimetric test based on the HRP-catalyzed oxidation of 2,2'-azino-bis(3-ethylbenzothiazoline-6-sufonic acid (ABTS) in the presence of hydrogen peroxide. The surface coverage of active HRP was estimated to be Γ = 7.3 × 10(13) molecules cm(-2). The use of the functionalized diamond surface as an optical sensor for the detection of hydrogen peroxide with a detection limit of 35 nM was demonstrated.     

Amperometric biosensors based on alumina nanoparticles-chitosan-horseradish peroxidase nanobiocomposites for the determination of phenolic compounds

A horseradish peroxidase (HRP) biosensor based on alumina (Al(2)O(3)) nanoparticles-chitosan (CHIT) nanocomposites was developed for the detection of phenolic compounds. UV-Vis spectra and Fourier transform infrared spectra showed that HRP retained its original structure on the Al(2)O(3)/CHIT film. The surface morphologies of the composite films were characterized by scanning electron microscopy. Cyclic voltammetry and amperometry were used to study the proposed electrochemical biosensor. Optimization of the experimental parameters was performed with regard to pH, applied electrode potential and the concentration of hydrogen peroxide. The linear range, sensitivity and detection limit of the biosensor were investigated for eight phenolic compounds. In particular, the linearity of the biosensor for the detection of hydroquinone was obtained from 5 × 10(-9) M to 7 × 10(-5) M with a detection limit of 1 nM (based on the S/N = 3). The optimized biosensor for hydroquinone determination displayed a high sensitivity of 518.4 nA μM(-1) with a response time of ～5 s.     

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.