Electrochimica Acta 55 (2010) 23632367

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Electrochimica Acta
journal homepage: www.elsevier.com/locate/electacta

Simple sensor for the determination of phenol and its derivatives in water based on enzyme tyrosinase
Juliusz Adamski a, , Pawe Nowak b , Jolanta Kochana a
a b

Department of Analytical Chemistry, Faculty of Chemistry, Jagiellonian University, Ingardena 3, 30-060 Cracow, Poland Institute of Catalysis and Surface Chemistry PAS, Niezapominajek 8, 30-239 Cracow, Poland

a r t i c l e

i n f o

a b s t r a c t
Design of a new sensor, based on enzyme tyrosinase, suitable for phenol determination in water was described. The inuence of measuring parameters such as pH of the sample and enzyme concentration on analytical performance of the sensor was evaluated. Calibration curves were constructed for catechol, 4-methylcatechol, 4-tertbutylcatechol, phenol, 4-chlorophenol, 2-tertbutylphenol and cresols (p and m isomers of cresol were treated separately) and sensitivity as well as LOD and LOQ were estimated for these phenols. Additionally, the inuence of sample matrix components on the electrode response was studied according to the PlackettBurman experimental design. The following potential interferents which are usually met in waters were taken into account in the examination: Mg2+ , Ca2+ , HCO3 , SO4 2 , Cl as well as Cu2+ . It was found that among tested ions only Cu2+ directly affected the electrode response. Determination of phenol index in tap water samples together with recovery studies was also performed. Obtained results suggest that developed sensor can be successfully used for the determination of phenols in concentration range covering its environmental levels. 2009 Elsevier Ltd. All rights reserved.

Article history: Received 26 August 2009 Received in revised form 29 November 2009 Accepted 30 November 2009 Available online 4 December 2009 Keywords: Phenol determination Tyrosinase sensor Water analysis Amperometric detection Experimental design

1. Introduction Phenolic compounds are important contaminants in ground and surface waters. Due to their toxicity and persistence in the environment phenols are considered priority pollutants. They are introduced to the environment in variety of ways like wastes from paper manufacturing, agriculture, petrochemical industry, coal processing, as municipal wastes and many others. In drinking water even at low concentration they give off strong taste and odor. Moreover, during chlorination process, various chlorophenols and chlorinated p-benzoquinones are produced, some of them thought to be mutagenic [1]. Consequently, the detection of phenols at low concentrations is of environmental concern. Different analytical techniques were proposed for the determination of phenol and its derivatives. The most widely used are: gas chromatography [25], high performance liquid chromatography [68], different types of capillary electrophoresis [9,10] spectrophotometry [11,12], and electrochemical methods of analysis [13 and references therein]. These methods, however, involve complicated and time-consuming procedures and require expensive instrumentation. They are also not suitable for rapid in situ monitoring.

Corresponding author. Tel.: +48 012 663 20 14; fax: +48 012 634 05 15. E-mail address: adamskij@chemia.uj.edu.pl (J. Adamski). 0013-4686/$ see front matter 2009 Elsevier Ltd. All rights reserved. doi:10.1016/j.electacta.2009.11.099

Direct electrooxidation of phenol takes place at high overpotential through a complex process during which electrode surface is covered by polymeric lms that impede the following charge transfer reactions [14]. Some procedures have been described attempting to diminish the problems caused by these lms. Cellulose or Naon membrane-covered electrodes for adsorption stripping voltammetry [15], electrochemical pretreatment [16,17] or modication of the electrode surface with different types of coatings [1820] were proposed. Surfactants [21] or EDTA [22] inuence on phenol determination was also investigated. Phenol conversion into more convenient form which can be electrochemically detected at lower potentials with signicantly reduced electrode fouling can be achieved using enzymatic catalysis. Tyrosinase (polyphenol oxidase, E.C. 1.14.18.1) is an enzyme that catalyzes phenol o-hydroxylation yielding o-diphenol (monophenolase activity), which is subsequently oxidized to o-quinone (diphenolase activity) [14]. This quinone can be then reduced at the electrode surface at low potentials giving o-diphenol. This concept was used in the construction of amperometric biosensors based on tyrosinase entrapped in different matrices such as alumina [23] or titania [24] gel, Naon [25], laponitechitosan [26], calcium carbonate [27] nanoparticles, conducting polymers such as polypyrrole or polyaniline [28,29] or different carbon composites [16,30]. However such constructions suffer from inherent disadvantages. Contrary to glucose oxidase enzyme most widely used in biosensor construction tyrosinase is not very stable. In the case of the application in the determination of phenolic compounds its

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stability is further diminished due to the poisoning by the products of phenol oxidation. The other factors inuencing negatively the performance of tyrosinase-based biosensors are leaching of enzyme to the solution and fouling of the electrode surface by the products of phenol oxidation. Therefore, biosensors based on this enzyme exhibit usually poor long-term stability. In the case of environmental samples the probability of enzyme deactivation is even higher because such samples may accidentally contain species that are poisonous towards tyrosinase. Moreover, most of the proposed methods of biosensor manufacturing described in the literature are timeconsuming and consists of many steps, which result in difculties in obtaining sensors with comparable sensitivity. In this article construction of a simple sensor that can be used for phenol determination in water samples was proposed. Optimization as well as analytical characterization of proposed sensor toward different phenolic compounds was performed. Additionally, the inuence of sample matrix components on the electrode response was studied according to the PlackettBurman experimental design. The following potential interferents which are usually met in waters were taken into account in the examination: Mg2+ , Ca2+ , HCO3 , SO4 2 , Cl as well as Cu2+ . Determination of phenol index in tap water samples together with recovery studies was also performed. 2. Experimental 2.1. Chemicals and reagents Tyrosinase from mushroom (polyphenol oxidase, E.C. 1.14.18.1, 5370 U/mg) was purchased from Sigma (Germany). KH2 PO4 was obtained from POCh (Poland); Na2 HPO4 2H2 O and phenolic compounds: catechol, 4-methylcatechol, 4-tertbutylcatechol, phenol, 4-chlorophenol, 2-tertbutylphenol, m-cresol and p-cresol were from Merck (Germany). All reagents were of analytical reagent grade purity. Solutions were prepared using double-distilled water. Buffer solutions were prepared by mixing appropriate volumes of 0.5 M KH2 PO4 and 0.5 M Na2 HPO4 . 2.2. Apparatus BM857 with Bs Software Data Recording System (Brymen, Thailand) and Elpan (Poland) EP21 potentiostat were used for chronoamperometric measurements. Glassy carbon (Goodfellow carbon vitreous foil 2 mm) was shaped to the form of a disc (d = 1.0 cm), glued to poly(methyl metacrylate) block using epoxy resin, and used as working electrode after polishing with 0.3 m alumina (Buehler Micropolish, USA). Electrical contact to the back of the electrode was achieved via copper wire and conducting glue. Ag/AgCl electrode was prepared by anodizing silver wire in an HCl solution. To increase the area of the electrode the wire was made in the shape of a coil. Combined glass electrode ERH-11 (HYDROMED, Poland) together with pH-meter CP-501 (Elmetron, Poland) were used for pH measurements. 2.3. Sensor construction Fig. 1 shows the idea of the sensor construction. A small droplet of the analyzed solution (in our case the volume of the solution was 100 l) containing phenol and enzyme was placed on the surface of a circular glassy carbon electrode. Glass tubing ended with the glass frit of the at surface and the diameter close to the diameter of the GC electrode was positioned over the GC electrode in such a manner that the solution formed a thin layer between the surface of the GC electrode and the surface of the glass frit. Glass tubing was previously lled with saturated KCl solution and

Fig. 1. Scheme of experimental setup. (1) WE (GC disc), (2) poly(methyl metacrylate) block, (3) Cu wire, (4) glass tubing, (5) Ag/AgCl/sat. KCl electrode, and (6) sample.

a high-area Ag/AgCl electrode was placed inside the tubing. Both electrodes (GC and Ag/AgCl) were connected through a resistor and the current owing in the circuit was measured as a potential drop on the resistor. The resistance of the resistor (R = 100 k ) was chosen in a way to make the potential drop on the resistor negligible (in our case usually less than 10 mV). The density of the glass frit was chosen in such a manner as to prevent KCl leaching from glass tubing to the analyzed solution on one hand (it was checked that high KCl concentrations decreased the activity of the enzyme), but to give negligible potential drop at the currents owing during measurements, on the other hand. Note, that the potential 0 V vs. Ag/AgCl/KClsat is close to the optimum potential at which current is usually registered in tyrosinase-based biosensors with amperometric detection [24,26]. Such a construction not only makes the sensor very simple but also reduces the electrical noises in the system because no electronic power supply is present. The main factor limiting the sensitivity of amperometric biosensors (besides the activity of the enzyme) is the background current, registered in the absence of the analyte. That current is mainly due to the presence of the traces of other electroactive substances. In the case of presented construction the background current is minimized due to the lack of convective transport and limited volume of the solution (and, hence, limited amount of the substance). To increase further the sensitivity of the sensor current was integrated during predetermined period of time (usually 10 min of integration was applied in our case) and the value correlated with the phenol concentration was the charge, not the instantaneous current. Such operation increases the sensitivity of the sensor in two ways. First the inuence of background current is minimized, because each molecule of interfering substances reacts only once whereas phenol molecules reacts many times due to the nature of the process (see Fig. 2 for explanation). The other reason emerges from the fact that the amperometric curves for monophenols and diphenols have different shapes. Monophenols must be rst oxidized to diphenols before they can enter the reduction/oxidation self-sustaining process (see Fig. 2). Therefore, in the

Fig. 2. Schematic representation of processes occurring during measurement.

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Fig. 3. Comparison of sensor response for catechol and phenol (both about 90 nM) obtained in 0.05 M phosphate buffer of pH 7.0 for enzyme concentration of 50 g/ml.

Fig. 4. Inuence of WE potential on background ( ) and response ( ) signal recorded in pure 0.05 M phosphate buffer and buffer solution containing catechol (90 nM) and tyrosinase (50 g/ml), respectively; ( ) represents the difference in both signals.

case of monophenols amperometric curve has a minimum and maximum, whereas in the case of diphenols no such a phenomena are observed (see Fig. 3). So, integration over sufciently long time solves the problem of choosing the optimal moment of time of current registration. 2.4. Measuring procedure for phenols determination Each sample was prepared using the same procedure. 90 l of phenol solution of appropriate concentration in double-distilled water was spiked with 10 l of 0.5 M phosphate buffer solution (pH 7.0) and carefully mixed. Then 1 l of tyrosinase solution (5 mg/ml) was added and solution was shaken during 1 min. Then it was poured into 0.5 mm crevice between Ag/AgCl electrode and GC electrode. Chronoamperometric response (t = 10 min, T = 294 1 K) was recorded and numerically integrated. Calibration curves were constructed as the Qcorr = f (cphenol ) dependence, where Qcorr was charge corrected for blank signal. Between measurements electrode surface was cleaned with double-distilled water and methanol. 3. Results and discussion 3.1. Inuence of pH of sample solution on sensor response Inuence of pH on sensor response was measured for pH range 6.08.0 with interval of 0.5 pH unit using phosphate buffer solution and 57 M catechol solution. The measuring procedure was the same as described in Section 2.4 though buffers of different pH were added to analyzed samples, and current recorded after 200 s and corrected for background signal for the same pH, was taken as sensor response. The highest response was obtained for pH 7.0 and this value was selected for further studies. 3.2. Inuence of enzyme concentration on sensor response Dependence of sensor response to amount of enzyme added to analyzed sample was evaluated for 8.54 mM catechol in 0.05 M phosphate buffer solution at pH 7.0 within the range of enzyme concentration in analyzed sample from 1.8 to 266 g/ml. It was observed (data not shown) that increase of signal (current measured after 200 s) with the increase of enzyme concentration was signicant up to about 50 g/ml. Further increase of enzyme con-

centration did not affect sensor response greatly therefore this concentration was chosen as the optimum value. 3.3. Inuence of crevice size on sensor response The inuence of the distance between the surface of GC electrode and the front surface of the glass tubing containing Ag/AgCl electrode on the measured charge was also investigated. No inuence for the distance range from 0.25 to 0.75 mm was observed. The 0.50 mm crevice size was used in further studies. 3.4. Inuence of working electrode potential on sensor response Additional experiments were performed in the aim to check the possibility of sensor response increase by changing the WE potential. The electrode was polarized vs. Ag/AgCl in two-electrode conguration in the range from 100 to +200 mV (with 20 mV interval) and current was recorded after 2 min in pure 0.05 M phosphate buffer and buffer solution containing catechol (0.9 M) and tyrosinase (50 g/ml) as well. Obtained results (see Fig. 4) conrmed that the potential the working electrode (GC electrode) assumes being short-circuited to the Ag/AgCl electrode (0 V vs. Ag/AgCl electrode) is the optimal potential for the amperometric detection of catechol. Fortunately, the Ag/AgCl/sat. KCl electrode showed the potential at which the signal/noise ratio was optimal (Fig. 4) so that electrode was chosen in our design. 3.5. Analytical characterization of the sensor 3.5.1. Precision and stability of sensor response The precision of optimized sensor was evaluated in terms of repeatability and intermediate precision. Repeatability studies were conducted by performing two series of experiments in 0.468 M catechol solution, each containing 5 experiments, with repolishing of electrode surface between these series as to check the inuence of electrode pretreatment on analytical response. The coefcients of variation, CV = RSD 100%, obtained for these series were 3.2 and 3.7%, and this values were much lower than the Horowitz accepted values for such an analyte concentration [32]. Qcorr for these series was equal to x1 = 0.1033 (s1 = 0.00369) and x2 = 0.1038 (s2 = 0.00454) mC, respec-

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Table 1 Sensitivity, LOD and LOQ of developed sensor for different phenols. Evaluated range [ M] Sensitivity [mC/ M] Detection limit [nM] Phenol Catechol p-Cresol m-Cresol 4-Methylcatechol 4-Tertbutylcatechol 4-Chlorophenol 2-Tertbutylphenol 00.629 00.631 00.615 00.693 00.656 00.650 00.621 00.615 0.256 0.005 0.213 0.004 0.182 0.005 0.167 0.004 0.128 0.002 0.126 0.005 0.123 0.005 Not detected 13.7 16.5 19.4 20.9 27.5 27.9 28.6 [ g/L] 1.29 1.82 2.10 2.26 3.41 4.64 3.68 Quantication limit [nM] 27.5 33.0 38.8 41.8 51.1 55.8 57.2 [ g/L] 2.59 3.63 4.20 4.52 6.84 9.28 7.35 0.9981 0.9983 0.9968 0.9949 0.9982 0.9915 0.9913 R2

tively. Based on F-test, no statistically signicant differences were obtained between response variations in each group (Fcalc = crit 2 2 s1 /s2 = 1.51, F4,4,0.05 = 6.39). The difference of mean values of sensor response was also statistically insignicant which was con 2 2 cluded based on t-test (tcalc = ( x1 x2 / s1 + s2 ) n = 0.16, n =
crit 5, t8,0.05 = 2.306). Intermediate precision was estimated by comparison of sensor response obtained in three different days in catechol solutions of similar concentration, ccatechol (about 0.5 M). The sensor responses, expressed as Qcorr /ccatechol , were: 0.2208, 0.2052 and 0.2101 mC (CV = 3.8%). The stability of the sensor response was evaluated by performing 17 successive measurements in more concentrated (0.906 M) catechol solution. This concentration was chosen as to check the possibility of electrode surface fouling during experiments. No signicant signal decrease was observed (Qcorr /ccatechol , were 0.3748 and 0.3705 mC for the rst and seventeenth measurements, respectively) which led to the conclusion that chosen cleaning procedure (see Section 2.4) was sufcient to preserve electrode activity.

lower than for catechol itself. This behavior was additionally conrmed by comparison of charge consumed during measurements, which was about ten times greater than those calculated from Faradays law, taking into account the amount of phenol in the analyzed sample. It was also conrmed that greater sensitivity and lower detection limits could be achieved if charge was used in the place of usually applied current as a sensor response. Limited number of experiments was also performed with the longer time of integration. They showed that the sensitivity of the sensor increases, when the time of integration increases. 3.5.3. Interferences Six potential interferents, which are usually met in waters, were selected: Mg2+ , Ca2+ , HCO3 , SO4 2 , Cl and Cu2+ and PlackettBurman experimental design was applied in this study [24]. The copper ions were taken into account since they can be introduced into drinking water from copper pipes. The PlackettBurman factorial is a two level orthogonal design [31] that minimizes the number of experiments required to allow exploratory study of a large number of factors to see whether they exhibit signicant effect on the response. The main factors effects are determined independently from each other. In the examination each potential interferent occurred at two concentration levels, higher (+) and lower (). The actual values of six tested factors (concentrations of interferents) are shown in Table 2. The higher concentration levels of Mg2+ , Ca2+ , HCO3 , SO4 2 and Cl were correlated to concentrations of these ions in natural waters of medium purity. The experimental design (concentrations of interferents) is presented in Table 3 together with response values (mean phenol concentration obtained from two parallel determinations carried out on the corresponding solution). The main effect of variable i (interferent), Ei , was calculated as the difference between the average of responses measured at higher settings (+) and such the average measured at lower settings () of the variable i. Signicance of the effects of interferents was estimated using Students t-test. The Students t-values, ti (i = 1, . . ., 6), presented in Table 3 (bottom), were obtained by dividing the effects by their standard error, SE (the same for all effects): ti = Ei /SE. The error SE was calculated from the following formula: SE = S/ N, where S = 2.686, the response standard deviation, was estimated from differences between the pairs of results of repeated determinations, and N = 8 number of trials. For 8 degrees of freedom the critical t-values are t(8,0.01) = 3.36 and t(8,0.05) = 2.31 at signicance levels = 0.01 and 0.05, respectively. From Table 3 it is seen that at 0.01 signicance level only Cu2+ ions inuenced (increase, t(Cu) > 0) sensor response. More detailed study of these problem lead to conclusion that this phenomenon can be connected with increase of background signal in the presence of Cu2+ ions. 3.5.4. Determination of phenolic compounds in water samples Real tap water samples were analyzed with the optimized sensor to evaluate its potential applicability. No special sample

3.5.2. Sensitivity, LOD and LOQ Analytical characterization of developed sensor was performed by measuring the response signal for different concentrations of eight phenols: catechol, 4-methylcatechol, 4-tertbutylcatechol, phenol, 4-chlorophenol, 2-tertbutylphenol and cresols (p and m isomers). Linear dependence between sensor response and analyte amount was obtained in evaluated concentration ranges for each phenol. Based on obtained calibration curves, detection limits, LOD = 3Sx a1 and quantication limits, LOQ = 6Sx a1 , were calculated for each analyte (Table 1). Sx was standard deviation of signal (n = 10) obtained in buffer solution (Sx = 0.00117 mC) and a was the slope of the calibration curve. From the presented results it may be concluded that differences in sensor sensitivity for different phenolic compounds may be attributed mainly to the inuence of substituent in benzene ring. Substituted phenols showed lower response than phenol, and chlorophenol showed the lowest among studied compounds, most probably due to the deactivating inuence of Cl substituent on benzene ring. No signal was recorded in the case of 2-tertbutylphenol, which may be due to the blocking of both p positions by spacious substituent. Similarly the sensitivity for substituted catechols was
Table 2 Levels of interferences concentration assumed in the PlackettBurman plan [mg/L]. Factor Concentration levels [mg/L] 1 (lower) Mg2+ Ca2+ Cu2+ HCO3 SO4 2 Cl 8 30 0 100 50 100 +1 (upper) 40 155 260 800 200 300

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Table 3 PlackettBurman design for 6 factors (concentration of interferents) and 8 trials. Actual concentration of phenol: 20 g/L. In each solution 0.05 M phosphate buffer solution (pH 7.0) and enzyme (50 g/ml) were present. Solution Factors (coded concentrations of interferents) Mg2+ 1 2 3 4 5 6 7 8 E |t| +1 +1 1 1 +1 1 +1 1 0.09 0.09 Ca2+ +1 1 1 +1 1 +1 +1 1 2.89 3.04 Cu2+ +1 +1 +1 1 1 +1 1 1 9.48 9.98 HCO3 1 +1 +1 +1 1 1 +1 1 2.49 2.62 SO4 2 +1 1 +1 +1 +1 1 1 1 1.13 1.19 Cl 1 +1 1 +1 +1 +1 1 1 2.32 2.45 40.92 38.13 29.12 17.40 32.67 15.84 40.21 13.74 Response [ g/L]

E: main effect of a factor; t: t-Students value for a factor (see Section 3.5.3); critical t-values for f = 8 degrees of freedom at signicance levels = 0.01 and = 0.05: t(8,0.01) = 3.36 and t(8,0.05) = 2.31.

pretreatment was performed, just water samples were mixed with concentrated (0.5 M) phosphate buffer solution pH = 7.0 to its nal concentration of 0.05 M. The uncertainty of phenol concentration (c) was estimated using following formula: u(c, Q ) = (Sxy /a) 1/p + 1/n + (c c ) /
2 n (c i=1 i

tant parameter in water quality management and construction of portable device that could be used for in situ monitoring. References
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c ) [33] where Sxy is resid-

ual standard deviation, p = 2 is number of repetitions, n = 5 is number of calibration points. c is the mean of all concentration values of the calibration experiment and a is a slope of calibration curve. Two samples of tap water were analyzed using standard addition method and the average content of phenolic compounds, expressed as phenol concentration, was found to be 6.8 1.9 g/L. To carry out the recovery study tap water sample was spiked with 10.0 g/L of phenol and the sample was analyzed. For this sample phenol concentration was found to be 17.7 1.4 g/L. Based on these results, the recovery factor was found to be ((17.7 6.8)/10) 100% = 109%. Though the recovery factor was only slightly differ than 100%, the results can be considered satisfactory as they fulll the needs of monitoring of natural waters contaminants at very low levels. 4. Conclusions A new sensor construction suitable for phenol determination in water was proposed. The inuence of some measuring parameters, such as pH of the analyzed sample and enzyme concentration on analytical performance of the sensor was evaluated. Taking into account great simplicity of measuring setup, which does not need any external voltage controller, excellent results were obtained. The other advantages of the proposed design are low amount of the sample needed to analysis (90 l were used but this value may be easily lowered) and low cost of the analysis. Note also that described sensor is much less susceptible to deactivation than sensors with immobilized enzyme because surface of the electrode may be easily renewed and the enzyme may be stored at low temperature. Among potential interferents, usually met in waters (Mg2+ , Ca2+ , HCO3 , SO4 2 , Cl as well as Cu2+ ) only Cu2+ ions directly affected the electrode response. Presented results suggest that developed sensor can be successfully used for the determination of phenols in concentration ranges covering its environmental levels. Proposed sensor was successfully used for the determination of phenol in tap water samples. Further research will be focused on the improvement of analytical parameters of proposed sensor, checking its ability to estimate phenol index an impor-