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Lateral and Vertical Nanophase Separation in Langmuir-Blodgett Films of Phospholipids and Semifluorinated Alkanes Mounir Maaloum, Pierre Muller, and Marie Pierre Krafft* Institut Charles Sadron (UPR CNRS 22), 6 rue Boussingault, 67083 Strasbourg Cedex, France Received July 28, 2003. In Final Form: November 20, 2003 It has recently been found that monodisperse surface micelles (hemimicelles) were formed in Langmuir monolayers of the semifluorinated alkane C8F17C16H33 (F8H16) after transfer onto silicon wafers. Grazing incidence X-ray diffraction studies have demonstrated that compression of mixed Langmuir monolayers made from combinations of dipalmitoyl phosphatidylethanolamine (DPPE) and diblock F8H16 in various molar ratios resulted in the complete expulsion of the diblock molecule at high surface pressure. F8H16 then formed a second layer on top of a DPPE-only monolayer, demonstrating a novel type of reversible, pressure-induced, vertical phase separation. Using atomic force microscopy and X-ray reflectivity, we show now that mixed DPPE/F8H16 (1:1.3) Langmuir-Blodgett films transferred onto silicon wafers below 10 mN m-1 are laterally phase separated and consist of domains of F8H16 surface micelles in coexistence with a monolayer of DPPE. The density of the network of F8H16 surface micelles increases when the surface pressure of transfer increases. Around 10 mN m-1, the F8H16 surface micelles start to glide on the DPPE monolayer, progressively overlying it, until total coverage is achieved.
Introduction A current objective in materials and biological sciences is the design and development of materials and devices with complex, multiscaled architectures. Fluorinated compounds, due to their combined hydro- and lipophobia,1 provide valuable tools for generating complexity in the organization and compartmentalization of molecular systems into segregated nano- to mesometric phases with controlled sizes and specific properties.2 Segregated micelles,3 multicompartment polymeric micelles,4,5 liposomes containing separated fluorinated domains within their bilayer membrane,6,7 and patterned phase-separated bidimensional films8,9 have been obtained by combining fluorinated and hydrogenated amphiphiles. Potential applications of such compartmentalized systems include cell mimicking and surface nanopatterning for microfluidics, nanolubrication, and molecular sensing. Using atomic force microscopy (AFM), we have recently shown that, when transferred onto silicon wafers, Langmuir monolayers of C8F17C16H33 (F8H16 diblocks) consist of highly monodisperse and stable surface micelles.10 X-ray reflectivity measurements determined that the hydrogenated segments of the F8H16 diblocks are directed toward the substrate, while the fluorinated segments are pointing toward air. We have established that the shape of these hemimicelles can be modeled by a simple disk model. Their * Corresponding author. Tel: (33) 3 88 41 40 60. Fax: (33) 3 88 40 41 99. E-mail:
[email protected]. (1) Riess, J. G. Tetrahedron 2002, 58, 4113. (2) Krafft, M. P., Ed. Curr. Opin. Colloid Interface Sci. Section on Fluorinated Colloids and Interfaces. 2003, 8, 213. (3) Mukerjee, P.; Yang, A. Y. S. J. Phys. Chem. 1976, 80, 1388. (4) Sta¨hler, K.; Selb, J.; Candau, F. Langmuir 1999, 15, 7565. (5) Laschewsky, A. Curr. Opin. Colloid Interface Sci. 2003, 8, 274. (6) Elbert, R.; Folda, T.; Ringsdorf, H. J. Am. Chem. Soc. 1984, 106, 7687. (7) Riess, J. G. J. Drug Targeting 1994, 2, 455. (8) Barriet, D.; Lee, T. Curr. Opin. Colloid Interface Sci. 2003, 8, 236. (9) Krafft, M. P.; Goldmann, M. Curr. Opin. Colloid Interface Sci. 2003, 8, 243. (10) Maaloum, M.; Muller, P.; Krafft, M. P. Angew. Chem., Int. Ed. 2002, 41, 4331.
size is controlled by the density mismatch between the fluorinated and hydrogenated segments and is a simple function of the length of these segments. Despite the fact that many studies have been conducted on Langmuir monolayers of semifluorinated alkanes,11-13 the existence of such surface micelles, as revealed by AFM on transferred monolayers, had not been anticipated. In parallel, it has been reported that a pressure-induced vertical phase separation occurs in mixed Langmuir monolayers of combinations of dipalmitoyl phosphatidylethanolamine (DPPE) and F8H16 spread on the water surface.14 Grazing incidence X-ray diffraction experiments demonstrated that compression of DPPE/F8H16 monolayers results in the complete expulsion of the diblock molecule at high surface pressure with the fluorinated segments pointing toward the air. The expulsed F8H16 diblocks then form a second layer on top of a DPPE-only monolayer. The phenomenon is reversible; that is, F8H16 respreads onto the water surface upon expansion. However, the structure of the mixed monolayer that forms at low surface pressures and the structure of the F8H16 layer that forms on top of the DPPE monolayer at high surface pressure had not been determined. We have now used AFM and X-ray reflectivity to investigate the phenomenon of ejection of the F8H16 molecules from the DPPE monolayer and the structure of the uppermost F8H16 monolayer in DPPE/F8H16 mixed Langmuir-Blodgett (LB) films transferred onto silicon wafers. The effects of the surface pressure of transfer on the film topology have been determined. Experimental Section Materials. F8H16 was synthesized according to ref 15 and was thoroughly purified by column chromatography. Its chemical (11) Gaines, G. L. Langmuir 1991, 7, 3054. (12) Huang, Z.; Acero, A. A.; Lei, N.; Rice, S. A.; Zhang, Z.; Schlossman, M. L. J. Chem. Soc., Faraday Trans. 1996, 92, 545. (13) El-Abed, A.; Pouzet, E.; Faure´, M.-C.; Sanie`re, M.; Abillon, O. Phys. Rev. E 2000, 62, R5895. (14) Krafft, M. P.; Giulieri, F.; Fontaine, P.; Goldmann, M. Langmuir 2001, 17, 6577. (15) Brace, N. O. J. Org. Chem. 1973, 38, 3167.
10.1021/la030312q CCC: $27.50 © 2004 American Chemical Society Published on Web 02/19/2004
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Figure 1. Molecular area vs surface pressure isotherms of monolayers made from F8H16 (a), DPPE (b), and DPPE/F8H16 mixtures (1:1.3) (c). purity (>99%) was determined using gas chromatography (GC), thin-layer chromatography (TLC), NMR, and elemental analysis. DL-R-DPPE was obtained from Sigma and used without further purification. Spreading solutions of DPPE and F8H16 (0.5 mmol L-1) were prepared in chloroform/methanol (9:1 v/v). Spreading solutions of DPPE/F8H16 mixtures were prepared by cosolubilization of the two compounds in chloroform/methanol (9:1). Water was purified using a Millipore system (surface tension, 72.1 mN m-1 at 20 °C; resistivity, 18 MΩ cm). Monolayer Isotherms: Preparation of the LB Films. Surface pressure versus molecular area isotherms were recorded on a Langmuir minitrough (Riegler & Kirstein, Germany) equipped with two movable barriers (compression speed, 0.1 nm2 min-1). The surface pressure π was measured using the Wilhelmy plate method. The temperature was regulated at 20.0 ( 0.5 °C. Aliquots of 25 µL of DPPE or F8H16 solutions or DPPE/F8H16 mixtures were spread on the water surface. The monolayers were transferred onto a silicon wafer, previously treated with a piranha solution (concentrated H2SO4 + 30% H2O2 3:1), using the Langmuir-Blodgett technique (one monolayer transferred), prior to being studied by AFM and X-ray reflectivity. Atomic Force Microscopy. The transferred films were analyzed with an atomic force microscope (NanoScope III) in tapping mode. The cantilever used here was a cantilever from Olympus with a very sharp tip (5 nm), a resonance frequency of 300 kHz, and a spring constant of 42 mN m-1. X-ray Reflectivity. The grazing incidence X-ray studies of the transferred films were performed with an EXPERT-MPD device from Philips (divergence slit 1/32°, parallel plate collimator, flat Ge monochromator and Xe detector). A Cu KR beam at wavelength 0.1542 nm was used. The data were analyzed using Parratt32 software (version 1.6.0).16
Figure 2. AFM images (1 × 1 µm) of the surface micelles observed in a F8H16 Langmuir monolayer transferred onto a silicon wafer at 3 and 7 mN m-1 (panels a and b, respectively).
Figure 1 depicts surface pressure/molecular area (π/A) isotherms for F8H16, DPPE, and mixed DPPE/F8H16 (1: 1.3) monolayers. This particular molar ratio has been investigated because it corresponds to the threshold at which the F8H16 surface concentration allows a dense monolayer to form on top of a dense DPPE monolayer.14 At 20 °C and at a nonzero surface pressure, DPPE (isotherm b) forms a monolayer in the liquid condensed (LC) state that is stable up to ∼47 mN m-1. As π increases, the DPPE molecules straighten progressively on the water surface (tilted L2d phase). Above a transition that is just
visible on the isotherm and occurs at 35 mN m-1, the DPPE molecules are perpendicular to the water surface (LS untilted phase).17 F8H16 (isotherm a) forms a stable monolayer up to ∼10 mN m-1 with a limiting area of ∼30 Å2, corresponding to the cross section of a perfluorinated chain, which is larger than that of a hydrogenated chain (∼20 Å2). The isotherm is reversible without hysteresis upon compression-expansion cycles. The isotherm of the DPPE/F8H16 (1:1.3) mixture (isotherm c) presents one transition at ∼10 mN m-1, and the limiting area at high π is very similar to that of pure DPPE. Grazing incidence X-ray diffraction (GIXD) on such DPPE/F8H16 (1:1.3) monolayers has demonstrated that the F8H16 diblocks were totally ejected at high π and formed a second monolayer on top of the DPPE monolayer.14 It was proposed that the transition observed on the isotherm corresponds to the onset of the ejection of the diblocks from the DPPE monolayer. Several key questions remained unanswered, however, such as the miscibility of the DPPE and F8H16 diblock molecules within the mixed monolayer at low π, the mechanism of ejection of the F8H16 diblocks from the water surface at intermediate π, and the organization of the uppermost F8H16 monolayer at high π. To get more information, mixed DPPE/F8H16 (1:1.3) monolayers were transferred onto silicon wafers and
(16) Braun, C. Parratt32 Fitting routine for reflectivity data; HMI: Berlin, 1997-1999.
(17) Bo¨hm, C.; Mo¨hwald, H.; Leiserowitz, L.; Als-Nielsen, J.; Kjaer, K. Biophys. J. 1993, 64, 553.
Results and Discussion
Nanophase Separation in LB Films
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Figure 3. AFM images (1 × 1 µm) of mixed 1:1.3 DPPE/F8H16 monolayers transferred (a) at 0.5 mN m-1, (b) on the plateau at 10 mN m-1 (cf. Figure 1), (c) at 20 mN m-1, and (d) at 30 mN m-1.
investigated by AFM and X-ray reflectivity. Figure 2a,b shows AFM images of monolayers of F8H16 alone transferred on a silicon wafer at 3 and 7 mN m-1. At both surface pressures of transfer (πt), the diblock molecules organize into two-dimensional monodisperse hemimicelles with a characteristic diameter of 300 Å and an average height of 23 ( 5 Å.10 As πt increases, the surface micelles become more dense and keep the same size, and at 7 mN m-1 they pack in an hexagonal array (Figure 2b). No coalescence of the micelles was observed. Figure 3a is an AFM image of the (1:1.3) DPPE/F8H16 LB film transferred at low π (0.5 mN m-1). It can be seen that domains consisting of circular surface micelles coexist with flat monolayer domains. The diameter of the micelles is very uniform. Although the topology of the surface micelles and that of the monolayer strongly differ, their heights, 23 ( 5 Å and 25 ( 5 Å, respectively, are very similar. The height of the flat monolayer domain, 25 ( 5 Å, corresponds to the thickness of a DPPE LB film, as assessed by X-ray reflectivity (see below). Many holes can be seen in the flat monolayer, indicating that it is not complete. Large voids can also be seen between the surface micelles because of the lack of surface pressure. It is very likely that the surface micelles visualized in this AFM image are mostly constituted of F8H16 diblocks because they have the exact same aspect as those observed in LB films of pure F8H16 and because they have the same size (300 Å) and height (23 ( 5 Å). It cannot be excluded, however, that some DPPE molecules are present in or
between the surface micelles. Likewise, the monolayer visualized on the AFM image is likely composed in majority of DPPE, though some F8H16 molecules may be present in the DPPE monolayer as well. In any case, the DPPE/ F8H16 LB films clearly present a phase-separated topology with a monolayer domain coexisting with domains of monodisperse molecular clusters. The influence of πt on the topology of the 1:1.3 DPPE/ F8H16 LB film is visible in Figure 3b-d. The AFM image of a film transferred at 10 mN m-1, that is, on the plateau of the isotherm, is shown in Figure 3b. The image depicts higher and lower regions. The topology of the higher regions is strongly reminiscent of that of surface micelles of pure F8H16. Here again, their diameter is consistently equal to 300 Å. In addition, the height of the molecular clusters above the underlying layer is the same as that of F8H16 surface micelles, that is, 23 ( 5 Å. We can thus reasonably infer that the molecular clusters forming the uppermost layer, visualized on Figure 3b, are surface micelles of F8H16. The lower regions are very similar to the image of Figure 3a and consist of flat DPPE-rich monolayer domains coexisting with F8H16 hemimicelles still in contact with water. The AFM image of the film transferred at 20 mN m-1 is shown in Figure 3c. When compared to Figure 3b, where πt was 10 mN m-1, Figure 3c clearly evidences that the proportion of the surface covered by F8H16 surface micelles on the DPPE monolayer increases with πt. At an even higher πt (30 mN/m), it can be seen (Figure 4d) that
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Maaloum et al. Scheme 1. Schematic Representation of the Physical States of Mixed DPPE/F8H16 (1:1.3) Langmuir Monolayers Transferred onto Silicon Wafers for Various Surface Pressures of Transfer (πt)a
Figure 4. Experimental X-ray reflectivity curves obtained from a monolayer of DPPE transferred onto a silicon wafer at 20 mN m-1 (open squares) and from mixed 1:1.3 DPPE/F8H16 monolayers transferred onto a silicon wafer at 5 mN m-1 (solid circles) and 15 mN m-1 (crosses).
the surface micelles totally cover the DPPE monolayer. The topology of the resulting LB film is then indistinguishable from that of surface micelles observed in monolayers of pure F8H16 transferred at 7 mN m-1. The 1:1.3 DPPE/F8H16 LB films transferred at various surface pressures (5, 15, and 40 mN m-1) and that of pure DPPE transferred at 20 mN m-1 have been investigated by X-ray reflectivity (Figure 4). The thickness of the monolayers was determined from the position of the first minimum of the curves. The results show that only the mixed LB film transferred at 5 mN m-1 has a thickness comparable to that of the monolayer of pure DPPE (25 Å at 20 mN m-1). On the other hand, all the other mixed films, which were transferred at higher surface pressures (15 and 40 mN m-1), exhibit a thickness over twice as large (55 Å). The reflectivity curve of the LB film transferred at 40 mN m-1 has not been plotted on Figure 4 for sake of clarity, but the position of its first minimum coincides with that of the curve of the film transferred at 15 mN m-1. The height of the F8H16 surface micelles in transferred monolayers of pure F8H16 has been determined by X-ray reflectivity to be 29.3 Å.10 This value is in good agreement with the height measured by AFM on transferred monolayers of pure F8H16 or of 1:1.3 DPPE/F8H16 mixtures, that is, 23 ( 5 Å. It is also in agreement with the length of a fully extended F8H16 molecule, lC, as calculated from refs 18 and 19: lC ) 1.3n + 1.265m + 2.58 ) 33.2 Å (n and m being the number of fluorinated and hydrogenated carbons, i.e., 8 and 16). The height of the surface micelles was found to be independent of πt. Therefore, the thickness determined by X-ray reflectivity for the mixed films for surface pressures varying from 15 to 40 mN m-1, that is, 55 Å, strongly supports the hypothesis of F8H16 surface micelles covering the DPPE monolayer at high surface pressures. The fact that the mixed LB film has the same thickness at 5 mN m-1 as the DPPE LB film supports the view that below the transition (∼10 mN m-1), the film is (18) Tanford, C. The Hydrophobic Effect: Formation of Micelles and Biological Membranes, 2nd ed.; John Wiley & Sons: New York, 1980. (19) Lo Nostro, P.; Chen, S. J. Phys. Chem. 1993, 97, 6535.
a (a) At low πt, a DPPE monolayer coexists with surface micelles of F8H16 in contact with the water surface. (b) When πt reaches 10 mN m-1, some of the surface micelles of F8H16 start gliding on top of the DPPE monolayer, while some others remain on the water surface. (c) At higher πt, the surface micelles of F8H16 totally cover the DPPE monolayer.
made of organized surface micelles of F8H16 coexisting with a monolayer primarily constituted of DPPE. Scheme 1 schematically summarizes the lateral and vertical phase separations found in LB films of DPPE/ F8H16 monolayers. Below 10 mN m-1, F8H16 hemimicelles coexist with a monolayer primarily constituted of DPPE (a). When surface pressure increases, the hemimicelles progressively glide onto the DPPE monolayer (b), until the DPPE monolayer is totally covered (c). Conclusions We report here on the original structure of mixed LB films made from combinations of a phospholipid (DPPE) and a semifluorinated alkane (F8H16) in 1:1.3 molar ratio. Owing to their respective cross sections, 1.3 molecules of F8H16 for 1 molecule of DPPE is the maximal ratio for which a F8H16 monolayer can be accommodated on top of a close-packed DPPE monolayer. These monolayers were transferred onto silicon wafers at various surface pressure values and were studied by AFM and X-ray reflectivity. At low surface pressure of transfer, the LB film is laterally phase separated with domains of monodisperse surface micelles of F8H16 diblocks coexisting with a DPPE monolayer. Upon compression (at around 10 mN m-1), the surface micelles progressively glide onto the DPPE monolayer until total coverage is achieved. The surface micelles of F8H16 in the DPPE/F8H16 mixtures have a characteristic diameter of 300 Å and a height of 30 Å, that is, similar to the diameter and height of the surface micelles obtained from pure F8H16. These observations demonstrate that the formation of hemimicelles from semifluorinated alkanes does not depend on the nature of the substrate, which can be either hydrophilic or lipophilic. Semifluorinated alkanes thus represent new tools for achieving surface nanopatterning on various substrates. Acknowledgment. The authors thank AtoFina for the gift of fluorinated precursors and the Centre National de la Recherche Scientifique (CNRS) for financial support. LA030312Q
