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Elaboration and Characterization of Phospholipid Langmuir-Blodgett Films J. M. Solletti,† M. Botreau,‡,§ F. Sommer,*,§ W. L. Brunat,§ S. Kasas,| Tran Minh Duc,‡ and M. R. Celio† Institute of Histology, University of Fribourg, Pe´ rolles, CH-1700 Fribourg, Switzerland, CENATS, University Claude Bernard, 43 Bd. Du 11 Novembre 1918, F-69622 Villeurbanne Cedex, France, BIOPHY RESEARCH, Novacite´ alpha, 43 Bd. Du 11 Novembre 1918, BP 2131, F-69603 Villeurbanne, France, and Tokyo Institute of Technology, Faculty of Biosciences, 4259 Nagatsuta, Midoriku, Yokohama 227, Japan Received July 14, 1995. In Final Form: June 6, 1996X In order to model biological membranes, DPPE (1,2-dihexadecanoyl-sn-glycero-3-phosphoethanolamine) and DPPC (1,2-dihexadecanoyl-sn-glycero-3-phosphocholine) Langmuir-Blodgett (LB) films were deposited on hydrophilic mica and hydrophobic highly ordered pyrolitic graphite (HOPG), and subsequently characterized by atomic force microscopy (AFM) and X-ray photoelectron spectroscopy (XPS). AFM was used to determine the topography and to monitor the stability of the deposited films in air and under water at varying experimental parameters. Angle-resolved XPS was used to measure the mean thickness and the orientation of the deposit. Monolayer transfer pressure was determined at 40 mN/m with molecular area at 0.42 ( 0.02 nm2 for DPPE and 0.55 ( 0.02 nm2 for DPPC. Nanometric resolution was achieved on DPPE crystal and LB films and allowed the determination of the corresponding lattice constants a ) 0.64 ( 0.03 nm, b ) 0.91 ( 0.07 nm, and c ) 5.6 ( 0.2 nm and a ) 0.68 ( 0.02 nm, b ) 0.93 ( 0.05 nm, and c ) 5.6 ( 0.2 nm, respectively. The DPPE monolayer on mica was measured to be 2.7 ( 0.2 nm thick and the surface was hydrophobic. The reorganization of DPPE bilayer when going from the water to the air was directly observed to form monolayer and trilayer domains. On HOPG, the outermost layer of DPPE was partly hydrophilic and partly hydrophobic. Under the same conditions of deposition, DPPC formed a mixed structure of flat domains and vesicles. In water, monolayer aliphatic tail ending is less compact and presents a higher friction coefficient than polar heads as measured by lateral force AFM.
Introduction Transmembraneous proteins play a crucial role in the operation of living cells. Despite their importance, these proteins and their interactions with the cell membrane are still poorly understood. The classical way to study biological membranes and transmembraneous protein structures or functions consists in the isolation of the cell membrane.1 However, this technique requires considerable care to maintain structure and biochemical activity. This is why more and more researchers simulate cell membranes by using phospholipids ordered as LangmuirBlodgett (LB) films or liposomes.2,3 Most of the LB films characteristics (topography, stacking structure, etc.) can be studied using a recent technique: atomic force microscopy (AFM). This microscope has shown its ability to give information on enzymes,4 poly(amino acids),5 fatty acids,6 phospholipid LB films,7-9 or liposomes.10 Until now, only very few AFM images on phospholipid monolayer or bilayer LB films have been published. * To whom correspondence should be addressed. † Institute of Histology, University of Fribourg. ‡ CENATS, University Claude Bernard. § BIOPHY RESEARCH. | Tokyo Institute of Technology. X Abstract published in Advance ACS Abstracts, September 1, 1996. (1) Shannon Moore, M.; Mahaffey, D. T.; Brodsky, F. M.; Anderson, R. G. W. Science 1987, 236, 558. (2) Chapman, D. Langmuir 1993, 9, 39. (3) Ahlers, M.; Mu¨ller, W.; Reichert, A.; Ringsdorf, H.; Venzmer, J. Angew. Chem., Int. Ed. Engl. 1990, 29, 1269. (4) Alexandre, S.; Dubreuil, N.; Fiol, C.; Malandain, J.-J.; Sommer, F.; Valleton, J.-M. Microsc. Microanal. Microstruct. 1994, 5, 359. (5) Auduc, N.; Ringenbach, A.; Stevenson, I.; Jugnet, Y.; Tran Minh Duc Langmuir 1993, 9, 3567. (6) Zasadinski, J. A. N.; Viswanathan, R.; Madsen, L.; Garnaes, J.; Schwartz, D. K. Science 1994, 263, 1726. (7) Singh, S.; Keller, D. J. Biophys. J. 1991, 60, 1401. (8) Zasadinski, J. A. N.; Helm, C. A.; Longo, M. L.; Weisenhorn, A. L.; Gould, S. A. C.; Hansma, P. K. Biophys. J. 1991, 59, 755.
S0743-7463(95)00579-8 CCC: $12.00
Molecular resolution on DMPE bilayer deposited on mica and observed in liquid medium has been achieved8 as also on DMPG monolayer deposited on a alkylated mica.9 Moreover, it is known that there is reorganization of fatty acid films after transfer in air.11 This reorganization is faster for molecules having short hydrocarbon chains such as cadmium palmitate than for longer molecules.6 The final aim of the present research is to characterize transmembraneous proteins incorporated in a phospholipid LB bilayer. A thorough understanding of the physical properties of the supporting LB film is thus of great importance for the study of reconstituted proteins by AFM. The protein we are interested in is the Ca-ATPase present in the plasma membrane of human erythrocytes. The human erythrocyte membrane is composed of DPPE (1,2dihexadecanoyl-sn-glycero-3-phosphoethanolamine) and DOPE (1,2-Di[(cis)-9-octadecanoyl]-sn-glycero-3-phosphoethanolamine) as 12.9% and 18.1% of total phosphatidylethanolamine by weight, respectively, and DPPC (1,2dihexadecanoyl-sn-glycero-3-phosphocholine) as 31.2% of total phosphatidylcholine by weight.12 In this paper therefore, we present a XPS (X-ray photoelectron spectroscopy) and AFM study of DPPE and DPPC L-B films. Results involving mixtures of non-miscible-saturated (DPPE and DPPC) and unsaturated (DOPE) phospholipid LB films are presented elsewhere.13 XPS provides us with complementary information to AFM on the elemental composition, chemical bonding, thickness, and orientation (angle resolved XPS) of LB films.14-17 AFM allows determination of the stability, morphology, and structure (9) Egger, M.; Ohnesorge, F.; Weisenhorn, A. L.; Heyn, S. P.; Drake, B.; Prater, C. B.; Gould, S. A. C.; Hansma, P. K.; Gaub, H. E. J. Struct. Biol. 1990, 103, 89. (10) Kasas, S.; Hofmann, F.; Celio, M. R.; Carafoli, E. Scanning 1992, 14, 276. (11) Schwartz, D. K.; Garnaes, J.; Viswanathan, R.; Zasadzinski, J. A. N. Science 1992, 257, 508. (12) Dodge, J. T.; Phillips, G. B. J. Lipid Res. 1967, 8, 667. (13) Solletti, J.-M.; Botreau, M.; Sommer, F.; Brunat, W. L.; Minh Duc, T.; Celio, M. R. J. Vac. Sci. Technol. B 1996, 14-2, 1492.
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of the monolayer and bilayer LB films. In this way the crystallographic unit cell of both crystal and LB film and the thickness and the size of the topographic molecular domains were measured. Experimental Section Langmuir-Blodgett Films. DPPE and DPPC (99% pure) were purchased from Sigma Chemical Co. and used without further purification. Just before the deposition, DPPE was dissolved in chloroform or in a chloroform/methanol solution (9/1 in volume) and DPPC was dissolved in chloroform. The typical concentration used was 1 mg/mL. LB films were transferred onto freshly cleaved mica and graphite HOPG (highly ordered pyrolytic graphite). For AFM study under liquid, mica was glued on a larger piece of PTFE (Teflon) in order to keep water on the mica during transfer to the AFM. The preparation of the Langmuir-Blodgett films was performed using a KSV 5000 thermoregulated Langmuir-Blodgett trough with subphase temperature at 23 ( 1 °C. The subphase was high-purity water (Milli Q, electrical resistivity > 18 MΩ cm) at pH 6.8. The interfacial pressure was measured with a Wilhelmy balance using a platinum plate. Phospholipid solution was spread on water, drop by drop, with a microsyringe fixed on a support. The compression barrier speed was 5 mN/m/min and the speed of dipping was 1 mm/min for all the experiments. Hydrophilic substrates (mica) were lifted (for a monolayer) and, for a bilayer, dipped. Hydrophobic substrates (HOPG) were dipped (for a monolayer) and lifted for a bilayer. The orientation of the substrate was either vertical or horizontal depending on which medium (air or liquid) the film will be imaged. In order to evaluate the transfer ratio during horizontal deposition, we recorded the barrier position before and after deposition. To investigate the change in the morphology of the LB films, films were transferred onto mica and HOPG at different surface pressures for DPPE and for DPPC and subsequently observed with AFM and XPS. X-ray Photoelectron Spectroscopy. XPS has been successfully applied to determine the quantitative chemical composition, chemical bonding, thickness, and molecular orientation of LB films.13,15,16 As the only difference between DPPE and DPPC is the hydrophilic head (ethanolamine and choline respectively), these two phospholipids cannot be resolved by XPS since their chemical functions are similar. This is why we will only discuss the DPPE results. The sampling depth of XPS is the distance, normal to the surface, from which a specified percentage of the signal is detected. For XPS, this percentage varies exponentially with distance from the surface, then 95% of the detected signal originates from a depth of 3λ cos θ, where λ is the photoelectron mean free path in the material and θ is the detection angle with respect to normal to the surface, while 63% of the signal arises from λ cos θ. Moreover, by using angle-resolved XPS, this sampling depth can be varied proportionally to cos θ. At normal detection angle (θ ) 0°), this sampling depth is 3λ. Since the thickness of a DPPE monolayer is about 2.8 nm, and because the mean free paths are in the 2-4 nm range, it is possible to investigate the entire film plus the substrate. At the grazing detection angle (75°), the sampling depth is more shallow, 0.78λ, and only the end groups pointing outward from the LB film surface are sampled. Thus valuable information on the molecular orientation of LB films can be obtained from angle-resolved XPS experiments. XPS experiments were performed using the imaging photoelectron spectrometer ESCASCOPE from Fisons Instruments.18 Spectra were obtained using a non-monochromatic Al KR source operated at 200 W and the area analyzed was 1200 µm in diameter (fwmh). The X-ray spot size was always larger than the angledependent area analyzed in order to ensure that the whole area (14) Atanasoska, L.; Cammarata, V.; Stallman, B. J.; Kwan, W. S. V.; Miller, L. L. Surf. Interface Anal. 1992, 18, 163. (15) Heens, B.; Gre´goire, Ch.; Pireaux, J. J.; Corrnelio, P. A.; Gardella, J. A. J. Appl. Surf. Sci. 1991, 47, 163. (16) Linton, R.; Guarisco, V.; Lee, J.; Hagenhoff, B.; Benninghoven, A. Thin Solid Films 1992, 210-211, 565. (17) Nakayama, Y.; Takahagi, T.; Soeda, F.; Ishitani, A.; Shimomura, M.; Kunitake, T. J. Colloid Interface Sci. 1988, 122, 464. (18) Coxon, P.; Krizek, J.; Humpherson, M.; Waerdell, I. R. M. J. Electron Spectrosc. Relat. Phenom. 1990, 52, 821.
Solletti et al. analyzed was flooded by X-rays. Angle profiles were obtained by sample rotation, the photon source and collection lens being held fixed during the experiments. No charge compensation has been used, and binding energies were referenced to aliphatic C-C bonds at 285 eV. Atomic Force Microscopy. The general principle of the technique is to scan the sample surface with a sharp probing tip, fixed at the end of a cantilever, and, using an optical method, to measure the deflection of the cantilever due to the forces between it and the sample surface during the scan. The displacement in three dimensions is provided by a highly sensitive piezoelectric scanner. We used three operating modes: the constant force, the friction, and the tapping modes. In the contact mode, the vertical displacement of the cantilever is used to visualize the tip-sample interaction forces. This imaging mode can give nanometric resolution on flat and fairly hard samples. But the additional friction and capillary forces also participate in the image formation process and increase the load charge of the cantilever, limiting the resolution and deforming the sample. The friction mode shows the lateral deflection of the cantilever during the scan. However this deflection can be due to the topography of the sample. To ensure that it is not the case, friction images were taken during the trace and retrace direction and then subtracted. This deflection can be used to investigate frictional (and mechanical) properties of the sample. In the tapping mode (TM-AFM) the tip is forced to oscillate in the z direction above the sample and touches its surface periodically. This way, the friction forces are eliminated and the tip-sample interaction decreases. In this mode very soft samples can be analyzed without damage,19 but so far atomic resolution has been difficult to obtain. Therefore, the LB films were first analyzed on the micrometer scale in tapping mode and then on the nanometer scale in contact mode in order to image the lattice of the deposited molecules. The AFM used was a Nanoscope III from Digital Instruments. In contact mode, samples were studied in air or in water. Images were taken with 120, 14, and 1.3 µm piezoelectric scanners with silicon nitride tips having a spring constant of 0.06 N/m. Tapping mode tips were in silicon with a spring constant of 25-64 N/m and a resonance frequency between 270 and 370 kHz depending on the tip. Images at high resolution were taken on the center of the sample to avoid edge effect due to the deposit of the film, effects which were confirmed by XPS analysis on the LB films.
Results and Discussion Isotherm Curves of Compression and Film Deposition. Isotherm compression curves were determined for the two phospholipids during the Langmuir-Blodgett experiments providing valuable information regarding the morphology and the stability of the transferred films. These curves are obtained by plotting the variation of the surface pressure (mN/m) as a function of the area per molecule (nm2). If the linear portion of the phase transition is extrapolated to zero surface pressure, the intercept gives the area per molecule that would be expected for the hypothetical state of an uncompressed close-packed layer. Another accessible parameter for characterizing the quality of the transfer is the transfer ratio between the areas respectively of the film transferred onto the substrate and the film previously on water. A typical isotherm compression curve of DPPE is presented in Figure 1. The optimum surface pressure determined by trial and error for our application was 40 mN/m. For the transfer and deposition of a monolayer LB film on mica, the mean molecular area was calculated from eight isotherm curves to be 0.42 ( 0.02 nm2 for DPPE and 0.55 ( 0.02 nm2 for DPPC (at 40 mN/m). The collapse of the DDPE film at the liquid interface occurs at 50 mN/m, where we obtained a transfer ratio of 4.5. This high value is due to the reordering of the monolayer at the liquid interface into a multilayer structure20 and correspondingly to the transfer (19) Sommer, F.; Tran Minh Duc; Coleman, A. W.; Skiba, M.; Wouessidjewe, D. Supramol. Chem. 1993, 3, 19. (20) Birdi, K. S.; Vu, D. T. Langmuir 1994, 10, 623.
Phospholipid Films
Figure 1. Isotherm compression curve of DPPE: the transfer pressure at 40 mN/m corresponds to a mean molecular area of 0.42 nm2, the collapse pressure occurs at 50 mN/m.
of this latter on mica. The mean molecular areas and also the collapse pressures as computed above are in good agreement with previous published values for these molecules.21 The mean area occupied by the DDPC molecule is found to be larger than that occupied by the DDPE due to the filling of the space by the trimethyl group within the polar head. The transfer ratio was typically equal to 1 ( 0.1 for a DDPE monolayer film transferred on mica and 2 ( 0.1 for the same film transferred onto HOPG. These results suggest that as expected a monolayer LB film in which the polar head is bound to a hydrophilic mica surface is much more stable than the same monolayer transferred to a hydrophobic HOPG surface. In the latter case a reorganization of the film occurs in the transferred phase and it may form multilayer domains as indicated by a larger transfer ratio (g2) and as discussed later at several points in this paper. In addition, we were unable to transfer more than two layers of DPPE. When a trilayer was deposited, the transfer ratio was close to 1 for the first two vertical dippings, but at the third vertical dipping, we obtained a negative transfer ratio close to -1. This clearly suggests not only that a third layer was not deposited but also that the second layer has been removed. This phenomenon has already been reported in the literature.22 Thickness and Stoichiometric Determinations with XPS. XPS results allow us to check the amount of phospholipids transferred, to measure the thickness of the transferred films, and to elucidate their molecular orientation. Freshly cleaved mica substrate was first analyzed and showed the expected silicon, aluminum, potassium, and oxygen peaks with a slight amount of carbon contamination. The XPS spectra after deposition of one monolayer of DPPE at 40 mN/m showed a high increase in carbon signal and in correlation a decrease in silicon, aluminum, and potassium demonstrating clearly that phospholipids were actually transferred. The C(1s) signal was used to trace the LB film and the K(2p) peak the underlying substrate, while the C(1s) to K(2p) ratio was used for estimating the film thickness. Three chemical states fit the C(1s) envelope and are in very close agreement with those generally assigned to C-C and C-H bonds at binding energy Eb 285.0 eV, C-O bonds at Eb 286.6 eV, and OdC-O bonds at Eb 288.8 eV. The relative concentrations for these functional groups determined at detection angle θ ) 0° relative to the sample (21) Sellstro¨m, A° .; Gustafson, I.; Ohlsson, P.-A° .; Olofsson, G.; Puu, G. Colloids Surf. 1992, 64, 289. (22) Hasmonay, H.; Caillaud, M.; Dupeyrat, M. Biochim. Biophys. Res. Commun. 1979, 89, 338.
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Figure 2. Plot of ln(IKLB(θ)/IKSub(θ)) versus 1/cos(θ) for a monolayer and a bilayer of DPPE transferred onto mica as measured by XPS, respectively. The slope of the straight lines indicates the film thickness expressed in units of the electron path (λ). Table 1. Chemical Composition of Carbon Functional Groups Expressed as Percentage of Carbon for a Monolayer and Bilayer of DPPE Transferred onto Mica As Determined by XPS at Normal Angle C-C and C-H
C-O
OdC-O
theory experiment theory experiment theory experiment monolayer bilayer
81
86
14
11
5
3
81
83
14
12
5
5
normal are given in Table 1. The XPS stoichiometry indicates an excess of C-C and C-H functions and conversely a lack of OdC-O and C-O functions in comparison with the theoretical DPPE composition. Since XPS is sensitive to the outermost surface layer, this result fits in well with the molecular orientation of the monomolecular film pointing the hydrocarbon chain outward from the surface, thus favoring the C-C and C-H signals. Reorganization of the transferred bilayer into a mixture of monolayer and trilayer as discussed later should reduce the contribution of the C-C and C-H components in the bilayer XPS signals relative to the monolayer and conversely increase the C-O and O-CdO signals (see Table 1). To evaluate the thickness of the deposited films on mica, the attenuation of the K(2p) signal between a clean substrate without LB films (lifted from the trough just before adding phospholipids) and the substrate covered by the LB films was measured as a function of detection angle. If IKLB(θ) and IKSub(θ) are the potassium intensities measured at the detection angle θ for the mica surface respectively covered by a LB film of thickness d and uncovered, then the absorption ratio IKLB(θ)/IKSub(θ) can be expressed as
IKLB(θ) IKSub(θ)
) exp(-d/λ cos θ)
where λ is the electron mean free path of K(2p) photoelectrons in the LB layer. A plot of ln(IKLB(θ)/IKSub(θ)) versus 1/cos(θ) for a flat and uniform layer will give a straight line with a slope equal -d/λ. This slope was used to determine the relative thickness (in units of λ), as shown in Figure 2 for respectively a monolayer and a bilayer of DPPE at 40 mN/m dried a few minutes at room temperature before analysis. A linear regression was made for each plot and the slopes were determined by least-squares fitting. The experimental values of the slopes are -1.17 and -2.42 for the monolayer and bilayer, respectively, i.e., a ratio of 2.07 (2.42/1.17) close to the expected value of 2. It must be noticed that the determined thickness is only a mean
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Figure 3. Ratio of C-C and C-H functions (labeled C1) over the C-O and C-N (labeled C2) and OdC-O functions (labeled C3), i.e., C1/(C2 + C3) versus the detection angle for (a) DPPE monolayer on mica, (b) DPPE bilayer on mica, (c) DPPE monolayer on HOPG, and (d) DPPE bilayer on HOPG. Depositions on mica show clearly a hydrophobic surface, whereas on HOPG, the surface is rather hydrophilic. The structural models for each stacking structure are shown in the corresponding inserts.
thickness on a much larger scale (about 2 mm2) than the one locally probed by AFM as discussed later. Moreover, the AFM results presented here will show that the bilayer is not homogeneous but rather forms domains of flip-flap layers on the first monolayer. XPS cannot be used to control the homogeneity of the transferred bilayer, but we measured that twice the material was really on the substrate for a bilayer than for a monolayer. XPS results on thickness may be compatible with the configuration of a monolayer covered by small bilayer domains as represented in the insert of Figure 3b. Molecular Orientation. The molecular orientation of DPPE monolayer and bilayer LB films deposited on mica and HOPG at a pressure of 40 mN/m was also investigated by angle-resolved XPS. After elaboration, the films were dried and immediately introduced into the instrument. The relative variation in intensity of the three C(1s) chemical states was recorded as a function of the detection angle. The C-C and C-H functions (labeled C1) are specific of the aliphatic hydrocarbon chain whereas the C-O and C-N (labeled C2) and OdC-O functions (labeled C3) are specific for the polar head group. The evolution of the intensity ratio C1/(C2 + C3) as a function of detection angle is thus characteristic of the molecular orientation of DPPE on the substrate. This ratio will increase with the detection angle if the film presents its
Solletti et al.
aliphatic tail pointing outward from the surface. Figure 3 shows the evolution of C1/(C2 + C3) for DPPE monolayer on mica (a), bilayer on mica (b), monolayer on HOPG (c), and bilayer on HOPG (d), respectively. It is very clear that DPPE monolayer on mica, as anticipated above, is well ordered, as shown in the insert of Figure 3a with the hydrophobic tail localized at the surface according to the variation with detection angle of the C1/(C2 + C3) ratio. On the other hand the DPPE bilayer undergoes a reorganization in air with a reordering of the second layer into a bilayer in a head to head structure (see insert in Figure 3b), so a bilayer with the polar head groups outward from the surface is not observed by XPS but instead a hydrophobic tail is observed on the surface (Figure 3b). This XPS result fits a structural model determined by AFM with a mixture of monolayer and trilayer domains as shown in the insert of Figure 3b. One has to be cautious in interpreting the XPS C(1s) data for DPPE monolayer and bilayer on HOPG, since this peak includes an interference component due to the C(1s) peak from the HOPG substrate. It seems that these films are less stable and that the stacking is not well ordered since no pronounced variations are detected in the C1/(C2 + C3) ratio and no large differences between monolayer (Figure 3c) and bilayer (Figure 3d) signals rise. Moreover, hydrophilic, as well as some hydrophobic parts, are found on the monolayer surface (see insert of Figure 3c), suggesting that some additional monolayer partially cover head to head the underlying monolayer film. These results are consistent with the AFM images indicating that on HOPG the DPPE film is formed of a relatively homogeneous layer covered by some monolayer domains. For the DPPE bilayer transferred onto HOPG, XPS detects, as for the monolayer, a mixed hydrophobic and hydrophilic surface and this result may fit the structural model with reordering in domains of bilayers and double bilayers (see insert of Figure 3d). One can argue that, as an ultravacuum technique, XPS could modify the stacking structure. In fact, DPPE LB films observed with AFM in the air before and after XPS analysis show no significant modification of the topography. Structural Models of LB Films from AFM Crystallographic Unit Cell Structure of DPPE Crystal and LB Films. First we aim to determine by AFM whether the LB films present the same crystallographic unit cell structure as the phospholipid crystals. Thus DPPE single crystals were grown and imaged together with DPPE LB films in order to compare the two unit cells. Micrometric scale crystals were obtained by partially dissolving DPPE in chloroform. Since DPPE is not entirely soluble in chloroform, single microcrystals could be deposited onto mica by using the LB technique (at 26 mN/m). In this way we obtained a sample containing both LB film and single crystals with a size on the order of tens of micrometers or below as shown in Figure 4. Interestingly it is observed that DPPE single crystals are formed by stacks of head to head bilayers as shown by the height of the steps measured to be 5.6 ( 0.2 nm corresponding to a molecular bilayer (TM-AFM data). Bilayers with head to head structure are thus confirmed to be the preferential orientation for DPPE. Nanometric resolution was achieved on such a microcrystal (contact mode data) as shown in Figure 5. Two-dimensional Fourier transformation (FT) analysis showed a parallelogram unit cell with lattice constants a ) 0.64 ( 0.03 nm and b ) 0.91 ( 0.07 nm and an angle of 80° (mean of four images).
Phospholipid Films
Figure 4. DPPE single crystal imaged in tapping mode AFM (height mode). Note the step height to be 5.6 ( 0.2 nm.
Figure 5. Nanometric resolution imaging of the DPPE single crystal surface of Figure 4 indicating a crystal monoclinic cell unit of 0.64 ( 0.03 nm × 0.91 ( 0.07 nm with an 80° angle. The image was taken in constant height mode at 61 Hz (z scale 0.0018 nm).
Images at nanometric resolution with regular lattice could not be obtained on a DPPE monolayer LB film transferred on mica at 40 mN/m. The molecular resolution in Figure 6 could only be obtained in contact mode in the case of a bilayer film and on the hydrophobic trilayer domain surface showing a regular hydrocarbon chain pattern as predicted by the flip-flap process of the structural model in the insert of Figure 3b. FT analysis yields a rectangular unit cell containing two molecules with the following lattice constants: a ) 0.68 ( 0.02 nm and b ) 0.93 ( 0.05 nm and an angle of 90° between the two directions (mean of 11 images). The data are consistent with X-ray diffraction data for the similar phosphatidylethanolamine.23,24 However the unit cell area (0.32 ( 0.06 nm2) is quite different from the area of a single molecule (0.42 ( 0.02 nm2) estimated from the compression isotherm at 40 mN/m. The better resolution obtained on the trilayer domains than on the monolayer is probably due to a lower flexibility of the outermost hydrocarbon chain in the latter. We assume the strength of interaction of the polar head with the mica surface to be lower than the one between polar heads, and the matching is also expected to be more loose, so the (23) Suwalksy, M.; Tapia, J.; Knight, E.; Duk, L.; Seguel, C. G.; Neira, F. Macromol. Chem., Macromol. Symp. 1986, 2, 105. (24) Hauser, H.; Pascher, I.; Pearson, R. H.; Sundell, S. Biochem. Biophys. Acta 1981, 650, 21.
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Figure 6. Nanometric resolution (constant force mode) on a DPPE bilayer LB film transferred on mica obtained in air contact mode AFM (61 Hz), showing the hydrophobic tails of the phospholipids, as XPS suggests (see text). A rectangular unit cell with a ) 0.68 ( 0.02 nm and b ) 0.93 ( 0.05 nm is observed.
compactness of the monolayer film may be lower than the trilayer domain. We expect then that the monolayer should be softer and will present a higher friction coefficient than bilayers and trilayers when probed under water with AFM lateral imaging mode. This is actually observed as shown later in the paper. It is worth noting also that the crystallographic unit cell has been observed only on the hydrophobic aliphatic surface of bilayers. Morphology of Transferred Film Investigated by AFM. The morphological structures and the stability of the different DPPE and DPPC LB films were then investigated by AFM in both contact and tapping modes. Except for the study at the collapse pressure, all the films were transferred at 40 mN/m. DPPE Monolayer. Nanometric resolution was achieved on the hydrophobic part of the monolayer deposited on mica but without obtaining a regular lattice. We actually observed undulations separated by a distance of 0.8-1 nm, as previously described in the literature for this molecule in liquid medium.25 We did not measure the height of the monolayer because the film observed in air uniformly covered the mica. On the monolayer LB film deposited on HOPG, a mixture of monolayer together with small bilayer domains and defects was observed (Figure 7). The monolayer was not homogeneous, with holes of 100 ( 5 nm in diameter. The depth of the holes (2.8 ( 0.4 nm) was found to be quite constant and corresponded actually to a monolayer thickness. An upper layer was detected with equally a monolayer thickness of 2.7 ( 0.2 nm, and then the stacked bilayer presented a thickness of 5.5 ( 0.2 nm. These findings support the structural model proposed in Figure 3c. In addition, linear-shaped defects of height 9.5 ( 0.5 nm covered the film. We assign these defects to DPPE trilayers. DPPE Bilayer. In Figure 8 we present images of bilayers transferred onto mica as observed with the tapping mode in air. We observed flat and relatively large bilayer domains 5.6 ( 0.2 nm thick covering a homogeneous monolayer. This observation is in accordance with the structure of a monolayer and trilayer mixture according to the structural model represented in the inset of Figure 3d resulting from the reorganization of the (25) Fare, T. L.; Palmer, C. A.; Silvestre, C. G.; Cribbs, D. H.; Turner, S. L.; Brandow, D. C.; Gabert, B. P. Langmuir 1992, 8, 3116.
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Figure 7. Monolayer of DPPE transferred onto HOPG observed in tapping mode AFM (height mode). It was not homogeneous; it had 100 nm size holes, superimposed monolayer domains, and defects. On the other hand, the transferred monolayer onto mica was completely homogeneous.
Figure 8. Typical monolayer and trilayer domains of a DPPE LB film transferred onto mica observed in tapping mode AFM (height mode). The first layer is homogeneous; the second one forms flip-flap domains.
bilayer during the drying process.11 Holes with a depth of 5.6 nm are present particularly on the largest domains and are certainly due to stress during elaboration of the film.26 The reorganization of the bilayer discussed above occurs in the hydrophobic air environment and should not occur in a hydrophilic environment such as in water. To clarify this issue, we observed then the morphology of the bilayer under water both in contact and friction modes. In Figure 9, one can observe that the film was not completed but rather was a mixture of approximately 50% monolayer and 50% bilayer. The upper layer was measured to be actually 2.6 ( 0.2 nm thick, correspondingly to a monolayer. We also observed that the two types of layers present very different mechanical surface properties. The bilayer shows a hydrophilic surface with polar head outward and is characterized by low friction, whereas the monolayer shows a hydrophobic surface with hydrocarbon chain outward and high friction coefficient. We tentatively attribute this difference in friction to be due to the different chemical and molecular composition probed by the tip. The higher friction coefficient quoted for the monolayer can also be ascribed to the lower compactness of the monolayer as discussed earlier in the paper. (26) Frommer, S. Angew. Chem., Int. Ed. Engl. 1992, 31, 1298.
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DPPE Monolayer Transferred at Collapse Pressure. As we reported in the section on LB films, it is difficult to obtain more than three layers by vertical deposition. In order to deposit more layers, one possibility is to transfer films at the collapse pressure. The drawback of this technique is that it makes it impossible to control the quality of deposition. An image of DPPE LB film transferred onto a mica sheet at 50 mN/m has also been achieved and is shown in Figure 10. At this pressure, as previously described for LB films,20 domains of various dimensions, (between 100 nm up to several micrometers) are superimposed. In Figure 10 up to five superimposing domains are observed. The thickness of each domain is equal to the height of the bilayer. Stability with Aging. The stability with time of the LB film plays an important role, especially in applied science or technology. To measure the stability as a function of time, we used the tapping mode AFM on a DPPE film deposited on mica. A DPPE transferred bilayer was analyzed in air immediately after deposition and after intervals of 1 day up to 2 months. To make sure that we were analyzing the same place (an area of 4 µm2), a reference mesh was stuck on the reverse side of the mica. Between two consecutive observations, the sample was kept in a dry atmosphere at the temperature of the laboratory (23 ( 2 °C). At this scanning scale, we did not observe any modification of the morphology of the bilayer domains. DPPC Bilayer on Mica. DPPC LB films are expected to be not as stable as DPPE since in DPPC the polar head includes a trimethyl group which should reduce the hydrophilicity. Figure 11 shows the AFM image of DPPC bilayer LB films deposited on mica. One can clearly see two kinds of morphological structure: flat domains and liposomes deposited on a homogeneous monolayer. The thickness of the domains is estimated to be 5.9 ( 0.1 nm and is higher than the thickness measured for the DPPE bilayer and is in accordance with the literature.7 This difference of 0.3 nm higher compared to the DPPE can be attributed to the additional trimethyl group fixed at the polar head of the DPPC. The reorganization of the DPPC bilayer LB films is also stronger than that of DPPE, since for DPPC in self-assembling vesicles the average diameter was observed to be around 50 nm. Concluding Remarks The phospholipids presently investigated belong to the ethanolamine and choline classes previously studied, but the combination of XPS and AFM studies has allowed us to obtain a deeper understanding of the morphology, the chemical structure, interaction with the substrate, stacking orientation, and the stability of monolayer and bilayer phospholipid LB films. The LB parameters for obtaining reproducible and good quality films have been defined for pure saturated phospholipids (DPPE and DPPC) and also for unsaturated DOPE and their mixture (ref 13). The chemical composition, the molecular orientation, and the thickness of the monolayer and bilayer DPPE LB films are in accordance with the theoretical expectations. We obtained nanometric resolution AFM imaging of DPPE crystal and monolayer and bilayer LB films. Moreover the direct observation of the unit cell was achieved with contact mode in air on the DPPE crystal and on the hydrophobic part of the bilayer LB film transferred onto mica. The measured lattice constants are mutually consistent and in good accordance with X-ray diffraction data. DPPE monolayer deposited on hydrophilic mica surface was verified to present the hydrophobicity, configuration,
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Figure 9. DPPE bilayer transferred on mica imaged in liquid contact mode (height mode and lateral friction mode). The left image represents topography consisting of a mixture of monolayer and bilayer domains. The height of the upper layer is 2.6 ( 0.2 nm. The right image was scanned simultaneously in lateral force mode. The lateral friction was weaker on the bilayer (dark zone on the image) than on the monolayer (bright zone of the image).
Figure 10. DPPE film transferred at collapse pressure imaged in tapping mode AFM (height mode). Films with domains of five bilayers, 5.6 nm high each, were transferred.
and thickness as theoretically expected. In the case of DPPE bilayer on mica, the flip-flap reorganization of the film when transferred into air was directly monitored. The bilayer film was first observed under water and found
to be partly incomplete with the upper half of the bilayer missing. When transferred into air this upper monolayer is flip-flapping to form bilayer domains superimposed on a homogeneous monolayer. In this way the LB film
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organized. The same is found for DPPC LB bilayers deposited on mica which reorganize into self-assembling vesicles and bilayer domains on a homogeneous monolayer. Friction force measurements show that the upper layer in a DPPE bilayer LB film on mica is stiffer than the underlying monolayer. We may expect that lateral forces depend on the molecular nature of the terminal groups of the LB film surfaces. The potentiality of lateral force friction mapping for resolving the molecular surface structure is currently under investigation. The next step in this research will be the investigation of the incorporation of the Ca-ATPase in the phospholipid bilayer LB films.
Figure 11. DPPC LB film imaged in tapping mode AFM (height mode) showing formation of vesicles with 50 nm diameter and a bilayer 5.9 ( 0.1 nm thick.
presents a hydrophobic surface. DPPE films transferred onto hydrophobic HOPG surfaces are less stable and less
Acknowledgment. This work was supported by the EMDO-Zu¨rich Foundation, the Roche Foundation, the Sandoz Foundation, and the Swiss National Foundation 31-33753.92 (J.-M.S., S.K.). LA950579S
