Assessing the Molecular Structure of Alkanethiol Monolayers in Hybrid

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Langmuir 1998, 14, 1604-1611

Assessing the Molecular Structure of Alkanethiol Monolayers in Hybrid Bilayer Membranes with Vibrational Spectroscopies Curtis W. Meuse,* Gediminas Niaura,† Mary L. Lewis,‡ and Anne L. Plant Biotechnology Division, Chemical Science and Technology Laboratory, National Institute of Standards and Technology, Gaithersburg, Maryland 20899 Received January 21, 1997. In Final Form: January 12, 1998 Hybrid bilayer membranes (HBMs), consisting of a lipid monolayer covering a self-assembled alkanethiol monolayer on a metal surface, are useful models for studying the structure and function of cell membranes. Surface-enhanced Raman spectroscopy (SERS) and reflection absorption infrared spectroscopy (RAIRS) are used to study HBMs with various alkanethiol and lipid components. Together, these two techniques clearly indicate that the lipid forms a well-ordered, non-interdigitated layer on the alkanethiol with the head groups of the lipids oriented away from the metal surface. Both techniques reveal that the formation of an HBM produces small changes in the alkanethiols, which are similar to those caused by a reduction in temperature, indicative of an increase in order. The small magnitude of the perturbations in the alkanethiol monolayer upon addition of the lipid layer will simplify the further study of HBMs.

Introduction The importance of cell membranes in biology and the interest in employing cell membrane components in applications such as sensors, electronics, and bioprocessing provide incentive for the development of membrane mimetic models. Examples of planar-supported bilayer membranes include spontaneously assembled bilayers on hydrophilic surfaces,1,2 transfer of lipid monolayers to glass surfaces made hydrophobic with a lipid layer,3 and fabrication of phospholipid-containing bilayers on gold through the use of thiol-terminated supporting layers.4-14 The formation of a well-defined hydrophobic surface by the self-assembly of alkanethiols on metal surfaces is wellknown and is well-characterized.15-18 We add a layer of phospholipid to the alkanethiol monolayer either by the * To whom correspondence should be addressed. † On leave from Institute of Chemistry, Vilnius, Lithuania. ‡ Current address: University of Denver, Denver, CO. (1) Tamm, L. K.; McConnell, H. M. Biophys J. 1985, 47, 105. (2) Tien, H. T. Adv. Mater. 1990, 2, 316. (3) Kalb, E.; Frey, S.; Tamm, L. K. Biochim. Biophys. Acta 1992, 1103, 307. (4) Lang, H.; Duschl, C.; Gratzel, M.; Vogel, H. Thin Solid Films 1992, 210/211, 818. (5) Spinke, J.; Yang, J.; Wolf, H.; Liley, M.; Ringsdorf, H.; Knoll, W. Biophys. J. 1992, 63, 1667. (6) Florin, E.-L.; Gaub, H. E. Biophys. J. 1993, 64, 375. (7) Plant, A. L. Langmuir 1993, 9, 2764. (8) Stelzle, M.; Weissmuller, G.; Sackman, E. J. Phys. Chem. 1993, 97, 2974. (9) Lang, H.; Duschl, C.; Vogel, H. Langmuir, 1994, 10, 197. (10) Erdelen, C.; Haussling, L.; Naumann, R.; Ringsdorf, H.; Wolf, H.; Yang, J.; Liley, M.; Spinke, J.; Knoll, W. Langmuir 1994, 10, 1246. (11) Plant, A. L.; Gueguetchkeri, M.; Yap, W. Biophys. J. 1994, 67, 1126. (12) Duschl, C.; Liley, M.; Corradin, G.; Vogel, H. Biophys. J. 1994, 67, 1229. (13) Naumann, R.; Jonczyk, A.; Kopp, R.; van Esch, J.; Ringsdorf, H.; Knoll, W.; Graber, P. Angew. Chem., Int. Ed. Engl. 1995, 34, 2056. (14) Duschl, C.; Liley, M.; Lang, H.; Ghandi, A.; Zakeeruddin, S. M.; Stahlberg, H.; Dubochet, J.; Nemetz, A.; Knoll, W.; Vogel, H. Mater. Sci. Eng. 1996, C4, 7. (15) Nuzzo, R. G.; Fusco, F. A.; Allara, D. L. J. Am. Chem. Soc. 1987, 109, 2358. (16) Porter, M. D.; Bright, T. B.; Allara, D. L.; Chidsey, C. E. D. J. Am. Chem. Soc. 1987, 109, 3559. (17) Bain, C, D.; Troughton, E. B.; Tao, Y.-T.; Evall, J.; Whitesides, G. M.; Nuzzo, R. G. J. Am. Chem. Soc., 1989, 111, 321.

S0743-7463(97)00067-X

Figure 1. Schematic representation of a hybrid bilayer membrane (HBM) consisting of an alkanethiol layer selfassembled at a metal surface and a lipid layer assembled on the hydrophobic surface of the alkanethiol layer.

spontaneous reorganization of phospholipid vesicles or by horizontal transfer of lipids from an air-water interface to produce a hybrid bilayer membrane (HBM) (Figure 1). The hydrophobicity of the alkanethiol monolayer provides the driving force for the organization of the phospholipids. This process must involve a significant driving force, since the phospholipids are highly stable in vesicles in aqueous solution yet will spontaneously reorganize to form a bilayer at the alkanethiol surface. The amphiphilic character of the phospholipids determines their orientation, which is controlled by the interaction of the polar head groups with water and the affinity between the hydrophobic alkane chains of the two layers. The configuration depicted in Figure 1 has been confirmed by a number of studies in our laboratory including electrochemistry, surface plasmon (18) Ulman, A. An Introduction to Ultrathin Organic Films, From Langmuir-Blodgett to Self-Assembly; Academic Press: San Diego, CA, 1991.

This article not subject to U.S. Copyright. Published 1998 by the American Chemical Society Published on Web 02/20/1998

Alkanethiol Monolayers in Hybrid Bilayer Membranes

resonance, atomic force microscopy, ellipsometry, infrared spectroscopy, and neutron reflectivity.7,11,19,20 The electrical insulating characteristics of the HBMs are consistent with those of natural and artificial lipid bilayer membranes and are affected by anesthetics, cations,19 and the peptide toxin melittin.11 HBMs are easily formed on an electroactive surface and are rugged enough to remain stable for weeks. These characteristics make them a desirable model system for examining lipid and membrane protein structure and function, as well as making them potentially suitable for practical applications. Careful characterization of the molecular structure of HBMs is necessary to confirm its structural similarity to lipid bilayer membranes. The structure and activity of membrane proteins in this matrix are likely to be relevant to other model systems and to natural membranes. In this study, surface-enhanced Raman spectroscopy (SERS) and reflection-absorption Fourier transform infrared spectroscopy (RAIRS) are employed to characterize HBMs. These techniques have been used extensively to characterize alkanethiol monolayers. SERS has been used to determine the number and location of gauche conformers, the dissociation of the S-H bond upon self-assembly, and the orientation of C-S and C-C bonds in alkanethiol monolayers.21-23 RAIRS has been used to determine the orientation and packing of many different monolayers.16,24,25 In addition, polarization modulation RAIRS has been used to determine that both the solvent and the initial structure (chain length) affect the alkanethiol monolayer structure.26,27 It is valid to compare RAIRS data collected from smooth surfaces and SERS data from roughened surfaces, since studies using SERS,21 contact angle measurements,17 and STM28 have indicated that large scale roughness has little influence on the structure of alkanethiol monolayers. In this first report on the vibrational spectroscopic characterization of HBMs, SERS and RAIRS are employed in concert to analyze the effect that the phospholipid layer has on the structure of the alkanethiol monolayer. Each technique has specific advantages, and the comparison of the two provides a more complete picture of the structure of HBMs formed by two techniques on different surfaces and in different environments. For example, SERS is primarily sensitive to the alkanethiol layer which is in intimate contact with the metal substrate. In addition, since water is a weak Raman scatterer, interference from the solution phase is minimal; thus, samples can be examined in an aqueous environment. In contrast, the RAIRS measurements are much more difficult in water but provide good structural detail of both the alkanethiol and phospholipid layers in air. (19) Plant, A. L., Gueguetchkeri, M. Proceedings of the 13th Southern Biomedical Engineering Conference, University of the District of Columbia, Washington, DC, April 16-17, 1994. (20) Meuse, C. W.; Krueger, S.; Majkrzak, C. F.; Dura, J. A.; Fu, J.; Connor, J. T.; Plant, A. L. accepted for publication in Biophys. J. (21) Bryant, M. A.; Pemberton, J. E. J. Am. Chem. Soc. 1991, 113, 546. (22) Bryant, M. A.; Pemberton, J. E. J. Am. Chem. Soc. 1991, 113, 8284. (23) Pemberton, J. E. Characterization of organic thin films; A. Ulman (Ed.) Butterworth-Heinemann: Boston, 1995; p 87. (24) Nuzzo, R. G.; Korenic, E. M.; Dubois, L. H. J. Chem. Phys. 1990, 93, 767. (25) Dubois, L. H.; Zegarski, B. R.; Nuzzo, R. G. J. Chem. Phys. 1993, 98, 678. (26) Anderson, M. A.; Evaniak, M. N.; Zhang, M. Langmuir 1996, 12, 2327. (27) Anderson, M. A.; Gatin, M. Langmuir 1994, 10, 1638. (28) Creager, S. E.; Hocket, L. A.; Rowe, G. K. Langmuir 1992, 8, 854.

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Materials and Methods29 Alkanethiol monolayers were prepared by immersing the metal substrates in 0.001 mol/L thiol (hexanethiol (Aldrich, 95%), decanethiol (Aldrich, 97%), or octadecanethiol (Aldrich, 97%)) solutions in ethanol (Warner Graham Co. Cockeysville, MD) for a minimum of 12 h. Fluorinated thiol was a gift from Tonya M. Herne and Michael J. Tarlov at the National Institute of Standards and Technology. The phospholipids used in these studies were either fully hydrogenated or fully deuterated acyl chain analogues of dimyristoylphosphatidylcholine (DMPC or DMPC-d54) or dipalmitoylphosphatidylcholine (DPPC or DPPC-d62) from Avanti Polar Lipids (Alabaster, AL). Vesicle solutions were formed by injecting the lipid in isopropyl alcohol (50 µL per 2 µmol of lipid) into 5-150 mmol/L phosphate buffer solutions which were also 150 mmol/L NaCl. For the SERS experiments, the planar bilayers were produced by adding the vesicle solution to the cell containing the thiol-coated substrates at least 1 h prior to making measurements. SERS measurements were taken after the cell was rinsed with buffer solution to remove excess lipid. For SERS, the substrates were 3-mm-diameter rods of silver (Adlrich, 99.98%), which were polished with alumina powder (0.5-mm grit), rinsed, and sonicated for 2 min to remove any residual powder. Electrochemical roughening was used to produce surfaces suitable for SERS measurements. The procedure consisted of a single cycle in a 1 mol/L NaClO4 solution from -500 mV to +600 mV at a rate of 100 mV/s. Hold times of 30 s at the lower potential and 10 s at the higher potential were used. The electrodes were removed from the roughening solutions and rinsed with water and ethanol before they were placed in the thiol solutions. Measurements were taken using Raman excitation provided by a He-Ne laser (Aerotech #LSR30P) at 632.8 nm. Solution Raman spectra of phospholipids vesicles were measured in a 1-mm optical quartz spectrometer cell (Uvonic) thermostated with a circulating bath. Excitation energy was provided by the 514.5-nm line from an Ar+ laser (Lexel #95) (50 mW of power at the sample). SERS measurements were performed in a cylindrical sealed spectroelectrochemical cell. The metal rod substrate served as the working electrode and was placed in a Teflon sleeve, leaving the front surface open to solution. A platinum wire served as the counter electrode. A saturated Ag/AgCl electrode was used as the reference electrode. The cell potential was controlled using a Princeton Applied Research 174A polarographic analyzer. All potentials cited are referenced to the saturated Ag/AgCl electrode. Raman scattering from both cells was collected at 90° by an f/1.2 camera lens (Nikon) and focused into the entrance slit (200500 mm) of a double monochromator (Spex 14018) converted to single-stage use. Elastic scattering was eliminated with a holographic laser line filter (Kaiser Optical Systems, Inc.). A Spectrum One liquid-nitrogen-cooled CCD (Spex) was used for detection. Spectra were obtained by adding 50-300 1-s scans. Data acquisition was performed using SPEX DM3000 software. The gold surfaces for RAIRS were prepared by the thermal evaporation of chromium and then gold (99.9%) at a base pressure of ∼1 × 10-4 Pa onto silicon (1 0 0) wafers (Virginia Semiconductor) to a nominal thickness of 2000 Å. For RAIRS, a solution of lipid vesicles was placed onto thiol-coated substrates for 1.5 h; the solution was then partially removed and diluted with water. Several such cycles were performed, so that the water never overflowed the surface and the surface was never dry. After extensive rinsing, excess water was removed all at once by blotting the surface with a stack of filter paper. Other samples for RAIRS were prepared by the transfer of phospholipid from the airwater interface using a Nima 2011 Langmuir trough (Coventry, England). Thiol-coated substrates were lowered horizontally until they contacted the interface; then they were raised back into air.20 This procedure frequently resulted in the transfer of two monolayers of lipid with water trapped between them. The outer lipid layer and the associated water were removed by blotting the wet sample onto filter paper. The success of this process was determined by spectroscopic ellipsometry, which confirmed the addition of a single molecular layer of phospholipid. (29) The specification of commercial products is for clarity only, and does not constitute endorsement by NIST.

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with the hexanethiol layer and phospholipid directly in contact with the metal surface through holes in the monolayer. To determine the position of the phospholipids, we evaluated our SERS spectra for contributions from each source. The relative intensity expected of the SERS bands from phospholipids adsorbed on top of the hexanethiol layer (IONHT) compared with those from phospholipids adsorbed in the pinholes (IINHOLES) can be expressed as

IONHT/IINHOLES ∼ SONHTNONHT/SINHOLESNINHOLES

Figure 2. Raman spectra in the C-D stretching region: (a) SERS spectrum from hydrogenated hexanethiol adsorbed on a Ag surface; (b) SERS spectrum from a hexanethiol/DMPC-d54 bilayer on a Ag surface; (c) Raman spectrum of bulk solution DMPC-d54 vesicles. Contact angle measurements were also used to monitor the presence of the lipid layer, since the polar head groups of the phospholipid make the surface hydrophilic relative to the alkane chains of the thiols. Comparison of the RAIRS spectra from these two preparations showed very similar spectral characteristics, suggesting that any differences in the two methods of fabrication are subtle.20 The FTIR spectra were obtained on a Mattson Research Series 2 spectrometer (Madison, WI) with a Harrick (Ossing, NY) 75° reflection accessory with a built in polarizer. Four data sets, each consisting of 1000 sample scans and 1000 background scans at 4-cm-1 resolution, were averaged to produce representative spectra. All spectral analysis was performed using Grams 386 software (Galactic Industries, Salem, NH).

Results and Discussion In this study, we examine HBMs composed of alkanethiols with different chain lengths and DMPC, DPPC or their deuterated analogues, using both SERS and RAIRS. The RAIRS data are taken in dry air. SERS spectra are obtained from HBMs formed by the addition of phospholipid vesicles to alkanethiol monolayers in aqueous solution. Our previous studies of HBMs by impedance spectroscopy7,11 have indicated that the addition of vesicles to alkanethiol monolayers results in an impedance response which is consistent with the formation of a single bilayer consisting of a monolayer of alkanethiol plus a monolayer of phospholipid. In interpreting the impedance response, we have assumed that the alkanethiol layer is unchanged by the addition of the phospholipid. Vibrational spectroscopy allows us to examine the validity of that assumption. Phospholipid Monolayer. SERS Results. Figure 2 shows the C-D stretching bands30 of the in situ SERS spectrum which suggest the presence of DMPC-d54 near the surface after the interaction of DMPC-d54 vesicles with a hexanethiol monolayer on silver. These C-D stretching bands have two possible origins, phospholipid associated (30) Bunow, M. R.; Levin, I. W. Biochim. Biophys. Acta 1977, 489, 388.

where SONHT and SINHOLES describe the amount of surface area occupied by and free of hexanethiol, respectively, and NONHT and NINHOLES denote the surface enhancement of Raman scattering for molecules adsorbed on the hexanethiol layer and in the pinholes, respectively. The ratio SONHT/SINHOLES was evaluated using Cl- ions as a probe for pinholes. Cl- ions strongly interact with a silver electrode under conditions of open circuit or slightly more negative potentials and give rise to an intense lowfrequency ν(Ag-Cl) band at ∼240 cm-1.31,32 The experiments were performed in 0.1 mol/L NaF + 0.01 mol/L NaCl solution with bare and hexanethiol monolayercovered Ag electrodes at 0.0 V (vs Ag/AgCl reference electrode). To reduce the uncertainty related to the chemical enhancement mechanism,33 the electrodes were subjected to deactivation at E ) -1.2 V, for 2 min prior to the experiments. The intensity of the ν(Ag-Cl) band decreased more than 470 times with the formation of the hexanethiol monolayer. Assuming that the intensity of the ν(Ag-Cl) band is proportional to the surface coverage of the Cl- ions, SONHT/SINHOLES is >470. The difference in the enhancement ratio, NONHT/NINHOLES, of CD3 and CD2 stretching bands is probably due to differences in the electromagnetic surface enhancement because chemical interaction of these groups with the surface is unlikely. Previously, the electromagnetic enhancement factor was shown to decrease by a factor of ∼1.3 with an increase of the distance from surface of ∼1 nm, the length of the hexanethiol layer.34-37 This value depends on the dimensions of the surface roughness and can vary several times. We assume that in our system the ratio NONHT/NINHOLES is at least 1.3-1. Combining terms, IONHT must be at least 50 times greater than IINHOLES. This analysis, although based on several assumptions, suggests that the observed C-D bands originate almost entirely from phospholipid adsorbed on the hexanethiol monolayer. This analysis is confirmed by comparing the C-D stretching intensities from the phospholipids with the intensities associated with the C-H stretching intensities from the alkanethiol monolayer. Since the intensities of the C-D bands are approximately 5% of the intensities of the C-H bands of the alkanethiols, this suggests that (31) Wetzel, H.; Gerisher, H.; Pettinger, B. Chem. Phys. Lett. 1981, 78, 392. (32) Pettinger, B.; Philpott, R. M.; Gordon II, J. G. J. Phys. Chem. 1981, 85, 2746. (33) Change, R. K. Ber. Bunsen-Ges. Phys. Chem. 1987, 91, 296. (34) Collins, R. W.; Allara, D. L.; Kim, Y.-T.; Lu, Y.; Shi, J. Characterization of organic thin films; A. Ulman (Ed.) Butterworth-Heinemann: Boston, 1995; p 35. (35) Pemberton, J. E.; Bryant, M. A.; Sobocinski, R. L.; Joa, S. L. J. Phys. Chem. 1992, 96, 3776. (36) Kovaks, G. J.; Loutfy, R. O.; Vincett, P. S.; Jennings, C.; Aroca, R. Langmuir 1986, 2, 689. (37) Cotton, T. M.; Uphaus, R. A.; Mobius, D. J. Phys. Chem. 1986, 90, 6071.

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Table 1. Components of the C-D Stretching Region of Raman and SERS Spectra of DMPC-d54 in Vesicles and in HBMs Raman shifta (cm-1)

relative intensity

SERS shifta (cm-1)

relative intensity

relative ISERS/IRaman ratio

assignmentb

2073 2106 2131 2160 2178 2200 2218

30 100 30 19 20 38 18

2068 2103 2123 2155 2171 2194 2215

46 100 32 17 18 15 16

1.5 1.0 1.1 0.9 0.9 0.4 0.9

νs(CD3) FR νs(CD2) νs(CD3) FR ν(C-C) + CD2 twist νas(CD2) νas(CD2) IR active νas(CD3)

a The frequency and the relative intensity of the bands have been obtained by decomposition of the observed spectrum into the Lorentzian components. The same number of bands was assumed for the solution and SERS spectra. b See text for band assignment references.

the interaction between the phospholipid and alkanethiols is probably not the result of interdigitation of deuterated phospholipid chains but that the phospholipid chains are on top of the hexanethiol. In addition, there is no indication of the presence of the choline head group in the SERS spectra which suggests that the phosphatidylcholine head groups are also far removed from the metal surface and that they are certainly not in contact with it. The spatial relationship between the alkanethiol layer and the phospholipid chains was further examined by comparing the SERS spectra from the HBMs with the Raman spectra from a solution of DMPC-d54 vesicles (see Figure 2 and Table 1). The relative intensity of the symmetric stretching band of the CD3 group at ∼2070 cm-1 compared to the CD2 symmetric stretching band at ∼2106 cm-1 is higher in the SERS spectrum than in the solution spectrum by a factor of 1.5. In the HBM SERS spectrum (Figure 2b), the decrease in the relative intensity of the asymmetric CD2 stretching vibration at ∼2200 cm-1 compared to the symmetric CD3 stretching band at ∼2070 cm-1 is readily apparent. This is consistent with the CD3 groups of the phospholipid chains being closer to the surface than the CD2 groups because SERS enhancement decreases with the distance from the surface.36,37 The vibrations of CD2 groups would be less enhanced than the vibrations of the CD3 groups if the lipid were oriented in a bilayer as shown in Figure 1. The relative intensity of the symmetric CD3 vibration at ∼2070 cm-1 compared to the asymmetric CD3 band at ∼2218 cm-1 is higher in the HBM SERS spectrum than in the solution spectrum (Table 1) such that

[I(νs(CD3))/I(νas(CD3))]SERS/[I(νs(CD3))/ I(νas(CD3))]SOL . 1.7 The intensity ratio of the symmetric to asymmetric methyl vibrations can be used to analyze the orientation of the adsorbed organic molecules.35 The method is based on the assumption that the major changes in the polarizability ellipsoids for the symmetric and asymmetric methyl vibrations are perpendicular and that the more perpendicular to the surface the changes in the polarizability ellipsoid are, the greater the surface enhancement.35,38 This method was successfully used for the study of the adsorption of molecules on silver and gold surfaces.35 For a deuterated alkane chain, the major change of the polarizability ellipsoid of the symmetric CD3 stretch is parallel to the CD2-CD3 bond. The [I(νs(CD3))/I(νas(CD3))]SERS ratio of the HBMs indicates that the angle between CD2-CD3 bonds and the surface normal is less than 45°. RAIRS Results. In SERS, scattering intensity is sensitive to the proximity of the molecules to the surface.36,37 In RAIRS, intensities do not decrease as a (38) Snyder, R. G. J. Chem. Phys. 1965, 42, 1744.

Figure 3. RAIRS spectrum of a hydrogenated DPPC layer ratioed to a self-assembled fluorinated thiol layer on gold. The fluorinated alkanethiols have negligible changes upon addition of the lipid, allowing the complete spectrum of the phospholipid layer of an HBM to be observed.

function of distance from the surface. RAIRS thus allows us to characterize both the lipid and the alkanethiol leaflets of HBMs. A RAIRS spectrum of the phospholipid portion of an HBM consisting of a hydrogenated phospholipid ratioed to a fluorinated alkanethiol (Figure 3) clearly shows bands from the alkane chains and the choline head groups of the phospholipids39,40 (Table 2) and confirms the presence of the phospholipid layer in the HBM. In addition, spectra of hydrogenated HBMs (Figure 3) reveal a series of CH2 wagging progression bands in the 10001500 cm-1 region. Since wagging progressions are too weak to be observed in alkanethiols,41 these progression bands are probably from the lipid layer. If the progression bands are from the lipid layer, they suggest that the phospholipid portion of the HBMs has a high degree of intramolecular order.42 However, it is possible that the addition of the lipid layer could increase the intensity of the wagging progression bands of the alkanethiols. Before in-depth analysis of the phospholipid structure can take place, it is essential to assess what effect the addition of phospholipid has on the structure of the underlying alkanethiol. We focus on this issue by examining the SERS and RAIRS spectra of alkanethiols in the presence and absence of an added layer of deuterated phospholipid. Alkanethiol Monolayer. SERS Spectral Changes in the Hexanethiol Monolayer Induced by the Formation of (39) Mendelsohn, R.; Mantsch, H. H. Progress in Protein-Lipid Interactions 2; Elsevier Science Publishers: New York, 1986; p 103. (40) Parikh, A. N.; Allara, D. L. J. Chem. Phys. 1992, 96, 927. (41) Parikh, A. H.; Liedberg, B.; Atre, S. V.; Ho, M.; Allara, D. L. J. Phys. Chem. 1995, 99, 9996. (42) Snyder, R. G.; Schachtschneider, J. H. Spectrochim. Acta, Part A 1963, 19, 85.

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Table 2. Assignments of Selected Infrared Modes of HBMs and Alkanethiol Monolayersa peak frequency (cm-1) mode assignment νas(CH3)3N+ νas(CH3) op νas(CH2) νs(CH3) νs(CH2) νs(CdO) δas(CH3)3N+ δ(CH2)n δs(CH3) CH3 wag (gtt) CH2 wag (tgtg*) CH2 wag progression (ttt)

octadecanethiol layer 2965 2918 2877 2850 ∼1468 ∼1383

νas(PO2-) νs(PO2-) νs(COsOsC) νas(CsN+sC) a

DPPC layer 3026 2965 2918 2878 2850 1737 1494 1465 1379 1346 1310 1330, 1297, 1264, 1223, 1199, 1180 1247 1100 1075 969

See text for assignment references.

Figure 4. (a) SERS spectra from hexanethiol adsorbed on Ag in the range 600-1150 cm-1 with (- - -) and without (s) a DMPC layer. (b) Difference spectrum (hexanethiol/DMPC HBM hexanethiol).

HBMs: 600-1200 cm-1 Region. The frequency region between 600 and 1200 cm-1 (including the C-C and C-S stretching vibrations and the CH2 and CH3 rocking modes) provides information about intrachain order. Monolayers of decanethiol and shorter chain lengths are incompletely ordered, as determined from X-ray reflection,43 SERS,21 RAIRS, and ellipsometric measurements.16 Several subtle changes in the SERS spectra of hexanethiol monolayers take place after DPPC vesicles are introduced into the solution (Figure 4a). For all SERS data, shifts in band frequencies and changes in full width at half maximum (fwhm) due to the addition of phospholipid were within the limits of experimental uncertainty ((2 cm-1), while (43) Fenter, P.; Eisenberger, P.; Liang, K. S. Phys. Rev. Lett. 1993, 70, 2447.

changes in relative intensities clearly occur. The difference spectrum (i.e. the hexanethiol SERS spectrum subtracted from the hexanethiol/DMPC SERS spectrum) is shown in Figure 4b. The relative intensities of the band due to trans (T) C-S bonds at 710 cm-1, the bands due to trans C-C bonds near 1042 cm-1, and the band due to trans terminal CH3 group rocking modes at 891 cm-1 increase, while the intensities of the band due to gauche (G) C-S bonds at 642 cm-1 and the band due to the rocking of gauche CH2 bonds at (877 cm-1) decrease. The increase in the relative intensity of the T versus G bands is shown in Table 3. Changes in the vicinity of the terminal CH3 groups of the alkanethiol monolayer upon addition of a layer of phospholipid are not surprising. During this thermodynamically favorable process, the aqueous environment around the CH3 group is replaced by alkanes. The effect of solvent changes on the structure of alkanethiol monolayers has been reported.26 The increase in I891/I877 ratio (Table 3) suggests that the addition of the phospholipid to the alkanethiol monolayer causes an increase in the number of CH3 trans conformers. Variable-temperature measurements also show that the number of CH3 trans conformers in alkanethiol monolayers usually increases at lower temperatures,22 and this change has been interpreted as indicating an increase in intrachain order. Figure 5 illustrates that, at 5 °C, the hexanethiol spectrum shows an increase in the intensity of the rocking vibration of trans CH3 groups at ∼891 cm-1. Increases in T/G ratios in the C-S bands have been reported to correlate to increasing chain length of alkanethiols44 and presumably are correlated with increased order. The observed increase in the T/G ratio in the C-S bands upon formation of a bilayer therefore suggests an increase in the intrachain order at the two C atoms which are proximal to the S atom. Thus, the presence of the phospholipid also appears to have a long-range effect on the structure of the short chain alkanethiol. SERS Spectral Changes in the Hexanethiol Monolayer Induced by the Formation of HBMs: 2800-3000 cm-1 Region. The C-H stretching region (2800-3000 cm-1) reflects both intrachain order and intermolecular chain interactions and shows a complex spectrum from the alkanethiol monolayers (Figure 6). More detailed analysis can be performed after deconvolution of the spectrum into the Lorentzian components. First we will consider the assignments of the bands, based on previous discussions.45-49 Two overlapped bands located at 2849 and 2859 cm-1 have been assigned to the symmetric stretching modes of the CH2 groups. The slightly higher frequency band has been associated with the CH2 group close to the terminal CH3 (ω-CH2).44 The symmetric methyl stretching band is known to be split by Fermi resonance with an overtone of the asymmetric methyl bending mode at about 1450 cm-1 into a pair of lines at 2874 cm-1 (νs(CH3 FR)) and 2936 cm-1 (νs(CH3 FR)). The asymmetric stretching mode of the CH2 groups has been observed at 2898 cm-1. The 2916-cm-1 band was assigned to the symmetric methylene stretching vibration in Fermi resonance with the CH2 deformation overtones (νs(CH2 (44) Bryant, M. A.; Pemberton, J. E. J. Am. Chem. Soc. 1991, 113, 3629. (45) Snyder, R. G.; Strauss, H. L.; Ellger, C. A. J. Phys. Chem. 1982, 86, 5145. (46) Hill, I. R.; Levin, I. W. J. Chem. Phys. 1979, 70, 842. (47) O’Leary, T. J.; Levin, I. W. J. Phys. Chem. 1984, 88, 1790. (48) Harrand, M. J. Mol. Struct. 1989, 214, 71. (49) Yahiaoui, B.; Masson, M.; Harrand, M. J. Chem. Phys. 1990, 93, 6047.

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Table 3. SERS Peak Height Intensity Ratios for Hexanethiol as a Monalayer and in HBMs system

a

mode

ratioa

hexanethiol, 23 °C

hexanethiol/ DPPC, 23 °C

hexanethiol 5 °C

ν(C-S) T/ν(C-S) G δ(CH3) T/δ(CH3) G νas(CH2)/νs(CH3) FR

I709/I642 I891/I877 I2898/I2936

5.7 2.5 0.75

8.3 2.8 0.78

10.7 3.7 0.85

Subscripts indicate frequency shifts in cm-1.

Figure 5. (a) SERS spectra from hexanethiol adsorbed on Ag in the range 625-950 cm-1 at 343 K (s) and at 278 K (- - -). (b) Difference spectrum (278 K - 343 K).

FR)). Finally, the asymmetric methyl stretching mode (νas(CH3)) was assigned to the clearly resolved band at 2964 cm-1. The most prominent changes in the SERS spectrum caused by the introduction of the phospholipid vesicles are the decreases in intensity of the νs(CH2 FR) band at 2916 cm-1 and the νs(CH3 FR) band at 2936 cm-1, which can be clearly seen in the difference spectrum (Figure 6b). These changes are similar to the perturbations induced in the alkanethiol monolayers by the lowering of the temperature (Figure 7). It has been noted42,44,45 that the νs(CH3 FR) mode and the νas(CH2 ) mode (near 2936 and 2898 cm-1, respectively) are particularly sensitive to intermolecular interactions; increases in interchain disorder cause an increase in the νs(CH3 FR) band intensity and a decrease in the νas(CH2 ) band intensity. It has been suggested that the increase in the νs(CH3 FR) band intensity when the chain intramolecularly disorders is related to the appearance of the infrared-active asymmetric methylene stretching mode in this frequency region.30,50 The νs(CH3 FR) band near 2936 cm-1 is clearly resolved in all spectra. The νas(CH2) band at 2898 cm-1 is not clearly resolved in our spectra, although it is somewhat more distinct after formation of the bilayer (Figure 6) or when the temperature is lowered (Figure 7). Therefore, the intensity of this band was estimated after decomposition (50) Bush, S. F.; Adams, R. G.; Levin, I. W. Biochemistry 1980, 19, 4429.

Figure 6. (a) SERS spectra from hexanethiol adsorbed on Ag in the range 2800-3000 cm-1 with (- - -) and without (s) a DMPC layer. (b) Difference spectrum (hexanethiol/DMPC HBM - hexanethiol).

of the spectrum into the components. Table 3 shows that lowering the temperature causes a decrease in the 2936cm-1 band and an increase in the I(2898 cm-1)/I(2936 cm-1) ratio. This change is consistent with an increase in interchain order at lower temperatures.21 The adsorption of the phospholipids induces the same effects in relative band intensities (Figure 5 and Table 3), leading us to conclude that interchain order in the hexanethiol monolayers increases when the HBM is formed. Thus, both intra- and interchain order increase when the phospholipid layer is added. Changes in the octadecanethiol monolayer are small in both regions of the spectrum. There are two reasons for the lack of large changes in the longer chain alkanethiol with the addition of phospholipid. The longer chains are initially more highly ordered, and SERS is less sensitive to changes that occur further away from the surface. RAIRS Analysis of the Spectral Changes in the Alkanethiol Monolayer Induced by the Formation of HBMs. Three alkanethiols of different chain lengths, hexane-, decane-, and octadecanethiol, were examined by RAIRS. Figure 8 shows the RAIRS spectra of hexanethiol, decanethiol, and octadecanethiol monolayers, before and after the addition of a DPPC-d62 monolayer from the airwater interface. The degree of conformational order in the alkane chains of the thiol monolayer is apparent from the peak position of the νa(CH2) bands, which varies according to the length of the chain.16 In our samples, these peaks occur at 2923, 2920, and 2918 cm-1 for

1610 Langmuir, Vol. 14, No. 7, 1998

Figure 7. (a) SERS spectra from hexanethiol adsorbed on Ag in the range 2800-3000 cm-1 at 343 K (s) and at 278 K (- - -). (b) Difference spectrum (278 K - 343 K).

Figure 8. Composite RAIRS spectra of the CH stretching contributions of octadecanethiol, decanethiol, and hexanethiol monolayers in the range 3100-2700 cm-1. The solid lines represent the spectra of the monolayers, and the dashed lines represent the spectra of the HBM consisting of hydrogenated alkanethiols and deuterated DPPC. The deuterated lipids have a negligible contribution in the CH stretching region, allowing the changes in the alkanethiols with the formation of the HBMs to be observed.

hexanethiol, decanethiol, and octadecanethiol, respectively, regardless of the presence of phospholipid. These frequencies indicate greater order for the longer chain alkanethiols. Like the SERS data, the RAIRS data also indicate subtle changes in the alkanethiol monolayers due to the presence of the phospholipid. To observe these differences, we

Meuse et al.

Figure 9. Composite RAIRS difference spectra obtained from the subtraction of alkanethiol monolayer spectra from spectra of DPPC-d62 HBMs. See the text for a full description of the subtraction procedure.

subtracted the normalized spectrum of each monolayer from the spectrum of its corresponding HBM prepared with deuterated lipid. Monolayer spectral intensities were normalized to the asymmetric CH3 stretching band of a bilayer spectrum at 2965 cm-1 by multiplying by 0.86. We chose to normalize spectral intensities at this peak for several reasons. First, models developed by Parikh and Allara predict that this peak is due almost entirely to gauche conformers in the terminal CH2-CH2 bonds and that the intensity of this peak is not significantly different for levels of terminal gauche conformers between 10% and 45%.40 This peak is clearly present in all our monolayer spectra, suggesting that our initial alkanethiol monolayers contain gauche conformers in this bond; however, we expect that if there are variations in the number of gauche conformers as a function of alkanethiol chain length, this would be difficult to detect. Second, there is less variation in the intensities of these bands when comparing samples prepared on different gold substrates than is observed for CH2 bands. In addition, the band is strong and narrow in all spectra, making it easier to compare the different alkanethiol monolayers. The difference spectra are shown in Figure 9. The RAIRS data indicate changes in the intensities of the CH2 bands, near 2850 and 2920 cm-1, and the CH3 bands, near 2960 and 2870 cm-1, upon addition of phospholipid. One explanation for these changes could be changes in the orientation of the alkanethiol monolayers, since RAIRS is only sensitive to the component of the transition moment perpendicular to the gold surface.51 This explanation seems unlikely, since it is unclear that the addition of phospholipid would alter the orientation of the alkanethiols by more than a few degrees. This small orientation change would induce several small intensity changes, each related to the orientation of its transition moment. While changes in the individual bands might be hard to detect, relative intensity changes, between, for example, the symmetric and asymmetric CH2 bands, should be detected. Analysis of the hexanethiol and octadecanethiol HBMs (51) Greenler, R. G.; J. Chem. Phys. 1966, 44, 310.

Alkanethiol Monolayers in Hybrid Bilayer Membranes

reveal that the ratios of the intensities of the symmetric and asymmetric CH2 bands do not change. Slight changes in the ratios of the decane and dodecanethiol HBMs may occur, but they are overwhelmed by the general increase in intensity that occurs upon addition of the phospholipid, as indicated by the positive deviation throughout the CH stretching region for each. Overall, the increases in the intensities of the HBMs upon addition of phospholipid appear to be a function of chain length. Previous studies of alkanes and alkanethiol monolayers at different temperatures indicate rather large increases in the intensities of both methyl and methylene stretching bands upon cooling the sample.24,52 This was observed for long chain, highly ordered, alkanethiols at temperatures between 80 and 380 K.24 In the case of the methylenes, these intensity changes were found to be a function of the number of methylenes involved.52 It has been proposed that the increased intensities are due to changes in the higher order dipole-derivative terms of the transition moments.52 The transition moments are altered when the amount of torsion about the C-C bonds changes as a function of different lattice spacing of the molecules at different temperatures.52 It is reasonable that the addition of the phospholipid could alter the lattice spacing of the alkanethiol. We observe changes in intensity upon addition of phospholipid, and the effect is found to be greater for longer chains, suggesting that our data may be attributed to torsional changes in alkanes and alkanethiols. This would lead us to conclude that the formation of an HBM is consistent with decreasing the temperature, contracting the lattice, and increasing the order in the methyl and methylene moieties. Also, like the SERS data, the RAIRS data indicate changes in the CH3 modes of the alkanethiol monolayers near 2960 and 2870 cm-1 upon formation of the HBMs (see Figure 9). These changes are not unexpected, since each alkanethiol has a methyl group directly in contact with the phospholipid layer. The changes are approximately the same for all chain lengths, each of which only contributes a single methyl group per chain. The origin of the methyl changes is unclear but may be attributed (52) Snyder, R. G.; Maroncelli, M.; Strauss, H. L.; Hallmark, V. M. J. Phys. Chem. 1986, 90, 5623.

Langmuir, Vol. 14, No. 7, 1998 1611

to one or more contributing factors including conformational, intermolecular, and torsional effects. The torsional changes described by Snyder et al. which we hypothesize describe the CH2 bands in Figure 9 are not a function of intermolecular or conformational changes.52 SERS indicates that the hexanethiol undergoes inter- and intrachain ordering upon addition of the phospholipid. In RAIRS, we do not observe changes in the νas(CH2) band, near 2920 cm-1, indicating that RAIRS is not sensitive to conformational changes that may occur in the SAMs when the phospholipid is added. If conformational changes were the source of the RAIRS changes, we would expect them to be large in the hexanethiol HBM and smaller in the longer chain HBMs. Since we observe an increase in the difference in intensity with chain length, we assign this effect to a decrease in intrachain torsion upon addition of the phospholipid, as described by Snyder et al.52 In both SERS and RAIRS experiments, the results support an increase in ordering upon the formation of an HBM, since the effects are consistent with what would be observed due to a decrease in temperature. Thus, it is apparent that the addition of phospholipid generally causes structural changes that are consistent with an overall ordering of the alkanethiol chains. Conclusions Phospholipid can be added to alkanethiol monolayers either by self-assembly from lipid vesicles or by transfer from the air-water interface, resulting in the formation of a bilayer. RAIRS and SERS confirm the presence of phospholipids in association with the alkanethiol layer in a well-ordered, non-interdigitated bilayer structure where the phospholipid head groups are oriented away from the support. Careful analysis of the vibrational spectroscopy of alkanethiol monolayers indicates spectral changes that are consistent with structural changes induced by lowering the temperature, suggesting increases in order in the monolayer accompany the formation of an HBM. The small magnitude of the perturbations in the alkanethiol monolayer upon formation of HBMs will simplify further the study of the lipid layer of HBMs. LA9700679