WO2025220405A1 - Composite particles and method for producing same - Google Patents

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

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.

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

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.

Japanese Patent Application Laid-Open No. 2022-30885

In the composite particles described in Patent Document 1, physiologically active substances are separated and purified by non-specifically adsorbing them to cellulose nanofibers containing carboxy 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. In order to capture or detect specific target compounds, it is necessary to introduce into the cellulose nanofibers a substance such as an antibody (protein) that specifically binds to the target compound. If it were possible to easily introduce into the cellulose nanofibers a carboxyl group-containing compound such as biotin or a protein as a substance that specifically binds to such target compounds, the convenience of the composite particles could be improved.

In view of the above, in one embodiment, the present invention aims to provide composite particles of cellulose nanofibers and magnetic particles into which a carboxy group-containing compound can be introduced, and a method for producing the same. In another embodiment, the present invention aims to provide novel composite particles in which a carboxy 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.

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

[3] Composite particles containing cellulose nanofibers and magnetic particles, the particles containing a carboxy group-containing compound bound to the cellulose nanofibers via an amide bond, which is a structure formed by dehydration condensation between _-NH2 of a group represented by the following general formula (1) possessed by the cellulose nanofibers and a carboxy group of the carboxy group-containing compound:
In the formula, R 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.

[6] A method for producing the composite particles according to [1] or [2],
Composite particles containing cellulose nanofibers having carboxy groups and magnetic particles;
hydrazine or an aliphatic diamine,
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] A method for producing composite particles having a carboxy group-containing compound introduced therein, comprising reacting the carboxy group of a carboxy group-containing compound with —NH ₂ of the group represented by the general formula (1) contained in the composite particles according to [1] or [2].
[8] The method for producing composite particles according to [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 [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.

In the above embodiment, a carboxy group-containing compound can be introduced into composite particles containing cellulose nanofibers and magnetic particles using the amino groups introduced into the cellulose nanofibers. Furthermore, novel composite particles into which a carboxy group-containing compound has been introduced can be provided, which can be used, for example, to capture or detect specific target compounds.

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

Composite particles (A) according to one embodiment contain cellulose nanofibers modified with amino groups and magnetic particles. The composite particles (A) are cellulose particles made of cellulose nanofibers combined with magnetic particles. The magnetic particles are integrated into a composite in a dispersed state with the cellulose nanofibers, and may be attached to the particle surface of the cellulose particles made of cellulose nanofibers or embedded inside.

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 Fe ₃ O ₄ and γ-Fe ₂ O _3, 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.

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.

Preferably, the cellulose nanofibers 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.

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

In other words, an aqueous dispersion of cellulose nanofibers with a solid content of 0.05 to 0.1% by mass is prepared, and this aqueous dispersion is cast onto a hydrophilically treated carbon film-coated grid to prepare a sample for observation with a transmission electron microscope (TEM). If the sample contains fibers with large diameters, a scanning electron microscope (SEM) image 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. 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 20 or more fibers intersect with this 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 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 a minimum of 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.

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

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

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 fiber length is defined as the length of the longest part of the fiber. The arithmetic mean of the fiber lengths thus obtained is calculated, and this is defined 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)

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)").

R ¹ in formula (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 number of carbon atoms in the divalent aliphatic group is more preferably 2 to 8, and even more preferably 2 to 6.

In one embodiment, 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.

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

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. These 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) it. 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). Oxidized cellulose nanofibers may also contain aldehyde or ketone groups in addition to the carboxyl groups.

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 processing a dispersion of cellulose fibers using, for example, a homomixer, high-pressure homogenizer, ultrasonic dispersion processor, beater, disk refiner, conical refiner, double-disc refiner, grinder, etc. at high speed, and a dispersion of cellulose nanofibers can be obtained.

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 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 a 0.1 mol/L aqueous hydrochloric acid solution, a 0.05 mol/L aqueous sodium hydroxide solution is added dropwise, and electrical conductivity is measured, continuing 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 group (mmol/g) = V (mL) × [0.05/mass of cellulose nanofiber (g)]

Aliphatic diamines with 1 to 10 carbon atoms are used as the aliphatic diamines to be reacted with the carboxyl group-containing cellulose nanofibers, thereby introducing functional group (1) into the cellulose nanofibers. The aliphatic diamine preferably has 2 to 8 carbon atoms, and more preferably 2 to 6 carbon atoms.

As the aliphatic diamine, saturated aliphatic diamines are preferred, alkylenediamines are more preferred, and straight-chain alkylenediamines are even more preferred. Specific examples of alkylenediamines include ethylenediamine, propylenediamine, trimethylenediamine, tetramethylenediamine, pentamethylenediamine, and hexamethylenediamine, and any of these may be used alone or in combination of two or more.

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 functional group (1) be introduced to at least the carboxy groups on the particle surface of composite particle (A). The amount of 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 cellulose nanofibers having functional group (1), similar to the amount of carboxy groups described above.

In the composite particles (A), the ratio of cellulose nanofibers having functional groups (1) 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.

The composite particles (A) are preferably micro-sized from the standpoint 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.

The composite particles (A) are formed from cellulose nanofibers and magnetic particles, and may consist solely of cellulose nanofibers and magnetic particles, but may also contain other components.

There are no particular restrictions on the method for producing the composite particles (A). For example, they can be produced by a method comprising spray-drying a dispersion containing carboxyl group-containing cellulose nanofibers and magnetic particles (Step 1), and reacting the carboxyl groups of the cellulose nanofibers contained in the composite particles obtained by spray-drying with hydrazine or an aliphatic diamine (Step 2).

In step 1, spray drying the dispersion liquid allows for the production of 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.

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).

Spray drying is a method of producing dry powder by spraying a dispersion into a gas and rapidly drying it, and can be carried out using a known spray dryer. There are no particular restrictions on the drying temperature in spray drying, and it can be, for example, 150 to 200°C.

The dispersion liquid used for spray drying may contain a crosslinking agent that reacts with carboxy groups. By including a crosslinking agent, the carboxy groups of the cellulose nanofibers react with the crosslinking agent during the spray drying process, resulting in composite particles with a crosslinked structure. This allows the composite particles to retain their shape well when redispersed in a liquid such as water, improving durability. The crosslinking agent is not particularly limited, and examples include amino resins, epoxy compounds, aziridine compounds, carbodiimide compounds, oxazoline compounds, polyisocyanate compounds, etc.

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 through 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 group (1) to be introduced more efficiently into the carboxy groups on the particle surface.

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.

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.

Examples of imidazole-based condensing agents include N,N'-carbonyldiimidazole and 1,1'-carbonyldi(1,2,4-triazole).

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.

Examples of phosphonium 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.

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, etc.

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.

These condensing agents may be used alone or in combination of two or more.

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

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 amount of carboxy groups in the cellulose nanofibers that make up the composite particles, for example. The amount of condensing agent is also not particularly limited, and 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, for example.

The composite particle (B) according to one embodiment is a composite particle containing cellulose nanofibers and magnetic particles, to which a carboxy group-containing compound has been introduced. Specifically, the composite particle (B) contains a carboxy group-containing compound bound to the cellulose nanofibers via an amide bond, which is a structure formed by dehydration condensation between the _-NH2 of the functional group (1) introduced into the composite particle (A) and the carboxy group of the carboxy group-containing compound.

Since the composite particles (A) have a primary amino group (—NH ₂ ), they 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 suitable for use as precursors for bonding a carboxyl group-containing compound such as a protein.

Preferred examples of carboxyl group-containing compounds include biotin and proteins. Examples of proteins include antibodies, peptides, protein A, protein G, protein L, streptavidin, and enzymes.

These carboxy group-containing compounds may have their carboxy groups converted into active esters, 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 include a method in which an additive such as N-hydroxysuccinimide, N,N'-disuccinimidyl carbonate, 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 described above.

There are no particular limitations on 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.

For example, if the carboxy group of the carboxy group-containing compound is not actively esterified, the carboxy group-containing compound is introduced into the composite particle (A) by mixing the composite particle (A), the carboxy group-containing compound, and the condensing agent in an organic solvent, thereby obtaining composite particle (B). If the carboxy group of the carboxy group-containing compound is actively esterified, the carboxy group-containing compound is introduced into the composite particle (A) by mixing the composite particle (A) with the actively esterified carboxy group-containing compound in an organic solvent, 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.

Composite particles (B) with biotin or protein introduced 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) with biotin introduced, examples of the target compound include avidin and streptavidin, which specifically bind to biotin. For example, in the case of composite particles (B) with an antibody introduced as the 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.

In one embodiment, a method for capturing or detecting a target compound involves mixing composite particles (B) into which biotin or protein has been introduced with a target compound that specifically binds to these particles in a liquid, thereby binding the biotin or protein to the target compound. More specifically, for example, the composite particles (B) are added to and mixed with a liquid that contains or is suspected of containing the target compound. 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 has bound can then be collected (recovered) from the liquid in which the composite particles (B) are dispersed by magnetic separation using a magnet.

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, allowing them to be easily separated 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 when washing composite particles before the amidation reaction in the method for producing composite particles (A), when separating and washing composite particles (A) from the reaction solution after the amidation reaction, when separating and washing composite particles (B) from the reaction solution after the introduction reaction in the step of introducing a carboxy group-containing compound, and when separating and washing composite particles (B) from the liquid after binding to the target compound in a method for capturing or detecting the target compound.

The composite particles according to this embodiment may be used, for example, in sandwich ELISA. Specifically, a composite particle (A) having a functional group (1) is reacted with an antibody (capture antibody) as a carboxy 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, causing the antigen to bind to the capture antibody. An enzyme-labeled antibody (detection antibody) is then added, causing the detection antibody to bind to the antigen bound to the capture antibody. A chromogenic substrate, colorimetric substrate, or luminescent substrate for the enzyme is then added, causing a reaction to develop color, color, or light, and the intensity of the color, color, or light emission is measured, allowing the target compound to be quantitatively detected.

The composite particles 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, biotin is introduced into composite particles (A) having functional group (1) by reacting them with biotin to produce composite particles (B). Next, the composite particles (B) are mixed with a liquid containing or suspected of containing enzyme-labeled avidin as the target compound, thereby binding the biotin and enzyme-labeled avidin. A color-developing substrate, color-developing substrate, or luminescent substrate for the enzyme is then added and reacted to produce color, color, or light. The intensity of the color, color, or light emission is measured, allowing the target compound to be quantitatively detected.

The following provides a more detailed explanation using examples, but the present invention is not limited to these.

[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, a sample was cast onto a hydrophilically treated 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.

(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.

(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.

(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.

(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.

<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)
_Fe3O4 particles 1: manufactured _by Toda Kogyo Co., Ltd. (average particle size: 10 nm)
_Fe3O4 particles 2: manufactured _by Toda Kogyo Co., Ltd. (average particle size: 300 nm)
- Oxazoline crosslinking agent (Epocross WS-700, manufactured by Nippon Shokubai Co., Ltd.)

<Preparation Example 1>
TOCN and the 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 with 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 the TOCN. Next, _Fe3O4 particles 1 were added to the aqueous dispersion to a concentration of 0.008 mass%, and similar ultrasonic dispersion was performed for 20 minutes to prepare an aqueous dispersion containing TOCN and _Fe3O4 particles ₁ ( _Fe3O4 /TOCN (mass ratio) = 1/10). The obtained aqueous dispersion was spray-dried using a spray dryer (Mini Spray Dryer B-290, manufactured by BUCHI Corporation) to crosslink the carboxy groups of the TOCN with the oxazoline-based crosslinking agent and simultaneously composite the TOCN with Fe ₃ O ₄ particles 1, thereby obtaining 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.

<Preparation Example 2>
The amount of Fe ₃ O ₄ particles 1 added was adjusted so that the Fe ₃ O ₄ /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.

<Preparation Example 3>
The procedure was otherwise the same as in 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, to obtain TOCN magnetic composite particles (TEFW-1-300) (average particle size: 2.59 μm) of Preparation Example 3.

[Preparation of Amino Group-Modified TOCN Magnetic Composite Particles]
The TOCN magnetic composite particles of Preparation Examples 1 to 3 were reacted with ethylenediamine to introduce amino groups 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 magnet stand (manufactured by Tamagawa Seiki Co., Ltd.), allowing the TOCN magnetic composite particles in the dispersion to collect on the side of the microtube, and then removing the DMF.
(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) 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, after which 0.5 mg (8 μmol) of ethylenediamine was added, dispersed ultrasonically, and stirred by end-over-end stirring at room temperature for 2 hours.
(5) After stirring by inversion, magnetic separation and washing using DMF was carried out in the same manner as in (2) above, thereby obtaining amino group-modified TOCN magnetic composite particles having a group represented by the general formula (1) above (where ^R1 is an ethylene group), as shown in the following reaction formula:

[Preparation of biotin-incorporated composite particles]
Using the amino group-modified TOCN magnetic composite particles obtained above, biotin-introduced composite particles were prepared according to the following procedures (1) to (3).
(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 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 method for preparing amino group-modified TOCN magnetic composite particles. As a result, composite particles with biotin incorporated were obtained, as shown in the following reaction formula.

[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 magnetic separation was performed 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 _MgCl , 0.1 mM _ZnCl , 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 5,000-fold, 10,000-fold, or 20,000-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 with a shaker at room temperature for 20 minutes.
(7) Then, the emission intensity at 460 nm was measured using a spectrophotometer.

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 procedures 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].

The results are shown in Figure 1. Avidin was detectable using the biotin-incorporated composite particles of the example (TEFW-0.1-10, TEFW-0.3-10, TEFW-1-300).

Note that the various numerical ranges described in this specification can each be combined with any upper and lower limit, and all such combinations are considered to be preferred numerical ranges and are described in this specification. Furthermore, a numerical range described as "X to Y" means between X and Y inclusive.

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 (9)

A composite particle comprising a cellulose nanofiber and a magnetic particle, wherein the cellulose nanofiber has a group represented by the following general formula (1):
In the formula, R ¹ represents a single bond or a divalent aliphatic group having 1 to 10 carbon atoms. The composite particles described in claim 1, wherein the average particle size of the magnetic particles is 1 to 500 nm. A composite particle comprising a cellulose nanofiber and a magnetic particle, the particle comprising a carboxy group-containing compound bound to the cellulose nanofiber via an amide bond, which is a structure formed by dehydration condensation between _-NH2 of a group represented by the following general formula (1) possessed by the cellulose nanofiber and a carboxy group of the carboxy group-containing compound:
In the formula, R ¹ represents a single bond or a divalent aliphatic group having 1 to 10 carbon atoms. The composite particle according to claim 3, wherein the carboxy group-containing compound is biotin or a protein. The composite particle described in claim 4, which is used to capture or detect a target compound that specifically binds to the biotin or the protein. A method for producing the composite particles according to claim 1 or 2,
Composite particles containing cellulose nanofibers having carboxy groups and magnetic particles;
hydrazine or an aliphatic diamine,
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. A method for producing composite particles having a carboxy group-containing compound introduced therein, comprising reacting a carboxy group of a carboxy group-containing compound with —NH ₂ of the group represented by general formula (1) contained in the composite particles according to claim 1 or 2. The method for producing composite particles according to claim 7, wherein the carboxy group-containing compound is biotin or a protein. A method for capturing or detecting a target compound, comprising mixing the composite particle according to 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.