Kulim Kht IMPROVED UNDERSTANDING OF LIPID MEMBRANES

IMPROVED UNDERSTANDING OF LIPID MEMBRANES THROUGH
COMBINED QCM-D AND EIS MEASUREMENTS
INTRODUCTION.

A current trend in the field of sensing is to use instruments in which several detection principles
can be combined in one experiment. Methods that provide a combination of structural and functional
information on biomolecules can provide crucial insights into the understanding of biomolecular
functions.

It is of great interest in many fields to investigate how peptide and protein
interactions change the propertiesof a lipid membrane. It iswell known that membrane damage
is fatal to cells and bacteria. Therefore,a common mechanism of toxins
is to disrupt the membrane or interfere with the transport of ions across
the membrane. Typically, antibacterial peptides incorporate into bacterial
membranes and destroy the ion gradient between the inside and the outside of the bacterium.
APPROACH AND EXPERIMENTAL SETUP
This application note reviews two examples of experiments where QCM-D
and Electrical Impedance Spectroscopy (EIS) were combined. The first
example deals with the disruption of a lipid membrane through the catalytic
action of the enzyme PLA2, found in insect and snake venom. PLA2
cleaves phospholipids into the corrsponding lysolipids and fatty acids by
catalyzing the hydrolysis of a phosphoester bond in the lipid molecule.
This cleavage disrupts the membrane.The second example demonstrates
the insertion of trans-membrane pores in a model membrane by the
addition of the peptide Gramicidin D.
The combination of QCM-D and electrochemistry is especially attractive
in these examples. The effect on the membrane can be sensitively detected
by the electrochemical method, whereas the strength of the QCM-D
method lies in its ability to characterize the membrane’s viscoelastic (soft
or rigid) properties.
RESULTS AND DISCUSSION
The formation of good quality lipid membranes was first ascertained by
both QCM-D and EIS. The formation of a lipid bilayer on the QCM-D
sensor was detected in real time by both frequency and dissipation shifts
and by single frequency impedance spectroscopy, as demonstrated in
Figure 1A. Bilayer formation follows the typical pathway via the adsoption
of a critical mass of liposomes, which eventually ruptures and forms
an extended lipid bilayer on the sur-face with the characteristic QCM-D
responses Δf = - 26 Hz and ΔD < 0.5, indicating that a layer a few nanometers
thick (typically 5 nm) and rigid (as indicated by the low ΔD) has formed
on the sensor surface. Note that bilayer formation as monitored by QCM-D
is already complete long before the impedance amplitude stabilizes. A
plausible explanation for this observation is that the fusion and rupture
of adsorbed liposomes is followed by a slower annealing process, resulting
from increased ordering of the lipid molecules in the lipid membrane