Noncovalent Assembly of Carbon Nanotubes and Single-Stranded DNA

Sep 4, 2008 - In this paper, we report the assembly of single-walled carbon nanotubes ... This novel sensing platform consists of a structure formed b...
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Anal. Chem. 2008, 80, 7408–7413

Noncovalent Assembly of Carbon Nanotubes and Single-Stranded DNA: An Effective Sensing Platform for Probing Biomolecular Interactions Ronghua Yang,†,‡ Zhiwen Tang,† Jilin Yan,† Huaizhi Kang,† Youngmi Kim,† Zhi Zhu,† and Weihong Tan*,† Department of Chemistry and Department of Physiology and Functional Genomics, Shands Cancer Center and University of Florida Genetics Institute, Center for Research at the Bio/Nano Interface, McKnight Brain Institute, University of Florida, Gainesville, Florida 32611-7200, and Biomedical Engineering Center, State Key Laboratory of Chemo/Biosensing and Chemometrics, College of Chemistry and Chemical Engineering, Hunan University, Changsha, 410082, China In this paper, we report the assembly of single-walled carbon nanotubes (SWNTs) and single-stranded DNA to develop a new class of fluorescent biosensors which are able to probe and recognize biomolecular interactions in a homogeneous format. This novel sensing platform consists of a structure formed by the interaction of SWNTs and dye-labeled DNA oligonucleotides such that the proximity of the nanotube to the dye effectively quenches the fluorescence in the absence of a target. Conversely, and very importantly, the competitive binding of a target DNA or protein with SWNTs for the oligonucleotide results in the restoration of fluorescence signal in increments relative to the fluorescence without a target. This signaling mechanism makes it possible to detect the target by fluorescence spectroscopy. In the present study, the schemes for such fluorescence changes were examined by fluorescence anisotropy and fluorescence intensity measurements for DNA hybridization and aptamer-protein interaction studies. Many approaches have been developed for probing biomolecular interactions such as DNA/RNA hybridization and protein interactions. The most versatile of these approaches include, for example, Taqman,1 molecular beacons,2,3 and fluorescence signaling aptamers,4,5 and utilize solution-based fluorescence hybridization. The selectivity of these probes is achieved by capitalizing on the highly specific molecular recognition ability of biomolecules, such as antibody-antigen binding and DNA base paring. Their effectiveness, however, is highly dependent on the ability to transduce a recognition event to a measurable fluorescence * To whom correspondence should be addressed. E-mail: tan@ chem.ufl.edu. Phone and fax: (+1) 352-846-2410. † University of Florida. ‡ Hunan University. (1) Holland, P. M.; Abramson, R. D.; Watson, R.; Gelfand, D. H. Proc. Natl. Acad. Sci. U.S.A. 1991, 88, 7276–7280. (2) Tyagi, S.; Kramer, F. R. Nat. Biotechnol. 1996, 14, 303–308. (3) Yang, C. J.; Medley, C. D.; Tan, W. H. Curr. Pharm. Biotechnol. 2005, 6, 445–452. (4) Jhaveri, S. D.; Kirby, R.; Conrad, R.; Maglott, E. J.; Bowser, M.; Kennedy, R. T.; Glick, G.; Ellington, A. D. J. Am. Chem. Soc. 2000, 122, 2469–2473. (5) Nutiu, R.; Li, Y. F. Methods 2005, 37, 16–25.

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signal. This is normally accomplished by labeling the same DNA oligonucleotide with a dye-quencher pair so that fluorescence resonance energy transfer (FRET) can take place. Binding with a target then changes the conformation of the oligonucleotide. This alters the distance between the dye and quencher and ultimately results in fluorescence restoration. There are, however, several variables that can compromise the increment of signal change upon interacting with the targets. These primarily include (1) selection of dye-quencher properties, (2) means of attachment of dye-quencher groups, (3) unidentifiable target binding sites, and (4) unforeseen conformational changes. Although great efforts6-11 have been made to find effective solutions to these problems, the results have, thus far, not been satisfactory. Nonetheless, researchers have recently been able to combine the specific molecular recognition ability of biomolecules with the unique structural and photophysical characteristics of inorganic nanomaterials, such as nanocrystals, nanotubes, and nanowires, to create new types of analytical tools for life science and biotechnology.12 Specifically, as a result of their unique chemical, electrical, and mechanical properties, carbon nanotubes have emerged as one of the most extensively studied nanomaterials13,14 with the potential for applications ranging from molecular electronics to ultrasensitive biosensors. Several investigators have recently reported on covalent or noncovalent functionalization of single-walled carbon nanotubes (SWNTs) with nucleic acids15-17 and proteins,18-21 making SWNTs attractive materials for interaction studies15,16 or for use as biosensors17–19 (6) Tyagi, S.; Marras, S. A. E.; Kramer, F. R. Nat. Biotechnol. 2000, 18, 1191– 1196. (7) Dubertret, B.; Calame, M.; Libchaber, A. J. Nat. Biotechnol. 2001, 19, 365– 370. (8) Kuhn, H.; Demidov, V. V.; Coull, J. M.; Fiandaca, M. J.; Gildea, B. D.; FrankKamenetskii, M. D. J. Am. Chem. Soc. 2002, 124, 1097–1103. (9) Wang, L.; Yang, C. J.; Medley, C. D.; Benner, S. A.; Tan, W. H. J. Am. Chem. Soc. 2005, 127, 15664–15665. (10) Cook, R. M.; Lyttle, M.; Dick, D. U.S. Patent 2001-US15082, 2001. (11) Yang, C. J.; Lin, H.; Tan, W. H. J. Am. Chem. Soc. 2005, 127, 12772–12773. (12) Niemeyer, C. M. Angew. Chem., Int. Ed. 2001, 40, 4128–4158. (13) Ajayan, P. M. Chem. Rev. 1999, 99, 1787–1799. (14) Tasis, D.; Tagmatarchis, N.; Bianco, A.; Prato, M. Chem. Rev. 2006, 106, 1105–1136. (15) Zhang, M.; Jagota, A.; Semke, E. D.; Bruce, A.; Diner, B. A.; Mclean, R. S.; Lustig, S. R.; Richardson, R. E.; Tassi, N. G. Nat. Mater. 2003, 2, 338– 342. 10.1021/ac801118p CCC: $40.75  2008 American Chemical Society Published on Web 09/04/2008

Figure 1. (A) Scheme for signaling biomolecular interactions by the assembly of SWNTs and dye-labeled ssDNA. The illustration of the dye-DNAfunctionalized SWNT is only a graphic presentation and does not represent the precise way that DNA binds on SWNTs. (B) Structures of FAM, the FAM-labeled oligonucleotides, P1 and P2, the perfect cDNA (T1) and one mismatched DNA (T2) of P1, and thrombin. Oligo represents the position of attachment of tethered oligonucleotides.

and drug transporters.20 Also, scatter examples of noncovalent interactions of SWNTs with organic dyes or dye-labeled biomolecules have now been reported.22-29 Photophysical studies have demonstrated that SWNTs can act collectively as quenchers for (16) 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, 196–199. (17) Tang, X. W.; Bansaruntip, S.; Nakayama, N.; Yenilmez, E.; Chang, Y. I.; Wang, Q. Nano Lett. 2006, 6, 1632–1636. (18) Chen, R. J.; Bangsaruntip, S.; Drouvalakis, K. A.; Kam, N. W. S.; Shim, M.; Li, M. Y.; Kim, W.; Utz, P. J.; Dai, H. J. Proc. Natl. Acad. Sci. U.S.A 2003, 100, 4984–4989. (19) So, H. M.; Won, K.; Kim, H.; Kim, B. K.; Ryu, B. H.; Na, P. S.; Kim, H.; Lee, J. O. J. Am. Chem. Soc. 2005, 127, 11906–11907. (20) Wang, N.; Kam, S.; Dai, H. J. J. Am. Chem. Soc. 2005, 127, 6021–6026. (21) Pantarotto, D.; Partidos, D.; Hoebeke, J.; Brown, F.; Kramer, E.; Briand, J. P.; Muller, S.; Prato, M.; Bianco, A. Chem. Biol. 2003, 10, 961–966. (22) For a review, see: Tournus, F.; Latil, S.; Heggie, M. I.; Charlier, J.-C. Phys. Rev. B 2005, 72, 075431. (23) Li, H. P.; Zhou, B.; Lin, Y.; Gu, L. R.; Wang, W.; Fernando, K. A. S.; Kumar, S.; Allard, L.; Sun, Y. P. J. Am. Chem. Soc. 2004, 126, 1014–1015. (24) Nakayama-Ratchford, N.; Bangsaruntip, S.; Sun, X. M.; Welsher, K.; Dai, H. J. J. Am. Chem. Soc. 2007, 129, 2448–2449. (25) Boul, P. J.; Cho, D. G.; Rahman, G. M. A.; Marquez, M.; Ou, P. Z.; Kadish, K. M.; Guldi, D. M.; Jonathan, L.; Sessler, J. L. J. Am. Chem. Soc. 2007, 129, 5683–5687. (26) Lu, Q.; Freedman, K. O.; Rao, R.; Lee, J.; Larcom, L. L.; Rao, A. M.; Ke, P. C. J. Appl. Phys. 2004, 96, 6772–6775. (27) Jeng, E. S.; Moll, A. E.; Roy, A. C.; Gastala, J. B.; Strano, M. S. Nano Lett. 2006, 6, 371–375. (28) Kim, N. W. S.; O’Connell, M.; Wisdom, J. A.; Dai, H. J. Proc. Natl. Acad. Sci. U.S.A. 2005, 102, 11600–11605. (29) Lin, S. J.; Keskar, G.; Wu, Y. N.; Wang, X.; Mount, A. S.; Klaine, S. J.; Moore, J. S.; Rao, A. M.; Chun Ke, P. Appl. Phys. Lett. 2006, 89, 143118.

dyes. Although this discovery provides a basis for fluorescent sensing, no quenching approaches have, thus far, been applied to analytical applications via fluorescence restoration.30,31 Therefore, in this report, we present an effective fluorescent sensing platform which is based on (1) the noncovalent assembly of SWNTs and dye-labeled single-stranded DNA (ssDNA) and (2) the ability of the SWNTs complex thus formed to both effectively quench and, in the presence of a target, restore the fluorescence signal to a state comparable to that without a target. To test the general feasibility of this approach, a 23-base oligonucleotide (P1, Figure 1) and a human R-thrombin (Tmb) binding aptamer (P2) were chosen as models. The two DNA oligonucleotides were labeled with a fluorescein derivative, FAM. The target DNA molecules, T1 and T2, were 23-mer long and were either perfectly complementary to the bases of P1 or contained one mismatch base with P1, respectively. Figure 1 shows the signaling scheme of this approach. Briefly, in DMF solution, the SWNTs exist in aggregate form; then, binding of the dye-labeled ssDNA with the SWNTs disperses the aggregates that are individual entities in the solution. This activity results in the formation of an ssDNA/SWNT structure in which the dye molecule is in close proximity to the nanotube, thus quenching the dye’s fluorescence. In addition, since the binding rate of DNA and the nanotube is lower than DNA hybridization, competitive binding of the target DNA with the (30) Britz, D. A.; Khlobystov, A. N. Chem. Soc. Rev. 2006, 35, 637–659. (31) Valca´rcel, M.; Ca´rdenas, S.; Simonet, B. M. Anal. Chem. 2007, 79, 4788– 4797.

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carbon nanotubes for the ssDNA strand reduces the interaction of the ssDNA and nanotube and thus the ability to disperse the nanotube’s aggregates. This results in an increase of fluorescence emission comparable to the fluorescence of the ssDNA/SWNT assembled complex. It should be noted that the selectivity of the approach is mainly determined by the specific interaction of the ssDNA and target and that there is a little improvement on the selectivity by the assembly. EXPERIMENTAL SECTION UV-visible-NIR absorption spectra were obtained with a Cary 6000i UV-visible-NIR spectrophotometer. Fluorescence measurements were performed on a Hitachi F-4500 fluorescence spectrofluorometer. Sonications were performed with a probe sonicator; the sonication mode was pulse per second at 20% of the maximum power. Fluorescence anisotropy was measured on a Fluorolog-3 model FL3-22 spectrofluorometer (JOBIN YVONSPEX Industries, Edison, NJ) using a 200 µL quartz cuvette. Transmission electron microscopy (TEM) was performed on a transmission microscope (Hitachi H-700). Data processing was performed on a Pentium IV computer with SigmaPlot software. All proteins were purchased from Aldrich or Sigma. Stock solutions of the samples were prepared by directly dissolving the materials in 0.01 mol/L NaCl and stored in a refrigerator at -20 °C. Standard protein solutions were prepared by serial dilution starting with 0.05 mol/L phosphate buffer (PBS, pH 7.4). All DNA synthesis reagents were purchased from Glen Research. All the DNA sequences were synthesized with an ABI3400 DNA/RNA synthesizer in our laboratory. FAM CPG was used for synthesis of P1 and P2. The products were purified by reversed-phase HPLC.32 The molar concentrations of DNA strands were determined according to the length of the oligonucleotides used. The samples for TEM analysis were prepared by pipetting ∼15 µL of the colloidal solutions onto standard holey carbon-coated copper grids. The grids were dried in air for >12 h before loading into the vacuum chamber of the electron microscope. In a typical dispersion experiment,28,33 the as-grown HiPCo SWNTs (Carbon Nanotechnologies, Inc., Houston, TX) were first sonicated in DMF for 5 h to give a homogeneous black solution. An aliquot of the freshly made SWNTs suspension (less than 3%, v/v) was then added to a PBS containing P1 or P2 and was allowed to incubate for around 15 min. An aliquot of the target (or water) was then added to the nanotube/ssDNA conjugate mixture. The volume of the mixture was around 1.0 mL. After incubation at room temperature for 2-3 h, the upper 70% of the clear solution was received. Undispersed SWNTs and ssDNA oligonucleotides were removed by ultracentrifugation (22 000g) and dialysis against PBS with a membrane (molecular weight cutoff 8000-14 400). In DMF solution, TEM images show that the SWNTs alone were not completely dispersed with sonication. However, in the presence of ssDNA in the DMF solution, well-dispersed, small bundles of the nanotube were observed.34 The mean length of these objects (32) Yang, C. Y.; Jockusch, S.; Vicens, M.; Turro, N. J.; Tan, W. H. Proc. Natl. Acad. Sci. U.S.A. 2005, 102, 17278–17283. (33) Vogel, S. R.; Kappes, M. M.; Hennrich, F.; Richert, C. Chem.sEur. J. 2007, 13, 1815–1820. (34) Yang, R. H.; Jin, J. U.; Chen, Y.; Shao, N.; Tang, Z. W.; Wu, Y. R.; Zhou, Z.; Tan, W. H. J. Am. Chem. Soc. 2008, 130, 8351–8358.

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Figure 2. UV-visible-NIR absorption spectra of the free P1 (50 nM, black), SWNTs (red), and P1 + SWNTs (blue) in PBS.

is typically on the order of micrometers with the diameters from a few nanometers to tens of nanometers. RESULTS AND DISCUSSION We first investigated the optical spectral properties of P1 and P2 in the presence of SWNTs. Figure 2 shows the UV-vis-NIR absorption spectra of the free state of P1, uncoated SWNTs, and P1-SWNTs. While P1 is characterized by absorption bands of the DNA sequence (261 nm) and FAM (472 nm), SWNTs display the characteristic semiconducting bands I (SI) and II (SII) of the pristine nanotube at around 1380 and 910 nm,35 respectively. Addition of SWNTs to the solution of P1 caused not only a hypochromic-red shift of the 472-nm Soret band of FAM but also significant perturbation of the SWNTs NIR absorption bands. This result indicates that there is notable electronic communication between the two π-systems of SWNTs and the dye in the ground state.23–25,36-38 Next, fluorescence spectra of the free P1 and P2 and their nanotube self-assembled complexes, P1-SWNTs and P2-SWNTs, were taken separately. Figure 3 shows the fluorescence emission spectra of P1 at different conditions. Spectrum a was measured in PBS in the absence of carbon nanotubes. Upon excitation at the maximal absorption wavelength of FAM, P1 shows strong fluorescence emission by FAM. However, in the presence of SWNTs, the fluorescence emission by FAM is ∼5% that of the free P1 (spectrum c), indicating that carbon nanotubes efficiently quench the fluorescence of the inner FAM moiety. This fluorescence quenching has been ascribed to the electron or energy transfer from the fluorophore to the nanotubes.22–25 In our study, we found that more than 90% of FAM’s fluorescence was quenched by SWNTs in the DNA probe concentrations of 50-150 nM. Further testing, however, revealed that higher probe concentrations result in a significant decrease of the fluorescence quenching efficiency. Dai et al. recently reported a 67% emission quenching by FAM for SWNTs in PBS.24 The phenomena support the strong adsorption of the ssDNA strand on SWNTs.29,34,38 (35) Satake, A.; Miyajima, Y.; Kobuke, Y. Chem. Mater. 2005, 17, 716–724. (36) Rahman, G. M. A.; Guldi, D. M.; Campidelli, S.; Prato, M. J. Mater. Chem. 2006, 16, 62–65. (37) Ma, Y. F.; Ali, S. R.; Wang, L.; Chiu, P. L.; Mendelsohn, R.; He, H. X. J. Am. Chem. Soc. 2006, 128, 12064–12065. (38) Zheng, M.; Jagota, A.; Strano, M. S.; Adelina, P.; Santos, A. P.; Barone, P.; Chou, S. G.; Diner, B. A.; Dresselhaus, M. S.; Mclean, R. S.; Onoa, G. B.; Samsonidze, G. G.; Semke, E. D.; Usrey, M.; Walls, D. J. Science 2003, 302, 1545–1548.

Figure 3. Fluorescence emission spectra (λex ) 480 nm) of P1 (50 nM) at different conditions: (a) P1 in PBS; (b) P1 + 300 nM T1; (c) P1 + SWNTs; and (d) P1 + SWNTs + 300 nM T1. Inset: fluorescence intensity ratio of P1 (b) and P1-SWNTs (9) with F/F0-1 plotted against the logarithm of the concentration of T1. Excitation was at 480 nm, and emission was monitored at 528 nm.

Taken together, these findings demonstrate that ssDNA fluorescence quenching can be highly efficient for use in probing biomolecular interactions. Specifically, the fluorescence of free P1 was scarcely influenced by the addition of excess of the perfectly cDNA T1, but significant emission enhancement was observed from P1-SWNTs by the same concentration of T1. Figure 3 inset illustrates the fluorescence intensity changes (F/ F0-1) of P1 and P1-SWNTs upon addition of different concentrations of T1, where F0 and F are FAM intensities at 528 nm in the absence and the presence of T1, respectively. In the absence of carbon nanotubes, no significant variation in the fluorescence intensity of P1 was found in the target concentration range. In the DNA concentration range of 5.0-600 nM, however, a dramatic increase of FAM’s fluorescence intensity was observed. These results suggest that the SWNT/DNA assembly approach is effective in probing biomolecular interactions because of the excellent signaling process. On the basis of their exceptional quenching capability to the proximate fluorescent dye, gold nanoparticles have been successfully used to construct fluorescent probes for DNA and protein targets.39-42 For example, Dubertret et al. previously developed DNA-functionalized gold nanoparticles to achieve efficient quenching (up to 99.96% under favorable conditions) and single-base mismatch detection by replacing Dabcyl with 1.4-nm gold clusters (nanogold).39 However, they followed the traditional molecular beacon2,3 design and used hairpin-shaped oligonucleotides by covalent labeling of the DNA strand with a dye and the nanoparticles. Such tedious processes involved in preparing the probe limit this application as a common approach for bioanalysis. While the present SWNTs self-assembled complex has high quenching efficiency and single-base mismatch detection ability equal to gold nanoparticles (see below), our design offers even more advantages, including simplicity of preparation and manipulation, as well as more stability. (39) Dubertret, B.; Calame, M.; Libchaber, A. J. Nat. Biotechnol. 2001, 19, 365– 370. (40) Maxwell, D. J.; Taylor, J. R.; Nie, S. J. Am. Chem. Soc. 2002, 124, 9606– 9612. (41) Seferos, D. S.; Gijohann, D. A.; Hill, H. D.; Prigodich, A. E.; Mirkin, C. A. J. Am. Chem. Soc. 2007, 129, 15477–15479. (42) Xue, X. J.; Wang, F.; Liu, X. G. J. Am. Chem. Soc. 2008, 130, 3244–3245.

Figure 4. Fluorescence emission spectra of P2 (50 nM, A) and P2-SWNTs (B) in the presence of different concentrations of Tmb. Black curves are the fluorescence emission of control probes (same volume of water added) without target. λex ) 480 nm.

Our studies also demonstrated that the interaction of P2-SWNTs with Tmb was effectively transduced by fluorescence enhancement, even in the presence of a low concentration of the target. Figure 4 shows the fluorescence emission spectra of P2 and P2-SWNTs in the presence of different concentrations of Tmb. No obvious emission change could be observed from free P2 upon addition of low concentrations of Tmb. Although P2 shows fluorescence enhancement by high concentration of Tmb due to change of the microenvironment of FAM by the target complex,43 the sensitivity (F/F0) is smaller than that of P2-SWNTs. The F/F0 of P2 at 528 nm is 1.24 in the presence of 100 nM Tmb, which is significantly lower than that observed from P2-SWNTs (F/F0 ) 4.93) by the same concentration of Tmb. The dynamic response range of P2-SWNTs for Tmb is 4.0-150 nM. The limit of Tmb detection, based on 3 times the signal-to-noise level, was estimated to be ∼1.8 nM, which is around 10-fold lower than that of the regular dye-quencher pair-labeled aptamers.4,5 This experiment clearly demonstrates that the aptamer/SWNT approach could be used as a sensitive approach for target protein detection. The kinetic behaviors of P1 and SWNTs, as well as P1-SWNTs with T1, were studied. Both the binding and subsequent DNA hybridization in the presence of carbon nanotubes are fundamentally different from the conventional DNA hybridization probes, which may be characterized by (1) relatively fast but (2) incomplete quenching. In contrast, ssDNA adsorption on the surface of carbon nanotubes is slow at room temperature. Figure 5 shows fluorescence quenching of P1 as a function of incubation time. In the absence of DNA target, the curve exhibits a rapid reduction in the first 0.5 h and a slow decrease over a 2-3-h period. It is hypothesized that the surface effect of the carbon nanotubes and the charge properties of the ssDNA may be the main contributors to this low adsorption rate.27,34,44 In the presence of target T1, a fluorescence decrease of P1 is also observed. However, formation of dsDNA reduces adsorption of P1 onto the SWNTs and thus fluorescence quenching efficiency. This, in turn, results in an overall fluorescence increase, which displays fluorescence enhancement comparable to that without a target. The best fluorescence response (F/F0) was obtained around 2.0 h, although shorter interaction time produced probes (43) Kurata, S.; Kanagawa, T.; Yamada, K.; Torimura, M.; Yokomaku, T.; Kamagata, Y.; Kurane, R. Nucleic Acids Res. 2001, 29, e34. (44) Jeng, E. S.; Barone, P. W.; Nelson, J. D.; Strano, M. S. Small 2007, 3, 1602–1609.

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Figure 5. Fluorescence quenching of P1 (50 nM) in PBS by SWNTs as a function of time, (a) in the absence of target and (b) in the presence of 300 nM T1. Fluorescence intensity was recorded at 528 nm with an excitation wavelength of 480 nm.

with lower F/F0. Strong adsorption of the ssDNA to the nanotube surface may account for the slow hybridization kinetics.27,44 However, the kinetics for both nanotube binding and DNA hybridization could be accelerated by sonication. To illustrate, about 90% fluorescence quenching was observed at room temperature upon continuous sonication for 10 min. We also found that the Tmb binding kinetics of P2 is dependent on both the protein concentration and nanotube surface property. The interaction equilibrium time of P2-SWNTs with Tmb is longer than that of P2 and Tmb, and the equilibrium time increases as Tmb concentrations decrease. The minimal enhancement in fluorescence emission of P2 by low concentration of Tmb can also be explained by the possibility that P2 interacts with Tmb but without fluorescence enhancement. To address this issue, we measured the fluorescence anisotropy (FA) changes of P2 and P2-SWNTs in the presence of Tmb. Anisotropy measurements are commonly used to probe molecular interactions and molecular diffusion in a solution or at a surface.45 In aqueous solution, FA is mainly dependent on the size and mass of the complex to which the dye is attached.45 Therefore, we could judge the binding interaction of P2 with Tmb by measuring FA. In the absence of SWNTs, the FA enhancement of P2 by 5.0 nM Tmb is only 0.002. In comparison, the FA enhancement of P2-SWNTs by 5.0 nM Tmb is 0.0205, a 10.2-fold increase. It is worth noting that FA changes are usually small in our spectrometer and that these changes are measurable and beyond the detection limit. Nevertheless, when combined with the fluorescence intensity changes, this result demonstrates that, while detection of low concentration of Tmb by the aptamer in the absence of SWNTs is ineffective, the limit of detection of the aptamer for Tmb could be reduced by the carbon nanotubes. Since enrichment of protein on the nanotube surface lowers the limit of detection of the analyte, our approach, which involves the binding of SWNTs with both ssDNA and protein,18,20 may itself be the main contributor to the lower limit of detection. We next tested the selectivity of the current approaches. Fluorescence responses of P1-SWNTs toward T1 and the singlebase mismatch target, T2, were determined. The F/F0 value of P1-SWNTs by 150 nM of T2 is ∼45% of that by the same concentration of T1. This DNA sequence specificity is slightly (45) Lakowicz, J. Principles of Fluorescence Spectroscopy, 3rd ed.; Springer: New York, 2006.

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Figure 6. FA and fluorescence intensity changes (F/F0) of P2-SWNTs by proteins (1.0 µM, x-axis markers). Bars represent the final fluorescence response (F) over the initial emission (F0). Excitation was at 480 nm, and emission was monitored at 528 nm.

lower than that of molecular beacons2,3 but still higher than that of linear DNA probes which cannot discriminate single-base mismatch targets. To understand the response behaviors of P2-SWNTs toward different proteins, both the fluorescence intensity and FA changes of P2-SWNTs by different proteins (Tmb, BSA, HSA, and IgG) were studied. As shown in Figure 6, the same enhancements of FA are observed for all proteins as a result of the nonspecific adsorption of the biomolecules on the nanotube surface. In contrast, the enhancements of fluorescence intensity (F/F0) differ among the proteins and follow the order of Tmb . HSA > BSA > IgG. Since only Tmb could change the conformation of the aptamer and, as a result, increase the dye’s fluorescence intensity, these results indicate that the interaction of the aptamer with Tmb is selective. To further characterize the binding specificity of P2-SWNTs for Tmb, the competitive complexes were also conducted in the presence of other proteins. To accomplish this, a titration of Tmb in the presence of 100 nM of BSA, HSA, and IgG gave response curves almost superimposable to the one obtained exclusively in the presence of Tmb. These results clearly indicate that P2-SWNTs are only sensitive and selective toward Tmb, even in the presence of other proteins. The contrasting results demonstrate that the carbon nanotubes are promising building blocks for DNA binding specificity. As the experimental data show, the proposed approach could work well for probing biomolecular interactions because of the high sensitivity and excellent selectivity. However, a key question that arises is precisely how the nanotube complexes interact with the target in order to produce fluorescence enhancement. The binding interactions of the ssDNA with its target could occur either at the nanotube surface or in solution. In the former case, the ssDNA strand adsorbed on the nanotube surface would undergo a change in conformation in response to interaction with its target. In the latter case, the ssDNA adsorbed on the carbon nanotube first comes out from the nanotube surface and then performs target complexation in solution. To determine whether the target interaction occurs on the surface of the carbon nanotube or in solution, the FA changes of P1 and P2 by SWNTs and their targets were respectively measured. As expected, the FA values of P1 and P2 were greatly enhanced by SWNTs, primarily indicating formation of the nanotube complexes. However, interactions of the nanotube complexes with their targets displayed a different trend in FA change (Figure 7). That is, when Tmb was added to the solution of P2-SWNTs, the

Figure 7. Fluorescence anisotropy changes of P1 and P2 (50 nM, x-axis markers) by SWNTs and the target (300 nM for T1; 1.0 µM for Tmb). Excitation was at 480 nm, and emission was monitored at 528 nm.

Figure 8. Fluorescence intensities of P1-SWNTs + T1 (150 nM) and P2-SWNTs + Tmb (100 nM) measured at different conditions: (a) the nanotube-target mixture without dialysis, (b) the mixture dialyzed against phosphate buffer to remove nonbinding DNA molecules, and (c) the dialyzing solution outside the membrane. Excitation was at 480 nm, and emission was monitored at 528 nm. The concentrations of P1 and P2 in the mixture are all 50 nM.

FA significantly increased relative to that of P2-SWNTs alone. This result indicates that complexation of the aptamer and Tmb might occur on the nanotube surface by forming a ternary complex among P2, the SWNT, and Tmb. In contrast, upon addition of T1 to P1-SWNTs, the FA was significantly reduced when compared to that of P1-SWNTs, which is almost the same as that of free P1 upon addition of T1. This result primarily indicates that the DNA hybrids form in solution. To further explain and confirm these apparently conflicting conclusions, we isolated the target complexes of P1-SWNTs and P2-SWNTs by dialysis against PBS with a membrane and measured the fluorescence intensity of the dialysis product and the nanotube complex. For P1-SWNTs, it was found that, while the nanotube complex inside the membrane is nonfluorescent, the product outside the membrane is highly fluorescent. On the contrary, for P2-SWNTs, we could find no significant

fluorescence of the product outside the membrane (Figure 8), implying that P2 is still adsorbed on the nanotube surface after interaction with Tmb. On the basis of these corroborative results, we can suggest that the DNA hybridization performs in solution, but that the interaction of the aptamer and protein occurs on the nanotube surface. In addition, we found that the fluorescence intensity of P1-SWNTs with a large excess of T1 (600 nM) is ∼70% that of free P1, which implies that, in the presence of SWNTs, the maximal amount of DNA hybridization by its perfectly complementary target is around 70%. We also observed that if the incubation time of P1 and SWNTs was more than 24 h, the P1 fluorescence intensity was only minimally affected by an excess of T1, indicating that, under normal conditions and at room temperature, the displacing interaction of P1-SWNTs by T1 hardly takes place by DNA hybridization. CONCLUSION In conclusion, a new sensing platform is proposed using a dyelabeled ssDNA and carbon nanotubes. The design is based on the attachment of the ssDNA on SWNTs, with the DNA strand interacting noncovalently with SWNTs by π-stacking between nucleotide bases and SWNT sidewalls. For probing biomolecular interactions, this platform possesses three excellent molecular engineering features. First, the DNA needs only one dye labeled to show high quenching efficiency by the nanotube, without the need for hairpin structure or peptide self-folding; thus, the application demonstrates superior sensitivity. Second, the interaction of the probe DNA and nanotubes improves the specificity for the target. Third, and most important, this approach can be applied to other types of molecular probes by simply changing the sequences of the ssDNA to a specific target. These features establish the universality and simplicity of the platform and could, therefore, provide the groundwork for the design of other nanodevices for biosensing applications. ACKNOWLEDGMENT We acknowledge financial support through NIH, NSF, and NSFC (Grants 20525518 and 20775005) grants. We also acknowledge Project 985 support and Major International (Regional) Joint Research Program of Natural Science Foundation of China (20620120107). SUPPORTING INFORMATION AVAILABLE Additional spectroscopic data, such as TEM imaging, influence of other substrates on fluorescence response, and kinetic data. This material is available free of charge via the Internet at http://pubs.acs.org. Received for review June 2, 2008. Accepted July 27, 2008. AC801118P

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