Layer-by-Layer Fabrication and Characterization of DNA-Wrapped

Kerstin Müller, John F. Quinn, Angus P. R. Johnston, Mathias Becker, Andreas Greiner, and Frank Caruso. Chemistry of Materials 2006 18 (9), 2397-2403...
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Layer-by-Layer Fabrication and Characterization of DNA-Wrapped Single-Walled Carbon Nanotube Particles Pingang He and Mekki Bayachou* Department of Chemistry, Cleveland State University, SR 397, Cleveland, Ohio 44115-2406 Received March 3, 2005 Carbon nanotubes have been proposed as support materials for numerous applications, including the development of DNA sensors. One of the challenges is the immobilization of DNA or other biological molecules on the sidewall of carbon nanotubes. This paper introduces a new fabrication of DNA-carbon nanotubes particles using the layer-by-layer (LBL) technique on single-walled carbon nanotubes (SWCNTs). Poly(diallyldimethylammonium) (PDDA), a positively charged polyelectrolyte, and DNA as a negatively charged counterpart macromolecule are alternatively deposited on the water-soluble oxidized SWCNTs. Pure DNA/PDDA/SWCNTs particles can be prepared and separated by simple unltracentrifugation. The characterization of DNA/PDDA/SWCNTs particles was carried out by scanning electron microscopy (SEM), transmission electron microscopy (TEM), UV-visible spectroscopy, Raman spectroscopy, and thermogravimetric analysis (TGA). An electrode modified by the DNA/PDDA/SWCNTs particles shows a dramatic change of the electrochemical signal in solutions of tris(2,2′-bipyridyl)ruthenium(II) ((Ru(bpy)32+) as a reporting redox probe. A preliminary application of the DNA-modified carbon nanotubes in the development of DNA sensors used in the investigation of DNA damage by nitric oxide is presented.

1. Introduction Nanostructured composite materials, either as such or as surface-supported architectures, are now the focus of intense fundamental and applied research in a number of areas. Particularly, areas of research which aim to develop the basic science needed for integrating biological materials into nonbiological nanoarchitectures are driving what seems to be the advent of a whole new set of nanoscale functional architectures with limitless applications.1 Integrating biological macromolecules in hybrid nanoarchitectures is of paramount importance; it not only provides prefabricated building blocks, which, when judiciously chosen, naturally bring with them built-in programs for directed assembly2a but also introduces desired functional features in the final composite materials.2b-c This article describes the fabrication process and characterization of DNA-modified single-walled carbon nanotubes (SWCNTs) particles by the layer-by-layer technique (LBL). DNA attachment to carbon nanotubes promises important applications in various fields including DNA sensing,3-5 separation of carbon nanotubes,6 and controlled deposition on conducting/semiconducting substrates.7 Covalent and noncovalent interactions between * Author to whom correspondence should be addressed. E-mail: [email protected]. (1) (a) Philip, D.; Stoddart, J. F. Angew. Chem., Int. Ed. Engl. 1996, 35, 1154. (b) Niemeyer, C. M. Angew. Chem., Int. Ed. 2001, 40, 4128 (c) Lee, B. S.; Lee, S. C.; Holliday, L. S. Biomed. Microdevices 2003, 5 (4), 269. (d) Katz, E.; Willner, I.; Wang, J. Electroanalysis 2004, 16, 19. (2) (a) Jaeger, L.; Westhof, E.; Leontis, N. B. TectoRNA: modular assembly units for the construction of RNA nano-objects. Nucleic Acids Res. 2001, 29, 455-463. (b) Whaley, S. R.; English, D. S.; Hu, E. L.; Barbara, P. F.; Belcher, A. M. Selection of peptides with semiconductor binding specificity for directed nanocrystal assembly. Nature 2000, 405 (6787), 665-668. (c) Hartgerink J. D.; Beniash E.; Stupp S. I. Selfassembly and mineralization of peptide-amphiphile nanofibers. Science 2001, 294 (5547), 1684-1688. (3) (a) Wang, J.; Liu, G.; Jan, M. R. J. Am. Chem. Soc. 2004, 126, 3010. (b) He, P.; Dai, L. Chem. Commun. 2004, 3, 348. (4) Li, J.; Ng, H. T.; Cassell, A.; Fan, W.; Chen, H.; Ye, Q.; Koehne, J.; Han, J.; Meyyappan, M. Nano Lett. 2003, 3, 597. (5) Cai, H.; Cao, X.; Jiang, Y.; He, P.; Fang, Y. Anal. Bioanal. Chem. 2003, 375 (2), 287.

DNA and carbon nanotubes were reported previously,8-11 as well as certain properties of carbon nanotubes-DNA systems.12-15 Other approaches for covalently binding DNA to oxidized SWCNTs by direct carbodiimide coupling or other multistep procedures were also reported.16-19 However, these methods rely on complicated procedures and yield to low coupling efficiency. Among assembly techniques used to build stable architectures, the layer-by-layer method is versatile and simple and has been used successfully and extensively to build thin films with thicknesses in the nanometer range on solid supports.20-23 This self-assembly strategy relies (6) Zheng, M.; Jagota, A.; Strano, S. M.; Santos, P. A.; Barone, P.; Chou, S. G.; Diner, A. B.; Dresselhaus, S. M.; Mclean, S. R.; Onoa, G. B.; Samsonidze, G. G.; Semke, D. E.; Usery, M.; Walls, J. D. Science 2003, 302, 1545. (7) Xin, H.; Woolley, T. A. J. Am. Chem. Soc. 2003, 125, 8710. (8) Gao, H.; Kong, Y.; Cui, D.; Ozkan, S. C. Nano Lett. 2003, 3, 471. (9) Gao, H. J.; Kong, Y. Ann. Rev. Mat. Res. 2004, 34, 123. (10) Moghaddam, M. J.; Taylor, S.; Gao, M.; Huang, S. M.; Dai, L. M.; McCall, M. J. Nano Lett. 2004, 4, 89. (11) Dwyer, C.; Guthold, M.; Falvo, M.; Washburn, M.; Superfine, R.; Erie, D. Nanotechnology 2002, 13, 601. (12) 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. (13) Guo, Z.; Sadler, P. J.; Tsang, S. C. Adv. Mater. 1998, 10, 701. (14) 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. (15) Hazani, M.; Naaman, R.; Hennrich, F.; Kappes, M. M. Nano Lett. 2003, 3, 153. (16) Buzaneva, E.; Karlash, A.; Yakovkin, K.; Shtogun, Y.; Putselyk, S.; Zherebetskiy, D.; Gorchinskiy, A.; Popova, G.; Prilutska, S.; Matyshevska, O.; Prilutskyy, Y.; Lytvyn, P.; Scharff, P.; Eklund, P. Mater. Sci. Eng., C 2002, 19, 41. (17) Guo, Z.; Sadler, J. P.; Tsang, C. S. Adv. Mater. 1998, 10, 701. (18) Dwyer, C.; Guthold, M.; Falvo, M.; Washburn, S.; Superfine, R.; Erie, D. Nanotechnology 2002, 13, 601. (19) Baker, S. E.; Cai, W.; Lassester, T. L.; Weidkamp, K. P.; Hamers, R. J. Nano Lett. 2002, 2, 1413. (20) Decher, G. Science 1997, 277, 1232. (21) Anzai, J.; Takeshita, H.; Kobayashi, Y.; Osa, T.; Hoshi, T. Anal. Chem. 1998, 70, 811. (22) Mamedov, A. A.; Kotov, N. A. Langmuir 2000, 16, 5530. (23) Mamedov, A. A.; Belov, A.; Giersig, M.; Mamedova, N. N.; Kotov, N. A. J. Am. Chem. Soc. 2001, 123, 7738.

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electrochemical signal in the solution of Ru(bpy)32+ as a reporting redox probe. 2. Experimental Section

Figure 1. Schematic description of fabrication of DNA-modified single-walled carbon nanotube particles by the LBL technique.

essentially on adsorbing a charged macromolecule on oppositely charged surface (e.g. oxidized graphite) and then repeating the process with another component oppositely charged; the latter can be a polyelectrolyte, a folded protein with a net surface charge, DNA, or colloids.20-23 The LBL self-assembly technique is a simple and versatile procedure24 and has been shown to have promising applications in areas such as chemical sensors or biosensors development,25 enzyme immobilization,26 production of hollow polyelectrolyte capsules,27 surface patterning,28 ultrathin membrane development,29 microporous films,30 nanocomposites, and nanoparticle-based materials.31-33 However, to the best of our knowledge, the LBL technology as applied to building a DNA film on SWCNTs has not been reported previously. In this paper, we introduce a new fabrication of DNA/ SWCNTs particles using the LBL technique. The LBL deposition of positively and negatively charged macromolecular species is an ideal method for constructing ultrathin films on the sidewall of CNTs. As shown (the procedure shown) in Figure 1, poly(diallyldimethylammonium), a positively charged polyelectrolyte, and DNA as the negatively charged component are alternatively deposited on water-soluble, negatively charged oxidized SWNTs. Pure DNA/PDDA/SWCNTs particles are prepared using this procedure and are separated by simple ultracentrifugation. The characterization of DNA/PDDA/ SWCNTs particles was carried by high-resolution transmission electron microscopy (HRTEM), UV-visible spectroscopy, Raman spectroscopy, and thermogravimetric analysis. In this work we also report preliminary results using the prepared DNA/PDDA/SWCNTs particles on electrodes to electrochemically detect DNA chemical damage. An electrode modified by the DNA/PDDA/ SWCNTs particles shows a dramatic change of the (24) Decher, G.; Hong, J. Makromol. Chem. Macromol. Symp. 1991, 46, 321. (25) Wang, B. Q.; Rusling, J. F. Anal. Chem. 2003, 75, 4229. (26) Sun, J. Q.; Sun, Y. P.; Wang, Z. Q.; Wang, Y.; Zhang, X.; Shen, J. C. Macromol. Chem. Phys. 2001, 202, 111. (27) Lvov, Y.; Antipov, A. A.; Mamedov, A.; Moehwald, H.; Sukhorukov, G. B. Nano Lett. 2001, 3, 125. (28) Zheng, H. P.; Lee, H.; Rubner. M. F.; Hammond, P. T. Adv. Mater. 2002, 14, 569. (29) Harris, J. J.; DeRose, P. M.; Bruening, M. L. J. Am. Chem. Soc. 1999, 121, 1978. (30) Zhang, H. Y.; Fu, Y.; Wang, D.; Wang, L.; Wang, Z. Q.; Zhang, X. Langmuir 2003, 19, 8497. (31) Kotov, N. A.; Dekany, I.; Fendler, J. H.J. Phys. Chem. 1995, 99, 13065. (32) Zhang, X.; Shi, F.; Yu, X.; Liu, H.; Fu, Y.; Wang, Z.; Jiang, L.; Li, X. J. Am. Chem. Soc. 2004, 126, 3064. (33) Jiang, C.; Markutsya, S.; Tsukruk, V. V. Adv. Mater. 2004, 16, 157.

2.1. Materials. Single-walled carbon nanotubes (50-70%, diameter 1.2-1.5 nm, length 2-5 µm) were purchased from Aldrich and shortened as described below in the section 2.2. Poly(diallyldimethylammonium) (Mw ) 200 000-350 000) was purchased from Aldrich. Double-stranded Calf thymus DNA (ds-CT DNA), single-stranded calf thymus DNA (ss-CT DNA), and polyguanine (PolyG) were purchased from Sigma-Aldrich. Water used in all experiments was purified in a Barnstead Nanopure purification system and had a resistivity higher than 18.1 MΩ‚ cm. Pyrolytic graphite used in electrochemical measurements was purchased from Advanced Ceramics, Cleveland, OH. Pyrolytic graphite was cut into small cylinders along the basal plane; these were inserted and sealed in glass tubes using nonconductive Huntington Labs resin so as to expose only the basal plane cross section. Inner contact with extension copper wires was secured with conductive epoxy (Huntington Labs). 2.2. Preparation of DNA-Modified SWCNTs Particles. Oxidation of SWCNTs. SWCNTs were purified and oxidized by following literature protocols.34-36 Typically, commercial SWCNTs (250 mg of SWCNTs) were refluxed in 500 mL of 2 M HNO3 for 2 days. Precipitation of SWCNTs as a solid was allowed to proceed overnight, and the clear solution above the suspension was then removed. About 15 mL of suspension was separated by ultracentrifugation (30 min at 14 000 rpm). The purified SWCNTs were further oxidized by treatment with 15 mL of 1:3 HNO3/ H2SO4 mixtures for 2 h in an ultrasonic bath. The suspension was diluted 10 times with water after removal of the clear solution over the precipitates. The solution was filtered on a 30 000 MW filtration membrane using Millipore model 8200 stirred ultrafiltration cell and further washed to neutral pH by water. The water was removed by a rotary evaporator, and the resulting sample was dried in a vacuum for about 4 h. The oxidized SWCNTs as prepared are water-soluble; they were aliquoted and stored as aqueous solutions of 10 mg/mL. Fabrication of DNA-Modified SWCNTs Particles. DNAwrapped SWCNTs particles were fabricated as described in the procedure shown in Figure 1. First, the PDDA polycation was deposited on the negatively charged oxidized SWCNTs as prepared above. The PDDA/SWCNTs system was prepared by slowly adding 100 µL of 10 mg/mL oxidized SWCNTs into 0.9 mL of 50 mM NaCl aqueous solution containing 10 mg/mL PDDA under strong stirring for 30 min, followed by centrifugation at 14 000 rpm for 20 min. The residue was washed with water 3 times. To further wrap DNA on the PDDA/SWCNTs, 1.0 mg of PDDA/SWCNTs particles was dissolved in 1.0 mL of 0.1 M tris(hydroxymethyl)aminomethane hydrochloride (tris-HCl) (trisHCl) (pH ) 7.0) buffer under sonication for 10 min. Subsequently, 100 µL of PDDA/SWCNTs solution was added into 100 µL of 0.1 M tris-HCl (pH ) 7.0) buffer solution containing 10 µg/mL DNA under strong stirring for 30 min. The resulting mixture was then centrifugated at 14 000 rpm for 20 min; the solid was washed 3 times with water to yield pure DNA/PDDA/SWCNTs particles. 2.3. Equipment and Measurements. Scanning Electron Microscopy (SEM). SEM images of SWCNTs, PDDA/SWCNTs, and DNA/PDDA/SWCNTs were taken using a Hitachi S-215 SEM. The samples were prepared by dropping 50 µL of aqueous (34) (a) Williams, K. A.; Veenhuizen, P. T. M.; Torre, B. G.; Eritja, R.; Dekker, C. Nature 2002, 420, 761. (b) Liu, Z.; Shen, Z.; Zhu, T.; Hou, S.; Ying, L. Langmuir 2000, 16, 3569. (c) Toebes, M. L.; van Heeswijk, J. M. P.; Bitter, J. H.; van Dillen, A. J.; de Jong, K. P. Carbon 2004, 42, 307. (35) (a) Lin, Y.; Rao, A. M.; Sadanadan, B.; Kenik, E. A.; Sun, Y. J. Phys. Chem. B 2002, 106, 1294. (b) Ajayan, P. M.; Ebbesen, T. W. Rep. Prog. Phys. 1997, 60, 1025. (c) Moon, J.-M.; An, K. H.; Lee, Y. H.; Park, Y. S.; Bae, D. J.; Park, G.-S. J. Phys. Chem. B 2001, 105, 5667. (36) (a) Kashiwagia T.; Dub, F.; Wineyc, K. I.; Grotha, K. M.; Shieldsa, J. R.; Bellayera, S. P.; Kimc, H.; Douglas, J. F. Polymer 2005, 46, 471 and references therein. (b) Shenogin; S.; Xue, L.; Ozisik, R.; Keblinskia, P. J. Appl. Phys. 2004, 95, 8136. (c) Further Raman characterization of end-residues of TGA in both cases of PDDA/SWCNTs and DNA/ PDDA/SWCNTs shows that the residues still contain intact SWCNTs as evidenced by their characteristic D-band and tangential stretch G-band (see Supporting Information Figure S2).

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Figure 2. SEM images of (a) oxidized SWCNTs, (b) PDDA/SWCNTs, and (c) DNA/PDDA/SWCNTs. Scale bars are 5 µm. suspensions containing 10 µg/mL of bare, or modified, SWCNTs particles on a fresh surface of mica; the sample was then allowed to dry at room temperature for 4 h. High-Resolution Transmission Electron Microscopy (HRTEM). HRTEM experiments were performed using a Tecnai F30 300 kV field-emission gun energy-filtering high-resolution scanning transmission electron microscope. The samples were dispersed in water or methanol and suspended on a perforated carbon film supported by Copper grids. Raman Spectroscopy. Raman spectra were obtained using a 647.1-nm excitation laser with 30 kW/cm2 power from Innova 400 krypton laser system and a back-illuminated CCD detector (model 1024 EHRB/l, Princeton Instruments) operating at 183 K; the Raman microscope used (Kaiser Optical Systems) was equipped with a 10× long working length objective (2 cm). The solid sample to be analyzed was placed on aluminum substrate. UV-Visible Spectra. The UV-visible spectra were taken using an Agilent 8453 UV-visible spectroscopy system (Agilent Technologies). A 1-cm quartz cell was used in all measurements. To take the UV-visible spectra, 10 µL of aqueous suspensions of purified DNA/PDDA/SWCNTs particles and 100 µL of the aqueous solution used in the prepration of DNA/PDDA/SWCNTs particles were diluted to 1.0 mL with 0.1 M tris-HCl (pH ) 7.0 buffer). Thermogravimetric Analysis (TGA). TGA was carried out using a TGA2950l thermogravimetric analyzer (TA Instrument). The sample was heated from room temperature to 800 °C at a rate of 20 °C/min in ultrapure N2 gas flow (8 mL/min). Prior to analysis, the samples were thoroughly dried in a vacuum for 4 h. Electrochemical Measurements. Square wave voltammetry (SWV) was performed on a BAS-100W electrochemical workstation (Bioanalytical System Inc.). A three-electrode system was used that included an Ag/AgCl reference electrode, a platinum wire as a counter electrode, and 3-mm diameter pyrolytic graphite (PG) homemade electrodes (bare or modified) as working electrodes. Except where otherwise specified, a potential range from 0.4 to 1.2 V was used in SWV experiments with 4 mV step height, 25 mV pulse height, and 15 Hz frequency, as working parameters. The electrolyte solution was 10 mM acetate buffer containing 50 mM NaCl (pH 5.5); 50 µM Ru(bpy)32+ was used as the reporting redox probe as described in the next section. Solutions were purged with pure N2 gas for 15 min prior to experiments, and a nitrogen blanket was maintained during the experiment. Modified PG electrodes were prepared using various particle systems, including poly(guanine)/PDDA/SWCNTs, ssCT-DNA/PDDA/SWCNTs, ds-CT-DNA/PDDA/SWCNTs, and chemically damaged ds-CT DNA/PDDA/SWCNTs. The modification procedure was carried out by dropping 10 µL of 10 µg/mL aqueous solutions of coated SWCNTs particles on the surface of PG electrode. Prior to modification, PG electrodes were polished with 0.3 µm alumina slurries, washed, sonicated for about 1 min, and then rinsed with water. Modified electrodes were allowed to dry for 4 h at room temperature. The dried electrode was further carefully rinsed with water prior to electrochemical measure-

ments. The reference (DNA/PDDA)3/PG electrode was prepared as described in the literature by Rusling et al.37

3. Results and Discussion 3.1. Preparation and Characterization. DNAmodified SWCNTs is prepared by the LBL deposition of positively charged polyelectrolyte PDDA on the sidewall of negatively charged oxidized SWCNTs, followed by a layer of negatively charged DNA. While the DNA/PDDA/ SWCNTs particles can be separated by ultracentrifugation at 10 000 rpm, ultracentrifugation at rotations as high as 14 000 rpm is necessary to separate particles of PDDA/ SWCNTs. On the other hand, while oxidized SWCNTs also tend to aggregate into small particles by electrostatic adsorption, these cannot be separated by ultracentrifugation. This is consistent with SEM images which show that the size of features increases from oxidized SWCNTs, to PDDA/SWCNTs and from the latter to DNA/PDDA/ SWCNTs, Figure 2. To further observe the surface morphology of the DNAmodified SWCNTs particles, we carried out HRTEM analysis of modified SWCNTs. Figure 3 presents typical HRTEM images of SWCNTs and coated SWCNTs. As shown in the magnification views in the insets of Figure 3a,b, the clear edge of the sidewalls of carbon nanotubes can be easily observed in the pristine SWCNTs (Figure 3a), as well as in oxidized SWCNTs (Figure 3b), although individual SWCNTs tend to cluster together due to high surface energies.38 However, in the images of PDDA/SWCNTs (Figure 3c) and DNA/PDDA/SWCNTs (Figure 3d), one cannot see the typical clear edge around the surface of SWCNTs; instead, the high-resolution images show ultrathin coating films of DNA and/or PDDA around the outer surface of oxidized SWCNTs. Figure 4 shows UV-visible absorption spectra of an aqueous suspension of DNA/PDDA/SWCNTs and of the solution from which DNA/PDDA/SWCNTs was separated out by ultracentrifugation. The DNA/PDDA/SWCNTs sample exhibits a typical DNA absorption band at 260 nm, Figure 4a. On the other hand, this feature is absent in the absorption spectrum of the solution from which DNA/PDDA/SWCNTs is separated out, Figure 4b. Together, these results indicate that DNA polyanion molecules in solution are efficiently adsorbed on the surface of the positively charged PDDA/SWCNTs particles. Raman spectroscopy is one of key analytical techniques used in the characterization of SWNTs; for instance, this (37) Zhou, L.; Rusling, F. Anal. Chem. 2001, 73, 4780. (38) He, P.; Lian, J.; Shi, D.; Wang, L.; Mast, D.; Ooij, W. J.; Schulz, M. Mater. Res. Soc. Symp. Proc. 2003, 740, I3.19.1

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Figure 3. HRTEM images of (a) pristine SWCNTs, (b) oxidized SWCNTs, (c) PDDA/SWCNTs, and (d) DNA/PDDA/SWCNTs. The inset on the bottom right of each image represents a magnification of the image.

spectroscopic technique has been successfully used in the characterization of SWCNTs/polymer composites.39-42 We also used Raman spectroscopy to monitor individual steps of the LBL fabrication process on the sidewalls of carbon nanotubes and to characterize the resulting DNA/PDDA/ SWCNTs particles. Figure 5 shows typical Raman spectra obtained with a 647.1 nm laser excitation at different steps of the LBL fabrication process. The disorder-induced D-band at ∼1328 cm-1 and the tangential stretch G-band at ∼1580 cm-1, which are the main features in Raman spectra of carbon nanotubes,43a are indeed observed for oxidized SWCNTs, PDDA/SWCNTs, and DNA/PDDA/ SWCNTs, Figure 5a-c. Interestingly, the PDDA/SWCNTs (39) Zhonghoua, Y.; Brus, L. E. J. Phys. Chem. A 2000, 104, 10995. (40) Hadjiev, V. G.; Lliv, M. N.; Arepalli, S.; Files, P. B. S. App. Phys. Lett. 2001, 78, 3193. (41) Chambers, G.; Carroll, C.; Farrell, F. G.; Dalton, B. A.; McNamara, M.; Panhuis, M.; Byrne, J. H. Nano Lett. 2003, 3, 843. (42) Bhattacharyya, B. A.; Sreekumar, V. T.; Lui, T.; Kumar, S.; Ericson, M. L.; Hauge, H. R.; Smalley, E. R. Polymer (London) 2003, 44, 2373.

and DNA/PDDA/SWCNTs exhibit tangential G-bands upshifted 16 and 22 cm-1, respectively, relative to that of bare oxidized SWCNTs at 1575 cm-1, Figure 5a-c. Similar upshifts in the tangential mode have been reported in SWCNTs/polymer composites, and they indicate the presence of polymer molecules wrapped around the sidewall of SWCNTs.40-42 The intensities of the typical disorder-induced D-band at ∼1328 cm-1 for both PDDA/SWCNTs and DNA/PDDA/ SWCNTs are markedly increased compared to the intensity of the same band in oxidized SWCNTs. This likely comes from the contribution of overlapping Raman features originating from added layers of PDDA and/or DNA on oxidized SWCNTs. In fact, Raman spectra of calf thymus DNA and PDDA indicate that both of these (43) (a) Dresselhaus, S. M.; Eklund, C. P. Adv. Phys. 2000, 49, 705. (b) The increased relative intensity of the disorder-induced D-band (∼1328 cm-1) in coated SWCNTs may also be inherently due to introduced DNA and/or PDDA on carbon nanotubes, which introduce structural defects, finite size effects, and symmetry breakdown on the nanotube surface (see ref 43a).

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Figure 4. UV-visible absorption spectra of suspension solutions of (a) DNA/PDDA/SWCNTs and (b) the solution after separating DNA/PDDA/SWCNTs particles by ultracentrifugation.

Figure 5. Raman spectra of (a) ds-calf thymus DNA/PDDA/ SWCNTs, (b) PDDA/SWCNTs, (c) oxidized SWNTs, (d) calf thymus DNA, and (e) PDDA taken at 647.1 nm laser excitation. Inset: Magnification of the Raman spectra of DNA/PDDA/ SWCNTs, PDDA/SWCNTs, and oxidized SWNTs from 1350 to 200 cm-1.

macromolecules exhibit overlapping Raman scattering bands in the 1200-1350 cm-1 range, Figure 5d,e.43b Careful analysis of Raman spectra gives further evidence for sequential PDDA and DNA adsorption on chemically oxidized SWCNTs. In fact, the inset of Figure 5 shows a magnification of the Raman spectra from 1350 to 200 cm-1 for bare SWCNTs, PDDA/SWCNT, and for DNA/PDDA/SWCNTs samples. Four Raman scattering peaks, which do not appear in the spectrum of oxidized SWCNTs, are clearly identified for DNA/PDDA/SWCNTs at 1130, 765, 615, and 410 cm-1 (Figure 5, inset a). These peaks originate from a number of Raman features of both PDDA and DNA in this range of the spectrum. As shown in Figure 5d, calf thymus DNA has Raman scattering peaks at ∼1336, ∼1092, ∼1141, ∼784, and ∼425 cm-1, for vibrations of various deoxynucleosides.44 In addition, as seen in Figure 5e, PDDA features Raman scattering peaks at 1326, 1105, 784, 580, and 440 cm-1. Together, these

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results give further evidence for the stepwise layer-bylayer preparation of DNA/PDDA/SWCNTs particles. The preparation is mainly driven by electrostatic adsorption of charged macromolecules, PDDA and DNA, on oppositely charged outer layer of SWCNTs and PDDA/SWCNTs, respectively. Thermogravimetric analysis (TGA) and its differential version (DTA) are complementary techniques that can reveal the composition and any change in thermal stability of modified SWCNTs. Figure 6A,B compares the TGA and DTA results of pristine SWCNTs, oxidized SWCNTs, PDDA/SWCNTs, and DNA/PDDA/SWCNTs. TGA of nontreated pristine SWCNTs shows that about 35.4 wt % residue is left after heating to 800 °C. This is probably due to nondegradable impurities originally present in nontreated SWCNTs sample. The purification and oxidative treatment of pristine single-walled carbon nanotubes likely removes most of these impurities35 as evidenced by the low end-residue after the TGA heating process. Also, the mere purification and chemical oxidation in H2SO4/ HNO3 of pristine SWCNTs increases significantly the temperature of the main weight transition relative to the loss of carbon nanotubes from 485 to 613 °C, Figure 6B (a, b) (see large transitions).36a On the other hand, the amount of residue decreases from 35.4% to 11.7%, Figure 6A (a, b), consistent with the expected outcome of the purification step.35 These results speak to the fact that oxidized SWCNTs have higher purity and higher thermal stability compared to nontreated pristine SWCNTs. When DNA and/or PDDA are deposited on the surface of oxidized SWCNTs, initial decomposition of the composites are recorded at relatively lower temperatures compared to oxidized SWCNTs. Typically, these initial transitions occur as low as 420 and 396 °C for PDDA/ SWCNTs and DNA/PDDA/SWCNTs, respectively, Figure 6B (c, d). Similar transitions below ca. 500 °C are observed for PDDA and DNA controls (Supporting Information Figure S1). On the other hand, assembly of DNA and/or PDDA coatings on oxidized SWCNTs have markedly increased the percentage of end-residues in both cases to over 50% as compared to bare SWCNTs (PDDA/SWCNTs, 51.62%; DNA/PDDA/SWCNTs, 58.42%). Raman characterization of the end-residue of PDDA/SWCNTs after TGA shows that the core SWCNTs still exist in the end-residue; see Supporting Information Figure S2. Whether decomposition products of DNA and/or PDDA coatings are contributing to the observed resistance to thermal degradation of the core SWCNTs in the end-residues remains to be addressed.36b-c At this point, however, it is clear that the DNA/PDDA layer-by-layer modification of oxidized SWCNTs cooperatively brings about an intrinsic thermal resistance of core nanotubes represented by more than ∼50% end-residue, with absence of the typical weight loss transition of the carbon nanotubes. 3.2. Electrocatalysis at DNA/PDDA/SWCNTs Particle Modified Electrodes. Ru(bpy)32+ as a diffusive redox probe has been extensively used for electrocatalytic sensing of DNA at electrode surfaces.45,46 The method relies on the catalytic oxidation of guanine sites in DNA by the free oxidized form of ruthenium complex generated at the electrode interface45,46 and offers a sensitive electrochemical detection of DNA. Rusling et al. utilized this technique (44) Deng, H.; Bloomfield, A. V.; Benevides, M. J.; Thomas, J. G. Biopolymers 1999, 50, 656. (45) Johnston, D. H.; Glasgow, K. C.; Thorp, H. H. J. Am. Chem. Soc. 1995, 117, 8933. (46) Sistare, F. M.; Codden, J. S.; Heimlich, G.; Thorp, H. H. J. Am. Chem. Soc. 2000, 122, 4742.

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Figure 6. Thermograms of (a) pristine SWCNTs, (b) oxidized SWCNTs, (c) PDDA/SWCNTs, (d) DNA/PDDA/SWCNTs: (A) TGA curves; (B) DTA curves.

Figure 7. SWV of different modified electrodes in 10 mM acetate buffer, pH 5.5, containing 50 µM Ru(bpy)32+ and 50 mM NaCl: (a) DNA/PDDA/SWCNTs/PG; (b) PDDA/SWCNTs/PG; (c) SWCNTs/PG; (d) (DNA/PDDA)3/PG; (e) PG.

to study DNA chemical damage on DNA-modified pyrolytic graphite electrodes. The technique is based on increased access of the redox probes to guanine sites as a result of structural change induced by the chemical damage.37,47 This method was successfully used to detect chemical damage of ds-DNA by styrene oxide in DNA-composite films formed by LBL adsorption of PDDA and ds-DNA on oxidized pyrolytic graphite (PG) flat electrodes. We wanted to investigate whether our DNA/PDDA/ SWCNTs particles can be used to modify solid electrodes. We also wanted to examine the performance of these modified electrodes in DNA chemical damage sensing using the sensitive electrocatalytic oxidation current of the Ru(bpy)32+ probe. Figure 7 shows the square wave voltammetry (SWV) responses of different modified PG electrodes in aqueous solution of 50 µM Ru(bpy)32+. While the three layers of the LBL film of ds-DNA adsorbed on the solid PG electrode give rise to an electrocatalytic oxidation current, an electrode modified with DNA/PDDA/ SWCNTs particles as described above shows relatively higher electrocatalytic response, Figure 7a. The observed larger electrocatalytic response recorded on electrodes modified with DNA/PDDA/SWCNTs particles relative to flat DNA films may speak to improved access of the (47) Zhou, L.; Yang, J.; Estavillo, C.; Stuart, D. J.; Schenkman, B. J.; Rusling, F. J. Am. Chem. Soc. 2003, 125, 1431.

ruthenium probe to guanine sites in the DNA-modified carbon nanotubes and/or relatively larger ratio of DNA to surface area. Control experiments with PDDA/SWCNTs/ PG and SWCNTs/PG modified electrodes show that the observed currents are larger than bare graphite electrode and electrodes modified with flat layers of DNA/PDDA; however, these currents are still smaller than the catalytic current measured on an electrode modified with DNAmodified SWCNTs, Figure 7b,c. The larger currents are recorded only in the presence of DNA-modified particles and at less positive potentials characteristic of electrocatalytic regeneration of the redox probe. We next wanted to exploit this sensitive DNA sensing strategy to detect structural damage in DNA-modified SWCNTs particles. We chose nitric oxide, NO, as the reactive metabolite in this preliminary study. In fact, emerging studies on chronic inflammation as it relates to carcinogenesis point to NO as a key player in cancer development.48 Nitric oxide is a molecule that is now widely known as being produced in living organisms including humans by enzymes called nitric oxide synthases (NOS). Although macrophage-derived NO is crucial for the normal immune response, cells and their constituents may become unsafely exposed to excessive amounts of NO especially during chronic inflammation, and it is believed that sustained high-level generation of NO provides a conceivable link between chronic inflammation and cancer initiation, growth, and progression.48 Detecting and quantifying direct DNA lesions induced by NO and derived reactive nitrogen species (RNS) under physiologic conditions is therefore invaluable in rationalizing these emerging hypotheses. As a preliminary step to use DNA-modified SWCNTs particles in the study of NO-induced DNA damage, we compared the square wave voltammetry response of various modified SWCNTs particles cast on pyrolitic graphite electrodes. Both particles coated with intact dsDNA and those with ds-DNA preincubated in NO solution were tested. The NO-treated ds-CT DNA was prepared by incubating 100 µg/mL of ds-CT DNA in NO for 30 min. The catalytic oxidation current in aqueous solution of Ru(bpy)32+ increases from intact ds-CT DNA, Figure 8a, to NO-treated ds-CT DNA, Figure 8b, possibly indicating (48) (a) Lala P.; Chakraborty C. Lancet Oncol. 2001, 2, 149. (b) Thomsen, L. L.; Lawton, F. G.; Knowles, R. G. Cancer Res. 1994, 54, 1352. (c) Thomsen, L. L.; Miles, D. W.; Happerfield, L. Br. J. Cancer 1995, 72, 41.

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Figure 8. SWV of different DNA/PDDA/SWCNTs modified electrodes in 10 mM acetate buffer, pH 5.5, containing 50 µM Ru(bpy)32+ and 50 mM NaCl: (a) intact ds-CT DNA; (b) NOtreated ds-CT DNA; (c) ss-CT DNA.

DNA structural damage upon reaction with NO. In fact, control experiment using ss-CT DNA-modified SWCNTs particles gave a larger catalytic current, consistent with the expected higher access of the redox probe to the guanine sites in ss-CT DNA-coated particles, Figure 8c.45 More work on detection of DNA structural changes induced by NO and derived reactive nitrogen species is in progress and will be presented in detail in another paper. 4. Conclusion Fabrication of new DNA/SWCNTs particles using the LBL technique on oxidized sidewalls of SWCNTs has been described. The particles were characterized by spectroscopic, thermogravimetric, and electrochemical tech-

He and Bayachou

niques. It was found that, during the process of the fabrication, the water-soluble oxidized SWCNTs can aggregate into small particles. Polycation PDDA and DNA as the negatively charged counterpart can be alternatively deposited on the sidewalls of oxidized SWCNTs. The resulting DNA/PDDA/SWCNTs particles have the distinctive UV-visible spectrum and Raman spectra of DNA. Thermogravimetric analysis shows that the outer shell (DNA/PDDA) of the particles brings about a thermal resistance to decomposition. An electrode modified by DNA/PDDA/SWCNTs particles exhibits larger electrocatalytic oxidation current in an aqueous solution of Ru(bpy)32+. Preliminary experiments have shown that these particles can be used as DNA carriers for sensitive electrochemical detection of DNA chemical damage, including NO-induced DNA structural damage. Acknowledgment. Authors wish to express their gratitude to Dr. Paul Carey and the Carey Laboratories at Case Western University for access and assistance with Raman characterization. We also thank Dr. Alan Riga for providing access to TGA instruments at CSU and Ling Li for help with some controls. This research is supported by a pilot grant from the American Cancer Society (Ohio Division, Cuyahoga office), by an EFFRD grant from CSU, and in part by a grant from the U.S. Department of Energy. Supporting Information Available: Thermogravimetric analysis controls of DNA and PDDA up to 800 °C (Figure S1) as well as Raman characterization of the end-residue after a typical TGA experiment of a PDDA/SWCNT sample (Figure S2). This material is available free of charge via the Internet at http://pubs.acs.org. LA050581B