Development of a new biosensor for superoxide radicals

A superoxide dismutase (SOD) biosensor for determination of superoxide radicals has been developed by immobilization of superoxide dismutase within gelatin (G) on a Pt electrode surface. The properties of the biosensor have been investigated and optimum conditions–enzyme concentration, glutaraldehyde concentration, and pH–were determined. The response of the G-SOD biosensor was proportional to concentration and the detection limit was 0.01 mmol L⁻¹ at a signal-to-noise ratio of 3. The biosensor retained 89% and 60% of its sensitivity after use for three and four weeks, respectively. Immobilization of SOD on gelatin provides a biocompatible microenvironment around the enzyme and stabilizes the activity of the enzyme very efficiently. The superoxide dismutase biosensor was used to determine the antioxidant properties of acetylsalicylic acid-based drugs and the anti-radical activity of healthy and cancerous human brain tissues.     

Studies towards an amperometric phosphate ion biosensor for urine and water analysis

An amperometric biosensor for phosphate ion is described that is based on a cobalt phthalocyanine modified screen-printed carbon electrode (CoPC-SPCE). The biosensor operation is based on the enzyme pyruvate oxidase (PyOd) which catalyses the oxidative decarboxylation of pyruvate, in the presence of inorganic phosphate and O₂, to acetyl phosphate, hydrogen peroxide (H₂O₂) and CO₂. The transducer allows the electrocatalytic oxidation of H₂O₂ in order to generate the analytical signal. The enzyme was immobilised onto the CoPC-SPCE using a sandwich format. The inner membrane was formed in situ by depositing an acetone solution containing cellulose acetate first onto the transducer surface. The enzyme and cofactors were then deposited onto this layer and allowed to dry; finally a second aliquot of the cellulose acetate solution was deposited onto the enzyme layer and allowed to dry. The biosensor was characterised by amperometry in stirred solution to produce current-voltage curves and for calibration studies. From these it was deduced that a reliable electrocatalytic response was obtained for phosphate ion; an operating potential of +0.4 V was selected for the analysis of urine samples. The precision of the response for urine analysis and recovery data for potable water suggests that the biosensor could have applications in clinical and environmental monitoring.     

Studies on simulated ageing of paper by photochemical degradation

The study of the ageing of two paper types was performed on monitoring it during a simulated process by means of the measure of the photocatalytic degradation of the paper cellulose. Such evaluation was possible due to the combined responses of a photosensor based on titanium dioxide in suspension, of an enzymatic biosensor based on superoxide dismutase (SOD), of a specific conductivity sensor and of an enzymatic biosensor based on glucose oxidase.     

Mathematical Model and Numerical Simulation of Conductometric Biosensor of Urea

In this article, a mathematical model was developed to describe and optimize the configuration of the urea biosensor. The biosensor is based on interdigitated gold microelectrodes modified with a urease enzyme membrane. The model presented here focuses on the enzymatic reaction and/or diffusion phenomena that occur in the enzyme membrane and in the diffusion layer. Numerical resolution of differential equations was performed using the finite difference technique. The mathematical model was validated using experimental biosensor data. The responses of the biosensor to various conditions were simulated to guide experiments, improve analytical performance, and reduce development costs.     

应用微生物传感器测定甲醛

将培养好的枯草芽孢杆菌与一定量辅料混合,将其涂布并夹入两片微孔纤维膜之间制成固定化微生物膜。将此微生物膜装在溶解氧电极的阴极(金电极)端面的聚四氟乙烯薄膜上,并使之固定即完成微生物传感器的组装。为测验此传感器对甲醛的响应,按方法的给定条件进行操作,记录加入含甲醛试液前后仪器的输出电流。有甲醛存在时,由于其与微生物之间的同化作用导致一定量氧消耗而使输出电流it与基线电流i0相比明显下降,且其下降程度Δi(i0-it)与甲醛质量浓度在0.005～0.20 g.L-1之间呈线性关系。在甲醛质量浓度为0.1 g.L-1条件下做精密度试验,测得其信号值的相对标准偏差(n=10)为1.13%。用标准加入法试验了方法的回收率,3次试验的平均回收率为107%。又经试验,所制备的干微生物固化膜在4℃冰箱中冷藏,可保存60d仍有效。     

PEDOT/Superoxide Dismutase Electrode Surface Modification for Superoxide Bioelectrochemical Sensing

An amperometric biosensor for the sensitive detection of superoxide was designed utilizing a drop‐coating approach for immobilizing the superoxide dismutase enzyme on Pt electrode modified with a thin layer of poly (3,4‐ethylenedioxythiophene) (PEDOT). The layer electrodeposited on Pt was characterized by cyclic voltammetry and atomic force microscopy (AFM). Then, drop‐coating procedure was chosen for the immobilization of superoxide dismutase (SOD), which was incorporated at the electrode surface using a solution containing SOD, glutaraldehyde and bovine serum albumin (optimized composition: SOD 0.1 % – BSA 2 % – GA 2.5 %.) This simple procedure allows forming a reproducible enzymatic biocomposite layer that allows optimal sensitivity and limit of detection for superoxide sensing. The synergistic effect integrates an effective conductivity and permselectivity attributed to the PEDOT layer, as well as the specificity and selectivity of SOD for the detection of superoxide. A high sensitivity (0.82±0.01 μA/μM) and a low detection limit of 11 nM were obtained, as well as good selectivity against main interfering biological compounds such as uric acid and ascorbic acid. Our results suggest that the biosensor could be used for the detection and quantification of in vitro and in vivo.     

Superoxide Dismutase Biosensors for Superoxide Radical Analysis

ABSTRACT

Some basic work has been performed on the development and optimisation of superoxide dismutase (SOD) biosensors for superoxide radical analysis. Initially we studied the possibility of obtaining a SOD biosensor using the Clark electrode as indicating sensor. However, the best results were obtained using as indicator a classical amperometric electrode for H₂O₂. In both cases the superoxide radical was generated in situ using the xanthine/xanthine oxidase (XOD) enzyme system, while the SOD was immobilised in kappa-carrageenan gel. The first application was realised by studying the effects in vitro on the superoxide radical of some molecules commonly accepted as radical scavengers.     

Optical Urea Biosensor Based On Ammonium Ion Selective Membrane

ABSTRACT

An optical urea biosensor was developed by immobilizing an urease enzyme layer on a thin ammonium-selective polymer membrane. The ammonium optical membrane utilized dichlorofluorescein octadecyl ester (DCFOE) as anionic chromophore and nonactin as neutral ionophore. The urease layer was coated on the top of the ammonium layer by gelatin entrapment combined with glutaradehyde cross-linking. Hydrolysis of urea catalyzed by urease produced ammonium ion, which was extracted into the-polymer film to form complexes with nonactin. A proton was released which resulted in a color change of the optical membrane due to charge neutrality principle. The biosensor     

The Effect of Diffusion Limitations on the Response of Amperometric Biosensors with Substrate Cyclic Conversion

A mathematical model of amperometric biosensors in which chemical amplification by cyclic substrate conversion takes place in a single enzyme membrane has been developed. The model involves three regions: the enzyme layer where enzyme reaction as well as mass transport by diffusion takes place, a diffusion limiting region where only the diffusion takes place, and a convective region where the analyte concentration is maintained constant. Using computer simulation the influence of the thicknesses of the enzyme layer and the diffusion region on the biosensor response was investigated. This paper deals with conditions when the mass transport in the exterior region may be neglected to simulate the biosensor response in a well-stirred solution. The digital simulation was carried out using the finite difference technique.     

Modelling carbon nanotube based biosensor

This paper presents a two-dimensional-in-space mathematical model of an amperometric biosensor based on an enzyme-loaded carbon nanotubes layer deposited on a perforated membrane. The developed model is based on non-linear non-stationary reaction-diffusion equations. By changing input parameters the output results are numerically analysed with a special emphasis to the influence of the geometry and the catalytic activity of the biosensor to its response. The numerical simulation at transition and steady state conditions was carried out using the finite difference technique. The mathematical model and the numerical solution were validated by experimental data. The obtained agreement between the simulation results and experimental data was admissible at different concentrations of the substrate and the mediator.     

Purification and Partial Properties of a Superoxide Dismutase from Agave Hybrid No.11648

A superoxide dismutase enzyme was extracted from the leaves of Agave Hybrid No.11648 using homogenisation, ammonium sulphate fractional precipitation, diethylaminoethyl cellulose ion exchange and Sephadex gel filtration chromatography. A superoxide dismutase parallelogram‐shaped crystal was formed after packing at 4 °C for two days. The optimum pH was 8, and the optimum temperature was 40 °C. The crystal was relatively stable at temperatures below 60 °C and at pH values from 5 to 10. The subunit size was determined to be approximately 27.5 kDa by sodium dodecyl sulphate‐polyacrylamide gel electrophoresis. It was also demonstrated that the enzyme is an iron‐containing superoxide dismutase based on its sensitivity to inhibitors of potassium cyanide, hydrogen peroxide and chloroform‐ethanol and based on the results of ultraviolet absorption spectroscopy.     

Modelling synergistic action of laccase-based biosensor utilizing simultaneous substrates conversion

The response of a laccase-based amperometric biosensor that acts in a synergistic manner was modelled digitally. A mathematical model of the biosensor is based on a system of non-linear reaction diffusion equations. The modelling biosensor comprises three compartments, an enzyme layer, a dialysis membrane and an outer diffusion layer. By changing input parameters the biosensor action was analysed with a special emphasis to the influence of the species concentrations on the synergy of the simultaneous substrates conversion. The digital simulation was carried out using the finite difference technique.     

Biosensor with Protective Membrane for the Detection of DNA Damage and Antioxidant Properties of Fruit Juices

With the purpose to prepare a DNA biosensor protected with an outer‐sphere membrane against high molecular weight interferences, a carbon film electrode was layer‐by‐layer modified with dsDNA and chitosan. Using cyclic and square‐wave voltammetry and impedance spectroscopy, the oxidative damage of DNA by the hydroxyl and superoxide anion radicals was detected which consists of opening of the helix structure followed by deep DNA chain degradation. The biosensor has been applied to the detection of the antioxidant effect of apple and orange juices. The investigation of the novel biosensor with a protective membrane represents a significant contribution to the field of DNA biosensors utilization.     

Subcutaneous microdialysis probe coupled with glucose biosensor for in vivo continuous monitoring

Microdialysis probes have been tested to evaluate the influence of flow-rate, probe dimensions and temperature when used as sampling systems inserted subcutaneously for in vivo monitoring of glucose. The probe was coupled with a glucose biosensor obtained from a thin layer electrochemical cell generally used as detector for liquid chromatography. Glucose oxidase was immobilized on a nylon net membrane and it was placed over an acetate cellulose membrane into the cell in contact with the platinum anode.     

Activation‐Based Catalase Enzyme Electrode and its Usage for Glucose Determination

Abstract

In this study, a novel type amperometric biosensor, which is based on the activation of catalase enzyme by glucose, was developed and used for the sensitive determination of glucose. For the preparation of the biosensor catalase enzyme was immobilized in gelatin by using cross‐linking agent glutaraldehyde and fixed on a pretreated teflon membrane of a dissolved oxygen probe. Glucose was used as an activator for the catalase enzyme and determination of glucose is based on the assay of the differences on the catalase activity of the biosensor on the oxygenmeter in the absence and the presence of glucose in the reaction medium. The responses of the activation based catalase biosensor have a linear relation to glucose concentrations and good measurement correlation between 0.5 and 5.0 µM with 2 min response time. In the optimization studies of the biosensor the most suitable catalase amount were found as 1324 U cm^?2 and also phosphate buffer (pH: 7.0; 50 mM) and 35°C were obtained as the optimum working conditions. For the characterization studies of the biosensor some parameters such as activator and interference effects of some substances on the biosensor response, reproducibility and operational stability were performed.     

流动注入式乳酸生物传感器

研制了一种测定L－乳酸的生物传感器,将乳酸氧化酶（LOD）通过共价键固定在尼龙钢上制备乳酸氧化酶膜,将制得的酶膜固定在氧电极上构成乳酸生物传感器;将透析膜放在氧化酶膜上产生对L－乳酸扩散高度限制来改变该生物传感器的响应;酶膜机械强度高,在氧电极上反复装卸而不损坏,所构成的乳酸生物传感器的校正曲线的乳酸定量上限达5mmol/L,响应时间小于30s;初步血样测试的结果显示该乳酸生物传感器用于临床血乳酸的测定具有可行性。     

Studies on the construction and operation of miniaturized potentiometric biosensors

This study aims at presenting the studies on construction and operation of solid-state miniaturized biosensor with potentiometric detection. The performance of sensors with layer which consisted of mixture of aliphatic thiols and thiols containing ferrocene terminal groups or the conductive polymer layer obtained by electrochemical polymerization of 3,4-ethylenedioxythiophene was compared. Both of the applied modifications proved to significantly affect the sensors’ performance, influencing the stability of their working parameters in time. Wherein, the greatest improvement in planar sensors’ operating parameters was achieved by applying conductive polymer layer. Subsequently, a system creating universal platform for the construction of a biosensor, dedicated to almost any analyte determination, depending on the composition of the affinity proteins used in the receptor layer, was developed. For the construction of proposed biosensor, alkaline phosphatase was chosen as a model enzyme, assuming either the role of direct component of receptor layer or a label in affinity biosensor. The affinity biosensor structure was characterized by a sandwich assay, using a highly specific interaction occurring between streptavidin and biotin. Aside from optimization of various components of the constructed sensor, the most adequate analysis conditions were designated in the course of presented research. As it turned out, the composition of buffer solution used has a significant influence on the activity of the applied enzyme as well as on the working parameters of the sensor. Tris buffer was, therefore, found as the one ensuring the best performance of sensors utilizing alkaline phosphatase.     

A new enzyme electrode based on ascorbate oxidase immobilized in gelatin for specific determination of l-ascorbic acid

A biosensor for the specific determination of l-ascorbic acid in fruit juices and vitamin C tablets was developed using ascorbate oxidase (EC 1.10.3.3) from cucumber (Cucumis sativus L.) in combination with a dissolved oxygen probe. Ascorbate oxidase immobilized with gelatin using glutaraldehyde and fixed on pretreated teflon membrane served as an enzyme electrode. The phosphate buffer (50 mM, pH 7.5) and 35 degrees C were established as providing the optimum conditions. The biosensor response depends linearly on l-ascorbic acid concentration between 5.0x10(-5) and 1.2x10(-3) M with a response time 45 s. The biosensor is stable for more than 2 months, while more than 200 assays were performed. The results obtained for fruit juices and tablets were compared with DCIP (2,6 dichlorophenolindophenol) method.     

The Study of a Disposable Reagentless Biosensor for Fast Test of Aspartate Aminotransferase

A reagentless biosensor for amperometric measurement of aspartate aminotransferase (AST) has been developed. The three‐electrode biosensor was modified with two enzyme membrane with an integrated planar Ag|AgCl reference electrode. With a cellulose membrane containing catalytic substance coated, the reagentless biosensor was used for measurement of serum AST. The optimal sensitive membrane composition and operation conditions were studied. With low polarizing potential (0 V vs. integrated Ag|AgCl) and appropriate substrate concentration (20 mM α‐ketoglutarate acid), the response of the biosensor to AST activity was linear over the range of 25–1000 U/L with good relativity (R=0.9904 and 0.9823 in standard AST solutions and AST serum respectively) and fast response time (120 s). The biosensor also indicated good stability of more than 90% of its original activity for 60 days stored in 4 °C, and 90% of the activity was retained after a storage period of 15 days at room temperature (25 °C). The biosensor is expected to be used for the diagnosis of heart and liver disease in the future.     

Determination of antioxidant properties of aromatic herbs,olives and fresh fruit using an enzymatic sensor

The aim was to experimentally evaluate the antioxidant capacity of different fresh aromatic herbs (field balm, marjoram, parsley, rosemary, sage, sweet basil), several varieties of olives from Central Italy ('Carboncello', 'Rosciolo', 'Olivastro', 'Coratello', 'Leccino', 'Frantoio') and several types of fresh fruit (apple, apricot, banana, cherry, fig, grape, medlar, melon, peach, pear, pineapple, plum, water melon, yellow plum) using a superoxide dismutase (SOD) biosensor developed by the present authors.Measurements were carried out by comparing the biosensor response to the concentration of superoxide radical produced in solution using a xanthine/xanthine oxidase system in the presence and absence of the antioxidant sample considered. Tests carried out on different samples of fruit and aromatic herbs showed that the homogenised samples had better antioxidant properties than the centrifuged ones (obtained by centrifuging the homogenate), which sometimes gave extremely low antioxidant capacity values.The reliability of the proposed method was checked by comparing the trend of some experimental results found using the SOD biosensor with those reported in the literature obtained using the classic (ORAC) method. The precision of this method of analysis was found to be good for samples of aromatic herbs (RSD% 相似文献