Anal. Chem. 1997, 69, 4108-4112
Reagentless Tyrosinase Enzyme Electrodes: Effects of Enzyme Loading, Electrolyte pH, Ionic Strength, and Temperature F. Daigle and D. Leech*
De´ partement de Chimie, Universite´ de Montre´ al, C.P. 6128, Succursale Centre-ville, Montre´ al, Que´ bec H3C 3J7, Canada
We have prepared a reagentless enzyme activity sensor based on the mediated reduction of oxygen by tyrosinase coimmobilized in an osmium redox polymer hydrogel on glassy carbon electrode surfaces. The activity of this sensor is shown to be influenced by the enzyme loading, yielding an optimum activity for 41.7% (w/w) enzyme in the deposition solution. The electroyte pH, ionic strength, and temperature also affect the electrode response by altering enzyme activity, charge transport rates, and mediator concentration in the films. The response of the sensor decreases by only 25% over a 6-h period. However, reproducible inhibition curves can be obtained by normalization of the sensor response. The resulting enzyme inhibition biosensor can detect levels of the enzyme inhibitor, azide, as low as 1.0 × 10-5 mol/dm3 in solution. The immobilized sensors can be utilized for the detection of modulators of tyrosinase enzyme activity, such as respiratory poison inhibitors. The use of enzyme electrodes is an inherently sensitive method for the detection of enzyme inhibitors and activators because of amplification by substrate turnover and the specificity of biological recognition.1-3 Previous research on enzyme inhibition electrodes has focused on cytochrome oxidase-, horseradish peroxidase (HRP)-, or tyrosinase-based enzyme sensors which are inhibited by the respiratory poisons.1-4 In an innovative approach Smit and Rechnitz3 reported that the natural substrate for the tyrosinase enzyme could be replaced with an electrochemically regenerable solution redox species, ferrocyanide, that can act as an electron donor in the enzymatic reduction of oxygen. Thus, the catalytic current for oxygen reduction is a measure of the tyrosinase electrode enzyme activity. The mediated reduction of solution tyrosinase by a copper(II) complex immobilized in a Nafion polymer film on a glassy carbon electrode has also been reported.4 Immobilization of both mediator and enzyme for the construction of a biosensor is necessary for a truly “reagentless” system that consumes only oxygen. In recent years, the coimmobilization of enzymes and redox mediators onto electrode surfaces for the development of enzyme * To whom correspondence should be addressed. E-mail: leechd@ ere.umontreal.ca. Fax: (514) 343 7586. (1) (a) Albery, W. J.; Cass, A. E. G.; Shu, Z. X. Biosens. Bioelectron. 1990, 5, 367. (b) Albery, W. J.; Cass, A. E. G.; Shu, Z. X. Biosens. Bioelectron. 1990, 5, 379. (c) Albery, W. J.; Cass, A. E. G.; Mangold, B. P.; Shu, Z. X. Biosens.Bioelectron. 1990, 5, 397. (2) Smit, M. H.; Cass, A. E. G. Anal. Chem. 1990, 62, 2429. (3) Smit, M. H.; Rechnitz, G. A. Anal. Chem. 1993, 65, 380. (4) Furbee, J. W., Jr.; Thomas, C. R.; Kelly, R. S.; Malachowski, M. R. Anal. Chem. 1993, 65, 1654.
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biosensors has received considerable attention.5-8 Heller’s group has successfully demonstrated the “molecular wiring” of glucose oxidase by coordinatively binding the enzyme in a hydrogel with an osmium redox mediator bound to a poly(vinylpyridine)5 or poly(vinylimidazole)6 backbone. This “wiring” has been extended to include other enzymes such as lactate oxidase7 and cellobiose oxidase.8 We have recently reported preliminary results on an immobilized reagentless sensor for enzyme modulators using the tyrosinase enzyme covalently attached to a redox hydrogel deposited on electrode surfaces.9 The system is based on the coimmobilization of tyrosinase and a redox polymer, [Os(bpy)2(PVI)10Cl]Cl (PVI-Os), where bpy is the 2,2′-bipyridine ligand and (PVI)10 is poly(N-vinylimidazole) indicating a ratio of coordinated redox sites to free pendant groups of 1:10, using the method of Ohara et al.6 The hydrogel enzyme electrode allows “reagentless” sampling of the enzyme activity, as depicted in Figure 1, by electrochemically “switching on” the mediated enzymatic reduction of oxygen. The observed current is sensitive to modulators of the enzyme activity, such as enzyme inhibitors or activators. Application of these sensors to the detection of the presence of respiratory poisons such as cyanide and azide, which inhibit oxygen binding to the enzyme active site, is proposed. In this paper, we report on the optimization of the response of the reagentless tyrosinase sensor. Optimized conditions for the operation of the sensors are obtained upon investigation of the effect of factors such as enzyme loading and electrolyte temperature, pH, and ionic strength on the mediated reduction of oxygen. The respiratory poison azide is selected as a model inhibitor to demonstrate the response of the sensors to enzyme modulators. EXPERIMENTAL SECTION Materials and Methods. Electrochemical experiments were carried out in 0.05 mol/dm3 phosphate buffer (PB), pH 7.4, at room temperature unless otherwise stated, using a BAS 100B/W potentiostat. Either a 0.5 cm-3 microcell or a 2-cm-3 onecompartment thermostated cell was used for the electrochemical studies. The electrochemical cell consisted of a Ag/AgCl reference electrode (BAS, 3 M KCl), a Pt wire counter electrode (Aldrich), and a 3-mm-diameter glassy carbon working electrode purchased from BAS or constructed in-house by embedding a (5) (a) Gregg, B. A.; Heller, A. Anal. Chem. 1990, 62, 258. (b) Gregg, B. A.; Heller, A. J. Phys. Chem. 1991, 95, 5970, 5976. (6) (a) Ohara, T. J.; Rajagopalan, R.; Heller, A. Anal. Chem. 1993, 65, 3512. (b) Ohara, T. J.; Rajagopalan, R.; Heller, A. Anal. Chem. 1994, 66, 2451. (7) Wang, D. L.; Heller, A. Anal. Chem. 1993, 65 1069. (8) Elmgren, M.; Nordling, M.; Lindquist, S.-E. Anal. Biochem. 1993, 215, 261. (9) Robinson, G.; Leech, D.; Smyth, M. R. Electroanalysis 1995, 7, 952. S0003-2700(97)00213-8 CCC: $14.00
© 1997 American Chemical Society
Figure 1. Catalytic scheme for the activity of the coimmobilized tyrosinase and osmium redox mediator in a hydrogel on a glassy carbon electrode surface.
glassy carbon rod (V-25, Atomergic Chemetals) in a glass tube using Spurr low-viscosity epoxy (Polyscience) and contacting the unexposed side to a copper wire with silver epoxy (EpoxyTechnology). Electrodes were abraded with successively finer grades of SiC paper and polished to a “mirrorlike” finish with 0.3and 0.05-µm alumina slurry (Buehler). Tyrosinase (polyphenol oxidase EC 1.14:18.1) from Agaricus bisporus was obtained from Sigma. Poly(N-vinylimidazole) was prepared by bulk free-radical polymerization of vacuum-distilled N-vinylimidazole (Polysciences) using azoisobutyronitrile (AIBN, Aldrich) as initiator. Synthesis and characterization of the PVIOs polymer was carried out according to literature methods.10,11 Redox polymer-modified electrodes containing covalently bound tyrosinase were prepared using poly(oxyethylene) bis(glycidyl ether) (PEG, Sigma) as a linker by pipetting quantities of the redox polymer (1 mg/mL in water), tyrosinase (2.5 mg/mL in PB), and PEG (2.5 mg/mL in water) onto the surface of a glassy carbon electrode and allowing the subsequent hydrogel to dry in vacuum for at least 48 h. RESULTS AND DISCUSSION Cyclic Voltammetry of Reagentless Mediating Films. The coimmobilization of both enzyme and electron donor in a stable film on the electrode surface can be achieved using an approach devised by Ohara et al.6 for the construction of mediated glucose biosensors. In this approach, PVI-Os and tyrosinase are crosslinked with PEG in phosphate buffer on the electrode surface. The cross-linker reacts with both the free imidazole nitrogen of the polymer and the enzyme amino groups to produce a stable hydrogel on the electrode surface. Cyclic voltammograms (CV) of a PVI-Os film containing immobilized tyrosinase in oxygenated and deoxygenated PB electrolyte are shown in Figure 2. In the absence of oxygen (dashed curve), the enzyme oxidationreduction cycle cannot function (see Figure 1), yielding voltammograms that are typical for redox polymer-modified electrodes.12 The formal potential, E°′, of the redox polymer is 220 mV (vs Ag/ AgCl), which is similar to that obtained for a model monomeric complex9 and for the PVI-Os polymer film alone, indicating that these layers are highly solvated.13 The tyrosinase electrodes in deoxygenated electrolyte exhibited a square root dependence of the current on the scan rate (v) for v > 20 mV/s, indicative of a (10) Lay, P. A.; Sargeson, A. M.; Taube, H. Inorg. Synth. 1986, 24, 291. (11) (a) Forster, R. F.; Vos, J. G. Macromolecules 1990, 23, 4372. (b) Forster, R. F.; Vos, J. G. J. Electrochem. Soc. 1992, 139, 1503. (12) Abrun ˜a, H. D. Coord. Chem. Rev. 1988, 86, 135. (13) Forster, R. F.; Vos, J. G. Langmuir 1994, 10, 4330.
Figure 2. Cyclic voltammograms (2 mV/s) of a PVI-Os/tyrosinase/ PEG (16.6:41.7:41.7% weight ratio in the deposition solution) sensor in the presence (solid curve) and absence (dashed curve) of oxygen. Osmium surface coverage 2.7 × 10-9 mol/cm2.
semi-infinite diffusion response.14 At slower scan rates, the peak currents scale linearly with v, as expected for a surface-immobilized redox couple.14 The peak-to-peak splitting of the voltammograms was, however, larger for the enzyme-containing films than for those observed for films of PVI-Os alone, as reported previously for osmium redox polymers containing glucose oxidase.15 Enzymatic reduction of oxygen mediated by the Os(II) redox couple is shown in Figure 2 (solid curve) with sigmoidal-shaped cyclic voltammograms typical of catalytic electron transfer observed.16,17 The magnitude of the current and the shape of the voltammogram depends on the oxygen tension in the electrolyte solution (not shown), as expected for a mediated reduction of this cosubstrate. The electrode in deoxygenated buffer (Figure 2, dashed curve), completely inhibited by azide (vide infra), or modified electrodes prepared by drop-coating the redox polymer alone or the redox polymer and the PEG crosslinker on the electrode surface, did not exhibit catalytic currents for the reduction of oxygen. Thus, the polymer-bound osmium complex can replace the natural enzyme substrate (phenols or catechols) as an electron donor in the mediated reduction of oxygen. Electrochemical “switching on” of the sensor is achieved by scanning the applied potential negative of the osmium complex formal potential. Catalytic currents were evaluated as the difference between the response observed at 0.0 V (vs Ag/AgCl) in the presence and absence of oxygen. The surface coverage of attached osmium complex (Γ, mol/ cm2) can be determined by integrating the charge passed upon electrolysis of the entire polymer film, estimated from the peak area under the voltammogram at slow (2 mV/s) scan rates. A linear dependence of the observed catalytic current at the modified electrodes on Γ is observed up to surface coverages of ∼2.5 × 10-9 mol/cm2. (14) Bard, A. J.; Faulkner, L. R. Electrochemical Methods: Fundamentals and Applications; Wiley: New York, 1980. (15) Elmgren, M.; Lindquist, S.-E.; Sharp, M. J. Electroanal. Chem. 1993, 362, 227. (16) Cass, A. E. G.; Davis, G.; Francis, G. D.; Hill, H. A. O.; Higgins, I. J.; Aston, W. J.; Plotkin, E. V.; Scott, L. D. L.; Turner, A. P. F. Anal. Chem. 1984, 56, 667. (17) Nicholson, R. S.; Shain, I. Anal. Chem. 1964, 36, 706.
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Figure 3. Dependence of the catalytic current (normalized to the maximum current obtained) on the fraction of tyrosinase in the films. The percent PEG was constant at 41.7% (w/w) and the ratio of PVIOs to tyrosinase was varied while maintaining a constant total mass added to the electrode surface. Other conditions as in Figure 4.
Figure 4. Dependence of response of the tyrosinase sensor, obtained in the presence (9) and absence (b) of oxygen, on NaCl. The response obtained for a modified electrode containing only PVIOs and PEG is also shown (2). Conditions as in Figure 2. The current is normalized to the maximum current obtained for each series of NaCl additions.
Enzyme, Osmium, and PEG Proportions. The effect of enzyme and osmium loading on the catalytic current for oxygen reduction was examined (Figure 3) by preparing a series of electrodes with various ratios of PVI-Os/tyrosinase while a constant percentage of PEG (41.7% w/w) was maintained. The catalytic currents show a gradual increase upon increasing percentage of tyrosinase in the deposition solutions, up to ∼40%. The catalytic currents are controlled by the enzyme activity in this region of the curve. Further increases in enzyme content results in a rapid decrease in catalytic currents, with the ratelimiting step proposed to be that of charge transport through the film, similar to that observed previously for enzyme mediation in an osmium-based redox hydrogel.5-8,15 This was examined by estimation of a charge transport diffusion coefficient, Dct, from CV experiments in deoxygenated buffer. The estimated Dct decreases dramatically with increasing proportion of enzyme in the film above 20% (w/w). This corresponds well with the results in Figure 3, where the decrease in catalytic current at higher tyrosinase percentages could be due to decreasing charge transport rates through the films. An optimal loading of ∼40% enzyme in the deposition solution is apparent from Figure 3. Thus, a tyrosinase/PVI-Os ratio of 41.7:16.6% (w/w) (2.5:1) was used for subsequent experiments. It should be noted that a Dct of (2.4 ( 0.5) × 10-10 cm2/s was estimated for the PVI-Os polymer and linker alone, which is similar in magnitude to the value obtained by Forster and Vos13 in 0.1 M p-toluenesulfonic acid. The effect on the catalytic currents of varying the level from 4 to 42% PEG in the deposition solution was also examined. An 11% relative standard deviation from the mean catalytic current (n ) 20) was obtained, with no systematic increase or decrease in catalytic current evident upon increasing the percent of PEG. This is similar to previous results, which showed no systematic variation in response with percent PEG for the PVP-Os/glucose oxidase system, where PVP is poly(4-vinylpyridine).5 A level of 41.7% PEG was selected for subsequent studies yielding a final PVI-Os/PEG/ tyrosinase ratio of 16.6:41.7:41.7% (w/w) (1:2.5:2.5). pH, Ionic Strength, Temperature, and Stability. The effect of electrolyte pH on each individual modified electrode was evaluated by measuring the slow-scan cyclic voltammetric catalytic current in the presence of oxygen followed by deoxygenation and
evaluation of Dct from CV peak currents at scan rates between 100 and 500 mV/s. The pH was varied by repeated additions of 0.05 M phosphoric acid to an initial solution of 0.05 M Na2HPO4. Dct values are steady from pH 9 to 6 but increase rapidly from pH 6 to 4, possibly because of protonation of the imidazole nitrogen and thus polymer swelling, facilitating charge transport, at these pH’s.5b,15 A maximum catalytic response in oxygenated electrolyte was observed between pH 5 and 6. This correlates well with that observed for the homogeneous reaction between tyrosinase and the model osmium complex (OsMeIm)9 and indicates that immobilization of the enzyme does not drastically alter its pH response characteristics. A pH of 7.4 was however used for all experiments to emulate the response of the sensor in physiological fluids. The effect of increasing ionic strength on the modified electrode response was evaluated by addition of aliquots of concentrated NaCl solution to the cell. The initial increase in current for the PVI-Os/PEG electrode containing no tyrosinase (Figure 4, 2) may be caused by increased compaction of the layers at higher ionic strengths, thus increasing COs, as postulated previously for osmium redox polymers.5,13 The dependence of the response of the electrode containing tyrosinase, obtained in the presence (9) and absence (b) of oxygen, on NaCl showed trends that are similar to each other. An initial increase in response was observed up to a NaCl concentration of ∼0.8 M, whereafter the response decreases gradually. This is interpreted by us as follows: at increasing ionic strength, the layers become more compact due to self-association of the film,5 thus increasing COs and hence the response. When the ionic strength is increased further, a combination of enzyme denaturation, decreased electron transfer rates from the redox couple to the enzyme,9 and decreased charge transport diffusion decreases the response. The tyrosinase enzyme electrodes are thus active in electrolyte concentrations of up to 1.0 M, in fact, yielding optimum responses in 0.4-0.8 M NaCl, which should prove useful for clinical or environmental analyses of enzyme modulators. The thermal stability of the modified electrode response was investigated by increasing the temperature of the cell and recording CVs in oxygenated and deoxygenated electrolyte. The catalytic current in oxygenated electrolyte increased with tem-
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Figure 5. Operating stability of a tyrosinase sensor, investigated by measuring the CV catalytic current every 10 min, with continuous cycling (9), holding at 0.5 V (b), and leaving at open circuit (2) in oxygenated electrolyte between the measurement cycle. Other conditions as in Figure 2.
perature for temperatures up to 60 °C, as expected for an Arrhenius-type temperature dependence. Above this temperature the rate (current) dropped off, indicative of enzyme denaturation. The currents in deoxygenated electrolyte also increased with increasing temperature up to 60 °C, reflecting an increase in the charge transport rates as expected. Figure 5 shows the operating stability of the sensor investigated by measuring the CV catalytic current every 10 min with continuous cycling (9), holding at 0.5 V (b), and leaving at open circuit (2) in oxygenated electrolyte between the measurement cycle. A gradual decrease in the response of the electrode was observed for all three methods, with a maximum decrease of 25% after 6 h of continuous cycling observed. The electrode left at open circuit in between each measurement cycle displayed the better response characteristics. An initial 10% decrease in response over 30 min was observed for this electrode, followed by a slow decrease (∼1.5% signal/h) to 17% of the initial signal after 6 h of operation. Problems with decreased response of the sensors can, however, be overcome by using a normalization of response as decribed in the next section. Reagentless Detection of Enzyme Modulators. We have selected the inhibitor sodium azide to test the utility of reagentless enzyme activity sensors for the detection of enzyme modulators. Representative cyclic voltammograms of an immobilized enzyme electrode before and after successive additions of the inhibitor, sodium azide, are shown in Figure 6. The magnitude of the catalytic current and the shape of cyclic voltammetric waves change upon increasing the inhibitor concentration, indicating inhibition of catalytic activity. Reproducible inhibition curves could be obtained from the voltammetric data from different polymer films by normalizing the inhibition response, as shown in Figure 7. Normalization is achieved by measuring the response in oxygenated and deoxygenated buffer and by assuming that the deoxygenated response represents 100% inhibition.
% inhibition )
Ioxy - Iinh × 100% Ioxy - Ideoxy
where Ioxy and Ideoxy are the currents in the presence and absence of oxygen and Iinh is the current observed upon inhibition in
Figure 6. Slow-scan (2 mV/s) cyclic voltammograms obtained at a tyrosinase sensor upon addition of 0 (solid), 3.3 × 10-4 (dashed), 1.0 × 10-3 (dotted), and 4.8 × 10-3 (dotted-dashed) M sodium azide to the cell. Other conditions as in Figure 2.
Figure 7. Normalized inhibition curves obtained at tyrosinase sensors for inhibition by sodium azide. See text for details. Inset shows enlarged portion of the graph for low azide concentrations. Conditions as in Figure 2.
oxygenated electrolyte. Changes in the catalytic current induced by inhibition of the tyrosinase enzyme by concentrations of azide as low as 1.0 × 10-5 M can be easily detected (inset, Figure 7), thus demonstrating the sensitivity of the reagentless sensors to the presence of enzyme modulators. The normalized inhibition curves from different sensors can be reproducibly overlayed. The reversibility of the sensor was investigated by placing the inhibited electrode in distilled water for a period of time. A decrease to 50% of initial current in CV was observed upon addition of 3.5 × 10-4 M sodium azide to the cell. No return to the initial current level was evident after prolonged washing with water, indicating irreversible inhibition of the sensor by azide, as described previously for a laccase-based azide sensor.18 We have investigated the selectivity of the system by adding possible inhibitors and interferents to the cell. Interference by direct electrochemical reaction at the electrode surface, mediated reduction by the osmium redox couple, or modulation of enzyme activity is possible. No change in the catalytic reduction currents were observed upon addition of up to 5 × 10-3 mol/dm3 LiClO4 (18) Trudeau, F.; Daigle, F.; Leech, D. Anal. Chem. 1997, 69, 882.
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or NaCl to the electrochemical cell. The catalytic currents decreased slightly (∼10%) upon addition of 5 × 10-3 mol/dm3 FeCl3 to the cell. Addition of 5 × 10-3 mol/dm3 ascorbic acid to the cell resulted in a large increase in oxidation currents which masked the mediated reduction of oxygen. NaF proved to be an effective inhibitor of tyrosinase activity as 5 × 10-3 mol/dm3 concentrations yielded ∼80% inhibition of the catalytic current. In summary, we have optimized the response of an immobilized tyrosinase sensor. The sensor is capable of mediating the reduction of oxygen in the electrolyte, thus yielding a reagentless system for addressing enzyme activity. Enzyme loading has been shown to affect charge transport rates and the catalytic currents observed. Electrolyte concentration, temperature, and pH also affect the signal observed for the mediated reduction of oxygen. Application of the sensor to the detection of enzyme modulators has been demonstrated, using the respiratory poison sodium azide as a model analyte. Normalized inhibition curves for this model analyte at various electrodes are
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reproducible even when the absolute catalytic currrents differ substantially or when the response of the sensor drifts with time. Thus, a reproducible calibration routine for our sensors is demonstrated. The use of the sensors as warning devices for the presence of inhibitors of oxygen binding to the copper-containing tyrosinase enzyme is proposed. Such inhibitors include the respiratory poisons CO, azide, cyanide, and H2S. ACKNOWLEDGMENT Funding for this research was provided by the Universite´ de Montre´al, FCAR Que´bec (Fonds pour la formation de Chercheurs et l’Aide a` la Recherche) and NSERC. The generous loan of Na2[OsCl6] by Johnson Matthey PLC is gratefully acknowledged. Received for review February 24, 1997. Accepted July 11, 1997.X AC970213F X
Abstract published in Advance ACS Abstracts, September 1, 1997.
