Graphene Fluorescence Resonance Energy Transfer Aptasensor for

Feb 24, 2010 - Department of Chemistry, Key Laboratory of Bioorganic Phosphorus Chemistry and Chemical Biology, Tsinghua University, Beijing 100084, P...
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Anal. Chem. 2010, 82, 2341–2346

Graphene Fluorescence Resonance Energy Transfer Aptasensor for the Thrombin Detection Haixin Chang,† Longhua Tang,† Ying Wang,† Jianhui Jiang,*,‡ and Jinghong Li*,† Department of Chemistry, Key Laboratory of Bioorganic Phosphorus Chemistry and Chemical Biology, Tsinghua University, Beijing 100084, People’s Republic of China, and State Key Laboratory for Chemo/biosensing and Chemo-metrics, College of Chemistry and Chemical Engineering, Hunan University, Changsha 410082, People’s Republic of China Combining nanomaterials and biomolecule recognition units is promising in developing novel clinic diagnostic and protein analysis techniques. In this work, a highly sensitive and specific fluorescence resonance energy transfer (FRET) aptasensor for thrombin detection is developed based on the dye labeled aptamer assembled graphene. Due to the noncovalent assembly between aptamer and graphene, fluorescence quenching of the dye takes place because of FRET. The addition of thrombin leads to the fluorescence recovery due to the formation of quadruplex-thrombin complexes which have weak affinity to graphene and keep the dyes away from graphene surface. Because of the high fluorescence quenching efficiency, unique structure, and electronic properties of graphene, the graphene aptasensor exhibits extraordinarily high sensitivity and excellent specificity in both buffer and blood serum. A detection limit as low as 31.3 pM is obtained based on the graphene FRET aptasensor, which is two orders magnitude lower than those of fluorescent sensors based on carbon nanotubes. The excellent performance of FRET aptasensor based on graphene will also be ascribed to the unique structure and electronic properties of graphene. Due to their unique optical, electrical, and catalysis properties, nanomaterials provide special opportunities for developing novel sensors with powerful functions.1-9 Graphene, a two-dimensional carbon crystal with only one atom thickness, attracts a lot attention * To whom correspondence should be addressed. E-mail: jhli@ mail.tsinghua.edu.cn (J.L.); [email protected] (J.J.). † Tsinghua University. ‡ Hunan University. (1) Chen, D.; Wang, G.; Li, J. H. J. Phys. Chem. C 2007, 111, 2351. (2) Kong, J.; Franklin, N. R.; Zhou, C.; Chapline, M. G.; Peng, S.; Cho, K.; Dai, H. Science 2000, 287, 622. (3) Cui, Y.; Wei, Q.; Park, H.; Lieber, C. M. Science 2001, 293, 1289. (4) Chang, H.; Yuan, Y.; Shi, N.; Guan, Y. Anal. Chem. 2007, 79, 5111. (5) Park, S. J.; Talon, T. A.; Mirkin, C. A. Science 2002, 295, 1503. (6) Michalet, X.; Pinaud, F. F.; Bentolila, L. A.; Tsay, J. M.; Doose, S.; Li, J. J.; Sundaresan, G.; Wu, A. M.; Gambhir, S. S.; Weiss, S. Science 2005, 307, 538. (7) Forzani, E. S.; Lu, D.; Leright, M. J.; Aguilar, A. D.; Tsow, F.; Iglesias, R. A.; Zhang, Q.; Lu, J.; Li, J. H.; Tao, N. J. Am. Chem. Soc. 2009, 131, 1390. (8) Zhang, L.; Zhang, Q.; Li, J. H. Adv. Funct. Mater. 2007, 17, 1958. (9) (a) Wu, Z.; Zhen, Z.; Jiang, J. H.; Shen, G. L.; Yu, R. Q. J. Am. Chem. Soc. 2009, 131, 12325. (b) Wang, J.; Musameh, M.; Lin, Y. H. J. Am. Chem. Soc. 2003, 125, 2408. 10.1021/ac9025384  2010 American Chemical Society Published on Web 02/24/2010

because of its unique structure and electronic properties.10 The special electrical and mechanical characteristics of graphene have been broadly studied and used in quantum electrical devices,11 nanocomposites,12 electromechanical resonators,13 and gas or pH sensors.14,15 Graphene is also an extraordinarily wonderful material for sensor applications because of its exceptionally low electrical noise, which results in ultrahigh individual molecule sensitivity and low-frequency noise.14,16 Graphene has been demonstrated to have great potentials in electrical biosensing for single bacterium detection.17 As a novel carbon nanomaterial, coupling biomolecules and graphene to develop high-performance sensors for biomolecular recognition is highly expected. A promising application of graphene in sensing technology is fluorescent detection due to its excellent capability of graphene in fluorescence resonance energy transfer (FRET).18 Graphene is a good energy acceptor in energy transfer due to its peculiar electronic properties. Photophysical calculations confirm the energy transfer from dyes to graphene, and graphene can be an excellent quencher of electronic excited states of dyes.18 Therefore, it is possible to realize sensitive fluorescent sensing using graphene as fluorescence quencher. However, there is still less reported study on graphene based FRET sensors. Aptamers are selected single-stranded oligonucleotides which are specific to proteins, small molecules, or ions and can be convenient sensing elements.19 Aptasensors have been broadly used in detection of cancer cells, drugs (cocaine, etc.), and a (10) (a) Novoselov, K. S.; Geim, A. K.; Morozov, S. V.; Jiang, D.; Zhang, Y.; Dubonos, S. V.; Grigorieva, I. V.; Firsov, A. A. Science 2004, 306, 666. (b) Zhang, Y.; Tan, Y. W.; Stormer, H. L.; Kim, P. Nature 2005, 438, 201. (11) (a) Xia, J.; Chen, F.; Li, J. H.; Tao, N. Nat. Nanotechnol. 2009, 4, 505. (b) Chen, F.; Qing, Q.; Xia, J.; Li, J. H.; Tao, N. J. Am. Chem. Soc. 2009, 131, 9908. (12) Stankovich, S.; Dikin, D. A.; Dommett, G. H. B.; Kohlhaas, K. M.; Zimney, E. J.; Stach, E. A.; Piner, R. D.; Nguyen, S. T.; Ruoff, R. S. Nature 2006, 442, 282. (13) Bunch, J. S.; van der Zande, A. M.; Verbridge, S. S.; Frank, I. W.; Tanenbaum, D. M.; Parpia, J. M.; Craighead, H. G.; McEuen, P. L. Science 2007, 315, 490. (14) Robinson, J. T.; Perkins, F. K.; Snow, E. S.; Wei, Z.; Sheehan, P. E. Nano Lett. 2008, 8, 3137. (15) Ang, P. K.; Chen, W.; Wee, A. T. S.; Loh, K. P. J. Am. Chem. Soc. 2008, 130, 14392. (16) Schedin, F.; Geim, A. K.; Morozov, S. V.; Hill, E. W.; Blake, P.; Katsnelson, M. I.; Novoselov, K. S. Nat. Mater. 2007, 6, 652. (17) Mohanty, N.; Berry, V. Nano Lett. 2008, 8, 4469. (18) (a) Swathi, R. S.; Sebastiana, K. L. J. Chem. Phys. 2008, 129, 054703. (b) Swathi, R. S.; Sebastiana, K. L. J. Chem. Phys. 2009, 130, 086101.

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Scheme 1. Schematic Demonstration of Graphene FRET Aptasensor and the Detection Mechanism for Thrombina

a Fluorescence of dye labeled aptamer is quenched when aptamer binds to graphene due to FRET between dyes and graphene. The fluorescence recovers while thrombin combines with aptamers to form quadruplex-thrombin complexes which have much less affinity to graphene, causing FAM far away from the graphene surface.

variety of proteins.20 Thrombin has great importance in molecular biology, such as in regulation of tumor growth, metastasis, and angiogenesis,21 and a lot of efforts have been done to develop sensitive sensors to detect thrombin. Aptamers based thrombin detection is one of mostly used strategies because of the excellent specificity of aptamers toward thrombin.22,23 Here, we developed a highly sensitive and selective aptasensor for thrombin detection via monitoring fluorescence quenching of dye labeled aptamers by graphene and subsequent fluorescence recovery induced by thrombin. The biosensing platform is constructed according to the noncovalent assembly of dye labeled aptamer on graphene which is induced by π-π stacking of DNA bases on graphene. The efficient construction of fluorescent aptasensors requires controllable proximity of dyes to the graphene surface and obvious fluorescence changes induced by targets. As shown in Scheme 1, the binding of aptamer to graphene guarantees the close proximity of dyes to graphene and the following FRET from dyes to graphene results in high efficiency quenching of fluorescence of the dyes. More importantly, the conformation of aptamer on graphene can be changed by quadruplex formation induced by thrombin. The weak binding between quadruplex-thrombin complexes and graphene surface makes the dye (fluorescein amidite, FAM) far away from the graphene surface, inducing the fluorescence recovery. Graphene FRET aptasensor can detect 31.3 pM thrombin with good selectivity. The sensitivity of the graphene FRET aptasensor is 2 orders of magnitude higher than those (19) (a) Zhang, Y.; Huang, Y.; Jiang, J. H.; Shen, G.; Yu, R. J. Am. Chem. Soc. 2007, 129, 15448. (b) Hermann, T.; Patel, D. J. Science 2000, 287, 820. (c) Rahman, M. A.; Son, J. I.; Won, M. S.; Shim, Y. B. Anal. Chem. 2009, 81, 6604. (20) (a) Huang, Y. F.; Chang, H. T.; Tan, W. Anal. Chem. 2008, 80, 567. (b) Willner, I.; Zayats, M. Angew. Chem., Int. Ed. 2007, 46, 6408. (c) Baker, B. R.; Lai, R. Y.; Wood, M. S.; Doctor, E. H.; Heeger, A. J.; Plaxco, K. W. J. Am. Chem. Soc. 2006, 128, 3138. (21) Nierodzik, M. L.; Karpatkin, S. Cancer Cell 2006, 10, 355. (22) (a) Xiao, Y.; Piorek, B. D.; Plaxco, K. W.; Heeger, A. J. J. Am. Chem. Soc. 2005, 127, 17990. (b) Pavlov, V.; Xiao, Y.; Shlyahovsky, B.; Willner, I. J. Am. Chem. Soc. 2004, 126, 11768. (c) Higuchi, A.; Siao, Y. D.; Yang, S. T.; Hsieh, P. V.; Fukushima, H.; Chang, Y.; Ruaan, R. C.; Chen, W. Y. Anal. Chem. 2008, 80, 6580. (23) (a) Zhao, Q.; Lu, X.; Yuan, C. G.; Li, X. F.; Le, X. C. Anal. Chem. 2009, 81, 7484. (b) Zhao, Q.; Li, X. F.; Shao, Y.; Le, X. C. Anal. Chem. 2008, 80, 7586. (c) Wilcox, J. M.; Rempel, D. L.; Gross, M. L. Anal. Chem. 2008, 80, 2365.

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similar carbon nanotube (CNT) based fluorescent sensors,24 showing excellent promising in biomolecule recognition and protein detection. EXPERIMENTAL SECTION Materials and Chemicals. Graphite powders (325 mesh) with purity of 99.95% were obtained from Alfa Aesar. Sodium dodecylbenzene sulfonate (SDBS) was from Guoyao Huashi (SCRC, China). Other regents were purchased from Beijing Chemical Company (China). All chemicals are of analytical grade except those noted otherwise. Deionized water was used for all solutions preparation. Thrombin aptamer was synthesized by Shanghai Biotech (China) and labeled at 5′ end with FAM dye. DNA sequence of thrombin aptamer was 5′-FAM-GGTTGGTGTGGTTGG-3′. Thrombin was bought from Sigma (U.S.A.). Bovine serum albumin (BSA) and human IgG antibody were from Dingguo Biotech (Beijing, China). Preparation of Surfactant Dispersed Graphene. Graphene was prepared from reduction of exfoliated graphite oxide. Graphite oxide was obtained through natural graphite oxidation based on Hummer’s method.25 Generally, the preoxidized graphite powders were put into concentrated H2SO4 at 0 °C with gradual addition of KMnO4 under stirring. The mixture was kept at 35 °C for several hours and diluted gradually in an ice bath with deionized water. The mixture was rediluted, followed by addition of H2O2. The mixture was filtered when the color changed to brilliant yellow and washed with HCl aqueous solution and deionized water. The obtained graphite oxide powder was dialyzed in graphite oxide dispersion. The graphite oxide was exfoliated under sonication for about 2 h to ensure most graphite oxide was exfoliated to single layer graphene oxide. The obtained homogeneous yellow solution of single layer graphene oxide was reduced by hydrazine to remove the oxygenous groups. Black precipitates were washed and dried as graphene, which were usually not soluble in water. To prepare SDBS dispersed graphene (SDBS-graphene) aqueous solution, SDBS was added to graphene and sonicated for about 2 h. Up to 1 mg/mL stable SDBS-graphene aqueous solution can be harvested in this way. The ratio of SDBS-graphene (wt.) was optimized in the range from 1 to 15. The excellent biocompatibility of SDBS provided a variety of possibilities for SDBS-graphene in biosensing applications in an aqueous environment. Characterizations. Powder X-ray diffraction (XRD) spectra were collected using Bruker D8-Advance X-ray powder diffractometer (Cu KR radiation; λ, 1.5418 Å). The high resolution transmission emission microscope (HRTEM) images of graphene were measured by JEM-2010 (Jeol) with the acceleration voltage of 120 kV. Tapping mode atomic force microscopy (AFM) characterizations were conducted on a Nanoscope III (Digital Instrument) scanning probe microscope. The AFM samples were prepared by casting 5-10 µL of graphene oxide aqueous solution on SiO2 glass. (24) Yang, R.; Tang, Z.; Yan, J.; Kang, H.; Kim, Y.; Zhu, Z.; Tan, W. Anal. Chem. 2008, 80, 7408. (25) Tang, L.; Wang, Y.; Li, Y.; Feng, H.; Lu, J.; Li, J. H. Adv. Funct. Mater. 2009, 19, 2782.

Figure 2. Fluorescence spectra of FAM-aptamer in the presence of various concentration of SDBS-graphene in 20 mM PBS buffer (pH 7.0). Inset: fluorescence intensity versus concentration of SDBS-graphene. FAM-aptamer concentration: 20 nM. Excitation wavelength: 470 nm.

Figure 1. (A) AFM image and depth profiles of as prepared single layer graphene oxide. Size: 15.3 × 15.3 µm. (B) High resolution TEM image of graphene from reduced graphene oxide. Inset: photograph of 1 mg/mL SDBS-graphene aqueous solution.

Fluorescence measurements were done with F-7000 Hitachi spectrometer. In a typical measurement, 0.1 mg/mL SDBSgraphene was added to 20 nM FAM-aptamer in 10 mM PBS buffer (pH 7.0) with 100 mM NaCl, 4 mM Mg2+. For detection of thrombin, a different concentration of thrombin was added to the above solution and incubated for 10-20 min before measurement. The time dependent experiments were conducted by monitoring fluorescence at different incubation times. In specificity studies, the graphene aptasensor was incubated with 1 µM BSA, 1 µM IgG, and 250 pM thrombin in PBS for the same incubation time, respectively. Goat and bovine serum (Dingguo biotechnology Co., Beijing) were used to study the performance of the graphene aptasensor in blood serum samples with 250 pM thrombin. The fluorescence was monitored at 517 nm with the excitation of 470 nm at room temperature. RESULTS AND DISCUSSIONS Characterization of Single Layer Graphene Oxide and Graphene. To confirm the single layer formation through exfoliation, graphene oxide was characterized by AFM on a SiO2 glass substrate. As shown in Figure 1A, the thickness of most graphene oxide is about 1.2 ± 0.2 nm. Considering the overestimation and the oxygen containing group, such thickness is reasonable for single layer graphene oxide.26 Interestingly, the wrinkles on single layer graphene oxide are clearly seen in the white area.

Depth profiles of lines 1 and 2 in Figure 1A show the height of wrinkles is about 2-10 nm, which is similar to those observed by other groups.17 Figure 1B shows the HRTEM image of graphene obtained from reduction of single layer graphene oxide. XRD data in Figure S1 in the Supporting Information shows that the (002) peak position increases after reduction, indicating the decreases of interplanar spacing after chemical reduction and the formation of graphene.25 The optical image in the inset of Figure 1B shows that the SDBS-graphene are well-dispersed in aqueous solution at a concentration of 1 mg/mL and are highly stable, which is used in the following fluorescence studies. FRET between Dye-Labeled Aptamer and Graphene. The fluorescence of FAM labeled aptamer can be efficiently quenched by SDBS-graphene due to FRET between FAM and graphene.18 The fluorescence quenching of the FAM aptamer based on the FRET between the FAM-aptamer and graphene was evaluated by fluorescence spectra. Figure 2 shows the fluorescence quenching of FAM-aptamer at various concentrations of graphene. The fluorescence intensity decreased rapidly when the graphene was added into the aptamer solution of 20 nM. Moreover, the fluorescence intensity decreased with increased concentration of graphene. Over 80% fluorescence was quenched by the addition of graphene of 0.1 mg/mL, and the fluorescence quenching efficiency by SDBS-graphene had good reproducibility. In three parallel experiments, the remained fluorescence of FAM-aptamer probes in the presence of 0.1 mg/mL SDBS-graphene were 13.1%, 14.6% and 18.8% with only ca. 3% standard deviation, indicating the good reproducibility of graphene quenching efficiency. The high efficiency quenching is considered as the direct consequence of noncovalent binding of aptamer on the graphene surface and the energy transfer from dyes to graphene. The π-π stacking of DNA bases with graphene guarantees the close proximity of FAM with graphene and efficient fluorescence quenching of dyes. The noncovalent binding of single stranded DNA (ssDNA) to aromatic nanocarbon surface has also been reported for ssDNA/CNTs assembly.27 Such strong binding between aptamer and graphene will cause the labeled fluorophores (26) Gmez-Navarro, C.; Weitz, R. T.; Bittner, A. M.; Scolari, M.; Mews, A.; Burghard, M.; Kern, K. Nano Lett. 2007, 7, 3499.

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Figure 3. Fluorescence spectra of FAM-aptamer (a), and FAMaptamer in the presence of thrombin (b), thrombin and SDBSgraphene in sequence (c), SDBS-graphene (d), SDBSgraphene and thrombin in sequence (e). FAM-aptamer concentration: 20 nM. Thrombin concentration: 250 pM. Excitation wavelength: 470 nm.

approach to graphene, and high efficiency energy transfer between the dyes and graphene will occur. Note that, unlike usual FRET sensors using two different fluorescent dyes, here only one dye (FAM) was applied in the graphene FRET aptasensor. Because graphene is a very good quencher of a large quantity of dyes, it is easier to construct a single and multiplex sensing FRET system by the use of graphene as the quencher. Fluorescence Recovery of Graphene FRET Aptasensor Induced by Thrombin. When adding thrombin to the graphene/ aptamer complex solution, the fluorescence recovered rapidly as shown in Figure 3. To confirm the fluorescence recovery was caused by thrombin, several control experiments were carried out. It is shown that the fluorescence intensity of FAM-aptamer nearly had no change by the addition of thrombin and SDBS, respectively (curve b in Figure 3 and Figure S2 in the Supporting Information). The fluorescence quenching efficiency was also marginal when aptamer forming quadruplex complex with thrombin before the addition of graphene (curve c in Figure 3). Such parallel experiments indicated that the fluorescence recovery of the graphene and FA-aptamer complex solution in the presence of thrombin was due to the formation of quadruplex-thrombin complex. It is reasonable that aptamers on graphene will combine with thrombin to form quadruplex because of the high affinity between aptamers and thrombin. It should be noted that the intensity of recovered fluorescence by thrombin was only about 50% to those without graphene (Figure 3e), which may be ascribed to graphene preventing the folding of aptamer to form quadruplex-thrombin complexes completely on the graphene surface. Fluorescence Quenching of Quadruplex-Thrombin Complexes by Graphene. To further understand the processes of thrombin induced fluorescence recovery, we conducted time dependent fluorescence quenching experiments for aptamer and quadruplex-thrombin complexes in the presence of SDBS(27) (a) Zheng, M.; Jagota, A.; Strano, M. S.; 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. (b) Zheng, M.; Jagota, A.; Semke, E. D.; Diner, B. A.; Mclean, R. S.; Lustig, S. R.; Richardson, R. E.; Tassi, N. G. Nat. Mater. 2003, 2, 338.

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Figure 4. Relative fluorescence of FAM-aptamer (4) and FAMaptamer quadruplex-thrombin complexes (3) via time with addition of SDBS-graphene. Relative fluorescence is calculated from the fluorescence intensity ratio of F/F0, where F0 and F correspond to the fluorescence intensity before and after addition of SDBS-graphene, respectively. FAM-aptamer concentration: 20 nM. Thrombin concentration: 40 nM. Excitation wavelength: 470 nm. Emission wavelength monitored: 517 nm.

graphene, respectively. As shown in Figure 4, the fluorescence quenching of aptamer by graphene is a very quick process. Fluorescence of FAM-aptamer was quenched by over 80% in less than 10 min, indicating the high strong binding affinity of aptamer to graphene. The largest fluorescence quenching was obtained in 25 min, and no significant fluorescence recovery was observed. However, for quadruplex-thrombin complexes, the fluorescence quenching process was slow when SDBS-graphene was added to quadruplex-thrombin complexes (Figure 4). Only less than 20% fluorescence quenching was observed in 60 min. Moreover, in comparison to aptamers, there was an increased fluorescence recovery for quadruplex-thrombin complexes in 60 min. These time dependent experiments clearly indicate that quadruplexthrombin complexes can prevent FAM-aptamer from being quenched by SDBS-graphene, and thrombin has higher binding affinity to aptamer than graphene. The phenomena can be explained by the structure difference between single stranded aptamer and quadruplex DNA. Therefore, once aptamers on the graphene surface interact with thrombin, they will transform to the quadruplex-thrombin complexes. The quadruplex-thrombin complexes have much lower binding ability to graphene and ease of disassociation from the graphene surface, as observed in the time dependent experiment. As a consequence, fluorophores are far away from the graphene surface and the energy transfer efficiency decreases (sometimes there is even no energy transfer at all), causing the fluorescence recovery. The fluorescence recovering is proved to be very sensitive to the existence of thrombin and can be designed as fluorescent sensing platforms for biomolecular recognition. Thrombin Detection with Graphene FRET Aptasensor. We developed one fluorescent biosensing platform for thrombin detection using the graphene FRET aptasensor. Figure 5A shows the fluorescence recovery of graphene FRET aptasensor in the presence of different concentrations of thrombin. The fluorescence

Figure 6. Relative fluorescence intensity of the graphene aptasensor incubated in blank PBS buffer; 1 µM BSA in PBS; 1 µM IgG in PBS; 250 pM thrombin in PBS, goat serum, and bovine serum, respectively. Relative fluorescence intensity (F/F0) is calculated by F/F0, where F0 and F are the fluorescence intensity without and with the presence of BSA, IgG, and thrombin, respectively. FAM-aptamer concentration: 20 nM. Excitation wavelength: 470 nm. Emission wavelength monitored: 517 nm.

Figure 5. (A) Fluorescence spectra of the graphene aptasensor via different concentrations of thrombin (0, 31.3, 62.5, 93.8, 125, 156.3, 187.5, 218.8, and 250 pM) in 20 mM PBS buffer (pH 7.0). (B) Relative fluorescence changes of FAM-aptamer and the graphene aptasensor via thrombin concentrations. Relative fluorescence changes are calculated by F/F0, where F0 and F are the fluorescence intensity without and with thrombin, respectively. FAM-aptamer concentration: 20 nM. Excitation wavelength: 470 nm.

intensity of the graphene aptasensor increased with the increasing concentration of thrombin. The plot of fluorescence intensity with thrombin concentration from 0 to 250 pM is shown in Figure 5B. The linear range is from 62.5-187.5 pM with linear equation y ) 1.053 x + 42, where y is fluorescence intensity and x is the concentration of thrombin (regression coefficient R ) 0.993). Detection limit was 31.3 pM or 31.3 fmol thrombin in a total 1 mL volume, which is of 2 orders of magnitude lower than those reported CNT fluorescent biosensors.24 Further, control studies showed no significant changes of fluorescence intensity when adding the same volume of thrombin to the aptamer without graphene (Figure 5B), coinciding with the above observation that the fluorescence recovery was induced by thrombin interacting with graphene FRET aptasensor. The recovered relative fluorescence has good reproducibility with standard deviation usually less than 5%. Several possible reasons may explain this excellent performance of graphene FRET aptasensor. First, the quenching and recovery of fluorescence are highly dependent on the energy transfer between dyes and graphene, which is critical for graphene FRET aptasensor. It is recently predicted through theoretical calculation that graphene is a superquencher and possessed long-term nano-

scale energy transfer properties which is quite different from CNTs.18,28 Of note, energy transfer between dyes and graphene is more like that between dyes and gold nanoparticles rather than that of CNTs.18b The high fluorescence quenching efficiency leads to the low background of signals and results in the high sensitivity. Second, random binding of aptamer on a two-dimensional graphene surface may be relatively weaker than helical interacting of aptamer with CNTs. Thus, the folding of aptamer will be more favorable on a two-dimensional sheet surface than that on a one-dimensional nanotube surface. The direct consequence of weak binding and folding is that the aptamers on the graphene surface are more sensitive to thrombin in fluorescence responses. Specificity of Graphene FRET Aptasensor. In our experiments, BSA and human IgG antibody were selected to study the specificity of graphene FRET aptasensor. In a typical experiment, the graphene aptasensor was incubated with 1 µM BSA, 1 µM IgG, and 250 pM thrombin in PBS, respectively. As shown in Figure 6, fluorescence of the graphene aptasensor changed less for BSA and IgG, while a significant fluorescence increase was observed for thrombin. The relative fluorescence F/F0 of the biosensor in the presence of thrombin is 3.25, which is much higher than that of 0.98 for BSA and 1.03 for IgG, where F0 and F are the fluorescence intensity of the graphene FAMaptamer solution without and with BSA, IgG, and thrombin, respectively. The good selectivity of graphene FRET aptasensor is attributed to the high specificity of aptamer.19,22 Therefore, the graphene FRET aptasensors can be applied in highly sensitive detection of thrombin with high specificity. We also investigated the specificity of the graphene FRET aptasensor for the detection of thrombin in blood serum. As shown in Figure 6, the relative fluorescence F/F0 of 250 pM thrombin in goat serum and bovine serum are 2.47 and 2.26, respectively. In other words, this graphene FRET sensor can detect at least picomolar thrombin in (28) (a) Nakayama-Ratchford, N.; Bangsaruntip, S.; Sun, X.; Welsher, K.; Dai, H. J. Am. Chem. Soc. 2007, 129, 2448. (b) Zhu, Z.; Tang, Z.; Phillips, J. A.; Yang, R.; Wang, H.; Tan, W. J. Am. Chem. Soc. 2008, 130, 10856.

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blood serum. Moreover, the good biocompatibility of graphene also shows practical analytical application in real commercial samples.29 CONCLUSIONS Graphene FRET aptasensor for thrombin detection has been constructed based on aptamer assembly on graphene. The aptasensor derives from fluorescence quenching of FAM-aptamer by graphene and the subsequent fluorescence recovery induced by the formation of quadruplex-thrombin complexes. The fluorescence recovery may be explained by the relative weaker binding of quadruplex-thrombin complexes to graphene compared with that of aptamer. This graphene FRET aptasensor is extraordinarily sensitive to the thrombin detection with high specificity in both buffer and blood serum. On the basis of their excellent performance, the graphene FRET aptasensor will be promising in cancer cell recognition or cocaine sensors by (29) Wang, Y.; Lu, J.; Tang, L.; Chang, H.; Li, J. H. Anal. Chem. 2009, 81, 9710.

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choosing suitable aptamers,19,22 which may also provide a platform for the optical or electrical applications of graphene. ACKNOWLEDGMENT This work was financially supported by the National Natural Science Foundation of China (No. 20675044 and No. 20975060) and National Basic Research Program of China (No. 2007CB310500 and No. 28-AZC0901). SUPPORTING INFORMATION AVAILABLE Figure S1 (XRD pattern of graphite oxide and graphene) and Figure S2 (SDBS influence on fluorescence of FAM labeled aptamer) as noted in text. This material is available free of charge via the Internet at http://pubs.acs.org.

Received for review November 6, 2009. Accepted February 12, 2010. AC9025384