Self-assembled biotinylated disulfide derivative monolayer on gold electrode for immobilizing enzymes

Based on self-assembled biotinylated disulfide derivative monolayer on gold electrode, the sensors immobilized monolayer or multilayer membranes composed of avidin and biotinlabeled glucose oxidase (B.GOD) or of avidin-B.GOD complex (ABC) and B.COD were prepared. The present technique may be useful for controlling the enzyme content of the sensors in molecular level by repeating the deposition of enzyme layers. The sensors have the characteristics of shorter response time, higher sensitivity. The linear range is from 6.0 x 10(-6) - 5.0 x 10(-3) M. The sensor can be used for more than 1 month and can be reactivated. The sensor was used to determine glucose in human blood serum, and the results are satisfactory.     

Amperometric enzyme sensor for glucose based on graphite paste-modified electrodes

Amperometric enzyme electrode for glucose is described based on the incorporation of glucose oxidase (GOD) into graphite paste modified with tetracyanoquinodimethane (TCNQ). The incorporated enzyme exhibits high activity and long-term stability over the earlier TCNQ-based glucose sensor (1). The sensor provides a linear response to glucose over a wide concentration range. The response time of the sensor is 15-50 sec, and the detection limit is 0.5 mM. Stable response to the substrate was obtained during a period of 35 d. Application of the sensor in the plasma analysis is reported.     

基于ZnO纳米线的螺旋线形跨尺度葡萄糖传感器

制备了一种基于螺旋线形跨尺度结构的酶传感器, 并对该传感器进行了表征和性能测试. 将ϕ 30 μm键合Au丝以螺旋线方式手工缠绕在ϕ 125 μm光纤纤芯上, 在该Au螺旋线上用水浴法合成ZnO纳米线, 得到螺旋线形跨尺度结构; 在ZnO纳米线上物理吸附葡萄糖氧化酶(GOD), 制备了葡萄糖传感器工作电极. 利用扫描电子显微镜(SEM)图像和MatLab图像处理算子分别对螺旋线形跨尺度结构表面形貌及其上活力为50 units/mg的GOD吸附效果进行了定性和定量表征, 分析了非高斯粗糙表面与GOD吸附效果的影响关系. 基于三电极体系采用循环伏安法和计时安培法测试了制备的12个工作电极的性能, 测得该类传感器的灵敏度为(1.410±0.665) μA·L/(mmol·cm²), 线性范围为0~(4.292±0.652) mmol/L, Michaelis-Menten常数为(3.571±1.280) mmol/L, 检出限为(14.085±8.393) μmol/L. 使用活力更高的GOD可以得到性能更好的螺旋线形跨尺度葡萄糖传感器. 该类传感器可广泛应用于医药、 生物、 食品加工及环境监测领域中尿酸、 尿素、 胆固醇、 过氧化氢和苯酚等的检测.     

Glucose oxidase-incorporated hydrogel thin film for fast optical glucose detecting under physiological conditions

Hydrogel biosensors usually suffer from a slow response, which severely hinders their practical applications. Here a new optical glucose biosensor was designed, using glucose-sensitive hydrogel films as both glucose-sensing material and Fabry-Perot cavity. The film was fabricated by layer-by-layer assembly from partially oxidized dextran (PO-Dex), chitosan, and glucose oxidase (GOD). The film responds to glucose because the incorporated GOD converts glucose to gluconic acid, and thus lowers the local pH in the film, and, in turn, triggers the pH-sensitive film to swell. The glucose-induced swelling causes a shift of Fabry?Perot fringes on the reflection spectra of the film, from which the glucose concentration can be reported. The new sensor works well under physiological conditions. Potential interferents, such as diols for phenylboronic acid-based sensors and electroactive compounds for electrochemical sensors, do not influence the new sensor. The sensor can respond reversibly over a wide range of glucose concentration. Particularly, it responds linearly within the clinically relevant glucose range (0–20 mM). More importantly, because the film is very thin, the new sensor can respond quickly, making it potential for real-time, continuous glucose monitoring.     

Amperometric biosensors for glucose based on carbon paste modified electrodes

The modification of carbon-paste electrodes by incorporation of the enzyme glucose oxidase (GOD) is described. The resulting probes can be operated as amperometric glucose sensors in the presence or absence of a mediator (1,1'-dimethylferrocene) mixed into the paste. Extended linear calibration ranges have been obtained up to 90 and 5OmM glucose respectively. The electrode responses were rapid, reaching steady-state values within 30-40 sec. Advantages of using a GOD-paste formulation are suggested. Plasma glucose assays were correlated with spectrophotometric determinations based on glucose oxidase (y = 1.07x - 0.16, r = 0.973, n = 17).     

纳米增强型毛细管酶柱用于葡萄糖液滴生物传感器的研究

葡萄糖的检测在临床医学以及食品工业等领域中十分重要.以往的检测方法主要包括化学发光法_{[1]、吸光光度法[2]、电化学法[3]和荧光法[4]等.固定化酶柱的制作是发展葡萄糖传感器的关键技术之一.传统的固定化方法主要是将具有生物活性的酶通过物理吸附、共价键合和交联的方法固定于载体基质上或包埋于有机聚合物的基质中.近期研究[5,6]表明,采用溶胶凝胶(Sol-gel)法将蛋白质和酶等生物活性物质包埋于无机陶瓷或玻璃材料内,保持生物组分的活性,且SiO2作为基质材料具有较好的坚固性、抗磨性、化学惰性以及高的光稳定性和透过性,但目前该法多用于电化学型生物传感器[7,8].本文利用纳米颗粒的比表面积大和吸附能力强等特点,将酶吸附在SiO2纳米颗粒表面,用易成膜的聚乙烯醇缩丁醛(PVB)作辅助基质在毛细管上固定酶,并采用分立式酶柱,克服了以往混合型酶柱普遍存在的酶促效率不高和使用寿命较短的局限性.所制得的酶柱具有表面反应活性高、表面活性中心多和催化效率高等特点.结合自行设计的液滴光化学传感装置[9,10],建立了一种高效、快速、微量的葡萄糖实时检测方法.}     

l-Glutamate biosensor for estimation of the taste of tomato specimens

An amperometric biosensor has been developed for measurement of Umami, or the taste based on the amount of L-glutamate, in tomato foods. The biosensor is based on an enzyme-mediator system in which L-glutamate oxidase is used for biochemical oxidation of L-glutamate and a tetrafulvalene-tetracyanoquinodimethane (TTF-TCNQ) paste, prepared from the mixture of TTF-TCNQ salt, graphite powder, and silicone oil, serves as the mediator. The limit of detection, calculated by use of a four-parameter logistic model, was 0.05 mmol L(-1), and the limit of quantification was 0.15 mmol L(-1). The correlation coefficient (R2) was 0.990 and the relative standard deviation was no more than 1% (n=5). The response time (tau (95)) was 20-50 s, depending on concentration. The repeatability of the sensor was better than 5% (n=10). The sensor developed was stable for more than ten days.     

Immobilized Fullerene C60‐Enzyme‐Based Electrochemical Glucose Sensor

A mixed‐valence cluster of cobalt(II) hexacyanoferrate and fullerene C60‐enzyme‐based electrochemical glucose sensor was developed. A water insoluble fullerene C60‐glucose oxidase (C60‐GOD) was prepared and applied as an immobilized enzyme on a glassy carbon electrode with cobalt(II) hexacyanoferrate for analysis of glucose. The glucose in 0.1 M KCl/phosphate buffer solution at pH = 6 was measured with an applied electrode potential at 0.0 mV (vs Ag/AgCl reference electrode). The C60‐GOD‐based electrochemical glucose sensor exhibited efficient electro‐catalytic activity toward the liberated hydrogen peroxide and allowed cathodic detection of glucose. The C60‐GOD electrochemical glucose sensor also showed quite good selectivity to glucose with no interference from easily oxidizable biospecies, e.g. uric acid, ascorbic acid, cysteine, tyrosine, acetaminophen and galactose. The current of H₂O₂ reduced by cobalt(II) hexacyanoferrate was found to be proportional to the concentration of glucose in aqueous solutions. The immobilized C60‐GOD enzyme‐based glucose sensor exhibited a good linear response up to 8 mM glucose with a sensitivity of 5.60 × 10² nA/mM and a quite short response time of 5 sec. The C60‐GOD‐based glucose sensor also showed a good sensitivity with a detection limit of 1.6 × 10^‐6 M and a high reproducibility with a relative standard deviation (RSD) of 4.26%. Effects of pH and temperature on the responses of the immobilized C60‐GOD/cobalt(II) hexacyanoferrate‐based electrochemical glucose sensor were also studied and discussed.     

Layer-layer assembly for extremely stable glucose sensors

A novel fabrication of an amperometric glucose sensor by layer after layer approach is described. The sensor electrode is fabricated by arranging a layer of Pt black, a layer of glucose oxidase (GOD) and a layer of stabilizer gelatin on a shapable electro-conductive (SEC) film surface. Finally, the dried layered-assembly is cross-linked by exposing to a diluted glutaraldehyde solution. The performance of the developed sensor is evaluated by a FIA system at 37°C and under a continuous polarization at 0.4 V (vs. Ag/AgCl). The sensitivity of the sensor was dependent on the amount of GOD loaded. The highest sensitivity (3.6 μA/mM cm⁻²) of the sensor was obtained at a GOD loading of 160 μg/cm², and the linear dynamic range was extended to 80 mM level when the sensor was covered with a polycarbonate membrane. The sensor shows an extremely stable response for several weeks and a storage stability of over 2 years.     

Application of polymaleimidostyrene as a convenient immobilization reagent of enzyme in biosensor

Sulfhydryl groups of glucose oxidase (GOD) were reacted with maleimide groups of polymaleimidostyrene (PMS) which was coated onto the porous carbon sheet, and the carbon sheet immobilized by GOD was combined with an oxygen electrode to fabricate a glucose sensor. The activity of thiolated GOD immobilized to PMS is much larger than that of native GOD immobilized to PMS. The good linear relationship of glucose and oxygen current response was obtained in a concentration range from 0.1 to 2 mM and upper limit of linear range was found to be 3.0 mM. The immobilized GOD activity is highly dependent on pH at immobilization and the maximum activity was obtained at pH 5.5, probably because the SH groups of GOD that are indispensable for generation of enzyme activity is not exposed at this pH. It was found that PMS is very effective reagent to immobilize enzyme strongly via covalent bond, because high density of maleimide groups of PMS can catch not only exposed SH groups but also buried SH groups.     

Preparation of the glucose sensor based on three-dimensional ordered macroporous gold film and room temperature ionic liquid

A novel type of glucose sensor was fabricated based on a glucose oxidase (GOD)-N,N-dimethtylformamide (DMF)-[BMIm][BF₄] composites modified three-dimensional ordered macroporous (3DOM) gold film electrode. The immobilized GOD exhibits a pair of well-defined reversible peaks in 50 mM pH 7.0 phosphate buffer solutions (PBS), which could be attributed to the redox of flavin adenine dinucleotide (FAD) in GOD. The research results show that ionic liquid ([BMIm][BF₄]), DMF and 3DOM gold film are crucial for GOD to exhibit a pair of stable and reversible peaks. It is believed that the large active area of 3DOM gold film can increase the amount of immobilized GOD. Simultaneously, the application of IL enhances the stability of GOD and facilitates the electron transfer between GOD and the electrode. The synergetic effect of DMF can help the GOD to maintain its bioactivity better. GOD immobilized on the electrode exhibits the favorable electrocatalytic property to glucose, and the prepared sensor has a linear range from 10 to 125 nM with a detection limit of 3.3 nM at a signal-to-noise ratio of 3σ. The apparent K _m (Michaelis- Menten constant) for the enzymatic reaction is 0.018 mM.     

Synthesis of Ag nanoparticle-decorated 2,4,6-tris(2-pyridyl)-1,3,5-triazine nanobelts and their application for H2O2 and glucose detection

The present paper reports on the first preparation of 2,4,6-tris(2-pyridyl)-1,3,5-triazine nanobelts (TPTNBs) by adjusting the pH value of the solution and the subsequent synthesis of Ag nanoparticle (AgNP)-decorated TPTNBs (AgNP-TPTNBs) by mixing an aqueous AgNO(3) solution with preformed TPTNBs without use of any external reducing agent. It is found that the resultant AgNP-TPTNBs exhibit notable catalytic performance for H(2)O(2) reduction. A glucose biosensor was fabricated by immobilizing glucose oxidase (GOD) onto a AgNP-TPTNBs-modified glassy carbon electrode (GCE) for glucose detection. The constructed glucose sensor has a wide linear response range from 3 mM to 20 mM (r: 0.999) with a detection limit of 190 μM. It is further shown that this glucose biosensor can be used for glucose detection in human blood serum.     

Development of glucose amperometric biosensor based on a novel attractive enzyme immobilization matrix: amino derivative of thiacalix[4]arene

Calixarenes and their derivatives may be a promising material for enzyme immobilization owing to their particular configuration, unique molecule recognition function and aggregation properties. In this paper, p-tert-butylthiacalix[4]arene tetra-amine (TC4TA) was first used as enzyme immobilization material. This attractive material was exploited for the mild immobilization of glucose oxidase (GOD) to develop glucose amperometric biosensor. GOD was strongly adsorbed on the TC4TA modified electrode to form TC4TA/GOD composite membrane. The adsorption mechanism was driven from the covalent bond between amino-group of TC4TA and carboxyl group of GOD and molecule recognition function of TC4TA. Amperometric detection of glucose was evaluated by holding the modified electrode at 0.60 V (versus SCE) to oxidize the hydrogen peroxide generated by the enzymatic reaction. The sensor (TC4TA/GOD) showed a relative fast response (response time was about 5 s), low detection limit (20 μM, S/N = 3), and high sensitivity (ca. 10.2 mA M⁻¹ cm⁻²) with a linear range of 0.08–10 mM of glucose, as well as a good operational and storage stability. In addition, optimization of the biosensor construction, the effects of the applied potential as well as common interfering compounds on the amperometric response of the sensor were investigated and discussed herein.     

The effect of the layer structure on the activity of immobilized enzymes in ultrathin films

The molecular engineering capability of the layer-by-layer (LbL) method for fabricating thin films has been exploited in order to immobilize glucose oxidase (GOD) in films with alternating layers of chitosan. Chitosan was proven a good scaffolding material, as GOD molecules preserved their catalytic activity towards glucose oxidation. Using electrochemical measurements, we showed that chitosan/GOD LbL films can be used to detect glucose with a limit of detection of 0.2 mmol l-1 and an activity of 40.5 microA mmol-1 L microg-1, which is highly sensitive when compared to other sensors in previous reports in the literature. The highest sensitivity of the LbL film was achieved when only the top layer contained GOD, thus indicating that GOD in inner layers did not contribute to glucose oxidation, probably because it hampers analyte diffusion and electron transport through the deposited layers. This may be explained by the dense packing of GOD molecules in the LbL films with chitosan, as inferred from estimates of the amount of GOD adsorbed per layer using a quartz crystal microbalance.     

Activated platinum electrodes as transducer for a glucose sensor using glucose oxidase in a photopolymer membrane

A planar platinum electrode was covered by a photopolymer membrane containing glucose oxidase (GOD) to construct an amperometric glucose sensor. The application of a photopolymer system in membrane formation gives the opportunity to manufacture cheap biosensors with good reproducibility by means of automated techniques and to miniaturise sensors using photolithography. The electrodes were pretreated mechanically and chemically resulting in a half-wave potential (E_1/2) of the H₂O₂ oxidation shifted towards more negative potentials. This shift allows the determination of glucose at a low working potential (300 mV vs. SCE) without addition of mediators. The important advantage of such applied potential decreasing lies in minimising the interference of oxidisable substances such as uric acid, bilirubin and paracetamol. The selectivity to ascorbic acid could also be proved without the application of additional protection layers. The glucose sensor developed has a high life-time, selectivity and sensitivity and a linear working concentration range from 0.05 up to 10 mmol/l of glucose. The sensor was used for the glucose determination in human serum samples with a very good correlation to a common photometric reference method. Received: 13 July 1996 / Revised: 11 September 1996 / Accepted: 14 September 1996     

Fully reversible fibre-optic glucose biosensor based on the intrinsic fluorescence of glucose oxidase

In a new type of glucose biosensor, the intrinsic green fluorescence of glucose oxidase (GOD) is used to provide the analytical information. It was found that the fluorescence of GOD changes during interaction with glucose. Fluorescence is excited at 450 nm and measured at ? 500 nm, which is a wavelength range that is compatible with glass and plastic fibres. The signal response is fully reversible because oxygen is a second substrate. A major feature of this sensor relies on the fact that the recognition element is identical with the transducer element.Enzyme solutions are entrapped at the fibre end within a semipermeable membrane. The change in fluorescence occurs over a small glucose concentration range (typically 1.5–2 mM), the signal at lower and higher glucose levels being unaffected by changes in glucose concentration. Response times of 2–30 min and regeneration times of 1–10 min are observed. Effects of pH and oxygen concentrations are also investigated. To achieve as extended analytical range (e.g., 2.5–10 mM) and shorter response times, kinetic measurements are suggested.     

纳米聚苯胺修饰石墨电极的葡萄糖双酶传感器

用循环伏安法在石墨电极上制得纳米纤维聚苯胺, 并在其上固定葡萄糖氧化酶(GOD)和辣根过氧化物酶(HRP)制备葡萄糖双酶传感器. 用交流阻抗、SEM等技术对其进行表征; 考察了各种因素对双酶电极响应电流的影响以及双酶电极的稳定性. 该传感器对葡萄糖响应电流的测定在0.05 V(vs SCE)下进行, 有效避免了电活性物质的影响, 线性响应范围为0.05-2.0 mmol·L-1.     

基于室温离子液体的有序多孔金膜葡萄糖传感器

将1-丁基-3-甲基咪唑四氟硼酸盐（[BMIm][BF4]）、N,N-二甲基甲酰胺（DMF）与葡萄糖氧化酶（GOD）的混合物修饰于三维有序大孔（3DOM）金膜电极上,构建了一种新型的葡萄糖传感器．固定的GOD在pH7．0的磷酸缓冲液（PBS）中展现出一对可逆性好的氧化还原峰,这归因于GOD的活性中心黄素腺嘌呤二核苷酸（FAD）的直接电化学行为．研究表明,离子液体（IL）、DMF以及3DOM金膜对GOD的直接电化学都起到了重要的作用．3DOM金膜修饰电极作为基底提高了酶的负载量,加速了GOD与电极表面的电子传递;IL的应用增加了固定GOD的电化学活性;DMF与IL、GOD的协同作用更好地保持了GOD的生物活性．固定在电极表面的GOD对葡萄糖显示出良好的催化性能,其检测线性范围为10～125nmol／L,检测限为3．3nmol／L（S／N=3）,酶催化反应的表观米氏常数Km为0．018mmol／L．     

Glucose oxidase electrodes of polyaniline,poly(o‐toluidine) and their copolymer as a biosensor: a comparative study

A simple technique is described for constructing a glucose sensor by the entrapment of glucose oxidase (GOD) in a polyaniline (PA), poly(o‐toluidine) (POT) and their copolymer poly(aniline‐co‐o‐toluidine) (PA‐co‐POT) thin films, which were electrochemically deposited on a platinum plate in phosphate and acetate buffer. The maximum current response was observed for PA, POT, and PA‐co‐POT GOD electrodes at pH 5.5 and potential 0.60 V (v. Ag/AgCl). The phosphate buffer gives fast response as compared to acetate buffer in amperometric measurements. PA GOD electrode shows the fastest response followed by PA‐co‐POT and POT GOD electrodes. Copyright © 2004 John Wiley & Sons, Ltd.     

Enzyme-entrapping membranes for enzyme sensors

A reliable method of physically immobilizing enzymes in cellulose triacetate (TAC) membranes was developed. The method has several advantages compared with analogous ones currently employed; it was possible to prepare enzyme sensors based on immobilized glucose oxidase (GOD) for determination of glucose in standard solutions and control sera, and based on GOD and invertase for determination of sucrose.