138
Anal. Chem. 1992, 6 4 , 138-142
Electrochemical Immobilization of Enzymes. 3. Immobilization of Glucose Oxidase in Thin Films of Electrochemically Polymerized Phenols P h i l i p N. Bartlett,*BLPeter Tebbutt, a n d Claire H. T y r r e l l Department of Chemistry, University of Warwick, Couentry, CV4 7AL Great Britain
Eiectrochemlcally polymerlsed f l h s of phenol and derivatlves of phenol can be used to immobilize glucose oxidase In a thin layer at a platinum electrode. The immobilized enzyme retalns its activity toward glucose oxldatlon, and hydrogen peroxide generated by the enzyme-catalyzed reaction of glucose with oxygen can be detected at 0.9 V vs SCE. The responses of the immoblllzed enzyme electrodes toward glucose are analyzed in terms of a thin-layer model. Of the flve phenols employed (phenol, 3-nltropheno1, pyrogallol, 4hydroxybenzenesulfonicacid, and bromophenol blue), phenol Itself is found to be the most suitable.
used (20-22). These films are typically grown from buffered aqueous solutions containing both the enzyme and the monomer. At neutral pH glucose oxidase carries an overall negative charge and it is probable that it is incorporated into the growing cationic polymer film as a counterion. When initially prepared, the pyrrole films are electronically conducting; thus, using this approach, thick films can be deposited, and the thickness of the final film can be controlled by the quantity of charge passed. Work by Belanger et al. (15) has shown that when oxygen is used as the mediator species, the hydrogen peroxide generated by the enzymecatalyzed reactions GOx(FAD) P-D-glucose GOx(FADH2) gluconolactone (1)
-
+
INTRODUCTION Immobilized enzymes are used in many applications both in synthetic and in analytical chemistry ( I , Z),and a range of methods exist for immobilization. Immobilization of an enzyme onto a solid support increases the rigidity of the enzyme molecule and, as a result, frequently improves the stability. Immobilization is also know to affect the enzyme kinetics, for example leading to changes in the pH optimum and/or apparent Michaelis constant for the enzyme-catalyzed reaction (3). The immobilization of enzymes at electrode surfaces has been much used to construct both amperometric and potentiometric enzyme electrodes (4). Many of these have used conventional immobilization techniques to localize the enzyme a t the electrode surface; examples include the physical entrapment of the enzyme behind a dialysis membrane (5,6), the covalent attachment of the enzyme to the electrode surface (7,8) or t,o a polymeric support (9), or the cross-linking of the enzyme in a protein matrix using glutaraldehyde (10, 11). Frequently, these approaches have been combined with the use of a discrete macroscopic membrane to reduce electrode fouling and to alleviate problems of interference caused by electroactive species present in the sample (12). Recently, the electropolymerization of heterocyclic compounds has been used to immobilize glucose oxidase at electrode surfaces. Electropolymerization possesses a number of advantages over more conventional methods for the immobilization of enzymes a t electrode surfaces. First, the electrochemical method is simple. Second, the deposition is readily controlled. Third, the deposition can be restricted to the electrode surface and thus the method is well suited to the immobilization of enzymes onto microelectrode structures where it may be desirable to coat a pair of adjacent electrodes with different enzymes. The majority of work in this area has concentrated on the use of electropolymerized films of pyrrole (13-17) or its derivatives (18,19),although some other monomers have been Present address: School of Chemistry, University of Bath, Bath, BA2 7AY U.K.
GOx(FADH2)
+0 2
-
+
GOx(FAD) + HZ02
(2) destroys the conductivity of the poly(pyrro1e) film. Similar behavior has been found for poly(N-methylpyrrole) f i i containing glucose oxidase. In this case a detailed kinetic analysis (18)shows that the hydrogen peroxide generated by the enzyme-catalyzed reaction is oxidized at the underlying platinum electrode surface and not on the poly(N-methylpyrrole) itself. Thus although the conductivity of the polymer is useful in that it allows the deposition of thick films, it is not essential for the application of the films as sensors. In this paper we present results for the immobilization of glucose oxidase in insulating electropolymerized films of phenols. The electrochemical polymerization of phenol and its derivatives is known to produce thin (between 50 and 100 nm), hydrophobic, insulating films a t the electrode surface (23). These films are produced by ortho or para coupling of phenolate radicals generated at the electrode surface leading to the deposition of a poly(pheny1ene oxide) f i i on the electrode. This film is generally insulating and blocks further polymerization. The uses of such films as coatings for corrosion protection (24), as permselective films (25-28), and as pH sensors (29) have been investigated; however to the best of our knowledge no detailed study of the use of such films for the immobilization of enzymes has appeared. Electrochemically deposited poly(pheny1ene oxide) films have a number of features which are attractive in this context. First, they can be grown from neutral aqueous solutions, second, a range of phenols is available, and third, the films are insulating but permselective. This could prove useful in reducing problems of electrode fouling and/or the effects of interferences from other electroactive species present in the analyte. In a recent study Sasso et al. (30) used electropolymerized f i of 1,2-diaminobenzeneto prevent interferences and fouling and to stabilize immobilized enzymes in an electrochemical biosensor. In this work the authors immobilized the enzyme at a platinized carbon electrode using glutaraldehyde cross coupling and then grew the electropolymerized film. Malitesta et al. (31)have extended this to use the electropolymerization of the 1,2-diaminobenzene itself to immobilize the enzyme. In the work presented here we have
0003-2700/92/0364-0138$03.00/00 1992 American Chemical Society
ANALYTICAL CHEMISTRY, VOL. 64, NO. 2, JANUARY 15, 1992
180
Table I. Conditions Used To Prepare Poly(pheno1ic) Films Containg GOx
monomer phenol
deposition conditions 25 mmol dm-3
v
appearance invisible
estimated thicknessa/nm
I /
Or
kKAa_e,l
I
38
M.945 .
pyrogallol
25 mmol dm-3 0-1.1 v 3-nitrophenol 25 mmol dm-3 0.2-1.2 v 4-hydroxybenzene- 100 mmol dm-3 sulfonic acid 0.45-1.65 V bromophenol blue 50 mmol dm-3 0-1.0 v
golden
280
yellow
280
irridescent
1200
yellow/green
1500
I
a Calculated from the total charge passed to grow the film and the following estimated molar volumes (cm3 mol-'): phenol 160; pyrogallol 160; 3-nitrophenol 540; 4-hydroxybenzenesulfonicacid 540; bromophenol blue 2400.
concentrated on the use of phenolic films. Our objective has been to establish the mechanism of the enzyme-catalyzed reaction in the film and to carry out a kinetic analysis of the process. We have done this for a number of different phenols in order to investigate the effects of the choice of phenol monomer.
EXPERIMENTAL SECTION All solutions were freshly prepared using water from a Whatman RO 50 water purification system. Phenol, 3-nitrophenol, 4-hydroxybenzenesulfonic acid, pyrogallol, and bromophenol blue were purchased from Aldrich and used without further purification. Tetraethylammonium tetrafluoroborate, TEATFB, (Aldrich) was recrystallized from methanol three times and dried under vacuum. Buffer solutions were prepared from disodium hydrogen orthophosphate (Fisons, Analytical Reagent), and the pH was adjusted to 7.0 or 7.2 by dropwise addition of 5 mol dm-3 hydrochloric acid. Glucose solutions were prepared from AnalaR D-glUCOSe and allowed to equilibrate at room temperature for 24 h before use. Glucose oxidase, GOx (EC 1.1.3.4 from Aspergillus niger) either was purchased from Sigma (type VII, 176.9 units mg-') or waa a gift from MediSense U.K. All electrochemical experiments were carried out using a conventional three-electrode system consisting of a large-area platinum-gauze counter electrode, a saturated calomel reference electrode (SCE), and a platinum-rotating-disk working electrode (Oxford Electrodes, area = 0.39 cm2). The working electrode was cleaned by hand-polishing with a slurry of 0.3-pm alumina (Banner Scientific) supported on a cotton wool pad. All measurements were carried out in a three-compartment, jacketed electrochemical cell thermostated at 25 i 0.1 OC. Polymeric films of the different phenols containing glucose oxidase were deposited by repetitive potential cycling at 50 mV s-l in an aqueous solution of the monomer containing 0.15 mol dm-3 phosphate, pH 7.0,O.l mol dm-3 TEATFB, and 1270 units cm-3 GOx. Full details for each phenol derivative are given in Table I. In order to allow adsorption of the enzyme onto the electrode surface, vide infra, the electrode was allowed to stand at open circuit in the deposition solution for 15 min before commencing potential cycling. After the deposition of the film the electmde was washed by rotating at 9 Hz in the background buffer for 15 min to remove any weakly adsorbed enzyme. The electrode was then transferred to a cell containing a measured volume of the buffer solution presaturated with oxygen at the desired concentration. The electrode was potentiostated at +0.90 V vs SCE, and when the background current had stabilized, typically after 10 min, aliquots of the stock glucose solution were added and the corresponding current response was recorded with the electrode rotating at 9 Hz.
THEORY The steady-state response of an electrode coated with a thin
I
Sm Figure 1. Plot of Is as a function of s ,showing the general pattern of behavior. The expressions for j S in the different regbns are shown. The plateau can arise either because the enzyme is saturated by the substrate = k,,e2/) or because the enzyme mediator kinetlcs are limiting = kK,a ,e
us us
f i i containing an immobilized enzyme can be modeled using the following kinetic scheme: in the film
E~ + A A
+B
at the electrode k'
B-A Where S is the substrate and P the product for the enzymecatalyzed reaction (in our case glucose and gluconolactone, respectively), Eland Ezare the oxidized and reduced forms of the enzyme, respectively, and A is the oxidized form of the mediator and B its reduced form (in our case oxygen and hydrogen peroxide, respectively). We assume that the electrode is held at a potential at which the oxidation of B to A is mass transport controlled so that we do not need to consider the electron-transfer kinetics in our theoretical treatment. Furthermore we assume that there is no B in the bulk solution. This problem is then a special case of the more general problem of coupled diffusion and reaction in an immobilized enzyme layer (9, 32, 33). If we assume that the polymer film is sufficiently thin that there is no concentration polarization of either S or A within the film, then, in the steady state, we can write
kcate1Kss-l jS
+
= K ~ s , KM
(3)
= kKAa,e21 (4) where Ks and KA, and s, and a, are the partition coefficients and bulk concentrations of S and A, respectively, el and e2 are the concentrations of the two forms of the enzyme within the film, and 1 is the thickness of the immobilized layer of enzyme. The total enzyme concentration in the film, e2, is given by e2 = el e2. Hence substitution in eq 3 and 4 and rearrangement gives the following expression for el
+
Substitution of eq 5 in eq 3 then gives
The general form of the behavior predicted by eq 6 is shown in Figure 1. At low substrate concentrations, when the term
140
ANALYTICAL CHEMISTRY, VOL. 64, NO. 2, JANUARY 15, 1992
II I
-201
0
I
I
0.2
0.4
0.6
0.8 E I V v s SCE
Figure 3. Growth of a poly(pheny1ene oxide) film containing glucose oxidase. Pbtinumdisk electrode cycled at 50 mV s-l in 0.15 mol dm-3 phosphate buffer, pH 7, containing 50 mmol dm-3 phenol. The first two cycles are shown.
2'.5t . .-
Flgure 2. Case diagram for is showing the interrelation between the three cases. The limiting expressions for is are shown. With increasing the concentration of mediator, a -, we move north and with increasing the concentration of substrate, s , we move east.
in k K A a J Min the denominator is dominant, j s is proportional to s., As the concentration of substrate is increased, either the term in kKAa,Kss, or the term in kcatKSSmbecomes dominant and j , becomes independent of substrate concentration. This can occur for two reasons: when kKAa,KsS, is dominant, js is limited by the saturated enzyme kinetics for consumption of substrate; when kCatKSs,is dominant, j s is limited by the rate of reoxidation of the enzyme by reaction with the mediator, under these circumstances j s depends on a,. The limiting expressions for j , are also shown. The interrelation between these three possible solutions is shown in Figure 2. In this figure we can move north/south by changing the bulk concentration of mediator, A, and we can move east/west by changing the bulk concentration of substrate, S. The limiting expressions for j , in each of the three cases are shown. The flux of substrate reacting within the film, is,is not necessarily the same as the flux of reduced mediator detected at the electrode, jobs, because some of the mediator will be lost to the bulk solution. The precise amount will depend on the efficiency of mass transport of B away from the electrode. When this is very efficient, so that the concentration of B is held at zero, at the outside of the film jobs
=.id2
If the experiment is carried out a t a rotating-disk electrode, the mass transport of B away from the electrode can be controlled and we can show (see Appendix) that jobs
(7)
=
and XD = 0.643DB,Soln1'3u~116W-1~~. Note that 1 < a < 2 , as expected. By combining eqs 6-8, we obtain an expression for the observed current. In order to analyze the experimental data, it is convenient to recast the expression for jobin the following form cy nFAa =--
jobs
i
KM - kcatKSsme +- l + 1 1 kcate 1 kKAa ,e
1
(9)
Thus, by measuring the current for the enzyme-coated electrode as a function of the concentrations of substrate and mediator and of the enzyme loading in the film, it is possible
E I V v s SCE Flgure 4. Growth of a polymer film of pyrogallol containing glucose oxidase. Phtinumdisk electrode cycled at 50 mV 5-l in 0.15 mol dm-3 phosphate buffer, pH 7, containing 50 mmol dm-3 pyrogallol. The first three cycles are shown.
to estimate the kinetics of the reactions of the immobilized enzyme.
RESULTS Deposition of Polymeric Films. Six different phenols were studied in this work. Five of these gave stable films containing GOx. Figure 3 shows typical results for the deposition of a film of poly(pheny1ene oxide) from a buffered solution containing the enzyme. Essentially identical results were obtained in the absence of the enzyme. On the first anodic cycle there is a peak a t around 0.65 V, which is suppressed on the second and subsequent cycles. This corresponds to the oxidation of the phenol to produce an insulating film of poly(pheny1ene oxide) at the electrode surface. From the amount of charge passed and an estimate of the molar volume we can obtain an estimate of the thickness of this film (Table I). Very similar results are obtained for 3-nitrophenol. In the case of 4-hydroxybenzenesulfonic acid, pyrogallol, and bromophenol blue the current decays more slowly and several cycles are required before the electrode passivates (Figure 4). Again, the thickness can be estimated from the total charge passed in the deposition of the film; the results are summarized in Table I. SEM studies of the coated electrodes are consistent with the deposition of thin continuous films. The ferro/ferricyanide redox couple was used as a probe to check for pinholes and gross defects in the films. For the films deposited from phenol and 3-nitrophenol, where the deposition passivates on the first cycle, the ferro/ferricyanide electrochemistry was almost completely suppressed (Figure 5b). This is consistent with the presence of a continuous, insulating film. The films coated from 4-hydroxybenzenesulfonic acid, pyrogallol, and bromophenol blue only partially suppress the ferro/ferricyanide electrochemistry (Figure 5c). For the films produced from phenol, 3-nitrophenol, 4hydroxybenzenesulfonic acid, and pyrogallol, the results are similar both with and without the enzyme present, for the bromophenol blue films the ferro/ferricyanide electrochem-
ANALYTICAL CHEMISTRY, VOL. 64, NO. 2, JANUARY 15, 1992
141
Table 11. Kinetic Data for the Different Immobilized GOx Films" monomer phenol O2sat. 3-nitrophenol pyrogallol bromophenol blue
5.8 X 6.2 X 5.1 x 3.1 x 1.9 x
lo4
1.1x 2.5 X 4.5 x 4.6 X 5.7 x 1.3 X 2.9 X
10" 10-7 10-7 10-7 7.3 x 10-8 8.2 X
4-hydroxybenzenesulfonicacid
1.9 x 10-5 4.0 x 10-5 8.8 X 15 x 10-5 30 x 10-5 18 x 10-5 35 x 10-5
10-10 10-11 lo-" 10-11
lo-" O2sat. "All data refer to air-saturated buffer solution, pH 7, W = 9 Hz,unless otherwise stated.
v
-24 0
0.2
I t
I t 0.4 E N
0
0.2
0.4
El V
0
0.2
0.4
El V
Flgure 5. Cyclic voltammograms of 2 mmol dm-3 K,Fe(CN)B in 0.15 mol dm-3 phosphate buffer, pH 7, recorded at 50 mV s-': (a) clean Pt electrode; (b) Pt modified with a poty(pheny!ene oxide) film Containing glucose oxidase; (c) Pt modified with a polymeric film of pyrogallol containing glucose oxidase. 0
20
60
40
80
[ g l ~ c o s e ] - ~ / m o l dm3 -~
Flgure 7. Plots of eq 9 for films of five different phenols containing immobiliied glucose oxidase. All currents were recorded at 9 k,+0.9 V vs SCE in 0.15 mol dm3 phosphate buffer, pH 7. The currents have been corrected for the background current, i,,, recorded In the absence of glucose. All solutions were air-saturated unless specified otherwise. The monomers used were (V)phenol, (V)phenol (0, saturated), (0)3-nltrophenyl, (0)4-hydroxybenzenesulfonlc acid, (M) 4hydroxybenzenesulfonicacid (0, saturated), (A)pyrogallol, and (0) bromophenol blue. The lines are the weighted least squares fits.
!'
in air- and oxygen-saturated solutions show that they are consistent, with K M /[ 1 kWt/kKAu,]increasing by a factor of -2 in both cases on going from air to oxygen saturation. Thus it is clear that for these two T i s , in air-saturated buffer, the plateau response at high glucose concentration is determined by the rate of reoxidation of the enzyme by oxygen.
+
t
Flgure 6. Typical responses to the addition of glucose in air-saturated buffer, 0.15 mol dm-3 phosphate, pH 7, recorded at +0.9 V vs SCE for a platinum electrode coated with a poly(pheny1ene oxide) film containing glucose oxidase. The electrode was rotated at 9 Hz. Each addition, marked by the arrows, corresponds to a change in glucose concentration of 13.3 mmol dm-3.
istry shows much less suppression when the enzyme is present. Responses t o Glucose. Figure 6 shows typical results for the addition of glucose to a solution containing a coated electrode held at +0.9 V vs SCE to detect hydrogen peroxide. The response times for the five different phenolic films were all similar, typically
