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Properties of glucose sensors based on the immobilization of glucose oxidase in N-substituted polypyrrole film
Sensors and Actuators B 66 Ž2000. 77–79 www.elsevier.nlrlocatersensorb
Properties of glucose sensors based on the immobilization of glucose oxidase in N-substituted polypyrrole film Mikito Yasuzawa) , Takashi Nieda, Tomoyuki Hirano, Akira Kunugi Department of Chemical Science and Technology, Faculty of Engineering, The UniÕersity of Tokushima, 2-1 Minamijosanjima, Tokushima 770-8506, Japan Received 30 July 1998; received in revised form 1 March 1999; accepted 31 May 1999
Abstract Amperometoric glucose sensors were prepared by the electropolymerization of 3-Ž1-pyrrolyl.propionic acid ŽPPA. in the presence of the enzyme, following the treatment with water-soluble carbodiimide Ž1-ethyl-3-Ž3-dimethylaminopropyl.carbodiimide, EDC. to provide covalent bonding between glucose oxidase ŽGOD. and polypyrrole derivatives. PolyŽ o-phenylenediamine. ŽPPD. and Nafion films were introduced as the inner film in the same electrodes. The EDC treatment was effective to improve the stability of the glucose response except on the electrode with Nafion inner film. Though neither electrode was influenced by the addition of urea or D-Žy.-fructose, the influence of ascorbic acid, acetaminophen and uric acid was not negligible even on the electrode with inner film. q 2000 Elsevier Science S.A. All rights reserved. Keywords: Glucose sensors; Electropolymerization; Pyrrole derivatives; Cross-linking
1. Introduction The importance of glucose determinations for the diagnosis and effective treatment of diabetes has been well recognized. Therefore, a large number of efforts have been devoted to make an effective sensor for a continuous estimation of glucose concentration in subcutaneous tissue w1–3x. The enzyme-immobilized electrode prepared by the electropolymerization of pyrrole in the presence of the enzyme is interesting in its simple preparation, miniaturization, and the precise localization of enzyme, but has a problem in electrode stability w4,5x. The authors have previously reported that the glucose oxidase ŽGOD.-immobilized glucose sensor prepared by electropolymerization and electrocopolymerization of pyrrole derivatives that have carboxyl and hydroxyl groups, 3-Ž1-pyrrolyl.propionic acid ŽPPA. and 3-Ž1-pyrrolyl. propanol, respectively, affords higher electrode stability and reproducibility compared with that of unsubstituted pyrrole w6,7x. However, when considering the ultimate purpose of this sensor is to provide a continuous estima-
)
Corresponding author. Fax: q81-886-55-7025. E-mail address: [email protected] ŽM. Yasuzawa.
tion of glucose, the lifetimes of the glucose sensors were not long enough. In this study, we investigated the properties of the GOD-immobilized electrode prepared by two steps; first, precise localization of GOD by the electropolymerization of PPA in the presence of GOD, second, firm immobilization of GOD by covalent bonding by the treatment with water-soluble carbodiimide. In addition, Dupont’s Nafion w perfluorinated sulfonated ion-exchange polymer film and electrochemically prepared polyŽ o-phenylenediamine. ŽPPD. film, which are both known to be effective in eliminating the influence of electroactive compounds on the sensor response w3,7–12x, were introduced as innermembrane, and the properties of the obtained electrodes were also investigated.
2. Experimental 2.1. Electrode fabrication GOD was immobilized on bare platinum electrode, Nafion dip-coated platinum electrode and PPD-electrodeposited platinum electrode. Firstly, the electrode was
0925-4005r00r$ - see front matter q 2000 Elsevier Science S.A. All rights reserved. PII: S 0 9 2 5 - 4 0 0 5 Ž 9 9 . 0 0 4 5 3 - 0
78
M. Yasuzawa et al.r Sensors and Actuators B 66 (2000) 77–79
Fig. 1. Calibration curves for GODrPPD-EDC electrode Ž`. and the electrode without the EDC treatment Žv ..
immersed in deaerated 0.1 mol dmy3 phosphate buffer solutions ŽpH 7.4. containing PPA Ž0.2 mol dmy3 ., GOD Ž2 mg cmy3 . and LiClO4 Ž0.1 mol dmy3 . and electrooxidized at a constant potential of 1.2 V Žvs. AgrAgCl. at 48C. Then, the electrode was immersed in the aqueous solution of 1-ethyl-3-Ž3-dimethylaminopropyl.carbodiimide ŽEDC. for 12 h at 258C in order to provide covalent bonding between GOD and polypyrrole derivatives. The electrodes prepared on bare electrode, Nafioncoated and PPD-deposited platinum electrode are referred to herein as ‘‘GODrEDC’’, ‘‘GODrNafion-EDC’’ and ‘‘GODrPPD-EDC’’, respectively.Response measurements Amperometric responses of the prepared electrodes to glucose were examined in a 0.1 mol dmy3 phosphate buffer solution of pH 7.4 by measuring the electrooxidation current at a potential of 0.65 V for GODrEDC and GODrNafion-EDC, and 0.45 V for GODrPPD-EDC.
3. Results and discussion 3.1. Glucose calibration curÕe Fig. 1 shows the calibration curve for the amperometric response of glucose at GODrPPD-EDC electrode and GODrPPD electrode. The results obtained here were performed after 30 days of use. The linear relationship between the glucose concentration and the response current
Fig. 2. Long-term stability of GODrEDC electrode Ž^. and the electrode without the EDC treatment Ž'..
Fig. 3. Long-term stability of GODrPPD-EDC electrode Ž`. and the electrode without the EDC treatment Žv ..
were found around 4.0 mmol dmy3 for both electrode. Similar behavior was observed in GODrEDC and GODrNafion-EDC electrodes. 3.2. Electrode stability The long-term stability of GODrEDC electrode was examined by determining 5.6 mmol dmy3 glucose and storing in phosphate buffer at 48C when not in use ŽFig. 2.. The results for the electrode without the treatment of EDC are included in Fig. 2 for comparison. Although the response current of GODrEDC electrode became lower due to the reduction of enzyme activity with the treatment, the response current for GODrEDC electrode was relatively constant for more than 150 days, after an initial change of signal over the first 10 days, whereas continuous gradual decrease of response current was observed on the electrode without the treatment. Similar result was obtained on the electrode with PPD inner film ŽFig. 3.. The response current for GODrPPD-EDC electrode was approximately constant for more than 70 days, after an initial change of signal over the first few days. The response current of the electrode without EDC treatment was almost eliminated after 50 days of measurement. However, the EDC treatment was not effective to improve the response stability of the electrode with Nafion inner film ŽFig. 4.. The response current for GODrNafion-EDC electrode gradually decreased and no response current was obtained after 40 days.
Fig. 4. Long-term stability of GODrNafion-EDC electrode.
M. Yasuzawa et al.r Sensors and Actuators B 66 (2000) 77–79
79
Table 1 Influence of interferent reagents on glucose response current Interferent Ascorbic acid Uric acid Urea D-Žy.-fructose Acetaminophen
Physiological concentration Žmmol dmy3 .
i Gq Iri Ga GODrEDC b
GODrPPD-EDC c
GODrNafion-EDC b
0.11 0.48 4.30 0.40 0.17
3.4 26.5 1.0 1.0 5.1
1.5 2.4 1.0 1.1 1.3
1.5 2.6 1.0 1.1 1.3
a i G : Response current of glucose Ž5.6 mmol dmy3 .. i GqI : Response current of glucose Ž5.6 mmol dmy3 . in the presence of interferent at physiological maximum. b Amperometric responses were measured at a potential of 0.65 V Žvs. AgrAgCl.. c Amperometric responses were measured at a potential of 0.45 V Žvs. AgrAgCl..
3.3. Interference of electroactiÕe compounds The interferences of electroactive compounds existing in biological fluids to the glucose response were examined in the presence of their physiological maximum levels with glucose concentration at 5.6 mmol dmy3 ŽTable 1.. Level of interference is expressed in Table 1 as i Gq Iri G : the ratio of the response current of glucose to the response current of glucose in the presence of the interferent. Neither electrode was influenced by the addition of urea or D-Žy.-fructose. Although the influence of the rest of the interferents were somewhat reduced by the introduction of Nafion and PPD, the influence was still high.
4. Conclusion This study demonstrates that the treatment with watersoluble carbodiimide ŽEDC. improved the response stability of the enzyme-immobilized electrodes prepared by the electropolymerization of PPA on bare platinum electrode and PPD-deposited platinum electrode, but such improvement could not be obtained on the electrode with Nafion inner film. Although the introduction of PPD and Nafion films as inner membrane were somewhat effective in
eliminating the influence of electroactive compounds, the influence of ascorbic acid, uric acid and acetaminophen still remained. References w1x V. Poitout, D. Moatti-Sirat, G. Reach, Y. Zhang, G.S. Wilson, F. Lemonnier, J.C. Klein, Diabetologia 36 Ž1993. 658. w2x Y. Zhang, G.S. Wilson, Anal. Chim. Acta 281 Ž1993. 513. w3x F. Moussy, D.J. Harrison, D.W. O’Brien, R.V. Rajotte, Anal. Chem. 65 Ž1993. 2072. w4x M. Umana, ˜ J. Waller, Anal. Chem. 58 Ž1986. 2979. w5x N.C. Foulds, C.R. Lowe, J. Chem. Soc., Faraday Trans. I 82 Ž1986. 143. w6x M. Yasuzawa, N. Matsushita, H. Satake, A. Kunugi, Sens. Actuators, B 13–14 Ž1993. 665. w7x M. Yasuzawa, H. Sasaki, A. Kunugi, H. Satake, in: M. Butler, A. Ricco, N. Yamazoe ŽEds.., Proc. Electrochem. Soc. Vol. 93-71993, p. 808. w8x D.S. Bindra, G.S. Wilson, Anal. Chem. 61 Ž1989. 2566. w9x S. Sasso, R.J. Pierce, R. Walla, A.M. Yacynych, Anal. Chem. 62 Ž1990. 2566. w10x Y. Zhang, Y. Hu, G.S. Wilson, D. Moatti-Sirat, V. Poitout, G. Reach, Anal. Chem. 66 Ž1994. 1183. w11x J.P. Lowry, K. McAteer, S.S. El Atrash, A. Duff, R.D. O’Neill, Anal. Chem. 66 Ž1994. 1754. w12x M. Yasuzawa, T. Matsuki, A. Kunugi, T. Nakaya, in: Proc. 3th East Asia Conf. Chem. Sensors, Seoul,1997, p. 355.
Properties of glucose sensors based on the immobilization of glucose oxidase in N-substituted polypyrrole film Mikito Yasuzawa) , Takashi Nieda, Tomoyuki Hirano, Akira Kunugi Department of Chemical Science and Technology, Faculty of Engineering, The UniÕersity of Tokushima, 2-1 Minamijosanjima, Tokushima 770-8506, Japan Received 30 July 1998; received in revised form 1 March 1999; accepted 31 May 1999
Abstract Amperometoric glucose sensors were prepared by the electropolymerization of 3-Ž1-pyrrolyl.propionic acid ŽPPA. in the presence of the enzyme, following the treatment with water-soluble carbodiimide Ž1-ethyl-3-Ž3-dimethylaminopropyl.carbodiimide, EDC. to provide covalent bonding between glucose oxidase ŽGOD. and polypyrrole derivatives. PolyŽ o-phenylenediamine. ŽPPD. and Nafion films were introduced as the inner film in the same electrodes. The EDC treatment was effective to improve the stability of the glucose response except on the electrode with Nafion inner film. Though neither electrode was influenced by the addition of urea or D-Žy.-fructose, the influence of ascorbic acid, acetaminophen and uric acid was not negligible even on the electrode with inner film. q 2000 Elsevier Science S.A. All rights reserved. Keywords: Glucose sensors; Electropolymerization; Pyrrole derivatives; Cross-linking
1. Introduction The importance of glucose determinations for the diagnosis and effective treatment of diabetes has been well recognized. Therefore, a large number of efforts have been devoted to make an effective sensor for a continuous estimation of glucose concentration in subcutaneous tissue w1–3x. The enzyme-immobilized electrode prepared by the electropolymerization of pyrrole in the presence of the enzyme is interesting in its simple preparation, miniaturization, and the precise localization of enzyme, but has a problem in electrode stability w4,5x. The authors have previously reported that the glucose oxidase ŽGOD.-immobilized glucose sensor prepared by electropolymerization and electrocopolymerization of pyrrole derivatives that have carboxyl and hydroxyl groups, 3-Ž1-pyrrolyl.propionic acid ŽPPA. and 3-Ž1-pyrrolyl. propanol, respectively, affords higher electrode stability and reproducibility compared with that of unsubstituted pyrrole w6,7x. However, when considering the ultimate purpose of this sensor is to provide a continuous estima-
)
Corresponding author. Fax: q81-886-55-7025. E-mail address: [email protected] ŽM. Yasuzawa.
tion of glucose, the lifetimes of the glucose sensors were not long enough. In this study, we investigated the properties of the GOD-immobilized electrode prepared by two steps; first, precise localization of GOD by the electropolymerization of PPA in the presence of GOD, second, firm immobilization of GOD by covalent bonding by the treatment with water-soluble carbodiimide. In addition, Dupont’s Nafion w perfluorinated sulfonated ion-exchange polymer film and electrochemically prepared polyŽ o-phenylenediamine. ŽPPD. film, which are both known to be effective in eliminating the influence of electroactive compounds on the sensor response w3,7–12x, were introduced as innermembrane, and the properties of the obtained electrodes were also investigated.
2. Experimental 2.1. Electrode fabrication GOD was immobilized on bare platinum electrode, Nafion dip-coated platinum electrode and PPD-electrodeposited platinum electrode. Firstly, the electrode was
0925-4005r00r$ - see front matter q 2000 Elsevier Science S.A. All rights reserved. PII: S 0 9 2 5 - 4 0 0 5 Ž 9 9 . 0 0 4 5 3 - 0
78
M. Yasuzawa et al.r Sensors and Actuators B 66 (2000) 77–79
Fig. 1. Calibration curves for GODrPPD-EDC electrode Ž`. and the electrode without the EDC treatment Žv ..
immersed in deaerated 0.1 mol dmy3 phosphate buffer solutions ŽpH 7.4. containing PPA Ž0.2 mol dmy3 ., GOD Ž2 mg cmy3 . and LiClO4 Ž0.1 mol dmy3 . and electrooxidized at a constant potential of 1.2 V Žvs. AgrAgCl. at 48C. Then, the electrode was immersed in the aqueous solution of 1-ethyl-3-Ž3-dimethylaminopropyl.carbodiimide ŽEDC. for 12 h at 258C in order to provide covalent bonding between GOD and polypyrrole derivatives. The electrodes prepared on bare electrode, Nafioncoated and PPD-deposited platinum electrode are referred to herein as ‘‘GODrEDC’’, ‘‘GODrNafion-EDC’’ and ‘‘GODrPPD-EDC’’, respectively.Response measurements Amperometric responses of the prepared electrodes to glucose were examined in a 0.1 mol dmy3 phosphate buffer solution of pH 7.4 by measuring the electrooxidation current at a potential of 0.65 V for GODrEDC and GODrNafion-EDC, and 0.45 V for GODrPPD-EDC.
3. Results and discussion 3.1. Glucose calibration curÕe Fig. 1 shows the calibration curve for the amperometric response of glucose at GODrPPD-EDC electrode and GODrPPD electrode. The results obtained here were performed after 30 days of use. The linear relationship between the glucose concentration and the response current
Fig. 2. Long-term stability of GODrEDC electrode Ž^. and the electrode without the EDC treatment Ž'..
Fig. 3. Long-term stability of GODrPPD-EDC electrode Ž`. and the electrode without the EDC treatment Žv ..
were found around 4.0 mmol dmy3 for both electrode. Similar behavior was observed in GODrEDC and GODrNafion-EDC electrodes. 3.2. Electrode stability The long-term stability of GODrEDC electrode was examined by determining 5.6 mmol dmy3 glucose and storing in phosphate buffer at 48C when not in use ŽFig. 2.. The results for the electrode without the treatment of EDC are included in Fig. 2 for comparison. Although the response current of GODrEDC electrode became lower due to the reduction of enzyme activity with the treatment, the response current for GODrEDC electrode was relatively constant for more than 150 days, after an initial change of signal over the first 10 days, whereas continuous gradual decrease of response current was observed on the electrode without the treatment. Similar result was obtained on the electrode with PPD inner film ŽFig. 3.. The response current for GODrPPD-EDC electrode was approximately constant for more than 70 days, after an initial change of signal over the first few days. The response current of the electrode without EDC treatment was almost eliminated after 50 days of measurement. However, the EDC treatment was not effective to improve the response stability of the electrode with Nafion inner film ŽFig. 4.. The response current for GODrNafion-EDC electrode gradually decreased and no response current was obtained after 40 days.
Fig. 4. Long-term stability of GODrNafion-EDC electrode.
M. Yasuzawa et al.r Sensors and Actuators B 66 (2000) 77–79
79
Table 1 Influence of interferent reagents on glucose response current Interferent Ascorbic acid Uric acid Urea D-Žy.-fructose Acetaminophen
Physiological concentration Žmmol dmy3 .
i Gq Iri Ga GODrEDC b
GODrPPD-EDC c
GODrNafion-EDC b
0.11 0.48 4.30 0.40 0.17
3.4 26.5 1.0 1.0 5.1
1.5 2.4 1.0 1.1 1.3
1.5 2.6 1.0 1.1 1.3
a i G : Response current of glucose Ž5.6 mmol dmy3 .. i GqI : Response current of glucose Ž5.6 mmol dmy3 . in the presence of interferent at physiological maximum. b Amperometric responses were measured at a potential of 0.65 V Žvs. AgrAgCl.. c Amperometric responses were measured at a potential of 0.45 V Žvs. AgrAgCl..
3.3. Interference of electroactiÕe compounds The interferences of electroactive compounds existing in biological fluids to the glucose response were examined in the presence of their physiological maximum levels with glucose concentration at 5.6 mmol dmy3 ŽTable 1.. Level of interference is expressed in Table 1 as i Gq Iri G : the ratio of the response current of glucose to the response current of glucose in the presence of the interferent. Neither electrode was influenced by the addition of urea or D-Žy.-fructose. Although the influence of the rest of the interferents were somewhat reduced by the introduction of Nafion and PPD, the influence was still high.
4. Conclusion This study demonstrates that the treatment with watersoluble carbodiimide ŽEDC. improved the response stability of the enzyme-immobilized electrodes prepared by the electropolymerization of PPA on bare platinum electrode and PPD-deposited platinum electrode, but such improvement could not be obtained on the electrode with Nafion inner film. Although the introduction of PPD and Nafion films as inner membrane were somewhat effective in
eliminating the influence of electroactive compounds, the influence of ascorbic acid, uric acid and acetaminophen still remained. References w1x V. Poitout, D. Moatti-Sirat, G. Reach, Y. Zhang, G.S. Wilson, F. Lemonnier, J.C. Klein, Diabetologia 36 Ž1993. 658. w2x Y. Zhang, G.S. Wilson, Anal. Chim. Acta 281 Ž1993. 513. w3x F. Moussy, D.J. Harrison, D.W. O’Brien, R.V. Rajotte, Anal. Chem. 65 Ž1993. 2072. w4x M. Umana, ˜ J. Waller, Anal. Chem. 58 Ž1986. 2979. w5x N.C. Foulds, C.R. Lowe, J. Chem. Soc., Faraday Trans. I 82 Ž1986. 143. w6x M. Yasuzawa, N. Matsushita, H. Satake, A. Kunugi, Sens. Actuators, B 13–14 Ž1993. 665. w7x M. Yasuzawa, H. Sasaki, A. Kunugi, H. Satake, in: M. Butler, A. Ricco, N. Yamazoe ŽEds.., Proc. Electrochem. Soc. Vol. 93-71993, p. 808. w8x D.S. Bindra, G.S. Wilson, Anal. Chem. 61 Ž1989. 2566. w9x S. Sasso, R.J. Pierce, R. Walla, A.M. Yacynych, Anal. Chem. 62 Ž1990. 2566. w10x Y. Zhang, Y. Hu, G.S. Wilson, D. Moatti-Sirat, V. Poitout, G. Reach, Anal. Chem. 66 Ž1994. 1183. w11x J.P. Lowry, K. McAteer, S.S. El Atrash, A. Duff, R.D. O’Neill, Anal. Chem. 66 Ž1994. 1754. w12x M. Yasuzawa, T. Matsuki, A. Kunugi, T. Nakaya, in: Proc. 3th East Asia Conf. Chem. Sensors, Seoul,1997, p. 355.