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Electrochemical biosensors for the detection of oxidative DNA damage induced by Fenton reagents in ionic liquid
Sensors and Actuators B 161 (2012) 274–278
Contents lists available at SciVerse ScienceDirect
Sensors and Actuators B: Chemical journal homepage: www.elsevier.com/locate/snb
Electrochemical biosensors for the detection of oxidative DNA damage induced by Fenton reagents in ionic liquid Yan Wang a , Huayu Xiong a , Xiuhua Zhang a , Shengfu Wang a,b,∗ a 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 b Key Laboratory of Analytical Chemistry for Biology and Medicine (Wuhan University), Ministry of Education, Wuhan 430072, PR China
a r t i c l e
i n f o
Article history: Received 8 August 2011 Received in revised form 22 September 2011 Accepted 13 October 2011 Available online 20 October 2011 Keywords: DNA damage Room temperature ionic liquid Co(bpy)3 3+ Fenton reaction Hydroxyl radical
a b s t r a c t 1-Butyl-3-methylimidazolium hexafluorophosphate ([BMIM][PF6 ]), a widely used hydrophobic room temperature ionic liquid (RTIL), has been applied as nonaqueous solvent for monitoring DNA damage. The generated hydroxyl radical by Fenton reagents (Fe2+ and H2 O2 ) in RTIL, which was validated by electron spin resonance spectroscopy, could induce oxidative damage to DNA. An electroactive indicator Co(bpy)3 3+ , which bonded to the intact DNA much more than the damaged DNA, was employed in the detection of DNA damage by square wave voltammetry (SWV). The oxidative peak current of Co(bpy)3 3+ decreased with the time of DNA film incubation in Fenton reagents. To further understand the role of DNA-associated metal in the Fenton reaction, comparison experiments were performed, in which EDTA, as a chelator for metal ion, was used to rinse the electrode. The mechanism was that Fe2+ in RTIL firstly associated with DNA and then engaged in the Fenton reaction. The experimental results testified that ascorbic acid could inhibit oxidative DNA damage. The method was promising for rapid, sensitive, and inexpensive detection of DNA damage. © 2011 Elsevier B.V. All rights reserved.
1. Introduction DNA, as an important functional biomolecule, has attracted considerable attention. Detection of DNA damage is comparative importance because of its critical role in mutagenesis, carcinogenesis and aging. In living organisms, there are much endogenous and exogenous origin damage to DNA, such as reactive oxygen species (ROS) [1], ionizing radiation and chemicals [2]. If the damaged DNA cannot be repaired duly, the induced gene mutation will result in cancer and tumor in DNA replication process [3]. Moreover, millions of new chemicals are produced every year, it is necessary for screening new chemicals for potential gene toxicity before they are marketed. Therefore, there is an extraordinary need for rapid detection of DNA damage and screening for the toxicity of chemicals.
Abbreviations: RTIL, room temperature ionic liquid; [BMIM][PF6 ], 1-butyl-3methylimidazolium hexafluorophosphate; SWV, square wave voltammetry; ROS, reactive oxygen species; • OH, hydroxyl radical; GCE, glassy carbon electrode; ESR, electron spin resonance; SCE, saturated calomel electrode; DMPO, 5,5-Dimethyl-1pyrroline N-oxide; AA, ascorbic acid. ∗ Corresponding author at: 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. Tel.: +86 27 50865309; fax: +86 27 88663043. E-mail address: [email protected] (S. Wang). 0925-4005/$ – see front matter © 2011 Elsevier B.V. All rights reserved. doi:10.1016/j.snb.2011.10.030
Up to now, varieties of analytical methods have been developed to detect DNA damage, including capillary liquid chromatography–mass spectrometry/mass spectrometry [4], fluorescence [5], 32 P-postlabeling [6], capillary zone electrophoresis [7], and photoelectrochemical [8]. These methods are generally performed at centralized laboratories, requiring long assay time and high cost. Recently, electrochemical methods offer an attractive alternative approach for simple, inexpensive and sensitive detection of DNA damage [9,10]. Usually, two kinds of electrochemical methods are adapted to detect DNA damage, which are based on the direct oxidative signals of DNA bases or indirect electrochemical indicators [9,11]. Many electrochemical indicators have been used to detect DNA damage, such as methylene blue [12], tris(2,2 bipyridyl) complex [9] and tris(1,10 -phenanthroline) complex [13]. In our study, square wave voltammetry (SWV) coupled with tris(2,2 bipyridyl) cobalt(III) perchlorate (Co(bpy)3 (ClO4 )3 ) was utilized to detect DNA damage. The Co(bpy)3 3+ binding site size for ds-DNA and ss-DNA is 3 base pairs [9,14]. The number of binding sites for Co(bpy)3 3+ bound to ds-DNA films was nearly 5-fold greater than that bound to ss-DNA films of similar architecture [9]. This difference in number of binding sites provides a basis for detecting chemical DNA damage, which partially unravels the ds-DNA and decreases the ability to bind the probe. The reaction between ROS and DNA attracts considerable interests due to the pathophysiologic significance of oxidative DNA damage by ROS. Such damage is implicated to be important in
Y. Wang et al. / Sensors and Actuators B 161 (2012) 274–278
cancer and aging [15,16]. ROS such as superoxide radical, singlet oxygen and hydroxyl radical (• OH) are formed by cellular metabolism and oxidative stress in the cells. In particular, the extremely reactive ROS of oxidative DNA damage seems to be • OH, which can be generated by the Fenton reaction [17]. The Fenton reaction is the reaction of metal ion (Fe2+ , Cu+ ) with H2 O2 , forming high reactive • OH. • OH attacks DNA to cause several ways of damage, including strand breakage and base release [18]. Given that • OH is a kind of very unstable and short lifetime species [19], it causes great difficulties in the direct detection • OH and the investigation of • OH-induced DNA damage. As the novel green non-aqueous media, ionic liquids (ILs) attract much attention for their negligible vapor pressure, extraordinarily high chemical and thermal stability, high conductivity and large viscosity [20]. These advantages, especially their inert nature, allow ILs to be designed for specific reaction system including radical chemistry. Buzzeo et al. [21] have studied the electrochemical generation of stable superoxide radical in two kinds of ILs, 1-ethyl-3-methylimidazolium bis((trifluoromethyl)sulfonyl)imide and hexyltriethylammonium bis-((trifluoromethyl)sulfonyl)imide. The results suggested that ILs had provided appropriate environment for the investigation of the radical. Strehmel et al. [22] have validated that the degree of free radical polymerization increased with increasing viscosity of ILs. Although follow-up chemistry may not be the same in ILs, two common effects of ILs are evident. First, mass transport rates (or diffusion coefficients) are smaller in ILs because of the viscous nature of the media. Second, it appears that heterogeneous electron transfer rate constants (k0 ) (in imidazolium-based ILs at least) can be smaller for processes controlled by outer-sphere dynamics [23]. In this paper, DNA/GCE was fabricated by a simple adsorption method. The produced • OH by the Fenton reaction in 1-butyl3-methylimidazolium hexafluorophosphate ([BMIM][PF6 ]) could cause DNA oxidative damage. The damage was monitored by SWV using Co(bpy)3 3+ as an electroactive indicator. Some mechanisms of DNA damage were also discussed. The experimental results indicated that the proposed method was reliable for the detection of DNA damage. 2. Experimental 2.1. Reagents and materials Double stranded calf thymus DNA was obtained from Sigma (USA) and dealt with 10 mM Tris–HCl containing 1 mM EDTA and 50 mM NaCl (pH 7.0). tris(2,2 -bipyridyl) cobalt(III) perchlorate (Co(bpy)3 (ClO4 )3 ) was prepared as described in the literature [14] and dissolved in 5 mM Tris–HCl (pH 7.0) containing 50 mM NaCl. 5,5-Dimethyl-1-pyrroline N-oxide (DMPO) was obtained from Sigma (USA). 0.1 M, pH 7.0 phosphate buffer solution (PBS) was comprised with Na2 HPO4 and KH2 PO4 , which were from TianJin Bodi Chemical Holding Co., Ltd. (TianJin, China). All other chemicals were of reagent grade. Doubly distilled water was used for preparing the solutions. [BMIM][PF6 ] was prepared as described in the literatures [24]. The purity of IL was checked by elemental analysis and 1 H NMR and 13 C NMR spectroscopy, and the residual water content was analyzed by standard Karl–Fisher titration to be below 0.07% (w/w). 2.2. The preparation of DNA modified electrode The clean glassy carbon electrode (GCE) was coated with 30 l of 1 mg/mL DNA solution, followed by air-drying overnight. Then the modified electrode was soaked in water for 4 h to remove the unadsorbed DNA. DNA/GCE was obtained.
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2.3. Incubation of sensors with Fenton’s reagent and antioxidant in [BMIM][PF6 ] Scheme 1 shows the detection approach of DNA damage induced by hydroxyl radicals in IL. DNA film on GCE was incubated in 1 mL of [BMIM][PF6 ] containing 0.416 mM FeSO4 , 12.5 mM H2 O2 (the cleavage agent) for the specified time (curve b). For the control experiments, DNA film was incubated in 1 mL of [BMIM][PF6 ] containing 0.416 mM FeSO4 or 12.5 mM H2 O2 , or blank solution (curve a). The sample and control incubations took place at 37 ◦ C with stirring. After the incubation, the modified electrode was rinsed with water. Before and after incubations, DNA/GCE was immersed in 150 M Co(bpy)3 3+ , and then analyzed by SWV. Herein, to correct the electrode-to-electrode or film-to-film variation for replicate experiments, the relative peak current ratio Ipt /Ip0 instead of absolute peak current, where Ipt and Ip0 were the peak currents after DNA film incubation with Fenton reagents in [BMIM][PF6 ] for t and 0 min, was employed to estimate the degree of damage to DNA. Each measurement was repeated atleast 3 times. The concentrations of Fe2+ and H2 O2 on DNA damage were investigated. And the optimal concentrations of Fe2+ and H2 O2 were 0.416 mM and 12.5 mM, respectively (data not shown). 2.4. Electron spin resonance measurement The electron spin resonance (ESR) spectra were operated at 9.78 GHz with a 100 kHz modulation frequency. The experimental conditions for ESR spin tripping experiments were as follow: sweep width, 8.0 mT; microwave frequency, 9.78 GHz; microwave power, 12.9 mW; modulation amplitude, 0.102 mT; conversion time, 40.96 ms; time constant, 81.92 ms; and receive gain, 8 × 104 . The ESR samples were placed in a quartz flat cell. In the experiment, FeSO4 and DMPO were continuously mixed in [BMIM][PF6 ]. The spin trapping signal was detected after the addition of hydroxyl peroxide. 2.5. Apparatus All the electrochemical experiments were conducted on a CHI 660 electrochemical workstation (CH Inc., USA). A standard threeelectrode system was used in the measurement, with DNA/GCE as a working electrode, a platinum wire as an auxiliary electrode, and a saturated calomel electrode (SCE) as a reference electrode. All potentials given were referred to SCE. The ESR spectra were performed on a Bruker ESP-300 spectrometer. 3. Results and discussion 3.1. ESR detection of • OH ESR was applied to detect and identify the short-life free radicals. DMPO had been widely employed as a spin-trapping agent. It was able to identify oxygen-derived free radicals, such as superoxide and hydroxyl radical [25]. In this experiment, ESR technique was carried out to detect • OH with DMPO as a spin trap. As shown in Fig. 1, Fenton reagents in IL in the presence of DMPO yielded a typical 1:2:2:1 quartet of lines in ESR spectra corresponding to the signal DMPO • OH adduct. The reaction involved that Fe2+ broke the weak O–O bond of H2 O2 to produce • OH [26], and the generated • OH was trapped by DMPO. 3.2. Detection of DNA damage Co(bpy)3 3+ , which was bonded to the intact DNA much more than the damaged DNA because it bonded to DNA mainly by
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Scheme 1. Schematic diagram of DNA damage.
Fig. 1. ESR spectra were obtained with 0.416 mM FeSO4 , 12.5 mM H2 O2 and 83.8 mM DMPO in 1 mL [BMIM][PF6 ].
electrostatic interaction with the negatively charged phosphate backbone region of the double helix [9], was used as the electroactive indicator. SWVs for DNA film in 150 M Co(bpy)3 3+ before and after incubations were shown in Scheme 1. The intact DNA film showed a good oxidative peak with peak potential of 0.064 V. After DNA film was incubated in the cleavage agent for half an hour, the peak current decreased distinctly. Fig. 2a shows that Ipt /Ip0 decrease apparently in the cleavage solvent. For the
Fig. 2. SWV oxidative peak current ratio Ipt /Ip0 of 150 M Co(bpy)3 3+ for DNA film after and before incubation with: (a) [BMIM][PF6 ] containing 0.416 mM FeSO4 , 12.5 mM H2 O2 , (b) [BMIM][PF6 ], (c) [BMIM][PF6 ] containing 0.416 mM FeSO4 , (d) [BMIM][PF6 ] containing 12.5 mM H2 O2 , and (e) PBS containing 0.416 mM FeSO4 , 12.5 mM H2 O2 .
control incubation experiments, the electrochemical responses did not show obvious change (Fig. 2 b–d). The decrease of peak currents after the incubation in the cleavage agent 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 Fenton reagents/IL system was that the formed • OH by Fenton reagents in [BMIM][PF6 ] disturbed DNA double helix structure or caused DNA strand break, thus leading to the decrease of Co(bpy)3 3+ oxidative peak current [27]. IL was expected to be suitable medium for free radicals. In order to enhance contrast between the two medias, DNA film incubation in PBS containing 0.416 mM FeSO4 , 12.5 mM H2 O2 was tested (Fig. 2e). After DNA film was incubated in PBS containing Fenton reagents, the peak current decreased less distinctly than in [BMIM][PF6 ]. It could be speculated that [BMIM][PF6 ] would provide appropriate environment for the investigation of DNA damage. It was thought the radical traveled slow because of the large viscosity of IL [23]. This might reduce the chance of interactive collision among the radicals, which led to prolong the lifetime of the radical. 3.3. Effect of Fe2+ on DNA damage The role of DNA-associated metals in the Fenton reaction was investigated. In the experiments, DNA sensor was firstly incubated in 1 mL of [BMIM][PF6 ] containing 0.416 mM FeSO4 for 10 min. After being rinsed with water, the sensor exposed to 12.5 mM H2 O2 in [BMIM][PF6 ] for 30 min. Then the film was placed into Co(bpy)3 3+ for SWV scans. Fig. 3a exhibits that the decrease in the electrochemical response of the indicator is close to the decrease corresponded to Fig. 2a. It indicated that Fe2+ bonded with the sensor film and participated in the Fenton reaction during the subsequent exposure to H2 O2 . The influence of EDTA, as a very strong chelator for metal ion, on the Fenton-induced DNA damage was studied. After its exposure to Fe2+ , the sensor was rinsed with 10 mM EDTA instead of water, followed by the same treatment with H2 O2 and Co(bpy)3 3+ . Fig. 3b shows that there was no evident decrease of the peak current. It could conclude that Fe2+ and EDTA formed a chelate, which removed the metal ion from DNA film, and thus eliminated the subsequent Fenton reaction with H2 O2 . The experimental results were in accordance with the previous report [8]. Therefore, it was deduced that Fe2+ in RTIL firstly associated with DNA and then engaged in the Fenton reaction. The generation of • OH close to DNA could attack both the DNA backbones and DNA bases to produce strand breaks or damaged based.
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of peak current was half of that in Fig. 3a. Control experiments were that DNA film incubated in [BMIM][PF6 ] containing 0.416 mM FeSO4 and 7.5 M AA or containing 12.5 mM H2 O2 and 7.5 M AA. As shown in Fig. 3d and e, no significant decrease of peak current was observed. It indicated that AA can protect DNA from oxidation efficiently. 3.5. Influence of incubation time
Fig. 3. Dependence of SWV oxidative peak current ratio Ipt /Ip0 in 150 M Co(bpy)3 3+ for DNA film after and before incubation with 1 mL of [BMIM][PF6 ] containing: (a) 0.416 mM FeSO4 for 10 min, water rinse, dry in air, and then 12.5 mM H2 O2 for 30 min, (b) 0.416 mM FeSO4 for 10 min, 10 mM EDTA rinse, dry in air, and then 12.5 mM H2 O2 for 30 min, (c) 0.416 mM FeSO4 , 12.5 mM H2 O2 and 7.5 M AA, (d) 0.416 mM FeSO4 and 7.5 M AA, and (e) 12.5 mM H2 O2 and 7.5 M AA.
The incubation time had obvious effect on the DNA damage induced by hydroxyl free radicals. The peak height decreased with incubation time at relatively larger rates for the first 5 min then at lower rates at longer time (Fig. 4A). The decrease trend was in accordance with the previous report [29]. Fig. 4B exhibits the effect of incubation time on Ipt /Ip0 in the absence and presence of 7.5 M AA. With the addition of AA, the decrease trend of Ipt /Ip0 was much slower (curve b), indicating the obvious protective effect of AA for DNA damage.
3.4. Influence of AA on DNA damage
4. Conclusion
As a strong antioxidant, ascorbic acid (AA) could scavenge various free radical, including • OH [28]. Fig. 3c shows Ipt /Ip0 of DNA film in the presence of 7.5 M AA. It was obvious that the decrease
The sensitive detection of dsDNA damage induced by free radical in the Fenton reagents/RTIL system was realized. [BMIM][PF6 ] provided appropriate environment for the generation of • OH and the investigation of DNA damage. Co(bpy)3 3+ as the electroactive indicator could be applied for the voltammetric detection of DNA damage. Making use of the surface reaction mode, Fe2+ bonded with DNA film on the sensors and took part in the following Fenton reaction with H2 O2 . It offered a useful platform to monitor DNA damage from metabolites. Future work would target to screen of genotoxic chemicals using the presented methodology.
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Acknowledgements This work was supported by the National Natural Science Foundation of China (No. 20875023), the Foundation of the Key Laboratory of Analytical Chemistry for Biology and Medicine (Wuhan University) and Ministry of Education (No. ACBM2010001).
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T/min Fig. 4. (A) SWV oxidative peak currents in 150 M Co(bpy)3 3+ for DNA film after incubation with [BMIM][PF6 ] containing 0.416 mM FeSO4 , 12.5 mM H2 O2 at different times. (B) Dependence of SWV oxidative peak current ratio Ipt /Ip0 of 150 M Co(bpy)3 3+ on incubation time for DNA film after and before incubated with 1 mL of [BMIM][PF6 ] containing (a) 0.416 mM FeSO4 , 12.5 mM H2 O2 ; (b) 0.416 mM FeSO4 , 12.5 mM H2 O2 and 7.5 M AA.
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Biographies Yan Wang is a graduate student in the Department of Chemistry and Chemical Engineering of Hubei University, PR China. She received BA degree in chemistry from Hubei University in 2007. Her current interest is in the development of biosensors based on bioelectrochemistry and room temperature ionic liquids. Huayu Xiong is a laboratory assistant in the College of Chemistry and Chemical Engineering of Hubei University. She received MSc degree in analytical chemistry in 2008 from Hubei University. Her research interest is in the development of electrochemical nonaqueous biosensors. Xiuhua Zhang is a professor in the College of Chemistry and Chemical Engineering of Hubei University. He received MSc degree in analytical chemistry and Dr. Eng. degree in materials science from Hubei University, in 2003 and 2008, respectively. His main current interest is in the development of sensors based on carbon nanotubes and on conducting polymers. Shengfu Wang is a professor in the College of Chemistry and Chemical Engineering of Hubei University. He received MSc and PhD in analytical chemistry from Wuhan University in 1992 and 2005, respectively. His main current interests are in bioelectrochemistry, nanoelectrochemistry, chemically modified electrodes, chemical sensors and biosensors.
Contents lists available at SciVerse ScienceDirect
Sensors and Actuators B: Chemical journal homepage: www.elsevier.com/locate/snb
Electrochemical biosensors for the detection of oxidative DNA damage induced by Fenton reagents in ionic liquid Yan Wang a , Huayu Xiong a , Xiuhua Zhang a , Shengfu Wang a,b,∗ a 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 b Key Laboratory of Analytical Chemistry for Biology and Medicine (Wuhan University), Ministry of Education, Wuhan 430072, PR China
a r t i c l e
i n f o
Article history: Received 8 August 2011 Received in revised form 22 September 2011 Accepted 13 October 2011 Available online 20 October 2011 Keywords: DNA damage Room temperature ionic liquid Co(bpy)3 3+ Fenton reaction Hydroxyl radical
a b s t r a c t 1-Butyl-3-methylimidazolium hexafluorophosphate ([BMIM][PF6 ]), a widely used hydrophobic room temperature ionic liquid (RTIL), has been applied as nonaqueous solvent for monitoring DNA damage. The generated hydroxyl radical by Fenton reagents (Fe2+ and H2 O2 ) in RTIL, which was validated by electron spin resonance spectroscopy, could induce oxidative damage to DNA. An electroactive indicator Co(bpy)3 3+ , which bonded to the intact DNA much more than the damaged DNA, was employed in the detection of DNA damage by square wave voltammetry (SWV). The oxidative peak current of Co(bpy)3 3+ decreased with the time of DNA film incubation in Fenton reagents. To further understand the role of DNA-associated metal in the Fenton reaction, comparison experiments were performed, in which EDTA, as a chelator for metal ion, was used to rinse the electrode. The mechanism was that Fe2+ in RTIL firstly associated with DNA and then engaged in the Fenton reaction. The experimental results testified that ascorbic acid could inhibit oxidative DNA damage. The method was promising for rapid, sensitive, and inexpensive detection of DNA damage. © 2011 Elsevier B.V. All rights reserved.
1. Introduction DNA, as an important functional biomolecule, has attracted considerable attention. Detection of DNA damage is comparative importance because of its critical role in mutagenesis, carcinogenesis and aging. In living organisms, there are much endogenous and exogenous origin damage to DNA, such as reactive oxygen species (ROS) [1], ionizing radiation and chemicals [2]. If the damaged DNA cannot be repaired duly, the induced gene mutation will result in cancer and tumor in DNA replication process [3]. Moreover, millions of new chemicals are produced every year, it is necessary for screening new chemicals for potential gene toxicity before they are marketed. Therefore, there is an extraordinary need for rapid detection of DNA damage and screening for the toxicity of chemicals.
Abbreviations: RTIL, room temperature ionic liquid; [BMIM][PF6 ], 1-butyl-3methylimidazolium hexafluorophosphate; SWV, square wave voltammetry; ROS, reactive oxygen species; • OH, hydroxyl radical; GCE, glassy carbon electrode; ESR, electron spin resonance; SCE, saturated calomel electrode; DMPO, 5,5-Dimethyl-1pyrroline N-oxide; AA, ascorbic acid. ∗ Corresponding author at: 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. Tel.: +86 27 50865309; fax: +86 27 88663043. E-mail address: [email protected] (S. Wang). 0925-4005/$ – see front matter © 2011 Elsevier B.V. All rights reserved. doi:10.1016/j.snb.2011.10.030
Up to now, varieties of analytical methods have been developed to detect DNA damage, including capillary liquid chromatography–mass spectrometry/mass spectrometry [4], fluorescence [5], 32 P-postlabeling [6], capillary zone electrophoresis [7], and photoelectrochemical [8]. These methods are generally performed at centralized laboratories, requiring long assay time and high cost. Recently, electrochemical methods offer an attractive alternative approach for simple, inexpensive and sensitive detection of DNA damage [9,10]. Usually, two kinds of electrochemical methods are adapted to detect DNA damage, which are based on the direct oxidative signals of DNA bases or indirect electrochemical indicators [9,11]. Many electrochemical indicators have been used to detect DNA damage, such as methylene blue [12], tris(2,2 bipyridyl) complex [9] and tris(1,10 -phenanthroline) complex [13]. In our study, square wave voltammetry (SWV) coupled with tris(2,2 bipyridyl) cobalt(III) perchlorate (Co(bpy)3 (ClO4 )3 ) was utilized to detect DNA damage. The Co(bpy)3 3+ binding site size for ds-DNA and ss-DNA is 3 base pairs [9,14]. The number of binding sites for Co(bpy)3 3+ bound to ds-DNA films was nearly 5-fold greater than that bound to ss-DNA films of similar architecture [9]. This difference in number of binding sites provides a basis for detecting chemical DNA damage, which partially unravels the ds-DNA and decreases the ability to bind the probe. The reaction between ROS and DNA attracts considerable interests due to the pathophysiologic significance of oxidative DNA damage by ROS. Such damage is implicated to be important in
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cancer and aging [15,16]. ROS such as superoxide radical, singlet oxygen and hydroxyl radical (• OH) are formed by cellular metabolism and oxidative stress in the cells. In particular, the extremely reactive ROS of oxidative DNA damage seems to be • OH, which can be generated by the Fenton reaction [17]. The Fenton reaction is the reaction of metal ion (Fe2+ , Cu+ ) with H2 O2 , forming high reactive • OH. • OH attacks DNA to cause several ways of damage, including strand breakage and base release [18]. Given that • OH is a kind of very unstable and short lifetime species [19], it causes great difficulties in the direct detection • OH and the investigation of • OH-induced DNA damage. As the novel green non-aqueous media, ionic liquids (ILs) attract much attention for their negligible vapor pressure, extraordinarily high chemical and thermal stability, high conductivity and large viscosity [20]. These advantages, especially their inert nature, allow ILs to be designed for specific reaction system including radical chemistry. Buzzeo et al. [21] have studied the electrochemical generation of stable superoxide radical in two kinds of ILs, 1-ethyl-3-methylimidazolium bis((trifluoromethyl)sulfonyl)imide and hexyltriethylammonium bis-((trifluoromethyl)sulfonyl)imide. The results suggested that ILs had provided appropriate environment for the investigation of the radical. Strehmel et al. [22] have validated that the degree of free radical polymerization increased with increasing viscosity of ILs. Although follow-up chemistry may not be the same in ILs, two common effects of ILs are evident. First, mass transport rates (or diffusion coefficients) are smaller in ILs because of the viscous nature of the media. Second, it appears that heterogeneous electron transfer rate constants (k0 ) (in imidazolium-based ILs at least) can be smaller for processes controlled by outer-sphere dynamics [23]. In this paper, DNA/GCE was fabricated by a simple adsorption method. The produced • OH by the Fenton reaction in 1-butyl3-methylimidazolium hexafluorophosphate ([BMIM][PF6 ]) could cause DNA oxidative damage. The damage was monitored by SWV using Co(bpy)3 3+ as an electroactive indicator. Some mechanisms of DNA damage were also discussed. The experimental results indicated that the proposed method was reliable for the detection of DNA damage. 2. Experimental 2.1. Reagents and materials Double stranded calf thymus DNA was obtained from Sigma (USA) and dealt with 10 mM Tris–HCl containing 1 mM EDTA and 50 mM NaCl (pH 7.0). tris(2,2 -bipyridyl) cobalt(III) perchlorate (Co(bpy)3 (ClO4 )3 ) was prepared as described in the literature [14] and dissolved in 5 mM Tris–HCl (pH 7.0) containing 50 mM NaCl. 5,5-Dimethyl-1-pyrroline N-oxide (DMPO) was obtained from Sigma (USA). 0.1 M, pH 7.0 phosphate buffer solution (PBS) was comprised with Na2 HPO4 and KH2 PO4 , which were from TianJin Bodi Chemical Holding Co., Ltd. (TianJin, China). All other chemicals were of reagent grade. Doubly distilled water was used for preparing the solutions. [BMIM][PF6 ] was prepared as described in the literatures [24]. The purity of IL was checked by elemental analysis and 1 H NMR and 13 C NMR spectroscopy, and the residual water content was analyzed by standard Karl–Fisher titration to be below 0.07% (w/w). 2.2. The preparation of DNA modified electrode The clean glassy carbon electrode (GCE) was coated with 30 l of 1 mg/mL DNA solution, followed by air-drying overnight. Then the modified electrode was soaked in water for 4 h to remove the unadsorbed DNA. DNA/GCE was obtained.
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2.3. Incubation of sensors with Fenton’s reagent and antioxidant in [BMIM][PF6 ] Scheme 1 shows the detection approach of DNA damage induced by hydroxyl radicals in IL. DNA film on GCE was incubated in 1 mL of [BMIM][PF6 ] containing 0.416 mM FeSO4 , 12.5 mM H2 O2 (the cleavage agent) for the specified time (curve b). For the control experiments, DNA film was incubated in 1 mL of [BMIM][PF6 ] containing 0.416 mM FeSO4 or 12.5 mM H2 O2 , or blank solution (curve a). The sample and control incubations took place at 37 ◦ C with stirring. After the incubation, the modified electrode was rinsed with water. Before and after incubations, DNA/GCE was immersed in 150 M Co(bpy)3 3+ , and then analyzed by SWV. Herein, to correct the electrode-to-electrode or film-to-film variation for replicate experiments, the relative peak current ratio Ipt /Ip0 instead of absolute peak current, where Ipt and Ip0 were the peak currents after DNA film incubation with Fenton reagents in [BMIM][PF6 ] for t and 0 min, was employed to estimate the degree of damage to DNA. Each measurement was repeated atleast 3 times. The concentrations of Fe2+ and H2 O2 on DNA damage were investigated. And the optimal concentrations of Fe2+ and H2 O2 were 0.416 mM and 12.5 mM, respectively (data not shown). 2.4. Electron spin resonance measurement The electron spin resonance (ESR) spectra were operated at 9.78 GHz with a 100 kHz modulation frequency. The experimental conditions for ESR spin tripping experiments were as follow: sweep width, 8.0 mT; microwave frequency, 9.78 GHz; microwave power, 12.9 mW; modulation amplitude, 0.102 mT; conversion time, 40.96 ms; time constant, 81.92 ms; and receive gain, 8 × 104 . The ESR samples were placed in a quartz flat cell. In the experiment, FeSO4 and DMPO were continuously mixed in [BMIM][PF6 ]. The spin trapping signal was detected after the addition of hydroxyl peroxide. 2.5. Apparatus All the electrochemical experiments were conducted on a CHI 660 electrochemical workstation (CH Inc., USA). A standard threeelectrode system was used in the measurement, with DNA/GCE as a working electrode, a platinum wire as an auxiliary electrode, and a saturated calomel electrode (SCE) as a reference electrode. All potentials given were referred to SCE. The ESR spectra were performed on a Bruker ESP-300 spectrometer. 3. Results and discussion 3.1. ESR detection of • OH ESR was applied to detect and identify the short-life free radicals. DMPO had been widely employed as a spin-trapping agent. It was able to identify oxygen-derived free radicals, such as superoxide and hydroxyl radical [25]. In this experiment, ESR technique was carried out to detect • OH with DMPO as a spin trap. As shown in Fig. 1, Fenton reagents in IL in the presence of DMPO yielded a typical 1:2:2:1 quartet of lines in ESR spectra corresponding to the signal DMPO • OH adduct. The reaction involved that Fe2+ broke the weak O–O bond of H2 O2 to produce • OH [26], and the generated • OH was trapped by DMPO. 3.2. Detection of DNA damage Co(bpy)3 3+ , which was bonded to the intact DNA much more than the damaged DNA because it bonded to DNA mainly by
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Scheme 1. Schematic diagram of DNA damage.
Fig. 1. ESR spectra were obtained with 0.416 mM FeSO4 , 12.5 mM H2 O2 and 83.8 mM DMPO in 1 mL [BMIM][PF6 ].
electrostatic interaction with the negatively charged phosphate backbone region of the double helix [9], was used as the electroactive indicator. SWVs for DNA film in 150 M Co(bpy)3 3+ before and after incubations were shown in Scheme 1. The intact DNA film showed a good oxidative peak with peak potential of 0.064 V. After DNA film was incubated in the cleavage agent for half an hour, the peak current decreased distinctly. Fig. 2a shows that Ipt /Ip0 decrease apparently in the cleavage solvent. For the
Fig. 2. SWV oxidative peak current ratio Ipt /Ip0 of 150 M Co(bpy)3 3+ for DNA film after and before incubation with: (a) [BMIM][PF6 ] containing 0.416 mM FeSO4 , 12.5 mM H2 O2 , (b) [BMIM][PF6 ], (c) [BMIM][PF6 ] containing 0.416 mM FeSO4 , (d) [BMIM][PF6 ] containing 12.5 mM H2 O2 , and (e) PBS containing 0.416 mM FeSO4 , 12.5 mM H2 O2 .
control incubation experiments, the electrochemical responses did not show obvious change (Fig. 2 b–d). The decrease of peak currents after the incubation in the cleavage agent 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 Fenton reagents/IL system was that the formed • OH by Fenton reagents in [BMIM][PF6 ] disturbed DNA double helix structure or caused DNA strand break, thus leading to the decrease of Co(bpy)3 3+ oxidative peak current [27]. IL was expected to be suitable medium for free radicals. In order to enhance contrast between the two medias, DNA film incubation in PBS containing 0.416 mM FeSO4 , 12.5 mM H2 O2 was tested (Fig. 2e). After DNA film was incubated in PBS containing Fenton reagents, the peak current decreased less distinctly than in [BMIM][PF6 ]. It could be speculated that [BMIM][PF6 ] would provide appropriate environment for the investigation of DNA damage. It was thought the radical traveled slow because of the large viscosity of IL [23]. This might reduce the chance of interactive collision among the radicals, which led to prolong the lifetime of the radical. 3.3. Effect of Fe2+ on DNA damage The role of DNA-associated metals in the Fenton reaction was investigated. In the experiments, DNA sensor was firstly incubated in 1 mL of [BMIM][PF6 ] containing 0.416 mM FeSO4 for 10 min. After being rinsed with water, the sensor exposed to 12.5 mM H2 O2 in [BMIM][PF6 ] for 30 min. Then the film was placed into Co(bpy)3 3+ for SWV scans. Fig. 3a exhibits that the decrease in the electrochemical response of the indicator is close to the decrease corresponded to Fig. 2a. It indicated that Fe2+ bonded with the sensor film and participated in the Fenton reaction during the subsequent exposure to H2 O2 . The influence of EDTA, as a very strong chelator for metal ion, on the Fenton-induced DNA damage was studied. After its exposure to Fe2+ , the sensor was rinsed with 10 mM EDTA instead of water, followed by the same treatment with H2 O2 and Co(bpy)3 3+ . Fig. 3b shows that there was no evident decrease of the peak current. It could conclude that Fe2+ and EDTA formed a chelate, which removed the metal ion from DNA film, and thus eliminated the subsequent Fenton reaction with H2 O2 . The experimental results were in accordance with the previous report [8]. Therefore, it was deduced that Fe2+ in RTIL firstly associated with DNA and then engaged in the Fenton reaction. The generation of • OH close to DNA could attack both the DNA backbones and DNA bases to produce strand breaks or damaged based.
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of peak current was half of that in Fig. 3a. Control experiments were that DNA film incubated in [BMIM][PF6 ] containing 0.416 mM FeSO4 and 7.5 M AA or containing 12.5 mM H2 O2 and 7.5 M AA. As shown in Fig. 3d and e, no significant decrease of peak current was observed. It indicated that AA can protect DNA from oxidation efficiently. 3.5. Influence of incubation time
Fig. 3. Dependence of SWV oxidative peak current ratio Ipt /Ip0 in 150 M Co(bpy)3 3+ for DNA film after and before incubation with 1 mL of [BMIM][PF6 ] containing: (a) 0.416 mM FeSO4 for 10 min, water rinse, dry in air, and then 12.5 mM H2 O2 for 30 min, (b) 0.416 mM FeSO4 for 10 min, 10 mM EDTA rinse, dry in air, and then 12.5 mM H2 O2 for 30 min, (c) 0.416 mM FeSO4 , 12.5 mM H2 O2 and 7.5 M AA, (d) 0.416 mM FeSO4 and 7.5 M AA, and (e) 12.5 mM H2 O2 and 7.5 M AA.
The incubation time had obvious effect on the DNA damage induced by hydroxyl free radicals. The peak height decreased with incubation time at relatively larger rates for the first 5 min then at lower rates at longer time (Fig. 4A). The decrease trend was in accordance with the previous report [29]. Fig. 4B exhibits the effect of incubation time on Ipt /Ip0 in the absence and presence of 7.5 M AA. With the addition of AA, the decrease trend of Ipt /Ip0 was much slower (curve b), indicating the obvious protective effect of AA for DNA damage.
3.4. Influence of AA on DNA damage
4. Conclusion
As a strong antioxidant, ascorbic acid (AA) could scavenge various free radical, including • OH [28]. Fig. 3c shows Ipt /Ip0 of DNA film in the presence of 7.5 M AA. It was obvious that the decrease
The sensitive detection of dsDNA damage induced by free radical in the Fenton reagents/RTIL system was realized. [BMIM][PF6 ] provided appropriate environment for the generation of • OH and the investigation of DNA damage. Co(bpy)3 3+ as the electroactive indicator could be applied for the voltammetric detection of DNA damage. Making use of the surface reaction mode, Fe2+ bonded with DNA film on the sensors and took part in the following Fenton reaction with H2 O2 . It offered a useful platform to monitor DNA damage from metabolites. Future work would target to screen of genotoxic chemicals using the presented methodology.
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Acknowledgements This work was supported by the National Natural Science Foundation of China (No. 20875023), the Foundation of the Key Laboratory of Analytical Chemistry for Biology and Medicine (Wuhan University) and Ministry of Education (No. ACBM2010001).
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T/min Fig. 4. (A) SWV oxidative peak currents in 150 M Co(bpy)3 3+ for DNA film after incubation with [BMIM][PF6 ] containing 0.416 mM FeSO4 , 12.5 mM H2 O2 at different times. (B) Dependence of SWV oxidative peak current ratio Ipt /Ip0 of 150 M Co(bpy)3 3+ on incubation time for DNA film after and before incubated with 1 mL of [BMIM][PF6 ] containing (a) 0.416 mM FeSO4 , 12.5 mM H2 O2 ; (b) 0.416 mM FeSO4 , 12.5 mM H2 O2 and 7.5 M AA.
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Biographies Yan Wang is a graduate student in the Department of Chemistry and Chemical Engineering of Hubei University, PR China. She received BA degree in chemistry from Hubei University in 2007. Her current interest is in the development of biosensors based on bioelectrochemistry and room temperature ionic liquids. Huayu Xiong is a laboratory assistant in the College of Chemistry and Chemical Engineering of Hubei University. She received MSc degree in analytical chemistry in 2008 from Hubei University. Her research interest is in the development of electrochemical nonaqueous biosensors. Xiuhua Zhang is a professor in the College of Chemistry and Chemical Engineering of Hubei University. He received MSc degree in analytical chemistry and Dr. Eng. degree in materials science from Hubei University, in 2003 and 2008, respectively. His main current interest is in the development of sensors based on carbon nanotubes and on conducting polymers. Shengfu Wang is a professor in the College of Chemistry and Chemical Engineering of Hubei University. He received MSc and PhD in analytical chemistry from Wuhan University in 1992 and 2005, respectively. His main current interests are in bioelectrochemistry, nanoelectrochemistry, chemically modified electrodes, chemical sensors and biosensors.