Biomedical subjects

A Brecht

Publications and source records attributed to A Brecht.

17 recordsLinked to original sources

PubMed2000

Label-free biochemical detection coupled on-line to liquid chromatography

The on-line coupling of a label-free optical biosensor to a HPLC system is described by combining the separation power of HPLC with the specificity of the biosensor system. A highly cross-reactive antibody against the pesticide isoproturon was used as model for affinity proteins. The binding strength of the antibody to the utilized pesticides was characterized with the biosensor, first. In the on-line coupling setup, the eluate of the HPLC was mixed continuously with the antibodies. The presence of antigens was detected by a reduction of the antibody binding to the transducer. This reduced binding was quantified by a differentiation of the sensor signal by applying a Savitzky-Golay algorithm. Limits of detection were found to be in the femtomole range without preconcentration, which is comparable to a study using fluorescence-based biochemical detection.

Journal Article↗

PubMed2000

A streptavidin surface on planar glass substrates for the detection of biomolecular interaction.

Based on the requirements of biomolecular interaction analysis on direct optical transducers, a streptavidin surface is examined. A general protocol was developed allowing the immobilization of biotinylated compounds using the rife biotin-streptavidin system. This type of surface modification can be applied to all biosensors using glass surfaces as sensor devices. Reflectometric interference spectroscopy (RIfS), a label-free, direct optical method was used to demonstrate the quality of the transducer surfaces. The surface modification is based on an aminofunctionalized polyethylene glycol layer covalently bound to the silica surface of the transducer and shows very little nonspecific binding. Biotin molecules can be easily coupled on such layers. Streptavidin followed by a biotinylated estrone derivative was immobilized by incubation of the biotinylated transducer surface. For the streptavidin layer we obtained interference signals corresponding to a protein monolayer. Finally, using a surface prepared as described above, biomolecular interaction experiments with an antibody against estrone were carried out to show the quality of the transducer surface. With RIfS all of the affinity-based surface modifications can be detected online and time resolved.

Antibodies↗

PubMed2000

A high-density poly(ethylene glycol) polymer brush for immobilization on glass-type surfaces.

Label-free heterogeneous phase detection critically depends on the properties of the interfacial layer. We have obtained high-density monomolecular poly(ethylene glycol) (PEG) layers by solvent-free coupling of homo-bifunctional PEGs (2,000 g/mol) at 75 degrees C to silica surfaces silanized with glycidyloxipropyltrimethoxysilane (GOPTS). Characterization by ellipsometry and contact angles revealed that PEG layers up to 3.4 ng/mm2 with low roughness and flexibility were obtained. Specific and non-specific binding at these PEG surfaces was monitored by reflectometric interference spectroscopy (RIfS). No significant non-specific adsorption upon incubation of 1 mg/ml ovalbumin was detectable (< 10 pg/mm2), and 150 pg/mm2 upon incubation of 10% calf serum, less than 10% of the amount adsorbed to the solely silanized surfaces. The terminal functional groups of the PEG layers were utilized to couple ligands and a protein. Specific protein interaction with these immobilized compounds was detected with saturation loadings in the range of protein monolayers (2-4 ng/mm2). The excellent functional properties, the high stability of the layers, the generic and practical coupling procedure and the versatility for immobilizing compounds of very different functionality make these PEG layers very attractive for application in label-free detection with silica or metal-oxide based transducers.

Animals↗

PubMed1999

Interaction of chemically modified antisense oligonucleotides with sense DNA: a label-free interaction study with reflectometric interference spectroscopy.

Antisense oligonucleotides (ON) are regarded as potential therapeutic agents for controlling gene expression at the mRNA level. The strength of the interaction with the target sequence is one critical factor for the therapeutic efficiency of an ON. Herein, the results of studies on antisense 15mer and 20mer ONs against mdr1b-mRNA are described. The mdr1b is a member of the group that encodes the P-glycoprotein (Pgp), responsible for the phenomenon of multidrug resistance. The effects of backbone modification (DNA, phosphorothioate (PTO)), terminal modifications (hexadecyl, cholesteryl, tocopherol, polyethylenglycol, 2'-O-methyl-modified RNA) and base sequence misalignments (1 to 3 bases) on interaction kinetics and binding strength were investigated. The interaction of an immobilized sense strand with the dissolved antisense ON was monitored with a label-free optical transducer based on thin film interference (RIfS). Association kinetics were detected at a low density of immobilized ON. Thermodynamics were investigated by homogeneous phase titration of sense and antisense ON and subsequent quantification of equilibrium concentrations of unbound ON at a transducer highly loaded with sense ON. Association rate constants varied from 3.1 (+/- 0.2) x 10(4) M-1 s-1 (poly(ethylene glycol)-modified DNA strand) to 4.3 (+/- 0.1) x 10(4) M-1 s-1 (hexadecyl-modified strand). Binding constants varied from 1.9 (+/- 0.1) x 10(8) M-1 (cholesteryl modification) to 5 (+/- 0.4) x 10(7) M-1 (tocopherol modification). Phosphorothioate ON showed a reduction in binding strength of more than 1 order of magnitude. The data presented give valuable information for the efficiency of modified antisense oligonucleotides.

ATP Binding Cassette Transporter, Subfamily B, Mem↗

PubMed1999

Integrated optical surface plasmon resonance immunoprobe for simazine detection.

This paper presents the detailed design and characterisation of a regenerable integrated optical surface plasmon resonance immunoprobe as a detector for the triazine herbicide simazine. A sensor design theoretically optimised for use in the aqueous environment is presented and its fabrication described. Experimental results on the sensitivity to changes in bulk refractive index of the analyte and on non-specific binding of ovalbumin are presented. Binding inhibition immunoassays were conducted for simazine and the lower limit of detection determined to be 0.16 microgram/l using anti-simazine IgG antibodies and 0.11 microgram/l using anti-simazine Fab fragments. A sample test cycle of 20 min was established.

Biosensing Techniques↗

PubMed1997

Studies on the Biotin-Avidin Multilayer Adsorption by Spectroscopic Ellipsometry.

Protein multilayers were prepared on silica surfaces by up to 20 alternating incubations of a biotin-protein conjugate and polymerized streptavidin. Spectroscopic ellipsometry (350-750 nm) was used to investigate the physical thickness and the dispersion of the protein layer after each incubation step. Both parameters could be determined independently for films formed by 5 to 15 incubations. A single homogeneous protein layer was assumed for evaluation. This determination of layer parameters was limited by high correlation coefficients for less than 5 incubation steps. Deviations from the homogeneous single layer model were found for more than 15 incubation steps. The growth of the layer was reproducible, with a thickness increase of about 18.75 nm per incubation. An almost constant refractive index nD of 1.384 +/- 0.002 was found for the multilayer system. A protein mass deposition of 4.74 ng/mm2 was calculated per incubation step. The protein concentration of the layer was estimated of about 0.27 g/ml. The affinity system investigated is quite simple and may well serve as a prototype system in the characterization of optical and other transducers for affinity reactions. Copyright 1997 Academic Press.

Journal Article↗

PubMed1997

Label-free monitoring of DNA-ligand interactions.

We report on the label- and isotope-free monitoring of DNA interactions with low-molecular-weight ligands. An optical technique based on interference at thin layers was used to monitor in real time binding of ligands at DNA which was immobilized by Coulomb interactions at a positively charged surface. Approximately 2 ng DNA/m2 was irreversibly bound to the surface, which remained stable over several days. This result was confirmed by characterization of the layer using spectroscopic ellipsometry. During incubation of immobilized DNA with a variety of intercalators and other DNA-binding compounds in a flow system, interactions were monitored by reflectometric interference spectroscopy. Binding effects between 10 and 400 pg/ mm2 were detected unambiguously. Nonspecific binding effects were excluded by using a negatively charged reference surface. Variation of intercalator concentration allowed the characterization of interaction with respect to kinetics and thermodynamics by the evaluation of binding rate and equilibrium coverage. The affinity constants were determined in the range between 10(5) and 10(6) M-1, in good agreement to those obtained by homogeneous phase assays. Association rate constants between 10(3) and 10(5) M-1 s-1 and dissociation rate constants between 10(-1) and 10(-2) s-1 were determined by evaluation of the binding curves. Both the fast and simple test format and a universal applicability make the new technique described attractive for detecting and characterizing interaction of low-molecular-weight molecules with DNA.

DNA↗

PubMed1997

Assessment of affinity constants by rapid solid phase detection of equilibrium binding in a flow system.

We present a method for the determination of affinity constants based on equilibrium binding between an analyte and an antibody in liquid phase by a heterogeneous phase detection scheme. Equilibrium concentration of free antibody binding sites was probed kinetically by direct optical detection of specific binding to an immobilised analyte derivative. The additional binding signal due to dissociation of the analyte-antibody complex during detection was minimised by the use of fast flow-through conditions. The concentration of free antibody binding sites was titrated by adding increasing analyte concentrations. The affinity constant was derived from the titration curve by a non-linear least square fit of a model function. The affinity of monoclonal triazine antibodies to several s-triazine pesticides and a relevant metabolite was investigated. Kinetic determination of equilibrium concentration of free binding sites was carried out by reflectometric interference spectroscopy (RIfS) using flow injection analysis. The capabilities of the model were investigated using different analyte-antibody pairs and various antibody concentrations. Both bivalent IgG and monovalent Fab fragments were used to compare different binding models. The applied model corresponds well to the titration curves for affinity constants of 10(7) M(-1) and higher. For lower affinity constants significant deviations due to dissociation of the analyte-antibody complex during detection were observed.

Animals↗

PubMed1997

Quartz crystal microbalances for quantitative biosensing and characterizing protein multilayers.

The use of quartz crystal microbalances (QCMs) for quantitative biosensing and characterization of protein multilayers is demonstrated in three case studies. Monolayers of QCM-based affinity biosensors were investigated first. Layers of a thiol-containing synthetic peptide constituting an epitope of the foot-and-mouse-disease virus were formed on gold electrodes via self-assembly. The binding of specific antibodies to epitope-modified gold electrodes was detected for different concentrations of antibody solutions. Oligolayers were studied in a second set of experiments. Dextran hydrogels were modified by thrombin inhibitors. The QCM response was used in a competitive binding assay to identify inhibitors for thrombin at different concentrations. Multilayers of proteins formed by self-assembly of a biotin-conjugate and streptavidin were investigated next. The QCM frequency response was monitored as a function of layer thickness up to 20 protein layers. A linear frequency decay was observed with increasing thickness. The decay per layer remained constant, thus indicating perfect mass coupling to the substrate. Frequency changes a factor of four higher were obtained in buffer solution as compared to measurements in dry air. This indicates a significant incorporation of water (75% weight) in the protein layers. This water behaves like a solid concerning the shear mode coupling to the substrate. The outlook discusses briefly the need for controlled molecular engineering of overlayers for subsequent QCM analysis, and the importance of an additional multiparameter analysis with other transducer principles and with additional techniques of interface analysis to characterize the mechanical coupling of overlayers as biosensor coatings. A promising trend concerns the use of QCM-arrays for screening experiments.

Biosensing Techniques↗

PubMed1997

Specific binding of low molecular weight ligands with direct optical detection.

The characterization of low molecular weight ligand interaction with receptor molecules is of importance for the investigation of biological processes and for drug research. We report on the investigation of the binding of low molecular weight ligands to immobilized receptors by label-free detection. Reflectometric interference spectroscopy, an optical transducer which allows the monitoring of a few picograms per square millimetre changes in surface coverage, was used to study two model systems. In both cases detection of the binding event was successful. High affinity binding of biotin to immobilized streptavidin was clearly detectable at receptor surface concentrations as low as 1-2 x 10(10) binding sites/mm2. Linear correlation between the receptor surface concentration and the response to biotin binding was observed. Using immobilized DNA, we investigated the binding of common intercalators with respect to kinetics and thermodynamics by evaluation of the association and the dissociation part of the binding curve. Bi-exponential increase and decrease of intercalator loading was observed, indicating complex interaction kinetics. The four structurally different intercalators showed significant distinction in binding kinetics and equilibrium signals. Improvement of experimental parameters is required to obtain more reliable kinetic data.

Adsorption↗

PubMed1996

Surface modification for direct immunoprobes.

The modification of glass-type surfaces by several hydrophilic polymers of different molecular masses and functional properties [chitosan, dextran, poly(oxyethylene), poly(ethyleneimine) and poply(acrylamide)] with respect to the application for direct immunoprobes was investigated. Activation of the surface was carried out by silanisation and the polymers were coupled to the surface via amide bonds. The carboxyl derivative of a hapten was attached to the functional groups of the polymers by carbodiimide-activated coupling. As a reference system, the ligand was directly coupled to the silanised surface. Non-specific protein adsorption, specific binding of antibodies and regeneration were monitored by evaluation of reflectance spectra obtained by white light interference at a thin silica layer (RifS). All polymer modified layers showed improved properties compared to those with direct attachment of the hapten. The non-specific adsorption was reduced to 5-50%. Binding of a specific antibody was significantly increased by the polymer modification: Mass transport limited binding of the specific antibody in low concentrations (30 nM) up to a surface coverage value of 2 ng/mm2 and a maximum surface coverage in the range of a monolayer of IgG (5-6 ng/mm2) was observed for most of the polymers. The surface coverage found for IgG bound specifically to the dextran-modified surface exceeded a protein monolayer.

Adsorption↗

PubMed1995

Optical probes and transducers.

Biosensors are by definition a combination of a biological receptor compound and a physical or physicochemical transducer. Therefore, the transducing structure is a critical part of every biosensor. In the development of new and improved biosensing layers the importance of the transducing structure is not restricted to the substrate to which biological structures have to be coupled. A field of even greater importance is the use of transducers as probes providing information on the structure and function of biosensing layers, and their relation to a transducer surface. The aim of this paper is to give an overview on optical transducer principles and optical (surface) analytical techniques relevant as part of biosensing structures as well as probes in the development and optimisation of biosensing layers. Categories discussed are basic optical effects, materials involved, surface chemistry, the principal and technological limits of spatial resolution, and sensitivity. The intimate relation between the spatial resolution of a probe, the resulting size of interaction areas, and the feasibility of array structures is pointed out. Two interferometric methods are presented in principle, and their application to biosensing and some results are discussed in detail. The necessity to characterise receptor layers to get detailed information about the interaction process is pointed out. The close relationship between optimal characterisation of layers by selection of adequate probe technologies and improvement of probe performance, and the development of new biosensing layers is discussed. Finally, an outlook is given for future aspects of improved spatial resolution and multianalyte detection.

Biosensing Techniques↗

PubMed1994

Characterization of biomembranes by spectral ellipsometry, surface plasmon resonance and interferometry with regard to biosensor application.

Phospholipid bilayers with transport proteins and antigen/antibody interfaces are considered to be suitable biosensor systems. The quality of such membranes or interfaces depends on the properties of the layers. Optical methods have proved to be an appropriate tool for characterizing those layers in situ and in a non-destructive manner. Two systems with potential for biosensor applications are characterized by some of these methods: phospholipid bilayer membranes spread from vesicle solution and protein-antigens both adsorbed on planar solid support. The results of spectral ellipsometry, surface plasmon resonance (SPR) and spectral interferometry are compared with respect to quality of characterization, expenditure of sample preparation and measurement, and time resolution. The phospholipid membranes adsorbed show a relatively low refractive index and a relatively high thickness. Bruggeman effective medium approximation is used to calculate the effective layer thickness. This result is compared to SPR measurements. A correlation between thickness and vesicle concentration may be detected. Further, the test protocol of an immunoassay is examined by spectral interferometry and SPR. Thicknesses determined are compared to results obtained by applying spectral ellipsometry. The data measured by ellipsometry are in agreement with the molecular dimensions of the immunoglobulins. Differences between details can be explained by physical considerations.

Biosensing Techniques↗

PubMed1993

beta'-COP, a novel subunit of coatomer.

Several lines of evidence favour the hypothesis that intracellular biosynthetic protein transport in eukaryotes is mediated by non-clathrin-coated vesicles (for a review see Rothman and Orci, 1992). The vesicles have been isolated and a set of their surface proteins has been characterized as coat proteins (COPs). These COPs exist in the cytosol as a preformed complex, the coatomer, which was prior to this study known to contain six subunits: four (alpha-, beta-, gamma- and delta-COP) with molecular weights between 160 and 58 kDa, and two additional proteins of approximately 36 and 20 kDa, epsilon- and xi-COP. Here we describe a novel subunit of the coatomer complex, beta'-COP. This subunit occurs in amounts stoichiometric to the established COPs both in the coatomer and in nonclathrin-coated vesicles and shows homology to the beta-subunits of trimeric G proteins.

Amino Acid Sequence↗

PubMed1991

A coat subunit of Golgi-derived non-clathrin-coated vesicles with homology to the clathrin-coated vesicle coat protein beta-adaptin.

Four high-molecular-weight proteins form the main subunits of the coat of Golgi-derived (non-clathrin) coated vesicles. One of these coat proteins, beta-COP, is identical to a Golgi-associated protein of relative mass 110,000 (110K) that shares homology with the adaptin proteins of clathrin-coated vesicles. This connection, and the comparable molecular weights of the coat proteins of Golgi-derived and clathrin-coated vesicles, indicates that they may be structurally related. The identification of beta-COP as the 110K protein explains the blocking of secretion by the drug brefeldin A.

Adaptor Protein Complex beta Subunits↗

PubMed1987

Purification and characterization of rat liver glutaminase.

Phosphate-dependent glutaminase (EC 3.5.1.2) from livers of starved rats was purified about 400-fold to near homogeneity. The specific activity of the final pool was more than 30 U/mg protein. For the rapid quantification of the enzyme activity a simple and sensitive assay, based on the determination of the produced ammonia with an o-phthalaldehyde reagent, was developed which avoids massive dilution of the samples. The enzyme preparation involved extraction of the enzyme from sonified isolated mitochondria after treatment with a brief hypotonic shock followed by ammonium sulphate precipitation, ion-exchange and hydroxyapatite chromatography. A major improvement was the stabilization of the enzyme by chymostatin protecting it from degradation by a protease of presumably lysosomal origin. In the presence of chymostatin or leupeptin the half-life of glutaminase in a crude mitochondrial preparation subsequent to mild treatment with digitonin could be increased to more than 200 h. The relative molecular mass of the protein (Mr 170,500) was estimated by sucrose gradient ultracentrifugation. The molecular mass of the subunits (Mr 57,000) was determined by sodium dodecyl sulphate/polyacrylamide gel electrophoresis. These results suggest a protein composed of three subunits of identical molecular mass. The molecular data clearly differentiate liver glutaminase from the phosphate-dependent glutaminase present in kidney.

Animals↗

PubMedDate not supplied

New donor-acceptor pair for fluorescent immunoassays by energy transfer.

A novel Förster donor-acceptor dye pair for an immunoassay based on resonance energy transfer (RET) is characterized with respect to its photophysical properties. As donor and acceptor, we chose the long-wavelength excitable cyanine dyes Cy5 and Cy5.5, respectively. Due to the perfect spectral overlap, an exceptionally high R(0) value of 68.7 A is obtained in solution. For biochemical applications, antibodies (IgG) are labeled with Cy5, while a tracer for competitive binding is synthesized by labeling bovine serum albumin (BSA) with an analyte derivative and Cy5.5. Binding the dyes to proteins at a low dye/protein ratio increases the fluorescence lifetimes and quantum yields, leading to an enhanced R(0) value of 85.2 A. At higher dye/protein ratios, the formation of nonfluorescent dimeric species causes a decrease in the fluorescence lifetime and quantum yield due to RET from monomeric dyes to dimers within one protein molecule. The Förster distances could be calculated using the dimer absorption spectra to 83.9 and 83.6 A for Cy5 and Cy5.5, respectively. Upon binding of the Cy5-labeled IgG to the tracer, efficient quenching of Cy5 fluorescence is observed. Steady-state and time-resolved measurements reveal that approximately 50% of the quenching results in Förster-type RET, while the residual quenching effect is caused by static quenching processes. The applicability of this dye pair is demonstrated in a homogeneous competitive immunoassay for the pesticide simazine.

Binding, Competitive↗