Lipid Bilayer Membrane with Atomic Step Structure: Supported Bilayer

Sep 12, 2008 - Fluorescence images of adsorbed vesicles and planar bilayer membranes were obtained using an epifluorescence microscope (Olympus) equip...
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Langmuir 2008, 24, 11567-11576

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Lipid Bilayer Membrane with Atomic Step Structure: Supported Bilayer on a Step-and-Terrace TiO2(100) Surface Ryugo Tero,*,† Toru Ujihara,‡ and Tsuneo Urisu† DiVision of Biomolecular Sensing, Institute for Molecular Science, Myodaiji, Okazaki, 444-8585, Japan, and Department of Crystalline Materials Science, Graduate School of Engineering, Nagoya UniVersity, Furo-cho, Chikusa-ku, Nagoya 464-8603, Japan ReceiVed April 7, 2008. ReVised Manuscript ReceiVed August 5, 2008 The formation of a supported planar lipid bilayer (SPLB) and its morphology on step-and-terrace rutile TiO2(100) surfaces were investigated by fluorescence microscopy and atomic force microscopy. The TiO2(100) surfaces consisting of atomic steps and flat terraces were formed on a rutile TiO2 single-crystal wafer by a wet treatment and annealing under a flow of oxygen. An intact vesicular layer formed on the TiO2(100) surface when the surface was incubated in a sonicated vesicle suspension under the condition that a full-coverage SPLB forms on SiO2, as reported in previous studies. However, a full-coverage, continuous, fluid SPLB was obtained on the step-and-terrace TiO2(100) depending on the lipid concentration, incubation time, and vesicle size. The SPLB on the TiO2(100) also has step-and-terrace morphology following the substrate structure precisely even though the SPLB is in the fluid phase and an ∼1-nm-thick water layer exists between the SPLB and the substrate. This membrane distortion on the atomic scale affects the phase-separation structure of a binary bilayer of micrometer order. The interaction energy calculated including DLVO and non-DLVO factors shows that a lipid membrane on the TiO2(100) gains 20 times more energy than on SiO2. This specifically strong attraction on TiO2 makes the fluid SPLB precisely follow the substrate structure of angstrom order.

Introduction Lipid bilayer membranes and membrane proteins play key roles in transferring materials, information, and energy into and out of cells. Many biological reactions are triggered by lateral and/or vertical structures and dynamics in the lipid bilayer, such as 2D domain formation,1 hydrophobic matching, and curvature stress.2 Lipid bilayer membranes deposited at the solid-water interface are called supported planar lipid bilayers (SPLBs).3-5 The SPLBs are used to investigate the basic material properties of lipid bilayers6-8 and are also used as a cell membrane model system in vitro.9-13 Lipid molecules freely diffuse in the SPLB in the liquid crystal (LR) or liquid ordered phase, and the SPLB retains its fluidity14-17 because lipid molecules do not directly adsorb on the substrate surface but are separated from it by a 1to 2-nm-thick water layer between the SPLB and the solid substrate.18-22 * To whom correspondence should be addressed. E-mail: [email protected]. † Institute for Molecular Science. ‡ Nagoya University.

(1) Anderson, R. G. W.; Jacobson, K. Science 2002, 296, 1821–1825. (2) Jensen, M. Ø.; Mouritsen, O. G. Biochim. Biophys. Acta 2004, 1666, 205– 226. (3) Tamm, L. K.; McConnell, H. M. Biophys. J. 1985, 47, 105–113. (4) Richter, R. P.; Berat, R.; Brisson, A. R. Langmuir 2006, 22, 3497–3505. (5) Castellana, E. T.; Cremer, P. S. Surf. Sci. Rep. 2006, 61, 429–444. (6) Dufreˆne, Y. F.; Lee, G. U. Biochim. Biophys. Acta 2000, 1509, 14–41. (7) Ratto, T. V.; Longo, M. L. Biophys. J. 2002, 83, 3380–3392. (8) Loi, S.; Sun, G.; Franz, V.; Butt, H. J. Phys. ReV. E 2002, 66, 031602. (9) Knoll, W.; Yu, F.; Neumann, T.; Schiller, S.; Naumann, R. Phys. Chem. Chem. Phys. 2003, 5, 5169–5175. (10) Dewa, T.; Sugiura, R.; Suemori, Y.; Sugimoto, M.; Takeuchi, T.; Hiro, A.; Iida, K.; Gardiner, A. T.; Cogdell, R. J.; Nango, M. Langmuir 2006, 22, 5412–5418. (11) Ataka, K.; Richter, B.; Heberle, J. J. Phys. Chem. B 2006, 110, 9339– 9347. (12) Mitomo, H.; Shigematsu, H.; Kobatake, E.; Furusawa, H.; Okahata, Y. J. Mol. Recognit. 2007, 20, 83–89. (13) Furuike, S.; Hirokawa, J.; Yamada, S.; Yamazaki, M. Biochim. Biophys. Acta 2003, 1615, 1–6. (14) Nollert, P.; Kiefer, H.; Ja¨hnig, F. Biophys. J. 1995, 69, 1447–1455. (15) Cha, T.; Guo, A.; Zhu, X.-Y. Biophys. J. 2006, 90, 1270–1274. (16) Morigaki, K.; Kiyosue, K.; Taguchi, T. Langmuir 2004, 20, 7729–7735.

It has been reported that, even through the existence of the water layer, the SPLB properties and structures are affected by the physical and chemical properties of solid substrates such as surface charges,15,23,24 chemical termination,25-28 material,29-36 and roughness.37 Recent studies showed unique substrate-induced phenomena of SPLBs, which do not occur in the free-standing membranes (e.g., decoupled phase transition38,39 and asymmetric molecular distribution4,29 between the upper and lower leaflets of an SPLB). Physical and chemical properties of the substrate (17) Tero, R.; Watanabe, H.; Urisu, T. Phys. Chem. Chem. Phys. 2006, 8, 3885–3894. (18) Kim, J.; Kim, G.; Cremer, P. S. Langmuir 2001, 17, 7255–7260. (19) Crane, J. M.; Kiessling, V.; Tamm, L. K. Langmuir 2005, 21, 1377–1388. (20) Ajo-Franklin, C. M.; Yoshina-Ishii, C.; Boxer, S. G. Langmuir 2005, 21, 4976–4983. (21) Kiessling, V.; Tamm, L. K. Biophys. J. 2003, 84, 408–418. (22) Johnson, S. J.; Bayerl, T. M.; McDermott, D. C.; Adam, G. W.; Rennie, A. R.; Thomas, R. K.; Sackmann, E. Biophys. J. 1991, 59, 289–294. (23) Cassier, T.; Sinner, A.; Offenha¨usser, A.; Mo¨hwald, H. Colloids Surf., B 1999, 15, 215–225. (24) Kim, Y.-H.; Rahman, M. M.; Zhang, Z.-L.; Misawa, N.; Tero, R.; Urisu, T. Chem. Phys. Lett. 2006, 420, 569–573. (25) Jenkins, A. T. A.; Bushby, R. J.; Evans, S. D.; Knoll, W.; Offenha¨usser, A.; Ogier, S. D. Langmuir 2002, 18, 3176–3180. (26) Tero, R.; Takizawa, M.; Li, Y. J.; Yamazaki, M.; Urisu, T. Langmuir 2004, 20, 7526–7531. (27) Tero, R.; Misawa, N.; Watanabe, H.; Yamamura, S.; Nambu, S.; Nonogaki, Y.; Urisu, T. e-J. Surf. Sci. Nanotechnol. 2005, 3, 237–243. (28) Isono, T.; Tanaka, H.; Ogino, T. e-J. Surf. Sci. Nanotechnol. 2007, 5, 99–102. (29) Rossetti, F. F.; Textor, M.; Reviakine, I. Langmuir 2006, 22, 3467–3473. (30) Rossetti, F. F.; Bally, M.; Michel, R.; Textor, M.; Reviakine, I. Langmuir 2005, 21, 6443–6450. (31) Reviakine, I.; Rossetti, F. F.; Morozov, A. N.; Textor, M. J. Chem. Phys. 2005, 122, 204711. (32) Reimhult, E.; Ho¨o¨k, F.; Kasemo, B. J. Chem. Phys. 2002, 117, 7401– 7404. (33) Suwalsky, M.; Schneider, C.; Mansilla, H. D.; Kiwi, J. J. Photochem. Photobiol. B 2005, 78, 253–258. (34) Reimhult, E.; Za¨ch, M.; Ho¨o¨k, F.; Kasemo, B. Langmuir 2006, 22, 3313– 3319. (35) Lei, S. B.; Tero, R.; Misawa, N.; Yamamura, S.; Wan, L. J.; Urisu, T. Chem. Phys. Lett. 2006, 429, 244–249. (36) Oleson, T. A.; Sahai, N. Langmuir 2008, 24, 4865–4873. (37) Seu, K. J.; Pandey, A. P.; Haque, F.; Proctor, E. A.; Ribbe, A. E.; Hovis, J. S Biophys. J. 2007, 92, 2445–2450.

10.1021/la801080f CCC: $40.75  2008 American Chemical Society Published on Web 09/12/2008

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surface also affect membrane fluidity,37 phase separation structures,35 the stability of the adsorbed vesicles,15,30,40 and the process and rate of the shape transformation from vesicles to planar bilayers.17,23-26,40 Our goal is to understand the interaction between SPLBs and solid surfaces on the atomic and molecular scales and to utilize the substrate surface functions to control the SPLB structures, properties, and molecular transportation. A correct understanding of the substrate effect will also be useful for the combination of SPLBs and surface micropatterning techniques16,30,41-47 and for avoiding the denaturing of bilayer membranes and membrane proteins when SPLBs are used as a cell membrane model system. The concept of a well-defined surface in the surface science field48,49 will be important in clarifying the substrate effect on the SPLB precisely. As a first step, we investigated the formation and structure of SPLBs on single-crystal TiO2(100) surfaces in this study. We prepared TiO2(100) surfaces with atomic steps and flat terraces by a wet treatment and annealing by a method taken from refs 50 and 51. Electron diffraction and noncontact atomic force microscopy studies showed that the wet-treated step-and-terrace TiO2 surfaces have (1 × 1) periodicity.51,52 We assume that the rutile TiO2 surface is appropriate for the investigation of the surface effect because it is the most deeply studied surface among metal oxide surfaces and there is an abundance of knowledge about the low-index surfaces of rutile TiO2.53,54 Additionally, TiO2 is a biocompatible material and is widely used at the interfaces where an inorganic material and the human body are in direct contact (e.g., bone implantation, sunburn protection, and cosmetics), thus the TiO2-bilayer interaction itself is an important research target.29-33,36,55-57 Interesting phenomena of the SPLBs on sputter-deposited TiO2 surfaces are reported by Reviakine and co-workers.29-31 Vesicles of a neutral lipid (phosphatidylcholine (PC)) remain intact on the TiO2 surface, and the transformation from vesicles to a planar membrane proceeds only when the vesicles contain more than 20% negatively charged lipids (phosphatidylserine (PS)) and Ca2+ ions exist in the buffer solution. After the planar membrane forms the PS, molecules selectively exist at the lower leaflet of the SPLB. In the present study, we found that the PC-SPLB forms on the step-and-terrace TiO2(100) from adsorbed vesicles depending on the lipid concentration and incubation time. The SPLB on the TiO2(100) has a step-and-terrace structure following (38) Keller, D.; Larsen, N. B.; Møller, I. M.; Mouritsen, O. G. Phys. ReV. Lett. 2005, 94, 025701. (39) Blanchette, C. D.; Orme, C. A.; Ratto, T. V.; Longo, M. L. Langmuir 2008, 24, 1219–1224. (40) Keller, C. A.; Kasemo, B. Biophys. J. 1998, 75, 1397–1402. (41) Groves, J. T.; Boxer, S. G. Acc. Chem. Res. 2002, 35, 149–157. (42) Jackson, B. L.; Groves, J. T. Langmuir 2007, 23, 2052–2057. (43) Parthasarathy, R.; Yu, C.-h.; Groves, J. T. Langmuir 2006, 22, 5095– 5099. (44) Nabika, H.; Sasaki, A.; Takimoto, B.; Sawai, Y.; He, S.; Murakoshi, K. J. Am. Chem. Soc. 2005, 127, 16786–16787. (45) Furukawa, K.; Sumitomo, K.; Nakashima, H.; Kashimura, Y.; Torimitsu, K. Langmuir 2007, 23, 367–371. (46) Han, X.; Critchley, K.; Zhang, L.; Pradeep, S. N. D.; Bushby, R. J.; Evans, S. D. Langmuir 2007, 23, 1354–1358. (47) Wang, Z.; Wilkop, T.; Cheng, Q. Langmuir 2005, 21, 10292–10296. (48) Vickerman, J. C. Surface Analysis: The Principal Techniques; John Wiley & Sons Ltd.: Chichester, U.K., 1997. (49) Henrich, V. E.; Cox, P. A. The Surface Science of Metal Oxides; Cambridge University Press: Cambridge, U.K., 1996. (50) Nakamura, R.; Ohashi, N.; Imanishi, A.; Osawa, T.; Matsumoto, Y.; Koinuma, H.; Nakato, Y. J. Phys. Chem. B 2005, 109, 1648–1651. (51) Yamamoto, Y.; Nakajima, K.; Ohsawa, T.; Matsumoto, Y.; Koinuma, H Jpn. J. Appl. Phys 2005, 44, L511–L514. (52) Namai, Y.; Matsuoka, O. J. Phys. Chem. B 2006, 110, 6451–6453. (53) Diebold, U. Surf. Sci. Rep. 2003, 48, 53–229. (54) Iwasawa, Y Surf. Sci. 1998, 404-404, 8–19. (55) Starr, T. E.; Thompson, N. L. Langmuir 2000, 16, 10301–10308. (56) Sinner, A.; Offenha¨usser, A. Thin Solid Films 1998, 327-329, 758–761. (57) Csu´cs, G.; Ramsden, J. J. Biochim. Biophys. Acta 1998, 1369, 61–70.

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the substrate morphology because of the strong van der Waals attraction of TiO2. This atomic-scale distortion affects the horizontal gel-LR phase separation structure on the micrometer scale.

Materials and Methods Substrate Cleaning and Preparation. Single-crystal wafers of rutile TiO2(100) (∼5 × 5 mm2, purchased from Furuuchi Chemical Corp., Japan) were cleaned before each lipid bilayer deposition by a wet process that was partially modified from refs 50 and 51, sonicated in acetone, methanol, and pure water (>18 MΩ cm-1, Millipore), boiled in the piranha solution (3:1 v/v concentrated H2SO4 and 30% H2O2;) for 5 s, immersed in 10% HF(aq) for 10 min, and annealed at 700-800 °C in a 1.0 L min-1 O2 flow for 1 h. Caution! Piranha solution Violently reacts with organic materials, and extreme care must be taken at all times during handling. The TiO2(100) wafers were cooled to room temperature and again sonicated in acetone, methanol, and water for 5 min each just before lipid bilayer deposition. The HF treatment and annealing almost completely eliminate contaminations such as Zn, Fe, Sn, Ca, and Si, which are not removed by organic solvents alone.51 After the lipid bilayer formation experiments, the TiO2(100) wafer was rinsed in pure water and ethanol and sonicated in a chloroform/ethanol mixture (3:1) to remove lipids from the surface. As-delivered Si(100) wafers were sonicated in acetone, methanol, and pure water for 5 min each before thermal oxidation. A thermally oxidized SiO2 layer with ∼100 nm thickness was obtained by annealing the SiO2(100) wafer at 900 °C in a 1.0 L min-1 O2 flow for 3 h. The thickness of the oxide layer was measured by an ellipsometer. The thermally oxidized SiO2 was boiled in piranha solution for 10 min and sonicated in 0.02 M KOH(aq) for 10 min just before lipid bilayer deposition. Preparation of Vesicle Suspension. Dipalmitoyl()dihexadecanoic, 16:0)-phosphatidylcholine (DPPC, gel-LR transition temperature (Tc) ) 41 °C), dipalmitoleoyl() di-9-cis-hexadecenoic, 16:1)-phosphatidylcholine (DPoPC, Tc ) -36 °C), and lissamine rhodamine B-labeled dioleoylphosphatidylethanolamine (Rb-DOPE, ex 557 nm/em 571 nm) were purchased from Avanti Polar Lipids Inc. (Alabaster, AL) and used without further purification. We chose two lipids with the same carbon number without (DPPC) and with (DPoPC) a double bond to distinguish gel-LR phase separation by the membrane thickness in AFM topographs. Chloroform solutions of these lipids were mixed in a glass vial in the required amounts and ratios, and the solvent was evaporated in an N2 flow followed by evacuation in a vacuum desiccator for more than 6 h. A multilamellar vesicle suspension of the phospholipids (0.1 mg mL-1) was prepared by agitating the vacuum-dried lipid films in a buffer solution (150 mM KCl, 10 mM HEPES/NaOH (pH 7.4), chemicals were purchased from Wako Pure Chemical Industries, Ltd., Japan). The suspension was frozen and thawed five times in liquid nitrogen and a 45 °C water bath and was extruded through a 100 nm polycarbonate filter (Liposo-Fast, Avestin, Inc.) to obtain unilamellar vesicles.58 The extruded vesicle suspension was further sonicated in a water bath ultrasonic cleaner to produce smaller vesicles. The extruded vesicles have diameters that are similar to the filter mesh size.58,59 Sonication using a general bath sonicator reduces the vesicle size to ∼30 nm,59 whereas much smaller vesicles can be made by a high-powered tip-type sonicator.60 We diluted the vesicle suspension to the required lipid concentration using the buffer solution before deposition onto the substrates. For the supported planar bilayer formation on the TiO2(100) surface, the TiO2(100) wafer was immersed in the vesicle suspension and incubated at 45 °C. The excess vesicles in the liquid phase were washed out by exchanging the suspension by the fresh buffer solution before the AFM and fluorescence microscopy observations. The incubation, washing, and (58) MacDonald, R. C.; MacDonald, R. I.; Menco, B. P. M.; Takeshita, K.; Subbarao, N. K.; Hu, L. Biochim. Biophys. Acta 1991, 1061, 297–303. (59) Lapinski, M. M.; Castro-Forero, A.; Greiner, A. J.; Ofoli, R. Y.; Blanchard, G. J. Langmuir 2007, 23, 11677–11683. (60) Reviakine, I.; Brisson, A. Langmuir 2000, 16, 1806–1815.

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Figure 1. (a, b) AFM topographies (2.0 × 2.0 µm2) and line profiles of the rutile TiO2(100) surface after wet treatment and oxygen annealing at (a) 700 °C and (b) 800 °C. (c) Sectional view of the bulk-terminated TiO2(100) structural model with a monolayer step (0.23 nm) and a bilayer step (0.46 nm) projected from the 001 direction. The smaller red spheres and larger blue spheres represent titanium and oxygen atoms, respectively. The plane directions of the two equivalent (110) and (11j0) microfacets are indicated by orange arrows and a green arrow, respectively.

AFM and fluorescence microscopy observations were performed in the same liquid cell without exposing the sample surface to air. Apparatuses. AFM observations were performed using PicoScan2500 (Agilent Technologies) in conventional tapping mode in air and a magnetic-ac mode61 in the buffer solution. The topography of the bare TiO2(100) surface was observed in air at room temperature. TiO2(100) after incubation in the vesicle suspension was observed in the buffer solution. Fluorescence images of adsorbed vesicles and planar bilayer membranes were obtained using an epifluorescence microscope (Olympus) equipped with a 40× water-immersion objective lens and a digital CCD camera (Nikon DS-2MBW). The sample was irradiated by a high-pressure mercury lamp through a 530-550 band-pass filter. The intensity of the excitation light from the mercury lamp was attenuated to 5.7 mW mm-2 for the observation through neutral density (ND) filters. The sample was irradiated by a brighter excitation light by removing the ND filters when photobleaching was performed.

Results and Discussion Surface Structure of Step-and-Terrace TiO2(100) Surfaces. Figure 1a shows the AFM topograph of the rutile TiO2(100) surface after the wet treatment and annealing under the oxygen flow at 700 °C. The surface consists of linear steps at even intervals and flat terraces. The height of the step is 0.226 nm (average of 20 steps), which corresponds to half the height of the TiO2(100) unit cell, 0.23 nm. This is the minimum step unit of TiO2(100) and thus the monolayer step of TiO2(100). The average width of the terraces is 136 nm (calculated from 73 terrace widths). (61) Raab, A.; Han, W. H.; Badt, D.; Smith-Gill, S. J.; Lindsay, S. M.; Schindler, H.; Hinterdorfer, P. Nat. Biotechnol. 1999, 17, 902–905.

TiO2(100) annealed at 800 °C has similar surface morphology with linear steps and flat terraces (Figure 1b), but the step height and terrace width are approximately double those in Figure 1a. The height of the step is 0.461 nm (average of 21 steps), which corresponds to a bilayer step, which means one unit cell height of the TiO2(100) (0.46 nm). The average width of the terrace is 235 nm. We call these TiO2(100) surfaces in Figure 1a,b monostep TiO2(100) and bistep TiO2(100), respectively, hereafter. It is reasonable from the viewpoint of the crystallographic structure that the monosteps appear on the TiO2(100) (Figure 1a). The bulk-terminated TiO2(100) surface consists of (110) microfacets as shown in Figure 1c.62 Cleaving between adjacent charge-neutral planes parallel to the (100) plane produces two equivalent surface structures: (110) and (11j0) microfacets. Their plane directions are indicated in Figure 1c by the orange arrows (110) and the green arrow (11j0). Only one of the (110) and (11j0) microfacet terraces stably exist on the monostep TiO2(100) because two terraces are energetically identical. No difference was observed between two neighboring terraces in AFM phaseshift mode (data not shown) or in the topograph, and two microfacet terraces cannot be recognized from the AFM images. Only one of the (110) and (11j0) microfacets is favored at 800 °C, which remains on the bistep TiO2(100). This is because of the difference in the step structure. If we assume that the 010 direction goes upstairs as in Figure 1c, then the step structures of the (110) and (11j0) microfacets are no longer the same (S1 in Supporting (62) The microfacet structure shown in Figure 1c is the same as the structural model of the TiO2(100)-(1 × 1) surface observed in ultrahigh vacuum.53 All of the exposed Ti atoms are 5-fold coordinated. You can choose a charge-neutral plane leaving 4-fold-coordinated Ti atoms, but they are energetically less stable and less preferable than the 5-fold ones.

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Figure 2. (a) Fluorescence microscope image (176 × 132 µm2) of monostep TiO2(100) incubated in the 0.013 mg mL-1 DPoPC + RbDOPE vesicle suspension at 45 °C for 30 min. Time course after a part of the sample (a) is photobleached: (b) 0 and (c) 50 s after photobleaching. (d) Fluorescence microscope image (176 × 132 µm2) of the thermally oxidized SiO2/Si after incubation under the same conditions as for sample a. Time course after a part of the sample (d) is photobleached: (e) 0 and (f) 180 s after photobleaching.

Information). We cannot judge which selectively appears in Figure 1b from the AFM topograph, and to our knowledge, there has been no report about the difference in the stability between these two bisteps either experimentally or theoretically. The morphology of the step-and-terrace TiO2(100) surfaces is basically similar to that in ref 51, in which the TiO2(100) wafer is annealed in air. In our study, oxygen annealing resulted in better reproducibility in obtaining the two kinds of step-andterrace surfaces. This may be because the surface stoichiometry is retained by oxygen annealing.63 We have confirmed that the monostep and bistep TiO2(100) surfaces remain stable both in air and water at least for 1 week, thus we can use these atomicstep surfaces for the substrate of SPLBs. SPLB Formation on Monostep TiO2(100). SPLB formation on the TiO2(100) surface was performed by the vesicle fusion method, which is efficient and widely used in SPLB studies.3-5 When a substrate is incubated in a vesicle suspension, spherical vesicles spontaneously transform to a planar membrane through the steps of vesicle adsorption, intervesicle fusion, and/or vesicle rupturing and spreading. The full-coverage SPLB can be easily obtained on the surfaces of oxides and ceramics such as mica, glass, quartz, and oxide layers on silicon wafers. On TiO2 surfaces, however, previous reports showed that there is difficulty in SPLB formation by the vesicle fusion method. Lipid vesicles of PC remain intact and do not transform to a planar membrane on sputter-deposited TiO2 surfaces.30-32,34 Figure 2a shows the fluorescence microscope image of the monostep TiO2(100) surface incubated in the 0.013 mg mL-1 DPoPC + Rb-DOPE (100:1) sonicated vesicle suspension for 30 min. It is a sufficiently high lipid concentration and long incubation time to form a fullcoverage SPLB on thermally oxidized SiO2/Si(100) (Figure 2d). (63) Wang, Y.; Warschkow, O.; Marks, L. D. Surf. Sci. 2007, 601, 63–67.

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Figure 3. (a-d) Fluorescence microscope images (176 × 132 µm2) of the monostep TiO2(100) surface incubated in the 0.025 mg mL-1 DPoPC + Rb-DOPE vesicle suspension at 45 °C. The incubation times are (a) 35 and (b-d) 60 min. (a) Planar membrane patches coexist with adsorbed vesicles. (b-d) The surface is fully covered by a planar bilayer, and the temporal change in FRAP is followed: (b) 0, (c) 97, and (d) 317 s after photobleaching. Full recovery indicates the formation of a continuous, fluid SPLB. (e) AFM topograph (1.0 × 1.0 µm2) of the SPLB on the monostep TiO2(100), prepared by the same incubation conditions as used in b-d.

The visual impression of Figure 2a is grainy compared with that of the SPLB on SiO2. After a part of the sample in Figure 2a was photobleached, the fluorescence did not recover (Figure 2b,c), whereas fluorescence recovery after photobleaching (FRAP) proceeded on the SPLB on SiO2 (Figure 2e,f). This indicates that the monostep TiO2(100) surface is covered by intact vesicles rather than a planar bilayer because the adsorbed vesicles on solid supports are immobile.14,15 These results correspond to previous studies that show that an intact vesicular layer forms on sputter-deposited TiO2 surfaces whereas the adsorbed vesicles lightly transform to the SPLB on SiO2.29-32,34 We confirmed that only the adsorbed vesicles were observed on the monostep TiO2(100) at this lipid concentration even after incubation for 120 min. We found, however, that adsorbed vesicles on the monostep TiO2(100) transform to a planar membrane at higher lipid concentration. When the monostep TiO2(100) was incubated in the 0.025 mg mL-1 vesicle suspension for 35 min, the surface was still mainly covered by the adsorbed vesicles, but smaller, darker domains appeared all over the surface (Figure 3a). These domains seemed smooth and uniform in brightness and were reasonably assigned to planar bilayer patches. The TiO2(100) surface was completely covered by a planar membrane after incubation for 60 min (Figure 3b-d). The FRAP in Figure 3b-d shows that the SPLB was fluid and continuous. Complete fluorescence recovery indicates that the SPLB adsorbs on the substrate binding a water layer between them and there is almost no immobile fraction in the SPLB. Figure 3a indicates that, for SPLB formation on TiO2(100), there is a critical vesicle coverage for vesicle decomposition, followed by self-promoting planar membrane formation as described in ref 40. SPLB formation on

Lipid Bilayer Membrane with Atomic Step Structure

TiO2(100) does not proceed through the single vesicle rupturing process,60,64 during which SPLB disks appears before the substrate surface is covered by adsorbed vesicles. The bright spots in Figure 3b-d are probably the vesicles adsorbed on the SPLB, and their density is 0.08 vesicles µm-1. On the monostep TiO2(100) surface, we often found that the adsorbed vesicles and second bilayer stably existed on the first SPLB (S2-a in Supporting Information), whereas usually only the first layer stably exists on SiO2 or mica surfaces.10,13,17,24,26,35,40,60 These adsorbed vesicles and second bilayers can be removed by a gently blowing throuth a pipet under the buffer solution, but the blowing generate defects in the first bilayer before all of the adsorbed vesicles are removed (S2 in Supporting Information). The vesicle size in the suspension was another factor that affected SPLB formation, in addition to the lipid concentration and incubation time. The vesicle suspension used in Figures 2 and 3 was sonicated after extrusion through a 100 nm polycarbonate filter to reduce the vesicle size to ∼30 nm.59 When we used the 100-nm-filtered or 50-nm-filtered vesicles without further sonication, the probability of planar bilayer formation decreased significantly, and intact vesicular layers such as those in Figure 2a almost always appeared. The intact vesicular layer formed if the lipid concentration of the sonicated vesicle suspension was below 0.015 mg mL-1, which is not dependent on incubation time at least up to 120 min. A full-coverage SPLB was obtained with good reproducibility when the lipid concentration was 0.025-0.05 mg mL-1. Higher concentration, however, leads to more adsorbed vesicles and second bilayers such as the SPLBs shown in Figure S2 in Supporting Information, thus the lipid concentration of 0.025 mg mL-1, adopted in Figure 3, was the best condition for SPLB formation. Incubation with a lipid concentration of 0.015-0.02 mg mL-1 resulted in a mixture of an intact vesicular layer and SPLB similar to that in Figure 3a. The higher lipid concentration in this range and the longer incubation time in the range of 30-120 min gave a larger SPLB ratio with respect to the vesicular layer, but usually a part of the vesicular layer remained. We also found that continuous incubation is necessary for SPLB formation. The adsorbed vesicles did not transform to a planar membrane even after the vesicular layer (Figure 2) was again incubated in the 0.025 mg mL-1 sonicated vesicle suspension. The adsorbed vesicles also stably existed after incubation in the buffer solution without vesicles at least up to 90 min. This indicates that the adsorption state of the vesicles irreversibly changes during the incubation, washing, and fluorescence microscopy observation (∼1 to 2 h). A vesicle newly adsorbed on the TiO2 surface is in a kind of transition state and gradually shifts to the intact adsorption state, which is inactive for the trigger of SPLB formation, such as intervesicle fusion and/or rupture.34,40,60 The behavior of the adsorbed vesicles on the step-and-terrace TiO2(100) surface is different from that previously reported on sputter-deposited SiO232 and on mica.60 On the former surface, smaller vesicles lead to faster SPLB formation, but eventually the SPLB forms independently of vesicle size. In SPLB formation on the latter surface, which proceeds through the single vesicle rupturing process, vesicles larger than a threshold radius (Rr, ∼75 nm) transform to planar membrane formation, and those smaller than Rr lead to intact vesicle adhesion at a lipid concentration similar to that in our study. When the suspension of vesicles close to Rr is used, higher lipid concentration tends to increase the planar membrane formation rate.60 The behavior of adsorbed vesicles and the SPLB formation mechanism on TiO2 seem very complicated and are deferred to future studies. (64) Seifert, U. AdV. Phys. 1997, 46, 13–137.

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Figure 3e shows the AFM topograph of TiO2(100) that is fully covered by DPoPC-SPLB as in Figure 3b-d. The step-andterrace structure that is similar to that of the monostep TiO2(100) substrate is clearly observed even on the SPLB. It is an interesting result that the SPLB follows the atomic-scale structure of the substrate, although a water layer exists between the SPLB and TiO2(100) and DPoPC-SPB is in the fluid LR phase (Tc) -36 °C). We do not assume that the step-and-terrace structure on the SPLB is an artificial one caused by AFM cantilever compression. The height of the step on the SPLB did not change with the applied force (oscillation amplitude of tapping) during the AFM observation in the range at which an AFM image is stably obtained. The step height on the SPLB should depend on the applied force in case the cantilever presses a flat membrane to the substrate and makes the substrate shape emerge on the membrane. Rather rigid gel-phase SPLBs also have step-andterrace morphology as described in the next section. The SPLB distortion caused by the 1D steps may cause anisotropic lipid diffusion between the directions parallel and perpendicular to the steps. However, we could not get clear evidence of the anisotropic diffusion. FRAP in Figure 3 is performed on too large a scale (bleached region of 100 µm diameter), because of the instrument limitation, to detect the effect of 140-nm-pitch substrate steps. Additionally, it has been reported that supported bilayers have diffusion coefficients reduced to half or less than half of those of free-standing membranes.65 The membrane distortion caused by the TiO2(100) monostep is only 5% of the membrane thickness, and the diffusion barrier caused by this distortion will be much smaller than the substrate-couplinginduced diffusion retardation. Phase Separation Structure of a Binary SPLB on TiO2(100). Figure 4 shows the AFM topograph of a 1:1 binary bilayer of DPoPC and DPPC observed at room temperature (RT, ∼20 °C). The AFM observation of the SPLB was performed 30 min after the sample was cooled to RT to avoid thermal drift, thus the sample in Figure 4 already reached the equilibrium state. The phase-separation structure shown in Figure 4 (and also in Figure 5) was stable and immobile. The gel-LR transition temperatures of DPoPC and DPPC are -36 and 41 °C, respectively, and the domains of the gel and LR phases coexist at RT. In the AFM topograph, DPPC-rich gel-phase domains and DPoPC-rich LRphase domains can be recognized from the height difference.38,39,66,67 Because both DPoPC and DPPC have C16-acyl chains, the thickness of the gel-phase bilayer, in which hydrocarbon chains of lipids are in an ordered all-trans conformation, is larger than that of the LR phase, in which hydrocarbon chains are disordered.68 Defects appeared in the phase-separated SPLB in Figure 4a as a result of the decrease in the molecular occupying area through the LR-gel transition because the SPLB is in the LR phase when it is formed at 45 °C and gel domains appear after the sample is cooled. The SPLB thickness can be measured from the bare TiO2(100) surface at the membrane defects: gel-phase and LR-phase domains measures 5.8 and 4.5 nm in height, respectively. The thickness difference between the gel and LR domains (1.3 nm, in Figure 4a) is larger than that obtained by X-ray diffraction (0.7 nm).68 Thus we assume that LR-SPLB is ∼10% compressed by the AFM cantilever whereas gel-SPLB undergoes a small amount of compression (65) Przybylo, M.; Sykora, J.; Humpolickova, J.; Benda, A.; Zan, A.; Hof, M. Langmuir 2006, 22, 9096–9099. (66) Blanchette, C. D.; Lin, W.-C.; Orme, C. A.; Ratto, T. V.; Longo, M. L. Langmuir 2007, 23, 5875–5877. (67) Kraft, M. L.; Weber, P. K.; Longo, M. L.; Hutcheon, I. D.; Boxer, S. G. Science 2006, 313, 1948–1951. (68) Nagle, J. F.; Tristram-Nagle, S. Biochim. Biophys. Acta 2000, 1469, 159– 195.

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Figure 4. (a) AFM topograph (2.0 × 2.0 µm2) and a line profile of the DPPC + DPoPC (1:1) binary SPLB on the monostep TiO2(100) surface observed at room temperature. Higher and lower domains are assigned to gel-phase and LR-phase domains, respectively. The line profile was taken at the dashed-dotted line drawn in the AFM topograph. (b) AFM topograph (2.0 × 2.0 µm2) of the DPPC + DPoPC binary SPLB (different sample from that in panel a). The contrast along the z axis is enhanced to facilitate visualization of the step traces on the SPLB. This image processing caused the artificial shadows around the gel-phase domain. The white-out protrusions are adsorbed vesicles on the SPLB. The gelphase domain edges running along the substrate steps are indicated by white dashed lines. (c) Schematic illustration of the cross-sectional view perpendicular to the substrate steps, like the black dashed-dotted line in panel b. The DPPC-rich gel-phase domain and DPoPC-rich LR-phase domains are separated at the substrate steps.

Figure 5. (a) AFM topograph (1.4 × 1.4 µm2) and line profile of the DPPC + DPoPC (1:3) binary SPLB on the bistep TiO2(100) surface observed at 5 °C. Three higher domains are gel-phase domains segregated in an LR-phase bilayer. (b) Traces of the substrate bisteps observed in panel a are indicated by dotted lines.

under our experimental conditions. Figure 4b is also an AFM topograph of the DPPC + DPoPC binary SPLB, but the contrast in the z axis is magnified. Both LR- and gel-phase domains have the substrate-induced step-and-terrace structure as in Figure 3e. We also found in Figure 4b that some of the gel domains run along the step of the TiO2(100) as illustrated in Figure 4c. When a gel-phase domain grows in an LR-phase bilayer, a lipid molecule at the gel-LR interface encounters an energy barrier due to the mismatching between the gel and LR domains.39 Bilayer distortion caused by the substrate step will enhance the mismatching and retard the domain growth, even though the distortion is not so large as to divide the bilayer or to stop the gel-phase domain

Tero et al.

growth completely. The gel domains can grow, crossing the steps as shown in Figure 4a,b, probably repeating slow growth above the steps and rather faster growth on the terraces. Thus we can find some domain edges in the middle of the terrace, also parallel to the steps. Figure 5 shows the DPPC and DPoPC 1:3 binary bilayer on the bistep TiO2(100). The SPLB on the bistep TiO2(100) also has the step-and-terrace structure precisely following the substrate morphology, and the traces of the steps are clearly recognized in the AFM topograph. The sample was cooled to 5 °C at a cooling rate of 30 K min-1. Under this lipid component and cooling condition, we obtained DPPC-rich gel phase domains of ∼200 nm width, which is comparable to the terrace width of the bistep TiO2(100) surface. When a gel-lipid cluster grows over the critical nucleus, growth across the step will experience a higher barrier as in Figure 4b, and also the lipid diffusion within the terraces will be more preferable than that crossing the steps. In Figure 5a, three gel-phase domains in an LR-phase bilayer are observed on the terraces (Figure 5b). The height difference between the gel- and LR-phase domains is 1.3 nm, which is almost the same as that in Figure 4a. Smaller protrusions in Figure 5 may also be small gel-phase domains, though the heights of these small protrusions do not seem as uniform as in the gel-phase domains because the smaller domains get more of an artificial effect from AFM cantilever compression than on the larger domains as a result of the larger contribution of the domain boundary. We did not find any preference for the presence of the gel-phase domains in the distorted region of the SPLB on the steps. We do not discuss further details of the nucleation process in this article, but one important point should be emphasized: the results in Figures 4 and 5 clearly show that atomic-scale distortion certainly exists in the SPLB on the step-and-terrace TiO2(100) surfaces and can affect the early process of the gel-LR phase separation (Figure 5b) and the lateral lipid organization on the order of micrometers (Figure 4b). Interaction Energy Between TiO2(100) and the Lipid Bilayer. The results in Figures 3-5 show that the SPLB follows the surface structures on the angstrom scale on the monostep and bistep TiO2(100) surfaces. This indicates that the distance between the SPLB and the TiO2 surface precisely determined in subangstrom order, even though the bilayer is in the fluid LR phase and a water layer exists between the SPLB and substrate. Previous reports showed that 2-3 molecular layers of ”structured water” exist in the vicinity of the oxide surfaces bound by hydrogen bond, and the thickness of this stable water layer is ∼0.5 nm.69-72 Thus the SPLBs, which generally exist at 1-2 nm from the surface,18-22 faces to rather fluid water which has similar property to bulk water. In addition, the bilayer membranes thermally fluctuate.73 It seems an amazing thing that the fluid SPLB precisely follows the atomic-scale structure of the substrate. To explain this distance-preciseness, we evaluated the interaction energy between the SPLB and the TiO2(100) surface. The interaction energy per unit area between two materials (Wtotal) is expressed as the summation of the van der Waals interaction (Wvdw), double-layer interaction (WDL), and hydration interaction (Whyd):73 (69) Michot, L. J.; Villie´ras, F.; Franc¸ois, M.; Bihannic, I.; Pelletier, M.; Cases, J.-M. C. R. Geoscience 2002, 334, 611–631. (70) Park, S.-H.; Sposito, G. Phys. ReV. Lett. 2002, 89, 085501. (71) Cheng, L.; Fenter, P.; Nagy, K. L.; Schlegel, M. L.; Sturchio, N. C. Phys. ReV. Lett. 2001, 87, 156103. (72) Catalano, J. G.; Park, C.; Zhang, Z.; Fenter, P. Langmuir 2006, 22, 4668– 4673. (73) Israelachvili, J. N. Intermolecular and Surface Forces, 2nd ed.; Academic Press: London, 1991.

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Wtotal)Wvdw+WDL+Whyd

(1)

Each of these three interaction energies can be expressed as a function of the separation distance, D. Nabika et al. has reported that this energy evaluation reasonably explains the interaction energy between lipid bilayer lobes during the self-spreading of PC bilayers.74 Here we calculate Wtotal as accurately as possible to exemplify the experimental conditions in this study, including the effect of substrate annealing and the TiO2 plane index. The van der Waals interaction energy between two parallel planar surfaces is expressed as

Wvdw ) -

A 12πD2

Table 1. Calculated Hamaker Constants and Relevant Physical Parameters of Media 1/3/2 Systems

medium 1 TiO2(100) TiO2(001) SiO2 medium 2 lipid medium 3 water

ε(0)

C

104.8a 120.8a 3.8b 2.04c 77.6c

5.162a 5.477a 1.098b 1.041c 0.762c

νj (s-1)

Aν)0 Aν>0 (10-21 J) (10-21 J)

1.20 × 1015b -0.44 1.20 × 1015b -0.64 3.21 × 1015b 2.67 2.64 × 1015c 3.17 × 1015c

8.73 9.15 1.99

a Anisotropic aspects are calculated following refs 78 and 79. b Reference 78. c Reference 76.

(2)

where A represents the Hamaker constant. Now we assume that two semi-infinite media 1 and 2 sandwich a planar slab of medium 3 of thickness D. The value of A in the media 1/3/2 system is obtained by

A132 ) Aν)0(2κD)e-2κD + Aν>0 Aν)0 )

(

)(

3kT ε1(0) - ε3(0) ε2(0) - ε3(0) 4 ε1(0) + ε3(0) ε2(0) + ε3(0)

Aν>0 )

3h 4π

(

Ci

where

∫0∞ R13(ν) R23(ν) dν C3

)

(4) (5)

+ Ci - C3 ν3 νi2 Ri3(V) ) 2 + Ci 2 + C3 2V4 2 + V + + Ci + C3 + 2 νi2ν32 ν32 νi2 (i ) 1, 2) (6) ν2

(

2

)

(3)

)

and κ, k, T, εj(0), h, and νj represent the inverse Debye length, Boltzmann constant, temperature, static dielectric constant of the medium j (j ) 1, 2, 3), Planck constant, and absorption frequency of medium j in the UV range, respectively. The oscillator parameter Cj is represented by Cj ) 4fj/νj, where fj is the oscillator strength of the absorption band and Cj can be experimentally obtained by a Cauchy plot.75-78 If we assume all of the media have the same absorption frequency νe, then eqs 5 and 6 are reduced to

AV>0 ) 3hVe

(n12 + n32)(n22 + n32)

2 2 1⁄2 2 2 1⁄2 2 2 1⁄2 2 2 1⁄2 8√2 (n1 + n3 ) (n2 + n3 ) {(n1 + n3 ) + (n2 + n3 ) } (7)

where nj is the refractive index of medium j and is represented as Cj ) nj2 - 1. This simplification is called the Tabor-Winterton (TW) approximation in the literature.77-79 The TW approximation gives a qualitatively good prediction of the effects of experimental parameters (e.g., salt concentration, surface charge density, temperature) on one media set74 but does not suit the comparison between different kinds of materials, especially if there is a significant difference among the νj values of the three media. It has been reported that the Hamaker constant (74) Nabika, H.; Fukasawa, A.; Murakoshi, K. Langmuir 2006, 22, 10927– 10931. (75) Hough, D. B.; White, L. R. AdV. Colloid Interface Sci. 1980, 14, 3–41. (76) Prieve, D. C.; Russel, W. B. J. Colloid Interface Sci. 1988, 125, 1–13. (77) Bergstro¨m, L. AdV. Colloid Interface Sci. 1997, 70, 125–169. (78) Knowles, K. M. J. Ceram. Process. Res. 2005, 6, 10–16. (79) Knowles, K. M.; Turan, S. Ultramicroscopy 2000, 83, 245–259.

Figure 6. (a) The van der Waals interaction energy of solid/water/lipid systems as a function of separation distance calculated for TiO2(100) and SiO2. Continuous lines are calculated by the Prieve and Russel (PR) method, and dashed lines are obtained from the Tabor-Winterton (TW) approximation. (b) Total interaction energy of solid/water/lipid systems calculated for TiO2(100), TiO2(001), and SiO2 as a function of separation distance. The SiO2 curve is magnified 5-fold to facilitate visualization. The inverse-sign undulation energy (Wundualtion) is also plotted for comparison.

of the TiO2/medium/TiO2 system is overestimated by the TW approximation because TiO2 has a significantly lower absorption frequency (1.20 × 1015 s-1) than that of SiO2, water, and hydrocarbons (∼3 × 1015 s-1).76-78 Therefore, we evaluated Wvdw more rigorously from eqs 4–6, following the Prieve and Russel (PR) approach76 in which experimentally obtained νi values are used for the respective media. The exact solution of eqs 5 and 6 can be obtained using the technique of partial fractions (Supporting Information). In addition, we also take into account the anisotropic dielectric constant and birefringence of the rutile TiO2 crystal, following Knowles’ works.78,79 The physical parameters used to calculate Aν)0 and Aν>0 and the calculated Aν)0 and Aν>0 values are summarized in Table 1. The parameters of vitreous SiO2 are also shown for comparison as the model of thermally oxidized SiO2/Si in Figure 2d-f. The thickness of this SiO2/Si is ∼100 nm and is large enough to satisfy the semi-infinite condition of medium 1. Figure 6a shows the Wvdw versus D plot calculated from eq 3 using the Aν)0 and Aν>0 values in Table 1 at T ) 300 K. The ε3(0) and C3 values

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Tero et al.

of water were substituted for those of the buffer solution, and κ ) 1.31 nm-1 was obtained from the ion strength of the buffer solution in this study. The TW approximation significantly overestimates the Aν>0 (44.3 × 10-21 J), thus overestimating Wvdw for the TiO2(100)/water/lipid system: at D ) 2.0 nm, Wvdw ) -57.8 µJ m-2 in the PR method but -294 µJ m-2 in the TW approximation. Therefore, the rigorous calculation of Wvdw by the PR method is necessary in this study because the overestimation by the TW approximation suppress the contribution from other two factors in eq 1. It is obvious, however, that even when we calculate Wvdw by the PR method the lipid membrane on TiO2(100) gains significantly more van der Waals energy than does that on SiO2 (Wvdw ) -13.6 µJ m-2 at D ) 2.0 nm). The difference in Wvdw between TiO2 and SiO2 is attributed to the second term in eq 3, specifically because the TiO2 surfaces have larger refractive indexes in the UV and visible regions than does SiO2. The effective refractive indexes obtained from C1 in Table 1 are 2.482 and 2.545 for TiO2(100) and TiO2(001) but 1.565 for SiO2. The negative Aν)0 values of the TiO2/water/lipid systems indicate that the permittivity-attributed component works as the repulsiVe van der Waals interaction. This is because TiO2 has a larger dielectric constant than does water. The sign of Aν)0 is determined by the magnitude correlation of the media’s εi(0) values. If medium 1 is a general oxide or a ceramic (e.g., SiO2, mica, or alumina), then the sequence is ε3(0) > ε1(0) > ε2(0) and hence Aν)0 is positive because of the large ε3(0) value of water attributed to its permanent dipole, whereas the sequence is ε1(0) > ε3(0) > ε2(0) in the TiO2/water/lipid system. In an electrolyte solution, however, the Aν)0 term is screened (eq 3) and makes only a small contribution to Wvdw. In a 1:1 electrolyte solution, the double-layer interaction energy, WDL, between the surfaces of media 1 and 2 with the surface potentials of Ψ1 and Ψ2 are obtained from the following equation:

( ) ( )

WDL ) 64 000NAkTI tanh

eΨ1 eΨ2 e-κD tanh 4kT 4kT κ

(8)

where NA, I, and e represent Avogadro’s number, the ion strength of the electrolyte solution in mol L-1, and the elementary charge, respectively. The surface potential Ψi in the unit of V is derived from the surface charge density σi by the Grahame equation

Ψi )

{

σi 2kT arcsin h 1 e (8000ε3ε0kTNAI) ⁄2

}

(i ) 1, 2) (9)

where ε0 is the electric constant. We took the surface charge density of a TiO2 surface without thermal treatment at pH 7.4 from that of suspended TiO2 particles, -0.062 C m-2.80 TiO2 surfaces usually have a negative net charge in the buffer solution of the present study (pH 7.4)81 because of the dissociation of surface hydroxyl (OH) groups. The OH groups thermally desorb above 600 °C under a dry environment, and ∼40 and ∼60% of the OH groups desorb at 700 and 800 °C.82 The thermal desorption of two OH groups leaves an oxygen defect site on the substrate surface, which is active with respect to the dissociative adsorption of oxygen.53 Thus, the OH desorption caused by oxygen annealing is irreversible, and the OH groups do not recover by immersing the annealed TiO2 in an aqueous solution. We simply assume that the surface charge density depends linearly on the surface OH density and estimate at σ1 ) -3.72 × 10-2 C m-2 and σ1 (80) Fukuzaki, S.; Urano, H.; Nagata, K. J. Ferment. Bioeng. 1996, 81, 163– 167. (81) Kosmulski, M. Chemical Properties of Material Surfaces; Marcel Dekker: New York, 2001. (82) Oosawa, Y.; Gratzel, M. J. Chem. Soc., Faraday Trans. 1 1988, 84, 197–205.

) -2.48 × 10-2 C m-2 for the monostep and bistep TiO2(100) surfaces, respectively, which are prepared by annealing at 700 and 800 °C, respectively. In the same way, we estimated the surface charge density of thermally oxidized SiO2/Si at σ1 ) -2.10 × 10-2 C m-2, from the charge density of SiO2 particles at pH 7.480 and the thermal desorption behavior of OH groups on SiO2.83,84 The thermal OH desorption from the SiO2 surface at 900 °C is also irreversible.84 The surface potentials (Ψ1) calculated from eq 9 are -37.9, 26.4, and -22.6 mV for the monostep TiO2(100), bistep TiO2(100), and SiO2 surfaces, respectively. The lipid membrane surface in this study is also negatively charged because 1% Rb-DOPE is mixed into the bilayer and the rhodamine (Rb) is negatively charged. We estimate at σ2 ) -2.5 × 10-3 C m-2 and Ψ2 ) -2.8 mV, assuming that the molecular occupying area of DPoPC is 0.64 nm.268 Nonnegligible WDL is given by only 1% Rb-DOPE using eq 8: WDL ) 13.7 and 8.7 µJ m-2 for TiO2 and SiO2 at D ) 2.0 nm, respectively. It should be noted that WDL in this study is attributed to the dye-labeled lipid molecule for the fluorescence microscope observation. If the lipid bilayer consists of only neutral lipids, such as synthetic phosphatidylcholine, then WDL is naturally zero, and repulsive interaction is dominated by the hydration term, Whyd.17 Apart from the two Derjaguin-Landau-Verwey-Overbeek (DLVO) forces, van der Waals and double-layer forces, it is well known that a short-range non-DLVO force works between two surfaces in a solvent. This is the source of the hydration energy, Whyd, in eq 1. Compared with the two DLVO energies, Wvdw and WDL, the origin of Whyd is rather complicated and is still controversial.73,85-87 Previous reports showed that the hydration energy is described in different ways between water/solid (e.g., metal oxides and ceramics) and water/soft material (e.g., lipid bilayers, proteins, and polymers) interfaces. At the interface between a hydrophilic solid surface and an aqueous solution, the hydration energy (Wsolid) is attributed to stable structured water layers hydrogen bonded to the solid surface. However, hydration repulsion energy between two lipid bilayer membranes (Wlipid) is dominated by entropic factors (i.e., microscopic and macroscopic thermal fluctuations). Previous studies of the hydration energy mainly focused on homogeneous interface sets, solid/ water/solid or soft material/water/softmaterial, and, at least to our knowledge, a quantitative expression of the hydration force at the heterogeneous interface sets, solid/water/softmaterial, has not been reported. Here we assume that Wsolid and Wlipid are independent of each other and thus assume that the hydration energy between a lipid bilayer membrane and a solid substrate (Whyd in eq 1) is the average of each hydration energy at the homogeneous interface sets:

Whyd )

Wsolid + Wlipid 2

(10)

Empirically, Wsolid follows the simple exponential decay function

Wsolid ) + W0e(-D/λ0)

(11)

m-2

Usually, the values of W0 is 3-30 mJ and λ0 is on the order of angstroms to nanometers, even though they strongly depend on environmental conditions such as pH, electrolyte type (83) Diaz, L.; Liauw, C. M.; Edge, M.; Allen, N. S.; McMahon, A.; Rhodes, N. J. Colloid Interface Sci. 2005, 287, 379–387. (84) Chuang, I.-S.; Maciel, G. E. J. Phys. Chem. B 1997, 101, 3052–3064. (85) Israelachvili, J.; Wennerstro¨m, H. Nature 1996, 379, 219–225. (86) Adler, J. J.; Rabinovich, Y. I.; Moudgil, B. M. J. Colloid Interface Sci. 2001, 237, 249–258. (87) Cherepanov, D. A. Phys. ReV. Lett. 2004, 93, 266104.

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Langmuir, Vol. 24, No. 20, 2008 11575

and concentration of the aqueous solution,88-92 and material, structure, and hydrophilicity of the substrate surface.90,93-96 In the present study, typical values of W0 ) 6.0 mJ m-2 and λ0 ) 0.3 nm are used to evaluate Wsolid from eq 11. The estimation of Wlipid is based on the thermal fluctuation forces between fluidlike surfaces,73 which consists of three thermal motions: protrusion, peristalsis, and undulation. The interaction energies due to these three forces are expressed as

Wprotrusion ) + 2.7ΓkTe(-RpD/kt) Wperistalsis ) + Wundulation ) +

(12)

2

(kT) 20kaD4

(13)

3π2(kT)2 128kBD3

(14)

where Γ, Rp, ka, and kb represent the surface density of protruding head groups, protruding energy, the area expansion modulus, and the bending modulus. We took the typical values of Rp ) 2.5 × 10-11 J m-1, ka ) 0.15 J m-2, and kb ) 10-19 J from ref 73 and used the value of Γ ) 1.6 nm-2 from the molecular occupying area of the lipid (0.64 nm2/lipid). It should be mentioned that the undulation force is drastically reduced or even eliminated when a membrane carries a surface charge or when it is under tension, hence we define

Wlipid ) Wprotrusion + Wperistaltic

(15)

and we will discuss the Wundulation factor independently afterward. There is another steric repulsive factor in the soft material/water/ soft material system as a result of the overlapping of thermally fluctuating species. This overlap force can be neglected in this study because it works only in the range where lipid headgroups of two bilayer membranes actually overlap, which means D < 0, whereas we consider only the noncontact region (D > 0). Substituting eqs 11-13 and 15 into eq 10, we obtain the expression of Whyd as follows:

Whyd )

{

}

1 (kT)2 W0e(-D⁄λ0) + + 2.7ΓkTe(-RpD/kT) (16) 2 20k D4 a

Figure 6b shows the Wtotal versus D plot calculated for TiO2(100)/water/lipid and SiO2/water/lipid systems from eq 1, estimating Wvdw from eqs 2-6 and the PR approach, WDL from eqs 8 and 9, and Whyd from eq 16. The lipid layers on the monostep (700 °C-annealed) and bistep (800 °C-annealed) TiO2(100) surfaces have sharp energy minima of -58.2 and -69.1 µJ m-2 at D ) 1.2 nm, respectively. In Figure 6b, we also plotted the Wtotal of the TiO2(001)/water/lipid because the anisotropy in permittivity and refractive index is considered in our calculation. The energy minimum of the TiO2(001) system appears at almost the same distance as that of the TiO2(100) system, although the (88) Horn, R. G.; Smith, D. T.; Haller, W. Chem. Phys. Lett. 1989, 162, 404– 408. (89) Pashley, R. M.; Israelachvili, J. N. J. Colloid Interface Sci. 1984, 101, 511–523. (90) Peschel, G.; Belouschek, P.; Mu¨ller, M. M.; Mu¨ller, M. R.; Ko¨nig, R. Colloid Polym. Sci. 1982, 260, 444–451. (91) Pashley, R. M. J. Colloid Interface Sci. 1981, 83, 531–546. (92) Ducker, W. A.; Senden, T. J.; Pashley, R. M. Langmuir 1992, 8, 1831– 1836. (93) Larson, I.; Drummond, C. J.; Chan, D. Y. C.; Grieser, F. J. Phys. Chem. 1995, 99, 2114–2118. (94) Larson, I.; Drummond, C. J.; Chan, D. Y. C.; Grieser, F. J. Am. Chem. Soc. 1993, 115, 11885–11890. (95) Larson, I.; Drummond, C. J.; Chan, D. Y. C.; Grieser, F. Langmuir 1997, 13, 2109–2112. (96) Pashley, R. M. AdV. Colloid Interface Sci. 1982, 16, 57–62.

former is deeper (-76.7 µJ m-2). The SiO2/water/lipid system has its energy minimum of -3.41 µJ m-2 at 2.8 nm, which is in good agreement with the previous theoretical study.97 The distance between the SPLB and the SiO2 surface is overestimated from the experimentally obtained values (1.3-1.7 nm).20-22 This is mainly due to the Wsolid value in the hydration repulsive term in eq 10. In the SiO2/water/lipid system, the energy gain from Wvdw and the energy loss from WDL and Whyd become comparable in the region of D ) 1-3 nm, thus the position of the energy minimum is quite sensitive to W0 and λ0. In the TiO2/water/lipid systems, however, the energy gain from Wvdw is significantly larger than Whyd, and the position of the energy minimum is not as affected by the W0 and λ0 values. In Figure 6b, Wundulation calculated by eq 14 is also plotted. The sign of Wundulation is inverted for comparison because the undulation force is repulsive and Wundulation has positive values. It should again be mentioned that the undulation force is drastically reduced under some specific conditions, such as the existence of surface charges; therefore, the plotted Wundulation is the hypothetical maximum estimation for an SPLB with no charge and no tension. The SPLB in this study contains 1% negatively charged RbDOPE, thus the effective undulation energy will be significantly smaller than that plotted in Figure 6b. Even compared with this maximum Wundulation, the energy gain of the lipid membrane on the TiO2 surfaces at the potential minimum is sufficiently larger, which means that the large Wvdw on TiO2 overcomes the thermal fluctuation of the SPLB. A sharp energy valley indicates that the distance between the SPLB and the TiO2(100) surface is accurately determined. We conclude that it is the strong van der Waals attraction of the TiO2 substrate that makes the SPLB follow the surface morphology faithfully on the atomic scale. The energy plot in Figure 6b also indicates that even under this strong attraction a water layer of 1.2 nm thickness exists between the SPLB and the TiO2(100). This agrees well with the FRAP result in Figure 3b-d. The membrane retains its lateral fluidity, but the distortion on the substrate step works as the barrier to molecular diffusion and affects the phase-separation structures as shown in Figures 4 and 5. In addition, we assume that the existence of adsorbed vesicles and second bilayers on the first SPLB on the TiO2(100) (Figure 3b-d and Figure S2-a in Supporting Information) can be explained by Figure 6b. The energy at the second bilayer position (D ) 7 nm) on TiO2 (-4.7 µJ m-2)98 is still lower than the potential minimum of the bilayer on SiO2 (-3.4 µJ m-2). It is possible that the strong attraction on TiO2 may seriously distort the bilayer structure and may cause some specific adsorption sites on the first SPLB, but we did not find any trace of such sites in AFM images. We always obtained the phaseshift image,99 a sensitive mapping to material properties such as viscoelasticity and chemical terminations, simultaneously when we observed AFM topographs and did not find inhomogeneity on the SPLBs on the TiO2 surfaces in the phase-shift image or in the topographs. The energy calculation in Figure 6b may seem to be inconsistent with the results in Figure 2 if we follow the adhesion-induced tension model (AT model)64 in which stronger adhesion leads to a larger distortion of the adsorbed vesicles and thus to easier vesicle rupturing and planar membrane formation. Stronger interaction on the SiO2 than on TiO2 was presumed from the easier SPLB formation on SiO2 than on TiO2 in previous reports about the vesicle behavior on sputter-deposited SiO2 and TiO2 (97) Swain, P. S.; Andelman, D. Phys. ReV. E 2001, 63, 051911. (98) The five-medium model (substrate/water/lipid/water/lipid) is necessary for the strict estimation of E2, but here we did not consider it for brevity. (99) Radmacher, M.; Tilmann, R. W.; Gaub, H. E. Biophys. J. 1993, 64, 735– 742.

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surfaces.30-32,34 It should be noted, however, that the AT model treats only the energetic aspect of static vesicle adsorption and that the transformation of adsorbed vesicles to SPLB is an irreversible and dynamic process largely containing kinetic aspect and other factors. This means, conversely, that the vesicle adsorption state (intact vesicle or SPLB formation) is not always valid as a barometer of the bilayer-substrate interaction energy in the equilibrium state. For example, in SPLB formation through the critical-vesicle-coverage mechanism described above, key factors are the intervesicle interaction and the interaction between the SPLB patches and adsorbed vesicles rather than the adsorption energy of individual vesicle. To our knowledge, there has been no direct measurement of the forces or interaction energy between lipid bilayers and the solid substrate; therefore, the theoretical calculation is important for evaluating the actual interaction energy. The AT model is based on the interaction energy of ∼103 µJ m-2 that exists between the bilayer membrane and the substrate (called strong adhesion64) on the condition of the general bilayer bending modulus, ∼10-19 J.64 Figure 6b indicates that the minimum interaction energies on the step-and-terrace TiO2(100) surfaces are -55-70 µJ m-2, which are still much smaller than that assumed in the AT model. Thus, it is energetically reasonable that the vesicles exist stably on the TiO2 surfaces (Figure 2a-c), unless SPLB formation is triggered. We do not have a clear explanation at present as to why SPLB formation proceeds more easily on SiO2 than on TiO2. From the viewpoint of the AT model, there may be the possibility that some factors of the interaction energy are missed in our calculation. In this case, some specific attractive energy should exist between the bilayer and the substrate because Wvdw on SiO2, without repulsive WDL and Whyd factors, is only -66.4 µJ m-2 even at D ) 1 nm (Figure 6a), which is too small to cause strong adhesion. Such a specific attractive force, however, was not found in the previously reported AFM force curve measured for adsorbed egg-PC vesicles.100 We also measured the force curve of PCSPLB and did not detect such a specific attraction (data not shown).101 These results again indicate that the bilayer-substrate interaction energy is not the only factor affecting on the vesicle-SPLB transformation. We do not discuss the behavior of the adsorbed vesicles and the SPLB formation mechanism further, but we believe that our energy evaluation between the bilayer and oxide substrates will be valuable in future research on these issues. There are some other substrate characteristics that may affect planar membrane formation from vesicles. We have hypothesized the same Whyd(D) for TiO2 and SiO2 in this study, but previous surface force studies indicated that hydration repulsion tends to be less effective on TiO2 than on SiO2,92-94 although the reason has not been clarified. It is also well known that TiO2 has a unique surface function, photoinduced superhydrophilicity.102 If TiO2 is exposed to UV light, for example, during UV-ozone cleaning,29-31 then the efficiency with respect to planar bilayer formation may decrease because of the increment of hydration repulsion associated with the expression of superhydrophilicity. It has been reported that photoinduced superhydrophilicity is (100) Liang, X.; Mao, G.; Simon Ng, K. Y. Colloids Surf., B 2004, 34, 41–51. (101) Si3N4 and Si cantilevers were used in ref 100 and our force curve measurement. Generally both surfaces are covered by a native silicon oxide layer. (102) Wang, R.; Hashimoto, K.; Fujishima, A.; Chikuni, M.; Kojima, E.; Kitamura, A.; Shimohigoshi, M.; Watanabe, T. Nature 1997, 388, 431–432.

Tero et al.

induced by the generation of metastable surface OH groups accompanying additional water adsorption on the surface.103,104 The surface OH group is one of the key factors in the interaction with biomaterials105,106 and probably plays an important role in the interaction between the TiO2 substrate and SPLBs, especially if membrane proteins are incorporated into the SPLBs. Furthermore, it is well known that each low index surface of TiO2 has specific structures and chemical properties,50,107-110 and these should also be considered in a future study. A detailed investigation of TiO2 surface effects on SPLBs and their dependence on plane directions is now underway.

Summary The formation of phosphatidylcholine-SPLB from adsorbed vesicles and its morphology and phase-separation structure on monostep and bistep TiO2(100) surfaces were investigated. The transformation from adsorbed vesicles to a planar bilayer on these step-and-terrace TiO2(100) surfaces had a larger barrier than on SiO2 but proceeded under higher lipid concentration and longer incubation time if the 100-nm-filtered and further sonicated vesicle suspension was used. We found that the adsorbed vesicles on the TiO2(100) surface had two competing irreversible pathways, transformation to a planar membrane and transition to a stable adsorption state. The SPLBs on the TiO2(100) surfaces had step-and-terrace morphology precisely following the substrate structure, and this angstrom-order membrane distortion affected the micrometer-order gel-LR phase separation structure horizontally. The distance preciseness between the SPLB and the step-and-terrace TiO2(100) surfaces was attributed to the specifically strong van der Waals attraction of TiO2. The calculated interaction energy showed that the lipid membrane on TiO2 gained 20 times more energy at its energy minimum than that on SiO2. Acknowledgment. This work was partially supported by a Grant-in-Aid for Young Scientists (B) no. 18750021, Scientific Research (A), no. 19201023, Scientific Research on Priority Areas, nos. 17034064 and 18059013, of the Ministry of Education, Culture, Sports, Science and Technology, NINS (National Institutes of Natural Sciences) Cooperative Project Bio-Molecular Sensor, the Cosmetology Research Foundation, and the Kao Foundation for Arts and Sciences. Supporting Information Available: TiO2(100) step structural models, fluorescence microscope images of the washing effect on the SPLB on the monostep TiO2(100), and details of the solutions of eqs 5 and 6. This material is available free of charge via the Internet at http://pubs.acs.org. LA801080F (103) Nakamura, R.; Ueda, K.; Sato, S. Langmuir 2001, 17, 2298–2300. (104) Sakai, N.; Fujishima, A.; Watanabe, T.; Hashimoto, K. J. Phys. Chem. B 2003, 107, 1028–1035. (105) Sousa, S. R.; Moradas-Ferreira, P.; Saramago, B.; ViseuMelo, L.; Barbosa, M. A. Langmuir 2004, 20, 9745–9754. (106) MacDonald, D. E.; Deo, N.; Markovic, B.; Stranick, M.; Somasundaran, P. Biomaterials 2002, 23, 1269–1279. (107) Ariga, H.; Taniike, T.; Morikawa, H.; Tero, R.; Kondoh, H.; Iwasawa, Y. Chem. Phys. Lett. 2008, 454, 350–354. (108) Kubo, T.; Sayama, K.; Nozoye, H. J. Am. Chem. Soc. 2006, 128, 4074– 4078. (109) Tero, R.; Fukui, K.; Iwasawa, Y. J. Phys. Chem. B 2003, 107, 3207– 3214. (110) Raza, H.; Pang, C. L.; Haycock, S. A.; Thornton, G. Phys. ReV. Lett. 1999, 82, 5265–5268.