Fluorescent cellulose particles

Abstract

The present invention provides fluorescent cellulose microparticles which are capable of reducing deployment failure by improving the deployability during deployment in immunochromatography, while maintaining sufficient color developability and dispersion stability if used for immunochromatography. The present invention relates to: fluorescent cellulose particles, each of which contains a cellulose particle, a fluorescent dye compound and a heterocyclic compound represented by general formula (1) (wherein R1 represents a functional group that has an affinity for a biological substance; and R2 represents an ether bond part bonded to the cellulose particle), and which are characterized in that, per 1 g of the fluorescent cellulose particles, the content of the cellulose particles is 30% by mass to 90% by mass, the content of the fluorescent dye compound is 1% by mass to 40% by mass, and the content of the heterocyclic compound is 3% by mass to 50% by mass; and a diagnostic agent and an immunochromatography kit, each of which comprises the fluorescent cellulose particles.

Description

Technical Field

[0001] The present invention relates to fluorescent cellulose particles, diagnostic agents using the same, and immunochromatography kits.

Background Art

[0002] Conventionally, as one of the immunoassay methods for detecting a substance to be detected consisting of a specific antigen or antibody by utilizing the specific reaction between an antigen and an antibody, an agglutination method in which the substance to be detected in a sample is specifically bound to an antibody or antigen supported on microparticles by an immunoreaction, and the agglutination state of the microparticles caused by the binding is measured, is a simple measurement method and is generally used particularly because visual determination is possible. In addition, as other immunoassay methods, radioimmunoassay, enzyme immunoassay, immunofluorescence assay, etc. are also widely used. Further, a method of using a substance that immunologically binds to a substance to be detected and combining the principles of immunoreaction and chromatography to detect the substance to be detected by visual determination is called an immunochromatography method or an immunochromatograph method, and has been widely used in recent years.

[0003] The immunochromatography method is a method in which an antibody (or antigen) against an antigen (or antibody) as a substance to be detected is immobilized on a chromatographic medium to prepare a reaction site on the chromatographic medium as a stationary phase, and a detection microparticle carrying an antibody (or antigen) capable of binding to the substance to be detected and a sample containing the substance to be detected are brought into contact with each other {by this contact, the antibody (or the antigen) on the detection microparticle for antibody sensitization (or antigen sensitization) reacts with the antigen (or antibody) in the sample, and a complex consisting of the detection microparticle - the antibody (or antigen) used for sensitization - the antigen (or antibody) in the sample is generated}, and by moving it on the chromatographic medium, the sample is brought into contact with the reaction site. As a result, at the reaction site, the complex is bound to the immobilized antibody (or immobilized antigen), and the detection microparticle is captured. Therefore, the presence of the substance to be detected in the sample can be determined by visually determining the presence or absence of the capture of the detection microparticle. A diagnostic agent kit utilizing this principle is called an immunochromatography kit.

[0004] In the above immunochromatography kit and agglutination method, colored fine particles are often used as detection fine particles to facilitate visual determination. As such detection fine particles, colloidal metal fine particles that naturally color depending on their particle size and preparation conditions, fine particles obtained by coloring latex fine particles made of synthetic polymers, and colored latex fine particles obtained by a method of polymerizing a monomer together with a coloring agent are known. Further, in Patent Document 1 below, highly color-developing colored fine particles made from cellulose fine particles are reported. However, these fine particles have problems such as being easily discolored and having limitations in color development, and further improvement in performance is desired. Therefore, in recent years, fluorescent nano fine particles have attracted attention as new detection fine particles.

[0005] Fluorescent reagents used for the detection and quantification of biomolecules using fluorescent nano fine particles have high color-developing properties and are used as highly sensitive reagents. For example, Patent Document 2 below discloses fluorescent latex fine particles in which a fluorescent dye compound is introduced into latex fine particles obtained by polymerizing styrene and acrylic acid. Further, 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.

[0006] However, these fluorescent nano fine particles have a problem that since the amount of the introduced fluorescent dye compound is small, the color-developing property satisfactory for an immunochromatography kit cannot be obtained, and aggregation occurs between the particles during storage of the particles, resulting in clogging and false positives when developed in an immunochromatography kit.

[0007] Aiming to solve such problems, Patent Document 4 below discloses fluorescent cellulose fine particles. When cellulose fine particles in a specific shape and particle size range contain a fluorescent dye compound in a specific content range, they become fluorescent cellulose fine particles with high color-developing properties and good dispersion stability of the particles, and further, it is reported that high-sensitivity of an immunochromatography kit can be achieved.

[0008] However, in Patent Document 4 below, the developability of the particles when used in immunochromatography has not been sufficiently studied, and the compatibility between sensitivity / dispersion stability and developability has not been mentioned.

Prior Art Documents

Patent Documents

[0009]

Patent Document 1

Patent Document 2

Patent Document 3

Patent Document 4

Patent Document 5

Patent Document 6

Summary of the Invention

Problems to be Solved by the Invention

[0010] In view of the problems of the above prior art, the problem to be solved by the present invention is to provide fluorescent cellulose microparticles that can improve the developability during immunochromatography and reduce poor development while maintaining sufficient color development and dispersion stability when used in immunochromatography. Furthermore, by improving the developability, the background coloring during development is improved, and a good S / N ratio can be achieved even near the limit detection concentration.

Means for Solving the Problems

[0011] As a result of intensive studies and repeated experiments to achieve the above problems, the inventors of the present invention surprisingly found that when a fluorescent dye compound binds to cellulose particles at a content within a specific range and further a compound having a heterocyclic structure binds to cellulose particles at a content within a specific range, when used in an immunochromatography, while maintaining sufficient color development intensity and dispersion stability of the particles, the developability during immunochromatography development is improved, and further the background coloring during development is improved, resulting in a good S / N ratio even near the limit detection concentration. Based on such findings, the present invention has been completed.

[0012] That is, the present invention is as follows. [1] Fluorescent cellulose particles containing cellulose particles, a fluorescent dye compound, and a heterocyclic compound represented by the following general formula (1): [Chemical formula] {In the formula, R 1 is a functional group having an affinity for a biological substance, and R 2 is an ether bond part with the cellulose particles.} The fluorescent cellulose particles are characterized in that, per 1 g of the fluorescent cellulose particles, the content of the cellulose particles is 30% by mass or more and 90% by mass or less, the content of the fluorescent dye compound is 1% by mass or more and 40% by mass or less, and the content of the heterocyclic compound is 3% by mass or more and 50% by mass or less. [2] The fluorescent cellulose particles according to [1] above, wherein R 1 of the heterocyclic compound is Cl and / or OH. [3] The fluorescent cellulose particles according to [1] or [2] above, wherein the average particle diameter of the fluorescent cellulose particles is 9 nm or more and 500 nm or less. [4] The fluorescent cellulose particles according to any one of [1] to [3] above, wherein the fluorescent dye compound is bonded to the OH group of the cellulose particles, and the heterocyclic compound is bonded to the OH group of the cellulose particles. [5] The fluorescent cellulose particles according to any one of [1] to [4] above, wherein the fluorescent dye compound is a europium complex. [6] The fluorescent cellulose particles according to any one of [1] to [5] above, wherein the biological substance is supported via physical adsorption. [7] The fluorescent cellulose particles according to [6] above, wherein the biological substance is a protein, a peptide or a nucleic acid. [8] The fluorescent cellulose particles according to [7] above, wherein the protein is an antigen or an antibody. [9] A diagnostic agent containing the fluorescent cellulose particles according to any one of [1] to [8] above.

[10] An immunochromatography kit containing the fluorescent cellulose particles according to any one of [1] to [8] above. [Advantages of the Invention]

[0013] When the fluorescent cellulose particles of the present invention are used as coloring particles for immunochromatography, they are excellent in developability while maintaining color developability and dispersion stability. Furthermore, by improving poor development and background coloring, a good S / N ratio can be achieved even near the limit detection concentration. [Brief Description of the Drawings]

[0014]

Figure 1

[0015] Hereinafter, the present invention will be described in detail. One embodiment of the present invention is a cellulose particle, a fluorescent dye compound, and the following general formula (1): [Chemical formula] [In the formula, R 1 is a functional group having an affinity for a biological substance, and R 2is an ether bond part with the cellulose particles. A fluorescent cellulose particle containing a heterocyclic compound represented by}, wherein the content of the cellulose particles is 30% by mass or more and 90% by mass or less, the content of the fluorescent dye compound is 1% by mass or more and 40% by mass or less, and the content of the heterocyclic compound is 3% by mass or more and 50% by mass or less per 1 g of the fluorescent cellulose particle. The fluorescent cellulose particle is characterized by the above. The cellulose particle content in the fluorescent cellulose of this embodiment is 30% by mass or more and 90% by mass or less, preferably 35% by mass or more and 85% by mass or less, per 1 g of the fluorescent cellulose particle.

[0016] In the general formula (1), preferably R 1 is a functional group having an affinity for a biological substance, and R 2 is an ether bond part with the cellulose particles. However, R 1 may be an ether bond part with the cellulose particles, and R 2 may be a functional group having an affinity for a biological substance.

[0017] Considering that the particle size may increase due to the modification of the fluorescent dye compound and the heterocyclic compound, the particle size of the raw material cellulose particles used in the production of the fluorescent cellulose particles of this embodiment is smaller than the particle size of the finally obtained fluorescent cellulose particles. Specifically, it is preferably 3 nm or more and 480 nm or less, and more preferably 6 nm or more and 460 nm or less.

[0018] The raw material of the fluorescent cellulose particles of this embodiment only needs to be cellulose, and the cellulose source is not particularly limited. Here, when using raw materials other than cellulose, when introducing a fluorescent dye compound, due to problems with chemical reactivity, a sufficient amount of the fluorescent dye compound cannot be introduced. For example, Patent Document 5 above states that when a colorant is included in latex particles in an amount exceeding 12% by mass, there is a high risk of clogging the pore portion of the immunochromatography kit. In addition, Patent Document 6 discloses fluorescent particles for diagnostic agents containing 10% by mass or more of an aggregating luminescent material, but the luminescent materials that can be used are limited. Originally, it is extremely difficult to introduce a large amount of a dye or a fluorescent chemical substance into latex. Even if a large amount could be introduced, the surface structure would collapse and the sphericity would become extremely poor. Therefore, latex is not preferable for introducing a large amount of a dye or a chemical substance. In contrast, cellulose can maintain its structure even when containing a large amount of a dye or a chemical substance. Therefore, it is precisely because of the cellulose rich in hydroxyl groups that high reactivity and a high content can be achieved. Therefore, cellulose is suitable as a material for detection particles for immunochromatography. In addition, the content of the fluorescent dye compound can be calculated from the weight change before and after the treatment with the fluorescent dye compound. Using the weight of the recovered particles after the treatment and the weight of the cellulose particles after thorough drying before the treatment, the ratio of the fluorescent dye compound component is calculated.

[0019] Also, when the weight of the cellulose particles before the treatment is unknown, the fluorescent cellulose particles are subjected to cellulase treatment, acid treatment, or base treatment to reduce the degree of polymerization. Then, the sample is dissolved in heavy water and analyzed by FT-NMR 13Perform measurements by 13C-NMR and calculate the degree of substitution. From the degree of substitution, the content of the fluorescent dye compound may be calculated. In this case, the cellulase, acid, and base used are not limited to any particular ones. For example, as the cellulase, there are Onozuka RS (manufactured by Yakult Pharmaceutical Industry Co., Ltd.), Cellsoft (manufactured by Novo Nordisk), and Maiserase (manufactured by Meiji Seika Kaisha, Ltd.). As the acid, there are hydrochloric acid, sulfuric acid, and nitric acid. As the base, there is alkali. Also, when the weight of the cellulose particles before treatment is unknown and the fluorescent dye compound contains a nitrogen atom, the nitrogen element content rate may be measured by a nitrogen quantifier CHN coder by luminescence analysis, and the content of the contained fluorescent dye compound may be calculated from the measured nitrogen element content rate.

[0020] The type of the fluorescent dye compound is not particularly limited. For example, there are fluoresceins, rhodamines, coumarins, cyanine compounds that emit fluorescence and have active substituents such as N-hydroxysuccinimide ester group, ester group, carboxyl group, maleimide group, isocyanate group, isothiocyanate group, cyano group, halogen group, aldehyde group, paranitrophenyl group, diethoxymethyl group, epoxy group, etc., and rare earth complexes containing europium. Specifically, as the fluorescent dye compound, there are fluorene, fluorene-9-acetic acid, fluorene-2-carboxaldehyde, 9-fluorene-1-carboxylic acid, 9-fluorene-4-carboxylic acid, 9-fluorenoxime, 9-fluorenemethyl succinimidyl carbonate, 9-fluorenetriphenylphosphonium bromide, 5-aminofluorescein, disodium 8-amino-1,3,6-naphthalenetrisulfonate hydrate, sulforhodamine 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 sodium, calcein, coumarin 102, coumarin 314, coumarin 343, AMCA, 5-carboxyfluorescein hydrate, 6-carboxyfluorescein hydrate, fluorescein chloride, 2’,7’-dichlorofluorescein, sodium 2’,7’-dichlorofluorescein, 2,3-diaminonaphthalene, dimidium bromide, 2,3-diphenylmaleK, fluorescein, uranine, 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, sulfonfluorescein, N-succinimidyl 7-methoxycoumarin-3-carboxylate, potassium tetrabromofluorescein, 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, 5 - (4,6 Dichlorotriazinyl) aminofluorescein (DTAF), Erythrosin 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 - NHS ester (above, Dy~ are manufactured by Dyomics), Trisodium [4’-(4’ - amino - 4 - biphenylyl) - 2,2’:6’,2’’ - terpyridine - 6,6’’ - diylbis(methyliminodiacetate)] europium(III) (ATBTA - Eu3+), BODYIPY650 / 665, ROX, TAMRA, CFSE, Cyto350, Cyto405, Cyto415, Cyto488, Cyto500LSS, Cyto505, Cyto510SS, Cyto514LSS, Cyto520LSS, Cyto532, Cyto546S, Cyto555, Cyto590, Cyto610, Cyto610, Cyto633, Cyto647, Cyto670, Cyto680, Cyto700, Cyto750, Cyto770, Cyto780, Cyto800 (above, Cyto~ are manufactured by Cytodaiagnostics), ATTO532, ATTORho6G, ATTO542, ATTO550, ATTO565, ATTORho3B, ATTORho11, ATTORho12, ATTOThio12, ATTORho101, ATTO590, ATTORho13, ATTO594, ATTO610, ATTO620, ATTORho14, ATTO633, ATTO647N, ATTO647, ATTO655, ATTOOxa12, ATTO665, ATTO680, ATTO700, ATTO725,Examples include ATTO740 (ATTO~ above is manufactured by ATTO-TEC). As the fluorescence wavelength of these fluorescent dye compounds, a range of 400 nm or more that does not overlap with the wavelengths of water or proteins during detection is preferred. There is no particular upper limit to the wavelength, and the higher the wavelength, the more preferred. More preferably, it is a fluorescent dye compound in the range of 500 nm or more. The fluorescent dye compound is more preferably a europium complex.,

[0021] Examples of the chemical bond between the fluorescent cellulose particles and the fluorescent dye compound include a method of directly linking the hydroxyl group of cellulose with the fluorescent dye compound and a method of linking through some compound as a spacer. When a large amount of the fluorescent dye compound is contained, there is a limit to simply direct linking, but by using a spacer, a large amount can be introduced. When linking using a spacer, the type of spacer is not particularly limited. For example, compounds having two or more moieties reactive with hydroxyl groups such as cyanuric chloride, epichlorohydrin, 2-chloroethanamine, 11-chloroundecanethiol, formalin, silane coupling agent, epoxy-modified silicone-based crosslinking agent, and glyoxal-based resin can be mentioned.

[0022] The content of the fluorescent dye compound in the fluorescent cellulose particles of this embodiment is 1% by mass or more and 40% by mass or less per 1 g of the fluorescent cellulose particles. If it is less than 1% by mass, sufficient color development cannot be obtained as the detection particles of the immunochromatography kit. On the other hand, by setting it to 40% by mass or less, concentration quenching derived from the fluorescent dye is suppressed, the fluorescence intensity is good, and the sensitivity as an immunochromatography kit is excellent. The preferred lower limit is 5% by mass, and the preferred upper limit is 35% by mass.

[0023] The heterocyclic compound contained in the fluorescent cellulose particles of this embodiment is represented by the following general formula (1):

Chemical formula

[0024] The content of the heterocyclic compound in the fluorescent cellulose particles of the present embodiment is 3% by mass or more and 50% by mass or less per 1 g of the fluorescent cellulose particles. By setting it to 3% by mass or more, the hydrophobic interaction between the cellulose developing film used in the immunochromatography kit and the particles is suppressed, the fluidity of the particles in the developing film is improved, and sufficient color developability can be obtained as the detection particles of the immunochromatography kit. On the other hand, by setting it to 50% by mass or less, the particles do not aggregate due to hydrophobic interaction, and there is no clogging or false positive occurrence during development. Therefore, sufficient sensitivity can be obtained as an immunochromatography kit. The preferable lower limit value of the content of the heterocyclic compound is 5% or more, and the preferable upper limit value is 45% or less.

[0025] When the weight of the cellulose particles before treatment is unknown, the fluorescent cellulose particles are subjected to cellulase treatment, acid treatment or base treatment to reduce the degree of polymerization. Then, the sample is dissolved in heavy water, and 13 measurement is performed by C-NMR using FT-NMR to calculate the degree of substitution. The contents of the fluorescent dye compound and the heterocyclic compound may be calculated from the degree of substitution. At this time, the cellulase, acid, and base used are not limited to any particular ones. For example, as the cellulase, Onozuka RS (manufactured by Yakult Pharmaceutical Industry Co., Ltd.), Cellsoft (manufactured by Novo Nordisk), Meicelase (manufactured by Meiji Seika Kaisha, Ltd.), as the acid, hydrochloric acid, sulfuric acid, nitric acid, and as the base, an alkali can be mentioned. Further, when the weight of the cellulose particles before treatment is unknown and the fluorescent dye compound contains a nitrogen atom, the nitrogen element content rate is measured by a nitrogen quantifier CHN coder by luminescence analysis, and the contents of the contained fluorescent dye compound and heterocyclic compound may be calculated from the measured nitrogen element content rate.

[0026] The particle size of the fluorescent cellulose particles or raw material cellulose particles of the present embodiment refers to the value obtained by measuring a cellulose particle dispersion in which the cellulose particles are dispersed in a liquid using a particle size distribution measuring device. The "average particle size" refers to the value of the volume average median diameter of the measured values. There are various particle size distribution measuring devices that apply various measurement principles, but in the present embodiment, a particle size distribution measuring device based on the dynamic light scattering method is used. As will be described later, in the examples, the "Nanotrac Particle Size Distribution Measuring Device UPA-EX150" manufactured by Nikkiso Co., Ltd. was used.

[0027] The average particle size of the fluorescent cellulose particles of the present embodiment is 9 nm or more and 500 nm or less. If the average particle size is within this range, aggregation due to long-term storage is less likely to occur, and it is also suitable for immunochromatography kits. When used as a diagnostic agent, it is preferably 20 nm or more and 500 nm or less. If it is 20 nm or more and 500 nm or less, it is possible to achieve both dispersion stability without aggregation and developability without clogging. However, in order to improve the sensitivity as an immunochromatography kit, two or more types of fluorescent cellulose particles with different average particle sizes may be mixed and used.

[0028] The fluorescent cellulose particles of the present embodiment can be used to carry a biological substance through physical adsorption. Examples of physical adsorption include, but are not limited to, ionic bonds, coordination bonds, metal bonds, hydrogen bonds, hydrophilic bonds, hydrophobic bonds, and van der Waals bonds. By carrying a biological substance on the fluorescent cellulose particles by various forces as described above, it is possible to prepare particles having functions not possessed by the fluorescent cellulose particles.

[0029] The "biomaterial" to be supported on the fluorescent cellulose particles of the present embodiment refers to various substances obtained from living organisms, and its type is not particularly limited. Examples thereof include collagen, gelatin, fibroin, heparin, hyaluronic acid, starch, chitin, chitosan, amino acids, peptides, proteins, nucleic acids, carbohydrates, fatty acids, terpenoids, carotenoids, tetrapyrroles, cofactors, steroids, flavonoids, alkaloids, polyketides, glycosides, enzymes, antibodies, antigens, carboxymethyl cellulose, carboxyethyl cellulose, methyl cellulose, and the like. By supporting them on the fluorescent cellulose particles, it becomes possible to improve the biocompatibility of the fluorescent cellulose particles and to use them as various bioassays and diagnostic agents.

[0030] In this embodiment, by supporting a substance that specifically binds to the analyte on the fluorescent cellulose particles, it becomes possible to use the fluorescent cellulose particles as a diagnostic agent. The analyte refers to the measurement target in tests such as immuno-serum tests, blood tests, cell tests, gene tests, etc., and its type is not particularly limited. For example, cancer markers, hormones, infectious diseases, autoimmunity, plasma proteins, TDM, coagulation / fibrinolysis, amino acids, peptides, proteins, genes, cells, etc. can be mentioned. More specifically, CEA, AFP, ferritin, β2 micro, PSA, CA19-9, CA125, BFP, elastase 1, pepsinogen 1·2, 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, type A influenza antigen, type A influenza antibody, type B influenza antigen, type B influenza antibody, rotavirus antigen, adenovirus antigen, rotavirus / adenovirus antigen, group A streptococcus, group B streptococcus, candida antigen, CD bacteria, cryptococcus antigen, cholera bacteria, meningococcus antigen, granular 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 collagen, hyaluronic acid, GHbA1c, various antigens, various antibodies, various viruses, various bacteria, various amino acids, various peptides, various proteins, various DNAs, various cells, etc. can be mentioned.

[0031] When using the fluorescent cellulose particles of this embodiment as a diagnostic agent, the fluorescent cellulose particles can be dispersed and used in various solutions. Preferably, a dispersion obtained by dispersing the fluorescent cellulose particles in a buffer solution with a pH of 5.0 or higher and 11.0 or lower is preferred. As the solution for dispersing the fluorescent cellulose particles, pure water or an organic solvent can be used. For example, phosphate buffer, glycine buffer, Tris buffer, borate buffer, citrate buffer, MES buffer, methanol, ethanol, acetone, tetrahydrofuran, etc. can be mentioned. The concentration of the buffer solution is not particularly limited, and various concentrations generally used as buffer solutions can be used. Also, the concentration of the fluorescent cellulose particles in the dispersion is not particularly limited, and it can be appropriately adjusted according to the type, nature, concentration, etc. of the substance to be tested. If the concentration of the fluorescent cellulose particles in the dispersion is too low, the detectability is poor and high sensitivity cannot be achieved. Therefore, 0.001% by mass or more, more preferably 0.002% by mass or more is preferred. On the other hand, if the concentration is too high, poor development due to concentration quenching or aggregation occurs and high sensitivity cannot be expected. Therefore, the concentration is preferably about 10% by mass or less, more preferably 1.0% by mass or less.

[0032] When using the fluorescent cellulose particles of this embodiment as a diagnostic agent, various sensitizers may be used to improve the measurement sensitivity and promote the antigen-antibody reaction. Also, a blocking agent or the like may be used to suppress non-specific adsorption caused by other substances present in the specimen. The fluorescent cellulose particles of this embodiment can be dispersed and used in an arbitrary liquid like a diagnostic agent, but it is also possible to disperse and use them in other arbitrary solids, or to immobilize the particles on the solid surface and use them, etc. Also, by coloring the fluorescent cellulose particles, it is possible to improve the visibility of the particles or improve the detection sensitivity.

[0033] The manufacturing method of the cellulose particles contained in the fluorescent cellulose particles of the present embodiment is not particularly limited. Although particles with a desired average particle size may be obtained by classification using a mechanical method such as wet grinding, in the present embodiment, cellulose is dissolved in its good solvent, and cellulose particles are prepared by using a coagulating liquid mixed with water, an organic solvent, ammonia, etc. By using this method, the particle size of the obtained cellulose particles can be adjusted by the composition of the coagulating liquid. Although it is not intended to limit the manufacturing method of the cellulose particle material contained in the fluorescent cellulose particles of the present embodiment, it is exemplified below as Manufacturing Methods 1 and 2.

[0034] [Manufacturing Method 1: Preparation of Cellulose Particles] Dissolve cellulose linters in a good solvent for cellulose. Use a copper-ammonia solution prepared by a known method as the good solvent. As the coagulating liquid, an organic solvent + water + ammonia mixed system is mainly used. While stirring this coagulating liquid, add the previously prepared copper-ammonia cellulose solution to carry out coagulation. Further, add sulfuric acid for neutralization and regeneration to obtain a slurry containing the target cellulose particles. At this time, the slurry is acidic due to the residual acid used for regeneration and further contains impurities such as ammonium salts generated by neutralization, so an operation to purify it into a cellulose dispersion composed of cellulose particles and a medium is required. As this purification operation, repeated treatment of centrifugation - decantation - dilution with a dispersion medium liquid is used. The type of the dispersion medium liquid used at this time is not particularly limited, and various hydrophilic solvents mentioned above can be used according to the purpose. The cellulose particles in the obtained cellulose particle dispersion may aggregate during the purification operation. In this case, a dispersion treatment such as shearing can be performed. As a means of applying shear, a high-pressure homogenizer is used. The cellulose particle dispersion thus obtained is measured for the average particle size and the CV value using a particle size distribution measuring device. The CV value is the abbreviation of Coefficient of Variation and represents the polydispersity in the particle size distribution of the cellulose particle dispersion on a volume basis and is defined by the following formula (1). The smaller this value, the sharper the particle size distribution, which means that the sizes of the cellulose particles are more uniform, and its unit is expressed in (%). CV value (%) = (standard deviation in the volume particle size distribution obtained from the particle size distribution measuring device) / (volume average median diameter obtained from the particle size distribution measuring device) × 100... Formula (1)

[0035] The obtained cellulose particle dispersion can also be used by adding a surfactant as needed. The cellulose particle dispersion can be used in a never-dry state as it is, or can be prepared into cellulose particles by drying as needed. The obtained cellulose particles are observed using an electron microscope, and the sphericity and aggregation constant are measured from the image. Further, the cellulose particles are dissolved in a cadoxen solution, and the average degree of polymerization is measured from the viscosity. Here, the average degree of polymerization of cellulose particles suitable for producing fluorescent cellulose particles is 30 or more and 700 or less. If the average degree of polymerization is 30 or more and 700 or less, the uniformity of the particles can be maintained, and the fluorescent dye compound can also be stably contained, so the quality is stable when used in an immunochromatography kit. Therefore, fluorescent cellulose particles can be produced into fluorescent cellulose particles suitable for an immunochromatography kit by controlling the degree of polymerization and the average particle size of the cellulose particles before dyeing within the scope of the present invention. In order to produce fluorescent cellulose particles, the lower limit value of the average degree of polymerization of the cellulose particles is preferably 35 or more, more preferably 40 or more. The preferable upper limit value is 650, more preferably 600.

[0036] [Production Method 2: Preparation of Fluorescent Cellulose Particles] The cellulose particles produced by the above manufacturing method 1 are added to an organic solvent and dispersed. These cellulose 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, cyclopentane, tetrahydrofuran, toluene, hexane, water, caustic soda, etc. It can be used as a mixture of one or more types according to the type of the fluorescent dye compound. Further, since the cellulose particles serving as the raw material of the fluorescent cellulose particles have a cellulose II crystal form, the degree of crystallinity is low, and thereby, the amount of the fluorescent dye introduced can be significantly increased as compared with the conventional latex particles and silica particles. Further, in order to increase the amount of the fluorescent dye introduced, cellulose may be physically or chemically modified to introduce an amino group or a thiol group, and then reacted with the fluorescent dye compound.

[0037] A fluorescent dye compound is added to the solution containing these cellulose particles, and then additives are appropriately added, the pH is adjusted, or heating or cooling is performed. Unreacted substances and by-products such as the fluorescent dye compound used in the reaction remain in the slurry, and an operation for purifying the fluorescent cellulose particles and the medium is required. As this purification operation, a repetition of centrifugation - decantation - dilution with a dispersion medium liquid is used. The type of the dispersion medium liquid used at this time is not particularly limited, and various hydrophilic or lipophilic solvents or solutions described above can be used according to the purpose. Further, a heterocyclic compound other than the fluorescent dye compound is added to the solution containing the fluorescent cellulose particles, and then additives are appropriately added, the pH is adjusted, or heating or cooling is performed. Unreacted substances and by-products such as the heterocyclic compound used in the reaction remain in the slurry, and an operation for purifying the fluorescent cellulose particles and the medium is required. The purification operation is as described above. Through the steps as described above, the fluorescent cellulose particles of the present embodiment can be manufactured.

Example

[0038] Hereinafter, the present invention will be specifically described with reference to Examples and Comparative Examples, but the present invention is not limited by the Examples. The main measured values in the Examples and Comparative Examples were obtained by the following methods.

[0039] <Content of fluorescent dye compound> The ratio of the fluorescent dye compound component to the fluorescent cellulose particles can be calculated from the weight change before and after the treatment with the fluorescent dye compound. Using the weight of the recovered particles after the treatment and the absolute dry weight of the cellulose particles before the treatment, the following formula (2): Fluorescent dye compound content (%) = 1 - {(weight of cellulose particles before treatment) / (weight of fluorescent cellulose particles after treatment with fluorescent dye compound)} × 100... Formula (2) was used to calculate the ratio of the fluorescent dye compound component.

[0040] (When the weight of the cellulose particles before treatment is unknown) After subjecting the fluorescent cellulose particles to cellulase treatment, acid treatment or base treatment, the sample is dissolved in heavy water to prepare a 3 - 5 mass% heavy aqueous solution, and 13 measurement is performed by C-NMR (Avance 400 MHz) to calculate the degree of substitution. The degree of substitution is calculated based on the peak area of C1 of cellulose and from the peak area of the fluorescent dye compound. From the degree of substitution and the molecular weight of the fluorescent dye compound, the content of the fluorescent dye compound is calculated.

[0041] (When the weight of the cellulose particles before treatment is unknown and the fluorescent dye compound contains a nitrogen atom) The nitrogen element content rate is measured by emission analysis using a nitrogen determination device CHN coder (manufactured by Yanaco Analytical Industries Co., Ltd.) under the following measurement conditions. From the measured nitrogen element content rate, the content of the contained fluorescent dye compound is calculated. Measurement method: Self-integration method Carrier gas: Helium Combustion-supporting gas: High-purity oxygen Combustion-supporting method: Helium-oxygen mixed method

[0042] <Content of heterocyclic compound> The ratio of the heterocyclic compound component to the fluorescent cellulose particles can be calculated from the weight change before and after the treatment of the fluorescent cellulose particles. Using the weight of the particles that could be recovered after the treatment and the weight of the cellulose particles after absolute drying before the treatment, the following formula (3): Heterocyclic compound content (%) = 1 - {(weight of the fluorescent cellulose particles before the treatment) / (weight of the fluorescent cellulose particles after the heterocyclic compound treatment)} × 100 … Formula (3) was used to calculate the ratio of the heterocyclic compound component.

[0043] (When the weight of the cellulose particles before the treatment is unknown) After subjecting the fluorescent cellulose particles after the heterocyclic compound treatment to cellulase treatment, acid treatment or base treatment, the sample is dissolved in heavy water to prepare a 3 - 5 mass% heavy aqueous solution, and then measured by FT-NMR 13 using C-NMR (Avance 400MHz) to calculate the degree of substitution. The degree of substitution is calculated based on the peak area of C1 of cellulose and from the peak area of the heterocyclic compound. From the degree of substitution and the molecular weight of the heterocyclic compound, the content of the heterocyclic compound is calculated.

[0044] (When the weight of the cellulose particles before the treatment is unknown and the fluorescent dye compound contains a nitrogen atom) The nitrogen element content rate is measured by emission analysis using a nitrogen determination device CHN coder (manufactured by Yanaco Analytical Industry Co., Ltd.) under the following measurement conditions. From the measured nitrogen element content rate, the content of the contained heterocyclic compound is calculated. When the fluorescent dye compound before the heterocyclic compound treatment also contains a nitrogen atom, it can be calculated based on the relative amount to it. Measurement method: Self-integration method Carrier gas: Helium Combustion-supporting gas: High-purity oxygen Combustion-supporting method: Helium-oxygen mixing method

[0045] <Method for measuring particle size> A slurry containing cellulose particles was diluted with distilled water so that the cellulose particles were 0.005% by mass and used for measurement. As a measuring instrument, measurement was carried out using the "Nanotrac Particle Size Distribution Measuring Device UPA-EX150" manufactured by Nikkiso Co., Ltd. which measures by the dynamic light scattering method.

[0046] <Method for Judging Sensitivity of Immunochromatographic Evaluation> For the method of judging color development, the color development intensity was evaluated using the fluorescence immunochromatography reader "DxCELL series HRDR-300" manufactured by Cellmic. Also, in Table 1 below, as the evaluation criteria for developability, when a UV lamp was applied to the immunochromatographic strip after development, for each of 4 mm upstream of the development shown in Figure 1 and the absorption pad, if no coloring was observed it was (-), if coloring was observed it was (+), and if coloring was observed and it was strong it was (++).

[0047] [Example 1] A copper-ammonia cellulose solution with a cellulose concentration of 0.37% by mass, a copper concentration of 0.13% by mass, and an ammonia concentration of 1.00% by mass was prepared. Further, a coagulating liquid with a tetrahydrofuran concentration of 87.5% by mass and a water concentration of 12.5% by mass was prepared. While slowly stirring 5000 g of the coagulating liquid using a magnetic stirrer, 500 g of the prepared copper-ammonia cellulose solution was added thereto. After continuing stirring for about 5 seconds, 1000 g of 10% sulfuric acid was added for neutralization and regeneration to obtain 6500 g of a slurry containing cellulose particles.

[0048] The obtained slurry was centrifuged at a speed of 10000 rpm for 10 minutes. The precipitate was taken out by decantation, ultra-pure water was poured in and stirred, and then centrifuged again. This operation was repeated several times until the pH reached 6.0 to 7.0, and then dispersion treatment was carried out using a high-pressure homogenizer to obtain 150 g of a cellulose particle dispersion liquid. As a result of measuring the average particle size of the obtained cellulose particles, it was 205 nm.

[0049] To a glass screw tube, 200 mg of sodium (III) europium (ATBTA-Eu) 3+ )(manufactured by Tokyo Chemical Industry Co., Ltd.) and 6 mL of sodium acetate buffer solution were added, and a solution prepared by dissolving 43 mg of cyanuric chloride (manufactured by Tokyo Chemical Industry Co., Ltd.) in 2.5 mL of acetone was added thereto. After reacting at room temperature for 1 hour, the reaction solution was added to 100 mL of acetone, and the precipitated solid: DTBTA-Eu 3+ was collected by centrifugation. Then it was washed twice with 50 mL of acetone and dried, and the dried product was dissolved in 100 mL of sodium carbonate buffer solution to obtain a DTBTA-Eu 3+ solution.

[0050] To an eggplant-shaped glass flask, 100 g of a slurry containing cellulose particles and 100 mL of the prepared DTBTA-Eu 3+ solution were added. A glass reflux tube was attached, and while refluxing and cooling with tap water, it was stirred with a magnetic stirrer at 60 °C for 3 hours. After that, using a centrifuge, decantation-dilution and washing with deionized water were repeated several times, and further dispersion treatment was performed with a high-pressure homogenizer to obtain 100 g of a fluorescent cellulose particle dispersion.

[0051] The obtained fluorescent cellulose particles were placed in an eggplant-shaped glass flask, 200 g of a 4% by mass aqueous sodium hydroxide solution was added as a dispersion medium, 12 g of cyanuric chloride (manufactured by Tokyo Chemical Industry Co., Ltd.) was added, a glass reflux tube was attached, and while refluxing and cooling with tap water, it was stirred with a magnetic stirrer at 60 °C for 3 hours. After that, using a centrifuge, decantation-dilution and washing with deionized water were performed 3 times. Then, dispersion treatment was performed with a high-pressure homogenizer to obtain 100 g of a slurry of modified fluorescent cellulose particles. As a result of measuring the average particle diameter of the obtained fluorescent cellulose particles, it was 281 nm.

[0052] [Example 2] The fluorescent cellulose particles were produced in the same manner as in Example 1, except that the amount of cyanuric chloride used for modifying the particles was changed so as to obtain the content shown in Table 1 below.

[0053] [Comparative Example 1] Particle staining was performed in the same manner as in Example 1, and fluorescent cellulose particles were produced without performing cyanuric chloride modification.

[0054] [Comparative Example 2] The fluorescent cellulose particles were produced in the same manner as in Example 1, except that the amount of cyanuric chloride used for modifying the particles was changed so as to obtain the content shown in Table 1 below.

[0055] [Measurement of Fluorescence Intensity of Fluorescent Cellulose Particle Dispersion] The obtained slurry-like fluorescent cellulose particles were diluted with distilled water so that the cellulose particles had a concentration of 0.002% by mass, and a sample for measuring fluorescence intensity was prepared. The sample was placed in a 1 cm square quartz cell, and measurement was performed at the excitation wavelength and fluorescence wavelength corresponding to the fluorescent substance using a spectrofluorometer (FP-8300 manufactured by JASCO Corporation).

[0056] [Developability Test of Test Strip Prepared Using Fluorescent Cellulose Particles] Using the obtained fluorescent cellulose particles, a test strip was prepared, and the developability of the particles on the developing membrane was evaluated. The preparation of the test strip is described below. 20 μL of a dispersion of fluorescent cellulose particles (Examples 1 to 2, Comparative Examples 1 to 2) having a concentration of 5 mg / ml (dispersion medium: distilled water) and 500 μL of distilled water were added to a microtube and gently stirred. Then, the mixture was centrifuged at 20,000 × g for 20 minutes, and the supernatant was removed. 526 μL of a storage buffer (50 mM borate buffer (pH 10.0), 10% trehalose) was added thereto to disperse the particles, and a fluorescent cellulose particle dispersion (0.038%) was obtained. 424 μL of the dispersion of the fluorescent cellulose particles was uniformly applied to a polyester conjugate pad (6613, manufactured by Ahlstrom) (10×160 mm). It was dried in a dryer at 37°C for 30 minutes to prepare a conjugate pad containing the fluorescent cellulose particles.

[0057] A sample pad (Microline CBSP097, manufactured by Asahi Kasei), the conjugate pad, a nitrocellulose membrane without antibody immobilization, and an absorption pad (Type A / B Extra Thick Glass Fiber 8×10 In, manufactured by PALL) were assembled in this order on a backing sheet (product name AR9020, manufactured by Adhesives Research), cut into strips 4 mm wide and 60 mm long to obtain test strips. Incidentally, both ends of each component member were overlapped with the adjacent member by about 2 mm and pasted.

[0058] 80 μL of the immunochromatographic developing solution was dropped onto the sample pad portion of the prepared test strip. After leaving it for 15 minutes, the test strip was observed with a UV light (wavelength: 375 nm). As shown in FIG. 1, coloring 4 mm upstream of the development and coloring in the absorption pad were confirmed. A strong coloring of the absorption pad indicates that many particles flowed to the absorption pad, that is, good developability. For each of 4 mm upstream of the development and the absorption pad, the case where no coloring was observed was evaluated as (-), the case where coloring was observed was evaluated as (+), and the case where coloring was observed and was strong was evaluated as (++). The results are shown in Table 1 below.

[0059]

Table 1

[0060] From the results shown in Table 1, for the fluorescent cellulose particles of Examples 1 and 2, no coloring was observed upstream of the development, and since the coloring of the absorption pad was strong, it was confirmed that they had good developability. In contrast, in Comparative Example 1, clogging occurred upstream of the development, resulting in poor development and no further development was possible, and almost no coloring of the absorption pad was observed. In Comparative Example 2, although coloring of the absorption pad could be confirmed, it was weaker compared to Examples 1 and 2, and coloring was also confirmed upstream of the development, so sufficient developability for immunochromatography could not be obtained.

[0061] [Examples 3, 4] Fluorescent cellulose particles were produced in the same manner as in Example 1, except that the amount of the fluorescent dye compound added and the amount of cyanuric chloride used to modify the particles were changed so as to have the contents shown in Table 2 below.

[0062] [Examples 5, 6] Fluorescent cellulose particles were produced in the same manner as in Example 1, except that the fluorescent dye compound was changed to 5-(4,6-dichlorotriazinyl)aminofluorescein (DTAF) (manufactured by Sigma-Aldrich), and the amount of the fluorescent dye compound added and the amount of cyanuric chloride used to modify the particles were adjusted so as to have the contents shown in Table 2 below.

[0063] [Comparative Example 3] Fluorescent cellulose particles were produced in the same manner as in Example 1, except that the amount of cyanuric chloride used to modify the particles was changed so as to have the content shown in Table 2 below.

[0064] [Comparative Examples 4 to 7] Fluorescent cellulose particles were produced in the same manner as in Example 1, except that the amount of the fluorescent dye compound added and the amount of cyanuric chloride used to modify the particles were changed so as to have the contents shown in Table 2 below.

[0065] [Measurement of Fluorescence Intensity of Fluorescent Cellulose Particle Dispersion] The obtained slurry-like fluorescent cellulose particles were diluted with distilled water so that the cellulose particles had a concentration of 0.002% by mass, and a sample for fluorescence intensity measurement was prepared. The sample was placed in a 1 cm square quartz cell and measured with a spectrofluorometer (FP-8300, manufactured by JASCO Corporation) at an excitation wavelength and a fluorescence wavelength suitable for the fluorescent substance.

[0066] [Color Intensity Test in Immunochromatography Kit Prepared Using Fluorescent Cellulose Particles] An immunochromatography kit was prepared using the obtained fluorescent cellulose particles, and the color intensity was evaluated. Hereinafter, the preparation of the immunochromatography kit will be described. 20 μL of a dispersion of fluorescent cellulose particles (Examples 1 to 6, Comparative Examples 1 to 7) with a concentration of 5 mg / ml (dispersion medium: distilled water) and 180 μL of 10 mM phosphate buffer (pH 7.0) were added to a 5 mL tube and gently stirred. 10 μL (5.8 mg / mL) of an anti-hCG antibody (Anti-hCG clone codes / 5008, manufactured by Medix Biochemica) was added to the 5 mL tube, and the mixture was incubated at 37°C for 2 hours to adsorb the anti-hCG antibody to the fluorescent cellulose particles. After incubation, a blocking buffer (100 mM boric acid (pH 8.5), 1% by weight casein) was added to the 5 mL tube, and the mixture was incubated at 37°C for 1 hour for blocking. The 5 mL tube after blocking was centrifuged at 20,000×g for 15 minutes to remove the supernatant. Next, a washing solution (50 mM boric acid buffer (pH 10.0)) was added thereto to disperse the particles. After dispersion, the mixture was centrifuged at 20,000×g for 15 minutes to remove the supernatant. A storage buffer (50 mM boric acid buffer (pH 10.0), 10% trehalose, 4% histidine, 0.4% casein) was added thereto so as to be 0.038% of the particle weight to disperse the particles, and a dispersion of composite particles of fluorescent cellulose particles / biomolecules was obtained. 424 μL of the dispersion of the composite particles was uniformly applied to a polyester conjugate pad (6613, manufactured by Ahlstrom) (10×160 mm). It was dried in a dryer at 37 °C for 30 minutes to prepare a conjugate pad containing the composite particles.

[0067] Hereinafter, the method for preparing the antibody-immobilized membrane will be described. Near the center (about 12 mm from the edge) of a membrane (length 25 mm, product name: Hi-Flow Plus120 membrane, manufactured by MILLIPORE), as a test line with a width of about 1 mm, a solution containing 1 mg / mL of anti-hCG antibody (alpha subunit of FSH (LH), clone code / 6601, manufactured by Medix Biochemica) ((50 mM KH2PO4, pH 7.0) + 5% sucrose) was applied at a coating amount of 0.75 μL / cm. Next, as a control line with a width of about 1 mm, a solution containing 1 mg / mL of anti-mouse IgG antibody (Anti Mouse IgG, manufactured by Dako) ((50 mM KH 2 PO 4 , pH 7.0) sugar-free) was applied at a coating amount of 0.75 μL / cm and dried at 50 °C for 30 minutes. The distance between the test line and the control line was 6 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 wt% sucrose, 0.1% sodium cholate) and left standing at room temperature for 30 minutes or more. The membrane was lifted up, placed on a paper towel and dried at room temperature overnight to prepare an antibody-immobilized membrane.

[0068] A sample pad (Microline CBSP097, manufactured by Asahi Kasei Corporation), the conjugate pad, the antibody-immobilized membrane, and an absorption pad (Type A / B Extra Thick Glass Fiber 8×10 In, manufactured by PALL Corporation) were assembled in this order on a backing sheet (product name AR9020, manufactured by Adhesives Research), cut into strips 5 mm wide and 60 mm long, and test strips were obtained. Furthermore, both ends of each component member were overlapped with an adjacent member by about 2 mm and attached. Recombinant hCG (manufactured by Roche Pharmaceutical Co., Ltd.) at the detection limit concentration (LOD) was dropped in an amount of 80 μL onto the sample pad portion of the prepared test strip. After leaving it for 15 minutes, the color development intensity of the test line was confirmed using a fluorescence immunochromatography reader "DxCELL series HRDR-300" manufactured by Cellmic. Furthermore, a sample containing no antigen was dropped in an amount of 80 μL, and the color development intensity of the test line was confirmed in the same manner. The color development confirmed with a sample containing no antigen is not the color development formed by the original antibody-antigen reaction, and thus becomes non-specific color development (noise). Then, the ratio of the non-specific color development to the test line at the detection limit concentration was calculated as the S / N ratio. The S / N ratio is the ratio of the signal to the noise, and the larger the value is, the more distinguishable it is from the noise. That is, it indicates that the antigen at the detection limit concentration can be detected. This time, it was determined that the detection was possible when the S / N ratio was 2 or more, and the detection was impossible when it was less than 2. Also, as shown in FIG. 1, a UV lamp was applied to the developed test strip, and for the coloring 4 mm upstream of the development and each absorption pad, the case where no coloring was observed was evaluated as (-), the case where coloring was observed was evaluated as (+), and the case where coloring was observed and was strong was evaluated as (++). The results are shown in Table 2 below.

[0069] From the results shown in Table 2 below, it can be seen that for the fluorescent cellulose particles of Examples 1 to 6, no coloring was observed upstream of the development, indicating good developability and S / N ratio. In contrast, in Comparative Example 1, clogging occurred upstream of the development, resulting in poor development and inability to detect the line. In Comparative Example 2, slight clogging occurred upstream of the development, and it was not possible to detect the same antigen concentration as in the other examples. In Comparative Example 3, although no coloring occurred upstream of the development, the coloring of the absorption pad was weaker than in the examples, and furthermore, the test line intensity was also weak, and it was not possible to detect the same antigen concentration as in the other examples, so it cannot be said to have sufficient developability for immunochromatography. In Comparative Example 4, clogging occurred upstream of the development, the coloring of the absorption pad was weaker than in the examples, and furthermore, the test line intensity was also weak and the S / N ratio was small, so it cannot be said to have sufficient developability for immunochromatography. In Comparative Example 5, strong coloring occurred from upstream of the development to the entire membrane. Although the value of the line intensity was high, non-specific coloring also became strong, resulting in a small S / N ratio. In Comparative Example 6, since the brightness of the particles was weak, it was not possible to detect the same antigen concentration as in the other examples. In Comparative Example 7, although no coloring was observed upstream of the development, concentration quenching occurred, the brightness of the particles became weak, and the sensitivity decreased, and it was not possible to detect the same antigen concentration as in the other examples.

[0070]

Table 2

Industrial Applicability

[0071] The fluorescent cellulose particles of the present invention and the immunochromatography kit using the same can highly sensitively detect the analyte contained in a biological sample, and thus can be suitably used for immunoassay methods in clinical tests and the like.

Claims

1. Cellulose particles, a fluorescent dye compound, and the following general formula (1): 【Chemical 1】 {In the formula, R 1 is a functional group having an affinity for a biological substance, and R 2 is an ether bond part with the cellulose particles.} A fluorescent cellulose particle containing a heterocyclic compound represented by the formula, wherein the content of the cellulose particles is 30% by mass or more and 90% by mass or less, the content of the fluorescent dye compound is 1% by mass or more and 40% by mass or less, and the content of the heterocyclic compound is 3% by mass or more and 50% by mass or less per 1 g of the fluorescent cellulose particle.

2. R of the complex cyclic compound 1 The fluorescent cellulose particles according to claim 1, wherein 1 is Cl and / or OH.

3. The fluorescent cellulose particles according to claim 1 or 2, wherein the average particle diameter of the fluorescent cellulose particles is 9 nm or more and 500 nm or less.

4. The fluorescent cellulose particles according to claim 1 or 2, wherein the fluorescent dye compound is bonded to the OH group of the cellulose particles, and the heterocyclic compound is bonded to the OH group of the cellulose particles.

5. The fluorescent cellulose particles according to claim 1 or 2, wherein the fluorescent dye compound is a europium complex.

6. The fluorescent cellulose particles according to claim 1 or 2, wherein a biological substance is supported via physical adsorption.

7. The fluorescent cellulose particles according to claim 6, wherein the biological substance is a protein, a peptide, or a nucleic acid.

8. The fluorescent cellulose particles according to claim 7, wherein the protein is an antigen or an antibody.

9. A diagnostic agent comprising the fluorescent cellulose particles according to claim 1 or 2.

10. An immunochromatography kit comprising the fluorescent cellulose particles according to claim 1 or 2.

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