JP5108568B2 - Nanomolecules capable of binding biomolecules and method for producing the same - Google Patents

Description

  The present invention relates to a biomolecule-bondable and water-soluble nanoparticle and a method for producing the same.

  In recent years, various technologies for detecting interactions between substances (hereinafter referred to as “sensing technologies”) have been developed and put into practical use due to advances in technologies such as biotechnology and nanotechnology. By using this sensing technology, it is possible to detect a very small amount of a substance present in the body or generated in the body with high sensitivity. As a result, it can be widely used in diagnosis and prediction of human health, drug discovery, and other fields. Examples of target substances to be detected by the sensing technology include nucleic acids such as genomic DNA and RNA, proteins, peptides, sugars, lipids, hormones, and the like. Currently, the mainstream method is to directly bind the labeling substance such as a fluorescent dye to the analyte to be examined, and determine the presence or absence of interaction between the analyte and the immobilized substance on the substrate from the fluorescence signal. .

  For example, a DNA chip in which a large number of probe nucleic acids are immobilized on a substrate is known as a means for detecting nucleic acids. A labeled nucleic acid extracted from a specimen or a replica thereof is reacted with a probe nucleic acid, hybridized and immobilized on a substrate. The signal of the label on the substrate is detected to detect the presence or absence of interaction between substances. Alternatively, there is a method in which a signal is detected by hybridizing unlabeled nucleic acid or a replica thereof and immobilizing it on a substrate, and then binding the nucleic acid by a chemical reaction or the like.

  Moreover, when detecting protein, it fixes on a base material using protein-protein interaction and nucleic acid-protein interaction. Thereby, a specific protein can be sensed.

  Currently, fluorescent dyes made of organic compounds are generally used as labels. However, when a very small amount of sample is used, detection sensitivity is insufficient, and it is difficult to clearly distinguish noise from weak signals. There was a problem.

  In order to overcome this problem, many attempts have been made to use high-luminance and highly stable semiconductor nanoparticles as a label for biomolecule detection (for example, Patent Document 1). A semiconductor nanoparticle (hereinafter sometimes abbreviated as “nanoparticle”) is a particle made of a semiconductor of nanometer order and emits fluorescence corresponding to band gap energy. Since it is an inorganic semiconductor, it is known to have high fluorescence characteristics such as being more stable than organic dyes. In addition, as the particles become smaller, the state of electrons in the material changes, and the phenomenon of the quantum size effect that absorbs and emits light of shorter wavelengths allows the emission of various wavelengths by changing the particle diameter. Can be obtained.

  Semiconductor nanoparticles are often synthesized in the organic phase, and many nanoparticles are hydrophobic. In order to use as a label for detecting the interaction of biomolecules, it is necessary to solubilize the semiconductor nanoparticles and introduce functional groups capable of binding to biomolecules on the surface.

  By coating the surface of the nanoparticle with a highly hydrophilic ligand, it becomes possible to make the nanoparticle water-soluble. However, depending on the compound adsorbed on the core surface of the nanoparticle, the fluorescence may be quenched. In order to suppress this quenching, a structure (core-shell structure) in which the core of the nanoparticles is coated with ZnS, ZnO, or the like is performed. However, with this core-shell structure, the particle size of the nanoparticles becomes very large compared to nucleic acids and proteins, which is not preferred as a label for detecting these.

  As the semiconductor nanoparticles, cadmium (Cd) -based nanoparticles (CdSe, CdTe, etc.) having extremely high luminance characteristics are cited as representatives, and are most commonly used. However, high toxicity is a major obstacle to commercialization. Therefore, in recent years, low toxicity semiconductor nanoparticles have been developed. As an example of a low toxicity nanoparticle, a nanoparticle that is made of zinc, a sulfide or an oxide containing a Group 11 element of the periodic table and a Group 13 element of the periodic table, and emits light at room temperature is known (Patent Document 2 and 3).

International Publication No. 00/017642 Pamphlet International Publication No. 06/009124 Pamphlet International Publication No. 07/026746 Pamphlet

  Surface modification is required for water-solubilization of nanoparticles and introduction of reactive groups with biomolecules, but the problem is that the emission intensity is significantly reduced by binding the surface-modified ligand to the core of the nanoparticles. It was. An object of the present invention is to provide nanoparticles that are water-soluble while maintaining emission intensity.

  As a result of intensive studies to solve the above problems, the present inventors have used amphiphilic substances having functional groups that react with the surface of the nucleus and functional groups that react with biomolecules using low-toxic nanoparticles. It has been found that by using a compound as a ligand, it becomes possible to water-solubilize nanoparticles having a reactive group with a biomolecule while maintaining the luminous ability, and the present invention has been completed. That is, the present invention is as follows.

That is, the present invention provides the following [1] to [15].
[1] Sulfide or oxide containing zinc, Group 11 element of Periodic Table and Group 13 element of Periodic Table, or containing Group 11 Element of Periodic Table and Group 13 of Periodic Table It is a nanoparticle containing a substance as a component, and on the surface of the nanoparticle,
General formula (1)
_{^{(R 1 -X) p - (}} Y) q - (OCH 2 CH 2) m -R 2
Wherein R ¹ is a nitrogen-containing functional group or sulfur-containing functional group, R ² is a functional group containing a reactive group capable of binding to biomolecules such as nucleic acids and proteins, and X is a heteroatom. And Y represents a divalent linking group, p represents 1 or 2, q represents 0 or 1, and m represents an integer of 1 to 6, respectively.
Nanoparticles comprising Compound 1 represented by
[2] The nanoparticle according to [1], wherein R ^{1 in the} general formula (1) is a substituent containing at least one of the group consisting of a mercapto group, a dithiocarboxyl group, and an amino group.
[3] R ^{2 in the} general formula (1) is a substituent containing at least one of the group consisting of a carboxyl group, a carboxyl group derivative, a biotinyl group, and a functional group containing a biotinyl group, either [1] or [2] Nanoparticles according to crab.
[4] The nanoparticle according to any one of [1] to [3], wherein X in the general formula (1) is a saturated aliphatic hydrocarbon group which may have an amide bond.
[5] Any one of [1] to [3], wherein X in the general formula (1) is — (CH ₂ ) ₁₁ — or — (CH ₂ ) ₁₀ —CONH— (CH ₂ ) ₂ —. Nanoparticles.
[6] The nanoparticle according to any one of [1] to [4], wherein m in the general formula (1) is 3 to 6.
[7] The nanoparticle according to any one of [1] to [6], wherein the Group 13 element of the periodic table is indium.
[8] The nanoparticle according to any one of [1] to [7], wherein the Group 11 element of the periodic table is silver.
[9] On the surface of the nanoparticles,
General formula (2)
R ³ — (CH ₂ ) n —R ⁴
(In the formula, R ³ represents a nitrogen-containing functional group or a sulfur-containing functional group, R ⁴ represents an ionic functional group, and n represents an integer of 1 to 11, respectively.)
The nanoparticle in any one of [1] to [8] containing the compound 2 represented by these.
[10] The nano according to any one of [1] to [9], wherein R ³ in the general formula (2) is selected from at least one member selected from the group consisting of a mercapto group, a pyridylthio group, a dithiocarboxyl group, and an amino group. particle.
[11] The nanoparticle according to any one of [1] to [10], wherein n in the general formula (2) is 2 or 3, and R ⁴ is SO ₃ ⁻ or N (CH ₃ ) ₃ ⁺ .
[12] A sulfide or oxide containing zinc, Group 11 element of Periodic Table and Group 13 element of Periodic Table, or a sulfide containing Group 11 Element of Periodic Table and Group 13 Element of Periodic Table or To nanoparticles that contain oxides,
General formula (1)
_{^{(R 1 -X) p - (}} Y) q - (OCH 2 CH 2) m-R 2
Wherein R ¹ is a nitrogen-containing functional group or sulfur-containing functional group, R ² is a functional group containing a reactive group capable of binding to biomolecules such as nucleic acids and proteins, and X is a heteroatom. And Y represents a divalent linking group, p represents 1 or 2, q represents 0 or 1, and m represents an integer of 1 to 6, respectively.
The manufacturing method of the nanoparticle including the process of making the compound 1 represented by these react.
[13] In the method for producing nanoparticles according to [12],
General formula (2)
R ³ — (CH ₂ ) n —R ⁴
(Wherein R ³ represents a nitrogen-containing functional group or a sulfur-containing functional group, R ⁴ represents an ionic functional group, and n represents an integer of 1 to 10, respectively),
A method for producing nanoparticles comprising:
[14] The method for producing nanoparticles according to [13], wherein n in the general formula (2) is 2 or 3, and R ₄ is SO ₃ ^— or N (CH ₃ ) ₃ ⁺ .

  A nanoparticle or zinc containing a sulfide or oxide containing a Group 11 element of the periodic table and a Group 13 element of the periodic table as a component, or a sulfide or oxide containing a Group 11 element of the periodic table and a Group 13 element of the periodic table By using the nanoparticles as a component and reacting the surface of the nanoparticles with a surface-modifying ligand having a functional group capable of reacting with biomolecules, while maintaining the luminous ability, It is possible to introduce a functional group capable of reacting. Furthermore, the water solubility of the nanoparticles can be further improved by allowing a highly hydrophilic functional group to coexist on the surface of the nanoparticles.

  Below, the suitable form for implementing this invention is demonstrated.

[Production of nanoparticles]
Nanoparticles serving as the core of the nanoparticles of the present invention are nanoparticles comprising a sulfide or oxide containing a Group 11 element and a Group 13 element of the periodic table, or zinc, Group 11 elements of the periodic table And a sulfide or oxide containing a Group 13 element of the periodic table as a component.

  Although it does not specifically limit as a periodic table 11 group element, Copper (Cu), silver (Ag), and gold (Au) are mentioned, Among these, Cu and Ag are preferable and Ag is especially preferable. Although it does not specifically limit as a periodic table group 13 element, Gallium (Ga), indium (In), and thallium (Tl) are mentioned, Among these, Ga and In are preferable and In is especially preferable.

  In the present invention, nanoparticles containing sulfides or oxides containing Group 11 elements of Periodic Table and Group 13 elements of Periodic Tables are used as nanoparticles (InAgS nanoparticles). Is preferred. Nanoparticles containing zinc, sulfides containing Group 11 elements and periodic table Group 13 elements as components are preferably nanoparticles containing Zn, indium, and silver sulfide (ZnInAgS nanoparticles). In the case of nanoparticles containing sulfide as a component, a small amount or a small amount of an oxide of the same element may be contained as long as the effects of the present invention are not impaired. In addition, the nanoparticle of the present invention includes a ligand for a minute amount or a small amount of impurities that may be mixed in the raw material of the core nanoparticle and the production stage, for example, a metal element, to the extent that the effects of the present invention are not impaired. Ingredients such as decomposition products may be contained.

  Preparation of nanoparticles containing the above-mentioned periodic table group 11 element and periodic table group 13 element as a component, and nanoparticles containing zinc as a component in the nanoparticles, It can be based on a method of mixing a raw material salt of a plurality of kinds of elements belonging to the ligand and a ligand having sulfur as a coordination element or a ligand having oxygen as a coordination element. Alternatively, a complex is prepared by mixing a raw material salt of zinc and a plurality of types of elements belonging to each group and a ligand having sulfur as a coordination element or a ligand having oxygen as a coordination element, and the complex It is possible to produce a thermal decomposition product by heating and heating the thermal decomposition product together with a fat-soluble compound, thereby obtaining nanoparticles coated with the fat-soluble compound.

  The conditions for thermally decomposing the complex vary depending on the elements used, but it is usually carried out in the range of 100 to 300 ° C, preferably in the range of 150 to 200 ° C. Moreover, although reaction time changes with reaction temperature, it is preferable to set in the range of 1 to 60 minutes.

  Further, the conditions for heating the pyrolysis product together with the fat-soluble compound are preferably in the range of 150 to 200 ° C., although depending on the elements used. Moreover, although reaction time changes with reaction temperature, it is preferable to set in the range of 1 to 60 minutes.

  The size of the nanoparticle is preferably 100 nm or less, more preferably 50 nm or less in consideration of the appearance of a quantum size effect, and further preferably 20 nm or less in consideration of the reaction with biomolecules.

  The fat-soluble compound only needs to be capable of binding to the particle surface containing the sulfide or oxide as a component. The bonding mode at that time is not particularly limited, and examples thereof include a covalent bond, an ionic bond, a coordination bond, a hydrogen bond, and a van der Waals bond. Specific examples of the fat-soluble compound include a sulfur-containing compound having a hydrocarbon group having 4 to 20 carbon atoms, a nitrogen-containing compound having a hydrocarbon group having 4 to 20 carbon atoms, and a hydrocarbon group having 4 to 20 carbon atoms. Examples thereof include oxygen-containing compounds. As hydrocarbon groups, saturated aliphatic hydrocarbon groups such as n-butyl group, isobutyl group, n-pentyl group, n-hexyl group, octyl group, decyl group, dodecyl group, hexadecyl group, octadecyl group; oleyl group, etc. An unsaturated aliphatic hydrocarbon group; an alicyclic hydrocarbon group such as a cyclopentyl group and a cyclohexyl group; and an aromatic hydrocarbon group such as a phenyl group, a benzyl group, a naphthyl group, and a naphthylmethyl group. Of these, saturated aliphatic hydrocarbon groups and unsaturated aliphatic hydrocarbon groups are preferred. Examples of the nitrogen-containing compound include amines and amides. Examples of the sulfur-containing compound include a group having a disulfide bond such as a mercapto group and an alkyldithio group, a group having a sulfide bond such as an alkylthio group, a pyridylthio group and a dithiocarboxyl group. Examples of the oxygen-containing compound include fatty acids. For example, alkylamines such as butylamine and hexylamine, and alkenylamines such as oleylamine are preferred.

  The ligand having sulfur as a coordination element is not particularly limited. For example, β-dithiones such as 2,4-pentanedithione; 1,2-bis (trifluoromethyl) ethylene-1, Dithiols such as 2-dithiol; and diethyldithiocarbamate. The ligand having oxygen as a coordination element is not particularly limited, and examples thereof include β-diketones such as acetylacetone and hexafluoroacetylacetone; and tropolone.

[Surface modifying ligand]
The surface-modifying ligand in the present invention means a ligand that modifies the surface of the nanoparticle, and typically represents an amphiphilic ligand having a reactive group with a biomolecule and a ligand for improving water solubility. . In the present invention, by reacting a nanoparticle with a surface-modified ligand having a functional group capable of reacting with a biomolecule, the functional group capable of reacting with the water molecule and reacting with the biomolecule while maintaining the luminous ability of the nanoparticle. Can be introduced. Furthermore, by introducing a ligand for improving the water solubility in addition to this, it is possible to further improve the water solubility.

(Amphiphilic ligands having reactive groups with biomolecules)
As an amphiphilic ligand having a reactive group with a biomolecule,
General formula (1)
_{^{(R 1 -X) p - (}} Y) q - (OCH 2 CH 2) m -R 2
The compound 1 represented by these can be mentioned.

In the general formula (1), R ¹ represents a nitrogen-containing functional group or a sulfur-containing functional group. These functional groups can react with the nanoparticle surface and are presumed to have a role of stably binding a ligand to the particle surface.
The nitrogen-containing functional group for R ¹ may be, for example, a functional group containing a nitrogen atom, and examples thereof include an amino group, an imino group, an amide group, and an imide group. The sulfur-containing functional group may be any functional group containing sulfur, and examples thereof include a mercapto group, a group having a disulfide bond, a group having a sulfide bond, a pyridylthio group, and a dithiocarboxyl group. Among these, a sulfur-containing functional group is preferably used in consideration of stability to particles.
Preferable examples of R ¹ include a mercapto group, an alkyldithio group, an alkylthio group, a pyridylthio group, a dithiocarboxyl group, and an amino group, and among these, a mercapto group is particularly preferable.

Moreover, in General formula (1), X shows the hydrocarbon group which may contain the hetero atom. Since the surface of the nanoparticles is stably coated with a fat-soluble compound, it is desirable that a fat-soluble group is present in the surface-modified ligand. Examples of the fat-soluble group include a saturated aliphatic hydrocarbon group, an unsaturated aliphatic hydrocarbon group, an alicyclic hydrocarbon group, and an aromatic hydrocarbon group, and among them, a saturated aliphatic hydrocarbon group is preferable. Examples of the hetero atom that may be contained as X include an oxygen atom, a nitrogen atom, and a sulfur atom, and a nitrogen atom and an oxygen atom are most preferable. The hetero atom is preferably contained as an amide bond (—CO—NH—), and the number of amide bonds is preferably 0 or 1. Examples of such X include saturated aliphatic hydrocarbon groups containing 0 or 1 amide bond. Carbon number of a hydrocarbon group is 1-24 normally, Preferably it is 1-18, More preferably, it is 6-15.
Preferred examples of X include — (CH ₂ ) ₂ —, — (CH ₂ ) ₆ —, — (CH ₂ ) ₁₁ —, and — (CH ₂ ) ₁₀ —CONH— (CH ₂ ) ₃ —.

  Y in the general formula (1) represents a divalent linking group. The linking group is preferably a hydrocarbon group having an ether bond and an aromatic ring.

A typical example of Y is the following general formula (Y-1).

R1 in the general formula (Y-1) represents an integer of 1 to 10, preferably 1 to 5, and most preferably 1. R2 in the general formula (Y-1) represents an integer of 0 to 10, preferably 0 to 5, and most preferably 0 or 1. R ₅ in the general formula (Y-1) represents a hydrocarbon group, preferably a saturated aliphatic hydrocarbon group (straight chain hydrocarbon group), and most preferably —C (CH ₃ ) ₂ —.

The most preferred specific examples of Y are shown in the following formulas (Y-2) and (Y-3).

  In general formula (1), p represents an integer of 1 or 2. Q represents an integer of 0 or 1. When p is 1, q is preferably 0, and when p is 2, q is preferably 1.

Further, this surface-modifying ligand contains an ethylene glycol group (OCH ₂ CH ₂ ). The ethylene glycol group is presumed to be useful for water-solubilization of nanoparticles as a hydrophilic group. The ethylene glycol group can be replaced with sulfonic acid and sulfonate, carboxyl group or carboxylate, quaternary amine or quaternary amine salt.
In general formula (1), m represents the number of repeating ethylene glycol groups, and m is an integer of 1 to 6. Preferably it is an integer of 3-6.

R ² in the general formula (1) represents a functional group containing a reactive group capable of binding to a biomolecule. The definition of the biomolecule is as described later. Although it does not specifically limit as a reactive group which has a binding ability with respect to a biomolecule, carboxyl group derivatives, such as a carboxyl group, a functional group containing a carboxyl group, and a functional group containing a derivative of a carboxyl group, an amino group, an amino group Derivatives, biotinyl groups, functional groups containing biotinyl groups and the like can be mentioned. Among these, in view of reactivity with biomolecules, a carboxyl group, a carboxyl group derivative, a biotinyl group, and a biotinyl group derivative are preferable. R ² may contain a group other than a functional group containing a reactive group capable of binding to a biomolecule, and examples thereof include an amide bond, an oxygen atom, and a hydrocarbon chain.

を挙げることができる。また、ビオチニル基を含む官能基としては、式（Ｒ²−２）

（ビオチニルアミノ基）を挙げることができる。 Specific examples of R ² include —OCH ₂ COOH as a functional group containing a carboxyl group. As a functional group containing a derivative of a carboxyl group, a functional group containing a succinimide ester, that is, the formula (R ² -1)

Can be mentioned. As the functional group containing a biotinyl group, the formula (R ² -2)

(Biotinylamino group).

Examples of the amphiphilic ligand having a reactive group with a biomolecule as described above include those represented by the following formulas (1-1) to (1-6).

(Ligand for improving water solubility; water-soluble ligand)
As a ligand for improving water solubility,
General formula (2)
R ³ — (CH ₂ ) _n —R ⁴
The compound 2 represented by these can be mentioned.

In the general formula (2), R ³ is preferably a functional group capable of reacting with the nanoparticle surface, and examples thereof include a nitrogen-containing functional group and a sulfur-containing functional group. Examples of nitrogen-containing functional groups include amide groups and imide groups. Among these, a sulfur-containing functional group is preferably used in consideration of stability to particles. Examples of sulfur-containing functional groups include mercapto groups, disulfide groups, sulfide groups, pyridyl sulfide groups, dithiocarboxyl groups, and the like. Of these, a mercapto group is preferable.

In the general formula (2), R ⁴ represents an ionic functional group. Although there is no restriction | limiting in particular as an ionic functional group, What is necessary is just a structure which has an ion pair in a structure and ionizes in water. Highly hydrophilic functional groups are desirable, and examples thereof include sulfonic acids and sulfonates, carboxyl groups and carboxylates, quaternary amines, and quaternary amine salts. Of these, sulfonates, quaternary amines and quaternary amine salts are preferred, and sulfonates are particularly preferred.
In General formula (2), n shows the integer of 1-18, it is preferable that it is 1-10, and it is especially preferable that it is 2 or 3.

Examples of the water-soluble ligand as described above include mercaptoethanesulfonic acid represented by the following formula (2-1) and mercaptopropanesulfonic acid represented by the formula (2-2).
Formula (2-1): Mercaptoethanesulfonic acid (MES)
_{HS- (CH 2) 2 -SO 3} - Na +
Formula (2-2): Mercaptopropanesulfonic acid (MPS)
_{HS- (CH 2) 3 -SO 3} - Na +

[Introduction of surface-modifying ligands on the surface of nanoparticles]
The method for introducing the surface-modified ligand into the nanoparticle is not particularly limited, but the surface-modified ligand may be added at the time of nanoparticle preparation, or it may be heated together with a thermally decomposed purified product of the above-mentioned complex or other fat-soluble compound. May be. Alternatively, after nanoparticle preparation, it may be mixed with a surface modifying ligand and introduced by surface substitution with a fat-soluble compound bonded to the surface. For example, as shown in step (A) of FIG. 1, an amphiphilic ligand having a reactive group with a biomolecule and a nanoparticle are mixed and stirred to introduce the ligand into the nanoparticle surface by a substitution reaction. It becomes possible. Next, as shown in step (B) of FIG. 1, the water solubility of the nanoparticles can be improved by adding and stirring the water-solubilizing ligand. The amphiphilic ligand having a reactive group with a biomolecule and the water-solubilizing ligand may be introduced by sequential reaction, or they are reacted using a solution in which these are mixed in advance, and the steps (A) and (B) are simultaneously performed. It may be what you do. In the case of sequential reaction, as a preferred form, when the affinity of the surface modifying ligand is weak or comparable to the affinity to the particle surface indicated by the pre-existing lipid-soluble compound on the surface of the nanoparticle, Add a large excess. It is better that the affinity of the surface-modifying ligand is stronger than the affinity of the pre-existing fat-soluble compound for the particle surface.

[Binding biomolecules to nanoparticles]
Biomolecules can be bound to the nanoparticles of the present invention. Examples of biomolecules include nucleic acids such as genomic DNA and RNA, proteins, peptides, sugars, lipids, hormones, etc. Among them, typical ones are nucleic acids and proteins.

  When the biomolecule is a nucleic acid, a reactive group is introduced into a part of the nucleic acid, and the reactive group and the functional group of the nanoparticle are reacted to enable binding to the nucleic acid. For example, a double-stranded nucleic acid having a terminal amino group can be prepared by performing a polymerase chain reaction (PCR) reaction using an oligonucleotide having a terminal amino group as a primer. By allowing the terminal amino group of the nucleic acid to undergo a condensation reaction with the carboxyl group introduced into the nanoparticle, the nanoparticle can be bound to the nucleic acid. The condensing agent for this condensation reaction is not particularly limited, and various condensing agents such as dicyclohexylcarbodiimide, N-ethyl-5-phenylisoxazolium-3'-sulfonate are used. Among these, 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide (EDC) is less toxic and relatively easy to remove from the reaction system. It is one of the most effective condensing agents for the reaction.

  When the biomolecule is a protein, the nanoparticle and the protein can be bound by a condensation reaction between the amino group of the lysine residue in the protein and the carboxyl group in the nanoparticle.

When the active ester contained in the carboxyl group derivative is introduced into the nanoparticles, the condensation reaction with the amino group can be performed without a condensing agent. Examples of this active ester include N-hydroxysuccinimide, p-nitrophenyl, pentafluorophenyl, and N-hydroxybenzotriazolyl.
Furthermore, when nanoparticles having a biotinyl group are used, the avidin molecule can be immobilized by the avidin-biotin interaction. In addition, since avidin has four binding sites in one molecule, for example, by first binding avidin to a nucleic acid into which a biotinyl group has been introduced, and then allowing nanoparticles having a biotinyl group to act on the unbound site of avidin The nucleic acid can be labeled with nanoparticles.

  EXAMPLES Examples will be given below to describe the present invention in detail, but the present invention is not limited to the examples.

Examples 1-1 to 1-3 and Reference Examples 1-4 to 1-6
(Preparation of InAgS nanoparticles)
Dithiocarbamate (1.126 g), silver nitrate (0.2124 g) and indium nitrate trihydrate (0.4436 g) are added to pure water in a test tube having an inner diameter of 16 mm, warmed to 30 ° C., and heated with a micro stirrer for 30 minutes. Stir. After stirring, the mixture was centrifuged with a tabletop small centrifuge (KUBOTA 2420, manufactured by Kubota) at a rotational speed of 2500 rpm, the supernatant was removed, washed with pure water, and this centrifugation and pure water washing operation was repeated four times. Thereafter, the operation of washing with methanol and removing the supernatant by centrifugation was repeated twice, and the precipitate was dried overnight in a desiccator to obtain a metal complex. Next, 50 mg of this metal complex powder was taken, put into an 18 mm long test tube, capped with a septum cap, and the inside of the test tube was replaced with argon three times. The test tube was heated in an oil bath at 180 ° C. for 3 minutes. Next, 3 ml of liquid oleylamine was added to the test tube and purged with argon. The test tube was heated in an oil bath at 180 ° C. for 3 minutes and allowed to cool for 30 minutes. The resulting suspension was centrifuged and the supernatant was collected and filtered through a 0.22 μm membrane filter. 3 ml of methanol was added to the filtrate to suspend the solution, and the supernatant was collected after centrifugation, and the washing step with methanol was repeated three times. Thereafter, 1 ml of chloroform was added to dissolve the nanoparticles.

(Introduction of surface-modified ligands into nanoparticles)
Six types of surface-modified ligands, monothiol alkane ethylene glycol (terminal carboxylated SPT0012a; ligand 1 (Example 1-1) , terminal succinimide SPT0012c; ligand 2 (Example 1-2) , terminal biotinylated SPT0012d; ligand 3 (Example 1-3)) , all manufactured by Senso Pass Technology, USA, dithiol rigid rod monodisperse ethylene glycol (one terminal carboxylated benzene ring SPT0014; ligand 4 (Reference Example 1-4)) , US Senso Manufactured by Path Technology), short-chain alkane ethylene glycol (Thio-dPEG ₄ ^TM- acid; Ligand 5 (Reference Example 1-5) , manufactured by Quanta Biodesign), dithiol rigid rod monodisperse ethylene glycol ( A chloroform solution (concentration 200 μM) was prepared for each of the terminal carboxylated benzene rings SPT0015; Ligand 6 (Reference Example 1-6)) , manufactured by Sensopath Technology, USA (see Table 1 for the structure of each ligand) . To 6 sample bottles and 200 μl each of the nanoparticle chloroform solution, 100 μl of each of the above 6 kinds of ligand solutions was added, and stirred for 16 hours with a micro stirrer.

  Subsequently, 400 μl of propanol and 400 μl of pure water were added and stirred for 16 hours. Thereafter, the reaction solution was transferred to an Eppendorf tube, centrifuged at 12,000 rpm for 12 minutes, the supernatant was removed, the residue was washed with chloroform, and dissolved in 2000 μl of pure water.

(Measurement of absorbance of water-soluble nanoparticles)
In order to confirm the water-solubilization of the nanoparticles, the light absorption of the aqueous nanoparticle solution was measured. The measurement was performed using an ultraviolet / visible spectrophotometer (UV-1650PC, manufactured by Shimadzu Corporation) using a cell having an optical path length of 1 cm. Table 2 shows the results of measuring the absorbance at a wavelength of 543 nm. In all six types of ligands 1-6, absorption at a wavelength of 543 nm due to the nanoparticles was observed, indicating that the nanoparticles can be water-solubilized in all six types.

(Introduction of water-soluble ligand to nanoparticles)
Nanoparticles into which six types of ligands 1 to 6 were introduced were prepared in the same procedure as described above. Next, 400 μl of propanol and 400 μl of 1M mercaptoethanesulfonic acid sodium salt (MES, manufactured by Tokyo Chemical Industry Co., Ltd., see Table 3 for the structure of MES) were added and stirred for 16 hours. Thereafter, the reaction solution was transferred to an Eppendorf tube, centrifuged using a tabletop centrifuge (CFM-200, manufactured by IWAKI) for 12 minutes at a rotational speed of 12000 rpm, the supernatant was removed, the residue was washed with chloroform, and 2000 μl of residue was washed. Dissolved in pure water. The absorbance of these nanoparticle aqueous solutions at a wavelength of 543 nm was measured with an ultraviolet / visible spectrophotometer. The results are shown in Table 4.

  As a result, as is apparent from Table 4, the absorbance at a wavelength of 543 nm due to the water-solubilized nanoparticles was increased by further surface modification with MES in all six types of ligands. From this, it can be seen that the water solubility is improved by modifying the nanoparticles with a water-soluble ligand in addition to the modification with an amphiphilic ligand having a reactive group with a biomolecule.

(Measurement of fluorescence intensity of nanoparticle aqueous solution)
In order to confirm the luminous ability of the water-soluble nanoparticles, the luminescence intensity of the nanoparticles in the aqueous solution was measured. The measurement was performed using a fluorescence spectrometer (FluoroMax-3, manufactured by Horiba) using a 4 mm square cell. The slit width was 3 nm, the excitation wavelength was 543 nm, and the fluorescence intensity (emission intensity) at a wavelength of 750 nm was measured. Table 5 shows the fluorescence intensity at a wavelength of 750 nm when fluorescence measurement was performed using the nanoparticle aqueous solution after surface modification with the ligands 1 to 6. From this, it was shown that all the water-solubilized nanoparticles have a light-emitting ability. Table 6 shows fluorescence intensity at a wavelength of 750 nm after surface modification with ligands 1 to 6 and surface treatment with MES. From these results, it was shown that, when the surface is modified with an amphiphilic ligand having a reactive group with a biomolecule, the luminous ability can be retained, and further, the luminous ability is retained after the surface treatment with the water-soluble ligand. .

Examples 2-1 to 2-3 and Reference Examples 2-4 to 2-6
(Preparation of ZnInAgS nanoparticles)
Using dithiocarbamate (1.126 g), zinc nitrate hexahydrate (0.104 g), silver nitrate (0.1826 g) and indium nitrate trihydrate (0.3815 g) in a test tube with an inner diameter of 16 mm Nanoparticles were prepared in the same procedure as in Examples 1-1 to 1-3 and Reference Examples 1-4 to 1-6 to obtain 1 ml of a nanoparticle chloroform solution.

(Introduction of surface-modified ligands into nanoparticles)
Using a chloroform solution of ZnInAgS nanoparticles, introduction of ligands 1-6 to the nanoparticle surface and pure water in the same procedure as in Examples 1-1 to 1-3 and Reference Examples 1-4 to 1-6 Water solubilization was performed. Furthermore, after water-solubilization of the nanoparticles by introducing the water-soluble ligand MES, the nanoparticles after surface modification with the ligands 1-6 in the same procedure as in Examples 1-1 to 1-3 and Reference Examples 1-4 to 1-6 And the absorbance of the aqueous nanoparticle after surface modification with ligands 1 to 6 and MES were measured for the absorbance at a wavelength of 543 nm and the emission intensity at a wavelength of 700 nm. The results of absorbance and emission intensity are shown in Table 2 and Table 5, respectively. From these results, it was shown that even when ZnInAgS nanoparticles were used, all of the ligands 1 to 6 could be water-solubilized while maintaining their luminous ability. Further, Table 4 shows the absorbance at a wavelength of 543 nm and Table 6 shows the fluorescence intensity at a wavelength of 700 nm of an aqueous solution obtained by surface modification with a ligand 1 to 6 and surface treatment with a water-soluble ligand and then dissolving nanoparticles in pure water. From these results, when surface modification with an amphipathic ligand having a reactive group with a biomolecule, it is possible to water-solubilize while maintaining the light-emitting ability, and further, while maintaining the light-emitting ability after surface modification with a water-soluble ligand. It was shown that the water-solubilization efficiency of the nanoparticles was improved by water-solubilization and surface modification with a water-solubilizing ligand.

Example 3
Mercaptopropanesulfonic acid (MPS, manufactured by Tokyo Chemical Industry Co., Ltd., see Table 3) was used as a ligand for improving water solubility. 5.346 g of MPS was dissolved in 30 ml of pure water.

It was performed introducing ZnAgInS nanoparticles produced and ligand 1 in the same manner as in Example 2 -1. Next, 400 μl of propanol and 400 μl of 1M MPS aqueous solution were added and stirred for 16 hours. Thereafter, the reaction solution was transferred to an Eppendorf tube, centrifuged at 12,000 rpm for 12 minutes, the supernatant was removed, the residue was washed with chloroform, and dissolved in 2000 μl of pure water. Table 7 shows the absorbance of this aqueous nanoparticle solution at a wavelength of 543 nm and the emission intensity at 700 nm when excited at a wavelength of 543 nm. From this, almost the same absorbance and emission intensity as those obtained when MES was used as the water-soluble ligand were obtained. (Comparison with Table 4 and Table 6)

Comparative Example 1
0.0131 g of 11-mercaptoundecanoic acid [MUA] (HS— (CH ₂ ) ₁₀ —COOH, Ligand 7, manufactured by Sigma) was dissolved in 300 ml of chloroform to a concentration of 200 μM.

  200 μl of the nanoparticle chloroform solution was added to 100 μl of the ligand 7 chloroform solution, and the mixture was stirred with a micro stirrer for 16 hours. Next, 400 μl of propanol and 400 μl of pure water were added and stirred for 2 hours. Thereafter, the reaction solution was transferred to an Eppendorf tube, centrifuged at 12,000 rpm for 12 minutes, the supernatant was removed, the residue was washed with chloroform, and dissolved in 2000 μl of pure water. Table 8 shows the absorbance of the aqueous nanoparticle solution obtained at a wavelength of 543 nm.

  From this, it was shown that the nanoparticles coated with MUA are hardly water-soluble.

FIG. 1 is an explanatory diagram of modification of a nanoparticle surface using a surface-modifying ligand.

Claims (9)

Zinc, a sulfide or oxide containing a Group 11 element of the periodic table and a Group 13 element of the periodic table, or a sulfide or oxide containing a Group 11 element of the periodic table and Group 13 of the periodic table And on the surface of the nanoparticle,
General formula (1)
_{^{(R 1 -X) p - (}} Y) q - (OCH 2 CH 2) m -R 2
Wherein R ¹ is a substituent containing at least one of the group consisting of a mercapto group, a disulfide group, a sulfide group, a dithiocarboxyl group and an amino group, and R ² is a carboxyl group, a carboxyl group derivative, an amino group, an amino group A substituent containing at least one of the group consisting of a derivative, a biotinyl group and a functional group containing a biotinyl group, X is — (CH ₂ ) ₁₁ — or — (CH ₂ ) ₁₀ —CONH— (CH ₂ ) ₂ - a, Y is .p showing respectively a divalent linking group represents 1 or 2, q is 0 or 1, m is an integer of 1 to 6, respectively).
Nanoparticles comprising Compound 1 represented by The nanoparticle according to claim 1 , wherein m in the general formula (1) is 3-6. The nanoparticle according to claim 1 or 2 , wherein the group 13 element of the periodic table is indium. The nanoparticle according to any one of claims 1 to 3 , wherein the Group 11 element of the periodic table is silver. On the surface of the nanoparticles,
General formula (2)
R ³ — (CH ₂ ) n —R ⁴
Wherein R ³ is a substituent containing at least one of the group consisting of mercapto group, disulfide group, sulfide group, pyridyl sulfide group, dithiocarboxyl group and amino group, and R ⁴ is sulfonic acid, sulfonate, carboxyl A substituent containing at least one of the group consisting of a group, a carboxylate, a quaternary amine, and a quaternary amine salt, and n represents an integer of 1 to 10, respectively.
The nanoparticle in any one of Claim 1 to 4 containing the compound 2 represented by these. The nanoparticle according to any one of claims 1 to 5 , wherein n in the general formula (2) is 2 or 3, and R ⁴ is SO ₃ ^- or N (CH ₃ ) ₃ ⁺ . Zinc, a sulfide or oxide containing a group 11 element of the periodic table and a group 13 element of the periodic table, or a sulfide or oxide containing a group 11 element of the periodic table and a group 13 element of the periodic table To the nanoparticles as a component,
General formula (1)
_{^{(R 1 -X) p - (}} Y) q - (OCH 2 CH 2) m -R 2
Wherein R ¹ is a substituent containing at least one of the group consisting of a mercapto group, a disulfide group, a sulfide group, a dithiocarboxyl group and an amino group, and R ² is a carboxyl group, a carboxyl group derivative, an amino group, an amino group A substituent containing at least one of the group consisting of a derivative, a biotinyl group and a functional group containing a biotinyl group, X is — (CH ₂ ) ₁₁ — or — (CH ₂ ) ₁₀ —CONH— (CH ₂ ) ₂ - a, Y is .p showing respectively a divalent linking group represents 1 or 2, q is 0 or 1, m is an integer of 1 to 6, respectively).
The manufacturing method of the nanoparticle including the process of making the compound 1 represented by these react. The method for producing nanoparticles according to claim 7 , further comprising:
General formula (2)
R ³ — (CH ₂ ) _n —R ⁴
Wherein R ³ is a substituent containing at least one of the group consisting of mercapto group, disulfide group, sulfide group, pyridyl sulfide group, dithiocarboxyl group and amino group, and R ⁴ is sulfonic acid, sulfonate, carboxyl A substituent comprising at least one of the group consisting of a group, a carboxylate salt, a quaternary amine and a quaternary amine salt, wherein n represents an integer of 1 to 10, respectively)
A method for producing nanoparticles comprising: The method for producing nanoparticles according to claim 8 , wherein n in the general formula (2) is 2 or 3, and R ₄ is SO ₃ ^- or N (CH ₃ ) ₃ ⁺ .

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