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Potentiometric studies at the glucose oxidase enzyme electrode
34 (1994)
195
Short communication Potentiometric studies at the glucose oxidase enzyme electrode Sangita D. Kumar a, A.V. Kulkarni ay*, R.G. Dhaneshwar a and SF. D’Souza b ‘Analytical Chemistry DiMon, Bhabha Atomic Research Centre, Trombay, Bombay 400 085 (India) b Food Technology and Enzyme Engineering Division, Bhabha Atomic Research Centre, Trombay, Bombay 400 085 (India) (Received 16 April 1993; in revised form 4 February 1994)
1. Introduction
Various transducers have been used together with biological systems for the fabrication of biosensors 111.A redox potentiometric enzyme electrode for glucose determination using a Pt disc as the base sensor and glucose oxidase immobilized on an extremely porous matrix, namely cheesecloth, is described in this communication. Wingard and coworkers [2,3] observed that a potential signal is developed across a glucose oxidase enzyme electrode and the reference electrode system in the buffered glucose solutions. The enzyme electrode response was Nemstian at glucose concentrations in the range 2.2-22.2 mM [4-71. They prepared the enzyme electrode by direct immobilization of glucose oxidase on the Pt electrode surface using polyacrylamide and glutaraldehyde cross-linking; this produced a lower potential signal due to high diffusional resistance to the transport of glucose through the membrane layer [8]. The earlier cyclic voltammetric studies from this laboratory showed that cheesecloth offers negligible diffusional resistance to the transport of glucose [9]. In addition it has high mechanical strength and flexibility. 2. Experimental 2.1. Materials
Glucose oxidase (Aspergillus niger, G-8135), glutaraldehyde (25%), catalase (bovine liver) and polyethylenimine (50%) were obtained from Sigma Chemicals
l
To whom correspondence
0302-4598/94/$7.00 SSDI 0302-4598(94)01709-A
should be addressed.
(USA). The cheesecloth (3.6 mg cmp2> used to immobilize the glucose oxidase was obtained from the local market. Other reagents used were of BDH AnalaR or E. Merck GR grade. Acids were of BDH Aristar grade purity. 2.2. Apparatus The electrochemical pretreatment of the Pt electrode was carried out on polarographic instruments (models 174A and 175) coupled to an x-y recorder (all manufactured by EG & G Princeton Applied Research Corporation, New Jersey). A digital pH meter-millivoltmeter (type EE 330A, EMCO, India) was used to perform the potentiometric measurements. A Pt disc and a Pt wire served as the working and auxiliary electrodes respectively, and a saturated calomel electrode (SCE) was used as the reference electrode. All experiments were performed at 25 f O.l”C. Oxygen-free nitrogen gas (IOLAR grade, Indian Oxygen Ltd., Bombay) was used to deaerate the solutions. 2.3 Electrochemical pretreatment In order to obtain reproducible surface conditions the Pt electrode was subjected to a rigorous electrochemical pretreatment. After polishing with a finegrained polishing paper (Orion Inc., USA), the Pt electrode was anodized for 30 s at + 1.4 V/SCE in 0.1 M H,SO, and then subjected to potential cycling between + 1.2 V and -0.5 V for 15 min. Finally, it was held at +0.2 V for 5 min. This preconditioning ensured that the Pt electrode had identical surface conditions for every experiment. The pretreatment was carried out at 25 + O.l”C under anaerobic conditions produced by bubbling nitrogen through the solution. 2.4. Method of standardization of hydrogen peroxide The hydrogen peroxide solution was standardized using the method described by Vogel [lo]. 2.5. Preparation of enzyme membrane Cheesecloth squares of dimensions 2 cm x 2 cm were washed and dried prior to soaking for 2 h in 0.2% polyethylenimine (PEI), at pH 7.0 (adjusted using HCl). The cloth squares were then rinsed with distilled water and dried in air. The PEI-coated cloth squares were then incubated in an aqueous solution of glucose oxidase (0.5 mg ml-‘) for 2 h. Glutaraldehyde (final concentration 0.2%) was then added. The cross-linking 0 1994 - Elsevier Science S.A. All rights reserved
196
S.D. Kumar et al. / Potentiometric studies at glucose oxidase enzyme electrode
was allowed to proceed for 1 h at room temperature with gentle shaking. The enzyme-bound cloth squares were washed with 0.05 M phosphate buffer (pH 6.8) and stored in the same buffer [ll-131. 2.4. Glucose oxidase assay The glucose oxidase activity on the cheesecloth was determined by measuring the rate of oxygen consumption using a Gilson Oxygraph model KM [14]. The assay system consisted of a cheesecloth square of known area, 2% glucose and excess catalase in 1.5 ml of 0.1 M phosphate buffer (pH 6.0). The glucose oxidase activity on cheesecloth was found to be 0.075-0.1 U cmm2. The optimum pH of bound glucose oxidase was 6.00. Over 90% of the activity could be detected at pH 6.8. Buffer molarity up to 0.4 M was not found to affect the activity of the enzyme. 2.7. Methods The potentiometric measurements were made in stirred solutions using bare Pt or glucose oxidase/Pt as the working electrode and an SCE as the reference electrode. The potentials were measured by adding aliquots of analyte solution (glucose or H,O,) to 0.05 M phosphate buffer (pH 6.8). After each measurement the electrodes and the cell were washed thoroughly with distilled water, and a fresh aliquot of analyte was added. The results are reported as the difference between the potential measured with a given glucose/H,O, concentration and the potential obtained in the buffer solution alone. When not in use the glucose oxidase/Pt electrode was stored in the buffer solution at 4°C. 3. Results and discussion The potentiometric response of glucose in the phosphate buffer (pH 6.8) at a bare Pt electrode was linear in the range 2.2-22.2 mM with a slope of - 18 mV per decade glucose concentration (Fig. 1, curve 1). Wingard et al. [S] have reported a similar slope of - 17 mV per decade glucose concentration at a bare Pt electrode. The mechanism for the response has been suggested to occur via adsorption and oxidation of glucose with concurrent reduction of a Pt oxide species [15-171. The potentiometric response for glucose in the phosphate buffer (pH 6.8) containing soluble glucose oxidase (2 pg ml-‘) was Nernstian with a slope of - 28 mV per decade glucose concentration (Fig. 1, curve 2) and much higher than that obtained in the absence of glucose oxidase (Fig. 1, curve 1). A Nernstian response was also observed for H,O, concentrations of 2.0-20.0 mM in the phosphate buffer (pH 6.8) in the absence of catalase using the bare Pt-SCE system (slope, -33.5 mV per decade glucose concentration; intercept, - 61 mV).
However, on adding an aliquot of H,O, (4 mM) to the phosphate buffer (pH 6.8) containing catalase (26 U ml-r) the potential signal for the buffer solution (+300 mV> decreased rapidly to + 220 mV in 30 s (mean of four observations), after which it increased to a steady value of +320 mV in about 7 min. The potentiometric response in the presence of catalase (20 mV) was much less than that obtained in the absence of catalase (80 mV>. The potentiometric response at the enzyme electrode was also Nernstian in the range 2.2-22.2 mM glucose concentration (slope, -30.8 mV per decade glucose concentration; intercept, -0.33 mV). This indicates that the electrode reaction occurring at the Pt electrode in the case of soluble glucose oxidase or enzyme electrode or H,O, in the absence of catalase is the same, and involves two electrons. Further, the negative slope in the above three cases suggests that a net reduction reaction is occurring at the indicator electrode. The response was quicker and higher at the enzyme electrode compared with that at the bare Pt electrode in the presence of soluble glucose oxidase (Fig. 2). The glucose oxidation reaction at the enzyme electrode occurs in the diffusion layer, producing H,O, at the electrode surface, while in the latter case H,O, is generated in the bulk of the solution and diffuses to the Pt electrode surface to produce a potential signal. The role of dissolved oxygen in the development of the potential signal at the enzyme electrode was ascertained by deoxygenating the buffer solution by purging
*20-
O-
$20
-
! E v) -4oL -60 -
I
I1111al
I 1
CONCENTRATION
IO
,
OF GLUCOSE/mM
Fig. 1. Potentiometric response of glucose at a bare Pt electrode in 0.05 M phosphate buffer (pH 6.8) at 25°C: (1) in the absence of glucose oxidase, slope of - 18.0 mV per decade (correlation coefficient 0.9691, potential response measured after 1 min; (2) in the presence of 2 pg ml-’ glucose oxidase, slope of -28.0 mV per decade glucose concentration (correlation coefficient 0.987), potential response measured after 2 min.
197
SD. Kumar et al. / Potentiometric studies at glucose oxidase enzyme electrode
O-
1
2
3
TIME /months
Fig. 3. Stability studies of the enzyme electrode in 2 mM glucose in 0.05 M phosphate buffer (pH 6.8) at 25°C. Each point represents a mean of five measurements.
-80 -
L
I
0
4
2
6
TIME/min
a
IO
12
-
Fig. 2. Potentiometric response in 0.05 M phosphate buffer (pH 6.8) at 25°C as a function of time: (1) at the bare Pt electrode with soluble glucose oxidase for 7.4 mM glucose concentration; (2) at the enzyme electrode for 7.4 mM glucose concentration.
it with high purity nitrogen gas. A marked fall in the signal was observed with increasing purging times (Table 1). However, Wingard et al. [12] reported an insignificant fall in the potential when the dissolved oxygen content of the buffer was reduced from 100% to 12% which is not comprehensible. These observations lead to the conclusion that the following O,/H,O, redox couple operates at the electrode surface: 0, + 2Hf+
2e- =
H,O,
(1)
for which the Nemst equation is E=E’+
y
( I [021
log [H2021
where E’ is a constant incorporating the standard potential of reaction (1) and the constant H+ concentration term. The behaviour of the potentiometric response for H,O, in the presence of catalase is explained by taking into account the catalytic decomposition of H,O, by TABLE 1. Effect of deoxygenation on potentiometric enzyme electrode
response at the
Deoxygenation time/min
Buffer potential/mV
Response for 7.4 mM [glucose] after 30 s/mV
0 10 15 20 30
300 260 232 212 178
67 40 23 13 1
Initial oxygen concentration
in air-saturated buffer, 0.24 mM.
catalase, producing oxygen, which results in the potential of the Pt electrode becoming more anodic (eqn. (2)). The stability and reproducibility of the enzyme electrode was studied for several months (Fig. 3). The cheesecloth immobilized with glucose oxidase had a long shelf life. Different membranes prepared under identical conditions produced similar potentiometric signals. Frequent pretreatment of the Pt electrode was not required when it was covered with cheesecloth membrane. 4. Conclusions Cheesecloth is a good membrane for the immobilization of glucose oxidase because of its high porosity, mechanical stability, flexibility and longer life-time as well as having a very low cost. The simplicity of the method and good response slopes towards glucose in the range 2.2-22.2 mM make it highly suitable for the fabrication of a biosensor for glucose. Co-immobilization of glucose oxidase and catalase is not desirable as a low potentiometric response is obtained in the presence of catalase. The O,/H,O, redox couple at the electrode surface is responsible for the development of the potentiometric response at the enzyme electrode. Acknowledgements
The authors thank Dr. S. Gangadharan, Associate Director, Analytical Chemistry Group, and Shri T.S. Krishnamoorthy, Head, Surface and Interstial Measurements Section, for their keen interest in and encouragement of the work. The authors also acknowledge Dr. T.P. Radhakrishnan for helpful discussions. References 1 A.P.F. Turner. I. Karube and G.S. Wilson (Eds.), Biosensors, Fundamentals and Applications, Oxford Science Publications, Oxford, 1987, p. 5.
198
S.D. Kkmar et al. / Potentiometric studies at glucose oxidase enzyme electrode
2 L.B. Wingard, CC. Liu and X.L. Nagda, Biotechnol. Bioeng., 13
3 4
5 6 7 8 9 10
(1971) 629. E.J. Lahoda, C.C. Liu and L.B. Wingard, Biotechnol. Bioeng., 17 (1975) 413. L.B. Wingard Jr., D. Ellis, S.J. Yao, J.G. Schiller, C.C. Liu, SK. Wolfson Jr. and A. L. Drash, J. Solid-Phase Biochem., 4 (1979) 253. L.B. Wingard Jr., C.C. Liu, S.K. Wolfson Jr., S.J. Yao and A.L. Drash, Diabetes Care, 5 (1982) 199. C.C. Liu, L.B. Wingard Jr., S.K. Wolfson Jr., S.J. Yao, A.L. Drash and G.J. Schiller, Bioelectrochem. Bioenerg., 6 (1979) 19. J.F. Castner and L.B. Wingard Jr., Anal. Chem., 56 (1984) 2981. L.B. Wingard Jr., J.F. Castner, S.J. Yao, SK. Wolfson Jr., A.L. Drash and C.C. Liu, Appl. Biochem. Biotechnol., 9 (1984) 95. S.D. Kumar, A.V. Kulkarni, S.F. D’Souza and R.G. Dhaneshwar, Bioelectrochem. Bioenerg., 27 (1992) 153. A.J. Vogel, A Textbook of Quantitative Inorganic Analysis, Longman, London, 1975, p. 29.5.
11 SF. D’Souza and N. Kamath, Appt. Microbial. Biotechnol., 29 (1988) 136. 12 N. Kamath, J.S. Melo and S.F. D’Souza, Appl. Biochem. Biotechnot., 19 (1988) 251. 13 K. Sankaran, S.S. Godbole and S.F. D’Souza, Enzyme Microbial. Technol., 11 (1989) 617. 14 SF. D’Souza and G.B. Nadkarni, Biotechnol. Bioeng., 22 (1980) 2179. 15 K. Heyns and H. Paulson in W. Forest (Ed.), Newer Methods of Preparative Organic Chemistry, Vol. 2, Academic Press, New York, 1963, p. 303. 16 M.L.B. Rao and R.F. Drake, J. Electrochem. Sot., 116 (1969) 334. 17 K. Popovic, A. Tripkovic, N. Markovic and R.R. Adzic, J. Electroanal. Chem., 295 (1990) 79. 18 L.B. Wingard Jr., J.G. Schiller, SK. Wolfson Jr., C.C. Liu, A.L. Drash and S.J. Yao, J. Biomed. Mater. Res., 13 (1979) 921.
195
Short communication Potentiometric studies at the glucose oxidase enzyme electrode Sangita D. Kumar a, A.V. Kulkarni ay*, R.G. Dhaneshwar a and SF. D’Souza b ‘Analytical Chemistry DiMon, Bhabha Atomic Research Centre, Trombay, Bombay 400 085 (India) b Food Technology and Enzyme Engineering Division, Bhabha Atomic Research Centre, Trombay, Bombay 400 085 (India) (Received 16 April 1993; in revised form 4 February 1994)
1. Introduction
Various transducers have been used together with biological systems for the fabrication of biosensors 111.A redox potentiometric enzyme electrode for glucose determination using a Pt disc as the base sensor and glucose oxidase immobilized on an extremely porous matrix, namely cheesecloth, is described in this communication. Wingard and coworkers [2,3] observed that a potential signal is developed across a glucose oxidase enzyme electrode and the reference electrode system in the buffered glucose solutions. The enzyme electrode response was Nemstian at glucose concentrations in the range 2.2-22.2 mM [4-71. They prepared the enzyme electrode by direct immobilization of glucose oxidase on the Pt electrode surface using polyacrylamide and glutaraldehyde cross-linking; this produced a lower potential signal due to high diffusional resistance to the transport of glucose through the membrane layer [8]. The earlier cyclic voltammetric studies from this laboratory showed that cheesecloth offers negligible diffusional resistance to the transport of glucose [9]. In addition it has high mechanical strength and flexibility. 2. Experimental 2.1. Materials
Glucose oxidase (Aspergillus niger, G-8135), glutaraldehyde (25%), catalase (bovine liver) and polyethylenimine (50%) were obtained from Sigma Chemicals
l
To whom correspondence
0302-4598/94/$7.00 SSDI 0302-4598(94)01709-A
should be addressed.
(USA). The cheesecloth (3.6 mg cmp2> used to immobilize the glucose oxidase was obtained from the local market. Other reagents used were of BDH AnalaR or E. Merck GR grade. Acids were of BDH Aristar grade purity. 2.2. Apparatus The electrochemical pretreatment of the Pt electrode was carried out on polarographic instruments (models 174A and 175) coupled to an x-y recorder (all manufactured by EG & G Princeton Applied Research Corporation, New Jersey). A digital pH meter-millivoltmeter (type EE 330A, EMCO, India) was used to perform the potentiometric measurements. A Pt disc and a Pt wire served as the working and auxiliary electrodes respectively, and a saturated calomel electrode (SCE) was used as the reference electrode. All experiments were performed at 25 f O.l”C. Oxygen-free nitrogen gas (IOLAR grade, Indian Oxygen Ltd., Bombay) was used to deaerate the solutions. 2.3 Electrochemical pretreatment In order to obtain reproducible surface conditions the Pt electrode was subjected to a rigorous electrochemical pretreatment. After polishing with a finegrained polishing paper (Orion Inc., USA), the Pt electrode was anodized for 30 s at + 1.4 V/SCE in 0.1 M H,SO, and then subjected to potential cycling between + 1.2 V and -0.5 V for 15 min. Finally, it was held at +0.2 V for 5 min. This preconditioning ensured that the Pt electrode had identical surface conditions for every experiment. The pretreatment was carried out at 25 + O.l”C under anaerobic conditions produced by bubbling nitrogen through the solution. 2.4. Method of standardization of hydrogen peroxide The hydrogen peroxide solution was standardized using the method described by Vogel [lo]. 2.5. Preparation of enzyme membrane Cheesecloth squares of dimensions 2 cm x 2 cm were washed and dried prior to soaking for 2 h in 0.2% polyethylenimine (PEI), at pH 7.0 (adjusted using HCl). The cloth squares were then rinsed with distilled water and dried in air. The PEI-coated cloth squares were then incubated in an aqueous solution of glucose oxidase (0.5 mg ml-‘) for 2 h. Glutaraldehyde (final concentration 0.2%) was then added. The cross-linking 0 1994 - Elsevier Science S.A. All rights reserved
196
S.D. Kumar et al. / Potentiometric studies at glucose oxidase enzyme electrode
was allowed to proceed for 1 h at room temperature with gentle shaking. The enzyme-bound cloth squares were washed with 0.05 M phosphate buffer (pH 6.8) and stored in the same buffer [ll-131. 2.4. Glucose oxidase assay The glucose oxidase activity on the cheesecloth was determined by measuring the rate of oxygen consumption using a Gilson Oxygraph model KM [14]. The assay system consisted of a cheesecloth square of known area, 2% glucose and excess catalase in 1.5 ml of 0.1 M phosphate buffer (pH 6.0). The glucose oxidase activity on cheesecloth was found to be 0.075-0.1 U cmm2. The optimum pH of bound glucose oxidase was 6.00. Over 90% of the activity could be detected at pH 6.8. Buffer molarity up to 0.4 M was not found to affect the activity of the enzyme. 2.7. Methods The potentiometric measurements were made in stirred solutions using bare Pt or glucose oxidase/Pt as the working electrode and an SCE as the reference electrode. The potentials were measured by adding aliquots of analyte solution (glucose or H,O,) to 0.05 M phosphate buffer (pH 6.8). After each measurement the electrodes and the cell were washed thoroughly with distilled water, and a fresh aliquot of analyte was added. The results are reported as the difference between the potential measured with a given glucose/H,O, concentration and the potential obtained in the buffer solution alone. When not in use the glucose oxidase/Pt electrode was stored in the buffer solution at 4°C. 3. Results and discussion The potentiometric response of glucose in the phosphate buffer (pH 6.8) at a bare Pt electrode was linear in the range 2.2-22.2 mM with a slope of - 18 mV per decade glucose concentration (Fig. 1, curve 1). Wingard et al. [S] have reported a similar slope of - 17 mV per decade glucose concentration at a bare Pt electrode. The mechanism for the response has been suggested to occur via adsorption and oxidation of glucose with concurrent reduction of a Pt oxide species [15-171. The potentiometric response for glucose in the phosphate buffer (pH 6.8) containing soluble glucose oxidase (2 pg ml-‘) was Nernstian with a slope of - 28 mV per decade glucose concentration (Fig. 1, curve 2) and much higher than that obtained in the absence of glucose oxidase (Fig. 1, curve 1). A Nernstian response was also observed for H,O, concentrations of 2.0-20.0 mM in the phosphate buffer (pH 6.8) in the absence of catalase using the bare Pt-SCE system (slope, -33.5 mV per decade glucose concentration; intercept, - 61 mV).
However, on adding an aliquot of H,O, (4 mM) to the phosphate buffer (pH 6.8) containing catalase (26 U ml-r) the potential signal for the buffer solution (+300 mV> decreased rapidly to + 220 mV in 30 s (mean of four observations), after which it increased to a steady value of +320 mV in about 7 min. The potentiometric response in the presence of catalase (20 mV) was much less than that obtained in the absence of catalase (80 mV>. The potentiometric response at the enzyme electrode was also Nernstian in the range 2.2-22.2 mM glucose concentration (slope, -30.8 mV per decade glucose concentration; intercept, -0.33 mV). This indicates that the electrode reaction occurring at the Pt electrode in the case of soluble glucose oxidase or enzyme electrode or H,O, in the absence of catalase is the same, and involves two electrons. Further, the negative slope in the above three cases suggests that a net reduction reaction is occurring at the indicator electrode. The response was quicker and higher at the enzyme electrode compared with that at the bare Pt electrode in the presence of soluble glucose oxidase (Fig. 2). The glucose oxidation reaction at the enzyme electrode occurs in the diffusion layer, producing H,O, at the electrode surface, while in the latter case H,O, is generated in the bulk of the solution and diffuses to the Pt electrode surface to produce a potential signal. The role of dissolved oxygen in the development of the potential signal at the enzyme electrode was ascertained by deoxygenating the buffer solution by purging
*20-
O-
$20
-
! E v) -4oL -60 -
I
I1111al
I 1
CONCENTRATION
IO
,
OF GLUCOSE/mM
Fig. 1. Potentiometric response of glucose at a bare Pt electrode in 0.05 M phosphate buffer (pH 6.8) at 25°C: (1) in the absence of glucose oxidase, slope of - 18.0 mV per decade (correlation coefficient 0.9691, potential response measured after 1 min; (2) in the presence of 2 pg ml-’ glucose oxidase, slope of -28.0 mV per decade glucose concentration (correlation coefficient 0.987), potential response measured after 2 min.
197
SD. Kumar et al. / Potentiometric studies at glucose oxidase enzyme electrode
O-
1
2
3
TIME /months
Fig. 3. Stability studies of the enzyme electrode in 2 mM glucose in 0.05 M phosphate buffer (pH 6.8) at 25°C. Each point represents a mean of five measurements.
-80 -
L
I
0
4
2
6
TIME/min
a
IO
12
-
Fig. 2. Potentiometric response in 0.05 M phosphate buffer (pH 6.8) at 25°C as a function of time: (1) at the bare Pt electrode with soluble glucose oxidase for 7.4 mM glucose concentration; (2) at the enzyme electrode for 7.4 mM glucose concentration.
it with high purity nitrogen gas. A marked fall in the signal was observed with increasing purging times (Table 1). However, Wingard et al. [12] reported an insignificant fall in the potential when the dissolved oxygen content of the buffer was reduced from 100% to 12% which is not comprehensible. These observations lead to the conclusion that the following O,/H,O, redox couple operates at the electrode surface: 0, + 2Hf+
2e- =
H,O,
(1)
for which the Nemst equation is E=E’+
y
( I [021
log [H2021
where E’ is a constant incorporating the standard potential of reaction (1) and the constant H+ concentration term. The behaviour of the potentiometric response for H,O, in the presence of catalase is explained by taking into account the catalytic decomposition of H,O, by TABLE 1. Effect of deoxygenation on potentiometric enzyme electrode
response at the
Deoxygenation time/min
Buffer potential/mV
Response for 7.4 mM [glucose] after 30 s/mV
0 10 15 20 30
300 260 232 212 178
67 40 23 13 1
Initial oxygen concentration
in air-saturated buffer, 0.24 mM.
catalase, producing oxygen, which results in the potential of the Pt electrode becoming more anodic (eqn. (2)). The stability and reproducibility of the enzyme electrode was studied for several months (Fig. 3). The cheesecloth immobilized with glucose oxidase had a long shelf life. Different membranes prepared under identical conditions produced similar potentiometric signals. Frequent pretreatment of the Pt electrode was not required when it was covered with cheesecloth membrane. 4. Conclusions Cheesecloth is a good membrane for the immobilization of glucose oxidase because of its high porosity, mechanical stability, flexibility and longer life-time as well as having a very low cost. The simplicity of the method and good response slopes towards glucose in the range 2.2-22.2 mM make it highly suitable for the fabrication of a biosensor for glucose. Co-immobilization of glucose oxidase and catalase is not desirable as a low potentiometric response is obtained in the presence of catalase. The O,/H,O, redox couple at the electrode surface is responsible for the development of the potentiometric response at the enzyme electrode. Acknowledgements
The authors thank Dr. S. Gangadharan, Associate Director, Analytical Chemistry Group, and Shri T.S. Krishnamoorthy, Head, Surface and Interstial Measurements Section, for their keen interest in and encouragement of the work. The authors also acknowledge Dr. T.P. Radhakrishnan for helpful discussions. References 1 A.P.F. Turner. I. Karube and G.S. Wilson (Eds.), Biosensors, Fundamentals and Applications, Oxford Science Publications, Oxford, 1987, p. 5.
198
S.D. Kkmar et al. / Potentiometric studies at glucose oxidase enzyme electrode
2 L.B. Wingard, CC. Liu and X.L. Nagda, Biotechnol. Bioeng., 13
3 4
5 6 7 8 9 10
(1971) 629. E.J. Lahoda, C.C. Liu and L.B. Wingard, Biotechnol. Bioeng., 17 (1975) 413. L.B. Wingard Jr., D. Ellis, S.J. Yao, J.G. Schiller, C.C. Liu, SK. Wolfson Jr. and A. L. Drash, J. Solid-Phase Biochem., 4 (1979) 253. L.B. Wingard Jr., C.C. Liu, S.K. Wolfson Jr., S.J. Yao and A.L. Drash, Diabetes Care, 5 (1982) 199. C.C. Liu, L.B. Wingard Jr., S.K. Wolfson Jr., S.J. Yao, A.L. Drash and G.J. Schiller, Bioelectrochem. Bioenerg., 6 (1979) 19. J.F. Castner and L.B. Wingard Jr., Anal. Chem., 56 (1984) 2981. L.B. Wingard Jr., J.F. Castner, S.J. Yao, SK. Wolfson Jr., A.L. Drash and C.C. Liu, Appl. Biochem. Biotechnol., 9 (1984) 95. S.D. Kumar, A.V. Kulkarni, S.F. D’Souza and R.G. Dhaneshwar, Bioelectrochem. Bioenerg., 27 (1992) 153. A.J. Vogel, A Textbook of Quantitative Inorganic Analysis, Longman, London, 1975, p. 29.5.
11 SF. D’Souza and N. Kamath, Appt. Microbial. Biotechnol., 29 (1988) 136. 12 N. Kamath, J.S. Melo and S.F. D’Souza, Appl. Biochem. Biotechnot., 19 (1988) 251. 13 K. Sankaran, S.S. Godbole and S.F. D’Souza, Enzyme Microbial. Technol., 11 (1989) 617. 14 SF. D’Souza and G.B. Nadkarni, Biotechnol. Bioeng., 22 (1980) 2179. 15 K. Heyns and H. Paulson in W. Forest (Ed.), Newer Methods of Preparative Organic Chemistry, Vol. 2, Academic Press, New York, 1963, p. 303. 16 M.L.B. Rao and R.F. Drake, J. Electrochem. Sot., 116 (1969) 334. 17 K. Popovic, A. Tripkovic, N. Markovic and R.R. Adzic, J. Electroanal. Chem., 295 (1990) 79. 18 L.B. Wingard Jr., J.G. Schiller, SK. Wolfson Jr., C.C. Liu, A.L. Drash and S.J. Yao, J. Biomed. Mater. Res., 13 (1979) 921.