JP7709837B2 - Nanocomposite particles, labeling substance, immunoassay method, immunoassay reagent, analyte measurement method, and analyte measurement kit - Google Patents

Description

The present invention relates to novel nanocomposite particles that can be preferably used for applications such as immunoassays, labeling substances that utilize the same, immunoassays, immunoassay reagents, analyte measurement methods, and analyte measurement kits.

Immunological measurement (also called "immunoassay") is a method for qualitatively and quantitatively analyzing trace components by utilizing a specific reaction between antigen and antibody, which is one type of immune reaction. Antigen-antibody reactions are widely used in the above fields due to their high sensitivity and selectivity. There are various immunoassay methods depending on the measurement principle. Examples include enzyme immunoassay (EIA), radioimmunoassay (RIA), chemiluminescence immunoassay (CLIA), fluorescence immunoassay (FIA), latex agglutination methods (LIA, PA), immunochromatography (ICA), hemagglutination (HA), hemagglutination inhibition (HI), etc. In addition to immunoassays, there are physical/chemical measurement methods and biological measurement methods.

In immunoassays, antigens or antibodies are qualitatively or quantitatively detected from the changes (changes in the concentrations of antigens, antibodies, or complexes) that occur when the antigens and antibodies react to form complexes. When detecting these, the detection sensitivity can be increased by binding a labeling substance to the antibody, antigen, or complex.
Therefore, the labeling ability of the labeling substance is an important factor that influences the detection ability in the immunoassay. In the immunoassays exemplified above, red blood cells (in the case of HA), latex particles (in the case of LIA), fluorescent dyes (in the case of FIA), radioactive elements (in the case of RIA), enzymes (in the case of EIA), chemiluminescent substances (in the case of CLIA), etc. are used as the labeling substances.

Incidentally, when colored particles are used as a labeling substance, it is expected that the detection can be confirmed visually without using a special analytical device, and therefore a simpler measurement can be performed. Examples of such colored particles include colloidal particles of metals and metal oxides, latex particles colored with dyes, etc. (Patent Document 1, Patent Document 2, etc.). However, the color tone of the above colloidal particles is determined by the particle size and preparation conditions, so it is difficult to obtain a desired clear and dark color tone, that is, there is a problem that visibility is insufficient.
In addition, the colored latex particles have a problem that the coloring effect of the dye is low and the visual judgment is insufficient. If the amount of coloring dye is increased to solve this problem, the dye covers the surface of the latex, damaging the original surface state of the latex particles, making it difficult to bind antigens or antibodies. In addition, there is a problem that increasing the amount of coloring agent of the dye to color deeply does not necessarily lead to improved performance, as the pores of the chromatographic medium such as a membrane filter are clogged or the latex particles undergo nonspecific aggregation.

In order to improve the visibility of the labeled substance, an immunochromatographic method has been disclosed in which, after an antibody bound to the labeled substance (labeled antibody) reacts with an antigen to form a complex, the labeled substance is further modified with another metal to amplify the detection sensitivity of the labeled substance. Also, an example in which gold colloid and colored latex are used in combination has been disclosed (Patent Document 3).
However, these methods require complicated operations, and stable amplification is difficult to achieve. In addition, they require special equipment and are costly to measure, so their applicable uses and environments are likely to be limited.

Resin particles to which metal nanoparticles are bonded have been disclosed as a solution to these problems in the prior art.
For example, a colored latex consisting of gold nanoparticles bound to the surface of polymer-based latex particles has been disclosed (Patent Document 4).
By binding gold nanoparticles to the surface of polymer-based latex particles, the gold nanoparticles themselves act as a colorant, helping to improve visual judgment and detection sensitivity. In addition, since the gold nanoparticles themselves have excellent binding properties to antigens or antibodies, it is believed that even if gold nanoparticles are bound to a level that results in a sufficiently dark color, a sufficient amount of antigen or antibody can be bound.

The applicant has also discovered and previously proposed a resin-metal composite in which a specific proportion of metal particles are present on the surface layer of resin particles (for example, Patent Document 5). This resin-metal composite is excellent in durability and visibility, particularly when used as an immunological measurement material, and enables highly sensitive determination without requiring any special equipment or additional work processes.

Japanese Patent Application Publication No. 5-10950 Japanese Patent Application Publication No. 3-206959 JP 2011-117906 A JP 2009-168495 A International Publication WO2016/002742

In conclusion, resin particles to which metal nanoparticles are bonded have excellent visibility and do not require any special equipment or additional work processes, and therefore are promising reagents for immunological assays.
However, in order to detect antigens of viruses and the like that are present in extremely small amounts in samples such as blood but that can have a significant impact on the health of patients who possess that blood, a different technical approach is required in addition to improving the visibility of the labeling substance alone.

In other words, the object of the invention is to provide nanocomposite particles that not only have excellent visibility in immunoassays, but also enable detection by magnetic concentration of analytes such as antigens and antibodies that are normally present in extremely small amounts below the detection limit in samples such as blood, urine, saliva, and environmental water, and to provide nanocomposite particles for immunoassays that enable highly sensitive determinations, for example, in immunoassays.

As a result of intensive research, the inventors discovered that the above problems could be solved by nanocomposite particles in which metal nanoparticles and magnetic nanoparticles are fixed to resin nanoparticles, and thus completed the present invention.

That is, the nanocomposite particles of the present invention are
Resin nanoparticles;
A plurality of metal nanoparticles having an average particle size of 1 to 100 nm, the particle size of which is relatively smaller than that of the resin nanoparticles;
A plurality of magnetic nanoparticles having an average particle size of 1 to 50 nm, the particle size of which is relatively smaller than that of the resin nanoparticles;
Equipped with
The average particle size is 50 to 1100 nm,
The metal nanoparticles and the magnetic nanoparticles are fixed to the resin nanoparticles.

The nanocomposite particles of the present invention may be particles of gold, silver, copper, palladium, platinum, tin, rhodium, iridium, or alloys thereof.

In the nanocomposite particles of the present invention, the magnetic nanoparticles may be iron, cobalt, _nickel , manganese, _Fe2O3 , _Fe3O4 , _AFe2O4 (where A means Mn, _Co , Ni, Cu or Zn), FePt, CoPt, FeNi, or _FeCo .

The nanocomposite particles of the present invention may be polymer particles in which the resin nanoparticles have a substituent in their structure capable of adsorbing metal ions.

In the nanocomposite particles of the present invention, at least some of the metal nanoparticles may be three-dimensionally distributed in the surface layer of the resin nanoparticles. In addition, 60 wt % to 100 wt % of the metal nanoparticles may be present in the surface layer. In addition, the magnetic nanoparticles may be fixed to the surface of the resin nanoparticles and/or the metal nanoparticles.

The labeling substance of the present invention is characterized by comprising any one of the nanocomposite particles described above. The nanocomposite particles may also be used by adsorbing an antigen or an antibody to the surface thereof.

The immunoassay method and the immunoassay reagent of the present invention are characterized by using the above-mentioned labeling substance.

The method for measuring an analyte of the present invention is a method for measuring an analyte, which detects or quantifies an analyte contained in a sample, comprising the steps of:
A lateral flow type chromatography test strip including a membrane and a determination part having a capture ligand that specifically binds to the analyte immobilized on the membrane is used, the following steps (I) to (IV):
Step (I): A step of contacting the analyte contained in a sample with a labeled antibody obtained by labeling an antibody that specifically binds to the analyte with any one of the nanocomposite particles described above to obtain a complex containing the analyte and the labeled antibody;
Step (II): recovering the complex containing the analyte and the labeled antibody by magnetic force;
Step (III): contacting the complex containing the analyte and the labeled antibody recovered in the step (II) with a capture ligand in the determination portion of the lateral flow chromatography test strip;
Step (IV): measuring the localized surface plasmon resonance of the nanocomposite particles and the color intensity resulting from light energy absorption due to electronic transition;
The method is characterized in that the method comprises the steps of:

The analyte measurement kit of the present invention is an analyte measurement kit for detecting or quantifying an analyte contained in a sample using a lateral flow type chromatography test strip,
A lateral flow type chromatography test strip including a membrane and a determination part in which a capture ligand that specifically binds to the analyte is immobilized on the membrane;
and a detection reagent comprising an antibody that specifically binds to the analyte and is labeled with any of the nanocomposite particles described above.

The nanocomposite particles of the present invention have metal nanoparticles and magnetic nanoparticles immobilized on the resin nanoparticles. In addition to the excellent visibility and the lack of need for additional special equipment or work processes, which are characteristics of resin particles bound to metal nanoparticles as exemplified in Patent Documents 4 and 5, the nanocomposite particles can detect antigens (antibodies) that are usually present in extremely small amounts in samples such as blood, urine, saliva, and environmental water, below the detection limit, by magnetically concentrating the antigen-antibody reaction. Therefore, the nanocomposite particles can be preferably used for purposes such as labeling substances and reagents for immunological assays that enable highly sensitive determinations, such as EIA, RIA, CLIA, FIA, LIA, PA, ICA, HA, and HI.

1 is a schematic diagram showing a cross-sectional structure of a nanocomposite particle according to one embodiment of the present invention. FIG. 1 is an explanatory diagram showing an overview of a method for measuring an analyte using a lateral flow chromatographic test strip according to one embodiment of the present invention. FIG. 1 is an explanatory diagram of a lateral flow type chromatography test strip used in the examples.

Hereinafter, the embodiment of the present invention will be described in detail with reference to the drawings as appropriate. FIG. 1 is a schematic cross-sectional view of a nanocomposite particle according to one embodiment of the present invention. The nanocomposite particle 100 includes a resin nanoparticle (hereinafter, also simply referred to as a "resin particle") 10, a metal nanoparticle 20, and a magnetic nanoparticle 70. In the nanocomposite particle 100, the metal nanoparticle 20 is fixed to the resin nanoparticle 10. The resin nanoparticle 10 is a particle relatively larger than the metal nanoparticle 20. That is, in the nanocomposite particle 100, a large number of metal nanoparticles 20 having a relatively small particle diameter are fixed to the resin nanoparticle 10 having a relatively large particle diameter. As shown in FIG. 1, the relationship between the particle diameter D1 of the entire nanocomposite particle 100, the particle diameter D2 of the resin nanoparticle 10, and the particle diameter D3 of the metal nanoparticle 20 is D1>D2>D3.
Further, a plurality of magnetic nanoparticles 70 are fixed to the nanocomposite particle 100. That is, in the nanocomposite particle 100, a large number of magnetic nanoparticles 70 are fixed to the resin nanoparticles 10 and/or the metal nanoparticles 20. As shown in Fig. 1, the relationship between the particle diameter D1 of the entire nanocomposite particle 100, the particle diameter D2 of the resin nanoparticles 10, and the particle diameter D4 of the magnetic nanoparticles 70 is D1>D2>D4.
In Fig. 1, the shape of the magnetic nanoparticles 70 is shown as a polygon and smaller than the metal nanoparticles 20, but this is for convenience of explanation only, and the shape and size are not limited. In Fig. 1, the magnetic nanoparticles 70 are bonded to the surfaces of the resin nanoparticles 10 and the metal nanoparticles 20, but this is merely an example, and the state of existence of the magnetic nanoparticles 70 is not limited, and they may be embedded inside the resin nanoparticles 10, for example.

The nanocomposite particles 100 have an average particle diameter of 50 to 1100 nm, the metal nanoparticles 20 constituting the nanocomposite particles 100 have an average particle diameter of 1 to 100 nm, and the magnetic nanoparticles 70 have an average particle diameter of 1 to 50 nm. When the average particle diameter of the nanocomposite particles 100 is less than 50 nm, the amount of the metal nanoparticles 20 carried tends to be smaller, so the coloring tends to be weaker than that of metal nanoparticles of the same size, and the magnetic concentration efficiency tends to decrease. When the average particle diameter of the nanocomposite particles 100 exceeds 1100 nm, when used as a labeling substance or reagent, the pores of a chromatographic medium such as a membrane filter tend to be clogged, and the dispersion stability tends to decrease. The average particle diameter of the nanocomposite particles 100 is preferably 100 nm, more preferably 250 nm, and even more preferably 300 nm, from the viewpoints of improving the dispersion stability when used as a labeling substance or reagent, obtaining high detection sensitivity when using the nanocomposite particles 100 in immunological measurements, and obtaining high magnetic concentration efficiency. On the other hand, the preferred upper limit is 700 nm, and more preferably 600 nm. Here, the particle diameter of the nanocomposite particle 100 means the particle diameter of the resin nanoparticle 10 plus the length of the protruding portion of the partially exposed particle 40 or the surface-adsorbed particle 50 described below, and can be measured by electron microscopy such as a scanning electron microscope (SEM) or a transmission electron microscope (TEM), a laser diffraction/scattering method, a dynamic light scattering method, or a centrifugal sedimentation method.

Furthermore, when the average particle diameter of the metal nanoparticles 20 (i.e., the average of the particle diameters D3 in FIG. 1) is less than 1 nm or exceeds 100 nm, localized surface plasmon resonance and light energy absorption due to electronic transition are less likely to occur, and thus the sensitivity tends to decrease.
When gold particles are used as the metal nanoparticles 20, the average particle size of the gold particles is preferably 1 nm or more preferably 3 nm in terms of obtaining high detection sensitivity as a labeling substance for immunoassay and a reagent for immunoassay, and is preferably 70 nm or more preferably 50 nm in terms of the upper limit.
When platinum particles are used as the metal nanoparticles 20, the average particle size of the platinum particles is preferably 1 nm, more preferably 2 nm, and even more preferably 3 nm from the viewpoint of obtaining high detection sensitivity as a labeling substance for immunoassay and a reagent for immunoassay, while the preferred upper limit is 50 nm, more preferably 20 nm, and even more preferably 15 nm.
Here, the average particle diameter of the metal nanoparticles 20 is measured by observation with a scanning electron microscope (SEM) or a transmission electron microscope (TEM).

In addition, when the average particle diameter of the magnetic nanoparticles 70 is less than 1 nm, the magnetic force exerted by the nanocomposite particles 100 is weak, so that the efficiency of magnetically recovering the analyte-labeled antibody complex after contacting the analyte and the labeled antibody (hereinafter referred to as "labeled antibody particle") labeled with the nanocomposite particles 100 in the specimen and forming a complex containing the analyte and the labeled antibody particle (hereinafter referred to as "analyte-labeled antibody complex"), that is, the efficiency of magnetic concentration, tends to decrease. On the other hand, when the average particle diameter exceeds 50 nm, the specific gravity of the nanocomposite particles 100 is large, so that the motility and diffusibility in the specimen are low, and the contact efficiency between the analyte and the labeled antibody particle in the specimen and the efficiency of magnetically recovering the analyte-labeled antibody complex tend to decrease. The preferred lower limit is 3 nm, more preferably 5 nm, and even more preferably 7 nm. On the other hand, the preferred upper limit is 30 nm, more preferably 20 nm, and even more preferably 15 nm.
Here, the average particle diameter of the magnetic nanoparticles 70 is measured by observation with a scanning electron microscope (SEM) or a transmission electron microscope (TEM).

The metal nanoparticles 20 do not include nanoparticles containing magnetic elements (Mn, Fe, Co, Ni) (however, it is acceptable for the particles to contain a small amount of magnetic elements as unavoidable impurities to the extent that they do not become magnetic). In other words, the metal nanoparticles 20 are nanoparticles of metals other than magnetic materials. In addition, from the viewpoint of ease of nano-size composite of the metal nanoparticles 20 and the resin nanoparticles 10, the material of the metal nanoparticles 20 is preferably gold, silver, copper, palladium, platinum, tin, rhodium, iridium, or alloys thereof. These metals can be used alone or in composites such as alloys. Here, for example, a gold alloy means an alloy consisting of gold and a metal species other than gold, containing 10% by weight or more, preferably 50% by weight or more, and more preferably 60% by weight or more of gold. In addition, for example, a platinum alloy means an alloy consisting of platinum and a metal species other than platinum, containing 1% by weight or more, preferably 10% by weight or more, more preferably 50% by weight or more, and more preferably 60% by weight or more of platinum. When used as a labeling substance for immunoassays and as a reagent for immunoassays, gold, platinum and palladium are more preferred because they have excellent visibility and are easy to immobilize antigens or antibodies on. These are preferred because they exhibit absorption derived from localized surface plasmon resonance. Gold and platinum are even more preferred.

For example, a gold-resin complex using gold particles as the metal nanoparticles 20 is less likely to aggregate when bound to a ligand such as an antibody, and has extremely excellent dispersibility, compared to a metal-resin complex having particles of other metal species. Furthermore, it has excellent visibility and allows easy immobilization of antigens or antibodies. In particular, by controlling the particle diameter of the gold particles and the interparticle distance between the gold particles, various colors such as red, purple, and blue can be produced. Therefore, when the gold-resin complex is used as a labeling substance for an immunochromatograph, a labeling substance of various colors can be obtained.

For example, a platinum-resin composite using platinum particles as the metal nanoparticles 20 is less likely to aggregate when bound to a ligand such as an antibody, and has extremely excellent dispersibility, compared to a metal-resin composite having particles of other metal species. In addition, platinum particles are resistant to deterioration such as oxidation, and have excellent storage stability. Furthermore, platinum particles exhibit absorption due to localized surface plasmon resonance in a wide range of wavelengths, for example, from 250 nm to 900 nm, and further exhibit a strong color close to black due to the expression of light energy absorption by electronic transition, so by using a platinum-resin composite as a labeling substance, high visibility can be obtained in immunological measurements, and the detection sensitivity of analytes can also be increased. In this case, by using platinum particles, excellent detection sensitivity can be obtained with a smaller loading amount compared to particles of other metal species. Therefore, if the average particle diameter is the same, the platinum-resin composite is preferable because it exhibits significantly higher detection sensitivity than resin composites having particles of other metal species.

The metal nanoparticles 20 may consist of only a single metal species, or may be an alloy of that metal species with another metal species. Here, in the case of metal nanoparticles 20 consisting of only a single metal species, inevitable impurities other than the metal species may be included. In the case of an alloy with another metal, the other metal species is not particularly limited, but for example, in the case of an alloy with gold, silver, copper, palladium, platinum, tin, rhodium, and iridium are preferred. In the case of an alloy with platinum, gold, silver, copper, palladium, tin, rhodium, and iridium are preferred.

In addition, from the viewpoints of ease of nano-composite formation of the magnetic nanoparticles 70 and the resin nanoparticles 10 at nano size and strength of magnetic force as the nanocomposite particles 100, the magnetic nanoparticles 70 are preferably iron, cobalt, nickel, manganese, _Fe2O3 , _Fe3O4 , _AFe2O4 (wherein A means Mn, _Co , Ni, Cu _or Zn), FePt, CoPt, FeNi or FeCo. More preferably, the magnetic _{nanoparticles 70} are _Fe3O4 or FeCo, which are soft magnetic materials with high saturation _magnetic flux density, and even more preferably _Fe3O4 .
As the magnetic nanoparticles 70, for example, commercially available products such as iron oxide magnetic nanoparticles (Sigma-Aldrich Japan LLC) can be preferably used.

In addition, the resin nanoparticles 10 are preferably polymer particles having a structure with a substituent capable of adsorbing metal ions, although the structure and composition are not limited. For example, a polymer capable of adsorbing anionic ions can be mentioned. As a polymer capable of adsorbing anionic ions, a nitrogen-containing polymer is particularly preferable. Nitrogen atoms in a nitrogen-containing polymer are preferable because they are easy to chemically adsorb anionic ions such as [AuCl ₄ ] ⁻ and [PtCl ₆ ] ^2−, which are precursors of metal nanoparticles 20 and have excellent visibility and are easy to immobilize antigens or antibodies. In this embodiment, the metal ions adsorbed in the nitrogen-containing polymer are reduced to form the metal nanoparticles 20, and a part of the generated metal nanoparticles 20 becomes the encapsulated particles 30 or the partially exposed particles 40 described later. When [AuCl ₄ ] ⁻ is used, gold particles are formed, and when [PtCl ₆ ] ²⁻ is used, platinum particles are formed. In addition, anionic ions such as silver, copper, palladium, tin, rhodium, and iridium can also be used.
In addition, carboxylic acid group-containing polymers such as acrylic acid polymers and sulfonic acid group-containing polymers such as polystyrene sulfonic acid (hereinafter collectively referred to as "polymers capable of adsorbing cationic ions") are preferred because they can chemically adsorb cationic ions such as Au ⁺ and Pt ²⁺ due to the carboxylic acid groups and sulfonic acid groups they contain. For example, it is possible to produce a structure similar to that of the nitrogen-containing polymer particles by reducing the chemically adsorbed Au ⁺ and Pt ²⁺ to form metal nanoparticles 20 (in this case, gold particles or platinum particles). In addition, cationic ions that are precursors of metals such as silver, copper, palladium, tin, rhodium, and iridium can be used.
On the other hand, for example, polystyrene can be used as the resin nanoparticles 10 other than the nitrogen-containing polymer having a substituent capable of adsorbing metal ions in its structure. However, in this case, it is relatively difficult to adsorb the metal ions inside the resin. As a result, most of the generated metal nanoparticles 20 become surface-adsorbed particles 50. Since the surface-adsorbed particles 50 have a small contact area with the resin nanoparticles 10, the adhesive force between the resin and the metal is small, and the metal nanoparticles 20 tend to be easily detached from the resin nanoparticles 10. The nitrogen-containing polymer is a resin having nitrogen atoms in the main chain or side chain, such as polyamine, polyamide, polypeptide, polyurethane, polyurea, polyimide, polyimidazole, polyoxazole, polypyrrole, polyaniline, melamine resin, etc. Preferably, it is a polyamine such as poly-2-vinylpyridine, poly-3-vinylpyridine, poly-4-vinylpyridine. In addition, when the side chain has a nitrogen atom, for example, acrylic resin, phenolic resin, epoxy resin, etc. can be widely used. The polymer capable of adsorbing the cationic ions is a resin having a carboxylic acid group, a sulfonic acid group, or the like in the main chain or side chain, and a wide variety of polymers, such as polyacrylic acid, vinyl carboxylate, polyvinyl acetate, polyvinyl sulfonic acid, polystyrene sulfonic acid, and cellulose, can be used.
The polymer capable of adsorbing anionic ions such as the nitrogen-containing polymer and the polymer capable of adsorbing cationic ions may be a copolymer with a known polymerizable monomer. Here, examples of the copolymer include random copolymers, block copolymers, alternating copolymers, and copolymers crosslinked with each other. In addition, two or more types of monomers may be copolymerized to form the resin nanoparticles 10, or a monomer may be reacted with a functional group present on the surface of the resin nanoparticles 10, and the monomer may be further polymerized as an active terminal for polymerization. The copolymerization composition is not limited, but it is preferable that the monomer containing a substituent capable of adsorbing the metal ion is 10 mol% or more. More preferably, it is 50 mol% or more.
The polymerizable monomer can be selected without limitation according to the application of the nanocomposite particle 100. For example, in the application of improving the shape, size uniformity and dispersion stability of the resin nanoparticles 10, a polymerizable monomer having properties as a surfactant can be used. Examples of such polymerizable monomers include polyethylene glycol methyl ether methacrylate and polyethylene glycol dimethacrylate. In addition, in the application of improving the mechanical strength and shape stability of the resin nanoparticles 10, a hydrophobic polymerizable monomer such as divinylbenzene or styrene can be used.
In addition, when the resin nanoparticles 10 have a hydrolyzable group such as an ester bond, they may be partially hydrolyzed by acid treatment or alkali treatment. Hydrolysis generates groups capable of adsorbing cationic ions such as carboxyl groups on the surface of the resin nanoparticles 10, which is preferable because it has the effect of adsorbing metal ions. As the acid, a known acid can be used. From the viewpoint of promoting the hydrolysis reaction, strong acidic hydrochloric acid, sulfuric acid, etc. are preferred. As the alkali, a known alkali can be used. From the viewpoint of promoting the hydrolysis reaction, strong alkaline potassium hydroxide, sodium hydroxide aqueous solution, etc. are preferred.

In addition, the nanocomposite particle 100 does not limit the dispersion state of the metal nanoparticles 20 in the resin nanoparticles 10. For example, the metal nanoparticles 20 may be two-dimensionally distributed on the surface of the resin nanoparticles 10, or the metal nanoparticles 20 may be encapsulated inside the resin nanoparticles 10. As one form of the former, a plurality of metal nanoparticles 20 may be in contact with each other on the surface of the resin nanoparticles 10 to form a continuous film. As one form of the latter, a core-shell structure may be formed in which the resin nanoparticles 10 are the shell and the metal nanoparticles 20 are the core. In addition, a portion of the metal nanoparticles 20 may be three-dimensionally distributed in the surface layer portion 60 of the resin nanoparticles 10. In this case, a portion of the three-dimensionally distributed metal nanoparticles 20 may be partially exposed to the outside of the resin nanoparticles 10, and the remaining portion may be encapsulated in the resin nanoparticles 10. Specifically, as shown in FIG. 1, metal nanoparticles 20 may include metal nanoparticles that are completely encapsulated in resin nanoparticles 10 (hereinafter also referred to as "encapsulated particles" 30), metal nanoparticles that have a portion embedded in the resin nanoparticles 10 and a portion exposed to the outside of the resin nanoparticles 10 (hereinafter also referred to as "partially exposed particles" 40), and metal nanoparticles that are adsorbed to the surface of the resin nanoparticles 10 (hereinafter also referred to as "surface-adsorbed particles" 50).
For example, when the nanocomposite particle 100 is used as a labeling substance for immunoassay or a reagent for immunoassay, an antigen, an antibody, or a blocking agent is immobilized on the surface of the resin nanoparticle 10 or on the surface of the partially exposed particle 40 or the surface-adsorbed particle 50. In this case, the antigen, the antibody, or the blocking agent is immobilized on the partially exposed particle 40 and the surface-adsorbed particle 50, while it is considered that it is difficult to immobilize on the encapsulated particle 30. However, since all of the partially exposed particle 40, the surface-adsorbed particle 50, and the encapsulated particle 30 exhibit light energy absorption due to electronic transition in addition to localized surface plasmon resonance, not only the partially exposed particle 40 and the surface-adsorbed particle 50, but also the encapsulated particle 30 contributes to improving the visibility of the labeling substance for immunoassay and the reagent for immunoassay. Furthermore, the partially exposed particle 40 and the encapsulated particle 30 have a larger contact area with the resin nanoparticle 10 than the surface-adsorbed particle 50, and in addition, an anchor effect due to the embedded state is exerted, so that the physical adsorption force is strong and it is difficult to detach from the resin nanoparticle 10. In addition, the antigen, antibody, or blocking agent adsorbed to the partially exposed particles 40 or the surface-adsorbed particles 50 is unlikely to be detached because it is coordinate-bonded with the metal, and therefore the durability and stability of the immunoassay labeling substance and the immunoassay reagent using the nanocomposite particles 100 can be improved.

In the nanocomposite particle 100, the encapsulated particle 30 has its entire surface covered by the resin that constitutes the resin nanoparticle 10. The partially exposed particle 40 has 5% or more and less than 100% of its surface area covered by the resin that constitutes the resin nanoparticle 10. From the viewpoint of durability of the immunoassay labeling substance and the immunoassay reagent, the lower limit is preferably 20% or more of the surface area, and more preferably 30% or more. The surface adsorbed particle 50 has more than 0% and less than 5% of its surface area covered by the resin that constitutes the resin nanoparticle 10.

The amount of metal nanoparticles 20 (the total of the encapsulated particles 30, the partially exposed particles 40, and the surface-adsorbed particles 50) carried by the nanocomposite particles 100 is preferably 3 to 80 wt% relative to the nanocomposite particles 100. In this range, the nanocomposite particles 100 have excellent visibility, visual judgment, and detection sensitivity as a labeling substance. If the amount of metal nanoparticles 20 carried is less than 3 wt%, the amount of immobilized antibody or antigen tends to decrease, and the detection sensitivity tends to decrease. On the other hand, if the amount of metal nanoparticles 20 carried exceeds 80 wt%, the particle diameter of the metal nanoparticles 20 tends to increase significantly, and the light absorption characteristics of the metal nanoparticles 20 tend to decrease. The lower limit of the amount of metal nanoparticles 20 carried is more preferably 10 wt%, and even more preferably 15 wt%. On the other hand, the upper limit is more preferably 70 wt%, and even more preferably 60 wt%.

The amount of magnetic nanoparticles 70 supported on the nanocomposite particles 100 is preferably 1 to 50 wt% relative to the nanocomposite particles 100. In this range, the nanocomposite particles 100 have an excellent balance between the strength of the magnetic force and the motility and diffusibility in the specimen. In other words, if the amount of magnetic nanoparticles 70 supported is less than 1 wt%, the magnetic force of the nanocomposite particles 100 is weak, so the efficiency of magnetically recovering the analyte-labeled antibody complex after the formation of the analyte-labeled antibody complex, that is, the efficiency of magnetic concentration, tends to decrease. On the other hand, if the amount exceeds 50 wt%, the specific gravity of the nanocomposite particles 100 is large, so the motility and diffusibility in the specimen are low, and the contact efficiency between the analyte and the labeled antibody particles in the specimen and the efficiency of magnetically recovering the analyte-labeled antibody complex tend to decrease. In addition, for example, in the measurement of the analyte performed using a test strip, the development speed of the analyte-labeled antibody complex tends to decrease, and the measurement efficiency tends to decrease. The more preferred lower limit of the amount of metal nanoparticles 20 supported is 5 wt%, and even more preferably 10 wt%. On the other hand, the more preferred upper limit is 40 wt%, and even more preferably 30 wt%.

It is also preferable that 10 to 90 wt % of the metal nanoparticles 20 are partially exposed particles 40 and surface-adsorbed particles 50. Within this range, a sufficient amount of antibody or antigen can be immobilized on the metal nanoparticles 20, and the sensitivity as a labeling substance is high. A more preferable lower limit of the proportion of partially exposed particles 40 and surface-adsorbed particles 50 is 20 wt %, and a more preferable upper limit is 80 wt %.

In addition, from the viewpoint of durability of the immunoassay labeling substance and the immunoassay reagent, it is preferable that the surface adsorption particles 50 be 20 wt % or less.

Furthermore, in order to obtain excellent detection sensitivity when using the nanocomposite particle 100 in an immunological measurement, it is preferable that 60 to 100 wt % of the metal nanoparticles 20 are present in the surface layer portion 60. A more preferable lower limit is 75 wt %, and even more preferable is 85 wt %.
In addition, it is preferable that 5 to 90 wt % of the metal nanoparticles 20 present in the surface layer 60 are partially exposed particles 40 or surface-adsorbed particles 50, since this ensures a sufficient amount of antibody or antigen immobilized on the metal nanoparticles 20, thereby increasing the sensitivity as a labeling substance. In other words, it is preferable that 10 to 95 wt % of the metal nanoparticles 20 present in the surface layer 60 are encapsulated particles 30.

Here, the "surface layer" refers to a range of 50% of the particle radius from the surface of the resin nanoparticle 10 in the depth direction, more preferably 40%, based on the outermost position of the nanocomposite particle 100 (i.e., the protruding end of the partially exposed particle 40 or the surface-adsorbed particle 50). The "three-dimensional distribution" means that the metal nanoparticles 20 are dispersed not only in the plane direction of the resin nanoparticle 10 but also in the depth direction. As described above, the encapsulated particles 30 also exhibit light energy absorption due to electronic transition in addition to localized surface plasmon resonance, so that not only the partially exposed particle 40 and the surface-adsorbed particle 50 but also the encapsulated particles 30 contribute to improving the visibility of the immunoassay labeling substance and the immunoassay reagent. From the viewpoint of such improved visibility, it is preferable that the encapsulated particles 30 of the nanocomposite particle 100 are distributed in a certain range in the depth direction from the surface of the resin nanoparticle 10, and that the encapsulated particles 30 are not substantially present near the center of the resin nanoparticle 10. More specifically, in order to effectively express the light energy absorption by electronic transition in addition to the localized surface plasmon resonance by the encapsulated particles 30, for example, when the particle diameter D2 of the resin nanoparticles 10 is 800 nm, 70 wt % or more, preferably 80 wt % or more, more preferably 90 to 100 wt % of the encapsulated particles 30 are present within a range of, for example, 0 to 200 nm from the surface of the resin nanoparticles 10 in the depth direction. In particular, when the region (encapsulated particle distribution region) in which all of the encapsulated particles 30 (100 wt %) are distributed is within a range of, for example, 0 to 100 nm from the surface of the resin nanoparticles 10, this is preferable because it is possible to maximize the expression of the light energy absorption by electronic transition in addition to the localized surface plasmon resonance by the encapsulated particles 30. In addition, the nanocomposite particle 100 may not have the encapsulated particles 30. For example, in the nanocomposite particle 100, all of the metal nanoparticles 20 may be fixed to the surface of the resin nanoparticles 10 without overlapping in the radial direction of the resin nanoparticles 10. In this case, the metal nanoparticles 20 are composed of partially exposed particles 40 and surface-adsorbed particles 50.

Furthermore, the nanocomposite particle 100 does not limit the dispersion state of the magnetic nanoparticles 70 in the resin nanoparticles 10. For example, the magnetic nanoparticles 70 may be distributed two-dimensionally or three-dimensionally on the surface of the resin nanoparticles 10, or the magnetic nanoparticles 70 may be encapsulated inside the resin nanoparticles 10.
As one form of the former, a plurality of magnetic nanoparticles 70 may be in contact with each other and form a continuous film on the surface of the resin nanoparticle 10 .
As one form of the latter, a core-shell structure may be formed in which the resin nanoparticles 10 serve as the shell and the magnetic nanoparticles 70 serve as the core. Also, some of the magnetic nanoparticles 70 may be three-dimensionally distributed inside the resin nanoparticles 10. In this case, some of the three-dimensionally distributed magnetic nanoparticles 70 may be partially exposed to the outside of the resin nanoparticles 10, and the remaining part may be encapsulated in the resin nanoparticles 10.
More preferably, from the viewpoint of storage stability, the magnetic nanoparticles 70 are fixed to the surface of a metal-resin composite consisting of the resin nanoparticles 10 and the metal nanoparticles 20 (i.e., the nanocomposite particles 100 excluding the magnetic nanoparticles 70).
In addition, when the magnetic nanoparticles 70 are fixed on the surface of the resin nanoparticles 10, the contact state between the magnetic nanoparticles 70 and the resin nanoparticles 10 is not particularly limited, and may be physical adsorption due to electrostatic interaction or chemical adsorption such as hydrogen bond, coordinate bond, covalent bond, etc. From the viewpoint of bond stability, it is preferable that the resin nanoparticles 10 or metal nanoparticles 20 and the magnetic nanoparticles 70 are in contact with each other via a chemical bond with a known linker.
The linker may be any known substance or bond, for example, an amide bond, an ether bond, or a saturated or unsaturated carbon-carbon bond.

[Method of manufacturing nanocomposite particles]
The method for producing the nanocomposite particles 100 is not particularly limited, but below will be described as a preferred method in which a metal-resin composite consisting of resin nanoparticles 10 and metal nanoparticles 20 is first produced, and then magnetic nanoparticles 70 are fixed to the metal-resin composite.

The metal-resin composite can be obtained, for example, by adding a solution containing metal ions to a dispersion of resin nanoparticles 10 produced by emulsion polymerization, so that the metal ions are adsorbed onto the resin nanoparticles 10 (hereinafter referred to as "metal ion-adsorbed resin particles"). Furthermore, by adding the metal ion-adsorbed resin particles to a reducing agent solution, the metal ions are reduced to generate metal nanoparticles 20, and a metal-resin composite can be obtained.

For example, when gold particles are generated as the metal nanoparticles 20, an example of a solution containing gold ions is an aqueous solution of chloroauric acid (HAuCl ₄ ). Also, a gold complex may be used instead of the gold ions.
Furthermore, when platinum particles are generated as the metal nanoparticles 20, examples of the solution containing platinum ions include an aqueous solution of chloroplatinic acid (H ₂ PtCl ₆ ), a platinum chloride (PtCl ₂ ) solution, etc. Also, a platinum complex may be used instead of platinum ions.

In addition, instead of water, a water-containing alcohol or alcohol such as methanol, ethanol, n-propanol, isopropanol, n-butanol, sec-butanol, or t-butanol, or an acid such as hydrochloric acid, sulfuric acid, or nitric acid may be used as a solvent for the solution containing metal ions.
Furthermore, additives such as various water-miscible organic solvents, for example, water-soluble polymer compounds such as polyvinyl alcohol, surfactants, alcohols; ethers such as tetrahydrofuran, diethyl ether, diisopropyl ether, etc.; polyols such as alkylene glycol, polyalkylene glycol, monoalkyl ethers or dialkyl ethers thereof, glycerin, etc.; and ketones such as acetone, methyl ethyl ketone, etc., may be added to the solution as necessary.
Such additives are effective in accelerating the reduction reaction rate of the metal ions and in controlling the size of the metal nanoparticles 20 that are produced.

The reducing agent may be any known agent. Examples of the reducing agent include sodium borohydride, dimethylamine borane, citric acid, sodium hypophosphite, hydrazine hydrate, hydrazine hydrochloride, hydrazine sulfate, formaldehyde, sucrose, glucose, ascorbic acid, erythorbic acid, sodium phosphinate, hydroquinone, Rochelle salt, etc. Among these, sodium borohydride, dimethylamine borane, and citric acid are preferred.
If necessary, a surfactant may be added to the reducing agent solution, or the pH of the solution may be adjusted using, for example, a buffer such as boric acid or phosphoric acid, an acid such as hydrochloric acid or sulfuric acid, or an alkali such as sodium hydroxide or potassium hydroxide.
Furthermore, the particle size of the generated metal nanoparticles 20 can be controlled by adjusting the reduction rate of the metal ions through the temperature of the reducing agent solution.

When reducing the metal ions in the metal ion-adsorbing resin particles to generate metal nanoparticles 20, the metal ion-adsorbing resin particles may be added to a reducing agent solution, or a reducing agent may be added to the metal ion-adsorbing resin particles. From the viewpoint of ease of generating the encapsulated particles 30 and the partially exposed particles 40, the former is preferred.

In order to maintain the dispersibility of the metal-resin composite in water, a dispersant such as citric acid, poly-L-lysine, polyvinylpyrrolidone, polyvinylpyridine, polyvinyl alcohol, DISPERBYK194, DISPERBYK180, or DISPERBYK184 (manufactured by BIGCEMIA JAPAN KK) may be added.
Furthermore, the pH can be adjusted with a buffer such as boric acid or phosphoric acid, an acid such as hydrochloric acid or sulfuric acid, or an alkali such as sodium hydroxide or potassium hydroxide to maintain dispersibility.

Next, the nanocomposite particles 100 are produced by fixing the magnetic nanoparticles 70 to the produced metal-resin composite. As described above, there are no particular limitations on the state of contact between the magnetic nanoparticles 70 and the resin nanoparticles 10. Below, we will explain an example of a method in which the resin nanoparticles 10 or metal nanoparticles 20 and the magnetic nanoparticles 70 are brought into contact with each other via chemical bonding with a known linker.

When an amide bond is used as the linker, an amino group is added to one of the metal-resin composite or the magnetic nanoparticle 70, and a carboxyl group is added to the other, and they are reacted to form an amide bond.
For example, a metal-resin composite is coated with a cationic polymer such as Poly-L-lysine, Poly-D-lysine, polyallylamine, or polyethyleneimine to synthesize a metal-resin composite having an amino group on the surface (hereinafter referred to as "amino group-containing composite particles"). On the other hand, a magnetic nanoparticle 70 is capped with a hydroxy acid such as citric acid, tartaric acid, or ascorbic acid to synthesize a magnetic nanoparticle 70 having a carboxyl group on the surface (hereinafter referred to as "carboxyl group-containing magnetic particles"). Then, the amino group-containing composite particles and the carboxyl group-containing magnetic particles are reacted to form an amide bond. Here, a known method can be used for the formation of the amide bond. For example, 1-ethyl-3-(-3-dimethylaminopropyl)carbodiimide hydrochloride (EDC) or N',N'-dicyclohexylcarbodiimide may be used as a crosslinking agent.
The magnetic nanoparticles 70 may be treated with a known amino group-containing silane coupling agent such as aminoalkyltrialkoxysilane to be used as magnetic nanoparticles 70 whose surfaces are modified with amino groups. The magnetic nanoparticles 70 whose surfaces are modified with amino groups may be treated with a known acid anhydride to be used as carboxyl group-containing magnetic particles.

The nanocomposite particle 100 having the above configuration can be preferably used as a material for immunoassay labeling substances or immunoassay reagents that are excellent in visual judgment, particularly in the low concentration range (high sensitivity range), by adsorbing an antigen or antibody to the surface of the metal nanoparticle 20. Furthermore, since the magnetic nanoparticles 70 are fixed to the resin nanoparticles 10 and/or metal nanoparticles 20, it is possible to detect antigens (antibodies) that are present in extremely small amounts in samples such as blood, urine, saliva, and environmental water, which are usually below the detection limit, by magnetically concentrating them after causing an antigen-antibody reaction. Therefore, it can be preferably used for purposes such as immunoassay labeling substances and immunoassay reagents that enable highly sensitive judgment, such as EIA, RIA, CLIA, FIA, LIA, PA, ICA, HA, and HI. In addition, there is no particular limitation on the form of the immunoassay labeling substance or immunoassay reagent, but it can be used, for example, as a dispersion liquid in which the nanocomposite particle 100 is dispersed in water or a buffer solution with an adjusted pH.

There is no particular limitation on the method for adsorbing the antigen or antibody onto the surface of the metal nanoparticles 20, and known physical adsorption and chemical adsorption methods can be used. Chemical adsorption methods are preferred because they strengthen the bond between the metal nanoparticles 20 and the antigen or antibody. Physical adsorption and chemical adsorption may be used in combination.
Examples of physical adsorption include a method of immersing the nanocomposite particle 100 in a buffer solution containing an antigen or antibody and incubating it, and a method of immersing the nanocomposite particle 100 in a buffer solution and then adding an antigen or antibody.
Examples of chemical adsorption include a method of introducing an SH group into an antigen or an antibody and reacting it with the nanocomposite particle 100 to form a metal-SH bond, and a method of introducing a carboxyl group onto the surface of the nanocomposite particle 100, then succinimidylating the antigen or antibody to form a chemical bond. Compounds that introduce a carboxyl group onto the surface of the nanocomposite particle 100 are suitable, for example, compounds having both a functional group that has a coordinate bond with a metal, such as an amino group, an SH group, a carbonyl, an amide, or an imide, and a carboxyl group. Because the coordinate bond with the metal is strong, an SH group is preferred as the functional group, and examples of compounds having an SH group and a carboxyl group at both ends include mercaptopropionic acid, mercaptoundecanoic acid, and mercaptolauric acid. In addition, polymer compounds having a plurality of functional groups that have a coordinate bond with a metal and a carboxyl group in one molecule are likely to obtain a stable bond with the nanocomposite particle 100. For example, polyglutamic acid and polyaspartic acid, which are polypeptides of glutamic acid and aspartic acid, are more preferable as polymer compounds for introducing carboxyl groups onto the surface of the nanocomposite particle 100 .

Next, we will explain a method for measuring an analyte using the nanocomposite particle 100 as a labeling substance, a test strip for lateral flow chromatography, and an analyte detection and quantification kit.

[Lateral flow type chromatography test strip]
First, a lateral flow type chromatographic test strip (hereinafter, simply referred to as a "test strip") according to one embodiment of the present invention will be described with reference to Fig. 2. This test strip 200 can be preferably used in the analyte measurement method according to one embodiment of the present invention, as described below.

The test strip 200 includes a membrane 110. The membrane 110 includes a sample application section 120, a determination section 130, and a liquid absorption section 140, which are arranged in this order in the direction of sample development.

<Membrane>
As the membrane 110 used in the test strip 200, materials used as membrane materials in general test strips can be applied. The membrane 110 is formed of an inactive material (a material that does not react with the analyte 160, various ligands, etc.) made of a microporous material that exhibits capillary action and develops the sample as soon as the sample is added. Specific examples of the membrane 110 include fibrous or non-woven fibrous matrices, membranes, filter paper, glass fiber filter paper, cloth, cotton, etc. made of polyurethane, polyester, polyethylene, polyvinyl chloride, polyvinylidene fluoride, nylon, cellulose derivatives, etc. Among these, membranes, filter paper, glass fiber filter paper, etc. made of cellulose derivatives or nylon are preferably used, and more preferably, nitrocellulose membranes, mixed nitrocellulose ester (a mixture of nitrocellulose and cellulose acetate) membranes, nylon membranes, and filter paper are used.

To facilitate easier operation, the test strip 200 preferably includes a support for supporting the membrane 110. The support may be made of, for example, plastic.

<Sample Addition Section>
The test strip 200 may have a sample addition section 120 for adding a sample containing an analyte 160. The sample addition section 120 is a portion for receiving a sample containing an analyte 160 in the test strip 200. The sample addition section 120 may be formed on the membrane 110 upstream of the determination section 130 in the direction in which the sample develops, or the sample addition section 120 may be formed by providing a sample addition pad made of a material such as cellulose filter paper, glass fiber, polyurethane, polyacetate, cellulose acetate, nylon, or cotton cloth on the membrane 110.

<Judgment section>
A capture ligand 131 that specifically binds to the analyte 160 is fixed to the determination section 130. Any capture ligand 131 can be used without particular limitations as long as it forms a specific bond with the analyte 160, and for example, an antibody against the analyte 160 can be preferably used. The capture ligand 131 is immobilized so as not to move from the determination section 130 even when a sample is provided to the test strip 200. The capture ligand 131 may be directly or indirectly fixed to the membrane 110 by physical or chemical binding, adsorption, or the like.

The determination unit 130 is not particularly limited as long as it is configured so that the complex 170 containing the labeled antibody 150 and the analyte 160 comes into contact with the capture ligand 131 that specifically binds to the analyte 160. For example, the capture ligand 131 may be fixed directly to the membrane 110, or the capture ligand 131 may be fixed to a pad made of cellulose filter paper, glass fiber, nonwoven fabric, or the like that is fixed to the membrane 110.

<Liquid absorption part>
The liquid-absorbing part 140 is formed by a pad of a water-absorbent material such as cellulose filter paper, nonwoven fabric, cloth, cellulose acetate, etc. The movement speed of the sample after the development front of the added sample reaches the liquid-absorbing part 140 varies depending on the material and size of the liquid-absorbing part 140. Therefore, the optimum speed for detection and quantification of the analyte 160 can be set by selecting the material, size, etc. of the liquid-absorbing part 140. The liquid-absorbing part 140 is of an optional configuration and may be omitted.

The test strip 200 may further include optional components such as a control section, if necessary.

<Control section>
Although not shown, the test strip 200 may have a control section formed on the membrane 110 downstream of the determination section 130 in the direction in which the sample develops, the control section having a capture ligand that specifically binds to the labeled antibody 150 fixed thereto. By measuring the color intensity in the control section as well as the determination section 130, it can be confirmed that the sample provided to the test strip 200 develops and reaches the reaction section and the determination section 130, and that the test has been performed normally. The control section can be produced in the same manner as the above-mentioned determination section 130, and can have the same configuration, except that a different type of capture ligand that specifically binds to the labeled antibody 150 is used instead of the capture ligand 131.

[Method of measuring analytes]
Next, a method for measuring the analyte 160 using the test strip 200 according to one embodiment of the present invention will be described.

The method for measuring analyte 160 according to the present embodiment is a method for measuring analyte 160 by detecting or quantifying analyte 160 contained in a sample. The method for measuring analyte 160 according to the present embodiment uses a test strip 200 including a membrane 110 and a determination part 130 having a capture ligand 131 that specifically binds to analyte 160 immobilized on the membrane 110. The method for measuring analyte 160 according to the present embodiment includes the following steps (I) to (IV);
Step (I): A step of contacting the analyte 160 contained in a sample with a labeled antibody 150 obtained by labeling an antibody that specifically binds to the analyte 160 with a nanocomposite particle 100;
Step (II): A step of recovering a complex 170 containing an analyte 160 and a labeled antibody 150 by magnetic force;
Step (III): A step of contacting the complex 170 containing the analyte 160 and the labeled antibody 150 recovered in step (II) with the capture ligand 131 in the determination portion 130 of the test strip 200;
Step (IV): A step of measuring the color intensity resulting from light energy absorption due to localized surface plasmon resonance and/or electronic transition of the nanocomposite particle 100;
may include.

Process (I):
Step (I) is a step of contacting the analyte 160 contained in the sample with the labeled antibody 150. As long as a complex 170 containing the analyte 160 and the labeled antibody 150 is formed, the manner of contact is not particularly limited. For example, the analyte 160 in the sample may be contacted with the labeled antibody 150 before the sample is provided to the test strip 200. As described above, the nanocomposite particle 100 of the present invention can detect antigens (antibodies) that are present in extremely small amounts in samples such as blood, urine, saliva, and environmental water, which are usually below the detection limit, by magnetically concentrating the antigens (antibodies) after causing an antigen-antibody reaction. Therefore, it is a preferred embodiment to contact the analyte 160 in the sample with the labeled antibody 150 in a sample having a low concentration of antigen (antibody). Of course, it goes without saying that the contact can be made in a sample having a medium to high concentration of antigen (antibody).

Process (II):
Step (II) is a step of recovering the complex 170 containing the analyte 160 and the labeled antibody 150 by magnetic force. For example, by applying magnetic force to a specimen containing the antigen (antibody) using a known magnet, the complex 170 containing the analyte 160 and the labeled antibody 150 can be locally concentrated in the specimen and recovered. Note that centrifugation may be used in combination during recovery.

Process (III):
In step (III), the complex 170 containing the analyte 160 and the labeled antibody 150 recovered in step (II) is provided to the sample application section 120 of the test strip 200 and developed on the test strip 200. In this manner, the complex 170 is brought into contact with the capture ligand 131 in the determination section 130 of the test strip 200. When the complex 170 is brought into contact with the capture ligand 131, the capture ligand 131 specifically binds to the analyte 160 of the complex 170. As a result, the complex 170 is captured in the determination section 130.

Note that since the capture ligand 131 does not specifically bind to the labeled antibody 150, when the labeled antibody 150 that is not bound to the analyte 160 reaches the determination section 130, the labeled antibody 150 that is not bound to the analyte 160 passes through the determination section 130. If the test strip 200 has a control section (not shown) to which another capture ligand that specifically binds to the labeled antibody 150 is fixed, the labeled antibody 150 that has passed through the determination section 130 continues to develop and binds to the other capture ligand in the control section. As a result, the labeled antibody 150 that has not formed a complex 170 with the analyte 160 is captured in the control section.

After step (III) and before step (IV) as necessary, a washing step may be performed to wash the test strip 200 with a buffer solution commonly used in biochemical tests, such as water, physiological saline, or phosphate buffer. The washing step can remove the labeled antibody 150 that has not been captured by the determination section 130 or the determination section 130 and the control section (the labeled antibody 150 that has not bound to the analyte 160 and has not formed a complex 170).

By carrying out the washing step, when measuring color development due to light energy absorption by localized surface plasmon resonance and/or electronic transition of the nanocomposite particle 100 in the determination section 130, or in the determination section 130 and the control section in step (IV), the background color development intensity can be reduced, the signal/background ratio can be increased, and the detection sensitivity and quantitation can be further improved.

Process (IV):
Step (IV) is a step of measuring the color intensity resulting from light energy absorption due to localized surface plasmon resonance and/or electronic transition of the nanocomposite particle 100. After carrying out the above step (III) or a washing step as necessary, the color intensity resulting from light energy absorption due to localized surface plasmon resonance and/or electronic transition of the nanocomposite particle 100 is measured in the test strip 200.

When a control section is formed in the test strip 200, the labeled antibody 150 is captured by another capture ligand in the control section in step (III) to form a complex. Therefore, in step (IV), coloring due to localized surface plasmon resonance and/or light energy absorption by electronic transition can be caused not only in the determination section 130 but also in the control section of the test strip 200. In this way, by measuring the coloring intensity in the control section as well as the determination section 130, it can be confirmed whether the sample provided to the test strip 200 has developed normally and reached the reaction section and the determination section 130.

Samples and Analytes
The sample in the analyte measurement method of this embodiment is not particularly limited as long as it contains a substance that can be an antigen such as a protein as the analyte 160. For example, a biological sample containing the target analyte 160 (i.e., whole blood, serum, plasma, urine, saliva, sputum, nasal or pharyngeal swab, cerebrospinal fluid, amniotic fluid, nipple secretion, tears, sweat, exudate from the skin, extracts from tissues, cells and feces, etc.) and food extracts can be mentioned. If necessary, in order to facilitate the specific binding reaction between the labeled antibody 150 and the capture ligand 131 and the analyte 160, the analyte 160 contained in the sample may be pretreated prior to the above step (I). Here, examples of the pretreatment include chemical treatment using various chemicals such as acids, bases, and surfactants, and physical treatment using heating, stirring, ultrasonic waves, etc. In particular, when the analyte 160 is a substance that is not usually exposed on the surface, such as influenza virus NP antigen, it is preferable to perform treatment using a surfactant or the like. As the surfactant used for this purpose, a non-ionic surfactant can be used in consideration of the binding reactivity between the capture ligand 131 and the analyte 160, such as a specific binding reaction, for example, an antigen-antibody reaction.

The sample may also be appropriately diluted with a solvent (such as water, saline, or a buffer solution) or a water-miscible organic solvent used in conventional immunological analysis methods.

The analyte 160 is not particularly limited and may be any known analyte, including those with strong anionic properties, those with strong cationic properties, and others. Examples of the analyte 160 include tumor markers, signal transduction substances, proteins such as hormones (including polypeptides, oligopeptides, etc.), nucleic acids (including single-stranded or double-stranded DNA, RNA, polynucleotides, oligonucleotides, PNA (peptide nucleic acid), etc.) or substances having nucleic acids, sugars (including oligosaccharides, polysaccharides, sugar chains, etc.) or substances having sugar chains, lipids, and other molecules, and are not particularly limited as long as they specifically bind to the labeled antibody 150 and the capture ligand 131. Examples of the analyte 160 include carcinoembryonic antigen (CEA), HER2 protein, prostate-specific antigen (PSA), CA19-9, α-fetoprotein (AF), and other molecules. P), immunosuppressant acidic protein (IAP), CA15-3, CA125, estrogen receptor, progesterone receptor, fecal occult blood, troponin I, troponin T, CK-MB, CRP, human chorionic gonadotropin (HCG), luteinizing hormone (LH), follicle-stimulating hormone (FSH), syphilis antibody, influenza virus human hemoglobin, chlamydia antigen, group A beta-hemolytic streptococcus antigen, HBs antibody, HBs antigen, rotavirus, adenovirus, albumin, glycated albumin, coronavirus (including SARS-CoV, MERS-CoV, SARS-CoV-2, their mutant strains, and their components), etc. Among these, antigens that are solubilized by nonionic surfactants are preferred, and antigens that form self-aggregates such as viral nucleoproteins are more preferred.

<Labeled antibody>
In step (I), the labeled antibody 150 is used to contact an analyte 160 contained in a sample to form a complex 170 containing the analyte 160 and the labeled antibody 150. The labeled antibody 150 is an antibody that specifically binds to the analyte 160, and is labeled with a nanocomposite particle 100 having a structure in which a plurality of metal nanoparticles 20 and magnetic nanoparticles 70 are immobilized on a resin nanoparticle 10. Here, "labeling" means that the nanocomposite particle 100 is directly or indirectly immobilized to the antibody by chemical or physical bonding, adsorption, or the like to such an extent that the nanocomposite particle 100 does not detach from the labeled antibody 150 in steps (I) to (IV). For example, the labeled antibody 150 may be one in which the nanocomposite particle 100 is directly bonded to the antibody, or one in which the nanocomposite particle 100 is bonded via an arbitrary linker molecule, or one in which each is immobilized to an insoluble particle.

In the present embodiment, the "antibody" is not particularly limited and may be any known antibody, including those with strong anionic properties, those with strong cationic properties, and others. For example, polyclonal antibodies, monoclonal antibodies, and antibodies obtained by genetic recombination, as well as antibody fragments that have the ability to bind to antigens [e.g., H chains, L chains, Fab, F(ab')2, etc.], may be used. The immunoglobulin may be any of IgG, IgM, IgA, IgE, and IgD. The animal species that produces the antibody may be humans or other animals (e.g., mice, rats, rabbits, goats, horses, etc.). Specific examples of antibodies include anti-PSA antibodies, anti-AFP antibodies, anti-CEA antibodies, anti-adenovirus antibodies, anti-influenza virus antibodies, anti-HCV antibodies, anti-coronavirus antibodies (here, coronaviruses include SARS-CoV, MERS-CoV, SARS-CoV-2, and their mutant strains), anti-IgG antibodies, anti-human IgE antibodies, etc. Anti-viral antibodies also include antibodies against their constituent components.

<Preferred method for producing labeled antibodies>
Next, a preferred method for producing the labeled antibody 150 will be described. The production of the labeled antibody 150 includes at least the following steps A:
Step A) includes a step of obtaining a labeled antibody 150 by mixing and binding the nanocomposite particle 100 with an antibody under a first pH condition, and preferably further includes step B;
Step B) may include treating the labeled antibody 150 with a second pH condition.

[Process A]
In step A, the nanocomposite particle 100 is mixed with an antibody under a first pH condition to obtain a labeled antibody 150. In step A, it is preferable to contact the solid nanocomposite particle 100 with the antibody in a state where the nanocomposite particle 100 is dispersed in a liquid phase.

The first pH condition is preferably in the range of pH 2 to 10, and more preferably in the range of pH 5 to 9, from the viewpoint of uniformly contacting the nanocomposite particles 100 with the antibody while maintaining the dispersion of the nanocomposite particles 100 and the activity of the antibody. If the conditions for binding the nanocomposite particles 100 with the antibody are less than pH 2, the antibody may be altered and inactivated due to strong acidity, and if the pH exceeds 10, the nanocomposite particles 100 and the antibody will aggregate when mixed, making dispersion difficult. However, if the antibody is not inactivated by strong acidity, processing is possible even at a pH of less than 2.

Step A is preferably performed in a binding buffer adjusted to the first pH condition. For example, a predetermined amount of nanocomposite particles 100 is mixed into the binding buffer adjusted to the above pH and thoroughly mixed. For example, a boric acid solution adjusted to a predetermined concentration can be used as the binding buffer. The pH of the binding buffer can be adjusted using, for example, hydrochloric acid, sodium hydroxide, etc.

Next, a predetermined amount of antibody is added to the resulting mixture, and the mixture is thoroughly stirred and mixed to obtain a labeled antibody-containing liquid. From the labeled antibody-containing liquid thus obtained, only the labeled antibody 150 can be separated as a solid portion using a solid-liquid separation means such as centrifugation.

[Process B]
In step B, the labeled antibody 150 obtained in step A is treated under a second pH condition to perform blocking for suppressing non-specific adsorption to the labeled antibody 150. In this case, the labeled antibody 150 separated by a solid-liquid separation means is dispersed in a liquid phase under the second pH condition.

The second pH condition is preferably, for example, in the range of pH 2 to 10 from the viewpoint of maintaining the activity of the antibody and suppressing aggregation of the labeled antibody 150, and more preferably in the range of pH 5 to 9 from the viewpoint of suppressing non-specific adsorption of the labeled antibody 150. If the blocking condition is less than pH 2, the antibody may be denatured and inactivated due to strong acidity, and if the pH exceeds 10, the labeled antibody 150 will aggregate and become difficult to disperse.

It is preferable to carry out step B using a blocking buffer in which the blocking agent is adjusted to the second pH condition. For example, a blocking buffer adjusted to the above pH is added to a predetermined amount of labeled antibody 150, and the labeled antibody 150 is uniformly dispersed in the blocking buffer. For example, a solution of a protein that does not bind to the analyte is preferably used as the blocking buffer. There is no particular restriction on the blocking agent that can be used in the blocking buffer, and known blocking agents can be used, including those with strong anionic properties, those with strong cationic properties, and other blocking agents. For example, proteins include bovine serum albumin, egg white albumin, casein, gelatin, and whey. More specifically, it is preferable to use a bovine serum albumin solution adjusted to a predetermined concentration. The pH of the blocking buffer can be adjusted using, for example, hydrochloric acid, sodium hydroxide, etc. It is preferable to use a dispersing means such as ultrasonic treatment to disperse the labeled antibody 150. In this way, a dispersion liquid in which the labeled antibody 150 is uniformly dispersed is obtained.

In the above steps A and B, the nanocomposite particle 100 having platinum nanoparticles as the metal nanoparticles 20 is less susceptible to aggregation due to pH and can be processed over a wide range of pH, from acidic to alkaline. Therefore, the nanocomposite particle 100 of the present invention using platinum nanoparticles also has the advantage of being less subject to restrictions on the production conditions of the labeled antibody.

In this manner, a dispersion of the labeled antibody 150 is obtained. From this dispersion, the labeled antibody 150 alone can be separated as a solid portion by solid-liquid separation means such as centrifugation. In addition, washing treatment, preservation treatment, etc. can be performed as necessary. The washing treatment and preservation treatment are described below.

(Cleaning treatment)
In the washing process, a washing buffer solution is added to the labeled antibody 150 separated by the solid-liquid separation means, and the labeled antibody 150 is uniformly dispersed in the washing buffer solution. For dispersion, it is preferable to use a dispersing means such as ultrasonic treatment. The washing buffer solution is not particularly limited, but for example, a Tris buffer solution, a glycine amide buffer solution, an arginine buffer solution, or the like, with a predetermined concentration adjusted to a pH range of 8 to 9, can be used. The pH of the washing buffer solution can be adjusted using, for example, hydrochloric acid, sodium hydroxide, or the like. The washing process of the labeled antibody 150 can be repeated multiple times as necessary.

(Preservation Treatment)
In the preservation treatment, a preservation buffer is added to the labeled antibody 150 separated by the solid-liquid separation means, and the labeled antibody 150 is uniformly dispersed in the preservation buffer. For the dispersion, it is preferable to use a dispersion means such as ultrasonic treatment. As the preservation buffer, for example, a solution in which a predetermined concentration of an anti-aggregation agent and/or a stabilizer is added to a washing buffer can be used. As the anti-aggregation agent, for example, sugars such as sucrose, maltose, lactose, and trehalose, and polyhydric alcohols such as glycerin and polyvinyl alcohol can be used. As the stabilizer, although it is not particularly limited, for example, proteins such as bovine serum albumin, egg white albumin, casein, and gelatin can be used. In this way, the preservation treatment of the labeled antibody 150 can be performed.

In each of the above steps, a surfactant or a preservative such as sodium azide or paraoxybenzoic acid ester can be used as necessary.

[Analyte measurement kit]
An analyte measurement kit according to one embodiment of the present invention is a kit for detecting or quantifying an analyte 160 contained in a sample, for example using a test strip 200, based on the analyte measurement method of this embodiment.

The analyte measurement kit of this embodiment includes:
A membrane 110;
A test strip 200 including a determination portion 130 having a membrane 110 and a capture ligand 131 that specifically binds to the analyte 160 immobilized thereon;
A detection reagent including a labeled antibody 150 in which an antibody that specifically binds to an analyte 160 is labeled with a nanocomposite particle 100;
The analyte measurement kit of this embodiment may further include other components (e.g., a developing solution, etc.) as necessary.

When using the analyte measurement kit according to this embodiment, the analyte 160 in the sample may be brought into contact with the labeled antibody 150 in the detection reagent to carry out steps (I) and (II), and then the sample may be provided to the sample application section 120 of the test strip 200 to carry out steps (III) and (IV) in sequence. Of course, when carrying out step (I) on a specimen having a medium to high concentration of antigen (antibody), it may be possible to recover the complex 170 containing the analyte 160 and the labeled antibody 150 by a known centrifugation method or the like without applying magnetic concentration. In that case, step (II) may be omitted.

In addition to being used as a labeling substance or a reagent for immunological assays, the nanocomposite particles 100 can also be used favorably as solid catalysts, pigments, paints, conductive materials, electrodes, sensor elements, recording materials, speaker and sealing materials that utilize their properties as a magnetic fluid, magnetic hyperthermia (magnetic hyperthermia therapy), MRI contrast agents, drug delivery systems, selective separation and collection of specific substances (medicines, antigens, antibodies, receptors, haptens, enzymes, proteins, peptides, sugars, nucleic acids, hormones, pathogens, toxins, etc.), cell manipulation, and blood purification (removal of pathogens).

Next, the present invention will be described in detail with reference to examples, but the present invention is not limited to these examples. Unless otherwise specified, the various measurements and evaluations in the following examples and comparative examples are as follows.

<Absorbance measurement of metal-resin composite>
The absorbance of the metal-resin composite was measured by placing a metal-resin composite dispersion (dispersion medium: water) prepared at 0.01 wt % in a quartz glass cell (optical path length 10 mm) and using a spectrophotometer (Shimadzu Corporation, UV3600) to measure the absorbance at 570 nm for the gold-resin composite and at 400 nm for the platinum-resin composite.

<Measurement of solid concentration and metal loading amount>
1 g of the dispersion before the concentration adjustment was placed in a porcelain crucible and dried for 3 hours at 70° C. The weights before and after drying were measured, and the solid content was calculated by the following formula.

Solid content (wt%) = [Weight after drying (g) / Weight before drying (g)] x 100

In addition, the sample after the above drying treatment was further heat-treated at 500°C for 5 hours, and the weights before and after the heat treatment were measured. The amount of metal and metal oxide supported in the nanocomposite particles (M) and the amount of metal supported in the metal-resin composite (M1) were calculated using the following formula.
Supported amount M or M1 (wt%)=
[Weight after heat treatment (g)/Weight before heat treatment (g)]×100

The amount of magnetic nanoparticles supported (M2) was calculated based on the above M and M1 using the following formula.
M2 (wt%) = [(M-M1)/(100-M1)] x 100

<Measurement of average particle size of resin nanoparticles, metal-resin composite particles, or nanocomposite particles>
A substrate was prepared by dropping a dispersion of resin nanoparticles, metal-resin composite particles, or nanocomposite particles onto a metallic mesh with a carbon support film, and the area-average diameter of any 100 resin nanoparticles, metal-resin composite particles, or nanocomposite particles was measured from an image observed with a field emission scanning electron microscope (FE-SEM; Hitachi High-Technologies Corporation, SU-9000).

<Measurement of the average particle size of metal nanoparticles>
A nanocomposite particle dispersion liquid was dropped onto a metallic mesh with a carbon support film to create a substrate, and an image was observed using a field emission scanning electron microscope (FE-SEM; Hitachi High-Technologies Corporation, SU-9000). The area average diameter of any 100 metal nanoparticles was measured from the image, and this was taken as the average particle diameter.

<Measurement of the average particle size of magnetic nanoparticles>
A nanocomposite particle dispersion liquid was dropped onto a metallic mesh with a carbon support film to create a substrate, and the area-average diameter of 100 random magnetic nanoparticles was measured from the image observed using a field emission scanning electron microscope (FE-SEM; Hitachi High-Technologies Corporation, SU-9000) to obtain the average particle diameter.

<Zeta potential measurement>
The zeta potential was measured by electrophoretic light scattering using a Zetasizer Nano-ZS manufactured by Malvern as a measuring device. The sample was diluted to 0.01 wt % with pure water and adjusted to each pH value of pH 3 to 10 using hydrochloric acid or NaOH aqueous solution to obtain a measurement sample. After measuring the pH using a pH meter (HORIBA LAQUA twin), the behavior of the change in the zeta potential at multiple pH points of pH 3 to 10 was measured by measuring the zeta potential. The change in the zeta potential was calculated from the maximum value of the zeta potential in the acidic region of pH 3 to 6 and the minimum value of the zeta potential in the alkaline region of pH 6 to 10. In addition, a linear function connecting an arbitrary zeta potential point close to 0 mV, an arbitrary zeta potential point greater than 0 mV, and an arbitrary zeta potential point less than 0 mV was determined, and the pH at which the zeta potential was 0 mV, i.e., the zero point of charge, was calculated.

[Preparation Example 1]
<Synthesis of resin nanoparticles>
After dissolving trioctylammonium chloride (1.50 g) and polyethylene glycol methyl ether methacrylate (10.00 g) in 300 g of pure water, 2-vinylpyridine (48.00 g) and divinylbenzene (2.00 g) were added and stirred under a nitrogen stream at 30 ° C. for 50 minutes, then at 60 ° C. for 30 minutes. After stirring, 2,2-azobis (2-methylpropionamidine) dihydrochloride (0.250 g) dissolved in 18.00 g of pure water was dropped and stirred at 60 ° C. for 3.5 hours to obtain resin nanoparticles A-1 with an average particle size of 359 nm. The mixture was precipitated by centrifugation (9000 rpm, 40 minutes), the supernatant was removed, and the mixture was dispersed again in pure water, and impurities were removed by dialysis. Thereafter, the concentration was adjusted to obtain a 10 wt% resin nanoparticle dispersion B-1.

[Example 1]
<Synthesis of platinum-resin composite particles>
54 g of pure water was added to B-1 (91.5 g), and then 400 mM aqueous chloroplatinic acid solution (100 g) was added and stirred at 30° C. for 3 hours. After leaving this mixture to stand for 24 hours, A-1 was precipitated by centrifugation (3000 rpm, 30 minutes), and excess chloroplatinic acid was removed by removing the supernatant. The concentration was then adjusted to obtain 5 wt % platinum ion-adsorbing resin particle dispersion C-1.

Next, C-1 (20.6 g) was added to 1392 g of pure water, and while stirring at 3 ° C., 132 mM dimethylamine borane aqueous solution (40 g) was dropped over 20 minutes, and then stirred at 3 ° C. for 1 hour and at room temperature for 3 hours to obtain platinum-resin composite particles D-1 with an average particle size of 367 nm. D-1 was concentrated by centrifugation, purified by dialysis, and the concentration was adjusted to obtain a 1 wt % platinum-resin composite particle dispersion E-1. The absorbance of platinum-resin composite particles F-1 in E-1 was 1.86. In addition, the average particle size of the platinum nanoparticles in F-1 was 3.5 nm, and the amount of platinum supported was 35.5 wt %.
When observed with a scanning transmission electron microscope (STEM), the platinum nanoparticles in this platinum-resin composite particle F-1 included encapsulated platinum particles that were completely encapsulated in the resin nanoparticles, and partially exposed platinum particles having a portion embedded in the resin nanoparticle and a portion exposed outside the resin nanoparticle, and at least a portion of the platinum nanoparticles were three-dimensionally distributed in the surface layer portion of the resin nanoparticles. Note that 97% of the platinum nanoparticles were present within a range of 40% of the particle radius in the depth direction from the surface of the resin nanoparticles.

Synthesis of Nanocomposite Particles
Platinum-resin composite particles F-1 were coated with ε-Poly-L-lysine (PLL) to obtain a PLL-coated platinum-resin composite. This PLL-coated platinum-resin composite has an amino group on the surface. Meanwhile, magnetic nanoparticles, superparamagnetic Fe ₃ O ₄ nanoparticles (average particle size about 15 nm), were reacted with citric acid to obtain citrate-capped Fe ₃ O ₄ nanoparticles (SPIONs). These citrate-capped Fe ₃ O ₄ nanoparticles have carboxyl groups on the surface. This PLL-coated platinum-resin composite and the citrate-capped Fe ₃ O ₄ nanoparticles were subjected to an EDC coupling reaction using 1-ethyl-3-(-3-dimethylaminopropyl)carbodiimide hydrochloride (EDC), and the Fe ₃ O ₄ nanoparticles were contacted with the platinum-resin composite particles F-1 through amide bonds to synthesize the immobilized nanocomposite particles G-1. The average particle size of the nanocomposite particles G-1 was 380 nm, and the amount of magnetic nanoparticles supported was 10 wt %.

[Example 2]
<Synthesis of Gold-Resin Composite Particles>
255 g of pure water was added to B-1 (91.5 g), and then 400 mM aqueous chloroauric acid solution (147 g) was added and stirred at room temperature for 3 hours. This mixture was centrifuged (3000 rpm, 30 minutes) to precipitate A-1, and the supernatant was removed to remove excess chloroauric acid. The concentration was then adjusted to obtain 2.5 wt % gold ion-adsorbing resin particle dispersion C-2.

Next, C-2 (43.3 g) was added to 1580 g of pure water, and while stirring at 3° C., 528 mM dimethylamine borane aqueous solution (10.0 g) was dropped over 2 minutes, and then the mixture was stirred at 3° C. for 1 hour and at room temperature for 3 hours to obtain gold-resin composite particles D-1 with an average particle size of 363 nm. D-1 was concentrated by centrifugation, purified by dialysis, and the concentration was adjusted to obtain a 1 wt % gold-resin composite particle dispersion E-2. The absorbance of gold-resin composite particles F-2 in E-2 was 1.51. The average particle size of the gold nanoparticles in F-2 was 30 nm, and the amount of gold supported was 48.1 wt %.
Observation with a scanning transmission electron microscope (STEM) revealed that the gold nanoparticles in this gold-resin composite particle F-2 included encapsulated gold particles that were completely encapsulated in the resin nanoparticles and partially exposed gold particles that had a portion embedded in the resin nanoparticle and a portion exposed outside the resin nanoparticle, and at least some of the gold nanoparticles were three-dimensionally distributed in the surface layer of the resin nanoparticles. Note that 97% of the gold nanoparticles were present within a range of 40% of the particle radius in the depth direction from the surface of the resin nanoparticle.

Synthesis of Nanocomposite Particles
Gold-resin composite particles F-2 were coated with ε-Poly-L-lysine (PLL) to obtain a PLL-coated gold-resin composite. This PLL-coated gold-resin composite has amino groups on the surface. Meanwhile, superparamagnetic Fe ₃ O ₄ nanoparticles (average particle size about 15 nm), which are magnetic nanoparticles, were reacted with citric acid to obtain citrate-capped Fe ₃ O ₄ nanoparticles (SPIONs). These citrate-capped Fe ₃ O ₄ nanoparticles have carboxyl groups on the surface. This PLL-coated gold-resin composite and the citrate-capped Fe ₃ O ₄ nanoparticles were subjected to an EDC coupling reaction using 1-ethyl-3-(-3-dimethylaminopropyl)carbodiimide hydrochloride (EDC), and the Fe ₃ O _{4 nanoparticles were contacted with the gold-resin composite particles F-2 via amide bonds to synthesize nanocomposite particles G-2 in which the Fe 3 O 4} nanoparticles were immobilized. The average particle size of the nanocomposite particles G-2 was 380 nm, and the amount of magnetic nanoparticles supported was 11 wt %.

[Test Example]
(Preparation of Labeled Antibody Dispersion)
After mixing 25 μg of anti-CRP monoclonal antibody (CalBioReagents #M353) and 0.45 mL of 100 mM boric acid aqueous solution (pH 8.5), a dispersion containing 500 μg of nanocomposite particles G-1 was added. The mixture was stirred by end-over-end at room temperature for 30 minutes to bind the anti-CRP monoclonal antibody to the nanocomposite particles G-1.
Next, the mixture was centrifuged at 3000 rpm for 5 minutes, the supernatant was removed, and 0.5 mL of a 1 wt % aqueous solution of Block Ace (manufactured by DS Biopharma) was added to the sediment, which was then subjected to ultrasonic dispersion treatment for 10 seconds. The mixture was then stirred by inversion at room temperature for 30 minutes, and a blocking treatment was performed.
Next, the mixture was centrifuged at 3000 rpm for 5 minutes, the supernatant was removed, and 0.5 mL of a 50 mM Tris aqueous solution (pH 8) containing less than 0.1 wt % of a surfactant was added to the sediment, followed by ultrasonic dispersion treatment for 10 seconds. This operation was repeated three times for washing.
Next, the mixture was centrifuged at 3,000 rpm for 5 minutes, and the supernatant was removed. 0.5 mL of a 50 mM Tris aqueous solution (pH 8) containing less than 0.1 wt % of a surfactant and 5 wt % of sucrose was then added to the sediment, followed by ultrasonic dispersion treatment for 10 seconds to obtain a labeled antibody dispersion in which the anti-CRP monoclonal antibody was labeled with the nanocomposite particle G-1.

(Preparation of immunochromatographic strips)
Anti-CRP monoclonal antibody (CalBioReagents #M354) was applied to the center of a nitrocellulose membrane 3 with a width of 25 mm, and a test line (TL) 4 was drawn as a judgment section. Furthermore, an anti-mouse IgG antibody was applied 3 mm downstream of the test line 4 to draw a control line 5 as a control section. After drying at 50°C, a support 1 (laminate film), a sample addition section 2 (glass fiber nonwoven fabric), and a liquid absorption section 6 (cotton nonwoven fabric) were laminated as shown in the cross-sectional view of the immunochromatographic strip in Figure 3. Finally, the immunochromatographic strip was prepared by cutting it to a width of 3.5 mm.

(Preparation of developing solution)
An aqueous solution (pH 7.1) containing 50 mM trishydroxymethylaminomethane, 150 mM sodium chloride, 1.0 wt % bovine serum albumin and 2.0 wt % Ceteth 20 was prepared as a developing solution.

[Test Example 1]
(Evaluation by immunochromatography)
A CRP positive control specimen solution was prepared by diluting the CRP antigen (BIO-RAD CRP Ag HP antigen) to a predetermined concentration using the developing solution. For the negative control specimen solution, a developing solution without the addition of antigen was used. 50 μl of the specimen solution was mixed with 3 μl of the labeled antibody dispersion solution, and 35 μl of the solution was dropped into the sample addition section 2 of the immunochromatographic strip to develop the sample. The color intensity of the test line 4 30 minutes after the specimen solution was dropped was measured using an immunochromatographic reader (manufactured by Hamamatsu Photonics). The color intensity of the test line 4 was judged as positive (+) for 13 mABS or more, pseudo positive (±) for 3 mABS or more and less than 13 mABS, and negative (-) for less than 3 mABS. The evaluation results by the immunochromatographic method are shown in the table below. As a result of the evaluation, it was possible to determine a positive result for CRP antigen concentrations up to 1.2 ng/ml by immunochromatography using an anti-CRP monoclonal antibody labeled with nanocomposite particle G-1.

The results of the immunochromatographic evaluation are shown in Table 1.

[Test Example 2]
(10-fold concentration by magnetic separation)
A mixture of the sample solution and the labeled antibody was prepared by mixing 60 μl of the labeled antibody dispersion with 10 ml of the sample solution of the CRP positive control and the negative control diluted to a predetermined concentration. Magnetic separation and concentration were performed using a magnetic separation column and a magnet to prepare 1 ml of a 10-fold concentrated solution of the sample solution and the labeled antibody.
(Evaluation by immunochromatography)
35 μl of 1 ml of the 10-fold concentrated solution was dropped onto the sample application section 2 of the immunochromatographic strip, and evaluation was performed in the same manner as in the above example. The evaluation results of the immunochromatographic method using 10-fold concentration by magnetic separation are shown in the table below. As a result of the evaluation, when using 10-fold concentration by magnetic separation, a positive determination was possible up to a CRP antigen concentration of 0.3 ng/ml.

The evaluation results using immunochromatography combined with 10-fold concentration by magnetic separation are shown in Table 2.

[Test Example 3]
(40-fold concentration by magnetic separation)
A mixture of the sample solution and the labeled antibody was prepared by mixing 60 μl of the labeled antibody dispersion with 40 ml of the sample solution of the CRP positive control and the negative control diluted to a predetermined concentration. Magnetic separation and concentration were performed using a magnetic separation column and a magnet to prepare 1 ml of a 40-fold concentrated solution of the sample solution and the labeled antibody.
(Evaluation by immunochromatography)
35 μl of 1 ml of the 40-fold concentrated solution was dropped onto the sample application section 2 of the immunochromatographic strip, and evaluation was performed in the same manner as in the above example. The evaluation results of the immunochromatographic method using 40-fold concentration by magnetic separation are shown in the table below. As a result of the evaluation, when 40-fold concentration by magnetic separation was used in combination, a positive determination was possible for CRP antigen concentrations up to 0.08 ng/ml.

The evaluation results using immunochromatography combined with 40-fold concentration by magnetic separation are shown in Table 3.

These results confirm that by using the resin nanoparticles of the present invention and applying magnetic concentration after the antigen-antibody reaction, it is possible to detect with high sensitivity analytes that are normally present in extremely small amounts in a specimen, below the detection limit.

The above describes in detail an embodiment of the present invention for illustrative purposes, but the present invention is not limited to the above embodiment and various modifications are possible.

1 Support 2 Sample application section 3 Membrane 4 Test line (TL)
5 control line 6 liquid absorbing section 10 resin nanoparticle 20 metal nanoparticle 30 encapsulated particle 40 partially exposed particle 50 surface adsorbed particle 60 surface layer 70 magnetic nanoparticle 100 nanocomposite particle 110 membrane 120 sample addition section 130 determination section 131 capture ligand 140 liquid absorbing section 150 labeled antibody 160 analyte 170 complex 200 test strip

Claims (12)

Resin nanoparticles;
A plurality of metal nanoparticles having an average particle size of 1 to 100 nm, the particle size of which is relatively smaller than that of the resin nanoparticles;
A plurality of magnetic nanoparticles having an average particle size of 1 to 50 nm, the particle size of which is relatively smaller than that of the resin nanoparticles;
Equipped with
The average particle size is 50 to 1100 nm,
A nanocomposite particle, characterized in that the metal nanoparticles are fixed to the resin nanoparticles, and the magnetic nanoparticles are fixed to the surface of the resin nanoparticles and/or the metal nanoparticles. The nanocomposite particles according to claim 1, wherein the metal nanoparticles are particles of gold, silver, copper, palladium, platinum, tin, rhodium, iridium, or alloys thereof. 3. The nanocomposite particles of claim 1 or 2 _{, wherein the magnetic nanoparticles are iron, cobalt, nickel, manganese, Fe2O3, Fe3O4, AFe2O4 ( where A} means Mn _{, Co} , Ni, Cu or Zn), FePt, CoPt, FeNi, or FeCo. The nanocomposite particles according to any one of claims 1 to 3, wherein the resin nanoparticles are polymer particles having a substituent in their structure capable of adsorbing metal ions. The nanocomposite particles according to any one of claims 1 to 4, wherein at least a portion of the metal nanoparticles are three-dimensionally distributed on the surface layer of the resin nanoparticles. The nanocomposite particle according to claim 5 , wherein 60 wt % to 100 wt % of said metal nanoparticles are present in said surface layer portion. A labeling substance comprising the nanocomposite particles according to any one of claims 1 to 6. The labeling substance according to claim 7, which is used by adsorbing an antigen or an antibody to the surface of the nanocomposite particle. An immunoassay method characterized by using the labeling substance according to claim 7 or 8. A reagent for immunoassays, characterized in that it uses the labeling substance according to claim 7 or 8. A method for measuring an analyte, which detects or quantifies an analyte contained in a sample, comprising the steps of:
A lateral flow type chromatography test strip including a membrane and a determination part having a capture ligand that specifically binds to the analyte immobilized on the membrane is used, the following steps (I) to (IV):
Step (I): A step of contacting the analyte contained in a sample with a labeled antibody obtained by labeling an antibody that specifically binds to the analyte with the nanocomposite particle according to claim 1 to obtain a complex containing the analyte and the labeled antibody;
Step (II): recovering a complex containing the analyte and the labeled antibody by magnetic force;
Step (III): A step of contacting the complex containing the analyte and the labeled antibody recovered in the step (II) with the capture ligand in the determination portion of the lateral flow type chromatography test strip;
Step (IV): measuring the localized surface plasmon resonance of the nanocomposite particles and the color intensity resulting from light energy absorption due to electronic transition;
A method for measuring an analyte, comprising carrying out the steps of: A kit for measuring an analyte for detecting or quantifying an analyte contained in a sample using a lateral flow type chromatography test strip, comprising:
A lateral flow type chromatography test strip including a membrane and a determination part in which a capture ligand that specifically binds to the analyte is immobilized on the membrane;
A detection reagent comprising an antibody that specifically binds to the analyte and is labeled with the nanocomposite particle according to claim 1;
An analyte measurement kit for detecting or quantifying an analyte comprising the compound.