Dopamine-, l-DOPA-, Adrenaline-, and Noradrenaline-Induced Growth

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Anal. Chem. 2005, 77, 1566-1571

Dopamine-, L-DOPA-, Adrenaline-, and Noradrenaline-Induced Growth of Au Nanoparticles: Assays for the Detection of Neurotransmitters and of Tyrosinase Activity Ronan Baron, Maya Zayats, and Itamar Willner*

Institute of Chemistry, The Hebrew University of Jerusalem, Jerusalem 91904, Israel

The neurotransmitters dopamine (1), L-DOPA (2), adrenaline (3), and noradrenaline (4) mediate the generation and growth of Au nanoparticles (Au-NPs). The plasmon absorbance of the Au-NPs allows the quantitative colorimetric detection of the neurotransmitters. Neurotransmitters 1, 2, and 4 are sensed with a detection limit of 2.5 × 10-6 M, whereas the detection limit for analyzing 3 corresponds to 2 × 10-5 M. The neurotransmitter-mediated growth of the Au-NPs is also used to probe the activity of tyrosinase. The later biocatalyst oxidizes tyrosine to L-DOPA that mediates the growth of the Au-NPs. The analysis of tyrosinase activity is important for detecting melanoma cells and Parkinson disease. Metal nanoparticles (NPs) are used as active components for the optical, electrical and piezoelectric sensing of biorecognition events.1 For example, the interparticle plasmon interactions of AuNPs functionalized with nucleic acids were used to analyze DNA and single-base mismatches.2 The interactions between the plasmon of Au-NPs and the plasmon wave associated with thin gold films on surfaces were employed as an amplification means for detecting antigen-antibody complexes,3 DNA hybridization,4 and biocatalytic transformations,5 using surface plasmon resonance (SPR) spectroscopy. Electrochemical detection of DNA was accomplished by the use of nucleic acid-functionalized metal NPs as labels.6 The dissolution of the NPs followed by the voltammetric analysis of the resulted ions enabled the amplified detection of * To whom correspondence should be addressed. Telephone: 972-2-6585272. Fax: 972-2-6527715. E-mail: [email protected]. (1) (a) Niemeyer, C. M. Angew. Chem., Int. Ed. 2001, 40, 4128-4158. (b) Katz, E.; Willner, I.; Wang, J. Electroanalysis 2004, 16, 19-44. (c) Katz, E.; Shipway, A. N.; Willner, I. In Nanoparticles-From Theory to Applications; Schmid, G., Ed.; Wiley-VCH: Weinheim, Germany, 2003; pp 368-421. (d) Katz, E.; Willner, I. Angew. Chem., Int. Ed. 2004, 43, 6042-6108. (2) (a) Storhoff, J. J.; Mirkin, C. A. Chem. Rev. 1999, 99, 1849-1862. (b) Elghanian, R.; Storhoff, J. J.; Mucic, R. C.; Letsinger, R. L.; Mirkin, C. A. Science 1997, 277, 1078-1081. (3) Lion, L. A.; Musick, M. D.; Natan, M. J. Anal. Chem. 1998, 70, 51775183. (4) He, L.; Musick, M. D.; Nicewarner, S. R.; Sallinas, F. G.; Benkovic, S. J.; Natan, M. J.; Keating, C. D. J. Am. Chem. Soc. 2000, 122, 9071-9077. (5) Zayats, M.; Pogorelova, S. P.; Kharitonov, A. B.; Lioubashevski, O.; Katz, E.; Willner, I. Chem. Eur. J. 2003, 9, 6108-6114. (6) (a) Wang, J.; Liu, G. D.; Merkoci, A. J. Am. Chem. Soc. 2003, 125, 32143215. (b) Wang, J.; Polsky, R.; Xu, D. K. Langmuir 2001, 17, 5739-5741. (c) Wang, J.; Liu, G. D.; Zhu, Q. Y. Anal. Chem. 2003, 75, 6218-6222.

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DNA. Similarly, nucleic acid-functionalized Au-NPs were used as “weight labels” for the microgravimetric quartz crystal microbalance analysis of DNA on piezoelectric crystals.7 The catalytic deposition of metals on metal NPs was used for the amplified biosensing of antigen-antibody complexes and DNA hybridization.9 For example, the catalytic enlargement of nucleic acidfunctionalized Au-NPs, acting as labels for DNA hyridization, was used to generate a conductive bridge between microelectrodes. Also, the catalytic deposition of metals on Au-NPs was used for the amplified microgravimetric quartz crystal microbalance analysis of DNA.10 Recently, the catalytic enlargement of Au-NPs was employed for the optical detection of aptamer-protein complexes on surfaces.11 The integration of enzymes with metal NPs is an almost unexplored field. The reconstitution of apo-glucose oxidase on a flavin adenine dinucleotide (FAD) cofactor associated with AuNP was used to electrically contact the enzyme with bulk electrodes.12 Also, the bioelectrocatalytic charging of the Au-NP in the presence of reconstituted glucose oxidase and glucose was used to sense glucose by following the surface plasmon resonance wave upon charging of the NPs.13 Recently, we applied several redox proteins for the biocatalyzed enlargement of metal NPs, e.g., Au-NPs. The dihydronicotinamide adenine dinucleotide (phosphate), NAD(P)H, cofactors were used to reduce Au(III) salts to Au0 in the presence of Au-NP seeds. The biocatalytic enlargement of the Au-NP seed was employed for the quantitative optical analysis of NAD(P)H and for the quantitative assay of biocatalytic transformation that included NAD+-dependent enzymes.14 Hydrogen peroxide, H2O2, was used as an alternative reducing agent of gold salts in the presence of Au-NPs.15 This (7) (a) Gao, X. H.; Nie, S. M. Trends Biotechnol. 2003, 21, 371-373. (b) Patolsky, F.; Ranjit, K. T.; Lichtenstein, A.; Willner, I. Chem. Commun. 2000, 1025-1026. (8) Velev, O. D.; Kaler, F. W. Langmuir 1999, 15, 3693-3698. (9) Park, S. J.; Taton, T. A.; Mirkin, C. A. Science 2002, 295, 1503-1506. (10) Willner, I.; Patolsky, F.; Weizmann, Y.; Willner, B. Talanta 2002, 56, 847856. (11) Pavlov, V.; Xiao, Y.; Shlyahovsky, B.; Willner, I. J. Am. Chem. Soc. 2004, 126, 11768-11769. (12) Xiao, Y.; Patolsky, F.; Katz, E.; Hainfeld, J. F.; Willner I. Science 2003, 299, 1877-1881. (13) Lioubashevski, O.; Chegel, V. I.; Patolsky, F.; Katz, E.; Willner I. J. Am. Chem. Soc. 2004, 126, 7133-7143. (14) Xiao, Y.; Pavlov, V.; Levine, S.; Niazov, T.; Markovitch, G.; Willner, I. Angew. Chem., Int. Ed. 2004, 43, 4519-4522. 10.1021/ac048691v CCC: $30.25

© 2005 American Chemical Society Published on Web 02/04/2005

Chart 1. Chemical Structures of the Neurotransmitters

enabled the use of oxidases, e.g., glucose oxidase, as biocatalysts for the in situ generation of H2O2 that enlarges the NP seeds. As the concentration of H2O2 is controlled by the concentration of the substrate (e.g., glucose), the enlargement of the particles was used for the quantitative optical detection of the respective substrate. The analysis of neurotransmitters is of substantial interest for the rapid and early detection of neural disorders. Different electrochemical16,17 and optical18 methods for the analysis of neurotransmitters were reported. Other reports have addressed the analysis of neurotransmitters by capillary electrophoresis19 and by mass spectrometry.20 Several studies21,22 have demonstrated the use of Au-NPs as active units for the electrochemical detection of neurotransmitters. Layered arrays of Au-NPs cross-linked by the biscyclo(paraquat-p-phenylene) electron acceptor receptor units were used to bind and preconcentrate the neurotransmitters in the receptor sites while the Au-NPs provided a conductive matrix for the electrochemical detection of the encapsulated neurotransmitters.21 Also, a layer consisting of biscyclo(paraquatp-phenylene) cross-linked Au-NPs assembled on the gate of an ion-selective field-effect transistor (ISFET) was used as an active sensing interface by controlling the gate potential of the ISFET device via the binding of π-donor neurotransmitters to the acceptor receptor sites.22 In the present study we report on the use of a series of neurotransmitters (L-DOPA, dopamine, adrenaline, and noradrenaline) as active reducing agents for the generation of AuNPs. The optical properties of the generated particles enable the quantitative analysis of the different neurotransmitters. The formation of the NPs in the systems proceeds without added AuNP seeds. We also use this process for the quantitative assay of tyrosinase activity. EXPERIMENTAL SECTION Materials. All chemicals, dopamine, L-DOPA, adrenaline, noradrenaline, cetyltrimethylammonium chloride (CTAC, 25% in (15) Zayats, M.; Baron, R.; Willner, I. Nano Lett. 2005, 5, 21-25. (16) (a) Chen, R.-S.; Huang, W.-H.; Tong, H.; Wang, Z.-L.; Cheng, J.-K. Anal. Chem. 2003, 75, 6341-6345. (b) Raj, C. R.; Okajima, T.; Ohsaka, T. J. Electroanal. Chem. 2002, 543, 127-133. (c) Huang, T.; Yang, L.; Gitzen, J.; Kissinger, P. T.; Vreeke, M.; Heller, A. J. Chromatogr., B 1995, 670, 323-327. (17) (a) Liu, T.; Li, M.; Li, Q. Talanta 2004, 63, 1053-1059. (b) Raj, C. R.; Ohsaka, T. J. Electroanal. Chem. 2001, 496, 44-49. (c) Wiedemann, D. J.; Kawagoe, K. T.; Kennedy, R. T.; Ciolkowski, E. L.; Wightman, R. M. Anal. Chem. 1991, 63, 2965-2970. (d) Kawagoe, K. T.; Zimmerman, J. B.; Wightman, R. M. J. Neurosci. Methods 1993, 48, 225-240. (18) Klegeris, A.; Korkina, L. G.; Greenfield, S. A. Free Radical Biol. Med. 1995, 18, 215-222. (19) (a) Britz-Mckibbin, P.; Wong, J.; Chen, D. D. Y. J. Chromatogr., A 1999, 853, 535-540. (b) Swanek, F. D.; Chen, G.; Ewing, A. G. Anal. Chem. 1996, 68, 3912-3916. (20) Moini, M.; Schultz, C. L.; Mahmood, H. Anal. Chem. 2003, 75, 6282-6287. (21) Lahav, M.; Shipway, A. N.; Willner, I. J. Chem. Soc., Perkin Trans. 2 1999, 1925-1931. (22) Kharitonov, A. B.; Shipway, A. N.; Willner, I. Anal. Chem. 1999, 71, 54415443.

water), hydrogen tetrachloroaurate (HAuCl4‚3H2O, 99.9%), and L-tyrosine were from commercial sources (Aldrich and Sigma) and were used without further purification. Tyrosinase (TR, from mushroom, 1.14.18.1, Fluka) was stored at -18 °C. Ultrapure water from NANOpure Diamond Barnstead source was used in all experiments. Experimental Conditions. Growth solutions consisted of HAuCl4, 2 × 10-4 M, CTAC, 2 × 10-3 M in 0.01 M phosphate buffer (PB), pH ) 7.2, and either different concentrations of the mentioned neurotransmitters or different concentrations of tyrosine and tyrosinase. All solutions were used within 1 h after preparation, and the experiments were performed at ambient temperature (25 ( 2 °C). Instrumentation. Absorbance measurements were performed using a spectrophotometer UV-2401PC (Shimadzu). Au-NPs were characterized by transmission electron microscopy, TEM, Tecnai F20 G2 (FEI) at 200 kV. The samples were prepared by placing a drop of the respective solution on a carbon-coated copper grid (300 mesh) and subsequent air-drying. RESULTS AND DISCUSSION The systems consist of an aqueous HAuCl4 solution, 2 × 10-4 M, that includes CTAC, 2 × 10-3 M, as stabilizer, pH ) 7.2. Upon addition of one of the neurotransmitters, dopamine (1), L-DOPA (2), adrenaline (3), or noradrenaline (4) (Chart 1), Au-NPs are formed, without the addition of any metal NP seeds. Figure 1A shows the spectra of the Au-NPs formed upon treatment with different concentrations of dopamine for a fixed time interval corresponding to 2 min. This time interval was selected since the absorbance of the Au-NPs reaches after this period an almost constant saturation value (for the time-dependent evolution of the absorbance spectra see Supporting Information). As the concentration of dopamine increases, the absorbance characteristic to the plasmon of the Au-NPs is intensified. Also, as the concentration of dopamine is elevated, the absorbance maxima are slightly blueshifted. The calibration curve corresponding to the absorbance of the resulting Au-NPs at variable concentrations of 1 is depicted in Figure 1B. To further understand the spectral features of the generated particles, we performed a transmission electron microscopy (TEM) analysis of the resulting Au-NPs. Figure 2 shows the TEM images of the Au-NPs formed in the presence of 1, (A and B) 1 × 10-5 M and (C) 5 × 10-5 M, respectively. The TEM images show that the Au-NPs generated at low and high concentrations of dopamine are within very similar particle dimensions (10-20 nm diameter). Typical TEM images for the lower concentration of 1 are shown in Figure 2A,B. The Au-NPs revealed a branched-like configuration or eventually a core structure decorated by Au nanoclusters. We do not observe particles larger than 20 nm in the resulting particle mixture formed in the presence of the higher concentration of 1. Figure 2C shows a typical image of the entire mixture of the NPs. In addition, to larger nanoparticles of ∼20 nm, very small and thin NPs of ca. Analytical Chemistry, Vol. 77, No. 6, March 15, 2005

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Figure 1. (A) Absorbance spectra of the Au-NPs formed in the presence of different concentrations of dopamine: (a) 0, (b) 2.5 × 10-6, (c) 5 × 10-6, (d) 8 × 10-6, (e) 1 × 10-5, (f) 1.5 × 10-5, and (g) 2 × 10-5 M. All systems include HAuCl4, 2 × 10-4 M, and CTAC, 2 × 10-3 M, in 0.01 M PB. Spectra were recorded after a fixed time interval of 2 min. Inset: Color images of the cuvettes containing Au-NPs formed in the presence of different concentrations of dopamine. (Labels correspond to the curve labels in the spectra.) (B) Calibration curve corresponding to the absorbance at λ ) 540 nm of the Au-NPs solution formed in the presence of variable concentrations of dopamine. (Experimental conditions as in A.)

Figure 2. HRTEM images of (A and B) typical Au-NPs formed after 2 min of reaction with dopamine, 1 × 10-5 M, in the presence of HAuCl4, 2 × 10-4 M, and CTAC, 2 × 10-3 M. (C) Au-NPs formed after reaction with dopamine, 5 × 10-5 M, HAuCl4, 2 × 10-4 M, and CTAC, 2 × 10-3 M, for a time interval of 2 min.

2-6 nm of low contrast are observed. The amount of these small particles is higher as the concentration of dopamine increases. These small-sized Au-NPs may either originate from the detachment of small Au nanoclusters from large particles or may be generated by the direct reduction of the gold salt by dopamine. Thus, the intensified plasmon absorbance bands observed at higher concentrations of dopamine originate from larger NPs, as well as from the increase in the content of generated NPs. The small NPs observed in the TEM image, Figure 2C, explain, however, the blue-shift in the absorbance spectra upon increase of the dopamine concentration. 1568

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The formation of the Au-NPs is attributed to the oxidation of dopamine by AuCl4-, Scheme 1. The mechanism and products of dopamine oxidation were discussed,23 and it was found that the two-electron oxidation of dopamine to the respective quinone is followed by a Michael-type cyclization that yields 5. Similar results are obtained for the analysis of the other neurotransmitters. Parts A-C of Figure 3 show the absorbance spectra of the resulting Au-NPs generated by different concentrations of L-DOPA (2), adrenaline (3), and noradrenaline (4), respectively (fixed reaction time corresponding to 2 min). The respective calibration curves for the spectroscopic analysis of the

Figure 3. (A) Absorbance spectra of the Au-NPs formed in the presence of different concentrations of L-DOPA: (a) 0, (b) 5 × 10-6, (c) 8 × 10-6, (d) 1 × 10-5, (e) 1.5 × 10-5, (f) 2 × 10-5, and (g) 3 × 10-5 M. All systems include HAuCl4, 2 × 10-4 M, and CTAC, 2 × 10-3 M, in 0.01 M PB. Spectra were recorded after a fixed time interval of 2 min. Inset: Calibration curve corresponding to the absorbance at λ ) 533 nm of the Au-NPs solution formed in the presence of variable concentrations of L-DOPA. (B) Absorbance spectra of the Au-NPs formed in the presence of different concentrations of adrenaline: (a) 0, (b) 4 × 10-5, (c) 5 × 10-5, (d) 7 × 10-5, (e) 8 × 10-5, and (f) 2 × 10-4 M. All systems include HAuCl4, 2 × 10-4 M, and CTAC, 2 × 10-3 M, in 0.01 M PB. Spectra were recorded after a fixed time interval of 2 min. Inset: Calibration curve corresponding to the absorbance at λ ) 542 nm of the Au-NPs solution formed in the presence of variable concentrations of adrenaline. (C) Absorbance spectra of the Au-NPs formed in the presence of different concentrations of noradrenaline: (a) 0, (b) 2.5 × 10-6, (c) 6.5 × 10-6, (d) 1 × 10-5, (e) 1.5 × 10-5, (f) 2 × 10-5, and (g) 3 × 10-5 M. All systems include HAuCl4, 2 × 10-4 M, and CTAC, 2 × 10-3 M, in 0.01 M PB. Spectra were recorded after a fixed time interval of 2 min. Inset: Calibration curve corresponding to the absorbance at λ ) 540 nm of the Au-NP solution formed in the presence of variable concentrations of noradrenaline.

different neurotransmitters by the Au-NPs are depicted in the Figure 3A-C insets. The detection limit for dopamine, L-DOPA, and noradrenaline is comparable and corresponds to 2.5 × 10-6 M, whereas the detection limit for adrenaline was found to be 2 × 10-5 M.

TEM images of the Au-NPs formed by the different neurotransmitters are similar to those described for dopamine. As the concentration of the neurotransmitter increases, NPs including a core that is decorated by an irregular shell of nanoclusters are observed. The surface deposition of the nanoclusters forming the Analytical Chemistry, Vol. 77, No. 6, March 15, 2005

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Scheme 1. Reduction of the Gold Salt by the Dopamine

Scheme 2. Oxidation of the Tyrosine by Tyrosinase and Further Reduction of the Gold Salt by L-DOPA

Figure 4. Absorbance spectra corresponding to the tyrosinasestimulated growth of Au-NPs in the presence of different concentrations of tyrosine: (a) 0, (b) 2 × 10-5, (c) 6 × 10-5, (d) 1 × 10-4, (e) 1.3 × 10-4, (f) 1.6 × 10-4, and (g) 2 × 10-4 M. In all systems the reaction mixture includes HAuCl4, 2 × 10-4 M, tyrosinase, 40 U mL-1, and CTAC, 2 × 10-3 M, in 0.01 M PB. Spectra were recorded after a fixed time interval of 10 min. Inset: Calibration curve corresponding to the absorbance at λ ) 520 nm of the Au-NPs formed in the presence of variable concentrations of tyrosine.

rough coating is enhanced as the concentration of the neurotransmitters increases. In addition to the rough Au-NPs, very small and thin separated Au nanoclusters (2-6 nm) are observed. These might originate from the detachment of Au nanoclusters associated with the surface of larger NPs or from the direct formation of small NP seeds by the neurotransmitters. The successful analysis of the different neurotransmitters by the formation of Au-NPs encouraged us to apply the process to analyze the activities of an enzyme that catalyzes the formation of a neurotransmitter. Tyrosinase, TR, stimulates the biocatalyzed hydroxylation of tyrosine (6) to 2 (Scheme 2). The analysis of the tyrosinase activity has important clinical implications because elevated amounts of the enzyme are found in malignant melanoma cells.24 Figure 4 shows the absorbance spectra of the Au-NPs generated within a fixed time interval of 10 min in the presence of a constant amount of TR and variable concentrations of tyrosine. This time interval was selected since the time-dependent evolution (23) (a) Arriagada, C.; Paris, I.; Sanchez de las Matas, M. J.; Martinez-Alvarado, P.; Cardenas, S.; Castan ˜eda, P.; Graumann, R.; Perez-Pastene, C.; Olea-Azar, C.; Couve, E.; Herrero, M. T.; Caviedes, P.; Segura-Aguilar, J. Neurobiol. Dis. 2004, 16, 468-477. (b) Shen, X.-M.; Dryhurst, G. Bioorg. Chem. 1996, 24, 340-357. (24) Angeletti, C.; Khomitch, V.; Halaban, R.; Rimm, D. L. Diagn. Cytopathol. 2004, 31, 33-37.

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of the absorbance of the Au-NPs tends to saturate after this reaction period (see Supporting Information). As the concentrations of tyrosine increase, the plasmon absorbance bands of the NPs are intensified and slightly blue-shifted. These results suggest, as expected, that as the concentration of tyrosine increases, larger particles are formed, and together with the enlargement of the NPs small Au nanoclusters are co-generated, causing the blue shifts in the spectra. The absorbance spectra of the generated Au-NPs do not show the sharp plasmon band characteristic of the Au-NPs formed by the neurotransmitters as reducing agents (cf. Figure 3A), but reveal the evolution of the Au-NP plasmon band on an absorbance tail that extends up to 750 nm. This absorbance tail is characteristic of tyrosinase. The TEM images of the Au-NPs formed in the presence of tyrosinase/tyrosine (after 10 min) are, however, very similar to those observed for the NPs generated by the pure neurotransmitters (e.g., Figure 2). From practical points of view, it is, however, interesting to monitor tyrosinase activities in a sample. Accordingly, we have monitored the absorbance spectra of the Au-NPs formed in the presence of different concentrations of TR and a fixed concentration of tyrosine, Figure 5. As the concentration of the enzyme increases, the absorbance of the Au-NPs is higher and blue-shifted, consistent with the fact that larger particles and small Au nanoclusters are formed as the product mixture. The Figure 5 inset shows the derived calibration curve. Tyrosinase is detected with a sensitivity limit of ∼10 units. This translates to ∼100 µg mL-1 of the enzyme in the sample.

complished (detection limit, 2.5 × 10-6 M for dopamine, L-DOPA, and noradrenaline and 2 × 10-5 M for adrenaline). The generation of Au-NPs by the neurotransmitters enables us to assay the activity of the enzyme tyrosinase that catalyzes the O2-induced hydroxylation of tyrosine to L-DOPA. The latter product stimulates the formation of the Au-NPs. The study has demonstrated the sensitive detection of tyrosinase activity and that the enzyme could be detected with a sensitivity limit of 10 units. Since tyrosinase is specifically expressed by melanocytes and melanoma cells and is viewed as a specific marker for these cells, this analytical protocol may be important for clinical diagnostics. The results clearly demonstrate the applicability of metal nanoparticles for new biosensing paths and specifically highlights that biocatalytic formation of nanoparticles has practical utility for enzyme assay. That is, the absorbance of the Au-NPs generated in the presence of a constant concentration of tyrosine may be an optical detection for the quantity of tyrosinase.

Figure 5. Absorbance spectra of Au-NPs formed by variable concentrations of tyrosinase: (a) 0, (b) 10, (c) 20, (d) 30, (e) 35, (f) 40, and (g) 60 U mL-1. All systems include tyrosine, 2 × 10-4 M, HAuCl4, 2 × 10-4 M, and CTAC, 2 × 10-3 M, in 0.01 M PB. Spectra were recorded after a fixed time interval of 10 min. Inset: Calibration curve corresponding to the absorbance at λ ) 520 nm of the AuNPs formed in the presence of variable concentrations of tyrosinase.

CONCLUSION The present study has demonstrated the development of optical detection means of neurotransmitters that are based on the seedless production of Au-NPs. The analytical arrays are rapid, and the sensitive detection of the neurotransmitters is ac-

ACKNOWLEDGMENT This research is supported by the German-Israeli Program (DIP). M.Z. acknowledges the Levi Eshkol fellowship, from the Israel Ministry of Science. SUPPORTING INFORMATION AVAILABLE Time-dependent evolution of the absorbance spectra of the AuNPs in the presence of L-DOPA and in the presence of tyrosinase. This material is available free of charge via the Internet at http:// pubs.acs.org.

Received for review November 30, 2004.

September

2,

2004.

Accepted

AC048691V

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