Electrochemical Sensor for Mercury (II) Based on Conformational

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Anal. Chem. 2009, 81, 5724–5730

Electrochemical Sensor for Mercury(II) Based on Conformational Switch Mediated by Interstrand Cooperative Coordination Si-Jia Liu, Hua-Gui Nie, Jian-Hui Jiang,* Guo-Li Shen, and Ru-Qin Yu State Key Laboratory for Chemo/biosensing and Chemometrics, College of Chemistry and Chemical Engineering, Hunan University, Changsha 410082, People’s Republic of China A novel electrochemical sensor was developed for sensitive and selective detection of mercury(II), based on thymine-Hg2+-thymine (T-Hg2+-T) coordination chemistry. This strategy exploited the cooperativity of proximate poly-T oligonucleotides in coordination with Hg2+. Ferrocene (Fc)-tagged poly-T oligonucleotides were immobilized on the electrode surface via self-assembly of the terminal thiol moiety. In the presence of Hg2+, a pair of poly-T oligonucleotides could cooperatively coordinate with Hg2+, which triggered a conformational reorganization of the poly-T oligonucleotides from flexible single strands to relatively rigid duplexlike complexes, thus drawing the Fc tags away from the electrode with a substantially decreased redox current. The response characteristics of the sensor were thoroughly investigated using capillary electrophoresis and electrochemical measurements. The results revealed that the sensor showed a sensitive response to Hg2+ in a concentration range from 1.0 nM to 2.0 µM, with a detection limit of 0.5 nM. Also, this strategy afforded exquisite selectivity for Hg2+ against a reservoir of other environmentally related metal ions, compared to existing anodic stripping voltammetry (ASV) techniques. In addition, this sensor could be implemented using minimal reagents and working steps with excellent reusability through mild regeneration procedure. It was expected that this cost-effective electrochemical sensor might hold considerable potential in on-site applications of Hg2+ detection. Mercury ions, which represent a widespread highly toxic contaminant in aquatic ecosystems, pose severe risk for human health and the environment.1,2 It is known that microbial biomethylation of mercury ions leads to accumulation of mercury in human body through food chain, which will cause serious and permanent brain damage and other chronic diseases.3,4 Therefore, sensitive and on-site detection of Hg2+ in aqueous media is of great concern in environmental and food monitoring, as well * To whom correspondence should be addressed. Tel.: 86-731-8821961; Fax: 86-731-8821916. E-mail: [email protected]. (1) Bolger, P. M.; Schwetz, B. A. New Engl. J. Med. 2002, 347, 1735–1736. (2) Stern, A. H. Environ. Res. 2005, 98, 133–142. (3) Morel, F. M. M.; Kraepiel, A. M. L.; Amyot, M. Annu. Rev. Ecol. Syst. 1998, 29, 543–566. (4) Onyido, I.; Norris, A. R.; Buncel, E. Chem. Rev. 2004, 104, 5911–5929.

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as clinical toxicology. Varying strategies for the detection of Hg2+ have been developed, including optical techniques based on organic molecular probes,5-9 metal/semiconductor nanoparticles,10,11 or conjugated polymers12,13 and electrochemical methods using anodic stripping voltammetry (ASV).14,15 Most of these procedures are based on simple coordination or redox chemistry properties of Hg2+, and their selectivity are heavily limited. Because trace levels of heavy metals often coexist with a million-fold excess of other ionic species, selectivity in metal ions detection is of crucial significance, and there is growing interest in developing highly selective and sensitive sensors for Hg2+ using exquisite biological components such as DNAzyme16 and metalloregulatory protein.17 Very recently, thymine-Hg2+-thymine (T-Hg2+-T) coordination chemistry and the resulting Hg2+-stabilized hybridization of oligonucleotides with T-T mismatches18,19 have been demonstrated as a selective and versatile strategy for Hg2+ detection. Prominent examples include colorimetric sensors based on Hg2+-mediated aggregation of gold nanoparticles20-22 or Hg2+-promoted amplification of HRP-mimicking DNAzyme,23 and fluorescence probes based on Hg2+-modulated resonance (5) Prodi, L.; Bargossi, C.; Montalti, M.; Zaccheroni, N.; Su, N.; Bradshaw, J. S.; Izatt, R. M.; Savage, P. B. J. Am. Chem. Soc. 2000, 122, 6769–6770. (6) Nolan, E. M.; Lippard, S. J. J. Am. Chem. Soc. 2003, 125, 14270–14271. (7) Zhu, X. J.; Fu, S. T.; Wong, W. K.; Guo, J. P.; Wong, W. Y. Angew. Chem., Int. Ed. 2005, 45, 3150–3154. (8) Yang, Y. K.; Yook, K. J.; Tae, J. J. Am. Chem. Soc. 2005, 127, 16760– 16761. (9) Nolan, E. M.; Lippard, S. J. J. Am. Chem. Soc. 2007, 129, 5910–5918. (10) Zhu, C.; Li, L.; Fang, F.; Chen, J.; Wu, Y. Chem. Lett. 2005, 34, 898–899. (11) Huang, C. C.; Chang, H. T. Anal. Chem. 2006, 78, 8332–8338. (12) Kim, I. B.; Bunz, U. H. F. J. Am. Chem. Soc. 2006, 128, 2818–2819. (13) Liu, X.; Tang, Y.; Wang, L.; Zhang, J.; Song, S.; Fan, C.; Wang, S. Adv. Mater. 2007, 19, 1471–1474. (14) Nolan, M. A.; Kounaves, S. P. Anal. Chem. 1999, 71, 3567–3573. (15) Wang, S. P.; Forzani, E. S.; Tao, N. J. Anal. Chem. 2007, 79, 4427–4432. (16) Hollenstein, M.; Hipolito, C.; Lam, C.; Dietrich, D.; Perrin, D. M. Angew. Chem., Int. Ed. 2008, 47, 4346–4350. (17) Chen, P.; He, C. J. Am. Chem. Soc. 2004, 126, 728–729. (18) 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. (19) Tanaka, Y.; Oda, S.; Yamaguchi, H.; Kondo, Y.; Kojima, C.; Ono, A. J. Am. Chem. Soc. 2007, 129, 244–245. (20) Lee, J. S.; Han, M. S.; Mirkin, C. A. Angew. Chem., Int. Ed. 2007, 46, 4093– 4096. (21) Xue, X. J.; Wang, F.; Liu, X. G. J. Am. Chem. Soc. 2008, 130, 3244–3245. (22) Liu, C. W.; Hsieh, Y. T.; Huang, C. C.; Lin, Z. H.; Chang, H. T. Chem. Commun. 2008, 2242–2244. 10.1021/ac900527f CCC: $40.75  2009 American Chemical Society Published on Web 06/12/2009

energy transfer24,25 or double-strand intercalation of fluorescent dyes.26,27 Electrochemical sensors using redox-tagged oligonucleotides constitute a potential platform for biological analysis. A fundamental mechanism for such sensors is the alteration of distance of redox labels from the electrode via analyte-induced conformational switch,28-32 strand dissociation,33 or surface hybridization34-36 of certain oligonucleotide probes that carry the electroactive reporters. Because of surface-selective mode in detecting the redox tags, these sensors can be implemented using minimal reagents and working steps together with high sensitivity. In this context, the development of such electrochemical sensors for Hg2+ detection is expected to be a significant advance, because of their superb compatibility with on-site applications, especially when these biosensors exhibit excellent selectivity for Hg2+ against a reservoir of other metal ions, in contrast to existing ASV techniques. Motivated by our previous observation that the surface-tethered proximate probes could work cooperatively to interact with target molecules,34 we reported herein, for the first time, a novel electrochemical sensor for Hg2+ based on the T-Hg2+-T coordination chemistry. This sensor relied on cooperative coordination with Hg2+ by short ferrocene (Fc)-tagged poly-T oligonucleotides immobilized on the electrode surface. Such cooperative interactions induced a conformational switch of the Fc-tagged oligonucleotides from single-strand to a duplexlike structure, which draws the ferrocenecarboxylic acid (Fc) tags away from the electrode with a substantially decreased redox current. The results revealed that the developed sensor afforded a highly selective and sensitive strategy for the detection of Hg2+, and the strategy was reusable and could be implemented with minimized reagents and working steps. EXPERIMENTAL SECTION Chemicals and Reagents. The synthesized oligonucleotides (the sequences are shown in Table 1), all HPLC-purified and lyophilized, were provided by TaKaRa Biotech. Co., Ltd. (Dalian, PRC). Ferrocenecarboxylic acid (Fc), mercury pernitrate, adenine, 1-ethyl-3-(3-dimethyl-aminopropyl) carbodiimide hydrochloride (EDC), and N-hydroxysulfosuccinimide (Sulfo-NHS) were pur(23) Li, D.; Wieckowska, A.; Willner, I. Angew. Chem., Int. Ed. 2008, 47, 3927– 3931. (24) Ono, A.; Togashi, Hi. Angew. Chem., Int. Ed. 2004, 43, 4300–4302. (25) Liu, J.; Lu, Y. Angew. Chem., Int. Ed. 2007, 46, 7587–7590. (26) Chiang, C. K.; Huang, C. C.; Liu, C. W.; Chang, H. T. Anal. Chem. 2008, 80, 3716–3721. (27) Wang, J.; Liu, B. Chem. Commun. 2008, 4759–4761. (28) Fan, C. H.; Plaxco, K. W.; Heeger, A. J. Proc. Natl. Acad. Sci., U.S.A. 2003, 100, 9134–9137. (29) Xiao, Y.; Lubin, A. L.; Heeger, A. J.; Plaxo, K. W. Angew. Chem., Int. Ed. 2005, 44, 5456–5459. (30) Radi, A. E.; Acero Sa′nchez, J. L.; Baldrich, E.; O’Sullivan, C. K. J. Am. Chem. Soc. 2006, 128, 117–124. (31) Xiao, Y.; Rowe, A. A.; Plaxco, K. W. J. Am. Chem. Soc. 2007, 129, 262– 263. (32) Willner, I.; Zayats, M. Angew. Chem., Int. Ed. 2007, 46, 6408–6418. (33) Wu, Z. S.; Guo, M. M.; Zhang, S. B.; Chen, C. R.; Jiang, J. H.; Shen, G. L.; Yu, R. Q. Anal. Chem. 2007, 79, 2933–2939. (34) Zhang, Y. L.; Huang, Y.; Jiang, J. H.; Shen, G. L.; Yu, R. Q. J. Am. Chem. Soc. 2007, 129, 15448–15449. (35) Huang, Y.; Zhang, Y. L.; Xu, X. M.; Jiang, J. H.; Shen, G. L.; Yu, R. Q. J. Am. Chem. Soc. 2009, 131, 2478–2480. (36) Zhang, Y. L.; Wang, Y.; Wang, H. B.; Jiang, J. H.; Shen, G. L.; Yu, R. Q.; Li, J. H. Anal. Chem. 2009, 81, 1982–1987.

Table 1. Synthesized Oligonucleotides (5′ f 3′) Used in the Experimentsa probe

oligonucleotide

1 2 3 4 5

SH-(CH2)6-TTT TTT TT-(CH2)6-NH2 SH-(CH2)6-CCC CCC CC-(CH2)6-NH2 SH-(CH2)6-TTT TTT TT-(CH2)6-FAM TTT TTT TT AAA AAA AA

a Probes 1 and 2 have a -NH2 moiety at the 3′ terminus for subsequent modification with Fc, probe 3 is used to determine the surface coverage, and probes 4 and 5 are used in capillary electrophoresis experiments.

chased from Sigma-Aldrich. All the other reagents were obtained from the Sinopharm Chemical Reagent Co., Ltd. (Shanghai, PRC). Ultrapure water with an electric resistance of >18 MΩ was supplied through a Milli-Q water purification system (Millipore, Billerica, MA). Labeling of Fc to NH2-Modified Oligonucleotides. The Fc label was conjugated to the 3′ NH2-moiety of the oligonucleotide (probe 1 or 2), using the succinimide coupling (EDCNHS) method.37 Briefly, 100 µL of 10 µmol oligonucleotide (probe 1 or 2) was mixed with 100 µL of 10 mM PBS (pH 7.4) containing 10 mmol of ferrocenecarboxylic acid, 1 mM EDC, and 5 mM Sulfo-NHS, followed by incubation for 2 h at 37 °C. The conjugate was dialyzed against 10 mM PBS (500 mL) for 24 h to remove excessive ferrocenecarboxylic acid. The resulting solution was stored at -20 °C in a freezer. Gold Electrode Treatment and Oligonucleotide Immobilization. Prior to the experiment, the working gold electrodes, 99.99% (w/w) polycrystalline with a diameter of ∼2 mm (CH Instruments, Inc.), were cleaned by immersing them in warm piranha solution (30% H2O2/concentrated H2SO4, 1:3 by volume) for 2 h three times and then washed with ultrapure water. Subsequently, the electrodes were polished on microcloth (Buehler) with a γ-alumina 0.05 µm suspension (CH Instruments, Inc.) for 3 min, followed by sonication in ultrapure water, ethanol, and ultrapure water for 5 min in each. Finally, the electrode were again rinsed thoroughly with ultrapure water and dried in a nitrogen stream. The roughness factor of the electrode surface was estimated to be 1.6 ± 0.1 via determination of the gold electrode area using the gold oxide stripping peak area obtained in cyclic voltammetry (CV) measurements in 0.5 M H2SO4.38 The immobilization of the Fc-tagged probe (probe 1 or 2) on the gold electrode was performed by adding 20 µL of a solution containing 0.5 µM Fc-tagged probe, 1.0 M NaCl in 10 mM PBS buffer (pH 7.4) dropwise on a gold electrode held upside-down. The self-assembly was allowed to proceed for 24 h at room temperature. The electrode then was rinsed thoroughly with a 33 mM PBS buffer (pH 6.9) solution containing 0.5 M NaCl and 0.1 M NaClO4 three times. The biosensor thus obtained was stored at 4 °C in the PBS buffer solution and was observed to maintain the activity for more than one month. The surface coverage of the surface-tethered oligonucleotides was determined using a synthesized probe (3) with an alkanethiol (37) Fahlman, R. P.; Sen, D. J. Am. Chem. Soc. 2002, 124, 4610–4616. (38) Salaun, P.; van den Berg, C. M. G. Anal. Chem. 2006, 78, 5052–5060.

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Scheme 1. Electrochemical Sensing Strategy for Hg2+ Based on Conformational Switch Mediated by T-Hg2+-T Coordinationa

a Poly(T) probe display flexible random-coil conformation in the absence of Hg2+, allowing Fc tags to approach the electrode with electron efficiently transferred. Duplexlike complex forms when two proximate probes cooperatively coordinate with Hg2+, inducing conformational switch and drawing Fc tags away from the electrode with decreased redox current.

moiety at the 5′ terminal and a FAM-label at the 3′ end. The fluorescent thiolated oligonucleotide 3 was assembled on the electrode under the aforementioned conditions, and the surface coverage of probe 3 was then determined using a procedure reported previously.39,40 Capillary Electrophoresis Detection. All the capillary electrophoresis experiments were performed using a capillary electrophoresis system equipped with UV absorption detection (P/ AGE MDQ, Beckman, Germany) under an applied potential of 15 kV, using a quartz capillary with a diameter of 75 µm (total length, 50 cm; effective length, 30 cm) in a 50 mM borate buffer (pH 8.3) solution. The temperature of the separation (15 °C) was controlled by immersion of the capillary in a cooling liquid circulating in the cartridge. Samples were injected into the capillary using the pressure injection mode at 0.5 psi for 5 s and detected at 254 nm for 30 min. Between successive runs, the capillary was rinsed with ultrapure water and running buffer for 15 min, respectively. Samples containing a 50 µM probe (4), Hg2+ of varying concentration (0, 50, 100, 300 µM), or a 25 µM probe (5), and an internal standard of 500 µM adenosine, were prepared using a 33 mM PBS buffer (pH 6.9) solution containing 0.5 M NaCl and 0.1 M NaClO4. Electrochemical Detection. To detect Hg2+ or other metal ions, the sensor was immersed in 50 µL of a 33 mM PBS buffer (pH 6.9) solution containing 0.5 M NaCl, 0.1 M NaClO4, 20 mM adenine, and Hg2+ of specified concentration or 10 µM other metal ions for 1.5 h at room temperature (∼25 °C), followed by washing for 1 min in 40 mL of a 33 mM PBS buffer (pH 6.9) solution containing 0.5 M NaCl and 0.1 M NaClO4. All electrochemical measurements including differential pulse voltammetry (DPV), cyclic voltammetry (CV), and electrochemical impedance spectroscopy (EIS) were performed at room temperature using a three-electrode system that consists of a KCl saturated calomel reference electrode (SCE), a platinum counter electrode, and the working electrode (gold electrode). DPV and CV were executed on an Autolab PGSTAT12/FRA2 potentiostat/ galvanostat system (Eco Chemie, Utrecht, The Netherlands). EIS was performed using a CH Instruments Model 760C electrochemical analyzer (Shanghai, PRC). DPV was recorded in a 33 mM PBS buffer (pH 6.9) solution containing 0.5 M NaCl and 0.1 M NaClO4 (39) Steel, A. B.; Levicky, R. L.; Herne, T. M.; Tarlov, M. J. Biophys. J. 2000, 79, 975–981. (40) Demers, L. M.; Mirkin, C. A; Mucic, R. C.; Reynolds, R. A., III; Letsinger, R. L.; Elghanian, R.; Viswanadham, G. Anal. Chem. 2000, 72, 5535–5541.

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within the potential range from 0 V to 0.5 V (vs SCE) under a modulation amplitude of 25 mV and a scan rate of 10 mV/s with a step potential of 1 mV. CV was performed in a 33 mM PBS buffer (pH 6.9) solution containing 0.5 M NaCl and 0.1 M NaClO4 within the potential range from -0.1 V to 0.5 V using a step potential of 0.01 V at the specified potential scan rate. EIS was performed in 67 mM PBS (pH 7.4) containing 5 mM Fe(CN)63-/Fe(CN)64- and 0.1 M KCl in the frequency range from 0.1 Hz to 100 kHz with 5 mV as the amplitude at a bias potential of 0.22 V (vs SCE). The reported DPV curves were background-subtracted using the GPES version 4.9.007 software (Eco Chemie, Utrecht, The Netherlands) through extrapolation to the baseline in the regions far from the peaks.41 To investigate the selectivity of the sensor, various metal salts including Hg(NO3)2 (1 µM), ZnNO3, Al2(SO4)3, Ca(NO3)2, MgCl2, Pb(NO3)2, CrCl3, Mn(CH3COO)2, Co(CH3COO)2, CdCl2, FeCl3, and CuCl2 (each at 10 µM concentration) were analyzed using the sensor. To evaluate the performance of the developed electrochemical strategy, eight samples were collected at eight sewage outfalls along the Xiang River (Changsha, PRC). These samples were diluted using equal volumes of a 33 mM PBS buffer (pH 6.9) solution containing 0.5 M NaCl, 0.1 M NaClO4, and 20 mM adenine. They were then filtered through a 0.22 µm membrane prior to analysis, using the described method and ICP-MS Sciex Elan 5000 spectrometer (Perkin-Elmer, Norwalk, CT). RESULTS AND DISCUSSION Analytical Principle of the Electrochemical Sensor. The developed electrochemical sensor relies on cooperative coordination with Hg2+ by a pair of short poly-T oligonucleotides with a concomitant conformational switch from single-strand to a duplexlike structure, as shown in Scheme 1. The sensor system comprises an eight-T oligonucleotide probe (1) with an alkanethiol moiety at the 5′-terminus and a ferrocene (Fc) tag at the 3′-end. The poly-T oligonucleotide is immobilized on a gold electrode surface via self-assembly through the thiol anchor. (The surface coverage is ∼6.1 × 1013 strands/cm2, as determined using a method previously reported.39,40) In the absence of Hg2+, the single-stranded oligonucleotides display a flexible random-coil conformation that allows the Fc labels to approach the electrode (41) Hirst, J.; Duff, J. L. C.; Jameson, G. N. L.; Kemper, M. A.; Burgess, B. K.; Armstrong, F. A. J. Am. Chem. Soc. 1998, 120, 7085–7094.

Figure 2. Electropherograms of (a) 50 µM poly-T, (b) 50 µM poly-T and 25 µM poly-A, (c) 50 µM poly-T and 50 µM Hg2+, (d) 50 µM poly-T and 100 µM Hg2+, and (e) 50 µM poly-T and 300 µM Hg2+. The internal standard (IS) was 500 µM adenosine. The sample buffer is a 33 mM PBS (pH 6.9) solution containing 0.5 M NaCl and 0.1 M NaClO4 in CV and CE experiments.

Figure 1. (A) Typical CV curves of the sensor in response to 0 M (solid line), 50 nM (dotted line), and 10 µM (dashed line) Hg2+, respectively (scan rate ) 100 mV/s). (B) Typical DPV curves of the sensor in response to 0 M (solid line), 50 nM (dotted line), and 10 µM (dashed line) Hg2+, respectively. DPV was recorded in a 33 mM PBS buffer (pH 6.9) solution containing 0.5 M NaCl and 0.1 M NaClO4 within the potential range from 0 V to 0.5 V (vs SCE) under a modulation amplitude of 25 mV and a scan rate of 10 mV/s with a step potential of 1 mV. (C) Typical CV curves of probe 2-modified electrode in response to 0 M (solid line) and 10 µM (dotted line) Hg2+, respectively (scan rate ) 100 mV/s).

and transfer the electrons efficiently.42 On introducing Hg2+ in the system, the poly-T oligonucleotides can cooperatively coordinate with Hg2+, because of their close proximity (∼1 nm for average interstrand distance) and form a duplexlike complex between two adjacent strands. The duplexlike complex is as relatively rigid as a normal DNA duplex and then undergoes a conformational reorganization that draws the Fc tags away from the electrode, resulting in a substantially decreased redox current attributed to the increased electron-tunneling distance. Typical voltammetric characteristics of the sensor, in response to Hg2+, are shown in Figure 1. One observed in Figure 1A shows that, in the absence of Hg2+, a couple of strong redox peaks (42) Anne, A.; Bouchardon, A.; Moiroux, J. J. Am. Chem. Soc. 2003, 125, 1112– 1113.

appeared at 0.162 and 0.273 V (vs SCE) in CVs, characteristic of the electrochemistry of Fc at the electrode. With the addition of Hg2+ up to 50 nM, substantially diminished redox peaks were observed. In the presence of Hg2+ at higher concentration (10 µM), the couple of redox peak almost disappeared. Both observations implied that the sensor was sensitively responsive to Hg2+. It was reported that Hg2+ could not remove thiolated oligonucleotides from the gold surface;20 then, one reasoned that the response was attributed to the interaction of the poly-T probe with Hg2+. Better resolution of these observations was achieved in DPV measurements, as shown in Figure 1B. It was observed that the sensor with a poly-T probe 1 gave a remarkable reduction peak (the standard deviation (SD) across 10 repetitive experiments was ∼2.4%) at ∼0.213 V (vs SCE) in the DPV curve in the absence of Hg2+, and the peak currents decreased appreciably by 40%, in response to 50 nM Hg2+, and by 96%, in response to 10 µM Hg2+ (the SD across 10 repetitive experiments was ∼5.8%), respectively. Moreover, a further control experiment with Fc-tagged poly-C oligonucleotide (2) was performed, and the results are depicted in Figure 1C. The sensor with poly-C probe 2 clearly also gave a remarkable reduction peak at ∼0.213 V (vs SCE) in the DPV curve in the absence of Hg2+; however, the introduction of Hg2+ had a negligible effect on the redox peaks, evidencing that the poly-T probe 1 interacted with Hg2+ selectively via the T-Hg2+-T coordination chemistry. Capillary Electrophoresis Characterization of Interaction between poly-T Probe and Hg2+. To characterize the interaction between poly-T probe and Hg2+, capillary electrophoresis experiments were performed. Figure 2 shows the electropherograms of poly-T probe 4 in the presence of Hg2+ of varying concentrations. It was observed that, in the absence of Hg2+, poly-T probe 4 only gave a strong peak (curve a in Figure 2). In the presence of an increasing Hg2+ concentration (50 and 100 µM), the poly-T peak became diminished, and one or two additional peaks appeared behind the poly-T peak (curves c and d in Figure 2). The poly-T peak almost disappeared (curve e in Figure 2) in the presence of a higher Hg2+ concentration (300 µM). It was also noticed that DNA duplexes between poly-T probe 4 and poly-A probe 5 exhibited lower electrophoretic mobility than poly-T (curve b in Figure 2). It was reported previously that the folded structure of poly-T probes mediated by T-Hg2+-T coordination Analytical Chemistry, Vol. 81, No. 14, July 15, 2009

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Figure 4. Nyquist plot (Zim vs Zre) for electrochemical impedance measurements in PBS buffer (1/15 M, pH 7.4) solution containing 5.0 mM Fe(CN)63-/4- (1:1 mixture) at a open-circuit potential of 0.24 V for (a) bare gold electrode, (b) the probe 1-modified gold electrode, (c) the probe 1-modified gold electrode after reaction with 100 nM Hg2+ ions, and (d) the probe 1-modified electrode after reaction with 1 µM Hg2+ ions. (Inset shows the equivalent circuit.) Table 2. Calculated Values for the Elements in the Equivalent Circuit

Figure 3. CV curves of the sensor in response to (A) 0 M Hg2+ and (B) 50 nM Hg2+ at varying potential scanning rates of 10, 40, 60, 80, 100, 120, and 140 mV/s. (C) Plot of the oxidation peak currents versus scan rates.

gave increased electrophoretic mobility.26 One then might infer that the two peaks that appeared in a poly-T and Hg2+ mixture were attributed to the duplexlike complexes between poly-T probes and Hg2+ with different numbers of T-Hg2+-T pairs. These findings gave immediate evidence for the formation of duplexlike complexes between poly-T probes and Hg2+, thus confirming the putative mechanism of the electrochemical sensor. A close electrophoretic inspection of a six-base poly-T in the absence or presence of Hg2+ revealed that no duplexlike complex between the poly-T probe and Hg2+ formed under the reaction conditions, implying that the two electrophoretic peaks for the eight-base poly-T probe and Hg2+ mixture were derived from the duplexlike complexes between poly-T probes and Hg2+ with 7 and 8 T-Hg2+-T pairs, respectively. Electrochemical Characterization of the Sensor. To study the controlled factor of the electrochemical process on the electrode surface, the effect of scan rates on the CV peak currents was investigated. As shown in Figures 3A and 3C, in the absence of Hg2+ the reduction peak currents of the sensor were observed to increase in linear correlation to the scan rate in 5728

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Hg2+ concentration

Rs (Ω)

Rct (Ω)

Rw (Ω s1/2)

CPE (µF/cm2)

0M 100 nM 10 µM

129.5 128.6 136.1

7260 4910 1814

2274 2180 1853

18.3 20.1 24.5

the range of 10-140 mV/s. A linear dependency of the reduction peak currents on the scan rates was also obtained for the sensor in the presence of Hg2+, as shown in Figures 3B and 3C. These observations manifested that the sensor demonstrated typical surface-bound electrochemical processes and the Fc tags were confined to the electrode surface. To investigate the surface interactions, electrochemical impedance measurements with the amino-modified poly(T) probe were performed. Figure 4 shows the impedance spectra in the form of a Nyquist plot obtained in the investigation. One can observe that the bare gold electrode behaved as an ideal conductor and the impedance spectra gave a linear plot (curve a in Figure 4). The oligonucleotide-modified electrode exhibited much larger impedance than the bare electrode (curve b in Figure 4). Interestingly, in the presence of Hg2+ of increasing concentration, the resistance decreased substantially (curves c and d in Figure 4). The equivalent circuit, as shown in the inset, was used to fit the EIS data. The components in the equivalent circuit included the solution resistance Rs, the charge-transfer resistance Rct, the constant phase element related to double layer capacitance (CPE), and the Warburg impedance (Zw, where Zw ) Rw/(jw)1/2 and Rw is the diffusion resistance).43 The calculated values for these elements are shown in Table 2. Clearly, as the Hg2+ concentration increased, the Warburg impedance and especially the charge-transfer resistance decreased substantially, with a slightly increased double layer capacitance. Such decreased impedance suggested that the interaction between poly-T probes and Hg2+ led to a less negatively charged surface. Because the amino proton of T was reported to be substituted by Hg2+ and no net charge alteration occurred in T-Hg2+-T coordination,18,19 one might deduce that the less negatively (43) Patolsky, F.; Lichtenstein, A.; Willner, I. J. Am. Chem. Soc. 2001, 123, 5194– 5205.

Figure 5. Effect of reaction temperature on DPV peak currents of the sensor, in response to 10 µM Hg2+. DPV conditions were as described previously in the text.

charged surface was arising from a reorganization of the poly-T probes into certain structures drawing the probes away from the surface, coinciding the conformation switch from random coil to duplexlike structure in the presumptive mechanism of the electrochemical sensor. The effect of temperature on the response of the sensor was also investigated. Figure 5 depicts the DPV current response of the sensor to 10 µM Hg2+ at varying reaction temperatures (in the range of 25-60 °C). One observed that, at higher reaction temperature, the DPV peak current gradually increase and the sensitivity of the sensor became declined. Therefore, the reaction temperature was set to room temperature (∼25 °C). Assay Performance of the Electrochemical Sensor. The DPV responses of the sensor to Hg2+ of varying concentration are shown in Figure 6. One observed dynamically decreased DPV peak currents in the presence of increasing Hg2+ concentration within the range from 1.0 nM to 2.0 µM. A quasi-linear response versus Hg2+ concentration with high-dose sensitivity was obtained in the range from 1.0 to 250 nM with a robust detection limit of 0.5 nM, amounting to 10 ppt. To our knowledge, such a low detection limit is the best among all sensors based on the T-Hg2+-T coordination chemistry,20-27 and is lower than the toxic level for Hg2+ defined by the United States Environmental Protection Agency (USEPA) in drinkable water (