NANO LETTERS
Binary Nanomaterials Based on Nanocarbons: A Case for Probing Carbon Nanohorns’ Biorecognition Properties
2003 Vol. 3, No. 8 1033-1036
Jin Zhu,*,† Daisuke Kase,‡ Kiyotaka Shiba,*,‡ Daisuke Kasuya,† Masako Yudasaka,† and Sumio Iijima†,§,| Carbon Nanotube Project, Japan Science and Technology Corporation, c/o NEC, 34 Miyukigaoka, Tsukuba 305-8501, Japan, Department of Protein Engineering, Cancer Institute, Kami-Ikebukuro, Toshima, Tokyo 170-8455, Japan, Department of Physics, Meijo UniVersity, Tenpaku-ku, Nagoya 468-8502, Japan, and NEC Corporation, 34 Miyukigaoka, Tsukuba 305-8501, Japan Received April 28, 2003; Revised Manuscript Received June 6, 2003
ABSTRACT A critical step toward the construction of complex architectures based on nanoscale carbonaceous materials is to interface these structures with other useful nanoscale building blocks. Herein we report the synthesis of a new class of binary nanomaterials from single-walled carbon nanohorns and nanoparticles by utilizing a bifunctional molecule as the bridging interconnect. Characterization of the materials by transmission electron microscopy, energy-dispersive X-ray spectroscopy, Raman spectroscopy, and thermogravimetric analysis unambiguously proves the formation of binary nanostructures. The strategy reported here is expected to be generic and readily applicable to carbon nanohorns’ interfacing with other nanoscale materials, such as Pt, in the construction of fuel cells. Significantly, with these binary nanomaterials, distinct differences in peptide recognition properties have been identified for carbon nanohorns treated under different conditions through a phage-display enzymelinked immunosorbent assay. Those peptide recognition motifs are important for exploiting this class of materials in bioassembly, bioseparation, and biosensing applications.
One of the most intriguing events in materials science during the last couple of decades has been the discovery of carbon nanotubes.1 Fundamental and technological investigations have been carried out on this fascinating class of materials.2 Recently, during the investigation of the carbon nanotube formation mechanism, a new type of graphene structures single-walled carbon nanohorns (SWNHs)swas discovered.3 As single-component materials, they have proven to be extremely useful in a variety of applications, such as gas adsorption and the construction of supercapacitors.4 SWNHs are a class of interesting spherical materials with substructures composed of nanoscale cone-shaped graphene sheets. A method for synthesizing more complex structures based on this type of material has yet to be developed. Increasing the compositional and functional diversities by interfacing with other material components is the key to probing the fundamental properties and extending the applications of * To whom correspondence should be addressed. E-mail:
[email protected]. go.jp;
[email protected]. † Japan Science and Technology Corporation. ‡ Cancer Institute. § Meijo University. | NEC Corporation. 10.1021/nl034266q CCC: $25.00 Published on Web 06/25/2003
© 2003 American Chemical Society
SWNHs.5 Herein we report an organic interconnect-mediated synthesis of a new class of SWNH/nanoparticle (specifically, magnetic nanoparticle, MNP) binary nanomaterials. An application of this type of material in the investigation of SWNHs’ biorecognition properties, through a phage-display method, is also discussed. SWNHs were generated by laser vaporization, as reported previously.3 Briefly speaking, a CO2 laser (wavelength 10.6 µm, powder density ∼20 kW/cm2, pulse width 500 ms, frequency 1 Hz) was used to hit the graphite target rod, which was rotated at ∼6 rpm in the presence of a buffer gas. The production of either “dahlia”- or “bud”-shaped structures could be easily controlled by changing the buffer gas. The carbon nanohorns used here are typically dahlia-like because they have interesting cone-shaped substructures. The synthesis of SWNH/MNP binary nanomaterials was carried out through a bifunctional bridging molecule: 1-pyrenebutanoic acid, succinimidyl ester (1).6 One side of 1 is a pyrene group that can interact with the graphene sheets of the nanohorn surfaces, and the other side is a succinimidyl ester group that can react with -NH2-functionalized surfaces. For this set of experiments, a two-step procedure was
Figure 1. TEM image (A) and EDX spectrum (B) of an SWNH/ MNP binary material.
employed: (1) functionalization of MNP surfaces with 1 and then (2) reaction with SWNHs. The first step was carried out in a mixed DMF/aqueous buffer solution, but the formation of SWNH/MNPs could be carried out in either an aqueous buffer solution or DMF.7 The reason for choosing this two-step sequential procedure is to ensure the efficient separation and purification of materials from the solution. After the reaction between MNPs and SWNHs, a change in the color of the dispersion from brownish to blackish could be easily observed (Supporting Information),8 and the application of a magnet could efficiently separate the binary nanomaterial from the solution supernatant. In this specific example, the advantage of a binary nanomaterial over two individual components is obvious: it combines the easy recoverability of MNPs and unique structural features of SWNHs, which is a key to probing SWNHs’ biorecognition properties (vide infra). Transmission electron microscopy (TEM) of the binary nanomaterials indeed shows the formation of binary materials (Figure 1A). Dark-contrasted MNPs are distributed along the SWNH structure. Correspondingly, nanoprobe energydispersive X-ray (EDX) spectroscopy shows the presence of Fe and O from the MNP (Figure 1B9), indicating the hybrid nature of the material. In the absence of a bridging molecule or when the coupling of the molecule to MNP surfaces fails, only MNPs were recovered after the separation and washing steps, demonstrating the key role played by the interconnecting species. The Raman spectrum of an SWNH/MNP hybrid shows two peaks characteristic of SWNHs (Figure 2A), indicating that the pristine graphene sheets were preserved after the 1034
Figure 2. Raman spectroscopy (A) and TGA (B) of an SWNH/ MNP binary material.
formation of binary nanomaterials because the formation of defects or a decrease in graphene sheet size will cause the shift of one peak from 1594 to 1620 cm-1.3b To characterize these materials further, thermogravimetric analysis (TGA) was carried out on a dried blackish sample, which revealed a typical SWNH weight loss within the 600-800 °C range (Figure 2B).10 After the TGA experiment, a red residue, presumably from the iron oxide of the MNPs, was left over. This technique, as a result, should be capable of providing quantitative information about the material’s composition.11 Taken together, all of the data presented above unambiguously prove the formation of binary nanostructures. The noncovalent method used here for the construction of SWNH-based binary nanomaterials preserves the original structural and functional features of SWNHs. This provides us with an opportunity to investigate various properties of SWNHs in solutions, such as their molecular recognition and catalytic capabilities.4a Here, we carried out a preliminary study on the biorecognition properties, (i.e., peptide binding abilities) of SWNHs treated under different conditions through a phage-display method. The recognition motifs, once identified, are important for exploiting this type of material in bioassembly, bioseparation, and biosensing applications.12 Phage-display enzyme-linked immunosorbent assay (ELISA) experiments, for characterizing interactions between biological species and SWNHs, were all carried out on SWNH/MNP samples with a specific phage clone NHD12-5-2 isolated out of a Ph.D.-12 phage-display peptide Nano Lett., Vol. 3, No. 8, 2003
Supporting Information Available: Optical properties of the SWNH/MNP binary nanomaterials in DMF before and after the application of a magnetic field. This material is available free of charge via the Internet at http:// pubs.acs.org. References
Figure 3. Biorecognition properties of SWNHs treated under different conditions through phage-display ELISA assays on SWNH/MNP samples.
library.13 As could be seen, the ELISA signature of as-grown SWNH is comparable to that of the control MNP sample (Figure 3), indicating that NHD-12-5-2 does not show any observable binding affinity for the as-grown SWNH. After treatment in O2,14 the binding of the peptide by the SWNH is significantly increased. The difference in binding affinity is probably due to the difference in the surface functionality. As-grown SWNH has only the graphene type of pristine structure on the surface, but O2 treatment of SWNHs would presumably generate polar functional groups on the surface. This is supported by the evolution of oxygen-related species H2O, CO2, and CO after O2 treatment from the thermogravimetric-mass spectrum analysis. The role of the surface polar sites is also evidenced by H2 treatment of O2-treated SWNHs,14 which would incur the reduction of such functional groups. After this step, the binding of NHD-12-5-2 for SWNHs was dramatically decreased. These data indicate that a simple change in the SWNHs’ surface functionality could significantly modify their biorecognition behaviors. In summary, the results in this communication suggest that the formation of binary nanomaterials based on nanocarbon materials can be mediated by organic interconnects. The synthetic scheme is expected to be generic and readily applicable to SWNHs interfacing with other nanoscale materials, such as Pt, in the construction of fuel cells. Such binary materials have proven useful in probing nanocarbons’ solution biorecognition properties, and SWNHs’ selective peptide binding capabilities have been demonstrated. The incorporation of biological interconnects into carbon nanostructures is likely to pave the way for more versatile placement and assembly strategies for this class of materials. Acknowledgment. We thank Elena Bekyarova for her help in the H2 treatment of carbon nanohorns. Nano Lett., Vol. 3, No. 8, 2003
(1) (a) Iijima, S. Nature 1991, 354, 56. (b) Iijima, S.; Ichihashi, T. Nature 1993, 363, 603. (2) For recent reviews, see (a) Iijima, S. Physica B 2002, 323, 1. (b) Ouyang, M.; Huang, J.-L.; Lieber, C. M. Acc. Chem. Res. 2002, 35, 1018. (c) Dai, H.; Kong, J.; Zhou, C.; Franklin, N.; Tombler, T.; Cassell, A.; Fan, S.; Chapline, M. J. Phys. Chem. B 1999, 103, 11246. (d) Carbon Nanotubes: Synthesis, Structure, Properties, and Applications; Dresselhaus, M. S., Dresselhaus, G., Avouris, Ph., Eds.; Springer-Verlag: Berlin, 2001. (3) (a) Iijima, S.; Yudasaka, M.; Yamada, R.; Bandow, S.; Suenaga, K.; Kokai, F.; Takahashi, K. Chem. Phys. Lett. 1999, 309, 165. (b) Kasuya, D.; Yudasaka, M.; Takahashi, K.; Kokai, F.; Iijima, S. J. Phys. Chem. B 2002, 106, 4947. (4) (a) Nisha, J. A.; Yudasaka, M.; Bandow, S.; Kokai, F.; Takahashi, K.; Iijima, S. Chem. Phys. Lett. 2000, 328, 381. (b) Bekyarova, E.; Kaneko, K.; Yudasaka, M.; Murata, K.; Kasuya, D.; Iijima, S. AdV. Mater. 2002, 14, 973. (c) Magnie, A.; Kasuya, D.; Yudasaka, M.; Iijima, S. Unpublished results. (5) For studies on binary heterostructures, see, for example, (a) Rajeshwar, K.; de Tacconi, N. R.; Chenthamarakshan, C. R. Chem. Mater. 2001, 13, 2765. (b) Feldheim, D. L.; Grabar, K. C.; Natan, M. J.; Mallouk, T. E. J. Am. Chem. Soc. 1996, 118, 7640. (c) Fogg, D. E.; Radzilowski, L. H.; Dabbousi, B. O.; Schrock, R. R.; Thomas, E. L.; Bawendi, M. G. Macromolecules 1997, 30, 8433. (6) For the use of 1 in the immobilization of proteins onto the side walls of carbon nanotubes, see (a) Chen, R. J.; Zhang, Y.; Wang, D.; Dai, H. J. Am. Chem. Soc. 2001, 123, 3838. For other attempts at the functionalization of nanotubes, see, for example, (b) Guo, Z.; Sadler, P. J.; Tsang, S. C. AdV. Mater. 1998, 10, 701. (c) Balavoine, F.; Schultz, P.; Richard, C.; Mallouh, V.; Ebberson, T. W.; Mioskowski, C. Angew. Chem., Int. Ed. 1999, 38, 1912. (7) Typically, 20 µL of -NH2-functionalized iron oxide MNP (Pierce Inc.) solution was added to 180 µL of sodium tetraborate buffer solution (0.1 M, pH 8.5). A solution of 1.0056 mg of 1 in 250 µL of dimethylformamide (DMF) was prepared separately and added to the previous MNP buffer solution. The reaction was carried on a vibrating mixer for 1 day. After that, a magnet was applied to separate the MNP from the supernatant, and MNP was washed with DMF (3 × 1 mL) and phosphate buffer solution (0.1 M, pH 8.5, 3 × 1 mL). The final MNP solution volume was adjusted to 400 µL. A phosphate buffer solution (250 µL) of 1.0579 mg of SWNH was added to 200 µL of MNP solution, mixed overnight, and washed with phosphate buffer solution (3 × 1 mL). (8) All characterizations except the biorecognition study were performed on buffer salt-free MNPs and SWNH/MNPs, which could be easily achieved through a solution/solvent exchange. All control experiments were carried out on 1-modified MNPs. (9) The Cu signature comes from the TEM grid. (10) TGA was carried out in an atmosphere of O2/Ar (1:99) from room temperature to 1000 °C at a heating rate of 5 °C/min. The curve has a positive slope, which was also observed in the MNP samples possibly because of the oxygenation of the inorganic minerals. (11) In this specific example, the total weight of dried SWNH/MNP was 0.509 mg, and the weight loss of SWNH was 0.297 mg. The composition of the binary nanomaterial is 58 wt % SWNH, 42 wt % MNP. (12) Although self-assembly has been explored in the synthesis of ordered materials, the number of useful methods and recognition elements is still very limited, partly because of the difficulty in producing rational designs for the chemical and biological ligands for such an effort, making combinatorial screening a relatively attractive alternative. For research endeavors on designed interlinks, see, for example, (a) Mirkin, C. A. Inorg. Chem. 2000, 39, 2258. (b) Brousseau, L. C., III; Novak, J. P.; Marinakos, S. M.; Feldheim, D. L. AdV. Mater. 1999, 11, 447. (c) Loweth, C. J.; Caldwell, W. B.; Peng, X.; Alivisatos, A. P.; Schultz, P. G. Angew. Chem., Int. Ed. 1999, 38, 1808. (d) Yun, C. S.; Khitrov, G. A.; Vergona, D. E.; Reich, N. O.; Strouse, G. F. J. Am. Chem. Soc. 2002, 124, 7644. For studies on 1035
the phage-display-based combinatorial screening of peptides against semiconductors, see (e) Whaley, S. R.; English, D. S.; Hu, E. L.; Barbara, P. F.; Belcher, A. M. Nature 2000, 405, 665. (f) Lee, S.; Mao, C.; Flynn, C.; Belcher, A. M. Science 2002, 296, 892. (13) The phage clones (a combinatorial library of random peptide 12mers fused to the N terminus of a minor coat protein pIII of the M13 phage, with a diversity of ∼2.7 × 109 sequences, Ph.D.-12 peptide library kit, New England Biolabs) that have specific binding affinities for HNO3-treated SWNHs have been selected through several rounds of biopanning (to be submitted for publication). The NHD-12-5-2 is one of those binders and was used in this study. The ELISA experiments were carried out by using the detection module recombinant phage antibody system (Amersham pharmacia biotech) following the manufacturer’s protocol. Briefly, the SWNH/MNP samples were suspended in TBS (50 mM TrisHCl, pH 7.5, 150 mM NaCl) to 2 mg of SWNH/mL. Aliquots of this 10-µL suspension (20 µg of SWNH based on the material composition from TGA) were placed into a 96-well flexible plate (353911, Faclon), and the conjugates were collected with a magnetic particle concentrator
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(MPC-96, Dynal). The conjugates were incubated with 200 µL of TBS containing 2% bovine serum albumin (BSA, Iwai chemicals) for 1 h at room temperature, washed three times with 200 µL of TBS containing 0.5% polyoxyethylenesorbitan monolaurate (Tween 20, Sigma), and incubated with 5 × 108 pfu of NHD-12-5 phage in 200 µL of TBS containing 2% BSA and 0.5% Tween 20 for 2 h at room temperature. Unbound phages were washed 10 times with 200 µL of TBS containing 0.5% Tween 20. The amount of bound phages was estimated by using the horseradish peroxidase conjugated mouse anti-M13 monoclonal antibody and 2,2′-azino-bis-(3-ethylbenzothioline-6-sulfonic acid), a substrate of peroxidase (Amersham pharmacia biotech). After 1 h of the peroxidase reaction, the absorption at 415 nm was measured with a microplate reader (model 550, BioRad). (14) O2 treatment was carried out at 420 °C for 10 min at a pressure of 760 Torr and a flow rate of 200 sccm. H2 treatment was carried out at 1000 °C for 1 h at a pressure of 760 Torr and a flow rate of 50 sccm.
NL034266Q
Nano Lett., Vol. 3, No. 8, 2003
