JPS63208753A - Immune sensor and immune detection - Google Patents

Abstract

PURPOSE:To permit selective detection of the dilute antigen material and antibody in an aq. soln. with high sensitivity by using an iridium oxide electrode covered with the antibody or antigen material as a working electrode and detecting the potential difference between said electrode and reference electrode. CONSTITUTION:Two sheets of electrodes are formed by immobilizing human IgG together with a monomolecular film of stearic acid by using a Langmuir- Blogett's technique on an electrode formed of the thin iridium oxide film formed to 500-1,000Angstrom film thickness by a sputtering method on a glass substrate, by which two sheets of electrodes are formed. The antigen of the one electrode is deactivated by projection of UV rays to form the reference electrode Ref. The aq. anti-human IgG antibody soln. is dropped to a phosphoric acid buffer soln. into which the working electrode of the non-deactivated antigen is immersed together with the reference electrode Ref. The potential generated on the surface of the IgG immobilized film is then measured by an amplifier A.

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to a novel immunosensor and an immunodetection method capable of detecting dilute concentrations of antigens or antibodies in a short time.

In recent years, field effect transistors have been used as sensors for detecting various minute chemical substances.
Chemical sensors using transistors (hereinafter abbreviated as FETs), such as ion-selective FETs and enzyme FETs, are being researched. These sensors have excellent high-speed response and high impedance compared to conventional glass PH electrodes, etc., and are also advantageous in terms of mass production and ultra-miniaturization due to the use of IC manufacturing technology.

Generally, FET sensors are formed from a substrate, a barrier film, and a sensitive film. A typical structure is a MO8FET substrate with the gate metal removed (hereinafter abbreviated as MO8FET substrate). Further, silicon oxide or silicon nitride is usually used for the barrier film. Furthermore, depending on the purpose, the sensitive membrane can be
For example, in the case of a PH sensor, aluminum oxide or tantalum oxide membranes are commonly used, and in the case of oxygen sensors, glucose oxidase, urease, etc. are used.

Although some of these FET sensors exhibit satisfactory characteristics in terms of response speed and detection sensitivity, they have a common problem: 1) They become sensitive to light if the light shielding effect of the gate part is insufficient. 2) When a normal metal thin film electrode with a light-shielding effect is provided at the gate part, the interfacial potential between the metal thin film surface and the solution in various sample solutions is not constant, and the antigen on the electrode surface is There were two problems: 3) minute potential changes associated with antibody reactions could not be detected; and 3) signal drift was observed during long-term use. These drawbacks pose a problem particularly when detecting dilute substances such as antigens and antibody proteins in aqueous solutions under natural light, and have prevented the realization of highly sensitive and stable immuno-FET sensors.

In addition, conventional methods for detecting antigens and antibodies include the EIA method using enzyme-labeled antibodies and the RIA method using antibodies labeled with radioactive elements, but the former requires time and effort in sample preparation. The latter had problems such as requiring facilities to handle radioactive elements. Another method is to immobilize antibodies and antigens on the surface of metal electrodes.
Although there are attempts to detect changes in surface potential associated with antibody reactions,
When a normal metal thin film electrode is provided, as already mentioned above,
There was a problem in that the interfacial potential between the surface of the metal thin film and the solution in various sample liquids was not constant, making it impossible to detect minute potential changes accompanying antigen-antibody reactions on the electrode surface.

In view of this situation, the inventors of the present invention have conducted extensive research into creating an FET sensor that does not have the above-mentioned drawbacks. By installing an iridium oxide film on the gate portion directly or via a conductor, the present inventors have found that the above-mentioned problems can be overcome. By obtaining an FET with almost no defects, and by providing an antibody or antigen substance layer on the iridium oxide film, it is possible to detect dilute antigen substances and antibodies in aqueous solutions with high sensitivity, selectively, and in small quantities. The present invention was achieved by discovering that the amount can be detected. That is, the present invention is an immunosensor that uses an iridium oxide electrode coated with a thin film of an antibody or an antigenic substance as a working electrode, and the working electrode is brought into contact with a reference electrode and a roughly combined antigenic substance or a solution containing an antibody. The potential change associated with the antigen-antibody reaction on the working electrode is
This is an immunodetection method characterized by detection as a change in potential, a change in current, or a change in the amount of charge.

More preferred embodiments of the present invention include: (1) An FET immunosensor in which iridium oxide is used as the gate metal of the MOSFET, and a thin film of an antibody protein or an antigen substance is provided thereon to serve as a working electrode.

Oi) An example is an FET immunosensor in which an iridium oxide electrode coated with a thin film of an antibody or an antigen substance is provided outside the gate region of the MOSFET and separated via a conductive wiring.

The iridium oxide film used as the electrode material or gate metal used in the present invention is usually formed by sputtering to a film thickness of 500 to 1000 mm. The membrane immobilizes a thin film of antibodies or antigenic substances on its surface, and
It can be used as an electrode to detect membrane potential changes associated with antibody reactions. As another embodiment, it is also possible to provide the above-mentioned iridium oxide film directly on the gate portion of the MOSFET or in a region other than the gate region via a conductor. M here
OSFET is a p-type or n-type silicon wafer doped with impurities of opposite sign (junction depth 5-10μ) to form source and drain electrodes, and a gate part on the surface between these electrodes. (Gate length; 10-1ooμ
, gate width: 100-500μ) and with the gate metal removed, usually
It consists of a 500-1000 thick silicon oxide layer and a 500-1000 thick silicon nitride layer formed thereon.

When the iridium oxide film is placed directly on the gate portion, it is often formed by sputtering at high temperatures, which may damage the MO8FET substrate, so it is preferable to provide an intermediate layer such as tantalum oxide. In addition, when an inridium oxide film is provided outside the gate region, the substrate for the film is preferably an insulator such as glass or plastic, or the same silicone substrate as the FET, and is connected to the gate portion via a metal conductor wire or a semiconductor conductor wire. be done. It is preferable to use iridium oxide as the conductive wire connecting the isolation gate to the gate portion in order to avoid the formation of a bonding interface with different metals.

Next, the antibodies and antigenic substances used in the present invention are related to immune reactions, have ionic groups in their molecules, and have 10
Immunoglobulins such as I (IG, IIJA, 10E, IoM, etc.) and chorionic gonadotropin (+- (CG), carcinoembryonic antigen (CEA), etc.) exhibiting a membrane potential of 0 μV or more, preferably 1 mV or more. Polyclonal or monoclonal antibodies against these antigens are used.

These antigen and antibody molecules are formed into a thin film, preferably a monolayer, either alone or in combination with other lipid molecules, and then
It is fixed on the iridium oxide electrode mentioned above. As a method for immobilizing antibodies and antigens on the electrode, immersion adsorption method, casting method, Langmuir-Prodgett method, etc. are employed.

Thus, when an element with an antibody or antigen immobilized on an iridium oxide electrode is immersed in an analyte solution containing the antigen or antibody, the surface membrane potential changes as a result of the antigen-antibody reaction on the electrode. do. As a result, antigens and antibodies can be detected by detecting the amount of change in membrane potential directly or by converting it into an electric current and detecting it through a low-noise amplification circuit.

Furthermore, in the case of a FET device, a current or voltage change between the source and drain is induced due to the antigen-antibody reaction on the gate electrode, making it possible to detect the antigen or antibody. The drain voltage employed in the above antigen-antibody reaction detection is 5-10V, and the gate voltage varies depending on the threshold of the membrane surface potential change of the antigen/antibody, but is 0-S
V is preferably determined experimentally in the range of 0-2v.

Detection of the above-mentioned antigen-antibody reaction is performed by reading the change in the amount of charge on the electrode surface or the change in current or voltage between the source and drain. The amount of change in current or voltage at that time depends on the antigen or antibody concentration in the sample fluid.

When the concentration of antigens and antibodies is dilute and the amount of change in current or voltage is small, antigens and antibodies can be detected by using a low-noise amplifier with a built-in bipolar or junction FET and shielding the electric field around the device. It becomes easier to detect electrical signals associated with reactions.

Furthermore, the time required to detect this antigen-antibody reaction depends on the concentration thereof, the area of the electrode surface, and the amount of immobilized antibody or antigen, but when compared under the same conditions, it is as shown in Example 4 below. Moreover, it is extremely quick compared to the conventional EIA method and RIA method.

Furthermore, in the present invention, several types of antibodies and/or antigens are immobilized on individual iridium oxide electrodes and are placed on the same solid substrate, thereby allowing multiple antigens and/or antibodies in the sample fluid to be immobilized on individual iridium oxide electrodes. It is also possible to detect them simultaneously.

The immunodetection method of the present invention described above is simpler and faster than conventional methods, is hardly affected by light or coexisting ions, and is extremely stable compared to conventional FET sensors. The industrial significance of this development is significant. EXAMPLES Hereinafter, the present invention will be explained in more detail with reference to Examples, but the present invention is not limited thereto.

Example 1 Human 1gG was stearinized on an iridium oxide thin film (2#1IllX7 shoes, a thickness: about 700 people) electrode formed by sputtering on a glass substrate (1cll x 1α) using the Langmuir-Blodgett method. It was immobilized with a molecular membrane in acid. Two sheets III created using the above method
IG-immobilized electrodes were prepared, and the antigen on one electrode was inactivated by ultraviolet irradiation to serve as a reference electrode, and the time response of the output voltage was measured using the circuit configuration shown in FIG. 1 via an amplifier (A). When an anti-human ICIG antibody aqueous solution (10 to 100 μm) is dropped into 10 μm of the phosphate buffer solution in which the electrode is immersed, a potential is generated on the surface of the TgG-immobilized membrane due to the antigen-antibody reaction. This potential causes a current to flow through a low impedance (1 to Ω) circuit containing an iridium oxide electrode and an amplifier,
-V (current-voltage) output by a converter amplifier. At this time, the amount of charge detected, that is, the time-integrated amount of current, is proportional to the amount of antibody added to the phosphate buffer. FIG. 2 shows the relationship between the detected charged acid and antibody concentration. With this measurement circuit system, it was possible to detect 1 x 10-7 mol/r1 of antibody.

Example 2 FIGS. 31 and 5 show the structure of the gate isolation type FET used in this example. First, 1 cm x 2 crn
After diffusing an n-type impurity of phosphorus into a p-type silicon wafer to form a source electrode (S') and a drain electrode (D'), the wafer surface was oxidized and 5iO
A z layer was formed. After that, an iridium oxide layer with a thickness of about 800 mm was formed by sputtering on the surface gate part (50 μm width x 1000 μm length) between the source and drain and the isolated gate electrode part (2#lIIIX7M) in FIG.
The above-mentioned surface gate part and separated gate electrode part were also connected with a reinforced iridium thin film.

Two identical immuno-FET electrodes thus fabricated on a silicon substrate were prepared, and anti-human IgG was immobilized on each of their separate gate electrodes by a dipping/adsorption method. After that, the antibody on one electrode is inactivated by ultraviolet irradiation,
After being used as a reference electrode, the above two separated gate electrodes were immersed in a phosphate buffer solution. Next, an aqueous solution of IgG of unknown concentration was dropped into this buffer solution, and then the change in membrane potential on the gate electrode surface due to the antigen-antibody reaction was measured.

In this measurement, the gate voltage was set to be constant (0 to 2 V) using a platinum electrode, and the drain voltage was 5. I (110
1 degree was 2×10″G mole/1.

Example 3 In Example 2, the gate portion made of an iridium oxide thin film was directly coated on the surface between the source and drain without separating it from the FET body, and anti-human 1 (IG antibody) was similarly immobilized on the surface. The antigen-antibody reaction was carried out in the same manner as in Example 2.The amount of change in the drain voltage after dropping the sample liquid became constant at about 70 mV after 30 seconds, and the I(
The IG concentration was 2,8 x 10'i mol/su. Add 0.1 potassium chloride as a new electrolyte to the above antibody solution.
- 0.5 wt% was added and the antigen-antibody reaction was performed in the same way, but the amount of change in drain voltage was about 70 mV after 30 seconds, which was a result with good reproducibility, as in the case above. It was done.

Furthermore, in this example, no influence of external light was observed in the detection of antigen-antibody reactions.

Example 4 This example shows that antigen-antibody reactions can be detected extremely quickly by using the FET device of the present invention compared to the EIA method.

3X on the surface of the iridium oxide electrode used in Example 1
Two sheets were prepared in which 10-''mol of anti-human I9G antibody was immobilized together with barium stearate by the Langmuir-Prodgett method, one of which was incorporated into the same charge measurement circuit as in Example 1. 10-7 mol/day human I
(IG could be detected in 30-40 seconds. On the other hand, another anti-human IaG fixed sample was used and immersed in a human IOG aqueous solution with the same concentration as above.

After soaking, peroxidase-labeled anti-human I (IG) was added to the surface.
After further reaction with the antibody, hydrogen peroxide solution and 0-phenylenediamine were added to the system and EIA was performed.
A total of 160 minutes was required to detect 7 mol/state of human IaG.

Comparative Example 1 This example shows that when a metal other than iridium oxide is used as the gate metal, a potential change accompanying an antigen-antibody reaction cannot be detected.

Instead of iridium oxide as the gate metal, an aluminum thin film with a thickness of 700 mm was used to immobilize anti-human I (IG antibody) in the same manner as in Example 3, and then an antigen-antibody reaction was carried out in the same manner as in Example 2. went.

The amount of change in the drain voltage after dropping the sample liquid varied depending on the electrolyte concentration in the sample liquid, and it was virtually impossible to detect an antigen-antibody reaction.

Comparative Example 2 This example shows the influence of external light when an antigen-antibody reaction is performed using a normal MOSFET with the gate metal removed.

In Example 2, when the antibody was directly immobilized on the gate insulating crotch without using a gate metal and the antigen-antibody reaction was similarly performed under external light irradiation of 350 lux, the change in drain voltage was 3.
It was not constant within -5 mV and varied depending on whether the external light was turned on or off.

[Brief explanation of the drawing]

FIG. 1 shows the configuration of the antigen-immobilized electrode and the measurement circuit. In the figure, ICIG stands for immunoglobulin G, Rer stands for a reference electrode, R stands for a resistance, A stands for an amplifier, and ■ stands for an electro-X meter. Further, 3 shield means a shielding jig against electrical signal noise caused by electric fields and magnetic fields. FIG. 2 shows the relationship between anti-IOG concentration and detected charge amount. In the figure, Q represents the amount of detected charge, and anti-IgG means an antibody against human IgG, which is an antigenic substance. FIG. 3 represents the immunoFET (top view). In the figure, S and S' represent the source and source electrode of the FET, D and D' represent the drain and drain electrode, respectively, and G represents the isolation gate. Further, C means a conductive wire made of iridium oxide that connects the gate portion of the FET and the isolation gate. Furthermore, A-A' and B-B' each represent a FET of the present invention.
represents a longitudinal and transverse cross section. FIG. 4 shows an immune FET (A-A' sectional view). In the figure, C represents iridium oxide and E represents epoxy resin. FIG. 5 shows an immune FET (B-8' sectional view). In the figure, S and S' represent the source and source electrode of the FET, respectively, and D and D' represent the drain and drain electrode, respectively. Also,
Patent applicant Teijin Ltd. C is characterized by a conducting wire made of iridium oxide that connects the gate part of the FET and the isolation gate.

Claims (1)

[Claims] 1. An immunosensor whose working electrode is an iridium oxide electrode coated with a thin film of an antibody or an antigen substance. 2. The immunosensor according to claim 1, comprising means for amplifying the potential difference between the working electrode and the reference electrode and detecting it as a voltage, current, or charge amount. 3. The immunosensor according to claim 2, wherein the reference electrode is one in which the antibody or antigen substance of the working electrode is inactivated. 4. The immunosensor according to any one of claims 1 to 3, wherein the working electrode is provided in a gate region on a MOSFET. 5. The immunosensor according to claim 4, wherein the working electrode is provided apart from the gate region of the MOSFET via a conductive wiring. 6. A working electrode made of an iridium oxide electrode coated with a thin film of an antibody or antigenic substance and a reference electrode are brought into contact with a solution containing the antigenic substance or antibody, and a potential change due to the antigen-antibody reaction on the working electrode is caused. , an immunodetection method that reads as a change in potential, a change in current, or a change in the amount of charge.