- Email: [email protected]
Amperometric enzyme electrode for l (+)-lactate determination using immobilized l (+)-lactate oxidase in poly(vinylferrocenium) film
Sensors and Actuators B 87 (2002) 8–12
Amperometric enzyme electrode for L(þ)-lactate determination using immobilized L(þ)-lactate oxidase in poly(vinylferrocenium) film ¨ zyo¨ru¨ka, Attila Yıldıza,* Gu¨lsev Aydına, Serdar S. C¸elebib, Haluk O a
Department of Chemistry, Hacettepe University, Beytepe, Ankara 06532, Turkey Department of Chemical Engineering, Hacettepe University, Beytepe, Ankara 06532, Turkey
b
Received 8 October 2001; received in revised form 10 March 2002; accepted 6 April 2002
Abstract The steady-state amperometric measurements of L(þ)-lactate was performed by an enzyme electrode which was developed by using immobilization of the L(þ)-lactate oxidase (LOx) in poly(vinylferrocenium) (PVFþ), film coated on a Pt electrode. The effects of temperature, pH, polymer film thickness and enzyme concentration were investigated. The linear concentration range was found to be between 0.05 and 0.60 mM for this enzyme electrode. The maximum current value obtained was higher than those of other studies. The enzyme electrode was found to be stable for about 15 days using the current values measured daily for the same substrate concentration. # 2002 Elsevier Science B.V. All rights reserved. Keywords: L(þ)-Lactate; Poly(vinylferrocenium); Horseradishperoxidase
1. Introduction L(þ)-Lactate is produced in tissues when oxygen is not available. Monitoring of the blood lactate level is an indicator of shock, respiratory insufficiency and heart diseases and of interest in sport medicine. Determination of L(þ)lactate is also important in some food and bioprocess analyses. Spectrophotometric [1] and amperometric methods [2] are based on the following reaction: LOx
lðþÞ-lactate þ O2 ! Pyruvate þ H2 O2 In amperometric methods either a hydrogen peroxide sensor which detects the increase in the H2O2 oxidation current or an oxygen sensor which measures the decrease in the O2 reduction current could be utilized. L(þ)-Lactate oxidase (LOx) which was cast on a platinized polyanion-doped polypyrrole film and crosslinked with glutaraldehyde was used as a sensor system and H2O2 oxidation current was measured [3]. LOx and horseradishperoxidase (HRP) were coimmobilized on a carbon paste electrode and the amount of the enzymatically produced H2O2 was monitored [4,5]. HRP allowed more sensitive determination of H2O2 at low applied potentials. Simultaneous determination of D() and L(þ)-lactate was accomplished by two enzyme modified carbon paste elec*
Corresponding author.
trodes, one using immobilized D()-lactatedehydrogenase (LDH) with a redox mediator, o-phenylenediamine and a cofactor, NADH, and the other one using coimmobilized LOx and HRP [6]. Oxygen consumption was monitored by an amperometric oxygen sensor in a study in which LOx and LDH were coimmobilized on a membrane tightly bound to an oxygen electrode [7]. LDH caused the catalytic regeneration of the lactate in the presence of NADH according to the following reaction: LDH
Pyruvate þ NADH þ Hþ ! lðþÞ-lactate þ NADþ Catalytic regeneration of L(þ)-lactate by LDH, increased the sensitivity of the sensor. Poly(vinylferrocenium) (PVFþ) modified Pt surface was used in previous studies as an immobilizing medium for several enzymes and enzyme electrodes for several substrates such as glucose, sucrose, and peroxides were developed [8–11]. The enzymes were immobilized in this redox polymer via anion-exchange process with a solution of corresponding enzyme(s), which has a pH value above the isoelectric point(s) of the enzyme(s). In the biosensors, PVFþ matrix acts also as a chemical oxidant for the enzymatically produced H2O2. The reduced form of the polymer (polyvinylferrocene, PVF) gets oxidized at the applied potential, catalytically regenerating the oxidized form of the polymer (PVFþ), producing a current response which is more intense than that of electroxidation
0925-4005/02/$ – see front matter # 2002 Elsevier Science B.V. All rights reserved. PII: S 0 9 2 5 - 4 0 0 5 ( 0 2 ) 0 0 1 6 7 - 3
G. Aydın et al. / Sensors and Actuators B 87 (2002) 8–12
of H2O2. Despite the great number of published and commercialized L(þ)-lactate electrodes, there is still a great necessity to improve the sensitivity and the long-term stability. In this study, the use of the PVFþ matrix as an immobilization medium of LOx for the determination of L(þ)-lactate is described. The optimum working conditions of this new amperometric sensor are discussed.
2. Experimental PVFþC1O4 modified Pt surface was prepared by electrooxidizing PVF at þ0.70 V versus Ag/AgCl electrode in a methylene chloride solution containing 0.1 M tetrabutylammonium perchlorate (TBAP). PVF was obtained using a method of chemical polymerization [12] of vinylferrocene (Alfa Products). The electroprecipitation of PVFþClO4 was carried out under nitrogen atmosphere. The purification of methylene chloride was accomplished according to the method proposed in literature in order to reduce the background current value at the applied potential for better sensitivity [13]. TBAP was prepared by the reaction of tetrabuthylammonium hydroxide (40% aqueous solution) (Merck) with HClO4 (BDH), crystallized from an ethyl alcohol–water mixture (9:1) several times under nitrogen atmosphere and vacuum dried at 120 8C. The buffer solutions were prepared using NaH2PO4 and Na2HPO4 (Merck) and NaOH (Merck) was used for pH adjustment. L(þ)-lactate (Sigma) solution was prepared in a 0.1 M buffer solution of pH ¼ 7:0. Enzyme stock solution was prepared by dissolving 1.5 mg L(þ)-lactate oxidase (Sigma, Cat. No: L0638) per milliliters of 0.1 M phosphate buffer solution of pH ¼ 7:0. LOx was incorporated into the polymer matrix by immersing PVFþC1O4 coated Pt electrode in enzyme solution for 90 min with stirring according to the following ion exchange process:
9
was then rinsed with 0.01 M phosphate buffer solution at working pH to remove the excess enzyme which was not immobilized electrostatically. The immobilized amount of the enzyme was determined by desorbing the enzyme into the blank solution at pH values below the isoelectric point. The activity of the enzyme electrode was determined in electrochemical cell with the jacket, which kept the solution at a desired temperature. Oxygen was supplied into the solution at a constant flow rate to obtain an oxygen saturated solution. Oxygen flow was continued above the solution to keep it at saturated condition during the measurements. Constant potential of þ0.70 V versus SCE was applied to enzyme electrode to measure the amperometric response due to the electrooxidation of H2O2 produced enzymatically. Steady-state back-ground current value was first measured at this potential with a blank buffer solution at working pH. After the steady-state back-ground current value was reached certain volumes of L(þ)-lactate solution of known concentration were added and the currents for each added amount of substrate were recorded. Electrochemical measurements of aqueous and nonaqueous solutions were carried out in a three-electrode cell. SCE was used as reference electrode for L(þ)-lactate determination. A Pt wire electrode (A ¼ 3:6 103 cm2) was used as the working electrode. A Pt spiral electrode was used as counter electrode in all electrochemical measurements. The reference electrode was Ag/AgCl during polymer electrooxidation in methylene chloride. The electrochemical instrumentation consisted of PAR Model 173 potentiostat–galvonastat equipped with PAR Model 175 universal programmer and Par Model 179 digical coulometer. Current curves were recorded on a Cole–Palmer X-t recorder. UV measurements were performed with a Unicam UV–VIS spectrophotometer.
PVFþ ClO4 þ LOx ! PVFþ LOx þ ClO4
3. Results and discussion
The enzyme is held electrostatically in the polymeric structure above the isoelectric point of pH 4.6. The enzyme electrode
During the preparation of PVFþC1O4 film on the Pt electrode, the amount of charge was controlled in order to
Fig. 1. The response of the L(þ)-lactate oxidase electrode as a function of film thickness (0.1 M buffer concentration, 1.26 mM L(þ)-lactate concentration, pH ¼ 7:0, 37 8C).
10
G. Aydın et al. / Sensors and Actuators B 87 (2002) 8–12
Fig. 2. The current–time responses of the L(þ)-lactate oxidase electrode to the additions of L(þ)-lactate solutions (0.1 M buffer concentration, pH ¼ 7:0, 37 8C, 30 mC film thickness).
obtain modified surfaces at different thicknesses. The activity of the enzyme electrode increased with the film thickness and remained constant after a certain value, which corresponded to charge of 30 mC (Fig. 1). This film thickness was used as an optimum value. The amount of the immobilized enzyme in the polymer was found to be 0.84 mg protein using UV spectrophotometer. Fig. 2 shows the current time responses of the enzyme electrode for different amounts of added L(þ)-lactate. The steady-state current value was reached within about 1 min after the stirring was stopped following each addition. The measured steady-state current values were used to construct the calibration plot (Fig. 3). Linear concentration range was found to be between 0.05 and 0.60 mM L(þ)lactate concentration. The response reached a saturation value of 13 mA at the concentration of 1.26 mM. Apparent Michealis–Menten constant, Km, for the immobilized enzyme was obtained from Lineweaver–Burke plot
and determined to be 1.38 mM L(þ)-lactate for the optimum thickness which corresponds to 30 mC. This value was found to increase proportionally as the thickness of the PVFþC1O4 film increased. The Km values were 0.17, 0.53 and 1.57 mM L(þ)-lactate for the film thicknesses which correspond to 15, 20 and 40 mC, respectively. Diffusion limitations in the polymer film become apparently increasingly significant expectedly, as the film gets thicker. It must be pointed out that the Km value of the free enzyme found from the Lineweaver–Burke plot obtained with the uncoated Pt electrode in the same buffer solution was 0.73 mM L(þ)-lactate. This value is about the same magnitude as that for the immobilized enzyme indicating that no major conformational changes occur in the structure of the enzyme as a result of immobilization. It is interesting to note that in thin films the diffusional limitations are minimal and the Km values of the immobilized enzyme are even smaller than that of the free enzyme, reflecting the effect of the
Fig. 3. The response of the enzyme electrode at different L(þ)-lactate concentrations (0.1 M buffer concentration, pH ¼ 7:0, 37 8C, 30 mC film thickness).
G. Aydın et al. / Sensors and Actuators B 87 (2002) 8–12
11
Fig. 4. The response of the enzyme electrode at different pHs of the buffer solution used (0.1 M buffer concentration, 1.26 mM L(þ)-lactate concentration, 37 8C, 30 mC film thickness).
catalytic process that takes place within the film. With thicker films the diffusion limitations become increasingly more important and the Km value starts to exceed that of the free enzyme. The response of the free and the immobilized enzymes at different temperatures were also investigated and found to reach a maximum value at about 37 8C in both cases. The denaturation of the enzyme becomes significant after this temperature. The measurements were carried out with a substrate concentration of 1.87 mM at which the responses of the electrodes were independent of the concentration of the substrate. The activation energy values obtained for the immobilized and free enzymes were 14.0 and 10.1 kcal/mol, respectively, again indicating that the immobilization caused no major changes in the structure of the enzyme. pH value is another important parameter on the response of the enzyme electrodes. The current values increased up to
pH value of 7.0 and decreased thereafter (Fig. 4). For this reason, the pH of the solutions was kept at 7.0 for all other measurements. pH 7.0 is also known to be the optimum pH for the activity of free LOx. The amount of the enzyme present in solution during the immobilization process also affects the current response. The response did not change significantly after about 0.375 mg protein/ml, when the duration of immobilization process was kept as 90 min. The preparation of the enzyme electrodes was carried out under these optimum conditions in all cases. The functional stability of the enzyme electrode was tested daily under the optimum operating conditions. The
response of the electrode showed the maximum value within few days and then decreased to about 60% of its initial value after 15 days.
4. Conclusions LOx electrode whose response characteristics described above offer a new possibility to measure the amount of L(þ)lactate. It is simple to prepare inexpensive and reliable. Its response time is comparable to those published in literature. Improved sensitivity of mA level stems from the catalytic nature of processes, which take place in the redox polymer, which also acts as an immobilization medium. The proposed mechanism for the operation of the lactate oxidase electrode in this study can be summarized as follows.
In this mechanism the first step involves the binding and oxidation of L-lactate by the enzyme, producing the reduced form of the enzyme and pyruvate. O2 then oxidizes the reduced form of the enzyme to produce electroactive H2O2. H2O2 can be oxidized electrochemically at the applied potential of 0.7 V versus SCE (8–10): H2 O2 ! 2Hþ þ O2 þ 2e H2O2 can also be oxidized chemically with PVFþ producing PVF and O2: PVFþ þ H2 O2 ! PVF þ 2Hþ þ O2
12
G. Aydın et al. / Sensors and Actuators B 87 (2002) 8–12
At the applied potential of the experiment PVF gets also electrooxidized to PVFþ: PVF ! PVFþ þ e Completing the catalytic cycle and thus, enhancing the current measured. References [1] A.N. Araujo, J.L.F.C. Lima, M.L.M.F.S. Saravia, E.A.G. Zagatto, A new approach to dialysis in sequential injection systems: spectrophotometric determination of L(þ)-lactate in wines, Am. J. Enol. Vitic. 48 (1997) 428–432. [2] M. Mascini, Analytical Uses of Immobilized Biological Compounds for Detection: Medical and Industrial Uses, D. Reidel Publishing Company, Dordrech, 1998, pp. 153–167. [3] G.F. Khan, W. Wernet, Design of enzyme electrodes for extended use and storage life, Anal. Chem. 67 (1997) 2682–2687. [4] U. Spohn, D. Narasaiah, L. Gorton, D. Pfeifer, A bienzyme modified carbon paste electrode for the amperometric detection of L-lactate at low potentials, Anal. Chim. Acta 319 (1996) 79–90. [5] U. Spohn, D. Narasaiah, L. Gorton, The influence of the carbon paste composition on the performance of an amperometric bienzyme sensor for L-lactate, Electroanalysis 8 (1996) 507–514.
[6] D. Narasaiah, U. Spohn, L. Gorton, Simultaneous determination of Land D-lactate by enzyme modified carbon paste electrodes, Anal. Lett. 29 (2) (1996) 181–201. [7] V. Casmiri, C. Burstein, Co-immobilized L-lactate oxidase and L-lactate dehydrogenase on a film mounted on oxygen electrode for higly sensitive L-lactate determination, Biosens. Biochem. 11 (1996) 783–789. ¨ zyo¨ ru¨ k, S.S. C¸elebi, A. Yıldız, Amperometric enzyme [8] H. Gu¨ lce, H. O electrode for aerobic glucose monitoring prepared by glucose oxidase immobilized in poly(vinylferrocenium), J. Electroanal. Chem. 394 (1995) 63–70. ¨ zyo¨ ru¨ k, A. Yildiz, Amperometric enzyme [9] H. Gu¨ lce, S.S. C¸elebi, H. O electrode for sucrose determination prepared from glucose oxidase and invertase co-immobilized in poly(vinylferrocenium), J. Electroanal. Chem. 397 (1995) 217 –223. ¨ zyo¨ ru¨ k, S.S. C¸elebi, A. Yıldız, Ampero[10] M. Gu¨ ndogˇ an-Paul, H. O metric enzyme electrode for hydrogen peroxide determination prepared with horseradish peroxidase immobilized in polyvinylferrocenium (PVFþ), Electroanalysis 14 (2001) 505–511. ¨ zyo¨ ru¨ k, A. Yıldız, Ampero[11] M. Gu¨ ndogˇ an-Paul, S.S. C¸elebi, H. O metric enzyme electrode for organic peroxides determination prepared from horseradish peroxidase immobilized in poly(vinylferrocenium) film, Biosens. Bioelectron. (2001) in press. [12] B.C. Aso, T. Kunitake, T. Nekashima, Cationic polymerization and copolymerization of vinylferrocene, Macromol. Chem. 124 (1969) 232–240. [13] D.D. Perrin, W.L.F. Armarego, Purification of Laboratory Chemicals, Pergamon Press, Oxford, 1980, p. 265.
Amperometric enzyme electrode for L(þ)-lactate determination using immobilized L(þ)-lactate oxidase in poly(vinylferrocenium) film ¨ zyo¨ru¨ka, Attila Yıldıza,* Gu¨lsev Aydına, Serdar S. C¸elebib, Haluk O a
Department of Chemistry, Hacettepe University, Beytepe, Ankara 06532, Turkey Department of Chemical Engineering, Hacettepe University, Beytepe, Ankara 06532, Turkey
b
Received 8 October 2001; received in revised form 10 March 2002; accepted 6 April 2002
Abstract The steady-state amperometric measurements of L(þ)-lactate was performed by an enzyme electrode which was developed by using immobilization of the L(þ)-lactate oxidase (LOx) in poly(vinylferrocenium) (PVFþ), film coated on a Pt electrode. The effects of temperature, pH, polymer film thickness and enzyme concentration were investigated. The linear concentration range was found to be between 0.05 and 0.60 mM for this enzyme electrode. The maximum current value obtained was higher than those of other studies. The enzyme electrode was found to be stable for about 15 days using the current values measured daily for the same substrate concentration. # 2002 Elsevier Science B.V. All rights reserved. Keywords: L(þ)-Lactate; Poly(vinylferrocenium); Horseradishperoxidase
1. Introduction L(þ)-Lactate is produced in tissues when oxygen is not available. Monitoring of the blood lactate level is an indicator of shock, respiratory insufficiency and heart diseases and of interest in sport medicine. Determination of L(þ)lactate is also important in some food and bioprocess analyses. Spectrophotometric [1] and amperometric methods [2] are based on the following reaction: LOx
lðþÞ-lactate þ O2 ! Pyruvate þ H2 O2 In amperometric methods either a hydrogen peroxide sensor which detects the increase in the H2O2 oxidation current or an oxygen sensor which measures the decrease in the O2 reduction current could be utilized. L(þ)-Lactate oxidase (LOx) which was cast on a platinized polyanion-doped polypyrrole film and crosslinked with glutaraldehyde was used as a sensor system and H2O2 oxidation current was measured [3]. LOx and horseradishperoxidase (HRP) were coimmobilized on a carbon paste electrode and the amount of the enzymatically produced H2O2 was monitored [4,5]. HRP allowed more sensitive determination of H2O2 at low applied potentials. Simultaneous determination of D() and L(þ)-lactate was accomplished by two enzyme modified carbon paste elec*
Corresponding author.
trodes, one using immobilized D()-lactatedehydrogenase (LDH) with a redox mediator, o-phenylenediamine and a cofactor, NADH, and the other one using coimmobilized LOx and HRP [6]. Oxygen consumption was monitored by an amperometric oxygen sensor in a study in which LOx and LDH were coimmobilized on a membrane tightly bound to an oxygen electrode [7]. LDH caused the catalytic regeneration of the lactate in the presence of NADH according to the following reaction: LDH
Pyruvate þ NADH þ Hþ ! lðþÞ-lactate þ NADþ Catalytic regeneration of L(þ)-lactate by LDH, increased the sensitivity of the sensor. Poly(vinylferrocenium) (PVFþ) modified Pt surface was used in previous studies as an immobilizing medium for several enzymes and enzyme electrodes for several substrates such as glucose, sucrose, and peroxides were developed [8–11]. The enzymes were immobilized in this redox polymer via anion-exchange process with a solution of corresponding enzyme(s), which has a pH value above the isoelectric point(s) of the enzyme(s). In the biosensors, PVFþ matrix acts also as a chemical oxidant for the enzymatically produced H2O2. The reduced form of the polymer (polyvinylferrocene, PVF) gets oxidized at the applied potential, catalytically regenerating the oxidized form of the polymer (PVFþ), producing a current response which is more intense than that of electroxidation
0925-4005/02/$ – see front matter # 2002 Elsevier Science B.V. All rights reserved. PII: S 0 9 2 5 - 4 0 0 5 ( 0 2 ) 0 0 1 6 7 - 3
G. Aydın et al. / Sensors and Actuators B 87 (2002) 8–12
of H2O2. Despite the great number of published and commercialized L(þ)-lactate electrodes, there is still a great necessity to improve the sensitivity and the long-term stability. In this study, the use of the PVFþ matrix as an immobilization medium of LOx for the determination of L(þ)-lactate is described. The optimum working conditions of this new amperometric sensor are discussed.
2. Experimental PVFþC1O4 modified Pt surface was prepared by electrooxidizing PVF at þ0.70 V versus Ag/AgCl electrode in a methylene chloride solution containing 0.1 M tetrabutylammonium perchlorate (TBAP). PVF was obtained using a method of chemical polymerization [12] of vinylferrocene (Alfa Products). The electroprecipitation of PVFþClO4 was carried out under nitrogen atmosphere. The purification of methylene chloride was accomplished according to the method proposed in literature in order to reduce the background current value at the applied potential for better sensitivity [13]. TBAP was prepared by the reaction of tetrabuthylammonium hydroxide (40% aqueous solution) (Merck) with HClO4 (BDH), crystallized from an ethyl alcohol–water mixture (9:1) several times under nitrogen atmosphere and vacuum dried at 120 8C. The buffer solutions were prepared using NaH2PO4 and Na2HPO4 (Merck) and NaOH (Merck) was used for pH adjustment. L(þ)-lactate (Sigma) solution was prepared in a 0.1 M buffer solution of pH ¼ 7:0. Enzyme stock solution was prepared by dissolving 1.5 mg L(þ)-lactate oxidase (Sigma, Cat. No: L0638) per milliliters of 0.1 M phosphate buffer solution of pH ¼ 7:0. LOx was incorporated into the polymer matrix by immersing PVFþC1O4 coated Pt electrode in enzyme solution for 90 min with stirring according to the following ion exchange process:
9
was then rinsed with 0.01 M phosphate buffer solution at working pH to remove the excess enzyme which was not immobilized electrostatically. The immobilized amount of the enzyme was determined by desorbing the enzyme into the blank solution at pH values below the isoelectric point. The activity of the enzyme electrode was determined in electrochemical cell with the jacket, which kept the solution at a desired temperature. Oxygen was supplied into the solution at a constant flow rate to obtain an oxygen saturated solution. Oxygen flow was continued above the solution to keep it at saturated condition during the measurements. Constant potential of þ0.70 V versus SCE was applied to enzyme electrode to measure the amperometric response due to the electrooxidation of H2O2 produced enzymatically. Steady-state back-ground current value was first measured at this potential with a blank buffer solution at working pH. After the steady-state back-ground current value was reached certain volumes of L(þ)-lactate solution of known concentration were added and the currents for each added amount of substrate were recorded. Electrochemical measurements of aqueous and nonaqueous solutions were carried out in a three-electrode cell. SCE was used as reference electrode for L(þ)-lactate determination. A Pt wire electrode (A ¼ 3:6 103 cm2) was used as the working electrode. A Pt spiral electrode was used as counter electrode in all electrochemical measurements. The reference electrode was Ag/AgCl during polymer electrooxidation in methylene chloride. The electrochemical instrumentation consisted of PAR Model 173 potentiostat–galvonastat equipped with PAR Model 175 universal programmer and Par Model 179 digical coulometer. Current curves were recorded on a Cole–Palmer X-t recorder. UV measurements were performed with a Unicam UV–VIS spectrophotometer.
PVFþ ClO4 þ LOx ! PVFþ LOx þ ClO4
3. Results and discussion
The enzyme is held electrostatically in the polymeric structure above the isoelectric point of pH 4.6. The enzyme electrode
During the preparation of PVFþC1O4 film on the Pt electrode, the amount of charge was controlled in order to
Fig. 1. The response of the L(þ)-lactate oxidase electrode as a function of film thickness (0.1 M buffer concentration, 1.26 mM L(þ)-lactate concentration, pH ¼ 7:0, 37 8C).
10
G. Aydın et al. / Sensors and Actuators B 87 (2002) 8–12
Fig. 2. The current–time responses of the L(þ)-lactate oxidase electrode to the additions of L(þ)-lactate solutions (0.1 M buffer concentration, pH ¼ 7:0, 37 8C, 30 mC film thickness).
obtain modified surfaces at different thicknesses. The activity of the enzyme electrode increased with the film thickness and remained constant after a certain value, which corresponded to charge of 30 mC (Fig. 1). This film thickness was used as an optimum value. The amount of the immobilized enzyme in the polymer was found to be 0.84 mg protein using UV spectrophotometer. Fig. 2 shows the current time responses of the enzyme electrode for different amounts of added L(þ)-lactate. The steady-state current value was reached within about 1 min after the stirring was stopped following each addition. The measured steady-state current values were used to construct the calibration plot (Fig. 3). Linear concentration range was found to be between 0.05 and 0.60 mM L(þ)lactate concentration. The response reached a saturation value of 13 mA at the concentration of 1.26 mM. Apparent Michealis–Menten constant, Km, for the immobilized enzyme was obtained from Lineweaver–Burke plot
and determined to be 1.38 mM L(þ)-lactate for the optimum thickness which corresponds to 30 mC. This value was found to increase proportionally as the thickness of the PVFþC1O4 film increased. The Km values were 0.17, 0.53 and 1.57 mM L(þ)-lactate for the film thicknesses which correspond to 15, 20 and 40 mC, respectively. Diffusion limitations in the polymer film become apparently increasingly significant expectedly, as the film gets thicker. It must be pointed out that the Km value of the free enzyme found from the Lineweaver–Burke plot obtained with the uncoated Pt electrode in the same buffer solution was 0.73 mM L(þ)-lactate. This value is about the same magnitude as that for the immobilized enzyme indicating that no major conformational changes occur in the structure of the enzyme as a result of immobilization. It is interesting to note that in thin films the diffusional limitations are minimal and the Km values of the immobilized enzyme are even smaller than that of the free enzyme, reflecting the effect of the
Fig. 3. The response of the enzyme electrode at different L(þ)-lactate concentrations (0.1 M buffer concentration, pH ¼ 7:0, 37 8C, 30 mC film thickness).
G. Aydın et al. / Sensors and Actuators B 87 (2002) 8–12
11
Fig. 4. The response of the enzyme electrode at different pHs of the buffer solution used (0.1 M buffer concentration, 1.26 mM L(þ)-lactate concentration, 37 8C, 30 mC film thickness).
catalytic process that takes place within the film. With thicker films the diffusion limitations become increasingly more important and the Km value starts to exceed that of the free enzyme. The response of the free and the immobilized enzymes at different temperatures were also investigated and found to reach a maximum value at about 37 8C in both cases. The denaturation of the enzyme becomes significant after this temperature. The measurements were carried out with a substrate concentration of 1.87 mM at which the responses of the electrodes were independent of the concentration of the substrate. The activation energy values obtained for the immobilized and free enzymes were 14.0 and 10.1 kcal/mol, respectively, again indicating that the immobilization caused no major changes in the structure of the enzyme. pH value is another important parameter on the response of the enzyme electrodes. The current values increased up to
pH value of 7.0 and decreased thereafter (Fig. 4). For this reason, the pH of the solutions was kept at 7.0 for all other measurements. pH 7.0 is also known to be the optimum pH for the activity of free LOx. The amount of the enzyme present in solution during the immobilization process also affects the current response. The response did not change significantly after about 0.375 mg protein/ml, when the duration of immobilization process was kept as 90 min. The preparation of the enzyme electrodes was carried out under these optimum conditions in all cases. The functional stability of the enzyme electrode was tested daily under the optimum operating conditions. The
response of the electrode showed the maximum value within few days and then decreased to about 60% of its initial value after 15 days.
4. Conclusions LOx electrode whose response characteristics described above offer a new possibility to measure the amount of L(þ)lactate. It is simple to prepare inexpensive and reliable. Its response time is comparable to those published in literature. Improved sensitivity of mA level stems from the catalytic nature of processes, which take place in the redox polymer, which also acts as an immobilization medium. The proposed mechanism for the operation of the lactate oxidase electrode in this study can be summarized as follows.
In this mechanism the first step involves the binding and oxidation of L-lactate by the enzyme, producing the reduced form of the enzyme and pyruvate. O2 then oxidizes the reduced form of the enzyme to produce electroactive H2O2. H2O2 can be oxidized electrochemically at the applied potential of 0.7 V versus SCE (8–10): H2 O2 ! 2Hþ þ O2 þ 2e H2O2 can also be oxidized chemically with PVFþ producing PVF and O2: PVFþ þ H2 O2 ! PVF þ 2Hþ þ O2
12
G. Aydın et al. / Sensors and Actuators B 87 (2002) 8–12
At the applied potential of the experiment PVF gets also electrooxidized to PVFþ: PVF ! PVFþ þ e Completing the catalytic cycle and thus, enhancing the current measured. References [1] A.N. Araujo, J.L.F.C. Lima, M.L.M.F.S. Saravia, E.A.G. Zagatto, A new approach to dialysis in sequential injection systems: spectrophotometric determination of L(þ)-lactate in wines, Am. J. Enol. Vitic. 48 (1997) 428–432. [2] M. Mascini, Analytical Uses of Immobilized Biological Compounds for Detection: Medical and Industrial Uses, D. Reidel Publishing Company, Dordrech, 1998, pp. 153–167. [3] G.F. Khan, W. Wernet, Design of enzyme electrodes for extended use and storage life, Anal. Chem. 67 (1997) 2682–2687. [4] U. Spohn, D. Narasaiah, L. Gorton, D. Pfeifer, A bienzyme modified carbon paste electrode for the amperometric detection of L-lactate at low potentials, Anal. Chim. Acta 319 (1996) 79–90. [5] U. Spohn, D. Narasaiah, L. Gorton, The influence of the carbon paste composition on the performance of an amperometric bienzyme sensor for L-lactate, Electroanalysis 8 (1996) 507–514.
[6] D. Narasaiah, U. Spohn, L. Gorton, Simultaneous determination of Land D-lactate by enzyme modified carbon paste electrodes, Anal. Lett. 29 (2) (1996) 181–201. [7] V. Casmiri, C. Burstein, Co-immobilized L-lactate oxidase and L-lactate dehydrogenase on a film mounted on oxygen electrode for higly sensitive L-lactate determination, Biosens. Biochem. 11 (1996) 783–789. ¨ zyo¨ ru¨ k, S.S. C¸elebi, A. Yıldız, Amperometric enzyme [8] H. Gu¨ lce, H. O electrode for aerobic glucose monitoring prepared by glucose oxidase immobilized in poly(vinylferrocenium), J. Electroanal. Chem. 394 (1995) 63–70. ¨ zyo¨ ru¨ k, A. Yildiz, Amperometric enzyme [9] H. Gu¨ lce, S.S. C¸elebi, H. O electrode for sucrose determination prepared from glucose oxidase and invertase co-immobilized in poly(vinylferrocenium), J. Electroanal. Chem. 397 (1995) 217 –223. ¨ zyo¨ ru¨ k, S.S. C¸elebi, A. Yıldız, Ampero[10] M. Gu¨ ndogˇ an-Paul, H. O metric enzyme electrode for hydrogen peroxide determination prepared with horseradish peroxidase immobilized in polyvinylferrocenium (PVFþ), Electroanalysis 14 (2001) 505–511. ¨ zyo¨ ru¨ k, A. Yıldız, Ampero[11] M. Gu¨ ndogˇ an-Paul, S.S. C¸elebi, H. O metric enzyme electrode for organic peroxides determination prepared from horseradish peroxidase immobilized in poly(vinylferrocenium) film, Biosens. Bioelectron. (2001) in press. [12] B.C. Aso, T. Kunitake, T. Nekashima, Cationic polymerization and copolymerization of vinylferrocene, Macromol. Chem. 124 (1969) 232–240. [13] D.D. Perrin, W.L.F. Armarego, Purification of Laboratory Chemicals, Pergamon Press, Oxford, 1980, p. 265.