Litcrosomal Nadph Oxidase and Hybrid Electrodes

Abstract

The NADPH oxidizing activity of rat liver microsomes was investigated and found to be mainly due to the cytochrome P-450 system. The XADPH oxidase was utilized for the development of several organelle electrodes. Gelatin membrane immobilized microsomes were combined with an O₂ membrane sensor for bioelectrochemical measurement of NADPH. The dependence of the current on substrate concentration was linear up to 1 mmol·1^?1 To assemble hybrid electrodes for determination of glucose-6-phoaphate, ATP and isocitrate pure enzymes were coimmobilized with the microsomal fraction.     

Solid state biosensors using thin-film electrodes

Solid-state technology was used to fabricate a new type of glucose sensor and a differential measurement system was incorporated with the sensor in order to suppress interfering signals. This glucose sensor was composed of film electrodes and an enzyme-immobilizing membrane. The hydrogen peroxide electrodes were fabricated by metallic film deposition. Glucose oxidase (GOD), an enzyme, was immobilized in photosensitive polyvinyl alcohol to enable the enzyme-immobilizing membrane to be formed by the usual photolithographic method. Two anodes and one common cathode were formed on a glass substrate. One of the anodes was coated with PVA containing active enzyme and the other with PVA containing deactivated enzyme. By setting up a differential output measurement system, it was possible to suppress the interfering signals that might be generated by some other reducing substances in a sample solution. With this measuring technique, the signal of ascorbic or uric acids up to 100 mg/dl was successfully cancelled. Glucose concentrations in the range 5 to 100 mg/dl could be detected by the present sensor.The fabrication processes of the present sensor were so compatible with IC technology that it should be possible to mass produce disposable glucose biosensors using IC fabrication techniques.     

A thin layer photocell based on a tris(2,2'-bipyridine)ruthenium complex immobilized in a membrane containing an electronically conductive polymer

A thin layer photocell was constructed in which tin oxide (Nesa glass) modified with a tris(2,2'-bipyridine)ruthenium complex immobilized in a polyvinylcinnamate membrane was used as the working electrode, platinum was used as the counter electrode, and an aqueous solution containing the disodium salt of ethylenediaminetetraacetic acid was placed between the electrodes. The photocell generated a photocurrent under visible light irradiation. The generation of photocurrent was considerably enhanced when the working electrode was modified by the addition of the tetracyanoquinodimethane salt of poly-4-vinylpyridine and neutral tetracyanoquinodimethane.     

Glass-based redox sensor

A new glass-based planar sensor for the determination of redox potentials is introduced. The sensor was fabricated by applying and sintering an iron-containing glass paste on an alumina substrate partially covered with a platinum layer. Regarding the condition of the functional glass layer, it was characterised using several physical and chemical methods. Furthermore, by means of the planar sensors, the determination of redox potentials of different redox-active substances was carried out. It could be shown that the measurement results obtained with these electrodes were comparable to those received with conventional noble metal-based redox electrodes. For sensors prepared by selected glass compositions, a significant pH dependence of the measurement signal was not detected. Moreover, the electrode could be used as working electrode for cyclic voltammetry, too.     

Neutral-carrier-type potassium ion-selective electrodes based on polymer-supported liquid-crystal membranes for practical use.

Polymer-supported liquid-crystal membranes have been designed for neutral-carrier-type potassium ion-selective electrodes, aiming for practical applications of high-performance liquid-crystalline membrane ion sensors. Two types of polymer-supported liquid-crystal membranes were tested for their usefulness; one is microporous poly(tetra fluoroethylene) (PTFE) membranes impregnated by thermotropic liquid-crystalline compounds, and another is poly(methyl methacrylate) (PMMA) membrane dispersing the same liquid-crystalline compounds. Both of the polymer-supported liquid-crystal membranes containing a liquid-crystalline benzo-15-crown-5 neutral carrier as well as a lipophilic anion excluder work well as ion-sensing membranes for potassium ion-selective electrodes, the ion selectivities of which can be switched by the measurement temperatures. Specifically, PTFE-impregnated liquid-crystal membranes are better than the PMMA-dispersed ones in the sensitivity and selectivity of the resulting ion electrodes. A potassium ion assay in blood sera has proved that neutral-carrier-type ion-selective electrodes based on the polymer-supported liquid-crystal membranes are reliable for practical uses.     

Use of the clark oxygen sensor with immobilized enzymes for determinations in flow systems

A small-volume cell has been constructed for amperometric flow measurements with a Clark oxygen sensor and its performance was tested. The Clark sensor can be combined with immobilized enzymes for determination of substances after enzymatic conversion during which oxygen is consumed or released. Two enzymes, glucose oxidase and tyrosinase, were used and two measuring techniques, employing the enzyme immobilized on the Clark sensor membrane and with the enzyme bound on a support in a preceding reactor, were tested and compared. It was found that, in the given system, measurement with the enzyme immobilized on the sensor membrane has better sensitivity, precision and response rate.     

Lipase biosensor for tributyrin and pesticide detection

Potentiometric biosensors based on Candida rugosa lipase was described for the detection of organophosphorus pesticide; methyl-parathion and tributyrin. Lipase was immobilized on the glass electrode by means of a gelatin membrane, which is then cross-linked with glutaraldehyde. The principle of the biosensor is based on the measurement of pH variation which was recorded in millivolts due to the enzymatic hydrolysis of tributyrin to butyric acid. For the inhibitor detection, biosensor responses were measured after pesticide treatment, which caused a drop in enzyme activity because of the irreversible inhibition. Reactivation conditions of the reused enzyme electrodes were also investigated by pyridine-2-aldoxime methiodide (2-PAM). The limit of detection for tributyrin was estimated as 93?µM for lipase sensor within the linear range of 65–455?µM.     

Hydrogel-based flexible micro-reference electrodes for use in alkaline and neutral pH solutions

Two types of nonbreakable, flexible micro-reference electrodes filled with gel-electrolytes were prepared for use in solutions with alkaline and neutral pH. The electrodes are intended for electrochemical measurements, in which chloride-free conditions are important. Due to the flexible, bendable construction of the electrodes, electrochemical experiments at locations difficult to access with common reference electrodes are enabled. Hg|HgO-type electrodes were prepared from amalgamated Au wires, followed by oxidation of the amalgam, which is mounted in a PTFE tube filled with 0.1M NaOH solution immobilized in a PAA-g-PEO gel. The potential of this type of electrode was found to be 0.162?±?0.002 V (SHE) at room temperature. Cu|CuSO₄ electrodes, consisting of a Cu wire immersed in a saturated CuSO₄ solution jellied with gelatin, showed a stable open-circuit potential of 0.312?±?0.001 V (SHE). Further characterization of the electrodes was performed in terms of electrochemical impedance spectroscopy and micro-polarization measurements. As an alternative to the flexible electrodes, rigid electrodes in glass enclosure were fabricated in analogy to the flexible-type electrodes.     

Measurement of pH by flow injection over a wide dynamic range with PVC/neutral carrier electrodes

Two pH-sensitive neutral carrier/PVC electrodes are used simultaneously for the measurement of pH by flow injection. One of these is based on the neutral carrier tridodecylamine and the other on octadecyl isonicotinate, and together they allow the pH range 1–13 to be covered. These electrodes have been used in a very low dispersion miniature potentiometric flow cell designed specifically for use in flow injection in conjunction with a multi-channel data acquisition system. The effects of the solution ionic strength and buffer capacity on the pH measurement are discussed. A flow-injection manifold is proposed which can be used for high-accuracy pH measurements without ionic strength adjustment of the sample and for simultaneous pH and ion concentration measurements with ionic strength adjustment. This has been tested on some synthetic samples for the simultaneous determination of potassium and calcium and the measurement of pH.     

Creatinine biosensor based on ammonium ion selective electrode and its application in flow-injection analysis

A new, highly sensitive, fast responding and stable potentiometric biosensor for creatinine determination is developed. The biosensor is based on an ammonium ion-selective electrode. Creatinine deiminase (EC 3.5.4.21) is chemically immobilized on the surface of the polymeric ion-sensitive membrane in the form of monomolecular layer using a simple, one-step carbodiimide covalent attachment method. The resulting enzyme electrodes are useful for measurement under flow injection analysis (FIA) conditions. The biosensors exhibit excellent operational and storage stability. The enzyme electrodes retain over 70% of initial sensitivity after ten weeks of work under FIA conditions. The storage stability at 4 °C is longer than half a year without loss of sensitivity. Under optimized conditions near 30 samples per hour can be analyzed and the determination range (0.02-20.0 mmol l⁻¹) fully covers creatinine concentrations important from clinical and biomedical point of view. The simple biosensor/FIA system has been successfully used for determination of creatinine in urine, serum and posthemodialysate samples.     

Dendrimer-encapsulated Pt nanoparticles on mesoporous silica for glucose detection

A hybrid system of mesoporous silica (MS) particle incorporated with poly(amidoamine) dendrimer-encapsulated platinum nanoparticles (Pt-DENs) was constructed in a neutral aqueous solution through electrostatic interaction. The MS/Pt-DENs composite particles immobilized with glucose oxidase (GOx) were used to modify a glassy carbon electrode for detecting the electrocatalytic response to the reduction of glucose. Pt-DENs can improve the conductivity of MS and enhance the electron transfer between redox centers in enzymes and electrode surfaces. The structure of composite particles and the performance of MS/Pt-DEN-modified electrodes were characterized by transmission electron microscopy, N₂ sorption characterization method, electrochemical impedance spectroscopy, cyclic voltammetry and amperometric measurements. The MS/Pt-DENs/GOx-modified electrodes, which had a fast response of GOx less than 3?s, could be used for the determination of glucose ranging from 0.02 to 10?mM. The detection limits were 4???M at signal-to-noise ratio of 3.     

Determination of Urinary Glucose by a Flow Injection Analysis Amperometric Biosensor and Ion-Exchange Chromatography

A practical biosensor system has been developed for the determination of urinary glucose using a flow-injection analysis (FIA) amperometric detector and ion-exchange chromatography. Glucose oxidase was immobilized onto porous aminopropyl glass beads via glutaraldehyde activation to form an immobilized enzyme column. On the basis of its negative charge at pH 5.5, endogenous urate in urine samples was effectively retained by an upstream anion-exchange resin column. The biosensor system possessed a sensitivity of 160 ±2.4 RU μM^-1 (RU or relative unit is defined as 2.86 μV at the detection output) for glucose with a minimum detection level of 10 μM. When applied for the determination of urinary glucose, the result obtained compared very well with that of the widely accepted hexokinase assay. The immobilized glucose oxidase could be reused for more than 1000 repeated analyses without losing its original activity. The reuse of the acetate anion-exchange column before replacement would be about 25–30 analyses. Acetaminophen and ascorbic acid were also effectively adsorbed by the acetate anion exchanger. The introduction of this type of anion exchanger thus greatly improved the selectivity of the FIA biosensor system and fostered its applicability for the determination of glucose in urine samples.     

Electrical manipulation of supported lipid membranes by embedded electrodes

Alkanethiol modified gold electrodes patterned over a silica surface provided a dual hydrophobic/hydrophilic surface suitable for phospholipid monolayer and bilayer formation over the alkylated gold and glass surfaces, respectively. The phospholipid monolayer and bilayer were connected, allowing free diffusion of lipids within both leaflets of the glass-supported bilayer over the alkanethiol/gold-to-glass interface. Application of large alternating current fields to these electrodes irreversibly switched the gold electrodes to diffusion barriers. Enclosure of the electrode devices within protein barriers revealed a resting state surface potential driven reorganization of the charged fluorescent probes. Application of lower magnitude direct current fields resulted in electrophoretic redistribution of the membrane probes and electro-osmotic reorganization of membrane associated proteins.     

Amperometric determination of urea using an NADH-dependent coupled enzyme

Enzyme electrodes for the amperometric measurement of urea were prepared by co-immobilizing l-glutamate dehydrogenase and urease onto an Immobilon-AV affinity membrane with attachment to a glassy carbon electrode. Reduced nicotinamide adenine dinucleotide (NADH) was used as the electroactive species. The electrochemical oxidation of NADH was monitored at +1.0 V vs. Ag/AgCl. The enzyme immobilized electrode was linear over the range of 2.0 x 10(-5) to 2 x 10(-4)M. The response time of the electrode was 3 min and the optimum pH of enzyme immobilized membrane was pH 7.4-7.6 (Dulbecco's buffer solution). It was stable for at least two weeks and 50 assays. There were no interferences from other physiological material, except for high levels of ascorbic acid.     

Determination of glucose in blood by flow injection analysis and an immobilized glucose oxidase column

A flow-injection system for glucose determination is described. Glucose oxidase is immobilized on controlled porosity glass (CPG) and used in a glass column (2.5 mm diameter × 2.5 cm). The hydrogen peroxide produced by the enzymatic reaction (? 1 × 10^?6 M) is detected by the current produced in a flow-through cell, with two platinum electrodes having a potential difference of 0.6 V. Glucose (0–20 mmol l^?1) can be determined in blood plasma either with a dialyser in the system or, better, by incorporating a column of copper(II) diethyldithiocarbamate on CPG before the enzyme column. The results compared well with those obtained by a conventional analyser system. The glucose oxidase column showed little change in activity over a 10-month period.     

Flow-through chemiluminescence sensor using immobilized histamine oxidase from Arthrobacter crystallopoietes KAIT-B-007 and peroxidase for selective determination of histamine.

A flow sensor with immobilized oxidases is proposed for the determination of histamine in fish meat. Chemiluminometric measurement of histamine was based on the luminol reaction with hydrogen peroxide produced by immobilized histamine oxidase (EC 1.4.3.-.) and peroxidase (EC 1.11.1.7.) within a flow cell. Histamine oxidase was found in cells of Arthrobacter crystallopoietes KAIT-B-007 isolated from soil. The oxidase and peroxidase were coimmobilized covalently on tresylated hydrophilic vinyl polymer beads and packed into transparent PTFE; the tubing was used as the flow cell. One assay for histamine was done at intervals of 2 min without carryover. The calibration curve for histamine was linear from 0.1 microM to 50 microM. The response was reproducible within 1.25% of the relative standard deviation for 115-replicate injections of 50 microM histamine. The sensor system was applied to the determination of histamine in fish meat extracts.     

Amperometric detection of superoxide dismutase at cytochrome c-immobilized electrodes: xanthine oxidase and ascorbate oxidase incorporated biopolymer membrane for in-vivo analysis.

Amperometric measurement of superoxide dismutase (SOD) was carried out at cytochrome c-immobilized monolayers and ascorbate oxidase (AOD)/xanthine oxidase (XOD)/cytochrome c- and (AOD, XOD)/cytochrome c-multilayers. Cytochrome c was covalently immobilized on mercaptopropionic acid-containing self-assembled monolayers on gold. A biopolymer membrane of poly-L-lysine confining XOD and AOD was cast on the monolayer of cytochrome c. While both the cytochrome c-immobilized monolayer and multilayer electrodes show anodic current responses to the generation of superoxide radical, the sensitivity of the multilayer system for the detection of superoxide radical was high relative to that of the monolayer system. In the case of the cytochrome c-multilayer electrodes, the generation of superoxide radical near the sensing element, cytochrome c, resulted in high sensitivity for the detection of superoxide. The use of a XOD and AOD-incorporated poly-L-lysine membrane enabled the detection of the generation of superoxide radical in the presence of L-ascorbic acid. Though L-ascorbic acid could scavenge superoxide radical, the biopolymer membrane confined with AOD will oxidize any L-ascorbic acid that permeated into the membrane. By using the multilayer electrodes, one could measure the activity of SOD in the presence of L-ascorbic acid.     

Determination of Urea in Undiluted Blood Samples by Flow Injection Analysis Using Optosensin

Abstract

A miniaturized Flow Injection system for the assay of urea in undiluted whole blood is described. Based on the optical determination of ammonia, the system incorporates a flow cell combining gas diffusion and optosensing, the separating barrier between the donor and accepting streams consisting of a sandwich of a hydrophobic gas permeable membrane and a hydrophilic membrane between which is contained a gel of covalently immobilized urease. The sample urea content is quantified by the colour change of an acid-base indicator contained within the acceptor stream. Within the physiological range the measurement is not affected by variations in the pH value, the buffering capacity or the hematocrit level of the sample solution, nor is it, due to the geometric construction of the flow cell, prone to interferences owing to the colour of the sample solution itself. The system is stable for a t least one week of continuous use.     

A novel method for the assay of alpha-glucosidase inhibitory activity using a multi-channel oxygen sensor.

The quantification of glucose by using a multi-channel dissolved oxygen (DO) meter (DOX96) with immobilized glucose oxidase (GOD) and mutarotase (MUT) was performed. An evaluation of the inhibitory activities for alpha-glucosidase (AGH) by modifying our batch-type pseudo-in vivo assay system [Oki et al.; Biol Pharm. Bull., 2000, 232, 1084] was also performed using a DOX96. When 45 U/well GOD and 18.75 U/well MUT were immobilized on the surface of a gelatin membrane on the electrodes, the response shown by the decrease percent of DO (%) obtained with 8 electrode wells in the same row was linear with the glucose concentration up to 3.3 mM and a correlation coefficient larger than 0.9. To estimate the AGH inhibitory activity, AGH-immobilized Sepharose supports in the well of a silent screen plate were used. The IC50 values of acarbose and 1-deoxynojirimycin, a medicinal inhibitor for diabetes, were 0.70 +/- 0.08 microM and 0.40 +/- 0.13 microM, respectively, and coincided well with those by a pseudo-in vivo assay.     

溶剂聚合膜pH电极作内电极的气敏电极的研究

研究了以中性载体为活性物的石墨内导平头溶剂聚合膜(SPM)pH电极作内电极的二氧化碳和氨气敏电极。电极的性能取决于中性载体的特性,其中以菸酸十八酯为内电极膜活性物的二氧化碳气敏电极和以二辛基十八胺为活性物的氨气敏电极性能较佳。二氧化碳气敏电极用作丙酮酸脱羧酶酶电极的原电极,其性能院于传统的以玻璃pH电极作内电极的二氧化碳气敏电极。