A detailed investigation of the formation kinetics and layer structure of poly(ethylene glycol) tether supported lipid bilayers† Stefan Kaufmann, a Georg Papastavrou, b Karthik Kumar, a Marcus Textor a and Erik Reimhult * a Received 30th January 2009, Accepted 7th April 2009 First published as an Advance Article on the web 8th June 2009 DOI: 10.1039/b901874c Supported lipid bilayers formed from liposomes containing phospholipids with covalently attached poly(ethylene glycol) (PEG-SLBs) potentially circumvent two current limitations of membrane sensor platforms, namely air-stability and limited aqueous space between the sensor substrate and the membrane. However, questions regarding the formation kinetics through self assembly and the presence of a PEG cushion underneath the lipid bilayer are still unanswered. Quartz crystal microbalance with dissipation monitoring and fluorescence recovery after photobleaching (FRAP) measurements show that PEG-SLB formation through self-assembly is possible in the mushroom regime while it is hindered in the brush regime. The observed dependence of the kinetics on grafting density and molecular weight of the PEG-lipids is attributed to the electrostatic and steric shielding effect of the enveloping PEG layer. In the mushroom phase, non-densely packed PEG does not completely screen lipid-surface interactions while in the brush phase, densely packed non-interacting PEG chains stabilise liposomes and prevent overall attractive interactions. Force–distance measurements are used to directly measure the thickness of the PEG-SLB structure and demonstrate the presence of PEG chains on both sides of the lipid bilayer. FRAP measurements support this finding by showing increased lipid mobility for increased PEG layer thickness through decoupling of the membrane from the substrate. Force–distance and FRAP measurements further reveal the mobility of the PEG-lipids and the novel detailed behavior of laterally mobile PEG mushrooms under mechanical compression. 1 Introduction Membrane proteins require incorporation into a suitable lipid bilayer environment to retain their tertiary structure and thereby their activity. Supported lipid bilayers (SLBs), which are extended planar lipid bilayers assembled at the liquid–solid interface, have been proposed as surface modifications for biosensors and other platforms for the study of membrane properties and, in particular, transmembrane protein function. 1–3 However, it has been reported that the close proximity of the SLB to the substrate causes pinning and partial denaturation of transmembrane proteins with hydrophilic domains. 4–7 In response, several groups have developed platforms where the lipid membrane is reconstituted using a poly(ethylene glycol) (PEG) spacer. 8–12 All these platforms essentially create a dense layer of covalently-linked short ethylene glycol spacers, which are functionalised with a lipid or cholesterol derivative that creates an amphiphilic sub-monolayer onto which an SLB can be formed. These tethered lipid membranes, however, have too short PEG spacers and too dense packing of immobilised PEG- lipids in the proximal leaflet to provide the ideal environment for large membrane proteins. They are also challenging to form, requiring ultraflat substrates and optimum conditions. 10 Cremer and coworkers demonstrated that SLBs can also be assembled from so-called PEG-liposomes or stealth lipo- somes. 7,13 PEG-liposomes comprise a percentage of lipids with a linear PEG chain attached to their headgroup and are commonly used in drug delivery research, as the stealth proper- ties of the PEG shell result in increased long circulation times in vivo compared to the non-PEGylated liposomes. 14 Formation of SLBs with low concentrations of PEG-lipids of much higher molecular weight than typically used for tethered SLBs was shown to be possible via self-assembly. 13 Enhanced stability upon air exposure was also demonstrated. 13 Furthermore, it was shown that size-discriminatory binding of proteins through the PEG shell could occur, meaning that proteins in a PEG-SLB would be accessible for ligand binding to study functionality. 13 The interaction of polymers with lipid bilayers in various configurations has been summarised by Tribet and Vial. 15 Yet several questions remain regarding the formation and structure of PEG-SLBs that are of great importance for their application. The most important questions are how they form, how their formation by self-assembly proceeds and whether a PEG layer is present between the lipid bilayer and the substrate to provide the desired additional spacer. Kuhl et al. 16 showed by SFA measurements that the interbilayer interactions are greatly modified by the insertion of polymers. They found that PEG bilayers do not adhere because of steric repulsion of the polymer chains. 16 PEG itself shows no interaction with SiO 2 or glass supports. Given the previously observed higher mechanical stability and the repulsion of PEG-lipid membranes, it is there- fore remarkable that PEG-SLBs seem to form although the a Department for Materials Science, ETH Zurich, Wolfgang-Pauli-Str. 10, Zurich, Switzerland; Fax: +41 44 633 10 27; Tel: +41 44 633 75 47 b Department of Inorganic, Analytical and Applied Chemistry, University of Geneva, Geneva, Switzerland † Electronic supplementary information (ESI) available: Supplementary Fig. S1–S2. See DOI: 10.1039/b901874c 2804 | Soft Matter , 2009, 5, 2804–2814 This journal is ª The Royal Society of Chemistry 2009 PAPER www.rsc.org/softmatter | Soft Matter