pubs.acs.org/Langmuir © 2010 American Chemical Society
Evolution of Supported Planar Lipid Bilayers on Step-Controlled Sapphire Surfaces Toshinari Isono,* Takayuki Ikeda, and Toshio Ogino Yokohama National University, 79-5 Tokiwadai, Hodogaya-ku, Yokohama, 240-8501, Japan Received January 14, 2010. Revised Manuscript Received March 11, 2010 Self-organized step/terrace structures on a sapphire surface were used to investigate interface properties between a solid surface and a supported planar lipid bilayer (SPB). We prepared random-stepped, single-stepped and multistepped sapphire surfaces. Some multistepped surfaces covered with crossing steps exhibit phase-separation into hydrophilic and hydrophobic domains. We studied evolution of self-spreading lipid bilayers that are subject to the atomic structures and chemical states on the surfaces. The growth direction of SPBs in the self-spreading method is regulated by the atomic steps. While the SPBs were apparently uniform after a 1 h self-spreading, a density gradient of the lipid molecules was observed even after 24 h spreading. We found that various patterns of the SPBs that depend on the density of the lipid molecules are self-assembled on the phase-separated surfaces. Although the SPB is supported on the sapphire surface via an about 1 nm water layer, the self-spreading direction and the morphology of the SPBs are affected by the atomic steps, whose height is much smaller than that of the water layer.
Introduction Artificial cell membranes, for example liposomes and black membranes, are widely used as a model system of the cell membranes as well as in the application to screening devises for drug discovery.1,2 Because membrane proteins, which are functional components of the cell membranes, are closely related to many diseases, they attract much attention as the target molecules of drug screening. While the membrane proteins denature in water without any support, they can retain their three-dimensional structures by incorporating into a lipid membrane. The lipid bilayers, therefore, play important roles in maintaining the functions of membrane proteins by providing an environment similar to the real cell membrane. A planar lipid bilayer membrane supported on a solid surface is a model system for in vitro studies of cell membranes and solidbiomaterial interfaces.3,4 This system is called a supported planar lipid bilayer (SPB). The SPBs have a well-defined unilamellar structure and are more stable than the black lipid membranes.5 Furthermore, the SPBs can be characterized using surface sensitive analytical methods which cannot be applied to the black membranes.6-14 In a SPB, the membrane is supported on the solid surface via a water layer. Many of the unique physical and biological features of the SPBs are generated by this water layer,
*Corresponding author. E-mail:
[email protected]. (1) Pioufle, B. L.; Suzuki, H.; Tabata, K. V.; Noji, H.; Takeuchi, S. Anal. Chem. 2008, 80, 328–332. (2) Shi, N.; Ye, S.; Alam, A.; Chen, L.; Jiang, Y. Nature 2006, 440, 570–574. (3) Sackmann, E. Science 1996, 271, 43–48. (4) Tamm, L. K.; McConnell, H. M. Biophys. J. 1985, 47, 105–113. (5) Castellana, E. T.; Cremer, P. S. Surf. Sci. Rep. 2006, 61, 429–444. (6) Scheuring, S.; Boudier, T.; Sturgis, J. N. J. Struct. Biol. 2007, 159, 268–276. (7) Lei, S. B.; Tero, R.; Misawa, N.; Yamamura, S.; Wan, L. J.; Urisu, T. Chem. Phys. Lett. 2006, 429, 244–249. (8) Jass, J.; Tj€arnhage, T.; Puu, G. Biophys. J. 2000, 79, 3153–3163. (9) Reviakine, I.; Brisson, A. Langmuir 2000, 16, 1806–1815. (10) Richter, R. P.; Brisson, A. R. Biophys. J. 2005, 88, 3422–3433. (11) Keller, C. A.; Kasemo, B. Biophys. J. 1998, 75, 1397–1402. (12) Ebara, Y.; Ebato, H.; Ariga, K.; Okahata, Y. Langmuir 1994, 10, 2267– 2271. (13) Ariga, K.; Okahata, Y. Langmuir 1994, 10, 2272–2276. (14) Tawa, K.; Morigaki, K. Biophys. J. 2005, 89, 2750–2758.
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such as lateral fluidity, which is an important character for understanding of the dynamics of the lipid bilayer membranes. The supported lipid bilayers are generally prepared by the vesicle fusion method,7-11,14-21 the Langmuir-Blodgett method,4,12,13 and the self-spreading method.22-30 The self-spreading is a simple method to form a single planar lipid bilayer, as illustrated in Figure 1. In this technique, the bilayer size is easy to control, because it simply depends on the spreading time. The spontaneous self-spreading at a water-solid interface in this system is expected to be a scientific and applicative methodology for the uniform SPB formation and the molecular transport along the substrate surface.23,26,27 Because the SPBs spontaneously self-spread due to a gain of attractive interaction energy between the lipid bilayer and the solid surface,22,26 the physical properties and the structure of the self-spreading SPBs are affected by the properties of the solid surface. Recently, micro- and nanostructured surfaces have been used in the fundamental studies and biological applications.23-25,27,28,30 The density of lipid molecules in the self-spreading SPB during growth is decreased from the center of (15) Groves, J. T.; Ulman, N.; Boxer, S. G. Science 1997, 275, 651–653. (16) Morigaki, K.; Kiyosue, K.; Taguchi, T. Langmuir 2004, 20, 7729–7735. (17) Isono, T.; Tanaka, H.; Ogino, T. e-J. Surf. Sci. Nanotech. 2007, 5, 99–102. (18) Tero, R.; Watanabe, H.; Urisu, T. Phys. Chem. Chem. Phys. 2006, 8, 3885– 3894. (19) Tero, R.; Ujihara, T.; Urisu, T. Langmuir 2008, 24, 11567–11576. (20) Tero, R.; Ujihara, T.; Urisu, T. Trans. Mater. Res. Soc. Jpn. 2009, 34(2), 183–188. (21) Johnson, J. M.; Ha, T.; Chu, S.; Boxer, S. G. Biophys. J. 2002, 83, 3371– 3379. (22) Nissen, J.; Gritsch, S.; Wiegand, G.; R€adler, J. O. Eur. Phys. J. B 1999, 10, 335–344. (23) Furukawa, K.; Nakashima, H.; Kashimura, Y.; Torimitsu, K. Lab Chip 2006, 6, 1001–1006. (24) Furukawa, K.; Sumitomo, K.; Nakashima, H.; Kashimura, Y.; Torimitsu, K. Langmuir 2007, 23, 367–371. (25) Werner, J. H.; Montano, G. A.; Garcia, A. L.; Zurek, N. A.; Akhadov, E. A.; Lopez, G. P.; Shreve, A. P. Langmuir 2009, 25, 2986–2993. (26) Nabika, H.; Fukasawa, A.; Murakoshi, K. Langmuir 2006, 22, 10927– 10931. (27) Takimoto, B.; Nabika, H.; Murakoshi, K. Jpn. J. Appl. Phys. 2006, 45, 6039–6042. (28) R€adler, J.; Strey, H.; Sackmann, E. Langmuir 1995, 11, 4539–4548. (29) Suzuki, K.; Masuhara, H. Langmuir 2005, 21, 537–544. (30) Suzuki, K.; Masuhara, H. Langmuir 2005, 21, 6487–6494.
Published on Web 03/26/2010
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Figure 1. Illustration of spontaneous self-spreading phenomenon of a SPB. The SPBs self-spread from an initial lipid source in a polar solution. A density gradient of lipid molecules in a self-spreading SPB occurs owing to the frictional force applied from the substrate surface. The density decreases with an increase in the distance from the initial lipid source.
the SPB to the edge by the frictional force applied from the substrate surface.22 In the region of a low density of lipid molecules, the influence of the surface structures to the SPBs is enhanced because the interaction between the individual lipid molecules, which maintains the bilayer structure, is reduced. To further study the interaction between the SPBs and the solid surface, we used well-ordered atomic structures on single-crystalline solid surfaces and investigated their effects on the shape and dynamics of the self-spreading lipid bilayers. Self-assembled step/ terrace structures on a vicinal surface are useful for fundamental study of the SPB formation. We used sapphire surfaces to control the biointerfaces.31 Regular step/terrace structures can be prepared by annealing polished sapphire substrates.32-35 The sapphire surfaces are chemically stable and their atomic structures can be retained even in air and liquid environment because of their chemically inert properties. In this work, we investigated the influence of the atomic structures on the step-controlled sapphire surfaces on behaviors of the SPBs and the dependence of the SPB patterns on the spatial lipid density.
Materials and Methods Preparation of Sapphire Surfaces. Single-crystalline sapphire (0001) substrates provided from Namiki Precision Jewel Co., Ltd. were used to support the lipid bilayers. In this work, we prepared four types of step-controlled sapphire surfaces. To form step/terrace structures, polished sapphire wafers with a miscut of 0.15 degree toward [1-100] were annealed in air. Upon annealing at 800 °C, a disordered step arrangement formed. We call this surface a random-stepped surface. For formation of straight single-layer steps, the sapphire surfaces were annealed at 1000 °C. We call this surface a single-stepped surface. Generally, step bunching occurs upon further high temperature annealing.35 Upon annealing at 1300 °C, the surface is covered with straight bunched steps of a few atomic layers. We call this surface a multistepped surface. When the miscut direction is slightly tilted from [1-100], the surface is covered with bunched steps and crossing steps generated by the tilting angle from the stable crystallographic orientation. We call this surface a crossstepped surface. These surfaces were sonicated in deionized water (>18 MΩ cm-1, Millipore) for 5 min using a bath-type sonicator. And they were cleaned by a mixture of H2SO4 (98%) and H2O2 (33%) (volume ratio of 3:1) at 80 °C for 10 min to remove organic contaminations and sonicated in deionized water for 5 min. Supported Lipid Bilayer Formation and Observation. Supported lipid bilayers were formed by the self-spreading method (31) Aoki, R.; Arakawa, T.; Misawa, N.; Tero, R.; Urisu, T.; Takeuchi, A.; Ogino, T. Surf. Sci. 2007, 601, 4915–4921. (32) Yoshimoto, M.; Maeda, T.; Ohnishi, T.; Koinuma, H.; Ishiyama, O.; Shinohara, M.; Kubo, M.; Miura, R.; Miyamoto, A. Appl. Phys. Lett. 1995, 67, 2615–2617. (33) Kurnosikov, O.; Van, L. P.; Cousty, J. Surf. Sci. 2000, 459, 256–264. (34) Shiratsuchi, Y.; Yamamoto, M.; Kamada, Y. Jpn. J. Appl. Phys. 2002, 41, 5719–5725. (35) Ribic, P. R.; Bratina, G. Surf. Sci. 2007, 601, 44–49.
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on the sapphire surfaces. 1,2-Dimyristoyl-sn-glycero-3-phosphocholine (DMPC) and fluorescently labeled 1-myristoyl-2-[12-{(7nitro-2-1,3-benzoxadiazol-4-yl)amino}dodecanoyl]-sn-glycero-3phosphocholine (NBD PC) were purchased from Avanti Polar Lipid Inc. and used without purification. Lateral interaction originating in the hydrophobic chains of lipid molecules is one of the important factors in SPBs. To clearly demonstrate the density effect of the SPBs, we chose DMPC molecules, whose hydrophobic chains are short. However, we also investigated selfspreading of DOPC, 1,2-dioleoyl-sn-glycero-3-phosphocholine, bilayers and obtained similar results. A mixture of the lipids (DMPC: NBD PC = 100: 1 w/w) was dissolved in chloroform. The fluorescently labeled lipid molecules were added to observe the spread SPBs by fluorescence microscopy. To obtain lipid thin films without the solvent, the lipid solution was dried in vacuum. A small amount of the dried lipid was deposited on a substrate surface as a lipid source, and a buffer solution (150 mM KCl, 1.0 mM CaCl2, 10 mM HEPES/NaOH, pH 7.4) was added. All reagents except lipids were purchased from KANTO CHEMICAL CO., INC. and used without further purification. To facilitate bilayer formation, temperature of the buffer solution should be kept above the phase transition temperature during the incubation. Since the phase transition temperature of the DMPC is 23 °C, the buffer solution was kept at 50 °C. The SPBs were incubated at 1 or 24 h. The 24 h incubation is enough long to generate a density gradient of the lipid molecules in a selfspreading SPB. The formed lipid bilayers were observed by an atomic force microscopy (AFM) (SII E-sweep) and a fluorescence microscopy (OLYMPUS BX51) in the buffer solution at room temperature. We used the cyclic contact mode and the contact mode in AFM imaging of the SPB and the sapphire surfaces, respectively. In AFM imaging of soft matters, the height measurement should be carefully done because the apparent height often depends on the loading force during the imaging. In the present measurement, the loading force was carefully chosen to measure the accurate height, especially at the edges of the region with a low-lipid density. In the cyclic-contact mode, the amplitude decay of the oscillating AFM tip is kept constant during obtaining the morphology. If the amplitude decay is too large, the SPBs are deformed by the pressure from the tip. We employed the minimum amplitude decay value within keeping attachment of the tip with the SPB surface. The height values of the SPBs in this paper were averaged data obtained from at least five edges of the SPB. Moreover, because the sapphire surfaces are atomically flat, the height of the SPBs can be measured without any influence from the substrate roughness. Therefore, the height data are believed to be reliable.
Results Step Control of the Sapphire Surfaces. Roughness of the random-stepped surfaces was about 0.13 nm in the root-meansquare value observed in AFM. Terrace regions of the single-, multi-, and cross-stepped surfaces were atomically flat. The step edges on the single- and multistepped surfaces were straight and parallel to each other toward the [1-100] direction. Figure 2a shows a typical AFM image of the cross-stepped sapphire surface. Hundred-nanometer-scaled terraces were grown on the areas surrounded by the bunched and the crossing steps. A single step height was 0.2 nm, whereas a height of the step bunch on the multi- and cross-stepped surfaces was about 1 nm, which corresponds to 5 atomic steps. The terraces of the cross-stepped surfaces consist of two domains with different hydrophilicity: the ellipsoidal areas at the center of the terraces exhibit lower hydrophilicity (domain A) than the other areas (domain B), as illustrated in Figure 2b. The random-, single-, and multi-stepped surfaces exhibit almost the same hydrophilicity as the domain B of the cross-stepped surface. Details of the formation process of the cross-stepped surfaces will be reported in separate papers. Langmuir 2010, 26(12), 9607–9611
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Figure 2. (a) AFM topography of the cross-stepped sapphire surface and its cross-sectional line profile. (b) Illustration of the domain structures featured by their hydrophilicity on the crossstepped sapphire surface. The main step bunches are directed toward [1-100], and small step bunches cross the terraces. On this surface, hydrophilicity of the ellipsoidal domains is smaller than the other areas.
Figure 3. Fluorescence microscopy image of a self-spreading SPB on the random-stepped surface after 1 h incubation. On the isotropic surfaces, the SPB exhibits a dendritic pattern that selfspreads from the initial lipid source, as observed in this image.
Behavior of Lipid Molecules on the Isotropic Surfaces. SPBs self-spread on the random-stepped surfaces and formed a dendritic pattern expanded from the initial source of the lipid source after 1 h incubation, as shown in Figure 3. In the fluorescence microscopy observation, contrasts of the fluorescence intensity depend on the layer number of SPBs.26 Under the present experimental conditions, single-bilayer membranes mainly formed and multibilayers also formed. The SPB height after 1 h incubation measured by AFM was about 4.9 nm. After 24 h incubation, a density gradient of lipid molecules appeared in the self-spreading SPBs on the random-stepped sapphire surfaces. Figure 4 shows an AFM image of the SPB after 24 h incubation in the region of a low density of lipid molecules. The height of the SPBs after 1 h incubation was about 5 nm, which is almost same as the ideal height, whereas that after 24 h incubation was about 2.8 nm. Moreover, voids were randomly generated in the SPB after 24 h incubation, as shown in Figure 4. These results indicate that the long incubation time for self-spreading induces a density gradient of the lipid molecules. On the low density regions, the formed SPBs were inclined from the vertical direction and their height was observed to be lower than the ideal one. Behavior of Lipid Molecules on the Anisotropic Surfaces. SPBs were formed by 1 h incubation on the single-, multi- and cross-stepped sapphire surfaces. On the stepped surfaces, the selfspreading direction of the SPBs was affected by the atomic steps, as shown in Figure 5. The SPBs self-spread along the step edges even when they were one-atomic height steps, as shown in Figure 5a. Though the height of the one-atomic step is much smaller than that of the SPB, the self-spreading direction is affected by the steps. The effect of the atomic steps on the growth Langmuir 2010, 26(12), 9607–9611
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Figure 4. AFM image of a self-spreading SPB on the randomstepped surface after 24 h incubation. Because the density of lipid molecules is low, the height of the SPBs is small compared with that in a high-lipid-density area.
direction is much larger in the case of a single-bilayer than that in a multibilayer. On the cross-stepped surface, the growth direction of the self-spreading SPBs was also affected by the steps. After 24 h incubation, SPBs that self-spread along the step edges were observed on the multistepped surfaces, as shown in Figure 6. The SPB height in Figure 6 was about 3.2 nm. A density gradient appeared in the same manner as that on the randomstepped surfaces for 24 h incubation. On the region of a low density of lipid molecules, the effect of scanning of the AFM tip was large and it was observed that the formed SPBs were dragged by the tip during the scanning, as shown in Figure 6. Even when the density of the lipid molecules was low, the planar SPBs selfspread along the step edges. Specific patterns of the SPBs, on the other hand, appeared on the cross-stepped surfaces, as shown in AFM images in Figure 7. The SPBs self-spread from the initial source, and the density of lipid molecules decreased as the distance from the initial spot is larger, as shown in Figure 7a-c. The density of the lipid molecules is significantly related to the stability of the bilayer structure in the SPBs. Therefore, the influence of the surface atomic structures upon the SPB morphology depends on the lipid densities. On the region of a high density of lipid molecules, a uniform SPB formed and atomic steps were observed as undulation of the SPB surface, as shown in Figure 7a. The SPB height measured by AFM was about 4.8 nm. Few defects were generated by the atomic steps on this region. On the region of an intermediate density of lipid molecules, distinctive patterns appeared in a manner of selfassembly, as shown in Figure 7b. The SPB islands preferentially formed on the terraces. Their height was about 1.8 nm. When the density of lipid molecules further decreased, voids formed around the center of the terraces, which correspond to the domain A, as shown in Figure 7c taken near the front of the self-spreading SPB. The SPB height in this region was about 1.7 nm.
Discussion Spontaneous self-spreading of SPBs occurs owing to a gain of the interaction energy at the water-solid interface and the hydrophobic interaction energy for formation of the bilayer structure of a lipid membrane. The former interaction energy is expressed as the summation of van der Waals energy, the electrostatic energy in the electric double layer, and the hydration energy. On the sapphire surfaces, the interaction energy is higher than the silicon dioxide one.20 A contribution of van der Waals interactions is the largest for the high gain of the interaction energy because the relative dielectric constant of sapphire, which is one of the elements of the Hamaker constant, is much larger than that of the silicon dioxide. Effects of the large van der Waals interaction are related to the small distance between a SPB and a DOI: 10.1021/la100179q
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Figure 5. Fluorescent microscopy images of self-spreading SPBs after 1 h incubation (a) on the single-stepped surface and (b) on the multistepped surface. (c) Illustration of the SPBs on the stepped surface. On the anisotropic surfaces, the self-spreading direction is affected by the surface structures. Dot lines indicate direction of the atomic steps. The SPBs spread along the steps, even when the step is one-atomic-layer height.
Figure 6. AFM image of self-spreading SPBs in a region of a low lipid density on the multistepped surface after 24 h incubation. On the region of low lipid density, the SPBs self-spread along the steps. According to scanning direction of the AFM tip, the formed SPBs were dragged by the tip during the scanning though the loading force of the AFM tip was carefully adjusted for suitable value.
sapphire surface and the sharp potential gradient near the potential minimum. Because of the short distance, the SPBs are more affected by the structure and chemistry on the sapphire surfaces than the silicon dioxide. On the sapphire surfaces, selfspreading phenomena of the SPBs are promoted by the large gain of the interaction energy at the SPB-solid interface. The SPBs, therefore, can self-spread in a wide area by a long incubation. The velocity of the self-spreading decreases with increase in the spreading time.22 In this case, a large density gradient of lipid molecules is induced. When the density becomes low, the SPB structure changes from the close-packed configuration to a loose one. Therefore, the hydrophobic interaction to retain the bilayer structures becomes weak. As a result, the SPBs cannot keep the upstanding structure, resulting in deformation to the inclined structure. When a silicon surface with line and space patterns fabricated by a dry-etching technique was used as a substrate, the selfspreading direction of SPBs was not affected by the patterns,24,25 though the vertical height of these patterns was 100 nm. The selfspreading direction on sapphire surfaces, on the other hand, was regulated by the atomic steps, though the height of a step bunch is about 1 nm, which is much smaller than the SPB height. These results can be explained by the curvature of the patterns. The curvature of a dry-etched pattern edge is much larger than the size of the lipid molecules, whereas the steps on the singlecrystalline sapphire surface are atomically sharp. This is the reason for the directional control of self-spreading of the SPBs on sapphire surfaces. When lipid molecules diffuse up or down the atomic steps, the height of the hydrophobic chains in the individual lipid molecules are misaligned. In this case, the hydrophobic chains cannot fully interact with the adjacent molecules. As a result, the SPBs self-spread on the same terraces, as 9610 DOI: 10.1021/la100179q
illustrated in Figure 5c. In the early stage of the self-spreading, a SPB swiftly spreads and forms circular or dendritic patterns, which do not depend on the atomic steps, because of a small frictional stress from the substrate surface and an entropy effect induced by the mass of the initial lipid source. In this stage, the kinetic effect is dominant. As the self-spreading of SPBs proceeds, the direction becomes aligned along the steps. Because the velocity of the spreading becomes slow, a thermodynamic effect induced by the hydrophobic interaction between the adjacent lipid molecules becomes dominant. The self-spreading of SPBs whose direction is aligned along the steps was observed also around the growth front or in the case where the initial amount of lipid molecules was small. This indicates that the atomic steps work as continuous barriers against the self-spreading even when the hydrophobic interaction is weakened owing to a lower density of lipid molecules. On the cross-stepped surfaces, specific patterns of the SPBs that depend on the distance from the initial lipid source appeared. When the density of lipid molecules is high, they are strongly bound to each other by the attractive interaction caused by the hydrophobic segment of the lipid molecule. Therefore, the planar bilayer structure can be stably retained also on the stepped surfaces in the region near the initial lipid source, as shown in Figure 7a, though the SPB is rippled by the steps. As a decrease in the density of lipid molecules, the attractive interaction between the individual lipid molecules becomes weak, and the morphology of the SPB is distorted owing to the stress applied from the sharp edge of the atomic steps. To reduce the deformation energy of this system, trenches are introduced in the SPBs. Since the trenches are preferentially generated around the atomic steps, the SPB domains divided by the steps form inside the flat terraces, as shown in Figure 7b. Two domains with different hydrophilicity coexist on the terraces of the cross-stepped sapphire surfaces. Though both domains are hydrophilic in a general sense, the ellipsoidal domains inside the terraces (domain A) are relatively hydrophobic in comparison with the other areas (domain B). Because the SPBs are more stable on the hydrophilic domain than the hydrophobic one, the SPB domains are preferentially assembled on the domain B when the density of lipid molecules becomes further low, as shown in Figure 7c. The SPBs self-spread with an energy gain from the interaction between the adjacent lipid molecules and that between the SPB and the substrate surface. It continues even when the SPBs can not retain the feature of a vertically standing bilayer. When a high density of voids is introduced for lack of lipid molecules, the spreading SPBs become discontinuous. However, in the present experimental results, the SPBs continue to self-spread in the region of a low molecular density on the sapphire surfaces. This result indicates that the energy gain from the interaction at the lipid-solid interface is dominant for the self-spreading, and that from the lateral interaction between the lipid molecules is subsidiary though it is Langmuir 2010, 26(12), 9607–9611
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Figure 7. AFM images of the self-spreading SPBs on the cross-stepped surface where the densities of lipid molecules are (a) relatively high, (b) intermediate, and (c) low. The SPB pattern depends on the density of lipid molecules. In region a, uniform SPBs formed though a few defects were generated by the steps. In region b, SPB islands preferentially formed on the terraces surrounded by the crossing steps. In region c, voids formed inside the SPB islands around the center of the flat terraces.
indispensable for maintaining the bilayer structure. By the strong interaction between the lipid molecules and the sapphire surface, the self-spreading of the SPBs is enhanced and distinctive SPB patterns are obtained. Finally, we discuss the advantage of the present technique to pattern the SPBs compare with the conventional technique. As shown in ref 15 and many papers, hydrophobic patterns on the hydrophilic surfaces formed by the lithographic technique are generally used as barriers to separate the lipid bilayers. In our paper, self-organized hydrophilic patters work as a barrier for the self-spreading. The big advantage in this technique is that the barrier can be made of the same material as the substrate. The atomic steps as barriers on the sapphire surfaces are very stable. Additionally, the edges of the barrier are atomically well-defined unlike the hydrophobic metals and resist patterns. As described in ref 24, when a silicon dioxide surface with line and space patterns fabricated by lithographic technique was used as a substrate, the self-spreading direction of SPBs was not affected by the patterns. Even when using a dry-etching technique, the curvature of the pattern edge is much larger than the lipid molecule size. On the other hand, the steps on the single-crystalline sapphire surface are atomically abrupt. Such atomically abrupt barriers can not be fabricated by the lithographic techniques. In the case of ref 16, the lipid bilayer patterns are formed using photolithographic polymerization of diacetylene-containing phospholipids. In this technique, however, species of the lipid molecules are limited. Therefore, the use of the atomic structure on the substrate surface is a unique technique to control the SPBs.
the abrupt feature of the atomic steps and the large energy gain from the interaction between the lipid molecules and the substrate surface, the spreading direction of SPBs can be controlled by the atomic step arrangement on the sapphire surfaces. On the cross-stepped surfaces, two domains with different hydrophilicity are self-organized, and specific SPB patterns that depend on the density of the lipid molecule appear. The present process is simple but applicable to fabrication of fine patterns. Since self-assembled SPB arrays can be applied to nanofluidic biochips that transport various materials with affinity to the SPBs, the stepped surfaces are applicable to fabrication of such devices by using directional spreading of the SPBs. Density control of SPBs can be used to separate and/or trap the specific materials that are carried through the SPBs. In face, we have succeeded to select or separate SPB species from a mixture on phase-separated sapphire surfaces, as will appear in a separate paper. Since the step arrangement can be artificially designed,36 the step-controlled sapphire surfaces are useful for the substrates of nanofluidic devices. Note Added after ASAP Publication. This article was published ASAP on March 26, 2010. Figure 2a has been modified. The correct version was published on April 28, 2010.
Conclusion
Acknowledgment. This work was partly supported by CREST/JST ant Grant-in-Aid for Scientific Research from the Ministry of Education, Culture, Sports, Science and Technology. The authors would like to thank Dr. Oya in Yokohama National University and Prof. Urisu and Dr. Tero in Institute for Molecular Science. The sapphire wafers were provided from Namiki Precision Jewel Co., Ltd.
We have observed behaviors of the SPBs that spontaneously self-spread on the step-controlled sapphire surfaces. Because of
(36) Ogino, T.; Hibino, H.; Homma, Y.; Kobayashi, Y.; Prabhakaran, K.; Sumitomo, K.; Omi, H. Acc. Chem. Res. 1999, 32, 447–454.
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