Biomedical subjects

Carsten Werner

Publications and source records attributed to Carsten Werner.

44 records · Page 3Linked to original sources

PubMed2003

Quantitative analysis of immobilized proteins and protein mixtures by amino acid analysis.

Biomolecular surface engineering of materials often requires precise, versatile and efficient quantification of immobilized proteins at solid surfaces. Acidic hydrolysis of surface-bound proteins and subsequent HPLC analysis of fluorescence-derivatized amino acids were adapted and critically evaluated for that purpose. Contaminations and concentration-dependent amino acid retrieval during HPLC were found to influence the accuracy of the method. In addition to the choice of adequate conditions for hydrolysis, derivatization and chromatographic separation extensions of the data evaluation were suggested to improve the accuracy of the approach when applied to single protein systems: comparing the experimentally obtained amino acid ratio to the protein constitution enabled to identify the properly separated and detected amino acids. Those amino acids were selected for a more precise calculation of the amount of immobilized protein. To further increase the accuracy of the method, the retrieval of amino acids corresponding to protein amounts in the range between 0.5 and 4.0 microg was analyzed for a variety of proteins of interest to derive protein-specific correction factors. The evaluation of amino acid data was furthermore applied to quantify binary protein mixtures at similar settings. This method was proven useful to detect the composition of protein mixtures throughout a wide range of absolute and relative concentrations.

Amino Acids↗

PubMed2003

Electrosurface phenomena at polymer films for biosensor applications.

Electrosurface phenomena at thin polymer films utilized in the preparation of biosensors have been studied by use of the Microslit Electrokinetic set-up. For the investigated polymer layers (aminodextran, carboxylated dextran, diaminopoly(ethylene glycol), dicarboxypoly(ethylene glycol), biotinylated poly(ethylene glycol), and streptavidin on biotinylated poly(ethylene glycol), the charge formation in aqueous electrolyte solutions was found to depend on the pH value, that is, OH- and H3O+ are the charge determining ions. The isoelectric points obtained from zeta potential versus pH plots could be utilized to draw conclusions on the introduction of acidic or basic groups and on the degree of molecular surface coverage, respectively. The hydrodynamically mobile charge reflected by the magnitude of the zeta potential contributed to only about 6% or less of the total surface conductivity of the polymer layers. The experimental determination of the total surface conductivity was found to provide valuable information on structural features of biosensor interfaces in aqueous environments.

Biosensing Techniques↗

PubMed2002

Low pressure plasma treatment of poly(3-hydroxybutyrate): toward tailored polymer surfaces for tissue engineering scaffolds.

Thin films of poly(3-hydroxybutyrate) were modified by microwave ammonia plasma treatment. The results of the modification were studied by means of contact angle goniometry, ellipsometry, Fourier transform infrared-attenuated total reflection spectroscopy, atomic force microscopy, and X-ray photoelectron spectroscopy. To prove the presence of amino groups on the poly(3-hydroxybutyrate) surface, chemical labeling with 4-trifluoromethyl benzaldehyde was performed before X-ray photoelectron spectroscopy analysis. Under the applied plasma conditions, a hydrophilic surface with a good long-term stability was obtained.

Electron Probe Microanalysis↗

PubMedDate not supplied

Maleic anhydride copolymers--a versatile platform for molecular biosurface engineering.

A platform of thin polymer coatings was introduced for the functional modulation of immobilized bioactive molecules at solid/liquid interfaces. The approach is based on covalently attached alternating maleic acid anhydride copolymers with a variety of comonomers and extended through conversion of the anhydride moieties by hydrolysis, reaction with functional amines, and other conversions of the anhydride moieties. We demonstrate that these options permit control of the physicochemical constraints for bioactive molecules immobilized at interfaces to influence important performance characteristics of biofunctionalized materials for medical devices and molecular diagnostics. Examples concern the impact of the substrate-anchorage of fibronectin on the formation of cell-matrix adhesions, the orientation of endothelial cells according to lateral anti-adhesive micropatterns using grafted poly(ethylene oxide), and the spacer-dependent activity of immobilized synthetic thrombin inhibitors.

Amines↗

PubMedDate not supplied

Thermo-responsive PNiPAAm-g-PEG films for controlled cell detachment.

A series of graft copolymers consisting of either poly(N-isopropylacrylamide) (PNiPAAm) or poly(N,N-diethylacrylamide) (PDEAAm) as a thermo-responsive component in the polymer backbone and poly(ethyleneglycol) (PEG) were immobilized as thin films and cross-linked on a fluoropolymer substrate using low-pressure argon plasma treatment. The surface-immobilized hydrogels exhibit a transition from partially collapsed to completely swollen, which is in the range of 32-35 degrees C and corresponds to the lower critical solution temperature of the soluble polymers. The hydrogels were used as cell carriers in culture experiments with L929 mouse fibroblast cells to probe for cell adhesion, proliferation, and temperature-dependent detachment of cell layers. The fibroblast cells adhere, spread, and proliferate on the hydrogel layers at 37 degrees C and become completely detached after reducing the temperature by 3 K. The cell release characteristics were further correlated to the swelling and collapsing behavior of the hydrogel films and the polymer solutions as measured in PBS solution and RPMI cell cultivation medium. It could be shown that, long before the swelling has completed upon temperature reduction, the cells detach. This can be attributed to the large content of PEG present in the hydrogel, which weaken the cell adhesion strength to the hydrogel layers.

Acrylic Resins↗

PubMedDate not supplied

Covalent immobilization of cellulose layers onto maleic anhydride copolymer thin films.

Thin films of cellulose are advantageous for analytical studies in aqueous environments to investigate various factors determining the performance of cellulose-based products. However, the weak fixation of cellulose layers on common carrier materials often limits this approach. To address this problem, we suggest a novel maleic anhydride copolymer precoating technique which allows for the covalent attachment of cellulose thin films through esterification. Maleic anhydride copolymers were deposited and covalently bound onto planar, aminosilane-modified glass or silicon oxide surfaces. Cellulose was subsequently immobilized on top of the copolymer precoatings by spin coating from N-methylmorpholine-N-oxide/dimethyl sulfoxide solutions. The resulting cellulose films were thoroughly characterized with respect to layer thickness, morphology, chemical constitution, and electrical charging. The stability of the layers against shear stress was demonstrated in aqueous solutions and the covalent attachment of the cellulose to the copolymer films was proven by means of dissolution experiments followed by ellipsometry and high-resolution X-ray photoelectron spectroscopy.

Cellulose↗

PubMedDate not supplied

In vitro reconstitution of fibrillar collagen type I assemblies at reactive polymer surfaces.

The reconstitution of fibrillar collagen and its assemblies with heparin and hyaluronic acid was studied in vitro. Fibril formation kinetics were analyzed by turbidity and depletion measurements in solutions containing varied concentrations of collagen and glycosaminoglycans. Fibril-forming collagen solutions were further applied for the coating of planar substrates which had been modified with alternating maleic anhydride copolymer films before. The immobilized collagen assemblies were characterized with respect to the deposited amount of protein using ellipsometry and acidic hydrolysis/HPLC-based amino acid analysis, respectively. AFM, SEM, and cLSM were utilized to gain information on structural features and patterns formed by surface-attached fibrils depending on the initial solution concentrations of collagen. The results revealed that the addition of heparin and hyaluronic acid affected both the fibril dimensions and the meshwork characteristics of the surface-bound fibrils.

Amino Acids↗

PubMedDate not supplied

Modulated fibronectin anchorage at polymer substrates controls angiogenesis.

A set of maleic anhydride copolymer thin films exhibiting well-defined differences in hydrophobicity and reactivity was compared with respect to the capability of supporting angiogenesis of human endothelial cells grown in contact. The physicochemical surface characteristics of the polymer substrates were found to modulate the anchorage of immobilized fibronectin. This was demonstrated to determine whether endothelial cells grow as a monolayer or form capillary networks. Enhanced reorganization of predeposited fibronectin into cell-matrix adhesions and slightly elevated levels of membrane-type matrix metalloproteinase 14 (MMP-14) occurred with weakly bound fibronectin layers where angiogenesis was most obvious. The key role of fibronectin-substrate binding for angiogenesis-under otherwise constant conditions-was further confirmed by the absence of variations in the expression of angiogenesis-related integrins (alpha(v)beta(3)) and in the secretion of the metalloproteinase MMP-2. Altogether, the results of this study point at the relevance of physicochemical surface characteristics of polymer materials for the stimulation of angiogenesis.

Cell Adhesion↗