日本分析科学Analytical Science

IntroductionPyridine derivatives are an important class of nitrogenous compounds. The pyrimidine bases (i.e . thymine and cytosine) are fundamental compounds in biological systems. They participate in processes as distinct as energy transduction, metabolic cofactors and cell signaling, and are essential building blocks of nucleic acids.1 A number of synthetic pharmacophores based upon the pyrimidyl structure exhibit antibacterial, antimicrobial, anticancer, anti-HIV-1 and anti-rubella virus activities.2 Among them, 2,4,6-triaminopyrimidine (TAP) (Fig. 1) is also an outstanding medicine raw material, which has been widely used to synthesize anti-cancer drugs, such as methotrexate and dyrenium. Rosowsky and coworkers 3 reported that a family of lipophilic small-molecule antifolates were synthesized using TAP, and had potential applications in the treatment and prophylaxis of AIDS-associated opportunistic microbial infections. What is more, TAP was reported to be a blocker of the cation-specific paracellular conductance pathways of several epithelia.4 The compound was also known as an inhibitor for sodium transport in frog skin,5 and affected the transmembrane potential and contractility in guinea-pig myocardium.6 Thus, the precise detection of TAP is of critical importance, not only in the field of biomedical chemistry and modern pharmacy, but also in pathological research. However, to the best of our knowledge, little literature has been reported on the determination of TAP so far, except for an IR spectrum method.7For all we known, carbon nanotubes (CNTs) have some excellent properties, such as a high-aspect ratio, electrical conductivity, chemical stability and an ability to promote electron-transfer reactions. In recent years, CNTs have been widely used in analytical chemistry, including the fabrication of electrochemical sensors and biosensors.8 For doing so, CNTs were homogeneously dispersed in a Nafion solution first, and then transferred onto the electrode surfaces for fabricating Nafion/CNTs film electrodes. These modified electrodes have been used as voltammetric sensors for the determination of trace heavy metals,9–11 dopamine,12 2-nitrophenol and 4-nitrophenol 13 and clenbuterol,14 respectively.In the present work, a Nafion/CNTs film modified GC electrode was fabricated by dispersing multi-wall carbon nanotubes (MWCNTs) in 1% Nafion solutions, and then transferred onto a GC surface. With this Nafion/MWCNTs film electrode, TAP showed electro-activity, and it gave an anodic peak at about 1.4 V . Based on this oxidizing reaction, a sensitive2010 © The Japan Society for Analytical Chemistry†T o whom correspondence should be addressed.E-mail: yebx@Electrochemical Behavior and Voltammetric Determination of 2,4,6-Triaminopyrimidine at Glassy Carbon Electrode Modified with Multi-Walled Carbon Nanotubes/NafionBaocheng Y ANG ,* Fei W ANG ,** Sujuan G UO ,* and Baoxian Y E *†* D epartment of Chemistry, Zhengzhou University, Zhengzhou 450001, P . R. China ** D epartment of Material and Chemistry Engineering, Henan Institute of Engineering, Zhengzhou 450007, P . R. ChinaA multi-wall carbon nanotubes (MWCNTs) composite with Nafion was modified on a glass carbon electrode. The modified electrode was then used as a voltammetric sensor in detecting 2,4,6-triaminopyrimidine (TAP). The surface morphology of the Nafion/MWCNTs composite film was characterized by atomic force microscopy (AFM), and the electrochemical behavior of TAP at this sensor was investigated in detail. The results indicated that the Nafion/MWCNTs modified electrode exhibited efficient electrocatalytic oxidation for TAP with relatively high sensitivity, stability and lifetime. Under the optimized condition using linear sweep voltammetry (LSV), the Nafion/MWCNTs modified electrode exhibited a linear voltammetric response for TAP in the concentration range of 2.0 × 10–7 to 3.6 × 10–5 mol L –1, with a detection limit of 5.0 × 10–8 mol L –1. The electrode was applied to detect TAP added to human blood serum, with an average recovery value of 101.3%.(Received May 28, 2010; Accepted June 24, 2010; Published October 10, 2010)Fig. 1 Chemical structure of 2,4,6-triaminopyrimidine.method for the determination of TAP was erected with satisfactory detection recovery.ExperimentalReagents and chemicals MWCNTs (purity ≥95%) were obtained from Nachen S&T Ltd. (Beijing, China). Nafion 117 (5%, w/v in alcoholic solution) was purchased from Alfa, and was diluted to 1% (w/v) with ethanol before use. TAP was purchased from J&KChemical Ltd. and used as received. A stock solution of TAP (1.0 × 10–3 mol L –1) was prepared using 0.01 mol/L hydrochloric acid, and stored under 4 C. All other chemicals used were of analytical grade and the experimental water used wasdouble-distilled. A mixed acid (MA: 0.04 mol L –1 H 3PO 4+ 0.04 mol L –1 HAc + 0.04 mol L –1 H 3BO 3) (pH 1.81) was used as a supporting electrolyte.Apparatus Electrochemical measurements were performed using a CHI650A electrochemical workstation (CHI Instrumental, Shanghai, China) coupled with a conventional three-electrode electrochemical cell. The three-electrode system was composedof a bare glassy carbon (GCE) (d = 3 mm) or modified GCE working electrode, an Ag/AgCl reference electrode and a platinum wire auxiliary electrode, respectively. All potentials in this paper are given against the Ag/AgCl (3 mol L –1 KCl). The UV-vis spectra were recorded by a Model UV-2102PC spectrophotometer (UNICO, Shanghai, China); 5500 atomic force microscopy (Agilent, USA) was used to observe the surface morphology of a film electrode modified by Nafion/MWCNTs.Preparation of film-modified GCEFor the preparation of a Nafion/MWCNTs-modified electrode, MWCNTs (3 mg) was added into a 5-mL Nafion ethanol solution (1%, w/v) and dispersed for 30 min under ultrasound.A Nafion/MWCNTs composite solution was obtained. Before modification, a bare GCE was first treated according to standard methods, and then cleaned ultrasonically with 1:1 nitric acid, alcohol and double-distilled water, sequentially. The Nafion/MWCNTs-modified GCE was prepared by casting 3 μL of a Nafion/MWCNTs composite solution onto a GCE surface, and dried at room temperature. A symbol of Nafion/MWCNTs/GCE was denoted for this film electrode, which was preserved indouble-distilled water in spacing interval experiments. For a comparison, a Nafion modified GCE was prepared using the same method and was named Nafion/GCE.Results and Discussion The morphology of film modified electrodesThe morphology of Nafion/MWCNTs/GCE and Nafion/GCE was observed using the atomic force microscope (AFM) technique. Nafion was evenly spread on the GCE surface, which formed a dense Nafion film (Fig. 2a). While theNafion/MWCNTs composite film was uniformly coated on theelectrode surface, it showed a strong interaction between MWCNTs and Nafion (Fig. 2b). The special surface morphology offered the Nafion/MWCNTs/GCE a much larger real surface area than that of the apparent geometric area.Electrochemical behavior of TAP Figure 3 shows the cyclic voltammetric responses of TAP (1.0 × 10–5 mol L –1) at bare GCE (Fig. 3a), Nafion/GCE (Fig. 3c) and Nafion/MWCNTs/GCE (Fig. 3d) in the MA medium. Figure 3b is a CV curve of Nafion/MWCNTs/GCE in a blank solution. Under the same conditions, no anodic peak of TAP was observed at the bare GCE, but a well-defined anodic peak of TAP was observed at the modified electrodes. Especiallyusing the Nafion/MWCNTs/GCE, the peak current was significantly higher than that at the Nafion/GCE. Moreover, compared with the Nafion/GCE, the anodic peak potential at the Nafion/MWCNTs/GCE was negatively shifted from 1401 to1340 mV . The reasons for the notable sensitivity of the TAP reaction at the Nafion/MWCNTs/GCE may be summarized asfollows: (1) TAP becomes positively charged in a MA medium,Fig. 2 AFM 2D topography images. (a) Nafion-modified mica sheet. (b) Nafion/MWCNTs film modified mica sheet. Scan area, 3 μm × 3 μm.and the Nafion/MWCNTs/GCE contains the cation exchanger of Nafion, which has an enriched ability due to an electrostatic interaction; (2) the interfusion of MWCNTs into Nafion would provide an effective means to improve the sensitivity and stability of Nafion-based sensors because the Nafion/MWCNTs composite film possesses more open structures and a larger surface area, allowing faster diffusion of the adsorbed TAP, and that MWCNTs could also adsorb TAP. Without any doubt, the synergetic functions of Nafion and MWCNTs make contributions to the higher current response of TAP.It also showed that no reduction peak was observed in the reverse scan, suggesting that the electrochemical reaction was an irreversible process. For a continuous cyclic scan, the anodic peak was only obtained in the first cycle, and no any peak appeared in following cycles (data are not shown). This result demonstrated that the oxidizing product of TAP was adsorbed on the electrode surface and the product was electro-inactive. Fortunately, after agitating the solution, the peak current would be revived as the first cycle in the next cycle. Thus, the oxidation peak current in the first anodic sweep was used in following studies.The optimal solution conditionThe electrode reaction might be affected by a supporting electrolyte and the pH of the solution. Some electrolytes, such as HCl, H 2SO 4, H 3PO 4, HNO 3, HAc (each 0.1 mol L –1) and MA, were investigated, and the best response was obtained using MA. The peak currents affected by the solution pH were investigated over the pH range of 1.81 – 4.78 (adjusted with 0.2 mol L –1 NaOH solution). As shown in Fig. 4, the peak currents decreased and peak potentials shifted negatively by increasing of the solution pH. When the solution pH was over 5, the anodic peak would disappear. MA was selected as the optimal solution.Abstracting the data from Fig. 4, the relationship between the peak potentials and the solution pH was linear with a slope of 0.0581. These data elucidated that the electron numbers and the proton numbers taking part in this reaction were equal, i.e. TAP oxidation occurs by the transformation of the same number of electrons and protons.The properties and mechanism of the electrode reactionThe number of electron transfer (n). The n value was determined using constant-potential electrolysis. In a TAP solution (5.0 × 10–5 mol L –1), a constant potential (1.5 V) was applied and continually kept until the electrolysis current decreased to 1% of the starting value. The time needed was about 15 h. The quantity of the electric charge was recorded and used to calculate the n according to the Faraday law. The obtained result was n = 1.Effect of scan rates . The effect of the scan rate (v ) on the peak currents and potentials was investigated over a wide range. Figure 5 shows CV curves using scan rates of 40, 60, 80, 100, 150, 200, 250 and 300 mV s –1, respectively. Obviously, the peak potentials shifted towards more positive, and the peak currents were markedly enhanced as the scan rates increased. The peak current showed a linear relationship with respecttoFig. 3 Cyclic voltammograms. (a) Bare GCE, (c) Nafion/GCE and (d) Nafion/MWCNTs/GCE in 1.0 × 10–5 mol L –1 TAP; (b)Nafion/MWCNTs/GCE in a blank solution. Supporting electrolyte,MA; scan rate, 100 mV/s; accumulating time, 300 s.Fig. 4 Cyclic voltammograms of 1.0 × 10–5 mol L –1 TAP at different solution pH. pH (curve a to f): 1.81, 2.21, 2.87, 3.78, 4.35, 4.78. The insert is the E p –pH relation. Supporting electrolyte, MA + NaOH (0.2 mol L –1); accumulating time, 300 s.Fig. 5 Cyclic voltammograms of TAP at different scan rates. Scan rates (curve a to h): 40, 60, 80, 100, 150, 200, 250, 300 mV s –1. The insert is the i p –v relation. Supporting electrolyte, MA + 2.0 × 10–5 mol L –1 TAP.the scan rates (inset in the Fig. 5) with an I p value of 1.187 + 25 v 1/2 (V s –1) (R = 0.998). These data indicate that TAP molecules were first absorbed at the electrode surface, and then underwent irreversible reactions. Similarly, the peak potentials had a linearly relationship with the logarithm of the scan rates, described by the following equation: E pa = 0.0421ln v + 1.502, (R = 0.9978). According to Laviron ’s theory, the slope was equal to RT /αnF , which indicated that the value of αn was 0.61. That is, the value of the transfer coefficient α, was 0.61 (n = 1).A number of studies reported about the protonation of aminopyrimidine derivatives.15–17 This protonation occurred at the pyrimidine ring nitrogen [N(1) and/or N(3)] and/or at the exocyclic amino group both in the gas phase and in solution.15 The amino group at the C2 position on the pyrimidine ring was electroactive.16,17 Constant-potential electrolysis experiments coupled with UV-vis were conducted to illuminate the mechanism of TAP oxidized at the Nafion/MWCNTs/GCE. The UV-vis spectra of TAP (before and after electrolysis) are shown in Fig. 6. The curves of a, b and c in Fig. 6 were obtained after electrolysis of 0, 3 and 15 h, respectively. TAP exhibited two main absorption peaks at 217 and 272 nm, respectively. By increasing the electrolysis time, the absorption peak at 217 nm was slightly decreased, but the peak at 272 nm finally disappeared (15 h). These data elucidated that the p-conjugated in TAP was destroyed after electrolysis. On the basis of these experimental results and the reported literature,16,17 a possible oxidation mechanism of TAP on the Nafion/MWCNTs/GCE is proposed as Scheme 1. In the scheme, an intermediate of positive nitrogen free radical 17 was formed by the oxidation of TAP on Nafion/MWCNTs/GCE. This intermediate would have passivated the electrode, and made no peak appearance after first cycle (see Electrochemical behavior of TAP section).Effect of the accumulation timeIt was important to fix the accumulation time for an adsorption-driven electrode process. In this investigation, the peak currents were linearly increment with the prolongation of the accumulation time. This phenomenon was up to 400 s in a 1.0 × 10–5 mol L –1 TAP solution. For balancing the detection limit and linearity range, an accumulation time of 50 s was selected for erecting an analytical method of TAP.Relationship between the peak currents and the TAP concentrationsThe linear sweep voltammetry (LSV) was used to investigate the relationship between the peak currents and the TAP concentrations. As shown in Fig. 7, LSV i –E curves exhibited a linear relationship between the anodic peak currents (i pa ) and the TAP concentrations within the range from 2.0 × 10–7 to 3.6 × 10–5 mol L –1, which can be described by the following linear regression equation: Y = 0.72X + 1.39, with a correlation coefficient R of 0.999. The detection limit was 5.0 × 10–8 mol L –1. To assess the stability of the Nafion/MWCNTs/GCE, cyclic voltammetric scanning were recorded on the first day and 35 days later and compared under exacting the same conditions. The anodic peak currents of TAP recorded 35 days later was decreased about 3% compared with that of recorded on the first day (data not shown). For investigating of the reproducibility, ten measurements of a 1.0 × 10–5 mol L –1 TAP solution wereperformed, and the calculated RSD was of 2.5%.Fig. 6 UV-vis spectra of TAP after different electrolysis times. (a), (b) and (c) for the electrolysis of 0, 3 and 15 h, respectively. Supporting electrolyte, MA + 5.0 × 10–5 mol L –1TAP.Fig. 7 Linear sweep voltammograms of different T AP concentrations.TAP concentrations (from a to j): 2.0 × 10–7, 2.0 × 10–6, 4.0 × 10–6, 8.0 × 10–6, 1.2 × 10–5, 1.6 × 10–5, 2.0 × 10–5, 2.6 × 10–5, 3.0 × 10–5, 3.6 × 10–5 mol L –1. The insert is an i p vs . C TAP plot. Scan rate, 100 mV s –1; accumulating time, 50 s; supporting electrolyte, MA +TAP.Scheme 1Table 1 Determination result of TAP in human blood serum (n = 3)Sample 10–6 Added/ mol L –110–6 Found/mol L –1RSD, %Recovery, %12341.05.010151.044.9810.314.82.81.32.93.1104.0 99.6103.0 98.6InterferencesUnder the optimized experimental conditions described above, the effects of some foreign species on the determination of TAP at the 2.0 × 10–6 mol L–1 level were evaluated in detail; 200-fold of Mg2+, Pb2+, Ca2+, Al3+, Fe3+, 100-fold of ascorbic acid, folic acid, uric acid and 15-fold of dopamine and epinephrine had almost no influence on the current response of TAP (signal change below 5%). All of these results indicated that the proposed method had good selectivity for the determination of TAP.Determination of TAP in human blood serumTo evaluate the practical applicability of the proposed method, it was employed for the detection of TAP in human blood serum obtained from 4 healthy volunteers. The results were that no distinct signal of TAP was observed. That is, the TAP content in healthy human blood serum is lower than the detection limit of this method. For evaluating the veracity, some TAP standard solution was added into blood serum before analysis. Also, the recovery was determined by the standard addition method. The results of determination are listed in Table 1. The average recovery value was of 101.3%.ConclusionsIn the present work, a multi-walled carbon nanotube-Nafion modified glassy carbon electrode (Nafion/MWCNTs/GCE) was successfully developed for the electrocatalytic oxidation of TAP in a MA solution. As a voltammetric sensor, Nafion/MWCNTs/GCE showed good sensitivity and reproducibility in the determination of TAP. The proposed method offers the advantages of accuracy and time saving as well as simplicity of the reagents and the apparatus. In addition, the results obtained in the analysis of TAP in a human blood serum sample demonstrated this applicability of this method for real sample analysis.AcknowledgementsFinancial support provided by National Natural Science Foundation of China (No. 20775073; 20875083) is greatly appreciated.References1. W. H. Brown, “Introduction to Organic and Biochemistry”,ed. Brooks Cole Publishing, 4th ed., 1987, Monterey, 418.2. M. Botta, F. Corelli, G. Maga, F. Manetti, M. Renzulli, andS. Spadari, Tetrahedron, 2001, 57, 8357.3. A. Rosowsky, H. Chen, H. N. Fu, and S. F. Queener,Bioorg. Med. Chem., 2003, 11, 59.4. R. H. Bowman, J. Arnow, and I. M. Weiner, J. Pharmacol.Exp. Ther., 1978, 206, 207.5. E. H. C. Verhaeren, A. M. Verbruggen, and J. A. J. M.Lemli, Pharmacy World & Science, 1981, 3, 815.6. M. Frank and L. L. Flom, J. Pharmacol. Exp. Ther., 1978,204, 175.7. S. Mohan and N. Sundaraganesan, Indian J. Pure Appl.Phys., 1991, 29, 807.8. Y. Yao and K. K. Shiu, Anal. Bioanal. Chem., 2007, 387,303.9. Y. C. Tsai, J. M. Chen, S. C. Li, and F. Marken, Electrochem.Commun., 2004, 6, 917.10. D. Sun and Z. M. Sun, J. Appl. Electrochem., 2008, 38, 1223.11. Y. C. Tsai, J. M. Chen, and F. Marken, Microchim. Acta,2005, 150, 269.12. K. B. Wu and S. S. Hu, Microchim. Acta, 2004, 144, 131.13. W. S. Huang, C. H. Yang, and S. H. Zhang, Anal. Bioanal.Chem., 2003, 375, 703.14. R. X. Guo, Q. Xu, D. Y. Wang, and X. Y. Hu, Microchim.Acta, 2008, 161, 265.15. (a) M. D. Threadgill, R. J. Griffin, M. F. G. Stevens, and S.K. Wong, J. Chem. Soc. Perkin. Trans. 1, 1987, 2229. (b) W. Stadeli, W. Philipsborn, A. Wick, and I. Kompis, Helv.Chim. Acta, 1980, 63, 504. (c) J. Riand, M. T. Chenon, and N. L. Bader, J. Am. Chem. Soc., 1977, 99, 6838. (d) R.Wagner and W. von. Philipsborn, Helv. Chim. Acta, 1970, 53, 299. (e) V. Q. Nguyen and F. Tureeek, J. Am. Chem.Soc., 1997, 119, 2280. (f) J. Riand, C. Coupry, and M. T.Chenon, J. Chem. Soc. Perkin. Trans. 2, 1981, 783. (g) D.V. Griffiths and S. P. Swetnam, J. Chem. Soc. Chem.Commun., 1981, 1224.16. A. C. F. Gorren, A. J. Kungl, K. Schmidt, E. R. Werner, andB. Mayer, Nitric Oxide, 2001, 5, 176.17. L. Gao, Y. J. Wu, J. X. Liu, and B. X. Ye, J. Electroanal.Chem., 2007, 610, 131.。