Nanoarchitectured air-stable supported lipid bilayer incorporating sucrose–bicelle complex system

Cell-membrane-mimicking supported lipid bilayers (SLBs) provide an ultrathin, self-assembled layer that forms on solid supports and can exhibit antifouling, signaling, and transport properties among various possible functions. While recent material innovations have increased the number of practically useful SLB fabrication methods, typical SLB platforms only work in aqueous environments and are prone to fluidity loss and lipid-bilayer collapse upon air exposure, which limits industrial applicability. To address this issue, herein, we developed sucrose–bicelle complex system to fabricate air-stable SLBs that were laterally mobile upon rehydration. SLBs were fabricated from bicelles in the presence of up to 40 wt% sucrose, which was verified by quartz crystal microbalance-dissipation (QCM-D) and fluorescence recovery after photobleaching (FRAP) experiments. The sucrose fraction in the system was an important factor; while 40 wt% sucrose induced lipid aggregation and defects on SLBs after the dehydration–rehydration process, 20 wt% sucrose yielded SLBs that exhibited fully recovered lateral mobility after these processes. Taken together, these findings demonstrate that sucrose–bicelle complex system can facilitate one-step fabrication of air-stable SLBs that can be useful for a wide range of biointerfacial science applications.

Electrically Controlling and Optically Observing the Membrane Potential of Supported Lipid Bilayers

Supported lipid bilayers are a well-developed model system for the study of membranes and their associated proteins, such as membrane channels, enzymes, and receptors. These versatile model membranes can be made from various components, ranging from simple synthetic phospholipids to complex mixtures of constituents, mimicking the cell membrane with its relevant physiochemical and molecular phenomena. In addition, the high stability of supported lipids bilayers allows for their study via a wide array of experimental probes. In this work, we describe a platform for supported lipid bilayers that is accessible both electrically and optically. We show that the polarization of the supported membrane can be electrically controlled and optically probed using voltage-sensitive dyes. Membrane polarization dynamics is understood through electrochemical impedance spectroscopy and the analysis of the equivalent electrical circuit. We also describe the effect of the conducting electrode layer on the fluorescence of the optical probe through metal-induced energy transfer. We conclude with a discussion on possible applications of this platform for the study of voltage-dependent membrane proteins and other processes in membrane biology and surface science.

Correlated diffusion in lipid bilayers

Lipid membranes are complex quasi–two-dimensional fluids, whose importance in biology and unique physical/materials properties have made them a major target for biophysical research. Recent single-molecule tracking experiments in membranes have caused some controversy, calling the venerable Saffman–Delbrück model into question and suggesting that, perhaps, current understanding of membrane hydrodynamics is imperfect. However, single-molecule tracking is not well suited to resolving the details of hydrodynamic flows; observations involving correlations between multiple molecules are superior for this purpose. Here dual-color molecular tracking with submillisecond time resolution and submicron spatial resolution is employed to reveal correlations in the Brownian motion of pairs of fluorescently labeled lipids in membranes. These correlations extend hundreds of nanometers in freely floating bilayers (black lipid membranes) but are severely suppressed in supported lipid bilayers. The measurements are consistent with hydrodynamic predictions based on an extended Saffman–Delbrück theory that explicitly accounts for the two-leaflet bilayer structure of lipid membranes.

Correlative microscopy reveals the nanoscale morphology of E. coli-derived supported lipid bilayers

Supported lipid bilayers (SLBs) made from reconstituted lipid vesicles are an important tool in molecular biology. A breakthrough in the field has come with the use of vesicles derived from cell membranes to form SLBs. These new supported bilayers, consisting both of natural and synthetic components, provide a physiologically relevant system on which to study protein-protein interactions as well as protein-ligand interactions and other lipid membrane properties. These complex bilayer systems hold promise but have not yet been fully characterised in terms of their composition, ratio of natural to synthetic component and membrane protein content. Here, we describe a method of correlative atomic force (AFM) with structured illumination microscopy (SIM) for the accurate mapping of complex lipid bilayers that consist of a synthetic fraction and a fraction of lipids derived from Escherichia coli outer membrane vesicles (OMVs). We exploit the enhanced resolution and molecular specificity that SIM can offer to identify areas of interest in these bilayers and the atomic scale resolution that the AFM provides to create detailed topography maps of the bilayers. We are thus able to understand the way in which the two different lipid fractions (natural and synthetic) mix within the bilayers, quantify the amount of bacterial membrane incorporated in the bilayer and directly visualise the interaction of these bilayers with bacteria-specific, membrane-binding proteins. Our work sets the foundation for accurately understanding the composition and properties of OMV-derived SLBs and establishes correlative AFM/ SIM as a method for characterising complex systems at the nanoscale.

A Pillar-Free Diffusion Device for Studying Chemotaxis on Supported Lipid Bilayers

Chemotactic cell migration plays a crucial role in physiological and pathophysiological processes. In tissues, cells can migrate not only through extracellular matrix (ECM), but also along stromal cell surfaces via membrane-bound receptor–ligand interactions to fulfill critical functions. However, there remains a lack of models recapitulating chemotactic migration mediated through membrane-bound interactions. Here, using micro-milling, we engineered a multichannel diffusion device that incorporates a chemoattractant gradient and a supported lipid bilayer (SLB) tethered with membrane-bound factors that mimics stromal cell membranes. The chemoattractant channels are separated by hydrogel barriers from SLB in the cell loading channel, which enable precise control of timing and profile of the chemokine gradients applied on cells interacting with SLB. The hydrogel barriers are formed in pillar-free channels through a liquid pinning process, which eliminates complex cleanroom-based fabrications and distortion of chemoattractant gradient by pillars in typical microfluidic hydrogel barrier designs. As a proof-of-concept, we formed an SLB tethered with ICAM-1, and demonstrated its lateral mobility and different migratory behavior of Jurkat T cells on it from those on immobilized ICAM-1, under a gradient of chemokine CXCL12. Our platform can thus be widely used to investigate membrane-bound chemotaxis such as in cancer, immune, and stem cells.