JP2022134172A - Nanocomposite particles, labeling substance, immunological assay, immunological assay reagent, analyte measurement method and analyte measurement kit - Google Patents

Abstract

To provide nanocomposite particles that in immunological assay, is not only excellent in visibility, but also can apply magnetic concentration to analytes such as antigens and antibodies that are normally present in extremely small amounts in samples such as blood, urine, saliva, and environmental water, which are usually below a detection limit, to detect the analytes.SOLUTION: A nanocomposite particle comprises: a resin nanoparticle; a plurality of metal nanoparticles having an average particle size of 1 to 100 nm, which is relatively smaller in particle size than the resin nanoparticle; and a plurality of magnetic nanoparticles having an average particle size of 1 to 50 nm, which is relatively smaller than the resin nanoparticle. The nanocomposite particles have an average particle size of 50 to 1100 nm, and the metal nanoparticles and the magnetic nanoparticles are immobilized on the resin nanoparticle.SELECTED DRAWING: None

Description

The present invention provides, for example, novel nanocomposite particles that can be preferably used for applications such as immunoassay, labeling substances using the same, immunoassay methods, immunoassay reagents, analyte assay methods, and an analyte measurement kit.

Immunological assay (also called "immunoassay") is a method of qualitatively and quantitatively analyzing trace components by using a specific reaction between antigen and antibody, which is one of immune reactions. . Antigen-antibody reactions are widely used in the above fields because of their high sensitivity and reaction selectivity. Immunoassay has various measurement methods depending on its measurement principle. For example, enzyme immunoassay (EIA), radioimmunoassay (RIA), chemiluminescence immunoassay (CLIA), fluorescence immunoassay (FIA), agglutination method such as latex (LIA, PA), immunochromatographic method ( ICA), hemagglutination method (HA), hemagglutination inhibition method (HI), and the like. In addition to immunoassay, there are physical/chemical measurement methods, biological measurement methods, and the like.

Immunoassays detect antigens or antibodies qualitatively or quantitatively from changes when antigens and antibodies react to form complexes (concentration changes of antigens, antibodies, or complexes). When detecting these, the detection sensitivity is increased by binding a labeling substance to the antibody, antigen or complex.
Therefore, it can be said that the labeling ability of a labeling substance is an important factor that influences the detection ability in immunoassays. 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), A chemiluminescent substance (in the case of CLIA) or the like is used.

By the way, when colored microparticles are used as the labeling substance, the detection can be visually confirmed without using a special analysis device, and thus it is expected that the measurement can be performed more easily. Examples of such colored fine particles include colloidal particles of metals and metal oxides, and latex particles colored with pigments (Patent Document 1, Patent Document 2, etc.). However, since the color tone of the colloidal particles is determined by the particle size and preparation conditions, it is difficult to obtain a desired clear and deep color tone, that is, the visibility is insufficient.
In addition, the above-mentioned colored latex particles have a problem that the effect of coloring with a dye is low and the visual judgment is insufficient. In order to solve this problem, if the coloring amount of the dye is increased, the dye covers the surface of the latex, impairing the original surface state of the latex particles, making it difficult to bind antigens or antibodies. was there. In addition, increasing the colorant of the dye to darken the color by clogging the pores of the chromatographic medium such as the membrane filter or causing non-specific aggregation of the latex particles does not necessarily lead to the improvement of the performance. There was also the problem of whether or not

In order to improve the visibility of the above-mentioned labeling substances, after the antibody bound to the labeling substance (labeled antibody) reacts with the antigen to form a complex, these labeling substances are further modified with other metals for labeling. An immunochromatographic method is disclosed that increases the sensitivity of detection of substances. Further, an example in which gold colloid and colored latex are used in combination is also disclosed (Patent Document 3).
However, these methods require complicated operations and are difficult to achieve stable amplification. In addition, it is considered that the applicable applications and operating environments are limited due to the high cost of measurement, such as the need for a special device.

As a solution to these problems in the prior art, resin particles to which metal nanoparticles are bound have been disclosed.
For example, a colored latex consisting of gold nanoparticles bound to the surface of polymeric latex particles has been disclosed (Patent Document 4).
By bonding the gold nanoparticles to the surface of the polymer-based latex particles, the gold nanoparticles themselves serve as a colorant to improve visual determination and detection sensitivity. In addition, since the gold nanoparticles themselves have excellent binding properties to antigens or antibodies, it is said that a sufficient amount of antigens or antibodies can be bound even if the gold nanoparticles are bound to a sufficiently dark color. there is

In addition, the present applicant found and previously proposed a resin-metal composite in which a specific proportion of metal particles is present in the surface layer of resin particles (eg, Patent Document 5). This resin-metal composite is excellent in durability and visibility, especially in the application of immunoassay materials, and enables high-sensitivity determination without requiring the addition of special equipment or work processes. is.

JP-A-5-10950 JP-A-3-206959 JP 2011-117906 A JP 2009-168495 A International publication WO2016/002742

As described above, the resin particles to which metal nanoparticles are bound are expected to be used as reagents for immunological measurements because they have excellent visibility and do not require special equipment or additional work steps.
However, in order to detect antigens such as viruses that are present in extremely small amounts in specimens such as blood but have a significant impact on the health of patients who possess the blood, it is necessary to improve the visibility of the labeling substance alone. In addition, another technical approach is required.

That is, the object of the invention is not only excellent visibility in immunoassay, but also antigens and antigens present in extremely small amounts in specimens such as blood, urine, saliva, environmental water, etc., which are usually below the detection limit. The purpose is to provide nanocomposite particles that can detect analytes such as antibodies by magnetically concentrating them, enabling highly sensitive determination, for example, in immunoassays. An object of the present invention is to provide nanocomposite particles for immunoassay.

As a result of intensive research, the present inventors have found that the above problems can be solved by nanocomposite particles in which metal nanoparticles and magnetic nanoparticles are immobilized on resin nanoparticles, and have 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, which are relatively smaller in particle size than the resin nanoparticles;
a plurality of magnetic nanoparticles having an average particle size of 1 to 50 nm and having a particle size relatively smaller than that of the resin nanoparticles;
with
an average particle size of 50 to 1100 nm,
The metal nanoparticles and the magnetic nanoparticles are immobilized on the resin nanoparticles.

In the nanocomposite particles of the present invention, the metal nanoparticles 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 are iron, cobalt, nickel, manganese, Fe ₂ O ₃ , Fe ₃ O ₄ , AFe ₂ O ₄ (where A is 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 capable of adsorbing metal ions in their structure.

In the nanocomposite particles of the present invention, at least a portion of the metal nanoparticles may be three-dimensionally distributed on the surface layer of the resin nanoparticles. Also, 60 wt % to 100 wt % of the metal nanoparticles may be present in the surface layer portion. Further, the magnetic nanoparticles may be immobilized on the surfaces of the resin nanoparticles and/or the metal nanoparticles.

A labeling substance of the present invention is characterized by comprising the nanocomposite particles according to any one of the above. Moreover, the surface of the nanocomposite particles may be adsorbed with an antigen or an antibody for use.

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

The analyte measurement method of the present invention is a method for detecting or quantifying an analyte contained in a sample, comprising:
The following steps (I) to (IV) using a lateral flow chromatographic test strip containing a membrane and a determination portion in which a capture ligand that specifically binds to the analyte is immobilized on the membrane;
Step (I): Contacting the analyte contained in the sample with an antibody that specifically binds to the analyte with a labeled antibody labeled with any of the above nanocomposite particles to label the analyte obtaining a complex comprising an antibody,
Step (II): a step of collecting a complex containing an analyte and a 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 a capture ligand in the determination section of the lateral flow chromatography test strip;
Step (IV): a step of measuring the intensity of color development derived from the localized surface plasmon resonance of the nanocomposite particles and the absorption of light energy by electronic transition;
characterized by performing a step including

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 chromatography test strip,
a test strip for lateral flow chromatography, comprising a membrane, and a determination section having a capture ligand that specifically binds to the analyte fixed to the membrane;
and a detection reagent comprising a labeled antibody labeled with any of the above nanocomposite particles, an antibody that specifically binds to the analyte.

In the nanocomposite particles of the present invention, the metal nanoparticles and the magnetic nanoparticles are immobilized on the resin nanoparticles. Therefore, the resin particles to which the metal nanoparticles are bound, as exemplified in Patent Documents 4 and 5, are characterized. In addition to the fact that it is highly visible and does not require the addition of special equipment or work processes, it is present in extremely small amounts in samples such as blood, urine, saliva, environmental water, etc., which are normally below the detection limit. Antigens (antibodies) can also be detected by magnetically concentrating them after causing an antigen-antibody reaction. Therefore, for example, EIA, RIA, CLIA, FIA, LIA, PA, ICA, HA, HI, etc. for the purpose of immunological measurement labeled substances, immunological measurement reagents, etc. that enable highly sensitive determination It can be preferably applied.

1 is a schematic diagram showing a cross-sectional structure of a nanocomposite particle according to an embodiment of the present invention; FIG. BRIEF DESCRIPTION OF THE DRAWINGS FIG. 1 is an explanatory diagram showing an overview of an analyte measurement method using a lateral flow chromatography test strip according to an embodiment of the present invention; FIG. 2 is an explanatory diagram of a test strip for lateral flow chromatography used in Examples.

BEST MODE FOR CARRYING OUT THE INVENTION Hereinafter, embodiments of the present invention will be described in detail with reference to the drawings as appropriate. FIG. 1 is a cross-sectional schematic diagram of a nanocomposite particle according to one embodiment of the present invention. The nanocomposite particle 100 includes resin nanoparticles (hereinafter also simply referred to as “resin particles”) 10 , metal nanoparticles 20 and magnetic nanoparticles 70 . A nanocomposite particle 100 has metal nanoparticles 20 immobilized on resin nanoparticles 10 . Resin nanoparticles 10 are particles relatively larger than metal nanoparticles 20 . That is, in the nanocomposite particle 100, many metal nanoparticles 20 having a relatively small particle size are fixed to the resin nanoparticles 10 having a relatively large particle size. As shown in FIG. 1, the relationship among the particle diameter D1 of the entire nanocomposite particles 100, the particle diameter D2 of the resin nanoparticles 10, and the particle diameter D3 of the metal nanoparticles 20 is D1>D2>D3.
A plurality of magnetic nanoparticles 70 are immobilized on the nanocomposite particle 100 . That is, in the nanocomposite particle 100 , a large number of magnetic nanoparticles 70 are immobilized on the resin nanoparticles 10 and/or the metal nanoparticles 20 . As shown in FIG. 1, the relationship among the particle diameter D1 of the entire nanocomposite particles 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 is shown to be smaller than the metal nanoparticles 20, but this is only for convenience of explanation, and the shape and size are limited. not a thing In addition, although FIG. 1 shows a mode in which the magnetic nanoparticles 70 are bound to the surfaces of the resin nanoparticles 10 and the metal nanoparticles 20, this is merely an example, and the state of existence of the magnetic nanoparticles 70 is limited. Instead, it 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. ~50 nm. When the average particle diameter of the nanocomposite particles 100 is less than 50 nm, the amount of metal nanoparticles 20 supported tends to be small, so the coloring tends to be weaker than metal nanoparticles of the same size, and the magnetic concentration efficiency is high. tends to decline. 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 easily clogged, and the dispersion stability tends to decrease. . The average particle diameter of the nanocomposite particles 100 is from the viewpoint of improving the dispersion stability when used as a labeling substance or reagent and obtaining high detection sensitivity when the nanocomposite particles 100 are used for immunological measurements. From the viewpoint of obtaining high magnetic concentration efficiency, the preferred lower limit is 100 nm, more preferably 250 nm, and even more preferably 300 nm. On the other hand, the upper limit is preferably 700 nm, more preferably 600 nm. Here, the particle diameter of the nanocomposite particles 100 means the particle diameter of the resin nanoparticles 10 plus the length of the protruding portion of the partially exposed particles 40 or the surface-adsorbed particles 50, which will be described later. It can be measured by electron microscopy, such as electron microscopy (SEM) or transmission electron microscopy (TEM), laser diffraction/scattering, dynamic light scattering, or centrifugal sedimentation.

In addition, when the average particle diameter of the metal nanoparticles 20 (that is, the average particle diameter 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 occur. Since it becomes difficult to express, sensitivity tends to decrease.
When gold particles are used as the metal nanoparticles 20, the preferred lower limit of the average particle size of the gold particles is 1 nm from the viewpoint of obtaining high detection sensitivity as a labeling substance for immunological measurement and a reagent for immunological measurement. , more preferably 3 nm. On the other hand, the preferred upper limit is 70 nm, more preferably 50 nm.
When platinum particles are used as the metal nanoparticles 20, the average particle size of the platinum particles preferably has a lower limit of 1 nm from the viewpoint of obtaining high detection sensitivity as a labeling substance for immunological measurement and a reagent for immunological measurement. , more preferably 2 nm, more preferably 3 nm. On the other hand, the upper limit is preferably 50 nm, more preferably 20 nm, and even more preferably 15 nm.
Here, the average particle size of the metal nanoparticles 20 is measured by scanning electron microscope (SEM) or transmission electron microscope (TEM) observation.

In addition, when the average particle diameter of the magnetic nanoparticles 70 is less than 1 nm, the magnetic force expressed in the nanocomposite particles 100 is weak. After forming a complex containing the analyte and the labeled antibody particles (hereinafter referred to as an "analyte-labeled antibody complex"), the analyte-labeled antibody complex is The efficiency of magnetic collection, that is, the efficiency of magnetic concentration tends to decrease. On the other hand, when it exceeds 50 nm, the specific gravity of the nanocomposite particles 100 is large, so the mobility and diffusibility in the specimen are low, and the contact efficiency between the analyte and the labeled antibody particles in the specimen and the analyte-labeled antibody The efficiency of collecting the complex by magnetism tends to decrease. A preferred lower limit is 3 nm, more preferably 5 nm, and still more preferably 7 nm. On the other hand, the upper limit is preferably 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 scanning electron microscope (SEM) or transmission electron microscope (TEM) observation.

The metal nanoparticles 20 do not contain nanoparticles containing magnetic elements (Mn, Fe, Co, Ni) (however, it is permissible to contain a small amount of magnetic elements as inevitable impurities to the extent that the particles do not become magnetized). ). That is, the metal nanoparticles 20 are metal nanoparticles excluding magnetic materials. In addition, the metal nanoparticles 20 are made of gold, silver, copper, palladium, platinum, tin, rhodium, and iridium, from the viewpoint of facilitating nano-sized composites of the metal nanoparticles 20 and the resin nanoparticles 10. or particles of these alloys. These metals can be used singly or in composites such as alloys. Here, for example, the gold alloy means an alloy composed of gold and a metal species other than gold and containing 10% by weight or more, preferably 50% by weight or more, more preferably 60% by weight or more of gold. Further, for example, a platinum alloy is composed of platinum and a metal species other than platinum and contains 1% by weight or more, preferably 10% by weight or more, more preferably 50% by weight or more, and still more preferably 60% by weight or more of platinum. means When used as a labeling substance for immunological measurement and a reagent for immunological measurement, gold, platinum and palladium, which are excellent in visibility and easily immobilized with antigens or antibodies, are more preferable. These are preferable because they exhibit absorption derived from localized surface plasmon resonance. More preferred are gold and platinum.

For example, a gold-resin composite using gold particles as the metal nanoparticles 20 does not aggregate when bound to a ligand such as an antibody, compared to a metal-resin composite having particles of other metal species. It is difficult to form and has excellent dispersibility. Furthermore, it has excellent visibility and facilitates immobilization of antigens or antibodies. In particular, various colors such as red, purple, and blue are exhibited by controlling the particle size of the gold particles and the inter-particle distance between the gold particles. Therefore, when the gold-resin complex is used as a labeling substance for an immunocytomatograph, a labeling substance of various colors can be obtained.

For example, a platinum-resin composite using platinum particles as the metal nanoparticles 20 does not aggregate when bound to a ligand such as an antibody, compared to a metal-resin composite having particles of other metal species. It is difficult to form and has excellent dispersibility. In addition, platinum particles are resistant to deterioration such as oxidation and have excellent storage stability. Furthermore, the platinum particles exhibit absorption derived from localized surface plasmon resonance in a wide wavelength range of, for example, 250 nm to 900 nm, and further exhibit light energy absorption due to electronic transition, thereby exhibiting a strong color near black. By using a platinum-resin complex as a labeling substance, high visibility can be obtained in immunoassays, and analyte detection sensitivity can also be increased. In this case, by using platinum particles, excellent detection sensitivity can be obtained with a small amount of support compared to particles of other metal species. Therefore, if the average particle size is the same, the platinum-resin composite is preferable because it exhibits significantly higher detection sensitivity than the resin composite containing particles of other metal species.

The metal nanoparticles 20 may consist of only a single metal species, or may be an alloy of the metal species and other 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 alloying with other metals, the species of the other metal is not particularly limited. For example, in the case of alloying with gold, silver, copper, palladium, platinum, tin, rhodium, and iridium are preferable. In the case of alloys with platinum, gold, silver, copper, palladium, tin, rhodium and iridium are preferred.

In addition, the magnetic nanoparticles 70 are the magnetic nanoparticles 70 and the resin nanoparticles 10 from the viewpoint of ease of nano-sized composite formation and the strength of the magnetic force of the nanocomposite particles 100. Particles 70 are iron, cobalt, nickel, _manganese , _{Fe2O3 , Fe3O4} , _{AFe2O4 (} where A means Mn, Co, Ni, Cu or Zn), FePt, CoPt, FeNi or FeCo are preferred. Fe ₃ O ₄ and FeCo, which are soft magnetic materials with high saturation magnetic flux densities, are more preferred, and Fe ₃ O ₄ is even more preferred.
As the magnetic nanoparticles 70, for example, commercially available products such as iron oxide magnetic nanoparticles (Sigma-Aldrich Japan LLC) can be preferably used.

Moreover, although the structure and composition of the resin nanoparticles 10 are not limited, they are preferably polymer particles having a substituent capable of adsorbing metal ions in their structure. Examples thereof include polymers capable of adsorbing anionic ions. Nitrogen-containing polymers are particularly preferred as polymers capable of adsorbing anionic ions. The nitrogen atoms in the nitrogen-containing polymer are anionic ions such as [AuCl ₄ ] ⁻ , [PtCl ₆ ] ^2- , which are precursors of the metal nanoparticles 20, which have excellent visibility and facilitate immobilization of antigens or antibodies. is preferred because it is easily chemisorbed. In the present embodiment, the metal ions adsorbed in the nitrogen-containing polymer are reduced to form the metal nanoparticles 20. Therefore, a part of the generated metal nanoparticles 20 are the included particles 30 or the partially exposed particles 40 described later. becomes. 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") contain carboxylic acid Groups and sulfonic acid groups are preferred as they allow chemisorption of cationic ions such as Au ⁺ , Pt ²⁺ . For example, by reducing chemisorbed Au ⁺ and Pt ²⁺ to form metal nanoparticles 20 (in this case, gold particles or platinum particles), it is possible to produce a structure similar to that of the nitrogen-containing polymer particles. be. 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, polystyrene, for example, can also 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 for the metal ions to be adsorbed 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 adhesion 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 a nitrogen atom in its main chain or side chain, and examples thereof include polyamine, polyamide, polypeptide, polyurethane, polyurea, polyimide, polyimidazole, polyoxazole, polypyrrole, polyaniline, melamine resin, and the like. be. Polyamines such as poly-2-vinylpyridine, poly-3-vinylpyridine and poly-4-vinylpyridine are preferred. Moreover, when the side chain has a nitrogen atom, it can be widely used, for example, an acrylic resin, a phenolic resin, an epoxy resin, and the like. In addition, the polymer capable of adsorbing cationic ions is a resin having a carboxylic acid group, a sulfonic acid group, or the like in its main chain or side chain. Acid, polystyrene sulfonic acid, cellulose, etc. can be widely used.
The polymer capable of adsorbing anionic ions such as the nitrogen-containing polymer and the polymer capable of adsorbing cationic ions may be copolymers with known polymerizable monomers. Examples of copolymers include random copolymers, block copolymers, alternating copolymers, and crosslinked polymers. Alternatively, the resin nanoparticles 10 may be formed by copolymerizing two or more types of monomers, or the functional groups present on the surface of the resin nanoparticles 10 may be reacted with the monomers, and the functional groups may be used as polymerization active terminals for further polymerization. can be Although the composition of the copolymer is not limited, it is preferable that the monomer containing the 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 particles 100 . For example, for the purpose of improving the shape and 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, for the purpose of improving the mechanical strength and shape stability of the resin nanoparticles 10, hydrophobic polymerizable monomers such as divinylbenzene and styrene can be used.
Moreover, when the resin nanoparticles 10 have hydrolyzable groups such as ester bonds, they may be partially hydrolyzed by acid treatment or alkali treatment. Hydrolysis is preferable because groups capable of adsorbing cationic ions such as carboxyl groups are generated on the surface of the resin nanoparticles 10, and the effect of adsorbing metal ions can be obtained. A known acid can be used as the acid. From the viewpoint of promoting the hydrolysis reaction, strongly acidic hydrochloric acid, sulfuric acid, and the like are preferable. A known alkali can be used as the alkali. From the viewpoint of promoting the hydrolysis reaction, strongly alkaline potassium hydroxide, sodium hydroxide aqueous solution and the like are preferable.

Moreover, in the nanocomposite particles 100, the state of dispersion of the metal nanoparticles 20 in the resin nanoparticles 10 is not limited. 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 included inside the resin nanoparticles 10. As one form of the former, a plurality of metal nanoparticles 20 may be in contact with each other to form a continuous film on the surface of the resin nanoparticles 10 . As one form of the latter, a core-shell structure may be formed in which the resin nanoparticles 10 are used as the shell and the metal nanoparticles 20 are used as the core. Moreover, part of the metal nanoparticles 20 may be three-dimensionally distributed in the surface layer portion 60 of the resin nanoparticles 10 . In this case, part of the three-dimensionally distributed metal nanoparticles 20 may be partially exposed outside the resin nanoparticles 10 , and the remaining part may be included in the resin nanoparticles 10 . Specifically, as shown in FIG. Metal nanoparticles having a portion embedded in and a portion exposed to the outside of the resin nanoparticles 10 (hereinafter also referred to as “partially exposed particles” 40) and metal nanoparticles adsorbed on the surface of the resin nanoparticles 10 (hereinafter also referred to as "surface adsorbed particles" 50) may be present.
For example, when the nanocomposite particles 100 are used as immunoassay labeling substances or immunoassay reagents, antigens, An antibody or blocking agent is immobilized and used. At that time, the antigen, antibody, or blocking agent is immobilized on the partially exposed particles 40 and the surface-adsorbed particles 50 , whereas it is difficult to immobilize it on the encapsulated particles 30 . However, since all of the partially exposed particles 40, the surface adsorbed particles 50, and the encapsulated particles 30 exhibit optical energy absorption due to electronic transition in addition to localized surface plasmon resonance, the partially exposed particles 40 and the surface adsorbed particles 50 In addition, the encapsulated particles 30 also contribute to improving the visibility of the labeling substance for immunological measurement and the reagent for immunological measurement. Furthermore, the partially exposed particles 40 and the encapsulated particles 30 have a larger contact area with the resin nanoparticles 10 than the surface adsorbed particles 50, and in addition, the anchor effect due to the embedded state is exhibited. It has a strong adsorptive power and is difficult to detach from the resin nanoparticles 10 . In addition, antigens, antibodies, or blocking agents adsorbed on the partially exposed particles 40 or the surface-adsorbed particles 50 are coordinated with the metal and are therefore difficult to detach. Therefore, the labeling substance for immunological measurement and the reagent for immunological measurement using the nanocomposite particles 100 can be made excellent in durability and stability.

In the nanocomposite particle 100 , the encapsulated particle 30 is entirely covered with the resin constituting the resin nanoparticle 10 . Moreover, 5% or more and less than 100% of the surface area of the partially exposed particles 40 is covered with the resin constituting the resin nanoparticles 10 . From the viewpoint of the durability of the labeling substance for immunological measurement and the reagent for immunological measurement, the lower limit is preferably 20% or more of the surface area, more preferably 30% or more. Moreover, more than 0% and less than 5% of the surface area of the surface adsorption particles 50 is covered with the resin constituting the resin nanoparticles 10 .

In addition, the amount of metal nanoparticles 20 (total of encapsulated particles 30, partially exposed particles 40 and surface-adsorbed particles 50) supported on nanocomposite particles 100 is 3 to 80 wt% with respect to nanocomposite particles 100. Preferably. Within this range, the nanocomposite particles 100 are excellent in visibility, visual determination, and detection sensitivity as a labeling substance. If the supported amount of the metal nanoparticles 20 is less than 3 wt %, the immobilized amount of the antibody or antigen tends to decrease and the detection sensitivity tends to decrease. On the other hand, when the supported amount of the metal nanoparticles 20 exceeds 80 wt %, the particle diameter of the metal nanoparticles 20 significantly increases, and the light absorption characteristics of the metal nanoparticles 20 tend to deteriorate. A more preferable lower limit of the supported amount of the metal nanoparticles 20 is 10 wt %, and more preferably 15 wt %. On the other hand, the upper limit is more preferably 70 wt%, more preferably 60 wt%.

Further, the amount of the magnetic nanoparticles 70 supported on the nanocomposite particles 100 is preferably 1 to 50 wt % with respect to the nanocomposite particles 100 . Within this range, the nanocomposite particles 100 have an excellent balance between the strength of the magnetic force and the motility and diffusivity in the specimen. That is, when the amount of the magnetic nanoparticles 70 supported is less than 1 wt %, the magnetic force of the nanocomposite particles 100 is weak, so the analyte-labeled antibody complex is magnetically collected after the analyte-labeled antibody complex is formed. The efficiency, that is, the efficiency of magnetic concentration, tends to decrease. On the other hand, when it exceeds 50 wt%, the specific gravity of the nanocomposite particles 100 is large, so the mobility and diffusibility in the specimen are low, and the contact efficiency between the analyte and the labeled antibody particles in the specimen and the analyte-labeling The efficiency of magnetic recovery of the antibody complex tends to decrease. In addition, for example, in the measurement of an analyte using a test strip, the development speed of the analyte-labeled antibody complex tends to decrease, resulting in poor measurement efficiency. A more preferable lower limit of the supported amount of the metal nanoparticles 20 is 5 wt %, more preferably 10 wt %. On the other hand, the upper limit is more preferably 40 wt%, more preferably 30 wt%.

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

Moreover, from the viewpoint of durability of the labeling substance for immunological measurement and the reagent for immunological measurement, it is preferable that the surface adsorption particles 50 are 20 wt % or less.

Moreover, when the nanocomposite particles 100 are used for immunological measurements, it is preferable that 60 to 100 wt % of the metal nanoparticles 20 are present in the surface layer portion 60 in order to obtain excellent detection sensitivity. A more preferable lower limit is 75 wt%, and more preferably 85 wt%.
In addition, 5 to 90 wt% of the metal nanoparticles 20 present in the surface layer portion 60 are partially exposed particles 40 or surface adsorbed particles 50, so that the amount of antibody or antigen immobilized on the metal nanoparticles 20 is sufficient. Since it can be secured, the sensitivity as a labeling substance is increased, which is preferable. In other words, 10 to 95 wt % of the metal nanoparticles 20 present in the surface layer portion 60 are preferably the encapsulated particles 30 .

Here, the “surface portion” refers to the surface of the resin nanoparticles 10 with reference to the outermost position of the nanocomposite particle 100 (that is, the protruding end of the partially exposed particle 40 or the surface-adsorbed particle 50). , means a range of 50%, more preferably 40%, of the particle radius in the depth direction. Moreover, the phrase “three-dimensionally distributed” means that the metal nanoparticles 20 are dispersed not only in the surface direction of the resin nanoparticles 10 but also in the depth direction. As described above, in addition to localized surface plasmon resonance, the encapsulated particles 30 also exhibit optical energy absorption due to electronic transition. It contributes to the improvement of the visibility of labeling substances for physical measurement and reagents for immunological measurement. From the viewpoint of such visibility improvement, in the nanocomposite particle 100, the encapsulated particles 30 are concentrated and distributed within a certain range in the depth direction from the surface of the resin nanoparticles 10, and the center of the resin nanoparticles 10 It is preferable that substantially no encapsulated particles 30 exist in the vicinity. More specifically, in order to effectively express optical energy absorption due to electronic transition in addition to 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 may be present, for example, within a range of 0 to 200 nm in the depth direction from the surface of the resin nanoparticles 10. . In particular, when the region where all (100 wt%) of the encapsulated particles 30 are distributed (encapsulated particle distribution region) is within the range of, for example, 0 to 100 nm from the surface of the resin nanoparticles 10, the localized surface by the encapsulated particles 30 In addition to plasmon resonance, it is preferable because it can maximize the expression of light energy absorption due to electronic transition. Moreover, 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 on 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 .

Moreover, in the nanocomposite particles 100, the dispersion state of the magnetic nanoparticles 70 in the resin nanoparticles 10 is not limited. 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 included inside the resin nanoparticles 10 .
As one form of the former, a plurality of magnetic nanoparticles 70 may be in contact with each other to form a continuous film on the surface of the resin nanoparticles 10 .
As one form of the latter, a core-shell structure may be formed in which the resin nanoparticles 10 are used as the shell and the magnetic nanoparticles 70 are used as the core. Moreover, part of the magnetic nanoparticles 70 may be three-dimensionally distributed inside the resin nanoparticles 10 . In this case, part of the three-dimensionally distributed magnetic nanoparticles 70 may be partially exposed outside 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 metal-resin composites composed of the resin nanoparticles 10 and the metal nanoparticles 20 (that is, the nanocomposite particles 100 excluding the magnetic nanoparticles 70 It is preferably immobilized on the surface of a substance).
In addition, when the magnetic nanoparticles 70 are immobilized 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 the like. , hydrogen bond, coordinate bond, covalent bond and the like. Preferably, from the viewpoint of bond stability, the resin nanoparticles 10 or the metal nanoparticles 20 and the magnetic nanoparticles 70 are in contact via chemical bonds with known linkers.
Here, a known substance or bond can be applied to the linker. Examples include amide bonds, ether bonds, and saturated or unsaturated carbon-carbon bonds.

[Method for producing nanocomposite particles]
The method for producing the nanocomposite particles 100 is not particularly limited, but as a preferred method hereinafter, first, a metal-resin composite composed of the resin nanoparticles 10 and the metal nanoparticles 20 is produced, and then the metal-resin composite A method for fixing the magnetic nanoparticles 70 to the body will be described.

The metal-resin composite is prepared, for example, by adding a solution containing metal ions to a dispersion liquid of resin nanoparticles 10 produced by an emulsion polymerization method to adsorb the metal ions on the resin nanoparticles 10 (hereinafter referred to as “metal ion "adsorbed resin particles"). Furthermore, by adding the metal ion-adsorbing resin particles to a reducing agent solution, metal ions are reduced to generate metal nanoparticles 20, thereby obtaining a metal-resin composite.

For example, when gold particles are generated as the metal nanoparticles 20, the solution containing gold ions includes a chloroauric acid (HAuCl ₄ ) aqueous solution. Alternatively, a gold complex may be used instead of the gold ion.
When platinum particles are generated as the metal nanoparticles 20, examples of solutions containing platinum ions include a chloroplatinic acid (H ₂ PtCl ₆ ) aqueous solution and a platinum chloride (PtCl ₂ ) solution. A platinum complex may be used instead of the platinum ion.

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

Moreover, a well-known thing can be used for a reducing agent. Examples of reducing agents include sodium borohydride, dimethylamine borane, citric acid, sodium hypophosphite, hydrazine hydrate, hydrazine hydrochloride, hydrazine sulfate, formaldehyde, sucrose, glucose, ascorbic acid, erythorbic acid, and phosphinic acid. sodium, hydroquinone, Rochelle salt and the like. Among these, sodium borohydride, dimethylamine borane, and citric acid are preferred.
If necessary, a surfactant can be added to the reducing agent solution, or the pH of the solution can be adjusted. The pH can 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 metal nanoparticles 20 to be generated can be controlled by adjusting the reduction rate of the metal ions by adjusting the temperature of the reducing agent solution.

Further, when the metal ions in the metal ion-adsorbing resin particles are reduced to generate the metal nanoparticles 20, the metal ion-adsorbing resin particles may be added to a reducing agent solution, or the reducing agent may be added to the metal ion-adsorbing resin particles. Although it may be added to the resin particles, the former is preferable from the viewpoint of ease of generation of the inclusion particles 30 and the partially exposed particles 40 .

In addition, in order to maintain the dispersibility in water of the metal-resin composite, for example, citric acid, poly-L-lysine, polyvinylpyrrolidone, polyvinylpyridine, polyvinyl alcohol, DISPERBYK194, DISPERBYK180, DISPERBYK184 (Big Chemie Japan) ) may be added.
Furthermore, the dispersibility can be maintained by adjusting the pH with buffers such as boric acid and phosphoric acid, acids such as hydrochloric acid and sulfuric acid, and alkalis such as sodium hydroxide and potassium hydroxide.

Next, nanocomposite particles 100 are produced by immobilizing magnetic nanoparticles 70 on the produced metal-resin composite. As described above, the contact state between the magnetic nanoparticles 70 and the resin nanoparticles 10 is not particularly limited. A method for bringing the resin nanoparticles 10 or the metal nanoparticles 20 and the magnetic nanoparticles 70 into contact with each other through chemical bonding with a known linker will be exemplified below.

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

The nanocomposite particles 100 having the above configuration are immunologically excellent in visual judgment, especially in a low concentration range (high sensitivity range), for example, by adsorbing an antigen or an antibody on the surface of the metal nanoparticles 20. It can be preferably applied as a material for a labeling substance for physical measurement or a reagent for immunological measurement. Furthermore, since the magnetic nanoparticles 70 are immobilized on the resin nanoparticles 10 and/or the metal nanoparticles 20, it is extremely effective for samples such as blood, urine, saliva, and environmental water, which are normally below the detection limit. Antigens (antibodies) present in only a small amount can also be detected by magnetically concentrating the antigen-antibody reaction. Therefore, for example, EIA, RIA, CLIA, FIA, LIA, PA, ICA, HA, HI, etc. for the purpose of immunological measurement labeled substances, immunological measurement reagents, etc. that enable highly sensitive determination It can be preferably applied. The form of the labeling substance for immunological measurement or the reagent for immunological measurement is not particularly limited. Available.

The method for adsorbing antigens or antibodies to the surface of the metal nanoparticles 20 is not particularly limited, and known methods based on physical adsorption and chemical adsorption can be used. A method using chemisorption is preferable because the binding between the metal nanoparticles 20 and the antigen or antibody becomes stronger. Physical adsorption and chemical adsorption may be used in combination.
Examples of physical adsorption include a method of immersing the nanocomposite particles 100 in a buffer containing an antigen or antibody and incubating, a method of immersing the nanocomposite particles 100 in a buffer, and then adding an antigen or an antibody. is mentioned.
As chemisorption, an SH group is introduced into the antigen or antibody and reacted with the nanocomposite particle 100 to form a metal-SH bond. Examples thereof include a method of forming a chemical bond by reacting with an amino group of an antigen or an antibody. Suitable compounds for introducing carboxyl groups onto the surface of nanocomposite particles 100 are compounds having both functional groups and carboxyl groups that have coordinative bonding with metals, such as amino groups, SH groups, carbonyls, amides, and imides. ing. SH groups are preferred as the functional groups because of their strong coordination bonds with metals, and examples of compounds having SH groups and carboxyl groups at both ends include mercaptopropionic acid, mercaptoundecanoic acid, and mercaptolauric acid. is mentioned. In addition, a polymer compound having a plurality of functional groups and carboxyl groups in one molecule having coordinative bonding with a metal is 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 nanocomposite particles 100 .

Next, an analyte measurement method using the nanocomposite particles 100 as a labeling substance, a test strip for lateral flow chromatography, and an analyte detection/quantification kit will be described.

[Test strips for lateral flow chromatography]
First, with reference to FIG. 2, a lateral flow chromatography test strip (hereinafter sometimes simply referred to as "test strip") according to an embodiment of the present invention will be described. As will be described later, this test strip 200 can be preferably used in the analyte measuring method of one embodiment of the present invention.

Test strip 200 includes membrane 110 . The membrane 110 is provided with a sample addition section 120, a determination section 130, and a liquid absorption section 140 in order in the sample development direction.

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

The test strip 200 preferably has a support that supports the membrane 110 for easier handling. As the support, for example, plastic or the like can be used.

<Sample addition part>
Test strip 200 may have sample application portion 120 for application of a sample containing analyte 160 . Sample application portion 120 is a portion for receiving a sample containing analyte 160 into test strip 200 . The sample application part 120 may be formed on the membrane 110 upstream of the determination part 130 in the direction in which the sample develops, or may be formed of, for example, cellulose filter paper, glass fiber, polyurethane, polyacetate, cellulose acetate, or nylon. A sample application pad made of a material such as cotton cloth may be provided on the membrane 110 to form the sample application section 120 .

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

Moreover, determination unit 130 is not particularly limited as long as it is configured such that complex 170 containing labeled antibody 150 and analyte 160 contacts capture ligand 131 that specifically binds to analyte 160 . For example, the capture ligand 131 may be directly immobilized on the membrane 110, or the capture ligand 131 may be immobilized on a pad made of cellulose filter paper, glass fiber, non-woven fabric, etc., immobilized on the membrane 110. .

<Liquid absorbing part>
The liquid absorbing portion 140 is formed of, for example, a pad of a water absorbing material such as cellulose filter paper, nonwoven fabric, cloth, cellulose acetate, or the like. The movement speed of the sample after the development front (front line) of the added sample reaches the liquid absorbing portion 140 varies depending on the material, size, and the like of the liquid absorbing portion 140 . Therefore, by selecting the material, size, etc. of the liquid absorbing portion 140, the optimum speed for detecting and quantifying the analyte 160 can be set. Note that the liquid absorbing portion 140 has an arbitrary configuration and may be omitted.

The test strip 200 may further include optional parts such as a control part, if desired.

<Control section>
Although illustration is omitted, the test strip 200 has a control portion in which a capture ligand that specifically binds to the labeled antibody 150 is immobilized on the membrane 110 downstream of the determination portion 130 in the direction in which the sample develops. may be formed. By measuring the color development intensity in the control unit as well as in the determination unit 130, the sample applied to the test strip 200 develops and reaches the reaction unit and the determination unit 130, confirming that the test was performed normally. be able to. The control section is prepared in the same manner as the determination section 130 described above, except that the capture ligand 131 is replaced with another type of capture ligand that specifically binds to the labeled antibody 150. configuration can be adopted.

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

The method for measuring analyte 160 of the present embodiment is a method for measuring analyte 160 for detecting or quantifying analyte 160 contained in a sample. The method for measuring the analyte 160 of the present embodiment uses a test strip 200 that includes a membrane 110 and a determination section 130 in which a capture ligand 131 that specifically binds to the analyte 160 is immobilized on the membrane 110 . Then, the method for measuring the analyte 160 of the present embodiment includes the following steps (I) to (IV);
Step (I): A step of contacting the analyte 160 contained in the sample with the labeled antibody 150 labeled with the nanocomposite particles 100 that specifically binds to the analyte 160;
Step (II): a step of magnetically recovering a complex 170 containing the analyte 160 and the labeled antibody 150;
Step (III): A step of contacting the complex 170 containing the analyte 160 and the labeled antibody 150 collected in the step (II) with the capture ligand 131 in the determination section 130 of the test strip 200;
Step (IV): a step of measuring the intensity of color development derived from light energy absorption by localized surface plasmon resonance and/or electronic transition of the nanocomposite particles 100;
can 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 the complex 170 containing the analyte 160 and the labeled antibody 150 is formed, the mode of contact is not particularly limited. For example, analyte 160 in the sample may be contacted with labeled antibody 150 prior to subjecting the sample to test strip 200 . As described above, the nanocomposite particles 100 of the present invention are particularly effective against antigens (antibodies) present in extremely small amounts in specimens such as blood, urine, saliva, environmental water, etc., which are usually below the detection limit. However, it is possible to detect the analyte 160 in the sample by magnetically concentrating it after causing the antigen-antibody reaction. Contacting with labeled antibody 150 is a preferred embodiment. Of course, it goes without saying that contact can be made in specimens with moderate to high concentrations of antigens (antibodies).

Step (II):
Step (II) is a step of magnetically collecting the complex 170 containing the analyte 160 and the labeled antibody 150 . For example, by applying a magnetic force using a known magnet to a specimen containing the antigen (antibody), the complex 170 containing the analyte 160 and the labeled antibody 150 is locally concentrated and collected in the specimen. can be done. In addition, you may use the centrifugation method together at the time of collection|recovery.

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

Since the capturing ligand 131 does not specifically bind to the labeled antibody 150, when the analyte 160 and the unbound labeled antibody 150 reach the determination unit 130, the analyte 160 and the unbound labeled antibody 150 passes through the determination unit 130 . Here, when the test strip 200 is formed with a control section (not shown) in which another capture ligand that specifically binds to the labeled antibody 150 is immobilized, the labeled antibody 150 that has passed through the determination section 130 is developed and bind to the other capture ligand at the control portion. As a result, labeled antibody 150 not forming complex 170 with analyte 160 is captured by the control section.

After step (III) and before step (IV) if necessary, the test strip 200 is washed with a buffer commonly used in biochemical tests, such as water, physiological saline, and phosphate buffer. A washing step may be performed. The washing step removes the labeled antibody 150 not captured by the determination unit 130 or the determination unit 130 and the control unit (the labeled antibody 150 that is not bound to the analyte 160 and does not form the complex 170). be able to.

By performing the washing step, in step (IV), coloration due to light energy absorption by localized surface plasmon resonance and/or electronic transition of the nanocomposite particles 100 in the determination unit 130 or in the determination unit 130 and the control unit When measuring , the intensity of background color development can be reduced, the signal/background ratio can be increased, and detection sensitivity and quantification can be further improved.

Step (IV):
Step (IV) is a step of measuring the intensity of color development resulting from optical energy absorption by localized surface plasmon resonance and/or electronic transition of the nanocomposite particles 100 . After performing the step (III) or washing step as necessary, the test strip 200 measures the intensity of color development derived from light energy absorption by localized surface plasmon resonance and/or electronic transition of the nanocomposite particles 100. do.

When the test strip 200 is formed with a control portion, the labeled antibody 150 is captured by another capturing ligand in the control portion to form a complex in step (III). Therefore, in the step (IV), in the test strip 200, not only the determination part 130 but also the control part can be colored by light energy absorption by localized surface plasmon resonance and/or electronic transition. In this way, by measuring the color development intensity in the control section as well as in the determination section 130, it can be confirmed whether the sample applied to the test strip 200 is normally developed and reaches the reaction section and the determination section 130.

<Sample and analyte>
The sample in the method for measuring an analyte of the present embodiment is not particularly limited as long as it contains a substance that can serve as an antigen, such as protein, as the analyte 160 . For example, a biological sample containing the analyte of interest 160 (i.e., whole blood, serum, plasma, urine, saliva, sputum, nasal or throat swabs, cerebrospinal fluid, amniotic fluid, nipple discharge, tears, sweat, exudates from the skin) , extracts from tissues, cells, and stool) and food extracts. If necessary, the analyte 160 contained in the sample is pretreated prior to the above step (I) in order to facilitate the specific binding reaction between the labeled antibody 150 and the capture ligand 131 and the analyte 160. may Examples of the pretreatment include chemical treatment using various chemicals such as acids, bases, and surfactants, and physical treatment using heating, stirring, ultrasonic waves, and the like. In particular, when the analyte 160 is a substance that is not normally exposed on the surface, such as an influenza virus NP antigen, it is preferable to perform treatment with a surfactant or the like. As the surfactant used for this purpose, a nonionic surfactant should be used in consideration of the specific binding reaction, for example, the binding reactivity between the capture ligand 131 and the analyte 160 such as an antigen-antibody reaction. can be done.

In addition, the sample may be appropriately diluted with a solvent (water, physiological saline, buffer solution, etc.) or a water-miscible organic solvent used in ordinary immunological analysis methods.

As the analyte 160, known ones can be used without any particular limitation. Examples of the analyte 160 include proteins such as tumor markers, signaling substances, and hormones (including polypeptides, oligopeptides, etc.), nucleic acids (single- or double-stranded DNA, RNA, polynucleotides, oligonucleotides, etc.). , PNA (peptide nucleic acid), etc.) or substances having nucleic acids, sugars (including oligosaccharides, polysaccharides, sugar chains, etc.) or substances having sugar chains, other molecules such as lipids, labeled antibodies 150 and Although it is not particularly limited as long as it specifically binds to the capture ligand 131, for example, carcinoembryonic antigen (CEA), HER2 protein, prostate specific antigen (PSA), CA19-9, α-fetoprotein (AFP), immune Inhibitory 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 β-hemolytic streptococcal antigen, HBs antibody, HBs antigen, rotavirus, adenovirus, albumin, glycated albumin, coronavirus (SARS-CoV, MERS-CoV , SARS-CoV-2, mutant strains thereof, and constituents thereof) and the like. 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>
The labeled antibody 150 is used in step (I) to contact the analyte 160 contained in the 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, labeled with the nanocomposite particles 100 having a structure in which a plurality of metal nanoparticles 20 and magnetic nanoparticles 70 are immobilized on the resin nanoparticles 10. It will be. Here, “labeling” means that the nanocomposite particles 100 are directly or indirectly attached to the antibody to the extent that the nanocomposite particles 100 are not detached from the labeled antibody 150 in steps (I) to (IV). It means that it is fixed by chemical or physical bonding, adsorption, or the like. For example, the labeled antibody 150 may be an antibody to which the nanocomposite particles 100 are directly bound, or an antibody to which the nanocomposite particles 100 are bound via an arbitrary linker molecule. may be immobilized on insoluble particles.

In addition, in the present embodiment, the "antibody" is not particularly limited and known ones can be used, and those with strong anionic properties, those with strong cationic properties, and others can also be used. For example, in addition to polyclonal antibodies, monoclonal antibodies, antibodies obtained by genetic recombination, antibody fragments [e.g., H chain, L chain, Fab, F(ab′)2, etc.] having antigen-binding ability can be used. can. Moreover, any of IgG, IgM, IgA, IgE, and IgD may be used as the immunoglobulin. Antibody-producing animal species include humans and non-human animals (eg, mice, rats, rabbits, goats, horses, etc.). Specific examples of antibodies include anti-PSA antibody, anti-AFP antibody, anti-CEA antibody, anti-adenovirus antibody, anti-influenza virus antibody, anti-HCV antibody, anti-coronavirus antibody (where coronaviruses are SARS-CoV, MERS -CoV, SARS-CoV-2, including mutant strains thereof), anti-IgG antibodies, anti-human IgE antibodies, and the like. The antiviral antibody also includes antibodies against its constituent components.

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

[Step A]
In step A, nanocomposite particles 100 are mixed with antibodies under a first pH condition to obtain labeled antibodies 150 . In step A, it is preferable to bring the solid nanocomposite particles 100 dispersed in a liquid phase into contact with the antibody.

The first pH condition is preferably a condition in the range of pH 2 to 10 from the viewpoint of uniformly contacting the nanocomposite particles 100 and the antibody while maintaining the dispersion of the nanocomposite particles 100 and the activity of the antibody. A pH range of 5 to 9 is more preferred. If the conditions for binding the nanocomposite particles 100 and antibodies are less than pH 2, the antibodies may be denatured and deactivated due to strong acidity, and if the pH exceeds 10, aggregation occurs when the nanocomposite particles 100 and antibodies are mixed. and difficult to disperse. However, if the antibody is not deactivated by strong acidity, the treatment can be performed even at pH less than 2.

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

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

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

The second pH condition is, for example, preferably 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 pH 5 from the viewpoint of suppressing non-specific adsorption of the labeled antibody 150. It is more preferably within the range of ~9. If the blocking condition is less than pH 2, the antibody may be denatured and deactivated due to strong acidity, and if the pH exceeds 10, the labeled antibody 150 aggregates and becomes difficult to disperse.

Step B is preferably performed using a blocking buffer having a blocking agent adjusted to a second pH condition. For example, a blocking buffer adjusted to the above pH is added to a predetermined amount of the labeled antibody 150, and the labeled antibody 150 is uniformly dispersed in the blocking buffer. As the blocking buffer, it is preferable to use, for example, a protein solution that does not bind to the substance to be detected. The blocking agent that can be used in the blocking buffer solution is not particularly limited and known ones can be used. For example, proteins include bovine serum albumin, ovalbumin, casein, gelatin, and whey. More specifically, it is preferable to use a bovine serum albumin solution or the like adjusted to a predetermined concentration. The pH of the blocking buffer can be adjusted using, for example, hydrochloric acid, sodium hydroxide, and the like. Dispersion means such as sonication is preferably used for dispersing the labeled antibody 150 . Thus, a dispersion liquid in which the labeled antibody 150 is uniformly dispersed is obtained.

In the above steps A and B, the nanocomposite particles 100 having platinum nanoparticles as the metal nanoparticles 20 are unlikely to aggregate due to pH, and can be treated in a wide range of pH from acidic to alkaline. Therefore, the nanocomposite particles 100 of the present invention using platinum nanoparticles also have the advantage of being less subject to restrictions on the conditions for producing labeled antibodies.

As described above, a dispersion liquid of the labeled antibody 150 is obtained. From this dispersion, only the labeled antibody 150 can be fractionated as a solid portion by solid-liquid separation means such as centrifugation. In addition, washing treatment, storage treatment, and the like can be carried out as necessary. The cleaning treatment and storage treatment will be described below.

(washing treatment)
In the washing process, a washing 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 washing buffer. For dispersion, it is preferable to use a dispersion means such as ultrasonic treatment. Although the washing buffer is not particularly limited, for example, Tris buffer, glycinamide buffer, arginine buffer, etc., having a predetermined concentration adjusted within the pH range of 8 to 9 can be used. can. The pH of the washing buffer can be adjusted using, for example, hydrochloric acid, sodium hydroxide, and the like. The washing treatment of the labeled antibody 150 can be repeated multiple times as necessary.

(preservation processing)
In the storage treatment, a storage buffer is added to the labeled antibody 150 collected by solid-liquid separation means, and the labeled antibody 150 is uniformly dispersed in the storage buffer. For dispersion, it is preferable to use a dispersion means such as ultrasonic treatment. As the storage buffer, for example, a solution obtained by adding a predetermined concentration of an anti-aggregation agent and/or a stabilizer to a washing buffer can be used. Examples of anti-aggregating agents that can be used include sugars typified by sucrose, maltose, lactose and trehalose, and polyhydric alcohols typified by glycerin and polyvinyl alcohol. Examples of the stabilizer include, but are not limited to, proteins such as bovine serum albumin, ovalbumin, casein and gelatin. In this manner, storage treatment of the labeled antibody 150 can be performed.

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

[Analyte measurement kit]
The analyte measurement kit according to one embodiment of the present invention detects or quantifies the analyte 160 contained in the sample based on the analyte measurement method of the present embodiment using, for example, the test strip 200. It is a kit for

The analyte measurement kit of the present embodiment includes
a membrane 110;
a test strip 200 including a determination section 130 in which a capture ligand 131 that specifically binds to the analyte 160 is immobilized on a membrane 110;
a detection reagent containing a labeled antibody 150 in which an antibody that specifically binds to the analyte 160 is labeled with the nanocomposite particles 100;
contains. The analyte measurement kit of the present embodiment may further contain other components (for example, a developing solution, etc.), if necessary.

In using the analyte measurement kit according to the present embodiment, the analyte 160 in the sample and the labeled antibody 150 in the detection reagent are brought into contact to carry out steps (I) and (II), A sample may be supplied to the sample application portion 120 of the test strip 200, and step (III) and step (IV) may be sequentially performed. Of course, for example, when performing the step (I) in the specimen having a medium to high concentration of antigen (antibody), the analyte 160 and the labeled antibody are separated by a known centrifugation method or the like without magnetic concentration. A composite 170 containing 150 and 150 may be recovered. In that case, step (II) can be omitted.

In addition, applications of the nanocomposite particles 100 other than labeling substances for immunological measurements or reagents for immunological measurements include solid catalysts, pigments, paints, conductive materials, electrodes, sensor elements, recording materials, and magnetic fluids. Speakers and sealing materials, magnetic hyperthermia (magnetic hyperthermia), MRI contrast agents, drug delivery systems, specific substances (drugs, antigens, antibodies, receptors, haptens, enzymes, proteins, peptides, sugars, nucleic acids , hormones, pathogens, toxins, etc.), manipulation of cells, purification of blood (removal of pathogens).

EXAMPLES Next, the present invention will be specifically described by way of Examples, but the present invention is not limited to these Examples. Various measurements and evaluations in the following examples and comparative examples are as follows unless otherwise specified.

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

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

Solid content concentration (wt%) = [weight after drying (g) / weight before drying (g)] × 100

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

The supported amount (M2) of the magnetic nanoparticles was calculated by the following formula based on the above M and M1.
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 prepared by dropping a dispersion of resin nanoparticles, metal-resin composite particles, or nanocomposite particles onto a metallic mesh with a carbon support film was observed with a field emission scanning electron microscope (FE-SEM; Hitachi High-Technologies Corporation). The area-average diameter of arbitrary 100 resin nanoparticles, metal-resin composite particles, or nanocomposite particles was measured from the image observed by SU-9000.

<Measurement of average particle size of metal nanoparticles>
A substrate prepared by dropping the nanocomposite particle dispersion onto a metallic mesh with a carbon support film was observed with a field emission scanning electron microscope (FE-SEM; manufactured by Hitachi High-Technologies Corporation, SU-9000). The area average diameter of arbitrary 100 metal nanoparticles was measured and taken as the average particle diameter.

<Measurement of average particle size of magnetic nanoparticles>
A substrate prepared by dropping the nanocomposite particle dispersion onto a metallic mesh with a carbon support film was observed with a field emission scanning electron microscope (FE-SEM; manufactured by Hitachi High-Technologies Corporation, SU-9000). The area average diameter of arbitrary 100 magnetic nanoparticles was measured and taken as the average particle diameter.

<Zeta potential measurement>
The zeta potential was measured by an electrophoretic light scattering method using Malvern's Zetasizer Nano-ZS as a measuring device. A sample is diluted with pure water to 0.01 wt % and adjusted to each pH value in the range of pH 3 to 10 using hydrochloric acid or an aqueous NaOH solution, which is used as a measurement sample. After pH measurement using a pH meter (HORIBA LAQUA twin), zeta potential measurement was performed to measure the behavior of changes in zeta potential at multiple pH points from pH 3 to 10. The width of change in zeta potential was calculated from the maximum value of zeta potential in the acidic region of pH 3-6 and the minimum value of zeta potential in the alkaline region of pH 6-10. In addition, the zeta potential is close to 0 mV, the zeta potential of any one point larger than 0 mV, and the zeta potential of any one point smaller than 0 mV is obtained, and the pH at which the zeta potential is 0 mV, that is, zero The zero point of charge was calculated.

[Production 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, The mixture was stirred at 30° C. for 50 minutes and then at 60° C. for 30 minutes under a nitrogen stream. After stirring, 2,2-azobis(2-methylpropionamidine) dihydrochloride (0.250 g) dissolved in 18.00 g of pure water was added dropwise and stirred at 60°C for 3.5 hours to obtain an average particle size. Resin nanoparticles A-1 with a diameter of 359 nm were obtained. It was precipitated by centrifugation (9000 rpm, 40 minutes), and after removing the supernatant, it was dispersed again in pure water, and impurities were removed by dialysis. Thereafter, the concentration was adjusted to obtain 10 wt % resin nanoparticle dispersion liquid B-1.

[Example 1]
<Synthesis of platinum-resin composite particles>
After adding 54 g of pure water to B-1 (91.5 g), 400 mM chloroplatinic acid aqueous solution (100 g) was added and stirred at 30° C. for 3 hours. After this mixture was allowed 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. After that, the concentration was adjusted to obtain 5 wt % platinum ion-adsorbing resin particle dispersion liquid C-1.

Next, C-1 (20.6 g) was added to 1392 g of pure water, and while stirring at 3° C., a 132 mM dimethylamine borane aqueous solution (40 g) was added dropwise over 20 minutes. By stirring for 3 hours, platinum-resin composite particles D-1 having an average particle size of 367 nm were obtained. After concentrating D-1 by centrifugation, it was purified by dialysis treatment and the concentration was adjusted to obtain 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 F-1, the average particle size of the platinum nanoparticles was 3.5 nm, and the amount of platinum supported was 35.5 wt%.
When observed with a scanning transmission electron microscope (STEM), in this platinum-resin composite particle F-1, the platinum nanoparticles consisted of the platinum nanoparticles completely encapsulated in the resin nanoparticles and the platinum nanoparticles embedded in the resin nanoparticles. Partially exposed platinum particles having an enclosed part and a part exposed outside the resin nanoparticles, and at least part of the platinum nanoparticles are three-dimensionally distributed in the surface layer part of the resin nanoparticles. rice field. Incidentally, 97% of the platinum nanoparticles were present in a range of 40% of the particle radius in the depth direction from the surface of the resin nanoparticles.

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

[Example 2]
<Synthesis of gold-resin composite particles>
After adding 255 g of pure water to B-1 (91.5 g), 400 mM chloroauric acid aqueous 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. After that, the concentration was adjusted to obtain a 2.5 wt % gold ion-adsorbing resin particle dispersion liquid C-2.

Next, C-2 (43.3 g) was added to 1580 g of pure water, and while stirring at 3°C, a 528 mM dimethylamine borane aqueous solution (10.0 g) was added dropwise over 2 minutes, followed by 1 hour at 3°C. , and stirred at room temperature for 3 hours to obtain gold-resin composite particles D-1 having an average particle size of 363 nm. After concentrating D-1 by centrifugation, it was purified by dialysis treatment and the concentration was adjusted to obtain 1 wt % gold-resin composite particle dispersion liquid E-2. The absorbance of gold-resin composite particles F-2 in E-2 was 1.51. In addition, the average particle size of the gold nanoparticles in F-2 was 30 nm, and the supported amount of gold was 48.1 wt%.
When observed with a scanning transmission electron microscope (STEM), in this gold-resin composite particle F-2, the gold nanoparticles consisted of the encapsulated gold particles completely encapsulated in the resin nanoparticles and the gold nanoparticles embedded in the resin nanoparticles. Partially exposed gold particles having an enclosed portion and a portion exposed to the outside of the resin nanoparticles, and at least a portion of the gold nanoparticles are three-dimensionally distributed in the surface layer portion of the resin nanoparticles. rice field. In addition, 97% of the gold nanoparticles were present in a range of 40% of the particle radius in the depth direction from the surface of the resin nanoparticles.

<Synthesis of nanocomposite particles>
The gold-resin composite particle F-2 was 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 its surface. On the other hand, citric acid was reacted with superparamagnetic Fe ₃ O ₄ nanoparticles (average particle diameter of about 15 nm), which are magnetic nanoparticles, to obtain citric acid-capped Fe ₃ O ₄ nanoparticles (SPIONs). The citrate-capped Fe ₃ O ₄ nanoparticles have carboxyl groups on the surface. This PLL-coated gold-resin composite and citric acid-capped Fe ₃ O ₄ nanoparticles were subjected to an EDC coupling reaction using 1-ethyl-3-(-3-dimethylaminopropyl)carbodiimide hydrochloride (EDC). , Fe ₃ O ₄ nanoparticles were brought into contact with gold-resin composite particles F-2 via amide bonds to synthesize immobilized nanocomposite particles G-2. The average particle size of the nanocomposite particles G-2 was 380 nm. 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 nanocomposite particles G-1 were bound to the anti-CRP monoclonal antibody by inversion stirring at room temperature for 30 minutes.
Next, centrifugation is performed at 3000 rpm for 5 minutes, and after removing the supernatant, 0.5 mL of a 1 wt% aqueous solution of Block Ace (manufactured by DS BioPharma) is added to the sediment, and ultrasonic dispersion treatment is performed for 10 seconds. gone. Block treatment was performed by performing inversion stirring for 30 minutes at room temperature.
Next, after centrifuging at 3000 rpm for 5 minutes and removing the supernatant, 0.5 mL of a 50 mM Tris aqueous solution (pH 8) containing less than 0.1 wt% surfactant was added to the sediment. Second ultrasonic dispersion treatment was performed. This operation was repeated three times to perform washing treatment.
Next, centrifugation was performed at 3000 rpm for 5 minutes, and after removing the supernatant, the sediment was treated with less than 0.1 wt % surfactant and 50 mM Tris aqueous solution (pH 8) containing 5 wt % sucrose. By adding 5 mL and performing ultrasonic dispersion treatment for 10 seconds, a labeled antibody dispersion liquid in which the anti-CRP monoclonal antibody was labeled with the nanocomposite particles G-1 was obtained.

(Preparation of immunochromatographic strip)
An anti-CRP monoclonal antibody (CalBioReagents #M354) was applied to the center of a nitrocellulose membrane 3 having a width of 25 mm, and a test line (TL) 4 was drawn as a judgment part. Further, 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 application portion 2 (glass fiber nonwoven fabric) and a liquid absorbing portion 6 (cotton nonwoven fabric) were laminated as shown in the cross-sectional view of the immunochromatographic strip shown in FIG. Finally, an immunochromatographic strip was produced by cutting into 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 sample solution was prepared by diluting the CRP antigen (BIO-RAD CRP Ag HP antigen) to a predetermined concentration using the developing solution. A developing solution to which no antigen was added was used as a negative control sample solution. 3 μl of the labeled antibody dispersion was mixed with 50 μl of the sample liquid, and 35 μl of the mixture was added dropwise to the sample application portion 2 of the immunochromatographic strip to develop the sample. Thirty minutes after dropping the sample solution, the intensity of color development in the test line 4 was measured using an immunochromatographic reader (manufactured by Hamamatsu Photonics Co., Ltd.). Regarding the color development intensity of test line 4, 13 mABS or more was determined as positive (+), 3 mABS or more and less than 13 mABS was determined as false positive (±), and less than 3 mABS was determined as negative (-). The evaluation results by immunochromatography are shown in the table below. As a result of the evaluation, by immunochromatography using a labeled antibody obtained by labeling an anti-CRP monoclonal antibody with nanocomposite particles G-1, positive determination was possible up to a CRP antigen concentration of 1.2 ng/ml.

Table 1 shows the evaluation results by immunochromatography.

[Test Example 2]
(10-fold concentration by magnetic separation)
60 μl of the labeled antibody dispersion was mixed with 10 ml of the CRP positive control and negative control sample solutions diluted to a predetermined concentration to prepare a mixed solution of the sample solution and the labeled antibody. 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 specimen solution and the labeled antibody.
(Evaluation by immunochromatography)
35 μl of 1 ml of the 10-fold concentrated solution was added dropwise to the sample addition portion 2 of the immunochromato strip and evaluated in the same manner as in the above examples. The evaluation results by immunochromatography combined with 10-fold concentration by magnetic separation are shown in the table below. As a result of evaluation, positive determination was possible up to a CRP antigen concentration of 0.3 ng/ml when 10-fold concentration by magnetic separation was used in combination.

Table 2 shows the evaluation results by immunochromatography combined with 10-fold concentration by magnetic separation.

[Test Example 3]
(40-fold concentration by magnetic separation)
60 μl of the labeled antibody dispersion was mixed with 40 ml of the CRP positive control and negative control sample solutions diluted to a predetermined concentration to prepare a mixed solution of the sample solution and the labeled antibody. 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 specimen solution and the labeled antibody.
(Evaluation by immunochromatography)
35 μl of 1 ml of the 40-fold concentrated solution was added dropwise to the sample addition portion 2 of the immunochromato strip and evaluated in the same manner as in the above examples. The evaluation results by immunochromatography combined with 40-fold concentration by magnetic separation are shown in the table below. As a result of evaluation, positive determination was possible up to a CRP antigen concentration of 0.08 ng/ml when 40-fold concentration by magnetic separation was used in combination.

Table 3 shows the evaluation results by immunochromatography combined with 40-fold concentration by magnetic separation.

From the above results, by using the resin nanoparticles of the present invention and magnetically concentrating after the antigen-antibody reaction, analytes that are usually below the detection limit and are present in extremely small amounts in the specimen can be detected. It was confirmed that it is possible to detect with high sensitivity.

Although the embodiments of the present invention have been described above in detail for the purpose of illustration, the present invention is not limited to the above embodiments and various modifications are possible.

REFERENCE SIGNS LIST 1 support 2 sample application section 3 membrane 4 test line (TL)
5 control line 6 liquid absorption part 10 resin nanoparticles 20 metal nanoparticles 30 encapsulated particles 40 partially exposed particles 50 surface adsorption particles 60 surface layer part 70 magnetic nanoparticles 100 nanocomposite particles 110 membrane 120 sample addition part 130 determination part 131 capture Ligand 140 Absorbing Portion 150 Labeled Antibody 160 Analyte 170 Complex 200 Test Strip

Claims (13)

resin nanoparticles;
a plurality of metal nanoparticles having an average particle size of 1 to 100 nm, which are relatively smaller in particle size than the resin nanoparticles;
a plurality of magnetic nanoparticles having an average particle size of 1 to 50 nm and having a particle size relatively smaller than that of the resin nanoparticles;
with
an average particle size of 50 to 1100 nm,
Nanocomposite particles, wherein the metal nanoparticles and the magnetic nanoparticles are immobilized on the resin nanoparticles. 2. The nanocomposite particle of claim 1, wherein said metal nanoparticles are particles of gold, silver, copper, palladium, platinum, tin, rhodium, iridium or alloys thereof. the magnetic nanoparticles are iron, cobalt, nickel, _manganese , _{Fe2O3 , Fe3O4} , _{AFe2O4 (} where A means Mn, Co, Ni, Cu or Zn), FePt, 3. Nanocomposite particles according to claim 1 or 2, which are CoPt, FeNi or FeCo. 4. The nanocomposite particle according to any one of claims 1 to 3, wherein the resin nanoparticles are polymer particles having in their structure substituents capable of adsorbing metal ions. The nanocomposite particle according to any one of claims 1 to 4, wherein at least part of the metal nanoparticles are three-dimensionally distributed on the surface layer of the resin nanoparticles. The nanocomposite particle according to any one of claims 1 to 5, wherein 60 wt% to 100 wt% of the metal nanoparticles are present in the surface layer portion. The nanocomposite particle according to any one of claims 1 to 5, wherein the magnetic nanoparticles are immobilized on the surface of the resin nanoparticles and/or the metal nanoparticles. A labeling substance comprising the nanocomposite particles according to any one of claims 1 to 7. 9. The labeling substance according to claim 8, which is used by adsorbing an antigen or an antibody to the surface of said nanocomposite particles. An immunoassay method, characterized by using the labeling substance according to claim 8 or 9. 10. An immunoassay reagent, characterized by using the labeling substance according to claim 8 or 9. A method for measuring an analyte for detecting or quantifying an analyte contained in a sample,
The following steps (I) to (IV) using a lateral flow chromatographic test strip containing a membrane and a determination portion in which a capture ligand that specifically binds to the analyte is immobilized on the membrane;
Step (I): The analyte contained in the sample is contacted with an antibody that specifically binds to the analyte with a labeled antibody labeled with the nanocomposite particles according to claim 1, and the analyte is obtaining a complex comprising light and said labeled antibody;
Step (II): a step of recovering a complex containing the analyte and the labeled antibody by magnetic force;
Step (III): The complex containing the analyte and the labeled antibody recovered in the step (II) is brought into contact with the capture ligand in the determination section of the lateral flow chromatography test strip. process,
Step (IV): a step of measuring the intensity of color development derived from the localized surface plasmon resonance of the nanocomposite particles and the absorption of light energy by electronic transition;
A method for measuring an analyte, characterized by performing a step comprising: An analyte measurement kit for detecting or quantifying an analyte contained in a sample using a lateral flow chromatography test strip,
a test strip for lateral flow chromatography, comprising a membrane, and a determination section having a capture ligand that specifically binds to the analyte fixed to the membrane;
a detection reagent comprising a labeled antibody labeled with the nanocomposite particles of claim 1, wherein the antibody that specifically binds to the analyte is labeled;
Analyte measurement kit for detecting or quantifying an analyte containing

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