Supported
lipid membranes by Ralf P. Richter, Josephine Lai Kee Him, and Alain Brisson
The current interest in reconstituting biological membranes on solid supports aims at linking the biological world, with its elaborate molecular architectures, properties, and functions, to the field of surface science, with its advanced technologies and sophisticated surface-sensitive analytical methods. Success in this enterprise would not only improve our understanding and description of basic cellular functions but also help developing biotechnological tools, biomedical devices, or biofunctional materials. To achieve this goal, reliable methods need to be developed for controlling the formation of solidsupported lipid membranes and the deposition, incorporation, and addressing of biological entities, from molecules to cells.
Laboratory of Molecular Imaging and NanoBioTechnology, IECB, CNRS-UMR 5471, University of Bordeaux 1, 16 Avenue Pey Berland, 33607 Pessac Cedex, France E-mail:
[email protected]
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November 2003
This review focuses on a versatile approach that combines the formation of solid-supported lipid bilayers (SLBs) by deposition of lipid vesicles with the adsorption of proteins and formation of ordered protein layers via specific interaction with ligands incorporated in the SLBs. This approach provides access to basic aspects of membrane biophysics, membrane-protein interactions, and molecular ordering in two dimensions. It may also constitute a strategy for the design of biofunctional surfaces at the nanoscale. Biological membranes play key roles in cell life, acting as permeability barriers and privileged sites of communication between the inside and outside of cellular worlds. These highly complex and dynamic assemblies, only a few nanometers thick, consist of two main components: a twodimensional space made of lipid molecules held together by hydrophobic interactions and self-assembled as a continuous bilayer; and proteins embedded within the membrane or attached to it. Our current knowledge of the molecular processes occurring at biological membranes is mainly based on studies performed on models of biological membranes, including liposomes and giant vesicles in solution, lipid monolayers at the air water-interface, black lipid films, membrane patches at pipettes, or solid-supported membranes (Fig. 1). Following the pioneering work of McConnell and collaborators1, (models of) biological membranes deposited on solid supports have become very popular, both for studying basic membrane processes and possible biotechnological
ISSN:1369 7021 © Elsevier Ltd 2003
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applications. The growing interest in the development of biofunctionalized surfaces has been nourished by the emergence of a multitude of surface-sensitive methods based on a variety of optical and mechanical sensing principles, e.g. fluorescence microscopy, Brewster angle microscopy, atomic force microscopy (AFM), surface plasmon resonance (SPR), ellipsometry, and quartz crystal microbalance, which enable the physico-chemical and/or structural characterization of thin molecular layers in aqueous media.
Fig. 1 Models of biological membranes: (a) liposomes: hollow spheres (25 nm to 100 µm in diameter) enveloped by a bilayer of lipid molecules; (b) lipid monolayers at the airwater interface; (c) black lipid membranes suspended over an aperture between two aqueous phases; (d) Langmuir-Blodgett method, which allows the transfer of lipid monoand multi-layers from the air-water interface to a solid support; (e) self-assembled monolayers (SAMs, e.g. thiols on Au or silanes on glass or silica), a second lipid layer can be deposited by spontaneous disruption of liposomes; (f) deposition of a polymer coating with tethers followed by the spontaneous spreading of liposomes, so that the polymer creates a cushion between support and bilayer; (g) spontaneous spreading of liposomes or membranes on mica, glass, and silica.
This review focuses on the formation of SLBs by spontaneous deposition of liposomes, on the adsorption of proteins, and the formation of two-dimensional ordered arrays of proteins.
Forming supported lipid membranes SLBs formed by the spontaneous deposition of liposomes in solution2-4 have already found widespread use as mimics of cell membranes, for the functional investigation of membrane (-bound) proteins, and as building blocks for biosensors5,6. However, a comprehensive physical understanding of the driving forces and structural intermediates in the SLBformation process started to emerge only recently. Both theoretical7 and experimental8-12 work during the last decade has considerably improved our understanding of the mechanisms underlying the formation of SLBs. A number of processes can occur when a liposome encounters a surface (Fig. 2 I)7,9,13. Adsorption is associated with membrane deformation (likely flattening). At sufficiently large deformations, a liposome may rupture and transform into a bilayer disk. Alternatively, neighboring vesicles may interact, or fuse, before rupturing into membrane patches. Bilayer disks or membrane patches may coalesce and/or induce the rupture of adsorbed vesicles. These events are governed by the interplay of membrane-support, intermembrane, and intra-membrane interactions. The relative contribution of these interactions is likely to depend on the nature of the support (its surface charge, structure, and roughness) and the lipid vesicles (their composition, charge, size, and physical state), as well as the aqueous environment (the pH and ionic strength). Using quartz crystal microbalance with dissipation monitoring (QCM-D), a technique pioneered by Kasemo’s group to monitor SLB-formation (see box)8, it is possible to characterize the role of electrostatics in the process of lipid deposition and SLB-formation (Fig. 2 II)11. Positively charged liposomes (N-[1-(2,3-dioleoyloxy)]-N,N,Ntrimethylammonium propane or DOTAP) exposed to silica, a negatively charged support, adsorb and rupture individually until the support is (almost) completely covered with an SLB. Conversely, liposomes of elevated negative charge (dioleoyl phosphatidylserine or DOPS) either do not adsorb (1,2dioloeyl-sn-glycero-3-phosphocholine or DOPC/DOPS, molar ratio 1:2), or do not rupture after adsorbing (DOPC/DOPS, molar ratio 1:1). Interestingly, around neutral liposome
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Fig. 2 Formation of SLBs by deposition of lipid vesicles. (I) Model of possible steps occurring between the adsorption (1) of lipid vesicles and the formation of continuous lipid bilayers. Vesicles can rupture individually (2), after interaction with other vesicles (3, 4) or with bilayer patches (5). (II) QCM-D data for the deposition of small unilamellar vesicles (SUVs) on silica. (A) SUVs made of positively charged DOTAP molecules rupture individually upon contact with the support (low dissipation). (B) SUVs made of 50% zwitterionic (DOPC) and 50% negatively charged (DOPS) lipids form a stable but incomplete vesicular layer (SVL) (high dissipation) (cf. text box on QCM-D). (C) For SUVs made of 80% DOPC and 20% DOPS, the QCM-D response shows a two-phase behavior. The initial phase resembles the formation of an SVL. In the second phase, surface bound vesicles transform into a continuous SLB as indicated by the decline in dissipation (indicating the transformation into a more rigid structure) and the decrease in frequency (in part due to the release of water from the surface bound lipid structure)11,13. The final frequency shift of -24.5 Hz corresponds to a film thickness of around 4.3 nm as expected for a continuous hydrated SLB. The low final dissipation (
