WO2019088287A1

WO2019088287A1 - Aggregated pigment particles, pigment-encasing particles, and fluorescent marking material

Info

Publication number: WO2019088287A1
Application number: PCT/JP2018/041026
Authority: WO
Inventors: 望月　誠; 北　弘志; 理枝櫻木; 康生宮田
Original assignee: コニカミノルタ株式会社
Priority date: 2017-11-06
Filing date: 2018-11-05
Publication date: 2019-05-09
Also published as: JPWO2019088287A1; JP7226329B2

Abstract

The present invention aims to provide aggregated pigment particles, pigment-encasing particles, and a fluorescent marking material that have high brightness and high vibration resistance. The present invention is capable of obtaining aggregated pigment particles, pigment-encasing particles, and a fluorescent marking material that: include aggregation-inducing luminescent molecules having a specific structure; and have high vibration resistance.

Description

Dye-aggregated particles, dye-containing particles, and fluorescent labeling material

The present invention relates to dye-aggregated particles, dye-containing particles, fluorescent labels, and methods for producing them.

In recent years, molecular imaging technology has attracted high attention in the clinical field and basic research. 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. In particular, due to its excellent detection sensitivity and operability, fluorescent imaging performed using a fluorescent substance is widely used to detect a trace substance in a living body.

In diagnosis and research using fluorescence imaging, a method has been proposed in which a fluorescent substance is bound to a biological substance to be detected as a labeling reagent and the fluorescence of the labeling reagent is detected with high sensitivity by irradiating a predetermined excitation light. ing. With the fluorescence signal obtained by such fluorescence imaging, quantification of biomolecular interactions, long-term dynamic observation of biomolecules, ultra-high sensitivity observation, etc. There is a need for a fluorescent labeling material that combines two properties.

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). However, 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. In addition, since 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.

In order to solve the problems as described above, there is a colloidal silica particle in which a fluorescent dye compound is dispersed, which is less prone to self-quenching than conventional fluorescent dyes, and fixes many fluorescent dye compounds in silica particles. Thus, high luminance can be obtained (Patent Document 2).

Furthermore, in recent years, 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. Although there are various explanations for the generation of high luminance of aggregated light emitting dyes, 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.

JP 2011-530187 gazette JP, 2010-112957, A US Patent Application Publication No. 2013/089889

However, with regard to the particles described in Patent Document 2, a fluorescent dye that can not be bonded to the silica, which is the matrix of the particle, or a fluorescent dye that is only weakly bonded to the silica, flows out of the particles. As a result, there is a problem that the fluorescence intensity when used for staining is impaired. Furthermore, when the inventors conducted an additional experiment on the pigment-aggregated particles described in Patent Document 3 described above, the particle disintegration progressed after the vibration process, and there was a problem that uneven brightness occurred when used for dyeing. It turned out that there was.

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.

That is, 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).

In the above formula (1), 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;

In the above formula (2), 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;

In the above formula (3), 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;

In the above formula (4), 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;

In the above formula (5), R and R ′ each independently represent a hydrophilic group, a hydrogen atom, an organic group or an organometallic group;

In the above formula (6), 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;

In the above formula (7), 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
When b to d are 2 or more, 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;

In the above formula (8), 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,
Among R ^A , R ^B and R ^C , 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 ;

In the above formula (9), R, R ′ and R ′ ′ are each independently a hydrophilic group, a hydrogen atom, an organic group or an organometallic group.
[Item 2] A dye-containing particle comprising a binder and at least one aggregation inducing luminescent molecule represented by the following general formulas (1) to (8).

In the above formula (1), 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;

In the above formula (2), 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;

In the above formula (3), 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;

In the above formula (4), 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 ;

In the above formula (5), 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;

In the above formula (6), 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;

In the above formula (7), 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
When b to d are 2 or more, 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;

In the above formula (8), 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;

In the above formula (9), 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.
[Item 3] The pigment aggregation particle according to Item 1, wherein the aggregation-induced light emitting molecule has a hydrophilic group.
[Item 4] The dye-containing particle according to Item 2, wherein the aggregation-induced light emitting molecule has a hydrophilic group.
[Item 5] 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. The fluorescent labeling material described.
[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.
[Item 12] A method for producing the dye-containing particle according to Item 2 or 4,
1) A process of dispersing aggregated light emitting molecules in a precursor of a binder 2) A method of producing dye-containing particles according to item 11, comprising forming a binder from a precursor of the binder by a sol-gel method and forming into a particle .

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.

As used herein, 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. However, the term “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.
<Pigmented particles>
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. In the present specification, when two or more same symbols in one formula exist, they may be the same as or different from each other.
<Aggregation-induced luminescent molecule>
The aggregation inducing luminescent molecules represented by the general formulas (1) to (8) will be described.

In Formula (1), 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.

In the above formula (2), 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.

In the above formula (3), 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.

In the above formula (4), 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 organic metal group.

The compound represented by the formula (4) is a compound having an ortho-carborane skeleton composed of C ₂ B ₁₀ . In the formula (4), black dots represent BH. Further, BH, which can not be illustrated, is omitted from the viewpoint of the three-dimensional structure.

In the formula (5), R and R ′ each independently represent a hydrophilic group, a hydrogen atom, an organic group or an organometallic group.

In the above formula (6), 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.

In the above formula (9), R, R ′ and R ′ ′ are each independently a hydrophilic group, a hydrogen atom, an organic group or an organometallic group.

Types of the hydrophilic group in the formula (1) to (9) in is not particularly limited, for example -OH, -SH, -COOH, -S ( = O) 2 OH, -S (= O) NH 2, _{_{-S (= O) 2 NH 2}} , -P (= O) (OH) 3, -P (= O) R (OH) 2, -P (= O) R 2 (OH), - P (OH) _{3, -P (= O) (} NH 2) 3, -P (= O) R (NH 2) 2, -P (= O) R 2 (NH 2), - P (NH 2) 3, -O (C = O) OH, -NH 2, -NHR, -NHCONH 2, -NHCONHR, -NHCOOH, -Si (OH) 3, -Si (R) (OH) 2, -Si (R) 2 OH, - _{Ge (OH) 3, -Ge (} R) (OH) 2, -Ge (R) 2 OH, -Ti (OH) 3, -Ti (R) (OH) 2, -Ti (R) 2 OH, - _{_{Si (NH 2) 3, -Si}} (R) _{_{NH 2) 2, -B (OH}} ) 2, -O-B (OH) 2, -B (NH 2) 2, -NHB (OH) 2 and the like. Each of R's independently represents hydrogen or an alkyl group having 1 to 20 carbon atoms. Besides, an NHS group, a maleimide group and the like can also be mentioned as a hydrophilic group exhibiting hydrophilicity.

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. Although 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.

In particular, 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. In the present invention, when 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. In addition, a metalloxane bond formed by polycondensation of a metal alkoxide group serves as a binder in the present invention.

In addition, when producing dye-containing particles, as described above, 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. Alternatively, various metal alkoxide monomers may be newly added and subjected to polycondensation. When various metal alkoxide monomers are newly added and polycondensed, 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.

When condensation-induced luminescent molecules having a metal alkoxide group are independently polycondensed, 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. In this way, the aggregation-induced luminescent molecules are in the form of dye-encapsulated particles immobilized three-dimensionally with metalloxane bonds while aggregating each other.

Even when aggregation-induced light emitting molecules having a metal alkoxide group are polycondensed first and then various metal alkoxide monomers are added to carry out additional polycondensation to produce dye-containing particles, while aggregation induced light-emitting molecules are aggregated together It takes the form of a dye-containing particle having a site immobilized three-dimensionally with metalloxane bonds.

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. Be

As an organic group which has the said hydrophilic group in said Formula (6), 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. And 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.

Aggregation-Induced Luminescent Molecules (1) to (6) and (9)>
When the aggregation-induced light emitting molecules represented by the above general formulas (1) to (6) and (9) are analyzed by single crystal structure analysis, the packing properties of the molecules (density and rigidity of the packing, etc.) It was found that it is greatly influenced by the molecular skeleton of the induced luminescent molecule. 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.

Moreover, in the carborane skeleton of the aggregation-induced light emitting molecule represented by (4), the effect of the boron atom becomes a three-dimensionally delocalized π-electron delocalized superaromatic molecule, so 3 It becomes a dimensionally strong packing. In addition, in 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.

Aggregation-Induced Luminescent Molecules (7) and (8)>
When single-crystal structural analysis is performed on the aggregation-induced luminescent molecules represented by the formulas (7) and (8), the packing properties of the molecules (such as the density and rigidity of the packing) indicate the molecules of the aggregation-induced luminescent molecules. It turned out that it is greatly affected by the skeleton. In the general formula (7), when a substituted aromatic group is introduced into the substituent R ¹ , it was confirmed that more robust packing can be obtained by laminating the substituted aromatic group portion in a form in which adjacent aromatic molecules are intermingled. . In addition, when a hydrophilic group other than OH is introduced into the substituent R ¹ , the hydrophilic group portion is gradually displaced, and aggregation-induced luminescent molecules are laminated in a form in which adjacent molecules are intermingled, resulting in a dense and strong packing. That was confirmed. It is considered that the flow of aggregation-induced luminescent molecules is suppressed by the packing of such compact and strong aggregation-induced luminescent molecules.

From the viewpoint of improving the vibration resistance, it is preferable that 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). In the case of using two or more types of aggregation-induced light emitting molecules, it is sufficient to select a combination of aggregation-induced light emitting molecules that respectively emit fluorescence of different wavelengths to produce dye aggregation particles. When using such two or more types of aggregation-induced light-emitting molecules, it is preferable to select one having the emission wavelength peaks separated by 100 nm or more.

Hereinafter, specific examples of the aggregation-induced luminescent molecules represented by the general formulas (1) to (8) which can be used in the present invention will be shown.

R ₁ and R ₂ are each independently selected arbitrarily from the above-mentioned formula (4) -1.

Dye-containing particles
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).

In the present invention, 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”. Examples of 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. In addition, as 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 agent binding group, R, R ′ and R ′ ′ in the formulas (5) and (9) are each independently a hydrophilic group, a hydrogen atom, Aggregation-induced light-emitting molecules that are organic groups, organometallic groups, or silane coupling agent binding groups, and when X in the above formula (6) is S, O or N, where X is O or S, R is ₄ is absent, Y is an electron withdrawing group or an electron donating group, and R ₁ , R ₂ , R ₃ and R ₄ each independently has an organic group or a hydrophilic group, an organic group, an organometallic group, or Represents a silane coupling agent-binding group, and R ₁ to R ₄ may be bonded to each other to form a ring structure; aggregation induced light emitting molecule, R ¹ in the above formula (7) is a substituted aromatic group, OH R ² to R ⁴ are each a hydrophilic group other than or a silane coupling agent binding group Are independently a hydrophilic group, an organic group or an organic metal group, and a to d are each independently an integer of 0 to 5, and when a is 2 or more, even if a plurality of R ¹ are identical And R ^{1 s} may be different from each other or R ^{1 s} may be bonded to each other to form a ring, and when b to d are 2 or more, R ^{2 s} , R ^{3 s} and R ^{4 s} may be the same. And an aggregation-induced light-emitting molecule in which 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, or 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, and 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. For example, 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.

As a result of STEM-EELS observation of the dye-containing particles of the present invention, it was confirmed that aggregation-induced light emitting molecules are aggregated and localized in the particles. Thus, aggregation of the aggregation-induced luminescent molecule in the particle is considered to suppress aggregation-induced emission of the molecule out of the particle. In addition, when single-crystal structure analysis is performed on aggregation-induced luminescent molecules that are aggregated and localized in particles, it is confirmed that aggregation-induced luminescent molecules are tightly packed by the same mechanism as dye-aggregated particles. The

Furthermore, when 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. .

Examples of the resin 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). Among them, 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.

Examples of the inorganic substance 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.

Furthermore, from the viewpoint of suppressing nonspecific adsorption due to hydrophobic binding, etc., the dye-containing particle is preferably hydrophilic. For example, 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. For example, 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). When two or more types of aggregation inducing luminescent molecules are used, it is sufficient to select a combination of aggregation inducing luminescent molecules that emit fluorescence of different wavelengths, respectively, to prepare the dye-containing particles. When using such two or more types of aggregation-induced light-emitting molecules, it is preferable to select one having the emission wavelength peaks separated by 100 nm or more.

<Fluorescent labeling material>
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.

<Targeting ligand>
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. .

For example, 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. When the target biological substance is a glycoprotein, a protein (for example, lectin) having a binding property with a sugar chain is preferably selected as the target-directed molecule.

Other target-directed molecules include, for example, organelle compatible substances, peptides and the like.

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. In the case of using 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.

<Method of producing pigment aggregated particles>
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 In the presence of the aggregation-induced luminescent molecule, 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. By performing the step (A) in the presence of the central nucleus, it is preferable to control the particle size variation coefficient and the average particle size of the pigment-aggregated particles. 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. Specifically, for example, 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 The method (flow-through method) to which microparticles | fine-particles are deposited is mentioned by mixing to (1).

As a micromixer that can be suitably used, 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. Specifically, for example, ether solvents such as tetrahydrofuran and dioxane, ketone solvents such as acetone and methyl ethyl ketone, and 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. Moreover, it is preferable to use 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. Moreover, you may dissolve an inorganic compound, a dispersing agent, etc. in a good solvent as needed.

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. For example, 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. Although it can be, it is not limited to these. From the viewpoint of facilitating removal, it is preferable that the poor solvent has a relatively low boiling point (eg, 40 ° C. to 120 ° C.) as a good solvent. Moreover, you may dissolve an inorganic compound etc. in a poor solvent as needed.

The 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.

Furthermore, 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.

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 For preparing the particles, for example, 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, It is preferable to select in the range of and irradiate a laser.

As 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. Here, 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.

Furthermore, in order to obtain pigment aggregate particles of a desired average particle diameter, ultrafiltration with an ultrafiltration membrane such as YM-10, YM-100 (manufactured by Millipore) is performed to remove particles with large particle diameters. It is also good.
<Method for producing dye-containing particles>
It is preferable that 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.

In addition, in the case of using a precursor of a binder that can be polycondensed by a sol-gel method as a binder when producing dye-containing 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.

When silica is used as the binder, 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.

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.

Moreover, when introducing an alkoxysilane to a part of aggregation induction light emission molecule by a silane coupling agent, first, an active group is introduce | transduced into the said aggregation induction light emission molecule, and it has a substituent which reacts to those activation groups subsequently By causing a reaction with a silane coupling agent and covalently bonding, 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.

Dyes with higher luminous efficiency by selecting 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. In this case, 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 γ-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.

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.). 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.

When the condensation induction light emitting molecule having the alkoxysilane group is independently polycondensed, there is no particular limitation, but it is possible to use a molecule in which one or more alkoxysilane groups are directly bonded to a part of the aggregation induction light emitting dye Preferably, it is more preferable to use a molecule in which two or more alkoxysilane groups are directly bonded.

The polycondensation reaction is preferably carried out in the presence of alcohol, water and ammonia. Here, examples of 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. Usually, the dye-containing particles of the present invention can be prepared by a reaction of several tens minutes to several tens hours.

By adjusting the concentration and reaction time of the aggregation inducing luminescent molecule having the alkoxysilane group to be used, 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.

In the case of producing a dye-containing particle by adding one or two or more kinds of silane compounds to the aggregation-induced light emitting molecule having the alkoxysilane group and performing polycondensation to produce a dye-containing particle, the silane compound is not particularly limited. Silane, tetraethoxysilane (TEOS), γ-mercaptopropyltrimethoxysilane (MPS), γ-mercaptopropyltriethoxysilane, γ-aminopropyltriethoxysilane (APS), 3-thiocyanatopropyltriethoxysilane, 3 And glycidyl oxypropyl triethoxysilane, 3-isocyanatopropyltriethoxysilane, and 3- [2- (2-aminoethylamino) ethylamino] propyl-triethoxysilane etc., and in particular TEOS, MPS or APS is preferred. 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. Here, examples of 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. Usually, 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). On the other hand, if the same process is repeated several times, larger silica particles can be prepared. Thus, the particle diameter (diameter) of the obtained dye-containing particles is, for example, in the order of nm to μm 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.

Moreover, 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. However, in a typical aspect, the dye-containing particles of the present invention can be obtained by a production method including the following polymerization step.

(Polymerization process)
As a polymerization step carried out in the method for producing dye-containing particles of the present invention,
(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. .

Here, 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.

In addition, 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 | numerator.

For example, when a melamine resin is used as the organic resin, 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. Also, if necessary, the polymerization reaction may be carried out in the presence of a suitable surfactant. Furthermore, while promoting the polycondensation reaction of a 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. For the synthesis of thermosetting resins such as melamine resins, the reaction temperature is usually 60 to 200 ° C., and the reaction time is usually 20 to 120 minutes. In addition, 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.

After completion of the polymerization reaction, 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. For example, 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.

On the other hand, when a thermoplastic resin such as a styrene resin is used as the organic resin, the thermoplastic resin can be synthesized according to known methods such as radical polymerization and ionic polymerization (anion polymerization, etc.). Although 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.

In the polymerization step in this case, typically, 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. For the synthesis of a thermoplastic resin, the reaction temperature is usually 20 to 150 ° C., and the reaction time is usually 10 to 240 minutes. Here, as 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.

In the present invention, 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 By further performing modification, 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.

Regardless of the type of binder, 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. Here, 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.

Furthermore, in order to obtain dye-containing particles having a desired average particle diameter, 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.

<Resin>
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 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. 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.

Specific examples of the thermosetting resin include melamine formaldehyde resin, urea formaldehyde resin, benzoguanamine formaldehyde resin, phenol formaldehyde resin and metaxylene formaldehyde resin. In the present invention, a melamine resin represented by a melamine / formaldehyde resin is preferable from the viewpoint of light emission intensity at the time of dye incorporation.

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. For example, in the production of melamine-formaldehyde resin, 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.

In the above 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. Such 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. In addition, 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.

Such a thermosetting resin can be synthesized according to a known method. For example, 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)
The 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.

Specific examples of the thermoplastic resin 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.

However, in the present invention, a styrene-based resin is preferable from the viewpoint of the light emission intensity at the time of aggregation-induced light emitting molecule encapsulation. Here, in the present invention, the "styrene-based resin" refers to a resin which is a homopolymer or copolymer of styrene which may or may not have a substituent.

The above-mentioned 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.

In the above-mentioned 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. Such 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).

Further, the above-mentioned 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). For example, by using a monomer such as glycidyl methacrylate having an epoxy group as a raw material, it is possible to prepare a resin particle for fluorescent labeling in which the epoxy group is oriented on the surface. This epoxy group can be converted to an amino group by reacting with excess ammonia water. Various biomolecules can be introduced to the thus formed amino group according to a known method (through a molecule serving as a linker, if necessary).

<Method for producing fluorescent labeling material>
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.

(I) 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. In particular, when 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.

(Ii) 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. In addition, this amino group can be bonded to an amino group possessed by a biomolecule or the like with a crosslinking agent such as glutaraldehyde. Furthermore, biomolecules and the like can be bound to the surface via an amide bond or a thiourea bond.

(Iii) binding a dye aggregation particle or dye-containing particle to a targeting ligand via specific binding by antigen-antibody reaction, biotin-avidin reaction, hybridization utilizing complementarity of base sequences, etc. it can. Specifically, for example, 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. .

(Surface modification of dye-containing particles)
In the present invention, 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.

Here, the surface modification that can be performed in the present invention is not particularly limited. However, when the dye-containing particles of the present invention are 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.

Here, 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. In the following description in the present specification, a functional group capable of forming a bond with another molecule may also be referred to as a reactive functional group.

As a method of introducing a functional group capable of forming a bond with another molecule, various conventionally known methods can be used.

For example, when the dye-containing particle obtained by the step (a-1) described above has a hydroxyl group on the surface, 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. For example, 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. In addition, 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.

Moreover, when the dye-containing particles obtained by the above-mentioned step (a-1) have an epoxy group on the surface, for example, an amino group can be introduced by treating such dye-containing particles with ammonia water. . In addition, 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.

In addition, even when 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.

In the method for producing a fluorescent labeling material of the present invention, a desired molecule may be bound to the surface by introducing an arbitrary acceptor group on the surface of the dye-containing particle. Examples of the acceptor group include an amino group, a hydroxyl group, a thiol group, a carboxyl group, a maleimide group, and a succinimidyl ester group.

For example, when an aggregation-induced light emitting molecule having an alkoxysilane group is polycondensed alone to produce a dye-containing particle, an OH group is present in the silica particle, and this may be used as an acceptor 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.

In addition, when 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.

When it is desired to introduce an acceptor group different from the acceptor group introduced on the surface by the polycondensed silane compound, this is achieved by additionally bonding a silane compound (silane coupling agent) having a desired group to the particle surface. can do.

Hereinafter, preferred embodiments of the present invention will be more specifically described based on examples, but the present invention is not limited to these examples.
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. 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.

0.1 g of the obtained pigment aggregate particles and 10 mL of sulfuric acid were mixed, and stirred at 50 ° C. for 1 hour to introduce SO ₃ groups on the particle surface.

Subsequently, referring to the amination reaction of aromatic sulfonate with sodium amide (Japan Chemical Society Journal 1974, (8), P. 1522, Nara et al.), 100 mg of pigment aggregate particles, 30 mg of sodium amide, 28 wt% ammonia water The sulfonic acid groups on the particle surface were substituted with amino groups by adding 0.5 mL of H 2 O, 5 mL of water, and reacting at 60 ° C. for 4 hours.

Subsequently, 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. Subsequently, purification was performed by column chromatography to obtain a compound of the following formula (12). Subsequently, 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. Subsequently, liquid separation purification was performed with water / ethyl acetate, and drying under reduced pressure was performed to obtain a compound of the following formula (14). Dye aggregated particles (6) were obtained in the same manner as in Example 1 except that a compound of the following formula (14) was used instead of the compound of the above formula (9).

[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 | transduced was obtained. 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. Subsequently, liquid separation purification was performed with water / ethyl acetate, and drying under reduced pressure was performed to obtain a compound of the following formula (16). 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 | transduced to the aromatic ring of the compound was obtained. 5.35 mg (0.22 mmol) of magnesium and 20 g of tetrahydrofuran are charged in a 100 mL four-necked flask equipped with a thermometer, a stirrer, and a reflux condenser, and 3.76 g of 1,2-dibromoethane while stirring under a nitrogen stream. (0.02 mol) was added to activate. A solution of 101.6 mg (0.2 mmol) of the chlorinated compound dissolved in 20 g of tetrahydrofuran was added dropwise thereto at 55 ° C. to prepare a corresponding Grignard reagent. This Grignard reagent was dropped into 91.3 g (0.6 mol) of tetramethoxysilane. The salt formed was filtered and distilled under reduced pressure to obtain a compound of the following formula (23).

10 mg of a compound of the following formula (23) was dissolved in 1 ml of dimethyl sulfoxide (DMSO), and stirred at room temperature (25 ° C.) for 1 hour. Subsequently, 5 ml of ethanol, 1.5 ml of distilled water and 100 μl of 28% by mass ammonia water were added to the reaction solution, and the reaction was allowed to proceed at room temperature for 24 hours.

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. Was concentrated. 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 | transduced Br group into the aromatic ring of was obtained.

Palladium acetate (3 mmol%, molecular weight 224.51) and xanthophos (3 mmol%, molecular weight 578.63) were placed in a 20 mL pressure test tube and purged with nitrogen. A solution of formic acid (0.035 mmol, molecular weight 46.03) and 119.2 mg (0.2 mmol) of the above bromo compound in 5 mL of DMF was added. The test tube was sealed and heated at 100 ° C. for 20 hours. After confirming the completion of the reaction by TLC, the reaction mixture was filtered and concentrated under reduced pressure. The crude product was purified by silica gel column chromatography to obtain the compound of formula (29) (J. Org. Chem., 2017, 82 (18), pp 9710-9714, Palladium-Catalyzed Carbonylative Transformation of Organic Halides with Formic Acid as a Coupling Partner and CO Source: Synthesis of Carboxylic Acids.

(Polycondensation of aggregated luminescent molecule introduced with NHS group)
5.6 mg of the compound of the formula (29) was dissolved in 1 ml of dimethyl sulfoxide (DMSO). To this was added 1.3 μl of APS, and the reaction was carried out at room temperature (25 ° C.) for 1 hour. To 50 μl of the reaction solution obtained, 3.95 ml of ethanol, 20 μl of MPS, 1 ml of distilled water and 100 μl of 28% by mass ammonia water were added and reacted at room temperature for 24 hours. The reaction solution was ultrafiltered with YM-100 (trade name, manufactured by Millipore). 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.

(Streptavidin modification of pigment aggregation particles)
100 mg of pigment aggregate particles, 30 mg of sodium amide, 0.5 mL of 28 wt% aqueous ammonia according to the amination reaction of aromatic sulfonate with sodium amide (Japan Chemical Society Journal 1974, (8), P. 1522, Nara et al.) By adding 5 mL of water and reacting at 60 ° C. for 4 hours, the sulfonic acid group on the particle surface was substituted with an amino group. Subsequently, purification was performed by ultrafiltration using pure water and YM-100 (manufactured by Millipore).

Subsequently, 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. -dodecaneethylene glycol ester (Thermo Scientific Inc.) was mixed and reacted at 5 ° C. for 1 hour.

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.

On the other hand, 40 μL of streptavidin (manufactured by Wako Pure Chemical Industries, Ltd.) adjusted to 1 mg / mL is added to 210 μL of borate buffer, and then 70 μL of 2-iminothiolane hydrochloride (manufactured by Sigma Aldrich) adjusted to 64 mg / mL is added. 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).

The above 0.04 mg of streptavidin having a thiol group added thereto and 740 μL of dye aggregated particles having a maleimide group introduced to the particle surface adjusted to 0.67 nM are mixed with PBS containing 2 mM of EDTA and reacted for 1 hour at room temperature I did. After that, the reaction is stopped by adding 10 mM mercaptoethanol, and the obtained solution is concentrated by a centrifugal filter, and unreacted streptavidin and the like are removed by a gel filtration column for purification, and the pigment aggregation particle is fluorescence modified with streptavidin. Label 1 was obtained.

[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.

On the other hand, 100 μg of anti-PD-L1 rabbit monoclonal antibody (Cell signaling Technology, Inc .; No. E1L3N) was dissolved in 100 μL of PBS. To this antibody solution, 0.002 mL (0.2 × 10 ^-5 mol) of 1 M 2-mercaptoethanol was added, and the reaction solution reacted at pH 8.5 and room temperature for 30 minutes was passed through a gel filtration column and excess Removal of 2-mercaptoethanol gave a solution of thiolated anti-PD-L1 rabbit monoclonal antibody.

Fluorescent labeling material 2 in which dye aggregate particles are modified with anti-PD-L1 rabbit monoclonal antibody by the same method as in Example 27 except that thiolated anti-PD-L1 rabbit monoclonal antibody is used instead of streptavidin having a thiol group added I got

[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. Furthermore, 1000 μL of a 10% aqueous solution of dodecylbenzenesulfonic acid (manufactured by Kanto Chemical Co., Ltd.) as a surfactant was added to this solution, and the mixture was heated and stirred at 70 ° C. for 50 minutes. Thereafter, the temperature was raised to 90 ° C., and heating and stirring were performed for 20 minutes.

In order to remove excess resin raw material and impurities from the resulting melamine resin dispersion of pigment aggregated particles (6), centrifuge for 15 minutes at 20000 G with a micro-cooling centrifuge 3740 (manufactured by Kubota Corporation). The steps of washing by centrifugation, adding ultrapure water, and applying ultrasonic waves for redispersion were repeated five times to obtain dye-containing melamine particles (27).

Dye-incorporated melamine particles (28) to (32) in the same manner except that the pigment aggregation particles (6) are replaced with pigment aggregation particles (8), (9), (11), (12) and (14) respectively. I got
[Example 30]
<Fluorescent labeling material 3>
With respect to the dye-containing melamine particles (27) to (32) produced in Example 29, a maleimide group was introduced to the surface of each particle by the following method.

0.1 mg of dye-containing particles are dispersed in 1.5 mL of ethanol, 2 μl 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.

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. for 1 hour, centrifuged at 10000 G for 20 minutes, and the supernatant removed Then, PBS containing 2 mM EDTA was added and washing was performed three times to disperse the precipitate, thereby obtaining dye-containing particles in which a maleimide group was introduced to the particle surface.

On the other hand, after adding a thiol group to streptavidin using a streptavidin (manufactured by Wako Pure Chemical Industries, Ltd.) and N-succimidyl-S-acetylthioacetic acid (SATA), gel filtration was performed.

The maleimide-modified dye-containing particles described above and streptavidin-containing thiol group are mixed in PBS containing 2 mM EDTA, and allowed to react at room temperature for 1 hour, whereby each maleimide group and thiol group are reacted. It was combined. After that, the reaction is stopped by adding 10 mM mercaptoethanol, concentrated with a centrifugal filter with φ = 0.65 μm, and then unreacted streptavidin is removed using a gel filtration column for purification, whereby the dye-containing melamine is eliminated. The particles were modified with streptavidin to obtain a fluorescent labeling material 3.

[Example 31]
<Dye-containing polystyrene particles (33) to (44)>
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. Add 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, and 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).

[Example 32]
<Fluorescent labeling material 4>
Streptavidin modification was carried out via the amino group derived from 4-aminostyrene on the surface of the 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). 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 above-described maleimide-modified dye-containing particle and a thiol group-added streptavidin are reacted in the same manner as described in Example 27 to obtain a fluorescent resin in which the dye-containing polystyrene resin particle is modified with streptavidin Label 4 was obtained.

[Example 33]
<Fluorescent labeling material 5>
The dye-containing polystyrene particles (33) to (44) were added to excess ammonia water to convert the epoxy groups on the particle surface into amino groups. The obtained particles are adjusted to 3 nM with PBS, and SM (PEG) ₁₂ (succinimidyl-[(N-maleimidopropionamide) -dodecaethylene glycol] ester, Thermo Scientific Co., Ltd. to a final concentration of 10 mM. (1), react at 120 ° C. for 1 hour, centrifuge at 10000 G for 20 minutes, remove the supernatant, and wash the dispersion with PBS containing 2 mM EDTA to disperse the precipitate three times. Thus, dye-containing polystyrene particles having a maleimide group introduced on the particle surface were obtained.

One hundred μg of anti-PD-L1 rabbit monoclonal antibody (Cell signaling Technology, Inc .; No. E1L3N) was dissolved in 100 μL of PBS. To this antibody solution, 0.002 mL (0.2 × 10 ^-5 mol) of 1 M 2-mercaptoethanol was added, and the reaction solution reacted at pH 8.5 and room temperature for 30 minutes was passed through a gel filtration column and excess Removal of 2-mercaptoethanol gave a solution of thiolated anti-PD-L1 rabbit monoclonal antibody.

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 μl of APS The reaction was performed at room temperature (25 ° C.) for 1 hour. 3.95 ml of ethanol, 20 μl of MPS, 1 ml of distilled water, 100 μl of 28% by mass ammonia water were added to 50 μl of the reaction solution, reacted at room temperature for 24 hours, and collected by ultrafiltration with YM-100 (manufactured by Millipore) The dispersion of the dye-containing particles was concentrated by ultrafiltration with YM-1 (manufactured by Millipore) to a concentration of 1/10 of the total amount. After concentration, dilution with distilled water and ultrafiltration with YM-1 are repeated four times to remove unreacted APS, ammonia, etc. contained in the dye-containing particle dispersion, thereby obtaining colloidal silica particles. The

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.
[Example 34]
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) / (initial luminance) is 0.95 or more BB: (luminance after vibration processing) / (initial luminance) is 0.85 or more and less than 0.95 CC: (vibration processing Later luminance) / (initial luminance) is 0.75 or more and less than 0.85 DD: (luminance after vibration processing) / (initial luminance) is less than 0.75

[Example 35]
<Vibration tolerance evaluation (room temperature)>
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. The

The results of the above Examples 34 and 35 are shown in Tables 2-1 and 2-2 below.

Claims

Dye aggregated particles comprising at least one aggregation inducing luminescent molecule represented by the following general formulas (1) to (9).

In the above formula (1), 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;

In the above formula (2), 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;

In the above formula (3), 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;

In the above formula (4), 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;

In the above formula (5), R and R ′ each independently represent a hydrophilic group, a hydrogen atom, an organic group or an organometallic group;

In the above formula (6), 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 organic group or a 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;

In the above formula (7), 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
When b to d are 2 or more, 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;

In the above formula (8), 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 of R A , R B and R C 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 ;

In the above formula (9), R, R ′ and R ′ ′ are each independently a hydrophilic group, a hydrogen atom, an organic group or an organometallic group.
Dye-containing particles comprising a binder and at least one aggregation-induced luminescent molecule represented by the following general formulas (1) to (9).

In the above formula (1), 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;

In the above formula (2), 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;

In the above formula (3), 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;

In the above formula (4), 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 ;

In the above formula (5), 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;

In the above formula (6), 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 organic metal group, or a silane coupling agent binding group, and R 1 , R 2 , R 3 , R 4 may be combined to form a ring structure;

In the above formula (7), 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
When b to d are 2 or more, 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;

In the above formula (8), 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;

In the above formula (9), R, R ′ and R ′ ′ are each independently a hydrophilic group, a hydrogen atom, an organic group, an organometallic group, or a silane coupling agent binding group.
The pigment aggregation particle according to claim 1, wherein the aggregation inducing luminescent molecule has a hydrophilic group.
The dye-containing particle according to claim 2, wherein the aggregation-induced light emitting molecule has a hydrophilic group.
The dye-containing particle according to claim 2 or 4, wherein the binder and the aggregation light emitting molecule form a covalent bond, and the binder forms a metalloxane bond. .
A fluorescent labeling material in which a targeting ligand is covalently bonded to the surface of the dye-aggregated particle according to claim 1 or 3.
A fluorescent labeling material in which a targeting ligand is covalently bonded to the surface of the dye-containing particle according to claim 2 or 4.
The fluorescence according to claim 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. Signs.
A fluorescent labeling material dispersion comprising the fluorescent labeling material according to any one of claims 6 to 8 and a buffer solution.
The method for producing dye-aggregated particles according to claim 1 or 3, comprising the step of bringing a solution of aggregation-induced luminescent molecules into contact with a poor solvent to aggregate the aggregation-induced luminescent molecules.
The method for producing dye-containing particles according to claim 2 or 4, comprising the step of dispersing the aggregation-induced light-emitting molecule in a binder or a precursor of the binder to form particles.
A method for producing the dye-containing particle according to claim 2 or 4,
1) A process of dispersing aggregated light emitting molecules in a precursor of a binder 2) A process of forming a binder from a precursor of a binder by a sol-gel method and making it into particles The production of dye-encapsulated particles according to claim 11 Method.