ANALYTICAL
BIOCHEMISTRY
94, 22-28 (1979)
Enzyme lmmunosensor III. Amperometric
MASUO
AIZAWA,”
Determination of Human Chorionic by Membrane-Bound Antibody
AYA MORIOKA,*
SHUICHI
SUZUKI,*
Gonadotropin
AND YOICHI
NAGAMURA?
*Research Laboratory of Resources Utilization, Tokyo Institute of Technology, Nagatsuta-cho, Midori-ku, Yokahama 227, and tDepartment of Biochemistry, School of Medicine, Fujita Gakuen University, Kutsukake, Toyoake, Aichi 470-11. Japan
Received June 27, 1978 An enzyme immunosensor was constructed for the determination of human chorionic gonadotropin (HCG), which is a hormone and an important diagnostic measure of pregnancy. An antibody to HCG was immobilized to a membrane. The antibody-bound membrane was placed onto an oxygen probe so as to react with HCG either specifically or selectively. Catalase, which catalyzes the decomposition of HZ02 into HZ0 and O,, was used to label HCG. Nonlabeled HCG to be assayed and catalase-labeled HCG were competitively reacted with the membrane-bound antibody of the sensor to form an antigen-antibody complex on the membrane surface. After the removal of nonspecifically adsorbed HCG, the sensor was contacted with a H,O, solution. The membrane-adsorbed catalase enzymatically generated oxygen with a resulting increase in cathodic current of the sensor. The HCG concentration was determined from the initial rate of the current increase. The enzyme immunosensor was applied to the determination of HCG in the concentration range of 2 x 10” to 102 IU/ml.
In the past decade enzyme sensors (electrodes) have been developed and employed to measure the concentration of substances of clinical importance (1-7). An enzyme electrode is the unique combination of an enzyme, which reacts sensitively and specifically with many organic and inorganic compounds in nature, with an electrochemical device. There have been, however, difficulties in the specific determination of proteins and peptides with enzyme sensors. Application of immunochemical methods to the measurement of substances of clinical importance has combined high sensitivity with exquisite specificity. Enzyme immunoassays have now become generally accepted as alternatives to and even substitutes for other immunoassays (8). An enzyme is used to label antigens and antibodies so that the conjugates are used in quantitative assays 0003-2697/79/050022-07$02.00/O Copyright All rights
6 1979 by Academic Press, Inc. of reproduction in any form reserved.
of soluble substances using the same methodology as radioimmunoassays (RIA).’ They offer great promise in the diagnostic assays for hormones, drugs, and antibodies. We applied the principle of enzyme immunoassay to the construction of a specific sensor for immunoglobulin G (9,lO). Such a sensor is named an enzyme immunosensor; its specificity depends upon immunochemical reaction, and its simple and rapid operation upon an electrochemical device. In the present investigation a new enzyme immunosensor was developed for the determination of human chorionic gonadotropin (HCG), which is a hormone and an important diagnostic measure of pregnancy. ‘Abbreviations used: RIA, radioimmunoassay; HCG, human chorionic gonadotropin. 22
ENZYME
IMMUNOSENSOR
The principle of the enzyme immunosensor is simple: A membrane-bound antibody is used to bind HCG either specifically or selectively on the membrane surface. The antibody-bound membrane is placed onto a conventional oxygen probe. Catalase is used to label HCG. Nonlabeled HCG to be assayed and catalase-labeled HCG competitively react with the membrane-bound antibody. The amount of nonlabeled HCG may be determined by the activity assay of the catalase complexed on the membrane after the removal of nonspecifically adsorbed species. EXPERIMENTAL Materials. Human chorionic gonadotropin (HCG) and an antiserum to HCG, the specific activity of which was 7000 IU/mg, were obtained from Teikoku Zoki Company, Ltd. (Tokyo). Antibody-bound membranes were prepared according to the following procedures. Acetylcellulose (150 mg) and bromoacetylcellulose (100 mg), which was prepared as reported in the previous paper (9), were dissolved in 5 ml of acetone with vigorous stirring at around 20°C. The
b
O-$-CH2Br 0
ACM-Br + NH2 (CH2)
,$JH2
u NH(CH2)6NH2 itACM-NH2
+ HZC-CH-C\H;CHZ \I 0 0 NH-CH2-$HC\H;CH2 OH 0
4 ACM-Epoxy
+ H*N+ff@ a f%f-+&CH-NHf ACM-Ab + ethanol
amine
0 Membrane-bound
antibody
FIG. 1. A postulated scheme for the immobilization of anti-HCG antibody onto the membrane surface.
23
resulting solution was cast on a glass plate. The membrane was peeled off and cut into small pieces (2 x 2 cm2 each). Each membrane was immersed in 20 ml of 0.05 M carbonate buffer at pH 9.7 containing 150 mg hexamethylenediamine with moderate stirring for about 15 h. After washing with carbonate buffer (pH 9.7), the membranes were added to 20 ml of 0.05 M carbonate buffer (pH 9.7) containing 100 ~1 butadienediepoxide. The reaction mixture was allowed to stand at around 20°C for 1 h. The membrane was washed with carbonate buffer (pH 9.0) and then contacted with 20 ml of 0.05 M phosphate buffer at pH 8.6 containing 10 ~1 antiserum to HCG with moderate stirring at around 4°C for about 15 h. The antibody-bound membrane was thoroughly washed with water, then contacted with 0.05 M ethanolamine in pH 8.6 phosphate buffer, and stirred at around 4°C for about 15 h so as to block the unreacted epoxy groups. A postulated scheme for the immobilization is presented in Fig. 1. Catalase-labeled HCG was prepared by conjugating catalase with HCG using glutaraldehyde. Catalase (10 mg) and HCG (8000 IU) were dissolved in 3 ml of 0.05 M carbonate buffer at pH 9.7. One-percent glutaraldehyde (100 ~1) was added to the solution. The solution was incubated at about 20°C for 20 min. The catalase-HCG conjugate was isolated from unreacted glutaraldehyde, catalase, and HCG by ultrafiltration. Further purification was conducted by gel filtration on Sephadex G-200. Assay procedures for the determination of HCG with the enzyme immunosensor.
The configuration of the enzyme immunosensor is shown in Fig. 2. The sensor is composed of the antibody-bound membrane and an oxygen probe, which is a set composed of a lead anode, a platinum cathode, an alkaline electrolyte, and an oxygen-permeable Teflon membrane. Dissolved oxygen diffuses into the Teflon membrane so as to be electrochemically reduced at the cathode surface. The galvanic
24
AIZAWA
current of the probe depends on the level of dissolved oxygen. The antibody-bound membrane is firmly fixed onto the Teflon membrane. The HCG is determined according to the following three steps, as schematically illustrated in Fig. 3. Step 1: Nonlabeled HCG to be assayed is added to a solution containing a known amount of the catalase-labeled HCG. Nonlabeled and labeled HCG competitively react with the membrane-bound antibody, resulting in formation of an antigenantibody complex on the membrane surface of the sensor. The reaction is continued at 37°C for 1 h. Step 2: The sensor is washed with saline to remove nonspecifically adsorbed HCG from the membrane. The antigen-antibody complex may remain stable on the membrane surface. Step 3: The sensor is contacted with a hydrogen peroxide solution for enzyme activity assay. The catalase complexed with the membrane-bound antibody decomposes hydrogen peroxide into oxygen and water. The enzymatically generated oxygen diffuses to the platinum cathode through the Teflon membrane and is electrochemically reduced there, with a resulting increase in cathodic current. The measurement is carried out with stirring. From the galvanic current of the sensor, the catalase activity is determined. According to a calibration curve for the catalase activity against the concentration of nonlabeled HCG at a constant amount of the catalase-labeled HCG, a sample may be assayed for its HCG concentration from the electrochemical data on the catalase activity assay. RESULTS
The catalase conjugated to HCG was assayed for its activity. The initial rates of the HCG-bound catalase were determined at a different concentration of HzOz and at
ET AL. Cathode
lead
Glass-tube I
Oxygen permeable membrane Antibody
Pb a&de
membrane
Pt cathode KOH electrolyte
Enzyme
Immunosensor
FIG. 2. The configuration sensor for HCG.
of the enzyme immuno-
30°C. The Lineweaver-Burk plots of the initial rate yielded a straight line for the HCG-bound catalase, as shown in Fig. 4. The kinetic parameters were calculated as 80 mM for Michaelis constant (K,) and 9.4 mmol/min for maximum velocity (V,,,). It indicated that the HCG-bound catalase retained enzymatic activity. A stock solution of the catalase-labeled HCG contained 4 units of HCG/ml, in which 1 unit would correspond to 1.4 x lo-’ g of HCG. In order to determine the response of the enzyme immunosensor the sensor was contacted with excess catalase-labeled HCG in the absence of nonlabeled HCG. One milliliter of the catalase-labeled HCG was added to 1 ml of 0.05 M phosphate buffer at pH 7.0. The sensor was immersed in the resulting solution, which was allowed to stand at 37°C for 30 min. We anticipated that the binding sites located on the membrane surface were occupied by the catalaselabeled HCG under these conditions. The sensor was rinsed with saline and was then inserted in 0.1 M phosphate buffer at pH 7.0 and 30°C. As the solution was saturated in advance with dissolved oxygen, the output current of the sensor remained constant when the solution was stirred at a constant speed. Hydrogen peroxide was added to the
ENZYME Competitive
IMMUNOSENSOR
immunochemical
25
reaction
Competitive reaction of the catalase-labeled and nonlabeled HCG with the membrane-bound antibody
Washing Separation of membranebound antigen-antibody complex from free HCG
Determination
of
Addition Cathodic
enzyme
of H202 current
B:Antibody
activity
of O2
a: HCG
FIG. 3. Three steps for the determination
solution so as to bring the final concentration to 5 mM. The output current of the sensor immediately increased, as presented in Fig. 5, by approximately 1 PA within 10 min. As the initial increasing rate of the current was found to sensitively depend on the amount of the membrane-attached 2.5
> \ r(
e:Catalase-labeled
HCG
of HCG with the enzyme immunosensor.
catalase, it was used as a measure of the enzyme assay. The membrane-bound antibody may be reversibly used for binding HCG in nature. I
1
r
1.0
I
J 0
0.5
5 Time
A
I 0
50
100 1 /
[H2021
# 150
200 ( l/M
250 )
FIG. 4. The Lineweaver-Burk plots of the initial rate for the HCG-bound catalase at 30°C.
10 ( min
15 )
FIG. 5. The time course of the enzyme immunosensor output. The sensor was contacted with 2 ml of the catalase-labeled HCG solution at pH 7.0 and 37°C for 30 min. After the removal of nonspecifically adsorbed species, the sensor was immersed in 10 ml of oxygen-saturated buffer at pH 7.0 and 30°C. At time 0, 50 ~1 of 3% H,O, solution was added.
AIZAWA
26
However, complete dissociation of the membrane-bound antibody-HCG complex is rather tedious and time consuming. In the present investigation, the antibody-bound membrane was changed for each measurement. Replacement of the membrane resulted in no serious effects on reproducibility. The effects of nonlabeled HCG on the response of the enzyme immunosensor were determined at a constant concentration of labeled HCG. Samples contained 0.4 III/ml labeled HCG and different concentrations of nonlabeled HCG. The enzyme immunosensor was reacted with a sample solution at 37°C for 30 min. After sufficient washing of the sensor, the catalase activity assay was carried out at pH 7.0 and 30°C. The enzyme immunosensor responded to H202 in a manner similar to that mentioned above. The correlation between the initial rate of current increase and the concentration of nonlabeled HCG is presented in Fig. 6. The initial rate was decreased by the addition of nonlabeled HCG. The antibody-bound membrane was replaced for each measurement. It was found that HCG was determined in the concentration range 0.02-1.0 IU/ml with an error of 25%. In a similar way, the standard curves were obtained up to lo2 IU of HCG, as presented in Fig. 7. The concentration of the catalase-labeled
(1) Immunochemical
.;r 6
10-2
KG
1.0
( 1.U.)
FIG. 6. The initial rate of current increase as a function of HCG concentration. One milliliter of the catalase-labeled HCG solution (0.4 IU/ml) was mixed with 1 ml of nonlabeled HCG solution at various concentrations. The sensor was reacted with the resulting solution at 37°C for 30 min. After thorough washing, the sensor was immersed in 10 ml of pH 7.0 phosphate buffer saturated with 0, at 30°C. The enzyme reaction was initiated by the addition of 50 pl of 3% H,O,.
HCG was maintained at 4 III/ml for the determination in the range of 0.2-10 IU HCG, and at 40 III/ml in the range of 2-100 IU HCG. Each standard curve gave a linear correlation through 10 units of HCG. DISCUSSION
The reactions involved in the present assay are characterized by the following.
antibody
-
HCG + membrane-bound
membrane-bound antibody
HCG-antibody
complex
-
membrane-bound (2) Enzymatic
10-l HCG
reaction
HCG + membrane-bound Catalase-labeled
ET AL.
catalase-antibody
reaction membrane-bound
(3) Electrochemical
reaction
catalase-HCG-antibody
complex
(cathode reaction) 0, + 2H,O + 4e -
40H-
> H,O,
+ ‘/O,
complex
ENZYME
IMMUNOSENSOR
27
of antigen-antibody complexes. Since the antibody reacts with the corresponding antigen (HCG) in a membrane-bound form, the antigen-antibody complex can be isolated from free antigen by simply washing the membrane. These advantages of solid-phase immunochemical reaction were effectively combined in the present sensor with the excellent operational characteristics of an amperometric device. When the membrane-bound .: ocatalase catalyzes the decomposition of 2 102 10 10-l 1.0 HzOz, part of the O2 generated may diffuse HCG (1.U.) away into the bulk solution. However, most FIG. 7. The initial rate of current increase as a oxygen molecules may penetrate into both function of HCG concentration at a fixed concentration antibody-bound and Teflon membranes and of catalase-labeled HCG. (a) W/ml and (b) 40 IU/ml. reach the cathode surface, as there must be a sharp gradient of oxygen molecules It should be evident that the enzyme across the membrane. immunosensor depends upon the immunoThis paper presents a possible determinachemical reaction for its specificity, because tion of HCG using a novel sensor. The an antibody binds specifically its corre- enzyme immunosensor was applied only to sponding antigen. The enzyme (catalase), HCG solutions. Further investigation should which was used to label the determinant be extended to serial assays on the sera of (HCG), works so as to transduce the pregnant women. Correlation with existing determinant into electrochemically meas- methods such as radioimmunoassay should urable substances (02) with prominent also be elucidated. Additionally, the problem amplification. Since the enzyme catalyzes of cross-reactivity to luteinizing hormone consecutively the generation of electroremains unsolved. chemically measurable substances at an appropriate turnover number, information CONCLUSION may be amplified at the membrane surface of the immunosensor. It may be termed The enzyme immunosensor was feasible chemical amplification, as information is in the determination of human chorionic enhanced by a chemical reaction. gonadotropin. The specificity and selectivity The conventional enzyme immunoassay of immunochemical reaction were coupled requires overnight incubation for the deterwith the simple and excellent operational mination of enzyme activity. In contrast, characteristics of an amperometric device. enzyme activity can be determined in several minutes with the enzyme immunosensor. REFERENCES The enzyme reaction can be continuously followed so that both initial rate and endI. Updike, S. J., and Hicks, G. P. (1967) Nature (London) 214, 986. point assays are applicable. In this paper, 2. Clark, L. C., Jr. (1972)Biorechnol. Bioeng. Symp. the results according to the initial rate 3, 377. assay have been described. 3. Cough, D. A., and Andrade, K. E. (1973) Science Another advantage of this sensor would 180, 380. be a simple procedure for the separation 4. Suzuki, S., Aizawa, M., and Karube, I. (1974)
28
AIZAWA
Immobilized Enzyme Technology (Weetall, H. H., and Suzuki, S., eds.), p. 253, Plenum, New York. 5. Rechnitz, G. A. (1975) Science 190, 234. 6. Guilbault, G. G. (1976) Handbook of Enzymatic Analysis, Academic Press, New York. 7. Suzuki, S., Karube, I., and Satoh, I. (1977) Biomedical Application of Immobilized Enzymes
ET AL. and Proteins (Chang, T. M. S., ed.), p. 177, Plenum, New York. 8. Wisdom, G. B. (1976) C/in. Chem. 22, 243. 9. Aizawa, M., Morioka, A., Matsuoka, H., Suzuki, S., Nagamura, Y., Shinohara, R., and Ishiguro, I. (1976) J. Solid-Phase Biochem. 1, 319. 10. Aizawa, M., Morioka, A., and Suzuki, S. (1978) J. Membrane Sci.
BIOCHEMISTRY
94, 22-28 (1979)
Enzyme lmmunosensor III. Amperometric
MASUO
AIZAWA,”
Determination of Human Chorionic by Membrane-Bound Antibody
AYA MORIOKA,*
SHUICHI
SUZUKI,*
Gonadotropin
AND YOICHI
NAGAMURA?
*Research Laboratory of Resources Utilization, Tokyo Institute of Technology, Nagatsuta-cho, Midori-ku, Yokahama 227, and tDepartment of Biochemistry, School of Medicine, Fujita Gakuen University, Kutsukake, Toyoake, Aichi 470-11. Japan
Received June 27, 1978 An enzyme immunosensor was constructed for the determination of human chorionic gonadotropin (HCG), which is a hormone and an important diagnostic measure of pregnancy. An antibody to HCG was immobilized to a membrane. The antibody-bound membrane was placed onto an oxygen probe so as to react with HCG either specifically or selectively. Catalase, which catalyzes the decomposition of HZ02 into HZ0 and O,, was used to label HCG. Nonlabeled HCG to be assayed and catalase-labeled HCG were competitively reacted with the membrane-bound antibody of the sensor to form an antigen-antibody complex on the membrane surface. After the removal of nonspecifically adsorbed HCG, the sensor was contacted with a H,O, solution. The membrane-adsorbed catalase enzymatically generated oxygen with a resulting increase in cathodic current of the sensor. The HCG concentration was determined from the initial rate of the current increase. The enzyme immunosensor was applied to the determination of HCG in the concentration range of 2 x 10” to 102 IU/ml.
In the past decade enzyme sensors (electrodes) have been developed and employed to measure the concentration of substances of clinical importance (1-7). An enzyme electrode is the unique combination of an enzyme, which reacts sensitively and specifically with many organic and inorganic compounds in nature, with an electrochemical device. There have been, however, difficulties in the specific determination of proteins and peptides with enzyme sensors. Application of immunochemical methods to the measurement of substances of clinical importance has combined high sensitivity with exquisite specificity. Enzyme immunoassays have now become generally accepted as alternatives to and even substitutes for other immunoassays (8). An enzyme is used to label antigens and antibodies so that the conjugates are used in quantitative assays 0003-2697/79/050022-07$02.00/O Copyright All rights
6 1979 by Academic Press, Inc. of reproduction in any form reserved.
of soluble substances using the same methodology as radioimmunoassays (RIA).’ They offer great promise in the diagnostic assays for hormones, drugs, and antibodies. We applied the principle of enzyme immunoassay to the construction of a specific sensor for immunoglobulin G (9,lO). Such a sensor is named an enzyme immunosensor; its specificity depends upon immunochemical reaction, and its simple and rapid operation upon an electrochemical device. In the present investigation a new enzyme immunosensor was developed for the determination of human chorionic gonadotropin (HCG), which is a hormone and an important diagnostic measure of pregnancy. ‘Abbreviations used: RIA, radioimmunoassay; HCG, human chorionic gonadotropin. 22
ENZYME
IMMUNOSENSOR
The principle of the enzyme immunosensor is simple: A membrane-bound antibody is used to bind HCG either specifically or selectively on the membrane surface. The antibody-bound membrane is placed onto a conventional oxygen probe. Catalase is used to label HCG. Nonlabeled HCG to be assayed and catalase-labeled HCG competitively react with the membrane-bound antibody. The amount of nonlabeled HCG may be determined by the activity assay of the catalase complexed on the membrane after the removal of nonspecifically adsorbed species. EXPERIMENTAL Materials. Human chorionic gonadotropin (HCG) and an antiserum to HCG, the specific activity of which was 7000 IU/mg, were obtained from Teikoku Zoki Company, Ltd. (Tokyo). Antibody-bound membranes were prepared according to the following procedures. Acetylcellulose (150 mg) and bromoacetylcellulose (100 mg), which was prepared as reported in the previous paper (9), were dissolved in 5 ml of acetone with vigorous stirring at around 20°C. The
b
O-$-CH2Br 0
ACM-Br + NH2 (CH2)
,$JH2
u NH(CH2)6NH2 itACM-NH2
+ HZC-CH-C\H;CHZ \I 0 0 NH-CH2-$HC\H;CH2 OH 0
4 ACM-Epoxy
+ H*N+ff@ a f%f-+&CH-NHf ACM-Ab + ethanol
amine
0 Membrane-bound
antibody
FIG. 1. A postulated scheme for the immobilization of anti-HCG antibody onto the membrane surface.
23
resulting solution was cast on a glass plate. The membrane was peeled off and cut into small pieces (2 x 2 cm2 each). Each membrane was immersed in 20 ml of 0.05 M carbonate buffer at pH 9.7 containing 150 mg hexamethylenediamine with moderate stirring for about 15 h. After washing with carbonate buffer (pH 9.7), the membranes were added to 20 ml of 0.05 M carbonate buffer (pH 9.7) containing 100 ~1 butadienediepoxide. The reaction mixture was allowed to stand at around 20°C for 1 h. The membrane was washed with carbonate buffer (pH 9.0) and then contacted with 20 ml of 0.05 M phosphate buffer at pH 8.6 containing 10 ~1 antiserum to HCG with moderate stirring at around 4°C for about 15 h. The antibody-bound membrane was thoroughly washed with water, then contacted with 0.05 M ethanolamine in pH 8.6 phosphate buffer, and stirred at around 4°C for about 15 h so as to block the unreacted epoxy groups. A postulated scheme for the immobilization is presented in Fig. 1. Catalase-labeled HCG was prepared by conjugating catalase with HCG using glutaraldehyde. Catalase (10 mg) and HCG (8000 IU) were dissolved in 3 ml of 0.05 M carbonate buffer at pH 9.7. One-percent glutaraldehyde (100 ~1) was added to the solution. The solution was incubated at about 20°C for 20 min. The catalase-HCG conjugate was isolated from unreacted glutaraldehyde, catalase, and HCG by ultrafiltration. Further purification was conducted by gel filtration on Sephadex G-200. Assay procedures for the determination of HCG with the enzyme immunosensor.
The configuration of the enzyme immunosensor is shown in Fig. 2. The sensor is composed of the antibody-bound membrane and an oxygen probe, which is a set composed of a lead anode, a platinum cathode, an alkaline electrolyte, and an oxygen-permeable Teflon membrane. Dissolved oxygen diffuses into the Teflon membrane so as to be electrochemically reduced at the cathode surface. The galvanic
24
AIZAWA
current of the probe depends on the level of dissolved oxygen. The antibody-bound membrane is firmly fixed onto the Teflon membrane. The HCG is determined according to the following three steps, as schematically illustrated in Fig. 3. Step 1: Nonlabeled HCG to be assayed is added to a solution containing a known amount of the catalase-labeled HCG. Nonlabeled and labeled HCG competitively react with the membrane-bound antibody, resulting in formation of an antigenantibody complex on the membrane surface of the sensor. The reaction is continued at 37°C for 1 h. Step 2: The sensor is washed with saline to remove nonspecifically adsorbed HCG from the membrane. The antigen-antibody complex may remain stable on the membrane surface. Step 3: The sensor is contacted with a hydrogen peroxide solution for enzyme activity assay. The catalase complexed with the membrane-bound antibody decomposes hydrogen peroxide into oxygen and water. The enzymatically generated oxygen diffuses to the platinum cathode through the Teflon membrane and is electrochemically reduced there, with a resulting increase in cathodic current. The measurement is carried out with stirring. From the galvanic current of the sensor, the catalase activity is determined. According to a calibration curve for the catalase activity against the concentration of nonlabeled HCG at a constant amount of the catalase-labeled HCG, a sample may be assayed for its HCG concentration from the electrochemical data on the catalase activity assay. RESULTS
The catalase conjugated to HCG was assayed for its activity. The initial rates of the HCG-bound catalase were determined at a different concentration of HzOz and at
ET AL. Cathode
lead
Glass-tube I
Oxygen permeable membrane Antibody
Pb a&de
membrane
Pt cathode KOH electrolyte
Enzyme
Immunosensor
FIG. 2. The configuration sensor for HCG.
of the enzyme immuno-
30°C. The Lineweaver-Burk plots of the initial rate yielded a straight line for the HCG-bound catalase, as shown in Fig. 4. The kinetic parameters were calculated as 80 mM for Michaelis constant (K,) and 9.4 mmol/min for maximum velocity (V,,,). It indicated that the HCG-bound catalase retained enzymatic activity. A stock solution of the catalase-labeled HCG contained 4 units of HCG/ml, in which 1 unit would correspond to 1.4 x lo-’ g of HCG. In order to determine the response of the enzyme immunosensor the sensor was contacted with excess catalase-labeled HCG in the absence of nonlabeled HCG. One milliliter of the catalase-labeled HCG was added to 1 ml of 0.05 M phosphate buffer at pH 7.0. The sensor was immersed in the resulting solution, which was allowed to stand at 37°C for 30 min. We anticipated that the binding sites located on the membrane surface were occupied by the catalaselabeled HCG under these conditions. The sensor was rinsed with saline and was then inserted in 0.1 M phosphate buffer at pH 7.0 and 30°C. As the solution was saturated in advance with dissolved oxygen, the output current of the sensor remained constant when the solution was stirred at a constant speed. Hydrogen peroxide was added to the
ENZYME Competitive
IMMUNOSENSOR
immunochemical
25
reaction
Competitive reaction of the catalase-labeled and nonlabeled HCG with the membrane-bound antibody
Washing Separation of membranebound antigen-antibody complex from free HCG
Determination
of
Addition Cathodic
enzyme
of H202 current
B:Antibody
activity
of O2
a: HCG
FIG. 3. Three steps for the determination
solution so as to bring the final concentration to 5 mM. The output current of the sensor immediately increased, as presented in Fig. 5, by approximately 1 PA within 10 min. As the initial increasing rate of the current was found to sensitively depend on the amount of the membrane-attached 2.5
> \ r(
e:Catalase-labeled
HCG
of HCG with the enzyme immunosensor.
catalase, it was used as a measure of the enzyme assay. The membrane-bound antibody may be reversibly used for binding HCG in nature. I
1
r
1.0
I
J 0
0.5
5 Time
A
I 0
50
100 1 /
[H2021
# 150
200 ( l/M
250 )
FIG. 4. The Lineweaver-Burk plots of the initial rate for the HCG-bound catalase at 30°C.
10 ( min
15 )
FIG. 5. The time course of the enzyme immunosensor output. The sensor was contacted with 2 ml of the catalase-labeled HCG solution at pH 7.0 and 37°C for 30 min. After the removal of nonspecifically adsorbed species, the sensor was immersed in 10 ml of oxygen-saturated buffer at pH 7.0 and 30°C. At time 0, 50 ~1 of 3% H,O, solution was added.
AIZAWA
26
However, complete dissociation of the membrane-bound antibody-HCG complex is rather tedious and time consuming. In the present investigation, the antibody-bound membrane was changed for each measurement. Replacement of the membrane resulted in no serious effects on reproducibility. The effects of nonlabeled HCG on the response of the enzyme immunosensor were determined at a constant concentration of labeled HCG. Samples contained 0.4 III/ml labeled HCG and different concentrations of nonlabeled HCG. The enzyme immunosensor was reacted with a sample solution at 37°C for 30 min. After sufficient washing of the sensor, the catalase activity assay was carried out at pH 7.0 and 30°C. The enzyme immunosensor responded to H202 in a manner similar to that mentioned above. The correlation between the initial rate of current increase and the concentration of nonlabeled HCG is presented in Fig. 6. The initial rate was decreased by the addition of nonlabeled HCG. The antibody-bound membrane was replaced for each measurement. It was found that HCG was determined in the concentration range 0.02-1.0 IU/ml with an error of 25%. In a similar way, the standard curves were obtained up to lo2 IU of HCG, as presented in Fig. 7. The concentration of the catalase-labeled
(1) Immunochemical
.;r 6
10-2
KG
1.0
( 1.U.)
FIG. 6. The initial rate of current increase as a function of HCG concentration. One milliliter of the catalase-labeled HCG solution (0.4 IU/ml) was mixed with 1 ml of nonlabeled HCG solution at various concentrations. The sensor was reacted with the resulting solution at 37°C for 30 min. After thorough washing, the sensor was immersed in 10 ml of pH 7.0 phosphate buffer saturated with 0, at 30°C. The enzyme reaction was initiated by the addition of 50 pl of 3% H,O,.
HCG was maintained at 4 III/ml for the determination in the range of 0.2-10 IU HCG, and at 40 III/ml in the range of 2-100 IU HCG. Each standard curve gave a linear correlation through 10 units of HCG. DISCUSSION
The reactions involved in the present assay are characterized by the following.
antibody
-
HCG + membrane-bound
membrane-bound antibody
HCG-antibody
complex
-
membrane-bound (2) Enzymatic
10-l HCG
reaction
HCG + membrane-bound Catalase-labeled
ET AL.
catalase-antibody
reaction membrane-bound
(3) Electrochemical
reaction
catalase-HCG-antibody
complex
(cathode reaction) 0, + 2H,O + 4e -
40H-
> H,O,
+ ‘/O,
complex
ENZYME
IMMUNOSENSOR
27
of antigen-antibody complexes. Since the antibody reacts with the corresponding antigen (HCG) in a membrane-bound form, the antigen-antibody complex can be isolated from free antigen by simply washing the membrane. These advantages of solid-phase immunochemical reaction were effectively combined in the present sensor with the excellent operational characteristics of an amperometric device. When the membrane-bound .: ocatalase catalyzes the decomposition of 2 102 10 10-l 1.0 HzOz, part of the O2 generated may diffuse HCG (1.U.) away into the bulk solution. However, most FIG. 7. The initial rate of current increase as a oxygen molecules may penetrate into both function of HCG concentration at a fixed concentration antibody-bound and Teflon membranes and of catalase-labeled HCG. (a) W/ml and (b) 40 IU/ml. reach the cathode surface, as there must be a sharp gradient of oxygen molecules It should be evident that the enzyme across the membrane. immunosensor depends upon the immunoThis paper presents a possible determinachemical reaction for its specificity, because tion of HCG using a novel sensor. The an antibody binds specifically its corre- enzyme immunosensor was applied only to sponding antigen. The enzyme (catalase), HCG solutions. Further investigation should which was used to label the determinant be extended to serial assays on the sera of (HCG), works so as to transduce the pregnant women. Correlation with existing determinant into electrochemically meas- methods such as radioimmunoassay should urable substances (02) with prominent also be elucidated. Additionally, the problem amplification. Since the enzyme catalyzes of cross-reactivity to luteinizing hormone consecutively the generation of electroremains unsolved. chemically measurable substances at an appropriate turnover number, information CONCLUSION may be amplified at the membrane surface of the immunosensor. It may be termed The enzyme immunosensor was feasible chemical amplification, as information is in the determination of human chorionic enhanced by a chemical reaction. gonadotropin. The specificity and selectivity The conventional enzyme immunoassay of immunochemical reaction were coupled requires overnight incubation for the deterwith the simple and excellent operational mination of enzyme activity. In contrast, characteristics of an amperometric device. enzyme activity can be determined in several minutes with the enzyme immunosensor. REFERENCES The enzyme reaction can be continuously followed so that both initial rate and endI. Updike, S. J., and Hicks, G. P. (1967) Nature (London) 214, 986. point assays are applicable. In this paper, 2. Clark, L. C., Jr. (1972)Biorechnol. Bioeng. Symp. the results according to the initial rate 3, 377. assay have been described. 3. Cough, D. A., and Andrade, K. E. (1973) Science Another advantage of this sensor would 180, 380. be a simple procedure for the separation 4. Suzuki, S., Aizawa, M., and Karube, I. (1974)
28
AIZAWA
Immobilized Enzyme Technology (Weetall, H. H., and Suzuki, S., eds.), p. 253, Plenum, New York. 5. Rechnitz, G. A. (1975) Science 190, 234. 6. Guilbault, G. G. (1976) Handbook of Enzymatic Analysis, Academic Press, New York. 7. Suzuki, S., Karube, I., and Satoh, I. (1977) Biomedical Application of Immobilized Enzymes
ET AL. and Proteins (Chang, T. M. S., ed.), p. 177, Plenum, New York. 8. Wisdom, G. B. (1976) C/in. Chem. 22, 243. 9. Aizawa, M., Morioka, A., Matsuoka, H., Suzuki, S., Nagamura, Y., Shinohara, R., and Ishiguro, I. (1976) J. Solid-Phase Biochem. 1, 319. 10. Aizawa, M., Morioka, A., and Suzuki, S. (1978) J. Membrane Sci.
