Clay

Anal. Chem. 2004, 76, 178-183

Subnanomolar Cyanide Detection at Polyphenol Oxidase/Clay Biosensors Dan Shan, Christine Mousty, and Serge Cosnier*

Laboratoire d’Electrochimie Organique et de Photochimie Re´ dox, UMR CNRS 5630, Institut de Chimie Mole´ culaire de Grenoble, FR CNRS 2607, Universite´ Joseph Fourier, Grenoble, France

A novel, inexpensive, and simple amperometric biosensor based on immobilization of polyphenol oxidase (PPO) into Zn-Al layered double hydroxides, also called anionic clays, is applied for determination of cyanide. The detection of cyanide was performed via its inhibiting action on the PPO electrode. Measurement was carried out with 3,4-dihydroxyphenylacetic acid as enzyme substrate, the enzymatically generated quinoid products being electroreduced at -0.2 V. An extremely sensitive detection limit (0.1 nM) was obtained for cyanide. Enzyme immobilization into an anionic exchanger clay seems to cause an increase in cyanide inhibition effects because of anion accumulation in the clay matrix. Cyanide is commonly used worldwide in industrial applications such as metalplating, metal mining, and plastics manufacture. As a consequence, accidental cyanide release in wastewater or rivers may lead rapidly to serious contamination of groundwater and even drinking water. Owing to its extreme toxicity, in particular its role in the suppression of oxygen transport, the presence of cyanide in drinking water is at the origin of serious human diseases that would have lethal issue. The World Health Organization fixed the maximum acceptable level of cyanide in drinking water at 1.9 µM. Several conventional methods such as titrimetric and spectrophotometric procedures, chromatographic methods, and electrochemical devices were employed for the cyanide determination, the detection limits being around 0.2-1 µM.1,2 The most sensitive approach (detection limit 8 nM) was based on fluorometric measurement combined with HPLC.3 Most of these methods, however, require several steps such as sample pretreatment and are not applicable for on-site cyanide monitoring. An attractive alternative consists of the design of portable cyanide biosensors. Electrochemical biosensors have been increasingly developed for continuous monitoring in environmental and health care applications. Such devices contain a biological sensing element intimately associated with a transducer that * Corresponding author: (e-mail) [email protected]; (fax) 0033476514267. (1) Ikebukuro, K.; Nakamusa, H.; Karube, I. In Handbook of Water Analysis; Nollet, L. M. L., Ed.; Marcel Dekker: New York, 2000; Vol. 102, 367-385. (2) Standard methods for the examination of water and wasterwater, 20th ed.; Clesceri, L. S., Greenberg, A. E., Eaton, A. D., Franson, M. A. H., Eds.; Water Environment Federation, American Public Health Association: Washington, DC, 1998. (3) Gamoth, K.; Sawamoto, H. Anal. Sci. 1988, 4, 665-666.

178 Analytical Chemistry, Vol. 76, No. 1, January 1, 2004

converts the specific biorecognition event to an electrical signal. The advantages of biosensors are the simple measurement procedure, short response time, and high selectivity by substrate specificity. These devices were applied to the direct monitoring of water pollutants, such as phenolic-based compounds and organophosphates.4 Moreover, the biodetection of toxins such as heavy metal ions, pesticides, or cyanides was carried out through inhibition processes of enzymatic reaction.5 The modulated biocatalytic activity resulting from the enzyme inhibition was commonly detected from the decreased amperometric response of the biosensor to its substrate. Cyanide biosensors were thus developed by inhibition of enzymes: horseradish peroxidase6-10 polyphenol oxidase11-14 cytochrome oxidase15 and microorganisms (Pseudomonas fluorescens NCIMB 11764),16,17 allowing with the most sensitive configuration to detect cyanide down to ∼0.02 µM. Taking into account that the latter were mainly used in batch experiments that required a sample dilution by a factor of 100 and assuming a detection range between 0.1 and 2 µM, the theoretical detection limit for a biosensor should be in the nanomolar range. As a consequence, the simple inhibition of a enzyme does not appear sensitive enough and should be associated to an amplification phenomenon or a preconcentration process to improve the detection limit of the resulting biosensor. With this aim in view, the present paper describes a novel biosensor for monitoring cyanide based on a polyphenol oxidase (PPO) entrapped in synthetic layered double hydroxides (LDHs). The LDHs consists of MII(OH)6 and MIII(OH)6 edge-sharing octahedra forming sheets similar to those of brucite.18 Net positive (4) Dong, S.; Wang, B. Electroanalysis 2002, 14, 7-16. (5) Evtugyn, G. A.; Budnikov, H. C.; Nikolskaya, E. B. Talanta 1998, 46, 465484. (6) Ruzgas, T.; Cso ¨regi, E.; Emmeús, J.; Gorton, L.; Marko-Varga, G. Anal. Chim. Acta 1996, 330, 123-138. (7) Smit, M. H.; Cass, A. E. Anal. Chem. 1990, 62, 2429-2436. (8) Tatsuma, T.; Oyama, N. Anal. Chem. 1996, 68, 1612-1615. (9) Park, T. M.; Iwuoha, E. I.; Smyth, M. R. Electroanalysis 1997, 9, 11201123. (10) Volotovsky, V.; Kim, N. Biosens. Bioelectron. 1998, 13, 1029-1033. (11) Smit, M. H.; Rechnitz, G. A. Anal. Chem. 1993, 65, 380-385. (12) Hu, X.; Leng, Z. Analyst 1995, 120, 1555-1557. (13) Besombes, J.-L.; Cosnier, S.; Labbe´, P.; Reverdy, G. Anal. Chim. Acta 1995, 311, 255. (14) Wang, J.; Tian, B.; Lu, J.; McDonald, D.; Wang, J.; Luo, D. Electroanalysis 1998, 15, 1034-1037. (15) Amine, A.; Alafandy, M.; Kauffmann, J. M.; Pekli, M. Anal. Chem. 1995, 67, 2882-2827. (16) Lee, J. I.; Karube, I. Anal. Chim. Acta 1995, 313, 69-74. (17) Lee, J. I.; Karube, I. Biosens. Bioelectron. 1996, 11, 1147-1154. 10.1021/ac034713m CCC: $27.50

© 2004 American Chemical Society Published on Web 11/25/2003

Table 1. Influence of Substrate Nature on Cyanide Inhibition at PPO/[Zn-Al-Cl] Electrode substrate (10 µM)

Kcat/KMa (s-1 mM-1)

substrate sensitivity (mA M-1 cm-2)

Inmax (%)

recovered activity (%)

detection limit (nM)

linear range (M)

catechol dopamine DOPAC L-DOPA

2925 200 294 134

7807 523 423 19

40 57 98 36

25 25 50 29

50 8 7 20

5 × 10-8-8 × 10-7 4.4 × 10-8-1 × 10-5 7 × 10-9-1.8 × 10-7 2 × 10-8-1.6 × 10-7

a

From ref 24.

charges of the layer are balanced by exchangeable anions intercalated between the sheets. Therefore, LDHs presents a lamellar structure and intercalation properties similar to that of cationic clays such as Laponite but with a higher charge density of the layers (anionic exchange capacity ∼2 mequiv g-1). These properties were recently exploited for the fabrication of enzyme electodes.19,20 Moreover, the positive charged layer may be an attractive point for preconcentration of negative analytes. In the literature, the preconcentration of inhibitors in the immobilization matrix was invoked to the spectacular low down of the detection limits.5,21 For cyanide, this effect was observed in poly(4-vinylpyridine-co-styrene) and in cationic amphiphilic polypyrrole.10,13 Similarly, we have previously shown that the inhibitor effect of tetraborate, an anionic compound, on the urease activity is stronger for a urease/LDH biosensor than for a urease/ Laponite biosensor, the latter matrix exhibiting cation exchange properties.19 The determination of cyanide was thus carried out through its inhibitory effect on the oxidase activity of PPO toward various diphenolic compounds. The role of the host matrix (LDHs) on the improvement of the biosensor performance was investigated by comparison between biosensors fabricated with LDHs and Laponite.

out in a conventional thermostated three-electrode cell (10 mL) at 30 °C. An Ag/AgCl electrode saturated with KCl solution was used as reference electrode, and a Pt wire was placed in a separate compartment containing the supporting electrolyte, as a counter electrode. The working electrode was a glassy carbon electrode (diameter 5 mm) polished with 1 µM diamond paste. Enzyme Immobilization. The clay colloidal suspension ([ZnAl-Cl] or Laponite, 2 mg mL-1) was dispersed overnight under stirring conditions in deionized and decarbonated water. PPO was dissolved in water with at a concentration of 8 mg mL-1. A defined amount of aqueous mixtures (for example, containing 25 µg of clay and 25 µg of PPO) was spread on the surface of the glassy carbon electrode. The coating was dried in air at room temperature. The resulting electrode was placed in saturated glutaraldehyde vapor for 15 min for cross-linking of the membrane. Finally, the PPO/clay biomembrane was rehydrated for 20 min into 0.05 M phosphate buffer solution (pH 6).

EXPERIMENTAL SECTION Materials. Polyphenol oxidase (EC 1.14.18.1) from mushroom (6050 units mg-1), catechol, 3,4-dihydroxyphenylacetic acid (DOPAC), dopamine, and 3-(3,4-dihydroxyphenyl)-L-alanine (L-DOPA) were purchased from Sigma. Laponite was received from Rockwood Specialties and glutaraldehyde from Fluka. The layered double hydroxide Zn3Al(OH)8Cl was synthesized by the coprecipitation method developed by De Roy.18 All other chemical reagents are of analytical grade. Water was doubly distilled in quartz apparatus. Since cyanide is a dangerous toxic compound, precautions must be taken to prevent any accidental release. Fresh solutions were prepared in pH 6, 0.05 M phosphate buffer. All the cyanide solutions were disposed of by adding an excess of sodium hypochlorite. Apparatus. The amperometric measurement was performed with a Tacussed PRG-DL potentiostat in conjuction with a Kipp and Zonen BP 91 XY/t recorder. All the experiments were carried

RESULTS AND DISCUSSION Influence of the Enzyme Substrate. PPO is a bifunctional copper protein that presents at least two distinct binding sites, one for aromatic compounds (substrate site) and the other for a metal-binding agent (oxygen site).22 The enzymatic oxidation diphenol derivatives provided o-quinones while molecular oxygen was reduced into water. The functioning mechanism of the amperometric PPO electrodes was therefore based on o-quinone reduction at -0.2 V. The influence of buffer nature, namely, phosphate buffer or Tris-HCl, and its concentration on the biosensor performance indicated an optimum biosensor response for 0.05 M phosphate buffer.23 The analytical performance of PPO/[Zn-Al-Cl] (25 µg/25 µg) electrodes was investigated in air-saturated 0.05 M phosphate buffer (pH 6.0) at -0.2 V for the detection of catechol and several other substituted diphenol substrates. The sensitivity of the PPO/ [Zn-Al-Cl] electrode follows the sequence catechol . dopamine > DOPAC . L-DOPA that is similar to the substrate specificity (Table 1) reported for the free enzyme in terms of Vmax and catalytic efficiency (Kcat/KM).24 Cyanide inhibition at PPO/[Zn-Al-Cl] electrodes was investigated by the following procedure. The enzyme substrate at a defined concentration was added to the electrolyte solution, and the current response was recorded at -0.2 V. After the stabiliza-

(18) De Roy, A.; Forano, C.; El Malki, K.; Besse, J.-P. In Expanded clays and other microporous solids; Occelli, M. L., Robson, H. E., Eds.; Van Nostrand Reinhold: New York, 1992; pp 108-169. (19) de Melo, J. V.; Cosnier, S.; Mousty, C.; Martelet, C.; Jaffrezic-Renault, N. Anal. Chem. 2002, 74, 4037-4043. (20) Shan, D.; Cosnier, S.; Mousty, C. Anal. Lett. 2003, 36, 907-920.

(21) Evtugyn, G. A.; Ivanov, A. N.; Gogol, E. V.; Marty, J.-L.; Budnokov, H. C. Anal. Chim. Acta 1999, 385, 13-21. (22) Duckworth, H. W.; Coleman, J. E. J. Biol. Chem. 1970, 1613-1625. (23) Shan, D.; Cosnier, S.; Mousty C. Anal. Chem. 2003, 75, 3872-3875. (24) Espıń, J. C.; Varoń, R.; Fenoll, L. G.; Gilabert, M. A.; Garcıá-Ruı´z, P. A.; Tudela, J.; Garcıá -Cańovas, F. Eur. J. Biochem. 2000, 267, 1270-1279.

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Figure 1. Inhibition of the amperometric response of PPO/[ZnAl-Cl] bioelectrode to substituted catechol substrates (10 µM) induced by increasing concentrations of cyanide: (a) DOPAC, (b) L-DOPA, (c) dopamine, (d) catechol, and (e) PPO/Laponite bioelectrode in 20 µM DOPAC. (Eapp -0.2 V, 30 °C; 0.05 M phosphate buffer pH 6).

tion of the steady-state current response, cyanide was added, inducing immediately a decrease in the biosensor response. The cyanide effect was quantified as an inhibition percentage (In/%) corresponding to the ratio of the current decrease (I°-I) versus the original current I° (no inhibition) in the steady state. The detection limit is based on a signal-to-noise ratio of 3. Figure 1 presents the influence of the substrate nature on the resulting calibration curves obtained at a PPO/[Zn-Al-Cl] (25 µg/25 µg) electrode for the inhibitive detection of cyanide. It appears that the inhibition efficiency is highest with DOPAC, intermediate with dopamine, and lowest with catechol and L-DOPA. As previously described for PPO/polypyrrole film,13 the substrate exhibiting the highest affinity for the active enzyme site (catechol, KM ) 0.3 mM)24 leads to a less efficient system for cyanide determination. Nevertheless, no relationship may be clearly established between the substrate affinities, illustrated by the substrate sensitivity (Table 1) and the efficiency of the inhibition process. These data are consistent with the reported cyanide effect that is competitive with molecular oxygen and noncompetitive with o-diphenol substrates.22 It should be noted that the binding of cyanide to the active binuclear copper site of PPO is reversible11,25 In contrast with the other substrates, cyanide can inhibit completely the biosensor response to DOPAC providing also the lowest detection limit (7 nM). To determine the recovered activity (RA), the biosensor was washed carefully with electrolyte after the inhibition process and the amperometric response toward the enzyme substrate was again measured in the absence of cyanide. The RA (%) is I°′/I°, where I° and I°′ correspond to the biosensor responses before and after cyanide determination, respectively. The RA are ∼25%, except for DOPAC (50%) (Table 1). This may be due to specific interactions between cyanide ions and the LDHs membrane. Indeed, layered double hydroxydes are known as anionic exchanger material that should induce an accumulation of cyanide anion within the biomembrane. The latter can still affect (25) Wilcox, D. E.; Porras, A. G.; Hwang, Y. T.; Lerch, K.; Winkler, M. E.; Solomon, E. I. J. Am. Chem. Soc. 1985, 107, 4015-4027.

180 Analytical Chemistry, Vol. 76, No. 1, January 1, 2004

the respiratory effect of the PPO enzyme and hence biosensor performance even in the absence of cyanide in solution. Taking into account the anionic nature of DOPAC, a competitive accumulation with cyanide can take place in the LDHs membrane leading to a better recovery (50%). As a consequence, DOPAC was chosen as the enzyme substrate in the following procedure. The reproducibility of the biosensor response to DOPAC was examined. Five electrodes constructed in the same procedure were tested independently for DOPAC amperometric response. The RSD value for the five electrodes was 3%. This indicates, in particular, an efficient and reproducible immobilization process of PPO within an LDH matrix. The long-term stability test was evaluated for 3 weeks under storage in the dry state at 4 °C. The biosensor retained ∼89% of its original response after 21 days.23 The dependence of DOPAC concentration on the inhibitory effect was also evaluated. It appears that a quasi-total inhibition (Inmax ≈ 100%) was obtained for all the investigated DOPAC concentrations (Table 2). Excepted for the highest DOPAC concentration (40 µM), all PPO/[Zn-Al-Cl]-DOPAC systems provided a similar RA (44-50%). However, the analytical characteristics of the resulting calibration curves for cyanide presented marked differences in terms of sensitivity, detection limit, and linear range (Table 2). The optimum system was based on 20 µM DOPAC, the sensitivity and detection limit being 9.13 A M-1 cm-2 and 0.1 nM, respectively. Since the increase in DOPAC concentration occurred in the linear part of the calibration curve, the part of the immobilized enzyme involved in the DOPAC detection increased for 10-40 µM DOPAC. As a consequence, the inhibitory effect of cyanide became more easily detectable as the part of PPO that interacted with DOPAC increased.5 On the other hand, as previously reported for PPO and flavin reductase, the electrochemical regeneration of the enzyme substrate induced an amplification of the biosensor response, the latter being inversely proportional to the substrate concentration.26-30 The effect of cyanide on the enzyme activity should be thus more important when the recycling phenomenon is maximum, namely, for the lowest substrate concentration. As a consequence, the optimum sensitivity for cyanide detection obtained with 20 µM DOPAC reflected a compromise between complex electroenzymatic effects. A similar effect of the DOPAC concentration on biosensor sensitivity for cyanide was observed with the Laponite-PPO configuration, the optimum configuration for the determination of cyanide corresponding to 20 µM DOPAC (Table 2). This corroborates the preceding assumptions that highlight the role of the electrochemical substrate recycling. It should be noted that the detection limit (0.1 nM) is markedly lower than the values commonly reported in the literature. In particular, the PPO/[Zn-Al-Cl]-DOPAC system appears to outclass the most sensitive inhibition biosensors based on HRP (1 × 10-7 M)10 and PPO (2 × 10-8 M)13 whereas the latter (26) Cosnier, S.; Innocent, C.; Allien, L.; Poitry, S.; Tsacopoulos, M. Anal. Chem. 1997, 69, 968-971. (27) Lisdat, F.; Wollenberger, U.; Makower, A.; Ho ¨rtnagl, H.; Pfeiffer; Scheller, F. W. Biosens. Bioelectron. 1997, 12, 1199-1211. (28) Bauer, C. G.; Eremenko, A. V.; Ku ¨ hn, A.; Ku ¨ rzinger, K.; Makower, A.; Scheller, F. W. Anal. Chem. 1998, 70, 4624-4630. (29) Coche-Gue´rente, L.; Desprez, V.; Diard, J. P.; Labbe´, P. J. Electroanal. Chem. 1999, 470, 53-60. (30) Coche-Guerente, L.; Labbe´, P.; Mengeaud, V. Anal. Chem. 2001, 73, 32063218.

Table 2. Influence of DOPAC Concentration on Cyanide Inhibition at PPO/Clay Electrode clay [Zn-Al-Cl]

Laponite

a

[DOPAC] (µM)

Inmax (%)


sensitivitya (mA M-1 cm-2)

detection limit (nM)

linear range (M)

1 10 20 40 10 20 40

100 98 97 98 100 98 92

46 50 44 15 65 68 51

40 801 9130 866 56 139 68

5 7 0.1 0.4 140 80 110

5 × 10-8-5.4 × 10-7 7 × 10-9-1.8 × 10-7 2 × 10-9-5 × 10-8 3.5 × 10-8-2.2 × 10-5 1.4 × 10-7-1.4 × 10-5 6 × 10-7-4 × 10-6 1.1 × 10-7-1.5 × 10-5

The sensitivity is calculated from the slope of the linear part of the curve obtained from the decreased current versus the inhibitor concentration.

Figure 2. Amperometric response of a PPO/[Zn-Al-Cl] bioelectrode upon successive additions of 4 nM cyanide in the presence of DOPAC (20 µM). Experimental conditions as in Figure 1.

exploited an anion accumulation in the polymeric matrix to increase the inhibitory effect of cyanide. In addition, this claybased biosensor responds more rapidly to cyanide than these previously cited biosensors. For example, Figure 2 shows that the response of the PPO/[Zn-Al-Cl] electrode, to successive additions of increasing concentration of cyanide using 20 µM DOPAC as substrate, was very fast (7 s), with good repeatability (RSD 9%). It should be noted that the presence of possible interfering anions (1 mM) such as Cl-, Br-, or NO3- did not alter the biosensor performance (in terms of sensitivity and detection limit) for the determination of cyanide. Influence of the Biomembrane Configuration. Since the performance of a biosensor device is strongly dependent on its configuration, the influence of the enzyme/LDHs ratio and the amount of adsorbed coating on the cyanide sensing was investigated with 20 µM DOPAC. When the PPO/[Zn-Al-Cl] ratio (wt/ wt) was decreased from 1 to 0.125, the sensitivity of the biosensors decreased sharply from 9130 to 0.5 mA M-1 cm-2 and the detection limits increased from 0.1 nM to 50 µM (Table 3). Although the use of an excess of LDHs facilitated the regeneration of the biosensor response and hence preserved the PPO activity, equal amounts of enzyme and clay appeared as the best configuration. The influence of the amount of deposited coating and hence the thickness of the clay film on the biosensor performance was examined by doubling the total amount of PPO and LDHs, while the PPO/LDHs ratio was maintained at 1 (PPO/ [Zn-Al-Cl] 50 µg/50 µg). The maximum inhibition and RA remain almost constant, but a higher sensitivity to cyanide and a lower detection limit are observed with the thinner coating (Table 3). An increase in the film thickness may increase the diffusion pathway from the clay solution to the electrode surface. Moreover,

the sensitivity of PPO-based biosensors was strongly related to the efficiency of the recycling phenomenon providing an amplification of the amperometric biosensor response at low substrate concentration. The immobilized PPO catalyzed the oxidation of diphenols to quinones while the electrochemical reduction of quinones regenerated the PPO substrate. The efficiency of this amplification and hence of the inhibiting action of cyanide depended on the proximity at the molecular level of PPO to the electrode surface. So, the thinner biocoating (25 µg/25 µg) was used for further experiments. Comparison between PPO/LDHs and PPO/Laponite Biosensor Performance. To ascertain the role played by the LDHs matrix in the inhibition process, another PPO biomembrane based on Laponite, a cationic clay, was also tested as a reference biosensor. We have thus compared the analytical performance of biosensors based on the immobilization of PPO within both clay matrixes for the determination of DOPAC. The sensitivity and Imax obtained at PPO/Laponite electrode (157 mA M-1 cm-2 and 151 µA cm-2) remained inferior to those recorded for a PPO/ [Zn-Al-Cl] electrode (446 mA M-1 cm-2 and 254 µA cm-2) (Figure 3). The better DOPAC sensitivity may be ascribed to a higher permeability of LDHs (2.5 × 10-2 cm s-1, determined with hydroquinone as electroactive permeant) compared to that of the Laponite membrane (6.6 × 10-3 cm s-1). To examine the role of the ion exchange properties of the host matrixes on the inhibition process, the effect of cyanide on the analytical characteristic of a biosensor based on PPO immobilized in both oppositely charged clays was investigated. A calibration curve of cyanide was recorded at PPO/Laponite (25 µg/25 µg) under the same conditions as those previously used for PPO/[Zn-Al-Cl] electrode ([DOPAC] ) 20 µM) (Figure 1, curve e). The sensitivity (139 mA M-1 cm-2), the detection limit (80 nM), and the linear range (range 6 × 10-7-4 × 10-6 M) were markedly less attractive than those obtained at the PPO/[ZnAl-Cl] electrode (Table 2). These results highlighted the specific electrostatic interaction of the host matrix that may induce an accumulation of the inhibitor within the biocoating. Moreover, the Laponite-based biosensor exhibited a better RA (68%) than the LDH one (44%). This more efficient regeneration of the biosensor activity after the inhibition process was more likely due to the cationic exchange properties of Laponite that prevented cyanide adsorption within the biomembrane. This effect corroborated the postulated accumulation of cyanide in LDHs. Apparent Michaelis-Menten constants (Kapp M ) were evaluated from the electrochemical Lineweaver-Burk plots of the calibration Analytical Chemistry, Vol. 76, No. 1, January 1, 2004

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Table 3. Influence of Coating Composition on Cyanide Inhibition at the PPO/[Zn-Al-Cl] Electrodea

a

PPO/[Zn-Al-Cl]

Inmax (%)


sensitivity (mA M-1 cm-2)

detection limit (µM)

linear range (M)

25 µg/25 µg 25 µg/50 µg 25 µg/200 µg 50 µg/50 µg

97 91 98 90

44 80 95 37

9130 142 0.5 592

0.0001 0.037 51 0.17

2 × 10-9-5 × 10-8 4.8 × 10-6-4 × 10-5 7 × 10-5-4 × 10-4 9.5 × 10-7-7 × 10-6

[DOPAC] ) 20 µM.

Figure 3. Calibration plot for DOPAC determination with (a) PPO/ [Zn-Al-Cl] and (b) PPO/Laponite bioelectrodes (25 µg/25 µg). Experimental conditions as in Figure 1.

Figure 4. (A) Lineweawer-Burk plots for the PPO/Laponite electrode without (a) and with 5 (b), 10 (c), and 20 µM cyanide (d). (B) Plots of 1/I versus cyanide concentration in the presence 10 (a), 15 (b), 30 (c), and 63 (d) µM DOPAC.

curves obtained with PPO/clay biosensors in the absence and presence of cyanide at various concentrations (Figures 4A and 5). For the PPO/Laponite electrode, the Lineweawer-Burk plots presented the same Kapp M value (∼0.7 mM) while the maximum current (Imax) decreased with the increase in inhibitor concentration, reflecting thus a typical noncompetitive inhibition of PPO by cyanide. An apparent inhibitor binding constant (Ki ) 6 µM) was determined for this biosensor by the Dixon plots from different DOPAC concentrations (Figure 4B). This value is in good agreement with the Ki value (14.7 µM) reported for the free enzyme.22 182 Analytical Chemistry, Vol. 76, No. 1, January 1, 2004

Figure 5. Lineweawer-Burk plots for the PPO/[Zn-Al-Cl] electrode in 0.05 M phosphate buffer solution without (a) and with 5 (b), 10 (c), and 20 (d) µM KCN.

In contrast, the electrochemical Lineweaver-Burk model of the calibration curves for DOPAC obtained at the PPO/ [Zn-Al-Cl] electrode clearly indicated an increase in Kapp M values and a decrease in Imax values with the increase in cyanide concentration (Figure 5). Such patterns of inhibition are characteristics of a mixed inhibition process,31 corroborating a different behavior between both biosensors toward the inhibiting action of cyanide. This may be ascribed to the combination of pure enzyme inhibition process and electrostatic cyanide accumulation in the microenvironment of enzymes entrapped in a LDHs matrix. Influence of the Incubation Time. To examine the influence of incubation time on the accumulation of cyanide within the LDHs matrix, we have investigated the effect of cyanide (1 µM) on the response of the PPO/[Zn-Al-Cl] electrode to DOPAC (10 µM) as a function of incubation time. After measurement of the initial response, the biosensor was washed and dipped into cyanide solution for varying incubation times. Then, the measurement of the inhibited response was recorded in the same DOPAC solution. The maximum inhibition effect was obtained after 10 min. This optimum incubation time was exploited to improve biosensor performance. It appears that the biosensor sensitivity (54.8 A M-1 cm-2) at low cyanide concentration (1-10 nM) is ∼6 times higher (31) Rawn, J. D. Traite´ de Biochimie; De Boeck-Wesmael s.a.: Bruxelles, 1990; pp 176-184.

than that determined without incubation. These results corroborate the important effect of cyanide accumulation into anionic clay matrix on the inhibition process. CONCLUSION In this work, we have described the development of a cyanide biosensor based on the entrapment of PPO into the layered double hydroxide membrane. Such devices offer a fast and sensitive response for cyanide. Furthermore, to our knowledge, the bioelectrode provides the lowest detection limit (0.1 nM) for cyanide reported for an electroenzymatic sensor. These attractive analytical characteristics were due to the high permeability and

the specific electrostatic interactions with cyanide displayed by the LDHs matrix. ACKNOWLEDGMENT D.S. gratefully acknowledges the Association Franco-Chinoise pour la Recherche Scientifique et Technique for her doctoral fellowship. C.M. thanks Pr. C. Forano (Universite´ Blaise Pascal of Clermont-Ferrand) for providing to us the [Zn-Al-Cl] phase. Received for review June 30, 2003. Accepted October 17, 2003. AC034713M

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Clay

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