Search PubMed⌕ Search

Biomedical subjects

B Liedberg

Publications and source records attributed to B Liedberg.

At least 19 recordsLinked to original sources
PubMed2001

Subtle differences in dissociation rates of interactions between destabilized human carbonic anhydrase II mutants and immobilized benzenesulfonamide inhibitors probed by a surface plasmon resonance biosensor.

The development of commercial biosensors based on surface plasmon resonance has made possible careful characterization of biomolecular interactions. Here, a set of destabilized human carbonic anhydrase II (HCA II) mutants was investigated with respect to their interaction kinetics with two different immobilized benzenesulfonamide inhibitors. Point mutations were located distantly from the active site, and the destabilization energies were up to 23 kJ/mol. The dissociation rate of wild-type HCA II, as determined from the binding to the inhibitor with higher affinity, was 0.019 s(-1). For the mutants, dissociation rates were faster (0.022-0.025 s(-1)), and a correlation between faster dissociation and a high degree of destabilization was observed. We interpreted these results in terms of increased dynamics of the tertiary structures of the mutants. This interpretation was supported by entropy determinations, showing that the entropy of the native structure significantly increased upon destabilization of the protein molecule. Our findings demonstrate the applicability of modern biosensor technology in the study of subtle details in molecular interaction mechanisms, such as the long-range effect of point mutations on interaction kinetics.

Biosensing Techniques↗
PubMed2001

Electrochemical evaluation of the interfacial capacitance upon phosphorylation of amino acid analogue molecular films.

An approach based on electrochemistry to differentiate between phosphorylated and nonphosphorylated amino acid analogues adsorbed on gold is presented. Analogues of serine, threonine, and tyrosine, containing thiohexadecyl headgroups, were synthesized and assembled on gold, and the surface capacitance was evaluated using electrochemical impedance spectroscopy. A procedure for deprotection of tert-butyl phosphate protecting groups, on the monolayer, is also described. Characterizations of the assembled analogues by cyclic voltammetry, infrared spectroscopy, and ellipsometry are used to confirm the insulating properties of the monolayers and the outcome of surface modifications. The results from cyclic voltammetry show good insulating properties for the monolayers even after phosphate deprotection. The infrared measurements reveal well-ordered monolayers, and the thickness from ellipsometry is in good agreement with expectations from molecular modeling. The impedance experiments show a capacitance increase up to 0.6 microF/cm2 as phosphate groups are introduced. The results in this study indicate the possibility of using a surface chemical and impedance spectroscopy approach to detect the kinase/phosphatase activity and kinetics involved in phosphorylation reactions.

Amino Acids↗
PubMed2001

Synthesis of a series of oligo(ethylene glycol)-terminated alkanethiol amides designed to address structure and stability of biosensing interfaces.

A strategy for the synthesis of a series of closely related oligo(ethylene glycol)-terminated alkanethiol amides (principally HS(CH(2))(m)CONH(CH(2)CH(2)O)(n)H; m = 2, 5, 11, 15, n = 1, 2, 4, 6, 8, 10, 12) and analogous esters has been developed. These compounds were made to study the structure and stability of self-assembled monolayers (SAMs) on gold in the prospect of designing new biosensing interfaces. For this purpose, monodisperse heterofunctional oligo(ethylene glycols) with up to 12 units were prepared. Selective monoacylation of the symmetrical tetra- and hexa(ethylene glycol) diols as their mesylates with the use of silver(I) oxide was performed. The synthetic approach was based on carbodiimide couplings of various oligo(ethylene glycol) derivatives to omega-(acetylthio) carboxylic acids via a terminal amino or hydroxyl function. SAM structures on gold were studied with respect to thickness, wettability (water contact angles approximately 30 degrees ), and conformation. A good fit was obtained for the relation between monolayer thickness (d) and the number of units in the oligo(ethylene glycol) chain (n): d = 2.8n + 21.8 (A). Interestingly, the corresponding infrared spectroscopy analysis showed a dramatic change in conformation of the oligomeric chains from all-trans (n = 4) to helical (n > or = 6) conformation. A crystalline helical structure was observed in the SAMs for n > 6.

Journal Article↗
PubMed2001

Protein adsorption to oligo(ethylene glycol) self-assembled monolayers: experiments with fibrinogen, heparinized plasma, and serum.

Low protein adsorption is believed advantageous for blood-contacting materials and ethylene glycols (EG)-based polymeric compounds are often attached to surfaces for this purpose. In the present study, the adsorption of fibrinogen, serum, and plasma were studied by ellipsometry on a series of well-defined oligo(EG) terminated alkane-thiols self-assembled on gold. The layers were prepared with compounds of the general structure HS-(CH2)15-CONH-EGn, where n = 2, 4, and 6. Methoxy-terminated tri(EG) undecanethiol and hydroxyl-terminated hexadecanethiol self-assembled monolayers (SAMs) were used as references. The results clearly demonstrate that the adsorption depends on the experimental conditions with small amounts of fibrinogen adsorbing from a single protein solution, but larger amounts of proteins from serum and plasma. The adsorption of fibrinogen and blood plasma decreased with an increasing number of EG repeats and was temperature-dependent. Significantly less serum adsorbed to methoxy tri(EG) than to hexa(EG) and more proteins remained on the latter surface after incubation in a sodium dodecyl sulfate (SDS) solution, indicating a looser protein binding to the methoxy-terminated surface. All surfaces adsorbed complement factor 3 (C3) from serum and plasma, although no surface-mediated complement activation was observed. The present study points to the importance of a careful choice of the protein model system before general statements regarding the protein repellant properties of potential surfaces can be made.

Adsorption↗
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↗
PubMed2000

Sensitivity deviation: instrumental linearity errors that influence concentration analyses and kinetic evaluation of biomolecular interactions.

Many scientific instruments utilise multiple element detectors, e.g. CCD's or photodiode arrays, to monitor the change in a position of an optical pattern. For example. instruments for affinity biosensing based on surface plasmon resonance (SPR) or resonant mirror are equipped with such detectors. An important and desired property of these bioanalytical instruments is that the calculation of the movement or change in shape follows the true change. This is often not the case and it may lead to linearity errors, and to sensitivity errors. The sensitivity is normally defined as the slope of the calibration curve. A new parameter is introduced to account for the linearity errors, the sensitivity deviation, defined as the deviation from the undistorted slope of the calibration curve. The linearity error and the sensitivity deviation are intimately related and the sensitivity deviation may lead to misinterpretation of kinetic data, mass transport limitations and concentration analyses. Because the linearity errors are small (e.g. 10 pg/mm2 of biomolecules on the sensor surface) with regard to the dynamic range (e.g. 30,000 pg/mm2), they can be difficult to discover. However, the linearity errors are often not negligible with regard to a typical response (e.g. 0-100 pg/mm2). and may therefore cause serious problems. A method for detecting linearity errors is outlined. Further on, this paper demonstrates how integral linearity errors of less than 1% can result in a sensitivity deviation of 10%, a value that in our opinion cannot be ignored in biospecific interaction analysis (BIA). It should also be stressed out that this phenomenon also occurs in other instruments using array detectors.

Kinetics↗
PubMed2000

Comparison of surface plasmon resonance and quartz crystal microbalance in the study of whole blood and plasma coagulation.

The coagulation of blood plasma and whole blood was studied with a surface plasmon resonance (SPR) based device and a quartz crystal microbalance instrument with energy dissipation detection (QCM-D). The SPR and QCM-D response signals were similar in shape but differing in time scales, reflecting differences in detection mechanisms. The QCM-D response time was longer than SPR, as a physical coupling of the sample to the substrate is required for molecules to be detected by the QCM-method. Change of sample properties within the evanescent field is sufficient for detection with SPR. Both the SPR signals and the QCM-D frequency and dissipation shifts showed dependency on concentrations of coagulation activator and sensitivity to heparin additions. The ratio of dissipation to frequency shifts, commonly considered to reflect viscoelastic properties of the sample, varied with the concentration of activator in blood plasma but not in whole blood. Additions of heparin to the thromboplastin activated whole blood sample, however, made the ratio variation reoccur. Implications of these observations for the understanding of the blood coagulation processes as well as the potential of the two methods in the clinic and in research are discussed.

Animals↗
PubMed2000

Suramin blocks nucleotide triphosphate binding to ribosomal protein L3 from Trypanoplasma borreli.

Ribosomal protein L3 (L3) has been demonstrated to participate in formation of the peptidyltransferase center and is essential for its catalytic activity. In the present study we show that L3 is able to bind nucleotide triphosphates with high and specific affinity in vitro. L3 was serendipitously identified by screening of a genomic phage library from a primitive kinetoplastid flagellate Trypanoplasma borreli with the ATPase domain of the topoisomerase II gene as a probe. The cloned gene was overexpressed and purified as a his-tag fusion protein in E. coli. Radioligand binding experiments, using [gamma-35S]ATP, showed that L3 is able to bind ATP but also GTP and UTP with similar high affinity (IC50 50-100 nM), while it has no ATPase activity. Furthermore, we showed that L3 has more than 500-fold higher affinity for nucleotide triphosphates compared to the corresponding nucleotide monophosphates and diphosphates. Molecular genetic and biochemical analyses allowed us to localize the NTP binding domain of L3 to the N-terminal 296 residues. Suramin, a polysulfonated naphthylamine derivative of urea, known for its chemotherapeutic effects completely inhibited the binding of [gamma-35S]ATP at subclinical levels. Results obtained with surface plasmon resonance technology showed that suramin both forms weak multimolecular complexes with L3 and binds strongly to L3 in nearly stoichiometric amounts.

Adenosine Triphosphatases↗
PubMed2000

Blood plasma coagulation studied by surface plasmon resonance.

A surface plasmon resonance (SPR) apparatus was used to investigate blood plasma coagulation in real time as a function of thromboplastin and heparin concentrations. The response curves were analyzed by curve fitting to a sigmoid curve equation, followed by extraction of the time constant. Clotting activation by thromboplastin resulted in increased time constant, as compared to spontaneously clotted plasma, in a dose dependent way. Addition of heparin to the thromboplastin-activated plasma counteracted this effect. Atomic force microscopy (AFM) pictures of sensor surfaces dried after completed clotting, revealed differences in fibrin network structures as a function of thromboplastin concentration, and the fiber thickness increased with decreased thromboplastin concentration. The physical reason for the SPR signal observed is ambiguous and is therefore discussed. However, the results summarized in the plots and the fibrin network properties observed by AFM correlate well with present common methods used to analyze blood coagulation.

Anticoagulants↗
PubMed1998

Immobilized chicken antibodies improve the detection of serum antigens with surface plasmon resonance (SPR).

Surface plasmon resonance (SPR) and other refractive index and mass sensitive methods are, due to complement activation by mouse monoclonal antibodies and with concomitant high background signal, only rarely used for the detection of antibody-antigen interactions in the blood serum milieu. In the present study chicken IgY and mouse IgG were immobilized to a sensor chip CM5 dextran matrix and compared for their background signal and detection of serum antigen. Ellipsometry with antibodies adsorbed to methylated silicon surfaces was used as a complementary detection method. As expected, fundamental differences in binding properties between the two kinds of antibodies were observed. Mouse antibodies bound large quantities of human serum. Human C1q was detected on mouse IgG and the complement system was activated, as seen from the rapid C3 and properdin depositions. Chicken antibodies bound low quantities of human serum and no human C1q. Moreover, C3 and properdin deposited only after prolonged serum incubations. Addition of EDTA to serum reduced the background signal modestly for both IgG and IgY. Serum samples with different concentrations of human C3 were injected over surfaces with immobilized chicken anti-C3, and the response was measured by SPR. Small concentration differences (< 1.25 micrograms/ml) in a physiologically relevant range (1-40 micrograms/ml after 100 times dilution) could then be detected reproducibly. The SPR signal was totally obscured when a mouse monoclonal anti-C3 antibody was used for the detection.

Animals↗
PubMed1998

Protein adsorption studies on model organic surfaces: an ellipsometric and infrared spectroscopic approach.

The development of accurate analytical tools to control the interfacial properties of solid substrates is of importance for the design of new biomaterials, as well as for the understanding of biomolecular interactions on surfaces. Considerable research efforts are presently devoted to this area on different levels of molecular complexity, i.e. both in the presence and in the absence of the biomolecules. In this contribution we review briefly applications of infrared reflection-absorption spectroscopy (IRAS) and ellipsometry as tools for analysis of the chemical properties of model surfaces, and their biological response in vitro when in contact with blood plasma or serum, respectively. The strength of the combination of the techniques is demonstrated by determination of protein adsorption patterns on a series of chemically well-defined so-called self-assembled alkanethiolate monolayers (SAMs) of 16-thiohexadecanol (HS-(CH2)16-OH) and n-hexadecanethiol (HS-(CH2)15-CH3) on gold. The protein adsorption patterns after incubations in plasma were determined by the specific binding of antibodies to the surfaces.

Adsorption↗
PubMed1996

Mapping of monoclonal antibody- and receptor-binding domains on human granulocyte-macrophage colony-stimulating factor (rhGM-CSF) using a surface plasmon resonance-based biosensor.

An automated surface plasmon resonance (SPR)-based biosensor system has been used for mapping antibody and receptor-binding regions on the recombinant human granulocyte-macrophage colony-stimulating factor (rhGM-CSF) molecule. A rabbit antimouse IgG1-Fc antibody (RAM.Fc) was coupled to an extended carboxymethylated-hydrogel matrix attached to a gold surface in order to capture an anti-rhGM-CSF monoclonal antibody (MAb) injected over the sensing layer. rhGM-CSF was subsequently injected and allowed to bind to this antibody. Multisite binding assays were then performed, by flowing sequentially other antibodies and peptides over the surface, and the capacity of the latter to interact with the entrapped rhGM-CSF in a multimolecular complex was monitored in real time with SPR. Eleven MAb (all IgG1K), were analyzed: respectively, four antipeptide MAb raised against three distinct epitopes of the cytokine (two clones against residues 14-24, that includes part of the first alpha-helix toward the N-terminal region; one clone against peptide 30-41, an intrahelical loop; and one clone against residues 79-91, including part of the third alpha-helix) and seven antiprotein MAbs raised against the entire rhGM-CSF, whose target native epitopes are still undetermined. In addition, the binding capacity to rhGM-CSF of a synthetic peptide, corresponding to residues 238-254 of the extracellular human GM-CSF receptor alpha-chain, endowed with rhGM-CSF binding activity, was tested. The results from experiments performed with the biosensor were compared with those obtained by a sandwich enzyme-linked immunosorbent assay (ELISA), using the same reagents. The features of the biosensor technology (fully automated, measure in real time, sharpened yes/no response, less background disturbances, no need for washing step or labeling of the reagent) offered several advantages in these studies of MAb immunoreactivity and epitope mapping, giving a much better resolution and enabling more distinct epitopes to be identified over ELISA.

Antibodies, Monoclonal↗
PubMed1995

Oral bolus kneading and shaping measured with chewing gum.

A masticatory test using two-colored chewing gum is presented and analyzed. Two separate parameters of chewing were studied on the same chewing gum bolus after 10 strokes of oral preparation, color mixing, and bolus shaping. The two parameters were evaluated in indices 1-5. A test series should preferably contain three chewing gums, and color mixing can be evaluated on six sides and shape-indexed for each bolus. The medians characterize the subjects' masticatory ability. Color mixing may relate, for example, to ensalivation of the food during chewing, and bolus shape may relate to preparation into a form suitable for swallowing. The tests were used on test subjects with different dental and denture status. Significant differences in both color mixing and shape indices were found between groups with different status. The method is discussed in relation to earlier types of tests, e.g., the comminution test mostly used hitherto.

Adult↗
PubMed1995

Mastication with and without removable partial dentures: an intraindividual study.

The chewing of 22 subjects, with a total of 27 removable partial dentures (RPD) that had been used for more than 6 months, was analyzed by four different methods. The study design was an intraindividual comparison of chewing, with and without the RPDs. Chewing tests were a color mixture of two-colored gums, chewing gum bolus shaping, comminution of Optosil silicone tablets, and number of chewing strokes to first swallow of an almond. The participants were also asked some standardized questions about their eating, biting, and chewing capacity. There was no significant difference in efficiency as measured by chewing gum color mixing, chewing gum shape, and pulverization of Optosil between chewing with and without the RPDs. Significantly more strokes were needed to swallow an almond without than with the RPD in place. With their RPDs in place, all subjects could chew a sandwich and an apple, and all but one could chew whole meat. The study shows that the chewing process should preferably not be analyzed by one method only.

Adult↗
PubMed1995

Biosensing with surface plasmon resonance--how it all started.

A subjective description is given of how the development of surface plasmon resonance for immunosensing began. The main differences between the initial experiments and a commercially available instrumentation are pointed out. For the practical use of surface oriented methods for biosensing it is noted that the arrangements around the optical system itself, such as the sensing chip or sample cell, are most important. It is concluded that the instrumentation developed can be used not only for immunosensing but also for "real time biospecific interaction analysis" in general. It is pointed out that the use of surface plasmon resonance for detection is only one possibility and that many new (optical) methods for real time biospecific interaction analysis have been and will be developed.

Biosensing Techniques↗
PubMed1994

In vitro plasma protein adsorption and kallikrein formation on 3-mercaptopropionic acid, L-cysteine and glutathione immobilized onto gold.

3-Mercaptopropionic acid (MPA), L-cysteine (L-cys), and glutathione (GSH) monolayers were immobilized onto gold and used in in vitro protein tests. The surfaces were characterized with ellipsometry, static contact angle measurements, atomic force microscopy, and Fourier transform infrared reflection absorption spectroscopy (FT-IRAS). After incubations in human plasma and antibody solutions, the surface antisera binding patterns were determined with ellipsometry. Using serum instead of plasma, complement activation was studied in the same fashion. Activated coagulation Factor XII and kallikrein formation on the surfaces and in the plasma were studied using a kallikrein-specific colorimetric assay. 3-Mercaptopropionic acid indicated contact activation of coagulation but L-cysteine did not. Glutathione displayed low deposition of plasma proteins, large deposition of proteins from serum, and did not promote kallikrein formation. None of the surfaces could be attributed complement activating properties, as determined by antibody deposition. The present study demonstrates that surface biology in complex model systems can be conveniently studied in vitro through systematic and well defined surface modifications.

3-Mercaptopropionic Acid↗