Fluorescence-enhancing nanoparticle comprising fluorescent dye and core-shell type metal nanoparticle

Abstract

To provide fluorescence-enhancing nanoparticles that do not reduce the fluorescence intensity of fluorescent dyes, but rather further enhance the fluorescence intensity.SOLUTION: Provided are fluorescence-enhancing nanoparticles comprising core-shell nanoparticles formed of a core particle and a shell, at least one fluorescent dye, and a spacer located between the core-shell nanoparticles and the fluorescent dye, the fluorescent dye having an excitation wavelength and a fluorescence wavelength, the core-shell nanoparticles having a maximum wavelength of localized surface plasmon resonance, the core-shell nanoparticles being metal nanoparticles composed of a combination of a silver core particle and a gold shell or a combination of a gold core particle and a silver shell, and the spacer being single-stranded DNA.SELECTED DRAWING: Figure 1

Description

[Technical Field]

[0001] The present invention relates to fluorescent dyes and fluorescence-enhancing nanoparticles comprising silver-core-gold-shell nanoparticles or gold-core-silver-shell nanoparticles. [Background technology]

[0002] Colorants in which fluorescent dyes are bound to metal nanoparticles are useful for bioimaging applications due to their color intensity, stability, and biocompatibility, and various combinations of metal particles and fluorescent dyes have been attempted. The emission intensity of colorants in which fluorescent dyes are bound to metal nanoparticles is enhanced or quenched by the localized surface plasmon resonance (LSPR) of the metal nanoparticles. It is known that the LSPR varies depending on the fluorescence wavelength of the fluorescent dye, the distance between the fluorescent dye and the metal nanoparticle, the size and shape of the metal nanoparticle, and the metal species of the metal nanoparticle. Patent Document 1 discloses labeled silica-coated gold nanorods, in which gold nanorods are coated with a silica layer and fluorescent substances are bound to the silica layer via functional groups (spacers). It is disclosed that by making the thickness of the silica layer at least a certain value, the distance between the fluorescent substance and the gold nanorod surface is maintained, improving the quenching effect of the gold nanorod on fluorescence intensity.

[0003] Patent Document 2 discloses a fluorescent nanostructure having a plasmonic nanostructure with a localized surface plasmon resonance wavelength, a spacer layer, and a fluorophore for enhancing the detection performance of biological assays. The structure's localized surface plasmon resonance wavelength is adjusted to match the maximum excitation wavelength of the fluorophore, resulting in enhanced fluorescence intensity greater than that of the fluorophore alone. Furthermore, the surface of the plasmonic nanostructure is covered with a spacer layer of a certain thickness, and sufficient distance is maintained between the plasmonic nanostructure and the fluorophore, thereby reducing or preventing quenching.

[0004] Non-Patent Document 1 discloses the metal-enhanced fluorescence effect of nanoparticles with gold core nanoparticles and silver shells of various thicknesses, using an avidin-biotin system as a spacer to bind fluorescent dyes to the nanoparticle surface. Although the avidin-biotin system functions as an inert spacer, providing sufficient distance from the metal surface to obtain strong fluorescence enhancement from the plasmonic field, fluorescence quenching is observed in silver-shell gold core nanoparticles.

[0005] These conventional techniques are limited to suppressing the quenching effect of fluorescence intensity by adjusting the distance between the core particle and the fluorescent substance, and are insufficient in terms of enhancing fluorescence by utilizing the localized surface plasmon resonance of the nanoparticles. Therefore, there is a need for the development of metal nanoparticles that can further enhance the fluorescence intensity of the fluorescent dyes used in measurements. [Prior art documents] [Patent documents]

[0006] [Patent Document 1] Japanese Patent Publication No. 2021-110734 [Patent Document 2] Special Publication No. 2022-512606 [Non-patent literature]

[0007] [Non-Patent Document 1] J.Phys.Chem.C, (US), 2018, Vol.122, p.28431-28438 Summary of the Invention [Problem to be solved by the invention]

[0008] It is desirable to design fluorescence-enhancing nanoparticles that do not reduce the fluorescence intensity of fluorescent dyes and can even enhance the fluorescence intensity. [Means for solving the problem]

[0009] In order to solve the above problems, the inventors have invented fluorescence-enhancing nanoparticles that do not reduce (quench) the fluorescence intensity of fluorescent substances, and that enhance the light-collecting effect of the localized surface plasmon resonance of metal nanoparticles, by appropriately selecting the distance between the fluorescent dye and the surface of the metal nanoparticles and controlling the maximum wavelength of the localized surface plasmon resonance of the metal nanoparticles, thereby enhancing the fluorescence intensity. Thus, one embodiment of the present invention includes the following.

[0010] [1] A fluorescence-enhancing nanoparticle comprising a core-shell nanoparticle consisting of a core particle and a shell, at least one fluorescent dye, and a spacer located between the core-shell nanoparticle and the fluorescent dye, the fluorescent dye has an excitation wavelength and a fluorescence wavelength; the core-shell nanoparticles have a maximum wavelength of localized surface plasmon resonance; the core-shell nanoparticles are metal nanoparticles consisting of a combination of a silver core particle and a gold shell, or a combination of a gold core particle and a silver shell; The spacer is a single-stranded DNA. Fluorescence-enhancing nanoparticles characterized by: [2] The fluorescence-enhancing nanoparticles according to [1], wherein the shape of the core particle of the core-shell nanoparticle is a nanocube, a nanoprism, a nanorod, a nanobipyramid, or a nanodecahedron. [3] The fluorescence-enhancing nanoparticles according to [1], wherein the core particles have an average diameter of 10 to 100 nm, and the shell portion has an average thickness of 1 to 10 nm. [4] The fluorescence-enhancing nanoparticles according to [1], wherein the fluorescent dye is a fluorescein dye, a rhodamine dye, or a cyanine dye. [5] The fluorescence-enhancing nanoparticle according to [1], wherein the distance from the surface of the shell of the core-shell nanoparticle to the fluorescent dye is 1 to 20 nm. [6] The fluorescence-enhancing nanoparticle according to [1], wherein the maximum wavelength of the localized surface plasmon resonance of the core-shell nanoparticle is between the excitation wavelength and the fluorescence wavelength of the fluorescent dye. [7] The fluorescence-enhancing nanoparticle according to [1], wherein the length of the spacer is 10 to 100 nm. [8] The fluorescence-enhancing nanoparticle according to [1], characterized in that the spacer is bound to the surface of the core particle of the core-shell nanoparticle. [9] A fluorescence-enhancing probe comprising the fluorescence-enhancing nanoparticle according to any one of [1] to [8] and having the function of specifically binding to a target substance. [Brief explanation of the drawings]

[0011] [Figure 1] FIG. 1 is a TEM image of gold-core silver-shell nanoparticles (core particle diameter 40 nm, cubic) of the present invention. [Figure 2] FIG. 2 is a TEM image of gold-core silver-shell nanoparticles (core particle diameter 40 nm, cubic) (AuNC / HEX) of the present invention to which 5′-end HEX-modified single-stranded DNA is bound. [Figure 3] Figure 3 is a graph showing the fluorescence properties of gold-core silver-shell nanoparticles (40 nm core particle, cubic) of the present invention to which 5'-end HEX-modified single-stranded DNA is bound. The left panel is a graph showing the maximum wavelength, and the extinction spectrum of the gold-core silver-shell nanoparticles and the wavelength dependence of the excitation (ex) and fluorescence (em) of the fluorescent dye, respectively. The right panel is a graph showing the relative fluorescence units (RFU) of the free [fluorescent dye-spacer] and the [fluorescent dye-spacer-gold core silver-shell nanoparticles] of the present invention in which a silver shell is formed at various AgNO3 concentrations. The AgNO3 concentration of 0 mM represents the RFU of the [fluorescent dye-spacer-gold nanoparticles] in which no silver shell is formed. [Figure 4]Figure 4 is a graph showing the fluorescence properties of gold-core silver-shell nanoparticles (core particle diameter 20 nm, cubic) of the present invention to which 5'-end HEX-modified single-stranded DNA is bound. The left panel is a graph showing the maximum wavelength, and the extinction spectrum of the gold-core silver-shell nanoparticles and the wavelength dependence of the excitation (ex) and fluorescence (em) of the fluorescent dye, respectively. The right panel is a graph showing the relative fluorescence units (RFU) of the free [fluorescent dye-spacer] and the [fluorescent dye-spacer-gold core silver-shell nanoparticles] of the present invention in which a silver shell is formed at various AgNO3 concentrations. The AgNO3 concentration of 0 mM indicates the RFU of the [fluorescent dye-spacer-gold nanoparticles] in which no silver shell is formed. [Figure 5] Figure 5 is a graph showing the fluorescence properties of gold-core silver-shell nanoparticles (core particle diameter 60 nm, cubic) of the present invention to which single-stranded DNA modified with Texas-Red at the 5' end is bound. The left panel is a graph showing the maximum wavelength, and the extinction spectrum of the gold-core silver-shell nanoparticles and the wavelength dependence of the excitation (ex) and fluorescence (em) of the fluorescent dye, respectively. The right panel is a graph showing the relative fluorescence units (RFU) of the free [fluorescent dye-spacer] and the [fluorescent dye-spacer-gold core silver-shell nanoparticles] of the present invention in which a silver shell is formed at various AgNO3 concentrations. The AgNO3 concentration of 0 mM represents the RFU of the [fluorescent dye-spacer-gold nanoparticles] in which a silver shell is not formed. [Figure 6] Figure 6 is a graph showing the fluorescence properties of gold-core silver-shell nanoparticles (core particle diameter 40 nm, cubic) of the present invention to which 5'-end FITC-modified single-stranded DNA is bound. The left panel is a graph showing the maximum wavelength, and the extinction spectrum of the gold-core silver-shell nanoparticles and the wavelength dependence of the excitation (ex) and fluorescence (em) of the fluorescent dye, respectively. The right panel is a graph showing the relative fluorescence units (RFU) of the free [fluorescent dye-spacer] and the [fluorescent dye-spacer-gold core silver-shell nanoparticles] of the present invention in which a silver shell is formed at various AgNO3 concentrations. The AgNO3 concentration of 0 mM indicates the RFU of the [fluorescent dye-spacer-gold nanoparticles] in which no silver shell is formed. [Figure 7]Figure 7 is a graph showing the fluorescence properties of gold-core silver-shell nanoparticles (core particle diameter 40 nm, spherical) to which 5'-end HEX-modified single-stranded DNA is bound. The left panel is a graph showing the maximum wavelength, and the extinction spectrum of the gold-core silver-shell nanoparticles and the wavelength dependence of the excitation (ex) and fluorescence (em) of the fluorescent dye, respectively. The right panel is a graph showing the relative fluorescence units (RFU) of the free [fluorescent dye-spacer] and the [fluorescent dye-spacer-gold core silver-shell nanoparticles] of the present invention in which a silver shell is formed at various AgNO3 concentrations. The AgNO3 concentration of 0 mM represents the RFU of the [fluorescent dye-spacer-gold nanoparticles] in which a silver shell is not formed. DETAILED DESCRIPTION OF THE INVENTION

[0012] As used herein, the term "fluorescence-enhanced nanoparticles" refers to nanoparticles in which at least one fluorescent dye is bound to a core-shell nanoparticle via a spacer, and in which the fluorescence is enhanced compared to that of the fluorescent dye alone. "Enhanced fluorescence" refers to the fact that the fluorescence intensity per particle is enhanced compared to that of the fluorescent dye alone. The fluorescence intensity per particle may be enhanced by binding multiple fluorescent dyes to a single core-shell nanoparticle, or the fluorescence intensity per fluorescent dye may be enhanced by localized surface plasmon resonance of the core-shell nanoparticle. Preferably, the fluorescence-enhanced nanoparticles have the fluorescence intensity per particle enhanced by binding multiple fluorescent dyes to a single core-shell nanoparticle, and the fluorescence intensity per fluorescent dye is enhanced by localized surface plasmon resonance of the core-shell nanoparticle. As used herein, the term "core-shell nanoparticles" refers to nanoparticles consisting of a core particle and a shell, and having a core-shell structure in which the surface of a core particle is coated with a shell made of another material. As used herein, the term "core particle" refers to a nano-sized metal core particle that serves as a template particle for core-shell nanoparticles. In this specification, the term "shell" refers to a layer made of a metal different from the metal of the core particle that covers the surface of the core particle in core-shell nanoparticles. In this specification, the "particle diameter" refers to the average particle diameter of 50 particles observed with a transmission electron microscope (TEM), and the "shell thickness" refers to the value obtained by subtracting the diameter of the core particle from the average particle diameter of 50 core-shell particles observed with a transmission electron microscope (TEM). As used herein, "seed particles" refer to particles with a diameter of approximately 10 nm that serve as starting points for growing gold nanocubes. In one embodiment of the present invention, these particles are synthesized from gold nanoclusters. In one embodiment of the present invention, gold nanoclusters or silver nanoclusters are used. As used herein, the "maximum wavelength" of a core-shell nanoparticle refers to the wavelength at which the extinction spectrum due to localized surface plasmon resonance (LSPR) of the core-shell nanoparticle exhibits a maximum value. The maximum wavelength is the wavelength showing the peak of the extinction spectrum obtained by UV-visible absorption measurement using a UV-visible spectrophotometer (Nanophotometer NP80, manufactured by Implen).

[0013] In the fluorescence-enhanced nanoparticles of the present invention, the core-shell nanoparticles are selected from the most suitable core particle material, shape, and size depending on the fluorescent dye to be used. The maximum wavelength due to localized surface plasmon resonance varies depending on the shape of the core particle, and therefore the optimal fluorescent dye also varies. In the present invention, by making the core particle anisotropic (cubic, prism, etc.), it is possible to select the optimal combination for any fluorescent dye. In one embodiment of the present invention, the maximum wavelength of the LSPR of the core particle of the fluorescence-enhanced nanoparticle and each wavelength of the fluorescent dye are expressed as follows: excitation wavelength of the fluorescent dye < maximum wavelength of the LSPR of the core particle < fluorescence wavelength of the fluorescent dye. That is, in one embodiment, the core-shell nanoparticle can be designed so that the maximum wavelength of the localized surface plasmon resonance (LSPR) is in the range between the excitation wavelength and the fluorescence wavelength of the fluorescent dye. The core particles of the present invention are metal particles, preferably gold core particles or silver core particles. The shell covering the surface of the core particle of the present invention is made of a metal material different from that of the core particle, and is preferably a gold shell or a silver shell. For example, a silver shell is used when the core particle is a gold core particle, and a gold shell is used when the core particle is a silver core particle. In one embodiment, the shape of the core particle is not particularly limited, but may be a nanocube, nanoprism, nanorod, nanobipyramid, or nanodecahedron, preferably a nanocube. In one embodiment, the diameter of the core particle of the core-shell nanoparticles is preferably 10 to 100 nm, 20 to 80 nm, or 30 to 70 nm, and particularly preferably 40 to 60 nm. In one embodiment, the thickness of the shell of the core-shell nanoparticles is preferably 1 to 100 nm, 2 to 80 nm, or 3 to 60 nm, and particularly preferably 4 to 40 nm. In one embodiment, the shell thickness of the core-shell nanoparticles can be controlled by the metal ion concentration in the shell formation step. The metal ion concentration in the shell formation step is preferably 0.1 to 1000 mM, or 0.5 to 100 mM. The average shell thickness is preferably 1 to 100 nm, 2 to 80 nm, or 3 to 60 nm, and particularly preferably 4 to 40 nm. In one embodiment, the fluorescent dye is preferably, but not limited to, one that emits visible to near-infrared light when excited by ultraviolet to near-infrared light. The fluorescent dye is not particularly limited, but known dyes can be used, including fluorescein dyes, rhodamine dyes, and cyanine dyes. Fluorescein dyes include FITC (Fluorescein Isothiocyanate) and HEX™, rhodamine dyes include Texas Red™, and cyanine dyes include Cyanine 7™. The excitation and fluorescence wavelengths of exemplary fluorescent dyes are shown in Table 1. In one embodiment, the maximum wavelength of LSPR in the fluorescence-enhanced nanoparticles of the core-shell nanoparticles is between the excitation and fluorescence wavelengths of the fluorescent dyes used, i.e., longer than the excitation wavelength and shorter than the fluorescence wavelength of the fluorescent dye.

[0014] [Table 1]

[0015] The spacer of the present invention binds the fluorescent dye to the surface of the core particle and adjusts the distance from the core particle surface to the fluorescent dye. The distance from the shell surface to the fluorescent dye is preferably within a range that does not cause the shell to quench the fluorescent dye and that allows for the light-harvesting effect due to the plasmon resonance of the core-shell nanoparticles to be obtained. In the present invention, the distance (d) from the shell surface to the fluorescent dye is the length (d=lt) obtained by subtracting the shell thickness (t) from the spacer length (l), and is preferably 1 to 20 nm. Therefore, the distance from the shell surface to the fluorescent dye can be changed by the length of the spacer and the thickness of the shell, and the length of the spacer is preferably 10 nm to 100 nm. In one embodiment, the spacer is single-stranded DNA. Single-stranded DNA is a chemically synthesized linear biopolymer, and its length is strictly proportional to the number of bases. Compared to the prior art that uses silica as a spacer, the length of single-stranded DNA can be strictly defined by the number of bases, and its ends can be chemically modified with functional groups, chromophores, etc.

[0016] The number of bases in the single-stranded DNA is selected depending on the desired spacer length. In one embodiment, commercially available single-stranded DNA can be used, and examples of sources include companies that provide oligo DNA synthesis services, such as FASMAC. In the present invention, the spacer is modified at its end so that it can bind to the surface of the core particle and to a fluorescent dye. In one embodiment, the single-stranded DNA is modified so that it has a thiol group at its 3' end and a fluorescent dye is attached to its 5' end. Commercially available single-stranded DNA to which a fluorescent dye has been attached can be used.

[0017] The core-shell nanoparticles having a gold nanoshell of the present invention can be used in a variety of industrially useful applications, such as color sensors and biomarkers that utilize plasmon resonance, photoelectric conversion materials and photothermal conversion materials that utilize the optical property of absorbing red to near-infrared light, near-infrared light-harvesting materials for photocatalysis in water splitting reactions, and carriers for photoupconversion.

[0018] Numerical values ​​expressing quantities of ingredients, attributes, and the like used in the present specification and claims may be interpreted as being modified by the modifier "about." The term "about" means that there may be errors or variations within the scope that do not change the essence of the present invention, and unless otherwise specified, the numerical values ​​may vary by ±10%, ±5%, ±3%, or ±1%. [Example]

[0019] The present invention will be described in more detail below with reference to examples. However, the scope of the present invention is not limited to the embodiments shown in the following examples. In the examples and comparative examples of this specification, unless otherwise specified, temperature refers to the liquid temperature. Furthermore, unless otherwise specified, % refers to % by weight.

[0020] In the examples herein, the following reagents and commercially available products were used unless otherwise specified. Chloroauric acid solution (HAuCl4, 29.0-31.0%) manufactured by Tanaka Kikinzoku Kogyo Co., Ltd. Hexadecyltrimethylammonium chloride (CTAC, ≥ 95.0%) manufactured by Fujifilm Wako Pure Chemical Industries, Ltd. Sodium tetrahydroborate (NaBH4, ≥95.0%) manufactured by Fujifilm Wako Pure Chemical Industries, Ltd. Ascorbic acid (AA, 99.6%) manufactured by Fujifilm Wako Pure Chemical Industries, Ltd. Sodium bromide (NaBr, 99.0-101.4%) manufactured by Fujifilm Wako Pure Chemical Industries, Ltd. Benzyl cetyl dimethyl ammonium chloride (BDAC, ≥ 90%), manufactured by Fujifilm Wako Pure Chemical Industries, Ltd. Hexadecyltrimethylammonium bromide (CTAB, ≥99%), manufactured by Sigma-Aldrich Co. LLC Dithiothreitol (DTT, 97.0%) Fujifilm Wako Pure Chemical Industries, Ltd.

[0021] The extinction spectrum of the nanoparticles described herein was measured from 200 nm to 900 nm using a UV-visible spectrophotometer (Nanophotometer NP80, manufactured by Implen). Fluorescence measurements For fluorescence measurement, 200 μL of a solution containing the fluorescent dye-attached core-shell nanoparticles prepared in the examples and comparative examples was placed in a 0.5 mL microtube, and the fluorescence intensity was measured using a fluorometer (DS-11, manufactured by DeNovix). TEM observation The shape and diameter of the core particles were observed using a transmission electron microscope (TEM). Specifically, 2 μL of the nanoparticle-containing solution was dropped onto a copper grid, dried under reduced pressure, and then observed using a TEM (JEOL Ltd., JEM2010).

[0022] [Preparation Example 1] Preparation of cubic gold core particles (AuNCs) Preparation of gold nanocluster solution 10 mM NaBH4 was prepared and immediately chilled in a -80°C freezer for 5 minutes. 500 μL of 200 mM CTAB aqueous solution and 500 μL of 0.5 mM HAuCl4 aqueous solution were mixed in a 1.5 mL tube. 90 μL of chilled 10 mM NaBH4 was added to this mixture, and the mixture was vigorously mixed using a vortex mixer for 1 minute. The stirred mixture was left to stand in a 30°C thermostatic bath for at least 1 hour to obtain a gold nanocluster solution. This gold nanocluster solution was used in the subsequent seed particle synthesis.

[0023] Seed particle synthesis 2000 μL of 200 mM CTAC aqueous solution, 1500 μL of 100 mM ascorbic acid (AA) aqueous solution, 50 μL of gold nanocluster solution, and 2000 μL of 0.5 mM HAuCl4 aqueous solution were added to a 20 mL vial, gently mixed, and then allowed to stand in a 30°C incubator for 30 minutes. This solution was centrifuged (20,000 g, 60 minutes), the supernatant was removed, and 1 mL of 10 mM CTAC was added to the remaining pellet and stirred to disperse it. The above centrifugation and redispersion steps may be repeated once or multiple times. This was performed twice in this example and this comparative example. This seed particle solution was used in the subsequent synthesis of cubic-shaped gold core particles.

[0024] Synthesis of gold core particles The temperature of the thermostatic water bath was set to 30°C, and a 20 mL vial containing a stir bar was placed in the water bath. 6,000 μL of 100 mM CTAC, 30 μL of 40 mM NaBr, 6 μL of seed particle solution (OD=5.0), and 62.4 μL of 100 mM AA were added to the vial and mixed, and stirring (500 rpm) was initiated. While stirring, 6,000 μL of 0.5 mM HAuCl4 was added to the mixture to initiate the gold core particle synthesis reaction. Stirring was continued for 30 minutes to complete the reaction. The solution was centrifuged (6,000 g, 10 minutes), the supernatant was removed, and 1 mL of 1 mM CTAC was added to the remaining pellet and stirred to disperse it. After repeating the washing step with CTAC twice, the mixture was centrifuged again, the supernatant was removed, and 100 μL of 1 mM CTAC was added to the remaining pellet. This solution was used as a gold nanoparticle-containing solution. The synthesized gold nanoparticles were cubic in shape, with each side measuring approximately 40 nm.

[0025] The resulting gold nanoparticles were observed under a transmission electron microscope (TEM) to examine the particle shape and core particle diameter. Specifically, 2 μL of the gold nanoparticle-containing solution was dropped onto a copper grid, dried under reduced pressure, and then observed under a TEM (JEM2100-Plus, manufactured by JEOL Ltd.).

[0026] [Preparation Example 2] Preparation of cubic gold core particles with fluorescent dye-modified single-stranded DNA added Gold nanoparticles with fluorescent dye-modified single-stranded DNA attached were prepared using a standard method used in the art. Specifically, single-stranded DNA with a thiol group modification at the 3' end and a fluorescent dye modification at the 5' end was added to the gold nanoparticles prepared in [Preparation Example 1] at a molar ratio of 10,000 times or more. This mixture was stirred at room temperature overnight to produce cubic gold nanoparticles with fluorescent dye-modified single-stranded DNA attached. Excess single-stranded DNA not bound to the gold nanoparticles was removed by centrifugation and the excess DNA was collected as the supernatant. The single-stranded DNA used was manufactured by FASMAC.

[0027] [Preparation Example 3] Preparation of core-shell nanoparticles Silver shell formation reaction To the cubic gold nanoparticles to which fluorescent dye-modified single-stranded DNA had been added, prepared in [Preparation Example 2], 0.1 to 100 mM silver nitrate was added, and the mixture was allowed to react at 60°C for 1 hour to form a silver shell on the cubic gold nanoparticles to which fluorescent dye-modified single-stranded DNA had been added, yielding gold core-silver shell nanoparticles.

[0028] [Example] [Example 1] Synthesis of 40 nm diameter AuNC / HEX (gold nanocube + fluorescent dye HEX) A total volume of 100 μL of mixed solution was prepared, with a final concentration of 1 nM of 40 nm AuNC synthesized in Preparation Example 1, a final concentration of 1 μM of 3'-end thiol-modified / 5'-end HEX-modified single-stranded synthetic DNA, and a final concentration of 1 mM dithiothreitol (DTT). This mixture was reacted overnight at room temperature with stirring at 500 rpm to obtain 40 nm AuNC bound to 5'-end HEX-modified single-stranded DNA. This mixture was centrifuged at 8,000 g for 5 minutes, and the supernatant was removed to remove excess 3'-end thiol-modified / 5'-end HEX-modified single-stranded synthetic DNA. Then, using 40 nm AuNC to which 5'-end HEX-modified single-stranded DNA was bound, gold core-silver shell nanoparticles to which 5'-end HEX-modified single-stranded DNA was bound were prepared according to the method of Preparation Example 3.

[0029] [Example 2] Synthesis of 20 nm diameter AuNC / HEX (gold nanocube + fluorescent dye HEX) A total of 100 μL of mixed solution was prepared, with a final concentration of 1 nM of 20 nm AuNCs synthesized in the preparation example, a final concentration of 1 μM of 3'-end thiol-modified / 5'-end HEX-modified single-stranded synthetic DNA, and a final concentration of 1 mM DTT. This was reacted overnight at room temperature with stirring at 500 rpm to obtain 20 nm AuNCs bound to 5'-end HEX-modified single-stranded DNA. This was centrifuged at 8,000 g for 5 minutes, and the supernatant was removed to remove excess 3'-end thiol-modified / 5'-end HEX-modified single-stranded synthetic DNA. Then, using 20 nm AuNC to which 5'-end HEX-modified single-stranded DNA was bound, gold core-silver shell nanoparticles to which 5'-end HEX-modified single-stranded DNA was bound were prepared according to the method of Preparation Example 3.

[0030] [Example 3] Synthesis of 60 nm diameter AuNC / Texas-Red (gold nanocubes + fluorescent Texas-Red) A total volume of 100 μL of mixed solution was prepared, with the final concentration of 60 nm AuNC synthesized in the preparation example at 1 nM, the final concentration of 3'-end thiol-modified / 5'-end Texas-Red-modified single-stranded synthetic DNA at 1 μM, and the final concentration of DTT at 1 mM. This was reacted overnight at room temperature with stirring at 500 rpm to obtain 60 nm AuNC bound to 5'-end Texas-Red-modified single-stranded DNA. This was centrifuged at 8,000 g for 5 minutes, and the supernatant was removed to remove excess 3'-end thiol-modified / 5'-end Texas-Red-modified single-stranded synthetic DNA. Then, using 60 nm AuNC with Texas-Red modified single-stranded DNA attached at the 5' end, gold core silver shell nanoparticles with Texas-Red modified single-stranded DNA attached at the 5' end were prepared according to the method of Preparation Example 3.

[0031] [Comparative Example 1] Synthesis of 40 nm diameter AuNC / FITC (gold nanocubes + fluorescent dye FITC) A total of 100 μL of mixed solution was prepared, with a final concentration of 1 nM of 40 nm AuNC synthesized in the preparation example, a final concentration of 1 μM of 3'-end thiol-modified / 5'-end FITC-modified single-stranded synthetic DNA, and a final concentration of 1 mM DTT. This was reacted overnight at room temperature with stirring at 500 rpm to obtain 40 nm AuNC bound to 5'-end FITC-modified single-stranded DNA. This was centrifuged at 8,000 g for 5 minutes, and the supernatant was removed to remove excess 3'-end thiol-modified / 5'-end FITC-modified single-stranded synthetic DNA. Then, using 40 nm AuNC with 5'-end FITC-modified single-stranded DNA bound thereto, gold core-silver shell nanoparticles with 5'-end FITC-modified single-stranded DNA bound thereto were prepared according to the method of Preparation Example 3.

[0032] Comparative Example 2 40nm diameter Cit-AuNS / HEX (citric acid coated gold nanospheres + fluorescent dye HEX) A 100μL mixture was prepared, containing 40nm spherical gold nanoparticles (Tanaka Kikinzoku Kogyo Co., Ltd.) at a final concentration of 1nM, 3'-end thiol-modified / 5'-end HEX-modified single-stranded synthetic DNA at a final concentration of 1μM, and DTT at a final concentration of 1mM. This mixture was allowed to react overnight at room temperature with stirring at 500 rpm to obtain 40nm Cit-AuNS bound to 5'-end HEX-modified single-stranded DNA. This mixture was centrifuged at 8,000g for 5 minutes, and the supernatant was removed to remove excess 3'-end thiol-modified / 5'-end HEX-modified single-stranded synthetic DNA. Then, using 40 nm Cit-AuNS bound to 5'-end HEX-modified single-stranded DNA, gold core-silver shell nanoparticles bound to 5'-end HEX-modified single-stranded DNA were prepared according to the method of Preparation Example 3.

[0033] [Measurement example] Optical properties of nanoparticles synthesized in Examples 1 to 3 and Comparative Examples 1 and 2 An ultraviolet-visible spectrophotometer (Nanophotometer NP80, manufactured by Implen) was used to obtain an extinction spectrum from 200 nm to 900 nm. The results are shown in Figures 3 to 7.

[0034] The measurement results showed that the cubic fluorescence-enhanced nanoparticles of Examples 1 to 3 of the present invention, in which the distance from the core particle surface to the fluorescent dye was appropriately adjusted, did not quench fluorescence, unlike the spherical nanoparticles of Comparative Example 2, and were excellent as detection agents, etc. This demonstrated the superiority of cubic core-shell particles, in which the distance can be finely adjusted by selecting a spacer of an appropriate length and shell thickness. Furthermore, it was confirmed that the nanoparticles of Examples 1 to 3, in which the maximum wavelength of localized surface plasmon resonance of the gold-core-silver-shell nanoparticles is between the excitation wavelength and fluorescence wavelength of the fluorescent dye, had a greater enhancement in fluorescence intensity than Comparative Example 1, in which the maximum wavelength of localized surface plasmon resonance did not satisfy the conditions, demonstrating that controlling the maximum wavelength of localized surface plasmon resonance is advantageous for enhancing fluorescence.

Claims

1. A fluorescence-enhancing nanoparticle comprising a core-shell nanoparticle consisting of a core particle and a shell, at least one fluorescent dye, and a spacer located between the core-shell nanoparticle and the fluorescent dye, the fluorescent dye has an excitation wavelength and a fluorescence wavelength; the core-shell nanoparticles have a maximum wavelength of localized surface plasmon resonance; the core-shell nanoparticles are metal nanoparticles consisting of a combination of a silver core particle and a gold shell, or a combination of a gold core particle and a silver shell; the spacer is single-stranded DNA; Fluorescence-enhancing nanoparticles characterized by:

2. The fluorescence-enhancing nanoparticle of claim 1 , wherein the core particle of the core-shell nanoparticle has a shape of a nanocube, a nanoprism, a nanorod, a nanobipyramid, or a nanodecahedron.

3. 2. The fluorescence-enhancing nanoparticles of claim 1, wherein the core particles have an average diameter of 10 to 100 nm, and the shell has an average thickness of 1 to 10 nm.

4. Fluorescence-enhancing nanoparticles according to claim 1, wherein the fluorescent dye is a fluorescein dye, a rhodamine dye, or a cyanine dye.

5. 2. The fluorescence-enhancing nanoparticle of claim 1, wherein the distance from the surface of the shell of the core-shell nanoparticle to the fluorescent dye is 1 to 20 nm.

6. 2. The fluorescence-enhancing nanoparticle of claim 1, wherein the maximum wavelength of the localized surface plasmon resonance of the core-shell nanoparticle is between the excitation wavelength and the fluorescence wavelength of the fluorescent dye.

7. 2. The fluorescence-enhancing nanoparticle of claim 1, wherein the length of the spacer is 10 to 100 nm.

8. The fluorescence-enhancing nanoparticle of claim 1 , wherein the spacer is attached to the surface of the core particle of the core-shell nanoparticle.

9. A fluorescence enhancing probe comprising the fluorescence enhancing nanoparticles according to any one of claims 1 to 8, and having the function of specifically binding to a target substance.

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