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Self-Assembled Monolayer Growth of Phospholipids on Hydrophobic Surface toward Mimetic Biomembranes: Scanning Probe Microscopy Study Zhiyong Tang, Weiguo Jing, and Erkang Wang* Laboratory of Electroanalytical Chemistry and National Analytical Research Center of Electrochemistry and Spectroscopy, Changchun Institute of Applied Chemistry, Chinese Academy of Science, Changchun, Jilin 130022, People's Republic of China Received October 22, 1998. In Final Form: October 12, 1999 Atomic force microscopy (AFM) and lateral force microscopy (LFM) were used simultaneously to analyze a model membrane bilayer structure consisting of a phospholipid outer monolayer deposited onto organosilane-derivatized mica surfaces, which were constructed by using painting and self-assembly methods. The phospholipid used as outer monolayer was dimyristoylphosphatidylcholine (DMPC). The hydrocarbon-covered substrate that formed the inner half bilayer was composed of a self-assembly monolayer (SAM) of octadecyltrichloroorganosilane (OTS) on mica. SAMs of DMPC were formed by exposing hydrophobic mica to a solution of DMPC in decane/isobutanol and subsequently immersing into pure water. AFM images of samples immersed in solution for varying exposure times showed that before forming a complete monolayer the molecules aggregated into dense islands (2.2-2.6 nm high) on the surface. The islands had a compact and rounded morphology. LFM, coupled with topographic data obtained with the atomic force mode, had made possible the distinction between DMPC and OTS. The rate constant of DMPC growth was calculated. This is the first systematic study of the SAM formation of DMPC by AFM and LFM imaging. It reveals more direct information about the film morphology than previous studies with conventional surface analytical techniques such as infrared spectroscopy, X-ray, or fluorescence microscopy.
Introduction One of the most significant challenges presently facing biologists is to define the two-dimensional structure of biological membranes. Phospholipids are the major components of biological membranes. Phospholipid monolayers and bilayers have been studied extensively because of the simplified biological membrane models. The previous studies mainly focus on the characterization of molecular order within phospholipid monolayers by spectroscopic techniques, such as internal and external reflection infrared spectroscopy,1-4 glancing incident-angle X-ray technique,5 and fluorescence microscopy.6-8 The above techniques are intrinsically limited by the fact that they require a large amount of crystallized materials, and they are all averaging or indirect techniques. Recently, scanning tunneling microscopy (STM) has gained much interest, because the method can directly acquire highresolution microscopic images. Since the fist STM image of a lipid film was demonstrated,9 some workers have reported STM images of phospholipids in various forms.10-16 Because the phospholipids are nonconducting, * To whom correspondence should be addressed. (1) Dluhy, R. A. J. Phys. Chem. 1986, 90, 1373. (2) Dluhy, R. A.; Cornell, D. G. In Fourier Transform Infrared Spectroscopy in Colloid and Interface Science; Scheuing, D. R., Ed.; ACS Symposium Series 447; American Chemical Society: Washington, DC, 1991; p 192. (3) Gregory, B. W.; Dluhy, R. A.; Bottomley, L. A. J. Phys. Chem. 1994, 98, 1010. (4) Tamm, L. K.; Tatulian, S. A. Biochemistry 1993, 32, 7720. (5) Jacquemain, D.; Wolf, S. G.; Leveiller, F.; Deutsch, M.; Kjaer, K.; Als-Nielsen, J.; Lahav, M.; Leiserowitz, L. Angew. Chem., Int. Ed. Engl. 1992, 31, 130. (6) McConnell, H. M. Annu. Rev. Phys. Chem. 1990, 42, 171. (7) Mohwald, H. Annu. Rev. Phys. Chem. 1990, 41, 441. (8) Knobler, C. M. Adv. Chem. Phys. 1990, 77, 397. (9) Smith, D. P. E.; Bryant, A.; Quate, C. F.; Rabe, J. P.; Gerber, Ch.; Swalen, J. D. Proc. Natl. Acad. Sci. U.S.A. 1987, 84, 969. (10) Lang, C. A.; Horber, J. K.; Hansch, T. W.; Heckel, W. M.; Mohwald, H. J. Vac. Sci. Technol., A 1988, 6, 368.
classical tunneling theory cannot interpret the contrast mechanism. The controversy over the contrast mechanism of STM images can be avoided by using atomic force microscopy (AFM).17 Because AFM uses atomic or molecular interaction forces between the cantilever and the sample as a probe, AFM offers an opportunity of imaging the surface on a molecular scale without the requirement of conducting samples, especially for biological materials. Since molecular-resolution AFM images of phospholipids were first reported respectively by Weisenhorn et al. and Egger et al.,18,19 AFM technique is extensively used to study the various structures of phospholipids, such as molecular-resolution and macroscale images of phospholipids by different preparation techniques,20-27 the mor(11) Voelker, M. A.; Hameroff, S. R.; He, J. D.; Dereniak, E. L.; McCuskey, R. A.; Schniker, C. W.; Chvapil, T. A.; Bell, L. S.; Weiss, L. B. J. Microsc. 1988, 152, 557. (12) Horber, J. K. H.; Lang, C. A.; Hansch, T. W.; Heckl, W. M.; Mohwald, H. Chem. Phys. Lett. 1988, 145, 151. (13) Luo, C.; Zhu, C.; Ruan, L.; Huang, G.; Dai, C.; Cheng, Z.; Bai, C.; Su, Yu.; Xu, S.; Lin, K.; Baldeswieler, J. D. J. Vac. Sci. Technol., A 1990, 8, 684. (14) Su, Y.-X.; Jiao, Y.-K.; Xu, S.-D.; Yao, J.-E.; Lin, K.-C. J. Vac. Sci. Technol., A 1990, 8, 695. (15) Niemi, H. E.-M.; Ikonen, M.; Levlin, J. M.; Lemmetyinen, H. Langmuir 1993, 9, 2436. (16) Heim, M.; Cevec, G.; Guckenberger, R.; Knapp, H. F.; Wiegrabe, W. Biophys. J. 1995, 69, 489. (17) Binning, G.; Quate, C. F.; Gerber, Ch. Phys. Rev. Lett. 1986, 56, 930. (18) Egger, M.; Ohnesorge, F.; Weisenhorn, A. L.; Heyn, S. P.; Drake, B.; Prater, C. B.; Gould, S. A. C.; Hansma, P. K.; Gaub, H. E. J. Struct. Biol. 1990, 103, 89. (19) Weisenhorn, A. L.; Drake, B.; Prater, C. B.; Gould, S. A.; Hansma, P. K.; Ohnesorge, F.; Egger, M.; Heyn, S. P.; Gaub, H. E. Biophys. J. 1990, 58, 1251. (20) Zasadzinski, J. A. N.; Helm, C. A.; Longo, M. L.; Weisenhorn, A. L.; Gould, S. A. C.; Hansma, P. K. Biophys. J. 1991, 59, 755. (21) Hansma, H. G.; Gould, S. A. C.; Hansma, P. K.; Gaub, H. E.; Longo, M. L.; Zasadzinski. J. A. N. Langmuir 1991, 7, 1051. (22) Hansma, H. G.; Weisenhorn, A. L.; Edmundson, A. B.; Gaub, H. E.; Hansma, P. K. Clin. Chem. 1991, 37, 1497.
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phologies of phospholipids under various conditions,28-31 the “liquid expanded”-“liquid condensed” coexisting phase of phospholipid LB films,32-35 and the phospholipids in the interaction with protein and DNA.36-40 Despite a large amount of research published about the surface structures of phospholipid monolayers and bilayers, little knowledge has been gained about the mechanism of their formation and growth. We propose, in this manuscript, a model system for such kinetic studies. We built up solid-supported phospholipid bilayers onto substrates using basically the well-known self-assembly of organosilane onto clean mica surfaces. The first monolayer was formed by self-assembly of organosilane on mica.41-44 The deposited monolayer then consisted of closely packed hydrocarbon chains, which made the mica hydrophobic and therefore well-suited for the attachment of a second phospholipid monolayer to obtain a bilayer system. This second monolayer was deposited onto the first one by the painted lipid membrane technique.45-47 AFM and lateral force microscopy (LFM) were used to image the second phospholipid monolayers. The microscopic morphology of the phospholipid monolayers at different painting times and adsorption kinetics are discussed. In this study, we did not utilize thiol-Au but organosilane-mica as hydrophobic substrates. This is because the atomically flat surface of mica may be several micrometers, and it is ideal for AFM and LFM studies. Otherwise, the atomic terrace of an Au single crystal is only several hundred nanometers. Experimental Section Materials. Dimyristoylphosphatidylcholine (DMPC) was obtained from Sigma, at >99% purity. Octadecyltrichloroorganosilane (OTS) was obtained from Aldrich, at >98% purity. The (23) Singh, S.; Keller, D. J. Biophys. J. 1991, 60, 1401. (24) Vikholm, I.; Peltonen, J.; Teleman, O. Biochim. Biophys. Acta 1995, 1233, 111. (25) Santesson, L.; Wong, T. M. H.; Taborelli, M.; Descouts, P.; Liley, M.; Duschl, C.; Vogel, H. J. Phys. Chem. 1995, 99, 1038. (26) Ohlsson, P.-K.; Tjarnhage, T.; Herbai, E.; Lofas, S.; Puu, G. Bioeletrochem. Bioenerg. 1995, 38, 137. (27) Muscatello, U.; Valdre, G.; Valdre, U. J. Microsc. 1996, 182, 200. (28) Mou, J.; Yang, J.; Shao, Z. Biochemistry 1994, 33, 4439. (29) Mou, J.; Yang, J.; Huang, C.; Shao, Z. Biochemistry 1994, 33, 9981. (30) Hui, S. W.; Viswanathan, R.; Zasadzinski, J. A.; Israelachvili, J. N. Biophys. J. 1995, 68, 171. (31) Fang, Y.; Yang, J. Biochim. Biophys. Acta 1997, 1324, 309. (32) Mikrut, J. M.; Dutta, P.; Ketterson, J. B.; MacDonald, R. C. Phys. Rev. B 1993, 48, 14479. (33) Yang, X.-M.; Xiao, S.-J.; Lu, Z.-H.; Wei, Y. Surf. Sci. 1994, 316, L1110. (34) Yang, X.-M.; Xiao, D.; Lu, Z.-H.; Wei, Y. Appl. Surf. Sci. 1995, 90, 175. (35) Yuan, C.; Yang, X.; Lu, Z.; Liu, J. Surf. Sci. 1996, 355, L381. (36) Weisenhorn, A. L.; Egger, M.; Ohnesorge, F.; Gould, S. A. C.; Heyn, S. P.; Hansma, H. G.; Sinsheimer, R. L.; Gaub, H. E.; Hansma, P. K. Langmuir 1991, 7, 8. (37) Burgess, J. D.; Jones, V. W.; Porter, M. D.; Rhoten, M. C.; Hawkridge, F. M. Langmuir 1998, 14, 6628. (38) Yang, J.; Tamm, L. K.; Tillack, T. W.; Shao, Z. J. Mol. Biol. 1993, 229, 286. (39) Mou, J.; Yang, J.; Shao, Z. J. Mol. Biol. 1995, 248, 507. (40) Fang, Y.; Yang, J. J. Phys. Chem. 1997, 101, 441. (41) Carson, G.; Granick, S. J. Appl. Polym. Sci. 1989, 37, 2767. (42) Kessel, C. R.; Granick, S. Langmuir 1991, 7, 532. (43) Schwartz, D. K.; Steinberg, S.; Israelachvili, J.; Zasadzinski, J. A. N. Phys. Rev. Lett. 1992, 69, 3354. (44) Bhushan, B.; Kulkarni, A. V.; Koinkar, V. N. Langmuir 1995, 11, 3189. (45) Florin, E.-L.; Gaub, H. J. Biophys. J. 1993, 64, 375. (46) Steinem, C.; Janshoff, A.; Ulrich, W.-P.; Sieber, M.; Galla, H.-J. Biochim. Biophys. Acta 1996, 1279, 169. (47) Ding, L.; Li, J.; Dong, S.; Wang, E. J. Electroanal. Chem. 1996, 416, 105.
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Figure 1. Illustration depicting the phospholipid model bilayer membrane structure examined in this paper: DMPC/OTS bilayer on mica, investigated by AFM and LFM. above two compounds were used as received. Hexadecane (C16H34), carbon tetrachloride (CCl4), chloroform (CHCl3), decane (C10H22), and isobutanol were purified by redistillation before use. Ultrapure water was distilled with an all-Pyrex distilling apparatus and then purified with a milli-Q ultrapure water system. Preparation of Substrates. The substrate was mica coated with OTS. For OTS treatment, mica must be hydroxylated.19-22 Then mica was dipped in a solution consisting of 100 mL of concentrated H2SO4, 5 mL of water, and 2 g of potassium bichromate for 10 min.44 After drying with nitrogen stream, it was dipped into a solution of 0.1 mL of OTS in 100 mL of solvent consisting of 80% C16H34, 12% CCl4, and 8% CHCl3. After the silylation reaction was completed, within about 1 min, the substrate was rinsed thoroughly with CHCl3 and again heated to 120 °C to remove noncovalently bound OTS molecules. OTS forms a self-assembly hydrocarbon monolayer on surface. OTS/ mica was used as substrate for the DMPC monolayer formation. Self-Assembly of Phospholipid Monolayers. The OTS/ mica substrates were submerged in a 1 mg/mL solution of DMPC in decane/isobutanol (10:1,v/v) and removed at intervals of 1, 5, 10, 30, 60, and 120 s. Upon removal the sample was dipped into ultrapure water for 10 min to remove extra DMPC and form bilayer membrane structure, and the samples were finally blown dry with a dry nitrogen stream. AFM and LFM. AFM and LFM measurements were performed with a TMX 2000 model from Topometrix Instruments (Santa Clara, CA) with a maximum scan size of 8 µm. A microfabricated Si3N4 cantilever with integrated pyramidal tips and a spring constant of 0.12N/m were used. For AFM measurements, the typical loading force was 10-9 N in air at room temperature. LFM measurements were performed simultaneously with AFM measurements. All images were recorded in 400 × 400 epixels at the scanning rate 2 µm/s.
Results and Discussion A model bilayer of DMPC/OTS on mica is constructed by painting a DMPC monolayer onto organosilanederivatized mica substrates. The results of DMPC/OTS model bilayer membranes systems are shown schematically in Figure 1. This bilayer membrane was used in our AFM and LFM studies. In the painted lipid membrane technique, the bilayer structure of the membrane was difficult to form in organic solvent because of the instability of the hydrophilic head of the lipid facing the hydrophobic organic solvent, and taking the DMPC/OTS out and immersing it into water were helpful in forming the bilayer structure. Therefore, the observed surface morphology only represents the structure of the DMPC monolayer formed in water, not the structure of the growing film in organic solvent. 1. AFM Images of Phospholipid Monolayer Growth. Figure 2a-g are typical AFM images of DMPC adsorbed on OTS/mica from the 1 mg/mL decane/isobutane solution at different immersion times. All images are
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Figure 2. AFM and LFM images of the same zone of the DMPC SAMs on OTS/mica produced by different immersion times in 1 mg/mL decane/isobutanol solutions. The immersion time is noted on the images. All images are 1 µm × 1 µm. (2a-g) AFM images: The brighter part on OTS/mica corresponds to the condensed DMPC phase (so-called liquid or solid phase), whereas the darker part corresponds to the dilute phase (so-called gas phase or bare OTS/mica). (2h-n) LFM images: The DMPC monolayer is characterized by a low friction. The brighter part corresponds to OTS; the darker corresponds to the DMPC SAMs.
typical of images obtained from at least five macroscopically separated regions on each sample and show typical behavior of coverage versus time that we have observed on five sets of samples. The control experiment of immersing the OTS/mica in bare solvent shows a micrometer-sized flat surface, and there is no surface damage as the force attains 50 nN exerted by the tip. The AFM clearly shows that DMPC molecules aggregate to form islands on the OTS/mica substrate during the early stages of DMPC monolayer formation. Repeated scans of the same area at 10-9 N reproducibly created the same image and no evidence of tip-induced damage to the samples. It shows that the bilayer structure has relatively high stability in air, which is consistent with recent observation by Yuan et al.48 It is also observed that the island is probably removed when the loading force of the tip attains 10 nN, and some aggregate and multilayer formation happen after the sample is exposed in air for 2 days. The heights of islands, obtained from the cross-
section of the original AFM images, are approximately 2.2-2.6 nm higher than the surrounding area (shown as Figure 3). Assuming that all trans-acyl chains are perpendicular to the phospholipid headgroup plane, the thickness of the DMPC monolayer should be approximately 2.4 nm.12,49 Therefore, the AFM image analysis shows that acyl chains orient nearly perpendicular to the bilayer plane, which is similar to the result of the supported bilayer obtained by the Langmuir-Blodgett technique.3 Let us now look at the details of DMPC monolayer formation. We find that those features of DMPC monolayer growth are not affected by immersion time in ultrapure water (1-30 min). The surface deposit is already inhomogeneous after 1s of immersion (Figure 2a). The outline of the islands, whose height and size are 2.2-2.6 nm and 60-150 nm, respectively, is clear. These islands are generally round. The fact that the preferred shape for the islands is round implies that there was a significant effective line between the islands and the bare area. It is
(48) Yuan, C.; Wu, Y.; Sun, Y.; Lu, Z.; Liu, J. Surf. Sci. 1997, 392, L1-L6.
(49) Watts, A.; Harlos, K.; Marsh, D. Biochim. Biophys. Acta 1981, 645, 91.
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Langmuir, Vol. 16, No. 4, 2000 1701 Table 1. Percentage of Surface Covered by DMPC Monolayer Islands from Sample Set of Figure 2
Figure 3. (a) AFM image of the DMPC SAMs on OTS/mica produced in 1 mg/mL decane/isobutanol solution at 2 s. (b) Crosssectional plots showing the height of the islands along line 1 and line 2 in (a).
also noticed that the round island is formed after removal from the organic solvent, and subsequent insertion and removal from water.50-52 Such a process affects the island structure and morphology, and some in situ techniques and new methods of forming films will be developed to observe the original morphology of the uncompleted film. After 2, 5, and 10 s of immersion, these islands increase in size, which are estimated to be 120-240 nm, 140-420 nm, and 140-480 nm respectively (Figure 2b-d). Over 50% of the substrate is covered with DMPC monolayer after an immersion time of 30 s (Figure 2e), whereupon the islands coalesce and turn into the continuous domains. Eventually, only small holes in the DMPC monolayer remain and gradually fill in after 60 s of immersion (Figure 2f). After 120 s of immersion (Figure 2g), the DMPC monolayer can be completely formed as large as 4 µm, and it also shows that the painting lipid membrane technique is a good method of forming the bilayer membrane. During the process of DMPC film formation, the DMPC selfassembly monolayer (SAM) exhibits nucleation-growthcollision process, not random adsorption. This indicates a rearrangement of the adsorbed DMPC molecules during the membrane formation process, especially during immersion in ultrapure water. The lateral interaction between DMPC molecular chains is large enough to induce such molecular rearrangement. Therefore the lateral diffusion of DMPC molecules on OTS/mica or the selective exchanges of DMPC molecules between surface and solution takes place within the duration of the membrane formation. As mentioned above, the features of DMPC monolayer growth are not affected by the immersion time in ultrapure water, so the diffusion and exchange are very fast. 2. LFM Images of Phospholipid Monolayer Growth. We use LFM here to identify the differences between chemical and frictional properties of DMPC and OTS/mica substrate. (50) Woodward, J. T.; Ulman, A.; Schwartz, D. K. Langmuir 1996, 12, 3626. (51) Woodward, J. T.; Schwartz, D. K. J. Am. Chem. Soc. 1996, 118, 7861. (52) Woodward, J. T.; Doudevski, I.; Sikes, H. D.; Schwartz, D. K. J. Phys. Chem. 1997, 101, 7535.
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Figure 2h-n are the LFM images of the DMPC membrane at different immersion times, which are recorded simultaneously with Figure 2a-g. To the LFM image, the light color represents the high friction zone, whereas the dark represents the low friction zone. Compared with AFM images, obviously DMPC film is characterized by a low friction and appears dark; on the contrary, the OTS/mica zone is characterized by a high friction and appears light. The LFM images also clearly show the growth process of DMPC, which is the same as results of AFM measurement. Referring to the bilayer membrane model (Figure 1), the parts of DMPC and OTS contacting with the tip are the hydrophilic choline head and the hydrophobic alkyl chain, respectively. It is interesting that the hydrophilic surface has higher friction than the hydrophobic surface in our observation. Valleton and colleagues also found that the hydrophilic surface has low friction force in the study of mixed Langmuir-Blodgett films consisting of amphiphilic molecules and protein by SPM.53-56 As we know, the composition of friction force is very complicated. On the molecular scale, it correlated not only with chemical properties but also with packing energy, packing density, elasticity, and local disorder.57,58 Packing energies mainly arise from hydrophobic attraction in acyl chains and probable tail group-tail group interaction, if there is hydrogen bonding in them. Besides the hydrophobic attraction in acyl chains, there are the strong hydrogen bonding interactions in the phosphate groups of DMPC molecules, which have been directly visualized by Yuan et al.48 The forces make the DMPC monolayer have higher packing energy and stronger capacity of defending penetration of the probe. Therefore the DMPC monolayer probably yields a lower friction force compared with the OTS monolayer. 3. Growth Kinetics of DMPC Monolayer. Known from the above discussion of Figure 2, the DMPC monolayer grows with immersion time. The area fraction of the DMPC monolayer on substrates could be estimated by the surface area-analyzing software of the TMX 2000, and the results are shown in the Table 1. The results are typical data of samples. According to a simple Langmuirian model, the monolayer coverage as a function of immersion time can be expressed:
θ ) 1 - exp(-ckt) where k is first-order rate constant, c is the bulk concentration of DMPC solution, t is immersion time, and θ is the fraction of monolayer coverage. Obviously plots of -ln(1 - θ) versus time should be linear, where the slope is proportional to the rate of monolayer formation. We tried to fit the data in Table 1 on the basis of the above discussion and the results are shown in Figure 4. We can (53) Alexandre, S.; Dubreuil, N.; Fiol, C.; Valleton, J. M. Microsc. Microanal. Microstruct. 1994, 5, 61. (54) Alexandre, S.; Dubreuil, N.; Fiol, C.; Malandain, J. J.; Sommer, F.; Valleton, J. M. Microsc. Microanal. Microstruct. 1994, 5, 539. (55) Dubreuil, N.; Alexandre, S.; Fiol, C.; Sommer, F.; Valleton, J. M. Langmuir 1995, 11, 2098. (56) Sommer, F.; Alexandre, S.; Dubreuil, N.; Lair, D.; Duc, T.; Valleton, J. M. Langmuir 1997, 13, 791. (57) Krim, J.; Scolina, D. H.; Chiarello, R. Phys. Rev. Lett. 1991, 66, 181. (58) Kim, H.; Koini, T.; Lee, T. R.; Perry, S. S. Langmuir 1997, 13, 7192.
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It is also found that there is a slightly larger slope at longer times, after closer inspection of Figure 4. It reveals that there is cooperative interaction during DMPC domain growth, which is different from thiol SAM on Au or OTS SAM on Ge.58-63 It is also consistent with the fact that the driving force of thiol and OTS SAM mainly comes from strong adsorbate-substrate interaction and partly comes from the adsorbate-adsorbate hydrophobic interaction, but the driving force of DMPC SAM comes more from strong hydrogen bonding interactions between the adsorptive DMPC molecules, besides the above interactions.
Figure 4. Plot of -ln(1 - θ) versus immersion time. The straight line shows the best fit to the experimental dots (slope ) 2.86 × 10-2; correlation factor r2 > 0.99).
calculate the intrinsic rate of the DMPC SAM to be 19.43 ( 2.11 s-1 M-1. The above fit only reflects the approximate kinetics parameter of adsorption. In fact, a systematic deviation from the fitting function can be seen in Figure 4, which demonstrates that the adsorption kinetics of DMPC is more complicated than the simple Langmuir kinetics would imply. Further work should be done to better fit the adsorption course. The SAM kinetics of amphiphilic molecules have recently been reported. The outstanding and excellent work has been done by Schwartz’s group. They have reported octadecylphosphonic acid (OPA) monolayer growth on mica in detail and conclude that the OPA growth obeys a complex diffusion-like kinetics.50-52 Suckenick and colleagues studied SAM of OTS on germanium by Fourier transform infrared spectroscopy/attenuated total reflectance (FTIR/ATR) and concluded that the OTS growth rate is 1.15 × 10-2 s-1 M-1.59 Condo et al. studied the assembly kinetics of thiol on Au, and the growth rate was about 250 s-1 M-1.60 Despite the error of various measurement methods, we should note that the intrinsic rate of DMPC SAM is less than that of thiol SAM on Au but larger than that of OTS SAM on Ge. The impact factors of molecular structures on the adsorption thermodynamics and kinetics include the energy of solvation of the molecules, adsorbate-substrate interaction, and the adsorbate-adsorbate interaction. To long-chain amphiphilic molecules, there is no large difference among the molecular solvation energy. The adsorbate-substrate interaction of DMPC mainly comes from van der Waals force of the acyl chains between DMPC and OTS molecules, which is much less than bonding energy of Au-S. It decides the difference of adsorption rate between DMPC on OTS/ mica and thiol on Au. To OTS SAM on Ge, the interaction of Ge-O is also larger than that between DMPC and OTS. Thus, the difference of adsorption rate is due to the adsorbate-adsorbate interaction. As in the above discussion, besides the hydrophobic attraction in acyl chains, there are strong hydrogen bonding interactions in the phosphate groups of DMPC molecules. All above interactions make DMPC molecules have a strong tendency to be adsorbed on the OTS surface and form and stabilize the structure of the bilayer membrane. (59) Cheng, S. S.; Scherson, D. A.; Sukenik, C. N. J. Am. Chem. Soc. 1992, 114, 5436. (60) Kondo, T.; Takechi, M.; Sato, Y.; Uosaki, K. J. Electroanal. Chem. 1995, 381, 203.
Conclusion We have found that monolayers of DMPC are adsorbed on hydrophobic OTS/mica to form a bilayer membrane. AFM images of partially formed monolayers created at different exposure times show that the molecules on the surface aggregate into rounded islands, implying that rearrangement takes place because of the lateral interaction between the adsorbed DMPC, and the islands have a line tension in water. The height of the island is 2.2-2.6 nm, implying that the alkyl chains orient nearly perpendicular to the bilayer plane. The whole process of forming a bilayer membrane is very fast and finishes in 2 min. Lateral force or friction mode in conjunction with the atomic force mode can help us to discriminate between DMPC and OTS domains, and a similar growth process is observed comparing AFM images. The rate constant of DMPC growth can be calculated to be 19.43 ( 2.11 s-1 M-1. The van der Waals force of acyl chains between OTS and DMPC molecules, the hydrophobic attraction in acyl chains, and strong hydrogen interaction in the phosphate head of DMPC molecules are considered to be responsible for the quick formation and high stability of the bilayer membrane. It must be pointed out that the present film-forming method precludes directly the observation of monolayer morphology as it grows in organic solvent. In fact, we do not gain the same observed structure without immersing the film in water. This makes our observation of DMPC SAM limited to the surface morphology formed in water. Here, rearrangement of DMPC molecules on OTS/mica may occur and the structure changes in water. It is also noticed that there are some disadvantages in AFM observation. First, for ex situ AFM, the observed morphologies of the uncompleted film may not represent the actual structure growing in the solution, and the structure arrangement probably happens during the insertion or removal of the sample from the solvent or solution; second, for in situ AFM, formation time of bilayer structure in our experiment is varied from 1 s ∼ 2 min, and it is too fast compared with the in situ AFM image capture rate. For better understanding of the course of the bilayer membrane formation, it is very important to understand the following two processes separately: the lateral diffusion and rearrangement of DMPC molecules on OTS/ mica. Future work is to develop a new method of forming film and fast in situ techniques for direct observation. Acknowledgment. The research was supported by the National Natural Science Foundation of China. Supporting Information Available: AFM images of bare mica and OTS monolayer on mica. This material is available free of charge via the Internet at http://pubs.acs.org. LA981491S (61) Bain, C. D.; Troughton, E. R.; Whiteside, G. M. J. Am. Chem. Soc. 1989, 111, 321. (62) Poirier, G. E.; Pylant, E. D. Science 1996, 272, 1145. (63) Pan, W.; Durning, C. J.; Turro, N. J. Langmuir 1996, 12, 4469.
