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WO2019088287A1 - Particules de pigment agrégées, particules contenant un pigment et matériau de marquage fluorescent - Google Patents

Particules de pigment agrégées, particules contenant un pigment et matériau de marquage fluorescent Download PDF

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Publication number
WO2019088287A1
WO2019088287A1 PCT/JP2018/041026 JP2018041026W WO2019088287A1 WO 2019088287 A1 WO2019088287 A1 WO 2019088287A1 JP 2018041026 W JP2018041026 W JP 2018041026W WO 2019088287 A1 WO2019088287 A1 WO 2019088287A1
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WIPO (PCT)
Prior art keywords
group
dye
independently
aggregation
particles
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PCT/JP2018/041026
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English (en)
Japanese (ja)
Inventor
望月　誠
北　弘志
理枝 櫻木
康生 宮田
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Konica Minolta Inc
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Konica Minolta Inc
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Priority to JP2019550513A priority Critical patent/JP7226329B2/ja
Publication of WO2019088287A1 publication Critical patent/WO2019088287A1/fr
Anticipated expiration legal-status Critical
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C—CHEMISTRY; METALLURGY
    • C09—DYES; PAINTS; POLISHES; NATURAL RESINS; ADHESIVES; COMPOSITIONS NOT OTHERWISE PROVIDED FOR; APPLICATIONS OF MATERIALS NOT OTHERWISE PROVIDED FOR
    • C09B—ORGANIC DYES OR CLOSELY-RELATED COMPOUNDS FOR PRODUCING DYES, e.g. PIGMENTS; MORDANTS; LAKES
    • C09B23/00—Methine or polymethine dyes, e.g. cyanine dyes
    • C09B23/14—Styryl dyes
    • C—CHEMISTRY; METALLURGY
    • C09—DYES; PAINTS; POLISHES; NATURAL RESINS; ADHESIVES; COMPOSITIONS NOT OTHERWISE PROVIDED FOR; APPLICATIONS OF MATERIALS NOT OTHERWISE PROVIDED FOR
    • C09B—ORGANIC DYES OR CLOSELY-RELATED COMPOUNDS FOR PRODUCING DYES, e.g. PIGMENTS; MORDANTS; LAKES
    • C09B57/00—Other synthetic dyes of known constitution
    • C—CHEMISTRY; METALLURGY
    • C09—DYES; PAINTS; POLISHES; NATURAL RESINS; ADHESIVES; COMPOSITIONS NOT OTHERWISE PROVIDED FOR; APPLICATIONS OF MATERIALS NOT OTHERWISE PROVIDED FOR
    • C09K—MATERIALS FOR MISCELLANEOUS APPLICATIONS, NOT PROVIDED FOR ELSEWHERE
    • C09K11/00—Luminescent, e.g. electroluminescent, chemiluminescent materials
    • C09K11/06—Luminescent, e.g. electroluminescent, chemiluminescent materials containing organic luminescent materials
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
    • G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
    • G01N21/64—Fluorescence; Phosphorescence
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/58—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances

Definitions

  • the present invention relates to dye-aggregated particles, dye-containing particles, fluorescent labels, and methods for producing them.
  • Molecular imaging is a technology that visualizes the movement of molecules in the living body that could not be visualized so far, and for example, analysis of biomolecules at the molecular level, research on the dynamics of viruses and bacteria that cause disease, drugs It is widely used for various purposes, such as the evaluation of the action of the compound on the living body.
  • fluorescent imaging performed using a fluorescent substance is widely used to detect a trace substance in a living body.
  • Conventional fluorescent labeling materials include, for example, commercially available organic fluorescent dyes and the like, which have high quantum yield but low luminance per molecule bound to a target molecule, and can be used At the time, due to the aggregation of the dye molecules, the functions such as the light emission efficiency, the color forming property, the photosensitivity and the photosensitivity are remarkably reduced, and there is a disadvantage that the inherent properties of the fluorescent dye are limited.
  • Another fluorescent labeling material is a quantum dot which is a nanoparticle having a high quantum yield and a high light resistance (Patent Document 1).
  • the composition of a quantum dot having a relatively high quantum yield has a problem that it can not be used for living cells or living organisms because it is a composition containing Cd having high biotoxicity.
  • the quantum dots are unstable in fluorescence, such as causing an unpredictable flicker phenomenon, and are particles with relatively large specific gravities, they interfere with the dynamics of labeled biological substances and interactions with other substances. For example, there is a drawback that accurate observation and quantification of biomolecules are difficult.
  • pigment aggregation particles have been developed in which aggregation induction light emitting molecules having a characteristic of emitting fluorescence by aggregation of pigment molecules are aggregated (Patent Document 3).
  • the dye-aggregated particles have the advantages of higher brightness than conventional fluorescent labeling materials and lower cytotoxicity.
  • the formation of fine particles in which aggregation induced light emitting molecules are packed at a high density suppresses rotation, vibration, conversion to thermal energy, etc. of partial structures of dye molecules.
  • the mechanism by which excitation light energy is effectively used for the light emission path to improve the quantum yield, and the mechanism by which the quantum yield is improved by packing the regular molecular stacks so that they do not emit excimer light. Etc. are considered.
  • the inventor of the present invention uses the aggregation-induced light emitting molecules constituting the pigment aggregation particles or the dye-containing particles as aggregation-induced light emitting molecules having a specific structure, thereby causing collapse of the pigment aggregation particles or aggregation-induced light emission in the dye-containing particles. It has been found that the outflow of sexual molecules can be suppressed.
  • the present invention provides the following analysis method.
  • [Item 1] Dye-aggregated particles comprising at least one aggregation-induced luminescent molecule represented by the following general formulas (1) to (9).
  • R 1 , R 2 , R 3 , R 4 , R 5 and R 6 are each independently a hydrophilic group, a hydrogen atom, an organic group or an organometallic group;
  • R 1 , R 2 , R 3 and R 4 are each independently a hydrophilic group, a hydrogen atom, an organic group or an organometallic group;
  • R 1 , R 2 and R 3 are each independently a hydrophilic group, a hydrogen atom, an organic group or an organometallic group, Y is an electron withdrawing group;
  • the open circles represent carbon atoms, and R 1 and R 2 are each independently a hydrophilic group, a hydrogen atom, an organic group or an organometallic group;
  • R and R ′ each independently represent a hydrophilic group, a hydrogen atom, an organic group or an organometallic group
  • X is S, O or N, and when X is O or S, R 4 is absent, Y is an electron withdrawing group or an electron donating group, R 1 , R 2 , R 3 and R 4 each independently represent an organic group or an organic group having an hydrophilic group, or an organic metal group, and R 1 , R 2 , R 3 and R 4 are each bonded to form a ring structure May take;
  • R 1 is a substituted aromatic group or a hydrophilic group other than OH
  • R 2 , R 3 and R 4 are each independently a hydrophilic group, an organic group or an organometallic group
  • a to d are each independently an integer of 0 to 5, and when a is 2 or more, a plurality of R 1 s may be the same or different, and a plurality of R 1 s combine with each other to form a ring May form a
  • plural R 2 's , R 3' s and R 4 's may be the same or different, R 1 and R 2 , R 2 and R 4 , R 3 and R 4 , R 3 and R 1 may be combined to form a ring;
  • R A is independently a hydrophilic group, a hydrogen atom or an organic group
  • a is independently an integer of 1 to 5
  • R B is independently an aromatic ring-containing group having an aromatic ring-containing organic group or a hydrophilic group
  • R C is independently a hydrophilic group, a hydrogen atom, an organic group or an organometallic group
  • at least one is a hydrophilic group or an aromatic ring-containing group having a hydrophilic group, wherein a tertiary amino group is not included in the groups constituting R B and R C ;
  • R, R ′ and R ′ ′ are each independently a hydrophilic group, a hydrogen atom, an organic group or an organometallic group.
  • a dye-containing particle comprising a binder and at least one aggregation inducing luminescent molecule represented by the following general formulas (1) to (8).
  • R 1 , R 2 , R 3 , R 4 , R 5 and R 6 are each independently a hydrophilic group, a hydrogen atom, an organic group, an organometallic group or a silane coupling agent bonding property Is a group;
  • R 1 , R 2 , R 3 and R 4 are each independently a hydrophilic group, a hydrogen atom, an organic group, an organometallic group or a silane coupling agent binding group;
  • R 1 , R 2 and R 3 are each independently a hydrophilic group, a hydrogen atom, an organic group, an organic metal group or a silane coupling agent binding group, Y is an electron withdrawing group;
  • white circles represent carbon atoms, and R 1 and R 2 are each independently a hydrophilic group, a hydrogen atom, an organic group, an organometallic group, or a silane coupling agent binding group ;
  • R and R ′ each independently represent a hydrophilic group, a hydrogen atom, an organic group, an organometallic group, or a silane coupling agent binding group;
  • X is S, O or N, and when X is O or S, R 4 is absent, Y is an electron withdrawing group or an electron donating group, R 1 , R 2 , R 3 and R 4 each independently represent an organic group or an organic group having an hydrophilic group, an organometallic group, or a silane coupling agent binding group, R 1 , R 2 , R 3 and R 4 may be combined to form a ring structure;
  • R 1 is a substituted aromatic group, a hydrophilic group other than OH, or a silane coupling agent binding group
  • R 2 , R 3 and R 4 are each independently a hydrophilic group, an organic group or an organometallic group
  • a to d are each independently an integer of 0 to 5, and when a is 2 or more, a plurality of R 1 s may be the same or different, and a plurality of R 1 s combine with each other to form a ring May form a
  • plural R 2 's , R 3' s and R 4 's may be the same or different, R 1 and R 2 , R 2 and R 4 , R 3 and R 4 , R 3 and R 1 may be combined to form a ring;
  • R A independently represents a hydrophilic group, a hydrogen atom, an organic group, an organic metal group, or a silane coupling agent binding group
  • a is independently an integer of 1 to 5
  • R B is independently an aromatic ring-containing organic group
  • R C is independently a hydrophilic group, a hydrogen atom, an organic group or an organometallic group
  • R, R ′ and R ′ ′ are each independently a hydrophilic group, a hydrogen atom, an organic group, an organic metal group, or a silane coupling agent binding group.
  • R, R ′ and R ′ ′ are each independently a hydrophilic group, a hydrogen atom, an organic group, an organic metal group, or a silane coupling agent binding group.
  • the pigment aggregation particle according to Item 1 wherein the aggregation-induced light emitting molecule has a hydrophilic group.
  • the dye-containing particle according to Item 2 wherein the aggregation-induced light emitting molecule has a hydrophilic group.
  • the dye-containing particle according to Item 2 or 4 characterized in that the binder and the aggregation light emitting molecule form a covalent bond, and the binder forms a metalloxane bond. Dye-containing particles.
  • [Item 6] A fluorescent labeling material in which a targeting ligand is bound to the surface of the dye-aggregated particle according to Item 1 or 3 via a covalent bond.
  • [Item 7] A fluorescent labeling material in which a targeting ligand is bound to the surface of the dye-containing particle according to Item 2 or 4 via a covalent bond.
  • [Item 8] The item 6 or 7, wherein the targeting ligand is one or more molecules selected from the group consisting of an antibody, an organelle affinity substance, and a protein having a binding property with a sugar chain.
  • [Item 9] A fluorescent label dispersion containing the fluorescent label according to any one of items 6 to 8 and a buffer solution.
  • [Item 10] A method for producing pigment aggregation particles according to Item 1 or 3, comprising the step of bringing a poor solvent into contact with a solution of aggregation-induced luminescent molecules to aggregate the aggregation-induced luminescent molecules.
  • [Item 11] A method for producing dye-containing particles according to Item 2 or 4, comprising the step of dispersing aggregation-induced light emitting molecules in a binder or a precursor of a binder to form particles.
  • the dye-aggregated particles, the dye-containing particles, and the fluorescent labeling material of the present invention are superior in durability to conventional fluorescent labeling materials, and maintain the dyeability unchanged from the time of production even after the process of distribution.
  • the term “aggregation-induced luminescent molecule” means that fluorescence is not emitted or fluorescence emission intensity is weak because the quantum yield is low when each molecule is dissolved or dispersed in a dilute solution.
  • fluorescent substance refers to a fluorescent substance having the property of increasing quantum yield and emitting strong fluorescence or increasing fluorescence intensity by aggregating to form an aggregate.
  • the “pigment-aggregated particle” of the present invention contains at least one aggregation-induced luminescent molecule represented by the following general formulas (1) to (9).
  • the aggregation inducing light emitting molecule contained in the pigment aggregation particle may be one kind or two or more kinds.
  • R 1 , R 2 , R 3 , R 4 , R 5 , and R 6 are each independently a hydrophilic group, a hydrogen atom, an organic group or an organic metal group.
  • R 1 , R 2 , R 3 and R 4 are each independently a hydrophilic group, a hydrogen atom, an organic group or an organometallic group.
  • R 1 , R 2 and R 3 are each independently a hydrophilic group, a hydrogen atom, an organic group or an organometallic group, Y is an electron withdrawing group.
  • the open circles represent carbon atoms
  • R 1 and R 2 are each independently a hydrophilic group, a hydrogen atom, an organic group or an organic metal group.
  • the compound represented by the formula (4) is a compound having an ortho-carborane skeleton composed of C 2 B 10 .
  • black dots represent BH.
  • BH which can not be illustrated, is omitted from the viewpoint of the three-dimensional structure.
  • R and R ′ each independently represent a hydrophilic group, a hydrogen atom, an organic group or an organometallic group.
  • X is S, O or N, and when X is O or S, R 4 is absent, Y is an electron withdrawing group or an electron donating group, R 1 , R 2 , R 3 and R 4 each independently represent an organic group or an organic group having an hydrophilic group, or an organic metal group, and R 1 , R 2 , R 3 and R 4 are each bonded to form a ring structure You may take it.
  • R 1 is a substituted aromatic group or a hydrophilic group other than OH
  • R 2 , R 3 and R 4 are each independently a hydrophilic group, an organic group or an organometallic group
  • a to d are each independently an integer of 0 to 5, and when a is 2 or more, a plurality of R 1 s may be the same or different, and a plurality of R 1 s combine with each other to form a ring May form a
  • plural R 2 's , R 3' s and R 4 's may be the same or different, R 1 and R 2 , R 2 and R 4 , R 3 and R 4 , R 3 and R 1 may be combined to form a ring;
  • R A is independently a hydrophilic group, a hydrogen atom or an organic group
  • a is independently an integer of 1 to 5
  • R B is independently an aromatic ring-containing group having an aromatic ring-containing organic group or a hydrophilic group
  • R C is independently a hydrophilic group, a hydrogen atom, an organic group or an organometallic group
  • at least one is a hydrophilic group or an aromatic ring-containing group having a hydrophilic group, wherein a tertiary amino group is not included in the groups constituting R B and R C ;
  • R, R ′ and R ′ ′ are each independently a hydrophilic group, a hydrogen atom, an organic group or an organometallic group.
  • the organic group in the formulas (1) to (9) is, for example, a hydrocarbon group having 1 to 20 carbon atoms, and examples thereof include an alkyl group, an alkenyl group, an alkynyl group, a cycloalkyl group and a cycloalkenyl group. And cycloalkynyl groups and aromatic groups.
  • One or more hydrogen atoms at any position in these molecules may be substituted with a hetero atom such as S, N, O or the like.
  • the organometallic group in the formulas (1) to (9) is, for example, a group having a metal atom by covalent bond or coordinate bond to a part of a hydrocarbon group having 1 to 20 carbon atoms, Those containing a covalent bond between a metal atom and an oxygen atom are preferred.
  • the metal atom is not limited, for example, magnesium, calcium, strontium, scandium, yttrium, ruthenium, laurenthium, lanthanum, titanium, zirconium, hafnium, cerium, vanadium, niobium, tantalum, chromium, molybdenum, tungsten, manganese, ruthenium, Cobalt, rhodium, iridium, nickel, platinum, palladium, copper, silver, gold, zinc, aluminum, gallium, indium, silicon, germanium and tin are preferred, with titanium, zirconium and silicon being preferred.
  • the organometallic group is preferably a metal alkoxide group such as a titanium alkoxide skeleton, a zirconium alkoxide skeleton, a silicon alkoxide skeleton or the like, and when these are hydrolyzed and polycondensed, a strong and stable metalloxane bond (metal atom and oxygen atom) Repeated covalent bonds). Furthermore, it is more preferable that the organometallic group have a silicon alkoxide skeleton, and the aggregation-induced luminescent molecule having such an organometallic group easily controls the reactivity.
  • the organometallic group of the aggregation-induced luminescent molecule is a metal alkoxide and polycondensation of the aggregation-induced luminescent molecule is performed
  • the metal alkoxide group is a binder precursor.
  • a metalloxane bond formed by polycondensation of a metal alkoxide group serves as a binder in the present invention.
  • aggregation-induced luminescent molecules having a metal alkoxide group as an organic metal group may be independently polycondensed as described above to form a stable metalloxane bond.
  • various metal alkoxide monomers may be newly added and subjected to polycondensation.
  • aggregation induction light emitting molecules having metal alkoxide groups and various metal alkoxide monomers may be simultaneously mixed and polycondensed, or aggregation induction having metal alkoxide groups
  • the light-emitting molecule may be first polycondensed and then various metal alkoxide monomers may be added to carry out additional polycondensation to produce dye-containing particles.
  • the aggregation-induced luminescent molecules are in the form of aggregated particles by adjusting the concentration, solvent polarity, etc., and then the polycondensation reaction is performed via the metal alkoxide group.
  • the aggregation-induced luminescent molecules are in the form of dye-encapsulated particles immobilized three-dimensionally with metalloxane bonds while aggregating each other.
  • Dye-containing particles produced by such a method can form strong and stable metalloxane bonds regardless of aggregation-induced light emitting molecules, binders, binders, and aggregation induced light-emitting molecules, and durability such as vibration resistance is achieved. Becomes higher.
  • the electron withdrawing group in the above formulas (3) and (6) means, for example, cyano group, nitro group, methoxy group, tosyl group, mesyl group, halogen, phenyl group, acyl group, keto group, carboxyl group, aldehyde Groups, ethoxycarbonyl group, methoxycarbonyl group, pyridyl group, pyrimidyl group, triazinyl group, triazolyl group, tetrazolyl group, dicyanomethyl group, cyanamide group and the like.
  • Examples of the electron donating group in the above formula (6) include a methoxy group, an alkoxy group, an amino group alkylamino group, a dialkylamino group, a trialkylamino group, an alkyl group and an aromatic group having a methoxy group moiety.
  • the group by which at least one of the hydrogen atoms which the said organic group has was substituted by the said hydrophilic group is mentioned, for example.
  • Examples of the aromatic ring-containing organic group in the formula (8) include phenyl group, 1-naphthyl group, 2-naphthyl group, pyrenyl group, anthracenyl group, anthraquinonyl group, tolyl group, benzyl group, trityl group, and styryl group.
  • a benzylidene group an aniline group, a pyridyl group, a quinolyl group, a tosyl group, a tetraphenylethylene group, a triphenylethylene group, a diphenylethylene group, a triazinyl group, a derivative to which these are linked, and a derivative to which a substituent is added.
  • the aggregation-induced light emitting molecules represented by the above general formulas (1) to (6) all have a heterocyclic skeleton, and the positional relationship between electron-rich N part, S part, O part and B part is specified It was packed to be a cycle of Among them, the molecular skeleton of the aggregation-induced light emitting molecule represented by (1) to (3) and (6) has high periodicity, and hetero atoms can be obtained by arranging N elements, S elements, and O elements in the same plane. Is the optimal arrangement, and it is presumed that a more robust packing is formed.
  • the effect of the boron atom becomes a three-dimensionally delocalized ⁇ -electron delocalized superaromatic molecule, so 3 It becomes a dimensionally strong packing.
  • the maleimide skeleton of the aggregation-induced light emitting molecule represented by (5) it is presumed that the interaction with the adjacent molecule is strengthened by hydrogen bonding of O of the carbonyl group and H of NH.
  • the aggregation inducing luminescent molecule has a hydrophilic group. It is considered that when the aggregation-induced light emitting molecule has a hydrophilic group, the electric double layer becomes thick and the particle form becomes more stable in an aqueous solvent such as a buffer.
  • the aggregation inducing luminescent molecule used for producing the pigment aggregation particle can be selected to emit fluorescence of a desired wavelength (color).
  • a desired wavelength color
  • R 1 and R 2 are each independently selected arbitrarily from the above-mentioned formula (4) -1.
  • the “pigment-containing particle” of the present invention is characterized by comprising a binder and at least one aggregation-induced luminescent molecule represented by the above general formulas (1) to (8).
  • the binder is “one that retains a certain form by including aggregation-induced luminescent molecules” or “one that retains a certain form by connecting aggregation-induced luminescent molecules”.
  • the binder that is “a substance that remains in a certain form by including aggregation-induced light-emitting molecules” include resins, inorganic substances, and the like, and can include aggregation-induced light-emitting molecules.
  • a binder which is "to be in a fixed form by tying aggregation-induced light emitting molecules together in the case of binding to an adjacent aggregation-induced light emitting molecule via a substituent which the aggregation-induced light emitting molecule has.
  • a bonding portion, a linker for binding aggregation-induced light emitting molecules to each other, and the like, and the aggregation-induced light emitting molecules can be fixed to each other to be in a fixed form.
  • the aggregation-induced luminescent molecules contained in the “pigment-containing particles” of the present invention are hydrophilic groups in which R 1 , R 2 , R 3 , R 4 , R 5 , and R 6 in the above formula (1) are each independently.
  • An aggregation-induced light-emitting molecule which is a hydrogen atom, an organic group, an organometallic group, or a silane coupling agent-binding group, R 1 , R 2 , R 3 and R 4 in the formula (2) are each independently Aggregation-induced light emitting molecules which are a hydrophilic group, a hydrogen atom, an organic group, an organometallic group, or a silane coupling agent binding group, R 1 , R 2 and R 3 in the above formula (3) are each independently Aggregation-inducing light-emitting molecule, wherein Y is an electron-withdrawing group, and is a white circle in the formula (4); and a hydrophilic group, a hydrogen atom, an organic group, an organometallic group, or a silane coupling agent binding group represents a carbon atom, hydrophilic group R 1 and R 2 are each independently hydrogen atom, an organic group, Aggregation-induced light emitting molecule which is an organic metal group or a silane coupling
  • R 1 and R 2 , R 2 and R 4 , R 3 and R 4 , and R 3 and R 1 may respectively combine to form a ring
  • R A in the above formula (8) is independently a hydrophilic group, a hydrogen atom, an organic group, an organometallic group, or a silane coupling agent binding group
  • a is independently an integer of 1 to 5
  • R Among aggregation-induced light-emitting molecules in which B is independently an aromatic ring-containing organic group, and R C is independently a hydrophilic group, a hydrogen atom, an organic group or an organic metal group Includes one or more.
  • the silane coupling agent binding group is not particularly limited.
  • N-hydroxysuccinimide (NHS) ester group maleimide group, isocyanate group, isothiocyanate group, aldehyde group, paranitrophenyl group, diethoxymethyl group , Epoxy group, cyano group, alkoxysilane group, halogen atom and the like.
  • the binder is not particularly limited, but is not particularly limited as long as it is a substance capable of aggregating aggregation-induced light emitting molecules with physical or chemical bonding force, and is preferably a resin or an inorganic substance.
  • the aggregation-induced light emitting molecule has a hydrophilic group
  • electrostatic interaction is caused with a compound such as a resin or an inorganic substance that forms a binder of particles, so that the outflow of the aggregation-induced light emitting molecule can be suppressed.
  • the resin examples include melamine resin, urea resin, benzoguanamine resin, phenol resin, xylene resin, styrene resin, (meth) acrylic resin, polyacrylonitrile, AS resin (acrylonitrile-styrene copolymer), ASA resin (acrylonitrile- Examples include various homopolymers and copolymers prepared using one or more monomers, such as styrene-methyl acrylate copolymer).
  • melamine resins and styrene resins are preferably used because particles containing aggregation-induced light emitting molecules can be easily produced and the emission intensity of the obtained dye-containing particles becomes high.
  • the inorganic substance examples include zirconium oxide, alumina, silica and the like. It is more preferable that it is a silica from a viewpoint of the improvement of the vibration tolerance at the time of normal temperature-ization, and reactivity control. Since silica is generally known to be chemically inert and to be easily modified, the dye-containing (silica) particles of the present invention using silica as a binder are also easily desired. Of molecules can be attached to the surface.
  • the dye-containing particle is preferably hydrophilic.
  • a hydrophilic substance such as a melamine resin is used as a binder to prepare a dye-containing particle, or the surface of the dye-containing particle produced with a hydrophobic substance is modified with a hydrophilic compound to be hydrophilic. Can be obtained.
  • the hydrophilic compound used to hydrophilize the surface of the dye-containing particle is not particularly limited.
  • linear hydrophilic polymers such as polyethylene glycol (PEG) and polypropylene glycol (PPG) are repeatedly used. It is preferable from the ease of adjustment of the molecular length depending on the number of units and easy preparation of a derivative having various functional groups etc. connected to the end or as a product.
  • the aggregation inducing luminescent molecule used for producing the dye-containing particles can be selected to emit fluorescence of a desired wavelength (color).
  • a desired wavelength color
  • the "fluorescent labeling material" of the present invention is characterized in that a targeting ligand is bound to the surface of the dye aggregation particle or the dye-containing particle via a covalent bond.
  • the form of the fluorescent labeling material for example, the form at the time of production, storage, and distribution is not particularly limited, but is preferably in the form of a dispersion using a known buffer such as PBS as a dispersion medium.
  • One embodiment of the present invention is in the form of a dispersion, that is, a fluorescent label dispersion, and the fluorescent label dispersion contains a fluorescent label and a buffer.
  • the targeting ligand used in the “fluorescent labeling material” of the present invention is a substance that specifically recognizes and binds to a target substance, and is a target substance that is a biological substance contained in a tissue or cell collected from an animal or the like, for example.
  • the substance is preferably a substance that specifically recognizes and binds as a target substance.
  • the target biological substance is not particularly limited, and examples include proteins, nucleic acids, sugar chains, lipids and the like.
  • the target biological material is preferably a biological material associated with any disease. Specifically, for example, marker proteins (for example, cancer-specific proteins, vascular endothelial cell-specific proteins, phosphorylated proteins, etc.) specifically expressed in cancer cells, inflammation-related proteins, etc., and immune-related proteins can be mentioned. .
  • the target biological substance when the target biological substance is a protein specifically expressed in a tumor tissue or a cancer cell, antibodies against these are preferably selected as a targeting ligand.
  • a protein for example, lectin
  • a binding property with a sugar chain is preferably selected as the target-directed molecule.
  • target-directed molecules include, for example, organelle compatible substances, peptides and the like.
  • an antibody When an antibody is selected as the targeting ligand, it is usually IgG or IgM, and IgG is preferably used.
  • the antibody may be a natural antibody such as full-length IgG, as long as it has the ability to specifically recognize and bind a target protein or a lower antibody, Fab, Fab ', F (ab' 2 ) It may be a non-naturally occurring antibody such as an artificial antibody which has been multifunctionalized (multivalented or multispecificized) using antibody fragments such as 2 , Fv and scFv, or antibody fragments thereof .
  • a primary antibody that recognizes and binds to a unique epitope on an antigen is preferably used.
  • a secondary antibody which is an antibody which recognizes and binds a unique epitope to a primary antibody as a targeting ligand, a target biological substance to which a primary antibody is bound in advance is used as a target substance.
  • the dye aggregation particle of the present invention preferably includes a step (A) of bringing a poor solvent into contact with a solution of aggregation-induced luminescent molecules to aggregate the aggregation-induced luminescent molecules, wherein the step (A) comprises a core
  • the solution of the aggregation-induced luminescent molecule may be brought into contact with the poor solvent to aggregate the aggregation-induced luminescent molecule.
  • the step other than the step (A) is not particularly limited, and, for example, a step of introducing a hydrophilic group into the aggregation-induced light emitting molecule, a step of introducing a hydrophilic group into the surface of the pigment aggregation particle, and the like are appropriately performed.
  • a step of introducing a hydrophilic group into the aggregation-induced light emitting molecule a step of introducing a hydrophilic group into the surface of the pigment aggregation particle, and the like are appropriately performed.
  • the central core may be premixed in the solution of aggregation-induced luminescent molecules, or may be premixed in the poor solvent.
  • the substance used as the central nucleus is not particularly limited, and for example, fine particles of organic molecules such as polystyrene and latex, and inorganic molecules such as silica are suitably used.
  • the nature and size of the central core can be selected according to the desired particle size of the dye aggregation particles and the nature of the aggregation inducing luminescent molecule used for preparation.
  • As the central nucleus one having an average particle diameter of 1 nm or more and 20 nm or less and a particle diameter variation coefficient of 5% or less is preferable.
  • the pigment aggregation particles in the present invention are prepared by using a solvent (good solvent) capable of dissolving aggregation-induced luminescent molecules and preparing a divided solution of the aggregation-induced luminescent molecules, followed by aggregation into a solution of aggregation-induced luminescent molecules. It can be prepared by the reprecipitation method of precipitating pigment aggregated particles by mixing the induced luminescent molecule with the poor solvent. By using such a reprecipitation method, it is possible to produce particles densely packed with aggregation-induced luminescent molecules.
  • a reprecipitation method using a mixer with a small inner diameter called a micromixer and pumping a good solvent and a poor solvent of aggregation-induced luminescent molecules into the micromixer, both rapidly and uniformly
  • fine-particles are deposited is mentioned by mixing to (1).
  • the inner diameter of the flow path of the mixing section for mixing the minute solution of aggregation-induced luminescent molecules and the poor solvent (when the cross section of the flow path is not circular, the cross-sectional area of the flow path
  • the diameter of the circle having the same area as that of the above is preferably 2 mm or less, and in order to mix the solution and the poor solvent more rapidly, the inner diameter of the flow path is preferably 1 mm or less. Further, in order to prevent the clogging of the flow path by the fine particles and to reduce the pressure loss inside the flow path, the inner diameter of the flow path is preferably 0.05 mm or more.
  • the good solvent of the present invention is not particularly limited as long as it exhibits good solubility in aggregation-induced light emitting molecules, and it is preferable to select one having good compatibility with the poor solvent described later.
  • ether solvents such as tetrahydrofuran and dioxane
  • ketone solvents such as acetone and methyl ethyl ketone
  • amides such as 1-methyl-2-pyrrolidinone, 1,3-dimethylimidazolinone and N, N-dimethylformamide
  • a system solvent, a sulfur-containing solvent such as dimethyl sulfoxide, or a mixed solvent of two or more of these can be suitably used.
  • a good solvent having a boiling point lower than the boiling point of the poor solvent from the viewpoint of preventing redispersion of aggregation-induced light emitting molecules.
  • the poor solvent of the present invention is not particularly limited as long as it has relatively low solubility in the aggregation-induced light emitting molecule, and it is preferable to select one having good compatibility with the above-mentioned good solvent.
  • water or an aqueous solution is preferable, and alcohol solvents such as methanol and ethanol, aliphatic solvents such as pentane, hexane and heptane, aromatic solvents such as benzene and toluene, or mixed solvents of two or more of them are used.
  • alcohol solvents such as methanol and ethanol
  • aliphatic solvents such as pentane, hexane and heptane
  • aromatic solvents such as benzene and toluene
  • mixed solvents of two or more of them are used.
  • the poor solvent has a relatively low boiling point (eg, 40 ° C. to 120 ° C.) as a good solvent.
  • reaction conditions for the reaction time and reaction temperature are not particularly limited as long as the dye aggregation particles satisfying the above conditions are produced, but a short time is required for efficiently forming aggregation-induced luminescent molecules into nanoparticles. It is preferable to mix a minute solution of aggregation-induced luminescent molecules and a poor solution rapidly, for example, under turbulent conditions such that the Reynolds number is 4,000 or more.
  • the present invention relates to a method (eg, JP 2005-238342 A) in which laser ablation is performed on a dispersion in which crystals of relatively coarse aggregation-induced luminescent molecules are dispersed in a poor solvent (particle size variation coefficient) Small agglomerated nanoparticles can be produced.
  • a method eg, JP 2005-238342 A
  • laser ablation is performed on a dispersion in which crystals of relatively coarse aggregation-induced luminescent molecules are dispersed in a poor solvent (particle size variation coefficient) Small agglomerated nanoparticles can be produced.
  • the laser ablation When the laser ablation is performed, various known lasers can be used as the laser, and a YAG laser, an excimer laser, a titanium-sapphire laser or the like is preferably used. As an irradiation laser, it is preferable to apply a pulse wave. Further, in order to prepare aggregated nanoparticles having a more uniform particle size distribution, it is preferable to adjust the concentration of the dispersion before performing laser application to 0.1 mg / L to 500 mg / L.
  • the irradiation power, pulse width, wavelength and irradiation time can be appropriately adjusted according to the type and size of the crystal of the aggregation-induced luminescent dye of interest, and the mixing ratio with the poor solvent, and aggregation nano size more uniform in particle size distribution
  • the power is 0.5 to 500 mJ / cm 2
  • the pulse width is 1 to 100 femtoseconds
  • the pulse width is 0.01 to 500 Hz
  • the irradiation time is 0.5 minutes to 5 hours
  • the poor solvent use may be made of water, alcohol solvents such as methanol and ethanol, aliphatic solvents such as pentane, hexane and heptane, aromatic solvents such as benzene and toluene, or a mixed solvent of two or more of them. But not limited thereto.
  • the laser ablation method can be performed, for example, with an apparatus set up by the method described in The Review of Laser Engineering, 33, 41-46.
  • the pigment-aggregated particles may be purified, if necessary, using a conventional method such as ultrafiltration. By purification, ions and unreacted substances in the reaction solution can be removed, and spherical or nearly spherical pigment aggregation particles can be obtained.
  • the particles having a shape close to spherical are specifically particles having a shape in which the ratio of the major axis to the minor axis is 2 or less.
  • the method for producing the dye-containing particles of the present invention includes the step of dispersing the aggregation-induced light emitting molecule in a binder or a precursor of the binder to form particles.
  • the method for producing the dye-containing particles is 1) A process of dispersing aggregated light emitting molecules in a precursor of a binder 2) It is preferable to include a process of forming a binder from a precursor of a binder by a sol-gel method and forming it into particles.
  • any method may be used as a method for producing the dye-containing particles, as long as it is a configuration of silica particles containing aggregation-induced light emitting molecules. Specifically, it can be obtained, for example, by a method of producing condensation-induced luminescent molecules having an alkoxysilane group and polycondensation.
  • the alkoxysilane group may be a monofunctional alkoxysilane group, a bifunctional alkoxysilane group or a trifunctional alkoxysilane group.
  • the polycondensation can usually be carried out by a sol-gel method.
  • the method for producing the aggregation-induced luminescent molecule having an alkoxysilane group is not particularly limited. For example, a method of directly introducing an alkoxysilane group into a part of the aggregation-induced luminescent dye, aggregation-induced luminescence by a silane coupling agent And a method of introducing an alkoxysilane to a part of the organic molecule.
  • an alkoxysilane group When an alkoxysilane group is directly introduced into the molecule of aggregation-induced light emitting molecule, it can be introduced at any position of the molecular skeleton of the aggregation-induced light emitting molecule, but by introducing an alkoxysilane group into the aromatic ring site Dye-containing particles with high luminous efficiency can be obtained.
  • an active group is introduce
  • an aggregation-induced luminescent molecule having an alkoxysilane group can be obtained.
  • the above-mentioned active group is not particularly limited, but N-hydroxysuccinimide (NHS) ester group, maleimide group, isocyanate group, isothiocyanate group, aldehyde group, paranitrophenyl group, diethoxymethyl group, epoxy group, cyano group, It can be selected from halogen atoms and the like.
  • NHS N-hydroxysuccinimide
  • N-hydroxysuccinimide (NHS) ester group or maleimide group as the active group to be introduced into aggregation-induced light emitting molecule and using a silane coupling agent having an amino group as a silane coupling agent Contained particles can be obtained.
  • the NHS ester group and the amino group of the silane coupling agent having an amino group form an amide bond (-NHCO-) to obtain an aggregation-induced luminescent molecule having an alkoxysilane group. That is, in the aggregation-induced light emitting molecule having the alkoxysilane group, the aggregation-induced light emitting molecule and the silica are bonded via an amide bond.
  • the silane coupling agent having an amino group is not particularly limited, and examples thereof include ⁇ -aminopropyltriethoxysilane (APS), 3- [2- (2-aminoethylamino) ethylamino] propyl-triethoxysilane, N And -2 (aminoethyl) 3-aminopropylmethyldimethoxysilane, 3-aminopropyltrimethoxysilane and the like, and APS is particularly preferable.
  • APS ⁇ -aminopropyltriethoxysilane
  • 3- [2- (2-aminoethylamino) ethylamino] propyl-triethoxysilane N And -2 (aminoethyl) 3-aminopropylmethyldimethoxysilane, 3-aminopropyltrimethoxysilane and the like
  • APS is particularly preferable.
  • the reaction between the aggregation-induced luminescent molecule having the NHS ester group and the silane coupling agent having the amino group is carried out after each of them is dissolved in a solvent such as DMSO (dimethyl sulfoxide) or DMF (N, N-dimethylformamide). It can be carried out by reacting under stirring at room temperature (eg, 25 ° C.).
  • a solvent such as DMSO (dimethyl sulfoxide) or DMF (N, N-dimethylformamide). It can be carried out by reacting under stirring at room temperature (eg, 25 ° C.).
  • the ratio of the aggregation-induced luminescent molecule to the silane coupling agent is not particularly limited, but a ratio of 1: 0.5 to 2 (molar ratio) is preferable, and a ratio of 1: 0.8 to 1.2 (molar ratio) Is more preferred.
  • the dye-containing particles of the present invention are subjected to polycondensation by adding an aggregation-induced light emitting molecule having an alkoxysilane group prepared by the above method polycondensation by itself or by adding one or more silane compounds. It can manufacture by a method.
  • condensation induction light emitting molecule having the alkoxysilane group is independently polycondensed
  • the polycondensation reaction is preferably carried out in the presence of alcohol, water and ammonia.
  • the alcohol include lower alcohols having 1 to 3 carbon atoms such as methanol, ethanol and propanol.
  • the ratio of water to alcohol in such a reaction system is not particularly limited, but preferably 0.5 to 20 parts by volume, more preferably 2 to 16 parts by volume, and still more preferably 4 to 10 parts by volume of alcohol to 1 part by volume of water. It is the range of the capacity part.
  • the amount of ammonia is also not particularly limited, but the concentration of ammonia is preferably 30 to 1000 mM, more preferably 60 to 500 mM, and still more preferably 80 to 200 mM.
  • This reaction can be carried out at room temperature, and is preferably carried out with stirring.
  • the dye-containing particles of the present invention can be prepared by a reaction of several tens minutes to several tens hours.
  • the size (diameter) of the aggregation inducing luminescent molecule having the alkoxysilane group can be appropriately adjusted, for example, the same If the process is repeated several times, larger silica particles can be prepared. Also, if necessary, dye-containing particles in a desired particle size distribution range can be prepared.
  • the silane compound is not particularly limited.
  • the ratio of the aggregation-induced light emitting molecule having an alkoxysilane group to the silane compound is not particularly limited, but a molar ratio of the silane compound to 1 mol of the aggregation-induced light emitting molecule having an alkoxysilane group is preferably 0.05 to 4000. 0.1 to 400 is more preferable, and 0.3 to 40 is more preferable.
  • the reaction of the aggregation inducing luminescent molecule having an alkoxysilane group with the silane compound is preferably carried out in the presence of alcohol, water and ammonia.
  • the alcohol include lower alcohols having 1 to 3 carbon atoms such as methanol, ethanol and propanol.
  • the ratio of water to alcohol in such a reaction system is not particularly limited, but preferably 0.5 to 20 parts by volume, more preferably 2 to 16 parts by volume, and still more preferably 4 to 10 parts by volume of alcohol to 1 part by volume of water. It is the range of the capacity part.
  • the amount of ammonia is also not particularly limited, but the concentration of ammonia is preferably 30 to 1000 mM, more preferably 60 to 500 mM, and still more preferably 80 to 200 mM.
  • This reaction can be carried out at room temperature, and is preferably carried out with stirring.
  • the dye-containing particles of the present invention can be prepared by a reaction of several tens minutes to several tens hours.
  • the size (diameter) of the dye-containing particles to be prepared can be appropriately adjusted by adjusting the concentration of the aggregation inducing luminescent molecule having an alkoxysilane group to be used or adjusting the reaction time.
  • Smaller silica particles can be prepared by reducing the concentration of silane compounds used or by shortening the reaction time (e.g. Blaaderen et al., "Synthesis and Characterization of Monodisperse Collio-Silicate Spheres See, "J. Colloid and Interface Science 156, 1-18. 1993).
  • Blaaderen et al. "Synthesis and Characterization of Monodisperse Collio-Silicate Spheres See, "J. Colloid and Interface Science 156, 1-18. 1993).
  • larger silica particles can be prepared.
  • the particle diameter (diameter) of the obtained dye-containing particles is, for example, in the order of nm to ⁇ m in the desired size, specifically, the dye-containing particles having a minute size such as 3 to 30 nm. It is possible to prepare the particles. Also, if necessary, dye-containing particles in a desired particle size distribution range can be prepared.
  • the dye-containing particle of the present invention is not particularly limited in the production method as long as the function required as a fluorescent label is not impaired.
  • the dye-containing particles of the present invention can be obtained by a production method including the following polymerization step.
  • (A-1) Polymerization step A step of polymerizing a resin raw material to be a raw material of the organic resin in the presence of the aggregation inducing luminescent molecule to produce a resin particle containing the aggregation inducing luminescent molecule can be mentioned. .
  • the resin raw material that can be used in the step (a-1) may be a monomer corresponding to the resin, or may be a prepolymer obtained from such a monomer.
  • Specific examples of such monomers and prepolymers include those described above in the section "Resin”.
  • the dye may be present from the beginning of the polymerization reaction in step (a-1), or may be added during the polymerization reaction.
  • the said polymerization reaction can be performed by conventionally well-known conditions and methods except performing in presence of the said aggregation induction light emission molecule
  • the aggregation inducing light emitting molecule and the antioxidant are encapsulated by adding formic acid to the mixed solution of the aggregation inducing light emitting molecule and the melamine resin and causing a polycondensation reaction.
  • Melamine resin particles can be obtained.
  • the reaction at this time can be performed, for example, in water.
  • the polymerization reaction may be carried out in the presence of a suitable surfactant.
  • thermosetting resin such as melamine resin
  • proton (H + ) is imparted to a functional group such as an amino group contained in the resin or aggregation-induced light emitting molecule to charge it.
  • a polymerization reaction accelerator such as an appropriate acid may be further added to the mixture of the dye and the melamine resin for the purpose of facilitating electrostatic interaction.
  • the conditions (temperature, time, etc.) of the polymerization reaction can be appropriately set in consideration of the type of resin, the composition of the raw material mixture, and the like.
  • the reaction temperature is usually 60 to 200 ° C.
  • the reaction time is usually 20 to 120 minutes.
  • the reaction temperature it is appropriate for the reaction temperature to be a temperature (within the heat resistant temperature range) at which the performance of the aggregation inducing luminescent molecule is not deteriorated.
  • the heating may be divided into a plurality of stages. For example, after reacting at relatively low temperature for a fixed time, the temperature may be raised and reacted at relatively high temperature for a fixed time.
  • impurities such as excess resin raw material, aggregation-induced light emitting molecule, surfactant and the like may be removed from the reaction liquid, and the generated dye-containing particles may be recovered and purified.
  • the reaction solution is centrifuged to remove the supernatant containing impurities, and then ultrapure water is added, and the mixture is irradiated with ultrasonic waves, dispersed again, and washed. It is preferable that these operations be repeated several times until the supernatant does not show absorption and fluorescence derived from the resin and the fluorescent dye.
  • thermoplastic resin such as a styrene resin
  • the thermoplastic resin can be synthesized according to known methods such as radical polymerization and ionic polymerization (anion polymerization, etc.).
  • radical polymerization and ionic polymerization anion polymerization, etc.
  • the resin particles for inclusion type fluorescent labeling using a thermoplastic resin can also be manufactured according to those methods, for example, it is preferable to manufacture by the polymerization process according to the soap free emulsion polymerization method.
  • the reaction mixture containing aggregation-induced light-emitting molecules, the resin raw material, and the polymerization initiator is heated to advance the polymerization reaction of the resin, and the aggregation-induced light-emitting molecules are It becomes the process of generating the resin particle to be included.
  • the polymerization initiator and the conditions (temperature, time, etc.) of the polymerization reaction can be appropriately set in consideration of the type of the resin and the like.
  • the reaction temperature is usually 20 to 150 ° C.
  • the reaction time is usually 10 to 240 minutes.
  • the polymerization initiator known ones such as benzoyl peroxide and azobisisobutyronitrile can be used, but when this polymerization step is carried out according to a soap-free emulsion polymerization method, 2,2'-azobis (A water soluble polymerization initiator such as 2-methyl propionamidine can be used.
  • the resin particle itself including the aggregation-induced light emitting molecule obtained in the step (a-1) described above may be used as the resin particle for fluorescent labeling according to the present invention, or the surface described later
  • resin particles having a functional group capable of forming a bond with another molecule may be used as the dye-containing particle according to the present invention.
  • the dye-containing particles of the present invention when producing the dye-containing particles of the present invention, if necessary, purification may be performed using a conventional method such as an ultrafiltration membrane. By performing purification, ions in the reaction solution and unreacted substances can be removed, and spherical or nearly spherical dye-containing particles can be obtained.
  • the particle having a shape close to a sphere is specifically a shape in which the ratio of the major axis to the minor axis is 2 or less.
  • ultrafiltration with an ultrafiltration membrane such as YM-10 or YM-100 (manufactured by Millipore) is performed to remove particles with large particle diameters. It is also good.
  • the resin constituting the dye-containing particles according to the present invention (the resin is also expressed as an organic resin in the present invention) functions as a container for containing aggregation-induced light emitting molecules described later.
  • the organic resin used in the present invention is not particularly limited as long as it does not impair the function of the aggregation inducing luminescent molecule, and may be a thermosetting resin or a thermoplastic resin.
  • Thermosetting resins that can be used as the organic resin in the present invention include, for example, melamine, urea, guanamines (including benzoguanamine and acetoguanamine), phenols (including phenol, cresol, xylenol and the like), xylene, and the like What contains the structural unit formed from the at least 1 type of monomer chosen from the group which consists of derivatives is mentioned.
  • melamine urea
  • guanamines including benzoguanamine and acetoguanamine
  • phenols including phenol, cresol, xylenol and the like
  • xylene and the like
  • What contains the structural unit formed from the at least 1 type of monomer chosen from the group which consists of derivatives is mentioned.
  • One of these monomers may be used alone, or two or more of these monomers may be used in combination. If desired, one or more comonomers other than the above compounds may be used in combination.
  • thermosetting resin examples include melamine formaldehyde resin, urea formaldehyde resin, benzoguanamine formaldehyde resin, phenol formaldehyde resin and metaxylene formaldehyde resin.
  • a melamine resin represented by a melamine / formaldehyde resin is preferable from the viewpoint of light emission intensity at the time of dye incorporation.
  • thermosetting resins As a raw material of these thermosetting resins, not only monomers as described above, but also prepolymers obtained by reacting in advance a monomer and a compound such as formaldehyde or another crosslinking agent may be used.
  • a monomer such as formaldehyde or another crosslinking agent
  • methylolmelamine which is generally prepared by condensing melamine and formaldehyde under alkaline conditions, is used as a prepolymer, and the compound is further alkyletherified (in water Or the like to improve the solubility in organic solvents, and the like.
  • thermosetting resin at least a part of hydrogen contained in the constituent unit may be replaced by a substituent having a charge or a substituent capable of forming a covalent bond.
  • a thermosetting resin can be synthesized by using, as a raw material, a (derivatized) monomer in which at least one hydrogen is replaced by the above-described substituent by a known method.
  • melamine resin, urea resin, benzoguanamine resin and the like usually have a cation naturally generated from an amino group or a site derived therefrom, and a phenol resin, a xylene resin and the like usually form an anion naturally produced from a hydroxyl group or a site derived therefrom Have.
  • thermosetting resin can be synthesized according to a known method.
  • a melamine / formaldehyde resin can be synthesized by heating and polycondensing methylolmelamine prepared in advance as described above, after adding a reaction accelerator such as an acid as necessary.
  • thermoplastic resin that can be used as the organic resin in the present invention is not particularly limited, but, for example, at least one kind of at least one selected from the group consisting of styrene, (meth) acrylic acid and its alkyl ester, acrylonitrile and derivatives thereof.
  • What contains the structural unit formed from a functional monomer (The group which participates in a polymerization reaction in one molecule, The monomer which has one vinyl group in the above-mentioned example) is mentioned.
  • One of these monomers may be used alone, or two or more of these monomers may be used in combination. If desired, one or more comonomers other than the above compounds may be used in combination.
  • thermoplastic resin examples include polystyrene, styrene-based resin consisting of styrene and other monomers, polymethyl methacrylate, acrylic-based resin consisting of (meth) acrylic acid and its alkyl ester and other monomers, polyacrylonitrile And acrylonitrile-based resins comprising AS resin (acrylonitrile-styrene copolymer), ASA resin (acrylonitrile-styrene-methyl acrylate copolymer), acrylonitrile and other monomers.
  • AS resin acrylonitrile-styrene copolymer
  • ASA resin acrylonitrile-styrene-methyl acrylate copolymer
  • a styrene-based resin is preferable from the viewpoint of the light emission intensity at the time of aggregation-induced light emitting molecule encapsulation.
  • the "styrene-based resin” refers to a resin which is a homopolymer or copolymer of styrene which may or may not have a substituent.
  • thermoplastic resin is, for example, a structural unit formed from a polyfunctional monomer such as divinylbenzene (a group participating in a polymerization reaction in one molecule, a monomer having two or more vinyl groups in the above example), ie, It may contain a crosslinking site.
  • a polyfunctional monomer such as divinylbenzene (a group participating in a polymerization reaction in one molecule, a monomer having two or more vinyl groups in the above example), ie, It may contain a crosslinking site.
  • thermoplastic resin at least a part of hydrogen contained in the constituent unit may be replaced by a substituent having a charge or a substituent capable of forming a covalent bond.
  • a thermoplastic resin can be synthesized by using, as a raw material, a monomer such as 4-aminostyrene in which at least one hydrogen is replaced by the above-described substituent (derivatized).
  • thermoplastic resin may contain a structural unit having a functional group for surface-modifying the resin particle for fluorescent labeling obtained in the step (a-1).
  • a monomer such as glycidyl methacrylate having an epoxy group as a raw material
  • This epoxy group can be converted to an amino group by reacting with excess ammonia water.
  • biomolecules can be introduced to the thus formed amino group according to a known method (through a molecule serving as a linker, if necessary).
  • One embodiment of the present invention is a method for producing a fluorescent labeling material, which comprises a step of binding a targeting ligand to the surface of the dye-aggregated particle or the dye-containing particle.
  • the dye-aggregated particles or the dye-containing particles and the targeting ligand may be directly bound or may be bound via a linker or the like.
  • the method for binding the targeting ligand to the surface of the dye-aggregated particles or the dye-containing particles is not particularly limited, and the following methods (i) to (iii) may be mentioned.
  • the dye-aggregated particle or the dye-containing particle having a thiol group on the surface can be bound to a targeting ligand via a disulfide bond, a thioester bond, or a thiol substitution reaction.
  • the targeting ligand has an amino group
  • the thiol group possessed by the dye aggregation particle or the dye-containing particle and the amino group possessed by the targeting ligand are succinimidyl-trans-4- (N- It may be coupled using a crosslinking agent such as maleimidyl methyl) cyclohexane-1-carboxylate (SMCC), N- (6-maleimidocaproyloxy) succinimide (EMCS) and the like.
  • SMCC maleimidyl methyl
  • EMCS N- (6-maleimidocaproyloxy) succinimide
  • the pigment-aggregated particle having an amino group on the surface or the pigment-containing particle is, as described above, bonded with the amino group and a thiol group possessed by a biomolecule or the like using a crosslinking agent such as SMCC or EMCS. Can.
  • this amino group can be bonded to an amino group possessed by a biomolecule or the like with a crosslinking agent such as glutaraldehyde.
  • biomolecules and the like can be bound to the surface via an amide bond or a thiourea bond.
  • the pigment aggregation particle and the targeting ligand are bound by the biotin-avidin reaction by reacting the pigment aggregation particle bound to biotin in advance and the targeting ligand to which avidin is bound. .
  • the dye-containing particles obtained by the step (a-1) described above may be used as they are for the fluorescent labeling material according to the present invention, but the dye-containing particles of the present invention may be surface-modified as required. It can be performed.
  • the surface modification that can be performed in the present invention is not particularly limited.
  • the dye-containing particles of the present invention when used as a fluorescent labeling material for immunostaining, the dye-containing particles of the present invention can be used in a mode in which a biorelevant binding substance according to the embodiment of immunostaining is linked. Become. Therefore, the surface modification that can be applied to the dye-containing particle of the present invention is preferably performed in the form of introduction of a functional group capable of forming a bond with another molecule.
  • examples of functional groups capable of forming bonds with other molecules include functional groups generally used in the field of biochemistry, and specific examples of such functional groups include a hydroxyl group, an amino group, a carboxyl group, A thiol group, a maleimide group, an aldehyde group etc. are mentioned.
  • a functional group capable of forming a bond with another molecule may also be referred to as a reactive functional group.
  • a silane coupling agent having a functional group capable of forming a bond with another molecule is reacted with the hydroxyl group
  • a functional group capable of forming a bond with the other molecule can be introduced.
  • a dye-containing particle having an amino group can be obtained by reacting a dye-containing particle having a hydroxyl group on the surface with a silane coupling agent having an amino group such as aminopropyltrimethoxysilane.
  • introduction of a functional group capable of forming a bond with another molecule to a dye-containing particle having a hydroxyl group on the surface is a suitable linker molecule having a functional group capable of forming a bond with another molecule. It can also be carried out by reacting with a hydroxyl group.
  • the introduction methods such as these can be suitably applied particularly to dye-containing particles formed by employing a melamine resin as the organic resin.
  • the dye-containing particles obtained by the above-mentioned step (a-1) have an epoxy group on the surface
  • an amino group can be introduced by treating such dye-containing particles with ammonia water.
  • it is possible to form a bond with the epoxy group by reacting an appropriate linker molecule having a functional group having reactivity with the epoxy group and a functional group capable of forming a bond with the other molecule with the epoxy group.
  • Functional groups can also be introduced.
  • the dye-containing particles do not have any reactive functional group on the surface, for example, hydroxyl groups etc. are once introduced on the particle surface by performing appropriate surface treatment such as plasma treatment and conventionally known, and then There are cases where the same method as the introduction of the “functional group capable of forming a bond with another molecule” into “the dye-containing particle” having a hydroxyl group on the surface may be applied. From the above, even when a resin is used as a binder, a targeting ligand can be bound in the same manner as in the case of surface modification of silica particles.
  • a desired molecule may be bound to the surface by introducing an arbitrary acceptor group on the surface of the dye-containing particle.
  • the acceptor group include an amino group, a hydroxyl group, a thiol group, a carboxyl group, a maleimide group, and a succinimidyl ester group.
  • an OH group is present in the silica particle, and this may be used as an acceptor group
  • a silane compound (silane coupling agent) having a desired group By bonding a silane compound (silane coupling agent) having a desired group to the surface, a dye-containing particle having an acceptor group capable of binding to a desired molecule may be formed on the surface.
  • the dye-containing particles are produced by adding one or more silane compounds to the aggregation-induced light emitting molecule having an alkoxysilane group and performing polycondensation, depending on the type of the polycondensed silane compound.
  • a dye-containing particle having an acceptor group capable of binding to a desired molecule on the surface can be obtained.
  • the relationship between the polycondensed silane compound (silane coupling agent) and the acceptor group formed on the surface of the dye-containing particle obtained thereby is shown in Table 1.
  • silane compound silane coupling agent
  • Example 1 Color-aggregated particles (1)
  • a compound 4,4′-Bis (1,2,2-triphenylvinyl) -1,1′-biphenyl (manufactured by Sigma-Aldrich Co., Ltd.) of the following formula (9) was dissolved in tetrahydrofuran so as to be 1 mM.
  • the above solution is sent at a flow rate of 1.0 mL / min using a pump (PU-1580, JASCO Corporation) in a stainless steel T-shaped micro mixer (MT1XCS6, manufactured by Valco) equipped with a flow path with an inner diameter of 0.15 mm.
  • MT1XCS6 stainless steel T-shaped micro mixer
  • the solution is mixed, and the two solutions are mixed in the micromixer by feeding ultrapure water at a flow rate of 74.0 mL / min using another pump (NS-KX-500, Japan Precision Science Co., Ltd.).
  • the pigment aggregation particles were precipitated.
  • the pressure at the time of mixing was 4 to 5 MPa, and blocking of the flow path by the pigment aggregation particles did not occur.
  • the Reynolds number at mixing was calculated to be about 12,000.
  • the mixture was treated with a centrifugal separator at 10000 rpm for 30 minutes to remove the supernatant and washed to obtain pigment aggregated particles (1).
  • Example 2 (Color-aggregated particles (2)) Example 1 and Example 1 except that the compound 4,4 '-(1,2-Diphenylethene-1,2-diyl) dibenzoic acid (manufactured by Sigma Aldrich) of the following formula (10) is used instead of the compound of the formula (9) Dye aggregated particles (2) were obtained in the same manner.
  • Example 3 (Color-aggregated particles (3))
  • Dye aggregated particles (3) were obtained in the same manner as in Example 1 except that a compound of the following formula (11) was used instead of the compound of the formula (9).
  • the compound of the following formula (11) was synthesized by the method described in Adv. Funct. Mater. 2014, 24, 3621.
  • Example 4 Color-aggregated particles (4)
  • 100 mg of 1,1,2,3,4,5-hexaphenyl-1H-silole manufactured by Sigma Aldrich
  • 30 mL of water, 30 mL of ethanol and 0.5 mL of concentrated sulfuric acid were mixed, and the mixture was stirred at 50 ° C. for 3 hours.
  • purification was performed by column chromatography to obtain a compound of the following formula (12).
  • pigment aggregated particles (4) were obtained in the same manner as in Example 1 except that a compound of the following formula (12) was used instead of the compound of the formula (9).
  • Example 5 (Color-aggregated particles (5))
  • the compound of the following formula (13) was synthesized by the synthesis method described in Organometallics, 2016, 35 (14), pp 2327-2332.
  • Dye aggregated particles (5) were obtained in the same manner as in Example 1 except that a compound of the following formula (13) was used instead of the compound of the formula (9).
  • Example 6 (Color-aggregated particles (6)) 5 mL of concentrated sulfuric acid and 5 mL of concentrated nitric acid were added to 0.1 mol of the compound of the formula (13), and the mixture was stirred for 1 hour to perform nitration to an aromatic ring. Subsequently, purification was performed by column chromatography to obtain a compound into which two nitro groups were introduced. Next, 0.1 g of tin powder and 10 mL of concentrated hydrochloric acid were added to a compound into which two 10 g of nitro groups were introduced, and the mixture was stirred for 1 hour.
  • Example 7 (Color-aggregated particles (7)) The compound of the following formula (15) was synthesized by the synthesis method described in Dalton Trans, 2013, 42, 3646-3652. Dye aggregated particles (7) were obtained in the same manner as in Example 1 except that a compound of the following formula (15) was used instead of the compound of the formula (9).
  • Example 8 (Color-aggregated particles (8)) 5 mL of concentrated sulfuric acid and 5 mL of concentrated nitric acid are added to 0.1 mol of the compound of the formula (15), and the mixture is stirred for 1 hour to perform nitration to an aromatic ring, followed by purification by column chromatography, The compound in which two nitro groups were introduce
  • Dye aggregated particles (8) were obtained in the same manner as in Example 1 except that a compound of the following formula (16) was used instead of the compound of the above formula (9).
  • Example 9 (Color-aggregated particles (9))
  • the compound of the following formula (17) was obtained by the synthetic method described in New J. Chem., 2007, 31, 2076-2082.
  • Dye aggregated particles (9) were obtained in the same manner as in Example 1 except that a compound of the following formula (17) was used instead of the compound of the above formula (9).
  • Example 10 (Color-aggregated particles (10)) By obtaining the carborane having a phenyl iodide group by the method described in Macromolecules 2009, 42, 1418-1420, and subsequently hydrogenating the iodine substituent by dehalogenation by the method described in WO 2009087994 A1 , The compound of following formula (18) was obtained.
  • Dye aggregated particles (10) were obtained in the same manner as in Example 1 except that a compound of the following formula (18) was used instead of the compound of the above formula (9).
  • Example 11 (Color-aggregated particles (11)) A compound of the following formula (19) was obtained by using 4-aminophenylacetylene instead of phenylacetylene in the method described in Macromolecules 2009, 42, 1418-1420.
  • Dye aggregated particles (11) were obtained in the same manner as in Example 1 except that a compound of the following formula (19) was used instead of the compound of the above formula (9).
  • Example 12 (Color-aggregated particles (12)) Chem. Lett. 2012, 41, 1445-1447, a compound of the following formula (20) was obtained.
  • Dye aggregated particles (12) were obtained in the same manner as in Example 1 except that a compound of the following formula (20) was used instead of the compound of the above formula (9).
  • Example 13 (Color-aggregated particles (13)) In the method described in Chem. Eur. J, 2013, 19, 4506-4512, a compound of the following formula (21) was obtained. Dye aggregated particles (13) were obtained in the same manner as in Example 1 except that a compound of the following formula (21) was used instead of the compound of the above formula (9).
  • Example 14 (Color-aggregated particles (14)) 100 mg of the compound of the above formula (21), 30 mL of water, 30 mL of ethanol, and 0.5 mL of concentrated sulfuric acid were mixed, and stirred at 50 ° C. for 3 hours. Subsequently, purification was performed by column chromatography to obtain a compound of the following formula (22). Dye aggregated particles (14) were obtained in the same manner as in Example 1 except that a compound of the following formula (22) was used instead of the compound of the above formula (9).
  • Example 15 (Color-containing particles (15)) 10 mg of iron powder, 10 mg of sodium acetate and 100 mL of THF are added to 100 mg of the compound of the above formula (13), stirred at room temperature for 1 hour under chlorine gas bubbling, and liquid separation purification with water / toluene The chlorinated compound which Cl group was introduce
  • the reaction solution was ultrafiltered with YM-100 (trade name, manufactured by Millipore).
  • the dye-incorporated silica particle dispersion liquid that has passed through the filter is recovered, and this is subjected to ultrafiltration with YM-1 (trade name, manufactured by Millipore) until the dye-incorporated silica particle dispersion liquid is reduced to one tenth of the total amount.
  • YM-1 trade name, manufactured by Millipore
  • the concentrated solution was diluted with distilled water and subjected to ultrafiltration again with YM-1. After concentration, dilution with distilled water and ultrafiltration were repeated four times to remove unreacted starting materials, ammonia and the like contained in the dye-incorporated silica particle dispersion, thereby obtaining dye-incorporated particles (15).
  • Example 16 (Color-containing particles (16)) The same as Example 15, except that the compound of the following formula (24) is synthesized using the compound of the above formula (15) instead of synthesizing the compound of the above formula (23) using the compound of the above formula (13) Dye-containing particles (16) were obtained by the following method.
  • Example 17 (Dye-containing particles (17)) The same as Example 15, except that the compound of the following formula (25) is synthesized using the compound of the above formula (17) instead of synthesizing the compound of the above formula (23) using the compound of the above formula (13) Dye-containing particles (17) were obtained by the following method.
  • Example 18 (Color-containing particles (18)) The same as Example 15, except that the compound of the following formula (26) is synthesized using the compound of the above formula (18) instead of synthesizing the compound of the above formula (23) using the compound of the above formula (13) Dye-containing particles (18) were obtained by the method of
  • Example 19 (Color-containing particles (19)) The same as Example 15, except that the compound of the following formula (27) is synthesized using the compound of the above formula (20) instead of synthesizing the compound of the above formula (23) using the compound of the above formula (13) Dye-containing particles (19) were obtained by the method of
  • Example 20 (Color-containing particles (20)) The same as Example 15, except that the compound of the following formula (28) is synthesized using the compound of the above formula (21) instead of synthesizing the compound of the above formula (23) using the compound of the above formula (13) Dye-containing particles (20) were obtained by the following method.
  • Example 21 (Dye-containing particles (21)) 10 mg of iron powder, 10 mg of sodium acetate, 10 mg of bromine and 100 mL of THF are added to 100 mg of the compound of the above formula (13), stirred at room temperature for 1 hour, and separated by water / toluene to obtain a compound of the above formula (13) The bromo compound which introduce
  • the fluorescent silica particle dispersion liquid that has passed through the filter is collected, and this is subjected to ultrafiltration with YM-1 (trade name, manufactured by Millipore), and the fluorescent silica particle dispersion liquid is concentrated to one tenth of the total amount. did.
  • the concentrated solution was diluted with distilled water and subjected to ultrafiltration again with YM-1. After concentration, the mixture was diluted with distilled water and subjected to ultrafiltration four times to remove unreacted APS, ammonia and the like to obtain dye-containing particles (21).
  • Example 22 (Dye-containing particles (22)) The same as Example 21, except that the compound of the following formula (30) is synthesized using the compound of the above formula (15) instead of synthesizing the compound of the above formula (29) using the compound of the above formula (13) Dye-containing particles (22) were obtained by the following method.
  • Example 23 (Color-containing particles (23)) The same as Example 21 except that the compound of the following formula (31) is synthesized using the compound of the above formula (17) instead of synthesizing the compound of the above formula (29) using the compound of the above formula (13) Dye-containing particles (23) were obtained by the method of
  • Example 24 (Color-containing particles (24)) The same as Example 21 except that the compound of the following formula (32) is synthesized using the compound of the above formula (18) instead of synthesizing the compound of the above formula (29) using the compound of the above formula (13) Dye-containing particles (24) were obtained by the following method.
  • Example 25 (Color-containing particles (25)) Instead of synthesizing the compound of the formula (29) using the compound of the formula (13), Example 21 is used except that a compound of the following formula (33) is synthesized using the compound of the formula (20) Dye-containing particles (25) were obtained in the same manner.
  • Example 26 (Color-containing particles (26)) Instead of synthesizing the compound of the formula (29) using the compound of the formula (13), Example 21 is used except that a compound of the formula (34) is synthesized using the compound of the formula (21) Dye-containing particles (26) were obtained in the same manner.
  • Example 27 ⁇ Fluorescent labeling material 1> The fluorescent dye was produced by labeling the dye-aggregated particles and dye-containing particles obtained in Examples 1 to 26 with streptavidin.
  • the treated aggregated nanoparticles are prepared into a 3 nM dispersion using PBS containing 2 mM EDTA, and SM (PEG) 12 (succinimidyl-[(N-maleimidopropionamido)] to a final concentration of 10 mM.
  • SM PEG 12
  • -dodecaneethylene glycol ester Thermo Scientific Inc.
  • the dispersion was centrifuged at 10,000 rpm for 20 minutes and then the supernatant was removed, and then PBS containing 2 mM EDTA was added to wash the precipitate three times to disperse the precipitate, whereby maleimide was applied to the particle surface.
  • Dye aggregated particles in which groups were introduced were obtained.
  • streptavidin manufactured by Wako Pure Chemical Industries, Ltd.
  • streptavidin adjusted to 1 mg / mL
  • borate buffer 210 ⁇ L
  • 2-iminothiolane hydrochloride manufactured by Sigma Aldrich
  • a thiol group was introduced into the amino group of streptavidin by reacting at room temperature for 1 hour, and this was desalted with a gel filtration column (Zaba Spin Desalting Columuns, Funakoshi).
  • Example 28 ⁇ Fluorescent labeling material 2> The fluorescent dye 2 was produced by labeling the dye-aggregated particles obtained in Examples 6, 8, 9, 11, 12 and 14 with an anti-PD-L1 antibody. (Pharmaceutical particle-modified antibody) Dye aggregated particles in which a maleimide group was introduced to the surface of each particle were obtained in the same manner as in Example 27.
  • Example 29 ⁇ Color-containing melamine particles (28) to (32)> 20.3 mg of the pigment aggregated particles (6) was added to 22 mL of water and dissolved. Thereafter, 2 mL of a 5% aqueous solution of an emulsifier for emulsion polymerization "Emulgen" (registered trademark) 430 (polyoxyethylene oleyl ether, manufactured by Kao Corporation) was added to this solution. The solution was heated to 70 ° C. while being stirred on a hot stirrer, and then 0.81 g of a melamine resin raw material “Nicalac MX-035” (manufactured by Nippon Carbide Industries Co., Ltd.) was added to the solution.
  • Emulgen registered trademark
  • dye-containing particles 0.1 mg are dispersed in 1.5 mL of ethanol, 2 ⁇ l of aminopropyltrimethoxysilane (LS-3150, manufactured by Shin-Etsu Chemical Co., Ltd.) is added, and the mixture is reacted for 8 hours to exist on the surface of dye-containing particles. The resulting hydroxyl group was converted to an amino group.
  • LS-3150 aminopropyltrimethoxysilane
  • the concentration of the dye-containing particles is adjusted to 3 nM with phosphate buffer saline (PBS) containing 2 mM ethylenediaminetetraacetic acid (EDTA) to give a final concentration of 10 mM, SM (PEG) 12 (Succinimidyl-[(N-maleimidopropionamide) -dodecaethylene glycol] ester, manufactured by Thermo Scientific Co., Ltd.) is mixed, reacted at 20 ° C.
  • PBS phosphate buffer saline
  • EDTA ethylenediaminetetraacetic acid
  • Dye-containing polystyrene particles (33) to (44) were produced by the soap-free emulsion polymerization method as follows.
  • Each compound is coupled to styrene by mixing each of the compounds of the above formulas (10) and (12) to (22) with 4-aminostyrene (manufactured by Tokyo Chemical Industry Co., Ltd.) at room temperature for 1 hour, dye bonding Styrene was made.
  • dye bonding Styrene was made.
  • 0.18 g of glycidyl methacrylate manufactured by Tokyo Kasei Kogyo Co., Ltd.
  • 0.05 g of styrene manufactured by Wako Pure Chemical Industries, Ltd.
  • 0.05 g of divinylbenzene 0.005 g of the above dye-bound styrene to 5 mL of pure water which has been subjected to argon bubbling.
  • the temperature was raised to 70 ° C. while stirring, 0.012 g of a water-soluble azo polymerization initiator V-50 (manufactured by Wako Pure Chemical Industries, Ltd.) was added, and reaction was performed for 12 hours.
  • the reaction solution was centrifuged at 10000 G for 20 minutes to recover particles. The collected particles were dispersed in pure water and again collected by centrifugation to perform purification to obtain dye-containing polystyrene particles (33) to (44).
  • the concentration of the dye-containing polystyrene particles was adjusted to 3 nM using phosphate buffered saline (PBS) containing 2 mM ethylenediaminetetraacetic acid (EDTA).
  • PBS phosphate buffered saline
  • EDTA ethylenediaminetetraacetic acid
  • SM (PEG) 12 succinimidyl-[(N-maleimidopropionamide) -dodecaethylene glycol] ester, Thermo Scientific Co., Ltd. to a final concentration of 10 mM for a dispersion of dye-containing polystyrene particles adjusted in concentration C.) and allowed to react at 20.degree. C. for 1 hour to obtain a mixed solution containing dye-containing polystyrene particles having a maleimide group introduced on the particle surface.
  • the mixture was centrifuged at 10000 G for 20 minutes, and after removing the supernatant, PBS containing 2 mM EDTA was added to disperse the precipitate, and centrifugation was performed again. After performing the above washing three times according to the same procedure, the dye-containing polystyrene particles modified with a maleimide group were recovered.
  • a thiol group-added streptavidin was prepared in the same manner as described in Example 27.
  • the dye-containing polystyrene resin particles were treated with anti-PD-L1 rabbit monoclonal in the same manner as in Example 27 except that dye-containing polystyrene particles having a maleimide group introduced on the particle surface and thiolated anti-PD-L1 rabbit monoclonal antibody were used. Obtained antibody-modified fluorescent labeling material 5
  • Comparative Example 1 (Color aggregate particles)
  • the compound of the following formula 35 was synthesized by the synthesis method described in US2013 / 089 889.
  • a dye aggregate was obtained by crystallizing the compound of the following formula 35 in a methanol / THF solution.
  • Comparative Example 2 Dissolve 5.6 mg of Y550-NHS ester (trade name, manufactured by Dyomics GmbH), which is a derivative of fluorescent dye Y550 which is not aggregation-induced luminescent molecule, in 1 ml of dimethyl sulfoxide (DMSO) and add 1.3 ⁇ l of APS The reaction was performed at room temperature (25 ° C.) for 1 hour.
  • DMSO dimethyl sulfoxide
  • Streptavidin-maleimide 0.5 mg (manufactured by Sigma) was added to 2 mg / mL ⁇ 1.5 mL of colloidal silica particles which were thiol-modified in the same manner as in Example 26, and a reaction was carried out at room temperature for 2 hours. After the reaction, unreacted streptavidin-maleimide was removed by dialysis in a conventional manner to obtain a fluorescent labeling material which is a streptavidin-modified colloidal silica particle.
  • Vibration resistance evaluation (refrigerated) A dispersion is prepared by dispersing the fluorescent labeling materials 1 to 5 and the dye aggregate of Comparative Example 1 and the colloidal silica particle dye of Comparative Example 2 in PBS so as to be 5 wt / wt%, and the respective luminances are measured. did. Subsequently, the dispersion liquid of each particle was subjected to vibration processing by reciprocating Tokyo-Fukuoka by Cool courier service (registered trademark) at 5 ° C., and the luminance after the vibration processing was measured. From the measured values of the initial brightness and the brightness after vibration treatment, vibration tolerance evaluation (refrigeration) was performed according to the following criteria.
  • AA luminance after vibration processing
  • BB luminance after vibration processing
  • CC luminance after vibration processing
  • DD luminance after vibration processing
  • Vibration resistance evaluation room temperature was performed according to the same procedures and evaluation criteria as in Example 31 except that Tokyo-Fukuoka was reciprocated by TA-Q-BIN (registered trademark) at room temperature instead of Cool TA-Q-BIN (registered trademark) at 5 ° C.
  • TA-Q-BIN registered trademark
  • Cool TA-Q-BIN registered trademark

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Abstract

La présente invention vise à fournir des particules de pigment agrégées, des particules contenant un pigment, et un matériau de marquage fluorescent qui ont une luminosité élevée et une résistance élevée aux vibrations. La présente invention est à même de produire des particules de pigment agrégées, des particules contenant un pigment, et un matériau de marquage fluorescent qui comprennent des molécules luminescentes induisant l'agrégation ayant une structure spécifique, et qui ont une résistance élevée aux vibrations.
PCT/JP2018/041026 2017-11-06 2018-11-05 Particules de pigment agrégées, particules contenant un pigment et matériau de marquage fluorescent Ceased WO2019088287A1 (fr)

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JPWO2021261398A1 (fr) * 2020-06-25 2021-12-30
JP2022059418A (ja) * 2020-10-01 2022-04-13 積水化学工業株式会社 重合体、検査薬、アナライト濃度測定法、及び、アナライト濃度測定装置
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JP2022546707A (ja) * 2019-08-30 2022-11-07 アラン、 マーク クラインフェルド、 生体体液中の非結合ビリルビンの濃度の測定のための単一工程方法、キット、およびシステム
US11945781B2 (en) 2020-11-18 2024-04-02 Southern Research Institute Compounds for the treatment of acute and chronic kidney disease
CN118033124A (zh) * 2024-02-01 2024-05-14 广东省大湾区华南理工大学聚集诱导发光高等研究院 一种双波长聚集诱导发光磁性编码微球及其制备方法与应用

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JP2022546707A (ja) * 2019-08-30 2022-11-07 アラン、 マーク クラインフェルド、 生体体液中の非結合ビリルビンの濃度の測定のための単一工程方法、キット、およびシステム
WO2021193909A1 (fr) * 2020-03-26 2021-09-30 積水化学工業株式会社 Polymère, agent de test, procédé de mesure de concentration d'analyte et instrument de mesure de concentration d'analyte
JP7765380B2 (ja) 2020-03-26 2025-11-06 積水化学工業株式会社 重合体、検査薬、及び、アナライト濃度測定法
JPWO2021193909A1 (fr) * 2020-03-26 2021-09-30
WO2021261398A1 (fr) * 2020-06-25 2021-12-30 コニカミノルタ株式会社 Nanoparticules électroluminescentes et matériau de marquage électroluminescent pour un diagnostic pathologique
JPWO2021261398A1 (fr) * 2020-06-25 2021-12-30
JP2022059418A (ja) * 2020-10-01 2022-04-13 積水化学工業株式会社 重合体、検査薬、アナライト濃度測定法、及び、アナライト濃度測定装置
JP7765173B2 (ja) 2020-10-01 2025-11-06 積水化学工業株式会社 重合体、検査薬、アナライト濃度測定法、及び、アナライト濃度測定装置
US20220153695A1 (en) * 2020-11-18 2022-05-19 Southern Research Institute Compounds for the Treatment Of Acute and Chronic Kidney Disease
US11753374B2 (en) * 2020-11-18 2023-09-12 Southern Reserach Institute Compounds for the treatment of acute and chronic kidney disease
US11945781B2 (en) 2020-11-18 2024-04-02 Southern Research Institute Compounds for the treatment of acute and chronic kidney disease
CN118033124A (zh) * 2024-02-01 2024-05-14 广东省大湾区华南理工大学聚集诱导发光高等研究院 一种双波长聚集诱导发光磁性编码微球及其制备方法与应用
WO2025161172A1 (fr) * 2024-02-01 2025-08-07 广东省大湾区华南理工大学聚集诱导发光高等研究院 Micro-sphère à codage magnétique à émission induite par agrégation à double longueur d'onde, et son procédé de préparation et son application

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