rXXXX American Chemical Society A dx.doi.org/10.1021/la2019132 | Langmuir XXXX, XXX, 000–000 ARTICLE pubs.acs.org/Langmuir Formation of Nanopore-Spanning Lipid Bilayers through Liposome Fusion Karthik Kumar, † Lucio Isa, † Alexander Egner, ‡,§ Roman Schmidt, ‡ Marcus Textor, † and Erik Reimhult* ,|| † Department of Materials, Laboratory for Surface Science and Technology, Swiss Federal Institute of Technology (ETH Z€ urich), CH-8093 Z€ urich, Switzerland ‡ Department of NanoBioPhotonics, Max-Planck Institute, D-37077 G€ ottingen, Germany ) Department of Nanobiotechnology, University of Natural Resources and Life Sciences (BOKU) Vienna, A-1190 Vienna, Austria b S Supporting Information 1. INTRODUCTION Understanding the workings of membrane proteins remains one of the open challenges of present day biology 1 and has a vast impact on drug design and development. 2,3 In vivo studies involve a high degree of complexity, and there is a strong drive toward studying membrane proteins in simpler, controlled model environments. Reconstituted lipid bilayers are good models of real cell membranes and can incorporate delicate membrane proteins in a native environment. This allows mem- brane protein functions to be directly studied with high-resolu- tion probes under controlled and reproducible conditions. 4 To incorporate membrane proteins with large hydrophilic domains and to enable access to the membrane from both sides, a common approach is to span lipid bilayers across several hundreds of micrometers wide apertures, forming the so-called black lipid membranes (BLM). 5 BLMs have several drawbacks that have prevented their implementation in biosensing applications, for which robustness and the use of delicate membrane proteins are generally required. Typically, BLMs are spread in the presence of solvents such as decane. 5,6 The large size of the apertures and the solvent remnants cause instabilities in the BLM, in turn causing these sensors to have too-short lifetimes. Moreover, the solvent remnants in the membrane affect the function of membrane proteins, therefore limiting the possibility of studying complex membrane proteins in native environments. 7 Alternatively, planar membranes bereft of nonpolar solvents can be formed by rupture and fusion of small or large unilamellar liposomes onto solid supports, forming a so-called supported lipid bilayer (SLB) as first reported by Tamm and McConnell. 8 SLBs are increasingly used due to their ease of formation, stability, and adaptability to most common sensing platforms. 9 The improved stability is however achieved at the price of losing the possibility to incorporate many membrane proteins in their native conformation due to steric hindrance and interactions with the solid support. Combining the ample space for protein incorporation and the small, defined sensor area of BLMs with the robustness, ease of formation, and longevity of supported lipid bilayers would con- stitute a major leap forward for membrane and membrane protein sensing. 7 Tiefenauer and co-workers demonstrated in 2007 that the lifetimes of solvent-spread BLMs continuously increased as the aperture size was decreased to 200 nm, 10 confirming previously shown long lifetimes of BLMs spread across nanosized pores. 6,11 H€ o€ ok et al. recently described how solvent-free SLBs preformed at physiological conditions on flat surfaces could be driven by shear flow in microfluidic channels to span nanoholes on another part of the substrate if the pH was increased to 9.5, 12 but it is uncertain whether this method can be applied with large membrane proteins which are pinned from moving at the surface. 13 Naumann and co- workers also recently placed giant vesicles over single nanopores to form nano-BLMs; 14 however, this manual approach does not allow incorporation of membrane proteins by proteoliposomes over large areas of pores. 15 Received: May 22, 2011 Revised: July 3, 2011 ABSTRACT: Self-assembly of nanopore-spanning lipid bi- layers (npsLBs) paves the way toward chip-based integrated membrane protein biosensing. We present a novel approach to analyze the formation of npsLB at individual nanopores using quantitative analysis of high-resolution microscopy images. From this analysis we derive necessary conditions for the formation of npsLBs on nanopore arrays by liposome fusion and discuss the limitations of the process as a function of nanopore geometry, lipid membrane properties, and surface interaction. Most importantly, applying liposomes with dia- meters larger than the nanopore is demonstrated to be a necessary but not sufficient condition for npsLB formation. A theoretical model is used to discuss and explain this experimental finding.