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Condensed Phases of Branched-Chain Phospholipid Monolayers Investigated by Scanning Force Microscopy S. Leporatti,* F. Bringezu, G. Brezesinski, and H. Mo¨hwald Max-Planck-Institute of Colloids and Interfaces, Rudower Chaussee 5, D-12489 Berlin, Germany Received July 8, 1998. In Final Form: October 9, 1998 Branched (triple- and quadruple-chain) phospholipid monolayers were characterized on different solid substrates (mica, silicon wafer, and glass) by scanning force microscopy. SFM in contact and tapping mode has revealed the presence of pinholes on submicrometer and nanometer scales for these lipids transferred by the Langmuir-Blodgett technique (vertical transfer) at room temperature. The transfer ratio was close to 1 in the case of triple-chain lipids and clearly lower in the case of the quadruple-chain lipid. Triple- and quadruple-chain phospholipids show different hole sizes and shapes in the monolayer depending on the deposition pressure and on the solid substrate. The tendency of decreasing their number with increasing surface pressure is maintained upon changing the substrate surface. At low pressure the quadruple-chain phospholipid has shown to have large holes while the triple-chain lipid has revealed condensed domains, which are probably induced by a surface-mediated condensation process, within the holes. In all cases the depth is only half that expected from molecular models.
Introduction Phospholipid monolayers provide suitable model systems for biological membranes. Therefore they have traditionally been used for studying the competitive interactions between polar hydrophilic headgroups and hydrophobic chains connected to the glycerol backbone.1-4 In the case of glycerophosphocholines (PC’s) substituted with 2-alkyl branched-chain fatty acids containing long branches, grazing incidence X-ray diffraction (GIXD) measurements have shown that the packing of the molecules in the monolayers is determined by the space requirements of the chains.5 However, for glycerophosphoethanolamines (PE’s) which are able to form a hydrogen-bonding network between adjacent headgroups there remain many open questions about the influence of lateral disturbances on the phase behavior in mono- and multilayers. Therefore triple-chain and quadruple-chain PE‘s and methylated analogues have been investigated in the present study. Because scanning force microscopy is a powerful tool for investigating a wide variety of materials with high spatial resolution, morphological information, such as topographic features of mono- and multilayer structures, height of structures and step sites, can be derived.6-9 The technique could also be used to locally measure friction and elastic compliance. There have been several SFM and * To whom correspondence should be addressed. (1) Mo¨hwald, H. Rep. Prog. Phys. 1993, 56, 653. (2) Menger, F. M.; Wood, M. G.; Zhou, Q. Z.; Hopkins, H. P.; Fumero, J. J. Am. Chem. Soc. 1988, 110, 1804. (3) Cevc, G. Chem. Phys. Lipids 1991, 57, 293. (4) Brezesinski, G.; Dietrich, A.; Struth, B.; Bo¨hm, C.; Bouwman, W. G.; Kjaer, K.; Mo¨hwald, H. Chem. Phys. Lipids 1995, 76, 145. (5) Brezesinski, G.; Dietrich, A.; Dobner, B.; Mo¨hwald H. Prog. Colloid Polym. Sci. 1995, 98, 255. (6) Binnig, G.; Roher, H.; Gerber, Ch. Phys. Rev. Lett. 1982, 49, 7. Binnig, G.; Quate, C.; Gerber, Ch. Phys. Rev. Lett. 1986, 56, 930. (7) Overney, R.; Meyer, E.; Frommer, J.; Brodbeck, D.; Lu¨thi, R.; Howald, L.; Gu¨ntherodt, H.-J.; Fujihira, M.; Takano, H.; Gotoh, Y. Nature 1992, 359, 133. Overney, R.; Meyer, E.; Frommer, J.; Gu¨ntherodt, H.J.; Fujihira, M.; Takano, H.; Gotoh, Y. Langmuir 1994, 10, 1281. (8) Leporatti, S.; Cavalleri, O.; Rolandi, R.; Tundo, P. Langmuir 1994, 10, 5. (9) Chi, L. F.; Anders, M. A.; Fuchs, H.; Johnston, R. R.; Ringsdorf, H. Science 1993, 259, 213.
AFM/LFM studies of self-assembled double-chain phospholipid monolayers,10 and also a large number of AFM images of double-chain phospholipid mono- or bilayer LB films have been published.11,12 However, until now no studies have been performed in which the hydrophobic part of the phospholipid molecule has been systematically varied (e.g. adding branched-chains) in order to understand the interplay between hydrophobic and hydrophilic interactions in the structure formation of a monolayer. Moreover, it was shown that there is a reorganization of fatty acid films after the transfer.13 This reorganization is faster for molecules having short hydrocarbon chains such as cadmium palmitate than it is for longer molecules.14 As for SPM imaging of hydrophobic groups, chemical force microscopy examination of hydrophilic and hydrophobic functional groups on Au-coated silicon substrates has been reported with the combined use of SFM and molecularly modified probe tips.15,16 Thiolate molecules or multilayers of amphiphilic molecules such as surfactants and lipids on solid substrates, prepared by either self-assembly or Langmuir-Blodgett (LB) methods, have been studied. In particular most of the work is focused on phase-separated systems: Overney et al.7 investigate domain structures of phase-separated mixed fluorocarbon films using lateral force microscopy (friction force microscopy), Leporatti et al. studied phase separation and microscopic textures of polymeric monolayers,8 and Chi et al. observed domain structures in stearic acid monolayers on a mica substrate9 with contact SFM. (10) Zasadzinski, J. A. N.; Helm, C. A.; Longo, M. L.; Weisenhorn, A. L.; Gould, S. A. C.; Hansma, P. K. Biophys. J. 1991, 59, 755. (11) Solletti, J. M.; Botreau, M.; Sommer, F.; Brunat, W. L.; Kasas, S.; Tran Minh Duc; Celio, M. R. Langmuir 1996, 12, 5379. (12) Mou, J.; Yang, J.; Shao, Z. Biochemistry 1994, 33, 9981. Czajkowsky, D. M.; Huang, C.; Shao, Z. Biochemistry 1995, 34, 12501. Hui, S. W.; Viswanathan, R.; Zasadzinski, J. A. N.; Israelachvili, J. Biophys. J. 1995, 68, 171. (13) Schwartz, D. K.; Garnaes, J.; Viswanathan, R.; Zasadzinski, J. A. N. Science 1992, 257, 508. (14) Zasadzinski, J. A. N.; Viswanathan, R.; Madsen, L.; Garnaes, J.; Schwartz, D. K. Science 1994, 263, 1726. (15) Frisbie, C. D.; Rozsnyai, L. F.; Noy, A.; Wrighton, M. S.; Lieber, C. M. Science 1994, 265, 2071. (16) Akari, S.; Schrepp, W.; Horn, D. Langmuir 1996, 12, 857.
10.1021/la980838o CCC: $15.00 © 1998 American Chemical Society Published on Web 11/25/1998
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It has been pointed out that the observation and characterization of microstructures of organic thin films, such as mono- and multilayers of phospholipids on solid substrates, are of particular importance for further practical applications of organic films as matrixes for functional organic or biomolecules in biosensors and biochemical probes. The final aim of the present research is to characterize condensed phases of branched-chain phospholipids after the transfer on solid support with combined SFM and LFM and to compare these results with the behavior at the air/water interface.17 A thorough understanding of the physical properties of LB films with increased hydrophobic tails is thus of great importance. We report results on SFM studies of phospholipids with different tail and headgroup structures after transfer onto different substrates by the Langmuir-Blodgett technique. Materials and Methods Langmuir-Blodgett Films. The triple-chain phospholipids 1-(2-hexadecylstearoyl)-2-hexadecylglycerophosphoethanolamine, 1(2C16-18:0)-2H-PE (I), and 1-(2-hexadecylstearoyl)-2hexadecylglycerophospho-N-methylethanolamine, 1(2C16-18:0)2H-NMe (II), as well as the quadruple-chain phospholipid 1,2di(2-hexadecylstearoyl)glycerophosphoethanolamine, 1,2-Di(2C1618:0)-PE (III), were synthesized according to ref 18. The corresponding chemical structures are presented in Figure 1a, c, and d. The lipids were purified by column chromatography. Highperformance liquid chromatography (HPLC) and electron spectroscopy have been used for analytical characterization. All lipids were spread from an approximately 1 mM p.a. grade chloroform (Merck, FRG) solution. The preparation of the LangmuirBlodgett films was performed using a circular trough with a subphase temperature of 20 °C. The subphase was ultrapure water (pH 5.5) with a specific resistance above 18 MΩ cm purified using a Millipore desktop unit. The pressure/area isotherms were recorded using a Wilhelmy quartz plate. Phospholipid solutions were spread on water using a microsyringe. After spreading, the monolayer was compressed to a desired surface pressure (varying between 3 and 40 mN/m) which was maintained constant within an accuracy of 0.1 mN/m. The compression speed was 2 Å2/(molecule‚min). The monolayers were transferred to solid substrates using the Langmuir-Blodgett technique with a dipping rate of approximately 4 mm/min. Mica substrates (Plano GmbH Marburg) were freshly cleaved with tape before dipping, while optical microscope cover glasses and Silicon wafers (SiO2, Wacker Chemitronic) with thermally grown oxide layers (400600 Å) were cleaned and prepared with a RCA cleaning procedure (SC-1).19 The substrates were ultrasonicated in 2-propanol and then placed into a quartz beaker filled with a solution of 5 volume parts ultrapure water, 1 volume part hydrogen peroxide (30% w/w nonstabilized, med. puriss, Merck), and 1 volume part hydrogen ammonium hydroxide (30% w/w, ACS reagent, Aldrich) at room temperature. This solution was heated to 80 °C and kept for 10 min at this temperature, after which the substrates were thoroughly rinsed with pure water. Then they were stored under water and used within 1 h. Grazing Incidence X-ray Diffraction (GIXD). X-ray experiments were performed using the liquid-surface diffractometer on the undulator beamline BW1 at HASYLAB, DESY, Hamburg, Germany. A monochromated synchrotron beam strikes the air/ water interface at a grazing incidence. The diffracted radiation is detected by a vertical linear position-sensitive detector (PSD) (OED-100-M, Braun, Garching, Germany) mounted behind a Soller collimator. As the monolayers are 2D powders consisting of 2D crystallites randomly oriented in the plane, Bragg rod scattering can be found merely by mapping the (Qxy, Qz) space. The lattice spacings dhk are related to the horizontal component (17) Mo¨hwald, H.; Dietrich, A.; Bo¨hm, C.; Brezesinski, G.; Thoma, M. Mol. Membr. Biol. 1995, 12, 29. (18) Paltauf, F.; Hermetter, A. Prog. Lipid Res. 1994, 33, 239. (19) Kern, W. J. Electrochem. Soc. 1990, 137, 1887.
Leporatti et al. Qxy of the scattering vector at maximum intensity. The Bragg rods in addition yield the tilt angle t and the tilt azimuth.20,21 Brewster Angle Microscopy. The morphology of the monolayer was visualized with a Brewster angle microscope from NFT, Go¨ttingen (BAM1).22,23 The images were digitized using a frame grabber and treated with image-processing software to correct the distortion resulting from the observation at the Brewster angle. Brewster angle microscopy is sensitive to both differences in the layer thickness and different orientations of the aliphatic chains with respect to the plane of incidence. In the first case only the intensity of the reflected light is different; in the second case also the polarization of the incident p-polarized light is changed. The resolution of the BAM1 is about 4 µm. The parts of the images shown here are 500 µm × 500 µm in size. Transfer Ratio. The difference ∆A between the total area of the lipid monolayer at the air /water interface before and after transfer was recorded. The ratio between ∆A and the area of the substrate (A) will be referred to as the “transfer ratio” and denoted R (R ) ∆A/A). A value of 1 reflects that the monolayer is properly transferred on the substrate and that no desorption of molecules occurs during the transfer. Scanning Force Microscopy. SFM images were obtained in air at room temperature (20-25 °C) using a Nanoscope III Multimode scanning force microscope (Digital Instruments, Inc., Santa Barbara, CA). This equipment enables the performance of contact AFM, lateral force microscopy (friction), and tapping mode measurements. Large size (120 µm, type J) and small size piezoscanner (12 µm, type E) scan heads were used. Samples of mica, glass, and silicon were cut to fit into the Nanoscope holder. Tips microlithographed on silicon nitride (Si3N4) cantilevers with a force constant of 0.58 N/m (Digital Instrument) were used for contact SFM, and silicon tips (Nanotips, DI) with a resonance frequency of ∼300 kHz and a spring constant of ∼40 N/m were used for tapping mode SFM. The contact force between the tip and the sample was kept as low as possible (∼1 nN) and images were acquired in constant-force mode (height mode) at a scan rate of 0.5-1 Hz. The sensitivity of the SFM detector was estimated using the slope of the loading curve. Tip checking and XY scanning calibration were routinely performed by imaging a diffraction grating. Z calibration was achieved in the micrometer range by measuring the diameter of Sephadex beads and in the nanometer range by measuring monomolecular mica steps. Scanning force microscopy (SFM) images were analyzed by using Nanoscope software and Image PC Software (Version beta 2, Scion Corporation) after a slight flattening (order 2, 3). The hole depth is determined using Bearing analysis of Nanoscope Software (Digital Instruments, Santa Barbara, CA) while the percentage of dark areas of holes is calculated using Image PC from statistics, obtained selecting three different thresholds, for each SFM picture (as shown in Figure 4a) and dividing the dark area calculated automatically (in pixel squared) by the whole image area (512 pixel)2. All the images presented are obtained in contact mode with a reduced force, as describe above. Tapping mode images are also repeated for several samples, and they always reveal similar features with regard to area and depth of holes. Therefore we used both types of images for calculating the values of Tables 1 and 2.
Results and Discussion Figure 1d shows the surface pressure-area isotherm of the 1(2C16-18:0)-2H-PE (I) monolayer at the air/water interface at 20 °C. A plateau region, usually attributed to a first-order phase transition, can be seen at about 9 mN/ m. The apparent transition enthalpy is very small (16.7 kJ/mol calculated using a modified Clausius-Clapeyron equation) and points to a transition between two condensed states. Grazing incidence X-ray diffraction (GIXD)24 (20) Als-Nielsen, J.; Jaquemain, D.; Kjaer, K.; Lahav, M.; Leveiller, F.; Leiserowitz, L. Phys. Rep. 1994, 246, 251. (21) Kjaer, K. Physica B 1994, 198, 100. (22) Ho¨nig, D.; Mo¨bius, D. J. Phys. Chem. 1991, 95, 4590. (23) Henon, S.; Meunier, J. Rev. Sci. Instrum. 1991, 62, 936. (24) Brezesinski, G.; Bringezu, F.; Weidemann, G.; Howes, P. B.; Kjaer, K.; Mo¨hwald, H. Thin Solid Films, in press.
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Figure 1. (a) Isotherm and chemical structure of compound II at 15 °C. The molecular areas (squares) calculated from GIXD data are additionally shown. (b) Contour plots of the corrected X-ray intensities as a function of the in-plane and out-of-plane scattering vector components of compound II at different lateral pressures (indicated) and 15 °C. (c) Isotherm and chemical structure of compound III at 20 °C. (d) Isotherm and chemical structure of compound I at 15 °C. BAM images (500 × 500 µm2) below and above the plateau region of the isotherm are additionally shown. The presence of holes can be seen in the monolayer on water at pressures below this plateau region.
measurements have shown that the monolayer exhibits a phase transition from a rectangular phase with NN
(nearest neighbor) tilted chains to a hexagonal arrangement of untilted chains. The insets in Figure 1d are
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Figure 2. Scanning force microscopy images of branched-chain phospholipid monolayers transferred on mica substrate below (a) and above (b) the transition region for compounds I (top) and III (bottom). The scale is indicated by the bar. Scan parameters: (a, top) scan rate 1 Hz, ζ range 8 nm; (b, top) scan rate 1 Hz, ζ range 5 nm; (a, bottom and b, bottom) scan rate 0.813 Hz, ζ range 10 nm.
Brewster angle microscopy (BAM) images. Above 13 mN/m (phase transition pressure) the monolayer is homogeneous even with an analyzer which is not parallel to the polarizer. The remaining inhomogeneities result from the intensity profile of the laser. Below 13 mN/m inhomogeneities result from anisotropy contrast, since they vanish with parallel polarizers. Below 10 mN/m additional inhomogeneities are observed even with parallel polarizers, indicating that holes are present in the monolayer.24 Figure 1a shows the pressure-area isotherm of compound II at 15 °C. A transition between two condensed phases can be detected as a change of slope. GIXD measurements were also performed at 15 °C. Figure 1b presents two contour plots of the corrected X-ray intensities versus the in-plane and out-of-plane scattering vector components Qxy and Qz at lateral pressures below and
above the kink in the isotherm. At 4 mN/m two diffraction peaks can be seen. One peak has its maximum at zero Qz and and the other one at higher Qz values, indicating a NN tilted rectangular phase. At 25 mN/m only one diffraction peak at zero Qz is detected, indicating a hexagonal packing of upright oriented chains. The phase transition occurs between 14 and 18 mN/m. Since the transition pressure has a slightly negative temperature dependence, the transition at 20 °C (the temperature for the transfer) is expected at lower lateral pressure. Figure 1a additionally shows the molecular areas calculated from the GIXD data (squares) at different surface pressures. These values are very similar to those measured by the pressure-area isotherm, indicating that the monolayer does not contain many defects. In general, the tilt angles at lower lateral pressures and the pressure of the
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Figure 3. Scanning force microscopy images of branched-chain phospholipid monolayers transferred on silicon substrate below (a) and above (b) the transition region for compounds I (top) and II (bottom). The scale is indicated by the bar. Scan parameters: (a, top) scan rate 1 Hz, ζ range 10 nm; b, top) scan rate 1 Hz, ζ range 4.5 nm; (a, bottom) scan rate 1 Hz, zeta range 16 nm; (b, bottom) scan rate 1 Hz, ζ range 5 nm.
transition to a hexagonal packing of upright oriented chains increase with increasing degree of methylation.24 This shows that, despite the large hydrophobic area of the triple-chain molecules, interactions of the hydrophilic headgroups must be taken into account. Figure 1c shows the isotherm of compound III at 20 °C together with the chemical structure of the quadruplechain phospholipid. From the temperature behavior of the isotherms a critical temperature Tc (above which the monolayer cannot be compressed into a condensed state) of 52 °C and a T0 value (above which a liquid-expanded phase LE appears at large molecular areas) of 28 °C were determined. At 15 °C GIXD measurements show that the tilting transition occurs between 20 and 27 mN/m. Defects in the monolayers of branched-chain phospholipids can be seen both at the air/water interface and after transfer onto different substrates. To directly compare SFM images, branched-chain lipids are transferred simultaneously under the same conditions (temperature
and pressure) at the same time onto two and in some cases onto three different substrates (mica, silicon, and glass). For each sample several images from different places are taken. Tips and cantilever are changed and tested before each experiment. At 5 mN/m the monolayer of compound I on mica shows round-shaped defects in a homogeneous condensed phase matrix (Figure 2a, top). These defects are partially, sometimes almost totally, covered with islands of condensed material which have the same height as the surrounding film.25 Obviously in this case there is a surface-mediated condensation which has also been observed for LB films transferred at very low speed.26 The monolayer (in the intact region) was pierced until the mica beneath the monolayer could be seen in order to demon(25) Leporatti, S.; Akari, S.; Bringezu, F.; Brezesinski, G.; Mo¨hwald, H. Appl. Phys. A 1998, 66, S 1245. (26) Spratte, K.; Riegler, H. Makromol. Chem., Macromol. Symp. 1991, 46, 113. Spratte, K.; Riegler, H. Langmuir 1994, 10, 3161.
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Figure 4. (a) Example of evaluation of height difference from a histogram of depth, with respect to the highest level plotted against hist %, the percentage of the film at a specified depth (right). The area considered (left) is the whole image (10 × 10 µm2). The difference between the two maxima gives the height difference. (b) Thresholding of an image for calculating the percentage of area covered (Image PC Beta 2 Scion Corporation).
strate that within the holes the substrate is imaged. Molecular resolution of the mica inside the artificially
produced holes can be obtained by increasing the operating force of the scanning force microscope over a smaller area.27
Condensed Phases of Phospholipid Monolayers Table 1. Percentage of Area Covered by Defects on a Mica Substrate (vertical Langmuir-Blodgett Transfer) Calculated from SFM Images and the Transfer Ratios for Compounds I, II, and III at the Surface Pressures Indicated mica
dark area of holes (%)
π ) 5 mN/m π ) 10 mN/m π ) 15 mN/m π ) 20 mN/m π ) 30 mN/m π ) 5 mN/m π ) 10 mN/m π ) 20 mN/m π ) 25 mN/m π ) 30 mN/m π ) 5 mN/m π ) 15 mN/m π ) 20 mN/m π ) 30 mN/m
1(2C16-18:0)-2H-PE (I) 13 ( 1 5(1 2(1 3(1
transfer ratio 0.95 ( 004 0.98 ( 0.04 1.07 ( 0.04 1.07 ( 0.04 0.94 ( 0.04
1(2C16-18:0)-2H-NMe (II) 12 ( 4 13 ( 5 4(1 3(1 4(1
0.95 ( 0.04 0.88 ( 0.04 1.04 ( 0.04 0.94 ( 0.04 0.98 ( 0.04
1,2-Di(2C16-18:0)-PE (III) 11 ( 2
0.76 ( 0.04
6(2 4(2
0.81 ( 0.04 0.90 ( 0.04
Table 2. Depth Difference between Holes and the Surrounding Monolayer for Different Substrates (Mica and Silicon) and Two Different Transfer Methods (the Vertical Langmuir-Blodgett Technique and the Horizontal Scooping-Up Technique25,29) at the Different Surface Pressures Indicated mica (vertical) π ) 5 mN/m π ) 10 mN/m π ) 15 mN/m π ) 20 mN/m π ) 30 mN/m π ) 5 mN/m π ) 10 mN/m π ) 20 mN/m π ) 25 mN/m π ) 30 mN/m π ) 40 mN/m
mica (horizontal)
1(2C16-18:0)-2H-PE (I) 0.80 ( 0.03 nm 0.92 ( 0.04 nm 1.00 ( 0.06 nm 1.22 ( 0.07 nm 1.2 ( 0.3 nm
silicon (vertical) 1.01 ( 0.05 nm 0.98 ( 0.07 nm
1(2C16-18:0)-2H-NMe (II) 1.01 ( 0.02 nm 0.93 ( 0.07 nm 0.83 ( 0.03 nm 0.77 ( 0.04 nm 1.0 ( 0.1 nm 1.1 ( 0.1 nm 1.1 ( 0.4 nm 0.8 ( 0.1 nm 0.80 ( 0.05 nm
1,2-Di(2C16-18:0)-PE (III) π ) 5 mN/m 1.1 ( 0.1 nm 1.2 ( 0.2 nm π ) 10 mN/m π ) 20 mN/m 1.2 ( 0.1 nm 0.9 ( 0.2 nm 0.81 ( 005 nm π ) 30 mN/m 1.0 ( 0.1 nm 0.90 ( 0.05 nm
The high softness and compressibility of this type of phospholipids allow an easy piercing of the monolayer during contact mode experiments. The monolayer of I transferred onto mica at 20 mN/m also exhibits pinholes, but the size and number of the defects are decreased. A small number of large (0.6-0.8 µm), roughly circular holes and a larger number of small (0.05-0.2 µm) holes are revealed (Figure 2b, top). Comparing with the image taken at 5 mN/m, the large defects of about 2-5 µm diameter have disappeared completely. The holes often contain either dust particles, probably due to exposition of the sample to air during the measurements, or lipid aggregates. At 5 mN/m, compound III on mica exhibits a heterogeneous structure (Figure 2a, bottom) formed by holes of different shapes whose size is up to 3 µm in diameter. The area covered by holes seems to be comparable to that of compound I. After compression and on transferring the (27) Hansma, H. G.; Gould, S. A. C.; Hansma, P. K.; Gaub, H. E.; Longo M.; Zasadzinski, J. A. Langmuir 1991, 7, 1051.
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sample at the increased lateral pressure of 20 mN/m, the area covered by holes is decreased. The hole diameter is reduced, and the shape is slightly changed. Again particles of dust or lipid aggregates are often revealed inside the holes. As mentioned above, the monolayer of compound (I) also shows a large number of holes at the air/water interface (Figure 1d). Higher resolution SFM on solid substrates revealed the presence of smaller defects not detectable by optical techniques (BAM resolution is in the range of 4 µm). On the other hand, there was no indication of defects in the monolayer of III on water. We have to assume that the defects observed on mica are generated due to a reorganization of the molecules during transfer. The space requirements of the large hydrophobic area could lead to a mismatch between the interactions of the headgroups with the substrate and the interactions between the chains. The results are reproducible, and the repetition of the measurements in different areas always leads to similar SFM pictures. A more precise examination of the condensed phase has revealed that the submicrometer defects are characteristics of these branched-chain lipids and are also present inside of LC domains when the transfer is performed in a phase-coexistence region (LE-LC) at higher temperature (30-35 °C).28 To demonstrate that the holes observed in transferred multiple-chain lipids are not only caused by the use of a specific substrate (mica), we also performed the transfer on other supports (silicon and glass). In this paper we present only the results obtained on silicon and mica. In Figure 3 the images of compounds I and II on silicon below (a) and above (b) the transition region are presented. The monolayer of compound I on silicon shows a large number of defects which cover a great part of the monolayer. Their shape is elongated and irregular. Their density and number are increased, but the size is reduced with respect to that of compound II. In a previous paper25 we reported results obtained for compounds I and II using two different types of transfer (horizontal and vertical) onto mica substrates. Comparing I and II at low transfer pressure, we found that II exhibits a smaller number of defects with a more elongated shape. This elongated shape is not due to the type of transfer. It could be due to the space requirement of the hydrophobic part, which leads to instabilities and defects in the condensed phase of branched-chain lipids during the transfer when the interaction of the headgroup with the underlying interface (water, mica, and silicon) is changed. For the monolayer of I the transfer onto silicon produces irregular defects without condensed material inside in contrast to the transfer onto mica. Upon compression the shape remains irregular, the number of holes is strongly reduced, and the density is smaller. A comparison of the transfer ratio and the percentage of area covered by gaps and defects can help to explain the appearance of such types of morphology. The transfer ratio is calculated for vertical transfer onto mica. The proportion of the surface covered by holes (%) is estimated by applying a threshold before processing them (see Figure 4b). The error of the measurements was deduced from the precision of the threshold and from the statistical analysis over several images and samples. The results are presented in Table 1. The transfer ratio of the quadruple-chain PE is significantly smaller compared to those of triple-chain (28) Leporatti, S.; Brezesinski, G.; Mo¨hwald, H. In preparation. (29) Kato, T.; Matsumoto, N.; Kawano, N.; Suzuki, T.; Iriyama, K. Thin Solid Films 1994, 242, 223.
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lipids if the transfer is performed in a monolayer phase with tilted chains. The transfer from the untilted state leads to comparable transfer ratios for all lipids investigated. One can clearly observe that the area covered by holes is much larger if the transfer starts in a tilted phase whereas the transfer from an untilted state leads to a drastic reduction of the defects in the transferred monolayer. Assuming that holes are due to the incomplete transfer does not explain the large dark area fractions measured for transfer at low pressure (10-15%). Presumably surface-mediated condensation leads to a lateral shrinking of the bright areas. For example, transfer at the molecular area near 70 Å2 and shrinking to a molecular area near 60 Å2, as expected for a triple-chain lipid, would cause a dark area fraction of 15%. Hence this is probably the most important process responsible for holes. Figure 4 shows an example of how to calculate the height difference between the covered part of the silicon and the defects in the monolayer of I performed on a 10 × 10 µm2 area (Figure 4a, left). The depth difference between the holes and the surrounding monolayer is calculated from a bearing analysis with the Nanoscope software. The hist %, a measure of the percentage of the film specifically at the given depth, is plotted against the depth, which is relative to an arbitrary zero point chosen above the monolayer (Figure 4a, right). By fitting two Gaussian curves to this plot, the step height difference between the two parts can be calculated from the difference of the two maxima. The values, taken from several images of various magnification comprising equal portions of holes and the background, are presented in Table 2. It is not clear why the heights, measured both in contact and tapping modes, differ so much from the theoretical value (2.8 nm) determined from the molecule length: this difference cannot be explained only by a tilt of the molecules, since this results in a tilt angle of 70°, which was not observed on water even at low pressure. Thus, another factor must be additionally taken into account. This could be the result of a quite large deformation of the monolayer during the measurement (caused by the tip pressure on the soft lipid surface). Preliminary tests of the layer thickness measured with different tip forces
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indicate a decreasing height with increasing force, but more systematic measurements are necessary to obtain sufficient statistics. This method was successfully applied to measure the thickness of bilayers in solution; however, it is not directly applicable to monolayers in air. The contact area is rather critical because increasing the set point (i.e. increasing force) can deform the surface, leading to an increased contact area and consequently to uncertainties in the real force applied. The film height is quite reproducible for most SFM pictures, and we think it is characteristic of the lipids studied and does not depend on the substrate used. Conclusion This paper demonstrated the existence of holes on a micrometer scale in branched-chain phospholipid monolayers on different solid supports. The analysis shows that monolayers transferred from a tilted state exhibit many more defects on the substrate compared to monolayers transferred from an untilted state. In the case of the quadruple-chain lipid, the small transfer ratio at low pressure clearly corresponds to the large area of holes on the substrate. Submicrometer defects are also detected in both contact and tapping modes and are characteristic for this type of lipids. This indicates a possible reorganization of the films during/after transfer onto a solid substrate. For triple- and quadruple-chain lipids, where the area requirement is determined by the aliphatic tails, a larger (compared to that observed for a double-chain lipid25) fraction of holes is observed, probably due to the mismatch between the interactions of the headgroups with the substrate and the interactions between the chains. For all lipids investigated the fraction of holes can be reduced by film compression. The height difference between lipidcovered areas and defects obtained from image analysis is generally less than expected from molecular models. Acknowledgment. Financial assistance from the Deutsche Forschungsgemeinschaft (DFG) and the EC through the Human Capital and Mobility program is gratefully acknowledged. We thank Dr. C. DeWolf for critical review of the manuscript. LA980838O
