Nanoscale Substrate Roughness Hinders Domain Formation in Supported Lipid Bilayers

Abstract

Supported Lipid Bilayers (SLBs) are model membranes formed at solid substrate surfaces. This architecture renders the membrane experimentally accessible to surface sensitive techniques used to study their properties, including Atomic Force Microscopy (AFM), optical fluorescence microscopy, Quartz Crystal Microbalance (QCM) and X-Ray/Neutron Reflectometry, and allows integration with technology for potential biotechnological applications such as drug screening devices. The experimental technique often dictates substrate choice or treatment, and it is anecdotally recognised that certain substrates are suitable for the particular experiment, but the exact influence of the substrate has not been comprehensively investigated. Here, we study the behavior of a simple model bilayer, phase separating on a variety of commonly used substrates, including glass, mica, silicon and quartz, with drastically different results. The distinct micron scale domains observed on mica, identical to those seen in free-floating Giant Unilamellar Vesicles (GUVs), are reduced to nanometer scale domains on glass and quartz. The mechanism for the arrest of domain formation is investigated, and the most likely candidate is nanoscale surface 2 roughness, acting as a drag on the hydrodynamic motion of small domains during phase separation.Evidence was found that the physico-chemical properties of the surface have a mediating effect, most likely due to changes in the lubricating interstitial water layer between surface and bilayer.

Impact of nanoscale hindrances on the relationship between lipid packing and diffusion in model membranes

“…There is already substantial evidence of a support's influence on bilayer diffusion [25][26][27][28] . To further assess the influence of support on other pivotal biophysical parameters and the relationships between them, we have selected four models that broadly represent the spectrum of designs extensively utilised.…”

“…However, few studies have ventured a comprehensive comparison of their biophysical properties as a function of the inherent parameters (a support presence, its material, model geometry etc.) 18,25,28,[40][41][42][43] . Indeed, a few studies have highlighted this concern by demonstrating altered protein functionality in different membrane models 44,45 .…”

Lipid exchange enhances geometric pinning in multicomponent membranes on patterned substrates

“…38 This could be ascribed to the following two possible mechanisms. First, as it was shown by Goodchild et al, 24 nanoscopic gel domains cannot fuse on a glass surface because of the roughness of the glass thereby hindering detection by confocal microscopy. Second, the mixtures lying on the SM/Chol axis are not ''pure'' in our experiment because of the inclusion of DOPE-PEG and rhodamine-PE necessary for the coating and identification of the LD phase, respectively.…”

“…This may result in preference for the mixed configuration, even in the absence of POPC. The use of MICA substrates in place of glass can alleviate this effect, 24,39 and allow for the formation of LD domains of circular shape, see also ESI, † Fig. S3.…”

“…19 While GUVs can change shape during the phase separation process, SLBs and SLVs are scaffolded. Therefore, they can mimic the supporting property of the actin cytoskeleton in cells and allow for the fabrication of stable bilayers for atomic force microscopy (AFM) studies, [20][21][22][23][24] for applications in biosensing, 25 in cell biology, 26 and in drug delivery. 27 Experiments with GUVs have shown that there is a correlation between positioning of soft domains and bilayer curvature.…”