Layered Double Hydroxides: An Attractive Material for Electrochemical

To prevent release of the enzyme molecules incorporated within the clay film, ..... compared the biosensors' performance to catechol detection at both...
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Anal. Chem. 2003, 75, 3872-3879

Layered Double Hydroxides: An Attractive Material for Electrochemical Biosensor Design Dan Shan, Serge Cosnier, and Christine Mousty*

Laboratoire d’Electrochimie Organique et de Photochimie Redox, UMR CNRS 5630, Institut de Chimie Mole´ culaire de Grenoble (FR CNRS 2607), Universite´ Joseph Fourier, 38041 Grenoble Cedex 9, France

Electrochemical biosensors for phenol determination were developed based on the immobilization of polyphenol oxidase (PPO) within two different clay matrixes, one anionic (layered double hydroxide, LDH) and the other cationic (Laponite). The biosensor based on the enzyme immobilized in [Zn-Al-Cl] LDH shows greater sensitivity (7807 mA M-1 cm-2) and maximum current (492 µA cm-2). Biosensor characteristics, such as MichaelisMenten constant, recycling constant, activation energy, and permeability highlight the advantages of LDH matrixes to immobilize PPO. It appears that LDH provides a favorable environment to PPO activity. The best PPO/ [Zn-Al-Cl] configuration was used to determine five different phenol derivatives reaching extremely sensitive detection limits (e1 nM). Electrochemical biosensors have been increasingly developed for continuous monitoring in environmental and health care applications. Such devices contain a biological sensing element connected to a transducer that converts the specific biorecognition event to an electrical signal. One of the key steps in the fabrication of biosensors consists of the effective immobilization of biomolecules onto the transducer surface. The objectives are to find an immobilization method that maintains the enzyme activity, increases its stability, and provides accessibility toward substrate. Numerous immobilization methods have been developed for electrochemical biosensor applications, such as covalent linkages, cross-linking methodologies, bioaffinity attachment, self-assembled multilayers, mixing in carbon composites, and entrapment within polymeric and inorganic matrixes.1 Among these methods, entrapment within hydrophilic gels such as sol-gel and clays appears to be an efficient and popular method.2,3 In our laboratory, we have developed several electrochemical biosensors based on clay-modified electrodes following an original and inexpensive strategy.4-9 Cationic clays are layered aluminosilicates with cationic exchange capacity. Due to their chemical * Corresponding author: [email protected]. Fax: 33.476.514.267. (1) Zhang, S.; Wright, G.; Yang, Y. Biosens. Bioelectron. 2000, 15, 273-282. (2) . Wang, J. Anal. Chim. Acta 1999, 399, 21-27. (3) Macha, S. M.; Fitch, A. Mikrochim. Acta 1998, 128, 1-18. (4) Cosnier, S.; Le Lous, K. Talanta 1996, 43, 331-337. (5) Cosnier, S.; Le Lous, K. J. Electroanal. Chem. 1996, 406, 243-246. (6) Cosnier, S.; Lambert, F.; Stoytcheva, M. Electroanalysis 2000, 12, 243260. (7) Poyard, S.; Jaffrezic-Renault, N.; Martelet, C.; Cosnier, S.; Labbe´, P.; Besombes., J-L. Sens. Actuators, B 1996, 33, 44-49.

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inertia, their mechanical and thermal stability, clays constitute a versatile material for modifying an electrode surface.3 Moreover, the unusual intercalation properties of clays have been exploited for the entrapment of biological macromolecules.10,11 Characteristics of clay coatings, such as porosity and swelling properties, induce an improvement in the activity and stability of the immobilized enzymes.8,9 The biosensor fabrication consists of the adsorption of an enzyme-clay aqueous mixture on a transducer surface. To prevent release of the enzyme molecules incorporated within the clay film, different stabilization processes were applied. The first one was carried out by physical constraints via the electrochemical generation of an organic polymer within the microchannels of the clay film.4-6 Another method to overcome this problem consists of the chemical cross-linking of the incorporated enzymes by glutaraldehyde.7-9 For instance, we reported very sensitive amperometric biosensor configurations applied to phenol determination. It consists of the entrapment of polyphenol oxidase (PPO) within a Laponite clay matrix associated with a chemical cross-linking process of the enzyme molecules.12,13 To improve the analytical characteristics of this type of biosensor, a redox mediator, azure B or polyazure B, was used for the mediated electrochemical determination of phenolic compounds. Since 1987, several publications have reported that the synthetic layered double hydroxides (LDH), also called anionic clays, were used to modify electrodes.14 The LDH consists of MII(OH)6 and MIII(OH)6 edge-sharing octahedra forming sheets similar to those of brucite.15 Net positive charges of the layer are balanced by exchangeable anions intercalated between the sheets. Therefore, LDH presents a lamellar structure and intercalation properties similar to that of cationic clays but with a higher charge density of the layers (anionic exchange capacity ∼2 mequiv g-1). The positive charged layers may be an attractive point to immobilize biomolecules, depending on their isoelectric point. (8) Poyard, S.; Jaffrezic-Renault, N.; Martelet, C.; Cosnier, S.; Labbe´, P. Anal. Chim. Acta 1998, 364, 165-172. (9) Mousty, C.; Cosnier, S.; Shan, D.; Mu, S. Anal. Chim. Acta 2001, 443, 1-8. (10) Naidja, A.; Violante, A.; Huang, P. M. Clays Clay Miner. 1995, 43, 647655. (11) Naidja, A.; Huang, P. M.; Bollag, J. M. J. Mol. Catal., A 1997, 115, 305316. (12) Shan, D.; Mousty, C.; Cosnier, S.; Mu, S. Electroanalysis, in press. (13) Shan, D.; Mousty, C.; Cosnier, S.; Mu, S. J. Electroanal. Chem, 2002, 537, 103-109, and references therein. (14) Therias, S.; Mousty, C. Appl. Clay Sci. 1995, 10, 147-162. (15) De Roy, A.; Forano, C.; El Malki, K.; Besse, J.-P. Expanded clays and other microporous solids; Occelli, M. L., Robson, H. E., Eds.; Van Nostrand Reinhold: New York, 1992; pp 108-169. 10.1021/ac030030v CCC: $25.00

© 2003 American Chemical Society Published on Web 06/20/2003

Very recently, we showed that this synthetic lamellar material can be used as an immobilization matrix of enzymes for biosensor applications.16,17 The aim of the present work was the use of this anionic clay to elaborate sensitive phenol amperometric biosensors. A comparative study of phenol biosensors based on the immobilization of PPO into synthetic clays, one Laponite Si8[Mg5.5Li0.5 H4O24]0.7‚ Na0.7+, a cationic clay, and the other LDH Znx2+Aly3+(OH)2x+3y-nz(Cl)z‚mH2O denoted [Zn-Al-Cl], an anionic clay, highlights the advantages of the latter material. This new biosensor configuration was used for the determination of five different phenol derivatives. EXPERIMENTAL SECTION Reagent. PPO (EC 1.14.18.1) from mushroom (6050 units mg-1), catechol, and phenol compounds were purchased from Sigma. LDH [Zn-Al-Cl] was synthesized by a coprecipitation method developed by De Roy.15 Laponite was obtained from Rockwood Specialities Inc (Princeton, NJ). All other reagents were of analytical grade and used as received without further purification. The LDH colloidal suspension was prepared in deionized and decarbonated water. Other aqueous solutions were prepared in deionized distilled water. Apparatus. The amperometric measurements were performed with a Tacussel PRG-DL potentiostat with a Kipp and Zonen BD 91 XY/t recorder. All electrochemical experiments were carried out in a conventional thermostated three-electrode cell. An Ag/ AgCl saturated KCl electrode was used as reference electrode and a Pt wire placed in a separated compartment containing the supporting electrolyte, as counter electrode. The working electrode was a glassy carbon electrode (diameter 5 mm), polished with 1 µM diamond paste. SEM photos were realized on JEOL 6400 apparatus. Spectrophotometric measurements were carried out with a Varian Cary 1 UV-visible spectrophotometer. Preparation of the Enzyme Electrodes. The LDH [Zn-AlCl] colloidal suspension (2 mg mL-1) was prepared by dispersing LDH in deionized and decarbonated water with stirring overnight. PPO was also dissolved in deionized and decarbonated water with a concentration of 8 mg mL-1. A defined amount of the aqueous mixtures (for instance, containing 25 µg of PPO and 25 µg of LDH) was spread on the surface of the glassy carbon electrode. The coating was dried in air at room temperature. The resulting electrode was then placed in saturated glutaraldehyde vapor for 15 min for cross-linking of the membrane. LDH gel formation was achieved by incubation of the modified electrode in phosphate buffer solution for 20 min. The amount of protein released in the buffer solution during this step was determined by spectrophotometry. Catecholase activity of released PPO was measured by the following procedure: 0.04 M potassium ferrocyanide was included in the reaction mixture containing 0.7 mM catechol and unknown PPO samples. Its oxidation by enzymatically generated o-quinone was monitored spectrophotometrically at 420 nm. The same procedure was repeated with commercial samples of PPO. The estimated activity of immobilized PPO molecules on clays was also measured by this spectrophotometric method. The PPO/ (16) de Melo, J. V.; Cosnier, S.; Mousty, C.; Martelet, C.; Jaffrezic-Renault, N. Anal. Chem, 2002, 74, 4037-4043. (17) Shan, D.; Cosnier, S.; Mousty, C. Anal. Lett. 2003, 36, 909-922.

clay coating was dispersed in the sampling solution. The results, corresponding to an average of two independent measurements, were compared to the activity of the same amount of free enzyme, used as a reference (100%). The activity of immobilized enzyme was also estimated from electrochemical assay with the PPO/ clay coating immersed into a catechol solution (pH 6). The o-quinone enzymatically formed at the PPO/clay coating was detected at a bare glassy carbon electrode (Eapp -0.2 V). The chronoamperometric response of the biosensor was compared to that obtained with a defined amount of free enzyme (0.6 µg). The experiment was performed with both biosensors PPO/LDH and PPO/Laponite, allowing a comparison of the estimated activity of the immobilized enzyme molecules into these two matrixes. Characteristics of PPO/LDH and PPO/Laponite biosensors were determined by measuring the steady-state current response of these bioelectrodes to five successive additions of a low catechol concentration (0.1 µM) in “air-saturated” 0.05 M phosphate buffer (pH 6) with the rotation velocity 500 rpm. The sensitivity obtained via amperometric experiment at 0.6 V was denoted SB. In contrast, after the thermal denaturation (70 °C for 15 min) of the immobilized enzymes, the current response at 0.6 V was denoted SC (see Discussion section, Table 3). RESULTS Optimization of PPO/[Zn-Al-Cl] Biosensor. PPO contains a coupled dinuclear copper active site that catalyzes the metabolic conversion of monophenols and diphenols to o-quinones. Enzymatically generated o-quinones can be detected via their electrochemical reduction at -0.2 V. Since catechol is generally reported as the best substrate for this enzyme, we used it as a model compound for the biosensor optimization study.18 The steady-state current responses to catechol at the rotating clay electrode were evaluated in 0.1 M phosphate buffer solution (pH 6.5) by holding the sensor at -0.2 V. The substrate concentration was increased stepwise by adding in 10-mL electrolyte solution defined volumes of concentrated stock solution. The linear part of the calibration curves, obtained this way, was fixed in order to obtain the highest correlation coefficient (R2 g 0.999) and allowed the determination of the sensitivity. The detection limit was defined as a signal-to-noise ratio of 3. A pseudoplateau in the current/concentration curve (Imax) occurs when the active sites of all enzyme molecules are saturated. The proportion between enzyme and LDH is the most significant process parameter for the performance of the biosensor. So the effect of PPO and LDH (w/w) ratio was studied, the amount of enzyme being fixed at 50 µg. The sensitivity and Imax increase with the PPO/LDH ratio from 0.25 to 1 and then decrease from 1 to 2 (Figure 1). A proportion of 1:1 PPO/LDH results in the best composition for this biomembrane. The effect of thickness of the biocoating on the performance of the biosensor was also studied (Table 1). The thickness of the film was adjusted by varying the total amount of PPO and LDH, while the PPO/ LDH ratio was maintained at 1. For the thickest film (PPO:LDH ) 100 µg:100 µg), a poor amperometric performance to catechol was obtained. It is probably due to the increase in diffusion pathway from the clay-solution interface to the electrode surface. (18) Brown, R. S.; Male, K. B.; Luong, J. H. T. Anal. Biochem. 1994, 222, 131139.

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Figure 1. Influence of PPO/HDL (w/w) ratio to the biosensor sensitivity (Eapp -0.2 V, in 0.1 M phosphate pH 6.5, at 30 °C).

Figure 2. Effect of temperature on PPO/HDL electrode response to catechol additions (0.1 µM) (0.05 M phosphate buffer solution pH 6, Eapp ) -0.2 V).

Table 1. Amperometric Performance of PPO/LDH Bioelectrodes with Different Coating Thicknesses thickness PPO/LDH (µg) 25:25 50:50 100:100 a

sensitivity (mA M-1 cm-2) 6438 ( 20 7300 ( 21 2615 ( 12

Imax (µA cm-2) 484 505 234

LR (M) 10-9-3.5

R2 a 10-5

1.5 × × 1.3 × 10-9-1.6 × 10-5 0.7 × 10-9-3.0 × 10-5

0.9997 0.9998 0.9994

Correlation coefficient of the linear range.

The two other biocoatings (50 µg:50 µg) and (25 µg:25 µg) exhibit almost the same satisfactory performance. However, the amount of PPO truly retained in the coating was determined by spectroscopy for these two biosensors, and it corresponds to 86 and 91%, respectively. The thinner coating (25 µg:25 µg) presents a better enzyme retention. Consequently, the specific enzyme activity (Imax/enzyme amount) is better with the smallest enzyme amount, 21 µA cm-2 µg-1 instead of 12 µA cm-2 µg-1. This configuration was therefore chosen for the further experiments. Optimization of Experimental Conditions. The various experimental parameters, which can affect the catechol determination, have been investigated, namely, the rotation velocity, temperature, pH value of solution, concentration of electrolyte and applied potential. The effect of the electrode rotation rate on the current response at 0.1 µM catechol was measured. At rotation rates ω above 500 rpm, the steady-state current is independent of ω, while below, the response decreases with decreasing rotation rate. The rotation velocity of 500 rpm was therefore chosen in order to prevent limitation of substrate mass transportation. The thermal stability of the sensor was also studied. Before the amperometric detection, PPO/LDH electrode was immersed into the buffer solution at different temperatures for 30 min. The effect of temperature on the biosensor response to 0.1 µM catechol is shown in Figure 2. The current first increases with the temperature from 5 to 35 °C. Maximum response appears at ∼35 °C; at higher temperature, the current decreases slowly due to the denaturation of the enzyme. To keep the stability of the biosensor, we chose 30 °C as the operating temperature of the experiments. The pH dependence of the PPO/LDH electrode was investigated over the pH range 5.5-8.5 in 0.1 M phosphate buffer in the presence of 0.1 µM catechol. Figure 3A shows the dependence 3874 Analytical Chemistry, Vol. 75, No. 15, August 1, 2003

Figure 3. PPO/HDL electrode response to catechol additions (0.1 µM) (A) Influence of pH (0.1 M phosphate buffer solution), (B) Influence of phosphate buffer concentration (pH 6.0) (Eapp ) -0.2 V, at 30 °C).

of the biosensor response on the electrolyte pH. The optimum biosensor response is achieved at pH 6, which is in agreement with the optimum value found for PPO/Laponite coating12,13 and for PPO/Al(OH)x-montmorillonite complexes.11 This optimum value is situated in the pH range (5-8) reported for the free enzyme.18 Even though clay particles are electrically charged and consequently could present interfacial pH effects, no pH shift in the optimum catalytic activity is observed when PPO is adsorbed on clays. The influence of buffer concentration was also evaluated between 0.025 and 0.2 M using buffer solution at pH 6.0 (Figure 3B). An initial increase in response was observed up to 0.05 M phosphate, whereafter the response decreases gradually. This fact confirms the drastic influence of ionic strength on the enzyme

Figure 4. Effect of Eapp on (a) the amperometric response to 0.1 µM catechol additions and (b) on background current of the PPO/ LDH electrode (0.05 M phosphate buffer solution, pH 6, at 30 °C).

activity and on charge transport diffusion.19 On one hand, we previously showed that electron transfer at LDH-modified electrodes depends on the electrolyte nature but also that an increase in electrolyte concentration improves its efficiency.20,21 On the other hand, adsorption of proteins on a solid surface is a complex phenomenon. The subtle balance between the intrinsic stability of the protein in solution, the physicochemical properties of the solid surface, and the pH and ionic strength of the solvent phase will have important effects on the state of an adsorbed enzyme.22 For instance, phosphate ions are reported to be a competitive sorbant for PPO adsorbed on Al(OH)x-montmorillonite complexes.10 In the present work, the optimum phosphate concentration is 0.05 M. Figure 4 illustrates the effect of operating potential on the background current and on the current response of the biosensor. The maximum response was obtained at -0.2 V while a decrease in biosensor response was observed for more negative potentials. At the same time, background current increased as the potential became more negative from -0.1 to -0.3 V. This may attributed to an increase in the direct oxygen reduction at the electrode surface leading to an oxygen depletion within the biocoating and hence to a decrease in the enzymatic rate. Biosensor Amperometric Characteristics. The steady-state current response of the PPO/LDH electrode to catechol was determined under these optimum conditions (Figure 5, curve a). The linear range spans the concentration of catechol from 1.7 nM to 13.3 µM with a correlation coefficient of 0.9996 (n ) 23). A low detection limit of 1.7 nM catechol was estimated at signal-to-noise ratio of 3. Its sensitivity and Imax are 7807 mA M-1 cm-2 and 492 µA cm-2, respectively. The apparent Michaelis-Menten constant (Kapp M ) 0.048 mM) was evaluated from the electrochemical Lineweaver-Burk plot analysis of the catechol calibration curve. The reproducibility of the current response of the PPO/LDH electrode was examined at a catechol concentration of 2 nM. The relative standard deviation (RDS) is 1% for n ) 9. The reproducibility of the analytical response obtained from different electrodes constructed by the same procedure was also analyzed. Eight (19) Daigle, F.; Leech, D. Anal. Chem. 1997, 69, 4108-41112. (20) Therias, S.; Mousty, C.; Forano, C.; Besse, J.-P. Langmuir 1996, 12, 49144920. (21) Therias, S.; Lacroix, B.; Schollhorn, B.; Mousty, C.; Palvadeau, P. J. Electroanal. Chem. 1998, 454, 91-97. (22) Quiquampoix, H. Soil Biochem. 2000, 10, 171-206.

Figure 5. Calibration curves of (a) PPO/LDH and (b) PPO/Laponite (25/25 µg/µg) for catechol, under the optimum conditions (0.05 M phosphate buffer solution pH 6, at 30 °C, Eapp ) -0.2 V).

Figure 6. Storage stability for PPO/LDH biosensor testing in 0.05 M phosphate buffer pH 6, at 30 °C, Eapp ) -0.2 V.

different electrodes were tested independently for catechol amperometric response. The RSD value for the all eight electrodes was 6.5%. This indicates, in particular, an efficient and reproducible immobilization process of PPO within the LDH matrix although the procedure used was a nonautomatic handmade process. The long-term stability test was evaluated for three weeks under storage at 4 °C (Figure 6). The electrode was tested weekly. The biosensor retained ∼89% of its original response after 21 days. This response decreases to 60% after 28 days of storage. This biosensor stability is better than that reported for a PPO/Laponitemodified electrode for which the residual activity is 47% after 21 days.12 Response Characteristics of the Biosensor to Various Phenolic Compounds. The substrate specificity of PPO enables the detection of a wide range of compounds. To test the performance of this PPO/LDH bioelectrode to the other phenolic compounds, four monophenolic compounds were determined (Table 2). The sensitivity follows the trends, 4-chlorophenol > 4-cresol > phenol > 2-cresol. The detection limit for phenol, 4-cresol, and 4-chlorophenol reaches values of 1.7, 0.7, and 1 nM, respectively. These analytical characteristics of the PPO/LDH biosensor can be compared to those previously reported for the PPO biosensor based on Laponite, a cationic clay matrix. In this latter case, we used redox mediators (azure B or polyazure B) in order to improve the analytical characteristics of the biosensors.12,13 Even with this mediated detection process, the sensitivities and detection Analytical Chemistry, Vol. 75, No. 15, August 1, 2003

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Table 2. Amperometric Characteristics of the PPO/LDH Bioelectrode to Phenolic Substrates (EApp ) -0.2 V, in pH 6, 0.05 M Phosphate)

a

compound

sensitivity (mA M-1 cm-2)

Imax (µA cm-2)

LR (M)

R2 a

DLb (nM)

phenol 4-cresol 2-cresol 4-chlorophenol

1488 ( 7 5273 ( 5 11.0 ( 0.1 8167 ( 15

210 140 19 296

1.7 × 10-9-3.8 × 10-5 1.7 × 10-9-1.6 × 10-6 7.6 × 10-7-7.7 × 10-5 1.1 × 10-9-6.6 × 10-6

0.9996 0.9999 0.9988 0.9999

1.7 0.7 760 1

Correlation coefficient of the linear range. b The detection limit determined from S/N ) 3.

Table 3. Comparison of Biosensor Characteristics to Catechol Determinationa

SA (mA M-1 cm-2) SB (mA M-1 cm-2) SC (mA M-1 cm-2) SA/(SC - SB) Pm (cm s-1) Imax (µA cm-2) Kapp M (µM) Ea (kJ mol-1)

PPO/LDH

PPO/Laponite

7807 ( 32 281 ( 4 791 ( 4 15 2.5 ( 0.2 × 10-2 492 48 24 ( 2

2729 ( 11 82.3 ( 0.7 510 ( 9 6 6.6 ( 0.1 × 10-3 322 95 35 ( 2

a S , biosensor sensitivity to catechol at -0.2 V; S , biosensor A B sensitivity to catechol at 0.6 V.; SC, biosensor sensitivity to catechol at 0.6 V after thermal denaturation of immobilized PPO.

limits of PPO/Laponite biosensors are worst for catechol and 4-chlorophenol or at least similar for phenol and 4-cresol than those reported with this new PPO biosensor based on the LDH matrix. Moreover, when dealing with the analysis of real samples of wastewater, the fabrication procedure of an enzyme electrode without mediator is simpler compared to the mediated one. It appears therefore that the layered double hydroxides provide an environment favorable to the PPO biosensor activity. DISCUSSION It is well known that biosensor activity depends on three main factors: the diffusion of substrate/product through the biomembrane, enzyme activity within the immobilization matrix, and efficiency of the electrochemical transduction step.23 To clarify the role played by the immobilization clay matrix on these three parameters, we compared the biosensors’ performance to catechol detection at both types of clay matrix: the [Zn-Al-Cl] anionic clay and the Laponite cationic clay with the same configuration (25 µg/25 µg of PPO/clay) and under the same experimental conditions. Figure 5 qualitatively confirms the best performance obtained with the PPO/LDH biosensor. The analytical performances of both electrodes are compared in Table 3. Permeability Study of the Biomembranes. The masstransfer process through PPO/clay biocoatings to the surface of an electrode can be examined by the use of rotating disk electrode voltammetry. Due to the high charge density of the LDH or Laponite layer, any charged molecule would affect the experiment results. So the rotating disk voltammograms were obtained at PPO/LDH and PPO/Laponite electrodes in an aqueous solution of a neutral molecule, hydroquinone (2 mM). The rotating disk (23) Eggins, B. R. Chemical Sensors and Biosensors; J. Wiley and Sons, Ltd.: Chichester, U.K., 2002.

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voltammograms were also recorded from the bare glassy carbon electrode as a control electrode. By comparing the ilim corresponding to the oxidation of hydroquinone at the three different electrodes, the PPO/Laponite biomembrane shows a higher diffusional resistance to the substrate than the PPO/HDL biomembrane (ilim(500 rpm) ) 0.992, 0.842, and 0.367 mA cm-2 for the bare, PPO/LDH, and PPO/Laponite electrodes, respectively). The permeability Pm of the biomembranes can be determined by using rotating disk electrode experiments at different rotating velocities. The results were treated through eqs 1-3, described by Gough and Leypoldt:24-26

1/ilim ) 1/0.62nFADs2/3C°ν1/6ω1/2 + δ/nFAC°KDm Pm ) KDm /δ

(1) (2)

1/ilim ) 1/0.62nFADs2/3C°ν1/6ω1/2 + 1/nFPmAC° (3)

The first term in eq 3 is the Levich current, which applies to current passed under the same conditions in the absence of clay coating. The permeability (Pm), in the second term, is the parameter to be extracted. A plot of 1/ilim versus 1/ω1/2 presents a linear behavior with the same slope as for a bare electrode with a positive intercept, whose value depends on the permeability Pm of the biomembrane. The estimated permeability of the PPO/ LDH biomembrane was 2.5 × 10-2 cm s-1, ∼10 times greater than that of the PPO/Laponite biomembrane (2.6 × 10-3 cm s-1). These results are in agreement with the values previously found for other biosensors based on a clay matrix, namely, urease/[Zn-Al-Cl] and urease/Laponite electrodes.16 As we have previously suggested, this effect may be due to the difference in clay morphology, in particular their particle size, namely, 200 × 200 × 20 nm and 40 × 10 × 1 nm for LDH and Laponite particles, respectively.3,15 SEM photographs of PPO/Laponite film coated on a glassy carbon electrode show a smooth and compact surface where the clay particles are completely merged into the enzyme film (Figure 7A). In the case of the PPO/[Zn-Al-Cl]-modified electrode, the LDH particles are still observable with the enzyme absorbed on the external surface of the layers (Figure 7B,C). The typical mesoporous structure called “sand roses” is certainly favorable to substrate diffusion to reach the immobilized enzyme within the whole LDH matrix. (24) Gough, D. A.; Leypoldt, J. K. Anal. Chem. 1979, 51, 439-444. (25) Gough, D. A.; Leypoldt, J. K. Anal. Chem. 1980, 52, 1126-1130. (26) Gough, D. A.; Leypoldt, J. K. J. Electrochem. Soc. 1980, 127, 1278-1286.

Scheme 1. Schematic Description of the Three Function Configurations of the PPO/Catechol Electrochemical System (A), Bioelectrodes Potentiostated at -0.2 V (B), and (C) Bioelectrodes Potentiostated at 0.6 Va

Figure 7. SEM photographs of PPO/Laponite (A) and PPO/[ZnAl-Cl] (B,C) modified glassy carbon electrodes.

Electroenzymatic Model for Characterizing the Biosensor. Cosnier and co-workers described a useful model to highlight detailed processes occurring in amperometric PPO-based sensors.27 In this biosensor process, the electrochemical transduction step corresponds to the reduction at -0.2 V of o-quinone, enzymatically formed within the biocoating. Moreover, catechol, the substrate, can be directly electrooxidized at 0.6 V and the immobilized enzyme can lose its activity after a thermal denaturation. Thus, depending on the applied potential (-0.2 or 0.6 V) and the reactive state of the enzyme (active or inactive), the immobilized PPO/catechol system can provide information on the enzyme and transduction efficiencies. Scheme 1 presents the three different efficient configurations of this system providing the parameters: (A) the current response (SA) for the diffusion of the enzymatically generated o-quinone recorded at -0.2 V, (B) the current response (SB) for the catechol (27) Besombes, J-L.; Cosnier, S.; Labbe´, P. Talanta 1996, 43, 1615-1619.

a PPO was inactivated in configuration C. S and P denoted respectively the catechol substrate and o-quinone enzymatically or electrochemically produced.

nonenzymatically oxidized during its diffusion through the biomembrane (Eapp ) 0.6 V), and (C) the current response (SC) for the catechol diffusion through the inactivated biomembrane. To keep this model simple, the diffusion of catechol in the biomembrane is considered identical to that of o-quinone and the supply of cosubstrate (O2) for PPO is supposed to be plentiful. Both PPO/LDH and PPO/Laponite biosensors were analyzed with the model. (Table 3). The analysis of the parameters gives some information about the PPO/catechol system involved in the biocatalytic process. For instance, for the same bioelectrode, the difference of the response sensitivities measured at 0.6 V (SB) for active and inactive (SC) biomaterial allows us to estimate the amount of catechol enzymatically oxidized during its permeation (Scheme 1B,C). Moreover, the efficiency of the bioelectrochemical detection can be evaluated via a comparison of the amounts of o-quinone electrochemically detected and enzymatically generated. This is illustrated by the Analytical Chemistry, Vol. 75, No. 15, August 1, 2003

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value of the SA/(SC - SB) ratio (Scheme 1A), SA and (SC - SB) being biosensor sensitivities corresponding to the electroreduction of enzymatically generated o-quinone and the enzymatic disappearance of catechol, respectively. This ratio indicates signal amplification due to the well-known substrate recycling.28 It is clear that the PPO/LDH biosensor induces a greater intrinsic amplification factor (15) than the PPO/Laponite biosensor (6) (Table 3). This fact can be due, at least partially, to the good permeability of the LDH host matrix. But it may be also due to an efficient electrochemical transfer. Indeed, a main drawback inherent to PPO-based sensors is the electrode fouling induced by polymerization of radicals formed during the electroenzymatic sequence. A more sensitive response is obtained when o-quinone is reduced via an electrocatalytic process.12,13 The [Zn-Al-Cl]-modified electrodes were reported to catalyze the catechol system.29 The charge-transfer kinetic and electrochemical reversibilities were improved, leading to a retention of electroactivity upon repetitive cycling. Hence, at PPO/[Zn-Al-Cl]-modified electrodes, the electrode fouling may be prevented and the regenerated catechol can be implied again in the recycling process. The same phenomenon was reported for an alumina sol-gel-derived PPO biosensor.30 The Al2O3 sol-gel surface displays an intrinsic electrocatalytic o-quinone response that offers a high-sensitivity (1814 mA M-1 cm-2) monitoring phenol. The value found for phenol in the present study is slightly lower (1488 mA M-1 cm-2). However, the sensitivity and detection limit values found, in particular for catechol, 4-cresol, and 4-chlorophenol, are better than that more recently reported with mediated polyazure B-PPO/ Laponite biosensors,13 with other PPO biosensors based on solgel matrixes31,32 or based on a polyaniline-polyacrylonitrile composite matrix,33 for example. Estimated Activity of Immobilized PPO. This better performance for catechol determination of the PPO/LDH biosensor is also confirmed by comparison of the apparent MichaelisMenten constant (Kapp M ), the maximum current (Imax), and the apparent activation energy (Ea) values reported in Table 3 for both biosensors. As expected for immobilized PPO, Kapp M values are lower than that found for the free enzyme (0.28 mM).18 The lowering of Kapp M values can be explained by the fact that o-quinone can enter into another enzymatic oxidation (Scheme 1A), providing a local increase in substrate concentration and an amplification of the electrode response.34 Even though the same amount of enzyme was immobilized in the two types of biosensors, the obtained Kapp M value was lower and Imax was higher for the PPO/LDH compared to the PPO/Laponite biosensor. In the same way, the Ea of the electroenzymatic reaction calculated according to the Arrhenius equation (ln i vs T-1) in the temperature range 1030°C is lower for the PPO/LDH than for the PPO/Laponite biosensor, namely, 24 and 35 kJ mol-1. The value of Ea, determined by the same method for free enzyme (7 kJ mol-1) is similar to the previously reported value (9 kJ mol-1).35 (28) Burestedt, E.; Narvaez, A.; Ruzgas, T.; Gorton, L.; Emme´us, J.; Dominguez, E.; Marko-Varga, G. Anal. Chem. 1996, 68, 1605-1611. (29) Shaw, B. R.; Creasy, K. E. J. Electroanal. Chem. 1988, 243, 209-217. (30) Liu, Z.; Liu, B.; Kong, J.; Deng, J. Anal. Chem. 2000, 72, 4707-4712. (31) Wang, B.; Zhang, J.; Dong, S. Biosens. Bioelectron. 2000, 15, 397-402. (32) Chen, X.; Cheng, G.; Dong, S. Analyst 2001, 126, 1728-1732. (33) Xue, H.; Shen, Z. Talanta 2002, 57, 289-295. (34) Cosnier, S.; Innocent, C. Bielectrochem. Bioeng. 1993, 31, 147-160.

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All these results induce that the diffusion barrier for the substrate is smaller in LDH and that LDH offers a better biocompatibility for the immobilized enzyme molecules. This latter fact suggests a higher amount of active enzyme after immobilization. Consequently, we have compared the residual activity of PPO immobilized on Laponite and on [Zn-Al-Cl] clay matrixes (see Experimental Section). The activity of PPO is 2 times higher when LDH (80%) is used as immobilization matrix instead of Laponite (40%). The same ratio (2) is obtained by the electrochemical assays. It means that LDH provides a better microenvironment for PPO, leading to a higher activity. Similarly, Inacio reported that LDH has less of an inhibition effect than montmorillonite, another cationic clay, for the enzymatic activity of chymotrypsin.36 Moreover, the presence of intercalated hydroxyaluminum into montmorillonite (Al(OH)xMte) modifies the adsorption capacity and activity of PPO.11 The immobilization of PPO on Camontmorillonite was not evident whereas the enzyme was immobilized on Al(OH)xMte. The amount of PPO immobilized on clay surfaces as well its specific activity increased with increasing level of hydroxyaluminum. These results suggest that specific interactions exist between the hydroxides materials and PPO. The isoelectric point (pI) of PPO (mushroom tyrosinase from Sigma) is reported to be 4.1.37 When the pH is higher than the pI, which is the case in this study (pH 6.0), the net charge of the enzyme is negative. Since the LDH sheets are positively charged at this pH, the electrostatic interaction between the enzyme and the matrix could be favorable to enzyme adsorption on the clay surface. However, preliminary XR diffraction and SEM measurements show no specific intercalation on the enzyme between LDH interlayer space, the enzyme being simply adsorbed on the external surface of the clay particles. Nevertheless, when proteins interact with a highly charged surface like cationic clay, electrostatic forces as well as hydrophilic interactions are important.22 Generally, adsorption of enzyme causes a drastic decrease of its activity due to a modification of the conformation of the protein. A detailed study of adsorption and enzymatic properties of PPO as a function of surface density of the [Zn-Al-Cl] layers, pH, and ionic strength would certainly be useful to understand better the specific behavior of PPO adsorbed at the LDH matrix. CONCLUSION In this work, we have described a comparative study between the properties of two different phenol biosensors based on the immobilization matrixes [Zn-Al-Cl] LDH, a anionic clay, and Laponite a cationic clay. The PPO/[Zn-Al-Cl] biosensor showed remarkable properties such as high sensitivity, low Kapp M , and good storage stability. The permeability of the biomembrane and the nature and the morphology of the clay clearly affect the biosensor performance. Due to the fact the LDH exit as synthetic phases, several parameters can be modulated during the coprecipitation process, for instance, the nature the metallic cations present in the layers, sheet charge density, and particle size.15 All these parameters would certainly influence diffusion within the biocoating, as well as the enzyme adsorption properties and hence its activity. Control of all these parameters will certainly (35) Duckworth, H. W.; Coleman, J. E. J. Biol. Chem. 1970, 1613-1625. (36) Inacio, J. Ph D. Thesis, Blaise Pascal University, Clermont-Ferrand, 2002. (37) Espin, J. C.; Wichers, H. J. J. Agric. Food Chem. 1999, 472, 638-2644.

contribute to improvement of the biosensor fabrication. This will be the main goal of our future investigation.

fellowship. C.M. thanks Pr. C. Forano (Universite´ Blaise Pascal of ClermontsFerrand) for providing the [Zn-Al-Cl] phase.

ACKNOWLEDGMENT

Received for review January 14, 2003. Accepted April 29, 2003.

D.S. gratefully acknowledges the Association Franco-Chinoise pour la Recherche Scientifique et Technique for her doctoral

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