JPH052940B2 - - Google Patents

Description

[Detailed description of the invention]

(Industrial Application Field) The present invention relates to a method for manufacturing a bioelectrochemical sensor using a field effect transistor type ion sensor (hereinafter referred to as ISFET). The bioelectrochemical sensor obtained by the manufacturing method of the present invention utilizes excellent substance identification functions such as enzymes, antibodies, and microorganisms to electrically measure the concentration of complex biological substances such as proteins, hormones, and viruses. It is converted into a quantity and output. To explain about immunosensors, which are a typical example of bioelectrochemical sensors, these sensors measure the concentration of antigens or antibodies in solutions based on immune reactions.Especially in the medical field, they measure various hormones and various proteinaceous substances in living bodies. There is a need for a miniaturized device that can measure accurately and quickly. Conventionally, known measuring methods of this type include radioimmunoassay, enzyme immunoassay, and fluorescent method. However, methods using these measurement principles have problems such as complicated operations, expensive reagents, and the need for expensive equipment, and have been subject to significant practical limitations. As a measuring method for solving the above-mentioned problems, an immunosensor using a field effect transistor type ion sensor was recently disclosed in Japanese Patent Laid-Open No. 161992/1983.
Such an immunosensor is a sensor in which the gate surface of an ISFET is coated with an insulating polymer and then chemical substances such as antigens and antibodies are chemically immobilized thereon. In principle, the material-sensitive insulating film of the ISFET used in the sensor must be coated thinly and uniformly to the extent that the electric field at the interface between the film and the measuring liquid sufficiently reaches the channel. This thickness is usually said to be several microns,
Plasma polymerization, sputtering, ultraviolet polymerization, parylene vapor deposition, and the like are conventionally known methods for producing a polymer insulating film with such a uniform thickness. However, the thin polymer film coated using the above method has a dense surface and many crosslinks, so it is difficult to immobilize antigens and antibodies using normal chemical reactions.
It is extremely difficult to introduce functional groups such as OH, COOH, and NH ₂ into the surface of a polymer membrane at high density, and the amount of sensitive substances that can be immobilized on a thin polymer membrane is also limited, making it difficult to obtain a highly sensitive sensor. was extremely difficult. The present inventors conducted extensive research on methods for immobilizing large amounts of biochemically sensitive substances such as antigens, antibodies, and enzymes on the surface of the polymer thin film coated on the gate surface of ISFET, and found that dissociative functional It was discovered that a large amount of biochemically sensitive substances could be immobilized on the surface of a thin polymer film by pre-treating the surface of the polymer film, which does not contain groups, with plasma of a gas containing ammonia molecules, and as a result of further investigation, the present invention was achieved. It has been reached. That is, the present invention covers the surface of a gate insulating film of a field effect transistor type ion sensor with an electrically insulating polymer thin film that does not contain a dissociative functional group, and then coats the polymer thin film with a gas plasma containing ammonia molecules. This is a method for producing a bioelectrochemical sensor, characterized in that a biochemically sensitive substance is immobilized on the surface of the plasma-treated polymer thin film. The reason why a large amount of biochemically sensitive substances can be immobilized by treating the polymer thin film with the plasma of the gas containing ammonia molecules is unknown;
It is presumed that by treating the surface of a thin polymer film that does not contain dissociative sensitive groups with plasma using a gas containing ammonia molecules, NH ₂ functional groups are introduced at a high density onto the surface of the thin polymer film. . The ISFET used in this invention is a normal MOSFET
The gate metal electrode is removed so that the gate part, which has a two-layer structure of silicon oxide and silicon nitride, is in direct contact with the electrolyte.
A structure in which the peripheral portion of the gate is completely insulated as shown in No. 54-66194 is preferably used. The polymer film covering the gate of this ISFET has good insulation properties and does not contain dissociative functional groups. These polymer films are coated on the gate by vacuum evaporation, sputtering, ultraviolet polymerization, plasma polymerization, glow discharge polymerization, electron beam polymerization, or the like. In particular, polyparaxylylene and its derivatives produced by vacuum deposition method, polytetrafluoroethylene film produced by sputtering method, polystyrene, polyparachlorostyrene, polyparachloromethylstyrene produced by ultraviolet polymerization method, polymethane, polystyrene, etc. produced by plasma polymerization method. is preferred. Among these, polyparachloromethylstyrene produced by ultraviolet polymerization is particularly preferred. The thickness of the film is preferably 10 μm or less so that an electric field effect can be applied across the film, and is usually 100 Å to 1 μm. If the film becomes too thin, defects such as pinholes will increase. In addition, the membrane prepared in this way has a pH of
The lower the sensitivity, the better, and the PH sensitivity is measured.
It is desirable that it be below 10mV/PH near PH. Only the surface of the polymer thin film produced in this way is treated with plasma generated by gas discharge of a gas containing ammonia molecules, which is easy to handle and control as a gas and has excellent functional group introduction efficiency. Introduce NH ₂ functional groups onto the surface. Alternatively, a mixed gas of the gas containing the ammonia molecules and an inert gas such as argon may be turned into plasma. In the plasma treatment, an ISFET whose gate part is coated with a thin polymer film is placed in a vacuum system, and the above-mentioned gas containing ammonia molecules (a mixed gas of the above gas and an inert gas such as argon gas may be used) is added to the ISFET at 10 ^-3 ~Ten
mmHg, high frequency or alternating current,
Alternatively, it may be carried out by discharging direct current and turning it into plasma. At this time, since the characteristics of the electrode portion of the ISFET may change due to discharge, it is preferable to cover it with an insulating resin or glass plate to prevent direct contact with the plasma. Additionally, the characteristics of the ISFET may change slightly after plasma treatment due to plasma charging. In order to restore this characteristic, it is effective to subject the sensor to heat treatment after plasma treatment. After plasma treatment in this manner, biochemically sensitive substances such as antibodies and antigens are immobilized on the surface of the polymer membrane by conventionally known methods. Many methods have been known for immobilizing such biochemically sensitive substances, but covalent bonding methods are suitable for the method of the present invention, such as methods using glutaraldehyde and methods using maleimide. The method is particularly suitable. Furthermore, instead of directly reacting these compounds with the functional groups on the surface of the polymer, the reaction can be carried out with an appropriate spacer in between. Biochemically sensitive substances immobilized on the surface of polymer membranes selectively coordinate or bind to specific biologically related substances and change the interfacial potential, such as antigens, antibodies, receptors, etc. . Antigens include autoimmune disease-related antigens such as cycloglobulin, cyclosome, thyroid-stimulating hormone receptor, and cell nucleus, cancer-specific antigens such as α-phetoprotein, and virus mimics such as Australian antigen. Antibodies include antibodies against proteins such as anti-albumin antibodies and anti-immunoglybulin antibodies, antibodies against hormones such as insulin, HCG, and steroid hormones, antibodies against cancer-specific antigens such as α-fetoprotein, and antibodies against pathogens such as Australia antigen. etc. can be mentioned. It is possible to create antibodies that specifically bind to biological substances to be measured. Other biochemically sensitive substances to be immobilized include various receptors such as thyroid hormone-binding protein and calcium-binding protein. ISFETs with biochemically sensitive substances immobilized on the gate insulating film have higher selectivity and sensitivity than conventional immunosensors. The present invention will be specifically explained below using Examples. Example 1 PH-sensitive ISFET (Tokuko Showa) whose entire circumferential surface in the gate part is coated with silicon oxide and silicon nitride
57-43863), and the ISFET is 1 mm in diameter.
Insert the tip of a nylon catheter so that the gate part protrudes, fill and fix resin at least between the electrode part and the inner wall of the catheter, excluding the gate part, and then attach the gate part to the DMF- Washed with methanol and then silanized with gamma-aminopropyltriethoxysilane. This ISFET was placed in a quartz cell, and after the system was evacuated, parachloromethylstyrene vapor (a commercially available first class reagent) was introduced into the cell. After that, the ISFET was irradiated overnight with a 100W high-pressure mercury lamp, and a photopolymerized film of polyparachloromethylstyrene with a thickness of about 4000 Å was obtained at the gate of the ISFET. The Vs value of this sensor in JIS buffer solutions PH9.18 and PH6.86 was 20 mV or less. Next, this sensor was sputtered with NH ₃ using a high-rate sputtering device (Japan Vacuum SBH-1104RE).
A plasma etching process was performed for 3 minutes. It is presumed that NH ₂ groups were introduced to the surface of the polyparachloromethylstyrene film by such plasma treatment. The NH ₃ pressure at this time is 0.005torr,
The output was 50W. The plasma-treated sensor was immersed in a 5% glutaraldehyde aqueous solution for 1 hour, washed with water, and immersed in anti-human albumin rabbit serum at 4° C. overnight to immobilize the anti-human albumin antibody on the sensor surface. Table 1 shows the results of measuring the source potential (Vs) by inserting this sensor into solutions with various albumin concentrations. Note that ΔVs is a source potential difference based on the source potential (0.481 V) for a solution with a human albumin concentration of 0 ppm. As is clear from Table 1, the output (-ΔVs) of the sensor increases as the human albumin concentration increases. Furthermore, the output for egg albumin is almost zero. This indicates that the sensor specifically responds to human albumin.

[Table] Example 2 and Comparative Examples 1 to 4 In the same manner as in Example 1, various thin films were prepared on the gate portion of the ISFET, and a CEA (carcinoembryonic antigen) antibody (rabbit) was immobilized thereon. Their sensitivity to CEA is shown in Table 2. In addition, the polymer thin film acrylamide and HEMA in Comparative Examples 2 and 3 in Table 2
To form a film on the gate surface of ISFET, instead of parachloromethylstyrene vapor in Example 1,
The vapors of acrylamide and hydroxyethyl methyl acrylate were introduced into a quartz cell, and photopolymerization was carried out under the same conditions as in Example 1. The photopolymerization of parachloromethylstyrene in Comparative Example 4 and Example 2 was carried out under the same conditions as in Example 1. Regarding plasma treatment, Comparative Examples 1, 2, 3, and Example 2 were performed under the same conditions as Example 1, and Comparative Example 4 was not subjected to plasma treatment. As is clear from Table 2, the ISFET gate is coated with an electrically insulating polymer thin film that does not contain dissociative functional groups, such as parachloromethylstyrene.
PH sensitivity is low (Comparative Example 4, Example 2). After plasma treatment of the above parachloromethylstyrene membrane in _NH3 , anti-CEA antibody was immobilized.
ISFET (Example 2) shows response to CEA, but without plasma treatment, anti-CEA response is obtained.
Even after immobilizing the antibody, sufficient CEA sensitivity was not obtained (Comparative Example 4). In addition, acrylamide and HEMA, which have dissociative functional groups, can be used as thin polymer films.
The PH sensitivity is high for those coated with
Even after immobilizing anti-CEA antibodies by NH ₃ plasma treatment, sufficient CEA sensitivity could not be obtained (Comparative Example 2,
3). Of course, there are also cases where the polymer thin film is not coated.
PH sensitivity is high and CEA sensitivity is low (Comparative Example 1). Although the thickness of the parachloromethylstyrene polymer thin film differs between Comparative Example 4 and Example 2, it is thought that this degree of difference in thickness does not affect the sensitivity of the sensor.

【table】

【table】

Claims (1)

[Claims] 1. After coating the surface of the gate insulating film of a field effect transistor type ion sensor with a thin polymer film containing no dissociative functional groups, the thin polymer film is treated with a plasma of a gas containing ammonia molecules, and then the plasma treatment is performed. A method for producing a bioelectrochemical sensor, characterized in that a biochemically sensitive substance is immobilized on the surface of a thin polymer film.