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Determination of Nitric Oxide by Glassy Carbon Electrodes Modified with Poly(Neutral Red)
Microchemical Journal 62, 377–385 (1999) Article ID mchj.1999.1737, available online at http://www.idealibrary.com on
Determination of Nitric Oxide by Glassy Carbon Electrodes Modified with Poly(Neutral Red) Tang Xiaorong, Fang Cheng, Yao Bing, and Zhang Wuming 1 Department of Chemistry, Wuhan University, Wuhan 430072, People’s Republic of China E-mail: [email protected] Received October 20, 1998; accepted March 17, 1999 Nitric oxide can be oxidized to NO 22 (then to NO 32) and reduced to N 2O at a glassy carbon electrode modified with the film of poly(neutral red). When the modified electrode is further coated with a thin Nafion film, the interference of NO 22 can be almost thoroughly eliminated, and the electrode can be employed to detect nitric oxide. © 1999 Academic Press Key Words: electropolymerized film; chemically modified electrode; neutral red; nitric oxide.
1. INTRODUCTION In 1987, it was discovered that vascular endothelial cells could synthesize the freeradical gas nitric oxide (NO) (1), and NO could act as a vasodilatory messenger (2). As the endothelial-derived relaxing factor (EDRF), NO is responsible for activating guanylate cyclase, the essential enzyme involved in the control of vasodilation. From then on, much attention was given to the study of NO in biological systems (3– 6), and many other important physiological functions of NO were discovered (8 –11). Therefore, the direct determination of NO has become attractive, especially in biological models. Unfortunately, due to its low stability and high fugacity, it is very difficult to measure NO directly. In fact, the half-life of NO is very short (ca. 6s). Moreover, NO is easily oxidized by O 2 or other oxidants to nitrite (NO 22) and even to nitrate (NO 32). Most measurement techniques for NO involve indirect chemical detection (12, 13). Because of their inherent speed and sensitivity, electrochemical methods are one of the most advantageous and well-suited methods for detection in biological media (14, 15). One of the electrochemical approaches to NO detection uses modified platinum electrodes, on which NO is directly oxidized. The modified membranes include chloroprene, nitrocellulose and silicone, and Nafion and cellulose acetate (16). Another approach is based on the electrocatalytic oxidation of NO on a carbon fiber electrode coated with nickel (II) tetrakis (3-methoxy-4-hydroxyphenyl) porphyrin (Ni-TMPP) and Nafion film (17–20) or modified with o-phenylenediamine (21). Maskus (22) reported electropolymerized films of (Cr(v-tpy) 2) 31 and their application to detecting cellular NO from Rhodobacter sphaeroides bacterial cells. NO has very complex redox behavior. The electrochemical behavior of NO on a rotating platinum electrode in 4 mol/liter H 2SO 4 has been studied (23). There are two steps in the process of NO oxidation: 1
To whom correspondence should be addressed. Fax: 86-027-87882661.
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HNO2 1 H 1 1 e 2 3 NO~g! 1 H2O.
(1)
NO is oxidized to HNO 2 via a kinetically fast read with a standard potential of 10.95 V (24). The product of the first step reaction, HNO 2, is afterwards oxidized to NO 32: 1 2 NO 2 3 NO 2 3 1 2H 1 2e 2 1 H2O.
(2)
On the other hand, there are also two steps in the process of NO reduction: 2NO 1 2H 1 1 2e 2 3 N2O 1 H2O.
(3)
Such a reaction is fast. In the second step, N 2O can be further reduced to NH 2OH, N 2H 4, or NH 3: N2O 1 4H 1 1 H2O 1 4e 2 3 2NH2OH.
(4)
N2O 1 6H 1 1 6e 2 3 N2H4 1 H2O.
(5)
N2O 1 8H 1 1 8e 2 3 2NH3 1 H2O.
(6)
However, the redox process of NO is very complicated. The following reaction may occur also: 2 2 2NO~aq! 1 NO 2 3 1 2OH 5 3NO 2 1 H2O.
(7)
In our laboratory, the glassy carbon electrodes modified with electropolymerized films of neutral red were studied comprehensively (25–27). Such an electropolymer has very good electrocatalytic activity toward the redox of small molecular compounds such as vitamin C, dopamine, and adrenaline (26). Reasoning that the film might also be active in the electrocatalytic redox of nitric oxide (NO), we have carried out such studies and have found that the poly(neutral red) film is quite active in the electrochemical redox of nitric oxide. 2. EXPERIMENTAL The glassy carbon electrodes modified with electropolymerized films of neutral red (PNR) were prepared as previously (26). All solutions were prepared with fresh doubledistilled water and were deoxygenated by purging with prepurified nitrogen gas for 20 min. High-purity nitric oxide (99.7%) was obtained from Sumitomo Seika Chemical Co. The standard saturated NO solution (typically containing 1.9 mmol/liter NO at 20°C) was obtained as follows: NO gas was first passed through a washing bottle containing 2 mol/L NaOH to remove possible NO 2 and other impurities, and then passed into pH 7.0 oxygen-free phosphate buffer solution (0.01 mol/L NaH 2PO 4 1 0.01 mol/L Na 2HPO 4) (PBS) for 30 min. The standard saturated NO solutions were used immediately under nitrogen atmosphere.
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FIG. 1. Cyclic voltammograms on bare glassy carbon electrode in 0.01 mol/liter NaH 2PO 4/0.01 mol/liter Na 2HPO 4 solution (pH 7.0) at a sweep rate of 50 mV/s in the presence of NO 22: (a) blank; (b) 2.0 3 10 23 mmol/liter; (c) 1.0 3 10 22 mmol/liter; (d), 5.0 3 10 22 mmol/liter.
Electrochemistry experiments were performed with a Model 366 Bi-Potentiostat (EG&G Princeton Applied Research, USA), a TYPE 3086 x–y Recorder (Sichuan 4th Instrument Co., China). Cyclic voltammetry was conducted with a three-electrode electrochemical cell. A glassy carbon electrode (area 5 0.07 cm 2) was used as the working electrode. The electrode was pretreated just like before (26). A platinum wire was applied as counter electrode. A homemade gas addition device was connected to the electrochemical cell. The whole cell was carefully designed to ensure that there was no gas leakage during the experiments. The potentials were expressed with reference to the aqueous SCE (saturated colomel electrode) in this paper. 3. RESULTS AND DISCUSSION 3.1. The Cyclic Voltammograms of NO 22 on Bare Glassy Carbon Electrode and on the PNR Electrode As mentioned earlier, since NO 22 is a serious interference in the detection of NO, we first investigated the electrochemical behavior of NO 22. The curves in Fig. 1 depict the voltammetric responses at bare glassy carbon electrode. It can be seen that NO 22 revealed a weak oxidation peak at 10.95 V, which is ascribed to the oxidation of NO 22 to NO 32 as shown in Eq. (2). This indicates that the bare glassy carbon electrode has some electrochemical activity toward the oxidation NO 22. It also can be seen that the bare glassy carbon electrode has little reduction activity toward NO 22. Compared with those shown in Fig. 1 (bare electrode), the voltammograms in Fig. 2 (PNR electrode) have a much higher oxidation peak at 10.95 V and an obvious reduction peak at 20.4 V whereas the bare glassy carbon electrode shows a negligible reduction peak at this place. This peak was due to the reduction of NO 22 to NO (Eq. (1)). It can be clearly seen that the PNR film is electroactive toward the redox of NO 22 in the PBS. There was a linear relationship between the oxidation currents at 10.95 V and the NO 22 concentrations in the range of 7.5 3 10 26–1.0 3 10 24 mol/liter with a correlation coefficient of 0.999. Thus, such a PNR electrode can be employed for the determination of NO 22 if other interference can be removed.
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FIG. 2. Cyclic voltammograms on PNR electrode in 0.01 mol/liter NaH 2PO 4/0.01 mol/liter Na 2HPO 4 solution (pH 7.0) at a sweep rate of 50 mV/s, in the presence of NO 22: (a) blank; (b) 5.0 3 10 24 mmol/liter; (c) 1.0 3 10 23 mmol/liter; (d) 1.5 3 10 23 mmol/liter; (e) 2.0 3 10 23 mmol/liter.
3.2. The Cyclic Voltammograms of NO on Bare Glassy Carbon Electrode and on the PNR Electrode The electrochemical activity of the PNR electrode for the oxidation and reduction of NO in PBS was evaluated by comparing to that on bare glassy carbon electrode, with both case under an N 2 atmosphere. Figure 3 depicts the cyclic voltammetric response on a bare glassy carbon electrode in pH 7.0 PBS. When NO was added into the solution, a small peak at 10.95 V appeared. At the same time, only a slight increase of the current was observed at the potential of 20.6 V. The peak at 10.95 V was ascribed to the oxidation of NO to NO 22 and then to NO 32 (Eqs. 1, 2). Figure 4 shows that the PNR electrode has the electrochemical activity toward the
FIG. 3. Cyclic voltammograms at a sweep rate of 50 mV/s on the bare glassy carbon electrode in 0.01 mol/liter NaH 2PO 4/0.01 mol/liter Na 2HPO 4 solution (pH 7.0) under a nitrogen atmosphere, in the presence of NO: (a) blank; (b) 95.0 mmol/liter.
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FIG. 4. Cyclic voltammograms at a sweep rate of 50 mV/s on PNR electrode in 0.01 mol/liter NaH 2PO 4/0.01 mol/liter Na 2HPO 4 solution (pH 7.0) under a nitrogen atmosphere, in the presence of NO: (a) blank; (b) 47.5 mmol/liter; (c) 95.0 mmol/liter; (d) 14.3 mmol/liter; (e) 190.0 mmol/liter.
oxidation and reduction of NO. Curve a in Fig. 4 is the typical voltammogram of the PNR electrode in PBS, and curves b, c, d, and e are the electrochemical responses upon the addition of NO. The appearance of a new peak at 10.95 V is ascribed to the oxidation of NO to NO 22 and even to NO 32, and the new peak at 20.6 V is ascribed to the reduction of NO to N 2O (Eq. 3). Compared with the voltammograms in Fig. 2, it should be noticed that NO 22 is reduced at 20.4 V (Eq. (1)), while NO is reduced at 20.6 V. There is a misty peak in 20.4V, which should respond to the reduction of NO 22 to NO. This part of NO 22 may come from the oxidation of NO at positive cycling or from the reaction of NO with other oxidants, it also may come from Eq. (7), in which NO 32 can be produced in the positive scanning process. After all, the peak is so small that we can deduce that the NO is quite stable in PBS under our experimental conditions. But the NO solution must be used immediately after it is prepared. 3.3. pH Dependent During the Redox of NO 22 The redox of nitrogen oxide (NO x ) is pH dependent. As shown in Fig. 5, in pH 2.0 PBS, a new reduction peak at 20.6 V appears, indicating that there are two steps in the reduction process of NO 22 on PNR electrode. The first is the reduction of NO 22 to NO at 20.4 V (Eq. (1)), and the second is the reduction of NO to N 2O at 20.6 V (Eq. (3)). This can be explained by Eq. (1). The acid solution promoted the two-step reduction, i.e., the reduction of NO 22 to NO, and NO can be further reduced to N 2O immediately. However, in pH 7.0 PBS, there was only one reduction peak at 20.4 V, which resulted from the reduction of NO 22 to NO. As depicted in Eq. (3), the further reduction of NO to N 2O is highly pH dependent. At weak acid PBS pH 7.0, the second reduction step (at 20.6 V) hardly happens.
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FIG. 5. Cyclic voltammograms in 0.01 mol/liter NaH 2PO 4/0.01 mol/liter Na 2HPO 4 solution (pH 2.0) at a sweep rate of 50 mV/s on PNR electrode, in the presence of NO 22: (a) blank; (b) 5.0 3 10 24 mmol/liter; (c) 1.0 3 10 23 mmol/liter; (d) 1.5 3 10 23 mmol/liter.
What should be pointed out is, NO in the reduction process may be on account of the reduction of NO 22 at 20.4 V, also may come from the disproportionation NO 22 of due to the high acid (pH 2.0). So we select PBS pH 7.0 as our blank solution. 3.4. Determination of NO with PNR Electrode As depicted in Eq. (1) and Eq. (2), NO can be oxidized to NO 22 and further to NO 32 at 10.95 V (Eqs. 1, 2). Although the oxidation current at 10.95 V increases upon the addition of NO, it is not a good method to use this oxidation peak current to determine the concentration of NO, because NO 22 seriously interferes with the determination of NO. Nevertheless, the reduction peak of NO at 20.6 V can be used to determine NO because there was no direct interference with the detection of NO at 20.6 V from NO 22, although the reduction potential of NO 22 (20.4 V) is very close to that of NO. 3.5. Determination of NO with PNR–Nafion Electrode However, note that the formation of NO 22 cannot be avoided completely (O 2 and others all can oxidize NO to NO 22). At the same time, the reduction potential of NO 22 to NO (20.4 V) is very close to the reduction potential of NO to N 2O (20.6 V). Therefore, it is still necessary to exclude the interference of NO 22. Nafion was used to further modify the PNR electrode. The modification was manipulated by placing 20 mL of a 0.5% (w/v) ethanol solution of Nafion over the surface of the electrode and drying the solvent in air. As shown in Fig. 6, curve a is the voltammogram for the PNR electrode in blank PBS
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FIG. 6. Cyclic voltammograms in 0.01 mol/liter NaH 2PO 4/0.01 mol/liter Na 2HPO 4 solution (pH 7.0) at a sweep rate of 50 mV/s under a nitrogen atmosphere, (a) NO 22 blank, PNR electrode; (b) NO 22 5.0 3 10 24 mmol/liter, PNR electrode; (c) NO 22 5.0 3 10 24 mmol/liter, PNR–Nafion electrode; (d) (c) 147.5 mmol/liter NO.
and curve b depicts the behavior of the electrode after the addition of 5.0 3 10 24 mmol/liter NO 22. At 10.95 V there is an oxidation peak, which is ascribed to the oxidation of NO 22 to NO 32, and at 20.4 V–20.6 V there is a wide reduction peak, which is ascribed to the reduction of NO 22 to NO, probably even including the reduction of NO to N 2O. Curve c is the voltammogram on the PNR–Nafion electrode in PBS containing 5.0 3 10 24 mmol/L NO 22. It can be clearly seen that on the PNR–Nafion electrode NO 22 can be neither oxidized nor reduced. Curve d depicts the response of the electrode in curve c upon the addition of NO. There are a clear oxidation peak at 10.95 V, which is ascribed to the oxidation of NO to NO 22 and then to NO 32, and a clear reduction peak at 20.6 V, which is ascribed to the reduction of NO to N 2O. Curve d proved that NO can be oxidized at 10.95 V and reduced at 20.6 V. Figure 7 depicts the linear relationship between the reduction currents and the concentration of NO by using a PNR–Nafion electrode under a nitrogen atmosphere. It was noticed that the scanning voltage range was 21.0 – 0.4 V. The reason was that there might exist some NO 22 in the modifying film when the electrode was polarized at high positive potential (10.95 V) due to the possibility of oxidation of NO and this part of NO 22 may interfere with the determination of NO. A good linear relationship ranging from 50 to 200 mmol/liter was obtained with a correlation coefficient of 0.995. The detection limit is as low as 10 mmol/liter. Of course, the oxidation peak also can be used to detect the concentration of NO. However, there are two steps in the oxidation process of NO, i.e., the oxidation of NO to NO 22 and the oxidation of NO 22 to NO 32. If NO can be completely oxidized to NO 32, or the oxidation reaction of NO 22 to NO 32 can be described quantitatively or cannot take place at all, detection through the oxidation peak will be advantageous in some way. Unfortunately, we cannot guarantee this. This means that there will be a great error if the oxidation peak is chosen to carry out the determination, because the intermediate product NO 22 can interfere with the determination badly. Thus, the reduction peak was selected to conduct the determination.
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FIG. 7. Cyclic voltammograms in 0.01 mol/liter NaH 2PO 4/0.01 mol/liter Na 2HPO 4 solution (pH 7.0) at a sweep rate of 50 mV/s on the PNR–Nafion electrode under a nitrogen atmosphere, in the presence of NO: (a) blank; (b) 47.5 mmol/liter; (c) 95.0 mmol/liter; (d) 143.0 mmol/liter; (e) 190.0 mmol/liter. The calibration curve is also shown.
4. CONCLUSION Since this is a successful conventional determination method, our further work will focus on the determination of NO in biological media when the sensitivity is improved and other interference is removed effectively. ACKNOWLEDGMENTS The support of the National Natural Science Foundation of China is greatly appreciated.
REFERENCES 1. Palmer, R. M. J.; Ferrige, A. G.; Moncada, S. Nature (London), 1987, 327, 524. 2. Lgnarro, L. I.; Buga, G. M.; Wood, K. S.; Byrns, R. E.; Chaudhuri, G., Proc. Natl. Acad. Sci. U.S.A., 1987, 84, 9265. 3. Fang, Z.; Jinyuan, M. J. Anal. Sci., 1998, 14, 74. [In Chinese] 4. Bredt, D. S.; Snyder, S. H. Proc. Natl. Acad. Sci. U.S.A., 1989, 86, 9030. 5. Hibbs, J. B., Jr.; Vavrin, Z.; Tainton, R. R. J. Immunol., 1987, 138, 550. 6. Vincent, S. R.; Hope, B. T. Trends Neurosci., 1992, 15, 108. 7. Burnett, A. L.; Lowenstein, C. J.; Bredt, D. S.; Chang, T. S. K.; Snyder, S. H. Science, 1992, 257, 401. 8. Radomski, M. W.; Palmer, R. M. J.; Moncada, S. Proc. Natl. Acad. Sci. U.S.A., 1990, 87, 5193. 9. Butler, A. R.; Williams, D. L. H. Chem. Soc. Rev., 1993, 233. 10. Schmidt, H. H. W.; Warner, T. D.; Ishii, K.; Muraud, F. Science, 1992, 255, 721. 11. Moncada, S.; Palmer, R. M. J.; Higgs, E. A. Biochem. Pharmacol., 1989, 38, 1709. 12. Snyder, S. H.; Bredt, D. S. Sci. Am., May 1992, 68. 13. Maraj, S. R.; Khan, S.; Cui, X. Y. et al., Analyst, 1995, 120, 699. 14. (a) Bedioui, F.; Trevin, S.; Devynck, J. J. Electroanal. Chem., 1994, 377, 295; (b) Malinski, T.; Taha, Z. et al., Anal. Chim. Acta, 1993, 279, 135. 15. Gomez, R.; Rodes, A.; Feliu, J. M. J. Electroanal. Chem., 1995, 393, 123.
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Pariente, F.; Alonso, J. L.; Abruna, H. D. J. Electroanal. Chem., 1994, 379, 191. Malinski, T.; Taha, Z. Nature, 1992, 358, 676. Lantoine, F.; Trevin, S. et al., J. Electroanal. Chem., 1995, 392, 85. Trevin, S.; Bedioui, F.; Devynck, J. J. Electroanal. Chem., 1996, 408, 261. Yu, C. H.; Su, Y. O. J. Electroanal. Chem., 1994, 368, 323. Friendemann, M. N.; Robinson, S. W.; Gerhardt, G. A. Anal. Chem., 1996, 68, 2621. Maskus, M.; Pariente, F.; Wu, Q.; Toffanin, A.; Shapleigh, J. P.; Abruna, H. D. Anal. Chem., 1996, 68, 3128. (a) Dutta, D.; Landolt, D. J. Electrochem. Soc., 1972, 119, 1320; (b) Ye, S.; Kita, H. J. Electroanal. Chem., 1993, 346, 489. Janssen, L. J. J.; Pieterse, M. M.; Barendrecht, E. Electrochim. Acta, 1977, 22, 27. Sun, Y.; Ye, B.; Wang, Y.; Tang, X.; Zhou, X. Microchem. J., 1998, 58, 182. Tang, X.; Yao, B.; Zhou, X. Wuhan Univ. J. Nat. Sci., 1997, 4, 479. Sun, Y.; Ye, B.; Zhang, W.; Zhou, X. Anal. Chim. Acta, 1998, 363, 75.
Determination of Nitric Oxide by Glassy Carbon Electrodes Modified with Poly(Neutral Red) Tang Xiaorong, Fang Cheng, Yao Bing, and Zhang Wuming 1 Department of Chemistry, Wuhan University, Wuhan 430072, People’s Republic of China E-mail: [email protected] Received October 20, 1998; accepted March 17, 1999 Nitric oxide can be oxidized to NO 22 (then to NO 32) and reduced to N 2O at a glassy carbon electrode modified with the film of poly(neutral red). When the modified electrode is further coated with a thin Nafion film, the interference of NO 22 can be almost thoroughly eliminated, and the electrode can be employed to detect nitric oxide. © 1999 Academic Press Key Words: electropolymerized film; chemically modified electrode; neutral red; nitric oxide.
1. INTRODUCTION In 1987, it was discovered that vascular endothelial cells could synthesize the freeradical gas nitric oxide (NO) (1), and NO could act as a vasodilatory messenger (2). As the endothelial-derived relaxing factor (EDRF), NO is responsible for activating guanylate cyclase, the essential enzyme involved in the control of vasodilation. From then on, much attention was given to the study of NO in biological systems (3– 6), and many other important physiological functions of NO were discovered (8 –11). Therefore, the direct determination of NO has become attractive, especially in biological models. Unfortunately, due to its low stability and high fugacity, it is very difficult to measure NO directly. In fact, the half-life of NO is very short (ca. 6s). Moreover, NO is easily oxidized by O 2 or other oxidants to nitrite (NO 22) and even to nitrate (NO 32). Most measurement techniques for NO involve indirect chemical detection (12, 13). Because of their inherent speed and sensitivity, electrochemical methods are one of the most advantageous and well-suited methods for detection in biological media (14, 15). One of the electrochemical approaches to NO detection uses modified platinum electrodes, on which NO is directly oxidized. The modified membranes include chloroprene, nitrocellulose and silicone, and Nafion and cellulose acetate (16). Another approach is based on the electrocatalytic oxidation of NO on a carbon fiber electrode coated with nickel (II) tetrakis (3-methoxy-4-hydroxyphenyl) porphyrin (Ni-TMPP) and Nafion film (17–20) or modified with o-phenylenediamine (21). Maskus (22) reported electropolymerized films of (Cr(v-tpy) 2) 31 and their application to detecting cellular NO from Rhodobacter sphaeroides bacterial cells. NO has very complex redox behavior. The electrochemical behavior of NO on a rotating platinum electrode in 4 mol/liter H 2SO 4 has been studied (23). There are two steps in the process of NO oxidation: 1
To whom correspondence should be addressed. Fax: 86-027-87882661.
377 0026-265X/99 $30.00 Copyright © 1999 by Academic Press All rights of reproduction in any form reserved.
378
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HNO2 1 H 1 1 e 2 3 NO~g! 1 H2O.
(1)
NO is oxidized to HNO 2 via a kinetically fast read with a standard potential of 10.95 V (24). The product of the first step reaction, HNO 2, is afterwards oxidized to NO 32: 1 2 NO 2 3 NO 2 3 1 2H 1 2e 2 1 H2O.
(2)
On the other hand, there are also two steps in the process of NO reduction: 2NO 1 2H 1 1 2e 2 3 N2O 1 H2O.
(3)
Such a reaction is fast. In the second step, N 2O can be further reduced to NH 2OH, N 2H 4, or NH 3: N2O 1 4H 1 1 H2O 1 4e 2 3 2NH2OH.
(4)
N2O 1 6H 1 1 6e 2 3 N2H4 1 H2O.
(5)
N2O 1 8H 1 1 8e 2 3 2NH3 1 H2O.
(6)
However, the redox process of NO is very complicated. The following reaction may occur also: 2 2 2NO~aq! 1 NO 2 3 1 2OH 5 3NO 2 1 H2O.
(7)
In our laboratory, the glassy carbon electrodes modified with electropolymerized films of neutral red were studied comprehensively (25–27). Such an electropolymer has very good electrocatalytic activity toward the redox of small molecular compounds such as vitamin C, dopamine, and adrenaline (26). Reasoning that the film might also be active in the electrocatalytic redox of nitric oxide (NO), we have carried out such studies and have found that the poly(neutral red) film is quite active in the electrochemical redox of nitric oxide. 2. EXPERIMENTAL The glassy carbon electrodes modified with electropolymerized films of neutral red (PNR) were prepared as previously (26). All solutions were prepared with fresh doubledistilled water and were deoxygenated by purging with prepurified nitrogen gas for 20 min. High-purity nitric oxide (99.7%) was obtained from Sumitomo Seika Chemical Co. The standard saturated NO solution (typically containing 1.9 mmol/liter NO at 20°C) was obtained as follows: NO gas was first passed through a washing bottle containing 2 mol/L NaOH to remove possible NO 2 and other impurities, and then passed into pH 7.0 oxygen-free phosphate buffer solution (0.01 mol/L NaH 2PO 4 1 0.01 mol/L Na 2HPO 4) (PBS) for 30 min. The standard saturated NO solutions were used immediately under nitrogen atmosphere.
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FIG. 1. Cyclic voltammograms on bare glassy carbon electrode in 0.01 mol/liter NaH 2PO 4/0.01 mol/liter Na 2HPO 4 solution (pH 7.0) at a sweep rate of 50 mV/s in the presence of NO 22: (a) blank; (b) 2.0 3 10 23 mmol/liter; (c) 1.0 3 10 22 mmol/liter; (d), 5.0 3 10 22 mmol/liter.
Electrochemistry experiments were performed with a Model 366 Bi-Potentiostat (EG&G Princeton Applied Research, USA), a TYPE 3086 x–y Recorder (Sichuan 4th Instrument Co., China). Cyclic voltammetry was conducted with a three-electrode electrochemical cell. A glassy carbon electrode (area 5 0.07 cm 2) was used as the working electrode. The electrode was pretreated just like before (26). A platinum wire was applied as counter electrode. A homemade gas addition device was connected to the electrochemical cell. The whole cell was carefully designed to ensure that there was no gas leakage during the experiments. The potentials were expressed with reference to the aqueous SCE (saturated colomel electrode) in this paper. 3. RESULTS AND DISCUSSION 3.1. The Cyclic Voltammograms of NO 22 on Bare Glassy Carbon Electrode and on the PNR Electrode As mentioned earlier, since NO 22 is a serious interference in the detection of NO, we first investigated the electrochemical behavior of NO 22. The curves in Fig. 1 depict the voltammetric responses at bare glassy carbon electrode. It can be seen that NO 22 revealed a weak oxidation peak at 10.95 V, which is ascribed to the oxidation of NO 22 to NO 32 as shown in Eq. (2). This indicates that the bare glassy carbon electrode has some electrochemical activity toward the oxidation NO 22. It also can be seen that the bare glassy carbon electrode has little reduction activity toward NO 22. Compared with those shown in Fig. 1 (bare electrode), the voltammograms in Fig. 2 (PNR electrode) have a much higher oxidation peak at 10.95 V and an obvious reduction peak at 20.4 V whereas the bare glassy carbon electrode shows a negligible reduction peak at this place. This peak was due to the reduction of NO 22 to NO (Eq. (1)). It can be clearly seen that the PNR film is electroactive toward the redox of NO 22 in the PBS. There was a linear relationship between the oxidation currents at 10.95 V and the NO 22 concentrations in the range of 7.5 3 10 26–1.0 3 10 24 mol/liter with a correlation coefficient of 0.999. Thus, such a PNR electrode can be employed for the determination of NO 22 if other interference can be removed.
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FIG. 2. Cyclic voltammograms on PNR electrode in 0.01 mol/liter NaH 2PO 4/0.01 mol/liter Na 2HPO 4 solution (pH 7.0) at a sweep rate of 50 mV/s, in the presence of NO 22: (a) blank; (b) 5.0 3 10 24 mmol/liter; (c) 1.0 3 10 23 mmol/liter; (d) 1.5 3 10 23 mmol/liter; (e) 2.0 3 10 23 mmol/liter.
3.2. The Cyclic Voltammograms of NO on Bare Glassy Carbon Electrode and on the PNR Electrode The electrochemical activity of the PNR electrode for the oxidation and reduction of NO in PBS was evaluated by comparing to that on bare glassy carbon electrode, with both case under an N 2 atmosphere. Figure 3 depicts the cyclic voltammetric response on a bare glassy carbon electrode in pH 7.0 PBS. When NO was added into the solution, a small peak at 10.95 V appeared. At the same time, only a slight increase of the current was observed at the potential of 20.6 V. The peak at 10.95 V was ascribed to the oxidation of NO to NO 22 and then to NO 32 (Eqs. 1, 2). Figure 4 shows that the PNR electrode has the electrochemical activity toward the
FIG. 3. Cyclic voltammograms at a sweep rate of 50 mV/s on the bare glassy carbon electrode in 0.01 mol/liter NaH 2PO 4/0.01 mol/liter Na 2HPO 4 solution (pH 7.0) under a nitrogen atmosphere, in the presence of NO: (a) blank; (b) 95.0 mmol/liter.
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FIG. 4. Cyclic voltammograms at a sweep rate of 50 mV/s on PNR electrode in 0.01 mol/liter NaH 2PO 4/0.01 mol/liter Na 2HPO 4 solution (pH 7.0) under a nitrogen atmosphere, in the presence of NO: (a) blank; (b) 47.5 mmol/liter; (c) 95.0 mmol/liter; (d) 14.3 mmol/liter; (e) 190.0 mmol/liter.
oxidation and reduction of NO. Curve a in Fig. 4 is the typical voltammogram of the PNR electrode in PBS, and curves b, c, d, and e are the electrochemical responses upon the addition of NO. The appearance of a new peak at 10.95 V is ascribed to the oxidation of NO to NO 22 and even to NO 32, and the new peak at 20.6 V is ascribed to the reduction of NO to N 2O (Eq. 3). Compared with the voltammograms in Fig. 2, it should be noticed that NO 22 is reduced at 20.4 V (Eq. (1)), while NO is reduced at 20.6 V. There is a misty peak in 20.4V, which should respond to the reduction of NO 22 to NO. This part of NO 22 may come from the oxidation of NO at positive cycling or from the reaction of NO with other oxidants, it also may come from Eq. (7), in which NO 32 can be produced in the positive scanning process. After all, the peak is so small that we can deduce that the NO is quite stable in PBS under our experimental conditions. But the NO solution must be used immediately after it is prepared. 3.3. pH Dependent During the Redox of NO 22 The redox of nitrogen oxide (NO x ) is pH dependent. As shown in Fig. 5, in pH 2.0 PBS, a new reduction peak at 20.6 V appears, indicating that there are two steps in the reduction process of NO 22 on PNR electrode. The first is the reduction of NO 22 to NO at 20.4 V (Eq. (1)), and the second is the reduction of NO to N 2O at 20.6 V (Eq. (3)). This can be explained by Eq. (1). The acid solution promoted the two-step reduction, i.e., the reduction of NO 22 to NO, and NO can be further reduced to N 2O immediately. However, in pH 7.0 PBS, there was only one reduction peak at 20.4 V, which resulted from the reduction of NO 22 to NO. As depicted in Eq. (3), the further reduction of NO to N 2O is highly pH dependent. At weak acid PBS pH 7.0, the second reduction step (at 20.6 V) hardly happens.
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FIG. 5. Cyclic voltammograms in 0.01 mol/liter NaH 2PO 4/0.01 mol/liter Na 2HPO 4 solution (pH 2.0) at a sweep rate of 50 mV/s on PNR electrode, in the presence of NO 22: (a) blank; (b) 5.0 3 10 24 mmol/liter; (c) 1.0 3 10 23 mmol/liter; (d) 1.5 3 10 23 mmol/liter.
What should be pointed out is, NO in the reduction process may be on account of the reduction of NO 22 at 20.4 V, also may come from the disproportionation NO 22 of due to the high acid (pH 2.0). So we select PBS pH 7.0 as our blank solution. 3.4. Determination of NO with PNR Electrode As depicted in Eq. (1) and Eq. (2), NO can be oxidized to NO 22 and further to NO 32 at 10.95 V (Eqs. 1, 2). Although the oxidation current at 10.95 V increases upon the addition of NO, it is not a good method to use this oxidation peak current to determine the concentration of NO, because NO 22 seriously interferes with the determination of NO. Nevertheless, the reduction peak of NO at 20.6 V can be used to determine NO because there was no direct interference with the detection of NO at 20.6 V from NO 22, although the reduction potential of NO 22 (20.4 V) is very close to that of NO. 3.5. Determination of NO with PNR–Nafion Electrode However, note that the formation of NO 22 cannot be avoided completely (O 2 and others all can oxidize NO to NO 22). At the same time, the reduction potential of NO 22 to NO (20.4 V) is very close to the reduction potential of NO to N 2O (20.6 V). Therefore, it is still necessary to exclude the interference of NO 22. Nafion was used to further modify the PNR electrode. The modification was manipulated by placing 20 mL of a 0.5% (w/v) ethanol solution of Nafion over the surface of the electrode and drying the solvent in air. As shown in Fig. 6, curve a is the voltammogram for the PNR electrode in blank PBS
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FIG. 6. Cyclic voltammograms in 0.01 mol/liter NaH 2PO 4/0.01 mol/liter Na 2HPO 4 solution (pH 7.0) at a sweep rate of 50 mV/s under a nitrogen atmosphere, (a) NO 22 blank, PNR electrode; (b) NO 22 5.0 3 10 24 mmol/liter, PNR electrode; (c) NO 22 5.0 3 10 24 mmol/liter, PNR–Nafion electrode; (d) (c) 147.5 mmol/liter NO.
and curve b depicts the behavior of the electrode after the addition of 5.0 3 10 24 mmol/liter NO 22. At 10.95 V there is an oxidation peak, which is ascribed to the oxidation of NO 22 to NO 32, and at 20.4 V–20.6 V there is a wide reduction peak, which is ascribed to the reduction of NO 22 to NO, probably even including the reduction of NO to N 2O. Curve c is the voltammogram on the PNR–Nafion electrode in PBS containing 5.0 3 10 24 mmol/L NO 22. It can be clearly seen that on the PNR–Nafion electrode NO 22 can be neither oxidized nor reduced. Curve d depicts the response of the electrode in curve c upon the addition of NO. There are a clear oxidation peak at 10.95 V, which is ascribed to the oxidation of NO to NO 22 and then to NO 32, and a clear reduction peak at 20.6 V, which is ascribed to the reduction of NO to N 2O. Curve d proved that NO can be oxidized at 10.95 V and reduced at 20.6 V. Figure 7 depicts the linear relationship between the reduction currents and the concentration of NO by using a PNR–Nafion electrode under a nitrogen atmosphere. It was noticed that the scanning voltage range was 21.0 – 0.4 V. The reason was that there might exist some NO 22 in the modifying film when the electrode was polarized at high positive potential (10.95 V) due to the possibility of oxidation of NO and this part of NO 22 may interfere with the determination of NO. A good linear relationship ranging from 50 to 200 mmol/liter was obtained with a correlation coefficient of 0.995. The detection limit is as low as 10 mmol/liter. Of course, the oxidation peak also can be used to detect the concentration of NO. However, there are two steps in the oxidation process of NO, i.e., the oxidation of NO to NO 22 and the oxidation of NO 22 to NO 32. If NO can be completely oxidized to NO 32, or the oxidation reaction of NO 22 to NO 32 can be described quantitatively or cannot take place at all, detection through the oxidation peak will be advantageous in some way. Unfortunately, we cannot guarantee this. This means that there will be a great error if the oxidation peak is chosen to carry out the determination, because the intermediate product NO 22 can interfere with the determination badly. Thus, the reduction peak was selected to conduct the determination.
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FIG. 7. Cyclic voltammograms in 0.01 mol/liter NaH 2PO 4/0.01 mol/liter Na 2HPO 4 solution (pH 7.0) at a sweep rate of 50 mV/s on the PNR–Nafion electrode under a nitrogen atmosphere, in the presence of NO: (a) blank; (b) 47.5 mmol/liter; (c) 95.0 mmol/liter; (d) 143.0 mmol/liter; (e) 190.0 mmol/liter. The calibration curve is also shown.
4. CONCLUSION Since this is a successful conventional determination method, our further work will focus on the determination of NO in biological media when the sensitivity is improved and other interference is removed effectively. ACKNOWLEDGMENTS The support of the National Natural Science Foundation of China is greatly appreciated.
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