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Analytical application for chemicals using an enzyme sensor based on an ISFET
578
Sensors and Actuators B, 13-14 (1993) 578-580
Analytical application for chemicals using an enzyme sensor based on an IWET Hiromitsu Sakai, Noriaki Kaneki and Hiroshi Hara Lkpartment of Compufw Science and Systems Engineering Faculty of Engineekzg
Mmmn Instituteof Technology,
27-l Mizumotcxho, Mummn 050 {lopan)
IIthOdWtlOll
Enviro~ental pollution by toxic chemicals has become one of the world’s most serious problems. In particular, monitoring by a sensor is more and more necessary for health-risk assessment of drinking water polluted by pesticides from golf links or agricultural land [l]. At present, the chemical analysis is mainly carried out by gas-chromatographic methods. However, this has many problems such as the complex procedures required for measuring chemicals, and large and expensive instruments. In this paper, we studied and discussed the characteristics and the application of an acetylcholine sensor based on ion-sensitive field-effect transistors (ISFETs) using acetylcholine esterase (AChE) as an effective and simple potentiometric sensor for detecting agricultural chemicals, as reversible and irreversible inhibitors. ExperiIlK.!lltal Reagents
Purified AChE and pyridine-Zaldoxime methiodide (PAM) was obtained from Sigma Chemical Co.; acetylcholine perchloride was obtained from Seikagaku Kogyo Co.; o-isopropoxyphenyl methylcarbamate (PHC), nicotine, trichlorfon (dimethyl 2,2,2-trichloro1-hydroxyethylphosphonate; DEP), Methomyl (Smethyl N-(methylcarbamoyloxy) thioacethnidate), Malathion and Dimethoate as insecticides were obtained from GL Science Co., and bovine serum albumin (BSA) was obtained from Toyobo Co., Ltd. All other chemicals employed were of analytical grade.
the ISFETs has been documented previously [3]. The AChE was coupled in albumin membrane following the procedure of the crosslinking method. Measurement of the potential by the acetylcholine sensor The measurement methods of the enzyme sensor
based on an ISFET have been described in previous studies [3-51. AChE catalyzes the hydrolysis of acetylcholine according to the reaction: (CH,),N +CH&H,OCOCH, + H,O (CH,),N+CH,CH~OH+HOCOCH,
the pH in the membrane is changed by the reaction. That is, the generated ions diffuse to the ISFET interface through the membrane and react electrochemically at the ISFET with a resulting increase in voltage. The measurement of chemicals using the acetylcholine sensor was carried and classified into three types of direct and indirect methods with various concentrations of inhibitors, as indicated in Fig. 1. From the difference Chenlcnl
Rnvnrrible
Anrhis by
inhibitor
Enzyme Sensor
Irrnverslble
PHC Nicotine
We fabricated a probe-type ISFET as a transducer following the methods of Matsuo and Esashi [2]. The ISFET was 1.8 mm wide, 500 pm thick and 9.6 mm long. The gate channel was approximately 1.5 mm long and 30 pm wide, with a channel depletion mode device. The procedure used to produce BSA membranes on
Inhibitor
Incubation in srwla for Msrrursnent
Fabrication of ISFET and procedure of a jired membmne
(1)
1 rfsrctivitv by 2-PAN
Ih
CB Me thorny DBP
I
@
Nsasursmmt
Dlmthosts Malathion
flex
t I” i ty
by Z-PAW
Fig. 1. Measurement procedure of chemicals by the enzyme sensor.
0 1993 - Elsevier Sequoia. All rights reserved
579
between the two steady states, i.e., before and after inhibition, the percentage of AChE inhibition was calculated. Dimethoate and Malathion were converted into their oxygen analogues by treating with bromine water since these chemicals as such do not show maximum toxicity. The reactivation of the AChE sensor was done by adding 1 mM PAM and 10 mM acetylcholine. 0
IO
20
30
40
50
Time
ResuIts and discussion Responseof the acetylcholinesensor using ISFET The characteristics of the ISFET with and without BSA membrane have been descriied in previous studies [4, 51. The detection range of acetylcholiie using the AChE-ISFET sensor was 0.05-10 mM at room temperature. The response of the enzyme sensor depended on pH (optimal pH 8.0) and the concentration of the buffer solution. The ouptput voltage was about - 48 mV/pH. In 10 mM HEPES buffer, no appreciable variation in the pH was observed during the measurement; hence, this buffer concentration was selected for the analysis of the toxic chemicals.
I (%) = ((EO- E)/E) x 100
(2)
where E, is the initial output voltage given by the sensor in the absence of the inhibitor and E is the potential response in the presence of the inhl%itor at the same substrate concentration. Figure 2 shows a typical response of the acetylcholine sensor in 10 mM HEPES buffer solutions containing 1OmMacetylcholine: the response (a) was in the absence of the inhibitor, and the response (b) was after having added PHC as reversible inhibitor. Figure 3 shows a calibration curve of PHC by the .sensor. On the other hand, typical responses of the sensor upon the addition of 10 mM acetylcholine for DEP and Methomyl of the irreversible inhibitors were almost in agreement to the previous report [3]. Figure 4 shows a typical response of the sensxx upon the acetylcholine solution: the response (a) was in the absence of the inhibitor, and the response (b) was after 1 h incubation (10-l M Methomyl) with the sensor at a particular concentration of the Methomyl.
70
80
Fig. 2. A typical response of the acetykholine sensor using ISFET for revexsible inhibitor in 10 mM acetylcholiie solution (pH-8.0): (a) in absence of the inhibitor, and (b) after addition of lo-’ M PHC.
10-3
IO-' PHC
Determinationof chemicaL.s by acetykholinesensor New data on the analysis of agricultural chemicals are reported using the present acetylcholine sensor based on the inhibition of the immobilized AChE. Determination of the toxic chemicals is based on the measurement of inhibition percentage (Z (%)) which is equal to the potential difference of the relative activity given by the sensor with and without the inhibitor for the same substrate concentration, and which can be depicted as follows:
60
(min)
concentration
CM)
Fig. 3. Calibration CUIVC of PHC by the acetylcholine sensor in 10 mM HEPES buffer solution with 10 mM acetylcholine.
s-“““““_
I
0
I
I
I
I
I
I
I
I
10 20 30 40 50 60 70 80 Time
(min)
Fig. 4. A typical response of the acetylcholine sensor using ISFET for irrexersible inhibitor in 10 mM acetyhzholinesolution (pH-8.0): (a) in absence of the inhibitor, 10-l M Methomyk (b) after incubation with the sensor, and (c) reactivity by 1 mM PAM.
Reactivation (c) of the enzyme was facilitated by adding 1 n&f PAM. Data on the quantitative analysis of Methomyl were recorded in terms of percentage inhibition as a function of inhibitor concentration for the substrate in Fig. 5. The rate of inhibition increase was proportional to the logarithm of the concent rations for chemicals. The detection of PHC and nicotine as reversible inhibitors, and DEP, Methomyl, Malathion and Dimethoate as irreversible inhibitors was found to be in the range of 10-6-10-1 M. However, in practical application, the low sensitivity of the sensor to chemicals must be improved by a selected receptor, immobilized-
580
sensor-inhibitionrates may be important as an index of biologicalsystems with regard to toxic matters. Conclusions
Fig. 5. Calibration curve of Methomyl by the acetylcholine sensor in 10 mM HEPES b&r solution with 10 mM acetylcboline.
TABLE 1. Comparison of chemical concentrations required for 50% inhibition of an acetylcholine esteras&mmobilid ISFET sensor with high enzyme activity Chemicals
Concentration for 50% inhibition
Methomyl DEP Dimethoate PHC Nicotine Malathion
1.2x 1.0x 5.0x 1.2x 3.6x 3.0x
10-l lo-* lo-’ lo-’ lo-’ 10-5
membrane methods, the fabrication of a more sensitive transducer and so on. Therefore, the inhibition to the enzyme of the urea sensor by the agriculturalchemicals was not observed in this study. The response of ISFET with the membrane was maintained for hvo weeks at the residual activity of more than 90%, and an new membrane was also easily formed. Table 1 shows the comparison of chemical concentrations for 50% inhibition (Z5J of the sensor. The concentrations of Iso were in the order Methomyl > DEP > Dimethoate > PHC > nicotine > Malathion, and their concentrations and order may show a toxic index. We will be able to apply their theoretical model to an enzyme sensor for a complex of toxic substances such as pesticides in the environment.Therefore, these
In this study, an acetylcholine sensor based on an ISFET as a pH sensor, using immobilizedacetylcholine esterase (AChE) in the BSA membrane is reported for its characteristics and the chemicals it measures. Data on the quantitative analysis of the insecticides are recorded in terms of percentage inhibition as a function of the inhibitor concentration for a fixed concentration of the substrate (10 mM) in pH=8.0 at room temperature. The detection of PHC and nicotine as reversibleinhibitors,and DEP, Methomyl,Malathion and Dimethoate as irreversible inhibitorswas found to be in the range of 10-6-10-’ M. The sensor was also easilyrenewed by 1 mM PAM solution and easilymade into a BSA membrane. A new technique for detecting toxic chemicalsin aquatic environmentsis expected for a faster, simpler, safer and inexpensivemethod which preserves the sensitivitiesof analysis. The present study was also developed for an electrochemical sensor for toxic substanceswhichworks by measuring the inhibition rates (e.g., Zm);the biosensor may be important as a biological index to biological systemswith regard to toxic matters. References 1 M. Morita and J. Terasawa, Physicochemical properties of pesticides, @I. I. Water Poll&. Res., 14(2) (1991) 75-78. 2 T. Matsuo and M. Eoashi, Methods of ISFET fabrication, Sensory and Actuators, 1 (1981) 77-96. 3 H. Sakai, IL Kaneki, H. Tanaka and H. Hara, Characteristics of acetylcboline sensor using an ISFlET and its application to chemical analysis, Sensors Mater., 3 (1992) 145-157. 4 H. Sakai, N. Kaneki and H. Hara, Availability and development of an enzyme immunomicrosensor based on an ISFET for buman immunoglobuiins, Anal. Chim. Acta, 230 (1990) 189-193. 5 H. Sakai, K Kaneki, H. Tanaka and H. Hara. Determination of heavy metal ions by urea sensor using ISFET, Senwrs Mater., 2 (1991) 217-227.
Sensors and Actuators B, 13-14 (1993) 578-580
Analytical application for chemicals using an enzyme sensor based on an IWET Hiromitsu Sakai, Noriaki Kaneki and Hiroshi Hara Lkpartment of Compufw Science and Systems Engineering Faculty of Engineekzg
Mmmn Instituteof Technology,
27-l Mizumotcxho, Mummn 050 {lopan)
IIthOdWtlOll
Enviro~ental pollution by toxic chemicals has become one of the world’s most serious problems. In particular, monitoring by a sensor is more and more necessary for health-risk assessment of drinking water polluted by pesticides from golf links or agricultural land [l]. At present, the chemical analysis is mainly carried out by gas-chromatographic methods. However, this has many problems such as the complex procedures required for measuring chemicals, and large and expensive instruments. In this paper, we studied and discussed the characteristics and the application of an acetylcholine sensor based on ion-sensitive field-effect transistors (ISFETs) using acetylcholine esterase (AChE) as an effective and simple potentiometric sensor for detecting agricultural chemicals, as reversible and irreversible inhibitors. ExperiIlK.!lltal Reagents
Purified AChE and pyridine-Zaldoxime methiodide (PAM) was obtained from Sigma Chemical Co.; acetylcholine perchloride was obtained from Seikagaku Kogyo Co.; o-isopropoxyphenyl methylcarbamate (PHC), nicotine, trichlorfon (dimethyl 2,2,2-trichloro1-hydroxyethylphosphonate; DEP), Methomyl (Smethyl N-(methylcarbamoyloxy) thioacethnidate), Malathion and Dimethoate as insecticides were obtained from GL Science Co., and bovine serum albumin (BSA) was obtained from Toyobo Co., Ltd. All other chemicals employed were of analytical grade.
the ISFETs has been documented previously [3]. The AChE was coupled in albumin membrane following the procedure of the crosslinking method. Measurement of the potential by the acetylcholine sensor The measurement methods of the enzyme sensor
based on an ISFET have been described in previous studies [3-51. AChE catalyzes the hydrolysis of acetylcholine according to the reaction: (CH,),N +CH&H,OCOCH, + H,O (CH,),N+CH,CH~OH+HOCOCH,
the pH in the membrane is changed by the reaction. That is, the generated ions diffuse to the ISFET interface through the membrane and react electrochemically at the ISFET with a resulting increase in voltage. The measurement of chemicals using the acetylcholine sensor was carried and classified into three types of direct and indirect methods with various concentrations of inhibitors, as indicated in Fig. 1. From the difference Chenlcnl
Rnvnrrible
Anrhis by
inhibitor
Enzyme Sensor
Irrnverslble
PHC Nicotine
We fabricated a probe-type ISFET as a transducer following the methods of Matsuo and Esashi [2]. The ISFET was 1.8 mm wide, 500 pm thick and 9.6 mm long. The gate channel was approximately 1.5 mm long and 30 pm wide, with a channel depletion mode device. The procedure used to produce BSA membranes on
Inhibitor
Incubation in srwla for Msrrursnent
Fabrication of ISFET and procedure of a jired membmne
(1)
1 rfsrctivitv by 2-PAN
Ih
CB Me thorny DBP
I
@
Nsasursmmt
Dlmthosts Malathion
flex
t I” i ty
by Z-PAW
Fig. 1. Measurement procedure of chemicals by the enzyme sensor.
0 1993 - Elsevier Sequoia. All rights reserved
579
between the two steady states, i.e., before and after inhibition, the percentage of AChE inhibition was calculated. Dimethoate and Malathion were converted into their oxygen analogues by treating with bromine water since these chemicals as such do not show maximum toxicity. The reactivation of the AChE sensor was done by adding 1 mM PAM and 10 mM acetylcholine. 0
IO
20
30
40
50
Time
ResuIts and discussion Responseof the acetylcholinesensor using ISFET The characteristics of the ISFET with and without BSA membrane have been descriied in previous studies [4, 51. The detection range of acetylcholiie using the AChE-ISFET sensor was 0.05-10 mM at room temperature. The response of the enzyme sensor depended on pH (optimal pH 8.0) and the concentration of the buffer solution. The ouptput voltage was about - 48 mV/pH. In 10 mM HEPES buffer, no appreciable variation in the pH was observed during the measurement; hence, this buffer concentration was selected for the analysis of the toxic chemicals.
I (%) = ((EO- E)/E) x 100
(2)
where E, is the initial output voltage given by the sensor in the absence of the inhibitor and E is the potential response in the presence of the inhl%itor at the same substrate concentration. Figure 2 shows a typical response of the acetylcholine sensor in 10 mM HEPES buffer solutions containing 1OmMacetylcholine: the response (a) was in the absence of the inhibitor, and the response (b) was after having added PHC as reversible inhibitor. Figure 3 shows a calibration curve of PHC by the .sensor. On the other hand, typical responses of the sensor upon the addition of 10 mM acetylcholine for DEP and Methomyl of the irreversible inhibitors were almost in agreement to the previous report [3]. Figure 4 shows a typical response of the sensxx upon the acetylcholine solution: the response (a) was in the absence of the inhibitor, and the response (b) was after 1 h incubation (10-l M Methomyl) with the sensor at a particular concentration of the Methomyl.
70
80
Fig. 2. A typical response of the acetykholine sensor using ISFET for revexsible inhibitor in 10 mM acetylcholiie solution (pH-8.0): (a) in absence of the inhibitor, and (b) after addition of lo-’ M PHC.
10-3
IO-' PHC
Determinationof chemicaL.s by acetykholinesensor New data on the analysis of agricultural chemicals are reported using the present acetylcholine sensor based on the inhibition of the immobilized AChE. Determination of the toxic chemicals is based on the measurement of inhibition percentage (Z (%)) which is equal to the potential difference of the relative activity given by the sensor with and without the inhibitor for the same substrate concentration, and which can be depicted as follows:
60
(min)
concentration
CM)
Fig. 3. Calibration CUIVC of PHC by the acetylcholine sensor in 10 mM HEPES buffer solution with 10 mM acetylcholine.
s-“““““_
I
0
I
I
I
I
I
I
I
I
10 20 30 40 50 60 70 80 Time
(min)
Fig. 4. A typical response of the acetylcholine sensor using ISFET for irrexersible inhibitor in 10 mM acetyhzholinesolution (pH-8.0): (a) in absence of the inhibitor, 10-l M Methomyk (b) after incubation with the sensor, and (c) reactivity by 1 mM PAM.
Reactivation (c) of the enzyme was facilitated by adding 1 n&f PAM. Data on the quantitative analysis of Methomyl were recorded in terms of percentage inhibition as a function of inhibitor concentration for the substrate in Fig. 5. The rate of inhibition increase was proportional to the logarithm of the concent rations for chemicals. The detection of PHC and nicotine as reversible inhibitors, and DEP, Methomyl, Malathion and Dimethoate as irreversible inhibitors was found to be in the range of 10-6-10-1 M. However, in practical application, the low sensitivity of the sensor to chemicals must be improved by a selected receptor, immobilized-
580
sensor-inhibitionrates may be important as an index of biologicalsystems with regard to toxic matters. Conclusions
Fig. 5. Calibration curve of Methomyl by the acetylcholine sensor in 10 mM HEPES b&r solution with 10 mM acetylcboline.
TABLE 1. Comparison of chemical concentrations required for 50% inhibition of an acetylcholine esteras&mmobilid ISFET sensor with high enzyme activity Chemicals
Concentration for 50% inhibition
Methomyl DEP Dimethoate PHC Nicotine Malathion
1.2x 1.0x 5.0x 1.2x 3.6x 3.0x
10-l lo-* lo-’ lo-’ lo-’ 10-5
membrane methods, the fabrication of a more sensitive transducer and so on. Therefore, the inhibition to the enzyme of the urea sensor by the agriculturalchemicals was not observed in this study. The response of ISFET with the membrane was maintained for hvo weeks at the residual activity of more than 90%, and an new membrane was also easily formed. Table 1 shows the comparison of chemical concentrations for 50% inhibition (Z5J of the sensor. The concentrations of Iso were in the order Methomyl > DEP > Dimethoate > PHC > nicotine > Malathion, and their concentrations and order may show a toxic index. We will be able to apply their theoretical model to an enzyme sensor for a complex of toxic substances such as pesticides in the environment.Therefore, these
In this study, an acetylcholine sensor based on an ISFET as a pH sensor, using immobilizedacetylcholine esterase (AChE) in the BSA membrane is reported for its characteristics and the chemicals it measures. Data on the quantitative analysis of the insecticides are recorded in terms of percentage inhibition as a function of the inhibitor concentration for a fixed concentration of the substrate (10 mM) in pH=8.0 at room temperature. The detection of PHC and nicotine as reversibleinhibitors,and DEP, Methomyl,Malathion and Dimethoate as irreversible inhibitorswas found to be in the range of 10-6-10-’ M. The sensor was also easilyrenewed by 1 mM PAM solution and easilymade into a BSA membrane. A new technique for detecting toxic chemicalsin aquatic environmentsis expected for a faster, simpler, safer and inexpensivemethod which preserves the sensitivitiesof analysis. The present study was also developed for an electrochemical sensor for toxic substanceswhichworks by measuring the inhibition rates (e.g., Zm);the biosensor may be important as a biological index to biological systemswith regard to toxic matters. References 1 M. Morita and J. Terasawa, Physicochemical properties of pesticides, @I. I. Water Poll&. Res., 14(2) (1991) 75-78. 2 T. Matsuo and M. Eoashi, Methods of ISFET fabrication, Sensory and Actuators, 1 (1981) 77-96. 3 H. Sakai, IL Kaneki, H. Tanaka and H. Hara, Characteristics of acetylcboline sensor using an ISFlET and its application to chemical analysis, Sensors Mater., 3 (1992) 145-157. 4 H. Sakai, N. Kaneki and H. Hara, Availability and development of an enzyme immunomicrosensor based on an ISFET for buman immunoglobuiins, Anal. Chim. Acta, 230 (1990) 189-193. 5 H. Sakai, K Kaneki, H. Tanaka and H. Hara. Determination of heavy metal ions by urea sensor using ISFET, Senwrs Mater., 2 (1991) 217-227.