Luminescent nanoparticles and luminescent labeling materials for pathological diagnosis

Abstract

The present invention addresses the problem of providing: light-emitting nanoparticles for enabling a high-luminous particle technique for bioimaging and also enabling a high-sensitivity imaging; and a light-emitting labeling material for pathological diagnosis use. The light-emitting nanoparticles according to the present invention comprise a first light-emitting compound and a second light-emitting compound, in which the first light-emitting compound has a function to be excited by the irradiation with light and transfer an energy generated by the excitation to the second light-emitting compound, the second light-emitting compound has a function to receive the energy generated by the excitation and emit light, and the content of the first light-emitting compound falls within the range from 4 to 90% by mass relative to the whole amount of the light-emitting nanoparticles.

Description

[Technical Field]

[0001] The present invention relates to luminescent nanoparticles and luminescent labeling materials for pathological diagnosis, and more particularly to luminescent nanoparticles and luminescent labeling materials for pathological diagnosis that realize high-brightness particle technology for bioimaging and enable highly sensitive imaging. [Background technology]

[0002] In "bioimaging" using luminescent compounds, a major challenge is to separate the luminescence of the luminescent compounds from the autofluorescence of cells in order to achieve high-sensitivity imaging that can determine the location and quantification of proteins by the bright spots of nanoparticles in the image. "Bioimaging" refers to the process of specifically adsorbing luminescent (e.g., fluorescent) probes such as tiny luminescent particles to targets such as cells or proteins, and then using the luminescence from the luminescent probes to observe the structure of the targets and their location and movement within the living body.

[0003] As a means for avoiding the adverse effect of autofluorescence on bioimaging, there are means utilizing phenomena such as near-infrared luminescence, long-Stokes shift luminescence, and delayed luminescence.

[0004] Long-Stokes-shift luminescence and delayed luminescence utilize luminescent compounds with structures containing donor and acceptor moieties, generating luminescence through intramolecular electron transfer upon excitation of either moiety. This limits the molecular design of the luminescent compounds, limiting control of excitation and emission wavelengths. In both cases, increasing the molar absorption coefficient of the luminescent compound to enhance brightness or extending the π-conjugated systems of the donor and acceptor moieties to extend the absorption wavelength can lead to reduced luminescence due to the promotion of electron transfer in the ground state or a lowering of the excited triplet level, making molecular design difficult.

[0005] Therefore, for long-Stokes-shift luminescent materials and delayed-luminescent materials, it is difficult to improve the molar extinction coefficient and design excitation wavelengths in the wavelength range (450 nm or higher) that do not cause cell degradation. Furthermore, delayed luminescence cannot be detected with general-purpose equipment, making it impractical for use in hospitals and clinics.

[0006] On the other hand, near-infrared emission is widely used in bioimaging because it is biotransparent and avoids cellular autofluorescence. However, emission in the near-infrared region has a fundamental problem: low quantum yield due to the energy gap law. Furthermore, in typical bioimaging in water, water solvation of the luminescent compound promotes the transition from the excited state to a charge-separated state at a lower energy level, resulting in a decrease in quantum yield.

[0007] In methods using luminescent nanoparticles that can mitigate the solvation effect of water, there is a problem of reduced luminescence due to aggregation quenching of the luminescent compound caused by packing the luminescent compound into the nanoparticles.Furthermore, the impact of aggregation quenching is extremely large in near-infrared emission, which has a low quantum yield due to the energy gap law.On the other hand, if the structure of the luminescent compound is made rigid in order to maintain the quantum yield, the maximum absorption wavelength and the maximum emission wavelength become close to each other, which causes the excitation light to become stray light and cause noise in imaging, hindering the realization of high-sensitivity imaging.

[0008] Patent Documents 1 and 2 disclose a technique in which two types of luminescent dyes (luminescent compounds) are introduced into nanoparticles, and the first component dye acts as an energy donor for photoexcitation, and then, via energy transfer, the second component dye acts as an energy acceptor to emit light. While these conventional techniques can avoid cellular autofluorescence and stray light from excitation light, they are designed to contain a very small amount of luminescent compound in the particles in order to maintain the quantum yield of the luminescent compound, which makes them sensitive to the effects of low absorbance and degradation of the luminescent compound on brightness, resulting in problems with particle brightness (= absorbance × quantum yield) in high-sensitivity imaging applications. [Prior art documents] [Patent documents]

[0009] [Patent Document 1] Patent No. 3773949 [Patent Document 2] Patent No. 5306714 Summary of the Invention [Problem to be solved by the invention]

[0010] The present invention has been made in consideration of the above problems and circumstances, and the problem to be solved is to realize high-brightness particle technology for bioimaging and to provide luminescent nanoparticles and luminescent labeling materials for pathological diagnosis that enable highly sensitive imaging. [Means for solving the problem]

[0011] In order to solve the above problems, the present inventors investigated the causes of the above problems and found that by incorporating a first luminescent compound, which is excited by light irradiation and has the function of transferring the energy generated by the excitation to a second luminescent compound, into a luminescent nanoparticle, and a second luminescent compound, which has the function of receiving the energy generated by the excitation of the first luminescent compound and emitting light, and by setting the content of the first luminescent compound relative to the total amount of the luminescent nanoparticle within the range of 4 to 90 mass%, the luminescent nanoparticle can realize a particle technology with high brightness for bioimaging and enable highly sensitive imaging, thereby completing the present invention. That is, the above problems according to the present invention are solved by the following means.

[0012] 1. A luminescent nanoparticle comprising a first luminescent compound and a second luminescent compound, The first luminescent compound is The compound has a structure represented by the following general formula (1d): a function of being excited by light irradiation and transferring the energy generated by the excitation to the second light-emitting compound; the second luminescent compound is Contains a compound having a structure represented by the following general formula (10): It has a function of receiving energy from the excitation and emitting light, and Luminescent nanoparticles, wherein the content of the first luminescent compound relative to the total amount of the luminescent nanoparticles is within a range of 4 to 90 mass %. [ka] [In formula (1d), a plurality of R 1 each independently represents a hydrogen atom or a substituent, and at least one represents a monovalent organic group having 3 to 30 carbon atoms. [ka] [In formula (10), X1 is O, CR 2 , SiR 2 , P(=O)R, or BR 2 Each R independently represents a hydrogen atom or a substituent. Y1 represents an amino group or a hydroxy group, and Y2 represents an ammonium group or an oxygen atom. R 30 represents a hydrogen atom or a substituent.] 2. A luminescent nanoparticle containing a first luminescent compound and a second luminescent compound, the first luminescent compound contains the following compound C-167, is excited by light irradiation, and has a function of transferring the energy generated by the excitation to the second luminescent compound; the second luminescent compound contains the following compound A-1, and has a function of receiving energy from the excitation and emitting light, and Luminescent nanoparticles, wherein the content of the first luminescent compound relative to the total amount of the luminescent nanoparticles is within a range of 4 to 90 mass %. [ka]

[0025] 3 Item 1, wherein the molar ratio of the second luminescent compound to the first luminescent compound is in the range of 1:2 to 1:200. or in paragraph 2 The luminescent nanoparticles described herein.

[0027] 4 Furthermore, items 1 to 3 containing a binder 3 The luminescent nanoparticles according to any one of claims 1 to 5.

[0028] 5 The surface of the luminescent nanoparticles has hydrophilic groups. 4 The luminescent nanoparticles according to any one of claims 1 to 5. Nanoparticles.

[0029] 6 .Items 1 to 5 5 1. A luminescent labeling material for pathological diagnosis, which uses the luminescent nanoparticles according to any one of claims 1 to 9. [Effects of the Invention]

[0030] The above-mentioned means of the present invention realizes a high-brightness particle technology for bioimaging, and provides luminescent nanoparticles and luminescent labeling materials for pathological diagnosis that enable highly sensitive imaging. This technology is particularly effective for highly sensitive biotransparent imaging in the near-infrared region, where the luminescence quantum yield is low.

[0031] The mechanism by which the effects of the present invention are manifested or the mechanism of action is not clear, but is speculated as follows.

[0032] The luminescent nanoparticles of the present invention contain at least two types of luminescent compounds, with their functions separated. In other words, in order to avoid cellular autofluorescence due to the large difference between the excitation wavelength and the emission wavelength, the control of the excitation and emission wavelength design, which is a problem that has arisen in conventional single-molecule luminescence imaging techniques, has become possible by using nanoparticles containing two or more types of luminescent compounds.

[0033] Specifically, in the luminescent nanoparticles of the present invention, the functions are separated into a first luminescent compound that is excited by light irradiation and a second luminescent compound that receives the excited energy of the first luminescent compound and emits light. More specifically, as the first luminescent compound, a luminescent compound that exhibits suppressed aggregation quenching at a high content of 4 to 90 mass % relative to the total amount of the luminescent nanoparticles was used.

[0034] This allows the first luminescent compound to be contained in the luminescent nanoparticles at a high concentration, maximizing absorbance and energy transfer efficiency. By receiving maximized energy, the second luminescent compound can emit light with high brightness, even at a trace content. As described below, typically, the relationship between the maximum emission wavelength of the first luminescent compound and the maximum absorption wavelength of the second luminescent compound is such that the maximum absorption wavelength of the second luminescent compound is longer than the maximum emission wavelength of the first luminescent compound. It is believed that this mechanism has enabled the realization of nanoparticle technology that satisfies high brightness and long Stokes shift emission in the present invention.

[0035] In particular, when the second light-emitting compound emits near-infrared light, the content of the second light-emitting compound can be kept low, thereby suppressing aggregation quenching and significantly maintaining the quantum yield.

[0036] In this way, the present invention is believed to realize a high-brightness particle technology for bioimaging and provide luminescent nanoparticles that enable highly sensitive imaging. DETAILED DESCRIPTION OF THE INVENTION

[0037] The luminescent nanoparticles of the present invention are luminescent nanoparticles containing a first luminescent compound and a second luminescent compound, wherein the first luminescent compound is excited by light irradiation and transfers the energy generated by the excitation to the second luminescent compound, the second luminescent compound receives the energy generated by the excitation and emits light, and the content of the first luminescent compound relative to the total amount of the luminescent nanoparticles is within the range of 4 to 90 mass%. This feature is a technical feature common to or corresponding to the following embodiments.

[0038] In the present invention, the first luminescent compound is preferably a luminescent compound having the maximum energy due to excitation (hereinafter also referred to as "excitation energy") when the content of the first luminescent compound relative to the total amount of the luminescent nanoparticles is within a range of 4 to 90 mass %. In other words, the first luminescent compound is preferably a compound such that a relatively large number of molecules of the first luminescent compound absorb excitation light and transfer the excitation energy thus obtained to the second luminescent compound with the maximum efficiency when the content of the first luminescent compound relative to the total amount of the luminescent nanoparticles is within a range of 4 to 90 mass %.

[0039] Furthermore, in the present invention, when the content of the second luminescent compound is set to a certain amount, it is preferable that the luminescence intensity of the second luminescent compound has a maximum value when the content of the first luminescent compound relative to the total amount of the luminescent nanoparticles is in the range of 4 to 90 mass%.

[0040] In an embodiment of the present invention, from the viewpoint of exerting the effects of the present invention, it is preferable that the first luminescent compound has a structure represented by the above general formula (1), general formula (2), or general formula (3). Furthermore, it is preferable that the first luminescent compound includes a compound represented by the above general formula (1c), general formula (1d), or general formula (1e). These compounds are preferable, for example, when their content relative to the total amount of luminescent nanoparticles is within a range of 4 to 90 mass%, because they have appropriate absorbance and maximum absorption wavelength for excitation light and exhibit good efficiency in energy transfer from excited molecules. They are luminescent compounds having a maximum excitation energy.

[0041] As an embodiment of the present invention, from the viewpoint of exerting the effects of the present invention, the molar ratio of the second luminescent compound to the first luminescent compound is preferably within the range of 1:2 to 1:200.

[0042] In an embodiment of the present invention, from the viewpoint of exerting the effects of the present invention and because light in the near-infrared region is biotransparent, it is preferable that the second light-emitting compound is a light-emitting compound that emits near-infrared light. For the same reason, it is also preferable that the second light-emitting compound is a xanthene dye.

[0043] In an embodiment of the present invention, from the viewpoint of exerting the effects of the present invention, it is preferable that the luminescent nanoparticles further contain a binder.

[0044] In an embodiment of the present invention, from the viewpoint of exerting the effects of the present invention, it is preferable that the surface of the luminescent nanoparticles has a hydrophilic group.

[0045] The luminescent labeling material for pathological diagnosis of the present invention is characterized by using the above-mentioned luminescent nanoparticles of the present invention.

[0046] The present invention, its components, and embodiments for carrying out the present invention will be described in detail below. In this application, the symbol "to" is used to mean that the numerical values ​​before and after it are included as lower and upper limits.

[0047] [Luminescent nanoparticles] The luminescent nanoparticles of the present invention are luminescent nanoparticles containing a first luminescent compound and a second luminescent compound, wherein the first luminescent compound is excited by light irradiation and has the function of transferring the energy generated by the excitation to the second luminescent compound, and the second luminescent compound has the function of receiving the energy generated by the excitation and emitting light, and the content of the first luminescent compound relative to the total amount of the luminescent nanoparticles is within the range of 4 to 90 mass%.

[0048] Here, when used alone, the first luminescent compound is a compound that can absorb predetermined excitation light according to the purpose, become excited, and emit light. Also, in a mutual relationship with the second luminescent compound, it is a compound that can transfer excitation energy to the second luminescent compound. Note that in the luminescent nanoparticles of the present invention, the energy excited in the first luminescent compound is transferred to the second luminescent compound, and the second luminescent compound emits light, so the first luminescent compound does not emit light.

[0049] The relationship between the maximum emission wavelength of the first luminescent compound and the maximum absorption wavelength of the second luminescent compound will be described below. In the following description, the maximum emission wavelength of the first luminescent compound and the maximum absorption wavelength of the second luminescent compound are the maximum emission wavelength and maximum absorption wavelength measured for the first luminescent compound and the second luminescent compound, respectively, independently.

[0050] As described above, the relationship between the maximum emission wavelength of the first luminescent compound and the maximum absorption wavelength of the second luminescent compound is typically expressed as follows: em1 , the maximum absorption wavelength of the second luminescent compound is λ ab2 When λem1 <λ ab2 It is preferable that:

[0051] The energy transfer between the first luminescent compound and the second luminescent compound is typically a Förster type energy transfer in which the emission spectrum of the first luminescent compound overlaps with the absorption spectrum of the second luminescent compound. Note that a Dexter type energy transfer may also occur simultaneously.

[0052] To make the Förster energy transfer efficient, λ ab2 -λ em1 λ shown by ab2 and λ em1 The difference is preferably 70 nm or less, more preferably 50 nm or less.

[0053] The maximum emission wavelength of the second light-emitting compound is λ em2 Denoted by λ em2 It is preferable that the wavelength is in the near-infrared region, because it has good biological permeability and the effects of the present invention are significant. In this specification, the near-infrared region refers to the region of 650 to 1800 nm. em2 More preferably, it is in the range of 650 to 1000 nm.

[0054] In the present invention, the term "luminescent nanoparticles" refers to particles containing a luminescent compound, and has an average particle size in the range of, for example, 1 to 1000 nm. The average particle size is preferably in the range of 30 to 500 nm, and more preferably in the range of 50 to 200 nm.

[0055] The average particle size of the luminescent nanoparticles can be measured by a method known in the art. Specifically, an electron micrograph is taken at an appropriate magnification using a scanning electron microscope (SEM), the cross-sectional area of ​​the luminescent nanoparticles is measured, and the measured value is taken as the area of ​​a circle, and the diameter (area-equivalent diameter) can be measured.

[0056] The average particle size (average particle size) and coefficient of variation of the particle size of a group of luminescent nanoparticles are calculated by measuring the particle sizes (particle sizes) of a sufficient number (e.g., 1,000) of luminescent nanoparticles as described above, and then calculating the average particle size as the arithmetic mean thereof, and the coefficient of variation using the formula: 100 × standard deviation of particle sizes / average particle size.

[0057] In the present invention, the coefficient of variation, which indicates the variation in particle size, is not particularly limited, but is usually 20% or less, and preferably 5 to 15%.

[0058] The luminescent nanoparticles of the present invention contain a first luminescent compound and a second luminescent compound as essential components. The luminescent nanoparticles of the present invention preferably further contain a binder as an optional component. Each component of the luminescent nanoparticles of the present invention will be described below in order.

[0059] <First luminescent compound> The first luminescent compound is a luminescent compound contained in an amount within the range of 4 to 90% by mass relative to the total amount of the luminescent nanoparticle of the present invention. The first luminescent compound has the property of absorbing light and becoming excited. The excitation energy is received by the second luminescent compound, which then emits light. The first luminescent compound is preferably a luminescent compound whose amount within the range of 4 to 90% by mass relative to the total amount of the luminescent nanoparticle of the present invention, has a maximum absorption wavelength with the maximum absorbance for a predetermined excitation light, and has a maximum excitation energy; in other words, has a maximum efficiency of energy transfer from excited molecules.

[0060] The content of the first luminescent compound is preferably within a range of 4 to 90 mass %, more preferably 10 to 80 mass %, based on the total amount of the luminescent nanoparticles of the present invention.

[0061] λ of the first luminescent compound em1 is not particularly limited, but may be the maximum absorption wavelength λ of the second luminescent compound ab2 and maximum emission wavelength λ em2 Considering the relationship between, for example, λ em2When the maximum absorption wavelength of the first light-emitting compound is in the near-infrared region, λ is preferably in the range of 500 to 900 nm, and more preferably in the range of 600 to 800 nm. ab1 Denoted by λ ab1 is not particularly limited, but may be, for example, λ em1 When is in the above range, it is preferably in the range of 500 to 700 nm, more preferably in the range of 550 to 650 nm.

[0062] Preferred specific examples of the first light-emitting compound according to the present invention are listed below, but these compounds may further have substituents or may have structural isomers, etc., and are not limited to the compounds exemplified below.

[0063] The first light-emitting compound preferably has a structure represented by the following general formula (1), general formula (2), or general formula (3). Hereinafter, a compound having a structure represented by general formula (1) will also be referred to as compound (1). The same applies to other compounds.

[0064] [ka]

[0065] In formula (1), multiple R 1 each independently represents a hydrogen atom or a substituent, and at least one represents a monovalent organic group having 3 to 30 carbon atoms. The benzene ring or naphthalene ring may further have a substituent, and * represents the position of the substituent that may be present on the benzene ring or naphthalene ring.

[0066] [ka]

[0067] In formula (2), R 2 represents a substituted or unsubstituted alkyl group, aryl group, or heteroaryl group. 3are each independently a hydrogen atom or a group having a structure represented by the following general formula (F1), and at least one of them represents a group having a structure represented by the following general formula (F1): The naphthalene ring may further have a substituent, and * represents the position of the substituent that may be present on the naphthalene ring.

[0068] [ka]

[0069] In formula (F1), Ar represents an aryl ring or a heteroaryl ring. 4 represents a substituent. When two or more groups represented by general formula (F1) are present, the two R 4 may be linked to each other. L represents a single bond, an oxygen atom, a sulfur atom, or -NR'-. R' represents a hydrogen atom, an alkyl group, an aryl group, or a heteroaryl group.

[0070] [ka]

[0071] In formula (3), R represents a luminescent compound skeleton. Each X independently represents an ionic substituent. L1 represents a single bond, an oxygen atom, a sulfur atom, a selenium atom, or an NH group. n represents an integer of 1 or greater.

[0072] (Compound (1)) Compound (1) is an imide derivative having a structure represented by the following general formula (1).

[0073] [ka]

[0074] In formula (1), multiple R 1each independently represents a hydrogen atom or a substituent, and at least one represents a monovalent organic group having 3 to 30 carbon atoms. The benzene ring or naphthalene ring may further have a substituent, and * represents the position of the substituent that may be present on the benzene ring or naphthalene ring. In compound (1), the substituent that may be present at the position indicated by * is not particularly limited.

[0075] Specifically, alkyl groups (e.g., methyl group, ethyl group, propyl group, isopropyl group, tert-butyl group, pentyl group, hexyl group, octyl group, dodecyl group, tridecyl group, tetradecyl group, pentadecyl group, etc.), cycloalkyl groups (e.g., cyclopentyl group, cyclohexyl group, etc.), alkenyl groups (e.g., vinyl group, allyl group, etc.), alkynyl groups (e.g., ethynyl group, propargyl group, etc.), aryl groups (e.g., phenyl group, p-chlorophenyl group, mesityl group, tolyl group, xylyl group, naphthyl group, anthryl group, azulenyl group, aryl group, cenaphthenyl group, fluorenyl group, phenanthryl group, indenyl group, pyrenyl group, biphenylyl group, etc.), heteroaryl group (for example, pyridyl group, pyrimidinyl group, furyl group, pyrrolyl group, imidazolyl group, benzimidazolyl group, pyrazolyl group, pyrazinyl group, triazolyl group (for example, 1,2,4-triazol-1-yl group, 1,2,3-triazol-1-yl group, etc.), pyrazolotriazolyl group, oxazolyl group, benzoxazolyl group, thiazolyl group, isoxazolyl group, isothiazolyl group, furazanyl group, thienyl group, quinolyl group, benzyl group, a benzofuryl group, a dibenzofuryl group, a benzothienyl group, a dibenzothienyl group, an indolyl group, a carbazolyl group, a carbolinyl group, a diazacarbazolyl group (which indicates a group in which one of the carbon atoms constituting the carboline ring of the carbolinyl group is replaced with a nitrogen atom), a quinoxalinyl group, a pyridazinyl group, a triazinyl group, a quinazolinyl group, a phthalazinyl group, etc.), a heterocyclic group (for example, a pyrrolidyl group, an imidazolidyl group, a morpholyl group, an oxazolidyl group, etc.), an alkoxy group (for example, a methoxy group, an ethoxy group, a propyloxy group, a pentyloxy group, a hexyloxy group, etc.), oxy group, octyloxy group, dodecyloxy group, etc.), cycloalkoxy group (for example, cyclopentyloxy group, cyclohexyloxy group, etc.), aryloxy group (for example, phenoxy group, naphthyloxy group, etc.), alkylthio group (for example, methylthio group, ethylthio group, propylthio group, pentylthio group, hexylthio group, octylthio group, dodecylthio group, etc.), cycloalkylthio group (for example, cyclopentylthio group, cyclohexylthio group, etc.), arylthio group (for example, phenylthio group, naphthylthio group, etc.), alkoxycarbonyl group (for example,methyloxycarbonyl group, ethyloxycarbonyl group, butyloxycarbonyl group, octyloxycarbonyl group, dodecyloxycarbonyl group, etc.), aryloxycarbonyl group (for example, phenyloxycarbonyl group, naphthyloxycarbonyl group, etc.), sulfamoyl group (for example, aminosulfonyl group, methylaminosulfonyl group, dimethylaminosulfonyl group, butylaminosulfonyl group, hexylaminosulfonyl group, cyclohexylaminosulfonyl group, octylaminosulfonyl group, dodecylaminosulfonyl group, phenylaminosulfonyl group, acyl groups (e.g., acetyl group, ethylcarbonyl group, propylcarbonyl group, pentylcarbonyl group, cyclohexylcarbonyl group, octylcarbonyl group, 2-ethylhexylcarbonyl group, dodecylcarbonyl group, phenylcarbonyl group, naphthylcarbonyl group, pyridylcarbonyl group, etc.), acyloxy groups (e.g., acetyloxy group, ethylcarbonyloxy group, butylcarbonyloxy group, octylcarbonyloxy group, dodecylcarbonyloxy group, phenylcarbonyl group, carbonyloxy group, etc.), amido group (for example, methylcarbonylamino group, ethylcarbonylamino group, dimethylcarbonylamino group, propylcarbonylamino group, pentylcarbonylamino group, cyclohexylcarbonylamino group, 2-ethylhexylcarbonylamino group, octylcarbonylamino group, dodecylcarbonylamino group, phenylcarbonylamino group, naphthylcarbonylamino group, etc.), carbamoyl group (for example, aminocarbonyl group, methylaminocarbonyl group, dimethylaminocarbonyl group, propylaminocarbonyl group, pentyl aminocarbonyl group, cyclohexylaminocarbonyl group, octylaminocarbonyl group, 2-ethylhexylaminocarbonyl group, dodecylaminocarbonyl group, phenylaminocarbonyl group, naphthylaminocarbonyl group, 2-pyridylaminocarbonyl group, etc.), ureido group (for example, methylureido group, ethylureido group, pentylureido group, cyclohexylureido group, octylureido group, dodecylureido group, phenylureido group, naphthylureido group, 2-pyridylaminoureido group, etc.), sulfinyl group (for example, methylsulfinyl group,ethylsulfinyl group, butylsulfinyl group, cyclohexylsulfinyl group, 2-ethylhexylsulfinyl group, dodecylsulfinyl group, phenylsulfinyl group, naphthylsulfinyl group, 2-pyridylsulfinyl group, etc.), alkylsulfonyl group (for example, methylsulfonyl group, ethylsulfonyl group, butylsulfonyl group, cyclohexylsulfonyl group, 2-ethylhexylsulfonyl group, dodecylsulfonyl group, etc.), arylsulfonyl group or heteroarylsulfonyl group (for example, phenylsulfonyl group, naphthylsulfonyl group, 2-pyridylsulfonyl group, etc.), amino group (for example, amino group, ethylamino group, dimethylamino group, diphenylamino group, group, diisopropylamino group, ditert-butyl group, cyclohexylamino group, butylamino group, cyclopentylamino group, 2-ethylhexylamino group, dodecylamino group, anilino group, naphthylamino group, 2-pyridylamino group, etc.), halogen atoms (for example, fluorine atom, chlorine atom, bromine atom, etc.), fluorohydrocarbon groups (for example, fluoromethyl group, trifluoromethyl group, pentafluoroethyl group, pentafluorophenyl group, etc.), cyano group, nitro group, hydroxy group, mercapto group, silyl group (for example, trimethylsilyl group, triisopropylsilyl group, triphenylsilyl group, phenyldiethylsilyl group, etc.), phosphono group, carboxy group, sulfo group, etc.

[0076] These substituents may be further substituted with the above-mentioned substituents. Furthermore, these substituents may be bonded to each other to form a ring. The cyclic structure formed by adjacent substituents may be an aromatic ring or an aliphatic ring, or may contain a heteroatom, and the cyclic structure may be a condensed ring of two or more rings.

[0077] Preferably, the * position has no substituent, or the substituent is an alkyl group, a halogen atom, a cyano group, a carboxylic acid anhydride formed by condensing two carboxylic acids, or a condensed ring formed by bonding the substituents together.

[0078] R 1R each independently represents a hydrogen atom or a substituent, and at least one represents a group having 3 to 30 carbon atoms. 1 The substituents represented by can be specifically selected from the above-mentioned substituents that * may have, but at least one of them is a group having 3 to 30 carbon atoms. By having a group having 3 to 30 carbon atoms, the carbonyl group of the imide and R 1 The phenyl group substituted on the nitrogen atom is oriented perpendicular to the naphthalene ring due to steric hindrance with the ortho-substituent R 1 can effectively shield the π-plane.

[0079] Also, R 1 Preferably, R has an oxygen atom or a sulfur atom in the carbon chain. More preferably, R has an oxygen atom in the carbon chain. By having an oxygen atom or a sulfur atom in the carbon chain, the structure becomes more flexible, and R 1 This can enhance the shielding effect of the π plane.

[0080] R 1is preferably an alkyl group (e.g., n-propyl group, isopropyl group, n-butyl group, sec-butyl group, isobutyl group, tert-butyl group, pentyl group, hexyl group, octyl group, dodecyl group, tridecyl group, tetradecyl group, pentadecyl group, 3-ethylpentyl group, etc.), a cycloalkyl group (e.g., cyclopentyl group, cyclohexyl group, cyclohexylethyl group, etc.), an alkenyl group (e.g., propenyl group, hexenyl group, etc.), an alkynyl group (e.g., propynyl group, hexynyl group, phenylethynyl group, etc.), an aryl group (e.g., phenyl group, p- chlorophenyl group, mesityl group, tolyl group, xylyl group, naphthyl group, anthryl group, azulenyl group, acenaphthenyl group, fluorenyl group, phenanthryl group, indenyl group, pyrenyl group, biphenylyl group, etc.), heteroaryl group (for example, pyridyl group, pyrimidinyl group, furyl group, pyrrolyl group, benzimidazolyl group, pyrazolyl group, pyrazinyl group, benzoxazolyl group, thienyl group, quinolyl group, benzofuryl group, dibenzofuryl group, benzothienyl group, dibenzothienyl group, indolyl group, carbazolyl group, carbolinyl group, diazacarbazolyl group (representing a group in which one of the carbon atoms constituting the carboline ring of the carbolinyl group is replaced with a nitrogen atom), quinoxalinyl group, pyridazinyl group, triazinyl group, quinazolinyl group, phthalazinyl group, etc.), heterocyclic group (for example, pyrrolidyl group, imidazolidyl group, morpholyl group, oxazolidyl group, etc.), alkoxy group (for example, pentyloxy group, hexyloxy group, octyloxy group, dodecyloxy group, 2-ethylbutyloxy group, etc.), cycloalkoxy group (for example, cyclopentyloxy group, cyclohexyloxy group, etc.), aryloxy group (for example, phenoxy group, noxy group, naphthyloxy group, etc.), alkylthio group (for example, pentylthio group, hexylthio group, octylthio group, dodecylthio group, etc.), cycloalkylthio group (for example, cyclopentylthio group, cyclohexylthio group, etc.), arylthio group (for example, phenylthio group, naphthylthio group, etc.), alkoxycarbonyl group (for example, butyloxycarbonyl group, octyloxycarbonyl group, dodecyloxycarbonyl group, etc.), aryloxycarbonyl group (for example, phenyloxycarbonyl group, naphthyloxycarbonyl group, etc.), sulfamoyl group (for example,butylaminosulfonyl group, hexylaminosulfonyl group, cyclohexylaminosulfonyl group, octylaminosulfonyl group, dodecylaminosulfonyl group, phenylaminosulfonyl group, naphthylaminosulfonyl group, 2-pyridylaminosulfonyl group, etc.), acyl groups (for example, butylcarbonyl group, pentylcarbonyl group, cyclohexylcarbonyl group, octylcarbonyl group, 2-ethylhexylcarbonyl group, dodecylcarbonyl group, phenylcarbonyl group, naphthylcarbonyl group, pyridylcarbonyl group, etc.), acyloxy groups (for example, butylcarbonyl group, pentylcarbonyl group, cyclohexylcarbonyl group, octylcarbonyl group, 2-ethylhexylcarbonyl group, dodecylcarbonyl group, phenylcarbonyl group, naphthylcarbonyl group, pyridylcarbonyl group, etc.), methylcarbonyloxy group, octylcarbonyloxy group, dodecylcarbonyloxy group, phenylcarbonyloxy group, etc.), amido group (e.g., propylcarbonylamino group, pentylcarbonylamino group, cyclohexylcarbonylamino group, 2-ethylhexylcarbonylamino group, octylcarbonylamino group, dodecylcarbonylamino group, phenylcarbonylamino group, naphthylcarbonylamino group, etc.), carbamoyl group (e.g., diethylaminocarbonyl group, propylaminocarbonyl group, pentylaminocarbonyl group, cyclohexyl aminocarbonyl group, octylaminocarbonyl group, 2-ethylhexylaminocarbonyl group, dodecylaminocarbonyl group, phenylaminocarbonyl group, naphthylaminocarbonyl group, 2-pyridylaminocarbonyl group, etc.), ureido group (for example, pentylureido group, cyclohexylureido group, octylureido group, dodecylureido group, phenylureido group, naphthylureido group, 2-pyridylaminoureido group, etc.), sulfinyl group (for example, butylsulfinyl group, cyclohexylsulfinyl group, 2-ethylhexylsulfinyl group, dodecylaminocarbonyl group, decylsulfinyl group, phenylsulfinyl group, naphthylsulfinyl group, 2-pyridylsulfinyl group, etc.), alkylsulfonyl group (for example, butylsulfonyl group, cyclohexylsulfonyl group, 2-ethylhexylsulfonyl group, dodecylsulfonyl group, etc.), arylsulfonyl group or heteroarylsulfonyl group (for example, phenylsulfonyl group, naphthylsulfonyl group, 2-pyridylsulfonyl group, etc.), amino group (for example, diphenylamino group, diisopropylamino group, cyclohexylamino group, butylamino group, cyclopentylamino group,2-ethylhexylamino group, dodecylamino group, anilino group, naphthylamino group, 2-pyridylamino group, etc.), fluorohydrocarbon group (e.g., decafluorobutyl group, pentafluorophenyl group, etc.), silyl group (e.g., triethylsilyl group, triisopropylsilyl group, triphenylsilyl group, phenyldiethylsilyl group, etc.),

[0081] R 1 is more preferably a bulky group, and examples thereof include aryl groups, heteroaryl groups, alkyl groups containing secondary or higher carbon atoms (e.g., secondary carbon: isobutyl group, cyclohexyl group, cyclopentyl group, cholesteryl group; tertiary carbon: tert-butyl group, adamantyl group, [2,2,2]bicyclooctyl group, etc.), tertiary amino groups (e.g., diethylamino group, diphenylamino group, etc.), tertiary silyl groups (e.g., triisopropylsilyl group, triphenylsilyl group, phenyldiethylsilyl group, etc.), etc. Such bulky groups can also be present at the terminals of alkyl groups, alkenyl groups, alkynyl groups, alkoxy groups, acyl groups, acyloxy groups, and amide groups.

[0082] The compound (1) is preferably a compound having a structure represented by any one of the following general formulas (2-1) to (2-6).

[0083] [ka]

[0084] (wherein a plurality of R 1 R each independently represents a hydrogen atom or a substituent, and at least one represents a group having 3 to 30 carbon atoms. 5 , R 6 and R 7 each independently represents a hydrogen atom, an alkyl group, an aryl group, a heteroaryl group, an alkenyl group, an alkynyl group, an alkoxy group, or an aryloxy group. R 1 is R in general formula (1) 1 is synonymous with. R 5 , R6 and R 7 each independently represents a hydrogen atom, an alkyl group, an aryl group, a heteroaryl group, an alkenyl group, an alkynyl group, an alkoxy group, or an aryloxy group. These groups have the same meanings as the alkyl group, aryl group, heteroaryl group, alkenyl group, alkynyl group, alkoxy group, or aryloxy group listed as the substituent that * may have in general formula (1).

[0085] In the present invention, a perylene bisimide derivative having a structure represented by general formula (2-2) is preferred, and the perylene bisimide derivative is preferably a compound (31) having a structure represented by the following general formula (31). Perylene bisimide derivatives are desirable because they not only exhibit high luminescence quantum yield but also high light resistance.

[0086] [ka]

[0087] In formula (31), multiple R 1 each independently represents a hydrogen atom or a substituent, and at least one represents a group having 3 to 30 carbon atoms. 5 R each independently represents a hydrogen atom, an alkyl group, an aryl group, a heteroaryl group, an alkenyl group, an alkynyl group, an alkoxy group, or an aryloxy group. 6 each independently represents a hydrogen atom, an alkyl group, an aryl group, a heteroaryl group, an alkenyl group, an alkynyl group, an alkoxy group, or an aryloxy group.

[0088] In the general formula (31), R 5 is preferably a phenoxy group or a group represented by the following general formula (2-2-1) (hereinafter also referred to as group (2-2-1)).

[0089] [ka]

[0090] In formula (2-2-1), R 12 represents a hydrogen atom or a substituent. The substituent has the same meaning as the substituent that may be present at the position indicated by * in general formula (1).

[0091] Compound (31) is more preferably a compound having a structure represented by the following general formula (1c), general formula (1d) or general formula (1e).

[0092] [ka]

[0093] In formula (1c) to formula (1e), a plurality of R 1 each independently represents a hydrogen atom or a substituent, and at least one represents a monovalent organic group having 3 to 30 carbon atoms. 21 R each independently represents a hydrogen atom or an ionic substituent, and at least one represents an ionic substituent. 1 is R shown in general formula (1) 1 and specific examples are as described above.

[0094] Compound (1c) is a compound represented by the formula (31) in which R 6 is a hydrogen atom, R 5 In compound (1c), R is a phenoxy group. 5 is a phenoxy group, which improves solubility and λ em1 Since it is possible to shift the wavelength of the light emitting compound to a longer wavelength, it is preferable as the first light emitting compound.

[0095] Compound (1d) is a compound represented by the formula (31) in which R 6 is a hydrogen atom, R 5 is the group (2-2-1) (wherein all R 12 is a hydrogen atom.) Compound (1d) is preferable as the first luminescent compound from the viewpoint of suppressing concentration quenching by reducing intermolecular interactions of the perylene moiety.

[0096] Compound (1e) is compound (1d) in which R in group (2-2-1) 12 Among them, R at the 4th position of the benzene ring 12 (In formula (1e), R 21 ) is a compound in which at least one of the following is substituted with an ionic substituent. Compound (1e) is preferable as the first luminescent compound from the viewpoints of improving solubility and suppressing concentration quenching due to repulsion of electrostatic forces due to the presence of an ionic substituent.

[0097] In compound (1e), R 21 The compound in which all of the substituents are substituted with ionic substituents is also classified as compound (3) described below, more specifically, compound (4). The ionic substituent in compound (1e) has the same meaning as in compound (3) described below. Specific examples of compound (1e) are described below as specific examples of compound (3).

[0098] (Compound (2)) The compound (2) is an imide derivative having a structure represented by the following general formula (2).

[0099] [ka]

[0100] In formula (2), R 2 represents a substituted or unsubstituted alkyl group, aryl group, or heteroaryl group. 3 are each independently a hydrogen atom or a group having a structure represented by the following general formula (F1), and at least one of them represents a group having a structure represented by the following general formula (F1) (hereinafter also referred to as substituent (F1)). The naphthalene ring may further have a substituent, and * represents the position of the substituent that may be on the naphthalene ring.

[0101] [ka]

[0102] In formula (F1), Ar represents an aryl ring or a heteroaryl ring. 4 represents a substituent. When two or more groups represented by general formula (F1) are present, the two R 4 may be linked to each other. L represents a single bond, an oxygen atom, a sulfur atom, or -NR'-. R' represents a hydrogen atom, an alkyl group, an aryl group, or a heteroaryl group.

[0103] In the compound (2), the ortho-substituent R of the aryl or heteroaryl ring represented by Ar in the substituent (F1) is 4 is oriented toward the perylene ring, effectively shielding the π plane, resulting in high quantum yield.

[0104] Ar represents an aryl ring or heteroaryl ring which may have a substituent, and examples of the aryl ring include a benzene ring, a naphthalene ring, an azulene ring, an anthracene ring, a phenanthrene ring, a naphthacene ring, and a pyrene ring.

[0105] Examples of the heteroaryl ring include a pyridine ring, a pyrimidine ring, a furan ring, a pyrrole ring, an imidazole ring, a benzimidazole ring, a pyrazole ring, a pyrazine ring, a triazole ring, a pyrazolotriazole ring, an oxazole ring, a benzoxazole ring, a thiazole ring, a thiophene ring, a quinoline ring, a benzofuran ring, a dibenzofuran ring, an indole ring, a quinoxaline ring, a triazine ring, etc. Ar preferably represents an aryl ring.

[0106] R 4 represents a substituent, which can be selected from the substituents that * may have in general formula (1).

[0107] The alkyl group, aryl group and heteroaryl group represented by R' have the same meanings as the alkyl group, aryl group and heteroaryl group exemplified as the substituent that * may have in general formula (1).

[0108] R 4is preferably an alkyl group (e.g., methyl, ethyl, propyl, butyl, pentyl, hexyl, isopropyl, tert-butyl, isobutyl, neopentyl), a cycloalkyl group (e.g., cyclopentyl, cyclohexyl), an aryl group (e.g., phenyl, naphthyl, anthryl), a heteroaryl group (e.g., pyridyl, carbazolyl), an alkenyl group (e.g., butenyl, pentenyl, hexenyl), an alkynyl group (e.g., propynyl, hexynyl, phenylethynyl, trimethylsilylethynyl), a silyl group (e.g., trimethylsilyl, triethylsilyl, triphenylsilyl), an alkoxy group (e.g., methoxy, tert-butyloxy), or an aryloxy group (e.g., phenoxy, naphthoxy).

[0109] R 2 represents a substituted or unsubstituted alkyl group, aryl group, or heteroaryl group. 2 R has the same meaning as the alkyl group, aryl group, and heteroaryl group exemplified as the substituent that * may have in general formula (1). 2 As R, a substituted or unsubstituted aryl group, particularly a substituted or unsubstituted phenyl group, is preferred. 2 As the group, a group represented by the structure of the following general formula (F2) is preferred.

[0110] [ka]

[0111] In formula (F2), multiple R 1 Each of R independently represents a hydrogen atom or a substituent, and at least one of R represents a group having 3 to 30 carbon atoms. The benzene ring may further have a substituent, and * represents the position of the substituent that the benzene ring may have. 1 and * are the substituents that may be present at the positions indicated by R in general formula (1), respectively. 1 and * are the same as the substituents listed as the optional substituents.

[0112] The compound (2) preferably has a structure represented by the following general formulas (7-1) to (7-4).

[0113] [ka]

[0114] In the formula, R 2 each independently represents a substituted or unsubstituted alkyl group, aryl group, or heteroaryl group. 3 R each independently represents a hydrogen atom or a group having a structure represented by the general formula (6), and at least one of them represents a group having a structure represented by the general formula (6). 8 and R 9 each independently represents a hydrogen atom, an alkyl group, an alkenyl group, an alkynyl group, an aryl group, a heteroaryl group, an alkoxy group, or an aryloxy group.

[0115] R 2 and R 3 is R in general formula (2) 2 and R 3 It is synonymous with R. 8 and R 9 The alkyl group, alkenyl group, alkynyl group, aryl group, heteroaryl group, alkoxy group, and aryloxy group represented by the following formula (1) have the same meanings as the alkyl group, alkenyl group, alkynyl group, aryl group, heteroaryl group, alkoxy group, and aryloxy group exemplified as the substituent that * may have in general formula (1).

[0116] In the present invention, a perylene bisimide derivative having a structure represented by general formula (7-1) is preferred, and the perylene bisimide derivative is preferably a compound (8) having a structure represented by the following general formula (8):

[0117] [ka]

[0118] In formula (8), multiple R 2 R each independently represents a substituted or unsubstituted alkyl group, aryl group, or heteroaryl group. 4 represents a substituent. 4 R may be linked to each other. 11 each independently represents a hydrogen atom, an alkyl group, an aryl group, a heteroaryl group, an alkenyl group, an alkynyl group, an alkoxy group, an aryloxy group, an amino group, an acyl group, an acyloxy group, an amido group, a carboxy group, or a sulfo group.

[0119] R 2 and R 4 are R in general formula (2), respectively. 2 and R 4 Furthermore, the imide derivative having the structure represented by general formula (8) is preferably an imide derivative having the structure represented by general formula (8A).

[0120] [ka]

[0121] In formula (8A), multiple R 2 R each independently represents a substituted or unsubstituted alkyl group, aryl group, or heteroaryl group. 4 represents a substituent. 4 R may be linked to each other. 2 and R 4 is R in general formula (8) 2 and R 4 is synonymous with.

[0122] Compound (8A) has four bay areas all substituted with R 4 is a phenoxy group having a substituent R 4 are desirable because they are oriented above and below the perylene ring, respectively, and enhance the shielding effect.

[0123] Furthermore, in the general formula (8), R 4It is desirable that any two of these be linked across the perylene ring. This linkage effectively inhibits the interaction between the perylene rings, resulting in a higher luminescence quantum yield.

[0124] Examples of the imide derivatives having the structures represented by the general formulae (1) to (8) of the present invention are given below, but the present invention is not limited thereto.

[0125] [ka]

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[0171] (Synthesis of Compound (1) and Compound (2)) Compound (1) and compound (2) can be synthesized by known methods, for example, by referring to Chem. Eur. J. 2004, 10, 5297-5310. As an example, the synthesis schemes of exemplified compounds C-53 (compound (1)) and C-45 (compound (2)) from the known compounds in the above literature are shown below. Other exemplified compounds can be synthesized in a similar manner. In the synthesis scheme, NMP represents N-methyl-2-pyrrolidone.

[0172] <Synthesis of exemplary compound C-53> [ka]

[0173] <Synthesis of exemplary compound C-45> [ka]

[0174] (Compound (3)) Compound (3) is a compound having a structure represented by the following general formula (3).

[0175] [ka]

[0176] In formula (3), R represents a luminescent compound skeleton. Each X independently represents an ionic substituent. L1 represents a single bond, an oxygen atom, a sulfur atom, a selenium atom, or an NH group. n represents an integer of 1 or greater.

[0177] In the compound (3), the luminescent compound skeleton represented by R preferably has a structure represented by any of the mother compounds shown below.

[0178] [ka]

[0179] In the above formula, R 20 represents a halogen atom or a cyano group.

[0180] Compound (3) is a compound in which n hydrogen atoms of the mother compound are substituted with the substituent enclosed in parentheses in the above formula (3) (hereinafter, sometimes referred to as the substituent (F3)). n is an integer of 1 or more and is appropriately selected depending on the structure of the mother compound. n is preferably 1 to 6, more preferably 2 to 4. For example, when the mother compound is perylene bisimide, n is preferably 2 to 6, particularly preferably 4. The substitution position of the substituent (F3) in the mother compound is not particularly limited, but a position where the influence of steric hindrance is large is preferred. For example, when the mother compound is perylene bisimide, the bay area is preferred.

[0181] The substituent (F3) is a substituent in which a biphenyl skeleton is bonded to L1 as a linking group, and the two benzene rings each have one ionic substituent X. Of the hydrogen atoms in the mother compound, hydrogen atoms not substituted with the substituent (F3) may be substituted with a substituent other than the substituent (F3).

[0182] In compound (3), specific examples of the ionic substituent represented by X include -OH, -SH, -COOH, -C(=O)H, -S(=O)2OH, -S(=O)NH2, -S(=O)2NH2, -P(=O)(OH)3, -P(=O)R(OH)2, -P(=O)R2(OH), -P(OH)3, -P(=O)(NH2)3, -P(=O)R(NH2)2, -P(=O)R2(NH2), -P(NH2)3, -O(C=O)OH, -NH2, Examples of the ionic substituent include -NHR, -NHCONH2, -NHCONHR, -NHCOOH, -Si(OH)3, -Si(R)(OH)2, -Si(R)2OH, -Ge(OH)3, -Ge(R)(OH)2, -Ge(R)2OH, -Ti(OH)3, -Ti(R)(OH)2, -Ti(R)2OH, -Si(NH2)3, -Si(R)(NH2)2, -B(OH)2, -OB(OH)2, -B(NH2)2, -NHB(OH)2, and polyethylene glycol groups. Each R independently represents hydrogen or an alkyl group having 1 to 20 carbon atoms. Other examples of the ionic substituent include an NHS group and a maleimide group.

[0183] The ionic substituent is preferably a sulfo group, a phosphate group, a sulfonate group, a phosphoric acid ester group, an ammonium group, a carboxy group, a phosphonium group, or a salt thereof. Among these, a sulfo group, a phosphate group, a sulfonate group, a phosphoric acid ester group, an ammonium group, or a salt thereof is more preferred, and a sulfo group or a salt thereof is particularly preferred. Specific examples include -SO3H, -SONa, -OSO3H, -OSONa, -SON3NH4, -PO4H2, -PO4Na, -OPO3H2, -OPO3Na2, -NMe3OH, and -NMe3Cl.

[0184] In compound (3), L represents a single bond, an oxygen atom, a sulfur atom, a selenium atom or an NH group, and is particularly preferably an oxygen atom.

[0185] It is preferable that the compound (3) has a structure represented by the following general formula (4) in that it has an excellent effect of suppressing concentration quenching.

[0186] [ka]

[0187] In formula (4), R represents a luminescent compound skeleton, X represents an ionic substituent, and L1 represents a single bond, an oxygen atom, a sulfur atom, a selenium atom, or an NH group.

[0188] In the general formula (4), R, X, and L1 have the same meanings as R, X, and L1 in the general formula (3).

[0189] Furthermore, it is preferable that the compound (4) has a structure represented by the following general formula (9) in terms of excellent concentration quenching suppression effect.

[0190] [ka]

[0191] In formula (9), X represents a sulfo group or a salt thereof. H in NH may be substituted with a substituent.

[0192] In the compound (9), when H in NH is substituted with a substituent, the substituent may be a substituted or unsubstituted alkyl group, aryl group, or heteroaryl group. 2 It can be the same as:

[0193] Specific examples of the light-emitting compound having the structure represented by the general formula (3) are shown below, but the invention is not limited to these.

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[0233] (Synthesis of compound (3)) The synthesis of compound (3) will be described using the case where X in compound (3) is a sulfo group as an example. Compound (3) where X is a sulfo group can be produced by, for example, sulfonating a compound (3) precursor of general formula (3) in which X is a hydrogen atom instead of an ionic substituent, thereby introducing one sulfo group into each benzene ring. This allows substitution with multiple ionic substituents at once, resulting in excellent production efficiency.

[0234] [ka]

[0235] In the formula, R represents a luminescent compound skeleton. L1 represents a single bond, an oxygen atom, a sulfur atom, a selenium atom, or an NH group. n represents an integer of 1 or more. In the formula, R has the same meaning as R in the general formula (3) described above.

[0236] <Synthesis Example of Exemplary Compound 1-1> The synthesis scheme for the exemplary compound 1-1 is shown below. The other exemplary compounds can be synthesized in the same manner. In the synthesis scheme, NMP represents N-methyl-2-pyrrolidone.

[0237] [ka]

[0238] The first luminescent compound has been described above. In the present invention, the first luminescent compound may be used singly or in combination of two or more. From the viewpoint of improving the transfer efficiency by limiting the energy transfer pathway, it is preferable to use one first luminescent compound singly. When two or more first luminescent compounds are used in combination, the content of the first luminescent compound in the luminescent nanoparticle is the total amount thereof.

[0239] <Second luminescent compound> The maximum emission wavelength λ of the second luminescent compound em2 On the other hand, the maximum absorption wavelength λ ab2 is the maximum emission wavelength λ of the first luminescent compound. em1 and the maximum emission wavelength λ of the second luminescent compound em2 Considering this, the wavelength is preferably in the range of about 500 to 900 nm, and more preferably in the range of 600 to 800 nm.

[0240] Furthermore, the molar ratio of the second luminescent compound to the first luminescent compound in the luminescent nanoparticles of the present invention (second luminescent compound:first luminescent compound) is preferably within the range of 1:2 to 1:200, more preferably 1:4 to 1:100, even more preferably 1:8 to 1:75, and even more preferably 1:16 to 1:50.

[0241] The content of the second luminescent compound in the luminescent nanoparticles of the present invention depends on the molar ratio of the contents of the first luminescent compound and the second luminescent compound, but is preferably in the range of approximately 0.05 to 1 mass %, and more preferably 0.1 to 0.5 mass %, relative to the total amount of the luminescent nanoparticles of the present invention.

[0242] The second light-emitting compound is preferably a xanthene dye from the viewpoint of chemical and optical stability of the compound. Typical examples of xanthene dyes include compounds represented by the structure of the following general formula (10):

[0243] [ka]

[0244] In formula (10), X1 represents O, CR2, SiR2, P(=O)R, or BR2. Each R independently represents a hydrogen atom or a substituent. Y1 represents an amino group or a hydroxy group, and Y2 represents an ammonium group or an oxygen atom. R 30 represents a hydrogen atom or a substituent.

[0245] Specific examples of R in CR2, SiR2, P(=O)R, and BR2 represented by X1, where R is a substituent, include the groups listed as the substituents that * may have in general formula (1). 30 The same applies when is a substituent.

[0246] When Y1 is an amino group, the amino group typically includes -NR2 (R is a hydrogen atom or a substituent). When R is a substituent, specific examples of R include the groups listed as the substituents that * in general formula (1) may have. Two R may be bonded to each other to form a ring.

[0247] When Y2 is an ammonium group, the ammonium group is typically =NR2 + (R is a hydrogen atom or a substituent). When R is a substituent, specific examples of R include the groups listed as the substituents that * may have in general formula (1). Two R may be bonded to each other to form a ring. Furthermore, R may be bonded to a carbon atom constituting the benzene ring to which the nitrogen atom is bonded to form a ring.

[0248] When Y2 is an ammonium group, compound (10) has a counter anion either intramolecularly or extramolecularly. When a counter anion is present intramolecularly, R 30 In this case, the counter anion is preferably COO - , SO3 - When compound (10) has a counter anion outside the molecule, the counter anion may be I - , F - , Br - , Cl - , PF6 - , BF4 - , ClO4 - etc.

[0249] Among the xanthene dyes, the following compounds are particularly preferred.

[0250] [ka]

[0251] As the second luminescent compound, in addition to the xanthene dyes described above, cyanine dyes, squarylium dyes, dipyrromethene dyes, azadipyrromethene dyes, terrylene dyes, perylene dyes other than the first luminescent compound, etc. Typical structures of these dyes are shown below.

[0252] [ka]

[0253] The second luminescent compound has been described above. In the present invention, the second luminescent compound may be used singly or in combination of two or more. From the viewpoint of improving the transfer efficiency by limiting the energy transfer pathway, it is preferable to use one type of second luminescent compound singly. When two or more types of second luminescent compounds are used in combination, the content of the first luminescent compound in the luminescent nanoparticles is the total amount thereof.

[0254] <Other luminescent compounds> The luminescent nanoparticles of the present invention may contain other luminescent compounds in addition to the first and second luminescent compounds, as needed, within the scope of not impairing the effects of the present invention. However, from the viewpoint of fully obtaining the effects of the present invention, it is preferable that the luminescent nanoparticles do not contain other luminescent compounds.

[0255] <Binder> The luminescent nanoparticles of the present invention preferably contain a binder having a fixing or binding action, since the binder can impart special functions to the particle surface.

[0256] When a binder is contained, the content of the binder relative to the total amount of the luminescent nanoparticles is the amount obtained by subtracting the total amount of the first luminescent compound, the second luminescent compound, and other luminescent compounds from the total amount of the luminescent nanoparticles, and is, for example, 9 to 95 mass%, preferably in the range of 10 to 95 mass%, more preferably 19 to 90 mass%, and even more preferably 20 to 90 mass%.

[0257] The binder is preferably an organic resin containing carbon atoms in the main chain and having a molecular weight of 300 or more, or a hydrolysis condensate of a metal alkoxide.

[0258] Specific examples of organic resins include polyolefin resins such as polypropylene, polymethylpentene, and polycyclohexylene dimethylene terephthalate (PCT), polyamide, polyacetal, polyethylene terephthalate, polybutylene terephthalate, polyphenylene sulfide, polycarbonate, ABS resin, AS resin, acrylic resin, amino resin, polyester resin, epoxy resin, mixed resin of acrylic resin and amino resin, and mixed resin of polyester resin and amino resin, cellulose resin, polyethylene resin, polypropylene resin, polystyrene resin, polyvinyl chloride resin, polymethyl methacrylate resin, polyacrylonitrile resin, polyacrylamide resin, and polyalcohol resin. , polyallyl acetate resin, polyoxymethylene resin, poly-n-butyl isocyanate resin, polyethylene oxide resin, 6-nylon resin, poly-β-oxypropionic acid ester resin, phenol resin, urea resin, melamine resin, alkyd melamine resin, unsaturated polyester resin, polyvinyl alcohol resin, poly(N-vinylformamide) resin, poly(N-vinylisobutylamide) resin, polyacrylic acid resin, poly(N-isopropylacrylamide) resin, poly(N-vinylpyrrolidinone) resin, polyhydroxyethyl methacrylate resin, polyoxyethylene methacrylate resin, polyethylene glycol dimethyl ether resin, polystyrene sulfonic acid resin, etc.

[0259] Specific examples of metals in metal alkoxides include 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.

[0260] The luminescent nanoparticles of the present invention preferably have hydrophilic groups on their surfaces. The hydrophilic groups on the surfaces of the luminescent nanoparticles may be hydrophilic groups of the first luminescent compound or the second luminescent compound, or may be hydrophilic groups of the binder. The luminescent nanoparticles of the present invention are preferred in that they have hydrophilic groups on their surfaces, which can suppress aggregation of particles and allow the particles to be dispersed in water.

[0261] (Binder having hydrophilic groups) The hydrophilic group of the binder is preferably an atomic group that has a strong interaction with water, and specific examples thereof include -OH, -SH, -COOH, -C(=O)H, -S(=O)2OH, -S(=O)NH2, -S(=O)2NH2, -P(=O)(OH)3, -P(=O)R(OH)2, -P(=O)R2(OH), -P(OH)3, -P(=O)(NH2)3, -P(=O)R(NH2)2, -P(=O)R2(NH2), -P(NH2)3, -O(C=O)OH, Examples of hydrophilic groups include NH, -NHR, -NHCONH, -NHCONHR, -NHCOOH, -Si(OH), -Si(R)(OH), -Si(R)OH, -Ge(OH), -Ge(R)(OH), -Ge(R)OH, -Ti(OH), -Ti(R)(OH), -Ti(R)OH, -Si(NH), -Si(R)(NH), -B(OH), -OB(OH), -B(NH), -NHB(OH), and polyethylene glycol groups. Each R independently represents hydrogen or an alkyl group having 1 to 20 carbon atoms. Other examples of hydrophilic groups that exhibit hydrophilicity include an NHS group and a maleimide group.

[0262] Specific examples of binders having a hydrophilic group include organic resins such as urea resins, melamine resins, polyvinyl alcohol resins, poly(N-vinylformamide) resins, poly(N-vinylisobutylamide) resins, polyacrylic acid resins, polyacrylamide resins, poly(N-isopropylacrylamide) resins, poly(N-vinylpyrrolidinone) resins, polyhydroxyethyl methacrylate resins, polyoxyethylene methacrylate resins, polyethylene glycol dimethyl ether resins, and polystyrene sulfonic acid resins.

[0263] Hydrolysis condensation products of metal alkoxides can also serve as binders having hydrophilic groups. Preferred metal alkoxides include titanium alkoxides, zirconium alkoxides, and silicon alkoxides. Titanium alkoxides, zirconium alkoxides, and silicon alkoxides produce hydrolysis condensation products such as titania, zirconia, and silica, respectively. In the luminescent nanoparticles of the present invention, melamine resin or silica is preferred as the binder.

[0264] (thermosetting resin) The binder according to the present invention may be a thermosetting resin. For example, from the viewpoint that the luminescent compound is less likely to be eluted in a permeation step using an organic solvent such as xylene, an organic resin containing a thermosetting resin such as a melamine resin, which can fix the luminescent compound inside a dense crosslinked structure, is preferred.

[0265] Examples of thermosetting resins include those containing structural units formed from at least one monomer selected from the group consisting of melamine, urea, guanamines (including benzoguanamine, acetoguanamine, etc.), and derivatives thereof. These monomers may be used alone or in combination of two or more. If desired, one or more comonomers other than the above compounds may also be used in combination.

[0266] Specific examples of thermosetting resins include melamine-formaldehyde resins and urea-formaldehyde resins.

[0267] The raw materials for these thermosetting resins can be not only the monomers themselves, as mentioned above, but also prepolymers obtained by reacting monomers with compounds such as formaldehyde or other crosslinking agents in advance. For example, in the production of melamine-formaldehyde resins, methylolmelamine, prepared by condensing melamine and formaldehyde under alkaline conditions, is generally used as the prepolymer, and this compound may be further alkyl-etherified (e.g., methylated to improve stability in water, or butylated to improve solubility in organic solvents).

[0268] The thermosetting resin may be one in which at least a portion of the hydrogen atoms contained in its structural units are substituted with a substituent having an electric charge or a substituent capable of forming a covalent bond. Such a thermosetting resin can be synthesized by using, as a raw material, a monomer in which at least one hydrogen atom has been substituted with the above-mentioned substituent (derivatized) by a known method.

[0269] Such thermosetting resins can be synthesized by known methods. For example, melamine-formaldehyde resins can be synthesized by polycondensing methylolmelamine, which has been prepared as described above, by heating it with the addition of a reaction accelerator such as an acid, if necessary.

[0270] (thermoplastic resin) The binder according to the present invention may be a thermoplastic resin. Examples of thermoplastic resins include those containing structural units formed from at least one monofunctional monomer (a group involved in a polymerization reaction in one molecule, such as a monomer having one vinyl group in the above example) selected from the group consisting of acrylic acid, methacrylic acid and their alkyl esters, acrylonitrile, and derivatives thereof. These monomers may be used alone or in combination of two or more.

[0271] If desired, one or more comonomers other than the above compounds may be used in combination. The thermoplastic resin may contain a structural unit, i.e., a crosslinking site, formed from a polyfunctional monomer (a group involved in a polymerization reaction in one molecule, such as a monomer having two or more vinyl groups in the above example), such as divinylbenzene. For example, a crosslinked product of polymethyl methacrylate may be mentioned.

[0272] Furthermore, the thermoplastic resin may contain a structural unit having a functional group for surface modification of the luminescent nanoparticles of the present invention. For example, by using a monomer such as glycidyl methacrylate having an epoxy group as a raw material, luminescent nanoparticles with the epoxy group oriented on the surface can be prepared. This epoxy group can be converted to an amino group by reacting it with excess aqueous ammonia. Various biomolecules can be introduced into the amino group thus formed according to known methods (via a linker molecule, if necessary).

[0273] <Method for producing luminescent nanoparticles> An example of a method for producing the luminescent nanoparticles of the present invention is a method of forming particles with a diameter on the order of nanometers, in which a luminescent compound (a luminescent compound comprising a first luminescent compound and a second luminescent compound; hereinafter, the term "luminescent compound" refers to a luminescent compound comprising the first luminescent compound and the second luminescent compound) is fixed inside or on the surface of a base body made of a binder.

[0274] The method for preparing these luminescent nanoparticles is not particularly limited, but for example, a method can be used in which a luminescent compound is added while (co)polymerizing a (co)monomer for synthesizing a binder (e.g., a thermoplastic resin or a thermosetting resin) that forms the matrix of the luminescent nanoparticles, and the luminescent compound is incorporated into the interior or surface of the (co)polymer. When the binder is a hydrolysis condensation product of a metal alkoxide, for example, a method can be used in which a luminescent compound is added while hydrolysis condensation of the metal alkoxide, and the luminescent compound is incorporated into the interior or surface of the hydrolysis condensation product.

[0275] The luminescent nanoparticles of the present invention can be produced by using a first luminescent compound and a second luminescent compound, and then, for example, following a known polymerization process or hydrolysis condensation process for various binders. Hereinafter, the production method will be described using an example in which the binder is an organic resin.

[0276] (Polymerization process) The polymerization process is a process in which a reaction mixture containing a light-emitting compound, a resin raw material (monomer, oligomer, or prepolymer), and preferably further a surfactant and a polymerization reaction accelerator is heated to cause the resin polymerization reaction to proceed, thereby producing resin particles containing the light-emitting compound.

[0277] The order of addition of the components contained in the reaction mixture is not particularly limited. A typical order is to add a surfactant to an aqueous solution of a luminescent compound, then add the resin raw material, and finally add a polymerization accelerator. Alternatively, the order may be to add the resin raw material to an aqueous solution of a surfactant, then add the polymerization accelerator, and allow the synthesis reaction of the resin particles to proceed while adding the aqueous solution of the luminescent compound. The concentration of the aqueous solution of the specific luminescent compound according to the present invention used in such a polymerization step can be adjusted to a range relatively higher than the concentration of aqueous solutions of conventional luminescent compounds (e.g., 2,500 to 10,000 μM).

[0278] The polymerization reaction conditions (temperature, time, etc.) can be appropriately set taking into consideration the type of resin, the composition of the raw material mixture, etc.

[0279] The polymerization method is not particularly limited as long as it is a known polymerization method. Examples of known polymerization methods include bulk polymerization, emulsion polymerization, soap-free emulsion polymerization, seed polymerization, and suspension polymerization. In the case of bulk polymerization, resin particles of a desired particle size can be obtained by crushing and then classifying the resulting particles. Emulsion polymerization is a polymerization method in which a medium such as water is mixed with a monomer that is poorly soluble in the medium and an emulsifier (surfactant), and a polymerization initiator that is soluble in the medium is added thereto. This method is characterized by small variations in the particle size obtained.

[0280] "Soap-free emulsion polymerization" is emulsion polymerization that does not use an emulsifier. It is characterized by the ability to obtain particles of uniform diameter. Seed polymerization is polymerization that is carried out by adding seed particles prepared separately at the start of polymerization. The particle size, particle size distribution, and amount (number) of the seed particles are arbitrarily determined for polymerization, and it is characterized by the ability to polymerize with the desired particle size and particle size distribution. Suspension polymerization is a polymerization method in which the monomer and water solvent are mechanically stirred and suspended. It is characterized by the ability to obtain particles with small and uniform particle sizes.

[0281] As a specific example, in the synthesis of a thermosetting resin such as melamine resin, the reaction temperature is usually 70 to 200°C, and the reaction time is usually 20 to 120 minutes. The reaction temperature should be set to a temperature (within the heat-resistant temperature range) at which the performance of the luminescent compound does not deteriorate. Heating may be carried out in multiple stages; for example, the reaction may be carried out at a relatively low temperature for a certain period of time, followed by heating and then a relatively lower temperature for a certain period of time.

[0282] After the polymerization reaction is complete, impurities such as excess resin raw material, luminescent compound, and surfactant are removed from the reaction solution, and the resulting luminescent nanoparticles are recovered and purified. For example, the reaction solution is centrifuged to remove the supernatant containing impurities, and then ultrapure water is added and the nanoparticles are redispersed and washed by ultrasonic irradiation. These operations are preferably repeated multiple times until the supernatant no longer exhibits light absorption or light emission due to the resin or luminescent compound.

[0283] The luminescent nanoparticles using thermosetting resin can basically be produced according to emulsion polymerization method, but preferably produced by the above-mentioned polymerization process using surfactant and polymerization reaction accelerator.In addition, in the luminescent nanoparticles obtained by such a production method, most of the luminescent compound is preferably fixed in the state where it is contained in the resin particle, but it is not excluded that some of the luminescent compound is fixed in the state where it is bonded or attached to the surface of the resin particle.

[0284] Furthermore, when the luminescent compound is contained, there is no limitation on the chemical or physical action by which the luminescent compound is fixed to the resin particles. In the present invention, prior to the polymerization step, it is not necessary to provide a derivatization step for previously covalently bonding the resin raw material and the luminescent compound or for introducing a positively charged substituent into the resin raw material (luminescent nanoparticles with excellent luminescence intensity and light resistance can be obtained without using such a step), but it is not excluded to use such a step in combination as desired.

[0285] (surfactant) As the surfactant, a known emulsifier for emulsion polymerization can be used. Surfactants include anionic (negative ionic), nonionic (nonionic), and cationic (cationic) types. When synthesizing a (cationic) thermosetting resin having a positively charged substituent or moiety, it is preferable to use an anionic or nonionic surfactant. Conversely, when synthesizing a (anionic) thermosetting resin having a negatively charged substituent or moiety, it is preferable to use a cationic or nonionic surfactant.

[0286] Examples of anionic surfactants include sodium dodecylbenzenesulfonate (product name: "Neopelex" series, Kao Corporation). Examples of nonionic surfactants include polyoxyethylene alkyl ether compounds (product name: "Emulgen" series, Kao Corporation), polyvinylpyrrolidone (PVP), and polyvinyl alcohol (PVA). Examples of cationic surfactants include dodecyltrimethylammonium bromide.

[0287] By adjusting the amount of surfactant added, it is possible to adjust the particle size of the resin particles and produce luminescent nanoparticles with a small coefficient of variation in particle size, i.e., uniform particle size. The amount of surfactant added is, for example, 10 to 60 mass% of the resin raw material, or 0.1 to 3.0 mass% of the entire raw material mixture. Increasing the amount of surfactant added tends to decrease the particle size, while decreasing the amount of surfactant added tends to increase the particle size.

[0288] (Polymerization reaction accelerator) The polymerization reaction accelerator accelerates the polycondensation reaction of thermosetting resins such as melamine resins, and also transfers protons (H + ) to charge the resin, facilitating electrostatic interactions. The reaction of thermosetting resins proceeds by heating alone, but the addition of a polymerization accelerator allows the reaction to proceed at lower temperatures, so it can be added within a range that allows control of the reaction and performance. Examples of such polymerization accelerators include acids such as formic acid, acetic acid, sulfuric acid, paratoluenesulfonic acid, and dodecylbenzenesulfonic acid. When the luminescent compound is a compound having a carboxy group or a sulfo group, the luminescent compound can also donate protons in the same way as the above-mentioned acids.

[0289] [Luminescent labeling material for pathological diagnosis] The luminescent labeling material for pathological diagnosis of the present invention is characterized by using the above-mentioned luminescent nanoparticles of the present invention. Specifically, the luminescent labeling material for pathological diagnosis of the present invention may be in the form in which a targeting ligand is bound to the surface of the above-mentioned luminescent nanoparticles of the present invention via a covalent bond.

[0290] The uses of the luminescent nanoparticles of the present invention are not particularly limited, but a typical example is their use as a luminescent labeling material for pathological diagnosis, for labeling a detection target substance contained in a sample (tissue section) so that it can be observed fluorescently in immunostaining. That is, the luminescent nanoparticles of the present invention as described above are preferably used as a complex (conjugate) by linking them with a targeting ligand according to the embodiment of immunostaining.

[0291] The substance to be detected is not particularly limited, but in pathological diagnosis, an antigen is generally selected according to the purpose. For example, in pathological diagnosis of breast cancer, HER2 can be used as the substance to be detected. Furthermore, the substance to be detected does not have to be inherent to the living body. For example, the substance to be detected can be a drug.

[0292] <Target-directed ligand> In the present invention, a "targeting ligand" refers to a molecule that has specific binding affinity to a specific tissue or cell (detection target substance). The targeting ligand of the present invention is preferably a molecule selected from the group consisting of antibodies, substances with affinity to organelles, and proteins that have binding affinity to sugar chains, in order to suppress nonspecific adsorption.

[0293] The type of targeting ligand is not particularly limited, and an optimum one can be selected depending on the purpose. Specific examples of targeting ligands include the following:

[0294] A first example of a targeting ligand is a primary antibody (an antibody that specifically binds to a substance to be detected). A luminescent labeling material for pathological diagnosis in which the targeting ligand is a primary antibody can directly bind to a substance to be detected and fluorescently label it (primary antibody method).

[0295] A second example of a targeting ligand is a secondary antibody (an antibody that binds to a primary antibody). For example, if the primary antibody is an antibody (IgG) produced from a rabbit, the secondary antibody would be an anti-rabbit IgG antibody. The targeting ligand is a secondary antibody, a luminescent labeling agent for pathological diagnosis, which binds to the primary antibody bound to the substance to be detected, thereby indirectly fluorescently labeling the substance to be detected (secondary antibody method).

[0296] A third example of the targeting ligand is avidin, streptavidin, or biotin. For example, when a luminescent labeling agent for pathological diagnosis whose targeting ligand is avidin or streptavidin is used, a secondary antibody-biotin complex is used in combination. The secondary antibody-biotin complex binds to a primary antibody bound to a substance to be detected, and the luminescent labeling agent for pathological diagnosis whose targeting ligand is avidin or streptavidin further binds to this complex, thereby indirectly fluorescently labeling the substance to be detected (biotin-avidin method or sandwich method). Conversely, a luminescent labeling agent for pathological diagnosis whose targeting ligand is biotin can also be used in combination with a secondary antibody-avidin complex or secondary antibody-streptavidin.

[0297] The primary antibody may be selected based on the specific binding capacity of the selected target substance. For example, if the target substance is HER2, an anti-HER2 monoclonal antibody can be used as the primary antibody. Such a primary antibody (monoclonal antibody) can be produced by a common method using mice, rabbits, cows, goats, sheep, dogs, chickens, etc. as immunized animals.

[0298] The secondary antibody can be selected based on the primary antibody selected, and can bind to it. For example, if the primary antibody is a rabbit anti-HER2 monoclonal antibody, an anti-rabbit IgG antibody can be used as the secondary antibody. Such secondary antibodies can also be produced by standard techniques.

[0299] Alternatively, the substance to be detected may be a nucleic acid molecule, and the corresponding target-directing ligand may be a nucleic acid molecule having a base sequence complementary to that of the nucleic acid molecule.

[0300] The luminescent labeling material for pathological diagnosis may be prepared by any known method. For example, amidation by the reaction of an amine with a carboxylic acid, sulfidation by the reaction of a maleimide with a thiol, imination by the reaction of an aldehyde with an amine, or amination by the reaction of an epoxy with an amine can be utilized. The functional group involved in such a reaction may be one that is pre-existing on the surface of the luminescent nanoparticle (a functional group derived from the raw material monomer of the binder), or may be a functional group obtained by converting a functional group present on the surface of the luminescent nanoparticle according to a known method, or a functional group introduced by surface modification, etc. An appropriate linker molecule may be utilized as needed.

[0301] In another aspect of the present invention, there is provided a kit for immunohistochemical staining using the luminescent nanoparticles of the present invention. This kit comprises at least the luminescent labeling material for pathological diagnosis of the present invention or the luminescent nanoparticles of the present invention, a targeting ligand, and reagents. This kit may further comprise, as necessary, a primary antibody, a secondary antibody, another targeting ligand (e.g., biotin) used in combination with the targeting ligand (e.g., streptavidin), reagents for forming the desired complex, other reagents used in immunohistochemical staining, etc.

[0302] <Method for producing luminescent marker for pathological diagnosis> In the technical field to which the present invention pertains, various techniques are known for producing luminescent labeling materials for pathological diagnosis by covalently binding luminescent labels (luminescent nanoparticles in the present invention) to targeting ligands or the like, and such techniques can also be used in the present invention.

[0303] For example, by utilizing the reaction occurring between reactive functional groups such as carboxyl groups, amino groups, aldehyde groups, thiol groups, and maleimide groups, a luminescent labeling material for pathological diagnosis (one reactive functional group present on its surface) can be covalently bonded to a targeting ligand (the other reactive functional group present in the molecule). Furthermore, if it is not possible to directly bond these functional groups, they can also be bonded via a "linker molecule" having predetermined functional groups at both ends of the molecule. Such a reaction can be carried out by adding the necessary reagents and allowing the reaction to proceed for a predetermined period of time.

[0304] A specific example is a method in which luminescent nanoparticles having hydroxyl groups on their surface are reacted with a silane coupling agent (e.g., aminopropyltrimethoxysilane) to introduce amino groups, while a thiol group-introducing reagent (e.g., N-succimidyl S-acetylthioacetate) is reacted with streptavidin to introduce thiol groups, and finally, a PEG (polyethylene glycol)-based linker molecule having maleimide groups at both ends that are reactive with both amino and thiol groups is reacted to link the luminescent nanoparticles to streptavidin.

[0305] Furthermore, when a resin (acrylic resin) is synthesized using glycidyl methacrylate as a raw material monomer, epoxy groups derived from the monomer are present on the surface of the luminescent nanoparticles. By adding aqueous ammonia to the luminescent nanoparticles, the epoxy groups are converted to amino groups, and the desired targeting ligands can be further linked to the amino groups. [Example]

[0306] The present invention will be specifically described below with reference to examples, but the present invention is not limited to these. In the examples, the terms "parts" and "%" are used, but unless otherwise specified, they represent "parts by mass" or "% by mass."

[0307] <Luminescent compounds> <Light-emitting compounds (excitable dyes) used in comparative examples> In a comparative example, a luminescent compound represented by the following structural formula was used in place of the first luminescent compound. Hereinafter, this luminescent compound is referred to as luminescent compound (Cf). In the luminescent compound (Cf), the maximum absorption wavelength λ ab1 is 338 nm, and the maximum emission wavelength λ em1 is 345 nm. The emission spectrum of the luminescent compound (Cf) does not overlap with the absorption spectrum of the second luminescent compound (A-1) shown below, so excitation energy is not transferred to the second luminescent compound. The absence of emission from the second luminescent compound in the resulting nanoparticles confirms that excitation energy is not transferred.

[0308] [ka]

[0309] <First Light-Emitting Compound Used in Examples> In the examples, the following compound (C-167) was used as the first light-emitting compound. In the compound (C-167), the maximum absorption wavelength λ ab1 is 570 nm, and the maximum emission wavelength λ em1 is 608 nm.

[0310] [ka]

[0311] <Second Light-Emitting Compound Used in Examples> In the examples, the following compound was used as the second luminescent compound. Hereinafter, this luminescent compound will be referred to as luminescent compound (A-1). In the luminescent compound (A-1), the maximum absorption wavelength λ ab2 is 655 nm, and the maximum emission wavelength λ em2 is 681 nm.

[0312] [ka]

[0313] <<Production of luminescent nanoparticles>> [Example 1 and Comparative Example] Preparation of luminescent nanoparticles using oil-soluble dyes <Preparation of Luminescent Nanoparticles Nos. (1-13) to (1-19)> [Example 1-13] Preparation of luminescent nanoparticles using oil-soluble dyes The first luminescent compound, compound (C-167), and the second luminescent compound, luminescent compound (A-1), were dissolved in 0.168 mL of dichloromethane to give concentrations of 25 mg / mL and 2.5 mg / mL, respectively, and 0.312 mL of Emulgen 430 (5% by mass aqueous solution) and 0.381 mL of DBS-Na (1.62% by mass aqueous solution) were added.

[0314] After cooling with ice water, ultrasonic waves were applied for 5 minutes using an ultrasonic homogenizer UH150. Dichloromethane was then removed using a vacuum pump while stirring at 300 rpm. The resulting solution was heated and stirred at 82°C for 15 minutes on a hot stirrer. Then, 0.07 mL of melamine resin (Nikarac MX-035 (Nippon Carbide Industries, Ltd., 50% solids aqueous solution) was added while stirring. After stirring for 2 minutes, 0.1 mL of a mixed aqueous solution of 1% by mass of DBS (dodecylbenzenesulfonic acid) and 0.33% by mass of TsOH (p-toluenesulfonic acid) was added and the mixture was heated and stirred for an additional 90 minutes. After heating and stirring, the mixture was heated in an autoclave at 121°C for 40 minutes.

[0315] The resulting dispersion was centrifuged at 18,500 G for 10 minutes, the supernatant removed, and then ultrapure water was added and the particles were redispersed using a homogenizer. This process of centrifuging, removing the supernatant, and redispersing in ultrapure water was repeated five times. Further washing was performed using an organic solvent instead of ultrapure water until the color of the supernatant disappeared, yielding luminescent nanoparticles No. (1-13).

[0316] [Comparative Examples 1-1 to 1-5, Examples 1-14 to 1-19] The first luminescent compound and the second luminescent compound shown in Table I were dissolved in 0.168 mL of dichloromethane to give the desired concentrations, and then luminescent nanoparticles Nos. (1-1) to (1-5) and (1-14) to (1-19) were obtained under the same conditions as in Example 1-13.

[0317] Table I lists the difference λ between the maximum emission wavelength of the first luminescent compound and the maximum absorption wavelength of the second luminescent compound. ab2 -λ em1 and the maximum emission wavelength λ of the second luminescent compound em2 showed.

[0318] <Calculation of the luminescent compound content of luminescent nanoparticles> Each of the luminescent nanoparticles Nos. (1-1) to (1-19) prepared above was dispersed in ultrapure water to a concentration of 0.0189 mg / mL, and the absorption spectra of the nanoparticles were measured at room temperature using a spectrophotometer (Hitachi High-Tech Science U-3300). The content of the luminescent compound in the nanoparticles was calculated from the maximum absorption wavelength corresponding to each luminescent compound and the molar extinction coefficient of each luminescent compound.

[0319] <Evaluation of luminescence intensity of luminescent nanoparticles> Luminescent nanoparticles were dispersed in ultrapure water to a concentration of 0.0189 mg / mL, and the emission spectrum of the dispersion was measured at room temperature using a fluorometer (Hitachi High-Technologies Corporation; F-7000) by exciting the nanoparticles at the maximum absorption wavelength of the first luminescent compound, except for Comparative Example 1-2, which was measured by exciting the nanoparticles at the maximum absorption wavelength of the second luminescent compound.

[0320] Among the luminescent nanoparticles prepared above, in the emission spectrum of Comparative Example 1-1, the maximum emission wavelength was located at the emission peak derived from the first luminescent compound, and no emission peak derived from the second luminescent compound was observed. In the emission spectra of Comparative Examples 1-2 to 1-5 and Examples 1-13 to 1-19, the maximum emission wavelength was located at the emission peak derived from the second luminescent compound.

[0321] The maximum emission wavelengths and the relative values ​​of emission intensities at the maximum emission wavelengths obtained from the emission spectra of Comparative Examples 1-2 to 1-5 and Examples 1-13 to 1-19 are shown in Table I. The emission intensity (relative value) of each particle is determined by setting the measured value of the luminescent nanoparticles of Comparative Example 1-2 to 1.

[0322] It can be seen that when the content of the first light-emitting compound is 4 to 90 mass %, the relative luminescence intensity is greater than that of the comparative example, and when the content is 30 mass %, the luminescence intensity reaches a maximum value.

[0323] [Table 1]

[0324] <<Preparation of luminescent labeling materials for pathological diagnosis>> [Example 2] Luminescent labeling material for pathological diagnosis consisting of luminescent nanoparticles No. (1-16) <Preparation of luminescent nanoparticles surface-modified with PEG chains bearing maleimide groups at their termini> 0.1 mg of the above-mentioned luminescent nanoparticles No. (1-16), which are melamine particles containing a luminescent compound, was dispersed in 1.5 mL of ethanol, and 2 μL of aminopropyltrimethoxysilane "LS-3150" (manufactured by Shin-Etsu Chemical Co., Ltd.) was added, and the mixture was allowed to react at room temperature with stirring for 8 hours to perform a surface amination treatment.

[0325] The concentration of the surface-aminated luminescent nanoparticles was adjusted to 3 nM using PBS (phosphate buffered saline) containing 2 mM EDTA (ethylenediaminetetraacetic acid). To this solution, the linker reagent "SM(PEG)12" (Thermo Scientific, cat. No. 22112) was added to a final concentration of 10 mM, mixed, and allowed to react at room temperature for 1 hour while stirring.

[0326] The reaction mixture was centrifuged at 10,000 G for 20 minutes, the supernatant was removed, and the precipitate was dispersed in PBS containing 2 mM EDTA. The mixture was then centrifuged again under the same conditions. After washing three times in the same manner, luminescent nanoparticles surface-modified with PEG chains bearing maleimide groups at their ends were obtained.

[0327] <Preparation of thiol group-introduced streptavidin> First, 70 μL of an aqueous solution of N-succinimidyl-S-acetylthioacetate (SATA, manufactured by Pirc) adjusted to 64 mg / mL was added to 40 μL of an aqueous solution of streptavidin (manufactured by Wako Pure Chemical Industries, Ltd.) adjusted to 1 mg / mL, and the mixture was allowed to react at room temperature for 1 hour to introduce a protected thiol group (-NH-CO-CH2-S-CO-CH3) onto the amino group of streptavidin.

[0328] Subsequently, the protected thiol groups were treated with hydroxylamine to generate free thiol groups (-SH), completing the process of introducing thiol groups (-SH) into streptavidin. The solution was desalted through a gel filtration column (Zaba Spin Desalting Columns, Funakoshi) to obtain thiol-introduced streptavidin.

[0329] <Preparation of streptavidin-modified luminescent nanoparticles> The prepared luminescent nanoparticles surface-modified with PEG chains bearing maleimide groups at their termini and the prepared thiol-introduced streptavidin were mixed in PBS containing 2 mM EDTA and allowed to react for 1 hour, thereby binding streptavidin to the luminescent nanoparticles via the PEG chains. 10 mM mercaptoethanol was added to the reaction solution to terminate the reaction. The resulting solution was concentrated using a centrifugal filter, and unreacted material was removed using a purification gel filtration column to obtain a luminescent labeling material for pathological diagnosis (streptavidin-modified luminescent nanoparticles).

[0330] [Comparative Example 2] Luminescent labeling material for pathological diagnosis consisting of luminescent nanoparticles No. (1-2) By the same experimental procedure as in Example 2, a luminescent labeling material for pathological diagnosis (streptavidin-modified luminescent nanoparticles) was obtained.

[0331] Example 3: Evaluation of luminescent labeling materials for pathological diagnosis <Tissue staining process> [Immunohistological staining] Immunostaining of human breast tissue was performed using a tissue stain containing the luminescent labeling material for pathological diagnosis made of the luminescent nanoparticles prepared in Example 2 and Comparative Example 2. The tissue stain was prepared using PBS buffer containing 1% BSA. Tissue array slides (Cosmo Bio, product number CB-A712) were used for staining sections.

[0332] The stained sections were pre-treated with the PathVision HER2 DNA probe kit (Abbott) to calculate the FISH score for each spot. The FISH score was calculated according to the procedure described in the document attached to the PathVision (registered trademark) HER2 DNA probe kit (Abbott Japan).

[0333] After deparaffinization, the tissue array slides were washed with water and autoclaved in 10 mM citrate buffer (pH 6.0) for 15 minutes for antigen retrieval. After antigen retrieval, the tissue array slides were washed with PBS buffer and then incubated with anti-HER2 rabbit monoclonal antibody (4B5) diluted to 0.05 nM in 1% BSA-containing PBS buffer for 2 hours. After washing with PBS, the slides were incubated with biotin-labeled anti-rabbit antibody diluted in 1% BSA-containing PBS buffer for 30 minutes. Immunohistochemically stained sections were obtained by further incubation with the above-mentioned tissue staining agent, i.e., the above-prepared luminescent labeling material for pathological diagnosis (luminescent nanoparticles containing streptavidin), for 2 hours, followed by washing. The immunohistochemically stained sections were then fixed by immersion in 4% neutral paraformaldehyde aqueous buffer for 10 minutes.

[0334] [Morphological staining] The immunohistochemically stained sections fixed as described above were subjected to HE staining, and the stained sections were dehydrated by immersing them in ethanol. The dehydrated sections were then further immersed in xylene and air-dried to perform clearing, resulting in double-stained sections.

[0335] [Enclosed] After the morphological staining, a xylene-based mounting medium, Entelaneu (Merck), was dropped onto the sections, which were then covered with a cover glass and mounted.

[0336] <Tissue sample evaluation> The shape of the cells (position of the cell membrane) was identified by image processing using stained images for morphological observation, and the image was superimposed on an immunostained image. A luminescent labeling agent for pathological diagnosis (streptavidin-modified luminescent nanoparticles consisting of luminescent nanoparticles) labeled with HER2 protein expressed on the cell membrane was then subjected to microscopic observation by irradiating it with excitation light. While bright spots could be confirmed with the nanoparticles prepared in Example 2, it was difficult to confirm bright spots with the nanoparticles prepared in Comparative Example 2, which had a Stokes shift of less than 50 nm, due to the influence of cellular autofluorescence. These results demonstrated that the luminescent nanoparticles of the present invention can be used as a luminescent labeling agent for pathological diagnosis. [Industrial Applicability]

[0337] According to the present invention, it is possible to realize a high-brightness particle technology for bioimaging, and to provide luminescent nanoparticles and luminescent labeling materials for pathological diagnosis that enable highly sensitive imaging.

Claims

1. A luminescent nanoparticle comprising a first luminescent compound and a second luminescent compound, the first luminescent compound contains a compound having a structure represented by the following general formula (1d), and has a function of being excited by light irradiation and transferring energy generated by the excitation to the second luminescent compound, the second luminescent compound contains a compound having a structure represented by the following general formula (10), and has a function of receiving energy from the excitation and emitting light, The content of the first luminescent compound relative to the total amount of the luminescent nanoparticles is within a range of 4 to 90 mass %. 【Chemistry 1】 [In formula (1d), multiple R 1 s each independently represent a hydrogen atom or a substituent, and at least one represents a monovalent organic group having 3 to 30 carbon atoms.] 【Chemistry 2】 [In formula (10), X1 represents O, CR 2 , SiR 2 , P(═O)R, or BR 2 . Each R independently represents a hydrogen atom or a substituent. Y1 represents an amino group or a hydroxy group, and Y2 represents an ammonium group or an oxygen atom. R 30 represents a hydrogen atom or a substituent.]

2. A luminescent nanoparticle containing a first luminescent compound and a second luminescent compound, the first luminescent compound contains the following compound C-167, is excited by light irradiation, and has a function of transferring energy generated by the excitation to the second luminescent compound; the second luminescent compound contains the following compound A-1, and has a function of receiving energy from the excitation and emitting light, and The content of the first luminescent compound relative to the total amount of the luminescent nanoparticles is within a range of 4 to 90 mass %. 【Transformation 3】

3. 3. The luminescent nanoparticles according to claim 1, wherein the molar ratio of the second luminescent compound to the first luminescent compound is in the range of 1:2 to 1:

200.

4. The luminescent nanoparticles according to any one of claims 1 to 3, further comprising a binder.

5. The luminescent nanoparticle according to claim 1 , wherein the surface of the luminescent nanoparticle has a hydrophilic group.

6. A luminescent labeling material for pathological diagnosis, which uses the luminescent nanoparticles according to any one of claims 1 to 5.

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