Nanocomposite particles, labeling substances, immunoassay methods, reagents for immunoassay, methods for measuring analytes, and kits for analyte measurement

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

Technical Field

[0001] The present invention relates to novel nanocomposite particles that can be preferably used for applications such as immunological measurements, a labeling substance using the same, an immunological measurement method, a reagent for immunological measurement, a method for measuring an analyte, and a kit for analyte measurement.

Background Art

[0002] Immunological measurement methods (also referred to as "immunoassays") are methods that utilize specific reactions between antigens and antibodies, which are one type of immune reaction, to qualitatively and quantitatively analyze trace components. Since the antigen-antibody reaction has high sensitivity and reaction selectivity, it is widely used in the above fields. There are various immunoassay methods depending on the measurement principle. For example, enzyme immunoassay (EIA), radioimmunoassay (RIA), chemiluminescent immunoassay (CLIA), fluorescence immunoassay (FIA), agglutination methods such as latex (LIA, PA), immunochromatography method (ICA), hemagglutination method (HA), hemagglutination inhibition method (HI), etc. can be mentioned. In addition to immunoassays, there are physical and chemical measurement methods, biological measurement methods, etc.

[0003] Immunoassays detect antigens or antibodies qualitatively or quantitatively from changes (concentration changes of antigens, antibodies, or complexes) when antigens and antibodies react to form a complex. 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 the labeling substance is an important factor that affects the detection ability in immunoassays. Also in the immunoassays exemplified above, red blood cells (in the case of HA), latex particles (in the case of LIA), fluorescent dyes (in the case of FIA), radioactive elements (in the case of RIA), enzymes (in the case of EIA), chemiluminescent substances (in the case of CLIA), etc. are used as the labeling substance.

[0004] By the way, when using colored fine particles as a labeling substance, it is expected that simpler measurement can be achieved because detection can be visually confirmed without using a special analyzer. Examples of such colored fine particles include colloidal particles of metals and metal oxides, latex particles colored with dyes, etc. (Patent Document 1, Patent Document 2, etc.). However, since the color tone of the above-mentioned colloidal particles is determined by the particle diameter and preparation conditions, it is difficult to obtain those with a desired distinct dark color tone, that is, there is a problem that the visibility is insufficient. In addition, the above-mentioned colored latex particles have a problem that the coloring effect by the dye is low and the visual determination property is insufficient. In addition, when trying to increase the amount of dye to solve this problem, the dye covers the surface of the latex, and the original surface state of the latex particles is impaired, so there is a problem that it becomes difficult to bind an antigen or an antibody. Also, there is a problem that increasing the coloring agent of the dye to color darkly does not necessarily lead to an improvement in performance, such as clogging in the pores of a chromatographic medium such as a membrane filter or non-specific aggregation of latex particles.

[0005] In order to improve the visibility of the above-mentioned labeling substance, an immunochromatographic method is disclosed in which after an antibody (labeled antibody) to which the labeling substance is bound reacts with an antigen to form a complex, the detection sensitivity of the labeling substance is amplified by further modifying these labeling substances with another metal. Also, an example of using gold colloid and colored latex together is disclosed (Patent Document 3). However, in these methods, the operation is complicated and stable amplification is difficult. In addition, since a special device is required and the measurement cost is high, the applicable applications and usage environments are considered to be limited.

[0006] As a solution to the problems in these prior arts, resin particles to which metal nanoparticles are bound are disclosed. For example, colored latex composed of gold nanoparticles bound to the surface of polymer-based latex particles is disclosed (Patent Document 4). By binding gold nanoparticles to the surface of polymer latex particles, the gold nanoparticles themselves serve as a coloring agent to improve visual judgment and detection sensitivity. Also, since the gold nanoparticles themselves have excellent binding properties to antigens or antibodies, it is said that a sufficient amount of antigen or antibody can be bound even when the gold nanoparticles are bound to such an extent that a sufficient dark color is obtained.

[0007] In addition, the present applicant has found a resin-metal composite in which a specific ratio of metal particles is present in the surface layer portion of resin particles and has proposed it previously (for example, Patent Document 5). This resin-metal composite is excellent in durability and visibility particularly in the use as an immunological measurement material, and enables highly sensitive determination without requiring the addition of a special device or working process.

Prior Art Documents

Patent Documents

[0008]

Patent Document 1

Patent Document 2

Patent Document 3

Patent Document 4

Patent Document 5

Summary of the Invention

Problems to be Solved by the Invention

[0009] From the above, the resin particles to which metal nanoparticles are bound are excellent in visibility and do not require the addition of a special device or working process, and thus are expected as a reagent for immunological measurement. However, in order to detect antigens such as viruses that exist in only extremely small amounts in specimens such as blood and have a great impact on the health of the patient carrying the blood, in addition to improving the visibility of the labeling substance alone, another technical approach is also necessary.

[0010] That is, the object of the invention is to provide, in immunoassay, not only excellent visibility but also a nano - composite particle capable of detecting analytes such as antigens and antibodies that exist in only extremely small amounts in specimens such as blood, urine, saliva, and environmental water, which are usually below the detection limit, by applying magnetic concentration. For example, in immunoassay, it is to provide nano - composite particles for immunoassay that enable highly sensitive determination.

Means for Solving the Problems

[0011] As a result of intensive research, the present inventors have found that metal nanoparticles and magnetic nanoparticles can solve the above problems by nano - composite particles immobilized on resin nanoparticles, and have completed the present invention.

[0012] That is, the nano - composite particles of the present invention include resin nanoparticles, a plurality of metal nanoparticles having an average particle diameter of 1 to 100 nm, which are relatively smaller in particle diameter than the resin nanoparticles, a plurality of magnetic nanoparticles having an average particle diameter of 1 to 50 nm, which are relatively smaller in particle diameter than the resin nanoparticles, and are characterized in that the average particle diameter is 50 to 1100 nm, the metal nanoparticles and the magnetic nanoparticles are immobilized on the resin nanoparticles.

[0013] In the nano - composite particles of the present invention, the metal nanoparticles may be particles of gold, silver, copper, palladium, platinum, tin, rhodium, iridium or alloys thereof.

[0014] The nano-composite particles of the present invention may be such that the magnetic nanoparticles may be iron, cobalt, nickel, manganese, Fe2O3, Fe3O4, AFe2O4 (where A means Mn, Co, Ni, Cu or Zn), FePt, CoPt, FeNi, or FeCo.

[0015] The nano-composite particles of the present invention may be such that the resin nanoparticles are polymer particles having a substituent capable of adsorbing metal ions in their structure.

[0016] The nano-composite particles of the present invention may be such that at least some of the metal nanoparticles are three-dimensionally distributed in the surface layer portion 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 surface of the resin nanoparticles and / or the metal nanoparticles.

[0017] The labeling substance of the present invention is characterized by comprising the nano-composite particles described in any of the above. Also, an antigen or an antibody may be adsorbed on the surface of the nano-composite particles for use.

[0018] The immunological measurement method and the immunological measurement reagent of the present invention are characterized by using the above labeling substance.

[0019] The method for measuring an analyte of the present invention is a method for measuring an analyte for detecting or quantifying an analyte contained in a sample, using a lateral flow type chromatographic test strip including a membrane and a determination unit in which a capture ligand specifically binding to the analyte is immobilized on the membrane, the following steps (I) to (IV); Step (I): A step of bringing the analyte contained in the sample into contact with a labeled antibody labeled with any of the above nano-composite particles and an antibody specifically binding to the analyte to obtain a complex containing the analyte and the labeled antibody, Step (II): A step of recovering the complex containing the analyte and the labeled antibody by magnetic force, Step (III): A step of bringing the complex containing the analyte and the labeled antibody, which was recovered in the step (II), into contact with the capture ligand at the determination unit of the test strip for the lateral flow chromatograph. Step (IV): A step of measuring the color development intensity derived from the localized surface plasmon resonance of the nano complex particles and the absorption of light energy by electron transfer. It is characterized by performing steps including this.

[0020] The kit for analyte measurement of the present invention is a kit for analyte measurement for detecting or quantifying an analyte contained in a sample, using a test strip for a lateral flow chromatograph, A test strip for a lateral flow chromatograph including a membrane and a determination unit in which a capture ligand that specifically binds to the analyte is immobilized on the membrane, A detection reagent including a labeled antibody in which an antibody that specifically binds to the analyte is labeled with any of the above nano complex particles.

Advantages of the Invention

[0021] In the nano complex particles of the present invention, since the metal nanoparticles and the magnetic nanoparticles are immobilized on the resin nanoparticles, in addition to the excellent visibility which is a feature of the resin particles to which the metal nanoparticles are bound, as exemplified in Patent Documents 4 and 5, and no additional special device or working process is required, even for an antigen (antibody) that is usually present in a very small amount in specimens such as blood, urine, saliva, environmental water, etc., which is below the detection limit, after an antigen-antibody reaction occurs, it can be detected by applying magnetic concentration. Therefore, for example, it can be preferably applied for the purpose of immunological measurement labeling substances, immunological measurement reagents, etc. that enable highly sensitive determination, such as EIA, RIA, CLIA, FIA, LIA, PA, ICA, HA, HI.

Brief Description of the Drawings

[0022]

Figure 1

Figure 2

Figure 3

Mode for Carrying Out the Invention

[0023] Hereinafter, embodiments of the present invention will be described in detail with reference to the drawings as appropriate. FIG. 1 is a schematic cross-sectional view of nano-composite particles according to an embodiment of the present invention. The nano-composite particles 100 include resin nano-particles (hereinafter, also simply referred to as "resin particles") 10, metal nano-particles 20, and magnetic nano-particles 70. In the nano-composite particles 100, the metal nano-particles 20 are immobilized on the resin nano-particles 10. The resin nano-particles 10 are particles relatively larger than the metal nano-particles 20. That is, in the nano-composite particles 100, a large number of metal nano-particles 20 with relatively small particle diameters are fixed to the resin nano-particles 10 with relatively large particle diameters. As shown in FIG. 1, the relationship between the particle diameter D1 of the entire nano-composite particles 100, the particle diameter D2 of the resin nano-particles 10, and the particle diameter D3 of the metal nano-particles 20 is D1 > D2 > D3. In addition, a plurality of magnetic nano-particles 70 are immobilized on the nano-composite particles 100. That is, in the nano-composite particles 100, a large number of magnetic nano-particles 70 are fixed to the resin nano-particles 10 and / or the metal nano-particles 20. As shown in FIG. 1, the relationship between the particle diameter D1 of the entire nano-composite particles 100, the particle diameter D2 of the resin nano-particles 10, and the particle diameter D4 of the magnetic nano-particles 70 is D1 > D2 > D4. In FIG. 1, the shape of the magnetic nano-particles 70 is shown as a polygon and is shown smaller than the metal nano-particles 20, but this is for the convenience of explanation only, and the shape and size are not limited. Also, in FIG. 1, the mode in which the magnetic nano-particles 70 are bonded to the surfaces of the resin nano-particles 10 and the metal nano-particles 20 is shown, but this is merely an example, and the state of existence of the magnetic nano-particles 70 is not limited, and for example, they may be embedded inside the resin nano-particles 10.

[0024] The average particle diameter of the nano-composite particles 100 is 50 to 1100 nm, the average particle diameter of the metal nanoparticles 20 constituting the nano-composite particles 100 is 1 to 100 nm, and the average particle diameter of the magnetic nanoparticles 70 is 1 to 50 nm. When the average particle diameter of the nano-composite particles 100 is less than 50 nm, the loading amount of the metal nanoparticles 20 tends to be small, so the coloring tends to be weaker than that of metal nanoparticles of the same size, and the magnetic concentration efficiency also tends to decrease. When the average particle diameter of the nano-composite particles 100 exceeds 1100 nm, when used as a labeling substance or reagent, it tends to clog easily in the pores of a chromatographic medium such as a membrane filter, and the dispersion stability also tends to decrease. The average particle diameter of the nano-composite particles 100 improves the dispersion stability when used as a labeling substance or reagent, and from the viewpoint of obtaining high detection sensitivity when the nano-composite particles 100 are used in immunological measurements, and further from the viewpoint of obtaining high magnetic concentration efficiency, the preferable lower limit is 100 nm, more preferably 250 nm, and even more preferably 300 nm. On the other hand, the preferable upper limit is 700 nm, and more preferably 600 nm. Here, the particle diameter of the nano-composite particles 100 means a value obtained by adding the length of the protruding portion of the partially exposed particles 40 or the surface adsorbed particles 50, which will be described later, to the particle diameter of the resin nanoparticles 10, and can be measured by an electron microscope method such as a scanning electron microscope (SEM) or a transmission electron microscope (TEM), a laser diffraction / scattering method, a dynamic light scattering method, or a centrifugal sedimentation method.

[0025] Also, when the average particle diameter of the metal nanoparticles 20 (that is, the average of the particle diameter D3 in FIG. 1) is less than 1 nm or exceeds 100 nm, the sensitivity tends to decrease because the localized surface plasmon resonance and the light energy absorption due to electron transition are difficult to occur. When gold particles are used as the metal nanoparticles 20, from the viewpoint of obtaining high detection sensitivity as a labeling substance for immunological measurements and a reagent for immunological measurements, the preferable lower limit of the average particle diameter of the gold particles is 1 nm, and more preferably 3 nm. On the other hand, the preferable upper limit is 70 nm, and more preferably 50 nm. When platinum particles are used as the metal nanoparticles 20, from the viewpoint of obtaining high detection sensitivity as a labeling substance for immunological measurement and a reagent for immunological measurement, the lower limit of the average particle diameter of the platinum particles is preferably 1 nm, more preferably 2 nm, and even 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 diameter of the metal nanoparticles 20 is measured by observation with a scanning electron microscope (SEM) or a transmission electron microscope (TEM).

[0026] Also, when the average particle diameter of the magnetic nanoparticles 70 is less than 1 nm, the magnetic force exhibited by the nano-composite particles 100 is weak. Therefore, after contacting an analyte in a sample with a labeled antibody (hereinafter referred to as "labeled antibody particles") labeled with the nano-composite particles 100 to form a complex containing the analyte and the labeled antibody particles (hereinafter referred to as "analyte-labeled antibody complex"), the efficiency of recovering the analyte-labeled antibody complex by magnetism, that is, the efficiency of magnetic concentration, tends to decrease. On the other hand, when it exceeds 50 nm, since the specific gravity of the nano-composite particles 100 is large, the motility and diffusibility in the sample are low, and the contact efficiency between the analyte and the labeled antibody particles in the sample and the efficiency of recovering the analyte-labeled antibody complex by magnetism tend to decrease. The lower limit is preferably 3 nm, more preferably 5 nm, and even 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 observation with a scanning electron microscope (SEM) or a transmission electron microscope (TEM).

[0027] The metal nanoparticles 20 do not contain nanoparticles containing magnetic elements (Mn, Fe, Co, Ni) (however, it is allowed that a trace amount of magnetic elements is contained as inevitable impurities to such an extent that the particles are not magnetic). That is, the metal nanoparticles 20 are nanoparticles of metals excluding magnetic materials. Also, from the viewpoint of ease of nano - size complexation between the metal nanoparticles 20 and the resin nanoparticles 10, the material is preferably particles of gold, silver, copper, palladium, platinum, tin, rhodium, iridium or alloys thereof. These metals can be used alone or as a composite such as an alloy. Here, for example, a gold alloy means an alloy composed of gold and a metal species other than gold and containing 10 wt% or more, preferably 50 wt% or more, more preferably 60 wt% or more of gold. Also, for example, a platinum alloy means an alloy composed of platinum and a metal species other than platinum and containing 1 wt% or more, preferably 10 wt% or more, more preferably 50 wt% or more, still more preferably 60 wt% or more of platinum. When used as a labeling substance for immunological measurement and a reagent for immunological measurement, more preferably, they are gold, platinum and palladium which are excellent in visibility and easy to immobilize antigens or antibodies. These are preferable because they exhibit absorption derived from localized surface plasmon resonance. Even more preferably, they are gold and platinum.

[0028] For example, a gold - resin composite using gold particles as the metal nanoparticles 20 is less likely to cause aggregation in a state of being bound to a ligand such as an antibody as compared with a metal - resin composite having particles of other metal species, and is extremely excellent in dispersibility. Furthermore, it is excellent in visibility and easy to immobilize antigens or antibodies. In particular, by controlling the particle diameter of the gold particles and the inter - particle distance between the gold particles, various colors such as red, purple, and blue can be exhibited. Therefore, when the gold - resin composite is used as a labeling substance for an immunochromatograph, labeling substances of various colors can be obtained.

[0029] For example, a platinum-resin composite using platinum particles as the metal nanoparticles 20 is less likely to aggregate when bound to a ligand such as an antibody compared to metal-resin composites having particles of other metal species, and has extremely excellent dispersibility. Also, platinum particles are resistant to alteration such as oxidation and have excellent storage stability. Furthermore, platinum particles exhibit absorption derived from localized surface plasmon resonance at a wide range of wavelengths from, for example, 250 nm to 900 nm, and furthermore, due to the expression of light energy absorption by electron transfer, exhibit a strong color close to black. Therefore, by using the platinum-resin composite as a labeling substance, high visibility can be obtained in immunological measurements, and the detection sensitivity of the analyte can also be enhanced. In this case, by using platinum particles, excellent detection sensitivity can be obtained with a smaller loading amount compared to particles of other metal species. Therefore, if the average particle diameter is equivalent, the platinum-resin composite is preferable because it exhibits significantly higher detection sensitivity compared to resin composites having particles of other metal species.

[0030] The metal nanoparticles 20 may consist of only a single metal species or may be an alloy of the metal species and another metal species. Here, in the case of the metal nanoparticles 20 consisting of only a single metal species, unavoidable impurities other than the metal species may be included. Also, in the case of an alloy with another metal, the other metal species is not particularly limited. For example, in the case of an alloy with gold, silver, copper, palladium, platinum, tin, rhodium, iridium are preferable. Also, in the case of an alloy with platinum, gold, silver, copper, palladium, tin, rhodium, iridium are preferable.

[0031] Also, from the viewpoints of the ease of nano-scale complexation between the magnetic nanoparticles 70 and the resin nanoparticles 10 and the strength of the magnetic force as the nano-composite particles 100, it is preferable that the magnetic nanoparticles 70 are iron, cobalt, nickel, manganese, Fe2O3, Fe3O4, AFe2O4 (where A means Mn, Co, Ni, Cu or Zn), FePt, CoPt, FeNi, or FeCo. More preferably, they are Fe3O4 and FeCo, which are soft magnetic materials with a high saturation magnetic flux density, and even more preferably Fe3O4. As the magnetic nanoparticles 70, commercially available products such as Iron Oxide Nanoparticles (Sigma-Aldrich Japan K.K.) can be preferably used, for example.

[0032] Further, the resin nanoparticles 10 are not limited in their structure and composition, but are preferably polymer particles having a substituent capable of adsorbing metal ions in their structure. For example, polymers capable of adsorbing anionic ions can be mentioned. As the polymer capable of adsorbing anionic ions, a nitrogen-containing polymer is particularly preferable. The nitrogen atom in the nitrogen-containing polymer is a precursor of the metal nanoparticles 20, [AuCl4], which is excellent in visibility and easy to immobilize an antigen or an antibody. - , [PtCl6] 2- etc. are preferable because they are easy to chemisorb anionic ions. In the present embodiment, in order to reduce the metal ions adsorbed in the nitrogen-containing polymer and form the metal nanoparticles 20, a part of the generated metal nanoparticles 20 becomes the encapsulated particles 30 or the partially exposed particles 40 described later. When [AuCl4] - is used, gold particles are formed, and when [PtCl6] 2- is used, platinum particles are formed. In addition, anionic ions such as silver, copper, palladium, tin, rhodium, and iridium can also be used. Also, 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") can chemisorb cationic ions such as Au + , Pt 2+ due to the contained carboxylic acid groups and sulfonic acid groups, and are therefore preferable. For example, by reducing the chemisorbed Au + , Pt 2+ and forming metal nanoparticles 20 (in this case, gold particles or platinum particles), it is possible to produce a structure similar to the above nitrogen-containing polymer particles. In addition, cationic ions that are precursors of metals such as silver, copper, palladium, tin, rhodium, and iridium can be used. On the other hand, as the resin nanoparticles 10 other than the nitrogen-containing polymer having a substituent capable of adsorbing metal ions in its structure, for example, polystyrene or the like can also be used. However, in this case, it is relatively difficult to adsorb the metal ions inside the resin. As a result, most of the generated metal nanoparticles 20 become surface-adsorbed particles 50. Since the surface-adsorbed particles 50 have a small contact area with the resin nanoparticles 10, the adhesive force between the resin and the metal is small, and the metal nanoparticles 20 tend to easily desorb from the resin nanoparticles 10. The above-mentioned nitrogen-containing polymer is a resin having a nitrogen atom in the main chain or side chain. For example, there are polyamine, polyamide, polypeptide, polyurethane, polyurea, polyimide, polyimidazole, polyoxazole, polypyrrole, polyaniline, melamine resin, etc. Preferably, it is a polyamine such as poly-2-vinylpyridine, poly-3-vinylpyridine, poly-4-vinylpyridine, etc. Also, when having a nitrogen atom in the side chain, for example, it can be widely used such as acrylic resin, phenol resin, epoxy resin, etc. Further, the polymer capable of adsorbing the cationic ion is a resin having a carboxylic acid group, a sulfonic acid group, etc. in the main chain or side chain. For example, polyacrylic acid, vinyl carboxylate, polyvinyl acetate, polyvinyl sulfonic acid, polystyrene sulfonic acid, cellulose, etc. can be widely used. The polymers capable of adsorbing anionic ions and the polymers capable of adsorbing cationic ions such as the above-mentioned nitrogen-containing polymer may be copolymers with known polymerizable monomers. Here, examples of the copolymer include random copolymers, block copolymers, alternating copolymers, and those in which polymers are crosslinked. Also, two or more types of monomers may be copolymerized to form the resin nanoparticles 10, or a monomer may be reacted with a functional group present on the surface of the resin nanoparticles 10 and further polymerized using it as a polymerization active end. The copolymer composition is not limited, but it is preferable that the monomer containing a substituent capable of adsorbing the metal ions is 10 mol% or more. More preferably, it is 50 mol% or more. The coincidence monomer can be selected without limitation according to the use of the nano composite particles 100. For example, for the use of improving the shape, size uniformity and dispersion stability of the resin nano particles 10, a polymerizable monomer having characteristics as a surfactant can be used. Such polymerizable monomers include, for example, polyethylene glycol methyl ether methacrylate and polyethylene glycol dimethacrylate. Also, for the use of improving the mechanical strength and shape stability of the resin nano particles 10, hydrophobic polymerizable monomers such as divinylbenzene and styrene can be used. In addition, when the resin nano particles 10 have a hydrolyzable group such as an ester bond, partial hydrolysis may be performed by acid treatment or alkali treatment. By hydrolysis, a group capable of adsorbing a cationic ion such as a carboxyl group is generated on the surface of the resin nano particles 10, and an effect of adsorbing metal ions can be obtained, which is preferable. As the acid, known acids can be used. From the viewpoint of promoting the hydrolysis reaction, strong acids such as hydrochloric acid and sulfuric acid are preferable. As the alkali, known alkalis can be used. From the viewpoint of promoting the hydrolysis reaction, strongly alkaline potassium hydroxide, sodium hydroxide aqueous solution, etc. are preferable.

[0033] In addition, the dispersion state of the metal nanoparticles 20 in the resin nanoparticles 10 of the nano-composite particles 100 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 encapsulated inside the resin nanoparticles 10. As one form of the former, a plurality of metal nanoparticles 20 may form a continuous film in contact with each other on the surface of the resin nanoparticles 10. Also, as one form of the latter, a core-shell structure with the resin nanoparticles 10 as the shell and the metal nanoparticles 20 as the core may be formed. Further, a 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, a part of the three-dimensionally distributed metal nanoparticles 20 may be partially exposed outside the resin nanoparticles 10, and the remaining part may be encapsulated in the resin nanoparticles 10. Specifically, as shown in FIG. 1, the metal nanoparticles 20 include metal nanoparticles completely encapsulated in the resin nanoparticles 10 (hereinafter also referred to as "encapsulated particles" 30), metal nanoparticles having a portion embedded in the resin nanoparticles 10 and a portion exposed outside 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). For example, when the nano-composite particles 100 are used as a labeling substance for immunological measurement or a reagent for immunological measurement, an antigen, an antibody, or a blocking agent is immobilized on the surface of the resin nanoparticles 10, the surface of the partially exposed particles 40, or the surface of the surface-adsorbed particles 50 for use. At this time, while the antigen, antibody, or blocking agent is immobilized on the partially exposed particles 40 and the surface-adsorbed particles 50, it is considered that they are difficult to be immobilized 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 light energy absorption by electron transfer in addition to localized surface plasmon resonance, not only the partially exposed particles 40 and the surface-adsorbed particles 50 but also the encapsulated particles 30 contribute to improving the visibility of the labeling substance for immunological measurement and the reagent for immunological measurement. Furthermore, in addition to the fact that 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, an anchor effect due to the embedding state is exhibited, so the physical adsorption force is strong and it is difficult to desorb from the resin nanoparticles 10. Also, the antigen, antibody, or blocking agent adsorbed on the partially exposed particles 40 or the surface-adsorbed particles 50 is difficult to desorb because it forms a coordination bond with the metal. Therefore, the durability and stability of the labeling substance for immunological measurement and the reagent for immunological measurement using the nano-composite particles 100 can be made excellent.

[0034] In the nano-composite particles 100, the entire surface of the encapsulated particles 30 is covered with the resin constituting the resin nanoparticles 10. Also, in the partially exposed particles 40, 5% or more and less than 100% of the surface area 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, more preferably 30% or more of the surface area. Also, in the surface-adsorbed particles 50, more than 0% and less than 5% of the surface area is covered with the resin constituting the resin nanoparticles 10.

[0035] Also, the loading amount of the metal nanoparticles 20 (the total of the encapsulated particles 30, the partially exposed particles 40, and the surface adsorbed particles 50) on the nanocomposite particles 100 is preferably 3 to 80 wt% with respect to the nanocomposite particles 100. Within this range, the nanocomposite particles 100 are excellent in visibility, visual judgment property, and detection sensitivity as a labeling substance. When the loading amount of the metal nanoparticles 20 is less than 3 wt%, the amount of immobilized antibody or antigen decreases, and the detection sensitivity tends to decrease. On the other hand, when the loading amount of the metal nanoparticles 20 exceeds 80 wt%, the particle diameter of the metal nanoparticles 20 significantly increases, and the light absorption characteristics by the metal nanoparticles 20 tend to decrease. The more preferable lower limit value of the loading amount of the metal nanoparticles 20 is 10 wt%, and more preferably 15 wt%. On the other hand, the more preferable upper limit value is 70 wt%, and more preferably 60 wt%.

[0036] Also, the loading amount of the magnetic nanoparticles 70 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 are excellent in the balance between the strength of the magnetic force and the motility and diffusibility in the specimen. That is, when the loading amount of the magnetic nanoparticles 70 is less than 1 wt%, the magnetic force of the nanocomposite particles 100 is weak, so after forming the analyte-labeled antibody complex, the efficiency of recovering the analyte-labeled antibody complex by magnetism, that is, the efficiency of magnetic concentration, tends to decrease. On the other hand, when it exceeds 50 wt%, since the specific gravity of the nanocomposite particles 100 is large, the motility and diffusibility in the specimen are low, and the contact efficiency between the analyte and the labeled antibody particles in the specimen and the efficiency of recovering the analyte-labeled antibody complex by magnetism tend to decrease. Also, for example, in the measurement of an analyte using a test strip, the development rate of the analyte-labeled antibody complex decreases, and the measurement efficiency tends to deteriorate. The more preferable lower limit value of the loading amount of the metal nanoparticles 20 is 5 wt%, and more preferably 10 wt%. On the other hand, the more preferable upper limit value is 40 wt%, and more preferably 30 wt%.

[0037] Also, it is preferable that 10 to 90 wt% of the metal nanoparticles 20 are partially exposed particles 40 and surface adsorbed particles 50. Within this range, the amount of antibody or antigen immobilized on the metal nanoparticles 20 can be sufficiently ensured, so the sensitivity as a labeling substance is high. A more preferable lower limit of the ratio occupied by the partially exposed particles 40 and the surface adsorbed particles 50 is 20 wt%, and a more preferable upper limit is 80 wt%.

[0038] Also, from the viewpoint of the durability of the labeling substance for immunological measurement and the reagent for immunological measurement, it is preferable that the surface adsorbed particles 50 are 20 wt% or less.

[0039] Also, when the nano-composite particles 100 are used for immunological measurement, in order to obtain excellent detection sensitivity, it is preferable that 60 to 100 wt% of the metal nanoparticles 20 are present in the surface layer portion 60. A more preferable lower limit is 75 wt%, and even more preferably 85 wt%. Also, it is preferable that 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, because the amount of antibody or antigen immobilized on the metal nanoparticles 20 can be sufficiently ensured, so the sensitivity as a labeling substance becomes high. In other words, it is good that 10 to 95 wt% of the metal nanoparticles 20 present in the surface layer portion 60 are encapsulated particles 30.

[0040] Here, the "surface layer portion" means a range of 50% of the particle radius, more preferably 40% of the particle radius, in the depth direction from the surface of the resin nanoparticles 10, based on the outermost position of the nano-composite particles 100 (that is, the protruding end portions of the partially exposed particles 40 or the surface-adsorbed particles 50). Further, the "three-dimensionally distributed" means that the metal nanoparticles 20 are dispersed not only in the plane direction of the resin nanoparticles 10 but also in the depth direction. As described above, since the encapsulated particles 30 also exhibit light energy absorption by electron transfer in addition to localized surface plasmon resonance, not only the partially exposed particles 40 and the surface-adsorbed particles 50 but also the encapsulated particles 30 contribute to the improvement of the visibility of the labeling substance for immunological measurement and the reagent for immunological measurement. From the viewpoint of such improvement in visibility, in the nano-composite particles 100, it is preferable that 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 encapsulated particles 30 are substantially absent near the center of the resin nanoparticles 10. More specifically, in order to effectively exhibit light energy absorption by electron transfer in addition to the localized surface plasmon resonance by the encapsulated particles 30, for example, when the particle diameter D2 of the resin nanoparticles 10 is 800 nm, 70 wt% or more, preferably 80 wt% or more, more preferably 90 to 100 wt% of the encapsulated particles 30 are present within a range of, for example, 0 to 200 nm in the depth direction from the surface of the resin nanoparticles 10. In particular, when the region (encapsulated particle distribution region) where all (100 wt%) of the encapsulated particles 30 are distributed is within a range of, for example, 0 to 100 nm from the surface of the resin nanoparticles 10, it is preferable because the expression of light energy absorption by electron transfer can be maximized in addition to the localized surface plasmon resonance by the encapsulated particles 30. Further, the nano-composite particles 100 may not have the encapsulated particles 30. For example, in the nano-composite particles 100, all of the metal nanoparticles 20 may be fixed to the surface of the resin nanoparticles 10 without overlapping in the radial direction of the resin nanoparticles 10. In this case, the metal nanoparticles 20 are composed of the partially exposed particles 40 and the surface-adsorbed particles 50.

[0041] In addition, the dispersion state of the magnetic nanoparticles 70 in the resin nanoparticles 10 of the nano-composite particles 100 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 encapsulated inside the resin nanoparticles 10. As one form of the former, a plurality of magnetic nanoparticles 70 may form a continuous film in contact with each other on the surface of the resin nanoparticles 10. As one form of the latter, a core-shell structure may be formed with the resin nanoparticles 10 as the shell and the magnetic nanoparticles 70 as the core. Also, a part of the magnetic nanoparticles 70 may be three-dimensionally distributed inside the resin nanoparticles 10. In this case, a 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 preferably immobilized on the surface of the metal-resin composite (that is, the one obtained by removing the magnetic nanoparticles 70 from the nano-composite particles 100) composed of the resin nanoparticles 10 and the metal nanoparticles 20. When immobilizing the magnetic nanoparticles 70 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 physical adsorption by electrostatic interaction or the like may be used, or chemical adsorption such as hydrogen bonding, coordination bonding, and covalent bonding may be used. Preferably, from the viewpoint of bonding stability, the resin nanoparticles 10 or the metal nanoparticles 20 and the magnetic nanoparticles 70 are preferably in contact via a chemical bond with a known linker. Here, a known substance or bond can be applied as the linker. For example, an amide bond, an ether bond, a saturated or unsaturated carbon-carbon bond can be mentioned.

[0042] [Method for manufacturing nano-composite particles] The method for manufacturing the nano-composite particles 100 is not particularly limited. However, as a preferred method below, first, a metal-resin composite composed of the resin nanoparticles 10 and the metal nanoparticles 20 is prepared, and then a method for fixing the magnetic nanoparticles 70 to the metal-resin composite will be described.

[0043] The metal-resin composite is obtained, for example, by adding a solution containing metal ions to a dispersion of resin nanoparticles 10 produced by an emulsion polymerization method and adsorbing the metal ions onto the resin nanoparticles 10 (hereinafter referred to as "metal ion-adsorbed resin particles"). Further, by adding the metal ion-adsorbed resin particles into a reducing agent solution, the metal ions are reduced to generate metal nanoparticles 20, and a metal-resin composite can be obtained.

[0044] For example, when producing gold particles as the metal nanoparticles 20, examples of the solution containing gold ions include an aqueous solution of chloroauric acid (HAuCl4). Also, a gold complex may be used instead of the gold ions. Also, when producing platinum particles as the metal nanoparticles 20, examples of the solution containing platinum ions include an aqueous solution of chloroplatinic acid (H2PtCl6), a solution of platinum chloride (PtCl2), etc. Also, a platinum complex may be used instead of the platinum ions.

[0045] Also, as the solvent of the solution containing metal ions, instead of water, water-containing alcohols or alcohols such as methanol, ethanol, n-propanol, isopropanol, n-butanol, sec-butanol, t-butanol, acids such as hydrochloric acid, sulfuric acid, nitric acid, etc. may be used. Also, additives such as water-soluble polymer compounds such as polyvinyl alcohol, surfactants, alcohols; ethers such as tetrahydrofuran, diethyl ether, diisopropyl ether; polyols such as alkylene glycol, polyalkylene glycol, their monoalkyl ethers or dialkyl ethers, glycerin; ketones such as acetone, methyl ethyl ketone, etc., various water-miscible organic solvents, etc. may be added to the solution as necessary. Such additives are effective in accelerating the reduction reaction rate of metal ions and controlling the size of the generated metal nanoparticles 20.

[0046] In addition, known reducing agents can be used. Examples of the reducing agent include sodium borohydride, dimethylamine borane, citric acid, sodium hypophosphite, hydrazine hydrate, hydrazine hydrochloride, hydrazine sulfate, formaldehyde, sucrose, glucose, ascorbic acid, erythorbic acid, sodium phosphinate, hydroquinone, Rochelle salt, and the like. Among these, sodium borohydride, dimethylamine borane, or citric acid is preferred. A surfactant can be added to the reducing agent solution and / or the pH of the solution can be adjusted as necessary. The pH adjustment can be performed using, for example, 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. Furthermore, the particle size of the produced metal nanoparticles 20 can be controlled by adjusting the reduction rate of metal ions according to the temperature of the reducing agent solution.

[0047] Also, when reducing the metal ions in the metal ion-adsorbing resin particles to produce the metal nanoparticles 20, the metal ion-adsorbing resin particles may be added to the reducing agent solution, or the reducing agent may be added to the metal ion-adsorbing resin particles. However, from the viewpoint of the ease of generating the encapsulated particles 30 and the partially exposed particles 40, the former is preferred.

[0048] In addition, in order to maintain the dispersibility of the metal-resin composite in water, a dispersant such as citric acid, poly-L-lysine, polyvinylpyrrolidone, polyvinylpyridine, polyvinyl alcohol, DISPERBYK 194, DISPERBYK 180, DISPERBYK 184 (manufactured by Big Chem Japan Co., Ltd.) may be added. Furthermore, the pH can be adjusted 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 to maintain the dispersibility.

[0049] Next, by immobilizing magnetic nanoparticles 70 on the fabricated metal-resin composite, nano-composite particles 100 are produced. As described above, the contact state between the magnetic nanoparticles 70 and the resin nanoparticles 10 is not particularly limited. Hereinafter, a method of bringing the resin nanoparticles 10 or the metal nanoparticles 20 into contact with the magnetic nanoparticles 70 through a chemical bond with a known linker will be exemplified.

[0050] When an amide bond is used as the linker, an amino group is imparted to one of the metal-resin composite or the magnetic nanoparticles 70, and a carboxyl group is imparted to the other, and they are reacted to form an amide bond. For example, the metal-resin composite is coated with a cationic polymer such as Poly-L-lysine, Poly-D-lysine, polyallylamine, or polyethyleneimine to synthesize a metal-resin composite having an amino group on the surface (hereinafter referred to as "amino group-containing composite particles"). On the other hand, 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 a carboxyl group on the surface (hereinafter referred to as "carboxyl group-containing magnetic particles"). Then, the amino group-containing composite particles and the carboxyl group-containing magnetic particles are reacted to form an amide bond. Here, a known method can be used to form the amide bond. For example, 1-ethyl-3-(-3-dimethylaminopropyl)carbodiimide hydrochloride (EDC) or N',N'-dicyclohexylcarbodiimide may be used as a crosslinking agent. Alternatively, the magnetic nanoparticles 70 may be treated with a known amino group-containing silane coupling agent such as aminoalkyltrialkoxysilane and used as magnetic nanoparticles 70 having a surface modified with an amino group. Further, the magnetic nanoparticles 70 having a surface modified with an amino group may be treated with a known acid anhydride and used as carboxyl group-containing magnetic particles.

[0051] The nano-composite particles 100 having the above configuration can be preferably applied, for example, as a labeling substance for immunological measurement or a material for a reagent for immunological measurement, which is excellent in visual determination in a low concentration range (high sensitivity range), by adsorbing an antigen or an antibody on the surface of the metal nanoparticles 20. Further, since the magnetic nanoparticles 70 are immobilized on the resin nanoparticles 10 and / or the metal nanoparticles 20, even for an antigen (antibody) that is usually present in a very small amount in specimens such as blood, urine, saliva, and environmental water, which is below the detection limit, after an antigen-antibody reaction occurs, it can be detected by applying magnetic concentration. Therefore, it can be preferably applied, for example, for the purpose of a labeling substance for immunological measurement, a reagent for immunological measurement, etc., which enable highly sensitive determination, such as EIA, RIA, CLIA, FIA, LIA, PA, ICA, HA, HI, etc. Also, although there is no particular limitation on the form of the labeling substance for immunological measurement or the reagent for immunological measurement, for example, it can be used as a dispersion in which the nano-composite particles 100 are dispersed in water or a buffer solution with adjusted pH.

[0052] The method for adsorbing an antigen or an antibody on the surface of the metal nanoparticles 20 is not particularly limited, and known methods by physical adsorption and chemical adsorption can be used. Since the binding between the metal nanoparticles 20 and the antigen or antibody becomes strong, the method by chemical adsorption is preferred. Physical adsorption and chemical adsorption may be used in combination. Examples of physical adsorption include a method of immersing the nano-composite particles 100 in a buffer solution containing an antigen or an antibody and incubating, a method of immersing the nano-composite particles 100 in a buffer solution and further adding an antigen or an antibody, etc. Examples of chemisorption include a method in which an SH group is introduced into an antigen or antibody and reacted with the nano-composite particles 100 to form a metal-SH bond, and a method in which a carboxyl group is introduced onto the surface of the nano-composite particles 100 and then succinimidylated and reacted with the amino group of an antigen or antibody to form a chemical bond. Examples of compounds for introducing a carboxyl group onto the surface of the nano-composite particles 100 include compounds having both a functional group capable of forming a coordination bond with a metal, such as an amino group, an SH group, a carbonyl group, an amide group, and an imide group, and a carboxyl group. Due to the strong coordination bond with the metal, an SH group is preferred as the functional group. Examples of compounds having an SH group at both ends include mercaptopropionic acid, mercaptoundecanoic acid, and mercaptolauric acid. In addition, a polymer compound having a plurality of functional groups capable of forming a coordination bond with a metal and carboxyl groups in one molecule is likely to obtain a stable bond with the nano-composite particles 100. For example, polyglutamic acid and polyaspartic acid, which are polypeptides of glutamic acid or aspartic acid, are more preferred as polymer compounds for introducing a carboxyl group onto the surface of the nano-composite particles 100.

[0053] Next, a method for measuring an analyte using the nano-composite particles 100 as a labeling substance, a test strip for lateral flow chromatography, and an analyte detection / quantification kit will be described.

[0054] [Test Strip for Lateral Flow Chromatography] First, with reference to FIG. 2, a test strip for lateral flow chromatography (hereinafter sometimes simply referred to as a "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 a method for measuring an analyte according to an embodiment of the present invention.

[0055] The test strip 200 includes a membrane 110. On the membrane 110, a sample addition part 120, a determination part 130, and a liquid absorption part 140 are provided in this order in the sample development direction.

[0056] <Membrane> As the membrane 110 used in the test strip 200, those generally used as membrane materials in common test strips are applicable. The membrane 110 is made of an inert substance (a substance that does not react with the analyte 160, various ligands, etc.) composed of a microporous material that exhibits, for example, capillary action and allows the sample to spread simultaneously when the sample is added. Specific examples of the membrane 110 include fibrous or non-woven fiber matrices, membranes, filter papers, glass fiber filter papers, cloths, cotton, etc. composed of polyurethane, polyester, polyethylene, polyvinyl chloride, polyvinylidene fluoride, nylon, cellulose derivatives, etc. Among these, membranes, filter papers, glass fiber filter papers, etc. composed of cellulose derivatives or nylon are preferably used, and more preferably nitrocellulose membranes, mixed nitrocellulose ester (a mixture of nitrocellulose and cellulose acetate) membranes, nylon membranes, and filter papers are used.

[0057] For easier operation, the test strip 200 preferably includes a support for supporting the membrane 110. As the support, for example, plastic or the like can be used.

[0058] <Sample addition part> The test strip 200 may have a sample addition part 120 for adding a sample containing the analyte 160. The sample addition part 120 is a part of the test strip 200 for receiving a sample containing the analyte 160. The sample addition part 120 may be formed on the membrane 110 upstream of the determination part 130 in the direction in which the sample spreads, or alternatively, a sample addition pad composed of a material such as cellulose filter paper, glass fiber, polyurethane, polyacetate, cellulose acetate, nylon, cotton cloth, etc. may be provided on the membrane 110 to constitute the sample addition part 120.

[0059] <Determination part> The determination unit 130 is fixed with a capture ligand 131 that specifically binds to the analyte 160. The capture ligand 131 can be used without particular limitation as long as it forms a specific binding 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 unit 130 even when a sample is provided to the test strip 200. The capture ligand 131 may be directly or indirectly fixed to the membrane 110 by physical or chemical bonding, adsorption, or the like.

[0060] Moreover, the determination unit 130 is not particularly limited as long as it is configured such that the complex 170 containing the labeled antibody 150 and the analyte 160 comes into contact with the capture ligand 131 that specifically binds to the analyte 160. For example, the capture ligand 131 may be directly fixed to the membrane 110, or the capture ligand 131 may be fixed to a pad made of cellulose filter paper, glass fiber, non-woven fabric, or the like that is fixed to the membrane 110.

[0061] <Liquid absorption part> The liquid absorption part 140 is formed by a pad of a water-absorbing material such as, for example, cellulose filter paper, non-woven fabric, cloth, or cellulose acetate. The moving speed of the sample after the development front line of the added sample reaches the liquid absorption part 140 will vary depending on the material, size, etc. of the liquid absorption part 140. Therefore, by selecting the material, size, etc. of the liquid absorption part 140, an optimal speed for detecting and quantifying the analyte 160 can be set. Note that the liquid absorption part 140 has an arbitrary configuration and may be omitted.

[0062] The test strip 200 may further include an arbitrary part such as a control part as needed.

[0063] <Control part> Although illustration is omitted, the test strip 200 may be formed with a control portion in which a capture ligand that specifically binds to the labeled antibody 150 is fixed to the membrane 110 on the downstream side of the determination portion 130 in the direction in which the sample develops. By measuring the color development intensity also in the control portion together with the determination portion 130, it can be confirmed that the sample applied to the test strip 200 has developed and reached the reaction portion and the determination portion 130, and that the inspection has been performed normally. The control portion can be produced in the same manner as the above-described determination portion 130 and can have the same configuration, except that another type of capture ligand that specifically binds to the labeled antibody 150 is used instead of the capture ligand 131.

[0064] [Method for Measuring Analyte] Next, a method for measuring the analyte 160 according to an embodiment of the present invention performed using the test strip 200 will be described.

[0065] The method for measuring the analyte 160 according to the present embodiment is a method for detecting or quantifying the analyte 160 contained in a sample. The method for measuring the analyte 160 according to the present embodiment uses a test strip 200 including a membrane 110 and a determination portion 130 in which a capture ligand 131 that specifically binds to the analyte 160 is fixed to the membrane 110. And the method for measuring the analyte 160 according to the present embodiment includes the following steps (I) to (IV); Step (I): A step of bringing the analyte 160 contained in the sample into contact with a labeled antibody 150 in which an antibody that specifically binds to the analyte 160 is labeled with the nano complex particles 100. Step (II): A step of recovering the complex 170 containing the analyte 160 and the labeled antibody 150 by magnetic force. Step (III): A step of bringing the complex 170 containing the analyte 160 and the labeled antibody 150 recovered in step (II) into contact with the capture ligand 131 at the determination portion 130 of the test strip 200. Step (IV): Measuring the color development intensity resulting from light energy absorption by the localized surface plasmon resonance and / or electron transition of the nanocomposite particles 100 may be included.

[0066] Step (I): Step (I) is a step of bringing the analyte 160 contained in the sample into contact with the labeled antibody 150. As long as a complex 170 containing the analyte 160 and the labeled antibody 150 is formed, the mode of contact is not particularly limited. For example, before supplying the sample to the test strip 200, the analyte 160 in the sample may be brought into contact with the labeled antibody 150. As described above, the nanocomposite particles 100 of the present invention can cause an antigen-antibody reaction even with an antigen (antibody) that is present in an extremely small amount in specimens such as blood, urine, saliva, and environmental water, which is usually below the detection limit. After that, it becomes possible to detect by applying magnetic concentration. Therefore, in a specimen having a low-concentration antigen (antibody), it is a preferred embodiment to bring the analyte 160 in the sample into contact with the labeled antibody 150. Of course, it goes without saying that it can be brought into contact in a specimen having a medium to high-concentration antigen (antibody).

[0067] Step (II): Step (II) is a step of recovering the complex 170 containing the analyte 160 and the labeled antibody 150 by magnetic force. For example, by applying magnetic force using a known magnet in the specimen having the antigen (antibody), the complex 170 containing the analyte 160 and the labeled antibody 150 can be locally concentrated and recovered in the specimen. Note that a centrifugation method may be used in combination during recovery.

[0068] Step (III): In step (III), the complex 170 containing the analyte 160 and the labeled antibody 150 recovered in step (II) is provided to the sample addition part 120 of the test strip 200 and developed on the test strip 200. In this way, in the determination part 130 of the test strip 200, the complex 170 is brought into contact with the capture ligand 131. When the complex 170 is brought into contact with the capture ligand 131, the capture ligand 131 specifically binds to the analyte 160 of the complex 170. As a result, the complex 170 is captured in the determination part 130.

[0069] Note that since the capture ligand 131 does not specifically bind to the labeled antibody 150, when the labeled antibody 150 that is not bound to the analyte 160 reaches the determination part 130, the labeled antibody 150 that is not bound to the analyte 160 passes through the determination part 130. Here, when a control part (not shown) in which another capture ligand that specifically binds to the labeled antibody 150 is fixed is formed on the test strip 200, the labeled antibody 150 that has passed through the determination part 130 continues to develop and binds to the another capture ligand in the control part. As a result, the labeled antibody 150 that does not form the complex 170 with the analyte 160 is captured in the control part.

[0070] After step (III) and, if necessary, before step (IV), for example, a washing step of washing the test strip 200 with a buffer solution commonly used in biochemical tests such as water, physiological saline, and phosphate buffer solution may be performed. By the washing step, the labeled antibody 150 (the labeled antibody 150 that is not bound to the analyte 160 and does not form the complex 170) that has not been captured in the determination part 130 or the determination part 130 and the control part can be removed.

[0071] By performing the washing step, in step (IV), when measuring the color development due to the localized surface plasmon resonance and / or electron transition of the nano-composite particles 100 in the determination unit 130, or in the determination unit 130 and the control unit, the background color development intensity can be reduced, the signal / background ratio can be increased, and the detection sensitivity and quantification can be further improved.

[0072] Step (IV): Step (IV) is a step of measuring the color development intensity derived from the localized surface plasmon resonance and / or light energy absorption due to electron transition of the nano-composite particles 100. After the above step (III) or the washing step is performed as necessary, in the test strip 200, the color development intensity derived from the localized surface plasmon resonance and / or light energy absorption due to electron transition of the nano-composite particles 100 is measured.

[0073] When the control unit is formed on the test strip 200, in step (III), the labeled antibody 150 is captured by another capture ligand in the control unit to form a complex. Therefore, in step (IV), in the test strip 200, not only in the determination unit 130 but also in the control unit, color development due to light energy absorption by localized surface plasmon resonance and / or electron transition can be caused. In this way, by measuring the color development intensity also in the control unit together with the determination unit 130, it can be confirmed whether the sample applied to the test strip 200 has developed normally and reached the reaction unit and the determination unit 130.

[0074] <Sample and Analyte> In the analyte measurement method of this embodiment, the sample is not particularly limited as long as it contains a substance that can be an antigen such as a protein as analyte 160. For example, biological samples containing the target analyte 160 (i.e., whole blood, serum, plasma, urine, saliva, sputum, nasal or throat swab, cerebrospinal fluid, amniotic fluid, nipple secretion, tears, sweat, extract from skin, extract from tissue, cells and feces, etc.) and extracts of foods, etc. may be mentioned. If necessary, prior to the above step (I), the analyte 160 contained in the sample may be pretreated in order to facilitate the specific binding reaction between the labeled antibody 150 and the capture ligand 131 and the analyte 160. Here, examples of the pretreatment include chemical treatment using various chemicals such as acids, bases, surfactants, etc., and physical treatment using heating, stirring, ultrasonic waves, etc. In particular, when the analyte 160 is a substance that is not usually exposed on the surface, such as influenza virus NP antigen, it is preferable to perform treatment with a surfactant or the like. As the surfactant used for this purpose, a nonionic surfactant can be used in consideration of the binding reactivity between the capture ligand 131 and the analyte 160 in a specific binding reaction, such as an antigen-antibody reaction.

[0075] Also, 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.

[0076] The analytes 160 are not particularly limited, and known ones can be used. Those with strong anionic properties, those with strong cationic properties, and others can also be used. Examples of the analytes 160 include proteins such as tumor markers, signal transduction substances, hormones (including polypeptides, oligopeptides, etc.), nucleic acids (including single-stranded or double-stranded DNA, RNA, polynucleotides, oligonucleotides, PNA (peptide nucleic acid), etc.) or substances having nucleic acids, sugars (including oligosaccharides, polysaccharides, sugar chains, etc.) or substances having sugar chains, lipids, and other molecules. As long as they specifically bind to the labeled antibody 150 and the capture ligand 131, they are not particularly limited. For example, carcinoembryonic antigen (CEA), HER2 protein, prostate-specific antigen (PSA), CA19-9, α-fetoprotein (AFP), immunosuppressive 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 streptococcus antigen, HBs antibody, HBs antigen, rotavirus, adenovirus, albumin, glycated albumin, coronavirus (SARS-CoV, MERS-CoV, SARS-CoV-2, their variants, and those containing their components), etc. Among these, antigens solubilized by nonionic surfactants are preferred, and antigens that form self-assemblies such as viral nucleoproteins are more preferred.

[0077] <Labeled Antibody> The labeled antibody 150 is used in step (I) to contact with 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 obtained by labeling an antibody that specifically binds to the analyte 160 with a nano-composite particle 100 having a structure in which a plurality of metal nanoparticles 20 and magnetic nanoparticles 70 are immobilized on resin nanoparticles 10. Here, "labeling" means that in steps (I) to (IV), the nano-composite particle 100 is directly or indirectly fixed to the antibody by chemical or physical bonding, adsorption, etc. to such an extent that the nano-composite particle 100 does not detach from the labeled antibody 150. For example, the labeled antibody 150 may be one in which the nano-composite particle 100 is directly bound to the antibody, or one in which the nano-composite particle 100 is bound via an arbitrary linker molecule, or one in which each is fixed to an insoluble particle.

[0078] In addition, in the present embodiment, as the "antibody", a known one can be used without particular limitation, and those with strong anionic properties, those with strong cationic properties, and others can also be used. For example, polyclonal antibodies, monoclonal antibodies, antibodies obtained by genetic recombination, and antibody fragments having a binding ability to an antigen [e.g., H chain, L chain, Fab, F(ab')2, etc.] can be used. Also, as the immunoglobulin, any of IgG, IgM, IgA, IgE, and IgD may be used. As the animal species for antibody production, in addition to humans, animals other than humans (e.g., mice, rats, rabbits, goats, horses, etc.) may be used. 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 (here, the coronavirus includes SARS-CoV, MERS-CoV, SARS-CoV-2, and their variants), anti-IgG antibody, anti-human IgE antibody, etc. Note that the anti-virus antibody also includes antibodies against its constituent components.

[0079] <Preferred method for preparing labeled antibody> Next, a preferred method for preparing the labeled antibody 150 will be described. The production of the labeled antibody 150 includes at least the following step A; Step A) A step of obtaining the labeled antibody 150 by mixing and binding the nano - composite particles 100 with an antibody under a first pH condition and preferably further includes step B; Step B) A step of treating the labeled antibody 150 under a second pH condition can be included.

[0080] [Step A] In step A, the nano - composite particles 100 are mixed with an antibody under a first pH condition to obtain the labeled antibody 150. In step A, it is preferable to bring the solid nano - composite particles 100 into contact with the antibody in a state where they are dispersed in a liquid phase.

[0081] From the viewpoint of uniformly contacting the nano - composite particles 100 and the antibody while maintaining the dispersion of the nano - composite particles 100 and the activity of the antibody, the first pH condition is preferably a condition within the range of pH 2 - 10, and more preferably, for example, within the range of pH 5 - 9. When binding the nano - composite particles 100 and the antibody, if the condition is below pH 2, the antibody may be denatured and inactivated due to strong acidity, and if it exceeds pH 10, aggregation may occur when the nano - composite particles 100 and the antibody are mixed, making dispersion difficult. However, if the antibody is not inactivated by strong acidity, treatment is also possible even below pH 2.

[0082] Step A is preferably carried out in a binding buffer adjusted to the first pH condition. For example, a predetermined amount of the nano - composite particles 100 is mixed with the binding buffer adjusted to the above - mentioned 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, etc.

[0083] Next, a predetermined amount of antibody is added to the obtained mixed solution, and the solution is sufficiently stirred and mixed to obtain a labeled antibody-containing solution. The labeled antibody-containing solution thus obtained can be separated by solid-liquid separation means such as centrifugation, etc., to collect only the labeled antibody 150 as the solid portion.

[0084] [Step B] In Step B, blocking is performed to suppress non-specific adsorption to the labeled antibody 150 by treating the labeled antibody 150 obtained in Step A under a second pH condition. In this case, the labeled antibody 150 separated by solid-liquid separation means is dispersed in the liquid phase under the second pH condition.

[0085] The second pH condition is preferably in the range of, for example, pH 2 to 10 from the viewpoint of maintaining the activity of the antibody and suppressing aggregation of the labeled antibody 150, and more preferably in the range of pH 5 to 9 from the viewpoint of suppressing non-specific adsorption of the labeled antibody 150. If the blocking condition is less than pH 2, the antibody may be denatured and inactivated due to strong acidity, and if it exceeds pH 10, the labeled antibody 150 will aggregate and dispersion will be difficult.

[0086] Step B is preferably carried out using a blocking buffer in which the blocking agent is adjusted to the second pH condition. For example, a predetermined amount of the labeled antibody 150 is added with the blocking buffer adjusted to the above pH, and the labeled antibody 150 is uniformly dispersed in the blocking buffer. As the blocking buffer, for example, it is preferable to use a solution of a protein that does not bind to the analyte. There is no particular limitation on the blocking agent that can be used in the blocking buffer, and known ones can be used. Those with strong anionic properties, those with strong cationic properties, and others can also be used. For example, if it is a protein, bovine serum albumin, ovalbumin, casein, gelatin, whey, etc. can be mentioned. More specifically, it is preferable to use a bovine serum albumin solution adjusted to a predetermined concentration. The pH of the blocking buffer can be adjusted using, for example, hydrochloric acid, sodium hydroxide, etc. For the dispersion of the labeled antibody 150, it is preferable to use a dispersion means such as ultrasonic treatment. In this way, a dispersion in which the labeled antibody 150 is uniformly dispersed is obtained.

[0087] In the above Step A and Step B, the nano-composite particle 100 having platinum nanoparticles as the metal nanoparticles 20 is less likely to aggregate due to pH and can be processed at a wide range of pH from acidic to alkaline. Therefore, the nano-composite particle 100 of the present invention using platinum nanoparticles also has the advantage of being less restricted by the production conditions of the labeled antibody.

[0088] As described above, a dispersion of the labeled antibody 150 is obtained. From this dispersion, only the labeled antibody 150 can be separated as a solid part by solid-liquid separation means such as centrifugation. Also, if necessary, washing treatment, storage treatment, etc. can be carried out. Hereinafter, the washing treatment and storage treatment will be described.

[0089] (Washing Treatment) For the washing treatment, 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 the dispersion, it is preferable to use dispersion means such as ultrasonic treatment. The washing buffer is not particularly limited, but for example, a Tris buffer, a glycine amide buffer, an arginine buffer, etc. at a predetermined concentration adjusted within the range of pH 8 to 9 can be used. The pH of the washing buffer can be adjusted using, for example, hydrochloric acid, sodium hydroxide, etc. The washing treatment of the labeled antibody 150 can be repeated a plurality of times as necessary.

[0090] (Storage treatment) For the storage treatment, a storage 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 storage buffer. For the dispersion, it is preferable to use 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 the washing buffer can be used. As the anti-aggregation agent, for example, saccharides represented by sucrose, maltose, lactose, trehalose, and polyhydric alcohols represented by glycerin, polyvinyl alcohol, etc. can be used. The stabilizer is not particularly limited, but for example, proteins such as bovine serum albumin, ovalbumin, casein, gelatin can be used. In this way, the storage treatment of the labeled antibody 150 can be performed.

[0091] In each of the above steps, if necessary, a surfactant and preservatives such as sodium azide and paraoxybenzoic acid ester can be used.

[0092] [Kit for analyte measurement] The kit for analyte measurement according to an embodiment of the present invention is a kit for detecting or quantifying the analyte 160 contained in a sample based on the analyte measurement method of this embodiment, for example, using the test strip 200.

[0093] The analyte measurement kit of this embodiment comprises a membrane 110, a test strip 200 including a determination unit 130 in which a capture ligand 131 that specifically binds to the analyte 160 is immobilized on the membrane 110, a detection reagent including a labeled antibody 150 in which an antibody that specifically binds to the analyte 160 is labeled with nano - composite particles 100, and includes. The analyte measurement kit of this embodiment may further include other components (such as a developing solution, etc.) as required.

[0094] When using the analyte measurement kit according to this embodiment, after bringing the analyte 160 in the sample into contact with the labeled antibody 150 in the detection reagent and performing steps (I) and (II), the sample may be supplied to the sample addition part 120 of the test strip 200, and steps (III) and (IV) may be sequentially performed. Of course, for example, when performing step (I) in a sample having a medium - to - high concentration of antigen (antibody), if the complex 170 containing the analyte 160 and the labeled antibody 150 can be recovered by a known centrifugation method or the like without applying magnetic concentration, step (II) can be omitted.

[0095] In addition, as applications other than the labeling substance for immunological measurement or the reagent for immunological measurement of the nano - composite particles 100, it can be preferably applied as a solid catalyst, pigment, paint, conductive material, electrode, sensor element, recording material, speaker and seal material taking advantage of the characteristics as a magnetic fluid, magnetic hyperthermia (magnetic thermotherapy), MRI contrast agent, drug delivery system, selective separation and collection of specific substances (such as pharmaceuticals, antigens, antibodies, receptors, haptens, enzymes, proteins, peptides, sugars, nucleic acids, hormones, pathogens, toxins, etc.), cell manipulation, and blood purification (removal of pathogens).

Example

[0096] Next, the present invention will be specifically described by way of examples, but the present invention is not limited in any way by these examples. Unless otherwise specified in the following examples and comparative examples, various measurements and evaluations are as follows.

[0097] <Measurement of absorbance of metal-resin composite> For the absorbance of the metal-resin composite, a metal-resin composite dispersion (dispersion medium: water) prepared to 0.01 wt% was placed in a quartz glass cell (optical path length 10 mm), and using a spectrophotometer (manufactured by Shimadzu Corporation, UV3600), the absorbance at 570 nm for the gold-resin composite and 400 nm for the platinum-resin composite was measured.

[0098] <Measurement of solid content concentration and metal loading> 1 g of the dispersion before concentration adjustment was placed in a magnetic crucible and dried at 70 °C for 3 hours. The weights before and after drying were measured, and the solid content concentration was calculated by the following formula.

[0099] Solid content concentration (wt%) = [weight after drying (g) / weight before drying (g)] × 100

[0100] In addition, the sample after the above drying treatment was further heat-treated at 500 °C for 5 hours, the weights before and after the heat treatment were measured, and the loading amount (M) of the metal and metal oxide in the nano-composite particles, or the metal loading amount (M1) in the metal-resin composite was calculated from the following formula, respectively. Loading amount M or M1 (wt%) = [weight after heat treatment (g) / weight before heat treatment (g)] × 100

[0101] The loading 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)] × 100

[0102] <Measurement of average particle diameter of resin nanoparticles, metal-resin composite particles or nano-composite particles> The area average diameter of any 100 resin nanoparticles, metal-resin composite particles, or nanocomposite particles was measured from an image obtained by observing 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 using a field emission scanning electron microscope (FE-SEM; SU-9000, manufactured by Hitachi High-Technologies Corporation).

[0103] <Measurement of the average particle diameter of metal nanoparticles> The area average diameter of any 100 metal nanoparticles was measured from an image obtained by observing a substrate prepared by dropping a nanocomposite particle dispersion onto a metallic mesh with a carbon support film using a field emission scanning electron microscope (FE-SEM; SU-9000, manufactured by Hitachi High-Technologies Corporation), and this was taken as the average particle diameter.

[0104] <Measurement of the average particle diameter of magnetic nanoparticles> The area average diameter of any 100 magnetic nanoparticles was measured from an image obtained by observing a substrate prepared by dropping a nanocomposite particle dispersion onto a metallic mesh with a carbon support film using a field emission scanning electron microscope (FE-SEM; SU-9000, manufactured by Hitachi High-Technologies Corporation), and this was taken as the average particle diameter.

[0105] <Zeta potential measurement> The zeta potential was measured by the electrophoresis light scattering method using a Zetasizer Nano-ZS manufactured by Malvern as the measuring device. The sample was diluted to 0.01 wt% with pure water, and the sample adjusted to each pH value in the range of pH 3 to 10 using hydrochloric acid or an aqueous NaOH solution was used as the measurement sample. After measuring the pH using a pH meter (HORIBA LAQUA twin), the zeta potential was measured to determine the behavior of the change in the zeta potential at multiple pH values from pH 3 to 10. The change width of the zeta potential was calculated from the maximum value of the zeta potential in the acidic region of pH 3 to 6 and the minimum value of the zeta potential in the alkaline region of pH 6 to 10. Also, a linear function connecting an arbitrary point of the zeta potential close to 0 mV, an arbitrary point of the zeta potential greater than 0 mV, and an arbitrary point of the zeta potential less than 0 mV was obtained, and the pH at which the zeta potential becomes 0 mV, that is, the zero point of charge, was calculated.

[0106] [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, and 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 the mixture was stirred at 60 °C for 3.5 hours to obtain resin nanoparticles A-1 with an average particle diameter of 359 nm. The precipitate was obtained by centrifugation (9000 rpm, 40 minutes), the supernatant was removed, and after redispersing in pure water, impurities were removed by dialysis treatment. Thereafter, the concentration was adjusted to obtain a 10 wt% resin nanoparticle dispersion B-1.

[0107] [Example 1] [Synthesis of Platinum-Resin Composite Particles] After adding 54 g of pure water to B-1 (91.5 g), 100 g of an aqueous solution of chloroplatinic acid (400 mM) was added, and the mixture was stirred at 30 °C for 3 hours. After allowing this mixed solution to stand for 24 hours, A-1 was precipitated by centrifugation (3000 rpm, 30 minutes), and the supernatant was removed to remove excess chloroplatinic acid. Thereafter, the concentration was adjusted to obtain a 5 wt% dispersion liquid C-1 of platinum ion adsorbing resin particles.

[0108] Next, C-1 (20.6 g) was added to 1392 g of pure water, and while stirring at 3 °C, an aqueous solution of dimethylamine borane (40 g, 132 mM) was added dropwise over 20 minutes. Then, the mixture was stirred at 3 °C for 1 hour and at room temperature for 3 hours to obtain platinum-resin composite particles D-1 with an average particle diameter of 367 nm. After concentrating D-1 by centrifugation, it was purified by dialysis treatment, and the concentration was adjusted to obtain a 1 wt% dispersion liquid E-1 of platinum-resin composite particles. The absorbance of the platinum-resin composite particles F-1 in E-1 was 1.86. Also, the average particle diameter of the platinum nanoparticles in F-1 was 3.5 nm, and the platinum loading was 35.5 wt%. When observed by a scanning transmission electron microscope (STEM), in this platinum-resin composite particle F-1, the platinum nanoparticles included encapsulated platinum particles completely encapsulated in the resin nanoparticles, partially exposed platinum particles having a part embedded in the resin nanoparticles and a part exposed outside the resin nanoparticles, and at least some of the platinum nanoparticles were three-dimensionally distributed in the surface layer portion of the resin nanoparticles. Incidentally, 97% of the platinum nanoparticles existed in the range of 40% of the particle radius in the depth direction from the surface of the resin nanoparticles.

[0109] <Synthesis of Nano-Composite Particles> Platinum-resin composite particles F-1 were coated with ε-Poly-L-lysine (PLL) to obtain PLL-coated platinum-resin composites. This PLL-coated platinum-resin composite has amino groups on its surface. On the other hand, superparamagnetic Fe3O4 nanoparticles (average particle diameter of about 15 nm), which are magnetic nanoparticles, were reacted with citric acid to obtain citric acid-capped Fe3O4 nanoparticles (SPIONs). This citric acid-capped Fe3O4 nanoparticle has a carboxyl group on its surface. This PLL-coated platinum-resin composite and the citric acid-capped Fe3O4 nanoparticles were subjected to an EDC coupling reaction using 1-ethyl-3-(-3-dimethylaminopropyl)carbodiimide hydrochloride (EDC), and Fe3O4 nanoparticles were contacted and immobilized on the platinum-resin composite particles F-1 by an amide bond to synthesize nano-composite particles G-1. The average particle diameter of the nano-composite particles G-1 was 380 nm. Also, the loading amount of the magnetic nanoparticles was 10 wt%.

[0110] [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 the mixture was stirred at room temperature for 3 hours. This mixed solution was centrifuged (3000 rpm, 30 minutes) to precipitate A-1, and the supernatant was removed to remove excess chloroauric acid. Then, the concentration was adjusted to obtain a 2.5 wt% gold ion-adsorbing resin particle dispersion C-2.

[0111] 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, and then stirred at 3 °C for 1 hour and at room temperature for 3 hours to obtain gold-resin composite particles D-1 with an average particle diameter of 363 nm. After concentrating D-1 by centrifugation, it was purified by dialysis treatment, and the concentration was adjusted to obtain a 1 wt% gold-resin composite particle dispersion E-2. The absorbance of the gold-resin composite particles F-2 in E-2 was 1.51. Also, the average particle diameter of the gold nanoparticles in F-2 was 30 nm, and the gold loading amount was 48.1 wt%. Observation with a scanning transmission electron microscope (STEM) revealed that in these gold-resin composite particles F-2, the gold nanoparticles included encapsulated gold particles completely encapsulated by the resin nanoparticles, partially exposed gold particles having portions embedded within the resin nanoparticles and portions exposed outside the resin nanoparticles, and at least some of the gold nanoparticles were three-dimensionally distributed in the surface layer of the resin nanoparticles. Incidentally, 97% of the gold nanoparticles were present in the range of 40% of the particle radius in the depth direction from the surface of the resin nanoparticles.

[0112] <Synthesis of Nanocomposite Particles> ε-Poly-L-lysine (PLL) was coated on the gold-resin composite particles F-2 to obtain a PLL-coated gold-resin composite. This PLL-coated gold-resin composite has amino groups on its surface. On the other hand, superparamagnetic Fe3O4 nanoparticles (average particle diameter of about 15 nm), which are magnetic nanoparticles, were reacted with citric acid to obtain citric acid-capped Fe3O4 nanoparticles (SPIONs). These citric acid-capped Fe3O4 nanoparticles have carboxyl groups on their surfaces. This PLL-coated gold-resin composite and the citric acid-capped Fe3O4 nanoparticles were subjected to an EDC coupling reaction using 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC), and nanocomposite particles G-2 in which the Fe3O4 nanoparticles were contacted and immobilized to the gold-resin composite particles F-2 by an amide bond were synthesized. The average particle diameter of the nanocomposite particles G-2 was 380 nm. Also, the loading amount of the magnetic nanoparticles was 11 wt%.

[0113] [Test Example] (Preparation of Labeled Antibody Dispersion) After mixing 25 μg of an anti-CRP monoclonal antibody (CalBioReagents #M353) and 0.45 mL of a 100 mM boric acid aqueous solution (pH 8.5), a dispersion containing 500 μg of nanocomposite particles G-1 was added. Inversion stirring was carried out at room temperature for 30 minutes to bind the anti-CRP monoclonal antibody to the nanocomposite particles G-1. Next, centrifugation was performed at 3000 rpm for 5 minutes. After removing the supernatant, 0.5 mL of a 1 wt% aqueous solution of Block Ace (manufactured by DS Biopharma) was added to the sediment, and ultrasonic dispersion treatment was carried out for 10 seconds. Inversion stirring was carried out at room temperature for 30 minutes to perform the blocking treatment. Next, centrifugation was performed at 3000 rpm for 5 minutes. After removing the supernatant, 0.5 mL of a 50 mM Tris aqueous solution (pH 8) containing a surfactant of less than 0.1 wt% was added to the sediment, and ultrasonic dispersion treatment was carried out for 10 seconds. This operation was repeated 3 times to perform the washing treatment. Next, centrifugation was performed at 3000 rpm for 5 minutes. After removing the supernatant, 0.5 mL of a 50 mM Tris aqueous solution (pH 8) containing a surfactant of less than 0.1 wt% and 5 wt% sucrose was added to the sediment, and ultrasonic dispersion treatment was carried out for 10 seconds to obtain a labeled antibody dispersion liquid in which the anti-CRP monoclonal antibody was labeled with nano composite particles G-1.

[0114] (Preparation of Immunochromatographic Strip) In the center of a 25-mm-wide nitrocellulose membrane 3, an anti-CRP monoclonal antibody (CalBioReagents #M354) was applied to draw a test line (TL) 4 as a determination 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 part. After drying at 50 °C, as shown in the cross-sectional view of the immunochromatographic strip in Fig. 3, a support 1 (laminate film), a sample addition part 2 (glass fiber non-woven fabric), and an absorbent part 6 (cotton non-woven fabric) were laminated. Finally, it was cut to a width of 3.5 mm to prepare an immunochromatographic strip.

[0115] (Preparation of Developing Solution) An aqueous solution (pH 7.1) containing 50 mM tris(hydroxymethyl)aminomethane, 150 mM sodium chloride, 1.0 wt% bovine serum albumin, and 2.0 wt% ceteth 20 was prepared as the developing solution.

[0116] [Test Example 1] (Evaluation by Immunochromatography) The test fluid of the CRP positive control was prepared by diluting the CRP antigen (BIO-RAD CRP Ag HP antigen) to a predetermined concentration using the developing solution. For the test fluid of the negative control, a developing solution without added antigen was used. 3 μl of the labeled antibody dispersion was mixed with 50 μl of the test fluid, and 35 μl of the mixture was dropped onto the sample application part 2 of the immunochromatography strip to develop the sample. The color development intensity of the test line 4 30 minutes after dropping the test fluid was measured using an immunochromatography reader (manufactured by Hamamatsu Photonics). Regarding the color development intensity of the test line 4, a value of 13 mABS or more was determined as positive (+), a value of 3 mABS or more and less than 13 mABS was determined as quasi-positive (±), and a value of less than 3 mABS was determined as negative (-). The evaluation results by the immunochromatography method are shown in the table below. As a result of the evaluation, by the immunochromatography method using the labeled antibody labeled with the anti-CRP monoclonal antibody with nano complex particles G-1, a positive determination was possible up to a CRP antigen concentration of 1.2 ng / ml.

[0117] The evaluation results by the immunochromatography method are shown in Table 1.

[0118]

Table 1

[0119] [Test Example 2] (10-fold Concentration by Magnetic Separation) To 10 ml of the test fluids of the CRP positive control and negative control diluted to a predetermined concentration, 60 μl of the labeled antibody dispersion was mixed to prepare a mixed solution of the test fluid and the labeled antibody. Magnetic separation concentration was performed using a magnetic separation column and a magnet to prepare 1 ml of a 10-fold concentrated solution of the test fluid and the labeled antibody. (Evaluation by Immunochromatography) 35 μl of the 10-fold concentrated solution was dropped onto the sample application part 2 of the immunochromatography strip, and evaluation was performed in the same manner as in the above example. The evaluation results by the immunochromatography method combined with 10-fold concentration by magnetic separation are shown in the table below. As a result of the evaluation, when combined with 10-fold concentration by magnetic separation, a positive determination was possible up to a CRP antigen concentration of 0.3 ng / ml.

[0120] Table 2 shows the evaluation results by the immunochromatography method combined with 10-fold concentration by magnetic separation.

[0121]

Table 2

[0122] [Test Example 3] (40-fold concentration by magnetic separation) To 40 ml of the test fluids of the CRP positive control and negative control diluted to a predetermined concentration, 60 μl of the labeled antibody dispersion was mixed to prepare a mixed solution of the test fluid and the labeled antibody. Magnetic separation concentration was performed using a magnetic separation column and a magnet to prepare 1 ml of a 40-fold concentrated solution of the test fluid and the labeled antibody. (Evaluation by immunochromatography method) 35 μl of the 1 ml of the 40-fold concentrated solution was dropped onto the sample addition part 2 of the immunochromatography strip, and evaluation was performed in the same manner as in the above example. The evaluation results by the immunochromatography method combined with 40-fold concentration by magnetic separation are shown in the following table. As a result of the evaluation, when combined with 40-fold concentration by magnetic separation, a positive determination was possible up to a CRP antigen concentration of 0.08 ng / ml.

[0123] Table 3 shows the evaluation results by the immunochromatography method combined with 40-fold concentration by magnetic separation.

[0124]

Table 3

[0125] From the above results, it was confirmed that by using the resin nanoparticles of the present invention and applying magnetic concentration after the antigen-antibody reaction, it is possible to highly sensitively detect an analyte that is usually present in an extremely small amount in a sample and is below the detection limit.

[0126] As described above, the embodiments of the present invention have been described in detail for illustrative purposes. However, the present invention is not limited to the above embodiments, and various modifications are possible.

Explanation of Reference Numerals

[0127] 1 Support 2 Sample addition section 3 Membrane 4 Test line (TL) 5 Control line 6 Liquid absorption section 10 Resin nanoparticles 20 Metal nanoparticles 30 Encapsulated particles 40 Partially exposed particles 50 Surface-adsorbed particles 60 Surface layer 70 Magnetic nanoparticles 100 Nanocomposite particles 110 Membrane 120 Sample addition section 130 Judgment section 131 Capture ligand 140 Liquid absorption section 150 Labeled antibody 160 Analyte 170 Complex 200 Test strip

Claims

1. Resin nanoparticles, a plurality of metal nanoparticles having an average particle diameter of 1 to 100 nm and a relatively smaller particle diameter than the resin nanoparticles, a plurality of magnetic nanoparticles having an average particle diameter of 1 to 50 nm and a relatively smaller particle diameter than the resin nanoparticles, comprising: having an average particle diameter of 50 to 1100 nm, wherein the metal nanoparticles are immobilized on the resin nanoparticles, and the magnetic nanoparticles are immobilized on the surface of the resin nanoparticles and / or the metal nanoparticles, a nano-composite particle.

2. The nano-composite particle according to claim 1, wherein the metal nanoparticles are particles of gold, silver, copper, palladium, platinum, tin, rhodium, iridium or an alloy thereof.

3. wherein the magnetic nanoparticles are iron, cobalt, nickel, manganese, Fe ２ O ３ , Fe ３ O ４ , AFe ２ O ４ (where A means Mn, Co, Ni, Cu or Zn), FePt, CoPt, FeNi, or FeCo, the nano-composite particles according to claim 1 or 2.

4. The nano-composite particle according to any one of claims 1 to 3, wherein the resin nanoparticles are polymer particles having a substituent capable of adsorbing metal ions in the structure.

5. The nano-composite particle according to any one of claims 1 to 4, wherein at least some of the metal nanoparticles are three-dimensionally distributed in the surface layer portion of the resin nanoparticles.

6. The nano-composite particle according to claim 5, wherein 60 wt% to 100 wt% of the metal nanoparticles are present in the surface layer portion.

7. A labeling substance comprising the nano-composite particle according to any one of claims 1 to 6.

8. The labeling substance according to claim 7, which is used by adsorbing an antigen or an antibody on the surface of the nano-composite particle.

9. An immunological measurement method characterized by using the labeling substance according to claim 7 or 8.

10. An immunological measurement reagent characterized by using the labeling substance according to claim 7 or 8.

11. An analite measurement method for detecting or quantifying an analite contained in a sample, using a lateral flow type chromatography test strip including a membrane and a determination unit in which a capture ligand specifically binding to the analite is immobilized on the membrane, the following steps (I) to (IV); Step (I): contacting the analite contained in the sample with a labeled antibody labeled with the nano-composite particle according to claim 1, which is an antibody specifically binding to the analite, to obtain a complex containing the analite and the labeled antibody, Step (II): recovering the complex containing the analite and the labeled antibody by magnetic force, Step (III): A step of bringing the complex containing the analite and the labeled antibody, which was recovered in the step (II), into contact with the capture ligand at the determination unit of the test strip for the lateral flow type chromatograph. Step (IV): A step of measuring the color development intensity derived from the localized surface plasmon resonance of the nano complex particles and the absorption of light energy by electron transfer. A method for measuring an analite, characterized by performing steps including the above.

12. An analite measurement kit for detecting or quantifying an analite contained in a sample, using a test strip for a lateral flow type chromatograph, A test strip for a lateral flow type chromatograph, including a membrane and a determination unit in which a capture ligand that specifically binds to the analite is immobilized on the membrane, A detection reagent containing a labeled antibody obtained by labeling an antibody that specifically binds to the analite with the nano complex particles according to claim 1, and An analite measurement kit for detecting or quantifying an analite, including the above.

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