Electrostatic Force Microscopy Analysis of Lipid Miscibility in Two

Mar 27, 2004 - Center for Biopolymers at Interfaces, University of Utah, Salt Lake City, Utah 84112, Department of Physics, University of Utah, Salt L...
0 downloads 0 Views 248KB Size
3684

Langmuir 2004, 20, 3684-3689

Electrostatic Force Microscopy Analysis of Lipid Miscibility in Two-Component Monolayers Thomas Goodman,† Ezra Bussmann,‡ Clayton Williams,†,‡ Mundeta Taveras,§ and David Britt*,†,§ Center for Biopolymers at Interfaces, University of Utah, Salt Lake City, Utah 84112, Department of Physics, University of Utah, Salt Lake City, Utah 84112, and Department of Biological Engineering, Utah State University, Logan, Utah 84322 Received December 15, 2003. In Final Form: February 20, 2004 Electrostatic force microscopy (EFM) was used to assess lipid miscibility and phase behavior in twocomponent Langmuir-Blodgett (LB) monolayers composed of cationic dioctadecyldimethylammonium bromide (DOMA) and nonionic methyl stearate (SME) lipids. The surface potential measurements were calibrated by applying known bias voltages to the sample during several line scans, thus creating surface potential “scale bars” on the images from which it was determined that circular domains were 50 mV more positive than the surrounding phase. As the spatially averaged surface potential of DOMA was over 400 mV more positive than that of SME, this 50-mV surface potential difference is too low to correspond to lipid phase separation (immiscibility) in the two-component film. Rather, the surface potential contrast was attributed to an increased packing density and a more orthogonal orientation of lipids in the domains resulting in a greater contribution of dipoles to the measured (normal) surface potential. Monolayers prepared by sequentially spreading the two lipids resulted in irregular domains that were 50-450 mV more positive than the surrounding phase, representing varying degrees of lipid mixing, restricted by two-dimensional diffusion at the interface. Fluorescent images of monolayers stained with negatively charged dye supported the EFM miscibility prediction and assignment of surface potential. These results demonstrate a new approach using EFM to quantitatively measure surface potential in order to assess the lateral distribution of components in thin films as well as predict adsorption patterns to heterogeneous interfaces.

Introduction Studies of the physical chemistry of single component and multicomponent monomolecular films at the air/water interface have revealed coexisting phases, such as liquid and gas or liquid and solid, that tend to break up into domains which are readily observed with fluorescence, Brewster angle, and atomic force microscopies.1 Of particular interest from both a theoretical and practical standpoint is how two lipids are distributed in the phases present in a binary monolayer, which when confined to the plane of an air/water interface represents a quasitwo-dimensional system having reduced degrees of freedom compared to bulk solutions.2 Monolayer films are also often investigated as simplified cell membrane mimetics, where membrane properties are readily controlled and observed using an array of surface-sensitive instrumentation. In cell membranes the miscibility and phase behavior of lipids contribute to the fluid mosaic nature, while influencing interfacial reactions such as protein adsorption and lateral distribution. It is becoming increasingly recognized that lipid- and cholesterol-rich domains, or “rafts”, present in cell membranes are implicated in intracellular trafficking of lipids,3 as well as playing varying roles in events such as platelet activation4 and *Corresponding author: e-mail, [email protected]; fax, 435-7971248. † Center for Biopolymers at Interfaces, University of Utah. ‡ Department of Physics, University of Utah. § Utah State University. (1) McConnell, H. M. In Micelles, microemulsions, and monolayers; Shah, D. O., Ed.; Marcel Dekker: New York, 1998; pp 387-393. (2) Crisp, D. J. In Surface Chemistry; Butterworth Scientific Publications: London, 1949; pp 17-22. (3) Mukherjee, S.; Maxfield, F. R. Traffic 2000, 1, 203-211.

amyloid β-protein binding in conjunction with Alzheimer’s disease5 and serving as entry and exit sites for microbial pathogens, such as influenza virus,6 measles virus,7 and HIV.8,9 The lateral distribution and interactions among the lipids and proteins of pulmonary surfactants have also been investigated in efforts to develop methods of preventing alveolar collapse in premature infants.10-12 In addition to biological membranes, surfactant mixtures are also of considerable interest in forming micelles and emulsions for particle separation, flocculation, and oil recovery and in food and dairy processing.13 Here the miscibility and phase behavior of twocomponent model lipid monolayers were characterized by electrostatic force microscopy (EFM) to map the electric surface potential and, using a method of constructing surface potential scale bars on the images, assign absolute differences in surface potential between domains and surrounding lipid phases. The lipids employed in this (4) Bodin, S.; Tronchere, H.; Payrastre, B. Biochim. Biophys. ActaBiomembr. 2003, 1610, 247-257. (5) Wood, W. G.; Eckert, G. P.; Igbavboa, U.; Muller, W. E. Biochim. Biophys. Acta-Biomembr. 2003, 1610, 281-290. (6) Scheiffele, P.; Rietveld, A.; Wilk, T.; Simons, K. J. Biol. Chem. 1999, 274, 2038-2044. (7) Manie, S. N.; Debreyne, S.; Vincent, S.; Gerlier, D. J. Virol. 2000, 74, 305-311. (8) Campbell, S. M.; Crowe, S. M.; Mak, J. J. Clin. Virol. 2001, 22, 217-227. (9) Mahfoud, R.; Garmy, N.; Maresca, M.; Yahi, N.; Puigserver, A.; Fantini, J. J. Biol. Chem. 2002, 277, 11 292-11 296. (10) Discher, B. M.; Maloney, K. M.; Grainger, D. W.; Sousa, C. A.; Hall, S. B. Biochemistry 1999, 38, 374-383. (11) Discher, B. M.; Maloney, K. M.; Schief, W. R. J.; Grainger, D. W.; Vogel, V.; Hall, S. B. Biophys. J. 1996, 71, 2583-2590. (12) Kaznessis, Y. N.; Kim, S.; Larson, R. G. J. Mol. Biol. 2002, 322, 569-582. (13) Shah, D. O., Ed. Micelles, microemulsions, and monolayers; Marcel Dekker: New York, 1998.

10.1021/la036366h CCC: $27.50 © 2004 American Chemical Society Published on Web 03/27/2004

Lipid Miscibility in Two-Component Monolayers

study, dioctadecyldimethylammonium bromide (DOMA) and methyl stearate (SME), have identical alkyl chain lengths and saturation and are thus not readily distinguishable in a two-component film using atomic force microscopy topography or lateral force imaging. However, a large electrical potential difference exists between these two lipids due to their respective cationic and nonionic headgroups and provides the basis for probing lipid miscibility with EFM. Although the lipid headgroup charge is a dominating contribution to the surface potential, the tilt-angle-dependent contribution of alkyl tail dipoles to the electric surface potential may be significant and is also considered. Electrostatic Force Microscopy EFM is a type of noncontact atomic force microscopy (AFM) sensitive to variations in the potential difference between a probe and the sample surface.14 The probe is a metal tip at the end of an AFM cantilever. The sample surface topography is first traced while either contacting the tip to the sample with an applied tip/sample voltage of zero or scanning the tip over the surface in intermittent contact (tapping) mode. The tip is then raised and scanned above the sample at a height of 25-75 nm, while retracing the topographic features. While in this “Lift Mode,” an ac voltage is applied between tip and substrate at the resonance frequency of the cantilever. As the cantilever is scanned across the surface, a deflection at the frequency of the applied voltage is produced which is proportional to the average potential difference between the tip and sample surface.15 The ac deflection of the cantilever is sensed by an optical beam deflection technique. The electrostatic deflection signal (proportional to the average surface potential difference) is the parameter recorded by the microscope to produce an EFM image. The images presented here have lateral spatial resolution below 100 nm (tip radius and probe-surface separation distance limited) and surface potential sensitivity (noise limited, 300 Hz bandwidth) of about 10 mV. This technique has been previously employed to investigate surface potential differences, attributed to dipole contributions, between different organosilanes within self-assembled monolayers on silicon substrates.16 Other means of mapping surface charge with sub-100nm resolution also involve scanning probe microscopy imaging modes such as force mapping, in which the electrostatic contribution is calculated by fitting the force data to the Derjaguin, Landau, Verwey, and Overbeek Theory (DLVO).17,18 This imaging mode has the advantage of working in aqueous solution where the ionic strength can be varied between scans to further probe the contribution of electrostatics. However, this method assumes a small (