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Langmuir 2005, 21, 5242-5246

Dip-Pen Patterning and Surface Assembly of Peptide Amphiphiles

Hongzhou Jiang and Samuel I. Stupp*,
Department of Materials Science and Engineering, Department of Chemistry, and Feinberg School of Medicine, Northwestern University, 2220 Campus Drive, Evanston, Illinois 60208-3108 Received January 21, 2005. In Final Form: April 11, 2005
This paper presents results on controlling the surface morphology of evaporation-driven self-assembly of peptide amphiphile (PA) nanofibers by dip-pen nanolithography. These PA nanofibers, which measure only a few nanometers in diameter, can be oriented perpendicularly to the receding edge of a solution. Dragging a meniscus of PA ink with an atomic force microscope (AFM) tip creates reproducibly aligned arrays of isolated and close-packed PA nanofiber patterns on silicon substrates, utilizing surface coating of poly(ethylene glycol) to suppress the self-assembly of nanofibers on AFM tips. We also demonstrate the ability to construct double-layer patterns of differing nanofiber orientations at the same position. This result could be important in producing a complex, multilayer pattern of these peptide-based supramolecular nanostructures.

Peptide amphiphile (PA) molecules have been shown to self-assemble into highly ordered, one-dimensional cylindrical nanostructures.1-5 These supramolecular objects measure only a few nanometers in diameter but can be hundreds to thousands of nanometers in length. The selfassembly of these molecules in aqueous solution is triggered by changes in pH1,2 or temperature,5 by the addition of multivalent ions,2,4 or simply by evaporating the solvent.2 The peptide segments assemble on the periphery of the nanostructure, enabling different chemical and biological functionalities to be displayed on their surfaces. PA nanofibers functionalized with bioactive sequences have exhibited exceptional properties, including the ability to template the crystallographic orientation in hydroxyapatite mineralization1 and to promote the selective differentiation of neural progenitor cells.4 Combined with surface patterning techniques, evaporation-driven self-assembly offers interesting opportunities to deposit these peptide-based nanostructures on surfaces in a controlled fashion. The ability to create localized, peptide-based patterns on surfaces could be useful in optimizing molecular recognition events for biosensing of analytes on nanofiber surfaces or to template the growth and orientation of nanocrystals by nucleation and mineralization on aligned nanofiber arrays. Toward achieving these goals, we have developed a dip-pen direct patterning technique for PA nanofibers. Dip-pen nanolithography (DPN) has been used previously to create high* To whom correspondence should be addressed. E-mail: Department of Materials Science and Engineering, Northwestern University. Department of Materials Science and Engineering, Department of Chemistry, and Feinberg School of Medicine, Northwestern University.
(1) Hartgerink, J. D.; Beniash, E.; Stupp, S. I. Science 2001, 294, 1684. (2) Hartgerink, J. D.; Beniash, E.; Stupp, S. I. Proc. Natl. Acad. Sci. U.S.A. 2002, 99, 5133. (3) Niece, K. L.; Hartgerink, J. D.; Donners, J. J. J. M.; Stupp, S. I. J. Am. Chem. Soc. 2003, 125, 7146. (4) Silva, G. A.; Czeisler, C.; Niece, K. L.; Beniash, E.; Harrington, D. A.; Kessler, J. A.; Stupp, S. I. Science 2004, 303, 1352. (5) Behanna, H. A.; Donners, J. J. J. M.; Gordon, A. C.; Stupp, S. I. J. Am. Chem. Soc. 2005, 127, 1193.

resolution surface patterns by depositing molecular inks with a modified atomic force microscope (AFM) tip.6-9 This method was originally developed for alkylthiol selfassembly on gold, but it has been subsequently extended to the patterning of complex molecules such as organic dyes,10-12 conjugated polymers,12-14 DNA,15-17 and peptides18,19 utilizing different combinations of AFM tip/ substrate modifications and ink composition. More interestingly, it has recently been demonstrated that proteins patterned in ordered nanoarrays by DPN can maintain their bioactivity to specific antibodies.20-25 Compared with these previously described systems, the patterning of PA nanofibers presents a unique challenge due to the additional complexity of controlling the in situ self-assembly of these molecular building blocks during the deposition process.
(6) Hong, S.; Mirkin, C. A. Science 2000, 288, 1808. (7) Hong, S.; Zhu, J.; Mirkin, C. A. Science 1999, 286, 523. (8) Piner, R. D.; Zhu, J.; Xu, F.; Hong, S.; Mirkin, C. A. Science 1999, 283, 661. (9) Ginger, D. S.; Zhang, H.; Mirkin, C. A. Angew. Chem., Int. Ed. 2004, 43, 30. (10) Su, M.; Dravid, V. P. Appl. Phys. Lett. 2002, 80, 4434. (11) Zhou, H.; Li, Z.; Wu, A.; Wei, G.; Liu, Z. Appl. Surf. Sci. 2004, 236, 18. (12) Noy, A.; Miller, A. E.; Klare, J. E.; Weeks, B. L.; Woods, B. W.; DeYoreo, J. J. Nano Lett. 2002, 2, 109. (13) Maynor, B. W.; Filocamo, S. F.; Grinstaff, M. W.; Liu, J. J. Am. Chem. Soc. 2002, 124, 522. (14) Lim, J.-H.; Mirkin, C. A. Adv. Mater. 2002, 14, 1474. (15) Nyamjav, D.; Ivanisevic, A. Adv. Mater. 2003, 15, 1805. (16) Demers, L. M.; Ginger, D. S.; Park, S.-J.; Li, Z.; Chung, S.-W.; Mirkin, C. A. Science 2002, 296, 1836. (17) Demers, L. M.; Park, S.-J.; Taton, T. A.; Li, Z.; Mirkin, C. A. Angew. Chem., Int. Ed. 2001, 40, 3071. (18) Cho, Y.; Ivanisevic, A. J. Phys. Chem. B 2004, 108, 15223. (19) Agarwal, G.; Sowards, L. A.; Naik, R. R.; Stone, M. O. J. Am. Chem. Soc. 2002, 125. (20) Lee, K.-B.; Kim, E.-Y.; Mirkin, C. A.; Wolinsky, S. M. Nano Lett. 2004, 4, 1869. (21) Lee, K.-B.; Park, S.-J.; Mirkin, C. A. J. Am. Chem. Soc. 2003, 125, 5588. (22) Lee, K.-B.; Park, S.-J.; Mirkin, C. A.; Smith, J. C.; Mrksich, M. Science 2002, 295, 1702. (23) Lim, J.-H.; Ginger, D. S.; Lee, K.-B.; Heo, J.; Nam, J.-M.; Mirkin, C. A. Angew. Chem., Int. Ed. 2003, 42, 2309. (24) Zhang, H.; Lee, K.-B.; Zhi, L.; Mirkin, C. A. Nanotechnology 2003, 14, 1113. (25) Hyun, J.; Ahn, S. J.; Lee, W. K.; Chilkoti, A.; Zauscher, S. Nano Lett. 2002, 2, 1203.

10.1021/la0501785 CCC: $30.25 2005 American Chemical Society Published on Web 05/10/2005

Chart 1. Structures of PA Molecules

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In this work, a dip-pen direct writing technique for PA nanofibers is demonstrated using the molecules shown in Chart 1, which were prepared as described previously using standard solid-phase peptide synthesis techniques.2 PA 1 and PA 2 were characterized by electrospray ionization mass spectrometry and were found to have the expected molecular weight (m/z ) 1206.6 and 1353.5 for PAs 1 and 2, respectively). PA 1 is soluble in water above pH 4, in which deprotonation of the acidic residues at the C terminus of the peptide results in electrostatic repulsion between the negatively charged molecules, thus, preventing aggregation. PA 2 is only soluble above pH 10, likely due to the addition of the aromatic moiety on the periphery of the molecule. AFM studies of the surface-assembled PA nanofibers were carried out to investigate the nanoscale structure of systems and to compare them with morphologies resulting from various patterning and alignment procedures. A total of 10 L of a 0.10 wt % aqueous solution of PA 1 was cast onto a (111) silicon surface with native oxidation layer (Silicon Quest International, Inc., Santa Clara, CA) cleaned by 15 min of ultrasonication in water (Millipore filtered, resistivity >18.2 Mcm) and 2-propanol (HPLC purity, Sigma-Aldrich Co.). After evaporation of water overnight, tapping mode atomic force microscopy was performed in ambient conditions using a MultiMode microscope from Digital Instruments (TESPA7-aluminumcoated, tapping-mode etched Si probes, Vecco Nanoprobes). As shown in Figure 1, we observed various morphologies, including isolated nanofibers, nanofiber networks, and close-packed bundles of nanofibers as well as partial monolayers, depending on local evaporation conditions within the same sample. The morphology of these nanostructures is similar to that observed previously by transmission electron microscopy (TEM) studies.1,2 The heights of these PA nanofibers as measured by AFM (see Figure 1) also agree with the previously reported diameter values of 6.0 ( 1 nm obtained by TEM. In a typical dip-pen patterning experiment, a conventional silicon nitride AFM cantilever (force constant ) 0.05 Nm-1) was first mounted on a ThermoMicroscopes CP AFM and then coated by dipping for 20 s into an aqueous solution containing 0.10 wt % PA 1 and 3.0 vol % glycerin, following methods reported previously for other types of inks.26 The addition of glycerin slowed the evaporation of water from the tip, preventing self-assembly of the PA molecules into supramolecular aggregates prior to deposition and allowing faster diffusion and migration of the molecules to the substrate. Even if the AFM tip
(26) Su, M.; Li, S.; Dravid, V. P. J. Am. Chem. Soc. 2003, 125, 9930.

coated with such a solution was left to dry for tens of minutes under ambient conditions, it could still retain the ability to pattern PA nanofibers, while patterning attempts without glycerin did not lead to deposition of nanofibers (results not shown). After the tip was coated in the PA ink, it was then brought into contact (2.5 nN) with the silicon substrate described above and patterned with a single stroke of pen motion at a writing speed of 0.1 m/s (see Figure 2a). These PA nanofibers exhibited morphologies and heights similar to those of the ones prepared from cast films, consistent with a mechanism of self-assembly that is not significantly altered by the patterning process. The width of the patterned area was 3-4 m, larger than typical DPN-patterned lines due to the oversized ink droplet needed to keep the PA molecules solvated, and it was not sensitive to a change of 200 times in the speed of AFM tips motion, as shown by Figure 2a,c. Because the microdroplets size depends on temperature and humidity,27 all the patterning was performed in a controlled environment at 20-23 C and a relative humidity of 45-55%. The length of the patterned area is limited only by the X-Y scanning range of the AFM tip, which for the instrument used was 100 m. However, if the AFM tip was retracted from the surface, part of the ink droplet would be left on the surface, leading to a much lower density of nanofibers in subsequent patterning. The angular distribution of the naonostructures was calculated and fitted with a Gaussian distribution, demonstrating effective alignment of PA nanofibers using the dip-pen patterning method described above, as shown in Figure 2b. Furthermore, the nanofibers in the center of the trace, as indicated by the green arrows, were better aligned along the patterning direction, while nanofibers on the edges of the trace, as indicated by the blue arrows, were tilted from the patterning direction to the center of the trace. This observation leads us to propose that the alignment effect observed arises from the tendency of the PA nanofibers to orient perpendicularly to the receding edge of the meniscus that is created by the AFM tip as it drags across the surface. Similar phenomena have been reported previously for DNA.28-31
(27) Israelachvili, J. N. Intermolecular and Surface Forces, 2nd ed.; Academic Press: London, 1992. (28) Bensimon, A.; Simon, A.; Chiffaudel, A.; Croquette, V.; Heslot, F.; Bensimon, D. Science 1994, 265, 2096. (29) Nakao, H.; Gad, M.; Sugiyama, S.; Otobe, K.; Ohtani, T. J. Am. Chem. Soc. 2003, 125, 7162. (30) Sasou, M.; Sugiyama, S.; Yoshino, T.; Ohtani, T. Langmuir 2003, 19, 9845. (31) Michalet, X.; Ekong, R.; Fougerousse, F.; Rousseaux, S.; Schurra, C.; Hornigold, N.; Slegtenhorst, M. v.; Jonathan Wolfe, S. P.; Beckmann, J. S.; Bensimon, A. Science 1997, 277, 1518.


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Figure 1. Tapping mode AFM images of PA 1 nanofibers after 0.1 wt % aqueous solutions were dried by evaporation on (111) silicon surfaces. (a) Topographical and (b) phase images of isolated PA nanofibers lying on top of a close-packed layer of nanofibers. In the height profile along the white line in part a, the height difference between the two markers is 5.7 nm (inset). (c) Topographical image of a random network of nanofibers. (d) Topographical image of patches of close-packed nanofibers monolayers, with isolated single fibers on top.

We also tested the possibility of constructing a more complex, multilayered pattern of PA nanofibers. This was accomplished by repeated dip-pen patterning onto the same area in a perpendicular fashion (see Figure 2c,d). In the center of the junction, fibers cross each other orthogonally, with double the height of individual PA nanofibers, indicating that the PA nanofibers are stably fixed on the surface and not destroyed by a second pattern being drawn over them with the AFM tip. This initial work suggests the possibility of constructing more complex patterns or even multilayer architectures of the selfassembled nanostructures. The formation of PA nanofibers can take place not only upon solvent evaporation but also in the ink solution or on the surface of the AFM tip. Because our target is to restrict self-assembly to the substrate, we introduced factors that could inhibit nanofiber formation in solution and on the AFM tip surface. Because the PA molecules used in this study are negatively charged at neutral pH, using the PA inks at pH > 10 dissolved the nanofiber network and kept the PA molecules more solvated in the ink.1,2 The second factor is the introduction of a poly-

(ethylene glycol) (PEG) coating on the AFM tip surface. PEG has been shown to inhibit protein adsorption,32-35 and we recently discovered that it can also inhibit PA nanofiber formation on the AFM tip (see Supporting Information). Combining the two techniques, an improved PA dippen patterning method was developed with a PEGmodified AFM tip, which was prepared by immersion in a 1.0 wt % methanol solution of 2-[methoxy poly(ethyleneoxy)propyl]-trimethoxysilane (molecular weight ) 400-532, Gelest, Inc., Morrisville, PA) for 1 h. The tip was then coated with PA by dipping into an aqueous solution containing 0.1 wt % PA 2, 3.0 vol % glycerin, and 10 mM ammonium hydroxide. Following the protocol described above, this tip was used to carry out the
(32) Ostuni, E.; Chapman, R. G.; Holmlin, R. E.; Takayama, S.; Whitesides, G. M. Langmuir 2001, 17, 5605. (33) Ostuni, E.; Chapman, R. G.; Liang, M. N.; Meluleni, G.; Pier, G.; Ingber, D. E.; Whitesides, G. M. Langmuir 2001, 17, 6336. (34) Prime, K. L.; Whitesides, G. M. Science 1991, 252. (35) Chapman, R. G.; Ostuni, E.; Liang, M. N.; Meluleni, G.; Kim, E.; Yan, L.; Pier, G.; Warren, H. S.; Whitesides, G. M. Langmuir 2001, 17, 1225.


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Figure 2. (a) Phase image of dip-pen patterned PA fibers. The black arrow indicates the dip-pen patterning direction. (b) Histogram of the angular distribution of nanofibers in part a fitted with a Gaussian distribution ( ) 42). (c) Topographical image of overlapping dip-pen patterns, with arrows representing their respective patterning directions. (d) Three-dimensional topography of the central area in part c; blue arrows indicate where fibers cross. The height at the point indicated by the right green marker on the white line is 10.7 nm, while that on the left marker is 4.7 nm. The pattern was created at a writing speed of 20 m/s, using 0.1 wt % PA 1 and 3.0 vol % glycerin in water on a bare Si3N4 AFM tip. The x and y axes in part d are 2.5 m, while the vertical scale is 20 nm.

patterning experiments, leading to patterns comprised of patches of close-packed nanofibers extending over micrometers in area (see Figure 3). These close-packed structures resembled what has been observed in samples prepared by evaporation of water from PA solution on surfaces (Figure 1d). As observed in the patterns described before, these patches contained nanofibers of varying orientation but exhibited good overall alignment. Because the self-assembly of PA molecules into nanofibers had been suppressed both in the bulk solution and on the AFM tip surface, we propose that such close-packed nanofiber structures originate from self-assembly that is partly mediated by the silicon substrate. The mechanism that leads to this highly ordered structure remains unclear; however, two important factors that are likely to play a role are surface charge screening and surface defectinduced nucleation of nanofibers. To test this hypothesis, we also applied the same patterning protocol on the freshly cleaved mica surface, where the formation of nanofiber was disturbed (see Supporting Information) probably due

to the high density of surface charges from dangling bonds on the mica surface.36 The close-packed nanofiber pattern extending over micrometers of area introduced the possibility of characterizing the surface pattern by a technique that lacks adequate lateral resolution and sensitivity to study single nanofibers. For example, we used time-of-flight secondary ion mass spectroscopy (ToF-SIMS, PHI TRIFT III from Physical Electronics Co., Chanhassen, MN) on these patterns. By using alignment marks on the substrate, we examined the same portion of the pattern probed by AFM (see Figure 3c). The CN- ion image showed the presence of CN- almost exclusively on the pattern, confirming the localization of PA molecules within the patterned area. We have reported the surface-driven self-assembly of PA molecules using DPN techniques to create reproducibly aligned, close-packed arrays of PA nanofiber patterns on silicon substrates. We also demonstrated the ability of the nanofibers on the surface to withstand multiple passes
(36) Heinz, W. F.; Hoh, J. H. Biophys. J. 1999, 76, 528.


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Figure 3. Topographical images of the dip-pen patterned traces with close-packed nanofibers patches, patterned at (a) 4 and (b) 2 m/s. The arrows indicate the dip-pen patterning direction. (c) ToF-SIMS image of CN- ions from the same area as part a. (d and e) Higher magnification topographical view of the central area in parts a and b. The scale bars in parts a and c measure 10 m.

of the dip-pen tip, enabling the formation of a more complex, multilayer pattern of these peptide-based supramolecular nanostructures. Acknowledgment. This work was supported by the U.S. Air Force Office of Scientific Research Grant F4962000-1-0283 and the U.S. Department of Energy Grant DEFG02-00ER45810. We thank the Nanoscale Integrated Fabrication, Testing and Instrumentation Center (NIFTI) for use of the AFM and Keck Interdisciplinary Surface Science Center (Keck-II) for use of its time-of-flight secondary ion mass spectrometer at Northwestern Uni-

versity. The authors are also grateful for useful discussions with Dr. Mukti Rao, Dr. Ming Su, Dr. Nick Wu, Dr. Yi Zhang, Dr. David Ginger, and Dr. Jung-Hyurk Lim of Northwestern University. Supporting Information Available: Scanning electron microscope images of the PA coating on silanized and unmodified AFM tips; an AFM image of the PA patterned on the freshly cleaved mica surface. This material is available free of charge via the Internet at