Groove-Spanning Behavior of Lipid Membranes on ... - ACS Publications

The groove-spanning behavior of membrane lobes is discussed in terms of a balance between adhesion and bending energies of lipid bilayers. View: PDF ...
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Langmuir 2005, 21, 6487-6494

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Groove-Spanning Behavior of Lipid Membranes on Microfabricated Silicon Substrates Kenji Suzuki* and Hiroshi Masuhara* Department of Applied Physics, Osaka University, 2-1 Yamadaoka, Suita, Osaka 565-0871, Japan Received January 20, 2005. In Final Form: April 19, 2005 We report on a spreading behavior of phospholipid membranes that arise from a lump of phospholipid (a lipid source) on topographically patterned substrates immersed in an aqueous solution. Microgrooves with well-defined shapes were prepared on Si(111) surfaces by anisotropic etching in an alkaline solution. A spreading front that consists of membrane lobes and a single lipid bilayer was observed on the patterned silicon substrates by utilizing fluorescence interference contrast (FLIC) microscopy. FLIC images indicate that the membrane lobes span the microgrooves, while the underlying single lipid bilayer spread along the surface of the microgrooves. In fact, fluorescent polystyrene nanoparticles could be encapsulated in the microgrooves that were completely covered with the membrane lobes. The groove-spanning behavior of membrane lobes is discussed in terms of a balance between adhesion and bending energies of lipid bilayers.

Introduction In aqueous solutions phospholipids form a variety of bilayer structures, such as long tubules, closed spheres (vesicles), and stacks of lamellae.1,2 When such a bilayer structure comes into contact with a water-solid interface, spontaneous spreading of phospholipid membranes can occur over the water-solid interface due to attractive interactions between the membranes and the solid.3-8 Recently, spreading of phospholipid membranes on a micropatterned substrate has received much attention in connection with its potential applications for preparing functional geometries of substrate-supported membranes, e.g., an array of bilayer patches for biological assays9 and lipid bilayer nanotube-vesicle networks for a novel microfluidic system.10 Spreading behaviors of phospholipid membranes on chemically patterned surfaces have been well studied from this point of view.11-16 However, despite the importance of surface topography,17 there have been * Corresponding authors. E-mail: [email protected].

[email protected],

(1) Bangham, A. D.; Standish, M. M.; Watkins, J. C. J. Mol. Biol. 1965, 13, 238. (2) Chapman, D.; Fluck D. J. J. Cell Biol. 1966, 30, 1. (3) Seifert, U.; Lipowsky, R. Phys. Rev. Lett. 1990, 42, 4768. (4) Lipowsky, R. Nature 1991, 349, 475. (5) Lipowsky, R.; Seifert, U. Langmuir 1991, 7, 1867. (6) Kaes, J.; Sackmann E. Biophys. J. 1991, 60, 1. (7) Ra¨dler, J.; Strey, H.; Sackmann, E. Langmuir 1995, 11, 4539. (8) Feder, T. J.; Weissmu¨ller, G..; Zˇ eksˇ, B.; Sackmann, E. Phys. Rev. E 1995, 51, 3427. (9) Groves, J. T.; Ulman, N.; Boxer, S. G. Science 1997, 275, 651 (10) Sott, K.; Karlsson, M.; Phil, J.; Hurtig, J.; Lobovkina, T.; Orwar, O. Langmuir 2003, 19, 3904. (11) Jenkins, A. T. A.; Bushby, R. J.; Boden, N.; Evans, S. D.; Knowles, P. F.; Liu, Q.; Miles, R. E.; Ogier, S. D. Langmuir 1998, 14, 4675. (12) Jenkins, A. T. A.; Bushby, R. J.; Evans, S. D.; Knoll, W.; Offenha¨usser, A.; Ogier, S. D. Langmuir 2002, 18, 3176. (13) Jenkins, A. T. A.; Boden, N.; Bushby, R. J.; Evans, S. D.; Knowles, P. F.; Miles, R. E.; Ogier, S. D.; Scho¨nherr, H.; Vancso, G. J. J. Am. Chem. Soc. 1999, 121, 5274. (14) Groves, J. T.; Ulman, N.; Cremer, P. S.; Boxer, S. G. Langmuir 1998, 14, 3347. (15) Kung, L. A.; Kam, L.; Hovis, J. S.; Boxer, S. G. Langmuir 2000, 16, 6773. (16) Bernard, A. L.; Guedeau-Boudeville, M. A.; Sandre, O.; Palacin, S.; di Meglio J. M.; Jullien, L. Langmuir 2000, 16, 6801. (17) Guedeau-Boudeville, M. A.; Jullien, L.; di Meglio, J. M. Proc. Natl. Acad. Sci. U.S.A. 1995, 92, 9590.

few studies on spontaneous spreading of lipid membranes on topographically patterned but chemically uniform surfaces, presumably because of the experimental difficulty in monitoring three-dimensional morphologies of lipid membranes formed on topographically patterned substrates. From scientific and practical viewpoints, it is indeed important and indispensable to clarify the influence of surface topography on spreading behaviors of lipid membranes. Anisotropic etching of a silicon substrate with an alkaline solution is a process that provides us patterned silicon surfaces with well-defined topography.18 Since the patterned silicon surfaces can be uniformly covered with an amorphous layer of SiO2 by chemical oxidation,19 the conventional silicon processing technique offers a topographically patterned surface with enough chemical uniformity. Furthermore, the oxidized silicon substrate provides high reflectance of a SiO2/Si interface which is another advantage. As a result, one can apply fluorescence interference contrast (FLIC) microscopy for the detection of the spatial distribution of separation distance between fluorescently labeled membranes and the oxidized silicon substrate using conventional incident-light fluorescence microscopy.20-25 Thus, we have expected that such a patterned silicon substrate can be used as one of the ideal substrates for monitoring three-dimensional morphologies of lipid membranes realized on topographically patterned surfaces. For the past decades, spontaneous spreading of phospholipid membranes has been studied mainly in relation to adhesion and fusion of lipid vesicles onto solid surfaces.8,9,11-16 However, spreading of individual vesicles starts randomly on solid surfaces and finishes in a moment. Recently, Ra¨dler and co-workers have reported another (18) Barycka, I.; Zubel, Irena. Sens. Actuators, A 1995, 48, 229. (19) Kern, W.; Puotinen, D. A. RCA Rev. 1970, 31, 207. (20) Lanbacher, A.; Fromherz, P. Appl. Phys. A 1996, 63, 207. (21) Braun, D.; Fromherz, P. Appl. Phys. A 1997, 65, 341. (22) Iwanaga, I.; Braun, D.; Fromherz, P. Eur. Biophys. J. 2001, 30, 17. (23) Lambacher, A.; Fromherz, P. J. Phys. Chem. B 2001, 105, 343. (24) Lambacher, A.; Fromherz, P. J. Opt. Soc. Am. B 2003, 19, 1435. (25) Parthasarthy, R.; Groves, J. T. Cell Biochem. Biophys. 2004, 41, 391.

10.1021/la050157a CCC: $30.25 © 2005 American Chemical Society Published on Web 06/03/2005

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Figure 1. A schematic illustration of the spreading front consisting of double bilayer lobes and a single lipid bilayer. The bold lines indicate lipid bilayers that form a double bilayer lobe and its underlying single lipid bilayer spreading from a lipid source. A more detailed illustration of the spreading front is shown in the upper right side.

type of spreading phenomena that starts from a lump of phospholipid (a lipid source) placed on a water-solid interface.7,26,27 Such a spreading phenomenon would be suitable for monitoring time evolution of lipid membranes on a limited area of patterned surfaces, since it can be initiated at an arbitrary position on the patterned substrate simply by placing a lipid sources on a desired position and can continue over several hours if there is sufficient amount of lipid in the lipid source. From this point of view, we have devoted our efforts to monitor such spreading phenomena on oxidized silicon wafers by utilizing FLIC microscopy in the past few years.28,29 As a result, a spreading front consisting of membrane lobes and a single lipid bilayer have been found to occur from a lipid source in the presence of NaCl. Most of the membrane lobes have proved to be double bilayer lobes that are very flat tubelike structures composed of two sheets of lipid bilayers as illustrated in Figure 1. In this study, we have investigated spreading of the double bilayer lobes and the underlying single lipid bilayer over microgrooves fabricated on Si(111) surfaces and revealed the influence of surface topography on spreading behaviors of lipid membranes. FLIC images of the spreading fronts suggest that the membrane lobes tend to span the microgrooves, while the underlying single lipid bilayer spread on the surface of microgrooves. The groove spanning of membrane lobes is strongly supported with the fact that fluorescent nanoparticles were encapsulated selectively in the microgrooves which were completely covered with the membrane lobes. Among a number of observed phenomena, we focus our attention on the groovespanning of the membrane lobes since it would be one of the most essential spreading behaviors of lipid membranes on topographically patterned surfaces and would provide a novel functional geometry of substrate-supported membranes. Materials and Methods Materials. The phospholipids used in this study were 99% L-R-phosphatidylcholine from egg yolk (Sigma, P-2772). Perylene

(Nacalai GR-26614), a commonly used fluorescence probe for labeling lipid membranes,30,31 was used after purification by (26) Nissen, J.; Gritsch, S.; Wiegand, G.; Ra¨dler, J. O. Eur. Phys. J. B 1999, 10, 335. (27) Nissen, J.; Jacobs, K.; Ra¨dler, J. O. Phys. Rev. Lett. 2001, 86, 1904. (28) Suzuki, K.; Masuhara, H. Chem. Lett. 2004, 33, 218. (29) Suzuki, K.; Masuhara, H. Langmuir 2005, 21, 537. (30) Johnson, D. A.; Nguyen, B.; Bohorquez, A. F.; Valenzuela, C. F. Biophys. Chem. 1999, 79, 1.

Suzuki and Masuhara sublimation. Optically flat silica glass substrates (Sigma Koki, OPSQ) and n-type (111) silicon wafers (Nilaco, SI-500443) were used after surface treatments that will be described later. The buffer solution (10 mM Tris-HCl, pH 8, 100 mM NaCl) was filtered through membrane filters (Whatman, Puradisk 25PP) and was bubbled with N2 gas for 1 h to reduce the dissolved oxygen that enhances the photobleaching of perylene fluorescence under fluorescence microscopy. The suspension of fluorescent polystyrene nanoparticles (Molecular Probes, F8784) with an actual diameter of 24 nm was mixed with the buffer solution in a volume ratio of 1:10 after its slight sonication for 30 s and was used in 1 h, since long storage of the mixture for periods of days results in the salt-induced aggregation of the nanoparticles. Preparation of Smooth Solid Substrates. The n-type silicon wafers were washed with a surfactant (Nacalai Tesque, SCATT) and rinsed with distilled water, and they were immersed in an aqueous solution of HF for removing the native oxide on the surfaces of the silicon wafers with contaminants. The silicon wafers were then immersed in a NH3-H2O2 solution (NH4OH: H2O2:H2O ) 1:1:10) at 80 °C for 15 min to form an oxide layer of less than 1 nm in thickness.19 The oxidized silicon wafers were rinsed with pure water and finally dried at 60 °C prior to use. The silica glass substrates were washed with the surfactant and rinsed with distilled water, and they were immersed in the NH3-H2O2 solution at 80 °C for 15 min. The silica glass substrates were then immersed in an aqueous solution of 5% HF for 5 min for reducing the sharpness of any scratches on the substrates. The silica glass substrates were rinsed with distilled water in an ultrasonic cleaner and finally dried at 60 °C prior to use. Preparation of Topographically Patterned Silicon Substrates. The n-type silicon wafers were oxidized in an electric furnace at 1100 °C. The thickness of the resulting oxide layers formed on the silicon wafers was about 120 nm. Microgrooves were fabricated on the silicon substrate with combination of the focused ion beam milling of the SiO2 layer using a focused ion beam system (FEI, Dual Beam 253 system) and the anisotropic etching of silicon by an aqueous solution of KOH and isopropyl alcohol.18 For each of different patterns, the etching time was adjusted so that the depth of microgrooves would become about 700 nm after the removal of the oxide layer of 120 nm thickness by HF. The depth of the microgrooves was then confirmed to be about 700 nm based on scanning electron microscopy (SEM) observation. The etched substrate was immersed in the NH3-H2O2 solution at 80 °C for 15 min to form a uniform oxide layer of less than 1 nm in thickness.19 The patterned silicon substrates were rinsed with pure water and finally dried at 60 °C prior to use. Figure 2 shows SEM images of representative examples of microgrooves fabricated on Si(111) surface. According to the SEM observation of sections of the microgrooves, curvature radii of the sharp edges of the microgrooves are around 50 nm. Observation of Spontaneous Spreading of Phospholipid Membranes. A chloroform solution of phospholipid (7.2 mg/mL) and perylene was prepared in a ratio of ca. [perylene];[lipid] ) 1:200. Two microliters of the chloroform solution was painted onto a glass rod with diameter about 0.1 mm in 1 min in air using a microsyringe. Some of the solvent evaporated during the process, and thin films of lipid were left on the surface of the glass rod. Immediately after that, the glass rod was stored in a desiccator connected to a rotary vacuum pump for 1 h for removing the solvent completely. The dried lipid films were stamped onto the clean surfaces of the dried substrates for depositing a line of phospholipid on the substrates as a lipid source. The substrate was placed into a well made on a slide glass, and the well was filled with the buffer solution (10 mM Tris-HCl, pH 8, 100 mM NaCl) by dropping a sufficiently large droplet of the aqueous solution onto the substrate. The well was then covered with a cover slip and was placed on the stage of an incident-light fluorescence microscope (Olympus, BX-50). The spreading fronts of phospholipid membranes arising on the substrates in the wells were observed using the incident-light fluorescence microscope with 20× and 40× objective lenses (Olympus, Uplan Apo), and their images were captured by a CCD camera (Olympus, DP-50). In most cases, fluorescence of perylene doped in lipid bilayers was detected with excitation at 404.7 and emission above 455 (31) Khan, T. K.; Chong, P. L.-G. Biophys. J. 2000, 78, 1390.

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Results

Figure 3. Characteristics of FLIC images of the typical spreading front observed on a smooth surface of an oxidized silicon wafer with thin oxide layer less than 1 nm in thickness. (a) An epifluorescence image of the spreading front on a silica glass substrate. The brightest region at the left corner of the image corresponds to the rim of a lipid source, and a spreading front that is composed of a single lipid bilayer and membrane lobes spreads from the left to the right. The fluorescence intensity of membrane lobes takes some discrete values, and the darkest membrane lobes have proved to be double bilayer lobes composed of two sheets of lipid bilayer. (b) A FLIC image of the spreading front on an oxidized silicon substrate with an oxide layer less than 1 nm in thickness. (c) Visualization of the single lipid bilayer on the oxidized silicon substrate with higher excitation intensity. The front of the single lipid bilayer is indicated with the white arrow. (d) A schematic illustration of the cross sectional view of the experimental system on an oxidized silicon substrate under incident-light fluorescence microscopy. (e) Theoretically calculated dependence of FLIC intensity of a fluorophore above an oxidized silicon wafer on the distance from the reflective SiO2/Si interface. This FLIC intensity is calculated under some simplifying assumptions (see Appendix for detail).

A. Spreading Behaviors of Membrane Lobes over Microgrooves. Figure 3 shows characteristics of FLIC images of the spreading front consisting of membrane lobes and a single lipid bilayer on smooth solid surfaces. An epifluorescence image of the spreading front on a smooth surface of silica glass substrates is depicted in Figure 3a. As already reported in our previous studies,28,29 there are few types of membrane lobes with different fluorescence intensity. The darkest membrane lobes correspond to the double bilayer lobes, whose thickness is in the range of several tens of nanometers. The advancement of membrane lobes has already proved to be driven by the spontaneous spreading of the underlying single lipid bilayer, which works like a conveyor belt for the membrane lobes. Figure 3b shows a representative example of FLIC images of such a spreading front on oxidized silicon substrates with a thin oxide layer less than 1 nm in thickness. Clearly the fluorescence intensity of the spreading front in the FLIC image is weaker than that in the epifluorescence image, although these two images were captured under the same conditions except for the substrates. Particularly, the image of the single lipid bilayer completely fades out in the FLIC image, though

illumination with higher excitation intensity makes it possible to confirm the existence of the single lipid bilayer as shown in Figure 3c. According to the principle of FLIC microscopy,20-25 optical interference between incident and reflected lights during fluorescence microscopy creates optical standing waves in front of the SiO2/Si interfaces as illustrated in Figure 3d. The decrease in fluorescence intensity specific to the FLIC images is due to the close proximity of the spreading front to the SiO2/Si interface that corresponds to the common end of and the first nodes of these optical standing waves. Figure 3e shows theoretically calculated dependence of FLIC intensity of a fluorophore over an oxidized silicon substrate on the distance from the SiO2/Si interface (see Appendix for details). The distance between these double bilayer lobes and the SiO2/Si interface should correspond to the region of the smaller FLIC intensity at the left side of the strong peak. Figure 4 shows typical spreading behaviors of double bilayer lobes on microgrooves of 0.7 µm depth and 2 µm width. Some of the fronts of membrane lobes branched at the ends of the microgrooves to avoid the microgrooves,

Figure 2. Scanning electron microscope images of microgrooves fabricated on a Si(111) surface: (a) a representative example of topographically patterned surfaces composed of alternatively arranged short and long microgrooves; (b) an enlarged image of an end of a microgroove; (c) an enlarged image of the end of the microgroove at an inclination angle of 45°. The bottom surfaces of the microgrooves are Si(111) surfaces parallel to the upper surface of the silicon substrate, although the walls of the microgrooves are not perpendicular to the upper surface. nm using a dichroic mirror unit (Olympus, U-MNV). In the case of the nanoparticle encapsulation test, lipid bilayers and nanoparticles were separately detected using a pair of other dichroic mirror units (Olympus, U-MNUA and U-MWG). All of the procedures and observations were performed in a clean room at 25 ( 1 °C.

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Figure 5. Successive FLIC images of fronts of double bilayer lobes spreading between long microgrooves at different delay times after their arrival at the ends of short microgrooves. After branching at the ends of the long microgrooves, the resulting three fronts of membrane lobes spread over the short microgrooves arranged between the long microgrooves.

Figure 4. Spreading behaviors of double bilayer lobes on a topographically patterned surface with both short and long microgrooves: (a) a light scattering image of the microgrooves without lipid membranes; (b) a FLIC image of fronts of membrane lobes spreading over the microgrooves; (c) a fluorescence intensity profile along the white broken line in the image (b).

while the others spread over the microgrooves with their fronts slightly branched. Here, we should notice that whenever the fronts of the membrane lobes spread over the microgrooves, they show a remarkable increase in their fluorescence intensity on the microgrooves. The fluorescence intensity of membrane lobes on these microgrooves is at least twice as large as that of membrane lobes on flat regions of the substrate. It is very interesting that although there would be a single lipid bilayer before the fronts of the membrane lobes, such a remarkable increase in fluorescence intensity of the single lipid bilayer was not observed in front of the membrane lobes. When fronts of the membrane lobes have already branched at the ends of such microgrooves, the fronts of the double bilayer lobes rather spread over the microgrooves than branch again to avoid the microgrooves as shown in Figure 5. A similar spreading of the double bilayer lobes was observed even if the membrane lobes spread perpendicularly to the long direction of such microgrooves as shown in Figure 6. In this case, the round fronts of membrane lobes were gradually transformed into flat fronts avoiding the microgrooves in Figure 6b, and in several tens of seconds, they suddenly recovered their rounding shapes to spread over the microgrooves (see Figure 6c,d). It is clear that the increase in fluorescence intensity of membrane lobes is observed regardless of the length and

Figure 6. Successive FLIC images of fronts of double bilayer lobes spreading across long microgrooves at different delay times after their arrival at the edges of the microgrooves: (a) a normal incident-light microscope image of the microgrooves without lipid membranes; (b-d) successive FLIC images of the membrane lobes spreading over these microgrooves.

orientation of microgrooves. However, in the case of wide microgrooves of the same depth and 7 µm width, some of double bilayer lobes do not show such an increase in their fluorescence intensity on the microgrooves as shown in Figure 7a. These results indicate that there are two different geometrical configurations for membrane lobes over microgrooves somewhat depending on aspect ratio of the microgrooves; one leads to the strong fluorescence and the other does not. As typically shown in Figure 3e, fluorescence intensity of FLIC images of fluorescently labeled membranes generally increases with the separation distance between the membranes and the SiO2/Si interface showing a rapidly

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Figure 7. Two different spreading modes of double bilayer lobes over wide microgrooves: (a) a FLIC image (60 µm × 70 µm) of membrane lobes spreading over wide microgrooves of 6.7 µm width; (b) a schematic illustration of a cross sectional view of a possible geometrical configuration of membrane lobes leading to the increase in fluorescence intensity; (c) a schematic illustration of a cross sectional view of a possible geometrical configuration of membrane lobes leading to weak fluorescence intensity.

Figure 8. Spreading behaviors of single lipid bilayer on narrow microgrooves: (a) a FLIC image of membrane lobes spreading toward a microgate that is composed of two microgrooves, the position of the microgate is indicated with the white broken lines; (b) a FLIC image captured with higher excitation intensity at the same position, a single lipid bilayer passes though the microgate without no deformation of its front line.

damped oscillation and finally levels off at a high level for sufficiently large separation distances over 0.5 µm due to a compensation of the different wavelengths in the spectrum and due to the angular spread of excitation and emission lights.20,21,23,25 Therefore, one of the most feasible geometrical configurations for the fronts that involve the increase in fluorescence intensity would be as illustrated in Figure 7b; the membrane lobes span the microgrooves, while the single lipid bilayer spreads along the surface of the microgrooves. In this geometrical configuration, the membrane lobe over the microgroove is sufficiently separated from the SiO2/Si interface to show high fluorescence intensity and the fluorescence intensity of the single lipid bilayer remains low because of its close proximity to the SiO2/Si interface. On the other hand, the absence of the increase in the fluorescence intensity of the membrane lobes on the wide microgrooves suggests that the membrane lobes fit themselves into the microgrooves as illustrated in Figure 7c. Figure 8 supports the notion that the single lipid bilayer spreads on the microgrooves as it does on smooth solid surfaces. A more direct evidence for the groove spanning of membrane lobes is shown in the next section. B. Encapsulation of Nanoparticles by the Groove Spanning of Membrane Lobes. To verify such a groovespanning behavior of membrane lobes, we have examined nanoparticle encapsulation in microgrooves. The groovespanning configuration we consider is shown in Figure 7b, where nanoparticles that are large enough not to pass through the thin water layer between the membrane lobe

Figure 9. Nanoparticle encapsulation in microgrooves: (a) a SEM image of microgrooves for the nanoparticle encapsulation test; (b) a FLIC image of double bilayer lobes spreading over the microgrooves, the fluorescence of perylene doped in lipid bilayers was detected with excitation at 360-370 nm and emission at 420-460 nm, a fluorescence intensity profile along the white broken line in the image (b) is inserted at the left side of the image; (c) a fluorescence image of the polystyrene nanoparticles observed over the same microgrooves in 1 min after Figure 9b was captured, the fluorescence of polystyrene nanoparticles labeled with nile red was detected with excitation at 515-550 nm and emission above 590 nm, a fluorescence intensity profile along the white broken line in the image (c) is inserted at the left side of the image. Encapsulation of nanoperticles was realized in the short microgroove which has been completely covered with a membrane lobe.

and the single lipid bilayer should be encapsulated in the microgroove that is completely covered with a membrane lobe. On the other hand, nanoparticles in a microgroove should leak from its open end when it is not completely covered with a membrane lobe. Fluorescent polystyrene nanoparticles with an actual diameter of 24 nm were used for the nanoparticle encapsulation test since the thickness of a thin water layer between lipid lamellae is generally less than a few nanometers in egg yolk phosphatidylcholine-water systems.32-33 Spreading of phospholipid membrane was initiated over a topographically patterned silicon substrate immersed in a buffer solution including 0.2% w/w of the nanoparticles. The patterned substrate was then transferred into a silicon rubber well that had filled with the buffer solution having no nanopartiles at 30 min after the initiation of spreading phenomena. During this solution exchange process, the surface of the substrate was not exposed to the air. After several minutes, the fluorescence of perylene doped in lipid bilayers was selectively detected with excitation at 360-370 nm and emission at 420-460 nm using the dichroic mirror unit (Olympus, U-MNUA), while the fluorescence of polystyrene nanoparticles left in the microgrooves was selectively detected with excitation at 515-550 nm and emission above 590 nm using the other dichroic mirror unit (Olympus, U-MWG). Figure 9a depicts a SEM image of the microgrooves that was used for the nanoparticle encapsulation test. As shown in Figure 9b, the increase in fluorescence intensity of the membrane lobes is in the same level for each of (32) Gulik-Krzywicki, T.; Tardieu, A.; Luzzati, V. Mol. Cryst. Liq. Cryst. 1969, 8, 285. (33) Shipley, G., G. in Biological Membrane Volume.2; Chapman, D. and Wallach, D., F., H., Ed.; Academic Press: London, 1973.

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Figure 10. Procedure of nanoparticle encapsulation and its two possible results depending on the groove length: (a, b) spreading of a membrane lobe over a short microgroove in the presence of nanoparticles; (c) nanoparticles encapsulated and remained in the short microgroove even after the solution was exchanged; (d, e) spreading of a membrane lobe over a long microgroove in the presence of nanoparticles; (f) leakage of nanoparticles in the long microgroove due to the solution exchange. Nanoparticle encapsulation is achieved for the short microgroove as the microgroove is completely covered with the membrane lobe, while nanoparticles leak from the long microgroove through the open end of the microgroove.

these microgrooves (cf. a fluorescence intensity profile across these microgrooves at the left side of the image). This indicates that the membrane lobes over these microgrooves are in the same geometrical configuration regardless of the groove length. Nevertheless, fluorescence from polystyrene nanoparticles in the short microgroove was clearly stronger than those in the long microgrooves as shown in Figure 9c. This means that even after the solution was exchanged, the nanoparticles has remained selectively in the short microgroove probably due to its complete coverage as illustrated in Figure 10a-c. In contrast, the nanoparticles in the long microgrooves might have completely leaked from their open ends as illustrated in Figure 10d-f. Therefore, this experimental result strongly supports the groove-spanning behavior of membrane lobes. Discussion A. Mechanism for the Groove Spanning of Membrane Lobes. The increase in fluorescence intensity of membrane lobes starts immediately after the arrival of the front of the membrane lobes at the edges of the microgrooves regardless of the length and orientation of microgrooves as shown in Figures 5 and 6. This means that at the edge of the microgrooves, the fronts of the membrane lobes rather detach from the surface of the underlying single lipid bilayer as illustrated in Figure 11a than spread together with the single lipid bilayer as illustrated in Figure 11b despite the attractive interaction between them. Such a local spreading behavior of membrane lobes at the sharp edges would be one of the most important phenomena leading to the groove spanning of membrane lobes. It is reasonable to anticipate that this local spreading behavior of membrane lobes is not due to the total morphology of the microgrooves but due to the local surface topography, particularly due to the sharp edge, of microgrooves. Therefore, we discuss here a balance between adhesion and bending energies of membrane lobes

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Figure 11. Schematic illustrations of geometrical configurations of membrane lobes: (a, b) cross sectional drawings of two possible geometrical configurations of fronts of membrane lobes at the edges of microgrooves; (c, d) schematic illustrations of top views of two possible geometrical configurations of membrane lobes over microgrooves, the broken lines indicate positions of microgrooves; (e) a cross sectional drawing of a possible geometrical configuration of lipid bilayer at the fringe of membrane lobes.

at the sharp edges for a better understanding of the local spreading behaviors of membrane lobes. The bending energy per unit area WB of a sheet of lipid bilayer on the sharp edge is approximately given by WB ) κ/(2R2), where κ is the bending rigidity of lipid bilayer and R is the curvature radius of the edges.3-5 In our experimental system, WB would be about 2 × 10-5 J m-2 since κ is believed to be 1 × 10-19 J4,5,34 and R was estimated to be about 5 × 10-8 m according to our SEM observations of the patterned substrates. This implies that the bending energy per unit area WBM for a double bilayer lobe consisting of two sheets of lipid bilayer would be around 2WB (≈4 × 10-5 J m-2). On the other hand, the adhesion energy between a membrane lobe and its underlying single lipid bilayer per unit area WAM would be about 1 × 10-5 J m-2 in the presence of 100 mM NaCl, assuming that WAM is equal to the adhesion energy between two lipid bilayers measured by using giant liposomes.35,36 Accordingly, it is reasonable to anticipate that in our system WBM is larger than WAM. In such a situation, the detachment of the membrane lobes from the curved surface of the single lipid bilayer would be energetically favorable due to the bending rigidity of the membrane lobes. Here we should notice that even if the bending front is more energetically favorable than the straight front owing to the adhesion energy on the flat surface of length L as illustrated in Figure 11b, the bending rigidity of the membrane lobes can still act at least as an energy barrier against the transformation of the straight front into the bending front. The coexistence of the two geometrical configurations of membrane lobes shown in Figure 7a may indicate that the groove-spanning configuration is energetically metastable over the wide microgrooves. (34) Sackmann, E. FEBS Lett. 1994, 346, 3. (35) Evans, E.; Metcalfe, M. Biophys. J. 1984, 46, 423. (36) Needham, D. Methods Enzymol. 1993, 220, 111.

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For the single lipid bilayer, its bending energy per unit area WBS would be given by WB ≈ 2 × 10-5 J m-2. On the other hand, there have been few studies on direct measurements of the adhesion energy per unit area WAS between a lipid bilayer and the surface of glass or oxidized silicon substrates probably because the strength of the adhesion is too large to be measured with conventional methods. Recently, Nissen et al. have studied spreading kinetics of a single lipid bilayer advancing from a lipid source over glass or oxidized silicon substrates, and they have estimated the spreading power S (i.e., the gain in free energy per unit area by the formation of membranesolid contact) at 10-4 J m-2.26 This implies that WAS ≈ 10-4 J m-2 and the same order of magnitude of WAS can be obtained also on the basis of theoretical estimation of van der Waals interactions between lipid bilayer and silica.26,27 Therefore WBS is clearly smaller than WAS, and thus it seems difficult for the single lipid bilayer to detach from the surface of the oxidized silicon substrate to span the microgrooves as the membrane lobes do. In other words, the presence of the underlying single lipid bilayer allows the membrane lobes to span the microgrooves by preventing the direct and strong adhesion of membrane lobes to the bare surface of oxidized silicon substrates. Finally, we will describe another factor which may play an important role in the local spreading behavior of membrane lobes. As shown in the images of membrane lobes, all of the membrane lobes have round fronts, indicating the presence of a considerable amount of an excess free energy per unit length along the fringe of a membrane lobe; we refer to the excess free energy per unit length along the fringe as the fringe energy. If there was no fringe energy, branching of membrane lobes as illustrated in Figure 11c would be more energetically favorable than the groove spanning of membrane lobes as schematically illustrated in Figure 11d, since the branching allows the membrane lobes to maximize their adhesion area with the single lipid bilayer. But in reality, most of the membrane lobes span the microgrooves to restrain the increasing of their fringe length. Therefore, the fringe energy would also play an indispensable role in the groove-spanning behavior of membrane lobes. Since the lipid bilayer composing the fringe of membrane lobes are considerably curved as illustrated in Figure 1, most part of the fringe energy might be derived from the bending energy of the lipid bilayer. As a matter of fact, the fringe of membrane lobes observed on oxidized silicon wafer is clearly brighter than its inside as depicted in Figure 3b, although the membrane lobes observed on silica glass substrate did not have such a bright fringe (see Figure 3a). Therefore, a more relevant configuration of the fringe of membrane lobes would be as illustrated in Figure 11e; the membrane lobes are bulging along their fringes due to the bending rigidity of the lipid bilayer. A similar bulging along fringes has already been reported for giant vesicles spreading on moderately adhesive surfaces.8 Recently a pore-spanning lipid bilayer has been formed on mesoporous alumina surfaces via vesicle fusion onto the nanometer-scale pores.37,38 In this case, the pore spanning highly relies on the selective adhesion of the positively charged lipid vesicles to the upper surface of the mesoporous alumina owing to the chemical modification of the upper surface with a negatively charged monolayer. Unlike such a pore spanning, the groove

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spanning of membrane lobes occurs on chemically uniform surfaces of the single lipid bilayer spreading along topographically patterned surfaces. Therefore, our results emphasize the importance of surface topography for spanning concave structures with lipid membranes. In this section we discussed the mechanism for the groove spanning of membrane lobes mainly by considering the balance between adhesion and bending energy of lipid membranes, although spreading kinetics of membrane lobes and other factors should be taken into account in order to reveal the phenomenon thoroughly. Since the competition between bending and adhesion of lipid bilayers can occur on a variety of nonsmooth surfaces, the groovespanning behavior of membrane lobes would be one of the most essential spreading behaviors of lipid membranes on topographically patterned surfaces. To establish the basis of spreading behaviors of lipid membranes on topographically patterned surfaces, further studies utilizing other types of bilayer structures such as giant vesicles and long tubules would be needed. B. Future Application Potential of the Groove Spanning of Membrane Lobes. From a practical point of view, immobilization of lipid bilayers on solid surfaces is of great importance for biofunctionalization of inorganic solid surfaces. However, direct attachment of the lipid bilayers onto solid surfaces is known to reduce the functionality of membrane-confined compounds such as receptor proteins and ion channels due to their strong interactions with the solid surfaces.37,38 Since the membrane lobes that span the microgrooves are sufficiently separated from the solid surface due to their specific geometrical configuration, they would be of great advantage to technological as well as biological applications as novel supported membranes for incorporation of large transmembrane proteins. In addition, the nanoparticle encapsulation in microgrooves suggests that the groovespanning membrane lobes are instrumental in encapsulation of biomacromolecules in the range of several tens of nanometers such as polynucleotides and relatively large water-soluble proteins. In contrast to the completely closed microspaces at the insides of liposomes, the microgrooves covered with a membrane lobe have thin water layers which can serve as a diffusion channel between the interior and exterior of microgrooves. Thus, such microgrooves can principally be used as a novel microreactor for continuous enzymatic reactions of encapsulated enzymes whose substrates are small enough to enter the microgrooves via the diffusion channel. Concluding Remarks We have revealed a groove-spanning behavior of membrane lobes on micrometer-scaled grooves with sharp edges. Indeed nanoparticles could be encapsulated in microgrooves that were completely covered with the membrane lobes. Our results emphasize the significance of surface topography, particularly of sharp edges, in spontaneous spreading of phospholipid membranes on micropatterned surfaces. The spreading behavior of the membrane lobes was discussed consistently in terms of the balance between adhesion and bending energies of lipid bilayers. From a practical point of view, the groove spanning of membrane lobes can be used for preparing a functional geometry of substrate-supported membranes for biofunctionalization of inorganic solid surfaces. Appendix

(37) Hennesthal, C.; Steinem, C. J. Am. Chem. Soc. 2000, 122, 8085. (38) Hennesthal, C.; Drexler, J.; Steinem, C. Chemphyschem 2002, 10, 885.

We consider FLIC intensity of a fluorophore above an oxidized silicon substrate in water following the theoretical

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Suzuki and Masuhara

framework by Lambacher and Fromherz.20,21,25 Generally, FLIC intensity, IFLIC, is proportional to the product of the excitation probability per unit time, Pex, and emission probability per unit time Pem

IFLIC ∝ PexPem

(1)

In our experimental system, the polydispersity in wavelengths of emission light (i.e., perylene spectrum from λ1 (455 nm) to λ2 (550 nm), selected with a dichroic mirror unit (U-MNV)) and the angular spread of excitation and emission lights cannot be negligible. On the other hand, we assume the excitation light to be monochromatic light, since a narrow emission line at 404.7 nm from a highpressure mercury lamp was used as a light source. Under these conditions, the excitation and emission probabilities are given by

Pex ∝

∫0

Θ

Uex(λex,Θ1in)

sin Θ1in cos Θ1in

Pem ∝

[

∫λ ∫0 λ2 1

Θmax

Uem(λem,Θ1

out

)

sin Θ1out cos Θ1out

Uex ) sin2 Θex|1 + rTE exp(iφin)|2 + sin2 Θex cos2 Θ1in|1 + rTM exp(iφin)|2 + 2 cos2 Θex sin2 Θ1in |1 + rTM exp(iφin)|2 (4) Uem ) sin2 Θem|1 + rTE exp(iφout)|2 + sin2 Θem cos2 Θ1out|1 - rTM exp(iφout)|2 + 2 cos2 Θem sin2 Θ1out|1 + rTM exp(iφout)|2 (5)

dΘ1in

(2) φin,out )

]

out

dΘ1

dichroic mirror unit, Uex(λex,Θ1in) is a function of both the wavelength, λex, of the excitation light and the angle, Θ1in, of incidence and reflection of excitation light, Uem(λem,Θ1out) is a function of both the wavelength, λem, of the emitted light and the angle, Θ1out, of direct and reflected components of emission light from the fluorophore. Neglecting optical path length in the thin oxide layer, the functions Uex(λex,Θ1in) and Uem(λem,Θ1out) are approximately given by

Bem(λem) dλem

(3)

Here, Θmax is given by the numerical aperture (NA ) nw sin Θmax ) 0.85) of the microscope, nw is the refraction index of water (1.34), Bem(λem) is the distribution of emission wavelengths accounting for both the perylene spectrum and the selection of wavelengths with the

4πnwz cos Θ1out λex,em

(6)

where z is the distance of the fluorophore from the reflective SiO2/Si interface and rTE and rTM are the Fresnel reflection coefficients at the interface for light polarized perpendicular and parallel to the plane of incidence, respectively (-rTE ≈ rTM ≈ 0.46).25 We have calculated the FLIC intensity curve shown in Figure 3e under a simplifying assumption that the transition dipole moments of the fluorophore are parallel to the SiO2/Si interface (Θex, Θem ) π/2). LA050157A