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glucose oxidase biosensor: Part II. Optimal preparation conditions for the biosensor
Biosensors & Bioe~ectronicsVol. 11, No. l/2, pp. 171-178, 19% 0 1996 Elsevier Science Limited Printed in Great Britain. All rights reserved 09565663/%/$15.M)
ELSEVIER ADVANCED TECHNOLOGY
Electrochemical characterization of polypyrrolelglucose oxidase biosensor: Part II. Optimal preparation conditions for the biosensor Min-Chol Department
Shin & Hak-Sung
Kim*
of Biotechnology,
Korea Advanced Institute of Science & Technology, 373-1, Kusung-dong, Yusung-ku, Taejon, 305-701, Korea. Tel: [82]-42-869-2616 Fax: [82]-42-869-2610 E-Mail: [email protected]. (Received
2 August 1994; revised 29 March 1995; accepted 3 April 1995)
Abstract:
The optimal conditions for the preparation of a polypyrrole/glucose oxidase biosensor were investigated. Response of the resulting biosensor was found to be critically dependent on preparation parameters such as pyrrole and enzyme concentrations in the electrodeposition solution, and the film thickness. It was also observed that the film thickness and the enzyme concentration have an equivalent effect on the response. Our results indicate that the analytical performance of the resulting biosensor can be improved by choosing the proper set of preparation parameters in terms of the sensitivity for glucose and the suppression of interference. Keywords: amperometric polypyrrole.
biosensor,
INTRODUCTION The
biosensor
has attracted
considerable
atten-
tion as the potential successor to a wide range of analytical techniques owing to its unique characteristic of specificity. The main feature of biosensors can be defined as the spatial unity of biological elements with an appropriate transducer (Lowe, 1985). With current emphasis on the miniaturization and mass production of biosensors, the efficient and reproducible immobilization of biological elements is regarded
* To whom correspondence
should be addressed
analytical performance,
glucose oxidase,
as one of the most important factors for design and development. Electrochemical immobilization within conducting polymers provides a promising alternative for the one-step loadings of biological elements over a small and defined area of the transducer element. The amount and spatial distribution of enzymes within the polymer grown on the electrode can be easily controlled by electrochemical immobilization. Extensive studies have shown that redox enzymes may be incorporated into polypyrrole (Foulds & Lowe, 1986; Umana & Waller, 1986), poly(N-methylpyrrole) (Bartlett & Whitaker, 1987b), polyaniline (Cooper & Hall, 1992), polyindole (Pandey, 1988), and polyphenol (Bartlett et al., 1992) films during electrochemical 171
Min-Chol
Shin & Hak-Sung
Kim
polymerizationof corresponding monomer in the presence of enzymes. Most research to date has concentrated on the physical entrapment of enzymes within electrochemically grown polypyrrole (B&anger et al., 1989; Fortier et al., 1990; Yon-Hin & Lowe, 1992) although other configurations of the film have been described, such as immobilization by covalent attachment with functionalized polymers (Schalkhammer et al., 1991; Schuhmann & Kittsteiner-Eberle, 1991) or by copolymerization with pyrrole-modified enzyme (Yon-Hin et al., 1993). Recently, approaches to enhance the electron transfer efficiency have been attempted including the coentrapment with an artificial mediator (Kajiya et al., 1991) and the direct electron transfer between enzyme and polypyrrole backbone (Koopal et al., 1992). The condition of preparing the polypyrrolei glucose oxidase (PPy/GOD) biosensor has been known to affect the performance of the resulting biosensor. Therefore, the effects of such parameters as pyrrole and enzyme concentrations in the electrodeposition solution, and film thickness were investigated. Polypyrrole (PPy) has been known to possess a size exclusion property which is effective in suppressing the interference by electro-oxidizable compounds in the sample (Wang et al., 1989; Hammerle et al., 1992; Cooper et al., 1993). However, the size exclusion effect is also dependent on the same parameters as above. In the first part of our work, we found that the enzyme concentration in the deposition solution affects significantly the electrochemical growth of PPy film and consequently the properties of the film. In this context, it is of great interest to investigate in detail the dependence of the analytical performance of the PPy/GOD biosensor on the parameters mentioned above. In this paper, we constructed a PPy/GOD biosensor as a model system and examined the effects of pyrrole concentration, enzyme concentration, and film thickness on the response of the resulting electrode. The condition of preparing the PPy/GOD biosensor was optimized in terms of the sensitivity for glucose and the suppression of interference. EXPERIMENTAL Materials Glucose oxidase (EC 1.1.3.4., type VII-S, 125 U/ mg from Aspergillus niger), /3-D( +)-glucose, 172
Biosensors & Bioelectronics
ascorbate, urate, and acetaminophen were purchased from Sigma Chemical Co. (St. Louis, USA). Pyrrole from Aldrich Chemical Co. was distilled in a vacuum prior to use and stored under nitrogen atmosphere. The platinum rods (99.999%) were purchased from Woosung Steel Co. (Korea) and the polyester from Choil Special Chemistry Industry Co. Ltd. (Korea). The distilled and deionized water used for all solutions was prepared using Autostill@ distiller and ion exchangers (Wheaton, USA). All solutions were prepared from distilled and deionized water. In particular, glucose stock solution was allowed to mutarotate overnight. All other chemicals were of analytical grade and were used without further purification. Instrumentation Voltammetric and amperometric measurements were performed with a potentiostat (EG&G Princeton Applied Research, Model 362). Output from the potentiostat was coupled to an IBM-PC compatible computer using a peripheral interface card (PC-LabCard, PCL 812, Advantech Co., Taiwan). The interface card consisted of a 16-channel analog-todigital (A/D) converters (12 bit) which was set up to receive + 1 V signals. After standardization, the resolution of A/D conversion was found to be 0.49069 mV bitt’. Data display and recording were supported by an operating software programmed in C language. Preparation of enzyme electrode All potentials were referred to an Ag/AgCl electrode (BAS, USA). The counter electrode was a platinum coil or disk. Working electrode was prepared by mounting a platinum rod (dia = 3 mm) in a polyester resin to expose a surface area of 0.070686 cm2. The electrode surfaces were mechanically polished with successively finer grades of diamond slurries and alumina slurry down to 0.3 pm (BAS, USA) and rinsed with distilled water in an ultrasonic cleaner for l-2 min. The electrodes were cleaned electrochemically by cycling the potential in 1 M H2S04 for 20 min between -200 and 1450 mV vs. Agl AgCl prior to use. The electropolymerization was performed in an undivided cell at room temperature using a platinum disk as an auxiliary electrode. The
Biosensors & Bioelectronics
Electrochemical
characterization of polypyrrolelglucose
platinum electrode was placed parallel to the working electrode for the uniform density of current during the electrochemical polymerization. Films were grown potentiostatically at 750 mV vs. Ag/AgCl in a stagnant and deoxygenated solution of 0.1 M KC1 (pH 7.0), containing 0.05 M pyrrole and glucose oxidase. The glucose oxidase concentration in the electrodeposition solution was varied from 0.25 to 5.0 mg ml-‘. The amount of charge transferred during electropolymerization was measured by on-line integration of anodic current according to Euler’s method. The thickness of the film was estimated by assuming that 45 mC cme2 of charge yields a film of 0.1 pm thickness as reported by Holdcroft & Funt (1988). After electropolymerization, the electrode was washed several times with distilled water to remove any loosely bound enzyme and pyrrole monomer, then stored in 0.1 M potassium phosphate buffer (pH 7.0). ElectrochemicaI
measurement
The polypyrrole/glucose oxidase biosensor was maintained at 700 mV vs. Ag/AgCl in 0.1 M potassium phosphate buffer (pH 7.0) and overoxidized in order to stabilize the background current. A platinum coil was used as an auxiliary electrode. The working volume of the electrochemical cell was 2 ml, and temperature was controlled using a thermostat (Laud, Wobblier GmbH, Germany). Unless otherwise stated, all experiments were carried out at 30°C. For the amperometric measurement of glucose and other electro-oxidizable substances, the steady state anodic current was measured at the potential of 700 mV vs. Ag/ AgCl in the 0.1 M potassium phosphate buffer (pH 7.0) after a spike of stock solution, The solution was stirred briefly by means of a micropipette.
RESULTS
oxidase biosensor: Part II
influences of monomer and enzyme concentrations in the electrodeposition solution. All films were grown to the thickness of 50 mC cmp2 to minimize a diffusional barrier, and the steady-state current was examined to 109 mM glucose. As shown in Fig. 1, the response to glucose was highest at 0.05 M pyrrole and gradually decreased with increasing concentration of pyrrole monomer regardless of the enzyme concentration. This result suggests that the electrochemical deposition at high pyrrole concentration does not facilitate the incorporation of enzyme, even though polypyrrole film is formed rapidly within a few minutes. At the pyrrole concentration lower than 0.05 M, the electrochemical polymerization proceeded very slowly, owing to low current density, and the film was hardly obtained. In this work, all films were prepared at the pyrrole concentration of 0.05 M. Another notable feature observed in Fig. 1 is that the PPyIGOD film prepared at high enzyme concentration exhibited highest response to glucose over the whole range of pyrrole concentration tested. This indicates that increase in the enzyme concentration in the electrodeposition solution results in an elevated amount of enzyme incorporated within the film, leading to an increased response, as reported elsewhere (Foulds & Lowe, 1986; Yabuki et al., 1989).
AND DISCUSSION
Effect of pyrrole and enzyme concentrations on the enzyme content within the PPyIGOD film 0.0
0.1
0.2
0.3
Pyrrole concentration
The activity of immobilized enzyme within the film is considered a crucial factor in the construction of PPyIGOD biosensor with high response. In consideration of this, the apparent activity of immobilized enzyme was evaluated by using an electrochemical method, to investigate the
0.4
t 5
(M)
Fig. 1. Effect of pyrrole concentration on the response of the PPyIGOD biosensor. The response was examined for 109 mM glucose. All films were grown to 50 mC crnp2 at different enzyme concentrations. Enzyme concentrations in the electrodeposition solution were: (W) 0.5 mg ml-‘; (A) 1.0 mg ml-‘; (0) 5.0 mg ml-‘. 173
Min-Chol Shin & Hak-Sung
Kim
Biosensors & Bioelectronics
Effect of film thickness on the response of PPy/ GOD biosensor The amperometric response of PPyIGOD biosensor depends on several factors, including the enzyme amount within the polymer film, mass transport of the substrates (glucose and oxygen), products (gluconate and H202) through the polymer layer, and the kinetics of enzymatic reaction. Initially, we investigated the effect of film thickness on the steady-state electrochemical response of the PPy/GOD biosensor prepared at different enzyme concentrations. As shown in Fig. 2, the optimum film thickness which exhibits maximum response was observed at a given enzyme concentration. In the case of the PPy/ GOD biosensor prepared at the enzyme concentration of 0.5 mg ml-‘, the response increased with increasing film thickness (due mainly to the elevated amount of enzyme incorporated in the film) and then decreased after an optimum thickness of 250 mC cmp2. A similar profile of the response was observed with the PPy/GOD biosensor fabricated at the enzyme concentration of 1.0 mg ml-‘, while the optimum thickness was moved down to a thinner region of 150 mC cm-*. In the case of the biosensor prepared at a high enzyme concentration of 5.0 mg mll’, however,
0.40 ,
0.001
0
7
7 100
1
200
7
Film thickness
c 300
’ 400
’
500
(mC/Cm’)
Fig. 2. Effect of film thickness on the response of PPyI GOD biosensor. The response was examined for 10 mM glucose. All films were prepared at different enzyme concentrations. The enzyme concentration in the deposition solution were: (m) 0.5 mg ml-t; (A) 1.0 mg ml-t; (0) 5.0 mg ml-r. The symbols and bars represent the mean and standard deviation of measurements in triplicate, Each experiment was conducted with a freshly prepared biosensors. 174
only a falling trend in the response profile was observed. Decrease in the response observed on the film thicker than the optimum can be explained by the internal diffusion of Hz02 rather than the internal diffusion of glucose. The diffusion of Hz02 within the film has a bidirectional characteristic: (i) the one contributes to the electrochemical response on the surface of the Pt electrode because the overoxidized polypyrrole used in this work is too nonconductive to oxidize H202 at its backbone; (ii) the other diffuses out into the bulk phase, resulting in a signal loss. As glucose diffuses into the inner part of polymer film from the film/solution interface, a local and progressive consumption of glucose influx occurs by the enzymatic reaction of glucose oxidase, assuming that glucose oxidase is distributed evenly throughout the film and there is no kinetic limitation by oxygen as a cosubstrate. At steady-state, the ratio of two opposite-directional fluxes of H202 could depend mainly on the spatial distribution of H202 generation within the film, since H202 is highly diffusible within the film. As the thickness of the film increases beyond the optimum, most of the glucose would be consumed by glucose oxidase before it reaches the surface of the electrode, resulting in a local ‘reaction zone’ and thereafter, ‘depletion zone’ in the film. In this situation, it is possible that H202 is no longer generated throughout the film and most of the H202 is produced within a localized part of the film, near the interface, leading to a large loss of H202 into the bulk and consequent decrease in response. This is consistent with the kinetic studies demonstrating that the enzyme reaction occurs mainly near the film/solution interface and within a small volume of the film near the interface (Bartlett & Whitaker, 1987a, b; Fortier et al., 1990; Janda & Weber, 1991; Marchesilello & Genies, 1992). On the basis of the above observations, it is evident that the optimum thickness is affected by enzyme concentration in the deposition solution. By increasing the film thickness at a given enzyme concentration, the total amount of enzyme within the film would increase, resulting in the depletion of glucose influx at the region closer to the interface and consequently a greater loss of H202 to the bulk. Therefore, the thickness that exhibits maximum response might be shifted down to a thinner region, as the enzyme concentration in the electrodeposition solution increases.
Biosensors & Bioelectronics
Electrochemical
characterization of polypyrrolelghcose
Effect of enzyme concentration on the response of PPy/GOD electrode To verify the influence of enzyme loading within the film to the response, 10 mM glucose was examined at different concentrations of enzyme at a given thickness of 250 mC cm-*. As shown in Fig. 3, maximum response was observed at the film prepared at 0.5 mg ml-’ enzyme concentration, showing a consistent agreement with the result found in Fig. 2. Increase in the response with increasing concentration of enzyme could be attributed to the elevated loading of enzyme within the film. Further increase in the enzyme showed a monotonical reduction in the response, and this might be caused by larger loss of H202 into the bulk phase because the production of H202 took place mostly at the region closer to the film/solution interface. This result reflects that the film thickness and enzyme concentration have an equivalent effect on the response of the PPy/GOD electrode. Effect of film thickness and enzyme concentration on the permeability of PPy/GOD film It is well known (Wang et al., 1989) that polypyrrole possessed size exclusion properties which are effective in suppressing the interference
0.401
I
i
I
I
,,’! 0.00
,, 0.50
1.00 Enzyme
1 150
concentration
2 00
2.50
(mg/ml)
Fig. 3. Effect of enzyme concentration on the response of PPyIGOD biosensor. The response was examined All films were grown to for 10 mM glucose. 250 mC cme2. The symbol and bar represent the mean and standard deviation of measurements in triplicate. Each experiment was conducted with a freshly prepared biosensor.
oxidase biosensor: Part II
by electro-oxidizable compounds in the sample. Size exclusion effect was found to be critically dependent upon the thickness and the permeability of the film. In particular, the film thickness can be controlled by varying the charge transferred during electropolymerization. Figure 4.4 shows the steady-state current for various interferants, and the relative current at PPy-modified electrode decreased to about 10% of its value observed at a bare Pt electrcde as the thickness increased to 400 mC crne2. Moreover, the suppression of interference was in good agreement with the increasing order of molecular size of the compounds: Urate (206.2 da) > Ascorbate (176.1) > Acetaminophen (151.2) Meanwhile, the current to Hz02, which is small and uncharged, was almost independent of the film thickness (data not shown). This observation indicates that the PPjr film ranging from 100 to 400 mC cmp2 can give a significant diffusional barrier against interferants. Because, as mentioned earlier, the permeability of PPy film was found to be affected significantly by the enzyme concentration, the current of PPy/ GOD biosensor was also examined by using the film prepared at different enzyme concentrations. Glucose itself cannot be oxidized on the electrode, therefore it is difficult to measure the film permeability. As an attempt to estimate the permeability, the steady-state current to ascorbate (as a substituent for glucose) was evaluated as a function of film thickness. The use of ascorbate is reasonable when considering that it is of comparable size and hydrophilicity to that of glucose (180.2 da). As shown in Fig. 4B, the response to ascorbate decreased with increasing thickness of the PPy/GOD film, regardless of the enzyme concentration. In addition, at a given thickness of the film, higher response was obtained as the enzyme content within the film became lower, strongly suggesting that the film with higher permeability can be achieved by preparing the polypyrrole film at low enzyme concentration in the electrodeposition solution. The change in the permeability of the film with the enzyme concentration could be attributed to the alteration in the morphology of the film, as described in the first part of our work (Shin & Kim, 1995). 175
Min-Chol Shin & Hak-Sung
Kim
Biosensors & Bioelectronics
Film thickness (mC/cm*)
(:1)
at given optimal conditions, such as the film thickness and the enzyme concentration, the sensitivity for glucose and the suppression of interference by ascorbate were investigated (Fig. 5). In the case of C, the sensitivity was enhanced by 37.6% compared to that of A, even though the film was thicker. Increase in sensitivity might be explained in terms of permeability of the film if the effective diffusivity of glucose in the film is assumed to be similar to that of ascorbate. In other words, higher flux of glucose in the case of C, which results from higher permeability of the film, produces more H202 enzymatically in the inner side of the film, increasing the portion of H202 directed to the electrode surface. Apart from the improvement of sensitivity, the suppression of interfering compounds is also expected by preparing thicker film at lower enzyme concentration, as in the case of C. As previously mentioned, ascorbate is a representative interferant in the amperometric assay of biological fluid. Response to ascorbate in the case of C was reduced by approximately 40% than that of A, mainly due to the effect of film thickness. Figure 6 shows the calibration curve for glucose using the PPyiGOD electrode fabricated under the condition of C. The response of the electrode was linear up to 10 mM glucose. The analytical performance of the PPy/GOD biosensor in terms of sensitivity for glucose and
;‘“-::&-%._ 100
200 300 Film thickness (mC/cm*)
400
Fig. 4. (A) Effect of film thickness on the steady-state current to 5 mM acetaminophen (A), 5 mM ascorbate (m), and 1.25 mM urate (Cl). All films were grown in the absense of enzyme. Current to each compound was normalized to its value obtained with a bare Pt electrode. (B) Effect of film thickness on the response of PPyl GOD biosensor to 5 mM ascorbate. All films were prepared from different enzyme concentrations. The enzyme concentrations in the deposition solution were: (W) 0.5 mg ml-‘; (A) 1.0 mg ml-‘; (0) 5.0 mg ml-‘. Current was normalized to its value obtained with a bare Pt electrode.
Analytical performance of PPy/GOD biosensor In the previous section, the optimal thickness of the film was found to exist, depending on the enzyme concentration in the deposition solution. In addition, polypyrrole film acts as a diffusional barrier against the interferants. To evaluate the analytical performance of the PPy/GOD biosensor 176
nr
r-10
7
Performance at each optimum condition
Fig. 5. Comparison of the performance of PPyIGOD biosensor prepared at each optimal condition: (A), film thickness of 10 mC cmm2 and 5.0 mg ml-’ GOD; (B), film thickness of 150 mC cme2 and I.0 mg ml-t GOD; (C), film thickness of 250 mC crnp2 and 0.5 mg ml-t GOD; Current to ascorbate was normalized to its value obtained with a bare Pt electrode.
Biosensors & Bioelectronics
Electrochemical
characterization of polypyrrolelglucose
oxidase biosensor: Part II
Schmidt, H.-L. (1993). Selectivity of conducting polymer electrodes and their application in flow injection analysis of amino acids. Biosensors & Bioelectron.,
8, 65-74.
Fortier, G., Brassard, E. & Belanger, D. (1990). Optimization of a polypyrrole oxidase biosensor.
600
Biosensors & Bioelectron.,
5, 473-490.
Foulds, N.C. & Lowe, CR. (1986). Enzyme entrapment in electrically conducting polymers. J. Chem. Sot., Faraday Trans., I, 82, 1259-1264.
Hammerle, M., Schumann, W. & Schmidt, H.-L. (1992). Amperometric polypyrrole enzyme electrodes: effect of permeability and enzyme location. Sensors & Actuators, B, 6, 106-112.
Fig. 6. Calibration curve of the PPvIGOD biosensor for glucose. The film was-grown to the thickness of 250 mC crnm2 in the presense of 0.5 mg ml-’ GOD. Insert indicates the calibration curve obtained at lower glucose concentration.
suppression of interference could be improved by proper combinations of the parameters (such as film thickness and enzyme concentration) in the deposition solution. The results observed in this work are expected to make a significant contribution to the design of the PPy/GOD biosensor.
Holdcroft, S. & Funt, B.L. (1988). Preparation and electrocatalytic properties of conducting films of polypyrrole containing platinum microparticles. J. Electroanal. Chem., 240, 89-103.
Janda, P. & Weber, J. (1991). Quinone-mediated glucose electrode with the enzyme immobilized on polypyrrole. J. Electroanal. Chem. 300, 119-127. Kajiya, Y., Sugai, C., Iwakura, C. & Yoneyama, H. (1991). Glucose sensitivity of pyrrole films containing immobilized glucose oxidase and hydroquinonesulphate ions. Anal. Chem., 63, 49-54. Koopal, C.G.J., Feiters, M.C., Nolte, R.J.M., de Ruiter, B. & Schasfoort, R.B.M. (1992). Glucose sensor utilizing polypyrrole incorporated in track-etch membranes as the mediator. Biosensors & Bioelectron.,
7, 461-471.
REFERENCES
Lowe, C.R. (1985). An introduction to the concept and technology of biosensors. Biosensors & Bioelectron., 1, 3-16. Marchesilello, M. & Genies, E.M. (1992). Glucose sensor: polypyrrole-glucose oxidase electrode in the presence of p-benzoquinone. Electrochim.
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., Tebbutt, P. & Tyrrell, C.H. (1992). Electrochemical immobilization of enzymes. 3. Immobilization of glucose oxidase in thin films of electrochemically polymerized phenols. Anal.
Pandey, P.C. (1988). A new conducting polymer-coated glucose sensor. J. Chem. Sot., Faraday Trans. 1, 84, 2259-2265. Schalkhammer, T., Mann-Buxbaun, E., Pittner, F. & Urban, G. (1991). Electrochemical glucose sensors on permselective nonconducting substituted pyrrole polymers. Sensors & Actuators, B, 4, 273-281. Schuhmann, W. & Kittsteiner-Eberle, R. (1991). Evaluation of polypyrrol/glucose oxidase electrodes in flow-injection analysis systems for sucrose determination. Biosensors & Bioelectron.,
Acta, 37, 1987-1992.
Chem., 64, 138-142.
Belanger, D., Nadreau, J. & Fortier, G. (1989). Electrochemistry of the polypyrrole glucose oxidase electrode. J. Electroanal. Chem., 274, 143-155.
Cooper, J.C. & Hall, E.A.H. (1992). Electrochemical response of an enzyme-loaded polyaniline film. Biosensors & Bioelectron.,
Cooper,
J.C.,
Hammerle,
M.,
7, 47-85.
Schuhmann,
W. &
6, 263-273.
Shin, M.-C. & Kim, H.-S. (1995). Electrochemical characterization of polypyrrole/glucose oxidase biosensor: Part I. Influence of enzyme concentration on the growth and properties of the film. Biosensors & Bioelectron. (accepted). Umaria, M. & Wailer, J. (1986). Protein-modified electrodes. The glucose oxidase/polypyrrole system. Anal. Chem., 58, 2979-2983. 177
Min-Chol Shin & Hak-Sung
Wang, J., Chen, S.-P. & Lin, MS. (1989). Use of different electropolymerization conditions for controlling the size-exclusion selectivity at polyaniline, polypyrrole and polyphenol films. J. Electroanal. Chem. , 273, 23 I-242. Yabuki, S., Shinohara, H. & Aizawa, M. (1989). Electroconductive enzyme membrane. J. Chem. Sot.,
Yon-Hin,
178
Chem. Commun.,
Biosensors & Bioelectronics
Kim
945-946.
B.F.Y. &Lowe, CR. (1992). Amperometric
response of polypyrrole entrapped bienzyme films. Sensors & Actuators, B, 7, 339-342.
Yon-Hin, B.F.Y., Smolander, M., Crompton, T. & Lowe, C.R. (1993). Covalent electropolymerization of glucose oxidase in pyrrole attachment to glucose oxidase on the performance of electropolymerized glucose sensors. Anal. Chem., 65, 2067-2071.
ELSEVIER ADVANCED TECHNOLOGY
Electrochemical characterization of polypyrrolelglucose oxidase biosensor: Part II. Optimal preparation conditions for the biosensor Min-Chol Department
Shin & Hak-Sung
Kim*
of Biotechnology,
Korea Advanced Institute of Science & Technology, 373-1, Kusung-dong, Yusung-ku, Taejon, 305-701, Korea. Tel: [82]-42-869-2616 Fax: [82]-42-869-2610 E-Mail: [email protected]. (Received
2 August 1994; revised 29 March 1995; accepted 3 April 1995)
Abstract:
The optimal conditions for the preparation of a polypyrrole/glucose oxidase biosensor were investigated. Response of the resulting biosensor was found to be critically dependent on preparation parameters such as pyrrole and enzyme concentrations in the electrodeposition solution, and the film thickness. It was also observed that the film thickness and the enzyme concentration have an equivalent effect on the response. Our results indicate that the analytical performance of the resulting biosensor can be improved by choosing the proper set of preparation parameters in terms of the sensitivity for glucose and the suppression of interference. Keywords: amperometric polypyrrole.
biosensor,
INTRODUCTION The
biosensor
has attracted
considerable
atten-
tion as the potential successor to a wide range of analytical techniques owing to its unique characteristic of specificity. The main feature of biosensors can be defined as the spatial unity of biological elements with an appropriate transducer (Lowe, 1985). With current emphasis on the miniaturization and mass production of biosensors, the efficient and reproducible immobilization of biological elements is regarded
* To whom correspondence
should be addressed
analytical performance,
glucose oxidase,
as one of the most important factors for design and development. Electrochemical immobilization within conducting polymers provides a promising alternative for the one-step loadings of biological elements over a small and defined area of the transducer element. The amount and spatial distribution of enzymes within the polymer grown on the electrode can be easily controlled by electrochemical immobilization. Extensive studies have shown that redox enzymes may be incorporated into polypyrrole (Foulds & Lowe, 1986; Umana & Waller, 1986), poly(N-methylpyrrole) (Bartlett & Whitaker, 1987b), polyaniline (Cooper & Hall, 1992), polyindole (Pandey, 1988), and polyphenol (Bartlett et al., 1992) films during electrochemical 171
Min-Chol
Shin & Hak-Sung
Kim
polymerizationof corresponding monomer in the presence of enzymes. Most research to date has concentrated on the physical entrapment of enzymes within electrochemically grown polypyrrole (B&anger et al., 1989; Fortier et al., 1990; Yon-Hin & Lowe, 1992) although other configurations of the film have been described, such as immobilization by covalent attachment with functionalized polymers (Schalkhammer et al., 1991; Schuhmann & Kittsteiner-Eberle, 1991) or by copolymerization with pyrrole-modified enzyme (Yon-Hin et al., 1993). Recently, approaches to enhance the electron transfer efficiency have been attempted including the coentrapment with an artificial mediator (Kajiya et al., 1991) and the direct electron transfer between enzyme and polypyrrole backbone (Koopal et al., 1992). The condition of preparing the polypyrrolei glucose oxidase (PPy/GOD) biosensor has been known to affect the performance of the resulting biosensor. Therefore, the effects of such parameters as pyrrole and enzyme concentrations in the electrodeposition solution, and film thickness were investigated. Polypyrrole (PPy) has been known to possess a size exclusion property which is effective in suppressing the interference by electro-oxidizable compounds in the sample (Wang et al., 1989; Hammerle et al., 1992; Cooper et al., 1993). However, the size exclusion effect is also dependent on the same parameters as above. In the first part of our work, we found that the enzyme concentration in the deposition solution affects significantly the electrochemical growth of PPy film and consequently the properties of the film. In this context, it is of great interest to investigate in detail the dependence of the analytical performance of the PPy/GOD biosensor on the parameters mentioned above. In this paper, we constructed a PPy/GOD biosensor as a model system and examined the effects of pyrrole concentration, enzyme concentration, and film thickness on the response of the resulting electrode. The condition of preparing the PPy/GOD biosensor was optimized in terms of the sensitivity for glucose and the suppression of interference. EXPERIMENTAL Materials Glucose oxidase (EC 1.1.3.4., type VII-S, 125 U/ mg from Aspergillus niger), /3-D( +)-glucose, 172
Biosensors & Bioelectronics
ascorbate, urate, and acetaminophen were purchased from Sigma Chemical Co. (St. Louis, USA). Pyrrole from Aldrich Chemical Co. was distilled in a vacuum prior to use and stored under nitrogen atmosphere. The platinum rods (99.999%) were purchased from Woosung Steel Co. (Korea) and the polyester from Choil Special Chemistry Industry Co. Ltd. (Korea). The distilled and deionized water used for all solutions was prepared using Autostill@ distiller and ion exchangers (Wheaton, USA). All solutions were prepared from distilled and deionized water. In particular, glucose stock solution was allowed to mutarotate overnight. All other chemicals were of analytical grade and were used without further purification. Instrumentation Voltammetric and amperometric measurements were performed with a potentiostat (EG&G Princeton Applied Research, Model 362). Output from the potentiostat was coupled to an IBM-PC compatible computer using a peripheral interface card (PC-LabCard, PCL 812, Advantech Co., Taiwan). The interface card consisted of a 16-channel analog-todigital (A/D) converters (12 bit) which was set up to receive + 1 V signals. After standardization, the resolution of A/D conversion was found to be 0.49069 mV bitt’. Data display and recording were supported by an operating software programmed in C language. Preparation of enzyme electrode All potentials were referred to an Ag/AgCl electrode (BAS, USA). The counter electrode was a platinum coil or disk. Working electrode was prepared by mounting a platinum rod (dia = 3 mm) in a polyester resin to expose a surface area of 0.070686 cm2. The electrode surfaces were mechanically polished with successively finer grades of diamond slurries and alumina slurry down to 0.3 pm (BAS, USA) and rinsed with distilled water in an ultrasonic cleaner for l-2 min. The electrodes were cleaned electrochemically by cycling the potential in 1 M H2S04 for 20 min between -200 and 1450 mV vs. Agl AgCl prior to use. The electropolymerization was performed in an undivided cell at room temperature using a platinum disk as an auxiliary electrode. The
Biosensors & Bioelectronics
Electrochemical
characterization of polypyrrolelglucose
platinum electrode was placed parallel to the working electrode for the uniform density of current during the electrochemical polymerization. Films were grown potentiostatically at 750 mV vs. Ag/AgCl in a stagnant and deoxygenated solution of 0.1 M KC1 (pH 7.0), containing 0.05 M pyrrole and glucose oxidase. The glucose oxidase concentration in the electrodeposition solution was varied from 0.25 to 5.0 mg ml-‘. The amount of charge transferred during electropolymerization was measured by on-line integration of anodic current according to Euler’s method. The thickness of the film was estimated by assuming that 45 mC cme2 of charge yields a film of 0.1 pm thickness as reported by Holdcroft & Funt (1988). After electropolymerization, the electrode was washed several times with distilled water to remove any loosely bound enzyme and pyrrole monomer, then stored in 0.1 M potassium phosphate buffer (pH 7.0). ElectrochemicaI
measurement
The polypyrrole/glucose oxidase biosensor was maintained at 700 mV vs. Ag/AgCl in 0.1 M potassium phosphate buffer (pH 7.0) and overoxidized in order to stabilize the background current. A platinum coil was used as an auxiliary electrode. The working volume of the electrochemical cell was 2 ml, and temperature was controlled using a thermostat (Laud, Wobblier GmbH, Germany). Unless otherwise stated, all experiments were carried out at 30°C. For the amperometric measurement of glucose and other electro-oxidizable substances, the steady state anodic current was measured at the potential of 700 mV vs. Ag/ AgCl in the 0.1 M potassium phosphate buffer (pH 7.0) after a spike of stock solution, The solution was stirred briefly by means of a micropipette.
RESULTS
oxidase biosensor: Part II
influences of monomer and enzyme concentrations in the electrodeposition solution. All films were grown to the thickness of 50 mC cmp2 to minimize a diffusional barrier, and the steady-state current was examined to 109 mM glucose. As shown in Fig. 1, the response to glucose was highest at 0.05 M pyrrole and gradually decreased with increasing concentration of pyrrole monomer regardless of the enzyme concentration. This result suggests that the electrochemical deposition at high pyrrole concentration does not facilitate the incorporation of enzyme, even though polypyrrole film is formed rapidly within a few minutes. At the pyrrole concentration lower than 0.05 M, the electrochemical polymerization proceeded very slowly, owing to low current density, and the film was hardly obtained. In this work, all films were prepared at the pyrrole concentration of 0.05 M. Another notable feature observed in Fig. 1 is that the PPyIGOD film prepared at high enzyme concentration exhibited highest response to glucose over the whole range of pyrrole concentration tested. This indicates that increase in the enzyme concentration in the electrodeposition solution results in an elevated amount of enzyme incorporated within the film, leading to an increased response, as reported elsewhere (Foulds & Lowe, 1986; Yabuki et al., 1989).
AND DISCUSSION
Effect of pyrrole and enzyme concentrations on the enzyme content within the PPyIGOD film 0.0
0.1
0.2
0.3
Pyrrole concentration
The activity of immobilized enzyme within the film is considered a crucial factor in the construction of PPyIGOD biosensor with high response. In consideration of this, the apparent activity of immobilized enzyme was evaluated by using an electrochemical method, to investigate the
0.4
t 5
(M)
Fig. 1. Effect of pyrrole concentration on the response of the PPyIGOD biosensor. The response was examined for 109 mM glucose. All films were grown to 50 mC crnp2 at different enzyme concentrations. Enzyme concentrations in the electrodeposition solution were: (W) 0.5 mg ml-‘; (A) 1.0 mg ml-‘; (0) 5.0 mg ml-‘. 173
Min-Chol Shin & Hak-Sung
Kim
Biosensors & Bioelectronics
Effect of film thickness on the response of PPy/ GOD biosensor The amperometric response of PPyIGOD biosensor depends on several factors, including the enzyme amount within the polymer film, mass transport of the substrates (glucose and oxygen), products (gluconate and H202) through the polymer layer, and the kinetics of enzymatic reaction. Initially, we investigated the effect of film thickness on the steady-state electrochemical response of the PPy/GOD biosensor prepared at different enzyme concentrations. As shown in Fig. 2, the optimum film thickness which exhibits maximum response was observed at a given enzyme concentration. In the case of the PPy/ GOD biosensor prepared at the enzyme concentration of 0.5 mg ml-‘, the response increased with increasing film thickness (due mainly to the elevated amount of enzyme incorporated in the film) and then decreased after an optimum thickness of 250 mC cmp2. A similar profile of the response was observed with the PPy/GOD biosensor fabricated at the enzyme concentration of 1.0 mg ml-‘, while the optimum thickness was moved down to a thinner region of 150 mC cm-*. In the case of the biosensor prepared at a high enzyme concentration of 5.0 mg mll’, however,
0.40 ,
0.001
0
7
7 100
1
200
7
Film thickness
c 300
’ 400
’
500
(mC/Cm’)
Fig. 2. Effect of film thickness on the response of PPyI GOD biosensor. The response was examined for 10 mM glucose. All films were prepared at different enzyme concentrations. The enzyme concentration in the deposition solution were: (m) 0.5 mg ml-t; (A) 1.0 mg ml-t; (0) 5.0 mg ml-r. The symbols and bars represent the mean and standard deviation of measurements in triplicate, Each experiment was conducted with a freshly prepared biosensors. 174
only a falling trend in the response profile was observed. Decrease in the response observed on the film thicker than the optimum can be explained by the internal diffusion of Hz02 rather than the internal diffusion of glucose. The diffusion of Hz02 within the film has a bidirectional characteristic: (i) the one contributes to the electrochemical response on the surface of the Pt electrode because the overoxidized polypyrrole used in this work is too nonconductive to oxidize H202 at its backbone; (ii) the other diffuses out into the bulk phase, resulting in a signal loss. As glucose diffuses into the inner part of polymer film from the film/solution interface, a local and progressive consumption of glucose influx occurs by the enzymatic reaction of glucose oxidase, assuming that glucose oxidase is distributed evenly throughout the film and there is no kinetic limitation by oxygen as a cosubstrate. At steady-state, the ratio of two opposite-directional fluxes of H202 could depend mainly on the spatial distribution of H202 generation within the film, since H202 is highly diffusible within the film. As the thickness of the film increases beyond the optimum, most of the glucose would be consumed by glucose oxidase before it reaches the surface of the electrode, resulting in a local ‘reaction zone’ and thereafter, ‘depletion zone’ in the film. In this situation, it is possible that H202 is no longer generated throughout the film and most of the H202 is produced within a localized part of the film, near the interface, leading to a large loss of H202 into the bulk and consequent decrease in response. This is consistent with the kinetic studies demonstrating that the enzyme reaction occurs mainly near the film/solution interface and within a small volume of the film near the interface (Bartlett & Whitaker, 1987a, b; Fortier et al., 1990; Janda & Weber, 1991; Marchesilello & Genies, 1992). On the basis of the above observations, it is evident that the optimum thickness is affected by enzyme concentration in the deposition solution. By increasing the film thickness at a given enzyme concentration, the total amount of enzyme within the film would increase, resulting in the depletion of glucose influx at the region closer to the interface and consequently a greater loss of H202 to the bulk. Therefore, the thickness that exhibits maximum response might be shifted down to a thinner region, as the enzyme concentration in the electrodeposition solution increases.
Biosensors & Bioelectronics
Electrochemical
characterization of polypyrrolelghcose
Effect of enzyme concentration on the response of PPy/GOD electrode To verify the influence of enzyme loading within the film to the response, 10 mM glucose was examined at different concentrations of enzyme at a given thickness of 250 mC cm-*. As shown in Fig. 3, maximum response was observed at the film prepared at 0.5 mg ml-’ enzyme concentration, showing a consistent agreement with the result found in Fig. 2. Increase in the response with increasing concentration of enzyme could be attributed to the elevated loading of enzyme within the film. Further increase in the enzyme showed a monotonical reduction in the response, and this might be caused by larger loss of H202 into the bulk phase because the production of H202 took place mostly at the region closer to the film/solution interface. This result reflects that the film thickness and enzyme concentration have an equivalent effect on the response of the PPy/GOD electrode. Effect of film thickness and enzyme concentration on the permeability of PPy/GOD film It is well known (Wang et al., 1989) that polypyrrole possessed size exclusion properties which are effective in suppressing the interference
0.401
I
i
I
I
,,’! 0.00
,, 0.50
1.00 Enzyme
1 150
concentration
2 00
2.50
(mg/ml)
Fig. 3. Effect of enzyme concentration on the response of PPyIGOD biosensor. The response was examined All films were grown to for 10 mM glucose. 250 mC cme2. The symbol and bar represent the mean and standard deviation of measurements in triplicate. Each experiment was conducted with a freshly prepared biosensor.
oxidase biosensor: Part II
by electro-oxidizable compounds in the sample. Size exclusion effect was found to be critically dependent upon the thickness and the permeability of the film. In particular, the film thickness can be controlled by varying the charge transferred during electropolymerization. Figure 4.4 shows the steady-state current for various interferants, and the relative current at PPy-modified electrode decreased to about 10% of its value observed at a bare Pt electrcde as the thickness increased to 400 mC crne2. Moreover, the suppression of interference was in good agreement with the increasing order of molecular size of the compounds: Urate (206.2 da) > Ascorbate (176.1) > Acetaminophen (151.2) Meanwhile, the current to Hz02, which is small and uncharged, was almost independent of the film thickness (data not shown). This observation indicates that the PPjr film ranging from 100 to 400 mC cmp2 can give a significant diffusional barrier against interferants. Because, as mentioned earlier, the permeability of PPy film was found to be affected significantly by the enzyme concentration, the current of PPy/ GOD biosensor was also examined by using the film prepared at different enzyme concentrations. Glucose itself cannot be oxidized on the electrode, therefore it is difficult to measure the film permeability. As an attempt to estimate the permeability, the steady-state current to ascorbate (as a substituent for glucose) was evaluated as a function of film thickness. The use of ascorbate is reasonable when considering that it is of comparable size and hydrophilicity to that of glucose (180.2 da). As shown in Fig. 4B, the response to ascorbate decreased with increasing thickness of the PPy/GOD film, regardless of the enzyme concentration. In addition, at a given thickness of the film, higher response was obtained as the enzyme content within the film became lower, strongly suggesting that the film with higher permeability can be achieved by preparing the polypyrrole film at low enzyme concentration in the electrodeposition solution. The change in the permeability of the film with the enzyme concentration could be attributed to the alteration in the morphology of the film, as described in the first part of our work (Shin & Kim, 1995). 175
Min-Chol Shin & Hak-Sung
Kim
Biosensors & Bioelectronics
Film thickness (mC/cm*)
(:1)
at given optimal conditions, such as the film thickness and the enzyme concentration, the sensitivity for glucose and the suppression of interference by ascorbate were investigated (Fig. 5). In the case of C, the sensitivity was enhanced by 37.6% compared to that of A, even though the film was thicker. Increase in sensitivity might be explained in terms of permeability of the film if the effective diffusivity of glucose in the film is assumed to be similar to that of ascorbate. In other words, higher flux of glucose in the case of C, which results from higher permeability of the film, produces more H202 enzymatically in the inner side of the film, increasing the portion of H202 directed to the electrode surface. Apart from the improvement of sensitivity, the suppression of interfering compounds is also expected by preparing thicker film at lower enzyme concentration, as in the case of C. As previously mentioned, ascorbate is a representative interferant in the amperometric assay of biological fluid. Response to ascorbate in the case of C was reduced by approximately 40% than that of A, mainly due to the effect of film thickness. Figure 6 shows the calibration curve for glucose using the PPyiGOD electrode fabricated under the condition of C. The response of the electrode was linear up to 10 mM glucose. The analytical performance of the PPy/GOD biosensor in terms of sensitivity for glucose and
;‘“-::&-%._ 100
200 300 Film thickness (mC/cm*)
400
Fig. 4. (A) Effect of film thickness on the steady-state current to 5 mM acetaminophen (A), 5 mM ascorbate (m), and 1.25 mM urate (Cl). All films were grown in the absense of enzyme. Current to each compound was normalized to its value obtained with a bare Pt electrode. (B) Effect of film thickness on the response of PPyl GOD biosensor to 5 mM ascorbate. All films were prepared from different enzyme concentrations. The enzyme concentrations in the deposition solution were: (W) 0.5 mg ml-‘; (A) 1.0 mg ml-‘; (0) 5.0 mg ml-‘. Current was normalized to its value obtained with a bare Pt electrode.
Analytical performance of PPy/GOD biosensor In the previous section, the optimal thickness of the film was found to exist, depending on the enzyme concentration in the deposition solution. In addition, polypyrrole film acts as a diffusional barrier against the interferants. To evaluate the analytical performance of the PPy/GOD biosensor 176
nr
r-10
7
Performance at each optimum condition
Fig. 5. Comparison of the performance of PPyIGOD biosensor prepared at each optimal condition: (A), film thickness of 10 mC cmm2 and 5.0 mg ml-’ GOD; (B), film thickness of 150 mC cme2 and I.0 mg ml-t GOD; (C), film thickness of 250 mC crnp2 and 0.5 mg ml-t GOD; Current to ascorbate was normalized to its value obtained with a bare Pt electrode.
Biosensors & Bioelectronics
Electrochemical
characterization of polypyrrolelglucose
oxidase biosensor: Part II
Schmidt, H.-L. (1993). Selectivity of conducting polymer electrodes and their application in flow injection analysis of amino acids. Biosensors & Bioelectron.,
8, 65-74.
Fortier, G., Brassard, E. & Belanger, D. (1990). Optimization of a polypyrrole oxidase biosensor.
600
Biosensors & Bioelectron.,
5, 473-490.
Foulds, N.C. & Lowe, CR. (1986). Enzyme entrapment in electrically conducting polymers. J. Chem. Sot., Faraday Trans., I, 82, 1259-1264.
Hammerle, M., Schumann, W. & Schmidt, H.-L. (1992). Amperometric polypyrrole enzyme electrodes: effect of permeability and enzyme location. Sensors & Actuators, B, 6, 106-112.
Fig. 6. Calibration curve of the PPvIGOD biosensor for glucose. The film was-grown to the thickness of 250 mC crnm2 in the presense of 0.5 mg ml-’ GOD. Insert indicates the calibration curve obtained at lower glucose concentration.
suppression of interference could be improved by proper combinations of the parameters (such as film thickness and enzyme concentration) in the deposition solution. The results observed in this work are expected to make a significant contribution to the design of the PPy/GOD biosensor.
Holdcroft, S. & Funt, B.L. (1988). Preparation and electrocatalytic properties of conducting films of polypyrrole containing platinum microparticles. J. Electroanal. Chem., 240, 89-103.
Janda, P. & Weber, J. (1991). Quinone-mediated glucose electrode with the enzyme immobilized on polypyrrole. J. Electroanal. Chem. 300, 119-127. Kajiya, Y., Sugai, C., Iwakura, C. & Yoneyama, H. (1991). Glucose sensitivity of pyrrole films containing immobilized glucose oxidase and hydroquinonesulphate ions. Anal. Chem., 63, 49-54. Koopal, C.G.J., Feiters, M.C., Nolte, R.J.M., de Ruiter, B. & Schasfoort, R.B.M. (1992). Glucose sensor utilizing polypyrrole incorporated in track-etch membranes as the mediator. Biosensors & Bioelectron.,
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