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Construction of an amperometric pyruvate oxidase enzyme electrode for determination of pyruvate and phosphate
Electrochimica Acta 52 (2007) 7972–7977
Construction of an amperometric pyruvate oxidase enzyme electrode for determination of pyruvate and phosphate Erol Akyilmaz ∗ , Emine Yorganci ˙ Department of Biochemistry, Faculty of Science, Ege University, 35100 Bornova-Izmir, Turkey Received 12 April 2007; received in revised form 21 June 2007; accepted 21 June 2007 Available online 27 June 2007
Abstract In this study an amperometric biosensor based on pyruvate oxidase was developed for the determination of pyruvate and phosphate. For construction of the biosensor pyruvate oxidase was immobilized with gelatin and insolubilized in film by forming cross-linked bonds with glutaraldehyde. The film was fixed on a YSI type dissolved oxygen (DO) probe, covered with a teflon membrane which is high-sensitive for oxygen. The working principle of the biosensor depends on detection of consumed DO concentration related to pyruvate concentration which is used in enzymatic reaction catalyzed by pyruvate oxidase. The biosensor response shows a linearity with pyruvate concentration between 0.0025 and 0.05 M and also response time of the biosensor is 3 min. In the optimization studies of the biosensor the most suitable enzyme activity was found as 2.5 U/cm2 for pyruvate oxidase, and also phosphate buffer (pH 7.0; 50 mM) and 35 ◦ C were established as providing the optimum working conditions. In the characterization studies of the biosensor some parameters such as reproducibility, substrate specificity, operational stability, determination of phosphate, and interference effects of some compounds on the pyruvate determination were investigated. Finally, the concentration of pyruvate was determined by using spectrophotometric method and the results obtained were compared to results obtained by the biosensor. © 2007 Elsevier Ltd. All rights reserved. Keywords: Pyruvate; Biosensor; Phosphate; Pyruvate oxidase; Enzyme electrode
1. Introduction Pyruvate is a key intermediate in the glycolytic and pyruvate dehydrogenase pathways, which are involved in biological energy production. It is widely found in living organisms and it is not an essential nutrient since it can be synthesized in the cells of the body. It may also have cardiac and skeletal muscle inotropic activity as well as bariatric activity, and also antioxidant activity [1]. Pyruvate may help some obese individuals lose weight. There is also the suggestion in current research that it might help some overweight individuals lower their blood pressure, and it may favorably modify lipid profiles [2,3]. It also appears to enhance exercise endurance in some and may be protective in others with cardiac ischemia. There is a suggestion in animal work that it might have an ability to reduce
∗
Corresponding author at: Ege University, Faculty of Science, Biochemistry Department, 35100 Bornova-˙Izmir/Turkey: +90 232 3438624. E-mail address: [email protected] (E. Akyilmaz). 0013-4686/$ – see front matter © 2007 Elsevier Ltd. All rights reserved. doi:10.1016/j.electacta.2007.06.058
insulin resistance [4]. According to the recent research, pyruvate in supraphysiological concentrations may have a role in cardiovascular therapy, as an inotropic agent [5–7]. Supraphysiological amounts of this dietary substance may also have bariatric and ergogenic applications [8,9]. Biosensor is a very useful tool that detects, records, and transmits information regarding a physiological change or the presence of various chemical or biological materials. It contains biologic sensitive materials and it is combined with a transducer to yield a measurable signal. Because they have simple analysis methods, short response time, sensitivity, specificity and cheapness, the biosensors are improved and offered to usage year by year. Different types of biosensors have been developed for the determination of pyruvate. Researchers have utilized different strategies and also transducer systems in the construction of these biosensors. An Au-electroplated glassy carbon electrode with a conductive redox polymer, poly (mercapto-pbenzoquinone) [10], a carbon paste electrode with a methylene green-mediator [11], an amperometric biosensor with a polyazetidine prepolymer matrix [12], oxygen electrode combined with
E. Akyilmaz, E. Yorganci / Electrochimica Acta 52 (2007) 7972–7977
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a porous acetylcellulose membrane [13], oxygen electrode based on chemical covalent immobilization of pyruvate oxidase [14], gold microelectrodes [15], a bienzyme modified carbon paste electrode [16], and a polyion complex membrane systems [17] have been developed for the determination of pyruvate. In addition, some biosensors have been developed for the determination of phosphate such as bilayer membrane systems with a poly (vinyl alcohol)/polyion complex- [18], a screen-printed electrode [19], a glassy carbon electrode with a nano-particle comprised poly - 5,2 :5 ,2 -terthiophene-3 -carboxylic acid, poly-TTCA (nano-CP) layers [20], a porous conductive carbon electrode in conjunction with a polyelectrolyte stabilized recombinant pyruvate oxidase [21], and chemiluminescent FIA systems [22,23]. In this study we have developed a new biosensor based on pyruvate oxidase for the determination of pyruvate and phosphate. For the construction of the biosensor we have selected an easy, inexpensive, and not-time consuming immobilization method that used gelatin and cross-linking reagent, glutaraldehyde. Basic transducer system is a Clark type dissolved oxygen electrode which is combined with YSI 58 model oxygenmeter. The biosensor provides both pyruvate and phosphate determination and on-site determination because of its portability. 2. Experiment 2.1. Chemicals Pyruvate oxidase (E.C 1.2.3.3) (100U), pyruvic acid sodium salt, KH2 PO4 , K2 HPO4 glutaraldehyde (%25), calf skin gelatin, and all other chemicals were purchased from Sigma Chemical Co.(USA). All solutions used in the experiments were prepared just before their use. 2.2. Apparatus In the experiments, a YSI model 58 Digital Oxygenmeter, YSI 5700 model dissolved oxygen (DO) probes, Gilson P100 and P1000 otomatic pipets (France), Pharmacia spectrophotometer (UK), Nuve model thermostat (TR), Yellow-Line magnetic stirrer (Germany) were used. 2.3. Preparation of the biosensor For the preparation of the biosensor a dissolved oxygen probe was covered with a high-sensitive teflon membrane by using an O-ring and then the teflon membrane which is selective for oxygen was pretreated with 0.5% SDS (sodiumdodecylsulphate) in phosphate buffer (50 mM, pH 7.0) to reduce the tension on the membrane surface of dissolved oxygen probe. After that, 210 l of pyruvate oxidase enzyme solution (2.5 U/cm2 in phosphate buffer pH 7.0) and gelatin (4.76 mg/cm2 ) were mixed and dissolved at 38 ◦ C for a few minutes (1), 200 l of the mixed solution was pipetted (2) and spread over the teflon membrane surface (3–4), and then allowed to dry at 4 ◦ C for 30 min. At the end of the time, the biosensor was cross-linked with glutaraldehyde solution (2.5%) (in phosphate buffer 50 mM, pH
Fig. 1. A The preparation steps of the biosensor based on pyruvate oxidase immobilized in gelatin. B Measurement system of the biosensor: (a) the biosensor, (b) injection valve, (c) magnetic stirrer, (d) YSI 58 Model oxygenmeter, (e) stirring bar.
7.0) by immersing for 4 min (5). Excess of the glutaraldehyde was romoved by washing with bidistilled water for couple of times (6). Finally, a bioactive layer was formed on the dissolved oxygen probe of the biosensor. Fig. 1 shows (a) the preparation steps of the biosensor based on pyruvate oxidase immobilized in gelatin and measurement system (b). 2.4. Measurements The detection principle of the biosensor is amperometric (polarographic). Our Clark type oxygen electrode has a gold electrode as cathode and it is made negative by −0.8 V with respect to a suitable reference anode (Ag/AgCl electrode in a half-saturated KCl solution (as electrolyte), so that any oxygen dissolved in the enzymatic reaction is reduced at the surface of the cathode. In polarographic sensors, the anode and cathode immersed in an electrolyte, into which oxygen permeates
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through a membrane. Polarographic electrodes are often made using silver, which requires a voltage in order to activate the oxygen permeation process. According to the reaction catalyzed by pyruvate oxidase, pyruvate is converted to acetly phosphate, carbondioxide and hydrogenperoxide in the presence of phosphate. Pyruvate oxidase
Pyruvate + Pi + O2 + H2 O−−−−−−−−→ Acetly phosphate + CO2 + H2 O2
The principle of the measurement is based on the determination of consumed dissolved oxygen concentration, which is related to pyruvate concentrations added into the reaction cell. The biosensor showed a steady-state dissolved oxygen concentration in the absence of pyruvate and when the pyruvate was injected into the reaction medium a decrease in the steady-state dissolved oxygen concentration related to pyruvate concentration was observed. In this case, a new equilibrium for dissolved oxygen concentration was formed. As a result, the differences between the first and the last steady state dissolved oxygen concentrations related to pyruvate concentrations were detected by the biosensor to obtain a calibration curve. All the measurements were done at 35 ◦ C by using a thermostatic reaction cells and the oxygen saturated phosphate buffer (50 mM, pH 7.0). 3. Results and discussion 3.1. Optimization of the bioactive surface of the biosensor 3.1.1. Effect of the enzyme activity on the biosensor response To detect the effect of the enzyme activity on the biosensor response different enzyme amounts were used in the construction of the biosensor. For this purpose, three biosensors were prepared. The biosensors contained 1.25, 2.5, and 5.0 U/cm2 activity of pyruvate oxidase, respectively and they were immobilized by gelatin (4.76 mg/cm2 ) and glutaraldehyde (2.5%). The measurements were made to obtain standard curves for pyruvate by using the biosensors prepared. The most useful calibration curve was obtained by using the biosensor, which was prepared with 2.5 U/cm2 activity of pyruvate oxidase. Pyruvate was detected with a linear range between 0.0025 and 0.05 M concentrations by this biosensor. When the pyruvate oxidase activity was increased from 2.5 to 5.0 U/cm2 higher biosensor responses were obtained but the linear range was detected between 0.0025 and 0.0375 M concentrations. From the results it can be said that, the most suitable biosensor responses and also a linear calibration curve were obtained by the biosensor, which contains 2.5 U/cm2 activity of pyruvate oxidase. 3.1.2. Detection of the effect of gelatin amounts on the biosensor response For the determination of the effect of gelatin amounts on the biosensor response different amounts of gelatin were
used in the construction of the biosensors. Biosensors contained 2.38, 4.76, and 9.52 mg/cm2 gelatin. The biosensors had 2.5 U/cm2 pyruvate oxidase activity and all of them were immobilized by glutaraldehyde (2.5%). The measurements were made to obtain standard curves for pyruvate by using the biosensors prepared. Decrease in the gelatin amount from 4.76 to 2.38 mg/cm2 created higher biosensor responses but the negative effect for the linearity of the standard curve. The reason of this effect was the reducing in the forming of cross-linked bonds between enzyme, gelatin, and glutaraldehyde. When the gelatin amounts were increased from 4.76 to 9.52 mg/cm2 , we detected increases in the biosensor responses but the linearity was swerved after the 0.0375 M concentration of pyruvate. In this case it can be said that, results obtained from the experiments showed that the most suitable biosensor responses were obtained by using 4.76 mg/cm2 gelatin. 3.1.3. Effect of the percentage of glutaraldehyde on the biosensor response To detect the effect of the glutaraldehyde amount on the biosensor responses, different percentage of glutaraldehyde were used in the construction of the biosensor. For this purpose we prepared biosensors, which contain 2.5 U/cm2 pyruvate oxidase and 4.76 mg/cm2 gelatin. On the other hand the biosensors were treated with 1.25, 2.5, and 5.0% (v/v) glutaraldehyde solution prepared in phosphate buffer (50 mM, pH 7.0) for the immobilization. After immobilization procedure, the experiments were made to obtain a standard curve for pyruvate by using the biosensors prepared. From the experiments the best linear range and the reliable responses were obtained when 2.5% glutaraldehyde was used. At this concentration the responses of the biosensor were reproducible and the linear curve was fairly smooth. Lower concentration of glutaraldehyde was not sufficient enough to allow adequate cross-linking of the enzyme and gelatin and this could be a possible explanation for this result. When 5.0% concentration of glutaraldehyde was used the linearity of the calibration curve was not suitable and this result probably due to the much more cross-linked bonds occured between the enzyme and gelatin. 3.2. Optimization of working conditions 3.2.1. Detection of the effect of pH on the biosensor response For the determination of the effect of pH value on the biosensor response different buffer systems were investigated. For this purpose 50 mM concentration of citrate (pH 5–6), phosphate (pH 7–8), and glycine (pH 9–10) buffers were used in the experiments. The optimum pH value was obtained as 7.0 (Fig. 2). Below and above this pH value decreases were observed on the biosensor responses. When we consider the optimum pH value of the free form of pyruvate oxidase (6–6.5) it can be said that immobilization procedure caused a little difference at the optimum pH value of the enzyme.
E. Akyilmaz, E. Yorganci / Electrochimica Acta 52 (2007) 7972–7977
Fig. 2. The detection of the optimum pH of the biosensor. The concentration of all buffers is 50 mM, T = 35 ◦ C.
3.2.2. Effect of temperature on the biosensor response Because of the nature of the biocomponent used in the biosensor construction and also its relation to temperature, detection of the effect of the temperature on the biosensor responses is one of the most important factors for the biosensor studies. For the detection of temperature effect on the biosensor responses, the experiments were carried out between 15 and 45 ◦ C. According to the results, the highest biosensor response was observed at 35 ◦ C. Below and above this degree decreases in the biosensor responses were observed (Fig. 3). 3.3. Analytical characteristics of the biosensor 3.3.1. Linear range of the biosensor After optimization of the bioactive surface and working conditions of the biosensor we obtained a standard or calibration curve for pyruvate under optimized conditions (Fig. 4). From the figure it can be said that the biosensor responses depend linearly on pyruvate concentration between 0.0025 and 0.05 M with a (y = 1.6638x + 0.1076) and R2 = 0.9983. 3.3.2. Reproducibility To detect the reproducibility of the biosensor some experiments were investigated for 0.025 M concentration of pyruvate
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Fig. 4. Standard curve for pyruvate determination (phosphate buffer; pH 7.0, 50 mM; T = 35 ◦ C). The percentage of glutaraldehyde, the amount of gelatine and the activity of pyruvate oxidase were kept constant at 2.5%, 4.76 mg/cm2 , and 2.5 U/cm2 , respectively.
(n = 10). From the experiments, the avarage value (¯x), standard deviation (S.D.) and variation coefficient of the biosensor (C.V.%) were calculated as 0.0247 M, ±0.00078 M and 3.14%, respectively. The results were compared to the results obtained from the modified enzymatic-spectrophotometric method (24) (Table 1). From the Table it can be said that pyruvate can be determined more sensitively by using the biosensor. 3.3.3. Determination of the concentration of phosphate In order to simultaneously determine the concentration of phosphate by the biosensor in the presence of pyruvate, the concentration of pyruvate was constant at 0.025 M and TrisHCI buffer (50 mM, pH 7.0) were used instead of phosphate buffer in the experiments. Results obtained from the experiments showed that phosphate was determined sensitively in a linear range between 1.0 and 10.0 M (Fig. 5). 3.3.4. Detection of specificity of the biosensor and interference effects of some substances on the biosensor responses In order to detect the substrate specificity of the biosensor some experiments were made by using 0.025 M concentration of pyruvate and various compounds such as glucose, ascorbic acid, lactic acid, oxalic acid, and citric acid. The biosensor response obtained for pyruvate was accepted as 100% and this was compared to other biosensor responses obtained for these compounds (Table 2). According to the results, the same biosenTable 1 Reproducibility comparison between two methods
Fig. 3. The optimum temperature of the biosensor (phosphate buffer; pH 7.0, 50 mM).
Average Standard deviation (S.D) Coefficient of varation (C.V %) a
The biosensora
The enzymaticspectrophotometrica
0.0247 M ±0.00078 M 3.14%
0.0261 M ±0.00198 M 7.57%
Concentration of pyruvate is 0.025 M and (n = 10 measurements)
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Fig. 5. Standard curve for phosphate determination (Tris-HCl buffer; pH 7.0, 50 mM; T = 35 ◦ C). The percentage of glutaraldehyde, the amount of gelatine and the activity of pyruvate oxidase were kept constant at 2.5%, 4.76 mg/cm2 , and 2.5 U/cm2 , respectively.
sor responses were obtained for glucose, ascorbic acid, and lactic acid. For oxalic and citric acid we didn’t observe any response. For investigation of the interference effects of the some substances on the pyruvate determination in the presence of pyruvate (0.025 M) some experiments were made. For this purpose the experiments were realized by using glucose, lactic acid, citric acid, ascorbic acid, and oxalic acid at the same concentration (0.025 M) with pyruvate. The biosensor response obtained was accepted as 100% for pyruvate in the absence of the other substances and this result was compared to other biosensor responses obtained for these compounds in the presence of pyruvate (Table 2). According to the results, it was detected that the substances showed an interference effect on pyruvate determination in the presence of pyruvate except oxalic and citric acid. Because they didn’t show any effect on the pyruvate determination, we have made some control experiments to investigate the nature of these responses by using the sensor prepared without the enzyme (blank sensor). From the experiments we have obtained responses for glucose, ascorbic and lactic acid by the blank sensor, and the responses were the same obtained with the biosensor. In addition, by using the blank sensor we have made the same assays for oxalic and citric acid but we have not obtained any responses for them. So, in this case it can be said that the sensor responses obtained for glucose, ascorbic acid and lactic acid can be related to a spontaneous process. Table 2 Substrate specificity and interference effects for the biosensor Substratea
Activity %
Substratea
Activity %
Pyruvate Glucose Ascorbic acid Lactic acid Oxalic acid Citric acid
100 25 25 25 0 0
Pyruvate (Pyruvate + Glucose) (Pyruvate + Ascorbic acid) (Pyruvate + Lactic acid) (Pyruvate + Oxalic acid) (Pyruvate + Citric acid)
100 75 87 75 100 100
a
Concentration: 0.025 M.
Fig. 6. Operational stability of the biosensor (phosphate buffer; pH 7.0, 50 mM; T = 35 ◦ C). The percentage of glutaraldehyde, the amount of gelatine and the activity of pyruvate oxidase were kept constant at 2.5%, 4.76 mg/cm2 , and 2.5 U/cm2 , respectively.
3.3.5. Operational stability In order to determine the operation stability of the biosensor, measurements were carried out periodically every hour to detect decreases in the biosensor responses. During this period enzymes used in the biosensor construction lost their activities as related to time. The biosensor prepared was used for only this purpose and during the period it was waited at 35 ◦ C. From the experiments it can be said that the remaining activity of the biosensor was more than 85% of its initial activity (Fig. 6). 4. Conclusion As a result of this work, the biosensor developed, based on pyruvate oxidase enzyme, was found to be more advantageous in comparison to other methods known, such as spectrophotometric, chemical, and fluorometric. The biosensor developed is specific, not time-consuming and also suitable for rutine analysis of pyruvate and phosphate at the same time. When we consider the determination limit of pyruvate it can be said that the biosensor is much more sensitive than the others developed by Zapata-Bacri and Burstein [14], Arai et al. [10], W. Bergmann et al. [16], Revzin et al. [15]. In addition, phosphate had been determined more sensitively than the other biosensors developed by Mak et al. [18], Ikebukuro et al. [23]. On the other hand, the immobilization procedure of the enzyme on the teflon membrane surface by using gelatin and glutaraldehyde is very easy and not time-consuming. Response time of the biosensor is only three minutes and more than 50 measurements can be done by the biosensor. Its operational stability is very high and the interference effects of some substances are negligible. As a result, the biosensor can be used for easy, sensitive, and accurate determination of pyruvate and phosphate. References [1] R.T. Stanko, D.L.J.E. Tietze, Am. J. Clin. Nutr. 56 (1992) 630. [2] D. Kalman, C.M. Colker, I. Wilets, J.B. Roufs, J. Antonio, Nutrition 15 (1999) 337.
E. Akyilmaz, E. Yorganci / Electrochimica Acta 52 (2007) 7972–7977 [3] R.T. Stanko, H.R. Reynolds, R. Hoyson, J.E. Janosky, R. Wolf, Am. J. Clin. Nutr. 59 (1994) 423. [4] J.L. Ivy, M.Y. Cortez, R.M. Chandler, H.K. Byrne, R.H. Miller, Am. J. Clin. Nutr. 59 (1994) 331. [5] R.T. Mallet, R. B¨unger, Biochim. Biophys. Acta 1224 (1994) 22. [6] W. Chen, R. London, E. Murphy, C. Steenbergen, Circ. Res. 83 (1998) 898. [7] A.V. Zima, J. Kocksk¨amper, R. Mejia-Alvarez, L.A. Blatter, J. Physiol. 550 (2003) 765. [8] R.T. Mallet, Exp. Biol. Med. 223 (2000) 136. [9] R.T. Mallet, J. Sun, Cardiovasc. Res. 42 (1999) 149. [10] G. Arai, T. Noma, H. Habu, I. Yasumori, J. Electroanal. Chem. 464 (1999) 143. [11] J. Kulys, L. Wang, N. Daugvilaite, Anal. Chim. Acta 265 (1992) 15. [12] M. Mascini, F. Mazzei, Anal. Chim. Acta 192 (1987) 9. [13] F. Mizutani, K. Tsuda, I. Karube, S. Suzuki, K. Matsumoto, Anal. Chim. Acta 118 (1980) 65. [14] A.M. Zapata-Bacri and C. Burstein, Biosensors 3 (1987-1988) 227.
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[15] A.F. Revzin, K. Sirkar, A. Simonian, M.V. Pishko, Sens. Actuators, B: Chem. 81 (2002) 359. [16] W. Bergmann, R. Rudolph, U. Spohn, Anal. Chim. Acta 394 (1999) 233. [17] F. Mizutani, S. Yabuki, Y. Sato, T. Sawaguchi, S. Iijima, Electrochim. Acta 45 (2000) 2945. [18] W.C. Mak, C. Chan, J. Barford, R. Renneberg, Biosens. Bioelectron. 19 (2003) 233. [19] R.C.H. Kwan, H.F. Leung, P.Y.T. Hon, H.C.F. Cheung, K. Hirota, R. Renneberg, Anal. Biochem. 343 (2005) 263. [20] M.A. Rahman, D.S. Park, S.C. Chang, C.J. McNeil, Y.B. Shim, Biosens. Bioelectron. 21 (2006) 1116. [21] V.G. Gavalas, N.A. Chaniotakis, Anal. Chim. Acta 427 (2001) 271. [22] H. Nakamura, K. Ikebukuro, S. McNiven, I. Karube, H. Yamamoto, K. Hayashi, M. Suzuki, I. Kubo, Biosens. Bioelectron. 12 (1997) 959. [23] K. IkebukuroA, R. Nishida, H. Yamamoto, Y. Arikawa, H. Nakamura, M. Suzuki, I. Kubo, T. Takeuchi, I. Karube, J. Biotechnol. 48 (1996) 67. [24] S¸.H. Baysal, A.H. Uslan, Art Cell. Blood Subst. Imm. Biotechnol. 30 (2002) 71.
Construction of an amperometric pyruvate oxidase enzyme electrode for determination of pyruvate and phosphate Erol Akyilmaz ∗ , Emine Yorganci ˙ Department of Biochemistry, Faculty of Science, Ege University, 35100 Bornova-Izmir, Turkey Received 12 April 2007; received in revised form 21 June 2007; accepted 21 June 2007 Available online 27 June 2007
Abstract In this study an amperometric biosensor based on pyruvate oxidase was developed for the determination of pyruvate and phosphate. For construction of the biosensor pyruvate oxidase was immobilized with gelatin and insolubilized in film by forming cross-linked bonds with glutaraldehyde. The film was fixed on a YSI type dissolved oxygen (DO) probe, covered with a teflon membrane which is high-sensitive for oxygen. The working principle of the biosensor depends on detection of consumed DO concentration related to pyruvate concentration which is used in enzymatic reaction catalyzed by pyruvate oxidase. The biosensor response shows a linearity with pyruvate concentration between 0.0025 and 0.05 M and also response time of the biosensor is 3 min. In the optimization studies of the biosensor the most suitable enzyme activity was found as 2.5 U/cm2 for pyruvate oxidase, and also phosphate buffer (pH 7.0; 50 mM) and 35 ◦ C were established as providing the optimum working conditions. In the characterization studies of the biosensor some parameters such as reproducibility, substrate specificity, operational stability, determination of phosphate, and interference effects of some compounds on the pyruvate determination were investigated. Finally, the concentration of pyruvate was determined by using spectrophotometric method and the results obtained were compared to results obtained by the biosensor. © 2007 Elsevier Ltd. All rights reserved. Keywords: Pyruvate; Biosensor; Phosphate; Pyruvate oxidase; Enzyme electrode
1. Introduction Pyruvate is a key intermediate in the glycolytic and pyruvate dehydrogenase pathways, which are involved in biological energy production. It is widely found in living organisms and it is not an essential nutrient since it can be synthesized in the cells of the body. It may also have cardiac and skeletal muscle inotropic activity as well as bariatric activity, and also antioxidant activity [1]. Pyruvate may help some obese individuals lose weight. There is also the suggestion in current research that it might help some overweight individuals lower their blood pressure, and it may favorably modify lipid profiles [2,3]. It also appears to enhance exercise endurance in some and may be protective in others with cardiac ischemia. There is a suggestion in animal work that it might have an ability to reduce
∗
Corresponding author at: Ege University, Faculty of Science, Biochemistry Department, 35100 Bornova-˙Izmir/Turkey: +90 232 3438624. E-mail address: [email protected] (E. Akyilmaz). 0013-4686/$ – see front matter © 2007 Elsevier Ltd. All rights reserved. doi:10.1016/j.electacta.2007.06.058
insulin resistance [4]. According to the recent research, pyruvate in supraphysiological concentrations may have a role in cardiovascular therapy, as an inotropic agent [5–7]. Supraphysiological amounts of this dietary substance may also have bariatric and ergogenic applications [8,9]. Biosensor is a very useful tool that detects, records, and transmits information regarding a physiological change or the presence of various chemical or biological materials. It contains biologic sensitive materials and it is combined with a transducer to yield a measurable signal. Because they have simple analysis methods, short response time, sensitivity, specificity and cheapness, the biosensors are improved and offered to usage year by year. Different types of biosensors have been developed for the determination of pyruvate. Researchers have utilized different strategies and also transducer systems in the construction of these biosensors. An Au-electroplated glassy carbon electrode with a conductive redox polymer, poly (mercapto-pbenzoquinone) [10], a carbon paste electrode with a methylene green-mediator [11], an amperometric biosensor with a polyazetidine prepolymer matrix [12], oxygen electrode combined with
E. Akyilmaz, E. Yorganci / Electrochimica Acta 52 (2007) 7972–7977
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a porous acetylcellulose membrane [13], oxygen electrode based on chemical covalent immobilization of pyruvate oxidase [14], gold microelectrodes [15], a bienzyme modified carbon paste electrode [16], and a polyion complex membrane systems [17] have been developed for the determination of pyruvate. In addition, some biosensors have been developed for the determination of phosphate such as bilayer membrane systems with a poly (vinyl alcohol)/polyion complex- [18], a screen-printed electrode [19], a glassy carbon electrode with a nano-particle comprised poly - 5,2 :5 ,2 -terthiophene-3 -carboxylic acid, poly-TTCA (nano-CP) layers [20], a porous conductive carbon electrode in conjunction with a polyelectrolyte stabilized recombinant pyruvate oxidase [21], and chemiluminescent FIA systems [22,23]. In this study we have developed a new biosensor based on pyruvate oxidase for the determination of pyruvate and phosphate. For the construction of the biosensor we have selected an easy, inexpensive, and not-time consuming immobilization method that used gelatin and cross-linking reagent, glutaraldehyde. Basic transducer system is a Clark type dissolved oxygen electrode which is combined with YSI 58 model oxygenmeter. The biosensor provides both pyruvate and phosphate determination and on-site determination because of its portability. 2. Experiment 2.1. Chemicals Pyruvate oxidase (E.C 1.2.3.3) (100U), pyruvic acid sodium salt, KH2 PO4 , K2 HPO4 glutaraldehyde (%25), calf skin gelatin, and all other chemicals were purchased from Sigma Chemical Co.(USA). All solutions used in the experiments were prepared just before their use. 2.2. Apparatus In the experiments, a YSI model 58 Digital Oxygenmeter, YSI 5700 model dissolved oxygen (DO) probes, Gilson P100 and P1000 otomatic pipets (France), Pharmacia spectrophotometer (UK), Nuve model thermostat (TR), Yellow-Line magnetic stirrer (Germany) were used. 2.3. Preparation of the biosensor For the preparation of the biosensor a dissolved oxygen probe was covered with a high-sensitive teflon membrane by using an O-ring and then the teflon membrane which is selective for oxygen was pretreated with 0.5% SDS (sodiumdodecylsulphate) in phosphate buffer (50 mM, pH 7.0) to reduce the tension on the membrane surface of dissolved oxygen probe. After that, 210 l of pyruvate oxidase enzyme solution (2.5 U/cm2 in phosphate buffer pH 7.0) and gelatin (4.76 mg/cm2 ) were mixed and dissolved at 38 ◦ C for a few minutes (1), 200 l of the mixed solution was pipetted (2) and spread over the teflon membrane surface (3–4), and then allowed to dry at 4 ◦ C for 30 min. At the end of the time, the biosensor was cross-linked with glutaraldehyde solution (2.5%) (in phosphate buffer 50 mM, pH
Fig. 1. A The preparation steps of the biosensor based on pyruvate oxidase immobilized in gelatin. B Measurement system of the biosensor: (a) the biosensor, (b) injection valve, (c) magnetic stirrer, (d) YSI 58 Model oxygenmeter, (e) stirring bar.
7.0) by immersing for 4 min (5). Excess of the glutaraldehyde was romoved by washing with bidistilled water for couple of times (6). Finally, a bioactive layer was formed on the dissolved oxygen probe of the biosensor. Fig. 1 shows (a) the preparation steps of the biosensor based on pyruvate oxidase immobilized in gelatin and measurement system (b). 2.4. Measurements The detection principle of the biosensor is amperometric (polarographic). Our Clark type oxygen electrode has a gold electrode as cathode and it is made negative by −0.8 V with respect to a suitable reference anode (Ag/AgCl electrode in a half-saturated KCl solution (as electrolyte), so that any oxygen dissolved in the enzymatic reaction is reduced at the surface of the cathode. In polarographic sensors, the anode and cathode immersed in an electrolyte, into which oxygen permeates
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E. Akyilmaz, E. Yorganci / Electrochimica Acta 52 (2007) 7972–7977
through a membrane. Polarographic electrodes are often made using silver, which requires a voltage in order to activate the oxygen permeation process. According to the reaction catalyzed by pyruvate oxidase, pyruvate is converted to acetly phosphate, carbondioxide and hydrogenperoxide in the presence of phosphate. Pyruvate oxidase
Pyruvate + Pi + O2 + H2 O−−−−−−−−→ Acetly phosphate + CO2 + H2 O2
The principle of the measurement is based on the determination of consumed dissolved oxygen concentration, which is related to pyruvate concentrations added into the reaction cell. The biosensor showed a steady-state dissolved oxygen concentration in the absence of pyruvate and when the pyruvate was injected into the reaction medium a decrease in the steady-state dissolved oxygen concentration related to pyruvate concentration was observed. In this case, a new equilibrium for dissolved oxygen concentration was formed. As a result, the differences between the first and the last steady state dissolved oxygen concentrations related to pyruvate concentrations were detected by the biosensor to obtain a calibration curve. All the measurements were done at 35 ◦ C by using a thermostatic reaction cells and the oxygen saturated phosphate buffer (50 mM, pH 7.0). 3. Results and discussion 3.1. Optimization of the bioactive surface of the biosensor 3.1.1. Effect of the enzyme activity on the biosensor response To detect the effect of the enzyme activity on the biosensor response different enzyme amounts were used in the construction of the biosensor. For this purpose, three biosensors were prepared. The biosensors contained 1.25, 2.5, and 5.0 U/cm2 activity of pyruvate oxidase, respectively and they were immobilized by gelatin (4.76 mg/cm2 ) and glutaraldehyde (2.5%). The measurements were made to obtain standard curves for pyruvate by using the biosensors prepared. The most useful calibration curve was obtained by using the biosensor, which was prepared with 2.5 U/cm2 activity of pyruvate oxidase. Pyruvate was detected with a linear range between 0.0025 and 0.05 M concentrations by this biosensor. When the pyruvate oxidase activity was increased from 2.5 to 5.0 U/cm2 higher biosensor responses were obtained but the linear range was detected between 0.0025 and 0.0375 M concentrations. From the results it can be said that, the most suitable biosensor responses and also a linear calibration curve were obtained by the biosensor, which contains 2.5 U/cm2 activity of pyruvate oxidase. 3.1.2. Detection of the effect of gelatin amounts on the biosensor response For the determination of the effect of gelatin amounts on the biosensor response different amounts of gelatin were
used in the construction of the biosensors. Biosensors contained 2.38, 4.76, and 9.52 mg/cm2 gelatin. The biosensors had 2.5 U/cm2 pyruvate oxidase activity and all of them were immobilized by glutaraldehyde (2.5%). The measurements were made to obtain standard curves for pyruvate by using the biosensors prepared. Decrease in the gelatin amount from 4.76 to 2.38 mg/cm2 created higher biosensor responses but the negative effect for the linearity of the standard curve. The reason of this effect was the reducing in the forming of cross-linked bonds between enzyme, gelatin, and glutaraldehyde. When the gelatin amounts were increased from 4.76 to 9.52 mg/cm2 , we detected increases in the biosensor responses but the linearity was swerved after the 0.0375 M concentration of pyruvate. In this case it can be said that, results obtained from the experiments showed that the most suitable biosensor responses were obtained by using 4.76 mg/cm2 gelatin. 3.1.3. Effect of the percentage of glutaraldehyde on the biosensor response To detect the effect of the glutaraldehyde amount on the biosensor responses, different percentage of glutaraldehyde were used in the construction of the biosensor. For this purpose we prepared biosensors, which contain 2.5 U/cm2 pyruvate oxidase and 4.76 mg/cm2 gelatin. On the other hand the biosensors were treated with 1.25, 2.5, and 5.0% (v/v) glutaraldehyde solution prepared in phosphate buffer (50 mM, pH 7.0) for the immobilization. After immobilization procedure, the experiments were made to obtain a standard curve for pyruvate by using the biosensors prepared. From the experiments the best linear range and the reliable responses were obtained when 2.5% glutaraldehyde was used. At this concentration the responses of the biosensor were reproducible and the linear curve was fairly smooth. Lower concentration of glutaraldehyde was not sufficient enough to allow adequate cross-linking of the enzyme and gelatin and this could be a possible explanation for this result. When 5.0% concentration of glutaraldehyde was used the linearity of the calibration curve was not suitable and this result probably due to the much more cross-linked bonds occured between the enzyme and gelatin. 3.2. Optimization of working conditions 3.2.1. Detection of the effect of pH on the biosensor response For the determination of the effect of pH value on the biosensor response different buffer systems were investigated. For this purpose 50 mM concentration of citrate (pH 5–6), phosphate (pH 7–8), and glycine (pH 9–10) buffers were used in the experiments. The optimum pH value was obtained as 7.0 (Fig. 2). Below and above this pH value decreases were observed on the biosensor responses. When we consider the optimum pH value of the free form of pyruvate oxidase (6–6.5) it can be said that immobilization procedure caused a little difference at the optimum pH value of the enzyme.
E. Akyilmaz, E. Yorganci / Electrochimica Acta 52 (2007) 7972–7977
Fig. 2. The detection of the optimum pH of the biosensor. The concentration of all buffers is 50 mM, T = 35 ◦ C.
3.2.2. Effect of temperature on the biosensor response Because of the nature of the biocomponent used in the biosensor construction and also its relation to temperature, detection of the effect of the temperature on the biosensor responses is one of the most important factors for the biosensor studies. For the detection of temperature effect on the biosensor responses, the experiments were carried out between 15 and 45 ◦ C. According to the results, the highest biosensor response was observed at 35 ◦ C. Below and above this degree decreases in the biosensor responses were observed (Fig. 3). 3.3. Analytical characteristics of the biosensor 3.3.1. Linear range of the biosensor After optimization of the bioactive surface and working conditions of the biosensor we obtained a standard or calibration curve for pyruvate under optimized conditions (Fig. 4). From the figure it can be said that the biosensor responses depend linearly on pyruvate concentration between 0.0025 and 0.05 M with a (y = 1.6638x + 0.1076) and R2 = 0.9983. 3.3.2. Reproducibility To detect the reproducibility of the biosensor some experiments were investigated for 0.025 M concentration of pyruvate
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Fig. 4. Standard curve for pyruvate determination (phosphate buffer; pH 7.0, 50 mM; T = 35 ◦ C). The percentage of glutaraldehyde, the amount of gelatine and the activity of pyruvate oxidase were kept constant at 2.5%, 4.76 mg/cm2 , and 2.5 U/cm2 , respectively.
(n = 10). From the experiments, the avarage value (¯x), standard deviation (S.D.) and variation coefficient of the biosensor (C.V.%) were calculated as 0.0247 M, ±0.00078 M and 3.14%, respectively. The results were compared to the results obtained from the modified enzymatic-spectrophotometric method (24) (Table 1). From the Table it can be said that pyruvate can be determined more sensitively by using the biosensor. 3.3.3. Determination of the concentration of phosphate In order to simultaneously determine the concentration of phosphate by the biosensor in the presence of pyruvate, the concentration of pyruvate was constant at 0.025 M and TrisHCI buffer (50 mM, pH 7.0) were used instead of phosphate buffer in the experiments. Results obtained from the experiments showed that phosphate was determined sensitively in a linear range between 1.0 and 10.0 M (Fig. 5). 3.3.4. Detection of specificity of the biosensor and interference effects of some substances on the biosensor responses In order to detect the substrate specificity of the biosensor some experiments were made by using 0.025 M concentration of pyruvate and various compounds such as glucose, ascorbic acid, lactic acid, oxalic acid, and citric acid. The biosensor response obtained for pyruvate was accepted as 100% and this was compared to other biosensor responses obtained for these compounds (Table 2). According to the results, the same biosenTable 1 Reproducibility comparison between two methods
Fig. 3. The optimum temperature of the biosensor (phosphate buffer; pH 7.0, 50 mM).
Average Standard deviation (S.D) Coefficient of varation (C.V %) a
The biosensora
The enzymaticspectrophotometrica
0.0247 M ±0.00078 M 3.14%
0.0261 M ±0.00198 M 7.57%
Concentration of pyruvate is 0.025 M and (n = 10 measurements)
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E. Akyilmaz, E. Yorganci / Electrochimica Acta 52 (2007) 7972–7977
Fig. 5. Standard curve for phosphate determination (Tris-HCl buffer; pH 7.0, 50 mM; T = 35 ◦ C). The percentage of glutaraldehyde, the amount of gelatine and the activity of pyruvate oxidase were kept constant at 2.5%, 4.76 mg/cm2 , and 2.5 U/cm2 , respectively.
sor responses were obtained for glucose, ascorbic acid, and lactic acid. For oxalic and citric acid we didn’t observe any response. For investigation of the interference effects of the some substances on the pyruvate determination in the presence of pyruvate (0.025 M) some experiments were made. For this purpose the experiments were realized by using glucose, lactic acid, citric acid, ascorbic acid, and oxalic acid at the same concentration (0.025 M) with pyruvate. The biosensor response obtained was accepted as 100% for pyruvate in the absence of the other substances and this result was compared to other biosensor responses obtained for these compounds in the presence of pyruvate (Table 2). According to the results, it was detected that the substances showed an interference effect on pyruvate determination in the presence of pyruvate except oxalic and citric acid. Because they didn’t show any effect on the pyruvate determination, we have made some control experiments to investigate the nature of these responses by using the sensor prepared without the enzyme (blank sensor). From the experiments we have obtained responses for glucose, ascorbic and lactic acid by the blank sensor, and the responses were the same obtained with the biosensor. In addition, by using the blank sensor we have made the same assays for oxalic and citric acid but we have not obtained any responses for them. So, in this case it can be said that the sensor responses obtained for glucose, ascorbic acid and lactic acid can be related to a spontaneous process. Table 2 Substrate specificity and interference effects for the biosensor Substratea
Activity %
Substratea
Activity %
Pyruvate Glucose Ascorbic acid Lactic acid Oxalic acid Citric acid
100 25 25 25 0 0
Pyruvate (Pyruvate + Glucose) (Pyruvate + Ascorbic acid) (Pyruvate + Lactic acid) (Pyruvate + Oxalic acid) (Pyruvate + Citric acid)
100 75 87 75 100 100
a
Concentration: 0.025 M.
Fig. 6. Operational stability of the biosensor (phosphate buffer; pH 7.0, 50 mM; T = 35 ◦ C). The percentage of glutaraldehyde, the amount of gelatine and the activity of pyruvate oxidase were kept constant at 2.5%, 4.76 mg/cm2 , and 2.5 U/cm2 , respectively.
3.3.5. Operational stability In order to determine the operation stability of the biosensor, measurements were carried out periodically every hour to detect decreases in the biosensor responses. During this period enzymes used in the biosensor construction lost their activities as related to time. The biosensor prepared was used for only this purpose and during the period it was waited at 35 ◦ C. From the experiments it can be said that the remaining activity of the biosensor was more than 85% of its initial activity (Fig. 6). 4. Conclusion As a result of this work, the biosensor developed, based on pyruvate oxidase enzyme, was found to be more advantageous in comparison to other methods known, such as spectrophotometric, chemical, and fluorometric. The biosensor developed is specific, not time-consuming and also suitable for rutine analysis of pyruvate and phosphate at the same time. When we consider the determination limit of pyruvate it can be said that the biosensor is much more sensitive than the others developed by Zapata-Bacri and Burstein [14], Arai et al. [10], W. Bergmann et al. [16], Revzin et al. [15]. In addition, phosphate had been determined more sensitively than the other biosensors developed by Mak et al. [18], Ikebukuro et al. [23]. On the other hand, the immobilization procedure of the enzyme on the teflon membrane surface by using gelatin and glutaraldehyde is very easy and not time-consuming. Response time of the biosensor is only three minutes and more than 50 measurements can be done by the biosensor. Its operational stability is very high and the interference effects of some substances are negligible. As a result, the biosensor can be used for easy, sensitive, and accurate determination of pyruvate and phosphate. References [1] R.T. Stanko, D.L.J.E. Tietze, Am. J. Clin. Nutr. 56 (1992) 630. [2] D. Kalman, C.M. Colker, I. Wilets, J.B. Roufs, J. Antonio, Nutrition 15 (1999) 337.
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