JPH02110363A - Working electrode for immunity sensor and manufacture thereof - Google Patents

Abstract

PURPOSE:To achieve high sensitivity by providing a conductive porous body as an electrode for a porous electrode to be used, and forming a device with antibody or antigen which is formed on the electrode and fixed to conductive macromolecules. CONSTITUTION:A porous electrode to be used is a conductive porous body as an electrode. The body is especially selected out of the following materials: a material wherein a metal thin film is provided on the surface and/or the inner hole wall of organic or inorganic porous body; net-shaped or sponge-shaped metal; and net-shaped carbon fiber. The body comprises conductive macromolecules formed on the electrode and antibody or antigen which is fixed in and/or on the surface of the conductive macromolecules. Any antibody or antigen can be used for the antibody or antigen which is fixed in the conductive macromolecule. In this way, the structure of a highly sensitive sensor characterized by excellent reproducibility and stability for a long time is obtained.

Description

[Detailed description of the invention] (1) Industrial application fields: The present invention relates to a novel working electrode for an immunosensor.

More specifically, the present invention relates to a working electrode for an immunosensor consisting of a conductive polymer formed on a porous electrode, and an antibody or antigen immobilized in and/or on the surface of the polymer, and a method for manufacturing the same.

(2) Prior Art: In the field of clinical medicine, immunological diagnosis using an antibody-antigen reaction, which is a very specific biochemical reaction, has been performed so far. As specific methods, sedimentation/aggregation methods, radioimmunoassay methods, enzyme immunoassay methods, fluorescence immunoassay methods, etc. are known and are in practical use. However, these methods have drawbacks such as requiring special equipment and expensive equipment, and being complicated to handle.

In recent years, immunosensors have been proposed as a convenient method for detecting antigens or antibodies. For example, Suzuki.

Aizawa et al. showed that it is possible to detect antigens or antibodies by immobilizing antibodies or antigens on cellulose acetate membranes and measuring the membrane potential.
, 2.125 (1977)), Sansui, 2 Morimura et al. detected antigens or antibodies by measuring the electrode potential using chemically modified electrodes on which antibodies or antigens were immobilized (J,
Immunol, Methods, 22.309
(197B)). In addition, Tamiya, Karube et al. fixed antibodies on the gate part of field effect transistors and detected antigens (5th Chemical Sensor Research Presentation Abstracts Z-4 (1988).
6)), Aiguchi et al. showed that antigens can be detected using polypyrrole, polythiophene, or polyfuran membranes on which antibodies are immobilized (Japanese Patent Application Laid-open No. 195346/1986, 6).
No. 3-50747). However, these methods have problems such as low detection sensitivity, poor reproducibility, and cannot withstand long-term use.

(3) Summary of the invention The present invention has been made in view of this situation. That is, the present invention was arrived at as a result of intensive research into constructing a highly sensitive sensor that is stable over a long period of time with excellent reproducibility.

(4) Structure of the present invention: That is, the present invention consists of: 1. A conductive polymer formed on a porous electrode, and an antibody or antigen immobilized in and/or on the surface of the conductive polymer. Working electrode for immunosensor 2. The porous electrode is selected from organic or inorganic porous material with a metal thin film provided on the surface and/or inner pore wall, reticular or sponge-like metal, and reticular carbon fiber. 3. An electrolytically polymerizable monomer capable of forming a conductive polymer is placed on the porous electrode in the presence of a supporting electrolyte and an antibody or an antigen. This is a method for producing a working electrode for an immunosensor, which is characterized by electrolytically polymerizing in an aqueous solution and entrapping an antibody or an antigen in the formed conductive polymer.

The porous electrode used in the present invention is a porous body having electrical conductivity as an electrode, and in particular, an organic or inorganic porous body provided with a metal thin film on the surface and/or inner pore wall, a net-like or sponge-like metal , and reticulated carbon fiber. The conductivity of these porous electrodes is O,Is/cm to 106 S/am, the average pore diameter is 0.1 μm to 1 mm, and the porosity is 20 to 80
% is desirable.

Roughly desirably, the conductivity is 1 S/c+n~106
S/cm.

The average pore diameter is 1 μm to 100 μm. The conductivity is 0. IS
/Cm or less, electrolytic polymerization of monomers that can be electrolytically polymerized to the porous electrode cannot be performed, and if the average pore diameter is less than 0.1 μm, it is impossible to form a polymer by electrolytic polymerization on the inner pore walls of the porous electrode. Can not.

The organic or inorganic porous body used in the present invention is not particularly limited, and any porous body can be used as long as it can form a metal thin film serving as an electrode on its surface and inner pore walls. Specific examples thereof include porous polymer films, inorganic filters, porous fibers, nonwoven fabrics, net-like materials, and the like. Materials for porous polymer films, fibers, nonwoven fabrics, and nettings include polystyrene, polyvinyl chloride, polytetrafluoroethylene, and polyvinylidene fluoride.

Examples include polyethylene, polypropylene, polyvinyl alcohol, polyacrylonitrile, poly(meth)acrylic acid ester, polysulfone, polyethersulfone, polyamide, polyimide, and copolymers and derivatives thereof.

The average pore diameter of these porous bodies used in the present invention is 1
It is desirable that the thickness is from nm to 1 mm. More preferably, the thickness is 10 nm to 10 μm. If the average pore diameter is less than 1 nm, it becomes impossible to form a metal thin film for an electrode on the inner pore walls of the porous body. On the other hand, if the pore diameter is larger than '+mm, the volume fraction occupied by the conductive polymer per unit volume of the porous electrode becomes significantly small, and the function as an electrode deteriorates. The porosity of the porous body is preferably 5 to 80%, preferably 20 to 80%. The metal provided on the surface and inner pore walls of the porous body used in the present invention is not particularly limited as long as it is a metal that is less likely to be altered or deteriorated by electrolytic polymerization, and may include gold and platinum.

Examples include palladium, nickel, copper, silver, and carbon.

Thin films of these metals can be formed, for example, by vacuum evaporation.

It is formed on the surface of the porous body and the inner pore walls by sputtering, chemical plating, ion plating, metal spraying, etc. The thickness of the metal thin film is preferably such that it does not block the inner pores of the porous body, and although it depends on the pore diameter of the porous body, it is
A suitable thickness is 10 μm to 10 μm.

The net-like or sponge-like metal used in the present invention is not particularly limited as long as it does not undergo alteration or deterioration in the electrolytic polymerization reaction for forming the conductive polymer, and includes gold, platinum, palladium, nickel, and copper.

Examples include silver.

The monomer used in the present invention is not particularly limited as long as it forms a conductive polymer by electrolytic polymerization, and includes, for example, pyrrole, aniline, and thiophene.

Mention may be made of furans and their derivatives. Among these monomers, pyrrole and aniline are particularly desirable, judging from the conductivity type, mechanical strength, chemical and thermal stability, and adhesion to porous electrodes of the resulting conductive polymer. Dopants used during electropolymerization vary depending on the type of monomer; in the case of pyrrole, thiophene, and furan, lithium perchlorate, tetraethylammonium perchlorate, tetraethylammonium borate, hexafluorophosphoric acid, etc. is preferably used. Further, in the case of aniline, PAm is preferably used, but the present invention is not limited thereto.

Any antibody or antigen can be immobilized on the conductive polymer in the present invention, such as I(IG).

Various immunoglobulins such as IgH, albumin, hCG
etc.

The antibody or antigen is encapsulated in a conductive polymer by electrolytically polymerizing a monomer that can be electrolytically polymerized on a porous electrode together with a supporting electrolyte and the antibody or antigen in an aqueous solution, and then applied to the surface of the porous electrode or/and the antibody or antigen. and fixed to the bore wall. The concentration of monomer in the solution during polymerization is generally 0.01 m
ol/1 to 0.5 mol/d, and the supporting electrolyte may be about 0.1 mol/rrdl.

Antibodies or antigens are dissolved in a solution, for example, in the case of human IgG, it is 0.01mMd to 0.5mq/
It is rd. Electrolytic polymerization can be performed using either a constant potential method or a constant current method.
~1.5V potential, 0.1mA/cm using constant current method
Electrolytic polymerization is carried out at a current density of 2 to 50 mA/cm2. The film thickness of the conductive polymer on which the antibody or antigen entrappingly immobilized thus obtained is desirably such that it does not block the inner pores of the porous electrode, and varies from 1 nm to 10 nm, depending on the pore diameter of the porous electrode.
μm is appropriate. Further, it is preferable that the conductive polymer entrapping and immobilizing the antibody or antigen in the porous electrode forms a continuous layer.

The porous electrode with the antibody or antigen immobilized thus obtained is soaked in a gel electrolyte placed in a suitable container.
Measure the change in potential difference relative to a reference electrode. As the reference electrode, it is desirable to use the above porous electrode on which no antibody or antigen is immobilized, or a porous electrode on which the antibody or antigen has been inactivated.

The detection response is a change in potential difference that occurs when a solution containing an antigen or antibody is dropped into the gel electrolyte.

(5) Effects of the invention: Thus, the immunosensor comprising the conductive polymer formed on the porous electrode of the present invention and the antibody or antigen immobilized in the polymer or/and on the surface has a small surface area per unit volume. is very large, and as a result, high sensitivity is achieved, making it possible to detect dilute concentrations of antigens or antibodies contained in sample fluids. In addition, even if left in an aqueous solution for a long period of time, the immobilized antibody or antigen does not come off and no deterioration of detection characteristics is observed. Surprisingly, after detecting an antigen or antibody, if the porous electrode is washed with distilled water and immersed in a solution containing the antigen or antibody, it is possible to detect the antigen or antibody again without any special regeneration treatment. It has also become clear in the present invention that

Hereinafter, the present invention will be explained in more detail with reference to Examples.

Example 1 A nonwoven fabric of polyethylene terephthalate (Japan Vilene ■ fF-135K) was chemically plated according to a conventional method.

That is, after being immersed in a tin chloride aqueous solution, it was immersed in a 0.1H palladium chloride aqueous solution of 0.18 hydrochloric acid, and then dried. Next, nickel ammonium sulfide 31.6
1J, sodium hypophosphite 42.4111, sodium tartrate 55.2g, ammonium sulfate 40.6 (
It was immersed in a solution prepared by dissolving 59.40% of boric acid in 11% of distilled water and adjusting the pH to 9 with an aqueous sodium hydroxide solution.

A filter plated with nickel using the above method was used as a porous electrode. The conductivity type of this porous electrode is 32, IS
/cm, and the average pore diameter was 12.5 μI. The above porous electrode was immersed in a propylene carbonate solution containing 0.1H tetraethylammonium verchlorate and 0.3M pyrrole, and the amount of electricity was 0.8 C/cm2 under constant potential conditions of 1.1 V with respect to the silver/silver chloride electrode. Electrolytic polymerization was carried out until the Rough 0.1) after drying and washing? potassium chloride and 0
．． IIJG with 15M virol and 0.25mM rail
0.65 for silver/silver chloride electrodes immersed in distilled water containing
Under constant potential conditions of V, the amount of electricity is 0. Electrolytic polymerization was carried out until IC/cm2 was reached. IOC according to scanning electron microscopy
It was observed that a thin film of polypyrrole containing polypyrrole was continuously formed on the surface of the nonwoven fabric and the inner pore walls, and did not block the inner pores of the porous electrode.

This porous electrode with immobilized IgG was immersed in 0.7% by weight agar mixed with 25n+H sodium phosphate buffer (1) 86.8) and bovine serum albumin. Potential difference measurements were made.

As a result, a potential difference change of 5.2 mV occurred for a concentration of 5 x 10-10 mol/d of anti-ICIQ labeled with peroxidase. 25ml) l of phosphate buffer (p 6.
After soaking in 8) for 7 days, similar measurements were performed.
×10-'Omol/d of peroxidase-labeled anti-I (
A potential difference change of 5.1 mV occurred with respect to JG, and no deterioration in response was observed.

Example 2 Platinum mesh (80 mesh) was mixed with 0.1) monopotassium chloride, 0.158 pyrrole, and 0.25 mg/d of IgG
0.6 for silver/silver chloride electrodes.
Under constant potential conditions of 5V, the amount of electricity is 0. Electrolytic polymerization was carried out until IC/Cm2 was reached. As a result of measurement in the same manner as in Example 1, 5xlO" mol/m of peroxidase-labeled anti-I
(A potential difference change of 4.0 mV occurred compared to JG.

Comparative Example Platinum was mixed with 0.18 potassium chloride and 0.158 pyrrole.
．． Silver/
Electrolytic polymerization was performed under constant potential conditions of 0.65 V with respect to a silver chloride electrode until the amount of electricity reached 0, IC/cm2. When the measurement was carried out in the same manner as in Example 1, only a 0.5 mV potential difference change occurred with respect to 5×10” Omol/d of peroxidase-labeled anti-ICIG.

Claims (3)

[Claims] (1) A working electrode for an immunosensor consisting of a conductive polymer formed on a porous electrode and an antibody or antigen immobilized in or/on the surface of the conductive polymer. (2) The porous electrode is selected from organic or inorganic porous bodies with a metal thin film provided on the surface and/or inner pore walls, net-like or sponge-like metals, and reticulated carbon fibers. Item 1. The working electrode for an immunosensor according to item 1. (3) On a porous electrode, an electrolytically polymerizable monomer capable of forming a conductive polymer is electrolytically polymerized in an aqueous solution in the presence of a supporting electrolyte and an antibody or an antigen. A method for producing a working electrode for an immunosensor, which comprises comprehensively immobilizing an antibody or an antigen in a molecule.