Label-Free Colorimetric Detection of Aqueous Mercury Ion (Hg2+)

Feb 19, 2009 - materials,14,15 and proteins.16 Among these sensors, many are constructed with .... G-quadruplex motif,34 which gives Hg2+ an access to...
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Anal. Chem. 2009, 81, 2144–2149

Label-Free Colorimetric Detection of Aqueous Mercury Ion (Hg2+) Using Hg2+-Modulated G-Quadruplex-Based DNAzymes Tao Li, Shaojun Dong, and Erkang Wang* State Key Laboratory of Electroanalytical Chemistry, Changchun Institute of Applied Chemistry, Chinese Academy of Sciences, Changchun, Jilin, 130022, China, and Graduate School of the Chinese Academy of Sciences, Beijing, 100039, China Mercury ion (Hg2+) is able to specifically bind to the thymine-thymine (T-T) base pair in a DNA duplex, thus providing a rationale for DNA-based selective detection of Hg2+ with various means. In this work, we for the first time utilize the Hg2+-mediated T-T base pair to modulate the proper folding of G-quadruplex DNAs and inhibit the DNAzyme activity, thereby pioneering a facile approach to sense Hg2+ with colorimetry. Two bimolecular DNA G-quadruplexes containing many T residues are adopted here, which function well in low- and high-salt conditions, respectively. These G-quadruplex DNAs are able to bind hemin to form the peroxidase-like DNAzymes in the folded state. Upon addition of Hg2+, the proper folding of Gquadruplex DNAs is inhibited due to the formation of T-Hg2+-T complex. This is reflected by the notable change of the Soret band of hemin when investigated by using UV-vis absorption spectroscopy. As a result of Hg2+ inhibition, a sharp decrease in the catalytic activity toward the H2O2-mediated oxidation of 2,2′azino-bis(3-ethylbenzothiazoline-6-sulfonic acid)diammonium salt (ABTS) is observed, accompanied by a change in solution color. Through this approach, aqueous Hg2+ can be detected at 50 nM (10 ppb) with colorimetry in a facile way, with high selectivity against other metal ions. These results indicate our introduced label-free method for colorimetric Hg2+ detection is simple, quantitative, sensitive, and highly selective. Mercury is a widespread heavy metal in the environment, with high toxicity and severe adverse effect on human health.1 The mercury pollution comes from diverse sources including nature and human activities. It is estimated that the total mercury released into the environment reaches to ∼7500 tons per year.2,3 Water-soluble divalent mercuric ion (Hg2+) is one of the most usual and stable form of mercury pollution. Therefore, envi* Corresponding author. Erkang Wang, State Key Laboratory of Electroanalytical Chemistry, Changchun Institute of Applied Chemistry, Chinese Academy of Sciences, Changchun, Jilin, 130022, China. Fax: (+86) 431-85689711. Phone: (+86) 431-85262003. E-mail: [email protected]. (1) Nolan, E. M.; Lippard, S. J. Chem. Rev. 2008, 108, 3443–3480. (2) Liu, J.; Lu, Y. Angew. Chem., Int. Ed. 2007, 46, 7587–7590. (3) Chiang, C. K.; Huang, C. C.; Liu, C. W.; Chang, H. T. Anal. Chem. 2008, 80, 3716–3721.

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ronmental monitoring of aqueous Hg2+ becomes an increasing demand. To meet this goal, a number of highly sensitive and selective Hg2+ sensors have been developed, based on gold nanoparticles,4-11 fluorophores,3,12 DNAzymes,2,8,13 polymer materials,14,15 and proteins.16 Among these sensors, many are constructed with thymine (T) containing oligonucleotides as the sensing elements. It is well-known that Hg2+ can bind to two T residues of DNA to form the T-Hg2+-T complex.17,18 This Hg2+-mediated T-T base pair is able to direct the folding of single-stranded DNAs into duplexes,3,7,8,14 strengthen DNA duplexes,4,5,9,10 and activate DNAzymes2 or DNA-based machines.8 Thus, it provides a rationale for the utilization of T-containing DNA sequences to specifically sense aqueous Hg2+ with diverse means.2-5,7-12,14 In most cases, an appropriate probe (e.g., gold nanoparticle or fluorophore) is tethered to oligonucleotides to indicate color or light intensity change. This means offers some advantages, but it is cost- and time-consuming. In contrast, a labelfree approach is simple and rapid, without any step of modification or labeling. So, it is interesting and significant to develop a facile effective method for aqueous Hg2+ detection. G-quadruplex-based DNAzymes formed by hemin and DNA G-quadruplexes are a kind of nucleic acid enzymes with the (4) Lee, J. S.; Han, M. S.; Mirkin, C. A. Angew. Chem., Int. Ed. 2007, 46, 4093– 4096. (5) Lee, J. S.; Mirkin, C. A. Anal. Chem. 2008, 80, 6805–6808. (6) Darbha, G. K.; Singh, A. K.; Rai, U. S.; Yu, E.; Yu, H. T.; Ray, P. C. J. Am. Chem. Soc. 2008, 130, 8038–8043. (7) Liu, C. W.; Hsieh, Y. T.; Huang, C. C.; Lin, Z. H.; Chang, H. T. Chem. Commun. 2008, 2242–2244. (8) Li, D.; Wieckowska, A.; Willner, I. Angew. Chem., Int. Ed. 2008, 47, 3927– 3931. (9) Xue, X.; Wang, F.; Liu, X. J. Am. Chem. Soc. 2008, 130, 3244–3245. (10) Ye, B. C.; Yin, B. C. Angew. Chem., Int. Ed. 2008, 47, 8386–8389. (11) He, S.; Li, D.; Zhu, C.; Song, S.; Wang, L.; Long, Y.; Fan, C. Chem. Commun. 2008, 4885–4887. (12) Wang, Z.; Lee, J. H.; Lu, Y. Chem. Commun. 2008, 6005–6007. (13) Hollenstein, M.; Hipolito, C.; Lam, C.; Dietrich, D.; Perrin, D. M. Angew. Chem., Int. Ed. 2008, 47, 4346–4350. (14) Liu, X. F.; Tang, Y. L.; Wang, L. H.; Zhang, J.; Song, S. P.; Fan, C.; Wang, S. Adv. Mater. 2007, 19, 1471–1474. (15) Zhao, Y.; Zhong, Z. J. Am. Chem. Soc. 2006, 128, 9988–9989. (16) Chen, P.; He, C. J. Am. Chem. Soc. 2004, 126, 728–729. (17) 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. (18) Tanaka, Y.; Oda, S.; Yamaguchi, H.; Kondo, Y.; Kojima, C.; Ono, A. J. Am. Chem. Soc. 2007, 129, 244–245. 10.1021/ac900188y CCC: $40.75  2009 American Chemical Society Published on Web 02/19/2009

peroxidase-like activity,19,20 which are able to effectively catalyze the H2O2-mediated oxidation of 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid)diammonium salt (ABTS)19-22 or luminol.23-25 To date, such DNAzymes have been utilized for the colorimetric and/or chemiluminescence detection of a number of analytes including metal ions,8,26 small molecules,27,28 DNA21,23,29,30 and proteins,22,24,31,32 showing their considerable application potential in the analytical field. In our previous work,32 a G-quadruplex DNA aptamer called AGRO100, which is originally selected as a potent anticancer aptamer,33 has proved to strongly bind hemin to form an excellent DNAzyme. Furthermore, we find this G-quadruplex aptamer is highly sensitive for K+ even at the submicromolar level.26 This implies that AGRO100 functions well under low-salt conditions in the presence of a very small amount of K+. In particular, in addition to G residues, AGRO100 contains many T residues with the ability to complex with Hg2+. According to these unique properties of AGRO100, we hypothesize that this G-quadruplex aptamer may be applicable to the DNAzyme-based detection of Hg2+. With this idea in mind, here we investigate the inhibitory effect of Hg2+ on the G-quadruplex-based DNAzyme via Hg2+-mediated formation of T-T base pairs, aiming to develop a novel method for aqueous Hg2+ detection. The oligonucleotide AGRO100, d(GGTGGTGGTGGTTGTGGTGGTGGTGG), is able to fold into a bimolecular G-quadruplex structure with many T loop residues.33 This enables Hg2+ to inhibit the proper folding of AGRO100 due to its specific and strong binding to T residues. In this case, the formation of hemin-AGRO100 DNAzyme found previously26,32 is no longer allowed. Another bimolecular G-quadruplex (herein referred to as G4-2S) with excellent DNAzyme function is also reported.34 This G-quadruplex is formed by two G-rich single-stranded DNAs d(ATAGGGACGGG) and d(TTTGTGGAGGG), designated S1 and S2, respectively. G4-2S has a T-rich DNA duplex flanking the G-quadruplex motif,34 which gives Hg2+ an access to modulate the hemin-G-quadruplex DNAzyme via Hg2+-mediated T-T base pairs. Furthermore, G4-2S binds K+ not so strongly as AGRO100, thus it is thought to be suitable for the detection of Hg2+ in high-salt aqueous solution. (19) Travascio, P.; Li, Y.; Sen, D. Chem. Biol. 1998, 5, 505–517. (20) Travascio, P.; Bennet, A. J.; Wang, D. Y.; Sen, D. Chem. Biol. 1999, 6, 779–787. (21) Li, T.; Dong, S.; Wang, E. Chem. Commun. 2007, 4209–4211. (22) Li, T.; Wang, E.; Dong, S. Chem. Commun. 2008, 3654–3656. (23) Pavlov, V.; Xiao, Y.; Gill, R.; Dishon, A.; Kotler, M.; Willner, I. Anal. Chem. 2004, 76, 2152–2156. (24) Li, T.; Wang, E.; Dong, S. Chem. Commun. 2008, 5520–5522. (25) Li, T.; Du, Y.; Wang, E. Chem. Asian J. 2008, 3, 1942–1948. (26) Li, T.; Wang, E.; Dong, S. Chem. Commun. 2009, 580–582. (27) Li, D.; Shlyahovsky, B.; Elbaz, J.; Willner, I. J. Am. Chem. Soc. 2007, 129, 5804–5805. (28) Elbaz, J.; Shlyahovsky, B.; Li, D.; Willner, I. ChemBioChem 2008, 9, 232– 239. (29) Xiao, Y.; Pavlov, V.; Niazov, T.; Dishon, A.; Kotler, M.; Willner, I. J. Am. Chem. Soc. 2004, 126, 7430–7431. (30) Xiao, Y.; Pavlov, V.; Gill, R.; Bourenko, T.; Willner, I. ChemBioChem 2004, 5, 374–379. (31) Shlyahovsky, B.; Li, D.; Katz, E.; Willner, I. Biosens. Bioelectron. 2007, 22, 2570–2576. (32) Li, T.; Shi, L.; Wang, E.; Dong, S. Chem.sEur. J. 2009, 15, 1036–1042. (33) Girvan, A. C.; Teng, Y.; Casson, L. K.; Thomas, S. D.; Juliger, S.; Ball, M. W.; Klein, J. B.; Pierce, W. M., Jr.; Barve, S. S.; Bates, P. J. Mol. Cancer Ther. 2006, 5, 1790–1799. (34) Li, T.; Wang, E.; Dong, S. Chem.sEur. J. 2009, 15, 2059–2063.

On the basis of the above rationale, in this work, we utilize AGRO100 and G4-2S to specifically sense aqueous Hg2+ with colorimetry. The interactions between G-quadruplex DNAs and hemin in the absence and presence of Hg2+ are investigated by using UV-vis absorption spectroscopy to indicate the changes of the hemin Soret band and thus reflect Hg2+ inhibition on hemin binding. The effect of Hg2+ is further characterized in the ABTS-H2O2 reaction system. The experimental observations confirm the feasibility of the quantitative analysis of aqueous Hg2+ via modulating G-quadruplex-based DNAzymes by Hg2+. High selectivity and sensitivity for labelfree Hg2+ detection are achieved.

EXPERIMENTAL SECTION Oligonucleotides and Chemicals. Purified oligonucleotides and hemin (from bovine) were obtained from Sangon Biotechnology Co., Ltd. (Shanghai, China). ABTS and 2,6-pyridinedicarboxylic acid (PDCA) were purchased from Sigma-Aldrich (St. Louis, MO). The used metal salts (Hg(Ac)2, Pb(Ac)2, CdCl2, CrCl3, FeCl3, FeSO4, Mg(Ac)2, Ca(Ac)2, Co(Ac)2, Zn(Ac)2, and Cu(Ac)2) and 30% H2O2 were purchased from Sinopharm Group Chemical Reagent Co., Ltd. (Shanghai, China). All reagents were used as received without further purification. Before use, the oligonucleotides were dissolved in 25 mM Tris-Ac buffer (pH 8.0) and quantified by using UV-vis absorption spectroscopy with the following extinction coefficients (ε260nm, M-1 cm-1): A ) 15 400, G ) 11 500, C ) 7 400, T ) 8 700. The stock solution of hemin (5 mM) was prepared in DMSO, stored in the dark at -20 °C, and diluted to the required concentration with aqueous buffer. Instrumentation. A Cary 500 scan UV-vis-NIR spectrophotometer (Varian) was used to record the Soret band of hemin and the absorption spectra of the cationic free radical ABTS•+ (the product of ABTS oxidation by H2O2) at room temperature. Preparation of the Hemin-G-Quadruplex Complexes Modulated by Hg2+. The AGRO100 solution (in 25 mM Tris-Ac, pH 8.0) was heated at 88 °C for 10 min and gradually cooled to room temperature. Then, to this solution was added an equal volume of different concentrations of Hg2+ (in Tris-Ac buffer). The mixture was allowed to incubate at room temperature for 20 min, followed by the addition of an equal volume of the lowsalt buffer (25 mM Tris-Ac, pH 8.0, 0.5 mM KAc, 0.05% (w/v) Triton X-100). After AGRO100 folding for 40 min, an equivalent of hemin was added and incubated with it for 1 h to form the hemin-AGRO100 complex. In another case, the solutions of S1 and S2 were mixed at a 1:1 molar ratio. After heated at 88 °C for 10 min and then gradually cooled to room temperature, this mixture was mixed with an equal volume of the high-salt buffer (25 mM Tris-Ac, pH 8.0, 20 mM KAc, 100 mM NaAc, 0.05% Triton X-100), allowing the folding of S1 and S2 for 40 min to form the bimolecular G-quadruplex G42S. Then, an equal volume of different concentrations of Hg2+ (in Tris-Ac buffer) was added, and this mixture was allowed to incubate further for 20 min. Finally, an equivalent of hemin was added and incubated for 1 h to form the hemin-G4-2S complex. Analytical Chemistry, Vol. 81, No. 6, March 15, 2009

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Scheme 1. Design and Process of the DNAzyme-Based Method for Colorimetric Hg2+ Detectiona

a AGRO100 is utilized to sense Hg2+ due to its T resides complexing with Hg2+. The formation of T-T base pairs inhibits AGRO100 folding into the G-quadruplex and thus no DNAzyme is formed, reflected by a decrease in the catalytic activity towards ABTS oxidation by H2O2.

Note that the adding sequence of Hg2+ and K+ does not influence the final results (see Figure S1 in the Supporting Information), although Hg2+ was added into the DNA solution before and after the addition of K+ in the above two cases. Spectroscopic Analysis of Hemin-DNA Interactions. The hemin-G-quadruplex complexes prepared above were diluted to appropriate concentrations with low- or high-salt buffer, respectively. With the use of the UV-vis spectrophotometer, the Soret band of hemin (centered at 396-405 nm) was recorded. The binding of G-quadruplex DNAs to hemin will be reflected by the hyperchromicity of the hemin Soret band.19,20 Colorimetric Measurement. The colorimetric detection of aqueous Hg2+ was performed at room temperature according to the procedure described previously26 with a little modification. Briefly, to 980 µL of 6 mM ABTS solution was added 10 µL of 15 µM DNAzymes. Then, 10 µL of 60 mM H2O2 was quickly added to initiate the ABTS-H2O2 reaction. The absorption spectra of the product ABTS•+ were recorded every 1 min by the UV-vis spectrophotometer in the wavelength range from 500 to 390 nm. Absorbance at 421 nm (the maximal absorption of ABTS•+, ε ) 3.6 × 104 L mol-1 cm-1) was used for quantitative analysis of Hg2+. Safety Considerations. As Hg2+ and most of tested metal ions are highly toxic and have adverse effects on human health, all experiments involving heavy metal ions should be performed with protective gloves. The waste solutions containing heavy metal ions should be collectively reclaimed to avoid polluting the environment. RESULTS AND DISCUSSION Utilizing AGRO100 for Aqueous Hg2+ Analysis. As Hg2+ is able to specifically bind to T residues of DNA, here we utilize the T-containing oligonucleotides (e.g., AGRO100) with excellent DNAzyme function to sense aqueous Hg2+. Scheme 1 depicts the design and procedure of this DNAzyme-based colorimetric method for Hg2+ detection. In the absence of coordination cation, AGRO100 is in the random coil state. Upon addition 2146

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Figure 1. Spectroscopic analysis of the inhibitory effect of Hg2+ on AGRO100 folding and DNAzyme forming. (A) UV-vis absorption spectra of the hemin-AGRO100 complex in the absence and presence of Hg2+: (a) 0.5 µM hemin; (b) incubation of 1 µM AGRO100 with solution in part a; (c) introduction of 10 µM Hg2+ into 1 µM AGRO100 prior to incubation with solution in part a. Experimental conditions: 25 mM Tris-Ac, pH 8.0, 0.5 mM KAc, 0.05% Triton X-100. (B) UV-vis absorption spectra (after 4 min) for analyzing the hemin-AGRO100 interaction in the absence and presence of Hg2+: (a) 0.15 µM hemin; (b) incubation of 0.15 µM AGRO100 with the solution in part a; (c) introduction of 2.5 µM Hg2+ into 0.15 µM AGRO100 prior to incubation with the solution in part a. Experimental conditions: 5.9 mM ABTS, 0.6 mM H2O2 in 25 mM Tris-Ac buffer (pH 8.0) containing 0.05% Triton X-100 and 0.5 mM KAc.

Figure 2. Utilization of the hemin-AGRO100 DNAzyme for spectroscopic analysis of different concentrations of Hg2+ with the ABTS-H2O2 colorimetry. (a) 0.15 µM hemin; (b) 0.15 µM heminAGRO100 DNAzyme; (c) solution in part b plus 0.125 µM Hg2+; (d) solution in part b plus 0.25 µM Hg2+; (e) solution in part b plus 0.5 µM Hg2+; (f) solution in part b plus 1.25 µM Hg2+; (g) solution in part b plus 2.5 µM Hg2+; (h) solution in part b plus 5 µM Hg2+. The inset shows the Hg2+ concentration-dependent change of absorbance at 421 nm (A421). Other experimental conditions are equal to those shown in Figure 1B.

of K+, it is able to fold into a bimolecular G-quadruplex, which strongly binds hemin to form the hemin-G-quadruplex DNAzyme with peroxidase-like activity.26,32 However, with the addition of Hg2+ prior to K+, the folding of AGRO100 is inhibited by the Hg2+-mediated formation of T-T base pairs and thus it is not able to fold into the G-quadruplex structure after addition of K+. As a result, the formation of hemin-AGRO100 DNAzyme is no longer allowed. This will be reflected by an absorbance decrease when monitored in the ABTS-H2O2 reaction system by using UV-vis absorption spectroscopy. As the contrast experiments, other metal ions are used in place of Hg2+ to test the selectivity of this DNAzyme-based method. The binding of DNA G-quadruplexes to hemin is known to cause an obvious hyperchromicity of the Soret band of hemin.19,20,32,34-36 This characteristic is here utilized to investigate the hemin-AGRO100 interaction and thus reveal the effect of

Hg2+ on AGRO100 folding. Figure 1A shows the UV-vis absorption spectra for analyzing the hemin-AGRO100 complex. The uncomplexed hemin has a Soret absorption band centered at 397 nm (curve a). After incubation with the folded AGRO100, a noticeable hyperchromicity is observed in the hemin Soret band (curve b). The absorption center shifts to 404 nm, accompanied by an observable increase in the absorption intensity. This is consistent with the binding of AGRO100 to hemin.32 However, in the presence of Hg2+, there is little hyperchromicity in the hemin Soret band (curve c), suggesting no binding interaction between AGRO100 and hemin. Similar phenomenon is also observed when the heminAGRO100 interaction in the absence or presence of Hg2+ is under investigation in the ABTS-H2O2 system (Figure 1B). The uncomplexed hemin exhibits a very low catalytic activity toward the H2O2-mediated oxidation of ABTS (curve a). After incubation with the folded AGRO100, there is a sharp increase in the catalytic activity (curve b), indicating the formation of hemin-AGRO100 DNAzyme. Upon the addition of Hg2+, the absorbance almost decreases to the background imparted by hemin catalysis (curve c). This suggests no DNAzyme formation in the presence of Hg2+. These experimental observations give evidence of Hg2+ inhibition on AGRO100 folding and DNAzyme formation. In addition, it is found that the effect of Hg2+ on the hemin-AGRO100 DNAzyme is primarily dependent on the concentration of K+ (see Figure S2 and related discussion in the Supporting Information). As the K+ concentration increases to 0.5 mM, the Hg2+ inhibition reaches to the maximum, whereas a decrease is observed at higher K+ concentrations. This can be well interpreted in terms of the intrinsic properties of the G-quadruplex. In general, DNA G-quadruplexes need coordination cations, especially K+ to keep stable.37 K+ can be located at the center cavity between two stacked G-tetrads and coordinates with eight carbonyl oxygen atoms of the G residues,38 significantly promoting the proper folding of Gquadruplex. This K+-binding characteristic enables several

Figure 3. Selectivity of utilizing 0.15 µM hemin-AGRO100 DNAzyme for aqueous Hg2+ detection. Hg2+ and other tested metal ions were all used at 2.5 µM. Introduction of PDCA is to overcome the potential interference. The inset shows the molecular structure of PDCA. Other experimental conditions are equal to those shown in Figure 1B. Analytical Chemistry, Vol. 81, No. 6, March 15, 2009

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Figure 4. Spectroscopic analysis of aqueous Hg2+ using the hemin-G4-2S DNAzyme. (A) UV-vis absorption spectra of the hemin-G4-2S complex in the absence and presence of Hg2+. (a) 0.5 µM hemin; (b) incubation of 0.5 µM G4-2S with the solution in part a; (c) introduction of 2.5 µM Hg2+ into 0.5 µM G4-2S prior to incubation with the solution in part a. Experimental conditions: 25 mM Tris-Ac, pH 8.0, 20 mM KAc, 100 mM NaAc, 0.05% Triton X-100. (B) Utilization of the hemin-G4-2S DNAzyme for spectroscopic analysis of different concentrations of Hg2+ with the ABTS-H2O2 colorimetry. (a) 0.15 µM hemin; (b) 0.15 µM hemin-G4-2S DNAzyme; (c) solution in part b plus 0.05 µM Hg2+; (d) solution in part b plus 0.125 µM Hg2+; (e) solution in part b plus 0.25 µM Hg2+; (f) solution in part b plus 0.5 µM Hg2+; (g) solution in part b plus 1.25 µM Hg2+; (h) solution in part b plus 2.5 µM Hg2+; (i) solution in part b plus 5 µM Hg2+. The inset shows the change of A421 with the Hg2+ concentration. Experimental conditions: 5.9 mM ABTS, 0.6 mM H2O2 in 25 mM Tris-Ac buffer (pH 8.0) containing 20 mM KAc, 100 mM NaAc, and 0.05% Triton X-100.

specific G-quadruplex DNAs including AGRO100 to sense K+.26,39,40 In contrast, Hg2+ can inhibit the proper folding of AGRO100 due to its ability to interact with the T residues of AGRO100 to form the T-Hg2+-T complex. Therefore, there is a potential competition between AGRO100 binding to K+ or Hg2+. An appropriate concentration of K+ is crucial for AGRO100 folding and DNAzyme formation (Figure S2A in the Supporting Information), whereas the Hg2+ inhibition on hemin-AGRO100 DNAzyme will be weakened if the K+ concentration becomes too high (Figure S2B in the Supporting Information). In this sense, AGRO100 is most suitable for the detection of Hg2+ in low-salt aqueous solutions. The presence of 0.5 mM K+ is enough to keep this DNA G-quadruplex functioning well. Under the optimal conditions, AGRO100 is utilized for quantitative analysis of aqueous Hg2+, as shown in Figure 2. In the absence of Hg2+, the hemin-AGRO100 DNAzyme exhibits modest catalytic activity (curve b). Upon addition of different concentrations of Hg2+, the DNAzyme activity is gradually 2148

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decreased (curves c-h). An obvious change in the absorption spectrum is observed when 250 nM of Hg2+ is added (curve d), indicating a detection limit of 250 nM for analyzing aqueous Hg2+ (S/N > 3). The inset of Figure 2 shows the dependence of absorbance at 421 nm on Hg2+ concentration, with a linear relationship (R2 ) 0.987) in the range of 250-1250 nM. To test the specificity of sensing Hg2+ by AGRO100, other metal ions are used in place of Hg2+. The contrast experiments reveal that this DNAzyme-based method for Hg2+ detection is subject to interference from several metal ions, especially Pb2+. The severe Pb2+ interference with Hg2+ detection has been reported previously whereas it can be overcome by addition of PDCA.8 In fact, in many Hg2+ sensors, PDCA is usually utilized to eliminate the main interference from Pb2+ and Cd2+ and thus provide the Hg2+-selective response.1 This chelating agent is also able to strongly complex with other metal ions (e.g., Zn2+, Cu2+, Co2+, Mn2+, Ni2+, Fe2+, and Fe3+) in aqueous solutions.41 Hence, PDCA is adopted here to improve the selectivity for Hg2+ detection. Figure 3 shows that, in the presence of PDCA, no interference from other metal ions is found. In particular, PDCA has no obvious influence on the target ion Hg2+, consistent with the previous report.8 Note that the appropriate dose of PDCA is dependent on interference ions. For Zn2+, Co2+, and Cr3+, an equivalent of PDCA is enough to thoroughly eliminate their interference whereas Pb2+ and Cd2+ need two equivalents. Other metal ions do not interfere with Hg2+ detection even in the absence of PDCA. G4-2S As Another Candidate for Colorimetric Hg2+ Detection. From the rationale point of view, the above DNAzymebased sensing system for Hg2+ detection is based on the specific interaction between Hg2+ and T residues. It means that other G-quadruplex DNAs containing T residues are able to sense aqueous Hg2+ as well. To demonstrate this issue, another bimolecular DNA G-quadruplex G4-2S is also chosen as the sensing element. The experimental procedure is largely similar to that shown in Scheme 1. Unlike AGRO100, G4-2S is found to need at least 20 mM K+ to keep stable. In addition, an appropriate concentration (100 mM) of Na+ slightly contributes to the stability of G4-2S and meanwhile reduces the background imparted by hemin catalysis. That is, G4-2S functions best in high-salt conditions. As the binding of DNA G-quadruplex to hemin can be reflected by the change of the hemin Soret band, UV-vis absorption spectroscopy is also utilized to investigate the interaction between hemin and G4-2S. Figure 4A shows G4-2S gives rise to a sharp hyperchromicity of the hemin Soret band, whereas the hyperchromicity becomes not so obvious upon adding Hg2+. This (35) Chinnapen, D. J.; Sen, D. Biochemistry 2002, 41, 5202–5212. (36) Mikuma, T.; Ohyama, T.; Terui, N.; Yamamoto, Y.; Hori, H. Chem. Commun. 2003, 1708–1709. (37) Sen, D.; Gilbert, W. Nature 1990, 344, 410–414. (38) Sundquist, W. I.; Klug, A. Nature 1989, 342, 825–829. (39) Ueyama, H.; Takagi, M.; Takenaka, S. J. Am. Chem. Soc. 2002, 124, 14286– 14287. (40) Nagatoishi, S.; Nojima, T.; Juskowiak, B.; Takenaka, S. Angew. Chem., Int. Ed. 2005, 44, 5067–5070. (41) Norkus, E.; Stalnioniene, I.; Crans, D. C. Heteroat. Chem. 2003, 14, 625– 632.

Figure 5. Selectivity of utilizing 0.15 µM hemin-G4-2S DNAzyme to sense aqueous Hg2+. Hg2+ and other tested metal ions were all used at 2.5 µM. Other experimental conditions are equal to those shown in Figure 4B.

phenomenon is similar to that observed in Figure 1A, mainly attributed to the inhibitory effect of Hg2+ on G4-2S folding. It confirms the feasibility of utilizing G4-2S to sense aqueous Hg2+. Figure 4B depicts the quantitative analysis of Hg2+ with colorimetry. The hemin-G4-2S DNAzyme exhibits a modest catalytic activity toward the ABTS oxidation by H2O2, whereas a gradual decrease in the enzyme activity is observed after addition of increasing Hg2+. An obvious change (S/N > 3) in the absorption spectrum is observed when 50 nM of Hg2+ is added (curve c). This means that the detection limit for analyzing aqueous Hg2+ corresponds to 50 nM (i.e., 10 ppb). The inset of Figure 4B shows the Hg2+ concentration-dependent change in the absorbance at 421 nm. A linear relationship (R2 ) 0.995) is observed in the range from 50 to 2500 nM. Furthermore, the contrast experiments reveal that, just like AGRO100, G4-2S is highly selective for Hg2+ over other metal ions, with assistance of the complexant PDCA (Figure 5). Applications. Hg2+ in two main water systems (freshwater and seawater) is usually determined for monitoring mercury pollution in the environment. Considering the working conditions, AGRO100 and G4-2S will be suitable for aqueous Hg2+ analysis in these two cases, respectively. To demonstrate this issue, we apply AGRO100 and G4-2S to analyzing the environmentally relevant samples (lake water). The experimental results indicate both AGRO100 and G4-2S can be utilized for analyzing the practical samples. However, for such a freshwater sample (low salt), AGRO100 is preferable to G4-2S. For more details, see Figure S3 and related discussion in the Supporting Information. It suggests that, with combined utilization of AGRO100 and G4-2S, our introduced DNAzyme-based method can be applied to facile detection of aqueous Hg2+ in most cases. CONCLUSION We have introduced a label-free colorimetric method for highly selective and sensitive detection of aqueous Hg2+ using Hg2+modulated G-quadruplex-based DNAzymes. Two bimolecular DNA G-quadruplexes with many T residues, AGRO100 and G4-

2S, are chosen as the sensing elements in low- and high-salt conditions, respectively. In the folded state, they are able to bind hemin to form the G-quadruplex-based DNAzymes with the peroxidase-like activities. Upon adding Hg2+, their proper folding is inhibited due to the Hg2+-mediated formation of T-T base pairs. This is confirmed by the notable change of the hemin Soret band when investigated by using UV-vis absorption spectroscopy. As a result, a sharp decrease in the catalytic activity toward ABTS oxidation by H2O2 is observed, accompanied by a change in solution color. This enables aqueous Hg2+ to be detected at 50 nM (10 ppb) with colorimetry in a facile way. With assistance of the complexant PDCA, this DNAzyme-based approach exhibits high selectivity for Hg2+ over other metal ions. Its application potential is demonstrated by applying AGRO100 and G4-2S to analyzing the practical sample (lake water). These experimental observations clearly reveal the noteworthy advantages of our introduced method for quantitative Hg2+ analysis, i.e., it is rapid, effective, and very easy to be performed and repeated. Another significance of this work lies in that it reveals that a small amount of Hg2+ is able to remarkably influence the G-quadruplex structure with T loop residues. Such a high-ordered structure can be found in the human telomere sequence. This may provide some insight into mercury toxicity in the human body. ACKNOWLEDGMENT This work is financially supported by the National Natural Science Foundation of China with the Grants 20675078 and 20735003, 973 Project 2009CB930100, and the Chinese Academy of Sciences Grant KJCX2.YW.H11. SUPPORTING INFORMATION AVAILABLE Additional information as noted in text. This material is available free of charge via the Internet at http://pubs.acs.org. Received for review November 2, 2008. Accepted January 29, 2009. AC900188Y

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