Combined AFM-SPR Analysis of Model Biomembrane Formation

DAZA MILLONE, M. A.; MATÉ, S.; HERLAX, V.; VELA, M. E.

La Plata

Workshop; Imaging Techniques for Biotechnology and Biomedical Applications Workshop; 2016

Cell membranes act as a barrier that separates the inner content from the outside environment and have a key role in several cell functions such as signaling, transport, scaffold, among others. Understanding the membrane structure and dynamics is of utmost importance for biological sciences and biomedical applications. In this sense, supported lipid bilayers (SLBs) and small unilamellar vesicles (SUVs) are model systems for cell membranes that allow biophysical studies with surface techniques. Among them, surface plasmon resonance (SPR) and atomic force microscopy (AFM) are powerful tools that give information on kinetics of biomolecular interactions and nanostructure.[1] Nevertheless, correct interpretation of these results relies on model system preparation, a technological challenge that involves surface nature, lipid composition and ionic strength conditions. In this work, we aim to provide a simple approach to functionalize SPR gold sensors with a good coverage and fluid properties of a lipid bilayer. Pure lipids (DMPC, POPC and DOPC), binary mixture (DOPC/Cho) or ternary mixtures (DOPC/24:1SM/Cho and DOPC/16:0SM/Cho) were employed to make SUVs.[2] Bare SPR gold sensors were modified with a 50 µM dithiothreitol (DTT) ethanolic solution during 30 min to achieve a DTT monolayer on the polycrystalline surface. [3] The vesicles were put in contact in situ with the SPR flow system at 5 µL/min during 15-20 min. The change in SPR signal for each mixture was used to determine surface coverage and whether fusion or vesicle adsorption was achieved. These results were further analyzed in the AFM fluid cell operating in contact mode. Since AFM imaging doesn?t allow to discriminate a full coverage of lipid from the bare gold substrate, force spectroscopy was employed to asses the nature of the surface. Force volume measurements with typical rupture forces allow to characterize the lipid bilayers and compare the values obtained for the same systems in mica.[1] Cooper, M. (2009) ?Label-Free Biosensors: Techniques and applications? London, Cambridge University Press[2] Maté, S; Vázquez, R; Herlax, V; Daza-Millone, M, A; Fanani, M. L; Maggio, B; Vela, M. E; Bakás, L. BBA-Biomembranes (2014) 1838, 1832-1841