Electroanalytical determination of peroxidases and laccases on carbon paste electrodes

213

Electroanalytical Determination of Peroxidases and Laccases on Carbon Paste Electrodes h o z a s Kulys, *' Alma Drungiliene, ' Ulla Wollenberger, Kastis Krikstopaitis, 'and Frieder Scheller" if

+

+ +

Laboratory of Enzyme Chemistry, Institute of Biochemistry, Mokslininku 12,2600 Vilnius, Lithuania University of Potsdam, Institute of Analytical Biochemistry and Molecular Physiology, c.10. Max-Delbriick Centrum, Robert Rossle Str.10, D-13122 Berlin, Germany

Received: May 9, 1996 Final version: October 4, 1996

Abstract Electrocatalytical reduction of hydrogen peroxide and oxygen was performed by horseradish peroxidase, recombinant Coprinus cinereus peroxidase, Polyporus pinsitus laccase and Botrytis cinerea laccase on carbon paste electrodes containing a paraffin soluble mediator. The electrocatalytical process proceeded at 0.OV (vs Ag/AgCl; in situ electrode). The electrode responses had maximum values at pH 6.4 and 5.5 for the horseradish peroxidase and laccases catalyzed reactions, respectively. The sensitivity for horseradish peroxidase and recombinant Coprinus cinereus peroxidase was 23.4 and 5.4pC cm-', respectively. A detection limit of 7.5 fmol horseradish peroxidase could thus be achieved. Systems based on laccases showed lower sensitivity. The model of electrocatalytical reduction of hydrogen peroxide and oxygen included a permanent liberation of mediator from the carbon paste and it's biocatalytical conversion in the diffusion layer. The model was analyzed by comparing kinetic data in homogeneous solution with the electrode response. Mass transport effects were studied by using rotating carbon paste electrodes.

I&-'

Keywords: Carbon paste electrode, Graphite, Chemically modified electrode, Horseradish peroxidase, Coprinus cinereus peroxidase, Polyporus pinsitus laccase, Botrytis cinerea laccase, l-(N,N-dimethylamine)-4-(4-morpholine)benzene

1. Introduction Oxidoreductases, especially horseradish peroxidase (HRF', EC 1.1 1.1.7), have been employed as the marker in a wide variety of biological recognition systems [l-41. HRP has the practical advantages of high stability, large catalytic activity and low unit cost. This heme glycoprotein is known to catalyze one-electron oxidation of a rather wide range of inorganic and organic substrates, usually phenols and anilines in the presence of hydrogen peroxide. Laccases (EC 1.10.3.2) also catalyze the oxidation of various aromatic compounds (mono-, di-, polyphenols, diamines and heterocyclic compounds) by reducing molecular oxygen to water [5,6]. As marker, these enzymes have not been used very often in comparison to peroxidases [7,8]. Because the mechanism of the peroxidase and laccases reaction is a redox process, electrochemical signal generation and consequently, employing enzymes for the analysis of biological molecular recognition by electronalytical technique is obvious. Direct eletrochemical communication between electrodes and adsorbed or covalently immobilized peroxidases and laccases has been described [9-121. In a mediatorless regime, only redox centers of oxidase molecules actually contacting the electrode surface may be electroreduced. Fast electron transfer has been achieved using mediators, i.e., an electron transfer shuttle between the enzyme active center and the working electrode. It was shown, that the sensitivity of mediatorless HRP based bioelectrodes is two orders of magnitude lower than that in presence of mediators 1131. Carbon paste consists of a mixture of graphite powder and a water-immiscible pasting liquid. The pasting liquid serves not only for filling up the crevices between the graphite particles, but also insulates the graphite from the contacting aqueous solution, and can act as a medium for dissolution of the mediator. Oxidases containing carbon pastes were employed for the determination of glucose and lactate and other metabolites [14,15]. In contrast, little attention has been given to the electrochemical measurement of peroxidase and laccase on carbon paste electrodes. The aim of this work was to study a mediator-modified carbon Electroanalysis 1997, 9, No. 3

paste electrode for the determination of minute amounts of peroxidase and laccase which is of particular importance for the development of a pseudohomogeneous amperometric enzyme immunoassay. As mediator, 1 -(N,N-dimethylamine)-4-(4-morpho1ine)benzene (AMB) was used. Recently this mediator was employed for the preparation of glucose bioelectrodes [ 151. AMB implies some advantages over other mediators: it is soluble in pasting oil, shows single electron transfer, and has a low oxidation reduction potential (Eb 0.39V vs. SHE [16]).

2. Experimental 2.1. Materials and Methods Horseradish peroxidase (HRP type I) containing RZ 3.2 was purchased from Sigma (USA) Cat. No. P-8250. Recombinant peroxidase from ink cap basidiomycetes Coprinus cinereus peroxidase (rCiP) containing RZ 2.8 and a new fungal laccase from Polyporus pinsitus (PPL) were received from Novo Nordisk A / S (Denmark); laccase from Botrytis cinerea (BCL) [5] was received from the State University of New York, College of Environmental Science and Forestry, Syracuse, USA. Molecular weight of rCiP and PPL were 39000 Da and 65000 Da, respectively. Graphite powder and paraffin oil were from Merck (Darmstadt, Germany), Cat. No.104206 and 107161, respectively. Spectroscopic grade graphite rods were obtained from Russia. 1-(N,Ndimethylamine)-4-(4-morpholine)benzene(AMB) was synthesized as described [16]. Other reagents were of analytical grade. All solutions were prepared with double distilled water. Measurements were carried out in 0.05 M Na-phosphate buffer solution (pH 5.6-8.0) or 0.05 M Na-acetate buffer solution (pH 3.6-5.8), containing 0.1 M NaC1. Concentration of Hz02 was determined using absorbance at 240nm (ez4" = 39.4 M-' cm-' [17]). Concentration of oxygen in buffer solutions was assumed to be 0.25 mM 1181.

0 VCH Verlagsgesellschaft mbH, 0-69469 Weinheim, 1997

1040-0397/97/0302-0213 $ 10.00+.25/0

J. Kulys et al.

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2.2. Preparation of Carbon Paste and Chemically Modified Graphite Electrodes Carbon paste (CP) containing 0.067-0.167% (w./w.) of mediator was prepared by dissolving 2-5 mg of AMB in 1 g of paraffin and mixing with 2 g of graphite powder. For preparing modified carbon paste electrodes (MCPE) two channels (diameter 2.3 mm) of poly(ethy1ene) rods (diameter 5.8 mm, length 40 mm) were filled with modified carbon paste. Two other channels in the same frame housed two silver electrodes prepared from silver wires of diameter 0.2 mni. Electric contacts were made by gold-plated copper pins. The electrode's scheme was depicted in [14]. For the preparation of chemically modified graphite electrodes (CMGE) the working surface of graphite rods (diameter 5.9 mm) was polished with emery paper (250pm). 25pL of 0.1-lOmM solution of AMB in methanol were dropped onto the graphite electrodes surface and dried in air for 4 h.

2.3. Electrochemical Measurements Electrochemical measurements were performed using a computercontrolled electroanalytical system in a three-electrode regime. The electrode's current was measured in a cell in such a manner that all the electrodes were facing upwards as described earlier [14]. In the case of MCPE the reference silverlsilver chloride electrode was prepared in situ immediately before every measurement [19]. The potential of the reference electrode was 45mV (vs. SCE). Each response was measured using a new electrode surface. The surface was renewed by cutting a slice of the polyethylene rod, containing the carbon paste, auxiliary and reference electrodes (thickness 0.3 mm) with a stainless steel knife. The procedure for sensor calibration included placing 50 pL Hz02 (for determination of HRP and rCiP) and enzyme onto the surface of the vertically orientated MCPE. In the following time interval (RT = 7-8 s), preparation of the reference electrode was done in situ. After a delay time ( D T ) the potential was applied lo the working electrodes. The current was integrated for a period of 10-30 s ( I T ) just after potential application or after integration delay time (IDT).The measurement regime is depicted in Figure 1. Two independent measurements from both channels were averaged and are shown in the figures as 'Response, pC'. The current of the chemically modified graphite electrodes was measured by facing the electrodes upwards. As a reference, a saturated calomel electrode (SCE, Radiometer, Denmark) and as an auxiliary electrode a platinum wire (diameter 0.2 mm, length 4 cm) mounted on the end of the reference electrode were used. Each experiment was performed using a new modified electrode. The

2.4. Kinetic Measurements The kinetics of AMB oxidation in homogeneous solution was measured by using a spectrophotometer Beckman DU-8 B at 25 2 0.1"C. The production of the oxidized form of AMB was followed at 604nm using an extinction coefficient of 9.8 mM-'cm-' [16]. Measurements were performed in 0.05 M phosphate buffer solution pH 7.0, containing 50 pM of H202. The concentration of HRP and rCiP was 2 nM, verified at 403 and 405 nm using extinction coefficients of 9.5 x lo4 and 1.04~ lo5 M-' cm-' for HRP and rCiP, respectively. Laccasescatalyzed AMB oxidation was performed in air saturated 0.05M Na-acetate buffer solution pH 5.3. Concentrations of PPL and BCL were 6.4 nM and 20 nM, respectively. For the determination of the concentration of laccases an extinction coefficient of 1.2 mL mg-'cm-' at 280 nm was used for both enzymes. Molecular weight of laccases was assumed 65 and 74kDa [5] for PPL and BCL, respectively. For the peroxidases and laccases the dependence of the initial reaction rate on substrate concentration was analyzed in a ping-pong scheme, following apparent k,, calculation as k,,/K,,, [16].

3. Results and Discussion

-

0

sequence of potentials applied to electrodes and the amount of buffer solution was the same a for MCPE electrodes. Cyclic voltammograms (CV) of both (MCPE and CMGE) electrodes were performed using a computer-controlled potentiostat (Model CS-2Ra, Cypress Systems, USA) in a three-electrode regime. CV for MCPE was obtained by applying 50pL of buffer solution to the electrode, as described above. CV of CMGE was recorded in a thermostated glass cell at 25 -+ 0.I"C by using the three-electrode system. A platinum plate (surface area 0.9 cm2) was used as an auxiliary electrode and SCE as a reference. Buffer volume was 5 mL. The bioelectrocatalytical current of the rotating MCPE was measured at 325-2249 min-' rotation by using homemade rotating electrode equipment. Carbon paste containing 1.67% of AMB was pressed into a Teflon holder and the electrode surface was polished with a filter paper. The geometrical surface of MCPE was 0.27 cm2. Before the measurements the electrode was immersed into a buffer solution of pH 7.0 containing 7.5 nM of HRP. After a potential of 46mV (vs. SCE) was applied to the electrode, the electrode was started to rotate when 1mM of hydrogen peroxide was introduced into the solution. The current at each rotation rate was expressed as an average between current at increasing and decreasing rotation rate.

3.1. Electrodes Response

g-looo 2 -500

*3

IDTResponse

IT

-1500

ADT

-2000 0

10

20

30

40

50

60

TIME, s Fig. 1. Time scheme of current measurement of carbon paste electrodes. ElectroanaIysis 1997, 9, No. 3

HRP-catalyzed electrocatalytical reduction of hydrogen peroxide in an AMB modified carbon paste electrode proceeded at an electrode potential ( E ) lower than 0.1V (vs. Ag/AgCl in situ) (Fig. 2). The electrocatalytical cathodic current exhibited constant values at E < 0.05 V. The electrode's response was 2.0 pC for 2 nM HRP at pH 7.0, electrode potential 0.0 V, integration time 30 s and 0.4mM hydrogen peroxide. At the same experimental conditions in buffer solution, the residual current of MCPE was less than 0.1 pC. The electrode's response did not change significantly in enzymefree buffer solution and in the presence of hydrogen peroxide solution; i.e., at 0.4mM hydrogen peroxide it was less than 0.2pC. The bioelectrocatalytical current depended on the hydrogen

Peroxidases and Laccases on CPE

715

Table 1 Sensitivity ot carbon paste and graphite electrodes Conditions ot medwrements electrode potential 0 0 V (vs Ag/AgCI in situ), DT 60 s, IDT Os, IT 1 0 ~ .and 0 4 m M hydrogen peroxide pH 7 0 for peroxidases or 0 25 mM oxygen pH 5 3 for laccases CP contained 0 167% AMB and graphite electrode was modified with 51 5 pg of AMB -

Enzvmc

-0.25

-0.15

-0.05

0.05

0.15

HRP rclP PPL BCL HRP

0.25

POTENTIAL, V vs Ag/AgCl in situ Fig. 2. Dependence of the integrated bioelectrocatalytical current of HRPcatalyzed reduction of hydrogen peroxide (1) and PPL-catalyzed reduction of oxygen (2) at a modified carbon paste electrode on the electrode potential at pH 7.0 (1) and pH 5.3 (2). Concentrations: HRP 3.9 nM (1); H2020.53 mM (1); PPL 120nM (2). DT = 60s; IT = 10s.

12t

~-

Electrodr

MCPE MCPE MCPE MCPE CMGE

-~~

Senritivitj ( 2 rd) [pc n N ' /

Jenutivriy per cm2

0 97 t 0 03 022+001 0 023 ? 0 002 0 016 Z 0 002 253t27

23 4 54 0 55 0 39 96 0

[ p c nM

~-

~

tnz-'/

.

i

n1

;8

86 a

3.5-

G 4

e:

v ~

4.5

5.5

6.5

-

7.5

8.5

SOLUTION pH

2 0 0

100

200

300

400

CONCENTRATION, n M Fig. 3. Dependence of the integrated cathodic current of MCPE on the concentration of HRP (l),rCiP (2) and PPL (3) at pH 7.0 (1,2) and pH 5.3 (3). Concentration of hydrogen peroxide 0.4 mM (1,2); DT = 60 s; IT = 10 s; potential 0.OV (vs. Ag/AgCl in situ).

peroxide concentration according to the Michaelis-Menten equation. In the range from 0.01 to 0.4mM hydrogen peroxide the electrode response showed a hyperbolic form. At 3.9 nM HRP the maximal integrated current (RmJ and an apparent Michaelis constant (K",) were 3.8 2 0.1 pC and 76.2 ? 7.2pM, respectively. The electrocatalytical oxygen reduction current by the PPLcatalyzed reaction was shifted to higher potentials, but approached a maximum at E < 0.1 V (Fig. 2). On the basis of the response/ potential dependence a potential of the working electrode of 0.0 V (vs. Ag/AgCl) was chosen to study the sensitivity for both peroxidases and laccases. At this potential the dependence of the integrated current on HRP concentration exhibits a sigmoidal form (Fig. 3). At 0.4mM of hydrogen peroxide the electrode showed linear response up to 10nM HRP and 30 nM rCiP. In air saturated solution the laccases calibration graph achieved a constant value at 200nM of PPL (Fig. 3). The calibration graph of BCL was similar to PPL (not shown). The sensitivity of the electrodes calculated as slope in the linear range of the calibration graph is depicted in Table 1. As shown the sensitivity for rCiP was 4.3 time lower than for HRP. The sensitivity for laccases was significant lower as compared to peroxidases, but the sensitivity for both laccases was similar (Table 1). A detection limit for HRP determination of 0.15nM was calculated from twice the standard deviation of the response of zero enzyme concentration. This value corresponds to 7.5 fmol of HRP in the measuring volume. The calculated detection limit for rCiP was 0.65nM. The detection limit for PPL and BCL were 5.9 nM and 8.6 nM, respectively. The dependence of the electrode response o n solution pH was bell shape with the maximum at pH 5.3-5.5 and pH 6.0-6.5 for

Fig. 4. Dependence of the integrated cathodic current of MCPE on solution pH. Concentrations: 200 nM PPL (1); 650 nM BCL (2); 5 nM HRP, 0.4 mM H20, (3). DT = 60s; IT = 10s; potential 0.OV (vs. Ag/AgCl in situ).

8,

I

I 0

30

60

90

120

150

180

DELAY TIME, s Fig. 5. Dependence of the integrated cathodic current (2,3) and residual current (1) of MCPE on the delay time in phosphate buffer solution pH 7.0 (1,2), 5.3 (3). Concentrations: HRP 7.5 nM (2); H202 0.4rnM (2); and PPL 200 nM (3). Potential 0.OV (vs. Ag/AgCl in situ), IT = 10s.

laccases and HRP, respectively (Fig. 4). Optimal activity for rCiP was shifted to the alkaline area about 2.3 units of pH in comparison to HRP, (data not presented in the text). Approximation of the activity dependence on pH by a scheme of single proton transfer (Eq. 1 ) gave apparent pK, values for PPL, BCL or HRP of 5.70 and 5.28, 4.69 and 6.29 or 4.84 and 7.42, respectively.

+

R = RLaX/(I [H+]/K,

+ Kz/[H+])

(1)

where Rkax is maximal response, [Hf] is hydrogen ions concentration, K , and K2 are apparent dissociation constants (pK, in text corresponded to -log(K,) and -log(K,)). The influence of DT on the electrodes current was studied between 0 to 180 s. The response of the electrodes increased when DT increased up to L J s (Fig. 5 ) and decreased at larger DT . For the comparison of the response of carbon paste electrodes (MCPE) with chemically modified graphite electrodes (CMGE) the dependence of bioelectrocatalytical current on HRP concentration Electroanalysis 1997, 9, No. 3

J. Kulvs et al.

216 3,

-3

I

I

'

100

0

200

300

I

I

400

POTENTIAL, V vs SCE

MEDIATOR, m M

Fig. 6. Cyclic voltammogram of MCPE in phosphate buffer solution pH 7.0 at a potential scan rate of 0.1 V/s. Other conditions are described in the text.

was investigated under the same conditions. It was found that the sensitivity of CMGE was almost 4 times larger in comparison to MCPE (Table 1).

3.2. Mediator Concentration on the Electrode and its Kinetic Parameters For the explanation of the mechanism of bioelectrocatalytical current generation, cyclic voltammetry experiments of modified electrodes were performed. In Figure 6 the cyclic voltammogram (CV) of MCPE is depicted. After incubation in buffer for 60s (DT = 60s) CV showed anodic (E,) and cathodic (E,) peaks at 183 C 3 m V and 133 C 4 m V (vs. SCE), respectively. The peak potential separation (AE) was 53 ? 4mV. This indicated that mediator dissolved in buffer solution and that electrochemical conversion of AMB on CPE is reversible. The calculated formal potential (ITo) of the mediator [E,= (E, Ec)/2] was 158 mV (vs. SCE). The CV of the chemically modified graphite electrode incubated for the same time in buffer solution showed larger peak currents and the calculated formal potential was 202 ? 13 mV (vs. SCE). The concentration of AMB in buffer solution was calculated from the anodic peak current (I,,) according to [20]

+

Ip = (2.69 x lo5)n3/2AD"2v1'2c where n is number of electrons, A is the geometrical surface area of the electrode, D is the diffusion coefficient (6.3 x lop6cm2/s [16]), v is the potential scan rate and c is the mediator Concentration. (Dimensions of parameters in the Equation 2 are expressed in cm, s, V and mol). The calculated concentration of mediator was 0.26 mM when MCPE was incubated in buffer solution for 6 0 s (Table 2). At the same incubation time the concentration of mediator on CMGE was significant larger. It increased proportionally to the amount of AMB used for graphite electrodes modification (Table 2). Thus, the

Fig. 7. Dependence of the integrated cathodic current of CMGE on AMB concentration in buffer solution after DT = 60 s. Concentration: HRP 7.5 nM; H20, 0.4 mM. IT = 10 s. Other experimental conditions are described in the text.

Table 3. Kinetic parameters (?sd) of AMB oxidation catalyzed by peroxidases and laccases at pH 7.0 and 5.3, respectively. Enzyme

KJpMl

HRP rCiP PPL BCL

13.6 C 1 2 76.7 C 8.9 137 ? 17 82.5 2 11

kd-'I

427 2 11 1030 C 58 218 C 13 46 C 3 ~ _ _ . _~ _ _ _

k d K ' s-

I

31.5 ? 3.6 13.4 t 2.3 1.6 t 0.3 0.55 t 0.11 ~ ~ _

_

_

sensitivity of CMGE was depended on the amount of mediator used for the electrodes modification (Fig. 7). The dependence of the CMGE response on the mediator concentration determined from cyclic voltammetry was analyzed by using Michaelis-Menten kinetics. The calculated R,, was 154 -+ 15pC and K , was 0.72 2 0.25mM at 7.5nM of HRP and sensitivity expressed as R,IK, was 28.5 pC/nM mM. Sensitivity divided to cm2 was 104 pC/nM mM cm2. The dependence of MCPE sensitivity on mediator concentration in CP was almost linear in the range 0.067-0.167%. Therefore the sensitivity expressed per mM of mediator was 90 pC/nM cm2 mM. Table 3 shows the results of comparative studies of peroxidasesand laccases-catalyzed AMB oxidation in homogeneous solution accomplished spectrophotometrically. An apparent K , for peroxidases-catalyzed A h 3 oxidation of 13.6 and 76.6pM and the apparent oxidation rate constant of 31.5 and 13.4pM-' s-' were obtained for HRP and rCiP, respectively (Table 3). The &,-values for laccases were little higher, but the apparent bimolecular constants were more than one order of magnitude smaller. Kinetic data of biocatalytic oxidation of AMB correlated well with the sensitivity of MCPE. The highest sensitivity was obtained for HRP (Table 1) and the oxidative constant was highest in reaction of HRP with AMB, too (Table 3). The sensitivity of rCiP decreased though k,,, for this peroxidase was 2.4 time lower. Also the sensitivity of laccases decreased proportionally to the decrease of kOx.

Table 2 Anodic peak current ( I p ) and concentration of mediator (L) in solution after 60 s incubation of the electrodes MCPE electrodes contained 0 167% of AMB, CMGE electrodes were modified with different amounts of 3.3. Mechanism of Bioelectrocatalytical Current Generation AMB _ ~ _ _ _ Kinetic measurements in solution showed that AMB was Eleclrode Amount of I,[pA] c[mM] oxidized in the enzyme (oxidases)-catalyzed process. Therefore, AMB[@gl ~

~

~~~

-

-

MCPE CMGE CMGE CMGE

24 85 159 338

I2 9 25 7 51 5

~~

0 26 14 26 56

-~~~~-

Elecjroanalysis 1997, 9, No. 3

~

~.

the scheme of bioelectrocatalyticaI current generation included the enzymes (E)-catalyzed mediator (&Ired) conversion and electrochemical reduction of oxidized mediator (Max):

(3)

_

Peroxidases and Laccases on CPE -k

-

217 Mred

(4)

CV experiments of the electrodes, which were incubated for a certain time in buffer solution, demonstrated mediator liberation into solution. The bioelectrocatalytical current was a function of mediator concentration on the electrode (Fig. 7). In the model of current generation it was possible to assume that the steady-state concentration of reduced mediator close to the electrode surface was constant due to a large excess of mediator in the pasting liquid. It dropped down to zero at the boundary of the diffusion layer (6). Steady-state change of mediator's concentration in the diffusion layer at saturating hydrogen peroxide or oxygen concentration was expressed by

At high enzyme concentration or large diffusion module, the calculated current (Eq.7) did not saturate. The reason for the current saturation (Fig. 3) was associated with hydrogen peroxide or oxygen limitation. This was confirmed in the experiments by using a rotating disk electrode. At 7.5 nM of HRP the current changed in the range 6.6, 6.2, 5.9,5.6,6.0pA at rotation 325, 715, 1031, 1314 and 2249 min-'. The rotation changed the thickness of diffusion layer which can be expressed by the Levich equation [20]: 6 = 1.61 ~ 1 / 3 ~ 1 / 6 ~ - 1 / 2

where v is a kinematic viscosity and w is the electrode rotation rate (rotations per s). Introduction of this equation into Equation 8 gave MCPE (CMGE) current dependence on the rotation rate: I = 0.53 nFAD'~3v'~6k,,[E][M],/w'~2

where D is the diffusion coefficient of mediator, [MIredand [MI,, are the concentrations of the mediator in oxidized and reduced form, [EJ is the enzyme concentration and x the distance from the electrode surface. At boundary conditions [MIred= [MI,, [MI,, = 0 at x = 0, and [MIred= [MI,, = 0 at x 2 6, the calculation of Equations 5 and 6 led to the electrode current [21]

I = nFAD(actanh(a6) - 6-'1 [MI,

(7)

where ' a = ko, [E]/D. At a6 5 1, the electrode current was expressed by the following equation: I = 0.33nFA6koX[E][M],

(9)

(10)

Experimentally, such dependence was observed at a low rotation rate: the current decreases with the rotation rate. However, at higher rotation the electrode current changes slightly. Some factors can alter this theoretical dependence (Eq. 10). The dominant factor, possibly, was an increasing hydrogen peroxide flux. Apparent K,,, for the stationary electrode was 76.2pM. However, it increased when the electrode started to rotate. At high rotation rates K , can exceed the hydrogen peroxide concentration used (1 mM). Therefore, the electrode current was dependent on the hydrogen peroxide concentration on the surface. It increased when the electrode rotation rate increased.

4. Conclusion

(8)

Equation 8 shows that the biocatalytical current is directly proportional to mediator concentration at the electrode surface. At low enzyme concentration or low diffusion module (as)the current is directly proportional to the enzyme concentration and diffusion layer thickness. The comparison of the electrode sensitivities (Table 1) with enzyme kinetic data (Table 3) indicated strong sensitivity/activity correlation. This indicated that at low enzyme concentration the electrodes acted in the kinetic regime (a65 1). Therefore the current of the mediator modified electrode is proportional to the enzyme concentration (Eq. 8). The similar sensitivity normalized to mediator concentration of MCPE and CMGE indicated that the same mediator amount was generated at the electrodes surface. When the electrode acted in the kinetic regime (a65 1) the dependence of the electrode response on pH corresponded to enzyme activity dependence. The activity was dependent on protonation of enzyme active center. The decrease of the enzyme activities at alkaline pH can be explained by deprotonation of the active center. Deprotonated enzyme was completely nonactive. However, activity decrease in acidic solution was related to protonation of AMB. It was determined that pK, of AMB protonation is 5.7 and that the protonated mediator is practically not oxidizable by oxidases. The apparent pK, values of AMB protonation determined from the response decreased in acidic solutions, however, were different for different oxidases. The reason of this difference is that apparent pK, (5.70, 4.69 and 4.84 for PPL, BCL and HRP, respectively) depended on the ratio of apparent K, and concentration of mediator on the surface. If this ratio is low, the electrode response is not sensitive to mediator protonation. In contrast when the ratio is high, for example due to higher K,, the response decrease following AMB protonation. Since K, was the largest for PPL (Table 3) the pK, determined from the response was similar to pK, of AMB.

High sensitive carbon paste based systems which are of particular importance for the development of pseudohomogeneous amperometric enzyme immunoassay for the determination of oxidoreductases were developed. As enzymes, horseradish peroxidase, recombinant Coprinus cinereus peroxidase, Polyporus pinsitus laccase and Botrytis cinerea laccase were used. The optimal conditions, such as electrode potential, pH and delay time of signal recording were determined. The macrokinetic model of oxidoreductases determination was build and for it validation necessary kinetic and mass transport effects were explored in homogeneous solution.

5. Acknowledgements The work was supported by the Bundesministeriurn fur Forschung und Technologie (BMFT), FRG (project No. 10903/95). We are grateful to A. Hjelholt Pedersen and K. Mondorf from Novo Nordisk A / S , Denmark and to Dr. S.W. Tanenbaum from State University of New York, College of Environmental Science and Forestry, Syracuse, USA for the gift of the enzyme samples.

6. References [ll L. Jin, X. Wei, J. Gomez, M. Datta, A. Birkett, D.L. Peterson, Anal. Biochem. 1995, 229 54. [21 S.H. Jenkins, J. Immunol. Merhods 1992, 150, 91. [3] T. Porstmann, S. T. Kiessig, J. Immunol. Methods 1992, 150, 5. 141 P. Tijssen, Practice and Theory of Enzyme Immunoassay, Elsevier, Amsterdam, 1985. [5] D. Slornczynski, J.P. Nakas, S.W. Tanenbaum, Appl. Environ. Microbid. 1995, 61, 907.

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218 161 A.Y. Yaropolov, O.V. Skorobogatko, S.S. Vartanov, S.D. Varfolomeyev, Appl. Biochem. Biotechnol. 1994, 49, 257. [7] A.L. Ghindilis, A. Makower, C.G. Bauer, F.F. Bier, F.W. Scheller, Anal. Chim. Acta 1995, 304, 25. [Sl O.V. Skorobogatko, A.L. Gindilis, E.N. Troitskaja, A.M. Shuster, A.I. Yaropolov, Anal. Lett. 1994, 27, 2997. [9] G.JSnsson, L.Gordon, Electroanalysis 1989, I, 465. [lo] J. Kulys, R.D. Schmid, Bioelectron. Bioenerg. 1990, 24, 305. [ I I] U. Wollenberger, V. Bogdanovskaya, S. Bobrin, F. Scheller, M. Tarasevich, Anal. Lett. 1990, 23, 1795. [12] S.D. Varfolomeev, I.V. Berezin, in Advances in Physical Chemi.rtry (Ed: Y.M. Kolotyrkin), MIR publishers, Moscow 1982, p. 60. 1131 M. Vreeke, R. Maidan, A. Heller, Anal. Chem. 1992, 64, 3084.

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.I Kulys . et al. [14] J. Kulys, H.E. Hansen, Anal. Chim. Acta 1995, 303, 285. [I51 J. Kulys, W. Schuhmann, H.-L. Schmidt, Anal. Left. 1992, 25, 1011. [161 J. Kulys, T. Buch-Rasmussen, K. Bechgaard, V. Razumas, J. Kazlauskaite, J. Marcinkeviciene, J.B. Christensen, H.E. Hansen, J. Mol. Cat. 1994, 91, 407. [17] D.P. Nelson, L.A. Kiesov, Anal. Biochem. 1972, 49, 474. [IS] W.H.Koppeno1, Adv. Free Rad. B i d . Med. 1985, I , 91. [ 191 J. Kulys, J.A. Munk, T. Buch-Rasmussen, H.E. Hansen, Electroanalysis 1994, 6, 945. [20] A.J. Bard, L.R. Faulkner, Electrochemical Methods. Fundamentals and Applications, Wiley, New-York 1980. L211 J. Kulys, in Advances in Biosensors, Vol.1 (Ed: A.P.F Turner), JAI Press, London 1990, p. 107.