Fabrication and electromechanical characterization of free-standing
asymmetric membranes
Authors: Liu Paige , Zabala-Ferrera Oscar , Beltramo Peter J. 
Published: October 2020

ABSTRACT
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. Here, we describe a new approach to fabricate and
characterize planar, free-standing, asymmetric membranes and use it to examine
the effect of headgroup charge on membrane stiffness. The approach relies on a
thin film balance used to form a freestanding membrane by adsorbing aqueous
phase lipid vesicles to an oil-water interface and subsequently thinning the oil
to form a bilayer. We validate this lipid-in-aqueous approach by analyzing the
thickness and compressibility of symmetric membranes with varying zwitterionic
DOPC and anionic DOPG content as compared to previous lipid-in-oil methods. We
find that as the concentration of DOPG increases, membranes become thicker and
stiffer. Asymmetric membranes are fabricated by controlling the lipid vesicle
composition in the aqueous reservoirs on either side of the oil. Membrane
compositional asymmetry is 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 15 to 80 mV. Introducing membrane charge asymmetry decreases both
the thickness and stiffness in comparison to symmetric membranes with the same
overall phospholipid composition. These initial successes demonstrate a viable
pathway to quantitatively characterize asymmetric bilayers that can be extended
to accommodate more complex membranes and membrane processes in the
future.

SIGNIFICANCE
A defining characteristic of the cell membrane is asymmetry in
phospholipid composition between the interior and exterior bilayer
leaflet. Although several methods have been used to artificially create
membranes with asymmetry, there has not been extensive characterization
of the impact of asymmetry on membrane material properties. Here, a
technique to fabricate free-standing asymmetric membranes is developed
which facilitates the visualization and electromechanical
characterization of the bilayer. Asymmetry in anionic phospholipid
concentration is quantified by measurements of membrane capacitance at
varying voltages, which also allows for determination of the membrane
compressibility. This method represents an advance in the development of
artificial biomembranes by reliably creating phospholipid bilayers with
asymmetry and facilitates the interrogation of more complex biological
processes in the future.