JP6606820B2 - Semiconductor nanoparticle-encapsulating resin particles, tissue-staining dye comprising the semiconductor nanoparticle-encapsulating resin particles, and tissue staining method - Google Patents

Description

  The present invention relates to resin particles encapsulating semiconductor nanoparticles used for a biological tissue stain.

  As fluorescent light-emitting materials, nanoparticles in which organic dyes are mainly accumulated at high density have been developed so far. Since these nanoparticles have high brightness and excellent solvent resistance, they are preferably used as a staining agent used in biological tissue examinations, etc., but there is a problem that the fluorescence signal gradually decreases when irradiated with excitation light. There was a need for further improvement in terms of light durability.

  In recent years, semiconductor nanoparticles have attracted attention in applications such as labeling agents or high-definition displays in the medical field. As a phosphor material having improved light durability using the semiconductor nanoparticles, Patent Document 1 describes a silica-based glass particle material in which fluorescent light-emitting semiconductor ultrafine particles are encapsulated in the central portion. The silica-based glass particle material of Patent Document 1 shows the properties of glass as a whole, is excellent in mechanical properties, heat resistance and chemical stability, and the encapsulated semiconductor fine particles are shielded from the external atmosphere, so that it has light resistance. Excellent in stability over time. However, the silica-based glass particle material of Patent Document 1 is used for applications such as a display panel. For example, a modification group or the like is introduced into the silica-based glass particle and applied to a biological tissue stain. It's not about tackling the issue.

  Patent Document 2 describes glass fine particles encapsulating semiconductor nanoparticles as a biological material fluorescent labeling agent. An amino compound having an amino group and a hydrophobic group is attached to the surface of the semiconductor nanoparticles, and then a glass precursor is used. Forming fine glass particles. Patent Document 2 shows that the relationship between the content and the emission intensity remains linear even in a region where the content of the semiconductor nanoparticles is low, and the semiconductor nanoparticle-containing glass fine particles are highly sensitive, and It can be seen that the solvent resistance is excellent. However, Patent Document 2 does not mention anything about the light durability of the semiconductor nanoparticle-containing glass fine particles.

Patent Document 3 discloses a tissue stain containing, as a staining component, resin particles of a thermosetting resin and dye resin particles having a fluorescent dye fixed to the resin particles, that is, a tissue in which the fluorescent dye is encapsulated in the resin particles. Staining agents are described. Patent Document 3 describes that the use of the tissue stain eliminates the problem of variations in bright spots, thereby improving the determination accuracy of the fluorescent signal. The dye resin particles have high brightness and It can be seen that the solvent resistance is excellent. However, when irradiated with excitation light, the determination accuracy of the fluorescence signal gradually decreases, and it is considered that the dye resin particles deteriorate due to the irradiation of excitation light.
Accordingly, there is a demand for the development of a tissue stain that has improved light durability in addition to high brightness and solvent resistance.

Japanese Patent No. 3755033 International Publication No. 2012/147429 International Publication No. 2014/136855

  The present invention provides semiconductor nanoparticle-containing resin particles having excellent light durability in addition to conventional properties of high luminance and solvent resistance, a tissue staining dye comprising the semiconductor nanoparticle-containing resin particles, and tissue staining The challenge is to provide a law.

The present invention solves the above-described problems in the prior art and includes the following items.
The semiconductor nanoparticle-encapsulating resin particle of the present invention comprises a semiconductor nanoparticle having a surface modifying group and a resin particle encapsulating the semiconductor nanoparticle having the surface modifying group, the semiconductor nanoparticle having the surface modifying group, and the It is characterized in that the resin particles have opposite charges.

It is preferable that the functional group of the resin particle has a positive charge.
The resin particles are preferably melamine resin particles.
The stain for tissue staining of the present invention is characterized by comprising the semiconductor nanoparticle-containing resin particles.
The tissue staining method of the present invention is characterized in that the tissue staining dye is used.

  According to the present invention, there are provided semiconductor nanoparticle-containing resin particles having excellent light durability in addition to high luminance and solvent resistance, a tissue staining dye comprising the semiconductor nanoparticle-containing resin particles, and a tissue staining method. can do.

  That is, in the semiconductor nanoparticle-containing resin particles of the present invention, the semiconductor nanoparticles having a surface modifying group and the resin particles have opposite charges. As a result, the bonding between the surface modification group in the semiconductor nanoparticles having the surface modification group and the resin particles becomes strong, and the semiconductor nanoparticle-containing resin particles are easily fixed in the resin particles. It is considered that the durability can be effectively maintained even after irradiation.

It is a figure showing the semiconductor nanoparticle inclusion resin particle. It is an enlarged view of the semiconductor nanoparticle in the semiconductor nanoparticle inclusion | inner_cover resin particle in FIG.

[Semiconductor nanoparticle-containing resin particles]
The semiconductor nanoparticle-encapsulating resin particle of the present invention comprises a semiconductor nanoparticle having a surface modifying group and a resin particle encapsulating the semiconductor nanoparticle having the surface modifying group.
Each component which comprises the said semiconductor nanoparticle inclusion resin particle is demonstrated in detail below.

(Semiconductor nanoparticles)
The semiconductor nanoparticles used in the present invention are not particularly limited as long as they are semiconductors such as III-V or II-VI direct transition semiconductors that emit light in the visible region. As the semiconductor nanoparticles, core-shell type semiconductor nanoparticles are usually used.

  The core-shell type semiconductor nanoparticles are multi-layered semiconductor nanoparticles composed of a core part (core part) and a shell part (covering part) covering the core part, and the particle size thereof is usually 20 nm or less, preferably 1 to 1 nm. 10 nm.

  Materials for forming the core include silicon (Si), germanium (Ge), indium nitride (InN), indium phosphide (InP), gallium arsenide (GaAs), aluminum selenide (AlSe), and selenide. Semiconductors such as cadmium (CdSe), aluminum arsenide (AlAs), gallium phosphide (GaP), zinc telluride (ZnTe), cadmium telluride (CdTe) and indium arsenide (InAs) are used, or raw materials forming these are used. It is done. Of these, indium phosphide (InP), cadmium telluride (CdTe) and cadmium selenide (CdSe) are preferred.

As a material for forming the shell portion, a II-VI group semiconductor, a III-V group semiconductor, a IV group compound semiconductor, or an oxide semiconductor is used. Specifically, silicon (Si), silicon dioxide (SiO ₂ ), germanium (Ge), germanium dioxide (GeO ₂ ), indium nitride (InN), indium phosphide (InP), gallium arsenide (GaAs), selenium Aluminum hydride (AlSe), cadmium selenide (CdSe), aluminum arsenide (AlAs), gallium phosphide (GaP), zinc sulfide (ZnS), zinc telluride (ZnTe), cadmium telluride (CdTe) and indium arsenide A semiconductor having a band gap larger than that of the core forming material and having no toxicity, such as (InAs), or a raw material for forming these is used.

  Of the above-described core part forming material and shell part forming material, when indium phosphide (InP), cadmium telluride (CdTe) or cadmium selenide (CdSe) is used as the core part forming material, sulfide is used as the shell part forming material. Zinc (ZnS) is preferably used.

In addition, when making a semiconductor nanoparticle react with the resin particle mentioned later, it is necessary to introduce | transduce a surface modification group into a semiconductor nanoparticle.
A liquid phase method is used for the method of manufacturing the semiconductor nanoparticles. Examples of the liquid phase method include a precipitation method, a coprecipitation method, a sol-gel method, a uniform precipitation method, and a reduction method. In addition, reverse micelle method, supercritical hydrothermal synthesis method and the like are also excellent methods for producing nanoparticles (for example, JP 2002-322468 A, JP 2005-239775 A, JP 10-310770 A). No. and JP-A-2000-104058).

When producing semiconductor nanoparticles by a liquid phase method, it is preferable to reduce a compound containing an element constituting the semiconductor nanoparticles (hereinafter referred to as “semiconductor precursor”) by a reduction reaction. The semiconductor precursor is, for example, silicon tetrachloride (SiCl ₄ ) when the semiconductor is silicon (Si). Other semiconductor precursors include indium trichloride (InCl ₃ ), tris (trimethylsilyl) phosphine (P (SiMe ₃ ) ₃ ), dimethyl zinc (ZnMe ₂ ), dimethyl cadmium (CdMe ₂ ), germanium tetrachloride (GeCl _4). ) And tributylphosphine selenide. The reaction of the semiconductor precursor is preferably performed in the presence of a surfactant.

The reaction temperature when producing semiconductor nanoparticles is not particularly limited as long as it is not lower than the boiling point of the semiconductor precursor and not higher than the boiling point of the solvent, but is preferably in the range of 70 to 110 ° C.
As the reducing agent used in the reduction reaction, various conventionally known reducing agents can be selected and used according to the reaction conditions. In the present invention, from the viewpoint of the strength of reducing power, lithium aluminum hydride (LiAlH ₄ ), sodium borohydride (NaBH ₄ ), sodium bis (2-methoxyethoxy) aluminum hydride, trihydride (sec- Reducing agents such as lithium (butyl) boron (LiBH (sec-C ₄ H ₉ ) ₃ ), potassium tri (sec-butyl) borohydride and lithium triethylborohydride are preferred. Of these, lithium aluminum hydride (LiAlH ₄ ) is particularly preferable because of its strong reducing power.

  Various conventionally known solvents can be used as the solvent, and alcohols such as ethanol, 2-butanol and 2-methyl-2-propanol, and hydrocarbon solvents such as toluene, decane and hexane can be used. preferable. In the present invention, a hydrophobic solvent such as toluene is particularly preferable as the solvent for dispersing the semiconductor precursor.

  As the surfactant, anionic, nonionic, cationic, or amphoteric surfactants known in the art can be used. Of these surfactants, quaternary ammonium salts such as tetrabutylammonium chloride, bromide or hexafluorophosphate, tetraoctylammonium bromide (abbreviation: TOAB), or tributylhexadecylphosphonium bromide are preferred, and tetraoctylammonium bromide is preferred. More preferred.

  The reaction by the liquid phase method varies greatly depending on the state of the compound containing the solvent in the liquid phase. Special care must be taken when producing nano-sized monodisperse particles. In the reverse micelle method, the size or state of the reverse micelle that becomes the reaction field varies depending on the concentration or type of the surfactant, and thus the conditions under which the nanoparticles are formed are limited. Therefore, an appropriate combination of surfactant and solvent is required.

(resin)
The resin constituting the semiconductor nanoparticle-containing resin particles of the present invention is not particularly limited as long as it can encapsulate semiconductor nanoparticles having a modifying group, but is usually a thermosetting resin.

  In the thermosetting resin, at least a part of hydrogen atoms contained in the structural unit is replaced by a substituent having a charge, or a part having a charge exists in a part of the chemical structure. Or it has a part which can couple | bond with the semiconductor nanoparticle which has a modification group in a part of the chemical structure. The “substituent or moiety having a charge” refers to a substituent or chemical structure that is positively (positively) or negatively (negatively) charged when dissolved in acidic, neutral or basic water. means.

  Examples of the thermosetting resin include a thermosetting resin formed from a monomer (or an oligomer thereof) selected from the group consisting of melamine, urea, guanamine, phenol, xylene, and derivatives thereof. Among these, melamine, urea, and guanamine are monomers that include a positively charged amino group (NH skeleton) as part of their structure. Phenol is a monomer containing a phenolic hydroxyl group having a negative charge as part of its structure. Since xylene is a monomer that does not contain a charged substituent or moiety, a portion of the hydrogen atoms contained in the structure is replaced with a positively charged or negatively charged substituent, and the semiconductor nanoparticles described later It can be used for chemical bonding.

  These thermosetting resins are known monomers that contain a substituent or moiety having a charge, or a monomer of a thermosetting resin that has a site capable of binding to a surface modifying group in a semiconductor nanoparticle having a surface modifying group. It can manufacture by polymerizing by the method of. Further, those containing a positively charged substituent such as melamine can be used without introducing a charged substituent.

  The thermosetting resin is not limited to a homopolymer, and may be a copolymer. If a specific example is given, the monomer which forms the said melamine resin will be copolymerized 1 type, or 2 or more types, or the copolymer obtained by copolymerizing combining the obtained oligomer, these monomers or oligomer, and other than that It may be a copolymer obtained by copolymerizing a monomer or an oligomer.

Here, examples of the substituent having a negative charge include a sulfonate group (—SO ₃ ⁻ ) and a carboxylate group (—COO ⁻ ). Examples of the positively charged substituent include a quaternary ammonium group (—NR ₃ ⁺ ) (R represents a hydrogen atom or an alkyl group).

  As a method of introducing a substituent having a charge into a thermosetting resin, there is a method of introducing the substituent into a monomer. Specifically, in the method of introducing a carboxylate group (carboxylate), an alkyl compound having a carboxylate, which is directly introduced into the monomer by a Friedel-Crafts reaction, and a monomer, a halide and a boronic acid, respectively. Or an alkyl compound having a carboxylate and a magnesium halide (MgX; X represents a chlorine atom, bromine atom or iodine atom) site introduced into the monomer. There is a method of introducing by Grignard reaction. In order to introduce the sulfonate group, the monomer is directly sulfonated with fuming sulfuric acid or chlorosulfuric acid, the alkyl compound having sulfonate and the monomer are converted into halide and boronic acid, respectively, and introduced by Suzuki coupling reaction. Or an alkyl compound having a sulfonate salt and a monomer in which a magnesium halide (MgX; X is as described above) moiety is introduced by a Grignard reaction. In order to introduce an amino group, the monomer is nitrated with fuming nitric acid, the nitro group is reduced to convert to an amino group, the monomer is halogenated, and then aminated with ammonia by direct reaction or Gabriel reaction. An alkyl compound having a group and a monomer are converted into a halide and a boronic acid and introduced by a Suzuki coupling reaction, respectively, and an alkyl compound having a carboxylate and a magnesium halide (MgX; X is as described above) ) There is a method of introducing a part introduced with a Grignard reaction.

  In the case of the above reaction, the carboxylate group, sulfonate group or amino group is introduced into the monomer in a form protected with a protective group as appropriate, and then deprotected to give a carboxylate group, sulfonate group or amino group. Also good.

  The molecular structure of the thermosetting resin is a three-dimensional network structure formed by cross-linking of polymers. For this reason, the semiconductor nanoparticles fixed in the thermosetting resin particles are not easily eluted to the outside of the resin particles even when exposed to a solvent, and the resulting semiconductor nanoparticle-containing resin particles or tissue staining dye is In the fluorescence observation, the effect of suppressing bright spot blurring and maintaining high brightness and solvent resistance is achieved.

(Semiconductor nanoparticle-containing resin particles)
As described above, the semiconductor nanoparticle-containing resin particles of the present invention are composed of semiconductor nanoparticles having a surface modifying group and resin particles containing the semiconductor nanoparticles.

  The semiconductor nanoparticles having the surface modification group and the resin particles have opposite charges. Such a semiconductor nanoparticle-containing resin particle is obtained by introducing a compound capable of imparting a surface modifying group onto the surface of the semiconductor nanoparticle, and then a semiconductor nanoparticle having the surface modifying group and a monomer or oligomer constituting the resin. It is manufactured by making it react.

The compound capable of imparting the surface modifying group is not particularly limited as long as it can impart a positive or negative charge as the modifying group to the surface of the semiconductor nanoparticles.
Examples of the surface modifying group capable of imparting a positive charge to the semiconductor nanoparticle surface include an amino group, a guanidino group, and an imidazolium group.

  Examples of the compound capable of imparting a positive charge include an amino compound having an amino group and a hydrophobic group (hereinafter also simply referred to as “amino compound”). The hydrophobic group in this amino compound refers to a hydrocarbon group, and specifically refers to an alkyl group and an aromatic hydrocarbon group. The alkyl group is preferably an alkyl group having 6 to 30 carbon atoms, and more preferably an alkyl group having 8 to 20 carbon atoms. Aromatic hydrocarbon groups include, for example, phenyl groups and naphthyl groups.

Specific examples of amino compounds include n-heptylamine, nonylamine, dodecylamine and hexadecylamine.
In order to introduce the amino compound into the surface of the semiconductor nanoparticles, the semiconductor nanoparticles and the amino compound may be mixed in an organic solvent and stirred.

Examples of the organic solvent include alcohols and ketones. Of these, alcohols are preferable, and lower alcohols having 1 to 4 carbon atoms are particularly preferable.
The amount of the semiconductor nanoparticles in the organic solvent is usually 0.001 to 1% by mass, preferably 0.01 to 0.1% by mass with respect to the organic solvent, and the content of the amino compound in the organic solvent is The amount of the organic solvent is usually 0.01 to 10% by mass, preferably 0.1 to 1% by mass.

Examples of the modifying group capable of imparting a negative charge to the semiconductor nanoparticle surface include a carboxylate group (—COO ⁻ ) or a sulfonate group (—SO ₃ ⁻ ).
Compounds that can impart a negative charge include, for example, sodium mercaptopropionate and sodium 3-mercapto-1-propanesulfonate.

In order to introduce the compound capable of imparting a negative charge to the surface of the semiconductor nanoparticles, the semiconductor nanoparticles and the compound may be mixed and stirred.
The semiconductor nanoparticle-containing resin particles of the present invention are produced, for example, according to the following steps (a) to (c).

A mixing step The mixing step is a step of mixing the semiconductor nanoparticles having the surface modification group and one or more of the monomers or oligomers forming the resin particles. In the step, in addition to the semiconductor nanoparticles having a surface modifying group and the monomer or oligomer constituting the resin particle, a surfactant, a proton supply agent or a proton acceptor, or a reaction accelerator is mixed as necessary. May be.

As the surfactant , for example, a surfactant having an emulsifying action is added in a range of 10 to 60% by weight with respect to the monomer or oligomer, whereby semiconductor nanoparticles having an arbitrary particle size can be obtained. it can. Increasing the proportion of surfactant makes it possible to produce even smaller particles, and reducing the proportion of surfactant makes it possible to produce even larger particles. Further, the surfactant at the time of particle production can be arbitrarily selected within the range of 0.1 to 3.0% by weight depending on the desired particle diameter.

  As the surfactant, all anionic, nonionic and cationic surfactants are used. Among these, the magnitude of the influence of the charge of the surfactant on the coefficient of variation of the particle size of the resin particles has a relationship of nonionic> anionic> cationic for monomers or oligomers having a positive charge. Therefore, it is preferable to use an anionic or nonionic surfactant. On the other hand, since the magnitude of the influence of the surfactant charge on the negatively charged monomer or oligomer has a relationship of nonionic> cationic> anionic, it is necessary to use a cationic or nonionic surfactant. preferable.

Examples of the anionic surfactant include sodium dodecylbenzenesulfonate. Examples of nonionic surfactants include polyethylene glycol and polyoxyethylene alkyl ether. Examples of the cationic surfactant include dodecyltrimethylammonium bromide.
The weight ratio between the monomer or oligomer and the surfactant is preferably set so that the monomer or oligomer: surfactant = 10: 1 to 10: 6.

When semiconductor nanoparticles are encapsulated in a proton supply agent or proton acceptor resin, protons (H ⁺ ) are positively attached to the positively charged substituent in the monomer or oligomer, or to the surface modifying group bonded to the semiconductor nanoparticles. It is also possible to use a proton supply agent that supplies a positive charge. Examples of proton donors include formic acid, acetic acid, and paratoluene sulfonic acid. When the substituent in the monomer or oligomer or the surface modifying group bonded to the semiconductor nanoparticle is a carboxyl group (—COOH) or a sulfonic acid group (—SO ₃ H), these groups themselves function as a proton supply agent. You can also

On the other hand, it is also possible to use a proton acceptor that positively extracts H ⁺ from a substituent that becomes a negative charge in the resin or a surface modifying group bonded to the semiconductor nanoparticles. Proton acceptors include, for example, bases such as sodium hydroxide.

As a reaction accelerator , for example, an acid is used. It is known that the reaction of melamine, urea, xylene and phenol is promoted by an acid catalyst. Examples of the acid include formic acid, acetic acid, sulfuric acid, hydrochloric acid, nitric acid, paratoluenesulfonic acid, and dodecylbenzenesulfonic acid. The reaction of the resin proceeds only by heating, but when the reaction accelerator is added, it proceeds even at a lower temperature, so that the reaction or performance can be added within a range that can be controlled.

(A) Polymerization step The polymerization step is a step in which a monomer or oligomer is thermoset, that is, polymerized with semiconductor nanoparticles having a surface modifying group to form semiconductor nanoparticle-containing resin particles. The reaction conditions (thermosetting temperature, polymerization time) in the polymerization step are determined from the composition of the monomer or oligomer to be polymerized and are in accordance with known methods. Here, the reaction temperature needs to be within the reaction conditions in which the performance of the semiconductor nanoparticle-containing resin particles does not deteriorate, that is, within the heat resistant temperature range of the semiconductor nanoparticles.

For example, when a melamine resin is selected as the resin, the melamine production reaction is usually 70 to 200 ° C, preferably 150 to 200 ° C.
By performing the polymerization step, the semiconductor nanoparticles contained therein are difficult to elute out of the resin particles even when exposed to a solvent or the like. If the semiconductor nanoparticles elute out of the resin particles due to inadequate polymerization, further heat treatment is performed within a range that does not adversely affect the semiconductor nanoparticles and the resin particles. And the elution of the semiconductor nanoparticles can be suppressed.

(C) Washing step The washing step is a step of removing excess semiconductor nanoparticles, resin, emulsifier and other impurities from the reaction solution of the semiconductor nanoparticle-containing resin particles after the completion of the reaction. Specifically, these excess components are centrifuged from the reaction solution, and after removing the supernatant, washing is performed by adding ultrapure water, irradiating with ultrasonic waves, and dispersing again. A series of washing operations of centrifugation, supernatant removal, and redispersion in ultrapure water is preferably repeated a plurality of times until the supernatant does not show any light absorption / fluorescence derived from the resin and semiconductor nanoparticles.

(Semiconductor nanoparticle-containing resin particles)
In the semiconductor nanoparticle-containing resin particles, the semiconductor nanoparticles having a surface modifying group and the resin particles have opposite charges. If the surface modification group attached to the semiconductor nanoparticle has a positive charge, the resin particle that encloses it has a negative charge, and if the surface modification group attached to the semiconductor nanoparticle has a negative charge, it encloses it. Resin particles have a positive charge. As a result, the semiconductor nanoparticles having the surface modifying group and the resin particles are strongly attracted by the electrostatic force, and the semiconductor nanoparticles are easily fixed in the resin particles. It is considered that it can be maintained across.

  The average particle size of the semiconductor nanoparticle-containing resin particles obtained as described above is usually 10 to 1000 nm, preferably 20 to 500 nm. The particle diameter of the resin particles encapsulating the semiconductor nanoparticles is the diameter obtained by taking an electron micrograph using a scanning electron microscope (SEM), measuring the cross-sectional area of the particles, and setting the measured value as the corresponding circle area. (Area circle equivalent diameter). Of the average particle size and variation coefficient of the semiconductor nanoparticle-containing resin particles, the average particle size is calculated as an arithmetic average of the particle diameters calculated as described above for a sufficient number (for example, 300 particles), and the variation coefficient Is calculated by 100 × standard deviation of particle diameter / average particle diameter.

[Staining for tissue staining and tissue staining method]
The stain for tissue staining of the present invention comprises the semiconductor nanoparticle-containing resin particles.
The stain for tissue staining is prepared by adding a linker for immunostaining to the semiconductor nanoparticle-containing resin particles. Examples of the linker added to the semiconductor nanoparticle-containing resin particles include, but are not limited to, a linker such as streptavidin-biotin.

  The addition of the streptavidin-biotin linker is performed, for example, as follows. That is, a thiol group is added to streptavidin by, for example, a thiol group introduction reagent. On the other hand, after introducing an amino group to the surface of the semiconductor nanoparticle-containing resin particles with an amino group introduction reagent, a linker such as PEG having an active ester that reacts with the amino group and a maleimide group that reacts with the thiol group at both ends is used. Thus, the semiconductor nanoparticle-containing resin particles and streptavidin are linked.

  Examples of the amino group introduction reagent include aminopropyltrimethoxysilane. The thiol group introduction reagent includes N-succinimidyl-S-acetylthioacetic acid, and the amino group introduction reagent and the addition itself can be performed by a known method.

  The tissue staining method of the present invention uses the staining agent for tissue staining, and is a fluorescent staining method for staining a biological material to be detected using the semiconductor nanoparticle-containing resin particles as a fluorescent label for immunostaining. is there. For example, in the case of immunostaining for a specific antigen, a method of producing a fluorescent label (conjugate) in which a semiconductor nanoparticle-containing resin particle and a primary antibody are directly bonded via the linker and staining the antigen (Primary antibody method), a method of producing a fluorescent label by directly binding a semiconductor nanoparticle-encapsulating resin particle and a secondary antibody, and using this fluorescent label for staining the primary antibody bound to the antigen (two Secondary antibody method), producing a fluorescent label by directly binding resin particles encapsulating semiconductor nanoparticles and biotin, and using the fluorescent label, a primary antibody and a secondary antibody modified with avidin or streptavidin are used as antigens. A method of staining the bound substance, and similarly, preparing a fluorescent label in which avidin or streptavidin is directly bound to the semiconductor nanoparticle-encapsulating resin particle, A method of staining with the union of secondary antibody biotin-modified -, or the like can be used (biotin avidin method or a sandwich method).

  Any primary antibody may be used for immunostaining, and it varies depending on the subject to be immunostained. For example, when performing immunostaining using HER2 as an antigen, an anti-HER2 antibody is used. In addition, any secondary antibody may be used, and varies depending on the primary antibody. Examples include anti-mouse, anti-rabbit, anti-cow, anti-goat, anti-sheep, anti-dog and anti-chicken antibodies.

  Any existing method may be used as the method for binding the semiconductor nanoparticle-containing resin particles to the antibody or biotin. For example, amidation by reaction of amine and carboxylic acid, sulfidation by reaction of maleimide and thiol, imination by reaction of aldehyde and amine, amination by reaction of epoxy and amine, etc. can be used. .

  The immunostaining described above is not limited to tissue staining, and can also be applied to cell staining. The biological substance to be detected is not particularly limited as long as a substance that specifically binds to the biological substance exists. Typically, a combination of the above-described antigen and antibody is used. For example, a combination of a nucleic acid molecule (oligonucleotide, polynucleotide) and a nucleic acid molecule having a sequence capable of hybridizing thereto can also be used.

(Fluorescence observation)
Fluorescence observation is performed by irradiating the tissue section subjected to tissue staining as described above with excitation light having an appropriate wavelength according to the type of semiconductor nanoparticles included in the semiconductor nanoparticle-containing resin particles. This is a method for observing emitted fluorescence. By detecting a predetermined biomolecule present in the tissue section by such a method, for example, it can be used as information for determining the necessity of application of an antibody drug such as Herceptin labeled with HER2. it can.

For irradiation with excitation light, the same irradiation means as in general fluorescence observation is used. For example, a predetermined wavelength may be selected from a laser light source provided in a fluorescence microscope as necessary.
The observation of fluorescence may be performed from a lens barrel of the fluorescence microscope, or may be performed by separately displaying an image captured by a camera installed in the fluorescence microscope on a display means (a monitor or the like). According to such a display means, although it depends on the type of semiconductor nanoparticles, even when fluorescence cannot be sufficiently observed by visual observation from the tube of the fluorescence microscope, it is possible to observe fluorescence through taking an image with a camera. May be possible. Moreover, you may use the filter which selectively passes a predetermined wavelength as needed.

EXAMPLES Hereinafter, although an Example demonstrates this invention in detail, this invention is not limited to this.
[Preparation Example] Preparation of CdSe / ZnS semiconductor nanoparticles Under argon flow, 7.5 g of tri-n-octylphosphine oxide was added with 2.9 g of stearic acid, 620 mg of n-tetradecylphosphonic acid, and 250 mg of cadmium oxide. The mixture was heated to 370 ° C. After allowing this to cool to 270 ° C., a solution of 200 mg of selenium dissolved in 2.5 mL of tributylphosphine was added, dried under reduced pressure, and a cadmium selenide (CdSe) core semiconductor nanoparticle coated with tri-n-octylphosphine oxide. Particles were obtained.

  To the obtained CdSe core semiconductor nanoparticles, 15 g of tri-n-octylphosphine oxide was added and heated. Subsequently, a solution of 1.1 g of zinc diethyldithiocarbamate dissolved in 10 mL of trioctylphosphine was added at 270 ° C., and CdSe / ZnS. A dispersion containing semiconductor nanoparticles was obtained.

[Example 1]
(Preparation of Melamine Resin Particles Encapsulating CdSe / ZnS Semiconductor Nanoparticles Having Carboxylate (Carboxylate Group) as Surface Modification Group (hereinafter referred to as “melamine resin nanoparticle 1”))
The CdSe / ZnS semiconductor nanoparticles obtained in the preparation examples were dispersed in decane so that the concentration was 5% by mass. Surface modification was performed by adding 0.5 mL of sodium propionate to 10 μL of this dispersion and stirring at room temperature. After adding 2.5 mL of pure water to the reaction mixture, the solution was stirred for 20 minutes while maintaining the temperature of the solution at 70 ° C. with a hot stirrer. To the solution after stirring, 1.5 g of melamine resin “Nicalak MX-035” (manufactured by Nippon Carbide Industries Co., Ltd.) was added, and the mixture was further heated and stirred under the same conditions for 5 minutes.

  100 μL of formic acid was added to the stirred solution and stirred for 20 minutes while maintaining the temperature of the solution at 60 ° C., and then the solution was left to cool to room temperature. The cooled solution was dispensed into a plurality of centrifuge tubes and centrifuged at 12,000 rpm for 20 minutes to precipitate the melamine resin nanoparticles contained in the solution as a mixture and remove the supernatant. Thereafter, the precipitated particles were washed with ethanol and water. Melamine resin nanoparticles 1 having an average particle size of 150 nm were prepared.

(Preparation of stain for tissue staining (hereinafter referred to as “stain 1”))
0.1 mg of surface melamine resin nanoparticle 1 treated with melamine resin nanoparticles 1 is collected and dispersed in 1.5 mL of ethanol, and 2 μL of aminopropyltrimethoxysilane (LS-3150, manufactured by Shin-Etsu Chemical Co., Ltd.) is added. In addition, an amination treatment was performed by converting the hydroxyl group present on the surface of the melamine resin nanoparticle 1 to an amino group by reacting for 8 hours.

  The concentration of the resulting melamine resin nanoparticles 1 was adjusted to 3 nM using phosphate buffered saline (abbreviation: PBS) containing 2 mM ethylenediaminetetraacetic acid (abbreviation: EDTA). SM (PEG) 12 (succinimidyl-[(N-maleimidopropionamide) -dodecaethylene glycol] ester; Thermo Fisher Scientific) to a final concentration of 10 mM with respect to the dispersion of the melamine resin nanoparticles 1 whose concentration has been adjusted. Fick Co., Ltd.) was mixed and reacted at 20 ° C. for 1 hour to obtain a mixed solution containing melamine resin nanoparticles 1 with maleimide at the end.

  The mixture was centrifuged at 10,000 G for 20 minutes, the supernatant was removed, PBS containing 2 mM EDTA was added to disperse the precipitate, and the mixture was centrifuged again. The above washing by the same procedure was performed three times to obtain a melamine resin nanoparticle 1 whose surface was maleimidized (hereinafter referred to as “maleimidated melamine resin nanoparticle 1”).

Streptavidin thiolated streptavidin (Wako Pure Chemical Industries, Ltd.) is subjected to thiol group addition using N-succinimidyl-S-acetylthioacetic acid (abbreviation: SATA) (Pierce), Thiolated streptavidin was obtained by removing excess reaction reagent by gel filtration.

Binding of Maleimide Melamine Resin Nanoparticle 1 and Thiolated Streptavidin After adding 10 nM maleimide melamine resin nanoparticle 1 and 1000 nM thiolated streptavidin into 1 mL PBS containing 2 mM EDTA, at room temperature Stir for 1 hour. Unreacted thiolated streptavidin and the like were removed using a gel filtration column for purification (column type; manufactured by Pierce) to obtain melamine resin nanoparticles bound to streptavidin (namely, staining agent 1).

[Comparative Example 1]
(Preparation of glass nanoparticles containing CdSe / ZnS semiconductor nanoparticles whose surface modifying molecule is dodecylamine)
The CdSe / ZnS semiconductor nanoparticles obtained in the preparation examples were dispersed in acetone as a poor solvent so that the concentration was 10% by mass, and CdSe / ZnS semiconductor nanoparticles were precipitated and separated by filtration. To 0.1 mg of this precipitate, 0.1 mg of dodecylamine and 1 mL of ethanol were added, and the surface was modified by vigorous stirring for 1 hour to obtain water-soluble semiconductor nanoparticles. By adding 0.1 mg of tetraethoxysilane (abbreviation: TEOS), 0.01 mL of water, and 0.03 mL of ammonia water to the water-soluble semiconductor nanoparticles, hydrolysis is performed, whereby glass nanoparticles having an average particle diameter of 160 nm ( Hereinafter, simply referred to as “glass nanoparticles”).
(Preparation of stain for tissue staining (hereinafter referred to as “stain 2”))
In Example 1, dyeing agent 2 was prepared in the same manner as in Example 1 except that glass nanoparticles were used instead of melamine resin nanoparticles 1.

[Comparative Example 2]
(Preparation of melamine resin nanoparticles encapsulating fluorescent dye with carboxylate)
After 2.5 mg of fluorescein (manufactured by Nacalai Tesque) was dissolved in 22.5 mL of pure water, the solution was stirred for 20 minutes while maintaining the temperature of the solution at 70 ° C. with a hot stirrer. To the solution after stirring, 1.5 g of melamine resin “Nicalak MX-035” (manufactured by Nippon Carbide Industries Co., Ltd.) was added, and the mixture was further heated and stirred under the same conditions for 5 minutes.

100 μL of formic acid was added to the stirred solution and stirred for 20 minutes while maintaining the temperature of the solution at 60 ° C., and then the solution was left to cool to room temperature. The cooled solution was dispensed into a plurality of centrifuge tubes and centrifuged at 12,000 rpm for 20 minutes to precipitate the melamine resin nanoparticles contained in the solution as a mixture and remove the supernatant. Thereafter, the precipitated particles were washed with ethanol and water to obtain melamine resin nanoparticles having an average particle size of 155 nm.
(Preparation of stain for tissue staining (hereinafter referred to as “stain 3”))
In Example 1, a staining agent 3 was prepared in the same manner as in Example 1 except that the melamine resin nanoparticles were used in place of the melamine resin nanoparticles 1.

[Example 2]
Breast cancer tissue HER2 staining was performed using the staining agent 1 obtained in Example 1.
A tissue array (USBiomax; BR243) in which a breast cancer tissue section is spotted on a slide is subjected to normal deparaffinization and hydrophilization treatment, and then reacted in the order of anti-HER2 antibody, biotinylated anti-mouse antibody, and stain 1. It was. Next, nuclear staining using hematoxylin was performed here. After dipping in the order of ethanol and xylene, it was sealed with a non-aqueous mounting medium to give an observation slide.

The presence or absence of coloring of the immersion liquid during xylene immersion was visually observed. The case where the coloring was “absent” was marked with “◯”, and the case where “colored” was marked “x”.
Using a fluorescent microscope “BX-53” (manufactured by Olympus Corporation), an immunostained image (400 ×) was photographed using a digital camera “DP73” (manufactured by Olympus Corporation) attached to the fluorescent microscope. Using. Corresponding to the Qdot 655 used as the phosphor, the wavelength of the excitation light to be irradiated is set to 415 to 455 nm using an optical filter for excitation light (“QD655-C” manufactured by Optline Co., Ltd.) provided in the fluorescence microscope. And the wavelength of the fluorescence to observe was set to 648-663 nm using the optical filter for fluorescence. The intensity of the excitation light at the time of observation and image photographing with a fluorescence microscope was such that the irradiation energy near the center of the visual field was 30 W / cm ² . The exposure time at the time of image shooting was adjusted within a range in which the luminance of the image was not saturated, and was set to 400 seconds, for example. Three fields of view were photographed per slide, and the fluorescence intensity was the average value thereof.

  The fluorescence intensity was measured by processing the captured image using image processing software “ImageJ” (open source). A region where the luminance of the fluorescence emitted from the phosphor is a predetermined value or more was extracted, and the total of the fluorescence intensities constituting the region was obtained. The case where the fluorescence intensity was 90 or more was marked with ◎, the case where it was 50 to 89 was marked with ○, and the case where it was 49 or less was marked with ×.

A change in fluorescence intensity before and after irradiation when irradiation with observation excitation light for 10 minutes, that is, a relative value of fluorescence intensity after irradiation with respect to before irradiation was obtained. The case where the relative value of the fluorescence intensity was 90 or more was evaluated as “◯”, the case of 60 to 89 as “Δ”, and the case of 59 or less as “X”.
The results are shown in Table 1.

Example 3
(Preparation of polylactic acid particles encapsulating CdSe / ZnS semiconductor nanoparticles having ammonium groups as surface modifying groups (hereinafter referred to as “polylactic acid nanoparticles”))
The CdSe / ZnS semiconductor nanoparticles obtained in the preparation examples were dispersed in decane so that the concentration was 5% by mass. Surface modification was performed by adding 0.5 mL of 2-aminoethanethiol hydrochloride to 10 μL of this dispersion and stirring at room temperature.

  100 mg of polylactic acid (Mn 4900, manufactured by Wako Pure Chemical Industries, Ltd.) was dissolved in 2 to 6 mL of acetone, and the surface-modified CdSe / ZnS semiconductor nanoparticles were added to this solution. This was allowed to stand at room temperature for 30 minutes, and then added to 40 mL of water with stirring using a pipette to prepare a suspension of polylactic acid nanoparticles. 1 mL of 0.5 M sodium citrate aqueous solution and 100 μL of Tween 80 [polyoxyethylene (20) sorbitan monooleate] aqueous solution (200 mg / mL) were added to the obtained suspension of polylactic acid nanoparticles. Thereafter, the polylactic acid nanoparticles were purified by subjecting the suspension of the polylactic acid nanoparticles to ultrafiltration (YM-50, manufactured by Amicon) and further filter filtration (5 μm; manufactured by Millipore). Polylactic acid nanoparticles having an average particle size of 160 nm were prepared.

(Preparation of stain for tissue staining)
In Example 1, instead of melamine resin nanoparticles 1, polylactic acid nanoparticles were used, and instead of aminopropyltrimethoxysilane (LS-3150, manufactured by Shin-Etsu Chemical Co., Ltd.) 2 μL, diaminoethane ( Polylactic acid resin nanoparticles bound with streptavidin in the same manner as in Example 1 except that 2 μL and 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide hydrochloride were used. (That is, Stain 5) was prepared.

Example 4
In Example 2, the breast cancer tissue HER2 staining and the fluorescence intensity were measured in the same manner as in Example 2 except that the stain 5 was used instead of the stain 1.
The results are shown in Table 1.

[Comparative Example 3]
In Example 2, the breast cancer tissue HER2 staining and the fluorescence intensity were measured in the same manner as in Example 2 except that the stain 2 was used instead of the stain 1.
The results are shown in Table 1.

[Comparative Example 4]
In Example 2, the breast cancer tissue HER2 staining and the fluorescence intensity were measured in the same manner as in Example 2 except that the stain 3 was used instead of the stain 1.
The results are shown in Table 1.

Example 5
A tissue array (BR243 manufactured by USBiomax) in which a breast cancer tissue section was spotted on a slide was subjected to normal deparaffinization and hydrophilization treatment, and then reacted in the order of anti-Ki67 antibody, biotinylated anti-mouse antibody, and stain 1. . Next, nuclear staining using hematoxylin was performed here. After dipping in the order of ethanol and xylene, it was sealed with a non-aqueous mounting medium to give an observation slide.

Using a fluorescence microscope (Olympus Co., Ltd.), a fluorescence image (immunostaining image (400 times)) was photographed in the same manner as in Example 2, and the fluorescence intensity was measured.
After the observation slide was left in a cool dark place for 6 months, a fluorescent image was taken again, the fluorescence intensity was measured, and the relative value to the fluorescence intensity before storage was determined.
The results are shown in Table 2.

Example 6
(Preparation of Melamine Resin Nanoparticles Encapsulating CdSe / ZnS Semiconductor Nanoparticles with Sulfonate (Sulfonate Group) as Surface Modification Group (hereinafter referred to as “melamine resin nanoparticle 2”))
The CdSe / ZnS semiconductor nanoparticles obtained in the preparation examples were dispersed in decane so that the concentration was 5% by mass. Surface modification was carried out by adding 0.5 mL of sodium 3-mercapto-1-propanoate sulfonate to 10 μL of this dispersion and stirring at room temperature. After adding 2.5 mL of pure water to the reaction mixture, the solution was stirred for 20 minutes while maintaining the temperature of the solution at 70 ° C. with a hot stirrer. To the solution after stirring, 1.9 g of melamine resin “Nicalak MX-035” (manufactured by Nippon Carbide Industries Co., Ltd.) was added, and the mixture was further heated and stirred under the same conditions for 5 minutes.

100 μL of formic acid was added to the stirred solution and stirred for 20 minutes while maintaining the temperature of the solution at 60 ° C., and then the solution was left to cool to room temperature. The cooled solution was dispensed into a plurality of centrifuge tubes and centrifuged at 12,000 rpm for 20 minutes to precipitate the melamine resin nanoparticles contained in the solution as a mixture and remove the supernatant. Thereafter, the precipitated particles were washed with ethanol and water. Melamine resin nanoparticles 2 having an average particle diameter of 150 nm were prepared.
(Preparation of stain for tissue staining (hereinafter referred to as “stain 4”))
In Example 1, dyeing agent 4 was prepared in the same manner as in Example 1 except that melamine resin nanoparticle 2 was used instead of melamine resin nanoparticle 1.

Example 7
In Example 5, the breast cancer tissue Ki67 staining and the fluorescence intensity were measured in the same manner as in Example 5 except that the stain 4 was used instead of the stain 1.
The results are shown in Table 2.

Claims (3)

A semiconductor nanoparticle having a surface modification group and a resin particle including the semiconductor nanoparticle , the surface of the semiconductor nanoparticle having the surface modification group has a negative charge, and the functional group of the resin particle is positive. Semiconductor nanoparticle-containing resin particles , wherein the resin particles are melamine resin particles. Tissue staining for stain consisting of semiconductor nanoparticles encapsulating resin particles of claim 1. A tissue staining method using the stain for tissue staining according to claim 2 .