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Miniature Clark-type oxygen electrode with a three-electrode configuration
Sensors and Acruarors B, 2 (1990) 297-303
297
Miniature Clark-type Oxygen Electrode with a Three-electrode Configuration HIROAKI SUZUKI*, AKIO SUGAMA and NAOMI KOJIMA Fujitsu Laboratories
Ltd.,
I@1 Morinosato- Wakamiya,
Atsugi 243-01 (Japan)
(Received April 28, 1990; in revised form June 25, 1990, accepted July 8, 1990)
Abstract
A Clark-type oxygen electrode with three electrodes has been fabricated using semiconductor techniques and its characteristics compared with those of double oxygen electrodes with a similar structure. The 90% response time is 40 s, the residual current at zero oxygen concentration is less than 7%, and the linearity is good. The oxygen electrode can be used for 25 h at -0.6 V. These characteristics are affected by electrochemical crosstalk between the electrodes and are improved by varying the electrode configuration. Introduction
Recent micromachining and photolithographic techniques have been used to fabricate miniature sensors. Even some chemical sensors have been made using these techniques [l-2]. Both independent and integrated sensors have been made. Sansen et al. combined a three-electrode sensor, a temperature sensor and a CMOS interface circuit [3]. The Clark-type oxygen electrode, however, needs an electrolyte and a gas-permeable membrane, and has fallen behind other sensors. It is used in fermentation control, clinical analysis and in various biosensors by immobilizing either enzymes or microorganisms which catalyse the oxidation of organic materials [4]. Clinical analysis and biosensors require inexpensive, disposable, miniature electrodes. The Clark-type oxygen electrode, which gives an amperometric output and operates in a liquid at room temperature, is essential in these fields. We previously developed a disposable miniature Clark-type electrode using a gel to hold the electrolyte solution and a directly formed gas-permeable membrane. We obtained satisfactory characteristics by using a conflguration with two gold electrodes [I. To improve the miniature oxygen electrode characteristics, we fabricated a miniature oxygen electrode with an Ag/AgCl anode [6]. Although *Author to whom correspondence should be addressed.
0925~4005/90/$3.50
some of its characteristics, such as response.time, residual current and linearity of the -calibration curve, were better than those of a similar oxygen electrode with a gold anode, it could only operate for a few hours because the Ag/AgCl anode was damaged by an electrochemical reaction that occurs during operation. This short lifetime could be extended to a practical length by changing from a two-electrode to a three-electrode configuration. In the latter the potentiostat circuit is designed so that little current flows through the reference, thus reducing the damage to the anode. Use of the three-electrode configuration could improve all the characteristics, including the stability. However, we may encounter other problems when integrating the three electrodes in a very small space. In this paper, we describe the fabrication of an oxygen electrode with a three-electrode confrguration and compare its characteristics with two-electrode ones.
Materials V-grooves were formed in non-doped ( 100) silicon wafers (2 in diameter, 350 pm thick, Japan Silicon Co.). 99.99% gold was used for the vacuum deposition. All chemicals were of reagent grade without further purification. Solutions were prepared with deionized water. A negative photoresist, OMR-83, which was used in the process and to define the sensitive area, was purchased from Tokyo Ohka Kogyo Co. Shin-etsu Silicone ES-1001 was used for the backside insulation and KR-5240 for the gas-permeable membrane. Procedure The characteristics of the oxygen electrodes were evaluated by immersing their sensitive areas in 50 ml of 10 mM phosphate buffer solution (pH 7.0) in a beaker, allowing them to equilibrate, reducing the oxygen concentration by adding sodium sulfite (Na,SO,) or by controlling the speed of agitation, and then monitoring the responses. The temperature of the buffer solution 0 Elsevier Sequoia/Printed
in The Netherlands
298
was held at 27 “C. Calibration curves were obtained by comparing the response with that of a dissolved-oxygen meter (TOA Electronics, Ltd., model DO-1B). The potential of the working electrode was held constant by a Hokuto Denko HA-5OlG potentiostat and the output current was recorded with a Yokogawa 3066 pen recorder. The applied voltage was -0.6 V or -0.8 V versus Ag/AgCl with reference to the result of the cyclic voltammogram. For comparison, the response of the oxygen electrode without the gel and the gas-permeable membrane whose sensitive area is directly exposed to a 0.1 M KC1 solution was examined by adding Na,SO,. Na$O, does not cause any electrochemical reactions on the working electrode in this configuration and at this voltage: a more negative voltage is needed. In our previous miniature oxygen electrodes with a gold cathode and anode, the completeness of the gas-permeable membrane was checked when the NazSO, was added to reduce oxygen concentration; a current increase was observed because of direct Na,SGs reduction on the cathode at the applied voltage. However, in the three-electrode system, we cannot use Na,SO, to check the completeness of the membrane because the reduction potential of the Na,SO, is more negative than that for oxygen. Instead, 200 ~1 of I&[ Fe( CN),] was added to the solution in the zero oxygen state. If the membrane was not complete, the current increased due to direct K3 [ Fe( CN),] reduction. As mentioned below (in the Stability Section), the output current of the oxygen electrode was affected by the dissolution of the reference electrode material. Therefore, the characteristics described in other Sections were taken at the tirst stable current level after the voltage was applied. The Oxygen Electrode Structure The oxygen electrode was fabricated on a 2 mm x 15 mm silicon chip using the process explained in ref. 6; its structure is shown in Fig. 1. The sensitive area was 0.2 mm x 2 mm. Only the counter electrode areas were anisotropically etched into V-grooves [7’j. A shallow groove was formed with the OMR-83 photoresist layer to fill the working electrode area and the reference electrode area with the gel. The resist layer also determined the active area. An 0.8 pm thick SiOz insulation layer was formed on the surface. The back of the wafer was covered with a hydrophobic insulator (ES-1001) over the SiOz layer. Figure 2 shows the sensitive areas of three-electrode (a) and two-electrode (b) configuration oxygen electrodes. The position and area of the working and counter electrodes were similar to those of our two-electrode contigurations for ease
Semitive area
C.E.
a
GE.
Electrolyte
I
a’ Fig. 1. Structure of the three-ecm6guration oxygen electrode. A cross section of the sensitive area is shown on the right. CE.(Au) \
Semitive area
sanaitive araa /
/
‘Anode (Au or AglAgU)
Imn
(4
(b)
Fig. 2. Sensitive area of the oxygen electrode with (a) the three-electrode and (b) the two-electrode cm6guration.
of comparison. The working and counter electrodes were made from 400 mn thick gold film and the reference electrode was made from 400 mn thick silver f&n. An intermediate chromium layer 40 nm thick was used to obtain good adhesion between these flhns and the SiOl layer. The working electrode area exposed to the electrolyte was 0.04 mm*, which was a little smaller than the cathode in the two-electrode configuration. The counter areas were the same as that of the anode for our double configuration oxygen electrode. The Ag/AgCl reference in the three-electrode system was placed between the working electrode and the counter electrode. A thin AgCl layer was formed just before the electrolyte was impregnated by immersing the sensitive area in 0.1 M FeCl, for 5 min. The stability of the Ag/AgCl electrode was confinned by applying + 0.8 V to the Ag/AgCl electrode against the gold working electrode in the two-electrode configuration. The voltage was applied for an additional 10 to 30 s if the potential was not stable. The groove was filled with calcium alginate gel containing a 0.1 M KC1 aqueous solution as an
299
electrolyte, then covered with a dip-coated gaspermeable membrane (KR-5240). The harder the gel, the better. We used 0.4% sodium alginate solution to make the gel layer. Although 0.2% sodium alginate was used in our last oxygen electrode [6], we could not make a good oxygen electrode with three electrodes using a gel of this concentration, as the dissolved AgCl immediately reached the warking electrode and was reduced there. The pads were made rather large to facilitate the connection to the potentiostat. The terminal of an IC socket can be attached to these pads and we do not have to bond gold or aluminium wires there. This made it easier to evaluate a large number of oxygen electrodes. Results and Discussion
Cyclic Voltammogram Figure 3 shows the cyclic voltammogram for a completed oxygen electrode with a gas-permeable membrane. Curve (a) shows the current for an initial oxygen-saturated state and curve (b) indicates that for zero oxygen concentration after Na,SO, was added. The curves were different below about -0.2 V versus Ag/AgCl. The variation in current reflects the decreased oxygen concentration. An oxygen sensor can be made by keeping the potential in this range. After the response was stabilized in the zero oxygen state, K3[ Fe( CN),] was added. However, there was no noticeable change in the Z-V curve, which indicated that the K3[ Fe(CN),] did not reach the surface of the working electrode. This confirmed that the KR-5240 membrane on the electrolyte worked as a gas-permeable membrane. Below about -0.6 V versus Ag/AgCl, a limiting current region was observed. If we set the
-0.8
-0.4
-0.2
(
Response Time The distribution of the 90% response time was statistically examined among the chips on a wafer. The response time we measured corresponds to the maximum response from oxygen saturation (7.9 ppm at 27 “C) to zero. Figure 4 shows distributions of the response time of the electrodes at -0.8 V and -0.6 V versus Ag/AgCl. The response time was between 20 s and 50 s. Table 1 shows the response time of the three types of oxygen electrodes. The response times of the
Iti 30
Response
time (s)
Response
0
90
(b) (a) Fig. 4. Distribution of the 90% response time for the oxygen concentration change from saturation (7.9 ppm) to zero, (a) at -0.8 V and (b) at -0.6V. Measurements were made in a 10 mM phosphate buffer (27 “C, pH 7.0).
i 40
160 Fig. 3. Cyclic voltammogram of the completed configuration oxygen electrode. Curve (a) is rated (7.9 ppm) initial state and curve (b) is state after Na,SO, was added. Measurements 10mM phosphate buffer (pH 7.0, 27 “C). 50 mV/s.
60
time (s)
TABLE 1. Comparison of the 90% response time and residual current for three types of oxygen electrodes. The residual current was expressed on a relative basis by dividing the residual current by the maximum current decrease from oxygen saturation (7.9 ppm at 27 “C) to zero. The compared data were those at -0.8 V for the three-electrode system and the Ag/AgCl anode two-electrode system and - 1.2 V for the two-gold-electrode system. Other experimental conditions were the same
Potential (v vs. Ag/AgCD -0.8
voltage in this region, the oxygen reduction current depends only on the oxygen concentration in the bulk of the inner electrolyte solution or, when there are no effects of electrochemical crosstalk in the electrolyte, in the solution outside the membrane. Although -0.6 V is slightly outside the region in Fig. 3, the current varies approximately in proportion to the oxygen concentration in the bulk, because even in this case the oxygen concentration in the vicinity of the working electrode is almost zero.
three-electrode the oxygen-satuthe zero oxygen were made in a Scan rate was
Configuration
90% response time (s)
Residual current (%)
Three-electrode
20-50 (average: 20-40 (average: 30-180 (average:
0.2-7 (average: l-20 (average: 20-60 (average:
Two-electrode (Ag/AgCl anode) Two-electrode (gold anode)
40) 30) 103)
3) 12) 37)
300
three-electrode and the two-electrode Ag/AgCl anodes were about the same. However, the response of the two-electrode gold anode was much slower than the other two. Liu et al. showed that H,O, produced on the cathode following the oxygen reduction affects the counter electrode [8]. With the gold anode, the H,O, helps to produce more oxygen, which can diffuse back to the cathode. This delays the oxygen concentration decrease when the concentration outside the membrane is decreased, and may be why the response of the gold anode oxygen electrode is slow. For comparison, we measured the response times of the triple oxygen electrodes without the gas-permeable membrane at -0.8 V and -0.6 V. The response time of the electrode was distributed around 15 s at both voltages. There seemed to be a delay before zero oxygen concentration was reached after adding Na,SO, . In the three-electrode system, the most dominant factor determining response time is the oxygen diffusion through the gas-permeable membrane. Although three types of oxygen electrodes with the same volume of electrolyte are compared here, the response time will also be affected by the electrolyte volume, because it takes more time to reduce the oxygen concentration in the electrolyte as the volume increases. The response time could also be affected by the hardness of the gel layer, because oxygen diffusion is delayed as the gel becomes harder. The response time for the reverse direction, i.e., for increasing oxygen concentration from zero to oxygen saturation, is difficult to measure accurately because there are no good ways to increase the oxygen concentration instantly to saturation. But if the oxygen electrode whose current indicated zero oxygen concentration level was transferred to another buffer solution where the oxygen was saturated, the current recovered to the oxygen-saturated level in about the same time as for the reverse direction. Residual Current The residual current at zero oxygen concentration should be as small as possible. This is particularly important if the oxygen electrode is used to determine oxygen concentration in a fermenter in which the oxygen concentration is relatively low. The residual current comes mainly from the electrochemical reduction of materials not related to oxygen reduction. Measurements were taken for all oxygen electrodes on the same wafer to evaluate the residual current. It did not change much when the potential was changed from -0.8 V to -0.6 V. This agrees with the result of the cyclic voltammogram. Figure 5 shows the distribution of the residual
Residual (a)
,Or...-
current
(nA)
Current
(nA)
Current
for
saturated
for
saturated
(nA)
_.__.~~_~
0 Residual
oxygen
current
50
100 oxygen
(nA)
(b)
Fig. 5. Distribution of the residual current at zero oxygen concentration and the current for saturated oxygen (7.9 ppm), (a) at -0.8 V and (b) at -0.6 V. Measurements were made in a 10 mM phosphate buffer (27 “C. pH 7.0).
current at -0.8 V and -0.6 V together with distribution of the current for saturated oxygen (7.9 ppm). For the three-electrode system, the current for saturated oxygen distributed from 50 to 100 nA at -0.8 V (average: 74 nA) and from 40 to 70 nA at --0.6 V (average: 57 nA). For the two applied voltages, the three types of oxygen electrodes gave about the same average level of current for saturated oxygen. The residual current was less than 7% of the maximum current variation in the response from oxygen saturation to zero (average: 2.6% at - 0.8 V and 2.1% at -0.6 V). This is as low as or a little lower than for the double electrode with Ag/AgCl anode (Table 1). (Although the average residual current is 12% for the Ag/AgCl anode two-electrode system, the value was raised because some of the electrodes gave a low current level for saturated oxygen and a high relative residual current. However, overall we can say that the residual current is not so bad compared with that of the three-electrode system.) The residual current for the three-electrode system gradually increased as it was operated for several hours and saturated at a level between 10 and 15 nA. However, it was significantly better than for the gold anode case, whose average residual current was 37%. Furthermore, the residual current of the three-electrode system was distributed in a narrow range and was more uniform than that of a two-electrode oxygen electrode with a gold or Ag/AgCl anode.
301
Although the three-electrode system has a gold counter electrode whose area is the same as the anode of the system with two gold electrodes, its response time and residual current were significantly better than those of the twogold-electrode system. This indicates that the voltage between the electrodes is effectively applied in the three-electrode system, decreasing inhibitive materials produced from the counter electrode. Figure 6 shows the residual current of the oxygen electrodes without the gas-permeable membrane. Even though the current varies over an order of magnitude, it does not depend on whether the oxygen electrode has a gas-permeable membrane. This indicates that electroactive materials which might be produced on the counter electrode do not have much effect on the response, even though the electrolyte is confined in a very small space and the electrodes are very close. Because the residual current is less than 10 nA and the current level for saturated oxygen is about 500 nA, the relative residual current is less than l%, which is negligible. The best way to reduce the residual current relatively is to improve the oxygen permeability of the membrane and to increase the current level for saturated oxygen. Calibration Curve A calibration curve for a three-electrode configuration oxygen electrode is shown in Fig. 7. Good linearity was obtained as easily as in the case of the Ag/AgCl anode two-electrode configuration. Electrochemical crosstalk often affects the linearity of the calibration curve. Such a good linearity has never been obtained in the gold anode two-electrode system. Deviations are always observed at low and high oxygen concentrations. This may be due to the oxygen produced on the gold anode.
0
(4
0
5 Residual
current
10
(nA)
0
(b)
5
80
0
2
Oxygen
4
6
concentration
8
10 bpm)
Fig. 7. Calibration curves of the three-electrode configuration oxygen electrode at -0.8 V and -0.6 V. Measurements were made in a 10 mM phosphate buffer (27 “C, pH 7.0).
Stability The gold anode oxygen electrode could operate until the content of the electrolyte solution changed significantly or the gas-permeable membrane broke. In one case, it operated for more than 40 h, but operation of the Ag/AgCl anode oxygen electrode was limited to 5 to 20 h because the anode was damaged during operation due to its thickness (400 nm) and the two-electrode configuration [6]. In the two-electrode configuration, electrochemical reaction (1) proceeds to the right on the anode throughout the electrode’s operation. Ag + Cl- z+AgCl+e-
(1)
Therefore, if the silver layer completely changes into AgCl, the current which compensates the cathodic reaction becomes almost zero, and the oxygen electrode cannot be used. Figure 8 shows the stability of the three-electrode configuration oxygen electrode in continuous operation. It does, of course, need a good reference to obtain a stable output current. The lifetime of the oxygen electrode is voltage dependent. When the applied voltage was -0.6 V ver-
10
Residual current (nA)
Fig. 6. Distribution of the residual current of the oxygen electrode without the gas-permeable membrane and the gel, and exposed to 0.1 M KCI, (a) at -0.8 V and (b) at -0.6 V. Measurements were made in a IOmM phosphate buffer (27 “C, pH 7.0).
0
5
10
15
20
25
30
Time (h)
Fig. 8. Stability of the output current of the three-electrode configuration oxygen electrode at -0.8 V and -0.6 V. The current for saturated oxygen (7.9 ppm) was monitored continuously in a 10 mM phosphate buffer (27 “C, pH 7.0).
sus Ag/AgCl, the electrode operated for about 25 h, and then a rapid increase in current was observed. When the voltage was -0.8 V, the oxygen electrode maintained a current level for about 15 h and rapid current increase was observed after that. However, the rapid decrease in current that was observed in the Ag/AgCl anode two-electrode configuration [6] was never seen. This indicated that the damage to the Ag/AgCl reference itself was negligible. However, the AgCl layer dissolved into the electrolyte solution, reached the working electrode, and was reduced to silver. The surface of the working electrode turned black after many hours of operation. This deposition of silver interfered with the oxygen reduction, which, in most cases, led to a gradual decrease in current. The H,O, which dissolves from the cathode can help dissolve the AgCl [8]. The current increase could also be ascribed to Cr.’ reduction on the working electrode. These are the major reasons for the instability seen in Fig. 8. Migration and diffusion of these ions also explain why the lifetime of the oxygen electrode is affected by the applied voltage: the higher the voltage, the shorter the lifetime. The gradient of the electric field and the concentration of the ions, both of which must be much more complicated than in an ordinary-scale system, could be large even in the bulk of the electrolyte and depend upon the applied voltage because the electrodes are placed very near to each other. In our previous oxygen electrodes, 0.2% sodium alginate was used to make the gel layer [6]. We could not obtain good results in the three-electrode case using the 0.2% concentration, especially in the case of stability of continuous operation. Even when 0.4% sodium alginate was used, a better stability was obtained if some of the water in the gel was released and the gel hardened. This shows that the gel layer reduced the contamination of the working electrode. Although the working electrode was 50 pm from the reference electrode, this distance was too short to give good stability. Based on the results obtained here, we can construct an improved miniature oxygen electrode by the following strategy. (1) The working electrode (cathode) and the counter electrode (anode) should be placed as far apart as possible. (2) Although in a usual electrochemical experiment the reference electrode is placed near to the working electrode, we should place the reference electrode as far away from the working electrode as possible. Ohmic drop will be negligible in the miniature electrode system even if we separate these electrodes. (3) To reduce electrochemical crosstalk, it is better to form each electrode in isolated grooves
and to connect these grooves by long narrow grooves. (4) The electrochemical crosstalk will be reduced by using an electrolyte impregnated in a gel. The gel layer suppresses diffusion of inhibitive electroactive materials. However, the hardness of the gel must be optimized, because it could delay the response. As mentioned before, the miniature two-electrode system with Ag/AgCl anode is not practical because of its short lifetime. The two-electrode gold anode system, however, could be upgraded like the three-electrode system by following the aforementioned strategy to eliminate the electrochemical crosstalk.
Conclusions A miniature Clark-type oxygen electrode with a three-electrode configuration was fabricated. By comparing its characteristics with those of twoelectrode configurations, it was found that electrochemical crosstalk affected the response time, residual current, linearity of the calibration curve and stability of continuous operation. These characteristics were significantly improved by using the three-electrode configuration.
Acknowledgements We thank Dr I. Karube and Dr E. Tamiya of the University of Tokyo for their helpful discussions in preparing this report.
References 1 K. Najafi, Integrated sensors in biological environments, Sensors and Aciuarors, Bl (1990) 4533459. R. S. Pickard, P. Wall. M. Ubeid, G. Ensell and K. H. Leong, Recording neural activity in the honeybee brain with micromachined silicon sensors, Sensors and Actuators, Bf (1990) 460-463. S. J. Tanghe, N. Najafi and K. D. Wise, A planar IrO multichannel stimulating electrode for use in neural prostheses. Sensors and Actuafors, Bl ( 1990) 464.-467. 2 0. Prohaska. in A. P. F. Turner, I. Karube and G. S. Wilson (eds.). Bio.~ensors-PFundamental.s and Applicarions, Oxford University Press, Oxford, 1987, p. 377. 3 W. Sansen, D. De. Wachter, L. Callewaert, M. Lambrechts and A. Claes. A smart sensor for the voltammetric measurement of oxygen or glucose concentrations, Sensors and Actuaiors, Bf (1990) 298 302. 4 I. Karube, in A. P. F. Turner, 1. Karube and G. S. Wilson (eds.), Biosensors -Fundamentals and Applications, Oxford University Press, Oxford, 1987, p. 471. L. C. Clark, Jr.. in A. P. F. Turner, I. Karube and G. S. Wilson (eds.), Biosensors ~- Fundamentak and Applications. Oxford University Press, Oxford, 1987, p. 3. 5 H. Suzuki. E. Tamiya and I. Karube, Development of the micro-oxygen electrode and its application to a micro-glu-
303 case sensor, Proc. Mater. Res. Sot. ht. Meet. Adwmced Materials, Tokyo, Japan, May 1988, pp. 115-120. H. Suzuki, A. Sugama and N. Kojima, Effect of anode materials on the characteristics of the miniature Clark-type oxygen electrode, Anal. Chitn. Acta, 233 (1990) 275-280. K.-k. Bean, Anisotropic etching of &on, IEEE Trans. Electron Devices. ED-25 f 1978) 1185-l 193. C. S. Cha, M. J: Shao add C’C. Liu, Problems associated with the miniaturization of a voltammetric oxygen sensor: chemical crosstalk among electrodes, Sensors and Actuators, B, 2 (1990) 277-281.
Biographies Hiroaki Suzuki received a M.E. degree in applied
physics from the University of Tokyo. In 1983, he joined Fujitsu Laboratories, Ltd. His current interest is the fabrication of miniature biosensors using micromachining techniques. Naomi Kojima received a B.S. degree in biochemistry from Tokyo Metropolitan University. In 1985, she joined Fujitsu Laboratories, Ltd. Akio Sugama received a B.S. degree in biochemistry from Tohoku University. In 1985, he joined Fujitsu Laboratories, Ltd.
297
Miniature Clark-type Oxygen Electrode with a Three-electrode Configuration HIROAKI SUZUKI*, AKIO SUGAMA and NAOMI KOJIMA Fujitsu Laboratories
Ltd.,
I@1 Morinosato- Wakamiya,
Atsugi 243-01 (Japan)
(Received April 28, 1990; in revised form June 25, 1990, accepted July 8, 1990)
Abstract
A Clark-type oxygen electrode with three electrodes has been fabricated using semiconductor techniques and its characteristics compared with those of double oxygen electrodes with a similar structure. The 90% response time is 40 s, the residual current at zero oxygen concentration is less than 7%, and the linearity is good. The oxygen electrode can be used for 25 h at -0.6 V. These characteristics are affected by electrochemical crosstalk between the electrodes and are improved by varying the electrode configuration. Introduction
Recent micromachining and photolithographic techniques have been used to fabricate miniature sensors. Even some chemical sensors have been made using these techniques [l-2]. Both independent and integrated sensors have been made. Sansen et al. combined a three-electrode sensor, a temperature sensor and a CMOS interface circuit [3]. The Clark-type oxygen electrode, however, needs an electrolyte and a gas-permeable membrane, and has fallen behind other sensors. It is used in fermentation control, clinical analysis and in various biosensors by immobilizing either enzymes or microorganisms which catalyse the oxidation of organic materials [4]. Clinical analysis and biosensors require inexpensive, disposable, miniature electrodes. The Clark-type oxygen electrode, which gives an amperometric output and operates in a liquid at room temperature, is essential in these fields. We previously developed a disposable miniature Clark-type electrode using a gel to hold the electrolyte solution and a directly formed gas-permeable membrane. We obtained satisfactory characteristics by using a conflguration with two gold electrodes [I. To improve the miniature oxygen electrode characteristics, we fabricated a miniature oxygen electrode with an Ag/AgCl anode [6]. Although *Author to whom correspondence should be addressed.
0925~4005/90/$3.50
some of its characteristics, such as response.time, residual current and linearity of the -calibration curve, were better than those of a similar oxygen electrode with a gold anode, it could only operate for a few hours because the Ag/AgCl anode was damaged by an electrochemical reaction that occurs during operation. This short lifetime could be extended to a practical length by changing from a two-electrode to a three-electrode configuration. In the latter the potentiostat circuit is designed so that little current flows through the reference, thus reducing the damage to the anode. Use of the three-electrode configuration could improve all the characteristics, including the stability. However, we may encounter other problems when integrating the three electrodes in a very small space. In this paper, we describe the fabrication of an oxygen electrode with a three-electrode confrguration and compare its characteristics with two-electrode ones.
Materials V-grooves were formed in non-doped ( 100) silicon wafers (2 in diameter, 350 pm thick, Japan Silicon Co.). 99.99% gold was used for the vacuum deposition. All chemicals were of reagent grade without further purification. Solutions were prepared with deionized water. A negative photoresist, OMR-83, which was used in the process and to define the sensitive area, was purchased from Tokyo Ohka Kogyo Co. Shin-etsu Silicone ES-1001 was used for the backside insulation and KR-5240 for the gas-permeable membrane. Procedure The characteristics of the oxygen electrodes were evaluated by immersing their sensitive areas in 50 ml of 10 mM phosphate buffer solution (pH 7.0) in a beaker, allowing them to equilibrate, reducing the oxygen concentration by adding sodium sulfite (Na,SO,) or by controlling the speed of agitation, and then monitoring the responses. The temperature of the buffer solution 0 Elsevier Sequoia/Printed
in The Netherlands
298
was held at 27 “C. Calibration curves were obtained by comparing the response with that of a dissolved-oxygen meter (TOA Electronics, Ltd., model DO-1B). The potential of the working electrode was held constant by a Hokuto Denko HA-5OlG potentiostat and the output current was recorded with a Yokogawa 3066 pen recorder. The applied voltage was -0.6 V or -0.8 V versus Ag/AgCl with reference to the result of the cyclic voltammogram. For comparison, the response of the oxygen electrode without the gel and the gas-permeable membrane whose sensitive area is directly exposed to a 0.1 M KC1 solution was examined by adding Na,SO,. Na$O, does not cause any electrochemical reactions on the working electrode in this configuration and at this voltage: a more negative voltage is needed. In our previous miniature oxygen electrodes with a gold cathode and anode, the completeness of the gas-permeable membrane was checked when the NazSO, was added to reduce oxygen concentration; a current increase was observed because of direct Na,SGs reduction on the cathode at the applied voltage. However, in the three-electrode system, we cannot use Na,SO, to check the completeness of the membrane because the reduction potential of the Na,SO, is more negative than that for oxygen. Instead, 200 ~1 of I&[ Fe( CN),] was added to the solution in the zero oxygen state. If the membrane was not complete, the current increased due to direct K3 [ Fe( CN),] reduction. As mentioned below (in the Stability Section), the output current of the oxygen electrode was affected by the dissolution of the reference electrode material. Therefore, the characteristics described in other Sections were taken at the tirst stable current level after the voltage was applied. The Oxygen Electrode Structure The oxygen electrode was fabricated on a 2 mm x 15 mm silicon chip using the process explained in ref. 6; its structure is shown in Fig. 1. The sensitive area was 0.2 mm x 2 mm. Only the counter electrode areas were anisotropically etched into V-grooves [7’j. A shallow groove was formed with the OMR-83 photoresist layer to fill the working electrode area and the reference electrode area with the gel. The resist layer also determined the active area. An 0.8 pm thick SiOz insulation layer was formed on the surface. The back of the wafer was covered with a hydrophobic insulator (ES-1001) over the SiOz layer. Figure 2 shows the sensitive areas of three-electrode (a) and two-electrode (b) configuration oxygen electrodes. The position and area of the working and counter electrodes were similar to those of our two-electrode contigurations for ease
Semitive area
C.E.
a
GE.
Electrolyte
I
a’ Fig. 1. Structure of the three-ecm6guration oxygen electrode. A cross section of the sensitive area is shown on the right. CE.(Au) \
Semitive area
sanaitive araa /
/
‘Anode (Au or AglAgU)
Imn
(4
(b)
Fig. 2. Sensitive area of the oxygen electrode with (a) the three-electrode and (b) the two-electrode cm6guration.
of comparison. The working and counter electrodes were made from 400 mn thick gold film and the reference electrode was made from 400 mn thick silver f&n. An intermediate chromium layer 40 nm thick was used to obtain good adhesion between these flhns and the SiOl layer. The working electrode area exposed to the electrolyte was 0.04 mm*, which was a little smaller than the cathode in the two-electrode configuration. The counter areas were the same as that of the anode for our double configuration oxygen electrode. The Ag/AgCl reference in the three-electrode system was placed between the working electrode and the counter electrode. A thin AgCl layer was formed just before the electrolyte was impregnated by immersing the sensitive area in 0.1 M FeCl, for 5 min. The stability of the Ag/AgCl electrode was confinned by applying + 0.8 V to the Ag/AgCl electrode against the gold working electrode in the two-electrode configuration. The voltage was applied for an additional 10 to 30 s if the potential was not stable. The groove was filled with calcium alginate gel containing a 0.1 M KC1 aqueous solution as an
299
electrolyte, then covered with a dip-coated gaspermeable membrane (KR-5240). The harder the gel, the better. We used 0.4% sodium alginate solution to make the gel layer. Although 0.2% sodium alginate was used in our last oxygen electrode [6], we could not make a good oxygen electrode with three electrodes using a gel of this concentration, as the dissolved AgCl immediately reached the warking electrode and was reduced there. The pads were made rather large to facilitate the connection to the potentiostat. The terminal of an IC socket can be attached to these pads and we do not have to bond gold or aluminium wires there. This made it easier to evaluate a large number of oxygen electrodes. Results and Discussion
Cyclic Voltammogram Figure 3 shows the cyclic voltammogram for a completed oxygen electrode with a gas-permeable membrane. Curve (a) shows the current for an initial oxygen-saturated state and curve (b) indicates that for zero oxygen concentration after Na,SO, was added. The curves were different below about -0.2 V versus Ag/AgCl. The variation in current reflects the decreased oxygen concentration. An oxygen sensor can be made by keeping the potential in this range. After the response was stabilized in the zero oxygen state, K3[ Fe( CN),] was added. However, there was no noticeable change in the Z-V curve, which indicated that the K3[ Fe(CN),] did not reach the surface of the working electrode. This confirmed that the KR-5240 membrane on the electrolyte worked as a gas-permeable membrane. Below about -0.6 V versus Ag/AgCl, a limiting current region was observed. If we set the
-0.8
-0.4
-0.2
(
Response Time The distribution of the 90% response time was statistically examined among the chips on a wafer. The response time we measured corresponds to the maximum response from oxygen saturation (7.9 ppm at 27 “C) to zero. Figure 4 shows distributions of the response time of the electrodes at -0.8 V and -0.6 V versus Ag/AgCl. The response time was between 20 s and 50 s. Table 1 shows the response time of the three types of oxygen electrodes. The response times of the
Iti 30
Response
time (s)
Response
0
90
(b) (a) Fig. 4. Distribution of the 90% response time for the oxygen concentration change from saturation (7.9 ppm) to zero, (a) at -0.8 V and (b) at -0.6V. Measurements were made in a 10 mM phosphate buffer (27 “C, pH 7.0).
i 40
160 Fig. 3. Cyclic voltammogram of the completed configuration oxygen electrode. Curve (a) is rated (7.9 ppm) initial state and curve (b) is state after Na,SO, was added. Measurements 10mM phosphate buffer (pH 7.0, 27 “C). 50 mV/s.
60
time (s)
TABLE 1. Comparison of the 90% response time and residual current for three types of oxygen electrodes. The residual current was expressed on a relative basis by dividing the residual current by the maximum current decrease from oxygen saturation (7.9 ppm at 27 “C) to zero. The compared data were those at -0.8 V for the three-electrode system and the Ag/AgCl anode two-electrode system and - 1.2 V for the two-gold-electrode system. Other experimental conditions were the same
Potential (v vs. Ag/AgCD -0.8
voltage in this region, the oxygen reduction current depends only on the oxygen concentration in the bulk of the inner electrolyte solution or, when there are no effects of electrochemical crosstalk in the electrolyte, in the solution outside the membrane. Although -0.6 V is slightly outside the region in Fig. 3, the current varies approximately in proportion to the oxygen concentration in the bulk, because even in this case the oxygen concentration in the vicinity of the working electrode is almost zero.
three-electrode the oxygen-satuthe zero oxygen were made in a Scan rate was
Configuration
90% response time (s)
Residual current (%)
Three-electrode
20-50 (average: 20-40 (average: 30-180 (average:
0.2-7 (average: l-20 (average: 20-60 (average:
Two-electrode (Ag/AgCl anode) Two-electrode (gold anode)
40) 30) 103)
3) 12) 37)
300
three-electrode and the two-electrode Ag/AgCl anodes were about the same. However, the response of the two-electrode gold anode was much slower than the other two. Liu et al. showed that H,O, produced on the cathode following the oxygen reduction affects the counter electrode [8]. With the gold anode, the H,O, helps to produce more oxygen, which can diffuse back to the cathode. This delays the oxygen concentration decrease when the concentration outside the membrane is decreased, and may be why the response of the gold anode oxygen electrode is slow. For comparison, we measured the response times of the triple oxygen electrodes without the gas-permeable membrane at -0.8 V and -0.6 V. The response time of the electrode was distributed around 15 s at both voltages. There seemed to be a delay before zero oxygen concentration was reached after adding Na,SO, . In the three-electrode system, the most dominant factor determining response time is the oxygen diffusion through the gas-permeable membrane. Although three types of oxygen electrodes with the same volume of electrolyte are compared here, the response time will also be affected by the electrolyte volume, because it takes more time to reduce the oxygen concentration in the electrolyte as the volume increases. The response time could also be affected by the hardness of the gel layer, because oxygen diffusion is delayed as the gel becomes harder. The response time for the reverse direction, i.e., for increasing oxygen concentration from zero to oxygen saturation, is difficult to measure accurately because there are no good ways to increase the oxygen concentration instantly to saturation. But if the oxygen electrode whose current indicated zero oxygen concentration level was transferred to another buffer solution where the oxygen was saturated, the current recovered to the oxygen-saturated level in about the same time as for the reverse direction. Residual Current The residual current at zero oxygen concentration should be as small as possible. This is particularly important if the oxygen electrode is used to determine oxygen concentration in a fermenter in which the oxygen concentration is relatively low. The residual current comes mainly from the electrochemical reduction of materials not related to oxygen reduction. Measurements were taken for all oxygen electrodes on the same wafer to evaluate the residual current. It did not change much when the potential was changed from -0.8 V to -0.6 V. This agrees with the result of the cyclic voltammogram. Figure 5 shows the distribution of the residual
Residual (a)
,Or...-
current
(nA)
Current
(nA)
Current
for
saturated
for
saturated
(nA)
_.__.~~_~
0 Residual
oxygen
current
50
100 oxygen
(nA)
(b)
Fig. 5. Distribution of the residual current at zero oxygen concentration and the current for saturated oxygen (7.9 ppm), (a) at -0.8 V and (b) at -0.6 V. Measurements were made in a 10 mM phosphate buffer (27 “C. pH 7.0).
current at -0.8 V and -0.6 V together with distribution of the current for saturated oxygen (7.9 ppm). For the three-electrode system, the current for saturated oxygen distributed from 50 to 100 nA at -0.8 V (average: 74 nA) and from 40 to 70 nA at --0.6 V (average: 57 nA). For the two applied voltages, the three types of oxygen electrodes gave about the same average level of current for saturated oxygen. The residual current was less than 7% of the maximum current variation in the response from oxygen saturation to zero (average: 2.6% at - 0.8 V and 2.1% at -0.6 V). This is as low as or a little lower than for the double electrode with Ag/AgCl anode (Table 1). (Although the average residual current is 12% for the Ag/AgCl anode two-electrode system, the value was raised because some of the electrodes gave a low current level for saturated oxygen and a high relative residual current. However, overall we can say that the residual current is not so bad compared with that of the three-electrode system.) The residual current for the three-electrode system gradually increased as it was operated for several hours and saturated at a level between 10 and 15 nA. However, it was significantly better than for the gold anode case, whose average residual current was 37%. Furthermore, the residual current of the three-electrode system was distributed in a narrow range and was more uniform than that of a two-electrode oxygen electrode with a gold or Ag/AgCl anode.
301
Although the three-electrode system has a gold counter electrode whose area is the same as the anode of the system with two gold electrodes, its response time and residual current were significantly better than those of the twogold-electrode system. This indicates that the voltage between the electrodes is effectively applied in the three-electrode system, decreasing inhibitive materials produced from the counter electrode. Figure 6 shows the residual current of the oxygen electrodes without the gas-permeable membrane. Even though the current varies over an order of magnitude, it does not depend on whether the oxygen electrode has a gas-permeable membrane. This indicates that electroactive materials which might be produced on the counter electrode do not have much effect on the response, even though the electrolyte is confined in a very small space and the electrodes are very close. Because the residual current is less than 10 nA and the current level for saturated oxygen is about 500 nA, the relative residual current is less than l%, which is negligible. The best way to reduce the residual current relatively is to improve the oxygen permeability of the membrane and to increase the current level for saturated oxygen. Calibration Curve A calibration curve for a three-electrode configuration oxygen electrode is shown in Fig. 7. Good linearity was obtained as easily as in the case of the Ag/AgCl anode two-electrode configuration. Electrochemical crosstalk often affects the linearity of the calibration curve. Such a good linearity has never been obtained in the gold anode two-electrode system. Deviations are always observed at low and high oxygen concentrations. This may be due to the oxygen produced on the gold anode.
0
(4
0
5 Residual
current
10
(nA)
0
(b)
5
80
0
2
Oxygen
4
6
concentration
8
10 bpm)
Fig. 7. Calibration curves of the three-electrode configuration oxygen electrode at -0.8 V and -0.6 V. Measurements were made in a 10 mM phosphate buffer (27 “C, pH 7.0).
Stability The gold anode oxygen electrode could operate until the content of the electrolyte solution changed significantly or the gas-permeable membrane broke. In one case, it operated for more than 40 h, but operation of the Ag/AgCl anode oxygen electrode was limited to 5 to 20 h because the anode was damaged during operation due to its thickness (400 nm) and the two-electrode configuration [6]. In the two-electrode configuration, electrochemical reaction (1) proceeds to the right on the anode throughout the electrode’s operation. Ag + Cl- z+AgCl+e-
(1)
Therefore, if the silver layer completely changes into AgCl, the current which compensates the cathodic reaction becomes almost zero, and the oxygen electrode cannot be used. Figure 8 shows the stability of the three-electrode configuration oxygen electrode in continuous operation. It does, of course, need a good reference to obtain a stable output current. The lifetime of the oxygen electrode is voltage dependent. When the applied voltage was -0.6 V ver-
10
Residual current (nA)
Fig. 6. Distribution of the residual current of the oxygen electrode without the gas-permeable membrane and the gel, and exposed to 0.1 M KCI, (a) at -0.8 V and (b) at -0.6 V. Measurements were made in a IOmM phosphate buffer (27 “C, pH 7.0).
0
5
10
15
20
25
30
Time (h)
Fig. 8. Stability of the output current of the three-electrode configuration oxygen electrode at -0.8 V and -0.6 V. The current for saturated oxygen (7.9 ppm) was monitored continuously in a 10 mM phosphate buffer (27 “C, pH 7.0).
sus Ag/AgCl, the electrode operated for about 25 h, and then a rapid increase in current was observed. When the voltage was -0.8 V, the oxygen electrode maintained a current level for about 15 h and rapid current increase was observed after that. However, the rapid decrease in current that was observed in the Ag/AgCl anode two-electrode configuration [6] was never seen. This indicated that the damage to the Ag/AgCl reference itself was negligible. However, the AgCl layer dissolved into the electrolyte solution, reached the working electrode, and was reduced to silver. The surface of the working electrode turned black after many hours of operation. This deposition of silver interfered with the oxygen reduction, which, in most cases, led to a gradual decrease in current. The H,O, which dissolves from the cathode can help dissolve the AgCl [8]. The current increase could also be ascribed to Cr.’ reduction on the working electrode. These are the major reasons for the instability seen in Fig. 8. Migration and diffusion of these ions also explain why the lifetime of the oxygen electrode is affected by the applied voltage: the higher the voltage, the shorter the lifetime. The gradient of the electric field and the concentration of the ions, both of which must be much more complicated than in an ordinary-scale system, could be large even in the bulk of the electrolyte and depend upon the applied voltage because the electrodes are placed very near to each other. In our previous oxygen electrodes, 0.2% sodium alginate was used to make the gel layer [6]. We could not obtain good results in the three-electrode case using the 0.2% concentration, especially in the case of stability of continuous operation. Even when 0.4% sodium alginate was used, a better stability was obtained if some of the water in the gel was released and the gel hardened. This shows that the gel layer reduced the contamination of the working electrode. Although the working electrode was 50 pm from the reference electrode, this distance was too short to give good stability. Based on the results obtained here, we can construct an improved miniature oxygen electrode by the following strategy. (1) The working electrode (cathode) and the counter electrode (anode) should be placed as far apart as possible. (2) Although in a usual electrochemical experiment the reference electrode is placed near to the working electrode, we should place the reference electrode as far away from the working electrode as possible. Ohmic drop will be negligible in the miniature electrode system even if we separate these electrodes. (3) To reduce electrochemical crosstalk, it is better to form each electrode in isolated grooves
and to connect these grooves by long narrow grooves. (4) The electrochemical crosstalk will be reduced by using an electrolyte impregnated in a gel. The gel layer suppresses diffusion of inhibitive electroactive materials. However, the hardness of the gel must be optimized, because it could delay the response. As mentioned before, the miniature two-electrode system with Ag/AgCl anode is not practical because of its short lifetime. The two-electrode gold anode system, however, could be upgraded like the three-electrode system by following the aforementioned strategy to eliminate the electrochemical crosstalk.
Conclusions A miniature Clark-type oxygen electrode with a three-electrode configuration was fabricated. By comparing its characteristics with those of twoelectrode configurations, it was found that electrochemical crosstalk affected the response time, residual current, linearity of the calibration curve and stability of continuous operation. These characteristics were significantly improved by using the three-electrode configuration.
Acknowledgements We thank Dr I. Karube and Dr E. Tamiya of the University of Tokyo for their helpful discussions in preparing this report.
References 1 K. Najafi, Integrated sensors in biological environments, Sensors and Aciuarors, Bl (1990) 4533459. R. S. Pickard, P. Wall. M. Ubeid, G. Ensell and K. H. Leong, Recording neural activity in the honeybee brain with micromachined silicon sensors, Sensors and Actuators, Bf (1990) 460-463. S. J. Tanghe, N. Najafi and K. D. Wise, A planar IrO multichannel stimulating electrode for use in neural prostheses. Sensors and Actuafors, Bl ( 1990) 464.-467. 2 0. Prohaska. in A. P. F. Turner, I. Karube and G. S. Wilson (eds.). Bio.~ensors-PFundamental.s and Applicarions, Oxford University Press, Oxford, 1987, p. 377. 3 W. Sansen, D. De. Wachter, L. Callewaert, M. Lambrechts and A. Claes. A smart sensor for the voltammetric measurement of oxygen or glucose concentrations, Sensors and Actuaiors, Bf (1990) 298 302. 4 I. Karube, in A. P. F. Turner, 1. Karube and G. S. Wilson (eds.), Biosensors -Fundamentals and Applications, Oxford University Press, Oxford, 1987, p. 471. L. C. Clark, Jr.. in A. P. F. Turner, I. Karube and G. S. Wilson (eds.), Biosensors ~- Fundamentak and Applications. Oxford University Press, Oxford, 1987, p. 3. 5 H. Suzuki. E. Tamiya and I. Karube, Development of the micro-oxygen electrode and its application to a micro-glu-
303 case sensor, Proc. Mater. Res. Sot. ht. Meet. Adwmced Materials, Tokyo, Japan, May 1988, pp. 115-120. H. Suzuki, A. Sugama and N. Kojima, Effect of anode materials on the characteristics of the miniature Clark-type oxygen electrode, Anal. Chitn. Acta, 233 (1990) 275-280. K.-k. Bean, Anisotropic etching of &on, IEEE Trans. Electron Devices. ED-25 f 1978) 1185-l 193. C. S. Cha, M. J: Shao add C’C. Liu, Problems associated with the miniaturization of a voltammetric oxygen sensor: chemical crosstalk among electrodes, Sensors and Actuators, B, 2 (1990) 277-281.
Biographies Hiroaki Suzuki received a M.E. degree in applied
physics from the University of Tokyo. In 1983, he joined Fujitsu Laboratories, Ltd. His current interest is the fabrication of miniature biosensors using micromachining techniques. Naomi Kojima received a B.S. degree in biochemistry from Tokyo Metropolitan University. In 1985, she joined Fujitsu Laboratories, Ltd. Akio Sugama received a B.S. degree in biochemistry from Tohoku University. In 1985, he joined Fujitsu Laboratories, Ltd.