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Simultaneous determination of adenine and guanine utilizing PbO2-carbon nanotubes-ionic liquid composite film modified glassy carbon electrode
Journal of Electroanalytical Chemistry 651 (2011) 216–221
Contents lists available at ScienceDirect
Journal of Electroanalytical Chemistry journal homepage: www.elsevier.com/locate/jelechem
Simultaneous determination of adenine and guanine utilizing PbO2-carbon nanotubes-ionic liquid composite film modified glassy carbon electrode Tao Liu a, Xiangbin Zhu b, Lin Cui a, Peng Ju a, Xiangjin Qu a,⇑, Shiyun Ai a,⇑ a b
College of Chemistry and Material Science, Shandong Agricultural University, Taian, Shandong 271018, China College of Resources and Environment, Shandong Agricultural University, Taian, Shandong 271018, China
a r t i c l e
i n f o
Article history: Received 15 June 2010 Received in revised form 5 September 2010 Accepted 21 November 2010 Available online 27 November 2010 Keywords: Carbon nanotubes Room temperature ionic liquid Nano-PbO2 Guanine Adenine Herring sperm DNA
a b s t r a c t A novel and reliable electrochemical sensor based on PbO2-carbon nanotubes-room temperature ionic liquid (i.e., 1-butyl-3-methylimidazolium hexafluorophosphate, BMIMPF6) composite film modified glassy carbon electrode (GCE) (PbO2–MWNT–RTIL/GCE) was proposed for simultaneous and individual determination of guanine and adenine. The guanine and adenine oxidation responses were monitored by differential pulse voltammetric (DPV) measurement. Compared with the bare electrode, the PbO2– MWNT–RTIL/GCE not only significantly enhanced the oxidation peak currents of guanine and adenine, but also lowered their oxidation overpotentials, suggesting that the synergistic effect of PbO2, MWNT and RTIL could dramatically improve the determining sensitivity of guanine and adenine. The PbO2– MWNT–RTIL/GCE showed good stability, high accumulation efficiency and enhanced electrocatalytic ability for the detection of guanine and adenine. Besides, the modified electrode also exhibited good behaviors in the simultaneous detection of adenine and guanine with the peak separation of 0.29 V in 0.1 M pH 7.0 phosphate buffer solution (PBS). Under the optimal conditions, the detection limit for individual determination of guanine and adenine was 6.0 10 9 M and 3.0 10 8 M (S/N = 3), respectively. The proposed method for the measurements of guanine and adenine in herring sperm DNA was successfully applied with satisfactory results. Ó 2010 Elsevier B.V. All rights reserved.
1. Introduction Deoxyribonucleic acid (DNA) is an important substance which plays a significant role in biological heritage information storage and in protein biosynthesis. Guanine (G) and adenine (A) are two important purine bases existed in DNA, which participate in processes as distinct as energy transduction, metabolic cofactors and cell signaling. The abnormal changes of guanine and adenine in DNA have been suggested to be related to the deficiency and mutation of the immunity system. And the concentration changes of guanine and adenine in DNA could be considered as an indication of various diseases such as carcinoma or liver diseases [1]. Hence, it is of great significance to establish sensitive methods for the analysis of these bases in DNA [2]. So far, a variety of methods have been established to investigate the concentration of the adenine and guanine, such as chemiluminescence, micellar electrokinetic chromatography with indirect laser-induced fluorescence detection, and electrophoresis coupled with electrochemical determination [3–6]. Although these techniques show high sensitivity and selectivity, it must be pointed ⇑ Corresponding authors. Tel.: +86 538 8247660; fax: +86 538 8242251. E-mail addresses: [email protected] (X. Qu), [email protected] (S. Ai). 1572-6657/$ - see front matter Ó 2010 Elsevier B.V. All rights reserved. doi:10.1016/j.jelechem.2010.11.026
out that they have severe setbacks for purines determination, such as time-consuming and expensive. Therefore, they are unsuitable for on-line or field monitoring. Owing to their high sensitivity, simple operation, fast response speed, low cost and real-time detection in situ condition, various kinds of chemically modified electrodes have been prepared to investigate the electrochemical oxidation of adenine and guanine [7]. Such as cobalt (II) phthalocyanine modified carbon paste electrode [8], b-cyclodextrin incorporated carbon nanotubes-modified electrode [9], and carbon ionic liquid electrode [10]. Although all the above modified materials show good improving oxidation signals, it is still a challenge to develop new and economical methods with high sensitivity and convenience for the determination of guanine and adenine. Carbon nanotubes (CNTs) have attracted a great deal of interest due to their unique properties such as fast electron transfer ability and catalysis effect. Wang et al. described a CNTs modified GCE, which improved the detection of DNA hybridization and greatly enhanced the guanine oxidation signal [11]. Ye and Ju prepared a MWNT modified screen-printed carbon electrode, which showed excellent catalytic characteristics for the direct electrochemical oxidation of guanine and adenine [12]. Meanwhile, the application of room temperature ionic liquid in the fields of analytical chemistry
T. Liu et al. / Journal of Electroanalytical Chemistry 651 (2011) 216–221
and electrochemical biosensors have attracted much attention, due to their specific characteristics such as good chemical and thermal stability, negligible vapor pressure, wide electrochemical windows and high ionic conductivity [13]. It has been demonstrated that the carbon nanotubes-ionic liquid composite exhibits a synergistic effect in the determination of chloramphenicol [14], cytochrome c [15], DNA hybridization [16] and glucose concentration [17]. It is also well known that PbO2 is one of the best electrode materials because of its good electrical conductivity, high oxygen overpotential, chemical inertness and good resistance to corrosion [18–21]. Nowadays, there is a great interest in the development of PbO2 and doped PbO2 as anode with high electro-catalytic activity for anodic oxygen transfer processes. Increasing attention has been paid on the modified electrodes with PbO2 and nanomaterial composite in hope for combining their unique advantages. In this study, the electrochemical oxidation behavior of guanine and adenine on the PbO2–MWNT–RTIL composite film modified electrode has been investigated utilizing differential pulse voltammetry (DPV) and cyclic voltammetry (CV). As a result of high electron transfer ability and good electrocatalytic ability of PbO2– MWNT–RTIL composite film, this type biosensor facilitated the electron transfer of guanine and adenine, resulting in the increase of oxidation signals. 2. Experimental 2.1. Apparatus and reagents Differential pulse voltammetry (DPV) and cyclic voltammetry (CV) measurements were carried out with BAS Epsilon Electrochemical Analyzer (Bioanalytical Systems, Inc., USA). A conventional three-electrode system was used comprising an Ag/AgCl (saturated KCl) reference electrode, a platinum auxiliary electrode and a working electrode based on bare or modified GCE. All potentials were referred against the Ag/AgCl reference electrode. X-ray diffraction (XRD) patterns of PbO2 were operated using D8 Advance X-ray diffractometer (Bruker). Scanning electron microscopy (SEM) observations was obtained using the JEOL JSM-7600F (Japan). Pb(NO3)2, (NH4)2S2O8 and polyvinyl pyrrolidone (PVP) were obtained from Aladdin. MWNT were purchased from Nachen S&T Ltd (Beijing, China) and treated according to reported procedure [22,23]. Room temperature ionic liquid (1-butyl-3-methylimidazolium hexafluorophosphate, BMIMPF6) was purchased from Cheng Jie Chemical Co., Ltd. (Shanghai, China). Guanine, adenine and herring sperm DNA (dsDNA) were purchased from Sigma and was used without further purification. Guanine and adenine stock solution were prepared by dissolving them into 0.1 M NaOH solution and kept in refrigerator at 4 °C. Working solutions were freshly prepared before use by diluting the corresponding stock solution. The phosphate buffer solution (PBS, 0.1 M) was prepared by 0.1 M KH2PO4 and 0.1 M K2HPO4, and adjusted the pH with 0.1 M H3PO4 and 0.1 M KOH solutions. Other chemicals were of analytical reagent grade and used without further purification. Doubly distilled deionized water from quartz was used throughout the work. All the measurements were carried out at room temperature (25 ± 0.5 °C). 2.2. Synthesis of PbO2 In a typical process [24], 2 mmol Pb(NO3)2 and 5 mmol PVP were put into 100 ml sodium hydroxide solution (1 M), the mixture was stirred strongly for 20 min to form a transparent solution. After that, 4 mmol (NH4)2S2O8 was added under constant stirring to form a homogeneous solution. Then the homogeneous solution
217
was transferred into a Teflon – lined autoclave, which was sealed and maintained at 90 °C for 10 h. Then, the autoclave was cooled to room temperature. The solid brown precipitate was filtered, and washed several times with distilled water and absolute ethanol to remove impurities, and then dried in vacuum at 50 °C for 4 h. 2.3. Preparation of the PbO2–MWNT–RTIL composite 1.0 mg MWNT and 7.03 mg RTIL were homogeneously dispersed in 1 mL DMF with the aid of ultrasonic agitation for 1 h. The 0.5 mg PbO2 was added to the obtained black suspension, and sonicated for 0.5 h. After that, a homogeneous, well-distributed mixed solution of PbO2–MWNT–RTIL composite was then obtained. 2.4. Electrode modification Before modification, the bare GCE was carefully polished to a mirror-like surface with 0.3, 0.05 lm alumina slurries followed by thorough rinsing with double distilled water, then sonicated in ethanol and double distilled water for 3 min, respectively. Finally, the GCE was dried under the stream of high purity nitrogen for further use. With a microinjector, 8 lL of the resulting mixture solution was deposited on the fresh prepared GCE surface until the solvent was evaporated. Afterwards, the electrode surface was thoroughly rinsed with double distilled water and dried in the air (noted PbO2–MWNT–RTIL/GCE). For comparison, the MWNT modified electrode (MWNT/GCE) and MWNT-RTIL modified electrode (MWNT-RTIL/GCE) were prepared with an analogous manner. The modified GCE was preserved in a refrigerator at 4 °C when it was not in use. 2.5. Preparation of DNA samples Thermally denaturation of herring sperm DNA was performed according to a previous report [10]. In brief, the DNA sample was dissolved in redistilled deionized water and then heated in a boiling water bath (100 °C) for about 10 min, and finally the sample was cooled rapidly in an ice-water bath. 2.6. Electrochemical measurements The accumulation procedures were carried out at the potential of +0.30 V for 240 s. After a quiet period of 2 s, the DPV measurements were performed in 0.1 M PBS (pH 7.0) with different concentrations of adenine or guanine or their mixtures by scanning from +0.30 to +1.20 V. The parameters are as follows: pulse amplitude 0.1 V, pulse width 0.05 s, and pulse period 0.2 s. CV was conducted in 0.1 M pH 7.0 PBS containing adenine or guanine with different scan rates. The electrode can be used for the next measurement after a continuous sweep for three cycles at the same potential range in the buffer solution and repeating the above assay procedure. 3. Results and discussion 3.1. Characterization of the synthesized PbO2 The morphology and microstructure of the as-prepared PbO2 was investigated by scanning electron microscope (SEM) as shown in Fig. 1, it can be seen that the insides of these spheres are hollow, which indicated that the as-obtained products were mainly composed of submicrometer hollow spheres. In addition, the crystal structures of the products were examined by XRD. Fig. 2 showed the XRD pattern of the prepared hollow spheres, which can be
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Fig. 1. SEM micrograph of nano-PbO2.
readily indexed to a relatively pure b-PbO2 because of few interference peaks. 3.2. Electrochemical characteristics of the modified electrode The influences of MWNT, RTIL and PbO2 on the direct electrochemistry of guanine oxidation were investigated based on DPV measurements at different electrodes (Fig. 3). In the absence of guanine in 0.1 M PBS (pH 7.0), no redox peak was observed in the range from +0.3 to +1.2 V at all modified electrodes, indicating that MWNT, RTIL and PbO2 are electroinactive in the scanned potential window. When in the presence of 33 lM guanine, the bare GCE (Fig. 3A-a) showed a small oxidation peak (6.1 lA). While the MWNT/GCE (Fig. 3A-b) and the MWNT-RTIL/GCE (Fig. 3A-c) resulted in excellent amplification of the guanine oxidation response, with peak current value of 15.2 and 19.4 lA, respectively. And a negative shift of peak potential as 50 ± 5 mV was obtained at the modified electrodes compared to that at the bare electrode. The highest oxidation peak current (31.8 lA) of guanine was observed at PbO2–MWNT–RTIL/GCE (Fig. 3A-d), which illustrated that the modified electrode possessed the highest electro-catalytic activity
[25,26]. The direct electrochemistry of adenine oxidation was carried out with an analogous manner and similar results were obtained (Fig. 3B). The results may be ascribed to the high conductivity, good antifouling property, fast electron transfer rate and high electrochemical activity of the MWNT, PbO2, and RTIL. As a result, the corresponding oxidation peak currents of guanine and adenine on the modified electrode increased greatly. The effect of pH on the oxidation response of PbO2–MWNT– RTIL/GCE towards the single determination of 33 lM guanine and 37 lM adenine was investigated in the range of 6.0–8.0. The results indicated that the peak potential (Epa) was depended on solution pH. Two good linear relationships were obtained between the Epa and pH, which obeyed the following equations, Epa = 0.063 pH + 1.2182 (R = 0.9965) and Epa = 0.0499 pH + 1.4219 (R = 0.9964) for guanine and adenine, respectively. The slope of 63 and 49 mV/pH implied that the electrons transfer was accompanied by an equal number of protons in electrode reaction process. Moreover, both the maximum current responses of guanine and adenine were obtained at pH 7.0. Therefore, pH 7.0 was chosen for the subsequent analytical experiments. The influence of accumulation time on the oxidation signals of 33 lM guanine and 37 lM adenine was investigated in pH 7.0 PBS under stirring. The oxidation peak currents of both guanine and adenine increased gradually with extending the accumulation time from 0 to 240 s, and then leveled off with the further increase of accumulation time, indicating the saturated adsorption of analytes. Considering both sensitivity and work efficiency, the optimal accumulation time of 240 s was employed in the further experiments. The effect of scan rate was also investigated in 33 lM guanine and 37 lM adenine by cyclic voltammetry, respectively. Only oxidation peaks were observed, indicating that the oxidation of guanine and adenine was a totally irreversible electrode process. As can be seen in Fig. 4, the oxidation peak currents of guanine and adenine were proportional to the scan rate in the range of 20– 100 mV s 1 and 10–150 mV s 1 respectively, and the linear regression equations can be expressed as Ipa = 0.5094 v 1.3926 (Guanine, R = 0.9959) and Ipa = 0.4578 v + 4.8812 (Adenine, R = 0.9963), indicating that the oxidation process was a typical adsorption-controlled process. In addition, the Epa of guanine and adenine shifted positively with increasing the scan rate.
Fig. 2. XRD pattern of nano-PbO2.
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Fig. 3. DPVs of 33 lM guanine (A) and 37 lM adenine (B) obtained at bare GCE (a), MWNT/GCE (b), MWNT-RTIL/GCE (c) and PbO2–MWNT–RTIL/GCE (d) in 0.1 M PBS (pH 7.0). Accumulation time: 240 s, accumulation potential: +0.30 V.
Fig. 4. (A) CVs of 33 lM guanine at various scan rates (from a to h: 100, 90, 80, 60, 50, 40, 30 and 20 mV s 1) in 0.1 M PBS (pH 7.0) at the PbO2–MWNT–RTIL/GCE. Insert: The relationship between peak current of guanine and scan rate. (B) CVs of 37 lM adenine at various scan rates (from a to h: 150, 100, 90, 70, 50, 30, 20, and 10 mV s 1) in 0.1 M PBS (pH 7.0) at the PbO2–MWNT–RTIL/GCE. Insert: The relationship between peak current of adenine and scan rate.
Fig. 5. (A) DPVs of different concentrations of guanine (from a to h: 0.07, 0.33, 1.3, 2.0, 2.6, 3.3, 5.3 and 20 lM) at the PbO2–MWNT–RTIL/GCE. Insert: The relationship between peak current of guanine and CG. (B) DPVs of different concentrations of adenine (from a to i: 0.37, 1.5, 1.8, 2.2, 3.0, 3.7, 7.4, 22 and 37 lM) at the PbO2–MWNT–RTIL/ GCE. Insert: The relationship between peak current of adenine and CA.
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Fig. 6. (A) DPVs for the oxidation signals of guanine (from a to e: 3.3, 6.6, 9.9, 13.2 and 19.8 lM) in 0.1 M pH 7.0 PBS containing 7.4 lM adenine at PbO2–MWNT–RTIL/GCE. (B) DPVs for the oxidation signals of adenine (from a to e: 3.7, 7.4, 11.1, 14.8 and 22.2 lM) in 0.1 M pH 7.0 PBS containing 6.6 lM guanine at PbO2–MWNT–RTIL/GCE.
3.3. Individual determination of adenine and guanine Under the optimal conditions, the calibration curve for guanine detection in pH 7.0 PBS was measured by DPV. The oxidation peak current of guanine was proportional to the concentration (c) in the range of 0.07–20 lM with the linear regression equation of Ipa (lA) = 1.7282 c + 2.8235 (R = 0.9979). The detection limit was calculated to be 6.0 10 9 M (S/N = 3) (Fig. 5A), with a sensitivity of 1.52 lA lM 1 cm 2. As for adenine detection, similar studies were also done as in the case of guanine and a linear relationship between Ipa and c was obtained from 0.37 to 37 lM. The regression equation was Ipa = 0.9537c + 0.8316 (R = 0.9993) and the detection limit was 3.0 10 8 M (S/N = 3) (Fig. 5B), with a sensitivity of 0.84 lA lM 1 cm 2. The detection limit and sensitivity were superior to some previous reports [27,28]. 3.4. Simultaneous determination of adenine and guanine The PbO2–MWNT–RTIL/GCE showed excellent ability for the simultaneous determination of guanine and adenine in the mixed solution. And the oxidation peak potentials located at +0.61 V and +0.90 V were attributed to that of guanine and adenine, respectively (Fig. 6). The determination of guanine and adenine in their mixtures was investigated when the concentration of one species changed, while the other species remained constant. Fig. 6A showed the DPV curves of different concentrations of guanine coexisting 7.4 lM adenine in 0.1 M pH 7.0 PBS. It can be seen that the oxidation signal of guanine increased with the increasing concentration and without the change of the response of adenine. Similarly, as shown in Fig. 6B, with the concentration of adenine changed by keeping the guanine concentration constant (6.6 lM), the oxidation peak current increased with increasing the concentration of adenine in the range of 3.7–22 lM. Moreover, the oxidation peak potential separation between the electrochemical signals of guanine and adenine was 0.29 V, which was enough for the simultaneous detection. Therefore, the proposed method can be applied to the simultaneous determination of guanine and adenine. 3.5. Reproducibility and stability of the modified electrode A series of eleven repetitive measurements of the oxidation of guanine and adenine gave reproducible results with the relative standard deviation (RSD) of 4.5% for guanine and 3.76% for adenine, indicating the excellent reproducibility of the modified electrode. After the modified electrode was stored at 4 °C for 20 days, only a small decrease of the oxidation peak current of guanine and ade-
nine was observed with the RSD of 3.45% (Guanine) and 2.51% (Adenine), which could be attributed to the excellent stability of the modified electrode. 3.6. Analytical applications In order to evaluate the validity of the proposed method, the fabricated electrode was used to detect the guanine and adenine concentration of thermally denatured herring sperm DNA. The determination of guanine and adenine concentrations was performed by standard addition methods according to previous reports [10]. According to the oxidation peak currents, the contents of guanine and adenine in thermally denatured DNA were calculated to be 22.3% and 27.6% (molar ratio, mol%), respectively. Therefore, the value of (G + C)/(A + T) for the thermally denatured DNA was calculated to be 0.81, which was close to the standard value of 0.77 [29]. 4. Conclusion Herein, a novel electrode based on PbO2–MWNT–RTIL composite for the electrochemical detection of guanine and adenine has been demonstrated. The results show that the modified electrode not only displays excellent amplification of the guanine and adenine oxidation responses but also decreases the oxidation peak potential remarkably. The proposed method was successfully applied to simultaneously determine guanine and adenine. In addition, the contents of guanine and adenine in herring sperm DNA were measured with satisfactory results. PbO2–MWNT–RTIL composite is thus promising material for the simultaneous determination of guanine and adenine, which will be useful in the related physiology process. Acknowledgements This work was supported by the National Natural Science Foundation of China (No. 20775044) and the Natural Science Foundation of Shandong province, China (Y2006B20). References [1] H.S. Wang, H.X. Ju, H.Y. Chen, Anal. Chim. Acta 461 (2002) 243–250. [2] J. Yang, G. Xu, H. Kong, Y. Zheng, T. Pang, Q. Yang, J. Chromatogr. B 780 (2002) 27–33. [3] N. Kuroda, K. Nakashima, S. Akiyama, Anal. Chim. Acta 278 (1993) 275–278. [4] H.C. Tseng, C. Dadoo, R.N. Zare, Anal. Biochem. 222 (1994) 55–58. [5] D.K. Xu, L. Hua, H.Y. Chen, Anal. Chim. Acta 335 (1996) 95–101.
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Contents lists available at ScienceDirect
Journal of Electroanalytical Chemistry journal homepage: www.elsevier.com/locate/jelechem
Simultaneous determination of adenine and guanine utilizing PbO2-carbon nanotubes-ionic liquid composite film modified glassy carbon electrode Tao Liu a, Xiangbin Zhu b, Lin Cui a, Peng Ju a, Xiangjin Qu a,⇑, Shiyun Ai a,⇑ a b
College of Chemistry and Material Science, Shandong Agricultural University, Taian, Shandong 271018, China College of Resources and Environment, Shandong Agricultural University, Taian, Shandong 271018, China
a r t i c l e
i n f o
Article history: Received 15 June 2010 Received in revised form 5 September 2010 Accepted 21 November 2010 Available online 27 November 2010 Keywords: Carbon nanotubes Room temperature ionic liquid Nano-PbO2 Guanine Adenine Herring sperm DNA
a b s t r a c t A novel and reliable electrochemical sensor based on PbO2-carbon nanotubes-room temperature ionic liquid (i.e., 1-butyl-3-methylimidazolium hexafluorophosphate, BMIMPF6) composite film modified glassy carbon electrode (GCE) (PbO2–MWNT–RTIL/GCE) was proposed for simultaneous and individual determination of guanine and adenine. The guanine and adenine oxidation responses were monitored by differential pulse voltammetric (DPV) measurement. Compared with the bare electrode, the PbO2– MWNT–RTIL/GCE not only significantly enhanced the oxidation peak currents of guanine and adenine, but also lowered their oxidation overpotentials, suggesting that the synergistic effect of PbO2, MWNT and RTIL could dramatically improve the determining sensitivity of guanine and adenine. The PbO2– MWNT–RTIL/GCE showed good stability, high accumulation efficiency and enhanced electrocatalytic ability for the detection of guanine and adenine. Besides, the modified electrode also exhibited good behaviors in the simultaneous detection of adenine and guanine with the peak separation of 0.29 V in 0.1 M pH 7.0 phosphate buffer solution (PBS). Under the optimal conditions, the detection limit for individual determination of guanine and adenine was 6.0 10 9 M and 3.0 10 8 M (S/N = 3), respectively. The proposed method for the measurements of guanine and adenine in herring sperm DNA was successfully applied with satisfactory results. Ó 2010 Elsevier B.V. All rights reserved.
1. Introduction Deoxyribonucleic acid (DNA) is an important substance which plays a significant role in biological heritage information storage and in protein biosynthesis. Guanine (G) and adenine (A) are two important purine bases existed in DNA, which participate in processes as distinct as energy transduction, metabolic cofactors and cell signaling. The abnormal changes of guanine and adenine in DNA have been suggested to be related to the deficiency and mutation of the immunity system. And the concentration changes of guanine and adenine in DNA could be considered as an indication of various diseases such as carcinoma or liver diseases [1]. Hence, it is of great significance to establish sensitive methods for the analysis of these bases in DNA [2]. So far, a variety of methods have been established to investigate the concentration of the adenine and guanine, such as chemiluminescence, micellar electrokinetic chromatography with indirect laser-induced fluorescence detection, and electrophoresis coupled with electrochemical determination [3–6]. Although these techniques show high sensitivity and selectivity, it must be pointed ⇑ Corresponding authors. Tel.: +86 538 8247660; fax: +86 538 8242251. E-mail addresses: [email protected] (X. Qu), [email protected] (S. Ai). 1572-6657/$ - see front matter Ó 2010 Elsevier B.V. All rights reserved. doi:10.1016/j.jelechem.2010.11.026
out that they have severe setbacks for purines determination, such as time-consuming and expensive. Therefore, they are unsuitable for on-line or field monitoring. Owing to their high sensitivity, simple operation, fast response speed, low cost and real-time detection in situ condition, various kinds of chemically modified electrodes have been prepared to investigate the electrochemical oxidation of adenine and guanine [7]. Such as cobalt (II) phthalocyanine modified carbon paste electrode [8], b-cyclodextrin incorporated carbon nanotubes-modified electrode [9], and carbon ionic liquid electrode [10]. Although all the above modified materials show good improving oxidation signals, it is still a challenge to develop new and economical methods with high sensitivity and convenience for the determination of guanine and adenine. Carbon nanotubes (CNTs) have attracted a great deal of interest due to their unique properties such as fast electron transfer ability and catalysis effect. Wang et al. described a CNTs modified GCE, which improved the detection of DNA hybridization and greatly enhanced the guanine oxidation signal [11]. Ye and Ju prepared a MWNT modified screen-printed carbon electrode, which showed excellent catalytic characteristics for the direct electrochemical oxidation of guanine and adenine [12]. Meanwhile, the application of room temperature ionic liquid in the fields of analytical chemistry
T. Liu et al. / Journal of Electroanalytical Chemistry 651 (2011) 216–221
and electrochemical biosensors have attracted much attention, due to their specific characteristics such as good chemical and thermal stability, negligible vapor pressure, wide electrochemical windows and high ionic conductivity [13]. It has been demonstrated that the carbon nanotubes-ionic liquid composite exhibits a synergistic effect in the determination of chloramphenicol [14], cytochrome c [15], DNA hybridization [16] and glucose concentration [17]. It is also well known that PbO2 is one of the best electrode materials because of its good electrical conductivity, high oxygen overpotential, chemical inertness and good resistance to corrosion [18–21]. Nowadays, there is a great interest in the development of PbO2 and doped PbO2 as anode with high electro-catalytic activity for anodic oxygen transfer processes. Increasing attention has been paid on the modified electrodes with PbO2 and nanomaterial composite in hope for combining their unique advantages. In this study, the electrochemical oxidation behavior of guanine and adenine on the PbO2–MWNT–RTIL composite film modified electrode has been investigated utilizing differential pulse voltammetry (DPV) and cyclic voltammetry (CV). As a result of high electron transfer ability and good electrocatalytic ability of PbO2– MWNT–RTIL composite film, this type biosensor facilitated the electron transfer of guanine and adenine, resulting in the increase of oxidation signals. 2. Experimental 2.1. Apparatus and reagents Differential pulse voltammetry (DPV) and cyclic voltammetry (CV) measurements were carried out with BAS Epsilon Electrochemical Analyzer (Bioanalytical Systems, Inc., USA). A conventional three-electrode system was used comprising an Ag/AgCl (saturated KCl) reference electrode, a platinum auxiliary electrode and a working electrode based on bare or modified GCE. All potentials were referred against the Ag/AgCl reference electrode. X-ray diffraction (XRD) patterns of PbO2 were operated using D8 Advance X-ray diffractometer (Bruker). Scanning electron microscopy (SEM) observations was obtained using the JEOL JSM-7600F (Japan). Pb(NO3)2, (NH4)2S2O8 and polyvinyl pyrrolidone (PVP) were obtained from Aladdin. MWNT were purchased from Nachen S&T Ltd (Beijing, China) and treated according to reported procedure [22,23]. Room temperature ionic liquid (1-butyl-3-methylimidazolium hexafluorophosphate, BMIMPF6) was purchased from Cheng Jie Chemical Co., Ltd. (Shanghai, China). Guanine, adenine and herring sperm DNA (dsDNA) were purchased from Sigma and was used without further purification. Guanine and adenine stock solution were prepared by dissolving them into 0.1 M NaOH solution and kept in refrigerator at 4 °C. Working solutions were freshly prepared before use by diluting the corresponding stock solution. The phosphate buffer solution (PBS, 0.1 M) was prepared by 0.1 M KH2PO4 and 0.1 M K2HPO4, and adjusted the pH with 0.1 M H3PO4 and 0.1 M KOH solutions. Other chemicals were of analytical reagent grade and used without further purification. Doubly distilled deionized water from quartz was used throughout the work. All the measurements were carried out at room temperature (25 ± 0.5 °C). 2.2. Synthesis of PbO2 In a typical process [24], 2 mmol Pb(NO3)2 and 5 mmol PVP were put into 100 ml sodium hydroxide solution (1 M), the mixture was stirred strongly for 20 min to form a transparent solution. After that, 4 mmol (NH4)2S2O8 was added under constant stirring to form a homogeneous solution. Then the homogeneous solution
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was transferred into a Teflon – lined autoclave, which was sealed and maintained at 90 °C for 10 h. Then, the autoclave was cooled to room temperature. The solid brown precipitate was filtered, and washed several times with distilled water and absolute ethanol to remove impurities, and then dried in vacuum at 50 °C for 4 h. 2.3. Preparation of the PbO2–MWNT–RTIL composite 1.0 mg MWNT and 7.03 mg RTIL were homogeneously dispersed in 1 mL DMF with the aid of ultrasonic agitation for 1 h. The 0.5 mg PbO2 was added to the obtained black suspension, and sonicated for 0.5 h. After that, a homogeneous, well-distributed mixed solution of PbO2–MWNT–RTIL composite was then obtained. 2.4. Electrode modification Before modification, the bare GCE was carefully polished to a mirror-like surface with 0.3, 0.05 lm alumina slurries followed by thorough rinsing with double distilled water, then sonicated in ethanol and double distilled water for 3 min, respectively. Finally, the GCE was dried under the stream of high purity nitrogen for further use. With a microinjector, 8 lL of the resulting mixture solution was deposited on the fresh prepared GCE surface until the solvent was evaporated. Afterwards, the electrode surface was thoroughly rinsed with double distilled water and dried in the air (noted PbO2–MWNT–RTIL/GCE). For comparison, the MWNT modified electrode (MWNT/GCE) and MWNT-RTIL modified electrode (MWNT-RTIL/GCE) were prepared with an analogous manner. The modified GCE was preserved in a refrigerator at 4 °C when it was not in use. 2.5. Preparation of DNA samples Thermally denaturation of herring sperm DNA was performed according to a previous report [10]. In brief, the DNA sample was dissolved in redistilled deionized water and then heated in a boiling water bath (100 °C) for about 10 min, and finally the sample was cooled rapidly in an ice-water bath. 2.6. Electrochemical measurements The accumulation procedures were carried out at the potential of +0.30 V for 240 s. After a quiet period of 2 s, the DPV measurements were performed in 0.1 M PBS (pH 7.0) with different concentrations of adenine or guanine or their mixtures by scanning from +0.30 to +1.20 V. The parameters are as follows: pulse amplitude 0.1 V, pulse width 0.05 s, and pulse period 0.2 s. CV was conducted in 0.1 M pH 7.0 PBS containing adenine or guanine with different scan rates. The electrode can be used for the next measurement after a continuous sweep for three cycles at the same potential range in the buffer solution and repeating the above assay procedure. 3. Results and discussion 3.1. Characterization of the synthesized PbO2 The morphology and microstructure of the as-prepared PbO2 was investigated by scanning electron microscope (SEM) as shown in Fig. 1, it can be seen that the insides of these spheres are hollow, which indicated that the as-obtained products were mainly composed of submicrometer hollow spheres. In addition, the crystal structures of the products were examined by XRD. Fig. 2 showed the XRD pattern of the prepared hollow spheres, which can be
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Fig. 1. SEM micrograph of nano-PbO2.
readily indexed to a relatively pure b-PbO2 because of few interference peaks. 3.2. Electrochemical characteristics of the modified electrode The influences of MWNT, RTIL and PbO2 on the direct electrochemistry of guanine oxidation were investigated based on DPV measurements at different electrodes (Fig. 3). In the absence of guanine in 0.1 M PBS (pH 7.0), no redox peak was observed in the range from +0.3 to +1.2 V at all modified electrodes, indicating that MWNT, RTIL and PbO2 are electroinactive in the scanned potential window. When in the presence of 33 lM guanine, the bare GCE (Fig. 3A-a) showed a small oxidation peak (6.1 lA). While the MWNT/GCE (Fig. 3A-b) and the MWNT-RTIL/GCE (Fig. 3A-c) resulted in excellent amplification of the guanine oxidation response, with peak current value of 15.2 and 19.4 lA, respectively. And a negative shift of peak potential as 50 ± 5 mV was obtained at the modified electrodes compared to that at the bare electrode. The highest oxidation peak current (31.8 lA) of guanine was observed at PbO2–MWNT–RTIL/GCE (Fig. 3A-d), which illustrated that the modified electrode possessed the highest electro-catalytic activity
[25,26]. The direct electrochemistry of adenine oxidation was carried out with an analogous manner and similar results were obtained (Fig. 3B). The results may be ascribed to the high conductivity, good antifouling property, fast electron transfer rate and high electrochemical activity of the MWNT, PbO2, and RTIL. As a result, the corresponding oxidation peak currents of guanine and adenine on the modified electrode increased greatly. The effect of pH on the oxidation response of PbO2–MWNT– RTIL/GCE towards the single determination of 33 lM guanine and 37 lM adenine was investigated in the range of 6.0–8.0. The results indicated that the peak potential (Epa) was depended on solution pH. Two good linear relationships were obtained between the Epa and pH, which obeyed the following equations, Epa = 0.063 pH + 1.2182 (R = 0.9965) and Epa = 0.0499 pH + 1.4219 (R = 0.9964) for guanine and adenine, respectively. The slope of 63 and 49 mV/pH implied that the electrons transfer was accompanied by an equal number of protons in electrode reaction process. Moreover, both the maximum current responses of guanine and adenine were obtained at pH 7.0. Therefore, pH 7.0 was chosen for the subsequent analytical experiments. The influence of accumulation time on the oxidation signals of 33 lM guanine and 37 lM adenine was investigated in pH 7.0 PBS under stirring. The oxidation peak currents of both guanine and adenine increased gradually with extending the accumulation time from 0 to 240 s, and then leveled off with the further increase of accumulation time, indicating the saturated adsorption of analytes. Considering both sensitivity and work efficiency, the optimal accumulation time of 240 s was employed in the further experiments. The effect of scan rate was also investigated in 33 lM guanine and 37 lM adenine by cyclic voltammetry, respectively. Only oxidation peaks were observed, indicating that the oxidation of guanine and adenine was a totally irreversible electrode process. As can be seen in Fig. 4, the oxidation peak currents of guanine and adenine were proportional to the scan rate in the range of 20– 100 mV s 1 and 10–150 mV s 1 respectively, and the linear regression equations can be expressed as Ipa = 0.5094 v 1.3926 (Guanine, R = 0.9959) and Ipa = 0.4578 v + 4.8812 (Adenine, R = 0.9963), indicating that the oxidation process was a typical adsorption-controlled process. In addition, the Epa of guanine and adenine shifted positively with increasing the scan rate.
Fig. 2. XRD pattern of nano-PbO2.
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Fig. 3. DPVs of 33 lM guanine (A) and 37 lM adenine (B) obtained at bare GCE (a), MWNT/GCE (b), MWNT-RTIL/GCE (c) and PbO2–MWNT–RTIL/GCE (d) in 0.1 M PBS (pH 7.0). Accumulation time: 240 s, accumulation potential: +0.30 V.
Fig. 4. (A) CVs of 33 lM guanine at various scan rates (from a to h: 100, 90, 80, 60, 50, 40, 30 and 20 mV s 1) in 0.1 M PBS (pH 7.0) at the PbO2–MWNT–RTIL/GCE. Insert: The relationship between peak current of guanine and scan rate. (B) CVs of 37 lM adenine at various scan rates (from a to h: 150, 100, 90, 70, 50, 30, 20, and 10 mV s 1) in 0.1 M PBS (pH 7.0) at the PbO2–MWNT–RTIL/GCE. Insert: The relationship between peak current of adenine and scan rate.
Fig. 5. (A) DPVs of different concentrations of guanine (from a to h: 0.07, 0.33, 1.3, 2.0, 2.6, 3.3, 5.3 and 20 lM) at the PbO2–MWNT–RTIL/GCE. Insert: The relationship between peak current of guanine and CG. (B) DPVs of different concentrations of adenine (from a to i: 0.37, 1.5, 1.8, 2.2, 3.0, 3.7, 7.4, 22 and 37 lM) at the PbO2–MWNT–RTIL/ GCE. Insert: The relationship between peak current of adenine and CA.
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Fig. 6. (A) DPVs for the oxidation signals of guanine (from a to e: 3.3, 6.6, 9.9, 13.2 and 19.8 lM) in 0.1 M pH 7.0 PBS containing 7.4 lM adenine at PbO2–MWNT–RTIL/GCE. (B) DPVs for the oxidation signals of adenine (from a to e: 3.7, 7.4, 11.1, 14.8 and 22.2 lM) in 0.1 M pH 7.0 PBS containing 6.6 lM guanine at PbO2–MWNT–RTIL/GCE.
3.3. Individual determination of adenine and guanine Under the optimal conditions, the calibration curve for guanine detection in pH 7.0 PBS was measured by DPV. The oxidation peak current of guanine was proportional to the concentration (c) in the range of 0.07–20 lM with the linear regression equation of Ipa (lA) = 1.7282 c + 2.8235 (R = 0.9979). The detection limit was calculated to be 6.0 10 9 M (S/N = 3) (Fig. 5A), with a sensitivity of 1.52 lA lM 1 cm 2. As for adenine detection, similar studies were also done as in the case of guanine and a linear relationship between Ipa and c was obtained from 0.37 to 37 lM. The regression equation was Ipa = 0.9537c + 0.8316 (R = 0.9993) and the detection limit was 3.0 10 8 M (S/N = 3) (Fig. 5B), with a sensitivity of 0.84 lA lM 1 cm 2. The detection limit and sensitivity were superior to some previous reports [27,28]. 3.4. Simultaneous determination of adenine and guanine The PbO2–MWNT–RTIL/GCE showed excellent ability for the simultaneous determination of guanine and adenine in the mixed solution. And the oxidation peak potentials located at +0.61 V and +0.90 V were attributed to that of guanine and adenine, respectively (Fig. 6). The determination of guanine and adenine in their mixtures was investigated when the concentration of one species changed, while the other species remained constant. Fig. 6A showed the DPV curves of different concentrations of guanine coexisting 7.4 lM adenine in 0.1 M pH 7.0 PBS. It can be seen that the oxidation signal of guanine increased with the increasing concentration and without the change of the response of adenine. Similarly, as shown in Fig. 6B, with the concentration of adenine changed by keeping the guanine concentration constant (6.6 lM), the oxidation peak current increased with increasing the concentration of adenine in the range of 3.7–22 lM. Moreover, the oxidation peak potential separation between the electrochemical signals of guanine and adenine was 0.29 V, which was enough for the simultaneous detection. Therefore, the proposed method can be applied to the simultaneous determination of guanine and adenine. 3.5. Reproducibility and stability of the modified electrode A series of eleven repetitive measurements of the oxidation of guanine and adenine gave reproducible results with the relative standard deviation (RSD) of 4.5% for guanine and 3.76% for adenine, indicating the excellent reproducibility of the modified electrode. After the modified electrode was stored at 4 °C for 20 days, only a small decrease of the oxidation peak current of guanine and ade-
nine was observed with the RSD of 3.45% (Guanine) and 2.51% (Adenine), which could be attributed to the excellent stability of the modified electrode. 3.6. Analytical applications In order to evaluate the validity of the proposed method, the fabricated electrode was used to detect the guanine and adenine concentration of thermally denatured herring sperm DNA. The determination of guanine and adenine concentrations was performed by standard addition methods according to previous reports [10]. According to the oxidation peak currents, the contents of guanine and adenine in thermally denatured DNA were calculated to be 22.3% and 27.6% (molar ratio, mol%), respectively. Therefore, the value of (G + C)/(A + T) for the thermally denatured DNA was calculated to be 0.81, which was close to the standard value of 0.77 [29]. 4. Conclusion Herein, a novel electrode based on PbO2–MWNT–RTIL composite for the electrochemical detection of guanine and adenine has been demonstrated. The results show that the modified electrode not only displays excellent amplification of the guanine and adenine oxidation responses but also decreases the oxidation peak potential remarkably. The proposed method was successfully applied to simultaneously determine guanine and adenine. In addition, the contents of guanine and adenine in herring sperm DNA were measured with satisfactory results. PbO2–MWNT–RTIL composite is thus promising material for the simultaneous determination of guanine and adenine, which will be useful in the related physiology process. Acknowledgements This work was supported by the National Natural Science Foundation of China (No. 20775044) and the Natural Science Foundation of Shandong province, China (Y2006B20). References [1] H.S. Wang, H.X. Ju, H.Y. Chen, Anal. Chim. Acta 461 (2002) 243–250. [2] J. Yang, G. Xu, H. Kong, Y. Zheng, T. Pang, Q. Yang, J. Chromatogr. B 780 (2002) 27–33. [3] N. Kuroda, K. Nakashima, S. Akiyama, Anal. Chim. Acta 278 (1993) 275–278. [4] H.C. Tseng, C. Dadoo, R.N. Zare, Anal. Biochem. 222 (1994) 55–58. [5] D.K. Xu, L. Hua, H.Y. Chen, Anal. Chim. Acta 335 (1996) 95–101.
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