NANO LETTERS
Confocal Fluorescence Imaging of DNA-Functionalized Carbon Nanotubes
2003 Vol. 3, No. 2 153-155
Miron Hazani,† Ron Naaman,*,† Frank Hennrich,‡ and Manfred M. Kappes‡ Department of Chemical Physics, Weizmann Institute, RehoVot 76100, Israel, and Forschungszentrum Karlsruhe, Institut fu¨ r Nanotechnologie, D-76021 Karlsruhe, Germany Received November 1, 2002; Revised Manuscript Received December 2, 2002
ABSTRACT Single-walled carbon nanotubes (SWNT) were covalently modified with DNA via carbodiimide-assisted amidation, yielding a highly watersoluble adduct. The specific and nonspecific interactions between DNA and SWNTs were examined by UV−vis spectroscopy and confocal fluorescence microscopy. Fluorescence imaging of individual bundles shows that the SWNT−DNA adducts hybridize selectively with complementary strands with minimal nonspecific interactions with noncomplementary sequences.
DNA functionalization of carbon nanotubes holds interesting prospects in various fields including solubilization in aqueous media, nucleic acid sensing, gene-therapy, and controlled deposition on conducting/semiconducting substrates.1,2 Noncovalent interactions between DNA and carbon nanotubes, as well as certain organizational properties of carbon nanotube-DNA systems were reported previously.1-3 Recently, strategies for covalent binding of DNA to oxidized SWNTs by direct carbodiimide coupling, or by a multistep procedure, involving a heterobifunctional linker, were reported.4,5 The carbodiimide assisted modification was characterized by radioisotope polyacrylamide gel, while the multistep procedure was characterized by NMR, XPS, and fluorescent measurements of SWNT-DNA dispersions. Here we report confocal fluorescence imaging of SWNTDNA adducts, obtained by carbodiimide-assisted coupling of amino-functionalized oligonucleotides to oxidized SWNTs, at resolutions of individual bundles. In the past, atomic force or scanning tunneling microscopes were used for visualization of SWNTs. These methods of imaging require special preparation of the sample (flatness of the substrate and limits on its size) and in addition may alter the sample during the measuring process. Despite the limited resolution of the confocal microscope, compared to AFM and STM, the ability to detect fluorescence makes it possible to visualize nanotubes with suboptical resolution. SWNTs were prepared by laser ablation of a carbon composite following the Smalley method using a 1:1 Ni/Co catalyst (1 at. % each) incorporated into the target. Details of the ablation setup have been described in a previous publication.6 This catalyst and procedure generates tube * Corresponding author. † Weizmann Institute. ‡ Institut fu ¨ r Nanotechnologie. 10.1021/nl025874t CCC: $25.00 Published on Web 12/12/2002
© 2003 American Chemical Society
bundles with individual tube diameters ranging from 1.2 to 1.4 nm. As prepared tubes were purified by refluxing in 2-3 M HNO3 for 48 h. This was followed by several ultracentrifugation and ultrafiltration steps using the surfactant Triton-X to further remove small particles. Purified SWNTs were shortened by treating with a 1:3 mixture of concentrated HNO3 (65 wt %, Sigma-Aldrich) and H2SO4 (95-98 wt %, Sigma-Aldrich) for 4 h in an ultrasonic bath and subsequently diluted and washed with deionized water in order to generate solid s-SWNTs after filtering and drying. A 0.5 mg portion of oxidized, shortened SWNTs was suspended in 5 mL 0.2% Triton-X and sonicated briefly. Carbodiimide-mediated amidations of SWNTs and MWNTs have been previously used to prepare protein-nanotube conjugates, nanocrystal-nanotube heterocyles, covalently functionalized nanotube probe tips, and polymer-wrapped soluble nanotubes.7-9 Huang and co-workers have found that sonication during the reactions could significantly increase the yield of carbodiimide mediated amidations.10 We have taken a two-step approach for covalently attaching aminomodified oligonucleotides to the nanotube surface-bound carboxylic groups. In the first step, oxidized SWNTs are sonicated in the presence of 1-ethyl-3-(3-dimethyl aminopropyl) carbodiimide and N-hydroxy succinimide in-order to form a labile intermediate. In the second step, amino modified oligonucleotides (C1, see Figure 1) are added to form an amide bond between the primary amine located at the 3′ of the capture oligonucleotide and the SWNT. Conducting the reaction in two successive steps was taken as a precaution against any sonication-induced damage to the DNA. Activation of the nanotube-bound carboxylic acids was achieved by sonicating the Triton-X suspension in the presence of 100 mM MES pH 6.0, 100 mM N-hydroxy succinimide (set to pH 6.0), and 100 mM 1-ethyl-3-(3-
Figure 2. Photograph of the supernatant fraction obtained from the coupling steps with C1.
Figure 1. Oligonucleotides used in the study. Two oligonuletides, C1 and C2 were used for carbodiimide-mediated modification of the SWNTs. C1 contains a primary amino modification located at its 3′, whereas C2 is unmodified. For hybridization studies, the oligonucleotides T1 and T2, both labeled at their 3′ with fluorescein isothiocyanate (FITC) were used.
dimethyl amino-propyl) carbodiimide, for 1 h at room temperature. Following the activation step, the pH was raised to 8.5, and amino-modified oligonucleotides were added to a final concentration of 5 µM. The reaction mixture was stirred overnight at room temperature. Following incubation, the reaction mixture was washed with deionized water and 0.5 M NaCl by several centrifugation cycles at 2500 rcf. After several cycles of washing, the samples were left to stand at room temperature for few hours, and the supernatant fractions were collected. The soluble fraction obtained from coupling to C1 consisted of a clear, transparent, blackish, homogeneous suspension as shown in Figure 2, whereas that obtained from reacting with unmodified oligonucleotides C2 (see Figure 1), under similar conditions, consisted of a colorless solution. Figure 3 shows the UV-vis spectra of the soluble fractions obtained from the coupling steps with oligonuleotides C1 and C2. The UV-vis spectra of the two conjugates clearly indicate that much more DNA is present in the soluble fraction resulting from the coupling with C1. The visual appearance and the UV-vis spectra of the different samples lead us to believe that the majority of the DNA present in the SWNT-C1 adduct is covalently bound rather than simply physisorbed. Furthermore, it seems that when the DNA is not bound covalently to the tubes, its concentration is not sufficient to promote solubility in water. To assess the ability of the SWNT-DNA adduct to specifically interact with complementary sequences, hybridization studies were carried out using complementary and 154
Figure 3. UV-vis spectra of the soluble fractions obtained form the coupling steps with oligonuleotides C1 (2) and C2 (b). The peak at 265 nm corresponds to the absorbance maximum of DNA.
noncomplementary strands, labeled at their 3′ with fluorescein. Reactions of the SWNT-C1 adduct with the fluorescent target sequences T1 or T2 (see Figure 1), final concentration 5 µM, were conducted in 50 mM Tris-HCl pH 7.4 supplemented with 300 mM NaCl. Each sample was allowed to hybridize at room temperature overnight. Following incubation, the samples were washed thoroughly with 50 mM TrisHCl buffer, pH 7.4, supplemented with 300 mM NaCl. Confocal images were acquired using a FLUOVIEW confocal laser scanning unit mounted on a BX50WI fixed stage upright microscope. The system enables simultaneous collection of fluorescent and DIC images. The 488 nm line from an argon laser was used for excitation, and fluorescence emission was detected using a 510 nm band-pass filter. Images were recorded using a ×60\1.4 oil immersion objective. As seen from Figure 4, the SWNT-DNA adducts hybridize efficiently with the complementary strands, with minimal Nano Lett., Vol. 3, No. 2, 2003
Figure 4. Confocal fluorescence and DIC images of the SWNT-C1 adduct reacted with the complementary (T1) and noncomplementary (T2) FITC labeled sequences. Figures A-C show images obtained from different regions of the samples. Figure 4D shows a 10× magnification of the circled areas in Figure 4C.
nonspecific interactions with noncomplementary sequences. This observation supports the notion that the majority of DNA present on the SWNT-C1 conjugate is covalently attached, rather than physisorbed to the nanotubes. The fluorescent images reveal features as small as 1 micron. We presume these features are small bundles, and possibly even single tubes. The homogeneous dispersions shown in figures 4A-C as well as the observation of distinguishable micron scale features, in Figure 4D are, we believe, a direct consequence of the aqueous solubility of the DNA functionalized SWNTs. The present work demonstrates the ability to obtain soluble SWNT and to visualize them using confocal microscopy. These abilities open new possibilities for using SWNT for various applications. Acknowledgment. M.H. is grateful for the Aron Zandman postdoctoral fellowship in organic chemistry. This work was supported by by the Israeli Ministry of Science, Culture, and Sport. Nano Lett., Vol. 3, No. 2, 2003
References (1) Tsang, S. C.; Guo, Z.; Chen, Y. K.; Green, M. L. H.; Hill, H. A.; Hambley, T. W.; Sadler, P. J. Angew. Chem., Int. Ed. Engl. 1997, 36, 2198. (2) Guo, Z.; Sadler, P. J.; Tsang, S. C. AdV. Mater. 1998, 10, 701. (3) Buzaneva, E.; Karlash, A.; Yakovkin, K.; Shtogun, Y.; Putselyk, S.; Zherebetskiy, D.; Gorchinskiy, A.; Popova, G.; Prilutska, S.; Matyshevska, O.; Prilutsky, Y.; Lytvyn, P.; Scharff, P.; Eklund, P. Mater. Sci. Eng., C. 2002, 19, 41. (4) Dwyer, C.; Guthold, M.; Falvo, M.; Washburn, S.; Superfine, R.; Erie, D. Nanotechnology 2002, 13, 601. (5) Baker, S. E.; Cai, W.; Lassester, T. L.; Weidkamp, K. P.; Hamers, R. J. Nano Lett. 2002, 2, 1413. (6) Lebedekin, S.; Schweiss, P.; Renker, B.; Malik, S.; Hennrich, f.; Neumaier, M.; Stoermer, C.; Kappes, M. M. Carbon 2002, 40, 417. (7) Banerjee, S.; Wong, S. S. Nano Lett. 2002, 2, 195. (8) Wong S. S.; Woolley, A. T.; Joselevich, E.; Cheung, C. L.; Lieber, C. M. J. Am. Chem. Soc. 1998, 120, 8557. (9) Huang, W.; Taylor, S.; Fu, K.; Lin, Y.; Zhang, D.; Hanks, T. W.; Rao, A. M.; Sun, Y. Nano Lett. 2002, 2, 311. (10) Huang, W.; Lin, Y.; Taylor, S.; Gaillard, J.; Rao, A. M.; Sun, Y. Nano Lett. 2002, 2, 231.
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