Biomedical Engineering Reference
In-Depth Information
BLMs have been extensively employed as matrixes for the in-
corporation of integral proteins, photoactive pigments and biomol-
ecules involved in biophysical, biochemical and physiological
studies. A major drawback of BLMs is their fragility, high sensi-
tivity toward vibrations and mechanical shocks, and low resistance
to electric fields; thus, they hardly last more than eight hours and
collapse under potential differences (transmembrane potentials)
greater than 100-150 mV between the solutions that bath the two
sides of the BLM; moreover, they do not lend themselves to inves-
tigations with surface-sensitive techniques.
A simple procedure for forming BLMs was recently described
by Poulos et al.
102
A squalene:decane (2:1) mixture containing 1%
DPhyPC is added to a 1 M KCl aqueous solution in a small glass
vial. Thanks to its immiscibility with water and its lower density,
the organic solvent floats on the aqueous phase. In half an hour, a
lipid monolayer is spontaneously formed at the organic/aqueous
interface, with the polar heads turned toward the aqueous phase. A
1 PL droplet of an aqueous solution is then dropped into the organ-
ic phase, reaching by gravity the organic/aqueous interface without
fusing with the aqueous phase. The lower, flattened portion of the
droplet surface in contact with the lipid monolayer becomes rapid-
ly coated by a lipid bilayer. Finally, an Ag/AgCl wire is immersed
into the droplet, allowing the closure of the electric circuit via a
further reference electrode immersed in the aqueous phase. This
system allowed the recording of single channel currents of grami-
cidin, alamethicin and D-hemolysin.
Recently, attempts have been made to form more robust lipid
bilayers (often referred to as
planar free-standing bilayers
) by
spanning them over nanopores interposed between two aqueous
and/or hydrogel phases. These free-standing bilayers have a fluidi-
ty probably comparable with that of biological membranes, thus
imparting lateral mobility to lipid molecules and membrane pro-
teins. The observed thirty times increase in stability by reducing
the pore size by a factor of four demonstrates the benefit from us-
ing nanopores. Besides stabilizing lipid bilayers, small apertures
increase the signal-to-noise ratio to an appreciable extent. Peptides
and small channel-forming proteins insert spontaneously into these
preformed bilayers. Bulky membrane proteins are incorporated
either by fusion of proteoliposomes to preformed bilayers or by
direct fusion of proteoliposomes to nanopores. Little is known
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