Preparation and Characterization of Mixed Monolayer Assemblies

Filled Nanoporous Surfaces: Controlled Formation and Wettability. Eyal Bittoun , Abraham Marmur , Mattias Östblom , Thomas Ederth and Bo Liedberg...
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Langmuir 1997, 13, 3210-3218

Preparation and Characterization of Mixed Monolayer Assemblies Composed of Thiol Analogues of Cholesterol and Fatty Acid Zhongping Yang,† Isak Engquist,† Mikael Wirde,§ Jean-Michel Kauffmann,‡ Ulrik Gelius,§ and Bo Liedberg*,† Laboratory of Applied Physics, Linko¨ ping University, S-581 83 Linko¨ ping, Sweden, Institute de Pharmcie, Universite Libre de Bruxelles, Campus Plaine CP205/6, B-1050 Brussels, Belgium, and Department of Physics, Uppsala University, Box 530, S-751 21 Uppsala, Sweden Received January 7, 1997. In Final Form: April 7, 1997X Mixed self-assembled monolayers provide an attractive model system for investigating the role of different molecules in biological membranes. This paper describes the preparation and characterization of a novel type of mixed monolayer assemblies composed of thiol analogues of cholesterol and fatty acid. The mixed monolayers are prepared by coadsorbing 11-mercaptoundecanoic acid (MUA) and thiocholesterol (cholest5-ene-3β-thiol, TC) from solution directly onto evaporated gold surfaces. The influence of TC on the molecular composition and conformation in the mixed monolayer is analyzed by using a combination of infrared reflection-absorption spectroscopy (IRAS), X-ray photoelectron spectroscopy (XPS), ellipsometry, contact angle measurement, and cyclic voltammetry. The results indicate that the TC molecules maintain their conformation in the mixed monolayers, whereas the MUA molecules display a significantly more disordered conformation as compared to the MUA molecules in the pure monolayer. Cyclic voltammetry shows that the mixed monolayers are more densely packed and less permeable than the pure TC and MUA monolayers. The kinetics of the coadsorption of TC and MUA from ethanol indicates that adsorption of TC initially is strongly preferred over MUA but that MUA dominates over TC at long coadsorption times. This is because there is a larger energy gain per unit area in forming monolayers with MUA. Further, it is also seen that the number of molecules per unit area changes with the molecular composition, as a consequence of the different sizes of TC and MUA. We present herein a method for calculating the mole fraction of TC on the gold surface, χTC, which accounts for this variation. As a consequence of the dissimilar size and shape of the two molecules, the wetting properties of the mixed monolayer are found to be mainly governed by the fractional area of TC, rather than by the molecular composition of TC, χTC.

1. Introduction The role of cholesterol in biological membranes is an issue of long-standing interest.1,2 A variety of model systems of biological membranes have been examined in order to improve the understanding of the direct and indirect cholesterol effects in the membranes. The most popular model systems are (i) Langmuir films, in which amphiphilic molecules are spread and compressed at an air/water interface3,4 and (ii) liposomes, i.e., self-assembled colloidal vesicles in which a phospholipid bilayer encapsulates a fraction of the surrounding aqueous medium.5-7 For example, studies of mixed monolayer films of cholesterol and phospholipids at an air/water interface suggest that cholesterol has a pronounced condensing effect;3,8,9 i.e., the area per molecule in the mixed film is less than the area per molecule in films of each of the compounds alone. The condensing effect leads to a reduction in the number of defects, and to a decrease in * To whom correspondence should be addressed. † Linko ¨ ping University. § Uppsala University. ‡ Universite Libre de Bruxelles. X Abstract published in Advance ACS Abstracts, May 15, 1997. (1) Yeagle, P. L. Biochim. Biophys. Acta 1985, 822, 267. (2) McMullen, T. P. W.; McElhaney, R. N. Curr. Opin. Colloid Interface Sci. 1996, 1, 83. (3) Demel, R. A.; Bruckdorfer, K. R.; Van Deenen, L. L. M. Biochim. Biophys. Acta 1972, 255, 311. (4) Mozaffary, H. Thin Solid Films 1994, 244, 847. (5) Lasic, D. D. Liposomes: From Physics to Applications; Elsevier: Amsterdam, 1993. (6) Parkes, J. G.; Watson, H. R.; Joyce, A.; Phadke, R. S.; Smith, I. C. P. Biochim. Biophys. Acta 1982, 691, 24. (7) Fernandez-Ballester, G.; Castresana, J.; Fernandez, A. M.; Arrondo, J.-L. R.; Ferragut, J. A.; Gonzalez-Ros, J. M. Biochemistry 1994, 33, 4065. (8) Tinoco, J.; McIntosh, D. J. Chem. Phys. Lipids 1970, 4, 72. (9) Vanderkooi, G. Biophys. J. 1994, 66, 1457.

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the permeability of the membrane.10 The effect of cholesterol on the characteristic properties of membranes depends greatly on its concentration and conformation in the membranes.1,2 However, the proper manipulation and characterization of the mole fraction and orientation of cholesterol are difficult using the above mentioned approaches. Mixed monolayers prepared by self-assembled monolayer (SAM) techniques,11,12 i.e., chemisorption of multicomponent ω-functionalized alkanethiols, HS(CH2)n-X, from solution directly onto gold surfaces, have attracted great attention during the last few years.13-27 The mole fraction and molecular orientation in the mixed SAMs (10) Demel, R. A.; Bruckdorfer, K. R.; Van Deenen, L. L. M. Biochim. Biophys. Acta 1972, 255, 321. (11) Ulman, A. An Introduction to ULTRATHIN ORGANIC FILMS From Langmuir-Blodgett to Self-Assembly; Academic Press Inc.: Boston, 1991. (12) Dubois, L. H.; Nuzzo, R. G. Annu. Rev. Phys. Chem. 1992, 437. (13) Bain, C. D.; Evall, J.; Whitesides, G. M. J. Am. Chem. Soc. 1989, 111, 7155. (14) Bain, C. D.; Whitesides, G. M. J. Am. Chem. Soc. 1989, 111, 7164. (15) Chidsey, C. E. D.; Bertozzi, C. R.; Putvinski, T. M.; Mujsce, A. M. J. Am. Chem. Soc. 1990, 112, 4301. (16) Bain, C. D.; Davies, P. B.; Ong, T. H.; Ward, R. N.; Brown, M. A. Langmuir 1991, 7, 1563. (17) Laibinis, P. E.; Nuzzo, R. G.; Whitesides, G. M. J. Phys. Chem. 1992, 96, 5097. (18) Folkers, J. P.; Laibinis, P. E.; Whitesides, G. M. J. Adhesion Sci. Technol. 1992, 6, 1397. (19) Folkers, J. P.; Laibinis, P. E.; Whitesides, G. M. Langmuir 1992, 8, 1330. (20) Bertilsson, L.; Liedberg, B. Langmuir 1993, 9, 141. (21) Chailapakul, O.; Crooks, R. M. Langmuir 1993, 9, 884. (22) Finklea, H. O.; Hanshew, D. D. J. Am. Chem. Soc. 1992, 114, 3173. (23) Zhang, L.; Lu, T.; Gokel, G. W.; Kaifer, A. E. Langmuir 1993, 9, 786.

© 1997 American Chemical Society

Monolayer Assemblies of Cholesterol and Fatty Acids

can be changed and manipulated in a controlled way. Such mixed SAMs provide excellent models of biological membranes for systematic studies of a wide range of biological interfacial phenomena including wetting,18,28,29 electron transfer,15,21,30 protein adsorption,31,32 polysiloxane,33 and Langmuir film deposition.34 So far, typical mixed SAMs have been prepared from binary mixtures of HS(CH2)nX/HS(CH2)m-Y onto gold surfaces with a wide variety of tail groups such as CH3, OH, COOH, CO2CH3, CN,13 and ferrocenyl15 and a variety of chain lengths, n and m.14,19,35 The generally accepted structural picture of SAMs is that mixed as well as single component SAMs form a hexagonal (x3 × x3) R30° overlayer structure on Au(111) crystal faces,36-38 where the average distance between pinning sites is about 5 Å. This distance fits very nicely the van der Waals diameter of a poly(methylene) chain (4.6 Å), which allows the preparation of densely packed and highly organized mixed SAMs with a predefined structure and composition. In this paper we describe a novel type of mixed SAM containing thiol analogues of lipids prepared by chemical coadsorption of thiocholesterol (cholest-5-ene-3β-thiol, TC) and 11-mercaptoundecanoic acid (HS(CH2)10COOH, MUA), Figure 1, from solution directly onto an evaporated gold surfaces. TC is a cholesterol molecule bearing a thiol moiety at the 3β-position (Figure 1a). The molecular structure is a nearly planar polycyclic steroid ring with a branched alkyl chain. SAMs of TC on evaporated gold have been prepared and characterized in a previous study.39 Infrared experiments showed that organized SAMs of TC were assembled on gold and the surface coverage was determined to be around 65% of that obtained for saturated alkanethiolate SAM.39 There is, however, some uncertainty about this value, as will be discussed below. Such a low coverage indicates that the TC SAM contains a large number of defects. These defects are probably an effect of the geometrical properties of the TC molecule, which are not well-matched with the pinning distance at the Au(111) lattice. Moreover, the defects could be filled by alkanethiols but not with the corresponding disulfide, suggesting that the defects are larger than 4.6 Å in diameter, but smaller than two alkyl chains, i.e., 4.6 × 9.2 Å, the minimum geometric size of a disulfide.39 The main purpose of this study is to investigate the mixing properties and structural characteristics of TC/ MUA SAMs. The monolayers have been independently characterized with a series of techniques including single wavelength ellipsometry, infrared reflection-absorption (24) Offord, D. A.; John, C. M.; Linford, M. R.; Griffin, J. H. Langmuir 1994, 10, 883. (25) Rowe, G. K.; Creager, S. E. Langmuir 1994, 10, 1186. (26) Stranick, S. J.; Parikh, A. N.; Tao, Y.-T.; Allara, D. L.; Weiss, P. S. J. Phys. Chem. 1994, 98, 7636. (27) Liedberg, B.; Tengvall, P. Langmuir 1995, 11, 3821. (28) Ulman, A.; Evans, S. D.; Shnidman, R.; Sharma, R.; Eilers, J. E. Adv. Colloid Interface Sci. 1992, 39, 175. (29) Engquist, I.; Lundstro¨m, I.; Liedberg, B. J. Phys. Chem. 1995, 99, 12257. (30) Hickman, J. J.; Ofer, D.; Laibinis, P. E.; Whitesides, G. M.; Wrighton, M. S. Science 1991, 252, 688. (31) Prime, K. L.; Whitesides, G. M. Science 1991, 252, 1164. (32) Lestelius, M.; Liedberg, B.; Lundstro¨m, I.; Tengvall, P. J. Biomed. Mater. Res. 1994, 28, 871. (33) Parikh, A. N.; Liedberg, B.; Atre, S. V.; Ho, M.; Allara, D. L. J. Phys. Chem. 1995, 99, 9996. (34) Evans, S. D.; Sanassy, P.; Ulman, A. Thin Solid Films 1994, 243, 325. (35) Atre, S. V.; Liedberg, B.; Allara, D. L. Langmuir 1995, 11, 3882. (36) Alves, C. A.; Smith, E. L.; Porter, M. D. J. Am. Chem. Soc. 1992, 114, 1222. (37) Samant, M. G.; Brown, C. A.; Gordon, J. G., II. Langmiur 1992, 8, 1615. (38) Poirer, G. E.; Tarlov, M. J. Langmuir 1994, 10, 2853. (39) Yang, Z.; Engquist, I.; Kauffmann, J.-M.; Liedberg, B. Langmuir 1996, 12, 1704.

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Figure 1. Space-filling models of (a) a TC and (b) a MUA molecule. The cross-sections of the molecules are shown at the bottom.

spectroscopy (IRAS), X-ray photoelectron spectroscopy (XPS), liquid drop contact angle measurements, and cyclic voltammetry. The mole fraction, χTC, of TC in the mixed SAMs has been manipulated by varying the molar fraction of TC, fTC, in the self-assembly solution (0.0 e fTC e 1.0). A new approach to determine the chemical composition is described under the condition that the mixed SAMs are formed from two different molecules, which have not only different terminal groups but also different molecular structures (poly(methylene) chain and steroid ring) and cross-sectional areas. To the authors knowledge, this is the first study where thiol analogues of lipids possessing distinctly different molecular geometries (cross-sections) at the pinning site have been used to prepare mixed SAMs with controlled composition on gold. It should be stressed, however, that mixed SAMs composed of different lipids (also cholesterol) attached to flexible tails containing SH anchoring groups have been prepared and characterized.40,41 2. Experimental Section 2.1. Materials. Gold (Au, A ¨ delmetall Stockholm, 99.99%), chromium (Cr, Balzers, 99.99%), titanium (Ti, Balzers, 99.99%), and single crystal silicon(100) wafers (Si, Okmetic) were used for the preparation of gold substrates. Hydrogen peroxide (H2O2, 30%), hydrochloric acid (HCl, 37%), and ammonia (NH3, 25%) were obtained from Merck and used for cleaning the substrates. Thiocholesterol (mp 97-99 °C) was purchased from Aldrich and used without further purification. 11-Mercaptoundecanoic acid was supplied by courtesy from Pharmacia AB (Uppsala, Sweden). Absolute ethanol (Kemetyl, Stockholm, 99.5%), cyclohexane (Fluka, 99.5%), and hexadecane (Fluka, 98%) were used without further purification. Potassium ferricyanide (K3Fe(CN)6, 99%), potassium nitrate (KNO3, 99%), and potassium dihydrogen phosphate (KH2PO4, 99.5%) were purchased from Merck. Milli-Q water (>18 MΩ with low organic content) was obtained from a Milli-Q purification system (Millipore, Bedford, MA). (40) Tam-Chang, S.-K.; Biebuyck, H. A.; Whitesides, G. M.; Jeon, N.; Nuzzo, R. G. Langmuir 1995, 11, 4371. (41) Williams, L. M.; Evans, S. D.; Flynn, T. M.; Marsch, A.; Knowles, P. F.; Bushby, R. J.; Boden, N. Langmuir 1997, 13, 751.

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2.2. Gold Film Preparation. Gold films were prepared as follows: Silicon(100) wafers were cut into 20 × 40 mm pieces and cleaned in a mixture of 50 mL of H2O, 10 mL of H2O2, and 10 mL of NH3 (TL1) at 80 °C for 10 min, and subsequently in a mixture of 60 mL of H2O, 10 mL of HCl, and 10 mL of H2O2 (TL2) at 80 °C for 5 min. The Si pieces were mounted in an electron beam evaporation system (Balzers UMS 500P) and coated with a 10-25 Å thick Cr or Ti layer at an evaporation rate of 1 Å/s, followed by 2000 Å Au at an evaporation rate of 3-5 Å/s. The Ti/Cr layer served to improve adhesion of the gold to the silicon substrate. The base pressure was always less than 2 × 10-9 Torr. Gold films produced in this manner have previously been characterized by X-ray diffraction, transmission electron microscopy, and scanning tunneling microscopy20 and were found to have grains comprised between 200-500 Å in diameter with large flat regions having a preferred (111) orientation. The gold films were stored in a laboratory atmosphere and cleaned in TL1 before use in adsorption experiments. Atomic force microscopy measurements showed that the grain size increased somewhat during the TL1 cleaning, after which grains up to 1000 Å in diameter were frequently seen. 2.3. Formation of Mixed SAMs. Solutions of TC (1 mM) and MUA (1 mM) were prepared in ethanol, which was always used as solvent unless otherwise stated in the text. Mixed TC/MUA solutions of the desired molar ratio and a total thiol concentration of 1.0 mM were freshly made before each adsorption. The molar fraction of TC in solution, denoted fTC, was varied from 0.0 to 1.0 in steps of 0.1, i.e., eleven compositions were investigated. Mixed SAMs were formed by immersing gold films, which were cleaned in TL1, into the mixed thiol solution (10 mL total volume) overnight (>16 h) at room temperature. After this modification, the samples were rinsed in ethanol followed by ultrasonic cleaning in ethanol for 10 min. Subsequently, the samples were immersed into 0.01 M HCl for 1 min, rinsed in water, blown dry with N2, and immediately analyzed. 2.4. Ellipsometry. Single wavelength ellipsometric measurements were performed with an automatic ellipsometer (Rudolph Research AutoEl III), equipped with a He-Ne laser (λ ) 632.8 nm) aligned at an incidence angle 70° from the surface normal. The complex refractive index (N ) n + ik) of a freshly TL1-cleaned gold surface was determined from ∆ and Ψ prior to modification. These values of ∆ and Ψ yielded n and k, which together with the refractive index for the thiolate SAMs (n ) 1.50 and k ) 0, regardless of composition13,39) were inserted into a calculation program (DAFIBM) to calculate the thickness of the SAM, following an algorithm by McCrackin42 and using a Au/thiol/air parallel slab model. 2.5. Contact Angle Measurements. Liquid drop contact angles were determined with a Rame´-Hart model 100-06 goniometer at room temperature and ambient relative humidity using water and hexadecane as the probing liquids. Advancing contact angles (θa) were measured by lowering a 1 µL drop onto the surface from a blunt-ended needle attached to a 2 mL syringe. The drop was then expanded until it began to spread across the surface. Both sides of two separate drops per surface were measured (four data points total) and averaged to obtain θa. This procedure gave reproducible contact angles to within (1°. In the case of hexadecane, the contact angle value was assumed to be zero for surfaces where the drop spread to an irregular shape (fingering). 2.6. IRAS Measurements. The FTIR measurements were performed on a Bruker IFS 113v spectrometer (42) McCrackin, F. L. A. NBS Technical Note 479; NBS: Washington, DC, 1969.

Yang et al.

operating at ≈10 Torr. A deuterated triglycine sulfate (DTGS) detector was used, and spectra with a resolution of 4 cm-1 were collected. Infrared reflection absorption spectroscopy (IRAS) measurements were performed with a single reflection accessory described previously,43 utilizing f/4 optics and an angle of incidence of 83° from the surface normal. The sample wheel was operated under computer control and enables eight samples to be analyzed at a time. For each sample and reference 1000 scans were collected with interchange of sample and reference after every 250 scans in order to avoid drift and long term instabilities. 2.7. XPS Measurements. The XPS measurements were carried out on a Scienta ESCA-300 spectrometer equipped with a monochromatized Al KR X-ray source (1486.6 eV) of high intensity and a 30 cm radius analyzer with a multichannel detector.44 The resolution, as determined from the shape of the Fermi edge of silver, was approximately 0.5 eV. The pressure in the sample analysis chamber during analysis was approximately 5 × 10-10 Torr. The photoelectrons were analyzed at a takeoff angle of 90° with respect to the sample surface. 2.8. Electrochemical Measurements. The electrochemical cell consists of a Teflon body machined to accept a Viton O-ring in a cylindrical cavity surrounding a hole on the side. The gold surface was pressed against the Viton O-ring to reproducibly define the active area exposed to the solution as the working electrode. An Ag/AgCl (3 M NaCl, BAS) electrode and a platinum wire were placed in the main compartment and used as the reference and counter electrodes, respectively. The electrochemical measurements were performed in deoxygenated 0.1 M KNO3, pH 7.0, buffered by 0.001 M phosphate at room temperature (20 ( 2 °C) using an Autolab electrochemical system (Eco chemie, the Netherlands). The electrochemical area of the gold electrode (cleaned in TL1 at 80 °C for 10 min) was measured by chronoamperometry in deoxygenated 0.1 M KNO3, pH 7.0, containing 1, 2, and 3 mM K3Fe(CN)6, respectively, and was found to be 0.26 ( 0.02 cm2.45 3. Results and Discussion 3.1. Ellipsometric Characterization. By ellipsometric characterization, the mixed SAMs of TC/MUA were found to have a thickness of 16 ( 1 Å over the entire solution composition studied. An ellipsometric thickness of 16 ( 1 Å was also observed for a pure TC SAM as reported previously.39 From a space-filling model, a fully extended TC molecule is around 20 Å. We can thus estimate a tilt angle of the steroid skeleton of approximately 37° with respect to the surface normal. The ellipsometric thickness of a pure MUA SAM is also 16 ( 1 Å, which is in good agreement with previous investigations46 and consistent with the theoretical value expected for an extended all trans poly(methylene) chain structure with an average tilt of about 30° from the surface normal.47 3.2. Infrared Characterization. Figure 2a shows a series of IRAS spectra of the mixed SAMs for the whole fTC range in the high-frequency region (2750-3050 cm-1). The molecular structure of TC is far more complex than (43) Ihs, A.; Liedberg, B.; Uvdal, K.; To¨rnkvist, C.; Bodo¨, P.; Lundstro¨m, I. J. Colloid Interface Sci. 1990, 140, 192. (44) Gelius, U.; Wannberg, B.; Baltzer, P.; Fellner-Feldegg, H.; Carlsson, G.; Johansson, C.-G.; Larsson, J.; Mu¨nger, P.; Vegerfors, G. J. Electron Spectrosc. Relat. Phenom. 1990, 52, 747. (45) Bard, A. J.; Faulkner, L. R. Electrochemical Methods: Fundamentals and Applications; John Wiley & Sons, Inc.: New York, 1980. (46) Smith, E. L.; Alves, C. A.; Anderegg, J. W.; Porter, M. D.; Siperko, L. M. Langmuir 1992, 8, 2707. (47) Bain, C. D.; Troughton, E. B.; Tao, Y.-T.; Evall, J.; Whitesides, G. M.; Nuzzo, R. G. J. Am. Chem. Soc. 1989, 111, 321.

Monolayer Assemblies of Cholesterol and Fatty Acids

Figure 2. (a) IRAS spectra of the mixed SAMs in the highfrequency region, shown for selected values of fTC (the mole fraction of TC in solution). The spectra were referenced against a CD3(CD2)19SH SAM on gold. (b) Variations in relative integrated intensity of the methyl asymmetric C-H stretching mode vibration of TC (2963 cm-1) as function of fTC.

that of long-chain alkanethiols. The protocols developed by Snyder et al.48 for poly(methylene)s are therefore not applicable to TC. Tentative assignments of the more prominent peaks in the IRAS spectrum of TC were, however, given in our previous study,39 and the main peaks at 2874 and 2963 cm-1 are assigned to the symmetric and asymmetric CH3 stretching modes (νs and νas), respectively. A number of studies concerning the IRAS spectra of singlecomponent SAMs of MUA on gold have also been published previously.46,49-51 In the high-frequency region, the symmetric (d+) and asymmetric (d-) methylene stretching modes observed in Figure 2a for fTC ) 0.0 at 2849 and 2919 cm-1 indicate clearly that the poly(methylene) chains assemble in a highly ordered state48,52,53 with a low number of gauche defects. It is clearly seen that the peak positions of the CH3 νas mode of TC are independent of fTC in solution but that the peak intensities are strongly dependent on fTC (Figure 2a). The possibility that the intensity change is due to a conformational rearrangement of TC as the mixed SAMs are formed was excluded in a previous study, where a mixed 11-mercaptodeuterioundecanoic acid (MDUA)/TC SAM was formed by postadsorption of MDUA,39 resulting in the same relative peak intensity pattern for TC in the CH stretching region as for a pure TC SAM. The TC/ (48) Snyder, R. G.; Schachtschneider, J. H. Spectrochim. Acta 1963, 19, 85. (49) Chidsey, C. E. D.; Loiacono, D. N. Langmuir 1990, 6, 682. (50) Sun, L.; Crooks, R. M.; Ricco, A. J. Langmuir 1993, 9, 1775. (51) Creager, S. E.; Clarke, J. Langmuir 1994, 10, 3675. (52) Porter, M. D.; Bright, T. B.; Allara, D. L.; Chidsey, C. E. D. J. Am. Chem. Soc. 1987, 109, 3559. (53) Nuzzo, R. G.; Dubois, L. H.; Allara, D. L. J. Am. Chem. Soc. 1990, 112, 558.

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Figure 3. (a) IRAS spectra of MUA in the mixed SAMs in the high-frequency region, obtained by subtracting the spectra of the pure TC SAM (fTC ) 1.0) with a subtraction factor equal to its relative integrated intensity as seen in Figure 3b from the mixed SAM spectra. (b) IRAS spectra of the mixed SAMs in the low-frequency region, shown for selected values of fTC. The spectra were referenced against a CD3(CD2)19SH SAM on gold.

MUA SAMs in this investigation are expected to behave similarly to the TC/MDUA SAM. This has been further confirmed in experiments with TC/MDUA SAMs for some selected fTC values (not shown). The 2963 cm-1 stretching mode intensity could therefore provide information about the TC content by comparison of the spectrum of the pure TC SAM with the mixed SAM spectra.26 The variation in the relative integrated intensity54 of the peak at 2963 cm-1 is illustrated in Figure 2b. The relative integrated intensities change rapidly between fTC ) 0.0 and 0.1 and for fTC g 0.8 but vary more slowly for 0.1 < fTC < 0.8. With the assumption that the mixed SAM spectra are mixtures of the pure TC and MUA SAM spectra, we can obtain the contribution of MUA to the spectra by subtracting the spectrum of pure TC (fTC ) 1.0) scaled by a factor, taken from Figure 2b, from the mixed SAM spectra. The resulting spectra are shown in Figure 3a. We note that the peak positions of the CH2 d+ and d- stretching modes are shifted from 2850 and 2919 cm-1 to 2854 ( 1 and 2924 ( 1 cm-1 for fTC g 0.2 and that the peaks become broader. This signifies an increased disordering of the poly(methylene) chains of MUA as compared to the pure MUA SAM, which, as mentioned above, possesses a high degree of intrachain order.48 Furthermore, an examination of the IRAS spectrum of MUA in the low-frequency region provides us with structural information about the chain terminus. The strong double peaks appearing at ∼1740 and ∼1714 cm-1 (Figure 3b, fTC ) 0.0) are assigned to the CdO stretching (54) In order to minimize the error from the interference of the neighboring CH2 νa peak of MUA, the integrated intensities of the CH3 νa vibration of TC are obtained by integrating half the CH3 νa peak, from 2963 to 3024 cm-1.

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vibration of non-hydrogen-bonded and laterally hydrogenbonded CdO, respectively.46,50,53,55 The detailed structure of the hydrogen-bonded CdO is unclear so far. However, at least two different forms of hydrogen bonding may exist in carboxylic acid terminated SAMs, namely, facing and sideways hydrogen bonds.46,53,56 Facing hydrogen bonds are not likely present in the pure MUA SAM because the dimer peak at 940 cm-1 that accompanies this structure is not observed.56 A “facing” structure would also imply bilayer formation, which can be excluded from the ellipsometric measurements. The hydrogen-bonding CdO peak at 1714 cm-1 is broad and asymmetric, which probably implies complex hydrogen-bonding interactions in the MUA SAM. Further, adsorbed water is observed on the MUA SAM surface by angle-dependent XPS, suggesting that hydrogen bonds between water and the CdO group also may contribute to the asymmetric shape of the peak at 1714 cm-1.55,57 In the mixed SAMs, the conformational disorder of MUA molecules should allow the carboxylic acid groups to have sufficient freedom to form a complete hydrogen-bonding network, including facing dimers, without free CdO groups. This would result in a single CdO stretching peak at approximately 17151700 cm-1.58 However, a free CdO stretching mode at 1740 cm-1 is still observed in Figure 3b. This result may imply that lateral distribution of the MUA molecules is similar to that observed for the pure MUA SAM and that the TC molecules act as a template to stabilize such a structure.39 Thus, some sort of microscopic islanding of MUA may occur, e.g. as lines (rows) or small clusters. However, it should be emphasized that we are not talking about phase segregation into macroscopic domains, since such structures are not expected to display methylene stretching frequencies (d+ and d-) indicative of disordered MUA chains. 3.3. X-ray Photoelectron Spectroscopy (XPS). Figure 4a presents a series of XPS spectra of O1s obtained from the mixed SAMs. The position of the photoelectron peaks provides useful structural information. The oxygen peak is split into two components, the O 1s (CdO) and the O 1s (COH) with binding energies at 532.8 and 533.8 eV, respectively. This confirms the existence of the carboxylic acid group in the mixed SAM. Figure 4b shows the relative integrated photoelectron intensity of the whole double peak plotted versus fTC. We see that the relative integrated XPS intensities oppositely follow the trend in Figure 2b. In general, IRAS or XPS relative integrated intensities can be directly used to estimate the mole fractions in mixed SAMs of multicomponent ω-terminated alkanethiols since the total number of molecules is assumed to be the same regardless of the nature of the tail group.13,26 However, in the mixed SAMs of TC/MUA, the TC molecular structure is completely different from that of MUA. It is therefore impossible to directly calculate the mole fraction in these mixed SAMs using the above results because the total number of chemisorbed MUA plus TC molecules will change with the mole fraction. Thus, we only present raw data here. A detailed analysis concerning the mole fractions in the mixed SAMs will be presented below. 3.4. Contact Angle Measurements. The wetting properties of the mixed SAMs are characterized by the variation in advancing contact angles (θa) and cos θa for water and hexadecane as a function of fTC (Figure 5). These data do not completely reflect the molecular composition (55) Nuzzo, R. G.; Zegarski, B. R.; Korenic, E. M.; Dubois, L. H. J. Phys. Chem. 1992, 96, 1355. (56) Ihs, A.; Liedberg, B. J. Colloid Interface Sci. 1991, 144, 282. (57) Engquist, I.; Lestelius, M.; Liedberg, B. J. Phys. Chem. 1995, 99, 14198-14200. (58) Duevel, R. V.; Corn, R. M. Anal. Chem. 1992, 64, 337.

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Figure 4. (a) O 1s peaks in the XPS spectra of mixed TC/MUA SAMs on gold, shown for selected values of fTC. (b) Variations in the relative integrated intensity of the O1s peaks as a function of fTC.

Figure 5. Variation of the advancing contact angles for (a) water and (b) hexadecane on the mixed SAMs as a function of fTC.

of the surface but do provide useful information about the chemical character of the SAM. For water and hexadecane on the pure SAM of TC (fTC ) 1.0), θa is 108° and 15°, respectively. The measured

Monolayer Assemblies of Cholesterol and Fatty Acids

values of θa on polyethylene and single component CH3terminated SAMs on gold are 103°, 113° for water and 0°, 47° for hexadecane, respectively.47 This indicates that a composite surface of methyl and methylene groups exists on the pure TC SAMs. For water and hexadecane on the pure SAM of MUA (fTC ) 0.0), θa is 15° and 0°, respectively. The nonzero water θa measured has also been observed in previous studies28,49,59 and has been explained as being due to reorganization of the tail group and/or rapid contamination of the high-energy SAM surface in the laboratory atmosphere. The variation in cos θa of water as a function of fTC can be separated into three regions (Figure 5a). When the fTC is below 0.1 or above 0.8, cos θa varies strongly with fTC, but between fTC ) 0.1 and 0.8, only a slow monotonic decrease is seen. These observations are different from those on mixed SAMs of CH3- and COOH-terminated thiols,13 for which cos θa decreases gradually with the increasing mole fraction of the CH3 component. The variation in the hexadecane contact angles was very small, as seen in Figure 5b, and did not yield any useful information for the compositional characterization of the SAMs. They display, however, evidence (increasing θHD) for an increasing population of CH3 group at the air/ vacuum interface33,35 for fTC > 0.8, which supports a model of highly oriented TC molecules in the mixed SAM, Figure 1. 3.5. Electrochemical Characterization. The ability of a SAM to block electron transfer between the gold surface and an electroactive probe in solution is a useful measure of the nature and extent of structural defects in SAMs. Compact SAMs, prepared by using alkanethiols on gold electrodes, have been reported to inhibit diffusion of electroactive probes to the gold surface, giving a very small response in cyclic voltammetry.52 Parts a and b of Figure 6 show cyclic voltammograms at a bare gold electrode and at a series of mixed SAMs modified gold electrodes in 0.1 M KNO3, pH 7, containing 1 mM K3Fe(CN)6. The voltammogram at the bare gold electrode (Figure 6a) shows a typical redox peak for Fe(CN)63-. The voltammograms (Figure 6b) at the single component and at the mixed SAM modified gold electrodes show that the SAMs block electron transfer between the Fe(CN)63- and the electrode surface to different degrees. The cyclic voltammogram at the TC SAM modified gold electrode (Figure 6b, fTC ) 1.0) displays a voltammetric feature similar to that of an array of ultramicroelectrodes,39,60 revealing that the TC assembly possesses a large number of defects, pinholes, of the order of less than 1 nm.39 The voltammogram of Fe(CN)63- at the MUA SAM modified gold electrode (Figure 6b, fTC ) 0.0) shows a different feature. The electron transfer is inhibited and the voltammogram exhibits a small response due to Fe(CN)63reduction at higher overpotentials. The voltammograms (Figure 6b, fTC ) 0.1-0.9) at the mixed SAM modified gold electrodes display a different voltammetric behavior as compared to the pure TC SAM even if the mixed SAM was formed in a high fTC (0.9) solution. This shows that for fTC < 1.0 the number of pinholes in the pure TC SAM are greatly reduced so that the voltammetric response of Fe(CN)63- is strongly inhibited. As a point of further interest, we note that the voltammetric response of Fe(CN)63- is less at the mixed SAM modified electrodes than at the pure MUA SAM modified electrode, even if the mixed SAM was formed in (59) Evans, S. D.; Sharma, R.; Ulman, A. Langmuir 1991, 7, 156. (60) Finklea, H. O.; Snider, D. A.; Fedyk, J.; Sabatani, E.; Gafni, Y.; Rubinstein, I. Langmuir 1993, 9, 3660.

Langmuir, Vol. 13, No. 12, 1997 3215

Figure 6. Cyclic voltammograms at (a) a bare gold electrode and (b) the mixed SAMs, shown for selected values of fTC. The modified gold electrodes were put in a 0.1 M KNO3 solution, pH 7, containing 1 mM K3Fe(CN)6. Scan rate: 100 mV/s.

low fTC (0.1) solution. This indicates that the mixed SAMs are more densely packed or less permeable to Fe(CN)63than both pure TC and pure MUA SAMs.61,62 This is an interesting observation that currently is subject to detailed investigations in our laboratory.63 3.6. Formation of the Mixed SAMs. The exact mechanism of mixed SAM formation is, so far, still not fully understood. The formation of a mixed SAM is influenced by many factors, some of which can be controlled relatively easily, such as molar fractions of components in solution, solvent, and temperature. Other factors that are inherent to the system are the rate of competitive reaction of the components with the surface, intermolecular interactions, reversibility of adsorption, and exchange of the components in the mixed SAMs. By examining the kinetics of the coadsorption of MUA and TC from ethanol solution onto gold and the influence of the solvent on the coadsorption, we obtained further clues about the formation of the mixed SAMs. We used ellipsometry and advancing contact angles with water to follow the mixed SAM formation as a function of time. Figure 7 shows the kinetics of the adsorption process from different fTC ethanol solutions. In the pure MUA (fTC ) 0.0) and TC (fTC ) 1.0) solutions, cos θa reaches stable values after 1 h and 30 s, respectively. No obvious differences in the ellipsometric thicknesses are observed during the formation of the pure SAMs. Furthermore, the variation of cos θa as a function of time in the fTC ) 0.3 and 0.7 solutions shows that TC is adsorbed preferentially onto gold. In both solutions, the SAM changes from hydrophobic to hydrophilic after a few hours of immersion. (61) Finklea, H. O.; Avery, S.; Lynch, M.; Furtsch, T. Langmuir 1987, 3, 409. (62) Miller, C.; Cuendet, P.; Gra¨tzel, M. J. Phys. Chem. 1991, 95, 877. (63) Yang, Z. P.; Engquist, I.; Liedberg, B.; Kauffmann, J.-M. J. Electroanal. Chem., Interfacial Electrochem. in press.

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peaks by iTC and iMUA, then the following equations apply:

Figure 7. Variation of cos θa (θa ) advancing contact angle for water) as a function of the time of immersion of the gold slides in 1 mM TC/MUA solution, shown for different fTC. The first data points were obtained by exposing the gold slides to thiol solution for 5 s and then immediately rinsing and ultrasonically cleaning them with ethanol.. The lines between data points are included simply as guides to the eye.

Figure 8. Comparison of cos θa (θa ) advancing contact angle for water) for mixed SAMs adsorbed from mixtures of MUA/TC dissolved in ethanol and cyclohexane.

The adsorption of TC onto gold in the TC/MUA system is also highly dependent on the nature of the solvent. In ethanol, the carboxylic group in MUA is capable of strong and specific interactions with ethanol, in particular, hydrogen bonding, which will result in very good solvation. TC, however, is not capable of interacting as strongly as MUA with ethanol, so its solubility is substantially lower than that of MUA, giving rise to monolayers containing a substantial amount of TC. In a similar manner, in TC/ MUA cyclohexane solutions where we expect TC to have a far better solubility than MUA, the resulting mixed SAMs were composed predominantly of MUA (Figure 8). This reveals that solvation is the most important factor in determining the preference of adsorption in the TC/ MUA system, consistent with observations made for coadsorption of two functionalized alkanethiols.13 In the following, however, we discuss only the characteristics of MUA/TC SAMs prepared from ethanolic solutions. 3.7. Surface Composition and Molecular Structures. We have pointed out that we could not easily evaluate the mole fractions of TC and MUA in mixed SAMs with either XPS or IRAS measurements alone because of the different sizes, shapes, and pinning geometries of the MUA and TC molecules. Nevertheless, on a unit area, A, of the gold substrate, the integrated intensities of peaks in IRAS and XPS spectra are proportional to the number of TC and MUA molecules in the SAMs, respectively. If we denote the number of TC and MUA molecules by nTC and nMUA and the integrated intensities of TC and MUA

iTC ) KTCnTC

(1)

iMUA ) KMUAnMUA

(2)

where KTC and KMUA are constants representing the crosssections in IRAS and XPS. In order to obtain an estimate of the mole fraction, we used the MUA SAM as a reference with a coverage of 100% on gold, since the IRAS and electrochemical measurements have shown that the pure MUA SAM is highly ordered and densely packed. Further, we assume that the coverage of the pure TC SAM is δ because the electrochemical measurements indicate that there is a larger number of pinhole defects in the TC SAM. For the pure SAMs, we obtain

i0TC ) KTCn0TC ) KTCAδ/A0TC

(3)

i0MUA ) KMUAn0MUA ) KMUAA/A0MUA

(4)

where the superscript “0” indicates pure TC or MUA SAMs, 0 0 and AMUA are the cross-sectional areas of TC and and ATC MUA. We use the cross-sectional areas instead of the effective surface in the SAMs since the overlayer structure of TC SAM is unknown. We assume that the geometrical cross-sectional area of the MUA and TC molecules, as determined by space-filling models, are 16.6 and 36.2 Å2, respectively. The area of TC is consistent with the crosssectional area of cholesterol monohydrate.35 We further denote the mole fractions of TC and MUA in the mixed SAMs by χTC and χMUA and define them as

χTC ) nTC/(nTC + nMUA)

(5)

χTC + χMUA ) 1

(6)

Using the above assumptions and data, this system of equations can easily be solved to yield χTC as functions of the relative integrated intensities ITC and IMUA of the peaks, i.e.,

χTC )

ITCK IMUA + ITCK

(7)

where

K)

A0MUAδ A0TC

(8)

ITC ) iTC/i0TC

(9)

IMUA ) iMUA/i0MUA

(10)

It is clearly seen that the variation of χTC as a function of fTC is dependent on the coverage, δ, of the pure TC SAM. However, the coverage is still not definitely measured so far. On one hand, in our previous investigation,39 we assumed that the coverage of the mixed SAM is the same as that of the pure MUA SAM (100%) and found the coverage of the pure TC SAM to be approximately 65% as compared to a fully covered MUA SAM. Based on this assumption, the variation of χTC as a function of fTC is illustrated in Figure 9, δ ) 0.65. On the other hand, we are still seeking alternative ways of determining the coverage. Another approach is to use XPS data. We used the carbon 1s peak in the XPS spectra obtained from the MUA and TC SAM. The ratio of the integrated intensities of this peak for TC compared to MUA is 1.19 ( 0.02 using a takeoff angle of 90°, which is

Monolayer Assemblies of Cholesterol and Fatty Acids

Langmuir, Vol. 13, No. 12, 1997 3217

Figure 9. Variation of χTC in mixed TC/MUA SAMs as a function of fTC. δ is the coverage of the pure TC SAM obtained by two different assumptions. χTC represents the mole fraction of TC in the mixed SAMs, calculated from relative integrated intensities obtained by IRAS and XPS characterization (see text for details).

consistent with the ratio of the number of carbon atoms per cross-sectional area for TC compared to MUA (1.13). If we assume that the attenuation of photoelectrons for the carbon atoms of TC and MUA is the same although the density of carbon atoms along the chain axes varies substantially between the two molecules, we obtain a result saying that the coverage of TC and MUA is approximately the same, i.e., δ ≈ 100%. The existence of pinhole defects in the TC SAM, which seems to contradict such a dense packing, could be explained by a lateral distribution of the irregular TC molecules. Based on this estimation, the variation of χTC as a function of fTC is illustrated in Figure 9, δ ) 1.0. Both the δ ) 0.65 and the δ ) 1.0 results show that χTC varies slowly within the fTC ) 0.1-0.8 region but changes more rapidly for fTC < 0.1 and fTC > 0.8. Obviously, there is a variation between the two sets of data, but the resulting deviation of χTC is less than 0.1 from δ ) 0.65 to 1.0. We are at present not able to exclude either of the two methods used for calculating χTC. We merely suggest that the true composition can be found between these two limiting curves. Importantly, Figure 9 indicates that χTC definitely is less than 0.5 for fTC < 0.85, meaning that the mixed SAMs are MUA-rich, when compared to the molar concentrations in the ethanol solution. This means that adsorption of MUA dominates over TC at longer adsorption times for most of the studied fTC, except, of course, for fTC ) 1.0. For a similar system of CH3/COOH-terminated alkanethiol SAMs,13 the adsorption of the CH3-terminated species was preferred at all concentrations in ethanol because the carboxylic acid is better solvated in ethanol. In the mixed SAMs of TC/MUA, the solvation of TC in ethanol is much lower than that of MUA. From this simple argument one would therefore expect a preferential adsorption of TC. Such a preferential adsorption of TC indeed occurs at the beginning of the assembly process, as can be seen in Figure 7, but the preference swings to MUA instead of TC when the coadsorption approaches equilibrium-like conditions. A possible explanation is offered by considering the crosssectional size of TC and MUA. The small cross-sectional size of MUA allows more than two times as many MUA thiolate bonds to be formed per unit gold area as TC thiolate bonds. Since the thiolate/gold chemisorption energy is quite large (∼44 kcal/mol) and is exothermic,12 there is a large energy gain per unit area in forming SAMs

Figure 10. Variation in cos θa for water as a function of χTC (a) and χTC area (b). The broken line represents Cassie’s equation (see text for details).

with MUA molecules as compared to TC molecules, and this energy difference can be expected to dominate the differences between the corresponding ethanol solvation energies. However, from this argument alone one would expect the mole fraction of TC to be practically zero at equilibrium-like conditions for all fTC g 0.1. The full explanation for the observed composition of the mixed TC/MUA SAMs must therefore also involve the van der Waals interaction between the TC and MUA molecules, and probably also other factors, which could be, for example, the kinetics of SAM formation, the solvation and the temperature. 3.8. Wettability. The wettability of the surfaces of model biomembranes is an important property which can explain certain interfacial phenomena, for example, the adsorption and binding of proteins and other organic molecules.64,65 The wetting by water of the mixed SAMs shows a nonlinear behavior with fTC (see Figure 5). The cos θa values plotted as a function of χTC are presented in Figure 10a, which are obviously dependent on the coverage of the pure TC SAM (δ). These plots are quite different from Figure 5 because χTC is used instead of fTC. It is also different from the corresponding plot for mixed CH3/ COOH-terminated alkanethiol SAMs.13 If the two components in the mixed CH3/COOHterminated alkanethiol SAMs would act independently and the effects of surface roughness and phase separation are neglected, then the contact angles would follow Cassie’s equation,66

cos θ ) χ1 cos θ1 + χ2 cos θ2

(11)

(64) DiMilla, P. A.; Folkers, J. P.; Biebuyck, H. A.; Ha¨rter, R.; Lo´pez, G. P.; Whitesides, G. M. J. Am. Chem. Soc. 1994, 116, 2225. (65) Kumar, A.; Whitesides, G. M. Science 1994, 263, 60. (66) Cassie, A. B. D. Disscuss. Faraday Soc. 1948, 3, 11.

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where χ1 and χ2 are the mole fractions of the two components and θ1 and θ2 are the contact angles of the two pure SAMs. Consequently, a graph of cos θ against the mole fraction of the hydrophobic component would be linear (Figure 10a, dashed line). In a study by Bain et al.,13 on mixed CH3/COOH-terminated alkanethiolate SAMs the water contact angles deviated strongly from Cassie’s equation because of specific H-bonding interactions, in which the surface develops a stronger hydrophilicity.13 The cos θa values for the mixed TC/MUA SAMs in Figure 10a are also seen to deviate considerably from the linear Cassie equation in both cases (δ ) 0.65 or 1.0). Although the mixed SAMs are dominated by hydrophilic MUA molecules, they appear more hydrophobic than one would expect. A tentative explanation is connected with the conformational disorder of the MUA molecules in the mixed SAMs. The disordered MUA molecules may reorganize to minimize the surface free energy, resulting in the more hydrophobic surface. The irregular TC molecules provide sufficient space for such a reorganization, which may reduce the exposure of the carboxylic acid groups to the contacting liquid. As an alternative, the mismatch in geometric cross-sectional size between MUA and TC may generate the same results. If we make plots of cos θa against the fractional area of TC (see Figure 10b), defined as

χTC area ) χTCA0TC/(χTCA0TC + χMUAA0MUA)

(12)

we find that the cos θa values now more closely follow Cassie’s equation. This indicates that the wettability may be ruled by the molecular surface area fractions, rather than the surface mole fractions of TC and MUA.67 This effect is not seen for mixed SAMs of alkanethiols, since the cross-sectional areas of the components are identical for these SAMs.28 However, the plots of cos θa against χTC area still deviate from Cassie’s equation. The deviation may indicate that molecular reorganization, geometric effects, and specific H-bonding interactions simultaneously affect the wetting properties on the mixed SAMs. On the other hand, the deviation is dependent on the value of the coverage (δ) of the TC SAM. A proper coverage of the pure TC SAM is the key for determining the structural (67) Whitesides, G. M. In Handbook of Surface Imaging and Visualization; Hubbard, A. T., Ed.; CRC Press, Boca Raton, FL, 1995; p 713.

features and properties of the mixed SAMs. Investigations aiming at clarifying the coverage using radio labeling techniques are in progress at our laboratories.68 4. Conclusion Mixed SAMs prepared from binary mixtures of TC and MUA provide an interesting model system for investigating the role of the molecular geometry on the properties of mixed SAMs. The effects of the molecular geometry on the compositions and conformations in the mixed SAMs are addressed by using a combination of IRAS, XPS, ellipsometry, contact angle measurements, and cyclic voltammetry. The results indicate that the irregular and rigid TC molecules maintain their orientation in the mixed SAMs, whereas MUA molecules display a significantly more disordered conformation as compared to the MUA molecules in the pure SAM. Furthermore, it is clearly seen that the number of molecules per unit area changes with the molecular composition, as a consequence of the different shape and size of TC and MUA molecules. A method to determine the composition of the mixed SAMs by combining the IRAS and XPS techniques is presented. The method utilizes the coverage, δ, as a parameter, which we presently are unable to determine unambiguously. Still, we can conclude that all mixed SAMs formed from solution with fTC e 0.8 are MUA-rich if prepared under equilibrium-like conditions; i.e., the mole fraction of MUA in these SAMs is always above 0.5. The resulting wettability of the mixed SAMs is primarily ruled by the mole fractions of the two molecules in combination with their cross-sectional area. In addition, the molecular conformation of MUA may also influence the wettability, but to a lesser extent. Acknowledgment. This work was supported by a grant from the Swedish Research Council for Engineering Sciences. We also thank the European Science Foundation (ESF) for providing an ABI (Artificial Biosensing Interfaces) Fellowship to Z.Y. (University of Brussels-ULBPharmaceutical Institute) in 1994 and ‘95. We are grateful to Mr. Martin Johansson for generating the space-filling models. LA970015J (68) Schlenoff, J. B.; Li, M.; Ly, H. J. Am. Chem. Soc. 1995, 117, 12528.