Voltammetric Sensor for Oxidized DNA Using Ultrathin Films of

Voltammetric Sensor for Oxidized DNA Using Ultrathin Films of Osmium and Ruthenium Metallopolymers .... Analytical Chemistry 2007 79 (5), 1897-1906...
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Anal. Chem. 2004, 76, 5557-5563

Voltammetric Sensor for Oxidized DNA Using Ultrathin Films of Osmium and Ruthenium Metallopolymers Amos Mugweru,† Bingquan Wang,† and James Rusling*,†,‡

Department of Chemistry, University of Connecticut, U-60, 55 North Eagleville Road, Storrs, Connecticut 06269-3060, and Department of Pharmacology, University of Connecticut Health Center, Farmington, Connecticut 06032 Films containing [Os(bpy)2(PVP)10Cl]+ and [Ru(bpy)2(PVP)10Cl]+ metallopolymers were assembled layer by layer on pyrolytic graphite electrodes to make sensors that selectively detect oxidized DNA. These films showed reversible, independent electrochemistry for electroactive Os3+/Os2+ and Ru3+/Ru2+ centers, with formal potentials of 0.34 and 0.76 V vs SCE, respectively. The combination of ruthenium and osmium metallopolymers in the films provided a catalytic Os square wave voltammetry (SWV) peak that is mainly selective for 8-oxoguanine and the detection of other oxidized nucleobases from the Ru peak. The method is applicable to measurements on DNA in solution or DNA incorporated into films. Using the Os SWV peak, 1 oxidized nucleobase in 6000 was detected. The sensor is simple and inexpensive, and the approach may be useful for the detection of oxidized DNA as a clinical biomarker for oxidative stress. Oxidative damage to DNA by reactive oxygen species (ROS) has been linked to cancer, aging,1 and neurological disease.2 ROS are generated from exposure to ionizing radiation, chemical reactions, and cellular metabolism and include singlet oxygen, superoxide radicals, and hydroxyl radicals.3 Reactions of ROS with DNA bases leads to formation of 7,8-dihydro-8-oxoguanine (8oxoguanine), 8-oxoadenine (8-hydroxyadenine), 5-hydroxycytosine, formamidopyrimidine derivatives, thymine glycol, and strand breaks and cross-links.3-5 8-Oxoadenine has been detected in * To whom correspondence should be addressed. E-mail: James.Rusling@ uconn.edu. † University of Connecticut. ‡ University of Connecticut Health Center. (1) Marnett, L. J. Carcinogenesis 2000, 21, 361-370. (b) Raha, S.; Robinson, B. H. Trends Biochem. Sci. 2000, 25, 502-508. (c) Lindahl, T. Nature 1993, 362, 709-715. (2) (a) Wiseman, H.; Halliwell, B. Biochem. J. 1996, 313, 17-29. (b) Freig, D. I.; Reid, T. M.; Loeb, L. A. Cancer Res. 1994, 54, 1890-1894. (3) (a) Halliwell, B.; Gutteridge, J. M. C. Biochem J. 1984, 219, 1-14. (b) Halliwell, B. Mutat. Res. 1999, 443, 37-52. (c) Cadet, J.; Delatour, T.; Douki, T.; Gasparutto, D.; Pouget, J.-P.; Ravanat, J.-L.; Sauvaigo, S. Mutat. Res. 1999, 424, 9-21. (d) Jaeschke, H.; Gores, G. J.; Cederbaum, A. I.; Hinson, J. A.; Pessayre, D.; Lemasters, J. J. Toxicol. Sci. 2002, 65, 166-176. (4) Pryor, W. A. Free Radical Biol. Med. 1988, 4, 219-223. (5) (a) Cheng, K. C.; Cahill, D. S.; Kasai, H.; Nishimura, S.; Loeb, L. A. J. Biol. Chem. 1992, 267, 166-172. (b) Olinski, R.; Gackowski, D.; Foksinski, M.; Rozalski, R.; Roszkowski, K.; Jaruga, P. Free Radical Biol. Med. 2002, 33, 192-200. (c) Floyd, R. A. Carcinogenesis 1990, 11, 1447-1450. 10.1021/ac049375j CCC: $27.50 Published on Web 08/07/2004

© 2004 American Chemical Society

human tumor tissue, suggesting possible involvement in carcinogenesis.6 Guanine is the most easily oxidized of the four DNA bases.7 The two-electron product of guanine oxidation is the highly mutagenic 8-oxoguanine (8-oxoG), which is considered an important biomarker for oxidative stress.8 Liquid chromatography (LC) with electrochemical (EC) or mass spectrometry (MS) detection can be used to determine 8-oxoG, but these methods require relatively expensive equipment, DNA hydrolysis, and long analysis times.8,9 8-OxoG is more easily oxidized than the DNA nucleobases and their primary oxidation products.10,11 When present in DNA, 8-oxoG is a preferred oxidation site. It also causes G-to-T transversions and A-to-C substitutions.5a,12,13 Major oxidation products of 8-oxoG are guanidinohydantoin and 2-amino-4,5,6trioxypyrimidine.11,14 Guanidinohydantoin in single-stranded DNA (ss-DNA) caused G-to-T transversions.15 8-OxoG in ss-DNA is more easily oxidized than in doublestranded (ds) DNA due to better accessibility to oxidants.14 Ropp (6) Olinski, R.; Zastawny, T.; Budzbon, J.; Skokowski, J.; Zegarski, W.; Dizdaroglu, M.; FEBS Lett. 1992, 309, 193-198. (7) Steenken, S.; Jovanovic S. V. J. Am. Chem. Soc. 1997, 119, 617-618. (8) (a) Shigenaga, M. K.; Ames, B. N. Free Radical Biol. Med. 1991, 10, 211216. (b) Lunec, J.; Holloway, K. A.; Cooke, M. S.; Faux, S.; Griffiths, H. R.; Evans, M. D. Free Radical Biol. Med. 2002, 33, 875-885. (c) Kasai, H. Mutat. Res. 1997, 387, 147-163. (d) Halliwell, B. Free Radical Biol. Med. 2002, 32, 968-974. (e) Gedik, C. M.; Boyle, S. P.; Wood, S. G.; Vaughan, N. J.; Collins, A. R. Carcinogenesis 2002, 23, 1441-1446. (9) (a) Floyd, R. A.; Watson, J. J.; Wong, P. K.; Altmiller, D. H.; Rickard, R. C. Free Radical Res. Commun. 1986, 1, 163-172. (b) Helbock, H. J.; Beckman, K. B.; Shigenaga, M. K.; Walter, P. B.; Woodall, A. A.; Yeo, H. C.; Ames, B. N. Proc. Natl. Acad. Sci. U.S.A. 1998, 95, 288-293. (c) Ravanat, J.; Turesky, R. J.; Gremaud, E.; Trudel, L. J.; Stadler, R. H., Chem. Res. Toxicol. 1995, 8, 1039-1045. (10) (a) Yanagawa, H.; Ogawa, Y.; Ueno, M. J. Biol. Chem. 1992, 267, 1332013326. (b) Shigenaga, M. K.; Park, J. W.; Cundy, K. C.; Gimeno, C. J.; Ames, B. N. Methods Enzymol. 1990, 186, 521-530. (11) (a) Goyal, R. N.; Jain, N.; Garg, D. K. Bioelectrochem. Bioenerg. 1997, 43, 105-114. (b) Duarte, V.; Muller, J. G.; Burrows, C. J. Nucleic Acids Res. 1999, 27, 496-502. (12) Cunningham, R. P., Curr. Biol. 1997, 7, R576-R579. (13) (a) Kuchino, Y.; Mori, F.; Kasai, H.; Inone, H.; Iwai, S.; Miure, K.; Ohtsuka, E.; Nishimura, S. Nature 1987, 327, 77-79. (b) Shibutani, S.; Takeshita, M.; Grollman, A. P. Nature 1991, 349, 431-434. (c) Moriya, M. Proc. Natl. Acad. Sci. U.S.A. 1993, 90, 1122-1126. (14) (a) Hickerson, R. P.; Prat, F.; Foote, C. S.; Burrows, C. J. J. Am. Chem. Soc. 1999, 121, 9423-9428. (b) Luo, W.; Muller, J. G.; Rachlin, E. M.; Burrows, C. J. Chem. Res. Toxicol. 2001, 14, 927-938. (15) Henderson, P. T.; Delaney, J. C.; Muller, J. G.; Neeley, W. L.; Tannenbaum, S. R.; Burrows, C. J.; Essigmann, J. M.Biochemistry 2003, 42, 9257-9262.

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and Thorp found that Os(bpy)33+ in solution oxidized 8-oxoG but not guanine.16 This group also reported conformationally controlled electron transfer with a 25-mer oligonucleotide labeled with Os(bpy)32+ on one end and 8-oxoG on the other,17 used Os(bpy)32+ to probe telomerase function in DNA using 8-oxoG placed at specific sites,18 employed two soluble ruthenium complexes to detect modified nucleobases,19 and detected guanine-containing DNA by ruthenium complexes in an immobilized metallopolymer.20 We recently used LC-EC to show that oxidation of free guanine and ds-DNA by hydroxyl radicals generated by Fenton reagent resulted in non-steady-state concentrations of 8-oxoG.21 LC-MS detected guanidinohydantoin as the product of 8-oxoG oxidation in these studies. The requirement for DNA hydrolysis in LC-EC or LC-MS contributes significantly to analysis time and expense, precluding routine clinical applications. Biosensor alternatives to detect DNA oxidation without hydrolysis or expensive instrumentation would be advantageous. Electrochemical methods provide simple, sensitive, inexpensive approaches to detect DNA damage and hybridization.22-27 In the present paper, we describe films of Os and Ru poly(vinylpyridines) constructed layer by layer on electrodes that catalyze the oxidation of 8-oxoguanine and guanine, respectively, at well-separated potentials. These biosensor electrodes were used to detect unhydrolyzed, oxidized DNA in solution or films by square wave voltammetry (SWV). In a complimentary approach, we recently utilized an osmium polymer with a higher oxidation potential to obtain electrochemiluminescence from oxidized DNA in thin films.28 EXPERIMENTAL SECTION Chemicals and Materials. Calf thymus DNA (CT) ds-DNA (type XV, 41.9% G/C and salmon testes ds-DNA (2000 average base pairs, 41.2% G/C) was from Sigma. Poly(sodium 4-styrenesulfonate) (PSS) and K2OsCl6 were from Aldrich. All other chemicals were reagent grade. Synthesis of [Os(bpy)2(PVP)10Cl]+ and [Ru(bpy)2(PVP)10Cl]+ followed literature procedures.29-31 Elemental analysis and UV-visible absorption spectra were consistent with the desired products. (16) Ropp, P. A.; Thorp, H. H. Chem. Biol. 1999, 6, 599-605. (17) Holmberg, R. C.; Tierney, M. T.; Ropp, P. A.; Berg, E. E.; Grinstaff, M. W.; Thorp, H. H. Inorg. Chem. 2003, 42, 6379-6387. (18) Szalai, V. A.; Singer, M. J.; Thorp, H. H. J. Am. Chem. Soc.2002, 124, 16251631. (19) Yang, I. V.; Ropp, P. A.; Thorp, H. H., Anal. Chem. 2002, 74, 347-353. (20) Ontko, A. C.; Armistead, P. M.; Kircus, S. R.; Thorp, H. H. Inorg. Chem. 1999, 38, 1842-1846. (21) White, B.; Smyth, M. R.; Stuart, J. D.; Rusling, J. F. J. Am. Chem. Soc. 2003, 125, 6604-6605. (22) Palecek, E.; Fojta, M.; Tomschik, M.; Wang, J., Biosens. Bioelectron. 1998, 13, 621-628. (23) Palecek, E. Electroanalysis 1996, 8, 7-14. (24) Thorp, H. H. Trends Biotechnol. 1998, 16, 117-121. (25) Palecek, E.; Fojta, M. Anal. Chem. 2001, 73, 74A-83A. (26) (a) Wang, J.; Rivas, G.; Ozsoz, M.; Grant, D. H.; Cai, X.; Parrado, C. Anal. Chem. 1997, 69, 1457-1460. (b) Wang, J. Chem. Eur. J. 1999, 5, 16811685. (27) Rusling J. F.; Zhang Z. In Biomolecular Films; Rusling J. F. Ed.; Marcel Dekker: New York, 2003; pp 1-64. (28) Dennany, L.; Forster, R. J.; White, B.; Smyth, M. R.; Rusling, J. F. J. Am. Chem. Soc., 2004, 126, 8835-8841. (29) Forster, R. J.; Vos, J. G. Langmuir 1994, 10, 4330-4338. (30) Buckingham, D. A.; Dwyer, F. P.; Goodwin, H. A.; Sargeson, A. M. Aust. J. Chem. 1964, 17, 325-336. (31) Forster, R. J.; Vos, J. G. Macromolecules 1990, 23, 4372-4377.

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Film Assembly. Alternate layer-by-layer electrostatic selfassembly32 was used to immobilize metallopolymers onto electrodes. First, a [Ru(bpy)2(PVP)10Cl)]+ layer (denoted hereafter as ClRu-PVP) was adsorbed on a disk electrode (A ) 0.017 cm2) of ordinary basal plane pyrolytic graphite (PG, Advanced Ceramics) by immersing in 1 mg mL-1 ClRu-PVP in 5% methanol/water for 15 min.33 The electrode was washed with methanol/water, then water, and then dried in a stream of nitrogen. It was then immersed in aqueous 2 mg mL-1 sodium poly(styrenesulfonate) (PSS, MW 70 000, Aldrich) in 50 mM NaCl for 15 min.34 It was then rinsed with water and immersed in 1 mg mL-1 Os(bpy)2(PVP)10 Cl)]Cl (denoted hereafter as ClOs-PVP) in methanol for 15 min, followed by ethanol and water rinses and drying in a stream of nitrogen. Film composition is denoted ClRu-PVP/PSS/ ClOs-PVP. This procedure was found to give more reversible and reproducible metallopolymer voltammetry compared to adsorbing mixed layers of ClRu-PVP/ClOs-PVP. Films containing DNA were fabricated by adsorbing PSS from 25-µL drops of 3 mg mL-1 PSS containing 0.5 M NaCl on the electrode surface and then washing with water after 15 min. These electrodes were then coated with 1 mg mL-1 ClRu-PVP in 5% methanol/water. By changing the polycation and polyanion solution successively, Os-PVP was absorbed from 1 mg mL-1 Os(bpy)2(PVP)10 Cl)]Cl in methanol, salmon testes DNA from 2 mg mL-1 pH 7.1 Tris buffer containing 0.5 M NaCl, and PDDA from 2 mg mL-1 PDDA in 0.05 M NaCl. Alternate adsorption cycles were repeated until the desired numbers of layers were made. Final films are denoted by order of layer assembly as PSS/ ClRu-PVP/PSS/ClOs-PVP/DNA/PDDA/DNA. The same procedure was used to make PSS/Ru-PVP/PSS/Os-PVP/poly [G]/PDDA/poly [G] films by replacing 2 mg mL-1 DNA with 0.5 mg/ mL poly[G] in pH 7.1 buffer + 0.5 M NaCl. Assembly of films was monitored with a quartz crystal microbalance (QCM, USI) using 9-MHz resonators (AT-cut, International Crystal Mfg.). To mimic the carbon electrode surface, a negative monolayer was made by treating gold-coated (0.16 ( 0.01 cm2) quartz resonators with 0.7 mM 3-mercapto-1-propanol and 0.3 mM 3-mercaptopropionic acid in ethanol.41 Films were as(32) (a) Lvov, Y. In Protein Architecture: Interfacing Molecular Assemblies and Immobilization Biotechnology; Lvov, Y., Mo ¨hwald, H., Eds.; Marcel Dekker: New York, 2000; pp 125-167. (b) Lvov, Y. In Handbook Of Surfaces And Interfaces Of Materials, Vol. 3. Nanostructured Materials, Micelles and Colloids; Nalwa, R. W., Ed.; Academic Press: San Diego, 2001; pp 170-189. (33) (a) Mugweru, A. M.; Rusling, J. F. Electrochem. Commun. 2001, 3, 406409. (b) Wang, B.; Rusling, J. F. Anal. Chem. 2003, 75, 4229-4235. (34) (a) Lvov, Y. M.; Lu, Z.; Schenkman, J. B.; Zu, X.; Rusling, J. F. J. Am. Chem. Soc. 1998, 120, 4073-4080. (b) Lvov, Y.; Ariga, K.;. Ichinose, I.; Kunitake; T. J. Am. Chem. Soc. 1995, 117, 6117-6123. (35) (a) Aruoma, O. I.; Halliwell, B.; Gajewski, E.; Dizdaroglu, M. J. J. Biol. Chem. 1989, 264, 20509-20512. (b) Walling, C. Acc. Chem. Res. 1975, 8, 125131. (36) (a) Rusling, J. F.; Zhang, Z. In Handbook of surfaces and interfaces of materials; Nalwa, H. S., Ed.; Academic Press: New York, 2001, (b) Lvov, Y.; Munge, B.; Giraldo, O.; Ichinose, I.; Suib, S. L.; Rusling, J. F. Langmuir 2000, 16, 8850-8857. (37) (a) Hirst, J.; Amstrong, F. A. Anal. Chem. 1998, 70, 5062-5071. (b) Laviron, E. J. Electroanal. Chem. 1979, 101, 19-28 (38) (a) Oliveira, A. M.; Piedade, J. A. P.; Serrano, S. H. P. Electroanalysis 2000, 12, 969-973. (b) Oliveira-Brett, A. M.; Vivan, M.; Fernades, I. R.; Piedade, I. A. P. Talanta 2002, 56, 956-970. (39) Henle, E. S.; Luo, Y.; Linn, S. Biochemistry 1996, 35, 12212-12219. (40) Luo, Y.; Henle, E. S.; Jin, R.; Chattopadhyaya, R.; Linn, S. Method Enzymol. 1994, 234, 51-59. (41) Zhou, L.; Rusling, J. F. Anal. Chem. 2001, 73, 4780-4786.

Figure 1. QCM frequency changes during monitoring of growth of (Ru-PVP/PSS/ClOs-PVP)2/PSS films on a gold-coated quartz resonator.

sembled as for PG electrodes and dried in a stream of nitrogen before measuring frequency change (∆F). Electrochemical Analysis. Voltammetry was done as described previously,33 with ohmic drop ∼98% compensated. SWV was at 4 mV step, 25 mV pulse, and 10 Hz. All solutions and buffers were made using water purified with a Hydro Nanopure System to specific resistance of >18 MΩ cm. Buffers were 20 mM acetate pH 5.5 or 20 mM tris pH 7.1 and included 50 mM NaCl. Solutions were purged for 5 min with purified nitrogen before voltammetry. Oxidation of DNA. DNA was oxidized by using Fenton reagent to generate hydroxyl radicals.35 DNA in 0.2 mg mL-1 solutions was incubated with Fenton reagent containing 0.15 mM FeSO4 and 50 mM H2O2 at 37° C with stirring. The 150-µL aliquots were taken from the reaction mixture and quenched with 1 mL of ethanol.21 The mixture was dried under nitrogen and then redissolved in buffer. PSS/Ru-PVP/PSS/Os-PVP/DNA/PDDA/DNA films were immersed in pH 7.1 buffer for 20 min, and then SWV was recorded. The same film was incubated in Fenton reagent containing 1 mM FeSO4 and 4 mM H2O2 at 37 °C. After a given reaction time, electrodes were rinsed with water and transferred into an electrochemical cell containing pH 7.1 buffer for SWV. RESULTS Film Characterization by QCM. Assembly of films was monitored by using QCM weighing at each adsorption step for films constructed on gold-coated quartz resonators.32 The mass increase [M(g)] for each adsorption step was estimated from the QCM frequency shifts [∆F(Hz)] of dry films by using the Saurbrey equation for the 9-MHz resonator characteristics:32,36

∆F ) (-1.83 × 108)M/A

(1)

where A is the area of the resonator electrode (0.16 cm2 on one side). Nominal film thickness (d) was estimated from32

d(nm) ≈ -(0.016 ( 0.002)∆F(Hz)

(2)

Figure 1 shows a linear decrease in QCM frequency with the number of adsorbed layers on a Au-coated resonator, consistent with reproducible layer formation. The average QCM frequency decrease for the ClRu-PVP layer was 194 Hz and was 212 Hz for ClOs-PVP. From eq 1, these

changes correspond to 1.17 × 10-10 mol cm-2 for ClOs-PVP and 0.97 × 10-10mol cm-2 for ClRu-PVP. From eq 2, nominal thickness was 3.1 for ClRu-PVP layers and 3.4 nm for ClOs-PVP layers. Total thickness of the film in Figure 1 was ∼25 nm. Voltammetry of Metallopolymer Films. Figure 2a shows cyclic voltammogram of a film containing one layer of each of the ClRu-PVP and ClOs-PVP metallopolymers. This film is ∼11 nm thick. Reversible pairs of oxidation-reduction peaks were found by cyclic voltammetry corresponding to the Ru3+/Ru2+ and Os3+/Os2+ redox centers. The formal potential as the midpoint between the oxidation and reduction peaks was 0.76 V versus SCE for Ru3+/Ru2+ and 0.34 V for Os3+/Os2+. Similar results were obtained regardless of whether ClRu-PVP or ClOs-PVP was adsorbed as the first layer on the electrode. Plots of peak current versus scan rate were linear at scan rates for the Os2+ (r ) 0.998) and Ru2+ peaks (r ) 0.999) for 0.01-0.40 V s-1 (Supporting Information, Figure S1) and the ratio of anodic to cathodic peak currents was unity, indicating chemical reversibility. Integration of low scan rate peaks of Os2+ gave surface concentration of 1.11 × 10-10 mol cm-2 and the Ru2+ peak gave 1.09 × 10-10 mol cm-2. Comparing these values with QCM results suggests that both catalysts are fully electroactive in these thin films. The separation between anodic and cathodic peak currents for Os3+/Os2+ at 0.10 V s-1 was ∼50 mV, for Ru3+/Ru2+ was 40 mV, and was essentially the same at lower scan rates. Oxidation and reduction peak potentials of Os and Ru sites varied in opposite directions as scan rate was increased above 100 mV s-1, with the net effect of increasing the peak separation. Peak width at half-height was greater than the theoretical 90 mV expected for reversible, one-electron reaction and increased significantly with scan rate. The CV results for both electroactive centers were consistent with nonideal, reversible, thin-layer electrochemistry.27 Figure 2b shows the influence of scan rate on oxidation-reduction peak separations for experimental data compared to calculated values based on a modified Butler-Volmer thin-film model. Following the suggestion of Hirst and Armstrong,37 the nearly constant peak separation of nonkinetic origin at low scan rates was subtracted from peak separations at higher scan rates. The corrected data gave reasonably good fits to the thin-film Butler-Volmer model (Figure 2b). Estimated apparent electron-transfer rate constant k° for Ru2+/Ru3+ was 11.5 ( 0.2 s-1 and for Os2+/Os3+ was 10.5 ( 0.1 s-1. Detection of Oxidized DNA in Solution. 8-OxoG forms when DNA reacts with hydroxyl radicals generated by Fenton reagent. The irreversible oxidation potential of 8-oxoG at a glassy carbon electrode is ∼0.45 V versus SCE.38 The Os3+/Os2+ redox couple in the metallopolymer film with formal potential 0.34 V was expected to catalytically oxidize 8-oxoG. Figure 3 shows a SWV of a ClRu-PVP/PSS/ClOs-PVP film in buffer and in the same buffer after addition of intact ds-CT-DNA. An increased peak was observed at the Ru oxidation potential corresponding to the catalytic oxidation of guanine (Figure 3, curve b).33 The Os peak remained unchanged, consistent with minimal 8-oxoG in the undamaged DNA. After incubation of ds-CT-DNA solutions with Fenton reagent, we found catalytic increases in peak current for both the Os and Ru peaks (Figure 3, curve c). Oxidized salmon testes DNA gave similar results. In control experiments where DNA was incubated Analytical Chemistry, Vol. 76, No. 18, September 15, 2004

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Figure 2. Voltammetric results for ClRu-PVP/PSS/ClOs-PVP/PSS films: (a) Cyclic voltammogram at 0.1 V s-1 in pH 5.5 buffer and (b) influence of scan rate on oxidation-reduction peak separation (∆Ep). Points are experimental data, and lines represent theory for a modified ButlerVolmer thin-film model at ks values shown, assuming transfer coefficient R ) 0.5 for ∆Ep compensated by subtracting average (constant) value below 100 V s-1.

Figure 3. SWV of ClRu-PVP/PSS/ClOs-PVP/PSS film (a) in pH 7.1 buffer, (b) in buffer containing 0.2 mg mL-1 CT ds-DNA, and (c) in buffer containing 0.2 mg mL-1 CT ds-DNA that had been incubated in Fenton reagent for 5 min.

Figure 4. Ratio of final to initial SWV peak currents of a ClRuPVP/PSS/ClOs-PVP/PSS film in solution of CT-DNA after incubated with Fenton reagent at different times: Os peaks (O), Ru peaks (b), and control incubated with H2O2 without FeSO4 (2). (Error bars represent standard deviations for three trials.)

with Fenton reagent and analyzed with a film containing only ClRuPVP, a catalytic current increase at the Ru peak was also found. Figure 4 shows the influence of reaction time of ds-CT-DNA with Fenton reagent on the ratio of final to initial peak current. Initial peak current is that before DNA incubation while final peak current is that after DNA incubation with Fenton reagent. A single 5560 Analytical Chemistry, Vol. 76, No. 18, September 15, 2004

electrode film was used to probe all solutions containing the same concentration of DNA but incubated at different times with Fenton reagent. The electrode was washed with water each time after analyzing each solution of DNA and the peak current monitored in fresh buffer to ensure that there was no change in the noncatalytic signals. The outer layer of negatively charged PSS on the films inhibits DNA adsorption. Peak current ratios for Os and Ru were monitored for DNA samples reacted with Fenton reagent collected after each minute. For the Os peak, the current increased up to ∼4 min and then decreased. For the Ru peak, there is a lag of 2 min, after which the peak current ratio increased. Error bars in Figure 4 reflect the reproducibility of the sensor in this application. Electrodes used in these studies showed less than 20% loss in Os peak current during three weeks of storage in buffer and less than 10% loss when stored in air for the same period. At longer oxidation times for ds-CT-DNA, salmon testes DNA (Supporting Information, Figure S2), and poly[G], we observed peak current ratios for Os that oscillated with a period of ∼10 min. Hydroxyl radicals are not produced unless FeSO4 and H2O2 are present.35 In control experiments, DNA was incubated with FeSO4 in the absence of H2O2 or with H2O2 but no FeSO4. The peak ratios for these control experiments showed no significant trends with reaction time. To establish the origins of the catalytic peak increases of the metallopolymer films, we investigated oxidized polynucleotides. Experiments on poly[G] showed the same peak current for the Os peak as in pure buffer, but after incubation with Fenton reagent, both the Os peak and the Ru peak increased (Figure 5). Controls in which poly[G] was incubated with H2O2 but without FeSO4 showed no significant peak increases. Other studies were done using polyadenine (Supporting Information, Figure S3) and polycytidine (Figure S4). In both cases, an increased catalytic peak current was observed for Ru when these polynucleotides were incubated with Fenton reagent. Peak currents for Os were not changed after incubation with Fenton reagent for poly[C], but an increased peak current for Os was found with oxidized poly[A]. The increase per mole of polynucleotide used was somewhat smaller than for poly[G] incubated under similar conditions. Detection of Oxidized DNA within Films. The peaks of the ClRu-PVP and ClOs-PVP were not stable in 50 mM H2O2,

Figure 5. SWV of ClRu-PVP/PSS/ClOs-PVP/PSS film (a) in pH 7.1 buffer, (b) in buffer containing 0.05 mg mL -1 Poly[G], and (c) in buffer containing 0.05 mg mL-1 Poly[G] that had been incubated in Fenton reagent for 5 min.

especially the ClOs-PVP peak. Linn studied DNA damage by Fenton reagent39 and found that the presence of oxygen decreased H2O2 consumption relative to Fe2+. Luo et al. observed measurable DNA damage using 2 mM H2O2 and 1 mM Fe2+.40 Thus, to minimize the degradation of metallopolymers by H2O2, we evaluated concentrations of 2-4 mM. We observed an Os peak increase at 2 mM H2O2 and 1 mM Fe2+, but a larger signal was found at 4 mM H2O2 and 1 mM Fe2+, which was used in all following studies. This level of H2O2 did not degrade the metallopolymer. PSS/ClRu-PVP/PSS/ClOs-PVP films showed the SWV Os peak at ∼0.25 V and the Ru peak at 0.70 V in pH 7.1 tris buffer (Figure 6). Previous QCM studies of DNA/PDDA layers suggest that assembly of DNA/PDDA/DNA layers on top of this film adds ∼5 nm.41 Combining this with QCM results on the metallopolymer layers, we estimate that PSS/ClRu-PVP/PSS/ClOs-PVP/DNA/ PDDA/DNA films were ∼17 nm thick. Figure 6a shows peak current at 0.7 V increased, consistent with the catalytic oxidation of intact guanine by the Ru2+/3+ redox couple.33 There is no significant difference at 0.25 V between the films with and without DNA, consistent with an unreactive Os site with respect to intact DNA. After the PSS/Ru-PVP/PSS/Os-PVP/DNA/PDDA/DNA film was incubated in Fenton reagent at 37 °C for 10 min, the film electrode showed much larger peaks at 0.25 and 0.7 V. Control experiments using 4 mM H2O2 alone for PSS/Ru-PVP/PSS/OsPVP/DNA/PDDA/DNA did not cause significant changes in the

Ru or and Os peaks (Figure 6b). Controls using 1 mM FeSO4 alone gave similar results as in Figure 6b. Figure 7 shows the Fenton reaction time dependence of the peak ratios. After incubation in the Fenton reagents, both Ru and Os peaks increased so that the ratios go through maximums at ∼5 min. Similar to the experiments in DNA solutions (cf. Figure 4), the Os peak ratio is larger than the Ru peak ratio, and the Ru peak ratio features a relative lag over the first few minutes. Control experiments using either H2O2 or FeSO4 alone did not cause a significant change in the Ru or Os peaks, even after 100-min incubation. We also studied the influence of Fenton reagent with poly[G] in the films. Figure 8 shows the peak ratio change of Os peaks during incubation of PSS/ClRu-PVP/PSS/ClOs-PVP/poly[G]/ PDDA/poly[G] films in Fenton reagent for different times. Again, a maximum was observed at ∼4 min. The Ru peak increased as well, but the Os peak was larger. DISCUSSION By comparing results from QCM and voltammetry (Figures 1 and 2a), we conclude that the immobilized metallopolymer catalysts are almost 100% reversibly electroactive. These 11-nmthick films feature only ∼5 nmol of ClRu-PVP and ∼6 nmol of ClOs-PVP. The two metallopolymers behaved electrochemically independent of one another in the films as observed by cyclic voltammetry (Figure 2) and had similar electrode reaction rate constants derived from a modified thin-film Butler-Volmer model (Figure 2b). Results in Figures 3 and 5-8, as well as Figures S3 and S4, show that the catalytic current increases for the Os2+ peaks for Fenton-oxidized oligonucleotides containing guanine. We previously documented the formation of 8-oxoG by LC-EC using the same Fenton reaction conditions as in the present work for DNA in solutions21 and in films.28 All these data support the key conclusion of the present work that the voltammetric Os peaks of ClOs-PVP respond to oxidized DNA and predominantly to the oxidation of 8-oxoG. Figure S3 shows a small response of the Os peak to oxidized poly[A] as well, but the predominant response for oxidized DNA is likely to be from the more easily formed 8-oxoG. We do not detect 8-oxoG by direct oxidation in the film environment because it is used up catalytically in the analysis or

Figure 6. Difference SWV of films in pH 7.1 buffer: (1) PSS/ClRu-PVP/PSS/ClOs-PVP film; (2) PSS/ClRu-PVP/PSS/ClOs-PVP/DNA/PDDA/ DNA film; (3) PSS/ClRu-PVP/PSS/ClOs-PVP/DNA/PDDA/DNA film (a) after 10-min incubation in Fenton reagent (4 mM H2O2 + 1 mM FeSO4); (b) PSS/ClRu-PVP/PSS/ClOs-PVP/DNA/PDDA/DNA film incubated in 4 mM H2O2 for 0 (solid line), 15 (dotted line), and 30 min (dashed line), all showing nearly identical voltammograms.

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Figure 7. Ratio of final to initial peak currents of films of PSS/ClRuPVP/PSS/ClOs-PVP/DNA/PDDA/DNA after incubation with Fenton reagent at different times for the Os (O) and Ru (b) peaks, and control experiments for Os peak for incubation in absence of H2O2 (4). (Error bars represent standard deviations for three trials.)

the voltammetric Os peak and LC-EC is consistent with the detection of 8-oxoG. Oxidized DNA in solution was easily detected by the Os catalytic peak after 1-min reactions with Fenton reagent (Figure 4). Previous LC-EC analysis of ds-DNA that was reacted with Fenton reagent under the same conditions and then hydrolyzed showed that 1 8-oxoG is formed in 1 min per 1250 guanines in the DNA.21 Since ds-CT-DNA has 21% guanine bases, assuming that the majority of the peak increase derives from 8-oxoG, we estimate that the ClOs-PVP sensors described here can detect 1 8-oxoG/6000 DNA bases. The invariance of the Os peaks in the presence of intact DNA (Figures 3 and 6) shows that guanine is not oxidized by the Os3+ sites in the films, most likely because of insufficient driving force. Furthermore, DNA controls incubated with Fe2+ or H2O2 alone did not yield catalytic peak increases for Os. Only incubation of DNA with Fenton reagent provided an increase of the Os peak. These results suggest that the Os peak increases in Figures 3 and Figure 6a were not caused by H2O2 or Fe2+ alone but by reaction products of DNA oxidation. All results are consistent with predominant oxidation of 8-oxoG by Os sites, and the major catalytic pathway for signal development at the Os peak is as follows:

[ClOs-PVP]2+ T [ClOs-PVP]3+ + e- (at electrode) (3) [ClOs-PVP]3+ + DNA(8-oxoG) f [ClOs-PVP]2+ + DNA(8-OxoG+) (4)

Figure 8. Ratio of final to initial peak currents for Os peak of PSS/ ClRu-PVP/PSS/ClOs-PVP/poly [G]/PDDA/poly[G] film after incubation with Fenton reagent at different times (b) control experiments which include buffer and H2O2 without FeSO4 (2). (Error bars represent standard deviations for three trials.)

the small amounts available fall below the detection limit for direct electrolysis. The maximums in the peak ratio versus reaction time data (Figures 4, 7, and 8) are also consistent with 8-oxoG as the species that is detected by the catalytic peak at the Os potential. These maximums are consistent with a competitive consecutive process in which guanine is oxidized by OH• to 8-oxoG, which is then rapidly oxidized by OH• to guanidinohydantoin. The latter oxidation product of 8-oxoG was confirmed as a major product of Fenton oxidation of 8-oxoG in our reactions by LC-MS.21 The overall process involves reaction of OH• with guanine as well as the initial reaction product 8-oxoG. This simple consecutive pathway typically leads to a maximum in the concentration of the initial reaction product,42 here 8-oxoG, consistent with the observed maximums in Os peak ratios. At longer reaction times, oscillations of the 8-oxoG concentration have been observed by LC-EC 21,28 that mirror the changes seen in time scales of 10-100 min for the SWV Os peak ratios with oxidized DNA (Figure S2). These oscillations in 8-oxoG concentration most likely involve complex coupled catalytic pathways.21 In any case, the full correspondence of results from (42) Zuman, P.; Patel, R. Techniques in Organic Reaction Mechanisms; Wiley: New York, 1984; pp 96-100.

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We previously showed that among the DNA nucleobases guanines are selectively oxidized by films of ClRu-PVP.33 Because of its low oxidation potential, 8-oxoG may also be oxidized at Ru sites. In the present work, we observed that intact and oxidized ds-DNA and poly[G], as well as oxidized poly[A] and poly[C], gave increased Ru oxidation peaks. These results suggest that the increased Ru peaks upon DNA oxidation result from oxidized cytosines, adenines, and guanines. ss-DNA also gave larger catalytic peaks with ClRu-PVP than ds-DNA because of better accessibility of the guanines to catalytic sites in the single-stranded material.33 Since Fenton reagent is known to produce strand breaks in DNA,4,5 it is possible that some single-stranded oligonucleotides are formed whose guanines react faster than in dsDNA to increase the catalytic current. Thus, we rationalize that the increase in the Ru peak upon DNA oxidation is caused by indiscriminate oxidation of altered nucleobases along with strand cleavage that increases the rate of reaction of Ru3+ sites with intact guanines. The above scenario is also consistent with the 2-min lag before the Ru peak current increases during the Fenton reaction, compared to the immediate increase in the Os peaks both in solution DNA reactions (Figure 4) and with DNA in films (Figure 7). Since guanines are by far the most easily oxidized nucleobases,7 8-oxoG is the predominant initial product of Fenton oxidation so that the Os peak increases immediately. After a number of guanines in the DNA have been oxidized, hydroxyl radicals begin to attack the other bases, additional oxidation products build up, and the Ru peak increases. In summary, films containing the catalytic metallopolymer [ClOs(bpy)2(PVP)10]2+ on electrodes can be used to selectively

detect oxidized DNA. The voltammetric measurement is simple and inexpensive, and the approach may be useful for the detection of oxidized DNA as a clinical biomarker for oxidative stress. The combination of ruthenium and osmium metallopolymers in the films allows for the detection of an Os signal that is mainly selective to 8-oxoG as well as detection of other oxidized nucleobases from the Ru peaks. This adds a measure of selfconsistency to the analyses. A related Os-PVP polymer with higher oxidation potential can generate electrochemiluminescence (ECL) with oligonucleotides containing 8-oxoG in thin films, providing an alternate method to detect oxidative stress.28 The present purely voltammetric approach is complementary to the ECL method. It requires no light detector, it is applicable to solution DNA measurements which are insensitive by ECL, and it requires a

lower applied potential than ECL, making the method less sensitive to possible oxidizable interferences. ACKNOWLEDGMENT Financial support was provided by U.S. PHS Grant ES03154 (J.R.) from the National Institute of Environmental Health Sciences (NIEHS), NIH. 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 April 27, 2004. Accepted July 1, 2004. AC049375J

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