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Simultaneous determination of phenolic compounds by using a dual enzyme electrodes system
Journal of
Electroanalytical Chemistry Journal of Electroanalytical Chemistry 566 (2004) 379–384 www.elsevier.com/locate/jelechem
Simultaneous determination of phenolic compounds by using a dual enzyme electrodes system Hideo Notsu, Tetsu Tatsuma
*
Institute of Industrial Science, University of Tokyo, Komaba, Meguro-ku, Tokyo 153-8505, Japan Received 19 September 2003; received in revised form 17 November 2003; accepted 18 November 2003
Abstract Simultaneous amperometric determination of two phenolic compounds was performed by using a tyrosinase (TYR) electrode and a TYR-peroxidase (POD) bienzyme electrode on the basis of differences in the selectivities for two phenolic compounds between the two enzyme electrodes. Catechol and p-cresol contained in the same solution were determined simultaneously with the present system. The response of the bienzyme electrode was greater than the sum of the response of the TYR electrode and that of a POD electrode. This amplification effect may be responsible for the differences in the selectivities. Amplification mechanisms were also discussed. Ó 2004 Elsevier B.V. All rights reserved. Keywords: Amperometric sensor; Biosensors; Chemically modified electrode; Catechol; Phenol
1. Introduction There are numerous phenolic compounds in our living environments. Most of them are generated artificially and found in wastewaters of chemical plants, exhaust gases of incinerators, sidestream smoke of cigarettes, etc [1]. Most phenolic compounds are toxic and harmful to our health [1,2]. The sensing of phenolic compounds is therefore important to evaluate the risk of those environmental samples. As sensing techniques for phenolic compounds, there have been spectroscopic [3,4] and electrochemical [5–11] methods. Among the electrochemical methods, those using enzyme electrodes with tyrosinase, laccase [9,12– 14] and peroxidase [15,16] are relatively sensitive. Phenolic compounds are enzymatically oxidized to the corresponding benzoquinones, which are electrochemi-
*
Corresponding author. Present address: Department of Applied Chemistry, Faculty of Engineering, University of Tokyo, Hongo, Bunkyo-ku, Tokyo 113-8656, Japan. Tel.: +81-3-5452-6336; Fax: +813-3812-6227/5452-6338. E-mail address: [email protected] (T. Tatsuma). 0022-0728/$ - see front matter Ó 2004 Elsevier B.V. All rights reserved. doi:10.1016/j.jelechem.2003.11.049
cally reduced in turn to give a quantitative response to the phenolic compounds. Usually, some different phenolic compounds (Fig. 1) coexist in a sample. Phenol, the simplest phenolic compound, as well as catechol and p-cresol are known to be harmful to the skin. Bisphenol-A (2,20 -bis(p-hydroxyphenyl)propane) is not only toxic for the skin but also suspected to be an endocrine disruptor. In addition, some phenolic compounds are reportedly carcinogenic. Since all these phenolic compounds exhibit different toxicities, measuring the total phenol concentration is not sufficient to evaluate the total toxicity of a sample. It may be important to determine concentrations of all the phenolic compounds contained. However, it is difficult to distinguish phenolic compounds with similar structures, because their absorbance wavelengths and redox potentials are close to each other. Therefore, in general, phenolic compounds are subjected to chromatographic separation before detection. However, the separation takes time, and often requires pre-concentration. Also, the equipment is expensive and is not generally portable. In this paper, simultaneous determination of phenolic compounds was examined by using different enzyme electrodes, which
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Fig. 1. Phenolic compounds used in the present work. (a) Phenol, (b) m-cresol, (c) p-cresol, (d) catechol and (e) pyrogallol.
have different selectivities. We expect that the present method will overcome the above-mentioned drawbacks. We used two enzymes, tyrosinase (TYR) and peroxidase (POD) in this study (preliminary results have been reported elsewhere [17,18]). Quite recently, Freire et al. [19] have reported simultaneous determination of phenolic compounds by using a laccase electrode and a TYR electrode. Although we had examined laccase also, the commercially available one is quite expensive and poorly active. On the other hand, TYR and POD are much less expensive and more active. As for the TYR system, TYR oxidizes monophenols to benzoquinones via diphenols, while reducing oxygen to water. On the other hand, POD oxidizes diphenols to benzoquinones, reducing hydrogen peroxide (H2 O2 ) to water. However, POD cannot oxidize monophenols to diphenols normally. If a sample solution contains a monophenol and a diphenol, these could be determined simultaneously by using a TYR electrode sensitive to both monophenols and diphenols and a POD electrode selective to diphenols, in principle. We also examined a bienzyme electrode modified with both TYR and POD (MIX electrode), because it has been reported that the responses of TYR electrodes to phenolic compounds are amplified by further modification with POD [20,21]. Moreover, selectivities to phenolic compounds of MIX electrodes may be different from those of TYR and POD electrodes.
2. Experimental An indium tin oxide (ITO) electrode, used as a substrate electrode, was well washed with acetone and sonicated in 1 M NaOH aqueous solution for 30 min to make the electrode surface hydrophilic. The hydrophilic ITO electrodes were soaked in a toluene solution containing 5% 3-aminopropyltriethoxysilane (APTES) and anhydrous sodium sulfate as a drying agent for 30 min and thus, amino groups were introduced onto them. After the treatment, the electrodes were rinsed with acetone and dried at room temperature.
A 5-ll aliquot of Mili-Q water containing 5 g/l TYR (from mushroom, Sigma) or 20 g/l POD (from horseradish, Sigma) was applied to the APTES modified ITO (5 mm 5 mm; defined by nitflon tape [Nitto Denko Corp.]) to obtain a TYR or POD electrode, respectively. In the case of a MIX electrode, both of the 5-ll aliquot of 5 g/l TYR solution and the 5-ll aliquot of 20 g/l POD solution were applied. Then, a 5-ll aliquot of 2.5% glutaraldehyde aqueous solution was added to the solution to cross-link the amino groups of APTES and enzyme molecules. Amperometric measurements were performed with a potentiostat/galvanostat (ALS, CHI model 900) using hand-made AgjAgCljKClsat and Pt coil as the reference and counter electrodes, respectively. A potential of )0.3 V vs. AgjAgCljKClsat was applied to the enzyme electrode in a continuously stirred 1/15 M phosphate buffer solution (30 ml). After a constant current was obtained, 5 or 10 ll of an acetonitrile solution containing an appropriate concentration of a phenolic compound was injected into the cell. The flow injection analysis (FIA) system used in the present study consisted of a binary pump, an autosampler, a thin-layer flow cell, an amperometric detector and a data acquisition system. The wall-jet-type flow cell consisted of a AgjAgCljKClsat reference electrode and a stainless steel counter electrode. A 0.5 mm thick silicone gasket was used as a spacer in the cell. The geometric area of the electrode in the cell was estimated to be 0.78 cm2 (diameter, 10 mm). The mobile phase was the phosphate-buffered solution described above, and the flow rate was 0.5–2.0 ml/min. The injection interval was 5 min, unless otherwise noted.
3. Results and discussion 3.1. Responses of the TYR electrode The TYR electrode exhibited cathodic current responses to all the phenolic compounds examined, phenol, m-cresol, p-cresol, catechol and pyrogallol, at )0.3 V vs. AgjAgCljKClsat (Fig. 2). In the TYR-catalyzed reactions, the monophenols (phenol, m-cresol and pcresol) are oxidized via the corresponding diphenols, and catechol and pyrogallol are oxidized directly, to the corresponding o-bezoquinones. The observed responses were due to electrochemical reduction of the generated o-benzoquinones. The magnitudes of the responses were as follows: catechol > p-cresol > pyrogallol > phenol > m-cresol. The sensitivity to catechol was highest, as described in previous reports [5,8–11]. Although phenol is oxidized to o-bezoquinone via catechol, the response to phenol was much smaller than that to catechol. Therefore, the first oxidation (phenol to catechol) should be the rate-limiting step in the TYR-catalyzed
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Fig. 2. Amperometric responses of the TYR electrode to (a) phenol, (b) m-cresol, (c) p-cresol, (d) catechol and (e) pyrogallol. Substrates were added (1–5 lM) every 100 s. Applied potential was )0.3 V vs. AgjAgCljKClsat in 1/15 M phosphate buffer solution, pH 7.4.
oxidation of phenol to o-benzoquinone. Pyrogallol (1,2,3-trihydroxybenzene) has three hydroxyl groups, and two neighboring hydroxyl groups might be oxidized to oxenes, to give 3-hydroxy-1,2-benzoquinone [22,23].
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compounds even in the absence of H2 O2 (Fig. 3). The responses were larger than those of the TYR electrode to the same compounds. As reported [5,8–11], the response of the MIX electrode was larger than the sum of the response of the TYR electrode and that of the POD electrode in the absence of H2 O2 . The order of sensitivity to the substrates examined was the same as that of the TYR electrode, except for pyrogallol: catechol > p-cresol > phenol > m-cresol > pyrogallol. However, the selectivity was different for the TYR and MIX electrodes. Table 1 shows the responses (to 5 lM substrates) relative to those to catechol. The amplification factor ( ¼ sensitivity of MIX electrode/sensitivity of TYR electrode) was >1.5 for catechol and >2 for p-cresol. This means that the simultaneous determination of two phenolic compounds contained in a sample solution is possible by using the TYR and MIX electrodes. The responses of the TYR and MIX electrodes showed good linearity with the substrate concentration in the range between 1 and 20 lM. The electrodes retained about 50% of their original sensitivity when they were stored in the refrigerator (4 °C) at 100% humidity
3.2. Responses of the POD electrode The POD electrode exhibited only small responses to the phenolic compounds examined (<10 nA/lM; less than one-fifth of those for the TYR electrode), in the absence of H2 O2 . The small responses were observed probably because a small amount of H2 O2 was generated by the reduction of dissolved oxygen at )0.3 V vs. AgjAgCljKClsat . In the presence of 0.1 mM H2 O2 , larger responses to the phenolic compounds were observed. The response to catechol (300 nA/lM) was larger than the responses to the other phenolic compounds examined (e.g. p-cresol: 30 nA/lM). Although POD catalyzes oxidization of catechol and pyrogallol to the corresponding quinones by H2 O2 , it does not catalyze the oxidation of monophenols to quinones. However, in the presence of POD and H2 O2 , monophenols are oxidized to phenoxy radicals [16], which may be reduced electrochemically in turn to give the cathodic response. In any event, the calibration curves (response–concentration curves) of the POD electrode showed poor linearity. Moreover, generated phenoxy radicals are easily polymerized causing electrode fouling and deactivation. Therefore, we have concluded that the POD electrode is not appropriate for the determination of phenolic compounds. 3.3. Responses of the MIX electrode Next, the MIX electrode was examined. It gave substantial cathodic current responses to the phenolic
Fig. 3. Amperometric responses of the MIX electrode to (a) phenol, (b) m-cresol, (c) p-cresol, (d) catechol and (e) pyrogallol. Substrates were added (1–5 lM) every 100 s. Applied potential was )0.3 V vs. AgjAgCljKClsat in 1/15 M phosphate buffer solution, pH 7.4.
Table 1 Responses of the TYR and MIX electrodes to 5 lM substrates relative to those to catechol Substrate
Catechol Phenol m-Cresol p-Cresol Pyrogallol
Relative response TYR electrode
MIX electrode
1.00 (std.) 0.10 0.07 0.25 0.12
1.00 (std.) 0.25 0.08 0.38 0.06
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or in dry air at room temperature for one month. These results indicate that the determination of the phenolic compounds is possible with both of the enzyme electrodes.
follows [28]. The iso-response curve for the response x is taken from Fig. 4(a) and that for the response y is taken from Fig. 4(b) (bold curves in Figs. 5(a) and (b), respectively). The intersection of the two curves gives the concentrations of catechol and p-cresol (Fig. 5(c)).
3.4. Responses of the enzyme electrodes in a flow system 3.6. The response amplification of the MIX electrode To measure many samples with various compositions, we applied the TYR and MIX electrodes to a FIA system. The electrode potential was )0.3 V vs. AgjAgCljKClsat , at which the current for the direct reduction of oxygen was negligible. The electrodes responded to the phenolic compounds, and the peak height decreased as the flow rate increased. This is because accumulation of the enzymatic reaction products, o-benzoquinone derivatives, is involved in the overall process. The reduction of a quinone gives the response, and enzymatic reoxidation of the catechol generated by the electrochemical reduction of the quinone (redox cycle) gives rise to response amplification [6,8,20,24–27]. Although a slow flow is thus advantageous in terms of the sensitivity, it results in a gradual increase of the response during repeated injections. Considering all these factors, we selected a flow rate of 1.5 ml/min. The differences between the relative responses of the TYR electrode and those of the MIX electrode were clearer in the flow system than in the batch system. Although the reason is unknown, a redox cycle may be involved. 3.5. Simultaneous determination of catechol and p-cresol Figs. 4(a) and (b) show the responses of the TYR and MIX electrodes, respectively, to solutions containing both catechol and p-cresol. By using these two plots, the concentrations of coexisting catechol and p-cresol can be determined if the sample contains no other phenolic compounds. For example, when the response of the TYR electrode was x and that of the MIX electrode was y, concentrations of the substrates can be evaluated as
As described above, the response of the MIX electrode was larger than the sum of the response of the TYR electrode and that of the POD electrode in the absence of H2 O2 . Although this amplification has been reported, the mechanism has not yet been elucidated [20,21,29]. There are some possible explanations, including generation of H2 O2 in the TYR-catalyzed system, which in turn oxidizes the phenolic compounds by POD-catalyzed reactions. Actually, there have been some reports describing H2 O2 generation in TYR-catalyzed oxidation of catechin, L -dopa, 4-methyl catechol (an intermediate generated in the oxidation of p-cresol), gallic acid, 6hydroxy dopamine and so on [30,31], although that in the oxidation of catechol has not yet been reported, to the best of our knowledge. Another possibility is promotion of the TYR-catalyzed reaction by a product or an intermediate of the POD-catalyzed reaction. It has been reported that some intermediates of TYR-catalyzed oxidation of L -tyrosine promotes the oxidation of L -tyrosine itself [32]. A similar process might proceed in the TYR–POD system. The difference in film structure is also one of the possible explanation. The film of the MIX electrode might be advantageous to mass transfer or to redox cycle-based amplification. To elucidate the mechanisms, we added catalase in the solution, after which, the responses of the MIX electrode to substrates became almost constant. However, no significant changes in the current were observed. This may not necessarily exclude the possibility that H2 O2 generation is involved in the response am-
Fig. 4. Responses to solutions containing catechol and/or p-cresol of (a) the TYR electrode and (b) the MIX electrode.
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Fig. 5. Iso-response curves for (a) the TYR electrode and (b) the MIX electrode taken from Fig. 3. The curve for the response ¼ 10 nA in (a) and that for the response ¼ 100 nA in (b) are shown in (c).
plification, because catalase (MW ¼ 240 000) is too large to fit inside the enzyme membrane. Then, to obtain further information, amperometric measurements were performed with two electrodes (the TYR or bare electrode and the POD or bare electrode) faced closely to each other (distance ¼ 160 lm). The potential of )0.3 V vs. AgjAgCljKClsat was applied to only one of the electrodes and the other one was opencircuited. After the background current became almost constant, 5 lM catechol was added to the electrolyte solution. If products from the open-circuited electrode cause amplification at the potential-applied electrode, the current will decrease when the former is replaced with a bare electrode. When the potential was applied to the TYR electrode, the current was not changed by replacing the open-circuited POD electrode with a bare electrode. On the other hand, when the potential was applied to the POD electrode, its cathodic current decreased when the open-circuited TYR electrode (230 nA) was replaced with a bare electrode (100 nA). However, the difference (130 nA) can be explained in terms of reduction of o-benzoquinone diffusing from the TYR electrode, because the potential-applied bare electrode exhibited a cathodic current of 120 nA, when it was coupled with the open-circuited TYR electrode.
Similar results were obtained in the case of p-cresol and 4-methyl catechol. Although the mechanism has not yet been revealed, our results suggest that the distance between TYR and POD should be less than 160 lm to give rise to the amplification.
4. Conclusions Simultaneous determination of two phenolic compounds (catechol and p-cresol) was possible by using the TYR and MIX electrodes. Simultaneous determination of three or more phenolic compounds might also be possible by using other enzymes, e.g. laccase and phenolase. For future development, introduction of the chemometric technique to a multiple enzyme electrode system and/or a multiple transduction system may be effective for the determination of multiple substrates.
Acknowledgements This study was supported in part by a Grant-in-Aid for Scientific Research of Priority Areas (No. 13022211 for
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T.T.) and a Grant-in-Aid for Encouragement of Young Scientists (for H.N.) both from the Ministry of Education, Culture, Sports, Science and Technology of Japan. References [1] S.E. Manahan, Environmental Chemistry, Lewis Publishers, Chelsea, USA, 1991. [2] M.-T. Huang, C.-T. Ho, C.Y.E. Lee, Phenolic Compounds in Food and their Effects on Health II, Antioxidants and Cancer Prevention, American Chemical Society, Washington, DC, 1992. [3] V. Janda, K. Krijt, J. Chromatogr. 283 (1984) 309. [4] S.S. Alimpiev, V.V. Mlynski, M.E. Belov, S.M. Nikiforov, Anal. Chem. 67 (1995) 181. [5] F. Ortega, E. Dominguez, G. Joensson-Pettersson, L. Gorton, J. Biotechnol. 31 (1993) 289. [6] P. Oennerfjord, J. Emneus, G. Marko-Varga, L. Gorton, F. Ortega, E. Dominguez, Biosens. Bioelectron. 10 (1995) 607. [7] A. Lindgren, T. Ruzgas, L. Stoica, F. Munteanu, L. Gorton, ACS Symp. Ser. 762 (2000) 113. [8] S. Cosnier, C. Innocent, Bioelectrochem. Bioenerg. 31 (1993) 147. [9] G. Marko-Varga, J. Emneus, L. Gorton, T. Ruzgas, Trends Anal. Chem. 14 (1995) 319. [10] J. Wang, L. Fang, D. Lopez, Analyst 119 (1994) 455. [11] M.A. Kim, W.-Y. Lee, Anal. Chim. Acta 479 (2003) 143. [12] R.S. Freire, N. Duran, J. Wang, L.T. Kubota, Anal. Lett. 35 (2002) 29. [13] A.I. Yaropolov, A.N. Kharybin, J. Emneus, G. Marko-Varga, L. Gorton, Anal. Chim. Acta 308 (1995) 137. [14] R.S. Freire, S. Thongngamdee, N. Duran, J. Wang, L.T. Kubota, Analyst 127 (2002) 258.
[15] S.S. Rosatto, L.T. Kubota, O.N.G. de, Anal. Chim. Acta 390 (1999) 65. [16] T. Ruzgas, J. Emneus, L. Gorton, G. Marko-Varga, Anal. Chim. Acta 311 (1995) 245. [17] H. Notsu, T. Tatsuma, in: Proceedings of the 35th Chemical Sens. Symposium, p. 109, 2002. [18] H. Notsu, T. Tatsuma, in: Proceedings of the 36th Chemical Sens. Symposium, p. 85, 2003. [19] R.S. Freire, M.M.C. Ferreira, N. Duran, L.T. Kubota, Anal. Chim. Acta 485 (2003) 263. [20] S. Cosnier, I.C. Popescu, Anal. Chim. Acta 319 (1996) 145. [21] S. Uchiyama, Y. Sano, Electroanalysis 12 (2000) 817. [22] C. Zhao, J. Song, J. Zhang, Talanta 59 (2003) 19. [23] J.-M. Zen, H.-H. Chung, A.S. Kumar, Anal. Chem. 74 (2002) 1202. [24] L. Coche-Guerente, V. Desprez, J.P. Diard, P. Labbe, J. Electroanal. Chem. 470 (1999) 53. [25] L. Coche-Guerente, V. Desprez, P. Labbe, S. Therias, J. Electroanal. Chem. 470 (1999) 61. [26] L. Coche-Guerente, P. Labbe, V. Mengeaud, Anal. Chem. 73 (2001) 3206. [27] E. Burestedt, A. Narvaez, T. Ruzgas, L. Gorton, J. Emneus, E. Dominguez, G. Marko-Varga, Anal. Chem. 68 (1996) 1605. [28] T. Tatsuma, D.A. Buttry, Anal. Chem. 69 (1997) 887. [29] S.C. Chang, K. Rawson, C.J. McNeil, Biosens. Bioelectron. 17 (2002) 1015. [30] Y. Jiang, P.W. Miles, Phytochemistry 33 (1993) 29. [31] G. Cohen, R.E. Heikkila, D. MacNamee, J. Biol. Chem. 249 (1974) 2447. [32] T. Tatsuma, K. Komori, H.H. Yeoh, N. Oyama, Anal. Chim. Acta 408 (2000) 233.
Electroanalytical Chemistry Journal of Electroanalytical Chemistry 566 (2004) 379–384 www.elsevier.com/locate/jelechem
Simultaneous determination of phenolic compounds by using a dual enzyme electrodes system Hideo Notsu, Tetsu Tatsuma
*
Institute of Industrial Science, University of Tokyo, Komaba, Meguro-ku, Tokyo 153-8505, Japan Received 19 September 2003; received in revised form 17 November 2003; accepted 18 November 2003
Abstract Simultaneous amperometric determination of two phenolic compounds was performed by using a tyrosinase (TYR) electrode and a TYR-peroxidase (POD) bienzyme electrode on the basis of differences in the selectivities for two phenolic compounds between the two enzyme electrodes. Catechol and p-cresol contained in the same solution were determined simultaneously with the present system. The response of the bienzyme electrode was greater than the sum of the response of the TYR electrode and that of a POD electrode. This amplification effect may be responsible for the differences in the selectivities. Amplification mechanisms were also discussed. Ó 2004 Elsevier B.V. All rights reserved. Keywords: Amperometric sensor; Biosensors; Chemically modified electrode; Catechol; Phenol
1. Introduction There are numerous phenolic compounds in our living environments. Most of them are generated artificially and found in wastewaters of chemical plants, exhaust gases of incinerators, sidestream smoke of cigarettes, etc [1]. Most phenolic compounds are toxic and harmful to our health [1,2]. The sensing of phenolic compounds is therefore important to evaluate the risk of those environmental samples. As sensing techniques for phenolic compounds, there have been spectroscopic [3,4] and electrochemical [5–11] methods. Among the electrochemical methods, those using enzyme electrodes with tyrosinase, laccase [9,12– 14] and peroxidase [15,16] are relatively sensitive. Phenolic compounds are enzymatically oxidized to the corresponding benzoquinones, which are electrochemi-
*
Corresponding author. Present address: Department of Applied Chemistry, Faculty of Engineering, University of Tokyo, Hongo, Bunkyo-ku, Tokyo 113-8656, Japan. Tel.: +81-3-5452-6336; Fax: +813-3812-6227/5452-6338. E-mail address: [email protected] (T. Tatsuma). 0022-0728/$ - see front matter Ó 2004 Elsevier B.V. All rights reserved. doi:10.1016/j.jelechem.2003.11.049
cally reduced in turn to give a quantitative response to the phenolic compounds. Usually, some different phenolic compounds (Fig. 1) coexist in a sample. Phenol, the simplest phenolic compound, as well as catechol and p-cresol are known to be harmful to the skin. Bisphenol-A (2,20 -bis(p-hydroxyphenyl)propane) is not only toxic for the skin but also suspected to be an endocrine disruptor. In addition, some phenolic compounds are reportedly carcinogenic. Since all these phenolic compounds exhibit different toxicities, measuring the total phenol concentration is not sufficient to evaluate the total toxicity of a sample. It may be important to determine concentrations of all the phenolic compounds contained. However, it is difficult to distinguish phenolic compounds with similar structures, because their absorbance wavelengths and redox potentials are close to each other. Therefore, in general, phenolic compounds are subjected to chromatographic separation before detection. However, the separation takes time, and often requires pre-concentration. Also, the equipment is expensive and is not generally portable. In this paper, simultaneous determination of phenolic compounds was examined by using different enzyme electrodes, which
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Fig. 1. Phenolic compounds used in the present work. (a) Phenol, (b) m-cresol, (c) p-cresol, (d) catechol and (e) pyrogallol.
have different selectivities. We expect that the present method will overcome the above-mentioned drawbacks. We used two enzymes, tyrosinase (TYR) and peroxidase (POD) in this study (preliminary results have been reported elsewhere [17,18]). Quite recently, Freire et al. [19] have reported simultaneous determination of phenolic compounds by using a laccase electrode and a TYR electrode. Although we had examined laccase also, the commercially available one is quite expensive and poorly active. On the other hand, TYR and POD are much less expensive and more active. As for the TYR system, TYR oxidizes monophenols to benzoquinones via diphenols, while reducing oxygen to water. On the other hand, POD oxidizes diphenols to benzoquinones, reducing hydrogen peroxide (H2 O2 ) to water. However, POD cannot oxidize monophenols to diphenols normally. If a sample solution contains a monophenol and a diphenol, these could be determined simultaneously by using a TYR electrode sensitive to both monophenols and diphenols and a POD electrode selective to diphenols, in principle. We also examined a bienzyme electrode modified with both TYR and POD (MIX electrode), because it has been reported that the responses of TYR electrodes to phenolic compounds are amplified by further modification with POD [20,21]. Moreover, selectivities to phenolic compounds of MIX electrodes may be different from those of TYR and POD electrodes.
2. Experimental An indium tin oxide (ITO) electrode, used as a substrate electrode, was well washed with acetone and sonicated in 1 M NaOH aqueous solution for 30 min to make the electrode surface hydrophilic. The hydrophilic ITO electrodes were soaked in a toluene solution containing 5% 3-aminopropyltriethoxysilane (APTES) and anhydrous sodium sulfate as a drying agent for 30 min and thus, amino groups were introduced onto them. After the treatment, the electrodes were rinsed with acetone and dried at room temperature.
A 5-ll aliquot of Mili-Q water containing 5 g/l TYR (from mushroom, Sigma) or 20 g/l POD (from horseradish, Sigma) was applied to the APTES modified ITO (5 mm 5 mm; defined by nitflon tape [Nitto Denko Corp.]) to obtain a TYR or POD electrode, respectively. In the case of a MIX electrode, both of the 5-ll aliquot of 5 g/l TYR solution and the 5-ll aliquot of 20 g/l POD solution were applied. Then, a 5-ll aliquot of 2.5% glutaraldehyde aqueous solution was added to the solution to cross-link the amino groups of APTES and enzyme molecules. Amperometric measurements were performed with a potentiostat/galvanostat (ALS, CHI model 900) using hand-made AgjAgCljKClsat and Pt coil as the reference and counter electrodes, respectively. A potential of )0.3 V vs. AgjAgCljKClsat was applied to the enzyme electrode in a continuously stirred 1/15 M phosphate buffer solution (30 ml). After a constant current was obtained, 5 or 10 ll of an acetonitrile solution containing an appropriate concentration of a phenolic compound was injected into the cell. The flow injection analysis (FIA) system used in the present study consisted of a binary pump, an autosampler, a thin-layer flow cell, an amperometric detector and a data acquisition system. The wall-jet-type flow cell consisted of a AgjAgCljKClsat reference electrode and a stainless steel counter electrode. A 0.5 mm thick silicone gasket was used as a spacer in the cell. The geometric area of the electrode in the cell was estimated to be 0.78 cm2 (diameter, 10 mm). The mobile phase was the phosphate-buffered solution described above, and the flow rate was 0.5–2.0 ml/min. The injection interval was 5 min, unless otherwise noted.
3. Results and discussion 3.1. Responses of the TYR electrode The TYR electrode exhibited cathodic current responses to all the phenolic compounds examined, phenol, m-cresol, p-cresol, catechol and pyrogallol, at )0.3 V vs. AgjAgCljKClsat (Fig. 2). In the TYR-catalyzed reactions, the monophenols (phenol, m-cresol and pcresol) are oxidized via the corresponding diphenols, and catechol and pyrogallol are oxidized directly, to the corresponding o-bezoquinones. The observed responses were due to electrochemical reduction of the generated o-benzoquinones. The magnitudes of the responses were as follows: catechol > p-cresol > pyrogallol > phenol > m-cresol. The sensitivity to catechol was highest, as described in previous reports [5,8–11]. Although phenol is oxidized to o-bezoquinone via catechol, the response to phenol was much smaller than that to catechol. Therefore, the first oxidation (phenol to catechol) should be the rate-limiting step in the TYR-catalyzed
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Fig. 2. Amperometric responses of the TYR electrode to (a) phenol, (b) m-cresol, (c) p-cresol, (d) catechol and (e) pyrogallol. Substrates were added (1–5 lM) every 100 s. Applied potential was )0.3 V vs. AgjAgCljKClsat in 1/15 M phosphate buffer solution, pH 7.4.
oxidation of phenol to o-benzoquinone. Pyrogallol (1,2,3-trihydroxybenzene) has three hydroxyl groups, and two neighboring hydroxyl groups might be oxidized to oxenes, to give 3-hydroxy-1,2-benzoquinone [22,23].
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compounds even in the absence of H2 O2 (Fig. 3). The responses were larger than those of the TYR electrode to the same compounds. As reported [5,8–11], the response of the MIX electrode was larger than the sum of the response of the TYR electrode and that of the POD electrode in the absence of H2 O2 . The order of sensitivity to the substrates examined was the same as that of the TYR electrode, except for pyrogallol: catechol > p-cresol > phenol > m-cresol > pyrogallol. However, the selectivity was different for the TYR and MIX electrodes. Table 1 shows the responses (to 5 lM substrates) relative to those to catechol. The amplification factor ( ¼ sensitivity of MIX electrode/sensitivity of TYR electrode) was >1.5 for catechol and >2 for p-cresol. This means that the simultaneous determination of two phenolic compounds contained in a sample solution is possible by using the TYR and MIX electrodes. The responses of the TYR and MIX electrodes showed good linearity with the substrate concentration in the range between 1 and 20 lM. The electrodes retained about 50% of their original sensitivity when they were stored in the refrigerator (4 °C) at 100% humidity
3.2. Responses of the POD electrode The POD electrode exhibited only small responses to the phenolic compounds examined (<10 nA/lM; less than one-fifth of those for the TYR electrode), in the absence of H2 O2 . The small responses were observed probably because a small amount of H2 O2 was generated by the reduction of dissolved oxygen at )0.3 V vs. AgjAgCljKClsat . In the presence of 0.1 mM H2 O2 , larger responses to the phenolic compounds were observed. The response to catechol (300 nA/lM) was larger than the responses to the other phenolic compounds examined (e.g. p-cresol: 30 nA/lM). Although POD catalyzes oxidization of catechol and pyrogallol to the corresponding quinones by H2 O2 , it does not catalyze the oxidation of monophenols to quinones. However, in the presence of POD and H2 O2 , monophenols are oxidized to phenoxy radicals [16], which may be reduced electrochemically in turn to give the cathodic response. In any event, the calibration curves (response–concentration curves) of the POD electrode showed poor linearity. Moreover, generated phenoxy radicals are easily polymerized causing electrode fouling and deactivation. Therefore, we have concluded that the POD electrode is not appropriate for the determination of phenolic compounds. 3.3. Responses of the MIX electrode Next, the MIX electrode was examined. It gave substantial cathodic current responses to the phenolic
Fig. 3. Amperometric responses of the MIX electrode to (a) phenol, (b) m-cresol, (c) p-cresol, (d) catechol and (e) pyrogallol. Substrates were added (1–5 lM) every 100 s. Applied potential was )0.3 V vs. AgjAgCljKClsat in 1/15 M phosphate buffer solution, pH 7.4.
Table 1 Responses of the TYR and MIX electrodes to 5 lM substrates relative to those to catechol Substrate
Catechol Phenol m-Cresol p-Cresol Pyrogallol
Relative response TYR electrode
MIX electrode
1.00 (std.) 0.10 0.07 0.25 0.12
1.00 (std.) 0.25 0.08 0.38 0.06
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or in dry air at room temperature for one month. These results indicate that the determination of the phenolic compounds is possible with both of the enzyme electrodes.
follows [28]. The iso-response curve for the response x is taken from Fig. 4(a) and that for the response y is taken from Fig. 4(b) (bold curves in Figs. 5(a) and (b), respectively). The intersection of the two curves gives the concentrations of catechol and p-cresol (Fig. 5(c)).
3.4. Responses of the enzyme electrodes in a flow system 3.6. The response amplification of the MIX electrode To measure many samples with various compositions, we applied the TYR and MIX electrodes to a FIA system. The electrode potential was )0.3 V vs. AgjAgCljKClsat , at which the current for the direct reduction of oxygen was negligible. The electrodes responded to the phenolic compounds, and the peak height decreased as the flow rate increased. This is because accumulation of the enzymatic reaction products, o-benzoquinone derivatives, is involved in the overall process. The reduction of a quinone gives the response, and enzymatic reoxidation of the catechol generated by the electrochemical reduction of the quinone (redox cycle) gives rise to response amplification [6,8,20,24–27]. Although a slow flow is thus advantageous in terms of the sensitivity, it results in a gradual increase of the response during repeated injections. Considering all these factors, we selected a flow rate of 1.5 ml/min. The differences between the relative responses of the TYR electrode and those of the MIX electrode were clearer in the flow system than in the batch system. Although the reason is unknown, a redox cycle may be involved. 3.5. Simultaneous determination of catechol and p-cresol Figs. 4(a) and (b) show the responses of the TYR and MIX electrodes, respectively, to solutions containing both catechol and p-cresol. By using these two plots, the concentrations of coexisting catechol and p-cresol can be determined if the sample contains no other phenolic compounds. For example, when the response of the TYR electrode was x and that of the MIX electrode was y, concentrations of the substrates can be evaluated as
As described above, the response of the MIX electrode was larger than the sum of the response of the TYR electrode and that of the POD electrode in the absence of H2 O2 . Although this amplification has been reported, the mechanism has not yet been elucidated [20,21,29]. There are some possible explanations, including generation of H2 O2 in the TYR-catalyzed system, which in turn oxidizes the phenolic compounds by POD-catalyzed reactions. Actually, there have been some reports describing H2 O2 generation in TYR-catalyzed oxidation of catechin, L -dopa, 4-methyl catechol (an intermediate generated in the oxidation of p-cresol), gallic acid, 6hydroxy dopamine and so on [30,31], although that in the oxidation of catechol has not yet been reported, to the best of our knowledge. Another possibility is promotion of the TYR-catalyzed reaction by a product or an intermediate of the POD-catalyzed reaction. It has been reported that some intermediates of TYR-catalyzed oxidation of L -tyrosine promotes the oxidation of L -tyrosine itself [32]. A similar process might proceed in the TYR–POD system. The difference in film structure is also one of the possible explanation. The film of the MIX electrode might be advantageous to mass transfer or to redox cycle-based amplification. To elucidate the mechanisms, we added catalase in the solution, after which, the responses of the MIX electrode to substrates became almost constant. However, no significant changes in the current were observed. This may not necessarily exclude the possibility that H2 O2 generation is involved in the response am-
Fig. 4. Responses to solutions containing catechol and/or p-cresol of (a) the TYR electrode and (b) the MIX electrode.
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Fig. 5. Iso-response curves for (a) the TYR electrode and (b) the MIX electrode taken from Fig. 3. The curve for the response ¼ 10 nA in (a) and that for the response ¼ 100 nA in (b) are shown in (c).
plification, because catalase (MW ¼ 240 000) is too large to fit inside the enzyme membrane. Then, to obtain further information, amperometric measurements were performed with two electrodes (the TYR or bare electrode and the POD or bare electrode) faced closely to each other (distance ¼ 160 lm). The potential of )0.3 V vs. AgjAgCljKClsat was applied to only one of the electrodes and the other one was opencircuited. After the background current became almost constant, 5 lM catechol was added to the electrolyte solution. If products from the open-circuited electrode cause amplification at the potential-applied electrode, the current will decrease when the former is replaced with a bare electrode. When the potential was applied to the TYR electrode, the current was not changed by replacing the open-circuited POD electrode with a bare electrode. On the other hand, when the potential was applied to the POD electrode, its cathodic current decreased when the open-circuited TYR electrode (230 nA) was replaced with a bare electrode (100 nA). However, the difference (130 nA) can be explained in terms of reduction of o-benzoquinone diffusing from the TYR electrode, because the potential-applied bare electrode exhibited a cathodic current of 120 nA, when it was coupled with the open-circuited TYR electrode.
Similar results were obtained in the case of p-cresol and 4-methyl catechol. Although the mechanism has not yet been revealed, our results suggest that the distance between TYR and POD should be less than 160 lm to give rise to the amplification.
4. Conclusions Simultaneous determination of two phenolic compounds (catechol and p-cresol) was possible by using the TYR and MIX electrodes. Simultaneous determination of three or more phenolic compounds might also be possible by using other enzymes, e.g. laccase and phenolase. For future development, introduction of the chemometric technique to a multiple enzyme electrode system and/or a multiple transduction system may be effective for the determination of multiple substrates.
Acknowledgements This study was supported in part by a Grant-in-Aid for Scientific Research of Priority Areas (No. 13022211 for
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