Gold Nanoparticle-Based Colorimetric and âTurn-Onâ Fluorescent

Anal. Chem. 2008, 80, 9021–9028

Gold Nanoparticle-Based Colorimetric and “Turn-On” Fluorescent Probe for Mercury(II) Ions in Aqueous Solution Hao Wang, Yongxiang Wang, Jianyu Jin, and Ronghua Yang* Beijing National Laboratory for Molecular Sciences, College of Chemistry and Molecular Engineering, Peking University, Beijing 100871, China An approach for visual and fluorescent sensing of Hg2+ in aqueous solution is presented. This method is based on the Hg2+-induced conformational change of a thymine (T)-rich single-stranded DNA (ssDNA) and the difference in electrostatic affinity between ssDNA and doublestranded (dsDNA) with gold nanoparticles. The dye-tagged ssDNA containing T-T mismatched sequences was chosen as Hg2+ acceptor. At high ionic strength, introduction of the ssDNA to a colloidal solution of the aggregates of gold nanoparticles results in color change, from blue-gray to red of the solution, and the fluorescence quenching of the dye. Binding of Hg2+ with the ssDNA forms the double-stranded structure. This formation of dsDNA reduces the capability to stabilize bare nanoparticles against salt-induced aggregation, remaining a blue-gray in the color of the solution, but fluorescence signal enhancement compared with that without Hg2+. With the optimum conditions described, the system exhibits a dynamic response range for Hg2+ from 9.6 × 10-8 to 6.4 × 10-6 M with a detection limit of 4.0 × 10-8 M. Both the color and fluorescence changes of the system are extremely specific for Hg2+ even in the presence of high concentrations of other heavy and transition metal ions, which meet the selective requirements for biomedical and environmental application. The combined data from transmission electron microscopy, fluorescence anisotropy measurements, and dialysis experiments indicate that both the color and the fluorescence emission changes of the DNA-functioned gold nanoparticles generated by Hg2+ are the results of the metal-induced formation of dsDNA and subsequent formation of nanoparticle aggregates. Heavy metal ion pollution poses severe risks in human health and the environment. Mercury(II) (Hg2+), widely distributed in the air, water, and soil,1 is considered to be a highly toxic heavy metal ion and is known as a hazardous pollutant with recognized accumulative character in the environment and biota.2-4 Mercury vapors and organic mercury derivatives, such as methylmercury, * To whom correspondence should be addressed. E-mail: [email protected]. Fax: +86-10-62751708. (1) Miller, J. R.; Rowland, J.; Lechler, P. J.; Desilets, M.; Hsu, L.-C. Water, Air, Soil Pollut. 1996, 86, 373–388. (2) Eisler, R. Environ. Geochem. Health 2003, 25, 325–345. (3) Wang, Q. R.; Kim, D.; Dionysiou, D. D.; Sorial, G. A.; Timberlake, D. Environ. Pollut. 2004, 131, 323–336. 10.1021/ac801382k CCC: $40.75  2008 American Chemical Society Published on Web 11/01/2008

affect many different areas of the brain and their associated functions. In addition, inorganic mercury can damage the heart, kidney, stomach, and intestines.5-8 These environmental and health problems of Hg2+ have prompted researchers to develop efficient methods for selective and sensitive assay of the metal to understand its distribution and pollution potential. Toward this goal, besides conventional methods, such as atomic absorption/ emission spectroscopy,9,10 inductively coupled plasma mass spectrometry (ICPMS),11 selective cold vapor atomic fluorescence spectrometry,12,13 and electrochemical and optical sensing devices,14-17 new molecule probes using organic small molecules,18-24 polymers,25,26 oligonucleotides,27-34 and proteins35 (4) Tchounwou, P. B.; Ayensu, W. K.; Ninashvili, N.; Sutton, D. Inc. Environ. Toxicol. 2003, 18, 149–175. (5) Hoyle, I.; Handy, R. D. Aquatic Toxicol. 2005, 72, 147–159. (6) Baughman, T. A. Environ. Health Perspect. 2006, 114, 147–152. (7) Kobal, A. B.; Horvat, M.; Prezelj, M.; Briski, A. S.; Krsnik, M.; Dizdarevic, T.; Mazej, D.; Falnoga, I.; Stibilj, V.; Arneric, N.; Kobal, D.; Sredkar, J. J. Trace Elem. Med. Biol. 2004, 17, 261–274. (8) Zalups, R. K. Pharmacol. Rev. 2000, 52, 113–144. (9) Gomez-Ariza, J.; Lorenzo, F.; Garcia-Barrera, T. Anal. Bioanal. Chem. 2005, 382, 485–492. (10) Han, F. X.; Dean Patterson, W.; Xia, Y. J.; Maruthi Sridhar, B. B.; Su., Y. J. Water, Air, Soil Pollut. 2006, 170, 161–171. (11) Fong, B.; Mei, W.; Siu, T. S.; Lee, J.; Sai, K.; Tam, S. J. Anal. Toxicol. 2007, 31, 281–287. (12) Geng, W.; Nakajima, T.; Takanashi, H.; Ohki, A. J. Hazard Mater. 2008, 154, 325–330. (13) Yu, Y. L.; Du, Z.; Wang, J. H. J. Anal. At. Spectrom. 2007, 22, 650–656. (14) Miguel, A. H.; Jankowski, C. M. Anal. Chem. 1974, 46, 1832–1834. (15) Bonfil, Y.; Brand, M.; Kirowa-Eisner, E. Anal. Chim. Acta 2000, 424, 65– 76. (16) Lerchi, M.; Ritter, E.; Simon, W.; Pretsch, E.; Chowdhury, D. A.; Kamata, S. Anal. Chem. 1994, 66, 1713–1717. (17) Sanchez-Pedren ˜o, C.; Ortun ˜o, J. A.; Albero, M. I.; Garcia, M. S.; Valero, M. V. Anal. Chim. Acta 2000, 414, 195–203. (18) Coronado, E.; Galan-Mascaros, J. R.; Marti-Gastaldo, C.; Palomares, E.; Durrant, J. R.; Vilar, R.; Gratzel, M.; Nazeeruddin, M. K. J. Am. Chem. Soc. 2005, 127, 12351–12356. (19) Coskun, A.; Akkaya, E. U. J. Am. Chem. Soc. 2006, 128, 14474–14475. (20) Ko, S. K.; Yang, Y. K.; Tae, J.; Shin, I. J. Am. Chem. Soc. 2006, 128, 14150– 14155. (21) Yang, Y. K.; Yook, K. J.; Tae, J. J. Am. Chem. Soc. 2005, 127, 16760– 16761. (22) Yoon, S.; Albers, A. E.; Wong, A. P.; Chang, C. J. J. Am. Chem. Soc. 2005, 127, 16030–16031. (23) Wang, J.; Qian, X.; Cui, J. J. Org. Chem. 2006, 71, 4308–4311. (24) Nolan, E. M.; Lippard, S. J. J. Am. Chem. Soc. 2007, 129, 5910–5918. (25) Wang, J.; Qian, X. Org. Lett. 2006, 8, 3721–3724. (26) Liu, X. F.; Tang, Y. L.; Wang, L. H.; Zhang, J.; Song, S. P.; Fan, C. H.; Wang, S. Adv. Mater. 2007, 19, 1471–1474. (27) Lee, J.-S.; Han, M. S.; Mirkin, C. A. Angew. Chem., Int. Ed. 2007, 46, 4093– 4096.

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have been developed. In addition, nanosensors based on metal nanoparticles,27,28,32,33,36-40 semiconductor quantum dots,41,42 single-walled carbon nanotubes,43 and nanoscale membrane44 or chip45 have attracted interest in recent years. Although these approaches have made great contributions toward a Hg2+ assay, limitations, such as insufficient resolution in water, irreversible Hg2+ complex, interference with other heavy metal ions, and sophisticated synthesis of the probe materials, still existed. Therefore, it should be desirable to develop a new method to solve these problems. The high specificity of interaction of oligonucleotides with metal ion makes the oligonucleotides the current tools for detection of a particular element in a mixture of analytes. It has been previously demonstrated that Hg2+ can selectively bind in between two DNA thymine (T) bases and promote these T-T mismatches to form stable T-Hg2+-T base pairs.46-48 This property has been applied to the design molecule sensors for Hg2+ in aqueous solution. The selectivity of the approach is achieved by capitalizing on the specific binding ability of thymine bases to Hg2+. Their effectiveness, however, is highly dependent on the ability to transduce the Hg2+ binding event to a measurable signal. Mirkin et al.27 recently designed a colorimetric sensor with DNAfunctionalized gold nanoparticles(Au-NPs) to achieve a Hg2+ detection limit of 100 nM by determining changes in the melting temperature of the metal complex. To avoid the electronic heating element, more recently, advanced approaches were developed by Liu et al.28 and Chang et al.32 through optimizing the structures of DNA strands so that they can operate at ambient temperature. Although both approaches have an advantage of being easily read with naked eye, they are limited with respect to detection sensitivity compared with fluorescence spectroscopy. (28) Liu, C.-W.; Hsieh, Y.-T.; Huang, C.-C.; Lin, Z.-H.; Chang, H.-T. Chem. Commun. 2008, 2242–2244. (29) Ono, A.; Togashi, H. Angew. Chem., Int. Ed. 2004, 43, 4300–4302. (30) Liu, J. W.; Lu, Y. Angew. Chem., Int. Ed. 2007, 46, 7587–7590. (31) Chiang, C.-K.; Huang, C.-C.; Liu, C.-W.; Chang, H.-T. Anal. Chem. 2008, 80, 3716–3721. (32) Xue, X. J.; Wang, F.; Liu, X. G. J. Am. Chem. Soc. 2008, 130, 3244–3245. (33) Li, D.; Wieckowsk, A.; Willner, I. Angew. Chem., Int. Ed. 2008, 47, 3927– 3931. (34) Liu, C.-W.; Huang, C.-C.; Chang, H. T. Langmuir 2008, 24, 8346–8350. (35) Wegner, S. V.; Okesli, A.; Chen, P.; He, C. J. Am. Chem. Soc. 2007, 129, 3474–3475. (36) Huang, C.-C.; Chang, H.-T. Anal. Chem. 2006, 78, 8332–8338. (37) Rex, M.; Hernandez, F. E.; Campiglia, A. D. Anal. Chem. 2006, 78, 445– 451. (38) Huang, C.-C.; Yang, Z.; Lee, K.-H.; Chang, H.-T. Angew. Chem., Int. Ed. 2007, 46, 6824–6828. (39) Darbha, G. K.; Singh, A. K.; Rai, U. S.; Yu, E.; Yu, H.; Ray, P. C. J. Am. Chem. Soc. 2008, 130, 8038–8043. (40) Darbha, G. K.; Ray, A.; Ray, P. C. ACS Nano 2007, 1, 208–214. (41) Chen, J. L.; Cao, Y. C.; Xu, X. B.; Wu, G. H.; Chen, Y. C.; Zhu, C. Q. Anal. Chim. Acta 2006, 577, 77–84. (42) Li, H.; Zhang, Y.; Wang, X.; Xiong, D.; Bai, Y. Mater. Lett. 2007, 61, 1474– 1477. (43) Gao, X. Y.; Xing, G. M.; Yang, Y. L.; Shi, X. L.; Liu, R.; Chu, W. G.; Jing, L.; Zhao, F.; Ye, C.; Yuan, H.; Fang, X. H.; Wang, C.; Zhao, Y. L. J. Am. Chem. Soc. 2008, 130, 9190–9191. (44) El-Safty, S. A.; Prabhakaran, D.; Kiyozumi, Y.; Mizukami, F. Adv. Funct. Mater. 2008, 18, 1739–1750. (45) Lee, J.-S.; Mirkin, C. A. Anal. Chem. 2008, 80, 6805–6808. (46) Katz, S. J. Am. Chem. Soc. 1952, 74, 2238–2245. (47) 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. (48) Tanaka, Y.; Oda, S.; Yamaguchi, H.; Kondo, Y.; Kojima, C.; Ono, A. J. Am. Chem. Soc. 2007, 129, 244–245.

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To develop a fluorescent sensor for Hg2+ based on this T-Hg2+-T coordination chemistry, Ono and Togashi29 designed a thymine-rich single-stranded DNA (ssDNA) with the 3′- and 5′end labeled with a dye and a quencher, respectively. Hg2+ binding brought the two ends close to each other, resulting in fluorescence quenching since fluorescence resonance energy transfer can take place between the dye and the quencher. Although the approach can efficiently detect Hg2+ from 40 to 100 nM, fluorescence quenching is disadvantageous for a high signal output upon complexation. To develop a “turn-on” fluorescent sensor, Lu et al. designed a catalytic beacon based on a uranium-specific DNAzyme.30 The beacon displays fluorescence enhancement in the presence of nanomolar concentration of Hg2+. However, the ingenious design of the beacon and double-labeling of the DNAzyme with a dye and quencher restrict its use as a common biosensing approach. To address these issues, we have employed here an entirely different designing strategy. Rothberg et al.49 found that ssDNA has strongly attractive electrostatic interactions with citrate-coated Au-NPs, which prevents the Au-NPs from salt-induced aggregation, but the double-stranded DNA (dsDNA) does not. By taking advantage of this phenomenon, colorimetric detection procedures have been introduced for analytes ranging from DNA to protein50 and metal ions.51,52 Further, based on the exceptional quenching capability of Au-NPs to the proximate fluorescent dye, Au-NPs have been successfully used to construct fluorescent probes for DNA and proteins by covalent modifying the nanoparticle’s surface with dye-labeled oligonucleotides.53,54 We reasoned that, if a thymine-rich ssDNA was labeled with a dye, binding of Hg2+ might change the conformation of the ssDNA to form a dsDNA. At a certain salt concentration, this formation of dsDNA will reduce the affinity with Au-NPs, and thus the ability to disperse the aggregated nanoparticles, which produces both color change and fluorescent signal enhancement. In this way, neither covalent modification of the Au-NPs with oligonucleotides nor double-labeling of oligonucleotide with a dye-quencher pair is required. Compared to the known molecular sensors for Hg2+ reported in the literature, our proposed approach possesses some remarkable features: (1) The DNA strand needs only one dye labeled, leading to less laborious and more cost-effective synthesis; (2) it could not only function in aqueous solution with good sensitivity for Hg2+ but also exhibit significant color and “turnon” fluorescence responses to the metal ion. The simultaneously colorimetric and fluorescent detections of Hg2+ provide advantages of the high sensitivity of fluorescence with the convenience and aesthetic appeal of a visual assay; and (3) most important, it displays extreme specificity toward Hg2+ even in the presence of a high concentration of other competitive heavy metal ions. In certain environmental samples, such as river water, the concentrations of some metal ions, such as Pb2+ and Fe3+/Fe2+, are (49) Li, H.; Rothberg, L. Proc. Natl. Acad. Sci. U. S. A. 2004, 101, 14036–14039. (50) Wei, H.; Li, B. L.; Li, J.; Wang, E. K.; Dong, S. J. Chem. Commun. 2007, 3735–3737. (51) Hua, W. L.; Fen, L. X.; Fang, H. X.; Ping, S. S.; Hai, F. C. Chem. Commun. 2006, 3780–3782. (52) Wei, H.; Li, B. L.; Li, J.; Dong, S. J.; Wang, E. K. Nanotechnology 2008, 19, 095501. (53) Maxwell, D. J.; Taylor, J. R.; Nie, S. J. Am. Chem. Soc. 2002, 124, 9606– 9612. (54) Dubertret, B.; Calame, M.; Libchaber, A. J. Nat. Biotechnol. 2001, 19, 365– 370.

Figure 1. (A) Schematic description of colorimetric and fluorescent sensing of Hg2+ based on the modification-free Au-NPs. The drawing of dye-DNA functionalized Au-NPs is only a graphic presentation and does not represent the precise way that DNA binds on the nanoparticles. (B) Structures of FAM, the FAM-labeled ssDNA, 1 and 2, the perfected cDNA 3, and 11-mer T-T mismatched bases DNA 4 of 2. Oligo represents the position of attachment of tethered oligonucleotides.

significantly higher than that of Hg2+; selective detection of Hg2+ in the presence of these metal interferents is a critical issue to the application of most common sensors.

EXPERIMENTAL SECTION Chemicals and Apparatus. HAuCl4 · 4H2O was purchased from Shenyang Research Institute of Nonferrous Metals, China. All olgionucleotides with different sequences (Figure 1) were synthesized by Beijing Augct Biological Technology Co., Ltd., China. The work solution of the oligonucleotide was obtained by diluting the stock solution with a 0.02 M Tris-HCl buffer (pH 7.4), which contains 0.1 M NaCl and 5 mM KCl. Metal ion solutions were prepared from nitrate salts. All other reagents were of analytical reagent grade and were used without further purification or treatment. All work solutions were prepared with Tris-HCl buffer solution. UV-visible absorption spectra were recorded on a Hitachi U-3010 UV/vis spectrophotometer (Kyoto, Japan). Fluorescence measurements were performed on a Hitachi F-4500 fluorescence spectrofluorometer. Fluorescence anisotropy measurements were conducted on an Edinburgh Instruments FLS 920. Transmission electron microscopy (TEM) was performed on a transmission microscope (Hitachi H-700). The samples for TEM analysis were prepared by pipetting 25 µL of the colloidal solutions onto standard holey carbon-coated copper grids. The grids were dried in air for >12 h before loading into the vacuum chamber of the electron microscope. The TEM samples were not subjected to heavy metal staining or other treatments. pH values were measured by model 868 pH meter (Orion). ICPMS experiments were performed on Agilent 7500 series.

Synthesis of Au-NPs. Au-NPs of ∼13 nm in diameter were synthesized using the method developed by Natan et al.55 The 100 mL of 1.0 mM HAuCl4 solution was boiled and stirred vigorously; 10 mL of 38.8 mM sodium citrate solution was then added into the boiling solution rapidly with concomitant color change from light yellow to wine-red. After boiling for 10 min and followed stirring for 15 min, the solution was allowed to reach room temperature. Then it was filtered through a 0.32-µM membrane filter and stored in a refrigerator at 4 °C before being used. The concentration of Au-NPs was estimated by UV/vis spectroscopy based on an extinction coefficient of 2.7 × 108 M-1 · cm-1 at λ ) 520 nm for 13-nm particles.56 Performance of Hg2+ Detection. For Hg2+ detection, 10 µL of the DNA probe solutions (1 or 2 and 4, 1.2 µM, respectively), 30 µL of metal solution (or blank buffer solution), and 100 µL of Au-NPs colloidal solutions as prepared were mixed and incubated for ∼0.5 h at room temperature. After 120 µL of 0.2 M NaCl solution was added, the UV/vis absorption and fluorescence emission spectra of the mixture were recorded immediately. RESULTS AND DISCUSSION Sensing Mechanism. The important insights in our design came from the interaction of Au-NPs and DNA, which are based on the following: (1) the color of gold colloid is very sensitive to the degree of aggregation of the nanoparticles in suspension, and the aggregation can be easily induced with electrolytes such as salt, (2) ssDNA adsorbs on negatively charged Au-NPs to reversibly disperse the Au-NP aggregates at a chosen ionic strength while dsDNA does not at the same ionic strength, and (3) when a dye-labeled olignucleotide sticks to the Au-NPs, the attendant proximity of the dye to the gold leads to fluorescence quenching of the dye. To test the general feasibility of the approach, two DNA oligonucleotides, 1 and 2, which contain rich thymine bases, were labeled with a fluorescent derivative, fluorescein (FAM) at the 3′-end. Oligonucleotide 1 contains two parts: the thymine-rich mercury-binding sequence and the linker sequence. It is desirable that mercury-mediated base pairs (T-Hg2+-T) of 1 could be formed between thymine residues from two Hg-binding sequences to give rise to a hairpin structure. 2 could not form a self-folded structure by Hg2+, but it could form a dsDNA with 4 in the presence of Hg2+. Figure 1A describes the mechanism of the present approach for colorimetric and fluorescent sensing of Hg2+, which is based on the discriminated effects of different DNA structures on the aggregations of AuNPs induced by the ionic strength of the solution.28,34,49 At certain NaCl concentrations and in the presence of a dye-labeled ssDNA, the strongly attractive electrostatic interactions of ssDNA with citrate-coated Au-NPs prevent the nanoparticles from salt-induced aggregation, showing pink-red in color of Au-NPs and quenched fluorescence of FAM. Binding of Hg2+ with the ssDNA forms a double-stranded structure, which reduces the affinity with nanoparticles so that Au-NPs aggregate readily, displaying a blue-gray color of nanoparticles and fluorescence restoration of FAM. (55) Grabar, K. C.; Freeman, R. G.; Hommer, M. B.; Natan, M. J. Anal. Chem. 1995, 67, 735–743. (56) Demers, L. M.; Mirkin, C. A.; Mucic, R. A.; Reynolds, R. L.; Letsinger, R.; Elghanian, R.; Viswanadham, G. Anal. Chem. 2000, 72, 5535–5541.


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Figure 2. (A) Effect of Hg2+ on the absorption spectra of 1 stabilized Au-NPs solution. (a) Absorption spectrum of the mixture solution of Au-NPs and 1 (pH 7.4, 0.1 M NaCl) in the absence of Hg2+; (b) absorption spectrum of the mixture solution in the presence of 6.0 µM Hg2+. (B) The corresponding color change of 1 stabilized AuNPs in the absence (a) and the presence of Hg2+ (b).

Effect of Different DNA Strands on Aggregation of Au-NPs in Salt Solution. Prior to application of the two DNA probes in fluorescence sensing of Hg2+, effects of different DNA strands on Au-NP aggregation were first studied in the Tris-HCl buffer solution (Figure S1, Supporting Information (SI)). Different adsorption propensities between ssDNA and dsDNA are realized as the different abilities to stabilize Au-NPs in the salt solution. The colloidal solutions of Au-NPs are stable at low salt concentration due to electrostatic repulsion from the negative capping agents; these stabilized gold colloids exhibit the nanoparticle’s characteristic surface plasma absorption peak at 520 nm and appear pink-red. Increasing the salt concentration of the solution (0.1 M NaCl), this 520-nm band is shifted to long wavelength concomitant with the color change of the solution, from pink-red to blue-gray. However, in the presence of ssDNA strand (2) in the colloidal solutions, the strong coordination interaction between the nitrogen atoms of the DNA strand and Au-NPs will enhance the Au-NPs’ stability against the salt-induced aggregation, displaying the intrinsic absorption peak at 520 nm and pink-red of the Au-NPs. To examine the effect of dsDNA on the Au-NP aggregations, 2 was first hybridized with its perfect cDNA (3). As for dsDNA, the colloidal solutions of the Au-NPs display blue-gray in color with a broad absorption between 600 and 800 nm, suggesting that the relatively rigid structure of dsDNA could not adsorb on the Au-NPs and lose the ability to protect the Au-NPs against saltinduced aggregation. Effect of Hg2+ Ions on the Interaction of Au-NPs and ssDNA. Figure 2 shows both the absorption spectrum and the color changes of the mixture solution of Au-NPs and 1 in 0.1 M NaCl caused by Hg2+. As expected from the original design, in the absence of Hg2+, the Au-NPs remain stable and display a maximal absorption band at 520 nm, indicating that at this salt concentration the free 1 exists in the single-stranded form and adsorbs on the surfaces of Au-NPs to protect the Au-NPs against salt-induced aggregation. While in the presence of Hg2+ ions, AuNPs display blue-gray concomitant with a broad absorption between 600 and 800 nm as those observed by dsDNA. This result implies that binding of Hg2+ with 1 forms a double-stranded structure, which reduces the interaction with Au-NPs and loses the ability to disperse the aggregated nanoparticles. 9024


Figure 3. Fluorescence emission spectra of 1 at different experiment conditions: (a) 40 nM 1 in Tris-HCl buffer (0.1 M NaCl); (b) a + 4.0 µM Hg2+; (c) a + 0.8 nM Au-NPs; (d) a + 0.8 nM Au-NPs + 4.0 µM Hg2+. Excitation wavelength was at 480 nm.

Consistent with the absorption spectra changes, FAM’s fluorescence of 1 is sensitive for Au-NPs and Hg2+. Figure 3 illustrates the results of a measurement comparing the fluorescence emission from 1 and 1-Hg2+ in the NaCl solution in the absence and the presence of Au-NPs. Upon exciting at the maximal absorption wavelength of FAM, the fluorescence emission of 1 was quenched a little by Hg2+ ions. However, the fluorescence of 1 was greatly decreased by Au-NPs. In our experiment, more than 95% quenching was observed for 1 concentration from 5 to 100 nM by ∼ 0.1 equiv of Au-NPs (Figure S2, SI). The observed quenching phenomenon largely originated from the free fluctuations of fluorescent molecules and intercollisions between the fluorophore and nanoparticles which leads to surface energy transfer. In addition, we suggest that the high quenching efficiency of FAM by Au-NPs could be a result of inner filter effect (IFE) mechanism. IFE is an alternative way to convert an absorbance signal into a fluorescence signal. In the approach, two dyes are employed, one absorbing, while the other is fluorescent. If the absorption spectrum of the absorbing dye possesses a complementary overlap region with the excitation or emission spectrum of the fluorescent dye, the fluorescence emission of the fluorophore is thus modulated by the absorber.57-60 The IFE is a source of errors in fluorometry, but it can be useful for optical chemical sensing by converting an absorption signal into a fluorescence signal.61-64 In the system of 1/Au-NPs, Au-NPs show strong absorption at 520 nm, while the maximum emission wavelength of FAM locates at 518 nm (Figure S3, SI). This main 518-nm emission band of the FAM overlaps nicely with the absorption band of Au-NPs. Thus, the effective emission intensity of FAM would be more decreased if the two materials coexist in a sensory device. Curve d of Figure 3 is the fluorescence emission spectrum of 1/Au-NPs in the presence of Hg2+; significant fluorescence emission enhancement is observed. The fluorescence intensity of 1 at 518 nm in the presence of 6.0 µM Hg2+ is 3.1-fold higher than that without Hg2+. This fluorescence enhancement is the (57) Holland, J. F.; Teets, R. E.; Kelly, P. M.; Timnick, A. Anal. Chem. 1977, 49, 706–710. (58) Leese, R. A.; Wehry, E. L. Anal. Chem. 1978, 50, 1193–1197. (59) Parker, C. A.; Barnes, W. J. Analyst 1957, 82, 606–617. (60) Parker, C. A.; Tees, W. T. Analyst 1962, 87, 83–111. (61) Gabor, G.; Walt, D. R. Anal. Chem. 1991, 63, 793–796. (62) He, H. R.; Li, H.; Mohr, G.; Kovacs, B.; Werner, T.; Wolfbeis, O. S. Anal. Chem. 1993, 65, 123–127. (63) Yuan, P.; Walt, D. R. Anal. Chem. 1987, 59, 2391–2394. (64) Yang, X. H.; Wang, K. M.; Guo, C. C. Anal. Chim. Acta 2000, 407, 45–52.

Figure 4. TEM images of 1/Au-NPs in 0.1 M NaCl solution in the absence (A) and the presence of 6.0 µM Hg2+ (B).

result of the formation of dsDNA of 1 by Hg2+, which hampers energy transfer between FAM and Au-NPs. For 2 and its T-mismatched 4, the same results were obtained by Hg2+ (Figure S4, SI). This Hg2+-induced color and fluorescence emission changes of ssDNA/Au-NPs colloidal solutions constitute the basis for colorimetric and fluorescent detections of Hg2+ proposed in this paper. Studies on Interaction of ssDNA and Hg2+. As mentioned, the present approach may work well for the sensing of Hg2+. However, before it could be practically applied for a Hg2+ assay, some questions must be answered. First, can Hg2+ bind to 1 to form a dsDNA so that the Au-NP aggregates are formed? The direct evidence for Hg2+-induced aggregation of Au-NPs in the presence of 1 is obtained from TEM. As shown in Figure 4, the TEM images of 1/Au-NPs in 0.1 M NaCl reveal uniform particles with an average size of ∼14 nm in diameter; however, after incubation with Hg2+ for 30 min, irregular Au-NP aggregates of a few hundred nanometers to micrometers in diameter were observed. Second, how does ssDNA interact with Hg2+ in the Au-NP colloidal solution? The binding of Hg2+ with the ssDNA can occur either in solution or at the surface of nanoparticles. With the former case, the DNA strand first comes out from the nanoparticle and then the metal binding take places in solution. With the latter case, however, the DNA strand absorbed on the Au-NPs, and the metal ion complexation is formed on the nanoparticle surface. This is where the ssDNA strand undergoes a change in conformation in response to interaction with Hg2+. Therefore, with this case, the dye end of the DNA molecule extends from the nanoparticle in such a way that it is no longer quenched, while the remaining DNA is still adsorbed to the nanoparticle. To understand whether the Hg2+ binding occurs on the AuNP surface or in solution, fluorescence anisotropy of (2 + 4) was determined. The fluorescence anisotropy of a dye reflects the molecule’s ability to rotate in its microenvironments, which include viscosity of the solution and the size and mass of the molecule to which the dye is attached.57 Therefore, fluorescence anisotropy can be used to judge whether the Hg2+ binding occurs on the Au-NP surface or in solution based on the difference of molecular weight of the fluorescent oligonucleotide. The fluorescence anisotropy of (2 + 4) in the Tris-HCl buffer is 0.013 ± 0.007, which was minimally increased by the addition of 6.0 µM Hg2+ (0.027 ± 0.011), but ∼5-fold enhancement was observed by Au-NPs (0.062 ± 0.014), indicating formation of the nanoparticle assembly complexes. However, when Hg2+ was added to the mixture solution of (2 + 4) and Au-NPs, the fluorescence anisotropy was 0.034 ± 0.005, which was reduced in comparison to (2 + 4) and Au-NPs. This is almost the same as that of (2 + 4) upon addition

Figure 5. Changes in the fluorescence emission spectra of 1/AuNPs in the Tris-HCl buffer (0.1 M NaCl) upon addition of different concentrations of Hg2+. The arrows indicate the signal changes as increases in Hg2+ concentrations (0, 0.08, 0.2, 0.4, 0.8, 1.6, 2.4, 4.0, and 6.0 µM). Excitation wavelength was at 480 nm. Inset: Response parameter (R) as a function of logarithm of Hg2+ concentration (M) at pH 7.4. The curve fitting of the experimental points (9) was calculated from eqs 1 and 2.

of Hg2+. It seems that the Hg2+ binding occurs in solution rather than on the nanoparticle surface. To further confirm this notion, we isolated the Hg2+-binding DNA by dialysis against Tris-HCl buffer with a membrane (molecular weight cutoff 8000-14 400) and measured the fluorescence intensity of the dialysis product and the nanoparticle complex. Specifically, we found the components inside the membrane are almost nonfluorescent, but the product outside the membrane is highly fluorescent. Collectively, these results demonstrate that the metal ion binding does take place in solution, not on the Au-NP surface. Third, how strong is the binding between thymine base and Hg2+? To estimate the association constant of 1 and Hg2+, fluorescence emission spectroscopy was followed as aliquots of Hg2+ were added to the solutions of 1/Au-NPs. Competitive binding of Hg2+ with Au-NPs for 1 causes the fluorescence change of FAM. Under the optimum conditions (pH 7.4, 0.1 M NaCl; nAu-NPs:n1 ) 1:50), the fluorescence response of 1/Au-NPs to different concentrations of Hg2+ is shown in Figure 5; fluorescence measurements were carried out at room temperature within 5 min. It is apparent that increase of the Hg2+ concentration causes a significant increase of the total emission of 1. If a 1:2 metal-to-ligand complex (T-Hg2+-T) is formed between Hg2+ and 1,44-46 the obtained emission intensities of 1

(65) Lakowicz, J. Principles of Fluorescence Spectroscopy, 3rd ed.; Springer: New York, 2006.


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at 518 nm as shown in Figure 5 were analyzed using the relation of Hg2+ concentration with the response parameter (R) that was established as previously reported elsewhere:66 1 1 1-R 2K [L]T R2

(1)

Fmax - F [L] ) LT Fmax - Fmin

(2)

[Hg2+] )

R)

where K is the association constant of the thymine base and Hg2+. [Hg2+] and [L] denote the free concentration of Hg2+ ions and the ligand 1, R is the ratio between the free ligand concentration, [L], and the initial concentration of the ligand, LT. F is the fluorescence intensity of 1 at 518 nm in the presence of different concentrations of Hg2+, and Fmin and Fmax are the limiting values of F at zero Hg2+ concentration and at final (plateau) Hg2+ concentration, respectively. In the inset of Figure 5, R is given as a function of the logarithm of Hg2+ concentration. The curve fitting for the experimental data points was calculated from eqs 1 and 2 with log K ) 5.71. The good correlation of the measured data with the theoretical predication confirms the validity of the proposed method. Finally, how reversible is the binding between 1 and Hg2+? EDTA is known to be a strong chelator of Hg2+. Because of the high stability constant of the EDTA-Hg2+ complex, it was anticipated that addition of EDTA will induce the decomplexation of Hg2+ from the Hg2+ complex of 1, thereby restoring the original photophysical properties of 1 and Au-NPs. With this intention, 1 equiv of EDTA solution was added to the Hg2+ complex of 1/Au-NPs. The change in the absorption spectrum of 1/Au-NPs-Hg2+ complex upon addition of EDTA solution is shown in Figure S5A (SI), which is compared to the original spectrum of 1/Au-NPs. The long wavelength absorption band of 600-800 nm nearly disappeared, and the spectrum matched that of 1/AuNPs where the original intensity of the band at 520 nm is regained. The fluorescence emission intensity is also decreased upon addition of the EDTA to the Hg2+ complexed solution (Figure S5B, SI). These results clearly indicate that the observed change in the absorption and fluorescence emission properties of 1/AuNPs with Hg2+ is due to the formation of a reversible complex between 1 and Hg2+. Kinetics and Thermodynamics of Hg2+ Binding. The kinetic and thermodynamic properties of Hg2+ binding with 1 were studied. Figure S6 (SI) shows absorbance changes of AuNPs and 1 at 750 nm as a function of incubation time. In the absence of Hg2+, the curve exhibits a rapid reduction in the first 0.5 h and a slow decrease over a 0.5-1.0 h period. This absorbance decrease is due to the interaction of the ssDNA with Au-NPs, which prevents the formation of aggregates of the nanoparticles. In the presence of Hg2+, formation of dsDNA does reduce adsorption of 1 onto the nanoparticles, which loses the ability to disperse the aggregates, and thus increases the absorbance at 750 nm. The best sensitivity, ∆A (∆A ) A - A0), is obtained around 0.5 h, where A0 and A are the absorbance of Au-NPs at 750 nm in the absence and the presence of Hg2+, respectively. (66) Yang, R. H.; Wang, K. M.; Long, L. P.; Xiao, D.; Yang, X. H. Anal. Chem. 2002, 74, 1088–1096.

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Figure 6. Effects of DNA sequences on the fluorescence response of 1 (9) or (2 + 4) (b) to Hg2+, where F0 and F are the FAM fluorescence intensity in the absence and the presence of different concentrations of Hg2+. Fluorescence emission was recorded at 518 nm in Tris-HCl buffer (pH 7.4, 0.1 M NaCl), and the excitation wavelength was at 480 nm. The structures of oligonucleotides 1, 2 and 4 are shown in Figure 1.

Shorter or longer interaction time produces the nanoparticles with correspondingly lower absorbance changes. At room temperature (17 °C), 1 exists as a single-stranded form. Increasing the incubating temperature accelerates the adsorption process of ssDNA to Au-NPs, and thus, the Au-NPs in solution are more stable at high temperature (Figure S7, SI). Moreover, the hairpin structure of 1 formed by Hg2+ would be dehybridized at high temperature, which decreases the absorbance at 750 nm. Thus, the absorption changes (∆A750) of the system by Hg2+ will decrease with increase of temperature. When the temperature reached 45 °C, the aggregated Au-NPs deposited as the strong thermodynamically interactions. The proposed approach thus works well at room temperature. Optimization of the Variables of the Measuring System. The influences of different DNA sequences on Hg2+ response were explored. The hybridized dsDNA would not help to stabilize Au-NPs at high salt solutions even in the presence of a high concentration of Hg2+. In Figure 6, the fluorescence enhancement, F/F0, for 1, and 2 and 4, at λex/λem ) 480 nm/518 nm, are plotted as functions of the Hg2+ concentrations in the Tris-HCl buffer solution (0.1 M NaCl), where F0 and F are the fluorescence intensity of FAM in the absence and the presence of Hg2+. Obviously, the sensitivity of the analytical system would be dependent on the length of the DNA strand. For 1, which contains 7-mer T-T mismatched base pairs for Hg2+ coordination, the best response sensitivity was obtained at lower Hg2+ concentration compared with that obtained by using 2 and 4; however, at high Hg2+ concentration, the response sensitivity is significantly smaller than that obtained using 2 and 4. We also employed five DNA strands that contain different thymine-rich mercury-binding sequences and the linker sequence; different response sensitivities for the Hg2+ were realized by using different DNA strands (Figure S8, SI).The flexibility of DNA sequence controlling allows us to monitor the Hg2+ concentration with appropriate adjustment of the number of T-T mismatched bases, so that the sample response falls within the most sensitive response region. The fluorescence response to Hg2+ was influenced by the acidity of the solution. Figure S9 (SI) depicts the pH dependence of fluorescence intensity enhancement of 1 by Hg2+ ions. The titration experiments were carried out by adding standard NaOH or H2SO4 solution to a solution of 1/Au-NPs as well as Hg2+. The

Figure 7. Effects of Au-NPs concentration on the fluorescence intensity of 1 (40 nM) in the absence (b) and the presence of 1.6 µM Hg2+ (1) in the Tris-HCl buffer solution containing 0.1 M NaCl. Inset: the emission enhancement of 1 by 1.6 µM Hg2+ as a function of AuNPs concentration, where F0 and F are FAM fluorescence intensity in the absence and the presence of 1.6 µM Hg2+. The excitation was at 480 nm, and emission was recorded at 518 nm.

fluorescence intensity ratio of the system, F/F0, increased with pH and reached a plateau when the pH is ∼7.0, where F0 and F are the FAM fluorescence intensity at λex/em ) 480/518 nm in the absence and the presence of Hg2+, respectively. It is obvious that the F/F0 is independent of pH in the range of 7.0-10.0. At a pH below 7.0, protonation of the nitrogen atoms of the thymine base reduces its affinity with Hg2+, while at relatively higher pH (>10.0), Hg2+ ions may be complexed by OH-, which, in turn, reduces its complex with the ssDNA. In our experiment, we chose Tris-HCl (pH 7.4) as buffer system. The fluorescence response of 1/Au-NPs to Hg2+ is strongly dependent on the relative amount of the ssDNA and Au-NPs. This can be seen in the variation of the response characteristics (working range and response sensitivity) due to the different amount ratios of 1 and Au-NPs. In Figure 7, the fluorescence intensity changes of 1 (40 nM) in the absence and the presence of Hg2+ are plotted as functions of the concentration of Au-NPs at pH 7.4 in 0.1 M NaCl solution. The fluorescence intensity of 1 is significantly decreased with increase in the nanoparticle concentration. In our experiment, 98.4% quenching was observed when the molar ratio of Au-NPs to 1 reaches 1:50. In the presence of Hg2+, formation of dsDNA does reduce adsorption of 1 onto the nanoparticles and thus increases the fluorescence signal compared with that without Hg2+ at the same conditions. Higher concentration of nanoparticles in the system results in the weaker blank fluorescence signal value, but the fluorescence recovery is also decreased; while at relatively lower nanoparticles concentration, the background fluorescence is high, and the response sensitivity will reduce even if a high concentration of Hg2+ ion was added to the solution. The best response sensitivity emerged when the concentration of Au-NPs was 0.5-1.5 nM (Figure 7, inset). Sensitivity and Selectivity. The fluorescence emission properties of FAM are sensitive to the presence of nanomolar concentrations of Hg2+. As shown in Figure 5, depending on the Hg2+ concentration range, there are different response sensitivities. The small but perceptible change at a low concentration of Hg2+ indicates that Hg2+ ions are capable of interacting with 1, even at very low concentration. The fitting curve of the inset of Figure 5 can serve as the calibration curve for the detection of Hg2+ concentration. A practically usable range for quantitative

Figure 8. Photographs of 1/Au-NP in the Tris-HCl buffer (0.1 M NaCl) in the presence of different metal ions. (A) In the absence of Hg2+; (B) in the presence of 6.0 µM Hg2+. The concentration of all other metal ions is 50 µM.

determination covers a range from 6.4 × 10-6 to 9.6 × 10-8 M (0.05 e R e 0.95).67 At Hg2+ concentrations more than 1.0 × 10-5 M, no further increase in fluorescence is observed and a plateau is reached. The detection limit that is taken to be three times the standard derivation in blank solution is found to be 4.0 × 10-8 M. In order to compare the detection sensitivity of fluorometry and absorptiometry in the present approach, the absorption change of 1/Au-NPs containing various concentrations of Hg2+ was obtained under the optimum conditions (pH 7.4, 0.1 M NaCl) (Figure S10, SI). Increase of the Hg2+ concentrations causes a significant decrease of the absorbance at 520 nm and dramatic increase of the absorbance at 750 nm. It is clear that the dynamic range of fluorometry shifts to low Hg2+ concentration range with respect to that of absorptiometry. At low concentration of Hg2+, there is no real change in the absorbance of Au-NPs while there is clearly a significant increase in the fluorescence intensity of 1 upon increasing Hg2+ concentration. The reduction of the limit of detection caused by fluorometry can be explained by the intrinsic relationship between fluorescence and absorbance. i.e., the changes in the absorption of the absorber translate into exponential changes in fluorescence. As a result, even small changes in absorbance cause a substantially large change in the effective intensity of fluorescence emission of the dye. An important criterion for a metal ion sensor is the ability to recognize a specific metal in the vicinity of other substances. The selectivity of 1/Au-NPs for Hg2+ was first evaluated by visual assay to environmentally relevant metal ions, including Cd2+, Fe3+, Mn2+, Cu2+, Zn2+, Ba2+, Mg2+, Ca2+, Ni2+, Pb2+, and Co2+, with the naked eye. In the buffer solution at room temperature, all others metal ions (50 µM) have turned a bright red even if 0.1 M NaCl was added (Figure 8A), whereas the Hg2+ (6.0 µM) sample displays blue-gray at the same conditions. Moreover, upon addition of Hg2+ to 1/Au-NPs solution containing of other metal ions, separately, the color of the solutions changed to blue-gray (Figure 8B). These results clearly indicate that the approach is not only insensitive to other metal ions, but also selective toward Hg2+ in their presence. We also investigated the changes in the fluorescence spectra of 1/Au-NPs that occurred by addition of some common anions, which show no obvious interference for detection of Hg2+. However, substrates that could interact with Hg2+, such as a high concentration of S2- and other thiol compounds, reduce the fluorescence enhancement. To quantify the Hg2+ specificity of the approach, complex experiments of the metal ions with different concentrations were (67) Choi, M. M. F.; Wu, X. J.; Li, Y. R. Anal. Chem. 1999, 71, 1342–1349.


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Table 1. Determination of Hg2+ in Water Samples Using the Proposed Method and ICPMS Hg2+(µM) sample

added

proposed method meana ± SDb

ICPMS mean ± SD

tap water 1 tap water 2 tap water 3 lake water 1 lake water 2 lake water 3

0.15 0.75 4.00 0 0.40 1.50

0.16 ± 0.01 0.75 ± 0.03 4.10 ± 0.18 c 0.42 ± 0.03 1.48 ± 0.14

0.18 ± 0.05 0.79 ± 0.06 4.27 ± 0.35 0.07 ± 0.01 0.42 ± 0.03 1.52 ± 0.11

a Mean of three determinations. b SD, standard deviation. c No Hg2+ concentration could be detected.

Figure 9. Fluorescence enhancements of 1/Au-NPs in the Tris-HCl buffer (0.1 M NaCl) by increasing concentrations of Ca2+, Pb2+, Co2+, Mn2+, Cu2+, Zn2+, Mg2+, Ni2+, Cd2+, Fe3+, and Hg2+, separately, as well as a mixture of metal ions (for concentrations of the metal ions, see text) with increasing concentrations of Hg2+. The excitation was at 480 nm, and emission was recorded at 518 nm.

performed by fluorescence spectroscopy. The fluorescence response of the system to Hg2+ ion presents excellent selectivity in comparison with other heavy metal ions. Figure 9 illustrates the fluorescence intensity increase, F/F0, upon additions of different concentrations of metal ions as well as a mixture containing 1.0 mM Mg2+ and Ca2+ and 50.0 µM Cu2+, Mn2+, Cd2+, Ni2+, Zn2+, Pb2+, and Co2+ with increasing amounts of Hg2+. All other metal ions could not induce any change in the 1 fluorescence even with a high concentration of the metal ion present in the sample solution. Additionally, a titration of Hg2+ in the presence of the interfering metal ions gives a curve almost superimposable on the one obtained exclusively in presence of Hg2+. No significant variations in the fluorescence intensity were found by comparison with those without metal ions other than Hg2+, which is important and helpful in validation of the method to meet the selectivity requirements of the Hg2+ assay in environmental and biological fields. Assay of Hg2+ Concentrations in Water Samples. The applications of the proposed method were evaluated for determination of Hg2+ in both tap and river water samples. For tap water, the sample was collected after discharging tap water for ∼20 min and boiled for 5 min to remove chlorine. River water sample was obtained from Weiming Lake on the campus of Peking University. The sample collected was first filtered through a column (packed with an anionic-exchange resin) to remove oils and other organic impurities. The response range of the proposed method is 9.6 × 10-8-6.4 × 10-6 M Hg2+, at pH 7.4, and thus not yet adequate for environment monitoring in the 10-8-10-9 M range.68 All the water samples were spiked with Hg2+ at different concentration levels, which were prepared on the basis of possible metal ions presenting in the environmental water and then analyzed with the method proposed. The results are summarized in Table 1 and show good agreement with the expected and found values. (68) The toxic level defined by U.S. Environmental Protection Agency.

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CONCLUSION We have developed a new colorimetric and fluorescent sensing system for homogeneous assay of Hg2+ in aqueous media with high selectivity and sensitivity. The metal recognition and transduction mechanism is one based on the metal-induced conformational change of thymine-rich ssDNA and the difference in electrostatic affinity between ssDNA and dsDNA with gold nanoparticles. For certain salt concentrations, ssDNA adsorbs on citrate-coated Au-NPs while dsDNA does not, and this fact can be exploited to different colors of the nanoparticles and quenched fluorescence of a dye-labeled ssDNA. The recognition of Hg2+ gave rise to major color change of the nanoparticles that was clearly visible to the naked eye, while the Hg2+ quantification could be achieved by fluorescence enhancement of dye-labeled ssDNA. This method requires no covalent modification of the AuNPs, and uses only commercially available materials, but it exhibits higher sensitivity and more convenient than similar approaches for Hg2+ detection. Although we have demonstrated here the detection of Hg2+ ions only, significantly, this method can in principle be used to detect different analytes, such as other metal ions, DNA, or proteins, by substituting this thymine-rich oligonucleotide with synthetic artificial bases that selectively bind the other analytes. Currently, intensive research using new oligonucleotides by substituting the thymine for other metal ions is being conducted in our laboratory, and the results will be reported in due course. ACKNOWLEDGMENT Financial support from the National Outstanding Youth Foundation of China (20525518) and the National Natural Science Foundation of China (20775005) is highly acknowledged. SUPPORTING INFORMATION AVAILABLE Additional spectroscopic data (absorption and color changes, influence of other substrates on fluorescence response, kinetic data, and anisotropy). This material is available free of charge via the Internet at http://pubs.acs.org. Received for review July 4, 2008. Accepted October 7, 2008. AC801382K

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