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Bioelectrochemistry and Bioenergetics, 24 (1990) 305-311 A section of J. Electroanal. Chem., and constituting Vol. 299 (1990) Elsevier Sequoia S.A., Lausanne

Mediatorless peroxidase electrode and preparation of bienzyme sensors

J. Kulys
Institute of Biochemistv, Lithuanian Academy of Sciences, Vilnius, Lithuania (U.S.S.R.)

R.D. S&mid
GBF, Gesellschaft ftir Biotechnologische (Received 5 February Forschung mbH, 3300 Braunschweig (F. R. G.) 1990; in revised form 21 May 1990)

ABSTRACT Fungal peroxidase (from Arthromyces ramosus (ARP)), covalently immobilized on a graphite electrode, catalyzes the mediatorless reduction of hydrogen peroxide. In the pH range 4.92-7.00 the enzyme electrode steady-state potential reached a value of 995-908 mV (SHE) which is similar to the compound I and compound II single-electron reduction potentials. The enzyme electrode operated under diffusionlimiting conditions, and at hydrogen peroxidase concentrations lower than 2.5 pM the sensitivity was 0.84 A/M. A mediatorless ARP electrode was used to prepare glucose, methanol- and choline-sensitive bienzyme electrodes. The sensitivity of the electrodes based on covalently immobilized peroxidase and glucose oxidase (GO) or peroxidase and alcohol oxidase (AO) was 2.6 and 0.6 mA/M, respectively. The steady-state potential of the ARP/GO electrode was similar to that of the ARP electrode. The sensitivity of the peroxidase/choline oxidase (ChO) electrode with entrapped ChO was 0.48 mA/M. The pH optima of the ARP/GO and ARP/ChO electrodes were 6.0 and 8.7, respectively. ARP, ARP/GO and ARP/ChO electrodes retained their efficiency for 2-7 days; however, ARP/AO electrodes were less stable.

INTRODUCTION

Amperometric enzyme electrodes have wide practical application [l]. The fundamental problem arising in the construction of enzyme electrodes concerns the mechanism of electron exchange between the enzyme active centre and the electrode. Two possible pathways can be postulated: (i) direct (mediatorless) electron transfer and (ii) electron transfer using a soluble or immobilized mediator [2]. Horse radish peroxidase (HRP), which catalyzes substrate oxidation by hydrogen peroxidase, has been used to prepare bienzyme electrodes [3]. Ferrocyanide was used as a mediator in these electrodes. Some experimental data on the direct electron transfer to the peroxidase active centre are available. The electrochemical reduction of ferriperoxidase on the surface-modified electrode has been investigated
0302-4598/90/$03.50 0 1990 - Elsevier Sequoia S.A.

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in detail [4]. Peroxidase adsorbed on carbon black [5] or organic metal [6] electrodes catalyzes the reduction of hydrogen peroxide. The latter system has been used to construct a mediatorless bienzyme electrode [3]. Since the reduction of hydrogen peroxidase was slow, the electrode action was limited by the peroxidase activity, and the sensitivity and stability of these electrodes were low. Recently, a novel peroxidase of fungal origin from Arthromyces rumosus (ARP) has been purified and crystallized [7]. The catalytic activity of ARP with respect to the substrate used is 2.9-540 times higher than that of HRP, but their molar masses are similar [8]. The high catalytic activity of ARP suggests that it can be used efficiently as a biocatalyst for the electrochemical reduction of hydrogen peroxidase. The aim of our work was to investigate the electrocatalytic function of ARP immobilized on a graphite electrode and to prepare bienzyme electrodes based on ARP and oxidases. Glucose oxidase (GO), alcohol oxidase (AO) and choline oxidase (ChO) were used. The last two oxidases were chosen, since they do not react directly with ferricinium ions [9,10] and no simple mediator-type [ll] sensor has been prepared to date.
EXPERIMENTAL

Electrode preparation Reagents: ARP - crystalline peroxidase (Arthromyces rarnosus; Suntory Ltd.) with a specific activity of 2110 U/mg when 2,2-azino-di(3-ethylbenzothiazoline-6sulphonic acid) (ABTS) was used as substrate [8]. GO (Aspergilh niger, 250 U/mg) and A0 (Candidu boidini, 8.1 U/mg) were both from Boehringer Mannheim GmbH, ChO (Ah&genes species, 11 U/mg) was obtained from Sigma, N-(3-dimethylamino-propyl)-N-ethyl-carbodiimide hydrochloride from Fluka and choline chloride from Sigma. All other reagents were obtained from Merck. ARP was immobilized on graphite rods (10 mm diameter, Ringsdorff-Werke GmbH), which had been prepared according to the following procedure: a copper wire (30 mm long) was fixed to one end of the graphite rod with silver epoxy. The side part of the electrode was isolated by a polyethylene foil. The other end of the graphite rod was polished with emery paper (220 pm) to form a convex (40 mm radius) or planar shape. After that the surface was washed twice with 50 ~1 of toluene and dried in air for 0.5 h. Enzyme immobilization The electrode was activated by application of water soluble carbodiimide-N-3(dimethylaminopropyl)-N-ethylcarbodiimide hydrochloride (20 mg/ml) in 0.1 M phosphate buffer, pH 4.5. After 0.5 h the electrode was washed with this buffer and 20 ~1 of ARP (10 mg/ml) in the same buffer was applied to the electrode surface, 0.5 h later the electrode was washed with phosphate buffer (pH 7.0) and stored at a temperature below 4O C in this buffer. The electrode based on ARP and GO was prepared similarly using a mixture of ARP (10 mg/rnl) and GO (20 mg/ml).

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The ARP/AO electrode was prepared by double immobilization; after the standard ARP i~obi~zation, the electrode was washed with phosphate buffer, pH 7.0, 20 ~1 of A0 solution (5 mg/ml) in phosphate buffer, pH 7.1 (containing 2.5% glutaraldehyde), was applied to the electrode and was allowed to dry. The electrode was washed with this buffer and was stored at below 4C in phosphate buffer, pH 7.1. To prepare the ARP/ChO electrode 20 ~1 of ChO (95mg/ml) in phosphate buffer, pH 7.0, was entrapped by a dialysis membrane (25 pm thickness when dry) against the face of the convex graphite electrodes on which ARP had been covalently immobilized. The membrane was held in place with a rubber ring.
Electrode operation

Operation of the electrodes was carried out at room temperature in 15 ml buffer solutions using a three-electrode voltammetric cell. All potentials were referred to a KC1 saturated Ag/AgCl electrode the potential of which was 226 mV (vs. SHE). The electrode was calibrated vs. SCE in the 0.1 M K-phosphate buffer using a high impedance voltmeter. The potential of the SCE was taken as 244 mV vs. SHE. The buffer solutions used were 0.1 M acetate, 0.1 M K-phosphate and 0.1 M glycine.
RESULTS AND DISCUSSION

The parameters of peroxidase and peroxidase / oxidme electrodes

The residual current of the ARP electrode in pH 7.0 phosphate buffer was negligibly small (smaller than 12 nA) at an electrode potential of 68 mV. Addition of hydrogen peroxide to the solution. increased the electrode cathodic current. The response time (to 90% steady-state current} was 12 s. A strong linearity between the electrode steady-state current and the hydrogen peroxide concentration was observed over the range O-27-2.46 PM (r = 0.99987). The electrode sensitivity observed was 0.84 A/M. The hydrogen peroxide detection limit was calculated to be 15 nM. At higher concentrations the calibration curve implies electrode saturation (Fig. 1). Lax and &,(app) were equal to 35 PA and 42 pM, respectively. The potential change kinetics of the ARP electrode, measured using a high impedance voltmeter, is shown in Fig. 2. The potential change rate is dependent on the H202 concentration. The steady-state potential depends on pH and at high H,O, concentrations, potentials of 682 mV (pH 7.0), 732 mV (PI-I 6.01) and 769 mV (pH 4.92) were observed. The response of the bienzyme ARP/GO electrode to glucose was also a cathodic current (Fig. 3). The electrode response time was 12 s. At an electrode potential of 130 mV in the presence of O-5-6.5 mM glucose the calibration curve was hyperbolic, I = 33 pA and Km(appj= 12.5 mM. Increasing the electrode potential resulted in amzecrease in the maximal current, but Km(appj was not affected greatly. At was estimated to be 11.1 and 9.8 mM, potentials of 530 and 630 mV lu,,,,, respectively. The sensitivity of the electrodes up to glucose concentrations of 2 mM

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L
Fig. 1. Peroxidase electrode current dependence K-phosphate buffer, electrode potential 68 mV. on hydrogen peroxide

10

15

t/mm

concentration.

pH 7.0, 0.1 M

Fig. 2. The peroxidase (l-4) and peroxidase/glucose oxidase (1,2) electrodes potential change kinetics. 0.1 M K-phosphate buffer; pH7.0 (1,2), 6.01 (l, 2, 3), 4.92 (4); H,O, concentration 1.3 PM (1) 0.32 mM (2-4); glucose concentration 0.6 (1) 1.8 mM (2).

was 2.6 mA/M

(E = 130 mv),

1.9 mA/M

(E = 530 mV) and 1.6 mA/M

(E = 630

mV). The potential of the ARP/GO electrode was positive and the potential change kinetics was dependent on glucose concentration (Fig. 2). The steady-state potential was calculated to be 691 mV and 726 mV using 0.6 and 1.8 mM glucose, respectively (pH 6.01). The bienzyme ARP/GO electrode generated a cathodic current in the pH range 4.5-10.5 (Fig. 4). The electrode exhibited the highest activity in the pH interval 5.5-8.0 and in phosphate buffer. The use of glycine or acetate buffers did not decrease the current significantly.

c/mM

Fig. 3. The peroxidase/glucose oxidase electrode current dependence on the glucose concentration. 6.01, 0.1 M K-phosphate buffer, electrode potential 630 (1) 530 (2) and 130 mV (3).

pH

Fig. 4. The peroxidase/choline oxidase (1) and peroxidase/glucose oxidase (2) electrode activity dependence on pH 0.1 M acetate buffer (A) 0.1 M K-phosphate buffer (0) and 0.1 M glycine buffer (0); electrode potential 70 (1) and 20 mV (2); choline chloride concentration 0.08 mM (l), glucose concentration 0.6 mM (2).

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The bienzyme ARP/AO electrode generated a cathodic current on the introduction of methanol into the buffer solution. The response time of this electrode increased, relative to that obtained with the ARP electrode, to 30 s. Up to 0.7 mM methanol a linear correlation between the electrode current and the substrate concentration was observed. The electrode sensitivity was 0.6 mA/M. The response time of the bienzyme ARP/ChO electrode increased, relative to that obtained with the ARP electrode, to 40 s, with a linear calibration up to 2.5 mM choline chloride. The electrode sensitivity was 0.48 mA/M. The pH optimum of the ARP/ChO electrode was 8.7 (Fig. 4). The electrode was more active in glycine buffer than in the other buffers tested. The pH dependence of the bienzyme electrode action may be expressed as the combination of ARP and oxidase pH-dependence. ARP was active in the pH range 3-11, with an optimum at pH 6-8 [7]. Thus, the pH optimum action of ARP/GO and ARP/ChO depends on the optimal activity of the GO and ChO sensors, which was estimated to be at pH 6.0 and 8.7, respectively. The electrodes investigated exhibited different long-term stabilities. The activity of the ARP and ARP/GO electrodes decreased by approximately 12-15% per day (over a period of 2-3 h assay per day) and after 7 days they retained 30-409~ of their initial activity. In the case of the ARP/AO electrode, a loss of activity averaging 36% was observed after 2 h usage. The next day it was possible to record only 5-7% of te initial activity, but the response to H,O, decreased by only 16%. The ARP/ChO electrodes were more stable and retained their efficiency for 2-3 days. During this period the response of these electrodes to H,O, decreased by up to 60%. The electrodes based on immobilized ARP and ARP/GO had the highest stability. This is consistent with the native enzyme stability. ChO and A0 are less stable. As far as the latter enzyme is concerned, other methods of immobilization can be used to stabilize it.
ELECTRODE ACTION MECHANISM

The electrodes based on peroxidase as well as peroxidase and oxidase generated a cathodic current. The steady-state potentials of the ARP and ARP/GO electrodes were extremely high. At the same pH (6.01) it is practically the same for both the ARP (H,O, action) and ARP/GO (glucose action) electrodes. A negligible difference in the potentials can be attributed to the slower kinetics of bienzyme electrodes. The results obtained permit the conclusion that the electrode potential depends on the peroxidase action. Hydrogen peroxide in the bienzyme electrodes was generated in the presence of oxidases: S + 0,-P H,Oz HRP, is reduced compound + H,O, (1)

by immobilized peroxidase. It is known that, under the action of I (E,) and compound II (E,) are formed [12]. One can assume,

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that immobilized E+H20,+E,+H,0 E, + e-+ E, + e-+ E, E

ARP acted in the following

way: (2) (3) (4)

Reactions (3) and (4) represent the mediatorless electron transfer to the enzyme active centre. If such a scheme is valid, the steady-state potential will be close to the single-electron transfer potential of compounds I and II. Redox potentials for ARP have not been determined, but for HRP compounds they are similar [12] and of the same order of magnitude as the ARP electrode steady-state potential. The maximum potential of the HRP-carbon black system was 1.24 V vs. a hydrogen electrode in the same buffer [5]. It was interpreted in terms of the mixed H,O, potential. At pH 7.00 this value was 82 mV lower than the potential of the ARP system. Obviously, low potential values were due to non-effective HRP action. The question arises why ARP catalyzed the electrochemical reduction of H,O, better than HRP. Evidently, this may be accounted for by the different enzyme structures. The carbohydrate content in ARP is 5% [7] and that of HRP is lS.l-18.2% [13]. The molar masses of the peroxidases are 41 kDa [7] and 40 kDa [13] for ARP and HRP, respectively, ARP and HRP isozymes contain similar charged amino acids. Hence from the differences in sugar content, it follows that ARP is a more hydrophobic protein and is therefore adsorbed more strongly on the electrode. Deglycosylation and stronger adsorption can diminish the electron transfer distance, thus, increasing the bioelectrocatalytic efficiency. The dependence of the electrocatalytic process efficiency on the electron transfer distance is illustrated by cytochrome c peroxidase CCP action data. Recently, Paddock and Bowden have shown that hydrogen peroxide reduction in the system CCP-graphite proceeds at 0.4 V overpotential [14]. Based on kinetic investigations in homogeneous solutions, the HRP electron transfer distance was calculated to be 5.8 A [15], whereas for CCP, estimated from crystal structure data, it was ca. 12 A [16]. It is possible that deglycosylation of ARP may also be responsible for the catalytic activity increase. The diffusion-limiting current of hydrogen peroxide reduction (electrode surface 1 cm*, diffusion layer thickness 30 pm) was calculated to be 0.67 PA per 1 I_IM H,O,. Hence it follows, that the ARP electrode operated under diffusion-limiting conditions. The sensitivity decrease on increasing the electrode potential may be explained by the electron transfer rate decrease. The sensitivity of bienzyme electrodes was about lo3 times lower than that of the ARP electrodes. This may be accounted for by the fact that the electrodes operated under kinetic conditions and their sensitivity (S) was greatly affected by both diffusion layer thickness (S = catalytic parameters (V,,,, K,) and the membrane nFAV,,,d/2K,); GO was the most active enzyme and ARP/GO electrodes exhibited the highest response. The electrodes studied can find practical application for highly sensitive electrochemical H,O, detection as well as in the preparation of electrochemical strips for

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ethanol (methanol) and choline determination. Investigations of the specificity to other electroactive compounds are necessary, however.
ACKNOWLEDGEMENTS

electrodes

We are grateful to I. Shinmen for the gift of Arthromyces ~U~OSWI peroxidase. We wish to thank Drs. U. Bilitewski, J. Bradley and V. Razumas for scientific discussions. J.K. expresses sincere thanks to the GBF for a two-months visiting professor scholarship. R.D.S. Wishes to thank the Fonds der Chemischen Industrie, Frankfurt, for financial support.
REFERENCES 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 R.D. Schmid and I. Karube in H.-J. Rehm and G. Reed (I%.), Biotechnology, Vol. 66, 1988, p. 317. J.J. Kulys and V.J. Razumas, Bioamperometry, Mokslas, Vilnius, 1986, p. 153 (in Russian). J.J. Kulys, M.V. Pesliakiene and A.S. Samalius, Bioelectrochem. Bioenerg., 8 (1981) 81. V.J. Razumas, A.V. Gudavicius and J.J. Kulys, J. Electroanal Chem., 151 (1983) 311. A.J. Jaropolov, V. Malovik, SD. Varfolomeev and I.V. Berezin, Dokl. Akad. Nauk USSR, 249 (1979) 1399. J.J. Kulys, A.S. Samalius and G.-J.S. Svirmickas, FEBS Lett., 114 (1980) 7. Y. Shinmen, S. Asami, T. Amachi, S. Shimizu and H. Yamada, Agric. Biol. Chem., 50 (1986) 247. Prospect: Novel Peroxidase of Fungal Origin from Arthromyces ramosus, Suntory Ltd. Institute for Fundamental Research, Mishima-gun, Osaka, Japan. W. Ktinnecke, personal communication, 1989. G. Davis in A.P.F. Turner, I. Karube and C.S. Wilson (Eds.), Biosensors. Fundamentals and Applications, Oxford University Press, Oxford, New York, Tokyo, 1987, pp. 247. N.K. Cenas and J.J. Kulys, Bioelectrochem. Bioenerg., 8 (1981) 103. Y. Hayashi and I. Yamazaki, J. Biol. Chem., 254 (1979) 91011. L.M. Shannon, E. Kay and J.Y. Lew, J. Biol. Chem., 241 (1966) 2166. R.M. Paddock and E.F. Bowden, J. Electroanal. Chem., 260 (1989) 487. V. Razumas, A. Gudavicius and J. Kulis, Khim. Fiz., 4 (1985) 1398. T.L. Poulos and J. Kraut, J. Biol. Chem., 255 (1980) 10322.