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J. Phys. Chem. 1995, 99, 1038-1045
Scanning Force Microscopy Characterization of Langmuir-Blodgett Films of Sulfur-Bearing Lipids on Mica and Gold Lars Santesson, Tim M. H. Wong, Mauro Taborelli, and Pierre Descouts GAP Biomfdicale, Universiti de Gentve, 20, rue de I'Ecole-de Mgdecine, CH-I21I , Gentve 4, Switzerland
Martha Liley, Claw Duschl, and Horst Vogel* Institut de Chimie Physique II, Ecole Polytechnique Ffdfrale de Lausanne, CH-1015 Lausanne, Switzerland Received: September 7, 1994; In Final Form: November I , 1994@
Physisorbed and covalently bound Langmuir-Blodgett (LB) monolayers of sulfur-bearing lipids on mica and on gold substrates, respectively, have been studied. Scanning force microscopy, in topography and friction modes as well as in contact and noncontact modes have been used to investigate these films in the region of coexisting phases. For the first time, a detailed and complex internal structure of solid-analogous monolayer domains, reflecting their symmetric morphology, has been observed. Comparisons of physisorbed monolayers on mica with chemisorbed monolayers on gold show distinct differences in the structure and stability of the films caused by the different substrate/film interactions. It is found that the use of a substrate to which the film can bind covalently does not result in changes to the morphology of solid-analogous domains in the films. The structure of covalently bound monolayers is well-preserved over long time periods and is stable at solidfluid interfaces. However, the covalently bound LB monolayers are more susceptible to damage by the SFM tip than the physisorbed films on mica.
Introduction In recent years, ultrathin organic films-prepared either by Langmuir-Blodgett (LB)' or by self-assembly (SA) methodshave attracted considerable interest.2 These films have great potential for technical application in many different fields ranging from nonlinear optics, wetting properties, and lubrication to biosensors and molecular electronic^.^ A crucial topic of research into these low-dimensional systems is their interactions with the solid substrates on which they are deposited. In this respect, two aspects seem to be especially important: first, the influence of the substrate on the internal structure of the films on scales reaching from molecular dimensions to the micron range, and second, the influence of the interaction between the organic molecules and the substrate on the stability of the film.4 In this paper, we investigate these issues using LB monolayers of lipids which bear a disulfide group (thiolipids). These thiolipids are members of a new class of synthetic lipids which are amphiphilic and which can covalently bind to the surface of a zerovalent metal (gold, silver, ~ o p p e r ) .Such ~ molecules allow the formation of stable, covalently bound monolayers on gold surfaces using either SA or LB techniques. On mica, thiolipid monolayers are a good example of a conventional, widely relevant LB system of amphiphile and substrate. Thus, it is possible to directly compare the properties of covalently bound LB monolayers of thiolipids on gold surfaces with those of noncovalently bound LB monolayers on other surfaces. Originally, the thiolipids were designed to form covalently bound monolayers to serve as building blocks for improved supported lipid bilayers. The thiolipids consist of two lipid molecules, each attached to a hydrophilic spacer of variable length and joined by a disulfide bond at the end of the two spacers. In a covalently bound monolayer, the spacers decouple the lipid layer from the rigid gold substrate and preserve a thin water film between lipid and gold. Addition of a second lipid
* To whom correspondence should be addressed. @Abstract published in Advance ACS Absrrucrs, December 15, 1994. 0022-365419512099-1038$09.0010
layer on top of the thiolipid monolayer results in a bilayer which is a good model for biological cell membranes and allows incorporation of membrane spanning proteins and peptides in the lipid b i l a ~ e r . ~Use of a combination of LB and SA techniques with the thiolipids allows the formation of twodimensionally structured covalently bound surface layers which can be selectively addressed either by chemical complex formation or by immuno reactions using specific antibodies6 The size of such lateral structures can, in principle, be reduced to the molecular scale, thus giving additional relevance to the use of scanning probe methods in their study. We have used scanning force microscopy (SFM) and fluorescence microscopy to investigate LB monolayers of the thiolipids. SFM has emerged as an important tool for studying ultrathin organic films because of its unprecedented ability to visualize in situ electrically insulating films with high lateral res~lution.~Samples can be observed in air and in liquids, which allows study of the outer hydrophilic side of biomembranes as well as active biological molecules. In addition to topographical imaging, SFM can also be used to measure the frictional interaction between the tip and the surface.* Such friction measurements on LB films have enabled differentiation between contrasting chemical components in the films,g the detection of film defects on perfectly flat bilayers of Cd arachidate,IOand the detection of friction anisotropy with respect to scan direction in lipid bilayers.I1 In recent years, fluorescence microscopy12has contributed considerably to the understanding of the rich phase behavior of lipid monolayers on the water surface. The introduction of dye-labeled lipids into the monolayer allows the visualization of the phase states due to the different solubilities of the fluorophore in the different phases.13 The observation of domain structures at the transition from fluidto solid-analogous phase states has triggered intensive research into this phenomenon, both experimental and theoretical. In this study, we report on investigations of thiolipid monolayers on gold (covalent binding) and on mica (physisorption). To ensure that the monolayer properties were preserved 0 1995 American Chemical Society
LB Films of Sulfur-Bearing Lipids during LB transfer from the water to the solid surface, fluorescence microscopy was used both on the water/& interface and on the mica surface. Once the successful transfer of monolayers in all phase states (including the coexistence region) to the mica surface was established, SFM investigations, using contact, noncontact, and friction force modes, were performed. A special effort was made to correlate the images obtained using all three modes of the SFM with each other and with the fluorescence images. The same strategy was then applied to films on gold surfaces. Since it is not possible to obtain fluorescence images of films on gold, the structures on mica were used as a reference for the interpretation of the results for these monolayers. This approach allows analysis of the influence of the substrates on the morphology and structure of the monolayers.
Experimental Section Preparation of Mica and Gold Surfaces. Mica (Balzers, Liechtenstein) was cleaved directly before the LB deposition of thiolipids. SFM analysis of the mica sample revealed an atomically flat surface for areas extending over tens of microns. Atomic lattice resolution could be routinely obtained. Contact angle measurements with water demonstrated a hydrophilic surface (0 5 10"). Gold (Advent Research Materials, Halesworth, Suffolk, United Kingdom, purity 99.99%) was electron beam evaporated (Leybold ESV 2, Koln, Germany) in high vacuum (5 x mbar) onto a freshly cleaved mica substrate. The mica was heated to 300 "C prior to and during the evaporation in order to obtain flat terraces on the gold surface.14 The evaporation rate was held at 0.1 n d s using a quartz balance (Edwards FTM5, West Sussex, United Kingdom). Typically, we evaporated a 150 nm thick gold layer. After the evaporation, the sample was slowly cooled down to room temperature. The gold surface (see Figure 8) had a root-mean-square (rms) roughness of about 4 nm over an area of 1 p m x 1 pm. The smooth grains had a diameter of 100-300 nm with atomically flat terraces about 30 nm wide. Atomic lattice resolution could be routinely obtained on the terraces. The freshly prepared gold surface exhibited a water contact angle of about 50". Auger electron spectroscopy showed the presence of carbon on the surface. No traces of mica (K, Si, Al) were observed, indicating that the gold completely covered the mica substrate. Thiolipid Molecule. The synthesis and characterization of the amphiphilic thiolipid bis(8-( 1,2-dipalmitoyl-sn-glycero-3phosphoryl)-3,6-dioxaoctyl)disulfide are described el~ewhere.~ N-(7-Nitrobenz-2-oxa-1,3-diazol-4-yl)-l,2-dimyristoyl-sn-glycero-3-phosphoethanolamine(NBD-PE)was purchased from Molecular Probes (Eugene, OR, USA) and used as a dye dopant for the fluorescence microscopy. The monolayers were prepared and transferred on a commercial Langmuir trough (RK 11, Riegler & Kirstein, Mainz, Germany15),mounted on the threedirectional stage of a fluorescence microscope (Zeiss Axiotron, Ziirich, Switzerland). The thiolipid was dissolved in chloroform together with 1 mol % NBD-PE and spread on doubly deionized water (Millipore, Volketswil, Switzerland, R = 18 MM cm-I). The gold substrates and the mica sheets were dipped into the subphase before spreading the lipid solution on the water surface. The film was compressed at a speed of approximately 0.1 A2 molecule-' s-l, and the formation of the domains was continuously observed. During the LB transfer, the applied lateral pressure was kept constant via a feedback loop. The samples were drawn out of the subphase at a transfer speed of 5 pm s-'. The structure of the thiolipid films deposited on mica deteriorated with time, with a typical time scale of several days.
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Area I Molecule [As]
Figure 1. Pressure-molecular area isotherms of the thiolipid (compression rate 0.03 nm2/min; subphase water; temperature 22 "C).
They were analyzed 1 day to 2 weeks after deposition. The films deposited on gold turned out to be more stable than those deposited on mica (see Results) which allowed SFM study of samples more than 1 month old. SFM Microscope. Normal contact-mode measurements were performed with a commercially available SFM system (Park Scientific Instruments, Sunnyvale, CA) on the thiolipids deposited on gold. V-shaped cantilevers (Park Scientific Instruments) with a normal spring constant of 0.067 N/m and a pyramidal Si3N4 tip were used in this study. An optical detection system was used to measure the deflection of the cantilever. We modified one of our SFM heads to enable friction and noncontact (ac) mode measurements.'6 The modified system is equipped with a four-quadrant photo diode which enables simultaneous probing of friction and topography.17The friction signal can couple into topography due to nonperfect alignment of the lever and the photodiode. We estimate that the coupling factor is less than 5% for our system. The friction force values were estimated in the following manner: From a previously obtained force vs distance curve, we calibrate the sensitivity of the photodiode to vertical deflection. The sensitivity of the photodiode in measuring the torsion is assumed to increase with respect to the sensitivity to the vertical deflection by a factor of L J h (where L, and & are the length of the cantilever and the length of the tip, respectively). Finally, the torsion force constant of the cantilever is calculated from the system geometry.'* This gives us a direct relation between the detected voltage difference in the photodiode and the friction force. The modified SFM system is controlled by a digital feedback systemlg which allows greater flexibility in acquiring data. It was used for the thiolipids deposited on mica.
Results Characterization of the LB Film with Fluorescence Microscopy on the LB Trough and on Mica. The pressure vs aredmolecule isotherm of the thiolipid is depicted in Figure 1. The isotherm of the thiolipid shows a clear discontinuity in the slope, indicating a fist-order phase transition from a fluid to a solid-analogous state at approximately 25 mN m-'. The area per molecule in the fully compressed state of the film of the thiolipid is 90 A* which compares well with the area occupied by four extended hydrocarbon chains aligned along the normal to the water surface. The fluorescent micrograph of the thiolipid (Figure 2) shows star-shaped solid-analogous domains which contain no fluorescent dopant and which emerge at the onset of the coexistence plateau of the phase transition. The superstructures in which the domains arrange themselves are indicative of a repulsive interaction between the domains. Under normal circumstances, the domains are very regular in shape and size. Fluorescence
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Figure 2. Fluarcsccnce rmcrmcopy picture 01 the thioliptd monolayer an the water surface at a surface pressure at 30 mN/m (well above the phase transition) at 22 "C. The photograph shown an area of approximately 80 x 60 pm?. micrographs taken after transfer of the films onto mica cannot be distinguished from those of films on the water surface. At a temperature of 22 "C and at a lateral pressure of 29 mN m-' the diameter of the domains is typically between IO and 20 pm depending on small variations in the purity of the thiolipid. To obtain smaller domains. the films were first compressed to the phase transition pressure. Next, the films were slightly expanded to a pressure 2 mN m-I below the phase transition and were kept at this pressure for 5 min before the transfer was begun. SFM Characterization of Thiolipids on Mica. Optical microscopy has a lateral resolution of around 0.5 pm. while the SFM at our institute have a maximal scan width of about 100 pm. Therefore, the two techniques have an overlapping working range which enables comparison between the fluorescence images and the SFM images. Figure 3A shows a typical SFM image of an LB-transferred thiolipid film on mica. Despite the different scales of the images, the similarities between the structures observed by SFM and fluorescence microscopy are striking. There is a distinct height difference between the solid-analogous and fluid phases in this image. In addition, a characteristic internal structure of the stars is revealed. Figure 3B is a friction image of the same film, in which the internal structure of the domains can clearly be distinguished. The internal structure consists of separate subdomains or crystallites for each segment of the star, each with uniform friction. In addition there is some less regular fine structure especially in the center of the stars. Figure 4 shows a thiolipid film which has been compressed far beyond the phase transition. At this pressure, the stars come into direct contact with each other. Preferential fracture of the stars at the subdomain boundaries can clearly he seen in this image. Brittle fracture of individual subdomains can also be observed. The topographical image of Figure 3A shows a typical solidanalogous star with six subdomains or "leaves" having welldefined straight or slightly curved edges.20,2' The long straight edges of the leaves indicate a long-range order in the star. The leaves often have a cleaved, W-shaped point. This point cleavage is more accentuated in highly compressed monolayers. In some exceptional cases, a star with seven leaves or more is observed (see Figure 4): this may be caused by the cleavage of the leaf point reaching the center of the star and forming two separate subdomains. Figure 3B shows the friction image
Figure 3.
( A . top) SFM ~ m ~ @ i .a thioiipid film I.H-deposmd on mica m the lluid/solid-;in;ilop,,"s corxistenct repion. The h - f d d starshaped solid-analogous domains are surrounded by a h i d areii. The stars are not in contact with each other. Deposition parameters: pressure of the LB film dunng sample upstroke 28.5 mN/m. subphase temperature 26 "C. (At this temperature the phase transition occurs at approximately 28 mN/m.) The image was taken 1 day after deposition. Height difference black to white (b/w) 5.5 nm; scan speed 0.4 Hz. (B, bottom) Fnction image of thiolipids on mica taken simultaneously with image A (white to black 2.2 x N)
taken simultaneously with the topographical image 3A. From the variation of the friction signal between forward and backward scan, we estimate the friction to be about N. This friction is independent of the normal force if the load is lower than 5 x N.22 Low normal forces were used to prevent deformation of the sample. In general. we see a higher friction in the fluid area than in the solid-analogous area. Moreover, we see a difference in friction between the different leaves in the star: adjacent leaves of the same star usually have different friction values, while opposite leaves have almost identical friction values. The friction contrast is the same on scanning in the opposite direction (left to right instead of right to left). To understand the origin of the friction difference between the two phases and between different leaves, we compared the force vs distance curves obtained from a tip approach and retraction on fluid areas and on different leaves of the solid-analogous stars. The retraction part of such a curve reveals that the tip separates from the surface at a higher pull off force in the fluid phase (see Figure 5 ) . This indicates a different tip-surface interaction in the two phases. No difference in the force-distance curve was observed for different leaves on the same star. We therefore assume that the molecules in each of the leaves are identically ordered but
LB Films of Sulfur-Bearing Lipids
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