Electrocatalytic Oxidation of Guanine and DNA on a Carbon Paste

Department of Chemistry, Shiraz University, Shiraz 71454, Iran. The electrochemical ..... 1998, 362, 194-200. (79) Stamford, J. A.; Justice, J. B. Ana...
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Anal. Chem. 2004, 76, 5690-5696

Electrocatalytic Oxidation of Guanine and DNA on a Carbon Paste Electrode Modified by Cobalt Hexacyanoferrate Films Abdolkarim Abbaspour* and Masoud Ayatollahi Mehrgardi

Department of Chemistry, Shiraz University, Shiraz 71454, Iran

With the discovery of the electroactivity of deoxyribonucleic acids in 1960,1 the interest of most researchers was to apply modern voltammetric methods in nucleic acid and DNA analysis.2 Electrochemical DNA sensing is a promising technique of nucleic acid analysis because of its quickness, high sensitivity, and low cost.3-9 Thorp and co-workers developed a catalytic guanine oxidation with a ruthenium complex, [Ru (bpy)3]2+, as an oxidation catalyst. They suggested a two-step electrochemical oxidation of [Ru (bpy)3]2+ to Ru(III) species, followed by the chemical oxidation of guanine in a redox reaction that this reaction serves to regenerate the [Ru (bpy)3]2+.10-17

In 2001, El-Maali and Wang18 by using an insoluble complex of ruthenium-tris(2, 2′-bipyridyl)dichloro-ruthenium(II) reported a chemically modified carbon paste electrode (CMCPE) for electrocatalytic detection of DNA. In contrast to carbon paste electrodes that have been widely used for stripping analysis of nucleic acids, this report and our previous study19 may be the first reports on the use of CMCPE for enhancing the voltammetric response of DNA. In this article, the continuation of previous study, we describe the application of cobalt hexacyanoferrate-modified carbon paste electrode for electrocatalytic detection of DNA in aqueous solutions. Chemically modified electrodes have attracted considerable interest over the past two decades as researchers have attempted to exert more direct control over the chemical nature of an electrode. Among the various reagents used for electrode modification, transition metal hexacyanoferrates (MHCFs) have attracted the attention of electrochemists as excellent electrontransfer mediators.20-25 These inorganic polymers have been widely used in electrochemistry to electrocatalyze the oxidation of dopamine,26 ascorbic acid,27 NADH,28 glutathione,29,30 cysteine,29 and H2O231 and the reduction of Fe3+,32 CO2,33 and hydrazine.34,35 When these inorganic complexes combine with glucose oxidase, MHCF-modified electrodes can also be applied as a biosensor for glucose.36-38

* To whom correspondence should be addressed. E-mail: abbaspour@ chem.susc.ac.ir. (1) Palecek, E. Nature 1960, 188, 656-657. (2) Wang, J.; Bollo, S.; Lopez-Paz, J. L.; Sahlin, E.; Mukherjee, B. Anal. Chem. 1999, 71, 1910-1913. (3) Palecek, E. Talanta 2002, 56, 809-819. (4) Wang, J.; Palecek, E.; Nielsen, P. E.; Rivas, G.; Cai, X.; Shiraishi, H.; Dontha, N.; Luo, D.; Farias, P. A. M. J. Am. Chem. Soc. 1996, 118, 7667-7670. (5) Mikkelsen, S. R. Electroanalysis 1996, 8, 15-19. (6) Yang, M.; McGovern, M. E.; Thompson, M. Anal. Chim. Acta 1997, 346, 259-275. (7) Wang, J. Anal. Chem. 1999, 71, 328R-332R. (8) Palecek, E.; Fojta, M. Anal. Chem. 2001, 73, 75A-83A. (9) Takenaka, S. Bull. Chem. Soc. Jpn. 2001, 74, 217-224. (10) Johntson, D. H.; Glasgow, K. C.; Thorp, H. H. J. Am. Chem. Soc. 1995, 117, 8933-8938. (11) Szali, V. A.; Jayawickamarajah, J.; Thorp, H. H. J. Phys. Chem. B 2002, 106, 709-716. (12) Holmberg, R. C.; Thorp, H. H. Anal. Chem. 2003, 75, 1851-1860. (13) Yang, I. V.; Ropp, P. A.; Thorp, H. H. Anal. Chem. 2002, 74, 347-354. (14) Armistead, P. M.; Thorp, H. H. Anal. Chem. 2001, 73, 558-564. (15) Arminstead, P. M.; Thorp, H. H. Anal. Chem. 2000, 72, 3764-3770. (16) Ontko, A. C.; Armistead, P. M.; Kircus, S. R.; Thorp, H. H. Inorg. Chem. 1999, 38, 1842-1846. (17) Johnstone, D. H.; Cheng, C. C.; Campbell, K. J.; Thorp, H. H. Inorg. Chem. 1994, 33, 6388-6390.

(18) El-Maali, N. A.; Wang, J. Sens. Actuators, B 2001, 76, 211-214. (19) Abbaspour, A.; Mehrgardi, M. A.; Kia, R. J. Electroanal. Chem. 2004, 568, 261-266. (20) Itaya, K.; Ataka, T.; Toshima, S. J. Am. Chem. Soc. 1982, 104, 4767-4772. (21) Siperko, L. M.; Kuwana, T. J. Electrochem. Soc. 1983, 130, 396-402. (22) Lin, C.; Bocarsly, A. B. J. Electroanal. Chem. 1991, 300, 325-345. (23) Zhou, J.; Wang, E. Electroanalysis 1994, 6, 29-35. (24) Hou, W.; Wang, E. J. Electroanal. Chem. 1991, 316, 155-163. (25) Zhou, D. M.; Ju, H. X.; Chen, H. Y. J. Electroanal. Chem. 1996, 408, 219223. (26) Jiang, M.; Zhou, X. Y.; Zhao, Z. F. Ber. Bunsen-Ges. Phys. Chem. 1991, 95, 720-724. (27) Cai, C. X.; Ju, H. X.; Chen, H. Y. Anal. Chim. Acta 1995, 310, 145-149. (28) Shi, G. Y.; Lu, J. X.; Xu, F.; Sun, W. L.; Jin, L. T.; Yamamoto, K.; Tao, S. G.; Jin, J. Y. Anal. Chim. Acta 1999, 391, 307-313. (29) Zhang, S.; Sun, W. L.; Zhang, W.; Qi, W. Y.; Jin, L. T.; Yamamoto, K.; Tao, S. G.; Jin, J. Y. Anal. Chim. Acta 1999, 386, 21-30. (30) Eftekhari, A. Microchim. Acta 2003, 141, 15-21. (31) Humphrey, B. D.; Sinha, S. J.; Bocarsly, A. B. J. Phys. Chem. 1987, 91, 586-593. (32) Ogura, K.; Nakayama, M.; Kusumoto, C. J. Electrochem. Soc. 1996, 143, 3606-3615. (33) Shankaran, D. R.; Narayanan, S. S. Russ. J. Electrochem. 2002, 38, 987991. (34) Golabi, S. M.; Noor-Mohammadi, F. J. Solid State Electrochem. 1998, 2, 30-37.

The electrochemical behavior of cobalt hexacyanoferrate complex adsorbed on a carbon paste electrode (CPE) and its application to the electrocatalytic oxidation of guanine and single-strand DNA (ss-DNA) in aqueous solution are investigated in this report. The modification of CPE by the adsorption of this complex results in excellent amplification of the guanine oxidation response of ss-DNA. The effects of paste composition, scan rate, DNA, and guanine concentration were studied. The detection limits of 52 and 920 ng mL-1 were obtained for guanine and ss-DNA, respectively.

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10.1021/ac049421f CCC: $27.50

© 2004 American Chemical Society Published on Web 08/25/2004

The modification on the electrode surface with these compounds is possible in different ways including electrodeposition, adsorption, and entrapping them in polymers39 or encapsulating in sol-gels.28,40 The immobilization of hexacyanoferrates as powder particles on an electrode surface is also used.41,42 These procedures may be divided into three main groups: (1) by immersing a conductive substrate surface in a solution containing hexacyanoferrates and transition metal ions, allowing formation of insoluble metal hexacyanoferrates by a simple chemical reaction43 or electrochemical reaction, cycling the electrode over a range of potentials;44-46 (2) by immersing the transition metal electrode surface or some plated conductive substrates with a transition metal, in a solution containing cyanoferrate ions, and then derivatizing the electrode surface to obtain insoluble metal cyanoferrates by chemical46-48 or electrochemical methods;23,49,50 (3) by immobilizing mechanically the metal hexacyanoferrates as microparticles on the surface of an appropriate electrode, e.g., a paraffin-impregnated graphite electrode,51-53 a carbon paste electrode (CPE), or a conductive polymer-coated electrode.54 Among the hexacyanoferrates, cobalt hexacyanoferrate (CoHCF) would appear to be especially attractive because of its interesting chemical and electrochemical properties.55-57 Electrodes modified with CoHCF have been prepared where two different types of film were reported. One type has a redox couple58 and the other has two redox couples,59 depending on the preparation procedure and nature of the electrode substrate and (35) Wang, J.; Zhang, X. J.; Prakash, M. Anal. Chim. Acta 1999, 395, 11-16. (36) Zhou, J. X.; Wang, E. K. J. Electroanal. Chem. 1992, 331, 1029-1043. (37) Milardovic, S.; Kruhak, I.; Ivekovic, D.; Rumenjak, V.; Tkalc´ec, M.; Grabaric, B. S. Anal. Chim. Acta 1997, 350, 91-96. (38) Kulesza, P. J.; Malik, M. A.; Zamponi, S.; Berrettoni, M.; Marassi, R. J. Electroanal. Chem. 1995, 397, 287-292. (39) Itaya, K.; Akahoshi, H.; Toshima, S. J. Electrochem. Soc. 1982, 129, 14981500. (40) Cai, C.; Ju, H.; Chen, H. J. Electroanal. Chem. 1995, 397, 185-190. (41) Arnet, D. J.; Hidalgo-Luangdilok, C.; Chun, J. K. M.; Bocarsly, A. B. J. Electroanal. Chem. 1992, 328, 295-310. (42) Pournaghi-Azar, M. H.; Razmi-Nerbin, H. J. Electroanal. Chem. 1998, 456, 83-90. (43) Honda, K.; Ochiai, J.; Hayashi, H. J. Chem. Soc., Chem. Commun. 1986, 168-170. (44) Guo, Y.; Guadalupe, A. R.; Resto, O.; Fonseca, L. F.; Weisz, S. Z. Chem. Mater. 1999, 11, 135-140. (45) Miecznikowski, K.; Cox, J. A.; Lewera, A.; Kulesza, P. J. J. Solid State Electrochem. 2000, 4, 199-204. (46) Schwudke, D.; Sto ¨sser, R.; Scholz, F. Electrochem. Commun. 2000, 2, 301306. (47) Schro ¨der, U.; Scholz, F. Inorg. Chem. 2000, 39, 1006-1015. (48) Pournaghi-Azar, M. H.; Razmi-Nerbin, H. Electroanalysis 2000, 12, 209214. (49) Bocarsly, A. B.; Sinha, S. J. Electroanal. Chem. 1982, 140, 157-162. (50) Dostal, A.; Meyer, B.; Scholz, F.; Schroder, U.; Bond, A. M.; Marken, F.; Shaw, S. J. J. Phys. Chem. 1995, 99, 2096-2103. (51) Zahkarchuk, N. F.; Meyer, B.; Henning, H.; Scholz, F.; Jaworski, A.; Stojek, Z. J. Electroanal. Chem. 1995, 398, 23-35. (52) Reddy, S. J.; Dostal, A.; Scholz, F. J. Electroanal. Chem. 1996, 403, 209212. (53) Ikeda, O.; Yoneyama, H. J. Electroanal. Chem. 1989, 265, 323-327. (54) Zhang, H. Q.; Lin, X. Q. Talanta 1997, 44, 2069-2073. (55) Kulesza, P. J.; Zamponi, S.; Malik, M. A.; Berrettonib, M.; Wolkiewicza, A.; Marassib, R. Electrochim. Acta 1998, 43, 919-923. (56) Kulesza, P. J.; Malik, M. A.; Berrettoni, M.; Giorgetti, M.; Zamponi, S.; Schmidt, R.; Marassi, R. J. Phys. Chem. B 1998, 102, 1870-1876. (57) Gao, Z.; Zhou, X.; Wang, G.; Li, P.; Zhao, Z. Anal. Chim. Acta 1991, 244, 39-48. (58) Joseph, J.; Gomathi, G.; Rao, P. J. Electroanal. Chem. 1991, 304, 263-269. (59) Cai, C.; Xue, K.; Xu, S. J. Electroanal. Chem. 2000, 486, 111-118.

supporting electrolyte used. CoHCF exhibits not only electrocatalytic or mediator activities via slow electrochemical reactions37,57,58 but also ion selectivity, the ability to store cations,60-66 electrochromic,67-69 thermochromic characteristics,70 solid-state batteries,71,72 and molecular magnetism.73,74 To our knowledge, there is no report on the modification of a carbon paste electrode using CoHCF and this paper describes the electrochemical properties of this modified electrode and mechanistic studies on the electrocatalytic oxidation of guanine and single-strand DNA (ss-DNA) at the surface of CMCPE. EXPERIMENTAL SECTION Instrumentation and Software. Voltammetric experiments were performed using a Metrohm electroanalyzer (model 757 VA Computrace) connected to a 633-MHz Pentium II computer. The system was operated and measurements were recorded using VA computrace version 2 (Metrohm) run under windows XP. The three-electrode system consists of the bare or chemically modified carbon paste electrode as working electrode, Ag|AgCl|3M KCl as a reference electrode, and a platinum wire as an auxiliary electrode. The body of the working electrode was a PTFE cylinder (1.9-mm i.d.) that was tightly packed with carbon paste. A copper wire inserted into the carbon paste provided the electrical contact. REAGENTS AND SOLUTIONS Herring sperm not highly polymerized deoxyribonucleic acid was obtained from TCI. Paraffin oil, graphite powder, guanine, potassium chloride, sodium chloride, sodium hydroxide, hydrochloric acid, potassium hexacyanoferrate, copper(II) nitrate, and cobalt(II) nitrate selected AR grade or better were from Fluka or Merck and were used as purchased, without further purifications. Doubly distilled deionized water was used throughout the work. A 50 µg mL-1 solution of guanine was prepared daily by dissolving appropriate amounts of guanine in 100 mL of alkali media (NaOH 0.1 M). This solution was diluted to the appropriate concentration, and its pH was adjusted by addition of acetic acid. The DNA stock solution was prepared by dissolving appropriate amounts of herring sperm not highly polymerized DNA in 5 mL of autoclaved water. The solution is heated to 94 °C, at which temperature the hydrogen bonds that hold together the two strands of double(60) Chen, S. M. Electrochim. Acta 1998, 43, 3359-3369. (61) Engel, D.; Grabner, E. W. Ber. Bunsen-Ges. Phys. Chem. 1985, 89, 98209825. (62) Krishnan, V.; Xidis, A. L.; Neff, V. D. Anal. Chim Acta 1990, 239, 7-12. (63) Kaneko, M.; Okada, T. J. Electroanal. Chem. 1988, 255, 45-52. (64) Lasky, S. J.; Buttry, D. A. J. Am. Chem. Soc. 1988, 110, 6258-6260. (65) Malik, M. A.; Miecznikowski, K.; Kulesza, P. J. Electrochim. Acta 2000, 45, 3777-3784. (66) Monk, P. M. S.; Mortimer, R. J.; Rosseinsky, D. R. Electrochromism, fundamentals and applications; VCH: Weinheim, 1995; Chapter 6. (67) Kellawi, H.; Rosseinsky, D. R. J. Electroanal. Chem. 1982, 131, 373-376. (68) Duek, E. A. R.; De Paoli, M. A.; Mastragostino, M. Adv. Mater. 1993, 5, 650-655. (69) Kulesza, P. J.; Malik, M. A.; Miecznikowski, A.; Wolkiewicz, S.; Zamponi, M.; Berrettoni, M.; Marrasi, R. J. Electrochem. Soc. 1996, 143, L10-L11. (70) Honda, K.; Hayashi, H. J. Electrochem. Soc. 1987, 134, 1330-1333. (71) Jayalaksmi, M.; Scholz, F. J. Power Sources 2000, 91, 217-223. (72) Kahn, O. Nature 1995, 378, 667-668. (73) Sato, O.; Einaga, Y.; Iyoda, T.; Fujishima, A.; Hashimoto, K. J. Phys. Chem. B 1997, 101, 3903-3905. (74) Shankaran, D. R.; Narayanan, S. S. Bull. Chem. Soc. Jpn. 2002, 75, 501505.

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Figure 1. Cyclic voltammogram for electrodeposition process of CoHCF films obtained from a solution mixture containing 0.5 M KCl, 1 mM CoCl2, and 0.5 mM, K3Fe(CN)6 at 100 mV s-1.

stranded DNA molecule are broken, causing the molecule to denature. Then the solution suddenly cooled using liquid nitrogen. The obtained solution was diluted to an appropriate concentration daily by acetate buffer solution (pH 5.1). Preparation of CMCPE. Carbon paste electrodes were prepared by mixing the graphite powder and paraffin oil (65:35 w/w), housed in a PTFE cylinder, and then polished with a weighing paper. For preparation of CMCPE, CoHCF films were electrodeposited with cyclic voltammetry from 0.0 to +1.1 V at 50 mV s-1 from a fresh solution mixture containing 0.5 M KCl, 1 mM CoCl2, and 0.5 mM K3Fe(CN)6. After 15 cycles, CPE was taken out and rinsed thoroughly with water. The area of these electrodes was 0.028 ( 0.003 cm2, which was calculated from chronoculometric experiments. RESULTS AND DISCUSSION Electrochemical Behavior of Cobalt HexacyanoferrateModified Carbon Paste Electrode. Figure 1 illustrates the cyclic voltammogram during the electrodeposition process. The progressively increasing currents for both anodic and cathodic peaks demonstrate that CoHCF films are deposited continuously on the electrode surface. Typical cyclic voltammetric (CV) responses of CoHCF/CPE obtained in 0.2 M KCl and 0.2 M NaCl at the potential scan rate of 50 mV s-1 are shown in Figure 2. The figure demonstrates that the number and position of the redox peaks are dependent on the nature of the cation of the supporting electrolyte. This behavior clearly indicates that the electrolyte cation should be responsible for maintaining the electroneutrality of the electrode surface, thus allowing redox reactions. To maintain the electroneutrality of the modified film during the electrochemical process, ions usually enter into or escape from the immobilized film. So ions have an extensive effect on the electrochemical performance of the modified electrodes. 5692 Analytical Chemistry, Vol. 76, No. 19, October 1, 2004

Figure 2. Cyclic voltammograms of (a) CoHCF-modified CPE in 0.5 M KCl solution and (b) as with (a) in 0.5 M NaCl. Scan rate, 50 mV s-1.

In a NaCl medium, the cyclic voltammogram exhibits two sets of reversible redox peaks with formal potentials (E°′ ) [(Ep,a + Ep,c)/2]) of about 0.43 and 0.83 V, which have been attributed to the transformations between Fe(II) and Fe(III) in NaCoII1.5FeII(CN)6 and Na2CoIIFeII(CN)6, respectively. Therefore, it is likely that two redox couples, including CoII1.5FeIII(CN)6/NaCoII1.5FeII(CN)6 and NaCoIIFeIII(CN)6/Na2CoIIFeII(CN)6, can be expected.35 Figure 3 shows the cyclic voltammograms corresponding to the response of a carbon paste electrode modified with adsorbed cobalt hexacyanoferrate complex in a 0.2 M NaCl solution at various scan rates over a range of 10-200 mV/s. The anodic and cathodic peak currents are directly proportional to the scan rate in the range below 100 mV s-1, as shown in Figure 3B for the second well-defined redox couple. The ratio of anodic to cathodic peak currents obtained at low scan rates was almost unity. The peak-to-peak potential separation (∆Ep) is small ( ∼20 mV for scan rate 10 mV s-1). Also, the formal potential E°′ ) (Epa + Epc)/2 is approximately independent of the potential scan rate for sweep rates below 100 mV s-1, suggesting facile chargetransfer kinetics over this range of sweep rate and a transfer coefficient (R) of ∼0.5. However, for sweep rates above 100 mV s-1, the peak separations begin to increase, emphasizing a limitation arising from charge-transfer kinetics. At higher sweep rates, the plot of peak current versus scan rate deviates from linearity and the peak current becomes proportional to the square root of the sweep rate, indicating diffusional behavior in charge transport at these sweep rates. The surface coverage can be evaluated from the equation Γ ) Q/nFA, where Q is the charge obtained by integrating the anodic peak under the background correction (at a low scan rate of 10 mV s-1) and other symbols have their usual meanings. In the present case, by assuming the involvement of one electron in the process, the calculated value of Γ is 5.73 × 10-9 mol cm-2.

Figure 3. (A) Cyclic voltammograms of modified electrode in 0.2 M NaCl solution at various scan rates over the range of 10-200 mV s-1 (increasing 10 mV s-1 in each step). (B) Plot of anodic and cathodic peak currents of the second well-defined redox couple of CoHCF versus scan rate.

The stability of the CoHCF-deposited carbon paste electrodes toward various influences was also examined and was found satisfactory. There were no changes in the height and separation of the cyclic voltammograms after 100 cycles of repetitive scanning in KCl solution. The stability toward repeated potential cycling in KCl solution is better than that in NaCl solution, which is probably due to the higher affinity of the complex for the K+ ion.31 In 0.2 M NaCl solution, the cycling of potential in the range from 0 to 1 V versus Ag | AgCl at a scan rate of 100 mV s-1 shows only just ∼10% decrease in peak current after 100 cycles of potential. Electrocatalytic Oxidation of Guanine and ss-DNA at the Surface of CoHCF-Modified CPE. The electrochemical reduction and oxidation of natural nucleic acids are irreversible and occur at highly negative and positive potentials, respectively. Guanine and adenine are the only nucleic acid bases that can be oxidized at carbon paste electrodes.75 These nucleic acid bases show well-defined oxidation peaks at +0.9 and +1.2 V versus Ag | AgCl, respectively.76 The oxidation peak of guanine is close to the second well-defined oxidation peak potential of CoHCF that appears in the NaCl medium; therefore, we expected an electrocatalytic mechanism initiated by electrochemical oxidation of the reduced form of the complex exist at the surface of the electrode and then completed by chemical oxidation of guanine, which also serves to regenerate the reduced form of the CoHCF. To reveal the electrocatalytic activity of CoHCF toward the oxidation of guanine, the voltammetric behavior of guanine was investigated at the surface of bare and CoHCF-modified carbon paste electrode. Figure 4 shows the cyclic voltammograms of CoHCF-modified CPE in a 0.2 M NaCl solution (a) and in the presence of 0.5 µg (75) Kafil, J.; Cheng, H. Y.; Last, T. Anal. Chem. 1986, 58, 285-289. (76) Wang, J.; Fernandes, J. R.; Kubota, L. T. Anal. Chem. 1998, 70, 36993702.

Figure 4. Cyclic voltammograms of modified electrode (a) in the absence and (b) in the presence of 0.5 µg mL-1 guanine in 0.2 M NaCl solution. (c) Cyclic voltammogram of 0.5 µg mL-1 guanine at the surface of the bare electrode in 0.2 M NaCl solution.

mL-1 guanine (b) (the signal of 0.5 µg mL-1 guanine at the surface of the bare electrode was subtracted) and also the cyclic voltammograms of 0.5 mg mL-1 guanine at the surface of the bare electrode (c). Similar results were obtained via the oxidation of ss-DNA that is shown in Figure 5. In this figure again, cyclic voltammogram a indicates the signal of the CoHCF-modified carbon paste electrode in 0.2 M NaCl solution, CV b shows the cyclic voltammogram related to 25 µg mL-1 ss-DNA (the signal of 25 µg mL-1 ss-DNA at the surface of the bare electrode was subtracted) and (c) illustrates the signal of 25 µg mL-1 ss-DNA at the surface of the bare electrode. The anodic peak current of Analytical Chemistry, Vol. 76, No. 19, October 1, 2004

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Figure 5. Cyclic voltammograms of modified electrode (a) in the absence and (b) in the presence of 25 µg mL-1 ss-DNA in 0.2 M NaCl solution. (c) Cyclic voltammogram of 25 µg mL-1 ss-DNA at the surface of the bare electrode in 0.2 M NaCl solution.

the CoHCF complex increased due to the presence of guanine or ss-DNA, whereas cathodic peak current of this complex decreased accordingly. Therefore, an enhancement of peak current is achieved in this system, which clearly demonstrates the occurrence of an electrocatalytic process. Since the current measure-

ment with modified electrode may be perturbed by the current from the possibly direct oxidation of guanine and ss-DNA, in Figures 4b and 5b, we subtract the currents of the oxidation of guanine and ss-DNA at the surface of the bare electrode from the currents at the surface of CoHCF-modified CPE. As Figure 4c and 5c show, the current related to direct oxidation of guanine and ss-DNA is not significant within the potential of the electrocatalytic oxidation. Hydrodynamic Voltammetric Studies at Rotating Disk Electrode. The hydrodynamic voltammograms of 2 µg mL-1 guanine on the surface of the CoHCF-modified carbon paste electrode were recorded at different rotation rates over range of 200-2600 rpm (Figure 6A). Using these hydrodynamic voltammograms, the limiting currents at different rotating rates versus the square root of the rotating rate were plotted as shown in Figure 6B (Levich plot). It could be expected from the Levich equation that this plot is linear, but as seen in Figure 6B, this is not linear and tends to level off at higher rotation rates. This nonlinearity suggests a kinetic limitation in the coupled chemical reaction. Hence, it can be concluded that the rate-determining step in this process is related to the oxidation of guanine at the modified carbon paste electrode. However, under these conditions, there is a linear correlation between the inverse of the limiting current and the inverse of the square root of the rotating speed (Koutecky-Levich equation) as illustrated in Figure 6C, and this equation can be formulated as following:

1 1 1 ) + Ilim Ik 0.62nFAν-1/6D2/3Cω1/2

where Ik is the plateau current limited by the kinetic step, C is the bulk concentration of guanine in solution, ν is the kinematic

Figure 6. (A) Hydrodynamic voltammograms of 2 µg mL-1 guanine at the surface of CoHCF-modified carbon paste electrode in 0.2 M NaCl solution at various rotating speeds over a range of 0-2600 rpm (increasing 200 rpm in each step and for each voltammogram, the potential scan rate was 20 mV s-1). (B) Levich plot (Ilim (µA) ) 12.99 ω1/2 (rps1/2) + 3.62, r2 ) 0.9992) and (C) Koutecky-Levich plot of the hydrodynamic voltammograms (Ilim-1 (µA-1) ) 0.062ω-1/2 (rps-1/2) + 0.002, r2 ) 0.9986). 5694

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Figure 7. (A) Cyclic voltammograms of various concentrations of guanine over range of 0-7.5 µg mL-1 guanine at the surface of a CoHCFmodified carbon paste electrode in 0.2 M NaCl solution. (B) Calibration curve obtained from these voltammograms. (Ip,a (µA) ) 22.50Cguanine (µg mL-1) +5.64, r2 ) 0.9989).

Figure 8. (A) Cyclic voltammograms of various concentrations of guanine over range of 0-60 µg mL-1 ss-DNA at the surface of a CoHCFmodified carbon paste electrode in 0.2 M NaCl. (B) Calibration curve obtained from these voltammograms. (Ip,a (µA) ) 0.256CDNA (µg mL-1) +2.61, r2 ) 0.9960).

viscosity, ω is the angular frequency and other symbols have their usual meanings. The diffusion coefficient of guanine at these conditions was calculated as 4.18 × 10-6 cm2 s-1, from the slope of Figure 3C (by considering two-electron mechanisms), which is comparable to those reported previously (The diffusion coefficients of purines and pyrimidines at all pH values varied between 2 × 10-6 and 4 × 10-6 cm2 s-1),77 and this would be evidence that confirms the two-electron mechanisms that follow: on CPE

2Na2CoIIFeII(CN)6 98 2NaCoIIFeIII(CN)6 + 2e (guanine)R + 2NaCoIIFeIII(CN)6 f (guanine)Ox + 2Na2CoIIFeII(CN)6

Calibration and Detection Limit Studies. Figure 7 displays the calibration curves for the different concentrations of guanine

ranging from 0 to 7.5 µg mL-1 at CoHCF-modified CPE that were obtained from the cyclic voltammograms. As can be seen in Figure 7B, the linear dynamic range for guanine is 0-4 µg mL-1. The voltammetric experiments were carried out 10 times on the blank solution, and the current was measured at the E ) 936 mV versus Ag|AgCl (peak potential of guanine oxidation) and the standard deviation of these measured currents calculated (sb). By using 3sb in the calibration equation, we calculated the detection limit concentration. The detection limit of 52 ng mL-1 (based on 3sb), and the sensitivity of 22.50 µA mL/µg was estimated. Also, the calibration curve for ss-DNA ranging from 0 to 60 µg mL-1 at the modified electrode is shown in Figure 8. Figure 8B shows that the linear dynamic range of this system for DNA is 0-25 µg mL-1. To determine the detection limit for DNA, the above procedure was performed but at the peak potential of DNA, and the detection (77) Faraggi, M.; Broitman, F.; Trent, J. B.; Klapper, M. H. J. Phys. Chem. 1996, 100, 14751-14761.

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limit of 920 ng mL-1 (based on 3sb) and the sensitivity of 0.256 µA mL/µg were estimated for ss-DNA. CONCLUSION In this report, the electrocatalytic activity of CoHCF-modified carbon paste electrode toward the oxidation of guanine and DNA in NaCl medium was demonstrated. As stated in the introduction, thin-film inorganic polymers have been widely used in electrochemistry to electrocatalyze many compounds, but these mediators have not been used for electrocatalytic oxidation of DNA. In this study, we show that the second well-defined oxidation peak potential of CoHCF that appears in NaCl medium can electrocatalyze and improve dramatically the oxidation signal of guanine and DNA. It should be mentioned that this proposed method is not only comparable with the previous report on detection of DNA using CPE modified by tris(2,2′-bipyridyl)dichlororuthenium(II)18 but also has the advantage that the cobalt hexacyanoferrate prompted cyclic voltammogram signal of the oxidation of guanine and DNA compared to a bare electrode more than tris(2,2′-bipyridyl)dichlororuthenium(II). (In the ruthenium complex-modified CPE, the signal was enhanced ∼5 times compared to bare CPE whereas (78) Schachl, K.; Alemu, H. M.; Kalcher, Moderegger, K. H.; Svancara, I.; Vytras, K. Fresenius J. Anal. Chem. 1998, 362, 194-200. (79) Stamford, J. A.; Justice, J. B. Anal. Chem. 1996, 68, 359A-366A. (80) Drummond, T. G.; Hill, M. G.; Barton, J. K. Nat. Biotechnol. 2003, 10, 11921199.

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CoHCF/CPE prompted the signal roughly 10 times more than bare CPE). Also, CoHCF is formed much less expensively than tris(2,2′-bipyridyl)dichlororuthenium(II). Comparing this sensor with those reported previously by the Thorp research group, who used the ITO electrode,10-17 it can be pointed out that a big advantage of pastelike electrode materials is the ease of modification in order to obtain higher selectivity, to lower the overpotential, or to achieve lower limits of detection and a wider range of linear response toward the analyte.78 In addition, CPE can be miniaturized easily79 whereas ITO material may or may not be amenable to microfabrication in a multiplexed configuration.80 Also in comparing CoHCF to Ru(bpy)3 as a probe for detecting of DNA,10-15 this sensor has the advantage of being a heterogeneous catalyst, while in Ru(bpy)3 probes, the catalyst is homogeneous (in solution) and the application of a homogeneous catalyst has a limitation in its practical utility for analytical voltammetry. ACKNOWLEDGMENT We gratefully acknowledge the support of this study by the Shiraz University Research Council.

Received for review April 17, 2004. Accepted July 20, 2004. AC049421F