RiosensorsL

~i~l~ni~

5 (1990) 473-490

Optimization of a Polypyrrole Glucose Oxidase Biosensor

G. Fortier, E. Brassard & D. Bklanger Groupe de recherche en enzymoiogie fondamentaie et appliqde (GREFA), D6partment de Chimie, Universith du Quebec BMontreal, C.P. 8888, Succ. A, Montrkal, Quebec, Canada, H3C 3P8 (Received 4 December 1989; revised version received 28 February 1990; accepted 3 March 1990)

An urn~~rnern-~glucose biosensor wasfab~~at~ by the ele&t~hemical po~rn~-ration of pyrrole onto a platinum electrodein the presence of the enzyme glucose oxidase in a KC1 solution at a potential of + 0 65 V versus SCE. The enzyme was entrapped into the polypyrrole film during the eiectropolymeri~ation process.Glucoseresponsesweremeasuredbypotentiostating the enzyme electrodeat a potential of + 0 7 V versusSCE in order to oxidize the hydrogengen~ated by the oxidation of glucoseby the enzyme in thepresenceof oxygen.Experimentswerepe.$ormedto determinethe optimal conditions of the polypyrrole glucose oxidase jlrn preparation (pywole and glucose oxidase concentrationsin the plating solution) and the responseto glucosefim such electrodeswas evaluatedas afinction ofjlm thickness,pH and temperature.It wasfound that a concentrationof 0 3 M pyrrole in the ~r~en~e of 65 U/~l ofglucose oxidase in 0. OI M KC1 were the optimal parameters for the fubrication of the biosensor, The optimal response was obtainedfor aJirm thickness of 0*17pm (75 mC/cm”, atpH 6 and at a temperatureof 313 K. The temperaturedependenceof the ampetvmetric responseindicated an activation energy of 41 kXmole. The linearity of the enzyme electrode response rang~~om I*0 mu to 7.5 mM glucose and kinetic parameters d~e~i~ed for the optimize biose~o~ were 33.4 mMfor the K, and 7-2 ItAfor the I,, It was demonstratedthat the internal dtfision of hydrogenperoxide through the polypyrtole layer to the 473 Biosmsom & Bioel&mnim 095~5663/90/$03.50 0 1990 Elsevier Science publishers Ltd, England Printed in Great Britain.

474

G. Fortier, E. Brassard & D. BtYanger platinum surface was the main limiting factor controlling the magnitude of the responseof the biosensorto glucose. The responsewasdirectlyrelatedto the enzyme loadingin the polypyrrolefilm. The sherflifeand the operational stability of the optimized biosensor exceed 500 days and 175 assays,

respectively.The substratespecificityof the entrappedglucoseoxidasewasnot altered by the immobilizationprocedure.

Key words: polypyrrole, amperometric, biosensor

glucose

oxidase,

enzyme

electrode,

INTRODUCTION Research in the field of enzyme electrodes is expanding very rapidly (Turner ec al., 1987) since the development of a glucose biosensor is of particular interest in biomedical analysis (Czaban, 1985). The determination of glucose in a variety of physiological fluids is one of the most frequent analyses performed in a clinical chemistry laboratory and the methods used for glucose analysis have been reviewed (Wingard, 1983; Turner et al., 1987). In addition, the self monitoring of blood glucose levels at home by diabetic patients is essential and critical for their insulin management (Skyler et al., 1978). Many of the methods for glucose evaluation in the presence of oxygen involve the soluble enzyme glucose oxidase that catalyzes the oxidation of glucose to gluconic acid and hydrogen peroxide. The reaction product, hydrogen peroxide, can be determined by the enzyme peroxidase that catalyzes the transfer of oxygen from hydrogen peroxide to a chromogenic oxygen acceptor such as o-toluidine (Blaedel & Hicks, 1962) odianisidine (Malmstad & Hicks, 1960) or 4-aminoantipyrine (Gallati, 1977). Hydrogen peroxide production can also be followed directly by the use of an electrochemical transducer (Gronow et al., 1985). Alternatively the immobilization of an enzyme into an inert matrix in such a way that its catalytic activity is retained has been described extensively over the years (Scouten, 1987). The basic design of a glucose oxidase sensor is essentially that glucose oxidase is immobilized in a polymer matrix and placed either onto a fiber optic (Abdel-Latif et aZ.,1988), an integrated-circuit temperature sensitive device (Muramatsu et al., 1987), a field effect transistor (Caras et al., 1985) or the surface of a platinum electrode (Updike & Hicks, 1967; Guilbault & Lubrano, 1973; Foulds & Lowe, 1986; Fortier et al., 1988). With these devices, glucose is quantified by measuring the color change (Abdel-Latif et al., 1988), the emitted light (Abdel-latif & Guilbault, 1988), the pH change (Caras d al.,

Optimization of a polypynoie glucose oxidase biosensor

475

1985), the temperature change (Muramatsu et al., 1987), the voltage produced (Updike & Hicks, 1967; Wingard d al., 1982) or the current generated (Guilbault & Lubrano, 1973; Foulds & Lowe, 1986; Fortier et al., 1988) when the reaction between the substrate, oxygen and the enzyme occurs. In the present paper, the description of an optimized procedure for the fabrication of a platinum/polypyrrole/glucose oxidase (Pt/PP/GOD) biosensor is described. It is well known that the electrochemical oxidation of pyrrole in aqueous solution produces an adherent ~nduc~g polymer coating at an electrode surface (~avapi~yanont et al., 1984). Recently, it was demonstrated that the amperometric response of such a polypyrrole enzyme electrode to glucose, as evaluated by measuring the amperometric oxidation of the hydrogen peroxide, was observed only when the polypyrrole film had lost its electroactivity (Belanger et al., 1989) and can be increased by incorporation of microparticles of platinum into the polymer fdm (Bellanger et al., 1990). Despite the loss of the conducting nature of the polypyrrole coating, the electrochemical preparation of such an enzyme/polymer electrode remains a very convenient technique since it is a simple one-step procedure. The effect of varying the con~ntmtion of the enzyme and of the pyrrole during the preparation of the Pt/PP/GOD electrode was investigated and also the effect of temperature, pH and film thickness on the electrode response. A calibration curve for glucose obtained with the optimized electrode and data concerning the shelf life, the operational stability and the substrate specificity are also presented. MATERIALS AND METHODS Reagents

Glucose oxidase, type VII-S (EC No. 1.1.3.4),was purchased from the Sigma Chemical (St Louis, USA) and the pyrrole from Aldrich (St Louis, USA). The pyrrole was distilled daily. The glucose solution was prepared in distilled water, filtered through a 0.22pm filter (Millipore, USA) and left at room temperature for 24 h before use to ensure the presence of the /&D-glucose form. Enzyme electrode fabrication The platinum electrode was fabricated as previously described (Fortier et al., 1988). Pt disc electrodes were cleaned according to standard

416

G. Fortier, E. Brassard & D. Bdanger

procedure (Gileadi et al., 1975) by anodic and cathodic treatments and polished with successively finer grades of diamond polishing compounds and aqueous alumina slurry down to O-5 pm (Buehler Ltd, USA). Electrochemical polymerization was performed in a one-compartment cell at room temperature with the platinum disc electrode (O-28 cm3 and a platinum flag as working and auxiliary electrodes respectively. To 2 ml of 10 mM degassed KC1 solution, pH 6, was added 0.2-O-7 M of freshly distilled pyrrole and 2.5-1250 U of GOD. PP/GOD films were grown potentiostatically at + 0.65 V versus SCE (saturated calomel electrode). The thickness of the films was controlled by the amount of charge passed during the electropolymerization procedure. The total charge, ranging from 3.2 to 820 mC/cm*, was evaluated from the area under the anodic current-time trace recorded during the deposition. Thereafter, the film thicknesses were estimated by assuming that 45 mC/cm* of charge yielded a film of 0.1 pm thickness (Holdcroft & Funt, 1988). As example, the times required for the growth of polypyrrole films of 0.1 and 0.3 pm thicknesses are 5 and 12 min, respectively. The electrode was washed for 3 min in PBS (phosphate buffered salts containing 125 mM NaCl, 2.7 mM KC1 and 10 mM phosphate buffer) solution, pH 7.5, under mild stirring to eliminate the enzyme which bound weakly to the glass and to the epoxy resin. Cyclic voltammetry was performed in 0.1 M aqueous KC1 to evaluate the state of electroactivity of the polypyrrole films. The electrode was stored in PBS solution at 277 K. Determination of glucose The amperometric response to glucose was evaluated in 5 ml of O-1M, pH 7, PBS solution by holding the enzyme electrode at the required potential of + 0.7 V versus SCE. The electrode was kept under gentle stirring at 200 rpm using a microprocessor-controlled stirring plate until the background current stabilized to a small constant value. The required amount of glucose stock solution was injected into the electrochemical cell and the current-time response was recorded over a 5 min period. The steady-state current was usually reached after 15 s to 1 min depending on the film thickness. The electrode was removed from the solution and washed with fresh PBS solution in order to be used for another assay. Instrumentation All electrochemical experiments were carried out in a conventional onecompartment cell. Potentials were applied to the cell with a bi-

Optimizationof a polypynole glucose oxidase biosensor

471

potentiostat (Pine Instruments Inc., USA) model RDE4. Current-time responses and cyclic voltammograms were recorded on a XYY’ recorder (Kipp & Zonen, USA) model BD91 equipped with a time base module. All potentials were measured and quoted against a saturated calomel electrode. Kinetic analysis The kinetic parameters, K, and ImaX,were obtained by iterative nonlinear curve fitting of raw data (current generated versus the glucose concentration) using the algorithm of Marquard available on EnzFitter software (Elsevier publ). The data fitted a modified Michaelis-Menten equation:

(1) where 1, is the observed current and I,,,,, is the maximal value for the current response. In addition, mass transport limitation of substrates (Horvath & Engasser, 1974; Goldstein, 1976) was determined qualitatively by deviation of the linearity on a Lineweaver-Burk plot (Lineweaver & Burk, 1934).

RESULTS AND DISCUSSION Cyclic voltammetry of the Pt/PP/GOD electrode Figure 1 shows cyclic voltammograms for the Pt/PP/GOD electrode obtained in O-1 M KC1 immediately after the electrochemical deposition and after a glucose assay at +0*7 V. The cyclic voltammogram for the asdeposited film () was slightly different from those previously reported for a polypyrrole/Clelectrode (Shimidzu et al., 1987; Hirai et al., 1988), indicating that glucose oxidase had some effect on the cyclic voltammogram. One of the slight differences, a redox wave at -0.48 V, is though to be due to polypyrrole units that are charge compensated with irreversibly oxidized polypyrrole units. This effect cannot be related to the flavine adenine dinucleotide (FAD), the cofactor of GOD, originating from denatured enzyme because the cyclic voltammogram is unaffected when the enzyme electrode is soaked in 0.5 mM FAD in KC1 solution (BClanger et al., 1989). Prior to the glucose assay, the electrode was potentiostated at +0.7 V and the large initial background current decayed rapidly during the first minute and then slowly reached a small stable value. Following the assay, the electroactivity of the polypyrrole film was lost as shown in Fig. 1 (- - --) despite the fact that

418

G. Fortier, E. Brassard % D. Bdanger I

I

I

I

I

I

I

I

.; 2

I

5 6

I -08

I

I

I -04 btential

I I 0 I V (versus SCE )

I 0.4

640pkm2 I

Fig. 1. Cyclic voltammograms for a Pt/PP/GOD electrode obtained in 0.1 M KC1 solution for the as-deposited film () and after glucose assay at a potential of 0.7 V (- - -). The scan rate was 100 mV/s.

reproducible responses to glucose were still obtained The voltammogram was identical to those obtained with a bare platinum electrode but smaller currents were observed. The lost of electroactivity has been related to a massive oxidation of the polypyrrole film which is now acting as an insulating film (Btlanger et al., 1989), reducing the effective electrode area, thus completely inhibiting the occurrence of a polypyrrole redox reaction (Asavapiriyanont d aZ., 1984). A full characterization of the electrochemical behaviour and the massive electrical and chemical oxidation of the polypyrrole film is essential in order to obtain a functional PP-based biosensor (Belanger et al., 1989). Effect of pyrrole concentration on the response of the enzyme electrode The effect of the pyrrole concentration in the electrodeposition solution, used to prepare the Pt/PP/GOD electrodes, on their amperometric responses to glucose is presented in Fig. 2. The total charge deposited on the platinum surface is 270 mC/cm* and corresponds to a film thickness of about 0.6pm. The electrode responses to 20 mM glucose were evaluated at +O-7 V and the current values at steady-state were used. The optimal concentration of pyrrole was O-3 M as indicated by the maximum current response observed (l-7 ,uA). At a lower concentration of 0.1 M pyrrole, very little current was passed and the film deposition did not occur in a reasonable period of time, i.e. less than half an hour. No

Optimizationof a polypyrroleglucose oxidase biosensor

479

15? 2 12E 0 t 3 09v

O3,_

Pyrrok (Ivl)

Fig. 2. The effect of pyrrole concentration on the steady-state response of the glucose electrode. The electrodes were prepared by electrodeposition at 0.65 V with varying concentrations of pyrrole (0.247 M) in the presence of 500 U/ml glucose oxidase in a solution of 10 mM KCl, pH 6. The charge deposited was 270 mC/cm2. The responses to 20 mM glucose were evaluated at a potential of @7 V in phosphate buffer, pH 7.

attempts were made to grow the film under these conditions. At concentrations higher than O-7 M pyrrole, a precipitate was formed in the aqueous solution and such solutions were not used to grow PP/GOD films due to their non-homogeneity. Thus, for all subsequent experiments, a concentration of 0.3 M pyrrole was used. Effect of fiim thickness on the response of the enzyme electrode The amperometric response of an enzyme electrode depends on several factors including the enzyme concentration in the polymer film, mass transport of the substrates (glucose and oxygen) and products (gluconolactone and hydrogen peroxide) through the polymer layer and the kinetics of the enzyme reaction. Firstly, we have investigated the effect of film thickness on the steady-state electrochemical response of the enzyme electrode. For these experiments, the concentration of glucose oxidase was kept constant at 500 U/ml in the deposition solution. The thickness of the film deposited at the surface of the electrode was evaluated using the relationship whereby 45 mC/cm2 is equivalent to a film thickness of 0.1 pm (Holdcroft & Funt, 1988). The electrode response diminished with increasing film thickness as depicted in Fig. 3. This is related to the fact that a large fraction of the hydrogen peroxide is lost in the solution and does not contribute to the measured current. An average charge of 75 mC/cm2, equivalent to a film thickness of 0.17 ,um, was chosen as the optimal thickness. With this charge, visual examination of the surface of the electrode revealed an homogeneously covered surface

480

G. Fortier, E. Brassard & D. BtYanger

0.5

1.5 1.0 Film thickness (km)

Fig. 3. The effect of film thickness on the steady-state response of the glucose electrode. The electrodes were prepared by varying the electrodeposition time at O-65 Vof a solution containing 0.3 M pyrrole, 500 U/ml glucose oxidase and 10 mM KCl, pH 6. The electrode surface was @28 cm’. The responses to 20 mM glucose were evaluated at a potential of O-7V in phosphate buffer, pH 7.

with a black polypyrrole film. In these conditions, the current generated by 20 mM glucose was sufficiently high and more reproducible. The shape of the curve in Fig. 3 is somewhat different from that of the poly(N-methylpyrrole)/glucose oxidase electrode (Bartlett & Whitaker, 1987a,b) for which an optimum film thickness was found at a lower concentration of GOD (125 U/ml). However, in the present case, the enzyme concentration is high (500 U/ml) and this would result in a shift of the current-thickness curve towards smaller thickness values if we assume that GOD is homogeneously distributed throughout the film. With a very thin film, the time required to reach a steady state diminishes and the magnitude of the response is then limited by the turnover number of the GOD (k,* = 1000/s) and the number of GOD molecules present in the film. For films thinner than O-1pm, the limitation of the response imposed by the diffusional constraint is minimized. Studies using a rotating disk-ring electrode are in progress in our laboratory to determine the exact contribution of the diffusional constraints (external and internal) and of the catalytic limitation, on the magnitude of the response of the biosensor for polypyrrole glucose oxidase tilms grown in various conditions. Effect of GOD concentration on the response of the enzyme electrode Glucose oxidase from Aspergillus niger has an isoelectric point of 4.2 and is therefore negatively charged at the pH 6 used for the electrodeposition

Optimizationof a polypywoleglucose oxidase biosensor

481

and could presumably be incorporated into the polymer as a counter ion of the polypyrrole during electropolymerization (Diaz et al., 1981). However, deposition at higher values of pH does not yield higher enzyme loading since the amperometric responses are unchanged with electrodes prepared under these conditions (results not shown). These experiments suggest that GOD is probably physically entrapped into the polypyrrole matrix during the electropolymerization and that chloride ions are incorporated as counter ions. As previously demonstrated, the amount of the enzyme GOD incorporated into the film of polypyrrole is proportional to its concentration in the deposition solution as evaluated by the 4aminoantipyrine assay (Foulds dz Lowe, 1986) or by the dimethoxybenzidine assay (Yabuki et al., 1989). The effect of the glucose oxidase concentration in the electrodeposition solution used to prepare the Pt/PP/GOD electrodes was evaluated for two film thicknesses (0.11 and 0.91 pm). The results are shown in Fig. 4 for an assay of 20 mM glucose in quiescent solution. For both films, the maximum responses were reached for a GOD concentration in the range of 25-100 U/ml. Due to the rapid increase and large variations in the response near the GOD concentration of 25 U/ml for both tilm thicknesses, a mid-range value of 65 U/ml was arbitrarily chosen. At this value, the response was still high and was more reproducible for both film thicknesses. For enzyme concentrations higher than 100 U/ml, the response decreased monotonically with

0.04.. 0

I..

loo

I.,

200

I.,

300

I..

400

.

u’

500

Glucose oxidase W/ml)

Fig. 4. The effect of glucose oxidase concentration on the steady-state response of the glucose electrode. The electrodes were prepared by electrodeposition of 0.3 M pyrrole at @65 V in the presence of various quantities of glucose oxidase (l-500 U/ml) in a solution of 10 mM KCl, pH 6. The charges deposited were 50 (0) and 410 mC/cm’ (a). The responses to 20 mM glucose were evaluated at a potential of @7 V in phosphate buffer, pH 7 without stirring.

G. Fortier, E. Brassard & D. B&anger

482

subsequent increase of GOD concentration. The decrease for the thicker film was more marked, about 40% of its optimal value, until the concentration of GOD reached 200 U/ml. At this concentration, the amperometric response to 20 mM glucose became independent of the enzyme concentration. This behaviour is related to

the internal diffusion of hydrogen

peroxide to the underlying platinum electrode and to the thickness of the film.

It can also be related to the spatial distribution of GOD in the film In this discussion, a homogeneous and a uniform distribution of the enzyme in the film is assumed. At a high concentration of GOD (500 U/ml), the hydrogen peroxide was mainly generated at the PP/solution interface. Alternatively at a low concentration of GOD, the hydrogen peroxide formation occurred more uniformly throughout the polypyrrole film. Consequently, the observed current was necessarily lower for the case where the enzyme reaction took place mainly at the polypyrrole/bulk solution interface. This is also values increased by 42% when stirring supported by the fact that the I,,,,, of the solution was stopped for a film prepared with 500 U/ml of GOD compared to 30% when lower enzyme concentrations were used as reported in Table 1. The data of Table 1 were obtained using a very thin film of about 0.1 pm. At this thickness, it is reasonable to assume that the film did not restrict peroxide diffusion to the underlying platinum electrode and that the variation observed in the current was directly related to enzyme loading. At 500 U/ml, an increase of 42% in the I,,, value when the stirring of the solution was stopped indicated clearly that the glucose was consumed mainly at the PP/solution interface. which influences the location of the glucose substrate consumption.

TABLE 1 Influence of GOD Concentration in the Electrodeposition Solution on the Kinetic Parameters of the Enzyme Electrode 200 rpm

Orpm (coo) (v/m0 1 25 65 250 500

K,,, AZSD Cm@ 25.7 + 24.9 + 31-8 + 23.9 f 32.9 f

0.9 0.5 0.4 1.1 2.3

I,,

f SD (Lw

0.85 + 0.01 8.35 If:0.07 7.57 + 0.04 5.01 * 0.09 4.69 f 0.14

K,,,+SD (mM 32.1 I!C1.5 29-5 I!Z0.9 33.0 & 1.0 29.6 + 1.3 31.0 rf:2.1

I,,,, f SD (b.4) 0.61 + 5-89 + 4.97 f 3.59 + 2-73 +

0.01 0.08 0.06 0.07 O-08

A I,,,(u(l cv 29 30 34 30 42

‘Percentage variation in Zmaxin the presence or absence of stirring in the solution

Optimizationof a polypytroleglucose oxidase biosensor

-005

0.05

0.15 l/glucose

025

035

483

045

(mM-I)

Fig. 5. Lineweaver-Burk type-plots showing the linear relation between the reciprocal of the current at steady-state with the reciprocal of the glucose concentration at a potential of 0.7 V for different amounts of enzyme in the electrodeposition solution for a film of 50 mC/cm* without stirring. For all straight lines, r* > O?I!98. The concentrations of enzyme were (A) 1 U/ml; (m) 25 U/ml; (Cl) 65 U/ml; (0) 250 U/ml and (0) 500 U/ml. We can therefore postulate that the catalytic current measured will decrease with both increasing the thickness and enzyme load due to a local and progressive depletion in glucose concentration which is consumed during its translocation through the inner part of the film. The Lineweaver-Burk type-plots are given in Fig. 5 for the film of 50 mC/cm2 loaded with different amounts of enzyme and the equation of the straight lines corresponds to: l/1, = l/1,,,

+ (l/PI)

(ZUZIn,X)

(2)

whereI, is the steady state current, I,,.,,, is the maximal current and S is the substrate concentration (Shu & Wilson, 1976). The coefficients of correlation (I-‘>were greater than 0998. TheK, values and the I,,,,, values for various amounts of enzyme in the deposition media, given in Table 1, were calculated by non-linear regression (eqn 1) of the curves of current generated versus the glucose concentration (not shown). From the Lineweaver-Burk plots obtained for a film of 0.11 pm loaded with various amounts of glucose oxidase, no deviation from linearity was observed either in the quiescent (Fig. 5) or turbulent (not shown) solutions, indicating that up to a concentration of 100 mM of glucose, the current was controlled by the enzymatic reaction and that no substrate inhibition occurred (Horvath & Engasser, 1974; Goldstein, 1976). The absence of small curvature at high glucose concentrations also suggests that there was no kinetic limitation by consumption and availability of the co-substrate, oxygen (Scheller et al., 1988).

434

G. Fortier, E. Brassard & D. Bklanger

Calibration curve for glucose A typical calibration curve for glucose ranging from 1 to 100 mM was obtained with the optimized Pt/PP/GOD electrode in the presence (-•-) or in the absence (-_o-) of stirring (200 r-pm) in the solution (Fig. 6(A)). The steady-state current method was used to evaluate the response of the electrode to glucose. The variation of the reproducibility of the measurements for one electrode was less than 3.5%. A linear relationship was observed for glucose concentrations ranging from 1 to 7.5 mM (9 > O-994,)for both cases and is given in Fig. 6(B). The K, values and the I,,,,, values were 33.4 + O-7 mM (SD) and 7.2 + 0*06pA

20

0

40 Glucose

2.0 Gluc~~

60 (mM)

80

6.0 (mM)

100

0,

Fig.6. (A) Calibration curves for glucose obtained with the optimized Pt/PP/GOD electrode. The electrode was prepared by electrodeposition of 0.3 M pyrrole at 0.65 V in the presence of 65 U/ml lucose oxidase in a solution of 10 mM KCl, pH 6. The charge deposited was 75 mC/cm Q. The glucose determinations were performed at a potential of 0.7 V in phosphate buffer, pH 7 and the current was recorded at steady state with (a) or without (0) stirring (200 x-pm). (B) The linear portion of the calibration curves of Fig. 6 (A) (3 = 096).

Optimizationof a polypyrroleglucose oxidase biosensor

435

respectively, in the quiescent solution and 36.8 k 2-Omu and 3.9 + 0.1 ruA, respectively, in the stirred solution. In both cases, the K,,, values in this study were very similar to the literature value of 33 mM for the soluble enzyme (Swoboda & Massey, 1965) and suggests that there was no limitation due to internal diffusion of the substrates. Similar K, values and I,,, values have been reported (Foulds & Lowe, 1986) for a Pt/PP/ GOD electrode prepared without chloride anions in the electrolyte during the deposition and for a polyindole/glucose oxidase electrode (Pandey, 1988). Effect of the pH on the enzyme electrode response The effect of pH on the response of the glucose electrode was determined by evaluating the steady-state current response from pH 4 to 8. However, it is important not to forget that the glucose assay is based on electrochemical oxidation of hydrogen peroxide which is itself a pHdependent redox process (Guilbault & Lubrano, 1973). Therefore a knowledge of the current potential curves is of crucial importance to establish the true pH dependence of the glucose electrode. The amperometric current for electrochemical oxidation of hydrogen peroxide at a platinum electrode, at a potential of +0.7 V, was found to be pH-independent in the range of our study. Thus, in our study, the variation of the current response was only related to a change in enzyme activity at the designated pH of the experiment. As is shown in Fig. 7, the polypyrrole/glucose oxidase electrode displayed an optimum response

-7

3

4

5

6

7

8

9

PH

Fig. 7. The effect of pH on the steady-state response of the Pt/PP/GOD electrode. The ektrodes were prepared as described in Fig. 6. The responses to 20 mu glucose we= evaluated with stirring (200 rpm) at a potential of O-7V using an acetate or a phosphate buffer.

486

G. Fortier E. Brassard & D. B&anger

at pH 6 and was similar to the value of pH 5.6 reported for soluble glucose oxidase (Swoboda & Massey, 1965). There was no significant shift of the optimal pH for GOD entrapped in polypyrrole film compared to the optimal pH of the soluble enzyme, as is often seen for immobilized enzymes. In the present case, the entrapment material is the polycationic polypyrrole with chloride as counter ion. Clearly, this does not give rise to a charged micro-environment that can affect both the structural and catalytic behaviour of the enzyme in such a way that a displacement of the optimal pH toward a more alkaline or a more acidic pH value (Goldstein et al., 1964). This is contrasted with glucose oxidase chemically bound to polyacrylic acid and polyacrylamide derivatives (Guilbault & Lubrano, 1973). Effect of temperature on the response of the enzyme electrode The effect of the temperature at which the assay is carried out on the response of the enzyme electrode to 20 IIIM glucose was studied from 277-323 K and is depicted in Fig. 8. The response of the electrode increased monotonically between 4 and 4O”C, reaching a maximum value at approximately 313 K after which the response decreased. The current response increased by 0.11 @/“C in the low temperature range. The optimal temperature of 313 K for the catalytic activity of glucose oxidase entrapped in the polypyrrole was similar to values obtained with other polymers (Szajani et al., 1987). The decrease in the amperometric current for the temperatures higher than 313 K was not related to an irreversible denaturation of the enzyme since the data in Fig. 8(A) were reproducible when the experiment was repeated with the same enzyme electrode, by starting at 277 K. The decrease in the response can be attributed to enhanced’disproportionation kinetics of hydrogen peroxide at higher temperatures which is favoured over electrochemical oxidation at the platinum electrode. The Arrhenius form of the temperature dependence of the amperometric current, 1, is shown in the following equation: I = I” exp (-E,/RT)

(3)

where I0 represents a collection of constants, R is the gas constant, T is the temperature in kelvin degrees and E, is the activation energy. In Fig. 8(B), I is plotted on a logarithmic scale versus the reciprocal of the temperature. The activation energy was calculated from the slope of the line of best-fit and was found to be 41 kJ/mol (r2 > 0995). This value compares well with that estimated (Ea= 31 kJ/mol) from the data of Fig. 6 from the paper by Guilbault and Lubrano (1973) for GOD

Optimizationof a polypywoleglucose oxidase biosensor 4.0

2 zi

487

(a)

3.0. .

+ s 2.0. k a 1.0.

273

3.2

263

3.3

293 303 Temperature

3.4 ‘IT

313 (K)

3 5

3.6

323

3.7

(K x 10-3)

Fig. 8. (A) The effect of temperature on the steady-state response of the Pt/PP/GOD electrode. The electrodes were prepared as described in Fig. 6. The effect of tempemture was evaluated from 4-50°C with stirring (200 rpm) at a potential of0.7 Vin phosphate buffer, pH 7 after the addition of 20 mM glucose. (B) Arrhenius type-plot showing the reciprocal of the temperature versus the logarithm of the response of the glucose electrode.

immobilized

into a gel of polyacrylamide,

for a GOD collagen electrode

(E,= 50kJ/mol) (Coulet ec al., 1980) and for a GOD graphite electrode (E,= 45 kJ/mol) (Ikeda et al., 1984). Specificity, shelf life and operational stability of the enzyme electrode The specificity of the Pt/PP/GOD electrode has been evaluated for different sugars such as sucrose, maltose, fructose and lactose up to 20 mM and no discernible signal was detected above the background current. This indicates that the immobilization procedure does not influence the substrate specificity of the glucose oxidase enzyme. The long term stability of the Pt/PP/GOD electrodes has been

488

G. Fortier, E. Brassard & D. B&anger TABLE 2

Operational Stability of the Response of the Enzyme Electrode to 20 mM Glucose Number of assays

37

54 32 52

Time”

Average response

(day)

(EL4f SD)

1 40 60 81

0.778 T!I0.045 0,770 + 0.076 0.914 5~0.178 O-889f @100

“Day after the preparation of the enzyme electrode when assays were performed.

evaluated with respect to their shelf life under storage in PBS at 277 K. These electrodes were prepared using 500 U/ml of GOD and a charge of 411 mC/cm*. The electrodes were tested periodically over 500 days by recording the current generated by 20 mM glucose. After a rapid decrease during the first 10 days, the current stabilized to about 70% of its initial value and remained fairly stable. The remarkable stability of our Pt/PP/ GOD electrode contrasts to that of other polypyrrole (Umana & Waller, 1986) and poly(N-methylpyrrole) (Bartlett & Whitaker, 19873) based glucose sensors that were characterized by very short (-5-21 days) lifetimes. Using an optimized enzyme electrode, the operational stability was evaluated in terms of the number of assays of 20 mM glucose and the results are summarized in Table 2. The current generated by 20 mM glucose remained fairly constant during the 175 assays performed during 4 days spanning over a period of 80 days. ACKNOWLEDGMENTS We would like to thank the Natural Sciences and Engineering Research Council of Canada and the ‘Action Structurante’ program of the Quebec government for financial support We would also like to thank Dr D. Bates for the revision of the manuscript and her comments. This work is in partial fulfilment of the MSc in Chemistry of E.B. REFERENCES Abdel-Latif, M. S. & Guilbault, G. G. (1988). Fiber-optic sensor for the determination of glucose using micellar enhanced chemiluminescence of the peroxyoxolate reaction. Anal. Chern., 60, 2671-4.

Optimizationof a polypynvle glucose oxidase biosensor

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Optimization of a polypyrrole glucose oxidase biosensor.

An amperometric glucose biosensor was fabricated by the electrochemical polymerization of pyrrole onto a platinum electrode in the presence of the enz...
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