KR20150097039A - Silica-silver core-shell nano particle for optical imaging in near-infrared region and its preparation method - Google Patents

Abstract

The present invention relates to a method for producing a silica-core and silver-shell nanoparticle for a near-infrared ray optical image, the nanoparticle produced by the same method, and an in-vivo optical detection method using the same. The method for producing a silica-core and silver-shell nanoparticle includes the step of dispersing silica particles and silver precursors in a diol compound, adding an aliphatic or aromatic amine compound, and making a reaction. With respect to 1 part by weight of the silica particle, 5-50 parts by weight of the silver precursors are mixed. According to the present invention, a nano-marker particle for a near-infrared ray optical image enables a user to check an optical image of subcutaneous tissue in vivo for example; has excellent surface enhanced Raman scattering intensity; has excellent biocompatibility and stability; and thus can be used as a nano-probe for obtaining a multi-optical image in vivo.

Description

Technical Field [0001] The present invention relates to a silica core and silver shell nanoparticles for near-infrared optical images,

The present invention relates to a silica core and silver shell nanoparticles for near infrared optical images for confirming optical images in vivo, a method for producing the same and an in vivo diagnostic method using the same.

Recently, it has been known that a high-sensitivity optical imaging method using metal nanoparticles can be applied to a cell-level image or a biological image, It is widely used in medical diagnostics.

In the case of fluorescent materials conventionally used to obtain optical images, it is possible to detect thousands of photons per molecule under appropriate conditions, so that detection of a single molecule level is theoretically possible. However, it is possible to directly substitute an element of an active ligand like a radioactive isotope There is a limitation in that a fluorescent substance must be connected by modifying a part that does not affect relatively activity through the structural activity relation. In addition, these fluorescent markers have a disadvantage in that their fluorescence intensity is weakened over time, and the wavelength of the excited light is very narrow and the wavelength of the emitted light is very wide and there is interference between different fluorescent materials. The number of fluorescent materials that can be used is extremely limited.

Inorganic quantum dots (QDs), which are semiconductor nanomaterials, are composed of CdSe, CdS, ZnS, ZnSe, and emit light of different colors depending on their size and type. Has a broad excitation wavelength and exhibits a narrow emission wavelength as compared with the organic fluorescent substance, so that the number of branches emitting different distinguishable colors is larger than that of the organic fluorescent substance. Due to these advantages, quantum dots are widely used as a method for overcoming the disadvantages of organic fluorescent materials. However, there is a disadvantage that it is toxic and it is difficult to mass-produce, and the number of branches is theoretically different, but the number actually used is very few, so biomedical application is limited.

As a method for solving these problems, surface enhanced Raman scattering (Raman scattering) with enhanced intensity of vibration signals of a label molecule by gold or silver nanoparticles, which maintains stability over a long period of time and is also excellent in biocompatibility, SERS) method is used. The surface enhancement Raman scattering is very narrow, the bandwidth of the optical signal is as small as 1 nm or less, the number of available Raman markers is very large, and it is advantageous to simultaneously detect multiple markers with one light source. Utilization is increasing.

Korean Patent Publication No. 10-2013-0118086 discloses a silica core particle; And a hollow metal nanoparticle layer surrounding the silica core particle, wherein the nanoparticle layer surrounds the silica core particle, the silver nanoparticle layer surrounds the silica core particle, and the hollow metal nanoparticle layer surrounds the silica core particle, Near-infrared optical images were obtained only when the silver nanoparticles were formed by hollow-silver and silver nanoparticles through a galvanic substitution reaction.

Korean Patent No. 10-0845010 discloses a polymer particle for NIR / MR dual mode molecular imaging. However, since NIR fluorescent material is used to obtain optical images in the near-infrared region, photo-blitting, There are limitations in application to diagnosis.

On the other hand, the shell nanoparticles have been produced through a seed-based seed-mediated growth method as shown in the upper part of FIG. 1 or a multilayer assembly method using a polymer electrolyte as shown in the lower part of FIG. In the above methods, a metal nanoparticle to be used as a seed for applying a silver shell to a center particle is first attached, and then a silver precursor solution is added again in the next step, or metal nanoparticles to be used as a seed are also first attached, In the next step, polymer electrolyte and silver nanoparticles with complementary charge were prepared by stacking the nanoparticles on the core material. All of these methods require multiple reaction steps to produce seed nanoparticles, time consuming due to repeated multi-step reactions, the reaction proceeds at relatively high temperatures, It is difficult to control the thickness of the shell easily and it is difficult to obtain a nanoparticle capable of enhancing SERS strength because nano irregularities are deeply formed on the silver shell surface.

The present invention provides a method for producing silica core and silver shell nanoparticles for near infrared optical images, which can confirm optical images in vivo using wavelengths in the near infrared region, has excellent surface enhanced Raman scattering intensity, and is excellent in biocompatibility and stability .

It is an object of the present invention to provide silica core and silver shell nanoparticles for near infrared optical images prepared by the above method.

An object of the present invention is to provide an in-vivo optical detection method of a substance capable of bonding to silica core and silver shell nanoparticles for near-infrared optical image using Raman scattering in the near-infrared region.

The present invention comprises a step of dispersing silica particles and a silver precursor in a diol compound and then adding an aliphatic or aromatic amine compound and reacting the mixture, wherein 5 to 50 parts by weight, preferably 5 to 50 parts by weight, And 8 to 20 parts by weight of a silver precursor are mixed with the silver nanoparticles.

When the weight of the silver precursor exceeds the upper limit, the thickness of the silver shell increases, the depth of the nano irregularities decreases, the surface enhancement Raman activity in the near infrared region weakens, and when the weight is less than the lower limit, the silica particles are blocked The formation of silver shells is not perfect and silver precursor content is lowered, so that the surface of silica particles is connected to a network having pores on the surface thereof, or silver nanoparticles are scattered sporadically, and the surface enhancement Raman activity in the near- .

The silica particles have a size of 50 to 500 nm, preferably 80 to 300 nm. If the silica particle size is less than the lower limit value, the Raman surface enhancement effect is deteriorated. If the upper limit is exceeded, the silica particle size may be restricted.

The silica particles are preferably prepared from at least one selected from the group consisting of tetraethyl orthosilicate and tetramethyl orthosilicate, but are not limited thereto.

The silica core particles can be prepared by methods commonly used in the art (W. Tober et al., Controlled growth of monodisperse silica spheres in micro size range, J. Colloid Interface Sci., 1968, 26, 62).

It is preferable that the silica particles have a functional group capable of binding to the silver precursor on the surface thereof. The functional group capable of binding to the silver precursor is, for example, a thiol group (-SH), an amine group (-NH ₂ ), a cyanide group (-CN), an isocyanide (-CNO), an isothiocyanide (-CNS) , and the like disulfide (-SS-), or azide group (-N _3), preferably a thiol group.

Wherein the precursor may be a _{AgNO 3, AgBF 4, AgPF 6} , Ag 2 O, CH 3 COOAg, AgCF 3 SO 3, AgClO 4, AgCl, Ag 2 SO 4, and CH ₃ COCH-COCH ₃ Ag silver salt is selected from And preferably AgNO ₃ .

In the present invention, the silica particles and the silver precursor should be dispersed in the diol compound, so that the silver shell on the surface of the silica particle can be easily formed even at a low reaction temperature of the present invention. When the silver particle is dispersed in a solvent such as alcohol or ether, have. The diol compound may be a linear diol compound having 2 to 8 carbon atoms, preferably a symmetrical diol compound, more preferably ethylene glycol.

The aliphatic or aromatic amine compound preferably has a pKa value of 5 or more, more preferably 5 to 15. When the pKa value of the amine compound is less than 5, the basicity of the amine compound is low, so that the reduction of the silver ion is insignificant. On the other hand, when the pKa value is more than 15, silver ion reduction in the bulk is expected. The aliphatic amine compound is preferably an amine compound having 2 to 20 carbon atoms, more preferably 4 to 12 carbon atoms, and is preferably a primary amine, a secondary amine or a tertiary amine. When an aliphatic amine having a carbon number less than the lower limit is used, electron donation is insufficient and there is little reduction effect. In the case of an aliphatic amine having a carbon number exceeding the upper limit, it is difficult to find a suitable solvent because of the difference in solubility with ions. The aliphatic amine may be a primary, secondary or tertiary amine.

The aromatic amine is preferably a heterocyclic amine compound having 5 to 20 carbon atoms. However, in the case of a heterocyclic amine compound having a carbon number exceeding the upper limit value described above, the aforementioned stereoregulatory hindrance increases, which is not preferable.

The method for producing silica core and silver shell nanoparticles for near-infrared optical images of the present invention is capable of forming silver shells even under mild conditions, unlike conventional seed-based multi-step growth methods or multi-layer assembly methods using polymer electrolytes, At a temperature of 5 to 50 DEG C, more preferably 10 to 40 DEG C, most preferably at 15 to 30 DEG C without separate heating. The reaction time is not particularly limited, but is preferably from 1 to 200 minutes, preferably from 10 to 180 minutes, more preferably from 30 to 160 minutes, and most preferably from 60 to 140 minutes. The reaction time depends on the ratio of the silica particles to the silver precursor, and if the reaction time is maintained for a sufficient time or longer, the thickness of the silver shell does not increase proportionally.

The thickness of the silver shell is a length from the interface of the silica particle and the silver shell to the outermost angle of the unevenness formed on the surface, and is in the range of 20 to 60 nm, preferably 30 to 55 nm, more preferably 35 to 45 nm. The surface enhancement Raman activity in the region is maximized.

The average depth of the nano irregularities formed on the silver shell is 1 to 40 nm, preferably 5 to 30 nm, more preferably 10 to 25 nm. When the average depth of the nano irregularities is less than the lower limit value, The surface enhancement Raman activity is very weak. When the upper limit is exceeded, the number of protrusions and protrusions formed in a hemispherical shape on the surface of silica particles may decrease, and the surface enhancement Raman activity in the near infrared region may be weakened.

The surface of the silver shell may be coated with a self-assembled monolayer made of a specific marking material. The labeling material is a material suitable for nano-labeled particles because it shows a Raman spectrum in a clear fingerprint area while having a simple structure.

The labeling substance may be selected from the group consisting of 4-mercaptotoluene (4-MT), 3,5-dimethylbenzenethiol (3,5-DMT), thiophenol (TP), 4-aminothiophenol BT), 4-bromobenzenethiol (4-BBT), 2-bromobenzenethiol (2-BBT), 4-isopropylbenzene thiol Dichlorobenzenethiol (3,4-DCT), 3,5-dichlorobenzenethiol (3,5-DCT), 4-chlorobenzenethiol (4-CBT), 2- chlorobenzenethiol , 2-fluorobenzene thiol (2-FBT), 4-fluorobenzene thiol (4-FBT), 4-methoxybenzenethiol (4-MOBT), 3,4-dimethoxybenzenethiol (2-MBI), 2-mercapto-1-methylimidazole (2-MMI) Amino-4-chlorobenzenethiol (2-ACBT), 3-mercaptoethanol (2-ATFT), benzylmercaptan (BZMT), benzyl disulfide (BZDSF) Benzoic acid (3-MBA), 1-phenyltetrazole-5-thiol (1-PTET), 5-phenyl-1,2,3-triazole- (2) a compound selected from the group consisting of aniline (2-IAN), phenylisothiocyanate (PITC), 4-nitrophenyl disulfide (4-NPDSF), 4-azido-2-bromoacetophenone , But it can be used without limitation as long as it is a chemical substance having high binding force with silver shell.

The chemical substance having a high binding force with the silver shell has a thiol group (-SH), an amine group (-NH ₂ ), a cyanide group (-CN), an isocyanide (-CNO), an isothiocyanide ), Disulfide (-SS-) or azide group (-N ₃ ).

In the silica core and silver shell nanoparticles for near-infrared optical images of the present invention, the silver shell may be surrounded by a shell layer of at least one selected from silica, protein, and polymer. The shell layer may include not only silica but also polymers and proteins. The polymer is not particularly limited as long as it is a hydrophilic polymer. For example, polyvinylpyrrolidone, polyethyleneimine, polyvinyl alcohol and the like can be used. Albumin, straptavidin and the like can be used as the protein. The shell layer may be in the form of a laminate of one or more shells. The shell enhances the stability of the silica core and silver shell nanoparticles, increases solubility in water, prevents nonspecific binding, removes autofluorescence, prevents the control of silica center and silver shell nanoparticles It is possible to inhibit aggregation, to inhibit the leakage of the labeling substance and the adsorption of undesired compounds onto the surface of the silica core and silver shell nanoparticles, and to introduce a ligand or a functional group which binds to a specific substance.

The silica shell may be prepared from a silica precursor, i.e., tetraethylorthosilicate or tetramethylorthosilicate, and preferably has a thickness of 5 to 10 nm. If the thickness of the silica shell is less than 5 nm, the silver shell coated with the labeling material can not be protected from the external environment. If the thickness is more than 10 nm, it may be a limitation in biological applications such as cells and living tissues It is preferable to adjust the thickness to the above range.

The present invention provides silica core and silver shell nanoparticles for near-infrared optical images, which are produced by the above-described method.

The present invention also relates to silica core particles having an average particle size of 50 to 500 nm; And a silver shell having an average thickness of 20 to 60 nm coated on the surface of the silica core particles so as not to be exposed on the surface, wherein an average depth of nano-irregularities formed on the silver shell is 1 to 40 nm. Image silica core and silver shell nanoparticles.

The maximum absorption wavelength of the nanoparticles is 500 to 1000 nm.

The silver shell of the nanoparticles may be coated with a labeling substance.

And may further comprise a shell layer formed of at least one selected from the group consisting of silica, protein and polymer surrounding the silver shell.

The present invention relates to a method for manufacturing a silver halide photographic light-sensitive material, which comprises the steps of injecting silica core and silver shell nanoparticles for infrared optical images into a living tissue of an animal other than human or human; And an in-vivo optical detection method of detecting a substance capable of binding to silica core and silver shell nanoparticles for near-infrared optical images injected into the living tissue using Raman scattering in a near-infrared region.

The wavelength of the near-infrared light source used in the detection method is preferably 650 to 900 nm, more preferably 750 to 850 nm, to obtain an optical image of a living tissue. Preferably, the excitation source for obtaining the wavelength of the near infrared region is a laser (LASER) having a wavelength of 2.5 to 50 mW, preferably 10 to 20 mW. When the wavelength is less than the lower limit, sufficient penetration depth And if it exceeds the upper limit value, it may affect the living tissue and destroy the cell, so that optical detection in vivo is difficult. The detection method is particularly suitable for detection in subcutaneous tissue, wherein the depth of penetration of the subcutaneous tissue to obtain surface enhanced Raman scattering is 1 to 10 mm.

In particular, the detection method of the present invention comprises the steps of: injecting the silica core and silver shell nanoparticles coated with two or more labeling substances, which bind to different substances and release Raman scattering, into living tissue of an animal other than human or human; And simultaneously detecting two or more substances bound to the two or more labeling substances using Raman scattering in the near infrared region.

The silica core and silver shell nanoparticles for near-infrared optical image of the present invention can confirm the optical image of the subcutaneous tissue in vivo using the wavelength in the near-infrared region, have excellent surface enhanced Raman scattering strength, have biocompatibility and stability Can be utilized as nano-probes for obtaining multi-optical images in vivo.

FIG. 1 is a schematic view showing a manufacturing process of silver shell nanoparticles of the conventional core shell type.
FIG. 2 is a schematic view of a process for preparing silica core and silver shell nanoparticles having nano irregularities formed on the silver shell surface according to one embodiment of the present invention.
FIG. 3 is a TEM photograph of nanoparticles prepared by varying the amount of silica particles from 50 mg to 5 mg in order to change the weight ratio of silver precursor to silica particles in Production Example 1. FIG.
FIG. 4A is a TEM photograph of nanoparticles prepared by varying the amount of silica particles from 4 mg to 1 mg in order to change the weight ratio of silver precursor to silica particles in Production Example 1, and on the left is enlarged a photograph on the right side. a) nanoparticles (AgNS-7.5) prepared using 4 mg of silica particles, b) 3 mg nanoparticles (AgNS-15), c) 2 mg nanoparticles (AgNS-22.5) (AgNS-30). The circles indicated by dotted lines inside the nanoparticles represent silica particles of about 150 nm.
FIG. 4B is a SEM photograph of the nanoparticles prepared by varying the amount of silica particles from 4 mg to 1 mg in order to change the weight ratio of silver precursor to silica particles in Production Example 1. FIG. a) nanoparticles (AgNS-7.5) prepared using 4 mg of silica particles, b) 3 mg nanoparticles (AgNS-15), c) 2 mg nanoparticles (AgNS-22.5) (AgNS-30).
5 is a graph showing surface enhanced Raman scattering (SERS) intensity at 1075 cm ^-1 of each nanoparticle when excited with a 785 nm laser source in Experimental Example 1. FIG.
6 is a photograph of elemental mapping of nanoparticles (AgNS-15) prepared by using 3 mg of silica particles in Production Example 1 in Experimental Example 2 using an energy dispersive X-ray spectrometer .
7A is an absorption band graph of nanoparticles prepared according to the weight ratio of silica particles and silver precursor in Experimental Example 3. FIG.
FIG. 7B is a photograph showing the color of the suspension of nanoparticles prepared according to the weight ratio of silica particles and silver precursor in Experimental Example 3. FIG.
FIG. 8A shows the surface enhanced Raman scattering (SERS) intensity at 1075 cm ^-1 of AgNS-7.5, AgNS-15, AgNS-22.5 and AgNS-30 nanoparticles when excited with a 785 nm laser source in Experimental Example 4 Graph.
8B is a graph showing the surface enhanced Raman scattering (SERS) intensity at 1075 cm ^-1 of each nanoparticle when AgNS-15 nanoparticles were excited with a source of 532, 647, and 785 nm in Experimental Example 4. FIG.
FIG. 9 is an image showing a discrete dipole approximation when the AgNS-15 nanoparticles were excited with a laser source of 532, 647, and 785 nm in Experimental Example 4. FIG.
10 is an image showing a discrete dipole approximation when the nano irregularities of the nano-irregularities of the flat nanoparticles and AgNS-15 nanoparticles of Comparative Example 2 were excited with a laser source of 785 nm.
11A is a TEM photograph of nanoparticles prepared by using anhydrous ethanol as a solvent in place of ethylene glycol in Comparative Example 3. FIG.
FIG. 11B is a TEM photograph of nanoparticles prepared by using ethylene glycol monoethyl ether as a solvent instead of ethylene glycol in Comparative Example 3. FIG.
12 is a graph showing the cyclic voltammogram when ethylene glycol and anhydrous ethanol were used as solvents in Comparative Example 3, respectively.
13A is a photograph showing the mapping of the intensity of surface enhanced Raman scattering at 1075 cm ^-1 of the AgNS-15 nanoparticles labeled with 4-fluorobenzenethiol in Experimental Example 5 and the corresponding SEM photographs .
13B is a surface enhanced Raman scattering spectrum of AgNS-15 nanoparticles labeled with 4-fluorobenzene thiol of FIG. 13A. FIG.
FIG. 13c shows a schematic representation of four Raman labeling materials, 4-fluorobenzenethiol (4-FBT), 4-bromobenzenethiol (4-BBT), 4-aminobenzenethiol (4-ABT) 4-CBT) labeled AgNS-15 particles.
14A is a Raman spectrum in liver, spleen, kidney, and heart after 3 days of intravenous administration of the nanoparticles of Preparation Example 2 in Experimental Example 6. FIG.
FIG. 14B is a photograph of H & E staining of liver, spleen, kidney and heart 3 days after intravenous administration of nanoparticles of Preparation Example 2 in Experimental Example 6. FIG.
15A is an image mapping the intensity of surface enhanced Raman scattering activity of a slide on which only A549 cells are fixed in Experimental Example 7. FIG.
FIG. 15B is an image obtained by mixing the A549 cells with the nanoparticles of Preparation Example 2 in Experimental Example 7, and then mapping the surface enhancement Raman scattering activity intensity of the slide in order to confirm intracellular intake of the nanoparticles into A549 cells.
15C is a photograph of an optical fiber sensor coupled to the injection site of the nanoparticles and the Raman probe in Experimental Example 7. FIG.
15D is a graph showing the spectrum of the surface enhanced Raman scattering activity at the nano-particle injection site in Experimental Example 7 with the passage of time.
FIG. 15E is a graph showing the peak intensity at 1075 cm ^-1 of the surface enhanced Raman scattering activity spectrum of the nanoparticle injection site in Experimental Example 7 over time. FIG.

Hereinafter, the present invention will be described in more detail with reference to preferred embodiments. It will be apparent to those skilled in the art, however, that these examples are provided to further illustrate the present invention, and the scope of the present invention is not limited thereto.

Preparation Example 1: Preparation of silica core and silver shell nanoparticles for near-infrared molecular image

First, 27% ammonia water (NH ₄ OH) of 3 ㎖ was added to absolute ethanol for 40 ㎖. Next, 1.6 ml of tetraethylorthosilicate (TEOS) was added to the mixed solution of ammonia water and anhydrous ethanol, and the mixture was stirred at 25 ° C for 20 hours. Centrifugation followed by washing with ethanol 5 times to synthesize silica core particles with an average size of 150 nm or less (J. Colloid Interface Sci., 1968, 26, 62).

After 300 mg of the silica core particles were dispersed in 6 ml of ethanol, 300 μl of 3-mercaptopropyl trimethoxysilane (MPTS) and 60 μl of 27% ammonia water were added thereto, Lt; / RTI > for 12 hours. This was centrifuged and washed several times with ethanol to synthesize silica particles with thiol groups introduced on the surface, that is, silica particles treated with MPTS.

5 mg of polyvinylpyrrolidone was mixed with 25 ml of ethylene glycol, and the MPTS-treated silica nanoparticles were added thereto at various concentrations. On the other hand, MPTS-treated silica nanoparticle-containing ethylene glycol solution prepared in the above various concentrations was added to 25 ml of ethylene glycol in which AgNO ₃ was dissolved and sufficiently mixed so that the final concentration of AgNO ₃ was 3.5 mM. To this mixed solution, 41.37 μl of octylamine was added thereto so as to have a concentration of 5 mM. The mixture was stirred at 25 ° C for 1 hour, and then centrifuged and washed with ethanol to remove the silica core having nano- Bumpy silver nanoshell particles were obtained.

FIG. 2 shows a schematic view of a process for producing the center of silica and the silver shell nanoparticles having the nano irregularities formed on the silver shell surface.

Table 1 shows the ratio of silica particles and silver precursor particles of the silica core and silver shell nanoparticles prepared by varying the concentration of the silica particles, and TEM photographs of the nanoparticles thus prepared are shown in FIGS. 3 and 4A Respectively.

(AgNS-22.5) using 2 mg of nanoparticles (AgNS-7.5) prepared using 4 mg of silica particles, b) 3 mg of nanoparticles (AgNS-15) , d) is 1 mg of nanoparticles (AgNS-30), and SEM photographs of the respective nanoparticles are shown in FIG. 4B.

Silver spheres / silica Silver sphere content Silica particle content The thickness of the silver shell 0.6 30 mg 50 mg - 1.2 30 mg 25 mg - 1.5 30 mg 20 mg - 2 30 mg 15 mg - 3 30 mg 10 mg - 6 30 mg 5 mg 25 nm 7.5 30 mg 4 mg 32 nm 10 30 mg 3 mg 39 nm 15 30 mg 2 mg 51 nm 30 30 mg 1 mg 76 nm

When the weight ratio of the silver precursor to the silica particles is less than 2, the silver nanoparticles are dispersed on the surface of the silica core particle or are aggregated by several, but when the weight ratio of the silver precursor to the silica particle is 3, The silver nanoparticles were connected to each other to form a network, and it was confirmed that the silver shell was formed from the silver precursor at a weight ratio of 6 or more to the silica particles. Also, it was found that nano irregularities having a depth of 20-30 nm were formed on the surface of the silver shell from FIGS. 4A to 4A.

Comparative Example 1: Preparation of nanoparticles in which hollow gold and silver nanoparticle layers were formed on silica core particles

1 mg / ml of nanoparticles prepared by using the silver precursor content of 30 mg and the silica particle content of 50 mg and nanoparticles in which silver nanoparticles were dispersed on the surface of the silica core particles were dispersed in an aqueous 8 wt% polyvinylpyrrolidone solution 5 And 0.1 mM HAuCl ₄ aqueous solution was injected into the dispersion solution at a rate of 0.75 ml / min using a micropump. Since the size of a hollow formed in the other hollow gold and silver nano-particles according to the amount of the aqueous solution of HAuCl ₄ was prepared in the addition amount of the aqueous solution of HAuCl ₄ with 1.5 ㎖.

After the HAuCl ₄ aqueous solution was injected, the mixture was stirred at 100 ° C for 10 minutes to stabilize the gold and silver nanoparticles, and then cooled to room temperature while stirring. Silver nanoparticles coated on the surface of the silica core particles are dark brown, but when HAuCl ₄ aqueous solution is added, the suspension becomes gray.

After washing the suspension with NaCl saturated solution to remove AgCl from the reaction mixture, the suspension was centrifuged and washed three times with distilled water and twice with ethanol to remove silver nanoparticles on the surface of the silica core particle through a hollow- And silver nanoparticles were prepared.

Experimental Example 1: Comparison of surface enhancement Raman scattering intensity in a near-infrared laser source

(AgNS) prepared using the silica particle contents of 50 mg, 10 mg and 3 mg respectively in Production Example 1, 1 mg of the hollow gold and silver nanoparticle-coated nanoparticles (Au / Ag HSA) of Comparative Example 1 , 1 ml of ethanol with a concentration of 4-fluorobenzene thiol of 2 mM Raman labeling substance was added and the mixture solution was stirred at 25 캜 for 1 hour to prepare a nanoparticle having a self-assembled layer of Raman labeling substance .

1 mg of each of the nanoparticles was added to 50 ml of ethanol and dispersed. When the nanoparticles were excited by a laser source at 785 nm, the surface enhanced Raman scattering (SERS) intensity of each nanoparticle was confirmed. For this purpose, the concentration of 4-fluorobenzenethiol was kept constant and the intensity of the spectrum was normalized to that of ethanol (882 cm ^-1 ).

5 is a graph showing the surface enhanced Raman scattering (SERS) intensity at 1075 cm ^-1 of each nanoparticle when excited with a 785 nm laser source.

Nanoparticles prepared using 50 mg and 10 mg of silica particles of Preparation Example 1 when excited with a near infrared ray laser source of 785 nm exhibited significantly lower surface enhancement than silica particles coated with the hollow gold and silver nanoparticles of Comparative Example 1 Raman scattering intensity.

However, when the true particle size of the silica particles was reduced to 3 mg, the galvanic substitution reaction for forming a separate hollow was not performed. However, as the silver shell having the nano irregularities around the silica particles was formed thick, the surface enhanced Raman scattering strength was lowered to the hollow silver and silver nanoparticle layers Was significantly increased to the level equivalent to that of the nanoparticles formed.

Experimental Example 2: Elemental analysis of silica core and silver shell nanoparticles

The nanoparticles (AgNS-15) prepared by using 3 mg of the silica particles of FIG. 4 (b) (b) showed that about 400 nanoparticles were found to be less than 1% in which the shells were not completely formed. This is shown in FIG. 6 by performing elemental mapping using energy dispersive X-ray spectroscopy.

According to the element mapping, only silver atoms were observed, and the central silicon atoms were not observed, and it was found that silver shells were formed thickly on the silica surface of the nanoparticles without voids.

Experimental Example 3: Absorption band of nanoparticles by weight ratio of silica particles and silver precursor

The addition amounts of silica particles in Preparation Example 1 were 4, 3, 2, and 1 mg, respectively, and the absorption bands and the maximum absorption wavelengths of silver core and silver shell nanoparticles formed with silver shells were observed, .

FIG. 7A is a graph showing the absorption bands of the nanoparticles, showing broad absorption bands from visible light to near-infrared region of 560 to 1000 nm in the silver-coated nanoparticles. Expansion of the plasmon absorption band was expected to be due to the nano-irregular structure of the silver shell.

FIG. 7B is a photograph showing the color of the particle suspension. As the thickness of the silver shell increases, the color changes from blue to brown.

Experimental Example 4: Surface enhancement Raman scattering activity of the near-infrared region of nanoparticles according to the weight ratio of silica particles and silver precursor

The surface enhancement Raman scattering activity in the near infrared region of silica core and silver shell nanoparticles having different silver shell thicknesses prepared by adding 4, 3, 2 and 1 mg of silica particles in Production Example 1 were compared.

As in Experimental Example 1, 1 ml of ethanol dissolved with 2 mM of 4-fluorobenzenethiol in 1 mg of nanoparticles was added, and the mixture solution was stirred at 25 DEG C for 1 hour to form a self-assembled layer of Raman labeling substance Nanoparticles were prepared.

FIG. 8A is a graph showing the surface enhanced Raman scattering (SERS) intensity at 1075 cm ^-1 of each nanoparticle when excited with a 785 nm laser source. The AgNS-15 nanoparticles having a silver shell thickness of 39 nm And exhibited the strongest surface enhanced Raman scattering activity.

The surface enhanced Raman scattering activity when the AgNS-15 nanoparticles were excited at 532, 647 and 785 nm, respectively, in order to confirm the relationship between excitation wavelength and surface enhanced Raman scattering activity is shown in FIG. Although the surface enhanced Raman scattering activity of AgNS-15 was expected to exhibit a broad absorption band at all wavelengths, the strongest surface enhanced Raman scattering activity appeared at 785 nm near-infrared excitation source, 1.7 times higher.

To confirm the relationship between the optical characteristics of the broad absorption band of the surface enhanced Raman scattering activity and the wavelengths from the visible light region to the near infrared region, discrete dipole approximation (excitation) of excitation with 532, 647 and 785 nm laser sources, respectively, The dipole approximation is used to compare the enhancement of the electric field and is shown in Fig.

The discrete dipole approximation was enhanced near the surface of the AgNS-15 nanoparticles at all wavelengths and was further enhanced as it moved from the visible to the near infrared region. Especially, when excited by a 785 nm laser source, a highly localized electric field was observed at the tip of the nano irregularities of the AgNS-15 nanoparticles, which was 2.3 times higher than the visible light region of 532 nm. As a result, it was confirmed that the AgNS-15 nanoparticles have the strongest surface-enhanced Raman scattering activity in the near-infrared excitation laser source.

Comparative Example 2: Synthesis of silica core and silver shell nanoparticles prepared using seed particles

The silica seeds and the silver shell nanoparticles were prepared using conventional seed particles, which were not the method and conditions of Production Example 1 of the present invention (N. J. Halas, J. Phys. Chem. B2001,105, 2743-2746)

The silica nanoparticles having the surface-bound gold nanoparticles of 1 to 3 nm were mixed with 0.15 mM silver nitrate solution and vigorously stirred. 50 [mu] l of 37% formaldehyde was added to the surface of the gold nanoparticles used as the seed particle so that the silver was reduced, and 50 [mu] l of twice distilled concentrated ammonia water was added. The aqueous ammonia rapidly raised the pH of the solution to induce reduction of silver ions and formation of silver shells, thereby preparing silica core and silver shell nanoparticles.

The silica core and silver shell nanoparticles prepared by using the seed particles were flat and free of nano irregularities on the surface. The silica core and silver shell nanoparticles prepared by using the seed particles were prepared by using 3 mg of the silica particles of Preparation Example 1, 10 shows a comparison of the electric field enhancement using a discrete dipole approximation when excitation is performed.

10 (a) is the AgNS-15 nanoparticles of Preparation Example 1, and b) is the nanoparticles prepared using the seed particles of Comparative Example 2. The nanoparticles are almost similar in size to 220 to 250 nm, 3 times more than the nanoparticles of Preparation Example 1.

Comparative Example 3: Center of silica and silver shell nanoparticles prepared by different dispersion solvents

The preparation of nanoparticles was carried out in the same manner as in the preparation of the silica core and silver shell nanoparticles by using 3 mg of the silica particles in Production Example 1 except that anhydrous ethanol and ethylene glycol monoethyl ether were used instead of ethylene glycol as a symmetric diol compound To prepare nanoparticles.

11A is a TEM photograph of nanoparticles prepared by using anhydrous ethanol as a solvent in place of ethylene glycol, and FIG. 11B is a TEM photograph of nanoparticles prepared by using ethylene glycol monoethyl ether as a solvent.

When used as anhydrous ethanol or ethylene glycol monoethyl ether instead of a symmetric diol compound, it was confirmed that no silver shell was formed on the surface of silica particles.

10 mM AgNO, Au working electrode _3, Pt counter electrode, Ag / Ag / Cl reference electrode into, and scan from -1.7 to 1.3V, respectively, the scanning speed glycol and absolute ethanol as solvent in 50 mVs ^-1 When used, the reduction potential of AgNO ₃ is compared with a cyclic voltammogram and is shown in FIG.

When ethylene glycol was used as a solvent, a complex was formed between the silver ion and the symmetric diol compound to increase the reduction potential of the silver ion, and the silver ion showed a rapid reduction rate even at room temperature. However, silver ion and ethanol form a complex The reduction rate of the difficult ion was relatively slow. This is because the silver ion (Ag ⁺ ) is dispersed in the symmetric diol compound to receive one electron from the amine compound and is easily reduced to form silver nuclei (Ag ⁰ ) near the silica surface, . ≪ / RTI >

Experimental Example 5: Surface enhanced Raman scattering activity of nanoparticles in a near-infrared laser source

In order to confirm the surface enhancement Raman scattering activity of nanoparticles, single particle surface enhanced Raman scattering experiments were carried out by surface enhancement Raman mapping and SEM photo correlation method.

The AgNS-15 nanoparticles of Preparation Example 1 were suspended in ethanol at a concentration of 0.1 mg / ml, dropped onto a patterned slide glass, and the surface enhanced Raman scattering intensity was measured with a power of 2.3 mW at 785 nm for 2 seconds in 0.5 탆 increments Respectively.

13A is a photograph showing the mapping of the intensity of surface enhanced Raman scattering of AgNS-15 nanoparticles labeled with 4-fluorobenzene thiol and the corresponding SEM photographs. The surface enhanced Raman scattering signal of the nanoparticles was clearly detected with a high signal detection ratio against noise, and the enhancement factor was about 3.8 ㅧ 10 ⁵ to 7.7 ㅧ 10 ^5, and the ratio of detectable nano-labeled particles was more than 80%.

The surface enhanced Raman scattering spectrum of the AgNS-15 nanoparticles labeled with the 4-fluorobenzenethiol is shown in FIG. 13B. From the above results, it was confirmed that the AgNS-15 nanoparticles labeled with 4-fluorobenzenethiol had a sufficient strength to be detected in the near-infrared region, that is, an enhancement factor of about 6.4 ㅧ 10 ^{5 on} average. These high enhancers were estimated to be due to the nano irregularities on the surface of the AgNS-15 nanoparticles, as can be seen in Comparative Example 2.

FIG. 13C shows the surface enhanced Raman scattering spectra of the AgNS-15 nanoparticles prepared using four different Raman labeling materials. It was confirmed that each of the AgNS-15 nanoparticles exhibits unique spectra without overlapping each other, Can be used as easy nano-labeled particles.

Production Example 2: PEG-coated silica center and silver shell nanoparticles

To 1 mg of AgNS-15 nanoparticles, 2 ml of 4-fluorobenzenethiol, 1 ml of ethanol was added. The mixture was stirred at 25 캜 for 1 hour, and then centrifuged and washed with ethanol to obtain a Raman labeling substance Nanoparticles with self-assembled layers were prepared.

1 mg of the nanoparticles in which the self-assembling layer of the Raman labeling substance was formed was added to 1 ml of ethanol containing 2 mM of methoxypoly (ethylene glycol) sulfhydryl (m-PEG-SH, molecular weight 5000) After stirring at 25 ° C for 1 hour, PEG-coated silica center and silver shell nanoparticles were prepared by centrifugation and washing with distilled water.

Experimental Example 6: In-vivo measurement of near-infrared optical image nanoparticles ( in vivo ) Toxicity

The in vivo toxicity of the PEG-coated silica center and silver shell nanoparticles of Preparation Example 2 was confirmed.

400 μl (0.5 nM) of the PEG-coated nanoparticles of Preparation Example 2 was dispersed in PBS buffer at pH 7.0 containing 3% BSA and administered at a dose of 50 mg / kg via the rat's tail vein . As a control group, 400 μl of PBS buffer solution was intravenously injected. The rats were sacrificed 3 days later, and blood was collected from the heart, and surface enhanced Raman scattering spectra were observed in liver, spleen, kidney and heart. 14A shows Raman spectra in each major organ. It was confirmed that the nanoparticles were mainly accumulated in the liver and spleen.

In addition, leukocyte, hemoglobin and platelet content were measured in blood, and the content of enzyme capable of confirming liver activity was shown in Table 2 and shown in Table 3.

division Control group Nanoparticle-administered group Leukocytes (counts / μl) 4600 ± 1800 5600 ± 1900 Hemoglobin (g / dl) 12.0 ± 1.2 10.9 ± 0.9 Platelets (number / l) 675700 ± 236400 564300 ± 201800

division Control group Nanoparticle-administered group Alkaline phosphatase (U / L) 61.7 ± 14.2 81.7 ± 5.9 Alanine aminotransferase (U / L) 46.7 ± 1.5 49.3 ± 2.4 Total bilirubin (mg / ㎗) 0.3 ± 0.0 0.3 ± 0.0 Albumin (g / ㎗) 3.1 ± 0.06 2.7 ± 0.15 Cholesterol (mg / ㎗) 97.0 ± 11.5 96.3 ± 11.6 Gamma glutamyltransferase (U / L) <5 <5

Hemoglobin, hemoglobin and platelets in the blood were not significantly different between the control group and the nanoparticle-treated group of Preparation Example 2, and no difference was found in various enzymes showing liver activity.

In Fig. 14 (b), the tissue damage, inflammation, and morphological changes of each organ were examined. From the above results, it was found that the nanoparticles of Preparation Example 2 showed no in vivo toxicity up to the test concentration.

Experimental Example 7: In-vivo optical imaging and confirmation of multiple detectability

First, it was confirmed whether the nanoparticles of Production Example 2 could be internalized into cells.

6 ⁵ 10 ⁵ A549 cells (adenocarcinomic human alveolar basal epithelial cells) were cultured on a Lab-tek chamber slide for 24 hours and mixed with the nanoparticle-containing solution of Preparation Example 2 for 12 hours. The slides were washed several times with PBS solution and the cells were fixed with 4% paraformaldehyde at 37 &lt; 0 &gt; C for 10 min. The slides were washed again, dried, and subjected to Raman spectral intensity mapping at 785 nm light-excitation with a 31 mW laser voltage and 1 second light acquisition time. As a control, a slide in which the mixing of the nanoparticles of Production Example 2 was omitted was used.

FIG. 15A shows only the A549 cells immobilized as a control group. FIG. 15B shows that the A549 cells were internally blended with the nanoparticles of Preparation Example 2. As a result, the nanoparticles of Preparation Example 2 showed intracellular uptake of A549 cells It was confirmed that it was internalized through.

In this experiment, A549 cells containing the nanoparticles of Preparation Example 2 were diluted with 100 [mu] l PBS and subcutaneously injected into the nude mouse hip muscle area at 8 weeks of age. 15C shows a photograph of an optical fiber sensor coupled to the injection site of the nanoparticle and the Raman probe

In addition, the surface enhanced Raman scattering activity spectrum of the nanoparticle injection site with the optical fiber sensor coupled to the portable Raman probe was performed at 785 nm light-excitation and light acquisition time of 30 mW with a 90 mW laser voltage, Respectively. The peak intensity at 1075 cm &lt; ^-1 &gt; is shown in Fig. 15E.

The Raman spectra could be confirmed immediately after the injection as well as after 2 days, but the peak intensity at 1075 cm ^-1 decreased from the third day.

Claims (24)

Silica particles and a silver precursor are dispersed in a diol compound, and then an aliphatic or aromatic amine compound is added and reacted,
Wherein the silver precursor is mixed with 5 to 50 parts by weight of silver particles per 1 part by weight of the silica particles.
The method according to claim 1, wherein 8 to 20 parts by weight of silver precursor is mixed with 1 part by weight of the silica particles.
The method according to claim 1, wherein the average particle size of the silica particles is 50 to 500 nm.
The method according to claim 1, wherein the silica particles have a functional group capable of binding to a silver precursor introduced into the surface thereof.
2. The method of claim 1, wherein the precursor is _{AgNO 3, AgBF 4, AgPF 6} , Ag 2 O, CH 3 COOAg, AgCF 3 SO 3, AgClO 4, AgCl, Ag 2 SO 4, and CH ₃ COCH-COCH ₃ Ag &Lt; / RTI &gt; wherein the silver halide is selected from the group consisting of silver nitrate and silver nitrate.
The method according to claim 1, wherein the diol compound is a linear diol compound having 2 to 8 carbon atoms.
7. The method according to claim 6, wherein the diol compound is a symmetric diol compound.
The method according to claim 1, wherein the aliphatic amine compound is an aliphatic amine compound having 4 to 12 carbon atoms.
The method according to claim 1, wherein the reaction is carried out at 5 to 50 ° C. 2. The method for producing silica core and silver shell nanoparticles for near-infrared optical images according to claim 1,
The method of claim 1, wherein the reaction is performed for 1 to 200 minutes.
The method according to claim 1, wherein the thickness of the silver shell is 20 to 60 nm.
12. The method according to claim 11, wherein the average depth of the nano irregularities formed on the silver shell of the silver shell is 1 to 40 nm.
The method of claim 1, further comprising immobilizing a labeling substance on the surface of the silver shell.
14. The method of claim 13, further comprising the step of forming at least one shell layer selected from the group consisting of silica, protein, and polymer surrounding the silver shell, wherein the silver core and silver shell nanoparticles for near- Gt;
A silica core and silver shell nanoparticle for near-infrared optical images, which is produced by the method of any one of claims 1 to 14.
Silica core particles having an average particle size of 50 to 500 nm; And a silver shell having an average thickness of 20 to 60 nm coated on the surface of the silica core particles so as not to be exposed on the surface, wherein an average depth of nano-irregularities formed on the silver shell is 1 to 40 nm. Image centered silica and silver shell nanoparticles.
17. The silica core and silver shell nanoparticles according to claim 16, wherein the maximum absorption wavelength of the nanoparticles is 500 to 1000 nm.
17. The silica core and silver shell nanoparticles according to claim 16, wherein the silver shell of the nanoparticles is coated with a labeling substance.
19. The method of claim 18, wherein the labeling material is selected from the group consisting of 4-mercaptotoluene (4-MT), 3,5-dimethylbenzenethiol (3,5-DMT), thiophenol (TP) ATP), benzene thiol (BT), 4-bromobenzene thiol (4-BBT), 2-bromobenzene thiol (2-BBT), 4- isopropylbenzene thiol 2-NT), 3,4-dichlorobenzenethiol (3,4-DCT), 3,5-dichlorobenzenethiol (3,5-DCT), 4-chlorobenzenethiol (2-CBT), 2-fluorobenzene thiol (2-FBT), 4-fluorobenzene thiol (4-FBT), 4-methoxybenzenethiol (4-MOBT), 3,4-dimethoxybenzene 2-mercapto-1-methylimidazole (2-MMI), 2-mercapto-5-methylbenzimidazole (2-mercapto- Amino-4-chlorobenzenethiol (2-ACBT), 2-amino-4- (trifluoromethyl) benzenethiol (2-ATFT), benzylmercaptan (BZMT), benzyl disulfide ), 3-mercaptobenzoic acid (3-MBA), 1-phenyltetrazole-5-thiol (1-PTET), 5-phenyl-1,2,3-triazole- (5-PTRT), 2-iodoaniline (2-IAN), phenylisothiocyanate (PITC), 4-nitrophenyldisulfide (4-NPDSF) and 4-azido-2-bromoacetophenone ABAPN). &Lt; / RTI &gt;&lt; Desc / Clms Page number 19 &gt;
17. The silica core and silver shell nanoparticles according to claim 16, further comprising a shell layer selected from the group consisting of silica, protein and polymer surrounding the silver shell.
A step of injecting silica core and silver shell nanoparticles for near-infrared optical image selected from claims 16 to 20 into a living tissue of an animal other than a human; And
And detecting a substance capable of binding to silica core and silver shell nanoparticles for near-infrared optical images injected into the living tissue using Raman scattering in a near-infrared region.
22. The in-vivo optical detection method according to claim 21, wherein the wavelength of the near infrared region is in the range of 650 to 900 nm.
23. The in-vivo optical detection method according to claim 22, wherein a laser (LASER) of 2.5 to 50 mW is used as an excitation source of the wavelength of the near-infrared region.
24. The in-vivo optical detection method according to claim 23, wherein the living tissue is a subcutaneous tissue.

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