Organization of Peptide Nanotubes into Macroscopic Bundles

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Organization of Peptide Nanotubes into Macroscopic Bundles Hiroshi Matsui*,† and Gary E. Douberly, Jr.‡ Department of Chemistry, Hunter College of The City University of New York, New York, New York 10021, and Department of Chemistry, University of Central Florida, Orlando, Florida 32816 Received June 18, 2001. In Final Form: September 18, 2001 A simple chemical method to assemble peptide nanotubes and simultaneously arrange them into macroscopic bundles is reported. The peptide nanotubes, self-assemblies of the bolaamphiphilic peptide monomer bis(N-R-amido-glycylglycine)-1,7-heptane dicarboxylate, appeared in acidic solution within 1 week. In the presence of Ni2+ ions, the bolaamphiphile peptide assembles into well-organized macroscopic bundles of the peptide nanotubes. Addition of EDTA to a suspension of peptide nanotube bundles resulted in disassembly of the bundles into peptide nanotubes with diameters between 40 and 140 nm. A majority of the free amide sites of the peptide nanotubes were involved in the intercalation of nickel ions, which act as bridges to bundle adjacent peptide nanotubes. The covalent bridge of Ni2+ ions with amide groups of the peptide nanotube was confirmed by Raman microscopy.

I. Introduction The organization of nanomaterials into macroscopic materials with practical uses is of recent interest. For example, the processing of single-walled carbon nanotubes (SWNTs) on macroscopic scales has recently been demonstrated.1,2 Ribbons and fibers of SWNTs were fabricated with diameters up to microns and lengths as high as 1 m. The nanotubes, suspended in a surfactant solution, were recondensed in the flow of a polymer solution, and the resulting macroscopic fibers consisted of SWNTs with preferential orientations.1 The use of transition metal ions in mediating molecular self-assembly has also proven useful in the formation of supramolecular structures. A well-ordered lipid bilayer stack was assembled when Cu2+ ions acted as bridges connecting lipid bilayers that contained iminodiacetic acid receptor sites along the bilayer surface.3 Dipalmitoyl-DLR-phosphatidyl-L-serine (DPPS) Langmuir-Blodgett films were deposited layer by layer with Pt2+ ions, and the ions stabilized the layer assembly since the ions formed coordinate covalent bonds between the serine headgroups.4 In addition, the coordination of zirconium to an organic monomer, N,N,N′,N′′-tetrasalicylidene-3,3′-diaminobenzidene, resulted in the formation of homogeneous metalcoordinated polymer ultrathin films.5 In each case, transition metal ions facilitated the formation and organization of supramolecular structures by interacting covalently with the molecular assemblies. This report examines whether this principle can be applied to facilitate the formation of macroscopic bundles of nanotubes. * To whom correspondence should be addressed. E-mail: [email protected]. † The City University of New York. ‡ University of Central Florida. (1) Vigolo, B.; Penicaud, A.; Coulon, C.; Sauder, C.; Pailler, R.; Journet, C.; Bernier, P.; Poulin, P. Science 2000, 290, 1331. (2) Gommans, H. H.; Alldredge, J. W.; Tashiro, H.; Park, J.; Magnuson, J.; Rinzler, A. G. J. Appl. Phys. 2000, 88, 2509. (3) Waggoner, T. A.; Last, J. A.; Kotula, P. G.; Sasaki, D. Y. J. Am. Chem. Soc. 2001, 123, 496. (4) Fanucci, G. E.; Backov, R.; Fu, R.; Talham, D. R. Langmuir 2001, 17, 1660. (5) Byrd, H.; Holloway, C. E.; Pogue, J.; Kircus, S.; Advincula, R. C.; Knoll, W. Langmuir 2000, 16, 10322.

Figure 1. (a) Chemical structure of the peptide monomer, bis(N-R-amido-glycylglycine)-1,7-heptane dicarboxylate. (b) Selfassembled structure of the peptide nanotube (more detailed information is available in ref 6). (c) Illustration of potential ion-chelating sites of the peptide nanotubes. Yellow arrows indicate that the atoms bind neighboring peptide monomers via hydrogen bonds.

The recent development of a peptide nanotube6 has found many important applications.7-11 Oligopeptide bolaamphiphile monomer, bis(N-R-amido-glycylglycine)1,7-heptane dicarboxylate (Figure 1a), was self-assembled (6) Matsui, H.; Gologan, B. J. Phys. Chem. B 2000, 104, 3383.

10.1021/la010910+ CCC: $20.00 © 2001 American Chemical Society Published on Web 11/10/2001

Organization of Peptide Nanotubes

to the peptide nanotubes in acidic solution within 2 weeks (Figure 1b).6 The peptide nanotubes were assembled in a long, aggregated form and broken down to individual nanotubes via sonication. These peptide nanotubes have diameters in the range from 20 nm to 1 µm, and their aspect ratios are determined by the degree of sonication.9 The peptide nanotube is assembled via intermolecular hydrogen bonds between amide groups and carboxylic acid groups of the bolaamphiphile peptide molecules (Figure 1c). One bolaamphiphile peptide molecule contains at least one free amide site in the tubule assembly. Since all proteins possess amide functional groups capable of interacting with the tubule free amide sites via hydrogen bonds, uniform coatings of protein molecules on the template tubules were observed.9 The similar hydrogen bond driven coating mechanism was also observed in coatings of carboxylic acid-thiol capped Au nanocrystals onto the peptide tubules.8 Here, a simple method for organizing peptide nanotubes into macroscopic bundles is reported. By use of the reactive amide sites on the peptide nanotubes (Figure 1c), the macroscopic bundles were fabricated as clusters of peptide nanotubes. The bundling of peptide nanotubes was facilitated by the coordinate covalent interactions between nickel ions and the reactive amide groups of the peptide nanotube. Recently, we produced protein-functionalized peptide nanotubes with various complementary receptors and anchored them on the acceptor/self-assembled monolayer.11 This new biological recognition driven, nonlithographic fabrication technique will create a new geometry for nanodevices in a simple and economical manner using the peptide nanotubes as building blocks. To apply the peptide nanotube based devices to harsh environments, the fabricated nanotubes are preferred to have a higher mechanical strength. The nanotube-bundling technique reported here will increase the potential for real-world applications of the peptide nanotubes. II. Experimental Section Materials. Bis(N-R-amido-glycylglycine)-1,7-heptane dicarboxylate was prepared according to previously published procedures.12 NiCl2‚6H2O was obtained through Aldrich and used as received. Fabrication of Bundled Peptide Nanotubes. A stock solution of the bolaamphiphile peptide monomer was prepared by adding 23.2 mg of the monomer to 6 mL of 30 mM NaOH. The pH was adjusted to 6.0 by the addition of 50 mM citric acid. The stock solution was added to NiCl2‚6H2O that resulted in the Ni2+ concentrations of 5 mM, 10 mM, 20 mM, 200 mM, and 2 M. The samples were allowed to sit at room temperature for 1 week. Bundled peptide nanotubes were washed with deionized water using a microcentrifuge (13 000 s-1). The stock solution was also added to appropriate amounts of NaCl to prepare 20, 40, 200, and 400 mM Na+ solutions. These solutions were also allowed to stand at room temperature for 1 week. Raman Spectroscopy. Glass slides were rinsed with hexane, and washed peptide nanotube/bundle solutions were deposited onto the slides and dried at room temperature. Raman spectra were acquired with a LabRam (Jobin Yvon/Horiba, Edison, NJ) (7) Matsui, H.; Pan, S.; Gologan, B.; Jonas, S. J. Phys. Chem. B 2000, 104, 9576. (8) Matsui, H.; Pan, S.; Douberly, G. E. J. J. Phys. Chem. B 2001, 105, 1683. (9) Douberly, G. E. J.; Pan, S.; Walters, D.; Matsui, H. J. Phys. Chem. B 2001, 105, 7612. (10) Matsui, H.; Gologan, B.; Pan, S.; Douberly, G. E. J. Eur. Phys. J. D 2001, 16, 403. (11) Matsui, H.; Porrata, P.; Douberly, G. E. J. Nano Lett. 2001, 461. (12) Kogiso, M.; Ohnishi, S.; Yase, K.; Masuda, M.; Shimizu, T. Langmuir 1998, 14, 4978.

Langmuir, Vol. 17, No. 25, 2001 7919 confocal Raman microscope. The 632.8 nm emission from an integrated He-Ne laser was spatially filtered and then injected into an integrated Olympus BX40 microscope by a holographic notch filter. A 100× working distance objective focused the beam to provide approximately 6 mW in a spot of less than 1 µm in diameter. The Raman scattering was collected at 180° geometry by the focusing objective, and the reflected excitation laser was rejected by a notch filter. The filtered radiation was focused at the entrance slit (250 µm) of a 0.3 mm spectrograph. An 1800 gr/mm grating dispersed the spectrum across a 1024 × 256 pixel TE-cooled CCD detector, providing a dispersion of approximately 1 cm-1/pixel. Scanning Electron Microscopy (SEM) and Energy Dispersive Spectrometry (EDS). Peptide nanotube and the bundle samples were washed thoroughly with deionized water and dried onto Si wafers. Prior to SEM and EDS analysis, all samples were dried in a vacuum oven overnight at room temperature. Scanning electron micrographs were obtained using a JEOL JSM-6400F scanning electron microscope at 10 kV accelerating voltage. A 4 nm layer of a Au/Pd alloy was deposited onto samples using a sputter coater prior to SEM imaging.

III. Results and Discussion Formation of Peptide Nanotube Bundles from IonContaining Peptide Monomer Solutions. All results described in this section are summarized in Figure 2. When the bolaamphiphile oligopeptide monomer was assembled in the presence of Ni2+ ions, macroscopic bundles were observed within 1 week with diameters of 100 µm and lengths up to 3 mm. The nanotube bundling was observed for the samples having concentrations between 20 and 200 mM Ni2+. Figure 3a is an optical micrograph of a cluster of peptide nanotubes, which we call a “peptide nanotube bundle”, assembled in the presence of 20 mM Ni2+. Within each bundle, the peptide nanotubes are preferentially aligned along the bundle’s major axis. Samples with Ni2+ concentrations less than 20 mM did not form bundles of the peptide nanotubes. The precipitation of the bolaamphiphile peptide monomer from solution was observed after 2-3 days for samples with concentrations above 200 mM. We also examined the nanotubebundling experiments in Cu2+ solutions between 5 mM and 2 M, and the nanotube bundling was observed in the concentrations less than 20 mM. The electrostatic effect of ions on the bundling process was investigated by comparing with the peptide nanotube assembly in a NaCl solution of the same ionic strength as the NiCl2 solution. After 1-2 weeks, the peptide nanotubes were observed, but bundling of nanotubes was not observed. The nanotubes do not appear to bundle regardless of the Na+ concentrations. The Raman spectrum of the peptide nanotubes obtained from NaCl solutions is identical to the Raman spectrum of peptide nanotubes grown in suspension without the addition of the ions. EDS of the nanotubes produced in the presence of Na+ ions did not show peaks corresponding to Na. To confirm the involvement of Ni2+ ions to the bundling process, Ni2+ ions were removed from the bundles by adding EDTA. If the peptide nanotubes are bundled under the influence of Ni2+ ions, removal of Ni2+ ions should disassemble the bundles to the peptide nanotubes. Upon addition of EDTA (50 mM in 50 mM NaOH) to a suspension of the bundles, the bundles lost their structural integrity and peptide nanotubes no longer had preferential alignment (Figure 3b). These smaller clusters of tubules in Figure 3b were further broken down to the diameters of 40-140 nm after sonicating them with EDTA for 1 h. Scanning electron micrographs of the disassembled peptide nanotubes and the individual peptide nanotube are shown in Figure 3c,d. The reassembly of disassembled

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Figure 2. Summary of the nanotube bundle production in various experimental conditions: (a) Peptide monomer solutions with moderate Ni2+ concentrations yield the nanotube bundles. (b) Peptide monomer solutions with low Ni2+ concentrations yield the individual nanotubes. (c) Peptide monomer solutions with high Ni2+ concentrations yield no individual nanotubes and no nanotube bundles. (d) Peptide monomer solutions with any Na2+ concentration yield the individual nanotubes. (e) Peptide nanotube bundles are disassembled after EDTA is added. (f) After Ni2+ ions are added to individual peptide nanotube solutions, no peptide nanotube bundles are observed.

peptide nanotubes was not observed in 2 weeks after sonication. When the peptide nanotube bundles with Cu2+ ions were disassembled by EDTA, higher EDTA concentrations (>80 mM) and longer sonication (>2 h) were necessary. From those results, the bundle assembly mechanism proposed is that the bundle formation is stabilized by coordinate covalent bonds between amide groups of the peptide nanotubes and Ni2+ ions. The formation of square planar bis(glycino) nickel complexes between the free amide groups and Ni2+ ions acts to bridge the peptide nanotubes together in solution (Figure 4). Since the free amide sites are distributed uniformly throughout the nanotube assembly, a very well ordered bundle of the peptide nanotubes seems to be formed, as shown in Figure 3a. The observation of peptide nanotube bundle disassembly with EDTA supports the proposed bundling mechanism that the Ni2+-amide interaction is a major force to bundle peptide nanotubes. High ionic concentrations did not produce any nanotubes. In the high ionic concentrations, it is possible that ions bind most of amide groups in the peptide monomers and there are no

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remaining free amide groups necessary to self-assemble the peptide monomers into the tubule structure. Therefore, the lack of amide groups in peptide monomers for the tubule assembly may shut down the production of peptide nanotubes in solutions with high ionic concentrations. Observation of the nanotube bundling in solutions with lower Cu2+ concentrations and the nanotube bundle disassembly in solutions with higher EDTA concentration may be explained by affinity of Cu2+ ions to ligands. Cu2+ shows higher affinity to bind ligands than Ni2+, and therefore it is reasonable that Cu2+ ions bundle peptide nanotubes in lower ionic concentrations than Ni2+ ions.13,14 Since stronger affinity of Cu2+ ties the nanotube bundles more strongly, higher EDTA concentrations seem to be required to disassemble the bundles. Within the wide concentration range tested, 20-400 mM, Na+ ions do not affect the final structures of the peptide nanotubes since Na+ cannot form the metal covalent bridges with the amide groups of the peptide nanotube. Therefore, it implies that the electrostatic effect of Ni2+ ions on the bundling process is nominal. Peptide nanotube bundling must be facilitated by covalent metal coordination of adjacent nanotubes. Neither bundling nor any significant aggregation of the peptide nanotubes was observed when the peptide nanotubes were produced in the pH 6.0 citric acid solution without Ni2+ ions first, and then the peptide nanotubes were mixed with the Ni2+ ion solutions. This observation suggests that the Ni2+ ion chelating effect is not strong enough to bring the peptide nanotubes together in the form of the bundles. Raman Microscopy in Peptide Nanotube Bundles. Raman microscopy and EDS were applied to the peptide nanotube bundles to confirm that the bundle formation is stabilized by coordinate covalent bonds between amide groups of the peptide nanotubes and Ni2+ ions. Figure 5a is the Raman spectrum for a peptide nanotube bundle assembled in 20 mM Ni2+. The Ni-O asymmetric stretch mode and the Ni-N symmetric stretch mode appeared at 280 and 440 cm-1, respectively. These assignments are consistent with vibrational frequencies of Ni-O and Ni-N stretching modes in bis(glycino) Ni complexes, 289 and 441 cm-1.14 EDS confirmed Ni on the bundle and showed that the intensity ratio of nickel KR and carbon KR peaks is 1:36. The observed Ni-O and Ni-N vibrational stretching modes in the Raman spectrum of the peptide nanotube bundle indicate that nickel ions bind covalently with the peptide molecules of the nanotube. This observation supports the proposed bundling mechanism. The three peaks between 1600 and 1700 cm-1 in the Raman spectrum of the bundle (Figure 5a) correspond to CdO stretch modes of amide groups with differing degrees of intermolecular hydrogen bonding. A peak at 1635 cm-1 corresponds to amide groups involved in amide-amide hydrogen bonding between adjacent bolaamphiphile peptide molecules in a single peptide nanotube.6 A peak at 1660 cm-1 corresponds to amide groups free from hydrogen bonds.6 These two CdO peaks of amides in the bundle Raman spectrum, the 1635 and the 1660 cm-1, are consistent with the CdO peaks observed in the Raman spectrum of the single peptide nanotube produced without the presence of Ni2+ ions.6 Therefore, these two CdO peaks originated from the peptide nanotubes in the bundle. However, a peak at 1647 cm-1 observed in the bundle spectrum is absent in the Raman spectrum for the peptide nanotube. (13) Jolly, E. L. Synthetic Inorganic Chemistry; Prentice Hall: Englewood Cliffs, NJ, 1960. (14) Nakamoto, K. Infrared and Raman spectra of inorganic and coordination compounds; John Wiley & Sons: New York, 1997; Part B.

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Figure 3. (a) Optical micrograph of the peptide nanotube bundle (i.e., the cluster of peptide nanotubes). (b) Optical micrograph of the smaller clusters of peptide nanotubes disassembled from the larger bundle (a) with EDTA. (c) Scanning electron micrograph of the peptide nanotubes disassembled further with EDTA and sonication. (d) Scanning electron micrograph of the single peptide nanotube in (c) with higher magnification.

Figure 5. Raman spectra of (a) the bundle and (b) the peptide nanotube disassembled from the bundle with EDTA.

Figure 4. Illustration of assembled structure of the peptide nanotube bundle.

The 1647 cm-1 peak was tentatively assigned as the CdO stretch of amide groups involved in amide-amide crossnanotube hydrogen bonding (Figure 4). The CdO stretching mode of the cross-nanotube hydrogen bond, the 1647 cm-1, is shifted to blue compared to the CdO mode of the single peptide nanotube involved in the intermolecular

hydrogen bonding, the 1635 cm-1. Therefore, the crossnanotube hydrogen bonding is considered weaker than the intermolecular hydrogen bonding within each peptide nanotube. A CdO peak of amide groups binding with Ni2+ should appear around 1589 cm-1.15 This peak was not observed in the Raman spectrum. This peak appears with very strong intensity in IR, but the Raman activity of this vibration seems to be much weaker.14 Adjacent peptide nanotubes, bridged by nickel ions along the length of the nanotubes, seem to undergo further stabilization of the bundle formation due to the regular, uniform cross(15) Kincard, J. R.; Nakamoto, K. Spectrochim. Acta 1976, 32A, 277.

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nanotube hydrogen bonding. Therefore, it is reasonable to consider that both Ni2+ bridging complexes and crossnanotube hydrogen bonds contribute in order to assemble peptide nanotubes into macroscopic bundles (Figure 4). Although cross-nanotube hydrogen bonding may further stabilize the nanotube bundle, the Ni2+-amide bond must be the dominant binding interaction since bundles do not form in the absence of Ni2+ and the bundles disassembled upon removal of Ni2+. The Raman spectrum of an individual peptide nanotube is shown in Figure 5b. The disassembled nanotubes were obtained via the addition of EDTA to the bundle suspension. The vibrational modes corresponding to Ni-O and Ni-N stretching modes were not observed. The peak at 330 cm-1 was assigned as a vibrational mode of the peptide nanotube from the comparison between spectra a and b of Figure 5. The signal-to-noise level of the Raman spectrum for the peptide nanotube in Figure 5b was lower than that of the bundle spectrum because the Raman scattering was weaker from much smaller peptide nanotubes. EDS for the peptide nanotube did not show peaks corresponding to nickel. This observation indicates that a majority of Ni2+ ions were bound to amide groups of the peptide nanotubes to bundle the nanotubes. IV. Conclusions In the presence of Ni2+ ions, macroscopic bundles of well-ordered peptide nanotubes were assembled. The organization of peptide nanotubes in the bundles seems to be driven by the ability of adjacent nanotubes to form metal coordination bridges. The cross-nanotube hydrogen bonding between amide groups of the peptide nanotubes was also observed, but its contribution to the bundling process is considered nominal. Since the locations of free amide groups are highly ordered along the length of the nanotubular assembly, well-organized macroscopic bundles

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of the peptide nanotubes with preferred alignments were observed. The interaction of Ni2+ ions with amide groups of peptide nanotubes is considered to be covalent since the peptide nanotube bundles were not observed in Na+ ion solution, which cannot form the metal coordinate covalent bridges with amides. This proposed binding scheme was also supported by Raman microscopy and energy dispersive spectrometry. Although the bundle was assembled from the Ni2+/ peptide monomer solution at pH 6.0 and Ni2+ ions were identified to locate between the peptide nanotubes in the bundles, we do not completely understand how Ni2+ ions are involved during the bundle assembly process. At this point, it is not clear why peptide monomers do not form single macrotubules under the influence of Ni2+ ions. This reason may be quite complicated due to various dynamic contributions to molecular assembly such as charge interactions and hydrogen bonds between the monomer and solvents. In addition to versatility of peptide nanotubes via various functionalizations,7-11 this bundling technique will add mechanical strength to peptide nanotube based devices such as sensors and electronics. A technical challenge for real-world applications of the nanotube bundles is to control the topology of nanotube bundles. Experimental conditions such as temperature, pressure, and solvents may control bundle dimensions via thermodynamics. Further bundling studies in various experimental conditions are necessary. Acknowledgment. This work was supported by Florida Hospital, the National Science Foundation NER program, and the Department of Energy, Office of Basic Energy Sciences. G.D. acknowledges Mr. Zia Ur Rahman for assistance with the SEM and EDS studies. LA010910+