[Microsystem technique for medical applications: overview of combined projects and prospects].
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Forecasting↗
Biomedical subjects
S P Heyn
Publications and source records attributed to S P Heyn.
Synthetic lipid-anchored receptors based on the binding site of a monoclonal antibody.
Highly specific ligand receptor interactions generally characterize molecular recognition at cell surfaces and other biological systems. In this study we simulate a membrane receptor by fusing a monoclonal antibody fragment to a phospholipid. A sulfhydryl group in the hinge region of a monoclonal antibody fragment, was covalently linked to derivatives of phosphatidylethanolamines and phosphatidylserine via three different hydrophilic spacer arms. We investigated and characterized these lipid-anchored Fab-fragments which we have named 'Fab-lipids' in liposomal and monolayer systems. Methods for the monomolecular assembling of such films at the air/water interface and techniques used for their manipulation are outlined. We describe two possibilities for building a monomolecular receptor layer, consisting of two-dimensional pattern of oriented Fab-fragments with their artificial hydrophobic anchor embedded in a lipid matrix. In the first method a monomolecular film at the air/water interface was allowed to form from a vesicular suspension and driven into a phase separation, resulting in protein rich domains embedded in a protein depleted phase. This film was transferred onto a solid support in such a way that the established pattern was preserved. Alternatively, a recognition pattern was formed by directly cross-linking the Fab-fragments to preformed planar membranes composed of the reactive spacer-lipids and an inert matrix lipid. Specificity as well as contrast of the binding activity of the receptor layers were qualified using micro-fluorimetry.
2,4-Dinitrophenol↗
Immobilized proteins in buffer imaged at molecular resolution by atomic force microscopy.
Samples of supported planar lipid-protein membranes and actin filaments on mica were imaged by atomic force microscopy (AFM). The samples were fully submerged in buffer at room temperature during imaging. Individual proteins bound to the reconstituted membrane were distinguishable; some structural details could be resolved. Also, surface-induced, self-assembling of actin filaments on mica could be observed. Monomeric subunits were imaged on individual actin filaments. The filaments could be manipulated on or removed from the surface by the tip of the AFM. The process of the decoupling of the filamentous network from the surface upon changing the ionic conditions was imaged in real time.
Actins↗
Two-dimensional recognition pattern of lipid-anchored Fab' fragments.
A two-dimensional pattern of oriented antibody fragments was formed at the air-water interface and transferred onto a solid support. The Fab'-fragments of a monoclonal antibody against the hapten dinitrophenyl (DNP) were covalently linked via a hydrophilic spacer to phospholipid vesicles. A monomolecular lipid-protein layer at equilibrium with these vesicles was allowed to form at the air-water interface. The monolayer was separated from the vesicle phase and transferred to a Langmuir-Blodgett trough. By cooling and compressing, the previously homogeneous lipid-protein film was driven into a two-dimensional phase separation resulting in protein-rich domains and a second phase consisting mainly of lipid. This film was transferred onto a solid support in a way that preserved the protein-lipid pattern. The specificity as well as the contrast in the binding activity of the two different separated phases were then quantified using microfluorometry. DNP conjugated to fluorescein-labeled bovine serum albumin (BSA) showed virtually no binding to the lipid regions, but gave a ratio of bound DNP-BSA to Fab'-lipid of greater than 50% in the protein-rich domains proving that the Fab'-moiety retained its biological activity. This demonstrates that the technique presented here is well suited to modify different solid surfaces with a pattern of a given biological function. The optional control of lateral packing and orientation of the components in the monolayer makes it a general tool for the reconstitution of supported lipid-protein membranes and might also open new ways for the two-dimensional crystallization of proteins at membranes.
Antibodies, Monoclonal↗
A miniaturized micro-fluorescence film balance for protein-containing lipid monolayers spread from a vesicle suspension.
In order to study protein-lipid monolayers at the air/water interface a miniaturized micro-fluorescence film-balance apparatus has been developed and combined with a modified technique of spreading and separating a monolayer from a vesicle suspension. The spreading method provides non-denaturing conditions for protein-lipids. When applied to protein-lipid vesicles, monolayers with incorporated proteins are obtained, and their thermodynamic parameters may be controlled in a well-defined way by film balance techniques. In the apparatus introduced, a movable microscope allows the observation of micro-fluorescence during the tracking of individual domains at the air/water interface of a fixed Langmuir trough. After the control of parameters such as subphase temperature, surface pressure and lateral molecule distribution, a monolayer may be transferred and immobilized on a planar solid support, making it accessible to optical surface-sensitive measuring methods as well as to electron microscopy and scanning probe techniques.
Lipids↗