JP6148033B2 - Cellulose microparticles containing fluorescent dye compounds - Google Patents

Description

  The present invention relates to a cellulose fine particle containing a fluorescent dye compound, a dispersion thereof, and a diagnostic agent using the same.

  Conventionally, as one of immunoassays for detecting a target substance comprising a specific antigen or antibody by utilizing a specific reaction by an antigen-antibody, the target substance in a sample is divided into an antibody or antigen carried on fine particles. An agglutination method that specifically binds by an immune reaction and measures the agglomeration state of fine particles generated by the binding is generally used because it is a simple measurement method and can be determined visually. As other immunoassays, radioimmunoassay, enzyme immunoassay, immunofluorescence assay, and the like are also widely used. In addition, a method that uses a substance that immunologically binds to a substance to be detected and combines the principle of immunological reaction and chromatography to detect the substance to be detected by visual judgment is called immunochromatography or immunochromatography. In recent years, it has been widely used.

  The immunochromatography method is a method in which an antibody (or antigen) against an antigen (or antibody), which is a substance to be detected, is immobilized on a chromatographic medium, and a reaction site is prepared on the chromatographic medium as a stationary phase. While the detection fine particles carrying an antibody (or antigen) capable of binding to a substance and the sample containing the detection object are brought into contact [the antibody on the detection fine particles for antibody sensitization (or antigen sensitization) by this contact] (Or the antigen) reacts with the antigen (or antibody) in the sample to form a complex consisting of the detection microparticles—the antibody (or antigen) used for sensitization—the antigen (or antibody) in the sample. Is a measurement method in which the sample is brought into contact with the reaction site by moving on the chromatographic medium. Thereby, at the reaction site, the complex is bound to the immobilized antibody (or immobilized antigen), and the detection microparticles are captured. Therefore, the presence or absence of capture of the detection microparticles is visually determined. Thus, the presence of the substance to be detected in the sample can be determined. A diagnostic kit using this principle is called an immunochromatography kit.

  In the immunochromatography kit and the aggregation method, colored fine particles are often used as detection fine particles in order to facilitate visual determination. Such detection fine particles can be obtained by colloidal metal fine particles that naturally color depending on the particle size or preparation conditions, fine particles colored latex fine particles made of a synthetic polymer, or a method of polymerizing monomers together with a colorant. Colored latex fine particles are known. Further, in the following Patent Document 1, the present inventors have reported colored fine particles having high color developability using cellulose fine particles as a raw material. However, these fine particles have problems such as fading easily and limit of color developability, and further improvement in performance is desired. Therefore, in recent years, fluorescent nanoparticles have attracted attention as new detection particles.

Fluorescent reagents used for detection and quantification of biomolecules using fluorescent nanoparticles are highly colored and are used as highly sensitive reagents. For example, Patent Document 2 below discloses fluorescent latex fine particles obtained by introducing a fluorescent dye compound into latex fine particles obtained by polymerizing styrene and acrylic acid.
Patent Document 3 below describes that fluorescent silica fine particles containing a fluorescent dye compound can be obtained by synthesizing a fluorescent dye compound, a silane coupling agent, and a silane compound.

  However, since these fluorescent nanoparticles are small in the amount of fluorescent dye compound introduced, color development satisfactory with an immunochromatography kit has not been obtained, and aggregation of particles occurs during storage of the particles, When developed with an immunochromatography kit, there is a problem that clogging or false positives are generated.

WO2011 / 062157A1 JP 2011-59003 A JP 2011-252819 A JP-A-10-206428

  In view of the above-described problems of the prior art, the problem to be solved by the present invention is to provide fluorescent cellulose fine particles having high color developability and good dispersion stability of particles. Further, by applying this to diagnostic agents, particularly lateral flow, it is to achieve high sensitivity of the immunochromatography kit.

  As a result of intensive investigations and repeated experiments to achieve the above-mentioned problems, the present inventors have found that when fine particles of a specific shape and particle size range contain a fluorescent dye compound in a specific range of content, The present inventors have found that the dispersion stability is improved and that the sensitivity of the immunochromatography kit is increased, and the present invention has been completed based on such knowledge.

That is, the present invention is as follows.
[1] The fluorescent dye compound is contained in a content of 0.01% to 95%, the average particle size is 9 nm to 700 nm, the CV value is 30% or less, and the average value of the major axis / minor axis is less than 10 der is, and fluorescent cellulose fine particles having an average degree of polymerization, characterized in der Rukoto 30 or more 700 or less.

  [2] The fluorescent cellulose fine particles according to [1] above, wherein components other than cellulose are supported via chemical bonding or physical adsorption.

  [3] The fluorescent cellulose fine particles according to [2], wherein the components other than the cellulose are substances that specifically interact with other components.

  [4] The fluorescent cellulose fine particles according to [2] or [3], wherein the component other than cellulose is a biomaterial.

  [5] The fluorescent cellulose fine particles according to [4], wherein the biomaterial is an antigen or an antibody.

  [6] A diagnostic agent comprising the fluorescent cellulose fine particles according to any one of [1] to [5].

  [7] The fluorescent cellulose fine particles according to any one of [2] to [4], wherein the component other than cellulose is a substance that specifically interacts with a test target substance. Diagnostics.

  [8] The diagnostic agent according to [6] or [7] for immunodiagnosis.

  [9] An in vitro sample detection method comprising a step of mixing the diagnostic agent according to any one of [6] to [8] and a sample, and detecting a test target substance in the sample.

  [10] An immunochromatography kit for detection by immunochromatography, comprising the diagnostic agent according to any one of [6] to [8].

  [11] A dispersion in which the fluorescent cellulose fine particles according to any one of [1] to [5] are dispersed in a liquid.

  [12] A molded article in which the fluorescent cellulose fine particles according to any one of [1] to [5] are fixed on a solid surface or dispersed in a solid.

  The fluorescent cellulose fine particles of the present invention have good dispersion stability of the particles in a storage state, and high sensitivity can be achieved by using such fluorescent cellulose fine particles in an immunochromatography kit.

Hereinafter, the present invention will be described in detail.
The fluorescent cellulose fine particles of the present invention contain a fluorescent dye compound. The “including” mode may be any of physical inclusion, adsorption, and chemical bonding. For example, when cellulose is dissolved in a good solvent and mixed with a coagulation liquid to produce fine particles, if the fluorescent dye compound is dispersed in the coagulation liquid, cellulose fine particles that physically contain the fluorescent dye compound are produced. be able to. On the other hand, in the case of chemically bonding, after forming cellulose into fine particles, a method of reacting a hydroxyl group of cellulose or a part thereof to covalently bond with a fluorescent dye compound, or binding with a fluorescent dye compound in advance. It can be produced by any method such as molding cellulose into fine particles. From the viewpoint of stability, a method in which cellulose fine particles and a fluorescent dye compound are chemically bonded is preferable.

  The type of the fluorescent dye compound is not particularly limited. For example, N-hydroxysuccinimide ester group, ester group, carboxyl group, maleimide group, isocyanate group, isothiocyanate group, cyano group, halogen group, aldehyde And fluoresceins, rhodamines, coumarins, and cyanines having active substituents such as a group, a paranitrophenyl group, a diethoxymethyl group, and an epoxy group. Specific examples of the fluorescent dye compound include fluorene, fluorene-9-acetic acid, fluorene-2carboxaldehyde, 9-fluorene-1-carboxylic acid, 9-fluorene-4-carboxylic acid, 9-fluorene oxime, and carbonic acid 9 Fluoromethylsuccinimidyl, 9-fluoretriphenylphosphonium bromide, 5-aminofluorescein, disodium 8-amino-1,3,6-naphthalene trisulfonate hydrate, sulfolodamine B, ethidium Bromide, 6-aminofluorescein, rhodamine B, rhodamine 6G, ammonium 8-anilino-1-naphthalenesulfonate, sodium 8-anilino-1-naphthalenesulfonate, magnesium 8-anilino-1-naphthalenesulfonate, 2,3-naphthalenedialdehyde, calcein Thorium, calcein, coumarin 102, coumarin 314, coumarin 343, AMCA, 5-carboxyfluorescein hydrate, 6-carboxyfluorescein hydrate, fluorescein chloride, 2 ', 7'-dichlorofluorescein, 2', 7'-dichloro Sodium fluorescein, 2,3-diaminonaphthalene, dimidium bromide, 2,3-diphenylmale K, fluorescein, uranin, fluorescein diacetate, coumarin-3-carboxylic acid, 7-hydroxycoumarin-3-carboxylic acid, 4- Dimethylaminoazobenzene-4'-carboxylic acid, 7-methoxycoumarin-3-carboxylic acid, pinacyanol chloride, pinacyanol iodide, pyranine, N- (1-pyrenyl) maleimide, rhodamine 6G, rhodamine B, sulfone Olecein, 7-methoxycoumarin-3-carboxylic acid N-succinimidyl, tetrabromofluorescein potassium, acid red 87, 2 ′, 4 ′, 5 ′, 7′-tetrabromo-3,4,5,6-tetrachlorofluorescein, 9H-fluoren-2-yl isocyanate, fluorescein 5-isothiocyanate, acid red 92, 3,4,5,6-tetrachlorofluorescein, tetraiodofluorescein, erythrosine B, 5- (6-) carboxytetramethylrhodamine -NHS ester, DYLIGHT-405-NHS ester, DY550-NHS ester, DY630-NHS ester, DY-631, DY-633, DY-635, DY-636, DY-650, DY-651-NHS ester, DY- 777-NH S ester (above, Dy ~ is manufactured by Dyomics) BODYIPY650 / 665, ROX, TAMRA, CFSE, Cyto350, Cyto405, Cyto415, Cyto488, Cyto500LSS, Cyto505, Cyto510SS, Cyto514L55, Cyto514LSS, Cyto514LSS, Cyto520L , Cyto 610, Cyto 633, Cyto 647, Cyto 670, Cyto 680, Cyto 700, Cyto 750, Cyto 770, Cyto 780, Cyto 800 (above, Cyto ~ is made by Cytodiagnotics). The fluorescence wavelength of these fluorescent dye compounds is preferably in the range of 400 nm or more that does not overlap with the wavelength of water or protein during detection. There is no particular upper limit to the wavelength, and the higher the wavelength, the better. More preferably, it is a fluorescent dye compound in the range of 500 nm or more.

  Examples of the chemical bond between the fluorescent cellulose fine particles and the fluorescent dye compound include a method of directly connecting a hydroxyl group of cellulose to the fluorescent dye compound and a method of connecting via a compound as a spacer. When a large amount of a fluorescent dye compound is contained, there is a limit to just connecting directly, but a large amount can be introduced through a spacer. When connecting using a spacer, the type of spacer is not particularly limited. For example, epichlorohydrin, 2-chloroethanamine, 11-chloroundecanethiol, formalin, silane coupling agent, epoxy-modified silicone crosslinking agent, Examples thereof include compounds having two or more moieties that react with hydroxyl groups, such as glioxal resins. Further, the fluorescent cellulose fine particles of the present invention may be colored.

  The content of the fluorescent dye compound in the fluorescent cellulose fine particles of the present invention is 0.01% or more and 95% or less. If it is less than 0.01%, sufficient color developability as a detection fine particle of an immunochromatography kit cannot be obtained. On the other hand, if it exceeds 95%, the particles are aggregated, and when developed with an immunochromatography kit, they become clogged and cannot be used. Many conventional fluorescent nanoparticles contain a fluorescent dye compound in a range of 0.001% or more and less than 0.01%, but in this range, sufficient sensitivity as an immunochromatography kit is not exhibited. The fluorescent cellulose fine particles of the present invention can achieve a suitable high sensitivity of an immunochromatography kit when the content of the fluorescent dye compound is around 0.01% or more and 0.03%. A preferable lower limit is 0.04% or more, and more preferably 0.05%. Moreover, a preferable upper limit is 90%, More preferably, it is 85%.

  The raw material of the fluorescent cellulose fine particles in the present invention is literally cellulose. Here, when raw materials other than cellulose are used, a sufficient amount of the fluorescent dye compound cannot be introduced due to the problem of chemical reactivity when the fluorescent dye compound is introduced. For example, Patent Document 4 describes that if the latex particles contain a colorant exceeding 12%, the pores of the immunochromatography kit are likely to be clogged. Originally, it is extremely difficult to introduce a large amount of dye or fluorescent chemical substance into latex, but even if a large amount can be introduced, the structure of the surface will collapse and the sphericity will become extremely bad. Latex is not preferred for introducing large amounts of chemical substances. On the other hand, Patent Document 1 suggests that cellulose maintains its structure even when it contains a large amount of dyes and chemical substances. Therefore, high reactivity and high content can be achieved because the cellulose is rich in hydroxyl groups. For this reason, cellulose is suitable as a material for detection fine particles for immunochromatography. The content of the fluorescent dye compound can be calculated from a change in weight before and after the treatment with the fluorescent dye compound. The ratio of the fluorescent dye compound component is calculated using the weight of the recovered particles after the treatment and the weight after the absolute drying of the cellulose fine particles before the treatment.

Moreover, when the weight of the cellulose fine particle before a process is unknown, fluorescent cellulose fine particles are subjected to cellulase treatment, acid treatment or base treatment to lower the degree of polymerization. Thereafter, the sample is dissolved in heavy water and measured by ¹³ C-NMR by FT-NMR to calculate the degree of substitution. The content of the fluorescent dye compound may be calculated from the degree of substitution. In this case, the cellulase, acid, and base to be used are not limited to any one. For example, as cellulase, Onozuka RS (manufactured by Yakult Pharmaceutical Co., Ltd.), Cellsoft (manufactured by Novo Nordics), Mecellase (Meiji Seika Co., Ltd.) The acid includes hydrochloric acid, sulfuric acid, nitric acid, and the base includes alkali. In addition, when the weight of the cellulose fine particles before treatment is unknown and the fluorescent dye compound contains nitrogen atoms, the nitrogen element content is measured by an emission analysis method with a nitrogen determination device CHN coder and measured. The content of the fluorescent dye compound contained may be calculated from the nitrogen element content.

  The particle size of the cellulose fine particles in the present invention refers to those obtained by measuring a cellulose fine particle dispersion in which cellulose fine particles are dispersed in a liquid using a particle size distribution measuring device. “Average particle diameter” refers to the value of the volume average median diameter of the measured values. Some particle size distribution measuring apparatuses apply various measurement principles. In the present invention, a particle size distribution measuring apparatus using a dynamic light scattering method is used. As will be described later, in the examples, “Nanotrack particle size distribution measuring device UPA-EX150” manufactured by Nikkiso Co., Ltd. was used. A value obtained by dividing the standard deviation of the obtained volume conversion particle size distribution by the average particle size is a CV value (abbreviation of Coefficient of Variation), which is used as an index representing the uniformity of fluorescent cellulose fine particles.

  The average particle diameter of the fluorescent cellulose fine particles in the present invention is 9 nm or more and 700 nm or less. If the average particle size is within this range, precipitation due to long-term storage hardly occurs, and it is also suitable for an immunochromatography kit. When used as a diagnostic agent, it is preferably 20 nm or more and 700 nm or less. When the average particle size is 20 nm or less, the particles aggregate to cause clogging when used in an immunochromatography kit, or sensitivity does not appear when used in an immunochromatography kit. On the other hand, when the thickness exceeds 700 nm, clogging occurs in the developed film when developed with an immunochromatography kit. The lower limit of the average particle diameter for achieving both dispersion stability without aggregation and development without clogging is more preferably 30 nm, and even more preferably 40 nm. The upper limit is preferably 500 nm, more preferably 400 nm. However, in order to improve sensitivity as an immunochromatography kit, two or more kinds of fluorescent cellulose nanoparticles having an average particle diameter may be mixed and used.

  The fluorescent cellulose fine particles in the present invention refer to those in which the ratio of the short diameter to the long diameter (major diameter / short diameter) of the fluorescent cellulose fine particles is sufficiently small in the image analysis of the fluorescent cellulose fine particles by the electron microscope. A rod-like, fiber-like or network-like one having this ratio too large is not included in the fluorescent cellulose fine particles. In order to exhibit the function as fluorescent cellulose fine particles for an immunochromatography kit, the average value of the major axis / minor axis of 100 fluorescent cellulose fine particles is less than 10.0. When this major axis / minor axis exceeds 10, when used in an immunochromatography kit, it does not develop and causes clogging or false positives. When the sphericity is high, it becomes easy to detect with high sensitivity. The average value of the major axis / minor axis of 100 fluorescent cellulose fine particles is preferably 5.0 or less, more preferably 3.0 or less, and still more preferably 2.0 or less. The smaller this value is, the closer the shape of the fluorescent cellulose fine particles is to a perfect sphere.

  The CV value of the fluorescent cellulose fine particles in the present invention is 30% or less. If the CV value exceeds 30%, the amount of the inspection object and the instability of the particle size change cannot be ignored, and the measurement accuracy is adversely affected. Preferably it is 25% or less, More preferably, it is 20% or less. Although it is possible to lower the CV value, it is not preferable that the CV value is too low from the viewpoint of productivity. More preferably, the CV value is 1% or more, and further preferably 3% or more.

  The fluorescent cellulose fine particles in the present invention can also be used by supporting components other than cellulose through chemical bonding or physical adsorption. Examples of chemical bonds or physical adsorption include, but are not limited to, covalent bonds, ionic bonds, coordinate bonds, metal bonds, hydrogen bonds, hydrophilic bonds, hydrophobic bonds, van der Waals bonds, and the like. By allowing the fluorescent cellulose fine particles to carry components other than cellulose by various forces as described above, it is possible to prepare fine particles having a function not found in the fluorescent cellulose fine particles. Even cellulose fine particles in which a fluorescent pigment is not introduced into cellulose can support components other than cellulose, but various types of components can be supported by arbitrarily changing the type of substituent.

  The “component other than cellulose” supported on the fluorescent cellulose fine particles in the present invention refers to various substances other than the fluorescent cellulose fine particles, and the kind thereof is not particularly limited. Examples of these include surfactants, inorganic fine particles, organic fine particles, biomaterials, dyes, ionic substances, water-soluble low molecules, water-soluble polymers, blocking agents, etc., but are not limited thereto. Absent. The “biological material” to be carried on the fluorescent cellulose fine particles in the present invention refers to various materials obtained from a living body, and the kind thereof is not particularly limited. Examples include collagen, gelatin, fibroin, heparin, hyaluronic acid, starch, chitin, chitosan, amino acids, peptides, proteins, nucleic acids, carbohydrates, fatty acids, terpenoids, carotenoids, tetrapyrrole, cofactors, steroids, flavonoids , Alkanoids, polyketides, glycosides, enzymes, antibodies, antigens, carboxymethylcellulose, carboxyethylcellulose, methylcellulose and the like. By supporting them on the fluorescent cellulose fine particles, the biocompatibility of the fluorescent cellulose fine particles can be improved and used as various bioassays and diagnostic agents.

In the present invention, the fluorescent cellulose fine particles can be used as a diagnostic agent by supporting the fluorescent cellulose fine particles with a substance that specifically binds to the substance to be examined.
The substance to be tested in the present invention refers to a measurement object in tests such as immune serum tests, blood tests, cell tests, genetic tests and the like, and the type thereof is not particularly limited. Examples thereof include cancer markers, hormones, infectious diseases, autoimmunity, plasma proteins, TDM, coagulation / fibrinolysis, amino acids, peptides, proteins, genes, cells, and the like. More specifically, CEA, AFP, ferritilin, β2 micro, PSA, CA19-9, CA125, BFP, elastase 1, pepsinogen 1 and 2, fecal occult blood, urinary β2 micro, PIVKA-2, urinary BTA, insulin , E3, HCG, HPL, LH, HCV antigen, HBs antigen, HBs antibody, HBc antibody, HBe antigen, HBe antibody, HTLV-1 antibody, HIV antibody, toxoplasma antibody, syphilis, ASO, influenza A antigen, influenza A Antibody, influenza B antigen, influenza B antibody, rota antigen, adenovirus antigen, rota adenovirus antigen, group A streptococci, group B streptococcus, candida antigen, CD fungus, cryptolocus antigen, cholera fungus, meningitis Fungal antigen, granule elastase, Helicobacter pylori antibody, O157 antibody, O157 antigen, Leptospira antibody, Aspergillus antigen, MRSA, RF, total IgE, LE test, CRP, IgG, A, M, IgD, transferrin, urinary albumin, urinary transferrin, myoglobin, C3 / C4, SAA, LP (a), α1-AC, α1-M, haptoglobin, microtransferrin, APR score, FDP, D dimer, plasminogen, AT3, α2PI, PIC, PAI-1, protein C, coagulation factor X3, type IV Examples include collagen, hyaluronic acid, GHbA1c, various antigens, various antibodies, various viruses, various bacteria, various amino acids, various peptides, various proteins, various DNAs, various cells, and the like.

  The substance that specifically interacts with the test target substance in the present invention refers to a substance that selectively adsorbs or binds to the test target substance, and the type thereof is not particularly limited. For example, antigen, antibody, amino acid, peptide, protein, base sequence and the like can be mentioned. In particular, when an antibody is used, it becomes possible to detect the presence of various antigens in an immune serum test. For example, if an antibody is used, its origin is not particularly limited, and either a polyclonal antibody or a monoclonal antibody may be used. Furthermore, the bonding method of these supporting substances is not particularly limited, and either physical adsorption or chemical bonding may be used. Physical adsorption is more preferable in view of the labor required for loading, and chemical bonding is more preferable in view of stability after loading.

  When the fluorescent cellulose fine particles are used as a diagnostic agent in the present invention, the fluorescent cellulose fine particles can be dispersed in various solutions, but preferably dispersed in a buffer solution having a pH of 5.0 to 11.0. The dispersed liquid is preferred. As the solution for dispersing the fluorescent cellulose fine particles, pure water or an organic solvent can be used. Examples include phosphate buffer, glycine buffer, Tris buffer, borate buffer, citrate buffer, MES buffer, methanol, ethanol, acetone, tetrahydrofuran, and the like. The concentration of the buffer solution is not particularly limited, and various concentrations generally used as a buffer solution can be used. Further, the concentration of the fluorescent cellulose fine particles in the dispersion is not particularly limited, and can be appropriately adjusted according to the type, nature, concentration, etc. of the substance to be inspected. If the concentration of fluorescent cellulose fine particles in the dispersion is too low, visibility is poor and high sensitivity cannot be achieved, so 0.001 or more, and more preferably 0.002% or more is preferable. On the other hand, if the concentration is too high, aggregation occurs and poor development occurs, and high sensitivity cannot be expected, so about 10% or less is preferable, and more preferably 1.0% or less.

In the present invention, when the fluorescent cellulose fine particles are used as a diagnostic agent, various sensitizers may be used for improving measurement sensitivity and promoting antigen-antibody reaction. Further, a blocking agent or the like may be used to suppress nonspecific adsorption caused by other substances present in the specimen.
The fluorescent cellulose fine particles in the present invention can be used by being dispersed in an arbitrary liquid like a diagnostic agent. However, the fluorescent cellulose fine particles can be used by being dispersed in any other solid, or the fine particles are immobilized on a solid surface. It is also possible. Further, by coloring the fluorescent cellulose fine particles, it is possible to improve the visibility of the particles or improve the detection sensitivity.

  The method for producing the raw material for fluorescent cellulose fine particles in the present invention is not particularly limited. Fine particles having a desired average particle diameter may be obtained by classification using a mechanical method such as wet grinding. In the present invention, cellulose is dissolved in a good solvent, and water, an organic solvent, ammonia, or the like is mixed. Cellulose fine particles are prepared by using a coagulation liquid. By using this method, the particle size of the cellulose fine particles obtained can be adjusted by the composition of the coagulation liquid. Although it does not intend to limit the manufacturing method of the raw material of the fluorescent cellulose fine particle of this invention, it illustrates as the manufacturing methods 1 and 2 below.

[Production Method 1: Preparation of Cellulose Fine Particles]
Cellulose linter is dissolved in a good solvent for cellulose. In the present invention, a copper ammonia solution prepared by a known method is used as a good solvent. As the coagulation liquid, an organic solvent + water + ammonia mixed system is mainly used. While the coagulation liquid is stirred, the prepared copper ammonia cellulose solution is added for coagulation. Furthermore, the slurry containing the target cellulose fine particle can be obtained by adding and neutralizing and regenerating sulfuric acid. At this time, the slurry is acidic due to the residue of the acid used for the regeneration, and further contains impurities such as ammonium salts generated by neutralization, and therefore it is necessary to perform an operation for purification into a cellulose dispersion composed of cellulose fine particles and a medium. Become. In the present invention, as this purification operation, centrifugation, decantation, and repetition of dilution with a dispersion medium liquid are used. The kind of the dispersion medium liquid used in this case is not particularly limited, and various hydrophilic solvents described above can be used depending on the purpose. Since the cellulose fine particles in the obtained cellulose fine particle dispersion may be aggregated in the course of the purification operation, in this case, a dispersion treatment by shearing or the like can be performed. In the present invention, a high-pressure homogenizer is used as means for applying shear. The cellulose fine particle dispersion thus obtained is measured for an average particle size and a CV value using a particle size distribution measuring device. The obtained cellulose fine particle dispersion can be used by adding a surfactant as required. The cellulose fine particle dispersion can be used as it is in a dry state, and can be prepared into cellulose fine particles by drying as necessary. The obtained cellulose fine particles are observed using an electron microscope, and the sphericity and the aggregation constant are measured from the image. Furthermore, cellulose fine particles are dissolved in a cadoxene solution, and the average degree of polymerization is measured from the viscosity. Here, the average degree of polymerization of cellulose fine particles suitable for production of fluorescent cellulose fine particles is 30 or more and 700 or less. Here, if the average degree of polymerization is less than 30, the uniformity of the particles is lost, so the CV value increases, and the quality is not stable when used in an immunochromatography kit. On the other hand, when the average degree of polymerization exceeds 700, the fluorescent dye compound is physically included, and even if chemical bonding is attempted, the crystalline crystallinity of the cellulose is high, so that the fluorescent dye compound is used up to the target introduction amount. Cannot be contained. Therefore, the fluorescent cellulose fine particles of the present invention can be produced for the first time by controlling the degree of polymerization of the cellulose fine particles before dyeing and the average particle size within the range of the present invention. . In order to produce fluorescent cellulose fine particles, the lower limit of the average degree of polymerization of the cellulose fine particles is preferably 35 or more, more preferably 40 or more. Moreover, a preferable upper limit is 650, More preferably, it is 600.

[Production Method 2: Preparation of fluorescent cellulose fine particles]
The cellulose fine particles produced by the production method 1 are added to an organic solvent and dispersed. The cellulose fine particles may be colored. Here, examples of the organic solvent include methanol, ethanol, isopropyl alcohol, butanol, pentanol, hexanol, diethyl ether, isopropyl ether, dichloromethane, chloroform, carbon tetrachloride, ethyl acetate, methyl acetate, methyl ethyl ketone, cyclohexane, Examples thereof include cyclopentane, tetrahydrofuran, toluene, hexane, water, and the like, and they can be used as one kind or a mixture of two or more kinds according to the kind of the fluorescent dye compound. In addition, since the cellulose fine particles used as the raw material of the fluorescent cellulose fine particles of the present invention are in the cellulose II crystal type, the degree of crystallinity is low, thereby significantly increasing the amount of fluorescent dye introduced into conventional latex particles or silica particles. Can be increased. In order to increase the amount of fluorescent dye introduced, cellulose may be physically or chemically modified to introduce an amino group or thiol group and then reacted with the fluorescent dye compound.

After adding the fluorescent dye compound to the solution containing the cellulose fine particles, an additive is appropriately added, the pH is adjusted, and the solution is heated and cooled. In the slurry, unreacted substances and by-products such as the fluorescent dye compound used in the reaction remain, and an operation for purifying the fluorescent cellulose fine particles and the medium is required. In the present invention, as this purification operation, centrifugation, decantation, and repetition of dilution with a dispersion medium liquid are used. The kind of the dispersion medium liquid used in this case is not particularly limited, and various hydrophilic or lipophilic solvents or solutions described above can be used depending on the purpose.
Through the above steps, the fluorescent cellulose fine particles of the present invention can be produced.

  EXAMPLES Hereinafter, although an Example is given and this invention is demonstrated concretely, this invention is not limited at all by an Example. The main measurement values in the examples were measured by the following methods.

<Measurement of average particle diameter (nm) and calculation method of CV value (%)>
The dispersion of cellulose fine particles and fluorescent cellulose fine particles before treatment was subjected to particle size distribution measurement using a particle size distribution meter Microtrac manufactured by Nikkiso Co., Ltd., and the average particle size was measured. The CV value is calculated by the following formula (1). The measurement was carried out with a measurement time of 30 seconds and an integration count of 99 times.
CV value (%) = (standard deviation in volume particle size distribution obtained from particle size distribution measuring device) / (volume average median diameter obtained from particle size distribution measuring device) × 100 Formula (1)

<Measurement and calculation method of average degree of polymerization>
The specific viscosity of a diluted cellulose solution in which cellulose fine particles are dissolved in cadoxen is measured with an Ubbelohde viscometer, and the value calculated from the intrinsic viscosity [η] by the following viscosity equation (2) and conversion equation (3) is average polymerization. (Reference: Eur. Polym. J., 1, 1 (1996)).
Intrinsic viscosity number [η] = 3.85 × 10 ⁻² × M _W ^0.76 formula (2)
Average degree of polymerization (DP) = _Mw / 162 Formula (3)

<Content of fluorescent dye compound>
The ratio of the fluorescent dye compound component to the fluorescent cellulose fine particles can be calculated from the weight change before and after the fluorescent dye compound treatment. The ratio of the fluorescent dye compound component was calculated from the following formula (4) using the weight of the recovered particles after the treatment and the weight after the absolute drying of the cellulose fine particles before the treatment.
Fluorescent dye compound content (%) = 1-{(weight of cellulose fine particles before treatment) / (weight of fluorescent cellulose fine particles after treatment of fluorescent dye compound)} × 100 Formula (4)

(When the weight of cellulose fine particles before treatment is unknown)
After subjecting the fluorescent cellulose fine particles to cellulase treatment, acid treatment or base treatment, the sample is dissolved in heavy water to prepare a 3-5 wt% heavy aqueous solution, and measured by ¹³ C-NMR (Avance 400 MHz) by FT-NMR, The degree of substitution is calculated. The degree of substitution is calculated from the peak area of the fluorescent dye compound on the basis of the C1 peak area of cellulose. From the degree of substitution and the molecular weight of the fluorescent dye compound, the content of the fluorescent dye compound is calculated.

(When the weight of cellulose fine particles before treatment is unknown and the fluorescent dye compound contains a nitrogen atom)
The nitrogen element content is measured by an emission analysis method under the following measurement conditions using a nitrogen determination device CHN coder (manufactured by Yanaco Analytical Industries). From the measured nitrogen element content, the content of the contained fluorescent dye compound is calculated.
Measurement method: Self-integration method Carrier gas: Helium Auxiliary gas: High-purity oxygen Auxiliary combustion method: Helium and oxygen mixing method

<Method of judging sensitivity of immunochromatographic evaluation>
As a method for determining color development, the membrane of the immunochromatography kit was exposed, excited using a laser diode, and the fluorescence profile of the membrane was obtained by receiving fluorescence with a photodiode. The sensitivity of the test line and the control line was evaluated from the obtained fluorescence profile. As the evaluation criteria, the case where color development was not recognized on the test line was (−), and the case where color development was recognized was (+).

[Example 1]
A copper ammonia cellulose solution having a cellulose concentration of 0.37 wt%, a copper concentration of 0.13 wt%, and an ammonia concentration of 1.00 wt% was prepared. Further, a coagulation liquid having a tetrahydrofuran concentration of 87.5 wt% and a water concentration of 12.5 wt% was prepared.
While stirring 5000 g of the coagulation liquid slowly using a magnetic stirrer, 500 g of the prepared copper ammonia cellulose solution was added thereto. After stirring for about 5 seconds, 1000 g of 10 wt% sulfuric acid was added to neutralize and regenerate to obtain 6500 g of a slurry containing cellulose fine particles.

  The resulting slurry was centrifuged for 10 minutes at a speed of 10,000 rpm. The precipitate was taken out by decantation, injected with ultrapure water, stirred, and centrifuged again. This operation was repeated several times until the pH reached 6.0 to 7.0, and then a dispersion treatment using a high-pressure homogenizer was performed to obtain 150 g of a cellulose fine particle dispersion. Dry cellulose fine particles were obtained by freeze-drying. As a result of measuring the average particle diameter of the obtained cellulose fine particles, it was 205 nm. The CV value was 18%. The obtained cellulose fine particle dispersion was centrifuged and dehydrated, and then freeze-dried by a metal contact method. At this time, the polymerization of the cellulose fine particles was 200, and the major axis / minor axis was 1.9.

  To an eggplant type glass flask, 50 g of tetrahydrofuran was added as a dispersion medium, 1.0 g of DY-651-NHS ester (manufactured by Dyomics) was added, and the mixture was stirred at 30 ° C. with a magnetic stirrer. Thereto was added 0.50 g of dried cellulose fine particles, and 1.0 ml of triethylamine (manufactured by Tokyo Chemical Industry Co., Ltd.) was added. Stir the mixture as it is, stop the stirring after 2 hours, repeat decantation and dilution with deionized water several times using a centrifuge, adjust the pH to 6.0 to 7.0, and disperse with a high-pressure homogenizer. To obtain 100 g of a fluorescent cellulose fine particle dispersion. As a result of measuring the average particle diameter of the obtained fluorescent cellulose fine particles, it was 208 nm. The CV value was 19% and the major axis / minor axis was 1.5. The content of the fluorescent pigment in the fluorescent cellulose fine particles after the treatment was 1.0%. The results are shown in Table 1 below.

[Examples 2 to 6]
The fluorescent cellulose fine particles were produced in the same manner as in Example 1 except that the average particle size of the cellulose fine particles was changed during the treatment with the fluorescent dye compound. The results are shown in Table 1 below.

[Examples 7 to 10]
Fluorescent cellulose fine particles were produced in the same manner as in Example 1 except that the amount of the fluorescent dye compound added was changed during the treatment with the fluorescent dye compound. The results are shown in Table 1 below.

[Examples 11 to 12]
The cellulose fine particles before introduction of the fluorescent dye compound obtained in Example 1 were added to a eggplant-shaped glass flask with 50 g of tetrahydrofuran and 1.0 g of a 10 wt% aqueous sodium hydroxide solution as a dispersion medium, and 2-chloroethanamine or 11-chloroundecane. 1.0 g of thiol was added, a glass reflux tube was attached, and the mixture was stirred with a magnetic stirrer at 60 ° C. for 3 hours while refluxing and cooling tap water. Thereafter, dilution and washing with decantation-deionized water were performed using a centrifuge. Subsequent washing and dispersion treatment was carried out in the same manner as in Example 1 to obtain modified cellulose fine particles. Thereafter, the fluorescent dye compound was introduced by the same method as in Example 1 to produce fluorescent cellulose fine particles. The results are shown in Table 1 below.

[Example 13]
At the time of the fluorescent dye compound treatment, tetrahydrofuran as a dispersion medium is added to an eggplant type glass flask, 10 g of DY-651-NHS ester and 10 g of γ-aminopropyltriethoxysilane are added, a glass reflux tube is attached, and tap water is added. While refluxing and cooling, the mixture was stirred with a magnetic stirrer at 60 ° C. for 3 hours. Thereto, 0.50 g of cellulose fine particles after drying treatment and 1.0 ml of triethylamine (manufactured by Tokyo Chemical Industry Co., Ltd.) were added and stirred for 2 hours. The mixed dispersion was centrifuged to remove the solvent, and then the eggplant flask was placed in an oven and heat treated at 120 ° C. for 5 minutes. Thereafter, dilution and washing with decantation-deionized water were performed using a centrifuge. Subsequent washing and dispersion treatment was carried out in the same manner as in Example 1. As a result of measuring the average particle diameter of the obtained fluorescent cellulose fine particles, the major axis / minor axis was 1.2 at 280 nm, and the CV value was 19%. The content of the fluorescent pigment in the fluorescent cellulose fine particles after the treatment was 20%. The results are shown in Table 1 below.

[Example 14]
Fluorescent cellulose fine particles were produced in the same manner as in Example 13, except that the amount of γ-aminopropyltriethoxysilane was changed during the treatment of the fluorescent dye compound. The results are shown in Table 1 below.

[Examples 15 to 19]
Fluorescent cellulose fine particles were produced in the same manner as in Example 11 except that the fluorescent dye compound was treated by changing the type of the fluorescent dye compound. The results are shown in Table 1 below.

[Examples 20 to 24]
Fluorescent cellulose fine particles were produced in the same manner as in Example 1 except that the degree of polymerization of the cellulose fine particles was changed during the treatment with the fluorescent dye compound. The results are shown in Table 1 below.

[Comparative Examples 1 and 2]
Fluorescent cellulose fine particles were produced in the same manner as in Example 1 except that the average particle diameter of the cellulose fine particles was changed outside the scope of the present invention during the treatment with the fluorescent dye compound. The results are shown in Table 1 below.

[Comparative Example 3]
Fluorescent cellulose fine particles were produced in the same manner as in Example 13, except that the average particle size of the cellulose fine particles was changed outside the scope of the present invention during the treatment with the fluorescent dye compound. The results are shown in Table 1 below.

[Comparative Examples 4 and 5]
Fluorescent cellulose fine particles were produced in the same manner as in Example 1, except that the amount of the fluorescent dye compound was changed outside the scope of the present invention during the treatment with the fluorescent dye compound. The results are shown in Table 1 below.

[Comparative Example 6]
Fluorescent cellulose fine particles were produced in the same manner as in Example 13 except that the amount of the fluorescent dye compound was changed outside the scope of the present invention during the treatment with the fluorescent dye compound. The results are shown in Table 1 below.

[Comparative Examples 7 and 8]
Fluorescent cellulose fine particles were produced in the same manner as in Example 1 except that the degree of polymerization of the cellulose fine particles was changed during the treatment with the fluorescent dye compound. The obtained particles were out of the scope of the present invention with respect to either the CV value (%) or the fluorescent dye compound content (%). The results are shown in Table 1 below.

[Comparative Example 9]
Fluorescent cellulose fine particles were produced in the same manner as in Example 13 except that the degree of polymerization of the cellulose fine particles was changed during the treatment with the fluorescent dye compound. The obtained particles were out of the scope of the present invention with respect to the fluorescent dye compound content (%). The results are shown in Table 1 below.

[Comparative Examples 10 and 11]
The fluorescent cellulose fine particles were produced in the same manner as in Example 1, except that the fluorescent cellulose fine particles were produced using a cellulose fine particle having a major axis / minor axis of 10 or more. The results are shown in Table 1 below.

[Dispersion stability test of fluorescent cellulose fine particles]
The fluorescent cellulose fine particles obtained in Examples 1 to 24 and Comparative Examples 1 to 11 were dispersed in pure water and adjusted to 1.0 wt% at a temperature of 25 ° C. in a polypropylene screw tube for 3 months in a dark place. The CV values of the samples stored for 6 months and 12 months were measured, and the dispersion stability test was performed. The results are shown in Table 1 below. From the results shown in Table 1, it can be seen that the fluorescent cellulose fine particles obtained in Examples 1 to 24 are generally better in dispersion stability than those obtained in Comparative Examples 1 to 11.

[Color intensity test in immunochromatography kit prepared using fluorescent cellulose fine particles]
Using the resulting fluorescent particles, an immunochromatography kit was prepared and the color intensity was evaluated.
Hereinafter, preparation of an immunochromatography kit will be described.
100 μL (dispersion medium: distilled water) of dispersion liquid of fluorescent cellulose fine particles having a concentration of 5 mg / ml (Examples 1 to 24, Comparative Examples 1 to 11) and 390 μL of 50 mM KH ₂ PO ₄ (pH 7.0) were added to the microtube. Stir gently. Add 10 μL (5.8 mg / mL) of anti-hCG antibody (Anti-hCG clone codes / 5008, manufactured by Medix Biochemica) to the microtube, mix gently at room temperature for 10 minutes, and add anti-hCG antibody to the fluorescent cellulose microparticles. Adsorbed.
The mixture was centrifuged at 12000 × g for 15 minutes and the supernatant was removed. To this, 1 mL of storage buffer (20 mM Tris-HCl (pH 8.2), 0.05% PEG20,000, 150 mM NaCl, 1% BSA, 0.1% NaN ₃ ) was added, centrifuged again, and the supernatant Removed. 500 μL of distilled water and 500 μL of coating buffer (20 mM Tris-HCl (pH 8.2), 0.05% PEG (molecular weight 20,000), 5% sucrose) were added thereto to disperse the particles. A colloid of composite particles was obtained (0.5 mg / mL × 1 mL).
0.8 mL of the colloid of the composite particles was uniformly applied to Glass Fiber Conjugate Pad (GFCP, manufactured by MILLIPORE) (8 × 150 mm). A conjugate pad containing composite particles was prepared by drying under reduced pressure overnight at room temperature in a desiccator.

Hereinafter, a method for producing an antibody-immobilized membrane will be described.
Anti-hCG antibody (alpha subunit of FSH (LH), clone) as a test line with a width of about 1 mm around the center (about 12 mm from the end) of the membrane (length 25 mm, trade name: Hi-Flow Plus120 membrane, manufactured by MILLIPORE) A solution ((50 mM KH ₂ PO ₄ , pH 7.0) + 5% sucrose) containing 1 mg / mL of code / 6601, manufactured by Medix Biochemica) was applied at a coating amount of 0.75 μL / cm.
Next, as a control line having a width of about 1 mm, 0.75 μL of a solution ((50 mM KH ₂ PO ₄ , pH 7.0) sugar free) containing 1 mg / mL of anti-mouse IgG antibody (Anti Mouse IgG, manufactured by Dako) was used. It was applied at a coating amount of / cm and dried at 50 ° C. for 30 minutes. The interval between the test line and the control line was 4 mm. Next, as a blocking treatment, the entire membrane was immersed in a blocking buffer (composition: 100 mM boric acid (pH 8.5), 1 wt% casein) at room temperature for 30 minutes.
The membrane was transferred to a membrane washing / stabilizing buffer (composition: 10 mM KH ₂ PO ₄ (pH 7.5), 1% by weight sucrose, 0.1% sodium cholate) and allowed to stand at room temperature for 30 minutes or more. The membrane was pulled up, placed on a paper towel and dried overnight at room temperature to prepare an antibody-immobilized membrane.

Backing sheet (trade name AR9020) of sample pad (Glass Fiber Conjugate Pad (GFCP), manufactured by MILLIPORE), the conjugate pad, the antibody-immobilized membrane, and the absorbent pad (Cellulose Fiber Sample Pad (CFSP), manufactured by MILLIPORE) , Manufactured by Adhesives Research Inc.) in this order and cut into strips having a width of 5 mm and a length of 60 mm to obtain test strips.
In addition, each constituent member is dropped by 100 μL of a predetermined concentration of recombinant hCG (manufactured by Rohto Pharmaceutical Co., Ltd.), which is pasted and overlapped with an adjacent member at about 2 mm on each sample pad portion of the prepared test strip for 20 minutes. After leaving, the sample pad and the conjugate pad of the test strip were peeled off to expose the membrane, excitation was performed using a laser diode, and fluorescence was received by the photodiode to obtain a fluorescence profile of the membrane. From the obtained fluorescence profile, the fluorescence intensity of the test line and the control line was evaluated as positive (+) or negative (−). The results are shown in Table 2 below.

[Color intensity test after storage for 3 months, 6 months, 12 months in an immunochromatography kit prepared using fluorescent cellulose fine particles]
The immunochromatography kit was prepared with the recombinant hCG at a concentration of 0.005 mIU / ml in the same manner as the above-described immunochromatography kit, and the color intensity test was performed after storage for 3 months, 6 months, and 12 months. did. The results are shown in Table 2 below.

From the results shown in Table 2, it can be seen that all of the fluorescent cellulose fine particles of Examples 1 to 24 exhibit high sensitivity and dispersion stability as developed particles of an immunochromatography kit.
On the contrary, in Comparative Example 1, although the sensitivity was high immediately after preparation of the immunochromatography kit, since the average particle size was too small, the particles aggregated from around 6 months and could not be detected.
In Comparative Examples 2 and 3, due to the large particle size, clogging occurred in the developed membrane, and the antigen could not be detected.
In Comparative Examples 4 and 5, since the content of the fluorescent dye compound was too small, almost no color was developed and sufficient sensitivity was not obtained.
In Comparative Example 6, aggregation occurred immediately after introduction of the fluorescent dye compound, and hardly developed when used in an immunochromatography kit.
In Comparative Example 7, the target sensitivity was achieved immediately after production, but the dispersion stability was poor, and after 3 months, no color was developed even at an antigen concentration of 0.002 mIU / ml that could be detected before storage. .
In Comparative Examples 8 and 9, the content of the fluorescent dye compound was too small, so that sufficient sensitivity could not be achieved.
In Comparative Example 10, false positives occurred.
In Comparative Example 11, clogging occurred immediately after deployment, and development failure occurred.

  The fluorescent cellulose fine particles of the present invention and the immunochromatography kit using the same are suitable for immunoassays in clinical tests and the like because they can detect a substance to be detected contained in a biological sample with high sensitivity and have high storage stability. Is available.

Claims (12)

The fluorescent dye compound is contained in a content of 0.01% to 95%, the average particle size is 9 nm to 700 nm, the CV value is 30% or less, and the average value of the major axis / minor axis is less than 10. And fluorescent cellulose fine particles having an average degree of polymerization of 30 or more and 700 or less .   The fluorescent cellulose fine particles according to claim 1, wherein components other than cellulose are supported via chemical bonding or physical adsorption.   The fluorescent cellulose fine particles according to claim 2, wherein the components other than cellulose are substances that specifically interact with other components.   The fluorescent cellulose fine particles according to claim 2 or 3, wherein the component other than cellulose is a biomaterial.   The fluorescent cellulose fine particles according to claim 4, wherein the biomaterial is an antigen or an antibody.   The diagnostic agent containing the fluorescent cellulose microparticle of any one of Claims 1-5.   The diagnostic agent according to claim 6, comprising the fluorescent cellulose fine particles according to any one of claims 2 to 4, wherein the component other than cellulose is a substance that specifically interacts with the substance to be examined.   The diagnostic agent according to claim 6 or 7, which is used for immunodiagnosis.   An in vitro sample detection method comprising a step of mixing the diagnostic agent according to any one of claims 6 to 8 and a sample, and detecting a test target substance in the sample.   An immunochromatography kit for detection by an immunochromatography method, comprising the diagnostic agent according to any one of claims 6 to 8.   A dispersion in which the fluorescent cellulose fine particles according to any one of claims 1 to 5 are dispersed in a liquid.   A molded article in which the fluorescent cellulose fine particles according to any one of claims 1 to 5 are fixed to a solid surface or dispersed in a solid.