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Voltammetric Response and Determination of DNA with a Silver Electrode
Analytical Biochemistry 271, 1–7 (1999) Article ID abio.1999.4102, available online at http://www.idealibrary.com on
Voltammetric Response and Determination of DNA with a Silver Electrode Chunhai Fan,* Haiyun Song,* Xiaofang Hu,* Genxi Li,* Jianqin Zhu,† Xianxiu Xu,* and Dexu Zhu* ,1 *Department of Biochemistry and National Laboratory of Pharmaceutical Biotechnology, and †Department of Biology and Biological Technology, Nanjing University, Nanjing 210093, People’s Republic of China
Received October 29, 1998
A current from DNA was obtained using a silver electrode with low overpotentials for the first time. Experimental results revealed that the voltammetric response of DNA was attributed to the redox reactions of purine bases. It was also shown that such a method provided a convenient and practical way to determine DNA. A linear dependence of the peak currents on ssDNA concentrations was observed in the range 0.5– 2.5 mg/mL. The relative standard deviation was 3.5% for six successive determinations at 0.5 mg/mL. The detection limit was 50 ng/mL. Influence of the structure and the length of the nucleic acids on their electrochemical behavior was discussed. In view of the merits of the silver electrode, this technique might provide new possibilities for further electrochemical research and determination of nucleic acids. © 1999 Academic Press
Key Words: electrochemistry; silver electrode; DNA.
The behavior of nucleic acids at charged biological interfaces (e.g., membranes) is of great importance in many biological processes. However, the complexity of their structures and properties has until now hindered a theoretical treatment of their behavior at electrically charged surfaces (1). The electrochemical system serves as a versatile and illuminating model of biological system despite obvious physical and chemical differences (1, 2). Systematic voltammetric studies of nucleic acids and their component units have provided much important information on the properties and conformation of such substances in solutions and on electrode surfaces. These include early electrochemical ev1 To whom correspondence should be addressed. Fax: (186)(25) 3607621. E-mail:[email protected].
0003-2697/99 $30.00 Copyright © 1999 by Academic Press All rights of reproduction in any form reserved.
idence for DNA premelting and the polymorphy of the DNA double helix, which have been widely accepted (1, 3). Most of these studies employed mercury as the electrode material (1, 2, 4 – 6). Solid electrodes have not been intensively explored due to large background current produced at these surfaces (7). However, a mercury electrode is toxic and has limited applications due to its fluid state. Solid electrodes have some advantages, which make them attractive as flow detectors following HPLC and capillary zone electrophoresis, various kinds of sensors, etc. (8). Recently, substantial interest has been shown in the study of the interfacial behavior of nucleic acids at different kinds of electrode materials (7–10); novel electrochemical DNA biosensors based on solid electrodes were also developed (11– 15). Several solid materials, such as gold (9), carbon paste (7, 8), glassy carbon (10), and graphite (16), were employed to carry out research on the electrochemical behavior of DNA. However, overpotentials of DNA at these reported electrodes are usually very high. For example, the anodic peak obtained at a carbon paste electrode, coming from the oxidation of guanine bases, is located at ca. 1.03 V (8). What’s more, voltammetric peaks are poorly developed; thus, the sensitivity of electrochemical analysis of DNA is generally low, except when a kind of highly sensitive new technique, potentiometric stripping analysis, was employed (7, 8, 11). Therefore, it still remains a central problem to find an appropriate interface for the study of DNA and to determine its concentration in solution. Many studies (1, 2, 7, 8, 16 –18) have shown that voltammetric responses of DNA at the surface of electrodes arise from the redox reactions of purine and/or pyrimidine bases. It was proposed that the peak potentials of DNA at carbon paste electrodes were characteristic of oxidation of the bound guanine (G) residues. 1
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FAN ET AL.
FIG. 1. Cyclic voltammograms obtained at a silver electrode after the addition of 2 mmol/L (a) adenine, (b) deoxyadenosine, (c) dATP in a 0.20 mol/L NaAc–HAc buffer solution at pH 5.5, and (d) in the same background solution. Scan rate: 40 mV/s.
Adenine (A) and guanine (G) bases can be oxidized at gold (9, 19), glassy carbon (10), or pyrolytic graphite electrodes (11), while adenine (A) and cytosine (C) bases are reduced at mercury electrodes (1, 2). Our previous studies (20) showed that coenzyme I, dihydronicotinamide adenine dinucleotide (NADH), 2 which had adenine as a moiety of its molecule, exhibited a good electrochemical response with low overpotentials at the surface of silver electrodes. Accordingly, electrochemistry of purine bases (adenine and guanine) was performed, confirming that purine bases and purine derivatives could show good electrochemical behavior on the surfaces of metallic silver. These results imply that a silver electrode might be also a suitable interface 2 Abbreviations used: CV, cyclic voltammetry; DPASV, differential pulse adsorptive stripping voltammetry; NADH, dihydronicotinamide adenine dinucleotide; dATP, deoxyadenosine 59-triphosphate; dCTP, deoxycytidine 59-triphosphate; dGTP, deoxyguanosine 59-triphosphate; dTTP, deoxythymidine 59-triphosphate; dNTP, deoxynucleotide 59-triphosphate.
to the electrochemical study of nucleic acids. For these reasons, silver electrodes were employed in the present research and the electrochemical behavior of DNA at the electrodes is discussed. To our knowledge, this is the first time that direct oxidation and reduction of DNA was achieved with low overpotentials at a solid electrode. MATERIALS AND METHODS
Materials. Salmon sperm DNA (Sodium Salt) and calf thymus DNA were purchased from Sigma Chemical Co. (St. Louis) and were further purified by extraction with phenol and chloroform and precipitation with ethanol. Reduction of the molecular weight of the nucleic acids was achieved through sonication to about 200 bp, estimated via agarose gel electrophoresis. Thermal denaturation was performed by heating DNA in a water bath at 100°C for 10 min and subsequently cooling it quickly in an ice bath. Deoxyadenosine 59-triphosphate (dATP), deoxycyti-
VOLTAMMETRIC RESPONSE AND DETERMINATION OF DNA
FIG. 2. Cyclic voltammograms obtained at a silver electrode in a 0.20 mol/L NaAc–HAc buffer solution at pH 5.5. (a) Before and (b) after 300 mg/L thermally denatured salmon sperm DNA (ssDNA) was added. Scan rate: 40 mV/s.
dine 59-triphosphate (dCTP), deoxyguanosine 59-triphosphate (dGTP), and deoxythymidine 59-triphosphate (dTTP) were obtained from Boehringer (Germany). Adenine and deoxyadenosine were from Sigma. Silver metal was obtained from the Shanghai Dian Guang Device Works. Its purity was 99.99%. Other chemicals were all of analytical grade. All solutions were prepared with double-distilled water and deaerated with high-purity nitrogen. Preparation of silver electrodes. The silver electrode was pretreated as follows. First, it was polished by rough and fine sand papers, respectively, and then polished to mirror smoothness with alumina (particle size of about 0.05 mm) water slurry on silk. Adsorbates on the electrode were removed by washing the electrode thoroughly with double-distilled water and then treating the electrode in an ultrasonic pool for about 5 min. Electrochemical measurements. Electrochemical measurements were performed with a Potentiostat/Galvanostat PAR 263 (EG&G, USA) using a three-electrode configuration. A Multi-Block Heater (LAB-LINE Instruments, Inc., USA) was employed to control the experimental temperature. A 2-mm-diameter silver disc was used as the working electrode. A saturated calomel electrode and a platinum electrode served as the reference electrode and the counter electrode, respectively. These three electrodes were placed in a glass electrochemical cell and connected to the PAR263. The cell was placed in the Multi-Block Heater to keep the experimental temperature at 20 6 0.5°C.
3
electrochemical research of DNA. The cyclic voltammetry of adenine was performed in a 0.20 mol/L NaAc– HAc buffer solution with pH 5.5 at the silver electrode, the result is shown in Fig. 1a. It’s apparent that a pair of redox peaks arose and thus proved the electroactivity of adenine in this potential range at the bare silver electrode. Two kinds of derivatives of adenine, deoxyadenosine and deoxyadenosine triphosphate (dATP), were also tested. The results obtained were similar to those for adenine and are shown separately in Figs. 1b and 1c. Figure 1d is the cyclic voltammogram (CV) obtained at a bare silver electrode; no redox wave could be seen. Electrochemistry research of DNA at the surface of silver electrode. Figure 2a is the CV obtained at a silver electrode in the background solution. Figure 2b displays the cyclic voltammogram of 300 mg/mL thermally denatured salmon sperm DNA (ssDNA, ;200 bp) in the same buffer solution. It can be observed that redox waves arose compared with Fig. 2a. The anodic and cathodic peak potentials are at about 0.24 and 0.13 V, respectively. Further experimental results revealed that the peak currents were proportional to DNA concentration. But the peak potentials didn’t change. Therefore, it can be deduced that ssDNA can be oxidized at a low overpotential and a reduction reaction occurs at the same time. As is shown in Fig. 3, plot of the anodic peak current of 300 mg/mL ssDNA in the 0.20 mol/L NaAc–HAc buffer solution against the scan rate is linear in the range of 20 –100 mV/s, which is in accord with the redox reaction of an adsorbed species (21). In fact, when an electrode which had been immersed in an ssDNA solution was transferred into a background solution, the peaks of DNA could be still observed in original scans, and would diminish in subsequent scans, which thus indicated weak adsorption at the electrode surface (Fig. 4). As documented previously, the voltammetric re-
RESULTS AND DISCUSSION
Electrochemistry behavior of adenine and its derivatives. The electrochemistry behavior of adenine and its derivatives was first studied as a basis for the
FIG. 3. Relationship between the cyclic voltammetric anodic peak current of ssDNA and the scan rate.
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FIG. 4. Cyclic voltammograms of 300 mg/mL (a) native DNA (double stranded) and (b) thermally denatured DNA (single stranded) in a 0.20 mol/L NaAc–HAc buffer solution at pH 5.5. Scan rate: 40 mV/s.
sponses of DNA at the surface of electrodes are due to the redox reactions of the purine and/or pyrimidine bases. It was confirmed here that electrochemistry of
DNA at a silver electrode was via the redox reactions of purine bases, i.e., A and G. In order to avoid the problem of low solubility of bases in water, four kinds of dNTPs were used instead. Figure 5 is the cyclic voltammograms of 2 mmol/L (a) dATP, (b) dTTP, (c) dCTP, and (d) dGTP in the background solution. It showed that electrochemical behavior of these substances was quite different. Pyrimidine-derivative compounds, dCTP and dTTP, would give no faradaic signal. On the contrary, purine-derivative compounds, dATP and dGTP, could exhibit current responses at the silver electrode with peak potentials close to those of DNA. The mixture of (a) dATP and dGTP, (b) dCTP and dTTP, and (c) four kinds of dNTPs (0.5 mmol/L for each dNTP) was also examined. As is shown in Fig. 6, the mixture of dCTP and dTTP could not give a current response, while the other two could. It was further shown that the current response of nucleic acids mainly came from the moiety of nucleic acidic bases. The cyclic voltammograms of 2 mmol/L (a) adenine, (b) deoxyadenosine, and (c) dATP in the background solution were shown in Fig. 1. The three figures are simi-
FIG. 5. Cyclic voltammograms of 2 mmol/L (a) dATP, (b) dTTP, (c) dCTP, and (d) dATP in a 0.20 mol/L NaAc–HAc buffer solution at pH 5.5. Scan rate: 40 mV/s.
VOLTAMMETRIC RESPONSE AND DETERMINATION OF DNA
5
FIG. 6. Cyclic voltammograms of the mixture of (a) dATP and dGTP, (b) dCTP and dTTP, and (c) four kinds of dNTPs (0.5 mmol/L for each dNTP) dATP in a 0.20 mol/L NaAc–HAc buffer solution at pH 5.5. Scan rate: 40 mV/s.
lar, which therefore suggests that redox reactions of bases determine the electrochemical response of nucleic acids. This result was in agreement with previous studies using surface enhanced raman spectra, which suggested that for the oligo- and polynucleotides, the adenine base was the adsorbing moiety to the electrode surface, while the phosphate–sugar backbone chain remained directed toward the solution (22). Denatured and native DNA have different electrochemical behaviors at the electrode surface. With denatured DNA it was much easier to exhibit current response than with native DNA. Figures 4a and 4b are the CV curves of 300 mg/mL native DNA (double stranded) and 300 mg/mL thermally denatured DNA (single stranded), respectively, in the background solution. It could be observed that the peak current of native dsDNA was much less than that of thermally denatured ssDNA, and the peak form of denatured DNA is better. Moreover, when the concentration of DNA was lowered to 10 mg/mL, the current response of ssDNA could be still observed, while no apparent peaks for dsDNA could be seen (no preconcentration proce-
dure was employed here). It is proposed that such a large difference might be attributed to the double-helix structure of native DNA. As described above, purine bases are the electroactive part of DNA at the silver electrode. In native dsDNA, the primary redox sites of adenine and guanine are hidden in the interior of the double-helical molecule, forming a part of the Waston– Crick hydrogen bonding system, while in ssDNA these sites are freely accessible for interaction with the environment. There was also a measurable difference between small fragments of ssDNA (ca. 200 bp, obtained via sonication as described under Materials and Methods) and intact ssDNA (several kb). Experimental results revealed that the electrochemical response of small fragments of ssDNA was not only higher but also more stable. The better response of fragmented ssDNA might also be due to the decrease of steric hindrance. The effect of different origins of DNA on their electrochemical behaviors was tested. Calf thymus DNA and salmon sperm DNA were used under the same conditions as a comparison, but no apparent difference
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FAN ET AL.
The detection limits of dsDNA and ssDNA employing UV absorbance are 0.5 and 0.4 mg/mL, respectively. Therefore, the detection limit of dsDNA of this voltammetric method is close to that of UV absorbance, while the detection limit of ssDNA of the voltammetric method is lowered nearly 10-fold. The difference in the detection limit of dsDNA and ssDNA could be used to selectively determine ssDNA in the presence of native dsDNA. It was observed that in a DPASV determination, the peak current of 1.0 mg/mL ssDNA in the presence of 1.0 mg/mL dsDNA just slightly increased (,10%) compared with that of 1.0 mg/mL ssDNA in the absence of dsDNA. FIG. 7. Effect of accumulation time on the DPASV (differential pulse adsorptive stripping voltammetric) peak current of 1 mg/mL ssDNA at the silver electrode. In DPASV, a 0.20 mol/L NaAc–HAc buffer (pH 5.5) was used as the background solution (scan rate, 60 mV/s; time step, 200 ms; pulse height, 50 mV; pulse width, 50 ms; initial potential, 20.10 V; final potential, 0.40 V).
could be observed. This implies that the structure complexity and length of DNA play an essential role in the electrochemical response of it while the difference in the base sequence is a minor factor. Determination of traces of DNA. A kind of sensitive electrochemical technique, differential pulse adsorptive stripping voltammetry (DPASV), was employed to determine traces of DNA in the range of 20.10 – 0.40 V. Because nucleic acids adsorbed to the silver electrode surface, the preconcentration of DNA onto the silver electrodes could be used as an effective way to improve determination sensitivity. The effect of accumulation time (t acc) and accumulation potential (E acc) on the current response of the DNA was examined. Figure 7 shows the dependence of the anodic peak current on the accumulation time, which showed that the peak current would reach a maximum after 5 min of accumulation. The variation of accumulation potentials in the range 0.10 – 0.4 V did not have much influence on the height of peak currents, so 0.25 V was chosen as the accumulation potential here. DPASV was thus performed under such optimum experimental conditions. Fig. 8a displays the plot of the anodic peak current for a series of ssDNA concentrations. A linear dependence of the peak currents on ssDNA concentrations was observed in the range 0.5–2.5 mg/mL. The relative standard deviation was 3.5% for six successive determinations at 0.5 mg/mL. The detection limit was 50 ng/mL (estimated from three times the signal-tonoise). Similar experiments were made to determine traces of dsDNA. Fig. 8b displays calibration data for dsDNA in the range of 2.5–15 mg/mL. The relative standard deviation was 4.2% for six successive determinations at 2.5 mg/mL. The detection limit was 300 ng/mL.
CONCLUSIONS
Electrochemical techniques have not been extensively exploited in recent years in nucleic acid analysis compared with electrophoretic, chromatographic, and
FIG. 8. Calibration plots for ssDNA (a) and dsDNA (b). In DPASV, accumulation time was 5 min and accumulation potential was 0.30 V. Others conditions were the same as those described in the legend to Fig. 7.
VOLTAMMETRIC RESPONSE AND DETERMINATION OF DNA
spectroscopic methods (23). Part of the reason might be the lack of suitable interfaces for the electron transfer of DNA. As is discussed here, the silver electrode provides an appropriate interface for the research of DNA. Determination of traces of DNA was carried out and good results were obtained. The selectivity of singlestranded and double-stranded DNA at the silver electrode also provides a convenient way to determine ssDNA in the presence of native dsDNA. Because the structure and length exert influence on the electrochemical behavior of DNA, it might be useful in detecting structure variations of DNA and the interactions of DNA with drugs, enzymes, etc. Also, this technique, as a basic research of DNA electrochemical biosensors, might open the door to modern nucleic acidic probes and detectors. ACKNOWLEDGMENT We greatly appreciate the support of the Science Foundation of Jiangsu Province, People’s Republic of China, for this research.
REFERENCES 1. 2. 3. 4.
Palecek, E. (1986) Bioelectrochem. Bioenerg. 15, 275–295. Palecek, E. (1988) Bioelectrochem. Bioenerg. 20, 179 –194. Palecek, E. (1976) Prog. Nucleic Acid Res. 18, 151–167. Wang, J., Grant, D. H., Ozsoz, M., Cai, X., Tian, B., and Fernandes, J. R. (1997) Anal. Chim. Acta 349, 77– 83. 5. Fojta, M., Teijeiro, C., and Palecek, E. (1994) Bioelectrochem. Bioenerg. 34, 69 –76. 6. Jelen, F., Fojta, M., and Palecek, E. (1997) J. Electroanal. Chem. 427, 49 –56.
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7. Wang, J., Cai, X., Wang, J., and Jonsson, C. (1995) Anal. Chem. 67, 4065– 4070. 8. Cai, X., Rivas, G., Farias, P. A. M., Shiraishi, H., Wang, J., Fojta, M., and Palecek, E. (1996) Bioelectrochem. Bioenerg. 40, 41– 47. 9. Pang, D., Qi, Y., Wang, Z., Cheng, J., and Wang, J. (1995) Electroanalysis 7, 774 –777. 10. Brett, C. M. A., Brett, A. M. O., and Serrano, S. H. P. (1994) J. Electroanal. Chem. 366, 225–231. 11. Wang, J., Palecek, E., Nielsen, P. E., Rivas, G., Cai, X., Shiraishi, H., Dontha, N., Luo, D., and Farias, P. A. M. (1996) J. Am. Chem. Soc. 118, 7667–7670. 12. Millan, K. M., and Mikkelsen, S. R. (1993) Anal. Chem. 65, 2317–2323. 13. Hashimoto, K., Ito, K., and Ishimori, Y. (1994) Anal. Chem. 66, 3830 –3833. 14. Hashimoto, K., Ito, K., and Ishimori, Y. (1994) Anal. Chim. Acta 286, 219 –224. 15. Liu, S., Ye, J., He, P., and Fang, Y. (1996) Anal. Chim. Acta 335, 239 –243. 16. Brabec, V., and Dryhurst, G. (1978) J. Electroanal. Chem. 89, 161–173. 17. Kafil, J., Cheng, H. Y., and Last, T. (1986) Anal. Chem. 58, 285–289. 18. Sequaris, J. M., and Valenta, P. (1987) J. Electroanal. Chem. 227, 11–20. 19. Lindsay, S. M., Tao, N. J., DeRose, J. A., Oden, P. I., and Lyubchenko, Y. L. (1992) Biophys. J. 61, 1570 –1584. 20. Li, G., Zhu, J., Fang, H., Chen, H., and Zhu, D. (1996) J. Electrochem. Soc. 143, L141–142. 21. Laviron, E. (1979) J. Electroanal. Chem. 100, 263–270. 22. Ervin, K. M., Koglin, E., Sequaris, J. M., Valenta, P., and Nurnberg, H. W. (1980) J. Electroanal. Chem. 114, 179 –194. 23. Palecek, E., and Fojta, M. (1994) Anal. Chem. 66, 1566 –1571.
Voltammetric Response and Determination of DNA with a Silver Electrode Chunhai Fan,* Haiyun Song,* Xiaofang Hu,* Genxi Li,* Jianqin Zhu,† Xianxiu Xu,* and Dexu Zhu* ,1 *Department of Biochemistry and National Laboratory of Pharmaceutical Biotechnology, and †Department of Biology and Biological Technology, Nanjing University, Nanjing 210093, People’s Republic of China
Received October 29, 1998
A current from DNA was obtained using a silver electrode with low overpotentials for the first time. Experimental results revealed that the voltammetric response of DNA was attributed to the redox reactions of purine bases. It was also shown that such a method provided a convenient and practical way to determine DNA. A linear dependence of the peak currents on ssDNA concentrations was observed in the range 0.5– 2.5 mg/mL. The relative standard deviation was 3.5% for six successive determinations at 0.5 mg/mL. The detection limit was 50 ng/mL. Influence of the structure and the length of the nucleic acids on their electrochemical behavior was discussed. In view of the merits of the silver electrode, this technique might provide new possibilities for further electrochemical research and determination of nucleic acids. © 1999 Academic Press
Key Words: electrochemistry; silver electrode; DNA.
The behavior of nucleic acids at charged biological interfaces (e.g., membranes) is of great importance in many biological processes. However, the complexity of their structures and properties has until now hindered a theoretical treatment of their behavior at electrically charged surfaces (1). The electrochemical system serves as a versatile and illuminating model of biological system despite obvious physical and chemical differences (1, 2). Systematic voltammetric studies of nucleic acids and their component units have provided much important information on the properties and conformation of such substances in solutions and on electrode surfaces. These include early electrochemical ev1 To whom correspondence should be addressed. Fax: (186)(25) 3607621. E-mail:[email protected].
0003-2697/99 $30.00 Copyright © 1999 by Academic Press All rights of reproduction in any form reserved.
idence for DNA premelting and the polymorphy of the DNA double helix, which have been widely accepted (1, 3). Most of these studies employed mercury as the electrode material (1, 2, 4 – 6). Solid electrodes have not been intensively explored due to large background current produced at these surfaces (7). However, a mercury electrode is toxic and has limited applications due to its fluid state. Solid electrodes have some advantages, which make them attractive as flow detectors following HPLC and capillary zone electrophoresis, various kinds of sensors, etc. (8). Recently, substantial interest has been shown in the study of the interfacial behavior of nucleic acids at different kinds of electrode materials (7–10); novel electrochemical DNA biosensors based on solid electrodes were also developed (11– 15). Several solid materials, such as gold (9), carbon paste (7, 8), glassy carbon (10), and graphite (16), were employed to carry out research on the electrochemical behavior of DNA. However, overpotentials of DNA at these reported electrodes are usually very high. For example, the anodic peak obtained at a carbon paste electrode, coming from the oxidation of guanine bases, is located at ca. 1.03 V (8). What’s more, voltammetric peaks are poorly developed; thus, the sensitivity of electrochemical analysis of DNA is generally low, except when a kind of highly sensitive new technique, potentiometric stripping analysis, was employed (7, 8, 11). Therefore, it still remains a central problem to find an appropriate interface for the study of DNA and to determine its concentration in solution. Many studies (1, 2, 7, 8, 16 –18) have shown that voltammetric responses of DNA at the surface of electrodes arise from the redox reactions of purine and/or pyrimidine bases. It was proposed that the peak potentials of DNA at carbon paste electrodes were characteristic of oxidation of the bound guanine (G) residues. 1
2
FAN ET AL.
FIG. 1. Cyclic voltammograms obtained at a silver electrode after the addition of 2 mmol/L (a) adenine, (b) deoxyadenosine, (c) dATP in a 0.20 mol/L NaAc–HAc buffer solution at pH 5.5, and (d) in the same background solution. Scan rate: 40 mV/s.
Adenine (A) and guanine (G) bases can be oxidized at gold (9, 19), glassy carbon (10), or pyrolytic graphite electrodes (11), while adenine (A) and cytosine (C) bases are reduced at mercury electrodes (1, 2). Our previous studies (20) showed that coenzyme I, dihydronicotinamide adenine dinucleotide (NADH), 2 which had adenine as a moiety of its molecule, exhibited a good electrochemical response with low overpotentials at the surface of silver electrodes. Accordingly, electrochemistry of purine bases (adenine and guanine) was performed, confirming that purine bases and purine derivatives could show good electrochemical behavior on the surfaces of metallic silver. These results imply that a silver electrode might be also a suitable interface 2 Abbreviations used: CV, cyclic voltammetry; DPASV, differential pulse adsorptive stripping voltammetry; NADH, dihydronicotinamide adenine dinucleotide; dATP, deoxyadenosine 59-triphosphate; dCTP, deoxycytidine 59-triphosphate; dGTP, deoxyguanosine 59-triphosphate; dTTP, deoxythymidine 59-triphosphate; dNTP, deoxynucleotide 59-triphosphate.
to the electrochemical study of nucleic acids. For these reasons, silver electrodes were employed in the present research and the electrochemical behavior of DNA at the electrodes is discussed. To our knowledge, this is the first time that direct oxidation and reduction of DNA was achieved with low overpotentials at a solid electrode. MATERIALS AND METHODS
Materials. Salmon sperm DNA (Sodium Salt) and calf thymus DNA were purchased from Sigma Chemical Co. (St. Louis) and were further purified by extraction with phenol and chloroform and precipitation with ethanol. Reduction of the molecular weight of the nucleic acids was achieved through sonication to about 200 bp, estimated via agarose gel electrophoresis. Thermal denaturation was performed by heating DNA in a water bath at 100°C for 10 min and subsequently cooling it quickly in an ice bath. Deoxyadenosine 59-triphosphate (dATP), deoxycyti-
VOLTAMMETRIC RESPONSE AND DETERMINATION OF DNA
FIG. 2. Cyclic voltammograms obtained at a silver electrode in a 0.20 mol/L NaAc–HAc buffer solution at pH 5.5. (a) Before and (b) after 300 mg/L thermally denatured salmon sperm DNA (ssDNA) was added. Scan rate: 40 mV/s.
dine 59-triphosphate (dCTP), deoxyguanosine 59-triphosphate (dGTP), and deoxythymidine 59-triphosphate (dTTP) were obtained from Boehringer (Germany). Adenine and deoxyadenosine were from Sigma. Silver metal was obtained from the Shanghai Dian Guang Device Works. Its purity was 99.99%. Other chemicals were all of analytical grade. All solutions were prepared with double-distilled water and deaerated with high-purity nitrogen. Preparation of silver electrodes. The silver electrode was pretreated as follows. First, it was polished by rough and fine sand papers, respectively, and then polished to mirror smoothness with alumina (particle size of about 0.05 mm) water slurry on silk. Adsorbates on the electrode were removed by washing the electrode thoroughly with double-distilled water and then treating the electrode in an ultrasonic pool for about 5 min. Electrochemical measurements. Electrochemical measurements were performed with a Potentiostat/Galvanostat PAR 263 (EG&G, USA) using a three-electrode configuration. A Multi-Block Heater (LAB-LINE Instruments, Inc., USA) was employed to control the experimental temperature. A 2-mm-diameter silver disc was used as the working electrode. A saturated calomel electrode and a platinum electrode served as the reference electrode and the counter electrode, respectively. These three electrodes were placed in a glass electrochemical cell and connected to the PAR263. The cell was placed in the Multi-Block Heater to keep the experimental temperature at 20 6 0.5°C.
3
electrochemical research of DNA. The cyclic voltammetry of adenine was performed in a 0.20 mol/L NaAc– HAc buffer solution with pH 5.5 at the silver electrode, the result is shown in Fig. 1a. It’s apparent that a pair of redox peaks arose and thus proved the electroactivity of adenine in this potential range at the bare silver electrode. Two kinds of derivatives of adenine, deoxyadenosine and deoxyadenosine triphosphate (dATP), were also tested. The results obtained were similar to those for adenine and are shown separately in Figs. 1b and 1c. Figure 1d is the cyclic voltammogram (CV) obtained at a bare silver electrode; no redox wave could be seen. Electrochemistry research of DNA at the surface of silver electrode. Figure 2a is the CV obtained at a silver electrode in the background solution. Figure 2b displays the cyclic voltammogram of 300 mg/mL thermally denatured salmon sperm DNA (ssDNA, ;200 bp) in the same buffer solution. It can be observed that redox waves arose compared with Fig. 2a. The anodic and cathodic peak potentials are at about 0.24 and 0.13 V, respectively. Further experimental results revealed that the peak currents were proportional to DNA concentration. But the peak potentials didn’t change. Therefore, it can be deduced that ssDNA can be oxidized at a low overpotential and a reduction reaction occurs at the same time. As is shown in Fig. 3, plot of the anodic peak current of 300 mg/mL ssDNA in the 0.20 mol/L NaAc–HAc buffer solution against the scan rate is linear in the range of 20 –100 mV/s, which is in accord with the redox reaction of an adsorbed species (21). In fact, when an electrode which had been immersed in an ssDNA solution was transferred into a background solution, the peaks of DNA could be still observed in original scans, and would diminish in subsequent scans, which thus indicated weak adsorption at the electrode surface (Fig. 4). As documented previously, the voltammetric re-
RESULTS AND DISCUSSION
Electrochemistry behavior of adenine and its derivatives. The electrochemistry behavior of adenine and its derivatives was first studied as a basis for the
FIG. 3. Relationship between the cyclic voltammetric anodic peak current of ssDNA and the scan rate.
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FAN ET AL.
FIG. 4. Cyclic voltammograms of 300 mg/mL (a) native DNA (double stranded) and (b) thermally denatured DNA (single stranded) in a 0.20 mol/L NaAc–HAc buffer solution at pH 5.5. Scan rate: 40 mV/s.
sponses of DNA at the surface of electrodes are due to the redox reactions of the purine and/or pyrimidine bases. It was confirmed here that electrochemistry of
DNA at a silver electrode was via the redox reactions of purine bases, i.e., A and G. In order to avoid the problem of low solubility of bases in water, four kinds of dNTPs were used instead. Figure 5 is the cyclic voltammograms of 2 mmol/L (a) dATP, (b) dTTP, (c) dCTP, and (d) dGTP in the background solution. It showed that electrochemical behavior of these substances was quite different. Pyrimidine-derivative compounds, dCTP and dTTP, would give no faradaic signal. On the contrary, purine-derivative compounds, dATP and dGTP, could exhibit current responses at the silver electrode with peak potentials close to those of DNA. The mixture of (a) dATP and dGTP, (b) dCTP and dTTP, and (c) four kinds of dNTPs (0.5 mmol/L for each dNTP) was also examined. As is shown in Fig. 6, the mixture of dCTP and dTTP could not give a current response, while the other two could. It was further shown that the current response of nucleic acids mainly came from the moiety of nucleic acidic bases. The cyclic voltammograms of 2 mmol/L (a) adenine, (b) deoxyadenosine, and (c) dATP in the background solution were shown in Fig. 1. The three figures are simi-
FIG. 5. Cyclic voltammograms of 2 mmol/L (a) dATP, (b) dTTP, (c) dCTP, and (d) dATP in a 0.20 mol/L NaAc–HAc buffer solution at pH 5.5. Scan rate: 40 mV/s.
VOLTAMMETRIC RESPONSE AND DETERMINATION OF DNA
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FIG. 6. Cyclic voltammograms of the mixture of (a) dATP and dGTP, (b) dCTP and dTTP, and (c) four kinds of dNTPs (0.5 mmol/L for each dNTP) dATP in a 0.20 mol/L NaAc–HAc buffer solution at pH 5.5. Scan rate: 40 mV/s.
lar, which therefore suggests that redox reactions of bases determine the electrochemical response of nucleic acids. This result was in agreement with previous studies using surface enhanced raman spectra, which suggested that for the oligo- and polynucleotides, the adenine base was the adsorbing moiety to the electrode surface, while the phosphate–sugar backbone chain remained directed toward the solution (22). Denatured and native DNA have different electrochemical behaviors at the electrode surface. With denatured DNA it was much easier to exhibit current response than with native DNA. Figures 4a and 4b are the CV curves of 300 mg/mL native DNA (double stranded) and 300 mg/mL thermally denatured DNA (single stranded), respectively, in the background solution. It could be observed that the peak current of native dsDNA was much less than that of thermally denatured ssDNA, and the peak form of denatured DNA is better. Moreover, when the concentration of DNA was lowered to 10 mg/mL, the current response of ssDNA could be still observed, while no apparent peaks for dsDNA could be seen (no preconcentration proce-
dure was employed here). It is proposed that such a large difference might be attributed to the double-helix structure of native DNA. As described above, purine bases are the electroactive part of DNA at the silver electrode. In native dsDNA, the primary redox sites of adenine and guanine are hidden in the interior of the double-helical molecule, forming a part of the Waston– Crick hydrogen bonding system, while in ssDNA these sites are freely accessible for interaction with the environment. There was also a measurable difference between small fragments of ssDNA (ca. 200 bp, obtained via sonication as described under Materials and Methods) and intact ssDNA (several kb). Experimental results revealed that the electrochemical response of small fragments of ssDNA was not only higher but also more stable. The better response of fragmented ssDNA might also be due to the decrease of steric hindrance. The effect of different origins of DNA on their electrochemical behaviors was tested. Calf thymus DNA and salmon sperm DNA were used under the same conditions as a comparison, but no apparent difference
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FAN ET AL.
The detection limits of dsDNA and ssDNA employing UV absorbance are 0.5 and 0.4 mg/mL, respectively. Therefore, the detection limit of dsDNA of this voltammetric method is close to that of UV absorbance, while the detection limit of ssDNA of the voltammetric method is lowered nearly 10-fold. The difference in the detection limit of dsDNA and ssDNA could be used to selectively determine ssDNA in the presence of native dsDNA. It was observed that in a DPASV determination, the peak current of 1.0 mg/mL ssDNA in the presence of 1.0 mg/mL dsDNA just slightly increased (,10%) compared with that of 1.0 mg/mL ssDNA in the absence of dsDNA. FIG. 7. Effect of accumulation time on the DPASV (differential pulse adsorptive stripping voltammetric) peak current of 1 mg/mL ssDNA at the silver electrode. In DPASV, a 0.20 mol/L NaAc–HAc buffer (pH 5.5) was used as the background solution (scan rate, 60 mV/s; time step, 200 ms; pulse height, 50 mV; pulse width, 50 ms; initial potential, 20.10 V; final potential, 0.40 V).
could be observed. This implies that the structure complexity and length of DNA play an essential role in the electrochemical response of it while the difference in the base sequence is a minor factor. Determination of traces of DNA. A kind of sensitive electrochemical technique, differential pulse adsorptive stripping voltammetry (DPASV), was employed to determine traces of DNA in the range of 20.10 – 0.40 V. Because nucleic acids adsorbed to the silver electrode surface, the preconcentration of DNA onto the silver electrodes could be used as an effective way to improve determination sensitivity. The effect of accumulation time (t acc) and accumulation potential (E acc) on the current response of the DNA was examined. Figure 7 shows the dependence of the anodic peak current on the accumulation time, which showed that the peak current would reach a maximum after 5 min of accumulation. The variation of accumulation potentials in the range 0.10 – 0.4 V did not have much influence on the height of peak currents, so 0.25 V was chosen as the accumulation potential here. DPASV was thus performed under such optimum experimental conditions. Fig. 8a displays the plot of the anodic peak current for a series of ssDNA concentrations. A linear dependence of the peak currents on ssDNA concentrations was observed in the range 0.5–2.5 mg/mL. The relative standard deviation was 3.5% for six successive determinations at 0.5 mg/mL. The detection limit was 50 ng/mL (estimated from three times the signal-tonoise). Similar experiments were made to determine traces of dsDNA. Fig. 8b displays calibration data for dsDNA in the range of 2.5–15 mg/mL. The relative standard deviation was 4.2% for six successive determinations at 2.5 mg/mL. The detection limit was 300 ng/mL.
CONCLUSIONS
Electrochemical techniques have not been extensively exploited in recent years in nucleic acid analysis compared with electrophoretic, chromatographic, and
FIG. 8. Calibration plots for ssDNA (a) and dsDNA (b). In DPASV, accumulation time was 5 min and accumulation potential was 0.30 V. Others conditions were the same as those described in the legend to Fig. 7.
VOLTAMMETRIC RESPONSE AND DETERMINATION OF DNA
spectroscopic methods (23). Part of the reason might be the lack of suitable interfaces for the electron transfer of DNA. As is discussed here, the silver electrode provides an appropriate interface for the research of DNA. Determination of traces of DNA was carried out and good results were obtained. The selectivity of singlestranded and double-stranded DNA at the silver electrode also provides a convenient way to determine ssDNA in the presence of native dsDNA. Because the structure and length exert influence on the electrochemical behavior of DNA, it might be useful in detecting structure variations of DNA and the interactions of DNA with drugs, enzymes, etc. Also, this technique, as a basic research of DNA electrochemical biosensors, might open the door to modern nucleic acidic probes and detectors. ACKNOWLEDGMENT We greatly appreciate the support of the Science Foundation of Jiangsu Province, People’s Republic of China, for this research.
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