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The bioelectrochemical response of the polyaniline sarcosine oxidase électrode
Journal of Electroanalytical Chemistry 415 (1996) 7 I-77
The bioelectrochemical response of the polyaniline sarcosine oxidase electrode Yifei Yang, Shaolin Mu * Depurtment
of Chemistry,
Teacher’s
College,
Ymgzhou
University,
Yungzhou
22.5002,
People’s
Republic
o/‘Chinu
Received 27 September 1995; revised 1I March I996
Abstract Sarcosine oxidase has been immobilized in the polyaniline film by the electrochemical doping method. The response current of the polyaniline sarcosine electrode is a function of the applied potential, and increases with increasing pH in the region 7.0 to 8.75 and with increasing ionic strength of the buffer. The optimum pH of the immobilized sarcosine oxidase is 8.75. The optimum temperature is 39.6”C. The activation energy of the enzyme-catalyzed reaction is 32.0kJmol- ‘. The apparent Michaelis-Menten constant Kk is 1.7mM. The enzyme electrode has a high operational stability. The response current of the enzyme electrode increases linearly with increasing concentration of sarcosine in the range below l.OmM, and is not affected by formaldehyde at concentrations less than 0.01 mM. Thus, the polyaniline sarcosine oxidase electrode can be used to determine sarcosine concentration. Keywords: Immobilization; Sarcosine oxidase; Polyaniline film; Bioelectrochemical response
1. Introduction The determination of creatinine levels in human blood serum is a valuable indicator for estimating renal function [I]. Creatine is present in skeletal muscle and also at low concentrations in the urine of women and children. It is an important reservoir of high energy phosphate groups [2], and its levels in urine and serum are used clinically as parameters of muscle damage. In general, spectrophotometric methods are used for routine determinations of creatine and creatinine [3,4]. Recently, the determination of creatine and creatinine using an enzyme electrode has been reported. The enzyme electrode for creatine determination was prepared by immobilization of creatine amidinohydrolase and sarcosine oxidase, at one end of a glass tube, using glutaraldehyde [51. The enzyme electrode for creatinine determination was prepared by co-immobilization of creatinine amidohydrolase, creatine amidinohydrolase and sarcosine oxidase to the surface of the polypropylene membrane using glutaraldehyde [6]. The determination of the response currents for both enzyme electrodes is based on the formation of hydrogen peroxide during the catalysis of sarcosine by sarcosine oxidase. These enzyme electrodes have a fast response time, low costs, and a high reliability. However, a quite large amount of sarcosine
* Corresponding author. 0022.0728/96/Sl5,00 PI1 SOO22-0728(96)04702-X
oxidase was contained in both enzyme electrodes, since the activity of the sarcosine oxidase is rather low. Conducting polymers, such as polypyrrole and polyaniline, have received a great deal of interest as new support materials for the immobilization of enzymes [7-171. This is due to the fact that they have a high conductivity and stability in air and aqueous solutions, and the enzyme can be immobilized directly into the polymer film to form the enzyme electrode without using any agency. Therefore, we tried to immobilize sarcosine oxidase by the electrochemical doping of polyaniline film. In this paper, we report the bioelectrochemical response of the polyaniline sarcosine oxidase electrode, the kinetics of the enzyme-catalyzed reaction. and the effects of the ionic strength, pH, potential and formaldehyde, which is one of the products of the enzyme-catalyzed reaction, on the response current of the enzyme electrode.
2. Experimental 2.1. Preparation trode
of the polyaniline
sarcosine oxiduse elec-
Polyaniline film was obtained using electrolysis of 0.2 M aniline in a 1 M hydrochloric acid aqueous solution at a constant potential of 0.70 V, as described elsewhere [ 121. A
Copyright 0 1996 Elsevier Science S.A. All rights reserved.
72
Yiftii Yung. Shaolin
Mu/.iourd
nf Elrcfrocmulyrictrl
PAR Model 178 potentiostat-galvanostat with a Model 179 digital coulometer was used for the electrochemical polymerization of aniline. The thickness of the film on the platinum foil (4 X 4mm’) was controlled by the charge consumed in the electrolysis, which was 0.03 C in this case. All potentials given are referred to the SCE. The sarcosine oxidase (EC. 1.5.3.1 from the Arfhrobacter species) used for preparing the enzyme electrode was obtained from Sigma Chemical Co. (its activity was determined at pH 8.3 and 37°C). Recently, we found that the amount of enzyme doped into the film of conducting polymers is affected strongly by the ionic strength of the buffer [ 181, since there is competition between the negatively charged enzyme and the anions of the buffer during the doping process. In order to increase the amount of enzyme in the film, i.e. to enhance the response current of the enzyme electrode, a lower concentration of phosphate buffer was used to prepare the sarcosine oxidase electrode in this work. The solution used to prepare the enzyme electrode consisted of 1.Omg sarcosine oxidase and 5.0 ml of 0.05 M phosphate buffer with pH 8.0. First the polyaniline film was immersed in the phosphate buffer of pH 4.5 and reduced at - 0.5OV for 20min, in order to remove chloride ions in the electrode materials as completely as possible. Then, the reduced polyaniline film was moved into the buffer containing sarcosine oxidase and oxidized at 0.6OV for 20 min. The purpose of this was to increase the content of sarcosine oxidase in the polyaniline film. A charge of 0.0067C was passed in the incorporation of sarcosine oxidase. Sarcosine oxidase was doped in the polyaniline film to form the enzyme electrode during the oxidation process. 2.2. Measurement
of the response current
The principle of the determination of the response current is based on the formation of hydrogen peroxide during the enzyme-catalyzed reaction, which is as follows: CH,-NH-CH,-COOH
+ H,O + 0, +
H,O, + HCHO + NH ,-CH ,-COOH
(1)
Chemistry
415 119961 71-77
the measured value at the same potential. Therefore, the response current I of the enzyme electrode is equal to the difference between the measured currents I, and I,, i.e. Z = Z, - I,. The background current of the enzyme electrode, which depends on the thickness of the film, the ionic strength of the buffer, the temperature, the applied potential and the operational time, was about 20nA in the 0.05 M phosphate buffer with pH 8.27 at 0.40 V and 25°C. The determination of the response current was carried out by adding an aliquot of stock substrate into the buffer. After brief stirring (2 s) by means of a magnetic stirrer, the current in the quiescent solution was recorded [7,20,21]. It is clear that the purpose of the brief stirring before the determination of the response current is to keep a uniform concentration of the substrate throughout the solution. In our case, as described above, the solution containing sarcosine did not need stirring. The apparatus used for determining the current was a PC-l potentiostat and a YEW 3066 pen recorder. The pH value of all buffers was 8.27, except in the experiment for the effect of pH on the response current.
3. Results and discussion 3.1. Cyclic voltammetry The cyclic voltammetry of both the polyaniline film electrode and the polyaniline sarcosine oxidase electrode in 0.05M phosphate buffer with pH 8.27 was carried out using a DH-1 potentiostat-galvanostat and a 3036 X-Y recorder. The scan rate was 48mV s-‘. Before carrying out cyclic voltammetry, a bare polyaniline film was reduced in the phosphate buffer with pH 4.5 at - 0.50 V for 20min, the purpose being to remove chloride ions in the film as described above. Then, the bare polyaniline film electrode was immersed in the buffer with pH 8.0 to be oxidized at 0.6OV for 20min. The bare polyaniline film was cycled first (curve 1, Fig. l), and this film was reduced in the phosphate buffer with pH 4.5 at -0.5OV for 20min and finally moved into the buffer containing the
The hydrogen peroxide is detected by the amperometric current method [ 191 at the enzyme electrode: H,O,
+ 0, + 2Ht+
2e-
(2)
The cell used to determine the response current consisted of a polyaniline sarcosine oxidase electrode, a platinum electrode, an SCE and 0.05M phosphate buffer containing sarcosine. The determination of the response current for the enzyme electrode is described as follows. The background current I,, which was allowed to decay to a steady state, was first determined in the buffer at a given potential. Then the enzyme electrode was moved immediately into a separate buffer containing a known concentration of sarcosine. The steady state current Z, on the current-time curve in the quiescent solution was taken as
0. 08
0
1
t
0. 1
0. a
I
L
0. 3 0. 4 E/V
(18.
1
I
0.6
0. 6
I
0.7
SCEl
Fig. 1. Cyclic voltammograms: (1) polyaniline film electrode, (2) polyaniline sarcosine oxidase electrode in 0.05 M phosphate buffer with pH 8.27. Scan rate 48 mV s- ’ ; room temperature.
Yifei Yung. Shuolin
Mu / Journcrl
oj’Electroanulyticcr1
Chemistry
415 (1996)
73
71-77
OF----8 PH
9
Fig. 2. Effect of pH on the response current of the polyaniline sarcosine oxidase electrode at 0.4OV in 0.05 M phosphate buffer containing I mM sarcosine at 25°C.
Fig. 3. Effect of pH on the response current of the polyaniline sarcosine oxidase electrode at 0.4OV in 0.05 M phosphate buffer containing 0.025 mM H,O, at 25°C.
sarcosine oxidase to be oxidized at 0.60 V, i.e. the enzyme electrode was prepared using the same piece of polyaniline film. After that, the polyaniline sarcosine oxidase electrode was cycled in the buffer (curve 2, Fig. 1). It is clear that the current of the cyclic voltammogram for the polyaniline sarcosine oxidase electrode is much less than that for the polyaniline film electrode in the range 0.18 to 0.70 V. This result is similar to that for a polyaniline glucose oxidase electrode [22], and indicates that sarcosine oxidase was doped into the film. There was some difference in the treatment of the enzyme electrode, being reduced twice at - OSOV and oxidized twice at 0.60 V. This different treatment may cause a small change in the CV of the polyaniline film, but the effect of the incorporation of the enzyme on the CV is predominant, since the polyaniline film was in kinetic equilibrium for such a long reduction and oxidation time. The cyclic voltammogram of the polyaniline film in 0.05 M phosphate buffer containing 1 mM sarcosine with pH 8.27 is the same as that of curve 1 in Fig. 1, since sarcosine was not reduced or oxidized at the polyaniline film in the potential region 0.18 to 0.7 V.
hydrogen peroxide as a function of pH was carried out at 0.4OV. Fig. 3 shows the change in response current with pH in the region 7.0 to 9.0 in the 0.05 M phosphate buffer containing 0.025mM H,O,. The response current increased first with increasing pH from 7 to 8, and then changed a little as the pH increased further. This result is different from that in Fig. 2, where there is a maximum current at pH 8.75. This means that the maximum current in Fig. 2 was caused by sarcosine oxidase itself, since its optimum pH is close to that of the free enzyme as mentioned previously. Comparing the curve in Fig. 2 with the curve in Fig. 3, we can see that the behaviour of the pH dependence of the response current of the enzyme electrode in the buffer containing sarcosine is similar to that in the buffer containing hydrogen peroxide alone below pH 8.75. This means that one of the causes of the decrease in response current with decreasing pH below 8.75 (Fig. 2) is the sensitive electrochemistry of hydrogen peroxide. Curves 1 and 2 in Fig. 4 show the changes in response current of the enzyme electrode with time in the phosphate buffer containing 0.025 mM H,O, with pH 7.0 at 0.40 and 0.5OV respectively. The currents first increased with time,
3.2. Effect of pH on the response current In 0.05 M phosphate buffer containing 1 mM sarcosine the response currents of the enzyme electrode at 0.4OV as a function of pH are shown in Fig. 2. A maximum current response occurs at about pH 8.75, which is the optimum pH of the immobilized sarcosine oxidase. The optimum pH for the free sarcosine oxidase is in the range pH 8 to 9 [5,23]. This indicates that the optimum pH value was not affected by the support material, polyaniline, and therefore the electronic state and properties of the enzyme protein were not affected by polyaniline. We repeated the experiment of the effect of pH on the response current of the enzyme electrode, which indicates that the response current of the enzyme electrode is reversible in the range pH 7 to 9.
Since the electrochemical oxidation of hydrogen peroxide is also pH sensitive, an experiment for the response current of the enzyme electrode in the buffer containing
0.60
h
2
0.40 t\
4
=a.30 c-(
\\ '-
O.zO~ 0
1
1 t/mill
Fig. 4. Time dependence of the response current of the polyaniline sarcosine oxidase electrode at 0.40 V in 0.05 M phosphate buffer containing 0.025mM H,O, at 25°C: (1) 0.40, (2) 0.5OV.
74
Y@
Yang. Shmlm
Mu/Journul
of Electrounulyticul
then a current peak formed on the Z-r curve. The response time for reaching a maximum current was about 7 s at 0.40 V (curve 1, Fig. 4). From curves 1 and 2, we can see that the decrease in response current with time at 0.5OV is faster than that at 0.4OV. The latter result is to be expected, since the potential of the diffusion-controlled plateau for hydrogen peroxide was about 0.7 V [7]. As shown in Fig. 4, the results are due to the oxidation of hydrogen peroxide at the platinum surface. Since the fibril diameter of the polyaniline film is very large (about 2000.$ [24], hydrogen peroxide can reach the platinum surface by diffusion through the polyaniline film. The evidence is that the response time on the I-r curve increases with increasing thickness of the polyaniline film, and hydrogen peroxide was not oxidized at the polyaniline film itself in the potential range 0.40 to 0.7OV. The latter result was obtained by determination of the response current of the polyaniline film deposited electrochemically on the glassy carbon substrate. Also, no response current of the glassy carbon electrode was observed in the 0.05 M phosphate buffer containing hydrogen peroxide with pH 8.27 in the above potential range. 3.3. Effect of potential
on the response current
The changes in the response currents of the enzyme electrode at various potentials with time in 0.05 M phosphate buffer containing 1 mM sarcosine with pH 8.27 are recorded in Fig. 5. The response current first increases with time, and then a current plateau is formed on the Z-t curves. The response time for reaching a maximum current is about 30s. It is clear that the results in Fig. 5 are very different from that in the buffer containing hydrogen peroxide (Fig. 41, where there is a current peak and the response time is very short. Both the increase in the response time and the occurrence of the current plateau (Fig. 5) may be caused by the enzyme-catalyzed reaction. We detected the signal caused by an oxidation of hydrogen peroxide, generated by the enzyme-catalyzed reaction that
01 0
2
I
Chemistry
4/S (19961
71-77
d.zo 0.30 0.40 o.rio 0.00 0.70 E/V (VE. SCE) Fig. 6. The relationship between the response current and the potential of the enzyme electrode in 0.05 M phosphate buffer containing I mM sarcosine with pH 8.27 at 25°C.
preceded the electron transfer at the electrode. The enzyme-catalyzed reaction needed a definite time, even though very short, which delayed the response time; hydrogen peroxide was supplied continuously to the electrode because of the enzyme-catalyzed reaction, a depletion zone of hydrogen peroxide at the electrode could not be established, therefore a plateau on the Z-E curve occurred instead of a peak. The curve in Fig. 6 shows the relationship between the response current and the applied potential. The response current first increases with increasing potential, and a maximum current response occurs at 0.5OV. When the potential was over 0.5OV, the response current decreased with further increase of the potential. In general, the potential for detection of hydrogen peroxide formed during the catalytic oxidation of glucose at the glucose oxidase electrode was controlled at 0.6OV [7,25], and when the potential was over 0.6OV the response remained almost the same [ 121. In order to discover why the diffusion-controlled potential of the oxidation of hydrogen peroxide formed during the catalytic oxidation of sarcosine was less than that of the glucose oxidase electrode and the response current decreased slightly with further increase of the potential over 0.5OV, an experiment on the cyclic voltammetry of 1 mM formaldehyde in 0.05 M phosphate buffer with pH 8.27 was carried out. The working electrode for the experiment was a platinum foil (4 X 4mm’). Fig. 7 shows the cyclic voltammograms of formaldehyde. Curves
3
t/mill Fig. 5. Time dependence of the response current of the polyaniline sarcosine oxidase electrode at various potentials: (I) 0.25, (2) 0.35, (3) 0.40, (4) 0.60 V in 0.05 M phosphate buffer containing I mM sarcosine with pH 8.27 at 25°C.
0.20 03
n.40 nm
0.60 0.7~ 0.80
E/V(vs.SCE) Fig. 7. Cyclic voltammograms of I mM formaldehyde in 0.05M phosphate buffer with pH 8.27: (I) first cycle, (2) second cycle. Scan rate 48 mV s- ’ ; room temperature.
Yi$el Yung, Shaolin
Mu /Journal
of
EIectroanulyticul
1 and 2 show the results of the first and second cycle respectively. We can see that the response current for the first cycle increases rapidly with increasing potential over 0.3 V, and then changes little from 0.6 to 0.8 V. This means that the formaldehyde was oxidized over 0.3 V; the oxidation current for the second cycle (curve 2) decreased markedly compared with that of the first cycle (curve 1). The results of the cyclic voltammetry of the enzyme electrode in the above solution (the cyclic voltammograms are omitted here) are similar to those shown in Fig. 7. The above results imply that the product from the oxidation of formaldehyde was adsorbed on the electrode surface, leading to a decrease in the current. Also, formaldehyde is produced during the enzyme-catalyzed reaction. It is expected that formaldehyde will be oxidized over 0.40 V, and its product adsorbed on the enzyme electrode, leading to a decrease in both the potential of the diffusion-controlled plateau for hydrogen peroxide and the response current of the enzyme electrode over OSOV with further increasing the potential. Considering the results of Fig. 6, in which the difference between the response current at 0.4OV and that at 0.5OV is very small, we preferred to set the polyaniline sarcosine oxidase electrode at 0.4OV as an amperometric sarcosine sensor. 3.4. Effect of formaldehyde
on the response current
Formaldehyde was formed during the enzyme-catalyzed reaction (Q. (1)). In order to understand the effect of formaldehyde on the properties of the enzyme electrode, a new enzyme electrode was immersed in 0.05 M phosphate buffers containing 0.01, 0.1 and 1.OmM formaldehyde with pH 8.27. The current of the enzyme electrode in 0.01 mM formaldehyde at 0.40 V decreased continually with time, i.e. no current plateau as recorded in Fig. 5 was observed. The response currents in 0.1 and 1.OmM formaldehyde were 7 and 63 nA at 0.40 V respectively. However, the response current of the enzyme electrode at 0.40 V was 2 132 nA, in the 0.05 M phosphate buffer containing 0.1 mM hydrogen peroxide with pH 8.27. This indicates that hy-
0
0.2 0.4 0.0 0.8 Tonic strength/M
1.0 I.2
Fig. 8. Effect of the ionic strength of the buffer containing I mM sarcosine on the response current of the enzyme electrode at O&IV with pH 8.27 at 25°C.
Chemistry
415 (1996)
71-77
75
[Sarcosine]/mM Fig. 9. The relationship between the response current of the enzyme electrode at 0.4OV and the sarcosine concentration in 0.05M phosphate buffer with pH 8.27 at 25°C.
drogen peroxide was oxidized more readily than formaldehyde at 0.4OV. It is clear that the response current of formaldehyde is negligible compared with that of hydrogen peroxide. In the 0.05 M phosphate buffer containing 1 mM sarcosine and 0.01 mM formaldehyde with pH 8.27, the response current of the enzyme electrode at 0.4OV is equal to that in the phosphate buffer containing 1 mM sarcosine alone. The above results indicate that the contribution of formaldehyde to the whole measured response current is small, in agreement with the results obtained by Yamato et al. [26]. 3.5. Eflect of ionic strength on the activity of the immobilized enzyme In the buffer containing 1 mM sat-cosine at 0.4OV, the response current changes with ionic strength of the phosphate buffer and then changes a little with further increase in the ionic strength. The pH value around the electrode decreases during the electrochemical oxidation of hydrogen peroxide. Thus, the decrease in response current with decreasing ionic strength (Fig. 8) was mainly caused by the decrease in local pH at the enzyme electrode compared with the results of pH dependence of the response current in Fig. 2 (because of the low buffering capacity in the region of low ionic strength). We found the response current of the enzyme electrode after determination at 0.01 M phosphate buffer, i.e. its ionic strength at 0.03 M can be restored to its initial value at 0.05 M phosphate buffer. This means that the stability of the enzyme electrode was not affected by the ionic strength in the region 0.03 to 1.2M. The enzyme electrode was used repeatedly to determine 1 mM sarcosine in the ionic strength of 0.15 M phosphate, the results are as follows: 0.067, 0.069, 0.070, 0.069 and 0.069 p,A. The operational reproducibility is good, but the storage stability of the enzyme electrode changed slowly with time. The reproducibility of the enzyme electrode was not improved in buffers of ionic strength above 0.15 M phosphate. According to the results
Yifei Yung, Shuolin
76
Mu/Journal
~fElectroun&iccrl
Chemistry
415 (19961
71-77
1.6 2 1.,7 c 2 1.6 g - 1.5 1.4 I
0.(H)0
0.6
1.6
1.0
le33.3
I 3.4
[SarcosineJ-x/mM-x constant Kb,
as shown in Fig. 8, 0.05 M phosphate buffer was used in our experiments. The calculation of the ionic strength is based on the concentration of Na,HPO,. concentration
I 3.5
I
I 3.6
IO~T-~/K-~
Fig. 10. Determination of the apparent Michaelis-Menten for the polyaniline sarcosine oxidase electrode.
3.6. .!Cffect of substrate current
I
Fig. 12. Plot of log I vs. l/ T in the temperature range 5.3 to 28.4“C.
Michaelis-Menten constant Kk were calculated from the intercept and slope of the straight line. The maximum current response was 130 nA and K& was 1.7 mM, which is close to the 2.1 mM for the free sarcosine oxidase relating to sarcosine [27].
on the response 3.7. Effect of temperuture sarcosine
The relationship between the response current of the enzyme electrode and the sarcosine concentration at pH 8.27 is shown in Fig. 9. The enzyme electrode potential was set at 0.4OV. The response current increases linearly with increasing concentration of sarcosine in the region 0.1 to l.OmM, and then increases slowly with further increase of the sarcosine concentration. The linear region between the response current and the substrate concentration is coincident with the results obtained by Motonaka et al. [5] and Nguyen et al. [6]. A plot of I-’ vs. [sarcosine]- ‘, based on the experimental data of Fig. 9, is shown in Fig. 10. The maximum current response I,,, and the apparent
on the catalyzed reaction of
The relationship between temperature and the response current of the enzyme electrode at 0.4OV for the buffer containing 1 mM sarcosine is shown in Fig. 11. The response current increases with increasing temperature between 5.3 and 35”C, and then increases more slowly with further increase of the temperature. The maximum current response appears at 39.6”C, which is the optimum temperature for sarcosine oxidase immobilized on the polyaniline film. We must point out that the reproducibility of the magnitude of the response current is still very good at the optimum temperature. Since the electrode surface area, amount of enzyme, and substrate concentration are constant, the response current is proportional to the rate constant k. We can then replace log k with log I in the Arrhenius formula. The relationship between log I and l/T at temperatures in the range 5.3 to 28.4”C is plotted in Fig. 12. The activation energy E was calculated from the slope of the straight line to be 32.0 kJ mol- ’ .
4. Conclusion
20'
5
L 10
I 15
I 1 20 25 t/T
I 30
I 35
1 40
Fi g. II. Effect of temperature on the response current of the enzyme electrode in 0.05 M phosphate buffer containing 1mM sarcosine with pH 8.27 at 0.4OV.
Sarcosine oxidase has been immobilized in polyaniline film by electrochemical doping. The enzyme electrode prepared in this manner needed only a very small amount of sarcosine oxidase. The optimum pH of the immobilized sarcosine oxidase is close to that of the free enzyme; its optimum temperature is higher than 37°C at which the activity of the free sarcosine oxidase was determined, as
mentioned previously. This means that the properties of the immobilized sarcosine oxidase were hardly affected by polyaniline. The enzyme electrode has a good operational stability, and its response current increases linearly with increasing concentration of sarcosine in the region 0.1 to l.OmM. Thus the polyaniline sarcosine oxidase electrode can be used to determine the sarcosine concentration.
References [I] N. Tietz, Fundamentals of Clinical Chemistry, Sanders, Philadelphia, 2nd edn., 1976, pp. 994-998. [z] G. Zubay, Biochemistry, Addison-Wesley, Massachusetts, 1983. p. 883. [31 O.Z. Folin, Phys. Chem., 41 (1904) 223. [4] H. Muller. Z. Fres. Anal. Chem., 332 (1988) 464. [51 J. Motonaka, H. Takabayashi, S. lkeda and N. Tanaka, Anal. Lett.. 23 (1990) 1981. [61 Vu.K. Nguyen, Ch.M. Wolff, J.L. Seris and J.P. Schwing, Anal. Chem., 63 (1991) 61 I. [71 N.C. Foulds and C.R. Lowe, J. Chem. Sot. Faraday Trans. 1, 82 (1986) 1259. [8] M. Umana and J. Wailer, Anal. Chem., 58 (1986) 2979. [9] P.N. Bartlett and R.G. Whitaker, Biosensors, 3 (1987-88) 359.
[IO] E. Tamiya and 1. Karube, Sensors and Actuators, 18 (1989) 297. [I 11 D. Belanger, J. Nadrean and G. Fortier. J. Electroanal. Chem., 274 (1989) 143. [I21 S.L. Mu, H.G. Xue and B.D. Qian, J. Electroanal. Chem.. 304 (1991) 7. [ 131 H. Shinohara, T. Chiba and M. Aizawa, Sensors and Actuators, 13 (1988) 79. [14] J.C. Cooper and E.A.H. Hall, Biosensors, 7 (1992) 473. [151 S.L. Mu, J.Q. Kan and J.B. Zhou, J. Electroanal. Chem., 334 (1992) 121. [16] P.N. Bartlett and J.M. Cooper. J. Electroanal. Chem., 362 (1993) I. [17] S.L. Mu, J. Electroanal. Chem.. 370 (1994) 135. (181 H.G. Xue and S.L. Mu. J. Electroanal. Chem.. 397 (1995) 241. [I91 W.J. Albery, P.N. Bartlett, A.E.G. Cass, D.H. Craston and B.G.D. Haggett, J. Chem. Sot. Faraday Trans. I, 82 (1986) 1033. [20] P.C. Pandey, J. Chem. Sot. Faraday Trans. I, 84 (1988) 2259. [2l] Y. Kajiya, H. Matsumoto and H. Yoneyama, J. Electroanal. Chem., 319 (1991) 185. [22] S.L. Mu and H.G. Xue, Sensors and Actuators B, in press. [23] K. Rikitake, 1. Oka, M. Ando, T. Yoshimoto and D.J. Tsuru, J. B&hem., 86 (I 979) I 109. [24] W.S. Huang. B.D. Humphrey and A.G. MacDiarmid, J. Chem. Sot., Faraday Trans. I, 82 (I 986) 2385. [25] P.N. Bartlett and D.J. Caruana, Analyst, I I7 (1992) 1287. [26] H. Yamato, M. Ohwa and W. Wemet, Anal. Chem., 67 (1995) 2776. [27] S. Hayashi, M. Suzuki and S. Nakamura, Biochem. Biophys. Acta, 742 ( 1983) 630.
The bioelectrochemical response of the polyaniline sarcosine oxidase electrode Yifei Yang, Shaolin Mu * Depurtment
of Chemistry,
Teacher’s
College,
Ymgzhou
University,
Yungzhou
22.5002,
People’s
Republic
o/‘Chinu
Received 27 September 1995; revised 1I March I996
Abstract Sarcosine oxidase has been immobilized in the polyaniline film by the electrochemical doping method. The response current of the polyaniline sarcosine electrode is a function of the applied potential, and increases with increasing pH in the region 7.0 to 8.75 and with increasing ionic strength of the buffer. The optimum pH of the immobilized sarcosine oxidase is 8.75. The optimum temperature is 39.6”C. The activation energy of the enzyme-catalyzed reaction is 32.0kJmol- ‘. The apparent Michaelis-Menten constant Kk is 1.7mM. The enzyme electrode has a high operational stability. The response current of the enzyme electrode increases linearly with increasing concentration of sarcosine in the range below l.OmM, and is not affected by formaldehyde at concentrations less than 0.01 mM. Thus, the polyaniline sarcosine oxidase electrode can be used to determine sarcosine concentration. Keywords: Immobilization; Sarcosine oxidase; Polyaniline film; Bioelectrochemical response
1. Introduction The determination of creatinine levels in human blood serum is a valuable indicator for estimating renal function [I]. Creatine is present in skeletal muscle and also at low concentrations in the urine of women and children. It is an important reservoir of high energy phosphate groups [2], and its levels in urine and serum are used clinically as parameters of muscle damage. In general, spectrophotometric methods are used for routine determinations of creatine and creatinine [3,4]. Recently, the determination of creatine and creatinine using an enzyme electrode has been reported. The enzyme electrode for creatine determination was prepared by immobilization of creatine amidinohydrolase and sarcosine oxidase, at one end of a glass tube, using glutaraldehyde [51. The enzyme electrode for creatinine determination was prepared by co-immobilization of creatinine amidohydrolase, creatine amidinohydrolase and sarcosine oxidase to the surface of the polypropylene membrane using glutaraldehyde [6]. The determination of the response currents for both enzyme electrodes is based on the formation of hydrogen peroxide during the catalysis of sarcosine by sarcosine oxidase. These enzyme electrodes have a fast response time, low costs, and a high reliability. However, a quite large amount of sarcosine
* Corresponding author. 0022.0728/96/Sl5,00 PI1 SOO22-0728(96)04702-X
oxidase was contained in both enzyme electrodes, since the activity of the sarcosine oxidase is rather low. Conducting polymers, such as polypyrrole and polyaniline, have received a great deal of interest as new support materials for the immobilization of enzymes [7-171. This is due to the fact that they have a high conductivity and stability in air and aqueous solutions, and the enzyme can be immobilized directly into the polymer film to form the enzyme electrode without using any agency. Therefore, we tried to immobilize sarcosine oxidase by the electrochemical doping of polyaniline film. In this paper, we report the bioelectrochemical response of the polyaniline sarcosine oxidase electrode, the kinetics of the enzyme-catalyzed reaction. and the effects of the ionic strength, pH, potential and formaldehyde, which is one of the products of the enzyme-catalyzed reaction, on the response current of the enzyme electrode.
2. Experimental 2.1. Preparation trode
of the polyaniline
sarcosine oxiduse elec-
Polyaniline film was obtained using electrolysis of 0.2 M aniline in a 1 M hydrochloric acid aqueous solution at a constant potential of 0.70 V, as described elsewhere [ 121. A
Copyright 0 1996 Elsevier Science S.A. All rights reserved.
72
Yiftii Yung. Shaolin
Mu/.iourd
nf Elrcfrocmulyrictrl
PAR Model 178 potentiostat-galvanostat with a Model 179 digital coulometer was used for the electrochemical polymerization of aniline. The thickness of the film on the platinum foil (4 X 4mm’) was controlled by the charge consumed in the electrolysis, which was 0.03 C in this case. All potentials given are referred to the SCE. The sarcosine oxidase (EC. 1.5.3.1 from the Arfhrobacter species) used for preparing the enzyme electrode was obtained from Sigma Chemical Co. (its activity was determined at pH 8.3 and 37°C). Recently, we found that the amount of enzyme doped into the film of conducting polymers is affected strongly by the ionic strength of the buffer [ 181, since there is competition between the negatively charged enzyme and the anions of the buffer during the doping process. In order to increase the amount of enzyme in the film, i.e. to enhance the response current of the enzyme electrode, a lower concentration of phosphate buffer was used to prepare the sarcosine oxidase electrode in this work. The solution used to prepare the enzyme electrode consisted of 1.Omg sarcosine oxidase and 5.0 ml of 0.05 M phosphate buffer with pH 8.0. First the polyaniline film was immersed in the phosphate buffer of pH 4.5 and reduced at - 0.5OV for 20min, in order to remove chloride ions in the electrode materials as completely as possible. Then, the reduced polyaniline film was moved into the buffer containing sarcosine oxidase and oxidized at 0.6OV for 20 min. The purpose of this was to increase the content of sarcosine oxidase in the polyaniline film. A charge of 0.0067C was passed in the incorporation of sarcosine oxidase. Sarcosine oxidase was doped in the polyaniline film to form the enzyme electrode during the oxidation process. 2.2. Measurement
of the response current
The principle of the determination of the response current is based on the formation of hydrogen peroxide during the enzyme-catalyzed reaction, which is as follows: CH,-NH-CH,-COOH
+ H,O + 0, +
H,O, + HCHO + NH ,-CH ,-COOH
(1)
Chemistry
415 119961 71-77
the measured value at the same potential. Therefore, the response current I of the enzyme electrode is equal to the difference between the measured currents I, and I,, i.e. Z = Z, - I,. The background current of the enzyme electrode, which depends on the thickness of the film, the ionic strength of the buffer, the temperature, the applied potential and the operational time, was about 20nA in the 0.05 M phosphate buffer with pH 8.27 at 0.40 V and 25°C. The determination of the response current was carried out by adding an aliquot of stock substrate into the buffer. After brief stirring (2 s) by means of a magnetic stirrer, the current in the quiescent solution was recorded [7,20,21]. It is clear that the purpose of the brief stirring before the determination of the response current is to keep a uniform concentration of the substrate throughout the solution. In our case, as described above, the solution containing sarcosine did not need stirring. The apparatus used for determining the current was a PC-l potentiostat and a YEW 3066 pen recorder. The pH value of all buffers was 8.27, except in the experiment for the effect of pH on the response current.
3. Results and discussion 3.1. Cyclic voltammetry The cyclic voltammetry of both the polyaniline film electrode and the polyaniline sarcosine oxidase electrode in 0.05M phosphate buffer with pH 8.27 was carried out using a DH-1 potentiostat-galvanostat and a 3036 X-Y recorder. The scan rate was 48mV s-‘. Before carrying out cyclic voltammetry, a bare polyaniline film was reduced in the phosphate buffer with pH 4.5 at - 0.50 V for 20min, the purpose being to remove chloride ions in the film as described above. Then, the bare polyaniline film electrode was immersed in the buffer with pH 8.0 to be oxidized at 0.6OV for 20min. The bare polyaniline film was cycled first (curve 1, Fig. l), and this film was reduced in the phosphate buffer with pH 4.5 at -0.5OV for 20min and finally moved into the buffer containing the
The hydrogen peroxide is detected by the amperometric current method [ 191 at the enzyme electrode: H,O,
+ 0, + 2Ht+
2e-
(2)
The cell used to determine the response current consisted of a polyaniline sarcosine oxidase electrode, a platinum electrode, an SCE and 0.05M phosphate buffer containing sarcosine. The determination of the response current for the enzyme electrode is described as follows. The background current I,, which was allowed to decay to a steady state, was first determined in the buffer at a given potential. Then the enzyme electrode was moved immediately into a separate buffer containing a known concentration of sarcosine. The steady state current Z, on the current-time curve in the quiescent solution was taken as
0. 08
0
1
t
0. 1
0. a
I
L
0. 3 0. 4 E/V
(18.
1
I
0.6
0. 6
I
0.7
SCEl
Fig. 1. Cyclic voltammograms: (1) polyaniline film electrode, (2) polyaniline sarcosine oxidase electrode in 0.05 M phosphate buffer with pH 8.27. Scan rate 48 mV s- ’ ; room temperature.
Yifei Yung. Shuolin
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oj’Electroanulyticcr1
Chemistry
415 (1996)
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OF----8 PH
9
Fig. 2. Effect of pH on the response current of the polyaniline sarcosine oxidase electrode at 0.4OV in 0.05 M phosphate buffer containing I mM sarcosine at 25°C.
Fig. 3. Effect of pH on the response current of the polyaniline sarcosine oxidase electrode at 0.4OV in 0.05 M phosphate buffer containing 0.025 mM H,O, at 25°C.
sarcosine oxidase to be oxidized at 0.60 V, i.e. the enzyme electrode was prepared using the same piece of polyaniline film. After that, the polyaniline sarcosine oxidase electrode was cycled in the buffer (curve 2, Fig. 1). It is clear that the current of the cyclic voltammogram for the polyaniline sarcosine oxidase electrode is much less than that for the polyaniline film electrode in the range 0.18 to 0.70 V. This result is similar to that for a polyaniline glucose oxidase electrode [22], and indicates that sarcosine oxidase was doped into the film. There was some difference in the treatment of the enzyme electrode, being reduced twice at - OSOV and oxidized twice at 0.60 V. This different treatment may cause a small change in the CV of the polyaniline film, but the effect of the incorporation of the enzyme on the CV is predominant, since the polyaniline film was in kinetic equilibrium for such a long reduction and oxidation time. The cyclic voltammogram of the polyaniline film in 0.05 M phosphate buffer containing 1 mM sarcosine with pH 8.27 is the same as that of curve 1 in Fig. 1, since sarcosine was not reduced or oxidized at the polyaniline film in the potential region 0.18 to 0.7 V.
hydrogen peroxide as a function of pH was carried out at 0.4OV. Fig. 3 shows the change in response current with pH in the region 7.0 to 9.0 in the 0.05 M phosphate buffer containing 0.025mM H,O,. The response current increased first with increasing pH from 7 to 8, and then changed a little as the pH increased further. This result is different from that in Fig. 2, where there is a maximum current at pH 8.75. This means that the maximum current in Fig. 2 was caused by sarcosine oxidase itself, since its optimum pH is close to that of the free enzyme as mentioned previously. Comparing the curve in Fig. 2 with the curve in Fig. 3, we can see that the behaviour of the pH dependence of the response current of the enzyme electrode in the buffer containing sarcosine is similar to that in the buffer containing hydrogen peroxide alone below pH 8.75. This means that one of the causes of the decrease in response current with decreasing pH below 8.75 (Fig. 2) is the sensitive electrochemistry of hydrogen peroxide. Curves 1 and 2 in Fig. 4 show the changes in response current of the enzyme electrode with time in the phosphate buffer containing 0.025 mM H,O, with pH 7.0 at 0.40 and 0.5OV respectively. The currents first increased with time,
3.2. Effect of pH on the response current In 0.05 M phosphate buffer containing 1 mM sarcosine the response currents of the enzyme electrode at 0.4OV as a function of pH are shown in Fig. 2. A maximum current response occurs at about pH 8.75, which is the optimum pH of the immobilized sarcosine oxidase. The optimum pH for the free sarcosine oxidase is in the range pH 8 to 9 [5,23]. This indicates that the optimum pH value was not affected by the support material, polyaniline, and therefore the electronic state and properties of the enzyme protein were not affected by polyaniline. We repeated the experiment of the effect of pH on the response current of the enzyme electrode, which indicates that the response current of the enzyme electrode is reversible in the range pH 7 to 9.
Since the electrochemical oxidation of hydrogen peroxide is also pH sensitive, an experiment for the response current of the enzyme electrode in the buffer containing
0.60
h
2
0.40 t\
4
=a.30 c-(
\\ '-
O.zO~ 0
1
1 t/mill
Fig. 4. Time dependence of the response current of the polyaniline sarcosine oxidase electrode at 0.40 V in 0.05 M phosphate buffer containing 0.025mM H,O, at 25°C: (1) 0.40, (2) 0.5OV.
74
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Yang. Shmlm
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then a current peak formed on the Z-r curve. The response time for reaching a maximum current was about 7 s at 0.40 V (curve 1, Fig. 4). From curves 1 and 2, we can see that the decrease in response current with time at 0.5OV is faster than that at 0.4OV. The latter result is to be expected, since the potential of the diffusion-controlled plateau for hydrogen peroxide was about 0.7 V [7]. As shown in Fig. 4, the results are due to the oxidation of hydrogen peroxide at the platinum surface. Since the fibril diameter of the polyaniline film is very large (about 2000.$ [24], hydrogen peroxide can reach the platinum surface by diffusion through the polyaniline film. The evidence is that the response time on the I-r curve increases with increasing thickness of the polyaniline film, and hydrogen peroxide was not oxidized at the polyaniline film itself in the potential range 0.40 to 0.7OV. The latter result was obtained by determination of the response current of the polyaniline film deposited electrochemically on the glassy carbon substrate. Also, no response current of the glassy carbon electrode was observed in the 0.05 M phosphate buffer containing hydrogen peroxide with pH 8.27 in the above potential range. 3.3. Effect of potential
on the response current
The changes in the response currents of the enzyme electrode at various potentials with time in 0.05 M phosphate buffer containing 1 mM sarcosine with pH 8.27 are recorded in Fig. 5. The response current first increases with time, and then a current plateau is formed on the Z-t curves. The response time for reaching a maximum current is about 30s. It is clear that the results in Fig. 5 are very different from that in the buffer containing hydrogen peroxide (Fig. 41, where there is a current peak and the response time is very short. Both the increase in the response time and the occurrence of the current plateau (Fig. 5) may be caused by the enzyme-catalyzed reaction. We detected the signal caused by an oxidation of hydrogen peroxide, generated by the enzyme-catalyzed reaction that
01 0
2
I
Chemistry
4/S (19961
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d.zo 0.30 0.40 o.rio 0.00 0.70 E/V (VE. SCE) Fig. 6. The relationship between the response current and the potential of the enzyme electrode in 0.05 M phosphate buffer containing I mM sarcosine with pH 8.27 at 25°C.
preceded the electron transfer at the electrode. The enzyme-catalyzed reaction needed a definite time, even though very short, which delayed the response time; hydrogen peroxide was supplied continuously to the electrode because of the enzyme-catalyzed reaction, a depletion zone of hydrogen peroxide at the electrode could not be established, therefore a plateau on the Z-E curve occurred instead of a peak. The curve in Fig. 6 shows the relationship between the response current and the applied potential. The response current first increases with increasing potential, and a maximum current response occurs at 0.5OV. When the potential was over 0.5OV, the response current decreased with further increase of the potential. In general, the potential for detection of hydrogen peroxide formed during the catalytic oxidation of glucose at the glucose oxidase electrode was controlled at 0.6OV [7,25], and when the potential was over 0.6OV the response remained almost the same [ 121. In order to discover why the diffusion-controlled potential of the oxidation of hydrogen peroxide formed during the catalytic oxidation of sarcosine was less than that of the glucose oxidase electrode and the response current decreased slightly with further increase of the potential over 0.5OV, an experiment on the cyclic voltammetry of 1 mM formaldehyde in 0.05 M phosphate buffer with pH 8.27 was carried out. The working electrode for the experiment was a platinum foil (4 X 4mm’). Fig. 7 shows the cyclic voltammograms of formaldehyde. Curves
3
t/mill Fig. 5. Time dependence of the response current of the polyaniline sarcosine oxidase electrode at various potentials: (I) 0.25, (2) 0.35, (3) 0.40, (4) 0.60 V in 0.05 M phosphate buffer containing I mM sarcosine with pH 8.27 at 25°C.
0.20 03
n.40 nm
0.60 0.7~ 0.80
E/V(vs.SCE) Fig. 7. Cyclic voltammograms of I mM formaldehyde in 0.05M phosphate buffer with pH 8.27: (I) first cycle, (2) second cycle. Scan rate 48 mV s- ’ ; room temperature.
Yi$el Yung, Shaolin
Mu /Journal
of
EIectroanulyticul
1 and 2 show the results of the first and second cycle respectively. We can see that the response current for the first cycle increases rapidly with increasing potential over 0.3 V, and then changes little from 0.6 to 0.8 V. This means that the formaldehyde was oxidized over 0.3 V; the oxidation current for the second cycle (curve 2) decreased markedly compared with that of the first cycle (curve 1). The results of the cyclic voltammetry of the enzyme electrode in the above solution (the cyclic voltammograms are omitted here) are similar to those shown in Fig. 7. The above results imply that the product from the oxidation of formaldehyde was adsorbed on the electrode surface, leading to a decrease in the current. Also, formaldehyde is produced during the enzyme-catalyzed reaction. It is expected that formaldehyde will be oxidized over 0.40 V, and its product adsorbed on the enzyme electrode, leading to a decrease in both the potential of the diffusion-controlled plateau for hydrogen peroxide and the response current of the enzyme electrode over OSOV with further increasing the potential. Considering the results of Fig. 6, in which the difference between the response current at 0.4OV and that at 0.5OV is very small, we preferred to set the polyaniline sarcosine oxidase electrode at 0.4OV as an amperometric sarcosine sensor. 3.4. Effect of formaldehyde
on the response current
Formaldehyde was formed during the enzyme-catalyzed reaction (Q. (1)). In order to understand the effect of formaldehyde on the properties of the enzyme electrode, a new enzyme electrode was immersed in 0.05 M phosphate buffers containing 0.01, 0.1 and 1.OmM formaldehyde with pH 8.27. The current of the enzyme electrode in 0.01 mM formaldehyde at 0.40 V decreased continually with time, i.e. no current plateau as recorded in Fig. 5 was observed. The response currents in 0.1 and 1.OmM formaldehyde were 7 and 63 nA at 0.40 V respectively. However, the response current of the enzyme electrode at 0.40 V was 2 132 nA, in the 0.05 M phosphate buffer containing 0.1 mM hydrogen peroxide with pH 8.27. This indicates that hy-
0
0.2 0.4 0.0 0.8 Tonic strength/M
1.0 I.2
Fig. 8. Effect of the ionic strength of the buffer containing I mM sarcosine on the response current of the enzyme electrode at O&IV with pH 8.27 at 25°C.
Chemistry
415 (1996)
71-77
75
[Sarcosine]/mM Fig. 9. The relationship between the response current of the enzyme electrode at 0.4OV and the sarcosine concentration in 0.05M phosphate buffer with pH 8.27 at 25°C.
drogen peroxide was oxidized more readily than formaldehyde at 0.4OV. It is clear that the response current of formaldehyde is negligible compared with that of hydrogen peroxide. In the 0.05 M phosphate buffer containing 1 mM sarcosine and 0.01 mM formaldehyde with pH 8.27, the response current of the enzyme electrode at 0.4OV is equal to that in the phosphate buffer containing 1 mM sarcosine alone. The above results indicate that the contribution of formaldehyde to the whole measured response current is small, in agreement with the results obtained by Yamato et al. [26]. 3.5. Eflect of ionic strength on the activity of the immobilized enzyme In the buffer containing 1 mM sat-cosine at 0.4OV, the response current changes with ionic strength of the phosphate buffer and then changes a little with further increase in the ionic strength. The pH value around the electrode decreases during the electrochemical oxidation of hydrogen peroxide. Thus, the decrease in response current with decreasing ionic strength (Fig. 8) was mainly caused by the decrease in local pH at the enzyme electrode compared with the results of pH dependence of the response current in Fig. 2 (because of the low buffering capacity in the region of low ionic strength). We found the response current of the enzyme electrode after determination at 0.01 M phosphate buffer, i.e. its ionic strength at 0.03 M can be restored to its initial value at 0.05 M phosphate buffer. This means that the stability of the enzyme electrode was not affected by the ionic strength in the region 0.03 to 1.2M. The enzyme electrode was used repeatedly to determine 1 mM sarcosine in the ionic strength of 0.15 M phosphate, the results are as follows: 0.067, 0.069, 0.070, 0.069 and 0.069 p,A. The operational reproducibility is good, but the storage stability of the enzyme electrode changed slowly with time. The reproducibility of the enzyme electrode was not improved in buffers of ionic strength above 0.15 M phosphate. According to the results
Yifei Yung, Shuolin
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~fElectroun&iccrl
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415 (19961
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1.6 2 1.,7 c 2 1.6 g - 1.5 1.4 I
0.(H)0
0.6
1.6
1.0
le33.3
I 3.4
[SarcosineJ-x/mM-x constant Kb,
as shown in Fig. 8, 0.05 M phosphate buffer was used in our experiments. The calculation of the ionic strength is based on the concentration of Na,HPO,. concentration
I 3.5
I
I 3.6
IO~T-~/K-~
Fig. 10. Determination of the apparent Michaelis-Menten for the polyaniline sarcosine oxidase electrode.
3.6. .!Cffect of substrate current
I
Fig. 12. Plot of log I vs. l/ T in the temperature range 5.3 to 28.4“C.
Michaelis-Menten constant Kk were calculated from the intercept and slope of the straight line. The maximum current response was 130 nA and K& was 1.7 mM, which is close to the 2.1 mM for the free sarcosine oxidase relating to sarcosine [27].
on the response 3.7. Effect of temperuture sarcosine
The relationship between the response current of the enzyme electrode and the sarcosine concentration at pH 8.27 is shown in Fig. 9. The enzyme electrode potential was set at 0.4OV. The response current increases linearly with increasing concentration of sarcosine in the region 0.1 to l.OmM, and then increases slowly with further increase of the sarcosine concentration. The linear region between the response current and the substrate concentration is coincident with the results obtained by Motonaka et al. [5] and Nguyen et al. [6]. A plot of I-’ vs. [sarcosine]- ‘, based on the experimental data of Fig. 9, is shown in Fig. 10. The maximum current response I,,, and the apparent
on the catalyzed reaction of
The relationship between temperature and the response current of the enzyme electrode at 0.4OV for the buffer containing 1 mM sarcosine is shown in Fig. 11. The response current increases with increasing temperature between 5.3 and 35”C, and then increases more slowly with further increase of the temperature. The maximum current response appears at 39.6”C, which is the optimum temperature for sarcosine oxidase immobilized on the polyaniline film. We must point out that the reproducibility of the magnitude of the response current is still very good at the optimum temperature. Since the electrode surface area, amount of enzyme, and substrate concentration are constant, the response current is proportional to the rate constant k. We can then replace log k with log I in the Arrhenius formula. The relationship between log I and l/T at temperatures in the range 5.3 to 28.4”C is plotted in Fig. 12. The activation energy E was calculated from the slope of the straight line to be 32.0 kJ mol- ’ .
4. Conclusion
20'
5
L 10
I 15
I 1 20 25 t/T
I 30
I 35
1 40
Fi g. II. Effect of temperature on the response current of the enzyme electrode in 0.05 M phosphate buffer containing 1mM sarcosine with pH 8.27 at 0.4OV.
Sarcosine oxidase has been immobilized in polyaniline film by electrochemical doping. The enzyme electrode prepared in this manner needed only a very small amount of sarcosine oxidase. The optimum pH of the immobilized sarcosine oxidase is close to that of the free enzyme; its optimum temperature is higher than 37°C at which the activity of the free sarcosine oxidase was determined, as
mentioned previously. This means that the properties of the immobilized sarcosine oxidase were hardly affected by polyaniline. The enzyme electrode has a good operational stability, and its response current increases linearly with increasing concentration of sarcosine in the region 0.1 to l.OmM. Thus the polyaniline sarcosine oxidase electrode can be used to determine the sarcosine concentration.
References [I] N. Tietz, Fundamentals of Clinical Chemistry, Sanders, Philadelphia, 2nd edn., 1976, pp. 994-998. [z] G. Zubay, Biochemistry, Addison-Wesley, Massachusetts, 1983. p. 883. [31 O.Z. Folin, Phys. Chem., 41 (1904) 223. [4] H. Muller. Z. Fres. Anal. Chem., 332 (1988) 464. [51 J. Motonaka, H. Takabayashi, S. lkeda and N. Tanaka, Anal. Lett.. 23 (1990) 1981. [61 Vu.K. Nguyen, Ch.M. Wolff, J.L. Seris and J.P. Schwing, Anal. Chem., 63 (1991) 61 I. [71 N.C. Foulds and C.R. Lowe, J. Chem. Sot. Faraday Trans. 1, 82 (1986) 1259. [8] M. Umana and J. Wailer, Anal. Chem., 58 (1986) 2979. [9] P.N. Bartlett and R.G. Whitaker, Biosensors, 3 (1987-88) 359.
[IO] E. Tamiya and 1. Karube, Sensors and Actuators, 18 (1989) 297. [I 11 D. Belanger, J. Nadrean and G. Fortier. J. Electroanal. Chem., 274 (1989) 143. [I21 S.L. Mu, H.G. Xue and B.D. Qian, J. Electroanal. Chem.. 304 (1991) 7. [ 131 H. Shinohara, T. Chiba and M. Aizawa, Sensors and Actuators, 13 (1988) 79. [14] J.C. Cooper and E.A.H. Hall, Biosensors, 7 (1992) 473. [151 S.L. Mu, J.Q. Kan and J.B. Zhou, J. Electroanal. Chem., 334 (1992) 121. [16] P.N. Bartlett and J.M. Cooper. J. Electroanal. Chem., 362 (1993) I. [17] S.L. Mu, J. Electroanal. Chem.. 370 (1994) 135. (181 H.G. Xue and S.L. Mu. J. Electroanal. Chem.. 397 (1995) 241. [I91 W.J. Albery, P.N. Bartlett, A.E.G. Cass, D.H. Craston and B.G.D. Haggett, J. Chem. Sot. Faraday Trans. I, 82 (1986) 1033. [20] P.C. Pandey, J. Chem. Sot. Faraday Trans. I, 84 (1988) 2259. [2l] Y. Kajiya, H. Matsumoto and H. Yoneyama, J. Electroanal. Chem., 319 (1991) 185. [22] S.L. Mu and H.G. Xue, Sensors and Actuators B, in press. [23] K. Rikitake, 1. Oka, M. Ando, T. Yoshimoto and D.J. Tsuru, J. B&hem., 86 (I 979) I 109. [24] W.S. Huang. B.D. Humphrey and A.G. MacDiarmid, J. Chem. Sot., Faraday Trans. I, 82 (I 986) 2385. [25] P.N. Bartlett and D.J. Caruana, Analyst, I I7 (1992) 1287. [26] H. Yamato, M. Ohwa and W. Wemet, Anal. Chem., 67 (1995) 2776. [27] S. Hayashi, M. Suzuki and S. Nakamura, Biochem. Biophys. Acta, 742 ( 1983) 630.