Biomedical subjects

J Emnéus

Publications and source records attributed to J Emnéus.

14 recordsLinked to original sources

PubMed2006

Electroenzymatic reactions with oxygen on laccase-modified electrodes in anhydrous (pure) organic solvent.

The electroenzymatic reactions of Trametes hirsuta laccase in the pure organic solvent dimethyl sulfoxide (DMSO) have been investigated within the framework for potential use as a catalytic reaction scheme for oxygen reduction. The bioelectrochemical characteristics of laccase were investigated in two different ways: (i) by studying the electroreduction of oxygen in anhydrous DMSO via a direct electron transfer mechanism without proton donors and (ii) by doing the same experiments in the presence of laccase substrates, which display in pure organic solvents both the properties of electron donors as well as the properties of weak acids. The results obtained with laccase in anhydrous DMSO were compared with those obtained previously in aqueous buffer. It was shown that in the absence of proton donors under oxygenated conditions, formation of superoxide anion radicals is prevented at bare glassy carbon and graphite electrodes with adsorbed laccase. The influence of the time for drying the laccase solution at the electrode surface on the electroreduction of oxygen was studied. Investigating the electroenzymatic oxidation reaction of catechol and hydroquinone in DMSO reveals the formation of various intermediates of the substrates with different electrochemical activity under oxygenated conditions. The influence of the content of aqueous buffer in the organic solvent on the electrochemical behaviour of hydroquinone/1,4-benzoquinone couple was also studied.

Biosensing Techniques↗

PubMed2003

Screen-printed multienzyme arrays for use in amperometric batch and flow systems.

Screen-printing technology for electrode fabrication enables construction of amperometric devices suitable for combination of several enzyme electrodes. To develop a biosensor array for characterisation of wastewaters, tyrosinase and horseradish peroxidase (HRP) or cholinesterase-modified electrodes were combined on the same array. The behaviour of the tyrosinase-modified electrode in the presence of hydrogen peroxide (required co-substrate for the HRP-modified electrode) and acetylthiocholine chloride (required co-substrate for cholinesterase) was studied. Performance of bi-enzyme biosensor arrays in the batch mode and in the flow-injection system are discussed.

Acetylcholinesterase↗

PubMed2002

GDH biosensor based off-line capillary immunoassay for alkylphenols and their ethoxylates.

The application of a quinoprotein glucose dehydrogenase modified thick-film sensor as label detector in a capillary immunoassay (CIA) for xenoestrogens is presented. The detection of the alkylphenols and their ethoxylates is based on the competition between the analyte and tracer molecules for the binding sites of anti-alkylphenol ethoxylate antibodies. This assay is performed off-line in small disposable PVC capillaries coated with immobilized antibodies. This format allows the combination of the assay with a small portable device potentially useful for on-site environmental monitoring. Beside high amplification the utilization of beta-galactosidase as enzyme label allows the direct combination with a GDH biosensor at optimal pH conditions. The bioelectrocatalytic properties of this biosensor offer an additional amplification and thus allow a very sensitive quantification of 4-aminophenol, generated by the beta-galactosidase. Detection limits of the analytes in the microg/l range were obtained, while other phenolics and surfactants showed no or very little cross reactivity.

Biosensing Techniques↗

PubMed2002

Detection of Escherichia coli in water by culture-based amperometric and luminometric methods.

The application of amperometric biosensor- and chemiluminiscence based methods for rapid detection of viable E. coli in water has been investigated. An amplification of the amperometric signal by a factor of 4 was obtained when the cellobiose dehydrogenase (CDH) biosensor was used instead of a plain graphite electrode for detection of b-galactosidase (b-GAL) activity at 22.5 degrees C. A linear correlation was demonstrated for detection time (DT) vs. initial concentrations (logarithmic units) of E. coli IT1 and E. coli in environmental samples, respectively, by use of the CDH biosensor or a chemiluminometric technique. The study has shown that an E. coli concentration > or = 10(4) cfu/100 mL in environmental samples was determined by the CDH biosensor within one working day. However, further reduction of the DT can be obtained, e.g. by increasing the signal amplification factor using other biosensors.

Biosensing Techniques↗

PubMed2001

Antibody-based methods for surfactant screening.

This brief overview summarises the immunoassay-based results obtained in the course of two years of the European INCO-Copernicus project BIOTOOLS. The project is aimed at simplifying the procedures for detection of surface active compounds (SAC) using, among others, antibody-based methods, i.e., microtiter plate-based enzyme-linked immunosorbent assays (ELISA), polarisation fluoro immunoassays (PFIA), and enzyme flow injection immunoassays (FIIA). Thirty-three rabbits were immunised with five different sulphophenyl moieties and three p-hydroxyphenyl moieties conjugated to protein immunogens to produce analytical antibodies against linear alkylbenzene sulphonates (LAS) and nonylphenol (NP). Although most of the antibodies exhibited binding reaction in indirect ELISA, only a few showed the required assay sensitivity. The best antibodies for LAS exhibited a 50% binding inhibition at IC50 19.8 microg L(-1) in indirect ELISA. Similar inhibition was observed for direct ELISA using peroxidase tracers. Antibodies against NP allowed the establishment of an indirect assay operating in the mg L(-1) range. A rapid and simple protocol for the screening of NP and LAS using homogeneous PFIA is described. The assay time for 10 samples was 7 minutes, thus allowing fast detection of the selected SAC at the mg L(-1) level. A generic competitive FIIA system, using a protein G column for separation of free and antibody-bound beta-galactosidase (beta-Gal) tracer, was developed for the screening of LAS, NP, and nonylphenol decaethoxylate (NPEO10). The FIIA had a sample throughput (STP) of 5-10 samples per hour, with limits of detection (LOD) for LAS, NP, and NPEO10 of 19.5, 52, and 2.4 microg L(-1), respectively. The developed FIIAs were applied to spiked rain and surface water.

Animals↗

PubMed2000

Flow immunochemical bio-recognition detection for the determination of interleukin-10 in cell samples.

On- and off-line heterogeneous non-competitive flow immunoassays for the determination of Interleukin-10 are described. The sample containing IL-10 is mixed, either on-line in a reaction coil or off-line in a test tube, with fluorescent labelled anti-IL-10 antibodies to form an antibody-antigen complex. The labelled unbound antibodies are trapped on an immobilized IL-10 column whereas the IL-10-antibody complexes are eluted and detected downstream by a fluorescence detector. The optimization of the systems was performed with respect to choice of affinity support, flow rate, carrier buffer additives, pH and antibody-antigen association. Both bio recognition assays were tested with a spiked cell medium and the IL-10 detection limits in this matrix was found to be 8 fmol using the off-line incubation mode and 40 fmol using the on-line incubation mode. The sample through-put was 26 and 40 samples per hour in the on-line and off-line incubation modes, respectively. IL-10 identification in the sample fractions was achieved using MALDI-TOF MS.

Antigen-Antibody Reactions↗

PubMed2000

An enzyme flow immunoassay that uses beta-galactosidase as the label and a cellobiose dehydrogenase biosensor as the label detector.

The aim was to develop a fast generic enzyme flow immunoassay (EFIA) using a beta-galactosidase (beta-GAL) label in combination with colorimetric detection as well as with a new amperometric biosensor as the label detector. The amperometric biosensor was previously developed within the group for the determination of diphenols in surface water samples. Antigen (Ag, analyte), tracer (Ag*, antigen labeled with beta-GAL), and antibody (Ab) were incubated off-line. After the equilibrium was reached, the sample was introduced into the flow system. The antibody complexes, AgAb and Ag*Ab, were trapped in a protein G column while the free unbound tracer was eluted and detected by an amperometric biosensor downstream after substrate reaction. The enzyme label beta-GAL converted the substrate 4-aminophenyl-beta-D-galactopyranoside (4-APG) into 4-aminophenol (4-AP), which subsequently was detected by a cellobiose dehydrogenase (CDH) modified solid graphite electrode. 4-AP was first oxidized at the electrode surface at +300 mV vs Ag/AgCl, and the formed 4-imino quinone (4-IQ) was reduced back to 4-AP by the CDH in the presence of cellobiose. By combining the EFIA with the CDH biosensor, the overall signal of one tracer molecule is amplified at two occasions, i.e., one enzyme label converts the substrate into many 4-AP molecules, and second these are further amplified by the CDH biosensor. The optimum conditions for the EFIA in terms of the molar ratio between tracer and beta-GAL, temperature, flow rate, etc., was investigated with colorimetric detection, using 2-nitrophenyl-beta-D-galactopyranoside (2-NPG) as the beta-GAL substrate. The performance of both the colorimetric and CDH biosensor detection was investigated and both methods were applied for determination of the model compound atrazine in spiked surface water samples. Detection limits of 0.056 +/- 0.008 and 0.038 +/- 0.007 microg L(-1) and IC50 values of 2.04 +/- 0.294 and 0.42 +/- 0.08 microg L(-1) were obtained for colorimetric and CDH detection, respectively. Matrix effects were less pronounced with the CDH biosensor than with colorimetric detection.

Biosensing Techniques↗

PubMed2000

Development of an offline noncompetitive flow immunoassay for the determination of interleukin-8 in cell samples.

A noncompetitive flow immunoassay system (FIA) for the analysis of interleukin-8 (IL-8) in cell samples was developed. Affinity interaction assays based on offline incubation of excess labeled antibodies and antigen (IL-8) were carried out. The residual unbound labeled antibody was trapped in an immunoaffinity column with immobilized IL-8 while the immunocomplex, labeled antibody/IL-8, was detected by a fluorescence detector. Two fluorophores, FLUOS and Cy5.5, were conjugated with IL-8 antibody. Optimization and comparison between the two fluorescent labeled antibodies were performed with regard to pH, antibody concentration, flow rate, injection volume, and association time. Additionally, a horseradish peroxidase enzyme label was used for the conjugation to the anti-IL-8. The enzyme substrate reaction was optimized with respect to temperature and length of the substrate reaction coil. The detection limits were found to be 200 amol using the FLUOS-labeled anti-IL-8 and 1 fmol using the Cy5.5 fluorescence label. The developed FIA technique was applied for the analysis of IL-8 in cell samples. Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry was used to identify IL-8 in the cell samples.

Antibodies↗

PubMed1998

On-line coupling of microdialysis sampling with liquid chromatography for the determination of peptide and non-peptide leukotrienes.

An automated on-line sampling method was developed using microdialysis as the simultaneous sampling and sample pre-treatment technique. The extraction fraction values of microdialysis probes sampling different eicosanoids were investigated. The impact of cyclodextrins in the perfusion liquid used for sampling hydrophobic eicosanoids in biological systems was also studied. The total time for one analysis was 7.6 min allowing seven measurements per hour for monitoring kinetic changes in biological systems.

Autoanalysis↗

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

Fluorescence polarisation for immunoreagent characterisation.

Antibodies were characterised using fluorescence polarisation, a homogeneous assay technique in which all reagents are in solution. Kinetic studies on the association and dissociation of the immunocomplex were performed. A competitive assay was used and the sensitivities, operational linearities, as well as the specificities of the immunoassays were experimentally determined for various antibody preparations with specificity for triazines. Detection limits for atrazine in water samples were determined to be within the range of 0.08-0.4 ng ml(-1) using a 5-min incubation time and a 0.5-ml sample volume.

Animals↗

PubMed1998

High sample throughput flow immunoassay utilising restricted access columns for the separation of bound and free label.

A flow immunodetection system with high sample throughput capacity is described for the screening of various analytes. The immunochemical detection principle is based on the chromatographic separation of the formed immunocomplex (AbAg or AbAg*) and the free antigen (Ag) by a restricted access (RA) column, utilising size-exclusion and reversed-phase mechanism. A fluorescein labelled analyte (Ag*) was used in the competitive assay format with fluorescence detection. The speed and simplicity of the assay were the greatest advantages, allowing measurement of the analyte to be carried out in less than 1 min. The biocompatibility and capacity of the restricted access material allowed multiple injections of up to 5000, without any breakthrough of the fluorescent tracer molecule and thus need for regeneration. The flow immunoassay was developed using the well-known atrazine herbicide and some transformation products as model compounds, due to their human toxicity and widespread use. The sample throughput was 80 samples per hour and the detection limits were 1.4 nM (300 pg/ml) for atrazine (Ab I) and 2.3 nM (500 pg/ml) for the sum of triazines (Ab II-III). Different sample matrices, PBS buffer, creek water, and urine were successfully applied in the flow system without the need for any sample handling step. For plasma samples an additional clean-up step using solid-phase extraction had to be included. The resulting detection limits for atrazine in plasma and water samples using this clean-up and trace enrichment procedure were found to be 2 ng/ml and 20 pg/ml, respectively. The analysis could be performed at a sample throughput rate of 400 per 6-h working shift.

Atrazine↗

PubMed1998

Optimisation of a heterogeneous non-competitive flow immunoassay comparing fluorescein, peroxidase and alkaline phosphatase as labels.

Off- and on-line strategies for a non-competitive heterogeneous flow immunoassay were developed comparing three different labels. The samples, containing the model compounds digoxin or digoxigenin, were either pre-incubated off-line or on-line in a mixing coil with excess of labelled anti-digoxigenin Fab-fragments. The excess of Fab-fragments was then separated from the digoxin bound Fab-fragments by passing the sample through a column with immobilised digoxin. The off-line immunochemical detection system is suitable for sensitive high through-put screening of the analytes, whereas the on-line system is more suitable for coupling as a post-column detection unit to liquid chromatography. The digoxin and digoxigenin content in the sample were quantified using fluorescein (F) and enzyme (peroxidase (POD), alkaline phosphatase (AP)) labelled Fab-fragments. The fluorescein label was directly measured with the fluorescence detector, whereas a fluorescent enzyme product was measured in the two enzyme based systems, using 3-(p-hydroxyphenyl)-propionic acid (HPPA) and hydrogen peroxide for POD and, and 4-methylumbelliferyl phosphate (4-MUP) for AP. The highest sensitivity and lowest limit of detection (LOD) was obtained with the Fab-POD system with LODs for digoxin and digoxigenin in the off- and on-line configurations of 0.025 and 0.01 nM, respectively. The sample through-put for the off- and on-line systems were 43 and 32 samples per hour, respectively.

Alkaline Phosphatase↗

PubMed1998

A flow immunoassay for studies of human exposure and toxicity in biological samples.

This paper describes a heterogeneous competitive flow immunoassay with a high sample throughput which can be used for the screening of smaller analytes in various samples. The method is based on off-line incubation of the analyte (Ag), a fluorescent labelled tracer (Ag*) and the corresponding antibody (Ab). The separation of bound (Ab-Ag*) and free tracer (Ag*) is based on a size exclusion and reversed phase mechanism utilizing a restricted access (RA) column. The column traps the free unbound tracer (Ag*) in its hydrophobic (C18) inner cavity but excludes the large Ab-Ag* complex, which is passed on and measured by the fluorescence detector. The flow immunoassay was developed using the triazine herbicide atrazine as a model compound owing to its human toxicity and widespread use. A sample throughput of 80 samples per hour and a detection limit of 300 pg ml-1 in water were obtained. Urine samples were successfully applied for direct injections into the flow system, while for human plasma samples an additional clean-up step using solid phase extraction was efficiently included where pure extract is obtained with the highly stable and biocompatible extracting column material. The resulting detection limits for atrazine in plasma and water samples using this clean-up and trace enrichment procedure were found to be 2 ng ml-1 and 20 pg ml-1 respectively.

Animals↗