Langmuir 2008, 24, 1989-1999
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Floating Lipid Bilayers Deposited on Chemically Grafted Phosphatidylcholine Surfaces Arwel V. Hughes,*,† Jonathan R. Howse,‡,| Aleksandra Dabkowska,§ Richard A. L. Jones,‡ M. Jayne Lawrence,§ and Stephen J. Roser£ ISIS, Rutherford Appleton Laboratory, Chilton, Didcot, OX11 OPU, U.K., Department of Physics and Astronomy, UniVersity of Sheffield, Hounsfield Road, Sheffield, S3 7RH, U.K., School of Pharmacy, Kings College London, London, SE1 9NN, U.K., and Department of Chemistry, UniVersity of Bath, ClaVerton Down, Bath, BA2 7AY, U.K. ReceiVed July 10, 2007. In Final Form: NoVember 7, 2007 Floating supported bilayers (FSBs) are new systems which have emerged over the past few years to produce supported membrane mimics, where the bilayers remain associated with the substrate, but are cushioned from the substrates constraining influence by a large hydration layer. In this paper we describe a new approach to fabricating FSBs using a chemically grafted phospholipid layer as the support for the floating membrane. The grafted lipid layer was produced using a Langmuir-Schaeffer transfer of acryloyl-functionalized lipid onto a pre-prepared substrate, with AIBN-induced cross-polymerization to permanently bind the lipids in place. A bilayer of DSPC was then deposited onto this grafted monolayer using a combination of Langmuir-Blodgett and Langmuir-Schaeffer transfer. The resulting system was characterized by neutron reflection under two water contrasts, and we show that the new system shows a hydrating layer of ∼17.5 Å in the gel phase, which is comparable to previously described FSB systems. We provide evidence that the grafted substrate is reusable after cleaning and suggest that this greatly simplifies the fabrication and characterization of FSBs compared to previous methods.
Introduction The lipid bilayer is the basic structural framework of biological membranes. It houses membrane proteins, which regulate various cellular and subcellular functions and activities, and are active in structures such as ion channels or pumps, ATPases, and membrane receptors. It is estimated that between 15% and 30% of the human genome codes accounts for membrane proteins (MP),1 and often, many of these proteins are functionally inactive or structurally compromised when removed from the membrane environment. Therefore, to make progress in the study of MP, there is great interest in developing model phospholipid bilayers that are experimentally tractable but preserve the main functionality of natural membranes. The phospholipid membrane in its native state is difficult to study. Generally, natural membranes are composed of several lipids, as well as sterols, sphingolipids, and often many hundreds of membrane proteins and peptides.2 The understanding of the complex phase behavior which exists in such systems is poor, and a robust experimental system is required where the parameters of the system are accessible to manipulation, such that its behavior may be probed in detail. Given the number of long-established techniques that exist for investigating soft matter on surfaces, a biomimetic membrane deposited on a substrate, while retaining its salient features, would clearly be an advantage. A number of approaches to solving this problem have been proposed, and various fabrication schemes have been utilized * To whom correspondence should be addressed. E-mail: a.v.hughes@ rl.ac.uk. † Rutherford Appleton Laboratory. ‡ University of Sheffield. § Kings College London. £ University of Bath. | Current address: Department of Chemical and Process Engineering, Sheffield University, Sheffield. (1) Ahram, M.; Litou, Z. I.; Fang, R.; Al-Tawallbeh, G. In Silico Biol. 2006, 6, 36. (2) Sackmann, E. Science 1996, 271, 43-48.
including Langmuir-Blodgett techniques, spontaneous fusion of unilamellar vesicles on un- and precoated surfaces, and selfassembled tethered bilayers.3-6 However, the common flaw of these approaches is that, in being localized on the substrate surface, the component lipids essentially become fixed and their freedom within the plane of the membrane is limited. A natural biomembrane is a dynamic, fluid system, where the component molecules have considerable translational freedom, and this fluidity is central to the behavior of the system. In a deposited bilayer, however, the loss of translational freedom means that the bilayer is, at best, only a poor approximation of the real system and suitable for probing only the simplest of interactions. The early attempts to preserve fluidity in supported bilayers were centered on single lipid bilayers on Si/SiO2 and quartz surfaces.6 It was shown that, under conditions of sufficient hydration, a thin water layer (
