Strategy for Molecular Beacon Binding Readout: Separating Molecular

Nov 9, 2009 - A new strategy for molecular beacon binding readout is proposed by using separation of the molecular recognition element and signal repo...
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Anal. Chem. 2009, 81, 9703–9709

Strategy for Molecular Beacon Binding Readout: Separating Molecular Recognition Element and Signal Reporter Yongxiang Wang,† Jishan Li,‡ Jianyu Jin,† Hao Wang,† Hongxing Tang,‡ Ronghua Yang,*,†,‡ and Kemin Wang*,‡ Beijing National Laboratory for Molecular Sciences, College of Chemistry and Molecular Engineering, Peking University, Beijing 100871, China, and State Key Laboratory of Chemo/Biosensing and Chemometrics, College of Chemistry and Chemical Engineering, Hunan University, Changsha, 410082, China A new strategy for molecular beacon binding readout is proposed by using separation of the molecular recognition element and signal reporter. The signal transduction of the target binding event is based on displacing interaction between the target DNA and a competitor, the signal transducer. The target-free capture DNA is first interacted with the competitor, forming an assembled complex. In the presence of a target DNA that the affinity is stronger than that of the competitor, hybridization between capture DNA and the target disassembles the assembled complex and releases the free competitor to change the readout of the signal reporter. To demonstrate the feasibility of the design, a thymine-rich oligonucleotide was examined as a model system. Hg2+ was selected as the competitor, and mercaptoacetic acid-coated CdTe/ZnS quantum dots served as the fluorescent reporter. Selective binding of Hg2+ between the two thymine bases of the capture DNA forms a hairpin-structure. Hybridization between the capture DNA and target DNA destroys the hairpin-structure, releasing Hg2+ ions to quench the quantum dots fluorescence. Under the optimal conditions, fluorescence intensity of the quantum dots against the concentration of perfect cDNA was linear over the concentration range of 0.1-1.6 µM, with a limit of detection of 25 nM. This new assay method is simple in design, avoiding any oligonucleotide labeling. Furthermore, this strategy is generalizable since any target binding can in principle release the signal transducer and be detected with separated signal reporter. Selective detection of specific single-stranded (ss-)DNA sequences is gaining central importance in the fields of modern life science, environmental science, and microbiology.1-3 Several * To whom correspondence should be addressed. E-mail: [email protected] (R.Y.); [email protected] (K.W.). Fax: +86-731-8882 2523. † Peking University. ‡ Hunan University. (1) Santangelo, P.; Nitin, N.; Bao, G. Ann. Biomed. Eng. 2006, 34, 39–50. (2) Marras, S. A. E.; Tyagi, S.; Kramer, F. R. Clin. Chim. Acta 2006, 363, 48–60. (3) Mart, A. A.; Jockusch, S.; Stevens, N.; Ju, J. Y.; Turro, N. Acc. Chem. Res. 2007, 40, 402–409. 10.1021/ac901906w CCC: $40.75  2009 American Chemical Society Published on Web 11/09/2009

methods have been developed for DNA detection, making using of optical transduction,4,5 electrochemistry,6-8 spectrochemistry,9-12 and gravimetry,13 etc. Among them the solution-based hybridization assay by using synthetic DNA probes has received a great deal of attention in the early years.14-16 For example, the wellknown molecular beacons (MBs) belong to this category.17-19 MBs are ssDNA hybridization probes that form a hairpin-structure. To obtain the signaling communication between the DNA hybridization event and outside word, a fluorophore-quencher pair is covalently linked at 5′- and 3′- termini of the hairpin-structure respectively, to form a quenched approach. Upon their hybridization to complementary targets, MBs brightly fluoresce since the MBs adopt an open conformation in which the labels are spatially separated (Figure 1A). While the unique thermodynamics and specificity of MBs have demonstrated several advantages in DNA detection, such as it is not necessary to isolate the probe-target hybrids and they easily discriminate single-nucleotide polymorphisms (SNPs), the great challenge still remains of simplifying the probe synthesis and optimization procedures. Double-labeling of an oligonucleotide (4) Ihara, T.; Uemura, A.; Futamura, A.; Shimizu, M.; Baba, N.; Nishizawa, S.; Teramae, N.; Jyo, A. J. Am. Chem. Soc. 2009, 131, 1386–1387. (5) Zheng, W. M.; He, L. J. Am. Chem. Soc. 2009, 131, 3432–3433. (6) Kerman, K.; Saito, M.; Morita, Y.; Takamura, Y.; Ozsoz, M.; Tamiya, E. Anal. Chem. 2004, 76, 1877–1884. (7) Fan, C. H.; Plaxco, K. W.; Heeger, A. J. Proc. Natl. Acad. Sci. U.S.A. 2003, 100, 9134–9137. (8) Xiao, Y.; Lubin, A. A.; Baker, B. R.; Plaxco, K. W.; Heeger, A. J. Proc. Natl. Acad. Sci. U.S.A. 2006, 103, 16677–16680. (9) Elghanian, R.; Storhoff, J. J.; Mucic, R. C.; Letsinger, R. L.; Mirkin, C. A. Science 1997, 277, 1078–1081. (10) Pan, B. F.; Ao, L. M.; Gao, F.; Tian, H. Y.; He, R.; Cui, D. X. Nanotechnology 2005, 16, 1776–1780. (11) Li, H. X.; Rothberg, L. Proc. Natl. Acad. Sci. U.S.A. 2004, 101, 14036– 14039. (12) Qian, X. M.; Peng, X. H.; Ansari, D. O.; Yin-Goen, Q.; Chen, G. Z.; Shin, D. M.; Yang, L. L.; Young, A. N.; Wang, M. D.; Nie, S. M. Nat. Biotechnol. 2008, 26, 83–90. (13) Liu, J. H.; Fan, J. B.; Gu, Z.; Cui, J.; Xu, X. B.; Liang, Z. W.; Luo, S. L.; Zhu, M. Q. Langmuir 2008, 24, 5241–5244. (14) Ho, H.-A.; Najari, A.; Leclerc, M. Acc. Chem. Res. 2008, 41, 168–178. (15) Thomas, S. W.; Joly, G. D.; Swager, T. M. Chem. Rev. 2007, 107, 1339– 1386. (16) Lakowicz, J. R. Principles of Fluorescence Spectroscopy, 3rd ed; Springer: New York, 2006. (17) Tyaji, S.; Kramer, F. R. Nat. Biotechnol. 1996, 14, 303–308. (18) Marras, S. A. E. Methods Mol. Biol. 2006, 335, 3–16. (19) Yang, C. Y.; Medley, C.; Tan, W. H. Curr. Pharm. Biotechnol. 2005, 6, 445–452.

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Figure 1. Comparison of the double-labeled MB (A) and label-free MB (B) for detection of cDNA oligonucleotide. In the case of part A, the target DNA binding separates the fluorophore and quencher, and fluorescence of the labeled fluorophore occurs. In the case of part B, the target DNA binding releases Hg2+ ions, and interaction of Hg2+ ions with a signal reporter (QDs) produces a signal readout.

with a fluorophore-quencher pair requires elaborate design of the synthetic routes and many heavy and complicated synthetic steps. These primarily include (1) selection of fluorophore-quencher properties, (2) means of attachment of fluorophore-quencher groups, and (3) qualities of labeling and purification of the product. What is more, covalent modification of an oligonucleotide with other functions may lead to significant loss in its affinity and specificity. Although DNA-intercalating dyes have been used to detect double-stranded (ds-) DNA in solution, thus giving a simple and label-free probe design,20-22 this method suffers from intrinsic limitation in the case of MB, because the dyes can intercalate in the stem part of the MB to produce high background. In this respect, design of an alternative MB-based optical approach that avoids any oligonucleotide labeling should be of general interest and widely applicable. One of label-free “chemosensing ensemble” approaches is the use of competitive displacement interaction.23,24 This general strategy was first proposed by Anslyn et al. for colorimetrical detection of citrate,25 tartrate,26 phosphate,27 amino acids,28,29 and carboxylates,30 then successfully developed by Gale et al.,31 Fabbrizzi et al.,32 and Akkaya et al.33 for fluorescent sensing of (20) Lerman, L. S. J. Mol. Biol. 1961, 3, 18–80. (21) Guo, Q.; Lu, M.; Marky, L. A.; Kallenbach, N. R. Biochemistry 1992, 31, 2451–2455. (22) Lee, K.; Maisel, K.; Rouillard, J.; Gulari, E.; Kim, J. Chem. Mater. 2008, 20, 2848–2850. (23) Wiskur, S. L.; Ait-Haddou, H.; Lavigne, J. J.; Anslyn, E. V. Acc. Chem. Res. 2001, 34, 963–972. (24) Nguyen, B. T.; Anslyn, E. V. Coord. Chem. Rev. 2006, 250, 3118–3127. (25) Metzger, A.; Anslyn, E. V. Angew. Chem., Int. Ed. 1998, 37, 649–652. (26) Lavigne, J. J.; Anslyn, E. V. Angew. Chem., Int. Ed. 1999, 38, 3666–3669. (27) Tobey, S. L.; Jones, B. D.; Anslyn, E. V. J. Am. Chem. Soc. 2003, 125, 4026–4027. (28) Leung, D.; Folmer-Andersen, J. F.; Lynch, V. M.; Anslyn, E. V. J. Am. Chem. Soc. 2008, 130, 12318–12327. (29) Leung, D.; Anslyn, E. V. J. Am. Chem. Soc. 2008, 130, 12328–12333. (30) Zhu, L.; Zhong, Z. L.; Anslyn, E. V. J. Am. Chem. Soc. 2005, 127, 4260– 4269. (31) Gale, P. A.; Twyman, L. J.; Handlin, C. I.; Sessler, J. L. Chem. Commun. 1999, 1851–1852. (32) Hortala´, M. A.; Fabbrizzi, L.; Marcotte, N.; Stomeo, F.; Taglietti, A. J. Am. Chem. Soc. 2003, 125, 20–21.

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halide anions, amino acids, and ATP in solution. Recently, Fan et al.34 and Ho et al.35 have applied such technology in aptamerbased optical probes to develop new methods for target binding readout. When the acceptor and competitor are carefully chosen, selective sensing event can be communicated through displacement of the signaling reporter from the bonded acceptor by the analyte. However, no attempt, so far, has been made to exploit such a competitive assay to MBs. Therefore, we present here a new strategy for MB-target binding readout based on such a competitive assay between the target DNA and a signal transducer. Our proposed approach contains three structural motifs. An unmodified oligonucleotide (here we selected a thymine-rich oligonucleotide) that is specific to the target serves as the captured probe, a competitor (Hg2+) that partially binds to the capture DNA as the signal transducer, and a fluorescent probe (quantum dots) that is sensitive to the competitor rather than to the capture DNA or target DNA serves as a signaling reporter. As shown in Figure 1B, in the absence of targets, Hg2+ ions bind to the capture DNA to form a hairpin-structure. However, in the presence of a target DNA, the bound-Hg2+ ions are displaced from the hairpin-structure by the target to change the optical readout of the quantum dots. Compared with the common MB, the present design possesses three excellent molecular engineering features. First, by separation of the molecular recognition element and signal reporter, one could successfully avoid the double-labeled processes, which usually lead to relatively complex operation and lose the affinity and specificity of the oligonucleotide. Second, the approach can achieve amplified signal function. From Figure 1B, one target DNA molecule that hybridizes with the capture DNA will release several Hg2+ ions to interact with the signal reporter, and amplification of the detection signals could be realized. Third, the metal-dependent stem forming makes this approach be flexible to adjust the sensitivity and the selectivity. For common MB, modulation of the sensitivity and the selectivity can be achieved by variation of the stem length and sequence, but this requires the synthesis of several different MBs. For the present approach, due to dependence of stem-forming on the competitor, it is the competitor that allows tuning of the selectivity and sensitivity of the MB toward target DNA sequences. This property may be of advantage in optimization studies. EXPERIMENTAL SECTION All oligonucleotides were synthesized by TaKaRa Biotechnology Co., Ltd., (Dalian, China). They were dissolved in highly pure water (sterile Minipore water, 18.2 MΩ) as stock solution and the concentration was identified according to UV absorption at 260 nm. Mercaptoacetic acid-coated CdTe/ZnS quantum dots (MAA-QDs) were purchased from Wuhan Jiayuan Quantum Dots Co., Ltd. (Wuhan, China). All other reagents were of analytical reagent grade and were purchased from Fluka (Switzerland). Stock solutions of metal ions were prepared from analytical grade nitrate salts and were dissolved in deionized water. All work (33) Atilgan, S.; Akkaya, E. U. Tetrahedron Lett. 2004, 45, 9269–9271. (34) Wang, J.; Wang, L. H.; Liu, X. F.; Ling, Z. Q.; Song, S. P.; Li, W. X.; Li, G. X.; Fan, C. H. Adv. Mater. 2007, 19, 3943–3946. (35) Li, N.; Ho, C.-M. J. J. Am. Chem. Soc. 2008, 130, 2380–2381.

Table 1. Design of Probes and Target Oligonucleotides

a

type

sequence

capture DNA (1) pc-ssDNA (2)a sm-ssDNA(3)b FAM/Dabcyl-labeled LN (4)c FAM/Dabcyl-labeled LN (5)

5′-TTTTTTACTAAATCACTATGGTCGCTTTTTT-3′ 5′-GCGACCATAGTGATTTAGT-3′ 5′-GCGACCATAATGATTTAGT-3′ 5′-Dabcyl-TTTTTTACTAAATCACTA TGGTCGCTTTTTT-FAM-3′ 5′-Dabcyl-CCTAGCACTAAATCACTATGGTCGCCGATCC-FAM-3′

Perfectly complementary target. b Single-base mismatched target. c Linear DNA that forms a hairpin-structure induced by Hg2+.

solutions were prepared with phosphate buffer solution (PBS, pH 7.4, 2.5 mM Mg2+). UV-visible absorption spectra were recorded on a Hitachi U-3010 UV-vis spectrophotometer (Kyoto, Japan). All fluorescence measurements were performed on a Hitachi F-7000 fluorescence spectrofluorometer (Kyoto, Japan). The pH was measured by a model 868 pH meter (Orion). Temperature was controlled by PolyScience 9112 refrigerating/heating circulators. RESULTS AND DISCUSSION Choices of the Capture DNA and Competitor. The operation principle of our design is based on competitive displacement between the target DNA and the competitor. One basic requirement for this approach is that the captured DNA is coupled to the competitor with an appropriate association constant (K0) for the complex equilibrium. In particular, if a target binding with capture DNA displays an association constant, K1, and an interfering agent substrate gives its own K2 value, the best situation for discrimination of the target and interfering agents is expressed by the inequality of K1 > K0 . K2. Under these conditions, only the envisaged target, but not the interfering agents even at higher concentration, will be able to displace the competitor from the capture DNA. To demonstrate the feasibility of this design for DNA detection, a thymine(T)-rich ssDNA (1, Table 1) which contains 19-mers in the middle together with 12 thymine (T) bases at the lateral portions was selected as the capture probe, and Hg2+ was selected as the competitor. It has been well established that Hg2+ can selectively bind between two DNA thymine bases and promote these T-T mismatches to form stable T-Hg2+-T base pairs.36-39 The target DNA molecules, 2 and 3, were 19-mers long and were either perfectly complementary to the 19 mers in the middle of 1 or contained one mismatch with the bases, respectively. As shown in Figure 1B, formation of T-Hg2+-T structures of 1 and Hg2+ facilitates 1 to fold into a hairpin structure (1-Hg2+). When 1-Hg2+ interacts with 2, the target opens the hairpin by forming a rigid DNA duplex, thereby leaving free Hg2+ ions. To signal the released Hg2+ ions in the solution, MAA-QDs were chosen as the fluorescent reporter. It has been reported that the fluorescence emission of many organic molecules40-43 and QDs44-46 are sensitive to Hg2+. Since one target molecule can release stoichiometric Hg2+ ions from (36) Ono, A.; Togashi, H. Angew. Chem., Int. Ed. 2004, 43, 4300–4302. (37) Miyake, Y.; Togashi, H.; Tashiro, M.; Yamaguchi, H.; Oda, S.; Kudo, M.; Tanaka, Y.; Kondo, Y.; Sawa, R.; Fujimoto, T.; Machinami, T.; Ono, A. J. Am. Chem. Soc. 2006, 128, 2172–2173. (38) Lin, Y. W.; Ho, H. T.; Huang, C. C.; Chang, H. T. Nucleic Acids Res. 2008, 36, e123. (39) Yang, R. H.; Jin, J. Y.; Long, L. P.; Wang, Y. X.; Wang, H.; Tan, W. H. Chem. Commun. 2009, 322–324.

1-Hg2+, the fluorescence changes of MAA-QDs will be proportional to target DNA concentration. To confirm the formation of a hairpin-structure of 1-Hg2+, 1 was labeled with a fluorophore, carboxyfluorescein (FAM), and a quencher, 4-([4- (dimethylamino)phenyl]azo) benzoic acid (Dabcyl)47 at the 3′- and 5′-end, respectively, to obtain 4. To compare, oligonucleotide 5 was also labeled with FAM and Dabcyl at 3′- and 5′-end, respectively. As shown in Table 1, 5 is also 31 bases long and the 19 bases in the middle are identical to those of 1, but 5 does not contain the thymine bases at the lateral portions and thus could not form a hairpin structure by Hg2+. We expected that the interaction of Hg2+ with 4 rather than 5 will lead to the FAM fluorescence quenching (see below). Interactions of MAA-QDs with Metal Ions. The detection sensitivity of the proposed approach for DNA hybridization assay depends on the interaction of Hg2+ ions and MAA-QDs. Effect of Hg2+ on the fluorescence of MAA-QDs was thus first studied. In the PBS, MAA-QDs emit strong fluorescence with a maximum emission wavelength at 592 nm (see Figure 3). With the addition of increasing concentrations of Hg2+ to the buffer solution of MAA-QDs, a gradual and Hg2+-concentration dependent decrease in fluorescence intensity was observed (Figure S1 in the Supporting Information). When Hg2+ concentration was up to 6.0 µM, no significant MAA-QDs fluorescence emission could be detected. The linear response range was 0-2.0 µM Hg2+ (y ) 0.429C - 0.404, R2 ) 0.969), the detection limit that is taken to be 3 times the standard deviation in blank solution and was estimated to be ∼0.5 nM. Both the intensity decrease and wavelength shift of MAA-QDs fluorescence emission by Hg2+ are due to the interaction of the metal with the QDs, which are ascribed to (1) replacement of Cd2+ ions from the core of the QDs by Hg2+,48,49 (2) effective electron transfer from the MAA moiety on the surface of QDs to Hg2+ ions since the carboxyl groups can coordinate with the metal (40) Descalzo, A. B.; Martinez-Manez, R.; Radeglia, R.; Rurack, K.; Soto, J. J. Am. Chem. Soc. 2003, 125, 3418–3419. (41) Guo, X. F.; Qian, X. H.; Jia, L. H. J. Am. Chem. Soc. 2004, 126, 2272–2273. (42) Caballero, A.; Martinez, R.; Lloveras, V.; Ratera, I.; Vidal-Gancedo, J.; Wurst, K.; Tarraga, A.; Molina, P.; Veciana, J. J. Am. Chem. Soc. 2005, 127, 15666– 15667. (43) Zhang, X. L.; Xiao, Y.; Qian, X. H. Angew. Chem., Int. Ed. 2008, 47, 8025– 8029. (44) Chen, B.; Yu, Y.; Zhou, Z. T.; Zhong, P. Chem. Lett. 2004, 33, 1608–1609. (45) Long, Y. F.; Jiang, D. L.; Zhu, X.; Wang, J. X.; Zhou, F. M. Anal. Chem. 2009, 81, 2652–2657. (46) Han, B. Y.; Yuan, J. P.; Wang, E. K. Anal. Chem. 2009, 81, 5569–5573. (47) Marras, S. A.; Kramer, F. R.; Tyagi, S. Nucleic Acid. Res. 2002, 30, e122. (48) Haesselbarth, A.; Eychmueller, A.; Eichberger, R.; Giersig, M.; Mews, A.; Weller, H. J. Phys. Chem. 1993, 97, 5333–5340. (49) Son, D. H.; Hughes, S. M.; Yin, Y.; Alivisatos, A. P. Science 2004, 306, 1009–1012.

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Figure 2. Fluorescence decreases of 50 nM MAA-QDs upon the additions of various metal ions, anions, or the mixtures of metal ions (x-axis markers, the concentrations of Cu2+, Ag+, and Hg2+ were 100 µM, respectively, other metals and anions were 10.0 mM, respectively). Fluorescence intensities were recorded at 596 nm with an excitation wavelength of 388 nm.

Figure 3. Fluorescence emission spectra of MAA-QDs (50 nM, λex ) 388 nm) at different experimental conditions: (a) MAA-QDs in PBS; (b), 2.0 µM Hg2+ + MAA-QDs; (c), 2.0 µM Hg2+ + 1.0 µM 1 + MAAQDs; (d), the mixture from part c + 2.0 µM 2.

ions,50 and (3) the facilitating nonradiative recombination of excited electrons (e-) in the conduction band and holes (h+) in the valence band.44 From the fluorescence titration data, we can determine the stoichiometry and the association constant of the QD with Hg2+ ions using the curve fitting method published before.51,52 If the complex equilibrium between a metal ion (Mn+) and a fluorescent ligand (QD) forms a m:n of metal-to-ligand complex, Mm(QD)n, the corresponding apparent association constant, K0, can be expressed as follows: [Mn+]m )

1 1-R 1 nK0 [QD]Tn-1 Rn

(1)

where [QD], [Mn+], and [Mm(QD)n] are the concentrations of the respective species, and R can be determined from the fluorescence changes of the QDs in the presence of different concentrations of Hg2+: R)

Fmax - F Fmax - Fmin

(2)

where Fmax and Fmin are the limiting fluorescence values for R ) 1 (in the absence of Hg2+) and R ) 0 (QD is completely complexed with Hg2+), respectively. F denotes the fluorescence intensity of QDs in the presence of different concentrations of Hg2+. The experimental data were fitted to eqs 1 and 2 by adjusting the K0 value and m to n, which shows the formation of a 2: 1 metal-to-ligand complex and gives a corresponding association constant for K0 ) 2.1 × 1012 M-2 (Figure S2 in the Supporting Information). The stoichiometric combination and relative high affinity of MAA-QDs with Hg2+ promote us to use the QDs as the fluorescent reporter in the present approach. Next, we examined the effects of biologically related metal ions or anions on the fluorescence emission of MAA-QDs. All titration (50) Kim, Y.; Johnson, R. C.; Hupp, J. T. Nano Lett. 2001, 1, 165–167. (51) Yang, R. H.; Wang, K. M.; Long, L. P.; Xiao, D.; Yang, X. H.; Tan, W. H. Anal. Chem. 2002, 74, 1088–1096. (52) Yang, R. H.; Chan, W. H.; Lee, A. W. M.; Xia, P. F.; Zhang, H. K.; Li, K. A. J. Am. Chem. Soc. 2003, 125, 2884–2885.

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Figure 4. Fluorescence signal changes of 1-Hg2+/MAA-QDs as a function of the concentration of 2 in the PBS. The concentrations of 1, Hg2+, and MAA-QDs were 0.2, 1.0, and 50 nM, respectively. Fluorescence intensities were recorded at 596 nm with an excitation wavelength of 388 nm.

studies were conducted using a 50 nM MAA-QDs. Reaction of MAA-QDs with Hg2+, Cu2+, and Ag+ leads to obvious fluorescence decreases (Figure 2), but the degree of fluorescence quenching by Cu2+ or Ag+ is smaller than that by Hg2+. Among other metal ions and anions investigated, alkali, alkaline earth ions, and anions have hardly an effect on the MAA-QDs fluorescence emission, while other transition metal ions show little quenching of MAA-QDs fluorescence. To further characterize the binding specificity of MAA-QDs for Hg2+, the competition experiments were also conducted in which MAAQDs were first mixed with the selected competing metal ion and Hg2+ was then added to the mixture. As shown in Figure 2, except for Cu2+, no significant variations in the fluorescence intensity of MAA-QDs were found by comparison with those without other substrates rather than Hg2+. The relatively high specificity of the QDs for Hg2+ indicates MAA-QDs could be used as Hg2+ fluorescent probes in biological systems. Formation of Hairpin-Structure between Hg2+ and T-Rich ssDNA. To test if there is interaction between 1 and Hg2+ and if it forms a hairpin-structure, we investigated the effect of Hg2+ at various concentrations on the FAM fluorescence of 4 and 5 in the PBS. As expected, upon addition of increasing concentrations of Hg2+ ions to the solution of 4, the FAM’s fluorescence

initially decreased rapidly (Figure S3 in the Supporting Information), as a result of enhanced fluorescence resonance energy transfer process between the FAM and Dabcyl moieties.47 In our experiment, more than 90% quenching was observed for 4 in the concentration from 0.1 to 2.0 µM by ∼5.0-fold excess of Hg2+. On the contrary, the fluorescence of 5 was slightly affected by Hg2+ under the same conditions. These results suggest that mercury-mediated base pairs of the ssDNA are formed between thymine residues to form a stable hairpin structure, and that the folded DNA structure was more stable in the presence of higher concentrations of Hg2+. To support this hypothesis, we conducted melting temperature (Tm) measurements; here, we define Tm as the temperature at which the fluorescence of FAM reaches 50% of the value without Hg2+. The free 4 showed no melting transition between 15 and 80 °C, indicating no hairpin structure. In the presence of Hg2+, the fluorescence intensity increased with increasing in the temperature as a result of breaking the T-Hg2+-T bonds (Figure S4 in the Supporting Information). With the concentration of 4 fixed at 100 nM and a change of the Hg2+ concentration from 0.2 to 5.0 µM, the Tm of the Hg2+ complex increased from 20 to 54 °C. When the Hg2+ concentration was 10 µM, the Tm value of 4-Hg2+ is about 63 °C. This dependence of 4-Hg2+ stability on Hg2+ concentration further supports the formation of a hairpin-structure by Hg2+ which provides a means to the design of functional MB so that it works well either at low temperature or at high temperature by simply changing the Hg2+ concentration. The sensing capability of 1 for a target DNA depends on the interplay of the complexes formed between Hg2+ and thymine bases and between the DNA sequence in the loop and the tested DNA. To confirm the validity of the proposed approach for a DNA competitive assay, it is necessary to demonstrate how strong is the binding between the thymine base and Hg2+. If a 1:2 metal-to-ligand complex (T-Hg2+-T) is formed between Hg2+ and thymine bases, the obtained emission intensities of 4 at 518 nm as shown in Figure S3 in the Supporting Information were analyzed using the relation of Hg2+ concentration with the response parameter (R):51,52

[Hg2+] )

1 1 1-R 2K1 [L]T R2

(3)

Figure S5 in the Supporting Information shows R as a function of the logarithm of Hg2+ concentration. The curve fitting for the experimental data points was calculated from eq 3 with K1 ) 3.4 × 106 M-2. This association strength of T-Hg2+-T could be able to compensate for two or three hydrogen bonds otherwise found in the natural base pairs.53 From the 12 thymine bases contained in 1, the maximum association constant of 1 and Hg2+ (all T bases were combined with Hg2+) was around 2.1 × 107 M-2. The appropriate affinity between 1 and Hg2+ ensures the performance of the DNA competitive hybridization assay proposed in this article. Influence of DNA Sequences on the Interaction of Hg2+ and MAA-QDs. The influence of different DNA sequences on the interaction of Hg2+ and MAA-QDs was studied in the PBS. (53) Meggers, E.; Holland, P. L.; Tolman, W. B.; Romesberg, F. E.; Schultz, P. G. J. Am. Chem. Soc. 2000, 122, 10714–10715.

The introduction of a random ssDNA (5′-ATACTTGGGCACACCCGCA-3′) to the solution of MAA-QDs leads to a little increase of the emission intensity (data not shown) due to a change in the microenvironment of the QDs.54,55 When Hg2+ was added to the solution of the mixture of MAA-QDs and the random DNA, strong fluorescence quenching was observed and the quenching efficiency, F/F0 (F0 and F are the QDs fluorescence intensity in the absence and the presence of Hg2+, respectively), is similar to that without a DNA sequence, demonstrating that the interaction of MAA-QDs and Hg2+ was hardly affected by a random DNA sequence. Curve c of Figure 3 shows the fluorescence emission spectrum of MAA-QDs in the presence of Hg2+ and 1, which was obtained by adding the QDs to the mixture of 1 and Hg2+. It is worth noting that, when Hg2+ was first interacted with 1, the metal had only a slight effect on the QDs fluorescence emission. The quenching efficiency of MAA-QDs at 598 nm by 2.0 µM Hg2+ in the presence of 1.0 µM 1 (F/F0 ) 0.91) is around 0.28-fold that in the absence of 1 (F/F0 ) 0.25), indicating there are low free Hg2+ ions in the solution due to the formation of T-Hg2+-T structure. On the contrary, if 1 was first hybridized with 2 in the PBS to form a duplex and introduction of the same amount of Hg2+ and MAA-QDs to the dsDNA solution, MAA-QDs exhibit enhanced fluorescence quenching in comparison with that without 2 (curve d, Figure 3). This difference in the DNA-induced fluorescence changes of MAA-QDs and Hg2+ constitutes the basis for the fluorescent assay of DNA hybridization proposed in this article. DNA Hybridization Assay. Taken together, these findings demonstrate that 1-Hg2+ can be efficient for the DNA hybridization assay by using a separated molecular recognition element and fluorescent reporter. The molecular recognition ability of 1-Hg2+/MAA-QDs was thus studied by examining the DNA-induced MAA-QDs fluorescence change in the PBS. It is a useful feature that MBs recognize their targets with higher specificity than linear DNA probes.17-19 Notwithstanding this performance, we expect that the increase in thermostability of the stem by Hg2+ and the basic competition between unimolecular hairpin reaction and probe-target hybridization of 1-Hg2+ will have an improvement in the selectivity for the DNA target, thus outperforming common MBs. Perfectly matched target 2 and single-base mismatched target 3 were used to compare the SNP detection capability of 1-Hg2+. Table S1 in the Supporting Information compares the fluorescence signal changes of 1-Hg2+ in the presence of the same concentration of 2 and 3. Both targets decrease the fluorescence emission of MAA-QDs, but the fluorescence signal change by 3 is about 57% compared with same amount of 2. The results reveal that the single-base mismatch detection ability of 1-Hg2+ is comparable or better than that of common MB, for which single-base mismatches result in a 50-90% signal decrease.17-19 Because of the flexibility of the metal-modulated stem-forming, the SNP detection capability of the approach could be further improved by reversible discomposing of the DNA hybrids by Hg2+ to form the T-Hg2+-T structure. To test this hypothesis, two (54) Liu, X. J.; Tan, W. H. Anal. Chem. 1999, 71, 5054–5059. (55) Chen, X. C.; Deng, Y. L.; Lin, Y.; Pang, D. W.; Qing, H.; Qu, F.; Xie, H. Y. Nanotechnology 2008, 19, 235105.

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aliquots of 1 were separately hybridized with 2 and 3 in the PBS. Two aliquots of Hg2+ and MAA-QDs were then added one after the other to the two duplex solutions. As shown in Table S1 in the Supporting Information, the fluorescence of MAA-QDs in the presence of a duplex formed by 2 and 1 was greatly quenched by Hg2+ (F/F0 ) 0.41) but that in the presence of a duplex formed by 3 and 1 was lightly decreased (F/F0 ) 0.7) by the same amount of Hg2+ ions, of which the Hg2+ quenching efficiency is about 32% in comparison with that in the case of 1 and 2. These results clearly indicate that the dsDNA formed between the capture DNA and single-base mismatched target rather than the perfectly complementary target could be discomposed by reversibly forming a T-Hg2+-T structure. This metal-modulating selectivity provides an alternative method that avoids variation of the stem length and sequence, which requires the synthesis of several different MBs. However, as it is expected, substrates that interact with Hg2+ would interfere with the DNA assay. The sensing feature of the approach depends on the amounts of the capture DNA, Hg2+ ions, and MAA-QDs presented in the solution. First, to couple a DNA hybridization event to be a measurable signal of the signaling reporter, the amount of the capture DNA should be optimized. Because of the high sensitivity of MAA-QDs to Hg2+, it needs a little 1 (∼20 nM) to be disrupted to induce MAA-QDs fluorescence quenching. However, since the response ranges of MAA-QDs for Hg2+ and thus for the DNA target depend on the amount of 1, a too low concentration of 1 leads to a very narrow Hg2+ response range, 0.2 µM 1 was used as an optimized concentration. Next, to realize the best response, optimization of the metal ion concentration was determined, at which the concentration of 1 remains constant and the amounts of Hg2+ vary. The Hg2+ fluorescence quenching efficiency in the absence and the presence of 2 are plotted as functions of the concentration of Hg2+ at pH 7.4 (Figure S6 in the Supporting Information). In the absence of 2, the formation of 1-Hg2+ complex leads to slight decrease of MAA-QDs fluorescence intensity. When the molar ratio of Hg2+ to 1 reaches around 8:1, an ∼24% fluorescence decrease was observed. In the presence of 2, formation of a duplex does displace the metal ions from 1-Hg2+ and thus enhances fluorescence quenching under the same conditions. With the molar ratio of metal-to-1 under 5.0, the quenching efficiency increases with the Hg2+ concentration and a further increase in Hg2+ concentration leads to lower quenching efficiency. We reasoned that at high Hg2+ concentration, the fluorescence of QDs will be quenched by the metal ion even if in the presence of a high concentration of 2, which, in turn, reduces the signal change. With the fixed capture DNA and Hg2+, the fluorescence quenching efficiency increases with the increase of the amount of MAA-QDs in the concentration range of 0-60 nM. When the MAA-QDs concentration exceeds 60 nM, the quenching efficiency decreases with an increase of the amount of QDs. In this regard, we should choose a minimum amount of QDs. However, a low QDs concentration leads to the small blank fluorescence and, thus, narrow response range for Hg2+. Taking into account the response sensitivity and dynamic range, 50 nM MAA-QDs was used in the final solution. 9708

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Figure 5. Advancement of the interaction of 1-Hg2+ with 2 (a) or a random ssDNA (5′-ATACTTGGGCACACCCGCA-3′) (b), respectively, as a function of time. The transition between each regime is marked with an arrow. The concentration of the target DNA was 1.5 µM, and the fluorescence intensities were recorded at 596 nm with an excitation wavelength of 388 nm.

Figure 4 shows the fluorescence signal decrease of 1-Hg2+/ MAA-QDs as a function of the concentrations of 2. Titration experiments were carried out by adding increasing amounts of 2 to the mixture of 1 and Hg2+ in the PBS. After hybridization for 10 min, 50 nM MAA-QDs was added into each solution. At these conditions, the MAA-QDs fluorescence intensity at 598 nm continued to decrease following the increase of the 2 concentration. When the 2 concentration was about 1.8 µM, the fluorescence intensity gradually leveled off (about an 83% decrease). There was a good linear relationship between the fluorescence change and the 2 concentration range from 0.1 to 1.6 µM (y ) 0.411 + 123.5C, R2 ) 0.988). The detection limit was estimated to be ∼25 nM. This DNA detection sensitivity is similar or slightly lower than those of common MBs, for which nanomolar DNA targets can be efficiently detected.22-24 However, the dependence of quenching efficiency and dynamic range on the amount of QDs allows one to tune the sensitivity of the present system. For example, when 10 nM MAAQDs was used, the detection limit was decreased to be around 2.0 nM under the same conditions. Figure 5 shows the real-time record of the interactions 1-Hg2+ with 2 and a random DNA in PBS using the MAA-QDs fluorescence emission as a function of time. Upon addition of a random DNA (5′-ATACTTGGGCACACCCGCA-3′) to the solution of 1-Hg2+ and MAA-QDs, the fluorescence intensity of MAA-QDs at 598 nm keeps almost constant (decrease around 6% within 25 min), indicating that QDs are of photostability. On the contrary, an obvious decrease of the QDs fluorescence intensity is observed with the addition of 2 to the solution. It can be seen from Figure 5 that the fluorescence quenching of MAA-QDs by 1.5 µM 2 is completed within 20 min, which is 2-3-fold longer than that in the case of common MBs.22,23 Moreover, it was observed that the DNA hybridization kinetics is related to the amount of Hg2+.39 A low Hg2+ concentration caused a rapid kinetic response, and an increase of the Hg2+ concentration reduced the DNA hybridization rate due to the high energy barrier of opening the T-Hg2+-T stem. We also studied the effect of temperature on the DNA hybridization assay. As shown in Figure S7 in the Supporting Information, the relative fluorescence change (F0/F) was only

slightly affected by the change in temperature in the range of 15-50 °C because of the increase in thermostability of the T-Hg2+-T stem, making 1-Hg2+ efficiently function under 50 °C. However, higher temperature may either destroy the T-Hg2+-T structure or affect the stability of the QDs and thus reduce the detection sensitivity. CONCLUSIONS In conclusion, we proposed here a strategy for MB-target binding readout using a separated molecular recognition element and signal reporter. The obtained sensitivity as far as SNPs discrimination is concerned and compares well with those of common MBs. This novel assay method is simple in design, avoiding any oligonucleotide modification. Moreover, this strategy is generalizable since any DNA-binding events can in principle release the signal transducer and be detected with a separated signal reporter. Although we used this approach to detect DNA hybridization based on a MAA-QDs fluorescence decrease, the strategy is versatile by selecting not only different types of competitors, such as cations56,57 and oligonucleotide,58 to avoid the toxic Hg2+ ions but also different types of signal reporters, such as the metal ion-induced fluorescence enhanced probes,59-61 (56) Ono, A.; Cao, S. Q.; Togashi, H.; Tashiro, M.; Fujimoto, T.; Machinami, T.; Oda, S.; Miyake, Y.; Itaru Okamotoa, I.; Tanaka, Y. Chem. Commun. 2008, 4825–4827. (57) Bourdoncle, A.; Estevez Torres, A.; Gosse, C.; Lacroix, L.; Vekhoff, P.; Le Saux, T.; Jullien, L.; Mergny, J.-L. J. Am. Chem. Soc. 2006, 128, 11094– 11105. (58) Grossmann, T. N.; Ro ¨glin, L.; Seitz, O. Angew. Chem., Int. Ed. 2007, 46, 5223–5225. (59) Chae, M. Y.; Czarnik, A. W. J. Am. Chem. Soc. 1992, 114, 9704–9705. (60) Ye, B. C.; Yin, B. C. Angew. Chem., Int. Ed. 2008, 47, 8386–8389. (61) Liu, L.; Zhang, G. X.; Xiang, J. F.; Zhang, D. Q.; Zhu, D. B. Org. Lett. 2008, 10, 4581–4584.

so that either signal-off or signal-on detections can be realized. However, it is worth noting that there is still plenty of room for improvement in comparison with common MBs. For example, since the signal transduction mechanism of a common MB relies on a very specific binding-induced conformational change, a fluorescence change is therefore only triggered upon hybridization of the specific DNA target to the loop, which allows applications such as in situ imaging and PCR monitoring. In addition, because of the single signal transducer (Hg2+) and signal reporter used, it is difficult to perform a multiplexing assay using this approach. Nevertheless, in view of the advantages, we expect this strategy will offer a new class of easily tunable DNA detection approaches. Further investigation is in progress in our laboratory to improve its molecular recognition characteristic by choosing an appropriate competitor and signal reporter. ACKNOWLEDGMENT Financial support from the National Outstanding Youth Foundation of China (Grant No. 20525518) and the National Natural Science Foundation of China (Grant No. 20775005) is highly acknowledged SUPPORTING INFORMATION AVAILABLE Details for the experimental section, additional spectroscopic data, and association constants. This material is available free of charge via the Internet at http://pubs.acs.org.

Received for review August 24, 2009. Accepted October 21, 2009. AC901906W

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