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Voltammetric Determination of Dopamine in the Presence of Ascorbic Acid at a Chemically Modified Electrode Jylz-Myng Zen* and I-Lang Chen Department of Chemistry, National Chung-Hsing University, Taichung, Taiwan 402, R.O.C. Received November 15, 1996 Final version: January 7, 1997

Abstract A Nafiodruthenium oxide pyrochlore chemically modified electrode (CME) was used for the selective determination of dopamine (DA) in the presence of a high concentration of ascorbic acid by square-wave voltammetry. Compared to a bare glassy carbon electrode, the CME exhibits an apparent shift of the oxidation potentials in cathodic direction and a marked enhancement of the current response. The selective sensing of DA is achieved by combining the electrocatalytic function of the ruthenium oxide pyrochlore catalyst with the charge-exclusion and preconcentration features of Nafion. With a preconcentration time of 60 s at a potential of -0.3 V (vs. Ag/AgCI), linear calibration plots are obtained for dopamine in 0.1 M, phosphate buffer (pH 7.4) over 0-20 pM with a detection limit (30) of 0.1 pM. Keywords: Nafion, Dopamine, Square-wave voltammetry, Ruthenium-oxide pyrochlore, Chemically modified electrode

1. Introduction Sensitive and selective methods are needed for the detection and determination of dopamine (DA) due to its importance in brain chemistry [l]. The electrochemical detection of DA in the extracellular fluid of the central nervous system has received much attention. However, attempts to measure neurotransmitters, particularly DA, in brain with voltammetric methods require the use of several strategies to improve the detection [2-201. The main problem is that the basal DA concentration is very low (0.01-1 pM) while the concentration of interfering anions such as ascorbic acid (AA) is much higher (about 0.1 mM) [17]. At a bare glassy carbon electrode (GCE), the two species oxidize at similar potentials [18, 191, preventing reliable measurement of the DA response. Among the strategies reported to address this problem, a convenient way is to coat the working electrode with an anionic film such as Nafion to protect the surface from the interferences [17, 201. The reason for the selective sensing of DA is obvious. At physiological pH of 7.4, AA exists in the anionic form (pK, = 4.10) while DA is in the cationic form (pKb= 8.87). Consequently, the Nafion film repels the negatively charged AA and the selective sensing of DA is achieved. Considering the model mentioned above, we report here a Nafiodruthenium oxide pyrochlore chemically modified electrode (CME) for the selective determination of DA in the presence of high concentrations of AA at physiological pH. This CME has been used for the determination of dissolved oxygen, hydrazine, caffeine, acetaminophen, and uric acid with excellent sensitivity in our previous studies [21-241. Significant advantages have been achieved by combining the electrocatalytic function of the ruthenium oxide pyrochlore catalyst with the charge-exclusion and preconcentration features of Nafion. In this article, the CME is further applied to the determination of DA in the presence of a high concentration of AA and the optimal experimental conditions are thoroughly investigated.

2. Experimental Dopamine and L-ascorbic acid was purchased from Sigma. Nafion perfluorinated ion-exchange powder, 5 wt.% solution in a mixture of lower aliphatic alcohols and 10 % water, was obtained from Aldrich. All the other compounds used in this work were prepared from ACS-certified reagent grade chemicals without Electroanalysis 1991, 9, No. I

further purification in double distilled deionized water. A 0.1 M phosphate buffer, pH 7.4, was used for all electrochemical measurements, unless otherwise noted. Electrochemistry was performed on a Bioanalytical Systems BAS 50W electrochemical analyzer. A BAS Model VC-2 electrochemical cell was employed in these experiments. The threeelectrode system consisted of a Nafiodruthenium-oxide pyrochlore CME working electrode, a AglAgCl reference electrode (Model RE-5,BAS), and a platinum wire auxiliary electrode. The glassy carbon disk electrode (3mm diameter, BAS) was polished on a polishing cloth sequentially with diamond powder of decreasing particle size (15, 3, 1, and 0.05 pm) to a shiny surface. It was then rinsed with deionized water and further cleaned ultrasonically in 1:1 nitric acid and deionized water successively. The preparation of the Nafionhthenium-oxide pyrochlore CME was described elsewhere [21]. Solutions of DA and AA were prepared daily using deionized water and used directly for detection under open air at room temperature, The DA quantitation was achieved by measuring the oxidation peak current from squarewave (SW) voltammograms taken between -0.30 and +0.60V in 0.1 M, pH 7.4 phosphate buffer.

3. Results and Discussion The catalytic function of the Nafiodruthenium oxide pyrochlore CME at pH 7.0 is demonstrated in Figure 1 for 0.5 mM DA recorded at a bare GCE, a Nafion-coated GCE, and the Nafiodruthenium oxide pyrochlore CME by SW voltammetry with a 2 s of quiet time. As can be seen, on scanning from -0.30V toward a positive potential at a bare GCE, a relatively small anodic peak at +0.35 V was observed (Fig. 1, curve a); while, a slightly increases in anodic peak was observed at +0.50V when a Nafion-coated GCE was used (Fig. 1, curve b). Note that the DA response for the Nafion-coated GCE can be further increased if a preconcentration period is applied. The slight increase in DA response for the Nafion-coated GCE is an indication of the ion-exchange process between Nafion and DA. Most important of all, the use of the Nafiodruthenium oxide pyrochlore CME resulted in a large enhancement in current response at +0.25 V (Fig. 1, curve c). The CME exhibits a clear shift of the oxidation potential in cathodic direction for DA. The results of lowered overpotential and increased current response are clear evidence of the catalytic effect.

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Fig. 1. SW voltammograms for 0.5 mM DA in 0.1 M, pH 7.0 phosphate buffer at a bare GCE (a), a Nafion-coated GCE (b) and the Nafiodruthenium oxide pyrochlore CME (c). SW amplitude, 25 mV; SW frequency, 15 Ha; step height, 4mV.

Further investigation was made to the transport characteristics of DA in the Nafiodruthenium oxide pyrochlore CME. The linear scan voltammetry current response obtained at the CME was found to be linearly proportional to the square root of the scan rate, which illustrated that the process was diffusion-controlled. More evidence for the nonadsorptive behavior of DA was demonstrated by the following experiment. When the CME was switched to a medium containing only pH 7.4 buffer solution after being used in measuring a DA solution, no voltammetric peak signal was observed at all. Both the electrode and the detection aspects should be considered to arrive at the optimum conditions for DA determination. As to the electrode aspect, the optimum conditions generally follow those used in previous studies [20-231. In order to determine the optimum range over which the film is permselective, the effect of pH on the voltammetric response of the Nafionhthenium oxide pyrochlore CME was studied first. The dependence of the peak current

for 10pM DA on the pH of the analyte solutions is shown in Figure 2. As can be seen, the CME shows an optimum performance between pH 6.5 and pH 8.0. This is in effect a perfect result for the selective sensing of DA in the presence of AA. As mentioned earlier, the Nafion film attracts the DA while it repels the AA since the amine group of DA is positively charged, whereas the hydroxyl next to the carbonyl group of AA is negatively charged at physiological pH of 7.4 (pKb= 8.87 and ply, = 4.10, respectively). A 0.1 M phosphate buffer, pH 7.4, was used for all subsequent electrochemical measurements. The effects of preconcentration potential and preconcentration time on the SW response for DA were studied next and the results obtained are shown in Figures 3A and B, respectively. As can be seen in Figure 3A, the peak current increases as the potential of the electrode becomes more negative between +0.1 and -0.3 V. This behavior is explained by the fact that DA bears a positive charge at pH 7.4; as a result, the accumulation of DA is favored at more negative potentials. However, the peak current drops rapidly as the potential is more negative than -0.3 V. A preconcentration potential of -0.3 V was therefore chosen in all subsequent work. As to the effect of preconcentration time, for 10 pM of DA, the peak current increases as the preconcentration time increases and starts to level off around 60 s as shown in Figure 3B. Note that it takes longer time for the peak current to level off for a lower concentration of DA (not shown). This phenomenon is as expected and further confirms the ion-exchange process between Nafion and DA. Therefore, to increase the sensitivity of detection, a longer preconcentration time is needed for the lower concentration of DA. The peak current obtained in SW voltammetry is dependent on various instrumental parameters such as SW amplitude, SW frequency, and step height. These parameters are interrelated and affect the response, but here only the general trends will be examined. It was found that these parameters had little effect on the peak potential. When the SW amplitude was varied among 20 and 90 mV, the peak currents were increased with increasing amplitude. I

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Fig. 3. Effect of A) preconcentration potential and B) preconcentration time on the peak current of l 0 p M DA obtained at the Nafionlruthenium oxide pyrochlore CME. SW amplitude, 70 mV; SW frequency, 50 Hz; step height, 4 mV.

Determination of Dopamine

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However, the peak width was also increasing at the same time, in particular when the amplitude was greater than 70 mV. Hence 70 mV was chosen as the SW amplitude. The step height together with the frequency define the effective scan rate. Hence an increase with either the frequency or the step height results in an increase in the effective scan rate. The response for DA increases with SW frequency up to 50 Hz, above which the peak current was unstable and obscured by a large residual current. By maintaining the SW frequency as 50Hz, the effect of step height was studied. At step heights greater than 8 mV, too few points are sampled, thus affecting the reproducibility of the detection; whereas at step height of 4 mV the response is more accurately recorded. Overall, the optimized parameters can be summarized as follows: SW frequency, 50Hz; SW amplitude, 70mV; step height, 4mV. Under optimum conditions, Figure 4 shows the SWVs obtained for DA immediately after the CME is immersed into the test

solutions with concentrations ranging from 0 to 70pM with (Fig. 4A) and without (Fig. 4B) the presence of I mM AA. A linear calibration plot (Fig. 4C) was obtained in 0.1 M, pH 7.4 phosphate buffer over 0-70pM and the detection limit (30) and correlation coefficient were 0.18pM and 0.9986 for DA alone. While, in the presence of lmh4 AA, a linear calibration plot (Fig. 4C) was obtained over 5-70 pM and the detection limit (3u)and correlation coefficients were 0.15 pM and 0.9990. Same experiments were repeated with a preconcentration time of 60 s at a preconcentration potential of -0.3V. Figure 5 shows the SW voltammograms obtained for DA with concentrations ranging from 0 to 20 pM with (Fig. 5A) and without (Fig. 5B) the presence of 1 mM AA. As can be seen, a linear calibration plot (Fig. 5C) was obtained over 020 pM and the detection limit (35) and correlation coefficient were 0.10 pM and 0.9986 for DA alone. While, in the presence of 1 mM AA, a linear calibration plot (Fig. 5C) was obtained over 0-10pM 60

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Fig. 5. A) SW voltammograms for a) 0, b) 0, c) 2.5, d) 5, e) 7.5, and f) 10pM DA obtained at the Nafiontruthenium oxide pyrochlore CME with the presence of 0.1 mM AA (a) and 1 mM AA (b-0. B) SWVs for a) 0, b) 2.5, c) 5, d) 7.5, e) 10, and f) 20 pM DA. C) Calibration plots. All the measurements were done with a preconcentration time of 60 s at a preconcentration potential of -0.3 V (vs. Ag/AgCl). Other conditions, as in Figure 3. Electroanalysis 1997, 9,No. I

540 and the detection limit (3a) and correlation coefficients were 0.11 pM and 0.9984. It is noteworthy that (i) no peak was observed for 0.1 mM AA alone as shown in Figure 5A, curve a and (ii) the appearance of a small peak for 1mM AA as shown in Figure SA, curve b apparently did not affect the linear range. Overall, the preconcentration step can increase the sensitivity but at the expense of the linear range. To characterize the reproducibility of the CME, repetitive measurement-regeneration cycles were carried out in 10 pM DA and 1 mM AA. The results of 15 successive measurements showed a relative standard deviation of 1.3 %. Thus, the electrode renewal gives a good reproducible surface. Finally, the stability of the Nafiodruthenium-oxide pyrochlore CME was also evaluated. The CME shows an excellent long-term stability. No decrease in DA response about either peak potential or peak current was observed after the electrode was repeatedly used and stored in I M NaOH solution for more than 2 months. Furthermore, since the process is diffusion-controlled as discussed previously, the response time for the CME is very fast. All the measurements can be done immediately after the CME is immersed into the test samples.

4. Conclusions The Nafiodruthenium oxide pyrochlore CME exhibit strong discrimination for DA over AA enabling quantitation of DA in the presence of a large excess of AA. The modification procedure is reproducible, the resultant attachment is stable and the modified electrodes have faster response times compared to those of unmodified electrodes. These features suggest that the Nafiodruthenium oxide pyrochlore CME could be developed as useful probes for the in vivo monitoring of neurotransmitters.

Electruanalysis 1997. 9, No. 7

J.-M. Zen, I-L. Chen

5. Acknowledgement The authors gratefully acknowledge financial support from the National Science Council of the Republic of China under grant NSC 86-21 13-M-005-021.

6. References [ I ] J.A. Stamford, J.B. Justice Jr., Anal. Chem. 1996, 69, 359A. [2] P. Capella, B. Ghasemzadeh, K. Mitchell, R.N. Adams, Electruanalysis 1990, 2, 175. [3] F.G. Gonon, C.M. Fombarlet, M.J. Buda, J.F. Pujol, Anal. Chem. 1981, 53, 1386. [4] A.G. Ewing, M.A. Dayton, M.R. Wightman, Anal. Chem. 1981, 53, 1842. [5] L. Falat, H.-Y. Cheng, Anal. Chem. 1982,54, 2108. [6] J.A. Stamford, Anal. Chem. 1986, 58, 1033. [7] G.N. Kainau, J.F. Rusling, Electruanalysis 1994, 6, 445. [8] G.A. Gerhardt, A.F. Oke, G. Nagy, B. Moghaddam, R.N. Adams, Brain Res. 1984,290, 390. [9] E.W. Kristensen, W.G. Kuhr, M.R. Wightman, Anal. Chern. 1987, 59, 1752. [lo] J.-X. Feng, M. Brazell, K. Renner, R. Kasser, R.N. Adams, Anal. Chem. 1987,59, 1863. 1111 Y.Y. Lau, J.B. Chien, D.K.Y. Wong, A.G. Ewing, Electruanulysis 1991.3, 87. 1121 0. Niwa, M. Morita, H. Tabei, Electroanalysis 1994, 6, 237. [I31 M.B. Gelbert, D.J. Curran, Anal. Chem. 1986,58, 1028. [14] G.E. Glynn, B.K. Yamamoto, Brain Res. 1989, 481, 235. [15] F. Malem, D. Mandler, Anal. Chem. 1993, 65, 37. 1161 M.R. Wightman, L.J. May, A.C. Michael, Anal. Chem. l988,60,769A. [17] A.J. Downard, A.D. Roddick, A.M. Bond, Anal. Chim.Acta 1995, 31 7, 303. 1181 J. Wang, L.D. Hutchins, Anal. Chim. Acta 1985, 167, 325. 1191 M.R. Deakin, P.M. Kovach, K.J. Stutts, M.R. Wightman, Anal. Chem. 1986, 58, 1474. [20] M.E. Rice, A.F. Oke, C.W. Bradberry, R.N. Adams, Brain Res. 1985,340, 151. 1211 J.-M. Zen, C.-B. Wang, J. Electruanal. Chem. 1994, 368, 251. [22] J.-M. Zen, Tang, Anal. Chem. 1995, 67, 208. [23] J.-M. Zen, J.-S. Tang, Anal. Chem. 1995, 67, 1892. 1241 J.-M. Zen, Y . 4 . Ting, Anal. Chim.Acta in press.

Voltammetric determination of dopamine in the ...

Jylz-Myng Zen* and I-Lang Chen. Department of Chemistry ..... The authors gratefully acknowledge financial support from the. National Science Council of the ...

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