CROSS-REFERENCE TO RELATED APPLICATION

This application is a 371 application of the international PCT application serial no. PCT/JP2015/068758, filed on Jun. 30, 2015, which claims the priority benefit of Japan application no. 2014-136355, filed on Jul. 1, 2014. The entirety of each of the abovementioned patent applications is hereby incorporated by reference herein and made a part of this specification.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates to a resin-metal composite applicable to, for example, markers and so on applicable to immunoassay, a marker utilizing same, an immunoassay method, an immunoassay reagent, a method for measuring analyte, an analyte measurement kit, and a lateral-flow chromatographic test strip.

2. Description of Related Art

Innumerable chemical substances exist in an organism, so the qualitative and quantitative analysis of specific trace components in the organism is an extremely important technique. In fields, such as medical care, pharmacy, health food, biotechnology and environment, drugs and food having effects only on specific parts (chemical substances) in organisms, analysis devices and diagnostic drugs and the like detecting slight changes in organisms develop along with the technique.

One of the analysis techniques is immunoassay. This technique is also called immunological assay, which is a method that qualitatively and quantitatively analyzes trace components by utilizing antigen-antibody specific reaction as one of immunoreactions. Because of high sensitivity or reaction selectivity, the antigen-antibody reaction is widely applied in the fields. Various assay methods exist in immunoassay according to assay principles. Examples are listed as follows: enzyme immunoassay (EIA), radioimmunoassay (RIA), chemiluminescent immunoassay (CLIA), fluorescence immunoassay (FIA), agglutination of latex and so on (latex immunoassay (LIA), particle agglutination (PA)), immunochromatography (ICA), hemagglutination (HA) and hemagglutination inhibition (HI). Moreover, besides immunoassay, there are physical and chemical assays, biological assay and so on.

Immunoassay qualitatively or quantitatively assays an antigen or an antibody according to change in the reaction between the antigen and the antibody for forming the composite (concentration change of the antigen, the antibody or a composite). When these are assayed, the antibody, the antigen or the composite are bound with a marker, and thereby assay sensitivity is increased.

Therefore, it can be said that the marking capability of the marker is a significant factor affecting assay capability in immunoassay. In the foregoing illustrated immunoassays, erythrocytes (the case of HA), latex particles (the case of LIA), fluorochromes (the case of FIA), radioactive elements (the case of RIA), enzymes (the case of EIA) and chemiluminescent substances (the case of CLIA) and so on can be adopted as markers.

However, when colored microparticles are used as a marker, assay can be determined visually without using a special analysis device, so it can be expected that simpler assay may be realized. As such colored microparticles, examples are listed as follows: colloidal particles of metals and metal oxides and latex particles colored by utilizing a pigment (patent document 1, patent document 4, etc.). However, because the color of the colloidal particles is determined according to particle sizes and preparation conditions, there exists a problem that it is hard to obtain a desired bright rich color, that is, visual recognizability is insufficient.

In addition, the colored latex particles have the problem of low coloring effect of the utilized pigment and insufficient visual determinability. Furthermore, if the coloring amount of the pigment is increased in order to solve the problem, then the pigment covers the surface of the latex, the original surface state of the latex particles is impaired, and as a result, there exists a problem that it is hard to bind the antigen or the antibody. In addition, there also exist the following problems: blockage in pores of a chromatographic medium such as a membrane filter, or nonspecific agglutination produced by the latex particles or uncertain association between rich coloration resulting from the increase in the coloring amount of the pigment and an increase in performance.

In order to improve the visual recognizability of the marker, following immunochromatography is disclosed: after an antibody (marked antibody) bound with a marker react with an antigen to form a composite, other metals modify the marker, and thereby the assay sensitivity of the marker is increased (patent document 2 and patent document 5). However, in the method, operation is complex, and it is hard to achieve a stable increase. In addition, it is considered that assay cost is consumed due to the fact that a special device is required, so applicable purposes and application environment are limited.

In addition, a coloring latex containing gold nanoparticles bound with the surfaces of polymeric latex particles (patent document 3) is disclosed.

By binding the surfaces of the polymeric latex particles with the gold nanoparticles, the gold nanoparticles as colorant itself can help to increase visual determinability or assay sensitivity. On the other hand, the gold nanoparticles itself are excellent for the binding of an antigen or an antibody, so, even if the gold nanoparticles are bound until a degree of sufficiently rich color, an enough amount of antigen or antibody can be bound.

γrays irradiate a dispersion of styrene-acrylic acid copolymer latex and a precursor of gold nanoparticles (i.e. HAuCl), so that the surface of the latex is bound with the gold nanoparticles, and thereby the coloring latex is formed. However, since the gold nanoparticles are only bound with the surface of the latex, not only is the quantity of carried gold particles manifesting surface plasmon absorption limited, but also the gold nanoparticles can easily come off. As a result, the visual recognizability or sensitivity of it as an immunoassay reagent may not be sufficient. In addition, because of the irradiation of electromagnetic radioactive rays such as γrays, the latex may be injured. Further, although the description of patent document 3 discloses preferred ranges of latex size or gold nanoparticle size, whether it has been verified within these preferred ranges in the embodiments is not clear, so a specified basis of preferred ranges does not exist.

In addition, patent document 4 discloses a metal gold-coated polymer latex particle, and suggests the application of a reagent applicable to microscopic examination and immunoassay.

However, the material or particle size of the metal gold-coated polymer latex particle is not disclosed. Further, the effect of it as a reagent applicable to immunoassay has not been verified. Therefore, the effect of it as a reagent in metal gold and polymer latex particles is not clear.

In addition, non-patent document 1 discloses a microgel which is prepared by carrying gold nanoparticles on poly-2-vinylpyridine latex particles, and determines the pH responsibility of the particle size of the microgel according to change in the behavior of localized surface plasmon absorption of the gold nanoparticles. However, the microgel carries the gold nanoparticles on a single layer near the surfaces of the latex particles. Therefore, it can be considered that the quantity of carried the gold nanoparticles is low, and as a result, a rich color which is effective for immunoassay cannot be obtained. In addition, research on the material, structure and composition of the microgel has not been made, so the effect of specific applications, such as an immunoassay reagent, is not clear.

According to what is mentioned above, although the latex particle bound or coated with the gold nanoparticle is expected as an immunoassay reagent, in the prior art, durability or visual recognizability is not sufficient. In addition, even if visual recognizability is high, applicable purposes and application environment are limited.

DOCUMENTS OF THE PRIOR ART

Patent Documents

Patent Document 1: Japanese Unexamined Patent Application Publication No. Hei-5-10950Patent Document 2: Japanese Unexamined Patent Application Publication No. 2011-117906Patent Document 3: Japanese Unexamined Patent Application Publication No. 2009-168495Patent Document 4: Japanese Unexamined Patent Application Publication No. Hei-3-206959Patent Document 5: Japanese Unexamined Patent Application Publication No. 2009-192270

Non-Patent Document

Non-patent Document 1: K. Akamatsu, M. Shimada, T. Tsuruoka, H. Nawafune, S. Fujii and Y. Nakamura,Langmuir,2010, 26, 1254-1259

SUMMARY OF THE INVENTION

Problem to be Solved by the Invention

The present invention is directed to provide a resin-metal composite which is excellent in durability and visual recognizability and can realize high-sensitivity determination without requiring the addition of a special device or operation steps in applications such as a material for immunoassay.

Means for Solving the Problem

As the result of the research effort of the inventor and the others, it is discovered that a resin-metal composite with a specific structure can solve the problem, and thereby the present invention is achieved.

That is, the resin-metal composite of the present invention comprises a structure which is formed by immobilizing a plurality of metal particles on a resin particle, and is characterized in that the plurality of metal particles includes:

first particles, having a portion exposed from the resin particle; and

second particles, completely encased in the resin particle; and

among the first particles and the second particles, at least portions of the particles are three-dimensionally distributed in a surface section of the resin particle.

In the resin-metal composite of the present invention, relative to the weight of the resin-metal composite, the quantity of the carried metal particles can be within a range from 5 wt % (percentage by weight) to 70 wt %.

In the resin-metal composite of the present invention, 60 wt % to 100 wt % of the metal particles can exist in the surface section, and 5 wt % to 90 wt % of the metal particles existing in the surface section can be the first particles.

In the resin-metal composite of the present invention, the resin particle can be a polymer particle which is provided with a substituent group capable of adsorbing metal ions in the structure.

In the resin-metal composite of the present invention, the average particle size of the metal particles can be within a range from 1 nm to 80 nm.

The average particle size of the resin-metal composite of the present invention can be within a range from 100 nm to 1000 nm.

In the resin-metal composite of the present invention, the metal particles can be gold particles.

A marker of the present invention comprises any one of the resin-metal composites.

The marker of the present invention can be used in adsorbing an antigen or an antibody on the surface of the resin-metal composite.

An immunoassay method of the present invention uses any one of the markers.

An immunoassay reagent in the present invention comprises any one of the resin-metal composites.

A method for measuring analyte in the present invention is a method for measuring analyte which can assay or quantify an analyte contained in a sample.

The method for measuring analyte in the present invention uses a lateral-flow chromatographic test strip which comprises a membrane and a determination portion formed by immobilizing capturing ligands specifically bound with the analyte on the membrane.

Moreover, the method for measuring analyte in the present invention is characterized by carrying out steps including Step (I) to Step (III) hereinafter:

Step (I): Step of making the analyte contained in the sample contact with a marked antibody formed by utilizing any one of the resin-metal composite to mark an antibody specifically bound with the analyte.

Step (II): Step of making the composite containing the analyte and the marked antibody formed in Step (I) contact with the capturing ligands in the determination portion.

Step (III): Step of determining colored intensity derived from the localized surface plasmon resonance of the resin-metal composite.

An analyte measurement kit in the present invention is an analyte measurement kit which uses the lateral-flow chromatographic test strip and is used to assay or quantify an analyte contained in a sample.

The analyte measurement kit in the present invention comprises: a lateral-flow chromatographic test strip, comprising a membrane and a determination portion formed by immobilizing capturing ligands specifically bound with the analyte on the membrane; and

an assay reagent, containing a marked antibody formed by utilizing any one of the resin-metal composite to mark an antibody specifically bound with the analyte.

A lateral-flow chromatographic test strip in the present invention is a lateral-flow chromatographic test strip which is used to assay or quantify an analyte contained in a sample.

The lateral-flow chromatographic test strip in the present invention comprises:

a membrane;

a determination portion, formed by immobilizing capturing ligands specifically bound with the analyte on the membrane in the spreading direction of a sample; and

a reaction portion, comprising an marked antibody formed by utilizing any one of the resin-metal composite to mark an antibody specifically bound with the analyte further upstream than the determination portion.

Effects of the Present Invention

At least portions of the metal particles of the resin-metal composite of the present invention are three-dimensionally distributed in the surface section of the resin particle, moreover, portions of the three-dimensionally distributed metal particles are partially exposed from the resin particle, and the rest portions are encased in the resin particle. Therefore, the quantity of the carried metal particles manifesting localized surface plasmon absorption on the resin particle is high. In addition, the metal particles can hardly come off from the resin particle. Therefore, as a material with excellent durability, visual recognizability, visual determinability and assay sensitivity, the resin-metal composite of the present invention can be preferably applied in purposes, such as a marker for immunoassay (e.g. EIA, RIA, CLIA, FIA, LIA, PA, ICA, HA and HI), an immunoassay reagent, a solid catalyst, a pigment, a coating, a conductive material, an electrode and a sensor component. In particular, when the resin-metal composite of the present invention is used as a material for immunoassay, the durability and visual recognizability are excellent, and high-sensitivity determination can be realized without requiring the addition of a special device or operation steps.

