Biomacromolecules 2005, 6, 2919-2922
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Supramolecular Conjugates of Carbon Nanotubes and DNA by a Solid-State Reaction Dhriti Nepal,† Jung-Inn Sohn,† Wilhelm K. Aicher,‡ Seonghoon Lee,§ and Kurt E. Geckeler*,† Department of Materials Science and Engineering, Gwangju Institute of Science and Technology (GIST), 500-712 Gwangju, South Korea, Basic Science Research Laboratories, Center of Orthopedic Surgery, University of Tu¨bingen, Tu¨bingen, Germany, and Department of Chemistry and Molecular Engineering, Seoul National University, Seoul, South Korea Received June 6, 2005; Revised Manuscript Received August 26, 2005
DNA-wrapped nanotubes of both multiwalled and single-walled carbon nanotubes were obtained by a solidstate mechanochemical reaction. Scanning electron microscopic images show that the nanotubes were cut into shorter lengths and were fully covered with DNA, which was further confirmed by fluorescence microscopy. This resulted in a high aqueous solubility of the products with a stability of >6 months. The results show that nanotubes were cut also with uniform distribution where >90% of the multiwalled products were 500 nm to 3 µm and 80% of the single-walled products were 250 nm to 1 µm in length, respectively. UV-vis spectra and gel electrophorosis show that the DNA in the product is intact. This facile technique for obtaining supramolecularly masked, water-soluble carbon nanotubes by a solid-state reaction has a great potential for both biological and nonbiological applications of nanotubes. 1. Introduction Carbon nanotubes (CNTs) have already been recognized as some of the most fascinating materials because of their unique combination of mechanical, electrical, thermal, and optical properties.1 However, because of their existence in aggregated or bundled states (by high attractive energy due to tube-tube van der Waals interactions and their axial geometery)2 and the lack of flexible groups or functionalities to interact with the surrounding solvent, this material exhibits a poor solubility in most of the common solvents. Therefore, the dispersion and solubilization3 of CNTs are of prime interest.4 This eases the process of using this fascinating material for making devices1 as well as preparing nanocomposites.5 A wide range of polymers and surfactants have been studied in conjunction with CNTs to obtain highly dispersed nanotubes, which include surfactants,6 synthetic polymers,7 and biopolymers.8 Despite significant progress, challenges still remain in developing simple and effective techniques for large-scale production. A monodisperse solution of short and thin CNTs having a reliable control over the interfacial chemistry is the key requirement for ideal CNT-polymer composite devices.9 In addition, recently, the potential of nanotubes for biochemical and biomedical applications10,12 has been realized. Though the exploration of their interactions with biological materials remains at a very early stage,11 aqueous solubilization and dispersion of nanotubes is of great interest, and their combination with biopolymers have found strong attention.8,11 Special focus has been on DNA, which * E-mail:
[email protected]. † Gwangju Institute of Science and Technology (GIST). ‡ University of Tu ¨ bingen. § Seoul National University.
because of its unique structure allows us to attain remarkable properties for both biological and nonbiological applications. These include the visualization of multiwalled carbon nanotubes (MWNTs) by platinated oligonucleotides,12a covalently bound DNA with single-walled carbon nanotubes (SWNTs),12b sonication-assisted dispersion of SWNTs in DNA,8b and DNA-assisted chiral separation of SWNTs.8a To the best of our knowledge, all the studies to date on DNA-stabilized nanotubes are based on sonication treatment and only centered on SWNTs. Here, we report a simple technique based on a solid-state mechanochemical reaction (MCR) to obtain supramolecular adducts13 of DNA-CNT conjugates14 from both MWNTs and SWNTs. In this process, highly reactive centers are generated by the mechanical energy in the solid phase enabling the nanotubes to interact with DNA (Scheme 1). This results in nanotube products with a very good dispersion property in the aqeuous phase with a uniform length distribution. This technique has been previously used for alcohol-functionalized nanotubes15 and debundlization of nanotubes.16 Interestingly, our soluble CNT products via a noncovalent approach appeared to be fully wrapped with DNA, which is different from the previous report on a mechanochemical reaction.15,16 The products were characterized by UV-visible spectroscopy (UV-vis), field emission scanning electron microscope (FE-SEM), transmission electron microscopy (TEM), gel electrophoresis, and fluorescence microscopy. 2. Experimental Section MWNTs were prepared by Fe-catalyzed MWNT growth over silicon by pulsed laser deposition.17 The purity of the
10.1021/bm050380m CCC: $30.25 © 2005 American Chemical Society Published on Web 10/28/2005
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Scheme 1. Formation of Supramolecular Conjugates of CNT and DNA by a Solid-State Mechanochemical Reaction
sample was analyzed by SEM, TGA, and TEM and found to be ∼95%. For the SWNTs, commercially available, purified SWNTs (∼90% purity, obtained from Ilzin Korea) were used. Double-stranded DNA as supplied by Sigma (from Herring testes, sodium salt) was used for all the experiments. A quantity (5 mg) of MWNTs was taken in a ball-milling apparatus (agate: length, 4 cm; diameter: 1.7 cm), 80 mg of DNA was added, and the mixture was milled for 1 h at room temperature (20 Hz, 220 V). A very fine, homogeneous black powder was obtained, which was readily soluble in water (MWNT-DNA) without any precipitation. The same process was repeated with SWNTs, but the milling time was decreased to 30 min. The black powder was dissolved in water to get a dark black solution. It was centrifuged in a Vision centrifuge (model VS-15000N) for 90 min at 16 000g, and the supernatant product (SWNT-DNA) was collected. For control, both the MWNTs and SWNTs were milled for 1 h and 30 min, respectively, in the absence of DNA for the products c-MWNT and c-SWNT. Instruments. UV-vis spectra were recorded on a Varian 1E spectrophotometer. SEM images were obtained with a FE-SEM S-4700 (operating voltage 15 kV). The samples for SEM were prepared by dropping 10 µL of each solution on a clean silicon substrate and drying in an oven at 70 °C. For the starting materials (MWNTs and SWNTs), the powder was used by mounting on a carbon tape. High-resolution TEM (Philips) was used with an accelerating voltage of 120 kV for the TEM measurement. The samples were prepared by placing a few drops on a 200 mesh copper grid with a holey carbon film prior to the examination. For the gel electrophoresis, the agarose gel (1%) prepared in a TAE buffer was subsequently soaked in an ethidium bromide solution and destained in pure water. After loading the wells, the gel was run for 13 min at a potential of 200 V and photographed under a UV transilluminator. For fluorescence microscopye, the IRBE inverse microscope equipped with a DC200 camera was used under UV irradiation (Hg ultrahighpressure lamp, N2.1 filter, long pass LP 590). The samples were stained with ethidium bromide (EtBr) by incubating in 200 µL of a 2.5 mg/mL EtBr solution for 5 min. Free EtBr was removed by washing with 50% ethanol in water by centrifugation and transferred onto microscope slides. Pictures were captured immediately from the wet samples.
Figure 1. UV-vis spectra of (A) DNA (a) and MWNT-DNA conjugates (b) (the inset shows the band broadening of the conjugates); (B) SWNT-DNA conjugates.
Figure 2. SEM images and length distribution of MWNT-DNA. (a) Pristine MWNT, (b) MWNT-DNA conjugates, (c) length distribution, and (d) photographs of solutions of MWNT-DNA conjugates: 5 mg/mL, 0.25 mg/mL, pristine MWNT in water, and DNA (from left to right).
3. Results and Discussion Both products obtained, the MWNT-DNA and SWNTDNA, were highly water soluble with a solubility of 5.6 mg/mL for the product. No precipitation was observed even after 6 months (Figure 2d). This high solubility is due to the π-π interactions between the backbones of DNA with the nanotube surfaces, which provide a strong binding, and the phosphate side groups constitute the solubilization. In contrast, the control samples c-MWNT and c-SWNT did not show any dispersion in water. Similarly, the stirring of the
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Figure 3. SEM images of (a) SWNT, (b and c) SWNT-DNA conjugates, and (d) length distribution.
nanotubes in a DNA solution for a week did not cause any change of the supernatant. Figure 1a shows the UV-vis spectrum of the MWNT-DNA, in which the characteristic absorption peaks at 774, 895, and 1019 nm are observed. These are attributed to the semiconducting band of MWNTs.18,19 The spectrum also shows the characteristic DNA peak at 260 nm, where the pristine DNA also shows the λmax at the same wavelength as in aqueous solution. This indicates that DNA remains intact under solid-state milling conditions. The UV-vis spectrum of SWNT-DNA (Figure 1b) clearly shows the van Hove transitions of metallic and semiconducting SWNTs. The peaks, centered in a range from ∼440 to ∼600 nm, are assigned to the first van Hove transition of metallic SWNTs (M11), and the peaks centered at ∼600 nm to ∼800 nm are the second van Hove singularity of semiconducting SWNTs (S22).19 These absorption peaks also confirm that the electronic properties of nanotubes are preserved in the product. Figure 2 shows the SEM images of the pristine MWNT and the MWNT-DNA conjugates. The image (Figure 2b) shows the thick DNA coverage on the sidewalls of short MWNTs. In contrast, no coverage was present on the pristine MWNTs (Figure 2a). A similar observation was found for the SWNT-DNA product (Figure 3b,c). The CNTs were cut mechanically and simultaneously in the solid-state process, and nonspecific interactions occurred between the CNTs and the amphiphilic DNA. The high binding affinity of DNA to the nanotube backbones due to π-stacking8a led to the wrapping with DNA. Since the amount of DNA was higher than that of the CNTs, the CNTs could be mostly wrapped in a complete manner. Figure 2c shows the length distribution of the MWNT-DNA product as calculated from the SEM and TEM analyses. The results show that more than 90% of the nanotubes are within 500 nm to 3 µm, which indicates that the solid-state milling process cut the nanotubes with an almost uniform distribution. Figure 3 shows the SEM images of the SWNT-DNA conjugates and their length distribution. Here, we can clearly see short-length nanotubes covered with DNA, which look very different from the starting SWNTs (Figure 3a). The length distribution (Figure 3d) shows that ∼80% of the tubes are within the range of
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250 nm to 1 µm, of which more than 50% are within a range of 500 nm to 1 µm. Since the starting SWNTs have shorter lengths (1-5 µm) compared to those of MWNTs (1020 µm), the reaction time was decreased to 30 min. The shorter reaction time could have also led to the narrower length distribution of the SWNT product compared to that of the MWNTs. However, in the case of MWNTs, decreasing the reaction time to 30 min led to longer MWNTs, but no full dissolution of the product was attained. With centrifugation, the SWNT reaction gave ∼5% residue; in contrast, no residue was obtained from the MWNT product. That clearly explains that in the solid-state MCR the reaction time plays a vital role for the interaction with the species as well as for the shortening of the length of the CNTs. Additionally, to further confirm the DNA coating of the CNT surface, fluorescence microscopy was used to image the conjugates. The result showed the typical orange fluorescence due to ethidium bromide staining of DNA on several spots of the substrate; however, it was absent in the control CNTs. Finally, to check the status of DNA in the final product, gel electrophoresis was run for the pristine DNA, MCRtreated DNA (milling under similar conditions, but in the absence of MWNT), and MWNT-DNA. The results show 287, 183, and 287 base pairs corresponding to pristine DNA, MCR-treated DNA, and the MWNT-DNA conjugate, respectively. This clearly indicates that the DNA was stable in the presence of MWNTs under these reaction conditions. However, the control DNA sample, which was milled in the absence of MWNTs, underwent cleavage. A similar result was obtained for the SWNT-DNA product. This might be due to a protection effect of the CNTs. A further study is underway in our laboratory to elucidate the role of the CNTs under these conditions. 4. Conclusion We have described a facile, one-pot synthetic method for preparing short-length, highly water soluble, DNA-covered CNTs by a mechanochemical reaction. This represents an aqueous stabilization of MWNTs by DNA as well as a visible observation of supramolecularly masked water-soluble CNTs by a solid-state reaction. Since water-soluble, short-length CNT products are very high in demand to enhance their compatibility with biological systems, this straightforward method to obtain supramolecular adducts paves the way for further surface functionalization (enabling the adsorption or attachment of various molecules or antigens), which subsequently can be delivered to the desired target sites. In addition, the versatility of this facile approach can also be easily utilized to upscale the production for CNTs for both other biological and nonbiological applications. Acknowledgment. Financial support from the Ministry of Education for the “Brain Korea 21” (BK21) project is gratefully acknowledged. Supporting Information Available. TEM, gel electrophorosis, and fluorescence microscope image of MWNT-
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DNA. This material is available free of charge via the Internet at http://pubs.acs.org. References and Notes (1) Haddon, R. C. Acc. Chem. Res. 2002, 35, 997. (2) Girifalco, L. A.; Hodak M.; Lee, R. S. Phys. ReV. B 2000, 62, 13104. (3) (a) Tasis, D.; Tagmatarchis, N.; Georgakilas, V.; Prato M. Chem. Eur. J. 2003, 9, 4000. (b) Chichak, K. S.; Star, A.; Altoe´, M. V.; Stoddart, J. F. Small 2005, 1, 452. (4) Nepal, D.; Kim, D. S.; Geckeler, K. E. Carbon 2005, 43, 660. (5) Lake, R.; Gun’ko, Y. K.; Coleman, J.; Cadek, M.; Fonseca, A.; Nagy, J. B.; Blau, W. J. J. Am. Chem. Soc. 2004, 126, 10226. (6) Moore, V. C.; Strano, M. S.; Haroz, E. H.; Hauge, R. H.; Smalley, R. E.; Schmidt, J.; Talmon, Y. Nano Lett. 2003, 3, 1379. (7) (a) Dalton, A. B.; Stephan, C.; Coleman, J. N.; McCarthy, B.; Ajayan, P. M.; Lefrant, S.; Bernier, P.; Blau, W. J.; Byrne, H. J. J. Phys. Chem. B 2000, 104, 10012. (b) Sinani, V. A.; Gheith, M. K.; Yaroslavov, A. A.; Rakhnyanskaya, A. A.; Sun, K.; Mamedov, A. A.; Wicksted, J. P.; Kotov, N. A. J. Am. Chem. Soc. 2005, 127, 3463. (8) (a) Wang, S.; Humphreys, E. S.; Chung, S.-Y.; Delduco, D. F.; Lustig, S. R.; Wang, H.; Parker, K. N.; Rizzo, N. W.; Subramoney, S.; Chiang, Y.-M.; Jagota, A. Nat. Mater. 2003, 2, 196. (b) Nakashima, N.; Okuzono, S.; Murakami, H.; Nakai, T.; Yoshikawa, K. Chem. Lett. 2003, 32, 456. (c) Zorbas, V.; Ortiz-Acevedo, A.; Dalton, A. B.; Yoshida, M. M.; Dieckmann, G. R.; Draper, R. K.; Baughman, R. H.; Jose-Yacaman, M.; Musselman, I. H. J. Am. Chem. Soc. 2004, 126, 7222. (d) Star, A.; Steuerman, D. W.; Heath, J. R.; Stoddart, J. F. Angew. Chem., Int. Ed. 2002, 41, 2508. (e) Numata, M.; Asai, M.; Kaneko, K.; Hasegawa, T.; Fujita, N.; Kitada, Y.; Sakurai, K.; Shinkai, S. Chem. Lett. 2004, 33, 232. (f) Numata, M.; Asai, M.; Kaneko, K.; Bae, A.-H.; Hasegawa, T.; Sakurai, K.; Shinkai, S. J. Am. Chem. Soc. 2005, 127, 5875.
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