Fluorescent particles and method for producing fluorescent particles

Abstract

The purpose of one aspect of the present invention is to provide fluorescent particles having excellent dispersibility. One aspect of the present invention relates to a fluorescent particle (100) having: a core particle (10A) with a first charge, the first charge being a positive charge or a negative charge; at least one polymer layer (12) and at least one fluorescent layer (11A, 11B) on the core particle, the polymer layer containing a polymer and carrying a second charge, the second charge being a positive charge or a negative charge; and a fluorescent layer containing metal nanoclusters (13) having fluorescent properties and carrying a third charge, the sign of which is opposite to that of the second charge.

Description

Technical Field

[0001] This invention relates to fluorescent particles and methods for manufacturing fluorescent particles. Background Technology

[0002] In the field of diagnostics utilizing techniques such as immunochromatography, colloidal gold labeled with antibodies and colored by absorbing light of a specific wavelength is sometimes used to detect target antigens. However, because the color development of colloidal gold is relatively weak, methods using colloidal gold suffer from insufficient detection sensitivity.

[0003] To improve detection sensitivity in diagnostics, fluorescent particles that can be used as alternatives to colloidal gold have been developed. As such a technology, for example, Non-Patent Document 1 describes a fluorescent silica particle formed by loading fluorescent gold nanoclusters onto porous silica particles via amide bonds.

[0004] Existing technical documents

[0005] Non-patent literature Non-patent literature 1: Y. Yuan, et al., Cell Prolif, 2021, 54, e13008. Summary of the Invention

[0006] The problem that the invention aims to solve However, in the technology described in Non-Patent Document 1, the active carboxyl groups on the proteins coated with gold nanoclusters not only form amide bonds with the amino groups on the surface of porous silica particles, but also form amide bonds with the amino groups on the surrounding protein molecules, so the resulting fluorescent silica particles are prone to aggregation.

[0007] One aspect of the present invention is to provide a fluorescent particle with excellent dispersibility.

[0008] Methods for solving problems To address the aforementioned issues, one aspect of the present invention relates to a fluorescent particle having: a core particle carrying a first charge, which is either positive or negative; at least one polymer layer on the core particle and at least one fluorescent layer, the polymer layer comprising a polymer and carrying a second charge, which is either positive or negative; the fluorescent layer comprising fluorescent metal nanoclusters and carrying a third charge, which is opposite in sign to the second charge.

[0009] Additionally, one aspect of the present invention relates to a method for manufacturing fluorescent particles, comprising: at least one polymer layer forming step, wherein the polymer layer comprising a polymer and having a second charge on a core particle having a first charge, the first charge being positive or negative, and the second charge being positive or negative; and at least one fluorescent layer forming step, wherein the fluorescent layer comprising fluorescent metal nanoclusters and having a third charge on the core particle, the third charge being opposite in sign to the second charge.

[0010] The effects of the invention According to one aspect of the present invention, fluorescent particles with excellent dispersibility can be provided. Attached Figure Description

[0011] Figure 1 This is a schematic cross-sectional view illustrating fluorescent particles according to one embodiment of the present invention.

[0012] Figure 2 This is a schematic cross-sectional view illustrating fluorescent particles according to another embodiment of the present invention.

[0013] Figure 3 This is a graph showing the fluorescence spectra of the particles in Examples 1 and 2 and Reference Examples 1 and 2.

[0014] Figure 4 These are SEM images of the particles in Examples 1 and 2 and Reference Examples 1 and 2.

[0015] Figure 5 It is a graph showing the particle size distribution of the particles in Examples 1 and 2 and Reference Examples 1 and 2.

[0016] Figure 6 This is a graph representing the Zeta potential of the particles in Examples 1 and 2 and Reference Examples 1 and 2.

[0017] Figure 7 This is a graph showing the fluorescence spectrum of the fluorescent particles in Example 3.

[0018] Figure 8 This is a SEM image of the fluorescent particles in Example 3.

[0019] Figure 9 This is a graph showing the fluorescence spectrum of the fluorescent particles in Example 5.

[0020] Figure 10 This is a SEM image of the fluorescent particles from Example 5. Detailed Implementation

[0021] [Fluorescent particles] The following is a detailed description of one embodiment of the present invention. In this specification, unless otherwise specified, "A~B" means above A and below B. In this specification, a new numerical range obtained by combining the upper limit of any of the listed numerical ranges with the lower limit of another numerical range can also be interpreted as being described herein.

[0022] One aspect of the present invention relates to a fluorescent particle having: a core particle carrying a first charge, the first charge being positive or negative; at least one polymer layer on the core particle and at least one fluorescent layer, the polymer layer comprising a polymer and carrying a second charge, the second charge being positive or negative; the fluorescent layer comprising fluorescent metal nanoclusters and carrying a third charge, the third charge being opposite in sign to the second charge.

[0023] [Core Particle] In one aspect of the invention, the core particle can be any inorganic particle, organic particle, or organic-inorganic composite particle, but is not limited thereto. Examples of inorganic particles include: metal oxide particles such as silicon dioxide particles and titanium dioxide particles; metal particles such as gold particles, silver particles, and platinum particles; and alloy particles composed of multiple metals. Examples of organic particles include: polymer particles such as polystyrene particles and polymethacrylic acid particles; and semiconductor particles.

[0024] From the viewpoint of making it easier to load the fluorescent layer and polymer layer onto the core particles, the core particles are preferably selected from metal oxide particles, metal particles, and polymer particles, and more preferably selected from silica particles, polymer particles, and metal particles. Furthermore, from the viewpoint of reducing wavelength shifts in the fluorescence wavelengths of the metal nanoclusters and ensuring that the resulting fluorescent particles also exhibit the original fluorescence wavelengths of the metal nanoclusters, the core particles are more preferably silica particles.

[0025] The core particle carries a first charge, which can be positive or negative. The surface of the core particle may have cationic or anionic functional groups through which it acquires the first charge. Alternatively, the core particle may be implanted with cations, anions, or electrons, through which it acquires the first charge.

[0026] Generally, if the surface of the core particle has cationic functional groups, the first charge is positive; if the surface of the core particle has anionic functional groups, the first charge is negative. Examples of cationic functional groups include primary amine (i.e., amino), secondary amine, tertiary amine, and quaternary amine (i.e., ammonium cations); and phosphonium (i.e., phosphorus cations). Examples of anionic functional groups include carboxyl and sulfonyl groups. From the viewpoint of stably loading the fluorescent layer onto the core particle, the surface of the core particle preferably has functional groups selected from amine and phosphonium groups.

[0027] The average particle size of the core particles can be appropriately selected based on the intended use of the fluorescent particles, and there are no particular restrictions. For example, it can be 100 nm or larger and 100 μm or smaller, 300 nm or larger and 50 μm or smaller, or 500 nm or larger and 10 μm or smaller. Furthermore, in this specification, the average particle size refers to the average particle size calculated using SEM images.

[0028] [Polymer layer] The polymer layer contains a polymer, for example, it may contain a polymer as a main component. Furthermore, in this specification, "main component" refers to the component that constitutes 50% or more by mass in the polymer layer.

[0029] Examples of polymers included in the polymer layer include cationic polymers and anionic polymers. Examples of cationic polymers include amino-containing polymers such as chitosan and polylysine; and quaternary ammonium-containing polymers such as sulfobetaine polymers. Examples of anionic polymers include sulfonated polymers such as polystyrene sulfonic acid; and carboxyl-containing polymers such as alginate.

[0030] In addition to classifying polymers based on ionicity as described above, polymers can also be classified according to their main structural backbone. Examples of polymers include: polysaccharide polymers such as chitosan and alginate; olefin polymers such as polyacrylic acid and polystyrene sulfonic acid; and proteins such as gelatin and polylysine. Furthermore, regarding polylysine, it is not limited to polylysine composed of 50 or more lysine molecules and classified as a protein; polylysine composed of 49 or fewer lysine molecules and therefore classified as a peptide in this specification can also be used as a polymer.

[0031] From the viewpoint of improving the coating rate of the polymer layer on the core particles and ensuring the stable loading of the polymer layer onto the core particles through entanglement between polymer chains, the weight-average molecular weight of the polymer is preferably 1,000 or more, more preferably 5,000 or more. Furthermore, from the viewpoint of further improving the dispersibility of the fluorescent particles, the weight-average molecular weight of the polymer is preferably 1,000,000 or less, more preferably 500,000 or less. In this specification, the weight-average molecular weight of the polymer is measured by gel permeation chromatography.

[0032] Regarding the polymer contained in the polymer layer, from the viewpoint of reducing the wavelength shift that occurs in the fluorescence wavelength from the metal nanoclusters, it is preferable that the polymer does not contain an aromatic ring in the repeating unit of the polymer, more preferably at least one selected from chitosan and polylysine, and even more preferably chitosan.

[0033] The polymer layer may also contain additives such as counterions that are opposite to the anions or cations of the polymer contained in the polymer layer.

[0034] The polymer layer carries a second charge, which can be positive or negative. The second charge is determined based on the type of polymer contained in the polymer layer. Generally, if the polymer layer contains a cationic polymer, the second charge is positive; if the polymer layer contains anionic polymers, the second charge is negative.

[0035] [Fluorescent layer] The fluorescent layer comprises fluorescent metal nanoclusters. In this specification, a metal nanocluster is a particle formed by the aggregation of at least one selected from metal atoms and metal oxide molecules. In one aspect of the invention, the metal nanoclusters are not particularly limited as long as they are fluorescent. Regarding the total number of atoms and molecules constituting the metal nanoclusters, it can be, for example, 2 or more and 1000 or less; from the viewpoint of further improving fluorescence intensity, it is preferably 5 or more and 100 or less, more preferably 10 or more and 50 or less. As for the average particle size of the metal nanoclusters, it can be, for example, 0.5 nm or more and 10 nm or less.

[0036] Examples of metal atoms constituting metal nanoclusters include gold, silver, copper, platinum, palladium, iridium, and iron. Metal nanoclusters can be composed of a single metal atom or a combination of multiple metal atoms. The metal atoms can be appropriately selected based on the fluorescence intensity, fluorescence wavelength, and biotoxicity required for the intended use of the fluorescent particles. From the viewpoint of easily synthesizing metal nanoclusters with excellent fluorescence properties, the metal atom is preferably selected from at least one noble metal, more preferably from at least one selected from gold and silver. In other words, metal nanoclusters are more preferably nanoclusters containing gold, silver, or both.

[0037] Examples of metal oxide molecules that constitute metal nanoclusters include silicon dioxide, aluminum oxide, titanium oxide, and iron oxide.

[0038] In one aspect of the invention, the metal nanoclusters are preferably coated with a coating agent. The coating agent is a compound that binds to the metal atoms constituting the metal nanoclusters. The coating agent protects the metal nanoclusters from further aggregation of metal atoms, thereby reducing excessive growth of the metal nanoclusters. Additionally, the coating agent sometimes also functions to impart a third charge to the metal nanoclusters; metal nanoclusters coated with such an agent are suitable for stacked structures formed using electrostatic interactions.

[0039] The coating agent has functional groups that can bind to metal atoms, such as thiol-containing compounds. Furthermore, from the viewpoint of maintaining dispersibility in the aqueous phase while simultaneously coating metal nanoclusters, the coating agent is typically hydrophilic. Examples of coating agents include: amino acids such as cysteine ​​and histidine; peptides such as glutathione; proteins such as bovine serum albumin (BSA), gelatin, and trypsin; aromatic ring compounds such as 4-mercaptobenzoic acid (p-MBA), 2-mercaptobenzoic acid (m-MBA), and captopril; and fatty chain compounds such as 11-mercaptoundecanoic acid (11-MUA), but are not limited to these. In this specification, a peptide refers to a compound composed of 2 to 49 amino acid molecules, while a protein refers to a compound composed of 50 or more amino acid molecules.

[0040] From the viewpoint of high biocompatibility, easy availability, and excellent effect of imparting negative charge as a third charge to metal nanoclusters, the coating agent is preferably at least one of amino acids, peptides, and proteins. Among these, in order to enable the reduction reaction to proceed under mild conditions and inhibit particle aggregation, the coating agent is more preferably a protein or peptide, and even more preferably BSA or glutathione.

[0041] The fluorescent layer carries a third charge, which has the opposite sign to the second charge. Here, the third charge only needs to have the opposite sign to the second charge; there is no particular restriction on the absolute value of the third charge.

[0042] [Stacked structure of fluorescent particles] Fluorescent particles can have a stacked structure, formed by stacking at least one polymer layer and at least one fluorescent layer on a core particle. Here, the polymer layer and fluorescent layer are preferably arranged on the core particle such that adjacent layers have opposite charges to the particle. For example, when the charge of the core particle (first charge) has the same sign as the charge of the polymer layer (second charge), a fluorescent layer with a third charge, which has the opposite sign to both the first and second charges, is directly disposed on the core particle, and a polymer layer is disposed on the side of this fluorescent layer facing away from the core particle. In other words, when the first charge has the same sign as the second charge, the fluorescent particle preferably has at least one fluorescent layer between the core particle and the polymer layer.

[0043] The fluorescent particles can have one or more fluorescent layers, but from the viewpoint of further improving fluorescence intensity, two or more are preferred. When there are two or more fluorescent layers, a polymer layer can be placed between the two fluorescent layers to alternately stack the polymer layers and the fluorescent layers.

[0044] When there are two or more fluorescent layers, the composition of the metal nanoclusters contained in each fluorescent layer can be the same or different. For example, the fluorescence properties of the entire fluorescent particle can be adjusted by making the fluorescent layers contain metal nanoclusters with different compositions for each fluorescent layer.

[0045] The number of polymer layers and fluorescent layers in fluorescent particles can be the same or different. Here, by appropriately setting the number of fluorescent layers in the fluorescent particles, any fluorescence intensity can be obtained. For example, from the viewpoint of further improving fluorescence intensity, at least two fluorescent layers can be used.

[0046] The fluorescent particle can consist of any one of the polymer layers and the outermost fluorescent layer. For example, the fluorescent particle may have at least one fluorescent layer outside the outermost layer of the polymer layers. According to this stacking structure, the fluorescence intensity of the fluorescent particle is further enhanced because it is the outermost layer among all polymer layers and fluorescent layers. Alternatively, the fluorescent particle may have at least one polymer layer outside the outermost layer of the fluorescent layers. According to this stacking structure, the fluorescent particles repel each other due to a second charge because they are the outermost layer among all polymer layers and fluorescent layers, thus further improving the dispersion of the fluorescent particles.

[0047] In addition to the polymer layer and fluorescent layer mentioned above, fluorescent particles may also have other layers. Examples of these other layers include a base layer and a protective layer. There are no particular restrictions on the arrangement of these other layers in the stacked structure of the fluorescent particles; they can be placed inside or outside all polymer and fluorescent layers, or between one polymer layer and one fluorescent layer. For example, a base layer can be placed inside all polymer and fluorescent layers (i.e., on the core particle side). Another example is that a protective layer can be placed outside all polymer and fluorescent layers (i.e., on the opposite side of the core particle side).

[0048] Furthermore, regarding the polymer layer, fluorescent layer, and other layers (if present) of the fluorescent particles, it is preferable that each layer directly or indirectly coats the entire circumference of the core particle, but this is not limited to this; as long as it coats at least a portion of the core particle, it is acceptable. The higher the coating rate (the ratio of the surface area covered by each layer to the total surface area of ​​the core particle) of each layer on the core particle, the stronger the dispersion of the fluorescent particles due to the polymer layer and the fluorescence of the fluorescent particles due to the fluorescent layer. The coating rate of each layer can be, for example, 10% or more, 20% or more, 30% or more, 40% or more, 50% or more, 60% or more, 70% or more, 80% or more, 90% or more, or 100%, preferably 100%. In one aspect of the invention, in the method for manufacturing fluorescent particles, the coating rate can be controlled by changing the amounts of the polymer and metal nanoclusters added to the solution, and by changing the contact time between the core particle and the solution.

[0049] [Characteristics of fluorescent particles] There are no particular limitations on the fluorescence properties of fluorescent particles. For example, when excited by excitation light in the wavelength range of 200 nm to 1000 nm, the intensity peak in the fluorescence spectrum can be in the wavelength range of 400 nm to 2000 nm. In order to provide fluorescent particles whose fluorescence wavelength is at a wavelength that makes it easy to identify fluorescence from fluorescent particles in biological samples, this intensity peak is preferably in the wavelength range of 600 nm to 1500 nm.

[0050] From the viewpoint of improving the processability of fluorescent particles, the average particle size of the fluorescent particles is preferably 100 nm or more, more preferably 200 nm or more, and even more preferably 300 nm or more. Furthermore, from the viewpoint of reducing optical scattering caused by fluorescent particles, the average particle size of the fluorescent particles is preferably 100 μm or less, more preferably 50 μm or less, and even more preferably 1000 nm or less.

[0051] One aspect of the present invention relates to fluorescent particles that exhibit excellent dispersibility. Here, dispersibility can be evaluated by observing SEM images, etc., and can also be expressed as the ratio of the average particle size of the fluorescent particles to the average particle size of the core particles. It can be considered that the lower this ratio, the closer the average particle size of the fluorescent particles is to that of the core particles used as raw materials, and the less aggregation of the core particles occurs during the manufacturing process of the fluorescent particles. The ratio of the average particle size of the fluorescent particles to the average particle size of the core particles is preferably 2.0 or less, more preferably 1.5 or less, and even more preferably 1.25 or less.

[0052] The shape of the fluorescent particles is not particularly limited; for example, they can be spherical, flat, or rod-shaped. However, from a processability point of view, the shape of the fluorescent particles is preferably spherical, and more preferably perfectly spherical. The shape of the fluorescent particles can be adjusted by appropriately selecting the shape of the core particles used in their manufacture; for example, spherical fluorescent particles can be manufactured by using spherical core particles.

[0053] From the viewpoint of further improving the dispersion stability of fluorescent particles, the zeta potential on the surface of the fluorescent particles is preferably 10 mV or more, more preferably 20 mV or more. Alternatively, from the viewpoint of further improving the dispersion stability of fluorescent particles, the zeta potential on the surface of the fluorescent particles is preferably -10 mV or less, more preferably -20 mV or less. In this specification, the zeta potential is measured by electrophoresis.

[0054] [Fluorescent particles involved in one embodiment] Below, refer to Figure 1 The fluorescent particles involved in one embodiment of the present invention will be described. Figure 1This is a schematic cross-sectional view illustrating a fluorescent particle 100 according to one embodiment of the present invention. The fluorescent particle 100 comprises: a positively charged core particle 10A, a negatively charged fluorescent layer 11A, a positively charged polymer layer 12, and a negatively charged fluorescent layer 11B; the fluorescent layer 11A, polymer layer 12, and fluorescent layer 11B are sequentially stacked on the core particle 10A. In this embodiment, the core particle 10A is a silica particle, which carries a positive charge due to the amino groups present on its surface. The fluorescent layers 11A and 11B are each composed of fluorescent metal nanoclusters 13. The metal nanoclusters 13 are coated with BSA, thereby carrying a negative charge. The polymer layer 12 contains chitosan, which carries a positive charge due to the amino groups present in the chitosan. According to this embodiment, a fluorescent particle 100 possessing both fluorescence and dispersibility is provided. Furthermore, the fluorescent particle 100 exhibits stronger fluorescence intensity because it is the outermost fluorescent layer 11B. In addition, the silica particles, which are the core particles 10A, do not easily cause the fluorescence wavelength of the metal nanoclusters 13 to shift. Therefore, the fluorescent particles 100 can directly display the fluorescence wavelength of the metal nanoclusters 13.

[0055] Reference Figure 2 The fluorescent particles involved in another embodiment of the present invention will be described. Figure 2 This is a schematic cross-sectional view illustrating a fluorescent particle 101 according to one embodiment of the present invention. The fluorescent particle 101 comprises: a negatively charged core particle 10B, a positively charged polymer layer 12 sequentially stacked on the core particle 10B, and a negatively charged fluorescent layer 11. In this embodiment, the core particle 10B is a silica particle, which carries a negative charge due to carboxyl groups present on its surface. The fluorescent layer 11 is composed of fluorescent metal nanoclusters 13.

[0056] according to Figure 1 and Figure 2 The fluorescent particles 100 and 101 illustrated respectively can be understood to mean that, in one aspect of the present invention, the stacking order of the core particles, polymer layer and fluorescent layer can be appropriately selected according to the charge of each component.

[0057] [Methods for manufacturing fluorescent particles] Other aspects of the present invention will now be described. Furthermore, for ease of explanation, components having the same function as those described above will not be described again.

[0058] One aspect of the present invention relates to a method for manufacturing fluorescent particles, comprising: at least one polymer layer forming step, wherein the polymer layer comprising a polymer and having a second charge on a core particle having a first charge, the first charge being positive or negative, and the second charge being positive or negative; and at least one fluorescent layer forming step, wherein the fluorescent layer comprising fluorescent metal nanoclusters and having a third charge on the core particle, the third charge being opposite in sign to the second charge.

[0059] [Electrified process] The method for manufacturing fluorescent particles may also include a charging process, which involves obtaining core particles through charging treatment prior to the polymer layer formation process and the fluorescent layer formation process. For example, the charging process can be performed by feeding uncharged core particle raw materials to a charging treatment. Examples of charging treatment include chemical treatment that modifies the surface of the raw material using cationic or anionic functional groups, and physical treatment that involves feeding the raw material to an electron beam irradiation method or an ion implantation method, but are not limited to these.

[0060] [Polymer layer formation process] The polymer layer formation process is a process of forming at least one polymer layer on a core particle. For example, the polymer layer formation process can be performed by directly or indirectly contacting the core particle with a solution containing a polymer. Here, at least one layer, such as a fluorescent layer and other layers, can be pre-formed on the core particle. In this case, the polymer layer formation process can be performed by indirectly contacting the core particle with the polymer solution by contacting that layer. To achieve contact, methods known in the art, such as spraying, coating, and dipping, can be used.

[0061] The solvent for the polymer solution can be appropriately selected depending on the type of polymer; for example, water can be used. The polymer concentration in the polymer solution can be 1 mg / L or more, preferably 10 mg / L or more. Alternatively, the polymer concentration in the polymer solution can be 100 g / L or less, preferably 10 g / L or less.

[0062] In the polymer layer formation process, the amount of polymer used (the amount of polymer contained in the polymer solution) relative to 1 g of core particles used can be 1 mg or more, preferably 10 mg or more. The amount of polymer used relative to 1 g of core particles used can be 100 mg or less, preferably 50 mg or less.

[0063] In the polymer layer formation process, the contact time between the core particles and the polymer solution can be, for example, 0.5 hours or more and 10 hours or less, or 1 hour or more and 5 hours or less. Furthermore, the contact temperature is preferably 0°C or more and 100°C or less, more preferably 20°C or more and 50°C or less. Based on this preferred contact temperature, denaturation of the coating agent (such as protein) covering the metal nanoclusters can be prevented, thus preventing changes in the fluorescence properties and particle size distribution of the metal nanoclusters due to denaturation.

[0064] [Fluorescent layer formation process] The fluorescent layer formation process is the process of forming at least one fluorescent layer on the core particles. For example, the fluorescent layer formation process can be performed by directly or indirectly contacting the core particles with a dispersion solution of metal nanoclusters. Here, at least one layer, such as a polymer layer or other layers, can be pre-formed on the core particles. In this case, the fluorescent layer formation process can be performed by indirectly contacting the core particles with the dispersion solution through contact with that layer. To achieve contact, methods known in the art, such as spraying, coating, and dipping, can be used.

[0065] Water can be used as a solvent for the dispersion solution. The concentration of metal nanoclusters in the dispersion solution can be 0.25 g / L or more, preferably 0.5 g / L or more. Alternatively, the concentration of metal nanoclusters in the dispersion solution can be 5 g / L or less, preferably 1 g / L or less.

[0066] In the fluorescent layer formation process, the amount of metal nanoclusters used (the amount of metal nanoclusters contained in the dispersion solution) relative to 1 g of core particles can be 1 mg or more, preferably 5 mg or more. The amount of metal nanoclusters used relative to 1 g of core particles can be 50 mg or less, preferably 25 mg or less.

[0067] In the fluorescent layer formation process, the contact time between the core particles and the dispersion solution can be, for example, 0.5 hours or more and 10 hours or less, or 1 hour or more and 5 hours or less. Furthermore, the contact temperature is preferably 0°C or more and 100°C or less, more preferably 20°C or more and 50°C or less. Based on this preferred contact temperature, denaturation of the coating agent (such as protein) covering the metal nanoclusters can be prevented, thus preventing changes in the fluorescence properties and particle size distribution of the metal nanoclusters due to denaturation.

[0068] [A set of processes] Regarding the manufacturing method of fluorescent particles, it is sufficient to include at least one polymer layer formation step and one fluorescent layer formation step, and fluorescent particles can be obtained by performing each of these steps at least once. Alternatively, the manufacturing method may include any one of the above steps or two of each of the above steps. For example, it may include a step of forming one polymer layer formation step and one fluorescent layer formation step as a group of steps, and repeating this group of steps at least twice. Here, in this step of repeating at least twice, the polymer layer formation step and the fluorescent layer formation step are performed alternately. According to this configuration, a layer-by-layer (LBL) self-assembly method can be used, which utilizes the electrostatic interaction between the second charge and the third charge, to obtain fluorescent particles composed of two or more polymer layers and fluorescent layers stacked alternately.

[0069] In methods for manufacturing fluorescent particles, the polymer layer formation step and the fluorescent layer formation step are usually performed alternately, but there is no particular restriction on which step is performed first. The step can be selected based on the charge of the core particle. For example, when the charge of the core particle (first charge) is the same as the charge of the polymer layer (second charge), the manufacturing method can include the fluorescent layer formation step first, that is, include at least one fluorescent layer formation step before the polymer layer formation step.

[0070] The method for manufacturing fluorescent particles may also include other layer formation processes. These other layer formation processes can be performed using methods known in the art, depending on the type of the other layers and the required configuration of those other layers.

[0071] 〔Summarize〕 A first aspect of the present invention relates to a fluorescent particle having: a core particle carrying a first charge, the first charge being positive or negative; at least one polymer layer on the core particle and at least one fluorescent layer, the polymer layer comprising a polymer and carrying a second charge, the second charge being positive or negative; the fluorescent layer comprising fluorescent metal nanoclusters and carrying a third charge, the third charge being opposite in sign to the second charge.

[0072] According to the first aspect above, a second aspect of the present invention relates to a fluorescent particle, characterized in that at least one fluorescent layer is provided on the outermost side of the polymer layer.

[0073] According to the first aspect above, a third aspect of the present invention relates to a fluorescent particle, characterized in that at least one polymer layer is located on the outermost side of the fluorescent layer.

[0074] According to any one of the first to third aspects above, a fourth aspect of the present invention relates to a fluorescent particle, characterized in that the first charge has the same sign as the second charge, and at least one fluorescent layer is present between the core particle and the polymer layer.

[0075] According to any one of the first to fourth aspects above, a fifth aspect of the present invention relates to a fluorescent particle, characterized in that the first charge is a positive charge, and the surface of the core particle has functional groups selected from amino and phosphonium groups.

[0076] According to any one of the first to fifth aspects above, a sixth aspect of the present invention relates to a fluorescent particle, characterized in that the core particle is selected from silica particles, polymer particles and metal particles.

[0077] According to any one of the first to sixth aspects above, the seventh aspect of the present invention relates to a fluorescent particle, characterized in that the average particle size of the fluorescent particle is 100 nm or more and 100 μm or less.

[0078] According to any one of the first to seventh aspects above, the eighth aspect of the present invention relates to a fluorescent particle, characterized in that the polymer comprises chitosan.

[0079] According to any one of aspects 1 to 8 above, a ninth aspect of the present invention relates to a fluorescent particle, characterized in that the metal nanoclusters are coated with a coating agent, said coating agent being at least one of amino acids, peptides and proteins.

[0080] A tenth aspect of the present invention relates to a method for manufacturing fluorescent particles, comprising: at least one polymer layer forming step, wherein the polymer layer comprising a polymer and having a second charge on a core particle having a first charge, the first charge being positive or negative, and the second charge being positive or negative; and at least one fluorescent layer forming step, wherein the fluorescent layer comprising fluorescent metal nanoclusters and having a third charge on the core particle, the third charge being opposite in sign to the second charge.

[0081] According to the 10th aspect above, the 11th aspect of the present invention relates to a method for manufacturing fluorescent particles, characterized in that the first charge has the same sign as the second charge, and at least one fluorescent layer formation step is included before the polymer layer formation step.

[0082] According to the 10th or 11th aspect above, the 12th aspect of the present invention relates to a method for manufacturing fluorescent particles, characterized in that it further includes a charging step, which obtains the core particles by charging treatment before the polymer layer formation step and the fluorescent layer formation step.

[0083] According to any one of aspects 10 to 12 above, the 13th aspect of the present invention relates to a method for manufacturing fluorescent particles, characterized in that the polymer layer formation step and the fluorescent layer formation step are performed at a temperature above 0°C and below 100°C.

[0084] [Notes] This invention is not limited to the embodiments described above. Various modifications can be made within the scope of the claims. Embodiments obtained by appropriately combining the technical means disclosed in different embodiments are also included within the technical scope of this invention.

[0085] Example The following describes one embodiment of the present invention. In this embodiment, fluorescent silica particles are prepared using the LBL method.

[0086] [Reference Example 1] Preparation of monolayer-loaded fluorescent silica particles (NH2-Silica / BSA-Au NCs particles) An aqueous solution was prepared with a final concentration of 0.36 mM BSA, 4.77 mM chloroauric acid (HAuCl4), and 47.6 mM sodium hydroxide (NaOH). The aqueous solution was then stirred overnight at 37°C to obtain an aqueous dispersion of BSA-coated gold nanoclusters (BSA-Au NCs) (gold atom concentration: 4.77 mM). Silica particles (NH2-Silica) (average particle size: 325 nm, surface functional group: amino) were used as the core particles. 0.25 mL of the silica particle aqueous dispersion (particle concentration: 0.1 g / mL) was added to a 1.5 mL EP tube. Next, 0.25 mL of an aqueous dispersion of BSA-coated gold nanoclusters with a gold atom concentration of 4.77 mM was added to the aqueous dispersion of silica particles, and the mixture was stirred at room temperature for 1 hour using a vibratory mixer (MT-400, manufactured by TOMY Medico). The resulting solution was washed three times (3000 g, 5 minutes, 4°C) using a centrifuge (MDX-110, manufactured by TOMY Industries, Ltd.), and the residue was redispersed with 0.25 mL of ultrapure water to obtain an aqueous dispersion of fluorescent particles monolayer loaded with silica particles / fluorescent layer.

[0087] [Example 1] Preparation of bilayer-loaded fluorescent silica particles (NH2-Silica / BSA-Au NCs / CS particles) Prepare a 0.25 mL chitosan (CS) solution (Fujifilm and Koko Pure Chemical Industries, Ltd.) (chitosan concentration: 1 mg / mL, 10% v / v aqueous acetic acid) and add it to the aqueous dispersion obtained from Reference Example 1. Stir the solution at room temperature for 1 hour using a vibratory mixer (MT-400, TOMY Medico, Ltd.). Wash the resulting solution three times (3000 g, 5 minutes, 4°C) using a centrifuge (MDX-110, TOMY Industries, Ltd.), and redisperse the residue with 0.25 mL of ultrapure water to obtain an aqueous dispersion of fluorescent silica particles with a bilayer loading of silica particles / fluorescent layer / polymer layer.

[0088] [Example 2] Preparation of three-layer supported fluorescent silica particles (NH2-Silica / 2BSA-Au NCs / CS particles) 0.25 mL of an aqueous dispersion of BSA-coated gold nanoclusters (gold atom concentration of 4.77 mM, prepared using the same method as in Reference Example 1) was added to the aqueous dispersion obtained in Example 1. The mixture was stirred at room temperature for 1 hour using a vibratory mixer (MT-400, manufactured by TOMY Medico Co., Ltd.). The resulting solution was washed three times (3000 g, 5 minutes, 4°C) using a centrifuge (MDX-110, manufactured by TOMY Industries, Ltd.), and the residue was redispersed with 0.25 mL of ultrapure water to obtain an aqueous dispersion of fluorescent silica particles with a three-layer loading of silica particles / fluorescent layer / polymer layer / fluorescent layer.

[0089] [Fluorescence Spectroscopy Measurement] Using aqueous dispersions of fluorescent particles obtained from Reference Example 1, Example 1, and Example 2, and aqueous dispersions of silica particles used in Reference Example 1 (hereinafter sometimes referred to as "Reference Example 2"), and measured by a NanoDrop spectrophotometer... TM The fluorescence spectrum was measured using a 3300 (manufactured by Thermo Fisher Scientific) (excitation wavelength: 365 nm). The results of the fluorescence spectrum measurement are as follows: Figure 3 As shown.

[0090] like Figure 3 As shown, the fluorescent particles of Examples 1 and 2 exhibit fluorescence. Furthermore, the fluorescent particles of Example 2, consisting of a three-layer structure (fluorescent layer / polymer layer / fluorescent layer), exhibited approximately five times the fluorescence intensity compared to the single-layer fluorescent particles of Reference Example 1. This demonstrates that a stacked structure with a fluorescent layer as the outermost layer contributes to higher fluorescence intensity.

[0091] In addition, such as Figure 3 As shown, the intensity peaks in the fluorescence spectra of Examples 1 and 2 are at 663 nm and 659 nm, respectively, which are close to the original intensity peak (667 nm) of the BSA-coated gold nanoclusters. This indicates that by using silica particles as the core particles, the fluorescent particles can be endowed with the original fluorescence wavelength of the metal nanoclusters.

[0092] [Observation of particle shape] The shapes of the silica particles in Reference Example 2, and the fluorescent particles in Reference Example 1, Example 1, and Example 2 were observed using an electron microscope. SEM images were acquired using a JSM-7800F Prime electron microscope (JEOL Ltd.) at an accelerating voltage of 10.0 kV, a working distance (WD) of 9.5 mm, and a magnification of 20,000x. The acquired SEM images are shown below. Figure 4 As shown.

[0093] like Figure 4 As shown, the fluorescent particles of Examples 1 and 2 have a spherical shape similar to the core particle, i.e., the silica particle of Reference Example 2. This demonstrates that the stacked structure formed by the LBL method does not affect the shape of the obtained fluorescent particles, thus allowing for the production of fluorescent particles with shapes similar to the core particles and excellent dispersibility.

[0094] [Particle size distribution measurement] The particle size distribution of silica particles in Reference Example 2, and fluorescent particles in Reference Example 1, Example 1, and Example 2 was measured using dynamic light scattering (DLS). The measurements were performed using an ELSZ-2000 Zeta potential / particle size / molecular weight measurement system (Otsuka Electronics Co., Ltd.). The measurement results are as follows: Figure 5 As shown.

[0095] like Figure 5 As shown, the particles have similar particle size distributions. Therefore, it can be concluded that the fluorescent particles of Examples 1 and 2 have excellent dispersibility, suppressing particle aggregation during the manufacturing process and fully maintaining the dispersion state brought about by the raw material silica particles (refer to the silica particles of Example 2).

[0096] In addition, the average particle size was measured using SEM images obtained from particle shape observation. Five particles were randomly selected from the SEM images, and the average particle size of these five particles was calculated to measure the average particle size. The average particle size of the silica particles in Reference Example 2 was 327 nm, the average particle size of the fluorescent particles in Reference Example 1 was 327 nm, the average particle size of the fluorescent particles in Example 1 was 327 nm, and the average particle size of the fluorescent particles in Example 2 was 347 nm. The measurement results of the average particle size also show that the fluorescent particles of Examples 1 and 2 can maintain the average particle size of the raw silica particles well, and therefore have excellent dispersibility.

[0097] [Measurement of the Zeta potential of particles] The zeta potentials of the silica particles in Reference Example 2, and the fluorescent particles in Reference Example 1, Example 1, and Example 2 were measured. The measurements were performed using an ELSZ-2000 zeta potential / particle size / molecular weight measurement system (manufactured by Otsuka Electronics Co., Ltd.). The measurement results are as follows: Figure 6 As shown.

[0098] like Figure 6 As shown, the Zeta potential alternately increases or decreases in the order of silica particles from Reference Example 2, fluorescent particles from Reference Example 1, fluorescent particles from Example 1, and fluorescent particles from Example 2. This indicates that a stacked structure consisting of a negatively charged fluorescent layer and a positively charged polymer layer is well formed. Furthermore, the Zeta potential of the fluorescent particles from Example 1 is greater in the positive direction than that of the silica particles from Reference Example 2, and, as... Figure 3 As shown, the fluorescence intensity of the fluorescent particles in Example 2 is much greater than that of the fluorescent particles in Reference Example 1, which indicates that the larger the absolute value of the Zeta potential, the greater the loading of the layer further formed on it.

[0099] [Example 3] Preparation of three-layer supported fluorescent silica particles (NH2-Silica / BSA-Au NCs / CS / GSH-AuNCs particles) An aqueous solution was prepared with a final concentration of 6.8 mM glutathione (GSH), 4.5 mM chloroauric acid (HAuCl4), and 90 mM sodium hydroxide (NaOH). The aqueous solution was then stirred overnight at 37°C to obtain an aqueous dispersion of GSH-coated gold nanoclusters (GSH-Au NCs) (gold atom concentration: 5 mM).

[0100] 0.25 mL of an aqueous dispersion of GSH-coated gold nanoclusters (gold atom concentration: 5.0 mM) was added to an aqueous dispersion of NH2-Silica / BSA-Au NCs / CS particles obtained by the same method as in Example 1. The mixture was stirred at room temperature for 1 hour using a vibratory mixer (MT-400, manufactured by TOMY Medico Co., Ltd.). The resulting solution was washed three times (3000 g, 5 minutes, 4°C) using a centrifuge (MDX-110, manufactured by TOMY Industries, Ltd.), and the residue was redispersed with 0.25 mL of ultrapure water to obtain an aqueous dispersion of fluorescent silica particles with a three-layer structure: silica particles / fluorescent layer (BSA-Au NCs) / polymer layer (Cs) / fluorescent layer (GSH-AuNCs).

[0101] [Fluorescence Spectroscopy Measurement] The fluorescence spectrum of the fluorescent particles in Example 3 at 365 nm UV was measured using an absolute PL quantum yield measurement device (C11347-01, manufactured by Hamamatsu Photonics). The measurement results of the fluorescence spectrum are as follows: Figure 7 As shown. Figure 7 As shown, the fluorescence peak of the fluorescent particles of Example 3 was observed in the near-infrared region at 824 nm.

[0102] [Observation of particle shape] The shape of the fluorescent particles in Example 3 was observed using an electron microscope. SEM images were acquired using a JSM-7800F Prime electron microscope (manufactured by Nippon Electron Co., Ltd.) at an accelerating voltage of 10.0 kV, a working distance of 10.0 mm, and a magnification of 10,000x. The acquired SEM images are shown below. Figure 8 As shown. Figure 8 As shown, the fluorescent particles of Example 3 have a spherical shape similar to the raw material silica particles, and, due to Figure 8 The particles shown are fully dispersed, which also indicates that the fluorescent particles have excellent dispersibility.

[0103] [Reference Example 3] Preparation of monolayer-loaded fluorescent silica particles (NH2-Silica / GSH-Au NCs particles) The core particles used were silica particles (NH2-Silica) (average particle size: 325 nm, surface functional group: amino). 5 mL of an aqueous dispersion of silica particles (particle concentration: 0.01 g / mL) was added to a 20 cc spiral flask. Next, 1 mL of an aqueous dispersion of GSH-coated gold nanoclusters (gold atom concentration of 5 mM, prepared using the same method as in Example 3) was added to the silica particle dispersion, and the mixture was stirred at room temperature for 5 hours using a magnetic stirrer (HERACLES-16G, manufactured by Koike Precision Instruments Co., Ltd.). The resulting solution was washed three times (3000 g, 3 minutes, 4°C) using a centrifuge (MDX-110, manufactured by TOMY Industrial Co., Ltd.), and the residue was redispersed with 3 mL of ultrapure water to obtain an aqueous dispersion of monolayer fluorescent particles such as silica particles / fluorescent layer (GSH-Au NCs).

[0104] [Example 4] Preparation of bilayer-loaded fluorescent silica particles (NH2-Silica / GSH-Au NCs / CS particles) Prepare a 1.5 mL chitosan (CS) solution (Fujifilm and Koko Pure Chemical Industries, Ltd.) (chitosan concentration: 1 mg / mL, 10% v / v aqueous acetic acid), and add it to the 1.5 mL aqueous dispersion obtained from Reference Example 3. Stir at room temperature for 5 hours using a magnetic stirrer (HERACLES-16G, manufactured by Koike Precision Instruments Co., Ltd.). Wash the resulting solution three times (3000 g, 3 minutes, 4°C) using a centrifuge (MDX-110, manufactured by TOMY Industries, Ltd.), and redisperse the residue with 3 mL of ultrapure water to obtain an aqueous dispersion of fluorescent silica particles with a bilayer loading of silica particles / fluorescent layer (GSH-Au NCs) / polymer layer (CS).

[0105] [Example 5] Preparation of three-layer supported fluorescent silica particles (NH2-Silica / GSH-Au NCs / CS / GSH-AuAgNCs particles) An aqueous solution was prepared with a final concentration of 5.8 mM GSH, 3.5 mM chloroauric acid (HAuCl4), 0.9 mM silver nitrate (AgNO3), and 70 mM sodium hydroxide (NaOH). The aqueous solution was then stirred overnight at 37°C to obtain an aqueous dispersion of GSH-coated gold-silver alloy nanoclusters (GSH-AuAg-NCs) (gold atom concentration: 5 mM).

[0106] Two mL of an aqueous dispersion of GSH-coated gold-silver alloy nanoclusters (gold atom concentration: 5.0 mM) was added to the aqueous dispersion obtained in Example 4. The mixture was stirred at room temperature for 5 hours using a magnetic stirrer (HERACLES-16G, manufactured by Koike Precision Instruments Co., Ltd.). The resulting solution was washed three times (3000g, 3 minutes, 4°C) using a centrifuge (MDX-110, manufactured by TOMY Industrial Co., Ltd.), and the residue was redispersed with 3 mL of ultrapure water to obtain an aqueous dispersion of fluorescent silica particles with a three-layer structure: silica particles / fluorescent layer (GSH-Au NCs) / polymer layer (CS) / fluorescent layer (GSH-AuAg-NCs).

[0107] [Fluorescence Spectroscopy Measurement] The fluorescence spectrum of the fluorescent particles in Example 5 at 365 nm UV was measured using a microplate reader (Infinite M200 PRO, manufactured by Tecan Group Ltd.). The measurement results of the fluorescence spectrum are as follows: Figure 9 As shown. Figure 9 As shown, the fluorescence peak of the fluorescent particles of Example 5 was observed in the near-infrared region at 750 nm.

[0108] [Observation of particle shape] The shape of the fluorescent particles in Example 5 was observed using an electron microscope. SEM images were acquired using a JSM-7800F Prime electron microscope (JEOL Ltd.) at an accelerating voltage of 10.0 kV, a working distance of 10.0 mm, and a magnification of 20,000x. The acquired SEM images are shown below. Figure 10 As shown. Figure 10 As shown, the fluorescent particles of Example 3 have a spherical shape similar to the raw material silica particles, and, due to Figure 10 The particles shown are fully dispersed, which also indicates that the fluorescent particles have excellent dispersibility.

[0109] [Particle size distribution measurement] The particle size distribution of the fluorescent particles in Example 5 was measured using dynamic light scattering (DLS). The measurement was performed using an ELSZ-2000 Zeta potential / particle size / molecular weight measurement system (Otsuka Electronics Co., Ltd.). The measurement results (not shown) indicate that the average particle size of the fluorescent particles in Example 5 is 345.1 ± 34.8 nm. This value is approximately the same as the average particle size of the raw silica particles (325 nm), and this result also indicates that the fluorescent particles in Example 5 have excellent dispersibility.

[0110] Industrial availability This invention can be applied to the diagnostic field that utilizes techniques such as immunochromatography.

[0111] Explanation of symbols 10A, 10B: Core particles 11, 11A, 11B: Fluorescent layers 12: Polymer layer 13: Metal nanoclusters 100, 101: Fluorescent particles

Claims

1. A fluorescent particle, comprising: a core particle having a first charge, the first charge being a positive charge or a negative charge; at least one polymer layer on the core particle, the polymer layer comprising a polymer and having a second charge, the second charge being a positive charge or a negative charge; and at least one fluorescent layer on the core particle, the fluorescent layer comprising a metal nanocluster having a fluorescent property and having a third charge, the third charge being opposite in sign to the second charge.

2. The fluorescent particle according to claim 1, wherein: the at least one fluorescent layer is outside of an outermost one of the polymer layers.

3. The fluorescent particle according to claim 1, wherein: the at least one polymer layer is outside of an outermost one of the fluorescent layers.

4. The fluorescent particle according to claim 1, wherein: the first charge is the same charge in sign as the second charge, and the core particle has the at least one fluorescent layer between the core particle and the polymer layer.

5. The fluorescent particle according to claim 4, wherein: the first charge is a positive charge, and the core particle has a functional group selected from an amine group and a phosphonium group on a surface of the core particle.

6. The fluorescent particle according to claim 4 or 5, wherein: the core particle is selected from a silica particle, a polymer particle, and a metal particle.

7. The fluorescent particle according to any one of claims 1 to 5, wherein: the fluorescent particle has an average particle diameter of 100 nm or more and 100 μm or less.

8. The fluorescent particle according to any one of claims 1 to 5, wherein: the polymer comprises chitosan.

9. The fluorescent particle according to any one of claims 1 to 5, wherein: the metal nanocluster is coated with a coating agent, and the coating agent is at least one of an amino acid, a peptide, and a protein.

10. A method for producing a fluorescent particle, comprising: at least one polymer layer forming step of forming at least one polymer layer comprising a polymer and having a second charge on a core particle having a first charge, the first charge being a positive charge or a negative charge, and the second charge being a positive charge or a negative charge; and at least one fluorescent layer forming step of forming at least one fluorescent layer comprising a metal nanocluster having a fluorescent property and having a third charge on the core particle, the third charge being opposite in sign to the second charge.

11. The method for producing a fluorescent particle according to claim 10, wherein: the first charge is the same charge in sign as the second charge, and the at least one fluorescent layer forming step is included before the at least one polymer layer forming step.

12. The method for producing a fluorescent particle according to claim 11, further comprising: a charging step of obtaining the core particle by a charging treatment before the at least one polymer layer forming step and the at least one fluorescent layer forming step.

13. The method for producing a fluorescent particle according to any one of claims 10 to 12, wherein: the at least one polymer layer forming step and the at least one fluorescent layer forming step are each performed at 0°C or higher and 100°C or lower. ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​

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