ARTICLE pubs.acs.org/JPCC
Supported Phospholipid Monolayers. The Molecular Structure Investigated by Vibrational Sum Frequency Spectroscopy Jonathan F. D. Liljeblad,†,^ Vincent Bulone,‡,^ Mark W. Rutland,†,§,^ and C. Magnus Johnson*,†,^ †
School of Chemistry, Division of Surface and Corrosion Science, KTH Royal Institute of Technology, SE-100 44 Stockholm, Sweden School of Biotechnology, Division of Glycoscience, KTH Royal Institute of Technology, SE-106 91 Stockholm, Sweden § YKI, Institute for Surface Chemistry, Box 5607, SE-114 86 Stockholm, Sweden ^ Swedish Center for Biomimetic Fiber Engineering (Biomime), KTH Royal Institute of Technology, SE-106 91 Stockholm, Sweden ‡
bS Supporting Information ABSTRACT: The molecular structure, packing properties, and hydrating water of LangmuirBlodgett monolayers of the phospholipids 1,2-distearoyl-sn-glyercophosphatidylcholine (DSPC, 18:0 PC), its deuterated analogue (18:0 PC-d83), and 1,2-distearoyl-sn-glyerco-phosphatidylserine (DSPC, 18:0 PS) deposited on planar calcium fluoride (CaF2) substrates have been investigated using the surfacespecific nonlinear optical technique vibrational sum frequency spectroscopy (VSFS). Compression isotherms were recorded before the deposition of the monolayers at a surface pressure of 35 mN/m, mimicking the conditions of biological cell membranes. The CH and CD stretch regions, the water region, and the lower wavenumber region, containing phosphate, ester, carboxylate, and amine signals, thus partly covering the fingerprint region, were probed to obtain a complete map of the molecules. The data indicate that all deposited monolayers formed a well-ordered and stable film, and probing the water region revealed significant differences in hydration for the different headgroups. In addition, the tilt angle of the aliphatic chains relative to the surface normal was estimated to be approximately 4 to 10 based on orientational analysis using the antisymmetric methyl stretching vibration. Orientational analysis of the ester CdO groups was also performed, and the result was consistent with the estimated tilt angle of the aliphatic chains.
’ INTRODUCTION Phospholipids are one of the key constituents of cell membranes and thus of immense importance for life. Because of the fragility of cells and the extreme complexity of biological membranes, in situ spectroscopic studies are rarely feasible. Biomimetic membranes, consisting of Langmuir monolayers1,2 or supported mono- or bilayers formed by deposition3,4 or vesicle fusion,5 and whose composition is limited to a few components, are a way to circumvent the problems associated with studying living cells. Such membranes can be used to investigate various properties associated with the membrane itself, such as transbilayer movement6 (“flip-flop”) of phospholipid molecules, packing properties,7 or oxidation8 as well as interactions between the membrane and proteins9,10 or polypeptides, such as AMPs11 (antimicrobial peptides). Of great biological importance is the understanding of the mode of packing and molecular interactions between lipids that form differentiated structures in cell membranes, for example lipid rafts. Such microdomains are lateral patches in the membrane that exhibit particular enrichment in sterols, (glyco)sphingolipids, and phospholipids that carry saturated fatty acids.12 The physicochemical properties of the rafts, and to some extent their biological function (e.g., intracellular sorting and exocytotic membrane transport),13 are determined by their r 2011 American Chemical Society
specific lipid composition.12 However, lipid rafts remain poorly characterized especially with respect to their molecular packing and the corresponding lipid/lipid and lipid/protein interactions. However, because lipid rafts represent complex structures that are difficult to study spectroscopically in their natural state, the use of simplified model systems is necessary. The preparation and characterization of biomimetic membranes with adapted compositions of gradually increasing complexity, starting from single component monolayers, is expected to provide an insight into the properties of the components of the lipid rafts, which can eventually be expanded to the rafts themselves. However, in all experimental approaches based on artificial structures, it is important to assess that the biomimetic membranes mimic cell membranes sufficiently well before extrapolating the data to cellular membranes. The research presented here represents a starting point to the validation and use of such biomimetic membranes, by exploiting the power of vibrational sum frequency spectroscopy (VSFS) combined with Langmuir monolayers to investigate further the spectral properties of a selection of saturated phospholipids that are enriched in the rafts. The type Received: December 6, 2010 Revised: April 12, 2011 Published: May 06, 2011 10617
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The Journal of Physical Chemistry C of data obtained provides insightful information on the physicochemical properties, comprising the molecular structure, orientation of hydrocarbon chains and other functional groups, and spectral features and assignments, of membranes and their individual constituents. Vibrational sum frequency spectroscopy, VSFS, is highly suited to the study of model membranes, as it is intrinsically surface specific and possesses submonolayer sensitivity.1416 It can be applied at the solid/air interface of deposited monolayers as well as the solid/liquid interface, provided that the substrate is transparent to the frequencies of the laser beams employed. In addition, VSFS can be combined with the Langmuir trough to perform in situ measurements of, for instance, hydration,17,18 oxidation of unsaturated phospholipids,8 and surface pressuredependent properties.1,19,20 Here VSFS was used to study deposited monolayers of the phospholipids 1,2-distearoyl-sn-glycero-phosphatidylcholine (DSPC, 18:0 PC), its deuterated analogue (DSPC-d83, 18:0 PCd83), and 1,2-distearoyl-sn-glycero-phosphatidylserine (DSPS, 18:0 PS), both possessing identical 18-carbon-atom long aliphatic chains that, together with 16-carbon-atom chains, are the most prevalent chain length in nature for saturated phospholipids.21 Although phospholipid monolayers have been frequently studied using VSFS during the past decade, most of the analyses have focused on the CH and OH stretching regions, and the lower wavenumber region (1000 cm1 to 1850 cm1) has, with the exception of the symmetric phosphate stretch,17,22 not yet been fully explored. An improved understanding of the spectral features in this region is useful when investigating protein and polypeptide interactions with membrane surfaces as well as headgroup hydration and orientation. Indeed, the lower wavenumber region mainly carries signals from the headgroups of the phospholipids and can reveal substantially more information than the probing of signals arising from the hydrocarbon moieties alone. Only a few materials are useful as substrates for supported lipid layers studied by VSFS. If the interface between water and the supported lipid bilayer, which is the biologically most relevant interface, is to be studied, the substrate must be transparent to the incident visible and IR beams. Silica,3 CaF2,11 and quartz4 have been used to this end. However, because silica is only transparent in the CH and OH stretching regions, and because one of the future aims of our research is to investigate the lower wavenumber region of supported bilayers, the use of silica is effectively limited. Additionally, the high surface charge at biological pH of silica may perturb the deposited lipid bilayer and has been shown to strongly influence its hydration by aligning the water molecules and introducing a long-range order that extends far away from the interface.11 The range of the disturbance depends upon the Debye length, a measure of the ionic strength, and ranges from a few nanometers to hundreds of nanometers. The surface potential is a useful indication of the influence of charge. For silica, it varies over a range between 50 and 100 mV around pH 7 for ionic strengths relevant to this work.23,24 The ordered water molecules also generate a strong water signal in the VSF spectra which interferes with the CH stretching vibrations and thus complicates the deconvolution of the spectral features.11 The water signal can be transferred to another spectral region by replacing the water with D2O to suppress the interference with the CH signal, but this neither alleviates the perturbation of the monolayer nor the influence on hydration. A possible problem associated with CaF2 is that it is slightly
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soluble in water,25 but the kinetics appears to be slow26 and the solubility is most pronounced at alkaline conditions. Because calcium ions are known to interact with phospholipids, however, in particular the phosphate group17,27 as well as the carboxylate group of the PS headgroup, this issue must be acknowledged. To conclude, CaF2 represents an interesting alternative to silica because the charging is considerably lower (surface potential approximately þ35 mV at pH 7.5),26 and it is transparent in a wider wavenumber range. For these reasons, we have chosen to use it as the substrate for the deposition of the phospholipid monolayers. For a spectroscopic signal to be generated in VSFS, the local inversion symmetry must be broken. Typically, bilayers will generate a very low, if any, signal because of their natural symmetry. However, asymmetric bilayers possessing one deuterated leaflet can be prepared by LangmuirBlodgett/LangmuirSchaefer (LB/LS) deposition. Because of this, the symmetry of bilayers can be broken, thereby allowing the detection of the CH signal from the nondeuterated leaflet.3,28 Because of this, we have chosen to include the deuterated analogue of 18:0 PC, which is 18:0 PC-d83, in our study. To enable interpretation of the data from experiments performed on asymmetric bilayers, detailed knowledge of the spectral features arising from a monolayer is essential. One of the main objectives of this study was to obtain and expand this knowledge.
’ THEORY: VIBRATIONAL SUM FREQUENCY SPECTROSCOPY (VSFS) The theoretical framework behind sum frequency generation was first described in 1962 by Bloembergen and Pershan,29 but it was not until 1987 that pioneering experimental work was performed by Shen.30 Nowadays, VSFS is an established technique, and the theories needed for its application are described elsewhere in great detail.3133 Briefly, when two laser beams, one at fixed visible frequency and one tunable infrared, of sufficient intensity to generate a nonlinear response, overlap in space and time at an interface, a third beam with the sum of the frequencies of the incident beams is generated by the induced nonlinear surface polarization. The intensity of the generated sum frequency signal is proportional to the intensities of the incident beams as well as the square of the second-order nonlinear susceptibility, χ(2). ð2Þ
ISF jχeff j2 Ivis IIR
ð1Þ
In eq 1, χ(2) eff is the effective second-order susceptibility corrected with the Fresnel factors to account for the local fields at the interface. Along with the angles of incidence and the refractive index of the two bulk media surrounding the interface, the Fresnel factors contain n0 , which is the refractive index of the thin surface layer where the SF signal is generated.31 Because this surface layer is in general only a few monolayers thick, and in the case of our experiments only consist of the phospholipid monolayer, its refractive index is difficult to measure. Generally, it is assumed to be different from the bulk refractive index of the material forming the surface layer and between the indices of the bulk materials on each side of the interface.31 A necessary condition under the electric dipole approximation is that for χ(2) to be nonzero the inversion symmetry of the matter must be broken, as is always the case at an interface but not in the bulk of amorphous materials or crystals possessing inversion symmetry. This makes VSFS intrinsically surface specific, 10618
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The Journal of Physical Chemistry C
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Figure 1. Molecular structures of the investigated phospholipids.
which is one of the main advantages of the technique compared to conventional IR and Raman spectroscopy. In addition, VSFS is very sensitive to the overall molecular order at the studied interface, and a completely disordered monolayer does not give rise to any signal at all. The susceptibility can be separated into nonresonant (χ(2) NR) and resonant contributions (χ(2) R,n) for each vibration. The resonant contributions are given by the orientational average (denoted by brackets) of the second-order hyperpolarizability, β(2), which describes the molecular properties: χð2Þ n, xyz
Ns ð2Þ ¼ Æβn, abc æ ε0
ð2Þ
In eq 2, ε0 is the permittivity of free space and Ns is the number density of molecules at the interface. It can thus be realized that the sum frequency intensity scales with the square of the number density. Transformation between the molecular- (abc) and surface- (xyz) oriented coordinate systems is performed by Euler angle transformation.34 The expression for the hyperpolarizability (eq 3) contains both the Raman tensor, Rab, and the IR transition moment, μc. This is the background for the selection rule that a vibration must be both IR and Raman active to generate a VSFS signal. Rab μc ð2Þ ð3Þ βabc ¼ ωn ωIR iΓ In eq 3, ωn is the vibrational frequency, ωIR is the frequency of the IR beam, and Γ is a damping constant that accounts for the homogeneous broadening. In the case of the picosecond system used here, a spectrum is recorded by overlapping a fixed visible and a tunable infrared beam in space and time on the sample surface. The intensity of the SF signal is recorded as a function of the IR wavenumber while tuning the IR beam through the desired wavenumber range. When the IR wavenumber coincides with the resonance frequency of a vibration, the SF signal is resonantly enhanced (see eq 3) and a peak is detected in the spectrum. By selectively polarizing the incident and emitted beams with S- (perpendicular to the plane of incidence) or P- (parallel to the plane of incidence) polarization, the recorded data can be used to estimate the orientation of functional groups in the investigated
molecule. This orientational analysis involves the determination of the ratios of the experimentally measured intensities of a certain vibrational mode in different polarization combinations and comparison with calculated values.31,32,35 The polarization combinations that may give rise to a spectroscopic signal for the case of an isotropic surface are SSP, PPP, PSS, and SPS, where the letters denote the polarization of the sum frequency, visible, and IR beams, respectively. PSS and SPS (which only differ by a constant related to the Fresnel factors) and SSP each probe one of the 27 χ(2)-tensor elements, respectively, whereas PPP probes an admixture of several elements. As VSFS is a coherent technique where both constructive and destructive interference can occur both between observed vibrational modes, as well as between the vibrational modes and the nonreseonant background, a model function is commonly fitted to the spectrum to deconvolute it and to extract the peak amplitudes needed for the orientational analysis. The secondorder susceptibility is modeled by a wavenumber-dependent term for each resonance and a frequency independent complex constant, χ(2) NR, that accounts for the nonresonant background (see eq 4). For dielectrics χ(2) NR is real, as no absorption takes place. ð2Þ
χð2Þ χNR þ ISF ðωIR Þ ¼ ANR þ
∑n
∑n χð2Þ R n
2 An ωn ωIR iΓn
ð4Þ
ð5Þ
The spectrum is modeled by fitting eq 5 (which describes a set of Lorentzian line shapes) to the experimental data, constraining Γn to positive values. Often several local minima generating a reasonable fit exist, and great care must be taken to ensure that the selected fit agrees with known experimental findings such as peak positions from IR and Raman spectra. The model can be extended further by assuming a Gaussian distribution of vibrational frequencies to account for inhomogeneous broadening,36 but the system studied here features sufficiently narrow peaks in the cases where fitting was performed for eq 5 to give the required accuracy.
’ EXPERIMENTAL SECTION Materials. The phospholipids 1,2-distearoyl-sn-glycero-3phosphatidylcholine (18:0 PC), 1,2-distearoyl-D70-sn-glycero3-phosphatidylcholine-1,1,2,2-D4-N,N,N-trimethyl-D9 (18:0 PC-d83), and 1,2-distearoyl-sn-glycero-3-phosphatidyl-L-serine (18:0 PS) dissolved in CHCl3 or as lyophilized powders were purchased from Avanti Polar Lipids (Alabaster, AL) and used without further purification. The molecular structures of the lipids are shown in Figure 1. For preparation of lipid solutions, CHCl3 (Sigma-Aldrich, g99.8%, stabilized with amylene) and CH3OH (Sigma-Aldrich, spectrophotometric grade, g99.9%) were used as received. IR-grade CaF2 windows, 5 mm thick with 60/40 surface finish used as substrates for the monolayers were obtained from CeNing Optics Co. Ltd., Fujian, China. All water used was purified using a Millipore system featuring constant monitoring of the conductivity (>18.2 MΩ 3 cm) and organic content (
