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Fluorescence Resonance Energy Transfer between Quantum Dots and Graphene Oxide for Sensing Biomolecules Haifeng Dong,† Wenchao Gao,† Feng Yan,*,‡ Hanxu Ji,† and Huangxian Ju*,† Key Laboratory of Analytical Chemistry for Life Science (Ministry of Education of China), Department of Chemistry, Nanjing University, Nanjing 210093, Jiangsu Institute of Cancer Prevention and Cure, Nanjing 210009, P.R. China This work designed a novel platform for effective sensing of biomolecules by fluorescence resonance energy transfer (FRET) from quantum dots (QDs) to graphene oxide (GO). The QDs were first modified with a molecular beacon (MB) as a probe to recognize the target analyte. The strong interaction between MB and GO led to the fluorescent quenching of QDs. Upon the recognition of the target, the distance between the QDs and GO increased, and the interaction between target-bound MB and GO became weaker, which significantly hindered the FRET and, thus, increased the fluorescence of QDs. The change in fluorescent intensity produced a novel method for detection of the target. The GO-quenching approach could be used for detection of DNA sequences, with advantages such as less labor for synthesis of the MB-based fluorescent probe, high quenching efficiency and sensitivity, and good specificity. By substituting the MB with aptamer, this strategy could be conveniently extended for detection of other biomolecules, which had been demonstrated by the interaction between aptamer and protein. To the best of our knowledge, this is the first application of the FRET between QDs and GO and opens new opportunities for sensitive detection of biorecognition events. The integration of unique structural characters of inorganic nanomaterials such as nanocrystals, nanotubes, and nanowires with highly specific recognition ability of biomolecules has created many new-style tools for bioanalysis. Some carbon nanostructures, such as carbon tubes,1 carbon nanodots,2 and carbon nanofibers,3 have been used extensively for development of biosensors.4 In recent years, graphene has attracted considerable attention in different fields due to its unique and interesting electronic, * Corresponding author. Phone/Fax: +86-25-83593593. E-mail: (H.J.)hxju@ nju.edu.cn, (F.Y.)
[email protected]. † Nanjing University. ‡ Jiangsu Institute of Cancer Prevention and Cure. (1) Cheng, W.; Ding, L.; Lei, J. P; Ding, S. J.; Ju, H. X. Anal. Chem. 2008, 80, 3867–3872. (2) Fu, C. C.; Lee, H. Y.; Chen, K.; Lim, T. S.; Wu, H. Y.; Lin, P. K.; Wei, P. K.; Tsao, P. H.; Chang, H. C.; Fann, W. Proc. Natl. Acad. Sci. U.S.A. 2007, 104, 727–732. (3) Hao, C.; Ding, L.; Zhang, X. J.; Ju, H. X. Anal. Chem. 2007, 79, 4442– 4447. (4) Yang, R. H.; Jin, J. Y.; Chen, Y.; Shao, N.; Kang, H. Z.; Xiao, Z. Y.; Tang, Z. W.; Wu, Y. R.; Zhu, Z.; Tan, W. H. J. Am. Chem. Soc. 2008, 130, 8351– 8358. 10.1021/ac100852z 2010 American Chemical Society Published on Web 06/04/2010
thermal, and mechanical properties.5-9 It can act as a quencher to quench the fluorescence of organic dye, which is labeled to ssDNA, by strong interaction between ssDNA and graphene oxide (GO).10-12 The quenching effect has produced a biosensing platform for the detection of DNA based on the conformation alteration of dye-labeled DNA for its release from the GO upon the recognition binding with the target molecule.10 Compared with conventional organic dyes, the photoluminescence (PL) of quantum dots (QDs) possesses a variety of advantages, such as high quantum yield; narrow, symmetric and stable fluorescence; and size-dependent and tunable absorption and emission.13 Thus, QDs have been used as excellent fluorescent labels for biological imaging, sensing, and diagnostics.14-16 The broad absorption and narrow emission spectra of the QDs have also made them excellent donors of fluorescence resonance energy transfer (FRET). Based on FRET, a series of QDs-based biosensors have been developed.17 By integrating CdTe QDs with different biomolecules, such as molecular beacon (MB) and aptamer, FRET could be conveniently used for design of novel sensing strategy. (5) Novoselov, K. S.; Geim, A. K.; Morozov, S. V.; Jiang, D.; Katsnelson, M. I.; Grigorieva, I. V.; Dubonos, S. V.; Firsov, A. A. Nature 2005, 438, 197– 200. (6) Novoselov, K. S.; Jiang, Z.; Zhang, Y.; Morozov, S. V.; Stormer, H. L.; Zeitler, U.; Maan, J. C.; Boebinger, G. S.; Kim, P.; Geim, A. K. Science 2007, 315, 1379. (7) Li, X. L.; Wang, X. R.; Zhang, L.; Lee, S. W.; Dai, H. J. Science 2008, 319, 1229–1232. (8) 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, 282–286. (9) Dikin, D. A.; Stankovich, S.; Zimney, E. J.; Piner, R. D.; Dommett, G. H. B.; Evmenenko, G.; Nguyen, S. T.; Ruoff, R. S. Nature 2007, 448, 457–460. (10) Lu, C. H.; Yang, H. H.; Zhu, C. L.; Chen, X.; Chen, G. N. Angew. Chem., Int. Ed. 2009, 48, 4785–4787. (11) Varghese, N.; Mogera, U.; Govindaraj, A.; Das, A.; Maiti, P. K.; Sood, A. K.; Rao, C. N. R. ChemPhysChem 2009, 10, 206–210. (12) Mohanty, N.; Berry, V. Nano Lett. 2008, 8, 4469–4476. (13) Wu, X.; Liu, H.; Liu, J.; Haley, K. N.; Treadway, J. A.; Larson, J. P.; Ge, N.; Peale, F.; Bruchez, M. P. Nat. Biotechnol. 2003, 21, 41–46. (14) Howarth, M.; Liu, W.; Puthenveetil, S.; Zheng, Y.; Marshall, L. F.; Schmidt, M. M.; Wittrup, K. D.; Bawendi, M. G.; Ting, A. Y. Nat. Methods 2008, 5, 397–399. (15) Somers, R. C.; Bawendi, M. G.; Nocera, D. G. Chem. Soc. Rev. 2007, 36, 579–591. (16) 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, 538–44. (17) Medintz, I. L.; Clapp, A. R.; Mattoussi, H.; Goldman, E. R.; Fisher, B.; Mauro, J. M. Nat. Mater. 2003, 2, 630–638.
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MB is a single-stranded oligonucleotide hybridization probe with a stem-and-loop structure in which the loop contains a probe sequence that is complementary to a target sequence and the annealing of self-complementary 5′ and 3′ ends forms a stem.18 In general, the two ends are labeled with a fluorophore and a quencher, and the binding of the probe to a complementary target causes disruption of the stem and thereby restores the fluorescence of the fluorophore.4 This probe has been extensively applied in real-time quantitative PCR,19,20 the study of enzymatic processes,21,22 and messenger-RNA monitoring in living cells.23,24 However, MB often suffers from some significant flaws.4,25 For instance, the variable residual fluorescence greatly limits the detection sensitivity, and the endogeneous nuclease degradation and nonspecific binding result in false-positive signal.4 An important approach to improve the performance of MB is the development of a new efficient signal-transduction strategy and novel fluorophores and quenchers.26-30 In addition to conventional organic dyes, QDs have been used as the fluorophores of MB for sequence-specific DNA detection by coupling with FRET from QDs and a gold nanostructure as the quencher.31-33 This work shows for the first time efficient FRET from QDs to GO. By the surface modification of QDs with MB, the FRET between QDs and GO and the strong interaction between the ssDNA of MB loop structure and GO were combined to develop a novel sensitive and selective platform for fluorescence-quenching detection of DNA. As shown in Scheme 1, upon the recognition of the MB to the target, the increasing QDs-GO distance and the weakened DNA-GO interaction significantly hindered the FRET and, thus, increased the fluorescence of QDs. The increase depended on the concentration and sequence of target analyte, leading to the sensitive detection of DNA concentration and sequence. This novel detection platform needed only QDs as a fluorophore to label MB, which reduced the labor for the labeling of quencher to another end of the MB and maintained the high DNA-binding specificity. The high quenching efficiency of GO to (18) Tyagi, S.; Kramer, F. R. Nat. Biotechnol. 1996, 14, 303–308. (19) Sokol, D. L.; Zhang, X.; Lu, P.; Gewirtz, A. M. Proc. Natl. Acad. Sci. U.S.A. 1998, 95, 11538–11543. (20) Li, X. M.; Huang, Y.; Guan, Y.; Zhao, M. P.; Li, Y. Z. Anal. Chem. 2006, 78, 7886–7890. (21) Li, J. J.; Geyer, R.; Tan, W. H. Nucleic Acids Res. 2000, 28, e52. (22) Tang, Z. W.; Wang, K. M.; Tan, W. H.; Ma, C. B.; Li, J.; Liu, L. F.; Guo, Q. P.; Meng, X. X. Nucleic Acids Res. 2005, 33, e97. (23) Medley, C. D.; Drake, T. J.; Tomasini, J. M.; Rogers, R. J.; Tan, W. H. Anal. Chem. 2005, 77, 4713–4718. (24) Wu, Y. R.; Yang, C. Y. J.; Moroz, L. L.; Tan, W. H. Anal. Chem. 2008, 80, 3025–3028. (25) Wang, K. M.; Tang, Z. W.; Yang, C. Y. J.; Kim, Y. M.; Fang, X. H.; Li, W.; Wu, Y. R.; Medley, C. D.; Cao, Z. H.; Li, J.; Colon, P.; Lin, H.; Tan, W. H. Angew. Chem., Int. Ed. 2009, 48, 856–870. (26) Dubertret, B.; Calame, M.; Libchaber, A. J. Nat. Biotechnol. 2001, 19, 365– 370. (27) Yang, C. Y. J.; Lin, H.; Tan, W. H. J. Am. Chem. Soc. 2005, 127, 12772– 12773. (28) Du, H.; Strohsahl, C. M.; Camera, J.; Miller, B. L.; Krauss, T. D. J. Am. Chem. Soc. 2005, 127, 7932–7940. (29) Yang, C. Y. J.; Pinto, M.; Schanze, K.; Tan, W. H. Angew. Chem., Int. Ed. 2005, 44, 2572–2576. (30) Du, H.; Disney, M. D.; Miller, B. L.; Krauss, T. D. J. Am. Chem. Soc. 2003, 125, 4012–4013. (31) Cady, N. C.; Strickland, A. D.; Batt, C. A. Mol. Cell. Probe 2007, 21, 116– 124. (32) Dyadyusha, L.; Yin, H.; Jaiswal, S.; Brown, T.; Baumberg, J. J.; Booy, F. P.; Melvin, T. Chem. Commun. 2005, 3201–3203. (33) Oh, E.; Hong, M. Y.; Lee, D.; Nam, S. H.; Yoon, H. C.; Kim, H. S. J. Am. Chem. Soc. 2005, 127, 3270–3271.
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Scheme 1. Schematic Representation of GO-Induced Fluorescence Quenching of MB-QDs and Biosensing Mechanism
the fluorescence of QDs led to high sensitivity for FRET-based biosensing, which was further demonstrated by combining with the specific interaction between protein and its aptamer. The results show the extended application of the detection platform and opened new opportunities for sensitive detection of biorecognition events.
EXPERIMENTAL SECTION Materials and Reagents. Graphite power and sodium borohydride of analytical grade were from Sinopharm Chemical Reagent Co. Ltd. (China). Thrombin and 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC) were purchased from Sigma-Aldrich, Inc. (U.S.A.). Mercaptoacetic acid (MPA) and cadmium chloride hemipentahydrate (CdCl2 · 2.5H2O) were from Alfa Aesar China Ltd. Ultrapure water obtained from a Millipore water purification system (g18 MΩ, Milli-Q, Millipore, Billerica, MA) was used in all runs. All other reagents were of analytical grade. The oligonucleotides were purchased from Sangon Biological Engineering Technology & Co. Ltd. (Shanghai, China) and purified using high-performance liquid chromatography. Their sequences were cyclin MB: 5′-NH2(A)10-TGGAGTTGTCGGTGTAGACTCCA3′; complementary strand: 5′-TGGAGCTACACCGACAACTCCA3′; single-base mismatch stand: 5′-TGGAGCTACAGCGACAACTCCA-3′; three-base mismatch stand: 5′-TGGA GCTAGAGCCACAACTCCA-3′; thrombin aptamer: 5′-TCTCTCAGTCCGTGGTAG GGCAGGTTGGGGTGACT-3′. Buffers. DNA hybridization buffer (HB) was phosphatebuffered saline (137 mM NaCl, 2.5 mM Mg2+, 10 mM Na2HPO4, and 2.0 mM KH2PO4, pH 7.4). DNA was stored in Tris-HCl (10 mM, pH 8.0) containing 1 mM ethylenediaminetetraacetic acid. Tris-borate (TB) (90 mM, pH 7.4) was used for modification of QDs with MB or aptamer. Synthesis of Graphene Oxide. GO was synthesized from graphitic power according to Hummer’s method with some modi-
fication.34,35 Briefly, graphite powder (3 g) was put into an 80 °C solution consisting of concentrated H2SO4 (12 mL), K2S2O8 (2.5 g), and P2O5 (2.5 g) and reacted for 4.5 h. The mixture was then diluted with 0.5 L water and left at 80 °C for 12 h. After residual acid was removed by filtrating and washing with water, the product was dried under ambient conditions overnight and added to concentrated H2SO4 (120 mL). Successively, KMnO4 (15 g) was added gradually under stirring, while keeping the temperature less than 20 °C. Next, this mixture was stirred at 40 °C for 30 min and 90 °C for 90 min. Afterward, the mixture was diluted with water (250 mL) and kept at 105 °C for 25 min. After the resulting mixture was stirred for 2 h, 0.7 L of water and 20 mL of 30% H2O2 were added to end the reaction. For purification, the mixture was filtered and washed with 1:10 HCl aqueous solution and water many times. Finally, the product was further purified by dialysis for 1 week to remove the remaining metal species. By sonicating dispersion under ambient conditions for 20 min, the homogeneous GO suspension (0.01 mg mL-1) was obtained, which was stable for several months. Preparation of MB-QDs. The MPA-capped CdTe QDs were prepared according to our previous report36 with a reflux time of 8 h at 80 °C. MB-QDs were prepared by mixing the QDs with 6 equiv of cyclin MB in TB buffer containing EDC (104-105-fold more than the QDs). The mixture was allowed to react for 6-8 h at room temperature. Successively, the unreacted MB and excess EDC were removed from the MB-QDs by filtration in Millipore Microcon 50 000 molecular weight cutoff spin filters at 5000g for 5 min. The product was twice spun and washed with TB buffer. The final product was redissolved in HB and stored at 4 °C. It could be diluted to appropriated concentration before detection. The aptamer-QDs were prepared with the same procedure by substituting 6 equiv of MB with 6 equiv of aptamer. Apparatus. The morphologies of GO and MB-QDs adsorbed GO were examined with an Agilent 5500 atomic force microscopy (AFM, U.S.A.) and a JEM 2100 high-resolution transmission electron microscope (HRTEM). The ultraviolet-visible (UV-vis) absorption spectrum of the QDs was recorded with a Lambda 35 UV/vis spectrometer (Perkin-Elmer Instruments, USA). The PL spectra were recorded at room temperature with RF-5301 PC (Shimdzu), and fluorescence anisotropy was examined with a F900 fluorescence spectrometer (Edinburgh Instruments Ltd., U.K.). The FT-IR spectrum was recorded on a Nicolet 400 Fourier transform infrared spectrometer (Madison, WI). Raman spectra were measured with Renishaw-inVia Raman microscope (Renishaw, U.K.). RESULTS AND DISCUSSION Characterization of GO. Figure 1A shows the AFM image of GO in tapping mode to simultaneously collect height and phase data. The sample used for AFM observation was prepared by depositing a droplet of GO dispersion (2 µL, 0.01 mg/mL) on a freshly cleaved mica surface and dried under vacuum at room (34) Hummers, W. S.; Offeman, R. E. J. Am. Chem. Soc. 1958, 80, 1339–1339. (35) Xu, Y. X.; Bai, H.; Lu, G. W.; Li, C.; Shi, G. Q. J. Am. Chem. Soc. 2008, 130, 5856–5857. (36) Ge, C. W.; Xu, M.; Liu, J.; Lei, J. P.; Ju, H. X. Chem. Commun. 2008, 4, 450–452.
Figure 1. Tapping mode AFM image of GO sheets deposited on mica substrate (A), HRTEM images of GO sheets (B), and MB-QDs adsorbed on GO (C).
Figure 2. FT-IR spectrum of as-prepared GO (A) and Raman spectra of raw graphite (a) and GO (b) (B).
temperature. From the cross-sectional view of the typical AFM image, the average thickness of GO sheet was about 2 nm, which was somewhat larger than the interlayer spacing of 0.78 nm. A similar result for the thickness of single GO sheet was also observed in a previous report.35 The cross-sectional view of the typical HRTEM image of as-prepared GO showed that the GO sheet was about 1 × 1 µm in size with occasional folds, crinkles and rolled edges (Figure 1B). The FT-IR and Raman spectra of GO further provided the information of the successful synthesis of GO. As shown in Figure 2A, the FT-IR spectrum gave the characteristic vibrations of GO, as those reported in previous work,35 including a broad and intense peak of O-H group at 3400 cm-1, a CdO peak at 1743 cm-1, an O-H deformation peak at 1411 cm-1, a C-OH stretching peak at 1250 cm-1, an C-O stretching peak at 1070 cm-1, and a peak attributed to the vibrations of unoxidized graphitic skeletal domains and the adsorbed water molecules at 1627 cm-1. Different from the Raman spectrum of raw graphite, which showed a strong peak assigned to the vibration of sp2-bonded carbon atoms at 1580 cm-1 (G band) and a very weak peak assigned to the vibration of carbon atoms with dangling bonds in plane terminations of disordered graphite at 1350 cm-1 (D band) (Figure 2B, curve a), the Raman spectrum Analytical Chemistry, Vol. 82, No. 13, July 1, 2010
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Figure 3. UV-vis absorption spectrum of as-prepared CdTe QDs (a), and PL spectra of diluted QDs (b) and MB-QDs (c). Inset: UV-vis absorption spectrum of as-prepared GO.
of GO showed the well-documented D and G bands (Figure 2B, curve b). This phenomenon agreed well with that reported previously and indicated the formation of some sp3 carbon in GO.37 Characterization of MB-QDs. The UV-vis absorption spectrum of the as-prepared CdTe QDs showed a well-resolved maximum absorption at 537 nm (Figure 3, curve a), indicating a sufficiently narrow size distribution of the QDs. The result was also verified by the PL spectrum with a half-wave width less than 60 nm (Figure 3, curve b). The mean size of the QDs and concentration of the QD solution were estimated from the adsorption peak by Peng’s empirical equation38 to be 3.09 nm and 1.33 × 10-5 mol/L, respectively. The characteristic peak of QDs in the PL spectrum of MB-QDs showed some red shift (Figure 3, curve c), which was mainly attributed to the change of the surface charge status and charge transfer upon MB modification.39,40 Using rhodamine B as a standard, the PL quantum yield of MB-QDs was calculated to be 8.83%. Kinetic Characteristics of Quenching Reaction. After the GO (0.1 µg/mL) was added to MB-QDs solution (150 nM in HB), the fluorescence intensity of MB-QDs reduced rapidly to 10% of the original intensity (Figure 4, curve a), indicating the strong quenching effect of GO on the fluorescence of QDs. This fluorescence quenching could be ascribed to the energy transfer from the QD to the GO (Scheme 1), as in the case of the fluorescence quenching of fluorophore by the nanotubes.41,42 This conclusion could be confirmed from the change of fluorescence decay time from 32.57 to 26.99 ns before and after addition of GO (inset in Figure 4). The quenching reaction reached equilibrium almost in 1 min due to the fast adsorption of MB-QDs on GO, which was mainly contributed to the π- π stacking interaction between the GO and the base, and hydrogen bonding interaction present between -OH or -COOH groups of GO and -OH or (37) Shen, J. F; Hu, Y. Z.; Shi, M.; Lu, X.; Qin, C.; Li, C.; Ye, M. G. Chem. Mater. 2009, 21, 3514–3520. (38) Yu, W. W.; Qu, L.; Guo, W.; Peng, X. G. Chem. Mater. 2003, 15, 2854– 2860. (39) Chen, Y. F.; Ji, T. H.; Rosenzweig, Z. Nano Lett. 2003, 3, 581–584. (40) Hua, F. J.; Swihart, M. T.; Ruckenstein, E. Langmuir 2005, 21, 6054–6062. (41) 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. (42) Nakayama-Ratchford, N.; Bangsaruntip, S.; Sun, X. M.; Welsher, K.; Dai, H. J. J. Am. Chem. Soc. 2007, 129, 2448–2449.
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Figure 4. Fluorescence quenching of MB-QDs (150 nM) by GO (0.1 µg/mL) in HB buffer before (a) and after (b) being incubated with target (800 nM) as a function of time. Fluorescence intensity was recorded at 587 nm with an excitation wavelength of 330 nm. Inset: Decay curves of QD (50 nM) (a) and QD (50 nM) in the presence of GO (0.05 µg/mL) (b).
-NH2 groups of the ssDNA.43 The strong interactions between the GO and MB and the good stability and water-solubility of GO led to a choice of GO as the quencher. The adsorption of MB-QDs on GO could be observed from the HRTEM image (Figure 1C). However, the fluorescence intensity-time plot upon addition of GO (0.1 µg/mL) to MB-QDs solution (150 nM in HB) after incubation with target (800 nM) at 92 °C for 2 min and then 42 °C for 60 min showed a slow decrease in the intensity (Figure 4, curve b), indicating a decrease in the adsorption rate of dsDNA-QDs on GO. The formation of dsDNA-QDs reduced the surface charge of the DNA molecules and the exposure of the base. The latter weakened the π-π stacking and hydrogen bonding interactions. More importantly, the fluorescence quenching efficiency of GO decreased greatly. After addition of GO for 1 h, the fluorescence intensity still remained at 64.7% of the original intensity. The weakened fluorescence quenching efficiency was due to the longer distance of “donor-acceptor” pair44,45 and their weaker interactions (Scheme 1). The distances between the MB-QDs and GO before and after hybridization were calculated to be 3.24 and 9.36 nm, respectively. The corresponding quenching efficiencies were 90% and 35.3%. The distances of less than 10.0 nm and good overlap between PL spectrum of QDs and absorption spectrum of GO (inset in Figure 3) suggested the quenching process followed an energy transfer mechanisms,46 FRET, or surface energy transfer (SET).47 At distances shorter than 7.0 nm, the quenching was mainly attributed to the FRET mechanism, whereas it was mainly due to the SET mechanism at distances longer than 7.0 nm.47 When the distance was longer than 10 nm, the FRET process was completely attributed to the SET mechanism. From the distance of MB-QDs and GO before hybridization and the highly sensitive change of (43) Yang, X.; Zhang, X.; Liu, Z.; Ma, Y.; Huang, Y.; Chen, Y. J. Phys. Chem. C 2008, 112, 17554–7558. (44) Swathi, R. S.; Sebastian, K. L. J. Chem. Phys. 2009, 130, 086101. (45) Swathi, R. S.; Sebastian, K. L. J. Chem. Phys. 2008, 129, 054703. (46) Biju, V.; Itoh, T.; Baba, Y.; Ishikawa, M. J. Phys. Chem. B 2006, 110, 26068– 26074. (47) Yun, C. S.; Javier, A; Jennings, T.; Fisher, M.; Hira, S.; Peterson, S.; Hopkins, B.; Reich, N. O.; Strouse, G. F. J. Am. Chem. Soc. 2005, 127, 3115–3119.
Figure 5. Change of fluorescence anisotropy of MB-QDs (50 nM) upon addition of (a) GO (0.1 µg/mL) and then target (800 nM) and (b) target (800 nM) and then GO (0.1 µg/mL). Excitation was at 330 nm, and emission was monitored at 587 nm.
the fluorescence intensity, the change of FRET provided a stratagy for sensitive biosensing of biomolecules. Fluorescence Anisotropy. The fluorescence anisotropy of a fluorophore reflects the ability of molecule to rotate in its microenvironment.48 Anisotropy measurements are commonly used to investigate molecular interactions. As shown in Figure 5, the fluorescence anisotropy of free MB-QDs in HB was 0.024, and it increased 7.17-fold after addition of GO, indicating that the MB-QDs were adsorbed on the GO surface. However, the fluorescence anisotropy decreased by 2.32-fold after further addition of the target into the mixture of GO and MB-QDs, indicating the hybridization of MB with the target decreased the adsorption of MB-QDs on GO, whereas in the absence of GO, the hybridization slightly increased the fluorescence anisotropy of MB-QDs, and after addition of GO this value was further increased to the level similar to that for addition of GO and then the target. These results further demonstrate the hybridization process of MB-QDs with target and the adsorption of MB-QDs on GO for producing the FRET and fluorescence quenching. Fluorescence Quenching Efficiency. Figure 6 shows the fluorescence quenching of different QDs by GO. The free QDs show strong fluorescence emission in HB (Figure 6A, curve a), which decreases to 20% after addition of GO (Figure 6A, curve b), indicating GO can quench the fluorescence of QDs by FRET due to the adsorption of QDs on GO. Different from the free QDs, the MB-QDs (120 nM) shows a fluorescence quenching by more than 97.6% in HB upon addition of 0.1 µg/mL GO (Figure 6B), and only 2.4% of the original fluorescence can be maintained, indicating the high quenching efficiency. The quenching efficiency is much higher than the typical efficiency of 85-97% for static or contact quenching in MB-based detection.25 The higher quenching efficiency would lead to a higher signal-to-background ratio and thus better sensitivity and a greater dynamic range for target detection. When the MB-QDs were hybridized with target sequence to form dsDNA, the quenching efficiency became 61.3% (Figure 6C). The difference between the quenching efficiencies of GO to MB-QDs and dsDNA-QDs produced a method for sensing target DNA. (48) Lakowicz, J. Principles of Fluorescence Spectroscopy, 3rd ed.; Springer: New York, 2006.
Figure 6. Fluorescence emission spectra of (A) QDs (120 nM), (B) MB-QDs (120 nM), and (C) MB-QDs incubated with target (800 nM) in HB buffer before (a) and after (b) adding GO (0.1 µg/mL) for 5 min.
Figure 7. Fluorescence emission spectra of MB-QDs (120 nM) at GO concentrations of 0, 0.02, 0.04, 0.06, 0.08, 0.1, and 0.12 µg/mL (a-g) (A), and plot of fluorescent intensity ratio F0/F upon hybridization with 800 nM target DNA vs MB-QD concentration at 0.1 µg/mL GO (B). Inset in A: fluorescent intensity ratio of MB-QDs upon addition of GO at different concentrations.
To examine the advantage of GO over other carbon nanostructures for sensing target DNA, single-walled carbon nanotubes (SWNTs) were used as a control system. A 0.1 µg/mL portion of SWNTs showed a quenching efficiency of 95% on the MB-QDs (120 nM) in HB, slightly lower than the GO. The similar result could be found by comparing their quenching efficiency on the PL of fluorescein-based dye.4,10 This was attributed to the 2D structure of the GO, which provided a larger area for contact of ssDNA. Fluorescent Analysis of Target DNA. The concentrations of both GO and MB-QDs strongly influenced the detection of target DNA. As shown in Figure 7A, with the increasing concentration of GO, the fluorensence intensity of the MB-QDs decreased and trended to a minimum value at 0.1 µg/mL (inset in Figure 7A). Thus, 0.1 µg/mL GO was used for analytical purposes. From the dependence of fluorescent intensity of MB-QDs on GO concentration, the fluorescence quenching could be described by the Stern-Volmer equation with a quenching Analytical Chemistry, Vol. 82, No. 13, July 1, 2010
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Figure 8. Fluorescence emission spectra of MB-QDs (50 nM) after incubation with 50, 150, 500, 800, 1200, and 1500 nM target (a-f), and then addition of GO (0.1 µg/mL) for 5 min. Inset: plot of fluorescence intensity ratio F0/F vs logarithm of target concentration.
constant of 1.1 × 105 (µg/mL)-1, indicating a dynamic quenching process.49 Meanwhile, the change of quenching efficiency of 0.1 µg/mL GO upon hybridization depended on the MB-QD concentration. At the target DNA concentration of 800 nM, the maximum change upon hybridization occurred at the MB-QD concentration of 50 nM (Figure 7B), where F and F0 are the fluorescence intensities of MB-QDs after and before hybridization in the presence of GO at 587 nm, respectively. Thus 50 nM MB-QDs was used for following detection. With the increasing concentration of target DNA used for hybridization prior to addition of GO, the fluorescence emission intensity increased (Figure 8). The plot of the fluorescence intensity ratio (F0/F) vs the logarithm value of target concentration showed a linear calibration in the range from 50 to 1500 nM. The limit of detection (LOD) for target DNA was 12 nM at 4 times the standard deviation of the control (free of target DNA). Although it was 3-fold higher than the previous report,4 a lower LOD could be obtained in the proposed method by reducing the concentration of the MB-QDs used for obtaining the calibration curve. These results suggested that the GOquenching approach was potentially appropriate for quantification of nucleic acid. A significant advantage of MB is the unique specificity18 in comparison with linear DNA probe. The specificity of MB-QD probe was studied using three kinds of DNA sequences, including perfectly complementary target, single-base mismatched strand, and three-base mismatched strand. As shown in Figure 9, the MB-QD probe presented good performance to discriminate the perfectly complementary target and the mismatched stand. Upon addition of GO, the perfectly complementary target showed a F0/F value of 2.5 times that for single-base mismatch sequence (Inset, histograms a and b), indicating good selectivity. The response to the three-base mismatch stand was only 15% of that for the perfectly complementary target (inset, histogram c), which was close to the blank control (inset, histogram d). These results demonstrated that the proposed approach was (49) He, Y.; Wang, H. F.; Yan, X. P. Anal. Chem. 2008, 80, 3832–3837.
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Figure 9. Fluorescence emission spectra of MB-QDs (50 nM) after incubation with (a) target (800 nM), (b) single-base mismatch stand (800 nM), (c) three-base mismatch stand (800 nM), and (d) no target and then addition of GO (0.1 µg/mL) for 5 min. Inset: fluorescence intensity ratio F0/F for four cases.
Figure 10. Fluorescence intensity ratio F0/F for aptamer-QDs (50 nM) after incubation with thrombin at different concentrations and then addition of GO (0.1 µg/mL) for 5 min.
able to detect effectively the target with high specificity and had potential application in single nucleotide polymorphism analysis. Application in Detection of Protein. To demonstrate the extended practicality of the suggested system, a thrombin binding aptamer was chosen as the model to study the application of the platform in the detection of protein. After apamter-QDs (50 nM) were hybridized with different concentrations of thrombin at 42 °C for 60 min, 0.1 µg/mL GO was added into the mixture to examine the fluorescence quenching effect. The typical fluorescence intensity ratios (F0/F) in the presence of thrombin with different concentrations upon addition of GO are shown in Figure 10. Upon increasing the thrombin concentration, the ratio increased, indicating that the proposed approach could be applied potentially in detection of protein. It is worth mentioning that when the thrombin concentration further increased to 200 nM, fluorescence self-quenching by the thrombin was observed, which was possibly attributed to the aggregation of the aptamer-QDs at a high concentration of thrombin.
CONCLUSION In conclusion, a novel sensing platform for biomolecules based on the fluorescence quenching of MB-QDs by GO was proposed. This strategy was based on the change in binding affinity of GO to MB-QDs upon recognition to target DNA. The quenching efficiency was much higher than the typical efficiency used in MB-based detection, which was an advantage for improving the sensitivity and dynamic range. The quenching effect resulted from the strong interactions between the ssDNA of the MB loop structure and GO, which led to the adsorption of MB-QDs on GO and the FRET from QDs to GO. The interactions had been demonstrated using quenching kinetic analysis, HRTEM, and fluorescence anisotropy. The proposed system showed high sensitivity and good selectivity and could be used for both the quantification of nucleic acid and single nucleotide polymorphism analysis. Furthermore, it could be developed for detection of other
aptamer-specific biomolecules, thus promising application in different fields. ACKNOWLEDGMENT This work was financially supported by the National Natural Science Foundation of China (20845005, 20710087, 90713015, 20875044, 20821063), the National Basic Research Program (2010CB732400), the Important National S&T Specific Project (2009ZX10004-313), the Outstanding Medical Talents Program (RC2007069) from the Department of Health of Jiangsu, and the Natural Science Foundation of Jiangsu (BK2008014).
Received for review February 2, 2010. Accepted May 25, 2010. AC100852Z
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