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

Mediatorless peroxidase electrode and preparationof bienzyme sensorsJ. KulysI nstitute of Biochemistv, Li thuanian Academy of Sciences, Vil nius, Li thuania (U .S.S.R.)R.D. S&midGBF, Gesell schaft fti r Bi otechnologische For schung mbH, 3300 Braunschweig (F . R. G.)(Received 5 February 1990; in revised form 21 May 1990)

ABSTRACT

Fungal peroxidase (from Arthromyces ramosus (ARP)), covalently immobilized on a graphite elec-trode, catalyzes the mediatorless reduction of hydrogen peroxide. In the pH range 4.92-7.00 the enzymeelectrode steady-state potential reached a value of 995-908 mV (SHE) which is similar to the compoundI and compound II single-electron reduction potentials. The enzyme electrode operated under diffusion-limiting conditions, and at hydrogen peroxidase concentrations lower than 2.5 pM the sensitivity was0.84 A/M. A mediatorless ARP electrode was used to prepare glucose, methanol- and choline-sensitivebienzyme electrodes. The sensitivity of the electrodes based on covalently immobilized peroxidase andglucose oxidase (GO) or peroxidase and alcohol oxidase (AO) was 2.6 and 0.6 mA/M, respectively. Thesteady-state potential of the ARP/GO electrode was similar to that of the ARP electrode. The sensitivityof the peroxidase/choline oxidase (ChO) electrode with entrapped ChO was 0.48 mA/M. The pHoptima of the ARP/GO and ARP/ChO electrodes were 6.0 and 8.7, respectively. ARP, ARP/GO andARP/ChO electrodes retained their efficiency for 2-7 days; however, ARP/AO electrodes were lessstable.

INTRODUCTION

Amperometric enzyme electrodes have wide practical application [l]. The funda-mental problem arising in the construction of enzyme electrodes concerns themechanism of electron exchange between the enzyme active centre and the elec-trode. Two possible pathways can be postulated: (i) direct (mediatorless) electrontransfer and (ii) electron transfer using a soluble or immobilized mediator [2].Horse radish peroxidase (HRP), which catalyzes substrate oxidation by hydrogenperoxidase, has been used to prepare bienzyme electrodes [3]. Ferrocyanide wasused as a mediator in these electrodes. Some experimental data on the directelectron transfer to the peroxidase active centre are available. The electrochemicalreduction of ferriperoxidase on the surface-modified electrode has been investigated0302-4598/90/$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] electrodescatalyzes the reduction of hydrogen peroxide. The latter system has been used toconstruct a mediatorless bienzyme electrode [3]. Since the reduction of hydrogenperoxidase was slow, the electrode action was limited by the peroxidase activity, andthe 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 tothe substrate used is 2.9-540 times higher than that of HRP, but their molar massesare similar [8]. The high catalytic activity of ARP suggests that it can be usedefficiently as a biocatalyst for the electrochemical reduction of hydrogen peroxidase.

The aim of our work was to investigate the electrocatalytic function of ARPimmobilized on a graphite electrode and to prepare bienzyme electrodes based onARP 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 directlywith ferricinium ions [9,10] and no simple mediator-type [ll] sensor has beenprepared to date.EXPERIMENTAL

Electrode preparationReagents: ARP - crystalline peroxidase (Arthromyces rarnosus; Suntory Ltd.)

with a specific activity of 2110 U/mg when 2,2-azino-di(3-ethylbenzothiazoline-6-sulphonic 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 MannheimGmbH, ChO (Ah&genes species, 11 U/mg) was obtained from Sigma, N-(3-di-methylamino-propyl)-N-ethyl-carbodiimide hydrochloride from Fluka and cholinechloride from Sigma. All other reagents were obtained from Merck.

ARP was immobilized on graphite rods (10 mm diameter, Ringsdorff-WerkeGmbH), which had been prepared according to the following procedure: a copperwire (30 mm long) was fixed to one end of the graphite rod with silver epoxy. Theside part of the electrode was isolated by a polyethylene foil. The other end of thegraphite rod was polished with emery paper (220 pm) to form a convex (40 mmradius) or planar shape. After that the surface was washed twice with 50 ~1 oftoluene 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 Mphosphate buffer, pH 4.5. After 0.5 h the electrode was washed with this buffer and20 ~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 atemperature below 4O C in this buffer.

The electrode based on ARP and GO was prepared similarly using a mixture ofARP (10 mg/rnl) and GO (20 mg/ml).

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The ARP/AO electrode was prepared by double immobilization; after thestandard ARP i~obi~zation, the electrode was washed with phosphate buffer, pH7.0, 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 electrodewas washed with this buffer and was stored at below 4C in phosphate buffer, pH7.1.To prepare the ARP/ChO electrode 20 ~1 of ChO (95mg/ml) in phosphatebuffer, 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 beencovalently immobilized. The membrane was held in place with a rubber ring.Elect rode operat i onOperation of the electrodes was carried out at room temperature in 15 ml buffersolutions using a three-electrode voltammetric cell. All potentials were referred to aKC1 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 highimpedance voltmeter. The potential of the SCE was taken as 244 mV vs. SHE. Thebuffer solutions used were 0.1 M acetate, 0.1 M K-phosphate and 0.1 M glycine.RESULTS AND DISCUSSION

The parameters of peroxi dase and peroxi dase / oxi dme el ectr odesThe residual current of the ARP electrode in pH 7.0 phosphate buffer wasnegligibly small (smaller than 12 nA) at an electrode potential of 68 mV. Additionof hydrogen peroxide to the solution. increased the electrode cathodic current. Theresponse time (to 90% steady-state current} was 12 s. A strong linearity between theelectrode steady-state current and the hydrogen peroxide concentration was ob-served over the range O-27-2.46 PM (r = 0.99987). The electrode sensitivity ob-served was 0.84 A/M. The hydrogen peroxide detection limit was calculated to be15 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 highimpedance voltmeter, is shown in Fig. 2. The potential change rate is dependent onthe 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) wereobserved.The response of the bienzyme ARP/GO electrode to glucose was also a cathodiccurrent (Fig. 3). The electrode response time was 12 s. At an electrode potential of130 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 inamzecrease in the maximal current, but Km(appj was not affected greatly. Atpotentials of 530 and 630 mV lu,,,,, was estimated to be 11.1 and 9.8 mM,respectively. The sensitivity of the electrodes up to glucose concentrations of 2 mM

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Fig. 1. Peroxidase electrode current dependenceK-phosphate buffer, electrode potential 68 mV.

on hydrogenL

5 10 15 t/mmperoxide 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 = 630mV).

respectively (pH 6.01). The bienzyme ARP/GO electrodecurrent in the pH range 4.5-10.5 (Fig. 4). The electrodeactivity in the pH interval 5.5-8.0 and in phosphate buffer.acetate buffers did not decrease the current significantly.

The potential of the ARP/GO electrode was positive and the potential changekinetics was dependent on glucose concentration (Fig. 2). The steady-state potentialwas calculated to be 691 mV and 726 mV using 0.6 and 1.8 mM glucose,

generated a cathodicexhibited the highestThe use of glycine or

2 4 c/mM

Fig. 3. The peroxidase/glucose oxidase electrode current dependence on the glucose concentration. pH6.01, 0.1 M K-phosphate buffer, electrode potential 630 (1) 530 (2) and 130 mV (3).

Fig. 4. The peroxidase/choline oxidase (1) and peroxidase/glucose oxidase (2) electrode activitydependence 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), glucoseconcentration 0.6 mM (2).

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that immobilized ARP acted in the following way:E+H20,+E,+H,0 (2)E, + e-+ E, (3)E, + e-+ E (4)Reactions (3) and (4) represent the mediatorless electron transfer to the enzymeactive centre. If such a scheme is valid, the steady-state potential will be close to thesingle-electron transfer potential of compounds I and II. Redox potentials for ARPhave not been determined, but for HRP compounds they are similar [12] and of thesame order of magnitude as the ARP electrode steady-state potential.

The maximum potential of the HRP-carbon black system was 1.24 V vs. ahydrogen electrode in the same buffer [5]. It was interpreted in terms of the mixedH,O, potential. At pH 7.00 this value was 82 mV lower than the potential of theARP 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 enzymestructures. The carbohydrate content in ARP is 5% [7] and that of HRP islS.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 similarcharged amino acids. Hence from the differences in sugar content, it follows thatARP is a more hydrophobic protein and is therefore adsorbed more strongly on theelectrode. Deglycosylation and stronger adsorption can diminish the electron trans-fer distance, thus, increasing the bioelectrocatalytic efficiency. The dependence ofthe electrocatalytic process efficiency on the electron transfer distance is illustratedby cytochrome c peroxidase CCP action data. Recently, Paddock and Bowden haveshown that hydrogen peroxide reduction in the system CCP-graphite proceeds at0.4 V overpotential [14]. Based on kinetic investigations in homogeneous solutions,the HRP electron transfer distance was calculated to be 5.8 A [15], whereas forCCP, estimated from crystal structure data, it was ca. 12 A [16]. It is possible thatdeglycosylation of ARP may also be responsible for the catalytic activity increase.

The diffusion-limiting current of hydrogen peroxide reduction (electrode surface1 cm*, diffusion layer thickness 30 pm) was calculated to be 0.67 PA per 1 I_IMH,O,. Hence it follows, that the ARP electrode operated under diffusion-limitingconditions. The sensitivity decrease on increasing the electrode potential may beexplained by the electron transfer rate decrease.

The sensitivity of bienzyme electrodes was about lo3 times lower than that of theARP electrodes. This may be accounted for by the fact that the electrodes operatedunder kinetic conditions and their sensitivity (S) was greatly affected by bothcatalytic parameters (V,,,, K,) and the membrane diffusion layer thickness (S =nFAV,, ,d/2K,) ; GO was the most active enzyme and ARP/GO electrodesexhibited the highest response.

The electrodes studied can find practical application for highly sensitive electro-chemical 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 electrodesspecificity to other electroactive compounds are necessary, however.ACKNOWLEDGEMENTS

We are grateful to I. Shinmen for the gift of Arthromyces ~U~OSWI peroxidase. Wewish to thank Drs. U. Bilitewski, J. Bradley and V. Razumas for scientific discus-sions. J.K. expresses sincere thanks to the GBF for a two-months visiting professorscholarship. R.D.S. Wishes to thank the Fonds der Chemischen Industrie, Frank-furt, for financial support.REFERENCES

1 R.D. Schmid and I. Karube in H.-J. Rehm and G. Reed (I%.), Biotechnology, Vol. 66, 1988, p. 317.2 J.J. Kulys and V.J. Razumas, Bioamperometry, Mokslas, Vilnius, 1986, p. 153 (in Russian).3 J.J. Kulys, M.V. Pesliakiene and A.S. Samalius, Bioelectrochem. Bioenerg., 8 (1981) 81.4 V.J. Razumas, A.V. Gudavicius and J.J. Kulys, J. Electroanal Chem., 151 (1983) 311.5 A.J. Jaropolov, V. Malovik, SD. Varfolomeev and I.V. Berezin, Dokl. Akad. Nauk USSR, 249 (1979)

1399.6 J.J. Kulys, A.S. Samalius and G.-J.S. Svirmickas, FEBS Lett., 114 (1980) 7.7 Y. Shinmen, S. Asami, T. Amachi, S. Shimizu and H. Yamada, Agric. Biol. Chem., 50 (1986) 247.8 Prospect: Novel Peroxidase of Fungal Origin from Arthromyces ramosus, Suntory Ltd. Institute for

Fundamental Research, Mishima-gun, Osaka, Japan.9 W. Ktinnecke, personal communication, 1989.

10 G. Davis in A.P.F. Turner, I. Karube and C.S. Wilson (Eds.), Biosensors. Fundamentals andApplications, Oxford University Press, Oxford, New York, Tokyo, 1987, pp. 247.

11 N.K. Cenas and J.J. Kulys, Bioelectrochem. Bioenerg., 8 (1981) 103.12 Y. Hayashi and I. Yamazaki, J. Biol. Chem., 254 (1979) 91011.13 L.M. Shannon, E. Kay and J.Y. Lew, J. Biol. Chem., 241 (1966) 2166.14 R.M. Paddock and E.F. Bowden, J. Electroanal. Chem., 260 (1989) 487.15 V. Razumas, A. Gudavicius and J. Kulis, Khim. Fiz., 4 (1985) 1398.16 T.L. Poulos and J. Kraut, J. Biol. Chem., 255 (1980) 10322.