ARTICLE pubs.acs.org/ac
Bifunctional Colorimetric Oligonucleotide Probe Based on a G-Quadruplex DNAzyme Molecular Beacon Libing Zhang,†,‡ Jinbo Zhu,†,‡ Tao Li,†,‡ and Erkang Wang*,† †
State Key Laboratory of Electroanalytical Chemistry, Changchun Institute of Applied Chemistry, Chinese Academy of Sciences, Changchun, Jilin, 130022, P. R. China ‡ Graduate School of the Chinese Academy of Sciences, Beijing, 100039, P. R. China
bS Supporting Information ABSTRACT: A label-free bifunctional colorimetric oligonucleotide probe for DNA and protein detection has been developed on the basis of a novel catalytic molecular beacon consisting of two hairpin structures and a split G-quadruplex DNAzyme in the middle. The two loops of this molecular beacon consist of thrombin aptamer sequence and the complementary sequence of target DNA, which are utilized to sense single-stranded DNA and thrombin. The G-quadruplex DNAzyme can effectively catalyze the H2O2-mediated oxidation of 3,30 ,5,50 -tetramethylbenzidine sulfate to generate colorimetric signal. Upon addition of the target, the DNA or protein combines with one loop of the hairpin structures, and meanwhile drives the middle G-quadruplex DNAzyme to dissociate. This results in a decrease of catalytic activity, enabling the separate analysis of DNA and thrombin.
D
NAzymes are single-stranded DNA molecules of a particular sequence that possess specific catalytic activities and serve as attractive options to achieve signal amplification in DNA detection.1 They are a kind of artificial enzyme that has exhibited surprising potential as new biocatalyst.2,3 DNAzymes have been widely applied to numerous biochemical reations such as DNA or RNA cleavage,47 porphyrin metalation,8 and DNA self-modification.911 G-quadruplex DNAzyme, as one of DNAzymes, has received considerable attention in recent years. G-quadruplexes result from the association of four oligonucleotides strands held together by the hydrophobic stacking of large, planar, hydrogenbonded DNA base quartets coordinated to a monovalent cation.12 The four-stranded DNA structures are stabilized by coordination cations, e.g., K+ and Na+.13 In particular, the potassium cation can be located in the cavity between two adjacent G-tetrads of a G-quadruplex due to its appropriate size, binding to eight carbonyl oxygen atoms from the G-tetrads.14 This coordination contributes to the highest efficiency of K+ at stabilizing G-quadruplexes. Although the basic features of G-quartets have been known for more than 40 years,15 it is only in the past decade that the interest in these peculiar structures has increased, due to growing evidence for the implication of G-quadruplex structures in key biological processes.1618 Interestingly, a few of K+-stabilized G-quadruplexes (with hemin as a cofactor) exhibit superior peroxidase-like activity and effectively catalyze the H2O2-mediated oxidation of 2,20 azinobis(3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt (ABTS),1924 3,30 ,5,50 -tetramethylbenzidine sulfate (TMB),2529 or luminal,3033 accompanied by a color change or chemiluminescence emission. In comparison with protein peroxidases, the G-quadruplex-based DNAzyme can be facilely tethered to DNA r 2011 American Chemical Society
sequences or other targets,3436 serving as a novel kind of catalytic label or beacon. With this advantage, they have been employed to develop many colorimetric and/or chemiluminescence probes for the sensitive and specific detection of proteins, DNA, and other biomolecules,3437 indicating their great potential in biological applications. Molecular beacons (MBs) are oligonucleotide probes that become fluorescent upon binding to a complementary sequence of single-stranded nucleic acid.38,39 To maintain quencherfluorophore proximity in the free state, MBs were initially designed with a hairpin shape, featuring a stem-and-loop structure. The stem portion usually involves WatsonCrick hydrogen bonding between natural DNA base pairs. Since their discovery in 1996,38 MBs have been extensively used in a broad spectrum of biological applications.4045 Conventional MB design involves considerations related to both fluorescence detection and molecular recognition. An efficient detection requires choosing an appropriate fluorescent dye/quencher combination to maximize the alteration of the fluorescence emission upon target binding.46,47 Generally, the 50 - and 30 -ends of a molecular beacon are respectively labeled with the fluorophore and the quencher. However, the employment of fluorescent label and quencher tethered to MB results in not only a high cost of operation but also potentially complex processes. In this paper, we combine G-quadruplex DNAzyme with the MB technique to develop a novel bifunctional colorimetric oligonucleotide probe for DNA and thrombin detection. It contains two hairpin Received: March 17, 2011 Accepted: October 21, 2011 Published: October 21, 2011 8871
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Analytical Chemistry loops that serve as the sensing elements, and a split G-quadruplex DNAzyme is employed as the reporter, which avoids any labeling of oligonucleotides with other indicators (see Scheme 1). The oligonucleotide forms double stem-loops and a G-quadruplex structure in the absence of targets, yielding a catalytically active DNAzyme that promotes a process that leads to generation of a colorimetric signal. In the presence of targets, the loops of the hairpin structures are combined and disrupted, which induces the G-quadruplex DNAzyme to dissociate and causes a decrease of catalytic activity. The two loops of bifunctional DNAzyme molecular beacon consist of a complementary sequence for target DNA and thrombin aptamer sequence, which acts as an active assembly for the separate analysis of nucleic acids and thrombin.
’ EXPERIMENTAL SECTION Materials and Apparatus. All oligonucleotides were purchased from Sangon Biotechnology Co., Ltd. (Shanghai, China), and their sequences are listed in Table 1. The stock solution of oligonucleotides were prepared in 25 mM Tris-Ac buffer and accurately quantified using UVvis absorption spectroscopy with the following extinction coefficients (ε260 nm, M‑1 cm‑1) for each nucleotide: A = 15 400, G = 11 500, C = 7400, T = 8700. Hemin was purchased from Sangon Biotechnology Co., Ltd. (Shanghai, China). The stock solution of hemin (5 μM) was prepared in dimethyl sulfoxide (DMSO), stored in darkness at 20 °C. Before use, the oligonucleotides and hemin solutions were diluted to required concentrations with the working buffer. Thrombin, bovine serum albumin (BSA), lysozyme, Triton X-100, and TMB were purchased from Sigma-Aldrich (St. Louis, MO). Other chemicals were reagent grade
Scheme 1. Schematic Representation of the Bifunctional Oligonucleotide Probe for Colorimetric Analysis of Target DNA or Thrombina
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and were used without further purification. Solutions were prepared with deionized water processed with a Milli-Q ultrahigh purity water system (Millipore, Bedford, MA). UVvis absorbance measurements were performed on a Cary 500 Scan UVvisNIR spectrophotometer (Varian). Procedure of Assay. In a typical DNA assay, the DNAzyme molecular beacon (50 μL, 1.0 μM) was hybridized with the target in 25 mM Tris-Ac buffer, and the solution was heated at 88 °C for 10 mi and gradually cooled to room temperature. An equal volume of the 2 HEPES buffer (50 mM HEPES, pH 7.4, 40 mM KCl, 400 mM NaCl, 0.1% Triton X-100, 2% DMSO) was added to the DNA solutions, and the DNA sequences were allowed to fold for 40 min at room temperature. Then, 10 μL of hemin (5.0 μM) solutions was added, and the mixture was incubated at room temperature for 1 h to form the heminG-quadruplex DNAzymes. In the thrombin assay, the DNAzyme molecular beacon (50 μL, 1.0 μM) was heated at 88 °C for 10 min and gradually cooled to room temperature. Then, an appropriate concentration of thrombin was added into the DNA solution, and the mixture was incubated at 37 °C for 40 min, allowing thrombin to bind specifically to its aptamer properly. An equal volume of the 2 HEPES buffer (50 mM HEPES, pH 7.4, 40 mM KCl, 400 mM NaCl, 0.1% Triton X-100, 2% DMSO) was added to the DNA solutions, and the DNA sequences were allowed to fold for 40 min at room temperature. Then, 10 μL of hemin (5.0 μM) solutions was added, and the mixture was incubated at room temperature for 1 h to form the heminG-quadruplex DNAzymes. Colorimetric Analysis. Colorimetric analysis utilizing G-quadruplex DNAzyme was performed in the TMBH2O2 reaction system at room temperature. In a typical experiment, the peroxidation reaction was initiated by the addition of 390 μL of buffer (26.6 mM citrate, 51.4 mM disodium hydrogen phosphate, 20 mM KCl, pH 5.0), 5 μL of 0.5% (w/v) TMB, and 5 μL of 3% (w/v) H2O2 to the prepared assay solution. After 5 min, 400 μL of 2.0 M H2SO4 was added into the solution to stop the reaction. UVvis spectra were collected and the photos were taken immediately after stopping the reaction.
’ RESULTS AND DISCUSSION
a
Region I is the complementary sequence for target DNA, region II is the thrombin aptamer sequence, and region III is the G-rich DNA sequence.
Principle of Operation. To test the feasibility of this approach, the catalytic ability of the bifunctional oligonucleotide probe is tested in different conditions. The DNAzyme molecular beacon fold into double stem-loops and forms G-quadruplex structure in solution, and the formed quadruplex structure binds hemin with high affinity,30 resulting in a complex that mimics the horseradish peroxidase and catalyzes the H2O2-mediated oxidation of TMB to generate color change. As shown in Figure 1, curve a is the UVvis
Table 1. Oligonucleotide Sequences Used in This Work
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Figure 1. UVvis absorption spectra of (a) P1 catalyzes TMBH2O2 in the free state, (b) P1 catalyzes TMBH2O2 in the presence of 1.0 μM target DNA, (c) P1 catalyzes TMBH2O2 in the presence of 5.0 μM thrombin, and (d) TMBH2O2 system. The inset is the corresponding photographs which show the color change.
absorbance spectra of the DNAzyme molecular beacon in the absence of target. Photograph a shows the color of the solution in the same situation. Whereas, in the presence of target, the DNA or thrombin combines with one loop of the hairpin structures and meanwhile drives the middle G-quadruplex DNAzyme to dissociate, resulting in a decrease of catalytic activity. Curves b and c are the UVvis absorbance spectra of the DNAzyme molecular beacon in the presence of 1.0 μM DNA and 5.0 μM thrombin, respectively. It is well-known that thrombin binds with its aptamer to form G-quadruplex structure, but this cannot influence the experimental results due to the low catalytic ability of the formed G-quadruplex structure (see Figure S1 in the Supporting Information). This is attributed to the complexes formed by hemin and parallel G-quadruplexes having much higher peroxidase activity than those formed by hemin and antiparallel G-quadruplexes.48,49 Curve d is the background signal of the H2O2TMB system. By comparing the results, we can conclude that UVvis absorbance changes of the system induced by the analyte constitute the basis for colorimetric assay using DNAzyme molecular beacon. Affect of the P1 Concentration and Reaction Time on Absorbance. For evaluation of validity of the proposed principle, the effect of different concentrations of P1 (Table 1) on the absorbance was first investigated. Absorbance (A450) was recorded in the presence of different concentrations of P1 from 10 to 300 nM (see Figure S2 in the Supporting Information). A450 is the absorbance of the H2O2TMB system in the presence of P1 at 450 nm. As the concentration of P1 increases, the absorbance is increased. This indicates that P1 forms a G-quadruplex structure which binds hemin with high affinity, resulting in a complex that mimics the horseradish peroxidase and catalyzes the H2O2-mediated oxidation of TMB. However, the concentration of P1 is greater than 100 nM, and the absorbance is not increased obviously. Therefore, 100 nM P1 was used in the whole experiments. To obtain the optimum responding time for colorimetric assay, we have recorded the relationship between A0 450 (A450/ Abackground,450) and responding time. Abackground,450 is the absorbance of the H2O2TMB system at 450 nm. As shown
Figure 2. Utilization of P1 for colorimetric DNA analysis in the TMBH2O2 system: (a) UVvis absorption spectra for utilizing P1 to analyze different concentrations of target DNA: 0 nM (curve a), 10 nM (curve b), 50 nM (curve c), 100 nM (curve d), 500 nM (curve e), and 1.0 μM (curve f). The inset is the corresponding photographs, which show the color change. (b) The relationship between the absorbance and the concentration of DNA. The inset shows the dependence of the absorbance at 450 nm (A450) on the logarithm of DNA concentration; the linear range is from 108 to 106 M.
in Figure S3 (in the Supporting Information), we can see that the A0 450 increased quickly and then decreased obviously. There is a high value at 5 min; therefore, 5 min is chosen as the optimum responding time for colorimetric assay in the whole experiment. Colorimetric Analysis of Nucleic Acids. For the target DNA detection using the bifunctional oligonucleotide probe, in the G-quadruplex DNAzyme catalyzed TMBH2O2 reaction system, different concentrations of target DNA T1 (Table 1) were analyzed via monitoring the change in absorption spectrum of the colored product TMB+, which has a maximal absorption of 450 nm (see Figure 2a). In the absence of T1, DNAzyme molecular beacon has a superior catalytic activity toward the H2O2-mediated oxidation of TMB, reflected by a strong absorbance (see curve a in Figure 2a). Upon the addition of an increasing amount of T1, there is a gradual decrease in readout signal. This result indicates that the target DNA combines with one loop of the hairpin structures and disrupts the G-quadruplex DNAzyme formed in the 8873
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Figure 3. Selectivity of the DNAzyme molecular beacon analyzing target DNA T1 (1.0 μM) over other mismatched DNA (1.0 μM): T2 (one-base mismatched target), T3 (two-base mismatched target), and T4 (four-base mismatched target). The inset is the corresponding photographs, which show the color change.
middle, resulting in a decrease of catalytic activity. An obvious absorbance change is observed when 10 nM target DNA is added and incubated (see Figure 2a). The detection limit, which is defined as 3 times the standard deviation of the blank measurements, is estimated to be 5.4 nM. Figure 2b outlines the relationship between the target DNA concentration and absorbance at 450 nm, indicating a dependence of A450 on the target DNA concentration. From this plot, it is observed that the absorbance decreases relatively obviously as target DNA concentration increases up to 106 M. The inset shows the calibration curve for quantitative analysis of target DNA, the absorbance is linearly dependent on the logarithm of target DNA concentration in the range from 108 to 106 M (R = 0.994). The above observations demonstrate that the G-quadruplex DNAzyme molecular beacon can serve as a novel colorimetric oligonucleotide probe for analysis of target DNA. To test the specificity of colorimetric oligonucleotide probe for analysis of target DNA, the similar DNA strands with only one (T2), two (T3), and four (T4) nucleotides mismatches were also investigated. The sequences of these mismatched DNA strands are shown in Table 1. Figure 3 shows the A00 450 (A450 Abackground,450) of target DNA and other mismatched DNA strands. In the presence of T1, the A00 450 decreased obviously, this is because T1 can hybridize with binding site and disrupt the G-quadruplex structure, which prevents catalyzing the H2O2mediated oxidation of TMB to generate color change. In contrast, other mismatched DNA make the A00 450 decrease a little. These data demonstrate that the approach shows a high selectivity toward the target DNA, and even one-nucleotide mismatched could be distinguished. Additionally, the length of DNA can be detected with this bifunctional oligonucleotide probe; here we test a series of DNA with different number of bases (Table S1 in Supporting Information). Figure S4 (in Supporting Information) shows the A00 450 of the bifunctional oligonucleotide probe response to different DNA and their corresponding single-base mismatched DNA strands. It is observed that the absorbance of DNA with 1721 bases can be distinguished with their corresponding singlebase mismatched DNA strands. It means that if the single-base
Figure 4. Utilization of P1 for colorimetric thrombin analysis in the TMBH2O2 system: (a) UVvis absorption spectra for utilizing P1 to analyze different concentrations of thrombin: 0 nM (curve a), 50 nM (curve b), 100 nM (curve c), 200 nM (curve d), 500 nM (curve e), 1.0 μM (curve f), 2.0 μM (curve g) and 5.0 μM (curve h). The inset is the corresponding photographs, which show the color change. (b) The relationship between the absorbance and the concentration of thrombin. The inset shows dependence of the absorbance at 450 nm (A450) on the logarithm of thrombin concentration; the linear range is from 5 108 to 5 106 M.
mismatch happened in DNA with a length in this range, it can be detected using this technique. Colorimetric Detection of Thrombin. To demonstrate the versatility of this designed oligonucleotide probe, we used the bifunctional oligonucleotide probe to detect thrombin via thrombin specifically bound to its aptamer,50 disrupting the G-quadruplex structure. For the sensitivity study, different concentrations of thrombin solution were investigated. Figure 4a shows the absorbance of DNAzyme molecular beacon in the presence of thrombin from 5 108 to 5 106 M. As the concentration of thrombin increases, the absorbance is decreased obviously. This result illuminates that the thrombin combines with its aptamer, which consisted of one loop of the hairpin structures and dissociated the G-quadruplex DNAzyme formed in the middle, resulting in the loss of catalytic activity. However, after the 50 nM thrombin was added and incubated, an obvious absorbance drop at 450 nm was observed (see curve b in Figure 4a), and the detection limit is 20.5 nM (3 times the standard deviation of the blank measurements). 8874
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Analytical Chemistry Figure 4b shows the relationship between the absorbance and the concentration of DNA. Upon the addition of an increasing amount of thrombin, the absorbance is decreased obviously. The inset shows the calibration curve for quantitative analysis of thrombin. The absorbance is linearly dependent on the logarithm of thrombin concentration in the range from 5 108 to 5 106 M (R = 0.997). The data demonstrate that the G-quadruplex DNAzyme molecular beacon can serve as a novel colorimetric oligonucleotide probe for detection of thrombin. To test the selectivity of the DNAzyme molecular beacon for thrombin analysis, control experiments were done using BSA and lysozyme. As shown in Figure 5, only thrombin makes the A00 450 decrease obviously. In the presence of other two proteins, there is nearly on absorbance change, implying that other proteins not interact with thrombin aptamer and thus not interfere with the detection of thrombin. This result indicates that thrombin binds to its aptamer specifically and the bifunctional oligonucleotide probe shows a high selectivity toward thrombin, G-quadruplex DNAzyme molecular beacon can serve as a novel bifunctional oligonucleotide probe for detection of thrombin. Differentiation of the Optical Signal. The introduced labelfree bifunctional oligonucleotide probe can analyze separately the two different analytes, target DNA and thrombin. Both targets create the same type of optical signal, and the bifunctional oligonucleotide probe is unable to determine whether the signal is induced by the presence of DNA or thrombin, or a mixture of both. However, it should be noted that the introduction of a fully complementary oligonucleotide sequence (Table S1 in Supporting Information) corresponding to T1 that folds into a doublestranded structure can inhibit the target DNA by combining with one loop of P1, generating half of a self-blocked bifunctional oligonucleotide probe (process 1). Moreover, thrombin will be denatured after heat treatment, resulting in the loss of the activity. The denatured thrombin cannot combine with its aptamer, generating another half of a self-blocked bifunctional oligonucleotide probe (process 2). The results are shown in Figure S5 (in Supporting Information). Through the further two processing, the bifunctional oligonucleotide probe is able to determine the signal induced in the presence of DNA or thrombin, or a mixture of both. When the signal got down after the addition of sample, we could first treat it according to process 1. If the absorbance returned to a high value, it would show that the sample only contains target DNA. However, if the absorbance remained at a low level, it would ensure that there is thrombin in it. To figure out the existence of target DNA in it, we should heat another sample with probe in the way of process 2. If the absorbance reverted to a high level, it would show that the sample only contains thrombin; if the absorbance is still low, it shows the sample contains a mixture of DNA and thrombin.
’ CONCLUSION This study has introduced a label-free bifunctional oligonucleotide probe for the separate colorimetric analysis of two different analytes, target DNA and thrombin, since the two loops of bifunctional oligonucleotide probe consist of complementary sequence for target DNA and thrombin aptamer sequence. In the strategy, the DNAzyme molecular beacon forms two hairpin structures and a split G-quadruplex DNAzyme in the middle, yielding an active DNAzyme with high catalytic activity. The presence of target promotes the dissociation of the G-quadruplex DNAzyme, resulting in the loss of catalytic activity. Through this
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Figure 5. Selectivity of the DNAzyme molecular beacon analyzing thrombin (5.0 μM) over BSA (10 μM) and lysozyme (10 μM). The inset is the corresponding photographs, which show the color change.
detection strategy, limits of detection of 5.4 nM for target DNA and 20.5 nM for thrombin analysis are achieved, respectively. The bifunctional oligonucleotide probe showed a high selectivity toward target DNA and thrombin over their corresponding analogs. In addition, this bifunctional colorimetric oligonucleotide probe has some unique features: (1) it can separate analysis of DNA and thrombin simply and rapidly and (2) it avoids the fluorescence label for oligonucleotides, with G-quadruplex DNAzyme as a catalytic unit to oxidize the H2O2TMB system for signal readout. The results of this study should substantially broaden the perspective for future development of oligonucleotide probe for analysis of other analytes.
’ ASSOCIATED CONTENT
bS
Supporting Information. Additional information as noted in the text. This material is available free of charge via the Internet at http://pubs.acs.org.
’ AUTHOR INFORMATION Corresponding Author
*Fax: +86-431-85689711. Tel: +86-431-85262003. E-mail: ekwang@ ciac.jl.cn.
’ ACKNOWLEDGMENT The work was supported by the National Natural Science Foundation of China with grant numbers of 21075120 and 20890020, the 973 Project 2009CB930100 and 2010CB933600. ’ REFERENCES (1) Fu, R. Z.; Li, T. H.; Lee, S. S.; Park, H. G. Anal. Chem. 2011, 83, 494–500. (2) Breaker, R. R. Nat. Biotechnol. 1997, 15, 427–431. (3) Emilsson, G. M.; Breaker, R. R. Cell. Mol. Life Sci. 2002, 59, 596–607. (4) Burmeister, J.; von Kiedrowski, G.; Ellington, A. D. Chem. Int. Ed. Engl. 1997, 36, 1321–1324. (5) Carmi, N.; Balkhi, S. R.; Breaker, R. R. Proc. Natl. Acad. Sci. U. S. A. 1998, 95, 2233–2237. (6) Li, J.; Zheng, W.; Kwon, A. H.; Lu, Y. Nucleic Acids Res. 2000, 28, 481–488. 8875
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Analytical Chemistry (7) Santoro, S. W.; Joyce, G. F. Proc. Natl. Acad. Sci. U. S. A. 1997, 94, 4262–4266. (8) Li, Y.; Sen, D. Nat. Struct. Biol. 1996, 3, 743–747. (9) Li, Y.; Breaker, R. R. Proc. Natl. Acad. Sci. U. S. A. 1999, 96, 2746–2751. (10) Li, Y.; Liu, Y.; Breaker, R. R. Biochemistry 2000, 39, 3106–3114. (11) Sheppard, T. L.; Ordoukhanian, P.; Joyce, G. F. Proc. Natl. Acad. Sci. U. S. A. 2000, 97, 7802–7807. (12) 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. (13) Williamson, J. R. Annu. Rev. Biophys. Biomol. Struct. 1994, 23, 703–730. (14) Sundquist, W. I.; Klug, A. Nature 1989, 342, 825–829. (15) Gellert, M.; Lipsett, M. N.; Davies, D. R. Proc. Natl. Acad. Sci. U. S. A. 1962, 48, 2013–2018. (16) Rangan, A.; Fedoroff, O. Y.; Hurley, L. H. J. Biol. Chem. 2001, 276, 4640–4646. (17) Duquette, M. L.; Handa, P.; Vincent, J. A.; Taylor, A. F.; Maizels, N. Genes Dev. 2004, 18, 1618–1629. (18) Paeschke, K.; Simonsson, T.; Postberg, J.; Rhodes, D.; Lipps, H. J. Nat. Struct. Mol. Biol. 2005, 12, 847–854. (19) Li, T.; Dong, S. J.; Wang, E. K. Chem. Commun. 2007, 4209–4211. (20) Travascio, P.; Bennet, A. J.; Wang, D. Y.; Sen, D. Chem. Biol. 1999, 6, 779–787. (21) Li, T.; Wang, E. K.; Dong, S. J. Chem. Commun. 2008, 3654–3656. (22) Travascio, P.; Li, Y.; Sen, D. Chem. Biol. 1998, 5, 505–517. (23) Li, T.; Wang, E. K.; Dong, S. J. Chem. Commun. 2009, 580–582. (24) Li, T.; Wang, E. K.; Dong, S. J. J. Am. Chem. Soc. 2009, 131, 15082–15083. (25) Li, T.; Li, B. L.; Wang, E. K.; Dong, S. J. Chem. Commun. 2009, 3551–3553. (26) Li, B. L.; Du, Y.; Li, T.; Dong, S. J. Anal. Chim. Acta 2009, 651, 234–240. (27) Yang, X.; Li, T.; Li, B. L.; Wang, E. K. Analyst 2010, 135, 71–75. (28) Zhu, J. B.; Li, T.; Hu, J. M.; Wang, E. K. Anal. Bioanal. Chem. 2010, 397, 2923–2927. (29) Du, Y.; Li, B. L.; Guo, S. J.; Zhou, Z. X.; Zhou, M.; Wang, E. K.; Dong, S. J. Analyst 2011, 136, 493–497. (30) Pavlov, V.; Xiao, Y.; Gill, R.; Dishon, A.; Kotler, M.; Willner, I. Anal. Chem. 2004, 76, 2152–2156. (31) Li, T.; Wang, E.; Dong, S. Chem. Commun. 2008, 5520–5522. (32) Niazov, T.; Pavlov, V.; Xiao, Y.; Gill, R.; Willner, I. Nano Lett. 2004, 4, 1683–1687. (33) Xiao, Y.; Pavlov, V.; Gill, R.; Bourenko, T.; Willner, I. ChemBioChem 2004, 5, 374–379. (34) Li, D.; Shlyahovsky, B.; Elbaz, J.; Willner, I. J. Am. Chem. Soc. 2007, 129, 5804–5805. (35) Shlyahovsky, B.; Li, D.; Katz, E.; Willner, I. Biosens. Bioelectron. 2007, 22, 2570–2576. (36) Xiao, Y.; Pavlov, V.; Niazov, T.; Dishon, A.; Kotler, M.; Willner, I. J. Am. Chem. Soc. 2004, 126, 7430–7431. (37) Cheglakov, Z.; Weizmann, Y.; Basnar, B.; Willner, I. Org. Biomol. Chem. 2007, 5, 223–225. (38) Tyagi, S.; Kramer, F. R. Nat. Biotechnol. 1996, 14, 303–308. (39) Marras, S. A. E. Methods Mol. Biol. 2006, 335, 3–16. (40) Broude, N. E. Trends Biotechnol. 2002, 20, 249–256. (41) Fang, X. H.; Mi, Y. M.; Li, J. W. J.; Beck, T.; Schuster, S.; Tan, W. H. Cell Biochem. Biophys. 2002, 37, 71–81. (42) Li, J. W. J.; Fang, X. H.; Tan, W. H. Biochem. Biophys. Res. Commun. 2002, 292, 31–40. (43) Tan, W.; Wang, K.; Drake, T. J. Curr. Opin. Chem. Biol. 2004, 8, 547–553. (44) Roy, S.; Kabir, M.; Mondal, D.; Ali, I. K.; Petri, W. A., Jr.; Haque, R. J. Clin. Microbiol. 2005, 43, 2168–2172. (45) Yang, R. H.; Jin, J. Y.; Long, L. P.; Wang, Y. X.; Wang, H.; Tan, W. H. Chem. Commun. 2009, 322–324.
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(46) Dubertret, B.; Calame, M.; Libchaber, A. Nat. Biotechnol. 2001, 19, 365–370. (47) Marras, S. A. E.; Kramer, F. R.; Tyagi, S. Nucleic Acids Res. 2002, 30, e122. (48) Cheng, X. H.; Liu, X. J.; Bing, T.; Cao, Z. H.; Shangguan, D. H. Biochemistry 2009, 48, 7817–7823. (49) Kong, D. M.; Yang, W.; Wu, J.; Li, C. X.; Shen, H. X. Analyst 2010, 135, 321–326. (50) Tasset, D. M.; Kubik, M. F.; Steiner, W. J. Mol. Biol. 1997, 272, 688–698.
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