Glassy carbon paste electrodes modified with polyphenol oxidase

Analytica Chimica Acta 459 (2002) 43–51

Glassy carbon paste electrodes modified with polyphenol oxidase Analytical applications Marcela C. Rodr´ıguez, Gustavo A. Rivas∗ INFIQC, Departamento de F´ısico Qu´ımica, Facultad de Ciencias Qu´ımicas, Universidad Nacional de Córdoba, Ciudad Universitaria, 5000 Córdoba, Argentina Received 24 July 2001; received in revised form 17 December 2001; accepted 5 February 2002

Abstract The electrochemical behavior of a glassy carbon paste electrode (GCPE) is evaluated in comparison to that of graphite paste electrode (gPE) and glassy carbon electrode (GCE). Important shifting in the peak potentials and increases in the peak currents for catechol, ascorbic acid, dopamine and hydroquinone were obtained for the GCPE and its usefulness for the development of phenol and catechol biosensors was also evaluated. Both, pure mushroom polyphenol oxidase (PPO) and fresh mushroom tissues were used as biorecognition elements. The effect of the binder percentage in the composite material was also studied. The bioelectrode was used for the determination of dopamine and acetaminophen in pharmaceutical formulations and for the detection of polyphenols in wine and tea. The bioelectrode demonstrated to be very stable as the response remained around 90% after four months at 4 ◦ C. © 2002 Elsevier Science B.V. All rights reserved. Keywords: Glassy carbon microparticles; Glassy carbon paste electrode; Composite electrodes; Enzymatic biosensors; Phenol biosensors; Catechol biosensors

1. Introduction Carbon materials that have been widely used in the preparation of solid electrodes, include glassy carbon, carbon fibers, carbon black, and several forms of graphite, from the highly orientated pirolytic graphite to the graphite powder used in the preparation of the well-known composite carbon paste [1]. The carbon materials that have probably received most attention due to their particular properties are glassy carbon and graphite paste. Among these materials, glassy carbon has been widely used for electroanalytical purposes. Since the polished material presents some problems connected with the polishing step such as ∗ Corresponding author. Tel.: +54-3514334169; fax: +54-3514334188. E-mail address: grivas@fisquim.fcq.unc.edu.ar (G.A. Rivas).

irreproducibility or slow electron transfer, many pretreatments have been proposed [1]. These not only improve the reproducibility but also largely improve the charge transfer with different electroactive compounds due to the resulting increase of the surface oxygenated groups density [1–5]. The effectiveness of the pretreatment depends not only on the nature of the activation procedure but also on the nature of the analyte [6]. Graphite powder has been used to prepare the classical composite carbon paste electrodes (gPE), which have received enormous attention especially in the last two decades due to their well-known advantages [7,8]. Girault et al. [9] proposed a composite based on glassy carbon microparticles and a polystirene polymer. These authors demonstrated that the resulting electrode possesses electrochemical properties similar to other glassy carbon electrodes (GCE), although they presented a strong dependence on the

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carbon-to-polymer ratio of the ink ranging from insulator behavior to a very active surface for electrochemical reactions. The goal of the present work was to show some unique properties of glassy carbon paste electrodes (GCPE) prepared with glassy carbon microparticles and mineral oil, as well as to demonstrate the analytical applications of this material for the preparation of enzymatic biosensors for phenols and catechols using polyphenol oxidase (PPO) as recognition layer. In the following sections we discuss the electrochemical behavior of the GCPE in comparison with regular GCE and gPE in different supporting electrolytes as well as the effect of usual pretreatments on the behavior of the electrodes towards several conventional redox couples. The analytical performance of the GCPE containing PPO for the quantification of phenols and catechols in different samples is also discussed. 2. Experimental 2.1. Reagents Ascorbic acid was from Fluka. Dopamine, catechol and mushroom PPO (or Tyrosinase E.C. 1.14.18.1, 1030 U mg−1 ) were from SIGMA. Other chemicals were reagent grade and used without further purification. Ultrapure water (ρ = 18 M) from a MilliporeMilliQ system was used for preparing all the solutions. A 0.050 M phosphate buffer solution pH 7.4 was employed as supporting electrolyte.

The gPE was prepared in the regular way by mixing in an agata mortar graphite powder (Fisher grade # 38) and mineral oil (Aldrich). The GCPE was prepared in a similar way by mixing glassy carbon microparticles (carbon spherical powder 0.4–12 ␮m, type 2, Alfa AEsar) with mineral oil. Carbon paste electrodes containing PPO were prepared in the following way: the desired amount of enzyme (0.7% (w/w)) was mixed with mineral oil (usually 30.8% (w/w)) in an agata mortar for 3 min followed by the incorporation of graphite or glassy carbon powder and mixing for 15 additional minutes. In case of using mushroom tissues as PPO source, a portion of mushroom tissue (15% (w/w)) was scratched in the mortar followed by the incorporation of mineral oil, mixing for 3 min until homogeneous aspect. Finally, the carbon (either graphite powder or glassy carbon microparticles) was incorporated and mixed for 30 additional minutes. A portion of the resulting paste was packed firmly into the cavity (3.0 mm diameter) of a Teflon tube. The electric contact was established through a stainless steel screw. A new surface was obtained by smoothing the electrode onto a weighing paper. 2.3. Procedure All the experiments were conducted at room temperature. Amperometric experiments were carried out by applying the desired potential and allowing the transient current to decay prior to addition of the analyte and subsequent current monitoring.

3. Results and discussion 2.2. Apparatus The measurements were performed with an EPSILON potentiostat (BAS). The electrodes were inserted into the cell (BAS, Model MF-1084) through its Teflon cover. A platinum wire and Ag/AgCl, 3 M NaCl (BAS, Model RE-5B) were used as counter and reference electrode, respectively. All the potentials are referred to the latter. A BAS Cell-Stand C3 gave the convective transport during the amperometric determinations. Impedance spectroscopy measurements were performed with an impedance analyzer Zahner Im5d. GCE (CH Instruments), gPE and GCPE have the same geometric area.

Fig. 1 shows cyclic voltammograms performed at 0.100 V s−1 in a deoxygenated 0.050 M phosphate buffer solution pH 7.4 at different carbon electrodes: GCE (a), gPE (b), and GCPE (c). The potential was first scanned in the positive direction from 0.00 to 2.50 V. The negative scan was then performed from 2.50 to −2.50 V. After that, the potential was scanned to the initial value (0.00 V). As it can be seen in this figure, the wider potential window is obtained at GCE (a). At this electrode the oxygen evolution starts at potentials higher than 1.50 V. In agreement with previous results [10], two current peaks are observed in the negative scan at around −0.6 and −1.8 V which

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Fig. 1. Cyclic voltammograms obtained in a deoxygenated 0.050 M phosphate buffer solution pH 7.4 on GCE (a), gPE (b) and GCPE (c) electrodes; v = 0.100 V s−1 . The potential was first scanned from 0 to 2.5 V. The negative scan was then performed up to −2.50 V to return finally to the initial potential (0 V).

correspond to the reduction of the graphite oxide formed after incursion in potentials at which oxygen evolution is produced, and to the reduction of oxygen itself, respectively. The reduction of the solvent takes place at potentials more negative than −2.00 V. At gPE (b) the oxygen evolution occurs at potentials higher than 1.10 V. In the negative scan there are two broad and not well defined peaks, one at around 0 V and the other at around −0.8 V. These peaks appear only if potentials more positive than 1.10 V are reached. The solvent reduction starts at −2.20 V. In the case of GCPE (c), as it can be clearly seen in Fig. 1, the capacitive as well as the faradaic currents are much higher than those at gPE and GCE. On the other hand, the oxygen evolution starts at around 1.0 V, that is, at potentials 500 and 100 mV less positive than at GCE and at gPE, respectively. The reduction of the graphite oxide and oxygen are less defined than in the case of the other two electrodes while the solvent reduction occurs at potentials similar to those at gPE. Thus, the window potential is between −0.40 and 1.00 V. Hence, in both composite electrodes the potential window is shorter than at GCE. The capacitances for GCPE, gPE and GCE were obtained from impedance spectroscopy experiments. Impedance spectra performed between 10 mHz and

10 MHz in 0.050 M phosphate buffer solution pH 7.4 demonstrated that a capacitive behavior is obtained for potentials (E) 0.50 V < E < 1.0 V. Consequently, the capacitances were obtained from impedance spectra performed at 0.50 V by fitting the data with an equivalent circuit that includes the solution resistance, the double layer capacitance and another resistive component. The values of capacitance are the following: 1.01 × 10−5 F (error 1.6%), 6.69 × 10−7 F (error 0.6%) and 4.00 × 10−7 F (error 1.7%) for GCPE, gPE and GCE, respectively. These results indicate that the highest area would correspond to GCPE, followed by gPE and GCE. Chronoamperometric experiments using catechol 5.0 × 10−4 M in 0.050 M phosphate buffer solution pH 7.4 by applying a potential step between open circuit and 0.350 V, also demonstrated that the largest area was that for GCPE. The behavior of GCPE in a 0.50 M sulfuric acid solution was also evaluated (not shown). The oxygen evolution started at around 1.30 V while the reduction of the solvent appeared at potentials more negative than −1.00 V. As in the case of phosphate buffer, the GCPE demonstrated to be the most active and presented the shortest potential window, compared to gPE and GCE.

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In order to evaluate the electrochemical behavior of different electroactive compounds at the three electrodes, CVs for 5.0 × 10−4 M ascorbic acid, catechol and dopamine in phosphate buffer solution pH 7.4 were performed. Fig. 2 shows the CVs at 0.100 V s−1

Fig. 2. Cyclic voltammograms for 5.0×10−4 M catechol obtained at GCE (A), gPE (B) and GCPE (C) v = 0.050 V s−1 . Supporting electrolyte: 0.050 M phosphate buffer solution pH 7.4. Initial potential: −0.40 V.

for 5.0 × 10−4 M catechol at gPE (A), GCE (B) and GCPE(C). CV parameters obtained at GCE, gPE and GCPE for the three compounds evaluated are depicted in Table 1a–c. Table 1a shows that the Ep for catechol at GCPE is 156 and 198 mV smaller than those at gPE and at GCE, respectively, while the ipa /ipc ratio decreases from 2.2 at GCE to 1.8 at gPE and 1.2 at GCPE. In the case of dopamine behavior (Table 1b), a decrease of 41 and 93 mV in the Ep is observed at GCPE in comparison with gPE and GCE, respectively, while an important decrease in the currents ratio is also obtained. Another aspect to remark is that the reduction of the dopaminequinone electrochemically generated as well as the reduction of the dopaminechrome chemically formed [11] are processes poorly defined at gPE and GCE. On the contrary, at GCPE these processes were more clearly defined (not shown). In the case of ascorbic acid (Table 1c) the oxidation peak potential at GCPE decreases 83 mV compared to gPE and 146 mV compared to GCE. The oxidation current at GCPE increases 51 and 136% in comparison with gPE and GCE, respectively.In summary, the most important features of the electrochemical behavior of GCPE compared to gPE and GCE are the following: (a) The potential window is the smallest; (b) the background current is the highest; (c) the oxygen evolution starts at the least positive potentials while the reduction of the solvent starts at the least negative potentials; (d) the reduction of graphite oxide is less evident than at the other carbon materials, even when the electrode is pretreated. It is interesting to remark that the presence of glassy carbon as microparticles embedded in mineral oil, allows one to improve notoriously the electroactivity of the electrode probably due to the exposure of a higher density of surface oxygenated groups and to the resulting array of the microparticles in the composite material. It is known that pretreatments have a crucial role in the behavior of GCE. Based on this knowledge, several pretreatments were performed on GCPE to evaluate their effectiveness on its electrochemical behavior. Three compounds were taken as model: ascorbic acid, dopamine and catechol. In all cases, the electrodes were pretreated in the given media (either H2 SO4 or phosphate buffer or NaOH), then they were transferred to a supporting electrolyte solution (0.050 M

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Table 1 Voltammetric parameters obtained from cyclic voltammograms for 5.0 × 10−4 M Ep (V)

Ipa (␮A)

ipa /ipc

(a) Catechol at glassy carbon GCE, gPE and GCPE GCE 0.305 gPE 0.280 GCPE 0.164

0.268 0.226 0.070

10.9 14.9 17.3

2.2 1.8 1.2

(b) Dopamine at GCE, gPE and GCPE GCE 0.227 gPE 0.195 GCPE 0.190

0.146 0.094 0.053

14.7 20.5 38.3

5.0 4.5 1.7

Electrode

Epa (V)

(c) Ascorbic acid at GCE, gPE and GCPE GCE 0.343 gPE 0.280 GCPE 0.197

7.3 11.4 17.2

Scan rate: 0.050 V s−1 .

phosphate buffer solution pH 7.4) to obtain the blank response, to be finally transferred to the analyte solution (ascorbic acid, dopamine or catechol). Four pretreatments were evaluated, potentiodynamic activation in 0.050 M phosphate buffer solution between −2.00 and 2.00 V at 0.100 V s−1 ; potentiostatic pretreatments either in phosphate buffer solution at 1.50 V, or in 0.050 M H2 SO4 at 1.50 V, or in 1.0 M NaOH at 1.20 V. The only pretreatment that shows some advantageous results at GCPE was the one performed in alkaline solution. All the others showed negative effects, with large capacitive currents and very small oxidation or reduction peaks currents for the three analytes evaluated. So, at variance with GCE where a positive effect was observed after performing all these pretreatments, in the case of GCPE, the electrochemical behavior of the three compounds at the pretreated electrodes, with exception of those activated in NaOH solutions, is negatively affected. It is known [2,5,10] that during the electrochemical pretreatments an oxide graphite layer is grown at the surface of the carbon electrodes which is thicker in acidic solutions and it is dissolved in highly alkaline medium. In the case of GCPE, a larger amount of the surface oxide layer is formed during the oxidation, due to the higher exposed surface that could act as an insulator film. In agreement with this assumption, the only pretreatment that resulted effective on the electrochemical behavior of catechol, was the one performed in NaOH solution where the graphite oxide layer is thinner [4,10]. Therefore, from the analytical point of view, it is better to use a polished

surface of GCPE to get a more sensitive response (higher faradaic and smaller capacitive currents). 3.1. Analytical applications Carbon paste electrodes prepared with graphite powder have been largely used for the preparation of biosensors [8,12]. In this section, we will discuss the usefulness of glassy carbon microparticles for the preparation of enzymatic biosensors for phenols and catechols, using mushroom PPO. PPO is widely distributed in nature and it catalyzes the oxidation of phenols to catechols as well as the oxidation of catechols to the corresponding quinones [13].Taking into account that a quinone is generated as a consequence of the enzymatic reaction between phenols and catechols and PPO, the voltammetric response of hydroquinone at GCPE and gPE was evaluated. Fig. 3 shows cyclic voltammograms for 5.0 × 10−4 M hydroquinone (in 0.050 M phosphate buffer solution pH 7.4) at gPE (a) and GCPE (b). As expected, according to Fig. 2, the electrochemical behavior of hydroquinone appears like more reversible at GCPE. A decrease of 246 mV in Ep compared to gPE is observed (Ep of 323 and 77 mV at GCPE and gPE, respectively) while the ipa /ipc ratio decreases from 1.5 to 1.2. As in the other cases before described, GCPE demonstrated to be the most active also for the quinone reduction. From a hydrodynamic voltammogram performed at GCPE–PPO for 1.0 × 10−5 M catechol, a potential of −0.100 V was selected as the optimum for measuring

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Fig. 3. Cyclic voltammograms for 5.0 × 10−4 M hydroquinone at gPE (a) and GCPE (b); v = 0.050 V s−1 . Initial potential: 0.40 V. Supporting electrolyte: 0.050 M phosphate buffer solution pH 7.4.

the reduction of the enzymatically generated quinone. Despite more negative potentials gave a more sensitive response, the noise was higher at those potentials and the stabilization of the base current took longer times (not shown). One important aspect to be considered concerning the analytical usefulness of composite biosensors is the amount of binder, since most of the properties of the material are connected with this parameter. Employing the percentage of oil commonly used to prepare gPEs (30% (w/w)), the resulting GCPE presents a wet consistency that makes it very difficult to manipulate it. Fig. 4 shows calibration plots for catechol at GCPE modified with 0.70% (w/w) PPO and different amounts of mineral oil: 20% (w/w) (a), 15% (w/w) (b), 10% (w/w) (c) and 5% (w/w) (d), obtained from amperometric recordings at −0.100 V. The corresponding sensitivities are (6.4 ± 0.3) × 106 nA M−1 (r = 0.998); (9.7±0.4)×106 nA M−1 (r = 0.997); (2.41± 0.07) × 107 nA M−1 (r = 0.9990) and (4.7 ± 0.1) × 107 nA M−1 (r = 0.9990), respectively. Thus, as expected from the decrease in the percentage of the non conductive phase, a faster electron transfer is obtained at the GCPE biocomposites containing the smallest amount of mineral oil. However and despite the highest sensitivity was obtained for pastes containing 5% (w/w) mineral oil, they present the disadvantage that the reproducibility is not good enough probably due to some inhomogeneity produced as a consequence of

Fig. 4. Calibration plots obtained from amperometric recordings at −0.100 V for successive additions of 5.0 × 10−6 M catechol. Working electrode: GCPE containing 0.7% (w/w) PPO purified from mushrooms and variable amounts of mineral oil: 20% (w/w) (a), 15% (w/w) (b), 10% (w/w) (c) and 5% (w/w) (d). The plot is the average of the data obtained from four amperometric recordings performed with different surfaces.

the small amount of binder. Thus, the best compromise between sensitivity and reproducibility was obtained with a paste containing 10% (w/w) mineral oil. The response of a GCPE containing PPO towards different substrates, including drugs such as acetaminophen, neurotransmitters as dopamine and pollutants as phenol, was evaluated. The analytical parameters of the bioelectrode for these substrates are shown in Table 2. As it was previously reported [14], the most sensitive response was obtained for the unsubstituted substrates, phenol and catechol compared to that for the corresponding substituted molecules. Table 2 Analytical parameters obtained from amperometric recordings at −0.100 V for different substrates at GCPE containing 10% (w/w) mineral oil and 0.70% (w/w) PPO Compound

Linear range (up to/M)

Catechol Dopamine l-Dopa Acetaminophen l-Tyrosine Phenol Gentisic acid

3.5 1.1 4 7.0 9.0 7.0 7.5

× × × × × × ×

10−5 10−5 10−5 10−5 10−5 10−5 10−5

Sensitivity (nA M−1 ) 2.4 3.6 1.0 1.5 3.8 9.0 1.9

× × × × × × ×

107 106 106 107 105 106 105

Detection limit (M) 4.1 2.4 1.6 7.8 1.5 1.7 2.0

× × × × × × ×

10−7 10−6 10−6 10−6 10−5 10−6 10−5

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Fig. 5. (A) Current—time profiles performed at GCPE containing 10% (w/w) mineral oil and 15% (w/w) fresh mushroom tissue for successive additions of 5.0 × 10−6 M catechin. (B): (a) calibration plot obtained from the data shown in Fig. 5A; (b) for comparison it is shown the calibration plot obtained from amperometric recordings at −0.100 V for successive additions of 5.0 × 10−6 M catechin at gPE electrode containing 10% (w/w) mineral oil and 15% (w/w) fresh mushroom tissue.

During the 80’s the field of biosensors was revolutionized when it was demonstrated that it is possible to prepare enzymatic carbon paste electrodes not only by incorporating pure enzymes, but also by using tissues as enzymatic source [15,16]. Taking into account this fact, the response of a GCPE containing mushroom tissue, instead of pure PPO as enzymatic source, was evaluated. Fig. 5A shows the amperometric response at GCPE–PPO at −0.100 V for successive additions of the polyphenol catechin 5.0 × 10−6 M, at GCPE–PPO

prepared with 10% (w/w) mineral oil and 15% (w/w) muhsroom tissue (catechin is a polyphenol present in red wine and green tea). Fig. 5B(a) shows the calibration plot obtained from the data of Fig. 5A. The corresponding slope for the linear range is (5.9±0.1)×107 (r = 0.998) nA M−1 , with a dynamic linear range up to 3.5 × 10−5 M. For comparison, the calibration plot obtained under the same conditions by using gPE prepared with 10% (w/w) mineral oil and 15% (w/w) fresh mushroom tissue is also shown. The sensitivity

Table 3 Determination of acetaminophen in two pharmaceutical formulations using a GCPE containing 10% (w/w) mineral oil and 15% fresh mushroom tissue Name of the pharmaceutical formulation

Composition

Compound tested

Vent-3 (Vent-3 Lab)

Acetaminophen 10 g% Excipients

Acetaminophen

Multin (Lazar Lab)

Acetaminophen 120 mg ml−1 Dipirona 400 mg ml−1 Excipients

Acetaminophen

Working potential: −0.100 V.

Values found (g%) 9.36 9.59 9.49 9.93 10.20 10.97 128.9 117.6 124.2 124.8

Average

Relative error (%)

9.9 ± 0.6 g%

−1.0

124 ± 5 mg ml−1

+3.3

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at this bioelectrode is (2.07±0.05)×107 (r = 0.998). As expected, the sensitivity at this electrode is smaller (almost three times) than that at GCPE. Short-term stability was evaluated by performing successive calibration plots for catechol using the same surface. The values for the sensitivities are the following: (4.1 ± 0.1) × 107 nA M−1 ; (4.2 ± 0.2) × 107 nA M−1 ; (4.1 ± 0.3) × 107 nA M−1 ; (4.4 ± 0.2) × 107 nA M−1 ; (4.4 ± 0.2) × 107 nA M−1 ; (4.4 ± 0.3) × 107 nA M−1 ; (4.3 ± 0.3) × 107 nA M−1 ; (4.4 ± 0.3) × 107 nA M−1 ; (5.5 ± 0.4) × 107 nA M−1 ; (5.3 ± 0.5) × 107 nA M−1 ; the average is (4.5 ± 0.5) × 107 nA M−1 , and the R.S.D., 10.8%. It is important to remark that if just 8 calibrations are performed with the same surface, the R.S.D. is 3.2%. The time for performing the eight calibrations (twelve additions of catechol in each case) was around 150 min, so after 2.5 h of almost continuous use, the bioelectrode allows one to obtain very reproducible results. The reproducibility using different surfaces was also evaluated and the results gave an average of (4.1 ± 0.6) × 107 nA M−1 with an R.S.D. of 10.7%. The long-term stability was checked by performing calibration plots at different times employing an electrode stored under dry conditions at 4 ◦ C. The sensitivity of the electrode after 4 months was almost 90% of the original value. The practical usefulness of the bioelectrode was evaluated by measuring the content of a given drug in pharmaceutical formulations. The GCPE–PPO (using fresh mushroom tissue) was used to check the content of dopamine in Inotropin (Bagó). The results obtained gave an average of (98 ± 9) mg per 5.00 ml, with a relative error of −2%. The relative error was obtained by comparison with the amount that the label claims (100 mg every 5.00 ml). The bioelectrode was also used to determine acetaminophen in two pharmaceutical formulations. The results are shown in Table 3. As it can be seen, the results obtained using this methodology are very close to those indicated in the pharmaceutical formulations and taken as the true values. Relative errors of −1.0 and +3.3% were found for the acetaminophen concentrations in Vent-3 (Vent-3 Lab) and Multin (Lazar Lab), respectively. Fig. 6 shows the amperometric response of the bioelectrode to additions of two kinds of red wine. The red wine A is “Colón”, grape Borgoña, Graffigna

winecellar, San Juan, Argentina while the red wine B is “Michel Torino”, grape Borgoña, La Rosa winecellar, Salta, Argentina. As it can be seen, after additions of red wine, since they have polyphenols that

Fig. 6. Amperometric recordings performed at GCPE containing 10% (w/w) oil and 15% (w/w) fresh mushroom tissue for successive additions of 100 ␮l of wine and 500 ␮l tea: (A) Col´on Borgogna, Graffigna, San Juan, Argentina; (B) Michel Torino, La Rosa, Cafayate, Salta, Argentina, (C) green tea “Hierbas del Oasis”.

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can be oxidized to the corresponding quinones by PPO and reduced at the working potential, a fast cathodic response is obtained from the reduction of those quinones. Similar experiments using white wine were performed, and since the content of polyphenols is very small, the response is poor (not shown). The response of the biosensor to additions of green tea “Hierbas del Oasis”, Argentina was also evaluated and it is shown in Fig. 6C. As this tea also has polyphenols like catechin, a similar response is obtained, although less sensitive than that obtained in the case of red wine (note that the green tea used in these experiments is not the one original from Orient). In summary, this electrode could also be used to obtain more information about the quality (bouquet) of the red wine or tea from catechin determination.

4. Conclusions The foregoing experiments showed the advantages of using GC microparticles for preparing a new biocomposite material which demonstrated to be more electroactive than gPE and GCE. Some properties of these electrodes are shared. Others, like the effect of pretreatments, are different. At variance with gPE and GCE, the pretreatments do not improve the electrochemical behavior of different redox compounds with the exception of the pretreatment in alkaline solutions. The new material demonstrated to be useful for preparing phenol and catechol enzymatic biosensors either with pure mushroom PPO or fresh mushroom tissue. This bioelectrode is suitable for the determination of dopamine and acetaminophen in pharmaceutical formulations and polyphenols in real samples. Other applications of this bioelectrode are currently being examined successfully in our laboratory.

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Acknowledgements The authors thank Fundación Antorchas, Consejo Nacional de Investigaciones Cient´ıficas y Técnicas de Argentina (CONICET), CONICOR, Agencia Córdoba Ciencia, Secretar´ıa de Ciencia y Tecnolog´ıa de la Universidad Nacional de Córdoba (SECyT) and Asociación de Bioqu´ımicos de la Provincia de Córdoba (ABC) for financial support. M.C.R. thanks CONICOR and CONICET for the fellowships received. The authors also want to thank Dr. Mar´ıa José Esplandiú for the fruitful discussions about impedance experiments. References [1] R.L. McCreery, K.K. Cline, in: Peter T. Kissinger, William R. Heineman (Eds.), Laboratory Techniques in Electroanalytical Chemistry, 2nd Edition, Marcel Dekker, NY, 1996 (Chapter 10). [2] C. Barbero, R. Kötz, J. Electrochem. Soc. 140 (1993) 1. [3] A. Beilby, A. Carlsson, J. Electroanal. Chem. 248 (1988) 283. [4] D.M. Anjo, M. Kahr, M.M. Khodabakhsh, S. Nowinsky, M. Wanger, Anal. Chem. 61 (1989) 2603. [5] L.J. Kepley, A.J. Bard, Anal. Chem. 60 (1988) 1459. [6] P. Chen, M.A. Fryhing, R.L. Mc Creery, Anal. Chem. 67 (1995) 3115. [7] M. Rice, Z. Galus, R.N. Adams, J. Electroanal. Chem. 143 (1983) 89. [8] L. Gorton, Electroanalysis 7 (1995) 23. [9] B.J. Deddon, M.D. Osborne, G. Lagger, R.A.W. Drufe, U. Loyall, H.H. Schäfer, H.H. Girault, Electrochim. Acta 42 (1997) 1883. [10] A.L. Beilby, T.A. Sasaki, M. Stern, Anal. Chem. 67 (1995) 976. [11] A. Brun, R. Rosset, J. Electroanal. Chem. 49 (1974) 287. [12] M.A.T. Gilmartin, J.P. Hart, Analyst 120 (1995) 1029. [13] S.G. Burton, Catal. Today 22 (1994) 459. [14] E.S. Forzani, G.A. Rivas, V.M. Sol´ıs, J. Electroanal. Chem. 435 (1997) 77. [15] G.A. Rechnitz, Science 214 (1981) 287. [16] J. Wang, M.S. Lin, Anal. Chem. 60 (1988) 1545.