Anal. Chem. 1998, 70, 3952-3956
A Bienzyme Electrode (Alkaline Phosphatase-Polyphenol Oxidase) for the Amperometric Determination of Phosphate Serge Cosnier,*,† Chantal Gondran,† Jean-Christophe Watelet,† Wagner De Giovani,‡ Rosa P. M. Furriel,‡ and Francisco A. Leone‡
Laboratoire d’Electrochimie Organique et de Photochimie Re´ dox, UMR CNRS 5630, Universite´ Joseph Fourier Grenoble 1, 301 rue de la Chimie, BP 53, 38041 Grenoble Cedex 9, France, and Departamento de Quimica, Faculdade de Filosofia, Cieˆ ncias e Letras de Ribeira˜o PretosUSP, 14040-901 Ribeira˜o Preto, SP Brazil
The electropolymerization of an alkaline phosphatase (AP)-amphiphilic pyrrole ammonium mixture previously adsorbed on the electrode surface provides efficient entrapment of AP in a polypyrrole film. The resulting biosensor was applied to the detection of phenyl phosphate via amperometric oxidation at 0.6 V vs SCE of the enzymically generated phenol. The sensitivity and detection limit of the biosensor were 862 µA M-1 cm-2 and 20 µM, respectively. The co-immobilization of polyphenol oxidase and AP leads to a bienzyme electrode for the determination of phenyl phosphate on the basis of amperometric detection of the enzymically generated oquinone at -0.2 V. An amplification factor of 9.7 was calculated as the ratio of the bienzyme to monoenzyme electrode sensitivities. In the presence of 5 mM MgCl2, the sensitivity and detection limit of the biosensor for phenyl phosphate were 36.82 mA M-1 cm-2 and 0.2 µM, respectively. Phosphate competitively inhibited the hydrolysis of phenyl phosphate. Consequently, its determination is based on the decrease of the biosensor response to phenyl phosphate. The sensitivity and detection limit of the biosensor for phosphate were 1.27 mA M-1 cm-2 and 2 µM, respectively.
using various enzyme electrodes.5-13 Their functioning principles are based either on the inhibitory effect of phosphate on the alkaline phosphatase activity5,12 or on the use of phosphate as cosubstrate of pyruvate oxidase9,13 and nucleoside phosphorylase.6-8,10,11 The amperometric detection of phosphate was carried out by monitoring the consumption of dissolved dioxygen or the production of hydrogen peroxide. The disadvantages of these different biosensors are their poor stability and their low sensitivity. In addition, the overpotential for the oxidation of hydrogen peroxide or the fluctuation of dioxygen concentration could generate interfering effects on the phosphate measurement. In this context, we report here the construction and characterization of a phosphate amperometric biosensor based on the original association of alkaline phosphatase (AP) and polyphenol oxidase (PPO). The measurement of phosphate is based on the inhibition by phosphate of the AP activity14 for the hydrolysis of phenyl phosphate. The resulting phenol is then catalytically oxidized by PPO to o-quinone15 AP
phenyl phosphate + H2O 98 phenol + HPO42PPO
phenol + O2 98 o-quinone + H2O Phosphate is a well-known contaminant of groundwater and streamwater. Widespread pollution by inorganic phosphate is an environmental problem illustrated by the eutrophication of lakes. Furthermore, the presence of phosphate in drinking water is a health hazard. Consequently, the determination and control of phosphate constitutes a priority for water quality. A conventional spectrophotometric method has been employed for the determination of phosphate.1 However, this procedure is time-consuming and not applicable to on-site monitoring. Owing to some advantages over other methods, including high selectivity, simple use and the possibility to develop portable analyzers, biosensors are used in many fields of analytical chemistry.2-4 Hence, phosphate analysis has been carried out †
Universite´ Joseph Fourier. USP. (1) Lowry, O. H.; Lopez, J. A. J. Biol. Chem. 1946, 162, 421-428. (2) Turner, A. P. F., Karube, I., Wilson, G. S., Eds. Biosensors: Fundamentals and Applications; Oxford University Press: New York, 1987. ‡
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(3) Guilbault, G. G., Mascini, M., Eds. Uses of Immobilized Biological Compounds; NATO ASI Series 252; Kluwer Acamedic Publishers: New York, 1993. (4) Wang, J. Anal. Chem. 1995, 67, 487R-492R. (5) Guilbault, G. G.; Nanjo, M. Anal. Chim. Acta 1975, 78, 69-74. (6) Watanabe, E.; Endo, H.; Toyama, K. Biosensors 1988, 3, 297-306. (7) Haemmerli, S. D.; Suleiman, A. A.; Guilbault, G. G. Anal. Biochem. 1990, 191, 106-109. (8) Urso, E. M.; Coulet, P. R. Anal. Chim. Acta 1990, 239, 1-5. (9) Kubo, I.; Inagawa, M.; Sugawara, T.; Arikawa, Y.; Karube, I. Anal. Lett. 1991, 24, 1711-1727. (10) Wollenberger, V.; Schubert, F.; Scheller, F. W. Sens. Actuators B 1992, 7, 412-415. (11) Kinoshita, H.; Yoshida, D.; Miki, K.; Usui, T.; Ikeda, T. Anal. Chim. Acta 1995, 303, 301-307. (12) Su, Y.; Mascini, M. Anal. Lett. 1995, 28, 1359-1378. (13) Ikebukwo, K.; Wakamura, H.; Karube, I.; Kubo, I.; Inagawa, M.; Sugawara, T.; Arikawa, Y.; Suzuki, M.; Takeuchi, T. Biosens. Bioelectron. 1996, 11, 959-965. (14) Say, J. C.; Furriel, R. P. M.; Ciancaglini, P.; Jorge, J. A.; Lourdes, M.; Polizeli, T. M.; Pizauro, J. M.; Terenzi, H. F.; Leone, F. A. Phytochemistry 1996, 41, 71-75. (15) Kertesz, D.; Zito, R. Biochim. Biophys. Acta 1965, 96, 447-462. S0003-2700(98)00125-5 CCC: $15.00
© 1998 American Chemical Society Published on Web 08/19/1998
Figure 1. Structure of the 1 amphiphilic pyrrole monomer.
Therefore, the decrease in phenol formation is monitored by potentiostating the biosensor at -0.2 V vs SCE to detect amperometrically the biocatalytically generated o-quinone.15 In addition, the measuring principle of the bienzyme electrode involves an amplification of the biosensor response based on a substrate recycling phenomenon.16-18 To obtain the bienzyme electrode, we used an original procedure of enzyme immobilization based on the electrochemical polymerization of an amphiphilic pyrrole monomer (1)-enzyme mixture previously adsorbed on an electrode surface19 (Figure 1). The analytical characteristics of the resulting poly 1-APPPO electrode have been investigated for the determination of phenyl phosphate and inorganic phosphate. EXPERIMENTAL SECTION Reagents. Polyphenol oxidase (EC 1.14.18.1, from mushroom, 3400 units mg-1) and 2-amino-2-methylpropan-1-ol (AMPOL) were purchased from Sigma. Phenyl phosphate disodium salt was obtained from Aldrich. The amphiphilic pyrrole monomer [12(pyrrol-1-yl)dodecyl]triethylammonium tetrafluoroborate (1) was synthesized according to the literature19 as follows. This compound was obtained by refluxing for 16 h in ethanol an excess of triethylamine with 12-(pyrrol-1-yl)dodecyl p-toluenesulfonate. The latter was prepared by reaction in anhydrous pyridine between 12-(pyrrol-1-yl)dodecan-1-ol and tosyl chloride. Water was doubly distilled in a quartz apparatus. Alkaline Phosphatase Preparation. Alkaline phosphatase (AP) was purified to homogeneity from Neurospora crassa conidia as described elsewhere.14 The specific activity of the enzyme was 1.96 units mg-1 when assayed in 50 mM AMPOL buffer, pH 9.4, containing 2 mM MgCl2 and phenyl phosphate as substrate. One enzyme unit was defined as the amount of enzyme needed to hydrolyze 1.0 µmol of phenyl phosphate per minute at 37 °C. Phenylphosphatase activity of the enzyme was measured continuously by following the liberation of phenol (pH9.4 ) 6000 M-1 cm-1) at 268 nm, at 37 °C, in a U3000 Hitachi spectrophotometer equipped with thermostated cells. The KM (0.74 mM) of the free enzyme was determined under the conditions described above, and phenyl phosphate varied from 10-5 to 3.5 10-3 M. At pH 9.4 and in the presence of Mg2+, the hydrolysis of phenyl phosphate is competitively inhibited by phosphate (Ki ) 0.84 mM). Enzyme Electrode Preparation. The poly 1-AP electrodes were prepared according to the two-step procedure previously reported.19 The amphiphilic pyrrole 1 was ultrasonically dispersed in pure water into a stable, optically transparent solution (3 mM). Then, 5 µL of water containing 25 µg of AP was mixed with 10 µL of the monomer 1 solution. The resulting mixture was spread on the surface of a glassy carbon disk electrode (diameter 3 mm), and the water was removed under reduced pressure. The (16) Cosnier, S.; Innocent, C. Bioelectrochem. Bioenerg. 1993, 31, 147-160. (17) Ortega, F.; Dominguez, E.; Jo ¨nsson-Petterson, G.; Gorton, L. J. Biotechnol. 1993, 31, 289-300. (18) Besombes, J.-L.; Cosnier, S.; Labbe´, P. Talanta 1996, 43, 1615-1619. (19) Cosnier, S. Electroanalysis 1997, 9, 894-902.
resulting modified electrodes were transferred into a cell containing aqueous 0.1 M LiClO4 solution. Polymerization of the adsorbed 1-AP coating was then performed by controlledpotential electrolysis for 20 min at 0.76 V. The bienzyme electrodes (poly 1-PPO-AP) were prepared first by the elaboration of a poly 1-PPO film following the preceding procedure, except that the lyophilized PPO (68 µg) was directly added to the monomer 1 solution. The preceding 1-AP (25 µg) mixture was then spread, dried, and electropolymerized on the poly 1-PPO electrodes. The resulting bienzyme electrodes were made of an inner poly 1 film containing PPO and an outer poly 1 film containing AP. The resulting enzyme electrodes were thoroughly rinsed in distilled water. These rotating electrodes were then transferred in 0.1 M Tris-HCl buffer (pH 8.8) and allowed to rotate for 20 min at 300 rpm in order to remove the adsorbed enzyme molecules. Electrochemical Measurements. The electrochemical polymerization of the monomer 1-enzyme coatings was performed with a Princeton Applied Research model 173 potentiostat equipped with a model 179 digital coulometer and a model 175 universal programmer in conjunction with a Kipp and Zonen BD 91 XY/t recorder. The amperometric measurements of phenyl phosphate and phosphate were performed with a Tacussel PRG-DL potentiostat. All experiments were carried out in a conventional thermostated three-electrode cell. A saturated calomel electrode (SCE) and a platinum wire were used as reference and counter electrodes, respectively. The working electrodes were glassy carbon disks (diameter 3 mm) polished with 1 µm diamond paste. Assays. The electrochemical measurements were performed in an air-saturated 0.1 M Tris-HCl buffer solution (pH 8.8), the rotating rate of the biosensors being 500 rpm. The poly 1-AP response to phenyl phosphate was monitored amperometrically by measuring the steady-state current for the oxidation of the enzymically generated phenol at 0.6 V vs SCE. The poly 1-PPOAP response to phenyl phosphate was monitored amperometrically by measuring the steady-state current for the reduction of the enzymically generated o-quinone at - 0.2 V. RESULTS AND DISCUSSION As reported previously,19 an original strategy of enzyme entrapment in polymer films was based on the unusual properties of monomer 1 in water. After dispersion of the poorly soluble amphiphilic monomer by ultrasonication and addition of AP, the aqueous 1-AP mixture was adsorbed on an electrode surface. The oxidative electropolymerization of the adsorbed 1-enzyme coating at 0.76 V induces the entrapment of AP in the resulting polypyrrolic film. It should be noted that AP has an isoelectric point of 4.0 ( 0.1.14 As a consequence, the protein surface displays large negative charges at neutral pH which promote its association in aqueous solution with the cationic monomer 1. It should be noted that the procedure of enzyme entrapment in poly 1 films can also be carried out with nonpurified enzyme preparations. In addition, owing to the high permeability of the resulting polypyrrole film, the presence of side components such as buffers in the enzyme-1 mixture does not influence the polymer structure. Owing to its phosphohydrolytic activity, AP catalyzes the hydrolysis of phenyl phosphate into phenol. Therefore, the amperometric detection of phenyl phosphate was assayed in 0.1 Analytical Chemistry, Vol. 70, No. 18, September 15, 1998
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Figure 2. (A) Calibration plot of the poly 1-AP electrode for phenyl phosphate. Applied potential, 0.6 V vs SCE in 0.1 M Tris-HCl buffer (pH 8.8) thermostated at 30 ( 0.1 °C; electrode rotation rate, 500 rpm. (B) Electrochemical Lineweaver-Burk plot for the poly 1-AP electrode (correlation coefficient, 0.9995).
M Tris-HCl buffer (pH 8.8) by holding the poly 1-AP electrode at 0.6 V vs SCE in order to oxidize the enzymically generated phenol. Figure 2A presents the amperometric response current of the poly 1-AP electrode as a function of phenyl phosphate concentration. The calibration curve was linear with phenyl phosphate concentration up to 0.8 mM and curved gradually at higher concentrations. The sensitivity of the biosensor (determined as the slope of the initial linear part of the calibration curve) and its detection limit (based on a signal-to-noise ratio of 3) are 862 µA M-1 cm-2 and 20 µM, respectively. The KMapp of the poly 1-AP electrode for phenyl phosphate was determined from a Michaelis-Menten analysis of the phenyl phosphate plot. The data fitted the electrochemical LineweaverBurk form of the Michaelis-Menten equation:20
Figure 3. (A) Influence of pH on the phenyl phosphate sensitivity of the poly 1-AP electrode. Applied potential was determined as the oxidation peak potential of phenol. (B) pH dependence of the oxidation peak potential of phenol at the poly 1 electrode.
Figure 4. Schematic representation of the enzymatic reaction sequences for the amplified amperometric detection of phenyl phosphate.
app
KM 1 1 1 ) + Is Imax [phenyl phosphate] Imax
where Is is the recorded current and Imax the maximum current at saturating substrate conditions (Figure 2B). The KMapp value (5 mM) for phenyl phosphate is slightly higher than that measured for phenyl phosphate with the free enzyme (KM ) 0.74 mM). It should be noted that similar KM values (1.38, 5.7, and 0.31 mM) have already been reported for free alkaline phosphatases.21,22 Nevertheless, this slight increase may indicate the presence of diffusional constraints due to the polymerization or adsorption of the oxidation product.23 To determine the optimum pH for the bienzyme electrode (poly 1-PPO-AP), the effect of the pH on the activity of the immobilized AP has been examined in the pH range 7-9.5. For this purpose, the sensitivity of the poly 1-AP electrode to phenyl phosphate was determined at different pH values (Figure 3A). It should be noted that the potential applied to the biosensor at each pH value was previously determined as the potential value of the oxidation peak of phenol at a poly 1 electrode (Figure 3B). It appears that the biosensor sensitivity increases with increasing pH value from 7 to 9 and then decreases slightly, the maximum (20) Kamin, R. A.; Wilson, G. S. Anal. Chem. 1980, 52, 1198-1205. (21) Jackson, S. D.; Halsall, H. B.; Pesce, A. J.; Heineman, W. R. Fresenius’ J. Anal. Chem. 1993, 346, 859-862. (22) Roig, M. C.; Bello, J. F.; Rodriguez, S.; Cachaza, J. M.; Kennedy, J. F. J. Mol. Catal. 1994, 93, 105-117. (23) Rosen, I.; Rishpon, J. J. Electroanal. Chem. 1989, 258, 27-39.
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sensitivity being at pH 9.0. This optimum pH value differs from that (9.8) reported for the free AP.14 As previously reported for the immobilization of galactose oxidase,19 this indicates that the optimum pH value is affected by the poly 1 host matrix. Since the poly 1 film is positively charged, protons are repelled, leading to a pH value higher in the polymer than in the bulk solution. To develop a more sensitive biosensor for phenyl phosphate and, hence, for phosphate, we have co-immobilized AP and PPO in the poly 1 host matrix. The functioning principle of the resulting bienzyme electrode is based on the enzymatic hydrolysis of the AP substrate (phenyl phosphate) followed by the enzymatic oxidation of phenol into o-quinone, which is then detected by electroreduction at the electrode surface (Figure 4). A heterogeneous enzyme location has been made with PPO in the inner poly 1 layer and AP in the outer poly 1 layer. This particular biosensor configuration has been fabricated to obtain an amplification of the amperometric detection of phenyl phosphate. Indeed, the sensitivity of PPO-based biosensors is strongly related to the efficiency of a recycling phenomenon. In fact, the entrapped PPO catalyzes the oxidation of phenol to o-quinone, while the amperometric detection generates by reduction catechol, another PPO substrate. The latter can undergo successive cycles of enzymatic oxidation-electrochemical reduction, inducing an amplification of the biosensor response.16-18 As reported previously for the free enzyme, an irreversible loss of PPO activity was observed beyond pH 9.3,15 while its activity at pH 8 dropped to 80% of its maximum value. Furthermore, the poly 1-PPO electrode presents an optimum pH range between
Figure 5. (A) Calibration plot of the poly 1-PPO-AP electrode for phenyl phosphate. Applied potential, -0.2 V vs SCE in 0.1 M TrisHCl buffer (pH 8.8) in the absence (a) and presence (b) of 5 mM MgCl2; experimental conditions are as described in Figure 2. (B) Electrochemical Lineweaver-Burk plot for the poly 1-PPO-AP electrode in the absence (a) and presence (b) of 5 mM MgCl2 (correlation coefficient, (a) 0.9973 and (b) 0.9988).
5.0 and 8.0 (85% of activity), the maximum current response being obtained at pH 6.5.16 Owing to the optimum range for the poly 1-AP electrode, the poly 1-PPO-AP electrodes have been tested for the detection of phenyl phosphate at pH 8.8. The amperometric detection of phenyl phosphate was assayed in 0.1 M Tris-HCl buffer thermostated at 30 ( 0.1 °C by holding the bienzyme electrode at -0.2 V. Figure 5A presents the amperometric current response of the bienzyme electrode as a function of phenyl phosphate concentration. The calibration curve was linear with phenyl phosphate concentration up to 20 µM and curved gradually at higher concentrations, a pseudoplateau being reached above 200 µM, reflecting saturation of the bienzymatic system. The comparison of the phenyl phosphate sensitivity of the bienzyme electrode (8.4 mA M-1 cm-2) with that of the poly 1-AP electrode clearly indicates an amplification phenomenon for low phenyl phosphate concentrations. An amplification factor of 9.7 was calculated as the ratio of the bienzyme to monoenzyme electrode response. The KMapp of the bienzyme electrode for phenyl phosphate was determined from the electrochemical Lineweaver-Burk plots (Figure 5B). The KMapp value (60 µM) for phenyl phosphate is markedly lower than that previously obtained with the poly 1-AP electrode (5 mM). However, it should be noted that this KMapp value for phenyl phosphate obtained with the bienzyme electrode is similar to that (50 µM) determined for catechol with the poly 1-PPO electrode.16 Since magnesium ions stimulated the AP activity,14 the influence of 5 mM MgCl2 on the biosensor response to phenyl phosphate was examined. Figure 5A clearly indicates a great enhancement of the current response of the bienzyme electrode. The sensitivity of the biosensor and its detection limit are 36.82 mA M-1 cm-2 and 2 × 10-7 M, respectively. It appears that the biosensor sensitivity is 4.4-fold higher in the presence of MgCl2. This result is in good accordance with the factor of 3.5 reported for the increase in the specific activity of the free AP in the presence of MgCl2.14 It should be noted that the KMapp value (60
Figure 6. (A) Steady-state current-time responses of a poly 1-PPO-AP electrode (diameter 3 mm, prepared with 68 µg of PPO and 25 µg of AP) to successive increments (20 and 100 µM) of phosphate concentration in the presence of 60 µM phenyl phosphate and 5 mM MgCl2. (B) Calibration plot for phosphate; experimental conditions are as described in Figure 5.
µM) is not affected by the presence of MgCl2, whereas the maximum current density increases from 0.64 to 3 µA cm-2 (Figure 5). These features could indicate that the limiting step in the bienzymatic system is the reaction catalyzed by AP. The amperometric response of the poly 1-PPO-AP electrode to phenyl phosphate in the presence of MgCl2 is relatively fast (response time 50 s) and reproducible. The prolonged series of repetitive injections of 1 µM phenyl phosphate yielded a relative standard deviation of 3.1%. The poly 1-PPO-AP electrodes were also examined for their operational and storage stability. The operational stability was evaluated by recording the current response with increasing concentration of phenyl phosphate in the linear part of the calibration curve. No loss of activity was observed after 50 analyses, while a 36% decrease in phenyl phosphate sensitivity appeared after 120 analyses. The biosensor activity dropped by 33% upon storage in a dry state at -20 °C for 5 weeks. The fabrication of the bienzyme electrode is also quite reproducible. Four different poly 1-PPO-AP electrodes were prepared by following identical adsorption and electropolymerization steps, and their responses toward phenyl phosphate were investigated. The comparison of the sensitivity determined from the resulting calibration curves indicates that the relative standard deviation is only 9.5%. The hydrolysis of phosphate esters by AP to give inorganic phosphate and a phenolic leaving group is inhibited by phosphate, which is a competitive inhibitor.14 Consequently, the effect of phosphate on the amperometric response of the poly 1-PPOAP electrode was investigated with phenyl phosphate as substrate (60 µM). Figure 6A presents the steady-state current response of the biosensor to two successive increments (20 and 100 µM) of phosphate concentration, illustrating the relatively fast response time (∼40 s) of the biosensor. The addition of phosphate was carried out, yielding a completely inhibited biosensor. The biosensor was then transferred to clean Tris-HCl buffer, and its response to phenyl phosphate was compared to the initial biosensor sensitivity. It appears that the loss of activity was only 6%, illustrating the reversible nature of the inhibition process by phosphate. Analytical Chemistry, Vol. 70, No. 18, September 15, 1998
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Figure 6B shows the “inhibition current” of the bienzymatic electrode as a function of phosphate concentration. The inhibition current corresponds to the current decrease of the amperometric biosensor response to 60 µM phenyl phosphate. The calibration curve was linear with phosphate concentration up to 84 µM and curved gradually at higher concentrations, reflecting the complete inhibition of AP. The sensitivity of the biosensor to phosphate and its detection limit are 1.27 mA M-1 cm-2 and 2 µM, respectively. It should be noted that the value of the detection limit (2 µM) is similar (4 µM)12 or more sensitive (50 µM)24 than those previously obtained with biosensors based on AP. The amperometric response of the poly 1-PPO electrode without AP to 60 µM catechol displayed no decrease in current as a result of successive injections of phosphate under the same operative conditions. Hence, it appears that the response observed at the biosensor requires the presence of AP and is phosphate-specific. Most of the biosensors proposed are based on the amperometric detection of enzymically generated hydrogen peroxide or phenol. However, the direct oxidation of these products suffers from the interference of more easily oxidizable species such as ascorbate or urate.12,21 In contrast, the functioning principle of the poly 1-PPO-AP electrode was based on the amperometric
detection of generated o-quinone via its electroreduction at -0.2 V. Consequently, it is to be expected that the electrochemical source of interferences should be considerably diminished. The response of the poly 1-PPO-AP electrode to 20 µM ascorbate and 20 µM urate was examined. No appreciable change in the steady-state current response of the biosensor was observed, illustrating the absence of interference.
(24) Katsu, T.; Kayamoto, T. Anal. Chim. Acta 1992, 265, 1-4.
AC980125A
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CONCLUSION In this report, we have shown for the first time the coimmobilization of polyphenol oxidase and alkaline phosphatase at the surface of an electrode using an electropolymerizable amphiphilic pyrrole ammonium. The resulting bienzyme electrode, exhibiting a heterogeneous enzyme distribution, represents a sensitive system for accurate phosphate determination. ACKNOWLEDGMENT The authors thank Dr. A. Deronzier for his interest in this work. This work was supported in part by the USP-COFECUB program (project UC/16). Received for review February 5, 1998. Accepted May 27, 1998.
