JP2008247713A - Fluorescent coloring material-containing nano-silica particle, and method for preparing the same - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a new method for efficiently and stably preparing fluorescent coloring material-containing nano-silica particles having high fluorescent intensity, to provide fluorescent coloring material-containing nano-silica particles obtained by the method, and to provide its application as a detection reagent. <P>SOLUTION: The fluorescent coloring material-containing nano-silica particles are prepared through the following stages (a) to (d): a stage (a) where a fluorescent coloring material bonding group is introduced into the surface of each nano-silica particle via the OH group thereof; a stage (b) where each nano-silica particle obtained by the above stage is reacted with a compound having fluorescent coloring material molecules, so as to bond a fluorescent coloring material to the surface of each nano-silica particle; a stage (c) where each fluorescent coloring material-bonded nano-silica particle obtained by the above stage is reacted with a silica compound, so as to introduce the silica compound into the fluorescent coloring material-bonded group in the surface of each fluorescent coloring material-bonded nano-silica particle; and a stage (d) where each nano-silica particle obtained by the above stage is reacted with a silane compound, so as to form a silica film on the surface of each nano-silica particle. <P>COPYRIGHT: (C)2009,JPO&INPIT

Description

  The present invention relates to a fluorescent dye-containing nanosilica particle useful as a detection reagent and a method for preparing the same. More specifically, the present invention relates to a fluorescent dye-containing nanosilica particle having a high fluorescence intensity while having a particle size of several tens of nm or less, particularly 30 nm or less or 10 nm or less, and a method for preparing the same.

  In recent years, various biochemical examination methods using fine particles containing a fluorescent dye as a detection reagent have been studied. For example, the alpha screen technique has been commercialized by Packard (Non-Patent Document 1, Patent Documents 1-7). ). This is made using latex donor beads (registered trademark) and acceptor beads (registered trademark) with a diameter of 250 nm. After the donor beads and acceptor beads are combined, The singlet oxygen molecule is generated by the fluorescence from the fluorescent molecule, and this chemically reacts with the fluorescent substance in the acceptor bead to generate chemiluminescence, and this is observed.

  In addition, detection reagents using silica spheres as the beads (fine particles) have been studied, and various methods for producing silica spheres with fluorescent dye molecules in the interior have been proposed. Such silica spheres have a form in which fluorescent dye molecules held inside are surrounded by silica, and as a result, quenching by external factors (for example, absorption of excitation energy by biochemical polymers) can be suppressed. Therefore, it is expected to be applied to various tests as a highly sensitive detection reagent.

  As a typical method for preparing this reagent, APS-FITC [N, wherein fluorescein isothiocyanate (FITC) is directly bonded to 3- (aminopropyl) triethoxysilane (APS: 3- (aminopropyl) triethoxysilane] in advance. There is a method in which -1- (3-triethoxysilylpropyl) -N'-fluoresceyl thiourea] is reacted with N-tris (hydroxylmethyl) methyl-2-aminoethane sulfonic acid (TES) in an aqueous ethanol solution containing ammonia (non-patent literature) 2). According to this method, a high concentration of fluorescent dye molecules (FITC) can be retained inside the silica sphere, but at the maximum concentration, it has also been reported that quenching occurs inside the silica sphere (non-) Patent Document 2). In addition, such a method has the disadvantages that FITC and APS are not so high in bonding ability and have poor production efficiency, and that only a single particle size and a silica sphere larger than several hundred nanometers can be obtained.

As a method for solving such a problem, the present inventors chemically bonded a fluorescent molecule to a silica compound having an amino group such as APS, using a succinimidyl ester compound in which a fluorescent dye such as FITC is bonded to succinimide, Next, a method for preparing silica particles containing a fluorescent dye by polymerizing a silica compound such as tetraethoxysilane (TEOS) has been proposed (Patent Document 8). This method is suitable for the preparation of relatively large fluorescent dye-containing silica particles. However, when silica particles of 10 nm or less are used as raw materials, only one or two fluorescent dye molecules can be introduced. This method is not suitable as a method for preparing a reagent having high fluorescence intensity using silica particles having a small particle diameter.
US 4918200 A WO 917087 A1 EP 502060 A1 Japanese Patent Laid-Open No. 5-501611 US 5252743 A US 5451683 A US 5482867 A WO 2006/070582 Analytica Chimica Acta 1998, 367, 159 A. Imhof, et al., “Spectroscopy of Fluorescein (FITC) Dyed Colloidal Silica Spheres”, J. Phys. Chem. B 1999,103, 1408-1415

  As a fluorescent agent for modifying micromolecules such as DNA, (1) the particle size is a few tens of nm or less, particularly 30 nm or less or 10 nm or less, (2) high fluorescence intensity, and (3) For example, a fluorescent agent using CdSe that does not cause toxicity is required. However, as described above, when a fluorescent dye-containing nanosilica particle having a particle size of 10 nm or less is prepared by a conventional method (for example, Patent Document 8), the fluorescence intensity is greatly reduced, and a highly sensitive reagent (fluorescent agent) is produced. There was a problem that it could not be obtained. This is because the volume of silica particles to be prepared is too small, so there is little space for introducing fluorescent dye molecules when polymerizing a low molecular silica compound, and only about 1 or 2 fluorescent dye molecules per silica particle enter. This is thought to be the cause.

  It is an object of the present invention to provide fluorescent dye-containing nanosilica particles having a high fluorescence intensity while having a particle size of 30 nm or less, particularly 10 nm or less, which is difficult to prepare by such conventional methods. . Furthermore, an object of the present invention is to provide a method for efficiently and stably preparing such fluorescent dye-containing nanosilica particles.

The inventors of the present invention have been diligently studying day and night to achieve the above object. As a result, an extremely small silica particle (nanosilica particle) having a particle size of several nm is used as a nucleus, and an amino group (—NH ₂ ) is formed on the surface. , A silane coupling reagent having a carboxyl group (—COOH), an isothiocyanate group (—N═C═S) or an ester group (—COOR) is bonded to the surface of the silica particle via these groups. To the amino group (—NH ₂ ), carboxyl group (—COOH), isothiocyanate group (—N═C═S) or ester group (—COOR) that is not bound to the fluorescent dye on the surface. By reacting the silica compound to form a silica film on the surface of the silica particles labeled with the fluorescent dye, the fluorescent dye-containing nanosilica particles suitable for the above purpose, that is, the particle size is several tens nm or less, particularly 30 nm or less. And sizes below 10nm A fluorescent dye-containing nanosilica particles having a high fluorescence intensity, yet is, moreover fluorescent dye on the surface of nano silica particles was confirmed that an indicator reagent comprising held stably (fluorescent probe) is obtained. In the method, before binding the fluorescent dye to the surface of the nanosilica particle, by binding a molecule that does not react with the fluorescent dye (also referred to as “spacer molecule” in the present invention) to the surface of the nanosilica particle, It was confirmed that the binding amount of the fluorescent dye can be adjusted and concentration quenching (decrease in fluorescence intensity) based on the increase in the binding amount can be prevented. The present invention has been completed based on such findings.

The present invention includes the following embodiments:
(I) Method for preparing fluorescent dye-containing nanosilica particles (I-1). A method for preparing fluorescent dye-containing nanosilica particles, comprising the following steps (a) to (d);
(a) introducing a fluorescent dye binding group into the surface of the nanosilica particles (1) via the OH group;
(b) reacting the nanosilica particles (2) obtained in the above step with a compound (3) having a fluorescent dye molecule to bind the fluorescent dye to the surface of the nanosilica particles;
(c) reacting the fluorescent dye-bonded nanosilica particles (4) obtained in the above step with a silica compound (5) to introduce the silica compound (5) onto the surface of the nanosilica particles (4); and
(d) A step of reacting the nanosilica particles (6) obtained in the above step with a silane compound (7) to form a silica film on the surfaces of the nanosilica particles (6).
(I-2). “Succinimidyl ester compound (3)” in which a fluorescent dye molecule and succinimide are bonded via an ester bond (—CO—O—) as the “compound having a fluorescent dye molecule (3)”,
Using the `` silica compound (5) having a trialkoxysilyl group '' as the `` silica compound (5) '' and the `` tetraalkoxysilane (7) '' as the `` silane compound (7) '',
(I-1) The preparation method of the fluorescent-dye containing nano silica particle described.
(I-3). After the step (a), the following step (a ′) is included, and then the step (b) is performed on the nanosilica particles (2 ′) obtained in the step (a ′). -1) or preparation method of the fluorescent dye-containing nanosilica particles described in (I-2):
(a ′) The compound (8) that binds to the fluorescent dye-binding group is reacted with the nanosilica particle (2) obtained in the step (a) to obtain one of the fluorescent dye-binding groups on the surface of the nanosilica particle (2). Protecting the part.
(I-4). The method for preparing fluorescent dye-containing nanosilica particles according to any one of (I-1) to (I-3), wherein the fluorescent dye-binding group is an amino group, a carboxyl group, an isothiocyanate group, or an ester group.

(II) Preparation method of fluorescent dye-containing nanosilica particles (II-1). Fluorescent dye-containing nanosilica particles in which fluorescent dye molecules are bonded to nanosilica particles,
The nano silica particles have a particle size of 20 nm or more and less than 30 nm, and
Fluorescent dye-containing nanosilica particles characterized in that 100 or more fluorescent dye molecules are bonded to one nanosilica particle.
(II-2). Fluorescent dye-containing nanosilica particles in which fluorescent dye molecules are bonded to nanosilica particles,
Fluorescent dye-containing nanosilica particles, wherein the nanosilica particles have a particle shape of 10 nm or more and less than 20 nm, and 50 or more fluorescent dye molecules are bonded to one nanosilica particle.
(II-3). Fluorescent dye-containing nanosilica particles in which fluorescent dye molecules are bonded to nanosilica particles,
The nanosilica particles have a particle size of less than 10 nm, and
Two or more fluorescent dye molecules are bonded to one nanosilica particle, and the fluorescent dye-containing nanosilica particles are characterized in that
(II-4). Fluorescent dye-containing nanosilica particles in which fluorescent dye molecules are bonded to nanosilica particles,
The fluorescence intensity in the dispersed aqueous solution is the same as the concentration of the fluorescent dye molecules bound to the nanosilica particles, and the fluorescence in the aqueous solution in which free fluorescent dye molecules not bound to the nanosilica particles are dispersed. Fluorescent dye-containing nanosilica particles characterized by higher than strength.
(II-5). The fluorescent dye-containing nanosilica particles according to any one of (II-1) to (II-4), wherein the fluorescent dye molecules bonded to the nanosilica particles are further covered with a silica layer.
(II-6). The fluorescent dye-containing nanosilica particles according to any one of (II-1) to (II-5), which are prepared by any one of the methods (I-1) to (I-4).

  In addition, a nanoparticle generally means a particle having a particle size of about 1 to 100 nm which is nano-order. Nanosilica particles targeted by the present invention also mean those having a normal particle size of about 1 to 100 nm according to such technical common sense, but preferably less than 100 nm, preferably 50 nm or less, more preferably 30 nm or less, more preferably 20 nm or less, Particularly preferred are particles of 10 nm or less. As a method for measuring the particle size (diameter) of the particles, a transmission electron microscope in which the target particles are dispersed in pure water and then dropped on a copper grid on which carbon is vapor-deposited and measured with an electron beam. The law can be mentioned.

  According to the present invention, it is possible to provide fluorescent dye-containing nanosilica particles having high fluorescence intensity while having a particle size of a few tens of nm or less, particularly 30 nm or less or 10 nm or less. Furthermore, according to the method of the present invention, such fluorescent dye-containing nanosilica particles can be prepared efficiently and stably.

  The fluorescent dye-containing nanosilica particles provided by the present invention can be effectively used as a highly sensitive labeling reagent (fluorescent probe) that can be used in a minute region (for example, a micro region such as a cell) based on the above characteristics. Can do. For example, the fluorescent dye-containing nanosilica particles of the present invention can be used alone or in combination with a ligand to assay biological substances such as haptens, antigens, antibodies, enzymes, nucleic acids (DNA, RNA), etc. Or research), affinity purification and cell separation. In addition, it is useful as a fluorescent probe for detecting and identifying various gene polymorphisms whose association with diseases is becoming clear.

  Labeling reagents that compete with the fluorescent dye-containing nanosilica particles provided by the present invention in the market include nanoparticles using organic dyes, and nanoparticles using CdSe (cadmium selenide) (for example, “Qdot (Quantum Dot) Registered trademark) ”and the like. Since organic dyes have problems in fluorescence efficiency and fading, the market is being replaced by the latter “Qdot (registered trademark)”, but CdSe is a highly toxic substance, so there are concerns about environmental problems. . In contrast, the fluorescent dye-containing nanosilica particles of the present invention are a labeling reagent that is excellent in luminance and fading property, and has high safety and low environmental impact because it uses a non-toxic oxide material. In addition, by preparing commercially available nanosilica particles having a particle size adjusted in advance as raw materials, it is possible to provide homogeneous fluorescent dye-containing nanosilica particles having a uniform particle size. In addition, by selecting the particle size of the nanosilica particles to be used as a raw material, it is possible to create reagents for nanoparticles with the same color emission and different sizes, and to provide users with labeling reagents suitable for the application. .

  Table 1 below shows the superiority of the fluorescent dye-containing nanosilica particles of the present invention (product of the present invention) in comparison with organic dyes and “Qdot (registered trademark)”.

(1) Method for Preparing Fluorescent Dye-Containing Nanosilica Particles The method for preparing the fluorescent dye-containing nanosilica particles of the present invention is a method using the core nanosilica particles (1) as a raw material, and includes the following (a) to (d): It has the process.
(a) introducing a fluorescent dye binding group into the surface of the nanosilica particles (1) via the OH group;
(b) reacting the nanosilica particles (2) obtained in the above step with a compound (3) having a fluorescent dye molecule to bind the fluorescent dye to the surface of the nanosilica particles;
(c) A fluorescent dye that is not bound to a fluorescent dye on the surface of the fluorescent dye-bonded nanosilica particle (4) by reacting the fluorescent dye-bonded nanosilica particle (4) obtained in the above step with a silica compound (5) Introducing the silica compound (5) into a bonding group; and
(d) A step of reacting the nanosilica particles (6) obtained in the above step with a silane compound (7) to form a silica film on the surfaces of the nanosilica particles (6).

  Hereinafter, the preparation method of the present invention will be described for each step.

Step (a)
The particle diameter (diameter) of the nanosilica particles (1) used as a raw material in the preparation of the fluorescent dye-containing nanosilica particles of the present invention is not particularly limited. For the purpose of the present invention to prepare fine fluorescent dye-containing nanosilica particles having a particle size (diameter) of 100 nm or less, preferably 50 nm or less, more preferably 30 nm or less, more preferably 20 nm or less, particularly preferably 10 nm or less, usually 30 nm. Less than, preferably less than 20 nm, more preferably less than 10 nm, still more preferably 9 nm or less, 8 nm or less, 5 nm or less, and particularly preferably 3 nm or less.

Nano silica particles having such a particle size can be obtained commercially. For example, LUDOX® TM-50 colloidal silica (average particle size 22 nm, specific surface area 140 m ² / g or less, 50 wt% suspension), LUDOX® HS-40 colloidal silica (average particle size 12 nm, ratio Surface area 220 m ² / g or less, 40 wt% suspension), LUDOX® HS-30 colloidal silica (average particle size 7.2 nm, specific surface area 220 m ² / g or less, 30 wt% suspension), and LUDOX (registered) (Trademark) AS-40 colloidal silica (average particle size 19.2 nm, specific surface area 135 m ² / g or less, 40 wt% suspension) (all of which are manufactured by LUDOX) (for example, BH Milosavlievic, SM Pimblot) , and D. Meisel, J. Phys. Chem. B 108, 6996-7001, 2004.)).

The nanosilica particles (1) may be previously treated with sodium silicate (Na ₂ O · nSiO ₂ : n = 0.5 to 4) before introducing a fluorescent dye-binding group described later on the surface thereof, By performing such treatment, the surface area of the nanosilica particles (1) can be increased and more fluorescent dye-binding groups can be introduced onto the surface. As the fluorescent dye binding group becomes a binding site with the fluorescent dye molecule as the name suggests, more fluorescent dye binding groups are introduced to bind more fluorescent dyes to the surface of the nanosilica particles. As a result, the fluorescence intensity can be enhanced (sensitized).

The sodium silicate used here (Na ₂ O · nSiO ₂ : n = 0.5-4) is an alkaline aqueous solution containing about 20-30% by weight of SiO ₂ , specifically, sodium silicate manufactured by Aldrich. An aqueous solution (Sodium silicate solution, Contains ~ 14% NaOH, ~ 27% SiO ₂ ) can be mentioned. The treatment of the nanosilica particles (1) with sodium silicate is not limited, but as an example, an aqueous solution containing nanosilica particles (1), for example, an aqueous sodium silicate solution diluted to about 0.54% by weight, has a concentration of 0.06 to 0.05. The mixture can be added at a ratio of about wt% and mixed and stirred at room temperature or about 60 ° C. for 10 minutes to 1 hour.

Step (a) is a step of introducing a fluorescent dye-binding group onto the surface of the nanosilica particle (1) via its OH group. The fluorescent dye-binding group is not limited as long as it is a group that binds to the fluorescent dye, but is usually an amino group (—NH ₂ ), a carboxyl group (—COOH), an isothiocyanate group (—N═C═S), and an ester. And a group (-COOR). An amino group is preferred.

For example, as a method for introducing an amino group (—NH ₂ ) as a fluorescent dye binding group onto the surface of the nanosilica particle (1), the nanosilica particle (1) and 3- (aminopropyl) triethoxy are represented by the following formula: Silanes or amino such as 3- [2- (2-aminoethylamino) ethylamino] propyl-triethoxysilane, N-2 (aminoethyl) 3-aminopropylmethyldimethoxysilane, 3-aminopropyltrimethoxysilane The method of making it react with the silica compound which has group can be mentioned.

  The reaction between the nanosilica particles (1) and the silica compound having an amino group can be carried out using a water-soluble solvent by mixing and stirring at room temperature or under heating conditions of about room temperature to 60 ° C. Moreover, the ratio of the nano silica particle (1) used for the reaction and the silica compound having an amino group is not limited, but is preferably 1 to 0.6 mol of the silica compound having an amino group with respect to 1 mol of the nano silica particle (1). Can exemplify a range of 0.5 to 0.3 mol.

  As a method for introducing an isothiocyanate group (-N = C = S) as a fluorescent dye binding group onto the surface of the nanosilica particle (1), 3-thiocyanatopropyltriethoxysilane (3- Examples thereof include a method in which a silica compound having an isothiocyanate group such as Thiocyanato propyl triethoxy silane) is reacted. In the same manner as described above, this reaction can be carried out by mixing and stirring with a water-soluble solvent at room temperature or under heating conditions of about room temperature to 60 ° C. Moreover, the ratio of the nano silica particle (1) used in the reaction and the silica compound having an isothiocyanate group is not limited, but 1 to 0.6 mol of the silica compound having an isothiocyanate group with respect to 1 mol of the nano silica particle (1). Preferably, a range of 0.5 to 0.3 mol can be exemplified.

  Moreover, as a method for introducing a carboxyl group (—COOH) as a fluorescent dye binding group onto the surface of the nanosilica particle (1), the above-described nanosilica particle (1) and 3-thiocyanatopropyltriethoxysilane (3- A compound having an amino group and a carboxyl group such as L-alanine after reacting with a silica compound having an isothiocyanate group such as Thiocyanato propyl triethoxy silane) and introducing an isothiocyanate group (-N = C = S) on the surface Can be mentioned. Thus, the isothiocyanate group on the surface of the nanosilica particle (1) reacts with the amino group of the compound to introduce a carboxyl group into the particle. This reaction can be carried out by mixing and stirring at room temperature or from about room temperature to 60 ° C. under a heating condition using a water-soluble solvent as described above. Moreover, the ratio of the nano silica particle (1) used for the reaction and the silica compound having a carboxyl group is not limited, but it is preferably 3 to 1 mol of the silica compound having a carboxyl group with respect to 1 mol of the nano silica particle (1). Can be exemplified by a range of 2.6 to 1.5 mol.

  Furthermore, as a method for introducing an ester group (—COOR) as a fluorescent dye binding group onto the surface of the nanosilica particle (1), the nanosilica particle (1), the nanosilica particle (1) described above, and 3-thiocyanato It reacts with a silica compound having an isothiocyanate group such as propyltriethoxysilane (3-Thiocyanato propyl triethoxysilane) to bond an isothiocyanate group (-N = C = S) to the surface, and then a carboxyl such as L-alanine. Silica having an ester group such as N-hydroxysucinimide after reacting the amino group of the compound having a group and an amino group with the isothiocyanate group on the surface of the nanosilica particle (1) to introduce a carboxyl group An example is a method in which a compound is esterified by bonding a carboxyl group on the surface using WSC as a catalyst. Similarly to the above, this reaction can also be performed by mixing and stirring at room temperature or under heating conditions of about room temperature to 60 ° C. using a water-soluble solvent. Moreover, the ratio of the nano silica particle (1) used for the reaction and the silica compound having an ester group is not limited, but it is preferably 3 to 1 mol of the silica compound having an ester group with respect to 1 mol of the nano silica particle (1). Can be exemplified by a range of 2.6 to 1.5 mol.

Step (b)
In the step (b), the nanosilica particles (2) having a fluorescent dye binding group on the surface prepared in the above step (a) are reacted with the compound (3) having a fluorescent dye molecule, and the surface of the nanosilica particles is reacted. This is a step of binding a fluorescent dye.

  As an example of the compound (3) having a fluorescent dye molecule, a succinimidyl ester compound (3) in which a fluorescent dye molecule and succinimide are bonded via an ester bond (—CO—O—) can be given. Here, examples of the succinimidyl ester compound (3) include compounds represented by the following general formula.

  Here, R means a fluorescent dye molecule. More specifically, R can be bonded to succinimide via an ester bond (—CO—O—) as shown in the following formula.

(In the formula, R represents a fluorescent dye molecule, and R ′ represents a hydrogen atom or an arbitrary group.)
Specifically, as the above R, as shown in the above formula, a carboxylic acid or a derivative thereof is formed by bonding a —COOR ′ group (R ′ means a hydrogen atom or an arbitrary group) as a side chain. Can be listed. Examples of the carboxylic acid or derivative thereof shown as compound (0) in the above include, for example, 5-carboxy-fluorescein, 6-carboxy-fluorescein, 5 (6) -carboxy-fluorescein, 6-carboxy-2 ′, 4,4 ', 5', 7,7'-Hexachlorofluorescein, 6-carboxy-2 ', 4,7,7'-tetrachlorofluorescein, 6-carboxy-4', 5'-dichloro-2 ', 7'-dimethoxy Fluorescein, 5-carboxy-rhodamine, 6-carboxy-rhodamine, 5 (6) -carboxy-rhodamine, Alexa Fluor 350 carboxylic acid, Alexa Fluor 405 carboxylic acid, Alexa Fluor 430 carboxylic acid, Alexa Fluor 488 carboxylic acid, Alexa Fluor 500 Carboxylic acid, Alexa Fluor 514 carboxylic acid, Alexa Fluor 532 carboxylic acid, Alexa Fluor 546 carboxylic acid, Alexa Fluor 555 carboxylic acid, Alexa Fluor 568 carboxylic acid, Alexa Fluor 594 carboxylic acid, Alexa Fluor 610 carboxylic acid, Alexa Fluor 633 carboxylic acid, Alexa Fluor 647 carboxylic acid, Alexa Fluor 660 carboxylic acid, Alexa Fluor 680 carboxylic acid, Alexa Fluor 700 carboxylic acid, Alexa Fluor 750 carboxylic acid, Alexa Fluor 790 carboxylic acid, and the like. Preferred are red pigments such as 5-carboxy-rhodamine, 6-carboxy-rhodamine and 5 (6) -carboxy-rhodamine.

  The succinimidyl ester compound (3) used in the step (b) of the present invention is obtained by esterifying a carboxylic acid or a derivative thereof [compound (0)] and N-hydroxysuccinimide according to a conventional method as shown in the above formula. It can be prepared by reacting. However, it can also be obtained commercially for convenience.

  Specifically, as the succinimidyl ester compound (3), 5-succinimidyl ester-fluorescein, 6-succinimidyl ester-fluorescein, 5 corresponding to the carboxylic acid or derivative thereof [compound (1)] (6) -succinimidyl ester-fluorescein, 6-succinimidyl ester-2 ', 4,4', 5 ', 7,7'-hexachlorofluorescein, 6-succinimidyl ester-2', 4,7 , 7'-tetrachlorofluorescein, 6-succinimidyl ester-4 ', 5'-dichloro-2', 7'-dimethoxyfluorescein, 5-succinimidyl ester-rhodamine, 6-succinimidyl ester-rhodamine, 5 (6) -succinimidyl ester-rhodamine, succinimidyl ester-Alexa Fluor 350, succinimidyl ester-Alexa Fluor 405, succinimidyl ester-Alexa Fluor 430, Cuccinimidyl ester-Alexa Fluor 488, Succinimidyl ester-Alexa Fluor 500, Succinimidyl ester-Alexa Fluor 514, Succinimidyl ester-Alexa Fluor 532, Succinimidyl ester-Alexa Fluor 546, Succinimidyl ester-Alexa Fluor 555, succinimidyl ester-Alexa Fluor 568, succinimidyl ester-Alexa Fluor 594, succinimidyl ester-Alexa Fluor 610, succinimidyl ester-Alexa Fluor 633, succinimidyl ester-Alexa Fluor 647, succin Imidyl ester-Alexa Fluor 660, Succinimidyl ester-Alexa Fluor 680, Succinimidyl ester-Alexa Fluor 700, Succinimidyl ester-Alexa Fluor 750, Succinimidyl ester-Alexa Fluor 790 or DY-495 / from DYOMICS 5-MHS-Ester, DY-495 / 6-NHS-Ester, DY-500-XL-NHS-EsterDY-505 / 5-NHS-Ester, DY-505 / 6-NHS-Ester, DY -550-NHS-Ester, DY-555-NHS-Ester, DY-610-NHS-Ester, DY-615-NHS-Ester, DY-630-NHS-Ester, DY-631-NHS-Ester, DY-633 -NHS-Ester, DY-635-NHS-Ester, DY-636-MHS-Ester, DY-650-NHS-Ester, DY-651-NHS-Ester, DYQ-660-NHS-Ester, DYQ-661-NHS -Ester, DY-675-NHS-Ester, DY-676-NHS-Ester, DY-680-NHS-Ester, DY-681-NHS-Ester, DY-700-NHS-Ester, DY-701-NHS-Ester , DY-730-NHS-Ester, DY-731-NHS-Ester, DY-750-NHS-Ester, DY-751-MHS-Ester, DY-776-NHS-Ester, DY-781-NHS-Ester, DY -782-NHS-Ester, etc. Preferably, Rhodamin Red-X, succinimidylester (Molecular Probes R6160, Invitorgen) can be exemplified.

  The reaction between the succinimidyl ester compound (3) and the nanosilica particles (2) can be performed by dissolving in a solvent such as DMSO or water and then reacting with stirring at room temperature. The ratio of the succinimidyl ester compound (3) and the nanosilica particles (2) used in the reaction is not particularly limited, but preferably the succinimidyl ester compound (3) 3 per 1 mol of the nanosilica particles (2). A proportion of ˜1 mol, more preferably 2.6 to 1.5 mol can be mentioned.

When the nanosilica particle (2) has an amino group (—NH ₂ ) on the surface, as shown in the following formula, the carbonyl group of the succinimidyl ester compound (3) and the amino group of the nanosilica particle (2) Amide bonds (—NH—CO—) bind the fluorescent dye molecules, and nanosilica particles (4) with the fluorescent dye bonded to the surface are generated.

When the nanosilica particle (2) has an amino group (-NH ₂ ) on the surface, in addition to the amide bond (-NH-CO-) above, via a urethane bond (-NH-CO-O-) Fluorescent dye molecules can also be immobilized.

  In this case, specifically, the silica group (2) is produced by the urethane bond (—NH—CO—O—) between the amino group of the nanosilica particle (2) and the carbonyl group of the succinimidyl ester compound (3). Fluorescent dye molecules are bonded to the surface of the fluorescent dye-binding nanosilica particles (4). More specifically, after introducing and fixing an amino group on the surface of the silica particles by the method described above, the succinimidyl ester compound (3) is added to the aqueous solution, and the succinimidyl ester compound (3 ) 3 to 1 mol, more preferably 2.6 to 1.5 mol, and the mixture is stirred at room temperature or room temperature to 60 ° C. for 10 minutes to 1 hour, whereby urethane bonds (—NH Fluorescent dye molecules are immobilized via -CO-O-).

  Similarly, when the nanosilica particle (2) has an isothiocyanate group (-N = C = S) on the surface, the fluorescent dye molecule having the isothiocyanate group (-N = C = S) and an amino group By the thiourea bond (-NH-CS-NH-), the fluorescent dye molecules are bonded to form nanosilica particles (4) having the fluorescent dye bonded to the surface. Specifically, 3-Thiocyanato propyl triethoxy silane is mixed by the same method as described above to fix the isothiocyanate group on the nanosilica particle surface, and then the amino group-containing compound is added to 1 mol of the nanosilica particle. The compound having an amino group is added in an amount of 3 to 1 mol, more preferably 2.6 to 1.5 mol, and the mixture is stirred at room temperature or from room temperature to about 60 ° C. for 10 minutes to 1 hour, so that the silica particle surfaces are mixed. A fluorescent dye molecule having an amino group is immobilized through a thiourea bond (-NH-CS-NH-).

  Similarly, when the nanosilica particles (2) have a carboxyl group or an ester group on the surface, N-hydroxysuccinimide is bonded to the carboxyl group using a condensing agent such as carbodiimide (for example, WSCI). After the introduction of the ester group, the fluorescent dye molecule having an amino group undergoes an amide bond (—NH—CO—), whereby the fluorescent dye molecule is bonded to form nanosilica particles (4) having the fluorescent dye bonded to the surface. Specifically, by a method similar to the method described above, N-hydroxysuccinimide is bonded using a condensing agent such as carbodiimide (for example, WSCI), a succinimidyl ester group is introduced, and the compound having an amino group is then converted to nano silica. Add 3 to 1 mole of compound having an amino group to 1 mole of particles, more preferably 2.6 to 1.5 mole, and mix and stir for 10 minutes to 1 hour at room temperature or under heating conditions of room temperature to about 60 ° C. The fluorescent dye molecule having an amino group is immobilized on the silica particle surface via an amide bond (—NH—CO—).

  The reaction in the step (b) can be performed directly on the nanosilica particles (2) obtained in the step (a), but the surface of the nanosilica particles (2) obtained in the step (a) A molecule serving as a spacer may be introduced, and the step (b) may be performed on the nanosilica particles (2 ′) into which the spacer molecule has been introduced. By introducing a spacer molecule on the surface of the nanosilica particle (2), concentration quenching (decrease in fluorescence intensity) caused by dense binding of fluorescent dyes to the surface of the nanosilica particle (2) can be prevented. .

Spacer molecules include fluorescent dye binding groups on the surface of nanosilica particles (2), specifically amino groups (-NH ₂ ), carboxyl groups (-COOH), isothiocyanate groups (-N = C = S) or esters. The compound (8) which reacts with group (-COOR) and couple | bonds with these groups can be mentioned. Further, the compound binds to the fluorescent dye-binding group, thereby reacting with the fluorescent dye-binding group from the reaction with the compound (3) having a fluorescent dye molecule (succinimidyl ester compound (3)) in the next step (b). It is preferable that it is a compound which can protect.

For example, the spacer molecule used when the nanosilica particle (2) has an amino group (—NH ₂ ) on the surface includes a compound having a succinimidyl ester group and not emitting fluorescence. be able to. Specific examples of such compounds include compounds in which a succinimidyl ester group is introduced by binding N-hydroxysuccinimide to a benzoic acid having a carboxyl group (—COOH) using a condensing agent such as carbodiimide (for example, WSCI). can do. In addition, spacer molecules used when the nanosilica particles (2) have carboxyl groups (—COOH) on the surface include amino groups such as aminopropylethoxysilane (APS), aminoazobenzene, and aminobenzoic acid. The compound which has can be mentioned. These compounds can be fixed on the surface of the nanosilica particles by adding the carboxyl group (—COOH) and N-hydroxysuccinimide on the surface of the nanosilica particles after condensing with a condensing agent such as carbodiimide (for example, WSCI). As the spacer molecule used when the nanosilica particles (2) have an isothiocyanate group (-N = C = S) or an ester group (-COOR) on the surface, aminopropylethoxysilane (APS) And compounds having an amino group such as aminoazobenzene and aminobenzoic acid.

  The compound having a fluorescent dye molecule may be a compound containing a group having a property of binding to the surface of silica particles or a group binding electrostatically.

Step (c)
Step (c) is a step of reacting the fluorescent dye-bonded nanosilica particles (4) prepared in the step (b) with the silica compound (5). By this treatment, fluorescent dye-binding groups [amino group (—NH ₂ ), carboxyl group (—COOH), isothiocyanate group (—N) that have not reacted with the fluorescent dye on the surface of the fluorescent dye-bonded nanosilica particles (4). = C = S) or ester group (-COOR)], the silica compound (5) can be introduced. The silica compound (5) includes a silica compound (5) having a trialkoxysilyl group [—Si (OR) ₃ ], an aqueous sodium silicate solution (˜27% SiO ₂ ), and 3- (trimethoxysilyl). ) Propyl methacrylate.

The silica compound (5) having a trialkoxysilyl group [—Si (OR) ₃ ] is preferred. Here, the trialkoxysilyl group [—Si (OR) ₃ ] is preferably a triethoxysilyl group (TEOS group).

For example, as a method of introducing the silica compound (5) into the amino group (—NH ₂ ) remaining on the surface of the nanosilica particle (4), as shown by the following formula, the nanosilica particle (4) is added with 3-thiocyanate. A method of reacting a silica compound (5) having a —Si (OEt) ₃ group, such as topropyltriethoxysilane (CNS), can be mentioned. The silica compound (5) having a -Si (OR) ₃ group is not limited to the above-mentioned 3-thiocyanatopropyltriethoxysilane (CNS), but also γ-mercaptopropyltriethoxysilane, aminopropyltriethoxysilane ( APS), 3-glycidyloxypropyltriethoxysilane, 3-isocyanatopropyltriethoxysilane, 3- [2- (2-aminoethylamino) ethylamino] propyl-triethoxysilane, or the like can also be used.

  Such a reaction can be performed by mixing and stirring at room temperature or from room temperature to about 60 ° C. under a heating condition using a water-soluble solvent. The ratio of the nanosilica particles (4) and the silica compound (5) used in the reaction is not particularly limited, but preferably 3 to 1 mol of the silica compound (5) with respect to 1 mol of the nanosilica particles (4). Preferably, a proportion of 2.6 to 1.5 mol can be mentioned.

As a method of introducing the silica compound (5) into the carboxyl group (—COOH) remaining on the surface of the nanosilica particle (4), N-hydroxysuccinimide is added to the nanosilica particle (4), and carbodiimide (for example, WSCI) is used. A method of reacting a silica compound having an amino group and a —Si (OR) ₃ group, for example, APS, after making a succinimidyl ester with the above condensing agent can be mentioned. Such a reaction can be performed by mixing and stirring at room temperature or under a heating condition of about room temperature to 60 ° C. using a water-soluble solvent. The ratio of the nanosilica particles (4) and the silica compound (5) used in the reaction is not particularly limited, but preferably 3 to 1 mol of the silica compound (5) with respect to 1 mol of the nanosilica particles (4). Preferably, a proportion of 2.6 to 1.5 mol can be mentioned.

As a method of introducing a silica compound into the isothiocyanate group (-N = C = S) remaining on the surface of the nanosilica particle (4), -Si (OR) such as APS having an amino group in the nanosilica particle (4). A method of reacting the silica compound (5) having _three groups can be mentioned. Such a reaction can be performed by mixing and stirring at room temperature or under a heating condition of about room temperature to 60 ° C. using a water-soluble solvent. The ratio of the nanosilica particles (4) and the silica compound (5) used in the reaction is not particularly limited, but preferably 3 to 1 mol of the silica compound (5) with respect to 1 mol of the nanosilica particles (4). Preferably, a proportion of 2.6 to 1.5 mol can be mentioned.

Furthermore, as a method of introducing the silica compound (5) into the ester group (-COOR) on the surface remaining in the nanosilica particle (4), N-hydroxysuccinimide is added to the nanosilica particle (4) and condensation such as carbodiimide (eg WSCI) is performed. An example is a method in which a succinimidyl ester is formed by an agent, and then a silica compound having an amino group and a —Si (OR) ₃ group, for example, APS is reacted. Such a reaction can be carried out by mixing and stirring at room temperature or under a heating condition of about room temperature to 60 ° C. using a water-soluble solvent. The ratio of the nanosilica particles (4) and the silica compound (5) used in the reaction is not particularly limited, but preferably 3 to 1 mol of the silica compound (5) with respect to 1 mol of the nanosilica particles (4). Preferably, a proportion of 2.6 to 1.5 mol can be mentioned.

Thus, nanosilica particles (6) having a silyl group, particularly a trialkoxysilyl group (—Si (OR) ₃ group) can be prepared while binding a fluorescent dye to the surface of the nanosilica particle (4). .

Step (d)
In the step (d), the silyl group formed on the surface of the nanosilica particles (6) prepared in the above step (c), particularly preferably a trialkoxysilyl group [—Si (OR) ₃ ] is added to a silane compound (7 ) Is reacted. Preferred examples of the silane compound (7) include 3- (trimethoxysilyl) propyl methacrylate and tetraalkoxysilane [Si (OR) ₄ ]. The reaction in this case is represented by the following formula. Here, the tetraalkoxysilane (7) is preferably tetraethoxysilane (TEOS).

As a result of this reaction, the surface of the nanosilica particles (4) [nanosilica particles (6)] bound with a fluorescent dye (indicated by “R ₁ ” in the above formula) is coated on the surface of the silica dye so as to cover the fluorescent dye layer. Can be formed.

  The ratio of the nanosilica particles (6) and the silane compound (7) used in the reaction is not particularly limited, but can be appropriately set according to the particle diameter of the nanosilica particles serving as the nucleus and the thickness of the silica layer. . That is, it is defined by the size of the nano silica particles used as the nucleus (raw material) and the thickness of the silica layer formed around the nano silica particles. For example, as a molar ratio of tetraalkoxysilane (7) to 1 mol of nanosilica particles (6), numerical values as shown in the table can be given.

  This reaction is carried out in the presence of alcohol, water and ammonia. Examples of the alcohol include lower alcohols having 1 to 3 carbon atoms such as methanol, ethanol, and propanol. The ratio of water and alcohol in such a reaction system is not particularly limited, but preferably 0.5 to 8 parts by volume, preferably 1 to 5 parts by volume, more preferably 1 to 2 parts by volume of alcohol with respect to 1 part by volume of water. A range can be mentioned. Although the amount of ammonia is not particularly limited, for example, a ratio of 1 to 30 moles, preferably 3 to 24 moles, more preferably 6 to 18 moles, with respect to 1 mole of tetraalkoxysilane (7) to be reacted. Can be mentioned. In this reaction, sodium silicate may be contained in these reaction solutions.

  This reaction can be performed at room temperature or under heating conditions of room temperature to about 60 ° C. Moreover, although it does not restrict | limit, performing with stirring is preferable. Usually, a silica film can be formed by a reaction of several tens of minutes to several tens of hours, and nanosilica particles containing the fluorescent dye molecules targeted by the present invention can be prepared.

  In addition, in the said process (d), the magnitude | size (diameter) of the silica sphere to prepare can be suitably adjusted by adjusting the density | concentration of the tetraalkoxysilane (7) to be used, or adjusting reaction time. Larger nanosilica particles can be prepared by increasing the concentration of tetraalkoxysilane (7) used and increasing the reaction time (eg, Blaaderen et al., “Synthesis and Characyerization of Monodisperse Collidal Organo -silica Spheres ”, J. Colloid and Interface Science 156, 1-18.1993). Thus, according to the method of the present invention, the particle diameter (diameter) of the obtained fluorescent dye-containing nanosilica particles can be freely adjusted to a desired size, for example, from the order of several tens of nm to the order of several tens of nm. it can. Specifically, according to the method of the present invention, as shown in the examples described later, fluorescent dye-containing nanosilica particles having a size of several to several tens of nanometers, specifically, a minute size of 10 nm or less to 30 nm. It is also possible to prepare. Further, if necessary, it can be adjusted to a desired particle size distribution by a fractionation operation using a subsequent ultrafiltration membrane or the like, thus obtaining nanosilica particles in a desired particle size distribution range. You can also

  For example, for a fluorescent dye-containing nanosilica particle having a particle size of 30 nm or less, an ultrafiltration apparatus equipped with an ultrafiltration filter (disk) such as a filtration membrane Amicon YM100 (fractional molecular weight 100 kDa) (manufactured by Nihon Millipore) is used. Can be obtained. The fluorescent dye-containing nanosilica particles having a particle size of 10 nm or less include, for example, the fluorescent dye-containing nanosilica particles recovered by ultrafiltration using the above filtration membrane Amicon YM100 (fractional molecular weight 100 kDa) (manufactured by Nihon Millipore). It can be obtained by processing with an ultrafiltration apparatus equipped with a filtration membrane Amicon YM10 (fractional molecular weight: 10 kDa) (manufactured by Nihon Millipore).

  The fluorescent dye-containing nanosilica particles obtained in this manner may be purified by removing a coexisting ion or an unnecessary coexisting matter using a conventional method such as dialysis as necessary.

  As shown in the examples to be described later, according to the method of the present invention, the molecular weight of the fluorescent dye that can be blended per particle as compared to the method of sequentially polymerizing the silica compound chemically bonded to the fluorescent dye (chemical bonding method). Therefore, a labeling reagent (fluorescent probe) having a significantly high fluorescence intensity can be prepared. In addition, according to the method of the present invention, particularly the method of the present invention having a spacer molecule introduction step [(a ′) step], a large number of fluorescent dye molecules can be attached to the silica particle surface without causing self-quenching (concentration quenching). Can be fixed to. Therefore, according to the method of the present invention, it is possible to provide a highly sensitive detection reagent that can be used even in a minute region.

(2) Fluorescent dye-containing nanosilica particles The present invention provides fluorescent dye-containing nanosilica particles having high fluorescence intensity. Such fluorescent dye-containing nanosilica particles are obtained by binding fluorescent dye molecules to nanosilica particles (hereinafter referred to as “nuclear particles” for convenience). Furthermore, the fluorescent dye-containing nanosilica particles of the present invention are characterized in that the fluorescent dye molecules bonded to the core particles are further covered with a silica layer.

  The fluorescent dye-containing nanosilica particles include nanosilica particles having a core particle size of less than 30 nm and having one or more fluorescent dye molecules bonded to one core particle. Specifically, the fluorescent dye-containing nanosilica particles have a core particle size of 20 nm or more and less than 30 nm, and one core particle has 100 or more, preferably 120 or more, preferably 120 or more fluorescent dye molecules. Nano silica particles formed by bonding at a ratio of 200 molecules or more, more preferably 500 molecules or more are included. The upper limit of the number of bonds of the fluorescent dye molecule is not limited, but can include about 1500 molecules.

  In addition, the fluorescent dye-containing nanosilica particles of the present invention include nanosilica particles having a core particle size of less than 20 nm and having a fluorescent dye molecule bound at a ratio of 50 or more to one core particle. It is. Specifically, the fluorescent dye-containing nanosilica particles have a core particle size of 10 nm or more and less than 20 nm, and one core particle particle contains 50 or more fluorescent dye molecules, preferably 60 or more molecules, preferably Nano silica particles formed by bonding at a ratio of 70 molecules or more, more preferably 75 molecules or more are included. The upper limit of the number of bonds of the fluorescent dye molecule is not limited, but can be about 1000 molecules, preferably about 100 molecules.

  Further, the fluorescent dye-containing nanosilica particles of the present invention include nanosilica particles having a core particle diameter of less than 10 nm and having two or more fluorescent dye molecules bonded to one core particle. It is. Specifically, the fluorescent dye-containing nanosilica particles have a core particle diameter of 2 nm or more and less than 10 nm, and two or more fluorescent dye molecules, preferably 4 or more molecules, preferably 1 core particle. Nano silica particles formed by bonding at a ratio of 6 molecules or more, more preferably 10 molecules or more are included. The upper limit of the number of bonds of the fluorescent dye molecule is not limited, but can be about 100 molecules, preferably about 50 molecules.

The number of molecules of the fluorescent dye bonded to one core particle, in other words, the number of molecules of the fluorescent dye contained in one particle of the fluorescent dye-containing nanosilica particles is determined by an aqueous solution (water) in which the nanosilica particles are dispersed. Concentration of the nanosilica particles in the dispersion) ([SiO ₂ ] _p ) and the concentration of the fluorescent dye molecule calculated from the Lambert-Beer law based on the peak absorbance obtained by measuring the absorption spectrum of the aqueous dispersion (C) can be determined according to the following equation (1).

  Such fluorescent dye-containing nanosilica particles of the present invention have free fluorescent dye molecules (that is, bonded to nanosilica particles) at the same concentration as that of the fluorescent dye molecules bonded to the nanosilica particles. The fluorescent intensity is higher than that in an aqueous solution in which a non-fluorescent dye molecule is dispersed. This is because the fluorescence intensity of one particle of the fluorescent dye-containing nanosilica particles of the present invention is higher than the fluorescence intensity of a free fluorescent dye having the same number of molecules as the number of molecules of the fluorescent dye contained in the one particle. means.

  Such fine and highly sensitive fluorescent dye-containing nanosilica particles are not limited, but can be prepared, for example, by the method of the present invention described above.

  The fluorescent dye-containing nanosilica particles thus prepared preferably have a particle size of 50 nm or less, and are filtered by a method using a membrane having a desired pore size (such as an ultrafiltration membrane). The fluorescent dye-containing nanosilica particles having a desired particle diameter can be adjusted. For example, as a specific method, filtration is performed using the method described in the examples, that is, using an ultrafiltration apparatus equipped with an ultrafiltration membrane having a molecular weight cut off of 100 kDa (filtration membrane Amicon YM100, manufactured by Nihon Millipore). As a result, fluorescent dye-containing nanosilica particles having a particle size of 30 nm or less can be obtained. In addition, an ultrafiltration apparatus equipped with the thus prepared fluorescent dye-containing nanosilica particles (particle size of 30 nm or less) and an ultrafiltration membrane (filtration membrane Amicon YM10, manufactured by Nihon Millipore) having a molecular weight cut-off of 10 kDa By using and filtering, fluorescent dye-containing nanosilica particles having a particle size of 10 nm or less can be obtained.

  Silica particles are generally known to be chemically inert and easy to modify. The fluorescent dye-containing nanosilica particles of the present invention can also easily bind desired molecules to the surface, and the surface can be made mesoporous or smooth.

  Hereinafter, examples will be described to describe the present invention in detail. However, the present invention is not limited to these examples.

In addition, the meaning of the following term used in the following Example is as follows:
CNS: 3-thiocinatopropyltriethoxysilane
APS: Aminopropylethoxysilane
TEOS: Tetraethoxysilane
YM100: Filtration membrane Amicon YM100 (fractional molecular weight 100 kDa) (manufactured by Nihon Millipore)
YM10: filtration membrane Amicon YM10 (fractional molecular weight 10 kDa) (manufactured by Nihon Millipore)
DMSO: dimethyl sulfoxide
HS-30: 7nm nano silica particles used as core particles (LUDOX HS-30 colloidal silica, 30% suspension)
HS-40: 12nm nano silica particles used as core particles (LUDOX HS-40 colloidal silica, 40% suspension)
TM-50: 22 nm nano silica particles used as core particles (LUDOX TM-50 colloidal silica, 50% suspension)
WSCI: 1-Ethyl-3- (3-dimethylaminopropyl) carbodiimide, hydrochloride
NHS: N-hydroxy-succinimide
Example 1
(1) Preparation of fluorescent dye-containing nanosilica particles Suspension of commercially available nanosilica particles [(1) particle size 7 nm: LUDOX HS-30 colloidal silica, 30% suspension, (2) particle size 12 nm: LUDOX HS-40 1 μl of 10-fold diluted aqueous solution of colloidal silica, 40% suspension, (3) particle size 22 nm: LUDOX TM-50 colloidal silica, 50% suspension] was added to 1 ml of distilled water, and then sodium silicate An aqueous solution [NaSi _x O _2-x (0.54% SiO ₂ ) aqueous solution] was added in an amount of 10 to 100 μl and mixed and stirred at about 60 ° C. to activate the surface of the nanosilica particles. To this nanosilica particle-containing solution, 1 μL of a mixed solution of 3-thiocinatopropyltriethoxysilane (CNS) and aminopropylethoxysilane (APS) (20 μL of a mixture of CNS and APS at a volume ratio of 1: 1) and DMSO 1 μL of 200 μL of the mixed solution 220 μL) was added, 10 μL of the spacer solution was added and stirred, and the mixture was stirred and reacted at about 60 ° C. for 10 minutes to 1 hour. In addition, as a spacer solution, a solution containing benzoic acid silicate (= spacer) prepared by adding 3.7 μl of benzoic acid, WSCI 1 mg, NHS 3.7 mg, and APS 1.8 μl to DMSO 1 ml and stirring reaction at room temperature for 1 hour is used. It was.

  Next, 50 μL (75 μg) of Rhodamin Red-X, N-hydroxysuccinimidyl ester (Molecular Probes Co. Ltd.), in which rhodamine and succinimide are bonded via an ester bond, is heated at about 60 ° C. The mixture was stirred and reacted for 10 minutes to 1 hour. Further, 4 ml of ethanol, 10 μl of tetraethoxysilane (TEOS) and 100 μl of aqueous ammonia were added thereto and stirred at room temperature for 24 hours.

  The rhodamine-containing nanosilica particles thus obtained were filtered using an ultrafiltration apparatus equipped with an ultrafiltration membrane having a molecular weight cut off of 100 kDa (filtration membrane Amicon YM100, manufactured by Nihon Millipore). Rhodamine-containing nanosilica particles of 30 nm or less were obtained. Next, the fluorescent dye-containing nanosilica particles thus prepared were further filtered using an ultrafiltration apparatus equipped with an ultrafiltration membrane having a molecular weight cut-off of 10 kDa (filtration membrane Amicon YM10, manufactured by Nihon Millipore) Rhodamine-containing nanosilica particles having a particle size of 10 nm or less were obtained.

(2) Evaluation of nano-silica particles containing fluorescent dye
(2-1) (1) Nanosilica particles with a particle size of 7 nm (HS-30), (2) Nanosilica particles with a particle size of 12 nm (HS-40), and (3) Nanosilica particles with a particle size of 22 nm (TM-50) An aqueous solution (water dispersion) prepared by dispersing rhodamine-containing nanosilica particles (particle size of 30 nm or less and particle size of 10 nm or less) in water as a raw material was prepared as described above, and (a) the aqueous dispersion Concentration of rhodamine-containing nanosilica particles ([SiO ₂ ] p), (b) Rhodamine concentration in the aqueous dispersion ([Rhodamin]), (c) Number of rhodamine dye molecules per rhodamine-containing nanosilica particle And (d) The fluorescence intensity at the particle concentration was measured. Note that (d) fluorescence intensity was measured as follows, and from this (b) the concentration of rhodamine ([Rhodamin]) in the aqueous dispersion was calculated from Lambert-Beer's law. ) To determine the number of rhodamine dye molecules per particle of (c) rhodamine-containing nanosilica particles.

<Measurement method of fluorescence intensity>
Collect 0.1 ml of a dispersion solution of fluorescent dye-containing nanosilica particles in a container and measure the fluorescence spectrum using a fluorescence spectrophotometer (Shimadzu RF5300PC) (measuring condition: SLIT (EX / EM) = 1.5NM / 1.5NM , Low sensitivity), the fluorescence intensity of the maximum peak is obtained. The fluorescence intensity is not limited to the fluorescence spectrophotometer, but can be measured using a 1 μL fluorescence spectrometer (for example, ND-3300 manufactured by Nanodrop). In this case, 1 μL of a dispersed aqueous solution of fluorescent dye-containing nanosilica particles is used as a test sample.

  The results are shown in Table 3.

  In addition, about the rhodamine (free rhodamine) used as a fluorescent pigment | dye above, it shows in FIG. 1 which shows the relationship between the rhodamine density | concentration in the aqueous solution which melt | dissolved it in water, and fluorescence intensity. From this graph, the fluorescence intensity of 0.098 μM rhodamine is 1.7706.

  (2-2) Place the aqueous dispersion of fluorescent dye-containing nanosilica particles (particle size of 30 nm or less) prepared in (1) into a quartz cell with an optical path length of 1 cm, and run a 1500 W Xe lamp (Nihon bunko FP-6500) for 120 minutes. Fluorescence intensity (excitation wavelength: 570 nm, measurement wavelength: 595 nm) was measured during irradiation. The results are shown in FIG. In the figure, “TKR-1” is (1) 7 nm nano silica particles (HS-30), “TKR-2” is (2) 12 nm nano silica particles (HS-40), and “TKR-3” "(3) shows the results of rhodamine-containing nanosilica particles prepared by using nanosilica particles (TM-50) having a particle diameter of 22 nm as raw materials. As shown in the figure, the fluorescent dye-containing nanosilica particles prepared by the method of the present invention are extremely stable against light because the fluorescence intensity is maintained at 92% or more even after light irradiation for 120 minutes. It can be seen that it is.

  (2-3) Rhodamine-containing nanosilica particles (particle size of 30 nm or less) prepared using nanosilica particles (TM-50) with a particle size of 22 nm as raw materials were mixed with adult thymocytes, and the fluorescence intensity was Measurement was performed using a flow cytometer. The results are shown in FIG. In FIG. 3, the horizontal axis represents fluorescence intensity (FL3-H), and the vertical axis represents the number of particles (Counts). In FIG. 3, the right figure shows the results of experiments conducted using the rhodamine-containing nanosilica particles of the present invention, and the left figure shows negative control results measured without adding the rhodamine-containing nanosilica particles.

As shown in FIG. 3, by reacting rhodamine-containing nanosilica particles with adult thymocytes, the peak value (horizontal axis (fluorescence intensity when the count value on the vertical axis is maximum)) is 5 to 20 of Negative control.
Increased to. From this, it was considered that the fluorescence intensity increased as a result of adsorption and concentration of the fluorescent dye-containing nanosilica particles of the present invention on adult thymocytes.

  (2-4) (1) Nanosilica particles with a particle size of 7nm (HS-30), (2) Nanosilica particles with a particle size of 12nm (HS-40), and (3) Nanosilica particles with a particle size of 22nm (TM-50) Rhodamine-containing nanosilica particles (particle size of 30 nm or less) prepared using the above as raw materials were subjected to agarose gel electrophoresis. Specifically, the mobility of the nanosilica particles was measured by dropping these fluorescent dye-containing nanosilica particles on the negative electrode side of the agarose gel, applying a high voltage (100 V), and detecting the fluorescence of rhodamine. (Use BioRad molecular Imager FX Pro). The results are shown in FIG. As shown in FIG. 4, any rhodamine-containing nanosilica particles moved to the positive electrode side (left side) of the agarose gel. From this, the rhodamine-containing nanosilica particles prepared above are all negatively charged, that is, the surface is formed as particles coated with silica having a —Si—O— bond (negatively charged). You can see that The mobility usually depends on the size of the particle, but this time, the reason why there was almost no difference in the moving distance of the particle was considered that the particle was too small.

  (2-5) Table 4 shows the fluorescence intensity (measured with Shimadzu spectrophotometer RF5300PC) at each particle concentration (μM). For comparison, the fluorescence intensity at each particle concentration (μM) of nanoparticles (Qdot525, Qdot605: both manufactured by QuantmDot, “Qdot” is a registered trademark) using a commercially available organic dye (CdSe) is also shown.

  From this result, it can be seen that the fluorescence intensity (fluorescence intensity relative to the particle concentration) of the fluorescent dye-containing nanosilica particles of the present invention is superior at the same size as Qdot or less.

  (2-6) (1) Nanosilica particles with a particle size of 7 nm, (2) Nanosilica particles with a particle size of 12 nm, and (3) Fluorescent dye-containing nanosilica particles prepared using nanosilica particles with a particle size of 22 nm as raw materials (particle size of 30 nm (Below) About 1 mL was added to a 1.5 mL tube, then left in a light shielding box (1000 hours). Measure the fluorescence spectrum of each fluorescent dye-containing nanosilica particle over time (measurement conditions: (Slit (Ex / Em) = 1.5 nm / 1.5 nm, Low sensitivity, excitation wavelength 573 nm, measurement wavelength 585 nm)) Intensities were calculated, and the results are shown in Fig. 5 (vertical time on the vertical axis and fluorescence intensity on the horizontal axis).

  As shown in this figure, all the fluorescent dye-containing nanosilica particles prepared by the method of the present invention were stable for 1000 hours.

Example 2
(1) Preparation of fluorescent dye-containing nanosilica particles Nanosilica particles having a particle size of about 3 nm and nanosilica particles having a particle size of 22 nm were used as raw materials, and instead of rhodamine used in Example 1 as a fluorescent dye, fluorescein, DY485, DY521 ( As described above, using DYOMICS) or Alexa647 (Molecular Probes), the same reaction was carried out according to the method of Example 1 to prepare fluorescent dye-containing nanosilica particles. The chemical formulas of DY485 and DY521 are shown below.

  The “compound having fluorescent dye (3)” (specifically, succinimidyl ester compound (3)) used in the present invention is an esterification reaction of DY485 and DY521 with N-hydroxysuccinimide. Can be prepared.

  For the nanosilica particles having a particle size of 22 nm, a commercially available suspension of nanosilica particles (particle size 22 nm: LUDOX TM-50 colloidal silica, 50% suspension) was used. A 22 nm nanosilica particle suspension (particle size 22 nm: LUDOX TM-50 colloidal silica, 50% suspension) was filtered with YM-100, and the eluate was further filtered with YM-10 (2.6 nm pore size). Prepared from the solution that was filtered and remained unfiltered.

(2) Evaluation of fluorescent dye-containing nanosilica particles The properties of the fluorescent dye-containing nanosilica particles thus obtained (number of molecules of fluorescent dye per particle, fluorescent intensity, etc.) were determined according to the method of Example 1. The results are shown in Table 5.

  The results for fluorescent dye-containing nanosilica particles prepared using nanosilica particles with a particle size of 22 nm as the raw material are shown in the upper part of the table, and the results for the fluorescent dye-containing nanosilica particles prepared using nanosilica particles with a particle size of about 3 nm as the raw material are shown in the lower part of the table Show. As can be seen from these results, we have prepared fluorescent dye-containing nanosilica particles containing four types of fluorescent dyes, Fluorescein, DY-485XL, DY-485XL + DY-521XL, and Alexa647, using nanosilica particles with a particle size of 22 nm. . In addition, fluorescent dye-containing nanosilica particles were prepared by blending three types of fluorescent dyes, Fluorescein, DY-521XL, and Alexa647, using nanosilica particles with a particle size of 3 nm as a raw material. In addition, it can be seen that in the fluorescent dye-containing nanosilica particles having a nucleus of 22 nm in diameter, 200 or more fluorescent dye molecules are fixed in one particle except in the case of Alexa647. Since the fixed number per particle by the conventionally known method (WO2006 / 070582) is 100 molecules, the above result corresponds to more than twice this. In addition, the fluorescent dye-containing nanosilica particles having a core with a particle size of 3 nm contained two or more fluorescent molecules in one particle. This corresponds to more than twice the conventional method (WO2006 / 070582).

Example 3
Suspension of commercially available nano silica particles [(1) particle size 7 nm: LUDOX HS-30 colloidal silica, 30% suspension, (2) particle size 12 nm: LUDOX HS-40 colloidal silica, 40% suspension, ( 3) Particle size 22nm: LUDOX TM-50 colloidal silica, 50% suspension] is diluted 10 times and 1 μl of aqueous solution is collected and added to 900 μL or 1 ml of distilled water, followed by sodium silicate aqueous solution [NaSi _x O _2-x (0.54% SiO ₂ ) aqueous solution] was added with 10 to 100 μl and 1 μL spacer solution 10 μL and stirred, and mixed and stirred at room temperature to activate the surface of the nanosilica particles. In addition, as a spacer solution, a solution containing benzoic acid silicate (= spacer) prepared by adding 3.7 μl of benzoic acid, WSCI 1 mg, NHS 3.7 mg, and APS 1.8 μl to DMSO 1 ml and stirring reaction at room temperature for 1 hour is used. It was. Next, 1 μl of aminopropylethoxysilane (APS) and 2 μl of 3-Thiocyanato propyl triethoxysilane (CNS) were added thereto, and the mixture was stirred and reacted at room temperature for 10 minutes to 1 hour.

  Next, Rhodamin Red-X, N-hydroxysuccinimidyl ester (Molecular Probes Co. Ltd.) formed by binding rhodamine and succinimide through an ester bond is added at 50 μL (75 μg) and heated at room temperature or about 60 ° C. The mixture was stirred for 10 minutes to 1 hour to cause the reaction. Thereafter, the mixture was filtered with YM-10, washed with distilled water, and subjected to fluorescence spectrum (Shimadzu fluorescence spectrophotometer RF5300PC) and absorption spectrum measurement (Shimadzu UV-1700). Samples prepared in this way (No. 73: using raw material HS-30, No. 75: using raw material HS-40, No. 76: using raw material TM-50) are fluorescent dyes on the surface of each raw nanosilica particle. (Rhodamine) and spacer molecule (CNS) are only fixed, and no silica layer is formed on the surface.

  Next, 4 ml of ethanol, 10 μl of tetraethoxysilane (TEOS) and 100 μl of aqueous ammonia were added to this sample and stirred at room temperature for 24 hours (formation of a silica layer). The rhodamine-containing nanosilica particles thus obtained are filtered using an ultrafiltration apparatus equipped with an ultrafiltration membrane (filtration membrane Amicon YM-100, manufactured by Nihon Millipore) having a molecular weight cut off of 100 kDa, Fluorescent dye-containing nanosilica particles having a diameter of 30 nm or less were obtained. After that, it was filtered with YM10, washed with distilled water, and the fluorescence spectrum and absorption spectrum of the aqueous dispersion prepared by dispersing this in distilled water were measured (No.73 ': raw material HS-30 used, No.75 ': Raw material HS-40 used, No. 76': Raw material TM-50 used).

From the results of the absorption spectrum measurement, the concentration of rhodamine in the aqueous dispersion ([Rhodamin]) is calculated according to Lambert-Beer's law, and the graph shows the fluorescence intensity at that concentration plotted on the horizontal axis and on the vertical axis. It is shown in FIG. According to the above-mentioned formula (1), the number of molecules ([Rhodamin] / [SiO ₂ ] p) of the fluorescent dye (rhodamine) contained in one rhodamine-containing nanosilica particle was determined.

The results are shown below. [SiO ₂ ] p is the concentration of the nanosilica particles contained in the aqueous dispersion of rhodamine-containing nanosilica particles:
Number of rhodamine molecules contained in one rhodamine-containing nanosilica particle ([Rhodamin] / [SiO ₂ ] p)
No.73 '(using raw material HS-30): 2
No.75 '(using raw material HS-40): 12
No.76 '(using raw material TM-50): 190.

  From this result, it can be seen that the fluorescence intensity is increased by coating the surface of the fluorescent dye-containing nanosilica particles with a silica layer. The reason is considered to be that rhodamine molecules are protected from the outside (aqueous solution) by covering the surface of the rhodamine molecules with a silica layer.

Example 4
For rhodamine-containing nanosilica particles No. 73 ′ (using raw material HS-30) prepared in Example 3 above, (1) particle size and (2) fluorescence lifetime in an aqueous dispersion were evaluated.

(1) Particle size Requested by Department of Morphology Research, Toray Research Center Co., Ltd. Obtained a transmission electron microscope image (TEM image) of rhodamine-containing nanosilica particles No. 73 '(using raw material HS-30) and TEM image Was negatively stained (PTA (phosphotungstic acid) staining). The result is shown in FIG. As can be seen from FIG. 7, it was observed that the particles have many branches and are aggregated in a bead shape. The primary particle diameter was about 5 nm to 13 nm, and it was irregular. Subsequently, about the obtained TEM image, particle | grains were discriminate | determined visually and the particle size distribution was analyzed. The results are shown in FIG. The primary particle size was 8.2 ± 0.5 nm (n = 146).

(2) Fluorescence lifetime in aqueous dispersion
The fluorescence lifetime of the aqueous dispersion of rhodamine-containing nanosilica particles No. 73 '(using raw material HS-30) was measured at the Toray Research Center, Inc. [Measuring device: NAES-1100 time-resolved spectrophotometer (Horiba Manufacturing), excitation wavelength 550 nm, integration time 6 hours]. The results are shown in FIG.

As can be seen from the figure, the fluorescence lifetime obtained by the one-component analysis was 4.09 ± 0.02 nsec. This value was longer than the fluorescence lifetime of 1.9 nsec in the aqueous dispersion of the rhodamine dye molecule itself and longer than the fluorescence lifetime of 2.9 nsec in ethanol (FL Arbeloa et al., J. of Luminescence, 44 , 1989, 105). This indicates that in the rhodamine-containing nanosilica particles No. 73 ', the rhodamine fluorescent molecules bound and accumulated on the silica nanoparticle (HS-30) surface exist without quenching and energy transfer. ing.

Example 5 Preparation of Rhodamine-Containing Silica Nanoparticles Incorporating Carboxyl Groups Suspension of commercially available nanosilica particles [(1) particle size 7 nm: LUDOX HS-30 colloidal silica, 30% suspension, (2) particle size 12 nm: LUDOX HS-40 colloidal silica, 40% suspension, (3) Particle size 22 nm: LUDOX TM-50 colloidal silica, 50% suspension] was diluted 10-fold, and 1 μl of 10 mM citric acid was collected. Add 10 μl, 5 μl, 10 μl, or 100 μl, add to 900 μL or 1 ml of distilled water, then add 10 to 100 μl of sodium silicate aqueous solution [NaSixO ₂ -x (0.54% SiO ₂ ) aqueous solution] and 10 μL of 1 μL spacer solution and stir The surface of the nano silica particles was activated by mixing and stirring at room temperature. In addition, as a spacer solution, a solution containing benzoic acid silicate (= spacer) prepared by adding 3.7 μl of benzoic acid, WSCI 1 mg, NHS 3.7 mg, and APS 1.8 μl to DMSO 1 ml and stirring reaction at room temperature for 1 hour is used. It was. Next, 1 μl of aminopropylethoxysilane (APS) and 2 μl of 3-Thiocyanato propyl triethoxysilane (CNS) were added thereto, and the mixture was stirred and reacted at room temperature for 10 minutes to 1 hour.

  Next, Rhodamin Red-X, N-hydroxysuccinimidyl ester (Molecular Probes Co. Ltd.) formed by binding rhodamine and succinimide through an ester bond is added at 50 μL (75 μg) and heated at room temperature or about 60 ° C. The mixture was stirred for 10 minutes to 1 hour to cause the reaction. Thereafter, the mixture was filtered with YM-10, washed with distilled water, and subjected to fluorescence spectrum (Shimadzu fluorescence spectrophotometer RF5300PC and nanodrop 1 μ fluorescence spectrometer) and absorption spectrum measurement (Shimadzu UV-1700). Samples prepared in this way (No. 73: using raw material HS-30, No. 75: using raw material HS-40, No. 76: using raw material TM-50) are fluorescent dyes on the surface of each raw nanosilica particle. (Rhodamine) and spacer molecule (CNS) are only fixed, and no silica layer is formed on the surface.

  Next, 4 ml of ethanol, 10 μl of tetraethoxysilane (TEOS) and 100 μl of aqueous ammonia were added to this sample and stirred at room temperature for 24 hours (formation of a silica layer). The rhodamine-containing nanosilica particles thus obtained have citric acid on the surface. The nanosilica particles are filtered using an ultrafiltration device equipped with an ultrafiltration membrane having a fractional molecular weight of 100 kDa (filtration membrane Amicon YM-100, manufactured by Nihon Millipore), and a fluorescent dye having a particle size of 30 nm or less Contained nanosilica particles were obtained. Thereafter, the mixture was filtered with YM10, washed with distilled water, and dispersed in distilled water to measure the fluorescence spectrum of an aqueous dispersion prepared.

  Fig. 10 shows the relative fluorescence of nanosilica particles measured for rhodamine-containing nanosilica particles prepared using raw material HS-30, with the amount of 10 mM citric acid (μl) used on the horizontal axis and the nanodrop 1μ fluorescence spectrometer on the vertical axis. The quantity (RFU) was plotted. As seen in FIG. 10, the relative fluorescence intensity was maximized when 10 μl of citric acid (10 mM) was used for the raw material HS-30.

FIG. 11 shows the rhodamine-containing nanosilica particles prepared using raw materials HS-30, HS-40, or TM-50, with the rhodamine molecule concentration (10 ⁻⁸ M) on the horizontal axis and the nanodrop 1 μ fluorescence spectrum on the vertical axis. Relative fluorescence intensity (RFU) measured with a meter was plotted. The added citric acid (10 mM) is 10 μl. As seen in FIG. 11, it can be seen that the rhodamine-containing nanosilica particles prepared using HS-40 as a raw material are minimized at the same rhodamine molecular concentration.

  Thus, the amount of citric acid and the size of the raw silica nanoparticles affect the fluorescence intensity, indicating that citric acid molecules are incorporated into the silica layer covering the fluorescent dye. Citric acid molecules are taken into the silica layer and have carboxyl groups (—COOH) on the surface.

Example 6 Preparation of nanosilica particles containing fluorescein
10 μL was taken from a solution of 2.0 mg of 5 (6) -Carboxy-fluorecein-N-hydroxy succinimide ester (FLUOS, Roche Co. Ltd.) in 1.5 ml of DMSO to prepare nanosilica particles containing fluorescein. It was. First, 1 mL of nanosilica particle suspension (particle size 7 nm: LUDOX HS-30 colloidal silica, diluted 1/10) was added and mixed with 1 mL of distilled water, and then an aqueous sodium silicate solution (NaSi _x O _2-x ( 0.54 wt% SiO ₂ ) aqueous solution) 10 μL, APS and CNS mixed solution (10 μL + 10 μL in 200 μL DMSO) 1 μL, spacer solution 10 μL were added and stirred, and further stirred and mixed while heating at about 60 ° C. for 15 minutes. In addition, as a spacer solution, a solution containing benzoic acid silicate (= spacer) prepared by adding 3.7 mg of benzoic acid, WSC 1 mg, NHS 3.7 mg, and APS 1.8 μl to DMSO 1 ml and stirring reaction at room temperature for 1 hour is used. It was.

  Thereafter, 4 mL of ethanol, 0.01 ml of TEOS solution and 0.1 ml of aqueous ammonia solution were added to the aqueous solution and stirred, and the mixture was further heated and stirred at about 60 ° C. for 15 minutes. The resulting solution was filtered and washed with YM-100 to obtain fluorescein-containing silica nanoparticles having a particle size of less than 10 nm.

Comparative example
Preparation of Rhodamine-Containing Silica Compound and Preparation of Nanosilica Particles [Rhodamine-Containing Nanosilica Particles] (Chemical Bond Type) Using This (1) Preparation of Rhodamine-Containing Silica Compound (Chemical Bond Type) Prepared.

[Wherein R ₃ means rhodamine (labeled molecule). In R ₃ , * means a bond with an ester group. ]
Specifically, as a succinimidyl ester compound, about 5 mg of 5-carboxyltetramethylrhodamin succinimidyl ester (9) (Molecular Probes) consisting of rhodamine and succinimide bonded via an ester bond is first dissolved in 1 ml of DMSO solution. Then, 3- (aminopropyl) triethoxysilane (APS) (2) as a silica compound having an amino group was added in an amount of about 1.2 μl so as to be equimolar with the succinimidyl ester compound (9). Rhodamine (labeled molecule) -containing silica compound (10) in which the carbonyl group of the succinimidyl ester compound (9) and the amino group of the silica compound (2) are amide-bonded by stirring with a stirrer piece for 1 hour. ) Was prepared.

(2) Preparation of rhodamine-containing nanosilica particles (chemically bonded type) Rhodamine-containing nanosilica particles (12) were prepared from the rhodamine-containing silica compound (10) according to the following formula.

[Wherein R ₃ represents rhodamine. ]
Specifically, 0.05 ml of tetraethoxysilane (TEOS), 1 ml of water and 3.95 ml of ethanol were added to 0.05 ml of the DMSO solution of the reaction solution [rhodamine-containing silica compound (10)] obtained above (ethanol: water). = 4: 1, volume ratio), 0.1 ml of about 30% ammonia water was added thereto, and the mixture was allowed to stand at room temperature with stirring for one day. The obtained solution (reaction completed solution) was subjected to ultrafiltration device [Amicon (registered trademark) stirring cell] (filter; UF disk YM100 Ultracell RC100K NMWL) (sales company: MILLIPORE) [Nominal Molecular Weight Limit (NMWL) : 100 kDa], and filtration and washing with distilled water were repeated several times to prepare rhodamine-containing nanosilica particles (11) (particle size 20 nm). The filtrate was further filtered and washed with YM-10 to prepare rhodamine-containing nanosilica particles having a particle size of about 10 nm.

  The number of fluorescent dye molecules and the fluorescence intensity of each of the fluorescent dye-containing nanosilica particles obtained in Example 5 and the fluorescent dye-containing nanosilica particles (chemically bonded type) obtained in Reference Example were measured (Shimadzu fluorescence spectroscopy). Photometer RF5300PC). The results are shown in Table 6 together with the fluorescent dye-containing nanosilica particles prepared in Example 1. For comparison, the fluorescence intensity of a commercially available CdSe labeling agent is also shown.

It is a graph which shows the relationship between the rhodamine density | concentration in the aqueous solution which melt | dissolved the free rhodamine in water, and fluorescence intensity. Fluorescence intensity (excitation wavelength: 570 nm) while irradiating the dispersion aqueous solution of the fluorescent dye-containing nanosilica particles (particle size of 30 nm or less) prepared in Example 1 (1) with a 1500 W Xe lamp (Nihon bunko FP-6500) for 120 minutes. , Measurement wavelength: 595 nm) is shown. In the figure, “TKR-1” is (1) 7 nm nano silica particles (HS-30), “TKR-2” is (2) 12 nm nano silica particles (HS-40), and “TKR-3” "(3) shows the results of rhodamine-containing nanosilica particles prepared by using nanosilica particles (TM-50) having a particle diameter of 22 nm as raw materials. Rhodamine-containing nanosilica particles (particle size of 30 nm or less) prepared from nanosilica particles (TM-50) having a particle size of 22 nm in Example 1 (1) as raw materials were mixed with adult thymocytes, and then the fluorescence intensity thereof was mixed. Shows the results of measurement using a flow cytometer. The horizontal axis represents fluorescence intensity (FL3-H), and the vertical axis represents the number of particles (Counts). In the figure, the right figure shows the results of experiments conducted using the rhodamine-containing nanosilica particles of the present invention, and the left figure shows the results of negative control measured without adding the rhodamine-containing nanosilica particles. In Example 1 (1), (1) nano silica particles (HS-30) having a particle size of 7 nm, (2) nano silica particles having a particle size of 12 nm (HS-40), and (3) nano silica particles having a particle size of 22 nm (TM- 50 shows the results of subjecting rhodamine-containing nanosilica particles (particle size of 30 nm or less) prepared using 50) as raw materials to agarose gel electrophoresis. In Example 1 (1), (1) nano silica particles (HS-30) having a particle size of 7 nm, (2) nano silica particles having a particle size of 12 nm (HS-40), and (3) nano silica particles having a particle size of 22 nm (TM- 50) shows the change in fluorescence intensity over time of nano-silica particles containing fluorescent dyes (particle size of 30 nm or less) prepared using raw materials (measurement conditions: (Slit (Ex / Em) = 1.5 nm / 1.5 nm, Low sensitivity Excitation wavelength 573 nm, measurement wavelength 585 nm) (vertical axis: standing time (hour), horizontal axis: fluorescence intensity). Rhodamine-containing nanosilica particles prepared in Example 3 (without silica layer coating; No. 73 (using raw material HS-30), No. 75 (using raw material HS-40), No. 76 (using raw material TM-50), and Silica layer coating; concentration of rhodamine in aqueous dispersion of No. 73 '(using raw material HS-30), No. 75' (using raw material HS-40), No. 76 '(using raw material TM-50)) The relationship between ([Rhodamin]) (horizontal axis) and fluorescence intensity (vertical axis) at that concentration is shown. It is a negative dyeing | staining (PTA (phosphotungstic acid) dyeing | staining) image of the transmission electron microscope image (TEM image) of the rhodamine containing nano silica particle No.73 '(raw material HS-30 use) prepared in Example 3 (Example 4). . The particle size distribution of rhodamine-containing nanosilica particles No. 73 '(using raw material HS-30) prepared in Example 3 is shown (Example 4). In the figure, “B” indicated by a bar graph means the number of rhodamine-containing nanosilica particles, and the result of Gaussian approximation of the distribution of the particles is “fit” indicated by a line graph in the figure. The result of evaluating the fluorescence lifetime in the aqueous dispersion of Rhodamine-containing nanosilica particles No. 73 ′ (using raw material HS-30) prepared in Example 3 is shown (Example 4). For the rhodamine-containing nanosilica particles prepared using the raw material HS-30, the 10 mM citric acid amount (μl) used on the horizontal axis and the relative fluorescence (RFU) of the nanosilica particles measured with a nanodrop 1 μ fluorescence spectrometer on the vertical axis (Example 5) which plotted this. Each rhodamine-containing nanosilica particle prepared using raw material HS-30, HS-40, or TM-50 was measured with a rhodamine molecule concentration (10 ^-8 M) on the horizontal axis and a nanodrop 1 μ fluorescence spectrometer on the vertical axis. It is the figure which plotted the relative fluorescence intensity (RFU) (Example 5).

Claims (9)

A method for preparing fluorescent dye-containing nanosilica particles, comprising the following steps (a) to (d);
(a) introducing a fluorescent dye binding group into the surface of the nanosilica particles (1) via the OH group;
(b) reacting the nanosilica particles (2) obtained in the above step with a compound (3) having a fluorescent dye molecule to bind the fluorescent dye to the surface of the nanosilica particles;
(c) reacting the fluorescent dye-bonded nanosilica particles (4) obtained in the above step with a silica compound (5) to introduce the silica compound (5) onto the surface of the nanosilica particles (4); and
(d) A step of reacting the nanosilica particles (6) obtained in the above step with a silane compound (7) to form a silica film on the surfaces of the nanosilica particles (6). “Succinimidyl ester compound (3)” in which a fluorescent dye molecule and succinimide are bonded via an ester bond (—CO—O—) as the “compound having a fluorescent dye molecule (3)”,
Using the `` silica compound (5) having a trialkoxysilyl group '' as the `` silica compound (5) '' and the `` tetraalkoxysilane (7) '' as the `` silane compound (7) '',
A method for preparing the fluorescent dye-containing nanosilica particles according to claim 1. The step (b) is performed on the nanosilica particles (2 ') obtained in the step (a') after the step (a), and then the step (a '). Method for preparing fluorescent dye-containing nanosilica particles described in 1 or 2:
(a ′) The compound (8) that binds to the fluorescent dye-binding group is reacted with the nanosilica particle (2) obtained in the step (a) to obtain one of the fluorescent dye-binding groups on the surface of the nanosilica particle (2). Protecting the part.   The method for preparing fluorescent dye-containing nanosilica particles according to any one of claims 1 to 3, wherein the fluorescent dye-binding group is an amino group, a carboxyl group, an isothiocyanate group or an ester group. Fluorescent dye-containing nanosilica particles in which fluorescent dye molecules are bonded to nanosilica particles,
The nano silica particles have a particle size of 20 nm or more and less than 30 nm, and
Fluorescent dye-containing nanosilica particles characterized in that 100 or more fluorescent dye molecules are bonded to one nanosilica particle. Fluorescent dye-containing nanosilica particles in which fluorescent dye molecules are bonded to nanosilica particles,
Fluorescent dye-containing nanosilica particles, wherein the nanosilica particles have a particle shape of 10 nm or more and less than 20 nm, and 50 or more fluorescent dye molecules are bonded to one nanosilica particle. Fluorescent dye-containing nanosilica particles in which fluorescent dye molecules are bonded to nanosilica particles,
The nanosilica particles have a particle size of less than 10 nm, and
Two or more fluorescent dye molecules are bonded to one nanosilica particle, and the fluorescent dye-containing nanosilica particles are characterized in that Fluorescent dye-containing nanosilica particles in which fluorescent dye molecules are bonded to nanosilica particles,
The fluorescence intensity in the dispersed aqueous solution is the same as the concentration of the fluorescent dye molecules bound to the nanosilica particles, and the fluorescence in the aqueous solution in which free fluorescent dye molecules not bound to the nanosilica particles are dispersed. Fluorescent dye-containing nanosilica particles characterized by higher than strength. The fluorescent dye-containing nanosilica particles according to any one of claims 5 to 8, wherein the fluorescent dye molecules bonded to the nanosilica particles are further covered with a silica layer.

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