AFM and electrochemical characterization of fluidity and continuity of DMPC supported bilayers on Hydroxilated Thiol modified gold electrodes

CRECZYNSKI-PASA, T. B.; DAZA MILLONE, M. A.; MUNFORD, M. L.; R. DE LIMA, V.; VIEIRA, T. O.; BENITEZ, G.; PASA, A. A.; SALVAREZZA, R. C.; VELA, M. E.

Workshop; XXI. Annual Linz Winter Workshop; 2019

The formation of fluid dimyristoylphosphatidylcholine (DMPC) bilayer from vesicle fusion has been performed on a self-assembled monolayer of dithiothreitol (DTT) on Au(111). XPS and electrochemical data indicate a DTT surface coverage of θ ≈ 0.16 with two S head-Au covalent bonds per DTT molecule. The DTT monolayer turns the Au surface hydrophilic enough to allow by vesicle fusion as revealed by in situ atomic force imaging. In order to determine the presence of the lipidic layer we have used a lithographic step by applying an additional force to the cantilever probe. This procedure removes the material present in the scanned region opening a clear squared window with a thickness of about 5 nm. We have also been able to image the self-healing of this layer by lateral motion of phospholipids indicating that it is not rigid at 22 ºC. The sequential AFM images allow making a rough estimation of the lateral diffusion coefficient (D) of the phospholipids of the window (see Figure). Taking the front displacement L for the time interval Δt and using L2 = 2DΔt we obtained D ≅ 10-11 cm2 s-1.This value is somewhat lower than that reported for DMPC bilayers supported on glass at this temperature (2.10-10 cm2 s-1). The discrepancy can be explained either by a self-limiting spreading or by an increased interaction of DMPC with the underlying DTT/Au with respect to the glass substrate.The DMPC lateral motion points toward the maintenance of membrane dynamics, which is a signal that this system could be adequate for fundamental studies in general phenomena of natural membranes, such as the incorporation of biomolecules (enzyme cofactors, proteins, drugs, etc.). Concerning this point, we have tested the behavior of our DMPC bilayer-DTT-Au arrangement when it was exposed to two electrochemically active molecules: Methylene Blue (MB), a lipophilic molecule able to penetrate biological membranes, and Flavin Adenine Dinucleotide (FAD), an electron carrier, that it is known that cannot diffuse across the membrane. The electrochemical behavior of these molecular probes demonstrates the continuity and fluidity of our supported bilayers verifying that it is both fluid and continuous.