Polarization Modulation Infrared Reflection Absorption Spectroscopy

The spectra were calculated from optical constants of DMPC. ... However, their contribution to the IR spectrum as compared to 24 methylene units in th...
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Polarization Modulation Infrared Reflection Absorption Spectroscopy Investigations of Thin Silica Films Deposited on Gold. 2. Structural Analysis of a 1,2-Dimyristoyl-sn-glycero-3-phosphocholine Bilayer Izabella Zawisza,*,† Gunther Wittstock,† Rabah Boukherroub,‡ and Sabine Szunerits*,‡,§ Center of Interface Science, Department of Pure and Applied Chemistry, and Institut of Chemistry and Biology of the Marine EnVironment, Carl Von Ossietzky UniVersity of Oldenburg, D-26111 Oldenburg, Germany, Institut de Recherche Interdisciplinaire (IRI), USR CNRS 3078, and Institut d’Electronique, de Microe´ lectronique et de Nanotechnologie (IEMN), CNRS-8520, Cite´ Scientifique, AVenue Poincare´ , BP 60069, 59652 VilleneuVe d’Ascq, France, and Laboratoire d’Electrochimie et de Physicochimie des Mate´ riaux et des Interfaces (LEPMI), CNRS-INPG-UJF, 1130 rue de la piscine, BP 75, 38402 St. Martin d’He` res Cedex, France ReceiVed NoVember 22, 2007. In Final Form: January 8, 2008 In this paper we report on the structural analysis of bilayers of 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) using polarization modulation infrared reflection absorption spectroscopy (PM IRRAS). The lipid bilayers were formed on SiO2|Au and Au surfaces using the Langmuir-Blodgett and Langmuir-Schaeffer techniques. As we showed in part 1 (Zawisza, I.; Wittstock, G.; Boukherroub, R.; Szunertis, S. Langmuir 2007, 23, 9303-9309), SiO2 layers of 7 nm thickness, synthesized by plasma-enhanced chemical vapor deposition on 200 nm thick gold covered glass slides, allow PM IRRAS investigations. Only minor changes in the order and structure of the lipid bilayer are observed when deposited on SiO2|Au and Au surfaces. The choline moiety in the leaflet directed toward the SiO2 surface exists in trans conformation and shows a tilt of 28° with the surface normal of the CN bond. On the silica surface in the second leaflet directed toward air and in two layers deposited on the Au surface, trans and gauche isomers of the choline moiety are present and the tilt of the CN bond increases to 55° with respect to the surface normal. The order and molecular orientation in the DMPC bilayers on SiO2 and Au surfaces are not affected by time. The analysis of the phosphate stretching mode on the Au surface shows slight dehydration of this group and reorientation of the phosphate moiety.

1. Introduction Lipid bilayers are the basic structural framework of biological membranes. In the native state, phospholipid membranes are difficult to study as the natural system is complex in its composition, and cells and vesicles are not in a state where they can be easily manipulated for investigation. Artificial model lipid membranes can be however generated on a solid support, and the structural similarity of these supported bilayers to living cell membranes makes them highly interesting biometric models for studying different functional cell processes.1,2 Various fabrication schemes have been used for the preparation of lipid bilayers on solid substrates including Langmuir-Blodgett3 and Langmuir-Schaefer4-6 techniques, spin coating,7 self-organized growth due to vesicle fusion,8-12 or covalent linking to thiopeptide layers.13 * To whom correspondence should be addressed. (I.Z.) E-mail: [email protected]. Fax: 0049-441-79 83 971. (S.S.) E-mail: [email protected]. Fax: 0033-4-76 82 65 52. † Carl Von Ossietzky University of Oldenburg. ‡ IRE and IEMN. § LEPMI. (1) Sackmann, E. Science 1996, 271, 43. (2) Chapman, D. Langmuir 1993, 9, 39. (3) Silvestro, L.; Axelsen, P. H. Chem. Phys. Lipids 1998, 96, 69-80. (4) Charitat, T.; BelletAmalric, E.; Fragneto, G.; Graner, F. Eur. Phys. J. B 1999, 8, 583-593. (5) Steinem, C.; Janshoff, A.; Ulrich, W.-P.; Sieber, M.; Galla, H.-J. Biochim. Biophys. Acta 1996, 1279, 169-180. (6) Hughes, A. V.; Goldar, A.; Gerstenberg, M. C.; Roser, S. J.; Bradshaw, J. Phys. Chem. Chem. Phys. 2002, 4, 2371-2378. (7) Perino-Gallice, L.; Fragneto, G.; Mennicke, U.; Salditt, T.; Rieutord, F. Eur. J. Phys. E 2002, 8, 275-282. (8) Cremer, P. S.; Boxer, S. G. J. Phys. Chem. B 1999, 103, 2554-2559. (9) Pierrat, O.; Lechat, N.; Bourdillon, C.; Laval, J.-M. Langmuir 1997, 13, 4112-4118. (10) Kalb, E.; Frey, S.; Tamm, L. K. Biochim. Biophys. Acta 1992, 1103, 307.

Such supported lipid bilayers can be regarded as self-assembled two-dimensional fluid systems whose structural properties are tightly controlled by a complicated balance among electrostatic, hydration, steric, and van der Waals forces. The lateral fluidity of the supported bilayer is essential for membrane function in a cell. Thus, the interaction of the lipid layers with the solid support plays an important and crucial role, especially in studies of biocompatible materials such as silica or titania. The early attempts to preserve the fluidity in supported bilayers were centered on single lipid bilayers on Si|SiO2 and quartz.14 It is now well-established that exposure of SiO2 to lipids leads to the formation of planar, stable, and fluid bilayers,15,16 and the formation of lipid monolayers and bilayers on pure and modified silicon has thus been widely employed.4,12,17-19 It was shown that, under conditions of sufficient hydration, a thin water layer (about 1 nm) forms between the bilayer and the hydrophilic surface, acting as a cushion for retaining the fluidity of the membrane.20 Another successful approach to prepare a model (11) Baumgart, T.; Kreiter, M.; Naumann, R.; Jung, G.; Jonczyk, A.; Offenhausser, A.; Knoll, W. J. Colloid Interface Sci. 2003, 258, 298. (12) Reimhult, E.; Larsson, C.; Kasemo, B.; Hook, F. Anal. Chem. 2004, 76, 7211. (13) Bunjes, N.; Schmidt, E. K.; Jonczyk, A.; Rippmann, F.; Beyer, D.; Ringsdorf, H.; Graber, P.; Knoll, W.; Naumann, R. Langmuir 1997, 13, 61886194. (14) Johnson, S. J.; Bayerl, T. M.; McDermott, D. C.; Adam, G. W.; Rennie, A. R.; Thomas, R. K.; Sackmann, E. Biophys. J. 1991, 59, 289-294. (15) Keller, C. A.; Glasmastar, C. A.; Kasemo, B. Phys. ReV. Lett. 2000, 84, 5443. (16) Reviakine, I.; Brisson, A. Langmuir 2000, 16, 1806. (17) Hollinshead, C. M.; Hanna, M.; Barlow, D. J.; De Biasi, V.; Bucknall, D. G.; Camilleri, P.; Hutt, A. J.; Lawrence, M. J.; Lu, J. R.; Su, T. J. Biochim. Biophys. Acta 2001, 1511, 49-59. (18) Tamm, L. K.; McConnell, H. M. Biophys. J. 1985, 47, 105. (19) Keller, C. A.; Kasemo, B. Biophys. J. 1998, 75, 1397. (20) Groves, J. T.; Ulman, N.; Bower, S. G. Science 1997, 275, 651-653.

10.1021/la703651n CCC: $40.75 © 2008 American Chemical Society Published on Web 03/08/2008

PM IRRAS InVestigation of Silica Films on Gold

lipid membrane on a solid support is the use of tethered bilayers.21-23 These membranes are long-term stable, laterally homogeneous on the molecular length scale, and electrically sealing and contain an electrolyte reservoir on both sides. Supported lipid bilayers can be easily investigated by techniques which are available for soft-matter surface science such as scanning probe, surface-sensitive methods, for example, surface plasmon resonance,9,11-13,24 quartz microbalance,12,25 and scattering6,17 and spectroscopic13,26,27 methods. FTIR spectroscopy belongs to the handful of spectroscopic methods frequently used in lipid chemistry as it provides structural information on a submolecular level.26-28 Photon polarization modulation infrared reflection absorption spectroscopy (PM IRRAS) is in this respect a particularly powerful alternative for the quantitative analysis of structural changes in organic layers and even in particular parts of an organic molecule adsorbed at the reflecting interface.18,19,22,24,26-28 This improvement is mainly due to the elimination of absorption from the sample environment (liquid reservoir and gas in the light path). Parameters such as the crystal packing, the tilt angle, and the physical state of the hydrocarbon chains, the degree of hydration, the hydrogen-bonded structures, and formation of chemical bonds are successfully analyzed by means of PM IRRAS.19,22-24,27,28 The knowledge of these parameters is crucial to understanding how organic material arranges at various interfaces. Recently, PM IRRAS has been used to characterize 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) bilayers deposited on Au(111) electrode surfaces.29-31 IRRAS measurements are routinely performed on highly reflecting materials such as gold, silver, and platinum or on weakly reflecting materials such as water. Silica is IR absorbing and not suitable for reflectance infrared spectroscopic analysis.17,18,20,21,29 However, the possibility of depositing an ultrathin transparent oxide film on an IR-reflecting surface would allow the exploitation of surface selection rules and thus PM IRRAS measurements.32,33 The deposition of surfactant-modified TiO2 (anatase) nanocrystallites (2 nm diameter) on a glassy carbon substrate allowed characterization using IRRAS.33 We have recently shown that using thin films of SiO2 deposited on gold can reduce the light absorption and preserve the reflectivity of gold.34 In this paper, the second part of this series, we want to demonstrate in detail the advantage of this interface for the structural characterization of DMPC bilayers formed on the Au|SiO2 and Au substrates using the Langmuir-Blodgett and Langmuir-Schaeffer techniques. PM IRRAS is an invaluable tool for the observation of changes in the tilt angle and packing (21) Guidelli, R.; Aloisi, G.; Becucci, L.; Dolfi, A.; Moncelli, M. R.; Tadini Bouninsegni, F. J. Electrochem. Chem. 2001, 504, 1-28. (22) McGillivray, D. J.; Valincius, G.; Vanderah, D. J.; Febo-Ayala, W.; Woodward, J. T.; Heinrich, F.; Kasianowicz, J. J.; Loesche, M. Biointerphases 2007, 2, 21-33. (23) Reference deleted in proof. (24) Naumann, R.; Schiller, S. M.; Giess, F.; Grohe, B.; Hartman, K. B.; Karcher, I.; Koper, I.; Lubben, J.; Vasilev, K.; Knoll, W. Langmuir 2003, 19, 5435-5443. (25) Glasmastar, C. A.; Larsson, C.; Hook, F.; Kasemo, B. J. Colloid Interface Sci. 2002, 246, 40. (26) Gauger, D. R.; Pohle, W. J. Mol. Struct. 2005, 744-747, 211-215. (27) Lewis, R. N. A. H.; McElhaney, R. N. In Infrared Spectroscopy of Biomolecules; Mantsch, H. H., Chapman, D., Eds.; Wiley-Liss: New York, 1996; p 159. (28) Pohle, W.; Gauger, D. R.; Fritzsche, H.; Rattay, B.; Selle, C.; Binder, H.; Bohlig, H. J. Mol. Struct. 2001, 563-564, 463. (29) Bin, X.; Zawisza, I.; Lipkowski, J. Langmuir 2005, 21, 330-347. (30) Zawisza, I.; Bin, X.; Lipkowski, J. Langmuir 2007, 23, 5180-5194. (31) Garcia-Araez, N.; Brosseau, C. L.; Rodriguez, P.; Lipkowski, J. Langmuir 2006, 22, 10365. (32) Ehrley, W.; Butz, R.; Mantl, S. Surf. Sci. 1991, 248, 193-200. (33) Marguerettaz, X.; Fitzmaurice, D. Langmuir 1997, 13, 6769-6779. (34) Zawisza, I.; Wittstock, G.; Boukherroub, R.; Szunerits, S. Langmuir 2007, 23, 9303-9309.

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of acyl chains, changes of the hydration of the polar head groups, and temperature-controlled phase transition on the hydrocarbon chain region of amphiphilic molecules. The results will be compared to those of DMPC deposited on Au surfaces of the same roughness. Furthermore, the stability of lipid bilayers is described. 2. Experimental Section 2.1. Materials. DMPC and chloroform were purchased from Sigma-Aldrich (Germany) and used without further purification. 2.2. Preparation of Au|SiO2 Composite Slides. Substrate electrodes were prepared by vacuum deposition of 5 nm of titanium and 200 nm of gold onto cleaned glass slides (76 × 26 × 1 mm3, n ) 1.58 at λ ) 633 nm, CML, France). Prior to silica film deposition, the gold samples were first degreased in 2-propanol and acetone in an ultrasound bath at room temperature, rinsed copiously with Milli-Q water, and dried under a stream of nitrogen. The gold slides were then heated in a plasma chamber at 300 °C at a pressure of 0.005 Torr for 1 h. SiO2 layers were synthesized using plasma-enhanced chemical vapor deposition (PECVD) technique in a Plasmalab 800Plus (Oxford Instruments, U.K.). The growth conditions used were as follows: substrate temperature, 300 °C; gas mixture, SiH4 (3% in N2) and N2O (the gas flow was 260 and 700 sccm for SiH4 and N2O, respectively); total pressure in the reactor, 1 Torr; power, 10 W at 13.56 MHz. Under these experimental conditions, the deposition rate was 414 Å min-1 and the silica films display a refractive index of 1.48. We have adjusted the silica film thickness by varying the deposition time.35,36 2.3. Formation of Lipid Layers. A few drops of a DMPC stock solution (1 mg mL-1 phospholipid in chloroform) were spread at the water surface in a Langmuir-Blodgett trough, equipped with a movable barrier and a Wilhelmi plate (KSV, Finland) to form a monolayer. The trough was controlled by a computer using KSV LB Instruments software. The temperature of the subphase was kept at 21 °C. Lipid monolayers were transferred from the air|water interface onto the Au|SiO2 surface at constant surface pressure π ) 40 mN m-1 (liquid-condensed (LC) state). The corresponding mean molecular area is equal to 0.46 ( 0.1 nm2. Langmuir-Blodgett (LB) vertical withdrawal was used to produce the DMPC monolayer on the silica surface. A combination of the Langmuir-Blodgett and Langmuir-Schaeffer (LS) techniques was employed to form phospholipid bilayers on the Au|SiO2. The monolayer was transferred using the LB method by vertical withdrawal of the Au|SiO2 and Au substrates at a speed of 35 mm min-1. The transfer ratio was 1.0 ( 0.1. After transfer and drying of the first lipid layer the monolayer formed on the silicate surface was horizontally brought into contact with the preformed monolayer at the air|water interface (LangmuirSchaeffer technique). The combined LB-LS transfer results in a Y-type bilayer on the silicate surface. 2.4. Instrumentation: PM IRRAS. All the PM IRRAS spectra were recorded using a Bruker Vertex 70 spectrometer with the polarization modulation set (PMA 50) equipped with a photoelastic modulator and demodulator (Bruker, Germany, Hinds Instruments). All spectra were recorded with a resolution of 4 cm-1. The PEM maximum efficiency was set for the half-wave retardation at 2900 cm-1 for analysis of CH stretching bands and at 1500 cm-1 for analysis of the CdO and CH bending modes. Each spectrum contains 10 000 averaged spectra. The angle of incident light was set to 80°. The PM IRRAS spectra were processed using the OPUS program (Bruker, Germany). All IR spectra were collected in a dry-air atmosphere after 1 h of incubation in the chamber. The enhancement of the electric field at the Au|SiO2|air interface and the PM IRRAS spectra of monolayers and bilayers for randomly distributed molecules were calculated using a home-written program. The spectra were calculated from optical constants of DMPC. The (35) Szunerits, S.; Boukherroub, R. Langmuir 2006, 22, 1660-1663. (36) Szunerits, S.; Boukherroub, R. Electrochem. Commun. 2006, 8, 439.

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Figure 2. PM IRRAS spectra in the CH stretching mode region of the DMPC bilayers transferred onto the (solid line) SiO2 and (dotted line) Au substrates at a surface pressure of 40 mN m-1. Figure 1. Structure of the DMPC molecule with marked groups analyzed by PM IRRAS. The schemes show the orientation of the analyzed group and directions of the transition dipole moments of each analyzed frequency. thickness of the lipid bilayer (5.4 nm) was taken from ref 37. The stratified system was composed of Au|7 nm SiO2|lipid film|air. The angle of incident light was 80°. For calculations of the PM IRRAS spectra of randomly distributed molecules in the DMPC films an surface coverage of Θ ) 0.88 was used.

3. Results and Discussion We have recently calculated and discussed the enhancement of the electric field of the p- and s-polarized IR light and the phase shift and reflectivity at the Au(200 nm)|SiO2 (0-120 nm)|air interface as a function of the angle of incidence of the IR light.34 It was shown that thin SiO2 layers deposited on a reflecting substrate such as gold can be used for IRRAS experiments. Subsequent structural analysis of organic layers deposited on the Au|SiO2 interface was used as an example to show the interest of this interface for the PM IRRAS analysis. In the present paper we focus on the qualitative and quantitative analysis of phospholipid (DMPC) bilayers transferred onto a Au|SiO2 substrate. The discussion of the PM IRRAS data is relatively complicated as the following factors may influence the structure of the lipid layer on the solid substrate: (i) DMPC has a gel and liquid crystalline transition temperature of 24 °C,38 which is very close to the 21 °C temperature used in the experiment; (ii) the surface roughness may affect molecular packing and order; (iii) the stability of the lipid bilayer at the Au|SiO2|air interface can vary with time. 3.1. PM IRRAS Analysis of the DMPC Bilayers on Au|SiO2 and Au Surfaces of the Same Roughness. DMPC (Figure 1) has a polar head group, a negatively charged phosphate moiety, a positively charged quaternary ammonium group, and an intermediate glycerol backbone containing two methylene groups and two hydrophobic hydrocarbon chains attached to this backbone by ester linkages. Each acyl chain contains 12 methylene groups. The PM IRRAS technique was used to analyze the structure, degree of hydration, and stability of the DMPC bilayers on Au and Au|SiO2 surfaces of the same surface roughness. The hydrophobic hydrocarbon chains were analyzed from the CH (37) Small, D. M. The Physical Chemistry of Lipids: From Alkanes to Phospholipids; Plenum Press: New York, 1986; Vol. 4. (38) Hauser, H.; Pascher, I.; Pearson, R. M.; Sundell, S. Biochim. Biophys. Acta 1981, 650, 21-51.

stretching and bending modes. In the glycerol moiety two methylene groups are present. However, their contribution to the IR spectrum as compared to 24 methylene units in the acyl chains is negligible. In the polar head group region, the ester and choline groups were analyzed from the CdO stretching modes of the ester group and the CH bending and CN stretching bands in the choline moiety, respectively. The phosphate stretching modes are observable only on the Au surface, while on the silica surface they are overlapped with the strong SiO2 stretching mode.39-41 To facilitate discussion of PM IRRAS, data schemes of each analyzed group with the direction of the transition dipole moments are shown in Figure 1. DMPC shows a transition from the liquid-crystalline phase to the gel phase at 24.1 °C.38 The Langmuir isotherm of DMPC at the air|water interface recorded at 21 °C shows a transition to the gel phase at a surface pressure of 42 mN m-1.30 When the surface pressure increases to 40 mN m-1, the area per molecule is reduced to 0.46 nm2, resulting in close packing of the acyl chains. The surface coverage is equal to 0.88. Under these conditions, the lipid molecules exist in the LC state and/or ripple phase, observed prior to transition to the gel phase.42-44 A surface pressure (π) between 40 and 70 mN m-1 corresponds to the typical physiological conditions for a lipid membrane. A surface pressure of 40 mN m-1 was chosen to transfer the DMPC bilayers onto Au and Au|SiO2 substrates. 3.1.1. Hydrocarbon Chains. Figure 2 shows the PM IRRA spectra of DMPC bilayers transferred at a surface pressure of 40 mN m-1 onto Au|SiO2 and Au substrates of the same roughness. Four absorption bands centered at ∼2964, 2922, 2878, and 2853 cm-1 are observed and attributed to νas(CH3), νas(CH2), νs(CH3), and νs(CH2), respectively. The shoulders at ∼2934 and ∼2900 cm-1 originate from the Fermi resonance between the νs(CH2) and methylene bending modes and between the νs(CH2) and CH3 bending modes, respectively.45 The asymmetric and symmetric methyl stretching modes are located at 2964.2 ( 0.2 and 2875.7 ( 0.2 cm-1. The positions of the symmetric and the (39) Hess, P.; Lambers, J. Microelectron. Eng. 2004, 72, 201-206. (40) Lisovskii, I. P.; Litovchenko, V. G.; Lozinskii, V. G.; Steblovskii, G. I. Thin Solid Films 1992, 213, 164-169. (41) Tolstoy, V. P.; Chernyshova, I. V.; Skryshevsky, V. A. Handbook of Infrared Spectroscopy of Ultrathin Films; John Wiley & Sons: Hoboken, NJ, 2003; pp 416-475. (42) Alaouie, A. M.; Smirnov, A. I. Langmuir 2006, 22, 5563-5565. (43) Kaasgaard, T.; Leidy, C.; Crowe, J. H.; Mouritsen, O. G.; Jorgensen, K. Biophys. J. 2003, 85, 350-360. (44) Sengupta, K.; Raghunathan, V. A.; Katsaras, J. Phys. ReV. E 2003, 68, 031710-1-031710-12. (45) MacPhail, R. A.; Strauss, H. L.; Snyder, R. G.; Elliger, C. A. J. Phys. Chem. 1984, 88, 334-341.

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asymmetric methylene stretching modes are however different on both substrates. On the Au surface νas(CH2) is located at 2925.5 ( 0.3 cm-1 and νs(CH2) at 2853.4 ( 0.2 cm-1. The full width at half-maximum (fwhm) is equal to 32.0 and 15.5 cm-1 for the νas(CH2) and νs(CH2) modes, respectively. On the SiO2 surface the νas(CH2) mode is located at 2920.9 ( 0.2 cm-1 and νs(CH2) at 2852.3 ( 0.2 cm-1. The fwhm is slightly reduced to 28.5 and 14.5 cm-1 for the respective methylene stretching modes. These results prove that the acyl chains in the DMPC bilayer formed on the Au and SiO2 surfaces of the same roughness (1.8-2.0 nm)35 exist in two different physical states. On the gold surface the position of the methylene stretching modes indicates that the hydrocarbon chains exist in the liquid phase, having a large number of gauche conformations.46,47 When the DMPC bilayer is transferred onto the Au|SiO2 surface, the methylene stretching modes are blue-shifted to the frequencies typical for hydrocarbon chains existing in the ripple phase, pretransition to the gel phase.48,49 This phase is characterized by the presence of well-ordered acyl chains in the lipid molecule with predominantly all trans conformations containing few gauche kinks.50,51 The number of gauche conformations in the hydrocarbon chain region is small, and the tilt of the hydrocarbon chain in the bilayer can be calculated. Knowing the integral intensity (Afilm) of a given vibration, the angle (θ) between the transition dipole moment of vibration and the electric field can be calculated:52,53

cos2 θ )

1 Afilm 3 Arandom

(1)

The transition dipole moments of the methylene asymmetric and symmetric stretches lie in one plane and are orthogonal to each other54 (Figure 1), and they make a right angle with the direction of the hydrocarbon chain. The tilt angle (θtilt) of the chains can be calculated:55

cos2 θνas + cos2θνs + cos2 θtilt ) 1

(2)

where θνas and θνs are the angles between the transition dipole moment and the electric field for asymmetric and symmetric methylene stretches, respectively. The procedure to calculate θνas and θνs angles from the integrated intensities of the abovereported IR absorption modes is described in detail elsewhere.52,53,56,57 The calculated tilt angle of the hydrocarbon chains in the DMPC bilayer on Au|SiO2 at a surface pressure of 40 mN m-1 is equal to 28.8° ( 1.0° with respect to the surface normal. On the gold surface the tilt of the hydrocarbon chains cannot be provided quantitatively since the acyl chains contain many gauche conformations. The integral intensities of the methylene stretching modes in both DMPC bilayers are however comparable, suggesting similar tilts and thicknesses of the DMPC bilayer on both substrates. (46) Dluhy, A. R.; Stephens, S. M.; Widayati, S.; Williams, A. D. Spectrochim. Acta A 1995, 51, 1413-1447. (47) Mitchell, M. L.; Dluhy, R. A. J. Am. Chem. Soc. 1988, 110, 712-718. (48) Cameron, D. G.; Casal, H. L.; Gudgin, E. F.; Mantsch, H. H. Biochim. Biophys. Acta 1980, 596, 463-467. (49) Snyder, R. G.; Hsu, S. L.; Krimm, S. Spectrochim. Acta 1977, 34A, 395406. (50) Brandenburg, K.; Snyder, R. G. Z. Naturforsch. 1986, 41C, 453. (51) Heimburg, T. Biophys. J. 2000, 78, 1154-1165. (52) Allara, D. L.; Nuzzo, R. G. Langmuir 1985, 1, 52-66. (53) Allara, D. L.; Swalen, J. D. J. Phys. Chem. 1982, 86, 2700-2704. (54) Fringeli, P. U. Z. Naturforsch. 1977, 32c, 20-45. (55) Umemura, J.; Kamata, T.; Kawai, T.; Takenaka, T. J. Phys. Chem. 1990, 94, 62-67. (56) Sun, F. Biophys. J. 2002, 2511-2519. (57) Zawisza, I.; Lachenwitzer, A.; Zamlynny, V.; Horswell, S. L.; Goddard, J. D.; J. L. Biophys. J. 2003, 86, 4055-4075.

Figure 3. PM IRRAS spectra in the CH bending mode region of the DMPC bilayers transferred onto the (solid line) SiO2 and (dotted line) Au substrates at a surface pressure of 40 mN m-1. Inset: deconvoluted spectrum in the CH bending mode region.

The expected tilt of the hydrocarbon chains (θexpectedtilt) can be calculated using eq 3,56 where Σ is the area of a single hydrocarbon chain, which in the gel phase is equal to 0.195 nm2, and A is the mean molecular area.56,58 The Langmuir isotherm

A cos θexpectedtilt ) 2Σ

(3)

of DMPC at π ) 40 mN m-1 gives a mean molecular area A ) 0.46 ( 0.01 nm2. This results in θexpectedtilt equal to 26.0° ( 1.6°. The very small difference of 2° in the tilt angles of the hydrocarbon chains can be due to slightly different orientations of the DMPC molecule on the solid and water surfaces. It is known that the surface roughness influences the orientation and physical state of the hydrocarbon chain in organized organic monolayers.59 The aqueous subphase has an average surface roughness of 0.3 nm,60 and SiO2 and Au substrates have average surface roughnesses of about 2.0 nm.35 The PM IRRAS spectra depicted in Figure 3 show the CH bending mode region in the DMPC bilayers. This busy spectral region is composed of five absorptions centered at 1493.0 ( 0.5, 1483.0 ( 0.5, 1468.0 ( 0.3, 1457.8 ( 0.8, and 1380 cm-1 where the two high-frequency bands originate from the CH3 bending modes of the choline group δ(N+(CH3)3), the next frequency originates from the methylene bending δ(CH2) mode, and the last two bands originate from the methyl bending modes: δas(CCH3) and δs(CCH3).54 From the inset in Figure 3 one can also see that the absorptions, arising from the methyl and methylene bending modes in the hydrocarbon chain region and the CH bending modes in the choline moiety, are overlapping. δ(CH2) is found at 1468.0 ( 0.3 cm-1, typical for hydrocarbon chains in the liquid phase, and points to a hexagonal or triclinic arrangement of the acyl chains.48,58 The transition dipole moment of the methylene bending mode lies along the bisector of the CH2 group and is overlapped with the dipole moment of the symmetric methylene stretching mode.54 The analysis of the δ(CH2) mode results in an angle between the dipole moment and the surface normal equal to 61.2° and 58.5° on the Au|SiO2 and Au surfaces, respectively. Indeed, these angles have the same values as those determined for the νs(CH2) band and confirm that the spectral analysis was done correctly. Concluding, our results indicate (58) Small, D. M. J. Lipid Res. 1984, 25, 1490-1500. (59) More, S. D.; Graaf, H.; Baune, M.; Wang, C.; Urisu, T. Jpn. J. Appl. Phys. 2002, 41, 4390-4394. (60) Braslau, A.; Deutsch, M.; Pershan, P. S.; Weiss, A. H.; Als-Nielsen, J.; Bohr, J. Phys. ReV. Lett. 1985, 54, 114-117.

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Table 1. Position, Integral Intensity, and Angle between the Transition Dipole Moment and the Surface Normal of the CH Bending Mode in the Choline Group in the DMPC Bilayers and Monolayer Transferred at a Surface Pressure of 40 mN m-1 on the Au and SiO2 Substrates

substrate SiO2 Au SiO2

DMPC film/ symmetry of δ(N+(CH3)3) bilayer A1 E bilayer A1 E monolayer A1 E

peak position/ cm-1

integrated intensity/cm-1

θ/ deg

1493.0 1482.4

0.01508 0.00896

53.0 64.5

1492.8 1481.4

0.01894 0.01004

50.3 52.5

1493.0 1483.0

0.01352 0.00491

28.0 63.2

that ordered lipid bilayers are formed and suggest similar tilts of the acyl chains close to 30° with respect to the surface normal. 3.1.2. Polar Head Group Region. Spectra in Figure 3 contain two absorptions originating from the polar group of the DMPC molecule, namely, the asymmetric CH3 bending modes in the choline moiety.54 These two bands in bilayers deposited on SiO2 and Au are located at 1493.0 ( 0.5 and 1483.0 ( 0.5 cm-1. The two δ(N+(CH3)3) absorption bands have different symmetries. The δ(N+(CH3)3) mode centered at 1490 cm-1 corresponds to the in-phase symmetric bending mode (A1), while the peak at 1481 cm-1 corresponds to the out-of-phase degenerative bending mode (E).54 The transition dipole moments are orthogonal to each other. The dipole moment of the mode with A1 symmetry lies along the C3V symmetry axis, thus along the CN bond. The dipole moment of the mode with E symmetry is oriented perpendicularly to the CN bond in the choline group (Figure 1).54,57 Table 1 summarizes the positions, integral intensities, and angles between the dipole moment for a given CH bending mode in the choline group and the surface normal in DMPC films on SiO2 and Au substrates. In both DMPC bilayers all determined angles are close to 55° with respect to the surface normal, characteristic of random distribution.53 In the DMPC bilayer prepared on the SiO2 surface deviation by ca. 10° is observed for the E mode of δ(N+(CH3)3). It is interesting to compare the results obtained for the choline group in the DMPC monolayer transferred at a surface pressure of 40 mN m-1 onto the silicate surface. The CN bond makes a small angle of 28° with respect to the surface normal. The band with E symmetry results in an angle of 63°. In the monolayer assembly on SiO2, an arrangement of the choline group different from that in the bilayer is found. In the leaflet directed toward the silicate surface the small tilt of the C-N bond is due to the interactions between the negatively charged silica surface61 and the positively charged choline group. Analysis of the CN stretching modes provides information on the conformation of the choline moiety, facilitating interpretation of the above-discussed tilt angles of the CN bond. The CN stretching modes are located between 970 and 800 cm-1.54 In this region the SiO2 surface also exhibits absorption of IR light; moreover, the cutoff of the PEM is around 1000 cm-1. Therefore, the IR spectra can only be used for qualitative not quantitative analysis. Figure 4 shows the spectra of DMPC bilayers on the SiO2 and Au surfaces. The two bands at 970 and 955 cm-1 originate from the asymmetric C-N stretching mode.54 The weaker bands located at lower wavenumbers originate from the symmetric C-N stretching modes. The number and position of

these modes are dependent on the conformation, trans or gauche, of O-C-C-N of the choline moiety.54,62 In the trans conformation of the choline group the νs(CN) peaks are observed at 925 and 875 cm-1.54 In the gauche conformation of the choline group the IR absorption due to the νs(CN) mode appears at 905 and 860 cm-1.54 In the DMPC bilayer and monolayer on SiO2 the νs(CN) (top two panels in Figure 4) IR bands are found at 920 and 870 cm-1, indicating the trans conformation of the choline moiety. In the bilayer transferred onto the Au surface in the symmetric CN stretching mode region, three bands centered at 925, 906, and 875 cm-1 are observed. In the bilayer on the Au surface (bottom panel) the choline moiety exists in the trans and gauche conformations. The above-discussed results agree with results reported by Fringeli.54 In the trans form, the average tilt of the CN bond is in the range 32-39° with respect to the bilayer normal. In the gauche conformation of the choline group this tilt increases to 52-54° with respect to the bilayer normal.54 In the DMPC bilayers the angle of 54° is close to the random distribution, which suggests that the choline moiety can freely rotate and change its transgauche conformation, confirmed by the CN stretching mode analysis. In the monolayer on SiO2, only the trans conformer is present and the average tilt of the choline group is typical for this isomer. Concluding, the interaction with the negatively charged silicate surface influences the conformation and tilt of the choline group in the leaflet facing toward the substrate. In the second leaflet of the bilayer the choline group has conformational freedom. 3.1.3. Ester Group. Figure 5 shows the PM IRRAS spectra of DMPC bilayers in the ν(CdO) stretching band region. In bilayers formed on SiO2 and Au surfaces this band has an absorption maximum at 1738.7 ( 0.2 cm-1. The ν(CdO) band is unsymmetric and can be deconvoluted into two bands centered at 1742.0 ( 0.2 and 1729.5 ( 0.3 cm-1. The band at higher frequency has an fwhm equal to 18.5 cm-1, while the lower frequency peak is broader and the fwhm is equal to 23.0 cm-1. The position and width of the ν(CdO) in various lipids are dependent on the hydration of the ester group. In the case of a dry ester group the maximum of absorption of the ν(CdO) band is centered at 1744-1740 cm-1.63-66 A red shift together with

(61) Liebau, F. Structural Chemistry of Silicates; Springer Verlag: Berlin, 1985.

(62) Akutsu, H. Biochemistry 1981, 20, 7359-7366. (63) Blume, A.; Hubner, W.; Messer, G. Biochemistry 1988, 27, 8239-8249.

Figure 4. PM IRRAS spectra in the CN stretching mode region of the DMPC monolayer and bilayer transferred onto the SiO2 surface (top panels) and onto the Au surface (bottom panel) with deconvolution of the CN stretching modes shown.

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Langmuir, Vol. 24, No. 8, 2008 3927

Figure 5. PM IRRAS spectra in the CdO stretching mode region of the DMPC bilayers transferred onto the (solid line) SiO2 and (dotted line) Au substrates at a surface pressure of 40 mN m-1.

band broadening of the ν(CdO) band is characteristic of hydrated ester moieties.65,66 In investigated lipid bialyers the carbonyl group of the DMPC molecule on SiO2 as well as on Au exists in both dry and hydrated states. The transition dipole moment of ν(CdO) lies along the CdO bond (Figure 1) and provides directly the tilt angle of the CdO double bond in the ester group.54 As shown in Figure 5, the intensities of the ν(CdO) mode in the studied lipid bilayers are comparable. On the SiO2 surface the angle between the transition dipole moment of the ν(CdO) mode and the surface normal is equal to 58.7°. In the bilayer prepared on the gold surface this angle is equal to 56.8°. The crystal structure of the DMPC molecules67,68 shows that the angle between the CdO bond and the direction of the hydrocarbon chains is close to normal. Indeed, the tilt of ν(CdO) close to 58° and the tilt of the hydrocarbon chains close to 30° sum to a right angle. This result shows that the lipid molecules in both bilayers have similar arrangements and long range orders. The interaction with the negatively charged silica surface as was shown influences the orientation of the polar head group region of the lipid molecule. 3.2. Stability of Lipid Bilayers on SiO2 and Au Substrates. The stability of DMPC bilayers deposited on the SiO2 and Au substrates was investigated. After transfer, the lipid bilayers were kept in air, and PM IRRAS spectra were taken within 6 weeks after transfer. Figure 6 shows the PM IRRAS spectra of DMPC bilayers immediately after transfer and after six weeks on the SiO2 (A) and Au (B) surfaces. The CH2 asymemetric methylene stretching mode shifts with time from 2920.7 to 2922.9 cm-1. The position of the symmetric methylene stretching mode shifts only by 0.6 cm-1 to higher wavenumbers. The red shift of the methylene stretching modes indicates that aging of the DMPC bilayer is connected with a fluidification and as a consequence slightly increasing disorder in the acyl chain region. In the CH stretching mode region, the integrated intensity of the CH stretching mode also slightly increases. The increase of the integral intensity of the CH2 methylene stretching modes corresponds to an increase of the tilt angle of the hydrocarbon chains to 31° with the surface normal, thus by 2° on the SiO2 surface and by 3° on the Au surface. (64) Green, P. M.; Mason, J. T.; O’Leary, J.; Levin, I. W. J. Phys. Chem. 1987, 91, 5099-5103. (65) Lewis, R. N. A. H.; McElhaney, R. N.; Pohle, W.; Mantsch, H. H. Biophys. J. 1994, 67, 2367-2375. (66) Popova, A. V.; Hincha, D. K. Biophys. J. 2003, 85, 1682-1690. (67) Aussenac, F.; Laguerre, M.; Schmitter, J. M.; Dufourc, E. J. Langmuir 2003, 19, 10468-10479. (68) Pearson, R. H.; Pascher, I. Nature 1979, 281, 499-501.

Figure 6. PM IRRAS spectra in the 3000-2800 and 1800-1350 cm-1 regions of the DMPC bilayers transferred onto the (A) SiO2 and (B) Au surfaces taken immediately after transfer (solid line) and after six weeks (dashed line).

The low-frequency region of the PM IRRAS spectra shows the CdO stretching mode and the CH bending modes in the choline and hydrocarbon chain regions. In this spectral region in a time of six weeks very small changes in the absorption bands are observed. An increase of the intensity of the ν(CdO) mode indicates slightly larger inclination of the CdO bond toward the bilayer plane. In the CH bending mode region the changes in the spectrum are insignificant when the DMPC bilayer is deposited on the Au surface. On the silica surface changes in the CH bending modes are not detected, confirming strong charge-charge interactions and a frozen conformation of the choline moiety. On the SiO2 surface the 1300-1000 cm-1 spectral region is dominated by strong absorption caused by the νas(SiOSi) mode39-41 not allowing for analysis of the phosphate group stretching modes in the lipid molecule. As a consequence, the orientation and hydration of the phosphate group cannot be studied. On the Au surface however this spectral region is free of absorption, and the analysis of the phosphate group can be provided. Figure 7 shows the PM IRRAS region in the phosphate stretching mode region of DMPC bilayers freshly transferred onto the Au surface and after 2 and 6 weeks from transfer. The phosphate asymmetric stretching mode νas(PO2-) is split into three absorptions with maxima located at 1232.5, 1255.0, and 1280.0 cm-1. The band at 1232 cm-1 has an fwhm equal to 18.5 ( 0.5 cm-1 and corresponds to a well-hydrated, evenly hydrogenbonded phosphate group. The most intense band located at 1255 cm-1 has an fwhm equal to 14.8 ( 0.3 cm-1. This position corresponds to a weakly hydrated phosphate group. The weak absorption at 1280.0 cm-1 has an fwhm equal to 7.3 ( 0.2 cm-1. This band arises from a dry phosphate group. The wide range

3928 Langmuir, Vol. 24, No. 8, 2008

Figure 7. (A) PM IRRAS spectra in the phosphate group stretching mode region of DMPC bilayers on Au surfaces immediately after transfer (solid line) and two (dotted line) and six (dashed line) weeks after transfer. (B) Scheme of the phosphate group with marked directions of the transition dipole moments of the asymmetric and symmetric phosphate stretching modes and their relation to the OPO line of the esterified phophate group.

of absorptions in the asymmetric phosphate stretching region indicates that the phosphate group in the DMPC bilayer has different degrees of hydration from well-hydrated and hydrogenbonded to dry.69 Time does not affect the positions of the νas(PO2-) modes. The symmetric phosphate stretching mode is located at 1100.1 ( 0.3 cm-1, and its position is independent of time. The νs(PO2-) mode is a single peak, and its position is not much affected by hydration changes.69 The integral intensities of the phosphate stretching modes vary with time. The integral intensity of νas(PO2-) changes only slightly. Detailed analysis of deconvoluted peaks shows that the bands arising from the two absorptions, originating from the hydrated phosphate group, decrease in intensity. The intensity arising from the dry phosphate group remains constant in time. This analysis suggests that with time some water is lost by the phosphate group. Small changes of the integrated intensity of the νas(PO2-) mode may indicate that rearrangements in the phosphate group are compensated by hydration changes. However, the integral intensity of the νs(PO2-) mode decreases 2-fold with time. This is more than can be induced exclusively by hydration changes and points to reorientations in the phosphate group region. The transition dipole moment of the symmetric phosphate stretching mode lies along the bisector of the phosphate nonesterified group.54,69 The asymmetric stretching mode lies in the same plane and makes a normal angle to the direction of the dipole moment of the symmetric stretching mode (Figure 7B). Schematically, the arrangement of the phosphate group and directions of the dipole moments of νas(PO2-) and (69) Shimanouchi, T.; Tsuboi, M.; Kyogoko, Y. Infrared Spectra of Nucleic Acids and Related Compounds; Interscience: New York, 1964; Vol. 7, pp 467499.

Zawisza et al.

νs(PO2-) are shown in Figure 7B. The phosphate group is a tetrahedron, where the esterified line of the phosphate group, the [C]OPO[C] line, is normal to the directions of both asymmetric and symmetric dipole moments. On the basis of geometrical considerations, eq 2 can be used to calculate the tilt of the phosphate esterified OPO group in the DMPC molecule. In the freshly formed lipid bilayer this angle is equal to 43.0° with respect to the surface normal. After two weeks the tilt angle decreases to 37° and after six weeks to 36° with respect to the surface normal. The change in the tilt angle of the phosphate esterified group is close to 7°. The phosphate group stays more vertical in the old lipid bilayer. This indicates a decrease in the area per molecule, a consequence of the dehydration of the lipid bilayer. On the silica surface of the same roughness minor changes in the order and structure of the lipid bilayer are observed. The results show that the order in the lipid bilayer is not much affected by time. The DMPC bilayer loses some of its water, but the long-range order does not change. These findings are in agreement with recent neutron reflectivity studies of lipid (DMPA) multibilayers deposited on a quartz substrate.70 The exposition of lipid multi-bilayers to a dry atmosphere does not affect the order in the lipid film. The reduction of the bilayer spacing was detected, due to the loss of water. The thickness of the hydrophobic part of the molecule was constant, and the thickness of the polar head group was reduced by 1.5 Å.70 Concluding, similar orientations of the lipid molecule and similar stabilities of the lipid bilayer can be expected in both Au and SiO2 substrates of the same roughness.

4. Conclusions DMPC bilayers transferred onto SiO2 and Au substrates of the same roughness show similar structures. PM IRRAS analysis of the CH2 stretching modes shows that the acyl chains in the DMPC molecule exist in the ripple phase on the transition to the gel phase. On the Au surface slightly larger disorder in the hydrophobic hydrocarbon chain region is found. The tilt of the hydrocarbon chains is close to 28° with respect to the surface normal. The CdO double bond in DMPC bilayers on Au and SiO2 shows the same tilt angle of ∼57° with respect to the surface normal. The differences in the conformation and tilt of the CN bond of the choline group are found on the silica and gold surfaces. In the first layer of DMPC deposited directly on the silica surface only the trans conformation of the N-C-C-O moiety is found (Figure 8). The tilt of the CN bond is small and equal to 28° with respect to the surface normal. When a bilayer is deposited on the gold surface, the trans conformation of the choline group predominates, but the average tilt of the CN bond increases to 53° with respect to the surface normal. A similar tilt of the CN bond is found on the Au surface, but the presence of two isomers, trans and gauche, is reported. The gauche conformation of the choline group is reported to be present predominantly in the liquid-crystalline and crystal states of lipid bilayers.54,71,72 In lipids existing in the liquid phase more trans conformations of the choline group are found.54 The calculations show that the conformation energies of the trans and gauche forms of choline do not differ much and both isomers of phosphatidylcholine may exist.73 Indeed, experimentally it was confirmed that the choline possesses a flexible conformation, which may be dependent on the temperature, ions present in the solution, or charge ac(70) Haas, H.; Steitz, R.; Fasano, A.; Liuzzi, G. M.; Polverini, E.; Cavatorta, E.; Riccio, P. Langmuir 2007, 23, 8491-8496. (71) Akutsu, H.; Nagamori, T. Biochemistry 1991, 30, 4510-4516. (72) Gally, H. U.; Niederbeger, W.; Seelig, J. Biochemistry 1975, 14, 36473652. (73) Terui, Y. J. Chem. Soc., Perkin Trans. 2 1975, 118-127.

PM IRRAS InVestigation of Silica Films on Gold

Figure 8. Schemes of the orientation of lipid molecules in DMPC bilayer on Au and SiO2 substrates with the marked conformation of the choline group adsorbed directly on the SiO2 surface and on the Au and directed toward air.

Langmuir, Vol. 24, No. 8, 2008 3929

charged polymer surface is different from the structure of the next lipid layer deposited in the second leaflet, directed toward air.75 Time does not affect the structure and order in the lipid bilayers on the SiO2 and Au substrates. The hydrocarbon chains preserve their conformation, order, and tilt. The intermediate ester group does not change its hydration and tilt in DMPC bilayers. The phosphate group is the only moiety which shows time-induced changes in the PM IRRAS spectra. This is indeed the group which in the crystal state possesses water of hydration.38,68 These water molecules are strongly bonded. Indeed, the spectra of the phosphate asymmetric stretching mode show absorption originating from the hydrogen-bonded group even after six weeks of storage in air. The orientation of the phosphate esterified line changes from 43° to 36° with respect to the surface normal. The esterified part of the phosphate group in the bilayer stands more vertically with time. This points out the reduction of the area per molecule in the bilayer, being in agreement with water loss. While the stability and the conformation of transmembrane proteins introduced into the lipid bilayer on the silica surface remain unknown, our results show long-range order in the bilayer and long-time stability and retention of water of hydration in the polar parts of the lipid molecule, fulfilling the requirements of the implant material. Acknowledgment. The Agence Nationale de la Recherche (ANR), the Centre National de la Recherche Scientifique (CNRS), the Institut National Polytechnique de Grenoble (INPG), and the Nord-Pas-de Calais region are gratefully acknowledged for financial support. We thank Dr. V. Zamlynny, Acadia University Canada, for providing the software for calculating reflectivity and the electric field at the interfaces. We also like thank Berthome´ Gre´gory for the XPS analysis. S.S. thanks the Hanse Wissenschaftskollege for a 3.5 month fellowship.

cumulated on the membrane.71,72,74 On the silica surface the charge-charge interaction between the negatively charged silica and positively charged choline guarantees a stiff orientation of the choline group and a frozen tilt angle of the CN bond (Figure 8). This is also reflected in the large stability of the orientation of the choline group in the DMPC bilayer on the SiO2 surface. The absorption arising from the CH bending modes does not change in time over six weeks. It was already reported that the structure of the first lipid monolayer deposited directly on the

LA703651N

(74) Seelig, J.; Macdonald, P. M.; Scherer, P. G. Biochemistry 1987, 26, 75357541.

(75) Wang, L.; Scho¨nhoff, M.; Mo¨chwald, H. J. Phys. Chem. B 2004, 108, 4767-4774.