JP2007112679A - Nanoparticles and production method thereof - Google Patents

Abstract

Nanoparticles having enhanced dispersibility and reactivity in aqueous media are provided.
A nanoparticle composed of a core portion and a surface organic layer covering the surface thereof, wherein the surface organic layer has an R ¹ O—L ¹ — group and a Y—L ² — group (wherein L ¹ and L ² each represent a divalent linking group satisfying a length relationship of L ¹ <L ^2. R ¹ represents an alkyl group having 1 to 3 carbon atoms or a hydrogen atom, and Y represents a reaction with a labeling substance. A nanoparticle having a surface-reactive group represented by:
[Selection figure] None

Description

  The present invention relates to a nanoparticle and a method for producing the same, and more particularly to a nanoparticle composed of a core portion and a surface organic layer covering the surface thereof and a method for producing the same.

Semiconductor nanoparticles are nanoparticles composed of inorganic, crystalline semiconductor materials and have inherent photophysics, photochemistry and non-linear optical properties resulting from quantum size effects. Therefore, its application as a biomolecule detection reagent in the life science field and as a useful material in the field of photocatalyst, charge transfer device and analytical chemistry is attracting attention.
Since semiconductor nanoparticles are hydrophobic, when used in such applications, they must be water soluble or at least water dispersible. For this reason, methods have been proposed for imparting good water dispersibility to semiconductor nanoparticles using various organic coatings.

For example, in Patent Document 1, a surfactant monomer is used as a dispersant, the nanoparticles interact with the hydrophobic region of the surfactant, and the hydrophilic region has an affinity for an aqueous medium. A method for stabilizing an aqueous suspension of is described. In US Pat. No. 6,057,059, a monomeric compound of formula HS— (CH ₂ ) _n —X, where n is preferably ≧ 10 and X is a carboxylate or sulfonate, is a monomeric surfactant. Instead it is disclosed.

  On the other hand, since nano-sized particles may have insufficient dispersion stability and non-specific adsorption of the surface, Non-Patent Document 1 discloses that hydrophobicity is obtained by modifying the surface of zinc oxide with methoxysilane or silanol. The water dispersibility of nanocrystals is improved. This technique replaces the original phosphine / phosphine oxide surfactant layer with a more polar ligand. However, this method has the disadvantage that the quantum yield is considerably reduced and it is never fully recovered, and the colloidal stability of particles dispersed in water is not sufficient.

In addition, it is generally known that modifying the surface of metal nanoparticles with a long-chain PEG improves the water dispersibility of the metal nanoparticles, and has little effect on antigenicity and the human body. For example, Non-Patent Document 2 discloses a surface modification technique for improving the water dispersibility of metal nanocrystals by modifying the zinc oxide surface with PEG with SiCl ₃ added to the end in order to take advantage of the above-mentioned advantages. is doing. In Non-Patent Document 3, as a surface modifier for metal nanoparticles, trifluoroethyl ester-PEG-terminated silane in which a reactive organic group is added to the end of a long-chain PEG having a molecular weight of 600 is used. Improves water dispersibility. Thereby, the water dispersibility is improved.

J.Phys.Chem, 2001, Vol.105, pp.8861-8871 Biomed.Microdevices, 1998, Vol.1, pp.81-89 J.Am.Chem.Soc., 2004, Vol.126, pp.7206-7211 International Publication No. 00/17655 Pamphlet International Publication No. 00/17656 Pamphlet

However, when long-chain PEG is surface-modified to improve water dispersibility, it is not possible to introduce a sufficient number of long-chain PEG molecules to form a sufficient hydration layer on the surface. Sex cannot be obtained. In order to solve this problem, even when a surface modifier with a reactive organic group added to the end of the long chain PEG is linked, the long chain PEG itself inhibits the reactivity of the organic group. Therefore, the water dispersibility cannot be effectively improved.
Accordingly, an object of the present invention is to provide nanoparticles with enhanced dispersibility and reactivity in aqueous media.

The nanoparticle of the present invention is a nanoparticle composed of a core portion and a surface organic layer covering the surface of the core portion. The surface organic layer has an R ¹ O—L ¹ — group and a YL ² — group ( In the formula, L ¹ and L ² represent a divalent linking group satisfying the length relationship of L ¹ <L ^2. R ¹ represents an alkyl group having 1 to 3 carbon atoms or a hydrogen atom, and Y represents a label. It is characterized by having a surface reactive group represented by (representing a group reactive with a substance).
In the nanoparticle, the surface organic layer includes a first surface treatment agent represented by the following general formula (I), a second surface treatment agent represented by the following general formula (II), and the following general formula ( It is preferably formed by a surface treatment with a third surface treatment agent represented by III).
Formula (I)
M- (R ² ) ₄
Formula (II)
R ¹ O-L ¹ -X
General formula (III)
X-L ² -Y
(In the above formula, M represents a Si or Ti atom, and R ² represents an organic group. R ² may be the same or different, but at least one of R ² is different from other organic groups. L ¹ and L ² represent a divalent linking group satisfying a length relationship of L ¹ <L ² , R ¹ represents an alkyl group having 1 to 3 carbon atoms or a hydrogen atom. X represents a substituent capable of forming a bond with the functional group on the particle surface, and Y represents a group reactive with the labeling substance.)

In the nanoparticle, the core part is preferably composed of a metal oxide.
In the nanoparticle, the labeling substance is preferably a biological molecule.

The method for producing nanoparticles according to the present invention is a method for producing nanoparticles comprising a core portion and a surface organic layer covering the surface thereof, and the first surface treatment represented by the following general formula (I) Forming a precursor particle composed of the core part and the surface organic layer by using an agent, a second surface treatment agent represented by the following general formula (II) and the following for the precursor particle: Performing a surface treatment with a third surface treating agent represented by the general formula (III) to form a surface reactive group represented by an R ¹ O—L ¹ — group and a YL ² — group, It is characterized by including.
Formula (I)
M- (R ² ) ₄
Formula (II)
R ¹ O-L ¹ -X
General formula (III)
X-L ² -Y
(In the above formula, M represents a Si or Ti atom, R represents an organic group. R ² may be the same or different, but at least one of R ² reacts with another organic group. L ¹ and L ² represent a divalent linking group satisfying a length relationship of L ¹ <L ² , R ¹ represents an alkyl group having 1 to 3 carbon atoms or a hydrogen atom. X represents a substituent capable of forming a bond with a functional group on the particle surface, and Y represents a group reactive with the labeling substance.

Nanoparticles according to the present invention, the surface organic layer on the nanoparticle, since L ¹ <length by satisfying the length relationship of L ² has two different kinds of surface reactive groups, the L ² Water dispersibility can be improved by the long-chain surface-reactive group contained, and reactivity with the labeling substance can be improved by the short-chain surface-reactive group containing L ¹ . Thereby, coexistence at a high level of water dispersibility and reactivity can be achieved.

  According to the present invention, nanoparticles having enhanced dispersibility and reactivity in an aqueous medium can be provided.

The nanoparticle of the present invention is a nanoparticle composed of a core portion and a surface organic layer covering the surface of the core portion. The surface organic layer has an R ¹ O—L ¹ — group and a YL ² — group ( In the formula, L ¹ and L ² represent a divalent linking group satisfying the length relationship of L ¹ <L ^2. R ¹ represents an alkyl group having 1 to 3 carbon atoms or a hydrogen atom, and Y represents a label. A surface-reactive group represented by (representing a group reactive with a substance).
In such nanoparticles, the surface organic layer preferably has a first surface treatment agent represented by the following general formula (I) and a second surface treatment agent represented by the following general formula (II). And a surface treatment with a third surface treatment agent represented by the following general formula (III).

  Since the nanoparticles of the present invention have surface-reactive groups with different lengths on the surface, the dispersibility and reactivity in an aqueous medium can be enhanced. This preserves the quantum efficiency of the original particles, maintains colloidal stability, and makes dispersibility of hydrophobic semiconducting nanocrystals in aqueous media while avoiding or minimizing any change in particle size distribution. Can be granted. Also, such properties are not affected by the type of nanoparticles, and are semiconducting nanoparticles, other types of nanoparticles having a hydrophobic surface (e.g., semiconducting nanoparticles that need not be crystalline, The same applies to metallic nanoparticles treated with other surface modifiers.

[1] First surface modifier The first surface modifier according to the present invention is a compound represented by the following general formula (I) or a decomposition product thereof. By modifying the surface of the core portion with such a surface modifier, the dispersibility of the nanoparticles in water or a hydrophilic solvent can be improved, and elution of the nanoparticle phosphors by body fluids or quenching of the fluorescence can be prevented. Furthermore, there is an advantage that it becomes easy to bind a molecular probe for detecting a target molecule.
Formula (I)
M- (R ² ) ₄

In the above formula, M represents a silicon (Si) atom or a titanium (Ti) atom, and is preferably a silicon atom from the viewpoint of water dispersibility.
R ² represents an organic group. R ² may be the same as or different from each other, but at least one of R ² represents a functional group reactive with another organic group.
Among the organic groups represented by R ² , functional groups having reactivity with other organic groups include a vinyl group, an allyloxy group, an acryloyl group, a methacryloyl group, an isocyanato group, a formyl group via a linking group L. Group, epoxy group, maleimide group, mercapto group, amino group, carboxyl group, halogen and the like are bonded. Among these reactive groups, those having an amino group at the terminal are particularly preferred from the viewpoint of reactivity with biopolymers.
Here, other organic groups capable of reacting with R ² include X in the general formula (III) described later, in addition to the labeling substance.

As the linking group L, for example, an alkylene group (eg, methylene group, ethylene group, trimethylene group, tetramethylene group, hexamethylene group, propylene group, ethylethylene group, cyclohexylene group, etc., having 1 to 10 carbon atoms, terminal From the viewpoint of the reactivity of the functional group, preferably 1 to 8 chain or cyclic).
The linking group L may have an unsaturated bond. Examples of unsaturated groups include alkenylene groups (eg, vinylene, propenylene, 1-butenylene, 2-butenylene, 2-pentenylene, 8-hexadecenylene, 1,3-butanedienylene, cyclohexenylene, etc. From the viewpoint of the reactivity of the terminal functional group, the number is preferably 1 to 10, and preferably 1 to 8 in the form of a chain or cyclic), an arylene group (eg, phenylene group, naphthylene group, etc., 6 to 10 carbon atoms, terminal From the viewpoint of the reactivity of the functional group, 6 phenylene groups) are preferred.

  The linking group L may have one or two or more heteroatoms (meaning any atom other than a carbon atom such as a nitrogen atom, an oxygen atom, or a sulfur atom). The hetero atom is preferably an oxygen atom or a sulfur atom, and the oxygen atom is most preferable from the viewpoint of water dispersibility. The number of heteroatoms is not particularly limited, but is preferably 5 or less, more preferably 3 or less, from the viewpoint of water dispersibility.

The linking group L may contain a functional group containing a carbon atom adjacent to the heteroatom as a partial structure. Such functional groups include ester groups (including carboxylic acid esters, carbonic acid esters, sulfonic acid esters, and sulfinic acid esters), amide groups (including carboxylic acid amides, urethanes, sulfonic acid amides, and sulfinic acid amides), ether groups, and thioethers. Group, disulfide group, amino group, imide group and the like. The above functional group may further have a substituent, and a plurality of these functional groups may be present in L. When two or more exist, they may be the same or different.
From the viewpoint of reactivity, the functional group is preferably an alkenyl group, ester group, amide group, ether group, thioether group, disulfide group or amino group, more preferably an alkenyl group, ester group or ether group.

The other organic group represented by R ² includes any group, but from the viewpoint of reactivity, methoxy group, ethoxy group, isopropoxy group, n-propoxy group, t-butoxy group, n- An alkoxy group such as a butoxy group and a phenoxy group; These alkoxy groups and phenoxy groups may further have a substituent, but those having a total carbon number of 8 or less are desirable. The surface modifier used in the present invention may be one in which an amino group, a carboxyl group or the like forms a salt with an acid or base.

Although the specific example of the surface modifier used for this invention is enumerated, in this invention, it is not limited to these compounds. In addition, these may be used independently and may be used in combination of 2 or more type.
N- (2-aminoethyl) -3-aminopropylmethyldimethoxysilane, N- (2-aminoethyl) -3-aminopropyltrimethoxysilane, N- (2-aminoethyl) -3-aminopropyltriethoxysilane 3-aminopropyltrimethoxysilane, aminophenyltrimethoxysilane, 3-aminopropyltriethoxysilane, bis (trimethoxysilylpropyl) amine, N- (3-aminopropyl) -benzamidotrimethoxysilane, 3-hydrazidepropyl Trimethoxysilane, 3-maleimidopropyltrimethoxysilane, (p-carboxy) phenyltrimethoxysilane, 3-carboxypropyltrimethoxysilane, 3-aminopropyltitanium tripropoxide, 3-aminopropylmethoxyethyl titania Diethoxide, 3-carboxypropyl titanium trimethoxide.

Of these, the following are preferable from the viewpoint of reactivity.
N- (2-aminoethyl) -3-aminopropylmethyldimethoxysilane, N- (2-aminoethyl) -3-aminopropyltrimethoxysilane, N- (2-aminoethyl) -3-aminopropyltriethoxysilane 3-aminopropyltrimethoxysilane, 3-aminopropyltriethoxysilane.

[2] Second and third surface modifiers The second and third surface modifiers according to the present invention are compounds represented by the following general formulas (II) and (III), or decomposition products thereof, respectively. .

Formula (II)
R ¹ O-L ¹ -X
General formula (III)
X-L ² -Y
The organic group represented by X is a substituent capable of forming a bond with a functional group on the particle surface. The term “functional group on the particle surface” as used herein includes not only the surface of the core part but also functional groups formed in the surface organic layer on the surface of the core part by the surface modifier. R ^{2 in the} agent is also applicable.
Examples of the organic group represented by X include vinyl group, allyloxy group, acryloyl group, methacryloyl group, isocyanato group, formyl group, epoxy group, maleimide group, mercapto group, amino group, carboxyl group, halogen, amino group From the viewpoint of the reactivity with the group, preferred are a carboxyl group, a maleimide group and an amino group, and most preferred is a carboxyl group.
The organic group represented by Y is a group having reactivity with the labeling substance. Examples of the organic group represented by Y include a maleimide group, a carboxyl group, an aldehyde group, a methanethiosulfate group, and the like, and from the viewpoint of reactivity with the SH group, a maleimide group and an aldehyde group are most preferable. Maleimide group.

The organic group represented by R ¹ represents an alkyl group having 1 to 3 carbon atoms or a hydrogen atom, and among these, a hydrogen atom is preferable from the viewpoint of water dispersibility.
The linking groups L ¹ and L ² are divalent linking groups that satisfy a length relationship of L ¹ <L ² . Thus by vary the length by L ¹ and L ^2, it is possible to increase both the reactivity and the water-dispersible.
L ¹ and L ² include, for example, an alkylene group (eg, methylene group, ethylene group, trimethylene group, tetramethylene group, hexamethylene group, propylene group, ethylethylene group, cyclohexylene group and the like having 1 to 10 carbon atoms, From the viewpoint of the reactivity of the organic group, preferably 1 to 8 chain or cyclic ones) may be mentioned, and these may be used singly or in combination.

Further, the linking groups L ¹ and L ² may have an unsaturated bond. Examples of unsaturated groups include alkenylene groups (eg, vinylene, propenylene, 1-butenylene, 2-butenylene, 2-pentenylene, 8-hexadecenylene, 1,3-butanedienylene, cyclohexenylene, etc. A linear or cyclic group having 1 to 10, preferably 1 to 8, and an arylene group (eg, a phenylene group having 6 to 10 carbon atoms, preferably 6 such as a phenylene group or a naphthylene group). These may be used singly or in combination, and may be further combined with the above alkylene group or the like.

Among these, on condition that the length relationship of L ¹ <L ² is satisfied, L ^{1 as a} whole has 1 to 10 carbon atoms, more preferably 1 to 4 carbon atoms from the viewpoint of the reaction inhibition of the organic group. A chain or cyclic thing can be mentioned. On the other hand, the entire L ² is preferably a chain or cyclic group having 50 to 5000 carbon atoms, more preferably 50 to 300 carbon atoms from the viewpoint of water dispersibility.
The size of L ¹ and L ² may be any relative relationship as long as the relationship of L ¹ <L ² is satisfied, but preferably from the viewpoint of compatibility between the reaction inhibition of the organic group and water dispersibility. , L ² can be 50 times to 5000 times, more preferably 50 times to 300 times as the length of the main chain of L ¹ . As such a combination, for example, L ¹ is an alkylene group having 1 to 4 carbon atoms or a polyoxyethylene group having 2 to 10 carbon atoms, and L ² is a polyoxyethylene group having 50 to 300 carbon atoms. Can be mentioned.

The linking groups L ¹ and L ² may have one or more heteroatoms (meaning any atom other than carbon atoms such as nitrogen atom, oxygen atom, sulfur atom). The hetero atom is preferably an oxygen atom or a sulfur atom from the viewpoint of stability, and most preferably an oxygen atom. The number of heteroatoms is not particularly limited, but is preferably 5 or less, more preferably 3 or less.

The linking groups L ¹ and L ² may contain a functional group containing a carbon atom adjacent to the hetero atom as a partial structure. Such functional groups include ester groups (including carboxylic acid esters, carbonic acid esters, sulfonic acid esters, and sulfinic acid esters), amide groups (including carboxylic acid amides, urethanes, sulfonic acid amides, and sulfinic acid amides), ether groups, and thioethers. Group, disulfide group, amino group, imide group and the like. The above functional group may further have a substituent, and a plurality of these functional groups may be present in each of the groups L ¹ and L ² . When there are a plurality of them, they may be the same or different as long as the above conditions are satisfied.

  The functional group is preferably an alkenyl group, an ester group, an amide group, an ether group, a thioether group, a disulfide group or an amino group, and more preferably an alkenyl group, an ester group or an ether group.

Specific examples of the second surface modifier are listed below, but are not limited to these compounds. In addition, these may be used independently and may be used in combination of 2 or more type.
Methoxyethoxyacetic acid, methoxyethoxypropionic acid, methoxyethoxybutyric acid, methoxyethoxyethoxyacetic acid, methoxyethoxyethoxypropionic acid, methoxyethoxyethoxybutyric acid, 3-hydroxypropionic acid, 3-hydroxybutyric acid, 3-hydroxypentanoic acid, 3 -Hydroxyhexanoic acid, 3-hydroxyheptanoic acid.

Specific examples of the third surface modifier are listed below, but are not limited to these compounds. In addition, these may be used independently and may be used in combination of 2 or more type.
Carboxy-PEG-amine, Carboxy-PEG-aldehyde, Carboxy-PEG-carboxylic acid, Carboxy-PEG-NHS, Carboxy-PEG-maleimide, Carboxy-PEG-thiol, of which, reaction with SH group From the viewpoint of sex, carboxy-PEG-NHS, carboxy-PEG-maleimide, and carboxy-PEG-thiol are preferable, and carboxy-PEG-maleimide is most preferable.

  Among these surface modifiers used in the present invention, decomposition products of those represented by the general formulas (I) to (III) are hydroxides obtained by hydrolysis of alkoxy groups, and dehydration condensation reaction between hydroxyl groups. The resulting low molecular weight oligomer (which may be any of a linear structure, a cyclic structure, a crosslinked structure, etc.), a dealcoholization condensation reaction product of a hydroxyl group and an unhydrolyzed alkoxy group, and these are further subjected to a dehydration condensation reaction. It refers to the formed sol and gel.

Any of the surface modifiers used in the present invention may be one in which the terminal NH ₂ group or COOH group forms a salt with an acid or base. Moreover, even if the surface modifier used for this invention coat | covers the whole surface of a nanoparticle, it may couple | bond with the one part. In the present invention, each surface modifier may be used alone or in combination.

  In addition to the above surface modifiers, known surface modifiers (for example, polyethylene glycol, polyoxyethylene (1) lauryl ether phosphate, lauryl ether phosphate, trioctyl phosphine, trioctyl phosphine oxide, sodium polyphosphate, Bis (2-ethylhexyl) sulfosuccinate sodium, etc.) may be present during or after the nanoparticle synthesis.

Further, the surface organic layer of the nanoparticles in the present invention, R ¹ O-L ¹ - group and Y-L ² - group forming possible, the surface modification treatment by the surface modifying agent (I) ~ (III) It is not limited to.

[3] Core Part The core part in the present invention may be any particle that emits fluorescence with a predetermined excitation light, and includes any one that has a so-called nano-sized number average particle diameter. As a number average particle diameter, Preferably it is 0.5-100 nm, More preferably, it is 0.5-50 nm, More preferably, it is 1-10 nm. The particle size distribution of the nanoparticles is preferably 0 to 50%, more preferably 0 to 20%, and still more preferably 0 to 10% in terms of variation coefficient. The coefficient of variation means a value (arithmetic standard deviation × 100 / number average particle size) obtained by dividing the arithmetic standard deviation by the number average particle size and expressing this as a percentage.

Preferable nanoparticles are metal oxide or metal sulfide nanoparticles. Examples of the metal constituting the metal oxide or metal sulfide include a group IIB such as Zn, a group IIIA such as Y, Eu, and Tb, a group IIIB such as Ga and In, a group IVA such as Zr and Hf, Si, Examples include IVB group such as Ge, VA group such as V and Nb, and VIA group such as Mo and W. Among these, Zn which is kind to a living body is particularly preferable. _{Further, Zn 2 SiO 4, CaSiO 3} , MgWO 4, YVO 4, may be a composite metal oxide such as Y ₂ SiO _5. Nanoparticles in the present invention can be manufactured stably, have little concern about toxicity, can be manufactured at low cost, have high monodispersibility of particles, can obtain strong luminescence, and have a wavelength range of emission spectrum for this purpose. From the viewpoint of easy matching, zinc oxide (ZnO) and zinc sulfide (ZnS) are particularly preferable, and zinc oxide (ZnO) is most preferable.

  Furthermore, it is also preferable that these metal oxide or metal sulfide nanoparticles contain a small amount of metal ions different from the metal in the metal oxide or metal sulfide constituting the metal oxide or metal sulfide. Examples of the metal ion include metal ions such as Mn, Cu, Eu, Tb, Tm, Ce, Al, and Ag. These metal ions are also preferably doped as a compound combining chloride ions and fluoride ions. The metal ion to be doped may be one kind of atom or a plurality of kinds of atoms. The concentration of the metal ions varies depending on the metal constituting the nanoparticle and the type thereof, but is preferably in the range of 0.001 to 10 atomic%, more preferably in the range of 0.01 to 10 atomic%.

  The nanoparticles according to the present invention are preferably excited by light in the ultraviolet region, more preferably 250 nm to from the viewpoint of separation of excitation light and signal fluorescence, utilization of an inexpensive light source, and simple detection system construction. Phosphor nanoparticles that use 380 nm ultraviolet light as excitation light are preferred. It is preferable that the excitation light emits light in the visible range, more preferably visible light of 400 nm to 700 nm. By emitting visible light, it is possible to excite the fluorescent dye in the visible range, and it is possible to develop the fluorescent dye with lower energy and reactivity, in particular, without reacting violently with the living body.

  The nanoparticles in the present invention preferably have a half-value width of light emission of 50 to 200 nm, and preferably 60 to 180 nm in order to detect light emission with high sensitivity using a simple device. Furthermore, it is preferable that the emission peak wavelength and the absorption peak wavelength are different as the fluorescent labeling material. In order to detect light emission with high sensitivity, the emission peak wavelength is more preferably separated from the absorption edge wavelength by 20 nm or more. A distance of 50 nm or more is particularly preferable. The phosphor nanoparticles having such a peak wavelength and half width of light emission are phosphor nanoparticles of a metal oxide or a metal sulfide, and those skilled in the art will know the constituent metals and the like as described above. It can obtain easily by selecting suitably.

[4] Method for Producing Nanoparticles and Dispersions thereof Nanoparticles composed of metal oxides are prepared by a sol-gel method for hydrolyzing organometallic compounds such as metal alkoxides and acetylacetonates, After adding alkali to the salt aqueous solution and precipitating it as hydroxide, dehydration and annealing hydroxide precipitation method, ultrasonic decomposition method using the above precursor solution of the metal, ultrasonic decomposition method, high temperature and high pressure It can be obtained by a liquid phase synthesis method such as a solvothermal method in which a decomposition reaction is carried out under low pressure, or a spray pyrolysis that is sprayed at high temperatures. Further, it can also be obtained by a vapor phase synthesis method such as a thermal CVD method or a plasma CVD method using an organometallic compound, a sputtering method or a laser ablation method using a target of the metal or the metal oxide.

  Nanoparticles composed of metal sulfides are composed of thermally decomposable metal compounds such as diethyldithiocarbamate compounds of metals, high-boiling organic solvents such as trialkylphosphine oxides, trialkylphosphines, and ω-aminoalkanes. Hot soap method for crystal growth, co-precipitation method for crystal growth by adding a sulfide solution such as sodium sulfide or ammonium sulfide to a solution of the metal salt, the raw material aqueous solution containing a surfactant as an alkane, ether In addition, it can be obtained by a liquid phase synthesis method such as a reverse micelle method in which a reverse micelle exists in a nonpolar organic solvent such as an aromatic hydrocarbon and crystal is grown in the reverse micelle. It can also be obtained by the same gas phase synthesis method as in the case of the metal oxide nanoparticles.

  The above-described surface modifier can be added at the time of synthesis of the nanoparticle, but is preferably added after synthesis, and is bonded to the nanoparticle by hydrolyzing at least a part thereof, so that at least the surface of the nanoparticle can be added. A part is coated (surface modified). The nanoparticles are washed and purified by conventional methods such as centrifugation and filtration, and then a solvent containing the surface modifier used in the present invention (preferably a hydrophilic organic such as methanol, ethanol, isopropyl alcohol, 2-ethoxyethanol). The solvent may be dispersed and coated.

  The addition amount of the surface modifier varies depending on the particle size of the nanoparticles, the concentration of the particles, the type (size, structure) of the surface modifier, etc. The first surface modifier is a metal oxide or metal sulfide. The amount is preferably 0.001 to 10 moles, more preferably 0.01 to 2 moles. The second surface modifier is preferably from 1 to 200 times mol, more preferably from 100 to 200 times mol, from the viewpoint of water dispersibility, relative to the metal oxide or metal sulfide. The agent is preferably 0.01 to 1 times mol from the viewpoint of water dispersibility, and more preferably 0.1 to 1 times mol from the viewpoint of reactivity with the labeling substance, relative to the second surface modifier. is there.

  As described above, in addition to the surface modifiers represented by the general formulas (I) to (III), a known surface modifier can be used in combination. The addition amount of the known surface modifier is not particularly limited, but is preferably 0.01 to 100 times mol, more preferably 0.05 to 10 times mol with respect to the metal oxide or metal sulfide. .

  In the nanoparticle dispersion liquid to which the surface modifier is bound, the concentration of the nanoparticle is not particularly limited because it varies depending on the fluorescence intensity, but is preferably 0.01 mM to 1000 mM, more preferably 0.1 mM to 100 mM. As the dispersion medium, in addition to the above alcohols, hydrophilic organic solvents such as DMF, DMSO, and THF, and water are preferable.

  In addition, it is confirmed by chemical analysis that the surface of the nanoparticles is coated with a surface modifier, and that a certain interval is observed between the particles when observed with a high-resolution TEM such as FE-TEM. Can do.

The nanoparticles of the present invention described above preferably form precursor particles composed of the core portion and the surface organic layer using the first surface treatment agent represented by the general formula (I). The surface treatment with the second surface treatment agent represented by the general formula (II) and the third surface treatment agent represented by the general formula (III) is performed on the precursor particles. Forming a surface-reactive group represented by an R ¹ O—L ¹ — group and a YL ² — group.

After the surface treatment is performed on the core portion with the first surface modifier in this way to form precursor particles (that is, non-surface reactive group-forming particles), the surfaces by the second and third surface modifiers By performing the modification treatment, the nanoparticles of the present invention can be efficiently produced.
At this time, the surface modification treatment with the second and third surface modifiers may be performed simultaneously, or one of them may be performed first.

  Since the nanoparticles of the present invention coated with the surface modifier represented by the general formulas (1) to (3) have the surface reactive group in the surface organic layer, amino which is the surface reactive group By reacting with a labeling substance such as a nucleic acid (monomer, oligonucleotide, etc.), antibody (monoclonal, other protein (amino acid), polysaccharide, etc.) By forming a bond, it becomes possible to act as a fluorescent labeling substance for a specific biological molecule.

  Examples of the substance to be labeled include biologically relevant molecules that can be detected in a living body and other related molecules, and among these, biologically relevant molecules are preferable. Labeled substances include antibodies, proteins, peptides, enzyme substrates, hormones, lymphokines, metabolites, receptors, antigens, haptens, lectins, avidin, streptavidin, toxins, carbohydrates, polysaccharides, nucleic acids, deoxynucleic acids, derived nucleic acids, derivatives Deoxynucleic acid, DNA fragment, RNA fragment, derived DNA fragment, derived RNA fragment, natural drug, virus particle, bacterial particle, viral component, yeast component, blood cell, blood cell component, bacteria, bacterial component, natural or synthetic lipid, drug And substances including poisons, environmental pollutants, polymers, polymer particles, glass particles, plastic particles, polymer films, etc. Among these, proteins, antibodies and peptides are preferred.

  The reactive group on the labeling substance that binds to the reactive group in the surface organic layer is not particularly limited depending on the combination with the reactive group in the surface organic layer, and examples include an amino group and an SH group. However, when an antibody is used as the labeling substance, an SH group is preferable. By utilizing the SH group, modification of the antigen recognition moiety can be avoided without reducing the specificity, and in addition, an increase in nonspecific adsorption can be avoided. In addition, as the SH group used for binding to the nanoparticles, the SH group obtained by cleaving the disulfide of the hinge part is not related to the antigen recognition site and does not impair the specificity of the antibody. preferable.

As used herein, the term “antibody” includes antibodies obtained from polyclonal and monoclonal preparations.
The term “monoclonal antibody” refers to an antibody composition having a heterogeneous antibody population. The term is not limited regarding the type or source of the antibody and is not intended to be limited by the manner in which it is made. Thus, this term includes murine hybridomas, as well as human monoclonal antibodies obtained using human hybridomas rather than murine hybridomas.

A functional antibody fragment can be generated from an antibody molecule by cleaving a constant region not responsible for antigen binding, eg, using pepsin, to produce an F (ab ′) ₂ fragment. These fragments contain two antigen-binding sites, but lack part of the constant region from each of the heavy chains. Similarly, Fab fragments (which contain a single antigen binding site) can be produced, for example, by digestion of polyclonal or monoclonal antibodies with papain. Functional fragments (including only heavy and light chain variable regions) can be produced using standard techniques such as recombinant production or preferential proteolytic cleavage of immunoglobulin molecules. These fragments are known as Fv.
The antibody used in the present invention is preferably a monoclonal antibody, more preferably a Fab ′ fragment.
As the antibody used in the present invention, it is particularly preferable if it is effective for blood diagnosis. For example, anti-AFP antibody, anti-HBs antibody, anti-HCV antibody, anti-CRP antibody, anti-TP antibody, anti-CEA antibody, anti-PSA antibody, anti-theophylline antibody And so on.

  The amidation reaction is carried out by condensation of a carboxyl group or a derivative group thereof (ester, acid anhydride, acid halide, etc.) and an amino group. When an acid anhydride or acid halide is used, it is desirable that a base coexists. When an ester such as methyl ester or ethyl ester of carboxylic acid is used, it is desirable to perform heating or decompression in order to remove the generated alcohol. When directly amidating a carboxyl group, amidation reagents such as DCC, Morpho-CDI, and WSC, condensation additives such as HBT, N-hydroxyphthalimide, p-nitrophenyl trifluoroacetate, 2,4,5- A substance that promotes an amidation reaction such as an active ester such as trichlorophenol may be allowed to coexist or may be reacted in advance. Further, during the amidation reaction, it is desirable to protect either the amino group or carboxyl group of the affinity molecule to be bound by amidation with an appropriate protecting group according to a conventional method, and to deprotect after the reaction.

Nanoparticles to which a target substance is bound by an amidation reaction are washed and purified by a conventional method such as gel filtration, and then dispersed in water or a hydrophilic solvent (preferably methanol, ethanol, isopropanol, 2-ethoxyethanol, etc.). use. The concentration of the nanoparticles in the dispersion is not particularly limited because it varies depending on the fluorescence intensity, but is preferably 10 ⁻¹ M to 10 ⁻¹⁵ M, more preferably 10 ⁻² M to ^{10 −10} M.

Moreover, when using protein, an antibody, and a peptide as a to-be-labeled substance, SH group in these molecules and the surface reactive group on the surface of a nanoparticle can also be combined. In this case, it can be easily coupled by using a hinge method or the like.
This reaction is a reaction using an SH group cleaved from the disulfide of the antibody hinge part, which is independent of the antigen recognition site. In other words, F (ab ′) ₂ is prepared by pepsin treatment, and further, a reducing agent is added to cleave the disulfide, and the Fab ′ obtained by reacting the SH group and the maleimide group can bind to each other. .

  Since the present invention has good dispersibility in aqueous media and has excellent colloidal and photophysical stability, it can be used in various fields (biology, analytical chemistry and combinatorial chemistry, medical diagnosis, and genetic analysis). Useful). In addition, using fluorescence specificity, it can be used as a fluorescent reagent or a light-emitting element in addition to a test reagent in the bio / medical field.

[Example 1]
Synthesis of Zinc Oxide Nanoparticles Dehydrated ethanol (250 ml) was added to zinc acetate dihydrate (5.49 g, 25 mmol), and gently refluxed for 2 hours while distilling off the solvent with a Dean-Steark dehydrator. . The solvent distilled off was 150 ml. 150 ml of dehydrated EtOH was again added to the white turbid reaction solution and heated to reflux, and the clarified reaction solution was cooled to room temperature.
Tetramethylammonium hydroxide (25% methanol solution, 11.4 ml, 28 mmol) was added to the reaction solution, and the mixture was stirred at room temperature for 4 hours.
Subsequently, 3-aminopropyltrimethoxysilane (4.7 ml, 25 mmol) and water (1.5 ml, 83.3 mmol) were added, and the mixture was stirred at 60 ° C. for 4 hours. A white solid precipitates 7 minutes after the start of the reaction. The reaction solution was water-cooled to room temperature, and the solid was suction filtered and washed with ethanol. When the obtained white powder was dried under reduced pressure, zinc oxide nanoparticles having an aminated surface were obtained. Yield 6.0g

200 mg of the particles were dissolved in 10 ml of distilled water, and gel filtration was performed using a column filled with Sephadex G25. All subsequent reactions used the desalted aqueous solution.
The nanoparticles synthesized by this formulation show a transparent and good dispersion state even at 10 wt%. The peak pattern of the hexagonal (wurtzite) zinc oxide sample belonging to the powder X-ray diffraction space group P6 ₃ mc coincided with the peak pattern, and the particle size was 3 nm as measured by TEM.
Hereinafter, the concentration of zinc oxide particles is estimated from the volume (14.13 × 10 ⁻²⁷ m ³ mol ⁻¹ ), molecular weight (81.38), and density (5.67) of zinc oxide having a particle size of 3 nm. .

[Example 2]
Giving water dispersibility to zinc oxide nanoparticles Add 5 mg each of WSC and N-hydroxysuccinimide to 1 ml of 0.1 M HEPES (pH 7.0) buffer, and then add 2.5 × 10 ⁻⁵ mol of methoxyethoxyacetic acid. And reacted at room temperature. After 30 minutes, 1 ml of nanoparticles (concentration: 14 mg / ml, dispersed in milliQ water) having the surface of zinc oxide particles having an average diameter of 3 nm coated with aminopropylsilane was added and reacted at room temperature for 7 hours. Gel filtration was performed with a PD-10 column (Pharmacia Bioscience). The particles were a colorless and good aqueous dispersion (particle dispersion A).
Particle dispersions B to D were obtained using methoxyethoxyethoxyacetic acid, 3-hydroxypropionic acid, and 3-hydroxybutyric acid instead of methoxyethoxyacetic acid. The optical properties of each particle dispersion are shown in Table 1.

[Example 3]
2. Hydrophilization and maleimidation of zinc oxide nanoparticles 50 mg of HCl.NH ₂ -PEG-COOH (MW.5000, carbon number 226; NEKTAR) was dissolved in 1 ml of 0.1 M (pH 8.0) HEPES buffer. 0 mg of N- (2-maleimidoethyloxy) succinide was added and reacted at room temperature for 6 hours.
1 ml of 0.1M HEPES (pH 7.0) buffer in 1 ml of nanoparticles (concentration: 14 mg / ml, dispersed in milliQ water) coated with aminopropylsilane on the surface of zinc oxide particles having an average diameter of 3 nm, 100 μl of the maleimidated solution (No. 1) 0.001 mmol) was added as a surface modifier of No. 3, and 5 mg each of WSC and N-hydroxysuccinimide were added and reacted at room temperature. After 30 minutes, 2.5 × 10 ⁻⁵ mol of methoxyethoxyacetic acid was added and reacted for 3 hours and 20 minutes. Purification with a PD-10 column (Pharmacia Bioscience) gave a colorless and transparent maleimidated zinc oxide nanoparticle dispersion (particle dispersion E).
Particle dispersions F to H using methoxyethoxyethoxyacetic acid, 3-hydroxypropionic acid, and 3-hydroxybutyric acid instead of methoxyethoxyacetic acid were obtained. The optical properties of these particle dispersions are shown in Table 2.

  An example of the nanoparticle synthesis process according to the above-described embodiment will be described below.

[Example 4]
Ligation of zinc oxide nanoparticles and anti-theophylline antibody 0.8 ml of Fab ′ fraction solution (100 mM HEPES buffer (pH 6.0); 0.96 mg / ml) obtained by purifying anti-theophylline antibody by pepsin treatment and mercaptoethylamine reduction, The mixture was mixed with 3.5 ml of the maleimidated zinc oxide nanoparticle dispersion (4 mg / ml) prepared in Example 3 and reacted at 4 ° C. overnight. After stirring, it was purified by gel filtration with Sephadex G100 (eluted with 0.1 M HEPES buffer (pH 7.4)) to obtain Fab′-linked PEGylated zinc oxide nanoparticles.

  Thus, in this example, a highly functional phosphor that maintains high reactivity by using highly durable semiconductor nanoparticles as a phosphor and a dispersion promoter other than a PEG chain having a short carbon number. It became possible to get.

Claims (9)

Nanoparticles composed of a core part and a surface organic layer covering the surface,
In the surface organic layer, R ¹ O—L ¹ — group and YL ² — group (wherein L ¹ and L ² represent a divalent linking group satisfying a length relationship of L ¹ <L ² . R ¹ represents an alkyl group having 1 to 3 carbon atoms or a hydrogen atom, and Y represents a group having reactivity with a labeling substance). The surface organic layer is represented by a first surface treatment agent represented by the following general formula (I), a second surface treatment agent represented by the following general formula (II), and the following general formula (III). The nanoparticle according to claim 1, wherein the nanoparticle is formed by a surface treatment with a third surface treatment agent.
Formula (I)
M- (R ² ) ₄
Formula (II)
R ¹ O-L ¹ -X
General formula (III)
X-L ² -Y
(In the above formula, M represents a Si or Ti atom, and R ² represents an organic group. R ² may be the same or different, but at least one of R ² is different from other organic groups. L ¹ and L ² represent a divalent linking group satisfying a length relationship of L ¹ <L ² , R ¹ represents an alkyl group having 1 to 3 carbon atoms or a hydrogen atom. X represents a group capable of forming a bond with R ² in the general formula (I), and Y represents a group reactive with the labeling substance.)   The nanoparticle according to claim 1 or 2, wherein the core part is composed of a metal oxide. The L ¹ is an alkylene group having 1 to 4 carbon atoms or a polyoxyethylene group having 2 to 10 carbon atoms, and L ² is a polyoxyethylene group having 50 to 300 carbon atoms. Nanoparticles.   The second surface treatment agent is methoxyethoxyacetic acid, methoxyethoxypropionic acid, methoxyethoxybutyric acid, methoxyethoxyethoxyacetic acid, methoxyethoxyethoxypropionic acid, methoxyethoxyethoxybutyric acid, 3-hydroxypropionic acid, 3-hydroxybutyl. The nanoparticle according to any one of claims 2 to 4, wherein the nanoparticle is at least one selected from an acid, 3-hydroxypentanoic acid, 3-hydroxyhexanoic acid, and 3-hydroxyheptanoic acid.   The third surface treatment agent is at least selected from carboxy-PEG-amine, carboxy-PEG-aldehyde, carboxy-PEG-carboxylic acid, carboxy-PEG-NHS, carboxy-PEG-maleimide and carboxy-PEG-thiol The nanoparticle according to claim 2, wherein the number is one.   The metal oxide is zinc oxide, and the first surface treatment agent is N- (2-aminoethyl) -3-aminopropylmethyldimethoxysilane, N- (2-aminoethyl) -3-aminopropyltrimethoxysilane And N- (2-aminoethyl) -3-aminopropyltriethoxysilane, 3-aminopropyltrimethoxysilane, and 3-aminopropyltriethoxysilane. The nanoparticle according to any one of 6.   The nanoparticle according to any one of claims 1 to 7, wherein the labeling substance is a bio-related molecule. A method for producing nanoparticles comprising a core portion and a surface organic layer covering the surface thereof,
Using the first surface treating agent represented by the following general formula (I) to form precursor particles composed of the core part and the surface organic layer;
Formula (I)
M- (R ² ) ₄
(In the above formula, M represents a Si or Ti atom, and R ² represents an organic group. R ² may be the same or different, but at least one of R ² is different from other organic groups. Indicates a functional group having reactivity.)
The precursor particles are subjected to a surface treatment with a second surface treatment agent represented by the following general formula (II) and a third surface treatment agent represented by the following general formula (III), and R ¹ Forming a surface reactive group represented by an O-L ¹ -group and a YL ² -group,
Formula (II)
R ¹ O-L ¹ -X
General formula (III)
X-L ² -Y
(In the above formula, L ¹ and L ² represent a divalent linking group satisfying the length relationship of L ¹ <L ^2. R ¹ represents an alkyl group having 1 to 3 carbon atoms or a hydrogen atom. X represents This represents a group capable of forming a bond with a functional group on the particle surface, and Y represents a group reactive with the labeling substance.
The manufacturing method of the nanoparticle characterized by including.