Anal. Chem. 1997, 69, 5087-5093

A Selective Voltammetric Method for Uric Acid and Dopamine Detection Using Clay-Modified Electrodes Jyh-Myng Zen* and Ping-Jyh Chen

Department of Chemistry, National Chung-Hsing University, Taichung, Taiwan 402

Electrochemically preanodized clay-modified electrodes were used for the detection of uric acid or dopamine in the presence of a high concentration of ascorbic acid by square wave voltammetry. The major obstacle of the overlapped oxidation potential of ascorbic acid could be overcome through the distinct ability of uric acid and dopamine to coordinate with the electrochemically preanodized clay-modified electrodes. The selective sensing of dopamine is further improved by the charge-exclusion and preconcentration features of Nafion. Linear calibration curves are obtained for 0.5-10 and 10-100 µM uric acid with 30 s preconcentration time in 0.1 M, pH 1.0 citrate buffer and for 0-6 µM dopamine in 0.1 M, pH 7.4 phosphate buffer with 20 s preconcentration time. The detection limits (3σ) are 0.2 µM and 2.7 nM for uric acid and dopamine, respectively. The practical analytical utility is illustrated by selective measurements of uric acid in human urine without any preliminary treatment. Uric acid (UA) is the primary end product of purine metabolism. It has been shown that extreme abnormalities of UA levels are symptoms of several diseases.1 Therefore, it is essential to develop simple and rapid methods for its determination in routine analysis. Earlier electrochemical procedures based on the oxidation of UA at carbon-based electrodes in acidic solutions suffer from interference from ascorbic acid (AA), which can be oxidized at a potential close to the oxidation potential of UA.2,3 Similarly, the electrochemical detection of dopamine (DA) is important in brain chemistry, and the ability to measure DA selectively in the presence of AA has been a major goal of electroanalytical research.4 Since the basal DA concentration is very low (0.01-1 µM), while the concentration of AA is much higher (about 0.1 mM),5 both sensitivity and selectivity are of equal importance in DA detection due to its neurochemical interest. Various methods, such as an adsorption/medium exchange approach,6,7 enzyme-based techniques,8-12 the use of polymer(1) Eswara Dutt V. S.; Mottola, H. A. Anal. Chem. 1974, 46, 1777. (2) Park, G.; Adams, R. N.; White, W. R. Anal. Lett. 1972, 5, 887. (3) Yao, T.; Taniguchi, Y.; Wasa, T.; Musha, S. Bull. Chem. Soc. Jpn. 1978, 51, 2937. (4) Stamford, J. A.; Justice, J. B., Jr. Anal. Chem. 1996, 69, 359A. (5) Capella P.; Ghasemzadeh, B.; Mitchell, K.; Adams, R. N. Electroanalysis 1990, 2, 175. (6) Wang, J.; Freiha, B. A. Bioelectrochem. Bioenerg. 1984, 12, 225. (7) Tatsuma, T.; Watanabe, T. Anal. Chim. Acta 1991, 242, 85. (8) Keedy, F. E.; Vadgama, P. Biosens. Bioelectron. 1991, 6, 491. (9) Gonzalez, E.; Pariente, F.; Lorenzo E.; Hernandez, L. Anal. Chim. Acta 1991, 242, 267. (10) Gilmartin, M. A. T.; Hart, J. P.; Birch, B. Analyst 1992, 117, 1299. S0003-2700(97)00356-9 CCC: $14.00

© 1997 American Chemical Society

modified electrodes with13 and without12,14,15 catalyst, and the use of electrochemically pretreated carbon paste electrode,16 were developed to solve the UA detection problem. Among these, our previous study demonstrated that a poly(4-vinylpyridine)-coated carbon paste electrode improves the electrochemical monitoring of UA due to the distinct ability of AA and UA to form hydrogen bonds with poly(4-vinylpyridine) in acidic solutions.15 A voltammetric method with electrochemically pretreated carbon paste electrode was reported to tolerate the interference of AA up to 30-fold excess with a detection limit of 1.2 × 10-8 M.16 Unfortunately, the electrode material was found to become swollen after more than 30 measurements. Meanwhile, among the several strategies reported to solve the DA detection problem,5,17-35 a convenient way is to coat the working electrode with an anionic film such as Nafion to protect the surface from the interferences.5,34,35 The reason is obvious: AA exists in the anionic form (pKa ) 4.10) while DA is in the cationic form (pKb ) 8.87) at the physiological pH of 7.4. However, the use of Nafion alone obviously cannot solve the sensitivity problem. Therefore, the addition of extra accumulation factors is necessary. For example, (11) Rocheleau, M. J.; Purdy, W. C. Electroanalysis 1991, 3, 935. (12) Miland, E.; Ordieres, A. J. M.; Blanco, P. T.; Smyth, M. R.; Fagain, C. O. Talanta 1996, 43, 785. (13) Zen, J.-M.; Tang, J.-S. Anal. Chem. 1995, 67, 1872. (14) Gandour, M. A.; Kasim, E.-A.; Amrallah, A. H.; Farghaly, O. A. Talanta 1994, 41, 439. (15) Zen, J.-M.; Chen, Y.-J.; Hsu, C.-T.; Ting, Y.-S. Electroanalysis, in press. (16) Cai, X.; Kalcher, K.; Neuhold, C.; Ogorevc, B. Talanta 1994, 41, 407. (17) Gonon, F. G.; Fombarlet, C. M.; Buda, M. J.; Pujol, J. F. Anal. Chem. 1981, 53, 1386. (18) Ewing, A. G.; Dayton, M. A.; Wightman, M. R. Anal. Chem. 1981, 53, 1842. (19) Falat, L., Cheng, H.-Y. Anal. Chem. 1982, 54, 2108. (20) Stamford, J. A. Anal. Chem. 1986, 58, 1033. (21) Kamau, G. N.; Rusling, J. F. Electroanalysis 1994, 6, 445. (22) Gerhardt, G. A.; Oke, A. F.; Nagy, G.; Moghaddam, B.; Adams, R. N. Brain Res. 1984, 290, 390. (23) Kristensen, E. W.; Kuhr, W. G.; Wightman, M. R. Anal. Chem. 1987, 59, 1752. (24) Feng, J.-X.; Brazell, M.; Renner, K.; Kasser, R.; Adams, R. N. Anal. Chem. 1987, 59, 1863. (25) Lau, Y. Y.; Chien, J. B.; Wong, D. K. Y.; Ewing, A. G. Electroanalysis 1991, 3, 87. (26) Niwa, O.; Morita, M.; Tabei, H. Electroanalysis 1994, 6, 237. (27) Gelbert, M. B.; Curran, D. J. Anal. Chem. 1986, 58, 1028. (28) Glynn, G. E.; Yamamoto, B. K. Brain Res. 1989, 481, 235. (29) Malem, F.; Mandler, D. Anal. Chem. 1993, 65, 37. (30) Wightman, M. R.; May, L. J.; Michael, A. C. Anal. Chem. 1988, 60, 769A. (31) Downard, A. J.; Roddick, A. D.; Bond, A. M. Anal. Chim. Acta 1995, 317, 303. (32) Wang, J.; Hutchins, L. D. Anal. Chim. Acta 1985, 167, 325. (33) Deakin, M. R.; Kovach, P. M.; Stutts, K. J.; Wightman, M. R. Anal. Chem. 1986, 58, 1474. (34) Rice, M. E.; Oke, A. F.; Bradberry, C. W.; Adams, R. N. Brain Res. 1985, 340, 151. (35) Zen, J.-M.; Chen, I.-L. Electroanalysis 1997, 9, 537.

Analytical Chemistry, Vol. 69, No. 24, December 15, 1997 5087

our group previously reported a Nafion/ruthenium oxide pyrochlore chemically modified electrode for the selective determination of DA in the presence of high concentrations of AA at physiological pH.35 Significant advantages have been achieved by combining the electrocatalytic function of the catalyst with the charge-exclusion and preconcentration features of Nafion. Combining the interesting concepts mentioned above, we report here an improved voltammetric method for the determination of UA or DA in the presence of a high concentration of AA using electrochemically pretreated clay-modified electrodes by square wave voltammetry (SWV). The modified electrodes were prepared on the surface of glassy carbon electrodes (GCEs) to prevent the swelling of electrode material after repetitive measurements. The clay used to construct the electrode in this study is nontronite, in which at least half of the aluminum ions within the clay are replaced by iron.36 According to Pearson’s principle, hard acids prefer to bind to hard bases, and soft acids prefer to bind to soft bases.37 Lighter transition metals in higher oxidation states, such as Fe3+, are hard acids, while UA or DA is apparently a hard base. Therefore, a preanodized procedure can, in effect, convert all of the iron in the nontronite into Fe3+ form or even higher oxidation state and results in a very strong complexing force with UA or DA. In this paper, the optimal experimental conditions are thoroughly investigated. The electrochemically pretreated clay-modified electrode is also applied to the selective determination of UA in urine samples. EXPERIMENTAL SECTION Chemicals and Reagents. Nafion perfluorinated ion-exchange powder, 5 wt % solution in a mixture of lower aliphatic alcohols and 10% water, was obtained from Aldrich. Standard clay mineral, nontronite (SWa-1, ferruginous smectite), was purchased from the Source Clay Minerals Repository (University of Missouri). UA (Sigma), DA (Sigma), AA (Wako), and all the other compounds (ACS-certified reagent grade) used in this work were prepared without further purification in doubly distilled deionized water. Apparatus. Electrochemistry was performed on a Bioanalytical Systems (West Lafayette, IN) BAS-50W electrochemical analyzer. A BAS Model VC-2 electrochemical cell was employed in these experiments. The three-electrode system consisted of an Ag/AgCl reference electrode (Model RE-5, BAS), a platinum wire auxiliary electrode, and one of the following working electrodes: a GCE, a Nafion-coated GCE, a nontronite-coated GCE, a Nafion/nontronite-coated GCE, or a Nafion-coated claymodified electrode. Since dissolved oxygen did not interfere with the anodic voltammetry, no deaeration was performed. Procedure. Clay colloids were prepared in the sodium form as previously described.38 Nafion-coated GCE was prepared by spin-coating 10 µL of 2 wt % Nafion on the GCE surface at 3000 rpm. In the preparation of the nontronite-coated GCE, the clay film was prepared by spin-coating 10 µL of a clay colloid (0.1 wt % in ethanol) onto a clean GCE surface at 3000 rpm. As for the Nafion/nontronite-coated GCEs, different ratios of the Nafion/ nontronite solution were first prepared based on the experimental requirement. The Nafion/nontronite solution (10 µL) was then spin-coated onto the GCE surface at 3000 rpm. A uniform thin (36) Jaynes, W. F.; Bigham, J. M. Clay. Clay Miner. 1987, 35, 440. (37) March, J. Advanced Organic Chemistry, 4th ed.; John Wiley & Sons: New York, 1992; pp 261-263. (38) Zen, J.-M.; Jeng, S.-H.; Chen, H.-J. J. Electroanal. Chem. 1996, 408, 157.

5088 Analytical Chemistry, Vol. 69, No. 24, December 15, 1997

Table 1. SW Responses for 10 µM UA and 6.05 µM DA at Variable Working Electrodes with/without Electrochemical Pretreatment working electrode

ip (µA)

GCEa Nafion-coated GCEa nontronite-coated GCEa Nafion/nontronite-coated GCEa Nafion/nontronite-coated GCEb GCEc nontronite-coated GCEc Nafion-coated clay-modified electrodec

2.9 3.9 6.2 20.2 34.0 50.5 75.0 90.1

a Experimental conditions: 10 µM UA in 0.1 M, pH 2.0 citrate buffer without electrochemical pretreatment; SW amplitude, 25 mV; SW frequency, 15 Hz; SW step, 4 mV; Pp ) -0.6 V; tp ) 30 s. b Electrochemical pretreatment conditions: Pa ) +1.8 V; ta ) 60 s. c Experimental conditions: 6.05 µM DA in 0.1 M, pH 7.4 phosphate buffer; SW amplitude, 25 mV; SW frequency, 15 Hz; SW step, 4 mV; Pa ) +1.8 V; ta ) 60 s.

film was formed after about 3 min of spinning. In the preparation of the nontronite-coated GCE, the clay film was prepared by dropping 6 µL of a clay colloid (0.5 g/L) on a clean GCE and dried under ambient conditions. For the Nafion-coated claymodified electrodes, 4 µL of a Nafion solution was spin-coated onto the previously mentioned nontronite-coated GCE at 3000 rpm. Unless otherwise stated, a 0.1 M, pH 1.0 citrate buffer and a 0.1 M, pH 7.4 phosphate buffer were used as the supporting electrolytes for UA and DA determination, respectively. Solutions of DA, UA, and AA were prepared daily using deionized water and used directly for detection under open air at room temperature. The accumulation step proceeded in constantly stirred (200 rpm) solution, and the voltage scanning step was performed after 2 s of quiet time. The detection limit (3σ) is defined as the concentration of the analyte resulting in a signal 3 times the standard deviation of the blank. Urine samples were obtained from laboratory personnel. After being filtered through membrane filters (0.45 µM), all samples were stored in the dark at 4 °C. To fit into the linear range, all urine samples used for detection were diluted by 100 times. The standard addition method was used to evaluate the content of UA in samples. RESULTS AND DISCUSSION Electrochemical Behavior. The advantage of the combination of Nafion and clay in UA detection is first demonstrated by SWV recorded at a bare GCE, a Nafion-coated GCE, a nontronitecoated GCE, and a Nafion/nontronite-coated GCE in 0.1 M, pH 2.0 citrate buffer without electrochemical preanodization. As shown in Table 1, the SWV current responses were found to increase in the sequence of GCE f Nafion-coated GCE f nontronite-coated GCE f Nafion/nontronite-coated GCE. The obviously increase in UA response from nontronite-coated GCE to Nafion/nontronite-coated GCE is a good indication of the excellent accumulation ability with the combination of Nafion and nontronite clay. Additional reasons for the combination of Nafion and nontronite clay in the fabrication of the clay-modified electrode for UA detection are as follows. First, even though AA is the major concern in UA detection, in real samples there are also other interferents. The Nafion membrane coating on the Nafion/ nontronite-coated GCE is primarily designated to protect the

surface from many surface-active compounds, since the nontronite clay alone cannot prevent the interference. Second, it is expected that even a Nafion-coated electrode can accumulate UA, as it has been shown previously that Nafion has a very high affinity for hydrophobic, organic cations. However, a preanodized procedure can, in effect, convert all of the iron in the nontronite into higher oxidation forms and results in a strong complexing force with UA. Besides, it is well known that electrochemical pretreatment of carbon-based electrodes is often used to enhance the analytical performance of electrodes.39,40 The anodic pretreatment can improve the performance of carbon-based electrodes in some respects: increased electrochemical activity, lowered overpotential, and increased wettability. The main effect was reported be due to the generation of hydrophilic electron-transfer-mediating groups by oxidation of the electrode material.39,40 The SW responses for 10 µM UA recorded at a Nafion/ nontronite-coated GCE with/without electrochemically pretreatment are listed in Table 1 for comparison. Evidently, the anodic polarization results in a large enhancement in current response. It is as expected, since both effects mentioned above would have a contribution in improving the sensitivity of UA detection. Note that a more detailed study to assess whether the clay is having an effect will be discussed later. Similarly, for the DA detection, the function of nontronite is first demonstrated by cyclic voltammetry (CV) for 2.42 × 10-4 M DA recorded at a nontronite-coated GCE with/without electrochemical pretreatment. As shown in Figure 1, a much smaller CV peak response with a ∆Ep of 272 mV was observed on scanning between -0.3 and +0.5 V without electrochemical pretreatment. Pretreatment at +1.8 V for 60 s leads to an obvious increase in CV peak response and a more reversible behavior for DA in CV, with an ∆Ep of only 48 mV. In view of the structure of DA, it contains both an amino group and a phenol group, which are classified as hard bases. Hence, it is expected that there is also a strong interaction between ferric ion and DA. In this study, however, DA is in its cationic state at pH 7.4, which makes it arguable whether the strong interaction still occurs. At pH 7.4,

the amino group is protonated to form an ammonium ion, but the phenol group still remains as a free form or a phenoxide ion. Although an ammonium ion is not a hard base, both the phenoxide ion and the free form of phenol are hard bases. Therefore, a strong response for the interaction between DA and preanodized nontronite still can be observed. It is well known that the sensitivity of SWV of adsorbed species is proportional to the degree of reversibility of the electrochemical reaction.41,42 Since the redox DA couple showed a more reversible behavior at the preanodized Nafion-coated clay-modified electrode, a clear advantage of using the electrode in the SW mode with respect to the sensitivity of DA detection is expected. By carrying out experiments for 2.42 × 10-4 M DA with SWV, differential pulse voltammetry (DPV), and linear scan voltammetry (LSV) at the same effective scan rate of 60 mV/s, the prediction agrees well with the observed behavior. Comparing the sensitivity between SWV and LSV, the more reversible DA behavior at the preanodized Nafion-coated clay-modified electrode shows an obvious improvement. In terms of background rejection, LSV gives the highest sloping baseline, which causes difficulty in the measurement of low concentrations. SWV competes with DPV for background rejection ability, with DPV having a smaller advantage in that respect. Overall, even though SWV has a small disadvantage in terms of background discrimination compared to DPV, it makes up for that with its superior sensitivity in practical applications. The advantage in the combination of Nafion and nontronite for DA detection was studied next. The SW responses for 6.05 µM DA recorded at a bare GCE, a nontronite-coated GCE, and a Nafion-coated clay-modified electrode with electrochemical pretreatment are shown in Table 1. The obviously increase in DA response from GCE to nontronite-coated GCE to Nafion-coated clay-modified electrode is a good indication of the proper function of each component. Further investigation was made into the transport characteristics of UA and DA in the clay-modified electrodes. Both the LSV current responses for UA obtained at the Nafion/nontronitecoated GCE and those for DA at the Nafion-coated clay-modified electrode were found to be linearly proportional to the scan rate, which is an indication of adsorption behavior. More evidence for the adsorptive behavior of UA and DA was demonstrated by the following experiment. When the Nafion/nontronite-coated GCE was switched to a medium containing only pH 1.0 citrate buffer solution after being used in measuring a UA solution, virtually the same voltammetric peak signal was observed. The same phenomenon was also observed for DA. Chronocoulometric experiments were also done to study the adsorption process, as presented in more detail in a later section for UA. Overall, the known facts are consistent with the results shown above. For the DA, the chronocoulometric experiments were done to study the adsorption process at the three different electrodes of GCE, nontronite-coated GCE, and Nafion-coated claymodified electrode with/without electrochemical pretreatment. Based on an Anson plot, the intercept of Q vs t1/2 can be used to evaluate the surface excess. Two important results can be extracted from the intercepts obtained, as shown in Table 2. First, compared to unpretreated electrodes, the intercepts increase rapidly when electrochemical pretreatment was applied in all three

(39) Engstrom, R. C. Anal. Chem. 1986, 58, 136. (40) Ravichandran, K.; Baldwin, R. P. Anal. Chem. 1984, 56, 1744.

(41) Lovric, M.; Branica, M. J. Electroanal. Chem. 1987, 226, 239. (42) Lovric, M.; Komorsky-Lovric, S. J. Electroanal. Chem. 1988, 248, 239.

Figure 1. Steady-state cyclic voltammograms for 2.42 × 10-4 M DA in 0.1 M, pH 7.4 phosphate buffer at a nontronite-coated GCE (A) with and (B) without electrochemical pretreatment at +1.8 V for 60 s. Scan rate was 100 mV/s.

Analytical Chemistry, Vol. 69, No. 24, December 15, 1997

5089

Table 2. Chronocoulometrya at GCE, Nontronite-Coated GCE, and Nafion-Coated Clay-Modified Electrode with/without Electrochemical Pretreatment working electrode

intercepta (µC)

interceptc (µC)

GCE GCEb nontronite-coated GCE nontronite-coated GCEb Nafion-coated clay-modified electrode Nafion-coated clay-modified electrodeb

1.71 2.17 1.86 2.95 2.63 4.35

-1.53 -1.82 -1.05 -2.32 -1.84 -2.98

a Experimental conditions: E ) 0 V; E ) +0.5 V; pulse, 50 ms; 20 i f s of preconcentration at open circuit. Subscripts a and c refer to anodic and cathodic responses, respectively. Intercepts were obtained from Anson plots. b Electrochemical pretreatment conditions: Pa ) +2.0 V; ta ) 60 s.

Table 3. Effects of the Coating Solutions Used in Preparing the Nafion/Nontronite-Coated GCE on the Peak Current of UA Determination and in Preparing the Nafion-Coated Clay-Modified Electrode on the Peak Current of DA Determinationa Nafion/nontronite-coated GCE

Nafion-coated clay-modified electrode

Nafion (wt %)

clay (wt %)

ip (µA)

Nafion (wt %)

ip (µA)

3.15 3.15 3.15 3.15 0.50 1.00 1.50 2.00 3.00 4.00

0.05 0.10 0.50 1.00 0.10 0.10 0.10 0.10 0.10 0.10

17.5 19.4 13.3 7.8 13.4 17.5 34.9 22.5 19.2 12.3

0.05 0.10 1.50 2.50 3.00 4.00 5.00

32.6 36.3 45.4 30.2 28.2 27.8 24.7

a

[UA] ) 10 µM; [DA] ) 6.05 µM.

electrodes. Second, the intercepts for the pretreated electrodes were found to increase in the sequence of GCE f nontronitecoated GCE f Nafion-coated clay-modified electrode. The results are quite consistent with the SW response of DA as shown in Table 1. In other words, the main contribution of the SW response was the large increase in DA adsorption after electrochemical pretreatment at the Nafion-coated clay-modified electrode. Optimization of Detection. To arrive at the optimum conditions for UA and DA determination, two aspects should be considered: the electrode and the detection. The major factors that should be considered are the Nafion/clay composition, the solution pH, the preanodization potential (Pa), the preanodization time (ta), the preconcentration potential (Pp), the preconcentration time (tp), and the SWV parameters. The Nafion/clay composition directly controls the electrode performance, and this condition was optimized by varying the ratio between Nafion and clay. The Nafion/nontronite-coated GCEs prepared from coating solutions that contain 1.7 mL of Nafion + 1 mL of nontronite (0.05, 0.1, 0.5, and 1 wt % in ethanol) were examined under identical conditions. As shown in Table 3, in all cases UA responses could be obtained, and the peak current reached a maximum when the content of nontronite was around 0.1 wt %. The accumulation ability of nontronite with UA functions properly in the Nafion/nontronite-coated GCE. The condition was 5090 Analytical Chemistry, Vol. 69, No. 24, December 15, 1997

Figure 2. Effects of (A) Pa and (B) ta at the Nafion/nontronite-coated GCE on the SW voltammetric response of 10 µM UA. SW parameters: modulation amplitude, 55 mV; modulation frequency, 55 Hz; step height, 4 mV. Conditions: (A) Pp ) -0.2 V; tp ) 40 s; ta ) 60 s. (B) Pp ) -0.2 V; tp ) 40 s; Pa ) +1.8 V.

further optimized by varying the amount of Nafion in the coating solutions as follows: 2-0.1 mL of Nafion + 1 mL of nontronite (0.1 wt % in ethanol). Also shown in Table 3, the results indicate that the optimum coating solution is 0.25 mL of Nafion + 1 mL of nontronite (0.1 wt % in ethanol). Electrodes prepared by the above combination of coating solution were used in all subsequent work for UA detection. For DA detection, the Nafion/clay composition was optimized by varying the amount of Nafion with fixed nontronite-coated GCE. The optimum composition depends on both the diffusion processes of the DA ions in the film and the maximum loading that does not affect the adhesion of the film to the GCE surface. The results indicate that the optimum coating solution is 1.5 wt % of Nafion (Table 3). Electrodes prepared by the above Nafion coating solution were thus used in the subsequent DA detection work. The effects of pH on the voltammetric oxidation of UA were studied at a Nafion/nontronite-coated GCE with electrochemical pretreatment. To ascertain the correctness of the pH effect, the preanodization was done in each pH buffer solution to avoid a pH change due to a medium exchange. The current response was found to increase rapidly at pH < 4. Obviously, UA gives a better response in the acidic environment. The stronger the acidic medium, the better is the performance of the Nafion/nontronitecoated GCE. The maximum response was seen at pH 1, where the peak potential decreased 60 mV/pH. The trend of the peak potential shifts linearly toward negative potentials with an increase in pH and indicates that the proton is directly involved in the rate determination step of the UA oxidation reaction. The effect of the Pa on the SW response for UA is shown in Figure 2A. As can be seen, the peak current increases as the Pa of the electrode becomes more positive between +1.4 and +2.2 V. The peak current drops rapidly as the Pa is more positive than +2.2 V, and the reason is not clear at this point. However, it is interesting to note that the increase in the peak current is, in effect, a reflection of the increase in the surface excess of UA, as will be discussed later. For practical use, since a more reproducible result was obtained with a Pa of +2.0 V, it is chosen for the subsequent experiments. The effect of ta on the SW response for UA is shown in Figure 2B. For 10 µM UA, the peak current increases as the ta increases and reaches a maximum around 200 s. For convenience, a ta of 60 s is chosen for the subsequent experiments. To increase the sensitivity of detection, a longer ta can be applied for a lower concentration of UA.

Figure 3. Effects of (A) Pp and (B) tp on the peak current in SWV for 10 µM UA at the Nafion/nontronite-coated GCE. Pa ) +1.8 V; ta ) 60 s. SW parameters as in Figure 2. Also shown in (A) is the effect of Pp on the intercept in chronocoulometry for 10 µM UA at the Nafion/ nontronite-coated GCE under the same experimental conditions. Table 4. Effects of Pa and ta at the Nafion-Coated Clay-Modified Electrode on the SW Voltammetric Response of 6.05 µM DA in the Presence of 6.05 mM AA with 20 s of Preconcentration at Open Circuita varying Pa

varying ta

Pa (V)

ta (s)

ip (µA)

Pa (V)

ta (s)

ip (µA)

1.4 1.6 1.8 2.0 2.2 2.4

60 60 60 60 60 60

55.5 60.8 117.9 132.9 63.2 28.8

2.0 2.0 2.0 2.0 2.0 2.0

20 40 60 80 100 120

58.6 84.9 132.9 102.8 49.9 38.4

a SW parameters: modulation amplitude, 45 mV; modulation frequency, 55 Hz; step height, 4 mV.

Using the preanodization conditions mentioned above, the effect of the Pp on the SW response for UA detection is shown in Figure 3A. It is interesting that, for 10 µM UA, the peak current reaches a maximum at a Pp of -0.2 V with a tp of 30 s. The large drop of peak current when the Pp was more negative than -0.2 V can be explained as follows. When the Pp was more negative than -0.2 V, Fe3+ was reduced to Fe2+. Based on Pearson’s principle, since Fe2+ is not as strong a hard acid as Fe3+, the adsorption of UA was thus decreased. Most important of all, the results clearly show that the clay does have a significant effect on the accumulation of UA. Also shown in Figure 3A are the intercepts measured for the chronocoulometric experiments under the same conditions at different Pp values. It is interesting that the trends of change are virtually the same for both cases. Obviously, the effect of the Pp on the SW response is mainly due to the adsorption of UA on the electrochemically pretreated Nafion/nontronite-coated GCE. The effect of the tp on the SW response for UA is shown in Figure 3B. For 10 µM UA, the peak current increases as the tp increases and starts to level off around 30 s. It takes an even longer time for the peak current to level off for a lower concentration of UA. Therefore, to increase the sensitivity of detection, a longer tp is needed for a lower concentration of UA. The effect of Pa on the SW response for DA is summarized in Table 4. As can be seen, the peak current increases as the Pa becomes more positive between +1.5 and +2.0 V. However, the peak current drops rapidly as the Pa is more positive than +2.0 V. For practical use, since a more reproducible result was obtained with a Pa of +1.8 V, it is chosen for the subsequent experiments. The effect of ta on the SW response for DA is also

shown in Table 4. For 6.05 µM DA, the peak current increases as the ta increases and reaches a maximum around 60 s. It is, therefore, chosen for the subsequent experiments. The SW parameters that were investigated are the frequency, the pulse height, and the pulse increment. These parameters are interrelated and have a combined effect on the response. The response for UA increases with SW frequency, but at frequencies higher than 45 Hz, sloping background current renders the measurement difficult. Increase in the pulse height causes an increase in the UA peak for up to 55 mV. The scan increment, together with the frequency, defines an effective scan rate; hence, an increase of either the frequency or the pulse increment results in an increase in the effective scan rate. Overall, the best signalto-background current characteristics can be obtained with the following instrumental settings: modulation amplitude, 55 mV; modulation frequency, 45 Hz; modulation step, 4 mV. The same evaluation was also done for the DA detection, and the best signalto-background current characteristics are as follows: modulation amplitude, 45 mV; modulation frequency, 55 Hz; modulation step, 4 mV. Analytical Characterizations. Under optimum conditions, Figure 4 shows the SW voltammograms obtained for 10 µM UA with/without the presence of 1 mM AA. As can be seen, virtually the same current responses were found for UA. The acceptable tolerance of concentration of AA for the determination of UA is, therefore, as high as 1 mM. Our previous study demonstrated a distinct ability of AA and UA to form hydrogen bonds with poly(4-vinylpyridine) in acidic solutions.15 The influence of the hydrogen bond is determined by a different strength between hydrogen bond donor and hydrogen bond acceptor (HBA). The correlation between hydrogen bond and molecular structure is noted by Taft et al.43 According to their studies, the HBA strength of an amide group is generally stronger than that of an ester group. It is evident that the amide group is more nucleophilic than the ester group. In other words, UA is a harder base than AA. The interference from AA can thus be attenuated due to the different nucleophilicity of UA and AA. Linear calibration curves are obtained over the 0.5-10 and 10100 µM ranges in 0.1 M, pH 1.0 citrate buffer solution with slopes (µA/µM) and correlation coefficients of 17.0, 0.9978 and 1.16, 0.9977, respectively. The detection limit (3σ) is 0.2 µM. Similarly, Figure 5 shows the dependence of the SWV peak current for DA with concentrations ranging from 0 to 25 µM with/without the presence of 6.05 mM AA, with a preconcentration time of only 20 s at open circuit. Linear calibration curve are obtained in 0.1 M, pH 7.4 phosphate buffer over 0-6 µM, and the detection limit (3σ) and correlation coefficients are 2.7 nM and 0.9918 for DA alone. Note that the detection limit is at least 1 order of magnitude lower than those for most of the other methods, with a much shorter preconcentration time.5,17-34 In the presence of 6.05 mM AA, linear calibration curves are obtained over 0-4 µM, and the detection limit and correlation coefficients are 4.7 nM and 0.9933. As can be seen, the presence of 6.05 mM AA only slightly affects the linear range and the detection limit. To characterize the reproducibility of the Nafion/nontronitecoated GCE, repetitive measurement-regeneration cycles were carried out in 10 µM UA. The Nafion/nontronite-coated GCE can be easily renewed by cleaning at +2.0 V for 60 s in 0.1 M, pH 1.0 citrate buffer solution. Actually, the preanodization procedure (43) Taft, R. W.; Berthelot, M.; Laurence, C.; Leo, A. J. CHEMTECH 1996, 20.

Analytical Chemistry, Vol. 69, No. 24, December 15, 1997

5091

Table 5. Influence of Potential Interferants on the Response of 10 µM UA interferant AA urea purine oxalate hydrazine sucrose glucose

concn (µM)

signal change (iUA ) 100%)

100 500 1000 100 100 100 100 500 500

-0.73 -4.27 -7.32 -2.22 +1.11 +0.43 +2.75 +4.94 +0.38

Table 6. Determination of UA in Urine Samples with the Nafion/Nontronite-Coated GCEa

original value (µM) spike (µM) after spike (µM) recovery (%) total value (ppm)

urine 1

urine 2

urine 3

26.23 ( 0.68 15 41.13 ( 1.29 99 440.93 ( 11.43

25.58 ( 0.78 15 40.28 ( 1.69 98 389.58 ( 11.88

27.41 ( 0.56 15 41.81 ( 2.08 96 460.76 ( 9.41

a Total value is obtained by multiplying the detected value by the dilution factor of 100. Number of sample assayed was five.

Figure 4. (A) SW voltammograms for 10 µM UA recorded at a Nafion/nontronite-coated GCE in 0.1 M, pH 1.0 citrate buffer with (a) and without (b) the presence of 1 mM AA. (B) Dependence of the SWV peak current on the concentration of UA. Pa ) +1.8 V; ta ) 60 s; Pp ) -0.2 V; tp ) 30 s. SW parameters are as in Figure 2.

Figure 5. Dependence of the SWV peak current on the concentration of DA with (9) and without (O) the presence of 6.05 mM AA. Concentration range: 0-25 µM DA. Preconcentration time was 20 s at open circuit. SW parameters: modulation amplitude, 45 mV; modulation frequency, 55 Hz; step height, 4 mV.

itself can result in a renewed electrode. The results of 15 successive measurements showed a 2.1% coefficient of variation. Meanwhile, to characterize the reproducibility of the Nafion-coated clay-modified electrode, repetitive measurement-regeneration cycles were carried out in 6.05 µM DA and 6.05 mM AA. The 5092 Analytical Chemistry, Vol. 69, No. 24, December 15, 1997

results of 15 successive measurements showed a relative standard deviation of 1.32%. Thus, both electrode renewals give good, reproducible surfaces. Various possible interfering substances, such as purine, glucose, sucrose, oxalate, hydrazine, urea, and AA, were examined for their effect on the determination of 10 µM UA. The results obtained are summarized in Table 5. It is well known that AA coexists with UA in many samples; therefore, its interference was investigated in more detail. As can be seen, most of these species do not interfere with the determination up to at least 100-fold excess. Since the acceptable tolerance of concentration of AA for the determination of UA is as high as 1 mM, the method is applicable to urine samples. Three human urine samples from laboratory personnel were determined with the method presented above. To fit into the linear range, all the samples used for detection were diluted by 100 times. The dilution process can actually help in reducing the matrix effect of the urine samples. The results obtained are listed in Table 6. To ascertain the correctness of the results, the samples were spiked with certain amounts of UA in about the same concentration as found in the samples themselves. The recovery rates of the spiked samples were determined and ranged between 96% and 99% for urine. Note that the uric acid contents found in urine samples are fairly close to those reported elsewhere.14,15 CONCLUSIONS The present study demonstrates the electrochemically preanodized clay-modified electrodes can improve electrochemical monitoring of organic analytes such as UA and DA. The major difficulty from the overlapped oxidation potential of AA could be overcome through the distinct ability of UA and DA to coordinate with the electrochemically preanodized clay-modified electrodes. The electrochemically preanodized Nafion/nontronite-coated GCEs can be applied to the detection of UA in urine samples with

excellent sensitivity and selectivity. The recovery of the spiked UA was observed to be good in urine samples. The electrode possesses good selectivity and can be regenerated easily by cleaning at +2.0 V in 0.1 M, pH 1.0 citrate buffer solution. Furthermore, the detection can be achieved without deoxygenating. On the other hand, the electrochemically preanodized Nafion-coated clay-modified electrodes exhibit strong discrimination for DA over AA, enabling quantitation of DA in the presence of a large excess of AA at the physiological pH of 7.4. The modification procedure is reproducible, and the resulting attachment is stable.

ACKNOWLEDGMENT The authors gratefully acknowledge financial support from the National Science Council of the Republic of China under Grant NSC 86-2113-M-005-021. The authors also express their thanks to Drs. Yao-Jung Chen and Han-Mou Gau for valuable discussion. Received for review April 2, 1997. Accepted August 19, 1997.X AC9703562 X

Abstract published in Advance ACS Abstracts, October 1, 1997.

Analytical Chemistry, Vol. 69, No. 24, December 15, 1997

5093