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Electrode processes on an enzyme embodied electrode
79
Sensors and Achutors B, 13-I4 (1993) 79-81
Electrode processes on an enzyme embodied electrode Shigeru Yamauchi, Masashi Yaoita, Futoshi Matsumoto and Takahiro Yokoyama Research instituteof National RehabilitationCenter, Namiki 4-1, Tokomzawa, Saitama 359 (Japan)
Yoshihito Ikariyama Depanmenr of BioengineeringTokyo Instituteof
Technology, Nagatsuda 4259, Midori-&u,Yokohama 227 (Japan)
Abstract An enzyme embodied electrode (EEE) is an electrochemical biosensing device fabricated by direct immobilization of enzyme on a platinized electrode. In order to elucidate the reaction scheme of an EEE, the pulse voltammettic technique wris employed, in which a potentiostatic pulse was applied after the preoxidation pulse and the following open circuit period, sequentially. A reaction scheme was proposed hy taking substrate and product diffusion in the micropores of platinum black into account, besides the enzymatic and electrochemical reactions. During the open circuit period the concentration profile of hydrogen peroxide was assumed to reach a steady state when the production of hydrogen peroxide equilibrated with the diffusion at the microporous electrode. Assuming a steady state concentration profile at the balanced state, an appropriate solution was deduced for a differential equation that expresses enzymatic production, electrochemical oxidation and diffusion of hydrogen peroxide to bulk solution. This expression explained well the observed transient current that decayed exponentially and the proportionality to the glucose concentration.
1. Introduction An enzyme embodied electrode (EEE) is fabricated by direct immobilization of enzyme on a platinized electrode. Our main target concerning this biosensing device is to develop an handy type glucose sensor for diabetic patients the utility of which is convenient glucose determination of blood glucose by themselves. Incorporation of enzyme molecules to a porous matrix of platinum black made it possible to fabricate an ultramicrobiosensing device with a fast response of less than 3 s, a wide dynamic range over five orders of magnitude, and a large signal to noise ratio [l-5]. However, the detailed reaction mechanism of signal transduction has not been fully elucidated. An intriguing application of the EEE sensor was to develop a pulse voltammetric device 13, 61 where a hydrostatic measurement was attained for a droplet of sample. ‘The planar electrochemical d&x, which consists of a working electrode of a glucose sensing EEE, an Ag/AgCl reference and an platinum auxiliary electrode, were assembled in a circle of 3 mm diameter. With one droplet of sample of this device a miniature hydrostatic electrochemical sensing was operated. A potentiostatic pulse of 0.6 V was applied and the resulting transient current was investigated. It was also found that keeping the sensor in an open circuit condition before the application of the pulsed potential
MS-4005/93/$6.00
was required for a reproducible amperometric response. The transient current increased with the prolongation of the open circuit period. The response was more reproducible when a preoxidation pulse was applied before every open circuit state. The pulse voltammetric technique provides not only a practical device but also useful information for the development of a portable biosensor. This paper reports the electrodic processes of the microbiosensing device with respect to transient current-based biosensing.
2. Experimental A platinum wire of 100 pm in diameter was sealed in a soda glass capillary tube. After the electrode surface was polished, the electrode was chemicallyetched to a depth of about 50 pm with aqua regia, following that platinum black was electrochemically deposited by the potentiostatic technique. Glucose oxidase (Asp.n&r, 113 u/mg, GOD) was immobilized by the immersion of the porous electrode in an enzyme solution. Pulse voltammetric measurement was carried out with a standard electrochemical cell employing an EEE as the working electrode, a reference electrode of Ag/ AgCl and a platinum auxiliary electrode in a 0.1 M phosphate buffered solution at 30 “C!. A potential of 0.6 V was supplied by a potentiostat for the measurement
0 1993- Elsevier Sequoia. All rights reserved
80
and the preoxidation. The resulting transient current was recorded by a digital memory.
3. Results and discussion 3.1. Reaction scheme in a micropore When an EEE is immersed in glucose solution, glucose diffuses into the micropores of the EEE as illustrated in Fig. l(a). Hydrogen peroxide is produced in an enzymatic oxidation of glucose at the electrode surface. Assuming that the reaction can be approximated by one dimensional diffusion in a single tube, the material balance of glucose is expressed by the following equation ac,(x)rn =D,(~zc*(x)/~2) - r(C,(x))
(1)
where C,(x) is the glucose concentration at x, D, is the diffusion coefficient of glucose, and r(C,(x)) represents the oxidation rate of glucose catalyzed by GOD. To solve eqn. (l), we have assumed that the oxidation rate is independent of the produced hydrogen peroxide and is proportional to the glucose concentration, r(C,(x))=k,C,(x) (kg is the rate constant). Based on this assumption the following equation is easily derived by putting the left-side term of eqn. (1) to zero at steady state. C,(x) = C,” cosh( la)/cosh( ti)
(2)
where C,” is the glucose concentration in the bulk solution, 1 is the thickness of the platinum black electrode, and K=&& The material balance of the hydrogen peroxide is expressed as follows, if the anodic oxidation rate of the hydrogen peroxide is proportional to the concentration of hydrogen peroxide,
~,(X)bf =Db(~2C&)/~Z)+ k&(x) - k&(x)
(3)
where C,,(x) is the concentration of hydrogen peroxide, D,, is the diffusion coefficient of hydrogen peroxide, and k, is the electrochemical rate constant. Under
steady state conditions, without an application of potential, the following equation is derived using eqn. (2) and the boundary condition of C,,(l) =0 and [K,,(x)/ hb_o=O C,(x)=(C,bD,lD,)(l-cosh(~)/~sh(~)
(4)
Upon application of an anodic pulsed potential of 0.6 V to this steady state, the hydrogen peroxide is electrochemically oxidized as shown in Fig. l(b). As the production of hydrogen peroxide can be neglected instantly after potential application, eqn. (3) can be approximated as follows. X,(x)/at = D,(W,(x)/~2)
-k&,(x)
(5)
The initial glucose concentration is represented by eqn. (2). Using the same boundary conditions employed in the derivation of eqn. (4), a newly derived eqn. (5) gives a solution for exponential response, i.e. yielding an expression for a transient current of I=2F
k,,C,, dr=ACzi B, exp(-C,,t) s n-0 Cl
In eqn. (6), A, B,, and C, represent A = ~~FS~~,,D,K~IIT~D,, B,, = l/(?n + 1)2(K2412+ ?r2(% + 1)‘)
C,,=k,+@1+1)‘0,/41~ where S is the cross-sectional area of a micropore. The exponential function in eqn. (6) converges rapidly and we can approximate it by taking the first term I =ACgb exp( - Bt)
(7)
with a constant B represented as B = k,, + ,rr2D,/412
Equation (7) states that transient current I is proportional to glucose concentration and decreases exponentially with the passage of time. The time constant B does not depend on glucose concentration. 3.2. Transient respon.se of pulse voltammetic determination of glucose
Fig. 1. Schematic representation of electrode processes in an EEE: (a) without potential; (b) under constant potential.
Edge effect also cannot be avoided in the electrochemical deposition of platinum black on a flat platinum electrode having a diameter of 100 pm. The depth (1) of the micropores of a platinized electrode is not constant but variable. Therefore, we fabricated an electrode where diffusion of substrate and product uniform tubes can be approximated, by chemically etching a platinum electrode. By this treatment a platinum electrode of
81
Fig. 2 The EEE sensor employed for glucose determination.
40
80 TimeIms
120
Fig. 3. Transient current of EEE sensor to a potentiostatic pulse of 0.6 V. Gluc~: 1 (O), 2 (0), 5 (D), 10 (O), 20 (A) mM.
o.,-
10 1 Glucose concentration / mM
Fig. 4. Dependence of response current on glucose concentration. Transient current at 40 (O), 60 (B), 80 (0) and 100 (0) ms.
depth 1 was manufactured, which was followed by platinization. The electrode thus prepared is schematically shown in Fig. 2. The depth of a micropore hole is thus estimated to be 1. Using this electrode, we measured the transient current accompanied by a potentiostatic pulse of 0.6 V. The transient currents observed at various concentrations of glucose are shown in Fig. 3. After a rapid decay of current (up to 40 ms in Fig. 3), every curve became a straight line in a semi-log plotting, i.e. every current decayed exponentially. This behavior is prea
dieted from eqn. (7). In addition, the slopes of these straight lines were the same, which confirmed that the time constant is independent of glucose concentration. These results agreed with our scheme of glucose consumption (hydrogen peroxide generation) and the electrochemical oxidation of hydrogen peroxide in a micropore. From the slopes of the response curve in Fig. 3, we obtained the value of constant B to be 4-5 s-‘. From this value the time constant of the diffusion coupled with the enzymatic reaction in a micropore of platinum black was estimated to be in the range 0.2-0.3 s. The response time of the EEE sensor was estimated to be less than l-3 s, when glucose determination was carried out by measuring the change in the steady current in a batch-type electrochemical cell or in a flow-type cell [2,3]. The present estimation has good agreement with previous results. 3.3. Dependence of response current on glucose concentration Equation (7) states that the current should be proportional to the glucose concentration. The dependence of the transient currents at 40, 60, %I and 100 ms on glucose concentration is given in Fig. 4. All these currents were proportional to glucose concentration as long as the glucose concentration was below 10 mM, irrespective of the time after application of the pulse. This result also supports eqn. (7). In our derivation of eqn. (5), we have neglected the production of hydrogen peroxide by GOD. The steady state current with this sensor was less than l/10 of the current at 100 ms. Since the steady state current of EEE represent the enzymatic production rate of hydrogen peroxide, we have enough reason to neglect the second term of eqn. (3). References 1 Y. Ikariyama, S. Yamauchi, T. Yukiashi and H. Ushioda, Micro-enzyme electrode prepared on platinized platinum, Anal. Left, 20 (1987) 1407-1416. 2 Y. Ikariyama, S. Yamauchi, M. Aizawa, T. Yukiashi and I-L Ushioda, High performance micro-enzyme sensor using platinized platinum, Bull. Chem. SW &IL, 61 (1988) 3525-3530. 3 S. Yamauchi, Y. Ikariyama and M. Yaoita, Chemical Sensor Techndogy, Vol. 2, Kodansha, Tokyo, 1989, pp. 205-223. 4 S. Yamauchi, A. Katayama, M. Yaoita and Y. Ikariyama, Tech. Digest,8th Sensor Sjwp, Tokyo,Japan, 1989, pp. 99-102. 5 S. Yamauchi, M. Yaoita, R. Nagai, Y. Yoshida and Y. Ikariyama, Tech. Digest,9th Sensor Symp., Tokyo,Japan, 1990, pp. 189-192. 6 Y. Ikariyama, N. Shimada, S. Yamauchi, T. Yukiashi and H. Ushioda, P&c voltammetric biosensing system for the rapid determination of glucose with micro-enzyme sensor, Ana.! Len., 21 (1988) 953-964.
Sensors and Achutors B, 13-I4 (1993) 79-81
Electrode processes on an enzyme embodied electrode Shigeru Yamauchi, Masashi Yaoita, Futoshi Matsumoto and Takahiro Yokoyama Research instituteof National RehabilitationCenter, Namiki 4-1, Tokomzawa, Saitama 359 (Japan)
Yoshihito Ikariyama Depanmenr of BioengineeringTokyo Instituteof
Technology, Nagatsuda 4259, Midori-&u,Yokohama 227 (Japan)
Abstract An enzyme embodied electrode (EEE) is an electrochemical biosensing device fabricated by direct immobilization of enzyme on a platinized electrode. In order to elucidate the reaction scheme of an EEE, the pulse voltammettic technique wris employed, in which a potentiostatic pulse was applied after the preoxidation pulse and the following open circuit period, sequentially. A reaction scheme was proposed hy taking substrate and product diffusion in the micropores of platinum black into account, besides the enzymatic and electrochemical reactions. During the open circuit period the concentration profile of hydrogen peroxide was assumed to reach a steady state when the production of hydrogen peroxide equilibrated with the diffusion at the microporous electrode. Assuming a steady state concentration profile at the balanced state, an appropriate solution was deduced for a differential equation that expresses enzymatic production, electrochemical oxidation and diffusion of hydrogen peroxide to bulk solution. This expression explained well the observed transient current that decayed exponentially and the proportionality to the glucose concentration.
1. Introduction An enzyme embodied electrode (EEE) is fabricated by direct immobilization of enzyme on a platinized electrode. Our main target concerning this biosensing device is to develop an handy type glucose sensor for diabetic patients the utility of which is convenient glucose determination of blood glucose by themselves. Incorporation of enzyme molecules to a porous matrix of platinum black made it possible to fabricate an ultramicrobiosensing device with a fast response of less than 3 s, a wide dynamic range over five orders of magnitude, and a large signal to noise ratio [l-5]. However, the detailed reaction mechanism of signal transduction has not been fully elucidated. An intriguing application of the EEE sensor was to develop a pulse voltammetric device 13, 61 where a hydrostatic measurement was attained for a droplet of sample. ‘The planar electrochemical d&x, which consists of a working electrode of a glucose sensing EEE, an Ag/AgCl reference and an platinum auxiliary electrode, were assembled in a circle of 3 mm diameter. With one droplet of sample of this device a miniature hydrostatic electrochemical sensing was operated. A potentiostatic pulse of 0.6 V was applied and the resulting transient current was investigated. It was also found that keeping the sensor in an open circuit condition before the application of the pulsed potential
MS-4005/93/$6.00
was required for a reproducible amperometric response. The transient current increased with the prolongation of the open circuit period. The response was more reproducible when a preoxidation pulse was applied before every open circuit state. The pulse voltammetric technique provides not only a practical device but also useful information for the development of a portable biosensor. This paper reports the electrodic processes of the microbiosensing device with respect to transient current-based biosensing.
2. Experimental A platinum wire of 100 pm in diameter was sealed in a soda glass capillary tube. After the electrode surface was polished, the electrode was chemicallyetched to a depth of about 50 pm with aqua regia, following that platinum black was electrochemically deposited by the potentiostatic technique. Glucose oxidase (Asp.n&r, 113 u/mg, GOD) was immobilized by the immersion of the porous electrode in an enzyme solution. Pulse voltammetric measurement was carried out with a standard electrochemical cell employing an EEE as the working electrode, a reference electrode of Ag/ AgCl and a platinum auxiliary electrode in a 0.1 M phosphate buffered solution at 30 “C!. A potential of 0.6 V was supplied by a potentiostat for the measurement
0 1993- Elsevier Sequoia. All rights reserved
80
and the preoxidation. The resulting transient current was recorded by a digital memory.
3. Results and discussion 3.1. Reaction scheme in a micropore When an EEE is immersed in glucose solution, glucose diffuses into the micropores of the EEE as illustrated in Fig. l(a). Hydrogen peroxide is produced in an enzymatic oxidation of glucose at the electrode surface. Assuming that the reaction can be approximated by one dimensional diffusion in a single tube, the material balance of glucose is expressed by the following equation ac,(x)rn =D,(~zc*(x)/~2) - r(C,(x))
(1)
where C,(x) is the glucose concentration at x, D, is the diffusion coefficient of glucose, and r(C,(x)) represents the oxidation rate of glucose catalyzed by GOD. To solve eqn. (l), we have assumed that the oxidation rate is independent of the produced hydrogen peroxide and is proportional to the glucose concentration, r(C,(x))=k,C,(x) (kg is the rate constant). Based on this assumption the following equation is easily derived by putting the left-side term of eqn. (1) to zero at steady state. C,(x) = C,” cosh( la)/cosh( ti)
(2)
where C,” is the glucose concentration in the bulk solution, 1 is the thickness of the platinum black electrode, and K=&& The material balance of the hydrogen peroxide is expressed as follows, if the anodic oxidation rate of the hydrogen peroxide is proportional to the concentration of hydrogen peroxide,
~,(X)bf =Db(~2C&)/~Z)+ k&(x) - k&(x)
(3)
where C,,(x) is the concentration of hydrogen peroxide, D,, is the diffusion coefficient of hydrogen peroxide, and k, is the electrochemical rate constant. Under
steady state conditions, without an application of potential, the following equation is derived using eqn. (2) and the boundary condition of C,,(l) =0 and [K,,(x)/ hb_o=O C,(x)=(C,bD,lD,)(l-cosh(~)/~sh(~)
(4)
Upon application of an anodic pulsed potential of 0.6 V to this steady state, the hydrogen peroxide is electrochemically oxidized as shown in Fig. l(b). As the production of hydrogen peroxide can be neglected instantly after potential application, eqn. (3) can be approximated as follows. X,(x)/at = D,(W,(x)/~2)
-k&,(x)
(5)
The initial glucose concentration is represented by eqn. (2). Using the same boundary conditions employed in the derivation of eqn. (4), a newly derived eqn. (5) gives a solution for exponential response, i.e. yielding an expression for a transient current of I=2F
k,,C,, dr=ACzi B, exp(-C,,t) s n-0 Cl
In eqn. (6), A, B,, and C, represent A = ~~FS~~,,D,K~IIT~D,, B,, = l/(?n + 1)2(K2412+ ?r2(% + 1)‘)
C,,=k,+@1+1)‘0,/41~ where S is the cross-sectional area of a micropore. The exponential function in eqn. (6) converges rapidly and we can approximate it by taking the first term I =ACgb exp( - Bt)
(7)
with a constant B represented as B = k,, + ,rr2D,/412
Equation (7) states that transient current I is proportional to glucose concentration and decreases exponentially with the passage of time. The time constant B does not depend on glucose concentration. 3.2. Transient respon.se of pulse voltammetic determination of glucose
Fig. 1. Schematic representation of electrode processes in an EEE: (a) without potential; (b) under constant potential.
Edge effect also cannot be avoided in the electrochemical deposition of platinum black on a flat platinum electrode having a diameter of 100 pm. The depth (1) of the micropores of a platinized electrode is not constant but variable. Therefore, we fabricated an electrode where diffusion of substrate and product uniform tubes can be approximated, by chemically etching a platinum electrode. By this treatment a platinum electrode of
81
Fig. 2 The EEE sensor employed for glucose determination.
40
80 TimeIms
120
Fig. 3. Transient current of EEE sensor to a potentiostatic pulse of 0.6 V. Gluc~: 1 (O), 2 (0), 5 (D), 10 (O), 20 (A) mM.
o.,-
10 1 Glucose concentration / mM
Fig. 4. Dependence of response current on glucose concentration. Transient current at 40 (O), 60 (B), 80 (0) and 100 (0) ms.
depth 1 was manufactured, which was followed by platinization. The electrode thus prepared is schematically shown in Fig. 2. The depth of a micropore hole is thus estimated to be 1. Using this electrode, we measured the transient current accompanied by a potentiostatic pulse of 0.6 V. The transient currents observed at various concentrations of glucose are shown in Fig. 3. After a rapid decay of current (up to 40 ms in Fig. 3), every curve became a straight line in a semi-log plotting, i.e. every current decayed exponentially. This behavior is prea
dieted from eqn. (7). In addition, the slopes of these straight lines were the same, which confirmed that the time constant is independent of glucose concentration. These results agreed with our scheme of glucose consumption (hydrogen peroxide generation) and the electrochemical oxidation of hydrogen peroxide in a micropore. From the slopes of the response curve in Fig. 3, we obtained the value of constant B to be 4-5 s-‘. From this value the time constant of the diffusion coupled with the enzymatic reaction in a micropore of platinum black was estimated to be in the range 0.2-0.3 s. The response time of the EEE sensor was estimated to be less than l-3 s, when glucose determination was carried out by measuring the change in the steady current in a batch-type electrochemical cell or in a flow-type cell [2,3]. The present estimation has good agreement with previous results. 3.3. Dependence of response current on glucose concentration Equation (7) states that the current should be proportional to the glucose concentration. The dependence of the transient currents at 40, 60, %I and 100 ms on glucose concentration is given in Fig. 4. All these currents were proportional to glucose concentration as long as the glucose concentration was below 10 mM, irrespective of the time after application of the pulse. This result also supports eqn. (7). In our derivation of eqn. (5), we have neglected the production of hydrogen peroxide by GOD. The steady state current with this sensor was less than l/10 of the current at 100 ms. Since the steady state current of EEE represent the enzymatic production rate of hydrogen peroxide, we have enough reason to neglect the second term of eqn. (3). References 1 Y. Ikariyama, S. Yamauchi, T. Yukiashi and H. Ushioda, Micro-enzyme electrode prepared on platinized platinum, Anal. Left, 20 (1987) 1407-1416. 2 Y. Ikariyama, S. Yamauchi, M. Aizawa, T. Yukiashi and I-L Ushioda, High performance micro-enzyme sensor using platinized platinum, Bull. Chem. SW &IL, 61 (1988) 3525-3530. 3 S. Yamauchi, Y. Ikariyama and M. Yaoita, Chemical Sensor Techndogy, Vol. 2, Kodansha, Tokyo, 1989, pp. 205-223. 4 S. Yamauchi, A. Katayama, M. Yaoita and Y. Ikariyama, Tech. Digest,8th Sensor Sjwp, Tokyo,Japan, 1989, pp. 99-102. 5 S. Yamauchi, M. Yaoita, R. Nagai, Y. Yoshida and Y. Ikariyama, Tech. Digest,9th Sensor Symp., Tokyo,Japan, 1990, pp. 189-192. 6 Y. Ikariyama, N. Shimada, S. Yamauchi, T. Yukiashi and H. Ushioda, P&c voltammetric biosensing system for the rapid determination of glucose with micro-enzyme sensor, Ana.! Len., 21 (1988) 953-964.