Biosensors & Bioelectronics 6 (1991) 143-149
Investigation of Polypyrrole/Glucose Oxidase Electrodes by Ellipsometric, Microgravimetric and Electrochemical Measurements Judith Rishpon* & Shimshon Gottesfeld* Electronics Research Group, Los Alamos National Laboratory, Los Aiamos NM 87545, USA (Received 27 February 1990; revised version received 5 September 1990; accepted 12 September 1990)
Abstract: We describe the simultaneous application of two in-situ techniques for
the study of the electrochemical growth of a conducting polymer film (polypyrrole) in the presence of an enzyme (glucose oxidase). The combination of optical (ellipsometric) and microgravimetric (QCMB) measurements employed in this study provides information on fundamental properties of the enzyme-containing film. including film thickness, mass and density. Our results show that incorporation of the enzyme results in changes in the apparent optical properties and in the apparent density of the electrochemically grown film which suggest mutual stabilization of the polypyrrole and the enzyme in the composite layer. Keywords: enzyme electrode, glucose sensor, ellipsometry, quartz crystal microbalance.
INTRODUCTION
procedure is simple and involves only the application of the appropriate potential in an aqueous solution containing the monomer and the enzyme. It has been shown that glucose oxidase can be incorporated into polypyrrole (Foulds & Lowe, 1986; Umana & Waller, 1986) or poly (N-methyl pyrrole) (Bartlett & Whitaker, 1987b) films electrochemically deposited on platinum electrodes. Conducting polymer enzyme electrodes have been employed for monitoring glucose concentration by following oxygen consumption (Shinohara et al., 1988), hydrogen peroxide formation (Pandy, 1988; Foulds & Lowe, 1986; Umana & Waller, 1986; Bartlett & Whitaker, 1987), or the oxidation of an artificial electron acceptor such as ferrocene (Foulds & Lowe, 1988). Bartlett & Whitaker (1987) have
Immobilization of enzymes by electrochemical polymerization has been suggested recently as a promising approach for the preparation of enzyme electrodes (Foulds & Lowe, 1986; Umana & Waller, 1986). In particular, immobilization of enzymes within conducting polymers, which takes place during the anodic oxidation of the monomer in the presence of the enzyme, is advantageous for the preparation of very small electrodes because better control of the amount of the enzyme and its spatial distribution may be achieved. In addition, the immobilization *On sabbatical leave from Department of Biotechnology, Tel-Aviv University, Israel. *To whom correspondence should be addressed. 143
Biosensors & Bioelectronics 0956-5663/91/$03.50© 1991 Elsevier Science Publishers Ltd, England. Printed in Great Britain
144
analysed the amperometric response of a glucose electrode based on hydrogen peroxide determination and have developed a kinetic model that showed electro-oxidation of the hydrogen peroxide occurring at the platinum surface and not at the conducting polymer backbone. Belanger et al. (1989) have discussed the possibility of a direct electronic communication between the enzyme and the conducting polymer. They have used cyclic voitametry to characterize the enzyme electrode in the presence of glucose and concluded that direct electronic communication is not likely to occur. However, the mode of enzyme entrapment in the polypyrrole matrix and the interactions between the conducting polymer backbone and the protein are still uncertain. Fundamental properties of the polymer/ enzyme film, such as the mass and the thickness of the composite layer, have to be known to allow systematic investigation of such enzyme electrodes. Additional non-electrochemical tools for the characterization of enzyme-/conductingpolymer-modified electrodes can provide such valuable information. The quartz crystal microbalance (QCMB) has been applied before for the microgravimetric characterization of electrochemical growth of polypyrrole films (Baker & Reynolds, 1988, 1989). Ellipsometry has also been used before for monitoring conducting polymer film growth (Gottesfeld, 1989; Hamnett et aL, 1989). In a recent publication, we have described a system which allows simultaneous ellipsometric and QCMB measurements (Rishpon etaL, in press). In the present communication, we describe the application of that system to study the growth of a film of polypyrrole in a solution containing glucose oxidase.
EXPERIMENTAL
Materials Pyrrole 99% was received from Aldrich Chemical Company, Milwaukee, WI and was purified by passing it over an alumina column until it became colourless. Glucose oxidase type VII-S (EC 1.1.3.4) derived from Aspergillus niger was obtained from Sigma Chemical Company, St Louis, Mo. Finally, glucose-dextrose anhydrous powder was obtained from J. T. Baker, Phillipsburg, NJ and was dissolved in phosphate buffer 0.1 M, pH = 7. This solution was prepared 24 h
Judith Rishpon, Shimshon Gottesfeld
before use. All other chemicals were of analytical grade and were used without further purification.
The experimental setup The system employed for the simultaneous ellipsometric and QCMB measurements has been described in more detail elsewhere (Rishpon et al.. 1989). The automatic ellipsometer employed is based on a photoelastic modulator operating at 50 kHz. The ellipsometric parameters ~ and A are derived from the 50 kHz, 100 kHz and d.c. components of the reflected intensity signal. The precision of the ellipsometer is 0-05 ° in ~, and 0. l o in A (Gottesfeld, 1989). The quartz crystal was a 5-MHz A-cut 0-5 in disc supplied by Valpey-Fisher, MA. Antisymmetric platinum keyhole patterns (150-nm-thick platinum film) were sputtered on both sides of the crystal using a 2-nm sputtered titanium interlayer for bonding platinum to quartz. One surface of the crystal faced the electrolyte solution and the platinum film on this side was used as a working electrode controlled by a home-built potentiostat. A platinum gauze was used as a counter electrode and a standard saturated calomel electrode (SCE) as a reference electrode. An Ag/AgNO3 reference electrode was used for experiments in acetonitrile.
The electropolymerization procedure Films were grown potentiostatically at 0-75 V versus SCE from 0.1 M phosphate buffer, pH = 7 solutions containing 0.2 M pyrrole and glucose oxidase concentrations of 0-1.4mg/ml. The growth time was 2-3 h. The ellipsometric parameters, ~ and A, the QCMB resonance frequency, and the current density were all collected automatically during film growth. A new sputtered platinum electrode was used for each growth experiment. After the deposition, the electrodes and the cell were washed three times with phosphate buffer 0" 1 M, pH = 7.
Glucose assay For glucose concentration measurements, the polypyrrole glucose oxidase electrode was held at 0.65 V versus SCE in 0"l M phosphate buffer, pH = 7; at the same time, aliquots of the stock 1 M glucose were added. The solution (volume = 70 ml) was stirred by a magnetic stirrer and the current was recorded.
145
Investigation of polypyrrole/glucose oxidase electrodes
RESULTS
3500
Figure 1 shows a typical response of a glucose electrode prepared according to the procedure described in the previous section. The electrode is active up to concentrations well above the K m of the soluble enzyme. The results shown in Fig. 1 analysed by a Lineweaver Burke type plot yield Km = 169mM for our electrode, whereas a Km= 33 mM has been reported for the soluble enzyme (Foulds & Lowe, 1986). This particular electrode was prepared by electropolymerization of pyrrole from a solution containing 0.2 M pyrrole and 20~tg/ml glucose oxidase. Figure 2 depicts the shift of the QCMB resonance frequency and the current recorded during the potentiostatic growth of this film. It can be seen from Fig. 2 that the current decays with time at constant potential. This behaviour suggests a selfinhibited film growth process, clearly reflected in the corresponding drop in the rate of change of
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Fig. 1. Response of PP-GO electrode to addition of glucose. Arrows show points in time when the glucose concentration was increased to the designated levels. 20
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Fig. 2. The variations of the QCMB resonance frequency and the anodic current during PP-GO film growth.
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Fig. 3. Variation of QCMB frequency with anodic charge during polypyrrole film growth. Lower curve:film grown at 0 75 V versus SCE from an aqueous solution of 0 2 M pyrrole and O~1 M phosphate buffer, pH = 7. Upper curve." film grown at 0 5 V versus Ag/AgNO ¢from a solution of 6t05 M pyrrole and ~ I u LiCl04 in acetonitrile.
the QCMB resonance frequency. The total QCMB frequency change for the growth of this particular film was 400 Hz. For the 5-MHz A-cut quartz crystal, a frequency change of 400 Hz corresponds to a mass increase of 6.7/lg/cm 2, or a total mass of the PP-GO film of approximately 2.3pg (electrode surface area is 0"34cm2). In Fig. 3, the shift of the QCMB frequency is plotted against the total anodic charge passed while the polypyrrole film was grown potentiostatically in phosphate buffer. For comparison, a similar plot is shown in the same figure (dashed curve) for the potentiostatic growth of polypyrrole in acetonitrile. The charge efficiency of polymer film growth in phosphate buffer obtained from this plot is about 8 g per Faraday. Assuming that a charge of 2 electrons is required for the formation of a single polypyrrole unit (molecular weight 65), this mass/charge ratio corresponds to a low faradaic growth efficiency of about 25%. The results shown for comparison (dashed curve) for the growth of the same polymer in CHaCN demonstrate that the low charge efficiency found in the phosphate buffer results from the use of this particular aqueous electrolyte. Such low growth efficiencies usually signify parallel anodic processes and result in polymeric films of lower density (Rishpon et al.. in press). Ellipsometric data are usually presented as a versus A plot. This form of presentation provides information on the optical properties of the film (Gottesfeld, 1989). Figure 4 shows a ~, versus A plot for the growth of polypyrrole in phosphate buffer both in the absence and in the presence of the enzyme, at low and high concentrations.
146
Judith Rishpon, Shimshon Gottesfeld 600
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Fig. 4. Experimental (individual points) and simulated (curves) ~t versus A plots obtained for the potentiostatic growth of a polypyrrole film in phosphate buffer, shown for enzyme concentrations of O (0), 20 pg/ml (A), 1.4 mg/ml ([3). The simulated curves were drawn for n = 1.51-0 13i. n = 1.71-t~ 19i and n = 1.83-~ 25ifor these three solution compositions, respectively.
Qualitatively, a ~ versus A plot associated with a larger curvature corresponds to a film with a higher optical density. In fitting these ellipsometric results to a model of film growth (the curves in Fig. 4), we assumed a 'uniform film growth model', i.e. a film with optical properties that do not vary with film thickness. The complex refractive index changes from 1.55-0-13i to 1.710"19i in the presence of the low concentration of enzyme (20 pg/ml) employed in the preparation of our electrodes. This increase in the refractive index and extinction coefficient suggests that denser polymer films are obtained in the presence of the enzyme. To check this possibility further, we measured the effect of a higher enzyme concentration on the optical properties of the film during polymerization. As seen in Fig. 4, the curve that best fits the experimental points at the highest enzyme concentration corresponds to a film with a complex refractive index of 1.83-0-25i. Deviations from the computer fits to a uniform film growth model, as seen in Fig. 4, are expected for such composite films that grow at poor charge efficiencies. These deviations correspond to an error bar of the order of 2-5% in the average optical properties evaluated by assuming uniform growth. Figures 5 and 6 show the QCMB readings taken simultaneously with the eilipsometric readings presented in Fig. 4. Figure 5 shows a plot of the QCMB resonance frequency shift versus optical thickness for the electrochemical growth
• AA 100
200
300 4OO 500 OPTICALTHICKNESS(A)
600
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Fig. 5. Variation of QCMB frequency versus optical thickness during potentiostatic growth of polypyrrole film in phosphate buffer, in the absence of the enzyme ( A) and in the presence of 1.4 mg/ml of the enzyme (0). 400
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Fig. 6. Variation of QCMB frequency plotted against optical thickness during potentiostatic growth of polypyrrolefilm in phosphate buffer in the presence of 20 I~g/ml of the enzyme.
of a polypyrrole film in the absence of enzyme (triangles) and in the presence of a relatively large concentration (1-4 mg/ml) of the enzyme (solid circles). There are quite significant variations in the slopes of these curves, but the average slope designated by the dashed lines is clearly different, and corresponds to 0-6 g/cm 3 and 1-67 g/cm 3 for the films grown in the absence and in the presence of the enzyme, respectively. The deviations of the experimental plots in Fig. 5 from perfect linearity most probably reflect some fluctuations of film density during growth, as expected for a composite film growing at such low charge efficiencies. Such fluctuations are averaged out when uniform film growth is assumed in the evaluation of the optical thickness. The conclusion on an increased density in films grown in the presence of the enzyme (Fig. 5) is in agreement with the higher
147
Investigation of polypyrrole/glucose oxidase electrodes
optical density evaluated for polypyrrole films grown in the presence of glucose oxidase (Fig. 4). One feature of the plots in Fig. 5 is a very small rate of mass increase (resonance frequency shift) at the earliest stages of film growth. This feature is highlighted in Fig. 6 for a polypyrrole film grown in the presence of 20 pg/ml of the enzyme. The more sensitive thickness scale used in Fig. 6 reveals an initial apparent film density of only 0-66 g/cm 3. As the film grows further the apparent density gradually increases, as reflected by the increasing slope of the plot, reaching 1-32 g/cm 3 at a film thickness of 400 A, which is similar to reported densities of conducting polymer films (Stilwell & Park, 1988).
DISCUSSION The in-situ QCMB measurements allow an estimate of enzyme enrichment in the composite film. Assuming a weight fraction of the enzyme in the film similar to that in solution (20/zg/g), the total expected weight of the enzyme in this film is calculated as 0-05 ng. This is much lower than the amount of enzyme in the film derived from its electrochemically measured activity. The specific activity of the solid enzyme employed was 129 units/mg, and this can be assumed as the upper limit of enzyme activity in the film. This activity corresponds to a maximum rate of H202 formation of 2" 15 btmol/mg GO and the measured current of 5 pA (Fig. 1) thus corresponds to 11 ng of GO enzyme in the composite film. Considerations of mass transport limitations within the film would make this number even larger. Thus, the enrichment factor: [GO] in the film/[GO] in solution is at least two orders of magnitude. The incorporated enzyme has a significant effect on the apparent optical properties and the apparent density of the composite film, as shown in Figs 4-6. The higher the concentration of the enzyme in solution the larger are the real and imaginary components of the complex refractive index of the composite layer. This trend reflects an increase in the degree of space filling during the earliest stages of polymer film growth as the enzyme concentration in the film is increased. The reason must be that some GO-polypyrrole interaction modifies the morphology of the film during the early stages of film growth. Further evidence for the effects of the enzyme on film morphology is obtained from the
combined results of the QCMB and the ellipsometer, as recorded during the growth of the composite film. To discuss these results, we should start from the rather peculiar finding of an apparent film density smaller than 1 g/cm 3 during the early stages of polypyrrole growth in the phosphate buffer solution (Figs 5-6). Observations of the opposite nature (surface films with apparent masses exceeding the maximum expected from coulometric data) have been made in previous QCMB studies of electrochemical film growth (Feldman & Melroy, 1987). These observations were interpreted as combined effects of electrochemically formed porous material and entrapped electrolyte on the resonance frequency of the QCMB. An apparent density smaller than l g/cm 3 derived from combined QCMB-ellipsometric measurements (Figs 5-6) can be explained if the QCMB does not sense the mass of electrolyte components filling spaces between growing polymer patches, whereas the ellipsometer measures the thickness of the composite polymer-electrolyte film as determined by the height of the growing patches (e.g. hemispherical patches) of the polymer. This might be the case for a certain geometry of a patchy growing polymer, with the separation between the patches (and their viscoelastic properties) determining the nature of the QCMB response. This interpretation for an apparent film density < 1 g,/cm 3 is schematically explained in Fig. 7. As the enzyme is added to the electrolyte solution, film growth becomes more "well behaved', that is, the apparent film density now corresponds to ordinary values at earlier stages of growth. This suggests a beneficial effect of the enzyme on space filling in the growing polymer, particularly during the earliest stages of growth. One possibility is that the enzyme adsorbs at the platinum surface (Bartlett & Whitaker, 1987) and Solution Thickness
Solution Film Melal
r
Film
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Fig. 7. Schematic mode/ of the polypyrrole film on the electrode in the absence (le3~) and in the presence (righO of the enzyme. In the absence of the enzyme, the po/ypyrrole seems to grow in phosphate buffer with very poor space filling and the QCMB seems to 'sense' only conducting polymer centres rather than a continuous film. Addition of the enzyme brings about densification and mutual stabilization such that the polymer/enzyme composite is sensed as a growing 's/ab'ofordinary density.
148 thus affects the 'wetting" characteristics of the metal electrode with respect to the polymer phase, which, in turn, affect the polymer nucleation process. The enzyme could thus act mainly as a substrate surface modifier. We have shown recently (Rubinstein et al., 1990) that a monolayer of a chemisorbed organic molecule can have significant effects on the morphology of electrochemically grown conducting polymer films. The possible effect of an adsorbed layer of the enzyme at the platinum surface further suggests a possible metal/enzyme/polymer structure for the composite film. The minimum overall mass of enzyme per square centimetre of electrode area, as deduced from the activity of the electrode, was calculated above as 33 ng/cm 2. This could, in fact, correspond to only a part of a single monolayer of this protein located fully at the film/metal interface. Our results cannot serve, however, as conclusive evidence for such a sharp concentration profile of the enzyme in the composite film. (One other factor that could bring about polymer-enzyme stabilization is the electrostatic interaction between a positively charged polymer and the negatively charged enzyme.) Reports by others (Belanger et al.. 1989) have shown that the polypyrrole loses its redox activity when exposed to the anodic potentials employed to monitor H202 (0.7 V versus SCE), and the po!ypyrrole thus seems to serve only as an encapsulating layer for the enzyme, Our results (Figs 4, 5) further suggest that there is a significant morphological effect caused by enzyme incorporation and, thus, some mutual stabilization of the polypyrrole and the enzyme in the composite layer. CONCLUSIONS I. Fundamental properties of electrochemically grown films with incorporated enzymes, such as the thickness and the weight of the film, can be obtained in situ for micron and submicron films by using optical and microgravimetric techniques. . Significant enrichment of GO enzyme in a matrix of electrochemically grown polypyrrole is concluded from film mass and GO activity measurements. The apparent enrichment can be explained, in principle, by adsorption of a part ofa monolayer of the enzyme on the platinum surface, but this configuration is not proved conclusively from our results.
Judith Rishpon. Shimshon Gottesfeld
3. Significant effects of the enzyme on the optical properties and the apparent density of the polypyrrole film have been detected. The incorporation of enzyme seems to improve the space filling in thin films of polypyrrole grown in phosphate buffer.
REFERENCES Baker. C. K. & Reynolds, J, R. (1988). A quartz microbalance study of the electrosynthesis of polypyrrole. J. Electroanal. Chem.. 251, 307-22. Baker, C. K. & Reynolds, J. R. (t989). Use of the quartz microbalance in the study of polyheterocycle electrosynthesis. Synth. Met.. 28, C21-26. Bartlett, P. N. & Whitaker, R. G. (1987a). Electrochemical immobilization of enzymes. Part I. Theory. J Electroanal. Chem.. 224, 27-35. Bartlett, P. N. & Whitaker, R. G. (1987b). Electrochemical immobilization of enzymes. Part II. Glucose oxidase immobilized in poly-N-methylpyrrole. J. Electroanal. Chem.. 224, 37-48. Bartlett. P. N. & Whitaker, R. G. (1987, 1988). Strategies for the development of amperometric enzyme electrode. Biosensors. 3, 359-79. Belanger, D., Nadreau, J. & Fortier. G. (1989). Electrochemistry of polypyrrole glucose oxidase electrode. J. Electroanal. Chem., 274, 143-55. Feldman, B. J. & Melroy, O. R. (1987). Ion flux during electrochemical charging of prussian blue films. J. Electroanal. Chem.. 234, 213-27. Foulds, N. C. & Lowe, C. R. (1986). Enzyme entrapment in electrically conducting polymers. Immobilization of glucose oxidase in polypyrrole and its application in amperometric glucose sensors. J. Chem. Soc. Faraday Trans.. 1, (82). 1259-64. Foulds, N. C. & Lowe. C. R. (1988). Immobilization of glucose oxidase in ferrocene-modified polymers. Anal, Chem,. 60, 2473-8. Gottesfeld, S. (1989). Ellipsometry: principles and recent application in electrochemistry. In Electroanalytical Chemistry. Vol, 15, ed. A. J. Bard. Marcel Dekker, New York. pp. 142-265. Hamnett, A., Higgins, S. J., Fisk, P. R. & Albery, J. W. (1989). An ellipsometric study of polypyrrole films on platinum. J. Electroanal. Chem.. 270, 479-88. Pandy. P. C. (1988). A new conducting polymer-coated glucose sensor. J. Chem. Soc. Faraday Trans.. 84(7), 2259-65. Rishpon, J., Redonod, A. & Gottesfeld, S. (in press). Simultaneous ellipsometric and microgravimetric measurements during the electrochemical growth of polyaniline. J. Electroanal. Chem. Rubinstein, I., Rishpon, J., Sabatani, E., Redondo, A. &
Investigation of polypyrrole/glucose oxidase electrodes Gottesfeld, S. (1990). Morphology control in electrochemically grown conducting polymer films (I) precoating the metal substrate with an organic monolayer.J. Am. Chem. Soc.. ! 12, 6135-6. Shinohara, H., Chiba, T. & Aizawa, M. (1988). Enzyme microsensor for glucose with an electrochemically synthesized enzyme-polyaniline film. Sens. Actuators. 13, 78-86.
149 Stilwell, D. E. & Su-Moon Park. (1988). Electrochemistry of conductive polymers. III. Some physical and electrochemical properties observed from electrochemically grown polyaniline. J. Electrochem. Soc.. 135, 2491-6. Umana, M. & Waller, J. (1986). Protein modified electrodes. Glucose oxidase/polypyrrole system. Anal Chem.. 58, 2979-83.
Investigation of Polypyrrole/Glucose Oxidase Electrodes by Ellipsometric, Microgravimetric and Electrochemical Measurements Judith Rishpon* & Shimshon Gottesfeld* Electronics Research Group, Los Alamos National Laboratory, Los Aiamos NM 87545, USA (Received 27 February 1990; revised version received 5 September 1990; accepted 12 September 1990)
Abstract: We describe the simultaneous application of two in-situ techniques for
the study of the electrochemical growth of a conducting polymer film (polypyrrole) in the presence of an enzyme (glucose oxidase). The combination of optical (ellipsometric) and microgravimetric (QCMB) measurements employed in this study provides information on fundamental properties of the enzyme-containing film. including film thickness, mass and density. Our results show that incorporation of the enzyme results in changes in the apparent optical properties and in the apparent density of the electrochemically grown film which suggest mutual stabilization of the polypyrrole and the enzyme in the composite layer. Keywords: enzyme electrode, glucose sensor, ellipsometry, quartz crystal microbalance.
INTRODUCTION
procedure is simple and involves only the application of the appropriate potential in an aqueous solution containing the monomer and the enzyme. It has been shown that glucose oxidase can be incorporated into polypyrrole (Foulds & Lowe, 1986; Umana & Waller, 1986) or poly (N-methyl pyrrole) (Bartlett & Whitaker, 1987b) films electrochemically deposited on platinum electrodes. Conducting polymer enzyme electrodes have been employed for monitoring glucose concentration by following oxygen consumption (Shinohara et al., 1988), hydrogen peroxide formation (Pandy, 1988; Foulds & Lowe, 1986; Umana & Waller, 1986; Bartlett & Whitaker, 1987), or the oxidation of an artificial electron acceptor such as ferrocene (Foulds & Lowe, 1988). Bartlett & Whitaker (1987) have
Immobilization of enzymes by electrochemical polymerization has been suggested recently as a promising approach for the preparation of enzyme electrodes (Foulds & Lowe, 1986; Umana & Waller, 1986). In particular, immobilization of enzymes within conducting polymers, which takes place during the anodic oxidation of the monomer in the presence of the enzyme, is advantageous for the preparation of very small electrodes because better control of the amount of the enzyme and its spatial distribution may be achieved. In addition, the immobilization *On sabbatical leave from Department of Biotechnology, Tel-Aviv University, Israel. *To whom correspondence should be addressed. 143
Biosensors & Bioelectronics 0956-5663/91/$03.50© 1991 Elsevier Science Publishers Ltd, England. Printed in Great Britain
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analysed the amperometric response of a glucose electrode based on hydrogen peroxide determination and have developed a kinetic model that showed electro-oxidation of the hydrogen peroxide occurring at the platinum surface and not at the conducting polymer backbone. Belanger et al. (1989) have discussed the possibility of a direct electronic communication between the enzyme and the conducting polymer. They have used cyclic voitametry to characterize the enzyme electrode in the presence of glucose and concluded that direct electronic communication is not likely to occur. However, the mode of enzyme entrapment in the polypyrrole matrix and the interactions between the conducting polymer backbone and the protein are still uncertain. Fundamental properties of the polymer/ enzyme film, such as the mass and the thickness of the composite layer, have to be known to allow systematic investigation of such enzyme electrodes. Additional non-electrochemical tools for the characterization of enzyme-/conductingpolymer-modified electrodes can provide such valuable information. The quartz crystal microbalance (QCMB) has been applied before for the microgravimetric characterization of electrochemical growth of polypyrrole films (Baker & Reynolds, 1988, 1989). Ellipsometry has also been used before for monitoring conducting polymer film growth (Gottesfeld, 1989; Hamnett et aL, 1989). In a recent publication, we have described a system which allows simultaneous ellipsometric and QCMB measurements (Rishpon etaL, in press). In the present communication, we describe the application of that system to study the growth of a film of polypyrrole in a solution containing glucose oxidase.
EXPERIMENTAL
Materials Pyrrole 99% was received from Aldrich Chemical Company, Milwaukee, WI and was purified by passing it over an alumina column until it became colourless. Glucose oxidase type VII-S (EC 1.1.3.4) derived from Aspergillus niger was obtained from Sigma Chemical Company, St Louis, Mo. Finally, glucose-dextrose anhydrous powder was obtained from J. T. Baker, Phillipsburg, NJ and was dissolved in phosphate buffer 0.1 M, pH = 7. This solution was prepared 24 h
Judith Rishpon, Shimshon Gottesfeld
before use. All other chemicals were of analytical grade and were used without further purification.
The experimental setup The system employed for the simultaneous ellipsometric and QCMB measurements has been described in more detail elsewhere (Rishpon et al.. 1989). The automatic ellipsometer employed is based on a photoelastic modulator operating at 50 kHz. The ellipsometric parameters ~ and A are derived from the 50 kHz, 100 kHz and d.c. components of the reflected intensity signal. The precision of the ellipsometer is 0-05 ° in ~, and 0. l o in A (Gottesfeld, 1989). The quartz crystal was a 5-MHz A-cut 0-5 in disc supplied by Valpey-Fisher, MA. Antisymmetric platinum keyhole patterns (150-nm-thick platinum film) were sputtered on both sides of the crystal using a 2-nm sputtered titanium interlayer for bonding platinum to quartz. One surface of the crystal faced the electrolyte solution and the platinum film on this side was used as a working electrode controlled by a home-built potentiostat. A platinum gauze was used as a counter electrode and a standard saturated calomel electrode (SCE) as a reference electrode. An Ag/AgNO3 reference electrode was used for experiments in acetonitrile.
The electropolymerization procedure Films were grown potentiostatically at 0-75 V versus SCE from 0.1 M phosphate buffer, pH = 7 solutions containing 0.2 M pyrrole and glucose oxidase concentrations of 0-1.4mg/ml. The growth time was 2-3 h. The ellipsometric parameters, ~ and A, the QCMB resonance frequency, and the current density were all collected automatically during film growth. A new sputtered platinum electrode was used for each growth experiment. After the deposition, the electrodes and the cell were washed three times with phosphate buffer 0" 1 M, pH = 7.
Glucose assay For glucose concentration measurements, the polypyrrole glucose oxidase electrode was held at 0.65 V versus SCE in 0"l M phosphate buffer, pH = 7; at the same time, aliquots of the stock 1 M glucose were added. The solution (volume = 70 ml) was stirred by a magnetic stirrer and the current was recorded.
145
Investigation of polypyrrole/glucose oxidase electrodes
RESULTS
3500
Figure 1 shows a typical response of a glucose electrode prepared according to the procedure described in the previous section. The electrode is active up to concentrations well above the K m of the soluble enzyme. The results shown in Fig. 1 analysed by a Lineweaver Burke type plot yield Km = 169mM for our electrode, whereas a Km= 33 mM has been reported for the soluble enzyme (Foulds & Lowe, 1986). This particular electrode was prepared by electropolymerization of pyrrole from a solution containing 0.2 M pyrrole and 20~tg/ml glucose oxidase. Figure 2 depicts the shift of the QCMB resonance frequency and the current recorded during the potentiostatic growth of this film. It can be seen from Fig. 2 that the current decays with time at constant potential. This behaviour suggests a selfinhibited film growth process, clearly reflected in the corresponding drop in the rate of change of
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Fig. 1. Response of PP-GO electrode to addition of glucose. Arrows show points in time when the glucose concentration was increased to the designated levels. 20
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Fig. 2. The variations of the QCMB resonance frequency and the anodic current during PP-GO film growth.
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o (mC/cm =t)
Fig. 3. Variation of QCMB frequency with anodic charge during polypyrrole film growth. Lower curve:film grown at 0 75 V versus SCE from an aqueous solution of 0 2 M pyrrole and O~1 M phosphate buffer, pH = 7. Upper curve." film grown at 0 5 V versus Ag/AgNO ¢from a solution of 6t05 M pyrrole and ~ I u LiCl04 in acetonitrile.
the QCMB resonance frequency. The total QCMB frequency change for the growth of this particular film was 400 Hz. For the 5-MHz A-cut quartz crystal, a frequency change of 400 Hz corresponds to a mass increase of 6.7/lg/cm 2, or a total mass of the PP-GO film of approximately 2.3pg (electrode surface area is 0"34cm2). In Fig. 3, the shift of the QCMB frequency is plotted against the total anodic charge passed while the polypyrrole film was grown potentiostatically in phosphate buffer. For comparison, a similar plot is shown in the same figure (dashed curve) for the potentiostatic growth of polypyrrole in acetonitrile. The charge efficiency of polymer film growth in phosphate buffer obtained from this plot is about 8 g per Faraday. Assuming that a charge of 2 electrons is required for the formation of a single polypyrrole unit (molecular weight 65), this mass/charge ratio corresponds to a low faradaic growth efficiency of about 25%. The results shown for comparison (dashed curve) for the growth of the same polymer in CHaCN demonstrate that the low charge efficiency found in the phosphate buffer results from the use of this particular aqueous electrolyte. Such low growth efficiencies usually signify parallel anodic processes and result in polymeric films of lower density (Rishpon et al.. in press). Ellipsometric data are usually presented as a versus A plot. This form of presentation provides information on the optical properties of the film (Gottesfeld, 1989). Figure 4 shows a ~, versus A plot for the growth of polypyrrole in phosphate buffer both in the absence and in the presence of the enzyme, at low and high concentrations.
146
Judith Rishpon, Shimshon Gottesfeld 600
I
I
I
l
I
/~1 /4
el e
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dapp = 1.67g cm"~
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Fig. 4. Experimental (individual points) and simulated (curves) ~t versus A plots obtained for the potentiostatic growth of a polypyrrole film in phosphate buffer, shown for enzyme concentrations of O (0), 20 pg/ml (A), 1.4 mg/ml ([3). The simulated curves were drawn for n = 1.51-0 13i. n = 1.71-t~ 19i and n = 1.83-~ 25ifor these three solution compositions, respectively.
Qualitatively, a ~ versus A plot associated with a larger curvature corresponds to a film with a higher optical density. In fitting these ellipsometric results to a model of film growth (the curves in Fig. 4), we assumed a 'uniform film growth model', i.e. a film with optical properties that do not vary with film thickness. The complex refractive index changes from 1.55-0-13i to 1.710"19i in the presence of the low concentration of enzyme (20 pg/ml) employed in the preparation of our electrodes. This increase in the refractive index and extinction coefficient suggests that denser polymer films are obtained in the presence of the enzyme. To check this possibility further, we measured the effect of a higher enzyme concentration on the optical properties of the film during polymerization. As seen in Fig. 4, the curve that best fits the experimental points at the highest enzyme concentration corresponds to a film with a complex refractive index of 1.83-0-25i. Deviations from the computer fits to a uniform film growth model, as seen in Fig. 4, are expected for such composite films that grow at poor charge efficiencies. These deviations correspond to an error bar of the order of 2-5% in the average optical properties evaluated by assuming uniform growth. Figures 5 and 6 show the QCMB readings taken simultaneously with the eilipsometric readings presented in Fig. 4. Figure 5 shows a plot of the QCMB resonance frequency shift versus optical thickness for the electrochemical growth
• AA 100
200
300 4OO 500 OPTICALTHICKNESS(A)
600
700
Fig. 5. Variation of QCMB frequency versus optical thickness during potentiostatic growth of polypyrrole film in phosphate buffer, in the absence of the enzyme ( A) and in the presence of 1.4 mg/ml of the enzyme (0). 400
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Fig. 6. Variation of QCMB frequency plotted against optical thickness during potentiostatic growth of polypyrrolefilm in phosphate buffer in the presence of 20 I~g/ml of the enzyme.
of a polypyrrole film in the absence of enzyme (triangles) and in the presence of a relatively large concentration (1-4 mg/ml) of the enzyme (solid circles). There are quite significant variations in the slopes of these curves, but the average slope designated by the dashed lines is clearly different, and corresponds to 0-6 g/cm 3 and 1-67 g/cm 3 for the films grown in the absence and in the presence of the enzyme, respectively. The deviations of the experimental plots in Fig. 5 from perfect linearity most probably reflect some fluctuations of film density during growth, as expected for a composite film growing at such low charge efficiencies. Such fluctuations are averaged out when uniform film growth is assumed in the evaluation of the optical thickness. The conclusion on an increased density in films grown in the presence of the enzyme (Fig. 5) is in agreement with the higher
147
Investigation of polypyrrole/glucose oxidase electrodes
optical density evaluated for polypyrrole films grown in the presence of glucose oxidase (Fig. 4). One feature of the plots in Fig. 5 is a very small rate of mass increase (resonance frequency shift) at the earliest stages of film growth. This feature is highlighted in Fig. 6 for a polypyrrole film grown in the presence of 20 pg/ml of the enzyme. The more sensitive thickness scale used in Fig. 6 reveals an initial apparent film density of only 0-66 g/cm 3. As the film grows further the apparent density gradually increases, as reflected by the increasing slope of the plot, reaching 1-32 g/cm 3 at a film thickness of 400 A, which is similar to reported densities of conducting polymer films (Stilwell & Park, 1988).
DISCUSSION The in-situ QCMB measurements allow an estimate of enzyme enrichment in the composite film. Assuming a weight fraction of the enzyme in the film similar to that in solution (20/zg/g), the total expected weight of the enzyme in this film is calculated as 0-05 ng. This is much lower than the amount of enzyme in the film derived from its electrochemically measured activity. The specific activity of the solid enzyme employed was 129 units/mg, and this can be assumed as the upper limit of enzyme activity in the film. This activity corresponds to a maximum rate of H202 formation of 2" 15 btmol/mg GO and the measured current of 5 pA (Fig. 1) thus corresponds to 11 ng of GO enzyme in the composite film. Considerations of mass transport limitations within the film would make this number even larger. Thus, the enrichment factor: [GO] in the film/[GO] in solution is at least two orders of magnitude. The incorporated enzyme has a significant effect on the apparent optical properties and the apparent density of the composite film, as shown in Figs 4-6. The higher the concentration of the enzyme in solution the larger are the real and imaginary components of the complex refractive index of the composite layer. This trend reflects an increase in the degree of space filling during the earliest stages of polymer film growth as the enzyme concentration in the film is increased. The reason must be that some GO-polypyrrole interaction modifies the morphology of the film during the early stages of film growth. Further evidence for the effects of the enzyme on film morphology is obtained from the
combined results of the QCMB and the ellipsometer, as recorded during the growth of the composite film. To discuss these results, we should start from the rather peculiar finding of an apparent film density smaller than 1 g/cm 3 during the early stages of polypyrrole growth in the phosphate buffer solution (Figs 5-6). Observations of the opposite nature (surface films with apparent masses exceeding the maximum expected from coulometric data) have been made in previous QCMB studies of electrochemical film growth (Feldman & Melroy, 1987). These observations were interpreted as combined effects of electrochemically formed porous material and entrapped electrolyte on the resonance frequency of the QCMB. An apparent density smaller than l g/cm 3 derived from combined QCMB-ellipsometric measurements (Figs 5-6) can be explained if the QCMB does not sense the mass of electrolyte components filling spaces between growing polymer patches, whereas the ellipsometer measures the thickness of the composite polymer-electrolyte film as determined by the height of the growing patches (e.g. hemispherical patches) of the polymer. This might be the case for a certain geometry of a patchy growing polymer, with the separation between the patches (and their viscoelastic properties) determining the nature of the QCMB response. This interpretation for an apparent film density < 1 g,/cm 3 is schematically explained in Fig. 7. As the enzyme is added to the electrolyte solution, film growth becomes more "well behaved', that is, the apparent film density now corresponds to ordinary values at earlier stages of growth. This suggests a beneficial effect of the enzyme on space filling in the growing polymer, particularly during the earliest stages of growth. One possibility is that the enzyme adsorbs at the platinum surface (Bartlett & Whitaker, 1987) and Solution Thickness
Solution Film Melal
r
Film
Y//////////////////,~,Metal
Fig. 7. Schematic mode/ of the polypyrrole film on the electrode in the absence (le3~) and in the presence (righO of the enzyme. In the absence of the enzyme, the po/ypyrrole seems to grow in phosphate buffer with very poor space filling and the QCMB seems to 'sense' only conducting polymer centres rather than a continuous film. Addition of the enzyme brings about densification and mutual stabilization such that the polymer/enzyme composite is sensed as a growing 's/ab'ofordinary density.
148 thus affects the 'wetting" characteristics of the metal electrode with respect to the polymer phase, which, in turn, affect the polymer nucleation process. The enzyme could thus act mainly as a substrate surface modifier. We have shown recently (Rubinstein et al., 1990) that a monolayer of a chemisorbed organic molecule can have significant effects on the morphology of electrochemically grown conducting polymer films. The possible effect of an adsorbed layer of the enzyme at the platinum surface further suggests a possible metal/enzyme/polymer structure for the composite film. The minimum overall mass of enzyme per square centimetre of electrode area, as deduced from the activity of the electrode, was calculated above as 33 ng/cm 2. This could, in fact, correspond to only a part of a single monolayer of this protein located fully at the film/metal interface. Our results cannot serve, however, as conclusive evidence for such a sharp concentration profile of the enzyme in the composite film. (One other factor that could bring about polymer-enzyme stabilization is the electrostatic interaction between a positively charged polymer and the negatively charged enzyme.) Reports by others (Belanger et al.. 1989) have shown that the polypyrrole loses its redox activity when exposed to the anodic potentials employed to monitor H202 (0.7 V versus SCE), and the po!ypyrrole thus seems to serve only as an encapsulating layer for the enzyme, Our results (Figs 4, 5) further suggest that there is a significant morphological effect caused by enzyme incorporation and, thus, some mutual stabilization of the polypyrrole and the enzyme in the composite layer. CONCLUSIONS I. Fundamental properties of electrochemically grown films with incorporated enzymes, such as the thickness and the weight of the film, can be obtained in situ for micron and submicron films by using optical and microgravimetric techniques. . Significant enrichment of GO enzyme in a matrix of electrochemically grown polypyrrole is concluded from film mass and GO activity measurements. The apparent enrichment can be explained, in principle, by adsorption of a part ofa monolayer of the enzyme on the platinum surface, but this configuration is not proved conclusively from our results.
Judith Rishpon. Shimshon Gottesfeld
3. Significant effects of the enzyme on the optical properties and the apparent density of the polypyrrole film have been detected. The incorporation of enzyme seems to improve the space filling in thin films of polypyrrole grown in phosphate buffer.
REFERENCES Baker. C. K. & Reynolds, J, R. (1988). A quartz microbalance study of the electrosynthesis of polypyrrole. J. Electroanal. Chem.. 251, 307-22. Baker, C. K. & Reynolds, J. R. (t989). Use of the quartz microbalance in the study of polyheterocycle electrosynthesis. Synth. Met.. 28, C21-26. Bartlett, P. N. & Whitaker, R. G. (1987a). Electrochemical immobilization of enzymes. Part I. Theory. J Electroanal. Chem.. 224, 27-35. Bartlett, P. N. & Whitaker, R. G. (1987b). Electrochemical immobilization of enzymes. Part II. Glucose oxidase immobilized in poly-N-methylpyrrole. J. Electroanal. Chem.. 224, 37-48. Bartlett. P. N. & Whitaker, R. G. (1987, 1988). Strategies for the development of amperometric enzyme electrode. Biosensors. 3, 359-79. Belanger, D., Nadreau, J. & Fortier. G. (1989). Electrochemistry of polypyrrole glucose oxidase electrode. J. Electroanal. Chem., 274, 143-55. Feldman, B. J. & Melroy, O. R. (1987). Ion flux during electrochemical charging of prussian blue films. J. Electroanal. Chem.. 234, 213-27. Foulds, N. C. & Lowe, C. R. (1986). Enzyme entrapment in electrically conducting polymers. Immobilization of glucose oxidase in polypyrrole and its application in amperometric glucose sensors. J. Chem. Soc. Faraday Trans.. 1, (82). 1259-64. Foulds, N. C. & Lowe. C. R. (1988). Immobilization of glucose oxidase in ferrocene-modified polymers. Anal, Chem,. 60, 2473-8. Gottesfeld, S. (1989). Ellipsometry: principles and recent application in electrochemistry. In Electroanalytical Chemistry. Vol, 15, ed. A. J. Bard. Marcel Dekker, New York. pp. 142-265. Hamnett, A., Higgins, S. J., Fisk, P. R. & Albery, J. W. (1989). An ellipsometric study of polypyrrole films on platinum. J. Electroanal. Chem.. 270, 479-88. Pandy. P. C. (1988). A new conducting polymer-coated glucose sensor. J. Chem. Soc. Faraday Trans.. 84(7), 2259-65. Rishpon, J., Redonod, A. & Gottesfeld, S. (in press). Simultaneous ellipsometric and microgravimetric measurements during the electrochemical growth of polyaniline. J. Electroanal. Chem. Rubinstein, I., Rishpon, J., Sabatani, E., Redondo, A. &
Investigation of polypyrrole/glucose oxidase electrodes Gottesfeld, S. (1990). Morphology control in electrochemically grown conducting polymer films (I) precoating the metal substrate with an organic monolayer.J. Am. Chem. Soc.. ! 12, 6135-6. Shinohara, H., Chiba, T. & Aizawa, M. (1988). Enzyme microsensor for glucose with an electrochemically synthesized enzyme-polyaniline film. Sens. Actuators. 13, 78-86.
149 Stilwell, D. E. & Su-Moon Park. (1988). Electrochemistry of conductive polymers. III. Some physical and electrochemical properties observed from electrochemically grown polyaniline. J. Electrochem. Soc.. 135, 2491-6. Umana, M. & Waller, J. (1986). Protein modified electrodes. Glucose oxidase/polypyrrole system. Anal Chem.. 58, 2979-83.
