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.     

Application of tubular tetrapod magnesium oxide in a biosensor for hydrogen peroxide

Tubular tetrapod magnesium oxide (tt-MgO) can be synthesized by thermal evaporation of Mg metal powder with a pre-grown tetrapod ZnO template. The morphology and structure of the tt-MgO were characterized by X-ray diffraction, scanning electron microscopy and transmission electron microscopy. A composite prepared from tt-MgO, nafion and horseradish peroxidase was employed to modify a gold electrode to result in an electrochemical biosensor for hydrogen peroxide that displays excellent sensitivity and rapid response in the presence of hydroquinone as a mediator. Its sensitivity is 335.4 μA mM^-1 cm^-2, its response is linear in the range from 1.0 to 450 μM, and the detection limit is 0.3 μM. These results demonstrate that tt-MgO provides a promising material for the designs of biosensors.

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.     

基于石墨烯-壳聚糖-辣根过氧化物酶的H2O2生物传感器的研制

制备了石墨烯-壳聚糖(GR-CS)纳米复合材料,并将之与辣根过氧化物酶(HRP)混合,构建了基于石墨烯-壳聚糖-辣根过氧化物酶的生物传感器(GR-CS-HRP/GC)。探针及循环伏安研究表明,该界面具有优异的电子传导能力、较大的比表面积和良好的生物相容性,对H2O2的还原显示出较好的电催化活性,在工作电位为-0.2 V,0.05 mol/L的磷酸盐缓冲盐溶液(PBS,pH 6.8)中,该酶传感器对过氧化氢响应灵敏度高,检测范围宽,测定H2O2的线性范围为5.0×10-7～2×10-3mol/L(相关系数为0.998)。检出限为2.0×10-7mol/L(S/N=3)。并且表现出良好的稳定性和高选择性。该电极用于实际样品中H2O2的测定,结果令人满意。     

Continuous measurement of glutamate and hydrogen peroxide using a microfabricated biosensor for studying the neurotoxicity of tributyltin.

We first measured the effects of trace levels of an endocrine disruptor, tributyltin (TBT), on the secretion response from nerve cells using a microfabricated biosensor designed for the continuous measurement of L-glutamate and hydrogen peroxide. We observed higher and long-lasting glutamate and hydrogen peroxide concentrations from the cells when cultured rat cortical neurons were exposed to TBT. Glutamate and hydrogen peroxide release was induced even when we reduced the TBT concentration to 10 nM. This concentration is about two orders of magnitude lower than the concentration that induced apoptosis-like cell death. We also report on the effects of NMDA and non-NMDA receptor antagonists, which can help us to understand the mechanism of TBT neurotoxicity.     

Ferrocene branched chitosan for the construction of a reagentless amperometric hydrogen peroxide biosensor

Chitosan was chemically branched with ferrocene moieties and further used as a support for the immobilization of horseradish peroxidase on a glassy carbon electrode. The reagentless biosensor device showed a linear amperometric response toward hydrogen peroxide concentrations between 35 x 10(-6) M and 2.0 x 10(-3) M. The biosensor reached 95% of the steady-state current in about 20 s and its sensitivity was 28.4 x 10(-3) microA x M(-1). The enzyme electrode retained 94% of its initial activity after 2 weeks of storage at 4 degrees C in 50 x 10(-3) M sodium phosphate buffer at pH 7.0.     

Amperometric enzyme biosensor for hydrogen peroxide via Ugi multicomponent reaction

A novel strategy based on the Ugi multicomponent reaction was employed for immobilizing horseradish peroxidase on sodium alginate-coated gold electrode. The electrode was employed for constructing an amperometric biosensor device using 1 mM hydroquinone as electrochemical mediator. The electrode showed linear response (poised at −300 mV vs Ag/AgCl) toward H₂O₂ concentration between 70 μM and 8.8 mM at pH 7.0. The biosensor reached 95% of steady-state current in about 12 s and its sensitivity was 33.8 mA/M cm². The electrode retained full initial activity after 30 days of storage at 4 °C in 50 mM sodium phosphate buffer, pH 7.0.     

An unmediated hydrogen peroxide biosensor based on hemoglobin incorporated in a montmorillonite membrane

Hemoglobin was incorporated in a montmorillonite membrane. Electrochemical and spectroscopic studies revealed that the electron transfer reactivity and peroxidase activity of the protein were both enhanced. Nevertheless, its structure was still maintained native-like in the membrane. An unmediated hydrogen peroxide biosensor was accordingly prepared. The calibration plot of this H202 sensor was linear in the range of 1.0 x 10(-6)-6.0 x 10(-4) mol L(-1). The relative standard deviation was 3.1% for six successive determinations at a concentration of 1.0 x 10(-4) mol L(-1). The detection limit was 6.0 x 10(-7) mol L(-1). Possible interferences in real sample analyses are discussed.     

基于自组装L-半胱氨酸与纳米金的H2O2生物传感器

通过在金电极表面自组装L-半胱氨酸,再分别吸附纳米金与辣根过氧化物酶(HRP)的方法,成功的制备了H2O2生物传感器.采用循环伏安法考察了传感器的电化学特性,电极对H2O2在浓度为2.1×10-6～3.6×10-3 mol/L的范围内呈线性,检出限为8.9×10-7 mol/L (S/N＝3).该传感器具有稳定性好,线性范围宽,检出限低等优点,同时具有一定的抗干扰能力.     

Detection of hydrogen peroxide produced at a liquid/liquid interface using scanning electrochemical microscopy

Scanning electrochemical microscopy (SECM) was used to monitor in situ hydrogen peroxide (H₂O₂) produced at a polarized water/1,2-dichloroethane (DCE) interface. The water/DCE interface was formed between a DCE droplet containing decamethylferrocene (DMFc) supported on a solid electrode and an acidic aqueous solution. H₂O₂ was generated by reducing oxygen with DMFc at the water/DCE interface, and was detected with a SECM tip positioned in the vicinity of the interface using a substrate generation/tip collection mode. This work shows unambiguously how the H₂O₂ generation depends on the polarization of the liquid/liquid interface, and how proton-coupled electron transfer reactions can be controlled at liquid/liquid interfaces.     

嗜中性粒细胞中过氧化氢及其释放过程的电化学检测

活性氧（ROS）是细胞代谢的重要产物之一,其浓度与多种生理、病理现象密切相关;H2O2是ROS的重要代表,对细胞内H2O2及其释放过程的测定有助于理解相关疾病的形成机制,因而具有重要的意义.本文报道一种检测细胞内H2O2浓度、监测其生成和释放过程的电化学方法,该方法利用固定在天然矿物凹土（attapulgite,全称凹凸棒石黏土）表面的血红蛋白（Hb）对H2O2还原的电催化作用实现H2O2的定量测定.将Hb固定到凹土表面制备了Hb-凹土（Hb-Atta）纳米复合材料,用透射电子显微镜（TEM）、原子力显微镜（AFM）、红外光谱（FTIR）、紫外-可见光谱（UV-vis）等对其进行了表征;将该复合物修饰到玻碳（GC）电极表面制成了Hb-Atta/GC电极,用伏安法考察了Hb在凹土表面的直接电子转移性能,结果表明,Hb能进行有效的直接电子转移反应,其伏安曲线上呈现出一对良好的氧化还原峰,式量电位（E0′）约为-350mV（vs.SCE,50mV/s）;进一步的结果表明,Hb-Atta对H2O2的电化学还原具有良好的催化性能,并能用于H2O2的定量测定,具有响应速率快、线性范围宽、检测限低等优点.将Hb-Atta/GC电极应用于嗜中性粒细胞内H2O2的测定,结果表明,该电极不仅能定量地检测细胞内的H2O2含量,还能监测细胞内H2O2生成和释放的过程,而且细胞内其他ROS和电活性物质如OCl-、NO.、ONOO-和抗坏血酸等不产生干扰.本文的结果为测量细胞内H2O2浓度、研究其生成及释放过程动力学以及进一步阐明由H2O2导致的相关疾病机制等提供了一个新的平台,可望在生物电化学、细胞生物学和病理生理学等领域得到应用.     

A novel amperometric hydrogen peroxide biosensor based on electrospun Hb-collagen composite

In this paper, the hemoglobin (Hb)-collagen microbelt modified electrode with three-dimensional configuration was fabricated via the electrospinning method. Direct electron transfer of the Hb immobilized into the electrospun collagen microbelts was greatly facilitated. The apparent heterogeneous electron transfer rate constant (k(s)) was calculated to be 270.6s?1. The electrospun Hb-collagen microbelt modified electrode showed an excellent bioelectrocatalytic activity toward the reduction of H?O?. The amperometric response of the biosensor varied linearly with the H?O? concentration ranging from 5 × 10??molL?1 to 30×10??molL?1, with a detection limit of 0.37 × 10??molL?1 (signal-to-noise ratio of 3). The apparent Michaelis-Menten constant (K(m)(app)) was 77.7 μmolL?1. The established biosensor exhibited fast amperometric response, high sensitivity, good reproducibility and stability.     

Preparation of Pt/polypyrrole-ferrocene hydrogen peroxide sensitive electrode for the use as a biosensor

A polypyrrole electrode with ferrocene mediator is prepared and its sensitivity to hydrogen peroxide is investigated. The polypyrrole is deposited upon a 0.5 cm² Pt plate by the polymerization of pyrrole by scanning the electrode potential between 0.0 and 0.9 V at a scan rate of 50 mV/s. The platinum/polypyrrole-ferrocene (Pt/PPy-Fc) electrode is prepared by adding ferrocene to the coverage medium. The electrode’s sensitivity to hydrogen peroxide is investigated at room temperature using 0.025 M phosphate buffer at pH 7. The working potential is 0.7 V, the concentrations of pyrrole and ferrocene are 0.2 M and 10 mM. Polypyrrole was coated on the electrode surface within 26 cycles. Published in Russian in Elektrokhimiya, 2006, Vol. 42, No. 2, pp. 160–164. The text was submitted by the authors in English.     

Catalase biosensor for the determination of hydrogen peroxide,fluoride and cyanide

The enzyme catalase, which catalyses the decomposition of hydrogen peroxide to oxygen and water, was immobilized in a membrane by entrapping it in polyacryl amide and contacted to a Clark-type oxygen electrode. With the resulting catalase biosensor it was possible to detect the substrate hydrogen peroxide and the inhibitors fluoride and cyanide in phosphate buffer.The sensor was integrated into a flow system. In the concentration range from 5–200 mg/l a linear dependence of the peak height on the hydrogen peroxide concentration was obtained. The average decrease in activity during 30 days of storage at 6 °C was 17%. Fluoride and cyanide could be determined by measuring the inhibition of the enzymatic reaction in the same flow system. The analysis was executed in three steps; namely determination of the original activity by pumping substrate solution, inhibition of the enzyme by pumping inhibitor solution, and determination of the activity after the inhibition.The decrease in activity correlated with the inhibitor concentration of the sample, but a linear dependence was not found. The inhibition of fluoride and cyanide was both reversible, the enzyme membrane could be reactivated completely by pumping substrate solution. The detection limit was 1 mg/l for fluoride and 1.5 mg/l for cyanide.     

碳纳米管/TiO2薄膜固定酶的生物传感器及电催化氧化H2O2的研究

采用酸性溶胶法在碳纳米管上负载纳米TiO2颗粒,并制备了CNT-TiO2薄膜固定辣根过氧化物酶的生物传感器。研究了该生物传感器在磷酸盐缓冲溶液中对H2O2的电催化氧化作用及实验条件的影响。结果表明该生物传感器对H2O2表现出良好的电催化性能,在最佳实验条件下,对H2O2检测的线性范围为:4.2×10-7mol/L~3.2×10-3mol/L。检出限为:7.22×10-8mol/L。     

Polyethyleneimine-AuNPs-copper protoporphyrin nanocomposite: a novel biosensor for sensitive detection of hydrogen peroxide in human serum

Journal of Solid State Electrochemistry - The detection of hydrogen peroxide (H2O2) is essential due to its extensive application in bio-medical and environment. Herein, we synthesized a novel...     

Bilayer lipid membrane biosensor with enhanced stability for amperometric determination of hydrogen peroxide

In this paper, a polydopamine (PDA) film is electropolymerized on the surface of bilayer lipid membrane (BLM) which is immobilized with horseradish peroxidase (HRP). The coverage of the PDA film on HRP/BLM electrode is monitored by electrochemical impedance spectroscopy (EIS). The electrocatalytic reduction of H₂O₂ at the PDA/HRP/BLM electrode is studied by means of cyclic voltammetry (CV). The biosensor has a fast response to H₂O₂ of less than 5 s and an excellent linear relationship is obtained in the concentration range from 2.5 × 10⁻⁷ to 3.1 × 10⁻³ mol L⁻¹, with a detection limit of 1.0 × 10⁻⁷ mol L⁻¹ (S/N = 3). The response current of BLM/HRP/PDA biosensor retains 84% of its original response after being stored in 0.1 mol L⁻¹ pH 7.0 PBS at 4 °C for 3 weeks. The selectivity, repeatability, and storage stability of PDA/HRP/BLM biosensor are greatly enhanced by the coverage of polydopamine film on BLM.     

Amperometric biosensor for hydrogen peroxide based on hemoglobin entrapped in titania sol-gel film

Hemoglobin (Hb) was entrapped in a titania sol-gel matrix and used as a mimetic peroxidase to construct a novel amperometric biosensor for hydrogen peroxide. The Hb entrapped titania sol-gel film was obtained with a vapor deposition method, which simplified the traditional sol-gel process for protein immobilization. The morphologies of both titania sol-gel and the Hb films were characterized using scanning electron microscopy (SEM) and proved to be chemically clean, porous, homogeneous. This matrix provided a biocompatible microenvironment for retaining the native structure and activity of the entrapped Hb and a very low mass transport barrier to the substrates. H₂O₂ could be reduced by the catalysis of the entrapped hemoglobin at −300 mV without any mediator. The reagentless H₂O₂ sensor exhibited a fast response (less than 5 s) and sensitivity as high as 1.29 mA mM⁻¹ cm⁻². The linear range for H₂O₂ determination was from 5.0×10⁻⁷ to 5.4×10⁻⁵ M with a detection limit of 1.2×10⁻⁷ M. The apparent Michaelis-Menten constant of the encapsulated hemoglobin was calculated to be 0.18±0.02 mM. The stability of the biosensor was also evaluated.     

Using novel polysaccharide-silica hybrid material to construct an amperometric biosensor for hydrogen peroxide

A new type of sol-gel organic-inorganic hybrid material was developed and used for the fabrication of an amperometric hydrogen peroxide biosensor. This material was prepared from natural chitosan and recently introduced completely water-soluble precursor, tetrakis(2-hydroxyethyl) orthosilicates (THEOS), by the sol-gel process without the addition of organic solvents and catalysts. The gelation time for the sol-gel transition and dynamic rheological properties of the resultant gel matrix could be modulated by the amount of added THEOS. The structure of the hybrid gel was made up of a network and spherical particles, as confirmed by SEM observation. By electrochemical experiments, it was found that such a hybrid gel matrix could retain the native biocatalytic activity of the entrapped horseradish peroxidase and provide a fast amperometric response to hydrogen peroxide. The linear range for the determination of hydrogen peroxide was found to be from 1.0 x 10(-6) to 2.5 x 10(-4) mol/L with a detection limit of 4.0 x 10(-7) mol/L. The apparent Michaelis-Menten constant was determined to be 2.198 mmol/L. To improve the analytical characteristics of the fabricated biosensor, the effects of applied potential and pH value on the steady-state current were studied. Under the optimized experimental conditions, the fabricated biosensor was found to have good analytical performance, reproducibility, and storage stability.