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Electrical communication of polyethylene glycol-modified glucose oxidase in carbon paste and its application to the assay of glucose
Sensorsand Actuators B, 13-14 (1993) M-168
166
Electrical communication of polyethylene glycol-‘modifiedglucose oxidase in carbon paste and its application to the assay of glucose Soichi Yabuki, Fumio Mizutani and Tatsuo Katsura National Instituteof Bioscience and Human-Technology l-l Higashi, Tsukubu, Ibaraki 305 (Japan)
AbShCt
An amperometric enzyme electrode was constructed by making use of polyethylene glyc&modified glucose oxidase (PEG-GOD) as a catalyst and carbon paste (CP) as a matrix. The electrode retained an enzyme activity of 20 mU cm-*, which’was higher than the CP containing unmodified GOD electrode. Electrical communication, i.e., electron transfer between the modified GOD and the CP matrix, was observed on the differential pulse voltammograms. The oxidation current of the CP/PEG-GOD electrode was increased by addition of glucose to a solution at the applied potential of 0.10 V versus Ag/AgCYand was proportional to the glucose concentration up to 50 n-M. CP/PEG-GOD was stable for a week, which was not improved by the modification of enzyme.
Introduction Although carbon paste (CP) is a useful material for constructing an amperometric enzyme electrode [l-3], there is some concern that the oil in CP may denature the hydrophilic enzyme incorporated into CP. To solve this problem, the modification of enzymes with polyethylene glycol (PEG) seems to be a suitable approach [4, 51. We have already reported an amperometric glucose-sensing electrode by incorporating PEG-modified glucose oxidase (GOD) and ferrocene, a mediator, into CP matrix [6]. Electrical communication between redox enzymes and electroconductive materials is an interesting pbenomenon. However, tbe construction of an amperometric sensor based on electrical communication is difficult because tbe current caused by the communication is very small [7-121: the protein shell of the enzyme tends to insulate the electro-active center of the enzyme from electroconductive materials. To use an electroconductive material as a matrix would be an excellent way for enhancing the rate of electrical communication. In this paper, CP is employed as an electroconductive material and PEG-modified GOD is incorporated into the matrix. Tbe modified-enzyme CP electrode is examined as a glucose sensing system based on tbe electrical communication.
Reagents GOD (from&q&s sp., EC 1.1.3.4.)was purchased from Toyobo (Grade II, 100 U mg-I). Metboxypoly-
0925-4005/93/$6.00
ethylene glywl-succinimidyl succinate (activated PEG) was obtained from Sigma Chemical and carbon paste (CP) from Bioanalytical Systems (Grade CP-0). All other reagents were of analytical grade. Preparation and characterization of PEG-modified GOD PEG-modified GOD was prepared as follows: GOD (10 mg) and activated PEG (30 mg) were dissolved in a 0.1 M phosphate buffer (2 ml, pH 7.0) solution and stirred for 2 b at 37 “C. Tbe solution was ultrafiltered through PTlX membrane (cut-off molecular weight, 30000, Millipore) to remove unreacted PEG. Lyophiliiation of the solution gave yellow powder. Enzyme activity of the powder was measured by using the peroxidase/o-dianisidine method and protein content of the powder was measured by the Bio-Rad protein assay kit (Bio-Rad Laboratories). Diflerential pulse voltammetry of CPlenzyme electrode CP (20 mg) and PEG-modified GOD (PEG-GOD, 2 mg) were thoroughly mixed in a mortar; then the mixture was used to fill a bole (3.2 mm in diameter, 4 mm in depth) at the end of an electrode body (Model 11-2010, Bioanalytical Systems) to construct a CP electrode containing modiied GOD (CPF’EG-GOD). To prepare a CP/unmodified GOD (CP/GOD) electrode, 0.8 mg unmodified GOD was employed instead of PEGGOD. Differential pulse voltammetry was performed on the enzyme electrode using a polarographic analyzer (Model 384B, Princeton Applied Research); the electrode was
0 1993 - Elsevier Sequoia. All rights reserved
167
immersed in a deoxygenated 0.1 M citrate buffer (pH 5.5), together with a counter electrode (Pt wire, 0.5 mm in diameter, 10 mm length) and a reference electrode (Ag/AgCl). Measurement of current response to glucose The CT/PEG-GOD electrode was immersed in the citrate buffer (pH 5.5) with counter and reference electrodes. The current increase was measured after the addition of glucose to the buffer solution under a constant potential of 0.10 V versus AglAgCl. Furthermore, the CPIPEG-GOD electrode was incorporated into the electrochemical flow detection system (Bioanalytical Systems) and the response to glucose was recorded: the flow-cell electrode (Model 11-1004, Bioanalytical Systems) has two holes (parallel), each hole was 3 mm in diameter, 5 mm in depth; potential applied on the electrode was 0 V versus Ag/AgC1; carrier solution was 0.1 M citrate buffer (pH 5.5); sample volume was 0.1 ml, flow rate was 0.5 ml min-‘.
Results and discussion PEG-modified GOD Characterization of the lyophilized yellow powder was performed. Enzyme activity of the powder was 20 mU mg-I. Protein content of the powder was 20%. The powder was soluble in organic solvents, for example, 1 mg of the powder was instantaneously dissolved in 1 ml of benzene. The result of SDS-polyacrylamide gel electrophoresis showed that the powder contained higher molecular weight protein than the unmodified GOD [6]. Enzyme activities of CP/PEG-GOD electrode and C!P/GOD electrode were determined to be 20 and 12 mU cmm2, respectively. The CT/PEG-GOD electrode had higher enzyme activity than CT/GOD, though the amount of native GOfi was much larger than that of PEG-GOD. This indicates that PEG-GOD retained a higher enzyme activity in CP than unmodified GOD: activity of the native GOD was reduced in CP and PEG is shown to be effective in protecting enzyme from oil. Electrical communication in CPIPEG-GOD electrode Electrical communication, i.e. electron transfer, from redox enzyme to CP was measured by the oxidative direction of differential pulse voltammetry with a CPI PEG-GOD electrode (Fig. 1). An oxidation peak was measured at -0.36 V versus AglAgCl (a reduction peak was also detected at -0.36 V versus AglAgCl in the reductive direction of DPV). In the absence of modified enzyme, there was no current peak around the potential. Oxidation and reduction current peaks
4
Ii-----l
3.2
si k
3
1
0
PI
-06
- 0.4
- 0.2
Pdential I V s.AgAgCl
Fig. 1. Oxidative direction of differential puke vokammograms of a CT/PEG-GOD electrode. Potential sweeping rate was 2 mV 6. Pulse interval was 1 s. Pulse height was 50 mV.
of CP/umnodified GOD in differential pulse voltammograms (DPVs) were located at -0.46 and -0.46 Vversus AglAgCl, respectively. Oxidation and reduction current peak potentials of flavin adenine dinucleotide (FAD) in DPVs were measured at -0.41 and -0.36 V versus AglAgCl, respectively. The redox peak potentials of FAD are similar to those of the CP/PEGGOD electrode. Hence, the oxidation peaks of PEGGOD are considered to be caused by the electrical communication from FAD in GOD to CP. Cunwt responre of CPIPEG-GOD electrode to glucose Oxidation current response of the CT/PEG-GOD electrode to glucose was measured. After the addition of 50 mM glucose, the oxidation current was gradually increased and reached a steady-state value after 3 min. The increased current in the steady state was 17 nA. The increase in the electrode oxidation current was not detected when the same amount of galactose or fructose was added to the solution. This indicates that the increased current was caused by the oxidation of reduced GOD in CP. The stability of thk current response with the electrode was measured: the response current to glucose decreased and became 50% of the initial value after a week. The decrease was caused by the leaching of the enzyme. The response decay of CP/ PEG-GOD was not improved by the modification of enzyme. Figure 2 shows the relationship between the increased current and glucose concentration. The increased current was proportional to the glucose concentration up to 50 mM. The CT/PEG-GOD electrode could thus be used for measuring glucose. Finally, the CT/PEG-GOD electrode was applied to a flow-sensing system. Figure 3 shows the current response with a CP/PEG-GOD electrode to 100 mM glucose. It should be noted that oxidation current increase was not detected when fructose or sucrose was added as a sample.
168
References
0100
50
0
GlucoseI
mM
Fig. 2. Relationship between increased oxidation current with a CF’/PEG-GODelectrode and glucose concentration. Electrode potential was 0 V vs. AglAgCl.
0
2 Time
I
min
Fig. 3. Gxidative current response to glucose with a CP/PEGGOD electrode, wbicb was applied to a flow cell. Sample gtucose was injected twice (at 0 and 2 min).
Conclusions
Modification of GOD with PEG was effective for protecting the enzyme from denaturation by oil contained in CP. The CP/PEG-GOD electrode retained higher enzyme activity than the WGOD electrode. The electrical communication, which could be detected by differential pulse voltammetry, was caused by the oxidation of FAD moieties in GOD. An anodic current increase due to the oxidation of reduced GOD (i.e., electrical communication from GOD to CP matrix) was observed in the presence of glucose. The increased oxidation current was proportional to the glucose concentration up to 50 mM. The CP/PEG-GOD electrode could also be applied to a flow-sensing system.
1 L. Gorton, H. I. Karan, P. D. Hale, T. Inagaki, Y. Okamoto and T. A. Skotbeim, A glucose electrode based on carbon paste chemically modified with ferrocznecontaining siloxane polymer and glucose oxidase, coated with a poly(ester-sulfonic acid) cation exchanger, AnuL Chtn. Acta, 228 (1990) 23-30. 2 J. Wang, J.-H. Wu, 2. Lu, R. Li and J. Sanchez, Mixed ferroc.enGglucose oxidase-carbon-paste electrode for amperometric determination of glucose, And Chim. Ado, 228 (1990) 251-2.57. 3 A. Amine, J.-M. Kauffmanand G. J. Patriarcbe, Amperometric biosensors for glucose based on carbon paste modified electrodes, Talanta, 38 (1991) 107-110. 4 F. M. Veronese, R. Largajolli, E. Boccu, C. A. Benassi and 0. Scbiavon, Surface modification of proteins. Activation of monometboxy-polyethyleneglycols by pbenylcbloroformates and modification of ribonuclease and superoxide dismutase, Appl. Biochem. BiotechnoL, 11 (1985) 141-152 5 Y. Inada, H. Nisbimura, K. Takabasbi, T. Yosbimoto, k R. Saba and Y. Saito, Ester synthesis catalyzed by polyethylene glycol-modified lipase in benzene, Biochem. Biophys. Res. Commun, I22 (1984) 845-850. 6 F. Mixutani, S. Yabuki, A. Okuda and T. Katsura, Glucosesensing electrode based on carbon paste containing fermcene and polyethylene glycol-modified enzyme, Bull. Chem Sot. Jpn.. 64 (1991) 2849-2851. 7 W. J. Albery, P. N. Bartlett and D. H. Craston, Amperometric enzyme electrodes. Part II. Conducting salts as electrode materials for the oxidation of glucose oxidase, 1. Electroanai Chem., I94 (198.5)223-235. 8 Y. Degani and A. Heller, Electrical communication between redox centers of glucose oxidase and electrodes via electrostatically and covalently bound redox polymers, J. Am. Chem. Sot., 111 (1989) 2357-2358. 9 S. Yabuki, H. Sbinobara and M. Aixawa, Elcctro-conductive enzyme membrane, 1. Chem. Sot., Chem Commun., (1989) 945-946.
10 P. D. T. D. Poet, S. Miyamoto,T. Murakami, I. Kbnura and I. Karube, Direct electron transfer with glucose oxidase immobilized in an electropolymerixed poly(h’-methylpyrrole) film on a gold microelectrode, Anal. Chbn. AC&Z,235 (1990) 255-263.
11 C. G. J. Koopal, B. Ruiter and R. J. M. Nolte, Amperometric biosensor based on direct communication between glucose oxidase and a conducting polymer inside the pores of a filtration membrane, 1. Chem. Sot., Chem. Commun., (1991) 1691-1692. 12 B. A. Gregg and A. Heller, Redox polymer films containing enxymes. 2. Glucose oxidase containing enxyme electrodes, J Phys. Chem., 95 (1991) 5976-5980.
166
Electrical communication of polyethylene glycol-‘modifiedglucose oxidase in carbon paste and its application to the assay of glucose Soichi Yabuki, Fumio Mizutani and Tatsuo Katsura National Instituteof Bioscience and Human-Technology l-l Higashi, Tsukubu, Ibaraki 305 (Japan)
AbShCt
An amperometric enzyme electrode was constructed by making use of polyethylene glyc&modified glucose oxidase (PEG-GOD) as a catalyst and carbon paste (CP) as a matrix. The electrode retained an enzyme activity of 20 mU cm-*, which’was higher than the CP containing unmodified GOD electrode. Electrical communication, i.e., electron transfer between the modified GOD and the CP matrix, was observed on the differential pulse voltammograms. The oxidation current of the CP/PEG-GOD electrode was increased by addition of glucose to a solution at the applied potential of 0.10 V versus Ag/AgCYand was proportional to the glucose concentration up to 50 n-M. CP/PEG-GOD was stable for a week, which was not improved by the modification of enzyme.
Introduction Although carbon paste (CP) is a useful material for constructing an amperometric enzyme electrode [l-3], there is some concern that the oil in CP may denature the hydrophilic enzyme incorporated into CP. To solve this problem, the modification of enzymes with polyethylene glycol (PEG) seems to be a suitable approach [4, 51. We have already reported an amperometric glucose-sensing electrode by incorporating PEG-modified glucose oxidase (GOD) and ferrocene, a mediator, into CP matrix [6]. Electrical communication between redox enzymes and electroconductive materials is an interesting pbenomenon. However, tbe construction of an amperometric sensor based on electrical communication is difficult because tbe current caused by the communication is very small [7-121: the protein shell of the enzyme tends to insulate the electro-active center of the enzyme from electroconductive materials. To use an electroconductive material as a matrix would be an excellent way for enhancing the rate of electrical communication. In this paper, CP is employed as an electroconductive material and PEG-modified GOD is incorporated into the matrix. Tbe modified-enzyme CP electrode is examined as a glucose sensing system based on tbe electrical communication.
Reagents GOD (from&q&s sp., EC 1.1.3.4.)was purchased from Toyobo (Grade II, 100 U mg-I). Metboxypoly-
0925-4005/93/$6.00
ethylene glywl-succinimidyl succinate (activated PEG) was obtained from Sigma Chemical and carbon paste (CP) from Bioanalytical Systems (Grade CP-0). All other reagents were of analytical grade. Preparation and characterization of PEG-modified GOD PEG-modified GOD was prepared as follows: GOD (10 mg) and activated PEG (30 mg) were dissolved in a 0.1 M phosphate buffer (2 ml, pH 7.0) solution and stirred for 2 b at 37 “C. Tbe solution was ultrafiltered through PTlX membrane (cut-off molecular weight, 30000, Millipore) to remove unreacted PEG. Lyophiliiation of the solution gave yellow powder. Enzyme activity of the powder was measured by using the peroxidase/o-dianisidine method and protein content of the powder was measured by the Bio-Rad protein assay kit (Bio-Rad Laboratories). Diflerential pulse voltammetry of CPlenzyme electrode CP (20 mg) and PEG-modified GOD (PEG-GOD, 2 mg) were thoroughly mixed in a mortar; then the mixture was used to fill a bole (3.2 mm in diameter, 4 mm in depth) at the end of an electrode body (Model 11-2010, Bioanalytical Systems) to construct a CP electrode containing modiied GOD (CPF’EG-GOD). To prepare a CP/unmodified GOD (CP/GOD) electrode, 0.8 mg unmodified GOD was employed instead of PEGGOD. Differential pulse voltammetry was performed on the enzyme electrode using a polarographic analyzer (Model 384B, Princeton Applied Research); the electrode was
0 1993 - Elsevier Sequoia. All rights reserved
167
immersed in a deoxygenated 0.1 M citrate buffer (pH 5.5), together with a counter electrode (Pt wire, 0.5 mm in diameter, 10 mm length) and a reference electrode (Ag/AgCl). Measurement of current response to glucose The CT/PEG-GOD electrode was immersed in the citrate buffer (pH 5.5) with counter and reference electrodes. The current increase was measured after the addition of glucose to the buffer solution under a constant potential of 0.10 V versus AglAgCl. Furthermore, the CPIPEG-GOD electrode was incorporated into the electrochemical flow detection system (Bioanalytical Systems) and the response to glucose was recorded: the flow-cell electrode (Model 11-1004, Bioanalytical Systems) has two holes (parallel), each hole was 3 mm in diameter, 5 mm in depth; potential applied on the electrode was 0 V versus Ag/AgC1; carrier solution was 0.1 M citrate buffer (pH 5.5); sample volume was 0.1 ml, flow rate was 0.5 ml min-‘.
Results and discussion PEG-modified GOD Characterization of the lyophilized yellow powder was performed. Enzyme activity of the powder was 20 mU mg-I. Protein content of the powder was 20%. The powder was soluble in organic solvents, for example, 1 mg of the powder was instantaneously dissolved in 1 ml of benzene. The result of SDS-polyacrylamide gel electrophoresis showed that the powder contained higher molecular weight protein than the unmodified GOD [6]. Enzyme activities of CP/PEG-GOD electrode and C!P/GOD electrode were determined to be 20 and 12 mU cmm2, respectively. The CT/PEG-GOD electrode had higher enzyme activity than CT/GOD, though the amount of native GOfi was much larger than that of PEG-GOD. This indicates that PEG-GOD retained a higher enzyme activity in CP than unmodified GOD: activity of the native GOD was reduced in CP and PEG is shown to be effective in protecting enzyme from oil. Electrical communication in CPIPEG-GOD electrode Electrical communication, i.e. electron transfer, from redox enzyme to CP was measured by the oxidative direction of differential pulse voltammetry with a CPI PEG-GOD electrode (Fig. 1). An oxidation peak was measured at -0.36 V versus AglAgCl (a reduction peak was also detected at -0.36 V versus AglAgCl in the reductive direction of DPV). In the absence of modified enzyme, there was no current peak around the potential. Oxidation and reduction current peaks
4
Ii-----l
3.2
si k
3
1
0
PI
-06
- 0.4
- 0.2
Pdential I V s.AgAgCl
Fig. 1. Oxidative direction of differential puke vokammograms of a CT/PEG-GOD electrode. Potential sweeping rate was 2 mV 6. Pulse interval was 1 s. Pulse height was 50 mV.
of CP/umnodified GOD in differential pulse voltammograms (DPVs) were located at -0.46 and -0.46 Vversus AglAgCl, respectively. Oxidation and reduction current peak potentials of flavin adenine dinucleotide (FAD) in DPVs were measured at -0.41 and -0.36 V versus AglAgCl, respectively. The redox peak potentials of FAD are similar to those of the CP/PEGGOD electrode. Hence, the oxidation peaks of PEGGOD are considered to be caused by the electrical communication from FAD in GOD to CP. Cunwt responre of CPIPEG-GOD electrode to glucose Oxidation current response of the CT/PEG-GOD electrode to glucose was measured. After the addition of 50 mM glucose, the oxidation current was gradually increased and reached a steady-state value after 3 min. The increased current in the steady state was 17 nA. The increase in the electrode oxidation current was not detected when the same amount of galactose or fructose was added to the solution. This indicates that the increased current was caused by the oxidation of reduced GOD in CP. The stability of thk current response with the electrode was measured: the response current to glucose decreased and became 50% of the initial value after a week. The decrease was caused by the leaching of the enzyme. The response decay of CP/ PEG-GOD was not improved by the modification of enzyme. Figure 2 shows the relationship between the increased current and glucose concentration. The increased current was proportional to the glucose concentration up to 50 mM. The CT/PEG-GOD electrode could thus be used for measuring glucose. Finally, the CT/PEG-GOD electrode was applied to a flow-sensing system. Figure 3 shows the current response with a CP/PEG-GOD electrode to 100 mM glucose. It should be noted that oxidation current increase was not detected when fructose or sucrose was added as a sample.
168
References
0100
50
0
GlucoseI
mM
Fig. 2. Relationship between increased oxidation current with a CF’/PEG-GODelectrode and glucose concentration. Electrode potential was 0 V vs. AglAgCl.
0
2 Time
I
min
Fig. 3. Gxidative current response to glucose with a CP/PEGGOD electrode, wbicb was applied to a flow cell. Sample gtucose was injected twice (at 0 and 2 min).
Conclusions
Modification of GOD with PEG was effective for protecting the enzyme from denaturation by oil contained in CP. The CP/PEG-GOD electrode retained higher enzyme activity than the WGOD electrode. The electrical communication, which could be detected by differential pulse voltammetry, was caused by the oxidation of FAD moieties in GOD. An anodic current increase due to the oxidation of reduced GOD (i.e., electrical communication from GOD to CP matrix) was observed in the presence of glucose. The increased oxidation current was proportional to the glucose concentration up to 50 mM. The CP/PEG-GOD electrode could also be applied to a flow-sensing system.
1 L. Gorton, H. I. Karan, P. D. Hale, T. Inagaki, Y. Okamoto and T. A. Skotbeim, A glucose electrode based on carbon paste chemically modified with ferrocznecontaining siloxane polymer and glucose oxidase, coated with a poly(ester-sulfonic acid) cation exchanger, AnuL Chtn. Acta, 228 (1990) 23-30. 2 J. Wang, J.-H. Wu, 2. Lu, R. Li and J. Sanchez, Mixed ferroc.enGglucose oxidase-carbon-paste electrode for amperometric determination of glucose, And Chim. Ado, 228 (1990) 251-2.57. 3 A. Amine, J.-M. Kauffmanand G. J. Patriarcbe, Amperometric biosensors for glucose based on carbon paste modified electrodes, Talanta, 38 (1991) 107-110. 4 F. M. Veronese, R. Largajolli, E. Boccu, C. A. Benassi and 0. Scbiavon, Surface modification of proteins. Activation of monometboxy-polyethyleneglycols by pbenylcbloroformates and modification of ribonuclease and superoxide dismutase, Appl. Biochem. BiotechnoL, 11 (1985) 141-152 5 Y. Inada, H. Nisbimura, K. Takabasbi, T. Yosbimoto, k R. Saba and Y. Saito, Ester synthesis catalyzed by polyethylene glycol-modified lipase in benzene, Biochem. Biophys. Res. Commun, I22 (1984) 845-850. 6 F. Mixutani, S. Yabuki, A. Okuda and T. Katsura, Glucosesensing electrode based on carbon paste containing fermcene and polyethylene glycol-modified enzyme, Bull. Chem Sot. Jpn.. 64 (1991) 2849-2851. 7 W. J. Albery, P. N. Bartlett and D. H. Craston, Amperometric enzyme electrodes. Part II. Conducting salts as electrode materials for the oxidation of glucose oxidase, 1. Electroanai Chem., I94 (198.5)223-235. 8 Y. Degani and A. Heller, Electrical communication between redox centers of glucose oxidase and electrodes via electrostatically and covalently bound redox polymers, J. Am. Chem. Sot., 111 (1989) 2357-2358. 9 S. Yabuki, H. Sbinobara and M. Aixawa, Elcctro-conductive enzyme membrane, 1. Chem. Sot., Chem Commun., (1989) 945-946.
10 P. D. T. D. Poet, S. Miyamoto,T. Murakami, I. Kbnura and I. Karube, Direct electron transfer with glucose oxidase immobilized in an electropolymerixed poly(h’-methylpyrrole) film on a gold microelectrode, Anal. Chbn. AC&Z,235 (1990) 255-263.
11 C. G. J. Koopal, B. Ruiter and R. J. M. Nolte, Amperometric biosensor based on direct communication between glucose oxidase and a conducting polymer inside the pores of a filtration membrane, 1. Chem. Sot., Chem. Commun., (1991) 1691-1692. 12 B. A. Gregg and A. Heller, Redox polymer films containing enxymes. 2. Glucose oxidase containing enxyme electrodes, J Phys. Chem., 95 (1991) 5976-5980.