An autoclavable glucose biosensor for microbial fermentation monitoring and control

The design, construction, and characterization of a prototype-regenerable glucose biosensor based on the reversible immobilization of glucose oxidase (GOx) using cellulose binding domain (CBD) technology is described. GOx, chemically linked to CBD, is immobilized by binding to a cellulose matrix on the sensor-indicating electode. Enzyme immobilization can be reversed by perfusing the cellulose matrix with a suitable eluting solution. An autocavable sensor membrane system is employed which is shown to be practical for use in real microbial fermentations. The prototype glucose biosensor was used without failure or deterioration during fed-batch fermentations of Escherichia coli reaching a maximum cell density of 85 g (dry weight)/L. Medium glucose concentration based on sensor output correlated closely with off-line glucose analysis and was controlled manually at 0.44 +/- 0.2 g/L for 2 h based on glucose sensor output. The sensor enzyme component could be eluted and replaced without interrupting the fermentation. To our knowledge, no other in situ biosensor has been used for such an extended period of time in such a high-cell-density fermentation. (c) 1995 John Wiley & Sons, Inc.     

Amperometric determination of glucose,based on the direct electron transfer between glucose oxidase and tin oxide

An amperometric glucose biosensor was designed for the detection of glucose in blood, urine, beverages, and fermentation systems. In typical glucose biosensors that employ enzymes, mediators are used for efficient electron transfer between the enzymes and the electrode. However, some of these mediators are known to be toxic to the enzymes and also must be immobilized on the surface of the electrode. We propose a mediator-free glucose biosensor that uses a glucose oxidase immobilized on a tin oxide electrode. Direct electron transfer is possible in this system because the tin oxide has redox properties similar to those of mediators. The method for immobilization of the glucose oxidase onto the tin oxide is also very simple. Tin oxide was prepared by the anodization and annealing of pure tin, and this provides a large surface area for the immobilization step because of its porosity. Glucose oxidase was immobilized onto the tin oxide using the membrane entrapment method. The proposed method provides a simple process for fabricating the enzyme electrode. Glucose oxidase immobilized onto the tin oxide, prepared in accordance with this method, has a relatively large current response when comparedto those of other glucose biosensors. The sensitivity of the biosensor was 19.55 μA/mM, and a linear response was observed between 0∼3 mM glucose. This biosensor demonstrated good reproducibility and stability.     

Novel redox surfactants and their interactions with glucose oxidase of Aspergillus niger

A number of novel redox surfactants (based on mixed bipyridine/dipyridylamine complexes of osmium (II) where the dipyridylamine ligands bears a saturated C(8), C(10), C(12), C(14), or C(16) alkyl chain) were synthesized and characterized electrochemically and biochemically as mediators for glucose oxidase (EC 1.1.3.4, GOD) of Aspergillus niger. These compounds exhibited critical micelle concentrations (CMCs) in phosphate-buffered saline solution (pH 7.4) in the range 10(-4) 10 10(-3) M, the value decreasing with increasing chain length. Dependence of a number of properties (speed of mediation, redox potential, denaturing action on the enzyme, adsorption on an electrode surface) on the length of the mediator alkyl chain was observed. The presence of an alkyl chain decreased the rate of mediation relative to otherwise similar nonsurfactant mediators, and the longer alkyl chain, the slower the rate of mediation. For each compound, mediation above the CMC was about tenfold slower than that observed below the CMC. However, for the cases of mediator absorbed on an electrode surface with GOD, longer chains give increased physisorption of mixed micelles of enzyme and mediator. The compounds were incidentally found to inhibit the glucose oxidase activity of GOD in a complex manner; inhibition increased with increasing chain length and the deactivation, for any given compound, was more pronounced below the CMC than above. Glucose oxidase activity assays and study of the action of surfactants and mediators on the fluorescent properties of carboxy-fluorescein-labeled GOD led to the consideration of a model for redox surfactant-GOD interaction where three mechanisms may operate: first, a selective interaction of mediators with the GOD active site; second, a nondenaturing association of short-chain (相似文献   

Optimization of a polypyrrole glucose oxidase biosensor

An amperometric glucose biosensor was fabricated by the electrochemical polymerization of pyrrole onto a platinum electrode in the presence of the enzyme glucose oxidase in a KCl solution at a potential of + 0·65 V versus SCE. The enzyme was entrapped into the polypyrrole film during the electropolymerization process. Glucose responses were measured by potentio-statting the enzyme electrode at a potential of + 0·7 V versus SCE in order to oxidize the hydrogen generated by the oxidation of glucose by the enzyme in the presence of oxygen. Experiments were performed to determined the optimal conditions of the polypyrrole glucose oxidase film preparation (pyrrole and glucose oxidase concentrations in the plating solution) and the response to glucose from such electrodes was evaluated as a function of film thickness, pH and temperature. It was found that a concentration of 0·3 M pyrrole in the presence of 65 U/ml of glucose oxidase in 0·01 M KCl were the optimal parameters for the fabrication of the biosensor. The optimal response was obtained for a film thickness of 0·17 μm (75 mC/cm²) at pH 6 and at a temperature of 313 K. The temperature dependence of the amperometric response indicated an activation energy of 41 kJ/mole. The linearity of the enzyme electrode response ranged from 1·0 mM to 7·5 mM glucose and kinetic parameters determined for the optimized biosensors were 33·4 mM for the K_m and 7·2 μA for the I_max. It was demonstrated that the internal diffusion of hydrogen peroxide through the polypyrrole layer to the platinum surface was the main limiting factor controlling the magnitude of the response of the biosensor to glucose. The response was directly related to the enzyme loading in the polypyrrole film. The shelf life and the operational stability of the optimized biosensor exceed 500 days and 175 assays, respectively. The substrate specificity of the entrapped glucose oxidase was not altered by the immobilization procedure.     

Shortening the biosensor assay by use of enzyme-containing temperature-sensitive hydrogels

To shorten the biosensor assay time, a new technique, based on the increasing of mass transfer rate between the enzyme-containing membrane and the bulk solution as a result of phase transition of stimuli-sensitive matrix (swelling-shrinkage processes) under the action of an outer signal, is proposed. For a model system – glucose oxidase immobilized into thermosensitive poly-N-isopropylacrylamide gel – the total time of the assay procedure can be halved.     

Enzyme-entrapping membranes for biosensors obtained by radiation-induced polymerization

A new method of physically immobilizing enzymes in poly(2-hydroxyethyl methacrylate) membranes was developed in order to obtain suitable biosensors. It was possible to prepare an enzyme sensor based on an oxygen Clark electrode and on glucose oxidase immobilized by low-temperature gamma radiation-induced polymerization. Temperature and pH effects on the activity of immobilized enzyme are described and the response characteristics of the resulting biosensor are summarized. The determination of glucose in standard solutions was carried out and a linear calibration curve, with an R² value of 0·9993, from the detection limit 5 × 10⁻⁵ to 1·2 × 10⁻³ was obtained. The biosensor was employed to analysis of control sera and the results were compared to those obtained by enzymatic-spectrophotometric detection.     

Stable mediated amperometric biosensors using a graphite electrode embedded with tetrathiafulvalene in silicone oil

A new method has been developed to incorporate the mediator, tetrathiafulvalene (TTF), to the electrode/solution interface of an amperometric biosensor. TTF was dissolved in methylphenyl polysiloxane (silicone oil) and embedded in a graphite disc electrode. The mediator was able to diffuse to the electrode surface at an electrocatalytically significant speed. The storage of TTF in the inert polysiloxane provided a long-lasting and stable mediator supply.

TTF-silicone oil electrodes with immobilized glucose oxidase, xanthine oxidase, or amino acid oxidase exhibited sensitive, fast and reproducible responses. The glucose oxidase electrode was very stable for at least 2 months when stored at 4°C. Together with flow injection analysis (FIA), the enzyme electrodes were reused for at least 500 repeated analyses during a 25 h operation without losing their initial activity.     

Immobilization of glucose oxidase in thin polypyrrole films: influence of polymerization conditions and film thickness on the activity and stability of the immobilized enzyme

Using monomers that polymerize to form electrically conducting polymers, one can control the thickness of the polymer film and the amount of enzyme that can be immobilized in the films. First, an investigation of the major variables that influence the immobilization of glucose oxidase by entrapment in polypyrrole films, prepared by electropolymerization from aqueous solutions containing the enzyme and monomer, was carried out. Then the optimized conditions were used to assess the effects of film thickness on the activity and stability of immobilized enzyme. For the films ranged in thickness from 0.1 mum to 1.6 mum, the resulting apparent activity and stability of the immobilized enzyme were found to be a strong function of the polymer film thickness. Above a thickness of 1.0 mum, the apparent activity of the immobilized enzyme increases linearly with increasing film thickness. The nonlinearity observed for films of thickness less than 1.0 mum can be attributed to the changes observed in the morphology of the resulting polypyrrole films. Furthermore, it was noted that when the glucose oxidase/polypyrrole films are stored in phosphate buffer, at 4 degrees C, the observed rate of loss in apparent activity of the immobilized enzyme is highest for the first few days, also being higher for the thinner films. However, after the loosely entrapped enzyme is leached from the polymer film, the rate of loss in activity is very low indicating that the well-entrapped enzyme, as well as the polypyrrole films, exhibit good stability. Finally, the reproducibility of the immobilization technique is excellent. (c) 1993 John Wiley & Sons, Inc.     

葡萄糖氧化酶在枯草芽孢杆菌芽孢表面的展示及其酶电极制备

葡萄糖生物传感器是目前最为常见的电化学生物传感器,绝大多数葡萄糖生物传感器采用在电极表面修饰葡萄糖氧化酶的方法来制备,但是,在电极的固定化过程中需要酶的纯化,使得成本增加,已成为固定化酶电极开发领域的瓶颈。文中主要以芽孢衣壳蛋白CotX为锚定蛋白将葡萄糖氧化酶(Glucose oxidase,GOD)展示到枯草芽孢杆菌芽孢表面,通过Western blotting分析、免疫荧光分析以及酶活检测均证明GOD在芽孢表面有效表达,发酵获得重组芽孢(Spore-GOD)。再采用滴涂法和电沉积法制备了氧化石墨烯/普鲁士蓝沉积膜修饰玻碳电极,将Spore-GOD固定在修饰电极表面,最后滴加一层Nafion溶液,制成了电化学生物传感器,用于葡萄糖的灵敏测定。葡萄糖在该酶电极传感器上的循环伏安图表明,该反应在0.42V处出现明显的氧化峰,并且氧化还原峰电流与葡萄糖浓度在0.1–7.0mmol/L之间具有良好的线性关系,校正曲线方程为:I=1.3047C_glucose+3.639(R²=0.9929),其检测限为7.5μmol/L(S/N=3)。此修饰电极具有...     

Development of a disposable glucose biosensor using electroless-plated Au/Ni/copper low electrical resistance electrodes

This paper presents a glucose biosensor, which was developed using a Au/Ni/copper electrode. Until now, research regarding the low electrical resistance and uniformity of this biosensor electrode has not been conducted. Glucose oxidase (GOD) immobilized on the electrode effectively plays the role of an electron shuttle, and allows glucose to be detected at 0.055 V with a dramatically reduced resistance to easily oxidizable constituents. The Au/Ni/copper electrode has a low electrical resistance, which is less than 0.01 Ω, and it may be possible to mass produce the biosensor electrode with a uniform electrical resistance. The low electrical resistance has the advantage in that the redox peak occurs at a low applied potential. Using a low operating potential (0.055 V), the GOD/Au/Ni/copper structure creates a good sensitivity to detect glucose, and efficiently excludes interferences from common coexisting substances. The GOD/Au/Ni/copper sensor exhibits a relatively short response time (about 3 s), and a sensitivity of 0.85 μA mM⁻¹ with a linear range of buffer to 33 mM of glucose. The sensor has excellent reproducibility with a correlation coefficient of 0.9989 (n = 100 times) and a total non-linearity error of 3.17%.     

Comparative study of hydrogel-immobilized l-glutamate oxidases for a novel thick-film biosensor and its application in food samples

Novel, thick-film biosensors have been developed for the determination of l-glutamate in foodstuffs. The sensors were prepared by immobilization of l-glutamate oxidase by using polycarbamylsulfonate-hydrogel on a thick-film sensor. l-Glutamate oxidases obtained from Streptomyces sp. with different degree of purification were compared with their characteristic response to l-glutamate at different conditions and for their specificity, inhibition, and storage properties. These sensors were applied to determine monosodium glutamate in soy sauce samples and show good correlation with colorimetric method.     

三种藻胆蛋白在复合累积LB膜中的能量传递

制备了R-藻红蛋白(R-PE)、C-藻蓝蛋白(C-PC)及变藻蓝蛋白(APC)的单组分累积LB膜及三组分复合累积LB膜. 吸收及荧光光谱测定表明三种蛋白的LB膜的光谱重叠性质与他们在水溶液中的性质相同. 结构测定表明, 三种蛋白在其LB膜中排列有序, 且由它们组装的复合累积LB膜结构类似于藻类植物中的藻胆体结构. 通过稳态荧光光谱及其光谱解叠, 观察到了三种蛋白在复合累积LB膜中的激发能量传递现象. 根据给体荧光峰的猝灭, 计算出在复合累积LB膜中从给体R-PE经C-PC到受体APC的能量传递效率为51%.     

NAD(P)H oxidase isoform Nox2 plays a prosurvival role in human leukaemia cells

The mechanism involved in the prosurvival effect of interleukin-3 on the human acute myeloid leukaemia cell line M07e is investigated. A decrease in intracellular reactive oxygen species (ROS) content, glucose transport activity and cell survival was observed in the presence of inhibitors of plasma membrane ROS sources, such as diphenylene iodonium and apocynin, and by small interference RNA for Nox2. Moreover, IL-3 incubation stimulated the synthesis of Nox2 cytosolic sub-unit p47phox and glucose transporter Glut1. Thus, the inhibition of ROS generation by Nox inhibitors stimulated apoptosis showing that ROS production, induced by IL-3 via Nox2, protects leukaemic cells from cell death. Also incubation with receptor tyrosine kinase inhibitors, such as anti-leukaemic drugs blocking the stem cell factor receptor (c-kit), showed similar effects, hinting that IL-3 transmodulates c-kit phosphorylation. These mechanisms may play an important role in acute myeloid leukaemia treatment, representing a novel therapeutic target.     

二茂铁－交联剂修饰的葡萄糖传感器

用年清白蛋白－戊二醛交联剂把葡萄糖氧化酶固定在Ｎａｆｉｏｎ－二茂铁修饰电极上,最后在电极上修饰一层Ｎａｆｉｏｎ膜,制备成葡萄糖传感器,电活物质如抗坏血酸,尿酸等对葡萄糖的测定无干扰。该传感器的线性范围为５．０×１０＾－＾４－１．３×１０＾－＾２ｍｏｌ／Ｌ,响应时间小于６０ｓ。     

二茂铁－交联剂修饰的葡萄糖传感器

用牛血清白蛋白-戊二醛交联剂把葡萄糖氧化酶固定在Nafion-二茂铁修饰电极上,最后在电极上修饰一层Nafion膜,制备成葡萄搪传感器,电活性物质如抗坏血酸、尿酸等对葡萄糖的测定无干扰.该传感器的线性范围为5.0×10^-4-1.3×10^-2mol/L,响应时间小于60s.