Journal of Electroanalytical Chemistry 648 (2010) 6066

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Journal of Electroanalytical Chemistry

journal homepage: www.elsevier.com/locate/jelechem

Boron doped diamond electrode pre-treatments effect on the electrochemical oxidation of dsDNA, DNA bases, nucleotides, homopolynucleotides and biomarker 8-oxoguanine
Severino Carlos B. Oliveira, Ana Maria Oliveira-Brett *
Departamento de Qumica, Faculdade de Cincias e Tecnologia, Universidade de Coimbra, 3004-535 Coimbra, Portugal

a r t i c l e

i n f o

a b s t r a c t
The effect of boron doped diamond (BDD) surface termination immediately after cathodic and anodic electrochemical pre-treatment, and the inuence of the pre-treatment in different supporting electrolytes, on the electrochemical oxidation potentials of dsDNA, DNA bases, nucleotides, homopolynucleotides and biomarker 8-oxoguanine was investigated, using differential pulse voltammetry in aqueous media at different pH. The anodic and cathodic BDD surface pre-treatments were performed in three different solutions, pH 4.5 0.1 M acetate buffer, pH 7.0 0.1 M phosphate buffer and pH 0.55 0.5 M sulphuric acid and the electrochemical response at the BDD surface varied as a function of the potential applied in the surface pre-treatment and also on the electrolyte used. All results demonstrated that the electrochemical properties of the BDD electrode are very dependent on the state of the surface termination, due to oxygen and hydrogen terminations. Concerning the compounds studied a better response was obtained when the BDD surface was pre-treated cathodically. The interaction and adsorption of the electrochemical species with the BDD surface pre-treated cathodically was facilitated due to a higher conductivity of the BDD electrode surface. On the other hand, after anodic pre-treatment a wider potential window of BDD surface was obtained enabling the detection of the pyrimidine bases. However, the hydroxyl radicals produced on BDD surface during the anodic pre-treatment are highly reactive, and consequently the BDD surface is not completely inert. 2010 Elsevier B.V. All rights reserved.

Article history: Received 23 March 2010 Received in revised form 17 June 2010 Accepted 25 June 2010 Available online 1 July 2010 Keywords: dsDNA DNA bases 8-oxoguanine Oxidation Boron doped diamond Reduced and oxidized BDD surface

1. Introduction The in vivo oxidation of DNA leads to multiple modications, including DNA strand breaks, sugar modications, base-free sites and oxidized bases causing cell damage, and is the central role in mutagenesis, carcinogenesis and various diseases involving inammatory processes [15], and exist great interest in sensitive determination and full characterization of the mechanisms involved in DNA oxidative damage. DNA oxidative damage in vivo occurs under various prooxidative conditions, including photosensitization reactions, Fentontype reactions and gamma irradiation, and several mutagenic products are generated within the double helix, such as 8-oxoguanine (8-oxoGua), 2,8-oxoadenine, 5-formyluracil and 5-hydroxicytosine. Fortunately, the importance of these lesions are underscored by the existence of mechanisms for their cellular repair, principally glycosylase activity that removes 8-oxoGua in both bacteria and yeast, and also in human cells [46].

* Corresponding author. Tel.: +351 239 835295. E-mail address: brett@ci.uc.pt (A.M. Oliveira-Brett). 1572-6657/$ - see front matter 2010 Elsevier B.V. All rights reserved. doi:10.1016/j.jelechem.2010.06.020

Among DNA bases, Fig. 1, guanine is known to be most readily oxidized and the product of oxidation in the C8 position, 8-oxoGua, Fig. 1, causes one of the major DNA damages induced by oxidation. The 8-oxoGua mutagenicity causes loss of DNA base pairing specicity [79], producing G to T transversions, and has been the subject of intensive research becoming widely accepted as a biomarker of in vivo DNA oxidative damage and cellular oxidative stress [10,11]. Oxidative stress in vivo is an imbalance between prooxidant and antioxidant reactions which causes disruption of the redox mechanisms. Elevated concentrations of 8-oxoGua were found in the urine and lung tissues of smokers [12] as well as in body uids and DNA from human tissues of patients with disorders such as cancer, atherosclerosis, chronic hepatitis, cystic brosis, diabetes, acquired immunodeciency syndrome, neurodegenerative, and age-related diseases [13]. As a result, various methods based on chemical and biochemical techniques have been developed and improved to detect and quantify 8-oxoGua in biological systems. These methods include high performance liquid chromatography associated with electrochemical detection (HPLCEC) [2,5,1416] and gas chromatography coupled to mass spectrometry (GCMS) [17,18]. Indirect measurements based on the use of antibodies, puried repair enzymes,

Severino Carlos B. Oliveira, A.M. Oliveira-Brett / Journal of Electroanalytical Chemistry 648 (2010) 6066

61

O N NH N NH2 N

NH2 N N

NH2 N

N H

N H

N H

Guanine (G)
O NH

Adenine (A)

Cytosine (C)
O

H N O O N H N

NH NH2

was observed that for some analytes cathodic pre-treatment on the BDD surface was more appropriate because a very low detection limit and high reproducibility of the data was obtained [37]. The great diversity of results is an evidence that clarication of the effect of anodic or cathodic electrochemical pre-treatment of the BDD surface is pertinent [39,41]. In this paper, in order to establish an appropriate methodology envisaging future applications for BDD electroanalytical determinations in biological uids, an electrochemical study of the mechanism of oxidation of dsDNA, DNA bases, nucleotides, homopolynucleotides and biomarker 8-oxoguanine, using differential pulse voltammetry, at a cathodically and anodically pre-treated BDD electrode surface, is presented.

N H

2. Experimental 2.1. Materials and reagents Guanine (G), adenine (A), thymine (T), cytosine (C), guanosine 5-monophosphate (GMP), adenosine 5-monophosphate (AMP), polyguanylic (poly[G]), polyadenylic (poly[A]), 8-oxoGua and calf thymus dsDNA, were obtained from SigmaAldrich and used without further purication. Analytical-grade reagents and puried water from a Millipore Milli-Q system (conductivity <0.1 lS cm1) were used for the preparation of pH 4.5 0.1 M acetate buffer, pH 7.0 0.1 M phosphate buffer and pH 0.55 0.5 M sulphuric acid electrolyte solutions. Stock solutions of 100 lM of all bases and 8-oxoGua, 300 lg mL1 of poly[G] and poly[G] and 800 lg mL1 of dsDNA, were prepared in deionised water and diluted to the desired concentration in buffer supporting electrolyte. To guarantee complete dissolution of guanine and 8-oxoGua, 5 lL of 6.5 M NaOH was added to each stock solution. Nano- and microvolumes were measured using an EP-10 Plus and an EP-100 Plus motorized microliter pipette (Rainin Instrument, Woburn, MA, USA). The pH was measured with a Crison Model micropH 2001 pH meter with an Ingold combined-glass electrode. All experiments were done at room temperature (25 1 C).

Thymine (T)

8-OxoGua

Fig. 1. Chemical structures of: guanine (G), adenine (A), cytosine (C), thymine (T) and 8-oxoguanine (8-oxoGua).

single-cell gel electrophoresis have been developed and improved to detect 8-oxoGua, but the specicity of these methods is still the subject of research [6,19]. Voltammetric techniques using glassy carbon electrodes have also been proposed to detect 8-oxoGua [4,20]. However, the values found by the different methods for the same sample source give results very different over several orders of magnitude. In general, the levels found for 8-oxoGua with HPLCEC are lower than then those found by GCMS, and higher than those determined by other methods [2,5,21,22]. The electrochemical behavior of DNA and its components at different types of electrochemical transducers has been investigated for a number of years, rst using mercury electrodes [23] and more recently solid electrodes, such as pyrolytic graphite electrodes [24,25] and glassy carbon electrodes [3,2628]. The electroreduction [23,29] and electrooxidation [3] of DNA occurs in the purine and pyrimidine bases, the sugar and phosphate groups being non-electroactive at carbon electrodes in aqueous solutions. The electrochemical oxidation behavior of DNA free purine, guanine and adenine, and pyrimidine, cytosine and thymine, bases was studied at carbon electrodes in aqueous solutions [3,24,25,27,28], and they all present a pH-dependent electron transfer mechanism. The oxidation of purine bases occurs at much lower positive potentials then that of the pyrimidine bases [3], which occurs at very high positive potentials, close to the potential of oxygen evolution, and consequently their oxidation peaks are more difcult to detect. The oxidation of DNA and DNA bases was studied at a BDD electrode with no pre-treatment or after anodic pre-treatment of the BDD surface [3033]. The properties of BDD thin-lm electrodes include a wide electrochemical potential window, a very low voltammetric background current, a high resistance to deactivation by fouling, a very high electrochemical stability and a relative insensitivity to dissolved oxygen. Consequently, BDD electrodes can be used at very high potential values, both negative and positive, without causing electrolyte decomposition [3436]. The as-prepared surface of commercial BDD electrodes is hydrophobic and different pre-treatments for preparing and activating the BDD surface for electrochemical measurements have been evaluated [3743]. A strong anodic pre-treatment in acid medium has been described in the literature and adopted as standard procedure [37]. On the other hand, the effect of a cathodic pre-treatment on the BDD electrochemical response has also been investigated and it

2.2. Voltammetric parameters and electrochemical cells Voltammetric experiments were performed using a lAutolab running with GPES 4.9 software, Metrohm, Autolab, Utrecht, The Netherlands. Differential pulse (DP) voltammetry conditions were pulse amplitude 50 mV, pulse width 70 ms and scan rate m = 5 mV s1. Measurements were carried out using a BDD working electrode, a Pt wire counter electrode, and an Ag/AgCl (3 M KCl) as reference, in a 2 mL one-compartment electrochemical cell. The BDD lms were prepared at the Centre Suisse de Electronique et de Microtechnique SA (CSEM), Neuchatel, Switzerland, on silicon wafers using the hot lament chemical vapour deposition (HF-CVD) technique with a lament temperature in the range 24402560 C and a gaseous mixture containing methane, H2 and trimethylboron. This HF-CVD process gives a columnar, randomly textured polycrystalline BDD lm with the surface dominated by facets. The nal boron content was of the order of 8000 ppm, 5.7 6.1 mm2 surface area, $1 lm thickness. Two different procedures for electrochemical pre-treatment of the BDD surface were carried out in different supporting electrolytes, pH 4.5 0.1 M acetate buffer, pH 7.0 0.1 M phosphate buffer and pH 0.55 0.5 M sulphuric acid, before every electrochemical assay:

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Severino Carlos B. Oliveira, A.M. Oliveira-Brett / Journal of Electroanalytical Chemistry 648 (2010) 6066

(1) Anodic pre-treatment: a positive potential was applied for 30 min, Eap = +3.0 V. The BDD surface was oxidized, OxBDD; together with long and extensive oxygen evolution. (2) Cathodic pre-treatment: a negative potential was applied for 30 min, Eap = 3.0 V. The BDD surface was reduced, RedBDD; together with long and extensive hydrogen evolution.

3. Results and discussion 3.1. Characterization of the BDD surface after anodic and cathodic pretreatments The effect of the anodic and cathodic pre-treatments on the electrochemical response of a BDD electrode and the inuence of the pre-treatment in different supporting electrolytes on the nal surface termination were investigated. The BDD anodic and cathodic pre-treatments were performed in three different solutions, pH 4.5 0.1 M acetate buffer, pH 7.0 0.1 M phosphate buffer and pH 0.55 0.5 M sulphuric acid. The electrochemical response of the BDD surface varied as a function of surface pre-treatment and also with the electrolyte used in the pre-treatment, no difference was found between acetate and phosphate buffers. The BDD surface was pre-treated in acetate buffer and sulphuric acid, as described in Section 2.2, and the Ox-BDD and Red-BDD surfaces were characterized by DP voltammetry at high positive potentials, Fig. 2. The DP voltammogram at an Ox-BDD surface pre-treated in acetate buffer exhibited the same surface activity when compared with the DP voltammogram at Ox-BDD pre-treated in sulphuric acid but a very small oxidation peak 1a, at E1 $ 1:3 V, appeared. pa Oxygen evolution occurred at both Ox-BDD surfaces at E $ +1.6 V.

The DP voltammogram at an Red-BDD surface pre-treated in sulphuric acid exhibited the highest surface activity and showed a big oxidation peak 1a, at E1 $ 0:7 V, and when compared with pa Red-BDD pre-treated in acetate buffer the peak 1a current decreased and was shifted to more a positive potential at E1 $ 1:2 V. Oxygen evolution occurred at both Red-BDD surfaces pa at E $ +1.4 V. The oxidation potential of peak 1a is due to the sp2 carbon incorporated into the diamond structure which provides the surface sites needed for the adsorption of reaction intermediates, including the reaction mechanisms of hydrogen and oxygen evolution and/or aromatics that can be responsible for a higher surface activity [44]. At the Ox-BDD surface, a high overpotential was observed for oxygen evolution with a low background current, compared with the current at Red-BDD surface, suggesting a more difcult electron transfer reaction at the Ox-BDD surface, because anodic pre-treatment led to the oxidation of the aromatic functional groups on the surface eliminating the sp2 carbon from the surface [45,46]. The cathodic pre-treatment causes an increase in the thickness of the adsorption layer formed by hydrogen termination on the Red-BDD surface, described by reactions (a), (b) and (c). In comparison to no electrochemical treatment, cathodic pretreatment only intensies but does not change the original BDD physical and chemical characteristics [46]. The rate of reaction (b) becomes limited by the rate of reaction (a), the equilibrium constant and the reaction rate are inuenced by proton concentration. When sulphuric acid is used for cathodic pretreatment, the rate of both reactions (a) and (b) increased, favouring H2 production. However, when acetate buffer was used in the cathodic pre-treatment the rate of both reactions (a) and (b) decreased and, due to the lower H2 production, a higher amount of boronhydrogen complex was obtained.

BDD H e  BDDH BDDH H e ! BDD H2

a b c

A
1a 1a
0.2 0.4 0.6 0.8 1.0 1.2 1.4 1.6

2BDDH ! 2BDD H2

1 A
1.8

Anodic pre-treatment causes the BDD hydrogen terminations to be converted to oxygen terminations (d), due to an internal transformation and/or most probably the presence of a adsorbed layer formed during the oxidation of water molecules, forming very reactive hydroxyl radicals on the Ox-BDD surface, thus changing its original physical and chemical characteristics [39,41,4649].

BDD H2 O ! BDD OH H e

E / V vs. Ag/AgCl

4 A
0.2 0.4 0.6 0.8 1.0 1.2 1.4 1.6 1.8

E / V vs. Ag/AgCl
Fig. 2. DP voltammograms in 4.5 0.1 M acetate buffer () at Ox-BDD surface and () at Red-BDD surface. BDD surface pre-treated: (A) in pH 4.5 0.1 M acetate buffer and (B) in pH 0.55 0.5 M sulphuric acid.

The kinetics of the H2O/OH redox couples (d) at Ox-BDD is pHdependent. Oxidation of the BDD surface by anodic pre-treatment was greater in sulphuric acid electrolyte solution and a thicker adsorption layer was formed due to the higher OH concentration produced and adsorbed on the BDD surface, decreasing the OxBDD surface conductivity [46]. An important property of hydroxyl radicals adsorbed on the OxBDD surface during anodic pre-treatment is their very high reactivity and hence being able to be involved in parallel reactions on the Ox-BDD surface, such as the oxidation of supporting electrolyte, hydrogen peroxide formation, and BDD corrosion. Thus the OxBDD surface is not completely inert [50,51]. The behavior after anodic pre-treatment is due to the presence of a discontinuous adsorption layer on the electrode surface, as discussed [46]. It was found that the surface resistance associated with this layer was over 300 X cm2 after anodic pre-treatment and decreased to about 4 X cm2 when cathodic pre-treatment was carried out [37,38]. It was shown by Raman spectra that no

Severino Carlos B. Oliveira, A.M. Oliveira-Brett / Journal of Electroanalytical Chemistry 648 (2010) 6066

63

AMP A C

T GMP 0.5 A

0.8

1.0

1.2

1.4

1.6

pA

E / V vs. Ag/AgCl pG

A
0.8 1.0 1.2 1.4

1 A
1.6

E / V vs. Ag/AgCl

B
G GMP
1 A

AMP

pG
0.6 0.8 1.0 1.2 1.4

E / V vs. Ag/AgCl
Fig. 3. DP voltammograms in pH 4.5 0.1 M acetate buffer of 5 lM guanine (G), 10 lM adenine (A), 20 lM of thymine (T) and 20 lM of cytosine (C) at: (A) Ox-BDD surface and (B) Red-BDD surface, both pre-treated in pH 4.5 0.1 M acetate buffer.

pA

important bulk structural differences are introduced in the BDD by cathodic pre-treatment, indicating that H-terminated sites play an important role in the electrochemical response of the Red-BDD electrodes [38]. 3.2. The effect of Ox-BDD and Red-BDD surface on the electrochemical oxidation of DNA bases, nucleotides, homopolynucleotides, 8-oxoguanine, and dsDNA The effect of Ox-BDD and Red-BDD surfaces and the inuence of the pre-treatment in different supporting electrolytes on the electrochemical oxidation of all DNA bases, nucleotides, GMP and AMP, and homopolynucleotides, poly[G] and poly[A], 8-oxoguanine and dsDNA, was studied using DP voltammetry in pH 4.5 and 7.0. 3.2.1. Oxidation of all DNA bases The DP voltammograms for 5 lM guanine, 10 lM adenine and 20 lM of cytosine and thymine in pH 4.5 at Ox- and Red-BDD surface, pre-treated in pH 4.5 0.1 M acetate buffer as described in Section 2.2, are shown in Fig. 3A and B. The electrochemical oxidation of the DNA bases at the Ox-BDD surface shows four well-dened oxidation peaks corresponding to guanine, at Epa = +0.9 V, adenine, at Epa = +1.24 V, thymine, at

2.0 A

0.8

1.0

1.2

1.4

1.6

E / V vs. Ag/AgCl
Fig. 4. DP voltammograms in pH 4.5 0.1 M acetate buffer of 25 lM guanosine 5-monophosphate (GMP), 25 lM adenosine 5-monophosphate (AMP), 150 lg mL1 polyguanylic (pG) and 150 lg mL1 polyadenylic (pA) at: (A) Ox-BDD surface and (B) Red-BDD surface, both pre-treated in pH 4.5 0.1 M acetate buffer.

Epa = +1.45 V, and cytosine, at Epa = +1.5 V, Fig. 3A. The easiest oxidizable DNA base is guanine. The Ox-BDD electrodes wide potential window in aqueous solution enables the detection of the thymine and cytosine peaks, which occur at high positive potentials in pH 4.5. The electrochemical oxidation of the DNA bases at the RedBDD surface shows only two well-dened oxidation peaks corresponding to guanine, at Epa = +0.88 V, and adenine, at Epa = +1.19 V, Fig. 3B. At the Red-BDD surface no oxidation peaks were observed for thymine and cytosine oxidation because of the smaller potential window in aqueous solution. The peaks occur at higher positive potentials in pH 4.5 solutions, coinciding with

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Severino Carlos B. Oliveira, A.M. Oliveira-Brett / Journal of Electroanalytical Chemistry 648 (2010) 6066

the potentials for oxygen evolution, the same as found at glassy carbon electrodes [3]. 3.2.2. Oxidation of DNA nucleotides In the DNA structure each guanine and adenine base is linked to a pentose-phosphate unit in the helix skeleton, the nucleotides guanosine 5-monophosphate (GMP), adenosine 5-monophosphate (AMP), and their electrochemical oxidation at the BDD electrode was studied and compared with the results obtained for the corresponding free DNA bases. The results at Ox-BDD and Red-BDD electrodes for 25 lM of GMP and AMP in pH 4.5 showed at the Ox-BDD surface the oxidation peak of GMP, at Epa = +1.1 V, an AMP, at Epa = +1.35 V, Fig. 4A, whereas at the Red-BDD surface the oxidation peaks occur for GMP, at Epa = +1.08 V, and AMP, at Epa = +1.33 V, Fig. 4B. The oxidation potentials of GMP, AMP are always shifted by $200 mV to more positive potentials in relation to those for free guanine and adenine bases in agreement with previous results [3]. The shift in the oxidation peak potentials of nucleotides relative to the corresponding base has been shown to be due to the inductive effect caused by the glycosidic bond on the p-system of the purine ring, making it more difcult to remove electrons from the bases [3]. The results at Ox-BDD and Red-BDD surfaces are summarised in Table 1. 3.2.3. Oxidation of DNA homopolynucleotides poly[G] and poly[A] The oxidation potentials for the homopolynucleotides, poly[G] and poly[A], at Ox-BDD and Red-BDD surfaces, in pH 4.5, showed at Ox-BDD the oxidation peak of poly[G], at Epa = +1.1 V, and of poly[A], at Epa = +1.3 V, Fig. 4A, whereas at Red-BDD the oxidation peaks occur at a lower potential for poly[G], at Epa = +1.0 V, and for poly[A], at Epa = +1.24 V, Fig. 4B. Relative to the free guanine and adenine bases, at Ox-BDD and Red-BDD surface, the oxidation peaks of poly[G] and poly[A] were always shifted to more positive potentials, Table 1, and lower peak currents and larger peak widths were obtained with the homopolynucleotides in comparison with the corresponding nucleotides, due to the difculty of electron transfer from the inside of quadruple and/or double helix structures of poly[G] and the double helix structure of poly[A] formed in acid media, at pH 4.5 on the electrode surface [52]. The above experiments were also performed in pH 7.0 solution at Ox-BDD and Red-BDD pre-treated in acetate buffer (not shown) and all potentials were shifted to lower values, Table 1. The oxidation peaks at Ox-BDD for GMP, AMP, poly[G] and poly[A], were all detected whereas at Red-BDD the oxidation peaks for AMP, poly[G] and poly[A] were not observed.

These experiments concerning the electrochemical oxidation of DNA nucleosides and nucleotides were also done in pH 4.5 0.1 M acetate buffer at Ox-BDD and Red-BDD pre-treated in pH 0.55 0.5 M sulphuric acid, Fig. 5, but no oxidation peak was obtained for thymine on the Ox-BDD surface, and at Red-BDD no oxidation peaks were obtained. The results at Ox-BDD pre-treated in sulphuric acid, Fig. 5, compared with the results at Ox-BDD pre-treated in acetate buffer, Figs. 3A and 4A, showed a decrease of the height of the oxidation peak currents. The choice of electrolyte for the electrochemical pre-treatment is very important, because it inuences the nal surface termination, the BDD conductivity, the electrochemical response and the reproducibility of experimental results. It can be concluded that acetate buffer is a better supporting electrolyte than sulphuric acid for performing either electrochemical treatment. 3.2.4. Oxidation of 8-oxoguanine The effect of the Ox- and Red-BDD surface on the electrochemical oxidation of 8-oxoGua was studied using DP voltammetry at pH 4.5 and 7.0. A saturated solution of concentration 1 mM, well above the solubility limit for 8-oxoGua, was prepared directly in the supporting electrolyte and the ltered supernatant was used for the voltammetric experiments.

A
C A

1 A

0.4

0.8

1.2

1.6

2.0

E / V vs. Ag/AgCl

AMP

Table 1 Oxidation potentials of guanine (G), adenine (A), thymine (T), cytosine (C), guanosine 5-monophosphate (GMP), adenosine 5-monophosphate (AMP), polyguanylic (pG), polyadenylic (pA) and 8-oxoguanine (8-oxoGua) in pH 4.5 and 7.0 electrolyte solutions at Ox-BDD and Red-BDD electrodes. Compounds Ox-BDD surface/E/V (vs. Ag/AgCl) pH 4.5 Guanine (G) Adenine (A) Thymine (T) Cytosine (C) Guanosine (GMP) Adenosine (AMP) Polyguanylic (pG) Polyadenylic (pA) 8-oxoguanine (8-oxoGua) 0.94 1.24 1.46 1.50 1.10 1.35 1.10 1.30 0.60 pH 7.0 0.91 1.20 1.45 1.46 0.99 1.30 1.01 0.62 Red-BDD surface/E/V (vs. Ag/AgCl) pH 4.5 0.89 1.20 1.08 1.33 1.03 1.24 0.49 pH 7.0 0.72 1.04 0.96 0.36

GMP

pA

pG

2 A

0.8

1.0

1.2

1.4

1.6

1.8

E / V vs. Ag/AgCl
Fig. 5. DP voltammograms in pH 4.5 0.1 M acetate buffer at Ox-BDD after surface pre-treatment in pH 0.55 0.5 M sulphuric acid: (A) of 10 lM guanine (G), 25 lM adenine (A) and 25 lM of cytosine (C) and (B) of 25 lM guanosine 5-monophosphate (GMP), 25 lM adenosine 5-monophosphate (AMP), 150 lg mL1 polyguanylic acid (pG) and 150 lg mL1 polyadenylic acid (pA).

Severino Carlos B. Oliveira, A.M. Oliveira-Brett / Journal of Electroanalytical Chemistry 648 (2010) 6066

65

The DP voltammograms for 8-oxoGua at Ox- and Red-BDD surface pre-treated in pH 4.5 0.1 M acetate buffer as described in Section 2.2, present, as expected, only one oxidation peak. Signicant differences in the electrochemical behavior, peak potential and current, of 8-oxoGua after Ox-BDD and Red-BDD surface pre-treatments were observed, Fig. 6. The peak currents also vary with sup-

+ 0.60 V

+ 0.62 V

5 nA

0.4

0.5

0.6

0.7

0.8

E / V vs. Ag/AgCl

+ 0.49 V

+ 0.36 V

20 nA

porting electrolyte pH, and at Red-BDD the currents were always higher than at Ox-BDD. In pH 4.5 buffer, the 8-oxoGua oxidation peak at Red-BDD occurs at Epa = +0.49 V, Fig. 5B, and at Ox-BDD is shifted to a higher oxidation potential Epa = +0.62 V, Fig. 6A. In pH 7.0 buffer, the 8-oxoGua oxidation peak at Red-BDD is shifted to a lower potential at Epa = +0.36 V, Fig. 6B, and at OxBDD is not pH dependent, Fig. 6A. The potential of +3.0 V applied for the anodic pre-treatment on BDD surface is in the region of water discharge at BDD electrodes, and involves a high rate of hydroxyl radical (OH ) production. At the BDD surface there is a weak interaction with hydroxyl radicals (OH ) but the hydroxyl radicals adsorbed on the surface or the products from their parallel reactions, due to their high reactivity, react with 8-oxoGua decreasing its signal. The big decrease at pH 4.5 in the oxidation current obtained at Ox-BDD for 8-oxoGua, in comparison with Red-BDD, and no peak for oxidation of 8-oxoGua when the sulphuric acid electrolyte solution was used for the anodic pre-treatment, is due to interaction of 8-oxoGua with the hy droxyl radicals (OH ) formed at the Ox-BDD surface. An electroanalytical procedure was developed for the determination of 8-oxoGua using DP voltammetry at BDD. A supporting electrolyte of pH 4.5 and cathodic pre-treatment were chosen, since higher oxidation peaks are obtained, Fig. 6A and B. In order to obtain reproducible results after the cathodic pre-treatment, the Red-BDD was placed in 4.5 0.1 M acetate buffer electrolyte and two consecutive DP voltammograms were recorded, between the potential limits of E1 = +0.25 V and E2 = +1.4 V. Nevertheless, it was not necessary to clean the electrode between measurements. DP voltammograms of 8-oxoGua, with three measurements for each concentration, are shown in Fig. 6C. The calibration curve was plotted for oxidation peak current of 8-oxoGua versus solution concentration of 8-oxoGua between 0 and 11.4 lM. A good linearity was found between peak height and concentration described by the equation: Ipa(nA) = 48.7 [8-oxoGua/lM] + 16.9 where r = 0.997, n = 8 and P < 0.0001. Other parameters to dene the sensitivity were calculated and the value obtained for the limit of detection, LOD = 1.0 lM, was based on three times the noise level, and the limit of quantication was LOQ = 3.2 lM, based on 10 times the noise level.

0.3

0.4

0.5

0.6

E / V vs. Ag/AgCl dAdo

dGuo

100 nA
0.4 0.5 0.6 0.7

0.5 A

E / V vs. Ag/AgCl
Fig. 6. Baseline-corrected DP voltammograms of 8-oxoGua in: () pH 4.5 0.1 M acetate buffer and () pH 7.0 0.1 M phosphate buffer: (A) at Ox-BDD surface, (B) at Red-BDD surface, and (C) successive DP voltammograms at Red-BDD of 1.6, 3.25, 4.9, 6.4, 8.1, 9.8 and 11.4 lM of 8-oxoGua, in pH 4.5 0.1 M acetate buffer electrolyte, scan rate m = 10 mV s1.

0.3

0.6

0.9

1.2

1.5

E / V vs. Ag/AgCl
Fig. 7. Baseline-corrected DP voltammograms of dsDNA in pH 4.5 0.1 M acetate buffer () at Ox-BDD surface and () at Red-BDD surface; (dGuo) desoxyguanosine and (dAdo) desoxyadenosine.

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Severino Carlos B. Oliveira, A.M. Oliveira-Brett / Journal of Electroanalytical Chemistry 648 (2010) 6066 [2] H.J. Helbock, K.B. Beckman, M.K. Shigenaga, P.B. Walter, A.A. Woodall, H.C. Yeo, B.N. Ames, Proc. Natl. Acad. Sci. USA 95 (1998) 288. [3] A.M. Oliveira-Brett, J.A.P. Piedade, L.A. Silva, V.C. Diculescu, Anal. Biochem. 332 (2004) 321. [4] A.M. Oliveira-Brett, J.A.P. Piedade, S.H.P. Serrano, Electroanalysis 12 (2000) 969. [5] J.-L. Ravanat, E. Gremaud, J. Markovic, R.J. Turesky, Anal. Biochem. 260 (1998) 30. [6] S. Loft, P.H. Danielsen, L. Mikkelsen, L. Risom, L. Forchhammer, P. Moller, Biochem. Soc. Trans. (2006) 1071. [7] A. Klungland, S. Bjelland, DNA Repair 6 (2007) 481. [8] S. Shibutani, M. Takeshita, A.P. Grollman, Nature 349 (1991) 431. [9] S. Bjelland, E. Seeberg, Mutat. Res.-Fund. Mol. M. 531 (2003) 37. [10] H. Kasai, Mutat. Res.-Rev. Mutat. 387 (1997) 147. [11] B. Halliwell, Mutat. Res.-Genet. Tox. En. 443 (1999) 37. [12] E.T. Borish, J.P. Cosgrove, D.F. Church, W.A. Deutsch, W.A. Pryor, Biochem. Biophys. Res. Co. 133 (1985) 780. [13] R. Olinski, D. Gackowski, M. Foksinski, R. Rozalski, K. Roszkowski, P. Jaruga, Free Radical Bio. Med. 33 (2002) 192. [14] I.A. Rebelo, J.A.P. Piedade, A.M. Oliveira-Brett, Talanta 63 (2004) 323. [15] S. Adachi, M. Zeisig, L. Moller, Carcinogenesis 16 (1995) 253. [16] K.E. Herbert, M.D. Evans, M.T.V. Finnegan, S. Farooq, N. Mistry, I.D. Podmore, P. Farmer, J. Lunec, Free Radical Bio. Med. 20 (1996) 467. [17] M. Dizdaroglu, Biochemistry 24 (1985) 4476. [18] M. Dizdaroglu, J. Chromatogr. A 295 (1984) 103. [19] E.M. Park, M.K. Shigenaga, P. Degan, T.S. Korn, J.W. Kitzler, C.M. Wehr, P. Kolachana, B.N. Ames, Proc. Natl. Acad. Sci. USA 89 (1992) 3375. [20] I. Rebelo, J.A.P. Piedade, A.M. Oliveira Brett, Bioelectrochemistry 63 (2004) 267. [21] B. Halliwell, M. Dizdaroglu, Free Radical Res. Commun. 16 (1992) 75. [22] A. Collins, J. Cadet, B. Epe, C. Gedik, Carcinogenesis 18 (1997) 1833. [23] E. Palecek, M. Fojta, F. Jelen, V. Vetterl, in: A.J. Bard, M. Stratmann (Eds.), The Encyclopedia of Electrochemistry, Wiley-VCH Verlag, Weinheim, Germany, 2002, pp. 365429. [24] G. Dryhurst, P.J. Elving, J. Electrochem. Soc. 115 (1968) 1014. [25] G. Dryhurst, Anal. Chim. Acta 57 (1971) 137. [26] T. Yao, T. Wasa, S. Musha, Bull. Chem. Soc. Jpn. 51 (1978) 1235. [27] A.M. Oliveira Brett, F.-M. Matysik, J. Electroanal. Chem. 429 (1997) 95. [28] A.M. Oliveira Brett, F.-M. Matysik, Bioelectrochem. Bioenergy 42 (1997) 111. [29] E. Palecek, F. Jelen, M.A. Hung, J. Lasovsky, Bioelectrochem. Bioenergy 8 (1981) 621. [30] T.A. Ivandini, K. Honda, T.N. Rao, A. Fujishima, Y. Einaga, Talanta 71 (2007) 648. [31] F.E. Karasz, C.-T. Jama, D. Delabouglise, P. Bouvier, T. Livache, P. Mailley, B. Marcus, M. Mermoux, J.P. Petit, S. Szunerits, E. Vieil, Electroanalysis 17 (2005) 517. [32] C. Prado, G.U. Flechsig, P. Grundler, J.S. Foord, F. Marken, C. R., Analyst 127 (2002) 329. [33] T.A. Ivandini, B.V. Sarada, T.N. Rao, A. Fujishima, Analyst 128 (2003) 924. [34] R.G. Compton, J.S. Foord, F. Marken, Electroanalysis 15 (2003) 1349. [35] V.A. Pedrosa, H.B. Suffredini, L. Codognoto, S.T. Tanimoto, S.A.S. Machado, L.A. Avaca, Anal. Lett. 38 (2005) 1115. [36] L. Codognoto, S.A.S. Machado, L.A. Avaca, Diam. Relat. Mater. 11 (2002) 1670. [37] H.B. Suffredini, V.A. Pedrosa, L. Codognoto, S.A.S. Machado, R.C. Rocha-Filho, L.A. Avaca, Electrochim. Acta 49 (2004) 40214026. [38] G.R. Salazar-Banda, L.S. Andrade, P.A.P. Nascente, P.S. Pizani, R.C. Rocha-Filho, L.A. Avaca, Electrochim. Acta 51 (2006) 4612. [39] T. Kondo, K. Honda, D.A. Tryk, A. Fujishima, Electrochim. Acta 48 (2003) 2739. [40] N. Simon, H. Girard, D. Ballutaud, S. Ghodbane, A. Deneuville, M. Herlem, A. Etcheberry, Diam. Relat. Mater. 14 (2005) 1179. [41] H. Girard, N. Simon, D. Ballutaud, M. Herlem, A. Etcheberry, Diam. Relat. Mater. 16 (2007) 316. [42] F.B. Liu, J.D. Wang, B. Liu, X.M. Li, D.R. Chen, Diam. Relat. Mater. 16 (2007) 454. [43] D.A. Tryk, K. Tsunozaki, T.N. Rao, A. Fujishima, Diam. Relat. Mater. 10 (2001) 1804. [44] T.A. Enache, A.-M. Chiorcea-Paquim, O. Fatibello-Filho, A.M. Oliveira-Brett, Electrochem. Commun. 11 (2009) 1342. [45] I. Duo, C. Levy-Clement, A. Fujishima, C. Comninellis, J. Appl. Electrochem. 34 (2004) 935. [46] S.C.B. Oliveira, A.M. Oliveira-Brett, Electrochim. Acta 55 (2010) 4599. [47] H.B. Suffredini, S.A.S. Machado, L.A. Avaca, J. Braz. Chem. Soc. 15 (2004) 16. [48] H.B. Martin, A. Argoitia, U. Landau, A.B. Anderson, J.C. Angus, J. Electrochem. Soc. 143 (1996) L133. [49] B. Marselli, J. Garcia-Gomez, P.A. Michaud, M.A. Rodrigo, C. Comninellis, J. Electrochem. Soc. 150 (2003) D79. [50] P.A. Michaud, M. Panizza, L. Ouattara, T. Diaco, G. Foti, C. Comninellis, J. Appl. Electrochem. 33 (2003) 151. [51] A. Kapalka, G. Foti, C. Comninellis, J. Electrochem. Soc. 155 (2008) E27. [52] S.C.B. Oliveira, O. Corduneanu, A.M. Oliveira-Brett, Bioelectrochemistry 72 (2008) 53.

3.2.5. Oxidation of dsDNA The effect of the anodic and cathodic pre-treatment on the electrochemical oxidation of dsDNA was studied, Fig. 7. The DP voltammograms for 200 lg mL1 of dsDNA in pH 4.5 at Ox- and Red-BDD surfaces obtained by pre-treatment in pH 4.5 0.1 M acetate buffer as described in Section 2.2, for 15 min, are shown in Fig. 7. The electrochemical oxidation of the dsDNA at Ox- and RedBDD shows two well-dened oxidation peaks corresponding to oxidation of desoxyguanosine (dGuo) and desoxyadenosine (dAdo), Fig. 7. However, using Red-BDD about two times higher peak currents at lower oxidation potentials than those at OxBDD, were obtained. The hydroxyl radicals adsorbed on the OxBDD surface during the anodic pre-treatment are highly reactive and consequently the Ox-BDD surface has a less good response because it is not completely inert. The results with dsDNA are in agreement with all the results described here for the DNA bases, nucleotides, homopolynucleotides and biomarker 8-oxoguanine. It is clear that the electrochemical properties of BDD electrodes are very dependent on the state of the surface termination, due to oxygen and hydrogen terminations, and a better response was obtained when the BDD surface was pretreated cathodically, Red-BDD, in comparison with the anodic pretreatment, Ox-BDD. The electron transfer reaction and adsorption of the electrochemical species with the BDD surface pre-treated cathodically was facilitated due to a higher conductivity of the BDD electrode. 4. Conclusions The results presented here clearly demonstrate that the electrochemical response of BDD electrode surface was inuenced by the anodic or cathodic electrochemical pre-treatment of the BDD surface. The choice of electrolyte for the electrochemical pre-treatment is also very important, because it inuences the nal surface termination, the BDD conductivity, the electrochemical response and the reproducibility of the experimental results. The hydroxyl radicals formed on the Ox-BDD surface during anodic pre-treatment are very highly reactive and can thence be involved in parallel reactions on the Ox-BDD surface. Hydrogen adsorption gives rise to H-terminated sites during the cathodic pre-treatment of the Red-BDD surface leading to a larger background current and narrower potential window of Red-BDD. As clearly demonstrated here, the BDD electrode surface termination caused by electrochemical pre-treatment very much affects the oxidation potentials and peak currents of dsDNA, DNA bases, nucleotides, homopolynucleotides and biomarker 8-oxoguanine. Acknowledgements Financial support from Fundao para a Cincia e Tecnologia (FCT), Ph.D. Grants SFRH/BD/27322/2006 (S.C.B. Oliveira), projects PTDC/QUI/65255/2006, PTDC/QUI/65732/2006, PTDC/QUI-QUI/ 098562/2008, POFC-QREN (co-nanced by the European Community Fund FEDER), and CEMUC-R (Research Unit 285), is gratefully acknowledged. References
[1] S. Fukuzumi, H. Miyao, K. Ohkubo, T. Suenobu, J. Phys. Chem. A 109 (2005) 3285.