Quantum dot dispersion for biomedical applications, method for producing the same, and composite quantum dot

Abstract

To provide a quantum dot dispersion for biomedical applications, in which water-soluble quantum dots are dispersed in water and exhibit excellent stability, high quantum yield, and low toxicity, as well as composite quantum dots for biomedical applications.SOLUTION: A quantum dot dispersion for biomedical applications comprises water-soluble quantum dots, a phosphine compound represented by the following formula (1), (2), (3), (4), or (5), and water (where, each R is independently selected from a hydroxyalkyl group, a carboxyalkyl group, an aminoalkyl group, a mercaptoalkyl group, or a cyanoalkyl group, each having 1-8 carbon atoms).SELECTED DRAWING: None

Description

[Technical field]

[0001] The present invention relates to a quantum dot dispersion for biomedical applications, a method for producing the same, and a composite quantum dot comprising a water-soluble quantum dot and an additive. [Background technology]

[0002] In recent years, the development of quantum dots as luminescent materials has progressed. Cadmium-based quantum dots such as CdSe, CdTe, and CdS are being developed as representative quantum dots due to their excellent luminescence properties. In addition, since cadmium is highly toxic and has a high environmental impact, the development of cadmium-free quantum dots such as InP, CuInS2, and ZnTeSe is expected.

[0003] Since quantum dots have a unique size of nanoparticles, it is necessary to stabilize them by suppressing particle aggregation and defects on the particle surface in order to maintain excellent luminescence properties. Ligands present on the particle surface play a major role in stabilizing quantum dots, and various methods have been considered. For example, Patent Document 1 describes that semiconductor nanocrystals (quantum dots) can be obtained by stirring a reaction mixture containing an inert solvent, a cationic precursor, an anionic precursor, and a surface stabilizing ligand at a specific temperature range, and describes that the nanocrystals are stabilized by using a first ligand and a second ligand as the surface stabilizing ligand.

[0004] Additionally, Patent Documents 2 and 3 use phosphine-based ligands to stabilize quantum dots, while Patent Document 4 uses multidentate ligands obtained by oligomerizing phosphine-based ligands. Furthermore, Patent Document 5 describes that by combining fluorescent semiconductor nanoparticles (quantum dots) with a stabilizing additive made of a phosphine-based compound, it is possible to improve dispersibility in a carrier fluid and prevent photodecomposition or thermal decomposition of the nanoparticles.

[0005] In the manufacture of quantum dots, non-aqueous organic solvents such as toluene and octane are generally used because of the ease of reaction control and preparation of quantum dots, the stability of the obtained quantum dots, and the high quantum yield, etc. The techniques described in Patent Documents 1 to 5 aim to achieve stabilization in non-aqueous organic solvents, but depending on the application, quantum dots dispersed in an aqueous solvent are required.

[0006] Regarding the development of quantum dots that can be dispersed in aqueous solvents, i.e., water-soluble quantum dots, methods are being considered for measuring biological substances such as proteins, viruses, and nucleic acids by using molecular recognition bodies bound to molecular recognition substances, taking advantage of the excellent luminescence properties of quantum dots. In order to use quantum dots as marker substances in vivo, it is necessary to make the quantum dots water-soluble by coating the surface of the quantum dots with water-soluble ligands, but such treatment causes problems in terms of stability and reduced quantum yield. In addition, low toxicity is also required for use in vivo. [Prior art documents] [Patent documents]

[0007] [Patent Document 1] International Publication No. 2010 / 083431 Brochure [Patent Document 2] International Publication No. 2007 / 095061 Brochure [Patent Document 3] JP 2010-9995 A [Patent Document 4] International Publication No. 2004 / 042784 Brochure [Patent Document 5] International Publication No. 2017 / 048608 Brochure Summary of the Invention [Problem to be solved by the invention]

[0008] To make quantum dots water-soluble, it is necessary to make the ligands present on the quantum dot surface have hydrophilic functional groups, but since the hydrophilic functional groups are easily changed in water, it is thought that this has a negative effect on the stability and quantum yield of quantum dots. In other words, if the behavior of the hydrophilic functional groups in water can be stabilized, it will be possible to achieve excellent stability and quantum yield even for water-soluble quantum dots, and the development of water-soluble quantum dots will progress.

[0009] In addition, when quantum dots are used in vivo, if they are highly toxic, cells will die, so a method to eliminate or reduce their toxicity is needed. However, water-soluble quantum dots that have excellent luminescence properties and low toxicity are still under development.

[0010] Therefore, an object of the present invention is to provide a quantum dot dispersion for biomedical applications in which water-soluble quantum dots are dispersed in water, which has excellent stability, excellent quantum yield, and low toxicity, and a composite quantum dot for biomedical applications. [Means for solving the problem]

[0011] As a result of intensive research to solve the above problems, the inventors have found that the oxidation of a functional group can be suppressed by having a reducing compound present in the vicinity of a hydrophilic functional group, and that the stability of the quantum dot itself is increased by coordinating a specific reducing compound to the surface of the quantum dot, which leads to the stability of the quantum dot in water and suppresses the decrease in quantum yield. In addition, the inventors have found that the use of a specific reducing compound makes it possible to obtain a water-soluble quantum dot dispersion liquid with low cytotoxicity, and have completed the present invention.

[0012] That is, the present invention provides a quantum dot dispersion for biomedical applications, which comprises a water-soluble quantum dot, a phosphine compound represented by the following formula (1), (2), (3), (4) or (5), and water.

[0013] [ka] (In the formula, each R independently represents a hydroxyalkyl group, an aminoalkyl group, a mercaptoalkyl group, or a cyanoalkyl group having 1 to 8 carbon atoms.)

[0014] The present invention also provides a method for producing a quantum dot dispersion for biomedical applications, which includes a ligand exchange step of adding hydrophilic ligands to a hydrophobic quantum dot dispersion and exchanging the ligands bonded to the surfaces of the quantum dots for hydrophilic ligands to obtain water-soluble quantum dots, and a dispersion step of dispersing the water-soluble quantum dots in water to obtain a water-soluble quantum dot dispersion, in which the phosphine compound is added to the water-soluble quantum dot dispersion.

[0015] The present invention also provides a composite quantum dot for biomedical applications, comprising a water-soluble quantum dot and the above-mentioned phosphine compound. Effect of the Invention

[0016] According to the present invention, it is possible to provide a quantum dot dispersion for biomedical applications, which has excellent stability, excellent quantum yield, and low toxicity, and a composite quantum dot for biomedical applications. [Brief description of the drawings]

[0017] [Figure 1] FIG. 13 is a graph showing the change in fluorescence intensity over time measured in a light irradiation resistance test of the quantum dot dispersion for biomedical applications obtained in Example 3. [Diagram 2] FIG. 13 is a graph showing the change in fluorescence intensity over time measured in a light irradiation resistance test of the quantum dot dispersion for biomedical applications obtained in Comparative Example 3. [Diagram 3] 4 shows the results of toxicity tests in Example 4 and Comparative Example 6. [Figure 4] 1A to 1C are confocal laser microscope images of Examples 5, 6, and Comparative Example 7. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0018] The quantum dot dispersion for biomedical applications of the present invention is used for biomedical applications such as bioimaging, in vitro diagnostics, flow cytometry, companion diagnostics, and photodynamic therapy, and comprises water-soluble quantum dots, a phosphine compound represented by the following general formula (1), (2), (3), (4) or (5), and water. [ka] (In the formula, each R independently represents a hydroxyalkyl group, an aminoalkyl group, a mercaptoalkyl group, or a cyanoalkyl group having 1 to 8 carbon atoms.)

[0019] As the alkyl group of the hydroxyalkyl group, aminoalkyl group, mercaptoalkyl group or cyanoalkyl group having 1 to 8 carbon atoms represented by R in the general formulas (1), (2), (3), (4) and (5), from the viewpoint of enabling the additive of the present invention to be stably present in the vicinity of the hydrophilic ligand, preferred examples thereof include linear or branched alkyl groups, and specific examples thereof include methyl, ethyl, n-propyl, n-butyl, 2-methylpropyl, n-pentyl, n-hexyl, n-heptyl and n-octyl.

[0020] Examples of linear or branched hydroxyalkyl groups having 1 to 8 carbon atoms include hydroxymethyl, 2-hydroxyethyl, 3-hydroxypropyl, 4-hydroxybutyl, 5-hydroxypentyl, 6-hydroxyhexyl, 7-hydroxyheptyl, 8-hydroxyoctyl, and 3-hydroxy-2-methylpropyl groups. Examples of linear or branched aminoalkyl groups having 1 to 8 carbon atoms include aminomethyl, 2-aminoethyl, 3-aminopropyl, 4-aminobutyl, 5-aminopentyl, 6-aminohexyl, 7-aminoheptyl, 8-aminooctyl, and 3-amino-2-methylpropyl groups. Examples of the linear or branched mercaptoalkyl group having 1 to 8 carbon atoms include a mercaptomethyl group, a 2-mercaptoethyl group, a 3-mercaptopropyl group, a 4-mercaptobutyl group, a 5-mercaptopentyl group, a 6-mercaptohexyl group, a 7-mercaptoheptyl group, an 8-mercaptooctyl group, and a 3-mercapto-2-methylpropyl group. Examples of the linear or branched cyanoalkyl group having 1 to 8 carbon atoms include a cyanomethyl group, a 2-cyanoethyl group, a 3-cyanopropyl group, a 4-cyanobutyl group, a 5-cyanopentyl group, a 6-cyanohexyl group, a 7-cyanoheptyl group, an 8-cyanooctyl group, and a 3-cyano-2-methylpropyl group. Among these, from the viewpoint of allowing the additive of the present invention to be stably present in the vicinity of a hydrophilic ligand, a linear hydroxyalkyl group having 1 to 8 carbon atoms, preferably 1 to 5 carbon atoms, is preferred.

[0021] In the general formula (1), the multiple Rs may be the same or different. In the phosphine compound represented by the general formula (1), R is a hydroxyalkyl group, an aminoalkyl group, a mercaptoalkyl group, or a cyanoalkyl group having 1 to 8 carbon atoms, preferably 1 to 5 carbon atoms. Among these, from the viewpoint of being present in the vicinity of a hydrophilic ligand and contributing to the stabilization of the water-soluble quantum dot, it is preferable that Rs are the same and are hydroxyalkyl groups having 1 to 5 carbon atoms. Examples of such phosphine compounds include tris(hydroxymethyl)phosphine, tris(2-hydroxyethyl)phosphine, tris(3-hydroxypropyl)phosphine, tris(4-hydroxybutyl)phosphine, and tris(5-hydroxypentyl)phosphine.

[0022] The phosphine compound represented by the general formula (1) can be produced by a known method. For example, it can be obtained by adding an alkene to a phosphine. The phosphine compound represented by the general formula (1) may be a commercially available product. For example, tris(3-hydroxypropyl)phosphine and the like are available from Nippon Chemical Industry Co., Ltd.

[0023] In the phosphine compound represented by the general formula (2), R is a hydroxyalkyl group, a carboxyalkyl group, an aminoalkyl group, a mercaptoalkyl group, or a cyanoalkyl group having 1 to 8 carbon atoms, preferably 2 to 6 carbon atoms. Among these, R is preferably a hydroxyalkyl group having 2 to 6 carbon atoms, from the viewpoint of being present in the vicinity of a hydrophilic ligand and contributing to the stabilization of the water-soluble quantum dot. Examples of such a phosphine compound include 1-(hydroxymethyl)phosphorane, 1-(2-hydroxyethyl)phosphorane, 1-(3-hydroxypropyl)phosphorane, 1-(4-hydroxybutyl)phosphorane, and 1-(5-hydroxypentyl)phosphorane.

[0024] The phosphine compound represented by the general formula (2) can be produced by a known method. For example, it can be obtained by an addition reaction between phosphorane and alkene. The phosphine compound represented by the general formula (2) may be a commercially available product.

[0025] In the phosphine compound represented by the general formula (3), R is a hydroxyalkyl group, a carboxyalkyl group, an aminoalkyl group, a mercaptoalkyl group, or a cyanoalkyl group having 1 to 8 carbon atoms, preferably 2 to 6 carbon atoms. Among these, R is preferably a hydroxyalkyl group having 2 to 6 carbon atoms, from the viewpoint of being present in the vicinity of a hydrophilic ligand and contributing to the stabilization of a water-soluble quantum dot. Examples of such a phosphine compound include 9-(hydroxymethyl)phosphabicyclo[4.2.1]nonane, 9-(2-hydroxyethyl)phosphabicyclo[4.2.1]nonane, 9-(3-hydroxypropyl)phosphabicyclo[4.2.1]nonane, 9-(4-hydroxybutyl)phosphabicyclo[4.2.1]nonane, and 9-(5-hydroxypentyl)phosphabicyclo[4.2.1]nonane.

[0026] The phosphine compound represented by the general formula (3) can be produced by a known method. For example, it can be obtained by subjecting phosphabicyclo[4.2.1]nonane to an addition reaction with an alkene. The phosphine compound represented by the general formula (3) may be a commercially available product.

[0027] The phosphine compound represented by the general formula (4) includes compounds in which R is a hydroxyalkyl group, an aminoalkyl group, a mercaptoalkyl group, or a cyanoalkyl group having 1 to 8 carbon atoms, preferably 2 to 6 carbon atoms. Among these, from the viewpoint of being present in the vicinity of a hydrophilic ligand and contributing to the stabilization of a water-soluble quantum dot, R is preferably a hydroxyalkyl group having 2 to 6 carbon atoms. Examples of such phosphine compounds include 9-(hydroxymethyl)phosphabicyclo[3.3.1]nonane, 9-(2-hydroxyethyl)phosphabicyclo[3.3.1]nonane, 9-(3-hydroxypropyl)phosphabicyclo[3.3.1]nonane, 9-(4-hydroxybutyl)phosphabicyclo[3.3.1]nonane, and 9-(5-hydroxypentyl)phosphabicyclo[3.3.1]nonane.

[0028] The phosphine compound represented by the general formula (4) can be produced by a known method. For example, it can be obtained by subjecting phosphabicyclo[3.3.1]nonane to an addition reaction with an alkene. The phosphine compound represented by the general formula (4) may be a commercially available product.

[0029] The phosphine compound represented by the general formula (5) is a compound in which one of the nitrogen atoms of hexamethylenetetramine is replaced with a phosphorus atom, specifically, 1,3,5-triaza-7-phosphaadamantane. The phosphine compound represented by the general formula (5) can be produced by a known method. For example, it can be obtained by reacting tris(hydroxymethyl)phosphine with hexamethylenetetramine. In addition, the phosphine compound represented by the general formula (5) may be a commercially available product, for example, available from Sigma-Aldrich Co., Ltd.

[0030] The quantum dot dispersion for biomedical applications of the present invention contains a phosphine compound represented by the general formula (1), (2), (3), (4) or (5), and when the phosphine compound is added to a dispersion of water-soluble quantum dots, the stability of the water-soluble quantum dots in water is excellent. As one of the reasons, the present inventors believe that the behavior of the quantum dots in water is stabilized because the phosphine compound is present in the vicinity of the hydrophilic functional group coordinated to the surface of the water-soluble quantum dots, and the oxidation is suppressed by the reduction action on the hydrophilic functional group. In addition, the present inventors believe that the stability of the quantum dots themselves is increased by the direct coordination of the phosphine compound to the surface of the quantum dots, which leads to the stability of the behavior of the quantum dots in water. Furthermore, since it is intended for biomedical applications, it cannot be used if it has strong cytotoxicity, but the phosphine compound not only stabilizes the behavior of the quantum dots in water, but also has low cytotoxicity, so it can be used for biomedical applications.

[0031] In view of the possibility of use in the living body, it is particularly preferable that the quantum dot dispersion liquid for biomedical applications of the present invention uses water as the solvent. However, as long as there are no problems in use, aqueous solvents such as methanol, ethanol, 2-propanol, ethylene glycol, propylene glycol, acetone, acetonitrile, N-methylpyrrolidone, dimethylformamide, dimethylsulfoxide, and tetrahydrofuran can also be used in combination.

[0032] The quantum dot dispersion for biomedical applications can be produced by adding a phosphine compound represented by the general formula (1), (2), (3), (4) or (5) to a dispersion of water-soluble quantum dots.

[0033] A dispersion of water-soluble quantum dots can be produced by a ligand exchange process in which hydrophilic ligands are added to a hydrophobic quantum dot dispersion and the ligands bonded to the surfaces of the quantum dots are exchanged for hydrophilic ligands to obtain water-soluble quantum dots, and a dispersion process in which the water-soluble quantum dots are dispersed in water to obtain a dispersion of water-soluble quantum dots.

[0034] The phosphine compound represented by the general formula (1), (2), (3), (4) or (5) is believed to suppress oxidation of the hydrophilic functional group of the ligand by being present in the vicinity of the hydrophilic ligand bonded to the surface of the water-soluble quantum dot, and to improve the stability of the quantum dot in water by being directly coordinated to the surface of the quantum dot. Therefore, by adding the phosphine compound to the dispersion of the water-soluble quantum dot obtained as described above, a water-soluble quantum dot dispersion for biomedical use that is stable and has excellent quantum yield can be obtained. Each step of producing a water-soluble quantum dot dispersion for biomedical applications will be described below.

[0035] <Hydrophobic quantum dot dispersion> The hydrophobic quantum dot dispersion liquid is a liquid in which hydrophobic quantum dots are dispersed in a solvent, and the hydrophobic quantum dots are quantum dots having hydrophobic ligands on their surfaces. The hydrophobic quantum dot dispersion liquid may be obtained by reacting raw material compounds consisting of elements constituting the hydrophobic quantum dots in a solvent, or may be a liquid in which commercially available hydrophobic quantum dots are dispersed in a solvent.

[0036] The solvent of the dispersion liquid is preferably a non-polar organic solvent, more preferably a solvent made of an aliphatic hydrocarbon. Examples of the aliphatic hydrocarbon include saturated hydrocarbons such as n-hexane, n-heptane, n-octane, n-nonane, n-decane, n-dodecane, n-hexadecane, and n-octadecane; and unsaturated aliphatic hydrocarbons such as 1-undecene, 1-dodecene, 1-tetradecene, 1-hexadecene, and 1-octadecene. The aliphatic hydrocarbon used as the solvent may be one type, or two or more types may be mixed together.

[0037] Examples of the quantum dots constituting the hydrophobic quantum dots include cadmium-based quantum dots such as CdSe, CdTe, and CdS, and cadmium-free quantum dots such as InP, CuInS, and ZnTeSe. In the present invention, the quantum dots are preferably of a core-shell structure in which a shell is formed on the surface of a core, and are particularly preferably of a core-shell structure in which the core has at least an InP-based quantum dot obtained by reacting a phosphorus source with an indium source, and the shell has a coating compound other than an InP-based compound.

[0038] Examples of the hydrophobic ligand that constitutes the hydrophobic quantum dots include carboxylic acid derivatives, phosphine derivatives, amine derivatives, and phosphonic acid.

[0039] The carboxylic acid derivative is preferably one in which the alkyl group in the molecule is linear and has 2 to 24 carbon atoms. Specific examples of carboxylic acids in which the alkyl group in the molecule is linear and has 2 to 24 carbon atoms include capric acid, lauric acid, myristic acid, palmitic acid, stearic acid, arachidic acid, behenic acid, elaidic acid, oleic acid, linolenic acid, and erucic acid. Among these, from the viewpoint of improving the quality of the obtained hydrophobic quantum dots, those in which the alkyl group in the molecule has 12 to 20 carbon atoms are particularly preferred, and oleic acid is the most preferred.

[0040] The phosphine derivative is preferably a primary to tertiary alkylphosphine, and the alkyl group in the molecule is preferably a straight chain having 2 to 18 carbon atoms. The alkyl groups in the molecule may be the same or different. Specific examples of the alkyl phosphine having a straight chain alkyl group having 2 to 18 carbon atoms include monoethylphosphine, monobutylphosphine, monodecylphosphine, monohexylphosphine, monooctylphosphine, monododecylphosphine, monohexadecylphosphine, diethylphosphine, dibutylphosphine, didecylphosphine, dihexylphosphine, dioctylphosphine, didodecylphosphine, dihexadecylphosphine, triethylphosphine, tributylphosphine, tridecylphosphine, trihexylphosphine, trioctylphosphine, tridodecylphosphine, and trihexadecylphosphine. Among these, from the viewpoint of improving the quality of the obtained hydrophobic quantum dots, those in which the number of carbon atoms in the alkyl group in the molecule is 4 to 12 are particularly preferred, those which are trialkylphosphines are preferred, and trioctylphosphine is most preferred.

[0041] The amine derivative is preferably a primary to tertiary alkylamine, and preferred examples thereof include linear alkylamines in which the alkyl group in the molecule has 2 to 18 carbon atoms, and aromatic alkylamines in which the alkyl group has 6 to 12 carbon atoms. The alkyl groups in the molecule may be the same or different. Specific examples of linear alkylamines in which the alkyl group has 2 to 18 carbon atoms include monoethylamine, monobutylamine, monodecylamine, monohexylamine, monooctylamine, monododecylamine, monohexadecylamine, diethylamine, dibutylamine, didecylamine, dihexylamine, dioctylamine, didodecylamine, dihexadecylamine, triethylamine, tributylamine, tridecylamine, trihexylamine, trioctylamine, tridodecylamine, and trihexadecylamine. Specific examples of aromatic alkylamines in which the alkyl group has 6 to 12 carbon atoms include aniline, diphenylamine, triphenylamine, monobenzylamine, dibenzylamine, tribenzylamine, naphthylamine, dinaphthylamine, and trinaphthylamine. The phosphonic acid is preferably a monoalkylphosphonic acid in which the alkyl group in the molecule is a straight-chain alkyl group having from 2 to 18 carbon atoms.

[0042] When the hydrophobic quantum dots used in the present invention have a core-shell structure, examples of coating compounds suitable for the shell include Al2O3, Ga2O3, SiO2, CdS, CdSe, CdSeS, CdTe, CdSeTe, CdTeS, ZnS, ZnSe, ZnSeS, ZnTe, ZnSeTe, ZnTeS, ZnO, ZnOS, ZnSeO, and ZnTeO, etc. In the present invention, it is preferable that the coating compound is obtained by reaction with at least a zinc source.

[0043] The hydrophobic quantum dot dispersion used in the present invention can be produced by a known method, for example, the method described in JP-A-2023-033157.

[0044] <Ligand exchange process> The ligand exchange reaction is a process in which hydrophilic ligands are added to a hydrophobic quantum dot dispersion liquid, and the ligands bonded to the surfaces of the quantum dots are exchanged for hydrophilic ligands to obtain water-soluble quantum dots.

[0045] Examples of hydrophilic ligands include thiols, amine-containing thiols, amines, alcohols, thiol-containing alcohols, selenols, phosphites, phosphinic acids, phosphonic acids, phosphoric acids, phosphine oxides, phosphine sulfides, phosphine selenides, carboxylic acids, thiol-containing carboxylic acids, thiocarboxylic acids, thionocarboxylic acids, dithiocarboxylic acids, sulfenic acids, sulfinic acids, and sulfonic acids. Among these, from the viewpoint of the stability of the water-soluble quantum dots and the improvement of the quantum yield, thiols, amine-containing thiols, amines, alcohols, thiol-containing alcohols, phosphonic acids, carboxylic acids, and thiol-containing carboxylic acids are preferred, and thiol-containing carboxylic acids are particularly preferred.

[0046] Examples of the thiol-containing carboxylic acid include thioglycolic acid, 2-mercaptopropionic acid, 3-mercaptopropionic acid, thiomalic acid, cysteine, acetylcysteine, homocysteine, penicillamine, etc. Among these, from the viewpoint of improving the water dispersibility and quantum yield of the quantum dots, thioglycolic acid, 3-mercaptopropionic acid, cysteine, and acetylcysteine ​​are preferred, and 3-mercaptopropionic acid is more preferred.

[0047] The temperature when adding hydrophilic ligands to the hydrophobic quantum dot dispersion is preferably 0°C to 350°C, more preferably 20°C to 300°C, and the treatment time is preferably 1 minute to 600 minutes, more preferably 5 minutes to 240 minutes. The amount of the ligand to be added depends on the type of the ligand, but is preferably 0.01 g / L to 100,000 g / L, more preferably 0.1 g / L to 2,000 g / L, relative to the reaction solution containing the quantum dots.

[0048] The atmosphere when adding the hydrophilic ligand to the hydrophobic quantum dot dispersion liquid is preferably a non-oxidizing atmosphere such as an inert gas atmosphere, reduced pressure, or vacuum, from the viewpoint of preventing the denaturation of the quantum dots and the by-production of oxides. The non-oxidizing atmosphere is preferably an inert gas atmosphere such as nitrogen gas or argon gas from the viewpoint of ease of operation. Through the above steps, a dispersion liquid containing water-soluble quantum dots is obtained.

[0049] Next, the water-soluble quantum dots are separated from the dispersion liquid containing the water-soluble quantum dots. Examples of the separation method include common methods such as centrifugation, decantation, and suction filtration. At this time, in order to remove unnecessary hydrophobic ligands, it is preferable to add a solvent capable of dissolving the ligands and repeat the operation of separating the water-soluble quantum dots by centrifugation or the like several times. Through the above steps, water-soluble quantum dots are obtained.

[0050] <Dispersion process> The dispersion step is a step of dispersing the water-soluble quantum dots in water to obtain a dispersion of the water-soluble quantum dots. The water-soluble quantum dots can be dispersed in water as they are. However, for example, when the hydrophilic ligand is a carboxylic acid containing a thiol, from the viewpoint of obtaining a uniform dispersion, it is preferable to react these with sodium hydroxide, potassium hydroxide, ammonia water, tetraethylammonium hydroxide, or the like to convert the carboxyl group into a salt form before dispersing in water, and it is preferable to convert it into an ammonium salt form before dispersing in water.

[0051] In addition, the water-soluble quantum dots before the dispersion process may contain by-products or unreacted impurities, so they may be subjected to a purification process. This purification process can be performed by adding an organic solvent such as methanol, ethanol, 2-propanol, acetone, acetonitrile, or tetrahydrofuran to a solution containing water-soluble quantum dots to precipitate the water-soluble quantum dots, separating the solution containing impurities from the water-soluble quantum dots, and dispersing the separated water-soluble quantum dots in water. The solution containing impurities and the water-soluble quantum dots can be separated by operations such as centrifugation, decantation, and suction filtration. Through the above steps, a dispersion of water-soluble quantum dots is obtained.

[0052] <Quantum dot dispersion for biomedical applications> The quantum dot dispersion for biomedical use of the present invention can be produced by adding a phosphine compound represented by the general formula (1), (2), (3), (4) or (5) to the obtained dispersion of water-soluble quantum dots. By adding the phosphine compound to the dispersion of water-soluble quantum dots, the phosphine compound can be easily arranged in close proximity to the water-soluble quantum dots, which makes it easier to improve the quantum yield and stability of the water-soluble quantum dots. The amount of the phosphine compound added depends on the type of the phosphine compound, but is preferably 10 molecules or more and 1,000,000 molecules or less, more preferably 100 molecules or more and 100,000 molecules or less, per water-soluble quantum dot particle in the dispersion.

[0053] The number of quantum dot particles can be determined, for example, by the following method. The average particle diameter r of each of the core and the core / shell is determined by transmission electron microscope measurement, and the shape is calculated by 4 / 3πr 3 The volume of the core is calculated by the radius of the shell, 4 / 3πr. 3The volume is calculated by subtracting the volumes of the core and shell inside from the volume calculated above. The calculated core and shell volumes are multiplied by the density of their constituent materials to convert them into mass, and these are added together to calculate the mass m of one core / shell quantum dot particle. In addition, the concentration of each element that makes up the core / shell quantum dot is quantified by ICP measurement to calculate the total mass M of quantum dots contained in 1 ml of solution. The quantum dot concentration (number of particles / ml) is calculated by dividing the total mass M per ml by the mass m per particle, and the number of particles is calculated from the quantum dot concentration and the volume of the quantum dot solution.

[0054] The method of adding the phosphine compound is not particularly limited, and examples thereof include a method of adding the phosphine compound directly to a dispersion of water-soluble quantum dots, and a method of adding the phosphine compound in a dissolved or dispersed state in water. Through the above steps, a quantum dot dispersion for biomedical applications is obtained.

[0055] In the quantum dot dispersion for biomedical applications, the phosphine compound represented by formula (1), (2), (3), (4) or (5) is arranged adjacent to or coordinated with the water-soluble quantum dots. That is, the quantum dot dispersion for biomedical applications is a dispersion in water of composite quantum dots containing water-soluble quantum dots and the phosphine compound represented by formula (1), (2), (3), (4) or (5).

[0056] The composite quantum dots have low cytotoxicity and improved stability in water, i.e., improved storage stability and light resistance over time, which allows the composite quantum dots to stably utilize their excellent luminescence function for a long period of time when used as a marker substance in a living body. EXAMPLES

[0057] The present invention will be described in more detail below with reference to examples, but the present invention is not limited thereto. The properties in the examples were measured by the following methods. (1) Emission spectrum, maximum fluorescence wavelength, full width at half maximum (FWHM) and quantum yield (PLQY) The obtained aqueous dispersion of quantum dots was measured using an absolute PL quantum yield measurement device (Quantaurus-QY, manufactured by Hamamatsu Photonics KK) under measurement conditions of an excitation wavelength of 400 nm and a measurement wavelength of 200 to 950 nm.

[0058] [Synthesis Example 1] Synthesis of orange InP / ZnSe / ZnS quantum dots 1.43 g of indium myristate was added to 14.25 g of 1-octadecene, and the mixture was heated to 120° C. under reduced pressure with stirring and degassed for 1.5 hours. After degassing, the mixture was returned to atmospheric pressure with nitrogen gas and cooled to 40° C. to obtain a 1-octadecene solution of indium myristate. The obtained 1-octadecene solution of indium myristate was kept at 40° C. under a nitrogen atmosphere, and 2.51 g of a trioctylphosphine solution containing 10% by mass of tris(trimethylsilyl)phosphine was added thereto, and the mixture was held for 10 minutes and then naturally cooled to 20° C. As a result, a yellow liquid containing an InP quantum dot precursor was obtained.

[0059] Next, the InP quantum dot precursor solution was heated to 280° C. to obtain a reaction solution of InP quantum dots. 7.54g of zinc oleate, 4.09g of zinc chloride, 5.40g of trioctylphosphine selenide, 8.90g of trioctylphosphine, 16.26g of oleylamine, and 16.26g of dioctylamine were mixed in a 200mL reaction vessel, heated to 120°C under reduced pressure with stirring, and degassed for 30 minutes. After degassing, the pressure was returned to atmospheric pressure with nitrogen gas, and the temperature was raised to 180°C under a nitrogen atmosphere, and 18.19g of the reaction solution of InP quantum dots obtained above was added, heated to 230°C and held for 30 minutes, and then further heated to 300°C and held for 60 minutes to obtain an oleylamine / dioctylamine dispersion of InP / ZnSe quantum dots having InP in the core and ZnSe in the shell. After the obtained dispersion was cooled to 240°C, 7.54 g of zinc oleate, 7.54 g of trioctylphosphine, and 4.35 g of trioctylphosphine sulfide were poured thereinto and held for 90 minutes to obtain an oleylamine / dioctylamine dispersion of multi-shell quantum dots having InP in the core and ZnSe and ZnS stacked in the shell.

[0060] After cooling the obtained dispersion to room temperature, 50 g of acetone was added, the mixture was stirred, and the InP / ZnSe / ZnS quantum dots were collected as a precipitate by centrifugation. The collected InP / ZnSe / ZnS quantum dots were suspended in 8.67 g of toluene to obtain a toluene dispersion of InP / ZnSe / ZnS quantum dots. 50 g of acetone was further added to this dispersion, the mixture was stirred, and the InP / ZnSe / ZnS quantum dots were collected as a precipitate by centrifugation. The collected InP / ZnSe / ZnS quantum dots were suspended in 7.03 g of octane to obtain an octane dispersion of purified InP / ZnSe / ZnS quantum dots. The emission wavelength of the obtained dispersion was 586 nm and showed orange color.

[0061] [Synthesis Example 2] Synthesis of red InP / ZnSe / ZnS quantum dots 1.59 g of indium myristate was added to 14.25 g of 1-octadecene, and the mixture was heated to 120° C. under reduced pressure with stirring and degassed for 1.5 hours. After degassing, the mixture was returned to atmospheric pressure with nitrogen gas and cooled to 40° C. to obtain a 1-octadecene solution of indium myristate. The obtained 1-octadecene solution of indium myristate was kept at 40° C. under a nitrogen atmosphere, and 2.51 g of a trioctylphosphine solution containing 10% by mass of tris(trimethylsilyl)phosphine was added thereto, and the mixture was held for 10 minutes and then naturally cooled to 20° C. As a result, a yellow liquid containing an InP quantum dot precursor was obtained.

[0062] Next, the InP quantum dot precursor solution was heated to 280° C. to obtain a reaction solution of the base InP quantum dots. In a separate reaction vessel, 2.39 g of indium myristate was added to 21.37 g of 1-octadecene, and the mixture was heated to 120°C under reduced pressure with stirring and degassed for 1.5 hours. After degassing, the mixture was returned to atmospheric pressure with nitrogen gas and cooled to 40°C to obtain a 1-octadecene solution of indium myristate.

[0063] The obtained 1-octadecene solution of indium myristate was kept at 40° C. under a nitrogen atmosphere, and 3.76 g of a trioctylphosphine solution containing 10% by mass of tris(trimethylsilyl)phosphine was added thereto, and the mixture was held for 10 minutes and then naturally cooled to 20° C. As a result, a yellow liquid containing an InP quantum dot precursor was obtained. The previously prepared base InP quantum dot reaction solution was reheated to 260° C., and the newly prepared InP quantum dot precursor solution was added over 60 minutes to obtain an InP quantum dot reaction solution.

[0064] 7.54g of zinc oleate, 4.09g of zinc chloride, 5.40g of trioctylphosphine selenide, 8.90g of trioctylphosphine, 16.26g of oleylamine, and 16.26g of dioctylamine were mixed in a 200mL reaction vessel, heated to 120°C under reduced pressure with stirring, and degassed for 30 minutes. After degassing, the pressure was returned to atmospheric pressure with nitrogen gas, and the temperature was raised to 180°C under a nitrogen atmosphere, and 15.79g of the reaction solution of InP quantum dots obtained above was added, heated to 230°C and held for 30 minutes, and then further heated to 300°C and held for 60 minutes to obtain an oleylamine / dioctylamine dispersion of InP / ZnSe quantum dots having InP in the core and ZnSe in the shell. After the obtained dispersion was cooled to 240°C, 7.54 g of zinc oleate, 7.54 g of trioctylphosphine, and 4.35 g of trioctylphosphine sulfide were poured thereinto and held for 90 minutes to obtain an oleylamine / dioctylamine dispersion of multi-shell quantum dots having InP in the core and ZnSe and ZnS stacked in the shell.

[0065] After cooling the obtained dispersion to room temperature, 50 g of acetone was added, the mixture was stirred, and the InP / ZnSe / ZnS quantum dots were collected as a precipitate by centrifugation. The collected InP / ZnSe / ZnS quantum dots were suspended in 8.67 g of toluene to obtain a toluene dispersion of InP / ZnSe / ZnS quantum dots. 50 g of acetone was further added to this dispersion, the mixture was stirred, and the InP / ZnSe / ZnS quantum dots were collected as a precipitate by centrifugation. The collected InP / ZnSe / ZnS quantum dots were suspended in 7.03 g of octane to obtain an octane dispersion of purified InP / ZnSe / ZnS quantum dots. The emission wavelength of the obtained dispersion was 619 nm, and it was red.

[0066] [Synthesis Example 3] Synthesis of yellow InP / ZnSe / ZnS quantum dots 1.20 g of indium myristate was added to 3.56 g of 1-octadecene, and the mixture was heated to 120° C. under reduced pressure with stirring and degassed for 1.5 hours. After degassing, the mixture was returned to atmospheric pressure with nitrogen gas and cooled to 40° C. to obtain a 1-octadecene solution of indium myristate. The obtained 1-octadecene solution of indium myristate was kept at 40° C. under a nitrogen atmosphere, and 2.51 g of a trioctylphosphine solution containing 10% by mass of tris(trimethylsilyl)phosphine was added thereto, and the mixture was held for 10 minutes and then naturally cooled to 20° C. As a result, a yellow liquid containing an InP quantum dot precursor was obtained.

[0067] Next, the InP quantum dot precursor solution was heated to 250° C. to obtain a reaction solution of InP quantum dots.

[0068] 7.54g of zinc oleate, 4.09g of zinc chloride, 5.40g of trioctylphosphine selenide, 8.90g of trioctylphosphine, 16.26g of oleylamine, and 16.26g of dioctylamine were mixed in a 200mL reaction vessel, heated to 120°C under reduced pressure with stirring, and degassed for 30 minutes. After degassing, the pressure was returned to atmospheric pressure with nitrogen gas, and the temperature was raised to 180°C under a nitrogen atmosphere, and 7.27g of the reaction solution of InP quantum dots obtained above was added, heated to 230°C and held for 30 minutes, and then further heated to 300°C and held for 60 minutes to obtain an oleylamine / dioctylamine dispersion of InP / ZnSe quantum dots having InP in the core and ZnSe in the shell. After the obtained dispersion was cooled to 240°C, 7.54 g of zinc oleate, 7.54 g of trioctylphosphine, and 4.35 g of trioctylphosphine sulfide were poured thereinto and held for 90 minutes to obtain an oleylamine / dioctylamine dispersion of multi-shell quantum dots having InP in the core and ZnSe and ZnS stacked in the shell.

[0069] After cooling the obtained dispersion to room temperature, 50 g of acetone was added, the mixture was stirred, and the InP / ZnSe / ZnS quantum dots were collected as a precipitate by centrifugation. The collected InP / ZnSe / ZnS quantum dots were suspended in 8.67 g of toluene to obtain a toluene dispersion of InP / ZnSe / ZnS quantum dots. 50 g of acetone was further added to this dispersion, the mixture was stirred, and the InP / ZnSe / ZnS quantum dots were collected as a precipitate by centrifugation. The collected InP / ZnSe / ZnS quantum dots were suspended in 7.03 g of octane to obtain an octane dispersion of purified InP / ZnSe / ZnS quantum dots. The emission wavelength of the obtained dispersion was 551 nm and it was yellow.

[0070] [Synthesis Example 4] Synthesis of green InP / ZnSe / ZnS quantum dots 1.20 g of indium myristate, 0.16 g of zinc myristate, and 0.06 g of zinc acetate were added to 14.25 g of 1-octadecene, and the mixture was heated to 120°C under reduced pressure with stirring and degassed for 1.5 hours. After degassing, the mixture was returned to atmospheric pressure with nitrogen gas and cooled to 40°C to obtain a 1-octadecene solution of indium zinc myristate. The obtained 1-octadecene solution of indium zinc myristate was kept at 40° C. under a nitrogen atmosphere, and 2.51 g of a trioctylphosphine solution containing 10% by mass of tris(trimethylsilyl)phosphine was added thereto, and the mixture was held for 10 minutes and then naturally cooled to 20° C. As a result, a yellow liquid containing an InP quantum dot precursor was obtained. Next, the InP quantum dot precursor solution was heated to 250° C. to obtain a reaction solution of InP quantum dots.

[0071] 7.54g of zinc oleate, 4.09g of zinc chloride, 5.40g of trioctylphosphine selenide, 8.90g of trioctylphosphine, 16.26g of oleylamine, and 16.26g of dioctylamine were mixed in a 200mL reaction vessel, heated to 120°C under reduced pressure with stirring, and degassed for 30 minutes. After degassing, the pressure was returned to atmospheric pressure with nitrogen gas, and the temperature was raised to 180°C under a nitrogen atmosphere, and 18.18g of the reaction solution of InP quantum dots obtained above was added, heated to 230°C and held for 30 minutes, and then further heated to 300°C and held for 60 minutes to obtain an oleylamine / dioctylamine dispersion of InP / ZnSe quantum dots having InP in the core and ZnSe in the shell. After the obtained dispersion was cooled to 240°C, 7.54 g of zinc oleate, 7.54 g of trioctylphosphine, and 4.35 g of trioctylphosphine sulfide were poured thereinto and held for 90 minutes to obtain an oleylamine / dioctylamine dispersion of multi-shell quantum dots having InP in the core and ZnSe and ZnS stacked in the shell.

[0072] After cooling the obtained dispersion to room temperature, 50 g of acetone was added, the mixture was stirred, and the InP / ZnSe / ZnS quantum dots were collected as a precipitate by centrifugation. The collected InP / ZnSe / ZnS quantum dots were suspended in 8.67 g of toluene to obtain a toluene dispersion of InP / ZnSe / ZnS quantum dots. 50 g of acetone was further added to this dispersion, the mixture was stirred, and the InP / ZnSe / ZnS quantum dots were collected as a precipitate by centrifugation. The collected InP / ZnSe / ZnS quantum dots were suspended in 7.03 g of octane to obtain an octane dispersion of purified InP / ZnSe / ZnS quantum dots. The emission wavelength of the obtained dispersion was 539 nm and showed green color.

[0073] [Example 1] (ligand exchange process) 2 ml of the octane dispersion of the orange InP / ZnSe / ZnS quantum dots obtained in Synthesis Example 1 and 10 ml of hexadecane were weighed into a 50 ml two-necked eggplant flask. The mixture was heated to 90°C under reduced pressure while stirring and degassed for 30 minutes. After degassing, the mixture was returned to atmospheric pressure with nitrogen gas and heated to 220°C under a nitrogen atmosphere, 4.0 ml of 3-mercaptopropionic acid was added, and the mixture was held for 10 minutes to obtain InP / ZnSe / ZnS quantum dots in which the ligands were substituted with 3-mercaptopropionic acid. After cooling to 160°C, 40 ml of octane was added and the mixture was cooled to room temperature. The supernatant was removed, 30 ml of hexane was added, and the mixture was stirred, and the supernatant was removed again. This was repeated three times to obtain a solid. The solid was suspended in 5 ml of ethanol, and the InP / ZnSe / ZnS quantum dots were collected as a precipitate by centrifugation. (Dispersion process) The recovered InP / ZnSe / ZnS quantum dot precipitate was suspended in 19 ml of pure water and 1 ml of 35% aqueous solution of tetraethylammonium hydroxide, and then insoluble impurities were removed by centrifugation to obtain a purified aqueous dispersion of InP / ZnSe / ZnS quantum dots. To this InP / ZnSe / ZnS quantum dot aqueous dispersion, 0.1 ml of a 40% aqueous solution of tris(3-hydroxypropyl)phosphine was added. The obtained aqueous dispersion was subjected to evaluation tests for storage stability and pH resistance.

[0074] [Example 2] The same procedure as in Example 1 was carried out except that the octane dispersion of red InP / ZnSe / ZnS quantum dots obtained in Synthesis Example 2 was used, to obtain an aqueous dispersion of purified water-soluble InP / ZnSe / ZnS quantum dots. To this InP / ZnSe / ZnS quantum dot aqueous dispersion, 0.1 ml of a 40% aqueous solution of tris(3-hydroxypropyl)phosphine was added. The obtained aqueous dispersion was subjected to evaluation tests for storage stability and pH resistance.

[0075] [Example 3] The same procedure as in Example 1 was carried out except that the octane dispersion of green InP / ZnSe / ZnS quantum dots obtained in Synthesis Example 4 was used, to obtain an aqueous dispersion of purified water-soluble InP / ZnSe / ZnS quantum dots. To this InP / ZnSe / ZnS quantum dot aqueous dispersion, 0.1 ml of a 40% aqueous solution of tris(3-hydroxypropyl)phosphine was added. The obtained aqueous dispersion was subjected to evaluation tests for storage stability, pH resistance, and light irradiation resistance.

[0076] [Comparative Example 1] For the InP / ZnSe / ZnS quantum dot aqueous dispersion in Example 1 to which the 40% aqueous solution of tris(3-hydroxypropyl)phosphine had not been added, evaluation tests were carried out on the storage stability and pH resistance.

[0077] [Comparative Example 2] For the InP / ZnSe / ZnS quantum dot aqueous dispersion in Example 2 to which the 40% aqueous solution of tris(3-hydroxypropyl)phosphine had not been added, evaluation tests were carried out on the storage stability and pH resistance.

[0078] [Comparative Example 3] For the InP / ZnSe / ZnS quantum dot aqueous dispersion in Example 3 to which the 40% aqueous solution of tris(3-hydroxypropyl)phosphine had not been added, evaluation tests were carried out on the storage stability, pH resistance, and light irradiation resistance.

[0079] [Comparative Example 4] In Example 1, 0.03 ml of trimethyl phosphate was added to the InP / ZnSe / ZnS quantum dot aqueous dispersion instead of the 40% aqueous solution of tris(3-hydroxypropyl)phosphine. The obtained aqueous dispersion was subjected to evaluation tests for storage stability and pH resistance.

[0080] [Comparative Example 5] In Example 1, 0.02 ml of dimethyl phosphite was added to the InP / ZnSe / ZnS quantum dot aqueous dispersion instead of the 40% aqueous solution of tris(3-hydroxypropyl)phosphine. The obtained aqueous dispersion was subjected to evaluation tests for storage stability and pH resistance.

[0081] [Rating 1] (1) Storage stability test Examples 1 to 3 and Comparative Examples 1 to 5 were subjected to a storage stability test. 0.006 ml of the water-soluble InP / ZnSe / ZnS quantum dot aqueous dispersion obtained in each Example and Comparative Example was placed in a 2 ml glass vial, and 1.2 ml of pure water was mixed to obtain a test solution. This test solution was left to stand in a cool, dark place at room temperature for one week, and the storage state was visually confirmed thereafter. In addition, the change in luminescence intensity due to irradiation with UV light was visually confirmed. The results are shown in Table 1.

[0082] (2) pH tolerance test Examples 1 to 3 and Comparative Examples 1 to 5 were subjected to a pH resistance test. 0.05 ml of the water-soluble InP / ZnSe / ZnS quantum dot aqueous dispersion obtained in each Example and Comparative Example was placed in a 1 ml glass vial, and 2 ml of the dilution solution shown in Table 3 was mixed to obtain a test solution. This was left to stand in a cool, dark place at room temperature for 1 hour, and the storage state thereafter was visually confirmed. In addition, the change in luminescence intensity due to irradiation with UV light was visually confirmed. The results are shown in Table 2.

[0083] (3) Light exposure resistance test Example 3 and Comparative Example 3 were subjected to a light irradiation resistance test. 60 μl of the green water-soluble InP / ZnSe / ZnS quantum dot aqueous dispersions obtained in Example 3 and Comparative Example 3 were placed in a 6 ml glass vial, and 3.0 ml of pure water was mixed to obtain test solutions. 2 The fluorescence intensity of the quantum dots was measured over time and the results are shown in Figures 1 and 2.

[0084]

Table 1

[0085]

Table 2

[0086] From the results of the storage stability test in Table 1, it can be seen that the InP / ZnSe / ZnS quantum dot aqueous dispersion of each example containing a specific phosphine compound has excellent storage stability because no precipitation is formed over a long period and there is no change in the emission intensity. Also, from the results of the pH tolerance test in Table 2, it can be seen that the InP / ZnSe / ZnS quantum dot aqueous dispersion of each example containing a specific phosphine compound maintains a stable solution state regardless of pH because no precipitation formation is confirmed and the emission intensity also remains stable. From the results of the light irradiation tolerance test in FIGS. 1 and 2, the green water-soluble InP / ZnSe / ZnS quantum dot aqueous dispersion of Comparative Example 3 shown in FIG. 2 shows a decrease in fluorescence intensity, while the green water-soluble InP / ZnSe / ZnS quantum dot aqueous dispersion of Example 3 shown in FIG. 1 has improved light irradiation tolerance because the decrease in fluorescence intensity under continuous irradiation with excitation light is suppressed.

[0087] [Example 4] <Preparation of a medium containing InP / ZnSe / ZnS quantum dots and tris(3-hydroxypropyl)phosphine> The dispersion process was carried out in the same manner as in Example 2 to obtain an InP / ZnSe / ZnS quantum dot aqueous dispersion. 150 μl of this InP / ZnSe / ZnS quantum dot aqueous dispersion was mixed with 300 μl of pure water, and centrifuged at 5,000×g for 15 minutes using a 30 kDa Amicon filter. After that, the Amicon filter was attached in the opposite direction, and centrifuged again at 5,000×g for 10 minutes to recover the InP / ZnSe / ZnS quantum dot aqueous dispersion. 13.89 μl of the recovered InP / ZnSe / ZnS quantum dot aqueous dispersion and 66.11 μl of pure water were mixed with a 5 mg / ml aqueous tris(3-hydroxypropyl)phosphine solution, shielded from light with aluminum foil, and incubated at 4°C for 24 hours to obtain a test solution consisting of an InP / ZnSe / ZnS quantum dot aqueous dispersion and an aqueous tris(3-hydroxypropyl)phosphine solution (InP / ZnSe / ZnS quantum dot concentration 5 μM, tris(3-hydroxypropyl)phosphine concentration 1 mg / ml). Next, 35 μl of a mixture of the test solution containing the six InP / ZnSe / ZnS quantum dot concentrations plotted in Figure 3 and pure water was added to 315 μl of medium (10% FBS / 1% Penicillin·Streptomycin / D-MEM (Low glucose)) that had been warmed in a thermostatic bath set to 37°C, to prepare a medium containing InP / ZnSe / ZnS quantum dots and tris(3-hydroxypropyl)phosphine. <Cell treatment> 50 μl of medium (10% FBS / 1% Penicillin·Streptomycin / D-MEM (Low glucose)) was added to each well of the 96-well plate and stored in an incubator at 37 °C in a 5% CO2 environment for 30 minutes. Then, 50 μl of NIH-3T3 cells (1×105 cells / ml) was seeded into each well of the plate. After incubation at 37 °C and 5% CO2 for 24 hours, the supernatant was removed and replaced with fresh medium and media (6 types) containing the InP / ZnSe / ZnS quantum dots and tris(3-hydroxypropyl)phosphine. Further, incubation was carried out at 37 °C and 5% CO2 for 24 hours. The wells replaced with fresh medium were used as control wells, and the wells replaced with the media containing the InP / ZnSe / ZnS quantum dots and tris(3-hydroxypropyl)phosphine were used as sample wells 1 to 6.

[0088] [Comparative Example 6] [Preparation of Medium Containing InP / ZnSe / ZnS Quantum Dots] Up to the dispersion step, the same method as in Example 2 was used to obtain an InP / ZnSe / ZnS quantum dot aqueous dispersion. 300 μl of pure water was mixed with 150 μl of this InP / ZnSe / ZnS quantum dot aqueous dispersion and centrifuged at 5,000×g for 15 minutes using a 30 kDa Amicon filter. Then, the Amicon filter was mounted in the reverse direction and centrifuged again at 5,000×g for 10 minutes to recover the InP / ZnSe / ZnS quantum dot aqueous dispersion. 86.11 μl of pure water was mixed with 13.89 μl of the recovered InP / ZnSe / ZnS quantum dot aqueous dispersion, shielded from light with aluminum foil, and incubated at 4 °C for 24 hours to obtain a test solution (InP / ZnSe / ZnS quantum dot concentration 7.88 μM) composed of the InP / ZnSe / ZnS quantum dot aqueous dispersion. Next, 35 μl of a mixture of the test solution with the seven InP / ZnSe / ZnS quantum dot concentrations plotted in Figure 3 and pure water was added to 350 μl of medium (10% FBS / 1% Penicillin-Streptomycin / D-MEM (Low glucose)) warmed in a thermostatic bath set to 37°C to prepare a medium containing InP / ZnSe / ZnS quantum dots. <Cell treatment> 50 μl of medium (10% FBS / 1% Penicillin·Streptomycin / D-MEM (Low glucose)) was added to each well of a 96-well plate, and the plate was stored in an incubator at 37°C and 5% CO2 for 30 minutes. Then, NIH-3T3 cells (1×10 5 50 μl of 100 μl of 100 μl of cells / ml was seeded. After incubation for 24 hours under conditions of 37°C and 5% CO2, the supernatant was removed and the medium was replaced with fresh medium and medium (seven types) containing the InP / ZnSe / ZnS quantum dots, and further incubated for 24 hours under conditions of 37°C and 5% CO2. The wells replaced with fresh medium were designated as control wells, and the wells replaced with medium containing InP / ZnSe / ZnS quantum dots were designated as sample wells 1 to 7.

[0089] [Rating 2] (1) Toxicity testing For Example 4 and Comparative Example 6, a toxicity test was carried out by calculating the cell viability. 100 μl of fresh medium was added to the wells without cells seeded for background measurement. The supernatants of the control wells and the sample wells were removed, and 100 μl of fresh medium was added. After repeating this procedure twice, the supernatant was removed and 100 μl of fresh medium was added. Then, 10 μl each of a solution of 5-{2,4-bis[(sodiooxy)sulfonyl]phenyl}-2-(2-methoxy-4-nitrophenyl)-3-(4-nitrophenyl)-3H-1,2λ5,3,4-tetrazol-2-ylium (CCK-8 solution, Dojindo Laboratories, 343-07623) was added to all the measurement wells, and the wells were incubated at 37 °C under 5% CO2 for 2 hours. Using a microplate reader (Varioskan VLBL00GD2, manufactured by Thermo Fisher), the absorbance at 450 nm, which is the absorption band of formazan in each well, was measured. The difference between the absorbances of the control wells and the sample wells and the absorbance of the background wells was taken, and the ratio of the sample wells to the control wells was converted to a percentage to calculate the cell viability of each well. The standard deviation was calculated from the results of 3 wells for each condition. The results are shown in Figure 3.

[0090] As is clear from Figure 3 as a result of the toxicity test, it can be seen that Example 4 containing a specific phosphine compound has a higher cell viability and lower cytotoxicity than Comparative Example 6.

[0091] [Example 5] <Preparation of a test solution consisting of InP / ZnSe / ZnS quantum dots and tris(3-hydroxypropyl)phosphine> The dispersion process was carried out in the same manner as in Example 2 to obtain an InP / ZnSe / ZnS quantum dot aqueous dispersion. 150 μl of this InP / ZnSe / ZnS quantum dot aqueous dispersion was mixed with 300 μl of pure water, and centrifuged at 5,000×g for 15 minutes using a 30 kDa Amicon filter. After that, the Amicon filter was attached in the opposite direction, and centrifuged again at 5,000×g for 10 minutes to recover the InP / ZnSe / ZnS quantum dot aqueous dispersion. 13.89 μl of the recovered InP / ZnSe / ZnS quantum dot aqueous dispersion and 66.11 μl of pure water were mixed with a 5 mg / ml aqueous tris(3-hydroxypropyl)phosphine solution, shielded from light with aluminum foil, and incubated at 4°C for 24 hours to obtain a mixed solution consisting of the InP / ZnSe / ZnS quantum dot aqueous dispersion and the tris(3-hydroxypropyl)phosphine aqueous solution (InP / ZnSe / ZnS quantum dot concentration 5 μM, tris(3-hydroxypropyl)phosphine concentration 1 mg / ml). This mixed solution was subjected to ultrasonic treatment while suspending, and passed through a 0.22 μm syringe filter to remove aggregated quantum dots. Then, 30.39 μl of a 1 mg / ml aqueous tris(3-hydroxypropyl)phosphine solution was added to 19.61 μl of the mixed solution to obtain a test solution consisting of InP / ZnSe / ZnS quantum dots and tris(3-hydroxypropyl)phosphine (InP / ZnSe / ZnS quantum dot concentration 1 μM, tris(3-hydroxypropyl)phosphine concentration 1 mg / ml). <Antibody modification> 5μl of the obtained test solution was ultrasonicated for 1 minute, and 3.84μl of an aqueous solution of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC, concentration 0.1mg / ml) and tris(3-hydroxypropyl)phosphine (concentration 0.1mg / ml) and 8.86μl of pure water were mixed thereto, and the mixture was shielded from light with aluminum foil and stirred at room temperature for 15 minutes. 2.3μl of an aqueous solution of N-hydroxysuccinimide (NHS, concentration 0.1mg / ml) and tris(3-hydroxypropyl)phosphine (concentration 0.1mg / ml) was added to the obtained mixed aqueous solution, suspended, shielded from light with aluminum foil, and stirred at room temperature for 15 minutes. The anti-EGFR antibody solution, which had been passed through a 0.22 μm low protein adsorption syringe filter in advance, was added to the InP / ZnSe / ZnS quantum dots so that the amount was 10 equivalents, and a phosphate buffer solution (10 mM, pH 7.6) was added so that the total amount of the solution was 50 μl, and the solution was shielded from light with aluminum foil and stirred at room temperature for 2 hours. 400 μl of 0.1 mg / ml tris(3-hydroxypropyl)phosphine aqueous solution was added to the obtained test solution to make the total amount 450 μl. The obtained test solution was centrifuged at 5,000 × g and 15 minutes using a 30 kDa Amicon filter to remove EDC and NHS, and then the Amicon filter was attached in the reverse direction and centrifuged at 5,000 × g and 10 minutes to recover the anti-EGFR antibody modified solution (InP / ZnSe / ZnS quantum dot concentration 100 nM, tris(3-hydroxypropyl)phosphine 0.1 mg / ml). After ultrasonic treatment for 1 minute, the mixture was protected from light with aluminum foil and stored at 4°C. <Cell treatment> 100 μl of medium (D-MEM (Low glucose) containing 10% FBS / 1% Penicillin-Streptomycin) was added to a multi-well glass bottom dish (D141400, Matsunami Glass Industry Co., Ltd.), and the medium containing A431 cells (6 × 10 5Cells were seeded by adding 100 μl of (cells / ml). Also, to suppress evaporation of the medium, 600 μl of 1×PBS(-) was added outside the well. After seeding, the cells were incubated at 37°C and 5% CO2 for 24 hours. Then, the supernatant was removed, and 125 μl of medium with 25 μl of the anti-EGFR antibody-modified solution added thereto was added to the well. Thereafter, after incubation at 37°C and 5% CO2 for 4 hours, the supernatant was removed, and the procedure of adding and removing fresh medium was repeated 3 times. Then, 200 μl of medium was added to obtain a sample for observation.

[0092] [Example 6] [Preparation of a test solution composed of InP / ZnSe / ZnS quantum dots] Up to the dispersion step, the same method as in Example 2 was used to obtain an InP / ZnSe / ZnS quantum dot aqueous dispersion. 300 μl of pure water was mixed with 150 μl of this InP / ZnSe / ZnS quantum dot aqueous dispersion, and centrifuged at 5,000×g for 15 minutes using a 30 kDa Amicon filter. Thereafter, the Amicon filter was mounted in the reverse direction and centrifuged again at 5,000×g for 10 minutes to recover the InP / ZnSe / ZnS quantum dot aqueous dispersion. 66.11 μl of pure water was mixed with 13.89 μl of the recovered InP / ZnSe / ZnS quantum dot aqueous dispersion, shielded from light with aluminum foil, and incubated at 4°C for 24 hours to obtain a solution composed of an InP / ZnSe / ZnS quantum dot aqueous dispersion (InP / ZnSe / ZnS quantum dot concentration: 5 μM). This solution was ultrasonically treated while being suspended, passed through a 0.22 μm syringe filter to remove aggregated quantum dots, and then 29.92 μl of pure water was added to 20.08 μl of this solution to obtain a test solution composed of InP / ZnSe / ZnS quantum dots (InP / ZnSe / ZnS quantum dot concentration: 1 μM). [Antibody modification] 5 μl of the obtained test solution was sonicated for 1 minute, and 3.84 μl of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC, concentration 0.1 mg / ml) aqueous solution and 8.86 μl of pure water were mixed thereto, and the mixture was shielded from light with aluminum foil and stirred at room temperature for 15 minutes. 2.3 μl of N-hydroxysuccinimide (NHS, concentration 0.1 mg / ml) aqueous solution was added to the obtained test solution, suspended, shielded from light with aluminum foil, and stirred at room temperature for 15 minutes. An anti-EGFR antibody solution that had been passed through a 0.22 μm low protein adsorption syringe filter in advance was added so that it was 10 equivalents to the InP / ZnSe / ZnS quantum dots, and a phosphate buffer solution (10 mM, pH 7.6) was added so that the total amount of the solution was 50 μl, and the mixture was shielded from light with aluminum foil and stirred at room temperature for 2 hours. 400 μl of pure water was added to the obtained test solution to make the total amount 450 μl. The obtained test solution was centrifuged at 5,000×g for 15 minutes using a 30 kDa Amicon filter to remove EDC and NHS, and then the Amicon filter was attached in the reverse direction and centrifuged at 5,000×g for 10 minutes to recover an anti-EGFR antibody modified solution (InP / ZnSe / ZnS quantum dot concentration 100 nM). After ultrasonic treatment for 1 minute, tris(3-hydroxypropyl)phosphine was added to a final concentration of 0.1 mg / ml, and then suspended to obtain an anti-EGFR antibody modified solution (InP / ZnSe / ZnS quantum dot concentration 100 nM, tris(3-hydroxypropyl)phosphine 0.1 mg / ml). The obtained anti-EGFR antibody modified solution was protected from light with aluminum foil and stored at 4°C. <Cell treatment> 100 μl of medium (D-MEM (Low glucose) containing 10% FBS / 1% Penicillin-Streptomycin) was added to a multi-well glass bottom dish (D141400, Matsunami Glass Industry Co., Ltd.), and the medium containing A431 cells (6 × 10 5The cells were seeded by adding 100 μl of 1× EGFR antibody modification solution (cells / ml) to the well. In addition, 600 μl of 1× PBS(-) was added to the outside of the well to suppress evaporation of the medium. After seeding, the wells were incubated at 37°C and 5% CO2 for 24 hours, after which the supernatant was removed and 125 μl of the medium was added with 25 μl of the anti-EGFR antibody modification solution to add to the well. After incubation at 37°C and 5% CO2 for 4 hours, the supernatant was removed and fresh medium was added and removed three times, after which 200 μl of medium was added to obtain a sample for observation.

[0093] [Comparative Example 7] A sample for observation was obtained by carrying out the same procedure as in Example 6, except that in the <antibody modification> of Example 13, tris(3-hydroxypropyl)phosphine was not added.

[0094] [Rating 3] (1) Observation with a confocal laser microscope For Example 5, Example 6 and Comparative Example 7, the fluorescence intensity was observed by a confocal laser microscope. Using a confocal laser microscope (Carl Zeiss, LSM800), the anti-EGFR antibody modified InP / ZnSe / ZnS quantum dots were excited with a 405 nm laser and observed with a 20x objective lens under the following observation conditions. The results are shown in Figure 4. [Observation conditions] Laser power 2% Detector power 850V Scan speed 5 Averaging 16 Threshold 50 (2) Luminance analysis For Example 5, Example 6 and Comparative Example 7, the luminance was analyzed. The image analysis software ImageJ (https: / / imagej.nih.gov / ij / ) was used to calculate the average brightness within the area where cells were present. The results are shown in Table 3.

[0095] [Table 3]

[0096] From the results of the luminance analysis shown in Table 3, Examples 5 and 6 containing a specific phosphine compound showed similarly high average luminance regardless of the timing of addition of the additive. On the other hand, the average luminance of Comparative Example 7 was low. Also, as shown in FIG. 4, it can be seen that Examples 5 and 6 showed high luminance in the images observed by a confocal laser microscope.

Claims

1. A quantum dot dispersion for biomedical applications comprising a water-soluble quantum dot, a phosphine compound represented by the following formula (1), (2), (3), (4) or (5), and water. 【Chemistry 1】 (In the formula, each R independently represents a hydroxyalkyl group, an aminoalkyl group, a mercaptoalkyl group, or a cyanoalkyl group having 1 to 8 carbon atoms.)

2. The quantum dot dispersion for biomedical applications according to claim 1, wherein R is a hydroxyalkyl group having 1 to 5 carbon atoms.

3. 2. The quantum dot dispersion for biomedical applications according to claim 1, wherein the phosphine compound is disposed in close proximity to the water-soluble quantum dots.

4. The water-soluble quantum dots are those in which a hydrophilic ligand selected from thiols, amine-containing thiols, amines, alcohols, thiol-containing alcohols, phosphonic acids, carboxylic acids, and thiol-containing carboxylic acids is coordinated to the surface of the quantum dots. Quantum dot dispersion for biomedical applications according to claim 1.

5. A ligand exchange step of adding hydrophilic ligands to the hydrophobic quantum dot dispersion to exchange the ligands bound to the surface of the quantum dots with hydrophilic ligands to obtain water-soluble quantum dots; and A dispersion step of dispersing the water-soluble quantum dots in water to obtain a dispersion of the water-soluble quantum dots, The method for producing a quantum dot dispersion for biomedical applications according to any one of claims 1 to 4, wherein a phosphine compound represented by the formula (1), (2), (3), (4) or (5) is added to the dispersion of the water-soluble quantum dots.

6. A composite quantum dot for biomedical applications comprising a water-soluble quantum dot and a phosphine compound represented by the following formula (1), (2), (3), (4) or (5): 【Chemistry 2】 (In the formula, each R independently represents a hydroxyalkyl group, an aminoalkyl group, a mercaptoalkyl group, or a cyanoalkyl group having 1 to 8 carbon atoms.)

7. The composite quantum dot for biomedical applications according to claim 6, wherein R is a hydroxyalkyl group having 1 to 5 carbon atoms.

8. The water-soluble quantum dots are those having ligands selected from thiols, amine-containing thiols, amines, alcohols, thiol-containing alcohols, phosphonic acids, carboxylic acids, and thiol-containing carboxylic acids coordinated to the surface of the quantum dots.

Images