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Polyvinylferrocenium modified Pt electrode for the design of amperometric choline and acetylcholine enzyme electrodes
Biosensors and Bioelectronics 19 (2004) 1261–1268
Polyvinylferrocenium modified Pt electrode for the design of amperometric choline and acetylcholine enzyme electrodes S. Sen, ¸ A. Gülce, H. Gülce∗ Department of Chemistry, Süleyman Demirel University, 32260 Isparta, Turkey Received 12 May 2003; received in revised form 20 October 2003; accepted 19 November 2003
Abstract A simple method of enzyme immobilization was investigated, which is useful for development of enzyme electrodes based on polyvinylferrocenium perchlorate coated Pt electrode surface. Enzymes were incorporated into the polymer matrix via ion exchange process by immersing polyvinylferrocenium perchlorate coated Pt electrode in enzyme solution for several times. Choline and acetylcholine enzyme electrodes were developed by co-immobilizing choline oxidase and acetylcholinesterase in polyvinylferrocenium perchlorate matrix coated on a Pt electrode surface. The amperometric responses of the enzyme electrodes were measured at +0.70 V versus SCE, which was due to the electrooxidation of enzymatically produced H2 O2 . The effects of the thickness of the polymeric film, pH, temperature, substrate and enzyme concentrations on the response of the enzyme electrode were investigated. The optimum pH was found to be pH 7.4 at 25 ◦ C. The steady-state current of these enzyme electrodes were reproducible within ±5.0% of the relative error. Response time was found to be 30–50 s and upper limit of the linear working portions was found to be 1.2 mM choline and acetylcholine concentrations in which produced detectable currents were 1.0 × 10−6 M substrate concentrations. The apparent Michaelis–Menten constant and the activation energy of this immobilized enzyme system were found to be 1.74 mM acetylcholine and 14.92 kJ mol−1 , respectively. The effects of interferents and stability of the enzyme electrodes were also investigated. © 2003 Elsevier B.V. All rights reserved. Keywords: Polyvinylferrocenium; Choline; Acetylcholine; Amperometric enzyme electrode
1. Introduction The determination of acetylcholine (ACh) and choline (ChO) are important clinically. ACh is neurotransmitter, and neurotransmitters play important roles in the brain as they are the key link in communication between neurons in the spiral chord and in nerve skeletal junctions in vertebrates. The activity of the enzyme choline acetyltransferase in the brain decreases, often significantly, with age. This is the enzyme responsible for the synthesis of acetylcholine. Determination of ACh in the brain is important for understanding the mechanisms of neurotransmission and neuroregulation, which in turn should facilitate early detection and effective treatment of neurodegradative diseases, such as Alzheimer’s disease (Campanella et al., 1989; Huang et al., 1993; Xin and Wightman, 1997; Tamiya et al., 1991; Mascini and Moscone, 1986; Hale et al., 1991; Ricny et al., 1989). Several methods for quantitative determination of ACh have re-
∗
Corresponding author. Fax: +90-246-2371106. E-mail address: [email protected] (H. Gülce).
0956-5663/$ – see front matter © 2003 Elsevier B.V. All rights reserved. doi:10.1016/j.bios.2003.11.011
cently been developed, including radio-labeling (Gilberstadt and Russell, 1984) and gas choromatographic techniques. In addition, experiments utilizing high-performance liquid choromatography (HPLC) with electrochemical detection have been reported. However, these are time consuming and expensive methods, and lengthy clean-up is needed before introduction of the sample for choromatographic methods (Hale and Wightman, 1988). Enzyme electrodes are becoming popular for the determination of specific substrates in clinical analysis. The rapid development of enzyme immobilization technology has assisted further development in this analytical field. The combination of immobilized enzymes with electrochemical sensors has produced many low-cost devices that are rapid and sensitive to use (Potter et al., 1983). Several acetylcholine sensors have been developed based on enzyme electrodes with potentiometric (Gibson and Guilbault, 1975) or amperometric detection (Campanella et al., 1989; Huang et al., 1993; Xin and Wightman, 1997; Tamiya et al., 1991; Mascini and Moscone, 1986; Hale et al., 1991; Hale and Wightman, 1988; Merz and Bard, 1978; Daum et al., 1980; Peerce and Bard, 1980a; Peerce and Bard, 1980b).
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ACh has been converted to choline (Ch) enzymatically using an enzyme, acetylcholinesterase (AChE), AChE
ACh + H2 O −−→ Ch + acetic acid followed by the production of H2 O2 from dissolved molecular O2 by a second enzyme, choline oxidase (ChO). ChO
Ch + 2H2 O −→ betaine + 2H2 O The first reaction was carried out with AChE in a solution (Campanella et al., 1989) and ChO was immobilized on cellulose triacetate membranes, co-immobilization of the two enzymes (AChE and ChO) (Huang et al., 1993; Xin and Wightman, 1997; Tamiya et al., 1991; Mascini and Moscone, 1986; Hale et al., 1991; Hale and Wightman, 1988; Doretti et al., 2000), co-immobilization of three enzymes (AChE, ChO and HRP) (Larsson et al., 1998) were used. The enzymes were immobilized in a matrix such as carbon–fiber microelectrode (Xin and Wightman, 1997; Tamiya et al., 1991; Hale and Wightman, 1988), carbon paste electrode (Larsson et al., 1998), vitreous carbon (Khayyami et al., 1998), nylon net (Mascini and Moscone, 1986), polyvinylalcohol cryogel membrane (Doretti et al., 2000) or in a conducting polymer such as 1,2-diaminobenzene and resorcinol (Huang et al., 1993). The amperometric response was obtained by either using O2 (Campanella et al., 1989) or H2 O2 (Mascini and Moscone, 1986; Tamiya et al., 1991; Huang et al., 1993; Ricci et al., 2003, Doretti et al., 2000) electrodes. A decrease in the reduction current of O2 or an increase in the oxidation current of H2 O2 was measured with O2 or H2 O2 amperometric sensors, respectively. An organic conducting salt, tetrathiafulvalene tetracyanoquinodimethane (TTF–TCNQ) (Xin and Wightman, 1997; Hale et al., 1991; Hale and Wightman, 1988) and Meldola Blue (Khayyami et al., 1998), were also employed as electron transfer mediator for ACh analysis in anaerobic conditions. The redox polymer polyvinylferrocenium perchlorate (PVF+ ClO4 − ) gives rise to some interesting electrochemical results when used as a layer on Pt surfaces. The preparation methods and the study of the electrochemical behavior of this redox polymer coated as a film on Pt surfaces have been the subject of several recent studies (Merz and Bard, 1978; Daum et al., 1980; Peerce and Bard, 1980a; Peerce and Bard, 1980b). This electroactive film can act as a modified surface through which an electron transfer between a substrate and a reactant can take place. Studies related to the reduction and oxidation of some reactants through this film-covered surface have also been reported (Merz and Bard, 1978; 1980; Gülce et al., 1994a; Gülce et al., 1994b; Gülce et al., 1995a). It is known that the reduced form of this redox polymer film, PVF, is a homogeneous compact film, whereas the oxidized form, PVF+ , is an inhomogeneous film (Inzelt and Bacskai, 1992). Pores or pinholes exist in PVF+ film, through which dissolved reactants could diffuse to the underlying metal surface (Peerce and Bard, 1980b).
Numerous studies have been reported concerning enzyme immobilization on various carriers for enzyme electrodes. These immobilization procedures often consist of multi-steps which generally require pretreatments of carrier and considerable times for immobilization. We have shown in an earlier study that the PVF+ ClO4 − matrix is sensitive to the anions present in the medium and some anions can be inserted into the polymer through an anion-exchange process (Gülce et al., 1995a). The interaction between the negatively charged enzyme species and the polymer matrix, ensures that the enzyme is homogeneously immobilized in the polymer matrix. In our laboratory, a mild method of general use for binding of enzymes on PVF+ ClO4 − modified electrodes has been developed and successfully applied to the design of the enzyme electrodes for glucose (Gülce et al., 1995b), sucrose (Gülce et al., 1995c), galactose (Gülce et al., 2002a), lactose (Gülce et al., 2002b), alcohols (Gülce et al., 2002c), and choline (Gülce et al., 2003). The corresponding enzymes for these amperometric enzyme electrodes were immobilized via an ion exchange process with a solution of enzyme(s), which has a pH value above the isoelectric point(s) of the corresponding enzyme(s). Various enzymes (E) could be immobilized electrostatically in this polymer, PVF+ ClO4 − + E− → PVF+ E− + ClO4 − We know quite well that enzyme molecules are immobilized through the ion exchange process. In a previous study, the amount of enzyme incorporated in the PVF+ ClO4 − coated Pt electrode was determined by following the decrease of the absorbance of the enzyme solution at 277 nm during the surface immobilization procedure. Furthermore, the amount of the enzyme immobilized within the polymer could then be determined using the same absorption peak after desorbing the enzyme in a solution that has a pH value less than the isoelectric point of the corresponding enzyme. The amount of the enzyme adsorbed onto the glass surface that is lost by washing was measured spectrophotometrically and found to be negligible (Gülce et al., 1995b). The immobilized enzyme produces electroactive H2 O2 as a result of a reaction with the substrate, E−
Substrate + O2 − → product + H2 O2 H2 O2 could be electrooxidized at the applied potential of +0.70 V versus SCE according to H2 O2 → O2 + 2H+ + 2e− The oxidation of H2 O2 could also occur chemically by the polyvinylferrocenium according to the following reaction. H2 O2 + 2PVF+ → O2 + 2PVF + 2H+ Amperometric response of the electrode was further enhanced as a result of the catalytic regeneration of PVF+ sites at the same applied potential PVF → PVF+ + e−
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H2 O2 can be oxidize both electrochemically at the applied potential and chemically by the PVF+ sites on the PVF+ coated electrodes. The role of molecular oxygen is to act as an oxidant for the regeneration of the oxidized form of the enzyme. The crucial role of O2 in this mechanism is evidenced from another independent experiment. It was found that the current value of the H2 O2 oxidation on enzyme loaded electrode was directly proportional to the concentration of dissolved O2 . It should be noted that the amperometric response of the same electrode was only 0.15% of the response of the aerobic conditions when the same experiment was carried out under anaerobic conditions, indicating the major role of the dissolved O2 in the mechanism (Gülce et al., 1995b). Such performances of this redox polymer could be used advantageously to develop amperometric enzyme electrodes (Gülce et al., 1995b; Gülce et al., 1995c; Gülce et al., 2002a; Gülce et al., 2002b; Gülce et al., 2002c; Gülce et al., 2003). In this paper, we report the use of the redox polymer PVF+ CIO4 − for the co-immobilization of AChE and ChO in order to determination of acetylcholine. The hydrogen peroxide production resulting from the sequentinal reactions of enzymes was detected amperometrically. The changes in the response of the enzyme electrode with buffer, substrate, enzyme concentrations, the film thickness, pH and temperature were measured and optimum working conditions were established. The results were compared with those obtained with free enzymes in solution using uncoated Pt electrode.
2. Experimental details 2.1. Materials Acetylcholine esterase (ACh, EC 3.1.1.7 from Electric Eel C 3389), choline oxidase (ChO, EC 1.1.3.17 from Alcaligenes species C 5896), choline chloride (C 1879), acetylcholine chloride (A 6625) were purchased from Sigma. PVF was prepared using a method of chemical polymerization (Smith et al., 1976) of vinylferrocene (Alfa products). Tetrabutylammonium perchlorate (TBAP) was prepared by the reaction of tetrabutylammonium hydroxide (Merck, 40% aqueous solution) with HClO4 (Merck), crystallized from an ethanol–water mixture (9:1) several times and kept under nitrogen atmosphere after vacuum drying at 120 ◦ C. The buffer solutions were prepared using NaH2 PO4 (Analar BDH) and NaOH (Merck). The purification of methylene chloride (Merck) was accomplished according to the method proposed in literature (Perrin and Armorego, 1980). 2.2. Preparation of the enzyme electrode PVF+ ClO4 − modified Pt surface was prepared by electrooxidizing PVF at +0.70 V versus Ag/AgCl electrode in a
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methylene chloride solution containing 0.10 M TBAP. The electroprecipitation of PVF+ ClO4 − was carried out in a dry and oxygen-free nitrogen (99.99%) atmosphere. The electrical charge passing during the eletroprecipitation was measured to prepare polymer-coated electrodes with varying thicknesses. The average thickness of the dry film was estimated from the charge, Q, passing during the electroprecipitation of the film as described by Bard (Peerce and Bard, 1980b). The enzyme electrode was prepared by immersing the PVF+ ClO4 − -coated Pt electrode in a solution of ACh and ChO. The polymer-coated electrode was kept in the enzyme solution for 30 min without stirring. The pH of the enzyme solution was kept at pH 7.4, which was well above the isoelectric points of ACh and ChO. The isoelectric points of the enzymes are between pH 4.5–5.5 for ChO (Campenella et al., 1985) and pH 5.35 for AChE (Nachmansohn, 1959). The enzymes exist in the form of anions under these conditions, facilitating its interaction with the oxidized polymer, PVF+ ClO4 − . The enzyme-attached electrode was rinsed with 0.01 M phosphate buffer solution at the working pH for 5 min to remove the remaining enzyme, which was held non-electrostatically by the surface of the polymer, and then stored in 0.01 M phosphate buffer solution (pH 7.4) at 4 ◦ C. 2.3. Measurements and apparatus The response of the enzyme electrode was determined with a jacketed electrochemical cell which kept the solution at the desired temperature. Oxygen was introduced into the solution in this cell at a constant flow rate to obtain an oxygen saturated solution. Oxygen flow was continued above the solution to keep it saturated with oxygen during the measurements. In order to determine the steady-state background current of the enzyme electrode, a potential of +0.70 V versus SCE was applied to the enzyme electrode, which was kept in buffer solution that did not contain acetylcholine. After the steady-state current value had been determined, known amounts of the acetylcholine were added to the cell from a stock acetylcholine solution and the solution was stirred for 5 s. The acetylcholine response of the enzyme electrode was measured at constant potential of +0.70 V versus SCE amperometically due to the electrooxidation of H2 O2 produced enzymatically. Electrochemical measurements were carried out in a three-electrode cell with separate compartments for the reference electrode and for the counter electrode (Pt spiral). The reference electrodes were non-aqueous Ag/AgCl in electroprecipitation and SCE for acetylcholine analysis. Pt disc electrode (2 mm diameter) was used as a working electrode. The electrochemical instrumentation consisted of PAR Model 362 Potentiostat–Galvanostat. Current–time curves were recorded on a Model 16100-II Linseis recorder.
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Fig. 1. Typical amperometric responses: (a) the acetylcholine enzyme electrode; (b) the choline enzyme electrode applying a potential step to +0.70 V vs. SCE for 0.15 mM choline and acetylcholine, respectively, in 0.10 M phosphate buffer solution (pH 7.4) at 25 ◦ C (film thickness: 1.46 × 10−6 mol cm−2 of PVF+ ).
3. Results and discussion 3.1. The response of the enzyme electrodes towards choline and acetylcholine The aim of this study is the preparation of a new enzyme electrode in order to determine the acetylcholine in the aqueous solution by means of AChE and ChO enzymes immobilized in PVF+ ClO4 − matrix on the surface of the Pt electrode. It was also prepared in an earlier study from this laboratory an enzyme electrode for the determination of choline. The choline enzyme electrode consisted of a PVF+ ClO4 − coated Pt surface onto which the choline oxidase enzyme was attached (Gülce et al., 2003). Fig. 1 shows the responses of the enzyme electrodes to acetylcholine and choline. Constant potential of +0.70 V versus SCE was applied to the enzyme electrodes to measure the amperometric response due to the electrooxidation of H2 O2 produced enzymatically. Steady-state background current was first measured at this potential with a blank buffer solution of working pH. After the steady-state background current value were reached, certain volumes of substrate solutions of known concentration were added and the currents for each added amount of substrate were recorded. The currents reached a steady-state value after the initial stirring was stopped in about 30–50 s following the addition of the substrate. These steady-state current values were used to construct the calibration plots. The response time of the enzyme electrodes developed in this study are comparable to those published in literature (Mascini and Moscone, 1986; Huang et al., 1993; Ricci et al., 2003; Doretti et al., 2000). 3.2. The effect of the enzyme concentration The amounts of the enzymes in the polymer matrix depends on the concentrations of the enzymes in solution used during immobilization process. The effect of choline
oxidase concentration on the response of the choline enzyme electrode based PVF+ ClO4 − coated-Pt electrode PVF+ ChO− was established previously (Gülce et al., 2003). It was found in this study that the maximum current was obtained if the PVF+ ChO− electrode was prepared using choline oxidase solutions that contained 7.24 mg ml−1 . By keeping the concentration of choline oxidase constant at this value and varying the acetylcholine esterase concentration in the bulk solution, the effect of the AChE concentration on the response of the acetylcholine enzyme electrode was determined. The pH, temperature and film thickness were kept constant in these experiments. The enzyme, AChE, concentrations varied between 5.0 and 10.0 mg ml−1 and the maximum activity was obtained at 7.24 mg ml−1 AChE concentration. When the enzyme, AChE, concentration was 5.0 mg ml−1 , the current of the enzyme electrode was measured as 0.65 A at 2.5 mM acetylcholine concentration. The enzyme electrode response increased to 0.85 A with the enzyme concentration up to a value corresponding to 7.24 mg ml−1 at the same substrate concentration, after which decrease of the immobilized enzyme, ChO, rather than immobilized enzyme, AChE, in the polymer matrix caused a decrease in the response. Enzyme concentrations of 7.24 mg ml−1 for ChO and 7.24 mg ml−1 for AChE were used in all of measurements. 3.3. The effect of the thickness of the polymeric film The effect of the thickness of the PVF+ ClO4 − film on the Pt electrode surface was examined. When the AChE and ChO concentrations were kept constant (7.24 mg ml−1 for ChO and 7.24 mg ml−1 for AChE), the charge that passed in the preparation of the polymer coated electrodes varied between 1.16 × 10−3 and 6.68 × 10−3 C corresponding to 3.83 × 10−7 mol cm−2 of PVF+ (dry thickness ∼0.82 m) and 2.2 × 10−6 mol cm−2 of PVF+ (dry thickness: ∼4.74 m). The enzyme electrode response
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Fig. 2. The effects of pH on the responses of the enzyme electrode and free enzymes in phosphate buffer solution at 25 ◦ C.
increased with the polymer thickness up to a value corresponding to the passage of a charge of 4.42 × 10−3 C (1.46 × 10−6 mol cm−2 of PVF+ , dry thickness: ∼3.14 m) during the electroprecipitation of the PVF+ ClO4 − . This behavior could be explained by the limited diffusion rate of substrate from the bulk solution into the inner regions of the polymer in thick film. The thickness of the PVF+ ClO4 − films were kept constant at this value for all measurements. 3.4. The effect of the pH The pH value is another variable which affects the response of the enzyme electrodes. The pH dependence of the response of the enzyme electrode was determined using a 0.10 M phosphate buffer solution, and the pH varied between 6.5 and 8.0. Fig. 2 shows that the maximum activity was obtained at pH 7.4. For each point in Fig. 2, a new enzyme electrode was prepared in order to eliminate the errors that might arise from the re-use the enzyme-loaded polymer surface. The data points are the averages of three measurements. The pH of the buffer solution was kept at pH 7.4 for all measurements. In order to compare this value with that obtained with free enzymes under the same solution conditions, the H2 O2 generated from solutions of different acetylcholine concentrations was measured electrochemically. The maximum current values obtained for the free enzymes using a bare Pt electrode were with a solution of pH 7.4 as seen in Fig. 2. The same optimum pH values of immobilized and free enzymes indicated that no structural changes in the structure of the immobilized enzyme occurred. 3.5. The effect of the temperature The temperature at which the measurements are carried out is also a critical factor in determining the activity of the enzyme electrode. The response of the acetylcholine enzyme electrode was measured at different temperatures varying from 20 to 70 ◦ C. Fig. 3 shows the results of this compar-
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Fig. 3. The effects of the temperature on the activity of the enzyme electrode and free enzymes in phosphate buffer solution (pH 7.4) at 25 ◦ C (film thickness: 1.46 × 10−6 mol cm−2 of PVF+ ).
ison. The activity of the immobilized enzymes reached a maximum value at about 55 ◦ C and remains almost constant between 55 and 65 ◦ C, after which denaturation of the enzymes caused a decrease in the response. The activation energy of this co-immobilized enzyme system was calculated to be 14.92 kJ mol−1 from the Arrhenius plot. The temperature, at which the free enzymes system gave the maximum response, and the activation energy were found to be 60 ◦ C and 76.95 kJ mol−1 , respectively (Fig. 3). The comparison of the two Ea values also confirm the fact that the immobilization of the enzymes causes no deformations in the structures of the enzymes in immobilized state. 3.6. The effect of the substrate concentrations Fig. 4(a) and (b) show the responses of the acetylcholine enzyme electrode as a function of acetylcholine concentration and a function of choline concentration, respectively. The calibration plots constructed using the optimum working conditions Fig. 4(a) and (b)) gave the linear working ranges up to a value of 1.2 mm of acetylcholine and choline concentrations. The minimum substrate concentrations that produced detectable currents were 1.0 × 10−6 M. The apparent Michaelis–Menten constant (KMapp ) for acetylcholine was calculated to be 1.74 mM according to the Linewaver–Burk graph of the Michaelis–Menten equation under the optimum conditions. The free enzymes in solution which produced a current response on an uncoated Pt electrode gave a KM value of 1.58 mM acetylcholine. KM values immobilized and free enzymes indicated no diffusional limitations in the immobilized state and the non-denaturating character of the procedure of enzyme anchoring. Fig. 5 shows the amperometric current response of the choline enzyme electrode as a function of choline concentration under the optimum conditions (Gülce et al., 2003). The upper limit of the linear working portion in calibration plot was found to be 1.2 mM choline. The data points in Figs. 4 and 5 are the averages of three measurements. Relative standard deviations were calculated equal to or <5%. The acetylcholine enzyme
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of both acetylcholine and choline in a single sample with two sensors, one selective for choline and other for acetylcholine, is possible. The current from the two biosensors can be converted into concentration signals with calibration data. The acetylcholine concentration can then be obtained by subtraction. 3.7. The interferences study
Fig. 4. Changes in the response of the acetylcholine enzyme electrode with the substrate concentration in 0.10 M phosphate buffer solution (pH 7.4) at 25 ◦ C (film thickness: 1.46 × 10−6 mol cm−2 of PVF+ ) for: (a) acetylcholine and (b) choline.
The interference signal due to the most common electrochemical intefering species was also evaluated. The signal for a fixed concentration of acetylcholine was compared with the current value obtained in the presence of the variable concentrations of the interfering species. Several interferants, such as ethanol, methanol, formaldehyde, lechitine, diethanolamine, triethanolamine and ascorbic acid were tested to study the relative response of the enzyme electrode. The relative responses obtained for most of these interferants under optimum working conditions were found to be negligible. The most important interference was caused by triethanolamine and ascorbic acid. No noticeable changes in current were detected in the concentration ranges of 0–0.16 mM triethanolamine and ascorbic acid in 1.2 mM acetylcholine solution which produced current values of 6% when acetylcholine response was taken as 100% for 0.16 mM interferants concentrations. The relative activity of the enzyme electrode was 25% of the 1.2 mM acetylcholine response when interferant concentrations were 1.5 mM. 3.8. Long-term stability of the enzyme electrode
electrode can detect not only acetylcholine signals but also choline signals while the choline enzyme electrode only responded to choline. Dynamic range and calibration sensitivity (i/C) values of acetylcholine enzyme electrode are approximately equal to acetylcholine and choline as shown in Fig. 4(a) and (b). Therefore, a simultaneous determination
Fig. 5. Changes in the response of the choline enzyme electrode with choline concentration in 0.10 M phosphate buffer solution (pH 7.4) at 25 ◦ C.
The enzyme electrode which was prepared under optimum conditions was tested at 25 ◦ C. A total of 255 measurements were made in 42 days. The enzyme electrode responses gradually decreased in the first 25 days, the activity remained constant thereafter, indicating good long term stability of the enzyme electrode. There is a noticeable decrease in the response during the first 25 days owing to the desorption of the enzyme, which is weakly held by the surface. The current response on the 30th day was 50% of the initial value (Fig. 6).
Fig. 6. The long-term stability of the acetylcholine enzyme electrode in 0.10 M phosphate buffer solution (pH 7.4) at 25 ◦ C.
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4. Conclusions It can be concluded that the redox polymer, PVF+ ClO4 − , possesses unique properties as an immobilizing medium for the enzymes and as a catalyst for H2 O2 oxidation for the development of a simple, sensitive, stable and low-cost amperometric acetylcholine enzyme electrode. The response time and the linear working range of the enzyme electrode developed in this study are better than those already proposed in the literature. The acetylcholine enzyme electrode described above offers the additional advantage of having a maximum response at physiological temperatures for in vivo applications. Furthermore, the higher sensitivity of the electrode also offers the possibility of using ultramicroelectrode designings for in vivo measurements. The electrode surface coated with PVF+ ClO4 − layer thus offers some novel application possibilities in the field of enzyme electrodes.
Acknowledgements The authors are grateful for financial support by The Scientific and Technical Research Council of Turkey (Project no: TBAG-1867) and The Administrative Units of The Research Projects of Süleyman Demirel University (Project no: SDUAF-226). References Campenella, L., Mascini, M., Palleschi, G., Tomassetti, M., 1985. Determination of choline-containing phospholipids in amniotic fluid by an enzyme sensor. Clin. Chim. Acta 151, 71–83. Campanella, L., Sammartino, M.P., Tomassetti, M., 1989. An enzyme sensor for determination of acetylcholine and choline in rat brain extracts. Anal. Lett. 22 (6), 1389–1402. Daum, P., Lenhard, J.R., Rolison, D., Murray, R.W., 1980. Diffusional charge transport through ultrathin films of radiofrequency plasma polymerized vinylferrocene at low temperature. J. Am. Chem. Soc. 102, 4649–4653. Doretti, L., Ferrara, D., Lora, S., Schiavon, F., Veronese, F.M., 2000. Acetylcholine biosensor involving entrapment of acetylcholinesterase and poly(ethylene glycol)-modified choline oxidase in a poly(vinyl alcohol) cryogel membrane. Enzyme Microb. Technol. 27, 279– 285. Gibson, K., Guilbault, G.G., 1975. Assay of cholinesterase using ion-selective electrodes. Anal. Chim. Acta 80, 245. Gilberstadt, M.L., Russell, J.A., 1984. Metabolism of acetylcholine in the nervous system of Aplysia californica. Anal. Biochem. 138, 78. Gülce, H., Özyörük, H., Yıldız, A., 1994a. Electrochemical reduction of anthracenes on poly(vinylferrocenium) coated Pt electrodes in acetonitrile. Ber. Bunsenges. Phys. Chem. 98, 228–233. Gülce, H., Özyörük, H., Yıldız, A., 1994b. Electrochemical oxidation of anthracenes on poly(vinylferrocenium) coated Pt electrodes in acetonitrile. Ber. Bunsenges. Phys. Chem. 98, 828–832. Gülce, H., Özyörük, H., Yıldız, A., 1995a. Electrochemical response of the poly(vinylferrocenium) coated Pt electrodes towards some anions in aqueous medium. Electroanalysis 7, 178–183.
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Gülce, H., Özyörük, H., Çelebi, S.S., Yıldız, A., 1995b. Amperometric enzyme electrode for aerobic glucose monitoring prepared by glucose oxidase immobilized in polyvinylferrocenium. J. Electroanal. Chem. 394, 63–70. Gülce, H., Özyörük, H., Çelebi, S.S., Yıldız, A., 1995c. Amperometric enzyme electrode for sucrose determination prepared from glucose oxidase and invertase co-immobilized in polyvinylferrocenium. J. Electroanal. Chem. 397, 217–223. Gülce, H., Ataman, I., Gülce, A., Co¸skun, H., Yıldız, A., 2002a. A new amperometric enzyme electrode for galactose determination. Enzyme Microb. Technol. 30, 41–44. Gülce, H., Gülce, A., Yıldız, A., 2002b. A novel two-enzyme amperometric electrode for lactose determination. Anal. Sci. 18, 1–3. Gülce, H., Gülce, A., Kavanoz, M., Co¸skun, H., Yıldız, A., 2002c. A new amperometric enzyme electrode for alcohol determination. Biosens. Bioelectron. 17, 517–521. Gülce, H., Akta¸s, Y.S., Gülce, A., Yıldız, A., 2003. Polyvinylferrocenium immobilized enzyme electrode for choline analysis. Enzyme Microb. Technnol. 32, 895. Hale, P.D., Wightman, R.M., 1988. Enzyme-modified tetrathiafulvalene tetracyanoquinodimethane microelectrodes: direct amperometric detection of acetylcholine and choline. Mol. Cryst. Liq. Cryst. 160, 269–279. Hale, P.D., Liu, L.F., Skotheim, T.A., 1991. Enzyme-modified carbon paste: tetrathiafulvalene electrodes for the determination of acetylcholine. Electroanalysis 3, 751–756. Huang, Z., Villarta Snow, R., Lubrano, G.J., Guilbault, G.G., 1993. Development of choline and acetylcholine Pt microelectrodes. Anal. Biochem. 215, 31–37. Inzelt, G., Bacskai, J., 1992. Electrochemical quartz crystal microbalance study of the swelling of polyvinylferrocene films. Electrochim. Acta 37, 647–654. Khayyami, M., Pita, M.T.P., Garcia, N.P., Johansson, G., Danielsson, B., Larsson, P.O., 1998. Development of an amperometric biosensor based on acetylcholine esterase covalently bound to a new support material. Talanta 45, 557–563. Larsson, N., Ruzgas, T., Gorton, L., Kokaia, M., Csöregi, E., 1998. Design and development of an amperometric biosensor for acetylcholine determination in brain microdialysates. Electrochim. Acta 43, 3541– 3554. Mascini, M., Moscone, D., 1986. Amperometric acetylcholine and choline sensors with immobilized enzymes. Anal. Chim. Acta 179, 439– 444. Merz, A., Bard, A.J., 1978. A stable surface modified platinum electrode prepared by coating with electroactive polymer. J. Am. Chem. Soc. 100, 3222–3223. Nachmansohn, D., 1959. Chemical and Molecular Basis of Nerve Activity. Academic Press, NY. Peerce, P.J., Bard, A.J., 1980a. Polymer films on electrodes. Part III. Digital simulation model for cyclic voltammetry of electroactive polymer film and electrochemistry of poly(vinylferrocene) on platinum. J. Electroanal. Chem. 114, 89–115. Peerce, P.J., Bard, A.J., 1980b. Polymer films on electrodes part film structure and mechanism of electron transfer with electrodeposited polyvinylferrocene. J. Electroanal. Chem. 112, 97–115. Perrin, D.D., Armorego, W.L.F., 1980. Purification of Laboratory Chemicals. Pergamon Press, Oxford, p. 265. Potter, P.E., Meek, J.L., Neff, N.H., 1983. Acetylcholine and choline in neuronal tissue measured by HPLC with electrochemical detection. J. Neurochem. 41, 188. Ricci, F., Amine, A., Palleschi, g., Moscone, D., 2003. Prussian Blue based screen printed biosensors with improved characteristics of long-term lifetime and pH stability. Biosens. Bioelectron. 18, 165–174. Ricny, J., Coupek, J., Tucek, S., 1989. Determination of acetylcholine and choline by flow-injection with immobilized enzymes and fluometric or luminometric detection. Anal. Biochem. 76, 221– 227.
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Smith, T.W., Kuder, J.E., Wychick, D., 1976. Voltammetric behavior of polyvinylferrocene. J. Polym. Sci. 14, 2433–2448. Tamiya, E., Sugiura, Y., Navera, E.N., 1991. Ultramicro acetylcholine sensor based on an enzyme-modified carbon fibre electrode. Anal. Chim. Acta 341, 43–51.
Xin, Q., Wightman, R.M., 1997. Enzyme modified amperometric sensors for choline and acetylcholine with tetrathiafulvalene tetracyanoquinodimethane as the electron-transfer mediator. Anal. Chim. Acta 341, 43–51.
Polyvinylferrocenium modified Pt electrode for the design of amperometric choline and acetylcholine enzyme electrodes S. Sen, ¸ A. Gülce, H. Gülce∗ Department of Chemistry, Süleyman Demirel University, 32260 Isparta, Turkey Received 12 May 2003; received in revised form 20 October 2003; accepted 19 November 2003
Abstract A simple method of enzyme immobilization was investigated, which is useful for development of enzyme electrodes based on polyvinylferrocenium perchlorate coated Pt electrode surface. Enzymes were incorporated into the polymer matrix via ion exchange process by immersing polyvinylferrocenium perchlorate coated Pt electrode in enzyme solution for several times. Choline and acetylcholine enzyme electrodes were developed by co-immobilizing choline oxidase and acetylcholinesterase in polyvinylferrocenium perchlorate matrix coated on a Pt electrode surface. The amperometric responses of the enzyme electrodes were measured at +0.70 V versus SCE, which was due to the electrooxidation of enzymatically produced H2 O2 . The effects of the thickness of the polymeric film, pH, temperature, substrate and enzyme concentrations on the response of the enzyme electrode were investigated. The optimum pH was found to be pH 7.4 at 25 ◦ C. The steady-state current of these enzyme electrodes were reproducible within ±5.0% of the relative error. Response time was found to be 30–50 s and upper limit of the linear working portions was found to be 1.2 mM choline and acetylcholine concentrations in which produced detectable currents were 1.0 × 10−6 M substrate concentrations. The apparent Michaelis–Menten constant and the activation energy of this immobilized enzyme system were found to be 1.74 mM acetylcholine and 14.92 kJ mol−1 , respectively. The effects of interferents and stability of the enzyme electrodes were also investigated. © 2003 Elsevier B.V. All rights reserved. Keywords: Polyvinylferrocenium; Choline; Acetylcholine; Amperometric enzyme electrode
1. Introduction The determination of acetylcholine (ACh) and choline (ChO) are important clinically. ACh is neurotransmitter, and neurotransmitters play important roles in the brain as they are the key link in communication between neurons in the spiral chord and in nerve skeletal junctions in vertebrates. The activity of the enzyme choline acetyltransferase in the brain decreases, often significantly, with age. This is the enzyme responsible for the synthesis of acetylcholine. Determination of ACh in the brain is important for understanding the mechanisms of neurotransmission and neuroregulation, which in turn should facilitate early detection and effective treatment of neurodegradative diseases, such as Alzheimer’s disease (Campanella et al., 1989; Huang et al., 1993; Xin and Wightman, 1997; Tamiya et al., 1991; Mascini and Moscone, 1986; Hale et al., 1991; Ricny et al., 1989). Several methods for quantitative determination of ACh have re-
∗
Corresponding author. Fax: +90-246-2371106. E-mail address: [email protected] (H. Gülce).
0956-5663/$ – see front matter © 2003 Elsevier B.V. All rights reserved. doi:10.1016/j.bios.2003.11.011
cently been developed, including radio-labeling (Gilberstadt and Russell, 1984) and gas choromatographic techniques. In addition, experiments utilizing high-performance liquid choromatography (HPLC) with electrochemical detection have been reported. However, these are time consuming and expensive methods, and lengthy clean-up is needed before introduction of the sample for choromatographic methods (Hale and Wightman, 1988). Enzyme electrodes are becoming popular for the determination of specific substrates in clinical analysis. The rapid development of enzyme immobilization technology has assisted further development in this analytical field. The combination of immobilized enzymes with electrochemical sensors has produced many low-cost devices that are rapid and sensitive to use (Potter et al., 1983). Several acetylcholine sensors have been developed based on enzyme electrodes with potentiometric (Gibson and Guilbault, 1975) or amperometric detection (Campanella et al., 1989; Huang et al., 1993; Xin and Wightman, 1997; Tamiya et al., 1991; Mascini and Moscone, 1986; Hale et al., 1991; Hale and Wightman, 1988; Merz and Bard, 1978; Daum et al., 1980; Peerce and Bard, 1980a; Peerce and Bard, 1980b).
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ACh has been converted to choline (Ch) enzymatically using an enzyme, acetylcholinesterase (AChE), AChE
ACh + H2 O −−→ Ch + acetic acid followed by the production of H2 O2 from dissolved molecular O2 by a second enzyme, choline oxidase (ChO). ChO
Ch + 2H2 O −→ betaine + 2H2 O The first reaction was carried out with AChE in a solution (Campanella et al., 1989) and ChO was immobilized on cellulose triacetate membranes, co-immobilization of the two enzymes (AChE and ChO) (Huang et al., 1993; Xin and Wightman, 1997; Tamiya et al., 1991; Mascini and Moscone, 1986; Hale et al., 1991; Hale and Wightman, 1988; Doretti et al., 2000), co-immobilization of three enzymes (AChE, ChO and HRP) (Larsson et al., 1998) were used. The enzymes were immobilized in a matrix such as carbon–fiber microelectrode (Xin and Wightman, 1997; Tamiya et al., 1991; Hale and Wightman, 1988), carbon paste electrode (Larsson et al., 1998), vitreous carbon (Khayyami et al., 1998), nylon net (Mascini and Moscone, 1986), polyvinylalcohol cryogel membrane (Doretti et al., 2000) or in a conducting polymer such as 1,2-diaminobenzene and resorcinol (Huang et al., 1993). The amperometric response was obtained by either using O2 (Campanella et al., 1989) or H2 O2 (Mascini and Moscone, 1986; Tamiya et al., 1991; Huang et al., 1993; Ricci et al., 2003, Doretti et al., 2000) electrodes. A decrease in the reduction current of O2 or an increase in the oxidation current of H2 O2 was measured with O2 or H2 O2 amperometric sensors, respectively. An organic conducting salt, tetrathiafulvalene tetracyanoquinodimethane (TTF–TCNQ) (Xin and Wightman, 1997; Hale et al., 1991; Hale and Wightman, 1988) and Meldola Blue (Khayyami et al., 1998), were also employed as electron transfer mediator for ACh analysis in anaerobic conditions. The redox polymer polyvinylferrocenium perchlorate (PVF+ ClO4 − ) gives rise to some interesting electrochemical results when used as a layer on Pt surfaces. The preparation methods and the study of the electrochemical behavior of this redox polymer coated as a film on Pt surfaces have been the subject of several recent studies (Merz and Bard, 1978; Daum et al., 1980; Peerce and Bard, 1980a; Peerce and Bard, 1980b). This electroactive film can act as a modified surface through which an electron transfer between a substrate and a reactant can take place. Studies related to the reduction and oxidation of some reactants through this film-covered surface have also been reported (Merz and Bard, 1978; 1980; Gülce et al., 1994a; Gülce et al., 1994b; Gülce et al., 1995a). It is known that the reduced form of this redox polymer film, PVF, is a homogeneous compact film, whereas the oxidized form, PVF+ , is an inhomogeneous film (Inzelt and Bacskai, 1992). Pores or pinholes exist in PVF+ film, through which dissolved reactants could diffuse to the underlying metal surface (Peerce and Bard, 1980b).
Numerous studies have been reported concerning enzyme immobilization on various carriers for enzyme electrodes. These immobilization procedures often consist of multi-steps which generally require pretreatments of carrier and considerable times for immobilization. We have shown in an earlier study that the PVF+ ClO4 − matrix is sensitive to the anions present in the medium and some anions can be inserted into the polymer through an anion-exchange process (Gülce et al., 1995a). The interaction between the negatively charged enzyme species and the polymer matrix, ensures that the enzyme is homogeneously immobilized in the polymer matrix. In our laboratory, a mild method of general use for binding of enzymes on PVF+ ClO4 − modified electrodes has been developed and successfully applied to the design of the enzyme electrodes for glucose (Gülce et al., 1995b), sucrose (Gülce et al., 1995c), galactose (Gülce et al., 2002a), lactose (Gülce et al., 2002b), alcohols (Gülce et al., 2002c), and choline (Gülce et al., 2003). The corresponding enzymes for these amperometric enzyme electrodes were immobilized via an ion exchange process with a solution of enzyme(s), which has a pH value above the isoelectric point(s) of the corresponding enzyme(s). Various enzymes (E) could be immobilized electrostatically in this polymer, PVF+ ClO4 − + E− → PVF+ E− + ClO4 − We know quite well that enzyme molecules are immobilized through the ion exchange process. In a previous study, the amount of enzyme incorporated in the PVF+ ClO4 − coated Pt electrode was determined by following the decrease of the absorbance of the enzyme solution at 277 nm during the surface immobilization procedure. Furthermore, the amount of the enzyme immobilized within the polymer could then be determined using the same absorption peak after desorbing the enzyme in a solution that has a pH value less than the isoelectric point of the corresponding enzyme. The amount of the enzyme adsorbed onto the glass surface that is lost by washing was measured spectrophotometrically and found to be negligible (Gülce et al., 1995b). The immobilized enzyme produces electroactive H2 O2 as a result of a reaction with the substrate, E−
Substrate + O2 − → product + H2 O2 H2 O2 could be electrooxidized at the applied potential of +0.70 V versus SCE according to H2 O2 → O2 + 2H+ + 2e− The oxidation of H2 O2 could also occur chemically by the polyvinylferrocenium according to the following reaction. H2 O2 + 2PVF+ → O2 + 2PVF + 2H+ Amperometric response of the electrode was further enhanced as a result of the catalytic regeneration of PVF+ sites at the same applied potential PVF → PVF+ + e−
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H2 O2 can be oxidize both electrochemically at the applied potential and chemically by the PVF+ sites on the PVF+ coated electrodes. The role of molecular oxygen is to act as an oxidant for the regeneration of the oxidized form of the enzyme. The crucial role of O2 in this mechanism is evidenced from another independent experiment. It was found that the current value of the H2 O2 oxidation on enzyme loaded electrode was directly proportional to the concentration of dissolved O2 . It should be noted that the amperometric response of the same electrode was only 0.15% of the response of the aerobic conditions when the same experiment was carried out under anaerobic conditions, indicating the major role of the dissolved O2 in the mechanism (Gülce et al., 1995b). Such performances of this redox polymer could be used advantageously to develop amperometric enzyme electrodes (Gülce et al., 1995b; Gülce et al., 1995c; Gülce et al., 2002a; Gülce et al., 2002b; Gülce et al., 2002c; Gülce et al., 2003). In this paper, we report the use of the redox polymer PVF+ CIO4 − for the co-immobilization of AChE and ChO in order to determination of acetylcholine. The hydrogen peroxide production resulting from the sequentinal reactions of enzymes was detected amperometrically. The changes in the response of the enzyme electrode with buffer, substrate, enzyme concentrations, the film thickness, pH and temperature were measured and optimum working conditions were established. The results were compared with those obtained with free enzymes in solution using uncoated Pt electrode.
2. Experimental details 2.1. Materials Acetylcholine esterase (ACh, EC 3.1.1.7 from Electric Eel C 3389), choline oxidase (ChO, EC 1.1.3.17 from Alcaligenes species C 5896), choline chloride (C 1879), acetylcholine chloride (A 6625) were purchased from Sigma. PVF was prepared using a method of chemical polymerization (Smith et al., 1976) of vinylferrocene (Alfa products). Tetrabutylammonium perchlorate (TBAP) was prepared by the reaction of tetrabutylammonium hydroxide (Merck, 40% aqueous solution) with HClO4 (Merck), crystallized from an ethanol–water mixture (9:1) several times and kept under nitrogen atmosphere after vacuum drying at 120 ◦ C. The buffer solutions were prepared using NaH2 PO4 (Analar BDH) and NaOH (Merck). The purification of methylene chloride (Merck) was accomplished according to the method proposed in literature (Perrin and Armorego, 1980). 2.2. Preparation of the enzyme electrode PVF+ ClO4 − modified Pt surface was prepared by electrooxidizing PVF at +0.70 V versus Ag/AgCl electrode in a
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methylene chloride solution containing 0.10 M TBAP. The electroprecipitation of PVF+ ClO4 − was carried out in a dry and oxygen-free nitrogen (99.99%) atmosphere. The electrical charge passing during the eletroprecipitation was measured to prepare polymer-coated electrodes with varying thicknesses. The average thickness of the dry film was estimated from the charge, Q, passing during the electroprecipitation of the film as described by Bard (Peerce and Bard, 1980b). The enzyme electrode was prepared by immersing the PVF+ ClO4 − -coated Pt electrode in a solution of ACh and ChO. The polymer-coated electrode was kept in the enzyme solution for 30 min without stirring. The pH of the enzyme solution was kept at pH 7.4, which was well above the isoelectric points of ACh and ChO. The isoelectric points of the enzymes are between pH 4.5–5.5 for ChO (Campenella et al., 1985) and pH 5.35 for AChE (Nachmansohn, 1959). The enzymes exist in the form of anions under these conditions, facilitating its interaction with the oxidized polymer, PVF+ ClO4 − . The enzyme-attached electrode was rinsed with 0.01 M phosphate buffer solution at the working pH for 5 min to remove the remaining enzyme, which was held non-electrostatically by the surface of the polymer, and then stored in 0.01 M phosphate buffer solution (pH 7.4) at 4 ◦ C. 2.3. Measurements and apparatus The response of the enzyme electrode was determined with a jacketed electrochemical cell which kept the solution at the desired temperature. Oxygen was introduced into the solution in this cell at a constant flow rate to obtain an oxygen saturated solution. Oxygen flow was continued above the solution to keep it saturated with oxygen during the measurements. In order to determine the steady-state background current of the enzyme electrode, a potential of +0.70 V versus SCE was applied to the enzyme electrode, which was kept in buffer solution that did not contain acetylcholine. After the steady-state current value had been determined, known amounts of the acetylcholine were added to the cell from a stock acetylcholine solution and the solution was stirred for 5 s. The acetylcholine response of the enzyme electrode was measured at constant potential of +0.70 V versus SCE amperometically due to the electrooxidation of H2 O2 produced enzymatically. Electrochemical measurements were carried out in a three-electrode cell with separate compartments for the reference electrode and for the counter electrode (Pt spiral). The reference electrodes were non-aqueous Ag/AgCl in electroprecipitation and SCE for acetylcholine analysis. Pt disc electrode (2 mm diameter) was used as a working electrode. The electrochemical instrumentation consisted of PAR Model 362 Potentiostat–Galvanostat. Current–time curves were recorded on a Model 16100-II Linseis recorder.
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Fig. 1. Typical amperometric responses: (a) the acetylcholine enzyme electrode; (b) the choline enzyme electrode applying a potential step to +0.70 V vs. SCE for 0.15 mM choline and acetylcholine, respectively, in 0.10 M phosphate buffer solution (pH 7.4) at 25 ◦ C (film thickness: 1.46 × 10−6 mol cm−2 of PVF+ ).
3. Results and discussion 3.1. The response of the enzyme electrodes towards choline and acetylcholine The aim of this study is the preparation of a new enzyme electrode in order to determine the acetylcholine in the aqueous solution by means of AChE and ChO enzymes immobilized in PVF+ ClO4 − matrix on the surface of the Pt electrode. It was also prepared in an earlier study from this laboratory an enzyme electrode for the determination of choline. The choline enzyme electrode consisted of a PVF+ ClO4 − coated Pt surface onto which the choline oxidase enzyme was attached (Gülce et al., 2003). Fig. 1 shows the responses of the enzyme electrodes to acetylcholine and choline. Constant potential of +0.70 V versus SCE was applied to the enzyme electrodes to measure the amperometric response due to the electrooxidation of H2 O2 produced enzymatically. Steady-state background current was first measured at this potential with a blank buffer solution of working pH. After the steady-state background current value were reached, certain volumes of substrate solutions of known concentration were added and the currents for each added amount of substrate were recorded. The currents reached a steady-state value after the initial stirring was stopped in about 30–50 s following the addition of the substrate. These steady-state current values were used to construct the calibration plots. The response time of the enzyme electrodes developed in this study are comparable to those published in literature (Mascini and Moscone, 1986; Huang et al., 1993; Ricci et al., 2003; Doretti et al., 2000). 3.2. The effect of the enzyme concentration The amounts of the enzymes in the polymer matrix depends on the concentrations of the enzymes in solution used during immobilization process. The effect of choline
oxidase concentration on the response of the choline enzyme electrode based PVF+ ClO4 − coated-Pt electrode PVF+ ChO− was established previously (Gülce et al., 2003). It was found in this study that the maximum current was obtained if the PVF+ ChO− electrode was prepared using choline oxidase solutions that contained 7.24 mg ml−1 . By keeping the concentration of choline oxidase constant at this value and varying the acetylcholine esterase concentration in the bulk solution, the effect of the AChE concentration on the response of the acetylcholine enzyme electrode was determined. The pH, temperature and film thickness were kept constant in these experiments. The enzyme, AChE, concentrations varied between 5.0 and 10.0 mg ml−1 and the maximum activity was obtained at 7.24 mg ml−1 AChE concentration. When the enzyme, AChE, concentration was 5.0 mg ml−1 , the current of the enzyme electrode was measured as 0.65 A at 2.5 mM acetylcholine concentration. The enzyme electrode response increased to 0.85 A with the enzyme concentration up to a value corresponding to 7.24 mg ml−1 at the same substrate concentration, after which decrease of the immobilized enzyme, ChO, rather than immobilized enzyme, AChE, in the polymer matrix caused a decrease in the response. Enzyme concentrations of 7.24 mg ml−1 for ChO and 7.24 mg ml−1 for AChE were used in all of measurements. 3.3. The effect of the thickness of the polymeric film The effect of the thickness of the PVF+ ClO4 − film on the Pt electrode surface was examined. When the AChE and ChO concentrations were kept constant (7.24 mg ml−1 for ChO and 7.24 mg ml−1 for AChE), the charge that passed in the preparation of the polymer coated electrodes varied between 1.16 × 10−3 and 6.68 × 10−3 C corresponding to 3.83 × 10−7 mol cm−2 of PVF+ (dry thickness ∼0.82 m) and 2.2 × 10−6 mol cm−2 of PVF+ (dry thickness: ∼4.74 m). The enzyme electrode response
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Fig. 2. The effects of pH on the responses of the enzyme electrode and free enzymes in phosphate buffer solution at 25 ◦ C.
increased with the polymer thickness up to a value corresponding to the passage of a charge of 4.42 × 10−3 C (1.46 × 10−6 mol cm−2 of PVF+ , dry thickness: ∼3.14 m) during the electroprecipitation of the PVF+ ClO4 − . This behavior could be explained by the limited diffusion rate of substrate from the bulk solution into the inner regions of the polymer in thick film. The thickness of the PVF+ ClO4 − films were kept constant at this value for all measurements. 3.4. The effect of the pH The pH value is another variable which affects the response of the enzyme electrodes. The pH dependence of the response of the enzyme electrode was determined using a 0.10 M phosphate buffer solution, and the pH varied between 6.5 and 8.0. Fig. 2 shows that the maximum activity was obtained at pH 7.4. For each point in Fig. 2, a new enzyme electrode was prepared in order to eliminate the errors that might arise from the re-use the enzyme-loaded polymer surface. The data points are the averages of three measurements. The pH of the buffer solution was kept at pH 7.4 for all measurements. In order to compare this value with that obtained with free enzymes under the same solution conditions, the H2 O2 generated from solutions of different acetylcholine concentrations was measured electrochemically. The maximum current values obtained for the free enzymes using a bare Pt electrode were with a solution of pH 7.4 as seen in Fig. 2. The same optimum pH values of immobilized and free enzymes indicated that no structural changes in the structure of the immobilized enzyme occurred. 3.5. The effect of the temperature The temperature at which the measurements are carried out is also a critical factor in determining the activity of the enzyme electrode. The response of the acetylcholine enzyme electrode was measured at different temperatures varying from 20 to 70 ◦ C. Fig. 3 shows the results of this compar-
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Fig. 3. The effects of the temperature on the activity of the enzyme electrode and free enzymes in phosphate buffer solution (pH 7.4) at 25 ◦ C (film thickness: 1.46 × 10−6 mol cm−2 of PVF+ ).
ison. The activity of the immobilized enzymes reached a maximum value at about 55 ◦ C and remains almost constant between 55 and 65 ◦ C, after which denaturation of the enzymes caused a decrease in the response. The activation energy of this co-immobilized enzyme system was calculated to be 14.92 kJ mol−1 from the Arrhenius plot. The temperature, at which the free enzymes system gave the maximum response, and the activation energy were found to be 60 ◦ C and 76.95 kJ mol−1 , respectively (Fig. 3). The comparison of the two Ea values also confirm the fact that the immobilization of the enzymes causes no deformations in the structures of the enzymes in immobilized state. 3.6. The effect of the substrate concentrations Fig. 4(a) and (b) show the responses of the acetylcholine enzyme electrode as a function of acetylcholine concentration and a function of choline concentration, respectively. The calibration plots constructed using the optimum working conditions Fig. 4(a) and (b)) gave the linear working ranges up to a value of 1.2 mm of acetylcholine and choline concentrations. The minimum substrate concentrations that produced detectable currents were 1.0 × 10−6 M. The apparent Michaelis–Menten constant (KMapp ) for acetylcholine was calculated to be 1.74 mM according to the Linewaver–Burk graph of the Michaelis–Menten equation under the optimum conditions. The free enzymes in solution which produced a current response on an uncoated Pt electrode gave a KM value of 1.58 mM acetylcholine. KM values immobilized and free enzymes indicated no diffusional limitations in the immobilized state and the non-denaturating character of the procedure of enzyme anchoring. Fig. 5 shows the amperometric current response of the choline enzyme electrode as a function of choline concentration under the optimum conditions (Gülce et al., 2003). The upper limit of the linear working portion in calibration plot was found to be 1.2 mM choline. The data points in Figs. 4 and 5 are the averages of three measurements. Relative standard deviations were calculated equal to or <5%. The acetylcholine enzyme
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of both acetylcholine and choline in a single sample with two sensors, one selective for choline and other for acetylcholine, is possible. The current from the two biosensors can be converted into concentration signals with calibration data. The acetylcholine concentration can then be obtained by subtraction. 3.7. The interferences study
Fig. 4. Changes in the response of the acetylcholine enzyme electrode with the substrate concentration in 0.10 M phosphate buffer solution (pH 7.4) at 25 ◦ C (film thickness: 1.46 × 10−6 mol cm−2 of PVF+ ) for: (a) acetylcholine and (b) choline.
The interference signal due to the most common electrochemical intefering species was also evaluated. The signal for a fixed concentration of acetylcholine was compared with the current value obtained in the presence of the variable concentrations of the interfering species. Several interferants, such as ethanol, methanol, formaldehyde, lechitine, diethanolamine, triethanolamine and ascorbic acid were tested to study the relative response of the enzyme electrode. The relative responses obtained for most of these interferants under optimum working conditions were found to be negligible. The most important interference was caused by triethanolamine and ascorbic acid. No noticeable changes in current were detected in the concentration ranges of 0–0.16 mM triethanolamine and ascorbic acid in 1.2 mM acetylcholine solution which produced current values of 6% when acetylcholine response was taken as 100% for 0.16 mM interferants concentrations. The relative activity of the enzyme electrode was 25% of the 1.2 mM acetylcholine response when interferant concentrations were 1.5 mM. 3.8. Long-term stability of the enzyme electrode
electrode can detect not only acetylcholine signals but also choline signals while the choline enzyme electrode only responded to choline. Dynamic range and calibration sensitivity (i/C) values of acetylcholine enzyme electrode are approximately equal to acetylcholine and choline as shown in Fig. 4(a) and (b). Therefore, a simultaneous determination
Fig. 5. Changes in the response of the choline enzyme electrode with choline concentration in 0.10 M phosphate buffer solution (pH 7.4) at 25 ◦ C.
The enzyme electrode which was prepared under optimum conditions was tested at 25 ◦ C. A total of 255 measurements were made in 42 days. The enzyme electrode responses gradually decreased in the first 25 days, the activity remained constant thereafter, indicating good long term stability of the enzyme electrode. There is a noticeable decrease in the response during the first 25 days owing to the desorption of the enzyme, which is weakly held by the surface. The current response on the 30th day was 50% of the initial value (Fig. 6).
Fig. 6. The long-term stability of the acetylcholine enzyme electrode in 0.10 M phosphate buffer solution (pH 7.4) at 25 ◦ C.
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4. Conclusions It can be concluded that the redox polymer, PVF+ ClO4 − , possesses unique properties as an immobilizing medium for the enzymes and as a catalyst for H2 O2 oxidation for the development of a simple, sensitive, stable and low-cost amperometric acetylcholine enzyme electrode. The response time and the linear working range of the enzyme electrode developed in this study are better than those already proposed in the literature. The acetylcholine enzyme electrode described above offers the additional advantage of having a maximum response at physiological temperatures for in vivo applications. Furthermore, the higher sensitivity of the electrode also offers the possibility of using ultramicroelectrode designings for in vivo measurements. The electrode surface coated with PVF+ ClO4 − layer thus offers some novel application possibilities in the field of enzyme electrodes.
Acknowledgements The authors are grateful for financial support by The Scientific and Technical Research Council of Turkey (Project no: TBAG-1867) and The Administrative Units of The Research Projects of Süleyman Demirel University (Project no: SDUAF-226). References Campenella, L., Mascini, M., Palleschi, G., Tomassetti, M., 1985. Determination of choline-containing phospholipids in amniotic fluid by an enzyme sensor. Clin. Chim. Acta 151, 71–83. Campanella, L., Sammartino, M.P., Tomassetti, M., 1989. An enzyme sensor for determination of acetylcholine and choline in rat brain extracts. Anal. Lett. 22 (6), 1389–1402. Daum, P., Lenhard, J.R., Rolison, D., Murray, R.W., 1980. Diffusional charge transport through ultrathin films of radiofrequency plasma polymerized vinylferrocene at low temperature. J. Am. Chem. Soc. 102, 4649–4653. Doretti, L., Ferrara, D., Lora, S., Schiavon, F., Veronese, F.M., 2000. Acetylcholine biosensor involving entrapment of acetylcholinesterase and poly(ethylene glycol)-modified choline oxidase in a poly(vinyl alcohol) cryogel membrane. Enzyme Microb. Technol. 27, 279– 285. Gibson, K., Guilbault, G.G., 1975. Assay of cholinesterase using ion-selective electrodes. Anal. Chim. Acta 80, 245. Gilberstadt, M.L., Russell, J.A., 1984. Metabolism of acetylcholine in the nervous system of Aplysia californica. Anal. Biochem. 138, 78. Gülce, H., Özyörük, H., Yıldız, A., 1994a. Electrochemical reduction of anthracenes on poly(vinylferrocenium) coated Pt electrodes in acetonitrile. Ber. Bunsenges. Phys. Chem. 98, 228–233. Gülce, H., Özyörük, H., Yıldız, A., 1994b. Electrochemical oxidation of anthracenes on poly(vinylferrocenium) coated Pt electrodes in acetonitrile. Ber. Bunsenges. Phys. Chem. 98, 828–832. Gülce, H., Özyörük, H., Yıldız, A., 1995a. Electrochemical response of the poly(vinylferrocenium) coated Pt electrodes towards some anions in aqueous medium. Electroanalysis 7, 178–183.
1267
Gülce, H., Özyörük, H., Çelebi, S.S., Yıldız, A., 1995b. Amperometric enzyme electrode for aerobic glucose monitoring prepared by glucose oxidase immobilized in polyvinylferrocenium. J. Electroanal. Chem. 394, 63–70. Gülce, H., Özyörük, H., Çelebi, S.S., Yıldız, A., 1995c. Amperometric enzyme electrode for sucrose determination prepared from glucose oxidase and invertase co-immobilized in polyvinylferrocenium. J. Electroanal. Chem. 397, 217–223. Gülce, H., Ataman, I., Gülce, A., Co¸skun, H., Yıldız, A., 2002a. A new amperometric enzyme electrode for galactose determination. Enzyme Microb. Technol. 30, 41–44. Gülce, H., Gülce, A., Yıldız, A., 2002b. A novel two-enzyme amperometric electrode for lactose determination. Anal. Sci. 18, 1–3. Gülce, H., Gülce, A., Kavanoz, M., Co¸skun, H., Yıldız, A., 2002c. A new amperometric enzyme electrode for alcohol determination. Biosens. Bioelectron. 17, 517–521. Gülce, H., Akta¸s, Y.S., Gülce, A., Yıldız, A., 2003. Polyvinylferrocenium immobilized enzyme electrode for choline analysis. Enzyme Microb. Technnol. 32, 895. Hale, P.D., Wightman, R.M., 1988. Enzyme-modified tetrathiafulvalene tetracyanoquinodimethane microelectrodes: direct amperometric detection of acetylcholine and choline. Mol. Cryst. Liq. Cryst. 160, 269–279. Hale, P.D., Liu, L.F., Skotheim, T.A., 1991. Enzyme-modified carbon paste: tetrathiafulvalene electrodes for the determination of acetylcholine. Electroanalysis 3, 751–756. Huang, Z., Villarta Snow, R., Lubrano, G.J., Guilbault, G.G., 1993. Development of choline and acetylcholine Pt microelectrodes. Anal. Biochem. 215, 31–37. Inzelt, G., Bacskai, J., 1992. Electrochemical quartz crystal microbalance study of the swelling of polyvinylferrocene films. Electrochim. Acta 37, 647–654. Khayyami, M., Pita, M.T.P., Garcia, N.P., Johansson, G., Danielsson, B., Larsson, P.O., 1998. Development of an amperometric biosensor based on acetylcholine esterase covalently bound to a new support material. Talanta 45, 557–563. Larsson, N., Ruzgas, T., Gorton, L., Kokaia, M., Csöregi, E., 1998. Design and development of an amperometric biosensor for acetylcholine determination in brain microdialysates. Electrochim. Acta 43, 3541– 3554. Mascini, M., Moscone, D., 1986. Amperometric acetylcholine and choline sensors with immobilized enzymes. Anal. Chim. Acta 179, 439– 444. Merz, A., Bard, A.J., 1978. A stable surface modified platinum electrode prepared by coating with electroactive polymer. J. Am. Chem. Soc. 100, 3222–3223. Nachmansohn, D., 1959. Chemical and Molecular Basis of Nerve Activity. Academic Press, NY. Peerce, P.J., Bard, A.J., 1980a. Polymer films on electrodes. Part III. Digital simulation model for cyclic voltammetry of electroactive polymer film and electrochemistry of poly(vinylferrocene) on platinum. J. Electroanal. Chem. 114, 89–115. Peerce, P.J., Bard, A.J., 1980b. Polymer films on electrodes part film structure and mechanism of electron transfer with electrodeposited polyvinylferrocene. J. Electroanal. Chem. 112, 97–115. Perrin, D.D., Armorego, W.L.F., 1980. Purification of Laboratory Chemicals. Pergamon Press, Oxford, p. 265. Potter, P.E., Meek, J.L., Neff, N.H., 1983. Acetylcholine and choline in neuronal tissue measured by HPLC with electrochemical detection. J. Neurochem. 41, 188. Ricci, F., Amine, A., Palleschi, g., Moscone, D., 2003. Prussian Blue based screen printed biosensors with improved characteristics of long-term lifetime and pH stability. Biosens. Bioelectron. 18, 165–174. Ricny, J., Coupek, J., Tucek, S., 1989. Determination of acetylcholine and choline by flow-injection with immobilized enzymes and fluometric or luminometric detection. Anal. Biochem. 76, 221– 227.
1268
S. S¸ en et al. / Biosensors and Bioelectronics 19 (2004) 1261–1268
Smith, T.W., Kuder, J.E., Wychick, D., 1976. Voltammetric behavior of polyvinylferrocene. J. Polym. Sci. 14, 2433–2448. Tamiya, E., Sugiura, Y., Navera, E.N., 1991. Ultramicro acetylcholine sensor based on an enzyme-modified carbon fibre electrode. Anal. Chim. Acta 341, 43–51.
Xin, Q., Wightman, R.M., 1997. Enzyme modified amperometric sensors for choline and acetylcholine with tetrathiafulvalene tetracyanoquinodimethane as the electron-transfer mediator. Anal. Chim. Acta 341, 43–51.