Composite particle and method for manufacturing the same

Abstract

To provide a composite particle of a cellulose nanofiber and magnetic particles, the composite particle being capable of easily introducing an amino group-containing compound such as a protein.SOLUTION: The composite particle according to an embodiment includes a cellulose nanofiber and magnetic particles, and the cellulose nanofiber has an active ester group. An amino group-containing compound such as amino group-containing biotin or a protein may be introduced into the composite particle by reacting the amino group of the amino group-containing compound with the active ester group.SELECTED DRAWING: Figure 1

Description

[Technical Field]

[0001] The present invention relates to composite particles containing cellulose nanofibers and magnetic particles, a method for producing the same, and a method for capturing or detecting a target compound using the composite particles. [Background technology]

[0002] Cellulose nanofibers are nano-sized cellulose fibers obtained by defibrating cellulosic raw materials such as wood. They are known to be used as adsorbents and carriers for biologically active substances such as proteins.

[0003] For example, Patent Document 1 discloses that cellulose / magnetic material composite particles are obtained by spray-drying a dispersion containing cellulose nanofibers and magnetic material particles, thereby improving the collectability of cellulose particles made of cellulose nanofibers. [Prior art documents] [Patent documents]

[0004] [Patent Document 1] Japanese Patent Publication No. 2022-30885 Summary of the Invention [Problem to be solved by the invention]

[0005] In the composite particles described in Patent Document 1, physiologically active substances are separated and purified by nonspecifically adsorbing them to cellulose nanofibers containing carboxyl groups. Therefore, it is not possible to capture or detect specific target compounds such as antigens using specific binding, such as an antigen-antibody reaction. To capture or detect specific target compounds, it is necessary to introduce a substance, such as an antibody (protein), that specifically binds to the target compound into the cellulose nanofibers. If it were possible to easily introduce an amino group-containing compound, such as a protein, into cellulose nanofibers as a substance that specifically binds to such target compounds, the convenience of the composite particles could be improved.

[0006] In view of the above, an object of the present invention is, in one embodiment, to provide composite particles of cellulose nanofibers and magnetic particles into which an amino group-containing compound can be easily introduced, and a method for producing the same. Also, in another embodiment, an object of the present invention is to provide novel composite particles in which an amino group-containing compound has been introduced into composite particles of cellulose nanofibers and magnetic particles, a method for producing the same, and a method for using the same. [Means for solving the problem]

[0007] The present invention includes the embodiments shown below. [1] A composite particle comprising a cellulose nanofiber and a magnetic particle, wherein the cellulose nanofiber has an active ester group.

[0008] [2] The composite particle described in claim 1, wherein the active ester group is formed by reacting the carboxy group of the cellulose nanofiber having a carboxy group with at least one condensing agent selected from the group consisting of a carbodiimide-based condensing agent, an imidazole-based condensing agent, a triazine-based condensing agent, a phosphonium-based condensing agent, a uronium-based condensing agent, and a haluronium-based condensing agent.

[0009] [3] The active ester group is represented by the following general formula (1): [ka] In the formula, X is any one of the following formulae (2) to (8), and in formula (3), M represents an alkali metal atom: [ka] The composite particle according to [1] or [2].

[0010] [4] The composite particles according to any one of [1] to [3], wherein the magnetic particles have an average particle size of 1 to 500 nm.

[0011] [5] Composite particles comprising cellulose nanofibers and magnetic particles, the composite particles comprising the amino group-containing compound bound to the cellulose nanofibers via an amide bond, which is a structure formed by dehydration condensation between a carboxy group of the cellulose nanofiber and an amino group of the amino group-containing compound. [6] The composite particle according to [5], wherein the amino group-containing compound is an amino group-containing biotin or protein. [7] The composite particle according to [6], which is used for capturing or detecting a target compound that specifically binds to the amino group-containing biotin or the protein.

[0012] [8] A method for producing composite particles containing cellulose nanofibers having active ester groups and magnetic particles, comprising: Composite particles containing cellulose nanofibers having carboxy groups and magnetic particles; at least one selected from the group consisting of N-hydroxysuccinimide, N,N'-disuccinimidyl carbonate, N-hydroxysulfosuccinimide alkali metal salts, 1-hydroxybenzotriazole, 1-hydroxy-7-azabenzotriazole, ethyl(hydroxyimino)cyanoacetate, pentafluorophenol, and nitrophenol; at least one condensing agent selected from the group consisting of carbodiimide-based condensing agents, imidazole-based condensing agents, triazine-based condensing agents, phosphonium-based condensing agents, uronium-based condensing agents, and haluronium-based condensing agents; in an organic solvent.

[0013] [9] A method for producing composite particles having an amino group-containing compound incorporated therein, the method comprising reacting the amino group of an amino group-containing compound with the active ester group of a composite particle comprising a cellulose nanofiber having an active ester group and a magnetic particle.

[10] The method for producing composite particles according to [9], wherein the amino group-containing compound is amino group-containing biotin or protein.

[0014]

[11] A method for capturing or detecting a target compound, comprising mixing the composite particle according to [6] with a target compound that specifically binds to the amino group-containing biotin or the protein in a liquid, and binding the amino group-containing biotin or the protein to the target compound. [Effects of the Invention]

[0015] In the above embodiment, an amino group-containing compound can be easily introduced into composite particles containing cellulose nanofibers and magnetic particles using an active ester group. Furthermore, novel composite particles into which an amino group-containing compound has been introduced can be provided, which can be used, for example, to capture or detect a specific target compound. [Brief explanation of the drawings]

[0016] [Figure 1] Graph showing the results of an avidin detection test in an example. DETAILED DESCRIPTION OF THE INVENTION

[0017] Composite particles (A) according to one embodiment contain cellulose nanofibers having active ester groups and magnetic particles. The composite particles (A) are obtained by combining magnetic particles with cellulose particles made of cellulose nanofibers. The magnetic particles are integrated into a composite in a dispersed state with the cellulose nanofibers, and may be attached to the surface of the cellulose particles made of cellulose nanofibers or embedded inside them.

[0018] The magnetic particles are not particularly limited as long as they are particles made of a ferromagnetic material that can be attracted by a magnet, and examples include particles of metal oxides such as iron oxides such as Fe3O4 and γ-Fe2O3, metals such as iron, manganese, nickel, cobalt, and chromium, or alloys thereof, metal salts such as various ferrites, ferritic or martensitic stainless steel, amorphous alloys, and silicon-iron soft magnetic crystals, and any one of these can be used alone or in combination of two or more.

[0019] The average particle size of the magnetic particles is not particularly limited, but is preferably 1 to 500 nm, more preferably 2 to 300 nm, more preferably 5 to 200 nm, more preferably 7 to 100 nm, and even more preferably 10 to 50 nm.

[0020] The cellulose nanofibers preferably have (a) a number average fiber diameter of 3 nm or more and 100 nm or less, (b) a cellulose type I crystal structure, and (c) an average aspect ratio of 2 or more and 5,000 or less.

[0021] The number average fiber diameter of (a) is more preferably 50 nm or less, further preferably 30 nm or less, and may be 10 nm or less. The number average fiber diameter can be measured as follows.

[0022] Specifically, an aqueous dispersion of cellulose nanofibers with a solid content of 0.05 to 0.1% by mass is prepared, and the aqueous dispersion is cast onto a hydrophilically treated carbon film-coated grid to prepare a sample for observation with a transmission electron microscope (TEM). When fibers with large diameters are included, scanning electron microscope (SEM) images of the surface cast onto glass may be observed. The sample for observation may also be negatively stained, for example, with 2% by mass uranyl acetate. The electron microscope image is then observed at a magnification of 5,000x, 10,000x, or 50,000x, depending on the size of the fibers. In this case, an arbitrary axis of vertical or horizontal width is assumed within the obtained image, and the sample and observation conditions (magnification, etc.) are adjusted so that at least 20 fibers intersect with the axis. After obtaining an observation image that satisfies these conditions, two random axes are drawn vertically and horizontally per image, and the fiber diameters of the fibers intersecting the axes are visually determined. In this way, at least three non-overlapping images of the surface area are taken with an electron microscope, and the fiber diameter values ​​of the fibers intersecting each of the two axes are read (thus, information on the diameters of at least 20 fibers x 2 x 3 = 120 fibers is obtained). The arithmetic mean of the fiber diameters obtained in this way is taken as the number-average fiber diameter.

[0023] The presence of the cellulose type I crystal structure (b) above can be identified by the presence of typical peaks at two positions, around 2θ=14° to 17° and around 2θ=22° to 23°, in the diffraction profile obtained by wide-angle X-ray diffraction image measurement.

[0024] The average aspect ratio of (c) above is more preferably 50 to 1000, further preferably 100 to 500, and may be 200 to 400. The average aspect ratio can be measured as follows.

[0025] That is, the number-average fiber diameter is calculated according to the method described above. The number-average fiber length of the cellulose nanofibers is also calculated from the same observation image. Specifically, the length from the start point to the end point of at least 10 fibers (fiber length) is visually read. For branched fibers, the length of the longest part of the fiber is taken as the fiber length. The arithmetic mean of the fiber lengths obtained in this way is calculated, and this is taken as the number-average fiber length. These values ​​are used to calculate the average aspect ratio according to the following formula. Average aspect ratio = number average fiber length (nm) / number average fiber diameter (nm)

[0026] Cellulose nanofibers having active ester groups are obtained by converting the carboxy groups of carboxy group-containing cellulose nanofibers into active esters.

[0027] Carboxy group-containing cellulose nanofibers are cellulose nanofibers that have carboxy groups, and examples include oxidized cellulose nanofibers obtained by oxidizing the hydroxyl groups of glucose units in cellulose molecules, and carboxymethylated cellulose nanofibers obtained by carboxymethylating the hydroxyl groups of glucose units in cellulose molecules.

[0028] Oxidized cellulose nanofibers include those in which the hydroxyl group at the C6 position of the glucose unit in the cellulose molecule has been selectively oxidized to a carboxyl group. The oxidized cellulose nanofibers are obtained by oxidizing natural cellulose, such as wood pulp, with a co-oxidant in the presence of an N-oxyl compound, followed by defibration (micronization). The N-oxyl compound used is a compound containing a nitroxy radical, which is commonly used as an oxidation catalyst, such as a piperidine nitroxyoxy radical. 2,2,6,6-tetramethylpiperidinoxy radical (TEMPO) or 4-acetamido-TEMPO is particularly preferred. TEMPO-oxidized cellulose nanofibers are generally referred to as TEMPO-oxidized cellulose nanofibers (TOCN). The oxidized cellulose nanofibers may also contain aldehyde or ketone groups in addition to the carboxyl groups.

[0029] Carboxy group-containing cellulose nanofibers may be obtained by defibration treatment. The defibration treatment may be carried out after or before the introduction of carboxy groups. The defibration treatment can be carried out by treating a dispersion of cellulose fibers using, for example, a homomixer, a high-pressure homogenizer, an ultrasonic dispersion processor, a beater, a disk refiner, a conical refiner, a double-disc refiner, a grinder, or the like under high-speed rotation, to obtain a dispersion of cellulose nanofibers.

[0030] The amount of carboxy groups in carboxy group-containing cellulose nanofibers is not particularly limited, and may be, for example, 0.5 to 3.0 mmol / g or 1.5 to 2.0 mmol / g per dry mass of carboxy group-containing cellulose nanofibers. The amount of carboxy groups can be determined according to the following formula: 60 mL of a cellulose nanofiber-containing slurry adjusted to a concentration of 0.1 to 1 mass % is prepared, the pH is adjusted to about 2.5 with 0.1 mol / L aqueous hydrochloric acid, a 0.05 mol / L aqueous sodium hydroxide solution is added dropwise, and electrical conductivity is measured. Measurement is continued until the pH reaches about 11, and the amount of sodium hydroxide (V) consumed in the neutralization stage of the weak acid, where the change in electrical conductivity is gradual, is used. Amount of carboxyl groups (mmol / g) = V (mL) × [0.05 / mass of cellulose nanofiber (g)]

[0031] Active ester groups are known as ester functional groups that are highly susceptible to nucleophilic attack. In this embodiment, the active ester groups can be formed by converting the carboxy groups of carboxy group-containing cellulose nanofibers into active esters. All or some of the carboxy groups of the carboxy group-containing cellulose nanofibers may be converted into active esters. For example, it is preferred that at least the carboxy groups on the particle surface of the composite particles (A) are converted into active esters. The amount of active ester groups is not particularly limited, and may be, for example, 0.5 to 3.0 mmol / g or 1.5 to 2.0 mmol / g per dry mass of the cellulose nanofibers having active ester groups, similar to the amount of carboxy groups described above.

[0032] In one embodiment, the active ester group may be formed by reacting the carboxy group of the carboxy group-containing cellulose nanofiber with at least one condensing agent selected from the group consisting of carbodiimide-based condensing agents, imidazole-based condensing agents, triazine-based condensing agents, phosphonium-based condensing agents, uronium-based condensing agents, and haluronium-based condensing agents. By reacting these condensing agents with the carboxy group, an active ester group represented by -COO-R (where R represents an activated substituent derived from a partial structure of the condensing agent) is formed.

[0033] Examples of carbodiimide condensing agents include 1-[3-(dimethylamino)propyl]-3-ethylcarbodiimide, 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride, N,N'-dicyclohexylcarbodiimide, and N,N'-diisopropylcarbodiimide.

[0034] Examples of the imidazole-based condensing agent include N,N'-carbonyldiimidazole and 1,1'-carbonyldi(1,2,4-triazole).

[0035] Examples of triazine-based condensing agents include 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride n-hydrate and (4,6-dimethoxy-1,3,5-triazin-2-yl)-(2-octoxy-2-oxoethyl)dimethylammonium trifluoromethanesulfonate.

[0036] Examples of phosphonium-based condensing agents include 1H-benzotriazol-1-yloxytris(dimethylamino)phosphonium hexafluorophosphate, 1H-benzotriazol-1-yloxytripyrrolidinophosphonium hexafluorophosphate, (7-azabenzotriazol-1-yloxy)tripyrrolidinophosphonium hexafluorophosphate, chlorotripyrrolidinophosphonium hexafluorophosphate, bromotris(dimethylamino)phosphonium hexafluorophosphate, and 3-(diethoxyphosphoryloxy)-1,2,3-benzotriazin-4(3H)-one.

[0037] Examples of uronium-based condensing agents include O-(benzotriazol-1-yl)-N,N,N',N'-tetramethyluronium hexafluorophosphate, O-(7-azabenzotriazol-1-yl)-N,N,N',N'-tetramethyluronium hexafluorophosphate, O-(N-succinimidyl)-N,N,N',N'-tetramethyluronium tetrafluoroborate, O-(N-succinimidyl)-N,N,N',N'-tetramethyluronium hexafluorophosphate, O-(3,4-dihydro-4-oxo -1,2,3-benzotriazin-3-yl)-N,N,N',N'-tetramethyluronium tetrafluoroborate, S-(1-oxido-2-pyridyl)-N,N,N',N'-tetramethylthiuronium tetrafluoroborate, O-[2-oxo-1(2H)-pyridyl]-N,N,N',N'-tetramethyluronium tetrafluoroborate, {{[(1-cyano-2-ethoxy-2-oxoethylidene)amino]oxy}-4-morpholinomethylene}dimethylammonium hexafluorophosphate, and the like.

[0038] Examples of halouronium condensing agents include 2-chloro-1,3-dimethylimidazolinium hexafluorophosphate, 1-(chloro-1-pyrrolidinylmethylene)pyrrolidinium hexafluorophosphate, 2-fluoro-1,3-dimethylimidazolinium hexafluorophosphate, and fluoro-N,N,N',N'-tetramethylformamidinium hexafluorophosphate.

[0039] These condensing agents may be used either alone or in combination of two or more.

[0040] In one embodiment, the active ester group is preferably represented by the following general formula (1): [ka]

[0041] X in formula (1) is any one of the following formulae (2) to (8), and M in formula (3) represents an alkali metal atom such as a sodium atom or a potassium atom. [ka]

[0042] The activated ester group in which X is represented by formula (2) can be obtained by reaction with, for example, N-hydroxysuccinimide (NHS), N,N'-disuccinimidyl carbonate, O-(N-succinimidyl)-N,N,N',N'-tetramethyluronium tetrafluoroborate, O-(N-succinimidyl)-N,N,N',N'-tetramethyluronium hexafluorophosphate, or the like.

[0043] The active ester group in which X is represented by formula (3) can be obtained, for example, by reacting with an alkali metal salt of N-hydroxysulfosuccinimide.

[0044] The activated ester group in which X is represented by formula (4) can be obtained by reaction with, for example, 1-hydroxybenzotriazole, 1H-benzotriazol-1-yloxytris(dimethylamino)phosphonium hexafluorophosphate, 1H-benzotriazol-1-yloxytripyrrolidinophosphonium hexafluorophosphate, O-(benzotriazol-1-yl)-N,N,N',N'-tetramethyluronium hexafluorophosphate, or the like.

[0045] The activated ester group in which X is represented by formula (5) can be obtained by reaction with, for example, 1-hydroxy-7-azabenzotriazole, (7-azabenzotriazol-1-yloxy)tripyrrolidinophosphonium hexafluorophosphate, O-(7-azabenzotriazol-1-yl)-N,N,N',N'-tetramethyluronium hexafluorophosphate, or the like.

[0046] The activated ester group in which X is represented by formula (6) can be obtained by reacting with, for example, ethyl(hydroxyimino)cyanoacetate, {{[(1-cyano-2-ethoxy-2-oxoethylidene)amino]oxy}-4-morpholinomethylene}dimethylammonium hexafluorophosphate, or the like.

[0047] The activated ester group in which X is represented by formula (7) can be obtained by reaction with, for example, pentafluorophenol.

[0048] The activated ester group in which X is represented by formula (8) can be obtained by reaction with, for example, nitrophenol.

[0049] Among these, X preferably has a structure of any one of formulas (2) to (6), and more preferably has a structure of formula (2) or (3) because it is stable enough to be isolated.

[0050] In the composite particles (A), the ratio of cellulose nanofibers having active ester groups to magnetic particles is not particularly limited, and the mass ratio M / C of magnetic particles (M) to cellulose nanofibers (C) may be 0.01 to 10, 0.05 to 2, 0.08 to 1, or 0.1 to 0.5.

[0051] The composite particles (A) are preferably microsized from the viewpoint of ease of handling. More specifically, the average particle size of the composite particles may be 1 to 30 μm, 1 to 20 μm, or 2 to 15 μm.

[0052] The composite particles (A) are formed from cellulose nanofibers and magnetic particles, and may be composed of only cellulose nanofibers and magnetic particles, or may contain other components.

[0053] The method for producing the composite particles (A) is not particularly limited. For example, the composite particles (A) can be produced by a method comprising spray-drying a dispersion containing carboxyl group-containing cellulose nanofibers and magnetic particles (step 1), and converting the carboxyl groups of the cellulose nanofibers contained in the composite particles obtained by spray-drying into active esters (step 2).

[0054] In step 1, spray drying the dispersion liquid allows for obtaining a dry powder without agglomerating the cellulose nanofibers, thereby enabling the cellulose nanofibers and magnetic particles to be composited. The dispersion liquid used for spray drying is prepared, for example, by using water as a solvent and uniformly mixing and dispersing the cellulose nanofibers and magnetic particles in the solvent. The concentration of cellulose nanofibers in the dispersion liquid is not particularly limited as long as cellulose particles can be formed by spray drying, and may be, for example, 0.005 to 5% by mass or 0.01 to 1% by mass. The concentration of magnetic particles is also not particularly limited, and may be, for example, 0.005 to 5% by mass or 0.01 to 1% by mass.

[0055] The ratio of cellulose nanofibers to magnetic particles in the dispersion may be the same as that in the composite particles (A), where M / C is the mass ratio of magnetic particles (M) to cellulose nanofibers (C), and may be 0.01 to 10, 0.05 to 2, 0.08 to 1, or 0.1 to 0.5.

[0056] Spray drying is a method of producing a dry powder by spraying a dispersion into a gas and rapidly drying it, and can be performed using a known spray dryer. The drying temperature in spray drying is not particularly limited and may be, for example, 150 to 200°C.

[0057] The dispersion liquid used for spray drying may contain a crosslinking agent that reacts with carboxy groups. By containing a crosslinking agent, the carboxy groups of the cellulose nanofibers react with the crosslinking agent during the spray drying process, resulting in composite particles having a crosslinked structure. This allows the composite particles to have excellent shape retention and increased durability when redispersed in a liquid such as water. The crosslinking agent is not particularly limited, and examples thereof include amino resins, epoxy compounds, aziridine compounds, carbodiimide compounds, oxazoline compounds, and polyisocyanate compounds.

[0058] In one embodiment, the active esterification method of step 2 preferably includes a step (step 2a) of mixing composite particles containing carboxy group-containing cellulose nanofibers and magnetic particles with a condensing agent and an additive for active esterification in an organic solvent. After preparing composite particles from carboxy group-containing cellulose nanofibers, the carboxy groups on the composite particles are then actively esterified using the composite particles, which allows the carboxy groups on the particle surface to be more efficiently actively esterified, making it easier to obtain composite particles (A) having active ester groups on the particle surface.

[0059] In step 2a, the condensing agent may be at least one selected from the group consisting of carbodiimide condensing agents, imidazole condensing agents, triazine condensing agents, phosphonium condensing agents, uronium condensing agents, and haluronium condensing agents. Specific examples of these condensing agents are as described above. The amount of condensing agent used is not particularly limited, and may be, for example, an amount equal to or greater than the amount of carboxyl groups in the cellulose nanofibers that make up the composite particles.

[0060] In step 2a, the additive for active esterification is preferably at least one selected from the group consisting of N-hydroxysuccinimide, N,N'-disuccinimidyl carbonate, N-hydroxysulfosuccinimide alkali metal salts, 1-hydroxybenzotriazole, 1-hydroxy-7-azabenzotriazole, ethyl(hydroxyimino)cyanoacetate, pentafluorophenol, and nitrophenol. This introduces active ester groups represented by the general formula (1) above, where X is one of the formulae (2) to (8). The amount of additive used is not particularly limited; for example, the additive may be added in an amount equal to or greater than the amount of carboxy groups in the cellulose nanofibers that make up the composite particles.

[0061] In step 2a, the organic solvent is not particularly limited, and examples thereof include dimethylformamide, dimethyl sulfoxide, tetrahydrofuran, dichloromethane, acetone, chloroform, toluene, 1,2-dichloroethane, 1,4-dioxane, and ethyl acetate.

[0062] The composite particles (B) according to one embodiment are composite particles containing cellulose nanofibers and magnetic particles to which an amino group-containing compound has been introduced. Specifically, the composite particles (B) contain the amino group-containing compound bound to the cellulose nanofibers via an amide bond, which is a structure formed by dehydration condensation between a carboxy group of the cellulose nanofiber and an amino group of the amino group-containing compound.

[0063] The composite particles (A) have active ester groups, which make them highly reactive with amino group-containing compounds such as proteins. Therefore, by reacting the amino group of an amino group-containing compound with the active ester group, composite particles (B) incorporating the amino group-containing compound can be easily obtained. That is, the amino group-containing compound can be easily bonded to the composite particles (A) via the amide bond formed by the reaction, thereby obtaining composite particles (B) incorporating the amino group-containing compound. Therefore, the composite particles (A) are suitable for use as precursors for bonding amino group-containing compounds such as proteins.

[0064] Preferred examples of amino group-containing compounds include amino group-containing biotin or proteins. Amino group-containing biotin is biotin with an amino group introduced therein, and examples include biotin-C5-amine, alkoxyamine-PEG-biotin, and biotin hydrazide. Examples of proteins include antibodies, peptides, protein A, protein G, protein L, streptavidin, and enzymes.

[0065] The method for reacting the amino group of an amino group-containing compound with the active ester group of a composite particle (A) containing cellulose nanofibers having active ester groups is not particularly limited. For example, the composite particle (A) and the amino group-containing compound are mixed in an organic solvent to obtain a composite particle (B) into which the amino group-containing compound has been introduced. The organic solvent used in this case is not particularly limited, and examples thereof include dimethylformamide, methyl sulfoxide, tetrahydrofuran, dichloromethane, acetone, chloroform, toluene, 1,2-dichloroethane, 1, 4-dioxane, ethyl acetate, etc. The amount of the amino group-containing compound used is not particularly limited, and for example, it may be added in an amount equal to or greater than the amount of carboxy groups in the cellulose nanofibers that make up the composite particles.

[0066] The composite particles (B) having amino group-containing biotin or protein introduced therein are suitable for use in capturing or detecting target compounds that specifically bind to the amino group-containing biotin or protein. For example, in the case of composite particles (B) having amino group-containing biotin introduced therein, examples of the target compound include avidin and streptavidin, which specifically bind to biotin. For example, in the case of composite particles (B) having an antibody introduced therein as a protein, the target compound is an antigen that specifically binds to the antibody. In this way, specific target compounds can be captured or detected by utilizing avidin-biotin interactions or antigen-antibody reactions.

[0067] One embodiment of a method for capturing or detecting a target compound involves mixing composite particles (B) containing amino-group-containing biotin or protein with a target compound that specifically binds to the composite particles (B) in a liquid, thereby binding the amino-group-containing biotin or protein to the target compound. More specifically, for example, the composite particles (B) are added to a liquid containing or suspected of containing the target compound and mixed. This allows the target compound to specifically bind to the amino-group-containing biotin or protein of the composite particles (B). The composite particles (B) with the bound target compound can then be collected (recovered) from the liquid in which the composite particles (B) are dispersed by magnetic separation using a magnet.

[0068] When collecting composite particles (B) by magnetic separation, for example, when composite particles (B) are dispersed in a liquid, a magnet can be used to attract and collect the composite particles (B) to the wall of a container using its magnetic force, thereby easily separating them from the liquid. The magnet may be a permanent magnet or an electromagnet. Such collection of composite particles by magnetic separation can be used in a variety of situations. Examples include washing composite particles before active esterification in the active esterification step in the method for producing composite particles (A), separating and washing composite particles (A) from a reaction solution after an active esterification reaction, separating and washing composite particles (B) from a reaction solution after an introduction reaction in an amino group-containing compound introduction step, and separating and washing composite particles (B) from a liquid after binding a target compound in a method for capturing or detecting a target compound.

[0069] The composite particles according to this embodiment may be used, for example, in sandwich ELISA. Specifically, composite particles (A) having active ester groups are reacted with an antibody (capture antibody) as an amino group-containing compound to produce composite particles (B) incorporating the capture antibody. Next, the composite particles (B) are mixed with a liquid containing or suspected of containing an antigen as the target compound, allowing the antigen to bind to the capture antibody. An enzyme-labeled antibody (detection antibody) is then added, allowing the detection antibody to bind to the antigen bound to the capture antibody. A chromogenic substrate, a colorimetric substrate, or a luminescent substrate for the enzyme is then added to cause a reaction, resulting in color development, color development, or light emission. The intensity of the color development, color development, or light emission is measured, allowing the target compound to be quantitatively detected.

[0070] The composite particle according to this embodiment may be used, for example, to detect avidin or streptavidin (hereinafter simply referred to as avidin) by utilizing the avidin-biotin interaction. Specifically, a composite particle (A) having an active ester group is reacted with biotin containing an amino group to produce a composite particle (B) into which biotin has been introduced. The composite particle (B) is then mixed with a liquid containing or suspected of containing enzyme-labeled avidin as a target compound, thereby binding the biotin and the enzyme-labeled avidin. Thereafter, a color-developing substrate, a color-developing substrate, or a luminescent substrate for the enzyme is added and reacted to cause color development, color generation, or light emission. The intensity of the color development, color generation, or light emission is measured, allowing the target compound to be quantitatively detected. [Example]

[0071] The present invention will be explained in more detail below with reference to examples, but is not limited to these.

[0072] [Preparation of TOCN magnetic composite particles] <Measurement method> (1) Number-average fiber diameter of cellulose nanofibers The number-average fiber diameter of cellulose nanofibers was observed using a transmission electron microscope (TEM) (JEM-1400, manufactured by JEOL Ltd.). Specifically, the sample was cast onto a hydrophilically treated carbon film-coated grid, and the number-average fiber diameter was calculated from the TEM image (magnification: 10,000x) negatively stained with a 2% by mass aqueous solution of uranyl acetate, according to the method described above.

[0073] (2) Average aspect ratio of cellulose nanofibers Using observation samples prepared in the same manner as for measuring the number average fiber diameter, the number average fiber length of cellulose nanofibers was calculated according to the method described above.The average aspect ratio was then calculated according to the above formula using the number average fiber diameter and number average fiber length values.

[0074] (3) Crystal structure of cellulose nanofibers The diffraction profile of the sample was measured using an X-ray diffractometer (Rigaku Corporation, RINT-Ultima3). If typical peaks were observed at two positions, around 2θ = 14° to 17° and 2θ = 22° to 23°, the sample was evaluated as having a crystalline structure (type I crystalline structure). If no peaks were observed, the sample was evaluated as having no crystalline structure.

[0075] (4) Carboxylic group content of cellulose nanofibers 0.25 g of the sample was dispersed in water to prepare 60 mL of an aqueous dispersion. The pH was adjusted to approximately 2.5 with 0.1 M aqueous hydrochloric acid, and then 0.05 M aqueous sodium hydroxide solution was added dropwise. Electrical conductivity was measured, and the amount of carboxyl groups was determined according to the method described above.

[0076] (5) Average particle size of composite particles and magnetic particles Photographs were taken at 5,000 to 20,000 magnifications using an SEM (scanning electron microscope: S-5000, manufactured by Hitachi High-Technologies Corporation, 20 kV), and 200 or more particles were randomly selected, their diameters were measured, and the average value was calculated.

[0077] <Raw materials> TOCN: TEMPO-oxidized cellulose nanofiber (Dai-ichi Kogyo Seiyaku Co., Ltd. "Rheocrysta I-2SX", cellulose concentration: 2% by mass, cellulose type I crystal structure: "present", number-average fiber diameter: 4 nm, average aspect ratio: 280, carboxyl group content: 1.9 mmol / g) Fe3O4 particles 1: Toda Kogyo Co., Ltd. (average particle size: 10 nm) Fe3O4 particles 2: Toda Kogyo Co., Ltd. (average particle size: 300 nm) Oxazoline crosslinking agent (Epocross WS-700 manufactured by Nippon Shokubai Co., Ltd.)

[0078] <Preparation Example 1> TOCN and the oxazoline crosslinker were mixed with pure water and ultrasonically dispersed for 20 minutes using an IKA T10 ULTRA-TU RRAX S0004 to prepare an aqueous dispersion with a TOCN solids concentration of 0.08% by mass and a 20 mol% oxazoline group content of the oxazoline crosslinker relative to the TOCN carboxyl group content. Next, FeO particles 1 were added to the aqueous dispersion to a concentration of 0.008% by mass, and ultrasonic dispersion was repeated for 20 minutes to prepare an aqueous dispersion containing TOCN and FeO particles 1 (FeO / TOCN (mass ratio) = 1 / 10). The obtained aqueous dispersion was spray-dried using a spray dryer (BUCHI, Mini Spray Dryer B-290) to crosslink the carboxyl groups of the TOCN with the oxazoline-based crosslinking agent and simultaneously composite the TOCN with Fe3O4 particles 1, yielding TOCN magnetic composite particles (TEFW-0.1-10) (average particle size: 2.73 μm) of Preparation Example 1. The spray drying was performed at an inlet temperature of 120°C, a liquid flow rate of 2.5 mL / min, and a gas flow rate of 6 L / min.

[0079] <Preparation Example 2> The amount of Fe3O4 particles 1 added was adjusted so that the Fe3O4 / TOCN (mass ratio) was 3 / 10, and the rest was the same as in Preparation Example 1 to obtain TOCN magnetic composite particles (TEFW-0.3-10) (average particle size: 2.92 μm) of Preparation Example 2.

[0080] <Preparation Example 3> The procedure for Preparation Example 3 was the same as that for Preparation Example 1, except that Fe3O4 particles 2 were used instead of Fe3O4 particles 1, and the amount of Fe3O4 particles 2 added was adjusted so that the Fe3O4 / TOCN (mass ratio) was 1 / 1. TOCN magnetic composite particles (TEFW-1-300) (average particle size: 2.59 μm) were obtained in Preparation Example 3.

[0081] [Preparation of NHS-modified TOCN magnetic composite particles] The TOCN magnetic composite particles of Preparation Examples 1 to 3 were subjected to active esterification with N-hydroxysuccinimide (NHS) according to the following procedures (1) to (5). (1) 5 mg of TOCN magnetic composite particles and 1 mL of N,N-dimethylformamide (DMF) were placed in a 1.5 mL microtube, and the TOCN magnetic composite particles were dispersed in the DMF. The TOCN magnetic composite particles were then washed by magnetic separation at room temperature for 5 minutes. The magnetic separation was performed by inserting the microtube into a magnetic stand (manufactured by Tamagawa Seiki Co., Ltd.) and allowing the TOCN magnetic composite particles in the dispersion to collect on the side of the microtube, after which the DMF was removed. (2) The washing procedure in (1) above was repeated three times. (3) 3.1 mg (16 μmol) of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDAC) and 0.9 mg (8 μmol) of N-hydroxysuccinimide (NHS) were each dissolved in 500 μL of DMF. (4) The EDAC solution and NHS solution were added to the magnetically separated TOCN magnetic composite particles obtained in (2) above, then dispersed by ultrasonic waves and mixed by inversion at room temperature for 2 hours. (5) After mixing by inversion, magnetic separation and washing using DMF was carried out in the same manner as in (2) above, thereby obtaining NHS-modified TOCN magnetic composite particles having an active ester group represented by the general formula (1) above (where X is represented by formula (2)), as shown in the following reaction formula. [ka]

[0082] [Preparation of biotin-incorporated composite particles] Using the NHS-modified TOCN magnetic composite particles obtained above, biotin-introduced composite particles were prepared according to the following procedures (1) to (5). (1) 2.6 mg (8 μmol) of biotin-C5-amine was dissolved in 500 μL of DMF in a 1.5 mL microtube. (2) 5 mg of NHS-modified TOCN magnetic composite particles was added to the biotin-C5-amine solution, dispersed by ultrasonic waves, and mixed by inversion at room temperature for 1 hour. (3) After mixing by inversion, magnetic separation and washing were performed three times using 1 mL of DMF. The magnetic separation and washing method was the same as in (1) of the preparation method for NHS-modified TOCN magnetic composite particles. (4) 1 mL of 1 M monoethanolamine DMF solution was added to the magnetically separated and washed particles, and the mixture was mixed by inversion at room temperature for 1 hour. (5) After mixing by inversion, magnetic separation and washing using DMF were carried out in the same manner as in (3) above, thereby obtaining biotin-incorporated composite particles as shown in the following reaction formula. [ka]

[0083] [Avidin detection test] Using the biotin-introduced composite particles obtained above, an avidin detection test was carried out according to the following procedures (1) to (7). (1) 2.5 mg of biotin-incorporated composite particles and 1 mL of buffer were placed in a 1.5 mL microtube and magnetically separated at room temperature for 5 minutes to wash the biotin-incorporated composite particles. Magnetic separation was performed by inserting the microtube into a magnetic stand (manufactured by Tamagawa Seiki Co., Ltd.), allowing the biotin-incorporated composite particles in the dispersion to collect on the side of the microtube, and then discarding the buffer. The buffer used was 0.1 M glycine-NaOH buffer (pH 10.3) containing 1 mM MgCl2, 0.1 mM ZnCl2, and 0.025% ovalbumin. (2) The washing procedure in (1) above was repeated three times. (3) After washing, the biotin-introduced composite particles were divided into 0.2 mg portions, and 200 μL of streptavidin-ALP (Promega) diluted 5000-fold, 10000-fold, or 20000-fold with the above buffer or buffer was added, and the mixture was mixed by inversion using a rotator at room temperature for 1 hour. (4) After mixing by inversion, magnetic separation was performed at room temperature for 5 minutes, and after removing all of the supernatant, 1 mL of buffer was added and pipetted. (5) The above (4) was repeated three times. (6) Then, 150 μL of Roche Diagnostics' CDP-STAR was added, and the mixture was stirred on a shaker at room temperature for 20 minutes. (7) Then, the emission intensity at 460 nm was measured using a spectrophotometer.

[0084] For comparison, NHS modification, biotinylation, and avidin detection were performed using commercially available carboxy-containing magnetic particles, "Magnosphere MS160 / Carboxyl" manufactured by JSR Life Sciences. Specifically, 500 μL of 10 mg / mL Magnosphere particles were placed in a 1.5 mL microtube, the microtube was inserted into a magnetic stand (manufactured by Tamagawa Seiki Co., Ltd.), and the Magnosphere particles in the dispersion were collected on the side of the microtube. The liquid was discarded, and 1 mL of DMF was added. Subsequent procedures were performed in the same manner as in (2) of [Preparation of NHS-modified TOCN magnetic composite particles], [Preparation of biotin-incorporated composite particles], and [Avidin detection test].

[0085] The results are shown in FIG. 1, and avidin was detectable with the biotin-introduced composite particles according to the examples (TEFW-0.1-10, TEFW-0.3-10, TEFW-1-300).

[0086] The various numerical ranges described in this specification can be arbitrarily combined with their upper and lower limits, and all such combinations are considered to be preferred numerical ranges described in this specification. Furthermore, a numerical range described as "X to Y" means from X to Y.

[0087] Although several embodiments of the present invention have been described above, these embodiments are presented as examples and are not intended to limit the scope of the invention. These embodiments can be implemented in various other forms, and various omissions, substitutions, and modifications can be made without departing from the spirit of the invention. These embodiments and their omissions, substitutions, modifications, etc. are included within the scope and spirit of the invention, as well as within the scope of the invention described in the claims and their equivalents.

Claims

1. A composite particle comprising a cellulose nanofiber and a magnetic particle, wherein the cellulose nanofiber has an active ester group.

2. The composite particle according to claim 1, wherein the active ester group is formed by reacting the carboxy group of the cellulose nanofiber having a carboxy group with at least one condensing agent selected from the group consisting of a carbodiimide-based condensing agent, an imidazole-based condensing agent, a triazine-based condensing agent, a phosphonium-based condensing agent, a uronium-based condensing agent, and a haluronium-based condensing agent.

3. The active ester group is represented by the following general formula (1): 【Chemistry 1】 In the formula, X represents any one of the following formulas (2) to (8), and in formula (3), M represents an alkali metal atom: 【Chemistry 2】 The composite particle according to claim 1 .

4. 2. The composite particles according to claim 1, wherein the magnetic particles have an average particle size of 1 to 500 nm.

5. A composite particle comprising a cellulose nanofiber and a magnetic particle, the composite particle comprising the amino group-containing compound bound to the cellulose nanofiber via an amide bond, which is a structure formed by dehydration condensation between a carboxy group of the cellulose nanofiber and an amino group of the amino group-containing compound.

6. The composite particle according to claim 5 , wherein the amino group-containing compound is an amino group-containing biotin or protein.

7. The composite particle according to claim 6, which is used for capturing or detecting a target compound that specifically binds to the amino group-containing biotin or the protein.

8. A method for producing composite particles containing cellulose nanofibers having active ester groups and magnetic particles, comprising: Composite particles containing cellulose nanofibers having carboxy groups and magnetic particles; at least one selected from the group consisting of N-hydroxysuccinimide, N,N'-disuccinimidyl carbonate, N-hydroxysulfosuccinimide alkali metal salts, 1-hydroxybenzotriazole, 1-hydroxy-7-azabenzotriazole, ethyl(hydroxyimino)cyanoacetate, pentafluorophenol, and nitrophenol; at least one condensing agent selected from the group consisting of carbodiimide-based condensing agents, imidazole-based condensing agents, triazine-based condensing agents, phosphonium-based condensing agents, uronium-based condensing agents, and haluronium-based condensing agents; in an organic solvent.

9. A method for producing composite particles having an amino group-containing compound incorporated therein, the method comprising reacting the amino group of an amino group-containing compound with the active ester group of a composite particle comprising cellulose nanofibers having active ester groups and magnetic particles.

10. The method for producing composite particles according to claim 9 , wherein the amino group-containing compound is amino group-containing biotin or a protein.

11. A method for capturing or detecting a target compound, comprising mixing the composite particle described in claim 6 with a target compound that specifically binds to the amino group-containing biotin or the protein in a liquid, and binding the amino group-containing biotin or the protein to the target compound.

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