Biosensors & Bioelectronics 17 (2002) 217 226 www.elsevier.

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Composite electrochemical biosensors: a comparison of three different electrode matrices for the construction of amperometric tyrosinase biosensors
B. Serra, S. Jime nez, M.L. Mena, A.J. Reviejo, J.M. Pingarro n *
Department of Analytical Chemistry, Faculty of Chemistry, Complutense Uni6ersity of Madrid, 28040 Madrid, Spain Received 7 November 2000; received in revised form 16 July 2001; accepted 7 August 2001

Abstract A comparison of the behaviour of three different rigid composite matrices for the construction of amperometric tyrosinase biosensors, which are widely used for the detection of phenolic compounds, is reported. The composite electrode matrices were, graphiteTeon; reticulated vitreous carbon (RVC)epoxy resin; and graphite ethylene/propylene/diene (EPD) terpolymer. After optimization of the experimental conditions, different aspects regarding the stability of the three composite tyrosinase electrode designs were considered and compared. A better reproducibility of the amperometric responses was found with the graphiteEPD electrodes, whereas a longer useful lifetime was observed for the graphite Teon electrodes. The kinetic parameters of the tyrosinase reaction were calculated for eight different phenolic compounds, as well as their corresponding calibration plots. The general trend in sensitivity was graphiteEPD \ graphite Teon  RVC epoxy resin. A correlation between sensitivity and the catalytic efciency of the enzyme reaction for each phenolic substrate was found. Furthermore, differences in the sensitivity order for the phenolic compounds were observed among the three biocomposite electrodes, which suggests that the nature of the electrode matrix inuences the interactions in the tyrosinase catalytic cycle. 2002 Elsevier Science B.V. All rights reserved.
Keywords: Composite electrode matrices; Tyrosinase biosensors; Phenolic compounds

1. Introduction Nowadays, the use of composite electrode matrices is well established as an efcient strategy to design robust electrochemical biosensors. These matrices are materials consisting of at least one conductor phase mixed with at least one insulator phase (Tallman and Petersen, 1990), and present several advantageous characteristics which can be of use in biosensors development. Probably, one of the most important features is the versatility that can be achieved with this type of approach. Thus, different species improving selectivity and/or sensitivity (biomolecules, cofactors, mediators) can be incorporated into the bulk of the electrode matrix. In this way, three-dimensional biocomponents reservoirs can be fabricated whose surface can be easily regenerated by
* Corresponding author. Tel.: + 34-91-394-4315; fax: + 34-91-3944329. E -mail address: pingarro@eucmax.sim.ucm.es (J.M. Pingarro n).

polishing. In addition, fast responses to the involved substrates can be expected because of the absence of supporting membranes on the electrode surface and the closeness of the biomolecules (and other components) to the electrode material. A lot of materials have been used to construct composite electrodes. In general, carbon-based matrices are ideal conductor phases to be used for the development of amperometric sensors (Ce spedes et al., 1996), whereas epoxy resins, silicone, polyurethane, metacrylate, Teon, etc. can be employed as insulator materials. The most popular group of bulk-modied bioelectrodes is carbonpaste biosensors (Byeld and Abuknestra, 1994; Gorton, 1995; Kalcher et al., 1995), but some problems associated with a lack of long-term stability, mainly under owing conditions, as well as of mechanization ability and compatibility with nonaqueous media, have led to the use of rigid composite matrices such as graphite epoxy (Wang and Varughese, 1990; Alegret et al., 1996) or graphite Teon (Wang et al., 1993).

0956-5663/02/$ - see front matter 2002 Elsevier Science B.V. All rights reserved. PII: S 0 9 5 6 - 5 6 6 3 ( 0 1 ) 0 0 2 6 9 - X

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The aim of this work is to compare the behaviour of three different rigid composite matrices, developed in our research group, for the construction of amperometric tyrosinase biosensors, which are widely used for the detection of phenolic compounds (Hedenmo et al., 1997; Ducey and Meyerhoff, 1998; Wang et al., 1994; Kotte et al., 1995; Nistor et al., 1995; Li et al., 1998; Narva ez et al., 1996; Daigle and Leech, 1997; Eggins et al., 1997; Cosnier et al., 1998). The composite electrode matrices to be compared were, graphite Teon; reticulated vitreous carbon (RVC) epoxy resin; and graphite ethylene/propylene/diene (EPD) terpolymer. The use of graphite Teon pellets for the fabrication of robust enzyme electrodes has been extensively documented by our group recently (Serra et al., 1999a,b; Cayuela et al., 1998; del Cerro et al., 1997; Ortiz et al., 1997), the bioelectrodes being prepared simply by inclusion of the enzyme(s) into the bulk of the graphite Teon pellet with no need of covalent attachments. Furthermore, we reported recently the fabrication and performance, in different aqueous and predominantly nonaqueous media, of a composite RVC amperometric peroxidase ferrocene electrode in which the insulator material used for lling the holes of the electrode matrix is an epoxy resin (Pen a et al., 1999). No covalent bindings were either needed to fabricate these composite polishable bioelectrodes. Finally, we have recently prepared a new composite material by mixing graphite and the EPD terpolymer (Alonso et al., 1999), a rubbery material with a 70 wt.% ethylene and a 4 wt.% 5-methylene-2-norbornene content. This material, with EPD contents around 2%, can be used as voltammetric electrode material as well as indicator electrode under owing conditions, and exhibits good signal-tobackground ratios for analytes of different hydrophobicity, and a very good resistance to fouling with no need of electrode surface pretreatment. Up to date, no data on the fabrication of enzyme electrodes by using this type of composite material are found in the literature. The comparison of capabilities of the amperometric composite tyrosinase biosensors are illustrated in this work by their response to different phenolic compounds (phenol, catechol, 4-chloro-3-methylphenol, 4-chloro-2-

methylphenol, 4-chlorophenol, 2,4-dimethylphenol, 2,3dimethylphenol, and 3,4-dimethylphenol), most of them included in the EPA pollutant list, in a phosphate buffer working solution. The enzyme reaction involves the catalytic oxidation of these compounds to the corresponding quinones, and the electrochemical reduction of these quinones was employed to monitor this reaction (Fig. 1).

2. Experimental

2.1. Apparatus, electrodes and electrochemical cells

Experiments were performed on a Metrohm (Herisau, Switzerland) 641 VA potentiostat connected to a Linseis (Serb, Germany) L6512 recorder. The electrochemical cell was a BAS (W. Lafayette, IN, USA) Model VC-2 cell with a BAS RE-1 Ag/AgCl/KCl (3 M) reference electrode and a platinum wire auxiliary electrode. Other apparatus used were a Metrohm 62810 rotating electrode connected to a E-510 Metrohm potentiostat, and a 728 Metrohm magnetic stirrer.

2.2. Composite tyrosinase electrodes

Graphite Teon tyrosinase composite electrodes were fabricated in the form of cylindrical pellets as described previously (Serra et al., 1999a). Regarding RVC epoxy resin tyrosinase electrodes, RVC cylinders were rstly chemically pretreated as described in a former article (Pen a et al., 1999). Then, the enzyme (from mushroom, EC 1.14.18.1, activity 3000 U per mg of solid, Sigma, St Louis, MO, USA) was immobilized by direct adsorption on the RVC by immersing the pretreated RVC cylinder for 30 min in a 0.05 mol l 1 phosphate buffer solution (pH 6.5) containing 3.0 mg of enzyme (9000 U). Next, the solvent was evaporated by passing an Ar stream through, and pores of RVC were lled with the epoxy resin (Araldit), as described in the above-mentioned article (Pen a et al., 1999). Graphite EPD (Aldrich, Milwaukee, WI, USA) tyrosinase composite electrodes were fabricated, also in the form of cylindrical pellets, as follows. Graphite (ultra-F purity; Carbon of America, Bay City, MI, USA), 0.540 g, and tyrosinase, 0.030 g, were accurately weighed and thoroughly mixed by mechanic stirring for 2 h in a 0.70 ml suspension of a 0.05 mol l 1 phosphate buffer solution of pH 6.5 at 4 C. Then, water was evaporated by passing an argon stream through the mixture, and 3.0 ml of a 1.0% (w/v) EPD solution in cyclohexane (Panreac, Barcelona, Spain) were added. The resulting mixture was thoroughly hand-mixed until cyclohexane was completely evaporated. Next, the mixture was pressed into pellets (1.3 cm diameter) by

Fig. 1. Schematic diagram displaying the enzyme and electrode reactions involved in the detection of phenolic compounds at composite tyrosinase electrochemical biosensors.

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Fig. 2. Current time recording obtained for successive additions of a stock solution of phenol (1.0 10 6 mol l 1) in phosphate buffer (0.05 M) of pH 6.5 at: (A) RVC epoxy resin tyrosinase, (B) graphite Teon tyrosinase and (C) graphite EPD tyrosinase electrodes. Applied potential, 0.15 V.

means of a Carver pellet press (Perkin Elmer, Norwalk, CT, USA) at 10 000 kg cm 2 for 10 min. Several 3.0 mm diameter cylindrical portions of the pellet were bored, and each portion was press-tted into a Teon holder. Electrical contact was made through a stainless steel screw.

2.3. Reagents and solutions

Other reagents used were phenol (Sigma), catechol (Sigma), 2,4-dimethylphenol (Aldrich), 2,3-dimethylphenol (Aldrich), 3,4-dimethylphenol (Aldrich), 4-chlorophenol (Aldrich), 4-chloro-2-methylphenol (Aldrich), 4-chloro-3-methylphenol (Aldrich), and methanol (Panreac). All chemicals were of analytical-reagent grade and the water used was obtained from a Milli-Q purication system (Millipore, Bedford, NA, USA). Stock solutions of the phenolic compounds, 0.10 mol l 1, were prepared in a 0.05 mol l 1 phosphate buffer of pH 6.5, or in methanol depending on the solubility of these compounds in water. More dilute standards were prepared by suitable dilution with the 0.05 mol l 1 phosphate buffer solution.

appropriate volume of the stock solution of the corresponding phenolic compound was added with a micropipete and amperometric measurements were carried out at the same potential and allowing the steady-state current to be reached. When the response obtained with any of the composite tyrosinase electrodes was signicantly lower than the original response (see below), regeneration of the electrode surface was performed in all cases by polishing for 5 s on a 150 grit SiC paper. After use, graphite Teon and RVC epoxy resin biocomposite electrodes were stored at 4 C in a refrigerator, whereas graphite EPD electrodes were stored at ambient temperature. Calculation of the bioelectrodes active area was accomplished by constructing rotating disc electrodes with each of the composite matrices, and recording amperograms after adding 50 ml of 0.1 mol l 1 potassium ferricyanide to the electrochemical cell containing 0.1 mol l 1 phosphate buffer (pH 7.0). These amperograms were registered at 0.20 V versus Ag/AgCl for different rotation rates of the electrodes. The value of the active area was obtained from the limiting current vs. the square root of the rotation rate plots.

2.4. Procedure
A similar experimental procedure was followed with all the three enzyme composite electrodes. Thus, amperograms in stirred solutions were recorded by immersing the biosensor, at room temperature, in the electrochemical cell containing 5.0 ml of the phosphate buffer solution which was mechanically stirred at a constant rate. Then, the selected potential was applied and the background current was allowed to stabilize. Next, the 3. Results and discussion Fig. 2 shows current-time recordings obtained for successive 1.0 10 6 mol l 1 phenol additions at the three composite tyrosinase electrodes. It can be seen that the graphite-EPD biosensor yielded the most sensitive response (almost 3-fold higher than that at the graphite Teon electrode and around 100-fold higher

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than that obtained with the RVC epoxy resin bioelectrode). The response times were comparable at the three electrodes, although somewhat longer at the RVC epoxy resin biosensor. Nevertheless, the steadystate current was always reached in no more than 3 min. Furthermore, the background current at the graphite Teon and graphite EPD electrodes was similar, whereas this current was much lower with the RVC epoxy electrode. As a consequence, the signal-tonoise ratio was comparable in the three cases.

3.1. Optimization of the composition of the electrode matrices

Concerning graphite Teon electrodes, both the percentage of Teon in the matrix (70%) and the amount of tyrosinase immobilized (90 000 U), were the same as those optimized previously by our group (Serra et al., 1999a). Regarding graphite EPD electrodes, composite pellets with EPD contents lower than 1% had poor compactness, whereas EPD contents above 7 8% showed poor conductivity (Alonso et al., 1999). Therefore, composite enzyme electrodes with EPD percentages between 1 and 7% were compared using 1.0 10 6 mol l 1 phenol as the substrate. Although the highest steady-state currents were achieved for EPD contents of 1% (0.90 9 0.07 mA), the mechanical consistency improved remarkably as the terpolymer percentage increased. Taking into account that not very big differences in the steady-state current were observed between 2% (0.75 9 0.05 mA) and 5% (0.68 9 0.02 mA) EPD, this later content was nally chosen to carry out further studies. Regarding the amount of enzyme entrapped into the electrode matrix, it was the same as for graphite Teon electrodes (90 000 U), since higher tyrosinase loading (a highest loading of 100 000 U was tested) did not improve the amperometric responses. Finally, concerning RVC epoxy resin tyrosinase electrodes, only the enzyme loading in the RVC matrix

was optimized, the remainder variables for the fabrication of the electrodes being the same as those optimized previously (Pen a et al., 1999). This was done by constructing three different biosensors containing tyrosinase loading of 4500, 9000 and 13 500 U, respectively. Sets of 10 amperometric measurements for 1.0 10 5 mol l 1 phenol at 0.15 V showed a high increase in the steady-state current when the enzyme content passed from 4500 (0.020 9 0.004 mA for a signicance level of 0.05 9000 U (0.040 9 0.006 mA), but only a slight increase from 9000 to 13 500 U (0.045 9 0.006 mA). Moreover, no signicant differences in stability were found for all of them. Consequently, an amount of 9000 U tyrosinase was selected for further work.

3.2. Optimization of other experimental conditions

The pH of the phosphate buffer working solution was optimized in the range 5.5 8.0. For the three composite enzyme electrodes, there was not a noticeable variation of the steady-state current between pH 5.5 and 6.5. However, the current decreased rapidly for higher pHs. Then, a 0.05 mol l 1 phosphate buffer solution of pH 6.5 was chosen for subsequent work. Regarding the inuence of the applied potential on the amperometric response, a similar behaviour was found for the three composite electrodes. A rapid increase of the current occurred for more reducing potentials than 0.10 V up to 0.15 V, following which the steady-state current remained practically constant. As an example, Fig. 3 shows the trend observed for the graphite EPD biosensor. A potential value of 0.15 V was selected because it is the less negative value in the current plateau, and, therefore, in this way, the number of potential interferents able to be reduced at the electrode can be minimized. Moreover, the background current and the stabilization time of the baseline also increased as the potential became more negative.

3.3. Stability of the composite tyrosinase biosensors

One of the most claimed advantages of the enzyme rigid composite electrodes is their stability and robustness, which is related in part to the simplicity of the electrode surface regeneration process, if needed. Different aspects regarding the stability of the three composite tyrosinase electrode designs were considered and compared. Firstly, the repeatability of successive amperometric measurements without regeneration of the electrode surface was evaluated. Sets of 10 measurements of 1.0 10-5 mol l 1 phenol, for graphite Teon and RVC epoxy resin electrodes, and of 1.0 10 6 mol l 1 phenol for the graphite-EPD electrode (the linear range of the response vs. the concentration plot was

Fig. 3. Inuence of the applied electrode potential on steady-state current obtained from a 1.0 10 6 mol l 1 phenol solution in phosphate buffer (0.05 M) of pH 6.5 at a composite graphite-EPD tyrosinase electrode.

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Fig. 4. Control charts constructed for: (A) graphite-Teon-tyrosinase, (B) graphite EPD tyrosinase and (C) RVC epoxy resin tyrosinase composite electrodes. Three measurements of different 1.0 10 5 mol l 1 (A and C) or 1.0 10 6 mol l 1 (B) phenol solutions daily. Eapp = 0.15 V.

obtained at lower concentrations for this electrode, as it will be shown below), yielded relative standard deviation (R.S.D.) values for the steady-state current of 4.0, 5.0, and 2.8%, respectively. Although, the graphite EPD biosensor offered a better repeatability, no big differences were observed for any electrode. On the other hand, the possibility of obtaining a fresh electrode surface simply by polishing is one of the most advantageous properties of the use of composite bioelectrodes. Thus, the reproducibility of the amperometric response was checked after regeneration of the electrode surfaces by polishing as described in the Section 2. Sets of 10 polishing, and three different measurements after each polishing, were carried out at the same phenol concentration levels commented above. R.S.D. values of 4.4, 5.6, and 2.4% were obtained for the ten steady-state current mean values of each series with the graphite Teon, the RVC epoxy resin, and the graphite EPD electrode, respectively. These results indicated that all the composite tyrosinase electrodes yielded reproducible amperometric responses after being subjected to the regeneration procedure, which means that the enzyme was uniformly distributed in the

electrode matrix. Nevertheless, again the R.S.D. was considerably lower for the graphite EPD biosensor, which can be attributed to the differences in the fabrication procedures. Actually, in the case of graphite EPD electrodes the insulator material was dissolved before mixing with graphite and the enzyme, which did not occur with the other enzyme electrodes. This can facilitate the obtention of a more homogeneous nal biomaterial. The useful lifetime of one single biosensor was also compared for the three composite biomaterials. This was done by performing everyday three measurements of different 1.0 10 5 mol l 1 phenol (or 1.0 10 6 mol l 1 in the case of the graphite EPD electrode) solutions. Control charts were constructed for the three electrodes (Fig. 4), considering as the central value the mean steady-state current calculated from the 10 successive measurements performed with no regeneration of the electrode surface mentioned above. In all cases, when a mean value was out of the lower limit (considered as the central value minus three times the standard deviation), the electrode surface was polished and the signal could then be restored inside the control limits. For the three electrode designs, it was necessary to polish the electrode surface at the beginning of each working day to recover the response. As can be seen in Fig. 4, the graphite Teon tyrosinase biosensor gave mean values of the amperometric signal inside the control limits over 30 days. However, the repetitive regeneration of the electrode surface resulted in a too thin composite pellet, and, consequently, the electrode became useless after approximately such a period of time. The control chart for the graphite EPD electrode shows a much shorter lifetime (5 days). This was due to the need of applying a longer polishing time (around 10 s) to recover the signal, which was attributed to a deeper fouling of the electrode matrix by the products of the enzyme reaction. Regarding the RVC epoxy resin tyrosinase biosensor, the useful lifetime of a single electrode was determined, in this case, by a loss of the enzyme activity, since from approximately 17 days, the amperometric response could not be recovered by polishing. We think that the enzyme activity is altered by the electrode matrix, as a consequence of a maturing process of the epoxy resin with time which could cause the enzyme deactivation. In any case, either because the electrode became useless after a repetitive regeneration of the sensing surface or due to a loss of the enzyme activity, the biocomposite electrode needed to be changed when the amperometric responses could not be restored by polishing. Therefore, the reproducibility of the analytical responses obtained with different electrodes constructed in the same manner for each design, is an important

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aspect to evaluate the real practical capabilities of the composite biosensors. In the case of graphite Teon and graphite EPD biosensors, this reproducibility was evaluated for four different electrodes, two of them fabricated from the same main pellet, and the other two from a different pellet, whereas in the case of the RVC epoxy resin design, the reproducibility was evaluated from the measurements obtained with six different electrodes. The results obtained are summarized in Table 1. The R.S.D. for the mean values of all the four electrodes was 5.0 and 4.3% for graphite Teon and graphite EPD electrodes, respectively, whereas it was 5.6% for the six RVC epoxy resin electrodes. These results indicate that the fabrication procedures of the composite tyrosinase biosensors are reliable in all cases, and allow the obtention of reproducible electroanalytical responses with different electrodes constructed in the same manner. Finally, it is important to remark that after 5 months of storage, at 4 C in a refrigerator, of graphite Teon and graphite EPD composite pellets, no signicant loss of the enzyme activity occurred, and similar (and reproducible) amperometric responses to those shown in Table 1 were obtained when the corresponding bioelectrodes were fabricated from such stored pellets. As a conclusion of all these studies, it can be said that a rather good stability and robustness of the three composite biosensor designs has been demonstrated. Nevertheless, it should be remarked the better reproducibility of the amperometric responses obtained with the graphite EPD electrodes in comparison with the other two biosensor designs, as well as the longer useful lifetime observed for the graphite Teon electrodes.

3.4. Kinetics of the enzyme reaction at the composite electrodes

Under the experimental conditions optimized for phenol, the kinetic parameters of the tyrosinase reaction were calculated and compared at the three composite matrices for the following compounds: phenol, catechol, 3,4-dimethylphenol, 4-chloro-3-methylphenol, 4-chlorophenol, 4-chloro-2-methylphenol, 2,4dimethylphenol and 2,3-dimethylphenol. The kinetics of the enzyme reaction for these phenolic compounds tted in all cases into a Michaelis Menten type kinetic, as demonstrated by the calculation of the parameter x from the Hills plots (log((imax/i ) 1] vs. the log of the substrate concentration). As can be seen in Table 2, this parameter was very close to 1 in all cases, indicating that the differences between the composite matrices did not affect the Michaelis Menten behaviour. Calculation of the apparent Michaelis Menten constants (Km, app.), and the maximum rate of the reaction (Vm) was accomplished from the corresponding Lineweaver Burk plots. The obtained values are shown in Table 2. When comparing Km, app. values obtained with each of the three tyrosinase composite biosensors developed, it can be observed that, in general, these values are lower for the graphite EPD electrode and higher for the RVC epoxy resin electrode. Regarding Vm, it could be found that this parameter was higher for the graphite EPD based biosensor and lower for the one constructed with RVC and epoxy resin. These trends depend mainly on two factors. On the one hand, they depend on the amount of immobilized enzyme on the electrode surface. Taking into account the bioelectrodes

Table 1 Reproducibility of the amperometric responses (n = 10) obtained with different electrodes Pellet Electrode I (mA) 1.76 9 0.04 1.80 9 0.08 1.83 9 0.07 1.97 9 0.07 0.66 9 0.01 0.68 9 0.02 0.69 9 0.02 0.73 9 0.02 0.039 0.040 0.044 0.038 0.043 0.040 R.S.D. (%) i( (mA) 1.8 9 0.1 R.S.D. (%)

GraphiteTeontyrosinase electrode 1 1 2 2 1 2 GraphiteEPDtyrosinase electrode 1 1 2 2 1 2 RVCepoxy resintyrosinase 1 2 3 4 5 6

4.8 5.6 5.5 4.8 2.8 3.2 3.8 4.1

5.0

0.69 9 0.05

4.3

0.041 9 0.002

5.6

1.0105 mol l1 phenol (graphiteTeon and RVCepoxi resin tyrosinase biosensors), and 1.0106 mol l1 phenol (graphiteEPD electrodes). Eapp = 0.15 V.

B. Serra et al. / Biosensors & Bioelectronics 17 (2002) 217 226 Table 2 Kinetic parameters of the tyrosinase reaction at composite tyrosinase electrodes: (1) graphiteTeon, (2) graphiteEPD and (3) RVCepoxy resin Compound Electrode x Vm, app (mA) 24.4 38.4 0.98 17.1 29.9 0.96 6.04 10.7 0.63 13.1 2.44 0.35 12.5 14.4 3.76 3.38 5.79 0.08 7.90 8.29 0.50 2.29 4.51 0.11 Km,app (M105) 7.76 6.26 24.0 4.99 5.63 20.0 0.788 1.04 11.0 3.76 0.089 6.80 6.90 2.53 97.0 17.2 5.80 120 53.3 22.9 64.0 435 36.1 1122

223

Phenol

Catechol

3,4- dimethylphenol

1 2 3 1 2 3 1 2 3 1 2 3 1 2 3 1 2 3 1 2 3 1 2 3

0.999 0.98 0.98 0.98 0.997 0.97 0.99 0.99 1.01 1.005 0.97 0.96 0.96 0.97 0.98 0.97 0.95 1.1 1.003 1.002 1.1 1.01 0.97 1.2

4-chloro-3-methylphenol

4-chlorophenol

4-chloro-2-methylphenol

2,4-dimethylphenol

2,3-dimethylphenol

Fig. 5. Current density vs. the square root of the rotation rate of: () graphite Teon tyrosinase, (") graphite EPD tyrosinase, and ( ) RVC epoxi resin tyrosinase electrodes. ( ) theoretical current density in the absence of enzyme recycling. Catechol 1.0 10 6 mol l 1.

On the other hand, and assuming that the rate limiting step of the reaction is in all cases the enzymatic oxidation of the phenolic compound, the Km, app values will depend on the phenolic compound recycling process at the electrodes surface. When the electrode active area is larger, such a process gives rise to an increase of the phenolic compound concentration in the diffusion layer. Then, assuming that the phenolic compound oxidation process is slower than the corresponding quinones electrode reduction, an enzyme saturation will be observed for lower concentrations of substrate, which implies lower Km, app values. As stated in the Section 2, the bioelectrodes active area was calculated by recording amperograms after adding 50 ml of a 0.1 mol l 1 potassium ferricyanide to the electrochemical cell containing 0.1 mol l 1 phosphate buffer (pH 7.0). These amperograms were registered at 0.20 V versus Ag/AgCl for different rotation rates of the electrodes, and the value of active area was obtained from the limiting current versus the square root of the rotation rate plots. These values were: 2.05 10 2 cm2, 7.00 10 2 cm2, and 5.96 10-3 cm2, for the graphite Teon, graphite EPD and RVC epoxy resin tyrosinase electrodes, respectively. Fig. 5 shows the plots of the current density (thus avoiding the active area contribution) vs. the rotation rate of enzyme electrodes constructed with each of the composite matrices studied, as well as a comparison with the theoretical current density (calculated from the Levich equation) when there is not enzyme recycling. It can be seen that the current density was 15.5-fold higher (for a rotation rate of 1500 rpm) at the graphite EPD bioelectrode, 12.7-fold higher at the graphite Teon electrode, and only 1.3-fold higher for the RVC epoxy resin biosensor than the current density obtained with no enzyme recycling. These differences indicate that the phenolic compound enzyme recycling was dependent on the electrode matrix used. On the other hand, when comparing the Km, app values obtained with the same composite electrode for the tested phenolic compounds, a higher Michelis Menten constant is always observed for those compounds with one ortho-position occupied; this means a lower afnity for the enzyme, and, therefore, lower responses than those of phenolic compounds substituted in para- and meta-positions.

3.5. Calibration plots and analytical characteristics

Table 3 summarises the characteristics of the calibration plots obtained for the phenolic compounds tested with the three composite tyrosinase electrodes under the optimised working conditions selected above. The corresponding limits of detection, calculated according to the 3sb/m criterium, where sb was estimated as the S.D. (n = 10) of the signals from different solutions of the

fabrication procedures, in all of which tyrosinase was rstly homogeneously adsorbed on the conductor phase, it can be assumed that a larger electrode active surface will imply a larger conductor material area, and, therefore, a higher amount of enzyme at the electrode surface. This factor will inuence mainly Vm.

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Table 3 Calibration data for the different phenolic compounds obtained at composite tyrosinase electrodes: (1) graphiteTeon, (2) graphiteEPD and (3) RVCepoxy resin Compound Phenol Electrode 1 2 3 1 2 3 1 2 3 1 2 3 1 2 3 1 2 3 1 2 3 1 2 3 Linear range, (mol l1) (0.125)106 (0.056)106 (0.024)105 (0.115)106 (0.058)106 (0.015)105 (0.16)106 (0.29)107 (0.013)105 (0.120)106 (0.23)107 (0.010.2)105 (0.748)106 (0.057)106 (0.018)105 (510)105 (230)106 (510)105 (0.7100)106 (150)106 (0.230)x105 (1.010)x104 (17)x105 (240)x104 r 0.997 0.998 0.9997 0.997 0.999 0.999 0.998 0.998 0.9997 0.999 0.998 0.999 0.998 0.999 0.999 0.998 0.999 0.998 0.998 0.999 0.997 0.999 0.998 0.999 Slope, (mA mol1 l) (2.79 9 0.09)x105 (6.2 9 0.2)x105 (3.4 9 3)x103 (3.0 9 0.1)x105 (6.6 9 0.1)x105 (4.11 9 0.09)x103 (5.2 9 0.2)x105 (1.2 9 0.1)106 (4.78 9 0.06)103 (2.91 9 0.07)105 (2.0 9 0.4)106 (5.9 9 0.9)103 (2.5 9 0.1)105 (4.6 9 0.1)105 (3.94 9 0.06)103 (6 9 5)103 (7.1 9 0.4)104 (2.1 9 0.4)102 (1.86 9 0.06)104 (3.3 9 0.1)104 (5.8 9 0.3)102 (4.0 9 0.8)102 (1.04 9 0.08)104 (7.7 9 0.4) LOD, mol l1 9.9108 2.63108 1.1107 1.0107 2.8108 9.2108 1.0107 8.7109 3.0108 3.1108 5.4109 3.5108 7.4108 2.3108 7.1108 2.3105 5.4109 4.2105 7.1107 6.7107 1.3106 6.7105 8.0106 8.1105

Catechol

3,4-dimethyl phenol

4-chloro-3-methylphenol

4-chlorophenol

4-chloro-2-methylphenol

2,4-dimethyl Phenol

2,3-dimethyl phenol

substrates at the concentration level corresponding to the lowest concentration of the calibration plot, are also given in Table 3. The condence intervals were calculated for a signicance level of 0.05. As expected, the general trend in sensitivity, when comparing the electrode matrices, was graphite EPD \ graphite Teon  RVC epoxy resin. As an example, and to allow a graphical view of the responses, Fig. 6 compares the calibration plots obtained for phenol, catechol and 4-chlorophenol with the three composite tyrosinase biosensors. As theoretically predicted, the sensitivity of the biosensors for each phenolic compound is higher as the corresponding Km, app is lower and the Vm value (which is proportional to the catalytic constant for the conversion of the enzyme-substrate complex into the product plus the enzyme) is higher. Thus, the ratio Vm/Km, app, the catalytic efciency (Fresht, 1980], gives an a priori indication of the substrates sensitivity trend. Furthermore, this parameter indicates the enzyme specicity for the different substrates (Peter and Wollenberger, 1997]. Table 4 collects the Vm/Km, app values for each of the enzyme biosensors developed. By comparing the slopes of the calibration plots (Table 3) with data of Table 4, it can be deduced that, actually, there is a correlation between sensitivity and the catalytic efciency of the enzyme reaction for each phenolic substrate.

Fig. 6. Calibration graphs obtained for phenol, 4-chlorophenol and catechol at: ( ) graphite Teon tyrosinase, () graphite EPD tyrosinase, and ( ) RVC epoxy resin tyrosinase electrodes. Eapp = 0.15 V.

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Table 4 Values of the catalytic efciency, Vm/Km, app, and slopes of the calibration graphs for the different phenolic compounds obtained at composite tyrosinase electrodes of graphiteTeon, graphiteEPD and RVCepoxy resin Electrode GraphiteTeontyrosinase Compound 3,4-DMPh Catechol 4-Cl-3-MPh Phenol 4-ClPh 2,4-DMPh 4-Cl-2-MPh 2,3-DMPh 4-Cl-3-MPh 3,4-DMPh Catechol Phenol 4-ClPh 4-Cl-2-MPh 2,4-DMPh 2,3-DMPh 4-Cl-3-MPh 3,4-DMPh Catechol 4-ClPh Phenol 2,4-DMPh 4-Cl-2-MPh 2,3-DMPh Slope, mA mol1 l 52104 30104 29104 28104 25104 1.9104 0.6104 0.04104 200x04 120104 66104 62104 46104 7.1104 3.3104 1.0104 0.59104 0.48104 0.41104 0.39104 0.34104 0.058104 0.021104 0.00077104 Vm/(Km, app105), mol l1 7.67 3.43 3.48 3.14 1.81 0.15 0.20 0.0053 27.4 10.3 5.31 6.13 5.69 1.00 0.36 0.125 0.052 0.057 0.048 0.039 0.041 0.0078 0.00067 0.000098

GraphiteEPD-tyrosinase

RVCepoxy resintyrosinase

4-Cl-3-MPh, 4-chloro-3-methylphenol; 3,4-DMPh, 3,4-dimethylphenol; 4-ClPh, 4-chlorophenol; 2,4-DMPh, 2,4-dimethylphenol; 4-Cl-2-MPh, 4-chloro-2-methylphenol; 2,3-DMPh, 2,3-dimethyl phenol.

On the other hand, it can be observed that the trend in sensitivity is not exactly the same for the three composite tyrosinase electrodes. Nevertheless, a general trend can be found for all of them. The three phenolic compounds with one ortho-position occupied are remarkably less sensitive than the others (and therefore they have lower Vm/Km, app. values). Moreover, the phenolic compounds with the positions 3 and 4 of the aromatic ring occupied show a high slope of their calibration graphs, probably because these substituents produce a stabilization of the enzyme-substrate link, which is corroborated by the low Km, app. values obtained for these compounds with the three composite electrodes. Catechol is also detected in all cases with a high sensitivity as a consequence of having a hydroxyl group in ortho position which facilitates the conversion to quinone, whereas phenol and 4-chlorophenol gave similar sensitive responses since both of them have the two ortho-positions free. The existing differences on the phenolic compounds sensitivity order for each biocomposite electrode suggest the inuence of other factors on the analytical responses, besides those commented above. As we have already stated, the nature of the electrode matrix inuences the interactions of the tyrosinase catalytic cycle. Apart from the afnity between the enzyme and the

substrate and the rate of the catalytic conversion, factors depending directly on the electrode matrix nature, such as the ability of accumulation of the analyte on the electrode surface, and the hydrophobicity of this electrode surface can promote differences in the amperometric responses obtained for the same compounds.

4. Conclusions From the comparison of the behaviour of the three tyrosinase composite electrode matrices, it can be concluded that all of them can be used for the monitoring of the phenolic compounds used as substrates. These composite matrices are reusable and show a rather good stability and robustness when compared with other tyrosinase biosensors designs found in the literature. Among the three composite biosensors tested, the graphite EPD tyrosinase electrodes show a better reproducibility of the amperometric measurements both with and without regeneration of the electrode surface by polishing. However, the graphite Teon electrode matrix exhibits a longer useful lifetime. The trend in sensitivity towards the different phenolic compounds tested is dependent on the nature of the electrode matrix used, and the limits of detection obtained are, in

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general, better than those found with other tyrosinase electrochemical biosensors designs. Actually, these composite biosensors can be used in the monitorization of phenolic compounds in industrial waste waters, in which the highest concentration level permitted is 2 mg l 1. The lowest limit of detections are obtained with the graphite EPD electrodes.

Acknowledgements The nancial support of Comunidad de Madrid, Project 07M/0033/99, and EC (Inco-Copernicus), Project PL 965022, is gratefully acknowledged.

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