Sum Frequency Generation (SFG) - American Chemical Society

Apr 16, 2010 - Phosphatidylethanolamine Hybrid Bilayer Membranes under Water ... has been used to study the structure of phosphatidylethanolamine...
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Sum Frequency Generation (SFG) Vibrational Spectroscopy of Planar Phosphatidylethanolamine Hybrid Bilayer Membranes under Water Peter J. N. Kett, Michael T. L. Casford, and Paul B. Davies* Department of Chemistry, University of Cambridge, Lensfield Road, Cambridge CB2 1EW, United Kingdom Received January 26, 2010. Revised Manuscript Received March 19, 2010 Sum frequency generation (SFG) spectroscopy has been used to study the structure of phosphatidylethanolamine hybrid bilayer membranes (HBMs) under water at ambient temperatures. The HBMs were formed using a modified Langmuir-Schaefer technique and consisted of a layer of dipalmitoyl phosphatidylethanolamine (DPPE) physisorbed onto an octadecanethiol (ODT) self-assembled monolayer (SAM) at a series of surface pressures from 1 to 40 mN m-1. The DPPE and ODT were selectively deuterated so that the contributions to the SFG spectra from the two layers could be determined separately. SFG spectra in both the C-H and C-D stretching regions confirmed that a monolayer of DPPE had been adsorbed to the ODT SAM and that there were gauche defects within the alkyl chains of the phospholipid. On adsorption of a layer of DPPE, methylene modes from the ODT SAM were detected, indicating that the phospholipid had partially disordered the alkanethiol monolayer. SFG spectra recorded in air indicated that removal of water from the surface of the HBM resulted in disruption of the DPPE layer and the formation of phospholipid bilayers.

Introduction The membrane of a biological cell plays a crucial role in preserving the cell structure, maintaining homeostasis within the cell, and facilitating intercell interactions.1 Biological membranes are complex environments containing a vast array of different phospholipids, proteins, carbohydrates, and sterols. This complexity has made it difficult to identify the role and function of individual components of the membrane, and so has necessitated the use of significantly simplified model systems to begin to understand the structure, function, and interactions between the different biomolecules.2,3 Several different model systems have been used to mimic biological membranes, including Langmuir-Blodgett films,4 black lipid membranes,5,6 and supported planar membranes.7 The success of these model systems is judged on a range of criteria, including the ease with which they can be formed, their robustness, their similarity to a real biological membrane, the simplicity with which other biomolecules can be introduced into them, and the range of techniques with which they can be studied. Supported planar lipid membranes, in which a monolayer or bilayer of phospholipid is supported on either a metal or nonmetal surface by a thin layer of water, polymer, or self-assembled monolayer (SAM), fulfill many of these criteria. Specifically, hybrid bilayer membranes (HBMs), in which a phospholipid monolayer is supported on a SAM, have been shown to be easy to form, are robust, and can be analyzed using a range of surface sensitive or surface specific techniques.3 HBMs consist of a phospholipid monolayer physisorbed onto a metal surface which has been rendered hydrophobic by an *To whom correspondence should be addressed. E-mail: [email protected]. (1) Alberts, B. M.; Johnson, A.; Lewis, J.; Raff, M.; Roberts, K.; Walter, P. Molecular Biology of the Cell, 5th ed.; Garland Science: New York, 2008. (2) Feigenson, G. W. Annu. Rev. Biophys. Biomol. Struct. 2007, 36, 63. (3) Castellana, E. T.; Cremer, P. S. Surf. Sci. Rep. 2006, 61, 429. (4) Mitchell, M. L.; Dluhy, R. A. J. Am. Chem. Soc. 1988, 110, 712. (5) Ries, R. S.; Choi, H.; Blunck, R.; Bezanilla, F.; Heath, J. R. J. Phys. Chem. B 2004, 108, 16040. (6) Montal, M.; Mueller, P. Proc. Natl. Acad. Sci. U.S.A. 1972, 69, 3561. (7) Tamm, L. K.; McConnell, H. M. Biophys. J. 1985, 47, 105.

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alkanethiol SAM. The phospholipid monolayer is transferred onto the SAM either from the air/water interface of a LangmuirBlodgett (LB) trough8 or by the fusion of phospholipid vesicles.9 The driving force for the adsorption of the phospholipid monolayer is the expulsion of water from the surface of the hydrophobic SAM.10 HBMs have proven to be a useful model for biological membranes as they retain mechanical stability over a period of days or weeks when kept under water, they are relatively easy to form, and other biomolecules such as cholesterol or membrane proteins can be readily introduced into them.3 Furthermore, the formation of HBMs can be studied using a range of surface specific or surface sensitive techniques including surface enhanced Raman scattering (SERS),8 surface plasmon resonance,9 sum frequency generation (SFG) spectroscopy,11 reflection absorption infrared spectroscopy (RAIRS),12 cyclic voltammetry,13 and capacitance measurements.10 Here we report an investigation into the structure of phosphatidylethanolamine (PE) HBMs in contact with a thin overlayer of water (thickness ∼ 1 μm). Specifically, the HBM consisted of a dipalmitoyl phosphatidylethanolamine (DPPE, Figure 1) monolayer physisorbed onto a hydrophobic octadecanethiol (ODT) SAM from the air/water interface of an LB trough. The packing density and fluidity of a covalently bound SAM are inherently different from that of the lower leaflet of a biological membrane. As such, it is important to fully characterize the relevant HBM over as wide a range of deposition pressure conditions as possible in order to understand what effect the deposition of the monolayer has on both the SAM and the resulting lipid monolayer. (8) Meuse, C. W.; Niaura, G.; Lewis, M. L.; Plant, A. L. Langmuir 1998, 14, 1604. (9) Fischer, T.; Senin, I. I.; Philippov, P. P.; Koch, K. W. Spectroscopy 2002, 16, 271. (10) Plant, A. L. Langmuir 1993, 9, 2764. (11) Anderson, N. A.; Richter, L. J.; Stephenson, J. C.; Briggman, K. A. J. Am. Chem. Soc. 2007, 129, 2094. (12) Meuse, C. W.; Krueger, S.; Majkrzak, C. F.; Dura, J. A.; Fu, J.; Connor, J. T.; Plant, A. L. Biophys. J. 1998, 74, 1388. (13) Peng, Z. Q.; Tang, J. L.; Han, X. J.; Wang, E. K.; Dong, S. J. Langmuir 2002, 18, 4834.

Published on Web 04/16/2010

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Figure 1. Molecular structures of DPPE and d62-DPPE.

Adsorbing the phospholipid from the LB trough allowed the surface pressure and area per molecule of the DPPE to be carefully controlled. HBMs can also be formed by the fusion of phospholipid vesicles to a hydrophobic SAM. Whilst no significant difference has been reported in the structure or properties of the HBMs formed using either technique, vesicle fusion does not allow for any control over the packing density of the lipid layer.12,14 DPPE layers were adsorbed for surface pressures ranging from 1 to 40 mN m-1, which encompasses both the liquidcondensed and liquid-expanded phases of the monolayer, and the most biologically relevant range from 30 to 40 mN m-1.15,16 The structure of the DPPE monolayer, or the underlying SAM, was investigated by selectively deuterating the upper or lower leaflet of the HBM. Model membranes containing DPPE were examined, as the use of PEs in HBMs has previously been limited in spite of them being an important class of phospholipids constituting the second most abundant group in eukaryotic cell membranes and the most common group in the cell membranes of bacteria.1 Furthermore, as PEs can be chemically tethered to a metal surface via a linker molecule, they can be used to form phospholipid bilayers on gold, representing an even more biologically relevant model system than the HBMs investigated here.9 This study therefore forms the beginning of a wider investigation into the formation of model biological membranes composed of a bilayer of DPPE, in which the lower leaflet of the phospholipid is chemically tethered to the gold surface via an 11-mercaptoundecanoic acid linker, creating a hydrophobic surface onto which the second layer of phospholipid can be adsorbed using the technique outlined in this study. The HBMs were investigated using SFG spectroscopy which is a laser based technique for obtaining the vibrational spectrum of molecules at an interface or on a surface. The spectrum is recorded by temporally and spatially overlapping a fixed frequency visible laser beam, and a tunable infrared beam on a surface or interface. Due to the nonlinear optical phenomenon of SFG, photons are emitted from the surface with a frequency at the sum of the two incident beams. Monitoring the SFG intensity as the infrared beam is swept through the relevant frequency range allows for the recording of the vibrational spectra of the molecules adsorbed to the surface. It is possible to determine the polar orientation, (14) Levy, D.; Briggman, K. A. Langmuir 2007, 23, 7155. (15) Demel, R. A.; Geurtsvankessel, W. S. M.; Zwaal, R. F. A.; Roelofsen, B.; Vandeenen, L. L. M. Biochim. Biophys. Acta 1975, 406, 97. (16) Nagle, J. F. J. Membr. Biol. 1976, 27, 233.

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conformation, and ordering of any adsorbed molecules from the SFG spectrum.17 Its surface specificity and conformational sensitivity have made it an ideal technique for investigating the adsorption of surfactants, polymers, and phospholipids onto surfaces and interfaces.18-22 Detailed descriptions of the theory of SFG spectroscopy can be found elsewhere.17,23,24 SFG spectroscopy is now a widely used technique for studying the structure of model biological membranes and the interactions between different biomolecules.25 It has been used to investigate the formation of model membranes on both metal and nonmetal surfaces as well as at the air/water interface.19,26,27 SFG spectroscopy has also been used to examine the phase transition temperature of phosphatidylcholine (PC) monolayers,11,28 interaction between PC and cholesterol,14,20 and dynamic membrane processes such as membrane lipid flip-flop.29,30 SFG spectroscopy has previously been used to examine the structure of HBMs containing phosphatidylcholine,11,31 but this is the first reported SFG spectroscopy study of a phosphatidylethanolamine HBM.

Experimental Section Octadecanethiol (h-ODT, 98%) and chloroform (g99.9%) were purchased from Aldrich. 1,2-Dipalmitoyl-sn-glycero-3-phosphatidylethanolamine (h-DPPE) and 1,2-dipalmitoyl-d62-sn-glycero-3-phosphatidylethanolamine (d-DPPE) were purchased from Avanti Polar Lipids (Alabaster, AL) and used as received. The substrates used were formed by the thermal evaporation under high vacuum (>10-5 mPa) of a 200 nm gold layer onto (17) Lambert, A. G.; Davies, P. B.; Neivandt, D. J. Appl. Spectrosc. Rev. 2005, 40, 103. (18) Kett, P. J. N.; Casford, M. T. L.; Yang, A. Y.; Lane, T. J.; Johal, M. S.; Davies, P. B. J. Phys. Chem. B 2009, 113, 1559. (19) Watry, M. R.; Tarbuck, T. L.; Richmond, G. I. J. Phys. Chem. B 2003, 107, 512. (20) Liu, J.; Conboy, J. C. Vib. Spectrosc. 2009, 50, 106. (21) Casford, M. T. L.; Davies, P. B. J. Phys. Chem. B 2008, 112, 2616. (22) Casford, M. T. L.; Davies, P. B.; Neivandt, N. J. Langmuir 2006, 22, 3105. (23) Shen, Y. R. The Principles of Nonlinear Optics; John Wiley & Sons: New York, 1984. (24) Bain, C. D. J. Chemi. Soc., Faraday Trans. 1995, 91, 1281. (25) Chen, X. Y.; Clarke, M. L.; Wang, J.; Chen, Z. Int. J. Mod. Phys. B 2005, 19, 691. (26) Liu, J.; Conboy, J. C. J. Am. Chem. Soc. 2004, 126, 8894. (27) Gurau, M. C.; Castellana, E. T.; Albertorio, F.; Kataoka, S.; Lim, S. M.; Yang, R. D.; Cremer, P. S. J. Am. Chem. Soc. 2003, 125, 11166. (28) Anderson, N. A.; Richter, L. J.; Stephenson, J. C.; Briggman, K. A. Langmuir 2006, 22, 8333. (29) Anglin, T. C.; Liu, J.; Conboy, J. C. Biophys. J. 2007, 92, L1. (30) Liu, J.; Conboy, J. C. Biophys. J. 2005, 89, 2522. (31) Petralli-Mallow, T.; Briggman, K. A.; Richter, L. J.; Stephenson, J. C.; Plant, A. L. Proc. SPIE 1999, 25.

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Figure 2. Surface pressure-area isotherm of DPPE on the surface of ultrapure water at 20 °C. precleaned silicon wafers, which had been primed with a thin layer of chromium (∼10 nm). The gold surfaces were rendered hydrophobic by placing them into methanolic solutions of h- or d-ODT (concentration in mM) for a minimum of 12 h. The solutions were prepared by sonicating a microdot amount of thiol in HPLCgrade methanol at 45 °C for 40 min. The silicon wafers, glassware, and stainless steel liquid cell were cleaned before use by placing them in a detergent solution (Decon 90) overnight, rinsing 20 times in ultrapure water (resistivity > 18.2 MΩ cm), leaving them overnight in concentrated nitric acid, and finally rinsing again in ultrapure water. The O-rings, fused silica, and CaF2 prisms were cleaned in detergent solution and rinsed in ultrapure water. The DPPE HBM was prepared by the transfer of a single layer of phospholipid from the air/water interface of a Nima 611 Langmuir-Blodgett (LB) trough (Coventry, U.K.) using a modified Langmuir-Schaefer (LS) method (see the Supporting Information). A custom built stainless steel liquid cell with a fused silica (when recording spectra in the C-H region) or CaF2 (when recording spectra in the C-D region) 60° equilateral prism attached was submerged into the ultrapure water subphase (resistivity > 18.2 MΩ cm) of the LB trough, and an isotherm was recorded to ensure that the water surface was clean. The DPPE was dissolved in chloroform (1 mg/mL) and gently warmed to approximately 30 °C to ensure the phospholipid had completely dissolved. An aliquot of solution was spread on the water surface of the LB trough and left for 30 min to allow the solvent to evaporate off. A surface pressure-area isotherm of DPPE was recorded (Figure 2), and the barriers of the trough were compressed to the required surface pressure. An ODT-covered gold substrate was held parallel to the water surface on the dipping mechanism of the LB trough by an aspirator pump. The sample was lowered onto the water surface at rate of 1 mm/min and contacted on the surface for 30 s. Instead of following the conventional LS technique and withdrawing the sample back into air, the substrate was slowly pushed through the DPPE monolayer into the water subphase. Once the sample was fully submerged, the remaining phospholipid was drawn off the water surface. The sample was slowly lowered into the awaiting liquid cell until there was an approximately micrometer thick layer of water between the sample surface and the prism. Once the sample had been pushed through the air/water interface, it remained submerged in the pure water subphase such that it was never exposed to air. The back plate of the liquid cell was screwed down to seal the cell. The liquid cell containing the sample was removed from the LB trough, and the sum frequency spectrum was recorded. All the spectra reported here were recorded under water except those shown in Figure 10. The SFG spectra recorded in the C-D region have a lower signal-to-noise ratio than those in the 9712 DOI: 10.1021/la1003512

Kett et al. C-H region due to a combination of both fundamental and experimental factors. In the C-D, region there is a reduction in the IR generation efficiency of the spectrometer and a reduction in the transmission of the SFG output through the bandpass filter of the photomultiplier tube. In addition to which, the C-D resonances are inherently weaker than the C-H resonances, leading to the significant decrease in the signal-to-noise ratio observed in these spectra. The samples were formed and the spectra recorded at 20 °C, which is well below the gel-liquid and lamellar-inverted hexagonal phase transition temperatures for DPPE in aqueous solution.32,33 The SFG spectra were recorded using the Cambridge Nanosecond spectrometer, the details of which can be found elsewhere.34 The visible and infrared laser beams were aligned in a counter propagating geometry, such that their angles of incidence at the gold/water interface were 60° and 65°, respectively. Spectra were recorded in both the PPP (SFG, visible, infrared) and SSP polarization combinations, in the C-H (2800-3000 cm-1) and C-D (2025-2250 cm-1) stretching regions. A minimum of 20 scans were averaged to produce the final spectra. The spectra were normalized and then modeled using a least-squares Levenberg-Marquardt algorithm to fit the resonance profiles to a Lorentzian description of the second-order susceptibilities.18,35 The spectra were repeated on at least two occasions to ensure that the results were satisfactorily reproducible.

Results The PPP and SSP SFG spectra in the C-H stretching region of an h-ODT SAM on gold under a thin layer of water were recorded prior to the adsorption of a layer of DPPE (Figure 3a and b). ODT forms a highly ordered crystalline SAM, with the alkyl chains adopting an all-trans conformation and tilting at ∼30° to the surface normal.36 The local centers of symmetry along the alkyl chains result in the methylene groups being SFG inactive, and the only contribution to the SFG spectra comes from the terminal methyl group. The three peaks in the PPP spectrum can be assigned to the asymmetric methyl stretch (r-, 2960 cm-1), the symmetric methyl stretch (rþ, 2869 cm-1), and its Fermi reso-1 17 nance (rþ FR, 2931 cm ). In the SSP spectrum, two dips can be modeled at 2871 and 2933 cm-1 due to the rþ and rþ FR modes. The r- mode is absent from the SSP spectrum, as its transition dipole moment lies principally parallel to the gold surface in the ODT SAM and in SSP spectra only vibrational modes with a component of their transition dipole moment along the surface normal can be detected. The change from peaks in the PPP spectrum of h-ODT to dips in the SSP spectrum is due to a difference in sign of the phase of the nonresonant susceptibility in the two polarization combinations.37 The PPP and SSP spectra of a d-ODT SAM on gold under water in the C-H stretching region were completely devoid of spectral features, and they are shown in the Supporting Information. The SFG spectra of h-ODT and d-ODT SAMs in the PPP and SSP polarization combinations were also recorded in the C-D region (Figure 3c and d). No resonances were observed for h-ODT in this region in either polarization combination (see the Supporting Information). For the d-ODT SAM in the PPP polarization combination, three peaks were present and modeled -1 (r ). In the at 2069 cm-1 (rþ), 2126 cm-1 (rþ FR), and 2222 cm

(32) Seddon, J. M.; Cevc, G.; Marsh, D. Biochemistry 1983, 22, 1280. (33) Huang, C. H.; Li, S. S. Biochim. Biophys. Acta, Rev. Biomembr. 1999, 1422, 273. (34) Lambert, A. G.; Neivandt, D. J.; Briggs, A. M.; Usadi, E. W.; Davies, P. B. J. Phys. Chem. B 2002, 106, 10693. (35) Casford, M. T. L.; Davies, P. B. ACS Appl. Mater. Interfaces 2009, 1, 1672. (36) Schreiber, F. Prog. Surf. Sci. 2000, 65, 151. (37) Ward, R. N.; Davies, P. B.; Bain, C. D. J. Phys. Chem. 1993, 97, 7141.

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Figure 3. Sum frequency spectra recorded of (a) h-ODT SAM on gold in the C-H stretching region and PPP polarization combination, (b) h-ODT SAM on gold in the C-H stretching region and SSP polarization combination, (c) d-ODT SAM on gold in the C-D stretching region and PPP polarization combination, and (d) d-ODT SAM on gold in the C-D stretching region and SSP polarization combination. All spectra were recorded with the SAMs in contact with a thin film of water.

SSP spectrum, two dips due to rþ (2061 cm-1) and rþ FR (2131 cm-1) were observed. The PPP SFG spectrum in the C-H region of a layer of h-DPPE physisorbed onto a d-ODT SAM from the air/water interface of a LB trough at 30 mN m-1 is shown in Figure 4a. The spectrum comprises a series of dips, which can be assigned to the vibrational modes of h-DPPE as d-ODT does not have any SFG active modes in this frequency region. The change from peaks in the spectrum of h-ODT (Figure 3a) to dips in the PPP spectrum of the HBM indicates a change in the sign of the phase of the resonant nonlinear susceptibility. This in turn implies a reversal in the polar orientation of the SF active groups of DPPE relative to h-ODT. In the SSP spectrum of an h-DPPE/d-ODT HBM (Figure 4b), two peaks can be modeled. The change from dips in the SSP spectrum of h-ODT (Figure 3b) to peaks in the SSP spectrum of h-DPPE on d-ODT confirms the reversal in polar orientation of the SF active groups of the phospholipid relative to those of ODT. Figures 5 and 6 show the PPP and SSP spectra in the C-H stretching region of a layer of h-DPPE deposited onto d-ODT at five surface pressures from 1 to 40 mN m-1. The HBMs with DPPE adsorbed between 10 and 40 mN m-1 correspond to a lipid layer in the liquid-condensed phase, while the HBM with the phospholipid layer adsorbed at 1 mN m-1 corresponds to a lipid layer in the liquid-expanded phase. For the spectra recorded in the liquid condensed phase (10-40 mN m-1), six resonances can be modeled at ∼2850, 2868, 2892, 2915, 2930, and 2961 cm-1, all appearing as dips. Aside from the resonance at 2850 cm-1, which showed some degree of variability between the spectra, the position of these spectral features did not change significantly with the surface pressure at which the DPPE was adsorbed. The Langmuir 2010, 26(12), 9710–9719

three dips at 2868, 2930, and 2961 cm-1 can be assigned to the three resonances of the two methyl groups of h-DPPE. The three remaining resonances, which are considerably weaker than the r resonances, can be assigned to the methylene antisymmetric stretch (d-, 2915 cm-1), symmetric stretch (dþ, 2850 cm-1), and -1 its Fermi resonance (dþ FR, 2892 cm ). The SSP spectra of the h-DPPE/d-ODT HBM could be modeled at all surface pressures at which the DPPE was adsorbed, with a peak at 2874 cm-1 due to the rþ resonance and a second weaker peak, with a more differential line shape, at 2933 cm-1 due to the rþ FR resonance (Figure 6). The two methyl peaks in the SSP spectra indicate that for all surface pressures at which the layer of DPPE was physisorbed, the methyl groups of the DPPE adopted the opposite polar orientation to those in ODT, for which the r resonances appeared as dips (Figure 3b). The r- resonance and methylene d resonances were absent from the SSP spectra of all the h-DPPE/d-ODT HBMs. The spectra recorded in the liquid expanded phase (1 mN m-1) show significant differences to those recorded at higher surface pressures, notably the almost complete absence of the dþ symmetric stretch and the markedly weaker intensity of all other resonances present in the spectrum. DPPE contains methylene groups both in its headgroup and two long alkyl chains, which, if they are in non-centrosymmetric environments, could be responsible for the d resonances observed in the SFG spectrum. To determine the contribution to the SFG spectra of the DPPE headgroups, spectra of d-DPPE/d-ODT HBMs in the C-H stretching region were recorded. Only the long alkyl chains of d-DPPE were perdeuterated leaving the headgroups perprotonated (Figure 1). It was not possible to model any resonances in the SFG spectra of the d-DPPE/d-ODT HBMs DOI: 10.1021/la1003512

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Figure 4. Sum frequency spectra recorded in the C-H stretching region of an h-DPPE/d-ODT HBM (a) in the PPP polarization combination and (b) in the SSP polarization combination. The h-DPPE was physisorbed at a surface pressure of 30 mN m-1, and all spectra were recorded with the HBM in contact with a thin film of water.

irrespective of the surface pressure at which the d-DPPE layer was adsorbed (see the Supporting Information). This implies that the methylene resonances in the SFG spectra of h-DPPE/ d-ODT HBMs (Figure 5) are solely due to CH groups in the polymethylene chains. To confirm that there were SF active methylene modes in the alkyl chains of the DPPE layer, SFG spectra of d-DPPE/h-ODT HBMs were recorded in the C-D region (Figure 7). In this HBM, the only groups which were deuterated and could contribute to the SFG spectra were in the long alkyl chains of the phospholipid. At each of the three surface pressures the DPPE was adsorbed to the h-ODT SAM, the PPP spectra could be modeled with six features which are spectral dips. The rþ resonance appeared as a strong spectral feature at 2063 cm-1, while the other two methyl resonances were somewhat weaker in intensity at 2130 cm-1 (rþ FR) and 2218 cm-1 (r-). The dþ and dþ FR resonances appeared as medium to weak intensity features at 2113 and 2138 cm-1, respectively, while the d- resonance appeared as a weak shoulder on the r- resonance at 2203 cm-1. The positions of these features and their relative intensities are in good agreement with other SFG studies on deuterated phospholipids.11,14 The presence of CD2 resonances in these spectra indicates that there are sum frequency active methylene modes in the long alkyl chains of DPPE, confirming the findings from the C-H region. The CD2 resonances in Figure 7 are more intense, relative to the methyl resonances, than those in the corresponding spectrum in the C-H region (Figure 5). 9714 DOI: 10.1021/la1003512

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Figure 5. Sum frequency spectra in the PPP polarization combination and the C-H stretching region of an h-DPPE/d-ODT HBM in contact with a thin film of water, with the h-DPPE physisorbed at a surface pressure of (a) 1 mN m-1, (b) 10 mN m-1, (c) 20 mN m-1, (d) 30 mN m-1, and (e) 40 mN m-1.

Such an increase in intensity of the methylene resonances in the C-D region has previously been observed.11,14,38 SFG spectra of d-DPPE/h-ODT HBMs in the C-D stretching region were also recorded in the SSP polarization combination. In these spectra, a strong peak at 2071 cm-1 along with a weaker peak at 2128 cm-1 could be modeled. The appearance of peaks in these spectra, as opposed to the dips modeled in the SSP spectrum of d-ODT, confirms the finding from the C-H region that the DPPE is physisorbed with the methyl groups pointing toward the ODT SAM and gold surface. SFG spectra were recorded in the PPP polarization combination of a d-DPPE and h-ODT HBM in the C-H region to determine the effect adsorption of the phospholipid layer had on the structure of the ODT SAM. Spectra were recorded for d-DPPE layers physisorbed from the air/water interface at three different surface pressures between 1 and 40 mN m-1 (Figure 8). Irrespective of the surface pressure at which the DPPE was adsorbed, the spectra displayed three strong resonances at 2869, 2928, and 2960 cm-1 which can be assigned to the three methyl stretches. However, two generally weaker peaks, at ∼2850 and 2907 cm-1, could also be modeled. These peaks must be due to resonances from the h-ODT SAM, as d-DPPE has no strong SFG resonances in the C-H region (Supporting Information Figure S3). (38) Yang, C. S. C.; Richter, L. J.; Stephenson, J. C.; Briggman, K. A. Langmuir 2002, 18, 7549.

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Figure 6. Sum frequency spectra in the SSP polarization combination in the C-H stretching region of an h-DPPE/d-ODT HBM in contact with a thin film of water, with the h-DPPE physisorbed at a surface pressure of (a) 1 mN m-1, (b) 10 mN m-1, (c) 20 mN m-1, (d) 30 mN m-1, and (e) 40 mN m-1.

These two peaks, which can be assigned to the dþ and dþ FR resonances, indicate that on adsorption of DPPE gauche defects are introduced into the alkyl chains of ODT. To confirm that the methylene resonances seen in Figure 8 were from the ODT SAM, the PPP SFG spectra of an h-DPPE/d-ODT HBM were recorded in the C-D region (Figure 9). For this HBM, contributions to the SFG spectra could only come from the deuterated SAM. In each of the spectra, three strong methyl -1 resonances at 2062 cm-1 (rþ), 2126 cm-1 (rþ FR), and 2216 cm (r ) could readily be modeled. Furthermore, in all the spectra, a peak could be modeled at 2095 cm-1 which can be assigned to the dþ resonance, and a weaker feature, which appeared as a shoulder on the r- resonance, could be modeled at 2196 cm-1 and assigned to the d- resonance. The appearance of methylene modes in the SFG spectrum confirmed that, on adsorption of a layer of DPPE, gauche defects are introduced into the alkyl chains of ODT.

Discussion The SFG spectra recorded using the PPP polarization combination in the C-H region of the h-ODT SAM and of the h-DPPE/d-ODT HBM confirmed that DPPE had been adsorbed to the SAM. In the PPP spectra, a reversal in the sign of the phase of the resonant susceptibility of the methyl groups of h-DPPE relative to h-ODT was observed. As this polarization combination probes four separate components of χ(2), the overall phase of the resonant susceptibility is dependent not only on the individual phases of the resonant susceptibilities but also on their relative magnitudes. The reversal in phase of the resonant susceptibility does not unambiguously prove that the methyl groups of ODT and DPPE have opposite polar orientations. Langmuir 2010, 26(12), 9710–9719

The absolute polar orientation of the DPPE layer can be unambiguously determined from the SSP spectrum, as this polarization combination probes only a single component of the nonlinear susceptibility, χ(2) yyz. Only vibrational modes that have a component of their transition dipole moment along the surface normal can contribute to the SSP spectrum. In the SSP spectrum of h-ODT, two dips could be modeled for the methyl symmetric stretch and its Fermi resonance. In the equivalent SSP spectrum of an h-DPPE/ d-ODT HBM, two peaks could be assigned to the resonances of the two methyl groups of the phospholipid. The change from dips in the h-ODT spectrum to peaks in the h-DPPE/d-ODT HBM spectrum indicates a change in the sign of the phase of the resonant susceptibility and hence a change in polar orientation of the methyl groups of DPPE relative to those of h-ODT. As the methyl groups of the hODT SAM are pointing away from the gold surface, the SSP spectra indicate that the methyl groups of the phospholipid in the HBM are oriented toward the ODT SAM and gold surface. The r- mode of DPPE was absent from the SSP spectra of the HBMs in both the C-H and C-D stretching regions, indicating that the component of the transition dipole moment of the rresonance along the surface normal was negligible. This implies that the alkyl chains of DPPE in the HBM are aligned with the surface normal, as the transition dipole moment of the methyl asymmetric stretch is perpendicular to the alkyl chain axis.39 The absence of a strong r- resonance in the SSP SFG spectrum has previously been used as evidence that the methyl groups are aligned with the surface normal for surfactants at the water/carbon tetrachloride interface,40 amino acids at the oil/water interface,41 and phospholipids at the vapor/water interface.19 The SSP and PPP spectra presented here are therefore consistent with the adsorption of a single layer of DPPE to the ODT SAM, in which the methyl groups of the phospholipid are in contact with the hydrophobic SAM, and the alkyl chains aligned with the surface normal. The PPP SFG spectra of d-DPPE/d-ODT HBMs in the C-H region and d-DPPE/h-ODT HBMs in the C-D region were used to determine the relative contributions to the SFG spectra from the headgroup and alkyl chains of DPPE. The absence of any detectable resonances in the spectra of d-DPPE/d-ODT HBMs in the C-H region indicated that the CH and CH2 stretching modes of the DPPE headgroup were either SFG inactive or their spectra were below the level of instrumental detection. The presence of CD2 resonances in the spectra of the d-DPPE/h-ODT HBMs in the C-D region indicated that there were SFG active methylene modes in the alkyl chains of DPPE. This implies that the alkyl chains of DPPE contain gauche defects. Weak methylene modes and hence the presence of gauche defects have previously been detected in the SFG spectra of phospholipids in HBMs42 at the liquid/liquid interface43 and at the air/water interface.44 Methylene (d) resonances were absent from the SSP spectra in both the C-H and C-D regions, indicating that the components of the transition dipole moments of the dþ and d- modes along the surface normal were negligible. This suggests that, even at gauche defects, the alkyl chains of DPPE were aligned with the surface normal and hence the methylene groups and their transition dipole moments were principally oriented parallel to the metal surface. (39) Allara, D. L.; Nuzzo, R. G. Langmuir 1985, 1, 52. (40) Conboy, J. C.; Messmer, M. C.; Richmond, G. L. J. Phys. Chem. B 1997, 101, 6724. (41) Watry, M. R.; Richmond, G. L. J. Phys. Chem. B 2002, 106, 12517. (42) Doyle, A. W.; Fick, J.; Himmelhaus, M.; Eck, W.; Graziani, I.; Prudovsky, I.; Grunze, M.; Maciag, T.; Neivandt, D. J. Langmuir 2004, 20, 8961. (43) Walker, R. A.; Gruetzmacher, J. A.; Richmond, G. L. J. Am. Chem. Soc. 1998, 120, 6991. (44) Can, S. Z.; Chang, C. F.; Walker, R. A. Biochim. Biophys. Acta, Biomembr. 2008, 1778, 2368.

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Figure 7. Sum frequency spectra in the C-D stretching region of a d-DPPE/h-ODT HBM in contact with a thin film of water, recorded in the PPP

polarization combination with the d-DPPE physisorbed at a surface pressure of (a) 1 mN m-1, (b) 20 mN m-1, and (c) 40 mN m-1, and recorded in the SSP polarization combination with the d-DPPE physisorbed at a surface pressure of (d) 1 mN m-1, (e) 20 mN m-1, and (f) 40 mN m-1.

The d resonances were however observed in the PPP spectra, as this polarization combination also probes vibrational modes with a component of their transition dipole moment parallel to the metal surface. The positions of the r, d-, and dþ FR resonances in the SFG spectra of the HBM formed with the lipid layer in the liquid-condensed phase were independent of the surface pressure at which the DPPE layer was adsorbed. The relative intensities of these peaks were markedly similar in each of the spectra. While the dþ resonance does appear reproducibly in the spectra of the 10-40 mN m-1 films, the exact position of this feature did show some variability between repeats and it is 9716 DOI: 10.1021/la1003512

therefore difficult to make any definitive structural interpretation of the HBM based solely on the shift of this resonance. The ratio of the intensity of the symmetric methylene stretch to the symmetric methyl stretch, dþ/rþ, can be used as a measure of the conformational order of the alkyl chains. For all the HBMs with the DPPE layer in the liquid-condensed phase, the ratio of the intensities of the symmetric stretches were ∼0.4, corresponding to a well-ordered film.45 There was (45) Ward, R. N.; Duffy, D. C.; Davies, P. B.; Bain, C. D. J. Phys. Chem. 1994, 98, 8536.

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Figure 8. Sum frequency spectra in the PPP polarization combination and the C-H stretching region of a d-DPPE/h-ODT HBM in contact with a thin film of water, with the d-DPPE physisorbed at a surface pressure of (a) 1 mN m-1, (b) 20 mN m-1, and (c) 40 mN m-1. The sum frequency spectrum of the h-ODT SAM prior to adsorption of a layer of DPPE is shown below the other spectra for ease of comparison.

no trend observed in the degree of conformational order of the alkyl chains of DPPE as the surface pressure was increased. This is not a surprising result, as the DPPE layer was in the liquid-condensed phase in each of these HBMs, which corresponds to an already well-ordered monolayer, and so significant changes in the alkyl chain conformation on further compression of the film are not anticipated. Compression of the film from 10 to 40 mN m-1 resulted in a reduction in area per molecule of just 9 A˚2 (∼20% reduction). The intensities of the peaks in the spectrum corresponding to a layer of DPPE adsorbed in the liquid-expanded phase were markedly weaker. This is likely to be due to the lipid layer having less overall order as a result of the lower packing density of the phospholipid molecules. Due to the weak overall intensity of the 1 mN m-1 spectrum, there is no significant evidence of the dþ resonance above the noise level at 2850 cm-1. It is therefore not possible to determine to any satisfactory degree of accuracy the dþ/rþ ratio for this HBM and consequently to directly compare the ordering of the alkyl chains of the lipid in the liquid-expanded and liquid-condensed phases. Theoretical calculations and infrared spectroscopic measurements of DPPE have shown that the alkyl chains of the phospholipid pack closely together due to the relatively small size of (46) Bouchet, A. M.; Frias, M. A.; Lairion, E.; Martini, F.; Almaleck, H.; Gordillo, G.; Disalvo, E. A. Biochim. Biophys. Acta, Biomembr. 2009, 1788, 918.

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Figure 9. Sum frequency spectra in the PPP polarization combination and C-D stretching region of an h-DPPE/d-ODT HBM in contact with a thin film of water, with the h-DPPE physisorbed at a surface pressure of (a) 1 mN m-1, (b) 20 mN m-1, and (c) 40 mN m-1. The sum frequency spectrum of the d-ODT SAM prior to adsorption of a layer of DPPE is shown below the other spectra for ease of comparison.

the headgroup and the strong electrostatic and hydrogen bonding interactions between neighboring molecules.46,47 A conclusion that is supported by the observed pressure area isotherm shown in Figure 2, which gives an area per molecule of ca. 35 A˚2 at the point of film collapse. In spite of this close packing, a recent molecular dynamics simulation of DPPE at 290 K has shown that approximately 10% of its C-C bonds are in a gauche conformation.48 A similar result was obtained for a phosphatidylcholine HBM by studying the spacing in a RAIRS spectrum between bands in the methylene wagging progression of the alkyl chains of the lipid.12 The conclusion from the SFG spectra that there are gauche defects in the alkyl chains of DPPE in the HBMs is therefore consistent with previous studies of this phospholipid. It was not possible to detect any contribution to the SFG spectra from the DPPE headgroups in spite of them containing CH and CH2 groups in non-centrosymmetric environments. Determining why the DPPE headgroup does not contribute to the SFG spectra is beyond the scope of this study, although it should be noted that the absence of methylene resonances from the headgroup in a SFG spectrum of a phospholipid has been previously reported, albeit for a phosphatidylcholine rather than a phosphatidylethanolamine monolayer.49 (47) Pimthon, J.; Willumeit, R.; Lendlein, A.; Hofmann, D. J. Mol. Struct. 2009, 921, 38. (48) Leekumjorn, S.; Sum, A. K. Biophys. J. 2006, 90, 3951. (49) Bonn, M.; Roke, S.; Berg, O.; Juurlink, L. B. F.; Stamouli, A.; Muller, M. J. Phys. Chem. B 2004, 108, 19083.

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Figure 11. Schematic showing (a) the structure of the HBM formed in air, (b) the reorientation of a layer of DPPE on drying the sample, (c) the mechanism by which a second layer of DPPE is pulled from the air/water interface.

Figure 10. Sum frequency spectra in the PPP polarization combination and C-H stretching region of h-DPPE/d-ODT HBMs recorded in air of samples dried by (a) evaporating the water in ambient laboratory conditions and (b) blotting the sample with filter paper.

The SFG spectra of d-DPPE/h-ODT HBMs in the C-H stretching region and h-DPPE/d-ODT HBMs in the C-D stretching region were recorded to determine the effect that adsorption of the phospholipid layer had on the ODT SAM structure. Weak methylene resonances which were not observable in the SFG spectrum of the pure ODT SAM in both the C-H and C-D regions could be modeled. These resonances were assigned to vibrational modes of ODT, as the only perdeuterated groups of the h-DPPE/d-ODT HBM or perprotonated groups of the d-DPPE/h-ODT HBM were in the SAM. The appearance of weak methylene modes from the ODT indicates that, on adsorption of a layer of DPPE, some gauche defects are introduced into the polymethylene chains of ODT and that the ordering of the SAM is reduced. This was most evident in the C-D region, where the d resonances are more intense relative to the r resonances than in the case of the C-H region, and therefore more readily detected. The ratio of the intensity of the dþ to rþ resonances in the SFG spectra of the d-DPPE/h-ODT HBMs in the C-H region is a measure of the disorder of the alkyl chains of the ODT SAM. For all three of the surface pressures at which DPPE was adsorbed, dþ/rþ ≈ 0.4, implying that there were only a small number of gauche defects in the ODT SAM and that the polymethylene chains in the alkanethiol layer are predominantly still in an alltrans conformation.45 On adsorption of the phospholipid layer, it is unlikely that there is significant interdigitation between the alkyl chains of DPPE and ODT because of the high packing density of the SAM. Interdigitation in an HBM between the lipid layer and the SAM has been shown to occur if the packing density (50) Cheng, W. L.; Han, X. J.; Wang, E.; Dong, S. J. Electroanalysis 2004, 16, 127.

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of the alkanethiol SAM is significantly lower.13,50 The gauche defects in the alkyl chains of the ODT SAM are most likely to occur at the chain ends.51 Therefore, on adsorption of the DPPE layer, a disordering of the terminal methyl groups resulting from the introduction of chain-end gauche defects is the most likely explanation for the appearance of the d resonances in the SFG spectra in Figures 8 and 9. Adsorption of a layer of lipid to an alkanethiol SAM has previously been shown to have an affect on some of the physical properties of the phospholipid layer including the phase transition temperature.28 It is possible that the interaction between the alkyl chains of the lipid layer and the alkanethiol SAM that results in the slight disordering of the ODT layer is responsible for these changes. Alternatively, we can speculate that, in contrast to studies carried out on atomically flat gold, the thermally evaporated gold samples used in this work contain a sufficient number of surface defects, such as height steps or strong curvature at grain boundaries, such that the adsorbed lipid film can interact sufficiently with the ODT SAM chain ends to give rise to the small degree of disordering of the ODT monolayer observed in these results. It has previously been assumed that the structure of the close packed and highly ordered ODT monolayer would not be affected by adsorption of a phospholipid layer.8,12,52 Infrared and Raman spectroscopic investigations along with molecular dynamics simulations of phosphatidylcholine containing HBMs led to the conclusion that there is no change in the ordering and structure of the ODT SAM on adsorption of the phospholipid.8,53 For HBMs containing a shorter chained alkanethiol, subtle changes in the infrared spectrum of the SAM on adsorption of the phospholipid have been noted, although different studies have interpreted this as either an ordering8 or a disordering54 of the monolayer. A previous SFG investigation into the formation of HBMs also (51) (52) (53) (54)

Maroncelli, M.; Strauss, H. L.; Snyder, R. G. J. Chem. Phys. 1985, 82, 2811. Plant, A. L. Langmuir 1999, 15, 5128. Tarek, M.; Tu, K.; Klein, M. L.; Tobias, D. J. Biophys. J. 1999, 77, 964. Leverette, C. L.; Dluhy, R. A. Colloids Surf., A 2004, 243, 157.

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concluded that the adsorption of a phospholipid monolayer left the structure of the underlying ODT SAM largely unaffected.11 However, the SFG spectrum of a d-DPPC/h-ODT HBM in the C-H region presented by Anderson et al. does show some evidence of methylene modes at approximately 2850 and 2900 cm-1, implying that the ODT SAM is disordered by the adsorption of the phospholipid layer, in agreement with our findings. Finally, it should be noted that although the HBMs remained stable under water for a number of days, removal of the water film in contact with the HBM resulted in a reordering of the lipid layer. Figure 10a is the SFG spectrum of an HBM in which the water overlayer has been allowed to thermally evaporate from the sample surface under ambient laboratory conditions. The appearance of the three methyl resonances as spectral peaks rather than dips is indicative of the formation of a phospholipid bilayer (Figure 11a).55 As only a single layer of DPPE was transferred onto the ODT SAM as it was pushed through the air/water interface, the bilayer must form as a result of the drying process. A mechanism for the reorientation of an LB film with a hydrophilic top surface as a result of the removal of water has been proposed by Ye and co-workers.55,56 A similar process could be occurring for the DPPE films dried in air. As the water evaporates from the substrate surface, some of the DPPE molecules could reorient on top of neighboring molecules, trapping a thin layer of water between the headgroups (Figure 11b). There was evidence for the water layer in RAIRS spectra recorded of the HBM after the drying process (not shown). An atomic force microscopy study by Solletti et al. of DPPE bilayers on hydrophilic mica noted a similar reorientation of the phospholipid molecules after the substrate had been removed from the water.57 While the importance of the water layer in maintaining the integrity of the HBM has been previously noted,10 there remain a number of examples in the literature of HBMs which have been studied in air and not in contact with a layer of water.8,12,54 In one such study by Meuse et al., the model biological membrane was formed using the conventional Langmuir-Schaefer technique of contacting a hydrophobic SAM onto a lipid layer at the water surface and then withdrawing the sample back into the air.8,12 Such a method leads to the formation of a lipid bilayer, as withdrawal of the substrate from the water surface results in the air/water interface curving back under the sample and pulling a second layer of phospholipid with it (Figure 11c).58 Meuse et al. removed the (55) Ye, S.; Noda, H.; Morita, S.; Uosaki, K.; Osawa, M. Langmuir 2003, 19, 2238. (56) Ye, S.; Noda, H.; Nishida, T.; Morita, S.; Osawa, M. Langmuir 2004, 20, 357. (57) Solletti, J. M.; Botreau, M.; Sommer, F.; Brunat, W. L.; Kasas, S.; Duc, T. M.; Celio, M. R. Langmuir 1996, 12, 5379. (58) Ulman, A. An Introduction to Ultrathin Films: From Langmuir-Blodgett to Self Assembly, 1st ed.; Academic Press Ltd.: London, 1991.

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upper phospholipid layer by blotting the sample with filter paper, and confirmed the presence of a monolayer of lipid by the use of spectroscopic ellipsometry and RAIRS. However, neither of these techniques can unambiguously prove that only a monolayer of lipid is present. The SFG spectrum of an HBM formed using the technique outlined by Meuse et al. contained three peaks which can be assigned to the methyl resonances of DPPE (Figure 10b). This suggests that even after blotting the sample with filter paper a bilayer of phospholipid remains. The formation of multilayer films of DPPE in air demonstrates the importance of the water overlayer in maintaining the structure of the HBM, and the consequences of exposing the model biological membrane to the air.

Conclusion SFG spectra of DPPE/ODT HBMs under water have been recorded in the C-H and C-D stretching regions which enabled the structures of the DPPE and ODT layers to be independently investigated. The spectra confirmed that a monolayer of DPPE had been physisorbed to the ODT SAM with the methyl groups of the phospholipid in contact with the hydrophobic SAM, and the alkyl chains aligned with the surface normal. The presence of methylene modes in the spectra of the phospholipid indicated that there were gauche defects in the alkyl chains of the DPPE at each of the surface adsorption pressures. No contribution to the SFG spectra from the headgroup of the phospholipid was detected. On adsorption of a layer of DPPE, methylene resonances arising from the ODT were observed which were not present in the spectrum of the pure SAM. This indicated that gauche defects were introduced into the alkyl chains of ODT on adsorption of the DPPE, implying that a slight disordering had occurred in the SAM. SFG spectra recorded in air indicated that removal of the over-layer of water in contact with the HBM resulted in a phospholipid bilayer being formed. This demonstrated (as previously reported in the literature) the importance that the water layer plays in maintaining a planar monolayer of phospholipid in the HBM and the potential complications that arise when trying to study HBMs in air. Acknowledgment. P.J.N.K. thanks the EPSRC for a studentship. Supporting Information Available: A more detailed description of the theory of SFG spectroscopy, a schematic showing how the HBMs were formed, and SFG spectra of h-ODT in the C-D region, d-ODT in the C-H region, and d-DPPE/d-ODT HBMs in the C-H region. This material is available free of charge via the Internet at http://pubs.acs.org.

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