KR101368076B1 - Surface-modified fluorescent silica nanoparticles with excellent aqeous dispersibility and preparing method thereof - Google Patents

Abstract

The present invention relates to fluorescent silica nanoparticles, a composition including the fluorescent silica nanoparticles, a preparing method of the fluorescent silica nanoparticles, and a detecting method of biomolecules using the fluorescent silica nanoparticles. Concretely, The present invention relates to fluorescent silica nanoparticles which is surface-modified by a surface modifier thereby having high aqueous dispersibility with fluorescent materials adapted, a manufacturing method of the fluorescent silica nanoparticles, and a detecting method of biomolecules using the fluorescent silica nanoparticles. The fluorescent silica nanoparticle can be used in various biological or medical fields such as display fields, fluorescence microscopes, image diagnosis probe, biochip, and biosensor because it has fluorescent materials adapted and joined antibodies, and besides, it is acidic in aqueous solutions. Besides, by the manufacturing method of the fluorescent silica nanoparticles according to the present invention, fluorescent silica nanoparticles with excellent aqueous dispersibility can be obtained and antibodies can be introduced into the flurescent silica nanoparticles through a simple step.

Description

Surface-modified fluorescent silica nanoparticles with excellent aqeous dispersibility and preparing method

The present invention relates to a fluorescent silica nanoparticles, a composition comprising the fluorescent silica nanoparticles, a method for producing the fluorescent silica nanoparticles, and a biomolecule detection method using the fluorescent silica nanoparticles, and more specifically, by a surface modifier. Fluorescent silica nanoparticles having a surface modified to have high dispersibility in aqueous solution, low photobleaching, and easy biomolecule binding, a method for preparing the fluorescent silica nanoparticles, and a living body using the fluorescent silica nanoparticles It relates to a molecular detection method.

Recently, nanoparticles into which light-emitting materials have been introduced have been attracting much attention as indicators and photon sources in the fields of medical biological imaging technology, biosensor technology, microarray, drug delivery and the like. Among them, the silica nanoparticles not only have high stability and biocompatibility, but also can introduce a large amount of fluorescent material, which can generate a strong fluorescent signal, and can prevent sudden photobleaching by oxygen, thereby improving photostability. It can be used as a substrate that can bind a variety of functional groups has the advantage that it is easy to introduce a variety of biomolecules or ligands.

However, in order to use the silica particles for medical purposes such as diagnosis or treatment, the nanoparticle size is preferably 100 nm or less.However, when the silica particles are made of nanoparticles, the silica particles aggregate to each other and grow to 500 nm or more, thereby stably transporting and dispersing them in an aqueous environment in vivo. As a result, the wider utilization of silica nanoparticles has not been achieved in nano biotechnology.

The technical problem to be solved by the present invention is a fluorescent silica nanoparticles, a composition comprising the fluorescent silica nanoparticles, the fluorescent, which can be usefully used as a probe for in vitro or in vivo diagnostics, testing by showing a significantly improved dispersibility in aqueous solution It is to provide a method for producing silica nanoparticles and a method for detecting biomolecules using the fluorescent silica nanoparticles.

In order to achieve the above technical problem, the present invention proposes a fluorescent silica nanoparticles having a high dispersion in aqueous solution and a fluorescent material is introduced.

In addition, the present invention proposes a contrast agent composition comprising the fluorescent silica nanoparticles and a pharmaceutically acceptable carrier.

In addition, the present invention proposes an immunoassay composition comprising the fluorescent silica nanoparticles and a pharmaceutically acceptable carrier.

In addition, the present invention (a) forming a fluorescent silica nanoparticle intermediate having a functional group on the surface; (b) forming a surface modifier using a linker and a surfactant; And (c) modifying the surface of the fluorescent silica nanoparticle intermediate formed in step (a) using a surface modifier in step (b), wherein the fluorescent silica nanoparticles are highly dispersed and have fluorescent materials introduced therein. We propose a method of manufacturing.

In addition, the present invention comprises the steps of (a) preparing the fluorescent silica nanoparticles according to any one of claims 1 to 9; (b) irradiating light after the fluorescent silica nanoparticles are added to a sample including a detection target material and reacted; And (c) proposes a biomolecule detection method comprising the step of measuring the fluorescence from the sample.

Since the fluorescent silica nanoparticles according to the present invention not only introduce fluorescent materials and conjugate molecules such as antibodies, but also exhibit high dispersibility in aqueous solution, detection of biomolecules and imaging of biomolecules, in particular, fluorescence microscopy It can be widely used in biomedical fields such as imaging diagnostic probe (contrast agent), biochip, biosensor.

In addition, according to the method for producing fluorescent silica nanoparticles according to the present invention, it is possible to produce fluorescent silica nanoparticles in which a fluorescent material is introduced and exhibit high dispersibility in an aqueous solution, and also through the simple reaction (one step) the fluorescent silica nanoparticles Antibodies can be introduced into the particles.

1 is a conceptual diagram of a process step performed in the synthesis (AF647 @ SiNP-NH ₂ ) of the fluorescent silica nanoparticle intermediate introduced with the amine group in Example 1 of the present application.
2 is a conceptual diagram of a process step performed during synthesis (PVP-GA) of polyvinylpyrrolidone (PVP) modified with an aldehyde group in Example 1 of the present application.
3 is a conceptual diagram of a process step performed in AF647 @ SiNP-NH ₂ surface modification (AF647 @ SiNP (PVP) -CHO) using PVP-GA in Example 1 of the present application.
4 is a conceptual diagram of a process of forming fluorescent silica nanoparticles conjugated with an antibody according to Example 4 of the present application.
5 is a particle size analysis result of the surface-modified fluorescent silica nanoparticles prepared in Examples 1 to 3 of the present application.
6 is an optical camera image of an aqueous solution in which surface-modified fluorescent silica nanoparticles prepared in Examples 1 to 3 of the present application are dispersed.

Hereinafter, the present invention will be described in detail.

Fluorescent silica nanoparticles according to the present invention has a surface is modified by a surface modifier to have a high dispersion in aqueous solution and the fluorescent material is introduced into the excitation state (excitation state) by light irradiation and then returned to the ground state (ground state) It has a fluorescence property that emits absorbed energy as light of a specific wavelength.

The fluorescent silica nanoparticles have a functional group on the surface to form a fluorescent silica nanoparticle intermediate in which a fluorescent material is introduced, and after forming a surface modifier prepared using a linker and a surfactant, the It can be prepared by modifying the surface of the fluorescent silica nanoparticle intermediate with the surface modifier.

The surface modifier is prepared using a linker and a surfactant, and the linker serves as a crosslinking agent between the fluorescent silica nanoparticle intermediate and the surfactant so that the surfactant can coat the surface of the fluorescent silica nanoparticle. And a functional group capable of reacting with a functional group on the surface of the fluorescent silica nanoparticle intermediate. For example, when the functional group on the surface of the fluorescent silica nanoparticle intermediate is an amine group, the linker may be a linker including an aldehyde group such as glutaraldehyde.

The surfactant is coated on the surface of the fluorescent silica nanoparticles according to the present invention so that the fluorescent silica nanoparticles do not aggregate in an aqueous solution and have a high dispersibility, preferably, an anionic surfactant or a nonionic surfactant. Specifically, polyvinylpyrrolidone (PVP: Polyvinylpyrrolidone), polyacrylic acid, polyimine, sulfo succinic acid, alkyl phosphate, polyoxyethylene fatty ether, polyoxyethylene phenyl ether, dodecyl benzene sulfonate (DBS), fatty acid amine Examples thereof include ether polyoxyethylene, sorbitan fatty acid ester, polyoxyethylene sorbitan fatty acid ester, fatty ether-containing polyoxyethylene, aromatic ether-containing polyoxyethylene and polyethylene glycol ester.

Specific examples of the surface modifier used in the present invention include a surface modifier represented by the following general formula (1) and prepared by reacting polyvinylpyrrolidone (PVP) and glutaaldehyde (GA).

(N = m = α: 1-α, and 0 <α <1)

In addition, the fluorescent material introduced into the fluorescent silica nanoparticles according to the present invention may be used by appropriately selecting a known form according to the application, as an example of the fluorescent material, Alexa fluor (350), 405, 430, 488, 500, 514, 633, 647, 660, 680, 700, cy3, cy5, cy7, Rupy (tris (2,2-bipyridyl) ruthenium (II)) FITC (fluoresein Isothiocyanate), rhodamine 6G ( rhodamine 6G), rhodamine B, 6-carboxy-tetramethyl-rhodamine (TAMRA), Texas red, DAPI (4,6-diamidino-2-phenylindole) and Coumarin. Among them, the fluorescent material used in the present invention preferably emits near infrared (NIR), because the near infrared is high in biotransmission and is suitable for bioimaging.

Regarding the amount of fluorescent material introduced into the silica nanoparticles, the nanoparticles generally contain 2 to 500 fluorescent molecules per fluorescent silica nanoparticle, although it is possible to lower the concentration because the fluorescent signal is stronger than that of ordinary dye molecules. Fluorescent silica nanoparticles are preferred, because if the content of the fluorescent substance is less than 2 per nanoparticle, the fluorescence is lowered, and if it is more than 500, the size of the nanoparticles becomes too large, which is not suitable for the use of the present invention. .

Furthermore, the fluorescent silica nanoparticles may conjugate other useful molecules, including biomolecues, to the fluorescent silica nanoparticles through functional groups such as aldehyde groups, amine groups, hydroxyl groups, and thiol groups introduced on the surface. have. At this time, the molecule to be conjugated to the fluorescent silica nanoparticles is not particularly limited, but is preferably a substance that can specifically bind to biomolecules, for example, antibodies, antigens, RNA, DNA, PNA, hapten (hapten) ), Radiation such as avidin, streptavidin, neutravidin, protein A, protein G, lectin, selectin, C ¹⁴ , I ¹²⁵ , P ³² and S ³⁵ Isotopically labeled substances.

On the other hand, the fluorescent silica nanoparticles are preferably 50 ~ 500nm in diameter. If the diameter is less than 50 nm, the nanoparticles are too small to be difficult to handle, and conjugation of other useful molecules is not easy, and if the diameter is larger than 500 nm, the fluorescent silica nanoparticles are too large to be used in membrane type diagnostic kits or biological systems. The problem arises.

Fluorescent silica nanoparticles according to the present invention described in detail above, the fluorescent material is introduced and the molecules such as antibodies are conjugated, as well as exhibit high dispersion in aqueous solution, it is effective for the detection of biomolecules and imaging of biomolecules Can be used.

More specifically, the fluorescent silica nanoparticles according to the present invention can be widely used in biomedical fields such as imaging diagnostic probes (contrast agents), biochips, biosensors using a fluorescence microscope.

For example, the fluorescent silica nanoparticles according to the present invention to which the antibody is conjugated are immunoassays such as enzyme-linked immunosorbent assay (ELISA), radioimmunoassay, immunoprecipitation, sandwich analysis, flow cytometry, and the like. Or it may be usefully used for the purpose of immunostaining (immunostaining).

On the other hand, the fluorescent silica nanoparticles according to the present invention is preferably supported on a suitable carrier to be used in combination with a lubricant, wetting agent, emulsifier, preservatives and the like. Here, the carrier is preferably a pharmaceutically acceptable carrier, specifically, water, ion exchange resins, alumina, aluminum stearate, lecithin, serum proteins, various buffer substances, magnesium trisilicate, polyvinylpyrrolidone, Cellulose matrix, polyethylene glycol, sodium carboxymethylcellulose, polyarylate, wax, polyethylene glycol, and the like. In addition, the composition may be administered in the body or in vitro samples according to a method of diagnosis or detection, when administered in the body may be administered without particular limitation according to methods commonly performed in the medical field, but rather than oral administration Parenteral administration is preferably performed with an injectable preparation or the like via an intravenous, intraperitoneal, intramuscular, subcutaneous or topical route.

Next, a method for preparing fluorescent silica nanoparticles into which a fluorescent substance having high dispersibility in an aqueous solution according to the present invention is introduced will be described.

Method for producing fluorescent silica nanoparticles in which a highly dispersible fluorescent substance is introduced in an aqueous solution according to the present invention, a) forming a fluorescent silica nanoparticle intermediate having a functional (functional) on the surface; (b) forming a surface modifier using a linker and a surfactant; And (c) modifying the surface of the fluorescent silica nanoparticles formed in step (a) using the surface modifier formed in step (b), which will be described in detail below.

In the step (a) of the production method to form a fluorescent silica nanoparticle intermediate having a functional (functional) on the surface as a specific execution condition is not particularly limited, it can be mentioned as an example performed by the following method.

That is, the step (a), (a-1) adding a fluorescent material and a functional group introducing material to the solvent and then stirring; (a-2) mixing the solution obtained in step (a-1) with a solution containing a silica precursor and a catalyst and then stirring; And (a-3) adding the functional group introduction material to the solution obtained in the step (a-2), followed by stirring and washing.

Specifically, step (a-1) is a step of synthesizing a material including a fluorescent material and a functional group to be introduced into the silica nanoparticles by reacting the fluorescent material and the functional group introducing material by reacting.

At this time, the solvent is preferably a polar organic solvent such as dimethylsulfoxide (DMSO), dimethylformamide (DMF), ethyl acetate, tetrahydrofuran (THF), ethyl acetate, acetone, acetonitrile, As already mentioned above, Alexa fluor and the like can be used.

In addition, the functional group introducing material is a precursor for introducing a functional group on the surface of the fluorescent silica nanoparticle intermediate prepared in step (a), preferably a precursor capable of introducing an amine group, an aldehyde group, a hydroxyl group or a thiol group, More preferably, as aminoalkyltrialkoxysilane into which an amine group can be introduced, 3-aminopropyltrimethoxysilane, 3-aminopropyltriethoxysilane, N-methyl-3-aminopropyltrimethoxysilane, N-methyl -3-aminopropyltriethoxysilane, N- (2-aminoethyl) -3-aminopropyltrimethoxysilane, N- (2-aminoethyl) -3-aminopropyltriethoxysilane, 4-aminocyclo Trimethoxysilane, 4-aminocyclotriethoxysilane, p-aminophenyltrimethoxysilane and p-aminophenyltriethoxysilane.

Step (a-2) is performed by combining the fluorescent material obtained in step (a-1) with a substance containing a functional group and a silica precursor in an appropriate solvent in the presence of a suitable catalyst to bind, hydrolyze and condense the silica precursor to a cluster. A step of synthesizing a silica nanoparticle intermediate into which a fluorescent material and a functional group are introduced by a gelation reaction through the reaction.

In this case, the solvent is not particularly limited, but it is preferable to mix the water such as deionized water (DI water), which plays a role of promoting the hydrolysis reaction of the silica precursor, and the silica and the silica precursor homogeneously, A mixed solvent of an alcohol that can be advanced is used. In the mixed solvent, the mixing ratio of water and alcohol is not particularly limited and may be suitably selected by those skilled in the art.

The silica precursor is preferably a known alkoxysilane such as tetramethoxy silane (TMOS), tetraethoxy silane (TEOS), or a mixture thereof.

On the other hand, the step (a-2) is preferably carried out under a catalyst in order to promote the hydrolysis reaction, the catalyst is selected from an acidic catalyst such as hydrochloric acid, acetic acid or basic catalyst such as ammonium chloride, potassium chloride, etc. Can be.

The step (a-3) is a step of additionally introducing a functional group on the surface of the fluorescent silica nanoparticle intermediate obtained in the step (a-2), a solution containing the fluorescent silica nanoparticle intermediate obtained in the step (a-2) It can be carried out by adding the functional group introduced material described above to the reaction through stirring.

In step (b) of the production method, a surface modifier is formed by using a linker and a surfactant, ultrapure water, distilled water, PBS (phosphate-buffered saline), various alcohols such as ethanol, methanol, propanol, butanol, and acetic acid A surface modifier can be prepared by adding a linker and a surfactant as described above in detail to an aqueous solvent such as acid) or formic acid, NaHCO ₃ buffer, and the like.

In the step (c) of the production method to prepare a fluorescent silica nanoparticles having a high dispersion in aqueous solution by modifying the surface of the fluorescent silica nanoparticle intermediate formed in the step (a) with a surface modifier in the step (b) to be.

Specifically, step (c) is performed by adding and stirring the surface modifier obtained in step (b) to a solution of fluorescent silica nanoparticles having functional groups on the surface obtained in step (a). By the functional groups on the surface of the silica nanoparticle intermediate react with the functional groups of the surface modifiers derived from the linker, the fluorescent silica nanoparticle intermediates are coated by the portion derived from the surfactant even in the surface modifier, more precisely the surface modifier, and finally the fluorescent silica Nanoparticles are prepared.

The fluorescent silica nanoparticles are coated with a surface modifier so that the total zeta potential is controlled to be in the range of -5 to -50 mV or less so that the dispersion properties can be maintained without causing entanglement of the nanoparticles in the aqueous solution have. The control of the zeta potential can be performed by appropriately adjusting the content of the surface modifier used in the present step. The control of the zeta potential of the fluorescent silica nanoparticles by controlling the content of the surface modifier can be easily carried out without undue trial and error can do.

On the other hand, the method for producing a fluorescent silica nanoparticles according to the present invention, after performing the step (c), (d) adding a step of introducing a functional group on the surface of the surface-modified fluorescent silica nanoparticles in the step (c) Can be done with This step is carried out by treating the surface of the fluorescent silica nanoparticles with a precursor corresponding to the functional group to be introduced, the specific method of execution is not particularly limited and can be carried out by a known method. At this time, the functional group is preferably an amine group, an aldehyde group, a hydroxyl group or a thiol group. For example, in order to introduce an aldehyde group to the surface of the fluorescent silica nanoparticles, a linker containing an aldehyde group, such as glutaraldehyde, may be used. As such, by introducing functional groups into fluorescent silica nanoparticles, it is possible to conjugate with other useful molecules such as biomolecules.

In addition, the method for producing fluorescent silica nanoparticles according to the present invention, (e) fluorescent silica silica containing other useful molecules through the functional group present on the surface of the fluorescent silica nanoparticles formed in step (d). Conjugating to the nanoparticles may be further included.

The molecule to be conjugated to the fluorescent silica nanoparticles according to the present invention is not particularly limited, but is preferably a substance that can specifically bind to biomolecules, for example, antibodies, antigens, RNA, DNA, PNA, hapten (hapten), avidin (avidin), streptavidin, neutravidin, protein A, protein G, lectin, selectin, radioisotope labeling substances, and the like.

In this step, the conjugation is performed by the functional group introduced in step (c) corresponding to the molecule to be conjugated, and in this case, a specific method of conjugating the biomolecule to the functional group of the fluorescent silicon nanoparticles is known. Techniques can be readily used by those skilled in the art. For example, in the case of fluorescent silicon nanoparticles having an aldehyde group on the surface, the aldehyde group can be conjugated with a protein such as an antibody, and the fluorescent silicon nanoparticles having an amine group convert an amine group to a carboxyl group with succinic anhydride or the like. After the reaction, it may be conjugated with a biomolecule containing an amine group by reacting with dicyclohexylcarbodiimide (EDC).

Next, the present invention describes a biomolecule detection method using the fluorescent silica nanoparticles.

Biomolecule detection method using a fluorescent silica nanoparticles according to the present invention, (a) preparing a fluorescent silica nanoparticles bonded molecules; (b) irradiating light after the fluorescent silica nanoparticles are added to a sample including a detection target material and reacted; And (c) measuring fluorescence from the sample.

Step (a) may be performed by preparing fluorescent silica nanoparticles according to the manufacturing method described in detail above. In the step (b) is irradiated with light of a wavelength corresponding to the absorption region of the fluorescent material contained in the silica nanoparticles. In the step (c), the fluorescence emitted from the fluorescent material reacted with the sample is detected through a fluorescence microscope.

EMBODIMENT OF THE INVENTION Below, this invention is demonstrated in detail based on an Example. The examples presented are exemplary and are not intended to limit the scope of the invention.

&Lt; Example 1 >

One) An amine group  Synthesis of Introduced Fluorescent Silica Nanoparticle Intermediates (AF647 @ SiNP-NH ₂ )

1 mg of Alexa Fluor 647 is dissolved in 100 uL of dimethylsulfoxide (DMSO), and then 10 uL of the mixture is mixed with a solution of 0.036 uL of ATPES (3-aminopropyltriethoxysilane) and 89.964 uL of DMSO (dimethylsulfoxide) The mixed solution was prepared such that Alexa Fluor 647 and ATPES had concentrations of 0.77M and 1.54M, respectively, and the mixed solution was stirred at 600 rpm for 3 hours at room temperature. Then, 15.3 mL of ethanol was added and 1 mL of TEOS (tetraethoxy silane) was added to the round flask, the mixed solution, 2 mL of water, and 0.2 mL of ammonia water were added sequentially, followed by stirring at 600 rpm for 16 hours at room temperature. After the addition and stirring at room temperature at 600 rpm for 12 hours at room temperature and washed three times with ethanol to synthesize a fluorescent silica nanoparticle intermediate having an amine group on the surface. For reference, a conceptual diagram of process steps performed in this step is shown in FIG.

2) With aldehydes  Formulated Of polyvinylpyrrolidone (PVP)  synthesis( PVP - GA )

Prepare 90 mL of sodium hydrogen carbonate (NaHCo ₃ ) solution (pH 9, 0.1M) as a buffer in a round flask and dissolve 2 g of PVP in it. In addition, 1.6 mL of glutaraldehyde was added to the round flask and stirred at 600 rpm for 12 hours at room temperature. Next, the stirred solution was dialyzed with distilled water in a semipermeable membrane of 40,000 Da or less, and then lyophilized and completely dried to prepare polyvinylpyrrolidone (PVP-GA) modified with an aldehyde group. For reference, a conceptual diagram of the process step performed in this step is shown in FIG.

3) PVP - GA AF647 @ SiNP-NH using ₂  Surface Modification (AF647 @ SiNP ( PVP ) - CHO )

₂ mL of the AF647 @ SiNP-NH ₂ solution prepared in 1) was collected, washed with sodium hydrogen carbonate (NaHCo ₃ ) solution (pH 9, 0.1M), concentrated to 1 mL, and the PVP prepared in 2) in a tube. -GA 2mg and the concentrated solution were put together and stirred to react at room temperature for 12hr. Next, 3 uL of glutaaldehyde was added to the reaction solution, followed by stirring for 12 hours, and then 0.3 mg of sodium borohydride (NaBH ₄ ) was added thereto, followed by gentle stirring for 3 hours. At this time, at the beginning of the reaction, the cap was intermittently opened to discharge the gas. Finally, three times washing with sodium hydrogen carbonate (NaHCo ₃ ) solution (pH 9, 0.1M) to prepare a surface-modified fluorescent silica nanoparticles. For reference, a conceptual diagram of the process step performed in this step is shown in FIG.

<Example 2>

In the synthesis of fluorinated silica nanoparticles (AF647 @ SiNP-NH ₂ ) introduced with amine groups, 1 mg of Alexa Fluor 647 was added to dimethyl sulfoxide (DMSO) such that Alexa Fluor 647 and ATPES had concentrations of 1.54M and 3.08M, respectively. In the same manner as in Example 1, except that a mixed solution consisting of 20 uL of a solution dispensed in 100 uL, 0.072 uL of ATPES (3-aminopropyltriethoxysilane) and 79.928 uL of DMSO (dimethylsulfoxide) was used. Surface-modified fluorescent silica nanoparticles were prepared.

<Example 3>

In the synthesis of fluorinated silica nanoparticles (AF647 @ SiNP-NH ₂ ) introduced with amine groups, 1 mg of Alexa Fluor 647 was added to dimethyl sulfoxide (DMSO) such that Alexa Fluor 647 and ATPES had concentrations of 3.08M and 6.16M, respectively. In the same manner as in Example 1, except that 40uL of the solution dispensed from 100uL of solution, 0.144uL of ATPES (3-aminopropyltriethoxysilane) and 59.856uL of DMSO (dimethylsulfoxide) were used. Surface-modified fluorescent silica nanoparticles were prepared.

<Example 4>

Using fluorescent silica nanoparticles prepared in Example 1 was prepared fluorescent silica nanoparticles conjugated to the antibody.

That is, 10ug (100uL) of antibody (FITC-Anti-mouseCD11B) was added to 1mL of sodium hydrogen carbonate (NaHCo ₃ ) buffer solution (pH9 0.1M) containing AF647 @ SiNP (PVP) -CHO prepared in Example 1 Stir slowly for hours. After stirring was completed, 1uL of ethanolamine was added to remove unreacted aldehyde groups, and the mixture was stirred by shaking for 3hr at room temperature. Finally, washing three times with PBS buffer (pH 7.4, 0.15M) to prepare fluorescent silica nanoparticles conjugated with antibodies. For reference, a conceptual diagram of a process of forming fluorescent silica nanoparticles conjugated with antibodies according to the present embodiment is shown in FIG. 4.

<Example 5>

Except for using the fluorescent silica nanoparticles prepared in Example 2, in the same manner as in Example 4, was prepared fluorescent silica nanoparticles conjugated to the antibody.

<Example 6>

Except for using the fluorescent silica nanoparticles prepared in Example 3, in the same manner as in Example 4, to prepare a fluorescent silica nanoparticle conjugated with an antibody.

< Experimental Example  1> Example  Surface manufactured at 1-3 Reformed  Particle Size Measurement of Fluorescent Silica Nanoparticles

The particle size of the surface modified fluorescent silica nanoparticles prepared in Examples 1 to 3 was measured by using Dynamic Light Scattering (DLS). As a result, the surface modified fluorescent silica nanoparticles prepared in Examples 1 to 3 were found to have an average particle size of about 85 nm, about 105 nm and about 110 nm, respectively (see FIG. 5). From the above results, it can be seen that the tendency to the nanoparticle size is maintained even after the modification by the surface modifier.

< Experimental Example  2> Example  Surface manufactured at 1-3 Reformed  Surface charge measurement of fluorescent silica nanoparticles

Zeta potential of the surface modified fluorescent silica nanoparticles prepared in Examples 1 to 3 was measured with a Zetapotentiometer. As a result, the surface-modified fluorescent silica nanoparticles prepared in Examples 1 to 3 were -26.76mV, -22.3mv, respectively, and it was confirmed that they had a negative charge. From this, it can be seen that the fluorescent silica nanoparticles according to the present invention has a high dispersion in the aqueous solution, which is also from Figure 6 showing the aqueous solution in which the surface-modified fluorescent silica nanoparticles prepared in Examples 1 to 3 dispersed. Supported.

< Experimental Example  3> Example  If the antibody prepared in 4-6 Bonded  PL for fluorescent silica nanoparticles photoluminescence ) Measure

Fluorescence analysis was performed on the surface modified fluorescent silica nanoparticles prepared in Examples 4 to 6 by PL (photoluminescence), and the fluorescence of 3937 (au), 7634 (au), and 6790 (au) were respectively measured at PMT 700V. The intensity value was obtained. From this, it can be seen that except in the case of Example 4, an almost similar amount of antibody (FITC-IgG) was immobilized on the surface-modified silicon nanoparticles (AF647 @ SiNP-PVP-CHO). In one step) it can be seen that the antibody can be introduced into the fluorescent silica nanoparticles.

Claims (23)

Fluorescent silica nanoparticles surface-modified with a surface modifier represented by the following Chemical Formula 1 and introduced with a fluorescent substance:
&Lt; Formula 1 >

(Wherein n: m = α: 1-α in the formula above and 0 <α <1). The method of claim 1, wherein the fluorescent material is Alexa fluor 350, 405, 430, 488, 500, 514, 633, 647, 660, 680, 700, cy3, cy5, cy7, Rupy (tris) (2,2-bipyridyl) ruthenium (II)), fluoresein Isothiocyanate (FITC), rhodamine 6G (rhodamine 6G), rhodamine B (rhodamine B), 6-carboxy-tetramethyl-rhodamine (TAMRA), Texas red (Texas) Red), DAPI (4,6-diamidino-2-phenylindole) and fluorescent silica nanoparticles, characterized in that at least one selected from the group consisting of. The fluorescent silica nanoparticles of claim 2, wherein the fluorescent silica nanoparticles comprise 2 to 500 molecules of the fluorescent substance per fluorescent silica nanoparticles. delete The fluorescent silica nanoparticle of claim 1, wherein at least one functional group selected from an aldehyde group, an amine group, a hydroxyl group and a thiol group is bonded to the surface. The fluorescent silica nanoparticles of claim 5, wherein the fluorescent particles are nanoparticles having a surface conjugated with the functional group. The method of claim 6, wherein the molecule is an antibody, antigen, RNA, DNA, PNA, hapten, avidin, streptavidin, neutravidin, protein A, protein G, lectin ( Fluorescent silica nanoparticles, characterized in that at least one selected from the group consisting of lectin), selectin and radioisotope labeling substances. The fluorescent silica nanoparticles of claim 1, wherein the diameter is 50 to 500 nm. The fluorescent silica nanoparticles of claim 1, wherein the zeta potential is -5 to -50 mV. A contrast medium composition comprising the fluorescent silica nanoparticles according to any one of claims 1 to 3 and 5 to 9 and a pharmaceutically acceptable carrier. An immunoassay composition comprising the fluorescent silica nanoparticles according to any one of claims 1 to 3 and 5 to 9 and a pharmaceutically acceptable carrier. (a) forming a fluorescent silica nanoparticle intermediate having functional groups on its surface;
(b) forming a surface modifier represented by Chemical Formula 1 using a linker and a surfactant
&Lt; Formula 1 >

(Wherein n: m = α: 1-α in the formula above and 0 <α <1); And
(c) modifying the surface of the fluorescent silica nanoparticle intermediate formed in step (a) with the surface modifier formed in step (b),
Step (a) is a method of producing a fluorescent silica nanoparticles introduced fluorescent material, characterized in that it comprises the following steps:
(a-1) adding a fluorescent material and a functional group introducing material to a solvent and then stirring the solvent;
(a-2) mixing the solution obtained in step (a-1) with a solution containing a silica precursor and a catalyst and then stirring; And
(a-3) adding a functional group introducing material to the solution obtained in step (a-2), stirring and washing. delete The method of claim 12, wherein the fluorescent material is Alexa fluor 350, 405, 430, 488, 500, 514, 633, 647, 660, 680, 700, cy3, cy5, cy7, Rupy (tris) (2,2-bipyridyl) ruthenium (II)), fluoresein Isothiocyanate (FITC), rhodamine 6G (rhodamine 6G), rhodamine B (rhodamine B), 6-carboxy-tetramethyl-rhodamine (TAMRA), Texas red (Texas) Red), DAPI (4,6-diamidino-2-phenylindole) and a method for producing fluorescent silica nanoparticles, characterized in that at least one selected from the group consisting of Coumarin. The method of claim 12, wherein the functional group introducing material is 3-aminopropyltrimethoxysilane, 3-aminopropyltriethoxysilane, N-methyl-3-aminopropyltrimethoxysilane, N-methyl-3-aminopropyl Triethoxysilane, N- (2-aminoethyl) -3-aminopropyltrimethoxysilane, N- (2-aminoethyl) -3-aminopropyltriethoxysilane, 4-aminocyclotrimethoxysilane, Method for producing fluorescent silica nanoparticles, characterized in that at least one selected from the group consisting of 4-aminocyclotriethoxysilane, p-aminophenyltrimethoxysilane and p-aminophenyltriethoxysilane. The method of claim 12, wherein the silica precursor is tetramethoxy silane (TMOS), tetraethoxy silane (TEOS), or tetramethoxy silane (TMOS), tetraethoxy silane (tetraethoxy silane) silane, TEOS) method for producing fluorescent silica nanoparticles. The method of claim 12, wherein step (b) is performed by adding a linker and a surfactant to the solvent and stirring the reaction. The method of claim 12, wherein the step (c) is performed by adding the surface modifier obtained in the step (b) to the solution of the fluorescent silica nanoparticle intermediate having the functional group on the surface obtained in the step (a) and stirring the reaction. Method for producing a fluorescent silica nanoparticles, characterized in that. The method of claim 12, further comprising (d) introducing a functional group to the surface of the fluorescent silica nanoparticles modified in step (c). 20. The method of claim 19, wherein the functional group introduced in step (d) is at least one selected from an amine group, an aldehyde group, a hydroxyl group and a thiol group. 20. The method of claim 19, comprising (e) conjugating the molecule to the fluorescent silica nanoparticles via a functional group present on the surface of the fluorescent silica nanoparticles formed in step (d). Method for producing silica nanoparticles. The method of claim 21, wherein the molecule is an antibody, antigen, RNA, DNA, PNA, hapten, avidin, streptavidin, neutravidin, protein A, protein G, lectin ( lectin), a selectin and a method for producing fluorescent silica nanoparticles, characterized in that at least one member selected from the group consisting of radioisotope labeling substances. (a) preparing the fluorescent silica nanoparticles according to any one of claims 1 to 3 and 5 to 9; (b) irradiating light after the fluorescent silica nanoparticles are added to a sample including a detection target material and reacted; And (c) measuring fluorescence from the sample.

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