JP7226329B2 - Dye Aggregate Particles, Dye Encapsulating Particles, and Fluorescent Labeling Materials - Google Patents

Description

TECHNICAL FIELD The present invention relates to dye-aggregated particles, dye-encapsulating particles, fluorescent labeling materials, and methods for producing them.

In recent years, molecular imaging technology has attracted a great deal of attention in the clinical field and basic research. Molecular imaging is a technology that visualizes the movements of molecules in vivo that could not be visualized until now. It is widely used for various purposes, such as evaluation of the effect of the substance on the living body. Fluorescence imaging, which uses fluorescent substances, is widely used for the detection of minute amounts of substances in living organisms, particularly because of its excellent detection sensitivity and operability.

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 it with a predetermined excitation light. ing. Fluorescence signals obtained by such fluorescence imaging can be used to quantify biomolecular interactions, to observe the dynamics of biomolecules over a long period of time, and to perform ultrahigh-sensitivity observations. There is a need for fluorescent labeling agents that combine the two properties.

Conventionally used fluorescent labeling agents include, for example, commercially available organic fluorescent dyes. In some cases, the aggregation of dye molecules remarkably reduces functions such as luminous efficiency, color development, photosensitivity, and photosensitivity, and has the drawback of limiting the original characteristics of the fluorescent dye.

Another fluorescent labeling material is a quantum dot, which is a nanoparticle with high quantum yield and high light resistance (Patent Document 1). However, since the composition of quantum dots with a relatively high quantum yield contains Cd, which is highly biotoxic, there is a problem that it cannot be used for living cells or living organisms. In addition, quantum dots have unstable fluorescence, such as causing unpredictable blinking phenomena, and because they are particles with a relatively large specific gravity, they interfere with the dynamics of labeled biological substances and interactions with other substances. There is a drawback that it is difficult to accurately observe and quantify biomolecules.

In order to solve the above-mentioned problems, there are colloidal silica particles in which fluorescent dye compounds are dispersed, which are less prone to self-quenching than conventional fluorescent dyes, and also allow many fluorescent dye compounds to be immobilized within the silica particles. Thus, high luminance can be obtained (Patent Document 2).

Furthermore, in recent years, pigment aggregate particles have been developed in which aggregation-inducing luminescent molecules are aggregated and have the property of emitting fluorescence when pigment molecules aggregate with each other (Patent Document 3). This pigment-aggregated particle has the advantage of being brighter than conventional fluorescent labeling materials and having low cytotoxicity. There are various theories about how the aggregation-induced luminescent dye produces high brightness. The mechanism is that the excitation light energy is effectively used for the emission path and the quantum yield is improved. etc. is considered.

Japanese Patent Publication No. 2011-530187 JP 2010-112957 A U.S. Patent Application Publication No. 2013/089889

However, with regard to the particles described in Patent Document 2, fluorescent dyes that could not bond with silica, which is the matrix of the particles, or fluorescent dyes that are only weakly bonded to silica flow out from the particles due to vibrations during distribution or the like. As a result, there is a problem that fluorescence intensity is impaired when used for staining. Furthermore, when the present inventors conducted a follow-up experiment on the pigment-aggregated particles described in Patent Document 3, the particles were disintegrated after the vibration process, and there was a problem that luminance unevenness occurred when used for dyeing. It turned out that there is

The present inventors have found that aggregation-induced luminescent molecules constituting dye aggregated particles and dye-encapsulating particles are aggregation-induced luminescent molecules having a specific structure, thereby disintegrating dye aggregated particles and causing aggregation-induced luminescence in dye-encapsulating particles. It was found that the outflow of sexual molecules can be suppressed.

That is, the present invention provides the following analysis method.
[Item 1] Aggregated pigment particles containing at least one aggregation-inducing luminescent molecule represented by the following general formulas (1) to (9).

前記式（１）中、Ｒ₁、Ｒ₂、Ｒ₃、Ｒ₄、Ｒ₅、およびＲ₆はそれぞれ独立に、親水基、水素原子、有機基または有機金属基である；

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 organometallic group;

前記式（２）中、Ｒ₁、Ｒ₂、Ｒ₃、およびＲ₄はそれぞれ独立に、親水基、 水素原子、有機基または有機金属基である；

In 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;

前記式（３）中、Ｒ₁、Ｒ₂、およびＲ₃はそれぞれ独立して親水基、水素原子、有機基または有機金属基であり、
Ｙは電子吸引性基である；

In 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;

前記式（４）中、白抜きの丸は炭素原子を示し、Ｒ₁およびＲ₂はそれぞれ独立して親水基、水素原子、有機基または有機金属基である；

In the above formula (4), an open circle represents a carbon atom, and R ₁ and R ₂ are each independently a hydrophilic group, a hydrogen atom, an organic group or an organometallic group;

前記式（５）中、ＲおよびＲ'はそれぞれ独立して親水基、水素原子、有機基または有機金属基である；

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

前記式（６）中、ＸはＳ、ＯまたはＮであり、ここでＸがＯまたはＳのときＲ₄は存在せず、
Ｙは電子吸引性基または電子供与性基であり、
Ｒ₁、Ｒ₂、Ｒ₃およびＲ₄はそれぞれ独立に有機基または親水基を有する有機基、有機金属基を表し、Ｒ₁、Ｒ₂、Ｒ₃およびＲ₄はそれぞれ結合して環構造を取っても良い；

In formula (6) above, X is S, O or N, where _R is absent when X is O or S,
Y is an electron-withdrawing or electron-donating group,
R ₁ , R ₂ , R ₃ and R ₄ each independently represent an organic group, an organic group having a hydrophilic group, or an organometallic group, and R ₁ , R ₂ , R ₃ and R ₄ each combine to form a ring structure. may take;

前記式（７）中、Ｒ¹は置換芳香族基または、ＯＨを除く親水基であり、
Ｒ²、Ｒ³、およびＲ⁴はそれぞれ独立して親水基、有機基または有機金属基であり、
ａ～ｄはそれぞれ独立して、０～５の整数であり、ａが２以上の場合、複数のＲ¹は同一であっても異なっていてもよく、複数のＲ¹が互いに結合して環を形成していてもよく、
ｂ～ｄが２以上の場合、複数のＲ²、Ｒ³、およびＲ⁴はそれぞれ同一であっても異なっていてもよく、
Ｒ¹とＲ²、Ｒ²とＲ⁴、Ｒ³とＲ⁴、Ｒ³とＲ¹はそれぞれ結合して環を形成していてもよい；

In the 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 may be the same or different, and a plurality of R ¹ are bonded to each other to form a ring may form
when b to d are 2 or more, a plurality of R ² , R ³ and R ⁴ 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 each combine to form a ring;

前記式（８）中、Ｒ^Aは独立に、親水基、水素原子または有機基であり、
ａは独立に１～５の整数であり、
Ｒ^Bは独立に芳香環含有有機基または親水基を有する芳香環含有基であり、
Ｒ^Cは独立に親水基、水素原子、有機基または有機金属基であり、
Ｒ^A、Ｒ^BおよびＲ^Cの内、少なくとも一つが親水基または親水基を有する芳香環含有基であり、ここでＲ^BおよびＲ^Cを構成する基の中に３級アミノ基は含まず；

In the formula (8), ^RA is independently a hydrophilic group, a hydrogen atom or an organic group,
a is independently an integer from 1 to 5,
^RB 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 the groups constituting R ^B and R ^C do not contain a tertiary amino group;

前記式（９）中、Ｒ、Ｒ'およびＲ’’はそれぞれ独立して親水基、水素原子、有機基または有機金属基である。
[項２] バインダと、下記一般式（１）～（８）で表される少なくとも一種の凝集誘起発光性分子とからなる、色素内包粒子。

In formula (9), R, R' and R'' are each independently a hydrophilic group, a hydrogen atom, an organic group or an organometallic group.
[Item 2] Pigment-encapsulating particles comprising a binder and at least one kind of aggregation-inducing luminescent molecule represented by the following general formulas (1) to (8).

前記式（１）中、Ｒ₁、Ｒ₂、Ｒ₃、Ｒ₄、Ｒ₅、およびＲ₆はそれぞれ独立に、親水基、水素原子、有機基、有機金属基、またはシランカップリング剤結合性基である；

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, an organometallic group, or a silane coupling agent-binding group. is a group;

前記式（２）中、Ｒ₁、Ｒ₂、Ｒ₃、およびＲ₄はそれぞれ独立に、親水基、水素原子、有機基、有機金属基、またはシランカップリング剤結合性基である；

In 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;

前記式（３）中、Ｒ₁、Ｒ₂、およびＲ₃はそれぞれ独立して親水基、水素原子、有機基、有機金属基、またはシランカップリング剤結合性基であり、
Ｙは電子吸引性基である；

wherein R ₁ , R ₂ , and R ₃ are each independently a hydrophilic group, a hydrogen atom, an organic group, an organometallic group, or a silane coupling agent-binding group;
Y is an electron-withdrawing group;

前記式（４）中、白抜きの丸は炭素原子を示し、Ｒ₁およびＲ₂はそれぞれ独立して親水基、水素原子、有機基、有機金属基、またはシランカップリング剤結合性基である；

In the above formula (4), each open circle represents a carbon atom, 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. ;

前記式（５）中、ＲおよびＲ'はそれぞれ独立して親水基, 水素原子、有機基、有機金属基、またはシランカップリング剤結合性基である；

In formula (5), 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;

前記式（６）中、ＸはＳ、ＯまたはＮであり、ここでＸがＯまたはＳのときＲ₄は存在せず、
Ｙは電子吸引性基または電子供与性基であり、
Ｒ₁、Ｒ₂、Ｒ₃およびＲ₄はそれぞれ独立に有機基または親水基を有する有機基、有機金属基、またはシランカップリング剤結合性基を表し、Ｒ₁、Ｒ₂、Ｒ₃およびＲ₄はそれぞれ結合して環構造を取っても良い；

In formula (6) above, X is S, O or N, where _R is absent when X is O or S,
Y is an electron-withdrawing or electron-donating group,
R ₁ , R ₂ , R ₃ and R ₄ each independently represents an organic group or an organic group having a hydrophilic group, an organometallic group, or a silane coupling agent-binding group, and R ₁ , R ₂ , R ₃ and R ₄ may be combined to form a ring structure;

前記式（７）中、Ｒ¹は置換芳香族基、ＯＨを除く親水基、またはシランカップリング剤結合性基であり、
Ｒ²、Ｒ³、およびＲ⁴はそれぞれ独立して、親水基、有機基または有機金属基であり、
ａ～ｄはそれぞれ独立して、０～５の整数であり、ａが２以上の場合、複数のＲ¹は同一であっても異なっていてもよく、複数のＲ¹が互いに結合して環を形成していてもよく、
ｂ～ｄが２以上の場合、複数のＲ²、Ｒ³、およびＲ⁴はそれぞれ同一であっても異なっていてもよく、
Ｒ¹とＲ²、Ｒ²とＲ⁴、Ｒ³とＲ⁴、Ｒ³とＲ¹はそれぞれ結合して環を形成していてもよい；

In the 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 may be the same or different, and a plurality of R ¹ are bonded to each other to form a ring may form
when b to d are 2 or more, a plurality of R ² , R ³ and R ⁴ 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 each combine to form a ring;

前記式（８）中、Ｒ^Aは独立に、親水基、水素原子、有機基、有機金属基、またはシランカップリング剤結合性基であり、
ａは独立に１～５の整数であり、
Ｒ^Bは独立に芳香環含有有機基であり、
Ｒ^Cは独立に親水基、水素原子、有機基または有機金属基である；

In the formula (8), ^RA 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 from 1 to 5,
^RB 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;

前記式（９）中、Ｒ、Ｒ'およびＲ’’はそれぞれ独立して親水基, 水素原子、有機基、有機金属基、またはシランカップリング剤結合性基である。
[項３] 前記凝集誘起発光性分子が親水基を有する、項１に記載の色素凝集粒子。
[項４] 前記凝集誘起発光性分子が親水基を有する、項２に記載の色素内包粒子。
[項５] 項２または４に記載の色素内包粒子であって、前記バインダと凝集発光性分子とが共有結合を形成しており、前記バインダがメタロキサン結合を形成していることを特徴とした色素内包粒子。
[項６] 項１または３に記載の色素凝集粒子の表面に標的指向性リガンドが共有結合を介して結合している蛍光標識材。
[項７] 項２または４に記載の色素内包粒子の表面に標的指向性リガンドが共有結合を介して結合している蛍光標識材。
[項８] 前記標的指向性リガンドが、抗体、細胞小器官親和性物質、および、糖鎖と結合性を有するタンパク質からなる群から選択される１種以上の分子である、項６または７に記載の蛍光標識材。
[項９] 項６～８のいずれか一項に記載の蛍光標識材と、緩衝液とを含む蛍光標識材分散液。
[項１０] 凝集誘起発光性分子の溶液に、貧溶媒を接触させ、凝集誘起発光性分子を凝集させる工程を含む、項１または３に記載の色素凝集粒子の製造方法。
[項１１] 凝集誘起発光性分子をバインダまたはバインダの前駆体中に分散させ、粒子化させる工程を含む、項２または４に記載の色素内包粒子の製造方法。
[項１２] 項２または４に記載の色素内包粒子を製造する方法であり、
１）凝集発光性分子をバインダの前駆体中に分散させる工程
２）ゾルゲル法によりバインダの前駆体からバインダを形成し、かつ粒子化させる工程を含む、項１１に記載の色素内包粒子の製造方法。

In 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.
[Item 3] The dye aggregated particles according to Item 1, wherein the aggregation-inducing luminescent molecule has a hydrophilic group.
[Item 4] The dye-encapsulating particles according to item 2, wherein the aggregation-inducing luminescent molecule has a hydrophilic group.
[Item 5] The dye-encapsulating particles according to Item 2 or 4, wherein the binder and the aggregated light-emitting molecules form a covalent bond, and the binder forms a metalloxane bond. Pigment-encapsulating particles.
[Item 6] A fluorescent labeling material in which a targeting ligand is covalently bound to the surface of the dye-aggregated particle according to item 1 or 3.
[Item 7] A fluorescent labeling material in which a targeting ligand is bound to the surface of the dye-encapsulating particles according to item 2 or 4 via a covalent bond.
[Item 8] Item 6 or Item 7, wherein the targeting ligand is one or more molecules selected from the group consisting of antibodies, organelle affinity substances, and proteins having binding properties to sugar chains. Fluorescent labeling agent as described.
[Item 9] A fluorescent labeling agent dispersion containing the fluorescent labeling agent according to any one of Items 6 to 8 and a buffer solution.
[Item 10] A method for producing aggregated dye particles according to Item 1 or 3, comprising the step of contacting a solution of aggregation-inducing luminescent molecules with a poor solvent to aggregate the aggregation-inducing luminescent molecules.
[Item 11] A method for producing dye-encapsulated particles according to Item 2 or 4, comprising the step of dispersing the aggregation-inducing luminescent molecules in a binder or a precursor of the binder to form particles.
[Item 12] A method for producing the dye-encapsulated particles according to Item 2 or 4,
Item 12. The method for producing dye-encapsulated particles according to Item 11, comprising the steps of: 1) dispersing the aggregated luminescent molecules in a binder precursor; and 2) forming a binder from the binder precursor by a sol-gel method and granulating the binder. .

The dye-aggregated particles, dye-encapsulating particles, and fluorescent labeling material of the present invention are superior in durability to conventional fluorescent labeling materials, and maintain the same dyeability as that at the time of production even after going through the process of distribution.

As used herein, the term “aggregation-induced luminescent molecule” means that when each molecule is dissolved or dispersed in a dilute solution, it does not emit fluorescence or has a weak fluorescence emission intensity due to its low quantum yield. However, it refers to a fluorescent substance that has the property of increasing the quantum yield and emitting strong fluorescence or increasing the fluorescence intensity by aggregating to form an aggregate.
<Dye aggregation particles>
The "aggregated pigment particles" of the present invention contain at least one aggregation-inducing luminescent molecule represented by the following general formulas (1) to (9). The aggregation-inducing luminescent molecules contained in the pigment aggregation particles may be of one type or two or more types. In this specification, when two or more of the same symbols are present in one formula, they may be the same or different.
<Aggregation-induced luminescent molecule>
Aggregation-induced luminescent molecules represented by general formulas (1) to (8) will be described.

前記式（１）中、Ｒ₁、Ｒ₂、Ｒ₃、Ｒ₄、Ｒ₅、およびＲ₆はそれぞれ独立に、親水基、水素原子、有機基または有機金属基である。

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 organometallic group.

前記式（２）中、Ｒ₁、Ｒ₂、Ｒ₃、およびＲ₄はそれぞれ独立に、親水基、 水素原子、有機基または有機金属基である。

In 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.

前記式（３）中、Ｒ₁、Ｒ₂、およびＲ₃はそれぞれ独立して親水基、水素原子、有機基または有機金属基であり、
Ｙは電子吸引性基である。

In 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.

前記式（４）中、白抜きの丸は炭素原子を示し、Ｒ₁およびＲ₂はそれぞれ独立して親水基、水素原子、有機基または有機金属基である。

In the above formula (4), each open circle represents a carbon atom, and R ₁ and R ₂ are each independently a hydrophilic group, a hydrogen atom, an organic group or an organometallic group.

The compound represented by formula (4) is a compound having an ortho-carborane skeleton composed of _{C2B10 .} In addition, in Formula (4), a black point represents BH. Moreover, BH, which cannot be illustrated from the viewpoint of the three-dimensional structure, is omitted.

前記式（５）中、ＲおよびＲ'はそれぞれ独立して親水基、水素原子、有機基または有機金属基である。

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

前記式（６）中、ＸはＳ、ＯまたはＮであり、ここでＸがＯまたはＳのときＲ₄は存在せず、
Ｙは電子吸引性基または電子供与性基であり、
Ｒ₁、Ｒ₂、Ｒ₃およびＲ₄はそれぞれ独立に有機基または親水基を有する有機基、有機金属基を表し、Ｒ₁、Ｒ₂、Ｒ₃およびＲ₄はそれぞれ結合して環構造を取っても良い。

In formula (6) above, X is S, O or N, where _R is absent when X is O or S,
Y is an electron-withdrawing or electron-donating group,
R ₁ , R ₂ , R ₃ and R ₄ each independently represent an organic group, an organic group having a hydrophilic group, or an organometallic group, and R ₁ , R ₂ , R ₃ and R ₄ each combine to form a ring structure. You can take it.

前記式（７）中、Ｒ¹は置換芳香族基または、ＯＨを除く親水基であり、
Ｒ²、Ｒ³、およびＲ⁴はそれぞれ独立して親水基、有機基または有機金属基であり、
ａ～ｄはそれぞれ独立して、０～５の整数であり、ａが２以上の場合、複数のＲ¹は同一であっても異なっていてもよく、複数のＲ¹が互いに結合して環を形成していてもよく、
ｂ～ｄが２以上の場合、複数のＲ²、Ｒ³、およびＲ⁴はそれぞれ同一であっても異なっていてもよく、
Ｒ¹とＲ²、Ｒ²とＲ⁴、Ｒ³とＲ⁴、Ｒ³とＲ¹はそれぞれ結合して環を形成していてもよい；

In the 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 may be the same or different, and a plurality of R ¹ are bonded to each other to form a ring may form
when b to d are 2 or more, a plurality of R ² , R ³ and R ⁴ 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 each combine to form a ring;

前記式（８）中、Ｒ^Aは独立に、親水基、水素原子または有機基であり、
ａは独立に１～５の整数であり、
Ｒ^Bは独立に芳香環含有有機基または親水基を有する芳香環含有基であり、
Ｒ^Cは独立に親水基、水素原子、有機基または有機金属基であり、
Ｒ^A、Ｒ^BおよびＲ^Cの内、少なくとも一つが親水基または親水基を有する芳香環含有基であり、ここでＲ^BおよびＲ^Cを構成する基の中に３級アミノ基は含まず；

In the formula (8), ^RA is independently a hydrophilic group, a hydrogen atom or an organic group,
a is independently an integer from 1 to 5,
^RB 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 the groups constituting R ^B and R ^C do not contain a tertiary amino group;

前記式（９）中、Ｒ、Ｒ'およびＲ’’はそれぞれ独立して親水基、水素原子、有機基または有機金属基である。

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

The types of the hydrophilic groups in the formulas (1) to (9) are not particularly limited, but examples include -OH, -SH, -COOH, -S(=O) ₂ OH, -S(=O)NH ₂ , -S(=O) _2NH2 , -P(=O)(OH) ₃ , -P(=O)R(OH) ₂ , -P( ₌ O)R2(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 , —OB(OH) 2 , —B(NH ₂ ) ₂ , —NHB(OH) ₂ and the _like mentioned. Each R independently represents hydrogen or an alkyl group having 1 to 20 carbon atoms. In addition, an NHS group, a maleimide group, and the like can also be mentioned as hydrophilic groups exhibiting hydrophilicity.

The organic group in the formulas (1) to (9) is, for example, a hydrocarbon group having 1 to 20 carbon atoms, such as an alkyl group, an alkenyl group, an alkynyl group, a cycloalkyl group, a cycloalkenyl group. , a cycloalkynyl group, an aromatic group, and the like. One or more of the hydrogen atoms at arbitrary positions in these molecules may be substituted with heteroatoms such as S, N, O and the like.

The organometallic group in the formulas (1) to (9) is, for example, a group having a metal atom in a part of a hydrocarbon group having 1 to 20 carbon atoms through covalent or coordinate bonding, particularly Those containing a covalent bond between a metal atom and an oxygen atom are preferred. The metal atoms are not limited, but are for example magnesium, calcium, strontium, scandium, yttrium, ruthenium, lawrencium, 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, preferably titanium, zirconium, silicon.

In particular, the organometallic group is preferably a metal alkoxide group such as a titanium alkoxide skeleton, a zirconium alkoxide skeleton, or a silicon alkoxide skeleton. can form a repeating covalent bond by Furthermore, the organometallic group more preferably has a silicon alkoxide skeleton, and the reactivity of the aggregation-induced luminescent molecule having such an organometallic group is easily controlled. In the present invention, when the organometallic group of the aggregation-inducing light-emitting molecule is a metal alkoxide and polycondensation of the aggregation-inducing light-emitting molecule is performed, the metal alkoxide group serves as a binder precursor. Moreover, the metalloxane bond formed by polycondensation of the metal alkoxide group serves as a binder in the present invention.

Further, when the dye-encapsulating particles are produced, aggregation-inducing light-emitting molecules having a metal alkoxide group as an organometallic group as described above may be singly polycondensed to form a stable metalloxane bond as described above. Alternatively, various metal alkoxide monomers may be newly added for polycondensation. When various metal alkoxide monomers are newly added and polycondensed, the aggregation-inducing light-emitting molecules having metal alkoxide groups and various metal alkoxide monomers may be mixed at the same time and polycondensed. The dye-encapsulating particles may be produced by first polycondensing the light-emitting molecules and then adding various metal alkoxide monomers for additional polycondensation.

When aggregation-inducing light-emitting molecules having metal alkoxide groups are subjected to polycondensation alone, the aggregation-inducing light-emitting molecules become aggregated particles by adjusting the concentration, solvent polarity, etc., and then undergo a polycondensation reaction via the metal alkoxide groups. As a result, the aggregation-inducing luminescent molecules take the form of dye-encapsulating particles that are three-dimensionally immobilized by metalloxane bonds while aggregating with each other.

When dye-encapsulating particles are produced by first polycondensing aggregation-inducing light-emitting molecules having a metal alkoxide group and then adding various metal alkoxide monomers for additional polycondensation, the aggregation-inducing light-emitting molecules are aggregated with each other. It takes the form of dye-encapsulating particles having sites three-dimensionally immobilized by metalloxane bonds.

The dye-encapsulating particles produced by such a method can form a strong and stable metalloxane bond between aggregation-induced light-emitting molecules, between binders, and between a binder and aggregation-induced light-emitting molecules. becomes higher.

The electron-withdrawing group in the formulas (3) and (6) includes, for example, a cyano group, a nitro group, a methoxy group, a tosyl group, a mesyl group, a halogen, a phenyl group, an acyl group, a keto group, a carboxyl group, an aldehyde group, 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 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 done.

Examples of the organic group having a hydrophilic group in the formula (6) include a group in which at least one hydrogen atom of the organic group is substituted with the hydrophilic group.

Examples of the aromatic ring-containing organic group in the formula (8) include a phenyl group, 1-naphthyl group, 2-naphthyl group, pyrenyl group, anthracenyl group, anthraquinonyl group, tolyl group, benzyl group, trityl group and styryl group. , a benzylidene group, an aniline group, a pyridyl group, a quinolyl group, a tosyl group, a tetraphenylethylene group, a triphenylethylene group, a diphenylethylene group, a triazinyl group, derivatives thereof, and derivatives to which a substituent is added.

<Aggregation-induced luminescent molecules (1) to (6) and (9)>
When the aggregation-induced luminescent molecules represented by the above general formulas (1) to (6) and (9) are analyzed by single crystal structure analysis, the packing property between molecules (packing density, strength, etc.) is determined by aggregation It was found that the molecular structure of the induced luminescence molecule had a great influence. Each of the aggregation-inducing luminescent molecules represented by the above general formulas (1) to (6) has a heterocyclic skeleton, and the positional relationship of the electron-rich N, S, O and B parts is specified. It was packed so as to have a period of Among them, the molecular skeleton of the aggregation-induced luminescent molecules represented by (1) to (3) and (6) has a high periodicity, and the N element, the S element, and the O element are arranged in the same plane, so that the heteroatom is the optimal arrangement, and it is presumed that a stronger packing is formed.

In addition, in the carborane skeleton of the aggregation-induced luminescent molecule represented by (4), the π electrons are three-dimensionally delocalized due to the effect of the boron atom, resulting in a hyperaromatic molecule. It results in dimensionally rigid packing. In addition, in the maleimide skeleton of the aggregation-inducing luminescent molecule represented by (5), hydrogen bonding between O of the carbonyl group and H of NH is presumed to strengthen interaction with adjacent molecules.

<Aggregation-induced luminescent molecules (7) and (8)>
When the aggregation-induced luminescent molecules represented by the formulas (7) and (8) are analyzed by single crystal structure analysis, it is found that the packing property between molecules (packing density, strength, etc.) is determined by aggregation-induced luminescent molecules. It was found to be greatly affected by the skeleton. In the general formula (7), it was confirmed that when a substituted aromatic group is introduced into the substituent R ¹ , the substituted aromatic group portion stacks in the form of interpenetrating adjacent molecules, resulting in stronger packing. . 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-inducing light-emitting molecules are stacked in such a way that they intertwine with adjacent molecules, resulting in dense and strong packing. was confirmed. Such dense and strong packing of aggregation-inducing light-emitting molecules is thought to suppress outflow of aggregation-inducing light-emitting molecules.

From the viewpoint of improving vibration resistance, the aggregation-inducing luminescent molecule preferably has a hydrophilic group. If the aggregation-induced luminescent molecule has a hydrophilic group, the electric double layer is thickened, and it is considered that the particle morphology becomes more stable in an aqueous solvent such as a buffer.

Aggregation-inducing luminescent molecules used for producing the dye-aggregated particles can be selected from those that emit fluorescence of a desired wavelength (color). When two or more aggregation-inducing light-emitting molecules are used, a combination of aggregation-inducing light-emitting molecules that emit fluorescence of different wavelengths may be selected to produce aggregated dye particles. When using two or more types of such aggregation-inducing light-emitting molecules, it is preferable to select those whose emission wavelength peaks are separated from each other by 100 nm or more.

Specific examples of aggregation-inducing luminescent molecules represented by general formulas (1) to (8) that can be used in the present invention are shown below.

Ｒ₁およびＲ₂は上記の式（４）‐１の中からそれぞれ独立に任意に選択される。

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

＜色素内包粒子＞
本発明の「色素内包粒子」は、バインダと、上記一般式（１）～（８）で表される少なくとも一種の凝集誘起発光性分子とからなることを特徴とする。

<Dye-Encapsulating Particles>
The "dye-encapsulating particles" of the present invention are characterized by comprising a binder and at least one kind of aggregation-inducing luminescent molecules represented by the general formulas (1) to (8).

In the present invention, the binder means "a substance that retains a certain form by encapsulating the aggregation-inducing luminescent molecules" or "a substance that retains a certain form by binding together the aggregation-inducing luminescent molecules". The binder, which is "a substance that retains a certain form by enclosing the aggregation-induced luminescent molecules", is a resin, an inorganic substance, or the like, and can include the aggregation-induced luminescent molecules. In addition, as a binder that "holds a fixed form by binding together aggregation-inducing luminescent molecules", a binder that binds to an adjacent aggregation-inducing luminescent molecule via a substituent possessed by the aggregation-inducing luminescent molecule is used. It is a binding part, a linker for binding aggregation-inducing light-emitting molecules to each other, and the like, and can be kept in a certain form by binding aggregation-inducing light-emitting molecules to each other.

In the aggregation-inducing luminescent molecule contained in the "dye-encapsulating particles" of the present invention, each of R ₁ , R ₂ , R ₃ , R ₄ , R ₅ and R ₆ in the formula (1) is independently a hydrophilic group _, a hydrogen atom, an organic group, _{an organometallic} group, or an aggregation-inducing light- _emitting molecule that is a silane coupling agent-binding group; _, a hydrophilic group, a hydrogen atom, an organic group, an organometallic group, or an aggregation _- inducing light-emitting molecule that _is a silane coupling agent-binding group; is a hydrophilic group, a hydrogen atom, an organic group, an organometallic group, or a silane coupling agent-binding group, and Y is an electron-withdrawing group; represents a carbon atom, 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; and R, R′ and R″ in (9) are each independently a hydrophilic group, a hydrogen atom, an organic group, an organometallic group, or a silane coupling agent-binding group; X in (6) is S, O or N, where when X is O or S, R ₄ is absent, Y is an electron-withdrawing or electron-donating group, and R ₁ , R ₂ , R ₃ and R ₄ each independently represents an organic group or an organic group having a hydrophilic group, an organometallic group, or a silane coupling agent-binding group, and R ₁ to R ₄ are each bonded to form a ring structure. Alternatively, the aggregation-inducing luminescent molecule, R ¹ in the above formula (7) is a substituted aromatic group, a hydrophilic group other than OH, or a silane coupling agent-binding group, and R ² to R ⁴ are each independently is 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 may be the same or different. a plurality of R ¹ may combine with each other to form a ring, and when b to d are 2 or more, a plurality of R ² , R ³ and R ⁴ 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 each combine to form a ring to form an aggregation-inducing luminescent molecule, or the above formula ( ^RA in 8) is independently a hydrophilic group, a hydrogen atom, an organic group, an organometallic group, or a silane coupling agent-binding group, and a is independently 1 to is an integer of 5, R ^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 organometallic group. .

The silane coupling agent-binding group is not particularly limited, but examples include N-hydroxysuccinimide (NHS) ester group, maleimide group, isocyanate group, isothiocyanate group, aldehyde group, paranitrophenyl group, diethoxymethyl group. , an epoxy group, a cyano group, an alkoxysilane group, a 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 assembling aggregation-inducing luminescent 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-encapsulating particles of the present invention, it was confirmed that the aggregation-inducing luminescent molecules were aggregated and unevenly distributed in the particles. Aggregation of the aggregation-inducing light-emitting molecules inside the particles in this way is thought to suppress the outflow of the aggregation-inducing light-emitting molecules to the outside of the particles. In addition, when the aggregation-induced luminescent molecules that are aggregated and unevenly distributed in the particles are analyzed by single crystal structure analysis, it is confirmed that the aggregation-induced luminescent molecules are tightly packed with each other by a mechanism similar to that of pigment aggregated particles. rice field.

Furthermore, since the aggregation-inducing luminescent molecule has a hydrophilic group, an electrostatic interaction occurs with a compound such as a resin or an inorganic substance that forms the binder of the particles, so that the outflow of the aggregation-inducing luminescent 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- styrene-methyl acrylate copolymer), and various homopolymers and copolymers made using one or more monomers. Among them, melamine resins and styrene resins are preferably used because particles encapsulating aggregation-inducing light-emitting molecules can be easily produced and the resulting dye-encapsulating particles have a high emission intensity.

Examples of the inorganic substance include zirconium oxide, alumina, silica, etc.
Silica is more preferable from the viewpoint of improving vibration resistance at room temperature and controlling reactivity. Silica is generally known to be chemically inert and easy to modify. of molecules can be attached to the surface.

Further, from the viewpoint of suppressing non-specific adsorption due to hydrophobic bonding, etc., the dye-encapsulating particles are preferably hydrophilic. For example, dye-encapsulated particles are produced using a hydrophilic substance such as melamine resin as a binder, or the surface of dye-encapsulated particles produced using a hydrophobic substance is modified with a hydrophilic compound to increase hydrophilicity. of dye-encapsulating particles can be obtained.

The hydrophilic compound used for hydrophilizing the surface of the dye-encapsulating particles is not particularly limited. It is preferable because it is easy to adjust the molecular length by adjusting the number of units, and because it is easy to prepare derivatives having various functional groups or the like linked to their ends and to obtain them as products.

Aggregation-inducing luminescent molecules used for producing dye-encapsulating particles can be selected from those that emit fluorescence of a desired wavelength (color). When two or more types of aggregation-inducing luminescent molecules are used, a combination of aggregation-inducing luminescent molecules that emit fluorescence of different wavelengths may be selected to produce dye-encapsulating particles. When using two or more types of such aggregation-inducing light-emitting molecules, it is preferable to select those whose emission wavelength peaks are separated from each other by 100 nm or more.

<Fluorescent labeling material>
The "fluorescent labeling agent" of the present invention is characterized in that a targeting ligand is bound to the surface of the dye-aggregated particles or the dye-encapsulating particles via a covalent bond. The form of the fluorescent labeling material, for example, the form during manufacture, storage, and distribution, is not particularly limited, but is preferably in the form of a dispersion using a known buffer solution such as PBS as a dispersion medium. One aspect of the present invention is in the form of a dispersion, ie, a fluorescent label dispersion, which includes 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. It is preferably a substance that specifically recognizes and binds to a substance as a target substance. The target biological substance is not particularly limited, and examples thereof include proteins, nucleic acids, sugar chains, lipids, and the like. Preferably, the target biological material is any disease-related biological material. Specific examples include marker proteins that are specifically expressed in cancer cells (e.g., cancer-specific proteins, vascular endothelial cell-specific proteins, phosphorylated proteins, etc.), pro-inflammatory proteins, and immune-related proteins. .

For example, when the biological substance of interest is a protein that is specifically expressed in tumor tissue or cancer cells, an antibody against these is preferably selected as the targeting ligand. When the target biological substance is a glycoprotein, the target-directing molecule is preferably selected from proteins (eg, lectins) having binding properties to sugar chains.

Other targeting molecules include, for example, organelle-affinity agents, peptides, and the like.

When an antibody is selected as the targeting ligand, it is usually IgG or IgM, with IgG being preferred. The antibody may be a natural antibody such as full-length IgG, or Fab, Fab', F(ab'), as long as it has the ability to specifically recognize and bind to the target protein or low-order antibody. ) ₂ , antibody fragments such as Fv and scFv, or non-natural antibodies such as artificial antibodies multifunctionalized (multivalent or multispecific) using antibody fragments thereof . A primary antibody that recognizes and binds to an epitope unique to the antigen is preferably used. When a secondary antibody, which is an antibody that recognizes and binds to a unique epitope to the primary antibody, is used as the targeting ligand, the target substance is used as the target substance after binding the primary antibody to the biological substance of interest.

<Method for producing aggregated pigment particles>
The dye aggregated particles of the present invention preferably include a step (A) of contacting a solution of the aggregation-inducing luminescent molecules with a poor solvent to aggregate the aggregation-inducing luminescent molecules, and the step (A) comprises a central nucleus It may be a step of contacting the solution of the aggregation-inducing luminescent molecules with a poor solvent in the presence of the aggregation-inducing luminescent molecules to aggregate the aggregation-inducing luminescent molecules. The steps other than the step (A) are not particularly limited, and for example, a step of introducing a hydrophilic group into the aggregation-inducing luminescent molecule, a step of introducing a hydrophilic group into the surface of the pigment aggregated particles, and the like are appropriately carried out. Carrying out the step (A) in the presence of the central nucleus is preferable because it facilitates control of the particle size variation coefficient and the average particle size of the pigment-aggregated particles. The central nucleus may be premixed in a solution of aggregation-inducing luminescent molecules, or may be premixed in a poor solvent.

The substance used as the central core is not particularly limited, and for example, fine particles made of organic molecules such as polystyrene and latex, and inorganic molecules such as silica are preferably used. The nature and size of the central nucleus can be selected according to the desired particle size of the dye aggregate particles and the properties of the aggregation-inducing luminescent molecules used for preparation. The central nucleus preferably has an average particle size of 1 nm or more and 20 nm or less and a particle size variation coefficient of 5% or less.

The dye aggregated particles in the present invention use a solvent (good solvent) capable of dissolving the aggregation-inducing luminescent molecules, prepare a separate solution of the aggregation-inducing luminescent molecules, and then add the aggregated luminescent molecules to the solution. It can be prepared by a reprecipitation method of precipitating pigment aggregate particles by mixing with a poor solvent for the induced luminescent molecule. By using such a reprecipitation method, particles packed with aggregation-inducing luminescent molecules at a higher density can be produced. Specifically, for example, it is a reprecipitation method using a mixer with a small inner diameter called a micromixer. a method (circulation method) of precipitating fine particles by mixing with

As a micromixer that can be suitably used, the inner diameter of the channel of the mixing part for mixing the separate solution of the aggregation-inducing luminescent molecule and the poor solvent (if the cross section of the channel is not circular, the cross-sectional area of the channel is preferably 2 mm or less, and the inner diameter of the channel is preferably 1 mm or less for rapid mixing of the solution and the poor solvent. Moreover, in order to prevent clogging of the channel by fine particles and to reduce pressure loss inside the channel, the inner diameter of the channel 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 for the aggregation-induced luminescent molecules, and it is preferable to select one that is well mixed with the below-described poor solvent. Specifically, for example, ether solvents such as tetrahydrofuran and dioxane, ketone solvents such as acetone and methyl ethyl ketone, amides such as 1-methyl-2-pyrrolidinone, 1,3-dimethylimidazolinone, and N,N-dimethylformamide A system solvent, a sulfur-containing solvent such as dimethylsulfoxide, or a mixed solvent of two or more thereof can be preferably used. Moreover, from the viewpoint of preventing re-dispersion of the aggregation-induced luminescent molecules, it is preferable to use a good solvent having a boiling point lower than that of the poor solvent. Moreover, you may dissolve an inorganic compound, a dispersing agent, etc. in a good solvent as needed.

The poor solvent used in the present invention is not particularly limited as long as it has a relatively low solubility for the aggregation-induced luminescent molecules, and it is preferable to select a solvent having good miscibility with the aforementioned good solvent. For example, water or an aqueous solution is preferable, and alcoholic 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 thereof are used. can be, but are not limited to. From the viewpoint of facilitating removal, the poor solvent preferably has a boiling point relatively lower than that of the good solvent (for example, 40° C. to 120° C.). Also, if necessary, an inorganic compound or the like may be dissolved in a poor solvent.

The reaction conditions such as the reaction time and the reaction temperature are not particularly limited as long as the dye-aggregated particles satisfying the above-mentioned conditions are produced. It is preferred that the separate solution and the poor solution of the aggregation-inducing luminescent molecule are rapidly mixed within, preferably under turbulent flow conditions such that the Reynolds number is 4,000 or more.

In addition, the present invention is a method of performing laser ablation on a dispersion liquid in which relatively coarse crystals of aggregation-induced luminescent molecules are dispersed in a poor solvent (Japanese Patent Application Laid-Open No. 2005-238342). can produce aggregated nanoparticles with small

When the laser ablation is performed, various known lasers can be used, and YAG laser, excimer laser, titanium-sapphire laser and the like are preferably used. As the irradiation laser, it is preferable to apply a pulse wave. In order to prepare aggregated nanoparticles with a more uniform particle size distribution, it is preferable to adjust the concentration of the dispersion to 0.1 mg/L to 500 mg/L before laser ablation. The irradiation power, pulse width, wavelength, and irradiation time can be appropriately adjusted according to the type and size of the crystals of the target aggregation-induced luminescent dye, and the mixing ratio with the poor solvent. For preparing 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 irradiate the laser by selecting within the range of .

As the poor solvent, alcoholic solvents such as water, 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 thereof can be used. can be, but are not limited to, Said laser ablation method can be carried out, for example, in an apparatus set up according to the method described in The Review of Laser Engineering, 33, 41-46.

The pigment-aggregated particles may be purified using a conventional method such as an ultrafiltration membrane, if necessary. Purification makes it possible to remove ions and unreacted substances in the reaction solution, and to obtain spherical or nearly spherical dye aggregate particles. Here, the near-spherical microparticles are specifically microparticles having a shape in which the ratio of the long axis to the short axis is 2 or less.

Furthermore, in order to obtain pigment aggregated particles having a desired average particle size, ultrafiltration is performed using an ultrafiltration membrane such as YM-10 or YM-100 (manufactured by Millipore) to remove particles having a large particle size. good too.
<Method for producing pigment-encapsulating particles>
The method for producing the dye-encapsulating particles of the present invention preferably includes a step of dispersing the aggregation-inducing luminescent molecules in a binder or a binder precursor to form particles.

Further, when a precursor of a binder that can be polycondensed by a sol-gel method is used as the binder when producing the dye-encapsulated particles, the method for producing the dye-encapsulated particles includes:
It is preferable to include the steps of 1) dispersing the aggregated luminescent molecules in a binder precursor, and 2) forming a binder from the binder precursor by a sol-gel method and granulating the binder.

When silica is used as the binder, any technique may be used as a method for producing the pigment-encapsulating particles as long as the silica particles encapsulating aggregation-inducing luminescent molecules are formed. Specifically, for example, it can be obtained by a method of preparing aggregation-induced luminescent molecules having an alkoxysilane group and subjecting them to polycondensation. A monofunctional alkoxysilane group, a bifunctional alkoxysilane group, or a trifunctional alkoxysilane group can be used as the alkoxysilane group. Polycondensation can usually be carried out by a sol-gel method.

A method for producing aggregation-induced luminescent molecules having an alkoxysilane group is not particularly limited, but examples include a method of directly introducing an alkoxysilane group into a part of molecules of an aggregation-induced luminescent dye, and aggregation-induced luminescence using a silane coupling agent. a method of introducing alkoxysilane into a part of the organic molecules, and the like.

When the alkoxysilane group is directly introduced into the aggregation-inducing light-emitting molecule, it can be introduced at any position on the molecular skeleton of the aggregation-inducing light-emitting molecule. Dye-encapsulating particles with high luminous efficiency can be obtained.

Further, when alkoxysilane is introduced into a part of the aggregation-inducing light-emitting molecules using a silane coupling agent, an active group is first introduced into the aggregation-inducing light-emitting molecule, and then a substituent that reacts with the active group is provided. Aggregation-inducing light-emitting molecules having an alkoxysilane group can be obtained by reacting with a silane coupling agent and forming a covalent bond.

Although the active group is not particularly limited, N-hydroxysuccinimide (NHS) ester group, maleimide group, isocyanate group, isothiocyanate group, aldehyde group, paranitrophenyl group, diethoxymethyl group, epoxy group, cyano group, It can be selected from halogen atoms and the like.

By selecting an N-hydroxysuccinimide (NHS) ester group or a maleimide group as the active group to be introduced into the aggregation-inducing luminescent molecule and using a silane coupling agent having an amino group as the silane coupling agent, a dye with higher luminous efficiency can be obtained. Encapsulated particles can be obtained. In this case, an NHS ester group and an amino group of a 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-inducing light-emitting molecule having the alkoxysilane group, the aggregation-inducing light-emitting molecule and silica are bonded via an amide bond.

The silane coupling agent having an amino group is not particularly limited, but examples include γ-aminopropyltriethoxysilane (APS), 3-[2-(2-aminoethylamino)ethylamino]propyl-triethoxysilane, N -2(aminoethyl)-3-aminopropylmethyldimethoxysilane, 3-aminopropyltrimethoxysilane, etc., and APS is particularly preferred.

The reaction between the aggregation-inducing luminescent molecule having the NHS ester group and the silane coupling agent having the amino group is performed after dissolving each in a solvent such as DMSO (dimethylsulfoxide) or DMF (N,N-dimethylformamide). , at room temperature (for example, 25° C.) with stirring. The ratio between the aggregation-inducing light-emitting molecule and the silane coupling agent is not particularly limited, but the ratio is preferably 1:0.5 to 2 (molar ratio), and the ratio is 1:0.8 to 1.2 (molar ratio). is more preferred.

The dye-encapsulating particles of the present invention can be produced by polycondensing the aggregation-inducing light-emitting molecules having an alkoxysilane group prepared by the above-described method "by polycondensation alone" or by adding one or more silane compounds and polycondensing them. method”.

When the aggregation-inducing light-emitting molecule having the alkoxysilane group is polycondensed alone, it is not particularly limited, but it is preferable to use a molecule in which one or more alkoxysilane groups are directly bonded to a part of the molecule of the aggregation-inducing light-emitting dye. More preferably, a molecule in which two or more alkoxysilane groups are directly bonded is used.

Also, this polycondensation reaction is preferably carried out in the presence of alcohol, water and ammonia. Examples of alcohols include lower alcohols having 1 to 3 carbon atoms such as methanol, ethanol and propanol. The ratio of water and 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 per 1 part by volume of water. This is the range of the capacity part. The amount of ammonia is also not particularly limited, but the concentration of ammonia is preferably 30-1000 mM, more preferably 60-500 mM, and even more preferably 80-200 mM. This reaction can be carried out at room temperature, and is preferably carried out with stirring. Usually, the dye-encapsulating particles of the present invention can be prepared by the reaction for several tens of minutes to several tens of hours.

By adjusting the concentration and reaction time of the aggregation-inducing light-emitting molecules having an alkoxysilane group to be used, the size (diameter) of the aggregation-inducing light-emitting molecules having an alkoxysilane group can be appropriately adjusted. Larger silica particles can be prepared by repeating the process multiple times. If necessary, dye-encapsulating particles having a desired particle size distribution range can be prepared.

When dye-encapsulating particles are produced by adding one or more silane compounds to the aggregation-inducing luminescent molecule having an alkoxysilane group and subjecting the molecules to polycondensation, the silane compound is not particularly limited. Silane, tetraethoxysilane (TEOS), γ-mercaptopropyltrimethoxysilane (MPS), γ-mercaptopropyltriethoxysilane, γ-aminopropyltriethoxysilane (APS), 3-thiocyanatopropyltriethoxysilane, 3 -glycidyloxypropyl triethoxysilane, 3-isocyanatopropyltriethoxysilane, and 3-[2-(2-aminoethylamino)ethylamino]propyl-triethoxysilane and the like, especially TEOS, MPS or APS is preferred. The ratio of the aggregation-inducing light-emitting molecules having an alkoxysilane group and the silane compound is not particularly limited, but the molar ratio of the silane compound to 1 mol of the aggregation-inducing light-emitting molecules having an alkoxysilane group is preferably 0.05 to 4000. , is more preferably 0.1 to 400, more preferably 0.3 to 40.

The reaction between the aggregation-inducing luminescent molecule having an alkoxysilane group and the silane compound is preferably carried out in the presence of alcohol, water and ammonia. Examples of alcohols include lower alcohols having 1 to 3 carbon atoms such as methanol, ethanol and propanol. The ratio of water and 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 per 1 part by volume of water. This is the range of the capacity part. Although the amount of ammonia is not particularly limited, the concentration of ammonia is preferably 30-1000 mM, more preferably 60-500 mM, and even more preferably 80-200 mM. This reaction can be carried out at room temperature, and is preferably carried out with stirring. Usually, the dye-encapsulating particles of the present invention can be prepared by the reaction for several tens of minutes to several tens of hours.

The size (diameter) of the dye-encapsulating particles to be prepared can be appropriately adjusted by adjusting the concentration of the aggregation-inducing light-emitting molecule having an alkoxysilane group to be used and adjusting the reaction time. Smaller silica particles can be prepared by using lower concentrations of silane compounds and shorter reaction times (see, for example, Blaaderen et al., "Synthesis and Characterization of Monodisperse Colloidal Organo-silica Spheres ", J. Colloid and Interface Science 156, 1-18.1993). On the other hand, if the same process is repeated multiple times, larger silica particles can be prepared. Thus, the particle size (diameter) of the obtained dye-encapsulated particles is adjusted to a desired size, for example, a size of the order of nm to μm, specifically, a fine size of 3 to 30 nm. Particles can be prepared. If necessary, dye-encapsulating particles having a desired particle size distribution range can be prepared.

Moreover, the production method of the dye-encapsulated particles of the present invention is not particularly limited as long as the necessary functions as a fluorescent label are not impaired. However, in a typical embodiment, the dye-encapsulating particles of the present invention can be obtained by a production method including the following polymerization steps.

(Polymerization process)
As the polymerization step performed in the method for producing the dye-encapsulating particles of the present invention,
(a-1) Polymerization step: a step of polymerizing a resin raw material, which is a raw material of the organic resin, in the presence of the aggregation-inducing light-emitting molecule to produce resin particles encapsulating the aggregation-inducing light-emitting molecule. .

Here, the resin raw material that can be used in the step (a-1) above may be a monomer corresponding to the above resin, or a prepolymer obtained from such a monomer. Specific examples of such monomers and prepolymers include those described above in the "Resins" section above.

The dye may be present from the beginning of the polymerization reaction in step (a-1), or may be added during the polymerization reaction.

The polymerization reaction can be carried out under conventionally known conditions and methods, except that it is carried out in the presence of the aggregation-inducing luminescent molecule.

For example, when a melamine resin is used as the organic resin, formic acid is added to the mixture of the aggregation-inducing light-emitting molecules and the melamine resin to cause a polycondensation reaction, thereby encapsulating the aggregation-inducing light-emitting molecules and the antioxidant. Melamine resin particles can be obtained. The reaction at this time can be performed, for example, in water. Moreover, 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 a melamine resin, a proton (H ⁺ ) is imparted to a functional group such as an amino group contained in the resin or the aggregation-inducing luminescent molecule to charge it, For the purpose of facilitating electrostatic interaction, a polymerization reaction accelerator such as a suitable acid may be further added to the mixture of the dye and melamine resin.

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 60-200° C. and the reaction time is usually 20-120 minutes. In addition, it is appropriate to set the reaction temperature to a temperature (within the heat resistant temperature range) at which the performance of the aggregation-induced luminescent molecules does not deteriorate. Heating may be performed in a plurality of stages, for example, after reacting at a relatively low temperature for a certain period of time, the temperature may be raised to react at a relatively high temperature for a certain period of time.

After completion of the polymerization reaction, impurities such as surplus resin raw materials, aggregation-induced luminescent molecules, and surfactants may be removed from the reaction solution, and the produced dye-encapsulating particles may be recovered and purified. For example, after centrifuging the reaction solution to remove the supernatant containing impurities, ultrapure water is added and ultrasonic waves are applied to re-disperse and wash. These operations are preferably repeated several times until no absorption or fluorescence originating from the resin or fluorescent dye is observed in the supernatant.

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 (anionic polymerization, etc.). Encapsulated resin particles for fluorescent labeling using a thermoplastic resin can also be produced according to these techniques, but are preferably produced by a polymerization process according to, for example, a soap-free emulsion polymerization method.

In the polymerization step in this case, typically, a reaction mixture containing aggregation-inducing luminescent molecules, a resin raw material, and a polymerization initiator is heated to advance the polymerization reaction of the resin, thereby producing aggregation-inducing luminescent molecules. This is a step of generating resin particles to be included.

The polymerization initiator and polymerization reaction conditions (temperature, time, etc.) can be appropriately set in consideration of the type of resin. For the synthesis of thermoplastic resins, the reaction temperature is usually 20-150° C. and the reaction time is usually 10-240 minutes. As the polymerization initiator, known ones such as benzoyl peroxide and azobisisobutyronitrile can be used. (A water-soluble polymerization initiator such as 2-methylpropionamidine can be used.

In the present invention, the resin particles themselves encapsulating the aggregation-inducing luminescent molecules obtained by the above-described step (a-1) may be used as the fluorescent labeling resin particles according to the present invention, or the surface Resin particles further having functional groups capable of forming bonds with other molecules by further modification can also be used as the dye-encapsulating particles according to the present invention.

Regardless of the type of binder, when producing the dye-encapsulating particles of the present invention, purification may be performed using a conventional method such as an ultrafiltration membrane, if necessary. Purification makes it possible to remove ions and unreacted substances from the reaction solution, and to obtain spherical or nearly spherical dye-encapsulating particles. Here, the near-spherical fine particles specifically have a shape in which the ratio of the long axis to the short axis is 2 or less.

Furthermore, in order to obtain dye-encapsulated particles with a desired average particle size, ultrafiltration is performed using an ultrafiltration membrane such as YM-10 or YM-100 (manufactured by Millipore) to remove particles having a large particle size. good too.

<Resin>
The resin constituting the dye-encapsulating particles according to the present invention (the resin is also expressed as an organic resin in the present invention) functions as a container for enclosing the aggregation-inducing luminescent molecules described later. The organic resin used in the present invention is not particularly limited as long as it does not impair the functions of the aggregation-induced light-emitting molecules, and may be a thermosetting resin or a thermoplastic resin.

(Thermosetting resin)
Examples of thermosetting resins that can be used as organic resins in the present invention include melamine, urea, guanamines (including benzoguanamine, acetoguanamine, etc.), phenols (including phenol, cresol, xylenol, etc.), xylene, and the like. Examples thereof include those containing structural units formed from at least one monomer selected from the group consisting of derivatives. Any one of these monomers may be used alone, or two or more thereof 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, melamine resins, typically melamine-formaldehyde resins, are preferred from the viewpoint of emission intensity when the pigment is encapsulated.

As raw materials for these thermosetting resins, not only the monomers themselves as described above, but also prepolymers obtained by reacting the monomers with compounds such as formaldehyde and other cross-linking agents in advance may be used. For example, in the production of melamine-formaldehyde resins, methylolmelamine, which is prepared by condensing melamine and formaldehyde under alkaline conditions, is generally used as a prepolymer. methylation for improving the stability of the polymer, butylation for improving the solubility in an organic solvent, etc.).

In the above thermosetting resin, at least part of the hydrogen atoms contained in the structural units thereof may be replaced with charged substituents or substituents capable of forming covalent bonds. Such a thermosetting resin can be synthesized by using, as a raw material, a monomer in which at least one hydrogen is replaced with the above substituent (derivatized) by a known method. Melamine resins, urea resins, benzoguanamine resins, etc. usually have cations naturally generated from amino groups or sites derived therefrom, and phenolic resins, xylene resins, etc. usually have anions naturally generated from hydroxyl groups or sites derived therefrom. have

Such thermosetting resins can be synthesized according to known techniques. For example, the melamine-formaldehyde resin can be synthesized by polycondensing the methylolmelamine prepared in advance as described above by adding a reaction accelerator such as an acid as necessary and then heating.

(Thermoplastic resin)
The thermoplastic resin that can be used as the organic resin in the present invention is not particularly limited. Those containing a structural unit formed from a functional monomer (a group that participates in the polymerization reaction in one molecule, in the above example, a monomer having one vinyl group) can be mentioned. Any one of these monomers may be used alone, or two or more thereof may be used in combination. If desired, one or more comonomers other than the above compounds may be used in combination.

Specific examples of thermoplastic resins include polystyrene, styrene resins composed of styrene and other monomers, polymethyl methacrylate, acrylic resins composed of (meth)acrylic acid and its alkyl esters, and other monomers, and polyacrylonitrile. , AS resin (acrylonitrile-styrene copolymer), ASA resin (acrylonitrile-styrene-methyl acrylate copolymer), acrylonitrile-based resins composed of acrylonitrile and other monomers.

However, in the present invention, a styrene-based resin is preferable from the viewpoint of the emission intensity when encapsulating the aggregation-induced luminescent molecule. In the present invention, the term "styrene-based resin" refers to a resin that is a styrene homopolymer or copolymer that may or may not have a substituent.

The above thermoplastic resin is, for example, a structural unit formed from a polyfunctional monomer such as divinylbenzene (a group involved in a polymerization reaction in one molecule, a monomer having two or more vinyl groups in the above example), that is, It may contain cross-linking sites.

Further, the above thermoplastic resin may be one in which at least part of the hydrogen contained in the structural units thereof is replaced with a substituent having an electric charge or a substituent capable of forming a covalent bond. Such a thermoplastic resin can be synthesized by using, as a starting material, a monomer such as 4-aminostyrene in which at least one hydrogen has been replaced with the above substituent (derivatized).

Further, the above thermoplastic resin may contain a structural unit having a functional group for surface modification of the fluorescent labeling resin particles 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 fluorescent labeling resin particles having epoxy groups oriented on the surface. This epoxy group can be converted to an amino group by reacting with excess aqueous ammonia. Various biomolecules can be introduced into the amino group thus formed according to a known technique (via a linker molecule if necessary).

<Method for producing fluorescent labeling material>
An embodiment of the present invention includes a method for producing a fluorescent labeling material, comprising the step of binding a targeting ligand to the surface of the dye-aggregated particles or the dye-encapsulating particles. The dye-aggregating particles or the dye-encapsulating particles and the targeting ligand may be bound directly 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-encapsulating particles is not particularly limited, and includes the following methods (i) to (iii).

(i) The dye-aggregated particles or the dye-encapsulating particles having thiol groups on their surfaces can be bound to targeting ligands via disulfide bonds, thioester bonds, or thiol substitution reactions. In particular, when the targeting ligand has an amino group, the thiol group of the dye-aggregating particles or the dye-encapsulating particles and the amino group of the targeting ligand are combined with succinimidyl-trans-4-(N- Cross-linking agents such as maleimidylmethyl)cyclohexane-1-carboxylate (SMCC), N-(6-maleimidocaproyloxy)succinimide (EMCS) may also be used for coupling.

(ii) For dye-aggregated particles or dye-encapsulating particles having amino groups on their surfaces, the amino groups and thiol groups of biomolecules or the like are bound to each other using a cross-linking agent such as SMCC or EMCS in the same manner as described above. can be done. In addition, this amino group and an amino group possessed by a biomolecule or the like can be combined with a cross-linking agent such as glutaraldehyde. Furthermore, biomolecules and the like can be bound to the surface via an amide bond or a thiourea bond.

(iii) The dye-aggregated particles or dye-encapsulated particles may be bound to the targeting ligand through specific binding by antigen-antibody reaction, biotin-avidin reaction, hybridization using base sequence complementarity, or the like. can. Specifically, for example, dye-aggregated particles previously bound to biotin are reacted with a targeting ligand bound to avidin, whereby the dye-aggregated particles and the targeting ligand are bound by a biotin-avidin reaction. .

(Surface modification of dye-encapsulating particles)
In the present invention, the dye-encapsulated particles obtained in the above-described step (a-1) may be used as they are as the fluorescent labeling material according to the present invention. It can be performed.

Here, the surface modification that can be performed in the present invention is not particularly limited. However, when the dye-encapsulated particles of the present invention are used as a fluorescent labeling material for immunostaining, the dye-encapsulated particles of the present invention are used in a manner in which a biologically relevant binding substance according to the embodiment of immunostaining is linked. Become. Therefore, the surface modification that can be applied to the dye-encapsulating particles of the present invention is preferably carried out in the form of introduction of functional groups capable of forming bonds with other molecules.

Here, the functional group capable of forming a bond with another molecule includes functional groups commonly used in the field of biochemistry. Specific examples of such functional groups include hydroxyl group, amino group, carboxyl group, A thiol group, a maleimide group, an aldehyde group, and the like can be mentioned. In the following description of this specification, a functional group capable of forming a bond with another molecule may also be called a reactive functional group.

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

For example, when the dye-encapsulating particles obtained by the above-described step (a-1) have hydroxyl groups on the surface, a silane coupling agent having a functional group capable of forming a bond with other molecules is reacted with the hydroxyl groups, A functional group capable of forming a bond with the other molecule can be introduced. For example, dye-encapsulating particles having hydroxyl groups on the surface can be reacted with a silane coupling agent having amino groups such as aminopropyltrimethoxysilane to obtain dye-encapsulating particles having amino groups. In addition, introduction of a functional group capable of forming a bond with other molecules into the dye-encapsulating particles having hydroxyl groups on the surface can be performed by using an appropriate linker molecule having an active ester and a functional group capable of forming a bond with other molecules. It can also be carried out by reacting with a hydroxyl group. These introduction methods are particularly suitable for dye-encapsulating particles employing melamine resin as the organic resin.

Further, when the dye-encapsulating particles obtained by the above step (a-1) have epoxy groups on the surface, amino groups can be introduced, for example, by treating such dye-encapsulating particles with aqueous ammonia. . In addition, by reacting an appropriate linker molecule having a functional group reactive with an epoxy group and a functional group capable of forming a bond with another molecule with the epoxy group, a bond can be formed with the other molecule. functional groups can also be introduced.

Further, even when the dye-encapsulating particles do not have any reactive functional groups on the surface, hydroxyl groups or the like are once introduced to the particle surfaces by subjecting them to a conventionally known suitable surface treatment such as plasma treatment. , a method similar to the introduction of a “functional group capable of forming a bond with another molecule” to a “dye-encapsulating particle” having a hydroxyl group on the surface may be applied. As described above, even when a resin is used as a binder, a targeting ligand can be bound in the same manner as in the surface modification of silica particles.

Further, in the method for producing a fluorescent labeling agent of the present invention, desired molecules may be bound to the surface by introducing an arbitrary acceptor group to the surface of the dye-encapsulating particles. Examples of the acceptor group include amino group, hydroxyl group, thiol group, carboxyl group, maleimide group, and succinimidyl ester group.

For example, when dye-encapsulating particles are produced by polycondensation of aggregation-inducing light-emitting molecules having alkoxysilane groups alone, silica particles have OH groups, which may be used as acceptor groups, and may be used as acceptor groups. By bonding a silane compound (silane coupling agent) having a desired group to the surface, the dye-encapsulating particles may have an acceptor group capable of bonding with a desired molecule on the surface.

Further, when dye-encapsulated particles are produced by polycondensing an aggregation-inducing luminescent molecule having an alkoxysilane group with one or more silane compounds added thereto, depending on the type of polycondensed silane compound, , a dye-encapsulating particle having on its surface an acceptor group capable of binding to a desired molecule. Table 1 shows the relationship between the polycondensed silane compound (silane coupling agent) and the acceptor groups formed on the surface of the resulting dye-encapsulating particles.

重縮合させたシラン化合物によって表面に導入されるアクセプター基とは異なるアクセプター基を導入したい場合には、追加で粒子表面に所望の基を有するシラン化合物（シランカップリング剤）を結合させることにより達成することができる。

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

Preferred embodiments of the present invention will be more specifically described below based on examples, but the present invention is not limited to these examples.
[Example 1]
(Dye aggregation particles (1))
A compound of the following formula (9), 4,4'-Bis(1,2,2-triphenylvinyl)-1,1'-biphenyl (manufactured by Sigma-Aldrich), was dissolved in tetrahydrofuran to 1 mM. The solution was sent at a flow rate of 1.0 mL/min using a pump (PU-1580, Jasco Co., Ltd.) to a stainless steel T-shaped micromixer (MT1XCS6, manufactured by Valco) equipped with a channel with an inner diameter of 0.15 mm. Then, another pump (NS-KX-500, Nippon Seimitsu Kagaku Co., Ltd.) is used to send ultrapure water at a flow rate of 74.0 mL / min to mix both liquids in the micromixer. The dye aggregated particles were precipitated by using a vacuum cleaner. The pressure during mixing was 4 to 5 MPa, and clogging of the flow channel by the pigment aggregated particles did not occur. The Reynolds number during mixing was calculated to be approximately 12,000.

0.1 g of the obtained pigment-aggregated particles and 10 mL of sulfuric acid were mixed and stirred at 50° C. for 1 hour to introduce SO ₃ groups onto the particle surfaces.

Subsequently, referring to the amination reaction of an aromatic sulfonate with sodium amide (Journal of the Chemical Society of Japan 1974, (8), P 1522, Nara et al.), 100 mg of pigment aggregate particles, 30 mg of sodium amide, and 28 wt% aqueous ammonia were prepared. and 5 mL of water were added and reacted at 60° C. for 4 hours to replace the sulfonic acid groups on the surface of the particles with amino groups.

Subsequently, by using a centrifugal separator at 10000 rpm for 30 minutes, the supernatant was removed and washed to obtain pigment aggregated particles (1).

［実施例２］
（色素凝集粒子（２））
式（９）の化合物の代わりに下記式（１０）の化合物4,4′-(1,2-Diphenylethene-1,2-diyl)dibenzoic acid（シグマアルドリッチ社製）を用いる以外は実施例１と同様の手法で色素凝集粒子（２）を得た。

[Example 2]
(Dye aggregation particles (2))
Same as Example 1 except that 4,4′-(1,2-Diphenylethene-1,2-diyl)dibenzoic acid (manufactured by Sigma-Aldrich), a compound of the following formula (10), was used instead of the compound of formula (9). Pigment Aggregated Particles (2) were obtained in a similar manner.

［実施例３］
（色素凝集粒子（３））
式（９）の化合物の代わりに下記式（１１）の化合物を用いる以外は実施例１と同様の手法で色素凝集粒子（３）を得た。下記式（１１）の化合物はAdv. Funct. Mater. 2014, 24, 3621.に記載の方法で合成した。

[Example 3]
(Dye aggregation particles (3))
Pigment Aggregated Particles (3) were obtained in the same manner as in Example 1, except that the compound of formula (11) below was used instead of the compound of formula (9). The compound of the following formula (11) was synthesized by the method described in Adv. Funct. Mater. 2014, 24, 3621.

［実施例４］
（色素凝集粒子（４））
1,1,2,3,4,5-Hexaphenyl-1H-silole（シグマアルドリッチ社製）１００ｍｇ、水３０ｍL、エタノール３０ｍL、濃硫酸０．５ｍLを混合し、５０℃で３時間撹拌した。続いて、カラムクロマトグラフィで精製を行い、下記式（１２）の化合物を得た。続いて、式（９）の化合物の代わりに下記式（１２）の化合物を用いる以外は実施例１と同様の手法で色素凝集粒子（４）を得た。

[Example 4]
(Dye aggregation 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 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 the compound of formula (12) below was used instead of the compound of formula (9).

［実施例５］
（色素凝集粒子（５））
Organometallics, 2016, 35 (14), pp 2327－2332に記載の合成法により下記式（１３）の化合物を合成した。式（９）の化合物の代わりに下記式（１３）の化合物を用いる以外は実施例１と同様の手法で色素凝集粒子（５）を得た。

[Example 5]
(Dye aggregation particles (5))
A compound of the following formula (13) was synthesized by the synthesis method described in Organometallics, 2016, 35 (14), pp 2327-2332. Pigment Aggregated Particles (5) were obtained in the same manner as in Example 1, except that the compound of formula (13) below was used instead of the compound of formula (9).

［実施例６］
（色素凝集粒子（６））
前記式（１３）の化合物０．１モルに対し、濃硫酸５ｍL、濃硝酸５ｍLを加え、１時間撹拌することで芳香環へのニトロ化を行った。続いて、カラムクロマトグラフィで精製することにより、ニトロ基が２つ導入された化合物を得た。次に、１０ｇのニトロ基が２つ導入された化合物に対して、スズ粉末０．１ｇおよび濃塩酸１０ｍLを加えて１時間撹拌した。続いて、水／酢酸エチルで分液精製し、減圧乾燥させることで、下記式（１４）の化合物を得た。前記式（９）の化合物の代わりに下記式（１４）の化合物を用いる以外は実施例１と同様の手法で色素凝集粒子（６）を得た。

[Example 6]
(Dye aggregation 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 nitrate the aromatic ring. Subsequently, a compound into which two nitro groups were introduced was obtained by purification by column chromatography. Next, 0.1 g of tin powder and 10 mL of concentrated hydrochloric acid were added to 10 g of the compound into which two nitro groups had been introduced, and the mixture was stirred for 1 hour. Subsequently, the compound of the following formula (14) was obtained by liquid separation purification with water/ethyl acetate and drying under reduced pressure. Pigment Aggregated Particles (6) were obtained in the same manner as in Example 1, except that the compound of the following formula (14) was used in place of the compound of the formula (9).

［実施例７］
（色素凝集粒子（７））
Dalton Trans, 2013, 42, 3646-3652に記載の合成法により下記式（１５）の化合物を合成した。式（９）の化合物の代わりに下記式（１５）の化合物を用いる以外は実施例１と同様の手法で色素凝集粒子（７）を得た。

[Example 7]
(Dye aggregation particles (7))
A compound of the following formula (15) was synthesized by the synthesis method described in Dalton Trans, 2013, 42, 3646-3652. Pigment Aggregated Particles (7) were obtained in the same manner as in Example 1, except that the compound of formula (15) below was used instead of the compound of formula (9).

［実施例８］
（色素凝集粒子（８））
前記式（１５）の化合物０．１モルに対し、濃硫酸５ｍL、濃硝酸５ｍLを加え、１時間撹拌することで芳香環へのニトロ化を行ない、続いて、カラムクロマトグラフィで精製することにより、ニトロ基が２つ導入された化合物を得た。次に、１０ｇのニトロ基が２つ導入された化合物に対して、スズ粉末０．１ｇおよび濃塩酸１０ｍLを加えて１時間撹拌した。続いて、水／酢酸エチルで分液精製し、減圧乾燥させることで、下記式（１６）の化合物を得た。前記式（９）の化合物の代わりに下記式（１６）の化合物を用いる以外は実施例１と同様の手法で色素凝集粒子（８）を得た。

[Example 8]
(Dye aggregation 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 nitrate the aromatic ring, followed by purification by column chromatography. A compound into which two nitro groups were introduced was obtained. Next, 0.1 g of tin powder and 10 mL of concentrated hydrochloric acid were added to 10 g of the compound into which two nitro groups had been introduced, and the mixture was stirred for 1 hour. Subsequently, the compound of the following formula (16) was obtained by liquid separation purification with water/ethyl acetate and drying under reduced pressure. Pigment Aggregated Particles (8) were obtained in the same manner as in Example 1, except that the compound of the following formula (16) was used in place of the compound of the formula (9).

［実施例９］
（色素凝集粒子（９））
New J. Chem., 2007, 31, 2076？2082に記載の合成法により下記式（１７）の化合物を得た。前記式（９）の化合物の代わりに下記式（１７）の化合物を用いる以外は実施例１と同様の手法で色素凝集粒子（９）を得た。

[Example 9]
(Dye aggregation particles (9))
New J. Chem., 2007, 31, 2076?2082 gave a compound of the following formula (17). Pigment Aggregated Particles (9) were obtained in the same manner as in Example 1 except that the compound of the following formula (17) was used in place of the compound of the formula (9).

［実施例１０］
（色素凝集粒子（１０））
Macromolecules 2009, 42, 1418-1420に記載の方法でヨウ化フェニル基を有するカルボランを取得し、続いて、WO 2009087994 A1に記載の方法で脱ハロゲン化することによりヨウ素置換基を水素化することで、下記式（１８）の化合物を得た。前記式（９）の化合物の代わりに下記式（１８）の化合物を用いる以外は実施例１と同様の手法で色素凝集粒子（１０）を得た。

[Example 10]
(Dye aggregation particles (10))
Macromolecules 2009, 42, 1418-1420 to obtain a carborane having a phenyl iodide group, followed by hydrogenation of the iodine substituent by dehalogenation as described in WO 2009087994 A1. , to obtain a compound of the following formula (18). Pigment Aggregated Particles (10) were obtained in the same manner as in Example 1, except that the compound of the following formula (18) was used in place of the compound of the formula (9).

［実施例１１］
（色素凝集粒子（１１））
Macromolecules 2009, 42, 1418-1420に記載の方法において、フェニルアセチレンの代わりに4-アミノフェニルアセチレンを用いることで、下記式（１９）の化合物を得た。

[Example 11]
(Dye aggregation 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.

Pigment Aggregated Particles (11) were obtained in the same manner as in Example 1, except that the compound of the following formula (19) was used instead of the compound of the formula (9).

［実施例１２］
（色素凝集粒子（１２））
Chem.Lett.2012, 41, 1445-1447に記載の方法において、下記式（２０）の化合物を得た。前記式（９）の化合物の代わりに下記式（２０）の化合物を用いる以外は実施例１と同様の手法で色素凝集粒子（１２）を得た。

[Example 12]
(Dye aggregation particles (12))
A compound of the following formula (20) was obtained by the method described in Chem.Lett.2012, 41, 1445-1447. Pigment Aggregated Particles (12) were obtained in the same manner as in Example 1, except that the compound of the following formula (20) was used instead of the compound of the formula (9).

［実施例１３］
（色素凝集粒子（１３））
Chem. Eur. J, 2013, 19, 4506-4512に記載の方法において、下記式（２１）の化合物を得た。前記式（９）の化合物の代わりに下記式（２１）の化合物を用いる以外は実施例１と同様の手法で色素凝集粒子（１３）を得た。

[Example 13]
(Dye aggregation particles (13))
A compound of the following formula (21) was obtained by the method described in Chem. Eur. J, 2013, 19, 4506-4512. Pigment Aggregated Particles (13) were obtained in the same manner as in Example 1 except that the compound of the following formula (21) was used instead of the compound of the formula (9).

［実施例１４］
（色素凝集粒子（１４））
前記式（２１）の化合物１００ｍｇ、水３０ｍL、エタノール３０ｍL、濃硫酸０．５ｍLを混合し、５０℃で３時間撹拌した。続いて、カラムクロマトグラフィで精製を行い、下記式（２２）の化合物を得た。前記式（９）の化合物の代わりに下記式（２２）の化合物を用いる以外は実施例１と同様の手法で色素凝集粒子（１４）を得た。

[Example 14]
(Dye aggregation particles (14))
100 mg of the compound of 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). Pigment Aggregated Particles (14) were obtained in the same manner as in Example 1, except that the compound of the following formula (22) was used instead of the compound of the formula (9).

［実施例１５］
（色素内包粒子（１５））
前記式（１３）の化合物１００ｍｇに対して鉄粉末１０ｍｇ、酢酸ナトリウム１０ｍｇ、ＴＨＦ１００ｍLを加え塩素ガスバブリング下、室温で１時間撹拌し、水／トルエンで分液精製することで上記式（１３）の化合物の芳香環にＣｌ基が導入されたクロロ化化合物を得た。温度計、攪拌機、および還流冷却管を備えた１００ｍＬの四つ口フラスコに，マグネシウム５．３５ｍｇ（０．２２ｍｍoｌ）およびテトラヒドロフラン２０ｇを仕込み，窒素気流下攪拌しながら１，２－ジブロモエタン３．７６ｇ（０．０２モル）を加えて活性化した。ここに、前記クロロ化化合物１０１．６ｍｇ（０．２ｍｍoｌ）をテトラヒドロフラン２０ｇに溶解した液を５５℃で滴下して対応するグリニャール試薬を調製した。このグリニャール試薬をテトラメトキシシラン９１．３ｇ（０．６モル）中に滴下した。生成した塩をろ過し、減圧蒸留することで、下記式（２３）の化合物を得た。

[Example 15]
(Dye-encapsulating 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 formula (13), and the mixture is stirred at room temperature for 1 hour under chlorine gas bubbling. A chlorinated compound was obtained in which a Cl group was introduced into the aromatic ring of the compound. 5.35 mg (0.22 mmol) of magnesium and 20 g of tetrahydrofuran were placed in a 100 mL four-necked flask equipped with a thermometer, stirrer, and reflux condenser, and 3.76 g of 1,2-dibromoethane was stirred under a nitrogen stream. (0.02 mol) was added to activate. A solution prepared by dissolving 101.6 mg (0.2 mmol) of the chlorinated compound in 20 g of tetrahydrofuran was added dropwise at 55° C. to prepare the corresponding Grignard reagent. This Grignard reagent was added dropwise to 91.3 g (0.6 mol) of tetramethoxysilane. The produced salt was filtered and distilled under reduced pressure to obtain a compound of the following formula (23).

10 mg of the compound of the following formula (23) was dissolved in 1 ml of dimethylsulfoxide (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 mixture was reacted at room temperature for 24 hours.

The reaction solution was ultrafiltered through YM-100 (trade name, manufactured by Millipore). The dye-encapsulating silica particle dispersion that has passed through the filter is collected, and this time, ultrafiltration is performed using YM-1 (trade name, manufactured by Millipore) until the amount of the dye-encapsulating silica particle dispersion becomes 1/10 of the total amount. was concentrated. The concentrated liquid was diluted with distilled water and subjected to ultrafiltration again with YM-1. After concentration, the operation of diluting with distilled water and performing ultrafiltration was repeated four times to remove unreacted raw materials, ammonia, etc. contained in the dye-encapsulating silica particle dispersion liquid to obtain dye-encapsulating particles (15).

［実施例１６］
（色素内包粒子（１６））
前記式（１３）の化合物を用いて前記式（２３）の化合物を合成する代わりに、前記式（１５）の化合物を用いて下記式（２４）の化合物を合成する以外は実施例１５と同様の手法で色素内包粒子（１６）を得た。

[Example 16]
(Dye-encapsulating particles (16))
The same as in Example 15 except that instead of synthesizing the compound of formula (23) using the compound of formula (13), the compound of formula (24) below is synthesized using the compound of formula (15). A dye-encapsulating particle (16) was obtained by the method of .

［実施例１７］
（色素内包粒子（１７））
前記式（１３）の化合物を用いて前記式（２３）の化合物を合成する代わりに、前記式（１７）の化合物を用いて下記式（２５）の化合物を合成する以外は実施例１５と同様の手法で色素内包粒子（１７）を得た。

[Example 17]
(Dye-encapsulating particles (17))
The same as in Example 15, except that instead of synthesizing the compound of formula (23) using the compound of formula (13), the compound of formula (25) below is synthesized using the compound of formula (17). A dye-encapsulated particle (17) was obtained by the method of .

［実施例１８］
（色素内包粒子（１８））
前記式（１３）の化合物を用いて前記式（２３）の化合物を合成する代わりに、前記式（１８）の化合物を用いて下記式（２６）の化合物を合成する以外は実施例１５と同様の手法で色素内包粒子（１８）を得た。

[Example 18]
(Particles encapsulating pigment (18))
The same as in Example 15, except that the compound of formula (18) is used to synthesize the compound of formula (26) below instead of using the compound of formula (13) to synthesize the compound of formula (23). A dye-encapsulated particle (18) was obtained by the method of .

［実施例１９］
（色素内包粒子（１９））
前記式（１３）の化合物を用いて前記式（２３）の化合物を合成する代わりに、前記式（２０）の化合物を用いて下記式（２７）の化合物を合成する以外は実施例１５と同様の手法で色素内包粒子（１９）を得た。

[Example 19]
(Dye-encapsulating particles (19))
The same as in Example 15, except that the compound of formula (20) is used to synthesize the compound of formula (27) below instead of using the compound of formula (13) to synthesize the compound of formula (23). A dye-encapsulated particle (19) was obtained by the method of .

［実施例２０］
（色素内包粒子（２０））
前記式（１３）の化合物を用いて前記式（２３）の化合物を合成する代わりに、前記式（２１）の化合物を用いて下記式（２８）の化合物を合成する以外は実施例１５と同様の手法で色素内包粒子（２０）を得た。

[Example 20]
(Dye-encapsulating particles (20))
The same as in Example 15, except that instead of synthesizing the compound of formula (23) using the compound of formula (13), the compound of formula (21) is used to synthesize the compound of formula (28) below. A dye-encapsulated particle (20) was obtained by the method of .

［実施例２１］
（色素内包粒子（２１））
前記式（１３）の化合物１００ｍｇに対して鉄粉末１０ｍｇ、酢酸ナトリウム１０ｍｇ、臭素１０ｍｇ、ＴＨＦ１００ｍＬを加え、室温で１時間撹拌し、水／トルエンで分液精製することで前記式（１３）の化合物の芳香環にＢｒ基を導入したブロモ化合物を得た。

[Example 21]
(Dye-encapsulating 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 formula (13), stirred at room temperature for 1 hour, and the compound of the formula (13) is purified by liquid separation with water / toluene. A bromo compound having a Br group introduced into the aromatic ring of was obtained.

Palladium acetate (3 mmol%, molecular weight 224.51) and xantphos (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 dissolved 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 a compound of formula (29) (J. Org. Chem., 2017, 82 (18), pp 9710-9714, Palladium-Catalyzed Carbonylative Transformation of Organic Halides with Formic Acid as the Coupling Partner and CO Source: Synthesis of Carboxylic Acids).

（ＮＨＳ基を導入した凝集発光性分子の重縮合化）
前記式（２９）の化合物５．６ｍｇを１ｍｌのジメチルスルホキシド（ＤＭＳＯ）に溶解した。ここに１．３μｌのＡＰＳを加え、室温（２５℃）で１時間反応を行った。得られた反応液５０μｌにエタノール３．９５ｍｌ、ＭＰＳ２０μｌ、蒸留水１ｍｌ、２８質量％アンモニア水１００μｌを加え室温で２４時間反応を行った。反応液をＹＭ－１００（商品名、ミリポア社製）で限外ろ過した。フィルターを透過した蛍光シリカ粒子分散液を回収し、今度はＹＭ－１（商品名、ミリポア社製）で限外ろ過を行い、全量の１０分の１量になるまで蛍光シリカ粒子分散液を濃縮した。濃縮した液を蒸留水で希釈して再度ＹＭ－１で限外ろ過を行った。濃縮後蒸留水で希釈し限外ろ過を行う操作を４回繰り返して行うことで未反応のＡＰＳやアンモニア等を除去することで色素内包粒子（２１）を得た。
［実施例２２］
（色素内包粒子（２２））
前記式（１３）の化合物を用いて前記式（２９）の化合物を合成する代わりに、前記式（１５）の化合物を用いて下記式（３０）の化合物を合成する以外は実施例２１と同様の手法で色素内包粒子（２２）を得た。

(Polycondensation of Aggregated Light-Emitting Molecules Introduced with NHS Groups)
5.6 mg of the compound of formula (29) was dissolved in 1 ml of dimethylsulfoxide (DMSO). 1.3 μl of APS was added thereto and reacted at room temperature (25° C.) for 1 hour. 3.95 ml of ethanol, 20 μl of MPS, 1 ml of distilled water, and 100 μl of 28 mass % aqueous ammonia were added to 50 μl of the resulting reaction solution, and the mixture was reacted at room temperature for 24 hours. The reaction solution was ultrafiltered through YM-100 (trade name, manufactured by Millipore). The fluorescent silica particle dispersion that has passed through the filter is recovered, and then subjected to ultrafiltration using YM-1 (trade name, manufactured by Millipore) to concentrate the fluorescent silica particle dispersion to 1/10 of the total volume. bottom. The concentrated liquid was diluted with distilled water and subjected to ultrafiltration again with YM-1. After the concentration, the operation of diluting with distilled water and performing ultrafiltration was repeated four times to remove unreacted APS, ammonia, and the like, thereby obtaining dye-encapsulated particles (21).
[Example 22]
(Dye-encapsulating particles (22))
The same as in Example 21, except that the compound of formula (15) is used to synthesize the compound of formula (30) below instead of using the compound of formula (13) to synthesize the compound of formula (29). A pigment-encapsulated particle (22) was obtained by the method of .

［実施例２３］
（色素内包粒子（２３））
前記式（１３）の化合物を用いて前記式（２９）の化合物を合成する代わりに、前記式（１７）の化合物を用いて下記式（３１）の化合物を合成する以外は実施例２１と同様の手法で色素内包粒子（２３）を得た。

[Example 23]
(Dye-encapsulating particles (23))
The same as in Example 21, except that the compound of formula (17) is used to synthesize the compound of formula (31) below instead of using the compound of formula (13) to synthesize the compound of formula (29). A pigment-encapsulated particle (23) was obtained by the method of .

［実施例２４］
（色素内包粒子（２４））
前記式（１３）の化合物を用いて前記式（２９）の化合物を合成する代わりに、前記式（１８）の化合物を用いて下記式（３２）の化合物を合成する以外は実施例２１と同様の手法で色素内包粒子（２４）を得た。

[Example 24]
(Dye-encapsulating particles (24))
The same as in Example 21 except that instead of synthesizing the compound of formula (29) using the compound of formula (13), the compound of formula (32) below is synthesized using the compound of formula (18). A dye-encapsulated particle (24) was obtained by the method of .

［実施例２５］
（色素内包粒子（２５））
前記式（１３）の化合物を用いて前記式（２９）の化合物を合成する代わりに、前記式（２０）の化合物を用いて下記式（３３）の化合物を合成する以外は、実施例２１と同様の手法で色素内包粒子（２５）を得た。

[Example 25]
(Dye-encapsulating particles (25))
Example 21 and Pigment-encapsulating particles (25) were obtained in a similar manner.

［実施例２６］
（色素内包粒子（２６））
前記式（１３）の化合物を用いて前記式（２９）の化合物を合成する代わりに、前記式（２１）の化合物を用いて下記式（３４）の化合物を合成する以外は、実施例２１と同様の手法で色素内包粒子（２６）を得た。

[Example 26]
(Dye-encapsulating particles (26))
Except for synthesizing the compound of the following formula (34) using the compound of the formula (21) instead of synthesizing the compound of the formula (29) using the compound of the formula (13), Example 21 and Pigment-encapsulating particles (26) were obtained in a similar manner.

［実施例２７］
＜蛍光標識材１＞
実施例１～２６で得られた色素凝集粒子および色素内包粒子をストレプトアビジンで標識することで蛍光標識材を作製した。

[Example 27]
<Fluorescent labeling material 1>
A fluorescent labeling material was produced by labeling the dye-aggregated particles and the dye-encapsulated particles obtained in Examples 1 to 26 with streptavidin.

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

Subsequently, the aggregated nanoparticles subjected to the above treatment were prepared as a 3 nM dispersion using PBS containing 2 mM EDTA, and SM(PEG) ₁₂ (succinimidyl-[(N-maleimidopropionamido) -dodecanethyleneglycol]ester; Thermo Scientific) was mixed and allowed to react at 5°C for 1 hour.

This dispersion was centrifuged at 10,000 rpm for 20 minutes, the supernatant was removed, and PBS containing 2 mM EDTA was added to disperse the sediment. A group-introduced pigment-aggregated particle was obtained.

On the other hand, after adding 40 μL of streptavidin (manufactured by Wako Pure Chemical Industries, Ltd.) adjusted to 1 mg / mL to 210 μL of borate buffer, 70 μL of 2-iminothiolane hydrochloride (manufactured by Sigma-Aldrich) adjusted to 64 mg / mL was added, By reacting at room temperature for 1 hour, a thiol group was introduced into the amino group of streptavidin, which was desalted using a gel filtration column (Zaba Spin Desalting Columns, Funakoshi Co.).

0.04 mg of the above thiol group-added streptavidin and 740 μL of dye-aggregated particles having a maleimide group introduced to the particle surface adjusted to 0.67 nM were mixed in PBS containing 2 mM of EDTA, and reacted at room temperature for 1 hour. let me After that, 10 mM mercaptoethanol was added to stop the reaction, the resulting solution was concentrated with a centrifugal filter, and unreacted streptavidin and the like were removed with a gel filtration column for purification. A labeling material 1 was obtained.

[Example 28]
<Fluorescent labeling material 2>
Fluorescent labeling material 2 was prepared by labeling the dye-aggregated particles obtained in Examples 6, 8, 9, 11, 12 and 14 with an anti-PD-L1 antibody.
(Dye Aggregate Particles-Antibody Modification)
In the same manner as in Example 27, dye-aggregated particles having a maleimide group introduced onto each particle surface were obtained.

On the other hand, 100 μg of anti-PD-L1 rabbit monoclonal antibody (Cell signaling Technology; No. E1L3N) was dissolved in 100 μL of PBS. 0.002 mL (0.2×10 ⁻⁵ mol) of 1 M 2-mercaptoethanol was added to this antibody solution and reacted at pH 8.5 at room temperature for 30 minutes. A solution of thiolated anti-PD-L1 rabbit monoclonal antibody was obtained by removing the 2-mercaptoethanol.

Fluorescent labeling material 2 in which dye-aggregated particles are modified with an anti-PD-L1 rabbit monoclonal antibody in the same manner as in Example 27, except that a thiolated anti-PD-L1 rabbit monoclonal antibody is used instead of streptavidin to which a thiol group is added. got

[Example 29]
<Pigment-encapsulating melamine particles (28) to (32)>
20.3 mg of the pigment aggregation particles (6) was added to 22 mL of water and dissolved. After that, 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. After heating this solution to 70° C. while stirring it on a hot stirrer, 0.81 g of melamine resin raw material “Nikalac MX-035” (manufactured by Nippon Carbide Industry Co., Ltd.) was added to this solution. Furthermore, 1000 μL of a 10% aqueous solution of dodecylbenzenesulfonic acid (manufactured by Kanto Kagaku Co., Ltd.) was added as a surfactant to this solution, and the mixture was heated and stirred at 70° C. for 50 minutes. After that, the temperature was raised to 90° C. and the mixture was heated and stirred for 20 minutes.

In order to remove surplus resin raw materials and impurities from the resulting melamine resin dispersion of the pigment aggregated particles (6), the centrifuge is centrifuged at 20,000 G for 15 minutes (manufactured by Kubota Shoji Co., Ltd.). The process of washing by centrifuging, adding ultrapure water, irradiating with ultrasonic waves and redispersing was repeated 5 times to obtain dye-encapsulating melamine particles (27).

Pigment-encapsulating melamine particles (28) to (32) in the same manner except that the pigment-aggregated particles (6) are replaced with pigment-aggregated particles (8), (9), (11), (12), and (14), respectively. got
[Example 30]
<Fluorescent labeling material 3>
Maleimide groups were introduced onto the surface of each of the dye-encapsulating melamine particles (27) to (32) produced in Example 29 by the following method.

0.1 mg of the dye-encapsulating particles were dispersed in 1.5 mL of ethanol, 2 μL of aminopropyltrimethoxysilane (LS-3150, manufactured by Shin-Etsu Chemical Co., Ltd.) was added, and the mixture was allowed to react for 8 hours to remove the pigment present on the surface of the dye-encapsulating particles. The hydroxyl group that was used was converted to an amino group.

Phosphate buffered saline (PBS) containing 2 mM ethylenediaminetetraacetic acid (EDTA) was used to adjust the concentration of the dye-encapsulated particles to 3 nM, and SM (PEG) was added to a final concentration of 10 mM. ₁₂ (Succinimidyl-[(N-maleimidopropionamide)-dodecaethylene glycol] ester, manufactured by Thermo Scientific) was mixed, reacted at 20°C for 1 hour, centrifuged at 10,000 G for 20 minutes, and the supernatant was removed. After that, PBS containing 2 mM EDTA was added and washing was performed three times to disperse the sediment, thereby obtaining dye-encapsulating particles having maleimide groups introduced onto the particle surface.

On the other hand, streptavidin (manufactured by Wako Pure Chemical Industries, Ltd.) and N-succimidyl-S-acetylthioacetic acid (SATA) were used to add a thiol group to streptavidin, followed by gel filtration.

The above maleimide-modified dye-encapsulating particles and thiol group-added streptavidin were mixed in PBS containing 2 mM EDTA and allowed to react at room temperature for 1 hour to convert the respective maleimide groups and thiol groups. combined. After that, 10 mM mercaptoethanol was added to stop the reaction, and after concentrating with a centrifugal filter of φ = 0.65 µm, unreacted streptavidin and the like were removed using a gel filtration column for purification, thereby obtaining pigment-encapsulated melamine. A fluorescent labeling material 3 having particles modified with streptavidin was obtained.

[Example 31]
<Dye-encapsulating polystyrene particles (33) to (44)>
Dye-encapsulating polystyrene particles (33) to (44) were prepared by a soap-free emulsion polymerization method as follows.

The compounds of the formulas (10) and (12) to (22) are mixed with 4-aminostyrene (manufactured by Tokyo Chemical Industry Co., Ltd.) for 1 hour at room temperature to bind each compound to styrene. Styrene was made. 0.18 g of glycidyl methacrylate (manufactured by Tokyo Chemical Industry 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 dye-bound styrene were added to 5 mL of argon-bubbled pure water. The temperature was raised to 70° C. while stirring, and 0.012 g of V-50 (manufactured by Wako Pure Chemical Industries, Ltd.), which is a water-soluble azo polymerization initiator, was added and reacted for 12 hours. The reaction solution was centrifuged at 10,000 G for 20 minutes to collect particles. The collected particles were dispersed in pure water and collected again by centrifugation for purification, thereby obtaining dye-encapsulating polystyrene particles (33) to (44).

[Example 32]
<Fluorescent labeling material 4>
The particle surfaces of the dye-encapsulating polystyrene particles (33) to (44) were modified with streptavidin via amino groups derived from 4-aminostyrene.

Phosphate-buffered saline (PBS) containing 2 mM ethylenediaminetetraacetic acid (EDTA) was used to adjust the concentration of the dye-encapsulated polystyrene particles to 3 nM. SM (PEG) 12 (succinimidyl-[(N-maleimidopropionamide)-dodecaethylene glycol] ester, Thermo Scientific Co., Ltd., was added to the dispersion liquid of dye-encapsulated polystyrene particles whose concentration was adjusted so that the final concentration was 10 mM. ) and allowed to react at 20° C. for 1 hour to obtain a mixed solution containing dye-encapsulated polystyrene particles having maleimide groups introduced on the particle surfaces.

This mixture was centrifuged at 10,000 G for 20 minutes, the supernatant was removed, PBS containing 2 mM EDTA was added to disperse the sediment, and the mixture was centrifuged again. After performing the above washing three times by the same procedure, the maleimide group-modified dye-encapsulating polystyrene particles were collected.

A thiol group-added streptavidin was prepared in a manner similar to that described in Example 27.

By reacting the maleimide-modified dye-encapsulating particles and streptavidin to which a thiol group is added in the same manner as described in Example 27, the dye-encapsulating polystyrene resin particles are streptavidin-modified fluorescence. A labeling material 4 was obtained.

[Example 33]
<Fluorescent labeling material 5>
The dye-encapsulating polystyrene particles (33) to (44) were added to excess aqueous ammonia to convert the epoxy groups on the particle surfaces to amino groups. The resulting particles were adjusted to 3 nM with PBS, and SM(PEG) ₁₂ (succinimidyl-[(N-maleimidopropionamido)-dodecaethylene glycol] ester, Thermo Scientific) was added to this solution to a final concentration of 10 mM. product), reacted at 120° C. for 1 hour, centrifuged at 10000 G for 20 minutes, removed the supernatant, added PBS containing 2 mM EDTA to disperse the sediment, and washed 3 times. Thus, dye-encapsulated polystyrene particles having maleimide groups introduced on the particle surface were obtained.

100 μg of anti-PD-L1 rabbit monoclonal antibody (Cell Signaling Technology; No. E1L3N) was dissolved in 100 μL of PBS. 0.002 mL (0.2×10 ⁻⁵ mol) of 1 M 2-mercaptoethanol was added to this antibody solution and reacted at pH 8.5 at room temperature for 30 minutes. A solution of thiolated anti-PD-L1 rabbit monoclonal antibody was obtained by removing the 2-mercaptoethanol.

Anti-PD-L1 rabbit monoclonal dye-encapsulated polystyrene resin particles were prepared in the same manner as in Example 27, except that the dye-encapsulated polystyrene particles having a maleimide group introduced onto the particle surface and the thiolated anti-PD-L1 rabbit monoclonal antibody were used. Antibody-modified fluorescent labeling material 5 was obtained.

[Comparative Example 1]
(Dye Aggregate Particles)
A compound of the following formula 35 was synthesized by the synthesis method described in US2013/089889. A dye aggregate was obtained by crystallizing the compound of formula 35 below in a methanol/THF solution.

［比較例２］
凝集誘起発光性分子ではない蛍光色素Ｙ５５０の誘導体である、Ｙ５５０－ＮＨＳエステル（商品名、Ｄｙｏｍｉｃｓ ＧｍｂＨ社製）５．６ｍｇを１ｍｌのジメチルスルホキシド（ＤＭＳＯ）に溶解し、１．３μｌのＡＰＳを加え、室温（２５℃）で１時間反応を行った。得られた反応液５０μｌにエタノール３．９５ｍｌ、ＭＰＳ２０μｌ、蒸留水１ｍｌ、２８質量％アンモニア水１００μｌを加え室温で２４時間反応させたものをＹＭ－１００（ミリポア社製）で限外ろ過して回収した色素内包粒子の分散液をＹＭ－１（ミリポア社製）で限外ろ過することで、全量の１０分の１量になるまで濃縮した。濃縮後蒸留水で希釈しＹＭ－１で限外ろ過を行う操作を４回繰り返して行い、色素内包粒子分散液に含まれる未反応のＡＰＳやアンモニア等を除去することで、コロイドシリカ粒子を得た。

[Comparative Example 2]
5.6 mg of Y550-NHS ester (trade name, manufactured by Dyomics GmbH), which is a derivative of the fluorescent dye Y550 that is not an aggregation-inducing luminescent molecule, was dissolved in 1 ml of dimethylsulfoxide (DMSO), and 1.3 μl of APS was added. , at room temperature (25° C.) for 1 hour. 3.95 ml of ethanol, 20 μl of MPS, 1 ml of distilled water, and 100 μl of 28 mass % aqueous ammonia were added to 50 μl of the resulting reaction mixture, and the mixture was allowed to react at room temperature for 24 hours. The resulting dispersion of the dye-encapsulating particles was subjected to ultrafiltration with YM-1 (manufactured by Millipore) to concentrate it to 1/10 of the total amount. The operation of diluting with distilled water after concentration and performing ultrafiltration with YM-1 is repeated four times to remove unreacted APS, ammonia, etc. contained in the dispersion of dye-encapsulating particles, thereby obtaining colloidal silica particles. rice field.

0.5 mg of streptavidin-maleimide (manufactured by Sigma) was added to 2 mg/mL×1.5 mL of thiol-modified colloidal silica particles in the same manner as in Example 26, and the mixture was reacted at room temperature for 2 hours. After the reaction, unreacted streptavidin-maleimide was dialyzed and removed by a conventional method to obtain a fluorescent labeling material, which was streptavidin-modified colloidal silica particles.
[Example 34]
<Vibration resistance evaluation (refrigeration)>
A dispersion was prepared by dispersing the fluorescent labeling materials 1 to 5, the dye aggregate of Comparative Example 1, and the colloidal silica particle dye of Comparative Example 2 at 5 wt/wt% in PBS, and the brightness of each was measured. bottom. Subsequently, vibration treatment was performed by reciprocating the dispersion liquid of each particle between Tokyo and Fukuoka by Cool Takkyubin (registered trademark) at 5° C., and the luminance after the vibration treatment was measured. Based on the measured values of initial luminance and luminance after vibration treatment, vibration resistance evaluation (refrigeration) was performed according to the following criteria.
AA: (Brightness after vibration processing) / (Initial brightness) is 0.95 or more BB: (Brightness after vibration processing) / (Initial brightness) is 0.85 or more and less than 0.95 CC: (Vibration processing After luminance)/(initial luminance) is 0.75 or more and less than 0.85 DD: (luminance after vibration treatment)/(initial luminance) is less than 0.75

[Example 35]
<Vibration resistance evaluation (room temperature)>
Vibration resistance evaluation (room temperature) was performed using the same procedure and evaluation criteria as in Example 31, except that courier service (registered trademark) at room temperature was used instead of courier service (registered trademark) at 5 ° C. to make a round trip between Tokyo and Fukuoka. rice field.

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

Claims (8)

A dye-encapsulating particle comprising a binder and an aggregation -inducing luminescent molecule represented by the following general formula (1) .

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, an organometallic group, or a silane coupling agent-binding group. is the basis,
The hydrophilic groups include -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(NH2 ₎ 2 _, -P( = O)R2 ₍ NH2 ₎ , -P(NH2 ₎ 3 _, -O(C=O)OH, -NH2 _, -NHR, -NHCONH2 _, -NHCONHR, -NHCOOH, -Si(OH) ₃ , -Si(R)(OH) ₂ , -Si(R) _2OH , -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 , —OB(OH) ₂ , —B(NH ₂ ) ₂ , —NHB(OH) ₂ , NHS group, or maleimide group, and each R is independently hydrogen or carbon number representing an alkyl group of 1 to 20,
The organic group is an alkyl group, an alkenyl group, an alkynyl group, a cycloalkyl group, a cycloalkenyl group, a cycloalkynyl group, or an aromatic group having 1 to 20 carbon atoms. one or more of the atoms may be substituted with a heteroatom such as S, N, O,
The organometallic group is a metal alkoxide group having a titanium alkoxide skeleton, a zirconium alkoxide skeleton, or a silicon alkoxide skeleton,
The silane coupling agent-binding group includes an N-hydroxysuccinimide (NHS) ester group, maleimide group, isocyanate group, isothiocyanate group, aldehyde group, paranitrophenyl group, diethoxymethyl group, epoxy group, and cyano group. , an alkoxysilane group, or a halogen atom . 2. The dye-encapsulating particles according to claim 1 , wherein said aggregation-inducing luminescent molecule has a hydrophilic group. 3. The dye-encapsulating particles according to claim 1 , wherein the binder and the aggregation -inducing luminescent molecule form a covalent bond, and the binder forms a metalloxane bond. encapsulated particles. 3. A fluorescent labeling material in which a targeting ligand is covalently bound to the surface of the dye-encapsulating particles according to claim 1 or 2 . 5. The fluorescent labeling material according to claim 4 , wherein the targeting ligand is one or more molecules selected from the group consisting of antibodies, substances with affinity for organelles, and proteins having binding properties to sugar chains. . A fluorescent labeling agent dispersion containing the fluorescent labeling agent according to claim 4 or 5 and a buffer solution. 3. The method for producing dye-encapsulating particles according to claim 1 , comprising dispersing the aggregation-inducing luminescent molecules in a binder or a precursor of the binder to form particles. A method for producing the dye-encapsulating particles according to claim 1 or 2 ,
1 ) A step of dispersing the aggregated luminescent molecules in a binder precursor; and 2) A step of forming a binder from the binder precursor by a sol-gel method and granulating the binder. Method.