A miniature Clark-type oxygen electrode using a polyelectrolyte and its application as a glucose sensor

Biosensors& Bioelectronics6 (1991) 395400

A miniature Clark-type oxygen electrode using a polyelectrolyte and its application as a glucose sensor* Hiroaki Suzuki, Akio Sugama, Fujitsu Laboratories

Naomi Kojima, Fumio Takei 81 Kasumi lkegami

Ltd., 10-l Morinosato-Wakamiya,

Atsugi, 243-01, Japan

(Received 4 May 1990; accepted 15 October 1990)

Abstract: A miniature Clark-type oxygen electrode was fabricated by anisotropically etching silicon. A two-gold-electrode configuration was used and a double-layered gas-permeable membrane was formed directly on the electrolyte, poly(vinyl+ethylpyridinium bromide) in the sensitive area. These materials improved the electrode’s stability in long-term storage and sterilization tolerance to a practical level. The 90% response time averaged 80 s and residual current lo%, with a good linear calibration curve. The oxygen electrode was also used to make an integrated sensor for the simultaneous determination of glucose and oxygen. The glucose sensor’s response time was 50-l 10 s, with good linearity in glucose concentrations between 56 PM and 1.1 mM at 37°C pH 7.0. Keywords: sensor.

Clark-type

oxygen electrode,

polyelectrolyte,

fermenter,

glucose

for example, and not be used with other patients. Oxygen electrodes currently available are not very small and are too expensive to be used once and discarded; an inexpensive miniature electrode is desired. In an attempt to respond to these requirements, several miniature oxygen electrodes have been developed including some Clark-type electrodes (Miyahara er al., 1983; Karagounis et al., 1986; Koudelka, 1986; Suzuki et al., 1988; Sansen et al., 1990; Suzuki et al., 1990a). The electrodes must meet two important criteria if they are to be used on a practical basis and have other than a very limited use: stability in long-term storage and sterilization tolerance. The electrode we developed previously broke easily because the gas-permeable membrane was on a soft gel and tended to be incompletely formed

INTRODUCTION

The Clark-type oxygen electrode is based on an electrochemical principle, in which the electrode produces a current following an electrochemical oxygen reduction on the cathode. The electrode does not suffer from fouling of the cathode and features room temperature operation, amperometric detection, and stable use in an aqueous solution, making it applicable in fields such as fermentation, clinical diagnosis and biosensing (Janata, 1990). In the clinical and biosensing fields, a miniature disposable oxygen electrode is needed that can be discarded after an operation, *Paper presented at Biosensors ‘90,Singapore, 2-4 May 1990. 395

Biosensors & Bioekcm~nics 0956-X63/91/$03.50 0 1991 Elsevier Science Publishers Ltd. England. Printed in Great Britain

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(Suzuki et al., 1990a). The problem was solved by using a polyelectrolyte and a double-layered gaspermeable membrane.

MATERIALS

anode was made in the grooves. The cathode and anode are made from 400 nm thick gold film with an intermediate 40 nm thick chromium layer to obtain good adhesion between the gold film and the SiOZ layer. The active area of the oxygen electrode was defined by an OMR-83 photoresist (Tokyo Ohka Kogyo) layer leaving a shallow ditch in the sensor area. The cathode area exposed to the electrolyte was 0.08 mm*. On the reverse side of the sensitive areas were pads where a constant voltage was applied and the oxygen reduction current detected. Apart from the pad areas of the cathode and the anode where the electrical contacts are made and the sensitive area, all other parts were covered with the OMR-83 negative photoresist layer to insulate the electrodes for use in an aqueous solution. The back of the chip was coated with hydrophobic ES-1001 silicone resin (Shin-Etsu Silicone, Tokyo) in addition to a thermally grown 0.8pm thick SiO2 layer. The groove and the shallow resist ditch were tilled with a polyelectrolyte (poly(vinyl+ ethylpyridinium bromide)) (Fig. 2) then covered with a dip-coated double-layered gas-permeable membrane. The polyelectrolyte was made by polymerizing 4-vinylpyridine with the help of benzoyl peroxide. The polymer was reacted with ethyl bromide in nitromethane and then purified. The polymer chain is somewhat movable and works as a polycation. The electrolyte also contains the B; ion as a carrier. The electrolyte shows ionic conductivity when water is absorbed by it, making oxygen reduction on the cathode possible. After the polyelectrolyte layer was

AND METHODS

Fabrication of the oxygen electrode The body of the electrode was made following the process described by Suzuki et al. (1990~). The miniature oxygen electrode (Fig. 1) was fabricated on a 2 mm X 15 mm nondoped Si (100) substrate (350 pm thick): the nondoped substrate was used to ensure enough insulation. The sensitive area, which contains essential elements of the Clarktype electrode such as the cathode and anode, electrolyte, and the membrane, was O-2 mm X 2 mm. Two deep V-grooves (0.2 mm X O-7 mm each) were formed in the anode areas alone by anisotropic etching (Lee, 1969; Bean, 1978; Petersen, 1982).Among materials that can be etched anisotropically, such as GaAs or rock crystal, silicon is best for making deep, three-dimensional structures because etching rates differ significantly among crystal surfaces and it provides an inexpensive substrate. In addition to anisotropic etching, the availability of techniques such as direct and anodic bonding, uniquely used with silicon, enabled us to make more advanced, practical electrodes (Lee, 1969). The cathode was formed on the flat area between the two grooves to place the cathode near to the gas-permeable membrane and to make the cathode and anode pattern formation easier. The P Sensitive area

I

‘Cathode

Gas-permeable membrane / (OMR-83 + KE-347T)

a -fSilicon

substrate

~-Insulator

: v)

(ES-1001) Cathode (Au)

350 pm

b’ _--

%

Pads

IL!L t!ll

Anode (Au)

2% Fig. 1. Oxygen electrode structure. Extended cross-sections of the sensitive area are shown on the right corresponding to the lines indicated by a-a’ and b-b’.

Miniature Clark-type oxygen electrode

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106 unit mg-‘, Toyobo, Tokyo) was dissolved in a mixed solution containing 1% BSA and 1% glutaraldehyde. The enzyme concentration was 5%. The solution was then immediately cast on the sensitive area (gas-permeable membrane) of one of two oxygen electrodes at room temperature, allowed to react at 4°C overnight, and rinsed in deionized water. The enzyme was thus crosslinked to the BSA matrix on the membrane. Fig. 2. The polyelectrolyte poly(vinyl-4-ethylpyridinium bromide) used in the oxygen electrode.

formed in the groove, the water in the electrolyte was completely removed by baking the electrode at 120°C for 10 min. The electrolyte layer was formed by casting the aqueous solution of the polymer on the groove area with a small rod. The gas-permeable membrane consists of the OMR-83 negative photoresist membrane which adheres to the electrolyte and the SiOz and another silicone rubber membrane (KE-347T, Shin-Etsu Silicone) which covers the first membrane. The gas-permeable membrane (OMR-83 and KE-347T) was formed on the dried electrolyte. The OMR-83 membrane was dip coated on the sensitive area, baked at 120°C for 15 min and exposed to UV radiation for 1 min. The KE-347T membrane was dip coated on the OMR-83 membrane and dried at room temperature overnight. The oxygen electrode does not produce a current if there is no water in the electrolyte. To activate the oxygen electrode, water essential for oxygen reduction was incorporated in the sensitive area by sterilizing the oxygen electrode in steam at 120°C and 1.2 atm for 15 min. The completed oxygen electrodes were stored in deionized water or dried. Fabrication of the glucose sensor An integrated miniature sensor was also made for determining oxygen and glucose. The principle of detection in the glucose sensor is based on the determination of the oxygen concentration variation following glucose oxidation by glucose oxidase. Before forming the enzyme-immobilized membrane, 98% y-aminopropyltriethoxysilane was cast on the membrane to make adhesion of the bovine serum albumin (BSA) matrix to the gas-permeable membrane stronger. The glucose oxidase enzyme (from Aspeeillus niger, activity:

Procedure The characteristics of the oxygen electrodes were evaluated by immersing their sensitive area in a well-stirred 10 mM phosphate buffer solution (pH 7.0,27”C), allowing them to equilibrate, and then monitoring their response to change. The oxygen concentration in the buffer solution was reduced by adding sodium sulfite (Naz SO3). The output of the oxygen electrode was calibrated with a dissolved-oxygen meter (TOA Electronics, model DO-1B). Except for the experiments related to the calibration curve, an excess of NazS03 was added to remove dissolved oxygen completely and responses to the change were studied. Voltages of -1.2 V were applied to the cathode against the anode. Characteristics of the glucose sensor were evaluated by a similar method in a 10 mM phosphate buffer solution at 37°C and pH 7.0. The applied voltage was the same as the oxygen electrode.

RESULTS AND DISCUSSION Response time and residual current Although the size of the sensitive area of our electrode is much smaller than that of oxygen electrodes now available, it produces a very clear response curve (Fig. 3) without appreciable noise. The 90% response time averaged 80 s. Oxygen, possibly produced on the anode, may delay the response if too much electrochemical crosstalk occurs between the cathode and anode. Before using the double-layered gas-permeable membrane, the same oxygen electrode with the same polyelectrolyte was made by using a single-layered gas-permeable membrane (OMR-83 or KE-347T). The response time of the electrode was 30 s on average. The response time with the doublelayered gas-permeable membrane became more

H. Suzuki

et al.

40 I - - -

Oxygensaturated

z L

z

E al 20 ::

=30E 2 2 200

10 5

‘O01

30

L

01 0

- - - Zero oxygen I

0

5

10

:/

15

Time (min)

Fig. 3. Response curve of the oxygen electrode afier Na2SOJ was added to reduce the oxygen concentration. The curve was obtained in a 10 mMphosphate buffer(pH 7.0,27”C).

than twice of that of the single-layered gaspermeable membrane electrode. Thus the response time of the electrode was mainly determined by oxygen diffusion through the gas-permeable membrane. Diffusion through the electrolyte layer was fast enough, as experienced in a previous study (Suzuki etal., 1990~) where the response time did not change when the distance between the cathode and the membrane was changed. In a practical oxygen electrode, the residual current at a zero oxygen concentration must be as small as possible. The residual current in the electrode in this study was about 10% of the maximum current decrease from oxygen saturation to zero. It was 37% when we used calcium alginate gel impregnated with 0.1 M KC1 and the same gold cathode and anode (Suzuki et al., 1990b). We suspect the difference was because the polyelectrolyte was harder than the gel and suppressed electrochemical crosstalk from the anode.

2

4

6

Oxygen concentration

a

10

(ppm)

Fig. 4. Calibration curve of the oxygen electrode. Experiments were carriedout in a 10 m,uphosphate buffer(pH 7.0, 27°C).

the calibration curve (Suzuki ec al., 1990b). These characteristics can be improved by separating the cathode and anode further. Stability in long-term storage

To use the oxygen electrode on a practical level, two of the most important characteristics are stability in long-term storage and sterilization tolerance. The dominant factor affecting these characteristics is the strength of the gas-permeable membrane. The stronger the membrane, the better are these characteristics. Figure 5 shows variations in the output of the oxygen electrode when different electrolyte and gas-permeable membrane materials were used. Both electrodes were stored in deionized water at room temperature when not in use. For the electrode with the polyelectrolyte, water had been incorporated in the membrane before the experiment was started. In our previous electrode, the electrolyte was a

1

Calibration curve

Good linearity (Fig. 4) was obtained over a wide range of oxygen concentrations below 7.9 ppm (at which the oxygen concentration is saturated at 27°C). Electroactive materials produced from the gold anode (major materials could possibly be 02 and H+) generate additional current when reduced on the cathode, causing residual current and decreased linearity. Even in this calibration curve, a slight deviation occurred at lower oxygen concentrations. As we found in a previous study, electrochemical crosstalk is not negligible in this electrode layout. The crosstalk degrades the response time, residual current, and linearity of

-----J 0

50

100

150

200

250

300

350

Time (days)

Fig. 5. Long-tetm stability of oxygen electrodes: 0, variation in the output current for saturated oxygen when the polyelectrolyte and the double-layer membrane wasformed; A. similar data obtained when the calcium alginate gel impregnated with 0.1 M KC1 electrolyte and a single membrane (KR-5240, Shin-etsu Silicone) were used. Experiments were carried out in a 10 rnMphosphate buffer (PH 7.0, 27°C).

Miniature Clark-type oxygen electrode

gel impregnated with 0.1 M KCl, with a singlelayered gas-permeable membrane. Malfunction of the oxygen electrode was mainly caused by deterioration of the membrane material and of adhesion between the membrane and the substrate. The membrane was easily degraded by water both in the electrolyte and in the outer solution used for measurement (Suzuki et al., 1990~~).This was because the electrode contained a soft gel, making it difficult for the membrane to bake at a high temperature. This oxygen electrode could not be used for more than 1 month because the membrane broke. However, output of the oxygen electrode with the polyelectrolyte and the doublelayered gas-permeable membrane was stable after 9 months of storage and variation in the output was small. The output current showed an increase in fluctuation after 250 days. We consider that this was caused not by the deterioration of the membrane but by the lifetime of the oxygen electrode cell. The electrode can be stored dry. A few oxygen electrodes were stored for a certain period without introducing water in the sensitive area; water was then incorporated just before the experiment and the responses to the variation from an oxygensaturated state to zero were examined. It showed a normal response after at least 6 months of storage. When used for a purpose in which the electrode is sterilized by steam and is discarded when measurement is finished, it is convenient to store the electrode in dry state and activate it by steam just before use.

399

permeable membrane layers cannot be formed selectively on the sensitive area. That makes simultaneous oxygen electrode fabrication on a wafer impossible and each electrode chip has to be made individually. The electrolyte and gaspermeable membrane materials need to be improved to make them more compatible with the process. Characteristics of the miniature glucose sensor The 90% response time of the glucose sensor was concentration dependent_ The smaller the variation in glucose concentration and the decrease in oxygen concentration, the faster was the response. For instance, the response time was 90-l 10 s for 0.5 mM glucose and was 50-70 s for 0.25 mM glucose for the same sensor. Because the response time of the aforementioned oxygen electrode was that which corresponded to the maximum oxygen concentration variation from oxygen saturation to zero, the response time of the glucose sensor was shorter in some cases. Figure 6 shows the calibration curve for the glucose sensor. A good linear relationship was observed for glucose concentrations between 56~~ and 1.1 mM at 37“C pH 7.0. Figure 7 shows the long-term stability of the

Sterilization tolerance The problems related to the long-term stability are closely associated with sterilization tolerance. When a single-layered gas-permeable membrane (either OMR-83 or KE-347T) was used with the same electrolyte, the membrane easily broke. Making the gas-permeable membrane more complete and double layered raised the tolerance to a point at which the oxygen electrode did not break after at least one sterilization. The stronger membrane led to a marked improvement in this behavior. . As shown in the results, characteristics such as sterilization tolerance and long-term storage stability were significantly improved by changing the electrolyte and gas-permeable membrane materials. Inconveniences arose in the process, however, because the electrolyte and gas-

00

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Glucose concentration (mM)

Fig. 6. Calibration curvefor theglucose sensor. Experiments werecarriedout in a IO mMphosphate buffer(pH 7.0.37”C).

20

30

Time (day)

Fig. 7. Long-term stability of the glucose sensor. Experiments werecarried out in a 1OmMphosphate bu#er(pH 7.0, 37°C). The glucose concentration was 4 mM.

400

glucose sensor. If the enzyme-immobilized BSA matrix was made on the gas-permeable membrane without any treatment, the BSA matrix detached from the membrane after only 3 days of storage. However, if the membrane surface was treated with y-aminopropyltriethoxysilane before forming the BSA matrix, the lifetime was extended to about 1 month. After that the BSA matrix also detached from the gas-permeable membrane. The glucose sensor and the oxygen electrode were integrated by immobilizing the enzyme on one of two oxygen electrodes. The decrease in oxygen concentration in the vicinity of the enzyme-immobilized membrane did not affect the response of the other oxygen electrode. The output of the glucose sensor, however, is affected by variation in the oxygen concentration because the principle of the sensor is based on the measurement of the decrease in oxygen concentration. However, if the variation in oxygen concentration is negligible over the response time of the glucose sensor, the integrated sensor can be used to monitor glucose and oxygen concentrations simultaneously. The above-mentioned problem could also be solved if oxygen electrodes which gave an output current of the same level were used and differential mode was employed.

CONCLUSIONS A miniature Clark-type oxygen electrode was fabricated using a polyelectrolyte and a doublelayered gas-permeable membrane. The long-term stability and sterilization tolerance were markedly improved. This enabled the oxygen electrode to be of practical use. The oxygen electrode was applied to fermentation monitoring and was used to fabricate a glucose sensor.

H. Suzuki et al.

ACKNOWLEDGMENT We thank Dr I. Karube and Dr E. Tamiya of the University of Tokyo for their helpful discussions in compiling this report.

REFERENCES Bean, K. E. (1978).Anisotropic etching of silicon. IEEE Trans. Electron Devices, ED-25, 1185-93. Janata, J. (1990). Principles of Chemical Sensors, Plenum,

New York. Karagounis. V., Lun, L. & Liu, C. C. (1986).A thick-film multiple component cathode three electrode oxygen sensor. IEEE Trans. Biomed. Eng., BME-33,108-12. Koudelka. M. (1986). Performance characteristics of a planar ‘Clark-type’ oxygen sensor. Sens. Actuators, 9, 249-58. Lee, D. B. (1969). Anisotropic etching of silicon. J. Appl. Phys., 40,4569-74. Miyahara, Y., Matsu, F., Shiokawa, S., Moriizumi, T., Matsuoka. H., Karube, I. & Suzuki, S. (1983). Biosensor using anisotropic etching of Si. In Proc. 3rd Sensor Symp., The Institute of Electrical Engineers of Japan, Tsukuba, Japan, pp. 21-6. Petersen, K E. (1982). Silicon as a mechanical material. Proc. IEEE, 70, 420-57. Sansen, W.. Wachter, D. D., Callewaert, L., Lambrechts, M. & Claes, A. (1990). A smart sensor for the voltammetric measurement of oxygen or glucose concentrations. Sens. Actuators, Bl, 298-302. Suzuki, H., Tamiya. E. & Karube, I. (1988). Fabrication of an oxygen electrode using semiconductor technology. Anal. Chem., 60, 1078-80. Suzuki, H., Kojima, N., Sugama. A. &Takei, F. (1990a). Development of a miniature Clark-type oxygen electrode using semiconductor techniques and its improvement for practical applications. Sens. Actuators, B2, 185-91. Suzuki, H., Sugama, A. & Kojima, N. (1990b). Effect of anode materials on the characteristics of the miniature Clark-type oxygen electrode. Anal. Chim. Acta, 233, 275-80.