KR101984623B1 - Vitamin D Binding Using Triazolidione-based Compound and Detection of Quantitative Determination of Vitamin D Using Fluorescence Supported Silica Nanoparticles - Google Patents

Abstract

The present invention relates to a method for immobilizing vitamin D by using a triazole dione-based compound, which is a chemical probe that can be used in an aqueous solution that is not a marker of vitamin D using existing proteins, D based on a triazole dione-based compound capable of quantitative detection even at a low concentration, and a method for detecting quantitative determination of vitamin D using fluorescence-supported silica nanoparticles. The present invention provides a chemical probe for fixing vitamin D comprising a triazole dione derivative.

Description

TECHNICAL FIELD The present invention relates to a method for detecting quantitative determination of vitamin D by using vitamin D binding and fluorescence-supported silica nanoparticles using a triazolidione-based compound (Vitamin D Binding Using Triazolidione-based Compounds and Detection of Quantitative Determination of Vitamin D Using Fluorescence Supported Silica Nanoparticles)

The present invention relates to a method for detecting quantitative determination of vitamin D by using vitamin D binding and fluorescence supported silica nanoparticles using a triazolidione-based compound, and more particularly, Based on a triazol dione-based compound, which is a usable chemical probe, it is possible to immobilize vitamin D and to use a triazole dione-based compound capable of quantitatively detecting vitamin D at low concentrations using fluorescence-loaded silica nanoparticles The present invention relates to a method for detecting quantitative determination of vitamin D using vitamin D binding and fluorescence supported silica nanoparticles.

Vitamin D is a precursor to lipid soluble steroids that are produced mainly by sun exposure or from dietary intake (mainly egg yolk, fish oil and plants). These vitamin Ds must be bound to two hydroxyl groups in the liver and kidney in order to be biologically active 1,25-dihydrowyvitamin D without biologically pharmacological action. The two main forms of vitamin D are vitamin D3 (cholecalciferol) and vitamin D2 (ergocalciferol). In the case of vitamin D3, it can be synthesized intramolecularly, but vitamin D2 can not be synthesized.

Vitamin D is mainly responsible for the metabolism of calcium and phosphorus. When it is deficient, calcium and phosphorus are not accumulated enough in the heat fluid to accumulate in the bone, so the skeleton weakens. As a result, the bone can not bend or break have. In other words, the amount of bone is normal, but the density of the bone is reduced, and the bone becomes soft and fragile. These symptoms are called rickets and osteomalacia. Unlike osteoporosis, which occurs mainly in postmenopausal women, it can occur in all ages.

In the human body, vitamin D3 and D2 bind to the plasma vitamin D binding protein and migrate to the liver, forming 25 (OH) D3 and hydroxylated. Since 25 (OH) D is the main storage form of vitamin D in the human body, 25 (OH) D is being measured to analyze the overall state of vitamin D. This rapid and accurate measurement of vitamin D concentration in the human body is a necessary technique for on-site diagnosis of various geriatric diseases associated with vitamin D deficiency.

Previous studies to detect existing vitamin D have been conducted to isolate and detect vitamin D using specific protein markers or surfactants. However, since the separation and detection of vitamin D using these proteins or surfactants proceed in the organic solvent, immobilized vitamin D is lost during the dissolution / separation process of the organic solvent, which has a problem in the accuracy of detection.

In order to solve the above-mentioned problems, the present invention relates to a method for immobilizing vitamin D by using a triazolidione-based compound, which is a chemical marker substance that can be used in an aqueous solution which is not a conventional protein marker, The present invention provides a method for detecting quantitative determination of vitamin D by using vitamin D binding and fluorescence supported silica nanoparticles using a triazole dione based compound capable of quantitatively detecting vitamin D at a low concentration.

In order to solve the above-mentioned problems, the present invention according to the first aspect provides a chemical probe for fixing vitamin D comprising a triazole dione derivative.

The triazole dione derivative may be 4-phenyl-1,2,4-triazole-3,5-dione (PTAD).

The present invention according to the second aspect provides the fluorescence-supported silica nanoparticles for quantitative detection through the above-mentioned vitamin D fixing chemical probe.

The silica nanoparticles may further carry a fluorescent substance.

Wherein the silica nanoparticles comprise: (a) forming a fluorescent silica nanoparticle intermediate; (b) conducting a surface modification represented by the following formula (1) using a linker and a surfactant,

≪ Formula 1 >

(In the above formula, n = 10 to 15 KDa); The step (a) may be produced by a method including the following steps.

(a-1) adding a fluorescent substance and a fluorescent substance precursor to a solvent and stirring the mixture; (a-2) mixing the solution obtained in the step (a-1) with a solution containing a silica precursor and a catalyst, stirring and washing.

The present invention according to the third aspect provides a method for quantitatively detecting vitamin D using the above-mentioned silica nanoparticles.

The method for detecting the quantitative determination of vitamin D by using the triazine dione-based compound of the present invention using the vitamin D-binding and fluorescence-supported silica nanoparticles enables accurate detection of the vitamin D even at a low concentration, It can be used for field diagnosis of geriatric diseases.

FIG. 1 is a schematic view of a fixing reaction of vitamin D using a chemical probe for fixing vitamin D according to an embodiment of the present invention.
FIG. 2 is a schematic view showing a method of carrying a fluorescent substance and a chemical probe through a sandwich technique using silica nanoparticles according to an embodiment of the present invention.
3 is a graph showing the results of HPLC analysis according to conjugation of a chemical probe and vitamin D according to an embodiment of the present invention.
4 is a graph showing the results of HPLC analysis according to conjugation of a chemical probe and vitamin D in an aqueous liquid phase reaction according to an embodiment of the present invention.
FIG. 5 is a graph showing a detection response test result of vitamin D according to an embodiment of the present invention. FIG.
FIG. 6 is a graph showing changes in fluorescence intensity due to a fluorescence pattern and a fluorescent precursor not contained in silica according to an injection timing of a fluorescent precursor according to an embodiment of the present invention.
FIG. 7 is a graph showing changes in size and fluorescence intensity according to an embodiment of the present invention.

Hereinafter, preferred embodiments of the present invention will be described in detail. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS Hereinafter, the present invention will be described in detail with reference to the accompanying drawings. Throughout the specification, when an element is referred to as " including " an element, it means that it can include other elements, not excluding other elements, unless specifically stated otherwise.

The present invention according to the first aspect relates to a chemical probe for fixing vitamin D comprising a triazole dione derivative.

In the conventional vitamin D detection method, vitamin D is immobilized using a protein probe or a surfactant. Since the detection is carried out using an organic solvent, a portion of the vitamin D bound to the protein is lost during washing and separation of the organic solvent I had a problem. However, in the present invention, it is possible to detect vitamin D in an aqueous solution by immobilizing vitamin D using a triazole dione derivative. Thus, accurate quantitative analysis is possible by minimizing the amount of vitamin D lost. The triazole dione derivative used herein is not particularly limited as long as it is a derivative capable of fixing vitamin D, but preferably 4-phenyl-1,2,4-triazole- 2,4-triazole-3,5-dione, PTAD). The 4-phenyl-1,2,4-triazole-3,5-dione can also directly fix vitamin D, but as shown in Figure 1, 1,3-dibromo-5,5- It is more preferable to activate vitamin D by using a 1,3-dibromo-5,5 dimethyihydantoin, and then fix the vitamin D using a Diels-Alder reaction.

Finally, a method for immobilization of magnetic nanoparticles is required for the detection of vitamin D in a sandwich assay. For the purpose of particle stability and uniform dispersion in aqueous solution, PEG and Biotin modified Dibenzocycloooctyne , DBCO) to bind PTAD-vitamin D immobilized probe through a click reaction.

The present invention according to the second aspect also relates to a fluorescence-supported silica nanoparticle for the quantitative detection of the vitamin D immobilized chemical probe. In the case of the above-mentioned vitamin D-immobilized chemical probe, it can not be used for detection alone. Therefore, the vitamin D-immobilized chemical probe is bound to the magnetic nanoparticle modified with a carboxyl group on the surface thereof, And can be used for the quantitative detection of vitamin D through the sandwich technique (FIG. 2).

The silica nanoparticles are additionally supported on the surface of the silica nanoparticles by surface modification with a surface modifier and have a high dispersibility in the aqueous solution. The silica nanoparticles are introduced into the excitation state by light irradiation, ) And emits the absorbed energy into light of a specific wavelength.

The fluorescent silica nanoparticles may be prepared by forming a fluorescent silica nanoparticle intermediate having a functional group on its surface with a fluorescent substance introduced thereinto, forming a surface modifier using a linker and a surfactant, And modifying the surface of the fluorescent silica nanoparticle intermediate with the surface modifier. In addition, various functional groups may be used for the surface functional group depending on a desired target substance, but it is preferable to use a triazol dione derivative as a chemical probe for fixing vitamin D in order to detect vitamin D of the present invention.

The surface modifier is prepared by using a linker and a surfactant. The linker bridges between the fluorescent silica nanoparticle intermediate and the surfactant so that the surfactant can coat the surface of the fluorescent silica nanoparticle. And has a functional group capable of reacting with the 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 containing 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 the aqueous solution and have a high dispersibility. Preferably, the surfactant is an anionic surfactant or a nonionic surfactant Specific examples thereof include polyvinylpyrrolidone (PVP), polyacrylic acid, polyimine, sulfosuccinic acid, alkylphosphate, polyoxyethylene fatty ether, polyoxyethylene phenyl ether, DBS (Dodecyl benzene sulfonate) Ether polyoxyethylene, consumptane fatty acid ester, polyoxyethylene succinic acid fatty acid ester, fatty ether-containing polyoxyethylene, aromatic ether-containing polyoxyethylene and polyethylene glycol ester, and the like.

Specific examples of the surface modifier used in the present invention include a surface modifier represented by the following formula (1), which is prepared by reacting hyaluronic acid.

≪ Formula 1 >

(Provided that n = 10 to 15 KDa in the above formula)

The fluorescent material to be introduced into the fluorescent silica nanoparticles according to the present invention can be appropriately selected in accordance with the intended use of the fluorescent material. Examples of the fluorescent material include Alexa fluor (AF) 350, 405, 4, 6, 680, 700, cy3, cy5, cy7, Rubpy (tris (2,2-bipyridyl) ruthenium (II)) FITC (fluoresein isothiocyanate), Rhodamine 6G (rhodamine 6G), rhodamine B, TAMRA (6-carboxytetramethyl-rhodamine), Texas Red, DAPI (4,6-diamidino-2-phenylindole) and Coumarin. In particular, the fluorescent material used in the present invention preferably emits near infrared (NIR) light because near infrared rays have high bio-transparency and are suitable for biological imaging.

Regarding the content of the fluorescent material to be introduced into the silica nanoparticles, it is usually possible to lower the concentration of the nanoparticles because the fluorescence signal is stronger than that of the ordinary dye molecules. However, since the fluorescent nanoparticles contain 2 to 500 fluorescent molecules per fluorescent nanoparticle Fluorescent silica nanoparticles are preferable because the fluorescence is lowered when the content of the fluorescent substance is less than 2 per one nanoparticle and the size of the nanoparticles is larger than 500, .

Further, the fluorescent silica nanoparticles can be conjugated to fluorescent nanoparticles with other useful molecules, including biomolecules, via functional groups such as aldehyde groups, amine groups, hydroxyl groups, and thiol groups introduced on the surface have. At this time, the molecules to be conjugated to the fluorescent silica nanoparticles are not particularly limited, but it is preferable that the molecule is capable of specifically binding to biomolecules. For example, the molecule is conjugated to fluorescent nanoparticles such as vitamin A, vitamin B, vitamin C, vitamin D, vitamin E, vitamin K, antibody, antigen, RNA, DNA, PNA, hapten, avidin, streptavidin, neutravidin, protein A, protein G, lectin, (selectin), C ¹⁴ , I ¹²⁵ , P ^32, and S ³⁵ .

On the other hand, the fluorescent silica nanoparticles preferably have a diameter of 50 to 500 nm. If the diameter is less than 50 nm, the nanoparticles are too small to be handled and it is not easy to conjugate other useful molecules, and if the diameter exceeds 500 nm, the fluorescent silica nanoparticles can be used in membrane type diagnostic kits or in biological systems There is a problem that it has.

The fluorescent silica nanoparticles according to the present invention described above in detail are not only bonded with fluorescent molecules and molecules such as antibodies, but also exhibit high dispersibility in an aqueous solution. Therefore, the fluorescent nanoparticles of the present invention can be effectively used for 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 probes (contrast agents), biochips, and biosensors using fluorescence microscopy.

For example, the antibody-conjugated fluorescent silica nanoparticles according to the present invention can be used for immunoassay such as enzyme-linked immunosorbent assay (ELISA), radioimmunoassay, immunoprecipitation, sandwich assay, flow cytometry, or the like And can be usefully used for immunostaining. Meanwhile, the fluorescent silica nanoparticles according to the present invention are preferably carried on a suitable carrier and used as a composition together with a lubricant, a wetting agent, an emulsifier, a preservative, and the like. The carrier is preferably a pharmacologically acceptable carrier. Specific examples of the carrier include water, an ion exchange resin, alumina, aluminum stearate, lecithin, serum proteins, various buffer substances, magnesium trisilicate, polyvinylpyrrolidone, Cellulose type substrate, polyethylene glycol, sodium carboxymethyl cellulose, polyarylate, wax, polyethylene glycol and the like.

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

The method for preparing a fluorescent silica nanoparticle into which a fluorescent substance having high dispersibility is introduced in an aqueous solution according to the present invention comprises the steps of: (a) forming a fluorescent silica nanoparticle intermediate; And (b) advancing the surface modification of the fluorescent silica nanoparticles using a linker and a surfactant. Each of the above steps will be described in detail below.

In the step (a) of the present production method, the fluorescent silica nanoparticle intermediate is formed, and the specific conditions for the preparation are not particularly limited, and examples thereof may be performed by the following method.

That is, the step (a) comprises the steps of: (a-1) adding a fluorescent substance and a fluorescent substance pretreatment agent to a solvent and stirring the mixture; (a-2) mixing the solution obtained in the step (a-1) with a solution containing a silica precursor and a catalyst, stirring and washing.

Specifically, the step (a-1) is a step of modifying the fluorescent material to be introduced into the silica nanoparticles by mixing and reacting the fluorescent substance and the fluorescent substance precursor.

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 described above, Alexa fluor (AF) or the like may be used.

Also, the fluorescent substance pretreatment agent may be at least one selected from the group consisting of 3-aminopropyltrimethoxysilane, 3-aminopropyltriethoxysilane, N-methyl-3-aminopropyltrimethoxysilane, , N- (2-aminoethyl) -3-aminopropyltrimethoxysilane, N- (2-aminoethyl) -3-aminopropyltriethoxysilane, 4-aminocyclotrimethoxysilane, Aminophenyltrimethoxysilane, p-aminophenyltrimethoxysilane, p-aminophenyltriethoxysilane, and preferably 3-aminopropyltrimethoxysilane (APTMS) can be used .

In the step (a-2), the modified fluorescent substance obtained in the step (a-1) and the silica precursor are mixed in an appropriate solvent in the presence of a suitable catalyst to form a gel precursor, And synthesizing a silica nanoparticle intermediate into which a fluorescent substance is introduced by a 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), tetraethoxysilane (TEOS) or a mixture thereof.

The catalyst may be appropriately selected from acidic catalysts such as hydrochloric acid, acetic acid and the like, or basic catalysts such as ammonium chloride and potassium chloride, etc., and used in the step (a-2) to promote the hydrolysis reaction. .

In step (b) of the present production method, the step of forming a surface modifier using a linker and a surfactant may be carried out using various kinds of alcohols such as ultrapure water, distilled water, phosphate-buffered saline, ethanol, methanol, propanol, The surface modifier may be prepared by reacting the linker and the surfactant described above in detail in an aqueous solvent such as an acid, formic acid or NaHCO3 buffer solution.

Specifically, the step (b) is carried out by adding a surface modifier to the surface obtained in step (a) and reacting with stirring. In this step, the functional group on the surface of the fluorescent silica nanoparticle intermediate is reacted with the functional group of the surface modifier derived from the linker By the reaction, the fluorescent silica nanoparticle intermediate is coated with the surface modifier, more specifically, the portion derived from the surfactant even in the surface modifier, to finally produce the fluorescent silica nanoparticle.

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.

The method for producing fluorescent silica nanoparticles according to the present invention is characterized in that after performing the step (b), (c) a chemical probe for fixing vitamin D is applied on the surface of the fluorescent silica nanoparticle surface-modified in the step (b) Can be carried out. This step is carried out by treating the surface of the fluorescent silica nanoparticles using a chemical probe for fixing vitamin D to be introduced, and the specific method of carrying out the method is not particularly limited and can be carried out by a known method.

In addition, the method for producing fluorescent silica nanoparticles according to the present invention may further comprise: (d) a step of immobilizing a fluorescent dye on the surface of the fluorescent silica nanoparticles formed in step (c) And further conjugating the other molecule to the fluorescent silica nanoparticles.

The molecule to be conjugated to the fluorescent silica nanoparticles according to the present invention is not particularly limited, but is preferably a substance capable of specifically binding to a biomolecule. Examples thereof include vitamin A, vitamin B, vitamin C, vitamin D , Vitamin E, vitamin K, antibody, antigen, RNA, DNA, PNA, hapten, avidin, streptavidin, neutravidin, protein A, protein G, lectin, , Selectin, and radioisotope labeling substances.

In this step, conjugation is carried out via the functional group introduced in the step (b) corresponding to the molecule to be conjugated, and a specific method of conjugating the biomolecule to the functional group of the fluorescent silicon nanoparticle is known Technique by a person skilled in the art. For example, in the case of fluorescent silica nanoparticles having an aldehyde group on their surface, they can be conjugated with an antibody-like protein by an aldehyde group. Fluorescent silica nanoparticles having an amine group can be converted into carboxyl groups by succinic anhydride or the like. , Which can then be conjugated with a biomolecule containing an amine group by reacting with EDC (dicyclohexylcarbodiimide).

Next, the present invention explains a vitamin D detection method using the fluorescent silica nanoparticles.

The method for detecting vitamin D using fluorescent silica nanoparticles according to the present invention comprises the steps of: (a) preparing fluorescence silica nanoparticles to which molecules are bonded; (b) injecting the fluorescent silica nanoparticles into a sample containing a detection target substance (vitamin D), reacting and irradiating light; And (c) measuring fluorescence from the sample.

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

The present invention will be described in more detail with reference to the following examples.

Example 1

Phenyl-4-methoxyphenyl-PEG chain coupled with 1,3-dibromo-5, 5-dimethyihydantoin on an organic solvent (acrylonitrile, acrylonitrile, ACN) After activation of 4-phenyl-1,2,4-triazole-3,5-dione (PTAD), vitamin D and Diels-Alder reaction Respectively. Thereafter, the binding between vitamin D and PTAD was confirmed by HPLC analysis.

As shown in FIG. 3, it was confirmed by HPLC analysis that the intrinsic retention peak of vitamin D at 30 minutes disappears, confirming that effective conjugation is proceeding.

Example 2

The above Example 1 was carried out in an organic solvent, but experiments were conducted in an aqueous solution in order to increase the detection accuracy of vitamin D and impart non-destructive properties in the detection process. ACN and water were mixed at a volume ratio of 50:50.

As shown in Fig. 4, it was confirmed that the intrinsic peak of vitamin D disappeared even within a reaction time of 1 minute, which indicates that conjugation of vitamin D is possible even in an environment close to an aqueous solution, and detection is also possible.

Example 3

The PTAD-based probe was combined with magnetic beads, and the detection response test of vitamin D was performed using the silica nanoparticles developed in this patent. As shown in FIG. 5, stable linear response characteristics were exhibited at the target D concentration (0.01 to 0.1 μg / mL).

Example 4

In order to improve the linear response characteristics of the PTAD-based probe, it is necessary to improve the fluorescence response of the fluorescent derivative (AF647, Invitrogen). Therefore, an experiment for synthesizing the optimized fluorescent derivative was carried out.

In order to increase the fluorescence efficiency of the existing AF647, the change of fluorescence according to each injection time was measured by changing the injection timing of the fluorescent derivative step by step. Through this, the fluorescent derivative (AF647) was induced to distribute to the outside of the silica particles.

As shown in FIG. 6, when the injection time of the fluorescent derivative was changed at intervals of 10 minutes, the fluorescence signal increased as the injection time of the fluorescent derivative was delayed, and it was confirmed that the maximum fluorescence occurred at about 60 minutes. As the final injection timing was delayed, the intensity of high fluorescence could be confirmed even with a small amount of fluorescent precursor, and it was considered that the synthesis of a fluorescent derivative more effective than before was possible.

Example 5

In order to confirm the stability of the fluorescent derivative synthesized by the method of Example 4, the change of fluorescence according to the temperature (4 ° C, 25 ° C) was observed. As shown in FIG. 7, it was confirmed that the fluorescence intensity and the particle size were hardly changed in both temperature conditions, and the stability problem would not occur even when the product was actually applied.

While the present invention has been particularly shown and described with reference to specific embodiments thereof, those skilled in the art will appreciate that such specific embodiments are merely preferred embodiments and that the scope of the present invention is not limited thereto will be. Accordingly, the actual scope of the present invention will be defined by the appended claims and their equivalents.

Claims (6)

Silica nanoparticles for quantitative detection of vitamin D immobilized chemical probes containing triazolidione derivatives. The method according to claim 1,
The triazole dione derivative is 4-phenyl-1,2,4-triazole-3,5-dione (PTAD). delete The method according to claim 1,
Wherein the silica nanoparticle further comprises a fluorescent substance. 5. The method of claim 4,
The silica nano-
(a) forming a fluorescent silica nanoparticle intermediate; And
(b) conducting a surface modification represented by the following formula (1) using a linker and a surfactant;
≪ Formula 1 >

(Provided that n = 10 to 15 KDa in the above formula)
Wherein step (a) comprises: preparing a silica nanoparticle prepared by a method comprising the steps of:
(a-1) adding a fluorescent substance and a fluorescent substance precursor to a solvent and stirring the mixture;
(a-2) mixing the solution obtained in the step (a-1) with a solution containing a silica precursor and a catalyst, stirring and washing. A method for detecting quantitative determination of vitamin D using the silica nanoparticles of claim 1.

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