| 1. |
- Samuelsson, Jörgen, 1971-
(författare)
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Development of Methods for Phase System Characterization in Liquid Chromatography
- 2008
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- The aim of this thesis is first and foremost to improve the fundamental knowledge of nonlinear and preparative separation theory by focusing on some of the remaining “white spots” on the theoretical chromatographic map. Secondly, the acquired knowledge is used to develop, validate and execute new methods for phase characterization in liquid chromatography. The methodology used in this thesis is a combination of experiments, fundamental nonlinear theory and systematic computer simulations. A fundamental knowledge of the molecular interactions between the compounds to be separated and the separation media requires the determination of adsorption isotherms over a broad concentration range to give a complete picture of all interactions in the separation system - weak as well as strong. In addition, such adsorption data is essential for optimization in preparative chromatography. For the first time, it has been experimentally shown that the injected molecules are not present in the detected peak when a small excess of molecules are injected into a chromatographic system equilibrated with a constant stream of identical molecules. Several experimental procedures for this method were developed such as (i) the optimal injection strategy and (ii) different labeling methods for visualizing the injected molecules. Remarkable phenomena in the single-component case, such as invisible peak deformation and deformed (invisible) frontal chromatograms, are reported, investigated, and explained. This phenomenon has asides from its future practical implementation, also a large didactic value. The accuracy of the ECP method is experimentally improved, and used to characterize the separation of protolytic compounds at different pH on modern commercially available silica and hybrid silica column packing materials. That investigation enables us to answer why basic compounds give a much more compact preparative peak profile at pH 11 than they yields at lower pH.
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| 2. |
- Sandberg, Tomas, 1973-
(författare)
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On the Development of Mucin-based Biomaterial Coatings
- 2008
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Owing to their key role in mucosal functioning as surface barriers with biospecific interaction potentials, the mucins are interesting candidates for use as surface modifiers in biomaterials applications. In this work, “mild” fractionation procedures were used to prepare mucins of bovine (BSM), porcine (PGM), and human (MG1) origin. Biophysicochemical analysis showed the prepared mucins to differ in size, charge, conformation, and composition. In turn, these factors were shown to govern mucin adsorption on hydrophilic and hydrophobic model surfaces. To enable for detailed coating analysis, methods for the qualitative and quantitative analysis of mucin-based coatings were developed. Of particular interest, a method for the determination of the fraction of surface-exposed, presumed bioactive proteins in a complex mucin coating was described. It was shown, using microscopy and activation assays, that mucin precoating effectively suppresses the neutrophil response towards a polymeric model biomaterial. Under optimal coating conditions, all mucins performed equally well, thus indicating them to be functionally similar. Coating analysis suggested that efficient mucin surface-shielding is critical for good mucin coating performance. Following a study on the complexation of albumin with preadsorbed mucin, we investigated the effect of mucin precoating on the conformation and neutrophil-activating properties of adsorbed host proteins. We found that mucin precoating greatly reduces the strong immune-response normally caused by adsorbed proinflammatory proteins (IgG and sIgA). Detailed coating analysis revealed that the fraction of surface-exposed protein in the mucin-protein composite influences the neutrophil response. Unexpectedly low neutrophil activation for composites containing near-monolayer concentrations of exposed IgG, suggested IgG to act synergistically with mucin on the surface. Conformational analysis supported this by showing that a preadsorbed mucin layer could stabilize adsorbed IgG through complexation. Our findings link well to the complex in vivo situation and suggest that functional mucosal mimics can be created in situ for improved biomaterials performance.
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| 3. |
- Fromell, Karin, 1972-
(författare)
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Nanoscale Reaction Systems
- 2007
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- The work presented in this thesis describes the use of polystyrene nanoparticles as model surfaces for bioanalytical work. Nanoparticles constitute convenient platforms for the attachment of bioactive agents, and receptor coated particles offer high local concentration of binding sites for specific ligands with minimal steric hindrance. However, it is not only the amount of bound protein that matters, the proteins must also be immobilized at the surface in such ways that they fully retain their activity, while at the same time protecting the surface from unspecific uptake of undesired components. The present work relates to the controlled immobilization of multiple types of active biomolecules onto nanoparticle surfaces to make them multifunctional. The surface expansion offered by the nanoparticles, in combination with the closeness between the reactants co-immobilized on the same particle, enables coupled reactions to be carried at a higher rate than otherwise possible. Thus, particle-decorated surfaces of this kind are highly suitable for miniaturized bioanalytical systems. Sensitive microarray systems are under development, including lectin-coated nanoparticles for glycoprotein mapping and a diagnostic device for Point-of-Care testing with a nanoparticle-based detection system.The full evaluation of protein attachment to nanoparticles requires precise analytical techniques for particle characterization, both in bare and coated form. The mass-sensitive SdFFF technique occupies a prominent position for particle characterization, as it offers both accurate determination of particle size and a quantification of adsorbed layers on small particles, whether of synthetic or biopolymeric nature. Here, this analytical technique is developed to precisely characterize nanoparticles that are sequentially coated with different layers, each rendering the particles a specific functionality. The thesis demonstrates how precise mass uptakes can be determined for each specific layer, and how control over the exact surface composition of the modified particles can be established for optimization of biological activity.
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| 4. |
- Ekeroth, Johan, 1974-
(författare)
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Phosphorylated Monomolecular Layers : Design, Synthesis, Characterization and Application
- 2002
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- This thesis describes the design, synthesis and characterization of organo-phosphatemonomolecular layers, so called self-assembled monolayers, using the strategy of linking organosulfur substances to a gold surface. Moreover there is also a paper included, which describes the development of synthetic pathways to various phospholipids, for which I have developed an interest in later projects.For the first study of the properties of phosphates, and also for concomitant application of developed systems, analogues of the amino acids serine, threonine and tyrosine, phosphorylated and non-phosphorylated, were synthesized. 16-Mercaptohexadecanamide based analogues were characterized as monolayers and large polar and charge effects were observed, introduced by the phosphate. Problems were, however, encountered as a post modification of phosphates was applied and a disordering of the monolayers observed. Moreover there were no expected effects observable upon interaction with Ca2+ and Mg2+.Based on the conclusions from the first study 3-mercaptopropionamide based analogues were used instead. Characterization of the analogues including counter ion exchange (H+,Na+, Ca2+ and Mg2+) revealed large electronic and possible structural differences depending on the counter ion.Again the charge and polar effects induced by the phosphate were investigated, and the study was also extended to interaction of the phosphate with Ca2+ and Mg2+. Strong effects were observed, which were also dependent on the ion concentration within a significant range. The polarity and charge properties of the phosphate layer were thus found to be variable.In the fourth study an additional Ca2+ and Mg2+ sensitive system was applied in combination with the phosphate surface. Phosphate surface/phospholipid vesicle interactions were studied under different conditions of Mg2+ (Ca2+) treatment. For comparison, the corresponding hydroxyl and sulphate surfaces were included and also the most well characterized surface concerning phospholipid vesicle interaction, SiO2. Most importantly, supported phospholipid bilayer (SPB) formation was found to be inducible on both the phosphate and sulphate surfaces, however at defined conditions of Mg2+ (Ca2+) treatment and concentration. The conclusion is that the processes of phospholipid vesicle adsorption and SPB formation are largely dependent on vesicle/surface interaction and vesicle stability.The last paper included in this thesis is a synthetic method development aimed at easy preparations of various phospholipid derivatives. In the future these methods could be used for synthesis of functionalized lipids and aid in the development of models mimicking biological systems such as artificial cell membranes.
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| 5. |
- Karlsson, Marjam, 1975-
(författare)
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Nano-porous Alumina, a Potential Bone Implant Coating
- 2004
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- This thesis describes a method of growing a highly adherent nano-porous alumina coating on titanium implant materials, a design which might be useful in hard tissue replacement. Alumina layers were formed by anodisation of aluminium, which had been deposited on titanium and titanium alloys by electron beam evaporation. Mechanical testing showed the coatings’ shear and tensile strength to be ~20MPa and ~10MPa respectively. Human osteoblasts were cultured on purchased membranes, produced in the same way with similar characteristics as the coating mentioned above. Cell viability, proliferation and phenotype were assessed by measuring redox reactions, DNA, tritiated thymidine incorporation and alkaline phosphatase production. Results showed normal osteoblastic growth patterns with increasing cell numbers the first two weeks after which cell growth decreased and alkaline phosphatase production increased, indicating that osteoblastic phenotype was retained on the alumina. Flattened cell morphology with filipodia attached to the pores of the material was seen. Implants frequently trigger inflammatory responses due to accumulation and activation of cells such as polymorphonuclear granulocytes (PMN), also called neutrophils. Activation and morphology of human PMN in response to nano-porous alumina with two pore sizes (20 and 200 nm) was investigated by luminol-amplified chemiluminescence, granule enzyme deposition measurement, optical and scanning electron microscopy. Activation was observed on both membrane types, however less pronounced on the 200 nm alumina. For both membranes a decrease in activation was seen after coating with fibrinogen, collagen I and serum (more pronounced for the two latter). On fibrinogen-coated alumina many flattened cells were observed, indicating frustrated phagocytosis. Finally when culturing osteoblasts on non-coated and collagen-coated membranes (after exposure to PMN) many more cells had established on the protein-coated surface after 24 h. The overall results indicate that it might be possible to produce a novel bone implant coating by anodisation of aluminium deposited on titanium and that this material will support osteoblast adhesion and proliferation. Furthermore neutrophil activation can be suppressed when coating the alumina with collagen I, which is beneficial considering the fact that this protein also is essential for bone formation.
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| 6. |
- Lindholm, Johan, 1974-
(författare)
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Development and Validation of HPLC Methods for Analytical and Preparative Purposes
- 2004
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- This thesis concerns the development and validation of high performance liquid chromatography (HPLC) methods aimed for two industrially important areas: (i) analysis of biotechnological synthesis and (ii) determination of adsorption isotherm parameters. There is today a lack of detailed recommendations for analytical procedures in the field of biotechnological production of drugs. Therefore, guidelines were given for analytical development and validation in this field; the production of 9α-hydroxyprogesterone was used as model. In addition, a rapid method using HPLC coupled with diode-array-detection (DAD) and mass spectrometry (MS), was developed for the preliminary identification and quantification of the product. In addition, requirements and recommendations were developed for the selection of the internal standard and for its inclusion in the process liquid. By using this approach the precision and accuracy of the quantitative method were considerably improved. Preparative chromatography is a powerful separation method for the purification of pure compounds from more or less complex sample mixtures. One such mixture can be the process liquid from a fermentation, another example can be a racemic mixture of compounds whose enantiomeric constituents must be isolated. Computer-assisted modeling can be used to optimize preparative chromatography. However, competitive adsorption isotherm parameters are required as input data for the computer simulations. In this thesis, a new injection technique, based on a firm theoretical basis, was developed for the peak perturbation (PP) method allowing the determination of binary competitive adsorption isotherm parameters from a broad concentration range. With the new method the determination of adsorption isotherm parameters from a quaternary mixture could be done for the first time. The profiles simulated with these parameters showed excellent agreement with the corresponding experimental profiles, validating the accuracy of the adsorption isotherm parameters derived by the new method.
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| 7. |
- Åsberg, Peter, 1973-
(författare)
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Hydrogels of conjugated polyelectrolytes for biosensor and biochip applications
- 2005
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- This thesis describes the use of conjugated polyelectrolytes (CPEs) in biosensor devices. The method is based on non-covalent assembly of the biomolecule of interest and the CPE functioning as the reporter, in one case as a transducer, of biomolecular events. Devices of these assemblies on solid supports that can operate in liquid solutions have been the focus. Polythiophenes, both semiconducting and conducting, is the class of materials that has been used in this work. The semiconducting polythiophenes have ionic side chains which makes them water soluble. This ionic side chain is capable of both forming electrostatic and hydrogen bonds, and when paired with the hydrophobic backbone of the polymer a great number of interactions with biomolecules are possible. The highly conducting polythiophene derivative PEDOT -PSS, (PEDOT) doped with ionic and water soluble PSS polyelectrolyte, was used as the conducting material in 3D-electrode. Both the semiconducting and conducting polymers described above forms hydrogels on solid supports if crosslinked with the appropriate ion, biomolecule or polymer. Evaluation of the CPEs, both with and without biomolecules, was performed in liquid, solid and hydrogel state using a number of techniques. This was done to understand how the CPEs behave when exposed to different buffer systems and various biomolecules.Hydrogels of conjugated polyelectrolytes combined with biomolecules are attractive as biosensors. The advantage with the hydrogel format is the high water content, the porous structure and the large capacity of binding molecules. High water content is important to preserve the biomolecules by providing the correct buffered environment. In this thesis we demonstrated a hydrogel of the highly conducting PEDOT -PSS polymer that was crosslinked on a solid support together with horseradish peroxidase (HRP) enzyme, forming an enzyme-enhanced electrode. Further studies of hydrogels were done using in situ quartz crystal microbalance with dissipation (QCM-D). POWT is a CPE withproperties well suited for biochip applications and readily forms hydrogels when exposed to water-based buffer solutions or biomolecule solutions. Detection ofcomplementary DNA and rejection of non-complementary DNA in a POWT hydrogel was demonstrated. The interaction between POWT and DNAoligonucleotides was also evaluated using fluorescence resonance energy transfer (FRET) in solution. Labeled DNA oligonucleotides with energy accepting or donating fluorophores allowed us to determine distance and binding stoichiometry in the non-covalent POWT-DNA complex.Patterning and anchoring of biomolecules and non-covalent assembled CPE-biomolecule complexes to a chip surface was studied; in the adsorbed state these complexes are hydrogels. Our novel method is based on the modification of the surface energy of a hydrophilic substrate surface using hydrophobic poly(dimethylsiloxane) (PDMS) elastomer stamp containing a relief pattern. Different conformations in biomolecules could be detected using fluorescence microscopy, where the CPEs acts as reporters and the PDMS modified substrates as discriminator. Also, excellent enzyme activity in patterned CPE/Horseradish peroxidase (HRP) enzyme was shown.Distances between the individual molecules in solid state devices of conjugated polymers can be small. In luminescence devices, such as light emitting diodes or fluorescence biosensors, there is a chance of interaction between conjugated molecules especially if more than one type of molecule is present. Quenching of the light and fluorescence energy transfer can occur and a simple approach to study this was developed.
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