Enzyme electrode for specific determination of L-lysine

L-Lysine alpha-oxidase from Trichoderma viride Y244-2 is immobilized in a gelatin support and fixed on a pO(2) sensor. The enzyme electrode obtained is used in a continuous flow system in order to measure the concentration of L-lysine in a fermentor. The sample oxygen-content dependance of the signal is minimized because of the enzyme support properties. The enzyme electrode response is set for lysine concentration from 0.2mM to 4mM. The specificity of lysine is tested with other amino acids. The enzyme membrane for lysine electrode can be used 3000 times or stored six months with good stability.     

Rapid determination of L-lysine with an enzyme electrode by steady state and kinetic measurement

Fast and simple methods of determination of L-lysine by a potentiometric enzyme electrode based on a CO₂ electrode and L-lysine decarboxylase are described. Measuring devices for manual and automated operation for steady state response measurement as well as kinetic measurement are compared. Sample frequency may be increased by decreasing the time of a measuring cycle.     

A specific enzyme electrode for l-glutamate-development and application

A biosensor for the specific determination of l-glutamate has been developed using l-glutamate oxidase in combination with a hydrogen peroxide indicating electrode. The biosensor response depends linearly on l-glutamate concentration between 0.001 and 1.0 mM. The measuring time is 2 min. The sensor is stable for more than 10 days during which more than 500 assays can be performed. The sensor has been applied to l-glutamate determination in liquid seasonings. Furthermore, transaminase activities have been determined by their catalytic l-glutamate production from alpha-ketoglutarate and l-alanine or l-aspartate. Also, the coimmobilization of glutaminase yielded a bienzyme electrode sensitive to l-glutamine.     

An enzyme electrode for on-line determination of ethanol and methanol

Since a stable alcohol oxidase with a high specific activity is not commercially available, we propose to produce and purify this enzyme from a strain of the yeast Hansenula polymorpha. This alcohol oxidase was immobilized into a gelatin matrix and its activity was estimated by a pO(2) sensor. The enzyme electrode obtained was then used in a continuous flow system to measure methanol or ethanol concentrations. The sample oxygen content dependence of the signal was minimized by the support properties. Measuring time for each sample were less than two minutes including response data treatment and rinsing step. The enzyme electrode response was set for ethanol from 0.5mM to 15mM and for methanol from 10mM to 300mM. On repeated use, the electrode signal for 10mM of ethanol was stable for at least 500 assays. Analysis have been performed in different beverages such as wine and beer, and the results compared to those obtained with classical methods of analysis.     

A new amperometric enzyme electrode for alcohol determination

A new enzyme electrode for the determination of alcohols was developed by immobilizing alcohol oxidase in polvinylferrocenium matrix coated on a Pt electrode surface. The amperometric response due to the electrooxidation of enzymatically generated H(2)O(2) was measured at a constant potential of +0.70 V versus SCE. The effects of substrate, buffer and enzyme concentrations, pH and temperature on the response of the electrode were investigated. The optimum pH was found to be pH 8.0 at 30 degrees C. The steady-state current of this enzyme electrode was reproducible within +/-5.0% of the relative error. The sensitivity of the enzyme electrode decreased in the following order: methanol>ethanol>n-butanol>benzyl alcohol. The linear response was observed up to 3.7 mM for methanol, 3.0 mM for ethanol, 6.2 mM for n-butanol, and 5.2 mM for benzyl alcohol. The apparent Michaelis-Menten constant (K(Mapp)) value and the activation energy, E(a), of this immobilized enzyme system were found to be 5.78 mM and 38.07 kJ/mol for methanol, respectively.     

A lysine dehydrogenase-based electrode for biosensing of L-lysine.

An amperometric biosensor for L-lysine based on the recently isolated enzyme lysine dehydrogenase is described. Immobilization of the enzyme onto a platinum electrode is achieved via entrapment within a gelatin support on a cellulose membrane. Anodic detection (at 0.4 V vs. Ag/AgCl) is facilitated by the presence of a redox-mediating ferricyanide ion. The effect of experimental variables such as pH, enzyme loading, applied potential, cofactor and mediator concentrations were evaluated in order to optimize the analytical performance. A detection limit of 7 x 10(-8) M, and linearity up to 7 x 10(-4) M are reported. The fast response permits adaptation for flow injection operation with good precision (RSD = 1.9%) and high sample throughout (40 samples per hour). The high specificity offered by this new enzyme is indicated by the lack of interference by other L-amino acids, alcohols or carbohydrates.     

Thin-film antimony-antimony-oxide enzyme electrode for penicillin determination

A potentiometric penicillinase electrode is reported in which the base pH transducer is a thin-film anti-mony-antimony-oxide electrode deposited by vacuum evaporation. Several enzyme immobilization procedures have been examined and a crosslinked protein film found to be the most appropriate to this type of sensor. The use of an adjacent antimony-antimony-oxide track as a pseudoreference electrode was successfully demonstrated. The overall response was shown to be independent of the stirring rate above 100 rpm, but the kinetics of the response were found to depend markedly on the stirring rate. The intrinsic linear response range was 3 x 10(-4)Mto 7 x 10(-3)M penicillin G. Linearizing transforms that extend the useful range were examined.     

An enzyme electrode for acetylcholine.

A new enzyme electrode is described to measure continuously acetylcholine concentration. A coating containing active acetylcholinesterase is produced on a pH-glass electrode. The mean thickness of the coating is 50 micrometer. Optimal operational conditions with respect to buffer concentration, ionic strength, linearity, stability, sensitivity, pH of the bulk solution, and response time are studied and discussed. The use of acetylcholinesterase-containing membranes as sensors could offer several novel advantages.     

An enzyme electrode for measurement of penicillin in fermentation broth: A step toward the application of enzyme electrodes in fermentation control

A penicillin sensitive enzyme electrode has been used to analyze the concentration of benzylpenicillin in fermentation broth. The electrode response time was in the region of 2 min and the response to penicillin concentration was linear within the range of 1 to 10mM. The buffering capacity of the medium influenced the sensitivity of the electrode. At low buffer capacity the sensitivity of the enzyme electrode to penicillin was very high, but then the sensitivity to small changes in buffer capacity was relatively large. At high buffer capacity the sensitivity to penicillin was reduced and the electrode became less dependent on changes to buffer capacity. Constant calibration curves were repeatedly obtained with the electrode when used for 2 hr daily in a fermentation medium over a six day period. Three methods devised to calibrate the electrode for use in fermentation media were investigated. Methods one and two, based on the relationship between electrode sensitivity and buffer capacity in phosphate buffer and in sterile media, gave rather high penicillin concentration values. The third method based on an internal standard was the most satisfactory.     

Amperometric enzyme electrode for determination of theophylline in serum.

This paper describes an amperometric enzyme electrode for the rapid determination of theophylline in serum. The method is based on the catalysed oxidation of theophylline by the haem-containing enzyme theophylline oxidase. Results are presented for two approaches. First, ferrocene monocarboxylic acid was used as a mediator. The second-order rate constant was 1.1 x 10(3) 1 mol-1 s-1. Secondly, the organic conducting salt NMP.TCNQ was used to construct enzyme electrodes. These electrodes were employed for the rapid (60 s) measurement of theophylline in serum at a working potential of +100 mV versus Ag/AgCl. Linear calibration curves were obtained over the clinically relevant range (y = 0.13x + 0.22, n = 8). Caffeine, theobromine and 3-methylxanthine at levels up to 100 mg l-1 do not interfere and 1-methylxanthine shows cross-reactivity at concentrations greater than 50 mg l-1.     

An enzyme electrode for amperometric measurement of D-amino acid

A carbon paste enzyme electrode has been developed for measurement of D-amino acids that employs a fatty acid modified FAD to prevent leaching of this essential cofactor to the surrounding aqueous environment and which serves as an enzyme stabilizing agent. The lower limit of detection is at least 10(-4) M and the electrode has a linear range of 10(-4) to 3 x 10(-3) M and a response time of 180 s. Twenty L-amino acids were tested and none of them elicited responses when electrodes were exposed to 0.5 mM concentration increases over a baseline level. On the other hand, some response was observed when exposed to 18 of 20 D-amino acids varying from 2 to 200% of the corresponding D-alanine response. Electrodes were shown to have longevities of over 30 days while maintaining 85% of their original sensitivity. Electrodes showed activity over a pH of 6.2-11.7 with a maximum at 9.2 and over temperatures of 10-47 degrees C with a maximum at 37 degrees C.     

Wireless enzyme sensor system for real-time monitoring of blood glucose levels in fish

Periodic checks of fish health and the rapid detection of abnormalities are thus necessary at fish farms. Several studies indicate that blood glucose levels closely correlate to stress levels in fish and represent the state of respiratory or nutritional disturbance. We prepared a wireless enzyme sensor system to determine blood glucose levels in fish. It can be rapidly and conveniently monitored using the newly developed needle-type enzyme sensor, consisting of a Pt-Ir wire, Ag/AgCl paste, and glucose oxidase. To prevent the effects of interfering anionic species, such as uric acid and ascorbic acid, on the sensor response, the Pt-Ir electrode was coated with Nafion, and then glucose oxidase was immobilized on the coated electrode. The calibration curve of the glucose concentration was linear, from 0.18 to 144mg/dl, and the detection limit was 0.18mg/dl. The sensor was used to wirelessly monitor fish glucose levels. The sensor-calibrated glucose levels and actual blood glucose levels were in excellent agreement. The fluid of the inner sclera of the fish eyeball (EISF) was a suitable site for sensor implantation to obtain glucose sample. There was a close correlation between glucose concentrations in the EISF and those in the blood. Glucose concentrations in fish blood could be monitored in free-swimming fish in an aquarium for 3 days.     

Disposable screen-printed enzyme sensor for simultaneous determination of starch and glucose

A disposable screen-printed biosensor with dual working electrodes was first established for simultaneous determination of starch and glucose. The electrochemical behavior of the sensor was assessed using cyclic voltammetry and chronoamperometry. The linear response ranges of the sensor were up to 0.4% (w/v) starch and 20 mmol glucose 1^–1. Real samples were analysed using the proposed method and the reference method and the correlation coefficient was 0.997.     

Extended-gate FET-based enzyme sensor with ferrocenyl-alkanethiol modified gold sensing electrode

We developed a field-effect transistor (FET)-based enzyme sensor that detects an enzyme-catalyzed redox-reaction event as an interfacial potential change on an 11-ferrocenyl-1-undecanethiol (11-FUT) modified gold electrode. While the sensitivity of ion-sensitive FET (ISFET)-based enzyme sensors that detect an enzyme-catalyzed reaction as a local pH change are strongly affected by the buffer conditions such as pH and buffer capacity, the sensitivity of the proposed FET-based enzyme sensor is not affected by them in principle. The FET-based enzyme sensor consists of a detection part, which is an extended-gate FET sensor with an 11-FUT immobilized gold electrode, and an enzyme reaction part. The FET sensor detected the redox reaction of hexacyanoferrate ions, which are standard redox reagents of an enzymatic assay in blood tests, as a change in the interfacial potential of the 11-FUT modified gold electrode in accordance with the Nernstian response at a slope of 59 mV/decade at 25 degrees C. Also, the FET sensor had a dynamic range of more than five orders and showed no sensitivity to pH. A FET-based enzyme sensor for measuring cholesterol level was constructed by adding an enzyme reaction part, which contained cholesterol dehydrogenase and hexacyanoferrate (II)/(III) ions, on the 11-FUT modified gold electrode. Since the sensitivity of the FET sensor based on potentiometric detection was independent of the sample volume, the sample volume was easily reduced to 2.5 microL while maintaining the sensitivity. The FET-based enzyme sensor successfully detected a serum cholesterol level from 33 to 233 mg/dL at the Nernstian slope of 57 mV/decade.     

Cell-bionics: tools for real-time sensor processing

The accurate monitoring of the physiological status of cells, tissues and whole organisms demands a new generation of devices capable of providing accurate data in real time with minimal perturbation of the system being measured. To deliver on the promise of cell-bionics advances over the past decade in miniaturization, analogue signal processing, low-power electronics, materials science and protein engineering need to be brought together. In this paper we summarize recent advances in our research that is moving us in this direction. Two areas in particular are highlighted: the exploitation of the physical properties inherent in semiconductor devices to perform very low power on chip signal processing and the use of gene technology to tailor proteins for sensor applications. In the context of engineered tissues, cell-bionics could offer the ability to monitor the precise physiological state of the construct, both during 'manufacture' and post-implantation. Monitoring during manufacture, particularly by embedded devices, would offer quality assurance of the materials components and the fabrication process. Post-implantation monitoring would reveal changes in the underlying physiology as a result of the tissue construct adapting to its new environment.     

The electrode adsorption method for determination of enzyme activity: a study of substrate requirements

The electrode adsorption method for the determination of enzyme activity requires substrates that, besides having good kinetics constants for the enzyme, also show good adsorption/desorption kinetics to the electrode surface and adsorb in such a way that they change the double-layer capacitance of the electrode. A series of peptide substrates containing one to three aromatic groups has been synthesized. Our results show that the aromatic groups are of crucial importance for the capacitance change caused by the adsorbing/desorbing substrate. Thus, the tripeptide substrate, Bz-Phe(NO2)-Val-Arg-pNA, with three aromatic groups is superior to the other synthesized substrates containing only one or two aromatic groups. Our desorption experiments show that several factors determine the rate of capacitance increase observed when thrombin is added to a substrate solution in equilibrium with a substrate-covered electrode. The kinetic constants of the substrate determine how the substrate concentration in the solution decreases and, consequently, determine the spontaneous desorption measured as capacitance increase. Thrombin does not seem to split adsorbed substrate molecules but it adsorbs to the substrate-covered surface and in that way causes a capacitance decrease counteracting the change caused by desorption of substrate.     

A method for the determination of enzyme mass loading on an electrode surface through radioisotope labelling

A direct method has been developed for the quantitation of the amount of immobilised enzymes on biosensor surfaces. This quantity is of key importance in establishing the activity, kinetics and optimal immobilisation conditions in the construction of both amperometric and optical biosensors. Recombinant L-lactate dehydrogenase incorporating both a biosynthetically introduced radiolabel, 3H-leucine, and a hexahistidine peptide tag was immobilised on a poly(aniline) composite film and then quantitated by liquid scintillation counting. It was found that enzyme mass loading was proportional to the concentration of LDH in solution, and also depended on the morphology of the composite film. The LDH mass loading on the composite film doubled when a surface cysteine containing variant was used, possibly due to the covalent attachment of the cysteine to the diiminoquinoid rings of the poly(aniline).     

A specific microbial sensor for formic acid

Summary A microbial sensor consisting of immobilized Clostridium butyricum, two gas permeable Teflon membranes and fuel cell type electrode was suitable for the determination of formic acid. When the sensor was inserted into the sample solution containing formic acid, the current increases to a steady state with a response time of 20 min. The relationship between the steady state current and the formic acid concentration is linear up to 1 000 mg l^–1. The currents are reproducible with an average relative error of 5%. Selectivity of the sensor is satisfactory. Results obtained with this sensor and by gas chromatography were in good agreement (regression coefficient; 0.98) when the cultivation medium of Aeromonas formicans was employed. Immobilized Clostridium butyricum is stable for more than 20 days.     

An electrochemical sensor for determination of calcium dobesilate based on PoPD/MWNTs composite film modified glassy carbon electrode

A poly-o-phenylenediamine and multi-wall carbon nanotubes composite (PoPD/MWNTs) modified glassy carbon electrode (GCE) was prepared by in situ electropolymerization using an ionic surfactant as the supporting electrolyte. The morphology of the resulting PoPD/MWNTs composite was characterized by TEM and the electrochemical properties of the modified electrode were characterized by cyclic voltammetry. The electrochemical behavior of calcium dobesilate on PoPD/MWNTs modified electrode was also investigated. The large current response of calcium dobesilate on PoPD/MWNTs modified electrode is probably caused by the synergistic effect of the electrocatalytic property of PoPD and MWNTs. The reductive peak current increased linearly with the concentration of calcium dobesilate in the range of 0.1–1.0 μmol/L and 4.0–400 μmol/L by square wave adsorptive stripping voltammetry, respectively. The detection limit (three times the signal blank/slope) was 0.035 μmol/L. The modified electrode could eliminate the interference of dopamine, norepinephrine and epinephrine at 100-, 90- and 70-fold concentration of 1.0 μmol/L calcium dobesilate, respectively. The proposed modified electrode provides a new promising and alternative way to detect calcium dobesilate.