Fab' fragments of a monoclonal antibody against a dinitrophenol hapten (DNP hapten) were covalently bound to a phospholipid. A monomolecular film of a mixture of this Fab-lipid with a pure phospholipid (DMPC) was allowed to form at the air-water interface starting from a vesicle suspension. This film was separated from the vesicle phase and driven into a two-dimensional phase separation by lateral compression. After large domains, rich in Fab-lipids, had formed, the monolayer was transferred onto a high refractive index glass prism which was precoated with a thin gold layer. The domain formation as well as their transfer was controlled by microfluorimetry. The domain pattern was shown to be preserved after the transfer procedure. Imaging by surface plasmon microscopy (SPM) confirmed that the film had monomolecular thickness. The measured height step between protein-rich domains and the surrounding phospholipid matrix agreed well with the data known for the dimensions of crystallized Fab' fragments. DNP-labeled bovine serum albumin (DNP-BSA) was used as an antigen and was allowed to bind to the supported membranes. The increase in the thickness of the film upon antigen binding was measured and compared with the nonspecific binding of BSA. Subsequent incubation with an anti-DNP antibody lead to an additional increase in the domain thickness as did the further incubation with an antimouse antibody. By means of this work we showed that phase-segregated lipid-protein membranes can be designed in such a way that they form a highly specific two-dimensional recognition pattern on solid surfaces. SPM is an extremely sensitive technique which, in combination with a recognition pattern, may also be applicable for sensor devices.

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.

Communication: An amperometric biosensor for glucose, based on an immunological recognition process between an antigen binding fragment (Fab, see Figure), immobilized in a planar bilayer lipid membrane, and glucose oxidase. The enzyme‐catalyzed formation of H2O2 in the presense of glucose can be detected amperometrically by means of an electrode. The principle of this sensor is also applicable for sensing a wide range of other substrates.magnified image

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.

An atomic force microscope (AFM) operating with the tip in contact with the surface has given molecular-resolution images of monomolecular Langmuir-Blodgett (LB) films consisting of the lipids DMPE (DL-alpha-dimyristoylphosphatidylethanolamine), DMPG (L-alpha-dimyristoylphosphatidylglycerol), DODAB (di-octadecyldimethylammonium bromide), or 1:1 DODAB/DPPG (L-alpha-dipalmitoylphosphatidylglycerol). All the LB films were submerged in a buffer solution during imaging. Precoated mica served as a substrate for the LB films, which were transferred onto it by vertical dipping. The molecular arrangement of the lipids in the LB films agrees with the known 3D crystal data to within the accuracy of our measurements, about 20%, and shows a clear difference from the substrate mica, whose hexagonal lattice could be seen by increasing the force applied by the AFM if the bottom monomolecular film was not covalently interlinked. The AFM also gave images of single-stranded DNA bound to LB films, thus illustrating the potential of LB films as substrates for binding and imaging macromolecules.

ADVERTISEMENT RETURN TO ISSUEPREVArticleNEXTLipid and lipid-protein monolayers spread from a vesicle suspension: a microfluorescence film balance studyS. P. Heyn, M. Egger, and H. E. GaubCite this: J. Phys. Chem. 1990, 94, 12, 5073–5078Publication Date (Print):June 1, 1990Publication History Published online1 May 2002Published inissue 1 June 1990https://pubs.acs.org/doi/10.1021/j100375a058https://doi.org/10.1021/j100375a058research-articleACS PublicationsRequest reuse permissionsArticle Views157Altmetric-Citations43LEARN ABOUT THESE METRICSArticle Views are the COUNTER-compliant sum of full text article downloads since November 2008 (both PDF and HTML) across all institutions and individuals. These metrics are regularly updated to reflect usage leading up to the last few days.Citations are the number of other articles citing this article, calculated by Crossref and updated daily. Find more information about Crossref citation counts.The Altmetric Attention Score is a quantitative measure of the attention that a research article has received online. Clicking on the donut icon will load a page at altmetric.com with additional details about the score and the social media presence for the given article. Find more information on the Altmetric Attention Score and how the score is calculated. Share Add toView InAdd Full Text with ReferenceAdd Description ExportRISCitationCitation and abstractCitation and referencesMore Options Share onFacebookTwitterWechatLinked InRedditEmail Other access options Get e-Alerts

We have employed an AFM to determine the structural properties of supported planar membranes and membrane-bound proteins in an aqueous environment. Images of an asymmetric Langmuir Blodgett film of a charged phospholipid show long range positional as well as orientational order; individual headgroups are resolved. In order to study biofunctional membranes we have employed a recently introduced technique that allows the controlled formation of planar lipid-protein membranes on solid supports from a vesicle suspension. Combining this technique with the AFM permits the nondestructive imaging of these models of cell membranes at molecular resolution under physiological conditions of ionic strength and temperature.

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.

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.