Conducting polymer based amperometric enzyme electrodes

The construction and the properties of conducting-polymer based amperometric enzyme electrodes are reviewed. The main aim is to focus on the properties of conducting polymer films which are important for the construction of amperometric enzyme electrodes. Additionally, the review is focused on electron-transfer pathways between conducting-polymer integrated immobilized enzyme molecules and the modified electrode using free-diffusing redox mediators as well as direct electron transfer via the conducting-polymer wires. Possible future applications using microstructured conducting-polymer films will be discussed.     

Determination of active diaphorase immobilized at electrode surfaces

The activity of diaphorase (from Bacillus stearothermophilus) immobilized on glassy carbon (GC) electrodes was determined by analyzing the catalytic currents for oxidation of the immobilized enzyme using digital simulation techniques, which gives the concentration of the active enzyme at the electrode surface. Results show that the immobilization by the cross-linking reaction with glutaraldehyde deactivates the enzyme and only about 10% of the total enzyme remains active at the electrode surface.     

Parameters important in fabricating enzyme electrodes using self-assembled monolayers of alkanethiols.

The fabrication of enzyme electrodes using self-assembled monolayers (SAMs) has attracted considerable interest because of the spatial control over the enzyme immobilization. A model system of glucose oxidase covalently bound to a gold electrode modified with a SAM of 3-mercaptopropionic acid was investigated with regard to the effect of fabrication variables such as the surface topography of the underlying gold electrode, the conditions during covalent attachment of the enzyme and the buffer used. The resultant monolayer enzyme electrodes have excellent sensitivity and dynamic range which can easily be adjusted by controlling the amount of enzyme immobilized. The major drawback of such electrodes is the response which is limited by the kinetics of the enzyme rather than mass transport of substrates. Approaches to bringing such enzyme electrodes into the mass transport limiting regime by exploiting direct electron transfer between the enzyme and the electrode are outlined.     

Simulation of Electrochemical Process for Glucose Oxidase Immobilized Conducting Polymer Electrodes

Abstract

Computer simulation of electrochemical processes that govern the operation of conducting polymer modified electrodes (CPME) have been reported in this paper. Comparison of the behaviour of a biocatalyst (GOX) in free solution and in the immobilized phase in conducting polymer modified electrodes (CPME) has been provided The output has been obtained using the Runga Kutta numerical method solved by programming in FORTRAN 77. The results point out that the catalytic current generated by an immobilized enzyme in layer is larger as compared to that for the enzyme in solution, and that it varies with the thickness of the diffusion layer.     

Bioelectrocatalysis by Hydrogenase Th. Roseopersicina Immobilized on Carbon Materials

Application of various carbon materials as substrates for immobilizing hydrogenase and creating a hydrogen enzyme electrode is studied. A stable, highly-active electrocatalyst for mediatorless oxidation–evolution of hydrogen is obtained. The electrocatalyst uses hydrogenase from Th. roseopersicina immobilized on an inactive carbon material (based on carbon cloth TVSh-300M) and retains more than 50% of its initial activity after a half year in storage. An equilibrium hydrogen potential is established on enzyme electrodes made of a graphitized cloth and graphite rods with hydrogenase immobilized on them.     

生物功能电极(Ⅴ)─葡萄糖氧化酶在环糊精聚合物中的固定化

将葡萄糖氧化酶(GOD)固定在α-环糊精聚合物中,而电子传递体分子被包含在环糊精腔穴中。固定化酶膜的FTIR测定表明,GOD与环糊精聚合物发生共价连接。制备了含电子传递体的不同GOD酶电极并比较了它们的性能。含四硫代富瓦烯的酶电极具有良好的电流响应特性,可望成为第二代葡萄糖酶电极的新构型。     

Catalytic activity of glucose oxidase after dielectrophoretic immobilization on nanoelectrodes

Dielectrophoresis (DEP) is an AC electrokinetic effect that is proven to be effective for the immobilization of not only cells, but also of macromolecules, for example, antibodies and enzyme molecules. In our previous work, we have already demonstrated the high catalytic activity of immobilized horseradish peroxidase after DEP. To evaluate the suitability of the immobilization method for sensing or research in general, we want to test it for other enzymes, too. In this study, glucose oxidase (GOX) from Aspergillus niger was immobilized on TiN nanoelectrode arrays by DEP. Fluorescence microscopy showed the intrinsic fluorescence of the immobilized enzymes flavin cofactor on the electrodes. The catalytic activity of immobilized GOX was detectable, but a fraction of less than 1.3% of the maximum activity that was expected for a full monolayer of immobilized enzymes on all electrodes was stable for multiple measurement cycles. Therefore, the effect of DEP immobilization on the catalytic activity strongly depends on the used enzyme.     

Activity of AC electrokinetically immobilized horseradish peroxidase

Dielectrophoresis (DEP) is an AC electrokinetic effect mainly used to manipulate cells. Smaller particles, like virions, antibodies, enzymes, and even dye molecules can be immobilized by DEP as well. In principle, it was shown that enzymes are active after immobilization by DEP, but no quantification of the retained activity was reported so far. In this study, the activity of the enzyme horseradish peroxidase (HRP) is quantified after immobilization by DEP. For this, HRP is immobilized on regular arrays of titanium nitride ring electrodes of 500 nm diameter and 20 nm widths. The activity of HRP on the electrode chip is measured with a limit of detection of 60 fg HRP by observing the enzymatic turnover of Amplex Red and H₂O₂ to fluorescent resorufin by fluorescence microscopy. The initial activity of the permanently immobilized HRP equals up to 45% of the activity that can be expected for an ideal monolayer of HRP molecules on all electrodes of the array. Localization of the immobilizate on the electrodes is accomplished by staining with the fluorescent product of the enzyme reaction. The high residual activity of enzymes after AC field induced immobilization shows the method's suitability for biosensing and research applications.     

纳米材料修饰电极上吸附态葡萄糖氧化酶的酶活性和电活性的比较

采用石英晶体微天平(QCM)技术, 监测了裸金(Au)电极、电沉积纳米金的金电极(Au_ed/Au)、多壁碳纳米管(MWCNTs)修饰的金电极(MWCNTs/Au)以及MWCNTs 修饰后再电沉积纳米金的金电极(Au_ed/MWCNTs/Au)上葡萄糖氧化酶(GOx)的吸附过程, 测算了吸附固定的GOx质量. 通过阳极恒电位检测吸附酶与葡萄糖发生酶反应所产生的过氧化氢, 考察了这些酶电极的安培响应, 并测算了各吸附态GOx的质量比生物活性(MSBAi).也通过循环伏安法研究酶的直接电化学, 测算了各吸附态GOx的电活性百分数(EAP_i). 实验结果表明, 酶吸附量和酶电极的安培响应满足MWCNTs/Au > Au_ed/MWCNTs/Au > Au_ed/Au > Au 的顺序; MSBA_i满足Au > Au_ed/MWCNTs/Au > Au_ed/Au > MWCNTs/Au的顺序; 而EAP_i则满足MWCNTs/Au > Au_ed/MWCNTs/Au > Au_ed /Au > Au的顺序. 根据酶和纳米材料的亲疏水作用以及酶的吸附量对实验结果进行了合理解释, 也定量验证了电极上吸附酶分子的总生物活性与酶电极的安培响应呈正相关关系, 所得数据和结论有助于纳米材料固定酶及其安培酶电极的研究.     

Enzyme sequence and competition electrodes based on immobilized glucose oxidase,peroxidase and catalase

The toxicologically important peroxidase substrates bilirubin and aminopyrine can be determined by combination of immobilized glucose oxidase, horseradish peroxidase and catalase, forming so-called enzyme sequence and enzyme competition electrodes. Bilirubin and aminopyrine are determined in the concentration range 5–50 μM.     

Fabricating and imaging carbon-fiber immobilized enzyme ultramicroelectrodes with scanning electrochemical microscopy.

The scanning electrochemical microscope (SECM) is used to image the activity of enzymes immobilized on the surfaces of disk-shaped carbon-fiber electrodes. SECM was used to map the concentration of enzymatically produced hydroquinone or hydrogen peroxide at the surface of a 33-microm diameter disk-shaped carbon-fiber electrode modified by an immobilized glucose-oxidase layer. Sub-monolayer coverage of the enzyme at the electrode surface could be detected with micrometer resolution. The SECM was also employed as a surface modification tool to produce microscopic regions of enzyme activity by using a variety of methods. One method is a gold-masking process in which microscopic gold patterns act as mask for producing patterns of chemical modification. The gold masks allow operation in both a positive or negative process for patterning enzyme activity. A second method uses the direct mode of the SECM to produce covalently attached amine groups on the carbon surface. The amine groups are anchors for attachment of glucose oxidase by use of a biotin/avidin process. The effect of non-uniform enzyme activity was investigated by using the SECM tip to temporarily damage an immobilized enzyme surface. SECM imaging can observe the spatial extent and time-course of the enzyme recovery process.     

Carbon film electrodes for oxidase-based enzyme sensors in food analysis

Carbon film resistor electrodes have been evaluated as transducers for the development of multiple oxidase-based enzyme electrode biosensors. The resistor electrodes were first modified with Prussian Blue (PB) and then covered by a layer of covalently immobilized enzyme. Electrochemical impedance spectroscopy was used to characterize the interfacial behaviour of the Prussian Blue modified and enzyme electrodes; the spectra demonstrated that the access of the substrates is essentially unaltered by application of the enzyme layer. These enzyme electrodes were used to detect the substrate of the oxidase (glucose, ethanol, lactate, glutamate) via reduction of hydrogen peroxide at +50 mV versus Ag/AgCl in the low micromolar range. Response times were 1-2 min. Finally, the glucose, ethanol and lactate electrochemical biosensors were used to analyse complex food matrices—must, wine and yoghurt. Data obtained by the single standard addition method were compared with a spectrophotometric reference method and showed good correlation, indicating that the electrodes are suitable for food analysis.     

Immobilization of histidine-tagged proteins on electrodes

The development of new enzyme immobilization techniques that do not affect catalytic activity or conformation of a protein is an important research task in biotechnology including biosensor applications and heterogeneous reaction systems. One of the most promising approaches for controlled protein immobilization is based on the immobilized metal ion affinity chromatography (IMAC) principle originally developed for protein purification. Here we describe the current status and future perspectives of immobilization of His-tagged proteins on electrode surfaces. Recombinant proteins comprising histidine-tags or histidine rich native proteins have a strong affinity to transition metal ions. For metal ion immobilization at the electrode surface different matrices can be used such as self-assembled monolayers or conductive polymers. This specific technique allows a reversible immobilization of histidine-tagged proteins at electrodes in a defined orientation which is an important prerequisite for efficient electron transfer between the electrode and the biomolecule. Any application requiring immobilized biocatalysts on electrodes can make use of this immobilization approach, making future biosensors and biocatalytic technologies more sensitive, simpler, reusable and less expensive while only requiring mild enzyme modifications.     

Integration of microfabricated needle-type glucose sensor devices with a novel thin-film Ag/AgCl electrode and plasma-polymerized thin film: mass production techniques

We developed an integrated array of needle-type biosensors employing a novel process of fabrication, comprising conventional semiconductor fabrication and micromachining technology. Amperometric sensing electrodes with plasma-polymerized films and a thin-film Ag/AgCl reference electrode were directly integrated on a glass substrate with thin-film process, e.g., sputtering. An enzyme was immobilized on the electrode via the plasma-polymerized film, which was deposited directly on the substrate using a dry process. The novel thin-film Ag/AgCl reference electrode showed stable potentials in concentrated chloride solutions for a long period. The plasma-polymerized film is considered to play an important role as an interfacial design between the sensing electrode and the immobilized enzyme considering that the film is extremely thin, adheres well to the substrate (electrode) and has a highly cross-linked network structure and functional groups, such as amino groups. The results showed increments of the sensor signal, probably because the plasma-polymerized film allowed a large amount of enzyme to be immobilized. The greatest advantage is that the process can permit the mass production of high-quality biosensors at a low cost.     

A Self‐Sampling‐and‐Flow Biosensor for Continuous Monitoring

A self‐sampling‐and‐flow biosensor was fabricated by sandwiching a nitrocellulose strip on the working electrode side of the double‐sided microporous gold electrodes and a wicking pad on the counter electrode side. The double‐sided microporous electrodes were formed by plasma sputtering of gold on a porous nylon substrate. Sample was taken up to the enzyme‐immobilized working electrode by the capillary action of the front nitrocellulose strip dipped into the sample solution, analyzed electrochemically at the enzyme‐immobilized electrode, and diffuses out to the backside wicking pad through the micropores of the electrodes, constituting a complete flow cell device with no mechanical liquid‐transporting device. Biosensor was formed by co‐immobilizing the glucose oxidase and electron transfer mediator (ferrocene acetic acid) on the thioctic acid self‐assembled monolayer‐modified working electrode. A typical response time of the biosensor was about 5 min with the sensitivity of 2.98 nA/mM glucose, providing linear response up to 22.5 mM. To demonstrate the use of self‐sampling‐and‐flow biosensor, the consumption rate of glucose in the presence of yeast was monitored for five days.     

Wavelet method for steady state immobilized enzyme kinetic model: an operational matrix approach

Journal of Mathematical Chemistry - This paper discusses a mathematical model of the diffusion and reaction in amperometric biosensor response with immobilized enzyme electrodes within a uniform...     

Application of the Ugi reaction for the preparation of enzyme electrodes

Glucose oxidase and catalase were immobilized via the Ugi reaction by means of cyclohexyl isocyanide and glutaraldehyde on a nylon net partially hydrolysed by hydrochloric acid. A specific enzyme sensor for D-glucose was made by fixing the nylon net with immobilized enzymes on the tip of a Clark-type oxygen sensor. For comparison purposes glucose oxidase and catalase were also co-immobilized in the absence of cyclohexyl isocyanide or only glucose oxidase was immobilized with and without cyclohexyl isocyanide. The prepared biosensors were characterized by the specific activity of glucose oxidase and its dependence on Ph and temperature and by the apparent Michaelis constant. The linear range of the biosensor response to the substrate concentration and the stability of the biosensor were determined. The long-term stabilities of the enzyme electrodes were compared and the advangtage of the developed method was demonstrated.     

Determination de l'Acide l-Ascorbique a l'Aide d'Electrodes Bacteriennes,Tissulaires et Enzymatiques

Abstract

L-ascorbic acid membrane electrodes based upon the use of four classes of biocatalysts immobilized at an oxygen electrode are evaluated and compared in terms of electrode properties and operating requirements. Isolated ascorbate oxydase enzyme in soluble form and in covalent binding matrices, peel of cucumber and living bacterial cells of Enterobacter agglomerans strain, respectively, are employed as biocatalytic layers. The physico-chemical factors, life times and interferences are discussed in details. The low stability of the soluble enzyme sensor does not allow its analytical utilization, but the immobilized enzyme, bacterial and tissue electrodes can be used, even in multivitamin pharmaceutical formulations. The common linear range of those three biosensors are of 4.10^?6 M to 7.10^?4 M with a precision and a reproducibility of ± 3%.     

Characterization and Potential Applications of Immobilized Glucose Oxidase and Polyphenol Oxidase

Pyrrole functionalized polystyrene (PStPy) was copolymerized with pyrrole to obtain a conducting copolymer, P(PStPy‐co‐Py) which is used as the immobilization matrix. Glucose oxidase and polyphenol oxidase enzymes were immobilized via the entrapment method by electrochemical polymerization. Enzyme electrodes were prepared by electrolysis at a constant potential using sodium dodecyl sulfate (SDS) as the supporting electrolyte during the copolymerization of PStPy with pyrrole. Maximum reaction rates (V_max) and enzyme affinities (Michaelis‐Menten constants, K_m) were determined for the enzyme entrapped both in polypyrrole (PPy) and P(PStPy‐co‐Py) matrices. Optimizations of enzyme electrodes were done by examining the effects of temperature and pH on enzymes' activities along with the shelf life and operational stability investigations. Glucose oxidase enzyme electrodes were used for human serum analysis and glucose determination in two brands of orange juices. Polyphenol oxidase enzyme electrodes were used for the determination of phenolics in red wines of Turkey.     

亲水纳米二氧化硅修饰丝网印刷碳糊电极的改进性能研究

研究了以铁氰化钾为电子传递剂,亲水纳米二氧化硅为固定化酶的载体与高分子成膜材料掺杂制作的生物敏感膜修饰丝网印刷碳糊电极葡萄糖生物传感器的改进特性,并从机理上分析了形成这种优化的原因。实验采用柠檬酸作为缓冲液,在高分子成膜材料、铁氰化钾、稳定剂、葡萄糖氧化酶中掺杂均相处理后的纳米二氧化硅制备生物敏感膜,并制成腔体,将其与未经过纳米二氧化硅掺杂制备的生物传感器进行对比实验。实验证明:用掺杂纳米二氧化硅制作的生物敏感膜修饰的丝网印刷碳糊电极与未修饰电极相比,灵敏度提高了2.6倍,线性检测范围为1.1~33.3 mmol/L,对测试范围内不同浓度的葡萄糖样本,相对标准偏差<5%,重现性和稳定性良好,具有较高的研究价值和应用前景。