Langmuir 2000, 16, 3927-3931
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Nanopatterning Phospholipid Bilayers Joseph W. Carlson,†,‡ Timothy Bayburt,‡,§ and Stephen G. Sligar*,†,‡,§ Center for Biophysics and Computational Biology, Department of Biochemistry, and Beckman Institute for Advanced Science and Technology, University of Illinois, Urbana, Illinois 61801 Received July 1, 1999. In Final Form: December 2, 1999
A nanolithography method for altering the composition of supported phospholipid bilayers has been developed. Although a number of lithography procedures for various chemical systems currently exist, these methods typically rely on covalent bonding to stabilize the resulting structure. The method described here is suitable for an entirely noncovalent system, where the pattern is maintained by specific hydrophobic and other noncovalent interactions. This lithography is achieved by the atomic force microscope, which is used to insert phospholipid molecules into a supported reconstituted high-density lipoprotein-phospholipid bilayer with 20 nm line widths. Lipid molecules are delivered to the supported bilayer through transient structural disruption, forcing the acceptance of new lipid monomers into the bilayer structure. Precoating the atomic force microscope tip with specific lipids is sufficient to deliver new lipid to the patterned areas. These results demonstrate a new method for controlling the structure of highly dynamic noncovalent surface assemblies.
Introduction Interest in molecule-based materials has expanded in recent years because of recent developments in their fabrication, control, and assembly. Self-organizing materials based on the assembly of molecular components have been constructed from inorganic, organometallic, and biological precursors (for a recent review, see ref 1). Biological precursors, such as lipids,2,3 proteins,4 and nucleic acids,5 as well as a number of organic precursors, such as triblock polymers,6 self-assemble into materials through highly specific noncovalent interactions, such as van der Waals contacts, hydrogen bonding, or π-π stacking. Controlling the composition of these materials at the nanometer scale has been an important goal. These methods could have particular impact in cell biology, where the organization of a cell membrane is heterogeneous and new techniques to probe and control this heterogeneity are especially needed.7 Thin film structure can be controlled in a number of ways. In negative lithography a self-assembled monolayer or chemical thin film is removed, either by such methods as beam lithography,8-10 micromachining,11,12 or scanning probe microscopy.13 Exposing a hydrophobic surface to a solution of phospholipid vesicles results in fusion of the * To whom correspondence may be addressed. Tel: 217/2447395. FAX: 217/244-7100. † Center for Biophysics and Computational Biology. ‡ Beckman Institute for Advanced Science and Technology. § Department of Biochemistry. (1) Alivisatos, A. P.; Barbara, P. F.; Castleman, A. W.; Chang, J.; Dixon, D. A.; Klein, M. L.; McLendon, G. L.; Miller, J. S.; Ratner, M. A.; Rossky, P. J.; Stupp, S. I.; Thompson, M. E. Adv. Mater. 1998, 10, 1297. (2) Sackmann, E. Science 1996, 271, 43. (3) Cornell, B. A.; Braachmaksvytis, V. L. B.; King, L. G.; Osman, P. D. J.; Raguse, B.; Wieczorek, L.; Pace, R. J. Nature 1997, 387, 580. (4) Douglas, K.; Clark, N. A.; Rothschild, K. J. Appl. Phys. Lett. 1990, 56, 692. (5) Elghanian, R.; Storhoff, J. J.; Mucic, R. C.; Letsinger, R. L.; Mirkin, C. A. Science 1997, 277, 1078. (6) Stupp, S. I.; LeBonheur, V.; Walker, K.; Li, L. S.; Huggins, K.; Keser, M.; Amstutz, A. Science 1997, 276, 321. (7) Brown, D. A.; London, E. Annu. Rev. Cell Dev. Biol. 1998, 14, 111.
vesicles with the surface.14 The atomic force microscope (AFM) can be used to generate frictional forces on a variety of substrates, allowing their structure to be modified.15,16 Thin film removal reveals the underlying substrate for subsequent functionalization. Positive lithography involves the addition of new molecules, through such techniques as microcontact printing17-19 or an atomic force microscope.20 Positive printing at the micrometer scale has used organothiols or organosilanes, where the resulting organothiol patterns are covalently linked to the gold surface.20 Supported phospholipid bilayers have unique patterning requirements in that their structure is noncovalent. They are therefore highly unstable when their hydrophobic exterior is exposed and will rapidly reorganize to sequester the lipid acyl chains. In this paper we describe the construction of supported phospholipid bilayers using the atomic force microscope. The AFM tip was used to induce phospholipid exchange with preexisting phospholipid bilayers in a controlled way to produce a homogeneous bilayer pattern consisting of a different phospholipid. The possible mechanism of phospholipid exchange with the surface is discussed. (8) Tiberio, R. C.; Craighead, H. G.; Lercel, M.; Lau, T.; Sheen, C. W.; Allara, D. L. Appl. Phys. Lett. 1993, 62, 476. (9) Sondag-Huethorst, J. A. M.; van Helleputte, H. R. J.; Fokkink, L. G. J. Appl. Phys. Lett. 1994, 64, 285. (10) Berggren, K. K.; Bard, A.; Wilbur, J. L.; Gillaspy, J. D.; Helg, A. G.; McClelland, J. J.; Rolston, S. L.; Phillips, W. D.; Prentiss, M.; Whitesides, G. M. Science 1995, 269, 1255. (11) Abbott, N. L.; Kumar, A.; Whitesides, G. M. Chem. Mater. 1994, 6, 596. (12) Abbott, N. L.; Rolison, D. R.; Whitesides, G. M. Langmuir 1994, 10, 2672. (13) Xu, S.; Liu, G.-Y. Langmuir 1997, 13, 127. (14) Keller, C. A.; Kasemo, B. Biophys. J. 1998, 75, 1397. (15) Hu, J.; Xiao, X. D.; Ogletree, D. F.; Salmeron, M. Surf. Sci. 1995, 327, 358. (16) Lio, A.; Morant, C.; Ogletree, D. F.; Salmeron, M. J. Phys. Chem. B 1997, 101, 4767. (17) Aizenberg, J.; Black, A. J.; Whitesides, G. M. Nature 1999, 398, 495. (18) Lahiri, J.; Ostuni, E.; Whitesides, G. M. Langmuir 1999, 15, 2055. (19) Jackman, R. J.; Brittain, S. T.; Adams, A.; Wu, H. K.; Prentiss, M. G.; Whitesides, S.; Whitesides, G. M. Langmuir 1999, 15, 826. (20) Piner, R. D.; Zhu, J.; Xu, F.; Hong, S.; Mirkin, C. A. Science 1999, 283, 661.
10.1021/la990860x CCC: $19.00 © 2000 American Chemical Society Published on Web 03/17/2000
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Langmuir, Vol. 16, No. 8, 2000
Experimental Section Preparation of Small Unilamellar Vesicles. Small unilamellar vesicles were prepared using ultrasonication.21 Solid dipalmitoyl phosphatidylcholine (DPPC, 16:0, Tm ) 41 °C), dimyristoyl phosphatidylcholine (DMPC, 14:0, Tm ) 23 °C), ditridecanoyl phosphatidylcholine (DTPC, 13:0, Tm ) 14 °C), and diarachidonoyl phosphatidylcholine (DAPC, 20:4, Tm ) -70 °C) were purchased from Avanti Polar Lipids (Alabaster, AL) and used without further purification. Stock solutions of the desired lipids were prepared in chloroform, and solvent was removed by filtered dry N2. The lipids were then resuspended by vortexing in water from a Millipore purification system to produce a solution with a typical concentration of 1 mg/mL. Probe sonication of the resuspended lipids was carried out for a 3 min on/off cycle with temperature equilibration, until the solution cleared. The solution was bathed in a water bath with a temperature greater than the transition temperature of the lipid mixture being used and maintained under filtered dry N2. After sonication the lipid solution was ultracentrifuged at 43 100 rpm in a Ti-70 rotor for 5 h to remove larger lipid aggregates. The temperature during centrifugation and in subsequent storage was maintained at 4 °C. Preparation of Reconstituted High-Density Lipoproteins. Reconstituted high-density lipoproteins (rHDL) with two apolipoprotein A-I molecules (apoA-I) incorporated per rHDL were prepared and characterized as described.22 Briefly, the sodium cholate dialysis method in a molar ratio of 150:1 (L-Rdipalmitoyl phosphatidylcholine/apoA-I) was used. Cholate was removed through exhaustive dialysis against 0.1 M Tris-HCl pH 8.0, 0.005% EDTA, 0.15 M NaCl, 1 mM NaN3. For dialysis, a Slide-A-Lyzer (Pierce Scientific, Rockford, IL), containing typically less than 1 mL of the apoA-I lipid mixture, was dialyzed against 1 L with a minimum of four changes. Dialysis was carried out at 37 °C. The size of the rHDL particles was determined by nondenaturing 8-25% polyacrylamide gel electrophoresis on a PHAST system (Pharmacia, Uppsala, Sweden). The dialysate was purified by gel filtration chromatography on a Superdex 200 HR 10/30 column on an FPLC system (Pharmacia, Uppsala, Sweden) pre-equilibrated with 0.1 M Tris-HCl pH 8.0, 0.005% EDTA, 0.15 M NaCl, 1 mM NaN3. Protein concentration was determined using the method of Lowry23 or through absorption spectroscopy, using an extinction coefficient of 280 ) 1.15 mL mg-1 cm-1 for apoA-I. Purified DPPC rHDL was stored at 4 °C for up to 2 months. Scanning Force Microscope Imaging and Manipulation. All images were taken in the liquid cell of a Nanoscope III AFM (Digital Instruments, Santa Barabara, CA). Care was taken to ensure that the phospholipid surfaces were never allowed to dry. Images were taken using the “E” scanner with oxide-sharpened silicon nitride tips (Digital Instruments, Santa Barbara, CA) on the thin, 200 µm cantilever with a nominal spring constant of 0.06 N/m. Tips were used as supplied, unless indicated. In some cases, tips were precoated by placing the chip holding the cantilever and tip into a 1 mg/mL solution of small unilamellar vesicles of DTPC for 5 h at room temperature and then rinsing to remove unadsorbed lipid. The nanometer size tip used by the AFM can generate an “image” with a variety of forces and scan speeds. By alternating between parameters optimized for imaging and those for manipulation, the tip can be used to both modify the surface and image the results. In the imaging mode, the scan speed was reduced (=2.0 µm/s), the force kept as low as possible (
