(197c) Fabrication and Characterization of Free-Standing Asymmetric Membranes

All biological cell membranes maintain an electric transmembrane potential of around 100 mV, due in part to an asymmetric distribution of charged phospholipids across the membrane. This asymmetry is crucial to cell health and physiological processes such as intracell signaling, receptor-mediated endocytosis, and membrane protein function. Experimental artificial membrane systems incorporate essential cell membrane structures, such as the phospholipid bilayer, in a controllable manner where specific properties and processes can be isolated and examined. Methods to fabricate asymmetric model membranes are in their infancy, and many do not allow for quantitative control over solvent inclusion, lipid composition, and/or the ability to dynamically change and monitor conditions on either side of the membrane, which is necessary to characterize membrane physicochemical properties. To address this experimental gap, a platform was developed to fabricate and characterize planar, free-standing, asymmetric membranes with control over the lipid composition and solvent. First, a thin film balance was used to form a freestanding membrane by adsorbing aqueous phase lipids to an oil-water interface and subsequently thinning the oil to form a bilayer. This lipid-in-aqueous approach resulted in the formation of membranes with similar thickness and compressibility as previous lipid-in-oil methods, based on membranes composed of zwitterionic DOPC and anionic DOPG. By controlling the lipid composition in the aqueous compartments on either side of the oil within the thin film balance, asymmetric membranes were formed. This asymmetry was qualitatively demonstrated using a fluorescence quenching assay and quantitatively characterized through voltage-dependent capacitance measurements. Stable asymmetric membranes with DOPC on one side and DOPC/DOPG mixtures on the other were created with transmembrane potentials ranging from 10 to 25 mV. Increasing membrane charge asymmetry increases the offset voltage and also increases the stiffness of the membrane. These initial successes demonstrate a viable pathway to quantitatively characterize asymmetric bilayers that can be extended to accommodate more complex membrane processes in the future.