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Atomic Force Microscopy Deposition of Poly-L-lysine Structures onto Lipid Bilayers Supported by Mica Henning Mueller,† Hans -Ju¨rgen Butt,*,‡ and Ernst Bamberg† Max-Planck-Institut fu¨ r Biophysik, Kennedyallee 70, D-60596 Frankfurt (Main), Germany, and Institut fu¨ r physikalische Chemie II, Universita¨ tsGH Siegen, D-57068 Siegen, Germany Received May 18, 2000. In Final Form: July 31, 2000 Creating patterns of microstructured arrays of functional material is of interest in many areas of current surface science and microsensing techniques. We have used the atomic force microscope to create micrometersized structures of poly-L-lysine on lipid bilayers. The entire process was performed in aqueous solution. The created structures were 0.8 ( 0.1 nm high and as small as 100 nm in diameter. Our results suggest that at least part of the poly-L-lysine molecules in these structures are anchored to the mica surface below the lipid bilayer. Thus, the presented method might also be used to design structures of poly- L-lysine and other biomolecules on mica.
Introduction Poly-L-lysine is commonly used as a coating for anorganic surfaces in the biosciences. It offers the possibility to attach proteins to the surface for further study1 and to enhance the adhesion of biomolecules and cells.2 A roughly homogeneous poly-L-lysine coated surface is easily prepared by unspecific adsorption from solution. Much more difficult is the preparation of patterns of functionalized areas on nonfunctionalized surfaces. Such structured surfaces are of considerable interest in (bio-)chemical sensing3 and the fabrication of micro- or nanoscale arrays. They offer the prospect of building functional supramolecular devices on the mesoscopic scale. The ability to combine “handmade” structures with the specific interactions and self-assembling properties of biological systems is especially intriguing. A requirement for functional biological systems is that the structures are stable in aqueous solution. Various techniques exist for the engineering of microscopic surface structures, such as microcontact printing,4,5 photolithography,6 and, recently, “dip-pen” lithography.7 * To whom correspondence should be addressed. (1) Jordan, C. E.; Frey, B. L.; Kornguth, S.; Corn, R. M. Langmuir 1994, 10, 3642. Subramanian A.; Kennel, S. J.; Oden, P. I.; Jacobson, K. B.; Woodward, J. Doktycz, M. J. Enzyme Microb. Technol. 1999, 24 (1-2), 26. Lu H. C.; Chen, H. M.; Lin, Y. S.; Lin, J. W. Biotechnol. Prog. 2000, 16 (1), 116. (2) James, C. D.; Davis, R.; Meyer, M.; Turner, A.; Turner, S.; Withers, G.; Kam, L.; Banker, G.; Craighead, H.; Isaacson, M.; Turner, J.; Shain, W. IEEE Transactions on Biomedical Engineering 2000, 47(1), 17. Mizutani F.; Sato, Y.; Yabuki, S.; Sawaguchi, T.; Iijima, S. Electrochim. Acta 1999, 44(21-22), 3833. (3) Dickert F. L.; Sikorski, R. Mater. Sci. Eng., C 1999, 10 (1-2), 39. Lang H. P.; Baller, M. K.; Berger, R.; Gerber, C.; Gimzewski, J. K.; Battiston, F. M.; Fornaro, P.; Ramseyer, J. P.; Meyer, E.; Gu¨ntherodt, H. J. Anal. Chim. Acta 1999, 393 (1-3), 59. (4) Wang, D. W.; Thomas, S. G.; Wang, K. L.; Xia, Y.; Whitesides, G. M. Appl. Phys. Lett. 1997, 70 1593. Jackman, R. J.; Wilbur, J. L.; Whitesides, G. M. Science 1995, 269, 664. Xia, Y.; Rogers, J. A.; Paul, K. E.; Whitesides, G. M. Chem. Rev. 1999, 99, 1823. Kumar, A.; Biebuyk, H. A.; Whitesides, G. M. Langmuir 1994, 10, 1498. (5) Xia, Y.; Whitesides, G. M. Langmuir 1997, 13, 2059. Evans, S. D.; Flynn, T. M.; Ulman A. Langmuir 1995, 11, 3811. (6) Wang, R.; Hashimoto, K.; Fujishima, A.; Chikuni, M.; Kojima, E.; Kitamura, A.; Shimohigoshi, M.; Watanabe, T. Nature 1997, 388, 431. Calvert, J. M. J. Vac. Sci. Technol., B 1993, 11 (6), 2155. Prucker, O.; Habicht, J.; Park, I.-J.; Ru¨he, J. Mater. Sci. Eng. C 1999, 8 -9, 291. (7) Hong, S.; Zhu, J.; Mirkin, C. A. Science 1999, 286, 523. Piner, R. D.; Zhu, J.; Xu, F.; Hong, S.; Mirkin, C. A. “Dip-Pen” Nanolithography. Science 1999, 283, 661. Jaschke, M.; Butt, H.-J. Langmuir 1995, 11, 1061.
In all these techniques, patterning is done in air and the patterned surfaces can then be used for biofunctionalization in solution by exploiting their chemical heterogeneity if a suitable biological adsorbant is available. If the goal is the direct and deliberate deposition of biological material, it is advantageous to perform the entire process in aqueous solution. The atomic force microscope (AFM) has been used for creating structures in lipid bilayers before. There, the AFM tip was used to remove a designated area of a supported lipid bilayer in the condensed phase by simply scraping out the lipids8,9 or by making them accessible to enzymes which would decompose them chemically.10 We report on the deposition of micrometer scale patterns of poly-L-lysine onto lipid bilayers in aqueous solution with an AFM. One advantage is that bilayers in the fluid phase are used. Homogeneous, solid-supported fluid bilayers are easy to form by vesicle fusion.8,11,12 We were also able to create patterns consisting of the peripheral membrane protein myelin basic protein (MBP). The process described might therefore serve as a suitable method for deliberate biomaterial deposition in physiological buffer. Experimental Section Reagents. Dioleoylphosphatidylcholine (DOPC) and dioleoylphosphatidylserine (DOPS) dissolved in chloroform were obtained from Avanti Polar Lipids (Alabaster, AL) and used without further purification. NaCl, KH2PO4, KCl, and KOH (Sigma, Steinheim, Germany) were used for preparation of buffer 1 (150 mM NaCl, 5 mM KH2PO4, pH 7.5 with KOH), buffer 2 (1 mM KCl, pH ≈ 6, uncontrolled), and buffer 3 (500 mM NaCl, pH ≈ 6, uncontrolled). Poly-L-lysine (Sigma, Steinheim, Germany) was used without further treatment. Atomic Force Microscopy. Experiments were carried out using a commercial AFM equipped with a quartz liquid cell and standard Si3N4 tips (Digital Instruments, Santa Barbara, CA). (8) Mou, J.; Yang, J.; Huang, C.; Shao, Z. Biochemistry 1994, 33, 9981. (9) Rinia H. A.; Demel, R. A.; van der Eerden, J. P. J. M.; de Kruijff, B. Biophys. J. 1999, 77 (3), 1683. (10) Clausenschaumann, H.; Grandbois, M.; Gaub, H. E. Adv. Mater. 1998, 10 (12), 949. Grandbois M.; Clausenschaumann, H.; Gaub, H.-E. Biophys. J. 1998, 74 (5), 2398. (11) Brian, A. A.; McConnell, H. M. Proc. Natl. Acad. Sci. U.S.A. 1984, 81, 6159. McConnell, H. M.; Watts, T. H.; Weis, R. M.; Brian, A. A. Biochim. Biophys. Acta 1986, 864, 95. (12) Mueller, H.; Butt, H.-J.; Bamberg, E. Biophys. J. 1999, 76, 1072. Mueller, H.; Butt, H.-J.; Bamberg, E. J. Phys. Chem. B 2000, 104, 4552.
10.1021/la000689l CCC: $19.00 © 2000 American Chemical Society Published on Web 11/03/2000
Poly-L-lysine Structure Formation by AFM
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Figure 1. Deposition of lipid bilayer: top, force curve on bare mica; bottom, force curve after presence of lipid vesicles in the glass cell. The prominent jump across approximately 4 nm was observed at any random point on the surface. Insets show large area scans of the surfaces at minimum imaging force. Both images are flat and without features. The AFM was operated in two modes: (1) in imaging mode, a designated area was scanned line by line and the deflection of the cantilever was recorded, thus creating a height image of the sample; (2) in force mode, surface forces were measured at a certain point on the surface by advancing the sample to and subsequently retracting it from the tip while recording tip deflection as a function of distance from contact.
Results and Discussion On bare mica, poly-L-lysine adsorbs unspecifically from solution and seems to cover the entire surface more or less homogeneously. To prevent this adsorption, a homogeneous lipid bilayer was deposited onto the mica. It has been shown that lipid bilayers prevent adsorption of some water-soluble macromolecules, such as bovine serum albumine (data not shown) or poly(ethylene glycol),13-14 to the liquid-solid interface. Lipid bilayers were formed by fusion of lipid vesicles with the mica surface.8,11,12 They consisted of 80 wt % DOPC and 20 wt % DOPS, resulting in a net negative surface charge due to the negative serine headgroups at physiological pH. The lipids dissolved in chloroform were mixed in the desired proportion, and the solvent was evaporated under N2. Buffer 1 was added to the obtained lipid film to produce a 5 mg/mL suspension which was thoroughly sonicated. The resulting vesicle suspension was injected into the liquid cell of the AFM and allowed (13) Ducker, W. A.; Clarke, D. R. Colloids Surf., A 1994, 94, 275. (14) Kuhl, T.; Berman, A. D.; Hui, S. W., Israelachvili, J. N. Macromolecules 1998, 31, 8250.
Figure 2. Two artificial geometric structures created by scanning of the designated area in the presence of 2 µg/mL poly-L-lysine in solution. Both structures are surrounded by smaller irregular patches which had formed spontaneously. Sizes denoted in white blocks refer to the scale bars. Deflection images are shown.
to adsorb onto freshly cleaved mica (Plano, Wetzlar, Germany) for approximately 1 h. Remaining vesicles were removed by rinsing the cell with buffer 2. Presence of a homogeneous lipid bilayer was verified by imaging and taking force curves before injecting the vesicle suspension and after rinsing with buffer 2 (Figure 1). Before vesicle injection, force curves recorded on various random points of the surface showed only short range (approximately 1 nm) repulsion upon approach and van der Waals adhesion upon retraction. After vesicles had been injected, a conspicuous jump across approximately 4 nm at forces between 4 and 12 nN was observed in all approaching force curves (Figure 1b). This is usually interpreted as a punch-through of the AFM tip through the lipid bilayer.12,13 Images in both cases showed flat, featureless surfaces (Figure 1, insets). Once a lipid bilayer had formed (Figure 1), 100 µL of poly-L-lysine dissolved in buffer 2 at a concentration of
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Figure 3. Height profile of a section through the lower third of the corresponding height image of Figure 2 (top). The structure exhibits a constant height of 0.85 nm (vertical distance between the two arrows) and an edge of 175 nm (horizontal distance between arrows).
2-5 µg/mL was added. To form lateral structures, the AFM tip was engaged and the imaging force adjusted to a value sufficient to penetrate the lipid bilayer (approximately 5-10 nN). The piezo was scanned through the desired structure at high speed (>30 Hz) for 5 -10 min. To image the formed structures, the liquid cell was flushed with 400 µL of buffer 3 followed by 2 mL of buffer 1. After 5 -10 min, the surface was imaged at lowest possible force (usually around 0.2 nN). Figure 2 shows two artificially created structures on a DOPC/DOPS bilayer after scanning an eight-pointed star (top) and a square (bottom) in the presence of poly-L-lysine in the solution. Both structures show clearly defined edges and exhibit a constant height of 0.8 ( 0.1 nm across their full extension (Figure 3). In both images, the structures are surrounded by patches of approximately equal height. Such patches were also observed in experiments where the scanning of a designated area had been omitted after injection of the poly-L-lysine solution (data not shown). We conclude that the geometric structures consist of poly-L-lysine which has been deposited on top of the lipid bilayer or (more probably) by penetration of the lipid bilayer onto the mica underneath. Irregular patches probably consist of spontaneously adsorbed aggregates of poly-L-lysine. In our system, some adsorption of the positively charged poly-L-lysine to the negative DOPC/ DOPS lipid bilayer was to be expected due to electrostatic attraction. The fact that adsorbed poly-L-lysine aggregates in patches (as opposed to a widely homogeneous distribution) might be explained by the idea that adsorbed poly-L-lysine molecules are able to diffuse on the lipid bilayer, which is in the liquid phase state. Individual molecules diffuse until they hit an obstacle and get stuck. Other poly-Llysine molecules then aggregate around such an nucleation site and form aggregates of monomolecular height. This “nucleation hypothesis” is supported by the observation that taking force curves at certain points on the surface results in the formation of patches at these points (Figure 4). The penetration of the lipid bilayer by the tip while recording force curves effectively seems to create nucleation sites. Poly-L-lysine spots created in this way exhibited diameters as small as 100 nm. Spontaneously formed patches aggregate around perturbations which were already present on the bilayer. These are probably contaminations of the homogeneous bilayer surface.
Figure 4. Sequence of deliberately created structures by penetration of the lipid bilayer in force mode. Image 1 shows a bare bilayer area without features. By taking force curves at designated points, patches of approximately 150 nm in diameter and 0.8 nm height could be formed. Deflection images 2-6 show one, three, four, five, and seven deliberately created patches, respectively.
Indeed, such irregularities (“specks”) are often observed on lipid bilayers created by vesicle fusion.15 Preliminary experiments suggest that the poly-L-lysine patterns remain even after removing the lipid bilayer with organic solvent. Furthermore it seems that in order to create a geometrical pattern, the force must be sufficient to penetrate the lipid bilayer. Thus we believe that at least some of the poly-L-lysine molecules forming the pattern are anchored to the mica underneath the lipid bilayer. If deposition conditions can be optimized in such a way that spontaneous formation of poly-L-lysine patches is reduced to a minimum, the presented method to create patterns of poly-L-lysine may offer a viable way for inscription of areas of functionalized surfaces in aqueous solution. Acknowledgment. Henning Mueller is supported by the Deutsche Forschungsgemeinschaft (grant Bu 701/13) and the Max-Planck-Gesellschaft zur Fo¨rderung der Wissenschaften. LA000689L (15) Ohlsson, P.; Tja¨rnhage, T.; Herbai, E.; Lo¨fås, S.; Puu, G. Bioelectrochem. Bioenerg. 1995, 38, 137.
