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

Abstract

A new method is presented for measuring sensitively the interactions between ligands and their membrane-bound receptors in situ using integrated optics, thus avoiding the need for additional labels. Phospholipid bilayers were attached covalently to waveguides by a novel protocol, which can in principle be used with any glass-like surface.In a first step, phospholipids carrying head-group thiols were covalently immobilized onto Si02-Ti02 waveguide surfaces. This was accomplished by acylation of aminated waveguides with the heterobifunctional crosslinker N-succinimidyl-3-maleimidopropionate, followed by the formation of thioethers 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 determination of optical thickness and one of the two refractive indices of the adsorbed organic layers. Surface coverages of 50-60% were calculated for thiolipid layers. Subsequent deposition of POPC resulted in an overall lipid layer thickness of 45-50 A , which corresponds to the thickness of a fluid bilayer membrane.Specific recognition reactions occurring at cell membrane surfaces were modeled by the incorporation of lipidanchored receptor molecules 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 in the outer POPC monolayer. Membrane binding of monoclonal antibodies, which were directed against the peptide moiety of the lipopeptide, was optically detected. The specific antibody binding correlated well with the lipopeptide concentration in the outer monolayer.

The Local Phospholipid Environment Modulates the Activation of Blood Clotting

“…Stoichiometric calculations of fX molecules bound/Nanodisc can be estimated from surface plasmon resonance signals when the bound molecules have equivalent impact on the refractive index. Previous studies have shown that phospholipid bilayers in the fluid phase have similar refractive indices compared with protein solutions (34,35). Therefore, at saturation, the molar ratio of fX to Nanodiscs equals (RU fX /RU discs )*(MW discs / MW fX ).…”

Instrumental biosensors: new perspectives for the analysis of biomolecular interactions

“…Lipid vesicles have been immobilised onto the CM5 sensor chip surface using thiol coupling, (28,29) biotin/steptavidin interaction, (30) covalent immobilisation using a photoactive cross-linker (31) or capture attachment using immobilised monoclonal antibodies, (32) or lipid-binding proteins. (33) Hydrophobic interaction has been used to immobilise lipids onto CM5 chips derivatised with alkylamine (31) or gold surfaces coated with a hydrophobic self-assembling monolayer of octadecanethiol, (34) although this surface appeared to have limited stability.…”

Arrays of lipid bilayers and liposomes on patterned polyelectrolyte templates

“…These membranes represent one of the major structural components of cells and are responsible for many vital cellular functions. Biomimetic interfaces that can mimic cell membranes and their functionalities have potential applications as biosensors and can also provide a platform for fundamental investigations of biomolecular behavior [1][2][3][4][5]. To mimic cell membranes, supported BLMs (sBLMs) have been formed on glass, silica and unfunctionalized metal surfaces [4,[6][7][8][9][10][11][12][13][14][15][16].…”