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.     

Detection of Hydrogen Peroxide and Glucose Based on Immobilized C60‐Catalase Enzyme

The interaction between fullerene C60 and catalase enzyme was studied with a fullerene C60‐coated piezoelectric (PZ) quartz crystal sensor. The partially irreversible response of the C60‐coated PZ crystal sensor for catalase was observed by the desorption study, which implied that C60 could chemically react with catalase. Thus, immobilized fullerene C60‐catalase enzyme was synthesized and applied in determining hydrogen peroxide in aqueous solutions. An oxygen electrode detector with the immobilized C60‐catalase was also employed to detect oxygen, a product of the hydrolysis of hydrogen peroxide which was catalyzed by the C60‐catalase. The oxygen electrode/C60‐catalase detection system exhibited linear responses to the concentration of hydrogen peroxide and amount of immobilized C60‐catalase enzyme that was used. The effects of pH and temperature on the activity of the immobilized C60‐catalase enzyme were also investigated. Optimum pH at 7.0 and optimum temperature at 25 °C for activity of the insoluble immobilized C60‐catalase enzyme were found. The immobilized C60‐catalase enzyme could be reused with good repeatability of the activity. The lifetime of the immobilized C60‐catalase enzyme was long enough with an activity of 93% after 95 days. The immobilized C60‐catalase enzyme was also applied in determining glucose which was oxidized with glucose oxidase resulting in producing hydrogen peroxide, followed by detecting hydrogen peroxide with the oxygen electrode/C60‐catalase detection system.     

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.     

Oxygen electrode-based enzyme immunoassay for the amperometric determination of hepatitis B surface antigen

Specific antibodies labelled with glucose oxidase are immobilized onto a gelatin membrane, which is fixed over an oxygen electrode. The sensor is immersed in a standard glucose solution and a signal is obtained by measuring the consumption of oxygen by the enzyme catalyzed reaction. The response increases linearly with increasing antigen concentration over the range 0.1–100 μg 1^?1. A microcomputer is used for data acquisition and processing.     

Amperometric response to reducing carbohydrates of an enzyme electrode based on oligosaccharide dehydrogenase. Detection of lactose and α-amylase

A mediated amperometric enzyme electrode, which was constructed by immobilizing oligosacharide dehydrogenase behind a dialysis membrane on the surface of a carbon paste electrode containing p-benzoquinone, showed a current response to d-xylose, d-galactose, d-mannose, lactose, maltose, maltotriose, maltopentaose and maltohexaose. The sensitivity of the current response to these carbohydrates was dependent on the kinetics of the immobilized enzyme reaction and/or the permeation rate of the substrate through the dialysis membrane. Hence the sensitivity could be varied by controlling the amount of the immobilized enzyme and the thickness of the dialysis membrane. The time dependence of the current response ofthe enzyme electrode with a large amount of the immobilized enzyme and a thicker dialysis membrane could be explained by an equation describing diffusion of the substrate in the membrane. The enzyme electrode was used to measure lactose in milk and to assay α-amylase in standard serum.     

An enzyme electrode forl-lactate with a chemically-amplified response

An enzyme electrode with a chemically-amplified response forl-lactate is constructed from an oxygen electrode and a layer containing immobilized lactate oxidase, to oxidizel-lactate, and lactate dehydrogenase, to regenerate thel-lactate. Regeneration enables oxygen to be consumed beyond the stoichiometric limitation, which results in an electrode response amplified 2–250 times according to the variation in the layer properties such as the V_max and K_m values of the immobilized enzymes and the thickness of the layer. The detection limit is as low as 5 × 10^?9 M. An equation is derived to relate the rate of oxygen consumption in the layer to the experimental parameters; the equation successfully explains the experimental results.     

环糊精聚合物与苯醌的分子包合作用及其在酶电极中的应用

研究了水溶性环糊精预聚合物的存在对苯醌/氢醌体系在铂电极上氧化还原行为的影响, 根据伏安曲线讨论了该预聚合物与苯醌的分子包合作用。环糊精预聚合物与戊二醛缩聚反应而形成的不溶性聚合物膜用于葡萄糖氧化酶的固定化, 以制得新型的第二代葡萄糖电极。由于分子包合作用, 作为电子受体的苯醌在含酶的环糊精聚合物膜中具有较高的浓度, 从而加速了固定化酶的电子传递。测定了酶电极上BQ反应的动力学参数。     

Immobilization of uricase to gas diffusion carbon felt by electropolymerization of aniline and its application as an enzyme reactor for uric acid sensor

Electropolymerizations of aniline and pyrrole solutions containing uricase at a neutral pH was performed to immobilize the enzyme on the surface of the gas diffusion carbon felt, and a selective uric acid sensor was fabricated by combining immobilized enzyme carbon felt and an oxygen electrode with oxygen gas permeable membrane. This carbon felt has a desirable feature for uricase sensing because an extremely efficient supply of oxygen for the enzymatic reaction can be realized due to its porosity permitting a transfer of oxygen. The current response time required from when sample is added until when current reaches the steady-state value is <5 min. The calibration graph of uric acid showed a linear line in a concentration range from 1x10(-5) to 4x10(-4) M with a detection limit of 4x10(-6) M.     

Glucose enzyme electrode with extended linearity: Application to undiluted blood measurements

An enzyme electrodes is described for glucose determination in unstirred, undiluted whole blood. The system comprises an H₂O₂-detecting electrode upon which is placed a membrane laminate incorporating glucose oxidase. The external membrane was pretreated with methyltrichlorosilane. The electrode response was linearly dependent on glucose concentration up to 50 mmol l^?1 glucose, it had a decreased dependence on dissolved oxygen concentrations and gave response times of 30–90 s. Whole blood glucose measurements correlated well with a routine spectrophotometric method.     

Characterization of mediated and non-mediated oxidase enzyme based glassy carbon electrodes

The performance and analytical characteristics of a glassy carbon glutaraldehyde immobilized glucose oxidase electrode have been established with regard to the direct detection of hydrogen peroxide produced from the reaction of glucose with oxygen. Measurements were performed at + 1.1 V vs. SCE, and selectivity was obtained by casting the surface with a cellulose acetate membrane. Results compared favorably with the classical platinum-enzyme probe. The mechanism of ascorbic acid interference in hydrogen peroxide detection is reported. Mediated detection was also investigated for oxidase enzymes (glucose oxidase and xanthine oxidase) immobilized on the bare glassy carbon electrode. The probes were characterized using a specific enzyme mediator in solution (phenazine methosulfate or dichlorophenol-indophenol) plus hexacyanoferrate(III) as an electrochemical mediator. The electrode was poised at + 0.36 V vs. SCE for the detection of hexacyanoferrate(II). The advantages of this dual mediator configuration include high stability and sensitivity of the electrochemical signal and the ability to use less positive potentials for increased selectivity. Application to other enzymes, such as hydrogenases, using such a binary redox configuration is suggested.     

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.     

Glucose sensor using a phospholipid polymer-based enzyme immobilization method

An electroenzymatic glucose sensor based on a simple enzyme immobilization technique was constructed and tested. The glucose sensor measures glucose concentrations as changes of oxygen concentrations induced by enzymatic reactions. The immobilizing procedure was developed with the purpose of producing wearable biosensors for clinical use. Two types of biocompatible polymers, 2-methacryloyloxyethyl phosphorylcholine (MPC) copolymerized with dodecyl methacrylate (PMD) and MPC copolymerized with 2-ethylhexyl methacrylate, were compared as a sensitive membrane of biosensors. The PMD enzyme membrane had a better response time. Linearity, reproducibility, effect of the concentrations of immobilized enzyme and drifts of sensor characteristics in long-term tests were also investigated. The linear characteristics were confirmed with glucose concentration from 0.01 to 2.00 mmol/l, with a coefficient of determination of 0.9999. The average output current for 1 mmol/l and the standard deviation were 0.992 and 0.0283 muA. Significant changes in the sensor's characteristics were not observed for 2 weeks when it was kept in a refrigerator at 4 degrees C. Because of the simple procedure, the enzyme immobilization method is not only useful for wearable devices but also other devices such as micro total analysis systems.     

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.     

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.     

Multi-walled carbon nanotubes for the immobilization of enzyme in glucose biosensors

A novel biosensor, comprised of electrode of gold/multi-walled carbon nanotubes–glucose oxidase (Au/MWNTs–GOD), has been developed. The MWNTs were produced by microwave plasma enhanced chemical vapor deposition. The enzyme of GOD was immobilized using MWNTs. Performance and characteristics of the fabricated glucose biosensor were assessed with respect to response time, detection limit, pH value and storage stability. The results show that the fabricated biosensor is sensitive and stable in detecting glucose, indicating that MWNTs are a good candidate material for the immobilization of enzyme in glucose biosensor construction.     

An enzyme electrode for the amperometric determination of glucose

An amperometric method utilizing a glucose electrode has been developed for the determination of blood glucose. The time of measurement is less than 12 s if a kinetic method is used and 1 min if a steady-state method is used. The long-term stability of the electrode is ca. 0.1% change from maximum response per day when stored at room temperature for over 10 months. The enzyme electrode determination of blood sugar compares favorably with commonly used methods with respect to accuracy, precision, and stability. The only reagent required for blood sugar determinations is a buffer solution. The electrode consists of a metallic sensing layer covered by a thin film of immobilized glucose oxidase held in place by means of cellophane. When poised at the correct potential, the current produced is proportional to the glucose concentration.     

Immobilized Glucose Oxidase in the Potentiometric Detection of Glucose

Previous work has shown that glucose oxidase can be immobilized on platinum to give an electrode that responds potentiometrically to glucose over the clinically useful range of about 10-250 mg glucose/100 mL. The present studies were carried out with electrochemically pretreated platinum and with gold or porous graphite substituted for the platinum support. The presence of the enzyme gave a significantly enhanced potentiometric response over that obtained with the bare support for both the pretreated platinum and the porous graphite, but not with gold. However, with platinum the potentiometric response became more negative with increasing glucose concentration. With porous graphite, the potential changed in the positive direction as the glucose concentration was increased. Hysteresis was demonstrated for the platinum-enzyme electrode. Mass transfer measurements with a rotating ring-disc electrode (RRDE) showed measurable diffusional resistances to the transport of a model electroactive compound (potassium ferrocyanide) through a matrix of immobilized enzyme attached to the disc of the RRDE. These results are part of a larger study to define the source of the potentiometric response by examining the roles of the support and the mass transfer resistances through the immobilized enzyme matrix.     

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.     

Amperometric biosensor with enzyme amplification fabricated using self-assembled monolayers of alkanethiols: the influence of the spatial distribution of the enzymes

The influence of the spatial distribution of the two enzymes in an amplified enzyme electrode is investigated by the step-wise fabrication of the enzyme interface. The two enzymes, glucose oxidase (GOD) and glucose dehydrogenase (GDH), are co-immobilised onto the surface of a gold electrode modified with the alkanethiol, 3-mercaptopropionic acid. Three geometries were fabricated: (1) a monolayer enzyme electrode where both enzymes were covalently attached to the alkanethiol layer, (2) a bilayer electrode where the inner layer was GOD and the outer layer was GDH, and (3) a bilayer sensor where the inner layer was GDH and the outer layer was GOD. The response of the three geometries was shown to vary with regard to linear range, sensitivity and hence gain. We believe this is the first demonstration of the spatial relationship between enzymes in a multi-enzyme system influencing the response of the resultant enzyme electrode.     

A coated-metal enzyme electrode for urea determinations

A potentiometric enzyme electrode is reported in which an enzyme immobilized in polyvinyl chloride is used to coat an antimony metal electrode to detect changes in pH when the electrode is immersed in a solution of the enzyme substrat. As an example, urea is determined in solution by using immobilized urease on an antimony electrode, giving a linear concentration range of 5.0 × 10^-4–1.0 × 10^-2 M urea with a slope of 44 mV per decade change in urea concentration. The response slope is stable for about 1 week, with response times in the range 1–2 min, but with absolute potential changes occurring from day to day.