Enhanced stability of freestanding lipid bilayer and its stability criteria

We present a new strategy to dramatically enhance the stability of freestanding lipid bilayers. We found that an addition of a water in oil emulsion stabilizer, SPAN 80 to a solvent phase guarantees nearly millimeter-scale stable freestanding lipid bilayers. The water permeability, bilayer area, contact angle, and interfacial tension were measured as a function of time and SPAN 80-to-lipid weight ratio (Φ_{SPAN 80}) with several different solvents. Surprisingly, the SPAN 80, instead of remaining in the bilayer, was moved out of the bilayer during the bilayer formation. Also we studied the effect of solvent on freestanding bilayer formation, and found that squalene was the only solvent that was not incorporated into the bilayer. The regime of stable bilayer formation was experimentally determined to be 3/1 < Φ_{SPAN 80} < 15/1, and we suggest general stability criteria for bilayer formation. This technique and the suggested stability criteria can be potentially helpful to many model membrane-based researches in life sciences, physical sciences and biomedical engineering fields.