JP7265315B2 - Metal nanoparticle-cellulose composite for immunological measurement, labeling substance, immunological measurement method, immunological measurement reagent, analyte measurement method, analyte measurement kit, and test strip for lateral flow chromatography - Google Patents

Description

The present invention provides, for example, a metal nanoparticle-cellulose composite that can be used for immunological measurement, a labeling substance using the same, an immunological measurement method, an immunological measurement reagent, an analyte measurement method, and an analyte measurement and a test strip for lateral flow chromatography.

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

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

By the way, when colored microparticles are used as the labeling substance, the detection can be visually confirmed without using a special analysis device, and thus it is expected that the measurement can be performed more easily. Examples of such colored fine particles include colloidal particles of metals and metal oxides, and latex particles colored with pigments (Patent Document 1, Patent Document 4, etc.). However, since the color tone of the colloidal particles is determined by the particle size and preparation conditions, it is difficult to obtain a desired clear and deep color tone, that is, the visibility is insufficient.
In addition, the above-mentioned colored latex particles have a problem that the effect of coloring with a dye is low and the visual judgment is insufficient. In order to solve this problem, if the coloring amount of the dye is increased, the dye covers the surface of the latex, impairing the original surface state of the latex particles, making it difficult to bind antigens or antibodies. was there. In addition, increasing the colorant of the dye to darken the color due to clogging in the pores of the chromatographic medium such as a membrane filter or causing non-specific aggregation of latex particles does not necessarily lead to an improvement in performance. There was also the problem of not sticking.

As a new material system that solves the above-mentioned material system problems, a colored latex composed of gold nanoparticles bound to the surfaces of polymer-based latex particles has been disclosed (Patent Document 2).
By binding gold nanoparticles to the surface of polymer-based latex particles, the gold nanoparticles themselves serve as a coloring agent to improve visual determination and detection sensitivity, while the gold nanoparticles themselves also have binding properties to antigens or antibodies. Therefore, it is considered that a sufficient amount of antigen or antibody can be bound even if gold nanoparticles are bound to such an extent that the color becomes sufficiently dark.

The colored latex is obtained by irradiating a dispersion of styrene-acrylic acid copolymer latex and HAuCl, which is a precursor of gold nanoparticles, with gamma rays to bind gold nanoparticles to the surface of the latex. However, in the colored latex, gold nanoparticles are bound only to the surface of the latex, so there is a limit to the amount of gold particles that cause surface plasmon resonance to be supported, and the gold nanoparticles are easily detached. As a result, the visibility and sensitivity as an immunoassay reagent may not be sufficient. In addition, since electromagnetic radiation such as gamma rays is applied, latex may be damaged. Furthermore, the specification of Patent Document 2 discloses the preferred ranges of the latex diameter and the gold nanoparticle diameter, but it is not clear whether these preferred ranges have been verified in Examples, and the preferred ranges are defined. There is almost no disclosure of conditions suitable for implementation, such as there is no basis for

In addition, US Pat. No. 6,200,000 discloses polymeric latex particles coated with metallic gold and suggests application to reagents that can be used in microscopy and immunoassay methods.

However, regarding the polymer latex particles coated with metallic gold, there is no disclosure of the material and particle size of the polymer latex particles. Furthermore, there is no verification of its effectiveness as a reagent that can be used in immunoassays. Therefore, its effectiveness as a reagent in metallic gold and polymer latex particles is unknown.

In addition, Patent Document 4 discloses a composite fine particle containing a metal core ultrafine particle and a polymer having a metal ion coordinating group and surrounding the metal core ultrafine particle. Application to various fields has been suggested. Further, in Examples, composite microparticles are disclosed in which gold nanoparticles obtained by reducing chloroauric acid are surrounded by crosslinked microparticles of a vinylpyridine-divinylbenzene copolymer. However, the detailed structure and physical properties of the composite microparticles have not been investigated, and their effects in specific applications such as immunoassay reagents are unknown.

From the above, resin particles bound or coated with metal nanoparticles are expected as reagents for immunological measurements, but the conventional techniques do not have sufficient durability and visibility. Moreover, even if the visibility is high, the applicable applications and usage environments are limited.

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

By the way, although the polymers constituting the resin particles disclosed in Patent Documents 2 to 5 are all excellent in chemical stability, there is concern about the difficulty of disposal at the time of disposal. Therefore, in the future, when resin particles are applied as materials for immunological measurements, it is expected that they will not only have excellent durability and visibility as reagents for immunological measurements, but also have a low environmental impact. considered to be an important factor.

A biodegradable polymer is an example of a resin material that has a low impact on the environment. Among the loads on the environment, the emission of carbon dioxide causes biodegradable polymers to be decomposed by microorganisms to produce carbon dioxide and water. Like synthetic polymers, biodegradable polymers also release carbon dioxide during their decomposition process, but the rate of release is slow, so the released carbon dioxide is absorbed by plants and biomass. As a result, it is less likely to cause global warming. In particular, natural biodegradable polymers such as plant-derived cellulose and crab shell-derived chitosan are considered carbon-neutral and do not affect the amount of carbon dioxide in the atmosphere when burned. It is attracting attention because it does not exist.
However, there have been no examples of applying biodegradable polymers as immunoassay reagents using resin particles bound or coated with metal nanoparticles.

JP-A-5-10950 JP 2009-168495 A JP-A-3-206959 JP 2009-227883 A International publication WO2016/002742

An object of the present invention is to provide a metal nanoparticle-resin composite for immunoassays, which not only has excellent durability and visibility but also has a low environmental load in immunoassays. It is in.

As a result of intensive research, the present inventors have found that the above problems can be solved by a metal nanoparticle-cellulose composite in which a plurality of metal nanoparticles are immobilized on cellulose particles, which is one of biodegradable polymers. He found the headline and completed the present invention.

That is, the metal nanoparticle-cellulose composite for immunological measurement of the present invention comprises cellulose particles and a plurality of metal nanoparticles relatively smaller than the cellulose particles, and the plurality of metal nanoparticles are It is characterized by being immobilized on the cellulose particles.

In the metal nanoparticle-cellulose composite for immunological measurement of the present invention, the average particle size of the metal nanoparticles may be in the range of 1 to 80 nm. In this case, the metal nanoparticle-cellulose composite may have an average particle size within the range of 50 to 1000 nm.

The metal nanoparticle-cellulose composite for immunological assays of the present invention may contain cellulose having a carboxyl group and/or an amino group as a substituent.

The metal nanoparticle-cellulose composite for immunological measurement of the present invention may contain a polysaccharide selected from the group consisting of chitin, chitosan, glucosamine, and pectin.

In the metal nanoparticle-cellulose composite for immunological determination of the present invention, the amount of the metal nanoparticles supported is in the range of 3 wt% to 70 wt% with respect to the weight of the metal nanoparticle-cellulose composite for immunological determination. may be

The labeling substance of the present invention comprises any of the metal nanoparticle-cellulose complexes for immunological measurement described above. In this case, an antigen or an antibody may be adsorbed on the surface of the metal nanoparticle-cellulose composite for immunological measurement.

The immunoassay method of the present invention uses any of the labeling substances described above.

An immunoassay reagent of the present invention comprises any of the metal nanoparticle-cellulose complexes for immunoassay described above.

The method for measuring an analyte of the present invention is a method for detecting or quantifying an analyte contained in a sample. This analyte measurement method uses a lateral flow chromatographic test strip that includes a membrane and a determination section in which a capture ligand that specifically binds to the analyte is immobilized on the membrane, and the following steps (I) to (III);
Step (I): The analyte contained in the sample and an antibody that specifically binds to the analyte are labeled with any of the metal nanoparticle-cellulose complexes for immunological measurement described above, and a labeled antibody. contacting;
Step (II): a step of contacting the complex formed in step (I) containing the analyte and the labeled antibody with a capture ligand in the determination unit;
Step (III): a step of measuring the intensity of color development derived from the localized surface plasmon resonance of the metal nanoparticle-cellulose composite and light energy absorption due to electronic transition;
characterized by performing a step including

The analyte measurement kit of the present invention is an analyte measurement kit for detecting or quantifying an analyte contained in a sample using a lateral flow chromatography test strip. This analyte measurement kit includes a lateral flow chromatographic test strip comprising a membrane, a determination section having a capture ligand that specifically binds to the analyte fixed to the membrane, and a test strip specific to the analyte. and a detection reagent containing a labeled antibody that binds to an antibody that is labeled with any of the metal nanoparticle-cellulose complexes for immunological measurement described above.

The lateral flow chromatography test strip of the present invention is for detecting or quantifying an analyte contained in a sample. This test strip for lateral flow chromatography comprises a membrane, a determination section in which a capture ligand that specifically binds to the analyte is immobilized on the membrane in the direction in which the sample develops, and On the upstream side, a reaction section containing a labeled antibody that specifically binds to the analyte is labeled with any of the metal nanoparticle-cellulose complexes for immunological measurement described above.

Since the metal nanoparticles for immunoassays of the present invention-cellulose contain many hydroxyl groups, they are excellent in dispersibility when bound to a ligand such as an antibody, and are less likely to aggregate. In addition, since it has a structure in which a plurality of metal nanoparticles are immobilized on cellulose particles, it is compared with composites in which metal nanoparticles or metal films are present on the surface of resin particles, as exemplified in Patent Documents 2 and 3. , the amount of metal nanoparticles supported is large. In addition, while cellulose is biodegradable, it does not contain organic dyes or organic pigments that can cause environmental pollution. can choose the method in which is small. Therefore, the metal nanoparticle-cellulose composite for immunological assays of the present invention can be used as a highly sensitive material that is excellent in handleability, durability, and visibility, and has a low environmental load, for example, EIA, RIA, It can be preferably applied for purposes such as labeling substances for immunological measurement such as CLIA, FIA, LIA, PA, ICA, HA, HI, and reagents for immunological measurement.

FIG. 2 is a schematic diagram showing the cross-sectional structure of a metal nanoparticle-cellulose composite according to one embodiment of the present invention. BRIEF DESCRIPTION OF THE DRAWINGS FIG. 1 is an explanatory diagram showing an overview of an analyte measurement method using a lateral flow chromatography test strip according to an embodiment of the present invention;

BEST MODE FOR CARRYING OUT THE INVENTION Hereinafter, embodiments of the present invention will be described in detail with reference to the drawings as appropriate. FIG. 1 is a schematic cross-sectional view of a metal nanoparticle-cellulose composite according to one embodiment of the present invention. The metal nanoparticle-cellulose composite 100 includes cellulose particles (hereinafter also simply referred to as “resin particles”) 10 and metal nanoparticles 20 . A metal nanoparticle-cellulose composite 100 has metal nanoparticles 20 immobilized on resin particles 10 . The resin particles 10 are particles relatively larger than the metal nanoparticles 20 . That is, in the metal nanoparticle-cellulose composite 100, a large number of metal nanoparticles 20 having a relatively small particle size are fixed to the resin particles 10 having a relatively large particle size. As shown in FIG. 1, the relationship between the particle diameter D1 of the entire metal nanoparticle-cellulose composite 100, which is a metal-resin composite, the particle diameter D2 of the resin particles 10, and the particle diameter D3 of the metal nanoparticles 20 is D1>D2>D3.
The metal nanoparticle-cellulose composite 100 is one embodiment of a metal-resin composite, and the metal-resin composite includes a gold-cellulose composite, a platinum-cellulose composite, a gold-resin composite, a platinum- Including resin composites and the like.

[Metal nanoparticle-cellulose composite]
In the metal nanoparticle-cellulose composite 100, part of the metal nanoparticles 20 may be three-dimensionally distributed in the surface layer portion 60 of the resin particle 10. In this case, part of the three-dimensionally distributed metal nanoparticles 20 may be partially exposed outside the resin particles 10 , and the remaining part may be included in the resin particles 10 . Specifically, as shown in FIG. Metal nanoparticles having an enclosed portion and a portion exposed to the outside of the resin particles 10 (hereinafter also referred to as "partially exposed particles 40") and metal nanoparticles adsorbed on the surface of the resin particles 10 (hereinafter, " Also referred to as "surface adsorbed particles 50") are preferably present.

When the metal nanoparticle-cellulose composite 100 is used as a labeling substance for immunological measurement or a reagent for immunological measurement, the surface of the resin particle 10 or the surface of the partially exposed particle 40 or the surface of the surface adsorbed particle 50 is coated with an antibody or Antigens are immobilized and used. At that time, the antibodies or antigens are immobilized on the partially exposed particles 40 and the surface-adsorbed particles 50 , but not on the encapsulated particles 30 . However, since all of the partially exposed particles 40, the surface adsorbed particles 50, and the encapsulated particles 30 exhibit optical energy absorption due to electronic transition in addition to localized surface plasmon resonance, the partially exposed particles 40 and the surface adsorbed particles 50 In addition, the encapsulated particles 30 also contribute to improving the visibility of the labeling substance for immunological measurement and the reagent for immunological measurement. Furthermore, the partially exposed particles 40 and the included particles 30 have a larger contact area with the resin particles 10 than the surface-adsorbed particles 50, and in addition, the anchor effect due to the embedded state is exhibited, so physical adsorption is achieved. It has a strong force and is difficult to detach from the resin particles 10 . Therefore, the labeling substance for immunological measurement and the reagent for immunological measurement using the metal nanoparticle-cellulose composite 100 can be made excellent in durability and stability.

In addition, the amount of metal nanoparticles 20 (total of encapsulated particles 30, partially exposed particles 40 and surface-adsorbed particles 50) supported on metal nanoparticles-cellulose composite 100 is the weight of metal nanoparticles-cellulose composite 100. On the other hand, it is preferably 3 wt % to 70 wt %. Within this range, the metal nanoparticle-cellulose composite 100 is excellent in visibility, visual determination and detection sensitivity as a labeling substance. If the supported amount of metal nanoparticles 20 is less than 3 wt %, the immobilized amount of antibody or antigen tends to decrease, resulting in a decrease in detection sensitivity. The supported amount of the metal nanoparticles 20 is more preferably 3 wt % to 70 wt %, still more preferably 3 wt % to 60 wt %.

Here, the “surface layer portion 60” refers to the outermost position of the metal nanoparticle-cellulose composite 100 (that is, the protruding end of the partially exposed particles 40 or the surface adsorbed particles 50), and the resin particles From the surface of 10, it means the range of 50% of the particle radius in the depth direction. The phrase “three-dimensionally distributed” means that the metal nanoparticles 20 are dispersed not only in the surface direction of the resin particles 10 but also in the depth direction.

Note that the metal nanoparticle-cellulose composite 100 may not have the encapsulated particles 30 . For example, in the metal nanoparticle-cellulose composite 100 , all of the metal nanoparticles 20 may be fixed on the surface of the resin particles 10 without overlapping in the radial direction of the resin particles 10 . In this case, the metal nanoparticles 20 are composed of partially exposed particles 40 and surface-adsorbed particles 50 .

Various substituents can be introduced into the cellulose constituting the resin particles 10 using hydroxyl groups in the molecule as reaction sites.
For example, a carboxyl group can be introduced into the resin particles 10 by reacting sodium chloroacetate with the hydroxyl group. Carboxyl groups can chemisorb cationic metal ions such as Au ³⁺ and Pt ²⁺ , which are precursors of metal nanoparticles 20 . Then, by reducing this to form metal nanoparticles 20, a metal nanoparticle-cellulose composite 100 having a structure in which a plurality of metal nanoparticles 20 are immobilized on the resin particles 10 can be produced. .
Further, for example, an amino group can be introduced into the resin particles 10 by reacting 2-chloroethylamine hydrochloride or 2-diisopropylaminoethyl chloride hydrochloride with the hydroxyl group. The amino group can chemisorb anionic metal ions such as [AuCl ₄ ] ⁻ , [PtCl ₆ ] ²⁻ , which are precursors of the metal nanoparticles 20 . Then, by reducing this to form metal nanoparticles 20, a metal nanoparticle-cellulose composite 100 having a structure in which a plurality of metal nanoparticles 20 are immobilized on the resin particles 10 can be produced. .
Since the cellulose constituting the resin particles 10 contains abundant hydroxyl groups in the molecule, by introducing the carboxyl groups and amino groups, the metal nanoparticles 20 can be stably added to the resin particles 10 in a sufficient amount. can be immobilized.

The degree of substitution of the carboxyl group or amino group (hereinafter collectively referred to as "metal ion adsorbing group") in the resin particles 10 is not limited as long as it can be used as a labeling substance, and the preferred range varies depending on the substituent. is preferably 0.1 to 2.5 when the substituent is a metal ion adsorbing group. Here, the degree of substitution represents the number of substituted hydroxyl groups per β-glucose unit (number of hydroxyl groups=3) of cellulose, and is the average value of all β-glucose units constituting the cellulose molecule. If the degree of substitution exceeds 2.5, the hydrophilicity of the metal nanoparticle-cellulose composite tends to be impaired, and the dispersibility in an aqueous solution tends to decrease. On the other hand, when the degree of substitution is less than 0.1, the amount of supported metal nanoparticles is insufficient, and the visibility as a labeling substance tends to decrease. A more preferable upper limit is 1.0. Moreover, a more preferable lower limit is 0.2.

In addition, saccharides such as glucose, galactose, mannose, xylose, N-acetylglucosamine, gluconic acid, galacturonic acid, mannuronic acid are α- or β-linked, and complexed or combined with known polysaccharides constituting main chains or side chains. You can use Such known polysaccharides include chitin, chitosan, glucosamine, pectin, etc. Chitosan is preferable because it has an acetylamino group, which is a metal ion-adsorbing group, in its molecule.

Also, the type of cellulose is not particularly limited. Known cellulose such as regenerated cellulose, purified cellulose, and natural cellulose can be used. Cellulose in which a part of these structures is modified may be used. Examples include cellulose, microfibrous cellulose, fermented cellulose, and methylcellulose, ethylcellulose, hydroxyethylcellulose, hydroxypropylmethylcellulose, hydroxypropylcellulose, carboxymethylcellulose, carboxyethylcellulose, and cellulose derivatives thereof such as sodium and calcium.

The above nitrogen-containing monomer forms a resin having a nitrogen atom in its main chain or side chain when polymerized. Examples include polyamine, polyamide, polypeptide, polyurethane, polyurea, polyimide, polyimidazole, It is a monomer that forms oxazole, polypyrrole and polyaniline. Further, when the side chain has a nitrogen atom, for example, a monomer constituting an acrylic resin, a phenol resin, or an epoxy resin can be applied.
In addition, the monomer capable of adsorbing cationic ions forms a resin having a carboxylic acid group, a sulfonic acid group, or the like on the main chain or side chain. Monomers that constitute vinyl, polyvinylsulfonic acid, polystyrenesulfonic acid can be applied.
Examples of copolymers with nitrogen-containing monomers or monomers capable of adsorbing cationic ions include random copolymers, block copolymers, alternating copolymers, and crosslinked polymers. Further, the resin particles may be formed by copolymerizing two or more types of monomers, or the functional groups present on the surface of the resin particles may be reacted with the monomers, and the functional groups may be used as polymerization active terminals for further polymerization.

Also, the metal nanoparticles 20 are preferably gold, platinum, palladium, silver, nickel, copper, or alloys thereof. These metals can be used singly or in composites such as alloys. Here, for example, a gold alloy means an alloy composed of gold and a metal species other than gold and containing 10% by weight or more of gold. Further, for example, a platinum alloy means an alloy composed of platinum and a metal species other than platinum and containing 1% by weight or more of platinum. More preferred are gold, platinum and palladium, which have excellent visibility and facilitate immobilization of antigens or antibodies. These are preferable because they exhibit absorption derived from localized surface plasmon resonance. More preferred are gold and platinum, which are excellent in detection sensitivity in immunoassay.

Also, the average particle diameter of the metal nanoparticles 20 measured by scanning electron microscope (SEM) observation (that is, the average particle diameter D3 in FIG. 1) is preferably 1 to 80 nm, for example. When the average particle size of the metal nanoparticles 20 is less than 1 nm or more than 80 nm, localized surface plasmon resonance and light energy absorption due to electronic transition are less likely to occur, and sensitivity tends to decrease.
When platinum particles are used as the metal nanoparticles 20, the average particle size of the platinum particles is preferably 1 nm or more and 50 nm or less from the viewpoint of obtaining high detection sensitivity as a labeling substance for immunological measurement and an immunological measurement reagent. more preferably 1 nm or more and 30 nm or less, still more preferably 1 nm or more and 20 nm or less, and most preferably 1 nm or more and 15 nm or less. In particular, when the average particle size of the metal nanoparticles 20 is 15 nm or less, particularly excellent detection sensitivity can be obtained when used as a labeling substance for immunochromatography.
Further, when gold particles are used as the metal nanoparticles 20, the average particle diameter of the gold particles is preferably 1 nm or more and 70 nm from the viewpoint of obtaining high detection sensitivity as a labeling substance for immunological measurement and a reagent for immunological measurement. less than, more preferably 1 nm or more and less than 50 nm.

Also, the average particle size of the metal nanoparticle-cellulose composite 100 (that is, the average particle size D1 in FIG. 1) is preferably, for example, 50 to 1000 nm. If the average particle size of the metal nanoparticle-cellulose composite 100 is less than 50 nm, for example, the amount of the metal nanoparticles 20 supported tends to be small, so the coloring tends to be weaker than metal nanoparticles of the same size. , the pores of a chromatographic medium such as a membrane filter tend to be clogged and the dispersibility tends to decrease when used as a labeling substance or reagent. The average particle size of the metal nanoparticle-cellulose composite 100 improves the dispersibility when used as a labeling substance or reagent, and provides high detection sensitivity when the metal nanoparticle-cellulose composite 100 is used for immunological measurements. From the viewpoint of obtaining the thickness, it is preferably 100 nm or more and 700 nm or less, more preferably 250 nm or more and 650 nm or less. In particular, when the average particle diameter of the metal nanoparticle-cellulose composite 100 is 250 nm or more, stable and excellent detection sensitivity can be obtained when the metal nanoparticle-cellulose composite 100 is used as a labeling substance for immunochromatography. . Here, the particle diameter of the metal nanoparticle-cellulose composite 100 means the value obtained by adding the length of the protruding portion of the partially exposed particle 40 or the surface adsorbed particle 50 to the particle diameter of the resin particle 10, and laser diffraction / can be measured by a scattering method, a dynamic light scattering method, or a centrifugal sedimentation method.

[Metal nanoparticle-cellulose composite for immunological measurement]
In the present specification, the metal nanoparticle-cellulose complex is for immunological measurement, at least the complex containing cellulose satisfies the requirement that it contains a metal nanoparticle having the property of binding to an antibody. Say.
In immunological measurements, binding between an antigen to be detected and an antibody bound to a metal nanoparticle causes a change, such as color development, that can be recognized by the senses or detected by an instrument.

[Method for producing metal-resin composite]
The method for producing the metal nanoparticle-cellulose composite 100, which is a metal-resin composite, is not particularly limited. For example, cellulose particles are synthesized, a known metal ion such as gold chloride or platinum chloride is added to the dispersion, and a known reducing agent such as sodium borohydride, hydrazine, alcohol, ascorbic acid, or dimethylamine borane is added. In addition, metal ions are reduced to deposit metal nanoparticles. As a result, the precipitated metal nanoparticles are immobilized on the cellulose particles.
Cellulose particles can be produced by known methods. For example, a coagulating liquid may be added to a cellulose solution to form microparticles, or the cellulose may be pulverized by a wet method or a wet method and classified to obtain microparticles.
When adding a coagulating liquid to a cellulose solution to micronize, for example, a cellulose linter is dissolved in a cuprammonium solution prepared by a known method to obtain a cuprammonium cellulose solution. Next, a mixture of an organic solvent, water and ammonia is prepared as a coagulating liquid, a cuprammonium cellulose solution is added to the coagulating liquid to coagulate, neutralized with sulfuric acid, etc., and dried to obtain particles. Obtainable.

Next, an analyte measurement method using the metal nanoparticle-cellulose composite 100 as a labeling substance, a test strip for lateral flow chromatography, and an analyte detection/quantitation kit will be described.

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

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

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

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

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

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

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

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

The test strip 200 may further include arbitrary parts such as a reaction part and a control part, if necessary.

<Reaction part>
Although not shown, the test strip 200 may have a reaction portion formed on the membrane 110 and containing the labeled antibody 150 . The reaction section can be provided upstream of the determination section 130 in the direction in which the sample flows. Note that the sample addition section 120 in FIG. 3 may be used as the reaction section. When the test strip 200 has a reaction portion, when a sample containing the analyte 160 is provided to the reaction portion or the sample application portion 120, the analyte 160 contained in the sample and the labeled antibody 150 can be brought into contact in the reaction portion. can. In this case, by simply providing the sample to the reaction section or the sample addition section 120, the complex 170 containing the analyte 160 and the labeled antibody 150 can be formed, so that so-called one-step immunochromatography is possible. Become.

The reaction section is not particularly limited as long as it contains the labeled antibody 150 that specifically binds to the analyte 160 , but the membrane 110 may be directly coated with the labeled antibody 150 . Alternatively, the reaction section may be formed by fixing a pad (conjugate pad) made of cellulose filter paper, glass fiber, non-woven fabric, or the like impregnated with the labeled antibody 150 to the membrane 110 .

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

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

The method for measuring analyte 160 of the present embodiment is a method for measuring analyte 160 for detecting or quantifying analyte 160 contained in a sample. The method for measuring the analyte 160 of the present embodiment uses a test strip 200 that includes a membrane 110 and a determination section 130 in which a capture ligand 131 that specifically binds to the analyte 160 is immobilized on the membrane 110 . Then, the method for measuring the analyte 160 of the present embodiment includes the following steps (I) to (III);
Step (I): Metal nanoparticles having a structure in which the analyte 160 contained in the sample and an antibody that specifically binds to the analyte 160 are immobilized on the resin particles 10- contacting with a labeled antibody 150 labeled with the cellulose composite 100;
Step (II): A step of contacting the complex 170 formed in the step (I) containing the analyte 160 and the labeled antibody 150 with the capture ligand 131 in the determination unit 130;
Step (III): a step of measuring the intensity of color development derived from the localized surface plasmon resonance of the metal nanoparticle-cellulose composite 100 and the absorption of light energy by electronic transition;
can include

Step (I):
Step (I) is a step of contacting the analyte 160 contained in the sample with the labeled antibody 150 . As long as the complex 170 containing the analyte 160 and the labeled antibody 150 is formed, the mode of contact is not particularly limited. For example, the sample may be provided to the sample application portion 120 or the reaction portion (not shown) of the test strip 200, and the analyte 160 may be brought into contact with the labeled antibody 150 in the reaction portion. , the analyte 160 in the sample may be contacted with the labeled antibody 150 .

The complex 170 formed in step (I) develops and moves on the test strip 200 to reach the determination section 130 .

Step (II):
In step (II), the complex 170 formed in step (I) and containing the analyte 160 and the labeled antibody 150 is brought into contact with the capture ligand 131 in the determination section 130 of the test strip 200 . When complex 170 is contacted with capture ligand 131 , capture ligand 131 specifically binds to analyte 160 of complex 170 . As a result, complex 170 is captured by determination unit 130 .

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

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

By performing the washing step, in the step (III), localized surface plasmon resonance of the metal nanoparticles-cellulose composite 100 in the determination unit 130, or the determination unit 130 and the control unit, and optical energy absorption due to electronic transition. When measuring color development with , the intensity of background color development can be reduced, the signal/background ratio can be increased, and detection sensitivity and quantification can be further improved.

Step (III):
Step (III) is a step of measuring the intensity of color development derived from the localized surface plasmon resonance of the metal nanoparticle-cellulose composite 100 and light energy absorption due to electronic transition. After performing the above step (II) or washing step as necessary, in the test strip 200, the localized surface plasmon resonance of the metal nanoparticle-cellulose composite 100 and the color intensity derived from light energy absorption by electronic transition to measure.

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

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

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

As the analyte 160, known ones can be used without any particular limitation. For example, proteins such as tumor markers, signaling substances, hormones (including polypeptides, oligopeptides, etc.), nucleic acids (single-stranded or double-stranded DNA, RNA, polynucleotides, oligonucleotides, PNA (peptide nucleic acids) etc.) or substances having nucleic acids, sugars (including oligosaccharides, polysaccharides, sugar chains, etc.) or substances having sugar chains, and other molecules such as lipids, which are specific to the labeled antibody 150 and the capture ligand 131 Although not particularly limited as long as it binds to, 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 streptococcal antigen, HBs antibody, HBs antigen, rotavirus, adenovirus, albumin, glycated albumin and the like. Among these, antigens that are solubilized by nonionic surfactants are preferred, and antigens that form self-aggregates like viral nucleoproteins are more preferred.

<Labeled antibody>
The labeled antibody 150 is used in step (I) to contact the analyte 160 contained in the sample to form a complex 170 containing the analyte 160 and the labeled antibody 150 . The labeled antibody 150 is obtained by labeling an antibody that specifically binds to an analyte 160 with a metal nanoparticle-cellulose composite 100 having a structure in which a plurality of metal nanoparticles 20 are immobilized on a resin particle 10. be. Here, “labeling” means that the metal nanoparticle-cellulose complex 100, which is a metal-resin complex, does not detach from the labeled antibody 150 in steps (I) to (III). It means that the particle-cellulose composite 100 is directly or indirectly fixed by chemical or physical bonding, adsorption, or the like. For example, the labeled antibody 150 may be obtained by directly binding the metal nanoparticle-cellulose complex 100 to the antibody, or the antibody and the metal nanoparticle-cellulose complex 100 are bonded via an arbitrary linker molecule. may be formed by combining with each other, or may be formed by fixing each of them to an insoluble particle.

In addition, in the present embodiment, the "antibody" is not particularly limited and known ones can be used, and those with strong anionic properties, those with strong cationic properties, and others can also be used. For example, in addition to polyclonal antibodies, monoclonal antibodies, antibodies obtained by genetic recombination, antibody fragments capable of binding to antigens [eg, H chain, L chain, Fab, F(ab') ₂ , etc.] can be used. can. Moreover, any of IgG, IgM, IgA, IgE, and IgD may be used as the immunoglobulin. Antibody-producing animal species include humans and non-human animals (eg, mice, rats, rabbits, goats, horses, etc.). Specific examples of antibodies include anti-PSA antibodies, anti-AFP antibodies, anti-CEA antibodies, anti-adenovirus antibodies, anti-influenza virus antibodies, anti-HCV antibodies, anti-IgG antibodies, anti-human IgE antibodies and the like.

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

[Step A]
In step A, labeled antibody 150 is obtained by mixing metal nanoparticle-cellulose composite 100 with an antibody under a first pH condition. In step A, the solid metal nanoparticle-cellulose composite 100 is preferably dispersed in a liquid phase and brought into contact with the antibody.

The first pH condition is within the range of pH 2 to 10 from the viewpoint of uniform contact between the metal nanoparticle-cellulose composite 100 and the antibody while maintaining the dispersion of the metal nanoparticle-cellulose composite 100 and the activity of the antibody. Conditions are preferred, and more preferably within the range of pH 5-9, for example. When the metal nanoparticle-cellulose complex 100 and the antibody are bound to each other, if the pH is less than 2, the antibody may be denatured and deactivated due to strong acidity, and if the pH exceeds 10, the metal nanoparticle-cellulose complex 100 and the antibody may be degraded. When mixed, it aggregates and becomes difficult to disperse. However, if the antibody is not deactivated by strong acidity, the treatment can be performed even at pH less than 2.

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

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

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

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

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

In steps A and B above, the metal nanoparticle-cellulose composite 100 having the metal nanoparticles 20 is unlikely to aggregate due to pH, and can be treated in a wide range of pH from acidic to alkaline. On the other hand, for example, in the case of a resin-gold composite having gold particles, in step A, the resin-gold composite tends to agglomerate under conditions exceeding pH 7, and in step B, under conditions exceeding pH 9, the resin - Gold complexes tend to agglomerate with each other. Therefore, the metal nanoparticle-cellulose composite 100 used in the present invention also has the advantage of being less subject to restrictions on the conditions for producing labeled antibodies.

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

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

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

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

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

The analyte measurement kit of the present embodiment includes
a membrane 110, a test strip 200 including a determination section 130 in which a capture ligand 131 that specifically binds to the analyte 160 is immobilized on the membrane 110;
a detection reagent containing a labeled antibody 150 in which an antibody that specifically binds to an analyte 160 is labeled with a metal nanoparticle-cellulose composite 100 having a structure in which a plurality of metal nanoparticles 20 are immobilized on a resin particle 10;
contains. The analyte measurement kit of the present embodiment may further contain other components as required.

In using the analyte measurement kit according to the present embodiment, the analyte 160 in the sample and the labeled antibody 150 in the detection reagent are brought into contact to carry out step (I), and then the test strip 200 reacts. The step (II) and the step (III) may be sequentially performed by providing the sample to the section or the sample addition section 120 . Alternatively, after applying a detection reagent to the upstream side of the determination portion 130 of the test strip 200 and drying it appropriately to form a reaction portion, the formed reaction portion or a position upstream of the reaction portion (for example, , the sample addition unit 120), and steps (I) to (III) may be sequentially performed.

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

<Measurement of Absorbance of Metal Nanoparticle-Cellulose Composite>
The absorbance of the metal nanoparticle-cellulose composite and gold colloid particles was measured using a metal nanoparticle-cellulose composite or gold colloid dispersion (dispersion medium: water ) was put in, and a spectrophotometer (Shimadzu Corporation, UV3600) was used to measure the absorbance at 570 nm for gold-cellulose composites or gold colloid particles and at 400 nm for platinum-cellulose composites.

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

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

Further, the sample after the drying treatment was further heat treated at 500° C. for 5 hours, the weight before and after the heat treatment was measured, and the amount of metal supported was calculated from the following formula.
Amount of metal supported (wt%) =
[Weight after heat treatment (g)/Weight before heat treatment (g)] × 100

<Measurement of average particle size of metal nanoparticles-cellulose composite>
It was measured using a disc centrifugal particle size distribution analyzer (CPS Disc Centrifuge DC24000 UHR, manufactured by CPS Instruments, Inc.). The measurement was performed with the metal nanoparticle-cellulose composite dispersed in water.

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

<Measurement of degree of substitution>
A 1H-NMR spectrum was measured using a nuclear magnetic resonance spectrometer JNM-ECX400 manufactured by JEOL under the following conditions to measure the degree of substitution.
Carboxylated cellulose microparticles: 11 wt% heavy water solution of heavy caustic soda was used as a measurement solvent, and the degree of substitution was measured from the ratio of the integral values of the protons bonded to the C1 position of the glucose skeleton and the methylene protons of the carboxymethyl group.
・ Aminated cellulose fine particles: A mixed solution of heavy water and heavy acetic acid (heavy water: heavy acetic acid = 4:6 to 5:3) was used as a measurement solvent, and the protons bonded to the C1 position of the glucose skeleton and the aminomethyl group The degree of substitution was measured from the ratio of the integrated value with methylene protons.

[Synthesis Example 1]
A cuprammonium cellulose solution prepared by a known method (cellulose 0.37 wt%, copper concentration 0.13 wt%, ammonia concentration 1.00 wt%) was added. After that, 1000 g of 10 wt % sulfuric acid was added for neutralization to obtain 6500 g of a suspension containing fine cellulose particles.
After centrifuging the obtained suspension and removing the supernatant, the operation of adding pure water and suspending again is repeated until the supernatant becomes neutral, and after dispersion treatment, the concentration is adjusted to obtain 1 180 g of a 0 wt % cellulose fine particle dispersion was obtained. The average particle diameter of the obtained cellulose fine particles was measured to be 265 nm, and the CV value (variation coefficient) was 17%.

20 g of the resulting 1.0 wt % cellulose fine particle dispersion was centrifuged to remove the supernatant, and then 100 g of a mixed solution of isopropyl alcohol and pure water adjusted to a weight ratio of 85:15 was added to suspend the cellulose fine particles. The cloudy liquid was stirred at 50° C. for 30 minutes. After that, 0.54 g of an 11 wt % aqueous sodium hydroxide solution was added, and the mixture was further stirred for 30 minutes. After that, sodium chloroacetate (560 mg) was added to the reaction solution, and the reaction solution was stirred under reflux for 3 hours. acidified. After centrifuging the obtained reaction solution and removing the supernatant, the operation of adding pure water and suspending again is repeated until the supernatant becomes neutral, and the dispersion treatment and the concentration adjustment are performed. 25 g of a 0 wt % carboxylated cellulose fine particle dispersion was obtained. The average particle size of the obtained carboxylated cellulose microparticles was measured and found to be 271 nm. Moreover, the CV value was 21%.
Furthermore, when the degree of substitution was calculated using a nuclear magnetic resonance spectrometer, the degree of substitution was 0.492.

[Synthesis Example 2]
After removing the supernatant by centrifuging 20 g of the 1.0 wt% cellulose fine particle dispersion prepared in Synthesis Example 1, 20 g of a 9.0 wt% sodium hydroxide aqueous solution was added to suspend the cellulose fine particles. , and stirred for 30 minutes. After that, 2-chloroethylamine hydrochloride (7.16 g) was added, and the mixture was stirred under reflux for 3 hours, cooled to 20°C, and 10% hydrochloric acid was added to acidify the reaction mixture. . After centrifuging the obtained reaction solution and removing the supernatant, the operation of adding pure water and suspending again is repeated until the supernatant becomes neutral, and the dispersion treatment and the concentration adjustment are performed. 23 g of a 0 wt % primary aminated cellulose fine particle dispersion was obtained. The obtained primary aminated cellulose microparticles had an average particle size of 270 nm, a CV value of 22%, and a degree of substitution of 0.350.
[Synthesis Example 3]
Tertiary aminated cellulose fine particles were obtained in the same manner as in Synthesis Example 2, except that 2-diisopropylaminoethyl chloride hydrochloride (12.36 g) was used as the reactant. The resulting tertiary aminated cellulose microparticles had an average particle size of 269 nm, a CV value of 24%, and a degree of substitution of 0.250.
[Example 1]
<Synthesis of gold-cellulose composite particles A>
A 400 mM chloroauric acid aqueous solution (2.5 g) was added to the carboxylated cellulose fine particles (20 g) obtained in Synthesis Example 1, and the mixture was stirred at 30°C for 3 hours. Excess gold chloride was removed by removing the supernatant from the mixture by centrifugation. After that, the concentration was adjusted to obtain 23 g of a 1.00 wt % gold ion-adsorbed carboxylated cellulose fine particle dispersion liquid A.

Next, 1.00 wt% gold ion-adsorbing carboxylated cellulose fine particle dispersion A (20 g) was added to 280 g of pure water, and while stirring at 3°C, a 30 mM dimethylamine borane aqueous solution (10.0 g) was added for 2 minutes. After dropping at 3° C. for 1 hour and then at room temperature for 3 hours, gold-cellulose composite particles A having an average particle size of 282 nm were obtained. After concentrating the reaction liquid by centrifugation, it was purified by dialysis treatment and the concentration was adjusted to obtain a gold-cellulose composite particle dispersion liquid A of 1 wt %. The absorbance of the gold-cellulose composite particles A in the 1 wt % gold-cellulose composite particle dispersion liquid A was 0.22. The average particle size of the gold nanoparticles in F-3 was 11 nm, and the amount of gold supported was 7.7 wt%.
[Example 2]
<Synthesis of platinum-cellulose composite particles B>
A 400 mM chloroplatinic acid aqueous solution (2.5 g) was added to the carboxylated cellulose microparticles (20 g) obtained in Synthesis Example 1, and the mixture was stirred at 30° C. for 3 hours. Excess platinum chloride was removed by removing the supernatant from this mixture by centrifugation. Thereafter, the concentration was adjusted to obtain 24 g of a 1.00 wt % platinum ion-adsorbed carboxylated cellulose fine particle dispersion liquid B.

Next, 1.00 wt% platinum ion-adsorbing carboxylated cellulose fine particle dispersion B (20 g) was added to 280 g of pure water, and while stirring at 3°C, a 7.5 mM dimethylamine borane aqueous solution (40 g) was added for 20 minutes. After dropping at 3° C. for 1 hour and then at room temperature for 3 hours, platinum-cellulose composite particles having an average particle size of 285 nm were obtained. After concentrating the reaction liquid by centrifugation, it was purified by dialysis treatment and the concentration was adjusted to obtain a 1 wt % platinum-resin composite particle dispersion liquid B. The absorbance of the platinum-cellulose composite particles B in the 1 wt % platinum-resin composite particle dispersion B was 0.35. The average particle size of the platinum nanoparticles was 3.0 nm, and the supported amount of platinum was 7.3 wt%.
[Example 3]
<Synthesis of gold-cellulose composite particles C>
A 400 mM chloroauric acid aqueous solution (2.5 g) was added to the primary aminated cellulose fine particles (20 g) obtained in Synthesis Example 2, and the mixture was stirred at 30°C for 3 hours. Excess chloroauric acid was removed by removing the supernatant from this mixture by centrifugation. Thereafter, the concentration was adjusted to obtain 40 g of a 1.00 wt % gold ion-adsorbed primary aminated cellulose fine particle dispersion liquid C.

Next, 1.00 wt% gold ion-adsorbing primary aminated cellulose fine particle dispersion C (20 g) was added to 280 g of pure water, and 30 mM of dimethylamine borane aqueous solution (10.0 g) was added thereto while stirring at 3°C. After the mixture was added dropwise over a period of minutes, the mixture was stirred at 3° C. for 1 hour and then at room temperature for 3 hours to obtain gold-cellulose composite particles C having an average particle size of 289 nm. After concentrating the reaction liquid by centrifugation, it was purified by dialysis treatment and the concentration was adjusted to obtain a 1 wt % gold-cellulose composite particle dispersion liquid. The absorbance of the gold-cellulose composite particles C in the 1 wt % gold-cellulose composite particle dispersion liquid C was 0.80. In addition, the average particle diameter of the gold nanoparticles was 25 nm, and the supported amount of gold was 29.0 wt %.
[Example 4]
<Synthesis of platinum-cellulose composite particles D>
A 400 mM chloroplatinic acid aqueous solution (2.5 g) was added to the primary aminated cellulose microparticles (20 g) obtained in Synthesis Example 2, and the mixture was stirred at 30° C. for 3 hours. Excess chloroplatinic acid was removed by removing the supernatant from this mixture by centrifugation. Thereafter, the concentration was adjusted to obtain 29 g of a 1.00 wt % platinum ion-adsorbed primary aminated cellulose fine particle dispersion liquid D.

Next, 1.00 wt% platinum ion-adsorbed primary aminated cellulose fine particle dispersion D (20 g) was added to 280 g of pure water, and 20 g of 7.5 mM dimethylamine borane aqueous solution (40 g) was added while stirring at 3°C. After dropping over a period of minutes, the mixture was stirred at 3° C. for 1 hour and at room temperature for 3 hours to obtain platinum-cellulose composite particles D having an average particle size of 287 nm. After concentrating the reaction liquid by centrifugation, it was purified by dialysis treatment and the concentration was adjusted to obtain a 1 wt % platinum-resin composite particle dispersion liquid D. The absorbance of the platinum-cellulose composite particles D in the 1 wt % platinum-resin composite particle dispersion D was 0.68. The average particle diameter of the platinum nanoparticles was 3.1 nm, and the amount of platinum supported was 14.3 wt%.
[Example 5]
<Synthesis of gold-cellulose composite particles E>
A 400 mM chloroauric acid aqueous solution (2.5 g) was added to the tertiary aminated cellulose microparticles (20 g) obtained in Synthesis Example 3, and the mixture was stirred at 30° C. for 3 hours. Excess chloroauric acid was removed by removing the supernatant from this mixture by centrifugation. Thereafter, the concentration was adjusted to obtain 31 g of a 1.00 wt % gold ion-adsorbed tertiary aminated cellulose fine particle dispersion liquid E.

Next, 1.00 wt% gold ion-adsorbing tertiary aminated cellulose fine particle dispersion E (20 g) was added to 280 g of pure water, and while stirring at 3°C, 30 mM aqueous dimethylamine borane solution (10.0 g) was added twice. After the dropwise addition over a period of minutes, the mixture was stirred at 3° C. for 1 hour and at room temperature for 3 hours to obtain gold-cellulose composite particles E having an average particle size of 278 nm. After concentrating the reaction liquid by centrifugation, it was purified by dialysis treatment and the concentration was adjusted to obtain a gold-cellulose composite particle dispersion E of 1 wt %. The absorbance of the gold-cellulose composite particles E in the 1 wt % gold-cellulose composite particle dispersion liquid E was 0.41. The average particle diameter of the gold nanoparticles was 22 nm, and the supported amount of gold was 15.1 wt %.
[Example 6]
<Synthesis of platinum-cellulose composite particles F>
A 400 mM chloroplatinic acid aqueous solution (2.5 g) was added to the tertiary aminated cellulose microparticles (20 g) obtained in Synthesis Example 3, and the mixture was stirred at 30° C. for 3 hours. Excess chloroplatinic acid was removed by removing the supernatant from this mixture by centrifugation. Thereafter, the concentration was adjusted to obtain 25 g of a 1.00 wt % platinum ion-adsorbed carboxylated cellulose fine particle dispersion liquid F.

Next, 1.00 wt% platinum ion-adsorbed primary aminated cellulose fine particle dispersion F (20 g) was added to 280 g of pure water, and 20 g of 7.5 mM dimethylamine borane aqueous solution (40 g) was added while stirring at 3°C. After the dropwise addition over a period of minutes, the mixture was stirred at 3° C. for 1 hour and then at room temperature for 3 hours to obtain platinum-cellulose composite particles F having an average particle size of 272 nm. After concentrating the reaction liquid by centrifugation, it was purified by dialysis treatment and the concentration was adjusted to obtain a 1 wt % platinum-resin composite particle dispersion liquid F. The absorbance of the platinum-cellulose composite particles F in the 1 wt % platinum-resin composite particle dispersion F was 0.38. The average particle diameter of the platinum nanoparticles was 2.8 nm, and the supported amount of platinum was 7.8 wt%.
[Example 7]
<Synthesis of gold-cellulose composite particles G>
While stirring 5000 g of a mixed solution in which tetrahydrofuran and pure water were adjusted to a weight ratio of 89:11, a cuprammonium cellulose solution prepared by a known method (cellulose 0.37 wt%, copper concentration 0.13 wt%, 500 g of ammonia concentration (1.00 wt %) and 2 g of chitosan were added. After that, 1000 g of 10 wt % sulfuric acid was added for neutralization to obtain 6500 g of a suspension containing cellulose-chitosan composite particles.
After centrifuging the obtained suspension and removing the supernatant, the operation of adding pure water and suspending again is repeated until the supernatant becomes neutral. 350 g of 00 wt % cellulose-chitosan fine particle dispersion was obtained.
20 g of the resulting 1.00 wt % cellulose-chitosan fine particle dispersion was centrifuged to remove the supernatant, and then 20 g of a 9.0 wt % sodium hydroxide aqueous solution was added to suspend the cellulose-chitosan fine particles. °C and stirred for 30 minutes. After that, 2-chloroethylamine hydrochloride (7.16 g) was added, and after stirring under reflux for 3 hours, the temperature of the reaction solution was cooled to 20°C, and 10% hydrochloric acid was added to acidify the reaction solution. made it After centrifuging the obtained reaction solution and removing the supernatant, the operation of adding pure water and suspending again is repeated until the pH of the supernatant becomes neutral, and the dispersion treatment and concentration adjustment are performed. 23 g of a primary aminated cellulose fine particle dispersion of 0.00 wt % was obtained. The obtained primary aminated cellulose-chitosan composite particles had an average particle diameter of 382 nm and a CV value of 25%.
A 400 mM chloroauric acid aqueous solution (2.5 g) was added to primary aminated cellulose-chitosan fine particles (20 g), and the mixture was stirred at 30° C. for 3 hours. Excess chloroauric acid was removed by removing the supernatant from this mixture by centrifugation. After that, the concentration was adjusted to obtain a gold ion-adsorbed cellulose-chitosan composite particle dispersion G of 1.00 wt %.

Next, 1.00 wt% gold ion-adsorbed primary aminated cellulose composite particle dispersion G (20 g) was added to 280 g of pure water, and stirred at 3°C to obtain a 30 mM dimethylamine borane aqueous solution (10.0 g). was added dropwise over 2 minutes, followed by stirring at 3° C. for 1 hour and then at room temperature for 3 hours to obtain gold-cellulose-chitosan composite particles G having an average particle size of 398 nm. After concentrating the reaction liquid by centrifugation, it was purified by dialysis treatment and the concentration was adjusted to obtain a 1 wt % gold-cellulose-chitosan composite particle dispersion liquid G. The absorbance of the gold-cellulose-chitosan composite particles G in the 1 wt % gold-cellulose-chitosan composite particle dispersion was 1.05. The average particle diameter of the gold nanoparticles was 23 nm, and the supported amount of gold was 35.0 wt%.

[Example 8]
<Synthesis of platinum-cellulose composite particles H>
A 400 mM chloroplatinic acid aqueous solution (2.5 g) was added to the primary aminated cellulose-chitosan fine particles (20 g) obtained in Example 7, and the mixture was stirred at 30° C. for 3 hours. Excess chloroplatinic acid was removed by removing the supernatant from this mixture by centrifugation. After that, the concentration was adjusted to obtain a 1.00 wt % platinum ion-adsorbed cellulose-chitosan composite particle dispersion H.

Next, 1.00 wt% platinum ion-adsorbed primary aminated cellulose composite particle dispersion H (20 g) was added to 280 g of pure water, and while stirring at 3°C, a 7.5 mM dimethylamine borane aqueous solution (40. 0 g) was added dropwise over 20 minutes, followed by stirring at 3° C. for 1 hour and then at room temperature for 3 hours to obtain platinum-cellulose-chitosan composite particles H with an average particle size of 393 nm. After concentrating the reaction solution by centrifugation, it was purified by dialysis treatment and the concentration was adjusted to obtain a platinum-cellulose-chitosan composite particle dispersion liquid H of 1 wt %. The absorbance of the platinum-cellulose-chitosan composite particles H in the 1 wt % platinum-cellulose-chitosan composite particle dispersion liquid H was 1.39. The average particle size of the platinum nanoparticles was 5 nm, and the amount of platinum supported was 29.8 wt%.

[Comparative Example 1]
<Synthesis of gold colloidal particles I>
Put 250 ml of 1 mM chloroauric acid aqueous solution in a 500 ml three-necked round-bottomed flask, bring to a boil with vigorous stirring using a heating reflux apparatus, add 25 ml of 38.8 mM sodium citrate aqueous solution after boiling, and the solution changes from pale yellow to thick. I confirmed that it changed to red. After continuing to heat for 10 minutes while stirring, the mixture was allowed to cool with stirring for about 30 minutes at room temperature. The solution was filtered using a membrane filter with a pore size of 2 μm to obtain colloidal gold particles I. The average particle size was 12.3 nm. The absorbance was 1.32 as a result of measuring according to the above method.

Next, the gold-cellulose composite particles obtained in Examples, the platinum-cellulose composite particles, and the colloidal gold particles I obtained in Comparative Examples were applied as labeling substances to carriers for diagnostic reagents, and their performance was evaluated. made an evaluation.

[Example 9]
An anti-influenza A monoclonal antibody was added to a mixture of 0.1 mL of 1 wt% gold-cellulose composite particle dispersion A and 100 mM borate buffer (pH 8.5), and the mixture was turned over at room temperature for 3 hours. Agitation was performed. Next, centrifugation is performed at 10,000 rpm, the supernatant is removed, 1 mL of blocking buffer is added to the solid residue, ultrasonic dispersion treatment is performed for 10 to 20 seconds, and the mixture is further subjected to 2 hours at room temperature. Inversion stirring was performed. Next, centrifugation was performed at 10000 rpm, the supernatant was removed, 1 mL of 5 mM Tris buffer (pH 8.5) was added to the solid residue, and ultrasonic dispersion was performed for 10 to 20 seconds. This operation was repeated three times, and was used as a washing treatment. Next, after centrifuging at 10000 rpm and removing the supernatant, 1 mL of a storage buffer (a 5 mM Tris solution with sucrose added to a concentration of 10% by weight) was added to the solid residue. Labeled antibody-containing liquid a was obtained by performing ultrasonic dispersion treatment for ~20 seconds.

<Performance evaluation>
Each row of 12 wells of a 96-well plate was filled with 3 μl of the labeled antibody dispersion liquid a, and influenza A inactivated antigens were added at concentrations of 2500, 1250, 625, 313, 156, 78, 40, 20 and 10FFU/ml, respectively. 100 μl of the liquid adjusted to the concentration was developed using an immunochromatographic kit prepared by a known method, and the coloration at the reaction site (test line) on the membrane filter after 5 minutes, 10 minutes, and 15 minutes. was visually observed. (-) indicates no coloration, (+) indicates coloration, (++) indicates clear coloration, and (+++) indicates strong coloration. Table 1 shows the results of the performance evaluation.

[Examples 10 to 17, Comparative Example 2]
Instead of gold-cellulose composite particle dispersion liquid A, platinum-resin composite particle dispersion liquid B, gold-cellulose composite particle dispersion liquid C, and platinum-resin composite particle dispersion liquid, which are also metal-resin composites. D, gold-cellulose composite particle dispersion E, platinum-resin composite particle dispersion F, gold-cellulose-chitosan composite particle dispersion G, platinum-cellulose-chitosan composite particle dispersion H, colloidal gold particles Labeled antibody-containing liquids b to i were obtained in the same manner as in Example 1 except that 1 mg/0.1 mL aqueous dispersion liquid I was used, and performance evaluation was performed on these liquids as carriers for diagnostic reagents. Table 1 shows the results of the performance evaluation.

10 Resin Particle 20 Metal Nanoparticle 30 Encapsulated Particle 40 Partially Exposed Particle 50 Surface Adsorbed Particle 60 Surface Layer Part 100 Metal Nanoparticle-Cellulose Composite 110 Membrane 120 Sample Addition Part 130 Judgment Part 131 Capturing Ligand 140 Liquid Absorption Part 150 Labeled Antibody 160 Analyte 170 Complex 200 Test strip

Claims (11)

Cellulose particles having carboxyl groups and/or amino groups introduced as substituents, and a plurality of metal nanoparticles relatively smaller than the cellulose particles,
The metal nanoparticles are immobilized on the cellulose particles by chemically adsorbing metal ions to the cellulose particles, and then adding a reducing agent to reduce and precipitate the metal ions, and have an average particle diameter of 1 to 80 nm. can be,
The metal nanoparticles are gold, platinum, palladium, or alloys thereof,
Metal nanoparticle-cellulose composite for immunoassay. The metal nanoparticle-cellulose composite for immunological measurement according to claim 1 , wherein the metal nanoparticles are platinum particles having an average particle size of 1 nm or more and 30 nm or less. The metal nanoparticle-cellulose composite for immunoassay according to claim 1 or 2 , wherein the metal nanoparticles are gold particles having an average particle size of 1 nm or more and 50 nm or less. The metal nanoparticle-cellulose composite for immunoassay according to any one of claims 1 to 3 , wherein the average particle size is in the range of 50 to 1000 nm. The metal nanoparticle-cellulose complex for immunological measurement chemisorbs the carboxyl group that chemisorbs the cationic metal ions that are the precursors of the metal nanoparticles and/or the anionic metal ions that are the precursors of the metal nanoparticles. The metal nanoparticle-cellulose composite for immunoassays according to any one of claims 1 to 4 , comprising cellulose having an amino group as a substituent. The immunological measurement according to any one of claims 1 to 5 , wherein the metal nanoparticle-cellulose complex for immunological measurement contains a polysaccharide selected from the group consisting of chitin, chitosan, glucosamine, and pectin. for metal nanoparticle-cellulose composites. The immunity according to any one of claims 1 to 6 , wherein the amount of the metal nanoparticles supported is in the range of 3 wt% to 70 wt% with respect to the weight of the metal nanoparticle-cellulose composite for immunological measurement. Metal nanoparticle-cellulose composite for scientific measurements. A labeling substance comprising the metal nanoparticle-cellulose complex for immunological measurement according to any one of claims 1 to 7 . 9. The labeling substance according to claim 8 , wherein an antigen or an antibody is adsorbed on the surface of said metal nanoparticle-cellulose composite for immunological measurement. An immunoassay method using the labeling substance according to claim 8 or 9 . An immunological measurement reagent comprising the immunological measurement metal nanoparticle-cellulose composite according to any one of claims 1 to 7 .

Images