WO2017099313A1 - Surface-enhanced raman scattering (sers) nanoparticles, method for preparing same, and applications thereof - Google Patents

Abstract

The present invention relates to surface-enhanced raman scattering (SERS) nanoparticles, a method for preparing the same, and applications thereof. The present invention is characterized by having a nanowell on the inner surface of a metal shell so that a raman active material can be placed in the nanowell whereby it is possible for the structure of the nanowell in the metal shell to cause further SERS. Thus, the nanoembosses according to the present invention are an effective approach for generating strong SERS signals, which allows for a platform for manufacturing novel structures for generating strong electromagnetic fields and also for sensitive and reliable biomedical applications.

Description

Surface-enhanced Raman Scattering (SERS) nanoparticles, preparation method thereof and application thereof

The present invention relates to surface enhanced Raman scattering (SERS) nanoparticles, methods for their preparation and applications thereof.

Raman scattering is an inelastic scattering in which the energy of incident light changes, and when light is applied to a specific molecule, light is generated with a slightly different wavelength from that irradiated by the intrinsic vibrational transition. Unlike infrared spectroscopy, Raman spectroscopy is not affected by water molecules, and thus is more suitable for detection of biomolecules such as proteins and genes.

Raman spectroscopy has very weak signal strength and low reproducibility. One way to overcome this problem is surface enhanced Raman scattering. Surface-enhanced Raman spectroscopy is a phenomenon in which the Raman signal of a molecule increases greatly when a molecule is present in the vicinity of the metal nanostructure. In understanding the surface enhancement Raman mechanism, there are electromagnetic enhancement effects and chemical enhancement effects. In order for Raman emission to be most effectively enhanced, there must be a collective vibration of free electrons on the metal surface between the metal and the laser, which is called surface plasmon, which is the basis of the electromagnetic enhancement effect. Surface plasmon resonance depends on the type of metal nanoparticles (usually Au, Ag, Cu, Pt, etc.), the size and shape, the solvent dispersed, the type of laser.

On the other hand, the significant enhancement of Raman scattering allows for single-molecule detection, thus providing one of the most sensitive optical signals. Surface-enhanced Raman scattering (SERS) is primarily due to electromagnetic field enhancement induced by excitation of localized surface plasmons in the vicinity of metallic nanostructures, thus designing and synthesizing metallic nanostructures that produce strong electromagnetic fields. Many efforts have been made to do this.

In order to provide functionality for detection and visualization, metallic nanoparticles capable of surface modification, easy synthesis, and excellent biocompatibility have been widely used as biological detection or contrast SERS substrates. When the metallic nanoparticles are adjacent to or in contact with each other, the plasmon coupling effect is synergistically increased in the vicinity of the contact point between the particles, so that the SERS is further enhanced by aggregation between the nanoparticles. The part showing significant enhancement of the electromagnetic field, such as a contact point between the nanoparticles, is called a hot-spot. However, in a cluster these Random generation and relatively low frequency of hot spots leads to uncertainty in scatter enhancement. On the other hand, by inducing strong electromagnetic field enhancement by forming structures that induce electromagnetic field enhancement such as pupils, sharp edges, nanolenses, reduced symmetry, and nanogap junction structures on planar substrates or on single nanoparticles, reliable SERS There are attempts to gain.

The planar substrate designed as described above can implement a nanostructure that can induce an increase in electromagnetic field with a relatively accurate and reproducible, but the use of the nanoparticle form than the planar substrate for use as a label for detection and image signal in biological medical applications Is high. Nanoparticles that produce highly augmented and reproducible Raman scattering near the surface needed for biological applications exhibit higher field enhancement than simple spherical particles. Nanoparticles include nanocrescent moons, nanorice and nanoshells. ), Nanostars and the like. In biomarkers, detection or labeling for medical imaging, the total intensity of the Raman signal from a single nanoparticle is as important as the enhancement of the signal. The total intensity of Raman scattering for biological analysis or detection is affected by the number of Raman reporters mounted on the nanoparticles as well as the electromagnetic field.

The nanoshell core structure, which has a relatively large number of reporter dyes inside the core, is one of the excellent candidates for providing strong Raman signals from a single nanoparticle. Thus, the Raman reporter dye may be mounted on the outer surface of the nanoshell and the core of the nanoshell core structure to increase the total SERS intensity. Significant improvement in SERS strength can be expected if the field-enhancing structure can be implemented at a level that does not compromise the ratio of the Raman reporter loading volume to the structure of the conventional nanoshell and the total volume. .

The present inventors have made diligent efforts to discover structures and / or materials that can significantly increase the SERS strength based on the particles of the nanoshell core structure, and as a result, nanoconvex on the surface using porous nanoparticles having two or more different sizes. In the case of introducing the metal shell after forming the core having the portion, the metal shell has a nano concave portion corresponding to the nano convex portion on the inner surface thereof, thereby forming a hot spot, and the Raman active material is formed in the pores of the nanoparticles. It was confirmed that it can support the excellent SERS effect was completed the present invention.

It is an object of the present invention to discover new locations exerting strong SERS in metal nanoshells and to design SERS particles capable of loading as many Raman active materials as possible in the nanoshells.

Nanoembosses according to the present invention are an effective approach for generating strong SERS signals, allowing for sensitive and reliable biomedical applications, as well as a platform for fabricating novel structures that produce strong electromagnetic fields.

1 is a schematic diagram illustrating a method of manufacturing a gold nanoshell having nanoembosses toward an inner surface according to one embodiment. (a) shows a transmission electron microscope (TEM) image of the flat silica core used in Example 1, (b) shows a TEM image of a nanoembossed silica core with gold seeds attached, and (c) An enlarged image of the box indicated by dotted lines in (b), (d) a TEM image of a gold nanoshell fabricated on a flat silica core, and (e) a TEM image of a nanoembossed silica core-gold nanoshell. Indicates. Scale bar is 100 nm.

FIG. 2 shows UV-Vis extinction spectra of (a) flat silica core-gold nanoshells and (b) nanoembossed silica core-gold nanoshells. Solid lines represent experimentally measured data and dashed lines represent spectra calculated in water using FDTD.

3 is a diagram showing an absorption spectrum of gold nanoshells nanoembossed in water according to the number of nanoembosses attached on the silica core. Spectra were calculated using FDTD.

FIG. 4 shows the 3-D FDTD calculation results of nanocore-embossed silica core-gold nanoshells at 633 nm excitation wavelength. Localized electromagnetic field distributions of nanoembossed silica core-gold nanoshells are shown. The radius of the silica core is 61.8 nm. The thickness of the shell is 28.7 nm. The number of nanoembosses (d = 15.7 nm) is 100. Scale bar is 100 nm. Electromagnetic field amplitudes are normalized to incident field amplitudes.

FIG. 5 shows a profile of a calculated electromagnetic field formed through nanoembossed silica core-gold nanoshells at 633 nm excitation wavelength. The radius of the silica core is 61.8 nm. The thickness of the shell is 28.7 nm. The number of nanoembosses (d = 15.7 nm) is 100. The field amplitude is normalized to the incident field amplitude.

FIG. 6 shows SERS intensity at 633 nm excitation wavelength as a function of shell thickness of nanoembossed silica core-gold nanoshells. FIG. (a) shows the SERS spectrum obtained from a single nanoembossed gold nanoshell and (b) shows the SERS intensity at 1487 cm ⁻¹ of the single nanoembossed gold nanoshell.

7 shows a comparison of SERS and fluorescence obtained at a 513 nm excitation wavelength. (a) shows SERS spectra from a single nanoembossed gold nanoshell and (b) shows fluorescence spectra obtained from about eight nanoembossed silica cores.

FIG. 8 shows the SERS signal at 633 nm excitation wavelength as a function of [Ru (bpy) ₃ ] ²⁺ concentration on a single flat silica core-gold shell. (a) 1487 cm ^-1 is a SERS spectra obtained after dispersing the dye in various concentrations (0.05 mM, 0.1 mM, 0.5 mM, and 1 mM) on the gold nano-shell, (b) as a function of dye concentration Indicate the SERS intensity.

9 is a diagram showing the optical emission of the aqueous solution of [Ru (bpy) _3] ²⁺ has a nano-embossing silica nanoparticles encapsulated.

10 shows the SERS spectrum of a single nanoembossed gold nanoshell at 785 nm excitation wavelength.

FIG. 11 is a diagram showing a SERS signal of a silica core-gold shell containing [Ru (bpy) ₃ ] ²⁺ . (a) shows the TEM image of the dye-sealed silica core-gold shell, (b) the UV / Vis absorption of the dye-sealed silica core-gold shell, and (c) the dye-sealed silica core-gold shell. Shows a SERS spectrum at 633 nm excitation wavelength.

12 is a diagram illustrating a comparison of absorption spectra of [Ru (bpy) ₃ ] ²⁺ and silica nanoparticles in a solution. (a) is [Ru (bpy) ₃ ] ²⁺ in water, (b) is [Ru (bpy) ₃ ] ²⁺ encapsulated nanoembossed silica nanoparticles, (c) is silica core, and (d) is [ Ru (bpy) ₃ ] ²⁺ nanoembossed silica cores, and (e) show results for [Ru (bpy) ₃ ] ²⁺ embedded silica cores.

13 is a view showing a comparison of the SERS signal according to the reporter dye encapsulation position. (a) and (b) represents the intensity at each SERS spectrum and 633 nm 1487 cm ^-1 for single AuNS according to the enclosed position of the reporter dye upon here.

FIG. 14 shows SEM of AuNS prepared on dye doped silica cores (132.4 ± 10.5 nm in diameter) with and without nanoembossing ((a) and (b), respectively) and nanoparticles on silica cores and dye doped silica cores. A diagram showing a TEM image of embossing. Scale bar is 100 nm.

15 is a view showing a structure generated when the shell is formed thinner than the nano-emboss. The right side shows a structure formed when the nanoembosses are densely positioned so that neighboring nanoembosses are in contact with each other, and the left side is formed when the nanoembosses are less densely positioned and the adjacent nanoembosses are spaced at predetermined intervals.

A first aspect of the invention provides a core having nanoconvex portions on a surface thereof; And a metal shell having a nano-concave portion corresponding to the nano-convex portion on an inner surface thereof, wherein the Raman active material is also supported on the nano-convex portion of the core, thereby providing surface enhanced Raman scattering (SERS) nanoparticles.

A second aspect of the present invention provides a core portion carrying a first Raman active material and having a (+) charge, and a nanoparticle carrying a second Raman active material and having a (-) charge formed on the surface of the core portion. A first core having a convex portion; Or a second carrier having a first Raman active material and having a (-) charged core portion and a nano convex portion having a second Raman active material and having a (+) charge formed thereon on the surface of the core portion. core; And it provides a surface-enhanced Raman scattering (SERS) nanoparticles comprising a metal shell having a nano-concave corresponding to the nano-convex portion on the inner surface side.

The third aspect of the present invention comprises the first step of preparing a silica core loaded with a Raman active material; A second step of modifying the surface of the silica core to carry a (+) or (−) charge; Silica core having nano convexities, having the nano-convex portion assembled by the assembly of the silica nanoparticles doped with the Raman active material on the surface of the silica core formed in the second step through electrostatic interaction, having an opposite charge to the silica core formed in the second step. Preparing a third step; Attaching metal nano seeds to the silica core formed in the third step; In a first or second aspect, the method comprises growing a nano seed in a growth solution to form a metal shell having a nano concave portion corresponding to the nano convex portion on an inner surface of the silica core having the nano convex portion. It provides a method for producing a surface surface enhanced Raman scattering (SERS) nanoparticles according.

A fourth aspect of the invention provides a Raman probe with surface enhanced Raman scattering (SERS) nanoparticles according to the first or second aspect.

A fifth aspect of the present invention provides a surface enhanced Raman scattering (SERS) substrate coated with surface enhanced Raman scattering (SERS) nanoparticles according to the first or second aspect on the substrate.

A sixth aspect of the present invention provides a process for a) functionalizing a biomolecule or compound capable of binding the surface of the surface enhanced Raman scattering (SERS) nanoparticles according to the first or second aspect with an analyte to be detected. step; b) exposing the functionalized SERS nanoparticles to a sample comprising one or more analytes; And c) identifying the analyte to which the SERS nanoparticles are bound using Raman spectroscopy.

The seventh aspect of the invention comprises the steps of: i) functionalizing a surface of the surface enhanced Raman scattering (SERS) nanoparticles according to the first or second aspect with a biomolecule or compound complementary to the nucleic acid to be detected; ii) performing hybridization by reacting the functionalized SERS nanoparticles with a sample that is expected to contain a nucleic acid to be detected; And iii) performing Raman spectroscopy to confirm the presence, amount or both of the nucleic acid to be detected to which the SERS nanoparticles are bound.

An eighth aspect of the invention provides a core having nano-convex portions on its surface; And it provides a surface enhanced Raman scattering (SERS) substrate comprising a metal shell having a nano-concave corresponding to the nano-convex portion on the inner surface side.

Hereinafter, the present invention will be described in detail.

In a core-shell structure, the inner surface of the shell is the first surface of the shell adjacent to the core, and the outer surface of the shell refers to the second surface opposite the first surface.

In the present specification, the emboss may provide nano convex portions on the surface of the core, and may provide nano concave portions corresponding to the nano convex portions on the inner surface side of the metal shell. Therefore, in this specification, embossing, a nano convex part, and a nano concave part are mixed.

The inventors have fabricated nanoshells with nanoembosses on the inner surface, where the strong field formed in the metal nanoshell layer induces substantial enhancement of Raman scattering, and through the FDTD calculation of these nanoparticles, In the nanoshells, there is a strong induced electromagnetic field. On the other hand, since the internal shape can be manipulated by forming a metal nanolayer around a core such as silica, a Raman active material such as a reporter dye can be included in extra space such as inside the nanoemboss and on the outer surface of the nanoparticle. have. In addition, the core may further include a Raman active material.

For example, the present invention can provide a nano-embossed silica core-metal nanoshell, with a core-shell structure with novel internal positions that form strong electromagnetic fields.

In one embodiment, smaller silica nanoparticles are electrostatically assembled around the silica core, followed by a wet process to grow gold nanoseeds on the core, thereby internally nanoembossed gold nanoshell with nanoembosses on the inner surface. Was prepared. The generation of strong electromagnetic fields (| E / E _in | _max. = 55 at 633 nm) at the sharp edges formed by the contact between the nanoembosses and the silica core was confirmed by FDTD calculations. The formation of the electromagnetic field was supported by the SERS signal measured for [Ru (bpy) ₃ ] ²⁺ , a Raman activator encapsulated in nanoembossed silica nanoparticles. In the present invention, SERS signals that are as strong as the corresponding fluorescent signals are obtained. Raman enhancement factors were estimated up to 10 ¹⁰ at 633 nm excitation, as well as comparable enhancers at 785 nm laser excitation. The total intensity from the nanoshell layer with nanoembosses is sufficiently high compared to the outer surface or the core of the nanoparticles, such that the nanoembossed metal nanoshells are biomedical applications as sensitive and reliable labeling particles.

Spherical gold nanoparticles is not suitable as the 830 nm, even though possible to improve the Raman signal to 10: ⁹ at the excitation wavelength, and because of the poor volatility of the irregularities and outside the effect of hot spots that cause the SERS enhancement reproducible SERS cover material which when agglomerated Is considered not. Unlike gold nanoparticle agglomerates, the SRES signal of a metal shell with nanoembosses is generated from nanoembosses containing Raman activators, protected by a nanoshell layer, thus creating a strong electromagnetic field that creates It is protected against and more stable.

Gold is a highly inert substance. Thus, the Raman reporter dye encapsulated in SiO ₂ nanoembosses can be physically and chemically stable. As a result, it was possible to stably generate the SERS signal from the nanoembossed gold nanoshell (AuNS). The present invention presents for the first time that a strong electromagnetic field is formed inside the nanoshell layer, thereby obtaining a strong SERS enhancement. As a result, structures bearing the Raman activator on the outer surface of the nanoshell, inside the nanoshell layer, and on the inner core of the nanoshell have a very high total SERS signal that can be detected with significantly improved analytical sensitivity without amplifying trace amounts of target biomolecules. It can increase the possibility of detection using a simplified Raman spectrometer without the aid of high-end equipment to measure low signals. Furthermore, various nanostructures with nanoembosses, which form much higher electromagnetic fields than those formed using spherical SiO ₂ cores, can be made into self-assembled molds of various structures.

In sum, the present invention has for the first time found that an inner surface with nanoconcave in a metal nanoshell can provide a novel location for generating a strong SERS signal as much as the surface or inside the core of the nanoshell. That is, the microstructure of the inner surface of the metal shell creates hot spots in which strong electromagnetic fields are generated in the local region, thereby enabling high SERS buildup.

Accordingly, the present invention is in the form of having a nano concave portion on the inner surface of the metal shell, it is possible to place the Raman active material in the nano concave portion, it is possible to exhibit additional SERS by the structure of the nano concave portion of the metal shell It is characteristic. This is based on the discovery that there is a strong induced electromagnetic field due to the inner surface with nano recesses in the metal nanoshells that can cause SERS enhancement.

<In accordance with the present invention Surface Enhancement Raman Scattering ( SERS ) Nanoparticles>

Surface-enhanced Raman scattering (SERS) nanoparticles according to the present invention is a core having a nano-convex portion on the surface while carrying a Raman active material; And a metal shell having a nano concave portion corresponding to the nano convex portion on an inner surface side thereof, and the Raman active material may be supported on the nano convex portion of the core.

In addition, the surface-enhanced Raman scattering (SERS) nanoparticles according to the present invention

A first core supporting a first Raman active material and having a (+) charge core portion and a nano-convex portion formed on the surface of the core portion and carrying a second Raman active material and having a (−) charge ; or

A second core supporting a first Raman active material and having a (-) charge core portion and a nano-convex portion supporting a second Raman active material and having a (+) charge on the surface of the core portion; ; And

It may include a metal shell having a nano concave portion corresponding to the nano-convex portion on the inner surface side.

The SERS nanoparticles according to the present invention may not only support the Raman active material inside the core having the nano convex portion, but may optionally support the Raman active material on the outer surface of the metal shell.

Raman activator refers to a substance that facilitates the detection and measurement of an analyte by a Raman detection device when the metal shell of the present invention adheres to one or more analytes. Raman actives preferably exhibit a distinct Raman spectrum. Raman activators show specific Raman spectra, allowing for more efficient analysis of biomolecules.

Raman active materials that can be used in Raman spectroscopy include organic or inorganic molecules, atoms, complexes or synthetic molecules, dyes, naturally occurring dyes (such as picoeryrin), organic nanostructures such as C60, buckyballs, carbon nanotubes, quantum dots, Organic fluorescent molecules and the like. Specifically, examples of the Raman active substance include FAM, Dabcyl, TRITC (tetramethyl rhodamine-5-isothiocyanate), rhodamine 6G, MGITC (malakit green isothiocyanate), XRITC ( X-Rhodamine-5-isothiocyanate), DTDC (3,3-diethylthiadicarbocyanine iodide), TRIT (tetramethyl rhodamine isothiol), NBD (7-nitrobenz-2-1 , 3-diazole), phthalic acid, terephthalic acid, isophthalic acid, para-aminobenzoic acid, erythrosine, biotin, digoxigenin, 5-carboxy-4 ', 5'-dichloro-2', 7 ' -Dimethoxy, fluorescein, 5-carboxy-2 ', 4', 5 ', 7'-tetrachlorofluorescein, 5-carboxyfluorescein, 5-carboxyrodamine, 6-carboxyrodamine, 6-carboxytetramethyl amino phthalocyanine, azomethine, cyanine (Cy3, Cy3.5, Cy5), xanthine, succinylfluorescein, aminoacrydine, quantum dots, carbon allotropes, cyanide, thiols, chlorine But this, bromine, methyl, phosphorus and sulfur, etc. are not limited. In this example, [Ru (bpy) ₃ ] ²⁺ was used as the Raman active material.

In the present invention, the core having the nano-convex portion on the surface while supporting the Raman active material preferably has an average diameter of 20 nm to 1000 nm, more preferably 50 nm to 500 nm, corresponding to its volume. If the diameter of the core is less than 20 nm, not only the capacity of the Raman activator that can be included, but also the Raman surface enhancement effect is lowered, if it exceeds 1000 nm is limited in biological applications. The shape of the core may be spherical or elliptical without considering the nano-convex portion, but may be any shape or irregular shape.

On the other hand, it is preferred that the nano-convex portion included therein has an average diameter of 5 to 50 nm, but is not limited thereto.

In addition, the thickness of the shell formed on the core having the nano-convex portion on the surface may be determined according to the size of the nano-convex portion, preferably 1 to 50 nm, but is not limited thereto.

The core having the nano convex portions on the surface preferably has sharp edges that enable the formation of electromagnetic fields at the bottom edge of the nano convex portions.

The core and its nano convex parts It is not limited to the material as long as it can support a Raman active substance. The core and its nano-convex portions are preferably porous so as to support the Raman active material.

The material of the core and its nano-convex portion is silica, gold sulfide, titanium dioxide, polymethyl methacrylate (PMMA), polystyrene, hydrogels or non-conductive materials. Combinations thereof, but is not limited thereto. The material of the nano-convex portion may be the same or different from that of the main core portion, and may be the same material but may be modified or unmodified differently. For example, in order to form a core having nanoconvex portions on its surface through self-assembly by electrostatic force, the main core portion and nanoconvex portions each independently have a positively charged silica and a negatively-charged silica. Can be used. The positively charged silica may be silica modified with amines, or the negatively charged silica may be silica modified with carboxyl groups.

According to the invention the core with nano convexities on the surface is

A first core supporting a first Raman active material and having a (+) charge core portion and a nano-convex portion formed on the surface of the core portion and carrying a second Raman active material and having a (−) charge ; or

A second core supporting a first Raman active material and having a (-) charge core portion and a nano-convex portion supporting a second Raman active material and having a (+) charge on the surface of the core portion; Can be.

In this case, the first Raman active material and the second Raman active material may be the same or different.

In the present invention, the core carrying the Raman active material may also serve as a template for forming a nanoscale microstructure of the inner surface of the metal shell. Therefore, the surface plasmon effect can be maximized by the microstructure of the inner surface of the metal shell formed by variously adjusting the size and shape of the core and the like. The microstructure of the inner surface of the metal shell according to the present invention can control the shape of the core used as a mold at the nanometer level, thereby producing a reproducible metal shell.

According to the present invention, a metal shell having nano recesses on an inner surface side creates a hot spot in which a strong electromagnetic field is generated in a local region corresponding to the nano recesses of the core, thereby providing an increased Raman signal.

According to the present invention, a metal shell having a nano concave portion on the inner surface side corresponding to the nano convex portion of the core through the nanoembossing technique is not only an alternative for forming a strong field, but also an alternative for supporting a sufficient number of Raman active materials. Can be. Furthermore, metal shells with various nanostructured nanoembosses on the inner surface can be made into various self-assembled molds to form much higher fields than those formed using spherical SiO ₂ cores.

Nanoembosses according to the present invention are an effective approach for generating strong SERS signals, allowing for sensitive and reliable biomedical applications, as well as a platform for fabricating novel structures that produce strong electromagnetic fields.

The thickness of the nanoshells may be 1 nm to 300 nm, more preferably 1 to 50 nm.

Experiments confirmed that the Raman scattering of the reporter dye, a Raman active material, rather decreased when the thickness of the shell increased to a level exceeding the upper limit of the above range.

The material of the metal shell may be gold, silver, copper, platinum or aluminum, but the material is not limited as long as it can function as a small antenna that enhances the effect of concentrating electromagnetic waves.

<In accordance with the present invention Surface Enhancement Raman Scattering ( SERS ) Manufacturing method of nanoparticles>

Surface enhanced Raman scattering (SERS) nanoparticles according to the present invention

A first step of preparing a silica core loaded with a Raman active material;

A second step of modifying the surface of the silica core to carry a (+) or (−) charge;

Silica core having nano convexities, having the nano-convex portion assembled by the assembly of the silica nanoparticles doped with the Raman active material on the surface of the silica core formed in the second step through electrostatic interaction, having an opposite charge to the silica core formed in the second step. Preparing a third step;

Attaching metal nano seeds to the silica core formed in the third step;

It can be provided by a manufacturing method comprising a fifth step of growing a nano seed in the growth solution to form a metal shell having a nano concave portion corresponding to the nano convex portion on the inner surface around the silica core having the nano convex portion.

1 is a schematic diagram illustrating a method of manufacturing a gold nanoshell having nanoembosses toward an inner surface according to one embodiment. First, negatively charged nanoprojected silica nanoparticles are assembled on a positively charged silica core. By reducing HAuCl ₄ on silica, the core seeds on the gold forms a gold nano-layer shell around the embossed nano silica core. By using dye-supported SiO ₂ nanoparticles arranged on the core particles to fabricate nanoembossing, the self-assembly method can easily adjust the size and number of nanoembosses according to the intended purpose.

The first step may be performed by a microemulsion method.

In the fifth step of forming the metal nanoshell on the surface of the core particle having the nano convex portions, the reaction may be performed at 10 to 100 ° C. in the growth solution containing the metal precursor.

If the reaction temperature is less than 10 ℃ takes too much time to form a metal shell, if it exceeds 100 ℃ may be a non-uniform metal shell. In the above, the reaction time may be adjusted to 1 minute to 24 hours depending on the reaction temperature.

The silver precursor may be AgNO ₃ or AgClO ₄ , and the gold precursor may be any compound containing Au ions such as HAuCl ₄ . The copper precursor may be Cu (NO ₃ ) ₂ , CuSO ₄ . Reducing agents required to convert silver ions or gold ions to gold or silver nanoshells include hydroquinone, sodium borohydride (NaBH ₄ ), sodium ascorbate, formaldehyde, and the like. The solvent of the growth solution may be purified water, an aqueous solution (eg, phosphate buffer). Additional stabilizers can be added for precise thickness control of the nanoshells.

<In accordance with the present invention Surface Enhancement Raman Scattering ( SERS Application of Nanoparticles

The present invention provides a Raman probe with SERS nanoparticles according to the present invention.

The present invention also provides a surface enhanced Raman scattering (SERS) substrate coated with the SERS nanoparticles according to the invention on the substrate. At this time, the SERS nanoparticle-containing coating layer may further induce electromagnetic field amplification between adjacent SERS nanoparticles by the access or contact of the SERS nanoparticles.

SERS nanoparticles according to the present invention can be used as a Raman probe that can be applied to detect a variety of biomolecules by functionalizing with biomolecules (molecules) or compounds that can recognize the analyte to detect the surface.

For example, analytes to be detected include amino acids, peptides, polypeptides, proteins, glycoproteins, lipoproteins, nucleosides, nucleotides, oligonucleotides, nucleic acids, sugars, carbohydrates, oligosaccharides, polysaccharides, fatty acids, lipids, Hormones, metabolites, cytokines, chemokines, receptors, neurotransmitters, antigens, allergens, antibodies, substrates, metabolites, cofactors, inhibitors, drugs, pharmaceuticals, nutrients, prions, toxins, toxins, explosives, pesticides, chemicals Inorganic agents, biohazardous agents, radioisotopes, vitamins, heterocyclic aromatic compounds, carcinogens, mutagens, anesthetics, amphetamines, barbiturates, hallucinogens, wastes or contaminants. In addition, if the analyte is a nucleic acid, the nucleic acid is a gene, viral RNA and DNA, bacterial DNA, fungal DNA, mammalian DNA, cDNA, mRNA, RNA and DNA fragments, oligonucleotides, synthetic oligonucleotides, modified oligonucleotides, single Stranded and double stranded nucleic acids, natural and synthetic nucleic acids.

As used herein, the term "biomolecule" refers to small molecules such as primary metabolites, secondary metabolites, and natural products, to macromolecules such as proteins, carbohydrates, lipids, and nucleic acids. It may refer to any molecule present in the living organisms included, and may include all biological materials in a conventional meaning. In addition, in addition to the endogenous material, it may also include all of the exogenous drugs that are natural, semi-synthetic or total synthetic material.

Non-limiting examples of biomolecules capable of binding to the surface of SERS nanoparticles according to the present invention that can recognize analytes include antibodies, antibody fragments, genetically engineered antibodies, single chain antibodies, receptor proteins, binding proteins, enzymes , Inhibitor proteins, lectins, cell adhesion proteins, oligonucleotides, polynucleotides, nucleic acids or aptamers.

In addition, the SERS nanoparticles according to the present invention, after the Raman active material is bonded to the surface, the whole nanoparticles may be further coated with an inorganic material. When the whole nanoparticles are coated with inorganic material, the structure is less likely to be deformed, so the structure of the nanoparticles can be stably maintained, which is more desirable for storage and use. Aggregation of nanoparticles can ensure reproducibility in inducing electromagnetic amplification between adjacent SERS nanoparticles. The inorganic material is not limited as long as it maintains the structure of the SERS nanoparticles and does not affect the Raman signal, and silica may be used as an example.

Surface-enhanced Raman scattering techniques are basically non-destructive for samples because they use low power lasers. In addition, with the development of the Raman probe manufacturing technology, it can be applied to the diagnosis of diseases in cells and in vivo using a material with excellent biocompatibility. In order to increase the mobility and biocompatibility of the Raman probe in cells and in vivo, the surface of the Raman probe may be coated with a biocompatible material such as a polymer ligand or silica, but is not limited thereto. In addition, specific reactions (DNA hybridization, antigen-antibody reactions, etc.) between biomaterials can be used for high-sensitivity diagnosis for specific diseases in cells and in vivo. It can also be used for identification, kinship, bacterial or cellular identification, or origin of flora and fauna, but its application is not limited thereto.

Therefore, the non-destructive surface enhancement Raman analysis technology using the SERS nanoparticles according to the present invention can be used in the field of real-time monitoring and therapeutic drug for specific diseases in living cells and in vivo.

Furthermore, since the SERS nanoparticles according to the present invention can secure a reproducible microstructure in which the SERS signal can be maximized, it can be used for a very reliable and useful ultra-high sensitivity biomolecular analysis method, and can also be used as an in vivo imaging technique in addition to in vitro diagnostic methods. Very useful.

In one embodiment, using the SERS nanoparticles according to the invention, a method for detecting or imaging an analyte is

a) functionalizing the surface of the SERS nanoparticles according to the invention with a biomolecule or compound capable of binding to the analyte to be detected;

b) exposing the functionalized SERS nanoparticles to a sample comprising one or more analytes; And

c) identifying the analyte to which the SERS nanoparticles are bound using Raman spectroscopy.

In another embodiment, using the SERS nanoparticles according to the present invention, a method for detecting a nucleic acid

i) functionalizing the surface of the SERS nanoparticles according to the invention with a biomolecule or compound complementary to the nucleic acid to be detected;

ii) performing hybridization by reacting the functionalized SERS nanoparticles with a sample that is expected to contain a nucleic acid to be detected; And

iii) performing Raman spectroscopy to confirm the presence, amount or both of the nucleic acid to be detected to which the SERS nanoparticles are bound.

In this case, the sample expected to contain the nucleic acid to be detected may be used as the sample itself, or the nucleic acid to be detected may be separated, purified or amplified therefrom.

Any known Raman spectroscopy can be used, preferably Surface Enhanced Raman Scattering (SERS), Surface enhanced resonance Raman spectroscopy (SERRS), Hyper-Raman and / or Ratio Coherent anti-Stokes Raman spectroscopy (CARS) can be used.

Surface Enhancement Raman Scattering (SERS) is a type of Raman scattering that occurs when adsorbed on a roughened metal surface or is located within a few nanometers (d≤10 nm). It is a spectroscopic method using the phenomenon that 10 ⁶ ~ 10 ⁸ times increase compared with the intensity of Surface augmented resonance Raman spectroscopy (SERRS) is a spectroscopy utilizing the resonance phenomenon of laser excitation wavelengths for adsorbates on SERS active surfaces. Non-coherent antistock Raman spectroscopy (CARS) is a spectroscopy method that injects two fixed laser light into a Raman active medium and measures the spectrum of anti-Stokes radiation obtained by combining them.

The nucleic acid detection method, using the SERS nanoparticles according to the present invention, can detect other information about the nucleic acid, such as one or more single base polymorphisms (SNPs) or other forms of genetic variation present in the sample, furthermore DNA It can also be applied to sequencing.

A surface enhanced Raman scattering (SERS) substrate, such as a Raman active substrate, coated with surface enhanced Raman scattering (SERS) nanoparticles according to the invention on a substrate may be operatively coupled with one or more Raman detection unit devices. Several methods for the detection of analytes by Raman spectroscopy are known in the art (eg, US Pat. Nos. 6,002,471, 6,040,191, 6,149,868, 6,174,677, 6,313,914). In SERS and SERRS, the sensitivity of Raman detection is enhanced to 10 ⁶ or higher for molecules absorbed on rough metal surfaces such as silver, gold, platinum, copper or aluminum surfaces.

Non-limiting examples of Raman detection devices are disclosed in US Pat. No. 6,002,471. As the excitation beam, a pulsed laser beam or a continuous laser beam can be used. The excitation beam passes through confocal optics and a microscope lens and is focused onto a Raman active substrate containing one or more analytes. Raman emission light from the analyte is collected by microscope lenses and confocal optics and combined with monochromators for spectral separation. Confocal optics include a combination of dichroic filters, blocking filters, confocal pinholes, objectives and mirrors to reduce background signals. Not only confocal optics but also standard full field optics can be used. Raman emission signals are detected by a Raman detector comprising a landslide photodiode interfaced with a computer that counts and digitizes the signal.

Any suitable form or configuration of Raman spectroscopy or related techniques known in the art can be used for analyte detection, including normal Raman scattering, resonance Raman scattering, surface enhanced Raman scattering, surface enhanced resonance Raman scattering, b. Coherent Vanstock Raman Spectroscopy (CARS), Stimulated Raman Scattering, Inverse Raman Spectroscopy, Stimulus Gain Raman Spectroscopy, Hyper-Raman Scattering, Molecular Optical Laser Examiner (MOLE) or Raman Microprobe or Raman Microscopy Or confocal Raman microspectral, three-dimensional or scanning Raman, Raman saturation spectroscopy, time resolved resonance Raman, Raman dissociation spectroscopy or UV-Raman microscopy.

The Raman detection device can be operatively coupled with the computer. Non-limiting examples of the computer may include a bus for exchanging information and a processor for processing information. The computer may further include RAM or other dynamic storage, ROM or other static storage and data storage, such as a magnetic disk or an optical disk and a corresponding drive. The computer may also include peripheral devices known in the art, such as display devices (e.g., cathode ray tube or liquid crystal displays), alphabet input devices (e.g., keyboards), cursor control devices (e.g., mice, trackballs, or cursor direction keys) and communication devices. (Eg, an interface device used to couple with a modem, network interface card or Ethernet, token ring or other type of network).

Data from the detection device may be processed by the processor and the data may be stored in the main memory. The processor can confirm the analyte type of the sample by comparing the emission spectra from the analyte on the Raman active substrate. The processor may analyze data from the detection device to determine the identity and / or concentration of the various analytes. Differently equipped computers can be used for specific implementations. After the data collection operation, the data will typically be sent to a data analysis operation. To facilitate the analysis task, the data obtained by the detection device will typically be analyzed using a digital computer as described above. Typically, a computer will be suitably programmed for analysis and reporting of collected data as well as for receiving and storing data from detection devices.

In addition, the present invention is a core having a nano-convex portion on the surface; And it can provide a surface enhanced Raman scattering (SERS) substrate comprising a metal shell having a nano-concave corresponding to the nano-convex portion on the inner surface side.

For example, when the thickness of the metal shell is smaller than the diameter of the nano-convex portion, since hot spots are formed at the interface between the metal shell and the nano-convex portion or the shell exposed to the outside, the Raman activity attached to the surface does not include the Raman active molecule therein. Since the signal of the material can be significantly improved, it can be usefully used as a surface enhanced Raman scattering substrate.

As described above, an example of the nanostructure provided when the shell is formed thinner than the diameter of the nano-convex portion is shown in FIG. 15. As shown in FIG. 15, the structure includes a Raman active molecule in the nanostructure because the structure may have a sharp structure formed not only on the inner surface side but also on the outer surface side of the shell to provide a hot spot with a significantly increased electromagnetic field. Even if not, since it can exhibit an effect of remarkably enhancing the signal of the Raman active molecule located near the tip of the shell formed on the outer surface of the nanostructure can be used as a surface-enhanced Raman scattering substrate as such.

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

Example 1 Synthesis of Nanoshells with Nanoembosses on the Inner Surface

First, a nanoembossed silica core was prepared as a template and the silica core was covered with a gold nanolayer. In FIG. 1A, a method of producing nanoembosses toward the inner surface of a nanoshell layer is schematically depicted through a wet process that is advantageous for mass production of nanoparticles.

Silica cores were synthesized by the Stover method and [Ru (bpy) ₃ ] ²⁺ was loaded as a Raman reporter dye by a modified water-in-oil microemulsion method. Specifically, 1.5 ml of tetraethyl orthosilicate (TEOS, Sigma) is added to 45 ml ethanol containing 2.8 ml of NH ₄ OH, and 0.1 M tris (dope) to dope Ru (bpy) to the silica core. 2,2-bipyridyl) dichlororuthenium (II) (Ru (bpy)) was added to the mixture and reacted for 8 hours. After 126 μl of amTESinopropyltrimethoxy silane (APTES) was added to the mixture and incubated for 8 hours, the APTES treated silica core was boiled for 2 hours. To remove unreacted reagent, the silica core was centrifuged at 2000 g for 30 minutes. After removing the supernatant, the pellet was redispersed in 50 ml of fresh ethanol for 10 minutes using a probe sonicator (VC 750, Sonics). Purification by centrifugation was repeated two more times. The aminated silica cores formed nanoprojected structures with Ru (bpy) doped smaller silica nanoparticles synthesized by the modified water-in-oil microemulsion method. Cores modified with amines by assembling through electrostatic interaction with negatively charged dye-doped silica nanoparticles (SiO ₂ NP; average diameter: 15.7 ± 2.1 nm, and ζ-potential: -17.47 ± 1.73) Average diameter: 123.6 ± 12.7 nm, and ζ-potential: 43.4 ± 0.76). The average distance between nanoembossed silica nanoparticles was calculated to be ~ 23.3 nm. After purification through three centrifugations, gold nanoseeds of 2-3 nm diameter were attached to the nanoembossed silica core by mixing with excess gold nanoseeds. The TEM images of FIGS. 1B and 1C clearly show the emboss structure and uniform adhesion of the gold nanoseeds on the silica core. Finally 6.7 μl of 30% formaldehyde was added in the growth solution to reduce HAuCl ₄ (0.0148%) to grow gold nanoseeds to form a gold nanolayer to cover around the nanoembossed silica core. The size of the synthesized nanoparticles was measured by TEM.

Example 2: Dye Doped Nanoembossed Silica Nanoparticles

Ru (bpy) 90 μl 0.1 M to 0.1 M aqueous solution of polyoxyethylene nonyl phenol ether (Sigma) was added to 10 ml, which was added to 29.6 wt% NH ₄ OH in 100 μl TEOS and 60 μl. After the mixture was stirred for 24 hours, TEOS and carboxyethylsilanetriol, sodium salt (25% by weight aqueous solution, Gelest) were added to the mixture. The mixture was further reacted for 24 hours and ethanol was added to break the microemulsion and recover the particles. The final nanoembossed silica nanoparticles were recovered after three centrifugation wash steps. Afterwards, nanoembossed silica nanointeractions were dispersed in ultrapure water.

Comparative Example 1: Synthesis of gold nanoshell (AuNS) with flat inner surface

Synthesis of the silica core by the stator technique in Example 1, but the inner surface side in the same manner as in Example 1, except that it does not impart nano-embossing by attaching negatively charged dye-doped silica nanoparticles Flat gold nanoshells (AuNS) were synthesized.

Experimental Example 1: Spectroscopic Characterization of Nanoshells with Nanoembosses on the Inner Surface

In FIG. 2, excitation spectra are shown for each of the gold nanoshells (AuNS) having a flat inner surface and the gold nanoshells (AuNS) having a nanoemboss at an inner surface. As shown in FIG. 2A, AuNS flat on the inner surface showed two distinct plasmon resonances (dipole at ˜763 nm and quadrupole plasmon resonance at 615 nm). Plasma resonance observed in AuNS with nanoembosses (FIG. 2b) reduced dipole plasmon resonance compared to AuNS flat toward the inner surface, but with long wavelength shifted (red-shifted) 830 nm compared to AuNS flat toward the inner surface. Characteristic dipole plasmon resonance and quadrupole resonance were shown at 630 nm, respectively. According to the FDTD calculation, the presence of nanoembosses led to the long wavelength shifted excitation spectrum of AuNS (FIG. 3). In addition, the roughness of the surface widened the plasmon spectrum and induced long wavelength shift. As a result, surface roughness and nanoembossing toward the inner surface resulted in a longer wavelength shift than expected spectrum.

Experimental Example  2: Inside surface Nano Emboss  have Nano Shell  Intestinal reinforcement effect by structure

The FDTD calculation confirmed the electromagnetic field enhancement inside the nanoembosses at the SERS excitation wavelength of 633 nm (FIG. 4). As shown in FIG. 4, a strong electromagnetic field was formed near the contact point between the nanoembosses and the core. In response to the distance between neighboring nanoembosses, in the case of AuNS having 100 nanoembosses, the strongest field (| E / E _in | _max. = 55) is formed by the contact of the nanoembosses with the core. It was obtained at two corners (Fig. 5). The shape of a single nanoemboss in contact with the core SiO ₂ NPs was similar to a nanocrescent with sharp rounded corners. The antenna effect is known to cause strong local electromagnetic field enhancement around the sharp rounded corners of the nanocrescent moon. Similarly, forming a nanoshell layer on the nanoembossed silica core can form sharp features and allow for the formation of strong fields around the contact site. To assess the contribution of nanoembosses to the internal SERS, Raman scattering of the reporter dye [Ru (bpy) ₃ ] ²⁺ inside each of the nanoembosses and the core SiO ₂ in AuNS synthesized according to Example 1 Was measured. Figure 6 shows the Raman spectrum of the dye from AuNS with nanoembosses on the inner surface according to shell thickness. Shell thickness was controlled by varying the amount of nanoembossed SiO ₂ NPs while maintaining the amount of HAuCl ₄ in the same volume. The synthesized particles were deposited on a glass substrate modified with APTES. After 2 hours, the substrate was washed three times with ultrapure water and the unattached particles were removed and carefully dried with nitrogen gas. Spots of the sample (focusing the 633 nm laser to 1.03 μm diameter) were scanned 30 times using a 3 second integration time per scan. As shown in FIG. 6, clear Raman scattering bands of the reporter dye were collected. The SERS of each nanoparticle was compared using Raman scattering per particle at 1487 cm ⁻¹ as the characteristic mode of the dye. The strongest Raman scattering signals were obtained from nanoshells with nanoembosses on the inner surface with a shell of ˜28.7 nm thick. In addition, it was confirmed that the Raman scattering of the reporter dye is reduced when having a thick shell of a certain thickness or more.

To assess the contribution of nanoembosses to SERS enhancement, the fluorescence was measured by excitation at 514 nm and the Raman scattering of the reporter dye inside SiO ₂ NP without gold nanoshell was very weak and difficult to measure, AuNS with nanoembosses toward the inner surface measured the SERS spectrum and compared the signal-to-noise ratio. As shown in FIG. 7, in the presence of nanoembosses, Raman scattering per single nanoshell was as strong as the fluorescent signal of the corresponding single nanoparticle without the nanoshell layer. In 514 nm fluorescence efficiency (fluorescence cross section, ~ 2.0 × 10 -19 cm -2) and Raman efficiency of the dye of the reporter ^{dye-Considering} the (Raman cross section, ~ 2.9 × 10 ^{-26 2} cm), the Raman signal enhancement Enhancement factor (EF) is 10 ⁷ level. λ _ex = from 633 nm SERS intensity λ _ex = 2 digits than the intensity at 514 nm The high but 633 nm, since laser output is one digit lower than the 514 nm laser, λ _ex = from 633 nm EF is at least 10 ¹⁰ Level Can be.

Furthermore, the SERS intensity of the dye doped AuNS with nanoembosses was compared with the SERS intensity of the dye inside the core SiO ₂ NP of AuNS without nanoembosses on the inner surface and on the flat outer surface, respectively (FIG. 8). As shown in FIG. 8, the SERS signal of AuNS with nanoembosses was similar to that of the other two structures. SERS on simple AuNS showed a tendency to saturate at a reporter dye concentration of 0.5 mM. The SERS intensity obtained from AuNS with nanoembosses was about 2 times higher than the signal from the reporter dye at 1 mM concentration on flat AuNS. Since the number of dye molecules on the AuNS surface and inside the nanoembosses is unclear, an accurate comparison of SERS enhancement caused by the two structures is currently impossible. However, these results indicate that nanoembosses enable internal Raman signal collection as strong as the signal on the nanoshell outer surface. Similar absorption spectra of the nanoembossed core SiO ₂ NP and the reporter dye doped core SiO ₂ NP confirmed that there was no significant difference in the number of dyes in the two structures (FIG. 9). Given the number of reporter dye molecules, the inner nanoshell layer was as efficient as the AuNS core to provide strong SERS signals. In addition, the nanoembossing technique may be an alternative to carrying a sufficient number of Raman reporter dyes, as well as an alternative to forming a strong field, compared to the other two techniques.

It was found that the inner shell layer of AuNS could provide a novel location for generating strong SERS signals, as much as on the surface or inside the core of the nanoshell. The internal structure of AuNS with nanoembosses resembles a series of arranged crescents. Similar to the crescent moon, the machined AuNS has a structure that produces a strong field. As a result, the structure was able to achieve 10 ¹⁰ SERS enhancement at 633 nm excitation wavelength. Similar SERS intensities at 785 nm and 633 nm excitation wavelengths (FIG. 10) indicate that AuNS with nanoembosses can be usefully used for detection and imaging of biological samples.

Finally, the inventors prepared to include reporter dyes at different locations of the nanoembossing structure and measured the SERS of the reporter dye (FIG. 13). The measured total SERS of the all-in-one structure was higher than three simple sums. The dye-doped silica cores used in the monolithic structures are larger than the dye-doped silica cores, so that a greater number of dye-doped embosses can be attached thereto (FIG. 14D), forming gold nanoshell structures. When the surface roughness is larger (Fig. 14b), as a result, it was possible to obtain a significantly higher SERS than the other three AuNS.

Claims (23)

1. A core having nano convex portions on the surface; And a metal shell having a nano concave portion corresponding to the nano convex portion on an inner surface thereof, wherein the Raman active material is also supported on the nano convex portion of the core, wherein the surface-enhanced Raman scattering (SERS) nanoparticle.
2. The SERS nanoparticle of claim 1, wherein the core having the nanoconvex portion on the surface has a sharp edge capable of forming an electromagnetic field at the bottom edge of the nanoconvex portion.
3. 2. The SERS nanoparticle of claim 1, wherein the metal shell having the nano recesses on the inner surface creates hot spots in which a strong electromagnetic field is generated in a local region corresponding to the nano recesses, thereby providing an increased Raman signal. .
4. The SERS nanoparticles according to claim 1, wherein the Raman active material is also supported on the outer surface of the metal shell.
5. The SERS nanoparticles of claim 1, wherein the core has an average diameter of 20 to 1000 nm.
6. According to claim 1, SERS nanoparticles, characterized in that the average diameter of the nano-convex portion is 5 ~ 50 nm.
7. The SERS nanoparticles of claim 1, wherein the nanoshells have a thickness of 1 to 50 nm.
8. The material of claim 1 wherein the core material is silica, gold sulfide, titanium dioxide, polymethyl methacrylate (PMMA), polystyrene, hydrogels, and combinations thereof. SERS nanoparticles characterized in that selected from the group consisting of.
9. The SERS nanoparticles of claim 1, wherein the core having nanoconvex portions on the surface is formed by self-assembly by electrostatic forces of (+)-charged silica and (-)-charged silica.
10. A first core supporting a first Raman active material and having a (+) charge core portion and a nano-convex portion formed on the surface of the core portion and carrying a second Raman active material and having a (−) charge ; or

    A second core supporting a first Raman active material and having a (-) charge core portion and a nano-convex portion supporting a second Raman active material and having a (+) charge on the surface of the core portion; ; And

    Surface-enhanced Raman scattering (SERS) nanoparticles comprising a metal shell having a nano-concave corresponding to the nano-convex portion on the inner surface side.
11. The SERS nanoparticle of claim 10, wherein the first Raman activator and the second Raman activator are the same or different.
12. 11. The SERS nanoparticle of claim 10, wherein the positively charged silica is silica modified with amine or the negatively charged silica is silica modified with carboxyl groups.
13. A first step of modifying the silica core to carry a (+) or (−) charge;

    Silica core having nano convex portions by assembling the silica nanoparticles doped with the Raman active material on the surface of the silica core formed in the first step through electrostatic interaction, having opposite charges to the silica core formed in the first step. Preparing a second step;

    A third step of loading the Raman active material on the silica core formed in the second step;

    Attaching metal nano seeds to the silica core formed in the third step;

    12. The method of claim 1, further comprising forming a metal shell having nano concave portions corresponding to the nano convex portions on the inner surface of the growth core by growing nano seeds. A method of making the surface enhanced Raman scattering (SERS) nanoparticles described.
14. The Raman probe provided with the surface-enhanced Raman scattering (SERS) nanoparticle in any one of Claims 1-12.
15. A surface enhanced Raman scattering (SERS) substrate coated with the surface enhanced Raman scattering (SERS) nanoparticles according to any one of claims 1 to 12 on the substrate.
16. 16. The SERS substrate of claim 15, wherein the SERS nanoparticle containing coating layer further induces electromagnetic field amplification between adjacent SERS nanoparticles by access or contact of the SERS nanoparticles.
17. a) functionalizing the surface of the surface enhanced Raman scattering (SERS) nanoparticles according to any one of claims 1 to 12 with biomolecules or compounds capable of binding to the analyte to be detected;

    b) exposing the functionalized SERS nanoparticles to a sample comprising one or more analytes; And

    c) identifying the analyte to which the SERS nanoparticles are bound using Raman spectroscopy.
18. i) functionalizing the surface of the surface enhanced Raman scattering (SERS) nanoparticles according to any one of claims 1 to 12 with a biomolecule or compound complementary to the nucleic acid to be detected;

    ii) performing hybridization by reacting the functionalized SERS nanoparticles with a sample that is expected to contain a nucleic acid to be detected; And

    iii) performing Raman spectroscopy to confirm the presence, amount or both of the nucleic acid to be detected to which the SERS nanoparticles are bound.
19. The method of claim 18,

    The sample that is expected to contain the nucleic acid to be detected is used as the collected sample itself, or the nucleic acid to be detected therefrom separated, purified or amplified.
20. 19. The nucleic acid detection method according to claim 18, wherein the nucleic acid detection method is a nucleic acid detection method for diagnosing a disease, identifying an identity, confirming a kinship relationship, identifying a bacterium or a cell, or confirming the origin of an animal or a plant.
21. 19. The nucleic acid detection method according to claim 18, wherein the nucleic acid detection method is a method for detecting single nucleotide polymorphism (SNP).
22. A core having nano convex portions on the surface; And a metal shell having a nano-concave portion corresponding to the nano-convex portion on an inner surface side thereof, surface enhanced Raman scattering (SERS) substrate.
23. 23. The surface enhanced Raman scattering (SERS) substrate of claim 22, wherein the thickness of the metal shell is smaller than the diameter of the nano-convex portion such that a hot spot is formed at the interface of the metal shell and the nano-convex portion or the shell exposed to the outside.

Images