JP2008231378A - Fluorescent polymer fine particles and production method thereof, complex for fluorescence detection, fluorescence detection method and fluorescence detection kit - Google Patents

Abstract

［一般式（Ｉ）中、Ｒ¹及びＲ^３はそれぞれ独立に水素原子又はアルキル基を表し、Ｒ^２及びＲ^４はそれぞれ独立に水素原子、アルキル基又はシアノ基を表し、Ｌ^１及びＬ^２はそれぞれ独立に２価の連結基を表す。ｎはエチレングリコール鎖の平均繰り返し数を表す。］
【選択図】なしDisclosed is a fluorescent polymer fine particle having high fluorescence intensity and good temporal stability.
A fluorescent polymer fine particle comprising a polymer fine particle having a core-shell structure consisting of a hydrophobic core and a hydrophilic shell, and a fluorescent lanthanoid dye incorporated in the polymer fine particle and containing a lanthanoid cation. In which the hydrophilic polymer forming the hydrophilic shell contains a hydrophilic unit represented by the following general formula (I) in the main chain, and the side chain is bonded with a polymerizable polymer that forms a hydrophobic core It is assumed to be a conductive polymer.
[Chemical 1]

[In General Formula (I), R ¹ and R ³ each independently represent a hydrogen atom or an alkyl group, R ² and R ⁴ each independently represent a hydrogen atom, an alkyl group, or a cyano group, and L ¹ and L ² Each independently represents a divalent linking group. n represents the average number of repeating ethylene glycol chains. ]
[Selection figure] None

Description

  The present invention relates to a fluorescent polymer fine particle and a production method thereof, a complex for fluorescence detection, a fluorescence detection method, and a fluorescence detection kit.

Various labels have been developed to visualize or quantify trace substances. Particularly in fields that require high sensitivity, radioisotopes are typical labels, and tritium, radioactive iodine, and the like are used as representative examples. However, the use of radioactive materials has many problems in disposal and handling after use, and a method to replace radioactive materials has been developed. Examples of such a technique include an enzyme labeling method (using peroxidase, alkaline phosphatase, glucose oxidase, or β-D-galactosidase), and a fluorescent substance labeling method (using fluorescein, rhodamine, etc.).
However, these methods have a drawback that the absolute sensitivity as a label is insufficient.

In order to increase the accuracy and sensitivity of the measurement, time-resolved fluorescence measurement has been developed as one development of the fluorescent substance labeling method (for example, Patent Document 1). This method irradiates a fluorescent substance with a long fluorescence quenching time represented by europium chelate with a pulsed excitation light, and measures the fluorescence after a certain period of time when the direct excitation light or the short fluorescence caused by the surrounding substance is quenched. This is a method for measuring europium-specific fluorescence.
Furthermore, in order to increase sensitivity, this europium chelate or dye is confined to polystyrene particles, and the surface of the polystyrene particles is coated with an antigen or antibody as a reagent, and the polystyrene particles immobilized by the antigen-antibody reaction are detected visually. Attempts have also been made (for example, Patent Document 2).

However, in the known method in which a dye or a fluorescent substance is enclosed in polystyrene particles and used as a labeling substance, a certain level of sensitivity can be obtained by a simple operation, but the sensitivity is not satisfactory, and a further high Sensitivity was required.
Furthermore, due to the characteristic that the surface is hydrophobic polystyrene, (1) after binding functional molecules such as antigens or antibodies to be coated, the non-binding surface is coated with proteins or similar substances of various biological materials, and polystyrene Various methods have been used such as a method of masking the hydrophobic property, or (2) a method of preventing the polystyrene particles from affecting each other by adding a surfactant to the liquid during the reaction. However, there were still cases where judgment errors occurred due to non-specific reactions.

On the other hand, in order to improve non-specific reactions caused by polystyrene particles, core-shell type fine particles were prepared by covering the surface of the core made of water-insoluble polymer compound with a water-soluble shell part having a reactive group in a brush shape. In addition, a technique for encapsulating a fluorescent dye in a core portion and a composition for fluorescence analysis having a high fluorescence intensity obtained thereby have been developed (for example, Patent Documents 3 and 4).
JP-A-61-128168. JP 2000-345052 A. International Publication No. 2002/097436 Pamphlet JP 2005-49207 A

However, this method using core-shell type fine particles can suppress noise due to non-specific reaction, but is not satisfactory in terms of fluorescence intensity. In addition, the stability over time is low, and the fluorescence intensity may decrease during the period of use.
Further, in the core-shell type fine particles described in Patent Document 3, since the hydrophobic core is bonded to the terminal of the hydrophilic unit, it is difficult to say that the temporal stability in the dispersed state is sufficient. In addition, since detection with ultraviolet light is necessary for detection, noise generation during detection may be a problem when a substance that absorbs ultraviolet light (for example, protein) exists in the analyte. It was.
An object of the present invention is to provide a fluorescent polymer fine particle having high fluorescence intensity and good temporal stability, and to detect a detection target substance with high sensitivity using such a fluorescent polymer fine particle, and is excellent. To provide a fluorescence detection complex, a fluorescence detection kit, and a fluorescence detection method having storage stability.

In order to achieve the above-mentioned object, the present inventor arrived at the present invention as a result of intensive studies on the hydrophilic polymer used in the synthesis of polymer fine particles.
That is, the present invention is as follows.
<1> A fluorescent polymer fine particle comprising a polymer fine particle having a core-shell structure comprising a hydrophobic core and a hydrophilic shell, and a fluorescent lanthanoid dye incorporated in the polymer fine particle and containing a lanthanoid cation. The hydrophilic polymer that forms the hydrophilic shell includes a hydrophilic unit represented by the following general formula (I) in the main chain, and a polymerizable polymer that forms a hydrophobic core is bonded to the side chain. Fluorescent polymer fine particles, characterized in that

[In General Formula (I), R ¹ and R ³ each independently represent a hydrogen atom or an alkyl group, R ² and R ⁴ each independently represent a hydrogen atom, an alkyl group, or a cyano group, and L ¹ and L ² Each independently represents a divalent linking group. n represents the average number of repeating ethylene glycol chains. ]

<2> The hydrophilic polymer has a main chain including the hydrophilic unit represented by the general formula (I) and a side chain including an ethylenically unsaturated bond group unit having an ethylenically unsaturated bond. <1> The fluorescent polymer fine particle according to <1>, which is derived from a precursor hydrophilic polymer.
<3> The precursor hydrophilic polymer includes a hydrophilic unit represented by the following general formula (I) and an ethylenically unsaturated bond group unit represented by the following general formula (II). The fluorescent polymer fine particles according to <2>.

[In General Formulas (I) and (II), R ¹ and R ³ each independently represent a hydrogen atom or an alkyl group, R ² and R ⁴ each independently represent a hydrogen atom, an alkyl group, or a cyano group; ¹ and L ² each independently represent a divalent linking group. n represents the average number of repeating ethylene glycol chains. R ⁵ represents a hydrogen atom or a methyl group. L ⁴ represents an oxygen atom or —NR ²¹ —. R ²¹ represents a hydrogen atom or an alkyl group having 1 to 6 carbon atoms. L ³ represents a divalent linking group, Z represents an allyl group, an allyloxyethyl group, and an atomic group represented by General Formula (III) or General Formula (IV).

In general formulas (III) and (IV), R ⁶ and R ⁷ each independently represent a hydrogen atom or a methyl group. m represents 1-12. ]

<4> The fluorescent polymer fine particle according to any one of <1> to <3>, wherein the hydrophilic polymer further includes a reactive functional group unit.
<5> The fluorescent polymer fine particle according to <4>, wherein the reactive functional group unit is an amino group unit represented by the following general formula (V).

[In the general formula (V), R ⁸ represents a hydrogen atom or a methyl group. ]

<6> Any of the above <1> to <5>, wherein the fluorescent lanthanoid dye has at least one nitrogen-containing heterocyclic ligand represented by the following general formula (LI) The fluorescent polymer fine particle according to item 1.

[In General Formula (LI), A ¹ , A ² , and A ³ are atomic groups represented by the following General Formulas (L-II) to (LV), or a hydroxyl group, an alkoxy group, and an aryloxy group. Represents an alkylamino group, a dialkylamino group, an arylamino group, or a diarylamino group, which may be the same as each other. B ¹ and B ² each independently represent a nitrogen atom or ═C (—R) —, and R represents a hydrogen atom or a substituent.

In each formula, R ^{11 to} R ¹⁹ each represent a hydrogen atom or a substituent. R ¹⁷ and R ¹⁸ , R ¹⁸ and R ¹⁹ and R ¹⁷ and R ¹⁹ may be combined with each other to form a ring, if possible. l represents 0, 1 or 2; G represents a carbon atom or a nitrogen atom which may have a substituent. Q represents an atomic group necessary for forming a 5-membered or 6-membered nitrogen-containing heterocyclic ring, and the nitrogen-containing heterocyclic ring may form a condensed ring. Ar ¹ represents an aromatic carbocyclic ring or an aromatic heterocyclic ring. In the general formulas (L-II) to (LV), # represents a position bonded to the nitrogen-containing heterocycle represented by the general formula (LI). ]

<7> A fluorescent polymer fine particle comprising a polymer fine particle having a core-shell structure composed of a hydrophobic core and a hydrophilic shell, and a fluorescent lanthanoid dye incorporated in the polymer fine particle and containing a lanthanoid cation. A manufacturing method comprising:
Radical polymerization in an aqueous solvent in the presence of a precursor hydrophilic polymer having a main chain containing a hydrophilic unit represented by the following general formula (I) and a side chain containing a polymerizable unit, and a radical generator. The core-shell type polymer fine particles obtained by dispersing and polymerizing monomers to form the core-shell type polymer fine particles in which the hydrophilic polymer that forms the hydrophilic shell is bonded to the polymerizable polymer that forms the hydrophobic core are obtained. A method for producing fluorescent polymer fine particles, comprising introducing the lanthanoid dye.

[In General Formula (I), R ¹ and R ³ each independently represent a hydrogen atom or an alkyl group, R ² and R ⁴ each independently represent a hydrogen atom, an alkyl group, or a cyano group, and L ¹ and L ² Each independently represents a divalent linking group. n represents the average number of repeating ethylene glycol chains. ]

<8> The method for producing fluorescent polymer fine particles according to <7>, wherein the polymerizable unit is an ethylenically unsaturated bond group unit having an ethylenically unsaturated bond as a side chain.

<9> The above <7> or <7>, wherein the precursor hydrophilic polymer further includes a reactive functional group precursor unit, and further includes generating a reactive functional group by activating the reactive functional group precursor unit. 8> The manufacturing method of the fluorescent polymer fine particle as described in 8>.

<10> For fluorescence detection, comprising the fluorescent polymer fine particles according to any one of <1> to <6>, and a linking substance capable of connecting the fluorescent polymer fine particles and a detection target substance. Complex.

<11> The detection target substance using the fluorescent polymer fine particle according to any one of <1> to <6> and a linking substance capable of connecting the fluorescent polymer fine particle and the detection target substance. Detecting fluorescence.
<12> A fluorescence detection kit comprising the fluorescent polymer fine particles according to any one of <1> to <6>.

  According to the present invention, fluorescent polymer fine particles having high fluorescence intensity and good temporal stability, and a detection target substance can be detected with high sensitivity using such fluorescent polymer fine particles, and good A complex for fluorescence detection, a fluorescence detection kit, and a fluorescence detection method having storage stability can be provided.

The fluorescent polymer fine particle of the present invention comprises a polymer fine particle having a core-shell structure composed of a hydrophobic core and a hydrophilic shell, and a fluorescent lanthanoid dye incorporated in the polymer fine particle and containing a lanthanoid cation. A fluorescent polymer fine particle comprising a hydrophilic polymer forming the hydrophilic shell having a hydrophilic unit represented by the general formula (I) in a main chain, and a hydrophobic core in a side chain. The polymerizable polymer to be formed is bonded.
In such a fluorescent polymer fine particle, the hydrophilic polymer forming the hydrophilic shell is grafted to the hydrophobic core, so that the hydrophilic polymer is branched to the hydrophobic core ( An example is shown below), and the dispersion stability of the fluorescent polymer fine particles can be further improved from the effect of steric hindrance.

Hereinafter, the present invention will be described in more detail.
<Hydrophilic polymer>
The hydrophilic polymer forming the hydrophilic shell of the polymer fine particles according to the present invention has a hydrophilic unit represented by the following general formula (I) in the main chain. Thereby, the dispersion stability of polymer fine particles can be improved.

In the general formula (I), R ¹ and R ³ each independently represent a hydrogen atom or an alkyl group. As said alkyl group, a C1-C3 alkyl group (for example, a methyl group, an ethyl group, a propyl group, an isopropyl group) is preferable. R ² and R ⁴ each independently represents a hydrogen atom, an alkyl group or a cyano group. As said alkyl group, a C1-C3 alkyl group (for example, a methyl group, an ethyl group, a propyl group, an isopropyl group) is preferable. L ¹ and L ² each independently represent a divalent linking group, for example, from the group consisting of a single bond, an alkylene group having 1 to 6 carbon atoms, an alkyleneoxy group having 1 to 6 carbon atoms, an ester bond and a carbonyl group. It represents a divalent linking group containing at least one selected and is preferably a divalent linking group containing at least one selected from the group consisting of an alkylene group having 1 to 6 carbon atoms, an ester bond and a carbonyl group. And a divalent linking group comprising an alkylene group having 1 to 6 carbon atoms and an ester bond or a carbonyl group.
n represents the average number of repeating ethylene glycol chains, preferably 5 to 1,000,000, more preferably 10 to 100,000, and still more preferably 20 to 10,000.

In the present invention, R ¹ and R ³ are each independently a hydrogen atom or an alkyl group having 1 to 3 carbon atoms, and R ² and R ⁴ are each independently a hydrogen atom or an alkyl group having 1 to 3 carbon atoms or It is a cyano group, and L ¹ and L ² are a divalent linking group comprising an alkylene group having 1 to 6 carbon atoms and an ester bond or a carbonyl group, and n is preferably 10 to 1000000.
The hydrophilic unit represented by the general formula (I) is more preferably a structure represented by the following general formula (VI) from the viewpoint of particle formability and hydrophilicity.

  In general formula (VI), n represents the average number of repeating ethylene glycol chains, and mm represents an integer of 1 to 10. n is preferably 5 to 1,000,000, more preferably 10 to 100,000, and still more preferably 20 to 10,000. Moreover, it is preferable that mm is an integer of 1-6, and it is more preferable that it is an integer of 2-4.

In the polymer fine particles according to the present invention, the polymerizable polymer forming the hydrophobic core is bonded to the side chain of the hydrophilic polymer. The hydrophilic polymer forming the polymer fine particles can be obtained using a precursor hydrophilic polymer including the hydrophilic unit represented by the general formula (I) and the polymerizable unit having a polymerizable group. In the preferred precursor hydrophilic polymer in the present invention, the polymerizable unit in the precursor hydrophilic polymer has a polymerizable group in the side chain.
Such a precursor hydrophilic polymer is dissolved in an aqueous solvent and reacts with radical species generated by the reaction of a radical polymerizable monomer described later and a radical generator. As a result, fine particles having a polymer of a radical polymerizable monomer as a hydrophobic core and a hydrophilic polymer as a hydrophilic shell are formed. Here, since the hydrophilic polymer has a polymerizable group in the side chain, when it is bonded to the particle surface, it can exhibit a good dispersion stabilizing effect of the particles due to steric repulsion.

  The polymerizable group unit in the precursor hydrophilic polymer is preferably an ethylenically unsaturated group unit represented by the following general formula (II) from the viewpoint of particle formability.

In general formula (II), R ⁵ represents a hydrogen atom or a methyl group. L ³ represents a divalent linking group, and is formed, for example, by a single bond, an alkylene group having 1 to 6 carbon atoms, an alkyleneoxy group having 1 to 6 carbon atoms, -COO-, -OCO-, or a combination thereof. Represents a divalent atomic group. L ⁴ represents an oxygen atom or —NR ³¹ —. R ³¹ represents a hydrogen atom or an alkyl group having 1 to 6 carbon atoms (methyl group, ethyl group, propyl group, butyl group, pentyl group, hexyl group), and may have a branched or cyclic structure.
Z represents an allyl group, an allyloxyethyl group, or an atomic group represented by general formula (III) or general formula (IV). In the following general formulas (III) and (IV), R ⁶ and R ⁷ each independently represent a hydrogen atom or a methyl group. In the general formula (III), m represents 1 to 12, and is preferably 1 to 6 from the viewpoint of particle formation.

The hydrophilic polymer according to the present invention preferably has a reactive functional group that can be linked to a linking substance [for example, a physiologically active substance (eg, antibody, enzyme, or nucleic acid)] described below.
The reactive functional group may be introduced as a unit in the hydrophilic polymer at the time of forming the precursor hydrophilic polymer and / or may be introduced into the hydrophilic polymer after being bonded to the core portion surface described later. . Introduction as a reactive functional group unit at the time of forming the precursor hydrophilic polymer is preferable because the introduction rate of the reactive functional group reactivity can be adjusted more easily.

These reactive functional groups are all stable in water (or an aqueous solvent), and furthermore, a linking substance [for example, a physiologically active substance (for example, a physiologically active substance (for example, , An antibody, an enzyme, or a nucleic acid)]], and the functional group is not particularly limited. Suitable as functional groups to be provided on the surface of the polymer fine particles are acid halide groups such as aldehyde groups, carboxy groups, mercapto groups, acid chloride groups, acid anhydride groups, ester groups, amide groups, maleimide groups, vinylsulfonyl groups, Methanesulfonyloxy group, thiol group, hydroxyl group, amino group, etc., among which aldehyde group, carboxy group, acid chloride group, acid anhydride group, maleimide group, thiol group, amino group, etc. are more preferred, aldehyde group, Maleimide groups, thiol groups and amino groups are most preferred. In addition, since an ester group can be easily converted into a carboxy group by hydrolysis, polymer fine particles having an ester group on the surface are useful intermediates.
When the functional group amount for binding these linking substances is expressed as the functional group equivalent per unit mass of the fluorescent polymer fine particles of the present invention, it is usually 0.001 to 0.5 meq / g, preferably 0.005 to 0.2 meq / gram, more preferably 0.01 to 0.1 meq / gram, more preferably 0.02 to 0.07 meq / gram, most preferably 0.03 to 0 .05 meq / gram.

  The reactive functional group unit for introducing an amino group is an amino group unit represented by the following general formula (V), which has a particle-forming property and a binding affinity with a physiologically active substance as a linking substance. From the viewpoint, this amino group unit is preferably derived from an amino group precursor unit represented by the following general formula (V-1) in the precursor hydrophilic polymer.

In the above general formula, R ⁸ represents a hydrogen atom or a methyl group, R ⁹ represents an acyl group, and is preferably a formyl group or an acetyl group.
By introducing the amino group precursor unit represented by the general formula (V) into the precursor hydrophilic polymer, a reactive group for binding, for example, an antigen or an antibody to the surface of the fluorescent polymer fine particle of the present invention. A large number of preferred amino groups can be introduced into the main chain.

In order to obtain the precursor hydrophilic polymer in the present invention, for example, radical polymerization of a compound represented by the following general formula (VII) and a known radical polymerizable monomer represented by the following general formula (VIII): By this, a polymer having a carboxylic acid can be obtained.
When the reactive functional group unit is introduced into the hydrophilic polymer, for example, a known radical polymerizable monomer represented by the following general formula (IX) is used in the same manner as described above to have the reactive functional group unit. A hydrophilic polymer can be obtained.

In the general formulas (VII), (VIII) and (IX), L ^{1 to} L ³ , R ^{1 to} R ⁵ , R ⁸ , R ⁹ and n are the same as those in the general formulas (I), (II) and (V ) Represents the same meaning as L ^{1 to} L ³ , R ^{1 to} R ⁵ , R ⁸ , R ⁹ and n. Moreover, q represents the integer of 2-1000.
The compound represented by the following general formula (VII) is a polymer polymerization initiator having a structure in which a plurality of polyethyleneoxy units are bonded via an azo group. The molecular weight of the polymer polymerization initiator represented by the general formula (VII) is preferably 1,000 to 1,000,000, more preferably 5,000 to 100,000, and still more preferably 10,000 to 50,000. The molecular weight of the polyethyleneoxy unit is preferably a number average molecular weight of 500 to 50,000, more preferably 1,000 to 10,000. Specific examples of such a polymer polymerization initiator include VPE-0201, VPE-0401, and VPE-0601 manufactured by Wako Pure Chemical Industries.

The amount of the compound represented by the general formula (VII) varies depending on the size and type of the target precursor hydrophilic polymer, but from the viewpoint of the hydrophilicity of the polymer and the particle dispersibility, the amount of the compound to be reacted is 5 to 90%. The range of mass% is preferable, 10 to 80 mass% is more preferable, and 20 to 80 mass% is particularly preferable.
The amount of the monomer represented by the general formula (VIII) varies depending on the size and type of the target precursor hydrophilic polymer, but from the viewpoint of the amount of ethylenically unsaturated bond introduction necessary for particle formation, all the monomers to be reacted The range of 0.1-30 mass% is preferable, 0.5-20 mass% is still more preferable, and 1-10 mass% is especially preferable.
The amount of the monomer represented by the general formula (IX) varies depending on the size and type of the target precursor hydrophilic polymer, but from the viewpoint of the amount of binding with a physiologically active substance, 1 to 99 mass in the total amount of monomers to be reacted. % Is preferable, 5 to 80% is more preferable, and 10 to 50 mol% is particularly preferable.

  In the polymerization reaction for forming the precursor hydrophilic polymer, as the solvent, a solvent for dissolving the polymer polymerization initiator and the radical polymerizable monomer for forming the hydrophilic polymer, for example, ethanol, 1-methoxy-2-propanol, Dimethylacetamide, water, dimethylsulfoxide and mixtures thereof can be used. The monomer concentration is preferably in the range of 10 to 50% by mass and more preferably in the range of 15 to 40% by mass with respect to the total mass of the monomer and the solvent from the viewpoints of molecular weight control and reaction stability. Although reaction temperature can be suitably set according to the decomposition temperature of the initiator to be used, 40 to 100 degreeC is preferable and 60 to 80 degreeC is more preferable.

Next, by reacting the carboxylic acid-containing polymer obtained above with a compound containing an ethylenically unsaturated group, a precursor hydrophilic polymer containing an ethylenically unsaturated bond can be synthesized.
For example, as a compound having an ethylenically unsaturated bond, an alcohol such as allyl alcohol, allyloxyethyl alcohol, hydroxyethyl acrylate, hydroxyethyl methacrylate, etc. is used, and the side chain of the polymer is ethylenic by an esterification reaction with carboxylic acid. Unsaturated bonds can be introduced. In this case, an acid such as sulfuric acid, hydrochloric acid, or toluenesulfonic acid can be used as a catalyst, and a carbodiimide compound such as 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide hydrochloride can also be used as a condensing agent. Furthermore, it is also possible to introduce an ethylenically unsaturated bond into the polymer side chain by an epoxide ring-opening reaction using an epoxy compound such as glycidyl methacrylate as the compound having an ethylenically unsaturated bond. In this case, a quaternary ammonium compound such as tetrabutylammonium chloride or tetrabutylammonium bromide, a quaternary phosphonium compound such as tetrabutylphosphonium bromide, N, N-dimethyl-n-dodecylamine, 1,8-diazabicyclo [ Amine compounds such as 5.4.0] -7-undecene and 4-dimethylaminopyridine can be used.

  The number average molecular weight of the precursor hydrophilic polymer according to the present invention is not particularly limited, but is preferably 500 to 20,000, more preferably 1,000 to 100,000 from the viewpoints of hydrophilicity and particle forming properties.

In order to synthesize a precursor hydrophilic polymer, in addition to the compounds and monomers represented by the above (VII) to (IX) for the purpose of adjusting hydrophilicity, solvent solubility, etc., a known radical polymerizable monomer Can also be copolymerized with the above monomers.
Examples of known radical polymerizable monomers include styrene monomers such as styrene, methylstyrene, chloromethylstyrene, 4-methoxystyrene, 4-acetoxystyrene, methyl (meth) acrylate, ethyl (meth) acrylate, ( Hydroxyethyl methacrylate, propyl (meth) acrylate, butyl (meth) acrylate, hexyl (meth) acrylate, octyl (meth) acrylate, 2-ethylhexyl (meth) acrylate, cyclohexyl (meth) acrylate (Meth) acrylates such as dodecyl (meth) acrylate, stearyl (meth) acrylate, phenyl (meth) acrylate, benzyl (meth) acrylate, 2-hydroxyethyl (meth) acrylate, vinyl acetate , Vinyl monomers such as vinylimidazole It can be mentioned. The copolymerization amount of these radically polymerizable monomers is preferably 0 to 30% by mass, more preferably 0 to 10% by mass with respect to the total hydrophilic polymer.

  Although the specific example (PI-P-14) of the precursor hydrophilic polymer concerning this invention is given to the following, this invention is not limited to these. In the formula, n represents the average number of repeating ethylene glycol chains, and x, y, z, and w represent the molar ratio of each repeating unit.

<Radically polymerizable monomer>
As the hydrophobic radical polymerizable monomer for forming the hydrophobic core of the fine particles in the present invention, a known radical polymerizable monomer that can be bonded to an ethylenically unsaturated bond group can be used. Those that are soluble in water or water-miscible solvents can also be used.
Known radical polymerizable monomers include, for example, styrene monomers such as styrene, methylstyrene, chloromethylstyrene, 4-methoxystyrene, 4-acetoxystyrene, methyl (meth) acrylate, ethyl (meth) acrylate, ( Meth) propyl acrylate, butyl (meth) acrylate, hexyl (meth) acrylate, octyl (meth) acrylate, 2-ethylhexyl (meth) acrylate, cyclohexyl (meth) acrylate, dodecyl (meth) acrylate, (Meth) acrylic acid stearyl, (meth) acrylic acid phenyl, (meth) acrylic acid benzyl, (meth) acrylic acid esters such as 2-hydroxyethyl, vinyl acetate, vinyl chloride, vinyl imidazole, vinyl List vinyl monomers such as pyridine Door can be. These monomers can be polymerized alone or two or more monomers may be copolymerized.

  Further, from the viewpoint of improving the strength of the particles, it is possible to introduce a crosslinked structure into the particle polymer. For this purpose, it is preferable to use a divalent radical polymerizable monomer such as divinylbenzene or ethylene glycol dimethacrylate. A bivalent radically polymerizable monomer can be used in 0.01-10 mass% with respect to all the radically polymerizable monomers which form particle | grains, and 0.1-20 mass% is still more preferable.

<Polymer fine particles>
Although there is no restriction | limiting in particular in the particle size of a polymer microparticle, Usually, it is set as the range of 0.01-20 micrometers as a volume average particle diameter. In particular, when used as a complex for fluorescence detection, the volume average particle diameter is preferably 0.01 to 2 μm. If this value is too large, it is not preferable for reasons such as sedimentation, and conversely if it is too small, it is not preferable in terms of light emission characteristics. For these reasons, the volume average particle size of the polymer fine particles of the present invention is more preferably 0.05 to 1 μm, still more preferably 0.05 to 0.5 μm, and most preferably 0.05 to 0.3 μm.

  The shape of the core part is not particularly limited, but is generally approximately spherical or approximately elliptical. Further, the size of the core portion is not particularly limited and can be appropriately changed according to the use, but the diameter of the substantially spherical core portion is generally about 5 nm to 200 nm.

  The ratio between the diameter of the core portion and the thickness of the shell portion can be appropriately changed according to the application. For example, the diameter of the core portion can be, for example, 5 nm to 200 nm, and the thickness of the shell portion can be, for example, 5 nm to 500 nm.

  The ratio of such a core part and a shell part can be suitably adjusted with the quantity ratio and kind of the hydrophilic polymer and radically polymerizable monomer to be used.

<Method for producing polymer fine particles>
The polymer fine particles according to the present invention can be formed by subjecting the radical polymerizable monomer to dispersion polymerization in an aqueous solvent in the presence of the precursor hydrophilic polymer and a radical generator.

In polymerization, a method is possible in which a radical polymerizable monomer and a precursor hydrophilic polymer are mixed and dissolved in an aqueous solvent, that is, water or a water-containing organic solvent, and a radical generator (polymerization initiator) is added. However, in order to control the reaction temperature and particle diameter, a method of gradually adding a polymerizable monomer and a radical generator into water or a water-containing organic solvent in which a precursor hydrophilic polymer is dissolved in advance is also preferable.
The precursor hydrophilic polymer can be used in the range of 1 to 200% by mass, more preferably 10 to 100% by mass, based on the total mass of the radical polymerizable monomer from the viewpoint of particle formation.

Suitable aqueous solvents include, for example, a mixture of water and a water-soluble organic solvent such as methanol, ethanol, acetone, N, N-dimethylformamide, N, N-dimethylacetamide, tetrahydrofuran and the like.
Typical radical generators are water-soluble radical generators, such as persulfates such as sodium persulfate, potassium persulfate, lithium persulfate, and ammonium persulfate, and are soluble in radical polymerizable monomers or water-containing organic solvents. For example, azo compounds such as 2,2′-azobisisobutyronitrile (AIBN), 2,2′-azobis (2,4-dimethylvaleronitrile), peroxides such as benzoyl peroxide, etc. However, different radical generators may be used in combination. The radical generator is preferably used in an amount of 0.01 to 5% by mass, more preferably 0.1 to 3% by mass, based on the total weight of the radical polymerizable monomer.
Although reaction temperature can be suitably set according to the decomposition temperature of the initiator to be used, 40 to 100 degreeC is preferable and 60 to 80 degreeC is more preferable.

  In addition, in the fluorescent polymer fine particles of the present invention, any additive, for example, an organic phosphorus compound such as trioctylphosphine oxide or tributylphosphine oxide, can be used as long as it does not significantly impair the spirit of the present invention. It is also possible to use an additive that can suppress a decrease in fluorescence intensity due to hydration or the like by coordination, and such an additive can be added in advance to the monomer solution at the time of polymerization. .

<Fluorescent lanthanoid dye>
The fluorescent lanthanoid dye used in the present invention is a fluorescent dye having at least one lanthanoid cation and an organic ligand. This fluorescent dye can be excited with visible light and exhibits a ligand sensitizing action (a phenomenon in which a lanthanoid cation emits light by the energy of light exciting the ligand).
As the ligand, it is preferable to use a nitrogen-containing heterocyclic ring-containing ligand represented by the general formula (LI) having a high fluorescence intensity and a long fluorescence lifetime.

In the general formula (LI), A ¹ , A ² and A ³ are atomic groups represented by the following general formulas (L-II) to (LV), or a hydroxyl group, an alkoxy group, an aryloxy group, Represents an alkylamino group, a dialkylamino group, an arylamino group, or a diarylamino group, which may be the same as each other; In the case of an alkoxy group, an aryloxy group, an alkylamino group, a dialkylamino group, an arylamino group, and a diarylamino group, each having 1 to 12 carbon atoms from the viewpoint of easy incorporation into polymer fine particles and solvent solubility. It is preferable that
A ¹ , A ² , and A ³ are the following general formula (L-II) from the viewpoints of fluorescence intensity, excitation wavelength adjustment, affinity adjustment with lanthanoid ions, ease of incorporation into polymer fine particles, and solvent solubility. It is preferable that it is an atomic group represented by-(LV).

In the formula, R ¹¹ and R ¹² each represent a hydrogen atom or a substituent. Examples of the substituent include an alkyl group, an aryl group, an amino group, a heterocyclic group, an alkylthio group, an arylthio group, an alkoxy group, an aryloxy group, Examples include amide groups, ureido groups, and halogen atoms (fluorine, chlorine, bromine, iodine). In the example of the substituent mentioned here, the number of atoms other than hydrogen atoms is preferably 1 to 50, more preferably 1 to 30, and most preferably 1 to 20. Further, when the substituent contains an alkyl group, it may be cyclic or chain-like, and in the case of a chain, it may be linear or branched and saturated. Or it may contain an unsaturated bond. When the alkyl group or aryl group has a substituent, examples of the preferred substituent include an alkyl group, an alkenyl group, an alkynyl group, an aryl group, a heterocyclic group, a silyl group, an alkoxy group, an amino group, and an alkylamino group. , Dialkylamino group, acylamino group, alkyl or arylsulfonylamino group, acyl group, alkyl or arylsulfonyl group, formyl group, alkoxycarbonyl group, carbamoyl group, sulfamoyl group, halogen atom, cyano group, sulfo group or carboxy group Can be mentioned.

R ^{13 to} R ¹⁶ each independently represent a hydrogen atom or a substituent. When R ^{13 to} R ¹⁶ represent a substituent, preferred examples of the alkyl group, alkenyl group, alkynyl group, aryl group, heterocyclic group, silyl group, alkoxy group, amino group, acylamino group, alkyl may be substituted. Or arylsulfonylamino group, acyl group, alkyl or arylsulfonyl group, formyl group, alkoxycarbonyl group, carbamoyl group, sulfamoyl group, halogen atom (fluorine, chlorine, bromine, iodine), cyano group, sulfo group or carboxyl group And more preferably an optionally substituted alkyl group, alkenyl group, alkynyl group, aryl group, acylamino group, alkyl or arylsulfonylamino group, halogen atom or cyano group.
Regarding R ^{13 to} R ¹⁶ , the number of atoms excluding hydrogen is preferably 1 to 60, more preferably 1 to 45, and most preferably 1 to 35.

R ¹⁷ , R ¹⁸ and R ¹⁹ may represent a hydrogen atom or a substituent, and R ¹⁷ and R ¹⁸ , R ¹⁸ and R ^19, and R ¹⁷ and R ¹⁹ may be bonded to each other to form a ring. Here, the substituent indicates a substituent selected from the group consisting of an alkyl group, an aryl group, a carboamide group, a sulfonamide group, an alkiothio group, a heterocyclic group, an alkoxy group, an aryloxy group, and combinations thereof. l represents 0, 1 or 2; Regarding R ^{17 to} R ¹⁹ , the number of atoms excluding hydrogen is preferably 1 to 60, more preferably 1 to 45, and most preferably 1 to 35.
G represents a carbon atom or a nitrogen atom. In the case of a carbon atom, G may have a substituent, and in this case, those exemplified in the examples of R ¹¹ and R ¹² can be used.
Q represents an atomic group necessary for forming a 5- or 6-membered nitrogen-containing heterocyclic ring, and the nitrogen-containing heterocyclic ring may form a condensed ring. Moreover, it may combine with R ¹⁷ , R ¹⁸ or R ¹⁹ to form a ring.

Examples of preferred heterocyclic rings as Q are given below. In the following examples, it represents a heterocyclic skeleton, which may be used as a partially saturated skeleton, and the position of the heteroatom can be appropriately selected in each ring system. You may condense in arbitrary positions. Moreover, the ring represented by the combination of these rings may be sufficient.
Pyrrole, pyrazole, imidazole, triazole, tetrazole, thiophene, furan, oxazole, thiazole, oxadiazole, thiadiazole, selenazole, pyridine, pyrimidine, pyrazine, pyridazine, triazine, tetrazine, oxazine, thiazine, oxadiazine, thiadiazine, pyrrolopyrrole, indole , Pyrrolopyrazole, pyrroloimidazole, pyrrolotriazole, pyrrolotetrazole, thienopyrrole, pyrrolooxazole, thienopyrrole, pyrroloxazole, pyrrolothiazole, pyrrolopyridine, pyrrolopyrimidine, pyrrolopyrazine, pyrrolopyridazine, pyrrolotriazine, pyrrolothazine, pyrrolothazine Pyrroloxazine, pyrrolothiadiazine, indazole, Zizimidazole, benzotriazole, benzothiophene, benzofuran, benzoxazole, benzothiazole, benzoxiadiazole, benzothiadiazole, benzoselenazole, quinoline, quinazoline, quinoxaline, phthalazine, benzotriazine, benzoxazine, benzothiazine, pyrazolopyrazole, pyra Zolooxazole, pyrazolothiadiazole, pyrazolopyridine, pyrazolopyrimidine, pyrazolopyrazine, pyrazolopyridazine, pyrazolotriazine, pyrazolooxazine, pyrazolothiazine, pyrazolothiadiazine, imidazolopyrazole, pyrazolotriazole, pyrazolotetrazole , Thienopyrazole, furopyrazole, pyrazolooxazole, imidazoloimidazole, imidazolotriazole, Midazolotetrazole, thienoimidazole, furimidazole, imidazolooxazole, thienoimidazole, imidazolooxadiazole, imidazolothiadiazole, imidazoloselenazole, imidazolopyridine, imidazolopyrimidine, imidazolopyrazine, imidazolopyridazine, imidazolo Triazine, imidazolooxazine, imidazolothiazine, imidazolooxadiazine, imidazolothiadiazine, triazolotriazole, thienotriazole, furotriazole, triazolooxazole, triazolothiazole, triazolooxadiazole, triazolothiadiazole, tria Zolopyridine, triazolopyrimidine, triazolopyrazine, triazolopyridazine, triazolotriazine, triazolooxadi , Triazolothiazine, triazolooxadiazine, triazolothiadiazine, tetrazolooxazole, tetrazolothiazole, tetrazolopyridine, tetrazolopyrimidine, tetrazolopyrazine, tetrazolopyridazine, tetrazoloxazine, tetrazolothia Gin, Thienothiophene, Thienofuran, Thienoxazole, Thienothiazole, Thienooxadiazole, Thienothiadiazole, Thienoselenazole, Thienopyridine, Thienopyrimidine, Thienopyrazine, Thienopyridazine, Thienotriazine, Thienotetrazole, Thienoxazine, Thienothazine, Thienothazine , Thienothiadiazine, furoxazole, furothiazole, furoxadiazole, furothiadiazole, furopyridine, furopirimidi , Flopyrazine, Flopyridazine, Furotriazine, Furoxazine, Flothiazine, Oxazolooxazole, Thiazolooxazole, Oxazolooxadiazole, Oxazolothiadiazole, Oxazolopyridine, Oxazolopyrimidine, Oxazolopyrazine, Oxazolopyridazine, Oxazolo Triazine, Oxazolooxazine, Oxazolothiazine, Oxazolooxadiazine, Oxazolothiadiazine, Thiazolothiazole, Thiazolooxadiazole, Thiazolooxadiazole, Thiazoloselenazole, Thiazolopyridine, Thiazolo Pyrimidine, thiazolopyrazine, thiazolopyridazine, thiazolotriazine, thiazolooxazine, thiazolothiazine, thiazoloxadiazine, thiazolothiadiazine, dithiol, dithiol Taxol, benzodithiol, benzodioxole.
Regarding Q, the number of atoms excluding hydrogen is preferably 4 to 70, more preferably 5 to 55, and most preferably 6 to 45.

B ¹ and B ² each independently represent a nitrogen atom or ═C (—R) —, and R represents a hydrogen atom or a substituent. It is preferable that at least one of B ¹ and B ² represents a nitrogen atom.
R represents a hydrogen atom or a substituent. When R represents a substituent, an alkyl group, alkenyl group, alkynyl group, aryl group, heterocyclic group, silyl group, alkoxy group, amino group, acylamino group, alkyl or arylsulfonylamino group, which may be substituted, An acyl group, an alkyl or arylsulfonyl group, a formyl group, an alkoxycarbonyl group, a carbamoyl group, a sulfamoyl group, a halogen atom, a cyano group, a sulfo group or a carboxyl group, more preferably an alkyl group, an alkenyl group, which may be substituted, An alkynyl group, an aryl group, an acyl group, an alkyl or arylsulfonyl group, an alkoxycarbonyl group, a carbamoyl group, a halogen atom or a cyano group. Regarding R, the number of atoms other than hydrogen atoms is preferably 1-30, more preferably 1-20, and most preferably 1-10.

Ar ¹ represents an aromatic carbocyclic ring or aromatic heterocyclic ring having 6 to 30 carbon atoms. Examples of aromatic carbocycles include benzene, thiophene, furan, pyrrole, pyrazole, triazole, thiazole, imidazole, oxazole, oxadiazole, and thiadiazole rings. Further, the ring may be condensed. When Ar ¹ has a substituent, examples of the substituent include the same groups as the examples of the substituent of R ¹¹ and R ¹² described above.
In the general formulas (L-II) to (LV), # represents a position bonded to the nitrogen-containing heterocycle represented by the general formula (LI).
The nitrogen-containing heterocyclic ligand according to the present invention is preferably one represented by the general formula (L-VI), more preferably from the viewpoint of fluorescence intensity, absorption wavelength, and ease of incorporation into polymer fine particles. What is represented by general formula (L-VII) is preferable.

[Wherein ^{A 1} has the same meaning as ^{A 1} in the general formula (L-I). R ^{11, R 12} and G have the same meanings as ^{R 11, R 12} and G in the general formula (L-II). ]

[Wherein R ¹¹ , R ¹² , R ¹⁷ , G and Q represent the same meaning as R ¹¹ , R ¹² , R ¹⁷ , G and Q in the general formulas (L-II) and (L-IV). . ]
The specific examples 1-32 of the nitrogen-containing heterocyclic ligand concerning this invention are shown below.

The exemplified compounds can be synthesized by using known compounds and reacting under known reaction conditions. The compound represented by the general formula (LI) can be synthesized preferably by a nucleophilic substitution reaction of A ^{1 to} A ³ atomic groups to cyanuric chloride.
For example, it can be synthesized by reacting a compound represented by the following general formula (LP-I) with a compound represented by the general formula (LP-III).

In the general formula (LP-I), A is the same as described for A ¹ , A ² , A ³ , E represents a leaving group or A, and in the general formula (LP-III), R ¹⁷ is the same as described above, and R ²⁰ represents an optionally substituted alkyl group or an optionally substituted aryl group (including a heteroaryl group), preferably having 1 to 30 carbon atoms. 1 to 20 is more preferable, and 1 to 12 is more preferable.
R ²¹ and R ²² each represent a hydrogen atom or a substituent, and R ²⁰ and R ²¹ and R ²¹ and R ²² may be bonded to each other to form a ring, if possible. When R ²¹ and R ²² represent a substituent, preferred substituents include alkyl, aryl, amino, heterocyclic, alkylthio, arylthio, alkoxy, aryloxy, amide, ureido and halogen. Atoms (fluorine, chlorine, bromine, iodine) and the like are preferred. In the example of the substituent mentioned here, the number of atoms excluding hydrogen is preferably 1 to 60, more preferably 1 to 45, and most preferably 1 to 35.
D represents a counter ion of an ammonium ion in the general formula (LP-III). Z represents an oxygen atom, —CR ²³ R ²⁴ —, NR ²⁵ , or a sulfur atom, and may combine with R ¹⁷ to form a ring if possible. Here, R ²³ , R ²⁴ , and R ²⁵ represent an alkyl group or an aryl group that may be substituted, and examples of the alkyl group preferably have 1 to 30 carbon atoms, more preferably 1 to 20 carbon atoms, and 1 to 10 carbon atoms. Is most preferable, it may be cyclic or chain-like, and in the case of a chain, it may be linear or branched, and it is saturated or contains an unsaturated bond It may be.

  Examples of the leaving group as E in the general formula (LP-I) include a halogen atom (fluorine, chlorine, bromine, iodine), an aryloxy group (phenoxy group, 4-nitrophenoxy group, etc.), an arylthio group ( Phenylthio group, 4-bromophenylthio group, etc.), sulfonyloxy group (p-toluenesulfonyloxy group, trifluoromethanesulfonyloxy group, etc.), carbamoyloxy group (dimethylcarbamoyloxy group, morpholinocarbonyloxy group, etc.) are preferred, halogen Atoms are most preferred.

  The counter ion as D in the general formula (LP-III) is preferably a halide ion (fluorine ion, chlorine ion, bromine ion, iodine ion), sulfate ion, perchlorate ion, nitrate ion, hydrogen sulfate ion, Examples include sulfonate ions (p-toluenesulfonate ions, p-chlorobenzenesulfonate ions, etc.), sulfate ester ions (monomethyl sulfate ions, etc.), tetrafluoroborate ions, hexafluorophosphate ions, and the like.

  The nitrogen-containing heterocyclic ring-containing ligand in the present invention is a mixture of a compound represented by the general formula (LP-I) and a compound represented by the general formula (LP-III), preferably using a solvent, Preferably, it can be obtained by carrying out the reaction at an appropriate temperature in the presence of a base.

  In this case, most bases can be used as long as they are usually used in organic synthesis, but preferably pyridines (pyridine, 2,6-lutidine, etc.), tertiary amines (triethylamine, N-ethyl). Diisopropylamine, N-methylmorpholine, tributylamine, etc.), guanidines (triphenylguanidine, 1,1,3,3-tetramethylguanidine, etc.), amidines (1,8-diazabicyclo [5.4.0]- 7-undecene, 1,5-diazabicyclo [4.3.0] -5-nonene, etc.), anilines (N, N-diethylaniline, etc.), potassium carbonate, sodium carbonate, sodium bicarbonate, sodium hydride, sodium Methoxide, sodium ethoxide, sodium-t-butoxide, potassium-t-butoxy De, potassium acetate, and the like can be preferably used sodium acetate, pyridine, tertiary amines, guanidines, amidines are more preferable.

As the solvent to be used, either a protic solvent or an aprotic solvent can be used, but an aprotic solvent is preferable, and acetonitrile, N, N-dimethylformamide, dimethyl sulfoxide, tetrahydrofuran, diethyl ether, N, N-dimethyl are used. Acetamide, N-methylpyrrolidone, 1,3-dimethyl-2-imidazolidinone and the like are preferable.
The reaction temperature should be set to an appropriate temperature depending on the reaction. In the present invention, generally, -20 ° C to 150 ° C is preferable, 0 ° C to 120 ° C is more preferable, and 0 ° C to 100 ° C is most preferable.
The reaction may be charged in any order, but the compound represented by the general formula (LP-I) and the compound represented by the general formula (LP-III) are dissolved or suspended in a solvent, and the base is added while stirring. It is preferable to add.

(Synthesis example) The synthesis | combination of the exemplary compound 4 For example, the said compound 4 is compoundable by the following method.
18.4 g of cyanuric chloride was dissolved in 100 mL of dimethylacetamide and 69.2 g of 3,5-dimethylpyrazole was added at room temperature. Subsequently, the reaction temperature was set at 80 ° C. for 2 hours.
After cooling, the reaction solution was poured into water, and the precipitated crystals were collected by filtration and recrystallized from dimethylformamide to obtain the following (LP-I-1). Yield 21.0 g, Yield 57.9%
nmr spectrum data (deuterated chloroform): 6.11 (3H, s), 2.81 (9H, s), 2.33 (9H, s)

419 mg of 6-chloro-5-cyano-1,3-diethyl-2-methyl-1H-benzimidazolium p-toluenesulfonate and 363 mg of the above compound (LP-I-1) were suspended in 8 mL of dimethyl sulfoxide. , 0.5 mL of tetramethylguanidine was added and reacted at 80 ° C. for 30 minutes.
After cooling, water was added and the precipitated crystals were filtered under reduced pressure, and the obtained crystals were purified by silica gel column chromatography. Furthermore, recrystallization was performed from a mixed solvent of methanol and ethyl acetate to obtain the desired product. Yield 287 mg, Yield 55.7%.
nmr spectral data (deuterated chloroform): 7.34 (1H, s), 7.21 (1H, s), 6.04 (2H, s), 5.31 (1H, s), 4.53 (2H, q), 4.39 (2H, q), 2.70 (6H, s), 2.33 (6H, s), 1.31 (3H, t), 1.28 (3H, t)
Other exemplary compounds can also be synthesized in the same manner as compound 4 by changing the portion corresponding to A ^{1 to} A ³ to the corresponding compound.
The nitrogen-containing heterocyclic ligand according to the present invention can be used in the range of 0.1 to 10 equivalents relative to the lanthanoid cation, and more preferably in the range of 1 to 5 equivalents. .
Examples of the lanthanoid cation include divalent to tetravalent cations, and specific examples thereof include Ce ³⁺ , Pr ³⁺ , Nd ³⁺ , Nd ⁴⁺ , Sm ²⁺ , Sm ³⁺ , Eu ²⁺ , Eu ³⁺ , Tb ³⁺ , ^{Dy 3+, Dy 4+, Ho 3+} , ER 13+, Tm 2+, Tm 3+, Yb 2+, Yb 3+ , and among ^{them, Pr 3+, Nd 3+, Sm} 3+, Eu 3+, Tb 3+, Dy 3+, Ho 3+ , Er ³⁺ , Tm ³⁺ , Yb ^{3+ and the} like are suitable because they emit fluorescence having characteristics such as ultraviolet to near-infrared region, long lifetime, narrow wavelength width, etc. Among them, Nd ³⁺ , Sm ³⁺ , Eu ³⁺ , Tb ³⁺ , Dy ³⁺ , and Tm ³⁺ are more preferable, and Eu ³⁺ and Tb ³⁺ are most preferable in terms of emission intensity.

In place of or in combination with a ligand other than the general formula (LI), a known organic ligand of a lanthanoid cation can be used from the viewpoint of fluorescence intensity and ease of synthesis of a lanthanoid dye.
Examples of other organic ligands include aromatic amines (Helv. Chim. Acta, Vol. 79, P. 789, 1966), β-diketones (Anal. Chem. Vol. 70, P. 596- 601, 1998), aromatic group-containing carboxylic acids (Chem. Mater., Vol. 10, 286-296, JP 2000-345052 A). Specific examples include 4,4,4-trifluoro-1- (2-thienyl) -1,3-butanedione, 4,4,4-trifluoro-1-phenyl-1,3-butanedione, 4-trifluoro-1- (2-naphthyl) -1,3-butanedione can be mentioned. Examples of the aromatic carboxylic acid include a dendron having a carboxylate group at a focal point and an aromatic ring at a repeating unit.

  In addition to these ligands, phosphine oxides, phosphate esters, sulfoxides, phosphites, phosphines, sulfides, amines, and nitrogen are included for the purpose of suppressing the decrease in fluorescence intensity of lanthanoid dyes. Aromatic heterocyclic compounds can be used simultaneously.

By adding a ligand solution containing at least the ligand of the present invention to the lanthanoid element-containing solution, the fluorescent lanthanoid complex, that is, the fluorescent lanthanoid dye of the present invention can be easily synthesized. When the fluorescent lanthanoid complex is precipitated from the solution, it can be filtered off. When the fluorescent lanthanoid complex does not precipitate, the solvent can be distilled off to obtain crystals, which can then be purified and used as necessary.
Although there is no restriction | limiting in the density | concentration of the fluorescent lanthanoid complex in the polymer fine particle of this invention, Usually, 0.01-50 mass% with respect to the mass of a polymer fine particle, Preferably it is 0.05-20 in terms of a brightness | luminance. The mass% is more preferably 0.1 to 10 mass.

<Method for producing fluorescent polymer fine particles>
The fluorescent polymer fine particles of the present invention can be produced by introducing (incorporating) a fluorescent lanthanoid dye after obtaining the core-shell polymer fine particles according to the present invention.
The pigment can be introduced as follows. The fine particles and the fluorescent lanthanoid dye are immersed in a solution containing an organic solvent (for example, acetone or toluene) that swells the water-insoluble polymer compound that forms the hydrophobic portion of the fine particles at a constant ratio. By the immersion, the water-insoluble polymer compound swells, and the fluorescent lanthanoid dye is taken into the fine particles along with the swelling. Subsequently, when the organic solvent is removed from the mixture, the water-insoluble polymer compound contracts, but the hydrophobic fluorescent lanthanoid dye cannot escape from the microparticles and is encapsulated in the microparticles. If desired, the fluorescent polymer fine particles of the present invention can be obtained by removing the fluorescent lanthanoid dye that has not been incorporated into the fine particles.

In the above production method, the method for removing the organic solvent from the mixture is not particularly limited, but for example, a method of evaporating the organic solvent by evaporation or the like, or a method of shrinking in a non-solvent ( For example, a method in which a solution containing an organic solvent is replaced with a solution not containing the organic solvent) can be used.
The ratio of the organic solvent in the organic solvent-containing solution used to swell the fine particles is such that the water-insoluble polymer compound can be swollen to the extent that the fluorescent lanthanoid dye is incorporated into the water-insoluble polymer compound. As long as it is, it will not specifically limit, For example, it can be 40-60 (vol / vol)%.
In addition to the above method, it is also possible to introduce a fluorescent lanthanoid dye simultaneously with the formation of fine particles by dissolving the fluorescent lanthanoid dye in a liquid containing a polymerizable monomer or macromonomer and then polymerizing it. is there.

When the hydrophilic polymer according to the present invention includes a reactive functional group unit, it further includes generating a reactive functional group by activating the reactive functional group precursor unit. Such activation can be appropriately selected depending on the type of the reactive functional group unit.
For example, when an amino group precursor structure is included as the reactive functional group precursor unit, an acid or alkaline treatment is performed on the polymer fine particles in order to convert the amino group precursor structure into an amino group. As the acid, inorganic acids such as hydrochloric acid, sulfuric acid and nitric acid, and organic acids such as p-toluenesulfonic acid and acetic acid can be used. The pH is preferably 5 or less, and more preferably 3 or less. As the alkali, an aqueous sodium hydroxide solution or an aqueous potassium hydroxide solution can be used. As pH, 9 or more are preferable and 11 or more are more preferable. As reaction temperature, 25-100 degreeC is preferable and 40-90 degreeC is more preferable.
Such activation treatment may be performed either before or after the step of introducing the fluorescent lanthanoid dye depending on the specific treatment to be applied.

<Fluorescence detection complex>
The complex for fluorescence detection of the present invention is composed of the fluorescent polymer fine particles of the present invention and a linking substance capable of linking the fluorescent polymer fine particles and the detection target substance. As a result, the target substance can be detected efficiently and with high sensitivity based on the fluorescence emission of the fluorescent polymer fine particles via the linking substance.
Examples of linking substances include antigens and antibodies. Examples of such antibodies include polyclonal antibodies such as rabbits and goats, mouse monoclonal antibodies IgG and IgM, or F (ab ′) ₂ , Fab and Fab ′ fractions obtained by enzymatic treatment or reducing agent treatment thereof. Is mentioned. On the other hand, examples of the antigen include various things such as proteins, polypeptides, steroids, polysaccharides, lipids, drugs, and pollen.

  Such antibody or antigen binding methods include a method in which the sugar chain of the antibody or antigen is bound to the amino group of the polymer fine particle using periodic acid, and the antibody or antigen is bound to the amino group of the polymer fine particle. Method of coupling amino group using glutaraldehyde, reaction with Sulfo-SMCC (sulfosuccinimidyl 4- (N-maleimidomethyl) cyclohexane-1-carboxylate) for amino group of polymer fine particles A method of introducing a maleimide group and binding with a mercapto group of an antibody can be exemplified. The binding amount is usually preferably 50 nanograms to 500 micrograms, more preferably 500 nanograms to 200 micrograms, as the mass per milligram of polymer fine particles. Thereby, it can be used as an immunoassay reagent having fluorescence ability (fluorescence immunoassay reagent).

In addition to antigens and antibodies, linking substances include allergens, enzymes, enzyme substrates, coenzymes, enzyme inhibitors, host compounds, hormones, hormone receptors, proteins, blood proteins, tissue proteins, cells, cell debris, Nuclear material, virus, virus particle, metabolite, neurotransmitter, hapten, drug, nucleic acid, metal, metal complex, microorganism, parasite, bacteria, biotin, avidin, lectin, sugar, bioactive substance, bioactive substance receptor In addition, environmental substances, chemical species, or derivatives thereof may be appropriately selected according to the detection target substance. For example, when the detection target substance can be avidinized in advance, biotin can be used as the linking substance. When the detection target substance is a predetermined ligand, it may be a receptor that specifically binds to the ligand and a fragment thereof. Further, when the target substance is a nucleic acid, it may be a protein or peptide (aptamer) that specifically binds to the nucleic acid.
The coupling method with these linking substances may be performed in the same manner as described above, or may be coupled by other known coupling methods.
Such a linking substance may be the detection target substance itself, or may be detected by the complex for fluorescence detection by binding to the detection target substance in the same manner as the fluorescent polymer fine particles. .

<Fluorescence detection method>
The fluorescence detection method of the present invention is to detect a detection target substance using a fluorescent substance fine particle, a linking substance capable of connecting the fluorescent polymer fine particle and the detection target substance, that is, a fluorescence detection complex. Including. Accordingly, the detection target substance can be detected with high sensitivity using the fluorescent polymer fine particles of the present invention having high fluorescence intensity and capable of detection with high sensitivity.
The above matters can be applied as they are to the complex for fluorescence detection.

  In the fluorescence detection method, the procedures and conditions in a known fluorescence detection method using a fluorescent substance can be applied as they are, and a detection sample is prepared by bringing a measurement target substance into contact with a fluorescence detection complex. Irradiating excitation light that excites the detection complex, and measuring fluorescence emitted by the irradiation.

  In addition, since the fluorescence detection method of the present invention uses a lanthanoid complex (especially europium, terbium, ruthenium) having a long emission lifetime as a fluorescent dye, a delayed fluorescence analysis method for detecting the target fluorescence after the background fluorescence disappears. It is preferable that For example, the emission lifetime of a europium complex is several hundred μsec to msec, which is 100,000 to 1 million times that of a general organic dye. Furthermore, it has characteristics such as a large Stokes shift of 250 nm or more and a sharp fluorescence peak. For analysis, the ligand of the lanthanoid ion is excited and the generated delay is measured with a time-resolved fluorescence measuring device.

In addition, the fluorescent lanthanoid dye in the present invention can be excited by visible light, for example, light of about 400 nm. For this reason, it is not necessary to use high-energy excitation light such as UV, and the range of selection of the excitation light source can be widened.
Thereby, the fluorescence detection by the low energy excitation light source can be performed with high sensitivity.
The fluorescence detection method is more preferably time-resolved fluorescence measurement from the viewpoint of measurement accuracy and sensitivity. Conditions in the time-resolved fluorescence measurement can be appropriately selected according to the selected metal ion.

<Fluorescence detection kit>
The fluorescence detection kit of the present invention includes the fluorescent polymer fine particles of the present invention.
As a result, the target substance can be detected efficiently and with high sensitivity based on the fluorescence emission of the fluorescent polymer fine particles via the linking substance.
The fluorescent polymer fine particles in the present fluorescence detection kit may be included together with a linking substance, or may be included as a complex for fluorescence detection.
Moreover, according to the kind of coupling | bonding substance, the reagent for making a detection target substance detectable with the composite_body | complex for fluorescence detection may be included. Examples of such a reagent include a reagent for avidinating a detection target substance when biotin is selected as a linking substance in the fluorescence detection complex.

  EXAMPLES The present invention will be specifically described below with reference to examples, but the present invention is not limited to these examples. Unless otherwise specified, “part” and “%” are based on mass.

(Example 1)
<Synthesis of Precursor Hydrophilic Polymer P-1>
Under a nitrogen stream, 10 g of VPE-0201 (manufactured by Wako Pure Chemical Industries, Ltd.), 38 g of N-vinylformamide (manufactured by Aldrich), 2 g of NK ester SA (manufactured by Shin-Nakamura Chemical Co., Ltd.), N, N-dimethyl After dissolving in 150 g of acetamide and stirring at 80 ° C. for 4 hours, the temperature was raised to 100 ° C. and stirring was performed for 2 hours. After cooling to room temperature, the reaction mixture was diluted with 100 g of N, N-dimethylacetamide. Next, this was poured into 4 L of ethyl acetate, and the precipitated polymer was suction filtered and dried in vacuum at room temperature. 46 g of polymer (PP-1) was obtained. When the acid value was measured by titration with a 0.1 mol / L potassium hydroxide aqueous solution, 0.13 mmol of acid was contained per 1 g of the polymer.
Next, 20 g of the polymer (PP-1) obtained above was dissolved in 100 g of dimethylacetamide, 1.8 g of allyl alcohol, 0.1 g of 4- (N, N-dimethylamino) pyridine, N, N′-diisopropyl. The carbodiimide 1.9g was added and it stirred at 40 degreeC for 4 hours. Next, 0.5 g of acetic acid and 10 g of ethanol were added, stirred at 40 ° C. for 2 hours, and cooled to room temperature. Next, this reaction mixture was poured into 3 L of ethyl acetate, and the precipitated polymer was subjected to suction filtration and then vacuum dried at room temperature to obtain 19 g of a precursor hydrophilic polymer P-1. In titration with a 0.1 mol / L aqueous potassium hydroxide solution, no acid was detected and it was found that the carboxylic acid in the polymer had disappeared by reaction with hydroxyethyl methacrylate. Moreover, the number average molecular weight Mn of this precursor hydrophilic polymer P-1 was 9500 from GPC measurement.

<Synthesis of Precursor Hydrophilic Polymer P-5>
10 g of the polymer (PP-1) obtained in the process of synthesizing the precursor hydrophilic polymer P-1 was dissolved in 50 g of dimethylacetamide, 4 g of 2-hydroxyethyl methacrylate, 4- (N, N-dimethylamino) 0.05 g of pyridine and 1 g of N, N′-diisopropylcarbodiimide were added and stirred at 40 ° C. for 4 hours. Next, 0.5 g of acetic acid and 10 g of ethanol were added, stirred at 40 ° C. for 2 hours, and cooled to room temperature. Next, this reaction mixture was poured into 3 L of ethyl acetate, and the precipitated polymer was subjected to suction filtration and then vacuum-dried at room temperature to obtain 23 g of precursor hydrophilic polymer P-5. In titration with a 0.1 mol / L aqueous potassium hydroxide solution, no acid was detected and it was found that the carboxylic acid in the polymer had disappeared by reaction with hydroxyethyl methacrylate. Moreover, the number average molecular weight Mn of this precursor hydrophilic polymer P-5 was 9600 from GPC measurement.

<Synthesis of fluorescent fine particle dispersion>
2 g of the precursor hydrophilic polymer P-1 (number average molecular weight 9500) synthesized above was dissolved in a mixed solution of 20 g of distilled water and 6 g of ethanol, and heated to 60 ° C. while flowing 50 mL of nitrogen per minute. Next, a solution consisting of 1 g of methyl methacrylate, 0.04 g of 2,2′-azobis (2,4-dimethylvaleronitrile) and 8 g of ethanol was dropped into this solution over 2 hours. After completion of dropping, the mixture was stirred at 60 ° C. for 5 hours, further stirred at 75 ° C. for 1 hour, and cooled to room temperature. The dispersion was purified by dialysis using distilled water for one day. Further, after separating the particles using a centrifuge, the particles were diluted and redispersed using distilled water to adjust the particle concentration to 4% by mass. When the particle size was measured using a particle size distribution analyzer (Coulter N4Plus, manufactured by Beckman Coulter, Inc.), the volume average particle size was 300 nm.
To 1 g of the particle dispersion, 2.5 mL of hydrochloric acid and 6.5 g of distilled water were added and mixed, followed by stirring at 25 ° C. for 14 hours to hydrolyze the formamide group in the hydrophilic polymer bonded to the particle surface and convert it to an amino group. did. After dialysis purification using distilled water for one day to remove water-soluble substances, the particles are concentrated using a centrifuge, and the dilution concentration is adjusted by adding distilled water to form a surface of 1.25% by mass. A fine particle dispersion having an amino group was obtained. When the zeta potential of this dispersion was measured, it showed a potential of +19 mV.
Next, 4 g of a fine particle dispersion having an amino group on the surface obtained above, 6.0 mg of tris (4,4,4-trifluoro-1- (2-thienyl) -1,3-butanediono) europium (III) Then, 3.8 mg of the ligand having the triazine ring of Compound Example 12 was dissolved in 1 g of methanol. Next, this was added to 4 g of a fine particle dispersion having an amino group on the surface synthesized above and stirred at room temperature for 3 hours. Thereafter, methanol was removed by dialysis, and the mixture was allowed to stand at 45 ° C. for 3 days, and then cooled to room temperature to obtain fluorescent fine particle dispersion-1 having an amino group on the surface. This fluorescent fine particle dispersion-1 showed strong fluorescence at 615 nm by irradiation with excitation light of 420 nm.

<Synthesis of biotin-linked fine particles>
The fluorescent fine particles (europium dye-stained particles) having amino groups on the surface synthesized above were dispersed in PBS buffer (pH 7 to 8.5) at a concentration of 10 mg / mL. 20 μL of 20 mM · EZ-Link · NHS-PEO4-Biotine (manufactured by PIERCE) was added to 2 mL of the fine particle dispersion and reacted at room temperature for 1 hour. Thereafter, purification was performed by dialysis to obtain a fluorescent fine particle solution having biotin on the surface. Further, concentration was performed to make the particle concentration 1% by mass.

<Detection using avidin binding plate>
The biotin-linked fine particle solution obtained above was diluted with purified water so as to be 0.001 mass%, 0.0001 mass%, and 0.00001 mass%. Next, 200 μL of each was spotted on an avidin-fixed 96-well microplate (Nunc, Nunc Immobilizer Streptavidin) and allowed to stand at room temperature for 2 hours. Next, it was washed with purified water. Water was removed, and the fluorescence amount of the microplate was measured using a plate reader ARBO (manufactured by PerkinElmer). 420 nm was used as the excitation wavelength, and fluorescence at 615 nm was measured in a time-resolved fluorescence measurement mode. As a result, delayed fluorescence depending on the fluorescent particle concentration was observed. Further, the fluorescent fine particle dispersion was allowed to stand for 2 weeks in an environment of 40 ° C., then spotted again on the microplate, and fluorescence measurement was performed in the same manner as described above. The fluorescence intensity of the microplate hardly changed, and it was confirmed that the fluorescence detection reagent had excellent storage stability.

(Examples 2 to 4)
In Example 1, fluorescent fine particles were prepared in the same manner as in Example 1 except that the hydrophilic polymer and radical polymerizable monomer shown in Table 1 below were used instead of the hydrophilic polymer P-1 and methyl methacrylate, respectively. . The average particle size is shown in Table 1. Further, in the same manner as in Example 1, particles bound with biotin were prepared and fluorescence measurement was performed. In any of the fluorescent fine particle dispersions, delayed fluorescence depending on the concentration of the fluorescent fine particles was observed. In any of the fluorescent fine particle dispersions, the fluorescent fine particle dispersion was allowed to stand in an environment of 40 ° C. for 2 weeks. The sample was spotted on a microplate, and fluorescence was measured in the same manner as described above. The fluorescence intensity of the microplate hardly changed, and it was confirmed that the fluorescence detection reagent had excellent storage stability.

(Comparative Example 1)
In accordance with the method described in Anal. Chem., 2003, 75, 6124-6132, radical polymerizable macromonomers each having an amino group and a polymerizable unsaturated bond unit at the polyethyleneoxy terminus represented by the following chemical formula were synthesized. . From the GPC measurement, the number average molecular weight was 7000.

Next, styrene (100 equivalents), the macromonomer (5 equivalents), and 1,4-divinylbenzene (2 equivalents) were coexisted with the radical generator V-65 (2 equivalents, manufactured by Wako Pure Chemical Industries, Ltd.). The mixture was heated to 70 ° C. for 6 hours in ethanol / distilled water = 4/1 (volume ratio) to perform radical polymerization. The reaction product was filtered, and the filtrate was purified by dialysis (fraction molecular weight = 12000-14000) using distilled water for one day to obtain a dispersion of fine particles having amino groups on the surface. When the particle size was measured using a particle size distribution analyzer (Coulter N4 Plus, manufactured by Beckman Coulter, Inc.), the average particle size was 130 nm. Diluted with distilled water to a particle concentration of 1.25% by mass.
Next, 4 g of a fine particle dispersion having an amino group on the surface obtained above, 6.0 mg of tris (4,4,4-trifluoro-1- (2-thienyl) -1,3-butanediono) europium (III) Then, 3.8 mg of the ligand having the triazine ring of Compound Example 12 was dissolved in 1 g of methanol. Next, this was added to 4 g of a fine particle dispersion having an amino group on the surface synthesized above and stirred at room temperature for 3 hours. Thereafter, methanol was removed by dialysis, and the mixture was allowed to stand at 45 ° C. for 3 days, and then cooled to room temperature to obtain a fluorescent fine particle dispersion-5 having an amino group on the surface.
This fluorescent fine particle dispersion-5 exhibited delayed fluorescence depending on the concentration of the fluorescent fine particles at 615 nm when irradiated with excitation light of 420 nm. However, when compared with Example 1, the fluorescence intensity was 50% of the fluorescence intensity of Example 1 at the same fluorescent fine particle concentration. Further, when the fluorescent fine particle dispersion liquid-5 was allowed to stand in an environment of 40 ° C. for 2 weeks and then subjected to fluorescence measurement again, particle aggregation was observed, and the fluorescence intensity further decreased to 50% before storage.

(Comparative Example 2)
In Comparative Example 1, a fluorescent fine particle dispersion-6 was obtained in the same manner as in Comparative Example 1, except that the ligand having a triazine ring of Compound 12 was not added.
In this fluorescent fine particle dispersion-6, no fluorescence was observed at 615 nm when irradiated with excitation light of 420 nm. When the excitation light of 340 nm was irradiated, delayed fluorescence depending on the concentration of the fluorescent fine particles was shown. However, when compared with Example 1, the fluorescence intensity was 50% of the fluorescence intensity of Example 1 at the same fluorescent fine particle concentration. Further, when the fluorescent fine particle dispersion liquid-6 was allowed to stand in an environment of 40 ° C. for 2 weeks and then subjected to fluorescence measurement again, particle aggregation was observed, and the fluorescence intensity further decreased to 50% before storage.

From the above, it can be seen that the fluorescent polymer fine particles of the present invention are fluorescent polymer fine particles having high fluorescence intensity and good temporal stability. On the other hand, it can be seen that the fluorescent polymer fine particles of the comparative example are inferior in fluorescence intensity and stability over time in a dispersion state as compared with the fluorescent polymer fine particles of the present invention.

Claims (12)

A fluorescent polymer fine particle comprising a polymer fine particle having a core-shell structure composed of a hydrophobic core and a hydrophilic shell, and a fluorescent lanthanoid dye incorporated in the polymer fine particle and containing a lanthanoid cation, The hydrophilic polymer that forms the hydrophilic shell contains a hydrophilic unit represented by the following general formula (I) in the main chain, and a polymerizable polymer that forms a hydrophobic core is bonded to the side chain. Fluorescent polymer fine particles characterized by the above.

[In General Formula (I), R ¹ and R ³ each independently represent a hydrogen atom or an alkyl group, R ² and R ⁴ each independently represent a hydrogen atom, an alkyl group, or a cyano group, and L ¹ and L ² Each independently represents a divalent linking group. n represents the average number of repeating ethylene glycol chains. ]   The hydrophilic polymer is a precursor hydrophilic having a main chain containing a hydrophilic unit represented by the general formula (I) and a side chain containing an ethylenically unsaturated bond group unit having an ethylenically unsaturated bond. The fluorescent polymer fine particle according to claim 1, wherein the fluorescent polymer fine particle is derived from a conductive polymer. The precursor hydrophilic polymer includes a hydrophilic unit represented by the following general formula (I) and an ethylenically unsaturated bond group unit represented by the following general formula (II). 2. The fluorescent polymer fine particle according to 2.

[In General Formulas (I) and (II), R ¹ and R ³ each independently represent a hydrogen atom or an alkyl group, R ² and R ⁴ each independently represent a hydrogen atom, an alkyl group, or a cyano group; ¹ and L ² each independently represent a divalent linking group. n represents the average number of repeating ethylene glycol chains. R ⁵ represents a hydrogen atom or a methyl group. L ⁴ represents an oxygen atom or —NR ³¹ —. R ³¹ represents a hydrogen atom or an alkyl group having 1 to 6 carbon atoms. L ³ represents a divalent linking group, Z represents an allyl group, an allyloxyethyl group, and an atomic group represented by General Formula (III) or General Formula (IV).

In general formulas (III) and (IV), R ⁶ and R ⁷ each independently represent a hydrogen atom or a methyl group. m represents 1-12. ]   The fluorescent polymer fine particle according to claim 1, wherein the hydrophilic polymer further includes a reactive functional group unit. 5. The fluorescent polymer fine particle according to claim 4, wherein the reactive functional group unit is an amino group unit represented by the following general formula (V).

[In the general formula (V), R ⁸ represents a hydrogen atom or a methyl group. ] The fluorescence according to any one of claims 1 to 5, wherein the fluorescent lanthanoid dye has at least one nitrogen-containing heterocyclic ligand represented by the following general formula (LI). Polymer fine particles.

[In General Formula (LI), A ¹ , A ² , and A ³ are atomic groups represented by the following General Formulas (L-II) to (LV), or a hydroxyl group, an alkoxy group, and an aryloxy group. Represents an alkylamino group, a dialkylamino group, an arylamino group, or a diarylamino group, which may be the same as each other. B ¹ and B ² each independently represent a nitrogen atom or ═C (—R) —, and R represents a hydrogen atom or a substituent.

In each formula, R ^{11 to} R ¹⁹ each represent a hydrogen atom or a substituent. R ¹⁷ and R ¹⁸ , R ¹⁸ and R ¹⁹ and R ¹⁷ and R ¹⁹ may be combined with each other to form a ring, if possible. l represents 0, 1 or 2; G represents a carbon atom or a nitrogen atom which may have a substituent. Q represents an atomic group necessary for forming a 5-membered or 6-membered nitrogen-containing heterocyclic ring, and the nitrogen-containing heterocyclic ring may further form a condensed ring. Ar ¹ represents an aromatic carbocyclic ring or an aromatic heterocyclic ring. In the general formulas (L-II) to (LV), # represents a position bonded to the nitrogen-containing heterocycle represented by the general formula (LI). ] A method for producing a fluorescent polymer fine particle comprising a polymer fine particle having a core-shell structure comprising a hydrophobic core and a hydrophilic shell, and a fluorescent lanthanoid dye incorporated in the polymer fine particle and containing a lanthanoid cation. There,
Radical polymerization in an aqueous solvent in the presence of a precursor hydrophilic polymer having a main chain containing a hydrophilic unit represented by the following general formula (I) and a side chain containing a polymerizable unit, and a radical generator. Dispersing and polymerizing monomers to form core-shell polymer fine particles in which a hydrophilic polymer that forms a hydrophilic shell is bonded to a polymerizable polymer that forms a hydrophobic core;
Introducing the lanthanoid dye into the obtained core-shell polymer fine particles,
The manufacturing method of the fluorescent polymer microparticle containing this.

[In General Formula (I), R ¹ and R ³ each independently represent a hydrogen atom or an alkyl group, R ² and R ⁴ each independently represent a hydrogen atom, an alkyl group, or a cyano group, and L ¹ and L ² Each independently represents a divalent linking group. n represents the average number of repeating ethylene glycol chains. ]   The method for producing fluorescent polymer fine particles according to claim 7, wherein the polymerizable unit is an ethylenically unsaturated bond group unit having an ethylenically unsaturated bond as a side chain.   The fluorescent property according to claim 7 or 8, wherein the precursor hydrophilic polymer further includes a reactive functional group precursor unit, and further includes generating a reactive functional group by activating the reactive functional group precursor unit. A method for producing polymer fine particles.   A fluorescent detection composite comprising the fluorescent polymer fine particle according to any one of claims 1 to 6 and a linking substance capable of connecting the fluorescent polymer fine particle and a detection target substance.   The detection target substance is detected using the fluorescent polymer fine particle according to any one of claims 1 to 6 and a connecting substance capable of connecting the fluorescent polymer fine particle and the detection target substance. And a fluorescence detection method. The fluorescence detection kit containing the fluorescent polymer fine particle of any one of Claims 1-6.