Covalent attachment of functionalized lipid bilayers to planar waveguides for measuring protein binding to biomimetic membranes

A new method is presented for measuring sensitively the interactions between ligands and their membrane-bound receptors in situ by using integrated optics, thus avoiding the need for addnl. labels. Phospholipid bilayers were attached covalently to waveguides by a novel protocol that can in principle be used with any glass-like surface. In a first step, phospholipids carrying head-group thiols were covalently immobilized onto SiO2-TiO2 waveguide surfaces. This was accomplished by acylation of aminated waveguides with the heterobifunctional crosslinker N-succinimidyl-3-maleimidopropionate, followed by the formation of thio ethers between the surface-grafted maleimides and the synthetic thiolipids. The surface-attached thiolipids served as hydrophobic templates and anchors for the deposition of a complete lipid bilayer either by fusion of lipid vesicles or by lipid self-assembly from mixed lipid/detergent micelles. The step-by-step lipid bilayer formation on the waveguide surface was monitored in situ by an integrated optics technique, allowing the simultaneous detn. of optical thickness and one of the two refractive indexes of the adsorbed org. layers. Surface coverages of 50-60% were calcd. for thiolipid layers. Subsequent deposition of POPC resulted in an overall lipid layer thickness of 45-50 .ANG., which corresponds to the thickness of a fluid bilayer membrane. Specific recognition reactions occurring at cell membrane surfaces were modeled by the incorporation of lipid-anchored receptor mols. into the supported bilayer membranes. (1) The outer POPC layer was doped with biotinylated phosphatidylethanolamine. Subsequent specific binding of streptavidin was optically monitored. (2) A lipopeptide was incorporated into the outer POPC monolayer. Membrane binding of monoclonal antibodies, which were directed against the peptide moiety of the lipopeptide, was optically detected. Specific antibody binding correlated well with the lipopeptide concn. in the outer monolayer. [on SciFinder (R)]