Can affinity sensors be used to detect food contaminants?
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Food Contamination↗
Biomedical subjects
U Bilitewski
Publications and source records attributed to U Bilitewski.
Development of a multichannel fluorescence affinity sensor system
A multichannel fluorometer is proposed for analysis of biochemical reactions. The sensor is based on the luminescence generation in the evanescent field of a totally reflected laser beam. For transduction, multiple reflection elements are used. Multichannel operation is realized, including the possibility of applying different solutions to each channel at the same time. First experimental results, obtained with fluorescein or Cy5 as labels in a model hybridization assay, demonstrate the applicability and allow the detection of 3-10 fmol injected fluorescently labeled oligonucleotide.
Journal Article↗
Electrochemical reduction of flavocytochromes 2B4 and 1A2 and their catalytic activity.
The present study shows that cytochromes P450 2B4 and 1A2 with a covalently attached riboflavin (semisynthetic flavocytochromes RfP450 2B4 and RfP450 1A2) can be reduced electrochemically on rhodium-graphite electrodes at a potential of -500 mV (vs Ag/AgCl). In the presence of substrates such as aminopyrine, aniline, 7-ethoxyresorufin, and 7-pentoxyresorufin, N-demethylation, p-hydroxylation, and O-dealkylation reactions proceeded, as was confirmed by product analysis. Rates of electrocatalytically driven reactions are comparable to those obtained using NAD(P)H as the source of reducing equivalents. These results suggest the practicality of developing flavocytochrome P450s as catalysts for oxidation reactions with different classes of organic substrates.
Alkylation↗
Enrichment of hydrophobic proteins via Triton X-114 phase partitioning and hydroxyapatite column chromatography for mass spectrometry.
Membrane proteins are the starting point of several signal transduction pathways. Therefore, the separation and identification of these proteins are of great interest in proteome analysis. However, the specific properties of membrane proteins seriously impede their analysis. We present an effective and highly reproducible method for the two-dimensional separation of extremely hydrophobic proteins and demonstrate the advantages of special preseparation procedures for the identification of proteins which have very similar Mr and p/. Using the example of the integral membrane protein very low density lipoprotein (VLDL) receptor (NCBI Acc. # 1730111) and the soluble heat shock protein (HSP) 90 (NCBI Acc. # 386786) we present the applicability of a phase-separation system with Triton X-114. Using matrix assisted laser desorption/ionization-time of flight-mass spectrometry (MALDI-TOF-MS) of the protein spots after 2-D separation of the hydrophilic and the strongly hydrophobic protein fraction of human endothelial cells (ECV cell line), we were able to distinguish both proteins.
Cell Line↗
Production and structure elucidation of glycoglycerolipids from a marine sponge-associated microbacterium species.
The bacterium Microbacterium sp., isolated from the sponge Halichondria panicea, produced four unusual cell-associated glycoglycerolipids and one diphosphatidylglycerol when grown on marine broth and on artificial seawater media. The lipids were isolated by chromatography on silica columns and their structures elucidated using a combination of multidimensional NMR and MS techniques. The main compound was 1-O-acyl-3-[alpha-glucopyranosyl-(1-3)-(6-O-acyl-alpha-mannopyranosyl )]glycerol (GGL.2) with 14-methyl-hexadecanoic acid and 12-methyl-tetradecanoic acid positioned at C-6 of the mannose unit and at the glycerol moiety. Glycolipid production was correlated with growth and reached a maximum value of 200 mg/L when grown on artificial seawater medium with 20 g/L glucose. The main compound decreased the surface tension of water down to 33 mN/m and the interfacial tension of the water/n-hexadecane system down to 5 mN/m. In addition to this good surface-active behavior, the main glycoglycerolipid showed antitumor activities.
Actinomycetales↗
Development of an automated microbial sensor system.
An automated whole cell biosensor system was developed by integration of immobilized microbial cells in a flow-through system with screen-printed flow-through electrodes as detectors. The detectors used were thick-film Pt-electrodes in a 3-electrode configuration constructed as sandwich flow-through cells with a volume of about 36 microliters polarized at -900 mV. The measuring principle was the determination of oxygen consumption due to the microbial metabolism. Fructose was used as model analyte. The microorganisms were immobilized on cellulose-acetate membranes and integrated into a newly created reaction chamber (membrane reactor). The microbial cells used were Rhodococcus erythropolis and Issatchenkia orientalis known to be suitable for the determination of biological oxygen demand.
Biosensing Techniques↗
Application of screen-printed electrodes as transducers in affinity flow-through sensor systems.
An affinity flow-through sensor system based on a heterogeneous competitive affinity assay for the determination of low molecular weight compounds is described using the examples of biotin and atrazine determination. The binding proteins, either streptavidin or a biotinylated monoclonal antibody, were immobilized on a biotinylated screen-printed electrode, where the competition between the analyte and an analyte-enzyme-conjugate took place. Determination of the bound enzyme was done through the supply of suitable enzyme substrates and electrochemical determination of an enzyme reaction product. In the assays described here, peroxidase was used as enzyme label. As hydrogen peroxide and hydroquinone were used as enzyme substrates, the amount of enzyme retained at the screen-printed graphite electrode was determined amperometrically at a reducing potential of -600 mV vs a screen-printed platinum electrode. The activation of the electrode by biotinylation was done in a batch procedure outside the system, before the electrode was inserted. All following steps of the assay were performed automatically in an unsegmented flow-through system through an appropriate delivery of required reagents. The system was optimized mainly through the determination of biotin. This assay was based on the competition between biotin and biotinylated peroxidase for the binding sites of streptavidin. The method showed a linear range from 0.045 to 2 micrograms/l (r2 = 0.9997, n = 7) with RSD lower than 3.8%. The system was modified further by using a biotinylated monoclonal antibody against atrazine for analyte recognition and performing a competitive assay between atrazine and a triazine-peroxidase-conjugate. The linear range was from 0.01 to 10 micrograms/l, with IC50 = 0.4 microgram/l and RSD lower than 4.6%. The method was also applied to atrazine spiked water samples. Regeneration of the sensor surface was based on removal of streptavidin in both assays.
Atrazine↗
Synthesis of mono- and bifunctional peptide-dextran conjugates for the immobilization of peptide antigens on ELISA plates: properties and application.
Dextran has been used as a carrier molecule for the synthesis of monofunctional peptide-dextran conjugates. The immunodetection of such carrier immobilized peptides on ELISA plates was compared to that of peptides adsorbed directly to immunoplates. The main features observed with peptide-dextran conjugates were as follows: only small amounts of peptide (1-2 mg) were necessary for coupling via alpha- or epsilon-amino groups to NaIO4-activated dextran (4 mg); the coupling yield was up to 68%; an amino acid analysis of the conjugate enabled the amount of carrier immobilized peptide to be calculated; an estimated 15-17 peptides were bound per dextran molecule (MW 73,500); using a carbohydrate as carrier reduces the possibility of non-specific interactions because no hydrophobic or ionic sites and no protein-like epitopes exist on the carrier apart from the peptide ligand. It can be assumed that some peptide ligands provide the forces for an interaction with the plate surface whereas other remain free for the interaction with the antibody. Thus, the detection with monoclonal anti-peptide antibodies allowed peptide-dextran conjugates to be used at coating concentrations of 1-3 nM peptide, corresponding to 0.6-2.6 ng peptide-dextran per well. In contrast, concentrations of 150-500 nM were required for coating with peptides. The applicability of monofunctional peptide-dextran conjugates was demonstrated by investigating the titer and specificity of a polyclonal anti-peptide serum developed against human gastrointestinal glutathione peroxidase. The introduction of biotin as a second ligand of the dextran conjugate permitted its capture on streptavidin coated plates. This synthesis of bifunctional peptide-biotin-dextran conjugates opens up additional possibilities for applications.
Adsorption↗
Real-time observation of affinity reactions using grating couplers: determination of the detection limit and calculation of kinetic rate constants.
The use of integrated optical grating couplers for the analysis of bioaffinity reactions in order to calculate kinetic rate constants was investigated. The specificity of the sensor surface was determined by adsorptive or covalent attachment of the specific ligands. As an evanescent field sensor, the specific interaction of the corresponding ligand could be observed in real time and without labels. The detection limit in terms of the molecular weight of the analyte was studied by the specific binding of biotinylated proteins of different molecular weights to avidin-loaded sensors. It was shown that grating coupler sensors allowed detection of compounds of at least 2000 daltons using high-affinity receptors, while the direct sensing of low molecular analytes, such as biotin, could not be significantly achieved. Association rate constants were calculated for the interaction of the different biotinylated proteins to avidin-covered sensors from single binding curves. Due to the strong binding between avidin and biotin, the dissociation of the formed complex could not be observed. Kinetic rate constants and equilibrium constants were determined by studying the interaction of human immunoglobulin with the immobilized receptor, protein G. For the four human immunoglobulin subclasses a high affinity to protein G was determined with affinity constants ranging from 3.3 to 8.4 x 10(8) M-1.
Antibody Affinity↗
A solid-phase enzyme linked immunosorbent assay using monoclonal antibodies, for the detection of African swine fever virus antigens and antibodies.
An improved solid-phase enzyme linked immunosorbent assay (ELISA) using monoclonal antibodies was developed to detect an African swine fever virus protein (VP73) in pig samples. The use of monoclonal antibodies against VP73 allowed a sensitive and specific sandwich ELISA. This assay detected a limiting antigen concentration of 0.05 microgram/ml of VP73, lower than the detection limit of 0.6 microgram/ml obtained by using polyclonal antibodies by the same ELISA. The whole virus particle was detected with this method to a limit of 2.3 x 10(2) PFU/ml. At the same time, an indirect ELISA was developed to detect ASFV antibodies. The results also indicate that this method may be a useful technique for epidemiological surveys.
African Swine Fever↗
Analysis of the biotin-binding protein actinavidin using affinity capillary electrophoresis.
Affinity capillary electrophoresis (ACE) was applied to study the bioaffinity of ligand-receptor interaction between the microbial biotin-binding protein actinavidin and biotin. The ACE method is based on short time incubation of a mixture of actinavidin and increasing concentrations of biotinylated oligonucleotide (bio-ON), which was found to be an effective affinity ligand. Separation of intermediate loading forms of actinavidin from unbound ligand in the presence of micellar phase and by capillary zone electrophoresis enabled the quantitation of free bio-ON, permitting the evaluation of the biotin-binding capacity of actinavidin in absence and presence of sodium dodecyl sulfate (SDS). Although in the latter case actinavidin lost a part of its binding capacity (not more than 12%), it was still possible to develop an indirect, noncompetitive assay for the determination of actinavidin in culture liquid, utilizing the combination of micellar electrokinetic capillary chromatography (MEKC) and ACE. Due to the affinity interaction, actinavidin in the sample decreases the amount of bio-ON added, enabling quantitation of the protein. SDS, which is required in this assay to prevent protein adsorption to the capillary wall, greatly enhances the reproducibility and peak shape. Actinavidin levels determined are in agreement with those obtained by commonly used solid-phase analysis. The limit of detection was about 500 ng/mL. Thus the proposed method was found to be well suited for the evaluation of actinavidin affinity and monitoring of its levels in cultivation process.
Biotin↗
Microbial biosensor for free fatty acids using an oxygen electrode based on thick film technology.
A microbial biosensor based on thick film technology was developed. The microorganisms, Arthrobacter nicotianae, were immobilized in Ca-alginate directly on the electrode surface. For the stability of the calcium alginate gel the addition of 0.5 mM CaCl2 to the assay buffer was necessary. The respiratory activity of the microorganisms was monitored by oxygen consumption at -600 mV vs. Ag/AgCl reference electrode. The sensor was used in a batch system and was applied to the determination of free fatty acids in milk. Short-chain fatty acids (C4:0-C12:0) were the preferential substrates, with butyric acid being the main substrate. Consequently, the concentration of free short-chain fatty acids was represented as the butyric acid equivalent. The sensor showed linearity over the concentration range 9.5-165.5 microM (correlation coefficient, r = 0.99920). The response time of the sensor was approximately 3 min. No additional dialysis membrane was necessary, which led to a high sensitivity of the sensor and fast response times. Recovery rates of 98-113% were found for butyric acid in milk samples using the sensor without any additional membrane and a sample dilution of 200 by the assay. Two widespread disadvantages of microbial sensors, long response times and long times to return to the baseline signal after use, could be overcome.
Acyl-CoA Oxidase↗
Optimization of biosensing using grating couplers: immobilization on tantalum oxide waveguides.
This paper presents a comparative study of immobilization strategies for integrated optical grating couplers using tantalum oxide waveguides. As a model system the affinity reaction between protein G and human IgG was investigated. The receptors were coupled to the waveguide by adsorption, covalent attachment and avidin-biotin bridges after modifying the sensor surface by silanization introducing epoxy and amino groups. In addition, the results obtained by a monolayer coverage of the sensors with the receptors were compared with those using a carboxymethyl-dextran matrix allowing the immobilization of an increased amount of protein. The different coupling procedures were assessed by the system response due to the specific binding of human IgG to the sensing layer and are discussed with respect to their effectiveness, stability and the absence of non-specific binding.
Adsorption↗
Gas phase detection of cocaine by means of immunoanalysis.
Immunoanalytical techniques based on an indirect competitive ELISA to determine cocaine in the gas phase are described. A test-gas generator was developed and evaluated by determining saturation vapour pressures and the sublimation enthalpy of cocaine base. To achieve quantitative recovery of the drug, the saturated air stream was sucked through a retention fluid. For validation of the test gas generator, samples were analysed with a microtitre plate cocaine-ELISA based on monoclonal antibodies. To simplify the sampling and analysis procedures by analysing the retention fluid directly in the sampling vessel, particle-based immunoassay formats were developed. According to the ELISA format, avidin was immobilized on the particles. The application of various solid particles consisting of different materials with a wide range of diameters (0.5-550 microns) to the analysis of cocaine standards and gas samples showed good correlation with the results obtained with the microtitre plate ELISA with an average IC50 value (end-point of the test; concentration at 50% binding) of 9.7 ng ml-1. The particle-based assays showed IC50 values in the range of 5-54 ng ml-1 and signal background ratios ranging from 2 to 11. The application of particle-based assays for direct analysis of cocaine in the sample fluid was successfully performed with glass beads, precoated with avidin and a biotin-cocaine conjugate. Recovery of cocaine from the gas phase depended on the volume of sample fluid and on the geometry of the sample tube.
Chromatography, Gas↗
Development and evaluation of a dipstick immunoassay format for the determination of atrazine residues on-site.
On the basis of a semi-quantitative dipstick immunoassay (IA) for atrazine with visual detection (Giersch, T., J. Agric. Food Chem., 1993, 41, 1006), a quantitative format suitable as a field assay for the analysis of pesticide residues in water and liquid food samples on-site is described. For antibody immobilization, different membranes and immobilization techniques were investigated. The measuring range for atrazine was 0.3-10 micrograms l-1 using reflectance detection. The total assay time was about 25 min with dipsticks previously coated with antibody. Atrazine-spiked water and liquid food samples were selected for assay evaluation. The samples could be measured directly without the need for any prior enrichment or clean-up steps. A satisfactory agreement was found between the results of the dipstick IA and HPLC or GC measurements of both the original and spiked samples.
Atrazine↗
On-line monitoring of monoclonal antibody production with regenerable flow-injection immuno systems.
In this paper two systems for the observation of the production of mouse-IgG during the cultivation of hybridoma cells in a perfusion reactor are presented. The direct immunosystem is based on the detection of changes in capacitance of a dielectric layer (tantalum oxide) on a metal surface (tantalum) when antibodies bind to immobilized anti-antibodies. The sensor consisted of a 25 nm tantalum oxide layer, electrochemically grown onto a laser patternized 1 micron thick tantalum layer. The indirect system is based on an automated fluorimetric sandwich ELISA system with beta-galactosidase conjugated secondary antibodies. Two cultivations of mouse hybridoma cells in a 2-1 perfusion reactor were performed. The first cultivation was monitored with the capacitance system, the second cultivation was monitored with the fluorimetric system.
Animals↗
Comparison of different biosensor systems suitable for bioprocess monitoring.
To achieve effective bioprocess monitoring, sensing systems are required which are suitable for an on-line determination of substrates, inhibitors, nutrients or products. Such devices may utilise biochemical principles, i.e. the specific interaction of biochemical receptors with their surroundings. They can be constructed either as in situ sensors or as flow-through sensors connected to the process via sampling devices. Hence, characteristic features of an in situ glucose electrode are described, e.g. analytical range, sensitivity and stability. The sensor was based on mediated electron transfer from the enzyme glucose oxidase to the graphite electrode, the mediators being tetrathiafulvalene (TTF) or dimethylferrocene (DMF). Additionally, various flow injection analysis (FIA) systems based on oxidases, which were immobilised either on controlled pore glass or in a membrane, were characterised with respect to analytical ranges and sensitivities and applied to glucose, lactate and glutamate determinations in off-line samples taken from an animal cell cultivation.
Biosensing Techniques↗
Control of microbial activity by flow injection analysis during high cell density cultivation of Escherichia coli.
The application of an automated flow injection analysis (FIA) system for on-line determination of microbial activity, during high cell density cultivations of Escherichia coli is reported. Based on a bioelectrochemical principle, the FIA method used a redox mediator (potassium hexacyanoferrate(III)) to facilitate electron transfer from the microorganisms to an electrochemical detector. Assays were carried out using a new sampling device which provided aseptic operation by use of a valve and chemical sterilisation. No sample dilution or pretreatment was necessary for biomass concentrations up to approx. 40 g l-1. The sample volume was 0.5 ml and the overall analysis time was 5 min. FIA signals were found to correlate well with the oxygen uptake rate (OUR). Changes in metabolic activity due to low substrate levels or high inhibitor concentrations in the cultivation medium became obvious from the FIA signals.
Bacteriological Techniques↗