- Email: [email protected]
Electrochemical polymerization of toluidine blue and its application for the amperometric determination of β-d -glucose
PII:
Electrochimica Acta, Vol. 43, Nos 12±13, pp. 1803±1809, 1998 # 1998 Elsevier Science Ltd. All rights reserved Printed in Great Britain S0013-4686(97)00297-1 0013±4686/98 $19.00 + 0.00
Electrochemical polymerization of toluidine blue and its application for the amperometric determination of b-D-glucose Dong-mei Zhou, Jian-Jun Sun, Hong-yuan Chen* and Hui-qun Fang Department of Chemistry, The State Key Laboratory of Coordination Chemistry, Nanjing University, Nanjing 210093, People's Republic of China (Received 2 April 1997; in revised form 18 June 1997) AbstractÐPoly(toluidine blue) modi®ed electrodes were successfully fabricated by cyclically sweeping the graphite electrode in the potential range ÿ0.3 to 1.3 V (vs SCE) in aqueous buer solutions containing 5.0 10ÿ3 M toluidine blue. The eect of pH on the polymerization process of toluidine blue and the electrochemical characteristics of the resulting polymer-modi®ed electrodes were studied in detail. The experimental results indicated that the polymerization of toluidine blue could be carried out in a wide pH region. Particularly, its polymerization process was somewhat similar to that of aniline in acidic media. These polymer-modi®ed electrodes showed redox activity in phosphate media and they have been further successfully used as mediators for the catalytic oxidation of reduced nicotinamide adenine dinucleotide (NADH). Meanwhile, a glucose enzyme electrode, constructed by immobilizing glucose dehydrogenase during the polymerization of toluidine blue, was developed. A good linear amperometric response to b-Dglucose in the range 5.0 10ÿ5±3.0 10ÿ3 M was obtained. # 1998 Elsevier Science Ltd. All rights reserved. Key words: electrochemical polymerization, toluidine blue, NADH, glucose dehydrogenase, amperometric determination, b-D-glucose.
INTRODUCTION Due to their unique use as coenzymes by over 300 dehydrogenases, b-nicotinamine adenine dinucleotide and its oxidized form (NAD+) have been considered interesting for understanding their redox activity and for further developing amperometric biosensors [1]. The direct oxidation of NADH is rather dicult because it has a large anodic overpotential at bare electrodes [2, 3]. Many eorts have been made to improve the kinetics of NADH oxidation. Chemically modi®ed electrodes, which were prepared by electrochemical pretreatment, mediator adsorption or self-assembly as well as immobilizing them in the polymer matrix, can meet this need [4±6]. Generally, these mediators included catechols, quinones, redox dyes, metal complexes and organic salts, etc. [7±12]. However, electrodes modi®ed only by simple adsorption *Author to whom correspondence should be addressed. E-mail: [email protected]
would easily lose their electrocatalytic activity because of unstable immobilization. Electrochemical polymerization is a good method to prepare the stable chemically modi®ed electrodes. Furthermore, electrodes modi®ed with redox polymer ®lms are very eective as electron-transfer mediators because of their three-dimensional distribution of mediators [13±15]. Some redox polymermodi®ed electrodes such as poly(3,4-dihydroxy benzaldehyde), poly(methylene blue) and poly(methylene green) have exhibited obvious electrocatalytic activity for the oxidation of NADH [16±21]. Dye molecules have been widely used as mediators to study the electrocatalytic oxidation of NADH. In general, the derivatives of dyes by a covalently bound dye molecule to an aromatic ring could decrease their proton-donor ability, resulting in improved catalytic activity [12]. Polymerization of dyes can also form such a cross-linked oligomer, which leads to the enhancement of its electrocatalytic ability [16±18].
1803
1804
D.-M. Zhou et al.
Here, toluidine blue was electrochemically polymerized at a graphite electrode surface by cyclic potential sweep. The eect of pH on the polymerization process was studied. The resulting polymers were electroactive and the catalytic oxidation of NADH was obtained at such polymer modi®ed electrodes. The enzyme electrode, prepared by immobilizing glucose dehydrogenase into the matrix of poly(toluidine blue), was further developed. Moreover, this enzyme electrode has been successfully applied to the amperometric determination of glucose in the presence of NAD+.
EXPERIMENTAL 1. Reagents and materials Toluidine blue (Chemical grade, Shanghai Chemical Co.) was used after crystallization three times in twice-distilled water. NADH, NAD+ as well as glucose dehydrogenase were purchased from Sigma Co. without further puri®cation. The standard buer solutions were obtained from Nanjing Chemical Factory of China. Phosphate buer solution (ca. pH 5.0±8.0) consisted of 5.0 10ÿ2 M K2HPO4 and 5.0 10ÿ2 M KH2PO4 containing 0.1 M KCl. The buer solution (pH 1 4.0) was prepared by using 5.0 10ÿ2 M KH2C6H5O7 adjusted with 0.1 M HCl; The buer (pH 1 9.0) was obtained using 5.0 10ÿ2 M Na2B4O71OH2O adjusted with 0.1 M NaOH. All solutions were prepared using twice-distilled water.
2. Apparatus Cyclic voltammetry experiments were performed in a traditional three-electrode system. Spectroscopic purity graphite electrodes (b = 5.0 mm) were used as working electrodes. The reference electrode and counter electrode were a saturated calomel electrode (SCE) and a platinum wire, respectively. All electrochemical experiments were carried out by a BAS 100B electrochemical analyzer at 25 2 18C. 3. The treatment and modi®cation of electrode Electrodes for the preparation of either polymer modi®ed or enzyme were treated by the following procedure. Firstly, they were polished with 400 mesh sandpaper and 0.05 mm aluminum slurry, sonicated in twice-distilled water for 5 min. Then, they were continuously cyclically swept from ÿ0.5 to +1.5 V at 50 mV sÿ1 in sulfuric acid (1.0 M) until constant background currents were observed. The polymerization of toluidine blue at the graphite electrode was carried out by cyclic potential sweep from ÿ0.3 to 1.3 V at 50 mVsÿ1 in 5.0 10ÿ3 M toluidine blue solution. The ®lm thickness depended on the cyclic sweep numbers. The resulting modi®ed electrodes were rinsed with phosphate buer solution and stored in the same solution for further use. The preparation of enzyme electrode was obtained by cyclic potential sweep in the range ÿ0.3±0.9 V in the phosphate buer (pH 7.0) containing 5.0 10ÿ3 M toluidine blue and 10.0 mgmLÿ1 glucose dehydrogenase for 15 cycles at the
Fig. 1. CVs obtained at the graphite electrode in a pH 4.0 buer containing 5.0 10ÿ3 M toluidine blue. Sweep rate: 50 mVsÿ1, cyclic numbers: 15.
Electrochemical polymerization of toluidine blue sweep rate of 50 mVsÿ1. The resulting electrode was rinsed with buer and stored in refrigerator (48C) for further use. RESULTS AND DISCUSSION 1. The electrochemical polymerization of toluidine blue Chemically modi®ed graphite electrode can be prepared conveniently by simple adsorption of mediator because of its special surface structure. The major advantage of such a modi®ed electrode is their ease-fabrication, but they would lose their electrochemical activity easily. Moreover, some substrates themselves cannot be used as mediators to catalyze special species due to their unsuitable redox potentials. However, polymer-modi®ed elec-
Scheme I. Proposed toluidine blue coupling scheme.
1805
trodes prepared by direct electrochemical polymerization of dye molecules such as methylene blue, thionine at the electrode surface can provide stable wide redox couples [16±18]. These couples usually exhibited a dierent formal potential compared with that of monomers and they were very eective to act as mediators of biomolecules. For such polymer-modi®ed electrodes, not only would the proton-donor ability be decreased compared with the monomers, but also they could be used as a matrix for the immobilization of enzymes at the electrode surface simultaneously. Toluidine blue is a redox dye and it can be adsorbed at the graphite electrode as a mediator for catalyzing the reduction of haemoglobin [22]. However, it was found that this modi®ed electrode was less active for the catalytic oxidation of NADH, which suggested the redox potential is not
1806
D.-M. Zhou et al.
suitable for acting as an electron-transfer mediator for NADH. The polymerization process of toluidine blue by cyclic potential sweep in a pH 4.0 buer solution containing 5.0 10ÿ3 M toluidine blue was shown in Fig. 1. If the potential sweep was con®ned to the region of TOB reduction, only the couple A was observed, corresponding to structures I and II (or I' and II') in Scheme I. However, when the anodic sweep was extended to 1.3 V, a shoulder (C) appeared in the cyclic voltammograms (CVs). It might be due to the formation of radical cations of toluidine blue (structures III and III' in Scheme I). That no corresponding reverse peak was observed suggested that the radical cation has undergone the following chemical reaction. Meanwhile, there was a wide-couple, which was marked as couple B (structures IV and V in Scheme I) in Fig. 1, appeared. Its formal potential was more positive than couple A in the monomer reaction. This couple (B) is ascribed to the redox of the polymer (structures IV and V in Scheme I). It can be also seen that the peak currents of couple B increased continuously with the cyclic numbers. Such a phenomenon could also be observed in the polymerization of some other similar dyes [23]. The dimer of toluidine blue corresponding to structures IV and V would further form radical cations and produce oligomers of toluidine blue. As shown in Scheme I, both the polymerization process and the corresponding redox couples of toluidine blue involved H+ participation [24]. It has been reported by Baudreay et al. [23] that the primary step of thionine ®lm deposition is a one-electron oxidation of the monomer, which formed a radical cation or dication, depending on the acidity. Similar to thionine, the polymerization rate and reaction pathways of toluidine blue might be dierent in the dierent pH media, which resulted in a dierent structure of the polymer formed at the electrode surface. In higher pH media, the polymerization process was found to be slower than that undergone in the lower one. some other changes about the shoulder were also observed. In neutral or base media, the shoulder's currents decreased rapidly with the continuous sweep and gradually disappeared after the sweep number up to 15, while the wide-couple (B) currents kept constant in the following cycles. Therefore, the three-dimensional mediator distribution was only limited in almost one monolayer or several monolayers thickness in such a polymer-modi®ed electrode [20]. However, the shoulder's currents decreased slowly in acidic media, as shown in Fig. 1. When toluidine blue was electropolymerized on a fresh electrode at a constant potential beyond +1.0 V, the electrochemical behavior of the resulting ®lm was similar to that obtained by cyclic sweep.
According to the above results, it can be concluded that the polymerization rate of toluidine blue would decrease with the increase of pH. The higher acidity of electrolytes would ensure the formation of some larger oligomer at the electrode surface than that in low ones during the polymerization process. It is well known that the polymerization rate of aniline is quick and a thick ®lm could be obtained only in acid media. In pH>4.0 aqueous solution, its polymerization was gradually dicult because such a resulting polymer possessed bad conductivity. So, it was suggested that the polymerization of toluidine blue was somewhat similar to that of aniline.
2. The characteristics of polymer and its electrocatalysis for NADH Figure 2 depicts that the CVs of polymer-modi®ed electrodes, which were prepared in dierent pH media, in the same buer solution (pH 6.8). There were two couples that appeared. The negative couples (couple II in Fig. 2) were alike for all of these modi®ed electrodes due to the monomer of TOB strongly adsorbed to the polymer, or a monomer-type conjugation in the polymer [25, 26]. However, for the positive couple (couple I in Fig. 2), the shapes were rather dierent and the dierences
Fig. 2. CVs at PTB-modi®ed electrodes in pH 6.8 buer. The electrodes were prepared at (a) pH 4.0, (b) pH 6.8 and (c) pH 9.0. Scan rate: 10 mVsÿ1.
Electrochemical polymerization of toluidine blue
1807
Fig. 3. CVs at PTB-modi®ed electrode (prepared in pH 4.0 media) in a buer solution (pH 6.8) at dierent sweep rates: (a) 20, (b) 40, (c) 60, (d) 80 and (e) 100 mVsÿ1. Right: Plot of the dependence of peak currents on the scan rates.
of its peak potential were smaller for the electrode prepared in acidic media than that in neutral or basic media. It suggested that the lower the pH of the polymerization media, the faster the surface reaction process of the resulting polymer.
Figure 3 showed the CVs of poly(toluidine blue)(PTB)-modi®ed electrode, which was prepared in pH 4.0 media, in pH 6.8 buer solution at dierent scan rates. The dependence of peak currents of couple I on scan rates was linear, which indicated a
Fig. 4. Cyclic voltammograms at PTB-modi®ed electrode (prepared in pH 4.0 media) in (a) pH 6.8 phosphate buer, (b) (a) +2.0 mM NADH, sweep rate: 20 mVsÿ1. Right: The calibration curve of NADH at the modi®ed electrode.
1808
D.-M. Zhou et al.
Fig. 5. Dependence of the DEp and catalytic peak currents on the pH value of the polymerization electrolytes.
surface reaction process occurred at this redox polymer-modi®ed electrode. The CVs of polymer-modi®ed electrode obtained in the absence and presence of NADH were shown in Fig. 4. It was obvious that this polymer-modi®ed electrode was eective for the catalytic oxidation of NADH. The oxidation overpotential of NADH was decreased for 400 mV compared with that obtained at the pretreated bare carbon electrode [2]. The increase in anodic current might be due to the fact that NADH in solution diused to the electrode surface and reacted with the oxidized state of the polymer and then the reduced state polymer was formed, which led to a current increase in the anodic region. From Scheme I, the electrode process includes 4eÿ, 4H+. So, the catalytic process can be expressed as: PTBred 4PTBox 4H 4eÿ PTBox 2NADH 2H 4PTBred 2NAD Since the dependence of the catalytic peak currents on the square root of scan rate was linear in the range as low as 10 0 80 mV sÿ1, the reaction was not a surface-controlled process. Also, the continuous cyclic voltammograms did not change in NADH solution for a long time, no fouling at this electrode could be observed. The electrocatalytic activity of the polymers obtained in various pH polymerization media to the NADH oxidation was also studied. It was found that the polymers prepared in acidic media were more ecient than those obtained in higher pH values (Fig. 5). Figure 4(b) showed the calibration curve of NADH at this polymer-modi®ed electrode. The dependence of the response currents on the concentration of NADH was linear in the range of 4.0 10ÿ5±6.0 10ÿ3 M. 3. The amperometric determination of glucose at the PTB(poly-toluidine blue)/GDH(glucose dehydrogenase) modi®ed electrode From the above experiments, it was shown that the polymer obtained in acidic media was more ecient for the catalytic oxidation of NADH than
Fig. 6. Amperometric response curves of glucose at the GDH/PTB modi®ed electrode in the pH 6.8 buer containing (a) 2.0 10ÿ3 M and (b) 4.0 10ÿ3 M NAD+.
that obtained in neutral or base media. However, it is well known that the enzyme would deactivate in acidic media. So, a pH 6.8 buer for the enzyme immobilization was chosen. The PTB/GDH modi®ed electrode was developed by immobilizing GDH into the matrix by means of electrochemical polymerization in the presence of toluidine blue and glucose dehydrogenase in pH 6.8 buer solution. When glucose was added into the buer solution containing NAD+, an increase in response current was observed at this polymer/enzyme electrode. It revealed that glucose was dehydrogenated by GDH, which transferred two electrons and a proton to NAD+ per converted glucose molecule. The NADH produced was oxidized at the enzyme electrode by the redox polymer that resulted in an increase in oxidation current. Figure 6 shows the calibration curve for glucose which was obtained with such a polymer/enzyme electrode. The response currents were dependent on the concentration of NAD+. According to Michaelis±Menten equation: Iss Imax ÿ K 0m Iss =CGlu ; where Iss represents the steady-state response current of glucose at GDH/PTB modi®ed electrode, K'ss is the Michaelis±Menten constant. The response current was consistent with the Michaelis±Menten mechanism. The results indicated that the apparent Michaelis constants for glucose and the maximum limiting currents were 2.0 10ÿ3 M and 0.150 mA for 2.0 10ÿ3 M NAD+, 3.3 10ÿ3 M and 0.230 mA for 4.0 10ÿ3 M NAD+, respectively. The linear response was obtained in the range of 5.0 10ÿ5±3.0 10ÿ3 M.
Electrochemical polymerization of toluidine blue CONCLUSIONS Toluidine blue was electrochemically polymerized at the graphite electrode in a wide pH range. The polymerization process showed that the radicals formed at a positive potential reacted with the monomer and formed the dimer or oligomer of toluidine blue. In acidic media, the polymerization process was somewhat similar to that of aniline. The resulting electroactive polymer-modi®ed electrodes could be used as mediators for the electrocatalytic oxidation of NADH. Meanwhile, the enzyme electrode by immobilizing glucose dehydrogenase into the polymer matrix has been further developed for the amperometric determination of glucose. ACKNOWLEDGEMENTS This project was supported by the National Natural Science foundation of China. REFERENCES 1. H. L. Schmidt, W. Schuhman, F. W. Scheller and F. Schubert, SensorsÐA comprehension (Edited by J. Gopel, J. Hesse and J. N. Zemel), Vol. 3, VCH, Weinhein (1992). 2. J. Moiroux and P. J. Elving, Anal. Chem 50, 1056 (1978). 3. H. Jaegfeldt, J. Electroanal. Chem. 110, 295 (1980). 4. B. Grundig, G. Wittstock, U. Rudel and B. Strehlitz, J. Electroanal. Chem 395, 143 (1995). 5. A. Silber, C. Brauchle and N. Hampp, J. Electroanal. Chem 390, 83 (1995). 6. H-Y. Chen, D-M. Zhou, J-J. Xu and H-Q. Fang, J. Electroanal. Chem., 422 (1/2) 21 (1997).
1809
7. N. K. Cenas, J. J. Kanapieniene and J. J. Kulys, Biochim. Biophys. Acta 767, 108 (1984). 8. K. Essaadi, B. Keita and L. Nadjo, J. Electroanal. Chem. 367, 275 (1994). 9. F-Q. Xu, H-W. Li, S. J. Cross and T. F. Guarr, J. Elecroanal. Chem. 368, 221 (1994). 10. M. D. Smith and C. L. Olson, Anal. Chem. 46, 1544 (1974). 11. F. Ni, H. Feng, L. Gorton and T. M. Cotton, Langmuir 6, 66 (1990). 12. B. Persson and L. Gorton, J. Electroanal. Chem. 292, 115 (1992). 13. P. R. Moses and R. W. Murray, J. Am. Chem. Soc. 98, 7435 (1976). 14. K. Itaya and A. J. Bard, Anal. Chem. 50, 1487 (1978). 15. D. D. Schlereth, E. Kaz and H. L. Schmidt, Electroanalysis 6, 725 (1994). 16. N. F. Atta, A. Galal, A. E. Karagozler, H. Zimmer, J. F. Rubison and H. B. Mark Jr, J. Chem. Soc. Chem. Commun. 1347, (1990). 17. T. Ohsaka, K. Tanaka and K. Tokuda, J. Chem. Soc. Chem. Commun. 222, (1993). 18. M. Somasundrum and J. V. Bannister, J. Chem. Soc. Chem. Commun. 1629, (1993). 19. A. A. Karyakin, E. E. Karyakina, W. Schuhmann, H. L. Schmidt and S. D. Varfolomeyev, Electroanalysis 6, 821 (1994). 20. D-M. Zhou, H-Q. Fang, H-Y. Chen, H-X. Ju and Y. Wang, Anal. Chim. Acta 329, 41 (1996). 21. W. Schuhmann, Mikrochim. Acta 121, 1 (1995). 22. R-G. Zhu, C-X. He, J-L. Han and H-Y. Chen, Chin. J. Anal. Chem. 24, 158 (1996). 23. J. M. Bauldreay and M. D. Archer, Electrochim. Acta 28(11), 1515 (1983). 24. B. Persson and L. Gorton, J. Electroanal. Chem. 292, 115 (1990). 25. D. D. Schlereth, W. Schumann and H. L. Schmidt, J. Electroanal. Chem. 381, 63 (1995). 26. A. A. Karyakin, A. K. Strakhova, E. E. Karyakina, D. Varfolomeyer and A. K. Y. Yatmirsky, Bioelectrochem. Bioenerg 32, 35 (1993).
Electrochimica Acta, Vol. 43, Nos 12±13, pp. 1803±1809, 1998 # 1998 Elsevier Science Ltd. All rights reserved Printed in Great Britain S0013-4686(97)00297-1 0013±4686/98 $19.00 + 0.00
Electrochemical polymerization of toluidine blue and its application for the amperometric determination of b-D-glucose Dong-mei Zhou, Jian-Jun Sun, Hong-yuan Chen* and Hui-qun Fang Department of Chemistry, The State Key Laboratory of Coordination Chemistry, Nanjing University, Nanjing 210093, People's Republic of China (Received 2 April 1997; in revised form 18 June 1997) AbstractÐPoly(toluidine blue) modi®ed electrodes were successfully fabricated by cyclically sweeping the graphite electrode in the potential range ÿ0.3 to 1.3 V (vs SCE) in aqueous buer solutions containing 5.0 10ÿ3 M toluidine blue. The eect of pH on the polymerization process of toluidine blue and the electrochemical characteristics of the resulting polymer-modi®ed electrodes were studied in detail. The experimental results indicated that the polymerization of toluidine blue could be carried out in a wide pH region. Particularly, its polymerization process was somewhat similar to that of aniline in acidic media. These polymer-modi®ed electrodes showed redox activity in phosphate media and they have been further successfully used as mediators for the catalytic oxidation of reduced nicotinamide adenine dinucleotide (NADH). Meanwhile, a glucose enzyme electrode, constructed by immobilizing glucose dehydrogenase during the polymerization of toluidine blue, was developed. A good linear amperometric response to b-Dglucose in the range 5.0 10ÿ5±3.0 10ÿ3 M was obtained. # 1998 Elsevier Science Ltd. All rights reserved. Key words: electrochemical polymerization, toluidine blue, NADH, glucose dehydrogenase, amperometric determination, b-D-glucose.
INTRODUCTION Due to their unique use as coenzymes by over 300 dehydrogenases, b-nicotinamine adenine dinucleotide and its oxidized form (NAD+) have been considered interesting for understanding their redox activity and for further developing amperometric biosensors [1]. The direct oxidation of NADH is rather dicult because it has a large anodic overpotential at bare electrodes [2, 3]. Many eorts have been made to improve the kinetics of NADH oxidation. Chemically modi®ed electrodes, which were prepared by electrochemical pretreatment, mediator adsorption or self-assembly as well as immobilizing them in the polymer matrix, can meet this need [4±6]. Generally, these mediators included catechols, quinones, redox dyes, metal complexes and organic salts, etc. [7±12]. However, electrodes modi®ed only by simple adsorption *Author to whom correspondence should be addressed. E-mail: [email protected]
would easily lose their electrocatalytic activity because of unstable immobilization. Electrochemical polymerization is a good method to prepare the stable chemically modi®ed electrodes. Furthermore, electrodes modi®ed with redox polymer ®lms are very eective as electron-transfer mediators because of their three-dimensional distribution of mediators [13±15]. Some redox polymermodi®ed electrodes such as poly(3,4-dihydroxy benzaldehyde), poly(methylene blue) and poly(methylene green) have exhibited obvious electrocatalytic activity for the oxidation of NADH [16±21]. Dye molecules have been widely used as mediators to study the electrocatalytic oxidation of NADH. In general, the derivatives of dyes by a covalently bound dye molecule to an aromatic ring could decrease their proton-donor ability, resulting in improved catalytic activity [12]. Polymerization of dyes can also form such a cross-linked oligomer, which leads to the enhancement of its electrocatalytic ability [16±18].
1803
1804
D.-M. Zhou et al.
Here, toluidine blue was electrochemically polymerized at a graphite electrode surface by cyclic potential sweep. The eect of pH on the polymerization process was studied. The resulting polymers were electroactive and the catalytic oxidation of NADH was obtained at such polymer modi®ed electrodes. The enzyme electrode, prepared by immobilizing glucose dehydrogenase into the matrix of poly(toluidine blue), was further developed. Moreover, this enzyme electrode has been successfully applied to the amperometric determination of glucose in the presence of NAD+.
EXPERIMENTAL 1. Reagents and materials Toluidine blue (Chemical grade, Shanghai Chemical Co.) was used after crystallization three times in twice-distilled water. NADH, NAD+ as well as glucose dehydrogenase were purchased from Sigma Co. without further puri®cation. The standard buer solutions were obtained from Nanjing Chemical Factory of China. Phosphate buer solution (ca. pH 5.0±8.0) consisted of 5.0 10ÿ2 M K2HPO4 and 5.0 10ÿ2 M KH2PO4 containing 0.1 M KCl. The buer solution (pH 1 4.0) was prepared by using 5.0 10ÿ2 M KH2C6H5O7 adjusted with 0.1 M HCl; The buer (pH 1 9.0) was obtained using 5.0 10ÿ2 M Na2B4O71OH2O adjusted with 0.1 M NaOH. All solutions were prepared using twice-distilled water.
2. Apparatus Cyclic voltammetry experiments were performed in a traditional three-electrode system. Spectroscopic purity graphite electrodes (b = 5.0 mm) were used as working electrodes. The reference electrode and counter electrode were a saturated calomel electrode (SCE) and a platinum wire, respectively. All electrochemical experiments were carried out by a BAS 100B electrochemical analyzer at 25 2 18C. 3. The treatment and modi®cation of electrode Electrodes for the preparation of either polymer modi®ed or enzyme were treated by the following procedure. Firstly, they were polished with 400 mesh sandpaper and 0.05 mm aluminum slurry, sonicated in twice-distilled water for 5 min. Then, they were continuously cyclically swept from ÿ0.5 to +1.5 V at 50 mV sÿ1 in sulfuric acid (1.0 M) until constant background currents were observed. The polymerization of toluidine blue at the graphite electrode was carried out by cyclic potential sweep from ÿ0.3 to 1.3 V at 50 mVsÿ1 in 5.0 10ÿ3 M toluidine blue solution. The ®lm thickness depended on the cyclic sweep numbers. The resulting modi®ed electrodes were rinsed with phosphate buer solution and stored in the same solution for further use. The preparation of enzyme electrode was obtained by cyclic potential sweep in the range ÿ0.3±0.9 V in the phosphate buer (pH 7.0) containing 5.0 10ÿ3 M toluidine blue and 10.0 mgmLÿ1 glucose dehydrogenase for 15 cycles at the
Fig. 1. CVs obtained at the graphite electrode in a pH 4.0 buer containing 5.0 10ÿ3 M toluidine blue. Sweep rate: 50 mVsÿ1, cyclic numbers: 15.
Electrochemical polymerization of toluidine blue sweep rate of 50 mVsÿ1. The resulting electrode was rinsed with buer and stored in refrigerator (48C) for further use. RESULTS AND DISCUSSION 1. The electrochemical polymerization of toluidine blue Chemically modi®ed graphite electrode can be prepared conveniently by simple adsorption of mediator because of its special surface structure. The major advantage of such a modi®ed electrode is their ease-fabrication, but they would lose their electrochemical activity easily. Moreover, some substrates themselves cannot be used as mediators to catalyze special species due to their unsuitable redox potentials. However, polymer-modi®ed elec-
Scheme I. Proposed toluidine blue coupling scheme.
1805
trodes prepared by direct electrochemical polymerization of dye molecules such as methylene blue, thionine at the electrode surface can provide stable wide redox couples [16±18]. These couples usually exhibited a dierent formal potential compared with that of monomers and they were very eective to act as mediators of biomolecules. For such polymer-modi®ed electrodes, not only would the proton-donor ability be decreased compared with the monomers, but also they could be used as a matrix for the immobilization of enzymes at the electrode surface simultaneously. Toluidine blue is a redox dye and it can be adsorbed at the graphite electrode as a mediator for catalyzing the reduction of haemoglobin [22]. However, it was found that this modi®ed electrode was less active for the catalytic oxidation of NADH, which suggested the redox potential is not
1806
D.-M. Zhou et al.
suitable for acting as an electron-transfer mediator for NADH. The polymerization process of toluidine blue by cyclic potential sweep in a pH 4.0 buer solution containing 5.0 10ÿ3 M toluidine blue was shown in Fig. 1. If the potential sweep was con®ned to the region of TOB reduction, only the couple A was observed, corresponding to structures I and II (or I' and II') in Scheme I. However, when the anodic sweep was extended to 1.3 V, a shoulder (C) appeared in the cyclic voltammograms (CVs). It might be due to the formation of radical cations of toluidine blue (structures III and III' in Scheme I). That no corresponding reverse peak was observed suggested that the radical cation has undergone the following chemical reaction. Meanwhile, there was a wide-couple, which was marked as couple B (structures IV and V in Scheme I) in Fig. 1, appeared. Its formal potential was more positive than couple A in the monomer reaction. This couple (B) is ascribed to the redox of the polymer (structures IV and V in Scheme I). It can be also seen that the peak currents of couple B increased continuously with the cyclic numbers. Such a phenomenon could also be observed in the polymerization of some other similar dyes [23]. The dimer of toluidine blue corresponding to structures IV and V would further form radical cations and produce oligomers of toluidine blue. As shown in Scheme I, both the polymerization process and the corresponding redox couples of toluidine blue involved H+ participation [24]. It has been reported by Baudreay et al. [23] that the primary step of thionine ®lm deposition is a one-electron oxidation of the monomer, which formed a radical cation or dication, depending on the acidity. Similar to thionine, the polymerization rate and reaction pathways of toluidine blue might be dierent in the dierent pH media, which resulted in a dierent structure of the polymer formed at the electrode surface. In higher pH media, the polymerization process was found to be slower than that undergone in the lower one. some other changes about the shoulder were also observed. In neutral or base media, the shoulder's currents decreased rapidly with the continuous sweep and gradually disappeared after the sweep number up to 15, while the wide-couple (B) currents kept constant in the following cycles. Therefore, the three-dimensional mediator distribution was only limited in almost one monolayer or several monolayers thickness in such a polymer-modi®ed electrode [20]. However, the shoulder's currents decreased slowly in acidic media, as shown in Fig. 1. When toluidine blue was electropolymerized on a fresh electrode at a constant potential beyond +1.0 V, the electrochemical behavior of the resulting ®lm was similar to that obtained by cyclic sweep.
According to the above results, it can be concluded that the polymerization rate of toluidine blue would decrease with the increase of pH. The higher acidity of electrolytes would ensure the formation of some larger oligomer at the electrode surface than that in low ones during the polymerization process. It is well known that the polymerization rate of aniline is quick and a thick ®lm could be obtained only in acid media. In pH>4.0 aqueous solution, its polymerization was gradually dicult because such a resulting polymer possessed bad conductivity. So, it was suggested that the polymerization of toluidine blue was somewhat similar to that of aniline.
2. The characteristics of polymer and its electrocatalysis for NADH Figure 2 depicts that the CVs of polymer-modi®ed electrodes, which were prepared in dierent pH media, in the same buer solution (pH 6.8). There were two couples that appeared. The negative couples (couple II in Fig. 2) were alike for all of these modi®ed electrodes due to the monomer of TOB strongly adsorbed to the polymer, or a monomer-type conjugation in the polymer [25, 26]. However, for the positive couple (couple I in Fig. 2), the shapes were rather dierent and the dierences
Fig. 2. CVs at PTB-modi®ed electrodes in pH 6.8 buer. The electrodes were prepared at (a) pH 4.0, (b) pH 6.8 and (c) pH 9.0. Scan rate: 10 mVsÿ1.
Electrochemical polymerization of toluidine blue
1807
Fig. 3. CVs at PTB-modi®ed electrode (prepared in pH 4.0 media) in a buer solution (pH 6.8) at dierent sweep rates: (a) 20, (b) 40, (c) 60, (d) 80 and (e) 100 mVsÿ1. Right: Plot of the dependence of peak currents on the scan rates.
of its peak potential were smaller for the electrode prepared in acidic media than that in neutral or basic media. It suggested that the lower the pH of the polymerization media, the faster the surface reaction process of the resulting polymer.
Figure 3 showed the CVs of poly(toluidine blue)(PTB)-modi®ed electrode, which was prepared in pH 4.0 media, in pH 6.8 buer solution at dierent scan rates. The dependence of peak currents of couple I on scan rates was linear, which indicated a
Fig. 4. Cyclic voltammograms at PTB-modi®ed electrode (prepared in pH 4.0 media) in (a) pH 6.8 phosphate buer, (b) (a) +2.0 mM NADH, sweep rate: 20 mVsÿ1. Right: The calibration curve of NADH at the modi®ed electrode.
1808
D.-M. Zhou et al.
Fig. 5. Dependence of the DEp and catalytic peak currents on the pH value of the polymerization electrolytes.
surface reaction process occurred at this redox polymer-modi®ed electrode. The CVs of polymer-modi®ed electrode obtained in the absence and presence of NADH were shown in Fig. 4. It was obvious that this polymer-modi®ed electrode was eective for the catalytic oxidation of NADH. The oxidation overpotential of NADH was decreased for 400 mV compared with that obtained at the pretreated bare carbon electrode [2]. The increase in anodic current might be due to the fact that NADH in solution diused to the electrode surface and reacted with the oxidized state of the polymer and then the reduced state polymer was formed, which led to a current increase in the anodic region. From Scheme I, the electrode process includes 4eÿ, 4H+. So, the catalytic process can be expressed as: PTBred 4PTBox 4H 4eÿ PTBox 2NADH 2H 4PTBred 2NAD Since the dependence of the catalytic peak currents on the square root of scan rate was linear in the range as low as 10 0 80 mV sÿ1, the reaction was not a surface-controlled process. Also, the continuous cyclic voltammograms did not change in NADH solution for a long time, no fouling at this electrode could be observed. The electrocatalytic activity of the polymers obtained in various pH polymerization media to the NADH oxidation was also studied. It was found that the polymers prepared in acidic media were more ecient than those obtained in higher pH values (Fig. 5). Figure 4(b) showed the calibration curve of NADH at this polymer-modi®ed electrode. The dependence of the response currents on the concentration of NADH was linear in the range of 4.0 10ÿ5±6.0 10ÿ3 M. 3. The amperometric determination of glucose at the PTB(poly-toluidine blue)/GDH(glucose dehydrogenase) modi®ed electrode From the above experiments, it was shown that the polymer obtained in acidic media was more ecient for the catalytic oxidation of NADH than
Fig. 6. Amperometric response curves of glucose at the GDH/PTB modi®ed electrode in the pH 6.8 buer containing (a) 2.0 10ÿ3 M and (b) 4.0 10ÿ3 M NAD+.
that obtained in neutral or base media. However, it is well known that the enzyme would deactivate in acidic media. So, a pH 6.8 buer for the enzyme immobilization was chosen. The PTB/GDH modi®ed electrode was developed by immobilizing GDH into the matrix by means of electrochemical polymerization in the presence of toluidine blue and glucose dehydrogenase in pH 6.8 buer solution. When glucose was added into the buer solution containing NAD+, an increase in response current was observed at this polymer/enzyme electrode. It revealed that glucose was dehydrogenated by GDH, which transferred two electrons and a proton to NAD+ per converted glucose molecule. The NADH produced was oxidized at the enzyme electrode by the redox polymer that resulted in an increase in oxidation current. Figure 6 shows the calibration curve for glucose which was obtained with such a polymer/enzyme electrode. The response currents were dependent on the concentration of NAD+. According to Michaelis±Menten equation: Iss Imax ÿ K 0m Iss =CGlu ; where Iss represents the steady-state response current of glucose at GDH/PTB modi®ed electrode, K'ss is the Michaelis±Menten constant. The response current was consistent with the Michaelis±Menten mechanism. The results indicated that the apparent Michaelis constants for glucose and the maximum limiting currents were 2.0 10ÿ3 M and 0.150 mA for 2.0 10ÿ3 M NAD+, 3.3 10ÿ3 M and 0.230 mA for 4.0 10ÿ3 M NAD+, respectively. The linear response was obtained in the range of 5.0 10ÿ5±3.0 10ÿ3 M.
Electrochemical polymerization of toluidine blue CONCLUSIONS Toluidine blue was electrochemically polymerized at the graphite electrode in a wide pH range. The polymerization process showed that the radicals formed at a positive potential reacted with the monomer and formed the dimer or oligomer of toluidine blue. In acidic media, the polymerization process was somewhat similar to that of aniline. The resulting electroactive polymer-modi®ed electrodes could be used as mediators for the electrocatalytic oxidation of NADH. Meanwhile, the enzyme electrode by immobilizing glucose dehydrogenase into the polymer matrix has been further developed for the amperometric determination of glucose. ACKNOWLEDGEMENTS This project was supported by the National Natural Science foundation of China. REFERENCES 1. H. L. Schmidt, W. Schuhman, F. W. Scheller and F. Schubert, SensorsÐA comprehension (Edited by J. Gopel, J. Hesse and J. N. Zemel), Vol. 3, VCH, Weinhein (1992). 2. J. Moiroux and P. J. Elving, Anal. Chem 50, 1056 (1978). 3. H. Jaegfeldt, J. Electroanal. Chem. 110, 295 (1980). 4. B. Grundig, G. Wittstock, U. Rudel and B. Strehlitz, J. Electroanal. Chem 395, 143 (1995). 5. A. Silber, C. Brauchle and N. Hampp, J. Electroanal. Chem 390, 83 (1995). 6. H-Y. Chen, D-M. Zhou, J-J. Xu and H-Q. Fang, J. Electroanal. Chem., 422 (1/2) 21 (1997).
1809
7. N. K. Cenas, J. J. Kanapieniene and J. J. Kulys, Biochim. Biophys. Acta 767, 108 (1984). 8. K. Essaadi, B. Keita and L. Nadjo, J. Electroanal. Chem. 367, 275 (1994). 9. F-Q. Xu, H-W. Li, S. J. Cross and T. F. Guarr, J. Elecroanal. Chem. 368, 221 (1994). 10. M. D. Smith and C. L. Olson, Anal. Chem. 46, 1544 (1974). 11. F. Ni, H. Feng, L. Gorton and T. M. Cotton, Langmuir 6, 66 (1990). 12. B. Persson and L. Gorton, J. Electroanal. Chem. 292, 115 (1992). 13. P. R. Moses and R. W. Murray, J. Am. Chem. Soc. 98, 7435 (1976). 14. K. Itaya and A. J. Bard, Anal. Chem. 50, 1487 (1978). 15. D. D. Schlereth, E. Kaz and H. L. Schmidt, Electroanalysis 6, 725 (1994). 16. N. F. Atta, A. Galal, A. E. Karagozler, H. Zimmer, J. F. Rubison and H. B. Mark Jr, J. Chem. Soc. Chem. Commun. 1347, (1990). 17. T. Ohsaka, K. Tanaka and K. Tokuda, J. Chem. Soc. Chem. Commun. 222, (1993). 18. M. Somasundrum and J. V. Bannister, J. Chem. Soc. Chem. Commun. 1629, (1993). 19. A. A. Karyakin, E. E. Karyakina, W. Schuhmann, H. L. Schmidt and S. D. Varfolomeyev, Electroanalysis 6, 821 (1994). 20. D-M. Zhou, H-Q. Fang, H-Y. Chen, H-X. Ju and Y. Wang, Anal. Chim. Acta 329, 41 (1996). 21. W. Schuhmann, Mikrochim. Acta 121, 1 (1995). 22. R-G. Zhu, C-X. He, J-L. Han and H-Y. Chen, Chin. J. Anal. Chem. 24, 158 (1996). 23. J. M. Bauldreay and M. D. Archer, Electrochim. Acta 28(11), 1515 (1983). 24. B. Persson and L. Gorton, J. Electroanal. Chem. 292, 115 (1990). 25. D. D. Schlereth, W. Schumann and H. L. Schmidt, J. Electroanal. Chem. 381, 63 (1995). 26. A. A. Karyakin, A. K. Strakhova, E. E. Karyakina, D. Varfolomeyer and A. K. Y. Yatmirsky, Bioelectrochem. Bioenerg 32, 35 (1993).