Fluorescent polymer particles, hydrophobized coated body, hydrophobized metal nanocluster, and methods for producing the same

Abstract

To provide novel polymer particles internally containing fluorescent metal nanoclusters.SOLUTION: A fluorescent polymer particle according to an embodiment includes: fluorescent metal nanoclusters; a hydrophobized coated body, including a coating agent covering the metal nanoclusters, and a hydrophobic group chemically bonded to the coating agent to hydrophobize it; and polymer particles internally containing the metal nanoclusters covered with the hydrophobized coated body.SELECTED DRAWING: Figure 1

Description

[Technical Field]

[0001] The present invention relates to fluorescent polymer particles, hydrophobic coatings, hydrophobic metal nanoclusters, and methods for producing them. [Background technology]

[0002] In the field of diagnostics using immunochromatography and the like, gold colloids that are labeled with antibodies and that develop color by absorbing light of a specific wavelength are sometimes used to detect target antigens. However, due to the weak color development of gold colloids, methods using gold colloids can sometimes lack detection sensitivity.

[0003] To improve detection sensitivity in diagnostics, fluorescent particles that can be used as an alternative to gold colloids have been developed. For example, Non-Patent Document 1 describes fluorescent polystyrene particles in which fluorescent gold nanoclusters are nonspecifically adsorbed onto the surface of polystyrene particles. [Prior art documents] [Non-patent literature]

[0004] [Non-Patent Document 1] T. Wu, et al., Biosens. Bioelectron., 2022, 215, 114575 Summary of the Invention [Problem to be solved by the invention]

[0005] However, in the technology described in Non-Patent Document 1, gold nanoclusters and aptamers for fixing the gold nanoclusters coat the polystyrene particle surface by non-specific adsorption. Therefore, the fluorescent polystyrene particles described in Non-Patent Document 1 can only be used for measurement targets to which gold nanoclusters present on the surface can bind and to which the gold nanoclusters do not detach from the polystyrene particles when used. This limits the applications of fluorescent polystyrene particles.

[0006] Therefore, one aspect of the present invention aims to provide novel polymer particles containing fluorescent metal nanoclusters. [Means for solving the problem]

[0007] In order to solve the above problems, a fluorescent polymer particle according to one embodiment of the present invention comprises a fluorescent metal nanocluster, a coating agent that coats the metal nanocluster, and a hydrophobic coating body that contains hydrophobic groups that chemically bond to the coating agent to hydrophobize the coating agent, and a polymer particle that encapsulates the metal nanocluster coated with the hydrophobic coating body.

[0008] In addition, a method for producing fluorescent polymer particles according to one embodiment of the present invention includes a hydrophobization step in which a hydrophobic group is bonded to a coating agent contained in a surface-coated metal nanocluster, which is formed by bonding a fluorescent metal nanocluster to a coating agent that coats the metal nanocluster, to obtain a hydrophobized metal nanocluster; an introduction step in which polymer particles are swelled and the hydrophobized metal nanocluster is introduced into the swollen polymer particles; and a shrinkage step in which the polymer particles into which the hydrophobized metal nanocluster has been introduced are shrunk to obtain fluorescent polymer particles.

[0009] Furthermore, a hydrophobic coating according to one embodiment of the present invention includes a coating agent that bonds to a metal atom, and a monovalent hydrophobic group chemically bonded to the coating agent and represented by the following formula (I'): [ka] In formula (I'), X is P or N, Y' is a divalent group that chemically bonds to the coating agent, n is an integer of 0 to 3, m is an integer of (3-n), and n R 1 are each independently an aryl group having 6 to 20 carbon atoms which may have a substituent, and m R 2 are each independently a hydrogen atom, an alkyl group which may have a substituent, an alkenyl group which may have a substituent, a heteroaryl group which may have a substituent, or an aralkyl group which may have a substituent, and * indicates the position at which the compound is chemically bonded to the coating agent.

[0010] Furthermore, a hydrophobic metal nanocluster according to one embodiment of the present invention is formed by combining a fluorescent metal nanocluster with a hydrophobic coating according to one embodiment of the present invention, which bonds to the metal atoms that constitute the metal nanocluster and coats the metal nanocluster.

[0011] Furthermore, a method for producing a hydrophobized coated body according to one aspect of the present invention includes a hydrophobization step of contacting a coating agent that bonds to metal atoms with a hydrophobizing agent represented by the following formula (I) to obtain a hydrophobized coated body in which the coating agent and a hydrophobic group derived from the hydrophobizing agent are bonded to each other, [ka] In formula (I), X is P or N, Y is a functional group that bonds to the coating agent, n is an integer of 0 to 3, m is an integer of (3-n), and n R 1 are each independently an aryl group having 6 to 20 carbon atoms which may have a substituent, and m R 2 are each independently a hydrogen atom, an alkyl group which may have a substituent, an alkenyl group which may have a substituent, a heteroaryl group which may have a substituent, or an aralkyl group which may have a substituent.

[0012] Furthermore, a method for producing a hydrophobized metal nanocluster according to one aspect of the present invention is a method for producing a hydrophobized coated body according to one aspect of the present invention, which further includes a coating step, prior to the hydrophobization step, of coating a fluorescent metal nanocluster composed of the metal atoms with the coating agent to obtain a surface-coated metal nanocluster, and in the hydrophobization step, the hydrophobizing agent hydrophobizes the surface-coated metal nanocluster to obtain a hydrophobized metal nanocluster. [Effects of the Invention]

[0013] According to one aspect of the present invention, it is possible to provide novel polymer particles containing fluorescent metal nanoclusters therein. [Brief explanation of the drawings]

[0014] [Figure 1] 1 is a graph showing the fluorescence spectra of BSA-coated gold nanoclusters, hydrophobized BSA-coated gold nanoclusters, fluorescent polystyrene particles, and polystyrene particles of Example 1. [Figure 2] 1 shows SEM images of fluorescent polystyrene particles and polystyrene particles in Example 1. [Figure 3] 1 is a graph showing EDX spectra of fluorescent polystyrene particles and polystyrene particles of Example 1. [Figure 4] 1 is a graph showing the fluorescence spectrum of an aqueous dispersion of BSA-coated gold nanoclusters-mixed polystyrene particles obtained in Reference Example 1. DETAILED DESCRIPTION OF THE INVENTION

[0015] [Fluorescent polymer particles] An embodiment of the present invention will be described in detail below. In this specification, "A to B" means A or more and B or less, unless otherwise specified. In this specification, a new numerical range obtained by combining the upper limit value of any numerical range and the lower limit value of any numerical range among the multiple numerical ranges listed can also be interpreted as being described in this specification.

[0016] A fluorescent polymer particle according to one embodiment of the present invention comprises a fluorescent metal nanocluster, a coating agent that coats the metal nanocluster, and a hydrophobic coating body that contains hydrophobic groups that chemically bond with the coating agent to hydrophobize the coating agent, and a polymer particle that encapsulates the metal nanocluster coated with the hydrophobic coating body.

[0017] [Metal Nanoclusters] As used herein, metal nanoclusters are particles formed by the aggregation of at least one type selected from the group consisting of metal atoms and metal oxide molecules. In one embodiment of the present invention, the metal nanoclusters are not particularly limited as long as they are fluorescent metal nanoclusters. The total number of atoms and molecules constituting the metal nanocluster may be, for example, 2 to 1,000, and from the viewpoint of further improving fluorescence intensity, preferably 5 to 100, and more preferably 10 to 50. The average particle diameter of the metal nanocluster may be, for example, 0.5 nm to 10 nm.

[0018] Examples of metal atoms constituting metal nanoclusters include gold, silver, copper, platinum, palladium, iridium, and iron. Metal nanoclusters may be composed of a single type of metal atom or a combination of multiple types of metal atoms. The metal atoms can be appropriately selected depending on the desired fluorescence intensity, fluorescence wavelength, and biotoxicity depending on the application of the fluorescent polymer particles. From the viewpoint of facilitating the synthesis of metal nanoclusters with excellent fluorescence, the metal atoms are preferably at least one type selected from the group consisting of gold and silver, and more preferably gold. In other words, the metal nanoclusters are more preferably gold nanoclusters.

[0019] Metal oxide molecules that make up metal nanoclusters include silica, alumina, titanium oxide, and iron oxide.

[0020] There are no particular limitations on the fluorescent properties of metal nanoclusters. The fluorescent wavelength of a metal nanocluster can be adjusted by adjusting the types and total numbers of metal atoms and metal oxide molecules that make up the metal nanocluster.

[0021] Furthermore, from the viewpoint of further improving the fluorescence intensity, the content of the metal nanoclusters in the fluorescent polymer particles is preferably 0.1 g or more, more preferably 1 g or more, per 1 g of polymer contained in the polymer particles that constitute the fluorescent polymer particles. There is no particular upper limit to the content of the metal nanoclusters in the fluorescent polymer particles, but it may be, for example, 10 g or less per 1 g of polymer.

[0022] [Hydrophobic coating] In one aspect of the present invention, the hydrophobic coating is a compound containing a coating agent that coats the metal nanoclusters and a hydrophobic group that bonds with the coating agent to hydrophobize the coating agent, and coats the metal nanoclusters to form hydrophobic metal nanoclusters. In the fluorescent polymer particles, the hydrophobic coating is encapsulated in the polymer particles, with at least a portion of the hydrophobic coating binding to the metal nanoclusters to coat them. Note that, in this specification, coating means coating at least a portion of the surface of the metal nanoclusters, and is not limited to coating the entire surface of the metal nanoclusters.

[0023] Generally, fluorescent metal nanoclusters are formed by the reduction of metal ions in solution, resulting in the aggregation of multiple metal atoms or metal oxide molecules. However, if the reduction reaction proceeds further and atoms or molecules further aggregate around the already formed metal nanoclusters, the metal nanoclusters may grow into larger metal particles, resulting in a loss of fluorescence. Therefore, to prevent the loss of fluorescence, coating agents are sometimes used to coat the metal nanoclusters and inhibit further aggregation of atoms or molecules. Hydrophilic substances are often used as coating agents to improve the dispersibility of metal nanoclusters in aqueous solutions.

[0024] While striving to solve the problems of the present invention, the inventors discovered that the hydrophilicity of the coating agent inhibits the encapsulation of metal nanoclusters into polymer particles. As a result of extensive research, the inventors came up with the idea of ​​realizing encapsulation by making the coating agent hydrophobic by bonding a hydrophobic group to the coating agent, and completed the present invention.

[0025] In the hydrophobic coating, the bond between the coating agent and the hydrophobic group is a chemical bond or a physical bond. Examples of chemical bonds include covalent bonds, coordinate bonds, and metallic bonds. Examples of physical bonds include bonds due to electrostatic interactions, bonds due to hydrophobic interactions, hydrogen bonds, and van der Waals forces. Among these, the bond is preferably a chemical bond, and more preferably a covalent bond. A hydrophobic coating in which the coating agent and the hydrophobic group are bonded by a chemical bond, particularly a covalent bond, has better dispersibility and is easier to handle than a coating formed by a physical bond.

[0026] The hydrophobic coating itself, which includes a coating agent that binds to metal atoms and a hydrophobic group that is bound to the coating agent, is also an aspect of the present invention. According to the hydrophobic coating of one aspect of the present invention, by appropriately hydrophobizing metal nanoclusters, the metal nanoclusters can be easily encapsulated in polymer particles. Furthermore, the hydrophobic coating of one aspect of the present invention can also be used in hydrogels, in addition to fluorescent polymer particles.

[0027] Another aspect of the present invention is a hydrophobic metal nanocluster formed by bonding a metal nanocluster to a hydrophobic coating that bonds to the metal atoms constituting the metal nanocluster and coats the metal nanocluster. According to the hydrophobic metal nanocluster according to one aspect of the present invention, the hydrophobic metal nanocluster can be easily encapsulated in a polymer particle, and therefore can be easily realized as a fluorescent polymer particle. Furthermore, the hydrophobic metal nanocluster according to one aspect of the present invention can also be used in display materials in addition to fluorescent polymer particles.

[0028] [Coating agent] In one embodiment of the present invention, the coating agent is a compound that bonds to the metal atoms that constitute the metal nanocluster, and the coating agent protects the metal nanocluster from further aggregation of metal atoms or metal oxide molecules. Note that in this specification, a substance formed by bonding a coating agent to a metal nanocluster is referred to as a surface-coated metal nanocluster.

[0029] The coating agent has a functional group capable of binding to the metal atom, and can be, for example, a thiol group-containing compound. Furthermore, the coating agent is typically hydrophilic in order to maintain the dispersibility of the metal nanoclusters in the aqueous phase. Examples of coating agents include, but are not limited to, amino acids such as cysteine ​​and histidine; peptides such as glutathione; proteins such as bovine serum albumin (BSA), gelatin, and trypsin; aromatic ring-containing compounds such as 4-mercaptobenzoic acid (p-MBA), 2-mercaptobenzoic acid (m-MBA), and captopril (Capt); and fatty chain-containing compounds such as 11-mercaptoundecanoic acid (11-MUA). As used herein, a peptide refers to a compound composed of 2 to 49 amino acid molecules bound together, and a protein refers to a compound composed of 50 or more amino acid molecules bound together.

[0030] The coating agent is preferably at least one of amino acids, peptides, and proteins, from the viewpoints of high biocompatibility and easy availability, and is more preferably a protein, and even more preferably BSA, in order to enable a reduction reaction and suppress particle aggregation in a mild environment.

[0031] [Hydrophobic group] The hydrophobic group is a functional group that bonds with the coating agent to hydrophobize the coating agent, i.e., to impart hydrophobicity to the coating agent. Examples of hydrophobic groups include, but are not limited to, alkyl groups having 6 to 30 carbon atoms, heteroalkyl groups having 6 to 30 carbon atoms, aryl groups having 6 to 20 carbon atoms which may have a substituent, heteroaryl groups having 5 to 20 carbon atoms which may have a substituent, arylalkyl groups having 7 to 30 carbon atoms, and heteroarylalkyl groups having 5 to 30 carbon atoms. The larger the number of carbon atoms in the hydrophobic group, the greater the effect of hydrophobizing the coating agent, and the smaller the number of carbon atoms, the easier it is to obtain a hydrophobizing agent.

[0032] From the viewpoint of increasing the content of metal nanoclusters in the fluorescent polymer particles and providing fluorescent polymer particles with higher fluorescence intensity, the hydrophobic group is preferably a monovalent group represented by the following formula (I').

[0033] [ka]

[0034] In formula (I'), X is P or N, preferably P. Y' is a divalent group that bonds to the coating agent. Examples of Y' include, but are not limited to, alkylene groups having 1 to 5 carbon atoms and alkenyl groups having 2 to 5 carbon atoms. Among these, Y' is preferably a methylene group or an ethylene group, and more preferably a methylene group, from the viewpoint of good affinity with aqueous solvents.

[0035] In formula (I'), n R 1 R are each independently an aryl group having 6 to 20 carbon atoms which may have a substituent. 1 The aryl group is preferably a phenyl group or a naphthyl group, and more preferably a phenyl group.

[0036] In formula (I'), m R 2R are each independently a hydrogen atom, an alkyl group which may have a substituent, an alkenyl group which may have a substituent, a heteroaryl group which may have a substituent, or an aralkyl group which may have a substituent. 2 The substituents which may be substituted include a butyl group, a cyclohexyl group, an ethylene group, a propylene group, a pyridinyl group, a quinolyl group, and a phenethyl group.

[0037] In formula (I'), n is an integer of 0 or more and 3 or less, preferably an integer of 1 or more and 3 or less, and more preferably 3. Furthermore, m is an integer of (3-n). * indicates the position at which the compound is chemically bonded to the coating agent.

[0038] When the hydrophobic coating is a monovalent group represented by the above formula (I'), the metal nanoclusters coated with the hydrophobic coating (hydrophobized metal nanoclusters) have higher hydrophobicity and higher affinity for polymer particles. Therefore, a high content of metal nanoclusters is encapsulated in the polymer particles, providing fluorescent polymer particles with excellent fluorescence intensity.

[0039] [Polymer particles] The polymer particles are particles that contain a polymer as a main component and act as a substrate in the fluorescent polymer particles. The polymer particles can be any polymer particles known in the art, and are not particularly limited. In this specification, the term "main component" refers to a component that accounts for 50% by mass or more of the polymer particles.

[0040] Examples of polymers contained in the polymer particles include aromatic ring-containing polymers such as polystyrene (PS), polyethylene terephthalate (PET), and polyaniline; olefin-based polymers such as polyethylene (PE), polypropylene (PP), and polylactic acid; and heterocyclic ring-containing polymers such as polythiophene. The aromatic ring-containing polymers are composed of structural units derived from aromatic vinyl compounds. Examples of aromatic vinyl compounds include styrene, vinylnaphthalene, divinylbenzene, vinyltoluene, vinylnaphthalene, vinylanthracene, and vinylpyridine. One type of polymer may be used alone, or two or more types may be used in combination.

[0041] The polymer particles containing structural units derived from aromatic vinyl compounds may also contain structural units derived from compounds other than aromatic vinyl compounds. Examples of such other monomers include polymerizable unsaturated carboxylic acids such as itaconic acid, maleic acid, and fumaric acid; polymerizable unsaturated sulfonic acids or their salts such as sodium styrenesulfonate; polymerizable carboxylic acid esters such as methyl methacrylate, ethyl methacrylate, n-butyl methacrylate, 2-hydroxyethyl methacrylate, glycidyl methacrylate, ethylene glycol-di-methacrylic acid ester, and tribromophenyl methacrylate; unsaturated carboxylic acid amides such as methacrylonitrile, methacrylamide, N-methylolmethacrylamide, methylenebismethacrylamide, butadiene, isoprene, vinyl acetate, vinylpyridine, N-vinylpyrrolidone, vinyl chloride, vinylidene chloride, and vinyl bromide; polymerizable unsaturated nitriles; vinyl halides; conjugated dienes; etc.

[0042] The polymer particles preferably contain an aromatic ring-containing polymer, and more preferably contain polystyrene. According to this configuration, the polymer particles have higher hydrophobicity and higher affinity for hydrophobized metal nanoclusters, so that a high content of metal nanoclusters is encapsulated in the polymer particles, providing fluorescent polymer particles with excellent fluorescence intensity. This effect is particularly pronounced when the hydrophobic group is a monovalent group represented by formula (I'), where n is an integer of 1 or greater. This is because π-π interactions occur between the aromatic rings contained in the polymer particles and the aromatic rings contained in the hydrophobic group, and the aromatic rings contained in the polymer and the P contained in the hydrophobic group are easily fused together. + or N + This is because a cation-π interaction occurs between the polymer particles and the hydrophobic metal nanoclusters, further enhancing the affinity between the polymer particles and the hydrophobic metal nanoclusters. In addition, polymer particles containing polystyrene are preferred because they are inexpensive, spherical particles can be easily produced, and they are easy to handle.

[0043] Here, polystyrene is a polymer containing styrene and / or a styrene derivative as a structural unit. Examples of styrene derivatives include alkylstyrenes such as methylstyrene, ethylstyrene, propylstyrene, t-butylstyrene, cyclohexylstyrene, dodecylstyrene, and dimethylstyrene; halogenated styrenes such as monochlorostyrene, dichlorostyrene, monobromostyrene, and dibromostyrene; alkoxystyrenes such as methoxystyrene, ethoxystyrene, propoxystyrene, and butoxystyrene; carboxystyrenes such as carboxystyrene; aminostyrenes such as 4-aminostyrene, dimethylaminostyrene, and diethylaminostyrene; hydroxystyrenes; and isomers thereof.

[0044] The polymer particles may further contain additives. Examples of additives include crosslinkers and surfactants. For example, the polymer contained in the polymer particles may be crosslinked with a crosslinker. By crosslinking the polymer, the swelling degree of the polymer particles can be controlled, and any amount of hydrophobic metal nanoclusters can be encapsulated in the polymer particles. Examples of crosslinkers include divinylbenzene. Examples of surfactants include sodium lauryl sulfate.

[0045] The polymer particles may also have functional groups on their surfaces. The functional groups can be appropriately selected depending on the intended use of the fluorescent polymer particles, and examples thereof include hydrophilic groups. When the fluorescent polymer particles are used for diagnostic purposes, the functional groups are preferably at least one hydrophilic group selected from the group consisting of sulfo, carboxy, hydroxy, and amino groups, in order to enhance compatibility with biological samples.

[0046] The number-average particle diameter of the polymer particles is not particularly limited, but from the viewpoint of ease of handling, it is preferably 10 nm or more, more preferably 50 nm or more, and even more preferably 100 nm or more. Furthermore, from the viewpoint of reducing optical scattering by the fluorescent polymer particles, the number-average particle diameter of the polymer particles is preferably 10,000 nm or less, more preferably 5,000 nm or less, and even more preferably 500 nm or less. In this specification, the number-average particle diameter is measured by dynamic light scattering (DLS).

[0047] [Other ingredients] The fluorescent polymer particles may contain other components in addition to the metal nanoclusters, the hydrophobic coating, and the polymer particles. The other components may be encapsulated in the polymer particles or may be present on the surface of the polymer particles. Examples of the other components include, but are not limited to, surfactants. The other components may be selected appropriately depending on the application of the fluorescent polymer particles.

[0048] [Characteristics of fluorescent polymer particles] The fluorescent properties of the fluorescent polymer particles are not particularly limited. For example, when excited with excitation light having a wavelength of 200 nm to 1000 nm, the intensity peak in the fluorescence spectrum may be within a wavelength range of 200 nm to 2000 nm. In order to provide fluorescent polymer particles having a fluorescence wavelength that allows easy identification of the fluorescence originating from the fluorescent polymer particles in a biological sample, the intensity peak is preferably within a wavelength range of 400 nm to 1500 nm.

[0049] There may be a wavelength difference between the intensity peak in the fluorescence spectrum of the fluorescent polymer particles and the intensity peak in the fluorescence spectrum of the hydrophobicized metal nanoclusters used as the raw material for the fluorescent polymer particles. This is because the interaction between the polymer and the hydrophobic groups of the hydrophobicized metal nanoclusters in the fluorescent polymer particles may affect the quantum size effect of the metal nanoclusters, thereby changing the wavelength of the fluorescence emitted by the metal nanoclusters. That is, in one embodiment of the present invention, the fluorescence wavelength of the resulting fluorescent polymer particles can be adjusted by changing either or both the hydrophobic groups and the type of polymer particles. For example, multicolor imaging may be achieved by varying the fluorescence spectrum of the fluorescent polymer particles. The wavelength difference between the intensity peaks of the fluorescent polymer particles and the hydrophobicized metal nanoclusters is preferably 0 nm to 500 nm, more preferably 0 nm to 250 nm.

[0050] From the viewpoint of ease of handling, the number-average particle diameter of the fluorescent polymer particles is preferably 10 nm or more, more preferably 50 nm or more, and even more preferably 100 nm or more, and from the viewpoint of reducing optical scattering by the fluorescent polymer particles, the number-average particle diameter of the fluorescent polymer particles is preferably 10,000 nm or less, more preferably 5,000 nm or less, and even more preferably 1,000 nm or less.

[0051] From the viewpoint of improving the dispersion stability of the fluorescent polymer particles, the zeta potential of the fluorescent polymer particle surface is preferably 10 mV or more, more preferably 20 mV or more. Alternatively, from the viewpoint of improving the dispersion stability of the fluorescent polymer particles, the zeta potential of the fluorescent polymer particle surface is preferably -10 mV or less, more preferably -20 mV or less. In this specification, the zeta potential is measured by electrophoresis.

[0052] [Method for producing fluorescent polymer particles] Other aspects of the present invention will be described below, and for the sake of convenience, the description of members having the same functions as those described above will not be repeated.

[0053] A method for producing a fluorescent polymer according to one embodiment of the present invention includes a hydrophobization step in which hydrophobic groups are bonded to the coating agent contained in a surface-coated metal nanocluster, which is formed by bonding a fluorescent metal nanocluster to a coating agent that coats the metal nanocluster, thereby obtaining a hydrophobized metal nanocluster; an introduction step in which polymer particles are swelled and hydrophobized metal nanoclusters are introduced into the swollen polymer particles; and a shrinkage step in which the polymer particles into which the hydrophobized metal nanoclusters have been introduced are shrunk to obtain fluorescent polymer particles.

[0054] [Coating process] The method for producing a fluorescent polymer may further include a coating step before the hydrophobizing step, in which fluorescent metal nanoclusters composed of metal atoms are coated with a coating agent to obtain surface-coated metal nanoclusters.

[0055] The coating step can be carried out by a conventionally known method. For example, the metal nanoclusters and the coating agent may be mixed together, and the metal nanoclusters may be coated with the coating agent. As another example, the coating agent may be mixed with a solution containing metal ions, and the reaction of forming metal nanoclusters by reducing the metal ions and the reaction of coating the formed metal nanoclusters with the coating agent may be carried out substantially simultaneously.

[0056] [Hydrophobizing step] The hydrophobization process is a process of obtaining a hydrophobized metal nanocluster by bonding a hydrophobic group to the coating agent contained in the surface-coated metal nanocluster, which is formed by bonding a metal nanocluster to a coating agent. Here, the surface-coated metal nanocluster may be commercially available, or may be obtained in the coating process if a coating process has been performed. Furthermore, the bond between the surface-coated metal nanocluster and the hydrophobic group is a chemical bond or a physical bond, and a chemical bond is preferred. The hydrophobization process can be carried out, for example, by mixing a hydrophobizing agent, which serves as a source of hydrophobic groups, with the surface-coated metal nanocluster in a solution and reacting them.

[0057] The hydrophobizing agent can be any compound that reacts with and bonds to a functional group, such as an amino group, possessed by the coating agent to form a hydrophobic group. Among these, a hydrophobizing agent represented by the following formula (I) is preferred. The hydrophobizing agent represented by the following formula (I) is a source of the hydrophobic group represented by the above-mentioned formula (I'). The hydrophobizing agent may be used in the form of a salt with an anion. Examples of the anion include chloride ions, bromide ions, and iodide ions.

[0058] [ka]

[0059] In formula (I), Y is a functional group that bonds with the coating agent. Y may be a group that yields Y' as described above for formula (I') by reaction, and may be a functional group represented as Y'-Z. Here, Z is a functional group that is eliminated by reaction with the coating agent, and examples thereof include, but are not limited to, a formyl group, a carboxy group, an amino group, and a thiol group.

[0060] In formula (I), X, R 1 , R 2 , n, and m may each have the same structure or value as in formula (I'). Alternatively, in formula (I), X, R 1 , R 2, n, and m may have a structure or value that can result in the same structure or value as formula (I') through additional reaction. In other words, in one embodiment of the present invention, the hydrophobizing agent represented by formula (I) may produce the hydrophobic group represented by formula (I') in a one-step reaction by reacting with a coating agent, or may produce the hydrophobic group in a multi-step reaction by reacting with a coating agent and then further reacting with other additives, etc. Using the hydrophobizing agent represented by formula (I), it is possible to obtain a hydrophobized metal nanocluster having a hydrophobic group represented by formula (I').

[0061] The hydrophobizing agent represented by formula (I) has good solubility in aqueous solvents such as water at room temperature, and thus its use allows the reaction (hydrophobization reaction) between the hydrophobizing agent and the surface-coated metal nanoclusters to occur in a homogeneous reaction system. Furthermore, the hydrophobizing agent has good reactivity with coating agents selected from amino acids, peptides, and proteins, and therefore can effectively hydrophobize surface-coated metal clusters having surface components derived from these coating agents. Furthermore, the hydrophobizing agent represented by formula (I) has good reactivity at room temperature, thereby reducing the denaturation of the coating agent.

[0062] An aqueous solvent can be used as the solvent. Without being limited thereto, when the coating agent is at least one of amino acids, peptides, and proteins, the use of an aqueous solvent as the solvent can reduce denaturation of the surface components of the surface-coated metal clusters derived from the coating agent. For example, in the hydrophobization step, a hydrophobizing agent dissolved in an aqueous solvent can be brought into contact with the surface-coated metal nanoclusters dispersed in the aqueous solvent to obtain hydrophobized metal nanoclusters. Examples of aqueous solvents include water, buffer solutions, and any mixtures thereof.

[0063] In the hydrophobization step, the amount of surface-coated metal nanoclusters added to the solvent may be, but is not limited to, a final concentration of 0.1 mM or more and 10 mM or less. From the viewpoint of improving reaction efficiency, the amount added is preferably 0.5 mM or more, and more preferably 1.0 mM or more. Furthermore, from the viewpoint of improving the dispersibility of the surface-coated metal nanoclusters, the amount added is preferably 5.0 mM or less, and more preferably 2.5 mM or less.

[0064] In the hydrophobization step, the amount of hydrophobizing agent added to the solvent may be, but is not limited to, a final concentration of 1.0 mM or more and 100 mM or less. From the viewpoint of improving the rate of introduction of hydrophobic groups, the amount added is preferably 10 mM or more, more preferably 25 mM or more. Furthermore, from the viewpoint of improving the dispersibility of the surface-coated metal nanoclusters, the amount added is preferably 75 mM or less, more preferably 50 mM or less, from the viewpoint of improving the dispersibility of the surface-coated metal nanoclusters.

[0065] In the hydrophobization step, the reaction temperature may be, but is not limited to, 0°C or higher and 100°C or lower. For example, the reaction temperature may be room temperature (20°C to 40°C) from the viewpoint of reducing denaturation of the surface components derived from the coating agent of the surface-coated metal nanoclusters. In the hydrophobization step, the reaction time may be, but is not limited to, 0.1 hours or higher and 2.0 hours or lower.

[0066] [Introduction process] The introduction step is a step of swelling polymer particles and introducing hydrophobic metal nanoclusters into the swollen polymer particles. For example, swelling of the polymer particles can be achieved by contacting the polymer particles with a solvent that swells the polymer particles, i.e., a swelling solvent, but this is not limiting. Furthermore, introduction of the hydrophobic metal nanoclusters can be achieved, for example, by dispersing the hydrophobic metal nanoclusters in the swelling solvent beforehand when swelling the polymer particles. In such an embodiment, the hydrophobic metal nanoclusters are incorporated into the polymer particles together with the solvent, and are thereby encapsulated within the polymer particles. As another example, introduction of the hydrophobic metal nanoclusters can be achieved by ultrasonic disruption.

[0067] Examples of swelling solvents include halogenated solvents such as chloroform and dichloromethane; and aromatic hydrocarbon solvents such as toluene. Among these, the swelling solvent is preferably a halogenated solvent, and more preferably chloroform, from the viewpoint of its high ability to swell polymer particles. When a halogenated solvent is used, the introduction step can be carried out, for example, by contacting hydrophobic metal nanoclusters dispersed in the halogenated solvent with polymer particles dispersed in an aqueous solvent. The swelling solvent may be selected depending on the type of polymer contained in the polymer particles.

[0068] In the introduction step, the polymer particles may be dispersed in an aqueous solvent before contacting with the swelling solvent. Here, the aqueous solvent that can be used in the hydrophobization step can also be used in the introduction step. In such an embodiment, the introduction step can be carried out by mixing the swelling solvent in which the hydrophobized metal nanoclusters are dispersed with the aqueous solvent in which the polymer particles are dispersed.

[0069] In the introduction step, the temperature at which the polymer particles are contacted with the hydrophobic metal nanoclusters dispersed in the swelling solvent may be from 0° C. to 50° C., or may be room temperature, and the contact time may be, but is not limited to, from 0.1 hours to 2.0 hours.

[0070] [Shrinkage process] The shrinking step is a step of shrinking the polymer particles into which the hydrophobic metal nanoclusters have been introduced to obtain fluorescent polymer particles. By shrinking the polymer particles, leakage of the hydrophobic metal nanoclusters from the polymer particles is reduced, and fluorescent polymer particles whose fluorescence intensity is maintained for a long period of time can be obtained.

[0071] The shrinking step can be carried out by replacing the solvent in which the polymer particles are dispersed with a non-swelling solvent. The non-swelling solvent can be appropriately selected depending on the type of polymer contained in the polymer particles, for example, an aqueous solvent. Here, as the aqueous solvent, one that can be used in the hydrophobization step can also be used in the shrinking step. When the solution in which the polymer particles are dispersed is a two-phase system, for example, a two-phase system of a halogen-based solvent phase and an aqueous solvent phase, and the polymer particles are dispersed in only one phase, the shrinking step can be carried out by removing both phases together and replacing them with a new aqueous solvent.

[0072] [Modification process] The method for producing fluorescent polymer particles may further include a modification step. The modification step is a step of modifying the surface of the polymer particles with hydrophilic groups. The modification step can be carried out by mixing the polymer particles with a modifier that provides hydrophilic groups such as sulfo groups, carboxy groups, hydroxy groups, or amino groups. The modification step may be carried out (1) before the introduction step, (2) after the introduction step and before the shrinkage step, or (3) after the shrinkage step.

[0073] [Method for producing hydrophobic coated body] A method for producing a hydrophobized coated body according to one embodiment of the present invention includes a hydrophobization step of contacting a coating agent that bonds to metal atoms with a hydrophobizing agent represented by formula (I) to obtain a hydrophobized coated body in which hydrophobic groups derived from the coating agent are bonded to the coating agent. The hydrophobization step included in the method for producing a hydrophobized coated body can be performed by using a coating agent instead of the surface-coated metal nanoclusters in the hydrophobization step described above for the method for producing fluorescent polymer particles.

[0074] [Method for producing hydrophobic metal nanoclusters] In the method for producing a hydrophobized coated body described above, a coating step is performed before the hydrophobization step in which fluorescent metal nanoclusters composed of metal atoms are coated with a coating agent to obtain surface-coated metal nanoclusters, and in the hydrophobization step, the hydrophobized metal nanoclusters are obtained by hydrophobizing the surface-coated metal nanoclusters with the hydrophobizing agent. In other words, the method for producing hydrophobized metal nanoclusters is also an aspect of the present invention. The coating step included in the method for producing hydrophobized metal nanoclusters can be performed in the same manner as the coating step described above for the method for producing fluorescent polymer particles.

[0075] 〔summary〕 The fluorescent polymer particles according to aspect 1 of the present invention comprise fluorescent metal nanoclusters, a coating agent that coats the metal nanoclusters, and a hydrophobic coating body that contains hydrophobic groups that chemically bond with the coating agent to hydrophobize the coating agent, and polymer particles that encapsulate the metal nanoclusters coated with the hydrophobic coating body.

[0076] The fluorescent polymer particles according to Aspect 2 of the present invention are the same as those of Aspect 1, wherein the hydrophobic group is a monovalent group represented by the following formula (I'): [ka] In formula (I'), X is P or N, Y' is a divalent group that chemically bonds to the coating agent, n is an integer of 0 to 3, m is an integer of (3-n), and n R 1are each independently an aryl group having 6 to 20 carbon atoms which may have a substituent, and m R 2 are each independently a hydrogen atom, an alkyl group which may have a substituent, an alkenyl group which may have a substituent, a heteroaryl group which may have a substituent, or an aralkyl group which may have a substituent, and * indicates the position at which the compound is chemically bonded to the coating agent.

[0077] The fluorescent polymer particles according to aspect 3 of the present invention are the same as those of aspect 2 above, characterized in that in formula (I'), n is an integer of 1 or more, and the polymer particles contain an aromatic ring-containing polymer.

[0078] The fluorescent polymer particles according to a fourth aspect of the present invention are characterized in that, in the third aspect described above, the aromatic ring-containing polymer contains polystyrene.

[0079] A fluorescent polymer particle according to a fifth aspect of the present invention is characterized in that, in any one of the first to fourth aspects described above, the metal nanoclusters are gold nanoclusters.

[0080] A fluorescent polymer particle according to a sixth aspect of the present invention is characterized in that, in any one of the first to fifth aspects described above, the coating agent is at least one of an amino acid, a peptide, and a protein.

[0081] A fluorescent polymer particle according to a seventh aspect of the present invention is characterized in that, in any one of the first to sixth aspects described above, the coating agent is bovine serum albumin (BSA).

[0082] The fluorescent polymer particles according to aspect 8 of the present invention are characterized in that, in any one of aspects 1 to 7 described above, the polymer particles have at least one group selected from the group consisting of a sulfo group, a carboxy group, a hydroxy group, and an amino group on their surface.

[0083] A method for producing fluorescent polymer particles according to aspect 9 of the present invention includes a hydrophobization step of bonding a hydrophobic group to a coating agent contained in a surface-coated metal nanocluster, which is formed by bonding a fluorescent metal nanocluster with a coating agent that coats the metal nanocluster, to obtain a hydrophobized metal nanocluster; an introduction step of swelling polymer particles and introducing the hydrophobized metal nanocluster into the swollen polymer particles; and a shrinkage step of shrinking the polymer particles into which the hydrophobized metal nanocluster has been introduced, to obtain fluorescent polymer particles.

[0084] A tenth aspect of the present invention relates to the method for producing fluorescent polymer particles of the ninth aspect, and in the hydrophobizing step, the surface-coated metal nanoclusters are brought into contact with a hydrophobizing agent represented by the following formula (I) to obtain the hydrophobized metal nanoclusters containing the hydrophobic groups derived from the hydrophobizing agent: [ka] (In formula (I), X is P or N, Y is a functional group that bonds to the coating agent, n is an integer of 0 or more and 3 or less, m is an integer of (3-n), and n R 1 are each independently an aryl group having 6 to 20 carbon atoms which may have a substituent, and m R 2 are each independently a hydrogen atom, an optionally substituted alkyl group, an optionally substituted alkenyl group, an optionally substituted heteroaryl group, or an optionally substituted aralkyl group.

[0085] A method for producing fluorescent polymer particles according to aspect 11 of the present invention is characterized in that, in the above-mentioned aspect 10, the coating agent is at least one of amino acids, peptides, and proteins, and in the hydrophobization step, the hydrophobizing agent dissolved in an aqueous solvent is brought into contact with the surface-coated metal nanoclusters dispersed in the aqueous solvent to obtain the hydrophobized metal nanoclusters.

[0086] A method for producing fluorescent polymer particles according to aspect 12 of the present invention is characterized in that, in any one of aspects 9 to 11 described above, the introduction step is a step of bringing the hydrophobic metal nanoclusters dispersed in a halogen-based solvent into contact with the polymer particles dispersed in an aqueous solvent to swell the polymer particles, and introducing the hydrophobic metal nanoclusters into the swollen polymer particles.

[0087] A method for producing fluorescent polymer particles according to aspect 13 of the present invention is characterized in that, in any one of aspects 9 to 12 described above, in the shrinking step, the solvent in which the polymer particles are dispersed is replaced with an aqueous solvent.

[0088] A hydrophobic coating according to a fourteenth aspect of the present invention comprises a coating agent that bonds to a metal atom, and a monovalent hydrophobic group represented by the following formula (I') that is chemically bonded to the coating agent: [ka] In formula (I'), X is P or N, Y' is a divalent group that chemically bonds to the coating agent, n is an integer of 0 to 3, m is an integer of (3-n), and n R 1 are each independently an aryl group having 6 to 20 carbon atoms which may have a substituent, and m R 2 are each independently a hydrogen atom, an alkyl group which may have a substituent, an alkenyl group which may have a substituent, a heteroaryl group which may have a substituent, or an aralkyl group which may have a substituent, and * indicates the position at which the compound is chemically bonded to the coating agent.

[0089] The hydrophobic metal nanocluster according to aspect 15 of the present invention is formed by combining a fluorescent metal nanocluster with the hydrophobic coating according to aspect 14 described above, which bonds to the metal atoms constituting the metal nanocluster and coats the metal nanocluster.

[0090] A method for producing a hydrophobized coated body according to a sixteenth aspect of the present invention includes a hydrophobization step of contacting a coating agent that bonds to metal atoms with a hydrophobizing agent represented by the following formula (I) to obtain a hydrophobized coated body in which the coating agent and a hydrophobic group derived from the hydrophobizing agent are bonded to each other: [ka] In formula (I), X is P or N, and Y is a functional group that bonds to the coating agent; n is an integer of 0 to 3, m is an integer of (3-n), and n R 1 are each independently an aryl group having 6 to 20 carbon atoms which may have a substituent, and m R 2 are each independently a hydrogen atom, an alkyl group which may have a substituent, an alkenyl group which may have a substituent, a heteroaryl group which may have a substituent, or an aralkyl group which may have a substituent.

[0091] A method for producing a hydrophobized metal nanocluster according to aspect 17 of the present invention is a method for producing a hydrophobized coated body according to aspect 16 described above, which further includes a coating step, prior to the hydrophobization step, of coating a fluorescent metal nanocluster composed of the metal atoms with the coating agent to obtain a surface-coated metal nanocluster, and in the hydrophobization step, the hydrophobizing agent hydrophobizes the surface-coated metal nanocluster to obtain a hydrophobized metal nanocluster.

[0092] [Additional Notes] The present invention is not limited to the above-described embodiments, and various modifications are possible within the scope of the claims. Embodiments obtained by appropriately combining the technical means disclosed in different embodiments are also included in the technical scope of the present invention. [Example]

[0093] An example of the present invention will be described below. In this example, hydrophobized BSA-coated gold nanoclusters were prepared and then introduced into polystyrene particles to produce fluorescent polystyrene particles containing gold nanoclusters.

[0094] Example 1 In Example 1, Ph3P was used as a hydrophobizing agent. + CH2CHO·Cl - Fluorescent polystyrene particles were prepared using the method described above. [Hydrophobizing step] (Preparation of BSA-coated gold nanoclusters) An aqueous solution was prepared with final concentrations of 0.36 mM BSA, 4.77 mM chloroauric acid (HAuCl), and 47.6 mM sodium hydroxide (NaOH), and then stirred overnight at 37 °C to obtain an aqueous dispersion of BSA-coated gold nanoclusters (Au-NCs).

[0095] (Preparation of hydrophobic BSA-coated gold nanoclusters) 1 mL of the BSA-coated gold nanocluster aqueous dispersion (gold atom concentration 3.8 mM) was added to a screw tube. Next, 50 mg of (formylmethyl)triphenylphosphonium chloride (Ph3P) was added as a hydrophobicity agent. + CH2CHO·Cl - (Tokyo Chemical Industry Co., Ltd.) was dissolved in 2 mL of ultrapure water. The BSA-coated gold nanocluster aqueous dispersion in the screw tube was stirred with a magnetic stirrer (HERACLES-16G, Koike Precision Machinery Works, Ltd.) while adding Ph3P + CH2CHO·Cl - The aqueous solution was slowly added and stirred for 1 hour at room temperature. While stirring the resulting reaction solution, 1 mL of a chloroform (Fujifilm Wako Pure Chemical Industries) solution containing 23.54 mg of 2-picoline borane (Tokyo Chemical Industry Co., Ltd.) was slowly added dropwise, and the mixture was stirred for 1 hour at room temperature to extract the chloroform layer. This extraction procedure resulted in the migration of a colored substance into the chloroform layer of the two-layer solution consisting of an aqueous layer and a chloroform layer, confirming the production of a chloroform dispersion of hydrophobic BSA-coated gold nanoclusters.

[0096] [Introduction process] Polystyrene particles (average particle diameter: 284 nm) were used as polymer particles. 25 mg of Pluronic® F-127 (Sigma-Aldrich) was added to 1 mL of a polystyrene particle aqueous dispersion (particle concentration: 12.6 mg / mL). Next, 0.1 mL of a hydrophobic BSA-coated gold nanocluster chloroform dispersion was added, and the mixture was stirred at room temperature for 1.5 hours using a shaker (MT-400, Tommy Medico Corporation) to introduce the hydrophobic gold nanoclusters into the polystyrene particles.

[0097] [Shrinkage process] The solution obtained in the introduction step was washed three times (3000 g, 5 min, 4°C) using a centrifuge (MDX-110, manufactured by Tomy Kogyo Co., Ltd.) to remove the chloroform, and the residue was redispersed in 1 mL of ultrapure water, causing the polystyrene particles to shrink and yielding fluorescent polystyrene particles.

[0098] Comparative Example 1 0.5 mL of a BSA-coated gold nanocluster aqueous dispersion (gold atom concentration 4.77 mM) prepared as in Example 1 was added to a screw tube, followed by 1 mL of ultrapure water. Next, 54.67 mg of hexadecyltrimethylammonium bromide (Fujifilm Wako Pure Chemical Industries, Ltd.) and 1.5 mL of methanol (Fujifilm Wako Pure Chemical Industries, Ltd.) were added sequentially to the screw tube. While stirring the BSA-coated gold nanocluster aqueous dispersion on a magnetic stirrer (HERACLES-16G, Koike Precision Machinery Manufacturing Co., Ltd.), the hexadecyltrimethylammonium bromide solution was slowly added and stirred for 5 minutes at room temperature. 0.5 mL of chloroform (Fujifilm Wako Pure Chemical Industries, Ltd.) was added dropwise to the resulting solution, which was then stirred for 5 minutes at room temperature, and the solution state was visually inspected.

[0099] The resulting solution was brown and cloudy throughout, and no migration of the BSA-coated gold nanoclusters into the chloroform layer was observed. These results indicate that the BSA-coated gold nanoclusters were not hydrophobicized. Therefore, in Comparative Example 1, the subsequent introduction step was not performed.

[0100] Comparative Example 2 0.5 mL of a BSA-coated gold nanocluster aqueous dispersion (gold atom concentration 4.77 mM) prepared as in Example 1 was added to a screw tube, followed by 1 mL of ultrapure water. Next, 25 mg of dodecylamine (Fujifilm Wako Pure Chemical Industries, Ltd.) and 1.5 mL of methanol (Fujifilm Wako Pure Chemical Industries, Ltd.) were added sequentially to the screw tube. While stirring the BSA-coated gold nanocluster aqueous dispersion on a magnetic stirrer (HERACLES-16G, Koike Precision Machinery Works, Ltd.), the dodecylamine solution was slowly added and stirred at room temperature for 1 hour. 0.5 mL of chloroform (Fujifilm Wako Pure Chemical Industries, Ltd.) was added dropwise to the resulting solution, which was then stirred at room temperature for 5 minutes, after which the solution state was visually inspected.

[0101] The resulting solution was cloudy throughout, and no migration of the BSA-coated gold nanoclusters into the chloroform layer was observed. These results indicate that the BSA-coated gold nanoclusters were not hydrophobized. Therefore, in Comparative Example 2, the subsequent introduction step was not performed.

[0102] [Fluorescence spectrum measurement] BSA-coated gold nanoclusters, hydrophobized BSA-coated gold nanoclusters, and fluorescent polystyrene particles obtained in Example 1, as well as the fluorescence spectrum of the polystyrene particles used in Example 1, and the NanoDrop spectrofluorometer TM Measurements were performed using a 3300 (Thermo Fisher Scientific) (excitation wavelength: 365 nm). The fluorescence spectrum measurement results are shown in Figure 1.

[0103] As shown in Figure 1, the hydrophobic BSA-coated gold nanoclusters exhibited a fluorescence spectrum with an intensity peak at 611 nm, demonstrating that fluorescent hydrophobic coatings were obtained. Furthermore, in the hydrophobic BSA-coated gold nanoclusters, the amino groups of BSA and Ph3P + CH2CHO·Cl -The acetyl groups of the BSA-coated gold nanoclusters are chemically bonded to the BSA-coated gold nanoclusters. This chemical bond is thought to have improved the dispersibility of the BSA-coated gold nanoclusters compared to bonds formed through electrostatic interactions, resulting in the production of the BSA-coated gold nanoclusters.

[0104] Furthermore, as shown in Figure 1, the fluorescent polystyrene particles of Example 1 exhibited a fluorescence spectrum with two intensity peaks at 475 nm and 519 nm, demonstrating that fluorescent polymer particles were obtained. The intensity peak of the fluorescent polystyrene particles was shifted by approximately 100 nm from the intensity peak of 611 nm exhibited by the hydrophobic BSA-coated gold nanoclusters before the introduction. This large shift is due to the difference between the phenyl groups in the polystyrene and the Ph3P in the hydrophobic BSA-coated gold nanoclusters. + This is thought to be due to interactions with the CH2 groups. This suggests that by appropriately selecting the hydrophobic groups in the hydrophobicized metal nanoclusters and the polymers in the polymer particles, it is possible to impart variation to the fluorescence spectra of the resulting fluorescent polymer particles.

[0105] [Observation of particle shape] The shapes of the fluorescent polystyrene particles obtained in Example 1 and the polystyrene particles used in Example 1 were observed using an electron microscope. SEM images were taken using an electron microscope JSM-7800F Prime (JEOL) under conditions of an accelerating voltage of 10.0 kV, a working distance of 10.0 mm, and a magnification of 10,000 times. The taken SEM images are shown in FIG. 2.

[0106] As shown in Figure 2, the fluorescent polystyrene particles had a spherical shape similar to that of polystyrene particles. This indicates that the introduction of hydrophobic metal nanoclusters into polymer particles does not affect the shape of the resulting fluorescent polymer particles, and therefore polystyrene particles with a shape similar to that of the polymer particles are obtained. Also, as shown in Figure 2, the polystyrene particles had a particle diameter of approximately 0.3 μm, while larger particles with a particle diameter of approximately 2 μm were present among the fluorescent polystyrene particles. This is thought to be due to the fact that the polystyrene chains that make up the polystyrene particles loosened and dissolved during the introduction process, causing multiple polystyrene particles to fuse together.

[0107] [Zeta potential measurement] The zeta potential of the fluorescent polystyrene particles obtained in Example 1 and the polystyrene particles used in Example 1 was measured. The measurements were performed using a Zeta Potential / Particle Size / Molecular Weight Measurement System ELSZ-2000 (Otsuka Electronics Co., Ltd.). The measurement results showed that the zeta potential of the fluorescent polystyrene particles was -32.59 mV, and the zeta potential of the polystyrene particles was -35.33 mV. The fact that both values ​​were similar suggested that the hydrophobicized metal nanoclusters, which have a positive charge due to the phosphonium cation of the hydrophobic group, were not adsorbed onto the surface of the polymer particles, but were encapsulated within the polymer particles.

[0108] [Elemental analysis using a transmission electron microscope] Elemental analysis of the fluorescent polystyrene particles obtained in Example 1 and the polystyrene particles used in Example 1 was performed using a transmission electron microscope (JEM-F200, manufactured by JEOL Ltd.). The measured EDX (Energy Dispersive X-ray Spectroscopy) spectrum is shown in Figure 3. The phosphorus atoms contained in the hydrophobizing agent were detected only in the spectrum of the fluorescent polystyrene particles (near 2 keV). The above results are thought to indicate that hydrophobized BSA-coated gold nanoclusters having phosphorus atoms in the hydrophobic groups were encapsulated within the polystyrene particles by the introduction process.

[0109] [Reference example 1] To verify that the hydrophobic metal nanoclusters are introduced into the polymer particles through swelling of the polymer particles during the introduction process, we conducted an experiment in which polymer particles and hydrophobic metal nanoclusters were mixed without swelling. Specifically, 25 mg of Pluronic® F-127 (Sigma-Aldrich) was added to 1 mL of an aqueous dispersion of polystyrene particles (particle concentration: 12.6 mg / mL). Next, 0.1 mL of an aqueous dispersion of BSA-coated gold nanoclusters (gold atom concentration: 1.6 mM) prepared as in Example 1 was added and stirred at room temperature for 1.5 hours using a shaker (MT-400, Tommy Medico Co., Ltd.). The resulting solution was washed three times (3000 g, 5 min, 4°C) using a centrifuge (MDX-110, Tommy Kogyo Co., Ltd.) and redispersed in 1 mL of ultrapure water.

[0110] The resulting aqueous dispersion of BSA-coated gold nanoclusters mixed with polystyrene particles was irradiated with a 365 nm UV lamp (Handy UV Lamp SLUV-6, AS ONE Corporation) and visually observed; no fluorescence was observed. The fluorescence spectrum for 365 nm UV was then measured using a microplate reader (Infinite® M200 PRO, Tecan Group Ltd.). The resulting fluorescence spectrum is shown in Figure 4. As shown in Figure 4, no fluorescence was observed in the visible light range. These results indicate that simply mixing BSA-coated gold nanoclusters and polystyrene particles without swelling results in the nanoclusters not being encapsulated within the particles but instead nonspecifically adsorbing to the surface and being removed by washing, resulting in the failure to produce fluorescent polystyrene particles. [Industrial Applicability]

[0111] The present invention can be used in the field of diagnosis using immunochromatography and the like.

Claims

1. A fluorescent metal nanocluster; a hydrophobized coating body including a coating agent that coats the metal nanoclusters and a hydrophobic group that chemically bonds with the coating agent to hydrophobize the coating agent; polymer particles encapsulating the metal nanoclusters and coated with the hydrophobic coating; having Fluorescent polymer particles.

2. The hydrophobic group is a monovalent group represented by the following formula (I'): 【Chemistry 1】 In formula (I'), X is P or N; Y' is a divalent group that chemically bonds to the coating agent, n is an integer of 0 to 3, m is an integer of (3-n), n R １ each independently represents an aryl group having 6 to 20 carbon atoms which may have a substituent, m R ２ each independently represents a hydrogen atom, an optionally substituted alkyl group, an optionally substituted alkenyl group, an optionally substituted heteroaryl group, or an optionally substituted aralkyl group, * indicates the position where the coating agent is chemically bonded. Fluorescent polymer particles according to claim 1 .

3. In the formula (I′), n is an integer of 1 or more, The polymer particles include an aromatic ring-containing polymer. Fluorescent polymer particles according to claim 2.

4. The aromatic ring-containing polymer includes polystyrene. Fluorescent polymer particles according to claim 3.

5. The metal nanoclusters are gold nanoclusters. Fluorescent polymer particles according to any one of claims 1 to 4.

6. The coating agent is at least one of an amino acid, a peptide, and a protein. Fluorescent polymer particles according to any one of claims 1 to 4.

7. The coating agent is bovine serum albumin (BSA). Fluorescent polymer particles according to any one of claims 1 to 4.

8. The polymer particles have at least one group selected from the group consisting of a sulfo group, a carboxy group, a hydroxy group, and an amino group on their surfaces. Fluorescent polymer particles according to any one of claims 1 to 4.

9. a hydrophobization step of bonding a hydrophobic group to the coating agent contained in the surface-coated metal nanocluster, which is formed by bonding a fluorescent metal nanocluster and a coating agent that coats the metal nanocluster, to obtain a hydrophobized metal nanocluster; an incorporation step of swelling polymer particles and incorporation of the hydrophobized metal nanoclusters into the swollen polymer particles; a shrinking step of shrinking the polymer particles having the hydrophobized metal nanoclusters introduced therein to obtain fluorescent polymer particles; Including, A method for producing fluorescent polymer particles.

10. In the hydrophobization step, the surface-coated metal nanocluster is contacted with a hydrophobizing agent represented by the following formula (I) to obtain the hydrophobized metal nanocluster containing the hydrophobic group derived from the hydrophobizing agent: 【Chemistry 2】 (In formula (I), X is P or N; Y is a functional group that bonds with the coating agent; n is an integer of 0 to 3, m is an integer of (3-n), n R １ each independently represents an aryl group having 6 to 20 carbon atoms which may have a substituent, m R ２ are each independently a hydrogen atom, an alkyl group which may have a substituent, an alkenyl group which may have a substituent, a heteroaryl group which may have a substituent, or an aralkyl group which may have a substituent. A method for producing the fluorescent polymer particles according to claim 9.

11. the coating agent is at least one of an amino acid, a peptide, and a protein; In the hydrophobization step, the hydrophobizing agent dissolved in an aqueous solvent is brought into contact with the surface-coated metal nanoclusters dispersed in the aqueous solvent to obtain the hydrophobized metal nanoclusters. A method for producing the fluorescent polymer particles according to claim 10.

12. The introduction step is a step of bringing the hydrophobized metal nanoclusters dispersed in a halogen-based solvent into contact with the polymer particles dispersed in an aqueous solvent to swell the polymer particles, and introducing the hydrophobized metal nanoclusters into the swollen polymer particles. The method for producing the fluorescent polymer particles according to any one of claims 9 to 11.

13. In the shrinking step, the solvent in which the polymer particles are dispersed is replaced with an aqueous solvent. The method for producing the fluorescent polymer particles according to any one of claims 9 to 11.

14. A coating agent that binds to metal atoms; and a monovalent hydrophobic group represented by the following formula (I') chemically bonded to the coating agent, 【Chemistry 3】 In formula (I'), X is P or N; Y' is a divalent group that chemically bonds to the coating agent, n is an integer of 0 to 3, m is an integer of (3-n), n R １ each independently represents an aryl group having 6 to 20 carbon atoms which may have a substituent, m R ２ each independently represents a hydrogen atom, an optionally substituted alkyl group, an optionally substituted alkenyl group, an optionally substituted heteroaryl group, or an optionally substituted aralkyl group, * indicates the position where the coating agent is chemically bonded. Hydrophobized coating.

15. A fluorescent metal nanocluster is bonded to the hydrophobic coating according to claim 14, which bonds to metal atoms constituting the metal nanocluster and coats the metal nanocluster. Hydrophobized metal nanoclusters.

16. The method includes a hydrophobization step of contacting a coating agent that bonds to metal atoms with a hydrophobizing agent represented by the following formula (I) to obtain a hydrophobized coating body in which the coating agent and a hydrophobizing agent are bonded to each other, 【Chemistry 4】 In formula (I), X is P or N; Y is a functional group that bonds with the coating agent; n is an integer of 0 to 3, m is an integer of (3-n), n R １ each independently represents an aryl group having 6 to 20 carbon atoms which may have a substituent, m R ２ each independently represents a hydrogen atom, an alkyl group which may have a substituent, an alkenyl group which may have a substituent, a heteroaryl group which may have a substituent, or an aralkyl group which may have a substituent; A method for producing a hydrophobic coating.

17. The method for producing a hydrophobic coating according to claim 16, The method further includes a coating step, prior to the hydrophobization step, of coating the fluorescent metal nanoclusters composed of the metal atoms with the coating agent to obtain surface-coated metal nanoclusters; In the hydrophobizing step, the hydrophobizing agent hydrophobizes the surface-coated metal nanoclusters to obtain hydrophobized metal nanoclusters. A method for producing hydrophobic metal nanoclusters.

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