Langmuir 2006, 22, 9089-9091
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Patterning Amyloid Peptide Fibrils by AFM Charge Writing Patrick Mesquida,*,† E. Macarena Blanco,‡ and Rachel A. McKendry‡ Department of Mechanical Engineering, King’s College London, Strand, London WC2R 2LS, United Kingdom, and London Centre for Nanotechnology and Department of Medicine, UniVersity College London, 5 UniVersity Street, London WC1E 6JJ, United Kingdom ReceiVed May 25, 2006. In Final Form: September 15, 2006 Surface charge patterns generated by atomic force microscopy-based charge writing were used to pattern amyloidlike peptide fibrils on a solid substrate. Fibrils of the short peptide TTR105-115 were encapsulated inside water droplets of a water-in-perfluorocarbon oil emulsion and retained their rod morphology. They were observed to deposit selectively with a lateral resolution of approximately 1 µm onto negatively charged patterns on a polymethyl-methacrylate substrate.
Introduction Self-assembling biomolecular structures have attracted much attention as potential building blocks for future bioinspired devices. The important advantage of biological systems lies in the ability to engineer their molecular structure precisely, allowing control over their physical, chemical, and biological properties. Together with the exploitation of biological design principles, this could lead to new methods for the fabrication of novel biosensors and biocompatible materials. Fibrillar structures of polypeptides, such as synthetic amyloid fibrils1 and cyclic D,Lpeptide nanotubes,2,3 have attracted considerable interest in recent years. Promising advances have been made with the incorporation of functional groups into artificial amyloid fibrils,4 the creation of responsive peptide gels,5 and the fabrication of hybrid polymer-peptide nanotubes.6 Furthermore, it has been shown that amyloid-like peptide fibrils are relatively easy to prepare in vitro and exhibit high thermal and chemical stability.4,7 The most attractive aspect is the increasing evidence that the ability to form amyloid-like peptide fibrils is a generic thermodynamic property of any polypeptide molecule, independent of the amino acid sequence.8 Beta-sheet-rich amyloid-like fibrils have been produced in vitro from a host of different natural and synthetic proteins and polypeptides.1,4,9 This offers interesting possibilities for applications in bio- and nanotechnology. For example, Kasai et al. and Kisiday et al. showed that biological functionality can be added to these fibrils by incorporating specific amino acid sequences promoting cell adhesion.10,11 Alternatively, * To whom correspondence
[email protected]. † King’s College London. ‡ University College London.
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(1) MacPhee, C. E.; Woolfson, D. N. Curr. Opin. Solid State Mater. Sci. 2004, 8, 141. (2) Ghadiri, M. R.; Granja, J. R.; Milligan, R. A.; McRee, D. E.; Khazanovich, N. Nature 1993, 366, 324. (3) Bong, D. T.; Clark, T. D.; Granja, J. R.; Ghadiri, M. R. Angew. Chem., Int. Ed. 2001, 40, 988. (4) MacPhee, C. E.; Dobson, C. M. J. Am. Chem. Soc. 2000, 122, 12707. (5) Aggeli, A.; Bell, M.; Boden, N.; Keen, J. N.; Knowles, P. F.; McLeish, T. C. B.; Pitkeathly, M.; Radford, S. E. Nature 1997, 386, 259. (6) Couet, J.; Jeyaprakash, J. D.; Samuel, S.; Kopyshev, A.; Santer, S.; Biesalski, M. Angew. Chem., Int. Ed. 2005, 44, 3297. (7) Adler-Abramovich, L.; Reches, M.; Sedman, V. L.; Allen, S.; Tendler, S. J. B.; Gazit, E. Langmuir 2006, 22, 1313. (8) Dobson, C. M. Philos. Trans. R. Soc. London, Ser. B 2001, 356, 133. (9) Jimenez, J. L.; Nettleton, E. J.; Bouchard, M.; Robinson, C. V.; Dobson, C. M.; Saibil, H. R. Proc. Natl. Acad. Sci. U.S.A. 2002, 99, 9196. (10) Kasai, S.; Ohga, Y.; Mochizuki, M.; Nishi, N.; Kadoya, Y.; Nomizu, M. Biopolymers 2004, 76, 27. (11) Kisiday, J.; Jin, M.; Kurz, B.; Hung, H.; Semino, C.; Zhang, S.; Grodzinsky, A. J. Proc. Natl. Acad. Sci. U.S.A. 2002, 99, 9996.
peptide fibrils have been utilized as templates to create metallic nanowires.12,13 Whereas to date much effort has focused on the synthesis of novel fibrils, relatively few studies have investigated methods to integrate these materials with micro- and nanotechnology. This ultimately requires the development of techniques to position the fibrils arbitrarily on technically relevant surfaces such as semiconductors and polymers. We recently showed that 2D microarrays of synthetic fibrils could be generated on solid surfaces by exploiting specific fibrilsurface electrostatic interactions.14 Our approach involved soft lithography with flexible silicone stamps whose potential limitation is that they require a casting mold with a predefined layout, which is relatively costly, requires multiple photolithography processing steps, and limits experimental flexibility. Therefore, we sought to investigate alternative, generic methods to create fibril patterns with high spatial resolution and specificity. Here, we report a promising alternative approach to creating peptide fibril microarrays based on atomic force microscopy charge writing (AFM-CW) and electrostatic deposition from solution.15 Briefly, nanoscale positive or negative surface charge patterns can be created by applying a voltage to an atomic force microscope (AFM) tip on insulating substrates such as polymers and silicon dioxide (Figure 1a).16-18 The patterned surface is then immersed in an ultrasonically produced water-in-oil emulsion in which the water microdroplets can be filled with nanoparticles and act as carriers to guide and deposit the particles onto the surface charge patterns via electrostatic attraction (Figure 1b).20 Perfluorinated oils are used as the continuous phase because of their excellent electrical insulation properties that avoid the decay of surface charge patterns. A range of different types of solid nanoparticles such as silica, metal, and polymer particles have been successfully (12) Reches, M.; Gazit, E. Science 2003, 300, 625. (13) Scheibel, T.; Parthasarathy, R.; Sawicki, G.; Lin, X.-M.; Jaeger, H.; Lindquist, S. L. Proc. Natl. Acad. Sci. U.S.A. 2003, 100, 4527. (14) Mesquida, P.; Ammann, D. L.; MacPhee, C. E.; McKendry, R. A. AdV. Mater. 2005, 17, 893. (15) Mesquida, P.; Stemmer, A. AdV. Mater. 2001, 13, 1395. (16) Stern, J. E.; Terris, B. D.; Mamin, H. J.; Rugar, D. Appl. Phys. Lett. 1988, 53, 2717. (17) Mesquida, P.; Knapp, H. F.; Stemmer, A. Surf. Interface Anal. 2002, 33, 159. (18) Morita, S.; Sugawara, Y.; Fukano, Y. Jpn. J. Appl. Phys. 1993, 32, 2983. (19) Nonnenmacher, M.; O’Boyle, M. P.; Wickramasinghe, H. K. Appl. Phys. Lett. 1991, 58, 2921. (20) Mesquida, P. Ph.D. Thesis no. 14854, ETH-Zurich, Switzerland, 2002 (available free of charge at http://e-collection.ethbib.ethz.ch/ show?type)diss&nr)14854).
10.1021/la061485t CCC: $33.50 © 2006 American Chemical Society Published on Web 09/29/2006
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Figure 1. Nanoparticle attachment by AFM-CW. (a) A bias voltage is applied to a conductive AFM tip, and an arbitrary charge pattern is created in a thin polymer film using a conductive substrate as the counter electrode. The inset shows the surface potential imaged by Kelvin-probe force microscopy19 (gray scale ) 6.6 V, dark ) negative potential) of 40-µm-long lines written on polymethyl-methacrylate using a tip voltage of -80 V with respect to the substrate. (b) An aqueous suspension of nanoparticles (grey background) is added to an oil (clear background). Small water droplets containing the solid nanoparticles are dispersed into the oil by ultrasonication. The water droplets are electrostatically attracted toward the surface charge pattern and deposit their contents.
deposited by this method.15,21,22 Recently, Naujoks et al. have reported the first experiments that show the compatibility of the method with biomolecules using the model biotin-avidin system.23 The aim of our work was to investigate whether this method could be used to generate 2D arrays of amyloid-like peptide fibrils on a typical polymer surface such as PMMA. Important aspects of this work were to investigate whether fibrils could be encapsulated in the water microdroplets, whether the emulsion is sufficiently stable to allow attachment, and how ultrasonication affects the fibril morphology. Our efforts have focused on the short, synthetic, amyloid-like peptide TTR105-115 because it readily self-assembles into rods of a few nanometers in diameter at low pH4 and because it is the only peptide whose molecular structure in the fibrillar state has been determined.24,25 Furthermore, it has also been shown that functional biological groups and different peptides can be incorporated into TTR105-115 fibrils and that they can serve as a scaffold to immobilize gold nanoparticles; this has attacted much attention as a novel biomaterial.4 Materials and Methods Substrate Preparation and Pattern Generation. As the substrate for AFM-CW, we used polymethyl-methacrylate (PMMA, Mw ) 996 000, Aldrich Chemical Co., Milwaukee, WI) deposited on polished Si wafers as the counter electrode. The 150-nm-thick PMMA films were prepared by spin coating (5 s at 500 rpm, followed by 45 s at 5000 rpm) a 3% PMMA solution in chlorobenzene and subsequently baking the film on a hot plate at 150 °C for 5 min. Charge writing on PMMA was performed with doped plain silicon and W2C-coated silicon AFM tips (MikroMasch Eesti OU, Tallinn, Estonia) using the nanolithography function of a Digital Instruments Multimode AFM (Veeco Metrology LLC, Santa Barbara, CA) to position the tip laterally during AFM-CW. Peptide Fibril Preparation and Attachment. The TTR105-115 peptide (YTIAALLSPYS) was purchased from CS Bio Company (Menlo Park CA). It was assembled into fibrils by dissolving 10 mg/mL peptide in a 20 vol % CH3CN solution at pH 2 as described (21) Mesquida, P.; Stemmer, A. Microelectron. Eng. 2002, 61-62, 671. (22) Naujoks, N.; Stemmer, A. Microelectron. Eng. 2005, 78-79, 331. (23) Naujoks, N.; Stemmer, A. Colloids Surf., A 2004, 249, 69. (24) Jaroniec, C. P.; MacPhee, C. E.; Astrof, N. S.; Dobson, C. M.; Griffin, R. G. Proc. Natl. Acad. Sci. U.S.A. 2002, 99, 16748. (25) Jaroniec, C. P.; MacPhee, C. E.; Bajaj, V. S.; McMahon, M. T.; Dobson, C. M.; Griffin, R. G. Proc. Natl. Acad. Sci. U.S.A. 2004, 101, 711.
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Figure 2. TTR105-115 fibrils on mica. Tapping-mode AFM images taken in air under ambient conditions: (a) z scale ) 20 nm, (b) z scale ) 10 nm; fibrils are drop cast on freshly cleaved mica, incubated for 1 min, rinsed with copious amounts of deionized water, and dried in a stream of nitrogen before imaging. elsewhere.14 A highly concentrated stock fibril suspension was obtained after a few days. The exact number concentration of fibrils, however, could not be determined because it is not known what percentage of the peptide is actually transformed into fibrils and because fibrils have different sizes. To transfer the fibrils into FC77 (Fluorinert FC77, 3M Company, St.Paul MN), a perfluorocarbon oil, the stock suspension was first diluted in deionized water at a volume ratio of 1:3, and then this diluted aqueous suspension was mixed with FC77 and dispersed by ultrasonication (standard ultrasonic bath Transonic T310, Camlab Ltd, Cambridge, United Kingdom). This procedure generated small water droplets that contained the peptide nanorods. The previously charge-patterned PMMA sample was then immersed in the emulsion, rinsed in pure FC77, and dried in a stream of nitrogen.
Results and Discussion Figure 2 shows the typical morphology of TTR105-115 fibrils adsorbed directly from the aqueous stock suspension on a freshly cleaved mica surface. It is observed that the fibrils have a striking rodlike morphology with individual lengths of up to approximately 1 µm (Figure 2a). However, a statistical analysis of the fibril length was not performed because it was often not possible to distinguish between single fibrils and several fibrils aligned behind each other. The fibril diameter cannot be determined directly from the lateral width in the AFM images because the finite size of the AFM tip leads to a lateral overestimation known as the tip convolution effect (Figure 2b). Under the assumption that the fibrils have a circular cross section and retain it upon adsorption on mica, the fibril diameter was determined from the height in the z direction and was found to be 9 ( 3 nm (from an analysis of 11 fibrils that could be discerned from the AFM images as individual fibrils as opposed to fibril bundles). With AFM-CW, charge patterns can be readily created on PMMA (inset of Figure 1a). No significant decay or lateral spreading of the charge patterns was observed either in air or after immersion of the charged sample in FC77 for 6 min. To investigate the ultrasonically generated water-in-FC77 emulsion, the droplets were observed by optical microscopy, and the emulsion turbidity was analyzed. Figure 3a shows a typical snapshot (Nikon TE2000U inverted optical microscope, 10× objective, NA 0.3) taken immediately after ultrasonication. Well-dispersed water droplets with sizes of up to approximately 5 µm diameter can be discerned. Because no surfactant was used, the dispersion was observed to break down after a few minutes. Figure 3b shows the decay of the optical density (measured at regular intervals in the visible spectrum (570 nm), EMax Precision Microplate Reader, Molecular Devices Corp, Sunnyvale, CA) of the emulsion whose optical turbidity caused by the micrometer-sized water droplets is a suitable indicator of the stability of the dispersion. The turbidity starts to decay
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Figure 3. Water-in-FC77 emulsion. (a) Optical microscopy image of a water-in-FC77 emulsion, 1 µL of deionized water is mixed with 1 mL of FC77 and ultrasonicated for a few seconds; (b) time decay of the turbidity of a water-in-FC77 emulsion, continuous line ) simple exponential fit ≈ exp(-t/tD), tD ) 4.15 min, 40 µL of deionized water is mixed with 2 mL of FC77 and ultrasonicated for a few seconds.
immediately after the ultrasonication is stopped, with an exponential decay time of approximately 4 min. The emulsion phase-separates, and after 20-30 min a single, large, clear water drop was visually observed floating on the clear FC77 (mass density of FC77 ) 1.78× the density of water).26 This short time was found to be sufficient for the fibril attachment process, which takes place on a time scale of a few seconds to a few minutes and facilitates the removal of excess material. Figure 4 shows TTR105-115 deposited on a pattern of three negatively charged lines on PMMA. These topographic AFM images clearly demonstrate that fibrillar peptide structures were directed to the charged line patterns with a lateral position accuracy of 1 to 2 µm (Figure 4a and b). In agreement with earlier studies, we have found that the resolution of this method is limited mainly by the size of the aqueous droplets.15,21 It can be seen that the fibrillar lines are not continuous, an effect that has also been observed with other nanoparticles15,21 and may be attributed to the variation of droplet size and the surface-wetting properties of PMMA. However, a notable strength of this method is the low nonspecific adsorption on the surrounding uncharged PMMA area (Figure 4a), which is very important for all potential applications of biomolecular microarrays. Figure 4b is an enlarged view of Figure 4a showing fibril fragments. The extended TTR105-115 nanorods are broken up, probably by the mechanical energy applied during the ultrasonication. Some larger agglomerates of fibrillar material can also be seen (Figure 4a). Repeatedly rinsing with pure FC-77 did not remove the fibrils from the surface.
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Figure 4. Microarray of TTR105-115 fibrils. Tapping-mode AFM images of topography, fibrils attached to negative line patterns (tipsample bias voltage pulses of -80 V applied in air under ambient conditions, pulse length ) 0.5 ms, pulse frequency ) 50 Hz) on PMMA (150 nm film on Si substrate). The sample is immersed for 30 s in 3 µL of an aqueous fibril suspension mixed with 1 mL of FC77: (a) z scale ) 300 nm and (b) enlarged view of the dashed square area of image a, z scale ) 150 nm.
To conclude, we have shown that amyloid-like peptide fibril micropatterned arrays can be created with a lateral position accuracy of approximatley 1 to 2 µm by AFM-CW and electrostatic deposition from a dispersion. We believe that the great advantage of this generic method for patterning soft materials on solid surfaces is that the functional units are enclosed in water droplet “containers”. Hence, the actual attachment process is relatively independent of the content of the droplets, which means that a wide range of nanosized objects and biological materials can be patterned. Furthermore, charge patterns can easily be generated at high resolution on a wide range of materials, making the method very widely applicable.27 Future studies will investigate the fundamental interfacial properties of fibrils dispersed in these emulsions to further improve the spatial resolution and accuracy of this technique and to use these peptide microarrays for biosensing applications. Acknowledgment. We thank Dr. Cait MacPhee, Cavendish Laboratory, University of Cambridge, U.K., and Professor Mike Horton, Department of Medicine, University College London, U.K., for providing the TTR105-115 peptide, fruitful discussions, and support. This research is funded by the Interdisciplinary Research Collaboration (IRC) in Nanotechnology and is further supported by the London Centre for Nanotechnology (LCN). R.A.M. is a Dorothy Hodgkin Royal Society Research Fellow. LA061485T
(26) Fluorinert FC77 product information, 3M Company, St. Paul, MN (available under “electronic materials” from http://www.3m.com/).
(27) Wright, W. M. D.; Chetwynd, D. G. Nanotechnology 1998, 9, 133.
