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Urea sensor based on an ammonium-ion-sensitive field-effect transistor
Sensors and Actuators
Urea
sensor
Yuji Miyahara*, Central
Research
287
B, 3 (1991) 287-293
based
on an ammonium-ion-sensitive
Keiji Tsukada
Laboratory,
Hitachi
field-effect
transistor
and Hiroyuki Miyagi
Ltd.,
l-280,
Higashikoigakubo,
Kokubunji,
Tokyo
185 (Japan)
Wilhelm Simon Department of Organic (Switzerland)
Chemisrry,
Swiss
Federal
Institute
of Technologv,
Universitatstrasse
16, CH-8092
Zurich
(Received March 1, 1991; in revised form June 11, 1991; accepted June 12, 1991)
Abstract A urea sensor based on an ammonium-ion-sensitive field-effect transistor has been realized in combination with an immobilized urease membrane. The effect of buffer concentrations on the response characteristics of the sensor is investigated. The lower detection limit, as given by the calibration curve, is strongly affected by the concentrations of potassium and sodium ions, while its slope in the appropriate buffer solution is independent of the buffer concentration in a limited urea concentration range. Although the slope in physiological saline solution is only 18 mV/dec, the dynamic range of the sensor covers the physiological range of urea in blood. The small slope and the narrow dynamic range are found to be due to poor selectivity of the ammonium-ion-selective membrane over sodium and potassium ions. It is, therefore, possible to improve the sensor response characteristics by improving the selectivity of the ammonium-ion-selective membrane.
1. Introduction
A number of studies on biosensors in which semiconductor devices are used as transducers have been reported over the last ten years. Among these semiconductor biosensors, enzyme-modified hydrogen-ion-sensitive field-effect transistors (pH-ISFETs), which are based on the potentiometric determination of H+* ions produced by an enzyme-substrate reaction, have been widely investigated [l-5]. The serious limitations and possible usefulness of the sensor were pointed out by experimental and theoretical studies [6-lo]. Due to the operating principle of an enzymemodified pH-ISFET, the sensor response is strongly dependent on the buffer capacity of the sample because the pH change produced by an enzyme-substrate reaction is suppressed by the buffer used, which leads to a narrow dynamic range and a loss in sensor sensitivity. Although a coulometric pH-ISFET combined with an actuator electrode has been proposed to keep the pH in the enzyme membrane at *Author to whom correspondence
0925-4005/91/$3.50
should be addressed.
the gate region constant [ll], it is in general difficult to overcome this problem as long as a pH-ISFET is used as a transducer in biosensors. If other ISFETs instead of pH-ISFETs are used in biosensors to detect concentration changes of other ions produced by an enzyme-substrate reaction, the sensor responses are not considered to be affected by the buffer capacity. The ammonium ion is known to be produced by several enzyme-substrate reactions such as urease-urea, creatininase-creatinine and amino acid oxidaseamino acids, and therefore corresponding enzyme electrodes have been constructed based on these reactions using either an ammoniumion-selective electrode [12,13] or an ammonia gas electrode [14, 151. In the present study, an ammonium-ionsensitive field-effect transistor (NH,+-ISFET) is used as an example for a urea sensor in combination with an immobilized urease membrane to detect ammonium ions produced by the urease-urea reaction. The fundamental characteristics of the urea sensor
0 1991 -
Elsevier Sequoia, Lausanne
based on an NH,+ -1SFET are presented its potential advantages are discussed.
and
2. Principle
3. Experimental
Urea is catalysed by urease according to the reaction CO(NH,), + 2H30 + = CO,+2NH,+
+H,O
(1)
In conventional urea sensors, pH electrodes [16], ammonium-ion-selective electrodes [12, 171, ammonia gas electrodes [14, 181 and carbon dioxide electrodes [ 191 have been used to detect hydrogen ions, ammonium ions, ammonia gas and carbon dioxide, respectively, which are produced by reaction (1). Some of these principles were adopted in so-called micro urea sensors in which a pH-ISFET [2, 3, 51 or an ammonia-gas-sensitive capacitor [20] was used. The conceptual structure of the urea sensor system studied in the present report is shown in Fig. 1. The system is composed of an urea FET which has an immobilized urease membrane at the gate, a reference FET with a blank membrane which does not contain urease and a reference electrode. When the concentration of urea in the sample solution changes, ammonium ions are produced in the immobilized urease membrane of the urea FET based on reaction (1). A concentration change of ammonium ions is detected by the NH,+-ISFET in a differential measurement with the reference FET. A differential measurement is often used in enzyme-modified Reference
Immobilized
ureass
NH,’
ion-satect
3.1. Ammonium-ion-selective
membrane
The materials and composition of the ammonium-ion-selective membrane used in the present study are listed in Table 1. Nonactin/ monactin, bis-(Zethylhexyl) sebacate and poly(vinylchloride) were used as ligand, plasticizer and matrix, respectively. These components were dissolved homogeneously in tetrahydrofuran. The mixture was injected onto the gate region of the FET by using a micro syringe and dried for two days to form a membrane. The thickness of the membrane was estimated to be 100 pm. 3.2. Immobilized urease membrane An immobilized urease membrane was prepared in a similar way to that described in ref. 21. First 2 mg of urease and bovine serum albumin were dissolved in 1 ml of 0.1 M pH 7.0 phosphate buffer solution. The mixture was injected onto the ammonium-ion-selective membrane at the gate region of the ISEET. After drying, 25% glutaraldehyde aqueous solution was introduced onto the gate region and the crosslinking reaction was allowed to proceed at 4 “C for 12 h. The membrane for the reference FET did not contain urease and was prepared only from bovine serum albumin and glutaraldehyde. Before use, both the urea FET and reference FET were cleaned with 0.1 M pH 7.0 phosphate buffer solution. 3.3. ISFET The ISFETs used in the present study were n-channel depletion-mode FETs, having as an insulator a 50 nm thick layer of silicon dioxide covered with a 120 nm thick layer of
electrode
TABLE 1. Composition and materials for ammonium-ionselective membrane
ive
lembrana
SbN4 SiO2
Urea FE1
FETs to detect only the ion concentration change produced by the enzyme-substrate reaction (see [l-5]).
Composition (wt.%)
Material
Rsfcrance
FE1
Fig. 1. Conceptual structure of the urea FET system.
Ligand Plasticizer Matrix
nonactinlmonactin bis-(Z-ethylhexyl) sebacate poly(vinylchloride)
1 33 66
289
Reference
‘3
Sample
tassium ions as metal cations was prepared by mixing aqueous solutions of dipotassium hydrogenphosphate and potassium dihydrogenphosphate (potassium phosphate buffer). The physiological saline solution was prepared from 4 mM potassium chloride, 140 mM sodium chloride, 0.6 mM magnesium chloride and 1.1 mM calcium chloride.
e I ect rode
Urea F,ET @T
u
solution
Fig. 2. Circuit diagram for measuring urea FET system.
the response
of the
silicon nitride. ISFET chips were mounted on printed circuit boards and encapsulated by epoxy resin (Araldite CY205 and Hardner HT972, Ciba-Geigy) except in the gate regions. The responses of the ISFET, the urea FET and the reference FET were measured by the circuit [22] shown in Fig. 2. The potential change produced at the ion-selective membrane could be read out directly with the drain current Z,, and the source-drain voltage V,, kept constant. The output signal of the circuit was connected to a differential amplifier, a low-pass filter, a multiplexer, a digital voltmeter and a computer. A double-junction calomel electrode of the type Hg, HgCl,; KCl(satd.) I1 M LiOCOCH3 I was used as a reference electrode. 3.4. Evaluation methods Response characteristics of the NH, +-ISFET were evaluated for slope and selectivity in comparison with those of classical NH,+ion-selective electrodes. The slope of the sensor response was determined by linear regression using four equidistant data points in the range 10-l to 10e4 M. Selectivity factors were determined by the separate solution method (SSM) using 0.1 M metal-chloride solutions. Calibration curves of the urea FET system were obtained in a phosphate buffer solution of pH 7.0 or in a physiological saline solution. The lower detection limit and slope of the calibration curve were evaluated. The phosphate buffer solution, which contained only sodium ions as metal cations, was prepared by mixing aqueous solutions of disodium hydrogenphosphate and sodium dihydrogenphosphate (sodium phosphate buffer), while the buffer solution which contained only po-
3.5. Reagents Poly(vinylchloride), bis-(2-ethylhexyl) sebacate and tetrahydrofuran were purchased from Fluka AG, Switzerland. Nonactin/monactin was purchased from Ciba-Geigy AG, Switzerland. Urease, bovine serum albumin and glutaraldehyde were purchased from Sigma Chemical Co., U.S.A. Water, doubly distilled in a quartz apparatus, and chemicals of highest available purity were used for all aqueous solutions. 4. Results and discussion 4.1. Characteristics of NH, + -ZSFE Ts A calibration curve of the NH,+-IFSET is shown in Fig. 3. The slope of the NH,+ISFET is 55.4 mV/dec, which is somewhat small compared with that of the NH,‘-ionselective electrode (ISE) (57.4 mV/dec). Selectivity factors of both the NH,+-ISFET and NH,+-ISE are shown in Fig. 4. The selectivity factors of the NH,+-ISFET are comparable to those of NH, + - ISEs. Potassium and sodium ions are the main interfering ions for selective detection of ammonium ions. The slope and the selectivity factor over sodium ion obtained
-650 I T
z aI ft 44
-
-900
-fOOO/ -950
-1050 1: 2
-1100
//
2 -1150
>”
* I
-1200
I -6.0
-5.0
Fig. 3. Calibration
-4.0
-3.0
log
aNHa’
curve
-2.0
-1.0
of the NH.,+-ISFET.
-
NH4
-
K+
-
Na+
-
H+
1
=
rg++
-
CB
NH,*-
I SFET
*
-NH4
-
K+
-
Na’
-
H’
-
t]g
-
c$*
NH,*-ISE
Fig. 4. Selectivity factors for the NH,+-ISFET ISE.
and NH,+-
Fig. 5. Effect characteristics
of sodium ion concentration of the urea FET system.
on response
I
for the NH,+- ISFET are similar to the reported values [23]. The typical drift of the NH,+-ISFET is approximately 0.2 mV/h, which would not be cancelled out completely even in the differential urea FET system, because the characteristics of all ISFETs are not the same. 4.2. Effect of the buffer solution on the response of the urea FET system Calibration curves of the urea FET system which were obtained in the sodium phosphate buffer solution are shown in Fig. 5. The concentrations of sodium ion, as background ion, change from 1 M to 10m3 M. When the concentration of the background sodium ion is low3 M, the slope of the calibration curve is 35.2 mV/dec in the range 10e4 M to lo-’ M. The highest slope is 55.8 mV/dec in the range lo-’ to lop4 M. As the concentration of sodium ions increases, the lower detection limit for urea moves towards higher concentrations and the dynamic range of the sensor is narrowed. However, when sodium buffer solutions in the range 10e3 to 10-l M are used, the slopes of the urea FET system are 35.2 mV/dec, 37.0 mV/dec and 32.5 mV/dec in lop3 M, lo-* M and 10-i M sodium phosphate buffers, respectively. It can be stated that the slope of the sensor is more or less independent of the concentration of the sodium phosphate buffer below 10-l M
Potassium Phosphate Buffer
pH 7.0
Fig. 6. Effect of potassium ion concentration characteristics of the urea FET system.
on response
for urea concentrations from 10m4 M to lo-* M. But when a 1 M buffer solution is used as a background, the slope of the urea FET system decreases, because the response is shifted to the transition region between the lower detection limit and the higher detection limit. Calibration curves of the urea FET system which were obtained in potassium phosphate buffer solutions are shown in Fig. 6. When a 10e4 M buffer solution was used, the slope of the urea FET system is 32.0 mV/dec in the range 10e4 M to lo-* M. As the concentration of the potassium phosphate buffer becomes higher, the lower detection limit moves towards the high concentration region of the calibration curve and as a result the
291
slope of the sensor becomes small. The urea sensor system becomes almost insensitive to urea when 10-l M potassium ions were in the buffer solution. Obviously the effect of potassium ion concentrations on the response of the urea FET system is more serious than that of sodium ion concentrations. In order to analyse the response characteristics of the urea FET system, an approach based on the theory of a conventional urea sensor with an NH,+ -1SE was used. According to ref. 24, the response of the urea sensor can be written as v= v, +s log([P] + (n’k,l(k, + kKWLd)~Sl
+
F
-
Physiological K’:
-
Nd:lLO
10-S
Saline
Solution
40 mM
v.7
Urea
mM
ld4
lo-3
Concentration
a2
16’
IM I
Fig. 8. Calibration curve of the urea FET system in physiological saline solution.
+k,
[Ql)
(2)
where V is the potential of the urea sensor; V, is the potential of the reference electrode; s is the slope sensitivity; [P] is the ammonium ion concentration; [S] the urea concentration; [Q] the concentration of interfering species; n’, kR, k,, KS, KM are constants. From this equation, the theoretical response of the urea sensor at equilibrium can be calculated. The relationship between the sensor potential and urea concentration is shown in Fig. 7. KJ KM = 0.015 mol/l, kR/k, = 0.0001786 x [E]([E] is the urease concentration) and s = 59.14 mV/ dec (at 298 K) were used for the calculation
log[SI
100 2,
IMI
Fig. 7. Theoretical calibration curves for a urea sensor. (a) Enzyme concentration, 200 g/l; (b) enzyme concentration, 10 gIl.
according to ref. 24 and ([P]+[Q])/n’ was changed from lo-’ to 10v3 mol/l. Figure 7(a) and (b) represents the calibration curves of the urea sensor with enzyme concentrations of 200 and 10 g/l, respectively. From these results, the lower detection limit of the sensor is found to be strongly affected by the concentrations of product and interfering species. When the concentration of interfering species present in the sample solution as background becomes higher, the dynamic range of the sensor is narrowed and, as a result, the apparent slope of the response of the sensor becomes smaller. Since the selectivity factor of ammonium-ion-selective membranes for potassium ions is bigger than for sodium ions, it is reasonable that the slope and the dynamic range of the urea FET system obtained in the presence of potassium ions are poorer than those obtained in the presence of sodium ions. Therefore, the NH,+-ISFET should be highly specific for ammonium ions to exhibit a wide dynamic range and to keep the response independent of the concentrations of other ions. In order to check the possible applicability of the urea FET system in blood samples, the response of the sensor was measured in physiological saline solutions. The calibration curve of the sensor is shown in Fig. 8. The slope of the sensor is 18 mV/dec in the range 10e4 to lo-’ M. The reference interval of urea in blood is 2-2.5 X 10m3 M [25] for a healthy person. Therefore, the urea sensor can be used to measure the concentration of urea in blood, although the slope of the calibration curve should be improved.
292
5. Conclusions The slopes of the response of urea FET systems based on NH,+-ISFETs were found to be unaffected by the concentration of buffer solutions in a limited range of urea concentrations, if appropriate buffer solutions were used. The lower detection limit was, however, strongly affected by the concentrations of potassium and sodium ions in the sample solution, because of the insufficient selectivity of the nonactin/monactin-based ammoniumion-selective membrane over potassium and sodium ions. In order to maintain a wide dynamic range and a high sensitivity, the selectivity of the ion-selective membrane should be improved. In the case of urea sensors based on a pH-ISFET, the response is affected by the buffer capacity. This problem is in principle due to suppression of the pH change produced by the enzyme-substrate reaction by the sample buffer solution. Therefore, it is difficult to improve the sensor as long as a pH-ISFET is used as a detector in enzyme-coupled semiconductor sensors. On the other hand, the response characteristics of enzyme-coupled semiconductor sensors based on NH,‘-ISFETs can be improved by improving the selectivity of the ammoniumion-selective membrane.
S J. Kimura, T. Kuriyama and Y. Kawana, An integrated SOS/FET multi-biosensor. Sensors and Actuators, 9
(1986) 373-387. 6 S. D. Caras, J. Janata, D. Saupe and K. Schmitt, pHbased enzyme potentiometric sensors. Part 1. Theory, Anal. Chem., 57 (1985) 1917-1920. 7 S. D. Caras, D. Petelenz and J. Janata, pH-based enzyme potentlometric sensors. Part 2. Glucose-sensitive field effect transistor, Anal. Chem., 57 (1985) 1920-1923. 8 S. D. Caras and J. Janata, pH-based enzyme potentiometric sensors. Part 3. Penicillin-sensitive field effect transistor, Anal. Chem., 57 (1985) 1924-1925. 9 M. J. Eddowes, D. G. Pedley and B. C. Webb, Response of an enzyme-modified pH-sensitive ion-selective device; experimental study of a glucose oxidase-modified ion-sensitive field-effect transistor in buffered and unbuffered aqueous solution, Sensors and Actuators, 7 (1985)
233-244.
10 M. J. Eddowes, Response of an enzyme-modified pHsensitive ion-selective device; analytical solution for the response in the presence of pH buffer, Sensors and Actuators, I1 (1987) 265-274. 11 B. H. van der Schoot, H. Voorthuyxen and P. Bergveld, The pH-static enzyme sensor: design of the pH control system, Sensors and Actuators, BI (1990) 546-549. 12 G. G. Guilbaut and J. G. Montalvo, An enayme electrode for the substrate urea, J. Am. Chem. Sot., 92 (1970)
2533-2538.
13 G. G. Guilbaut and E. Hrabankova, An electrode for determination of amino acids, Anal. Chem., 42 (1970) 1779-1783. 14 G. G. Guilbaut and M. Tarp, A specific enzyme electrode for urea, Anal. Chim. Acfa, 73 (1974) 355-365. 15 M. Meyerhoff and G. A. Rechnitz, An activated enzyme electrode for creatinine, Anal. Chim. Acta, 85 (1976) 277-285.
Acknowledgements The authors are grateful to Dr K. Yagi and Mr Y. Kawamoto in Hitachi Ltd. for their help in ISFET fabrication.
References S. Caras and J. Janata, Field effect transistor sensitive to penicillin, AnaL Chem., 52 (1980) 1935-1937. Y. Miyahara, T. Moriixumi, S. Shiokawa, H. Matsuoka, I. Karube and S. Suzuki, Micro urea sensor using semiconductor and enzyme-immobilizing technologies, 1. Chem. Sot. Jpn., 6 (1983) 823-830. Y. Miyahara, T. Moriizumi and K. Ichimura, Integrated
enzyme FETs for simultaneous determinations of urea and glucose, Sensors and Actuators, 7 (1985) l-10. Y. Hanaxato, M. Nakako, M. Maeda and S. Shiono, Glucose sensor based on field-effect transistor with a photolithographically patterned glucose oxidase membrane, Anal. Chim. Acta, I93 (1987) 87-96.
16 J. Ruzicka, E. H. Hansen, A. K. Ghose and H. A. Mottola, Enzymatic determination of urea in serum based on pH measurement with the flow injection method, Anal. Chem., 51 (1979) 199-203. 17 G. G. Guilbaut and J. G. Montalvo, Urea-specific enzyme electrode, _I. Am. Chem. Sot., 91 (1969) 2164-216s. 18 R. A. Llenado and G. A. Rechnitz, Automated serum urea determination using membrane electrodes, Anal. Chem., 46 (1974) 1109-1112. 19 G. G. Guilbaut and F. R. Shu, Enzyme electrodes based on the use of a carbon dioxide sensor. Urea and r-tyrosine electrodes, Anal. Chem., 44 (1972) 2161-2166. 20 F. Winquist, A. Spetz, I. Lundstrom and B. Danielsson, Determination of urea with an ammonia gas-sensitive semiconductor device in combination with urease, Anal. Chim. Acfa,
163 (1984)
143-149.
21 M. Mascini and G. G. Guilbaut, Urease coupled ammonia electrode for urea determination in blood serum, Anal. Chem., 49 (1977) 795-798. 22 H. Nakajima, M. Esashi and T. Matsuo, The pHresponse of organic gate ISFETs and the influence of macro-molecule adsorption,J. Chem. Sot. Jpn., 10 (1980) 1499-1508.
293 23 U. Oesch, S. Caras and J. Janata, Field effect transistor sensitive to sodium and ammonium ions, Anal. Chem., 53 (1981) 1983-1986. 24 W. E. Morf, Theoretical evaluation of performance of enzyme electrodes and enzyme reactors, Mikrochim. Acza,
2 (1980)
317-332.
25 N. Kosakai and M. Abe (eds.), Normal Examinations
Igaku-shoin,
1982, where he has been involved in the study of chemical sensors. He received the Ph.D degree from Tsukuba University in 1990.
and its Application
Value in Clinical to Clinical Medicine,
Tokyo, 3rd edn., 1983, p. 105.
Biographies Yuji Miyuharu was born in Tokyo in 1956. He received B.S., M.E. and Ph.D. degrees from Tokyo Institute of Technology in 1980, 1982 and 1985, respectively. He joined Hitachi Ltd. in 1985. From 1988 to 1989, he was at the Swiss Federal Institute of Technology (ETH) as a visiting researcher. His interest is in solid-state biochemical sensors.
Keiji Tsukada received the B.S. in natural science and an M.E. degree in scientific technology from Tsukuba University in 1979 and 1982, respectively. He joined Hitachi Ltd. in
Hiroyuki Miyagi received the B.S. and M.E. degrees in geo-science from Nagoya University in 1967 and 1969, respectively, after which he joined Hitachi Ltd. He has been engaged in environmental and medical measurements and he has been a department manager in the Central Research Laboratory, Hitachi Ltd. since 1989.
Wilhelm Simon received his diploma in chemistry at the Swiss Federal Institute of Technology (ETH) in Zurich in 1953 and his doctorate in 1956. He was appointed Privatdozent, Assistenzprofessor, ausserordentlicher Professor and ordentlicher Professor at the same school in 1960, 1965, 1967 and 1970 respectively. Since 1985 he has been full professor of analytical chemistry at ETH Zurich.
Urea
sensor
Yuji Miyahara*, Central
Research
287
B, 3 (1991) 287-293
based
on an ammonium-ion-sensitive
Keiji Tsukada
Laboratory,
Hitachi
field-effect
transistor
and Hiroyuki Miyagi
Ltd.,
l-280,
Higashikoigakubo,
Kokubunji,
Tokyo
185 (Japan)
Wilhelm Simon Department of Organic (Switzerland)
Chemisrry,
Swiss
Federal
Institute
of Technologv,
Universitatstrasse
16, CH-8092
Zurich
(Received March 1, 1991; in revised form June 11, 1991; accepted June 12, 1991)
Abstract A urea sensor based on an ammonium-ion-sensitive field-effect transistor has been realized in combination with an immobilized urease membrane. The effect of buffer concentrations on the response characteristics of the sensor is investigated. The lower detection limit, as given by the calibration curve, is strongly affected by the concentrations of potassium and sodium ions, while its slope in the appropriate buffer solution is independent of the buffer concentration in a limited urea concentration range. Although the slope in physiological saline solution is only 18 mV/dec, the dynamic range of the sensor covers the physiological range of urea in blood. The small slope and the narrow dynamic range are found to be due to poor selectivity of the ammonium-ion-selective membrane over sodium and potassium ions. It is, therefore, possible to improve the sensor response characteristics by improving the selectivity of the ammonium-ion-selective membrane.
1. Introduction
A number of studies on biosensors in which semiconductor devices are used as transducers have been reported over the last ten years. Among these semiconductor biosensors, enzyme-modified hydrogen-ion-sensitive field-effect transistors (pH-ISFETs), which are based on the potentiometric determination of H+* ions produced by an enzyme-substrate reaction, have been widely investigated [l-5]. The serious limitations and possible usefulness of the sensor were pointed out by experimental and theoretical studies [6-lo]. Due to the operating principle of an enzymemodified pH-ISFET, the sensor response is strongly dependent on the buffer capacity of the sample because the pH change produced by an enzyme-substrate reaction is suppressed by the buffer used, which leads to a narrow dynamic range and a loss in sensor sensitivity. Although a coulometric pH-ISFET combined with an actuator electrode has been proposed to keep the pH in the enzyme membrane at *Author to whom correspondence
0925-4005/91/$3.50
should be addressed.
the gate region constant [ll], it is in general difficult to overcome this problem as long as a pH-ISFET is used as a transducer in biosensors. If other ISFETs instead of pH-ISFETs are used in biosensors to detect concentration changes of other ions produced by an enzyme-substrate reaction, the sensor responses are not considered to be affected by the buffer capacity. The ammonium ion is known to be produced by several enzyme-substrate reactions such as urease-urea, creatininase-creatinine and amino acid oxidaseamino acids, and therefore corresponding enzyme electrodes have been constructed based on these reactions using either an ammoniumion-selective electrode [12,13] or an ammonia gas electrode [14, 151. In the present study, an ammonium-ionsensitive field-effect transistor (NH,+-ISFET) is used as an example for a urea sensor in combination with an immobilized urease membrane to detect ammonium ions produced by the urease-urea reaction. The fundamental characteristics of the urea sensor
0 1991 -
Elsevier Sequoia, Lausanne
based on an NH,+ -1SFET are presented its potential advantages are discussed.
and
2. Principle
3. Experimental
Urea is catalysed by urease according to the reaction CO(NH,), + 2H30 + = CO,+2NH,+
+H,O
(1)
In conventional urea sensors, pH electrodes [16], ammonium-ion-selective electrodes [12, 171, ammonia gas electrodes [14, 181 and carbon dioxide electrodes [ 191 have been used to detect hydrogen ions, ammonium ions, ammonia gas and carbon dioxide, respectively, which are produced by reaction (1). Some of these principles were adopted in so-called micro urea sensors in which a pH-ISFET [2, 3, 51 or an ammonia-gas-sensitive capacitor [20] was used. The conceptual structure of the urea sensor system studied in the present report is shown in Fig. 1. The system is composed of an urea FET which has an immobilized urease membrane at the gate, a reference FET with a blank membrane which does not contain urease and a reference electrode. When the concentration of urea in the sample solution changes, ammonium ions are produced in the immobilized urease membrane of the urea FET based on reaction (1). A concentration change of ammonium ions is detected by the NH,+-ISFET in a differential measurement with the reference FET. A differential measurement is often used in enzyme-modified Reference
Immobilized
ureass
NH,’
ion-satect
3.1. Ammonium-ion-selective
membrane
The materials and composition of the ammonium-ion-selective membrane used in the present study are listed in Table 1. Nonactin/ monactin, bis-(Zethylhexyl) sebacate and poly(vinylchloride) were used as ligand, plasticizer and matrix, respectively. These components were dissolved homogeneously in tetrahydrofuran. The mixture was injected onto the gate region of the FET by using a micro syringe and dried for two days to form a membrane. The thickness of the membrane was estimated to be 100 pm. 3.2. Immobilized urease membrane An immobilized urease membrane was prepared in a similar way to that described in ref. 21. First 2 mg of urease and bovine serum albumin were dissolved in 1 ml of 0.1 M pH 7.0 phosphate buffer solution. The mixture was injected onto the ammonium-ion-selective membrane at the gate region of the ISEET. After drying, 25% glutaraldehyde aqueous solution was introduced onto the gate region and the crosslinking reaction was allowed to proceed at 4 “C for 12 h. The membrane for the reference FET did not contain urease and was prepared only from bovine serum albumin and glutaraldehyde. Before use, both the urea FET and reference FET were cleaned with 0.1 M pH 7.0 phosphate buffer solution. 3.3. ISFET The ISFETs used in the present study were n-channel depletion-mode FETs, having as an insulator a 50 nm thick layer of silicon dioxide covered with a 120 nm thick layer of
electrode
TABLE 1. Composition and materials for ammonium-ionselective membrane
ive
lembrana
SbN4 SiO2
Urea FE1
FETs to detect only the ion concentration change produced by the enzyme-substrate reaction (see [l-5]).
Composition (wt.%)
Material
Rsfcrance
FE1
Fig. 1. Conceptual structure of the urea FET system.
Ligand Plasticizer Matrix
nonactinlmonactin bis-(Z-ethylhexyl) sebacate poly(vinylchloride)
1 33 66
289
Reference
‘3
Sample
tassium ions as metal cations was prepared by mixing aqueous solutions of dipotassium hydrogenphosphate and potassium dihydrogenphosphate (potassium phosphate buffer). The physiological saline solution was prepared from 4 mM potassium chloride, 140 mM sodium chloride, 0.6 mM magnesium chloride and 1.1 mM calcium chloride.
e I ect rode
Urea F,ET @T
u
solution
Fig. 2. Circuit diagram for measuring urea FET system.
the response
of the
silicon nitride. ISFET chips were mounted on printed circuit boards and encapsulated by epoxy resin (Araldite CY205 and Hardner HT972, Ciba-Geigy) except in the gate regions. The responses of the ISFET, the urea FET and the reference FET were measured by the circuit [22] shown in Fig. 2. The potential change produced at the ion-selective membrane could be read out directly with the drain current Z,, and the source-drain voltage V,, kept constant. The output signal of the circuit was connected to a differential amplifier, a low-pass filter, a multiplexer, a digital voltmeter and a computer. A double-junction calomel electrode of the type Hg, HgCl,; KCl(satd.) I1 M LiOCOCH3 I was used as a reference electrode. 3.4. Evaluation methods Response characteristics of the NH, +-ISFET were evaluated for slope and selectivity in comparison with those of classical NH,+ion-selective electrodes. The slope of the sensor response was determined by linear regression using four equidistant data points in the range 10-l to 10e4 M. Selectivity factors were determined by the separate solution method (SSM) using 0.1 M metal-chloride solutions. Calibration curves of the urea FET system were obtained in a phosphate buffer solution of pH 7.0 or in a physiological saline solution. The lower detection limit and slope of the calibration curve were evaluated. The phosphate buffer solution, which contained only sodium ions as metal cations, was prepared by mixing aqueous solutions of disodium hydrogenphosphate and sodium dihydrogenphosphate (sodium phosphate buffer), while the buffer solution which contained only po-
3.5. Reagents Poly(vinylchloride), bis-(2-ethylhexyl) sebacate and tetrahydrofuran were purchased from Fluka AG, Switzerland. Nonactin/monactin was purchased from Ciba-Geigy AG, Switzerland. Urease, bovine serum albumin and glutaraldehyde were purchased from Sigma Chemical Co., U.S.A. Water, doubly distilled in a quartz apparatus, and chemicals of highest available purity were used for all aqueous solutions. 4. Results and discussion 4.1. Characteristics of NH, + -ZSFE Ts A calibration curve of the NH,+-IFSET is shown in Fig. 3. The slope of the NH,+ISFET is 55.4 mV/dec, which is somewhat small compared with that of the NH,‘-ionselective electrode (ISE) (57.4 mV/dec). Selectivity factors of both the NH,+-ISFET and NH,+-ISE are shown in Fig. 4. The selectivity factors of the NH,+-ISFET are comparable to those of NH, + - ISEs. Potassium and sodium ions are the main interfering ions for selective detection of ammonium ions. The slope and the selectivity factor over sodium ion obtained
-650 I T
z aI ft 44
-
-900
-fOOO/ -950
-1050 1: 2
-1100
//
2 -1150
>”
* I
-1200
I -6.0
-5.0
Fig. 3. Calibration
-4.0
-3.0
log
aNHa’
curve
-2.0
-1.0
of the NH.,+-ISFET.
-
NH4
-
K+
-
Na+
-
H+
1
=
rg++
-
CB
NH,*-
I SFET
*
-NH4
-
K+
-
Na’
-
H’
-
t]g
-
c$*
NH,*-ISE
Fig. 4. Selectivity factors for the NH,+-ISFET ISE.
and NH,+-
Fig. 5. Effect characteristics
of sodium ion concentration of the urea FET system.
on response
I
for the NH,+- ISFET are similar to the reported values [23]. The typical drift of the NH,+-ISFET is approximately 0.2 mV/h, which would not be cancelled out completely even in the differential urea FET system, because the characteristics of all ISFETs are not the same. 4.2. Effect of the buffer solution on the response of the urea FET system Calibration curves of the urea FET system which were obtained in the sodium phosphate buffer solution are shown in Fig. 5. The concentrations of sodium ion, as background ion, change from 1 M to 10m3 M. When the concentration of the background sodium ion is low3 M, the slope of the calibration curve is 35.2 mV/dec in the range 10e4 M to lo-’ M. The highest slope is 55.8 mV/dec in the range lo-’ to lop4 M. As the concentration of sodium ions increases, the lower detection limit for urea moves towards higher concentrations and the dynamic range of the sensor is narrowed. However, when sodium buffer solutions in the range 10e3 to 10-l M are used, the slopes of the urea FET system are 35.2 mV/dec, 37.0 mV/dec and 32.5 mV/dec in lop3 M, lo-* M and 10-i M sodium phosphate buffers, respectively. It can be stated that the slope of the sensor is more or less independent of the concentration of the sodium phosphate buffer below 10-l M
Potassium Phosphate Buffer
pH 7.0
Fig. 6. Effect of potassium ion concentration characteristics of the urea FET system.
on response
for urea concentrations from 10m4 M to lo-* M. But when a 1 M buffer solution is used as a background, the slope of the urea FET system decreases, because the response is shifted to the transition region between the lower detection limit and the higher detection limit. Calibration curves of the urea FET system which were obtained in potassium phosphate buffer solutions are shown in Fig. 6. When a 10e4 M buffer solution was used, the slope of the urea FET system is 32.0 mV/dec in the range 10e4 M to lo-* M. As the concentration of the potassium phosphate buffer becomes higher, the lower detection limit moves towards the high concentration region of the calibration curve and as a result the
291
slope of the sensor becomes small. The urea sensor system becomes almost insensitive to urea when 10-l M potassium ions were in the buffer solution. Obviously the effect of potassium ion concentrations on the response of the urea FET system is more serious than that of sodium ion concentrations. In order to analyse the response characteristics of the urea FET system, an approach based on the theory of a conventional urea sensor with an NH,+ -1SE was used. According to ref. 24, the response of the urea sensor can be written as v= v, +s log([P] + (n’k,l(k, + kKWLd)~Sl
+
F
-
Physiological K’:
-
Nd:lLO
10-S
Saline
Solution
40 mM
v.7
Urea
mM
ld4
lo-3
Concentration
a2
16’
IM I
Fig. 8. Calibration curve of the urea FET system in physiological saline solution.
+k,
[Ql)
(2)
where V is the potential of the urea sensor; V, is the potential of the reference electrode; s is the slope sensitivity; [P] is the ammonium ion concentration; [S] the urea concentration; [Q] the concentration of interfering species; n’, kR, k,, KS, KM are constants. From this equation, the theoretical response of the urea sensor at equilibrium can be calculated. The relationship between the sensor potential and urea concentration is shown in Fig. 7. KJ KM = 0.015 mol/l, kR/k, = 0.0001786 x [E]([E] is the urease concentration) and s = 59.14 mV/ dec (at 298 K) were used for the calculation
log[SI
100 2,
IMI
Fig. 7. Theoretical calibration curves for a urea sensor. (a) Enzyme concentration, 200 g/l; (b) enzyme concentration, 10 gIl.
according to ref. 24 and ([P]+[Q])/n’ was changed from lo-’ to 10v3 mol/l. Figure 7(a) and (b) represents the calibration curves of the urea sensor with enzyme concentrations of 200 and 10 g/l, respectively. From these results, the lower detection limit of the sensor is found to be strongly affected by the concentrations of product and interfering species. When the concentration of interfering species present in the sample solution as background becomes higher, the dynamic range of the sensor is narrowed and, as a result, the apparent slope of the response of the sensor becomes smaller. Since the selectivity factor of ammonium-ion-selective membranes for potassium ions is bigger than for sodium ions, it is reasonable that the slope and the dynamic range of the urea FET system obtained in the presence of potassium ions are poorer than those obtained in the presence of sodium ions. Therefore, the NH,+-ISFET should be highly specific for ammonium ions to exhibit a wide dynamic range and to keep the response independent of the concentrations of other ions. In order to check the possible applicability of the urea FET system in blood samples, the response of the sensor was measured in physiological saline solutions. The calibration curve of the sensor is shown in Fig. 8. The slope of the sensor is 18 mV/dec in the range 10e4 to lo-’ M. The reference interval of urea in blood is 2-2.5 X 10m3 M [25] for a healthy person. Therefore, the urea sensor can be used to measure the concentration of urea in blood, although the slope of the calibration curve should be improved.
292
5. Conclusions The slopes of the response of urea FET systems based on NH,+-ISFETs were found to be unaffected by the concentration of buffer solutions in a limited range of urea concentrations, if appropriate buffer solutions were used. The lower detection limit was, however, strongly affected by the concentrations of potassium and sodium ions in the sample solution, because of the insufficient selectivity of the nonactin/monactin-based ammoniumion-selective membrane over potassium and sodium ions. In order to maintain a wide dynamic range and a high sensitivity, the selectivity of the ion-selective membrane should be improved. In the case of urea sensors based on a pH-ISFET, the response is affected by the buffer capacity. This problem is in principle due to suppression of the pH change produced by the enzyme-substrate reaction by the sample buffer solution. Therefore, it is difficult to improve the sensor as long as a pH-ISFET is used as a detector in enzyme-coupled semiconductor sensors. On the other hand, the response characteristics of enzyme-coupled semiconductor sensors based on NH,‘-ISFETs can be improved by improving the selectivity of the ammoniumion-selective membrane.
S J. Kimura, T. Kuriyama and Y. Kawana, An integrated SOS/FET multi-biosensor. Sensors and Actuators, 9
(1986) 373-387. 6 S. D. Caras, J. Janata, D. Saupe and K. Schmitt, pHbased enzyme potentiometric sensors. Part 1. Theory, Anal. Chem., 57 (1985) 1917-1920. 7 S. D. Caras, D. Petelenz and J. Janata, pH-based enzyme potentlometric sensors. Part 2. Glucose-sensitive field effect transistor, Anal. Chem., 57 (1985) 1920-1923. 8 S. D. Caras and J. Janata, pH-based enzyme potentiometric sensors. Part 3. Penicillin-sensitive field effect transistor, Anal. Chem., 57 (1985) 1924-1925. 9 M. J. Eddowes, D. G. Pedley and B. C. Webb, Response of an enzyme-modified pH-sensitive ion-selective device; experimental study of a glucose oxidase-modified ion-sensitive field-effect transistor in buffered and unbuffered aqueous solution, Sensors and Actuators, 7 (1985)
233-244.
10 M. J. Eddowes, Response of an enzyme-modified pHsensitive ion-selective device; analytical solution for the response in the presence of pH buffer, Sensors and Actuators, I1 (1987) 265-274. 11 B. H. van der Schoot, H. Voorthuyxen and P. Bergveld, The pH-static enzyme sensor: design of the pH control system, Sensors and Actuators, BI (1990) 546-549. 12 G. G. Guilbaut and J. G. Montalvo, An enayme electrode for the substrate urea, J. Am. Chem. Sot., 92 (1970)
2533-2538.
13 G. G. Guilbaut and E. Hrabankova, An electrode for determination of amino acids, Anal. Chem., 42 (1970) 1779-1783. 14 G. G. Guilbaut and M. Tarp, A specific enzyme electrode for urea, Anal. Chim. Acfa, 73 (1974) 355-365. 15 M. Meyerhoff and G. A. Rechnitz, An activated enzyme electrode for creatinine, Anal. Chim. Acta, 85 (1976) 277-285.
Acknowledgements The authors are grateful to Dr K. Yagi and Mr Y. Kawamoto in Hitachi Ltd. for their help in ISFET fabrication.
References S. Caras and J. Janata, Field effect transistor sensitive to penicillin, AnaL Chem., 52 (1980) 1935-1937. Y. Miyahara, T. Moriixumi, S. Shiokawa, H. Matsuoka, I. Karube and S. Suzuki, Micro urea sensor using semiconductor and enzyme-immobilizing technologies, 1. Chem. Sot. Jpn., 6 (1983) 823-830. Y. Miyahara, T. Moriizumi and K. Ichimura, Integrated
enzyme FETs for simultaneous determinations of urea and glucose, Sensors and Actuators, 7 (1985) l-10. Y. Hanaxato, M. Nakako, M. Maeda and S. Shiono, Glucose sensor based on field-effect transistor with a photolithographically patterned glucose oxidase membrane, Anal. Chim. Acta, I93 (1987) 87-96.
16 J. Ruzicka, E. H. Hansen, A. K. Ghose and H. A. Mottola, Enzymatic determination of urea in serum based on pH measurement with the flow injection method, Anal. Chem., 51 (1979) 199-203. 17 G. G. Guilbaut and J. G. Montalvo, Urea-specific enzyme electrode, _I. Am. Chem. Sot., 91 (1969) 2164-216s. 18 R. A. Llenado and G. A. Rechnitz, Automated serum urea determination using membrane electrodes, Anal. Chem., 46 (1974) 1109-1112. 19 G. G. Guilbaut and F. R. Shu, Enzyme electrodes based on the use of a carbon dioxide sensor. Urea and r-tyrosine electrodes, Anal. Chem., 44 (1972) 2161-2166. 20 F. Winquist, A. Spetz, I. Lundstrom and B. Danielsson, Determination of urea with an ammonia gas-sensitive semiconductor device in combination with urease, Anal. Chim. Acfa,
163 (1984)
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21 M. Mascini and G. G. Guilbaut, Urease coupled ammonia electrode for urea determination in blood serum, Anal. Chem., 49 (1977) 795-798. 22 H. Nakajima, M. Esashi and T. Matsuo, The pHresponse of organic gate ISFETs and the influence of macro-molecule adsorption,J. Chem. Sot. Jpn., 10 (1980) 1499-1508.
293 23 U. Oesch, S. Caras and J. Janata, Field effect transistor sensitive to sodium and ammonium ions, Anal. Chem., 53 (1981) 1983-1986. 24 W. E. Morf, Theoretical evaluation of performance of enzyme electrodes and enzyme reactors, Mikrochim. Acza,
2 (1980)
317-332.
25 N. Kosakai and M. Abe (eds.), Normal Examinations
Igaku-shoin,
1982, where he has been involved in the study of chemical sensors. He received the Ph.D degree from Tsukuba University in 1990.
and its Application
Value in Clinical to Clinical Medicine,
Tokyo, 3rd edn., 1983, p. 105.
Biographies Yuji Miyuharu was born in Tokyo in 1956. He received B.S., M.E. and Ph.D. degrees from Tokyo Institute of Technology in 1980, 1982 and 1985, respectively. He joined Hitachi Ltd. in 1985. From 1988 to 1989, he was at the Swiss Federal Institute of Technology (ETH) as a visiting researcher. His interest is in solid-state biochemical sensors.
Keiji Tsukada received the B.S. in natural science and an M.E. degree in scientific technology from Tsukuba University in 1979 and 1982, respectively. He joined Hitachi Ltd. in
Hiroyuki Miyagi received the B.S. and M.E. degrees in geo-science from Nagoya University in 1967 and 1969, respectively, after which he joined Hitachi Ltd. He has been engaged in environmental and medical measurements and he has been a department manager in the Central Research Laboratory, Hitachi Ltd. since 1989.
Wilhelm Simon received his diploma in chemistry at the Swiss Federal Institute of Technology (ETH) in Zurich in 1953 and his doctorate in 1956. He was appointed Privatdozent, Assistenzprofessor, ausserordentlicher Professor and ordentlicher Professor at the same school in 1960, 1965, 1967 and 1970 respectively. Since 1985 he has been full professor of analytical chemistry at ETH Zurich.