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Polyvinylbutyral resin membrane for enzyme immobilization to an isfet microbiosensor
Journal of Molecular
Catalysis,
37 (1986)
133 - 139
133
POLYVINYLBUTYRAL RESIN MEMBRANE FOR ENZYME IMMOBILIZATION TO AN ISFET MICROBIOSENSOR MASAO GOTOH Research
Department,
NOK
4 -3 -1
Corporation,
Tsujidoshinmachi,
Fujisawa
251
(Japan) EIICHI TAMIYA and ISA0 KARUBE* Research Laboratory of Resources Utilization, Midori-ku, Yokohama 227 (Japan) (Received January 16,1986;
Tokyo Institute
of Technology,
Nagatsuta,
accepted March 4,1986)
Summary A polyvinylbutyral resin membrane was applied to the surface of a silicon nitride-coated silicon wafer. The membrane exhibited good adhesive properties and was used for urease immobilization. The water content of the membrane was 27.6% with a thickness of 23 pm. The surface structure of the membrane was observed by scanning electron microscopy, which revealed a regular porous microfilter arrangement with a mean pore diameter of 0.25 pm. Immobilized urease activity in this membrane was approximately 24% higher than when a urease-cellulose triacetate membrane was used.
Introduction The ISFET (Ion Selective Field Effect Transistor) device was first reported by Bergveld in 1970 [ 11. Later, Matsuo et al. improved the ISFET [2, 31 by using silicon nitride, Si3N4, as the gate insulator, reporting that it could be used as a micro pH-sensitive device. In 1980, Caras and Janata reported a penicillin sensor that utilized an ISFET [4]. We previously reported a microbiosensor that utilized an ISFET [5]. The major properties required of a membrane for enzyme immobilization are that it should exhibit good adhesion to the silicon nitride insulated gate surface, possess high enzyme activity and be thin, hydrophilic and porous. Thus the diffusion of substrates and products in aqueous solution into the membrane are not limiting. Many immobilized enzyme, antibiotic and microbial carriers for use in ISFET microbiosensors have been developed. For example, cellulose triacetate [ 51, polyvinylchloride [6], and agar [7] have been utilized as membranes, but are, however, not ideally suited for biosensor use, since *Author to whom correspondence should be addressed. 0304-5102/86/$3.50
0 Elsevier Sequoia/Printed in The Netherlands
I.34
adhesion to the surface of the silicon nitride gate is, in practice, poor. The characteristics of the ISFET microbiosensor depend on the immobilized enzyme membrane, and are thus of great importance. In this paper, a novel membrane material for use in an ISFET microbiosensor is described, and its properties are discussed.
Experimental Materials Polyvinylbutyral resin Industries, Ltd. The structure
was purchased from Wako Pure Chemical of polyvinylbutyral resin is as follows:
It consists of vinylbutyral, vinyl alcohol and vinyl acetate moieties, in the approximate ratio 75:22:3, respectively. Urease (EC 3.5.1.5, from jack beans, 3.9 units mg-‘) was purchased from Sigma Chemical Company, dichloromethane and glutaraldehyde from Kanto. Chemical Company, 1,8diamino4-aminomethyloctane was a gift from Asahi Kasei Industries and the silicon wafer was obtained from Kyoto Ceramic Company. All other polymers and chemicals used were of analytical grade. All solutions were prepared in distilled water. Assessment of polymer adhesion to silicon nitride surface Twenty-two different species of polymer were examined for their adhesion to silicon. nitride. Silicon nitride was formed on a silicon wafer by the chemical vapor deposition method and was used as the substrate for the tests. 0.1 g of the polymers was dissolved in 10 ml of the respective solvent. The polymer solutions were then cast onto the substrate and formation accelerated by placing the substrate in an oven for 20 min at 60 “C. The substrate membranes were then placed into either distilled water, 1 M NaOH solution, or 1 M HCl solution for 2 h at 40 “C. After this treatment, the adhesive properties were examined by scratching the membrane off the substrate. Preparation of the polyvinylbutyral resin membrane 0.1 - 1.0 g polyvinylbutyral resin was dissolved in 10 ml dichloromethane and cast onto a glass plate at room temperature. After 8 h setting, the membrane formed was leached off in distilled water at room temperature for 2 h.
135
Measurement of membrane thickness and water content The thickness of the polyvinylbutyral resin membrane was estimated using a micrometer thickness gauge. The water content of the membrane was determined from the weight loss after drying at 100 “C. Observation of membrane surface by scanning electron microscopy 0.1 g polyvinylbutyral resin and 1 ml 1,8diamino-4aminomethyloctane were dissolved in 10 ml dichloromethane and cast onto a silicon nitride-coated silicon wafer. The specimen was then dried using a critical point dryer (Hitachi, HCP-2), and sputter-coated with gold. This coated specimen was then analysed in a scanning electron microscope (Hitachi, S-415). Immobilization of urease Polyvinylbutyral resin and cellulose triacetate membranes were used for enzyme immobilization. 0.1 g of polymer and 1 ml of 1,8diamino-4-aminomethyloctane were dissolved in 10 ml dichloromethane, and cast onto a glass plate at room temperature. After 8 h setting, the membranes formed were leached off in distilled water at room temperature for 2 h. The membranes obtained were cut (1 cm*) and immersed in 5% glutaraldehyde solution at 4 “C for 24 h. They were then placed into a 5 mg ml-’ urease solution in 5 mM Tris HCl buffer (pH 7.0) and incubated at 4 “C for 24 h, after which they were washed with a large volume of 5 mM Tris HCl buffer ‘and stored in 5 mM Tris HCl buffer (pH 7.0) at 4 “C. Enzyme assay Activity of the immobilized urease membrane was measured by a spectrophotometric method [8]. Experiments were performed at 37 ‘C, pH 7.0 and the absorbance change at 570 nm was recorded.
Results
Polymer adhesion to the silicon nitride surface Table 1 shows the result of the adhesion tests. Almost all the polymers were stripped in distilled water at 40 ‘C, except polyglutamate, polyester urethane, polyvinylbutyral resin, 6,6-nylon, cellulose acetate, cellulose triacetate and cellulose acetate butyrate membranes. These polymers were then analysed in a 1 M HCI solution at 40 “C. Only the polyester urethane, polyvinylbutyral resin and cellulose acetate membranes remained unstripped, and so these were further analysed in a 1 M NaOH solution at 40 “C. Only the polyvinylbutyral resin membrane was not stripped. These adhesion tests indicate that of all the polymers analysed, polyvinylbutyral resin revealed the greatest adhesion to the silicon nitride surface.
136 TABLE 1 Assessment
of polymer
Polymera
adhesion
to a silicon nitride
Solvent
surface Adhesion Hz6
polystyrene polysulfone polycarbonate polyarylate polyethersulfone polysulfone sulfonate aromatic polyamide polyglutamate polyvinyl chloride polyvinyl acetate polyvinyl alcohol polyacrylonitrile polyvinyl difloride polyester urethane polyvinylbu.tyral resin 6,6-nylon cellulose acetate cellulose triacetate cellulose acetate butyrate agar K carrageenan sodium alginate aPolymer concentration: bImmersion conditions: stripped.
CH$ON(CH& HCON(CH& CHzClz CHzClz HCON(CH& HCON(CH& CH$ON(CH& CHzClz C4HsO
CHCls Hz0
HCON(CH& CH$ON( CH3)2 HCON( CH3)2 CH2C12 HCOOH (CH3)zCO
CH2C12 CH2C12 H2’J H20 H20
0.1 g/10 ml solvent; 40 “C, 2 h; Adhesive
testb lMHC1
1 M NaOH
X X
X X X X X 0
X
X X X X X 0 0
0 0 0
0
0
X
0 X
0
0
X
X X
X X X heat treatment: 60 “C, 20 min. estimation: scratch test; X: stripped;
0: not
Membrane thickness and water content Table 2 shows the membrane thickness and water content of the polyvinylbutyral resin membrane formed at different polymer concentrations. At higher polymer concentrations, the thickness of the membrane is increased and its water content decreased. In view of the diffusion of substrates and products, it is desirable to utilize a membrane which possesses a minimum thickness and a high water content. Observation of membrane surface by scanning electron microscopy Figure 1 shows an electron micrograph of the surface structure of the gold-sputtered polyvinylbutyral resin membrane. Figure 2 shows the pore size distribution of the polyvinylbutyral resin membrane estimated from the electron micrograph; the pore characteristics are summarized in Table 3. These results indicate that the polyvinylbutyral resin membrane possesses a microfilter, porous structure, ideally suited for high-density enzyme immobilization and chemical diffusion.
137 TABLE 2 Membrane
thickness
and water content
of polyvinylbutyral
resin membrane
Polymer concentrationa (%)
Membrane thickness (@n)
Water contentb (%)
1 3 5 7 10
23 62 114 130 222
27.6 10.9 8.7 8.0 8.5
aSolvent : CH&la. bWater content
=
wt wet -
wtdry
x 100; drying conditions:
100 *C, 2 h.
wt wet
Fig. 1. Electron micrograph membrane. The membrane electron microscopy.
showing the surface was sputter-coated
structure of the polyvinylbutyral resin with gold and observed by scanning
TABLE 3 Pore properties
of polyvinylbutyral
Mean pore diameter (Erm)
Maximum diameter (pm)
0.25
0.75
aObservation
resin membranea Minimum diameter (pm)
pore
by SEM. Area observed:
0.03 8.1
x
lo-‘cm’.
pore
Number of pores (n cmm2) 3.2 x lo8
138 100
6 9 $ z F
-
50
L
0 0.05
: 0.53
0.29
Pore
d ameter
0.77
( urn )
Fig. 2. Pore size distribution of the polyvinylbutyral resin membrane. Only pores greater than 0.01 pm in diameter were counted from electron micrograph pictures of the polyvinylbutyral resin membrane. Area observed was 8.1 x 10e7 cm’.
TABLE 4 Comparison of urease activity Membrane
Enzyme activitya (ng urea min-’ cm+)
cellulose triacetate polyvinybutyral resin
159 198
Wrea concentration:
80 PM; 37 “C, pH 7.0
Enzymeactivity of cellulose triacetate and polyvinylbutyral resin membranes Table 4 gives a comparison of urease activity for the cellulose triacetate and polyvinylbutyral resin membranes. Cellulose triacetate membrane has been conventionally used as an immobilized urease membrane for the ISFET micro-urea sensor. The activity of immobilized urease polyvinylbutyral resin membrane was, however, approximately 24% higher than that of the immobilized urease cellulose triacetate membrane.
139
Discussion Of all the membranes analysed, polyvhiylbutyral resin membrane revealed the most tenacious adhesion to the silicon nitride surface under acidic, alkaline and neutral conditions at 40 “C, even greater than that of cellulose triacetate, polyvinyl chloride and agar membranes, which are conventionally used for the immobilized enzyme membrane ISFET microbiosensor. It is thought that the high adhesion to the silicon nitride surface results from repetitive vinyl alcohol sequences in the polymer, forming continuous hydroxy regions within its structure. The repetitive hydroxy regions in the polymer also contribute to its expected hydrophilic properties. Concomitantly, the polyvinylbutyral resin membrane is thin, has a high water content and a multipore structure. These properties distinguish the polyvinylbutyral resin membrane as being very well suited for the purposes of enzyme immobilization and the diffusion of substrates and products within it. The activity of the immobilized urease polyvinylbutyral resin membrane is approximately 24% higher than for the specific type of cellulose triacetate membrane used in this study. It is thought that this enhancement results from a reaction between glutaraldehyde and the vinyl alcohol hydroxy regions, contributing to a high immobilized enzyme density. In conclusion, the polyvinylbutyral resin membrane is highly suited for use as an ISFET microbiosensor membrane in view of its adhesion to silicon nitride substrates. Moreover, its structural properties and high enzyme activity could be further applied to other immobilized enzyme systems. Our intention is to develop the immobilized urease polyvinylbutyral resin membrane for its application in an ISFET micro-urea sensor. The results of this study will shortly be reported.
Acknowledgement We thank Mr. J. M. Dicks for helpful discussions.
References 1 2 3 4 5
P. Rergveld, IEEE Trans. Biomed. Eng., 17 (1970) 70. T. Matsuo and K. D. Wise, IEEE Trans. Biomed. Eng., 21 (1974) 485. T. Matsuo and M. Esashi, Sensors and Actuators, 1 (1981) 71. S. Caras and J. Janata, Anal. Chem., 52 (1980) 1935. Y. Miyahara, F. Matsu, T. Moriizumi, H. Matsuoka, I. Karube and S. Suzuki, in Proc. Znt. Meeting on Chemical Sensors, Kodansha, Tokyo, 1983, pp. 501 - 506. 6 S. D. Moss, J. Janata and C. C. Johnson, Anal. Chem., 47 (1975) 2238. 7 Y. Hanazato and S. Shiono, in Proc. Znt. Meeting on Chemical Sensors, Kodansha, Tokyo, 1983, pp. 513 - 518. 8 R. L. Searcy, J. E. Reardon and J. A. Foreman, Am. J. Med. Tech., 33 (1967) 15.
Catalysis,
37 (1986)
133 - 139
133
POLYVINYLBUTYRAL RESIN MEMBRANE FOR ENZYME IMMOBILIZATION TO AN ISFET MICROBIOSENSOR MASAO GOTOH Research
Department,
NOK
4 -3 -1
Corporation,
Tsujidoshinmachi,
Fujisawa
251
(Japan) EIICHI TAMIYA and ISA0 KARUBE* Research Laboratory of Resources Utilization, Midori-ku, Yokohama 227 (Japan) (Received January 16,1986;
Tokyo Institute
of Technology,
Nagatsuta,
accepted March 4,1986)
Summary A polyvinylbutyral resin membrane was applied to the surface of a silicon nitride-coated silicon wafer. The membrane exhibited good adhesive properties and was used for urease immobilization. The water content of the membrane was 27.6% with a thickness of 23 pm. The surface structure of the membrane was observed by scanning electron microscopy, which revealed a regular porous microfilter arrangement with a mean pore diameter of 0.25 pm. Immobilized urease activity in this membrane was approximately 24% higher than when a urease-cellulose triacetate membrane was used.
Introduction The ISFET (Ion Selective Field Effect Transistor) device was first reported by Bergveld in 1970 [ 11. Later, Matsuo et al. improved the ISFET [2, 31 by using silicon nitride, Si3N4, as the gate insulator, reporting that it could be used as a micro pH-sensitive device. In 1980, Caras and Janata reported a penicillin sensor that utilized an ISFET [4]. We previously reported a microbiosensor that utilized an ISFET [5]. The major properties required of a membrane for enzyme immobilization are that it should exhibit good adhesion to the silicon nitride insulated gate surface, possess high enzyme activity and be thin, hydrophilic and porous. Thus the diffusion of substrates and products in aqueous solution into the membrane are not limiting. Many immobilized enzyme, antibiotic and microbial carriers for use in ISFET microbiosensors have been developed. For example, cellulose triacetate [ 51, polyvinylchloride [6], and agar [7] have been utilized as membranes, but are, however, not ideally suited for biosensor use, since *Author to whom correspondence should be addressed. 0304-5102/86/$3.50
0 Elsevier Sequoia/Printed in The Netherlands
I.34
adhesion to the surface of the silicon nitride gate is, in practice, poor. The characteristics of the ISFET microbiosensor depend on the immobilized enzyme membrane, and are thus of great importance. In this paper, a novel membrane material for use in an ISFET microbiosensor is described, and its properties are discussed.
Experimental Materials Polyvinylbutyral resin Industries, Ltd. The structure
was purchased from Wako Pure Chemical of polyvinylbutyral resin is as follows:
It consists of vinylbutyral, vinyl alcohol and vinyl acetate moieties, in the approximate ratio 75:22:3, respectively. Urease (EC 3.5.1.5, from jack beans, 3.9 units mg-‘) was purchased from Sigma Chemical Company, dichloromethane and glutaraldehyde from Kanto. Chemical Company, 1,8diamino4-aminomethyloctane was a gift from Asahi Kasei Industries and the silicon wafer was obtained from Kyoto Ceramic Company. All other polymers and chemicals used were of analytical grade. All solutions were prepared in distilled water. Assessment of polymer adhesion to silicon nitride surface Twenty-two different species of polymer were examined for their adhesion to silicon. nitride. Silicon nitride was formed on a silicon wafer by the chemical vapor deposition method and was used as the substrate for the tests. 0.1 g of the polymers was dissolved in 10 ml of the respective solvent. The polymer solutions were then cast onto the substrate and formation accelerated by placing the substrate in an oven for 20 min at 60 “C. The substrate membranes were then placed into either distilled water, 1 M NaOH solution, or 1 M HCl solution for 2 h at 40 “C. After this treatment, the adhesive properties were examined by scratching the membrane off the substrate. Preparation of the polyvinylbutyral resin membrane 0.1 - 1.0 g polyvinylbutyral resin was dissolved in 10 ml dichloromethane and cast onto a glass plate at room temperature. After 8 h setting, the membrane formed was leached off in distilled water at room temperature for 2 h.
135
Measurement of membrane thickness and water content The thickness of the polyvinylbutyral resin membrane was estimated using a micrometer thickness gauge. The water content of the membrane was determined from the weight loss after drying at 100 “C. Observation of membrane surface by scanning electron microscopy 0.1 g polyvinylbutyral resin and 1 ml 1,8diamino-4aminomethyloctane were dissolved in 10 ml dichloromethane and cast onto a silicon nitride-coated silicon wafer. The specimen was then dried using a critical point dryer (Hitachi, HCP-2), and sputter-coated with gold. This coated specimen was then analysed in a scanning electron microscope (Hitachi, S-415). Immobilization of urease Polyvinylbutyral resin and cellulose triacetate membranes were used for enzyme immobilization. 0.1 g of polymer and 1 ml of 1,8diamino-4-aminomethyloctane were dissolved in 10 ml dichloromethane, and cast onto a glass plate at room temperature. After 8 h setting, the membranes formed were leached off in distilled water at room temperature for 2 h. The membranes obtained were cut (1 cm*) and immersed in 5% glutaraldehyde solution at 4 “C for 24 h. They were then placed into a 5 mg ml-’ urease solution in 5 mM Tris HCl buffer (pH 7.0) and incubated at 4 “C for 24 h, after which they were washed with a large volume of 5 mM Tris HCl buffer ‘and stored in 5 mM Tris HCl buffer (pH 7.0) at 4 “C. Enzyme assay Activity of the immobilized urease membrane was measured by a spectrophotometric method [8]. Experiments were performed at 37 ‘C, pH 7.0 and the absorbance change at 570 nm was recorded.
Results
Polymer adhesion to the silicon nitride surface Table 1 shows the result of the adhesion tests. Almost all the polymers were stripped in distilled water at 40 ‘C, except polyglutamate, polyester urethane, polyvinylbutyral resin, 6,6-nylon, cellulose acetate, cellulose triacetate and cellulose acetate butyrate membranes. These polymers were then analysed in a 1 M HCI solution at 40 “C. Only the polyester urethane, polyvinylbutyral resin and cellulose acetate membranes remained unstripped, and so these were further analysed in a 1 M NaOH solution at 40 “C. Only the polyvinylbutyral resin membrane was not stripped. These adhesion tests indicate that of all the polymers analysed, polyvinylbutyral resin revealed the greatest adhesion to the silicon nitride surface.
136 TABLE 1 Assessment
of polymer
Polymera
adhesion
to a silicon nitride
Solvent
surface Adhesion Hz6
polystyrene polysulfone polycarbonate polyarylate polyethersulfone polysulfone sulfonate aromatic polyamide polyglutamate polyvinyl chloride polyvinyl acetate polyvinyl alcohol polyacrylonitrile polyvinyl difloride polyester urethane polyvinylbu.tyral resin 6,6-nylon cellulose acetate cellulose triacetate cellulose acetate butyrate agar K carrageenan sodium alginate aPolymer concentration: bImmersion conditions: stripped.
CH$ON(CH& HCON(CH& CHzClz CHzClz HCON(CH& HCON(CH& CH$ON(CH& CHzClz C4HsO
CHCls Hz0
HCON(CH& CH$ON( CH3)2 HCON( CH3)2 CH2C12 HCOOH (CH3)zCO
CH2C12 CH2C12 H2’J H20 H20
0.1 g/10 ml solvent; 40 “C, 2 h; Adhesive
testb lMHC1
1 M NaOH
X X
X X X X X 0
X
X X X X X 0 0
0 0 0
0
0
X
0 X
0
0
X
X X
X X X heat treatment: 60 “C, 20 min. estimation: scratch test; X: stripped;
0: not
Membrane thickness and water content Table 2 shows the membrane thickness and water content of the polyvinylbutyral resin membrane formed at different polymer concentrations. At higher polymer concentrations, the thickness of the membrane is increased and its water content decreased. In view of the diffusion of substrates and products, it is desirable to utilize a membrane which possesses a minimum thickness and a high water content. Observation of membrane surface by scanning electron microscopy Figure 1 shows an electron micrograph of the surface structure of the gold-sputtered polyvinylbutyral resin membrane. Figure 2 shows the pore size distribution of the polyvinylbutyral resin membrane estimated from the electron micrograph; the pore characteristics are summarized in Table 3. These results indicate that the polyvinylbutyral resin membrane possesses a microfilter, porous structure, ideally suited for high-density enzyme immobilization and chemical diffusion.
137 TABLE 2 Membrane
thickness
and water content
of polyvinylbutyral
resin membrane
Polymer concentrationa (%)
Membrane thickness (@n)
Water contentb (%)
1 3 5 7 10
23 62 114 130 222
27.6 10.9 8.7 8.0 8.5
aSolvent : CH&la. bWater content
=
wt wet -
wtdry
x 100; drying conditions:
100 *C, 2 h.
wt wet
Fig. 1. Electron micrograph membrane. The membrane electron microscopy.
showing the surface was sputter-coated
structure of the polyvinylbutyral resin with gold and observed by scanning
TABLE 3 Pore properties
of polyvinylbutyral
Mean pore diameter (Erm)
Maximum diameter (pm)
0.25
0.75
aObservation
resin membranea Minimum diameter (pm)
pore
by SEM. Area observed:
0.03 8.1
x
lo-‘cm’.
pore
Number of pores (n cmm2) 3.2 x lo8
138 100
6 9 $ z F
-
50
L
0 0.05
: 0.53
0.29
Pore
d ameter
0.77
( urn )
Fig. 2. Pore size distribution of the polyvinylbutyral resin membrane. Only pores greater than 0.01 pm in diameter were counted from electron micrograph pictures of the polyvinylbutyral resin membrane. Area observed was 8.1 x 10e7 cm’.
TABLE 4 Comparison of urease activity Membrane
Enzyme activitya (ng urea min-’ cm+)
cellulose triacetate polyvinybutyral resin
159 198
Wrea concentration:
80 PM; 37 “C, pH 7.0
Enzymeactivity of cellulose triacetate and polyvinylbutyral resin membranes Table 4 gives a comparison of urease activity for the cellulose triacetate and polyvinylbutyral resin membranes. Cellulose triacetate membrane has been conventionally used as an immobilized urease membrane for the ISFET micro-urea sensor. The activity of immobilized urease polyvinylbutyral resin membrane was, however, approximately 24% higher than that of the immobilized urease cellulose triacetate membrane.
139
Discussion Of all the membranes analysed, polyvhiylbutyral resin membrane revealed the most tenacious adhesion to the silicon nitride surface under acidic, alkaline and neutral conditions at 40 “C, even greater than that of cellulose triacetate, polyvinyl chloride and agar membranes, which are conventionally used for the immobilized enzyme membrane ISFET microbiosensor. It is thought that the high adhesion to the silicon nitride surface results from repetitive vinyl alcohol sequences in the polymer, forming continuous hydroxy regions within its structure. The repetitive hydroxy regions in the polymer also contribute to its expected hydrophilic properties. Concomitantly, the polyvinylbutyral resin membrane is thin, has a high water content and a multipore structure. These properties distinguish the polyvinylbutyral resin membrane as being very well suited for the purposes of enzyme immobilization and the diffusion of substrates and products within it. The activity of the immobilized urease polyvinylbutyral resin membrane is approximately 24% higher than for the specific type of cellulose triacetate membrane used in this study. It is thought that this enhancement results from a reaction between glutaraldehyde and the vinyl alcohol hydroxy regions, contributing to a high immobilized enzyme density. In conclusion, the polyvinylbutyral resin membrane is highly suited for use as an ISFET microbiosensor membrane in view of its adhesion to silicon nitride substrates. Moreover, its structural properties and high enzyme activity could be further applied to other immobilized enzyme systems. Our intention is to develop the immobilized urease polyvinylbutyral resin membrane for its application in an ISFET micro-urea sensor. The results of this study will shortly be reported.
Acknowledgement We thank Mr. J. M. Dicks for helpful discussions.
References 1 2 3 4 5
P. Rergveld, IEEE Trans. Biomed. Eng., 17 (1970) 70. T. Matsuo and K. D. Wise, IEEE Trans. Biomed. Eng., 21 (1974) 485. T. Matsuo and M. Esashi, Sensors and Actuators, 1 (1981) 71. S. Caras and J. Janata, Anal. Chem., 52 (1980) 1935. Y. Miyahara, F. Matsu, T. Moriizumi, H. Matsuoka, I. Karube and S. Suzuki, in Proc. Znt. Meeting on Chemical Sensors, Kodansha, Tokyo, 1983, pp. 501 - 506. 6 S. D. Moss, J. Janata and C. C. Johnson, Anal. Chem., 47 (1975) 2238. 7 Y. Hanazato and S. Shiono, in Proc. Znt. Meeting on Chemical Sensors, Kodansha, Tokyo, 1983, pp. 513 - 518. 8 R. L. Searcy, J. E. Reardon and J. A. Foreman, Am. J. Med. Tech., 33 (1967) 15.