WO2020075879A1 - 라만 활성 나노입자 및 이의 제조방법 - Google Patents

라만 활성 나노입자 및 이의 제조방법 Download PDF

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WO2020075879A1
WO2020075879A1 PCT/KR2018/011873 KR2018011873W WO2020075879A1 WO 2020075879 A1 WO2020075879 A1 WO 2020075879A1 KR 2018011873 W KR2018011873 W KR 2018011873W WO 2020075879 A1 WO2020075879 A1 WO 2020075879A1
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metal
raman
nano
nanocore
nanoparticles
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PCT/KR2018/011873
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English (en)
French (fr)
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유은아
김완선
이태걸
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한국표준과학연구원
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Priority to US16/498,326 priority Critical patent/US12013393B2/en
Publication of WO2020075879A1 publication Critical patent/WO2020075879A1/ko

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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
    • G01N33/54313—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals the carrier being characterised by its particulate form
    • G01N33/54346—Nanoparticles
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
    • G01N33/551—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals the carrier being inorganic
    • G01N33/553—Metal or metal coated
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B82—NANOTECHNOLOGY
    • B82B—NANOSTRUCTURES FORMED BY MANIPULATION OF INDIVIDUAL ATOMS, MOLECULES, OR LIMITED COLLECTIONS OF ATOMS OR MOLECULES AS DISCRETE UNITS; MANUFACTURE OR TREATMENT THEREOF
    • B82B1/00—Nanostructures formed by manipulation of individual atoms or molecules, or limited collections of atoms or molecules as discrete units
    • B82B1/008—Nanostructures not provided for in groups B82B1/001 - B82B1/007
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B82—NANOTECHNOLOGY
    • B82Y—SPECIFIC USES OR APPLICATIONS OF NANOSTRUCTURES; MEASUREMENT OR ANALYSIS OF NANOSTRUCTURES; MANUFACTURE OR TREATMENT OF NANOSTRUCTURES
    • B82Y15/00—Nanotechnology for interacting, sensing or actuating, e.g. quantum dots as markers in protein assays or molecular motors
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B82—NANOTECHNOLOGY
    • B82Y—SPECIFIC USES OR APPLICATIONS OF NANOSTRUCTURES; MEASUREMENT OR ANALYSIS OF NANOSTRUCTURES; MANUFACTURE OR TREATMENT OF NANOSTRUCTURES
    • B82Y30/00—Nanotechnology for materials or surface science, e.g. nanocomposites
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B82—NANOTECHNOLOGY
    • B82Y—SPECIFIC USES OR APPLICATIONS OF NANOSTRUCTURES; MEASUREMENT OR ANALYSIS OF NANOSTRUCTURES; MANUFACTURE OR TREATMENT OF NANOSTRUCTURES
    • B82Y40/00—Manufacture or treatment of nanostructures
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B82—NANOTECHNOLOGY
    • B82Y—SPECIFIC USES OR APPLICATIONS OF NANOSTRUCTURES; MEASUREMENT OR ANALYSIS OF NANOSTRUCTURES; MANUFACTURE OR TREATMENT OF NANOSTRUCTURES
    • B82Y5/00—Nanobiotechnology or nanomedicine, e.g. protein engineering or drug delivery
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
    • G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
    • G01N21/65—Raman scattering
    • G01N21/658—Raman scattering enhancement Raman, e.g. surface plasmons

Definitions

  • the present invention relates to Raman active nanoparticles and their manufacturing methods, in detail,
  • SERS surface-enhanced Raman scattering
  • SERS spectroscopy is a measurement technology with high selectivity and high informationality.
  • Detection of SERS spectroscopy-based materials is mainly in the form of particulates or plasmon metal thin films.
  • hot-spot based on a plasmon metal on a wire KR2017-0129633
  • a hot-spot-based SERS substrate formed by contacting two or more plasmon particles.
  • the purpose of the present invention is to provide a free Raman active nanoparticle and its manufacturing method from the size limitation of the analyte, which has Raman activity, specifically surface-enhanced Raman scattering activity (hereinafter, SERS activity).
  • SERS activity specifically surface-enhanced Raman scattering activity
  • Another object of the present invention is that organic materials, including Raman reporters,
  • Another object of the present invention is to provide a Raman active nanoparticle and a method for manufacturing the Raman scattering signal, which are capable of generating a markedly improved Raman scattering signal because two or more different hot-spots are located on the particle itself.
  • Another object of the present invention is a wide range of localized surface plasmon (LSPR).
  • LSPR localized surface plasmon
  • Another object of the present invention is to provide a manufacturing method that can produce large quantities of active nanoparticles in a short period of time with excellent durability and high SERS activity.
  • the method of manufacturing Raman active nanoparticles according to the present invention is a) buffer solution (buffer
  • step b) a self-assembled monolayer of Raman reporter surrounding the metal nanocore may be formed.
  • the molar ratio of R1 divided by the number of moles of the buffer divided by the number of moles of the first metal precursor may be 200 to 750.
  • the second reaction solution may include a surfactant or a surfactant and an organic acid.
  • the complete solution is HEPES (4- (2-hydroxy ethyl) -1 -piperazineethanesulfonic acid), MES (4- (2-Hydroxyethy l) piperazine-1 -ethanesulfonic acid), PBS (Phosphated buffered saline), Tris (2-Amino-2hydroxymethyl propne- 1,3 -idol), PB (Phosphate buffer), MOPS (3- (N-morpholino) propanesulfonic acid ),
  • TAPS (3-[[l, 3-dihydroxy-2- (hydroxymethyl) propan-2-yl] amino] propane-l -sulfonic acid) and PIPES (piperazine-N, N'-bis (2-ethanesulfonic acid)) It may contain one or more selected from.
  • the metal of the precursor can be for show or as well.
  • ⁇ fixing a receptor that binds to the analyte to the metal well may further include.
  • the present invention is the Raman activity prepared by the method of manufacturing the Raman activity nanoparticles described above.
  • Raman activated nanoparticles according to the present invention are nano-star-shaped metal nanocores; above
  • It includes a self-assembled monolayer film comprising a Raman reporter fixed to a metal nano-core; and a metal well surrounding the self-assembled monolayer film; and has a striated structure by a nano-star-shaped protrusion.
  • the nanoparticles can have a shape corresponding to the nano-star shape.
  • the metal nano-core has a size of 10 to 5011111 in the center region and 5 to 7011111 in size and protrudes from the center region to the protruding direction. It may include an overhanging projection.
  • the Raman active nanoparticles may further include a receptor fixed to the metal shell and bound with an analyte.
  • the Raman active nanoparticle according to the present invention contains a nano-star-shaped metal nanocore, a strong hot-spot is located in the particle itself, and a nanogap of uniform size between the metal nanocore and the metal well Only (hot spot) is formed in the whole area of the active nanoparticles, and as the Raman reporter is located in the hot-spot nanocap, the very good Raman signal can be enhanced.
  • the Raman active nanoparticles according to the present invention are organic containing Raman reporters.
  • the components are wrapped and protected by a metal well, and the two functional groups of the Raman reporter, the metal nano-core and the self-assembled monolayer of the Raman reporter-the metal shell are strongly bonded, which has very good durability and physical / chemical stability. There are advantages.
  • the method of manufacturing Raman active nanoparticles according to the present invention is a very simple method of producing Raman active nanoparticles having the above-mentioned advantages, and mass production at room temperature in a short time. There are possible advantages.
  • Fig. 1 is an observation of a metal nanocore manufactured according to an embodiment of the present invention.
  • Fig. 2 shows the observation of the Au nanocore dispersion dispersed in the HEPES buffer solution.
  • FIG 3 is a view showing the measurement of light absorption of a metal nanocore manufactured according to an embodiment of the present invention.
  • Figure 4 observes the Raman active nanoparticles prepared according to an embodiment of the present invention
  • SERS surface-enhanced Raman scattering
  • the method of manufacturing active nanoparticles according to the present invention is a) buffer solution (buffer
  • the Raman reporter has a binding force to each of the first metal of the metal nanocore and the second metal of the metal shell.
  • the nano-star shape protrudes from a single central region and a central region, and one or more, specifically two or more, more specifically 2 to 10, more specifically 3 to 8 projections, which are reduced in the direction of projection. It may be a shape including.
  • the shape of the protrusion may be polygonal or conical, but it is not limited to this. If the metal nanocore has more than one protrusion, The shape or size of each protrusion (protrusion length) can be the same or different.
  • two or more protrusions with respect to the center area may have a symmetrical relationship, form a certain angle with each other, or the protrusions may be projected at random locations in the center area.
  • the method of manufacturing active nanoparticles according to the present invention described above is a buffer solution and a metal.
  • the surface of the nanocore is not in a state in which organic substances (such as surfactants) are strongly bound, but may be in a pure (bare) metal state.
  • organic substances such as surfactants
  • the Raman reporter in step b) is fixed, it is stable in all areas of the metal nanocore.
  • the reporter can be fixed only in the form of a self-assembled monolayer.
  • the first reaction solution may not contain a surfactant (organic surfactant), and further, the first reaction solution is a surfactant May not contain all organic acids.
  • the method of manufacturing active nanoparticles according to the present invention includes a buffer solution and
  • Metal nano-core formation using a liquid containing a metal precursor, Raman reporter attachment, and metal well-forming are used to manufacture Raman active nanoparticles, which has the advantage of producing large amounts of Raman active nanoparticles in a short time at low cost. .
  • the method of manufacturing Raman active nanoparticles according to the present invention is an organic substance containing Raman reporters, but is not exposed on the surface of the active nanoparticles and is wrapped by a metal shell, so that organic substances containing Raman reporters are included. It has the advantage that it can be stably protected from other environments.
  • the method of manufacturing active nanoparticles according to the present invention is made of metal nanocores.
  • Raman has the advantage of enhancing the scattering signal with only the active nanoparticle itself.
  • a hot-spot means a region where a very strong Korean small electric field is formed and localized surface plasmon resonance (LSPR) occurs.
  • LSPR localized surface plasmon resonance
  • signal augmentation When signal augmentation is caused by hot spots between components, signal augmentation can be achieved only when the analyte is located (or combined) in the nanogap region or near the nanogap region of the two components. These spatial constraints limit the size of the analyte. This makes it impossible to fit an analysis of biochemicals on the order of a few micrometers or dozens of micrometers.
  • the method of manufacturing active nanoparticles according to the present invention is made of metal nanocores.
  • nano-star shape it is possible to tune the LSPR wavelength in a very wide range by adjusting the shape or size of the nano-star (including the length of a branch).
  • the tuning of the LSPR wavelength can be achieved even in the 800 nm region, which is not possible in the spherical shape.
  • the LSPR wavelength that can be tuned above 800 nm is not visible, but near infrared ( NIR, 780nm ⁇ 1500nm) means that detection and analysis of analytes can be achieved by light irradiation in the band.
  • fluorescence may occur when visible light is irradiated to biomaterials containing biochemicals. Fluorescence intensity is stronger than Raman scattering, and only fluorescence occurs in a region similar to scattering, which causes the fluorescence peak. There is a problem in that it is difficult to obtain an obscure pure Raman spectrum. Therefore, the SERS analysis by light irradiation in the near-infrared band, not visible light, is very advantageous in the bio field because it is possible to obtain a Raman spectrum without influence (interference) of fluorescence.
  • the pH of the buffer solution can be controlled to control the shape, size, or shape and size of the metal nanocore by controlling one or more factors.
  • the buffer solution is usually used with conventional inorganic acids such as HC1, NaOH, etc. to adjust the pH.
  • conventional inorganic acids such as HC1, NaOH, etc.
  • it may contain a compulsory base or a mixture of these.
  • the molar ratio of the number of moles of the buffer in the first reaction solution divided by the number of moles of the first metal precursor may be 200 to 750.
  • a metal nanocore having a nano-star shape is produced. It can be manufactured, and nano-star-shaped metal nano-cores with a center region size of 10 to 50 nm and specifically 10 to 40 nm can be manufactured.
  • silver can be 500 to 750 days.
  • Nano-star metal nano-core having 3 or 8 protrusions, specifically 5 to 70 nm, specifically 5 to 50 nm, and more specifically 10 to 50 nm, by controlling 500 to 750 Can be manufactured.
  • the yarn R1 within the range of 200 to 750, it is possible to control the length of the protrusion (extrusion or branch) protruding from the center region of the metal nanocore.
  • the value of R1 within the range of 200 to 750 By increasing the length of the protrusion, the LSPR wavelength of the active nanoparticles (or metal nanocores) can be controlled by controlling the length of the protrusion, and by adjusting within the range of 200 to 750, the LSPR wavelength is 600 to 900 nm. This can be adjusted.
  • the pH of the buffer solution can be 5.0 to 7.5 days
  • the length of the protrusion can be increased by increasing the pH of the buffer solution.
  • Buffer solutions include HEPES (4- (2-hydroxyethyl) -1 -piperazineethanesulfonic acid), MES (4- (2-Hydroxyethyl) piperazine-1 -ethanesulfonic acid), PBS (Phosphated buffered saline), Tris (2-Amino-2hydroxymethyl propne-1,3-idol), PB (Phosphate buffer), MOPS (3- (N-morpholino) propanesulfonic acid),
  • TAPS (3-[[l, 3-dihydroxy-2- (hydroxymethyl) propan-2-yl] amino] propane-l-sulfonic acid) and PIPESipiperazine-N'-bis ⁇ -ethariesulfonic acid)
  • these buffers can act as weak reducing agents to reduce metals, and surfactants for stabilizing metal nanocores can be excluded by buffers, thus producing metal nanocores with pure metal surfaces.
  • the buffer solution aqueous solution
  • the first metal of the first metal precursor may be a metal in which surface plasmons are generated by interaction with light.
  • the first metal is silver, silver, platinum, palladium, nickel, aluminum, copper, or these. is a mixture or the like alloy of these.
  • the biological origin, in consideration of the stability of the substance or biochemical first metal is preferably gold or silver in. in the first metal according to an advantageous example, the first metal
  • the precursors are HAuCl 4 , HAuBr 4 , NaAuCl 4 , AuCl 3 .3H 2 0, NaAuCl 4 _2H 2 0, or mixtures thereof.
  • It may be a monetary sphere, or may be a precursor such as AgNO 3 , but it is of course not possible that the present invention cannot be limited by the type of sphere material of the metal precursor.
  • the first reaction solution is prepared by mixing the first metal precursor solution with the buffer solution, and the first reaction solution is reacted at a temperature of 15 to 40 O C to react the metal nanocore. It may include manufacturing steps.
  • the molar concentration of the buffer in the buffer solution may be 100 to 400 mM, and the first metal
  • the molar concentration of the first metal precursor may be 20 to 60 mM.
  • the concentration of the buffer solution and the concentration of the first metal precursor solution are not necessarily limited to the above-mentioned range.
  • the solutions can be mixed to satisfy R1 described above.
  • the reaction may proceed simultaneously with the mixing of the buffer solution and the first metal precursor solution, and the reaction may be 15 to 40 ° C, more specifically 15 to 35 ° C.
  • the temperature of 15 to 25 O C more specifically room temperature (21 to
  • the room temperature can mean the temperature without artificially applying thermal energy to the first reaction solution.
  • the reaction time is sufficient time to complete the synthesis of the metal nanocore. You can do it, specifically, 10 to 10 It may be 50 minutes, more specifically 20 to 40 minutes, but the present invention is not limited to this.
  • stirring of the first reaction solution may be selectively performed if necessary.
  • the reaction yield may be improved, but the shape or size of the manufactured metal nanocore may be stirred. It is virtually unaffected by conditions. Stirring needs to be between 500 rpm and 1500 rpm.
  • step a) al) preparing a first reaction solution by mixing the first metal precursor solution with a buffer solution, and reacting the first reaction solution at a temperature of 15 to 40 ° C to react the metal nanocore.
  • the metal nanocores are not separated and recovered from the first reaction solution, and stored in the state of the first reaction solution containing the metal nanocores, but can be stored at a temperature of 1 to 10 ° C, specifically at a temperature of 1 to 5 ° C.
  • step a) al) mixing the first metal precursor solution with the buffer solution to prepare a first reaction solution, and reacting the first reaction solution at a temperature of 15 to 40 ° C. Preparing a core; and a2) recovering the metal nanocore from the first reaction solution that has been completed and dispersing it in a buffer solution (separate buffer solution) to a temperature of 1 to 10 O C, specifically 1 to 5 O C And storing at a temperature.
  • the metal nanocore prepared in step al) is stored at a low temperature of 1 to 10 O C as a dispersion medium, the metal nanocore can be stored
  • the first reaction solution stabilizes and disperses the nanoparticles.
  • step al can be performed using only the buffer solution and the first metal precursor solution.
  • the manufacturing method is metal by simple mixing of two solutions and room temperature reaction of tens of minutes.
  • metal nanocores are very suitable for mass production.
  • step a) Raman reporter for metal nanocore
  • the fixing step can be performed.
  • the Raman reporter can mean an organic compound (organic molecule) containing an active molecule of Raman, and has a binding force with a metal of a metal nanocore, and an organic compound (organic molecule) containing an active molecule. Reporters are well known and can be used without limitations as long as they are widely used in this technical field.
  • the Raman reporter (molecule) has a binding force with the metal of the metal nanocore
  • the Raman reporter's self-assembled monomolecular film is applied to the metal nanocore where the pure metal surface is exposed. Can be formed.
  • Organic surfactants that can reduce growth, inhibit growth, induce growth in a specific direction, and / or stabilize nanoparticles are used for mainstream use.
  • organic acids are used, or organic acids that can replace surfactants.
  • the surface of the metal nanoparticles synthesized in this way combines a metal nanoparticle metal material with organic surfactants or organic acid-derived organic functional groups with very strong binding force. Due to this, there is a problem in that the surface of the metal nanoparticles is uniformly and completely wrapped (replaced) as a target function.
  • step a) in the state where organic acid or organic surfactant is excluded, a metal nanocore is completely prepared from the buffer solution and the metal precursor, and as described above, dispersion is maintained stably in the buffer solution.
  • Manufacturing metal nano-core can purely represent the surface state of the metal itself. Due to the surface state of the metal, the metal nano-core Eraman reporter (organic compound having binding force with the metal nano-core and containing Raman active molecules) ) Is uniformly and homogeneously and spontaneously combined, and even though the irregularity of nano-stars is severe, a self-assembled monolayer of the Raman reporter can be stably formed.
  • Raman active molecules can include surface-enhanced Raman active molecules, surface-enhanced resonance Raman active molecules, hyper Raman active molecules, or coherent anti-Stokes Raman active molecules. Or, it can have a Raman signal.
  • the Raman active molecule is cyanine, fluorescein, rhodamine, 7-nitrobenzene-2 -oxa-1,3-diazole
  • Examples of the cyanine include Cy3, Cy3.5, or Cy5 may be included.
  • Rhodamine 6G R6G
  • Isothiol tetramethyl rhodamine isothiol: TRIT
  • sulforhodamine 101 acid chloride Texas Red dye
  • the Raman active molecule may be a benzene ring type Raman active molecule, and the benzene ring type Raman active molecule may be 4-AminothiophenoI (4-ATP), 4-Mercaptobenzoic acid (4-MBA), Phenyl isothiocyanate (PITC), Benzenethiol (BT),
  • the Raman reporter is composed of a metal nanocore metal (first metal) and a metal shell.
  • the metal (second metal) may be an organic compound (organic molecule) that has a binding force with each and contains an active molecule.
  • the Raman reporter contains the Raman active molecule, but has an agent i function that voluntarily bonds with the first metal, and has a second metal (the second metal of the second metal precursor).
  • the second functional group is more smooth and uniform
  • the second metal shell and the Raman reporter can greatly improve the bonding force between the fixed metal nano-core, i.e., the Raman reporter is also used with the metal nano-core and with the metal well. As it has cohesion,
  • the self-assembled monolayer may be chemically bound to the metal nanocore by the first functional group and chemically bound to the metal shell by the second functional group.
  • the functional group (first functional group or second functional group) takes into account the metal and
  • any functional group known as spontaneous binding may be used.
  • the functional group is a thiol group (-SH), a carboxyl group. It may be (-COOH) or an amine group (-NH 2 ), but the invention is not limited by the specific type of functional group.
  • TPDT -Terphenyl-4,4 ”-dithiol
  • a Raman reporter self-assembled monomolecular film of the Raman reporter can be formed on the metal nanocore by spontaneously binding (fixing) the Raman active molecule having a binding force with the metal of the metal nanocore by the first functional group.
  • the Raman reporter Despite being an anisotropic shape, the Raman reporter has an extremely uniform thickness. 2020/075879 1 »(: 1 ⁇ 1 ⁇ 2018/011873 Allow a single molecular film to be formed homogeneously on the entire surface of the metal nanocore.
  • nanogaps of extremely uniform size thereby allowing a uniformly controlled hot spot to be formed in all areas of the active nanoparticle.
  • the length (size) of the Raman reporter in terms of hotspot formation in which stronger signal enhancement is achieved is urine pie, and specifically 0.5 to
  • the non-step of fixing the metal nanocore Raman reporter may include a step of preparing a mixed solution containing the metal nanocore and Raman reporter prepared in the step and stirring the ultrasonic wave.
  • 1 ) is a step of mixing the metal nanocores and Raman reporter prepared in step 1? 1) 3) so that the molar concentration is 0.01 to 11 ⁇ 1 and 10 to 1000. ; 10 to 30 minutes at room temperature with ultrasonic agitation; and ⁇ 4) separating and recovering the metal and nanocores (where the Raman reporter's self-assembled monolayer is formed) with a fixed Raman reporter.
  • Silver water system
  • It can be a mixed solution, and the mixed solution is used to prevent the aggregation of metal nanocores.
  • Water-soluble phosphorus aromatic compounds may be further included.
  • step I?) Is performed, 0) the nanocore and the second metal with the Raman reporter fixed
  • a step of forming a metal shell surrounding the fixed Raman reporter nanocore from the second reaction solution in which the precursor is mixed may be performed.
  • the nanocore may be a metal nanocore having a self-assembled monolayer of Raman reporter.
  • the metal shell formed in step 0) forms a metal nanocore and a nanogap by a Raman reporter fixed to the nanocore, it is a hot spot in the form of a surface corresponding to the surface of the metal nanocore, not a dot or line shape. This has the advantage of being able to acquire a stronger Raman scattering signal.
  • the size of the nanogap is determined by the self-assembled monolayer of the Raman reporter, by forming a self-assembled monolayer with a Raman reporter having a properly designed size, The size of the nanogap can be controlled uniformly and precisely in the nano-star region.
  • the second reaction solution is a nanocore (metal nanocore) with a fixed Raman reporter, a second metal
  • the nanoparticles have a shape that corresponds substantially to the shape of the nano-star-shaped metal nano-core, and forms a metal shell in the form of an extremely thin, uniform, and smooth film.
  • the receptor that specifically binds the analyte to the nanoparticles can be fixed more uniformly, and it is advantageous because more receptors can be fixed.
  • the surfactant contained in the second reaction solution may be a cationic surfactant.
  • the cationic surfactant may be a quaternary ammonium salt, but a quaternary ammonium salt
  • nitrogen compound it can contain not only a chain alkyl group but also a nitrogen heterocyclic compound containing a nitrogen-containing compound.
  • the cyclic compound may include pyridum salt, quinolium, imidazolium, or a mixture thereof.
  • the quaternary ammonium salt surfactant can only touch the following formula (1) so that it can be reduced to form a metal shell in a very smooth and thin dense film form.
  • R1, R2 and R3 are independently of each other a C1 C3 alkyl group
  • R4 is a C10 to C19, advantageously a C14 C18 alkyl group, which is a counterion of ammonium ions.
  • X may be halogen.
  • a quaternary ammonium salt surfactant satisfying Chemical Formula 1 hexadecyl-trimethylammonium chloride (CTAC),
  • CTAB Hexadecyl-trimethyl-ammonium bromide
  • the second reaction solution is quaternary.
  • Organic acids can contain more organic acids with ammonium salt surfactants.
  • Organic acids are acetic acid, propionic acid, fumaric acid, malic acid, succinic acid, glycolic acid, tartaric acid, citric acid, butyric acid, palmitic acid, formic acid; lactic acid, oxalic acid, oxalic acid, tartaric acid , Can be one or more selected from the group consisting of ascorbic acid, citric acid and isocitric acid, but is not limited to this.
  • the second metal of the second metal precursor also interacts with light, causing surface plasmon to
  • the metal may be generated, and the second metal may be gold, silver, platinum, palladium, nickel, aluminum, copper or a mixture thereof, or alloys thereof.
  • the second metal is the first metal in consideration of biostability. It is preferable that the metal is gold or silver independent of the metal.
  • the second metal precursor is HAuCl 4 , HAuBr 4 , NaAuCl 4 , AuCl 3 o 3H 2 0, NaAuCl 4 o 2H 2 0, Or it may be a gold precursor such as a mixture thereof, or, AgNO 3 and the like may be a precursor, but is not limited thereto. 2020/075879 1 »(: 1 '/ 10 ⁇ 2018/011873
  • step 0 the surfactant solution, the second metal precursor solution and Raman
  • a second reaction solution is prepared by mixing a metal nano-core dispersion in which the reporter is fixed, and the second reaction solution is reacted within 15 minutes at a temperature of 15 to 40 ° (: advantageously at room temperature, specifically 5 to 15 minutes)
  • the second reaction solution can be prepared by mixing a surfactant solution, a second metal precursor solution, a metal nanocore dispersion with a fixed Raman reporter solution, and an organic acid solution. .
  • the molar concentration of surfactant in the surfactant solution may be 0.1 to 0.3M
  • the molar concentration of the second metal precursor in the second metal precursor solution may be 1 to 2011 114, and the molar concentration of the metal nanocore in the metal nanocore dispersion in which the Raman reporter is fixed may be 0.01 to 0.511], and the organic acid molar concentration of the organic acid solution May be 0.01 to 0.31, but is not limited to this.
  • the surfactant solution and the second metal precursor solution so that the molar ratio of the number of moles of surfactant divided by the number of moles of the second metal precursor is 200 to 500 and specifically 300 to 500
  • the organic acid solution may be mixed so that the mole ratio of the surfactant divided by the mole number of the organic acid is 113 to 0.20, specifically 0.05 to 0.1.
  • the reduction of the second metal is spontaneously achieved at room temperature, the reduction is achieved at an appropriate rate, but the shape of the nano-star metal nanocore is virtually maintained, and the thin, dense, and smooth film of the second metal (metal shell) It is a suitable condition to be generated.
  • the metal nanocore dispersion may be mixed so that the molar ratio of the second metal precursor: metal nanocore is 1: 1x10 7 to 1x10. At this time, the metal nanocore (s) can be uniformly formed with metal wells. 2 After the metal precursor solution and the metal nanocore dispersion are first mixed, the surfactant solution or the surfactant solution and the organic acid solution may be mixed.
  • step 0), 01) use a second metal precursor solution and a metal nanocore dispersion.
  • the second reaction solution is prepared by mixing a surfactant-nanocore mixture with a surfactant solution or a surfactant solution and an organic acid solution, and the second reaction solution is within 15 minutes at a temperature between 15 and 40 ° Only by reacting to prepare the active nanoparticles; may include.
  • Raman active nanoparticles can be produced comprising a self-assembled monolayer, a metal well surrounding the self-assembled monolayer.
  • the Raman reporter's self-assembled monolayer is produced by forming a metal shell with a smooth surface with a very uniform and thin thickness in all areas of the nanocore.
  • Raman activated nanoparticles may have a protruding portion derived from the nano-star shape of the metal nanocore, and in fact, may have a shape corresponding to the nano-star shape of the metal nanocore. It has a shape that protrudes at a position substantially the same as the position of the protruding part of the nano-star of the nano-core, and can mean a shape protruding substantially the same as the number of protruding parts of the nano-star of the metal nano-core.
  • the average size is less than 150nm (similar volume conversion diameter)
  • Raman-active nanoparticles of sizes of practically 60 to 100 nm, more practically 65 to 80 nm can be produced.
  • the method of manufacturing active nanoparticles according to an embodiment of the present invention may further include; c) after step d) fixing a receptor that binds (specifically binds) the analyte to the metal shell; have. It is understood that step d) can be performed by mixing the prepared Raman active nanoparticle dispersion with a receptor, and can be carried out according to the protocol known for each receptor.
  • the receptor can be any substance known to be a complementary bond between the analyte and the enzyme-substrate, antigen-antibody, protein-protein or DNA, where the receptor is a functional group that voluntarily binds the second metal in the metal shell. (For example, thiol group, carboxyl group or amine group, etc.), and may be in a state of being spontaneously bound to a metal well by a functional group.
  • the analyte can be a living organism (including a virus) or a non-biologically derived material.
  • the organism-derived material can contain cellular components.
  • the analyte is a lesion-marking biomaterial, a lesion-marking material with lesion specificity. It can be a pathogen, a drug, an organic compound, a biochemical or a bio-derived substance.
  • Biochemicals or bio-derived substances can be peptides, carbohydrates, proteins, protein complexes, lipids, metabolites, antigens, antibodies, enzymes, substrates, amino acids, pressures These may include tamers, sugars, nucleic acids, fragments of nucleic acids, peptide nucleic acids (PNAs), cell extracts, or mixtures thereof, but are not limited thereto.
  • PNAs peptide nucleic acids
  • the produced Raman active particles may be for in-vivo or in-vitro.
  • the present invention includes Raman active nanoparticles prepared by the above-described manufacturing method.
  • nanoparticles metal nanocores, nano-stars, Raman reporters, self-assembled monolayers, metal shells, analytes, and receptors are the same as described above in the preparation method of active nanoparticles.
  • Raman activated nanoparticles include all of the above described in the manufacturing method of the Raman activated nanoparticles.
  • the Raman active nanoparticles according to the present invention include a nano-star-shaped metal nanocore; a self-assembled monolayer including a Raman reporter fixed to the metal nanocore; and the 2020/075879 1 »(: 1 ⁇ 1 ⁇ 2018/011873 A metal shell that wraps a self-assembled monolayer; and has a striated structure by the nano-star-shaped protrusion.
  • Raman activated nanoparticles according to the present invention may contain nano-star-shaped metal nanocores.
  • Nano-star-shaped nano-cores have a center area size of 10 to 50 11111 ,
  • Nanocores can have three or more protrusions, specifically three to eight protrusions.
  • the Raman-active nanoparticles contain nano-star-shaped nanocores, the Raman-active nanoparticles themselves can have a hot-spot, thereby enhancing Raman signaling with the Raman-active nanoparticles themselves.
  • the nano-star size and spherical shape can be easily tuned to the wavelength, and has a wide advantage in that the tuning range reaches 80011111. 11 wavelengths of 1 stone reaching 80011111 area, subject to analysis by near infrared irradiation
  • the only active nanoparticles according to the present invention are nanocores and nano-stars.
  • the Raman reporter is fixed to the nano-core in the form of a self-assembled monolayer, it has the advantage of exhibiting uniform and stable representational activity in all areas, and the Raman reporter is positioned as a hot spot. Enhancement of the signal can be achieved.
  • active nanoparticles according to the present invention are self-assembled monolayer membranes in metal shells.
  • Raman active nanoparticles according to the present invention are Raman reporter first metal (metal
  • Nanocore As it has a first functional group that voluntarily bonds with a 12th functional group that voluntarily bonds with a second metal (metal shell), it has a very strong bond between the nanocore-self-assembled monolayer film and the metal shell, resulting in excellent durability and stability. It has the advantage of having
  • the Raman active nanoparticles according to the present invention correspond to the thickness of the self-assembled monolayer between the metal well and the nanocore (corresponding to the size of the Raman reporter), as the Raman reporter is fixed to the nanocore in the form of a self-assembled monolayer.
  • a uniform sized nanogap (hot spot) is formed, which can increase the Raman signal.
  • the metal well in the state of being combined with the self-assembled monolayer by the Raman reporter's functional group may be 3 to 2011 111, specifically 5 to 1511111, and may be a thin and uniform thickness dense film.
  • Raman activated nanoparticles are made of metal.
  • It can have a shape that corresponds to the nano-star shape of the nanocore.
  • the nano-star shape of the nano-core corresponds to the nano-star.
  • Augmentation of the Raman signal can be achieved by a surprisingly enhanced by the synergistic action of the shaped metal shell structure.
  • Raman-activated nanoparticles can further contain a receptor that is bound to an analyte by being fixed to a metal well, and the receptor can contain a functional group that voluntarily binds to the metal shell.
  • a receptor that specifically binds to the analyte By this, only the analyte can be analyzed and detected by spectroscopy (SERS spectroscopy), and further, sensing or imaging of the analyte can be achieved.
  • SERS spectroscopy spectroscopy
  • Raman active nanoparticles described above may be used for in-vivo or in-vitro.
  • the prepared metal nanocore was stored at a temperature of 4 O C in a HEPES buffer solution of l40 mM concentration until observation or subsequent fixation of the Raman reporter.
  • the nano-star-shaped Au nanocore is manufactured, and the size of the center region is about 30 nm, and the length of the protrusion is about 20-30 nm, and the nano-star-shaped Au nanocore is manufactured. Can be seen.
  • Fig. 2 is an optical photograph of the Au nanocores stored in the HEPES buffer solution, as shown in Fig. 2, stably maintaining the dispersion of Au nanocores without the aid of a separate surfactant or organic dispersant. Can be checked
  • FIG. 3 is a view showing the light absorption of a metal nanocore manufactured according to an embodiment of the present invention.
  • Figure 4 shows the formation of a self-assembled monolayer of Show 11 nanocore Eraman reporter
  • the Raman active nanoparticles prepared by forming a show 11 shell as a second reaction solution were observed.
  • the nano-core is separated by centrifugation (8000 ⁇ ! 11, 10 minutes) (II [[ yeomyo 7.2)
  • the manufactured Raman active nanoparticles have a shape corresponding to the nano-star shape of the metal nanocore, and the metal well is very smooth and
  • the Raman reporter reliably wraps the entire area of the fixed metal nanocore in the form of a dense and uniform film.
  • FIG. 5 is a diagram showing the surface-enhanced Raman scattering spectrum of the prepared Raman active nanoparticles (sample in FIG. 4).
  • the 5 £ 1 spectrum was obtained by irradiating 51411111, 63311111 or 7851101 light with Raman activated nanoparticles using a micro Raman system 03 ⁇ 4.
  • FIG. As you can see in Figure 5, near-infrared light so that the band is remarkably by 7,850,111 light is a strong Raman signal is obtained, and can make up and 1100 (- near the first area and 1,550,011 - strong Raman signal is observed in the vicinity of the first region is a Raman reporter (061) It can be seen that it matches the unique signal.

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Abstract

본 발명에 따른 라만 활성 나노입자의 제조방법은 a) 완충용액 (buffer solution)에 제 1 금속 전구체가 혼합된 제 1반응액으로부터 나노-별 형상의 금속 나노코어를 제조하는 단계; b) 상기 금속 나노 코어에 라만 리포터를 고정하는 단계;및 c) 상기 라만 리포터가 고정된 나노 코어와 제 2금속전구체가 혼합된 제 2 반응액으로부터 라만 리포터가 고정된 나노코어를 감싸는 금속 쉘을 형성하는 단계;를 포함하며, 상기 라만 리포터는 상기 금속 나노코어의 제 1금속 및 상기 금속 쉘의 제 2 금속 각각에 결합력을 갖는다.

Description

2020/075879 1»（：1/10公018/011873 명세서
발명의명칭:라만활성나노입자및이의제조방법 기술분야
[1] 본발명은라만활성나노입자및이의제조방법에관한것으로,상세하게 ,
표면증강라만산란 (SERS)활성을갖는나노입자및이의제조방법에관한 정이다.
배경기술
[2] SERS(Surface-Enhanced Raman Scattering,이하 SERS)분광법은금，은등의
금속나노구조표면에분자가흡착될때라만산란의세기가급격히 10 6니 08배 이상증가되는현상을이용한분광법이다.현재아주빠른속도로발전하고있는 나노기술과결합하여단하나의분자를직접측정할수있는고감도의기술로， 특히메디컬센서로서긴요하게쓰일수있을것으로많은기대를받고있다.
[3] SERS분광법은고선택성및고정보성을갖는측정기술임과동시에 ,
초고감도의화학적/생물학적/생화학적분석을위한강력한분석방법임에따라, 고감도 DNA분석과더불어현재알쯔하이머병혹은당뇨병등을비롯한다양한 질병의초기진단을수행하려는연구가활발히진행되고있다.
[4] SERS분광기반물질의검출은주로플라즈몬금속박막에입자상또는
와이어상의플라즈몬금속이위치하여형성되는핫-스팟 (KR2017-0129633), 또는둘이상의플라즈몬입자들이접촉하여형성되는핫-스팟에기반한 SERS 기판을이용하는것이통상적이다.
[5] 그러나，이러한두구성요소간의접촉에의한핫-스팟은두구성요소사이의
나노갭이라는극히제한된크기와영역내에서만표면증강라만산란이
이루어질수있음에따라，수내지수십마이크로미터크기의
생화학물질 (바이오물질)의검출이어려운문제점이있다.
[6] 또한，생화학물질이나생체유래물질의경우가시광이조사시형광현상이
발생하여순수한라만스펙트럼을얻기어려운문제점이있다.
[7] 이에 ,가시광이아닌근적외선대역의광조사에의해 SERS분석이가능하며
분석대상물에크기제한없이신호의증강이이루어질수있는라만활성및 플라즈몬활성입자의개발이요구되고있다.
발명의상세한설명
기술적과제
[8] 본발명의목적은라만활성，구체적으로표면증강라만산란활성 (이하， SERS 활성)을가지며분석대상물의크기제한으로부터자유로운라만활성나노입자 및이의제조방법을제공하는것이다.
[9] 본발명의다른목적은라만리포터를포함한유기물이외부환경으로부터
안정적으로보호되는과만활성나노입자및이의제조방법을제공하는것이다. [10] 본발명의또다른목적은입자자체에서로상이한 2종이상의핫-스팟이 위치하여현저하게향상된라만산란신호를생성할수있는라만활성나노입자 및이의제조방법을제공하는것이다.
[11] 본발명의또다른목적은넓은범위로 LSPR(localized surface plasmon
resonance)파장의튜닝이가능한라만활성나노입자및이의제조방법을 제공하는것이다.
[12] 본발명의또다른목적은우수한내구성및높은 SERS활성을갖는라만활성 나노입자를단시간에대량생산할수있는제조방법을제공하는것이다.
과제해결수단
[13] 본발명에따른라만활성나노입자의제조방법은 a)완충용액 (buffer
solution)에제 1금속전구체가혼합된제 1반응액으로부터나노-별형상의금속 나노코어를제조하는단계 ; b)상기금속나노코어에라만리포터를고정하는 단계;및 c)상기라만리포터가고정된나노코어와제 2금속전구체가혼합된 제 2반응액으로부터라만리포터가고정된나노코어를감싸는금속쉘을 형성하는단계 ;를포함하며，라만리포터는상기금속나노코어의제 1금속및 상기금속웰의제 2금속각각에결합력을갖는다.
[14] 본발명의일실시예에따른라만활성나노입자의제조방법에있어，상기 b) 단계에서상기금속나노코어를감싸는라만리포터의자기조립단분자막이 형성될수있다.
[15] 본발명의일실시예에따른라만활성나노입자의제조방법에있어，상기 완충용액의완충제 (buffer agent)와상기제 1금속전구체의몰비;및상기 완충용액의 pH;중하나이상의인자 (factor)를제어하여나노코어의형상,크기 또는형상과크기를조절할수있다.
[16] 본발명의일실시예에따른라만활성나노입자의제조방법에있어，상기 완충제의몰수를상기제 1금속전구체의몰수로나눈몰비인 R1은 200내지 750일수있다.
[17] 본발명의일실시예에따른라만활성나노입자의제조방법에있어,상기제 2 반응액은계면활성제또는계면활성제와유기산을포함할수있다.
[1到 본발명의일실시예에따른라만활성나노입자의제조방법에있어，상기 완중용액은 HEPES(4-(2-hydroxy ethyl)- 1 -piperazineethanesulfonic acid), MES (4-(2-Hydroxyethy l)piperazine- 1 -ethanesulfonic acid), PBS(Phosphated buffered saline), Tris(2-Amino-2hydroxymethyl propne- 1,3 -idol), PB(Phosphate buffer), MOPS(3-(N-morpholino)propanesulfonic acid),
TAPS(3-[[l,3-dihydroxy-2-(hydroxymethyl)propan-2-yl]amino]propane-l -sulfonic acid)및 PIPES(piperazine-N，N'-bis(2-ethanesulfonic acid))에서선택되는하나 이상을함유할수있다.
[19] 본발명의일실시예에따른라만활성나노입자의제조방법에있어，상기금속 2020/075879 1»（：1^1{2018/011873 전구체의금속은 또는쇼용일수있다.
[2이 본발명의 일실시예에따른라만활성나노입자의제조방법에 있어,이단계 후,섟상기금속웰에분석대상물과결합하는수용체를고정하는단계;를더 포함할수있다.
[21] 본발명은상술한라만활성나노입자의제조방법으로제조된라만활성
나노입자를포함한다.
[22] 본발명에따른라만활성나노입자는나노-별형상의금속나노코어 ;상기
금속나노코어에고정된라만리포터를포함하는자기조립단분자막;및상기 자기조립단분자막을감싸는금속웰;을포함하며나노-별형상의돌출부에의해 돌줄구조를갖는다.
[23] 본발명의 일실시예에따른라만활성나노입자에 있어 ,상기라만활성
나노입자는상기나노-별형상에대응하는형상을가질수있다.
[24] 본발명의 일실시예에따른라만활성나노입자의제조방법에 있어，상기금속 나노코어는 10내지 5011111크기의중심영역및 5내지 7011111의크기를가지며 상기중심영역으로부터돌출되어돌출방향으로축경되는돌출부를포함할수 있다. '
[25] 본발명의 일실시예에따른라만활성나노입자의제조방법에 있어,상기라만 활성나노입자는상기금속쉘에고정되어분석대상물과결합하는수용체를더 포함할수있다.
발명의효과
[26] 본발명에따른라만활성나노입자는나노입자자체에핫-스팟이위치함에
따라,나노입자와분석대상물이단지결합하는것으로분석대상물의분석이 가능하여，분석대상물의크기제한이없는장점이 있다.
[27] 또한,본발명에따른라만활성나노입자는 8001«11영역까지아우르는매우
넓은 1 ¾파장튜닝범위를가져근적외선의조사에의해분석대상물의분석이 가능한장점이 있다.
[28] 또한，본발명에따른라만활성나노입자는나노-별형상의금속나노코어를 포함함에따라，입자그자체에강력한핫-스팟이위치하고，금속나노코어와 금속웰간균일한크기의나노갭（핫스팟）이라만활성나노입자의전영역에 형성되고,라만리포터가핫-스팟인나노캡에위치함에따라,매우우수한라만 신호의증강이이루어질수있는장점이 있다.
[29] 또한，본발명에따른라만활성나노입자는라만리포터를포함하는유기
구성성분이금속웰에의해감싸여보호되며,라만리포터의두작용기에의해 금속나노코어-라만리포터의자기조립단분자막-금속쉘이강하게결합되어 있음에따라，매우우수한내구성및물리적/화학적안정성을갖는장점이 있다.
[3이 또한,본발명에따른라만활성나노입자의제조방법은상술한장점들을갖는 라만활성나노입자를극히간단한방법으로상온에서단시간에대량생산 가능한장점이있다.
도면의간단한설명
[31] 도 1은본발명의일실시예에따라제조된금속나노코어를관찰한
주사전자현미경사진이다.
[32] 도 2는 HEPES완충용액에분산된 Au나노코어분산액을관찰한
광학사진이다.
[33] 도 3은본발명의일실시예에따라제조된금속나노코어의광흡수도를측정 도시한도면이다.
[34] 도 4는본발명의일실시예에따라제조된라만활성나노입자를관찰한
주사전자현미경사진이다.
[35] 도 5는본발명의일실시예에따라제조된라만활성나노입자의표면증강 라만산란 (SERS)스펙트럼을도시한도면이다.
[36]
발명의실시를위한형태
[3刀 이하첨부한도면들을참조하여본발명에따른라만활성나노입자및이의 제조방법을상세히설명한다.다음에소개되는도면들은당업자에게본발명의 사상이충분히전달될수있도록하기위해예로서제공되는것이다.따라서，본 발명은이하제시되는도면들에한정되지않고다른형태로구체화될수도 있으며，이하제시되는도면들은본발명의사상을명확히하기위해과장되어 도시될수있다.이때，사용되는기술용어및과학용어에있어서다른정의가 없다면,이발명이속하는기술분야에서통상의지식을가진자가통상적으로 이해하고있는의미를가지며,하기의설명및첨부도면에서본발명의요지를 불필요하게흐릴수있는공지기능및구성에대한설명은생략한다.특별히 한정하지않는한후술하는내용에서용액은탈이온수를이용한수용액을 의미하며，농도는몰농도를의미한다.
[3到 본발명에따른라만활성나노입자의제조방법은 a)완충용액 (buffer
solution)에제 1금속전구체가혼합된제 1반응액으로부터나노-별 (nano-star) 형상의금속나노코어를제조하는단계 ; b)상기금속나노코어에라만리포터를 고정하는단계;및 c)상기라만리포터가고정된나노코어와제 2금속전구체가 혼합된제 2반응액으로부터라만리포터가고정된나노코어를감싸는금속쉘을 형성하는단계 ;를포함하며，상기라만리포터는상기금속나노코어의제 1금속 및상기금속쉘의제 2금속각각에결합력을갖는다.
[39] 이때,나노-별형상은단일한중심영역및중심영역으로부터돌출되어돌출 방향으로축경되는하나이상，구체적으로둘이상，보다구체적으로 2내지 10개,보다더구체적으로 3내지 8개의돌출부를포함하는형상일수있다. 돌출부의구체형상으로다각뿔이나원뿔 (conical)등을들수있으나，반드시 이에한정되는것은아니다.금속나노코어가둘이상의돌출부를가질경우, 돌출부각각의형상이나크기 (돌출길이)는서로동일하거나상이할수있다. 금속나노코어가둘이상의돌출부를가질경우,중심영역을기준하여둘이상의 돌출부는대칭관계를갖거나서로일정각도를이루거나，중심영역에서랜덤한 위치에돌출부가돌출될수있다.
[4이 상술한본발명에따른라만활성나노입자의제조방법은완충용액과금속
전구체를함유하는액으로부터금속나노코어를제조함에따라，금속
나노코어의표면은유기물 (일예로계면활성제등)이강하게결합된상태가아닌, 아닌순수한 (bare)금속상태일수있음에따라， b)단계의라만리포터의고정시 금속나노코어전영역에서안정적으로자기조립단분자막의형태로라만 리포터가고정될수있다.
[41] 이에따라，본발명의일실시예에따른라만활성나노입자의제조방법에있어， 제 1반응액은계면활성제 (유기계면활성제)를함유하지않을수있으며，나아가， 제 1반응액은계면활성제와유기산을모두함유하지않을수있다.
[42] 또한，본발명에따른라만활성나노입자의제조방법은,완충용액과
금속전구체를함유하는액을이용한금속나노코어형성,라만리포터부착， 금속웰형성이라는간단한공정을이용하여라만활성나노입자를제조함에 따라,저비용으로단시간에라만활성나노입자를대량생산할수있는장점이 있다.
[43] 또한,본발명에따른라만활성나노입자의제조방법은라만리포터를포함한 유기물이라만활성나노입자의표면에노출되어있지않고,금속쉘에의해 감싸여있음에따라，라만리포터를포함한유기물이외부환경으로부터 안정적으로보호될수있는장점이있다.
[44] 또한,본발명에따른라만활성나노입자의제조방법은금속나노코어가
나노-별형상임에따라,금속나노코어자체에핫스팟이형성되어있어，라만 활성나노입자그자체만으로산란신호를증강시킬수있는장점이있다.
[45] 이때,알려진바와같이，핫스팟 (hot-spot)은매우강한국소전기장이형성되며 국부적표면플라즈몬공명 (LSPR; localized surface plasmon resonance)이 발생하는영역을의미한다.
[46] 나노입자간또는나노입자와다른구성요소간등과같이별개의두
구성요소간의핫스팟에의해신호증강이이루어지는경우，두구성요소의나노 갭영역이나나노갭인근영역에분석대상물이위치 (또는결합)하여야신호 증강이이루어질수있다.이러한공간적제약은분석대상물의크기를제한하게 되어수마이크로미터나수십마이크로미터크기의생화학물질의분석을 불가하게맞든다.
[47] 그러나，개별적으로독립된상태의단일한 (single)라만활성나노입자그
자체가핫스팟을갖는경우，분석대상물이라만활성나노입자와결합하는 것만으로신호증강이이루어질수있음에따라，실질적으로분석대상물의 크기에제한이없어，통상적으로크기가큰 (수백나노미터오더내지수십 마이크로미터오더)생화학물질을검출/분석하는데매우적합하다.
[48] 또한，본발명에따른라만활성나노입자의제조방법은금속나노코어가
나노-별형상임에따라,나노-별의구체형상이나크기 (튀어나온가지 (branch)의 길이등을포함함)를조절함으로써현저하게넓은범위로 LSPR파장의튜닝이 가능한장점이있다.
[49] 실질적인일예로,금속나노코어가나노-별형상인경우，구형에서는가능하지 않은 800nm영역대까지도 LSPR파장의튜닝이이루어질수있다.이러한 800nm 이상으로튜닝가능한 LSPR파장은가시광대역이아닌근적외선 (NIR, 780nm~1500nm)대역의광조사에의해분석대상물의검출및분석이이루어질 수있음을의미할수있다.
[50] 알려진바와같이생화학물질을포함한바이오물질에가시광이조사되는경우 형광현상이발생할수있다.형광의세기는라만산란에비해매우강하고, 형광이라만산란과유사한영역에서발생하기때문에형광피크에가려순수한 라만스펙트럼을얻기어려운문제점이있다.이에，가시광이아닌근적외선 대역의광조사에의한 SERS분석은형광의영향 (간섭)없이라만스펙트럼을 수득할수있어바이오분야에매우유리하다.
[51] 완충용액의완충제 (buffer agent)와상기제 1금속전구체의몰비;및상기
완충용액의 pH;중하나이상의인자 (factor)를제어하여금속나노코어의형상, 크기또는형상과크기를조절할수있다.이때,완충용액은 pH조절을위해 HC1등의통상의무기산， NaOH등와통상의무기염기또는이들의혼합물을 함유할수있음은물론이다.
[52] 실질적인일예로，제 1반응액에서완충제의몰수를상기제 1금속전구체의 몰수로나눈몰비인 R1은 200내지 750일수있다.이러한 R1의범위에서나노-별 형상을갖는금속나노코어가제조될수있으며,중심영역의크기가 10내지 50nm,구체적으로 10내지 40nm수준인나노-별형상의금속나노코어가제조될 수있다.
[53] 유리하게, 은 500내지 750일수있다. 을 500내지 750으로제어함으로써 3개이상의돌출부，구체적으로 3내지 8개의돌출부를가지며 5내지 70nm, 구체적으로 5내지 50nm,보다구체적으로 10내지 50nm크기의돌출부를갖는 나노-별형상의금속나노코어가제조될수있다.
[54] 또한， 200내지 750범위내에사 R1을조절함으로써금속나노코어의중심 영역에서돌출되는돌출부 (extrusion또는 branch)의길이를제어할수있다.보다 구체적인일예로， 200내지 750범위내에서 R1값을높여돌출부의길이를 증가시킬수있다.이때，돌출부의길이를제어하여라만활성나노입자 (또는 금속나노코어)의 LSPR파장이제어될수있으며， 200내지 750범위내에서 을조절함으로써 600내지 900nm로 LSPR파장이조절될수있다.
[55] R1과함께또는 R1과독립적으로 (고정된 R1하에),완충용액의 pH를
조절함으로써금속나노코어의중심영역에서돌출되는돌출부의길이를 2020/075879 1»（：1/10公018/011873 제어할수있다.상세하게，완충용액의 pH는 5.0내지 7.5일수있으며,
완충용액의 pH를증가시킴으로써돌출부의길이를증가시킬수있다.
[56] 완충용액 (또는완충제)은 HEPES(4-(2-hydroxyethyl)- 1 -piperazineethanesulfonic acid), MES (4-(2-Hydroxyethyl)piperazine- 1 -ethanesulfonic acid)， PBS(Phosphated buffered saline), Tris(2-Amino-2hydroxymethyl propne- 1,3-idol), PB (Phosphate buffer), MOPS(3-(N-morpholino)propanesulfonic acid),
TAPS(3-[[l,3-dihydroxy-2-(hydroxymethyl)propan-2-yl]amino]propane-l-sulfonic acid)및 PIPESipiperazine- N'-bis^-ethariesulfonic acid))에서선택되는하나 이상을함유할수있다.이러한완충제는금속을환원시키는약한환원제로 작용할수있고,완충제에의해금속나노코어의안정화를위한계면활성제를 배제시킬수있어，순수한금속표면을갖는금속나노코어를제조할수있다. 이때,상술한바와같이，완충용액 (수용액)은 pH조절을위한무기산및/또는 무기염기를더함유할수있음은물론이다.
[57] 제 1금속전구체의제 1금속은광과상호작용에의해표면플라즈몬이발생하는 금속일수있다.구체예로，제 1금속은금，은，백금,팔라디움，니켈，알루미늄, 구리또는이들의혼합물또는이들의합금등을들수있다.다만,생체유래' 물질이나생화학물질과의안정성을고려하여제 1금속은금또는은인것이 좋다.유리한일예에따른제 1금속에있어 ,제 1금속전구체는 HAuCl 4, HAuBr 4, NaAuCl 4, AuCl 3.3H 20, NaAuCl 4_2H 20,또는이들의혼합물등과같은
금전구체일수있으며，또는， AgNO 3등과같은은전구체일수있으나，본발명이 금속전구체의구체물질종류에의해한정될수없음은물론이다.
[58] 보다구체적으로， a)단계는,완충용액에제 1금속전구체용액을혼합하여제 1 반응액을제조하고， 15내지 40OC의온도에서제 1반응액을반응시켜금속 나노코어를제조하는단계를포함할수있다.
[59] 완충용액에서완충제의몰농도는 100내지 400mM일수있으며，제 1금속
전구체용액에서제 1금속전구체의몰농도는 20내지 60mM일수있다.이러한 몰농도의완충용액과제 1금속전구체용액을이용하는경우투입된제 1금속 전구체의대부분을금속나노코어로전환시킬수있어유리하고 10내지 50분의 반응시간내에반응 (금속나노코어합성)이완료될수있어유리하나，완충용액의 농도와제 1금속전구체용액의농도가반드시상술한범위로한정되는것은 아니다.완충용액과제 1금속전구체용액의혼합시，상술한 R1을만족하도록 용액들이혼합될수있음은물론이다.
[60] 완충용액과제 1금속전구체용액의혼합과동시에반응이진행될수있으며, 반응은 15내지 40°C의온도，구체적으로는 15내지 35°C의온도，보다
구체적으로는 15내지 25OC의온도，보다더구체적으로는상온 (21내지
23OC)에서수행될수있다.이때,상온은제 1반응액에인위적으로열에너지가 인가되지않은상태에서의온도를의미할수있음은물론이다.반응시간은금속 나노코어의합성이완료되기충분한시간이면무방하여,구체예로, 10내지 50분，보다구체예로 20내지 40분일수있으나본발명이이에한정되는것은 아니다.
[61] 제 1반응액의반응시,필요시선택적으로제 1반응액의교반이이루어질수 있다.반응액을교반하는경우반응수율을향상시킬수있으나，제조되는금속 나노코어의형상이나크기등은교반조건에거의영향을받지않는다.교반은 500rpm내지 1500rpm수준이면족하다.
[62] 또한, a)단계는, al)완충용액에제 1금속전구체용액을혼합하여제 1반응액을 제조하고， 15내지 40°C의온도에서제 1반응액을반응시켜금속나노코어를 제조하는단계 ;및 a2)반응이완료된제 1반응액을금속나노코어의분산매이자 보관액으로하여 , 1내지 10OC의온도,구체적으로 1내지 5°C의온도로보관하는 단계;를포함할수있다.즉, al)의반응이완료된후,반응이완료된
제 1반응액에서금속나노코어를분리회수하지않고，금속나노코어를함유하는 제 1반응액상태로보관하되， 1내지 10°C의온도，구체적으로 1내지 5°C의 온도로저온보관할수있다.또는,이와달리, a)단계는, al)완충용액에제 1금속 전구체용액을혼합하여제 1반응액을제조하고， 15내지 40°C의온도에서제 1 반응액을반응시켜금속나노코어를제조하는단계;및 a2)반응이완료된제 1 반응액으로부터금속나노코어를회수하고완충용액 (별도의완충용액)에 분산하여 1내지 10OC의온도,구체적으로 1내지 5OC의온도로보관하는단계;를 포함할수있다.
[63] 물을포함한다른분산매가아닌,반응이완료된반응액이나완충용액을
분산매로 al)단계에서제조된금속나노코어를 1내지 10OC의저온에서 보관하는경우수십일에이르는보관에도금속나노코어의
플라즈모닉-활성 (plasmonic-active)특성이변화되지않고안정적으로유지될수 있어좋다.
[64] 이때，상술한바와같이，제 1반응액은나노입자의안정화와분산성을
향상시킴과동시에환원제역할을수행할수있는계면활성제，유기산또는 계면활성제와유기산을함유하지않을수있으며， al)단계는오직완충용액과 제 1금속전구체용액만을이용하여수행될수있다.이와같이,본발명에따른
, 제조방법은 2가지용액의단순혼합및수십분의상온반응에의해금속
나노코어가합성될수있음에따라，금속나노코어를대량생산에매우적합하다.
[65] a)단계가수행된후， b)금속나노코어에라만리포터 (Raman reporter)를
고정하는단계가수행될수있다.
[66] 라만리포터는라만활성분자를포함하는유기화합물 (유기분자)을의미할수 있으며,금속나노코어의금속과결합력을가지며라만활성분자를포함하는 유기화합물 (유기분자)을의미할수있다.라만리포터는기공지된것이며，이 기술분야에서널리사용되는것이라면어느것이나제한없이사용할수있다.
[67] 라만리포터 (분자)가금속나노코어의금속과결합력을가짐에따라，순수한 금속표면이노출되는금속나노코어에는라만리포터의자기조립단분자막이 형성될수있다.
[68] 알려진바와같이금속나노입자화및설계된형상화를위해서는적절한
환원성을제공하면서도성장을억제하거나특정방향으로의성장을유도하거나 및/또는나노입자를안정화시킬수있는유기계면활성제가주지관용으로 사용되고있으며，이와함께유기산이사용되거나또는계면활성제를대체할수 있는유기산이사용되고있다,그러나，이러한방법으로합성된금속나노입자의 표면에는금속나노입자의금속물질과매우강한결합력으로유기계면활성제나 유기산유래유기작용기들이결합되어있다.이에，이미강하게결합되어있는 표면작용기들에의해，목적하는작용기로금속나노입자표면을균일하고 완전하게감싸기 (치환하기)어려운문제가있다.
[69] 그러나， a)단계에서유기산이나유기계면활성제가배제된상태에서，온전히 완충용액과금속전구체로부터금속나노코어가제조되며，상술한바와같이， 완충용액내에서안정적으로분산이유지됨에따라，제조된금속나노코어는 순수하게금속의자체의표면상태를나타낼수있다.이러한금속의표면 상태에의해,금속나노코어에라만리포터 (금속나노코어와결합력을가지며 라만활성분자를포함하는유기화합물)가균일하며균질하게자발적으로 결합하며，나노-별이라는요철이심한형상임께도,안정적으로라만리포터의 자기조립단분자막이형성될수있다.
R0] 라만활성분자는표면강화라만활성분자,표면증강공명라만활성분자, 하이퍼라만활성분자，또는코히런트반스토크스라만활성분자를포함할수 있다.라만활성분자는라만신호와형광신호를동시에갖거나,라만신호를 가질수있다.
[71] 구체예로,라만활성분자는시아닌 (cyanines),플루오레세인 (fluorescein), 로다민 (rhodamine), 7 -니트로벤젠- 2 -옥사- 1 ,3 -디아졸
(7-nitrobenz-2-oxa-l, 3-diazole: NBD),프탈산，테레프탈산,아이소프탈산,크레실 패스트바이올렛 (cresyl fast violet),크레실블루바이올렛 (cresyl blue violet), 브릴리언트크레실블루 (brilliant cresyl blue),파라아미노벤조산，에리쓰로신， 비오틴,디옥시제닌 (digoxigeniii)，프탈로시아닌,아조메틴，크산틴,
N，N-디에틸- 4-(5'-아조벤조트리아졸일)-페닐아민，아미노아크리딘，및이들의 조합으로이루어지는군으로부터선택될수있다.시아닌의예에는 Cy3，Cy3.5, 또는 Cy5가포함될수있다.플로오레세인의예에는,카복시플루오레세인 (carboxyfluorescein : FAM), 6 -카복시
Figure imgf000011_0001
핵사클로로플루오레세인
(6-carboxy-2’，4,4’，5’，7,7’-hexachlorofluorescein: HEX),
6 -카복시 -2',4,7,7'-테트라클로로플루오레세인
(6-carboxy-2/,4,7,7,-tetrachlorofluorescein: TET),
5 -카복시- 4'，5'-디클로로- 2', 7'-디메톡시플루오레세인， 6 -카복시- 4 -디클로로- 2 7’-디메톡시플루오레세인 (6-carboxy-4,,5,-dichloro-2,, T-dimethoxyfluorescein:
Joe) 5 -카복시- 2',4',5',7-테트라클로로플루오레세인, 5 -카복시플루오레세인, 2020/075879 1»（：1/10公018/011873 또는석신일플루오레세인이포함될수있다.로다민의예에는
테트라메틸로다민 (tetramethylrhodamine: Tamra), 5 -카복시로다민，
6 -카복시로다민로다민， 6G (Rhodamine 6G: R6G),테트라메틸로다민
이소티올 (tetramethyl rhodamine isothiol: TRIT),술포로다민 101산클로라이드 (sulforhodamine 101 acid chloride: Texas Red dye),카복시 - X-로다민
(carboxy-X-rhodamine: Rox),또는로다민 B (rhodamine 가포함될수있다.
[72] 다른구체예로，라만활성분자는벤젠고리형태의라만활성분자일수있으며, 벤젠고리형태의라만활성분자는 4-AminothiophenoI(4-ATP), 4-Mercaptobenzoic acid(4-MBA), Phenyl isothiocyanate(PITC), Benzenethiol(BT),
1 ,4-Benzenedithiol(BDT), Biphenyl-4, 4'-dithiol(BPDT), p
-Terphenyl-4,4"-dithiol(TPDT), 4-Bromobenzenethiol(4-BBT), 4-Chlorobenzenethiol (4-CBT), 3,3’-Diethylthiatricarbocyanine iodoide(DTTC)등을들수있다.
[73] 유리하게,라만리포터는금속나노코어의금속 (제 1금속)및금속쉘의
금속 (제 2금속)각각과결합력을가지며라만활성분자를포함하는유기 화합물 (유기분자)일수있다.
[74] 상세하게，라만리포터는라만활성분자를포함하되,제 1금속과자발적으로 결합하는제 i작용기를가지며제 2금속 (제 2금속전구체의제 2금속)과
자발적으로결합하는제 2작용기를갖는것이좋다.
[75] c)단계에서금속쉘이형성됨에따라제 2작용기는보다원활하고균일한
제 2금속웰의핵생성장소를제공할수있으며，제 2금속쉘과라만리포터가 고정된금속나노코어간의결합력을크게향상시킬수있어유리하다.즉,라만 리포터가금속나노코어와도，금속웰과도결합력을가짐에따라，
자기조립단분자막은제 1작용기에의해금속나노코어와화학결합하고， 제 2작용기에의해금속쉘과화학결합한상태일수있다.
[76] 작용기 (제 1작용기또는제 2작용기)는금속을고려하여해당금속과
자발적으로결합하는것으로알려진작용기이면무방하다.실질적인일예로, 제 1금속과제 2금속이서로독립적으로금또는은인경우，작용기 (제 1작용기 또는제 2작용기)는티올기 (-SH),카르복실기 (-COOH)또는아민기 (-NH 2)등일수 있으나,본발명이작용기의구체종류에의해한정되는것은아니다.
제 1작용기와제 2작용기를모두갖는라만리포터의실질적인예로,
1 ,4-Benzenedithiol(BDT), Biphenyl-4, 4'-dithiol(BPDT), p
-Terphenyl-4,4”-dithiol(TPDT)등을들수있으나,본발명이라만리포터의구체 물질에의해한정되는것은아니다.
기 상술한바와같이,제 1작용기에의해금속나노코어의금속과결합력을갖는 라만활성분자가금속나노코어에자발적으로결합 (고정)됨으로써금속 나노코어에는라만리포터의자기조립단분자막이형성될수있다.
[78] 이러한자기조립단분자막의형성은，금속나노코어가나노-별모양의큰
이방성을갖는형상임에도불구하고,극히균일한두께를갖는라만리포터의 2020/075879 1»（：1^1{2018/011873 단일한분자막이금속나노코어의전표면에균질하게형성될수있도록한다.
[79] 또한，라만리포터의자기조립단분자막형성은금속나노코어와금속쉘간
극히균일한크기의나노갭을형성할수있으며,이에따라，라만활성나노입자 전영역에균일하게제어된핫스팟이형성되도록할수있다.
[80] 금속나노코어에결합되는라만리포터에의해금속나노코어와금속웰간
나노갭 (핫-스팟)이형성됨에따라，보다강한신호증강이이루어지는핫스팟 형성측면에서라만리포터의길이 (크기)는뇨파이하，구체적으로 0.5내지
211111인것이좋다.
[81] 금속나노코어에라만리포터를고정하는비단계는 단계에서제조된금속 나노코어와라만리포터를함유하는혼합액을제조하고초음파교반하는 단계;를포함할수있다.
[82] 구체적으로 , 1))단계는 1?1) 3)단계에서제조된금속나노코어와라만리포터의 몰농도가 0.01내지 11企1과 10내지 1000나 이되도록혼합하여혼합액을 제조하는단계;초음파교반하며 10내지 30분간상온반응시키는단계;및匕4) 라만리포터가고정된 (라만리포터의자기조립단분자막이형성된)금속 , 나노코어를분리회수하는단계;를포함할수있다.이때，혼합액은수계
혼합액일수있으며，혼합액은금속나노코어의응집을방지하기위해
Figure imgf000013_0001
Figure imgf000013_0002
수용성인계방향족화합물을더 포함할수있다.
[83] I?)단계가수행된후， 0)상기라만리포터가고정된나노코어와제 2금속
전구체가혼합된제 2반응액으로부터라만리포터가고정된나노코어를감싸는 금속쉘을형성하는단계가수행될수있다.라만리포터가고정된
나노코어 (금속나노코어)는라만리포터의자기조립단분자막이형성된금속 나노코어일수있다.
[84] 0)단계에서형성되는금속쉘은나노코어에고정된라만리포터에의해금속 나노코어와나노갭을형성함에따라,점이나선형태가아닌금속나노코어의 표면에대응한형상의면형태로핫스팟이형성되어보다강한라만산란 신호를획득할수있는장점이 있다.또한,라만리포터의자기조립단분자막에 의해나노갭의크기가결정됨에따라，단지설계된적절한크기를갖는라만 리포터로자기조립단분자막을형성함으로써,나노-별전영역에서균일하고 정밀하게나노갭의크기가제어될수있다.
[85] 제 2반응액은라만리포터가고정된나노코어 (금속나노코어 )，제 2금속
전구체와함께，제 2금속전구체를환원시키는환원제의 역할및금속
나노코어와제조되는라만활성나노입자를안정화시키는안정화제의 역할을 수행할수있는계면활성제또는계면활성제와유기산을더함유할수있다.
[86] 계면활성제를이용한금속웰을형성하는경우，제조되는라만활성
나노입자가나노-별형상의금속나노코어의형상과실질적으로대응되는 형상을가질정도로，극히얇고균일하며매끈한막의형태로금속쉘을형성할 수있다.
[87] 이러한매끈한막 (표면거칠기가낮은막)형태의금속쉘은라만활성
나노입자에보다분석대상물과특이적으로결합하는수용체를보다균일하게 고정시킬수있어유리하며，보다다량의수용체를고정시킬수있어유리하다.
[88] 제 2반응액에함유되는계면활성제는양이온성계면활성제일수있다.
양이온성계면활성제는 4급암모늄염일수있으며, 4급암모늄염은함
질소화합물로서사슬형알킬과결합한것뿐만아니라고리형질소화합물의함 질소헤테로고리화합물또한포함할수있다.이때,함질소헤테로
고리화합물은피리둠염，퀴놀륨,이미다졸륨또는이들의혼합물을포함할수 있다.
[89] 유리하게,제 2금속전구체의제 2금속을자발적으로환원시키되,느리게
환원시켜매우매끈하고얇은치밀막형태로금속쉘이형성될수있도록， 4차 암모늄염계계면활성제는하기화학식 1을만촉할수있다.
[90] (화학식 1)
Figure imgf000014_0001
[92] 화학식 1에서, R1，R2및 R3는서로독립적으로 C1 C3의알킬기이며, R4는 C10~C19,유리하게는 C14 C18의알킬기이며，암모늄이온의카운터이온인
X는할로겐일수있다.화학식 1을만족하는 4차암모늄염계계면활성제의구체 예로, CTAC(hexadecyl-trimethylammonium chloride),
CTAB(hexadecyl-trimethyl-ammonium bromide)등을들수있으나，본발명이구체 4차암모늄염계계면활성제물질에의해한정되는것은아니다.
[93] 보다얇고표면조도가낮은금속쉘을형성하기위해，제 2반응액은 4차
암모늄염계계면활성제와함께유기산을더함유할수있다.유기산은아세트산, 프로피온산，푸마르산,말산，숙신산,그리콜산，주석산，구연산,부티르산, 팔미트산,포름산；젖산,옥살산，옥살아세트산，타타르산，아스코르브산, 시트르산및이소시트르산으로이루어진군에서선택된하나이상일수있으나， 반드시이에한정되는것은아니다.
[94] 제 2금속전구체의제 2금속또한광과상호작용에의해표면플라즈몬이
발생하는금속일수있으며，제 2금속은금,은，백금，팔라디움，니켈，알루미늄, 구리또는이들의혼합물또는이들의합금등을들수있다.다만,생체안정성을 고려하여제 2금속은제 1금속과독립적으로금또는은인것이좋다.유리한일 예에따른제 2금속에있어，제 2금속전구체는 HAuCl 4, HAuBr 4, NaAuCl 4, AuCl 3 o 3H 20, NaAuCl 4 o 2H 20,또는이들의혼합물등과같은금전구체일수있으며， 또는, AgNO 3등과같은은전구체일수있으나，이에한정되지않는다. 2020/075879 1»（：1'/10{2018/011873
[95] 보다구체적으로， 0）단계는，계면활성제용액，제 2금속전구체용액및라만
리포터가고정된금속나노코어분산액을혼합하여제 2반응액을제조하고， 15 내지 40ᄋ（:의온도，유리하게는상온에서제 2반응액을 20분이내，구체적으로는 5 내지 15분동안반응시켜금속쉘을제조하는단계를포함할수있다.보다더 구체적으로,제 2반응액은계면활성제용액,제 2금속전구체용액，라만리포터가 고정된금속나노코어분산액및유기산용액을혼합하여제조될수있다.
반응시격렬한교반이수행될수있으며,반응의종료는원심분리등을이용하여 고상을분리회수함으로써수행될수있다.
[96] 계면활성제용액에서계면활성제의몰농도는 0.1내지 0.3M일수있으며，
제 2금속전구체용액에서제 2금속전구체의몰농도는 1내지 2011114일수있고， 라만리포터가고정된금속나노코어분산액에서금속나노코어의몰농도는 0.01내지 0.511] 일수있으며，유기산용액의유기산몰농도는 0.01내지 0.31 일 수있으나，반드시 이에한정되는것은아니다.
[97] 제 2반응액제조시，계면활성제의몰수를제 2금속전구체의몰수로나눈몰비인 ]¾2가 200내지 500,구체적으로는 300내지 500이되도록계면활성제용액과 제 2금속전구체용액이혼합될수있으며,제 2반응액이유기산을더포함하는 경우，계면활성제의몰수를유기산의몰수로나눈몰비인 113가 0.01내지 0.20， 구체적으로는 0.05내지 0.1이되도록유기산용액이혼합될수있다.
[98] 이러한 112의몰비,나아가， 112와 의몰비는별도의에너지 인가없이
상온에서자발적으로제 2금속의환원이이루어지면서도적절한속도로환원이 이루어져나노-별형상의금속나노코어의형상이실질적으로거의그대로 유지되며얇고치밀하며매끈한제 2금속의막（금속쉘）이 생성되기에적합한 조건이다.
[99] 금속나노코어분산액은제 2금속전구체 :금속나노코어의몰비가 1 : 1x10 7 내지 1x10 이되도록혼합될수있다.이때,금속나노코어（들）에균일하게금속 웰이형성될수있도록，제 2금속전구체용액과금속나노코어분산액이먼저 혼합된후에계면활성제용액또는계면활성제용액과유기산용액이혼합될수 있다.
[100] 상세하게, 0）단계는， 01）제 2금속전구체용액과금속나노코어분산액을
혼합하여전구체-나노코어혼합액을제조하는단계; 02）전구체-나노코어 혼합액에 계면활성제용액또는계면활성제용액과유기산용액을혼합하여 제 2반응액을제조하고 15내지 40ᄋ（:의온도，유리하게는상온에서제 2반응액을 20분이내로반응시켜라만활성나노입자를제조하는단계;를포함할수있다.
[101] 0）단계에의해,금속나노코어，금속나노코어를감싸는라만리포터의
자기조립단분자막，자기조립단분자막을감싸는금속웰을포함하는라만활성 나노입자가제조될수있다.
[102] 라만리포터의자기조립단분자막이형성된늠속나노코어 전영역에서매우 균일하고얇은두께를갖는매끈한표면의금속쉘이형성됨에따라，제조되는 라만활성나노입자는금속나노코어의나노-별형상에서유래한돌출부를가질 수있으며,실질적으로，금속나노코어의나노-별형상과대응하는형상을가질 수있다.이때，대응하는형상은，금속나노코어의나노-별에서의돌출부위치와 실질적으로동일한위치에서돌출되어있는형상을가지며，금속나노코어의 나노-별에서의돌출부의개수와실질적으로동일한개수로돌출되어있는 형상을의미할수있다.
[103] 이단계에의해,평균 150nm이하의크기 (동일부피의구환산지름)，
구체적으로평균 lOOnm이하의크기，실질적으로 40내지 lOOnm，보다
실질적으로 60내지 lOOnm,보다더실질적으로 65내지 80nm크기의라만활성 나노입자가제조될수있다.
[104] 본발명의일실시예에따른라만활성나노입자의제조방법은 c)단계후， d) 금속쉘에분석대상물과결합 (특이적으로결합)하는수용체를고정하는단계;를 더포함할수있다. d)단계는제조된라만활성나노입자분산액에수용체를 혼합하여수행될수있으며，수용체별로알려진프로토콜에따라고정이수행될 수있음은물론이다.
[105] 수용체는분석대상물과효소-기질，항원-항체,단백질-단백질또는 DNA간의 상보적결합하는것으로알려진어떠한물질이든무방하다.이때，수용체는금속 쉘의제 2금속과자발적으로결합하는작용기 (일예로，티올기，카르복실기또는 아민기등)를포함할수있으며，작용기에의해금속웰에자발적으로결합된 상태일수있다.
[106] 분석대상물은생물 (바이러스를포함함)또는비생물유래물질일수있다.생물 유래물질은세포성분을포함할수있다.구체적으로，분석대상물은병변 특이성을갖는병변표지생체물질，병변지표물질,병원체,약물,유기화합물, 생화학물질또는생체유래물질등일수있다.생화학물질이나생체유래 물질은펩타이드,탄수화물，단백질,단백질복합체,지질,대사체,항원，항체， 효소，기질，아미노산,압타머，당，핵산，핵산단편， PNA(Peptide Nucleic Acid), 세포추출물또는이들의혼합물등을포함할수있으나,반드시이에한정되는 것은아니다.
[107] 제조되는라만활성입자는인-비보 (in-vivo)또는인-비트로 (in-vitro)용일수 있다.
[108] 본발명은상술한제조방법으로제조된라만활성나노입자를포함한다.
[109] 이하，본발명에따른라만활성나노입자를상술한다.이때,라만활성
나노입자에서금속나노코어，나노-별형상,라만리포터,자기조립단분자막, 금속쉘,분석대상물,수용체등은앞서라만활성나노입자의제조방법에서 상술한바와유사내지동일하다.이에，본발명에따른라만활성나노입자는 앞서라만활성나노입자의제조방법에서상술한모든내용을포함한다.
[110] 본발명에따른라만활성나노입자는나노-별형상의금속나노코어 ;상기 금속나노코어에고정된라만리포터를포함하는자기조립단분자막;및상기 2020/075879 1»（：1^1{2018/011873 자기조립단분자막을감싸는금속쉘;을포함하며상기나노-별형상의돌출부에 의해돌줄구조를갖는다.
[111] 본발명에따른라만활성나노입자는나노-별형상의금속나노코어를포함할 . 수있다.나노-별형상의나노코어는중심영역의크기가 10내지 5011111，
구체적으로 10내지 401^1수준이며，중심영역으로부터돌출되어돌출방향으로 죽경되는 5내지 7011111,구체적으로 5내지 50:1111，보다구체적으로 10내지 5011111 크기수준인돌출부를가질수있다.실질적인일예로,나노코어는 3개이상의 돌출부，구체적으로 3내지 8개의돌출부를가질수있다.
[112] 라만활성나노입자가나노-별형상의나노코어를포함함에따라，라만활성 나노입자자체가핫-스팟을가질수있다.이에의해라만활성나노입자자체로 라만신호증강이이루어질수있으며 ,분석대상물의크기제한이 없고， 나노-별의크기및구체형상에의해 요파장이용이하게튜닝될수있으며, 그튜닝범위가 80011111영역에·이르도록넓은장점이 있다. 80011111영역에 이르는 1石 11파장은，근적외선조사에의한분석대상
Figure imgf000017_0001
이루어질수있음을의미한다.
[113] 또한,본발명에따른라만활성나노입자는나노코어가나노-별이라는매우
이방성이큰복잡한형상임에도，라만리포터가자기조립단분자막형태로 나노코어에고정되어 있음에따라,전영역에서균일하고안정적인 표묘 활성을 나타낼수있는장점이 있으며，라만리포터가핫스팟에위치함에따라우수한 라만신호의증강이이루어질수있다.
[114] 또한,본발명에따른라만활성나노입자는，자기조립단분자막이금속쉘에
의해감싸여보호됨에따라,물리적/화학적으로취약한유기구성성분（라만 리포터）이외부환경으로부터안정적으로보호되는장점이 있다.
[115] 또한，본발명에따른라만활성나노입자는라만리포터가제 1금속（금속
나노코어）과자발적으로결합하는제 1작용기와제 2금속（금속쉘）과자발적으로 결합하는저 12작용기를가짐에따라,나노코어-자기조립단분자막-금속쉘간매우 강한결합을가져우수한내구성과안정성을갖는장점이 있다.
[116] 또한，본발명에따른라만활성나노입자는라만리포터가자기조립단분자막 형태로나노코어에고정됨에따라，금속웰과나노코어간자기조립단분자막의 두께（라만리포터의크기에상응）에해당하는균일한크기의나노갭（핫스팟）이 형성되어 ,보다큰라만신호의증강이 이루어질수있다.
[117] 라만리포터의작용기（제 2작용기）에의해자기조립단분자막과결합된상태인 금속웰은 3내지 2011111,구체적으로 5내지 1511111의두께일수있으며,얇고 균일한두께의치밀막일수있다.
[118] 이러한얇고균일한두께의금속웰에의해,라만활성나노입자는금속
나노코어의나노-별형상에대응하는형상을가질수있다.라만활성
나노입자가나노-별형상에대응하는형상을갖는경우나노코어의나노-별 형상-라만리포터의자기조립단분자막에의한나노갭-나노별에대응하는 형상의금속쉘구조에의한상승작용에의해놀랍도록향상된라만신호의 증강이이루어질수있다.
[119] 라만활성나노입자는금속웰에고정되어분석대상물과결합하는수용체를더 포함할수있으며，수용체는금속쉘에자발적으로결합하는작용기를포함할수 있다.분석대상물과특이적으로결합하는수용체에의해분석대상물이라만 분광 (SERS분광)에의해분석및검출될수있으며,나아가，분석대상물의 센싱이나이미징이이루어질수있다.
[120] 상술한라만활성나노입자는인-비보 (in-vivo)또는인-비트로 (in-vitro)용일수 있다.
[121] 도 1은본발명의일실시예에따라제조된금속나노코어를관찰한
주사전자현미경사진이다.
[122] 상세하게,도 1의금속나노코어는 40mM농도의 HAuCl 4용액 500[ 와 140mM 농도의 HEPES완충용액 (pH=7.2) 100mL을혼합 (Rl=700)하고상온에서
lOOOrpm으로 30분간교반하여제조한것이다.제조된금속나노코어는관찰이나 후속되는라만리포터고정전까지 l40mM농도의 HEPES완충용액에 4OC의 온도로보관되었다.
[123] 도 1에서알수있듯이，나노-별형상의 Au나노코어가제조됨을확인할수 있으며,중심영역의크기가약 30nm이고，돌출부의길이가약 20-30nm인 나노-별형상의 Au나노코어가제조됨을알수있다.
[124] 도 2는 HEPES완충용액에보관된 Au나노코어를관찰한광학사진으로，도 2에서알수있듯이，별도의계면활성제나유기분산제등의도움없이도 안정적으로 Au나노코어의분산이유지되는것을확인할수있다.
[125] 도 3은본발명의일실시예에따라제조된금속나노코어의광흡수도를측정 도시한도면이다.도 3의샘플들에서 R [HEPES/AU]=700(PH 7.2)는도 1의나노코어와 동일한방법으로제조하되 이 700이고 HEPES완충용액의 pH가 7.2인 조건에서제조된 Au나노코어를， R [HEPES/AU]=500(PH 7.¾는도 1의나노코어와 동일한방법으로제조하되 이 500이고 HEPES완충용액의 pH가 7.2인 조건에서제조된 Au나노코어를, R [HEPES/AU]=500(PH 5.2)는도 1의나노코어와 동일한방법으로제조하되 R1이 5W이고 HEPES완충용액의 pH가 5.2인 조건에서제조된 Au나노코어를의미한다.
[126] 주사전자현미경관찰을통해, R1과완충용액의 pH에따라나노-별의돌출부의 길이가달라지며 , R1이커질수록，또한, pH가높아질수록보다잘발달된 돌출부를갖는나노-별형상의 Au나노코어가제조됨을확인하였다.
[127] 도 3을통해，중심영역에서돌출된돌출부에의해핫-스팟이형성됨에따라, 이러한돌출부의발달정도에따라 LSPR파장이튜닝됨을확인할수있으며， 돌출부가잘발달될수록 LSPR파장이장파장으로이동함을확인할수있다. 또한, R [HEPES/AU]=700(PH 7.2)샘플에서확인할수있듯이， LSPR파장의튜닝이 근적외선영역까지가능함을알수있다. 2020/075879 1»（：1^1{2018/011873
[128] 도 4는쇼11나노코어에라만리포터의자기조립단분자막을형성한후，
제 2반응액으로쇼11쉘을형성하여제조된라만활성나노입자를관찰한
주사전자현미경사진이다.
[129] 상세하게 ,원심분리（8000印!11, 10분）로 나노코어（II [ ⑵여묘 7.2）
샘물）를반응액에서회수하고， 88 1＞（ 8（1）-811比01131；01）11611＞（1） 611 11511081）¾116
Figure imgf000019_0001
혼합하고 10분동안초음파처리하여 0.1：1 몰농도의쇼11나노코어분산액을제조하였다.쇼11나노코어분산액 411止와 10 몰농도의묘이따/내빼 선他 ） 200ᅣ止와혼합하고 10분동안초음파처리한후， 6000 111으로 10분간원심분리하여라만리포터인 601의자기조립단분자막이 형성된쇼11나노코어를회수하였다.회수된자기조립단분자막이형성된쇼11 나노코어를 41 의탈이온수에분산（0.111] 몰농도）시키고,분산액에 10
11 （：1 4 100此와혼합한후， 0.2 （: 용액 2 와 0.2 아스코르브산용액 0.21 를더혼합하여제 2반응액을제조하고 10분간교반하며반응시킨후 원심분리（5000 111, 10분）로제조된라만활성나노입자（도 4의라만활성 나노입자）를제조하였다.
[13이 도 4에서알수있듯이제조된라만활성나노입자가금속나노코어의나노-별 형상에대응하는형상을가짐을알수있으며，금속웰이매우매끈하고
치밀하며균일한두께의막형태로라만리포터가고정된금속나노코어전 영역을안정적으로감싸는것을알수있다.
[131] 도 5는제조된라만활성나노입자（도 4의샘플）의표면증강라만산란 표요幻 스펙트럼을도시한도면이다. 5£1 스펙트럼은마이크로라만시스템 0¾加幻을 이용하여 51411111, 63311111또는 7851101의광을라만활성나노입자에조사하여 수득하였다.
[132] 도 5에서알수있듯이，근적외선대역인 7850111광에의해놀랍도록강한라만 산란신호가얻어짐을확인할수있으며， 1100（ -1영역근처와 1550011 -1 영역근처에서관찰되는강한라만신호는라만리포터（061）고유의요묘요 신호와일치함을알수있다.
[133] 이상과같이본발명에서는특정된사항들과한정된실시예및도면에의해
설명되었으나이는본발명의보다전반적인이해를돕기위해서제공된것일 뿐，본발명은상기의실시예에한정되는것은아니며，본발명이속하는
분야에서통상의지식을가진자라면이러한기재로부터다양한수정및변형이 가능하다.
[134] 따라서，본발명의사상은설명된실시예에국한되어정해져서는아니되며, 후술하는특허청구범위뿐아니라이특허청구범위와균등하거나등가적변형이 있는모든것들은본발명사상의범주에속한다고할것이다.
[135]

Claims

2020/075879 1»（：1/10公018/011873 청구범위
[청구항 1] a)완중용액 (buffer solution)에제 1금속전구체가혼합된제 1
반응액으로부터나노-별형상의금속나노코어를제조하는단계;
b)상기금속나노코어에라만리포터를고정하는단계 ;및
c)상기라만리포터가고정된나노코어와제 2금속전구체가혼합된제 2 반응액으로부터라만리포터가고정된나노코어를감싸는금속웰을 형성하는단계 ;
를포함하며，
상기라만리포터는상기금속나노코어의제 1금속및상기금속쉘의제 2 금속각각에결합력을갖는라만활성나노입자의제조방법.
[청구항 2] 제 1항에 있어서,
상기 b)단계에서상기금속나노코어를감싸는라만리포터의 자기조립단분자막이형성되는라만활성나노입자의제조방법.
[청구항 3] 제 1항에 있어서,
상기완충용액의완충제 (buffer agent)와상기제 1금속전구체의몰비;및 상기완충용액의 pH;중하나이상의 인자 (factor)를제어하여나노코어의 형상，크기또는형상과크기를조절하는라만활성나노입자의제조방법 .
[청구항 4] 제 3항에 있어서，
상기완충제의몰수를상기제 1금속전구체의몰수로나눈몰비인 은 200내지 750인라만활성나노입자의제조방법 .
[청구항 5] 제 1항에 있어서，
상기제 2반응액은계면활성제또는계면활성제와유기산을포함하는 라만활성나노입자의제조방법.
[청구항 6] 제 1항에 있어서,
상기완충용액은 HEPES(4-(2-hydroxyethyl)-l-piperazineethanesulfonic acid), MES (4-(2-Hydroxyethyl)piperazine- 1 -ethanesulfonic acid), PBS(Phosphated buffered saline), Tris(2-Amino-2hydroxymethyl propne-1, 3-idol), PB (Phosphate buffer),
MOPS(3-(N-morpholino)propanesulfonic acid),
TAPS(3-[[l,3-dihydroxy-2-(hydroxymethyl)propan-2-yl]amino]propane-l-sulf onic acid)및 PIPES(piperazine-N,N,-bis(2-ethanesulfonic acid))에서
선택되는하나이상을함유하는라만활성나노입자의제조방법.
[청구항 7] 제 1항에 있어서， - 상기금속전구체의금속은 Au또는쇼은인라만활성나노입자의
제조방법.
[청구항到 제 1항에 있어서，
c)단계후, 2020/075879 1»（：1^1{2018/011873
(1)상기금속웰에분석대상물과결합하는수용체를고정하는단계;를더 포함하는라만활성나노입자의제조방법.
［청구항 9］ 제 1항내지제 8항중어느한항에따른라만활성나노입자의
제조방법으로제조된라만활성나노입자.
［청구항 1이 나노-별형상의금속나노코어 ;
상기금속나노코어에고정된라만리포터를포함하는
자기조립단분자막;및
상기자기조립단분자막을감싸는금속웰;
을포함하며상기나노-별형상의돌출부에의해돌출구조를갖는라만 활성나노입자.
［청구항 1 1］ 제 10항에있어서， ,
상기라만활성나노입자는상기나노-별형상에 대응하는형상을갖는 라만활성나노입자.
［청구항 12］ 제 10항에있어서，
상기금속나노코어는 10내지 5^01크기의중심영역및 5내지 7011111의 크기를가지며상기중심영역으로부터돌출되어돌출방향으로축경되는 돌출부를포함하는라만활성나노입자.
［청구항 13］ 제 10항에있어서,
상기라만활성나노입자는상기금속쉘에고정되어분석대상물과 결합하는수용체를더포함하는라만활성나노입자.
PCT/KR2018/011873 2018-10-08 2018-10-10 라만 활성 나노입자 및 이의 제조방법 WO2020075879A1 (ko)

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