KR20100123795A - Photoswitchable fluorescent silica nanoparticles and method for preparing the same - Google Patents

Abstract

PURPOSE: Silica nano particles in which a photoswitchable material is combined with a fluorescent material and a manufacturing method thereof are provided to usefully use silica nano particles as a contrast medium for biometric imaging. CONSTITUTION: A photoswitchable material and silica nano particles are mixed. The silica nano particles are collected. The nano particle is dispersed in ethanol. Amide coupling is formed between an amine and a fluorescent material on the surface of the silica nano particles. The silica nano particle in which the photoswitchable material is combined with the fluorescent material is produced.

Description

Silica nanoparticles in which a photochromic material and a fluorescent material are combined and a method for preparing the same {Photoswitchable Fluorescent Silica Nanoparticles and Method for Preparing the Same}

The present invention relates to silica nanoparticles having a photochromic substance and a fluorescent substance bonded to the surface of silica particles, and a method of manufacturing the same.

Photochromic phenomenon refers to a phenomenon in which the color of a compound or a product containing such a compound is reversibly changed by light when exposed to ultraviolet light or visible light.

For example, the diarylethene-based compound is a photochromic compound that changes color when exposed to ultraviolet light, and then returns to the original color of the compound when irradiated with light of different wavelengths. It has been known as a stable photochromic material which does not cause discoloration due to the occurrence of color change (Japanese Patent Laid-Open No. 86-263935), and a method for synthesizing various derivatives and applying them to optoelectronic materials has been published. 91-261782, 92-178383, 94-199846, 93-169820, 93-301873, 94-267071, 95-72567, 96-69083, 96- 245579, 97-61647, Masahiro Irie, Chem. Rev., 100: 1685, 2000, He Tian and Songjie Y., Chem. Soc. Rev. , 33:85, 2004).

Among them, fluorine-substituted diarylethene compounds are known to have higher stability and very fast photochromic properties (Scott B. et al., Org. Chem., 56: 373, 1991). Since most of the diarylethene-based compounds are dissolved in organic solvents, it is difficult to dissolve them in water, and thus the application of the diarylethene-based compounds to the bio-industrial field in an aqueous solution is extremely limited. (Takashi Hirose, et al., J. Org. Chem., 71: 7499, 2006), using such photochromic materials, has also synthesized functional groups capable of labeling biomolecules (Korean Patent Application No. 10-2007-0107245).

In addition to research on biomarkers using photochromic materials, studies on light converting materials in which optical properties are switched when irradiated with light having a specific wavelength, and light converting materials using photochromic materials and fluorescent materials Studies are also underway for use as markers (Andresen M. et al. , PNAS , 102: 13070, 2005, Soh N. et al., Chem. Commun. , 5206, 2007).

However, when the photochromic material and the photoconverting material are used as the labeling substance with a single molecule, the generated signal is weak and difficult to detect. The biomarking method using the conventional fluorescent material only has autofluorescence derived from the biomolecule. Due to the auto-fluorescence material, there was a problem in that the detection accuracy was lowered.

Accordingly, the present inventors have made efforts to develop biomolecule labeling materials using photochromic materials and fluorescent materials. As a result, the present inventors have prepared high absorbance and high sensitivity silica nanoparticles in which photochromic materials and fluorescent materials are combined, The present invention was completed by confirming that it can be used for detection of molecules and imaging of biomolecules.

It is an object of the present invention to provide silica nanoparticles having a photochromic material and a fluorescent material bonded to a surface thereof, and a method of manufacturing the same.

Another object of the present invention is to provide a method of detecting biomolecules using silica nanoparticles in which the photochromic material and the fluorescent material are combined.

In order to achieve the above object, the present invention provides silica nanoparticles in which a photochromic material represented by the following general formula (1) and a fluorescent material are bonded to silica nanoparticles having an ionic functional group exposed on its surface.

  [Formula 1]

Where P is a photochromic molecule, K is an atom or atom group, a peptide binding ligand, a DNA binding ligand, a PNA binding ligand, a protein binding ligand or a nucleic acid binding ligand, alkyl having 1 to 100 carbon atoms, and having 6 to 100 carbon atoms. Aryl, carbon number is 2 to 100 heterocyclic linker or (CH ₂ CH ₂ 0) _n , L is an atom or atomic group, except for the hydrogen atom (H), peptide binding ligand, DNA binding ligand , PNA binding ligand, protein binding ligand or nucleic acid binding ligand, alkyl having 1 to 100 carbon atoms, aryl having 6 to 100 carbon atoms, heterocyclic linker having 2 to 100 carbon atoms or (CH ₂ CH ₂ 0) _n (where n is an integer from 1 to 100), X is selected from the group consisting of O, S, NH and CH ₂ , and M is an ionic functional group exposed to the surface of the silica nanoparticles. It means a functional group to bind.

 The present invention also provides silica nanoparticles in which a photochromic material represented by the following Chemical Formula 10 and a fluorescent substance are bonded to silica nanoparticles having an ionic functional group exposed on a surface thereof.

[Formula 10]

상기 화학식 10에서 R은 결합선, 산소원자 또는 불소로 치환 또는 비치환된 C₁~C₃알킬렌기이고, Y는 각각 독립적으로 산소원자, 질소원자 또는 황원자이며, Z는 불소로 치환 또는 비치환된 메틸렌기이거나, 또는 카르보닐기이고, M 또는 M'는 수소 또는 실리카 나노입자의 이온성 기능기와 결합하는 작용기고, M과 M' 중 어느 하나는 실리카 나노입자의 이온성 기능기와 결합하는 작용기이며, n은 1~100의 정수를 의미한다.

In Formula 10, R is a C ₁ to C ₃ alkylene group unsubstituted or substituted with a bond line, an oxygen atom or fluorine, Y is independently an oxygen atom, a nitrogen atom or a sulfur atom, and Z is unsubstituted or substituted with fluorine Methylene group or carbonyl group, M or M 'is a functional group which binds to the ionic functional group of hydrogen or silica nanoparticles, and either M and M' is a functional group which binds to the ionic functional group of silica nanoparticles, n Means an integer of 1 to 100.

The present invention also provides a method for preparing a cationic silica precursor, comprising: (a) adding a silica precursor and a catalyst to a solvent to activate the silica precursor, and then adding and reacting a compound having a cationic functional group to prepare a cationic silica precursor; (b) adding distilled water to the cationic silica precursor prepared in step (a) and preparing silica nanoparticles in which the cationic functional group is exposed on the surface thereof; (c) combining the photochromic material to the cationic functional group exposed on the surface of the silica nanoparticles prepared in step (b) to prepare silica nanoparticles to which the photochromic material is bound; And (d) bonding the fluorescent material to the silica nanoparticles to which the photochromic material is bound to produce the highly sensitive silica nanoparticles to which the photochromic material and the fluorescent material are combined. To provide.

The present invention also provides a method for detecting biomolecules using silica nanoparticles in which the photochromic material and the fluorescent material are combined.

The silica nanoparticles in which the photochromic material and the fluorescent material are combined according to the present invention have high detection sensitivity and excellent light conversion properties, and thus may be usefully used as a bioimaging contrast agent.

As used herein, the term "photochromic material" has at least two isomeric forms that differ in optical properties, such as light absorption properties and refractive properties, and by light excitations at molecular intrinsic wavelengths. As a molecule that can change from one form to another, it refers to a molecule which is characterized by reversible color change upon exposure to ultraviolet light or visible light.

As used herein, the term “phosphorescent material” is photoexcitation at a wavelength inherent to the molecule, which, when excited, emits absorbed energy as light of a specific wavelength and returns to the ground level. It refers to a material characterized in that.

As used herein, the term "photoconversion" means that the photochromic material and the fluorescent material are located in close proximity, so that when the ultraviolet light is irradiated, the photochromic material that causes the structural change absorbs the fluorescence emitted from the adjacent fluorescent material, The emitted fluorescence cannot be detected or the intensity of the detected fluorescence is reduced, and upon irradiation with visible light, the photochromic material is changed into a structure that cannot absorb fluorescence, and the intensity of the fluorescence emitted from the fluorescent material is restored to its original state. It refers to the characteristic being.

As used herein, the term "contrast agent" refers to a compound that generates an image signal of a specific optical appearance contrasted with the surroundings so that the structure of cells or tissues and the location, extent and duration of expression of various biomolecules of interest can be visualized. do.

The present invention relates to silica nanoparticles in which a photochromic material represented by the following general formula (1) and a fluorescent substance are bonded to silica nanoparticles in which an ionic functional group is exposed on a surface thereof.

[Formula 1]

Where P is a photochromic molecule, K is an atom or atom group, a peptide binding ligand, a DNA binding ligand, a PNA binding ligand, a protein binding ligand or a nucleic acid binding ligand, alkyl having 1 to 100 carbon atoms, and having 6 to 100 carbon atoms. Aryl, carbon number is 2 to 100 heterocyclic linker or (CH ₂ CH ₂ 0) _n , L is an atom or atomic group, except for the hydrogen atom (H), peptide binding ligand, DNA binding ligand , PNA binding ligand, protein binding ligand or nucleic acid binding ligand, alkyl having 1 to 100 carbon atoms, aryl having 6 to 100 carbon atoms, heterocyclic linker having 2 to 100 carbon atoms or (CH ₂ CH ₂ 0) _n (where n is an integer from 1 to 100), X is selected from the group consisting of O, S, NH and CH ₂ , and M is an ionic functional group exposed to the surface of the silica nanoparticles. It means a functional group to bind.

)는 표 1에 나타낸 화합물 중에서 선택할 수 있으나 이에 국한되는 것은 아니다.In Formula 1, the photochromic molecule (

) May be selected from the compounds shown in Table 1, but is not limited thereto.

isomer A isomer B Formula 2

Formula 3

Formula 4

Formula 5

6

Formula 7

Formula 8

Formula 9

In Formulas 2 and 3, R is a C ₁ ~ C ₃ alkylene group unsubstituted or substituted with a bond line, an oxygen atom or fluorine, Y is independently an oxygen atom, a nitrogen atom or a sulfur atom, Z is a fluorine substituted or It means an unsubstituted methylene group or a carbonyl group.

The functional group that binds to the ionic functional group of the silica nanoparticles according to the present invention is a carboxyl group, a carboxylic acid derivative group (C (= O) -Cl, C (= O) -Br, C (= O) -O-succinimide) , Isocyanate group, isothiocyanate group, amine group, thiol group, hydroxyl group, iodine acetamino group, α-haloacetyl group and maleimide group may be selected.

In another aspect, the present invention provides a high sensitivity in which a photochromic material represented by the following Chemical Formula 10 and a fluorescent material are bonded to silica nanoparticles in which an ionic functional group is exposed on a surface thereof. It relates to silica nanoparticles.

       [Formula 10]

상기 화학식 10에서 R은 결합선, 산소원자 또는 불소로 치환 또는 비치환된 C₁~C₃알킬렌기이고, Y는 각각 독립적으로 산소원자, 질소원자 또는 황원자이며, Z는 불소로 치환 또는 비치환된 메틸렌기이거나, 또는 카르보닐기이고, M 또는 M'는 수소 또는 실리카 나노입자의 이온성 기능기와 결합하는 작용기고, M과 M' 중 어느 하나는 실리카 나노입자의 이온성 기능기와 결합하는 작용기이며, n은 1~100의 정수를 의미한다.

In Formula 10, R is a C ₁ to C ₃ alkylene group unsubstituted or substituted with a bond line, an oxygen atom or fluorine, Y is independently an oxygen atom, a nitrogen atom or a sulfur atom, and Z is unsubstituted or substituted with fluorine Methylene group or carbonyl group, M or M 'is a functional group which binds to the ionic functional group of hydrogen or silica nanoparticles, and either M and M' is a functional group which binds to the ionic functional group of silica nanoparticles, n Means an integer of 1 to 100.

The functional groups that bind to the ionic functional groups of the silica nanoparticles are a carboxyl group, a carboxylic acid derivative group (C (= O) -Cl, C (= O) -Br, C (= O) -O-succinimide), an isocyanate group, It may be selected from the group consisting of isothiocyanate group, amine group, aldehyde group, thiol group, hydroxyl group, iodine acetamine group, α-haloacetyl group and maleimide group.

The fluorescent material is Alexa555, bodipy TMR, Cy3, DY500, rhodamine Red-X, spectrum orange, fluorescein isothiocyanate, FITC ), Tetramethylrhodamine isothiocyanate (TRITC), alexa fluor, texas red, tetramethylrhodamine, cascade blue, DAPI ( It may be at least one selected from the group consisting of 4 ', 6-diamidino-2-phenylindole, coumarine, lucifer yellow and dansylamide.

The fluorescent material according to the present invention is 546 nm and 576 nm when the excitation and emission wavelength is Alexa545, Alexa555 is 555 nm and 580 nm, Bodipy TMR is 540 nm and 580 nm, Cy3 is 550 nm and 570 nm, and DY500 Is 500 nm and 575 nm, rhodamine Red-X is 580 nm and 590 nm, and spectrum orange is equal to 550 nm and 590 nm and the like.

As described above, in the present invention, since the photochromic material has an absorption wavelength band different from the excitation wavelength of the fluorescent material, light of the wavelength band for exciting the fluorescent material does not affect the photochromic material. can do.

When the photochromic material of the silica nanoparticles in which the photochromic material and the fluorescent material are combined is irradiated with ultraviolet rays (for example, ultraviolet rays of 320 nm), the photochromic material of the nanoparticles is in a closed form. When it is colored and absorbs fluorescence, and when light in the visible light region is irradiated, it becomes an open form and loses color because it does not absorb fluorescence. As shown in FIGS. 2 and 3, the nanoparticles are photoelectric. To shout.

The silica nanoparticles in which the photochromic material and the fluorescent material of the present invention are combined may maintain light conversion even after repeatedly performing visible and ultraviolet irradiation.

An ionic functional group is exposed on the surface of the silica nanoparticles, and the ionic functional group may be a cationic functional group or an anionic functional group.

The cationic functional group may be selected from the group consisting of an amine group, an aldehyde group, a hydroxyl group and a thiol group, and preferably may be an amine group.

When the ionic functional group remaining on the surface of the silica nanoparticles without binding to the photochromic material or the fluorescent material is an anionic functional group, the dispersibility of the silica nanoparticles to which the photochromic material and the fluorescent material are bound in an aqueous solution is improved. do.

Therefore, in the silica nanoparticles in which the photochromic material and the fluorescent material are combined according to the present invention, it is preferable that an anionic functional group is exposed on the surface where the photochromic material or the fluorescent material is not bonded. In the silica nanoparticles in which the photochromic material and the fluorescent material are combined, the silica nanoparticles in which the anionic functional group is exposed on the surface thereof can be prepared as follows. ① The surface of the silica nanoparticles in which the cationic functional group to which the photochromic substance or fluorescent substance is not bound is exposed to the surface is modified with an anionic functional group, and then the photochromic substance and the fluorescent substance are combined. The photochromic material and the fluorescent material are bonded to the silica nanoparticles having the functional group exposed on the surface thereof, and then the cationic functional group remaining on the surface of the silica nanoparticle without the photochromic material or the fluorescent material is bound to the anionic function. It can be prepared by modifying with a group.

The anionic functional group is a group consisting of carboxyl group, carboxylic acid derivative group (C (= O) -Cl, C (= O) -Br, C (= O) -O-succinimide), isocyanate group, isothiocyanate group It may be selected from, and is preferably a carboxyl group.

In order to modify the cationic functional group into an anionic functional group, succinyl anhydride may be used.

The photochromic material and the fluorescent material may be bonded to the surface of the silica nanoparticles by ionically or covalently bonding to a cationic functional group or a modified anionic functional group exposed to the surface of the silica nanoparticles.

Meanwhile, silica nanoparticles having a photochromic material and a fluorescent material bonded thereto may be prepared by combining a photochromic material and a fluorescent material with a cationic functional group of the silica nanoparticle having a cationic functional group exposed on a surface thereof, or the silica Cationic functional groups may be anionic modified on the surface of the nanoparticles, and then the photochromic material and the fluorescent material may be combined with the anionic functional group.

In another aspect, the present invention provides a method for preparing a cationic silica precursor, comprising: (a) adding a silica precursor and a catalyst to a solvent to activate the silica precursor, and then adding and reacting a compound having a cationic functional group to prepare a cationic silica precursor; (b) adding distilled water to the cationic silica precursor prepared in step (a) and preparing silica nanoparticles in which the cationic functional group is exposed on the surface thereof; (c) combining the photochromic material to the cationic functional group exposed on the surface of the silica nanoparticles prepared in step (b) to prepare silica nanoparticles to which the photochromic material is bound; And (d) bonding the fluorescent material to the silica nanoparticles to which the photochromic material prepared in step (c) is bonded, to a high sensitivity silica nanoparticles to which the photochromic material and the fluorescent material are combined. .

In another aspect, (a) adding a silica precursor and a catalyst to a solvent to activate the silica precursor, and then adding and reacting a compound having a cationic functional group to prepare a cationic silica precursor; (b) adding distilled water to the cationic silica precursor prepared in step (a) and preparing silica nanoparticles in which the cationic functional group is exposed on the surface thereof; (c) modifying the cationic functional group exposed to the surface of the silica nanoparticles in step (b) with an anionic functional group; (d) combining the photochromic material with the anionic functional group modified in step (c) to prepare silica nanoparticles to which the photochromic material is bound; And (e) bonding the fluorescent material to the silica nanoparticles to which the photochromic material prepared in step (d) is bound to produce silica nanoparticles to which the photochromic material and the fluorescent material are combined. The present invention relates to a method for preparing silica nanoparticles in which a photochromic material and a fluorescent material are combined.

Since the particles having an anion on the surface have high dispersibility in an aqueous solution, it is preferable to replace the cationic functional group on the surface of the nanoparticle with an anionic functional group to improve dispersibility.

In the case where the photochromic material is bonded to the cationic functional group, the photochromic material and the fluorescent material are combined with the photochromic material or the fluorescent material to improve dispersibility in the aqueous solution of silica nanoparticles. The method may further include the step of modifying a cationic functional group that is not used to an anionic functional group, and preferably, succinic anhydride may be added to replace the cationic functional group on the surface of the nanoparticle with a carboxyl group. When the substitution is not performed well in an aqueous solution because of less substitution with an anionic functional group, dispersibility may be increased by covalently binding a water-soluble polymer such as polyethylene glycol to the remaining amine group.

The anionic functional group is a group consisting of carboxyl group, carboxylic acid derivative group (C (= O) -Cl, C (= O) -Br, C (= O) -O-succinimide), isocyanate group, isothiocyanate group It may be selected from, and is preferably a carboxyl group.

The solvent of step (a) may be selected from the group consisting of ethanol, methanol, butanol, heptanol, hexanol, and mixtures thereof.

The catalyst is an aqueous ammonia solution. When the ammonia concentration of the ammonia solution is less than 10% by weight, the catalyst is less effective as a catalyst. When the catalyst is higher than 40% by weight, the chemical properties of the aqueous solution, such as pH, may change, causing unwanted byproducts. The ammonia concentration is preferably 10 to 40% by weight, most preferably 25 to 30% by weight.

The silica precursor of step (a) may be selected from the group consisting of tetraethyl orthosilicate, tetramethyl orthosilicate, tetrapropyl orthosilicate and mixtures thereof.

In the present invention, it is preferable to add a compound having a cationic functional group before the formation of the silica nanoparticles in order to form the silica nanoparticles and to expose the cationic functional group on the particle surface, and the cationic functional group is an amine group, an aldehyde group. It may be selected from the group consisting of a hydroxyl group and a thiol group, the compound having a cationic functional group is preferably composed of aminopropyltromethoxysilane, aminopropyltroethoxysilane, aminopropyl diethoxymethylsilane and mixtures thereof It may be selected from the group.

 The ratio of the cationic functional group per surface area of the silica nanoparticles is controlled according to the addition ratio of the silica precursor and the compound having a cationic functional group in the step (a), and different surfaces according to the ratio of the ionic functional group per surface area. Silica Nanoparticles with Dislocations

When the addition ratio of the silica precursor and the compound having a cationic functional group is added to the solvent at 1: 0.1 to 4, respectively, the surface of the silica nanoparticles prepared as the content of the silica precursor having the cationic functional group increases. There is a characteristic that the charge is increased, and the addition ratio of the compound having a cationic functional group to the silica precursor may be preferably 1: 0.5 to 2.

In the present invention, the photochromic material is added to the silica nanoparticles to be combined, and the photochromic material and the fluorescent material having various fluorescence intensities are combined by adding fluorescent molecules at various concentrations (for example, 0 to 20 µM). This bonded silica nanoparticles can be obtained.

The concentration of the photochromic material and the fluorescent material was determined by the succinic unhydrad of cationic functional groups on the surface of the silica nanoparticles which did not bind the photochromic material or the fluorescent material after the photochromic material and the fluorescent molecule were bonded to the silica nanoparticles. It can affect the tendency to disperse in aqueous solution when substituted with anion.

The photochromic material of the present invention, preferably, the photochromic material having a carboxyl group can be bonded to the surface of the silica nanoparticles through covalent or ionic bonding, so that the photochromic properties contained within the particles when the silica particles are formed. It has about 40 times higher absorbance and improved photochromic properties than silica particles (Jonas Folling et al., Small, 4: 134, 2008). In particular, when the carboxyl group of the photochromic material and the amine group on the surface of the nanoparticles are covalently linked, the bonding strength is strong, and the photochromic material may be stably present without being separated from the silica nanoparticles even if chemical treatment is additionally performed.

The photochromic material may change from colorless to colored when absorbing light in the ultraviolet region, absorb light in the visible region, and become colorless when absorbing light in the visible region. As shown in FIG. 5, silica nanoparticles having a photochromic material (based on a maximum of 25 μM) are colored when absorbing light in an ultraviolet region of about 320 nm, and have a light absorption region of 500 to 700 nm, and maximum at about 600 nm. When absorbed and exposed to visible light, it has reversible optical properties by ultraviolet light and visible light that become colorless again.

The silica nanoparticles are dispersed by condensation in distilled water, and the size of the silica nanoparticles formed at this time is generally about 20 nm, and may be 5 nm to 40 nm.

The silica nanoparticles in which the photochromic material and the fluorescent material are combined may bind a probe to an ionic functional group present on the surface of the silica nanoparticle, and when the nanoparticles combined with the probe are reacted with a biomolecular sample, the photochromic material may be changed. Alternatively, the target biomolecule in the sample can be detected by measuring the fluorescence.

Therefore, in another aspect of the present invention, (a) combining the ionic functional group of the silica nanoparticles with the photochromic material and the fluorescent material combined with a probe capable of binding to the biomolecule to be detected, wherein the A probe is bound to an ionic functional group that is not bound to a photochromic material or fluorescent material in the silica nanoparticles; (b) reacting the probe labeled with the nanoparticle with a sample containing the biomolecule to be detected, and then irradiating visible and ultraviolet rays, respectively; And (c) detecting the biomolecules to be detected by measuring fluorescence and photoconversion of the sample and detecting the biomolecules using the silica nanoparticles in which the photochromic material and the fluorescent material are combined.

The silica nanoparticles in which the photochromic material and the fluorescent material are combined in the step (a) are silica nanoparticles in which the photochromic material and the fluorescent material are bonded to the silica nanoparticles having the cationic functional group exposed on the surface thereof. Silica nanoparticles or surfaces in which the cationic functional group remaining without the photochromic material and the fluorescent material are not bonded to the surface of the silica nanoparticles in which the photochromic material and the fluorescent material are bonded to the anionic functional group are replaced with the anionic functional group. After the modification, it includes all of the silica nanoparticles combined with the photochromic material and the fluorescent material.

On the surface of the silica nanoparticles in which the photochromic material and the fluorescent material are combined, there is an ionic functional group in which the photochromic material and the fluorescent material are not combined. It may be combined with an ionic functional group that is not bound to a color changeable material or a fluorescent material.

The ultraviolet rays of step (b) are ultraviolet rays having a wavelength of about 365 nm, and the visible light includes light of an excitation wavelength band of the fluorescent material. The wavelength band region of the visible light to measure the fluorescence of step (c) is the fluorescent wavelength band region of the fluorescent material.

 Since the silica nanoparticles are combined with a plurality of photochromic materials and a plurality of fluorescent materials for each silica nanoparticle, they generate a detection signal thousands of times stronger than labeling a single photochromic material or a fluorescent molecule on a biomolecule. The highly sensitive, ionic functional groups on the surface of the silica nanoparticles can be combined with various biomolecules or probes, so that they can be directly labeled with the biomolecules to be detected or can be combined with proteins, peptides, nucleic acids, sugars and the ionic functional groups. Compound having a can be used as a probe that binds to the target biomolecule for detection of the biomolecule.

The combination of the probe and the target biomolecule capable of binding to the target biomolecule is composed of antigen-antibody, ligand-receptor, enzyme-substrate, biotin-avidin, hapten-antihapten, hormone-receptor and lectin-glycoprotein. It may be selected from the group.

In still another aspect, the present invention relates to a bioimaging contrast agent composition containing silica nanoparticles in which the photochromic material and the fluorescent material are combined.

The contrast agent composition is distinguished from background noise caused by naturally occurring fluorescent materials in vivo by using fluorescence and fluorescence conversion generated from fluorescent materials of the silica nanoparticles, thereby enabling reliable biometric imaging.

The contrast agent composition may additionally include a pharmaceutically acceptable carrier.

Carriers used in the contrast agent composition according to the present invention include carriers and vehicles commonly used in the pharmaceutical field, and specifically, ion exchange, alumina, aluminum stearate, lecithin, serum proteins (eg, human serum albumin), buffer substances ( E.g. several phosphates, glycine, sorbic acid, potassium sorbate, partial glyceride mixtures of saturated vegetable fatty acids), water, salts or electrolytes (e.g. protamine sulfate, disodium hydrogen phosphate, hydrogen phosphate, sodium chloride and zinc salts), Colloidal silica, magnesium trisilicate, polyvinylpyrrolidone, cellulose based substrates, polyethylene glycol, sodium carboxymethylcellulose, polyarylates, waxes, polyethylene glycols or wool, and the like. The contrast agent compositions of the present invention may also further comprise lubricants, wetting agents, emulsifiers, suspending agents or preservatives, etc., in addition to the above components.

In one embodiment, the contrast agent composition according to the invention can be prepared in an aqueous solution for parenteral administration. Preferably, a buffer solution such as Hanks' solution, Ringer's solution, or physically buffered saline may be used. Aqueous injection suspensions can be added with a substrate that can increase the viscosity of the suspension, such as sodium carboxymethylcellulose, sorbitol or dextran.

Another preferred embodiment of the contrast composition of the present invention may be in the form of sterile injectable preparations of aqueous or oily suspensions. Such suspensions may be formulated according to techniques known in the art using suitable dispersing or wetting agents (eg, Tween 80) and suspending agents. Sterile injectable preparations may also be sterile injectable solutions or suspensions (eg, solutions in 1,3-butanediol) in nontoxic parenterally acceptable diluents or solvents. Vehicles and solvents that may be used include mannitol, water, Ringer's solution, and isotonic sodium chloride solution. In addition, sterile, nonvolatile oils are conventionally employed as a solvent or suspending medium. For this purpose any non-irritating non-volatile oil can be used including synthetic mono or diglycerides.

The contrast agent composition according to the present invention may be administered to a living body or a sample, and the image may be obtained by detecting a signal emitted by the fluorescent material of the silica nanoparticles from the living body or the sample, and the background noise may be obtained by inducing light conversion by ultraviolet irradiation. Can be removed. Through this, it will be available for bio-imaging applications that can be applied to diagnose cancer and specific diseases, study mechanisms of biological signals, and study differentiation of stem cells through fluorescence of specific target sites.

The term "sample" as used above means a tissue or cell isolated from a subject to be diagnosed.

Injecting the contrast agent composition into a living body or a sample may be administered via a route commonly used in the pharmaceutical field, and parenteral administration is preferred, for example, intravenous, intraperitoneal, intramuscular, subcutaneous or topical routes. It can be administered through. In addition, when culturing experimental cells, silica nanoparticles can be added to the culture medium and introduced into the cells.In order to obtain important information for a specific study by observing particles introduced into the cells and defects in specific parts by using light conversion. Will be available.

In the above method, the signal emitted by the silica nanoparticles of the present invention is a fluorescence microscope that can detect a fluorescence signal, confocal fluorescence microscope, spectrophotometer, fluorimeter, CCD camera, real-time living cell observation microscope (Delta Vision) and a combination thereof may be detected by the selected equipment, but is not limited thereto.

Hereinafter, the present invention will be described in more detail with reference to Examples. These examples are only for illustrating the present invention, and it will be apparent to those skilled in the art that the scope of the present invention is not to be construed as being limited by these examples.

Example 1 Preparation of Silica Nanoparticles Exposed with Amine Group

10 mL of ethanol, 100 μL of tetraethyl orthosilicate, and 200 μl of an aqueous ammonia solution were mixed, reacted for 30 minutes, and 100 μl of aminopropyltriethoxysilane was added thereto, followed by stirring at room temperature for 12 hours. 20 mL of distilled water was added to the stirred solution, followed by further stirring for 2 hours to form silica nanoparticles in which the amine group was exposed to the surface of the particles, and washed three times with ethanol to remove unreacted chemicals, followed by nanoparticles. Was recovered. The recovered nanoparticles were dispersed in ethanol and the surface charge was measured to confirm the state in which the amine group was exposed to the nanoparticle surface.

Example 2 Preparation of Silica Nanoparticles Combining Photochromic Material and Fluorescent Material

The photochromic substance having a carboxyl group represented by the following formula (11) is dissolved in 0.5mM ethanol and an excess of 2.5 mM EDC (1-ethyl-3- (3-dimethylaminopropyl) carbodimide) and 2.5 mM NHS (N-hydroxy succinimide) are added. After reacting for 2 hours at room temperature, the silica nanoparticles prepared in Example 1 were mixed in a 50:50 ratio and stirred for 2 hours.

[Formula 11]

After washing three times with ethanol to remove the unreacted material, the silica nanoparticles to which the photochromic material is bound were recovered, and the nanoparticles were dispersed in ethanol. After binding 25 μM photochromic material to silica nanoparticles as described above, a high concentration of 20 mM Rhodamin B isothiocianate was reacted with a mixture of ethanol and THF (3: 2) for 3 hours while argon gas was injected at 0 ° C. Or NHS-Cy3 was reacted for 1 hour at room temperature according to the concentration (0, 0.5, 1, 2, 5, 10 and 20μM) to form an amide bond between the amine and the fluorescent material on the surface of the silica nanoparticles. After the reaction was completed three times with ethanol to remove the remaining Rhodamin B isothiocianate or NHS-Cy3 by centrifugation to prepare a silica nanoparticles combined with a high concentration of photochromic material and fluorescent material (Fig. 4 and Fig. 5).

Example 3 Confirming the Absorption Region of the Silica Nanoparticles Combining Photochromic Material and Fluorescent Material

The nanoparticles prepared in Example 2 were irradiated with ultraviolet light having a wavelength of 365 nm at room temperature for 10 minutes or visible light at room temperature for 1 hour, and then absorbance was measured in a region of 400 to 800 nm using a spectrophotometer. The absorption region of the fluorescent molecule Rhodamin B isothiocianate (maximum absorption 540 nm) or Cy3 (maximum absorption 560 nm, FIG. 6) and the absorption region of the photochromic molecules (maximum absorption 600 nm, FIG. 7) were confirmed.

In addition, the silica nanoparticles in which the photochromic material and the fluorescent molecule are combined are light compared to the case where the conventional photochromic material is contained in the silica particle (Jonas Folling et al., Small, 4: 134, 2008). While using less color changeable material, it was confirmed that the absorbance was improved by about 40 times (Table 2).

Absorbance analysis when the photochromic material is contained inside the silica particles (reference) and when bonded to the particle surface (the present invention) division Photochromic substances
Final molarity Maximum absorbance of photochromic material UV ON vs OFF absorbance difference A ^* Nested inside a particle
670 μM 0.02
(λ max = 640 nm)
0.02 B When bonded to the particle surface
25 μM 0.7
(λ max = 600 nm)
0.45

[A ^* Reference: Jonas Folling et al., Small, 4: 134, 2008]

Example 4: Confirmation of fluorescence characteristics of silica nanoparticles in which photochromic material and fluorescent material are combined

After irradiating ultraviolet rays or visible rays having 365 nm wavelength to the particles prepared in Example 2, the fluorescence characteristics were confirmed using a fluorophotometer. Silica nanoparticles combined with the photochromic material and the fluorescent material Rhodaimnin B isothiocianate showed a maximum fluorescence at 600 nm when excited at 540 nm (FIG. 8).

When the ultraviolet ray was irradiated for 10 minutes to induce the photochromic material to absorb the wavelength of 600nm, it was confirmed that the intensity of fluorescence was reduced by 85%. In addition, as shown in Figure 9, in the particles to which NHS-Cy3 is applied, it was confirmed that the fluorescence disappeared by 85 to 100% depending on the concentration of the fluorescent molecules. In particular, in the silica nanoparticles in which the photochromic material and the fluorescent molecule were combined to fluoresce up to 100%, fluorescence appeared and disappeared repeatedly when irradiated with UV and visible light (FIG. 10).

Example 5: Live Cell Image Detection of Silica Nanoparticles with Photochromic Materials and Fluorescent Molecules

Silica nanoparticles prepared by combining the photochromic material and the fluorescent molecules were added to the dendritic cell culture medium at 0.5% (v / v) and incubated for 4 hours using a biological cell imaging device (DeltaVision RT, Applied Precision). Was observed. As shown in Figure 11, by detecting the fluorescence in the 617nm region using the excitation wavelength of 555nm was able to confirm the nanoparticles introduced into the cell, the photovoltaic disappears when observing the cells again after irradiating UV light for 10 minutes The ringing characteristic was confirmed.

The specific parts of the present invention have been described in detail above, and it is apparent to those skilled in the art that such specific descriptions are merely preferred embodiments, and thus the scope of the present invention is not limited thereto. something to do. Thus, the substantial scope of the present invention will be defined by the appended claims and their equivalents.

Figure 1 shows a schematic diagram of the silica nanoparticles are combined photochromic material and fluorescent material according to the present invention.

2 is a light absorbance and photochromic properties before and after ultraviolet irradiation after bonding the rhodamine fluorescent material to the silica nanoparticles are bonded to the photochromic material prepared according to an embodiment of the present invention Absorbance (PC) for the material is shown.

Figure 3 shows the change in the fluorescence sensitivity before and after ultraviolet irradiation (Before) and after (UV) irradiation when the ultraviolet light to the photochromic material and rhodamine-coupled silica nanoparticles prepared according to an embodiment of the present invention .

Figure 4 is an image shown after combining the Cy3 fluorescent material with a concentration of 0, 0.5, 1, 2, 5, 10 and 20μM to silica nanoparticles combined with a photochromic material 25μM prepared according to an embodiment of the present invention (1: silica nanoparticles not bound to Cy3 fluorescent molecules; 2: silica nanoparticles bound to 0.5 μM Cy3 phosphors; 3: silica nanoparticles bound to 1 μM Cy3 phosphors; 4: 2 μM Cy3 phosphors) Silica nanoparticles bound to 5: 5 μM Cy3 phosphors; silica nanoparticles bound to 10 μM Cy3 phosphors; and silica nanoparticles bound to 20 μM Cy3 phosphors.

FIG. 5 is a view showing when 25 μM of photochromic material and Cy3 prepared according to one embodiment of the present invention are irradiated with UV light on silica nanoparticles having concentrations of 0, 0.5, 1, 2, 5, 10, and 20 μM. (1: silica nanoparticles not bound to the Cy3 phosphor; 2: silica nanoparticles bound to the 0.5 μM Cy3 phosphor; 3: silica nanoparticles bound to the 1 μM Cy3 phosphor; 4: 2 μM) Silica nanoparticles bound to Cy3 phosphors; 5: Silica nanoparticles bound to 5 μM Cy3 phosphors; 6: Silica nanoparticles bound to 10 μM Cy3 phosphors; and 7: Silica nanoparticles bound to 20 μM Cy3 phosphors) .

Figure 6 is a graph showing the absorbance before ultraviolet irradiation of the photochromic material prepared according to an embodiment of the present invention and the silica nanoparticles Cy3 is bonded for each concentration of 0, 0.5, 1, 2, 5, 10 and 20μM (1: silica nanoparticles bonded with 25 μM of photochromic substance and 0 μM of Cy3 fluorescent substance; 2: silica nanoparticles bound with 25 μM of photochromic substance and 0.5 μM of Cy3 fluorescent substance; 3: 25 μM with photochromic substance Silica nanoparticles bonded with 1 μM Cy3 phosphor; 4: Silica nanoparticles bound with 25 μM photochromic material and 2 μM Cy3 phosphors; 5: Silica nanoparticles combined with 25 μM photochromic material and 5 μM Cy3 phosphor; : Silica nanoparticles bonded with 25 μM photochromic substance and 10 μM Cy3 fluorescent substance; and 7: silica nanoparticles combined with 25 μM photochromic substance and 20 μM Cy3 fluorescent substance.

FIG. 7 shows absorbance after UV irradiation of silica nanoparticles in which photochromic material 25 μM and Cy 3 are bonded according to concentrations of 0, 0.5, 1, 2, 5, 10 and 20 μM according to an embodiment of the present invention. (1: silica nanoparticles bound with Cy3 fluorescent material 0 μM; 2: silica nanoparticles bound with Cy3 fluorescent material 0.5 μM; 3: silica nanoparticles bound with Cy3 fluorescent material 1 μM; 4: Cy3 fluorescent material) Silica nanoparticles bound to 2 μM; 5: silica nanoparticles bound to 5 μM of Cy3 phosphors; 6: silica nanoparticles bound to 10 μM of Cy3 phosphors; and silica nanoparticles bound to 20 μM of Cy3 phosphors.

8 is irradiated with an excitation wavelength of 540nm to the silica nanoparticles 25μM photochromic material prepared according to an embodiment of the present invention and Cy3 is bonded to each concentration of 0, 0.5, 1, 2, 5, 10 and 20μM Fluorescence measured as a graph (1: silica nanoparticles not bound to Cy3 fluorescent molecules; 2: silica nanoparticles bound to 0.5 μM Cy3 fluorescent material; 3: silica nanoparticles bound to 1 μM Cy3 fluorescent material; 4: Silica nanoparticles bound to 2 μM Cy3 phosphors; 5: Silica nanoparticles bound to 5 μM Cy3 phosphors; 6: Silica nanoparticles bound to 10 μM Cy3 phosphors; and 7: Silica bound to 20 μM Cy3 phosphors Nanoparticles).

9 is 540nm after irradiating UV light to the silica nanoparticles 25μM photochromic material prepared according to an embodiment of the present invention and Cy3 is bonded to each concentration of 0, 0.5, 1, 2, 5, 10 and 20μM Fluorescence measured as an excitation wavelength is shown graphically (1: silica nanoparticles not bound to Cy3 fluorescent molecules; 2: silica nanoparticles bound to 0.5 μM Cy3 fluorescent materials; 3: combined with 1 μM Cy3 fluorescent materials) Silica nanoparticles; 4: Silica nanoparticles bound to 2 μM Cy3 phosphors; 5: Silica nanoparticles bound to 5 μM Cy3 phosphors; 6: Silica nanoparticles bound to 10 μM Cy3 phosphors; and 7: 20 μM Cy3 phosphors And silica nanoparticles).

FIG. 10 is a graph showing absorbance after repeated irradiation of visible light and ultraviolet light on photochromic material and Cy3-coupled silica nanoparticles prepared according to an embodiment of the present invention.

FIG. 11 is a graph illustrating fluorescence changes before and after ultraviolet irradiation (10 min) after adsorption of photochromic material and Cy3-coupled silica nanoparticles prepared in accordance with one embodiment of the present invention to a dendritic cell. It is shown.

Claims (35)

High sensitivity silica nanoparticles in which a photochromic material represented by the following general formula (1) and a fluorescent substance are bonded to silica nanoparticles having an ionic functional group exposed on the surface thereof:   [Formula 1]

Where P is a photochromic molecule, K is an atom or atom group, a peptide binding ligand, a DNA binding ligand, a PNA binding ligand, a protein binding ligand or a nucleic acid binding ligand, alkyl having 1 to 100 carbon atoms, and having 6 to 100 carbon atoms. Aryl, carbon number is 2 to 100 heterocyclic linker or (CH ₂ CH ₂ 0) _n , L is an atom or atomic group, except for the hydrogen atom (H), peptide binding ligand, DNA binding ligand , PNA binding ligand, protein binding ligand or nucleic acid binding ligand, alkyl having 1 to 100 carbon atoms, aryl having 6 to 100 carbon atoms, heterocyclic linker having 2 to 100 carbon atoms or (CH ₂ CH ₂ 0) _n (where n is an integer from 1 to 100), X is selected from the group consisting of O, S, NH and CH ₂ , and M is an ionic functional group exposed to the surface of the silica nanoparticles. It means a functional group that binds. According to claim 1, wherein the photochromic molecules (

) Is a high-sensitivity silica nanoparticles are combined with a photochromic material and a fluorescent material, characterized in that selected from the group consisting of a compound represented by the following formula 2 to 9 and their isomers:        [Formula 2]

       (3)

       [Formula 4]

       [Chemical Formula 5]

       [Formula 6]

       [Formula 7]

       [Formula 8]

       [Formula 9]

In formulas (2) and (3), R is a C ₁ to C ₃ alkylene group unsubstituted or substituted with a bond line, oxygen atom or fluorine, Y is independently an oxygen atom, a nitrogen atom or a sulfur atom, and Z is a fluorine substituted or unsubstituted group It means a cyclic methylene group or a carbonyl group. The method of claim 1, wherein the functional group (M) which binds to the silica nanoparticles is a carboxyl group, a carboxylic acid derivative group (C (= O)-Cl, C (= O)-Br, C (= O)-O- succinimide), isocyanate group, isothiocyanate group, amine group, aldehyde group, thiol group, hydroxyl group, iodine acetamino group, α-haloacetyl group and maleimide group, characterized in that selected from the group consisting of Highly sensitive silica nanoparticles in which the material is bound. High sensitivity silica nanoparticles in which a photochromic material represented by the following Chemical Formula 10 and a fluorescent substance are bonded to silica nanoparticles having an ionic functional group exposed on its surface:        [Formula 10]

In Formula 10, R is a C ₁ to C ₃ alkylene group unsubstituted or substituted with a bond line, an oxygen atom or fluorine, Y is independently an oxygen atom, a nitrogen atom or a sulfur atom, and Z is unsubstituted or substituted with fluorine Methylene group or carbonyl group, M or M 'is a functional group which binds to the ionic functional group of hydrogen or silica nanoparticles, and either M and M' is a functional group which binds to the ionic functional group of silica nanoparticles, n Is an integer from 1 to 100. 5. The functional group of claim 4, wherein the functional group that binds to the ionic functional group of the silica nanoparticles is a carboxyl group, a carboxylic acid derivative group (C (= 0) -Cl, C (= 0) -Br, or C (= 0) -O. -succinimide), an isocyanate group, an isothiocyanate group, an amine group, an aldehyde group, a thiol group, a hydroxyl group, an iodine acetamine group, an α-haloacetyl group and a maleimide group And highly sensitive silica nanoparticles combined with fluorescent materials. The highly sensitive silica nanoparticles according to claim 1 or 4, wherein the ionic functional group is a cationic functional group or an anionic functional group. The method according to claim 6, wherein the cationic functional group is selected from the group consisting of an amine group, an aldehyde group, a hydroxyl group and a thiol group, the anionic functional group is a carboxyl group, a carboxylic acid derivative group (C (= O)-Cl, C (= O ) -Br, C (= O) -O-succinimide), a highly sensitive silica nanoparticles are combined with a photochromic material and a fluorescent material, characterized in that selected from the group consisting of isocyanate group, isothiocyanate group. The method of claim 1 or 4, wherein the ionic functional group on the surface of the silica nanoparticles is a cationic functional group, and the cationic functional group to which the photochromic material or fluorescent substance is not bonded on the surface of the silica nanoparticles is anionic. High-sensitivity silica nanoparticles are combined with a photochromic material and a fluorescent material characterized in that the functional group is modified. The method of claim 1 or 4, wherein the fluorescent material is Alexa555, bodiphy TMR, Cy3, DY500, rhodamine red-X, spectral orange, fluorescein isothiocyanate, tetramethyltamine isothiocyanate , Alexafluor, Texas red, tetramethyltamine, cascade blue, DAPI, coumarin, lucifer yellow, and at least one selected from the group consisting of single amide amide photochromic material and fluorescent material is combined High sensitivity silica nanoparticles. 5. The highly sensitive silica nanoparticles of claim 1, wherein the photochromic material has an absorption wavelength band different from the excitation wavelength of the fluorescent material. 6. 5. The highly sensitive silica nanoparticles according to claim 1, wherein the silica nanoparticles in which the photochromic material and the fluorescent material are bonded are photoconvertible. The photochromic material according to claim 1 or 4, wherein the silica nanoparticles in which the photochromic material and the fluorescent material are combined maintain the original photoconversion even after repeatedly performing visible and ultraviolet irradiation. Highly sensitive silica nanoparticles that combine materials and fluorescent materials. A method for preparing highly sensitive silica nanoparticles in which a photochromic material and a fluorescent material are bonded to a surface of silica nanoparticles including the following steps: (a) activating the silica precursor by adding a silica precursor and a catalyst to a solvent, and then adding and reacting a compound having a cationic functional group to prepare a cationic silica precursor; (b) adding distilled water to the cationic silica precursor prepared in step (a) and preparing silica nanoparticles in which the cationic functional group is exposed on the surface thereof; And (c) combining the photochromic material to the cationic functional group exposed on the surface of the silica nanoparticles prepared in step (b) to prepare silica nanoparticles to which the photochromic material is bound; And (d) bonding the fluorescent material to the silica nanoparticles to which the photochromic material is bound to produce high sensitivity silica nanoparticles to which the photochromic material and the fluorescent material are combined. A method for preparing highly sensitive silica nanoparticles in which a photochromic material and a fluorescent material are bonded to a surface of a nanoparticle including the following steps: (a) adding a silica precursor and a catalyst to a solvent to activate the silica precursor, and then adding and reacting a compound having a cationic functional group to prepare a cationic silica precursor; (b) adding distilled water to the cationic silica precursor prepared in step (a) and preparing silica nanoparticles in which the cationic functional group is exposed on the surface thereof; (c) modifying the cationic functional group exposed to the surface of the silica nanoparticles in step (b) with an anionic functional group; (d) combining the photochromic material with the anionic functional group modified in step (c) to prepare silica nanoparticles to which the photochromic material is bound; And (e) preparing high sensitivity silica nanoparticles in which the photochromic material and the fluorescent material are combined by bonding the fluorescent material to the silica nanoparticles to which the photochromic material is bound. 15. The method according to claim 13 or 14, wherein the solvent of step (a) is an alcohol, wherein the photochromic material and the fluorescent material are combined. 16. The method of claim 15, wherein the alcohol is selected from the group consisting of ethanol, methanol, butanol, heptanol, hexanol, and mixtures thereof. Way. The method according to claim 13 or 14, wherein the catalyst agent of step (a) is an aqueous ammonia solution, wherein the photosensitive material and the fluorescent material are combined. 15. The method according to claim 13 or 14, wherein the ammonia concentration of the aqueous ammonia solution is 10 to 40% by weight. 15. The photochromic material according to claim 13 or 14, wherein the silica precursor of step (a) is selected from the group consisting of tetraethyl orthosilicate, tetramethyl orthosilicate, tetrapropyl orthosilicate and mixtures thereof. Method for producing high sensitivity silica nanoparticles in which a substance and a fluorescent substance are combined. The compound according to claim 13 or 14, wherein the compound having a cationic functional group in step (a) is composed of aminopropyltromethoxysilane, aminopropyltroethoxysilane, aminopropyldiethoxymethylsilane, and mixtures thereof. Method for producing a highly sensitive silica nanoparticles are combined with a photochromic material and a fluorescent material, characterized in that selected from the group. 15. The method of claim 13 or 14, wherein the addition ratio of the silica precursor and the compound having a cationic functional group in the step (a) is 1: 0.1 to 4, characterized in that the photochromic material and the fluorescent material is combined Method for producing high sensitivity silica nanoparticles. The photochromic material and the fluorescent material of claim 13, further comprising the step of modifying the cationic functional group on the surface of the silica nanoparticles prepared in step (c) with an anionic functional group. Method for producing high sensitivity silica nanoparticles. 23. The method of claim 22, wherein the modification is performed by using succinic anhydride. 15. The method of claim 14, wherein the modifying in step (c) uses succinic anhydride, wherein the photosensitive material and the fluorescent material are combined. 15. The method according to claim 13 or 14, wherein the photochromic material is a compound represented by the following general formula (1):   [Formula 1]

Where P is a photochromic molecule, K is an atom or atom group, a peptide binding ligand, a DNA binding ligand, a PNA binding ligand, a protein binding ligand or a nucleic acid binding ligand, alkyl having 1 to 100 carbon atoms, and having 6 to 100 carbon atoms. Aryl, carbon number is 2 to 100 heterocyclic linker or (CH ₂ CH ₂ 0) _n , L is an atom or atomic group, except for the hydrogen atom (H), peptide binding ligand, DNA binding ligand , PNA binding ligand, protein binding ligand or nucleic acid binding ligand, alkyl having 1 to 100 carbon atoms, aryl having 6 to 100 carbon atoms, heterocyclic linker having 2 to 100 carbon atoms or (CH ₂ CH ₂ 0) _n (where n is an integer from 1 to 100), X is selected from the group consisting of O, S, NH and CH ₂ , and M is an ionic functional group exposed to the surface of the silica nanoparticles. It means a functional group that binds. The photochromic molecule according to claim 25,

) Is a method for preparing high-sensitivity silica nanoparticles are combined with a photochromic compound and a fluorescent material, characterized in that selected from the group consisting of compounds represented by the formula (2) to (9) and their isomers:        [Formula 2]

       (3)

       [Formula 4]

       [Chemical Formula 5]

       [Formula 6]

       [Formula 7]

       [Formula 8]

       [Formula 9]

In Formulas 2 and 3, R is a C ₁ to C ₃ alkylene group unsubstituted or substituted with a bond line, an oxygen atom or fluorine, Y is each independently an oxygen atom, a nitrogen atom or a sulfur atom, and Z is a fluorine substituted or unsubstituted It means a cyclic methylene group or a carbonyl group. The functional group of claim 25, wherein the functional group that binds to the ionic functional group of the silica nanoparticles is a carboxyl group, a carboxylic acid derivative group (C (= 0) -Cl, C (= 0) -Br, or C (= 0) -O. -succinimide), an isocyanate group, an isothiocyanate group, an amine group, an aldehyde group, a thiol group, a hydroxyl group, an iodine acetamino group, an α-haloacetyl group, and a maleimide group, Method for producing highly sensitive silica nanoparticles in which a fluorescent material is bound. 15. The method according to claim 13 or 14, wherein the photochromic material is a compound represented by the following Chemical Formula 10:        [Formula 10]

In Formula 10, R is a C ₁ to C ₃ alkylene group unsubstituted or substituted with a bond line, an oxygen atom or fluorine, Y is independently an oxygen atom, a nitrogen atom or a sulfur atom, and Z is unsubstituted or substituted with fluorine Methylene group or carbonyl group, M or M 'is a functional group which binds to the ionic functional group of hydrogen or silica nanoparticles, and either M and M' is a functional group which binds to the ionic functional group of silica nanoparticles, n Is an integer from 1 to 100. 29. The method of claim 28, wherein the functional groups that bind to the ionic functional group of the silica nanoparticles are a carboxyl group, a carboxylic acid derivative group (C (= O) -Cl, C (= O) -Br, C (= O) -O -succinimide), an isocyanate group, an isothiocyanate group, an amine group, an aldehyde group, a thiol group, a hydroxyl group, an iodine acetamine group, an α-haloacetyl group and a maleimide group Method for producing a highly sensitive silica nanoparticles are combined with a fluorescent material. The method according to claim 13 or 14, wherein the fluorescent material is Alexa555, bodipy TMR, Cy3, DY500, rhodamine red-X, spectral orange, fluorescein isothiocyanate, tetramethyltamine Photochromic materials and fluorescent materials characterized in that at least one selected from the group consisting of isothiocyanate, alexafluor, texas red, tetramethyltamine, cascade blue, DAPI, coumarin, lucifer yellow and monosilamide Method for producing the highly sensitive silica nanoparticles are bonded. A biomolecule detection method comprising the following steps: (a) binding a probe capable of binding to an ionic functional group of a silica nanoparticle to which a photochromic material and a fluorescent material of claim 1, 4 or 8 and a fluorescent substance is bound, and a biomolecule to be detected, wherein The probe is bound to an ionic functional group that is not bound to a photochromic material or fluorescent material in the silica nanoparticles; (b) reacting the probe labeled with the nanoparticle with a sample containing the biomolecule to be detected, and then irradiating visible and ultraviolet rays, respectively; And (c) detecting the biomolecule to be detected by measuring the fluorescence and light conversion of the sample. 32. The method of claim 31, wherein the ultraviolet rays of step (b) are ultraviolet rays of about 365 nm wavelength. 32. The method of claim 31, wherein the visible light of step (b) comprises light of an excitation wavelength band of the fluorescent material. The combination of claim 31, wherein the combination of the probe and the biomolecule to be detected is antigen-antibody, ligand-receptor, enzyme-substrate, biotin-avidin, hapten-antihapten, hormone-receptor and A detection method, characterized in that selected from the group consisting of lectin-glycoprotein. A bioimaging contrast agent containing silica nanoparticles in which the photochromic material and the fluorescent material of claim 1, 4 or 8 are bound.

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