Influence of remote plasma on PEDOT:PSS-coated carbon felt for improved activity of glucose oxidase

Increasing wettability of carbon felts is an important strategy to improve their efficiency in bio-electrochemical applications. Herein, influence of cold remote plasma (N₂ + O₂) treatment on surface properties of carbon felts with poly(3,4-ethylenedioxythiophene) polystyrene sulfonate (PEDOT:PSS) coating was tested, aiming to improve immobilizing of glucose oxidase enzyme (GOx). Spectra of N 1s and O 1s confirmed the integration of carbonyl and ether as well as amide and amine groups on bare carbon fiber surface, while on coated fibers, carbonyl groups were pre-dominant. S 2p spectra confirmed oxidation of PEDOT:PSS coating with reduction of (S⁻) compared to (SO³⁻) group. GOx immobilized on different samples showed highest activity for PEDOT:PSS coating subjected to plasma with 2% O₂, maintaining up to 60% after immobilization, and 37% of its activity after six cycles for some samples. Enzymes immobilized on samples without plasma treatment lost their activity after four cycles. © 2019 Wiley Periodicals, Inc. J. Appl. Polym. Sci. 2020 , 137, 48521.     

Porous Poly(vinyl alcohol) Composite Membranes for Immobilization of Glucose Oxidase

Particle loaded porous poly(vinyl alcohol) composite membranes were selected for immobilization of glucose oxidase (GOx) for their hydrophilicity and unique interactions with amino functional groups. GOx was immobilized on the membranes by adsorption at pH values between 3.5 and 7.1. The highest adsorption loading was observed at pH 7.1 and the highest catalytic activity was observed at pH 5.1. Infrared studies showed that the highest ratio of amide I to amide II at pH 5.1 is obtained for GOx immobilized on membranes loaded with amine-functionalized micro-particles, suggesting that the conformational changes of GOx on these membranes yield to higher catalytic activity than in other supports.     

Porous Poly(vinyl alcohol) Composite Membranes for Immobilization of Glucose Oxidase

Particle loaded porous poly(vinyl alcohol) composite membranes were selected for immobilization of glucose oxidase (GOx) for their hydrophilicity and unique interactions with amino functional groups. GOx was immobilized on the membranes by adsorption at pH values between 3.5 and 7.1. The highest adsorption loading was observed at pH 7.1 and the highest catalytic activity was observed at pH 5.1. Infrared studies showed that the highest ratio of amide I to amide II at pH 5.1 is obtained for GOx immobilized on membranes loaded with amine-functionalized micro-particles, suggesting that the conformational changes of GOx on these membranes yield to higher catalytic activity than in other supports.

     

Properties of the enzyme electrode fabricated with a film of polythiophene derivative and its application to a glucose fuel cell

A polymer electrode in the form of a thin film was prepared by electrochemical copolymerization of 3‐methylthiophene and thiophene‐3‐acetic acid. Glucose oxidase (GOx) was immobilized by covalent binding to the carboxyl groups on the electrode, and the GOx‐immobilized electrode (GOx‐electrode) was used as an anode in a glucose fuel cell. It was demonstrated by cyclic voltametry that in the presence of p‐benzoquinone (BQ), which was adopted as an electron mediator, the GOx‐electrode generated a significant glucose‐oxidation current depending on the concentrations of both glucose and BQ. A large surface area of the GOx‐electrode was considered to afford effective environment for the enzyme reaction and electron transfer. The fuel cell using the GOx‐electrode as an anode gave a power output of 42 μW/cm²‐anode at 30°C, when its anodic compartment contained 100 mM glucose and 10 mM BQ. The performance of the cell was influenced by the concentrations of glucose and BQ in the anodic compartment. © 2007 Wiley Periodicals, Inc. J Appl Polym Sci 2007     

Bioelectrochemistry and enzymatic activity of glucose oxidase immobilized onto the bamboo-shaped CNx nanotubes

The novel bamboo-shaped CN_x nanotubes, synthesized by nitrogen atoms doping into carbon nanotubes, were used for the immobilization of a relatively large enzyme glucose oxidase (GOx) and its bioelectrochemical studies. The morphologies and adsorptions of GOx immobilization onto CN_x nanotubes were clearly observed by transmission electron microscopy (TEM) and high-resolution transmission electron microscopy (HRTEM). Electrochemical impedance spectroscopy (EIS) was also used to feature the GOx adsorbed onto the surface of CN_x nanotubes. The immobilized GOx incorporated into CN_x nanotubes films exhibited a well-defined nearly reversible cyclic voltammetric peaks for the electroactive centers of GOx and a fast heterogeneous electron transfer rate with the rate constant (K_s) of 1.96 s⁻¹. The immobilized GOx onto the CN_x nanotubes exhibited its bioelectrocatalytic activity for the oxidation of glucose. The obtained results suggest that with a large amount of defective/active sites on the tube surfaces, a special bamboo structure and a suitable C-N microenvironment introduced by nitrogen doping, CN_x nanotubes could not only facilitate the direct electron transfer between the enzyme and electrode, but also retain the high enzyme loading and the enzymatic bioactivity.     

Two‐Stage Oxidation of Glucose by an Enzymatic Bioanode

The present study reports the design of a novel bioanode to deeply oxidize glucose in an enzymatic biofuel cell (EFC). This enzymatic glucose cell utilizes three co‐immobilized enzymes: NAD‐dependent glucose dehydrogenase (GDH), NAD(P)⁺‐dependent gluconate‐5‐dehydrogenase (Ga5DH), and diaphorase (DI). Glucose is oxidized to gluconate by NAD‐dependent GDH, gaining two electrons per glucose; the gluconate obtained as a by‐product is oxidized at the C5 carbon to 5‐keto‐gluconate by Ga5DH. Operation of our bioanode enabled the oxidation of glucose in two stages, resulting in the gain of four electrons. The three‐enzyme EFC provides a maximum power density of 10.51 ± 1.72 μW cm^–2, which is about 1.6 times higher than the maximum power density of an EFC using a bioanode based on the co‐immobilization of two enzymes (GDH and DI). Our results hold promise for increasing the current density of EFCs, and for application in glucose biosensor.     

Fabrication of bioanode by using electrically conducting polythiophene via entrapment technique

A glassy carbon electrode (GCE) was tailored with conducting polymer polythiophene and further immobilized by an enzyme glucose oxidase (GOx). A thin film of polymer was developed by electrochemical polymerization of thiophene monomer. During electrochemical polymerization of the monomer the enzyme GOx and the redox active mediator ferritin (Frt) were entrapped within this polymer matrix. In this novel approach, the entrapment of enzyme and mediators within a polymer matrix occurs without chemical reaction that could affect their activity. The entrapment of enzyme and mediator within the conducting polymer matrices increases the surface area of the electrode. The tailored GCE/Ptp/Frt/GOx electrode showed a high catalytic activity. The increased surface area causes a high rate of electron transfer between the electrode and Frt engaged as an electron transfer mediator. The electrochemical properties of the electrode were determined by cyclic voltammetry (CV) and linear sweep voltammetry (LSV). The fabricated bioanode showed a current density of 3.9mA cm^?2 at 1.0 V vs. Ag/AgCl in a 45 mM glucose solution and suggests proficient chances in biofuel cells (BFCs) applications.     

Improved catalytic activity by catalase immobilization using γ‐cyclodextrin and electrospun PCL nanofibers

Nanofibrous structures are promising for biocatalyst immobilization due to their large surface area which facilitates the enzyme attachment, stability, ease of separation, and fine porous structure. There is limited research available on the change in enzyme activity following interaction with cyclodextrin. In this study, catalase enzyme was immobilized into nanofibrous structures by various techniques, with and without γ‐CD addition, and the enzymatic activity of catalase was evaluated. In addition, catalase‐γ‐CD complex containing PEO polymer solution was electrospun in between PCL nanofibrous layers as a newly developed technique. The enzyme immobilized nanofibrous structures were characterized by SEM, XRD, and FT‐IR analysis methods. Among all the activity tests, best enzyme activity was recorded with catalase‐γ‐CD physical mixture encapsulated PCL nanofibrous layers. Moreover, the test results indicated that the use of cyclodextrin in immobilization process considerably improves the catalytic activity of the enzyme. © 2016 Wiley Periodicals, Inc. J. Appl. Polym. Sci. 2017 , 134, 44404.     

Polystyrene Attached Pt(IV)�CAzomethine, Synthesis and Immobilization of Glucose Oxidase Enzyme

Modified polystyrene with Pt(IV)–azomethine (APS–Sch–Pt) was synthesized by means of condensation and demonstrated to be a promising enzyme support by studying the enzymatic properties of glucose oxidase enzyme (GOx) immobilized on it. The characteristics of the immobilized glucose oxidase (APS–Sch–Pt–GOx) enzyme showed two optimum pH values that were pH = 4.0 and pH = 7. The insertion of stable Pt(IV)–azomethine spacers between the polystyrene backbone and the immobilized GOx, (APS–Sch–Pt–GOx), increases the enzymes’ activity and improves their affinity towards the substrate even at pH = 4. The influence of temperature, reusability and storage capacity on the free and immobilized glucose oxidase enzyme was investigated. The storage stability of the immobilized glucose oxidase was shown to be eleven months in dry conditions at +4 °C.     

UV-curable methacrylated/fumaric acid modified epoxy as a potential support for enzyme immobilization

The immobilization procedure of UV-curing coating is simple and causes less loss of enzymatic activity. UV-curable methacrylated/fumaric acid modified cycloaliphatic epoxide is here proposed as a rigid support material for covalent immobilization of α-amylase. The immobilized enzyme is analyzed in terms of bioactivity retention as a function of repeated use ability, pH, storage, as well as stability under various experimental conditions, taking starch as a substrate. The properties of immobilized enzyme were also compared with those of the free enzyme. The highest activity of free enzyme was obtained at pH 7.0 while this value was shifted to pH 7.5 for immobilized system. Optimum catalytic activity was observed at 30 °C, for both free and immobilized enzyme; however, the immobilized enzyme had a higher activity than the free one. The immobilized enzyme that was used 35 times in 8 h in repeated batch experiments demonstrated that about 73% of the enzyme activity was retained. The free enzyme lost all its activity with in 15 days. The retained activity of immobilized enzyme was found to be around 80%. The amount of bound α-amylase was found 94 mg per gram polymeric support material.     

Soybean oil based resin: A new tool for improved immobilization of α‐amylase

Acrylated epoxidized soybean resin has been utilized to immobilize the α‐amylase via UV‐curing technique. Among the numerous methods that exist for enzyme immobilization, entrapment and covalent binding are the focus of this study. The properties of immobilized enzyme were investigated and compared with those of the free enzyme. Upon immobilization by the two methods, the catalytic properties of the enzyme were not considerably changed as compared with that of nonimmobilized form; only the pH profile was broadened for the immobilized enzyme. The free enzyme lost its activity completely in 20 days, where as storage and repeated usage capability experiments demonstrated higher stability for the immobilized form. Immobilized enzyme prepared by attachment method possesses relatively higher activity compared with the activity of those obtained by entrapment method. © 2006 Wiley Periodicals, Inc. J Appl Polym Sci 100: 4757–4761, 2006     

Production and characterization of cellobiose dehydrogenase from <Emphasis Type="Italic">Phanerochaete chrysosporium</Emphasis> KCCM 60256 and its application for an enzymatic fuel cell

The enzyme cellobiose dehydrogenase (CDH), with high ability of electron transport, has been widely used in enzymatic fuel cells or biosensors. In this study, the cellobiose dehydrogenase gene from Phanerochaete chrysosporium KCCM 60256 was amplified and expressed in the methylotrophic yeast Pichia pastoris X-33. The recombinant enzyme (PcCDH) was purified using a metal affinity chromatography under non-denaturing conditions. The purified enzyme was analyzed by SDS-PAGE, confirming a corresponding band about 100 kDa. The enzyme activity of this purified PcCDH was determined as 1,845U/L (65mg/L protein). The enzyme showed the maximum activity at pH 4.5 and high activity in broad ranges of temperature from 30°C to 60°C. Moreover, the application of PcCDH to enzymatic fuel cell (EFC) was demonstrated. Lactose was used as the substrate in the EFC system; anode and cathode were immobilized with PcCDH and laccase, respectively. The cell’s open circuit voltage and maximum power density of the EFC system were, respectively, determined as 0.435 V and 314 μW/cm² (at 0.247 V) with 10 mM lactose.     

Effects of 1-butyl-3-methylimidazolium tetrafluoroborate on the oxidation of glucose catalyzed by glucose oxidase

The effects of room temperature ionic liquids (ILs) on the conformation and electrocatalytic activity of enzymes were studied using glucose oxidase (GOx) and 1-butyl-3-methylimidazolium tetrafluoroborate ([bmim]BF₄) as models. UV-vis and circular dichroic (CD) spectra indicated that [bmim]BF₄ did not affect the conformation of the enzyme, and the secondary structure of GOx in [bmim]BF₄-PBS mixtures (the content of the IL is from 0 to 20 vol%) was essentially the same as that of the native one. The Raman spectra showed that no interaction existed between glucose and [bmim]BF₄. The oxidation of glucose catalyzed by GOx was investigated under a substrate-saturated condition in [bmim]BF₄-PBS mixtures using ferroceneacetic acid (FcA) as a mediator. The voltammetric results showed that electrocatalytic current (i_cat) decreased with the increase of the content of [bmim]BF₄ in the mixtures. The reason causing the decrease of i_cat was analyzed. The reduction in the diffusion rate of FcA due to the increase of the viscosity after the addition of the IL was a key factor of causing the decrease of i_cat. The results presented here will be useful for the designing of the related biosensor used in ILs-containing system.     

介孔TiO2固定化葡萄糖氧化酶的直接电化学性能

采用非模板软化学法制备了一种孔径均一的介孔TiO₂材料（m-TiO₂）。XRD测试结果表明其晶型为锐钛矿,且结晶度高,通过N₂吸-脱附曲线可看出存在规整的介孔结构且具有高比表面,从FESEM和TEM可以看出制备的m-TiO₂由许多纳米粒子构成微米级大颗粒,且存在均匀分布的介孔。将此种TiO₂材料固定化葡萄糖氧化酶（GOx）后,通过红外光谱（FT-IR）分析可知m-TiO₂材料可以有效固定化GOx。将m-TiO₂作为固定化材料固定化GOx制备成Nafion/GOx/m-TiO₂/GC电极并进行相关电化学测试,测试结果表明m-TiO₂作为固定化材料固定化GOx后具有良好的催化活性,在无电子媒介体存在下能够实现反应电子与电极表面的直接电子传递,检测线性范围为0.1～1.2 mmol·L^-1葡萄糖,灵敏度为3.44μA·mmol^-1·L·cm^-2,在葡萄糖传感领域具有良好的应用前景。     

Photosynthetic membranes, 21. Immobilization of catalase by photochemically grafted ultrafiltration membranes

Catalase has been immobilized in membranes prepared by photoinduced grafting onto microporous polymeric supports and its catalytic activity on hydrogen peroxide decomposition has been studied under ultrafiltration conditions by means of a recirculation apparatus. The membranes showed a very good catalytic performance and the enzyme reaction took place exclusively within the membrane structure. Initial reaction rates measured in the temperature range 5 – 35°C as a function of both substrate concentration and enzyme amount immobilized per unit membrane surface indicate that the mechanism of action of catalase is not altered after immobilization.     

Immobilization of glucose oxidase and electron-mediating groups on the film of 3-methylthiophene/thiophene-3-acetic acid copolymer and its application to reagentless sensing of glucose

Enzyme electrodes were prepared by covalent immobilization of glucose oxidase (GOx) on the films of conducting copolymer obtained by electrochemical copolymerization of 3-methylthiophene and thiophene-3-acetic acid. Onto the enzyme electrodes, 2,5-dihydroxyphenyl (DHP) groups were introduced as redox mediators by the reaction of 2,5-dihydroxybenzaldehyde using alkylenediamines as linker molecules. The mediator-carrying enzyme electrodes were applied to reagentless glucose sensing system. It was found that response current to glucose was increased by introducing DHP groups, and the longer the alkylene chain of used alkylenediamine was, the higher the response current became. For a comparison, poly-l-lysine was linked onto the films of the conducting copolymer in advance, and immobilization of GOx and then introduction of DHP groups were carried out. The mediator-carrying electrode prepared thus gave higher amperometric response than those prepared with alkylenediamines, suggesting that amino groups of poly-l-lysine functioned effectively as the sites for binding GOx and DHP groups.     

Facile direct electron transfer in glucose oxidase modified electrodes

Glucose oxidase (GOx) is widely used in the glucose biosensor industry. However, mediatorless direct electron transfer (DET) from GOx to electrode surfaces is very slow. Recently, mediatorless DET has been reported via the incorporation of nanomaterials such as carbon nanotubes and nanoparticles in the modification of electrodes. Here we report GOx electrodes showing DET without the need for any nanomaterials. The enzyme after immobilization with poly-l-lysine (PLL) and Nafion^® retains the biocatalytic activities and oxidizes glucose efficiently. The amperometric response of Nafion-PLL-GOx modified electrode is linearly proportional to the concentration of glucose up to 10 mM with a sensitivity of 0.75 μA/mM at a low detection potential (−0.460 V vs. Ag/AgCl). The methodology developed in this study will have impact on glucose biosensors and biofuel cells and may potentially simplify enzyme immobilization in other biosensing systems.     

Continuous glucose monitoring using enzyme-immobilized platinized diamond microfiber electrodes

A sensitive and stable glucose biosensor for in vivo monitoring has been developed using boron-doped diamond microfiber (BDDMF) electrodes. The electrodes were modified with platinum nano-particles to detect H₂O₂, which was enzymatically produced by glucose oxidase (GOx) immobilized on the electrode surface. The platinum-modified BDDMF (Pt-BDDMF) electrodes exhibited much higher sensitivity compared to Pt-microfiber electrodes, Pt electrodes and Pt-modified diamond thin film electrodes. Deposition conditions for Pt nano-particles on the BDDMF electrodes and immobilization of GOx were optimized. GOx/overoxidized polypyrrole (OPPy)/Pt-modified BDDMF electrodes were applied for continuous interference-free glucose monitoring. Amperometric measurements of glucose showed a linear response in the range of 1-70 mM, with an R.S.D. of 3.7% for five injections of 100 μM glucose. The electrodes exhibited good stability over 3 months with no detected anodic current for ascorbic acid (AA), which is an interfering compound.     

α-淀粉酶在MCM-41介孔分子筛上的固定化研究

采用浸渍法将α-淀粉酶固定在介孔分子筛MCM-41上。考察了吸附时间、给酶量和pH对α-淀粉酶固定化性能的影响,并对固定化酶的活性、稳定性和载体结构等进行了研究。结果表明,在固定化时间为11 h,给酶量为70 mg.g-1,pH=5.9的条件下,固定化酶活性回收率可达48%。与游离酶相比,固定化酶的耐热能力增强,温度达到70℃时,固定化酶相对活性可达到75%,而游离酶只有14%;在pH=3.3～8.0的内,固定化酶相对活性为62%～100%,而游离酶的相对活性为5%～100%,固定化酶具有更宽的pH适应性;此外,固定化酶储存稳定性明显增强,并具有一定的可重复操作性,且固定后载体仍然保持了良好的介孔结构。     

以层柱黏土为载体固定辣根过氧化物酶

引 言 辣根过氧化物酶(HRP)能在较宽的温度、pH值、污染物浓度和盐度范围内将多种芳香性化合物催化氧化为酚氧自由基,它们之间可聚合生成溶解性较差的聚合物从溶液中沉淀出来,便于采用混凝法对其进行去除[1-2].许多学者将该酶用于含酚废水处理中,取得了较好的效果.然而,传统酶催化方法中使用的大多是溶液酶,不仅不能循环使用以节省费用,而且易受废水中其他污染物的影响,稳定性差,加上酶的费用较高,因此酶法在废水处理中尚未得到有效推广应用[3].国内外学者研究发现酶固定化后稳定性大大提高,可重复或连续使用,这样不仅降低了废水处理的成本,而且还避免了蛋白质的污染等问题[4].