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L Gorton

Publications and source records attributed to L Gorton.

At least 37 records · Page 2Linked to original sources
PubMed1999

Characterisation of a thermophilic L-glutamate dehydrogenase biosensor for amperometric determination of L-glutamate by flow injection analysis.

Carbon paste wax electrodes incorporating thermophilic L-glutamate dehydrogenase, NADP and a polymeric toluidine blue O (poly-TBO) mediator have been characterised for the amperometric determination of L-glutamate at 313-318 K in a flow injection analysis (FIA) system. The biosensors exhibit good sensitivity, mechanical stability and reproducibilty, unlike carbon paste- or carbon wax-based electrodes under the same conditions. The carbon paste wax electrode responds linearly to L-glutamate up to 40 mM, the detection limit is 0.3 mM and the RSD (n = 10) for 5 mM L-glutamate was 7.6%. The response to some potential interferents has been quantified. Addition of finely ground hexaammineruthenium (III) trichloride ([Ru(NH3)6]Cl3) to the carbon paste wax electrodes decreases the FIA peak width and increases the peak current. The metal complex appears to accelerate the rate of oxidation of NAD(P)H by poly-TBO.

Biosensing Techniques↗
PubMed1998

Oligosaccharide dehydrogenase-catalyzed assay for the determination of polysaccharides.

Oligosaccharide dehydrogenase (ODH), an enzyme known to have a broad selectivity for reducing sugars of low molecular weight, was investigated to determine its catalytic properties with larger polysaccharides. Six substrates were studied: pullulan standards with molecular weights of between 5,400 and 90,900, debranched starch, and dextran. In addition, maltotriose, isomaltotriose, maltose, and glucose were used as substrates for comparison. ODH catalyzed the oxidation of the large pullulans with a degree of polymerization of at least 560. Isomaltotriose and dextran were not oxidized. ODH activity for the pullulans, expressed as the rate constant Kps, was only three times lower than that for maltose. When the oxidation of sugars with ODH was coupled to a color-forming reaction, quantitative spectrophotometric determination of sugars was possible using either Meldola's blue or N-methylphenazinium as electron acceptors in combination with nitrotetrazolium blue. Linear calibration curves for maltose, maltotriose, and debranched starch were obtained using this ODH method and compared with curves from the conventional spectrophotometric copper sulfate method. This work demonstrates that ODH can be advantageously used for the determination of polysaccharides.

Carbohydrate Dehydrogenases↗
PubMed1998

Rapid alcohol determination in plasma and urine by column liquid chromatography with biosensor detection.

An enzyme based amperometric biosensor used as a selective and sensitive detection unit in column liquid chromatography for the determination of ethanol and methanol in biological fluids such as plasma and urine is described. The reagentless enzyme electrode is based on the co-immobilisation of alcohol oxidase and horseradish peroxidase in carbon paste. The selectivity of the biosensor was found to vary when four various alcohol oxidase enzyme preparations from Candida boidinii, Pichia pastoris, and Hansenula polymorpha were used in the biosensors described. High sensitivity could be obtained for a number of alcohols, organic acids, and aldehydes. Optimisation regarding the sensitivity and selectivity of the four alcohol oxidase co-immobilised biosensors are outlined. A fast and reliable liquid chromatographic separation system with a PLRP-S polymer based separation column used with a phosphate buffer as the mobile phase was optimised using the best biosensor which was based on alcohol oxidase from P. pastoris and which showed the highest turnover rate for alcohols, as the detector for the determination of ethanol and methanol in human urine and plasma samples. The selectivity and stability of the biosensor were retained by working at an applied potential of -50 mV versus Ag/AgCl, the optimal operational potential, and by the casting of a protective membrane on the electrode surface. High selectivity of the enzyme electrode was also found towards other easily oxidisable interfering species normally present in biological fluids. It was found that stable and reliable determinations of ethanol and methanol in plasma and urine could be performed with only a simple dilution and centrifugation step prior to injection into the liquid chromatographic system. An analysis time of 4 min was required for the assay, with a sample throughput of 13 samples h(-1).

Acids↗
PubMed1998

Bioelectrochemical monitoring of phenols and aromatic amines in flow injection using novel plant peroxidases.

An amperometric flow system combined with a glucose oxidase-mutarotase reactor was optimized and used to determine aromatic amines and phenols using peroxidase-modified graphite electrodes. An increase in currents upon injection of the analyzed substrate was shown to be approximated by a Michaelis-Menten type dependence. The detection limit was calculated as 3 times the noise, and the sensitivity was calculated as Imax/K(m)app. Commercially available horseradish peroxidase was compared with tobacco anionic and peanut cationic peroxidases for determination of aromatic amines and phenols. Detection limits of 10 nM for determination of o-aminophenol and o- and p-phenylenediamine achieved with a tobacco peroxidase-modified electrode give a promise for further improvements in sensitivities and detection limits of biosensors.

Aniline Compounds↗
PubMed1998

Optimal membrane choice for microdialysis sampling of oligosaccharides.

An analytical methodology based on microdialysis sampling, high-performance anion-exchange chromatography and integrated pulsed electrochemical detection for the monitoring of oligosaccharides in bioprocesses is presented. Amylopectin and model maltooligosaccharide standards; glucose, maltose, maltotriose, maltotetraose, maltopentaose, maltohexaose and maltoheptaose were used to demonstrate its versatility in view to sampling in enzymatic bioprocesses. The performance and characteristics of membranes with the same cut-off ranging between 3 and 100 kDa, were evaluated with respect to their extraction fraction (EF), permeability factors, temperature stability and protein (enzyme) interaction. All investigated membranes showed some non-specific interaction with enzymes. The EF and non-specific membrane-enzyme interactions were higher for the polysulfone membranes compared with the polyamide and polyethersulfone membranes. For all saccharides, the EF was independent of the concentration even for a 250-fold change in concentration. The EF and morphology of the membranes in their dehydrated state, as observed using scanning electron microscopy did not show any significant difference between membranes exposed to a 90 degrees C temperature for 3 and 24 h indicating their applicability to the study of high temperature bioprocesses.

Algorithms↗
PubMed1998

Enzymatic specificity and hydrolysis pattern of the catalytic domain of the xylanase Xynl from Rhodothermus marinus.

The catalytic domain of a xylanase from Rhodothermus marinus was produced in Escherichia coli. The catalytic domain belongs to glycosyl hydrolase family 10. The produced protein has a 22-amino acid leader peptide followed by a 411-amino acid truncated xylanase. The molecular mass was 48 kDa and the recombinant xylanase had a pI of 4.9. The pH and temperature optima for activity were determined to be 7.5 and 80 degrees C, respectively. At that temperature the enzyme had a half-life of 1 h 40 min. An addition of 1 mM calcium stabilized the activity of the enzyme at 80 degrees C. The xylanase had its highest specific activity on oat spelt xylan but was active also on other xylans and to a limited extent on some other polysaccharides (soluble glucans). No exo- or endo-cellulase activity was observed. Hydrolysis of xylo-oligomers and oat spelt xylan was studied and the predominant products of hydrolysis were xylobiose and xylotriose. The enzyme was inactive on xylobiose, xylotriose and on the soluble fraction from oat spelt xylan. The R. marinus xylanase is shown to have a strong preference for internal linkages and is therefore classified as an endo-xylanase.

Amino Acid Sequence↗
PubMed1996

Effect of liposome-encapsulation on immunomodulating and antiviral activities of interferon-gamma 1.

The effect of liposome-encapsulation on the immunomodulating and antiviral activities of interferon-gamma (IFN-gamma) was evaluated in this study. The immunomodulating activity was measured by increases in phagocytic activity and in nitric oxide production by peritoneal macrophages from mice treated with both free and LIP-IFN-gamma (4000 U/mouse, intraperitoneal injection). Resident peritoneal macrophages harvested from mice treated with free unencapsulated IFN-gamma or muramyl dipeptide showed significant increases in macrophage yield, and enhanced ability to phagocytize zymosan particles. In mice treated with liposome-encapsulated IFN-gamma (LIP-IFN-gamma), both macrophage yield and phagocytic activity further increased by 2-fold over unencapsulated IFN-Y. In addition, the activation of peritoneal macrophages with LIP-IFN-gamma showed enhanced production of NO when the cells were cultured ex vivo. Using a murine respiratory influenza infection model, intranasally administered LIP-IFN-gamma conferred protection to 70% in mice challenged intranasally with 10 LD50 doses of influenza A/PR/8 virus compared with a 20% survival rate using free IFN-gamma. Together these results suggest that liposome-encapsulation increases the immunomodulating and antiviral activities of IFN-gamma. Liposome-encapsulation of IFN-gamma may provide additional therapeutic advantages by reducing IFN-gamma toxicity while prolonging its body retention.

Animals↗
PubMed1995

Amperometric thin film biosensors based on glucose dehydrogenase and Toluidine Blue O as catalyst for NADH electrooxidation.

Amperometric glucose sensors were constructed based on solid graphite electrodes, surface-modified with NAD+ dependent glucose dehydrogenase (GDH), Toluidine Blue O (TBO), and protective ionic polymers. The electrocatalytic oxidation of NADH was evaluated from cyclic voltammetry with TBO dissolved, adsorbed, and electrostatically or covalently bound to polymers. The NADH and glucose sensors constructed were investigated and operated at 0 mV vs. Ag/AgCl using single potential step chronoamperometry. The operational stability of the glucose sensors was limited by leakage of NAD+. A glucose sensitivity much higher than carbon paste electrode was found. A sensitivity as high as 25 microA cm-2 mM-1 was achieved.

Biosensing Techniques↗
PubMed1994

Enzyme-based biosensor as a selective detection unit in column liquid chromatography.

A reagentless enzyme electrode based on co-immobilized alcohol oxidase and horseradish peroxidase was used as the working electrode in an amperometric flow-through cell connected to a column liquid chromatographic (CLC) system for the selective detection of methanol and ethanol. The enzymes were covalently immobilized in carbon paste (graphite-phenylmethylsilicone oil) in the presence of polyethylenimine. Electrodes prepared from the enzyme-modified carbon paste were optimized with respect to their sensitivity and selectivity. Different membranes were cast or electropolymerized directly on the surface of the electrode to increase the long-term stability of the biosensor. The compatibility with the reversed-phase chromatographic system was established. A PLRP-S polymer-based separation column was used with phosphate buffer as the mobile phase. The selectivity of the enzyme electrode was also determined by injecting some easily oxidizable and possibly interfering species normally present in biological samples. The enzyme electrode was also used in an on-line system, consisting of a microdialysis probe as the sampling unit, the CLC system and the biosensor detection device, for the selective following of the ethanol produced when a paper pulp industrial waste water was fermented with Saccharomyces cerevisiae.

Biosensing Techniques↗
PubMed1994

Optimization of enzyme ratios in a coimmobilized enzyme reactor for the analysis of D-xylose and D-xylulose in a flow system.

A coupled enzyme system for the detection of D-xylose and D-xylulose is presented. The system is based on three consecutive enzymatic steps. The enzymes xylose isomerase (XI), mutarotase (MT), and glucose dehydrogenase (GDH) are coimmobilized on controlled pore glass and packed in a bed reactor. The relative amount of enzymes, i.e., enzyme ratio, plays a critical role in driving the overall reaction, resulting in a system with linear response characteristics and an operational range of several orders of magnitude. Three different enzyme ratios are assayed to achieve maximum conversion efficiencies for xylose and xylulose. The highest enzyme unit ratio assayed, 13.4 of GDH to XI, gave the highest apparent pseudo-first-order rate constant showing the importance of the last enzymatic reaction in the coupled system to make the overall reaction thermodynamically favorable. A pH of 7.0 was found to be an optimum compromise for the multienzyme system. Sensitivity was dependent on NAD+ concentration. The study was carried out in a flow injection system. The optimized reactor has been applied for the catalytic detection of pentoses in flow injection analysis (FIA) and liquid chromatography (LC).

Aldose-Ketose Isomerases↗
PubMed1993

Amperometric biosensor for the determination of phenolic compounds using a tyrosinase graphite electrode in a flow injection system.

Selective and sensitive devices for the monitoring of phenol and phenolic compounds are required in clinical and environmental analysis. This paper describes a biosensor for the analysis of phenolic compounds in a flow injection system. The enzyme electrode is based on the use of immobilized tyrosinase and the amperometric detection of the enzymatic product at -50 mV vs. SCE. The enzyme is covalently immobilized on the surface of a carbodiimide-activated graphite electrode. The biosensor responds to a variety of phenolic substrates with different conversion efficiencies. The detection limit for phenol is 0.003 microM (S/N = 3), a quantification limit of 0.01 microM (rsd 3.7%), and an extended dynamic range up to 5 microM is achieved with a sample frequency of 110 samples per hour.

Biosensing Techniques↗
PubMed1993

A reagentless amperometric biosensor for alcohol detection in column liquid chromatography based on co-immobilized peroxidase and alcohol oxidase in carbon paste.

A reagentless carbon paste electrode chemically modified with covalently bound alcohol oxidase and horse-radish peroxidase was examined as a selective sensor in flow injection and column liquid chromatography. A combination of carbodiimide, glutaraldehyde, and polyethyleneimine was used for immobilizing the enzymes in the paste. The surface of the electrodes was protected by first forming a layer of electropolymerized ortho-phenylenediamine followed by deposition of a cation exchange membrane (Eastman AQ 29D). The electrodes were used for detection of hydrogen peroxide, methanol, ethanol, propanol, isopropanol, and butanol. Preliminary investigations of the use of this sensor for bioprocess control are reported.

Alcohol Oxidoreductases↗
PubMed1993

Study of a reagent- and mediator-less biosensor for D-amino acids based on co-immobilized D-amino acid oxidase and peroxidase in carbon paste electrodes.

A biosensor for the analysis of D-amino acids is described. Carbon paste (graphite/paraffin oil) was chemically modified with immobilized D-amino acid oxidase and either horse-radish peroxidase or fungal peroxidase from Arthromyces ramosus. The two enzymes dissolved in buffer, together with an amine containing oligomer or polymer, were adsorbed on dry graphite. Prior to immobilization, the graphite was heat treated at 700 degrees C for 15 s to promote an efficient electron transfer between graphite and the peroxidase. The mixture was dried before addition of the pasting liquid. The sensor is based on the fact that the hydrogen peroxide produced by the action of D-amino acid oxidase is electrocatalytically reduced through the action of the peroxidase. The amine containing compound acted as a stabilizer and activator of the enzymes in the paste. The enzyme electrode was investigated as a sensor for D-phenylalanine and hydrogen peroxide in a flow through electrochemical cell connected to a single line flow injection system. The influences on the response by different additives to the paste and pH are reported. Linear calibration curves were obtained between 0.1 and 1.4 mM for D-phenylalanine and 5 and 1000 microM for hydrogen peroxide at an applied potential of -50 mV vs. Ag/AgCl. The sensor was also active for the following D-amino acids: D-alanine, D-valine, D-leucine, D-isoleucine, D-serine, D-aspartic acid, D-glutamic acid, D-lysine, D-histidine, D-arginine, D-tryptophan, D-methionine, and D-proline.

Amino Acids↗
PubMed1990

Bioselective detection in liquid chromatography by the use of immobilized enzymes.

The combination of liquid chromatography and immobilized enzyme reactors (LC/IMER) is a rapidly developing field of research. The enzymes are used to catalyse chemical reactions and thereby facilitate selective detection. In this instance the chemical derivatizations are performed in the post-column mode. The selectivity is demonstrated with respect to interfering compounds present in complex samples.

Amino Acids↗
PubMed1990

Liquid chromatographic separation and stereoselective detection of L- and D-amino acids with catalytic reaction detection using immobilized enzymes.

A post-column LC detection system is described for the stereoselective detection of L- and D-amino acids. The effluent of the LC column passes an immobilized enzyme reactor (IMER) containing either L- or D-amino acid oxidase. The hydrogen peroxide formed in this reactor by the oxidation of the amino acids is then transported to a second IMER containing horse-radish peroxidase. With the addition of 4-aminophenazone and dichlorophenosulphonyl chloride to the carrier, the hydrogen peroxide is reacted to form a red-coloured complex which is detected in a flow-through photometric cell at 514 nm. Applications to the analysis of amino acids in bovine and human sera are described.

Amino Acid Oxidoreductases↗
PubMed1987

Enzyme electrodes and their application.

Starting from the state of the art, principles for improving the analytical characteristics of enzyme electrodes are discussed. Coupling of appropriate amperometric electrode processes with enzyme systems, e.g. urease or aminopeptidases, results in a simplification of operation. Optimal sample frequencies are realized on the basis of enzyme membranes, with both a small characteristic diffusion time and a high enzyme activity, applied in a well-designed sample-processing system. Coupled enzyme reactions of the sequence or competition type are successfully used for extension to new analytes, e.g. inhibitors, cofactors or alternative substrates. Cyclization of the analyte enhances the sensitivity of enzyme electrodes to the nanomolar concentration range. Enzymic anti-interference layers are a tool for improving the sensor specificity. The operational characteristics of enzyme electrodes are thus adaptable to any given analytical problem.

Biotechnology↗
PubMed1985

An amperometric glucose sensor made by modification of a graphite electrode surface with immobilized glucose oxidase and adsorbed mediator.

A membrane-free glucose sensor was made by covalent immobilization of glucose oxidase on graphite followed by adsorption of N-methyl-phenazinium ion (PMS+). The mediator was found to be necessary for the electron transfer between the enzyme and the electrode. beta-D-glucose was determined amperometrically at an applied potential of +50 mV vs SCE. The current was independent of the rotational speed which indicates a kinetically controlled response. The response was strictly linear from the detection limit, 0.5, to 150 microM and usable up to about 2 mM beta-D-glucose. The immobilized enzyme was stable over several months but the mediator had to be renewed daily.

Electrodes↗