Monitoring glucose consumption in an Escherichia coli cultivation with an enzyme electrode

An autoclavable enzyme electrode, the externally buffered glucose oxidase electrode, is used to monitor the glucose consumption during batch cultivations of E. coli. The electrode signal showed good correlation with data from a conventional procedure and was independent of the dissolved oxygen concentration in the fermentation broth.     

Effect of enzyme concentration on the dynamic behavior of a membrane-bound enzyme system

The dynamic behavior of the reaction-diffusion system, composed of glucose oxidase (EC 1.1.3.4) immobilized at a uniform concentration in a membrane, used as a glucose electrode is represented by a diffusion equation with a nonlinear reaction-term in one-dimensional space. The mathematical model is analyzed by computer simulation, that is, numerical integration of the equation under various initial and boundary conditions, to examine the effect of enzyme concentration on the response characteristics (responsiveness and linearity in response) of the electrode. The analysis of the responses of the system to stepwise changes in the boundary value (glucose concentration in simple solution) infers that the enzyme concentration governs the patterns of the spatial distributions of the substrates (glucose and dissolved oxygen) in steady states and transient responses. It is also revealed that the response characteristics of the electrode are optimized with concentration of immobilized enzyme and that the system establishes the steady states at the same spatial distributions of the substrates, regardless of the boundary value. The diffusion of the substrates and the oxygen concentration also have significant effects on the response characteristics of the electrode.     

Development of Anti-Interference Enzyme Layer for α -Amylase Measurements in Glucose Containing Samples

Abstract

A glucose oxidase-glucoamylase-bienzyme electrode has been developed and tested for determination of α-amylase activity. To eliminate interfering endogeneous glucose a glucose oxidase-catalase anti-interference layer was coupled with the bienzyme electrode. Linearity was obtained for the kinetic signal up to 1.0 I.U. α-amylase. Glucose was effectively eliminated up to 2 mM final concentration thus not influencing α-amylase determinations. A general concept for anti-interference enzyme layers is suggested.     

Penicillin fermentation in a 200-Liter tower fermentor using cells confined to microbeads

The scale-up of the penicillin fermentation through cell confinement in a 200-L tower fermentor is described.P. chrysogenum spores were adsorbed into Celite microbeads having diameters greater than 180 Μm. Fed-batch fermentations were performed using both free and confined cells. Cell growth and penicillin concentrations were measured during the fermentation. In addition, the oxygen transfer rate, the aeration rate, and the level of dissolved oxygen were also measured. Significant improvement in the mass transfer coefficient was found when the cells were anchored onto the microbeads. This improved oxygen transfer rate was accompanied by higher production of penicillin at a lower aeration rate. Besides the improved oxygen transfer rate into the mycelial broth, a reduction of the energy input for the oxygen transfer was observed. The confinement of the cells to this microcarrier furthermore allowed the in-termittent harvesting of fermentation broth without reducing the cell mass in the fermentor.     

Study on the United Glucose Oxidase Electrode Constructed by Composite Electrode

A novel type of united glucose oxidase (GOD) electrode was designed. Glucose oxidase and ferrocene (Fc), which was a mediator, were added into the composite electrode that was constructed by graphite powder, acetylene black, and epoxy resin. These three materials in composite electrode kept constant proportion in weight. And the optimum amounts of GOD and Fc among united enzyme electrode were 5% and 2%, respectively. The glucose was detected linearly in the concentration range 0.01–9.0 mM with a 20-s steady-state response time and 36 nA/mM of the sensitivity at 0.15 V applied potential. And electrode fouling problem and the response current from the interferents were avoided. The response current of the united GOD electrode had no obvious deterioration within 80 days when stored at 4°C in a refrigerator. The detecting results of human serum by the united GOD electrode had good consistency with that by standard enzyme method. The maximum deviation between these two detecting values was 5%. It might be used for detecting the blood sugar in clinical assay.     

Electrocatalytic and Biosensing Properties of Aerobic Carbon Monoxide Dehydrogenase from Hydrogenophaga Pseudoflava Immobilized on Au Electrode towards Carbon Monoxide Oxidation

Herein, an enzyme‐electrode based on the oxygen‐insensitive carbon monoxide dehydrogenase (CODH) containing molybdenum (Mo) and copper (Cu), flavin adenine dinucleotide (FAD) and two different [2Fe‐2 S] clusters as cofactors from the aerobic bacterium Hydrogenophaga pseudoflava, is proposed as a platform for dissolved CO concentration monitoring. The immobilized CODHs on Au electrode retain their catalytic activity and demonstrates changes to cyclic voltammetry and amperometry signals upon interactions with various dissolved CO concentrations. Cyclic voltammetry shows that CODHs are capable of direct electron transfer without any mediator use as oxidative current which starts around ?0.268 V (vs Ag/AgCl) is observed in the presence of CO. When CO‐saturated standard solution was spiked sequentially into the gas‐tight reactor, amperometry analysis shows current increased accordingly with response time within 5 s. Our study demonstrates that this enzyme‐electrode is promising to serve as platform for developing an on‐line dissolved CO concentration monitoring tool which is essential to fill in the gap for conventional technologies which are limited to off‐line measurement.     

Preparation and application of a new glucose sensor based on iodide ion selective electrode

An electrode for glucose has been prepared by using an iodide selective electrode with the glucose oxidase enzyme. The iodide selective electrode used was prepared from 10% TDMAI and PVC according our previous study. The enzyme was immobilized on the iodide electrode by holding it at pH 7 phosphate buffer for 10 min at room temperature. The H₂O₂ formed from the reaction of glucose was determined from the decrease of iodide concentration that was present in the reaction cell. The iodide concentration was followed from the change of potential of iodide selective electrode. The potential change was linear in the 4×10⁻⁴ to 4×10⁻³ M glucose concentration (75-650 mg glucose/100ml blood) range. The slope of the linear portion was about 79 mV per decade change in glucose concentration. Glucose contents of some blood samples were determined with the new electrode and consistency was obtained with a colorimetric method. The effects of pH, iodide concentration, the amount of enzyme immobilized and the operating temperature were studied. No interference of ascorbic acid, uric acid, iron(III) and Cu(II) was observed. Since the iodide electrode used was not an AgI-Ag₂S electrode, there was no interference of common ions such as chloride present in biological fluids. The slope of the electrode did not change for about 65 days when used 3 times a day.     

Glucose Biosensor Based on Oxygen Electrode Part III: Long-Term Performance of the Glucose Biosensor in Blood Plasma at Body Temperature

Abstract

A potentially implantable glucose biosensor for continuous monitoring of glucose levels in diabetic patients has been developed. The glucose biosensor is based on an amperometric oxygen electrode and Glucose Oxidase immobilized on carbon powder held in a form of a liquid suspension. The enzyme material can be replaced (the sensor recharged) without sensor disassembly. Glucose diffusion membranes from polycarbonate (PC) and from polytetrafluorethylene (PTFE) coated with silastic are used.

Sensors were evaluated continuously operating in phosphate buffer solution and in undiluted blood plasma at body temperature. Calibration curves of the sensors were periodically obtained. The sensors show stable performance during at least 1200 hours of operation without refilling of the enzyme. The PTFE membrane demonstrates high mechanical stability and is little effected by long-term operation in undiluted blood plasma.     

The Amperometric Glucose Enzyme Electrode by Immobilized Glucose Oxidase in Glucose Oxidase/N-ethyl-Poly (4-vinyl)pyridine Multilayer

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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.     

Preparation,characterization, and potential application of an immobilized glucose oxidase

A simple, one-step process, using 0.25Mp-benzoquinone dissolved in 20% dioxane at 50°C for 24 h was applied to the activation of polyacrylamide beads. The activated beads were reacted with glucose oxidase isolated fromAspergillus niger. The coupling reaction was performed in 0.1M potassium phosphate at pH 8.5 and 0–4°C for 24 h. The protein concentration was 50 mg/mL. In such conditions, the highest activity achieved was about 100 U/g solid. The optimum pH for the catalytic activity was shifted by about 1 pH unit in the acidic direction to pH 5.5. Between 35 and 50°C, the activity of the immobilized form depends on the temperature to a smaller extent than that of the soluble form. Above 50°C, the activity of immobilized glucose oxidase shows a sharper heat dependence. The enzyme-substrate interaction was not profoundly altered by the immobilization of the enzyme. The heat resistance of the immobilized enzyme was enhanced. The immobilized glucose oxidase is most stable at pH 5.5. The practical use of the immobilized glucose oxidase was tested in preliminary experiments for determination of the glucose concentration in blood sera.     

Enzymatic formation of ions and their detection at a three-phase electrode

An electrochemical method for the detection of enzymatically created anions is described that uses a thin-film electrode with decamethylferrocene as an electroactive redox probe. The enzymatic oxidation of glucose with enzyme glucose oxidase produces gluconic acid as a final product. The oxidation of decamethylferrocene dissolved in the thin-nitrobenzene film, that is spread on the working graphite electrode and submerged in the aqueous solution containing glucose and glucose oxidase, is followed by the up-take of gluconate anions from the aqueous phase to nitrobenzene. The peak currents of the square-wave voltammetric responses of that system are a linear function of the glucose concentration in the milimolar range from 0.1 mmol/L to 0.7 mmol/L (R²=0.994).     

Improved Blood Compatibility at a Glucose Enzyme Electrode Used for Ejctraoorpokeal Monitoring

Abstract

A glucose enzyme electrode based on glucose oxidase has been used in an extracorporeal flow system which allows standardization during monitoring. The detection limit for glucose was 0.02 mmol/l, and the linear range, which extended to 5 mmol/l glucose, allowed measurement of whole blood after dilution. Restandardization following whole blood exposure led to a loss of sensitivity which was attributed to coating of electrode membranes by blood constituents. The problem was largely overcome through use of silane-treated membranes and the operation of electrodes in a narrow channel flow cell.     

Which Parameters Affect the Response of the Channel Biosensor?

The important parameters in defining the response of the portable channel biosensor described previously are explored by connecting the portable flow cell to a gravity feed flow system and using a highly defined enzyme immobilization protocol which ensures the enzyme reaction is a surface reaction. The enzyme glucose oxidase (GOD) was immobilized by covalent attachment to a self‐assembled monolayer modified gold surface. As a glucose solution flowed down the rectangular duct defined by the flow cell, it passed over the enzyme layer where the enzyme reaction produced hydrogen peroxide. The hydrogen peroxide was swept further downstream to the detector electrode. The response of such an enzyme electrode was shown to be limited by mass transport of the cosubstrate oxygen to the enzyme layer. Increasing the amount of oxygen in the sample meant the response of the biosensor became limited by the enzyme kinetics. The influence of parameters such as flow rate, height of the channel, enzyme layer length and the gap between the enzyme layer and the detector electrode were explored.     

中性红掺杂SiO2纳米粒子修饰的酶阵列传感器同时测定葡萄糖、乳酸和L-谷氨酸的研究

本文以中性红为核，二氧化硅为壳，利用反相微乳液技术，通过正硅酸四乙酯的水解制备了掺杂有中性红的二氧化硅纳米粒子，并用TEM技术进行了表征。核中性红能够催化测定葡萄糖，乳酸和L-谷氨酸的反应，而壳二氧化硅不仅克服了电活性物质中性红易流失的缺点，且具有高的生物亲和性。分别与葡萄糖氧化酶、乳酸氧化酶以及L-谷氨酸氧化酶混合后，修饰在碳阵列电极表面。最后在该酶阵列电极表面滴加一层Nafion, 防止电活性物质抗坏血酸、尿酸等的干扰。该酶阵列传感器与流动注射分析技术(FIA)相结合，可应用于同时检测大鼠血样中的葡萄糖，乳酸和L-谷氨酸浓度。该方法无需通过传统的色谱柱的分离，大大简化了实验条件，为这一领域的研究提供了有效的分析方法。     

Determination of the Apparent Michaelis Constant of Glucose Oxidase Immobilized on a Microelectrode with Respect to Oxygen

We report determination of the apparent Michaelis constant of glucose oxidase (GOx) immobilized on a microelectrode with respect to oxygen. We used a GOx‐modified microelectrode as a probe for scanning electrochemical microscopy. We detected hydrogen peroxide generated by the enzyme reaction at the microelectrode under controlling the oxygen concentration using water electrolysis at an interdigitated array (IDA) electrode. The response depends on the oxygen concentration, which is regulated by the microelectrode position and the potential applied to the IDA electrode. We estimated the apparent Michaelis constant with respect to oxygen in this experimental condition to be about 0.28 mM.     

The Application of Engineered Glucose Dehydrogenase to a Direct Electron–Transfer‐Type Continuous Glucose Monitoring System and a Compartmentless Biofuel Cell

Abstract

Continuous glucose monitoring (CGM) is expected to become an ideal way to monitor glycemic levels in diabetic patients. On the other hand, biofuel cells can be used as an alternative energy source in future implantable devices, such as implantable glucose sensors in the artificial pancreas. Glucose dehydrogenase from Acinetobacter calcoaceticus, which harbors pyrroloquinoline quinone as the prosthetic group (PQQGDH), is one of the enzymes most attractive as a glucose sensor constituent and as the anode enzyme in biofuel cells, due to its high catalytic activity and insensitivity to oxygen. However, the application of PQQGDH for these purposes is inherently limited because an electron mediator is required for the electron transfer to the electrode.

We have recently reported on the development of an engineered enzyme, quinohemoprotein glucose dehydrogenase (QH‐GDH), in which the cytochrome c domain of the quinohemoprotein ethanol dehydrogenase (QH‐EDH) was fused with PQQGDH, to enable electron transfer to the electrode in the absence of an artificial mediator. In this study, we constructed a direct electron‐transfer‐type CGM system employing QH‐GDH. This CGM system showed sufficient current response and high operational stability. Furthermore, we successfully constructed a compartmentless biofuel cell employing QH‐GDH.     

纳米级金膜微电极的制作,表征及异相催化反应

报道了纳米级金膜微电极的制作方法，用ＸＰＳ及ＳＥＭ对电极表面进行了表征，考察了该电极的循环伏安及计时电流特性，在聚吡咯修饰微带金电极上成功地实现了葡萄糖氧化酶和电子传递媒体Ｆｅ（ＣＮ）６＾３－的同时固定，并研究了ＧＯＤ／Ｆｅ（ＣＮ）６＾３－／ＰＰｙ微酶电极对葡萄糖的响应，稳态响应电流与葡萄糖浓度之间存在Ｍｉｃｈｅａｌｉｓ－Ｍｅｎｔｅｎ动力学特征。     

扫描电化学显微镜直接电沉积法构建葡萄糖氧化酶微米点

利用自制的凹形电极在铂基底电极上直接构建了葡萄糖氧化酶微米点. 首先, 将电聚合和电化学刻蚀法相结合制备了凹形铂微米电极. 然后将此种电极作为参比及辅助电极, 基底铂电极作为工作电极, 利用葡萄糖氧化酶在合适的条件下(浓度、一定量Triton X-100存在、电极电位等)由于电极表面pH的降低可以在铂电极上电沉积这一特性, 将酶固定在铂基底电极上, 微修饰得到了具有活性的葡萄糖氧化酶微米点. 最终用扫描电子显微镜和扫描电化学显微镜对所得微米点进行了表征. 所得微米点直径约20 μm, 且具有催化活性. 该方法简便, 干扰因素较少.     

Amperometric L-glutamate sensor using a novel L-glutamate oxidase from Streptomyces platensis NTU 3304

Streptomyces platensis NTU 3304, isolated from soil samples, produces extracellular L-glutamate oxidase in liquid culture. Strains of this species have never been reported to be able to produce this enzyme. The purified enzyme was immobilized onto a cellulose triacetate membrane which was held at an oxygen electrode. The sensor was specific to L-glutamate in accordance with the properties of the novel L-glutamate oxidase. The time required for each assay in batch operation was less than 3 min. A linear relationship is observed between the decrease in dissolved oxygen and the concentration of L-glutamate between 20 and 140 mg l^?1 (ca. 0.12 and 0.84 mM). The sensor retained 95% of its original activity after 400 assays over a period of 3 weeks. The sensor was applied to determine the concentration of L-glutamate in broth samples during L-glutamic acid fermentation. Good correlations were achieved between results obtained with the sensor and by enzymatic analysis using glutamate dehydrogenase.