Anal. Chem. 2002, 74, 1202-1206

Correspondence

Selective Detection of o-Diphenols on Copper-Plated Screen-Printed Electrodes Jyh-Myng Zen,* Hsieh-Hsun Chung, and Annamalai Senthil Kumar

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

Selective detection of o-diphenols (e.g., catechol, dopamine, and pyrogallol) in the presence of simple phenols, m- and p- derivative diphenols, and ascorbic acid has been demonstrated on copper-plated screen-printed electrodes (CuSPEs) in pH 7.4 phosphate buffer solution. The CuSPE showed an unusual catalytic response at -0.05 V versus Ag/AgCl selectively to o-diphenolic compounds. The o-diphenols can thus be determined amperometrically through direct electrochemical oxidation in low potentials (∼0 V), where the CuSPE is much less subject to interfering reactions. Such a catalytic phenomenon cannot be observed on conventional Pt and glassy carbon electrodes. The selective mechanism is explained in terms of the formation of cyclic five-member complex intermediate (Cu(II)-o-quinolate). Most important of all, the common drawbacks of electrode fouling through polymerization were completely overcome in this system. Aromatic hydroxyl phenols are common chemicals used in industries as well as in clinical and biochemical applications. Such chemicals often exist as a mixture of polyphenols in the working matrix.1-4 Due to human exposure to aromatic hydrocarbons, various phenolic derivatives were found in clinical blood and urine samples.5,6 Among these, the catecholamines of o-diphenol derivatives are well known for their neurotransmitting action. Qualitative and quantitative assays of selective phenolic groups, especially o-diphenols, are thus of clinical and biochemical importance.7-11 Electrochemical determination of aromatic hydroxyl phenols is often performed with tyrosinase, a binuclear copper-containing monoxygenase enzyme, by detecting either O2 consumption or * Corresponding author: (e-mail) [email protected]. (1) Murga, R.; Ruiz, R.; Beltran, S.; Cabezas, J. L. J. Agric. Food Chem. 2000, 48, 3408-3412. (2) Koganow, M. M.; Dueva, O. V.; Tsorin, B. L. J. Nat. Prod. 1999, 62, 481483. (3) De Armas, R.; Martinez, M.; Vicente, C.; Legaz, M.-E. J. Agric. Food Chem. 1999, 47, 3086-3092. (4) Canizares, P.; Dominguez, J. A.; Rodrigo, M. A.; Villasenor, J.; Rodriguez, J. Ind. Eng. Chem. Res. 1999, 38, 3779-3785. (5) Norberg, J.; Emneus, J.; Johansson, J. A.; Mathiasson, L.; Burestedt, E.; Knustsson, M.; Marko-Vargaw, G. J. Chromatogr., B 1997, 701, 39-46. (6) Siren, H.; Karjalainen, U. J. Chromatogr., A 1999, 853, 527-533. (7) Lobbes, J. M.; Fitznar, H. P.; Kattner, G. Anal. Chem. 1999, 71, 30083012. (8) Morales, S.; Cela, R. J. Chromatogr., A 2000, 896, 95-104. (9) Sainthorant, C.; Morin, P.; Dreux, M.; Baudry, A.; Goetz, N. J. Chromatogr., A 1995, 717, 167-179. (10) Herberer, T.; Stan, H.-J. Anal. Chim. Acta 1997, 341, 21-34. (11) Uchimura, T.; Imasaka, T. Anal. Chem. 2000, 72, 2648-2652.

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quinone reduction12-14 at solid electrodes. However, tyrosinasebased electrodes can be used for the detection of both monophenols and o-diphenols;12,15-17 in other words, they are not specific to o-diphenols alone. Common prevailing problems such as degradation, inhibition, or structural changes of the enzyme and leaching of active components under hydrodynamic stress always cause a decrease in response and restrict the extended use of the enzyme assays. On the other hand, direct detection assays that used Pt, Pt/Au, steel, and carbon solid electrodes with some complexes (e.g., Co(II)-phthalocyanine),4,18-22 suffer from the following drawbacks. Higher detecting potentials open up the detection system for interfering reactions together with an increase in the background current and noise level. Moreover, direct electrochemical oxidation of phenols is coupled with fouling reactions due to the passivation of the surface through polymerization of the products.19,20 Recently, a laser-activated method was adopted by means of surface ablation using a Nd:YAG 532-nm laser to remove the passivated layer.19,20 The time-consuming procedures that had to be done with a complicated and expensive instrument make the technique unsuitable for rapid and routine analytical assays. We report here a simple and disposable copper-plated screenprinted carbon electrode (CuSPE) for the selective determination of o-diphenols (e.g., catechol, dopamine, and pyrogallol) in the presence of other isomers and phenols without any passivation problem. Note that no selective electroanalytical assays without the aid of a separation technique, such as HPLC and capillary electrophoresis (CE), for o-diphenols in the presence of other polyphenols were reported so far. Using the specific redox couple of Cu(II)/Cu(I) at -0.05 V versus Ag/AgCl, the CuSPEs provide a low-potential and selective detection method for o-diphenols. Furthermore, quinones are important to organic chemistry and represent one of the oldest and most basic redox processes.14,23-28 Electrosynthetic applications of selective oxidation of o-diphenols (12) Liu, Z.; Liu, B.; Kong, J.; Deng, J. Anal. Chem. 2000, 72, 4707-4712. (13) Burestedt, E.; Narvaez, A.; Ruzgas, T.; Gorton, L.; Emneus, J.; Dominguez, E.; Marko-Varga, G. Anal. Chem. 1996, 68, 1605-1611. (14) Klinman, J. P. Chem. Rev. 1996, 96, 2541-2561. (15) Ortega, F.; Dominguez, E. Biosen., Bioelectr. 1995, 10, 607-619. (16) Kaisheva, A.; Iliev., I.; Christov, S.; Kazareva, R. Sens. Actuators, B 1997, 44, 571-577. (17) Chioccara, F.; Chiodini, G.; Farina, F.; Orlandi, M.; Rindone, B.; Sebastiano, R. J. Mol. Catal. 1995, 97, 187-194. (18) Iotov, P. I.; Kalcheva, S. V. J. Electroanal. Chem. 1998, 442, 19-26. (19) Fulian, Q.; Compton, R. G. Analyst 2000, 125, 531-534. (20) Fulian, Q.; Compton, R. G. Anal. Chem. 2000, 72, 1830-1834. (21) Yaropolov, A. I.; Kharybin, A. N.; Emnus, J.; Marko-Varga, G.; Gorton, L. Anal. Chim. Acta 1995, 308, 137-144. (22) Mafatle, T.; Nyokong, T. Anal. Chim. Acta 1997, 354, 307-314. 10.1021/ac011012l CCC: $22.00

© 2002 American Chemical Society Published on Web 02/02/2002

Figure 1. CV responses of various diphenols and diphenolic derivatives on the CuSPE and SPE in pH 7.4 PBS at a scan rate of 5 mV/s. The asterisk (*) corresponds to the usual behavior of diphenols in CV experiments.

might be interesting in organic synthesis. In fact, the electrochemical method is very precise and informative in solving the intriguing mechanistic aspects. The basic reaction path is similar to the solution-phase studies with the Cu-o-diphenol complex mechanism.25,26 We believe that this enzymeless approach starts a new path in analytical, synthetic, and bioorganic fields. EXPERIMENTAL SECTION Chemicals and Reagents. Phenol (Ph), m-diphenol (resorcinol, Re), p-diphenol (hydroquinone, HQ), catechol (CA), dopamine (DA), pyrogallol (Py), and ascorbic acid (AA) were bought from Sigma (St. Louis, MO). All the other compounds used in this work were ACS-certified reagent grade. Distilled, deionized water was used for preparing the standard solutions. A 200 ppm Cu(II) solution in 0.1 M HNO3 was used for the platting experiments. Unless otherwise mentioned, all base electrolyte solutions are pH 7.4 phosphate buffer solution (PBS, I ) 0.1 M). Phenol standards in pH 7.4 PBS was prepared daily. (23) Demmin, T. R.; Swerdloff, M. D.; Rogic, M. M. J. Am. Chem. Soc. 1981, 103, 5795-5804. (24) Capdevielle, P.; Maumy, M. Tetrahedron Lett. 1982, 23, 1577-1576. (25) Rogic, M. M.; Demmin, T. R. J. Am. Chem. Soc. 1978, 100, 5472-5487. (26) Maumy, M.; Capdevielle, P. J. Mol. Catal. 1996, 113, 159-166. (27) Sayre, L. M.; Nadkarni, D. V. J. Am. Chem. Soc. 1994, 116, 3157-3158. (28) Balla, J. B.; Kiss, T.; Jameson, R. F. Inorg. Chem. 1992, 31, 58-62.

Apparatus. Cyclic voltammetric and chronoamperometric experiments were carried out in a CHI 900 electrochemical workstation (CH Instruments, Austin, TX). The three-electrode system consists of the CuSPE working electrode (0.196 cm2), an Ag/AgCl reference electrode, and a platinum auxiliary electrode. A BAS Pt and glassy carbon electrodes (GCE) of geometric surface area of 0.07 cm2 were used for comparative experiments. The flow injection analysis (FIA) system consisted of a ColeParmer microprocessor pump drive, a Rehodyne model 7125 sample injection valve (20-µL loop) with interconnecting Teflon tube, and a BAS thin-layer detecting electrochemical system. A semiautomatic screen printer was used to prepare disposable SPEs. The SPE, CuSPE, and the incorporation into thin-layer cells were similar to our earlier procedure.29 In brief, a Cu layer was electrochemically plated on a bare SPE in 200 mg/L Cu(NO3)2/ 0.1 M HNO3 at -0.7 V versus Ag/AgCl for 300 s.29 Procedure. The CuSPE was first washed thoroughly with deionized water and then dipped into working solution that contains pH 7.4 PBS for subsequence static experiments. For FIA, the CuSPE was equilibrated in pH 7.4 PBS carrier solution at -0.05 V until the current became constant. The quantification of o-diphenols was achieved by measuring the oxidation current from (29) Zen, J.-M.; Chung, H.-H.; Kumar, A. S. Analyst 2000, 125, 1633-1637.

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chronoamperometric signals. All experiments were performed at room temperature (25 °C). RESULTS AND DISCUSSION Cyclic voltammetric (CV) responses of the CuSPE and SPE under quasi-steady-state conditions (v ) 5 mV/s) with different diphenols and diphenolic derivatives are shown in Figure 1. As can be seen, Ph, Re (m-diphenol), and HQ (p-diphenol) all yielded normal oxidation peaks at high potentials (as indicated by the asterisk *) without altering the copper redox processes of A1/A2 and C1/C2 (A, anodic; C, cathodic). This oxidation reaction often ended with polymeric products and caused electrode fouling as reported earlier, and no exception was observed in this study.18 It is interesting to note that an unusual profound oxidation peak at ∼-0.05 V was observed only for o-diphenols (i.e., CA, DA, and Py). Parallel experiments on bare SPE, GCE, and Pt electrodes did not show such a catalytic behavior at -0.05 V toward o-diphenols. This observation clearly indicated that the selective oxidation reaction of o-diphenols is mediated by higher oxidation state of copper. On the basis of our previous CV studies on the CuSPE, the broad anodic shoulder (A2 and A1) at oxidation potential ∼-0.05 V versus Ag/AgCl comes from copper oxide (i.e., CuI2O and CuIIO) formation and the cathodic peaks C1 (CuIIO f CuI2O) and C2 (CuI2O f Cu0) are their corresponding reduction reactions.29 Note that the relatively higher depletion rate in C1 than that in C2 in the presence of o-diphenols seems to emphasize the mediation reaction by the CuIIO f CuI2O process. Meanwhile, since Py possesses a pair of 1,2-dihydroxy phenolic groups the obtained catalytic current for Py (276.30 µA/cm2) is comparatively higher than that for CA (70.94 µA/cm2). This is indeed important evidence in solving the reaction mechanism as will be discussed in detail later. Since monophenols, m-diphenols, and p-diphenols did not influence or alter the specific oxidation of o-diphenols, it is a clear advantage to construct a chemical sensor for selective detection of o-diphenols. The electrochemical oxidation reactions at high potentials always cause an electrode fouling behavior. A continuous CV response of DA (i.e., a derivative of o-diphenols) on the CuSPE under a wide potential window (-0.4 to +0.4 V) gave a diminishing peak current behavior, as shown in Figure 2a. This observation is also true for CA except with a relatively slower decreasing rate in current magnitude than that of DA. Similar behavior was reported earlier for CA and dihydroxybenzoic and caffeic acids in pH 7 buffer at GCE due to polymerization of reactive o-quinone intermediates.30 Simple monophenols and derivatives were also reported to get passivated on the working electrode surface due to polymerization by the phenoxy radical intermediate under such a high oxidation potential.18 Interestingly, experiments with restricted a CV window (i.e., -0.4 to +0.1 V) showed a catalytic oxidation signal at ∼-0.05 V without any degradation reaction on the CuSPE (Figure 2b). The peak currents observed with a number of cycles for the cases mentioned above are plotted together for comparison, as shown in Figure 2c. This result indicates the absence of any passivation effect and thus electrode fouling drawbacks at the low operating potential. Meanwhile, the CuSPE is highly stable in pH 7 PBS even under oxidation conditions. The continuous CV experiment of the CuSPE in blank pH 7 PBS for 60 min at v ) 100 mV/s under the potential window of +0.5 to -0.8 V did not show any variation in either peak

potential or current. One possibility is that the phosphate salt of copper is highly insoluble (Ksp ) 1.39 × 10-37)31 and hence the CuSPE is stable and reproducible in amperometric analytical measurements. This positive evidence for the selective oxidation of o-diphenols on the CuSPE ensures the development of a useful analytical method. A possible catalytic oxidation mechanism on the CuSPE is speculated on as shown in Scheme 1. In the mechanism, three major steps were proposed in the course of the selective oxidation: (i) o-diphenol chelating with Cu(II) to form a weak fivemember complex intermediate (i.e., Cu(II)-o-quinolate complex), (ii) electron-transfer and dehydrogenation reaction, and (iii) formation of o-quinone derivative with reduced Cu(I), which can be further reoxidized to Cu(II) at -0.05 V. Overall, the formation of a substrate-catalyst five-member complex intermediate is considered as an essential factor for the selectivity. Note that such behavior was well established by homogeneous solution-phase catalysis between the copper salts and quinone derivatives and now it is a mere extension to electroanalytical studies.25,26 Electrochemical observation of similar complexation phenomena were also noticed for amino acids at conventional copper electrodes near 0 V except with marked corrosion and dissolution of active material.32-34 The formation of more stable copper-amino acid complexes might be the reason for such irreversible corrosion

(30) Davis, J. D.; Vaughan, D. H.; Cardosi, M. F. Electrochim. Acta 1998, 43, 291-300 and references therein.

(31) Lide, D. R., Ed. CRC Handbook of Chemistry and Physics, 72 ed.; CRC Press: Boca Raton, FL, 1991.

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Figure 2. Continuous CV response (n ) 10) of the CuSPE with 2 mM DA under (a) wider and (b) restricted potential windows in pH 7.4 PBS at a scan rate of 5 mV/s. (c) The plot of corresponding anodic peak currents against number of cycles.

Scheme 1. Reaction Mechanism for the Selective Oxidation of o-Diphenol on the CuSPEa

a (i) Formation of five-member complex intermediate; (ii) dehydrogenation and electron-transfer reactions; (iii) formation of oxidative product. R ) H (CA), CH2CH2NH2 (DA), OH (Py).

and dissolution characteristics. Higher reproducibility of the CuSPE in the present case indicates a relatively weak binding of the copper(II)-o-quinolate complex intermediate and thus a reversible surface behavior. This observation is close to the enzyme catalysis system in terms of Michaelis-Menten-type kinetics, where the inner-sphere subtrate-catalyst intermediate is an important step for the reproducibility and selectivity.14,35 Thus, the low detection potential at the CuSPE is useful for selective and simple oxidation electroanalysis of o-diphenols. Figure 3 shows amperometric response at the Pt electrode, GCE, SPE, and CuSPE with 2 mM phenols and polyphenols at an applied potential of -0.05 V. It is obvious that almost negligible catalytic responses for Ph, HQ, Re, CA, DA, and Py are observed at the conventional Pt electrode, GCE, and SPE. As to the CuSPE, no response with Ph, HQ, and Re and a huge catalytic current for CA, DA, and Py was observed. These observations validate the selective electroanalytical assays of o-diphenols in the presence of other diphenolic derivatives. Furthermore, all biological liquids contain a variety of electrochemically easily oxidizable reactants, e.g., AA, which are oxidized at similar potentials and dramatically affect the biosensor selectivity. The interference due to AA was also completely arrested on the CuSPE. A simple FIA (without any separation technique) is also used to test the interference effect of other diphenols and phenols on (32) Kok, W. Th.; Hanekamp, H. B.; Bos, P.; Frei, R. W. Anal. Chim. Acta 1982, 142, 31-45. (33) Kok, W. Th.; Brinkman, U. A. Th.; Frei, R. W. J. Chromatogr. 1983, 256, 17-26. (34) Luo, P.; Zhang, F.; Baldwin, R. P. Anal. Chem. 1991, 63, 1702-1707. (35) Zen, J.-M.; Kumar, A. S. Acc. Chem. Res. 2001, 34, 772-780.

Figure 3. Typical amperometric hydrodynamic response for the CuSPE (A), SPE (B), GCE (C), and Pt electrode (D) with a spike of 2 mM various phenolic and o-diphenol derivatives in pH 7.4 PBS at an applied potential of -0.05 V (vs Ag/AgCl).

the detection assays of some important o-diphenols such as CA and DA as shown in Figure 4. Under optimized hydrodynamic Analytical Chemistry, Vol. 74, No. 5, March 1, 2002

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Figure 4. Quantification (a) and interferences (with respect to 100 µM analyte) (b) for catechol (A) and dopamine (B) by FIA at the CuSPE in pH 7.4 PBS. Applied potential -0.05 V vs Ag/AgCl and flow rate 0.3 mL/min.

conditions, the FIA signals show linearity up to 200 (r ) 0.9986) and 300 µM (r ) 0.9993) for CA and DA, respectively. The calculated detection limits (S/N ) 3) for CA and DA were 3 and 5 µM, respectively. Interestingly, 20 times higher concentrations of diphenolic interferences such as Ph, Re, and HQ showed only a ∼10% decrease in the FIA signal. In addition, the relative standard deviation was less than 3% for the measuring FIA signals. The observations support the selective detection of o-diphenols on the CuSPE at very low potentials. CONCLUSIONS A CuSPE was successfully applied to the selective detection of o-diphenols in the presence of other diphenol isomers and ascorbic acid in pH 7.4 PBS without the aid of a separation technique at the low oxidation potential of -0.05 V versus Ag/ AgCl. The common electrode surface passivation through polymerization by intermediate phenolic derivatives is eliminated. The 1206

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formation of a five-member complex intermediate (Cu(II)-oquinolate) is considered an essential step for the selective oxidation of o-diphenols. One can make specific sequencing analysis for whole polyphenolic compounds (i.e., aiming at the detection of both o-diphenols and phenols) by using a bipotentiostatic system coupled with separation techniques such as HPLC or CE. Numerous applications can be imagined, and further work is in progress. ACKNOWLEDGMENT The authors gratefully acknowledge financial support from the National Science Council of the Republic of China. Received for review September 19, 2001. Accepted December 5, 2001. AC011012L