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Electrochemical enhancement for flow-amperometric biosensing with an oxidase column
Sensors and Actuators B 91 (2003) 175–179
Electrochemical enhancement for flow-amperometric biosensing with an oxidase column Yasuhiro Iida, Takuya Kikuchi, Ikuo Satoh* Department of Applied Chemistry, Faculty of Engineering, Kanagawa Institute of Technology, 1030 Shimo-Ogino, Atsugi-shi, Kanagawa-ken 243-0292, Japan
Abstract An electrolytic device to enlarge a determination range for biosensing with use of oxidase was fabricated and introduced into a flowinjection amperometric system for L-ascorbate. The biosensing system was assembled with the plunger pump, the electrolytic device, an immobilized laccase column unit, a flow-through type of oxygen electrode and a pen recorder. The electrolytic device enabled to increase dissolved oxygen in the flow streams by electrolysis of the carrier, and thereby, the additional oxygen attributable to electrolysis should be provided to the laccase column and contribute an activation of the laccase reaction. L-Ascorbate solutions (20 ml) with various concentrations were injected to the system without the electrolytic device and amounts of dissolved oxygen enzymatically consumed were amperometrically monitored. A linear relationship was obtained in a range of 0.25–4.0 mM. On the other hand, a wider linear range of the L-ascorbic acid determination was obtained with use of the electrolytic device (0.25–10.0 mM). Not only laccase but other oxidases, such as ascorbate oxidase and glucose oxidase, were immobilized and introduced into this electrolytic FIA system. Determination ranges of the substrates were also enlarged by using the system. This study indicated that use of the electrolytic device enabled to extend the dynamic range of substrate for the oxidases without any mediators. # 2003 Elsevier Science B.V. All rights reserved. Keywords: Dissolved oxygen; Electrolytic device; Laccase; Mediatorless
1. Introduction Biosensors are widely used for quantitative determination of bio-related compounds and many kinds of enzyme are used as a recognition element. Among these sensors, immobilized oxidases are particularly used as a recognition element for these sensing system. Since the response of these sensing system is limited by the concentration of dissolved oxygen in buffer solution, the pre-dilution of samples is usually required [1]. Therefore, determination rage of the sensors depends on the amount of dissolved oxygen in the assay system. As another way to overcome this limitation of the response and extend the determination range, development of mediators for the enzyme reaction and an increase in dissolved oxygen by bubbling pure O2 gas have been attempted [2–5]. If the increase in dissolved oxygen can be achieved much more easily, the method will have many advantages over the use of mediators, for example, low cost, clean and applicable to many other oxidases, etc.
* Corresponding author. Tel./fax: þ81-46-291-3105. E-mail address: [email protected] (I. Satoh).
In the previous paper [6], we reported a novel method for generating oxygen gas by the electrolysis of buffer streams. In the method, an electrolytic device was developed and agar gel containing saturated KCl was used as a salt bridge which was a part of the device. This agar gel was used not to mix produced oxygen and hydrogen by electrolysis of carrier solution. However, sufficient current to produce oxygen by electrolysis could not be applied by using this agar type of salt bridge because of its high resistance. Therefore, in this report, a nobel electrolytic device was fabricated by using Nafion1 117 membrane instead of the salt bridge to make more large current applied. Then, the electrolytic device was introduced to a laccase column-FIA (flow-injection analysis) system for determination of L-ascorbic acid. L-Ascorbic acid, well known as vitamin C, is used in many fields such as food, cosmetics, medicine, etc. Therefore, the determination of L-ascorbic acid provides important information not only for quality control of food products in food industry but also for clinical chemistry [7]. So, a rapid and simple method for determining L-ascorbic acid is demanded [8–10]. In this study, we proposed an FIA system with the electrolytic device and demonstrated performance characteristics of the mediatorless system for the determination of
0925-4005/03/$ – see front matter # 2003 Elsevier Science B.V. All rights reserved. doi:10.1016/S0925-4005(03)00085-6
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Y. Iida et al. / Sensors and Actuators B 91 (2003) 175–179
L-ascorbic acid by using the laccase column as a recognition element. Furthermore, other immobilized oxidases were also applied into this FIA system with the electrolytic device.
2. Experiments 2.1. Materials Laccase (from Trametes sp. Ha 1) and ascorbate oxidase (from cucumber) were generously provided by Daiwa Kasei (Osaka) and Asahi Kasei (Tokyo), respectively. Glucose oxidase (from Aspergillus niger) was purchased from Sigma. Controlled-pore glass (CPG, mean pore diameter 24.2 nm, particle size 120–200 mesh) was purchased from Funakoshi (Tokyo). Perfluorinated ion exchange membrane, Nafion1 117 (0.007 in. thick), was purchased from SigmaAldrich. L-Ascorbic acid (substrate for a laccase and an ascorbate oxidase) and D(þ)-glucose (substrate for a glucose oxidase) were purchased from Merck (Darmstadt, Germany), and all other reagents were commercially available in analytical grades and were used without further purification. 2.2. Electrolytic device In order to extend the determination range of substrates of oxidase, additional oxygen in the carrier stream should be provided for the oxidase reaction. To generate oxygen electrolytically in the buffer stream, an electrolytic device was originally developed. Polymer plates made of acrylic resins (thickness: 2 mm) were pasted by dichloromethane solutions for constructing an electrolytic device and the diagram is schematically illustrated in Fig. 1. This electrolytic device was constructed with two compartments. One was equipped with two stainless tubings (2.0 mm (outer diameterÞ 1:0 mm (inner diameterÞ 30:0 mm (length); generously provided by ABLE, Tokyo) for carrier streams. A stainless tubing at upstream of the carrier streams was used as a working electrode to produce an oxygen in the
Fig. 1. Schematic drawing of the electrolytic device. WE: working electrode; CE: current electrode (platinum plate); RE: reference electrode (Ag/AgCl, in the KCl saturated); NM: Nafion1 117 membrane.
carrier streams. The other compartment was filled with carrier solution as an electrolyte, and a platinum plate which was used as a counter electrode and a reference electrode (Ag/AgCl, in the KCl saturated) were soaked in the solution. These compartments were connected with each other through a Nafion1 membrane. Therefore, oxygen and hydrogen gases were produced at different compartment each other by applying a constant current between the stainless tubing and platinum plate electrodes with the galvanostat. Dissolved oxygen in the carrier stream was increased by the electrolysis and provided to the laccase column. 2.3. Flow system and procedure Laccase was covalently immobilized onto alkylaminated CPG as previously described [11]. The immobilized preparations were packed into a small polymer column and then mounted in a water-jacketed holder. The enzyme column (0.3 ml packed volume) was used as a recognition element for L-ascorbic acid. A schematic diagram of the flow system is shown in Fig. 2. The system was assembled with a double-plunger pump (flow rate 1.0 ml/min, PU1508i; JASCO, Tokyo), an electrolytic device, a galvanostat (NPGS-2501; Nikko Keisoku, Atsugi) for applying constant current to the electrolytic device, an injection valve (Syringe Loading Sample Injector 7120, Rheodyne, California, USA) equipped with a 20 ml sample loop, the laccase column surrounded by the water jacket maintained at 303 K, the flow-through type of a polarographic oxygen electrode for monitoring the dissolved oxygen enzymatically consumed, the potentio-galvanostat (Potentiostat/Galvanostat HA-301; Hokuto Denko, Tokyo, Japan), the ammeter (Zero Shunt Ammeter HM-104; Hokuto Denko) and the pen recorder (Multi-Pen Recorder; RIKADENKI, Tokyo).
Fig. 2. Schematic drawing of the FIA system with the electrolytic device. (1) Carrier reservoir; (2) double-plunger pump (flow rate 1.0 ml/min); (3) electrolytic device; (4) galvanostat; (5) damper; (6) injection valve; (7) water jacket; (8) enzyme column; (9) O2 electrode; (10) waste; (11) electrometer; (12) potentio-galvanostat; (13) pen recorder.
Y. Iida et al. / Sensors and Actuators B 91 (2003) 175–179
Citrate buffer solution (50 mM) as the carrier was continuously pumped through the system. The sample solutions were introduced into the system through the rotary injection valve. The catalytic activity of the enzyme-packed column was assessed by injecting 20 ml of L-ascorbate solutions with various concentrations. Variation in the dissolved oxygen caused by the laccase-catalyzed reaction was monitored by the polarographic oxygen electrode and then recorded. To apply other oxidases to this FIA system, in the similar way, ascorbate oxidase and glucose oxidase were immobilized onto CPG separately, and packed into a small column, respectively. These column units were introduced into the FIA system instead of the laccase column unit. Fifty millimolar phosphate buffer (pH 6.0) and same concentration of phosphate buffer (pH 7.0) were used as a carrier in the case of using ascorbate oxidase and glucose oxidase, in this FIA system, respectively.
3. Results and discussions 3.1. Increasing of dissolved oxygen by the electrolytic device A determination range of substrates for oxidase by FIA system is limited by the concentration of oxygen in the carrier streams because oxidases, such as laccase, ascorbate oxidase and glucose oxidase, require dissolved oxygen. Therefore, in many cases, redox mediators have been used to raise these oxidase activities instead of addition of O2. In contrast, we proposed an electrochemical method instead of a chemical method to be employed to increase dissolved oxygen in the flow stream. To increase the amount of dissolved oxygen, the electrolytic device was introduced into the amperometric FIA system and various constant currents (0–10.0 mA) were applied to the electrolytic device. As shown in Fig. 3, the current output of the oxygen electrode increased with increment of the applied current. A linear relationship between the current of the oxygen electrode and the constant current up to 3 mA was obtained. It was shown that increasing of dissolved oxygen depended on the applied constant current because the current of the oxygen electrode increased with increasing of the applied
Fig. 3. Effect of current on the amperometric response.
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constant current. Dissolved oxygen in the carrier stream increased until 10 mA constant current was applied, and the current output of the oxygen electrode was about 8.0 mA. In the previous paper, the salt bridge made of agar gels containing saturated potassium chloride was used instead of Nafion1 membrane to connect with electrolytic compartments. In the case of using the salt bridge, constant current was not sufficiently applied and thereby, no more than 4.2 mA in the current value of the oxygen electrode was obtained. When a carrier solution of O2 saturated by O2 bubbling passed through the oxygen electrode, the current value of the oxygen electrode was about 9.0 (data was not shown). 3.2. Determination of L-ascorbic acid by using the laccase column Amperometric biosensing of an L-ascorbic acid was carried out by using a laccase column as a recognition element. Oxidation of L-ascorbate to dehydroascorbate catalyzed by laccase is as follows: 2 l-Ascorbate þ O2 ! 2 dehydroascorbate þ 2H2 O The enzyme activity is measured by detecting the consumption of dissolved oxygen in the above reaction. Twenty microliters of L-ascorbate solutions with various concentrations (0.25–20 mM) were injected into the sensing system,
Fig. 4. Time response curves to 20 ml injections of L-ascorbic acid with various concentrations.
Fig. 5. Responses to L-ascorbic acid solutions with various concentrations.
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Y. Iida et al. / Sensors and Actuators B 91 (2003) 175–179 Table 1 Determination ranges using the oxidases with or without the electrolytic device Immobilized enzyme Laccase Ascorbate oxidase Glucose oxidase
Fig. 6. Calibration graph for L-ascorbic acid with introduction of the electrolytic device. &: Determination of L-ascorbic acid by using the electrolytic device (5.0 mA applied); *: determination of L-ascorbic acid without the device.
and L-ascorbate was amperometrically monitored by using the oxygen electrode. Variation in the amount of dissolved oxygen followed by catalysis of the enzyme was continuously monitored and displayed on the pen-recorder. As shown in Fig. 4, sharp peaks from a stable baseline were reproducibly obtained. Repeatable measurements was illustrated in Fig. 5, when these concentrations of substrate solutions were introduced. The relative standard deviations of 10 repeated measurements of these samples were below 3.0%. A linear relationship was obtained in the range of 0.25–4.0 mM and the coefficient was calculated to be 0.999. To increase dissolved oxygen and enlarge the determination range of L-ascorbic acid, 5.0 mA constant current was applied to the laccase column-FIA system. Although microvesicles of oxygen gas were produced by applying the constant current, the oxygen bubbles were trapped inside the compartment and also in the damper which was placed at downstream of the compartment. As shown in Fig. 6, a wider linear range of L-ascorbate determination was obtained. The dynamic range of L-ascorbate measurement was extended from 4.0 to 10.0 mM. The coefficient was calculated to be 0.999 and the relative standard deviations for L-ascorbate determination with each concentration were below 3.0%. 3.3. Application of the electrolytic device to other oxidase column-FIA system As mentioned above, the electrolytic device could enlarge the determination range of L-ascorbic acid. To ensure this effect, this electrolytic device was applied to another oxidase column-FIA system. Ascorbate oxidase and glucose oxidase were selected and determination ranges of the substrates, Lascorbic acid and D-glucose, were measured. Taking into consideration of measurement of real samples, ascorbate oxidase has much advantages over laccase because of the selectivity. Needless to say, glucose oxidase is suitable for measurement of real samples. As shown in Table 1, both determination ranges of these substrates were extended from 4.0 to 9.0 mM by using the
Substrate
L-Ascorbate L-Ascorbate D-Glucose
Determination range (mM) Without the device
With the device
0.25–4.0 0.25–4.0 0.25–4.0
0.25–10.0 0.25–9.0 0.25–9.0
electrolytic device. It is not elucidated clearly why the similar extension of determination range can be exhibited among these columns. Because several factors were involved in the reason of agreement, for example, the size of FIA system, immobilization yield, activity of these immobilized column, sample injection volume, etc. However, it is indicated that about 2-fold wide determination range was obtained by introducing the electrolytic device, in all cases.
4. Conclusion In this study, we developed a novel system which could increase a dissolved oxygen in carrier solution of the FIA system by electrolysis of the flow streams. This electrolytic device could extend the determination range for L-ascorbate using the immobilized laccase column as a recognition element. This system was applicable not only to laccase but also to other oxidase (ascorbate oxidase and glucose oxidase). The system should be promising as a mediatorless biosensing with a wide dynamic range. References [1] M. Przybyt, Influence of anions on glucose electrode response: application to extending concentration range, Biosens. Bioelectron. 13 (1998) 471–477. [2] K. Li, F. Xu, K.-E.L. Eriksson, Comparison of fungal laccases and redox mediators in oxidation of a nonphenolic lignin model compound, Appl. Environ. Microbiol. 65 (1999) 2654–2660. [3] A. Majcherczyk, C. Johannes, Radical mediated indirect oxidation of a PEG-coupled polycyclic aromatic hydrocarbon (PAH) model compound by fungal laccase, Biochim. Biophys. Acta 1474 (2000) 157–162. [4] C. Johannes, A. Majcherczyk, Natural mediators in the oxidation of polycyclic aromatic hydrocarbons by laccase mediator systems, Appl. Environ. Microbiol. 66 (2000) 524–528. [5] B. Xie, B. Danielsson, An integrated thermal biosensor array for multianalyte determination demonstrated with glucose, urea and penicillin, Anal. Lett. 29 (1996) 1921–1932. [6] Y. Iida, T. Satoh, I. Satoh, Application of an electrolytic device to an FIA system for extension of the determination range of L-ascorbic acid, Electrochemistry 70 (2002) 37–39. [7] R.A. Verdini, C.M. Lagier, Voltammetric iodometric titration of ascorbic acid with dead-stop end-point detection in fresh vegetables and fruit samples, J. Agric. Food Chem. 48 (2000) 2812–2817. [8] C.W. Bradberry, R.N. Adams, Flow injection analysis with an enzyme reactor bed for determination of ascorbic acid in brain tissue, Anal. Chem. 55 (1983) 2439–2440.
Y. Iida et al. / Sensors and Actuators B 91 (2003) 175–179 [9] M. Giroux, B. Outtara, R. Yefsah, W. Smoragiewicz, L. Saucier, M. Lacroix, Combined effect of ascorbic acid and gamma irradiation on microbial and sensorial characteristics of beef patties during refrigerated storage, J. Agric. Food Chem. 49 (2001) 919–925. [10] Z. Ayhan, H.W. Yeom, Q.H. Zhang, D.B. Min, Flavor, color, and vitamin C retention of pulsed electric field processed orange juice in different packaging materials, J. Agric. Food Chem. 49 (2001) 669– 674. [11] I. Satoh, I. Sakurai, Use of a laccase-column for flow-injection calorimetry, Ann. NY Acad. Sci. 864 (1998) 493–496.
Biographies Yasuhiro Iida received his BEng, MEng and doctorate in Engineering degrees from Tokyo University of Agriculture and Technology in 1995, 1997 and 2000, respectively. He is a research associate of Kanagawa
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Institute of Technology since 2000. His current interests include analysis of biomaterials by using immobilized enzymes and development of flowinjection biosensing. Takuya Kikuchi received his BEng from Kanagawa Institute of Technology in 2002, and now a graduate student in the Graduate School of Yokohama National University. His current interest is development of SOFC (solid oxide fuel cell). Ikuo Satoh received his BEng degree from Waseda University in 1970 and MEng and doctorate in Engineering degrees from Tokyo Institute of Technology in 1972 and 1977, respectively. He is a professor of Kanagawa Institute of Technologysince 1995. His current interests include flow injection analysis of bio-related compounds in combination with immobilized biocatalysts, microdetermination of heavy metal ions based on apoenzyme reactivation methods and flow-injection enthalpimetry for enzyme-catalyzed reactions.
Electrochemical enhancement for flow-amperometric biosensing with an oxidase column Yasuhiro Iida, Takuya Kikuchi, Ikuo Satoh* Department of Applied Chemistry, Faculty of Engineering, Kanagawa Institute of Technology, 1030 Shimo-Ogino, Atsugi-shi, Kanagawa-ken 243-0292, Japan
Abstract An electrolytic device to enlarge a determination range for biosensing with use of oxidase was fabricated and introduced into a flowinjection amperometric system for L-ascorbate. The biosensing system was assembled with the plunger pump, the electrolytic device, an immobilized laccase column unit, a flow-through type of oxygen electrode and a pen recorder. The electrolytic device enabled to increase dissolved oxygen in the flow streams by electrolysis of the carrier, and thereby, the additional oxygen attributable to electrolysis should be provided to the laccase column and contribute an activation of the laccase reaction. L-Ascorbate solutions (20 ml) with various concentrations were injected to the system without the electrolytic device and amounts of dissolved oxygen enzymatically consumed were amperometrically monitored. A linear relationship was obtained in a range of 0.25–4.0 mM. On the other hand, a wider linear range of the L-ascorbic acid determination was obtained with use of the electrolytic device (0.25–10.0 mM). Not only laccase but other oxidases, such as ascorbate oxidase and glucose oxidase, were immobilized and introduced into this electrolytic FIA system. Determination ranges of the substrates were also enlarged by using the system. This study indicated that use of the electrolytic device enabled to extend the dynamic range of substrate for the oxidases without any mediators. # 2003 Elsevier Science B.V. All rights reserved. Keywords: Dissolved oxygen; Electrolytic device; Laccase; Mediatorless
1. Introduction Biosensors are widely used for quantitative determination of bio-related compounds and many kinds of enzyme are used as a recognition element. Among these sensors, immobilized oxidases are particularly used as a recognition element for these sensing system. Since the response of these sensing system is limited by the concentration of dissolved oxygen in buffer solution, the pre-dilution of samples is usually required [1]. Therefore, determination rage of the sensors depends on the amount of dissolved oxygen in the assay system. As another way to overcome this limitation of the response and extend the determination range, development of mediators for the enzyme reaction and an increase in dissolved oxygen by bubbling pure O2 gas have been attempted [2–5]. If the increase in dissolved oxygen can be achieved much more easily, the method will have many advantages over the use of mediators, for example, low cost, clean and applicable to many other oxidases, etc.
* Corresponding author. Tel./fax: þ81-46-291-3105. E-mail address: [email protected] (I. Satoh).
In the previous paper [6], we reported a novel method for generating oxygen gas by the electrolysis of buffer streams. In the method, an electrolytic device was developed and agar gel containing saturated KCl was used as a salt bridge which was a part of the device. This agar gel was used not to mix produced oxygen and hydrogen by electrolysis of carrier solution. However, sufficient current to produce oxygen by electrolysis could not be applied by using this agar type of salt bridge because of its high resistance. Therefore, in this report, a nobel electrolytic device was fabricated by using Nafion1 117 membrane instead of the salt bridge to make more large current applied. Then, the electrolytic device was introduced to a laccase column-FIA (flow-injection analysis) system for determination of L-ascorbic acid. L-Ascorbic acid, well known as vitamin C, is used in many fields such as food, cosmetics, medicine, etc. Therefore, the determination of L-ascorbic acid provides important information not only for quality control of food products in food industry but also for clinical chemistry [7]. So, a rapid and simple method for determining L-ascorbic acid is demanded [8–10]. In this study, we proposed an FIA system with the electrolytic device and demonstrated performance characteristics of the mediatorless system for the determination of
0925-4005/03/$ – see front matter # 2003 Elsevier Science B.V. All rights reserved. doi:10.1016/S0925-4005(03)00085-6
176
Y. Iida et al. / Sensors and Actuators B 91 (2003) 175–179
L-ascorbic acid by using the laccase column as a recognition element. Furthermore, other immobilized oxidases were also applied into this FIA system with the electrolytic device.
2. Experiments 2.1. Materials Laccase (from Trametes sp. Ha 1) and ascorbate oxidase (from cucumber) were generously provided by Daiwa Kasei (Osaka) and Asahi Kasei (Tokyo), respectively. Glucose oxidase (from Aspergillus niger) was purchased from Sigma. Controlled-pore glass (CPG, mean pore diameter 24.2 nm, particle size 120–200 mesh) was purchased from Funakoshi (Tokyo). Perfluorinated ion exchange membrane, Nafion1 117 (0.007 in. thick), was purchased from SigmaAldrich. L-Ascorbic acid (substrate for a laccase and an ascorbate oxidase) and D(þ)-glucose (substrate for a glucose oxidase) were purchased from Merck (Darmstadt, Germany), and all other reagents were commercially available in analytical grades and were used without further purification. 2.2. Electrolytic device In order to extend the determination range of substrates of oxidase, additional oxygen in the carrier stream should be provided for the oxidase reaction. To generate oxygen electrolytically in the buffer stream, an electrolytic device was originally developed. Polymer plates made of acrylic resins (thickness: 2 mm) were pasted by dichloromethane solutions for constructing an electrolytic device and the diagram is schematically illustrated in Fig. 1. This electrolytic device was constructed with two compartments. One was equipped with two stainless tubings (2.0 mm (outer diameterÞ 1:0 mm (inner diameterÞ 30:0 mm (length); generously provided by ABLE, Tokyo) for carrier streams. A stainless tubing at upstream of the carrier streams was used as a working electrode to produce an oxygen in the
Fig. 1. Schematic drawing of the electrolytic device. WE: working electrode; CE: current electrode (platinum plate); RE: reference electrode (Ag/AgCl, in the KCl saturated); NM: Nafion1 117 membrane.
carrier streams. The other compartment was filled with carrier solution as an electrolyte, and a platinum plate which was used as a counter electrode and a reference electrode (Ag/AgCl, in the KCl saturated) were soaked in the solution. These compartments were connected with each other through a Nafion1 membrane. Therefore, oxygen and hydrogen gases were produced at different compartment each other by applying a constant current between the stainless tubing and platinum plate electrodes with the galvanostat. Dissolved oxygen in the carrier stream was increased by the electrolysis and provided to the laccase column. 2.3. Flow system and procedure Laccase was covalently immobilized onto alkylaminated CPG as previously described [11]. The immobilized preparations were packed into a small polymer column and then mounted in a water-jacketed holder. The enzyme column (0.3 ml packed volume) was used as a recognition element for L-ascorbic acid. A schematic diagram of the flow system is shown in Fig. 2. The system was assembled with a double-plunger pump (flow rate 1.0 ml/min, PU1508i; JASCO, Tokyo), an electrolytic device, a galvanostat (NPGS-2501; Nikko Keisoku, Atsugi) for applying constant current to the electrolytic device, an injection valve (Syringe Loading Sample Injector 7120, Rheodyne, California, USA) equipped with a 20 ml sample loop, the laccase column surrounded by the water jacket maintained at 303 K, the flow-through type of a polarographic oxygen electrode for monitoring the dissolved oxygen enzymatically consumed, the potentio-galvanostat (Potentiostat/Galvanostat HA-301; Hokuto Denko, Tokyo, Japan), the ammeter (Zero Shunt Ammeter HM-104; Hokuto Denko) and the pen recorder (Multi-Pen Recorder; RIKADENKI, Tokyo).
Fig. 2. Schematic drawing of the FIA system with the electrolytic device. (1) Carrier reservoir; (2) double-plunger pump (flow rate 1.0 ml/min); (3) electrolytic device; (4) galvanostat; (5) damper; (6) injection valve; (7) water jacket; (8) enzyme column; (9) O2 electrode; (10) waste; (11) electrometer; (12) potentio-galvanostat; (13) pen recorder.
Y. Iida et al. / Sensors and Actuators B 91 (2003) 175–179
Citrate buffer solution (50 mM) as the carrier was continuously pumped through the system. The sample solutions were introduced into the system through the rotary injection valve. The catalytic activity of the enzyme-packed column was assessed by injecting 20 ml of L-ascorbate solutions with various concentrations. Variation in the dissolved oxygen caused by the laccase-catalyzed reaction was monitored by the polarographic oxygen electrode and then recorded. To apply other oxidases to this FIA system, in the similar way, ascorbate oxidase and glucose oxidase were immobilized onto CPG separately, and packed into a small column, respectively. These column units were introduced into the FIA system instead of the laccase column unit. Fifty millimolar phosphate buffer (pH 6.0) and same concentration of phosphate buffer (pH 7.0) were used as a carrier in the case of using ascorbate oxidase and glucose oxidase, in this FIA system, respectively.
3. Results and discussions 3.1. Increasing of dissolved oxygen by the electrolytic device A determination range of substrates for oxidase by FIA system is limited by the concentration of oxygen in the carrier streams because oxidases, such as laccase, ascorbate oxidase and glucose oxidase, require dissolved oxygen. Therefore, in many cases, redox mediators have been used to raise these oxidase activities instead of addition of O2. In contrast, we proposed an electrochemical method instead of a chemical method to be employed to increase dissolved oxygen in the flow stream. To increase the amount of dissolved oxygen, the electrolytic device was introduced into the amperometric FIA system and various constant currents (0–10.0 mA) were applied to the electrolytic device. As shown in Fig. 3, the current output of the oxygen electrode increased with increment of the applied current. A linear relationship between the current of the oxygen electrode and the constant current up to 3 mA was obtained. It was shown that increasing of dissolved oxygen depended on the applied constant current because the current of the oxygen electrode increased with increasing of the applied
Fig. 3. Effect of current on the amperometric response.
177
constant current. Dissolved oxygen in the carrier stream increased until 10 mA constant current was applied, and the current output of the oxygen electrode was about 8.0 mA. In the previous paper, the salt bridge made of agar gels containing saturated potassium chloride was used instead of Nafion1 membrane to connect with electrolytic compartments. In the case of using the salt bridge, constant current was not sufficiently applied and thereby, no more than 4.2 mA in the current value of the oxygen electrode was obtained. When a carrier solution of O2 saturated by O2 bubbling passed through the oxygen electrode, the current value of the oxygen electrode was about 9.0 (data was not shown). 3.2. Determination of L-ascorbic acid by using the laccase column Amperometric biosensing of an L-ascorbic acid was carried out by using a laccase column as a recognition element. Oxidation of L-ascorbate to dehydroascorbate catalyzed by laccase is as follows: 2 l-Ascorbate þ O2 ! 2 dehydroascorbate þ 2H2 O The enzyme activity is measured by detecting the consumption of dissolved oxygen in the above reaction. Twenty microliters of L-ascorbate solutions with various concentrations (0.25–20 mM) were injected into the sensing system,
Fig. 4. Time response curves to 20 ml injections of L-ascorbic acid with various concentrations.
Fig. 5. Responses to L-ascorbic acid solutions with various concentrations.
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Y. Iida et al. / Sensors and Actuators B 91 (2003) 175–179 Table 1 Determination ranges using the oxidases with or without the electrolytic device Immobilized enzyme Laccase Ascorbate oxidase Glucose oxidase
Fig. 6. Calibration graph for L-ascorbic acid with introduction of the electrolytic device. &: Determination of L-ascorbic acid by using the electrolytic device (5.0 mA applied); *: determination of L-ascorbic acid without the device.
and L-ascorbate was amperometrically monitored by using the oxygen electrode. Variation in the amount of dissolved oxygen followed by catalysis of the enzyme was continuously monitored and displayed on the pen-recorder. As shown in Fig. 4, sharp peaks from a stable baseline were reproducibly obtained. Repeatable measurements was illustrated in Fig. 5, when these concentrations of substrate solutions were introduced. The relative standard deviations of 10 repeated measurements of these samples were below 3.0%. A linear relationship was obtained in the range of 0.25–4.0 mM and the coefficient was calculated to be 0.999. To increase dissolved oxygen and enlarge the determination range of L-ascorbic acid, 5.0 mA constant current was applied to the laccase column-FIA system. Although microvesicles of oxygen gas were produced by applying the constant current, the oxygen bubbles were trapped inside the compartment and also in the damper which was placed at downstream of the compartment. As shown in Fig. 6, a wider linear range of L-ascorbate determination was obtained. The dynamic range of L-ascorbate measurement was extended from 4.0 to 10.0 mM. The coefficient was calculated to be 0.999 and the relative standard deviations for L-ascorbate determination with each concentration were below 3.0%. 3.3. Application of the electrolytic device to other oxidase column-FIA system As mentioned above, the electrolytic device could enlarge the determination range of L-ascorbic acid. To ensure this effect, this electrolytic device was applied to another oxidase column-FIA system. Ascorbate oxidase and glucose oxidase were selected and determination ranges of the substrates, Lascorbic acid and D-glucose, were measured. Taking into consideration of measurement of real samples, ascorbate oxidase has much advantages over laccase because of the selectivity. Needless to say, glucose oxidase is suitable for measurement of real samples. As shown in Table 1, both determination ranges of these substrates were extended from 4.0 to 9.0 mM by using the
Substrate
L-Ascorbate L-Ascorbate D-Glucose
Determination range (mM) Without the device
With the device
0.25–4.0 0.25–4.0 0.25–4.0
0.25–10.0 0.25–9.0 0.25–9.0
electrolytic device. It is not elucidated clearly why the similar extension of determination range can be exhibited among these columns. Because several factors were involved in the reason of agreement, for example, the size of FIA system, immobilization yield, activity of these immobilized column, sample injection volume, etc. However, it is indicated that about 2-fold wide determination range was obtained by introducing the electrolytic device, in all cases.
4. Conclusion In this study, we developed a novel system which could increase a dissolved oxygen in carrier solution of the FIA system by electrolysis of the flow streams. This electrolytic device could extend the determination range for L-ascorbate using the immobilized laccase column as a recognition element. This system was applicable not only to laccase but also to other oxidase (ascorbate oxidase and glucose oxidase). The system should be promising as a mediatorless biosensing with a wide dynamic range. References [1] M. Przybyt, Influence of anions on glucose electrode response: application to extending concentration range, Biosens. Bioelectron. 13 (1998) 471–477. [2] K. Li, F. Xu, K.-E.L. Eriksson, Comparison of fungal laccases and redox mediators in oxidation of a nonphenolic lignin model compound, Appl. Environ. Microbiol. 65 (1999) 2654–2660. [3] A. Majcherczyk, C. Johannes, Radical mediated indirect oxidation of a PEG-coupled polycyclic aromatic hydrocarbon (PAH) model compound by fungal laccase, Biochim. Biophys. Acta 1474 (2000) 157–162. [4] C. Johannes, A. Majcherczyk, Natural mediators in the oxidation of polycyclic aromatic hydrocarbons by laccase mediator systems, Appl. Environ. Microbiol. 66 (2000) 524–528. [5] B. Xie, B. Danielsson, An integrated thermal biosensor array for multianalyte determination demonstrated with glucose, urea and penicillin, Anal. Lett. 29 (1996) 1921–1932. [6] Y. Iida, T. Satoh, I. Satoh, Application of an electrolytic device to an FIA system for extension of the determination range of L-ascorbic acid, Electrochemistry 70 (2002) 37–39. [7] R.A. Verdini, C.M. Lagier, Voltammetric iodometric titration of ascorbic acid with dead-stop end-point detection in fresh vegetables and fruit samples, J. Agric. Food Chem. 48 (2000) 2812–2817. [8] C.W. Bradberry, R.N. Adams, Flow injection analysis with an enzyme reactor bed for determination of ascorbic acid in brain tissue, Anal. Chem. 55 (1983) 2439–2440.
Y. Iida et al. / Sensors and Actuators B 91 (2003) 175–179 [9] M. Giroux, B. Outtara, R. Yefsah, W. Smoragiewicz, L. Saucier, M. Lacroix, Combined effect of ascorbic acid and gamma irradiation on microbial and sensorial characteristics of beef patties during refrigerated storage, J. Agric. Food Chem. 49 (2001) 919–925. [10] Z. Ayhan, H.W. Yeom, Q.H. Zhang, D.B. Min, Flavor, color, and vitamin C retention of pulsed electric field processed orange juice in different packaging materials, J. Agric. Food Chem. 49 (2001) 669– 674. [11] I. Satoh, I. Sakurai, Use of a laccase-column for flow-injection calorimetry, Ann. NY Acad. Sci. 864 (1998) 493–496.
Biographies Yasuhiro Iida received his BEng, MEng and doctorate in Engineering degrees from Tokyo University of Agriculture and Technology in 1995, 1997 and 2000, respectively. He is a research associate of Kanagawa
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Institute of Technology since 2000. His current interests include analysis of biomaterials by using immobilized enzymes and development of flowinjection biosensing. Takuya Kikuchi received his BEng from Kanagawa Institute of Technology in 2002, and now a graduate student in the Graduate School of Yokohama National University. His current interest is development of SOFC (solid oxide fuel cell). Ikuo Satoh received his BEng degree from Waseda University in 1970 and MEng and doctorate in Engineering degrees from Tokyo Institute of Technology in 1972 and 1977, respectively. He is a professor of Kanagawa Institute of Technologysince 1995. His current interests include flow injection analysis of bio-related compounds in combination with immobilized biocatalysts, microdetermination of heavy metal ions based on apoenzyme reactivation methods and flow-injection enthalpimetry for enzyme-catalyzed reactions.