Composite particles and method for producing same

Abstract

Provided are composite particles which comprise: cellulose nanofibers into which a carboxy-group-containing compound such as a protein can be easily introduced; and magnetic particles. Composite particles according to an embodiment comprise cellulose nanofibers and magnetic particles, wherein each of the cellulose nanofibers has a group represented by general formula (1). In formula (1), R1 represents a single bond or a divalent aliphatic group having 1-10 carbon atoms.

Description

Composite particles and their manufacturing method

[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.

[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.

[0004] Japanese Patent Application Laid-Open No. 2022-30885

[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 carboxyl group-containing compounds, such as biotin or proteins, into cellulose nanofibers as substances that specifically bind 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 to provide, in one embodiment, composite particles of cellulose nanofibers and magnetic particles into which a carboxyl group-containing compound can be 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 a carboxyl 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.

[0007] The present invention includes the following embodiments: [1] Composite particles containing cellulose nanofibers and magnetic particles, wherein the cellulose nanofibers have a group represented by the following general formula (1): R in the formula １ represents a single bond or a divalent aliphatic group having 1 to 10 carbon atoms. [2] The composite particle according to [1], wherein the average particle size of the magnetic particles is 1 to 500 nm.

[0008] [3] Composite particles containing cellulose nanofibers and magnetic particles, wherein the —NH of the group represented by the following general formula (1) possessed by the cellulose nanofibers: ２ and the carboxy group-containing compound bound to the cellulose nanofibers via an amide bond, which is a structure formed by dehydration condensation between the carboxy group of the carboxy group-containing compound and the carboxy group of the carboxy group-containing compound, R in the formula １ represents a single bond or a divalent aliphatic group having 1 to 10 carbon atoms. [4] The composite particle according to [3], wherein the carboxy group-containing compound is biotin or a protein. [5] The composite particle according to [4], which is used for capturing or detecting a target compound that specifically binds to the biotin or the protein.

[0009] [6] A method for producing the composite particles according to [1] or [2], comprising mixing, in an organic solvent, composite particles containing cellulose nanofibers having carboxy groups and magnetic particles, hydrazine or an aliphatic diamine, and 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.

[0010] [7] The —NH group of the group represented by the general formula (1) contained in the composite particle according to [1] or [2]. ２ [8] The method for producing composite particles according to [7], wherein the carboxy group-containing compound is biotin or a protein.

[0011] [9] A method for capturing or detecting a target compound, comprising mixing the composite particle according to [4] with a target compound that specifically binds to the biotin or the protein in a liquid, thereby binding the biotin or the protein to the target compound.

[0012] In the above embodiment, a carboxy group-containing compound can be introduced into a composite particle containing a cellulose nanofiber and a magnetic particle by using the amino group introduced into the cellulose nanofiber. Furthermore, it is possible to provide a novel composite particle into which a carboxy group-containing compound has been introduced, which can be used, for example, to capture or detect a specific target compound.

[0013] Graph showing the results of an avidin detection test in an example.

[0014] Composite particles (A) according to one embodiment contain cellulose nanofibers modified with amino 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.

[0015] The magnetic particles are not particularly limited as long as they are made of a ferromagnetic material that can be attracted by a magnet. For example, Fe ３ O ４ , γ-Fe ２ O ３ Examples of suitable particles include metal oxides such as iron oxide, 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.

[0016] 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.

[0017] 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.

[0018] 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.

[0019] 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 observation sample may also be negatively stained, for example, with 2% by mass uranyl acetate. Then, electron microscope images are observed at magnifications of 5,000x, 10,000x, or 50,000x, depending on the size of the fibers constituting the sample. In this case, an arbitrary vertical or horizontal axis of the image width is assumed within the obtained image, and the sample and observation conditions (magnification, etc.) are adjusted so that 20 or more fibers intersect with the axis. After obtaining an observation image that satisfies these conditions, two random axes are drawn vertically and horizontally on the 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 portion 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 thus obtained is taken as the number-average fiber diameter.

[0020] 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 a diffraction profile obtained by wide-angle X-ray diffraction image measurement.

[0021] The average aspect ratio of (c) 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.

[0022] 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. Using these values, the average aspect ratio is calculated according to the following formula: Average aspect ratio = Number average fiber length (nm) / Number average fiber diameter (nm)

[0023] Cellulose nanofibers modified with amino groups are cellulose nanofibers having a group represented by the following general formula (1) (hereinafter referred to as "functional group (1)").

[0024] R in formula (1) １ represents a single bond or a divalent aliphatic group having 1 to 10 carbon atoms. The divalent aliphatic group may be a saturated aliphatic group or an unsaturated aliphatic group, but is preferably a saturated aliphatic group (i.e., an alkanediyl group). The alkanediyl group may be linear or branched, but is preferably linear. The divalent aliphatic group more preferably has 2 to 8 carbon atoms, and even more preferably has 2 to 6 carbon atoms.

[0025] In one embodiment, the cellulose nanofibers modified with amino groups are obtained by subjecting the carboxy groups of carboxy group-containing cellulose nanofibers to a dehydration condensation reaction with the amino groups of hydrazine or an aliphatic diamine.

[0026] 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.

[0027] 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, using a co-oxidant in the presence of an N-oxyl compound and then defibrating (refining) the cellulose. The N-oxyl compound used is a compound containing a nitroxy radical, which is commonly used as an oxidation catalyst. For example, a piperidine nitroxyoxy radical is used. 2,2,6,6-tetramethylpiperidinoxy radical (TEMPO) or 4-acetamido-TEMPO is particularly preferred. Cellulose nanofibers oxidized with TEMPO 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.

[0028] 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.

[0029] The amount of carboxy groups in the 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 the carboxy group-containing cellulose nanofibers. The amount of carboxy groups can be determined by preparing 60 mL of a cellulose nanofiber-containing slurry adjusted to a concentration of 0.1 to 1% by mass, adjusting the pH to about 2.5 with a 0.1 mol / L aqueous hydrochloric acid solution, adding a 0.05 mol / L aqueous sodium hydroxide solution dropwise, measuring the electrical conductivity, and continuing until the pH reaches about 11. The amount of carboxy groups can be determined according to the following formula from the amount of sodium hydroxide (V) consumed in the neutralization stage of the weak acid, where the change in electrical conductivity is gradual: Amount of carboxy groups (mmol / g) = V (mL) × [0.05 / mass of cellulose nanofiber (g)]

[0030] The aliphatic diamine to be reacted with the carboxyl group-containing cellulose nanofibers is an aliphatic diamine having 1 to 10 carbon atoms, thereby introducing functional group (1) into the cellulose nanofibers. The aliphatic diamine preferably has 2 to 8 carbon atoms, more preferably 2 to 6 carbon atoms.

[0031] The aliphatic diamine is preferably a saturated aliphatic diamine, more preferably an alkylenediamine, and even more preferably a linear alkylenediamine. Specific examples of the alkylenediamine include ethylenediamine, propylenediamine, trimethylenediamine, tetramethylenediamine, pentamethylenediamine, and hexamethylenediamine. These may be used alone or in combination of two or more.

[0032] The functional group (1) may be introduced to all or some of the carboxy groups of the carboxy group-containing cellulose nanofibers. For example, it is preferable that the functional group (1) is introduced to at least the carboxy groups on the particle surface of the composite particles (A). The amount of the functional group (1) 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 the functional group (1), similar to the amount of the carboxy groups described above.

[0033] In the composite particle (A), the ratio of the cellulose nanofibers having the functional group (1) to the magnetic particles is not particularly limited, and the mass ratio M / C of the magnetic particles (M) to the cellulose nanofibers (C) may be 0.01 to 10, 0.05 to 2, 0.08 to 1, or 0.1 to 0.5.

[0034] 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.

[0035] 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.

[0036] The method for producing the composite particles (A) is not particularly limited. For example, the composite particles (A) can be produced by a method including spray-drying a dispersion containing carboxy group-containing cellulose nanofibers and magnetic particles (Step 1), and reacting the carboxy groups of the cellulose nanofibers contained in the composite particles obtained by spray-drying with hydrazine or an aliphatic diamine (Step 2).

[0037] In step 1, by spray-drying the dispersion, a dry powder can be obtained without agglomerating the cellulose nanofibers, and the cellulose nanofibers and magnetic particles can be composited. The dispersion 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 the cellulose nanofibers in the dispersion 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 the 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.

[0038] The ratio of cellulose nanofibers to magnetic particles in the dispersion, similar to the ratio in the composite particles (A), may be 0.01 to 10, 0.05 to 2, 0.08 to 1, or 0.1 to 0.5, in terms of the mass ratio M / C of magnetic particles (M) to cellulose nanofibers (C).

[0039] 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.

[0040] 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.

[0041] In step 2, the method for reacting hydrazine or aliphatic diamine is not particularly limited, as long as it allows the carboxy groups of the carboxy group-containing cellulose nanofibers and the amino groups of the hydrazine or aliphatic diamine to form amide bonds by dehydration condensation. In one embodiment, it is preferable to include a step (step 2a) of mixing the composite particles obtained by the above-mentioned spray drying with hydrazine or aliphatic diamine and a condensing agent in an organic solvent. This allows functional groups (1) to be introduced more efficiently into the carboxy groups on the particle surfaces.

[0042] In step 2a, the condensing agent may be at least one 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.

[0043] 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.

[0044] Examples of the imidazole-based condensing agent include N,N'-carbonyldiimidazole and 1,1'-carbonyldi(1,2,4-triazole).

[0045] 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.

[0046] 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.

[0047] 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 S-(1-oxido-2-pyridyl)-N,N,N',N'-tetramethyluronium 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.

[0048] 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.

[0049] These condensing agents may be used either alone or in combination of two or more.

[0050] 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.

[0051] In steps 2 and 2a, the amount of hydrazine or aliphatic diamine is not particularly limited, and may be added in an amount equal to or greater than the molar amount of carboxy groups in the cellulose nanofibers that make up the composite particles.The amount of condensing agent is also not particularly limited, and may be added in an amount equal to or greater than the molar amount of carboxy groups in the cellulose nanofibers that make up the composite particles.

[0052] The composite particle (B) according to one embodiment is a composite particle containing cellulose nanofibers and magnetic particles, to which a carboxyl group-containing compound has been introduced. Specifically, the composite particle (B) is a composite particle containing cellulose nanofibers and magnetic particles, to which a carboxyl group-containing compound has been introduced. ２ and a carboxy group of the carboxy group-containing compound bound to the cellulose nanofiber via an amide bond, which is a structure formed by dehydration condensation between the carboxy group of the carboxy group-containing compound and the carboxy group of the cellulose nanofiber.

[0053] The composite particles (A) have a primary amino group (—NH ２ ), it can react with a carboxyl group-containing compound such as a protein to form an amide bond. This makes it possible to easily obtain composite particles (B) into which a carboxyl group-containing compound has been introduced. Therefore, the composite particles (A) are suitably used as a precursor to which a carboxyl group-containing compound such as a protein is bonded.

[0054] The carboxy group-containing compound is preferably, for example, biotin or a protein, such as an antibody, peptide, protein A, protein G, protein L, streptavidin, or enzyme.

[0055] These carboxy group-containing compounds may be those in which the carboxy group has been converted into an active ester, and the concept of the carboxy group of a carboxy group-containing compound encompasses such active esterified active ester groups. The method for converting into an active ester is not particularly limited, and examples thereof include a method in which an additive such as N-hydroxysuccinimide, N,N'-disuccinimidyl carbonate, an N-hydroxysulfosuccinimide alkali metal salt, 1-hydroxybenzotriazole, 1-hydroxy-7-azabenzotriazole, ethyl(hydroxyimino)cyanoacetate, pentafluorophenol, or nitrophenol is reacted with the carboxy group-containing compound using the condensing agent.

[0056] The method for reacting the primary amino group of the functional group (1) contained in the composite particle (A) with the carboxy group of the carboxy group-containing compound is not particularly limited.

[0057] For example, when the carboxy group of the carboxy group-containing compound is not actively esterified, the composite particle (A), the carboxy group-containing compound, and the condensing agent are mixed in an organic solvent to introduce the carboxy group-containing compound into the composite particle (A), thereby obtaining composite particle (B). When the carboxy group of the carboxy group-containing compound is actively esterified, the composite particle (A) and the actively esterified carboxy group-containing compound are mixed in an organic solvent to introduce the carboxy group-containing compound into the composite particle (A), thereby obtaining composite particle (B). The organic solvent used in these cases is not particularly limited, and examples include dimethylformamide, methyl sulfoxide, tetrahydrofuran, dichloromethane, acetone, chloroform, toluene, 1,2-dichloroethane, 1,4-dioxane, and ethyl acetate. The amount of the carboxy group-containing compound used is also not particularly limited, and may be added in an amount equal to or greater than the amount of primary amino groups in the cellulose nanofibers that make up the composite particle.

[0058] The composite particles (B) having biotin or protein introduced therein in this manner are suitable for use in capturing or detecting target compounds that specifically bind to biotin or protein. For example, in the case of composite particles (B) having 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, antigen-antibody reactions, or the like.

[0059] A method for capturing or detecting a target compound according to one embodiment includes mixing composite particles (B) containing biotin or a protein with a target compound that specifically binds to the composite particles (B) in a liquid, thereby binding the 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 biotin or protein of the composite particles (B). The composite particles (B) to which the target compound is bound can then be collected (recovered) from the liquid in which the composite particles (B) are dispersed by magnetic separation using a magnet.

[0060] When collecting the composite particles (B) by magnetic separation, for example, when the 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 making it easy to separate 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 the composite particles before the amidation reaction in the method for producing the composite particles (A), separating and washing the composite particles (A) from the reaction solution after the amidation reaction, separating and washing the composite particles (B) from the reaction solution after the introduction reaction in the step of introducing a carboxy group-containing compound, and separating and washing the composite particles (B) from the liquid after binding the target compound in a method for capturing or detecting the target compound.

[0061] The composite particle according to this embodiment may be used, for example, in a sandwich ELISA. Specifically, a composite particle (A) having a functional group (1) is reacted with an antibody (capture antibody) as a carboxyl group-containing compound to produce a composite particle (B) incorporating the capture antibody. The composite particle (B) is then mixed with a liquid containing or suspected of containing an antigen as the target compound, allowing the antigen and the capture antibody to bind. 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 and reacted to produce color, color, or light, and the intensity of the color, color, or light emission is measured, allowing the target compound to be quantitatively detected.

[0062] 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 a functional group (1) is reacted with biotin 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, coloration, or light emission. The intensity of the color development, coloration, or light emission is measured, allowing the target compound to be quantitatively detected.

[0063] The present invention will be explained in more detail below with reference to examples, but is not limited to these.

[0064] [Preparation of TOCN Magnetic Composite Particles] <Measurement Method> (1) Number Average Fiber Diameter of Cellulose Nanofibers The number average fiber diameter of the cellulose nanofibers was observed using a transmission electron microscope (TEM) (JEM-1400, manufactured by JEOL Ltd.) That is, a sample was cast onto a hydrophilized carbon film-coated grid, and then the number average fiber diameter was calculated from a TEM image (magnification: 10,000x) negatively stained with a 2% by mass aqueous solution of uranyl acetate, according to the method described above.

[0065] (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 the 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.

[0066] (3) Crystalline 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 crystal structure). If no peaks were observed, the sample was evaluated as having no crystalline structure.

[0067] (4) Amount of carboxyl groups in cellulose nanofibers 60 mL of an aqueous dispersion was prepared by dispersing 0.25 g of a sample in water, and the pH was adjusted to approximately 2.5 with 0.1 M aqueous hydrochloric acid solution. After that, 0.05 M aqueous sodium hydroxide solution was added dropwise, and electrical conductivity was measured, and the amount of carboxyl groups was determined according to the method described above.

[0068] (5) Average particle size of composite particles and magnetic particles Photographs were taken at a magnification of 5,000 to 20,000 times 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.

[0069] <Raw Materials> TOCN: TEMPO-oxidized cellulose nanofiber ("Rheocrysta I-2SX" manufactured by Daiichi Kogyo Seiyaku Co., Ltd., cellulose concentration: 2% by mass, cellulose type I crystal structure: "present", number average fiber diameter: 4 nm, average aspect ratio: 280, carboxyl group amount: 1.9 mmol / g) Fe ３ O ４ Particle 1: manufactured by Toda Kogyo Co., Ltd. (average particle size: 10 nm) Fe ３ O ４ Particle 2: manufactured by Toda Kogyo Co., Ltd. (average particle size: 300 nm) Oxazoline-based crosslinking agent (manufactured by Nippon Shokubai Co., Ltd., "Epocross WS-700")

[0070] Preparation Example 1 TOCN and an oxazoline-based crosslinking agent were mixed with pure water and subjected to ultrasonic dispersion (T10 ULTRA-TU RRAX S0004, manufactured by IKA Corporation) for 20 minutes to prepare an aqueous dispersion having a TOCN solids concentration of 0.08 mass % and an oxazoline group content of the oxazoline-based crosslinking agent of 20 mol % relative to the carboxyl group content of TOCN. ３ O ４ Particle 1 was added to a concentration of 0.008 mass %, and the same ultrasonic dispersion was carried out for 20 minutes to obtain a mixture of TOCN and Fe. ３ O ４ Aqueous dispersion containing particle 1 (Fe ３ O ４ The resulting aqueous dispersion was spray-dried using a spray dryer (Buchi Corporation, Mini Spray Dryer B-290) to crosslink the carboxyl groups of the TOCN with the oxazoline-based crosslinking agent, and simultaneously crosslink the TOCN and Fe. ３ O ４ Particle 1 was composited to obtain 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.

[0071] Preparation Example 2: Fe ３ O ４ / TOCN (mass ratio) was 3 / 10 ３ O ４ The amount of Particle 1 added was adjusted, and the other procedures were 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.

[0072] Preparation Example 3: Fe ３ O ４ Instead of particle 1, Fe ３ O ４ Particle 2 is used, and Fe ３ O ４ / TOCN (mass ratio) was 1 / 1 ３ O ４The amount of Particle 2 added was adjusted, and the other procedures were the same as in Preparation Example 1 to obtain TOCN magnetic composite particles (TEFW-1-300) (average particle size: 2.59 μm) of Preparation Example 3.

[0073] [Preparation of Amino Group-Modified TOCN Magnetic Composite Particles] Amino groups were introduced into the TOCN magnetic composite particles of Preparation Examples 1 to 3 by reacting them with ethylenediamine 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. The TOCN magnetic composite particles were dispersed in the DMF, and then magnetic separation was performed at room temperature for 5 minutes to wash the TOCN magnetic composite particles. Magnetic separation was performed by inserting the microtube into a magnetic stand (manufactured by Tamagawa Seiki Co., Ltd.), collecting the TOCN magnetic composite particles in the dispersion on the side of the microtube, and then removing the DMF. (2) The washing procedure described in (1) above was repeated three times. (3) 3.1 mg (16 μmol) of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDAC) was dissolved in 1 mL of DMF to prepare an EDAC solution. (4) The EDAC solution was added to the magnetically separated TOCN magnetic composite particles obtained in (2) above and mixed, and then 0.5 mg (8 μmol) of ethylenediamine was added, dispersed by ultrasonic waves, and stirred by inversion at room temperature for 2 hours. (5) After inversion stirring, magnetic separation and washing were carried out using DMF in the same manner as in (2) above. As a result, as shown in the following reaction formula, the group represented by the general formula (1) above (R in the formula) １ is an ethylene group), amino group-modified TOCN magnetic composite particles were obtained.

[0074] [Preparation of Biotin-Incorporated Composite Particles] Biotin-incorporated composite particles were prepared using the amino group-modified TOCN magnetic composite particles obtained above, following the steps (1) to (3) below. (1) 2.7 mg (8 μmol) of biotin-NHS (biotin N-hydroxysuccinimideester) was dissolved in 500 μL of DMF in a 1.5 mL microtube. (2) 5 mg of amino group-modified TOCN magnetic composite particles were added to the biotin-NHS solution, dispersed ultrasonically, and mixed by inversion at room temperature for 1 hour. (3) After 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 step (1) of the preparation method for amino group-modified TOCN magnetic composite particles. As a result, biotin-incorporated composite particles were obtained, as shown in the following reaction formula.

[0075] [Avidin Detection Test] Using the biotin-introduced composite particles obtained above, an avidin detection test was performed according to the following procedures (1) to (7). (1) 2.5 mg of biotin-introduced composite particles and 1 mL of buffer were placed in a 1.5 mL microtube, and magnetic separation was performed at room temperature for 5 minutes to wash the biotin-introduced composite particles. Magnetic separation was performed by inserting the microtube into a magnet stand (manufactured by Tamagawa Seiki Co., Ltd.), collecting the biotin-introduced composite particles in the dispersion on the side of the microtube, and then discarding the buffer. The buffer used was 1 mM MgCl ２ , 0.1 mM ZnCl ２0.1M glycine-NaOH buffer (pH 10.3) containing 0.025% ovalbumin was used. (2) The washing procedure (1) above was repeated three times. (3) After washing, the biotin-incorporated composite particles were divided into 0.2 mg portions, and 200 μL of streptavidin-ALP (Promega) diluted 5,000-fold, 10,000-fold, or 20,000-fold with the above buffer or buffer was added. The mixture was then mixed by inversion using a rotator at room temperature for 1 hour. (4) After inversion, the mixture was magnetically separated at room temperature for 5 minutes, and the supernatant was completely removed. 1 mL of buffer was then added and pipetted. (5) The above procedure (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.

[0076] For comparison, amino group modification, biotin introduction, 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.), 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 operations were performed in the same manner as in (2) of [Preparation of Amino Group-Modified TOCN Magnetic Composite Particles], [Preparation of Biotin-Introduced Composite Particles], and [Avidin Detection Test].

[0077] The results are shown in FIG. 1, and avidin was detectable when the biotin-introduced composite particles according to the examples (TEFW-0.1-10, TEFW-0.3-10, TEFW-1-300) were used.

[0078] The various numerical ranges described in this specification can be arbitrarily combined with their respective upper and lower limit values, 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.

[0079] 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. Composite particles containing cellulose nanofibers and magnetic particles, wherein the cellulose nanofibers have a group represented by the following general formula (1): R in the formula １ represents a single bond or a divalent aliphatic group having 1 to 10 carbon atoms.

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

3. Composite particles containing cellulose nanofibers and magnetic particles, wherein the cellulose nanofibers have a group represented by the following general formula (1) -NH ２ and the carboxy group-containing compound bound to the cellulose nanofibers via an amide bond, which is a structure formed by dehydration condensation between the carboxy group of the carboxy group-containing compound and the carboxy group of the carboxy group-containing compound, R in the formula １ represents a single bond or a divalent aliphatic group having 1 to 10 carbon atoms.

4. The composite particle according to claim 3, wherein the carboxy group-containing compound is biotin or a protein.

5. The composite particle according to claim 4, which is used to capture or detect a target compound that specifically binds to said biotin or said protein.

6. A method for producing composite particles according to claim 1 or 2, comprising mixing composite particles comprising cellulose nanofibers having carboxy groups and magnetic particles, hydrazine or aliphatic diamine, and 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.

7. The —NH group of the group represented by general formula (1) contained in the composite particle according to claim 1 or 2 ２ a method for producing composite particles having a carboxy group-containing compound introduced therein, the method comprising reacting a carboxy group of a carboxy group-containing compound with the 8. The method for producing composite particles according to claim 7, wherein the carboxy group-containing compound is biotin or a protein.

9. A method for capturing or detecting a target compound, comprising mixing the composite particle described in claim 4 with a target compound that specifically binds to the biotin or the protein in a liquid, thereby binding the biotin or the protein to the target compound.

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