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Supported Lipid Bilayer Self-Spreading on a Nanostructured Silicon Surface Kazuaki Furukawa,* Koji Sumitomo, Hiroshi Nakashima, Yoshiaki Kashimura, and Keiichi Torimitsu NTT Basic Research Laboratories, NTT Corporation, 3-1 Morinosato Wakamiya, Atsugi, Kanagawa, Japan 243-0198 ReceiVed October 4, 2006. In Final Form: December 1, 2006 We report on the self-spreading behavior of a supported lipid bilayer (SLB) on a silicon surface with various 100 nm nanostructures. SLBs have been successfully grown from a small spot of a lipid molecule source both on a flat surface and uneven surfaces with 100 nm up-and-down nanostructures. After an hour, the self-spreading SLB forms a large circle or an ellipse depending on the nanostructure pattern. The results are explained by a model that shows that a single-layer SLB grows along the nanostructured surfaces. The model is further supported by a quantitative analyses of our data. We also discuss the stability of the SLB on nanostructured surfaces in terms of the balance between its bending and adhesion energies.
Introduction A lipid bilayer is a basic component of a cell membrane. Recently, supported lipid bilayers (SLB) have attracted considerable attention1,2 both in terms of general interest as a fluidic membrane supported on a solid surface3-6 and for applications such as biosensors.7-12 Lipid bilayers are hosts for many transmembrane proteins,13-15 and SLBs are used to immobilize such proteins on solid surfaces. In addition to these biocompatible characteristics, SLBs are good electrical insulators.16,17 This makes the SLB a key material with regard to realizing novel devices that utilize biomolecule functions together with semiconductor electric devices. Some effective methods have been reported for obtaining a wide-area SLB on solid surfaces, namely, vesicle rapture18 and self-spreading.19 These techniques have been examined on surfaces with hydrophilic/hydrophobic patterns.5 When employed * To whom correspondence should be addressed. E-mail: furukawa@ nttbrl.jp. (1) Sackmann, E. Science 1996, 271, 43-48. (2) Kasemo, B. Surf. Sci. 2002, 500, 656-677. (3) Mou, J.; Yang, J.; Shao, Z. Biochemistry 1994, 33, 4439-4443. (4) Giocondi, M.-C.; Le Grimellec, C. Biophys. J. 2004, 86, 2218-2230. (5) Groves, J. T.; Boxer, S. G. Acc. Chem. Res. 2002, 35, 149-157. (6) Richter, R. P.; Be´rat, R.; Brisson, A. R. Langmuir 2006, 22, 3497-3505. (7) Cornell, B. A.; Braach-Maksvytis, V. L. B.; King, L. G.; Osman, P. D. J.; Raguse, B.; Wieczorek, L.; Pace, R. J. Nature 1997, 387, 580-583. (8) Fertig, N.; Tilke, A.; Blick, R. H.; Kotthaus, J. P.; Behrends, J. C.; ten Bruggencate, G. Appl. Phys. Lett. 2000, 77, 1218-1220. (9) Fertig, N.; Meyer, Ch.; Blick, R. H.; Trautmann, Ch.; Behrends, J. C. Phys. ReV. E 2001, 64, 040901. (10) Cheng, Y. L.; Bushby, R. J.; Evans, S. D.; Knowles, P. F.; Miles, R. E.; Ogier, S. D. Langmuir 2001, 17, 1240-1242. (11) Grane´li, A.; Rydstro¨m, J.; Kasemo, B.; Ho¨o¨k, F. Langmuir 2003, 19, 842-850. (12) Suzuki, H.; Tabata, K.; Kato-Yamada, Y.; Noji, H.; Takeuchi, S. Lab Chip 2004, 4, 502-505. (13) Salafsky, J.; Groves, J. T.; Boxer, S. G. Biochemistry 1996, 35, 1477314781. (14) Ide, T.; Takeuchi, Y.; Aoki, T.; Yanagida, T. Jpn. J. Physiol. 2002, 52, 429-434. (15) Glasma¨star, K.; Larsson, C.; Ho¨o¨k, F.; Kasemo, B. J. Colloid Interface Sci. 2002, 246, 40-47. (16) Wiegand, G.; Arribas-Layton, N.; Hillebrandt, H.; Sackmann, E.; Wagner, P. J. Phys. Chem. B 2002, 106, 4245-4254. (17) Rahman, M. M.; Nonogaki, Y.; Tero, R.; Kim, Y.-H.; Uno, H.; Zhang, Z.-L.; Yano, T.; Aoyama, M.; Sasaki, R.; Nagai, H.; Yoshida, M.; Urisu, T. Jpn. J. Appl. Phys. 2005, 44, L1207-L1210. (18) Groves, J. T.; Ulman, N.; Cremer, P. S.; Boxer, S. G. Langmuir 1998, 14, 3347-3350. (19) Ra¨dler, J.; Strey, H.; Sackmann, E. Langmuir 1995, 11, 4539-4548.
on such surfaces, the vesicle rapture technique results in an SLBpatterned surface because an SLB is adsorbed only on hydrophilic surfaces. The self-spreading also occurs only on hydrophilic surfaces;20-22 therefore, the self-spreading position and direction can be controlled using the hydrophobic patterns fabricated on a hydrophilic surface. This result has led us to propose a new type of microchannel device that employs a self-spreading SLB as a molecule-carrying medium.20 By contrast, SLB formation on structured surfaces fabricated with an identical material has yet to be investigated, although it appears to be interesting in terms of a basic understanding of self-spreading SLBs on topological surfaces. An effective way of covering a large and uneven solid surface with an SLB would also be very useful for further studies that combine micro- and nanoelectronic devices with biological research. For coating surfaces using SLBs, it has been examined on surfaces with a variety of roughenesses.6,23,24 With respect to semiconductor devices, SLBs are also expected to be used as passivation layers. For instance, if we wish to embed a semiconductor device, which usually has uneven surface structures, in a human body, then it may be advantageous to cover its surfaces with a biocompatible and highly insulating material such as a lipid bilayer. In this letter, we investigate how an SLB self-spreads on nanostructured surfaces. For this purpose, the nanostructures should have dimensions comparable to an SLB thickness of 4 nm. We examined the self-spreading of SLBs on uneven surfaces with 100 nm level nanostructures. Although the scale still has a gap of 4 nm, the nanostructures on this scale are available by X-ray photolithography in a controllable manner. We approached the subject by using confocal laser scanning optical microscopy observations. The technique clearly visualizes single SLBs containing a small number of dye-conjugated lipid molecules. Although optical microscopy does not have a resolution of 100 nm, namely, the scale of our nanostructures, we overcame this difficulty by growing a sufficiently large self-spreading SLB (20) Furukawa, K.; Nakashima, H.; Kashimura, Y.; Torimitsu, K. Lab Chip 2006, 6, 1001-1006. (21) Nissen, J.; Jacobs, K.; Ra¨dler, J. O. Phys. ReV. Lett. 2001, 86, 19041907. (22) Nabika, H.; Sasaki, A.; Takimoto, B.; Sawai, Y.; He, S.; Murakoshi, K. J. Am. Chem. Soc. 2005, 127, 16786-16787. (23) Cremer, P. S.; Boxer, S. G. J. Phys. Chem. B 1999, 103, 2554-2559. (24) Weng, K. C.; Stålgren, J. J. R.; Duval, D. J.; Risbud, S. H.; Frank, C. W. Langmuir 2004, 20, 7232-7239.
10.1021/la062911d CCC: $37.00 © 2007 American Chemical Society Published on Web 12/20/2006
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Figure 1. (Top) schematic illustrations of surface structure designs S1, S2, and S3. The black area is the ground level, and the white area is the +100 nm level. (Bottom) SEM images of S1, S2, and S3 used in the present study.
and analyzing time-lapse measurements. We show the observation results of the formation of large, stable SLBs on a variety of nanostructured surfaces. We propose and verify a self-spreading model of an SLB on such nanostructured surfaces on the basis of those observations. Experimental Section L-R-Phosphatidylcholine (L-R-PC) extracted from egg yolk was purchased from Sigma-Aldrich. Fluorescein-conjugated lipid (fluorescein-DHPE) was purchased from Invitrogen. Chloroform and sodium chloride were purchased from Kanto Chemical Co. Inc. A 1 mol/L Tris-HCl buffer (pH 7.6) was purchased from Nacalai Tesque. Deionized water (Millipore, >18 MΩ) was used throughout the work. Nanostructured surfaces were fabricated on a Si wafer by using synchrotron orbital radiation X-ray photolithography followed by a dry etching process. Substrate patterns S1, S2, and S3 are shown in Figure 1. S1 and S2 have a grating structure composed of lines that are 100 nm wide and 100 nm high. The lines are fabricated at intervals of 100 nm for S1 and 400 nm for S2, the sectional views of which are shown in Figure 1. S3 has 100 × 100 × 100 nm3 projections in every 500 × 500 nm2 square with 2-D tetragonal symmetry. The substrates were immersed in a 1:4 H2O2/H2SO4 solution for 5 min to obtain hydrophilic surfaces before use. A chloroform solution of fluorescein-DHPE was mixed with L-R-PC to prepare L-R-PC solutions with 5 mol % fluoresceinDHPE. The chloroform was evaporated with a nitrogen gas stream and dried in vacuum overnight to yield a sticky solid of lipid mixtures. A small amount of the mixture was transferred to the substrate using the tip of a glass capillary, and it adhered to the substrate surface. A self-spreading lipid bilayer membrane was developed by immersing the substrate gently in a buffer solution of 100 mM NaCl and 10 mM Tris-HCl (pH 7.6). The dye molecules were used to visualize the self-spreading behavior of the SLB with a confocal laser scanning microscope. An Olympus BX51-FV300 confocal laser scanning microscope with a 488 nm excitation laser was used for all observations. A 505-525 nm filter was used to detect the fluorescein fluorescence.
Because the samples have to be immersed in buffer solution throughout the observations, they were observed via a buffer solution that covered the surface for low-magnification (×10 objective lens) observations, and the buffer solution was employed between the sample and the objective lens (PlanApo 40×WLSM, Olympus) for high-magnification (×40) observations. All observations were carried out at room temperature.
Results and Discussion Time Evolution of SLB Growth. We first look at the appearance of the self-spreading SLB that grows from an initial point of a lipid molecule source to the final macroscopic structure in Figure 2. Figure 2A shows the initial spot of a lipid source adhering to S1 under atmospheric conditions. The lipid source usually has an undefined shape with dimensions of 100 µm. Figure 2B shows the same spot after it had been immersed in a buffer solution. In the initial spreading stage (from Figure 2A to B), the lipid spot seems to be spread more easily in a direction parallel rather than perpendicular to the gratings, which is affected by capillary action on the surface. The self-spreading of a singlelayer SLB has already started around the bright spot, as indicated by the arrow in Figure 2B. The initial shape of the self-spreading SLB is disordered, because it is affected by the initial shape of the source. However, as time passes the SLB adopts a circular or elliptical shape. This is most likely due to the stabilization of the membrane structure induced by intermolecular interactions. Parts C and D of Figure 2, respectively, are images of the front edge of an SLB that is self-spreading parallel and perpendicular to the grating direction. The edge appears to be serrated in Figure 2C and smooth in Figure 2D. After 4 h of self-spreading, a very large SLB has been grown as shown in Figure 2E. The outermost shape of the resulting SLB was an almost perfect ellipse in the center of which there remained a bright area containing an excess quantity of lipid molecules. This provides a source of molecules for further growth of the self-spreading SLB. To stop further growth, we transferred the sample into a large amount of buffer
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Figure 3. Typical macroscopic structures grown on S1, S2, and S3 and a flat surface (ref) as a reference. Scale bars: 1 mm. Doubleheaded arrows indicate the parallel direction of the S1 and S2 gratings.
Figure 2. Initial shape of the source of the lipid molecules (A) in the air on S1 and (B) just after immersion in the buffer solution. The edge of SLB self-spreading (C) in a parallel direction to the gratings and (D) in a perpendicular direction. (E) Self-spreading SLB after 4 h. There is excess lipid in the center of the SLB that exhibits significant luminescence. (F) The same SLB as in E after gentle washing for 2 h. The double-headed arrows indicate the parallel direction of the grating of S1. Scale bars: (A, B) 100 µm, (C, D) 20 µm for, and (E, F) 1 mm.
solution and shook it gently for about 2 h. Figure 2F shows the same SLB as that in Figure 2E after the washing process. Although most of the excess lipid in the center of the SLB is now removed, a single SLB remains with an area on the order of millimeters squared. The fluorescence intensity in the single-layer region of the SLB remains largely unchanged after the washing process, which supports the idea that the single-layer SLB is growing on nanostructured surfaces. It also indicates that the SLB supported directly on the surface is stable and is not removed by the washing process whereas the lipid molecules on the SLB are readily removed. Difference in SLB Shape by Means of Surface Nanostructures. Figure 3 shows self-spreading single SLBs on S1, S2, and S3. We also prepared an SLB in the same way on a flat surface without nanostructures as a reference. The SLBs were grown for 4 to 5 h and washed by using the above-mentioned process, thus all images are of single-layer SLBs. S1 and S2 are elliptical, and S3 and the reference are circular. Figures 2 and 3 show that the SLB grows not only in the direction parallel to the gratings but also in the perpendicular direction, although in the latter direction the SLB has to overcome the up-and-down structures in order to grow. This is in contrast to the fact that the self-spreading is controlled by the surface pattern formed by different materials such as a photoresist pattern on SiO2, where self-spreading occurs only on the hydrophilic
Figure 4. Growth model of a self-spreading SLB in a direction perpendicular to the grating on S1.
SiO2 surfaces.20-22 Although it is reported that self-spreading proceeds over a slope on the surface,23 the present results show for the first time that the self-spreading SLB grows over 100nm-scale periodic structures. We also observed a reproducible difference in shape. The structures must be related to the surface nanostructure. Because an elliptical or circular shape was formed on all of the substrates and the major and minor axes corresponded to directions parallel and perpendicular to the gratings, respectively, the self-spreading behavior can be analyzed on the basis of the separation of two variables. In this regard, the circle observed as a reference surface can be readily understood because it has no structures and is isotropic in all directions within the surface. The S3 surface is also isotropic as a result of its tetragonal symmetry nanostructures, and it forms a circle. However, we have to discuss self-spreading on S1 and S2 by considering an anisotropic spreading model. The spreading parallel to the grating direction should be the same as that of a flat surface because the surface is flat in that direction. Thus, the self-spreading behavior perpendicular to the grating direction should be considered. The growth model that we propose is shown schematically in Figure 4. The model assumes that the single-layer self-spreading SLB grows along the surface. The front-edge velocity is assumed to be the same as that in the parallel direction, independent of whether the SLB grows up, down, or laterally at the local time. According to the model, optical microscope observations suggest that growth in the perpendicular direction would require a longer time than that in the parallel direction. This is because the observations show only the change in the macroscopic shape of the SLB and do not recognize events on a 100 nm scale. The macroscopic shape is thus affected by the difference in the distances between the flat surface (the parallel direction) and the
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Figure 5. Double logarithmic plot of the front-edge velocity on S1 in the (A) parallel and (B) perpendicular directions as a function of time. The straight lines have a slope of -1/2.
up-and-down surface (the perpendicular direction). This ratio becomes 2:1 for S1 as shown in Figure 4 and 7:5 for S2 or 2.0 for S1 and 1.4 for S2. From the experimental results, the major/ minor axis ratios are 1.9 and 1.5, respectively, for the ellipses in Figure 3.25 The values are in good agreement with the ratio of 2.0 for the S1 surface and 1.4 for the S2 surface. The validity of our model is further supported by comparing our data with analyses of self-spreading SLBs on flat surfaces.19 Figure 5 shows the time dependence of the front-edge velocities spreading in directions parallel and perpendicular to the grating direction measured during the initial stage of the self-spreading.26 Both sets of data can be fit by straight lines with a slope of -1/2. The behavior is characteristic of a self-spreading SLB on a flat surface.19 The velocities observed in Figure 5, 0.1-0.01 µm s-1, are relatively slower than those reported on a smooth surface, 1-0.1 µm s-1,19 which indicates that the surfaces of the substrates used in the present study are rough rather than smooth, probably as a result of the nanofabrication processes. This is consistent with a previous report stating that the front-edge velocity is an order of magnitude smaller on a rough surface than on an atomically flat smooth surface such as mica.19 It is noteworthy, however, that the periodic edges of the nanostructures had no significant effect on the front-edge velocities. We also analyzed the front-edge velocities after a sufficient period of self-spreading. We performed time-lapse observations at positions in Figure 2C and D and obtained velocities of 2.7 and 1.3 µm min-1, respectively.27 The ratio of the velocities is 2.1. The velocities on S2 were also examined, and values of 1.1 and 0.7 µm min-1 were obtained,28 giving a ratio of 1.5. These results are in good agreement with the surface distance ratios of 2.0 for S1 and 1.4 for S2. Another feature of our observations is the serrated pattern observed in Figure 2C. Similar roughening of the front-edge shape has been observed for a self-spreading SLB on a rough hydrated surface such as glass.19 The roughening can be explained (25) The average ratios and standard deviations are 1.82 ( 0.15 (1.6-2.0, six samples) for S1 and 1.45 ( 0.13 (1.3-1.6, four samples) for S2. All samples give 1.0 for S3 (four samples). (26) Movies depicting the self-spreading behavior at the initial stage are supplied as supporting information. (27) The velocities are average values for 40 min. They are calculated from the alternate time-lapse observations (each of them is for 20 min) in two rectangular directions. (28) The difference in average velocities on S1 and S2 is due to the time when those velocities were observed. This is because the front-edge velocity is not constant over a long period. The velocity decreases as the time develops and the distance between the edge and the lipid source increases. This is also true for the velocity in the parallel direction.
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by the existence of pinning centers, namely, local sites with strong membrane-surface interactions. The serrated pattern that we observed is also explained by this idea. However, the edge was very smooth for the SLB spreading in the perpendicular direction as shown in Figure 2D. If the nanostructure edges work as pinning centers, then the front-edge shape perpendicular to the grating direction should be more serrated. However, our observations are the opposite, suggesting that the nanostructure edges do not work as pinning centers. The reason for the edge being so smooth is not perfectly understood at this moment. It is possible that the serrated pattern could be relieved by the topological effect of the up-and-down surface because the edge pattern on the wall surfaces (i.e., the surfaces vertical to the substrate surface) is observed to be flat in the top view. As discussed above, our model qualitatively explains the present results and corresponds with them quantitatively. Thus, we believe that our observations confirm the validity of our model. According to the model, the SLB on S3 most likely covers the projections. A question arises as to whether the SLB becomes unstable when it covers the edge of nanostructures. The edge is not very sharp as shown in Figure 1 and has a somewhat rounded shape on the molecular scale. Because the lipid bilayer is flexible and fluidic even when it is supported on a surface, the SLBs remain stable on such nanostructured surfaces and continue to grow over them without any significant hindrances. The bending energy per unit area, Eb, at the nanostructure edge could be estimated by Eb ) kb/(2R2), where kb is the bending modulus and R is the radius of curvature. A typical value of kb for L-R-PC is ∼10-20 J,29 and R is estimated to be 20 nm from Figure 1. Under these given parameters, Eb becomes ∼10-5 J m-2. The value should be compared with the adhesion energy of an SLB, which is the gain in free energy caused by forming an SLB. The adhesion energy has been estimated to be 10-4 J m-2 per unit area.30 Thus, the adhesion energy is larger than the bending energy Eb, which is consistent with the experimental results showing the stable formation of SLB on the present nanostructured surfaces. A comparison of the images in Figure 2E and F shows that there is no collapse or damage in the area where the single SLB is grown after washing. The estimated critical radius for L-R-PC of 11 nm,29 which is less than our estimated edge radius, also agrees with our results. This means, however, that the SLB would not remain stable when we use a surface with nanostructures possessing shaper edges or a lipid with a larger critical radius than that of L-R-PC. The former is a technical problem related to nanostructure fabrication, and the latter is a problem related to membrane stiffness. Therefore, it would be possible to control self-spreading with nanostructures fabricated by identical materials by using structures with sharper edges or by using different lipids that can form stiffer membranes.
Conclusions We observed the self-spreading behavior of a supported lipid bilayer on a silicon surface with a variety of 100 nm nanostructures. We found that self-spreading occurred successfully on these nanostructured surfaces to form a very large homogeneous single-layer SLB with an area on the order of millimeters squared. We also found that the macroscopic shape of SLB was circular or elliptical, on the basis of which we proposed and (29) Israelachvili, J. N. Intermolecular and Surface Forces, 2nd ed.; Academic Press: London, 1992; Chapter 17. (30) Nissen, J.; Gritsch, S.; Wiegand, G.; Ra¨dler, J. O. Eur. Phys. J. B 1999, 10, 335-344.
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confirmed the validity of a growth model. Although optical microscopy observations do not offer a resolution of 100 nm, our observations of these macroscopic structures made it possible for the first time to discuss self-spreading behavior on a nanostructured surface. Our data were collected by using sufficiently large self-spreading SLBs grown on the substrates, which makes our discussion solid and reliable. The formation
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of a single layer is further supported by quantitative analyses of the data. Supporting Information Available: AVI files of time-lapse observations of self-spreading behavior at the initial stage of spreading. This material is available free of charge via the Internet at http://pubs.acs.org. LA062911D
