Improved stability and altered selectivity of tyrosinase based graphite electrodes for detection of phenolic compounds

Analytica Chimica Acta 387 (1999) 309±326

Improved stability and altered selectivity of tyrosinase based graphite electrodes for detection of phenolic compounds Catalin Nistora, Jenny EmneÂusa,*, Lo Gortona, Anton Ciucub a

Department of Analytical Chemistry, Lund University, PO Box 124, S-221 00, Lund, Sweden b Department of Analytical Chemistry, Faculty of Chemistry, University of Bucharest, Bucharest sector 5, Sos. Panduri, Romania Received 20 July 1998; received in revised form 4 January 1999; accepted 9 January 1999

Abstract The operational and storage stability of tyrosinase biosensors were investigated for different tyrosinase modi®ed electrodes, i.e., plain bulk modi®ed carbon paste electrodes (CPEs), surface modi®ed by simple adsorption to solid graphite electrodes (SGEs), and surface modi®ed by the immobilisation in Eastman AQ, a poly ester±sulphonic acid cation exchanger, and Na®on, a per¯uorinated-sulphonated ionomer, on the surface of both CP and SGEs. Factors such as the pH of immobilisation, the enzyme loading, and the polymer concentration were investigated in regards to the in¯uence on the sensitivity, the limit of detection (LOD), the sample throughput (STP), and the operational and storage stability. Both storage and operational stability were improved by immobilisation of tyrosinase in either of the two polymer matrices. For 50 consecutive injections of 50 mM catechol, the response stayed the same for the optimal Eastman and Na®on modi®ed CP and SGEs. After about 42 days 80% and 75% of the original response for the Eastman and Na®on modi®ed tyrosinase electrodes remained, respectively, whereas the tyrosinase bulk-modi®ed CPE and adsorbed tyrosinase SGEs had lost virtually 100% of the original response. The selectivity for nine phenolic compounds were investigated and found to change with the introduction of the polymer membrane. The detection of especially monophenols was improved probably due to their preconcentration into the polymer membranes. The best performing biosensor in terms of sensitivity, LOD, STP, operational and storage stability was reached for one where tyrosinase was immobilised in Na®on, i.e., sensitivity: 11.51 nA/mM, LOD: 0.015 mM catechol, and STP: 36 samples/h. The phenolic content, expressed as catechol equivalents was evaluated in six waste water ef¯uents from tannery industries in Spain and Sweden. The operational stability after 90 consecutive injections of extremely contaminated waste waters showed that the Na®on sensor retained 70% of its initial response. # 1999 Elsevier Science B.V. All rights reserved. Keywords: Tyrosinase; Carbon paste electrode; Biosensor; Na®on; Eastman AQ; Phenols; Catechol; Waste water; Operational and storage stability; Selectivity

1. Introduction A considerable number of organic pollutants, widely distributed throughout the environment, have *Corresponding author. Tel.: +46-46-2224820; fax: +46-462224544; e-mail: [email protected]

a phenolic structure [1]. Phenol and substituted phenols such as chlorinated phenols and related aromatic compounds are known to be widespread as components of industrial waste (2). Organophosphorus and chlorinated phenoxyacids also yield chloro and nitrophenols as major degradation products [2,3]. Many of these phenolic compounds have toxic effects on

0003-2670/99/$ ± see front matter # 1999 Elsevier Science B.V. All rights reserved. PII: S0003-2670(99)00071-9

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animals and plants of which they easily penetrate the skin and membranes, determining a broad spectrum of genotoxic, mutagenic, and hepatotoxic effects, also affecting the biocatalysed reaction rates in respiration and photosynthesis [4±6]. Some phenolic compounds present in plants and in a variety of industrial products have shown to have oestrogen properties [7,8], e.g., p-nonylphenol (a phenol present in certain plastics and formed as a degradation product of detergents such as nonylphenol ethoxylate (NPE) [9]) and other alkylphenols [10±12]. For these reasons, many of the phenols have been included in the environmental legislation. The European Community directive (80/ 778/EEC), e.g., sets the maximum concentration permitted for all phenols in aquatic environments at 0.5 mg/l and at 0.1 mg/l for individual phenols. The detection of mono- and polyphenols is usually accomplished by means of chromatography and/or spectrometry [3,13±18]. However, these techniques do not easily allow continuous on-site monitoring, they are expensive, time-consuming, need skilled operators, and sometimes require preconcentration and extraction steps that increase the risk of sample loss. Biosensors represent promising tools to supplement already existing techniques, due to their unique characteristics such as selectivity, the relatively low cost of realisation and storage, the potential for miniaturisation and easy automation, and for the construction of simple portable devices for fast screening purposes and in-®eld/ on-site monitoring. It should, however, be emphasised that these tools cannot and should not be seen as a replacement of classical analytical techniques but rather as a complement to them. Numerous biosensors have been proposed for the detection of phenolic compounds based on primarily the phenol oxidising enzyme tyrosinase (E.C. 1.14.18.1) [19]. Tyrosinase is a phenol oxidase, which oxidises monophenols and o-diphenols into their corresponding o-quinones, at the expense of reducing oxygen to water. The conversion of monophenols by tyrosinase proceeds in two consecutive steps involving molecular oxygen, i.e., in the ®rst step monophenol is hydroxylated to its corresponding o-diphenol (the enzyme's hydroxylase activity), which in a second step is oxidised to its corresponding o-quinone, whereby the enzyme is oxidised by molecular oxygen back to its native form (the enzyme's catecholase activity) [20,21].

A general problem for many biosensors is the lack of the necessary operational and storage stability needed for commercial exploitation, and is currently a major obstacle to solve in the biosensor area. The instability of tyrosinase biosensors in pure standard solutions is mainly due to that: 1. quinones suffer from high instability in water, and 2. formation of intermediate radicals in both the enzymatic and electrochemical reactions may readily react and polymerise to polyaromatic compounds that can inactivate the enzyme and foul the electrode [22±27]. Several papers describe the addition of various additives and modifications of tyrosinase biosensors to influence both stability and activity of tyrosinase biosensors [27±30]. However, when dealing with the analysis of real samples such as surface- or waste waters, additional factors will influence the sensor performance, e.g., the presence of tyrosinase inhibitors, electrode fouling by deposition of various species on the sensor surface etc. This could necessitate a traditional pre-clean-up step, thus leading to more complex analysis schemes and partly diminishing the proposed advantage of using biosensor for fast screening purposes. The present work proposes a simple way for developing comparatively stable amperometric tyrosinase biosensors for phenol screening in waste water samples based on physical entrapment of tyrosinase in a protecting and biocompatible membrane on the surface of carbon paste electrodes (CPE) and solid graphite electrodes (SGEs), using two types of polymeric matrices, i.e., Eastman AQ ± a poly ester±sulphonic acid ± and Na®on ± a per¯uorinated-sulphonated ionomer. The operational and storage stability of these biosensors was studied in a ¯ow injection (FI) system with repetitive injections of a catechol standard, i.e., the compound most easily oxidised by tyrosinase and also the most prone to in¯uence the stability of tyrosinase biosensors. Fig. 1 presents, in a simpli®ed form, the function of the surface-modi®ed tyrosinasemembrane electrode. The in¯uence of different parameters, i.e., the number of polymer/enzyme layers, the enzyme loading, the pH of immobilisation, the polymer concentration, and the in¯uence of the polymer membranes on the selectivity for nine different

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Fig. 1. The principle of bioelectrocatalytic conversion of phenols for tyrosinase immobilised in a polymer film on the surface of an electrode. The phenolic substrate (S) diffuses through the polymer membrane where it is enzymatically oxidised to o-quinone (P) by tyrosinase under the consumption of O2. S is regenerated through electrochemical reduction of P, thus forming a bioelectrocatalytic amplification cycle.

phenolic compounds, were investigated. The optimal biosensors were then characterised in an optimised automated FI system in terms of sensitivity, limit of detection (LOD), sample through-put (STP), and precision. Finally, the standard addition method was used to investigate their performance for the screening of phenols in six heavily contaminated waste waters obtained from tannery industries in Barcelona (Spain) and Sweden. 2. Material and methods 2.1. Reagents Lyophilised mushroom tyrosinase (E.C. 1.14.18.1) powder was purchased from Sigma (St. Louis, MO, USA, LOT 24H9524, 4400 U/mg). One unit of tyrosinase will cause a decrease in A280nm of 0.001 minÿ1 at pH 6.5 and 258C in a 3 ml reaction mixture containing L-tyrosine. One hundred mM stock solutions of catechol (Merck, Darmstadt, Germany), phenol (Merck), 3,4 dihydroxy-hydrocinamic acid (Sigma), dihydroxy-benzaldehyde (Aldrich, Steinheim, Germany), p-cresol (Merck), p-chlorophenol (Merck), tetrachlorophenol (Sigma), m-chlorophenol (Merck), and p-aminophenol (Fluka Chemika-Biochemika, Buchs, Switzerland) were prepared in acetonitrile. The working solutions were prepared daily by dilution in 0.1 M, NaH2PO4/Na2HPO4 buffer at pH 6.5.

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Eastman AQ Polymer 29 D, a 30% (w/v) water dispersion, was purchased from Eastman Kodak (Kingsport, Tennessee, USA) and Na®on ± per¯uorinated ion-exchange powder, 5% (w/v) in a solution of 90% aliphatic alcohol/10% water mixture, was obtained from Aldrich (Cat. no. 27, 470-4). Graphite powder and paraf®n oil were purchased from Fluka. Graphite rods (RW001, 3.05 mm OD) were obtained from Ringsdorff-Werke, Germany. Water puri®ed in a Milli-Q system (Millipore, Milford, MA, USA) was used throughout all the experiments to prepare the different solutions and buffers. All solutions were degassed before use to prevent microbubbles appearing in the ¯ow system. In order to have suf®cient oxygen present when using SGEs, the buffers were saturated with air at 408C and then cooled to room temperature before use. 2.2. Biosensor preparation Unmodi®ed carbon paste (CP) was prepared as follows: 1 g of graphite powder and 400 ml of paraf®n oil were mortared for 20 min. The unmodi®ed CP was packed into 1 ml plastic syringes (ONCE, ASIK, Denmark), leaving about 4 mm of the outer tips empty for ®lling with bulk modi®ed tyrosinase CP (see below). To obtain electrical contact, a silver wire was inserted around the tip of the piston, which was pressed into the syringe containing unmodi®ed CP. The exposed geometric surface area of electrodes was 0.051 cm2. SGEs were cut and polished on wet ®ne emery paper, thoroughly washed with deionised water, and allowed to dry at room temperature before modi®cation. Different kinds of tyrosinase biosensors were prepared, i.e., bulk modi®ed tyrosinase CPEs (no polymer), tyrosinase adsorbed to the surface of SGEs (no polymer), surface modi®ed tyrosinase-polymer CPEs, and surface modi®ed tyrosinase-polymer SGEs, according to the following procedures: 1. Bulk modi®ed (non-polymer based) tyrosinase CPEs were prepared by mixing 3850 units of dry Sigma tyrosinase powder with 100 mg of graphite powder. Forty ml of paraf®n oil were added and the mixture mortared for 20 min. The tyrosinase modi®ed CP was then packed into the outer empty tips of the syringes prepared above. The exposed

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Fig. 2. Effect of Eastman AQ concentration on the operational stability of surface modified CPEs with one or two layers. Conditions: FI system 1 (see Section 2), Sigma tyrosinase, 300 unit/layer. 50 mM catechol. Each point was based on the mean value from three identically made electrodes.

outer electrode surfaces were then polished on a glass surface and then gently rubbed on a ®lter paper. 2. Surface modified (non-polymer based) tyrosinase SGEs were prepare by applying 15 ml of a 38 500 units/ml Sigma tyrosinase solution (580 units), prepared in 0.1 M phosphate buffer pH 6.5, on the surface of a polished graphite rod and let to adsorb and dry before use. 3. Surface modified Eastman AQ/tyrosinase CPEs and SGEs were prepared according to the following: different stock solutions of tyrosinase±Eastman AQ mixtures were prepared by mixing tyrosinase powder directly with different concentrations of Eastman AQ solution (0.5%, 1.0%, 1.5%, 2%, and 2.5%, see Fig. 2), giving a final tyrosinase concentration of 20 000 units/ml in the immobilisation mixtures. The Eastman stock solutions were prepared in 0.1 M phosphate buffer at pH 6.5, but the measured pH in the tyrosinase polymer mixtures were in all cases around pH 4.9. For some electrode preparations this pH was adjusted to 4.4, 6.2, 6.8, and 8.0 with NaOH or HCl,

see Fig. 3. 1±3 layers of each 15 ml Eastman AQ± tyrosinase mixture (300 units) were then applied on top of the unmodified CPEs or SGEs, see Fig. 4. 4. Surface modified Nafion/tyrosinase CPEs and SGEs were prepared according to two different procedures described below for types I and II electrodes. Type I. Na®on±tyrosinase immobilisation mixtures were prepared by mixing different amounts of tyrosinase dissolved in 0.1 M phosphate buffer at pH 6.5 with different concentrations of Na®on dissolved in methanol, giving tyrosinase concentrations of 3850, 7700 or 11 550 units/ml and Na®on concentrations of 0.1%, 0.25%, or 0.5%, in the ®nal immobilisation mixtures. In some experiments the methanol content of the tyrosinase±Na®on immobilisation mixtures was varied between 80% and 95% (see Fig. 5). Fifteen ml of these mixtures (60, 120, or 180 units tyrosinase) were then applied on top of unmodi®ed CPEs and SGEs (see Fig. 6). Type II. Tyrosinase powder was dissolved in 0.1 M phosphate buffer pH 6.5, giving ®nal enzyme concen-

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Fig. 3. Effect of pH in the Eastman AQ/tyrosinase mixture on the operational stability of surface modified CPEs. Conditions: FI system 1 (see Section 2) one layer of 300 units Sigma tyrosinase in 2% Eastman AQ; 50 mM catechol. Each point was based on the mean value from three identically made electrodes.

Fig. 4. Operational stability in relation to the number of Eastman/tyrosinase layers on the surface of CPEs. Conditions: FI system 1 (see Section 2), polymer concentration 2% (v/v); Sigma tyrosinase, 300 units/layer, 50 mM catechol. Each point was based on the mean value from three identically made electrodes.

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Fig. 5. Effect of methanol content upon the operational stability of the Nafion/tyrosinase (type I) modified carbon paste electrodes. Conditions: FI system 1 (see Section 2) polymer concentration: 0.5% (v/v); Sigma tyrosinase: 60 units. Each point was based on the mean value from three identically made electrodes.

trations of 19 250, 38 500 or 57 750 units/ml. In a ®rst step, 15 ml of these solutions (290, 580, 870 units) were placed on top of unmodi®ed CPEs and SGEs and let to adsorb and dry. In a second step, 10 ml of a mixture of Na®on/methanol containing either 0.1%, 0.25%, or 0.5% Na®on, were applied on top of the tyrosinase modi®ed CPEs and SGEs (see Fig. 6). All tyrosinase biosensors were dried for 20 h at 48C before use. 2.3. Equipment The tyrosinase electrodes, as described above, were ®tted into a PTFE holder and inserted into a ¯owthrough wall jet amperometric cell [31]. The tyrosinase electrodes were used as working electrodes, an Ag/AgCl (0.1 M KCl) electrode as the reference electrode, and a platinum wire served as the auxiliary electrode. The electrodes were connected to a three electrode potentiostat (ZaÈta Elektronik, Lund, Sweden) and the current was registered on a strip-chart recorder (Kipp&Zonen, The Netherlands, mod. BD111). A ¯ow injection system (FI system 1) was used to evaluate and compare different electrodes under the different conditions shown in Figs. 2±9 and Tables 1 and 2. The system consisted of a peristaltic pump

(Alitea, Sweden) and a six port injection valve (Rheodyne, model 7010) with a 20 ml injection loop, connected to the amperometric ¯ow-through cell and the potentiostat with PTFE tubings (ID 0.5 mm). All measurements with the amperometric biosensors were performed at an applied potential of ÿ0.05 V vs. Ag/AgCl [32] and a ¯ow rate of 1.0 ml/min, using 0.1 M phosphate buffer at pH 6.5 as the carrier [1]. The characteristics of the optimal biosensors as well as the analysis of waste water samples were evaluated in a fully automated and optimised ¯ow injection system (FI system 2) using a Gilson ASTED XL Autoinjector (20 ml loop, Gilson, Villiers-le-Bel, France) and controlled by Gilson Unipoint software. The general conditions were the same as in FI system 1, except that tubings made of peak (ID 0.25 mm) with minimised tube length was used as connection between injector and detector in order to reduce to minimum the sample dispersion in the carrier ¯ow. Operational stability experiments were performed by injecting 50 mM catechol into the FI systems. 2.4. Biosensor evaluation and analysis of waste water samples The apparent Michaelis±Menten constant Km,app and the maximum current Imax were evaluated by

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Fig. 6. Influence of enzyme loading in the Nafion film upon the response and operational stability of the Nafion/tyrosinase CPEs for continuous injection of 50 mM catechol. Conditions: FI system 1 (see Section 2), (a) type I. ± polymer concentration: 0.5% (v/v), methanol: 80% (v/v); (b) type II. ± polymer concentration: 0.25% (v/v). Each point was based on the mean value from three identically made electrodes.

®tting the experimental data to the Michaelis±Menten equation below: Iˆ

Imax jSj : Km;app ‡ jSj

The sensitivity was de®ned as the ratio between half the maximum current (Imax/2) and the Km,app for each biosensor. The LOD was calculated as the catechol concentration (S), giving a response equal to three times the noise.

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Fig. 7. Comparison between the operational stability of surface modified tyrosinase SGEs. Conditions: FI system 1 (see Section 2), Eastman AQ: 2% (v/v); Nafion (type II): 0.25% (v/v); Sigma tyrosinase: 580 units; 50 mM catechol. Each point was based on the mean value from three identically made electrodes.

Table 1 Sensitivity, limit of detection (LOD), and sample through-put (STP) of CPEs modified as a function of one or two layers of tyrosinase± Eastman AQ membrane and with different concentrations of Eastman AQ in the layers Number of layers

Polymer concentration (%)

Sensitivity (nA/mM)

LOD (mM)

STP (samples/h)

Bulk modified 1 1 1 1 1 2 2 2 2 2 3

± 0.5 1.0 1.5 2.0 2.5 0.5 1.0 1.5 2.0 2.5 2.0

± 0.62 0.52 0.38 0.60 0.67 2.11 1.99 0.29 0.65 0.82 0.36

± 0.10 0.20 0.75 0.50 0.75 0.25 0.25 1.00 1.00 1.00 5.00

100 60 48 38 33 30 40 33 32 29 27 22

Enzyme loading: 300 units/layer. FI system 1.

Six waste water samples, obtained from tannery industries in Spain and Sweden were ®ltered on 0.22 mm ®lters, buffered 1:1 with 200 mM phosphate buffer, and then diluted with 100 mM phosphate buffer (see Table 4) at pH 6.5, before injected into FI system 2. Standard addition calibration plots were obtained by injecting waste water samples also containing 0.1; 0.5; 1 and 2 mM catechol.

3. Results and discussion The terms ion-exchange polymer and ionomer are both used to describe membrane polymers which bear ®xed acidic and/or basic groups or their salts [33]. Eastman AQ is the commercial name of a group of poly (ester±sulphonic acid) cation exchangers available dissolved in water. Once dried it does not readily

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Fig. 8. Relative response for different tyrosinase-modified electrodes as a function of the storage time: (a) CPEs and (b) SGEs. Conditions as in Fig. 7.

dissolve in aqueous solution, a speci®c advantage for biosensor construction. Na®on is a linear copolymer derived from tetra¯uoroethylene and per¯uorosulphonic acid monomers. It is available as an aliphatic alcohol/water mixture. Over the last few years one important application of ion-exchange polymers has been as immobilisation matrices for different enzymes in the development of amperometric biosensors. Na®on- [34±36] and Eastman AQ-polymers [29,37±39] have been used for the

construction of amperometric biosensors with immobilised enzymes such as glucose oxidase [34±36], glucose dehydrogenase [38], galactose oxidase [40], lactate dehydrogenase [41], alcohol dehydrogenase [34], choline oxidase [40], L-aminoacid oxidase [40], tyrosinase [39,42] and peroxidase [36,43] onto the surface of glassy carbon [34,39], platinum disk electrodes [35,36,40] and CPEs [29,38,42±44]. These entrapment matrices were shown to contribute signi®cantly to several aspects of the biosensor perfor-

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Fig. 9. Relative selectivity of different types of tyrosinase-modified electrodes for 100 mM injections of nine phenolic compounds: (a) CPEs, and (b) SGEs. Conditions as in Fig. 7.

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Table 2 Sensitivity, limit of detection (LOD) and sample through-put (STP) of CPEs modified with one layer of Nafion±tyrosinase membrane as a function of polymer concentration, enzyme loading and organic modifier in the layer, FI system 1 Enzyme loading (units)

Methanol content (%)

Sensitivity (nA/mM)

LOD (mM)

STP (samples/h)

Nafion type I (%) 0.10 0.25 0.50 0.50 0.50 0.50 0.50 0.50

116 116 58 58 58 58 116 173

80 80 80 85 90 95 80 80

0.62 0.61 0.20 0.09 0.02 0.01 0.43 1.00

0.25 0.25 0.50 0.75 1.00 10.0 0.50 0.50

58 54 53 70 75 ± 53 51

Nafion type II (%) 0.10 0.25 0.25 0.25 0.25 0.50

580 870 580 290 116 580

5.72 6.30 5.32 2.24 0.62 3.38

0.05 0.03 0.03 0.1 0.25 0.07

36 36 36 51 54 39

mance, e.g., operational and long-term stability, response time, linear range, sensitivity, selectivity and biocompatibility [34,36,43±46]. The biosensor response time may be in¯uenced by several rate limiting steps: 1. the diffusion and solubility of the substrates (oxygen and phenols) in the membrane, 2. the enzymatic reaction kinetics, and 3. the electrochemical reduction of the o-quinone at the electrode surface. Previously, it was demonstrated that the response time of tyrosinase-modified CPEs was limited by the electrochemical reduction of the quinone back to the diphenolic compound [47]. The presence of a polymer layer on the surface of the working electrode reduces the diffusion coefficient of both the substrates and the reaction products through the membrane, which consequently increases the response time of the biosensor. The most important problem that appears in the construction of tyrosinase modi®ed electrodes, is their low operational and storage stability, especially for detecting o-diphenols [1,48]. A main objective of this work was to identify ways to improve the short and long term stability of tyrosinase biosensors. The main optimisations were performed by continuous injection

of catechol on surface modi®ed tyrosinase±polymer CPEs inserted into a FI system. In some instances bulk modi®ed non-polymer based tyrosinase CPEs and surface modi®ed non-polymer and polymer based tyrosinase SGEs were used for comparative reasons as will be shown below. CPEs were preferably used due to the low noise and background currents obtained and due to that the oxygen concentration did not have to be controlled in the same way as for SGEs. This is probably due to that enough oxygen is present inside the carbon paste that can diffuse freely to the immobilised tyrosinase [1,49,50]. The characteristics of a membrane biosensor (response time, sensitivity, stability, etc.) depend on the membrane thickness and on the amount of active enzyme in the catalytic ®lm. By adding several tyrosinase/polymer layers on the electrode surface, the membrane becomes thicker but at the same time the amount of the enzyme increases in the biosensor. A third important factor is that the environment surrounding the enzyme can in¯uence the enzyme conformation and thus both the activity and stability [28]. This surrounding environment can be in¯uenced by the density of the polymer (e.g., the initial polymer concentration), the presence of activators/stabilisers/ inhibitors (e.g., the purity of the enzyme preparation),

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the presence of organic modi®er, and the pH of immobilisation. Depending on the polymer used for biosensor construction (Eastman AQ or Na®on) some of the above factors were selected for investigation and the obtained sensitivity, limit of detection (LOD) and sample through-put (STP) were compared. The short and/or long term stability of a bulk modi®ed nonpolymer based tyrosinase CPEs, surface modi®ed non polymer based tyrosinase SGEs, and surface modi®ed Eastman AQ and Na®on±tyrosinase CP and SGEs were compared. The selectivity for nine different phenols was investigated for both surface modi®ed Eastman AQ and Na®on±tyrosinase CP and SGEs. Finally, the optimal biosensors were characterised (i.e., enzyme kinetics, sensitivity, LOD, dynamic range, and precision) in an optimised automated ¯ow injection system (FI system 2) as well as applied for the evaluation of the total phenol content in six highly contaminated waste water samples. 3.1. Surface modified Eastman AQ±tyrosinase CPEs Since the Eastman AQ polymer is soluble in aqueous media, surface modi®ed Eastman AQ±tyrosinase CPEs were constructed by dissolving the enzyme directly in the Eastman AQ polymer solution. This mixture was then applied on top of the CPE surface as described in Section 2. 3.1.1. Immobilisation pH The pH in the polymer/enzyme layer may in¯uence the stability and response characteristics of enzymatic biosensors, both in terms of enzyme activity and stability but also on the electrocatalytic transduction reaction [43,46]. The isoelectric point for tyrosinase is reported to lie between 4.75 and 4.95 [23]. At pHs higher or lower than the pI, the total charge of the tyrosinase molecule is negative or positive, respectively, which means that there could be some repulsive respective attractive forces between the charged polymer and enzyme that could in¯uence the conformation of the enzyme. On the other hand one should bear in mind that the optimal pH for tyrosinase is reported to be in the area of pH 6.5 [1]. In Fig. 3, the response for 50 mM catechol as well as the operational stability of the tyrosinase biosensor are reported as a function of the pH of immobilisation. Here it is clearly seen that the signal increases up to a

pH of 6.8, i.e., the area of the enzyme's pH optimum [1]. As seen, after 50 consecutive injections of catechol the operational stability is better at pH 4.9 than at pH 6.8, which can be due to that the former pH corresponds to the isoelectric point of the enzyme. The purpose of this work was to try and improve the operational and storage stability of tyrosinase biosensor why pH 4.9 was chosen for further investigations. 3.1.2. The number of Eastman AQ±tyrosinase layers (enzyme loading) Fig. 4 depicts how the application of 1±3 polymer/ tyrosinase layers on the CPE surface in¯uences the current response and operational stability of surfacemodi®ed tyrosinase±Eastman CPEs. After 45th injection, a 15% decrease in current response is seen for the electrode with one layer of Eastman AQ±tyrosinase mixture. By addition of a second layer, only a slight improvement in stability (10% decrease after 45 injections) can be seen, but with an accompanying increase in the overall response of the sensor due to the higher enzyme loading. The application of a third layer results in a decreased overall response, indicating that the thickness of the membrane has become limiting for the diffusion of the analyte. Similarly in Table 1, the in¯uence of the three different layers on the sensitivity and LOD can be seen for an Eastman AQ concentration of 2.0%. By applying a second layer the sensitivity is slightly increased, but the LOD is increased. However, the sensitivity decreases and the LOD increases drastically for a third layer. Thus, it can be concluded that even though the enzyme loading is increased by addition of a second and a third layer, the thickness of the ®lm and the diffusion through the membrane becomes rate limiting for the biosensors. 3.1.3. Polymer concentration By increasing the polymer concentration in the layer, the polymer network surrounding the enzyme becomes denser and could in¯uence the stability of the enzyme in the polymer matrix. In Fig. 2 the stability and in Table 1 the sensitivity and LOD for Eastman AQ±tyrosinase CPEs as a function of an increase in the polymer concentration and number of applied layers can be seen. The enzyme loading was kept constant for one respective two applied layers. As seen the highest sensitivity was obtained for two layers of either 0.5% or 1.0% Eastman AQ polymer, whereas the LOD and

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stability was best using one layer at a polymer concentration of 0.5%. The sample through-put (STP) is another aspect that should be considered, especially for development of fast screening methods. Table 1 clearly demonstrates that the application of one respective several enzyme± polymer layers on the electrode surface and in addition with an increase in the polymer concentration in the layers (s) drastically decreases the STP of the method. From the obtained results it can be concluded that the optimal surface modi®ed Eastman AQ±tyrosinase CPE in terms of stability, sensitivity, LOD, and STP is a balance between the number of enzyme/polymer layers, the ratio between the enzyme and polymer concentration and ®nally the total amount of enzyme present. The best surface modi®ed Eastman AQ±tyrosinase CPE obtained here in relation to stability, LOD, and STP seems to be one where tyrosinase was immobilised at pH 4.9 with an Eastman polymer concentration of 0.5% applied in one single layer. 3.2. Surface modified Nafion±tyrosinase CPEs (types I and II sensors) Owing to limited solubility of tyrosinase in the Na®on alcohol mixture two different electrode con®gurations were investigated. Type I: tyrosinase was dissolved in a Na®on alcohol mixture which then was applied on the surface of a CPE, and type II: tyrosinase was ®rst adsorbed on the surface of CPEs and then a Na®on membrane was applied on top. For surface modi®ed Na®on±tyrosinase CPEs factors such as concentration of methanol in the tyrosinase±Na®on polymer mixture (for type I electrodes only), enzyme loading (for types I and II), and Na®on polymer concentration (for types I and II) were investigated. 3.2.1. Concentration of methanol in the Nafion±tyrosinase film The properties of enzymes in organic solvents and water±organic mixtures have been intensively studied. One of the main reasons for such an interest was an increase in the solubility of hydrophobic compounds and thus a shift of the reaction equilibrium in favour of the desired products [34]. Tyrosinase is a well-known enzyme that can act even in high concentrations of organic solvents [51±55]. With higher concentration of organic solvent, the activity of the enzyme

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decreases because of strong alterations of the protein structure. Hydrophilic solvents and supports can in addition remove the ``essential water'' of the enzyme, i.e., in its absence the enzyme looses its biocatalytic properties [34,56±60]. Because Na®on per¯uorinated polymers are less soluble in aqueous solutions, a high concentration of organic solvent (more than 80%) was necessary in order to obtain a uniform membrane on the surface of the type I CPEs. In addition, it was not possible to apply several layers, as was done for Eastman CPEs, because the performances of the sensors was deteriorated by this process. Fig. 5 presents the in¯uence of different concentrations of methanol upon the response and operational stability of the type I biosensors. As can be seen, a small increase of the organic modi®er from 80% to 95% in the immobilisation mixture drastically decreased the response of the sensors. It is evident from Table 2 that the STP value was favourably affected by increasing amounts of organic modi®er, which can be due to that the membrane was thinner and thus the analyte diffusion barrier smaller and/or the analyte was better solubilised in the membrane with high organic content. The enzyme activity was, however, not favourably affected and hence the low sensitivity for catechol. As seen in Fig. 5, for 80% methanol it took about 25 injections before the maximum signal was reached, however, once reached the signal was quite stable, as was the case also for the other methanol concentrations. A methanol content of 80% was selected for all further experiments. 3.2.2. Enzyme loading The effect of the enzyme loading, upon the response and operational stability of the Na®on/tyrosinase modi®ed type I and type II CPEs, was investigated, see Fig. 6. The amount of enzyme that could be solubilised in the polymer alcohol mixture of type I sensors was limited, why these sensors had approximately ®ve times less enzyme loading than type II and hence also overall lower sensitivities and higher LODs (results not shown). For type I sensors it can be seen in Fig. 6(a) that the responses are all rather stable. For type II sensors (Fig. 6(b)), the best combination of good operational stability and response current was obtained for the sensor with 580 units of tyrosinase.

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3.2.3. Polymer concentration The Na®on concentration was varied between 0.1% and 0.5% for both type I and type II sensors. A Na®on concentration of 0.25% resulted in both instances in the most stable electrodes (graphs not shown). As a compromise in regards to stability, sensitivity, LOD, and also for economical reasons, a surface modi®ed type II sensor containing 580 units of tyrosinase and 0.25% Na®on polymer was selected for all further experiments. 3.3. Comparison of surface modified Eastman AQ± and Nafion±tyrosinase CPEs and SGEs with non-polymer based tyrosinase CPEs and SGEs 3.3.1. Stability A direct comparison of the operational stability of tyrosinase immobilised in an Eastman AQ or Na®on polymer on the surface of CPEs with purely adsorbed enzyme on a CPE surface was not possible. This was due to that the enzyme could not successfully be immobilised by adsorption on the CPE surface, why it had to be mixed into the paste (i.e., bulk modi®cation). The amount of enzyme at the surface of the polymer and non-polymer based electrodes would thus be different and not directly comparable. A comparison was performed with solid graphite electrodes (SGEs) using the same amount of tyrosinase immobilised either directly by adsorption or in Eastman AQ or Na®on polymer. In Fig. 7, the operational stability and response of the three kinds of modi®ed SGEs are presented. As seen the adsorbed tyrosinase SGE was extremely unstable, whereas both the Eastman AQ and Na®on (type II) electrodes showed very good stability even after 50 consecutive injections of catechol. Fig. 8 shows the comparison of the long-term stability of bulk and surface-modi®ed polymer and nonpolymer based tyrosinase CP (Fig. 8(a)) respective SGEs (Fig. 8(b)). The substantial improvement in long-term stability of the polymer based as compared to non-polymer based tyrosinase biosensors demonstrates the good biocompatibility between the enzyme and the two polymers. 3.3.2. Selectivity for different phenolic compounds The relative selectivity of tyrosinase-modi®ed electrodes for different phenolic compounds was clearly in¯uenced by the presence of a cation exchange

polymeric membrane on the surface of the sensor, as seen in Fig. 9(a) for CPEs and Fig. 9(b) for SGEs. Especially monophenolic substrates had a tendency to concentrate into the polymeric membrane, which is an aspect that can be exploited to improve the sensitivity and LOD for these type of compounds. The type of carbon electrode, i.e., CPE (Fig. 9(a)) or SGE (Fig. 9(b)), also seems to play an important role in this aspect. 3.4. Evaluation of biosensors characteristics Table 3 shows the most important characteristics of the optimal Eastman± and Na®on±tyrosinase biosensors tested in an automated and optimised ¯ow injection system (i.e., the inner diameter and the length of tubing used as connections in FI system were minimised). Even though the same amount of enzyme was immobilised in the two types of biosensors, the obtained Km,app value was lower and the maximum current (Imax) was higher for the Na®on± compared to the Eastman±tyrosinase biosensor. These results indicate that the diffusion barrier for the substrate as well as the polymer density is smaller in Na®on and that it has a better biocompatibility with immobilised tyrosinase (that leads to a higher amount of active enzyme after immobilisation), a fact also con®rmed by the higher sensitivity obtained using the Na®on±tyrosinase biosensors. 3.5. Applications to waste water samples The phenol content of six tannery waste waters from Spain and Sweden was estimated by standard addition analysis using the two different surface modi®ed polymer based tyrosinase CPEs. All samples analysed had a strong smelly odour and were black/brown/grey coloured to various degrees due to collection at different stages of the clean-up process. The results, in catechol equivalents, are presented in Table 4 with the analysis performed in the order of the least contaminated samples (the order of appearance in Table 4). Between analysis of each waste water sample a standard calibration was performed to control the sensor stability. As can be seen in Table 4, the slopes of the standard addition plots in waste water were in all cases lower than for the standard calibration, indicating the presence of tyrosinase inhibitors in the waste waters.

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Table 3 The main characteristics of three identically made Eastman± and Nafion±tyrosinase biosensors, respectively

Km,app (mM) Imax (mA) Sensitivity (nA/mM) LOD (mM) Dynamic range (mM) N n Repeatability of calibration (CV, %) Reproducibility (CV, %)

Eastman±Tyrosinase

Nafion±Tyrosinase

Electrode

Electrode

1

2

3

1

2

3

257 1498 2.91 0.030 0.05±150 15 3 7.61

225 1736 3.86 0.030 0.05±150 15 3 5.67 78.79

200 1847 4.62 0.025 0.05±200 15 3 7.17

170 3914 11.51 0.015 0.03±100 15 3 6.77

180 3774 10.48 0.015 0.03±100 15 3 6.13 64.13

175 3442 9.83 0.015 0.03±100 15 3 5.13

Nˆthe number of total standard injections per calibration, nˆthe number of repetitive injections for each concentration. Conditions as in Fig. 10.

Fig. 10. The decrease in the biosensor response for 1 mM catechol after standard addition analysis of six waste waters. Conditions: FI system 2. Eastman AQ biosensor, 0.5% (v/v) Eastman at pH 4.9 applied in one layer; Sigma tyrosinase, 870 units; Nafion biosensor (type II), 0.25% (v/v) Nafion, Sigma tyrosinase, 870 units; each value was obtained by injection of 1 mM catechol after 15 consecutive injections of the specified waste water sample (three injections for the buffered sample and three injections for each of the four spiking levels).

The degree of inhibition of the different waters could be evaluated using the ratio between the sensitivity of the standard addition calibration in the different waste water samples and the sensitivity of the standard calibration (Ssample/Sstandard), i.e., the closer the ratio is to 1 the lower the inhibition. Unfortunately the

inhibition of the sensors was irreversible in some of the waters which can be seen in Fig. 10 as a decrease in the responses for a catechol standard injected between each standard addition analysis. It can be seen that the Na®on±tyrosinase biosensor retained approximately 70% of its response after analysis of

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Sample description (dilution)

Standard BAR 3 secondary effluent (1:1) SWE 3 secondary effluent (1:1) BAR 2 primary effluent (1:25) SWE 2 primary effluent (1:1) SWE 1 untreated (1:100) BAR 1 untreated (1:250)

Notes

Phenols and surfactants30 mg/l ± TOC >150 mg/l, phenols and surfactants 10±100 mg/l ± ± TOC >150 mg/l, phenols and surfactants in mg/l

Sensitivity (nA/mM)

Ssample/Sstandard

Estimated catechol equivalents mM (mg/l)

Eastman

Nafion

Eastman

Nafion

Eastman

Nafion

4.53 4.28 1.54 1.09 0.19 1.30 0.89

11.34 10.81 5.28 4.87 2.77 9.90 5.48

1.00 0.95 0.42 0.43 0.11 0.84 0.59

1.00 0.96 0.47 0.47 0.26 0.90 0.56

± 0.19 0.41 0.84 0.46 3.59 11.87

± 0.12 0.15 1.07 0.31 2.80 14.18

(21) (45) (92) (51) (395) (1307)

(13) (15) (118) (34) (314) (1561)

TOCˆtotal organic carbon. The phenol content, expressed as the catechol equivalents, was estimated from the intercept of the calibration plot from five standard additions of catechol (Nˆ5) and three injections for each spiking level (nˆ3) to equal volumes of diluted waste sample. Conditions as in Fig. 10. TOC is the total organic content of the water sample.

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Table 4 Standard addition analysis of six waste water samples from tannery industries in Barcelona, Spain (BAR 1-3) and Sweden (SWE 1-3), collected at different stages of the clean-up process

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the six waste waters (i.e., a total injection of 90 waste waters on the sensor) whereas the Eastman±tyrosinase biosensor only retained about 30% of its initial response. These results show that the Na®on polymer not only is more biocompatible with tyrosinase but also seems to provide a better protection for the immobilised enzyme for the analysis of waste waters. Column two in Table 4 gives some notes given by the provider of what and in which concentration range contaminants can be expected to be present in the waste water samples from Barcelona (BAR 1-3) and Sweden (SWE 1-3). Inspite of the apparent inhibition of the sensors, the values obtained by standard addition analysis for both types of biosensors correlate rather well with each other as well as with the indicated range of phenols present in the samples. 4. Conclusions The results presented in this work demonstrate the possibility of using Na®on modi®ed tyrosinase electrodes for obtaining biosensors with improved stability for the screening of phenolic compounds in waste waters. The immobilisation of tyrosinase in cationic exchange membranes has some advantages, e.g., exclusion of anionic interferences and altered selectivity of the sensors due to preconcentration of certain monophenols into the membranes. The high ionic conductivity, the high solubility of oxygen, the biocompatibility, and the high stability of Na®on make it a particularly good matrix for tyrosinase immobilisation. The disadvantage of introducing a membrane is, however, the reduced sensitivity and LOD as compared to other previously developed tyrosinase biosensors [1,30,61,62]. However, in comparison with other tyrosinase CPEs, a LOD of 0.015 mM catechol, as obtained for the best Na®on sensor, was quite good and it was shown to be useful for screening of phenols in highly contaminated samples such as waste waters. Acknowledgements The authors kindly acknowledge the ®nancial support from the European Community, i.e., the EC Environment and Climate program EC No. ENV4-CT97-0476, the EC Tempus program S-JEP09227-95, the EC INCO-Copernicus program ERB

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