Exploiting the Interaction of Metal Ions and Peptide Nucleic Acids

Jan 7, 2010 - ... Paul S. Francis , Conor F. Hogan , Alan M. Bond , Gilles Gasser , and Leone Spiccia ... Gang Liang , Tao Li , Xiaohong Li , Xinhui L...
0 downloads 0 Views 206KB Size
Download PDF
Anal. Chem. 2010, 82, 1166–1169

Exploiting the Interaction of Metal Ions and Peptide Nucleic Acids-DNA Duplexes for the Detection of a Single Nucleotide Mismatch by Electrochemical Impedance Spectroscopy Congjuan Li,† Xiaohong Li,*,† Xinhui Liu,§ and Heinz-Bernhard Kraatz*,‡ Department of Chemistry and State Key Laboratory of Water Environment Simulation, School of Environment, Beijing Normal University, Beijing, 100875, China, and Department of Chemistry, University of Western Ontario, 1151 Richmond Street, London, N6A 5B7 Canada The interaction of the metal ions Mg2+, Zn2+, Ni2+, and Co2+ with DNA-peptide nucleic acid (PNA) films on a gold surface is studied by electrochemical impedance spectroscopy in the presence of [Fe(CN)6]3-/4- as the redox probe. Impedance data were analyzed with the help of a modified Randles’ equivalent circuit. Changes in the charge-transfer resistance, RCT, decreases in the order of Ni2+ > Co2+ > Zn2+ > Mg2+. We interpret these results in terms of stronger interactions for Ni2+ with the DNA-PNA film compared to the other metal ions, potentially involving interactions with the nucleobases, presumably with the N7 of purines or the N3 of pyrimidines. On the basis of these observations, Ni2+ was chosen to probe the detection of a C-T mismatch in 15-mer PNA-DNA films. Using Ni2+, it is possible to detect a single C-T mismatch. The resulting ∆RCT is larger for the PNA-DNA hybrid compared to that for the identical 15-mer DNA-DNA hybrid. Electrochemical biosensing platforms for DNA detection have been developed, which have enabled the detection of hybridization events and have allowed the discrimination of base pair mismatches in double-stranded DNA (ds-DNA), including singlenucleotide mismatches.1-6 Originally reported by Nielsen et al.,7-9 peptide nucleic acids (PNAs) are the most widely used DNA * Corresponding author. E-mail: [email protected] (X.L.); [email protected] (H.-B.K.). † Department of Chemistry, Beijing Normal University. § State Key Laboratory of Water Environment Simulation, Beijing Normal University. ‡ University of Western Ontario. (1) Zhang, X.; Ju, H.; Wang, J. Electrochemical sensors, biosensors and their biomedical applications; Academic Press: New York, 2008. (2) Chow, K.-F.; Mavre, F.; Crooks, R. M. J. Am. Chem. Soc. 2008, 130, 7544– 7545. (3) Yu, C. J.; Wan, Y.; Howanto, H.; Li, J.; Tao, C.; James, M. D.; Tan, C. L.; Blackburn, G. F.; Meade, T. J. J. Am. Chem. Soc. 2001, 123, 11155–11161. (4) Xia, Q.; Chen, X.; Liu, J.-H. Biophys. Chem. 2008, 136, 101–107. (5) Umek, R. M.; Lin, S. W.; Vielmetter, J.; Terbrueggen, R. H.; Irvine, B.; Yu, C. J.; Kayyem, J. F.; Yowanto, H.; Blackburn, G. F.; Farkas, D. H.; Chen, Y.-P. J. Mol. Diagn. 2001, 3, 74–84. (6) Treadwaya, C. R.; Hill, M. G.; Barton, J. K. Chem. Phys. 2002, 281, 409– 428. (7) Nielsen, P. E.; Egholm, M.; Berg, R. H.; Buchardt, O. Science 1991, 254, 1497–1500. (8) Nielsen, P. E. Pure Appl. Chem. 1998, 70, 105–110.

1166

Analytical Chemistry, Vol. 82, No. 3, February 1, 2010

analogues. Having a polyamide backbone instead of the polyanionic phosphodiester backbone, PNA lowers the electrostatic repulsion with an incoming oligonucleotide. PNA hybridization to a cDNA strand occurs with high affinity and specificity, and the resulting DNA-PNA duplex is more stable than the corresponding DNA-DNA duplex. As expected, differences in the electrostatic interactions between DNA-PNA and DNA-DNA duplexes are readily identifiable electrochemically by monitoring interactions with [Co(NH3)6]3+ as an external cationic redox probe.10 Importantly, DNA-PNA hybridization is severely affected by nucleobase mismatches. Even the presence of a single-nucleotide mismatch destabilizes a DNA-PNA duplex to a greater extent than the identical mismatch in a DNA-DNA duplex.11 It is in part because of these properties that PNA has found applications in PNA-based arrays,12,13 in antisense DNA targeting,14,15 in nucleic acid purification,16 and in mutation analysis.17 There are a number of reported methods available for single-nucleotide mismatch detection exploiting PNA as a capture probe.18-22 Metal ions, such as Cu2+, are known to interact with PNA molecules, presumably involving coordination (9) Nielsen, P. E.; Haaima, G. Chem. Soc. Rev. 1997, 26, 73–78. (10) Kerman, K.; Vestergaard, M.; Nagatani, N.; Takamura, Y.; Tamiya, E. Anal. Chem. 2006, 78, 2182–2189. (11) Egholm, M.; Buchardt, O.; Christensen, L.; Behrens, C.; Freier, S. M.; Driver, D. A.; Berg, R. H.; Kim, S. K.; Norden, B.; Nielsen, P. E. Nature 1993, 365, 566–568. (12) Weiler, J.; Gausepohl, H.; Hauser, N.; Jensen, O. N.; Hoheisel, J. D. Nucleic Acids Res. 1997, 25, 2792–2799. (13) Lukeman, P. S.; Mittal, A. C.; Seeman, N. C. Chem. Commun. 2004, 1694– 1695. (14) Larsen, H. J.; Bentin, T.; Nielsen, P. E. Biochim. Biophys. Acta 1999, 1489, 159–166. (15) Nulf, C. J.; Corey, D. Nucleic Acids Res. 2004, 32, 3792–3798. (16) Demidov, V. V.; Bukanov, N. O.; Frank-Kamenetskii, M. D. Curr. Issues Mol. Biol. 2000, 2, 31–35. (17) Orum, H; Nielsen, P. E.; Egholm, M.; Berg, R. H.; Buchardt, O.; Stanley, C. Nucleic Acids Res. 1993, 21, 5332–5336. (18) Sawata, S.; Kai, E.; Ikebukuro, K.; Iida, T.; Hoda, T.; Karube, I. Biosens. Bioelectron. 1999, 14, 397–404. (19) Corradini, R.; Feriotto, G.; Sforza, S.; Marchelli, R.; Gambari, R. J. Mol. Recognit. 2004, 17, 76–84. (20) Basile, A.; Giuliani, A.; Pirri, G.; Chiari, M. Electrophoresis 2002, 23, 926– 929. (21) Wang, J.; Nielsen, P. E.; Jiang, M.; Cai, X.; Fernandes, J. R.; Grant, D. H.; Ozsoz, M.; Beglieter, A.; Mowat, M. Anal. Chem. 1997, 69, 5200–5202. (22) Wittung-Stafshede, P.; Rodahl, M.; Kasemo, B.; Nielsen, P. E.; Norden, B. Colloid Surf., A 2000, 174, 269–273. 10.1021/ac902813y  2010 American Chemical Society Published on Web 01/07/2010

to the nucleobase.23 Chelating sites for metal ions are presumably the amide groups and the nucleobases, which might be competing.24 More recently, the use of quinone-modified PNAs made it possible to incorporate Cu2+ into the center of the base stack, and the properties of the resulting PNA-Cu complex are strongly affected by the presence of mismatches.25 Zn2+ was believed to increase the affinity of the modified PNA to complementary singlestranded DNA more significantly than to a single-nucleotide mismatch contained strand.26 Electrochemical impedance spectroscopy (EIS) measurements of DNA-PNA interactions exploiting an external redox probe, such as [Fe(CN)6]3-/4-, have grown in importance.27-29 In EIS studies of ds-DNA films on gold surfaces in the presence of [Fe(CN)6]3-/4-, we demonstrated that the presence of Zn2+ ions is beneficial for mismatch discrimination, presumably by enhancing diffusion of the anionic redox probe into the ds-DNA film. In contrast, the presence of mismatches alters the ability of the redox probe to diffuse into the film and EIS is highly sensitive to these differences, expressed as a chance in the resistance to charge transfer RCT.30 On the basis of a purely electrostatic argument, PNA-DNA films should also benefit from metal addition and differences in RCT between matched and mismatched PNA-DNA films should be measurable. However, to the best of our knowledge, such studies have not been reported in the literature and no information exists about the effects of metal ions on PNA-DNA films, nor are there reports on metal enhanced mismatch detection by EIS. Here, we reported the interaction of metal ions (Zn2+, Ni2+, Co2+, Mg2+) with DNA-PNA films in the presence of [Fe(CN)6]3-/4- as the redox probe. Our measurements indicate that metal interactions with the film decreases in the order of Ni2+ > Co2+ > Zn2+ > Mg2+. Therefore, Ni2+ was chosen to probe the detection of a C-T mismatch. Compared to ds-DNA-DNA films, films of PNA-DNA appear more sensitive to a single-nucleotide mismatch. EXPERIMENTAL SECTION Materials. The working gold electrodes, 99.99% (w/w) polycrystalline with a diameter 1 mm were purchased from Aida Instrument Inc. in Tianjin and cleaned prior to use. Gold electrodes were first polished on a microcloth (Buehler) with Gamma micropolish deagglomerated alumina suspension (0.05 µm) for 5 min, followed by ultrasonication in distilled ethanol for 5 min and then Milli-Q water for 5 min. Finally, the electrodes were electrochemically cleaned by cyclic voltammetry (CV) cycling in 1 M H2SO4 (0-1.5 V vs Ag/AgCl) to remove any remaining surface impurities. NaClO4, K3[Fe(CN)6], K4[Fe(CN)6], Na2HPO4, NaH2PO4, Zn(ClO4)2 · 6H2O, Ni(ClO4)2 · 6H2O, Co(ClO4)2 · 6H2O, and Tris (tris-(hydroxymethyl)-aminomethane) (23) Szyrwiel, J.; Młynarz, P.; Kozłowski, H.; Taddei, M. J. Chem. Soc., Dalton Trans. 1998, 1263–1264. (24) Popescu, D.-L.; Parolin, T. J.; Achim, C. J. Am. Chem. Soc. 2003, 125, 6354– 6355. (25) Watson, R. M.; Skorik, Y. A.; Patra, G. K.; Achim, C. J. Am. Chem. Soc. 2005, 127, 14628–14639. (26) Andriy, M.; Roland, K.; Helena, W. J. Am. Chem. Soc. 2004, 126, 6208– 6209. (27) Degefa, T. H.; Kwak, J. J. Electroanal. Chem. 2008, 612, 37–41. (28) Keighley, S. D.; Estrela, P.; Li, P.; Mighorato, P. Biosens. Bioelectron. 2008, 24, 906–911. (29) Liu, J. Y.; Tian, S. J.; Nielsen, P. E.; Knoll, W. Chem. Commun. 2005, 2969– 2971. (30) Li, X.; Lee, J. S.; Kraatz, H.-B. Anal. Chem. 2006, 78, 6096–6101.

Figure 1. Representative Nyquist plots (-Zim vs Zre) for films of 15mer DNA-PNA(I) (O); DNA-PNA(I) incubated with 0.4 mM divalent metal ions Mg2+ (0), Zn2+ (b), Co2+(9), and Ni2+(2) for 2 h. Measured data are shown as symbols with calculated fit to the equivalent circuit as solid lines. Inset: the measured data are fit to the equivalent circuit; Rs, solution resistance; Cfilm, capacitance of the DNA + PNA(I) film; RCT, charge-transfer resistance of DNA + PNA(I) film; Rx and CPE, resistance and nonlinear capacitor accounting for 6-mercaptohexanol film. E1/2 ) 250 mV vs Ag/AgCl, sinusoidal potential modulation of (5 mV; f: 0.1 Hz to 100kHz; 4 mM K3[Fe(CN)6]/K4[Fe(CN)6] (1:1).

were purchased from Aldrich and used without further purification. Mg(ClO4)2 was purchased from Fluka and used as received. Deionized water (18.2 MΩ · cm resistivity) from a Millipore Milli-Q system was used throughout this work. The following 15-mer DNA strand (Shanghai Sangon Biological Engineering Technology & Service Co. Ltd.), which is an unsymmetrical hexanol-DNA disulfide, and complementary 15-mer PNA sequences (PANAGENE Inc. Daejeon, Korea) were used in this study:

Film Preparation. Solutions of DNA-PNA hybrids were prepared by combining equimolar amounts of DNA and PNA strands in 20 mM PBS (pH 7.0) Since the solubility of PNA is poor in Tris-ClO4, PBS was only used to hybridize PNA and DNA to and from DNA-PNA duplexes. Hybridization of DNA and PNA(I) resulted in the formation of the matched DNA-PNA duplex. Combination of DNA and PNA(II) leads to a duplex possessing a single C-T mismatch. Films of DNA + PNA(I) and DNA + PNA(II) were formed by incubating clean gold electrodes with a diameter of 1 mm for 5 days in the prehybridized duplex solutions in PBS buffer. The modified electrodes were then washed with 20 mM Tris-ClO4 (pH ) 8.7) and then mounted into the electrochemical cell. EIS measurements were carried out to evaluate the capacitive and resistive properties of the PNA-DNA films on the gold electrodes in the absence and presence of 0.4 mM Mg2+, Zn2+, Ni2+, and Co2+ in 20 mM Tris-ClO4 (pH ) 8.7). Electrochemical Measurements. A conventional threeelectrode system was used, and all the measurements were carried out at room termperature in an enclosed and grounded Faraday cage. The reference electrode was constructed by sealing a Ag/AgCl wire Analytical Chemistry, Vol. 82, No. 3, February 1, 2010

1167

Table 1. Equivalent Circuit Element Values for 15-mer DNA + PNA(I) Films on a Gold Electrode in the Absence and Presence of Metal Ionsa equivalent circuit elements Rs (Ω · cm )

Cfilm (µF · cm )

RCT (Ω · cm2)

Rx (Ω · cm2)

CPE (µF · cm-2)

n

∆RCT (Ω · cm2)

5.5(0.1) 5.7(0.1) 5.8(0.1) 5.8(0.1) 5.8(0.1)

19.0(1.2) 19.2(1.1) 19.5(1.3) 18.1(1.2) 14.8(1.1)

5747(14) 4775(22) 3856(12) 3320(17) 2803(18)

3.4(0.2) 3.8(0.2) 5.2(0.3) 3.6(0.3) 3.4(0.3)

22.9(2.1) 13.7(1.3) 22.7(2.0) 14.3(1.2) 17.5(1.5)

0.9(0.01) 0.9(0.01) 0.9(0.01) 0.9(0.02) 0.9(0.04)

972(4) 1891(6) 2427(5) 2944(8)

2

DNA + PNA(I) +Mg2+ +Zn2+ +Co2+ +Ni2+ a

-2

The values in parentheses represent the standard deviations from at least five electrode measurements.

RESULTS AND DISCUSSION To begin our studies, we investigated the interaction of metal ions (Mg2+, Zn2+, Ni2+, Co2+) and matched 15-mer films of DNA + PNA(I) on gold electrodes. Representative Nyquist plots for the fully matched film of DNA + PNA(I) in the absence and presence of Mg2+, Zn2+, Ni2+, and Co2+ are shown in Figure 1. EIS results for bare gold electrodes in the absence and presence of metal ions are shown in the Supporting Information. The impedance spectra for all the systems were analyzed with the help of a modified Randles equivalent circuit shown in the inset to Figure 1. The results of this analysis are listed in Table 1. The solution resistance, Rs, is the resistance between the reference electrode and the film of DNA-PNA(I) on the gold electrode. For each measurement, the position of the two electrodes is kept the same. All measurements were carried out under identical conditions of electrolyte concentration (20 mM Tris-ClO4) and at room temperature to minimize variations in Rs, which ranged from 5.5 to 5.8 Ω · cm2. Cfilm accounts for the capacitance of the DNA-PNA(I) films on the gold electrodes. In the presence of Mg2+ and Zn2+, Cfilm is virtually unchanged. For Co2+, the decrease is negligible. However, in the presence of Ni2+, the film capacitance Cfilm decreases significantly from 19.0(1.2) to 14.8(1.1) µF · cm-2. The results could be rationalized that Ni2+ not only reduces the negative charge of the DNA-PNA(I) duplex but also interacts with the PNA backbone and the nucleobases involving the N7 of purines or the N3 of pyrimidines, which results in a decrease in the film capacitance Cfilm (see Supporting Information).31,32 The combination of Rx and the constant phase element (CPE) accounts for the behavior of the 6-mercaptohexanol groups on the electrode surfaces, which are the result of the interaction of the hexanol-DNA-disulfide conjugate with the gold electrode.30 CPE acts as a nonlinear capacitor accounting for the inhomogeneity of the films on the electrode surface with the

exponential modifier n ) 0.9.33 Mass transport is not a major contributor to the overall impedance signature of the system, as is apparent from the absence of any Warburg impedance, as shown in Figure 1. The charge-transfer resistance, RCT, is the result of the resistance to charge transfer from the solution-based redox probe [Fe(CN)6]3-/4- to the electrode surface. For DNA + PNA(I) film, RCT is larger than that in the presence of Zn2+, Ni2+, Co2+ and Mg2+. In the presence of Mg2+, RCT decreased from 5747(14) Ω · cm2 to 4775(22) Ω · cm2+ and the RCT difference (∆RCT) was 972(4) Ω · cm2+. In the presence of Zn2+, RCT decreased to 3856(12) Ω · cm2 and ∆RCT was 1891(6) Ω · cm2+. In the presence of Co2+, RCT decreased to 3320(17) Ω · cm2+ and ∆RCT was 2944(8) Ω · cm2+. For Ni2+, RCT decreased to 2803(18) Ω · cm2+ and ∆RCT was 2427(5) Ω · cm2+. Subsequently, DNA-PNA(I) modified gold electrodes were incubated in blank buffer solution for another 2 h, which should lead to decomplexation of the metal ions and a significant change in the EIS. However, no such changes were observed in the EIS, which suggests that the metal ions strongly bind with DNA-PNA(I) films and the binding order is Ni2+ > Co2+ > Zn2+ > Mg2+. This RCT change can be ascribed to metal ions reducing the negative charge of the DNA-PNA (I) film effectively and allowing the anionic redox probe to easily penetrate the film.34 On the other hand, X-ray photoelectron spectroscopy (XPS) results show that Zn2+, Ni2+, and Co2+ bind with nucleobases presumably involving the N7 of purines or the N3 of pyrimidines in addition to interactions with the PNA backbone (see Supporting Information). Both interactions are beneficial to electron transfer through the DNA-PNA(I) film and lead to a decreased charge-transfer resistance, RCT. Next, we explored the metal-duplex interactions for C-T mismatch detection by EIS focusing on Ni2+. Films of DNA + PNA(I) and DNA + PNA(II) were formed on surfaces, and EIS were recorded in the absence and presence of Ni2+. Representative Nyquist plots are shown in Figure 2, and the measured data were analyzed and fitted to the equivalent circuit shown earlier in Figure 1. The fitted results for matched and C-T mismatched films are list in Table 2 (see Supporting Information). For RCT, DNA-PNA(I) has a higher RCT in the absence of Ni2+ as compared to DNA-PNA(II). As reported, the presence of a single-nucleotide mismatch destabilizes a DNA-PNA duplex to a greater extent than a mismatch in a DNA-DNA duplex.10 C-T mismatch in DNA-PNA(II) films may potentially

(31) Hossain, Z.; Huq, F. J. Inorg. Biochem. 2002, 90, 97–105. (32) Fuente, M.; Hernanz, A.; Navarro, R. J. Biol. Inorg. Chem. 2004, 9, 973– 986.

(33) Dijksma, M.; Boukamp, B. A.; Kamp, B.; van Bennekom, W. P. Langmuir 2002, 18, 3105. (34) Bin, X.; Kraatz, H.-B. Anaylst 2009, 134, 1309–1313.

into a glass tube with a solution of 3 M KCl that was capped with a Vycor tip. The counter electrode was a platinum wire. Impedance spectra were measured using a potentiostat/frequency analyzer (EG&G 2273). The ac voltage amplitude was 5 mV, and the voltage frequencies used for EIS measurements ranged from 100 kHz to 100 mHz. The applied potential was 250 mV vs Ag/AgCl (formal potential of the redox probe [Fe(CN)6]3-/4- in the buffer solution). All measurements were repeated for a minimum of five times with a separate electrode to obtain statistically meaningful results.

1168

Analytical Chemistry, Vol. 82, No. 3, February 1, 2010

Figure 2. Representative Nyquist plots (-Zim vs Zre) for films of 15-mer duplex of DNA-PNA(I) (A) and DNA-PNA(II) (B). (A) Film of DNA-PNA(I) in the absence (O) and presence (b) of Ni2+; (B) film of DNA + PNA(II) in the absence (0) and presence (9) of Ni2+. Measured data are shown as symbols with calculated fit to the equivalent circuit as solid lines. Table 2. Rs, RCT, and Rx Values for Matched 15-mer DNA-PNA(I) and C-T Mismatched 15-mer DNA-PNA(II) Films in the Absence and Presence of Ni2+a equivalent circuit elements DNA + PNA(I) +Ni2+ DNA + PNA(II) +Ni2+

Rs(Ω · cm2)

RCT(Ω · cm2)

Rx(Ω · cm2)

∆RCT(Ω · cm2)

5.5(0.1)

5747(14)

3.4(0.2)

2944(8)

5.8(0.1) 5.8(0.1)

2803(18) 3587(15)

3.4(0.3) 2.9(0.1)

1464(10)

5.5(0.1)

2123(12)

2.9(0.2)

a

The values in parentheses represent the standard deviations from at least five electrode measurements.

induce a structure change that influences the structure of the film.35 Such a conformational change induced by a C-T mismatch will facilitate the electron transfer through the DNA-PNA(II)films.36 UponadditionofNi2+,RCT forDNA-PNA(I) decreases from 5747(14) to 2803(18) Ω · cm2. For the mismatched film (DNA-PNA(II)), RCT decreases from 3587(15) to 2123(12) Ω · cm2. In the presence of Ni2+, there is significantly less electrostatic repulsion and interaction between Ni2+ and PNA resulting in a lower RCT due to easy diffusion of the redox probe into the DNA-PNA film. As was reported earlier,30,34 the difference in charge-transfer resistance, ∆RCT, in the presence and absence of Ni2+ is a key parameter, which allows us to discriminate the fully matched DNA + PNA(I) from a C-T mismatched DNA + PNA(II). For matched DNA + PNA(I), ∆RCT is 2944(8) Ω · cm2, and for mismatched DNA + PNA(II), ∆RCT is 1464(10) Ω · cm2. Using this approach, we (35) Ratilainen, T.; Holme´n, A.; Tuite, E.; Haaima, G.; Christensen, L.; Nielsen, P. E.; Norde´n, B. Biochemistry 1998, 37, 12331–12342. (36) Komiyama, M.; Ye, S.; Liang, X.; Yamamoto, Y.; Tomita, T.; Zhou, J.-M.; Aburatani, H J. Am. Chem. Soc. 2003, 125, 3758–3762.

are able to discriminate a C-T mismatch without the prior labeling of the DNA target. In addition, the difference of ∆RCT between matched and mismatched DNA + PNA is 1480(5) Ω · cm2. In comparison, ∆RCT between matched and mismatched ds-DNA (see Supporting Information) is 940 (48) Ω · cm2, suggesting that the DNA + PNA assay is more sensitive in the detection of a single-nucleotide mismatch. In conclusion, we demonstrated that metal ions Mg2+, Zn2+, 2+ Co , and Ni2+ interact with 15-mer PNA-DNA duplex films and influence the impedance response of such films. Our results suggest that the interaction with the metal ions decreases in the following order Ni2+ > Co2+ > Zn2+ > Mg2+. We suggest that in addition to electrostatic interactions, the Ni2+ ion appears to be also interacting favorably with the nucleobases, presumably with the N7 of purines or the N3 of pyrimidines. Using Ni2+, it is possible to detect a single C-T mismatch. Other assays based on the interaction of metal ions and a DNA-PNA duplex for single-nucleotide mismatch detection by EIS are currently under way. ACKNOWLEDGMENT The work is supported by NSFC (Grant No. 20703006), MSBRDP (Grant No. 2009CB421605), and SRF for ROCS, SEM. We gratefully acknowledge financial support from the Natural Science and Engineering Research Council of Canada. 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 December 10, 2009. Accepted December 16, 2009. AC902813Y

Analytical Chemistry, Vol. 82, No. 3, February 1, 2010

1169