pubs.acs.org/Langmuir © 2009 American Chemical Society
Thermomechanical Manipulation of Aromatic Peptide Nanotubes Victoria L. Sedman, Stephanie Allen, Xinyong Chen, Clive J. Roberts, and Saul J. B. Tendler* Laboratory of Biophysics and Surface Analysis, School of Pharmacy, The University of Nottingham, Nottingham, NG7 2RD, United Kingdom Received May 7, 2009 Self-assembling aromatic dipeptides are among the smallest known biological materials which readily form ordered nanostructures. The simplicity of nanotube formation makes them highly desirable for a range of bionanotechnology applications. Here, we investigate the application of the atomic force microscope as a thermomechanical lithographic tool for the machining of nanotubes formed by two self-assembling aromatic peptides; diphenylalanine and dinapthylalanine. Trenches and indentations of varying depth and width were patterned into the peptide tubes with nanometer precision highlighting the ability to thermally machine and manipulate these robust and versatile nanotubes.
1. Introduction The great potential of bottom-up approaches to the generation of novel functional materials is related to the diversity of building blocks available which utilize self-assembling molecules (e.g., alkane thiols, carbon, DNA, RNA, and peptides)1-5 and which can be modified to direct a specific function. A central challenge to this emerging area of material science is the ability to control and manipulate both the building blocks and their assembled structures into functionalized materials through direct nanotechnology applications. In this study, we demonstrate the thermomechanical lithography of self-assembled biological nanostructures; the aromatic peptide nanotubes using a novel form of atomic force microscopy (AFM) with the ability to controllably heat the imaging probe. Interest in the use of aromatic peptide nanotubes for biotechnology applications arises, in part, from the mild and inexpensive conditions required for their self-assembly, but also more importantly from their robust physical properties.6-11 Here, we focus on the self-assembling aromatic peptide L-diphenylalanine (FF)12 and an analogue with higher aromatic content, di-D-2napthylalanine (di-D-2-Nal),7 both of which readily form tubular nanostructures.12,13 FF nanotubes are chemically and thermally robust,8,9 with widths of 100 nm to 2 μm with a central hollow bore of ∼20 A˚. *Corresponding author. Professor SJB Tendler. Laboratory of Biophysics and Surface Analysis, School of Pharmacy, The University of Nottingham, Nottingham, NG7 2RD, UK. Tel: +44-115-951-5101. Fax: +44-115-9515110. E-mail:
[email protected]. (1) Zhao, X.; Pan, F.; Lu, J. R. Prog. Nat. Sci. 2008, 18, 653–660. (2) Nuzzo, R. G.; Allara, A. L. J. Am. Chem. Soc. 1983, 105, 4481–4483. (3) Chworos, A.; Severcan, I.; Koyfman, A. Y.; Weinkam, P.; Oroudjev, E.; Hansma, H. G.; Jaeger, L. Science 2004, 306, 2068–2072. (4) Iijima, S. Nature 1991, 354, 56–58. (5) Winfree, E.; Liu, F.; Wenzler, L. A.; Seeman, N. C. Nature 1998, 394, 539–544. (6) Gorbitz, C. H. Chem. Commun. 2006, 2332–2334. (7) Reches, M.; Gazit, E. Phys. Biol. 2006, 3, S10–S19. (8) Adler-Abramovich, L.; Reches, M.; Sedman, V. L.; Allen, S.; Tendler, S. J. B.; Gazit, E. Langmuir 2006, 22, 1313–1320. (9) Sedman, V. L.; Adler-Abramovich, L.; Allen, S.; Gazit, E.; Tendler, S. J. B. J. Am. Chem. Soc. 2006, 128, 6903–6908. (10) Kol, N.; Adler-Abramovich, L.; Barlam, D.; Shneck, R. Z.; Gazit, E.; Rousso, I. Nano Lett. 2005, 5, 1343–1346. (11) Niu, L.; Chen, X.; Allen, S.; Tendler, S. J. B. Langmuir 2007, 23, 7443–7446. (12) Reches, M.; Gazit, E. Science 2003, 300, 625–627. (13) Song, Y.; Challa, S. R.; Medforth, C. J.; Qiu, Y; Watt, R. K.; Pe~na, D.; Miller, J. E.; van Swol, F.; Shelnutt, J. A. Chem. Commun. 2004, 1044–1045.
7256 DOI: 10.1021/la9016273
The tubes are single or multiwalled and display high persistence lengths and considerable rigidity and strength; an averaged point stiffness of 160 N/m and Young’s modulus of 19 GPa have been reported.10,11 The high level of stability exhibited by FF nanotubes is conferred by π-π stacking interactions and the extensive hydrogen bonding interactions of the peptide backbone.6,14 By comparison, di-D-2-Nal nanotubes display similar high persistence lengths but have smaller dimensions with widths of 50 nm to 1 μm, comprising single or multiwalled elongated tubes.7 The comparative mechanical properties of the di-D-2-Nal nanotubes have yet to be fully characterized. The functionalization of the FF nanotubes by decoration or impregnation with metal and chemical moieties has been demonstrated.7,13,15-18 Aromatic peptide nanotubes have been exploited for a range of applications including biocompatible hydrogels through chemical modification of the peptides for potential uses in tissue engineering;16 as nanoelectrodes for biosensors,17,18 and in microelectronics through decoration of the tubes with inorganic moieties to generate novel composite materials;13 or as coaxial nanocables15 or as degradable scaffolds for nanowire generation.7 To maximize the applicability of the aromatic peptide nanotubes, techniques for their controlled deposition or patterning have focused on the manipulation of their physical properties, for example, the use of magnetic fields to align FF nanotubes into ordered lateral arrays.19,20 Here, we focus on exploitation of their thermal stability8,9 for the machining of regular features in discrete peptide nanotubes utilizing the nanoscale precision of nanothermal AFM (NT-AFM). In NT-AFM, the traditional AFM imaging probe is replaced with a doped-silicon cantilever with a heated probe. This enables its use for probing nanoscale thermal events with nanometer precision crucial for investigating nanostructures.21 (14) Reches, M.; Gazit, E. Nano Lett. 2004, 4, 581–585. (15) Carny, O.; Shalev, D. E.; Gazit, E. Nano Lett. 2006, 6, 1594–1597. (16) Mahler, A.; Reches, M.; Rechter, M.; Cohen, S.; Gazit, E. Adv. Mater. 2006, 18, 1365–1370. (17) Yemini, M.; Reches, M.; Gazit, E.; Rishpon, J. Anal. Chem. 2005, 77, 5155–5159. (18) Yemini, M.; Reches, M.; Rishpon, J.; Gazit, E. Nano Lett. 2005, 5, 183–186. (19) Hill, R. J.; Sedman, V. L.; Allen, S.; Williams, P. M.; Paoli, M.; AdlerAbramovich, L.; Gazit, E.; Eaves, L.; Tendler, S. J. B. Adv. Mater. 2007, 19, 4474–4479. (20) Reches, M.; Gazit, E. Nat. Nanotechnol. 2006, 1, 195–200. (21) Nelson, B. A.; King, W. P. Rev. Sci. Instrum. 2007, 78, 023702.
Published on Web 06/04/2009
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NT-AFM cantilevers have a conductive coating through which an electrical current is passed to an integrated heater located directly above the probe. By varying the resistance of the circuit, the temperature of the heater can be controlled up to 500 °C. Following thermal calibration of the probe,22,23 when the heated tip is brought into contact with a sample surface the deflection of the cantilever is recorded versus temperature, and a thermal plot can be generated. Variations in the cantilever deflection reveal the occurrence of thermal phase transitions, melting or glass transitions of the material,21 as well as the nature of a material (amorphous versus crystalline).24,25 The thermal conductivity of a material can also be mapped and the topography of a surface imaged using the thermal probe.26,27 NT-AFM has been employed as a thermal lithography tool including the controlled decomposition and analysis of polymers22,28 and thermal dip pen lithography of metals and organic molecules.29,30 Furthermore, direct applications of heated AFM cantilevers in the “millipede” for thermomechanical writing of nanometer-sized digital data in polymer coatings has been explored for thermal AFM machining of erasable data storage devices.31
2. Experimental Section All peptide nanotubes used in this study were prepared using the 1,1,1,3,3,3-hexafluoro-2-propanol (HFIP)/water method developed by Gazit12 to a final concentration of 2 mg mL-1. Aliquot samples of the peptide solutions were placed onto freshly cleaved mica substrates and dried under nitrogen gas. To generate a localized melt of a nanotube, the probe was placed on a target nanotube, the raster scan and NT-AFM feedback mechanism were disabled, and the probe was heated with a steady heating rate of 10 °C s-1 for a predetermined period of time or until a dramatic drop in the cantilever deflection was observed. Nanothermal mechanical analysis was performed with a NanoTA2 system (Anasys Instruments, CA) using tapping-mode nanothermal AN2 probes (spring constant 0.7-2 N m-1 and resonant frequency 50-100 kHz) with nominal apex radii of 30 nm or less. A constant tip-sample load was maintained during contact. Probes and the NanoTA2 system were calibrated at the start of each experiment using polymer samples with known melting points. Images of the samples before and after heating were generated using a Digital Instruments Multimode AFM with a Nanoscope V controller (Digital Instruments, Veeco Metrology, CA).
3. Results and Discussion To establish the effectiveness of the NT-AFM for the controlled thermomechanical patterning of FF and di-D-2-Nal nanotubes, discrete full melts of the nanotubes were performed in which the probe was heated while in contact with the nanotube surface until a drop in cantilever deflection was observed. Illustrated in Figure 1 (22) King, W. P.; Saxena, S.; Nelson, B. A.; Weeks, B. L.; Pitchimani, R. Nano Lett. 2006, 6, 2145–2149. (23) Meyers, G.; Pastzor, A.Jr.; Kjoller, K. Am. Lab. 2007, 39, 9–14. (24) Harding, L.; King, W. P.; Dai, X.; Craig, D. Q. M.; Reading, M. Pharm. Res. 2007, 24, 2048–2054. (25) Royall, P. G.; Kett, V. L.; Andrews, C. S.; Craig, D. Q. M. J. Phys. Chem. B. 2001, 105, 7021–7026. (26) Haeberle, W.; Panteaa, M.; Hoerber, J. K. H. Ultramicroscopy 2006, 106, 678–686. (27) Kim, K. J.; Park, K.; Lee, J.; Zhang, Z. M.; King, W. P. Sens. Actuators, A 2007, 136, 95–103. (28) Fang, T.-H.; Chang, W.-J. Appl. Surf. Sci. 2005, 240, 312–317. (29) Nelson, B. A.; King, W. P.; Laracuente, A. R.; Sheehan, P. E.; Whitman, L. J. Appl. Phys. Lett. 2006, 88, 033104. (30) Sheehan, P. E.; Whitman, L. J.; King, W. P.; Nelson, B. A. Appl. Phys. Lett. 2004, 85, 1589–1591. (31) Binnig, G.; Despont, M.; Drechsler, U.; H€aberle, W.; Lutwyche, M.; Vettiger, P.; Mamin, H. J.; Chui, B. W.; Kenny, T. W. Appl. Phys. Lett. 1999, 74, 1329–1331.
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Figure 1. Nanothermal AFM imprinting of FF and di-D-2-Nal aromatic peptide nanotubes. Typical AFM topographic images of the peptide aromatic nanotubes following nanothermal heating events are shown in (a) for a FF tube and (b) for a di-D-2-Nal tube. Contrast height scale bars are presented next to the images.
are typical examples of the resultant damage incurred by the FF (Figure 1a) and di-D-2-Nal (Figure 1b) nanotubes following heating. Topography images of the damaged area reveal that the impact of the heated probe is limited to a localized area and that the probe has protruded completely through the nanotube to the visible mica beneath. The shape of the NT-AFM pyramidal tip is clearly observed in the thermal imprint of the FF nanotube (Figure 1a). An estimate for the average number of moles of peptide excavated from the FF nanotubes during an individual lithographic process is approximately 380 amol of material. The scale of this excavation demonstrates that the damage exhibited by the nanotube is localized to the nanosized tip contact area and not dissipated through the material. Previous studies utilizing the nanothermal probe for thermal analysis of pharmaceutical materials also demonstrated a similar localized heating of the sample with decomposition restricted to a nanometer-sized probe contact area.22 In contrast, and without exception, a trench was observed sectioning completely through the di-D-2-Nal nanotubes (Figure 1b). It is evident that there has also been movement of peptide material during heating to the side of the tube. This is most likely attributable to the softening of the di-D-2-Nal material surrounding the tip contact area during heating, resulting in flow and cooling of the material away from the contact area. This, in turn, would result in lateral drift of the heated probe generating a line across the tube. Although it should be remembered that the di-D2-Nal nanotubes have smaller dimensions (nominal NT-AFM probe apex ∼15 nm versus range of tube widths 50-350 nm) than the FF tubes, it is probable that the nanothermal heating of these nanotubes actually reveals a difference in the stability and packing of the peptide within the two types of nanotube. The higher aromatic content of the di-D-2-Nal peptide compared to FF tubes may contribute to a greater order and π-stacking interactions of the peptide within the nanotubes. However, the additional bulk contributed by these aromatic rings may have resulted in an altered (less favorable) packing in comparison to the that of the FF nanotubes, thus contributing to a changed thermal stability. DOI: 10.1021/la9016273
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Figure 2. Series of AFM topographic images of FF nanotube following incremental temperature increases of the nanothermal probe. Images were generated at room temperature, (a) preheating, and following nanothermal heating at (b) 80, (c) 90, (d) 100, (e) 110, and (f ) 120 °C. For clarity, the center of the boxed area highlights the location of the nanothermal probe contact. Cross-sectional line profiles across the boxed area are shown below the corresponding AFM image.
The application of NT-AFM to perform controlled thermal indentations of the aromatic nanotubes was further investigated by combining nanoscale high spatial precision with the ability to vary the temperature of the probe and time of contact, to generate a series of dots in close proximity and of varying depth. To this end, Figure 2 shows a series of AFM topographic images where the nanothermal probe has been moved with regular spacing along the axis of the same nanotube and the temperature raised in 10 °C increments from 80 to 120 °C at each location (Figure 2b-f ). The topographic images demonstrate the thermal softening behavior of the peptide nanotubes. The indentation and extent of thermal decomposition increased as the temperature of the probe was raised, until finally complete localized decomposition was observed at approximately 120 °C. Cross-sectional line profiles across the probe contact areas (region highlighted by the box in the corresponding image) demonstrate the damage incurred as the temperature of the probe was raised producing holes of increasing depth and width. A gradual increase in the damage can be observed up to approximately 110 °C followed by a 63% increase in depth upon further increases in temperature upon which the mica substrate was observed. Control images whereby the probe was not heated but remained in contact with the nanotube surface for an equivalent time period revealed no deformation of the tubes indicating that the observed holes and damage incurred during nanothermal measurements can be attributed to the increasing probe temperature. On exposing the FF nanotubes to probes with lower temperatures in the range 25-100 °C (Figure 2), no debris or deposited material was observed, thus producing clean discrete indentations in the tube surfaces. However, at higher temperatures displaced material was always observed either as residual debris in the surrounding area or as a mound in close proximity to the contact area (Figures 1a and 2f ). In a study by Niu et al., the elasticity of FF nanotubes with increasing temperature was investigated by AFM using the bending beam model; it was reported that the tubes retained stability but exhibited a gradual reduction in elasticity of 30% up to 100 °C, most likely attributable to an increased thermal motion of the FF peptides.11 This reduction in elasticity correlates well with the apparent softening of the nanotube material observed in this study; the heated probe leaves an imprint in the softened sample surface, but temperatures are insufficient for thermal transition or decomposition of the material to occur. However, on heating the tubes at higher temperatures of approximately 110 °C, there is loss of aromatic material 7258 DOI: 10.1021/la9016273
demonstrated by the dramatic increase in probe penetration and displacement of material. Increasing the temperature sufficiently weakens the peptide bonds and interactions which stabilize the packing and structure of the tubes resulting in the sublimation of phenylalanine fragments.9 The sublimed material may then begin to cool as a result of the decreasing temperature gradient away from the heated tip forming mounds or debris in the surrounding area. To further explore the effectiveness of the nanothermal AFM as a lithographic tool for the machining of soft biological material, the generation of nanoprecision lines in the peptide nanotube surfaces was also performed. A series of AFM topography images showing a FF nanotube with trenches thermally created perpendicular to the fibril axis is presented in Figure 3. In all, the varying factors were either time or temperature with the scan rate (0.1 Hz) remaining constant. Lines were drawn by zooming in to the nanotube surface, ensuring that the scan area was equal to that of the nanotube width. Consequently, the time of contact could be controlled by the number of raster scan lines employed. Figure 3a,b shows lines fabricated at 85 °C after 8, 20, and 30 s. From Figure 3a (line 1), it can be seen that an incision has been made across part of the nanotube, which as expected due to the low temperature did not shear through the complete nanotube depth. By comparison, after 20 s at 85 °C a line has been drawn across the entire width of the nanotube and a complete break occurred after 30 s (Figure 3b, lines 3 and 2, respectively). Interestingly, there was complete removal of the excavated material producing clean lines in the tube surfaces. Lines of varying thickness, depth, and length can be formed simply by altering either the contact time or temperature of the probe or both. A 3D representation (Figure 3c) of the nanotube surface and cross-sectional profile along the tube axis (Figure 3d) demonstrates the close spatial proximity of the lines that can be achieved without cross-damage between features. In this study, we observe good reproducibility for the generation of patterned aromatic peptide nanotubes with dots or lines thermally drawn at nanoscale proximity. However, it should be noted that several factors may directly affect the reproducibility of the dimensions of these patterned features and would require further modeling and refinements for future applications, namely, that the size of indentations is dependent on probe dimensions, as well as a precise calibration of the cantilever heater temperature. Future refinements of the manufacturing of these specialized silicon etched heater cantilevers or perhaps through the Langmuir 2009, 25(13), 7256–7259
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Figure 3. Thermal lithography of FF nanotubes by NT-AFM. AFM topography images of FF nanotubes following nanothermal line lithography. Lines were drawn across the width of the nanotube using the nanothermal probe heated to 85, 100, and 120 °C and with varying scan times as shown in the accompanying table. Shown in (a) is a topographic image of a partial line drawn across a nanotube and in (b) is a topographic image of lines drawn at 85 °C for 30 (top) and 20 (bottom) s. A 3D representation of the surface following the drawing of lines under different times and temperature (see accompanying table for line conditions) and a cross-sectional profile along the axis of the nanotube is presented in (c) and (d), respectively.
incorporation of carbon nanotubes may limit and improve the effect of probe apex variations. The accuracy of probe heater calibration is dependent on several factors including the rate of heat transfer between probe and sample and the heat conductance of the sample material versus that of the test polymers used in the calibration procedure.32,33 However, in this instance we found that the calibrated probes used throughout our experiments produce temperature transitions for the sublimation of the FF tubes that correlate well with those previously reported8,9 and that a range of calibrated tips provided holes and lines in the peptide nanotube surface of comparable dimensions.
4. Conclusion The simplicity and ease with which these aromatic nanotubes can be decomposed in a controlled manner at the nanoscale demonstrates the versatility of NT-AFM as a valuable tool for the generation of nanopatterned structures. These peptide nanotubes hold potential for the miniaturization of microprocessors and micromechanical systems which have been restricted to micrometer-sized machined silicon devices. Here, we have shown (32) Fischer, H. J. Therm. Anal. Calorim. 2008, 92, 625–630. (33) Nelson, B. A.; King, W. P. Sens. Actuators, A 2007, 140, 51–59.
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that indents and trenches can be thermally etched into the nanostructures, thus paving the way for their nanofabrication as nano barcodes.34,35 Combined with the proven ability to directly align these aromatic nanotubes into ordered arrays19,20 and decoration with, or internalization of, metal ions,12,13 the versatility and adaptability of the nanothermal patterning technique demonstrated here provides additional application opportunities for the nanotubes as thermally manipulatable biomaterials through nanoscale patterning and as thermally degradable scaffolds. Acknowledgment. We acknowledge the financial support of the EU BeNatural project (STRP 033256). Supporting Information Available: Additional experimental detail and AFM images of the nanotubes following nanothermal heating in which debris can be observed in the surrounding area. This material is available free of charge via the Internet at http://pubs.acs.org. (34) Nicewarner-Pe~na, S. R.; Freeman, R. G.; Reiss, B. D.; Lin, H.; Pe~na, D. J.; Walton, I. D.; Cromer, R.; Keating, C. D.; Natan, M. J. Science 2001, 294, 137–141. (35) Yan, H.; LaBean, T. H.; Feng, L.; Reif, J. H. Proc. Natl. Acad. Sci. U.S.A. 2003, 100, 8103–8108.
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