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Amperometric biosensor for the determination of phenolic compounds using a tyrosinase graphite electrode in a flow injection system
Journal of Biotechnology, 31 (1993) 289-300
289
© 1993 Elsevier Science Publishers B.V. All rights reserved 0168-1656/93/$06.00
BIOTEC 00967
Amperometric biosensor for the determination of phenolic compounds using a tyrosinase graphite electrode in a flow injection system F. O r t e g a a n d E. D o m l n g u e z Department of Analytical Chemistry, Faculty of Pharmacy, University of Alcald de Henares, Madrid, Spain
G. J 6 n s s o n - P e t t e r s s o n t a n d L. G o r t o n Department of Analytical Chemistry, University of Lund, P.O. Box 124, S-221 O0 Lund, Sweden
(Received 8 April 1992; revision accepted 13 December 1992)
Summary Selective and sensitive devices for the monitoring of phenol and phenolic compounds are required in clinical and environmental analysis. This p a p e r describes a biosensor for the analysis of phenolic compounds in a flow injection system. The enzyme electrode is based on the use of immobilized tyrosinase and the amperometric detection of the enzymatic product at - 5 0 mV vs. SCE. The enzyme is covalently immobilized on the surface of a carbodiimide-activated graphite electrode. The biosensor responds to a variety of phenolic substrates with different conversion efficiencies. The detection limit for phenol is 0.003 /~M ( S / N = 3), a quantification limit of 0.01 ~ M (rsd 3.7%), and an extended dynamic range up to 5 p~M is achieved with a sample frequency of 110 samples per hour. A m p e r o m e t r i c biosensor; Tyrosinase; Phenolic compounds
Correspondence to: F. Ortega, Department of Analytical Chemistry, Faculty of Pharmacy, University of Alcal~ de Henares, E-28871 Alcal~ de Henares, Madrid, Spain.
290
Introduction Phenolic compounds include a large variety of analytes with relevant significance in clinical analysis and in the control of environmental pollutants. Levodopa (L-3,4-dihydroxyphenylalanine), for instance, is routinely used for the treatment of Parkinson's disease and chlorinated phenols are widespread as insecticides, pesticides and disinfectants. The complexity of the samples and the low concentration of the analytes call for selective and sensitive detection systems to avoid matrix effects and sample preparation. Direct electrochemical oxidation of phenols takes place at large over-voltages: +500 mV and + 1000 mV vs. Ag/AgC1 under alkaline and acidic conditions, respectively (Nieminen and HeikkiRi, 1986). The irreversibility and complexity of the direct oxidation of phenolic compounds with the risk of the electrode fouling limit the use of naked electrodes for electrochemical detection. Moreover at these potentials there is a high risk for interferences due to the oxidation of many organic compounds and further fouling of the electrode. To circumvent these drawbacks, biochemical modification of the electrode with enzymes allows the indirect detection of these compounds through species, which can be detectable at lower potentials. In this sense, the enzyme makes possible to decrease the applied potential of the analysis to optimum values between - 100 mV and + 100 mV vs. SCE (Marko-Varga and Domlnguez, 1991) and, moreover, it introduces a further selective step inherent to enzymatic catalysis. Polyphenol oxidases (PhOD, EC 1.14.18.1) catalyze the oxidation of phenolic compounds via hydroxylation with molecular oxygen to catechols and subsequent dehydrogenation to o-quinone (Kazandjian, and Klibanov, 1985). These oxidases, also known as tyrosinase, are widely distributed in nature (Bendall and Gregory, 1963) and they contain copper which acts as built-in electron carrier undergoing reversible oxidation and reduction of the enzyme. As other oxidases containing copper as cofactor (Gorton et al., 1991), oxygen is the reoxidizing agent, being subsequently reduced to water. The strong oxidizing power of oxygen makes the overall reaction irreversible: Phenol + 0 2 Ph_~ODCatechol PhOD o-Quinone + H20 02
02
(1)
Quinones suffer from high instability in water and they readily polymerize to polyaromatic compounds which have proved to inactivate the enzyme (Horowitz et al., 1970). Quinones are electroactive species which can be reduced at low potentials (Rivas and Solis, 1991). By using the electrochemical reduction of quinones as detection reaction for the quantification of phenols with the tyrosinase electrode, three advantages may be predicted: (a) the low applied potential minimizes the risk of interferences opening the applicability of the sensor to complex samples, (b) the reaction inactivation is avoided by the electrochemical removal of the poisoning products, and subsequently (c) the risk for electrode fouling diminishes.
291 In previous work (Toyota et al., 1985) a tyrosinase enzyme electrode was reported based on the amperometric measurement of oxygen uptake during oxidation, for the determination of total protein serum. A carbon paste bioelectrode based on the use of banana tissue as the biocatalytic component has been described by Wang and Lin (1988) and used for the determination of dopamine. The banana tissue was entrapped in the slurry of the oil-graphite. Connor et al. (1989) have used a silicone-grease-based immobilization method for the preparation of a tyrosinase electrode. The biosensor responds to phenol with a sensitivity of 913 nA mM -1. Plant tissue and microorganisms, containing tyrosinase, have been incorporated into the micropores of reticulated vitreous carbon for the construction of biosensors (Wang and Naser, 1991). This paper describes another approach for making a biosensing probe for phenolic compounds. Covalent immobilization with carbodiimide and cross-linking of tyrosinase with glutaraldehyde on the surface of a graphite electrode is used to prepare a stable biosensor in a flow system. The characteristics and advantages of this tyrosinase graphite electrode are presented.
Materials and Methods
Chemicals Tyrosinase (polyphenol oxidase, PhOD, EC 1.14.18.1) from mushroom was purchased as a lyophilized powder (3430 U mg-~ protein, Sigma Cat. No. T 7755) and it was used as received. Water-soluble carbodiimide, 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride, was obtained from Sigma (Cat. No. E 6383). Glutaraldehyde was purchased as a 25% aqueous solution (Sigma Cat. No. G-5882), and prior to use the polymerized aldehyde was removed by addition of activated carbon. The mixture was centrifuged at 4°C and the supernatant was stored at -18°C. Phenol, chlorinated phenols, hydroquinone, catechol and paracetamol (Carlo-Erba), dopamine (Janssen), L-tyrosine, and terbutaline hemisulfate (Sigma), 3,4-dihydroxyphenylacetic acid, 4-hydroxybenzoic acid, 3-methoxyphenol, 3-hydroxybenzaldehyde, 3,4-dihydroxybenzoic acid, 2-chlorophenol, mcoumaric acid, 2,4-dihydroxybenzaldehyde, 2,4,5-trichlorophenol, 3-hydroxybenzaldehyde, cinnamic aldehyde, 4-methoxybenzoic acid, cinnamic acid, 2,6-dimethylphenol and caffeic acid, phosphate salts (Merck, Darmastadt, Germany) were used, without further purification. All chemicals were of analytical grade.
Preparation of the electrodes Rods of spectrographic graphite (RW 001, Ringsdorff-Werke GmbH) of 3.1 mm diameter were cut, polished on wet, fine emery paper, thoroughly washed with deionized water and allowed to dry at ambient temperature. They were then heated to 700°C for 90 s in a muffle furnace. They were cooled and stored in a desiccator until use.
292
Immobilization of tyrosinase Polished end of graphite rods were dipped into 2 ml of 0.05 M acetate buffer p H 4.8 containing 110 m M carbodiimide. An acidic pH, low ionic strength and an acetate buffer were chosen as optimum activation conditions of the carboxylic functionalities of the electrode material (Papisov et al., 1985; Dom~nguez et al., 1988). The activation was allowed to proceed for 2 h at ambient temperature. The activated electrodes were then carefully rinsed with buffer to eliminate the excess of carbodiimide which could inactivate the enzyme (Domfnguez et al., 1988). The coupling was made by dipping the activated electrodes into 0.1 ml of 0.1 M phosphate buffer p H 6 containing 1372 U of P h O D and 0.5% glutaraldehyde and allowing the reaction to proceed overnight at 4°C. The enzyme electrodes were carefully rinsed with buffer and stored dipped in a 0.1 M phosphate buffer at p H 6 in refrigerator at 4°C until use. When in use, the enzyme electrode was press-fitted into a Teflon holder so that only the flat circular end (0.0731 cm 2) was exposed to the flow.
Instrumentation Measurements were carried out in a single-channel flow injection system, containing a wall-jet flow through amperometric cell connected to a three-electrode potentiostat with a saturated calomel electrode (SCE) reference electrode and a Pt wire counter electrode. The immobilized enzyme graphite sensor was inserted as the working electrode in the cell. Samples of 25/xl were injected with a pneumatically operated valve (Cheminert type SVA) into the carrier, which consisted of a 0.1 M phosphate buffer at p H 6 delivered by a Gilson peristaltic pump. In all cases, the carrier was first filtered through 0 . 4 / x m pore diameter Millipore membranes, and degassed for 10 min by reduced pressure before use. The various parts of the flow injection system were connected with Teflon tubing (i.d. 0.5 mm) and Altex screw couplings. All measurements with the amperometric biosensor were performed with an applied potential of - 5 0 m V vs. SCE, if not otherwise stated.
Results and Discussion
Bioelectrocatalytic determination of phenol Phenol can be oxidized by tyrosinase in the presence of molecular oxygen, forming catechol as an intermediate product. Further catalytic oxidation of catechol yields o-quinone as the final product (see reaction 1). Quinones polymerize in water following a sort of non-enzyme-dependent chemical reactions. They can also be reduced electrochemically at the surface of graphite electrode at different potentials, according to the scheme proposed in Fig. 1. This enzyme electrode was inserted in a wall-jet cell and studied in the flow injection system shown in Fig. 2. The variation of the current with the applied potential for 25 Izl injections of 0.01 mM phenol is presented in Fig. 3. The flow rate was 0.7 ml min-1 and the carrier a 0.1 M phosphate buffer at p H 6. Reduction of quinone starts at + 150 mV vs. SCE
293
POLYMERIZATION
1
QUINONE
H2° 0 2 ~~__...
~'/
ne-
CATECHOL
GRAPHITE
e --
~
o2
PHENOL Fig. 1. Reaction detection scheme for the detection of phenol with a tyrosinase graphite electrode.
obtaining higher cathodic currents when the applied potential is lowered. Maxim u m current intensity was obtained at - 200 mV vs. SCE; at - 300 mV a decrease in the current intensity was registered probably due to the electrochemical reduction of molecular oxygen which may then become the limiting step in the overall reaction (see Fig. 1) or a potential dependence of the activity of the immobilized enzyme. When unmodified electrodes were inserted in the flow injection system, injections of 0.01 m M phenol gave no response between + 150 mV and - 300 mV vs. SCE. Further experiments were all carried out at - 5 0 m V vs. SCE, which was
Pump
Injector I
n~,~.,,,.
D,.,~,,,iostat
Recorder
i|ll~t
j
Fig. 2. Flow injection manifold.
294
2.5
~- 100
2
~- 8 0
0
Q.
1.5 <
60
~ 40
.
m
0.5
--~ IlJ 2 0 0 - 3 0 0 - 2 0 0 -100 0 100 E vs. SCE / mV
0 200
Fig. 3. Variation of the intensity current with the applied potential for a tyrosinase electrode in the flow injection system. Twenty-five /zl of 0.01 m M phenol were injected into the carrier stream. The carrier was 0.1 M phosphate buffer pH 6.0 and the flow rate was 0.7 ml min - I .
chosen as optimum working potential to achieve high current intensity and to preserve the enzyme from very low potentials. The mean peak current for 0.01 mM phenol was 1.6/xA with a range of _+0.4 IzA (20 electrodes), and for a flow rate of 0.7 ml min-1. The high current densities obtained after injections of phenol show the efficient coupling between the catalytic oxidation and the electrochemical reduction reactions in the tyrosinase graphite electrode. As stated above, quinones readily polymerize in aqueous solution, and this reaction may be responsible for inactivation of the enzyme. The fact that quinones are efficiently reduced at the surface of the graphite electrode allows the monitoring of the enzymatic reaction and subsequently a competition between the electrochemical reduction and the polymerization reactions is established (see Fig. 1). The instability in water of the produced quinone may define which reaction is favoured, that is to say that the chemical structure of the substrate and the operational conditions (pH and flow rate) will limit the stability and performance of the tyrosinase graphite electrode, considering that tyrosinase suffers from polymerization reaction inactivation. Attempts to increase the response by charging different amounts of the enzyme was not successful. Electrodes charged with 2042, 2712 and 3047 units of tyrosinase did not give higher current intensities.
Effect of pH The optimum pH in an enzyme electrode will be determined by the influence of this parameter on the catalytic activity and the electrochemical transducer. The dependence of the response of the tyrosinase graphite electrode on the pH of the carrier is shown in Fig. 4. An optimum is found at pH 6 when samples of phenol are injected into the flow injection system, and a sharper decrease is observed at higher pHs. This pH profile does not strictly correspond to the reported one (Horowitz el al., 1970) for the free enzyme, which shows a broad optimum pH range between 5 and 8 for the oxidation of dopamine and to our results with an
295
100 1.5 < 1 "
80
tO c~
60 40
i1)
°
0.5
° ~
20 I1) rr"
0 3.5
~
0 4.5
5.5
6.5 pH
7.5
8.5
Fig. 4. Variation of the intensity current with the pH in the flow injection system. Same experimental conditions as mentioned in Fig. 3. The applied potential was - 50 mV vs. SCE.
immobilized tyrosinase reactor in combination with photometric monitoring (unpublished results). Under these conditions, the optimum pH for the catalytic activity was found to be around 7 if the pH was kept constant in the spectrophotometric flow cell. When the pH was not adjusted in the flow cell, the activity increased up to pH 8, due to hyperchromic effect (unpublished results). According to these data, the lower optimum pH found for the tyrosinase graphite electrode results as a compromise between the catalytic activity and the electrochemical reduction of the quinone formed, which is also favoured by suppression of the polymerization reaction.
Effect of flow rate Figure 5 shows the variation of the response for a tyrosinase graphite electrode with the flow rate. Samples of 0.01 mM phenol were injected in the flow injection
3 100 2.5 tO
80 60
4O 2O n-
2 1.5 <::x 1
. m
0.5
0 0 0.1 0.3 0.5 0.7 0.9 1.1 1.3 1.5 Flow rate / ml min -1
Fig. 5. Variation of the iiatensity current with the flow rate. Same experimental conditions as mentioned in Fig. 3. The applied potential was - 50 mV vs. SCE.
296
3 2.5 ,< :=.
2.
'- 1.5 1
/
0.5 ,
0 0
Io
1
2
3
4 5 6 7 8 Phenol / I.=M
I P h e n II / IJM
9
10
11
Fig. 6. Calibration graph obtained for phenol from flow injection measurements. The injection volume was 25/zl, with a carrier of 0.1 M phosphate buffer at pH 6 and the flow rate was 0.6 ml min - I . The inserted graph shows the sensitivity/[phenol] 1 vs. the logarithm of phenol concentration.
system. The curve levels off to a plateau region starting at 1.1 ml m i n - t . Increasing the flow rate favours the mass transport of phenol to the catalytic surface and thus an increase in the current intensity could be expected if the enzyme is working under first order conditions. The plateau observed indicates that at high flow rates the kinetics of the overall catalytical and electrochemical reactions is the rate limiting step for the response. The faster the sample plug passes the electrode the smaller is the fraction of substrate oxidized and the faster the quinone is transported back into the bulk solution. It should be noticed that the plateau response is already obtained in the flow injection system at 1.1 ml rain -1 and represents approximately 40% of the maximum response achieved. Between 1 and 0.1 ml min-~ the response increases when the flow rate is decreased as a consequence of the longer time the sample is in contact with the enzyme. At 0.7 ml min-1 the peak response to injections of 0.01 mM phenol gave values corresponding to 29% of the steady-state value for the same flow. At this flow rate a residence time of 12 s was observed and a sample frequency of 110 samples per h was achieved.
Calibration A strictly linear calibration curve was obtained from 0.01/zM to 5 / z M at pH 6.0 in the flow injection system with 25/xl injections of phenol and 0.6 ml min -~ flow rate. The upper linear range limit can easily be extended by reducing the injection volume. The calibration is presented in Fig. 6. The average sensitivity was 0.49/zA /zM-1. Regression analysis gave a slope, intercept and a correlation coefficient of 0.3213/zA tzM-1, 0.0224/xA and 0.9985, respectively, (n = 9). The quantification limit was 0.01 /xM (cv = 3.7%, n = 23) and a detection limit of 0.003 /zM was determined using a S / N ratio of 3.
Electrode stability The stability of the tyrosinase graphite electrode depends on the compound tested. Different operational stabilities were observed with phenol and dopamine.
297 TABLE 1 Relative response of the tyrosinase graphite electrode for different phenolic compounds in the flow injection system Phenolic compounds, x
(i x / iphenoj)(Cohenol/
Catechol 4-Chlorophenol Phenol Dopamine Pyrogallol 3,4-Dihydroxyphenylacetic L-Dopa 3,4-Dihydroxycinnamicacid 2,4-Dihydroxybenzaldehyde L-Tyrosine Acetaminophen 3,4-Dihydrobenzoic acid 3-Methoxyphenol 3-Hydroxybenzaldehyde Resorcinol 2,5-Dichlorophenol 3,4-Dichlorophenol Hydroquinone Terbutaline sulphate 2-Chlorophenol m-Coumaric acid 4-Hydroxybenzoicacid 2,4,5-Trichlorophenol 3-Methoxybenzaldehyde Cinnamic aldehyde 4-Methoxybenzoicacid Cinnamic acid 2,6-Dimethylphenol Phenaeetin Phenylephrine
2.063 1.090 1.000 0.250 0.143 0.120 0.093 0.094 0.032 0.020 0.013 0.012 0.011 < 0.001 < 0.001 < 0.001 < 0.001 < 0.001 < 0.001 < 0.001 < 0.001 < 0.001 < 0.001 < 0.001 < 0.001 < 0.001 < 0.001 < 0.001 < 0.001 < 0.001
Cx)
Concentrations, c (mM) 0.003 0.010 0.010 0.040 0.100 0.100 0.100 0.050 0.010 0.100 0.100 0.040 0.040 0.040 0.100 0.200 0.200 0.200 0.200 0.050 0.050 0.020 0.050 0.050 0.050 0.050 0.050 0.050 0.050 0.050
The measurements were made by 25/zl injections of the indicated concentrations and related to the response to phenol. Carrier: 0.1 M phosphate buffer pH 6. Flow rate: 0.7 ml min -1.
A f t e r 50 consecutive injections of 0.01 m M p h e n o l , the response r e m a i n e d stable, while after 30 consecutive injections of 0.04 m M of d o p a m i n e a decrease of 10% in the r e s p o n s e was observed. T h e different stabilities may b e a t t r i b u t e d to the different q u i n o n e structure f o r m e d which can d e t e r m i n e the rate for b o t h electrochemical r e d u c t i o n a n d p o l y m e r i z a t i o n (see Fig. 1; O r t e g a et al., 1992). Moreover, the inactivation r e a c t i o n rate may also d e p e n d o n the chemical structure of the polymer. A higher c o n c e n t r a t i o n of d o p a m i n e was used to equal the conversion efficiency o b t a i n e d for p h e n o l (see T a b l e 1). T h e e n z y m e electrodes had to be kept in 0.1 M p h o s p h a t e buffer at p H 6 to r e m a i n stable. Otherwise, if they are kept u n d e r dry conditions, 100% of the activity is lost. T h e activity could n o t be recovered by flowing buffer solution. It
298
was observed that an air bubble, reaching the electrode surface in the flow system, could inactivate the enzyme electrode after 1 h of contact. Under storage conditions in 0.1 M phosphate buffer at pH 6 and 4°C, the biosensor is very stable. After 1 m o n t h o f storage, the electrodes keep the initial response. The covalent binding of tyrosinase to the graphite, via carbodiimide, stabilizes the active configurations of the enzyme preventing unfolding which can be expected when only adsorbed.
Relative activity Phenol oxidases are enzymes with a broad selectivity. The relative activity for different phenolic compounds is presented in Table 1. The values are related to the response obtained for injections of 0.01 mM phenol. The highest activity was found for catechol. The oxidation of this o-diphenol does not require the first hydroxylation step caused by the cresolase activity of the enzyme, and the final oxidation of catechol to o-quinone is catalyzed by the catecholase activity of the enzyme (Dawson and Magee, 1970). Different active centres in the enzyme control these activities (Burges, 1963). The ratio of the catecholase/cresolase activity is different for phenol oxidases of different origin and depends on the purification procedure of the enzyme, as an effect of the higher instability of the cresolase activity (Burges, 1963). However, the presence of a second - O H in the molecule is not the only requirement for a high response. Resorcinol and hydroquinone, diphenols with the hydroxyl groups in meta- and para-position did not show detectable response. The introduction of an hydroxyl group in ortho-position when there exists already one in meta or para position becomes difficult limiting the oxidation of these diphenols by tyrosinase. For L-tyrosine, p-hydroxyphenylalanine, the difficulties involved in the cresolase activity (introduction of hydroxyl group) are not as severe as in the case of hydroquinone. The response for the amino acid was 2% of the response obtained for phenol. With the acetamide group in para-position, as in the case of acetaminophen (paracetamol), 1% of the response for phenol was achieved. In this sense, the high response registered for 4-chlorophenol is remarkable. The response obtained by the substitution of the hydroxyl group in para position for a chlorine atom, gives a slightly higher response than for phenol. All these values are also affected by the second step (controlled by the catecholase activity), the formation of the o-quinone. This conclusion can be drawn from the relative response obtained for dopamine, 3,4-dihydroxyphenylacetic, L-Dopa, and 3,4-dihydroxycinnamic acid (caffeic acid), and 3,4-dihydroxybenzoic acid. All these compounds, which already contain a catechol skeleton, give a response 12%, 6%, 4.5%, 4.5% and 0.6%, respectively, in the relation to the one obtained for catechol. From these results, it can be inferred that the catecholase activity in tyrosinase decreases progressively by introduction in para position of the following groups: aminoethyl > acetic acid > 2-aminopropanoic acid = 2-propenoic acid > benzoic acid. The response found for pyrogallol demonstrates that the catecholase activity is also dependent on the presence of a group in meta-position. Pyrogallol, 1,2,3-trihydroxybencene, gave a response that represents 7% of that obtained for catechol.
299 It should be stated finally that the electrochemical reduction of the quinones formed will also contribute to the responses observed.
Conclusions In summary a biosensor based on covalently immobilized tyrosinase on the surface of graphite electrode has been described for the selective detection of phenol and several phenolic compounds in a flow system at a low applied potential which assures measurements unaffected by otherwise interfering species. The very short response time observed to phenol and other substrates for this biomodified electrode makes the carbodiimide-glutaraldehyde coupling a convenient method for the preparation of this biosensor. The performance of the tyrosinase graphite electrode presented here opens up the use of this sensor as a selective and sensitive detection system in liquid chromatography where broad enzyme selectivity and low detection limits are required. A wide variety of applications can be expected in the near future for the determination of phenolic compounds in clinical (Ortega et al., 1992), environmental, biotechnological and industrial samples based on biocatalytic electrodes. This may be implemented by the study of different polyphenoloxidases from diverse sources, which will extend the number of detectable phenolic compounds.
Acknowledgements Financial support from the Spanish Foreign office, The University of Alcal~ de Henares, The D G I C Y T (Direcci6n General de Investicaci6n Cientifica y T6cnica, PS90-0028), the Swedish Natural Science Research Council and the Swedish Board for Technical Development (STU and STUF) is gratefully acknowledged.
References Bendall, D.S. and Gregory, R.P.F. (1963) Purification of phenol oxidases. In: Pridham, J.B. (Ed.) Enzyme Chemistry of Phenolic Compounds. Pergamon, Oxford, pp. 7-24. Burges, N.A. (1963) Enzymes associated with phenols. In: Pridham, J.B. (Ed.) Enzyme Chemistry of Phenolic Compounds. Pergamon, Oxford, pp. 1-6. Connor, M.P., Sfinchez, J., Wang, J., Smyth, M.R. and Mannino, S. (1989) Silicone-grease-based immobilization method for the preparation of enzyme electrodes. Analyst, 114, 1427-1429. Dawson, Ch.R. and Magee, R.J. (1955) Plant tyrosinase. In: Colowick, S.P. and Kaplan, N.O. (Eds.) Methods in Enzymology,II. Academic Press, New York, pp. 817-827. Dom~nguez, E., Nilsson, M. and Hahn-H~igerdal, B. (1988) Carbodiimide coupling of /3-galactosidase Aspergillus oryzae to alginate. Enzyme Microb. Technol. 10, 606-610. Gorton, L., Cs6regi, E., Domlnguez, E., Emn6us, J. J6nsson-Pettersson, G., Marko-Varga, G. and Persson, B. (1991) Selective detection in flow analysis based on the combination of immobilized enzymes and chemicallymodified electrodes. Anal. Chim. Acta, 250, 203-248.
300 Horowitz, N.H., Fling, M. and Horn, G. (1970) Tyrosinase. In: Tabor, H. and Tabor, C.W. (Eds.) Methods in Enzymology XVII, A. Academic Press, New York, pp. 615-620. Kazandjian, R.Z. and Klibanov, A.M. (1985) Regioselective oxidation of phenols catalyzed by polyphenol oxidase in chloroform. J. Am. Chem. Soc. 107, 5448-5450. Marko-Varga, G. and Dom~nguez, E. (1991), Enzymes as analytical tools. Trends Anal. Chem. 10, 290-297. Nieminen, E. and Heikkil~i, P. (1986) Simultaneous determination of phenol, cresols and xylenols in work-place air, using a polystyrene-divinylbencene column and electrochemical detection. J. Chromatogr. 360, 271-278. Ortega, F., Cuevas, J.L., Centenera, J.I. and Domlnguez, E. (1992). Liquid chromatographic separation of phenolic related drugs using catalytic detection: comparison of an enzyme reactor and enzyme electrode. J. Pharm. Biomed. Anal. 10, 789-796. Papisov, M.I., Maksimenko, A.V. and Torchilin, V.P. (1985) Optimization of reaction conditions during enzyme immobilization on soluble carboxyl-containing carriers. Enzyme Microb. Technol. 7, 11-16. Rivas, G.A. and Solis, V.M. (1991) Indirect electrochemical determination of L-tyrosine using mushroom tyrosinase in solution. Anal. Chem. 63, 2762-2765. Toyota, T., Kuan, S.S. and Guilbault, G.G. (1985) Determination of total protein serum using a tyrosinase enzyme electrode. Anal. Chem. 57, 1925-1928. Wang, J. and Naser, N. (1991) Reticulated vitreous carbon-plant tissue composite bioelectrode. Anal. Chim. Acta 242, 259-265. Wang, J. and Lin, M.S. (1988) Mixed plant tissue-carbon paste bioelectrode. Anal. Chem. 60, 1545-1548.
289
© 1993 Elsevier Science Publishers B.V. All rights reserved 0168-1656/93/$06.00
BIOTEC 00967
Amperometric biosensor for the determination of phenolic compounds using a tyrosinase graphite electrode in a flow injection system F. O r t e g a a n d E. D o m l n g u e z Department of Analytical Chemistry, Faculty of Pharmacy, University of Alcald de Henares, Madrid, Spain
G. J 6 n s s o n - P e t t e r s s o n t a n d L. G o r t o n Department of Analytical Chemistry, University of Lund, P.O. Box 124, S-221 O0 Lund, Sweden
(Received 8 April 1992; revision accepted 13 December 1992)
Summary Selective and sensitive devices for the monitoring of phenol and phenolic compounds are required in clinical and environmental analysis. This p a p e r describes a biosensor for the analysis of phenolic compounds in a flow injection system. The enzyme electrode is based on the use of immobilized tyrosinase and the amperometric detection of the enzymatic product at - 5 0 mV vs. SCE. The enzyme is covalently immobilized on the surface of a carbodiimide-activated graphite electrode. The biosensor responds to a variety of phenolic substrates with different conversion efficiencies. The detection limit for phenol is 0.003 /~M ( S / N = 3), a quantification limit of 0.01 ~ M (rsd 3.7%), and an extended dynamic range up to 5 p~M is achieved with a sample frequency of 110 samples per hour. A m p e r o m e t r i c biosensor; Tyrosinase; Phenolic compounds
Correspondence to: F. Ortega, Department of Analytical Chemistry, Faculty of Pharmacy, University of Alcal~ de Henares, E-28871 Alcal~ de Henares, Madrid, Spain.
290
Introduction Phenolic compounds include a large variety of analytes with relevant significance in clinical analysis and in the control of environmental pollutants. Levodopa (L-3,4-dihydroxyphenylalanine), for instance, is routinely used for the treatment of Parkinson's disease and chlorinated phenols are widespread as insecticides, pesticides and disinfectants. The complexity of the samples and the low concentration of the analytes call for selective and sensitive detection systems to avoid matrix effects and sample preparation. Direct electrochemical oxidation of phenols takes place at large over-voltages: +500 mV and + 1000 mV vs. Ag/AgC1 under alkaline and acidic conditions, respectively (Nieminen and HeikkiRi, 1986). The irreversibility and complexity of the direct oxidation of phenolic compounds with the risk of the electrode fouling limit the use of naked electrodes for electrochemical detection. Moreover at these potentials there is a high risk for interferences due to the oxidation of many organic compounds and further fouling of the electrode. To circumvent these drawbacks, biochemical modification of the electrode with enzymes allows the indirect detection of these compounds through species, which can be detectable at lower potentials. In this sense, the enzyme makes possible to decrease the applied potential of the analysis to optimum values between - 100 mV and + 100 mV vs. SCE (Marko-Varga and Domlnguez, 1991) and, moreover, it introduces a further selective step inherent to enzymatic catalysis. Polyphenol oxidases (PhOD, EC 1.14.18.1) catalyze the oxidation of phenolic compounds via hydroxylation with molecular oxygen to catechols and subsequent dehydrogenation to o-quinone (Kazandjian, and Klibanov, 1985). These oxidases, also known as tyrosinase, are widely distributed in nature (Bendall and Gregory, 1963) and they contain copper which acts as built-in electron carrier undergoing reversible oxidation and reduction of the enzyme. As other oxidases containing copper as cofactor (Gorton et al., 1991), oxygen is the reoxidizing agent, being subsequently reduced to water. The strong oxidizing power of oxygen makes the overall reaction irreversible: Phenol + 0 2 Ph_~ODCatechol PhOD o-Quinone + H20 02
02
(1)
Quinones suffer from high instability in water and they readily polymerize to polyaromatic compounds which have proved to inactivate the enzyme (Horowitz et al., 1970). Quinones are electroactive species which can be reduced at low potentials (Rivas and Solis, 1991). By using the electrochemical reduction of quinones as detection reaction for the quantification of phenols with the tyrosinase electrode, three advantages may be predicted: (a) the low applied potential minimizes the risk of interferences opening the applicability of the sensor to complex samples, (b) the reaction inactivation is avoided by the electrochemical removal of the poisoning products, and subsequently (c) the risk for electrode fouling diminishes.
291 In previous work (Toyota et al., 1985) a tyrosinase enzyme electrode was reported based on the amperometric measurement of oxygen uptake during oxidation, for the determination of total protein serum. A carbon paste bioelectrode based on the use of banana tissue as the biocatalytic component has been described by Wang and Lin (1988) and used for the determination of dopamine. The banana tissue was entrapped in the slurry of the oil-graphite. Connor et al. (1989) have used a silicone-grease-based immobilization method for the preparation of a tyrosinase electrode. The biosensor responds to phenol with a sensitivity of 913 nA mM -1. Plant tissue and microorganisms, containing tyrosinase, have been incorporated into the micropores of reticulated vitreous carbon for the construction of biosensors (Wang and Naser, 1991). This paper describes another approach for making a biosensing probe for phenolic compounds. Covalent immobilization with carbodiimide and cross-linking of tyrosinase with glutaraldehyde on the surface of a graphite electrode is used to prepare a stable biosensor in a flow system. The characteristics and advantages of this tyrosinase graphite electrode are presented.
Materials and Methods
Chemicals Tyrosinase (polyphenol oxidase, PhOD, EC 1.14.18.1) from mushroom was purchased as a lyophilized powder (3430 U mg-~ protein, Sigma Cat. No. T 7755) and it was used as received. Water-soluble carbodiimide, 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride, was obtained from Sigma (Cat. No. E 6383). Glutaraldehyde was purchased as a 25% aqueous solution (Sigma Cat. No. G-5882), and prior to use the polymerized aldehyde was removed by addition of activated carbon. The mixture was centrifuged at 4°C and the supernatant was stored at -18°C. Phenol, chlorinated phenols, hydroquinone, catechol and paracetamol (Carlo-Erba), dopamine (Janssen), L-tyrosine, and terbutaline hemisulfate (Sigma), 3,4-dihydroxyphenylacetic acid, 4-hydroxybenzoic acid, 3-methoxyphenol, 3-hydroxybenzaldehyde, 3,4-dihydroxybenzoic acid, 2-chlorophenol, mcoumaric acid, 2,4-dihydroxybenzaldehyde, 2,4,5-trichlorophenol, 3-hydroxybenzaldehyde, cinnamic aldehyde, 4-methoxybenzoic acid, cinnamic acid, 2,6-dimethylphenol and caffeic acid, phosphate salts (Merck, Darmastadt, Germany) were used, without further purification. All chemicals were of analytical grade.
Preparation of the electrodes Rods of spectrographic graphite (RW 001, Ringsdorff-Werke GmbH) of 3.1 mm diameter were cut, polished on wet, fine emery paper, thoroughly washed with deionized water and allowed to dry at ambient temperature. They were then heated to 700°C for 90 s in a muffle furnace. They were cooled and stored in a desiccator until use.
292
Immobilization of tyrosinase Polished end of graphite rods were dipped into 2 ml of 0.05 M acetate buffer p H 4.8 containing 110 m M carbodiimide. An acidic pH, low ionic strength and an acetate buffer were chosen as optimum activation conditions of the carboxylic functionalities of the electrode material (Papisov et al., 1985; Dom~nguez et al., 1988). The activation was allowed to proceed for 2 h at ambient temperature. The activated electrodes were then carefully rinsed with buffer to eliminate the excess of carbodiimide which could inactivate the enzyme (Domfnguez et al., 1988). The coupling was made by dipping the activated electrodes into 0.1 ml of 0.1 M phosphate buffer p H 6 containing 1372 U of P h O D and 0.5% glutaraldehyde and allowing the reaction to proceed overnight at 4°C. The enzyme electrodes were carefully rinsed with buffer and stored dipped in a 0.1 M phosphate buffer at p H 6 in refrigerator at 4°C until use. When in use, the enzyme electrode was press-fitted into a Teflon holder so that only the flat circular end (0.0731 cm 2) was exposed to the flow.
Instrumentation Measurements were carried out in a single-channel flow injection system, containing a wall-jet flow through amperometric cell connected to a three-electrode potentiostat with a saturated calomel electrode (SCE) reference electrode and a Pt wire counter electrode. The immobilized enzyme graphite sensor was inserted as the working electrode in the cell. Samples of 25/xl were injected with a pneumatically operated valve (Cheminert type SVA) into the carrier, which consisted of a 0.1 M phosphate buffer at p H 6 delivered by a Gilson peristaltic pump. In all cases, the carrier was first filtered through 0 . 4 / x m pore diameter Millipore membranes, and degassed for 10 min by reduced pressure before use. The various parts of the flow injection system were connected with Teflon tubing (i.d. 0.5 mm) and Altex screw couplings. All measurements with the amperometric biosensor were performed with an applied potential of - 5 0 m V vs. SCE, if not otherwise stated.
Results and Discussion
Bioelectrocatalytic determination of phenol Phenol can be oxidized by tyrosinase in the presence of molecular oxygen, forming catechol as an intermediate product. Further catalytic oxidation of catechol yields o-quinone as the final product (see reaction 1). Quinones polymerize in water following a sort of non-enzyme-dependent chemical reactions. They can also be reduced electrochemically at the surface of graphite electrode at different potentials, according to the scheme proposed in Fig. 1. This enzyme electrode was inserted in a wall-jet cell and studied in the flow injection system shown in Fig. 2. The variation of the current with the applied potential for 25 Izl injections of 0.01 mM phenol is presented in Fig. 3. The flow rate was 0.7 ml min-1 and the carrier a 0.1 M phosphate buffer at p H 6. Reduction of quinone starts at + 150 mV vs. SCE
293
POLYMERIZATION
1
QUINONE
H2° 0 2 ~~__...
~'/
ne-
CATECHOL
GRAPHITE
e --
~
o2
PHENOL Fig. 1. Reaction detection scheme for the detection of phenol with a tyrosinase graphite electrode.
obtaining higher cathodic currents when the applied potential is lowered. Maxim u m current intensity was obtained at - 200 mV vs. SCE; at - 300 mV a decrease in the current intensity was registered probably due to the electrochemical reduction of molecular oxygen which may then become the limiting step in the overall reaction (see Fig. 1) or a potential dependence of the activity of the immobilized enzyme. When unmodified electrodes were inserted in the flow injection system, injections of 0.01 m M phenol gave no response between + 150 mV and - 300 mV vs. SCE. Further experiments were all carried out at - 5 0 m V vs. SCE, which was
Pump
Injector I
n~,~.,,,.
D,.,~,,,iostat
Recorder
i|ll~t
j
Fig. 2. Flow injection manifold.
294
2.5
~- 100
2
~- 8 0
0
Q.
1.5 <
60
~ 40
.
m
0.5
--~ IlJ 2 0 0 - 3 0 0 - 2 0 0 -100 0 100 E vs. SCE / mV
0 200
Fig. 3. Variation of the intensity current with the applied potential for a tyrosinase electrode in the flow injection system. Twenty-five /zl of 0.01 m M phenol were injected into the carrier stream. The carrier was 0.1 M phosphate buffer pH 6.0 and the flow rate was 0.7 ml min - I .
chosen as optimum working potential to achieve high current intensity and to preserve the enzyme from very low potentials. The mean peak current for 0.01 mM phenol was 1.6/xA with a range of _+0.4 IzA (20 electrodes), and for a flow rate of 0.7 ml min-1. The high current densities obtained after injections of phenol show the efficient coupling between the catalytic oxidation and the electrochemical reduction reactions in the tyrosinase graphite electrode. As stated above, quinones readily polymerize in aqueous solution, and this reaction may be responsible for inactivation of the enzyme. The fact that quinones are efficiently reduced at the surface of the graphite electrode allows the monitoring of the enzymatic reaction and subsequently a competition between the electrochemical reduction and the polymerization reactions is established (see Fig. 1). The instability in water of the produced quinone may define which reaction is favoured, that is to say that the chemical structure of the substrate and the operational conditions (pH and flow rate) will limit the stability and performance of the tyrosinase graphite electrode, considering that tyrosinase suffers from polymerization reaction inactivation. Attempts to increase the response by charging different amounts of the enzyme was not successful. Electrodes charged with 2042, 2712 and 3047 units of tyrosinase did not give higher current intensities.
Effect of pH The optimum pH in an enzyme electrode will be determined by the influence of this parameter on the catalytic activity and the electrochemical transducer. The dependence of the response of the tyrosinase graphite electrode on the pH of the carrier is shown in Fig. 4. An optimum is found at pH 6 when samples of phenol are injected into the flow injection system, and a sharper decrease is observed at higher pHs. This pH profile does not strictly correspond to the reported one (Horowitz el al., 1970) for the free enzyme, which shows a broad optimum pH range between 5 and 8 for the oxidation of dopamine and to our results with an
295
100 1.5 < 1 "
80
tO c~
60 40
i1)
°
0.5
° ~
20 I1) rr"
0 3.5
~
0 4.5
5.5
6.5 pH
7.5
8.5
Fig. 4. Variation of the intensity current with the pH in the flow injection system. Same experimental conditions as mentioned in Fig. 3. The applied potential was - 50 mV vs. SCE.
immobilized tyrosinase reactor in combination with photometric monitoring (unpublished results). Under these conditions, the optimum pH for the catalytic activity was found to be around 7 if the pH was kept constant in the spectrophotometric flow cell. When the pH was not adjusted in the flow cell, the activity increased up to pH 8, due to hyperchromic effect (unpublished results). According to these data, the lower optimum pH found for the tyrosinase graphite electrode results as a compromise between the catalytic activity and the electrochemical reduction of the quinone formed, which is also favoured by suppression of the polymerization reaction.
Effect of flow rate Figure 5 shows the variation of the response for a tyrosinase graphite electrode with the flow rate. Samples of 0.01 mM phenol were injected in the flow injection
3 100 2.5 tO
80 60
4O 2O n-
2 1.5 <::x 1
. m
0.5
0 0 0.1 0.3 0.5 0.7 0.9 1.1 1.3 1.5 Flow rate / ml min -1
Fig. 5. Variation of the iiatensity current with the flow rate. Same experimental conditions as mentioned in Fig. 3. The applied potential was - 50 mV vs. SCE.
296
3 2.5 ,< :=.
2.
'- 1.5 1
/
0.5 ,
0 0
Io
1
2
3
4 5 6 7 8 Phenol / I.=M
I P h e n II / IJM
9
10
11
Fig. 6. Calibration graph obtained for phenol from flow injection measurements. The injection volume was 25/zl, with a carrier of 0.1 M phosphate buffer at pH 6 and the flow rate was 0.6 ml min - I . The inserted graph shows the sensitivity/[phenol] 1 vs. the logarithm of phenol concentration.
system. The curve levels off to a plateau region starting at 1.1 ml m i n - t . Increasing the flow rate favours the mass transport of phenol to the catalytic surface and thus an increase in the current intensity could be expected if the enzyme is working under first order conditions. The plateau observed indicates that at high flow rates the kinetics of the overall catalytical and electrochemical reactions is the rate limiting step for the response. The faster the sample plug passes the electrode the smaller is the fraction of substrate oxidized and the faster the quinone is transported back into the bulk solution. It should be noticed that the plateau response is already obtained in the flow injection system at 1.1 ml rain -1 and represents approximately 40% of the maximum response achieved. Between 1 and 0.1 ml min-~ the response increases when the flow rate is decreased as a consequence of the longer time the sample is in contact with the enzyme. At 0.7 ml min-1 the peak response to injections of 0.01 mM phenol gave values corresponding to 29% of the steady-state value for the same flow. At this flow rate a residence time of 12 s was observed and a sample frequency of 110 samples per h was achieved.
Calibration A strictly linear calibration curve was obtained from 0.01/zM to 5 / z M at pH 6.0 in the flow injection system with 25/xl injections of phenol and 0.6 ml min -~ flow rate. The upper linear range limit can easily be extended by reducing the injection volume. The calibration is presented in Fig. 6. The average sensitivity was 0.49/zA /zM-1. Regression analysis gave a slope, intercept and a correlation coefficient of 0.3213/zA tzM-1, 0.0224/xA and 0.9985, respectively, (n = 9). The quantification limit was 0.01 /xM (cv = 3.7%, n = 23) and a detection limit of 0.003 /zM was determined using a S / N ratio of 3.
Electrode stability The stability of the tyrosinase graphite electrode depends on the compound tested. Different operational stabilities were observed with phenol and dopamine.
297 TABLE 1 Relative response of the tyrosinase graphite electrode for different phenolic compounds in the flow injection system Phenolic compounds, x
(i x / iphenoj)(Cohenol/
Catechol 4-Chlorophenol Phenol Dopamine Pyrogallol 3,4-Dihydroxyphenylacetic L-Dopa 3,4-Dihydroxycinnamicacid 2,4-Dihydroxybenzaldehyde L-Tyrosine Acetaminophen 3,4-Dihydrobenzoic acid 3-Methoxyphenol 3-Hydroxybenzaldehyde Resorcinol 2,5-Dichlorophenol 3,4-Dichlorophenol Hydroquinone Terbutaline sulphate 2-Chlorophenol m-Coumaric acid 4-Hydroxybenzoicacid 2,4,5-Trichlorophenol 3-Methoxybenzaldehyde Cinnamic aldehyde 4-Methoxybenzoicacid Cinnamic acid 2,6-Dimethylphenol Phenaeetin Phenylephrine
2.063 1.090 1.000 0.250 0.143 0.120 0.093 0.094 0.032 0.020 0.013 0.012 0.011 < 0.001 < 0.001 < 0.001 < 0.001 < 0.001 < 0.001 < 0.001 < 0.001 < 0.001 < 0.001 < 0.001 < 0.001 < 0.001 < 0.001 < 0.001 < 0.001 < 0.001
Cx)
Concentrations, c (mM) 0.003 0.010 0.010 0.040 0.100 0.100 0.100 0.050 0.010 0.100 0.100 0.040 0.040 0.040 0.100 0.200 0.200 0.200 0.200 0.050 0.050 0.020 0.050 0.050 0.050 0.050 0.050 0.050 0.050 0.050
The measurements were made by 25/zl injections of the indicated concentrations and related to the response to phenol. Carrier: 0.1 M phosphate buffer pH 6. Flow rate: 0.7 ml min -1.
A f t e r 50 consecutive injections of 0.01 m M p h e n o l , the response r e m a i n e d stable, while after 30 consecutive injections of 0.04 m M of d o p a m i n e a decrease of 10% in the r e s p o n s e was observed. T h e different stabilities may b e a t t r i b u t e d to the different q u i n o n e structure f o r m e d which can d e t e r m i n e the rate for b o t h electrochemical r e d u c t i o n a n d p o l y m e r i z a t i o n (see Fig. 1; O r t e g a et al., 1992). Moreover, the inactivation r e a c t i o n rate may also d e p e n d o n the chemical structure of the polymer. A higher c o n c e n t r a t i o n of d o p a m i n e was used to equal the conversion efficiency o b t a i n e d for p h e n o l (see T a b l e 1). T h e e n z y m e electrodes had to be kept in 0.1 M p h o s p h a t e buffer at p H 6 to r e m a i n stable. Otherwise, if they are kept u n d e r dry conditions, 100% of the activity is lost. T h e activity could n o t be recovered by flowing buffer solution. It
298
was observed that an air bubble, reaching the electrode surface in the flow system, could inactivate the enzyme electrode after 1 h of contact. Under storage conditions in 0.1 M phosphate buffer at pH 6 and 4°C, the biosensor is very stable. After 1 m o n t h o f storage, the electrodes keep the initial response. The covalent binding of tyrosinase to the graphite, via carbodiimide, stabilizes the active configurations of the enzyme preventing unfolding which can be expected when only adsorbed.
Relative activity Phenol oxidases are enzymes with a broad selectivity. The relative activity for different phenolic compounds is presented in Table 1. The values are related to the response obtained for injections of 0.01 mM phenol. The highest activity was found for catechol. The oxidation of this o-diphenol does not require the first hydroxylation step caused by the cresolase activity of the enzyme, and the final oxidation of catechol to o-quinone is catalyzed by the catecholase activity of the enzyme (Dawson and Magee, 1970). Different active centres in the enzyme control these activities (Burges, 1963). The ratio of the catecholase/cresolase activity is different for phenol oxidases of different origin and depends on the purification procedure of the enzyme, as an effect of the higher instability of the cresolase activity (Burges, 1963). However, the presence of a second - O H in the molecule is not the only requirement for a high response. Resorcinol and hydroquinone, diphenols with the hydroxyl groups in meta- and para-position did not show detectable response. The introduction of an hydroxyl group in ortho-position when there exists already one in meta or para position becomes difficult limiting the oxidation of these diphenols by tyrosinase. For L-tyrosine, p-hydroxyphenylalanine, the difficulties involved in the cresolase activity (introduction of hydroxyl group) are not as severe as in the case of hydroquinone. The response for the amino acid was 2% of the response obtained for phenol. With the acetamide group in para-position, as in the case of acetaminophen (paracetamol), 1% of the response for phenol was achieved. In this sense, the high response registered for 4-chlorophenol is remarkable. The response obtained by the substitution of the hydroxyl group in para position for a chlorine atom, gives a slightly higher response than for phenol. All these values are also affected by the second step (controlled by the catecholase activity), the formation of the o-quinone. This conclusion can be drawn from the relative response obtained for dopamine, 3,4-dihydroxyphenylacetic, L-Dopa, and 3,4-dihydroxycinnamic acid (caffeic acid), and 3,4-dihydroxybenzoic acid. All these compounds, which already contain a catechol skeleton, give a response 12%, 6%, 4.5%, 4.5% and 0.6%, respectively, in the relation to the one obtained for catechol. From these results, it can be inferred that the catecholase activity in tyrosinase decreases progressively by introduction in para position of the following groups: aminoethyl > acetic acid > 2-aminopropanoic acid = 2-propenoic acid > benzoic acid. The response found for pyrogallol demonstrates that the catecholase activity is also dependent on the presence of a group in meta-position. Pyrogallol, 1,2,3-trihydroxybencene, gave a response that represents 7% of that obtained for catechol.
299 It should be stated finally that the electrochemical reduction of the quinones formed will also contribute to the responses observed.
Conclusions In summary a biosensor based on covalently immobilized tyrosinase on the surface of graphite electrode has been described for the selective detection of phenol and several phenolic compounds in a flow system at a low applied potential which assures measurements unaffected by otherwise interfering species. The very short response time observed to phenol and other substrates for this biomodified electrode makes the carbodiimide-glutaraldehyde coupling a convenient method for the preparation of this biosensor. The performance of the tyrosinase graphite electrode presented here opens up the use of this sensor as a selective and sensitive detection system in liquid chromatography where broad enzyme selectivity and low detection limits are required. A wide variety of applications can be expected in the near future for the determination of phenolic compounds in clinical (Ortega et al., 1992), environmental, biotechnological and industrial samples based on biocatalytic electrodes. This may be implemented by the study of different polyphenoloxidases from diverse sources, which will extend the number of detectable phenolic compounds.
Acknowledgements Financial support from the Spanish Foreign office, The University of Alcal~ de Henares, The D G I C Y T (Direcci6n General de Investicaci6n Cientifica y T6cnica, PS90-0028), the Swedish Natural Science Research Council and the Swedish Board for Technical Development (STU and STUF) is gratefully acknowledged.
References Bendall, D.S. and Gregory, R.P.F. (1963) Purification of phenol oxidases. In: Pridham, J.B. (Ed.) Enzyme Chemistry of Phenolic Compounds. Pergamon, Oxford, pp. 7-24. Burges, N.A. (1963) Enzymes associated with phenols. In: Pridham, J.B. (Ed.) Enzyme Chemistry of Phenolic Compounds. Pergamon, Oxford, pp. 1-6. Connor, M.P., Sfinchez, J., Wang, J., Smyth, M.R. and Mannino, S. (1989) Silicone-grease-based immobilization method for the preparation of enzyme electrodes. Analyst, 114, 1427-1429. Dawson, Ch.R. and Magee, R.J. (1955) Plant tyrosinase. In: Colowick, S.P. and Kaplan, N.O. (Eds.) Methods in Enzymology,II. Academic Press, New York, pp. 817-827. Dom~nguez, E., Nilsson, M. and Hahn-H~igerdal, B. (1988) Carbodiimide coupling of /3-galactosidase Aspergillus oryzae to alginate. Enzyme Microb. Technol. 10, 606-610. Gorton, L., Cs6regi, E., Domlnguez, E., Emn6us, J. J6nsson-Pettersson, G., Marko-Varga, G. and Persson, B. (1991) Selective detection in flow analysis based on the combination of immobilized enzymes and chemicallymodified electrodes. Anal. Chim. Acta, 250, 203-248.
300 Horowitz, N.H., Fling, M. and Horn, G. (1970) Tyrosinase. In: Tabor, H. and Tabor, C.W. (Eds.) Methods in Enzymology XVII, A. Academic Press, New York, pp. 615-620. Kazandjian, R.Z. and Klibanov, A.M. (1985) Regioselective oxidation of phenols catalyzed by polyphenol oxidase in chloroform. J. Am. Chem. Soc. 107, 5448-5450. Marko-Varga, G. and Dom~nguez, E. (1991), Enzymes as analytical tools. Trends Anal. Chem. 10, 290-297. Nieminen, E. and Heikkil~i, P. (1986) Simultaneous determination of phenol, cresols and xylenols in work-place air, using a polystyrene-divinylbencene column and electrochemical detection. J. Chromatogr. 360, 271-278. Ortega, F., Cuevas, J.L., Centenera, J.I. and Domlnguez, E. (1992). Liquid chromatographic separation of phenolic related drugs using catalytic detection: comparison of an enzyme reactor and enzyme electrode. J. Pharm. Biomed. Anal. 10, 789-796. Papisov, M.I., Maksimenko, A.V. and Torchilin, V.P. (1985) Optimization of reaction conditions during enzyme immobilization on soluble carboxyl-containing carriers. Enzyme Microb. Technol. 7, 11-16. Rivas, G.A. and Solis, V.M. (1991) Indirect electrochemical determination of L-tyrosine using mushroom tyrosinase in solution. Anal. Chem. 63, 2762-2765. Toyota, T., Kuan, S.S. and Guilbault, G.G. (1985) Determination of total protein serum using a tyrosinase enzyme electrode. Anal. Chem. 57, 1925-1928. Wang, J. and Naser, N. (1991) Reticulated vitreous carbon-plant tissue composite bioelectrode. Anal. Chim. Acta 242, 259-265. Wang, J. and Lin, M.S. (1988) Mixed plant tissue-carbon paste bioelectrode. Anal. Chem. 60, 1545-1548.