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An electrochemical biosensor for the rapid detection of DNA damage induced by xanthine oxidase-catalyzed Fenton reaction
Sensors and Actuators B 181 (2013) 85–91
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Sensors and Actuators B: Chemical journal homepage: www.elsevier.com/locate/snb
An electrochemical biosensor for the rapid detection of DNA damage induced by xanthine oxidase-catalyzed Fenton reaction Huayu Xiong, Yang Chen, Xiuhua Zhang, Haoshuang Gu, Shengfu Wang ∗ Ministry of Education Key Laboratory for the Synthesis and Application of Organic Functional Molecules & College of Chemistry and Chemical Engineering, Hubei University, Wuhan 430062, PR China
a r t i c l e
i n f o
Article history: Received 28 October 2012 Received in revised form 20 December 2012 Accepted 18 January 2013 Available online 28 January 2013 Keywords: DNA damage Xanthine oxidase Co(bpy)3 3+ Hydroxyl radical Square wave voltammetry
a b s t r a c t Oxidative DNA damage is one of the most critical factors implicated in carcinogenesis and other disorders. Sensitive and reliable detection of oxidative DNA damage remains a significant challenge. In this work, a sensitive electrochemical biosensor based on a double stranded DNA immobilized on a xanthine oxidase (XOD)-modified glassy carbon electrode (denoted as DNA–XOD/GCE) has been developed to explore the rapid detection of DNA damage. Co(bpy)3 3+ was used as a redox indicator to monitor DNA damage induced by hydroxyl radical (• OH), which is a reactive oxygen species generated by a XOD-catalyzed Fenton reaction in xanthine/FeSO4 system. The produced • OH was validated by UV–vis spectroscopy. The electrochemical behavior of the underlying electrodes was characterized by square wave voltammetry and electrochemical impedance spectroscopy. Optimization of the concentrations of FeSO4 and XA, and the incubation time in terms of DNA damage was explored. Moreover, the protection of DNA from damage by antioxidants, such as ascorbic acid, aloe-emodin, and rutin was investigated. The conclusions demonstrate that the proposed electrochemical method is expected to be of use in further application for DNA damage studies. © 2013 Elsevier B.V. All rights reserved.
1. Introduction In recent decades, the detection of DNA damage has become one of the most important DNA research fields, because of the critical role of DNA in mutagenesis, carcinogenesis, and aging. It is well known that DNA in biological systems may be damaged by both exogenous and endogenous sources. Among them, the reactive oxygen species (ROS) have aroused great scientific interest. ROS play an important role in DNA damage, which is tightly related to mutagenesis and carcinogenesis [1]. If the damaged DNA cannot be repaired in time, the induced genetic mutation will result in a cancer or tumor during the DNA replication process [2]. Therefore, it is necessary to establish a sensitive, rapid, and inexpensive way to detect DNA damage. Up to now, varieties of analytical methods have been developed for detecting DNA damage, including capillary liquid chromatography/mass spectrometry [3], fluorescence [4], 32 P-postlabeling [5],
Abbreviations: XOD, xanthine oxidase; • OH, hydroxyl radical; ROS, reactive oxygen species; XA, xanthine; DNA–XOD/GCE, dsDNA and a XOD modified glassy carbon electrode; Co(bpy)3 (ClO4 )3 , Tris(2,2 -bipyridyl) cobalt(III) perchlorate; AA, ascorbic acid; AE, aloe-emodin; GCE, glassy carbon electrode; DNA/GCE, DNA modified GCE; XOD/GCE, XOD modified GCE; SWV, square wave voltammetry; MV, methyl violet. ∗ Corresponding author. Tel.: +86 27 50865309; fax: +86 27 88663043. E-mail address: [email protected] (S. Wang). 0925-4005/$ – see front matter © 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.snb.2013.01.033
and capillary zone electrophoresis [6]. These methods are generally performed at centralized laboratories, requiring long assay times and high costs. Electrochemical methods have aroused great interest among researchers, mainly because of their simplicity, fast response, relatively low cost, and low power requirements [7,8], and much work have been carried out. Usually, two kinds of electrochemical methods are adapted to detect DNA damage, which are based on the direct electrochemical signals of guanine and adenine bases [9–11], or indirect electroactive indicators which could specifically interact with DNA [12,13]. Our groups have detected the oxidative DNA damage by hydroxyl radical (• OH) using Co(bpy)3 3+ as electroactive probes, where • OH were generated by Fenton reagents [14]. In order to mimic the metal-mediated ROS generation pathway in vivo, H2 O2 could be produced in situ through the enzymatic reaction. A large number of studies have been reported about the electrochemical detection of DNA damage by the enzymatic reaction [15,16]. Recently, our group had performed the electrochemical detection of in situ DNA damage induced by glucose oxidase-catalyzed Fenton reaction [17]. Some references have reported that xanthine oxidase (XOD) could catalyze the oxidation of xanthine (XA) under aerobic conditions, and the produced H2 O2 reacted with ferrous ions in a Fenton-type reaction to generate • OH [16,18]. In this report, a sensitive electrochemical biosensor constructed from dsDNA and a XOD modified glassy carbon electrode (DNA–XOD/GCE) has been developed to explore the rapid detection
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of DNA damage. Tris(2,2 -bipyridyl) cobalt(III) perchlorate (Co(bpy)3 (ClO4 )3 ) was used as a redox indicator to monitor DNA damage induced by • OH in aqueous solutions. The results demonstrate that the proposed electrochemical method is likely to find further application in DNA damage studies. 2. Experimental 2.1. Chemicals and reagents Calf thymus DNA was purchased from Sigma–Aldrich. Solutions of DNA were prepared by dissolving it in 0.01 M Tris–HCl/0.001 M ethylenediaminetetraacetic acid/0.05 M NaCl (pH 7.0) to form the 1.0 mg mL−1 stock solution and stored at 4 ◦ C. XOD (EC 1.1.3.4, Type X-S, from Aspergillus niger) was obtained from Sigma–Aldrich. Solutions of XOD were dissolved in 0.1 M phosphate buffer solution (PBS, pH 7.0) to form 3.0 mg mL−1 solutions. XA were purchased from Sigma–Aldrich. Co(bpy)3 (ClO4 )3 was prepared as described in the literature [19] and dissolved in 0.005 M Tris–HCl (pH 7.0) containing 0.05 M NaCl. Ascorbic acid (AA) was supplied by the Hubei University Chemical Factory (Hubei, China). Rutin and aloeemodin (AE) were purchased from Shanghai Boyun Biotech Co., Ltd. (Shanghai, China). All solutions were prepared with doubly distilled water. Other reagents were analytical grade. 2.2. Apparatus Electrochemical measurements were performed on a model CHI 625A electrochemical workstation (CH Instruments, Chenhua Co., Shanghai, China). A standard three-electrode system, consisting of a film modified glassy carbon electrode (GCE) as the working electrode, a saturated calomel electrode as the reference electrode, and platinum foil as the auxiliary electrode, was used in the measurements. UV–vis spectroscopy was carried out with a UV 2300 spectrophotometer (Shanghai Tian Mei Scientific Instrument Co., Ltd., China). Atomic force microscopy (AFM) images were obtained on a PicoScan system (Molecular Imaging Inc.) operated in contact mode with commercially ultrasharpened Si3 N4 tips (MAClever II, Molecular Imaging Inc.). The AFM measurement parameters
were as follows: force constant, 0.12 N/m; number of scans, 8 times.
2.3. Preparation of the DNA–XOD/GCE In our work, 1 mg mL−1 DNA and 3 mg mL−1 XOD with a volume ratio of 1:1 were mixed thoroughly, and 30 L of this mixture was directly applied to the surface of the clean GCE, followed by airdrying overnight. Then, the modified electrode was incubated in a pH 7.0 PBS for 4 h to remove any non-adsorbed DNA. This electrode was hereafter referred to as DNA–XOD/GCE. For comparison, DNA modified GCE (DNA/GCE) and XOD modified GCE (XOD/GCE) were also prepared using a similar direct application technique.
2.4. Procedures Scheme 1 shows the detection approach of DNA damage induced by XOD-catalyzed Fenton reaction in xanthine/FeSO4 system. DNA–XOD film was incubated in pH 3.0 buffer containing 1 mM FeSO4 and 1.7 mM XA, with stirring for the specified time for DNA damage (curve b). Control experiments were that DNA–XOD film was incubated in pH 3.0 buffer containing FeSO4 or XA separately, and DNA film or XOD film were incubated in pH 3.0 buffer containing FeSO4 or XA or FeSO4 /XA (curve a). To investigate the effects of antioxidants, aliquots of antioxidant samples were added into the above system. That is, DNA–XOD film was incubated with FeSO4 and XA in a pH 3.0 buffer in the presence of AA, AE, or rutin. All incubations were conducted at room temperature with stirring for 20 min. After incubation, the modified electrode was rinsed with doubly distilled water and then transferred into pH 7.0 Tris–HCl containing 200 M Co(bpy)3 3+ for square wave voltammetry (SWV). Each measurement was repeated at least 3 times. To correct the electrode-to-electrode or film-to-film variation for replicative experiments, the SWV oxidation peak current ratio (Ipt /Ip0 ), instead of the absolute peak current (Ipt ), was used to evaluate the effect of DNA damage, where Ipt and Ip0 are the peak currents after DNA–XOD film incubation with FeSO4 and XA in a pH 3.0 buffer for t and 0 min.
Scheme 1. Schematic diagram showing working principle of SWV detection of in situ DNA damage on DNA–XOD film.
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Fig. 1. UV absorption spectrum of (a) XOD–DNA film incubated with MV + PBS (pH 3.0), (b) MV + PBS (pH 3.0), (c) MV + Fe2+ + PBS (pH 3.0), (d) MV + XA + PBS (pH 3.0), (e) MV + Fe2+ + XA + PBS (pH 3.0), and (f) XOD–DNA film incubated with MV + Fe2+ + XA + PBS (pH 3.0).
3. Results and discussion 3.1. Verification of hydroxyl radicals in Fenton-type system Methyl violet (MV) was introduced to measure the amount of with UV–vis spectroscopy in the present work. In MV, the carbon–carbon double bonds with high electron cloud density are easily attacked by hydroxyl free radicals, with an electrophilic addition reaction taking place, leaving MV to fade [20]. As you can see in Fig. 1a–f, the difference in the absorption bands of MV appearing at about 580 nm was employed to determine the generation of hydroxyl radicals. The results suggested that • OH was generated by the XOD-catalyzed Fenton reaction in XA/FeSO4 system. • OH
3.2. Morphology characterization of the fabricated films Fig. 2 shows a set of representative AFM topographs of DNA and DNA/protein films immobilized on mica disks. Fig. 2A presents the AFM image of DNA membrane. Many small but dispersed evenly particles could be observed. It is known that different structures of DNA molecules can be found depending on solution conditions. Typically, DNA molecules condense into toroids, rod-like shapes, or globules [21]. In this study, DNA exhibited the globules conformation and distributed evenly. After the addition of XOD, the surface morphology changed dramatically, showing a lot of clusters or agglomerates (Fig. 2B). The particles in the image should be induced by the cross-linked XOD molecules. The crater and valley topographic features existed. The difference between DNA and DNA/XOD indicated that the deposited enzyme and DNA formed a uniform layer.
Fig. 2. AFM images of (A) DNA, (B) DNA–XOD on the mica disks.
3.3. Scheme of the DNA damage 3.3.1. Detection of DNA damage induced by XOD/XA/FeSO4 system Co(bpy)3 3+ is mainly bonded to the double helix of DNA by electrostatic interaction with the negatively charged phosphate backbone. It was used as an electroactive indicator, which bonds more to intact DNA than damaged DNA [12]. SWVs for DNA–XOD film in Co(bpy)3 3+ before and after incubations were shown in Scheme 1. The intact DNA–XOD film showed a good oxidative peak with peak potential of 0.064 V. After DNA–XOD film was incubated with FeSO4 and XA in a pH 3.0 buffer at room temperature with stirring for 20 min, the peak current of Co(bpy)3 3+ decreased obviously (Fig. 3a). DNA damage degree induced by XOD-catalyzed Fenton
Fig. 3. Ipt /Ip0 of 200 M Co(bpy)3 3+ for DNA–XOD film after incubation with pH 3.0 buffer containing: (a) 1.7 mM XA and 1 mM FeSO4 ; (b) XA; (c) FeSO4 . For DNA film after incubation with pH 3.0 buffer containing: (d) XA; (e) FeSO4 ; (f) XA and FeSO4 . For XOD film after incubation with pH 3.0 buffer containing: (g) XA; (h) FeSO4 ; (i) XA and FeSO4 .
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Fig. 4. EIS spectra of (a) bare GCE; DNA–XOD/GCE (b) before and (c) after incubation with pH 3.0 buffer containing 1.7 mM XA and 1 mM FeSO4 in 5 mmol/L [Fe(CN)6 ]3−/4− containing 0.1 mol/L KCl.
reaction in xanthine/FeSO4 system was more severely than the reported damage degrees generated by a glucose oxidase-catalyzed Fenton reaction in glucose/FeSO4 system [17], the Fenton reagents in ionic liquid system [14]. To further investigate the effects of various reagents on DNA damage, Ipt /Ip0 ratios for different films incubated in different types of solutions were compared. For the control incubation experiments, the electrochemical response did not show obvious change within the incubation time (Fig. 3b–i). These results confirmed that, for DNA–XOD film, XA and FeSO4 were necessary for DNA damage. The decrease of peak currents after the incubation with FeSO4 and XA in a pH 3.0 buffer suggested that the ability of the film to bond with the probe was gradually losing and DNA damage was becoming more severe [13]. The possible mechanism of DNA damage induced by the XOD/XA/FeSO4 system was that XOD could catalyze the oxidation of XA under aerobic conditions, and the produced H2 O2 reacted with ferrous ions in a Fenton-type reaction to generate • OH. The formed • OH disturbed DNA double helix structure or caused DNA strand break, thus leading to the decrease of Co(bpy)3 3+ oxidative peak current [22]. 3.3.2. Validation of DNA damage in Fenton-type system It is well known that electrochemical impedance spectroscopy (EIS) is a simple and effective method for affording information on the impedance changes on the electrode surface during the modification process [23]. Fig. 4 shows the results of EIS at a bare electrode (curve a), a DNA–XOD modified electrode before (curve b) and after (curve c) incubated with FeSO4 and XA in a pH 3.0 buffer, respectively, in the presence of 5 mmol/L [Fe(CN)6 ]3−/4− solution. It can be seen that the bare electrode exhibits an almost straight line, which is characteristic of a diffusion limiting step of the electrochemical process. A pronounced semicircle was obtained in Fig. 4b, indicating that DNA–XOD film hindered the charge transfer. After incubation treatment with FeSO4 and XA in a pH 3.0 buffer for 20 min, charge-transfer resistance was furthermore increased, which was reflected in the apparent increase in the semicircular part of the spectrum, indicating the evidently attenuation of DNA conductibility induced by hydroxyl radicals [17,24]. The possible reason for this phenomenon is that • OH induce oxidative DNA damage, producing a variety of modifications on the DNA level, including base and sugar lesions, strand breaks, DNA–protein cross-links, and base-free sites. Then the ability of transfer electron of DNA film decreased. To investigate the extent of DNA damage, a gel electrophoresis experiment was performed (Fig. 5). In the experiment, 0.3 mg mL−1
Fig. 5. Agarose gel electrophoresis of different types of DNA. Lane 2: 0.3 mg mL−1 ds-DNA reacted with XOD/1.7 mM XA/1 mM FeSO4 , pH 3.0 PBS. Lane 3: ds-DNA, pH 7.0 PBS. Lane 4: ds-DNA, pH 3.0 PBS. Lane 5: ds-DNA reacted with XA/FeSO4 , pH 3.0 PBS (no XOD). Lane 1: -Hind III digest DNA Marker. Lane 6: DL2000 DNA Marker.
of ds-DNA was reacted with XOD/1.7 mM XA/1 mM FeSO4 . After the reaction, the DNA sample was run on an agarose gel (Lane 2), together with the markers (Lanes 1 and 6) and the controlled DNA samples which were obtained by immersing in pH 7.0 buffer (Lane 3), pH 3.0 buffer (Lane 4), or reacting with XA/FeSO4 (no XOD, Lane 5). In Fig. 5, there was a major band (length about 23,130 base pairs) for the control (Lanes 3–5) that was absent in the images for Lane 2. Instead, many weak bands representing different DNA lengths appeared for the latter, illustrating extensive DNA breakage. The difference between the controls and sample 2 indicates that XOD was active at pH 3.0 and induced DNA damage in the presence of XA and FeSO4 . The similarity between the two DNA samples reacted at pH 3.0 and 7.0 suggests that DNA sample in pH 3.0 is suitable for investigating the degree of damage as in the neutral solution.
3.4. Selection of the optimum experimental conditions 3.4.1. The optimum pH During the experiment, it was found that after DNA–XOD sensor was incubated in a 1 mM Fe2+ solution alone (without XA) if pH of the solution was higher than 3.0, the peak current of Co(bpy)3 3+ decreased greatly. Since the modified electrode was rinsed with doubly distilled water after the incubation and then scanned, the effect might come from the metal ions adsorbed on the sensor surface during the incubation step. The detrimental effect was eliminated completely when the pH of Fe2+ solution was reduced to 3.0 or lower. Considering that XOD has low activity in acidic solutions and Fe2+ suppresses the peak current at high pH values, pH 3.0 Fe2+ /XA solution was selected. As we known, even at pH 3.0, the activity of XOD is high enough to generate sufficient H2 O2 for the DNA damage reaction [25]. It is well known that protonation of purine residues and subsequent unpairing of base pairs are found to commence at pH 3.0. It is noticed that the referred objects are DNA solutions. In our work, dehydrated DNA molecules could be irreversibly adsorbed on the surfaces of GCE. The DNA-adsorbed layer on GCE was stable to pH 3.0 buffer, which was confirmed by Pang’s work [26]. The relatively reasonable explanations for this are as follows. When DNA solution on GCEs is evaporated to dryness, the bases of DNA which have dehydrated are exposed, thus the hydrophobic bases are strongly adsorbed flat on the electrode surfaces [26]. Once it is adsorbed, DNA is difficult to re-hydrate and not so sensitive to pH of solution.
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3.5. Protection of DNA from damage by antioxidants 3.5.1. Evaluation of the hydroxyl radical scavenging activities of AA, AE, and rutin The influences of the antioxidants on the damage of DNA were investigated in the presence of AA, AE or rutin. In Fig. 7A(b–d), Ipt /Ip0 were shown when different antioxidants were injected into the OHgenerating system. A significant increase in the current level after antioxidant injection was observed. The increase of Ipt /Ip0 implied an effective protection effect of AA, AE and rutin. The presence of antioxidants recovered the electrochemical signal. We supposed that the release of the bases and an interruption of the phosphodiester bonds induced by radicals were resisted by those antioxidants [29]. 3.5.2. Influence of the AA, AE and rutin concentration In order to study the optimal concentration of antioxidants, different concentrations of AE, AA and rutin were added into the damage system (Fig. 7B). At low concentrations of AE, AA and rutin, Ipt /Ip0 increased with their concentrations and then reached the maximum. Then the marker signals decreased with the concentrations of antioxidants. The optimal concentrations of antioxidants for AE, AA and rutin were 3.3 M. As we know, those small molecules reacted rapidly with oxidant that made them good antioxidants. At the same time, they had a strong reducing power for metal ions. The antioxidant and oxidant activity of the three small molecules were correlative with its concentration. Low concentrations of antioxidants were excellent free radical scavengers, whereas high concentrations could promote Fenton-type reaction to generate OH• [30]. The nature of shown in the experiment was a comprehensive result of free radical scavenging ability and reducing capacity. When reducing capacity of AA was stronger than the capacity of scavenging free radicals, AA could promote the reaction of Fe2+ and H2 O2 to produce OH• , leading to DNA damage [31].
Fig. 6. (A) Influence of concentrations of XA on Ipt /Ip0 of 200 M Co(bpy)3 3+ for DNA–XOD film after incubation with pH 3.0 buffer containing 1.7 mM XA and 1 mM FeSO4 . (B) Effect of the incubation time on Ipt /Ip0 of 200 M Co(bpy)3 3+ for DNA–XOD film after incubation with pH 3.0 buffer containing (a) FeSO4 ; (b) XA; (c) XA and FeSO4 .
3.4.2. Optimum concentration of Fe2+ and XA In the above reactions, iron acts as a catalyst but excess Fe2+ might cause scavenger effects. Increasing Fe2+ concentration from 50 M to 1 mM resulted in the production of • OH. Excess • OH would tend to react with other • OH at higher Fe concentrations [27]. 1 mM FeSO4 was selected at our experiment (data not shown). The detection limit for the metal ion was less than 50 M. With 1 mM FeSO4 and varying concentrations of XA, Ipt /Ip0 decreased rapidly as the concentration of XA was increased from 0.68 to 1.7 mM and then tended toward a constant value (Fig. 6A), revealing a dependence of the damage reaction on the XA concentration. 1.7 mM XA was selected in this work. The detection limit for XA was less than 0.68 mM. 3.4.3. The optimum incubation time Fig. 6B presents the influence of incubation time on the DNA damage induced by the Fenton-type reaction. No obviously trends were observed for the control incubation experiments (curves a and b). After DNA–XOD film incubated in the pH 3.0 buffer containing FeSO4 and XA, Ipt /Ip0 decreased with the incubation time within 20 min and then reached a plateau (curve c). So, 20 min was selected as the optimum incubation time. The decrease trend was in accordance with the previous report [28].
3.5.3. Influence of incubation time Fig. 7C exhibits the influence of incubation time on Ipt /Ip0 in the presence of 3.3 M AA, AE, and rutin. When DNA film was incubated in pH 3.0 buffer containing FeSO4 and XA in the presence of antioxidants, Ipt /Ip0 decrease rapidly for the first 20 min and then decrease slowly (curves a–c). But the decreasing trend of Ipt /Ip0 was much slower compared with that of damage process, indicating the obvious protective effect of AA, AE, and rutin toward DNA damage. 3.6. Comparison of the enzyme catalyzed reactions • OH generated by a XOD-catalyzed hypoxanthine (HX) reaction was also studied by the electrochemical techniques. Experimental results revealed that this system was similar to the xanthine/FeSO4 system. HX can be transformed to XA, then to uric acid, and then to H2 O2 through XOD under aerobic conditions. The producing • OH can cause DNA damage but the damage degree was much small (data not shown).
3.7. Reproducibility and stability Stability and fabrication reproducibility are important parameters for the modified electrode. The studies on sensor reproducibility were as follows: DNA–XOD film was incubated with FeSO4 and XA in a pH 3.0 buffer for 20 min. After that, the modified electrode was taken out and rinsed with doubly distilled water and then transferred into Co(bpy)3 3+ for 10 successive scanning, the relative standard deviation (R.S.D.) was 3%. Fabrication reproducibility was estimated with five different electrodes. Five DNA–XOD modified electrodes were incubated with FeSO4 and XA in a pH 3.0 buffer for 20 min. After that, the damage films were taken out and rinsed
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with doubly distilled water and then transferred into Co(bpy)3 3+ for scanning, R.S.D. was 9%. Moreover, the responses decreased to 92% and 95% of the original current for the film after 1 week of use and 1 week stored without using, respectively. As a result,
DNA–XOD/GCE exhibited good reproducibility and comparative stability. 4. Conclusion This paper utilized electrode with adsorbed DNA and XOD to investigate DNA oxidative damage and the free radical scavenging ability of AA, AE, and rutin using Co(bpy)3 3+ as the electrochemical probe. The detection of DNA damage by • OH, which was generated by the XOD-catalyzed Fenton reaction in XA/FeSO4 system, was realized by electrochemical methods. Their antioxidative mechanisms of AA, AE, and rutin were investigated. They simultaneously exhibited excellent antioxidant and reducing capacities, and which one played a dominant role dependence on their concentrations. It offered a useful platform to detect DNA damage from metabolites. Future work would target the screening of genotoxic chemicals using the presented methodology. Acknowledgements This work was supported by the National Natural Science Foundation of China (Nos. 20875023 and 51173038), the Ph.D. Programs Foundation of the Ministry of Education of China (No. 20114208130001), the Natural Science Fund for Creative Research Groups of Hubei Province of China (No. 2011CDA111), and the Foundation for Innovative Research Groups of the Education Department of Hubei Province (No. T201101). References
Fig. 7. (A) Ipt /Ip0 of 200 M Co(bpy)3 3+ for DNA–XOD film after incubation with pH 3.0 buffer containing: (a) 1.7 mM XA and 1 mM FeSO4 ; (b) (a) + 3.3 M AA; (c) (a) + 3.3 M AE; (d) (a) + 3.3 M rutin. (B) Influence of concentrations of different antioxidants on Ipt /Ip0 of 200 M Co(bpy)3 3+ for DNA–XOD films after incubation with pH 3.0 buffer containing XA and FeSO4 and (a) AA, (b) AE, and (c) rutin for 30 min. (C) Effect of the incubation time on Ipt /Ip0 of 200 M Co(bpy)3 3+ for DNA–XOD film after incubation with XA and FeSO4 containing (a) 3.3 M AA; (b) 3.3 M AE; (c) 3.3 M rutin.
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[29] H. Xiong, Y. Wang, X. Zhang, Y. Ye, S. Wang, Evaluation of antioxidative capacity via measurement of the damage of DNA using an electrochemical biosensor, Mikrochimica Acta 176 (2012) 479–484. [30] A. Carr, B. Frei, Does vitamin C act as a pro-oxidant under physiological conditions, FASEB Journal 13 (1999) 1007–1024. [31] B. Tian, Y.J. Hua, Concentration-dependence of prooxidant and antioxidant effects aloin and aloe emodin on DNA, Food Chemistry 91 (2005) 413–418.
Biographies Huayu Xiong is a laboratory assistant in the College of Chemistry and Chemical Engineering of Hubei University. She received her MSc degree in analytical chemistry in 2008 from Hubei University. Her research interest is in the development of electrochemical biosensors. Yang Chen is a post-graduate student in the department of chemistry and chemical engineering of Hubei University. Her current interest is in the development of biosensors based on free radical chemistry. Xiuhua Zhang is a professor in the College of Chemistry and Chemical Engineering of Hubei University. He received his MSc degree in analytical chemistry and his DrEng degree in materials science from Hubei University, in 2003 and 2008, respectively. His main current interest is in the development of sensors based on graphene and on conducting polymers. Haoshuang Gu is a professor in the College of Chemistry and Chemical Engineering of Hubei University. He received his PhD from Huazhong University of Science and Technology (China). His main current interests are in materials science and nanochemistry. Shengfu Wang is a professor in the College of Chemistry and Chemical Engineering of Hubei University. He received his MSc and PhD degrees in analytical chemistry from Wuhan University (China) in 1992 and 2005, respectively. His main current interests are in bioelectrochemistry, nanoelectrochemistry, chemically modified electrodes, chemical sensors, and biosensors.
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Sensors and Actuators B: Chemical journal homepage: www.elsevier.com/locate/snb
An electrochemical biosensor for the rapid detection of DNA damage induced by xanthine oxidase-catalyzed Fenton reaction Huayu Xiong, Yang Chen, Xiuhua Zhang, Haoshuang Gu, Shengfu Wang ∗ Ministry of Education Key Laboratory for the Synthesis and Application of Organic Functional Molecules & College of Chemistry and Chemical Engineering, Hubei University, Wuhan 430062, PR China
a r t i c l e
i n f o
Article history: Received 28 October 2012 Received in revised form 20 December 2012 Accepted 18 January 2013 Available online 28 January 2013 Keywords: DNA damage Xanthine oxidase Co(bpy)3 3+ Hydroxyl radical Square wave voltammetry
a b s t r a c t Oxidative DNA damage is one of the most critical factors implicated in carcinogenesis and other disorders. Sensitive and reliable detection of oxidative DNA damage remains a significant challenge. In this work, a sensitive electrochemical biosensor based on a double stranded DNA immobilized on a xanthine oxidase (XOD)-modified glassy carbon electrode (denoted as DNA–XOD/GCE) has been developed to explore the rapid detection of DNA damage. Co(bpy)3 3+ was used as a redox indicator to monitor DNA damage induced by hydroxyl radical (• OH), which is a reactive oxygen species generated by a XOD-catalyzed Fenton reaction in xanthine/FeSO4 system. The produced • OH was validated by UV–vis spectroscopy. The electrochemical behavior of the underlying electrodes was characterized by square wave voltammetry and electrochemical impedance spectroscopy. Optimization of the concentrations of FeSO4 and XA, and the incubation time in terms of DNA damage was explored. Moreover, the protection of DNA from damage by antioxidants, such as ascorbic acid, aloe-emodin, and rutin was investigated. The conclusions demonstrate that the proposed electrochemical method is expected to be of use in further application for DNA damage studies. © 2013 Elsevier B.V. All rights reserved.
1. Introduction In recent decades, the detection of DNA damage has become one of the most important DNA research fields, because of the critical role of DNA in mutagenesis, carcinogenesis, and aging. It is well known that DNA in biological systems may be damaged by both exogenous and endogenous sources. Among them, the reactive oxygen species (ROS) have aroused great scientific interest. ROS play an important role in DNA damage, which is tightly related to mutagenesis and carcinogenesis [1]. If the damaged DNA cannot be repaired in time, the induced genetic mutation will result in a cancer or tumor during the DNA replication process [2]. Therefore, it is necessary to establish a sensitive, rapid, and inexpensive way to detect DNA damage. Up to now, varieties of analytical methods have been developed for detecting DNA damage, including capillary liquid chromatography/mass spectrometry [3], fluorescence [4], 32 P-postlabeling [5],
Abbreviations: XOD, xanthine oxidase; • OH, hydroxyl radical; ROS, reactive oxygen species; XA, xanthine; DNA–XOD/GCE, dsDNA and a XOD modified glassy carbon electrode; Co(bpy)3 (ClO4 )3 , Tris(2,2 -bipyridyl) cobalt(III) perchlorate; AA, ascorbic acid; AE, aloe-emodin; GCE, glassy carbon electrode; DNA/GCE, DNA modified GCE; XOD/GCE, XOD modified GCE; SWV, square wave voltammetry; MV, methyl violet. ∗ Corresponding author. Tel.: +86 27 50865309; fax: +86 27 88663043. E-mail address: [email protected] (S. Wang). 0925-4005/$ – see front matter © 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.snb.2013.01.033
and capillary zone electrophoresis [6]. These methods are generally performed at centralized laboratories, requiring long assay times and high costs. Electrochemical methods have aroused great interest among researchers, mainly because of their simplicity, fast response, relatively low cost, and low power requirements [7,8], and much work have been carried out. Usually, two kinds of electrochemical methods are adapted to detect DNA damage, which are based on the direct electrochemical signals of guanine and adenine bases [9–11], or indirect electroactive indicators which could specifically interact with DNA [12,13]. Our groups have detected the oxidative DNA damage by hydroxyl radical (• OH) using Co(bpy)3 3+ as electroactive probes, where • OH were generated by Fenton reagents [14]. In order to mimic the metal-mediated ROS generation pathway in vivo, H2 O2 could be produced in situ through the enzymatic reaction. A large number of studies have been reported about the electrochemical detection of DNA damage by the enzymatic reaction [15,16]. Recently, our group had performed the electrochemical detection of in situ DNA damage induced by glucose oxidase-catalyzed Fenton reaction [17]. Some references have reported that xanthine oxidase (XOD) could catalyze the oxidation of xanthine (XA) under aerobic conditions, and the produced H2 O2 reacted with ferrous ions in a Fenton-type reaction to generate • OH [16,18]. In this report, a sensitive electrochemical biosensor constructed from dsDNA and a XOD modified glassy carbon electrode (DNA–XOD/GCE) has been developed to explore the rapid detection
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of DNA damage. Tris(2,2 -bipyridyl) cobalt(III) perchlorate (Co(bpy)3 (ClO4 )3 ) was used as a redox indicator to monitor DNA damage induced by • OH in aqueous solutions. The results demonstrate that the proposed electrochemical method is likely to find further application in DNA damage studies. 2. Experimental 2.1. Chemicals and reagents Calf thymus DNA was purchased from Sigma–Aldrich. Solutions of DNA were prepared by dissolving it in 0.01 M Tris–HCl/0.001 M ethylenediaminetetraacetic acid/0.05 M NaCl (pH 7.0) to form the 1.0 mg mL−1 stock solution and stored at 4 ◦ C. XOD (EC 1.1.3.4, Type X-S, from Aspergillus niger) was obtained from Sigma–Aldrich. Solutions of XOD were dissolved in 0.1 M phosphate buffer solution (PBS, pH 7.0) to form 3.0 mg mL−1 solutions. XA were purchased from Sigma–Aldrich. Co(bpy)3 (ClO4 )3 was prepared as described in the literature [19] and dissolved in 0.005 M Tris–HCl (pH 7.0) containing 0.05 M NaCl. Ascorbic acid (AA) was supplied by the Hubei University Chemical Factory (Hubei, China). Rutin and aloeemodin (AE) were purchased from Shanghai Boyun Biotech Co., Ltd. (Shanghai, China). All solutions were prepared with doubly distilled water. Other reagents were analytical grade. 2.2. Apparatus Electrochemical measurements were performed on a model CHI 625A electrochemical workstation (CH Instruments, Chenhua Co., Shanghai, China). A standard three-electrode system, consisting of a film modified glassy carbon electrode (GCE) as the working electrode, a saturated calomel electrode as the reference electrode, and platinum foil as the auxiliary electrode, was used in the measurements. UV–vis spectroscopy was carried out with a UV 2300 spectrophotometer (Shanghai Tian Mei Scientific Instrument Co., Ltd., China). Atomic force microscopy (AFM) images were obtained on a PicoScan system (Molecular Imaging Inc.) operated in contact mode with commercially ultrasharpened Si3 N4 tips (MAClever II, Molecular Imaging Inc.). The AFM measurement parameters
were as follows: force constant, 0.12 N/m; number of scans, 8 times.
2.3. Preparation of the DNA–XOD/GCE In our work, 1 mg mL−1 DNA and 3 mg mL−1 XOD with a volume ratio of 1:1 were mixed thoroughly, and 30 L of this mixture was directly applied to the surface of the clean GCE, followed by airdrying overnight. Then, the modified electrode was incubated in a pH 7.0 PBS for 4 h to remove any non-adsorbed DNA. This electrode was hereafter referred to as DNA–XOD/GCE. For comparison, DNA modified GCE (DNA/GCE) and XOD modified GCE (XOD/GCE) were also prepared using a similar direct application technique.
2.4. Procedures Scheme 1 shows the detection approach of DNA damage induced by XOD-catalyzed Fenton reaction in xanthine/FeSO4 system. DNA–XOD film was incubated in pH 3.0 buffer containing 1 mM FeSO4 and 1.7 mM XA, with stirring for the specified time for DNA damage (curve b). Control experiments were that DNA–XOD film was incubated in pH 3.0 buffer containing FeSO4 or XA separately, and DNA film or XOD film were incubated in pH 3.0 buffer containing FeSO4 or XA or FeSO4 /XA (curve a). To investigate the effects of antioxidants, aliquots of antioxidant samples were added into the above system. That is, DNA–XOD film was incubated with FeSO4 and XA in a pH 3.0 buffer in the presence of AA, AE, or rutin. All incubations were conducted at room temperature with stirring for 20 min. After incubation, the modified electrode was rinsed with doubly distilled water and then transferred into pH 7.0 Tris–HCl containing 200 M Co(bpy)3 3+ for square wave voltammetry (SWV). Each measurement was repeated at least 3 times. To correct the electrode-to-electrode or film-to-film variation for replicative experiments, the SWV oxidation peak current ratio (Ipt /Ip0 ), instead of the absolute peak current (Ipt ), was used to evaluate the effect of DNA damage, where Ipt and Ip0 are the peak currents after DNA–XOD film incubation with FeSO4 and XA in a pH 3.0 buffer for t and 0 min.
Scheme 1. Schematic diagram showing working principle of SWV detection of in situ DNA damage on DNA–XOD film.
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Fig. 1. UV absorption spectrum of (a) XOD–DNA film incubated with MV + PBS (pH 3.0), (b) MV + PBS (pH 3.0), (c) MV + Fe2+ + PBS (pH 3.0), (d) MV + XA + PBS (pH 3.0), (e) MV + Fe2+ + XA + PBS (pH 3.0), and (f) XOD–DNA film incubated with MV + Fe2+ + XA + PBS (pH 3.0).
3. Results and discussion 3.1. Verification of hydroxyl radicals in Fenton-type system Methyl violet (MV) was introduced to measure the amount of with UV–vis spectroscopy in the present work. In MV, the carbon–carbon double bonds with high electron cloud density are easily attacked by hydroxyl free radicals, with an electrophilic addition reaction taking place, leaving MV to fade [20]. As you can see in Fig. 1a–f, the difference in the absorption bands of MV appearing at about 580 nm was employed to determine the generation of hydroxyl radicals. The results suggested that • OH was generated by the XOD-catalyzed Fenton reaction in XA/FeSO4 system. • OH
3.2. Morphology characterization of the fabricated films Fig. 2 shows a set of representative AFM topographs of DNA and DNA/protein films immobilized on mica disks. Fig. 2A presents the AFM image of DNA membrane. Many small but dispersed evenly particles could be observed. It is known that different structures of DNA molecules can be found depending on solution conditions. Typically, DNA molecules condense into toroids, rod-like shapes, or globules [21]. In this study, DNA exhibited the globules conformation and distributed evenly. After the addition of XOD, the surface morphology changed dramatically, showing a lot of clusters or agglomerates (Fig. 2B). The particles in the image should be induced by the cross-linked XOD molecules. The crater and valley topographic features existed. The difference between DNA and DNA/XOD indicated that the deposited enzyme and DNA formed a uniform layer.
Fig. 2. AFM images of (A) DNA, (B) DNA–XOD on the mica disks.
3.3. Scheme of the DNA damage 3.3.1. Detection of DNA damage induced by XOD/XA/FeSO4 system Co(bpy)3 3+ is mainly bonded to the double helix of DNA by electrostatic interaction with the negatively charged phosphate backbone. It was used as an electroactive indicator, which bonds more to intact DNA than damaged DNA [12]. SWVs for DNA–XOD film in Co(bpy)3 3+ before and after incubations were shown in Scheme 1. The intact DNA–XOD film showed a good oxidative peak with peak potential of 0.064 V. After DNA–XOD film was incubated with FeSO4 and XA in a pH 3.0 buffer at room temperature with stirring for 20 min, the peak current of Co(bpy)3 3+ decreased obviously (Fig. 3a). DNA damage degree induced by XOD-catalyzed Fenton
Fig. 3. Ipt /Ip0 of 200 M Co(bpy)3 3+ for DNA–XOD film after incubation with pH 3.0 buffer containing: (a) 1.7 mM XA and 1 mM FeSO4 ; (b) XA; (c) FeSO4 . For DNA film after incubation with pH 3.0 buffer containing: (d) XA; (e) FeSO4 ; (f) XA and FeSO4 . For XOD film after incubation with pH 3.0 buffer containing: (g) XA; (h) FeSO4 ; (i) XA and FeSO4 .
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Fig. 4. EIS spectra of (a) bare GCE; DNA–XOD/GCE (b) before and (c) after incubation with pH 3.0 buffer containing 1.7 mM XA and 1 mM FeSO4 in 5 mmol/L [Fe(CN)6 ]3−/4− containing 0.1 mol/L KCl.
reaction in xanthine/FeSO4 system was more severely than the reported damage degrees generated by a glucose oxidase-catalyzed Fenton reaction in glucose/FeSO4 system [17], the Fenton reagents in ionic liquid system [14]. To further investigate the effects of various reagents on DNA damage, Ipt /Ip0 ratios for different films incubated in different types of solutions were compared. For the control incubation experiments, the electrochemical response did not show obvious change within the incubation time (Fig. 3b–i). These results confirmed that, for DNA–XOD film, XA and FeSO4 were necessary for DNA damage. The decrease of peak currents after the incubation with FeSO4 and XA in a pH 3.0 buffer suggested that the ability of the film to bond with the probe was gradually losing and DNA damage was becoming more severe [13]. The possible mechanism of DNA damage induced by the XOD/XA/FeSO4 system was that XOD could catalyze the oxidation of XA under aerobic conditions, and the produced H2 O2 reacted with ferrous ions in a Fenton-type reaction to generate • OH. The formed • OH disturbed DNA double helix structure or caused DNA strand break, thus leading to the decrease of Co(bpy)3 3+ oxidative peak current [22]. 3.3.2. Validation of DNA damage in Fenton-type system It is well known that electrochemical impedance spectroscopy (EIS) is a simple and effective method for affording information on the impedance changes on the electrode surface during the modification process [23]. Fig. 4 shows the results of EIS at a bare electrode (curve a), a DNA–XOD modified electrode before (curve b) and after (curve c) incubated with FeSO4 and XA in a pH 3.0 buffer, respectively, in the presence of 5 mmol/L [Fe(CN)6 ]3−/4− solution. It can be seen that the bare electrode exhibits an almost straight line, which is characteristic of a diffusion limiting step of the electrochemical process. A pronounced semicircle was obtained in Fig. 4b, indicating that DNA–XOD film hindered the charge transfer. After incubation treatment with FeSO4 and XA in a pH 3.0 buffer for 20 min, charge-transfer resistance was furthermore increased, which was reflected in the apparent increase in the semicircular part of the spectrum, indicating the evidently attenuation of DNA conductibility induced by hydroxyl radicals [17,24]. The possible reason for this phenomenon is that • OH induce oxidative DNA damage, producing a variety of modifications on the DNA level, including base and sugar lesions, strand breaks, DNA–protein cross-links, and base-free sites. Then the ability of transfer electron of DNA film decreased. To investigate the extent of DNA damage, a gel electrophoresis experiment was performed (Fig. 5). In the experiment, 0.3 mg mL−1
Fig. 5. Agarose gel electrophoresis of different types of DNA. Lane 2: 0.3 mg mL−1 ds-DNA reacted with XOD/1.7 mM XA/1 mM FeSO4 , pH 3.0 PBS. Lane 3: ds-DNA, pH 7.0 PBS. Lane 4: ds-DNA, pH 3.0 PBS. Lane 5: ds-DNA reacted with XA/FeSO4 , pH 3.0 PBS (no XOD). Lane 1: -Hind III digest DNA Marker. Lane 6: DL2000 DNA Marker.
of ds-DNA was reacted with XOD/1.7 mM XA/1 mM FeSO4 . After the reaction, the DNA sample was run on an agarose gel (Lane 2), together with the markers (Lanes 1 and 6) and the controlled DNA samples which were obtained by immersing in pH 7.0 buffer (Lane 3), pH 3.0 buffer (Lane 4), or reacting with XA/FeSO4 (no XOD, Lane 5). In Fig. 5, there was a major band (length about 23,130 base pairs) for the control (Lanes 3–5) that was absent in the images for Lane 2. Instead, many weak bands representing different DNA lengths appeared for the latter, illustrating extensive DNA breakage. The difference between the controls and sample 2 indicates that XOD was active at pH 3.0 and induced DNA damage in the presence of XA and FeSO4 . The similarity between the two DNA samples reacted at pH 3.0 and 7.0 suggests that DNA sample in pH 3.0 is suitable for investigating the degree of damage as in the neutral solution.
3.4. Selection of the optimum experimental conditions 3.4.1. The optimum pH During the experiment, it was found that after DNA–XOD sensor was incubated in a 1 mM Fe2+ solution alone (without XA) if pH of the solution was higher than 3.0, the peak current of Co(bpy)3 3+ decreased greatly. Since the modified electrode was rinsed with doubly distilled water after the incubation and then scanned, the effect might come from the metal ions adsorbed on the sensor surface during the incubation step. The detrimental effect was eliminated completely when the pH of Fe2+ solution was reduced to 3.0 or lower. Considering that XOD has low activity in acidic solutions and Fe2+ suppresses the peak current at high pH values, pH 3.0 Fe2+ /XA solution was selected. As we known, even at pH 3.0, the activity of XOD is high enough to generate sufficient H2 O2 for the DNA damage reaction [25]. It is well known that protonation of purine residues and subsequent unpairing of base pairs are found to commence at pH 3.0. It is noticed that the referred objects are DNA solutions. In our work, dehydrated DNA molecules could be irreversibly adsorbed on the surfaces of GCE. The DNA-adsorbed layer on GCE was stable to pH 3.0 buffer, which was confirmed by Pang’s work [26]. The relatively reasonable explanations for this are as follows. When DNA solution on GCEs is evaporated to dryness, the bases of DNA which have dehydrated are exposed, thus the hydrophobic bases are strongly adsorbed flat on the electrode surfaces [26]. Once it is adsorbed, DNA is difficult to re-hydrate and not so sensitive to pH of solution.
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3.5. Protection of DNA from damage by antioxidants 3.5.1. Evaluation of the hydroxyl radical scavenging activities of AA, AE, and rutin The influences of the antioxidants on the damage of DNA were investigated in the presence of AA, AE or rutin. In Fig. 7A(b–d), Ipt /Ip0 were shown when different antioxidants were injected into the OHgenerating system. A significant increase in the current level after antioxidant injection was observed. The increase of Ipt /Ip0 implied an effective protection effect of AA, AE and rutin. The presence of antioxidants recovered the electrochemical signal. We supposed that the release of the bases and an interruption of the phosphodiester bonds induced by radicals were resisted by those antioxidants [29]. 3.5.2. Influence of the AA, AE and rutin concentration In order to study the optimal concentration of antioxidants, different concentrations of AE, AA and rutin were added into the damage system (Fig. 7B). At low concentrations of AE, AA and rutin, Ipt /Ip0 increased with their concentrations and then reached the maximum. Then the marker signals decreased with the concentrations of antioxidants. The optimal concentrations of antioxidants for AE, AA and rutin were 3.3 M. As we know, those small molecules reacted rapidly with oxidant that made them good antioxidants. At the same time, they had a strong reducing power for metal ions. The antioxidant and oxidant activity of the three small molecules were correlative with its concentration. Low concentrations of antioxidants were excellent free radical scavengers, whereas high concentrations could promote Fenton-type reaction to generate OH• [30]. The nature of shown in the experiment was a comprehensive result of free radical scavenging ability and reducing capacity. When reducing capacity of AA was stronger than the capacity of scavenging free radicals, AA could promote the reaction of Fe2+ and H2 O2 to produce OH• , leading to DNA damage [31].
Fig. 6. (A) Influence of concentrations of XA on Ipt /Ip0 of 200 M Co(bpy)3 3+ for DNA–XOD film after incubation with pH 3.0 buffer containing 1.7 mM XA and 1 mM FeSO4 . (B) Effect of the incubation time on Ipt /Ip0 of 200 M Co(bpy)3 3+ for DNA–XOD film after incubation with pH 3.0 buffer containing (a) FeSO4 ; (b) XA; (c) XA and FeSO4 .
3.4.2. Optimum concentration of Fe2+ and XA In the above reactions, iron acts as a catalyst but excess Fe2+ might cause scavenger effects. Increasing Fe2+ concentration from 50 M to 1 mM resulted in the production of • OH. Excess • OH would tend to react with other • OH at higher Fe concentrations [27]. 1 mM FeSO4 was selected at our experiment (data not shown). The detection limit for the metal ion was less than 50 M. With 1 mM FeSO4 and varying concentrations of XA, Ipt /Ip0 decreased rapidly as the concentration of XA was increased from 0.68 to 1.7 mM and then tended toward a constant value (Fig. 6A), revealing a dependence of the damage reaction on the XA concentration. 1.7 mM XA was selected in this work. The detection limit for XA was less than 0.68 mM. 3.4.3. The optimum incubation time Fig. 6B presents the influence of incubation time on the DNA damage induced by the Fenton-type reaction. No obviously trends were observed for the control incubation experiments (curves a and b). After DNA–XOD film incubated in the pH 3.0 buffer containing FeSO4 and XA, Ipt /Ip0 decreased with the incubation time within 20 min and then reached a plateau (curve c). So, 20 min was selected as the optimum incubation time. The decrease trend was in accordance with the previous report [28].
3.5.3. Influence of incubation time Fig. 7C exhibits the influence of incubation time on Ipt /Ip0 in the presence of 3.3 M AA, AE, and rutin. When DNA film was incubated in pH 3.0 buffer containing FeSO4 and XA in the presence of antioxidants, Ipt /Ip0 decrease rapidly for the first 20 min and then decrease slowly (curves a–c). But the decreasing trend of Ipt /Ip0 was much slower compared with that of damage process, indicating the obvious protective effect of AA, AE, and rutin toward DNA damage. 3.6. Comparison of the enzyme catalyzed reactions • OH generated by a XOD-catalyzed hypoxanthine (HX) reaction was also studied by the electrochemical techniques. Experimental results revealed that this system was similar to the xanthine/FeSO4 system. HX can be transformed to XA, then to uric acid, and then to H2 O2 through XOD under aerobic conditions. The producing • OH can cause DNA damage but the damage degree was much small (data not shown).
3.7. Reproducibility and stability Stability and fabrication reproducibility are important parameters for the modified electrode. The studies on sensor reproducibility were as follows: DNA–XOD film was incubated with FeSO4 and XA in a pH 3.0 buffer for 20 min. After that, the modified electrode was taken out and rinsed with doubly distilled water and then transferred into Co(bpy)3 3+ for 10 successive scanning, the relative standard deviation (R.S.D.) was 3%. Fabrication reproducibility was estimated with five different electrodes. Five DNA–XOD modified electrodes were incubated with FeSO4 and XA in a pH 3.0 buffer for 20 min. After that, the damage films were taken out and rinsed
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with doubly distilled water and then transferred into Co(bpy)3 3+ for scanning, R.S.D. was 9%. Moreover, the responses decreased to 92% and 95% of the original current for the film after 1 week of use and 1 week stored without using, respectively. As a result,
DNA–XOD/GCE exhibited good reproducibility and comparative stability. 4. Conclusion This paper utilized electrode with adsorbed DNA and XOD to investigate DNA oxidative damage and the free radical scavenging ability of AA, AE, and rutin using Co(bpy)3 3+ as the electrochemical probe. The detection of DNA damage by • OH, which was generated by the XOD-catalyzed Fenton reaction in XA/FeSO4 system, was realized by electrochemical methods. Their antioxidative mechanisms of AA, AE, and rutin were investigated. They simultaneously exhibited excellent antioxidant and reducing capacities, and which one played a dominant role dependence on their concentrations. It offered a useful platform to detect DNA damage from metabolites. Future work would target the screening of genotoxic chemicals using the presented methodology. Acknowledgements This work was supported by the National Natural Science Foundation of China (Nos. 20875023 and 51173038), the Ph.D. Programs Foundation of the Ministry of Education of China (No. 20114208130001), the Natural Science Fund for Creative Research Groups of Hubei Province of China (No. 2011CDA111), and the Foundation for Innovative Research Groups of the Education Department of Hubei Province (No. T201101). References
Fig. 7. (A) Ipt /Ip0 of 200 M Co(bpy)3 3+ for DNA–XOD film after incubation with pH 3.0 buffer containing: (a) 1.7 mM XA and 1 mM FeSO4 ; (b) (a) + 3.3 M AA; (c) (a) + 3.3 M AE; (d) (a) + 3.3 M rutin. (B) Influence of concentrations of different antioxidants on Ipt /Ip0 of 200 M Co(bpy)3 3+ for DNA–XOD films after incubation with pH 3.0 buffer containing XA and FeSO4 and (a) AA, (b) AE, and (c) rutin for 30 min. (C) Effect of the incubation time on Ipt /Ip0 of 200 M Co(bpy)3 3+ for DNA–XOD film after incubation with XA and FeSO4 containing (a) 3.3 M AA; (b) 3.3 M AE; (c) 3.3 M rutin.
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Biographies Huayu Xiong is a laboratory assistant in the College of Chemistry and Chemical Engineering of Hubei University. She received her MSc degree in analytical chemistry in 2008 from Hubei University. Her research interest is in the development of electrochemical biosensors. Yang Chen is a post-graduate student in the department of chemistry and chemical engineering of Hubei University. Her current interest is in the development of biosensors based on free radical chemistry. Xiuhua Zhang is a professor in the College of Chemistry and Chemical Engineering of Hubei University. He received his MSc degree in analytical chemistry and his DrEng degree in materials science from Hubei University, in 2003 and 2008, respectively. His main current interest is in the development of sensors based on graphene and on conducting polymers. Haoshuang Gu is a professor in the College of Chemistry and Chemical Engineering of Hubei University. He received his PhD from Huazhong University of Science and Technology (China). His main current interests are in materials science and nanochemistry. Shengfu Wang is a professor in the College of Chemistry and Chemical Engineering of Hubei University. He received his MSc and PhD degrees in analytical chemistry from Wuhan University (China) in 1992 and 2005, respectively. His main current interests are in bioelectrochemistry, nanoelectrochemistry, chemically modified electrodes, chemical sensors, and biosensors.