WO2006001417A1

WO2006001417A1 - Functional silica particle and use thereof

Info

Publication number: WO2006001417A1
Application number: PCT/JP2005/011731
Authority: WO
Inventors: Hirokazu Miyoshi
Original assignee: Techno Network Shikoku Co., Ltd.
Priority date: 2004-06-28
Filing date: 2005-06-27
Publication date: 2006-01-05
Also published as: JPWO2006001417A1; JP4820963B2

Abstract

Disclosed is a functional silica particle which can be effectively used in nano-sized regions or micro-sized regions. Also disclosed is a method for producing such a functional silica particle. Specifically disclosed is a functional silica particle wherein one compound selected from the group consisting of methyl viologen, xylenol orange and 4-amino-(2,3,6,6-tetramethyl-1-piperidinyloxy) is contained in a silica particle. Such a functional silica particle can be produced by a method including a step wherein the above-mentioned one compound is mixed with (3-thiocyanatepropyl)triethoxysilane and then further mixed with tetraethylorthosilicate in the presence of ammonia.

Description

Specification

Functional silica particles and uses thereof

Technical field

[0001] The present invention relates to functional silica particles and uses thereof. More specifically, the present invention relates to a novel functional silica particle that expresses new characteristics by fixing a specific compound to the silica particle, and its use.

Background art

Conventionally, increasing the concentration of a dye molecule for obtaining signal intensity has caused the formation of aggregates and aggregates, which has caused undesirable properties that are different from the properties of the dye molecules themselves. For example, in the case of fluorescent molecules, concentration quenching, and in the case of dyes, absorption changes due to aggregation formation, and in the case of stable radicals, ESR peaks are broadened due to spin-spin interaction, resulting in intensity reduction ( For example, see Non-Patent Documents 1 to 3.)

[0003] As one method for increasing the concentration of dye molecules, a method of immobilizing some dye on a support can be considered. Various glass beads have been proposed as a strong support (see, for example, Non-Patent Document 4).

[0004] However, they cannot be adjusted to the desired size with particle sizes larger than micrometer! /, So for example, they cannot be used in the nano- or micro-region! There are disadvantages.

¥ af .l '■ Ferrer, ML; Monte, F.; Levy, D .. Rhodamine 19 Fluorescent Dimers Resulting from Dye aggregation on the Porous Surface of Sol-Gel Silica Glasses; Lan gmuir (19) 2003; pp2782-2786.

Patent Document 2: Wehry, E.L-; Rogers, L.B.In Fluorescence and Phosphorescence Analysis; Hercules, D.M..Ed .; Interscience: New York, 1966; pp81—149.

Non-Patent Document 3: Keith, AD; Snipes, W .; Metlhorn, RJ; Gunter, T. Factors Resrictin g Diffusion of Water-soluble Spin Labels; Biophysicaljournal 1977 (19); pp205-218. Non-Patent Document 4: Imhof, A.; Megens, M.; Engelberts, JJ; Lang, DTN ;; Sprik, R.; Vos, W 丄; Spectroscopy of Fluorescein (FITC) Dyed Colloidal Silica Spheres; J. Phys. Chem. B1999 (103); ppl408-1415.

Disclosure of the invention

Problems to be solved by the invention

[0005] The present invention is novel and powerful by using methyl viologen, xylenol orange, or 4-amino- (2,3,6,6-tetramethyl-1-piberidi-loxy) as the molecule. It aims at providing the functional material which expressed the special characteristic. In particular, an object of the present invention is to provide functional silica particles that can be used effectively in the nano-region and micro-region as the functional material.

Means for solving the problem

[0006] In order to achieve the above-mentioned object, the present inventor has intensively studied day and night. As a result, the silica particles are subjected to methyl viologen, xylenol orange, or 4-amino- (2,3,6,6-tetramethyl- By fixing compounds such as (1-piberidyl-loxy), silica particles having new functions that are inherent in these compounds can be obtained, or the functions of these compounds can be further improved. It has been found that silica particles that can be more effectively exhibited are obtained, and it has been confirmed that these are extremely useful materials as functional silica particles.

[0007] The present invention has been completed based on hard knowledge.

That is, the present invention firstly relates to a group consisting of methyl viologen, xylenolene diene, and 4-amino- (2,3,6,6-tetramethyl-1-piveridyl-loxy) in silica nanoparticles. The gist is functional silica particles containing one kind of compound whose force is also selected.

[0009] Powerful functional silica particles can be classified into the following (1) to (3):

(1) Functional silica particles containing methyl viologen in the silica particles (hereinafter referred to as “methyl viologen ZSiO particles” for convenience! /)

2

The functional silica nanoparticles have the following characteristics: (a) having fluorescence, (b) being colorless and transparent to yellow when irradiated with ultraviolet rays in the presence of oxygen, and (c) being excited by ultraviolet irradiation and having a long excitation Z fluorescence wavelength. It can be mentioned that the wavelength shifts to the wavelength side.

[0010] (2) Functional silica particles containing xylenol orange in silica particles (hereinafter referred to as feces 1)

The characteristics of the functional silica particles are as follows: (d) yellow under acidic conditions and red under alkaline conditions And (e) a purple color due to the presence of Fe ³⁺ under acidic conditions. The functional silica particles are red in the presence of alkali regardless of the presence or absence of Fe ³⁺ .

[0011] (3) Functional silica particles containing 4-amino- (2,3,6,6-tetramethyl-1-piberidinyloxy) in silica particles (hereinafter referred to as “4-amino” for convenience) Also called “TempoZSiO particles”)

2

The functional silica particles are characterized by having stable (f) radicals by stably including “4-amino Tempo” in the protected state in the silica layer. .

Furthermore, the present invention provides the following methods for producing the functional silica particles: methyl viologen, xylenol orange, and 4-amino- (2,3,6,6-tetramethyl-1- One compound selected from the group consisting of (Pyberidi-Loxy) is mixed with (3-thiocyanatepropyl) triethoxysilane, and then mixed with tetraethylorthosilicate in the presence of ammonia in The method for producing functional silica particles according to any one of the above (1) to (3).

Hereinafter, the present invention will be described.

[0014] (1) Methylviologen ZSiO particles

2

The methyl viologen Zsio particles of the present invention are fine particles (silica particles) formed from silica.

2

) Is fixed with a plurality of methyl viologen molecules. The immobilization mode is not particularly limited as long as methyl viologen molecules are incorporated in the silica particles. Silica particles are formed in layers of silica (SiO 2)

2

Forces that exist as a form of methyl viologen molecules in the silica particles

For example, as shown in FIG. 1, a mode in which a plurality of methylviologens are present between the layers of the silica particles can be exemplified. However, it is not limited to this.

[0015] The size of the methyl viologen ZSiO particles of the present invention is not particularly limited, depending on the purpose.

2

It can be adjusted appropriately. For example, how to produce methylviologen Zsio particles as described below

2

By adjusting the number of reactions with tetraorthosilicate in the presence of ammonia, or by adjusting the concentration of tetraorthosilicate used in the reaction, Ruviologen ZSiO particles with a nano-order particle size (diameter) of several nm to several hundred nm

2

The particle size can be freely adjusted as desired, for example, as a nanoparticle having a particle size, or as a microparticle having a particle size (diameter) on the order of several tens / zm to ten / zm. be able to. Preferred methyl viologen ZSiO particles have a nanoparticle diameter.

2

As such nanoparticles, for example, a diameter of 2 to 1000 nm, preferably a diameter of 2 to 100 nm, more preferably about 5 to 60 nm, and even more preferably a diameter of 10 to Silica nanoparticles having a diameter of about 40 nm, more preferably about 15 to 30 nm, and particularly preferably about 20 to 30 nm can be mentioned.

[0016] The silica particles may contain a plurality of methyl viologen molecules. For example, when silica nanoparticles having a diameter of 30 nm are taken as an example, 1 to 5 molecules, preferably 5 to 10 molecules, more preferably about 15 molecules of methyl viologen molecules can be contained in one particle. In the case of particles containing 15 molecules of methyl viologen in one particle, the concentration of the methyl viologen in the particle is about 1.8 mM.

[0017] Methyl viologen (also known as paraquat) is a known compound represented by the following formula.

[0018] [Chemical 1]

1.1 圆

[0019] The methyl viologen itself has the following properties (a) to (c) by allowing the silica particles to retain a force that is a colorless and transparent compound having no fluorescence. Become.

(a) having fluorescence

By emitting excitation light with a wavelength of 357 nm, light emission with a wavelength of 430 nm is generated.

(b) It turns from colorless and transparent to yellow when irradiated with ultraviolet light in the presence of oxygen.Methylviologen itself is colorless and transparent, and is irradiated with ultraviolet light in the presence of oxygen. No color change occurs. However, when it is irradiated with ultraviolet rays in the absence of oxygen, it turns from colorless and transparent to blue.

(C) Excitation by ultraviolet irradiation Z fluorescence wavelength shifts to longer wavelength side

As shown in Example 1 described later, when ultraviolet rays (peak wavelength 365) are irradiated, the maximum peak wavelength (357 nm Z430 nm) of the excitation light Z emission before irradiation is shifted to the longer wavelength side. The shift width toward the long wavelength side can be adjusted by changing the proportion of methylviologen retained in the silica particles.

[0020] As indicated above, the methyl viologen ZSiO particles of the present invention can be irradiated by ultraviolet irradiation.

2

Since it emits fluorescence in the visible light region, it can be effectively used as a fluorescent labeling agent. In addition, since it is possible to change the fluorescence wavelength continuously in the range of 400 to 500 by ultraviolet irradiation, it can be effectively used as a wavelength-selective fluorescent labeling agent for cells, etc. with respect to 400-500 nm fluorescence. it is conceivable that.

[0021] Furthermore, the reduced form of methyl viologen is called a methyl viologen radical, and it is known that an oxygen molecule is easily reduced in vivo to generate an active oxygen radical (Bahnemnn, DW; Fisher, Jan., E .; Henglein, A .; The Two-electron Oxidation of Methyl Viologen; J. Chem. Soc. Faraday Trans. 1, 1987 (83); pp 2559-2571.).

[0022] Generally, tumor radiotherapy involves irradiating a tumor site with γ-rays to destroy the tumor by radicals generated by radiolysis of water around the tumor or by high energy directly from γ-rays. is there. At this time, if the methyl viologen ZSiO 2 particles of the present invention, which is washed only with water, are taken into the tumor site, methyl viologen ZSiO particles in addition to radicals generated by the decomposition of water by the irradiated γ-rays. Methylviologen supported on

2

It is thought that the electrons are stored as a result of the reduction, and the reducing power makes it possible to attack the tumor. By doing so, it is possible to reduce the normal γ-ray irradiation dose, and to reduce the side effects caused by γ-ray irradiation. Thus, the methylviologen ZSiO particles of the present invention are used as a sensitizer for γ-ray irradiation in radiotherapy.

2

Is also expected.

[0023] In addition, ESR-CT is known as a device for non-invasively measuring radicals generated in vivo. By using this, ESR-CT is used to track radicals in living organisms. Can. Thus, for example, labeling a target drug with methylviologen Zsio particles

2

By doing so, it is possible to observe the metabolism in the body. It can also be used to observe in real time how radicals generated during radiation therapy are generated and disappeared, attacking tumors, or how normal cells are attacked and cause side effects. It is possible to use.

[0024] The methyl viologen ZSiO particles of the present invention first contain methyl viologen (3-thiocyanate).

2

Topropyl) triethoxysilane, and then mixed with tetraethylorthosilicate in the presence of an aqueous ammonia solution as a catalyst for silica particle formation.

[0025] By adopting a process in which methylviologen is mixed with (3-thiocyanatepropyl) triethoxysilane as the first process, a high concentration of methylviologen in the silica particles (for example, a nanoparticle having a particle diameter of 30 nm). The particles can be fixed at a ratio of 15 molecules / particle or more, and preferably a ratio of 5 molecules / particle or more to nanoparticles having a particle size of 20 nm. Specifically, this operation can be performed by dissolving methyl viologen in a dimethyl sulfoxide solution and mixing it with (3-thiocyanate propyl) triethoxysilane. In this case, the mixing ratio of methylviologen and (3-thiocyanatepropyl) triethoxysilane, for example, the mixing ratio of (3-thiocyanatepropyl) triethoxysilane to 2.5 mg of methylviologen is usually 0.5 to A ratio of about 1 μ1, preferably 1 to 21 and more preferably about 2.31 can be exemplified. In addition, when expressed as a molar ratio, the mixing ratio of (3-thiocyanatepropyl) triethoxysilane used per 1 mol of methylviologen is usually 0.5 to 1 mol, preferably 1 About 2 mol, more preferably about 1 mol can be mentioned.

Here, the mixing conditions are not particularly limited. For example, the room temperature can usually be raised as the mixing temperature. Other mixing conditions are not particularly limited, but it is preferable to mix well for about 20 to 24 hours while stirring.

Next, the obtained reaction solution is mixed with tetraethyl orthosilicate. The reaction in this case is a polycondensation reaction of tetraethylorthosilicate to form silica particles. 1S This reaction (silica particle formation) should be performed in the presence of ammonia as a catalyst. Is preferred. Further, this reaction is preferably carried out in a hydrous alcohol, preferably a hydrous ethanol, as a solvent. Here, the alcohol concentration in the reaction system is not limited, and preferably about 80% by volume.

[0028] The proportion of tetraethyl orthosilicate used in the reaction is not particularly limited, but is 400 to 500 times, preferably 500 to 600 times the amount of methyl viologen used in the previous step 8.5 μmol / ml. It is preferable to use at a ratio of 500 times to 550 times, more preferably 500 to 550 times. In terms of the molar ratio, 400 to 500 moles, preferably 500 to 600 moles, more preferably 500 to 550 moles may be cited as a blending ratio of tetraethyl orthosilicate used for 1 mole of methylviologen. Can do.

[0029] Powerful mixing conditions are not particularly limited, but examples thereof include a method of sufficiently mixing for 20 to 24 hours with stirring under conditions of 20 to 25 ° C, for example.

[0030] Next, the obtained reaction solution is subjected to solid-liquid separation using, for example, an ultrafiltration apparatus in which a membrane filter is set, and washed with water as necessary, whereby the methyl piorogen Zsio particles of the present invention are obtained. Can be obtained.

2

[0031] (2) Xylenol orange ZSiO particles

2

The xylenol orange / SiO particles of the present invention are fine particles formed from silica (silica

2

Particles) are fixed with a plurality of xylenol orange molecules. The manner of immobilization is not particularly limited as long as it exists in a form in which xylenol orange molecules are incorporated into silica particles! For example, the existence mode of xylenol orange molecules inside silica particles is the methylviologen Zsio particles described in (1).

2, a plurality of xylenol alcohols are formed between the layers of silica (SiO 2) forming the particles.

2

Force that can exemplify a mode in which a range exists [0032] The size of the xylenol orange ZSiO particles of the present invention is not particularly limited and depends on the purpose.

2

It can be adjusted accordingly. For example, xylenol orange Zsio particles described below

By adjusting the number of reactions with tetraorthosilicate in the presence of ammonia or adjusting the concentration of tetraorthosilicate used in the reaction according to the production method of 2, xylenol orange ZSiO particles Nano-order from several nm to several hundred nm They can be prepared as nanoparticles with a single particle size (diameter), or as microparticles with a particle size (diameter) on the order of several micrometers to several tens of micrometers. The particle size can be adjusted freely. Preferred xylenol orange / SiO grains

The twins are nanoparticles (silica nanoparticles) having a diameter of the order of nanometers, and as such nanoparticles, for example, a diameter of 2 to 1000 nm, preferably a diameter of 2 to 100 nm, more preferably about 5 to 60 nm, More preferred are silica nanoparticles having a diameter of about 10 to 40 nm, more preferably about 15 to 30 nm, and particularly preferably about 20 to 30 nm.

[0033] The silica particles may contain a plurality of xylenol orange molecules. For example, when silica nanoparticles having a diameter of 30 nm are taken as an example, 1 to 10 molecules, preferably 50 to 150 molecules, more preferably 100 to 150 molecules, and even more preferably 112 molecules of xylenol orange molecules are contained in one particle. Can be included. In the case of particles containing 112 molecules of xylenol orange in one particle, the concentration of xylenol orange in the particles is about 13 mM.

[0034] Xylenol orange is a known compound represented by the following formula.

[0035] [Chemical 2]

Xylenol orange is a compound that has different properties depending on pH changes (yellow under acidity, red under alkali) and itself has a use as a pH indicator. In addition, the silica nanoparticles have the same properties as shown in the following (d):

(d) yellow under acidic conditions (e.g. pH 2), red under alkali conditions (e.g. pH 9) As described above, the xylenol orange Zsio particles of the present invention are nano-order and

2

It can be adjusted to micro-order microparticles, and the force can be contained inside the xylenol orange in a concentrated form without impairing its properties. It can be effectively used as a pH indicator in the measurement of pH and changes in pH in the micro region such as cells, microcells and fine tissues.

(e) Under acidic conditions, purple due to the presence of Fe ³⁺

As mentioned above, xylenol orange ZSiO particles can be produced under acidic conditions, for example Fe ²⁺

2

In the absence also Fe ³⁺ in the case but the present exhibits a purple coordinated with F e ³⁺ in the presence of a force Fe ³⁺ that exhibits a yellow color. However, under alkaline conditions, Fe ³⁺ is red regardless of the presence or absence of Fe ³⁺ .

[0037] Such a phenomenon is caused by immobilizing xylenol orange on SiO particles to form Fe ³⁺ .

2

This is thought to be due to an increase in coordination ability. Xylenol orange of the present invention

/ SiO particles can be used as a detection agent for Fe ³⁺ in cells, etc.

2

wear.

[0038] Further, the xylenol orange ZSiO of the present invention has a radiation sensitivity as described below.

2

It can be used as a dye for light in chemical dosimeters for radiation-related personal exposure management, contamination management, medical exposure management, or radiation dosimetry:

When an aqueous solution containing Fe ²⁺ is irradiated with γ-rays, the radiolysis of water first occurs, generating radicals and hydrated electrons. These radicals react with dissolved oxygen to produce a large amount of hydrogen peroxide and soot radicals. When Fe ²⁺ coexists in these, they become oxidizing agents and convert Fe ²⁺ to Fe ³⁺ . And sour. Therefore, it is possible to measure the amount of radiation received from this oxidation amount (Fe ²⁺ → Fe ³⁺ ). The xylenol orange / SiO particles of the present invention have xylenol orange molecules in one SiO particle.

twenty two

Because it is contained in a high concentration (in other words, xylenol orange is concentrated and contained in one particle), it effectively captures Fe ³⁺ generated from Fe ²⁺ with a small dose A small dose can be qualitatively and quantitatively indicated by a color change (yellow to purple). [0039] The xylenol orange ZSiO particles of the present invention first have xylenol orange (3-thiol).

2

It can be prepared by mixing with cyanatepropyl) triethoxysilane and then mixing with tetraethylorthosilicate in the presence of aqueous ammonia as a catalyst for silica particle formation.

[0040] As a first step, xylenol orange is mixed with (3-thiocyanatepropyl) triethoxy silane to increase the concentration of xylenol orange in silica particles (for example, more than 112 molecules Z particles or more in 30 nm nanoparticles). Can be fixed in proportion). Specifically, such an operation can be carried out by dissolving xylenol orange in a dimethyl sulfoxide solution and mixing it with (3-thiocyanatepropyl) triethoxysilane. In this case, the mixing ratio of xylenol orange and (3-thiocyanatepropyl) triethoxysilane is, for example, xylenol orange 14 mol / ml, and (3-thiocyanatepropyl) triethoxysilane as a molar ratio of 0.5. A method of mixing at a ratio of ˜2, preferably 0.5 to 1.5, more preferably about 0.5 can be mentioned.

[0041] Here, the mixing conditions are not particularly limited. For example, the room temperature can usually be raised as the mixing temperature. Other mixing conditions are not particularly limited, but it is preferable to mix well for about 20 to 24 hours while stirring.

[0042] Next, the obtained reaction solution is mixed with tetraethyl orthosilicate. The reaction in this case is a polycondensation reaction of tetraethylorthosilicate to form silica particles. This reaction (silica particle formation) is preferably carried out in the presence of ammonia as a catalyst. Further, such a reaction is preferably performed in a hydrous alcohol, preferably hydrous ethanol, as a solvent. Here, the alcohol concentration in the reaction system is not limited, but preferably about 80% by volume.

[0043] The ratio of tetraethyl orthosilicate used in the reaction is not limited !, but it is preferably 200 to 500 times the amount of xylenol orange used in the previous step, 7 μmol / ml. Is preferably used in a ratio of 500 to 700 times, more preferably 600 to 650 times. When this is shown by molar ratio, the proportion of tetraethyl orthosilicate used per mole of xylenol orange is 200 to 500 moles, preferably 500 to 700 moles, more preferably 600 to 650 moles. [0044] Powerful mixing conditions are not particularly limited. For example, while stirring at 20 to 25 ° C, 2

A method of sufficiently mixing for about 0 to 24 hours can be exemplified.

[0045] The xylenol orange ZSiO particles of the present invention are then separated by solid-liquid separation of the obtained reaction liquid using, for example, an ultrafiltration device with a membrane filter set, and washing with water as necessary. Can be obtained.

2

[0046] (3) 4-Amino TempoZSiO particles

2

The 4-amino tempoZSiO particles of the present invention are fine particles (silica particles) formed from silica.

2

A plurality of 4-amino- (2,3,6,6-tetramethyl-1-piberidi-loxy) [4-amino tempo] molecules are fixedly attached to each other. The form of the anchor is not particularly limited as long as it is present in the form in which 4-amino Tempo molecules are incorporated inside the silica nanoparticles! / ヽ. For example, the presence mode of 4-amino tempo molecules in silica nanoparticles is similar to the methyl viologen ZSiO particles described in (1), and the SiO layer that forms the particles.

2 A force that can exemplify a mode in which a plurality of 4-amino tempos exist between two layers, but is not limited to this.

[0047] The size of the 4-amino tempoZSiO particles of the present invention is not particularly limited, and is suitable according to the purpose.

2

It can be adjusted. For example, in the production method of 4-amino TempoZSiO particles described later

2

Leave

By adjusting the number of reactions with tetraorthosilicate in the presence of ammonia and adjusting the concentration of tetraorthosilicate used in the reaction, 4-amino Tempo / SiO particles can be reduced to several nm to several hundred nm. Nano-particles with a nano-order particle size (diameter)

2

The particle size can be freely adjusted as desired, for example, as a microparticle, or as a microparticle having a microorder particle size (diameter) of several / zm to several tens / zm. The preferred 4-amino tempo / SiO particles are nano-sized in diameter.

2

Particles (silica nanoparticles), for example, having a diameter of 2 to 1000 nm, preferably 2 to 100 nm, more preferably about 5 to 60 nm, even more preferably about 10 to 40 nm, and even more preferably. Examples thereof include silica nanoparticles having a diameter of about 15 to 30 nm, particularly preferably a diameter of about 20 to 30 nm.

[0048] The silica particles can contain a plurality of 4-amino tempo molecules. example For example, taking silica nanoparticles with a diameter of 30 nm as an example, one particle can contain 1 to 100 molecules, preferably 100 to 200 molecules, more preferably about 150 molecules of 4-amino tempo molecules. In the case of particles containing 150 molecules of 4-amino tempo in one particle, the concentration of 4-amino tempo in the particles is about 17 mM.

[0049] 4-Amino Tempo (4-amino- (2,3,6,6-tetramethyl-1-piberidi-loxy)) is a known compound represented by the following formula.

[0050] [Chemical 3]

[0051] The 4_amino Tempo molecule is a radical molecule, and as such is a compound having a use as a labeling reagent. The 4-amino tempoZSiO particles of the present invention have 4-

2

By maintaining the amino Tempo molecule as a radical molecule stably and in a state of protecting it in the silica layer, it has the following properties (f):

(f) having radicals

These properties of 4-amino TempoZSiO particles, when administering radicals in vivo,

2

It can prevent oxidation and disappearance of radicals and can be used as an effective labeling substance.

[0052] The 4-amino tempo ZSiO particles of the present invention are prepared by first producing 4-amino tempo (3-thiocyanate).

2

It can be prepared by mixing with oral pill) triethoxysilane and then with tetraethylorthosilicate in the presence of aqueous ammonia as a catalyst for silica particle formation.

[0053] As the first step, 4-amino tempo is mixed with (3-thiocyanatepropyl) triethoxysilane to give a high concentration of 4-amino tempo (for example, as 30-particle particles). It can be fixed at a ratio of 150 molecules Z particles or more. This operation is specific Specifically, it can be carried out by dissolving 4-amino tempo in a dimethyl sulfoxide solution and mixing it with (3-thiocyanatepropyl) triethoxysilane. In this case, the blending ratio of 4-amino tempo and (3-thiocyanatepropyl) triethoxysilane is, for example, 7 mol / ml of 4-amino tempo molecule, (3-thiocyanatepropyl) triethoxysilane. Can be mentioned in a molar ratio of 0.5 to 2, preferably 0.5 to 1.5, more preferably about 1.

[0055] Next, the obtained reaction liquid is mixed with tetraethyl orthosilicate. The reaction in this case is a polycondensation reaction of tetraethylorthosilicate to form silica particles. This reaction (silica particle formation) is preferably carried out in the presence of ammonia as a catalyst. Further, such a reaction is preferably performed in a hydrous alcohol, preferably hydrous ethanol, as a solvent. Here, the alcohol concentration in the reaction system is not limited, but preferably about 80% by volume.

[0056] The ratio of tetraethyl orthosilicate used in the reaction is not limited, but it is 200 to 400 times, preferably 400 to 550, with respect to the amount of 4-amino tempo used in the previous step of 7 mol / ml. It is preferably used at a ratio of 550 to 600 times. In terms of molar ratio, the proportion of tetraethyl orthosilicate used relative to 4-amino tempol mole is 200 to 400 moles, preferably 400 to 550 moles, more preferably 550 to 600 moles. be able to.

[0057] Although vigorous mixing conditions are not particularly limited, for example, a method of sufficiently mixing for about 20 to 24 hours with stirring under conditions of 20 to 25 ° C can be exemplified.

[0058] Next, the obtained reaction solution is subjected to solid-liquid separation using, for example, an ultrafiltration apparatus in which a membrane filter is set, and washed with water as necessary, whereby the 4-amino tempo of the present invention is obtained. / SiO particles can be obtained.

2

The invention's effect

[0059] According to the present invention, methyl viologen, xylenol orange, or 4-amino- (2,3,6,6 -Tetramethyl-tobiberidi-loxy) can be retained stably and at a high concentration in silica particles, and as a result, a new function or characteristic that cannot be obtained by each of the above molecules alone can be obtained. It became possible.

Brief Description of Drawings

[0060] [Fig. 1] Expected morphology of methyl viologen ZSiO particles (methyl viologen in SiO particles)

twenty two

FIG.

FIG. 2 is a diagram comparing the excitation Z fluorescence spectra of methyl viologen ZSiO 2 particles before irradiation with ultraviolet rays (dotted line) and after irradiation with ultraviolet rays (solid line) in Example 1. The vertical axis represents fluorescence intensity (Intensity), and the horizontal axis represents wavelength (Wavelength: nm).

FIG. 3 shows an absorption spectrum of xylenol orange ZSiO particles in Example 2.

2

It is. The vertical axis represents the absorbance, and the horizontal axis represents the wavelength (wavelength).

[Fig. 4] In Example 2 (5), the Fe ²⁺ Z xylenol orange ZSiO reagent aqueous solution

2

This is a spectrum obtained by measuring the absorbance (520 to 700 nm) of the aqueous solution before and after irradiation of about 10 Gy (irradiation with a 60-Coy line irradiation device for 30 minutes). It can be seen that the absorbance at 600 nm increases by about 0.15 due to the color change caused by y-ray irradiation. b is the spectrum before γ-ray irradiation, and a is the spectrum after γ-ray irradiation.

[FIG. 5] In Example 2 (5), Fe ²⁺ Z xylenol orange ZSiO reagent and gelatin (0.2 g

2

This is a spectrum obtained by measuring the absorbance (300-800 nm) of the aqueous solution before and after γ-irradiation (irradiated with 60_Co y-ray irradiator for 30 minutes) to an aqueous solution containing 1 ml of gelatin / ml gelatin). . The dotted line is the spectrum of the aqueous solution before γ-ray irradiation, and the solid line is the spectrum of the aqueous solution after γ-ray irradiation.

[FIG. 6] Electron spin resonance (ESR) of 4-amino TempoZSiO particles prepared in Example 3

2

It is a figure which shows a spectrum.

BEST MODE FOR CARRYING OUT THE INVENTION

[0061] In the following, in order to clarify the configuration and effects of the present invention, examples will be described.

However, the present invention is not affected by these examples.

Example 1

[0062] Silica particles containing methyl viologen (methyl viologen ZSiO granule 1 (1) Methyl viologen n hydrate (manufactured by Wako Pure Chemical Industries, Ltd.) 2.1 mg was dissolved in 1 ml of dimethyl sulfoxide (DMSO), and (3-thiocyanatepropyl) triethoxysilane [( 3-Thiocyanatopropyl) triethoxy silane] stock solution diluted with DMSO 10 times was mixed with 181, and mixed at about 25 ° C for about 1 hour. Next, 3 ml of ethanol, 0.1 ml of tetraethylorthosilicate (TEOS), 1 ml of distilled water, and 0.1 ml of 30% by volume aqueous ammonia solution are added to 1 ml of this reaction solution, and stirred at about 25 ° C. for about 1 day. Mixed. Thereafter, the obtained reaction solution was filtered using an ultrafiltration apparatus in which an Amicon YM-100 membrane filter (Amicon, pore size: 15 nm) was set, and methyl viologen ZSiO particles (particle size: 20

2

˜30 nm) was filtered off and washed with distilled water.

[0063] (2) 260 nm of an aqueous solution obtained by diluting the obtained methylviologen ZSiO particles 30 times with water

2

The absorbance was measured and found to be 0.072. Since the molecular extinction coefficient is 204170, the concentration is 10.5 μΜ. Since 20 ml of this solution was obtained, it becomes 0.21 μmol per 20 ml. Therefore, as a 30 nm particle, the methyl viologen concentration in the particle is about 1.8 mM. Until now, there have been known examples of methyl viologen molecules immobilized on silica nanoparticles at such concentrations.

[0064] (3) The methyl viologen used as a raw material is a substance that does not emit fluorescence, whereas the methyl viologen ZSiO particles obtained above have fluorescence, and the slit width (Ex / Em) = 5 /Five

2

Broad emission was observed at 430 nm with excitation light at 357 nm at nm. From this fact, methyl viologen ZSiO particles are useful as fluorescent substances themselves.

2

It can be used as a fluorescent label that emits fluorescence in the visible light region.

[0065] (4) Next! In, in this 30-fold diluted aqueous solution of methyl viologen ZSiO particles, 500 W ultra high pressure

2

Using a mercury lamp (Ushio-500), UV light was irradiated for about 10 minutes, and fluorescence was measured again. Then, it was observed that the aqueous solution was colored yellow, and as shown in Table 1 and FIG. 2, the excitation light and the emission wavelength were shifted to the longer wavelength side.

[0066] [Table 1] Excitation / emission wavelength (SIU) before and after ultra-high pressure mercury lamp irradiation

)

[0067] As described above, when the methyl viologen ZSiO particles are irradiated with ultraviolet rays, the fluorescence wavelength is increased.

2

Since it shifts to longer (low energy side), methyl viologen

2

It is considered that ruviologen molecules overlap and the resulting electrons are delocalized. Although not limited, the methylviologen ZSiO particles obtained above are shown in FIG.

2

Thus, a plurality of methyl viologen molecular forces encapsulated between layers formed by SiO

2

It is thought to form a child.

[0068] Further, the forceful methyl viologen ZSiO particles are the amount of methyl viologen molecules to be encapsulated.

2

By changing, the shift wavelength of the fluorescence wavelength after UV irradiation can be changed and the amount of electrons captured per particle can be increased, so it is effective as a wavelength selective fluorescent label for cells, etc., for 400-500 nm fluorescence. It is thought that it can be used.

Example 2

[0069] Silica nanoparticles containing xylenol orange (xylenol orange SiO particles)

2 ¾1

(1) Xylenol orange (manufactured by Aldrich) 2. Dissolve lmg in 2ml of dimethyl sulfoxide (DMS 0) and add (3-Thiocyanatopropyl) triethoxysilane ((3-Thiocyanatop ropyDtriethoxy silane) stock solution Was adjusted so that the weight ratio of xylenolene range: (3-thiocyanatepropyl) triethoxysilane contained in the reaction solution was 2: 1, which was about 25 ° C. Then, 2 ml of this reaction solution was mixed with 6 ml of ethanol, 0.1 ml of tetraethylorthosilicate (TEOS), 2 ml of distilled water, and 0.2 ml of 30% by volume aqueous ammonia solution at about 25 ° C. The resulting reaction solution was filtered using an ultrafiltration device in which an Amicon YM-100 membrane filter (Amicon, pore size: 15 nm) was set, Xylenol orange ZSiO grains (Grain

(2 diameter 20-30 nm) was collected by filtration and washed with distilled water. Although not limited, the obtained xylenol orange ZSiO particles are the same as the methylviologen ZSiO particles.

twenty two

In addition, a plurality of xylenol orange molecules are formed between each layer of particles formed by SiO. Is considered to have a form of inclusion.

[0070] (2) 578 η of an aqueous solution obtained by diluting the obtained xylenol orange ZSiO particles 10 times with water

2

The absorbance at m was measured to be 2.173 (Fig. 3). Since the molecular extinction coefficient is 47000, 0.462 mM xylenol orange molecule was fixed in silica particles, that is, about 6.6% of xylenol orange used as a raw material was fixed in silica particles. confirmed. Therefore, the xylenol orange molecular concentration in the 30 nm particle is about 13 mM. To date, it is well known that xylenol orange molecules have been immobilized on silica nanoparticles at such concentrations.

[0071] (3) A 10-fold diluted aqueous solution of the above xylenol orange ZSiO particles is used as a raw material.

2

As with xylenol orange, the color was yellow under acidic conditions (pH 7) and red under alkaline conditions (pH> 7). Xylenol orange itself is a pH indicator (yellow under acidic conditions and red under alkaline conditions). From the above, xylenol orange can be used as a pH indicator even when it is supported on SiO and concentrated. Confirm that it can be used

2

Came. Furthermore, xylenol orange can be used as it is by supporting it on SiO.

2

It can be used for pH measurement in a specific area (for example, pH measurement of substances dispersed in an aqueous solution or cells). From this, according to the xylenol orange ZSiO particles of the present invention, for example, in vivo microparticles such as macrophages

2

It may be possible to measure the pH in the tissue.

[0072] (4) As described above, a 10-fold diluted aqueous solution of xylenol orange ZSiO particles is acidic.

2

It showed a yellow color under the conditions (pH 7). Next, when Fe ³⁺ was added thereto, the color changed to purple. By taking advantage of this phenomenon of xylenol orange / SiO particles, Si

2

Zero-protected xylenol orange can detect Fe ³⁺ in cells

2

It is.

[0073] (5) Xylenol orange Z silica particle aqueous solution 0.4ml, 10mM ammonium sulfate iron (Π) (1Ν) 0.1ml aqueous solution and 0.1ml IN sulfuric acid were mixed, and Fe ²⁺ Z xylenol orange An aqueous solution of / SiO reagent (acidic, pH 2, yellow) was prepared. Then this Fe ²⁺ / xyleno

2

Luorange ZSiO reagent aqueous solution is irradiated with about 10 Gy of γ-ray at room temperature (60-Co y-ray irradiation device

2

For 30 minutes). Then, it is observed that the aqueous solution changes its color to yellowish purple. It was done. This color change is thought to be caused by the coordination of Fe ³⁺ force xylenol orange produced by acidification of Fe ^{2+ in} aqueous solution by gamma irradiation. When the absorbance at 600 nm was measured, it was found that the absorbance increased by about 0.15 due to the color change caused by γ-ray irradiation (Fig. 4, change from spectrum b to spectrum a). In view of the above, the change in absorbance caused by γ-irradiation was observed by using the xylenol orange ZSiO particles of the present invention in which xylenol orange was fixed to silica nanoparticles.

2

It turns out that you can.

[0074] Next, 0.4 ml or 0.2 ml of an aqueous solution of xylenol orange-silica particles was mixed with 0.1 ml of 10 mM ammonium iron sulfate (IIX1N) solution, 0.1 ml of IN sulfuric acid, and 0.6-2.4 ml of distilled water. Prepare an aqueous solution of Fe ²⁺ / xylenol orange / SiO reagent (acidic, pH 2, yellow)

2

Thereafter, 0.2 g / ml gelatin was mixed at a rate of 1 ml to 3 ml, and these aqueous solutions were irradiated with about 10 Gy of γ rays (irradiated with a 60-Co y ray irradiation device for 30 minutes). As a result, a color change (change from yellow to purple) was observed as a result of coordination of Fe ³⁺ produced by Fe ²⁺ by γ-irradiation to xylenol orange as described above. In addition, when this color change was observed by measuring the absorbance, it was confirmed that the purple absorbance changed to the maximum when 1 ml of 0.2 g / ml gelatin was added (absorbance of 1.329 at 586 nm). From this value, according to the xylenol orange Z silica particles of the present invention, the absorbance of xylenol orange after irradiation is about 1.2 times that of the literature [Radiation Physics and Chemistry, 61 (2001) 433-435]. What I got was helping (Figure 5)

Example 3

[0075] Silica nanoparticles containing 4-amino- (2,3,6,6-tetramethyl-biperidinyloxy) "4-amino amino acid Terqpo SiO

2 ¾1

(1) 4-amino- (2,3,6,6-tetramethyl-1-piberidi-loxy) [hereinafter referred to as “4-amino Tempo”] (manufactured by Aldrich) 0.9 mg of lml of dimethyl sulfoxide ( In (DMSO), the (3-thioocyanatopropyl) triethoxysilane ((3-Thiocyanatopropyl) triethoxysilane) stock solution was mixed with a solution 18 1 diluted 10-fold with DMSO, and this was mixed. The mixture was stirred at 25 ° C for about 1 hour. Then the reaction solution 0.05 ml, ethanol 3.95 ml, tetraethylorthosilicate (TEOS) 0.05 ml, of distilled water lml, and 30 volume ^_0/0 aqueous ammonia 0.1ml pressurized The mixture was stirred at about 25 ° C. for about 1 day. Thereafter, the obtained reaction solution was filtered using an ultrafiltration apparatus in which an Amicon YM-100 membrane filter (Amicon, pore size: 15 nm) was set, and 4-amino TempoZSiO particles (particle size: 20- (30 nm)

2

Washed with distilled water.

(2) The obtained 4-amino amino TempoZSiO particles are dispersed in distilled water to prepare a colloidal state,

2

Of these, 170 1 was collected in a quartz flat cell for measuring aqueous solutions (JEOL DATUM LC12), and electron spin resonance (ESR) spectra were measured at room temperature (JEOL TE-300 [X-band ]use). The result is shown in FIG.

[0077] As shown in FIG. 6, the peak interval was 17.2 gauss, which was slightly larger than the peak interval (15 gauss) when 4-amino Tempo itself was measured in the same manner. This is because the free rotation of the radical (4-Amino Tempo) is suppressed, that is, 4-Amino Tempo is converted into SiO.

2 This indicates that it is fixed. Although not limited to these, the resulting 4-amino amino TempoZSiO particles are similar to the methylviologen ZSiO particles.

twenty two

A plurality of 4-amino tempo molecules are formed between each layer of particles formed by SiO layers.

2

It is considered to have a form that is enclosed and fixed.

[0078] Conventionally, when a radical (4-amino tempo) is administered in a living body, there is a problem that it is oxidized in the living body or a signal disappears. Dicar (4-amino Tempo) can be stably immobilized on SiO particles,

2

A radical signal that does not occur can be observed. Therefore, 4-amino TempoZ SiO particles are effective as a labeling reagent for evaluating the kinetics of drug metabolism in vivo.

2

It is considered that it can be used.

Claims

The scope of the claims

[1] Silica particles contain one compound with a selected group strength consisting of methyl viologen, xylenol orange, and 4-amino- (2,3,6,6-tetramethyl-1-piberidi-loxy) Thus, functional silica particles.

[2] The functional silica particle according to claim 1, which contains methyl viologen in the silica particle and has the following characteristics:

(a) having fluorescence,

(b) It turns from colorless and transparent to yellow when irradiated with ultraviolet light in the presence of oxygen.

(c) Excitation Z fluorescence wavelength shifts to longer wavelength side by ultraviolet irradiation.

[3] The functional silica particle according to claim 1, which contains xylenol orange in the silica particle and has the following characteristics:

(d) yellow under acid, red under alkali,

(e) It turns purple due to the presence of Fe ³⁺ under acidic conditions.

[4] The silica particle according to claim 4, wherein 4-amino- (2,3,6,6-tetramethyl-topiberidioxy) is stably contained in a state protected by a silica layer. Functional silica particles described in 1:

(f) Has a radio canore.

[5] The functional silica particles according to V of claim 1 to 4, wherein the functional silica particles are fine particles having a nano-order particle size.

[6] One compound selected from the group consisting of methylviologen, xylenol orange, and 4-amino- (2,3,6,6-tetramethyl-1-piberidi-loxy) is selected as (3-thio The method for producing functional silica particles according to claim 1, comprising a step of mixing with cyanate propyl) triethoxysilane and then mixing with tetraethylorthosilicate in the presence of ammonia.