WO2011071343A2 - Nanoparticle coeur-écorce hétérodimère dans laquelle des molécules actives à effet raman sont situées au niveau d'une partie de liaison d'une nanoparticule hétérodimère, utilisation de celle-ci, et procédé de préparation correspondant - Google Patents

Nanoparticle coeur-écorce hétérodimère dans laquelle des molécules actives à effet raman sont situées au niveau d'une partie de liaison d'une nanoparticule hétérodimère, utilisation de celle-ci, et procédé de préparation correspondant Download PDF

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WO2011071343A2
WO2011071343A2 PCT/KR2010/008862 KR2010008862W WO2011071343A2 WO 2011071343 A2 WO2011071343 A2 WO 2011071343A2 KR 2010008862 W KR2010008862 W KR 2010008862W WO 2011071343 A2 WO2011071343 A2 WO 2011071343A2
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core
shell
nanoparticle
dimer
oligonucleotide
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PCT/KR2010/008862
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Korean (ko)
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WO2011071343A3 (fr
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서영덕
남좌민
임동권
전기석
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한국화학연구원
서울대학교 산학협력단
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Priority to CN201080060485.3A priority Critical patent/CN102811943B/zh
Application filed by 한국화학연구원, 서울대학교 산학협력단 filed Critical 한국화학연구원
Priority to BR112012013999-1A priority patent/BR112012013999B1/pt
Priority to JP2012543027A priority patent/JP5701896B2/ja
Priority to CA2783788A priority patent/CA2783788C/fr
Priority to AU2010328768A priority patent/AU2010328768B2/en
Priority to US13/514,920 priority patent/US20130029360A1/en
Priority to EP10836238.5A priority patent/EP2511231B1/fr
Priority to RU2012129158/10A priority patent/RU2542386C2/ru
Publication of WO2011071343A2 publication Critical patent/WO2011071343A2/fr
Publication of WO2011071343A3 publication Critical patent/WO2011071343A3/fr
Priority to IL220294A priority patent/IL220294A/en
Priority to US15/069,063 priority patent/US20160266104A1/en
Priority to US16/906,093 priority patent/US20200385790A1/en

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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
    • G01N33/54366—Apparatus specially adapted for solid-phase testing
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    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6813—Hybridisation assays
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    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
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    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6813—Hybridisation assays
    • C12Q1/6834—Enzymatic or biochemical coupling of nucleic acids to a solid phase
    • 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
    • G01N33/5302—Apparatus specially adapted for immunological test procedures
    • 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
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    • C12Q2563/00—Nucleic acid detection characterized by the use of physical, structural and functional properties
    • C12Q2563/107—Nucleic acid detection characterized by the use of physical, structural and functional properties fluorescence
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    • C12Q2563/00—Nucleic acid detection characterized by the use of physical, structural and functional properties
    • C12Q2563/155—Particles of a defined size, e.g. nanoparticles
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    • C12Q2565/00—Nucleic acid analysis characterised by mode or means of detection
    • C12Q2565/60—Detection means characterised by use of a special device
    • C12Q2565/628—Detection means characterised by use of a special device being a surface plasmon resonance spectrometer
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    • C12Q2565/00—Nucleic acid analysis characterised by mode or means of detection
    • C12Q2565/60—Detection means characterised by use of a special device
    • C12Q2565/632—Detection means characterised by use of a special device being a surface enhanced, e.g. resonance, Raman spectrometer

Definitions

  • the present invention relates to a nanoparticle dimer prepared so that the Raman active molecule is located at the junction of a dimer composed of nanoparticles, and more specifically, is bonded to the surface of the core with an oligonucleotide, the gold or silver shell surrounding the core ( It relates to a core-shell nanoparticle dimer consisting of a shell).
  • the nanoparticles that form the core are gold or silver nanoparticles.
  • the present invention relates to the core-shell nanoparticle dimer, a preparation method and a use thereof.
  • Nanoparticles and chemicals labeled with specific materials have been widely used to study metabolism, distribution, and binding of substances and biological molecules.
  • Representative methods include radioisotopes, organic fluorescent materials, and inorganic quantum dots.
  • Radioactive labeling substance (Radioactive isotope) in the method using a radioactive isotope, 12 C, 31 P, 32 S , a 3 H, a radioactive isotope such as 127 I 14 C, 32 P, 35 S, 125 I and the like are widely used.
  • Radioactive isotopes have been used for a long time because they have almost the same chemical properties as non-radioactive isotopes, which can be arbitrarily substituted and have a relatively large amount of emission energy.
  • it is not easy to handle due to radiation harmful to the human body and the radioactivity of some isotopes has a disadvantage in that it is inconvenient for long-term storage or experiment due to the short half-life instead of large emission energy.
  • radioactive isotopes are organic fluorescent dyes. When fluorescent materials are activated by a specific wavelength, they emit light having a specific wavelength. In particular, as search methods become smaller, radioactive materials also exhibit detection limits, requiring a long time to search. In contrast, fluorescent materials can emit thousands of photons per molecule under the right conditions, making it possible to theoretically detect single molecules. However, there is a limitation in that it is not possible to directly substitute the element of the active ligand like the radioisotope, and to connect the fluorescent material by modifying the part which does not affect the activity relatively through the structure activity relationship.
  • these fluorescent markers have a disadvantage in that the fluorescence intensity becomes weaker with time (photobleaching), and the wavelength of light to be activated is very narrow and the wavelength of light to be emitted is very wide so that there is interference between different fluorescent materials.
  • the number of fluorescent materials that can be used is extremely limited.
  • the quantum dots which are semiconductor nanomaterials, are composed of CdSe, CdS, ZnS, ZnSe, and the like, and emit light of different colors depending on the size and type. Compared with organic fluorescent materials, they have broader active wavelengths and have narrower emission wavelengths. Therefore, there are more gadgets emitting different colors than organic fluorescent materials. Therefore, recently, quantum dots have been widely used as a method for overcoming disadvantages of organic fluorescent materials.
  • the toxicity is strong and mass production is difficult. In theory, the number of branches varies, but the number actually used is extremely limited.
  • SERS Surface Enhanced Raman Scattering
  • Raman spectroscopy can be developed into a highly sensitive technology that can directly measure a single molecule in combination with nanotechnology, which is currently developing at a very high speed, and is expected to be used as a medical sensor.
  • SERS surface enhanced Raman spectroscopy
  • Raman spectroscopy has several advantages over other analytical methods (infrared spectroscopy). Infrared spectroscopy can produce a strong signal for molecules with a change in the dipole moment of the molecule, while Raman spectroscopy can produce a strong signal for nonpolar molecules with a change in the induced polarization of the molecule. It has its own Raman Shift (cm -1 ). In addition, since it is not affected by the interference of water molecules, it is more suitable for the detection of biomolecules such as proteins and genes. However, the low signal strength did not reach a practical level despite the long research period.
  • SERS surface-enhanced Raman Scattering
  • the most dense sites (EF> 10 9 ) were 64 of a total of 1,000,000 sites, but they were reported to contribute 24% to the overall SERS intensity (Science, 2008, 321, 388). If a structure capable of maximizing the SERS signal can be reproducibly obtained, it can be a very reliable and useful ultra-high sensitivity biomolecular analysis method, and it may be very useful as an in vivo imaging technique in addition to in vitro diagnosis.
  • a hot spot or interstitial field which is a very strong electromagnetic field, is formed between two or more nanoparticles to enhance the SERS signal.
  • the hot spot predicts SERS enhancement of about 10 12 .
  • the enhanced sensitivity of Raman detection is clearly not uniform inside the colloidal particle aggregates, but depends on the presence of hot spots.
  • the physical structure of such hot spots, the range of distance from nanoparticles with enhanced sensitivity, and the spatial relationship between analyte and aggregate nanoparticles that enhance sensitivity have not been characterized. Aggregated nanoparticles are also inherently unstable in solution, adversely affecting the reproducibility of single particle analyte detection.
  • the present inventors have tried to develop a nanoparticle structure having a high sensitivity and reproducibility for single DNA detection. As a result, the present inventors have placed a single Raman active molecule at the junction of the dimeric nanoparticles of the core-shell structure, and the thickness of the shell.
  • the surface-enhanced Raman the dimer nanoparticles are very reinforced It showed a surface-enhanced Raman Scattering (SERS) signal and proved to be a highly reproducible hot spot nanoparticle, and by confirming that the Raman scattering enhancement of the nanoparticles showed SERS enhancer (E EM ) to 2.7 ⁇ 10 12 .
  • SERS surface-enhanced Raman Scattering
  • One object of the present invention is to provide a core-shell structured nanoparticle dimer wherein the Raman active molecule is located at the junction of the nanoparticle dimer.
  • Another object of the present invention to provide an analyte detection kit comprising the nanoparticle dimer.
  • the present invention provides a Raman active molecule at a junction of two core-shell nanoparticles consisting of a gold or silver core having an oligonucleotide bound thereto and a gold or silver shell surrounding the core.
  • the present invention relates to a nanoparticle dimer having a structure in which the two nanoparticles are linked through oligonucleotides.
  • the nanoparticle dimer of the present invention consists of two nanoparticles, each nanoparticle consisting of a core (gold or silver) and a shell (gold or silver) surrounding the core, each nanoparticle
  • the oligonucleotides are bonded to the core surface of the core and are exposed to the outside of the shell, and have a structure in which the oligonucleotides are linked through a hybrid bond between the two oligonucleotides.
  • Raman active molecules may be located at the junction where the hybrid bond is made.
  • each of the nanoparticles, one end of the oligonucleotide is bound to the surface of the core, a portion of the oligonucleotide is exposed to the outside of the shell, it is possible to form a dimer through the sequence of the exposed portion.
  • each of the two exposed oligonucleotides may be hybridized to each other, or may be hybridized to each other through an oligonucleotide capable of hybridizing to each of the two exposed oligonucleotides.
  • the term "core” means a metal particle having an oligonucleotide directly bonded to the surface thereof, and preferably gold or silver.
  • the term “shell” in the present invention is a metal coating layer surrounding the core, a portion of the oligonucleotides bonded to the core surface is located inside the shell. In the present invention, it is preferable to use gold or silver as the shell.
  • the present invention provides a method for preparing a nanoparticle dimer comprising: i) a nanoparticle dimer with two core-shell nanoparticles consisting of a gold core and a silver shell, ii) a nanoparticle dimer with two core-shell nanoparticles consisting of a silver core and a gold shell, iii) Nanoparticle dimer with two core-shell nanoparticles consisting of a gold core and a gold shell, iv) a nanoparticle dimer with two core-shell nanoparticles consisting of a silver core and a silver shell, and v) a core-shell consisting of a gold core and a silver shell A nanoparticle dimer selected from the group consisting of nanoparticle dimers to which nanoparticles and core-shell nanoparticles consisting of a silver core and a gold shell are linked.
  • the nanoparticle dimer of the present invention is a nanoparticle dimer with two core-shell nanoparticles consisting of a gold core
  • the core-shell structured nanoparticle dimer of the present invention as described above may exist as a homodimer or a heterodimer.
  • Homodimer is a two-particle nanoparticles of the same size and structure are connected to form a dimer
  • heterodimer is a two-particle nanoparticles of different size or structure are connected to form a dimer.
  • Core particles forming the center of the surface-enhanced Raman scattering nano-labeled particles according to the present invention preferably has a diameter of 5 nm to 300 nm, which is a problem that the Raman surface enhancement effect is lowered if the diameter of the core is less than 5 nm If it exceeds 300 nm, there is a problem that many restrictions in biological applications. More preferably, the diameter of the core is 10 nm to 40 nm.
  • the nanoparticles may be approximately spherical in shape, but nanoparticles of any shape or irregular shape may be used.
  • Nanoshells are introduced to the surface of the core particles, and the nanoshells provide enhanced Raman scattering effects on the surface of the core particles to facilitate analysis by Raman spectroscopy.
  • the core particles into which the nanoshell is introduced have a very large surface enhanced Raman scattering effect, a signal of any chemical can be obtained.
  • the shell thickness of the present invention is 1 nm to 300 nm, more preferably 1 to 20 nm.
  • the thickness of the shell can increase proportionally as the size of the core and the length of the DNA used increase.
  • the core particle surface of the present invention is characterized in that one or more oligonucleotides are combined and functionalized.
  • core A may be bound to one or more protective oligonucleotides whose 3 ′ ends are modified with thiols and also one target oligonucleotide whose 3 ′ ends are modified with thiols.
  • Core B may be bound to one or more protective oligonucleotides whose 5 ′ ends are modified with thiols, and also one target oligonucleotide whose 5 ′ ends are modified with thiols.
  • either of the target oligonucleotide modified on the surface of the core A and the target oligonucleotide modified on the surface of the core B is characterized in that the Raman active molecule is modified.
  • the present invention can also be completed by a bond using a 5 'end to Core A and a bond using a 3' end to Core B.
  • protecting oligonucleotide refers to an oligonucleotide that is bound to the surface of the core particle and functions to protect the surface while stabilizing the core particle so that the target oligonucleotide can be bound to the core surface.
  • target oligonucleotide refers to an oligonucleotide having a sequence complementary to a target oligonucleotide, wherein both the target oligonucleotide of Core A and the target oligonucleotide of Core B hybridize with one common target oligonucleotide ( hybridization) to form nanoparticle dimer structures.
  • target oligonucleotide includes a sequence complementary to both the target oligonucleotide of Core A and the target oligonucleotide of Core B, and hybridizes with each of the target oligonucleotides, thereby obtaining two target captures. It refers to a crosslinking role oligonucleotide that connects the oligonucleotide to form a nanoparticle dimer structure, and does not mean the final target analyte using the dimer structure.
  • the protective oligonucleotide and the target oligonucleotide of the present invention are modified with a compound having a surface binding functional group at the 3 'end or the 5' end and attached to the surface of the core particle through the surface binding function.
  • compound with surface binding functional group refers to a compound linked to the 3 'or 5' end of each oligonucleotide to attach the oligonucleotide to the surface of the core particle.
  • the type of compound having such surface binding functional groups is not limited as long as it produces small aggregates of nanoparticles that will not precipitate in solution. Methods for cross-linking nanoparticles via surface binding functional groups are known in the art (see Feldheim, The Electrochemical Society Interface, Fall, 2001, pp. 22-25).
  • One end of the compound having a surface binding functional group has a surface binding functional group attached to the surface of the core particle, preferably a sulfur-containing group such as a thiol group or a sulfhydryl (HS) group.
  • the reactor may be a compound having a formula of RSH containing sulfur in the oxygen site as a derivative of alcohol and phenol.
  • the reactor may be a thiol ester or a dithiol ester having a formula of RSSR 'or RSR', respectively.
  • the reactor may be an amino group (-NH 2 ).
  • the compound having the surface binding functional group is a reactive group for biomolecules such as DNA probes, antibodies, oligonucleosides and amino acids, such as -NH 2 , -COOH, -CHO, -NCO, and It can be connected with various reactors such as epoxide groups. Many such reactive groups are known in the art and can be used in the present methods and apparatus.
  • the opposite end of the compound having a surface-binding functional group in the oligonucleotide may comprise a spacer sequence which may be appropriate without the shell introduced at the core surface covering the target recognition sequence of the target oligonucleotide. Provides space to maintain shell thickness.
  • the spacer sequence A 10 -PEG was used as an example of the spacer sequence.
  • the term "raman active molecule” refers to a substance that facilitates the detection and measurement of an analyte by a Raman detection device when the nanoparticle dimer of the present invention is attached to at least one analyte.
  • either one of the target oligonucleotide modified on the surface of the core A and the target oligonucleotide modified on the surface of the core B is characterized in that the Raman active molecule is modified. Since the Raman active molecule shows a specific Raman spectrum, there is an advantage that can later analyze the biomolecule more effectively.
  • Raman active molecules that can be used in Raman spectroscopy are organic or inorganic molecules, atoms, complex or synthetic molecules, dyes, naturally occurring dyes (such as picoeryrin), organic nanostructures such as C 60 , buckyballs, carbon nanotubes, quantum dots , Organic fluorescent molecules and the like.
  • Raman active molecules include FAM, Dabcyl, TRITC (tetramethyl rhodamine-5-isothiocyanate), MGITC (malakit green isothiocyanate), XRITC (X-rhodamine-5-iso Thiocyanate), 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, fluorine Lecein, 5-carboxy-2 ', 4', 5 ', 7'-tetrachlorofluorescein, 5-carboxyfluorescein, 5-carboxyfluorescein,
  • the organic fluorescent molecules include Cy3, Cy3.5, Cy5 or FAM, Dabcyl, Rhodamine-based fluorescent molecules, which are cyanine-based fluorescent maintenance molecules.
  • Organic fluorescent molecules resonate with the excitation laser wavelength used in Raman analysis, which has the advantage of enabling detection of higher Raman signals.
  • Raman active molecules may be attached directly to the analyte or may be attached via various linker compounds.
  • the present invention relates to a method for producing a core-shell structured nanoparticle dimer labeled with the Raman active molecule.
  • the first step is to prepare Core A and Core B, the surfaces of which are bound to protective oligonucleotides and target oligonucleotides, respectively.
  • gold core particle A is one or more protective oligonucleotides, which are two kinds of oligonucleotide sequences whose 3 ′ ends are modified with thiols in order to control one target oligonucleotide sequence to be bound to the core particle surface.
  • Gold core particle B was also bound by two kinds of oligonucleotide sequences whose 5 'ends were modified with thiols.
  • the molar ratio of the two types of oligonucleotide sequences is 99: 1 for core A and 199: 1 for core B, based on the nanoparticle-size dependent loading capacity of the oligonucleotides on the surface of the gold core particles. Adjusted (FIG. 1A). Importantly, Cy3, FAM, or Dabcyl, which functions as a Raman signal, was bound to the end of the target capture oligonucleotide sequence bound to Core B.
  • Magnetic separation techniques can also be used to purify oligonucleotide modified cores A and B by removing monomer particles to which the target capturing sequence is not bound.
  • Tosyl group magnetic beads (1 ⁇ m in diameter, Invitrogen) may be substituted with target capture sequences of Core A or B, respectively, by amine-modified oligonucleotide complementary sequences. Only core particles bound with the target capture sequence are complementary to the magnetic beads, so that after the reaction, an external magnetic field is applied to the magnetic nanoparticles to separate the magnetic nanoparticles, and then to the melting point (Tm) of the double-stranded DNA sequence. The temperature may be elevated to separate core particles bound to the magnetic nanoparticles.
  • hybridization reactions are performed with magnetic microparticles having a sequence complementary to the target capture oligonucleotides of Core A and Core B, thereby targeting the targets in Core A and Core B.
  • the method may further include separating only the nanoparticles to which the capture oligonucleotides are bound.
  • the second step is to form a dimer by adding and hybridizing target oligonucleotides to Core A and Core B prepared above.
  • a solution of core particles A and B separated and purified by magnetic beads in the first step can synthesize the desired dimer nanoparticles, for example, by hybridizing with a sufficient amount of target oligonucleotide sequence in 0.3 M PBS.
  • the dimer synthesis method according to the present invention can synthesize dimers in a very high yield (70-80%).
  • the introduction of the nanoshell particles to the core particle surface is preferably introduced by, for example, reacting the gold core particles with the precursor of the silver nanoparticles at 10 to 100 ° C. in the presence of a solvent.
  • the precursor of the silver nanoparticles is preferably selected from AgNO 3 , or AgClO 4
  • the precursor of the gold nanoparticles can be used as a precursor any compound containing Au ions such as HAuCl 4 .
  • a reducing agent required to convert silver ions or gold ions into gold or silver nanoparticles may be hydroquinone, sodium borohydride (NaBH 4 ), sodium ascorbate, etc., but is not limited thereto. It is not.
  • an aqueous solution (purified water or phosphate buffer or the like may be present) is preferable. Additional stabilizers can be added for precise thickness control of the nanoshells. If the reaction temperature is less than 10 °C is a problem that takes too much time to form the silver nanoparticles, if it exceeds 100 °C there is a problem that the silver nano particles are formed non-uniformly reacting at a temperature in the above range desirable. The reaction time may be adjusted to 10 to 24 hours depending on the reaction temperature.
  • a biomolecule capable of recognizing an analyte to be detected on the surface of the nanoparticle dimer or the core is functionalized.
  • the present invention can be applied to detect various biomolecules.
  • 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.
  • 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.
  • biomolecules capable of binding to surfaces of nanoparticle dimers include antibodies, antibody fragments, engineered antibodies, single chain antibodies, receptor proteins, binding proteins, enzymes. , Inhibitor proteins, lectins, cell adhesion proteins, oligonucleotides, polynucleotides, nucleic acids or aptamers.
  • the entirety of the nanoparticle dimer may be coated with an inorganic material.
  • the structure is less likely to be deformed, so that the structure of the nanoparticle dimer can be stably maintained, which is more preferable for storage and use.
  • the inorganic material is not limited as long as it maintains the structure of the nanoparticle dimer and does not affect the Raman signal, and silica may be used as an example.
  • the present invention provides a method for detecting an analyte using the nanoparticle dimer of the present invention as described above.
  • 1) preparing a nanoparticle dimer of the present invention 2) functionalizing a biomolecule capable of recognizing an analyte to be detected on the surface of the nanoparticle dimer or the surface of the core; 3) exposing the nanoparticle dimer to a sample comprising one or more analytes; And 4) detecting and identifying one or more analytes using laser excitation and Raman spectroscopy.
  • the analytes of the present invention can be detected or identified by any known Raman spectroscopy, preferably Surface Enhanced Raman Scattering (SERS), Surface Enhanced Resonance Raman Spectroscopy (SERRS) resonance Raman spectroscopy), hyper-Raman and / or incoherent anti-Stokes Raman spectroscopy (CARS) can be used.
  • Raman spectroscopy preferably Surface Enhanced Raman Scattering (SERS), Surface Enhanced Resonance Raman Spectroscopy (SERRS) resonance Raman spectroscopy), hyper-Raman and / or incoherent anti-Stokes Raman spectroscopy (CARS) can be used.
  • SERS Surface Enhanced Raman Scattering
  • SERRS Surface Enhanced Resonance Raman Spectroscopy
  • CARS incoherent anti-Stokes Raman spectroscopy
  • the term "surface enhanced Raman scattering method (SERS)” is a type of Raman scattering generated when adsorbed on a rough metal surface or located within a few hundred nanometers, the intensity of Raman scattering is general It refers to spectroscopy using the phenomenon that 10 6 ⁇ 10 8 times increase compared with Raman intensity.
  • the term “surface enhanced resonance Raman spectroscopy” (SERRS) refers to spectroscopy using the resonance phenomenon of the laser excitation wavelength for an adsorbate on the SERS active surface.
  • SERRS surface enhanced resonance Raman spectroscopy
  • CARS coherent antistock coherent Raman spectroscopy
  • the analyte detection method of the present invention 1) preparing a nanoparticle dimer of the present invention; 2) functionalizing a biomolecule complementary to the nucleic acid to be detected on the surface of the nanoparticle dimer or the surface of the core; 3) extracting, purifying, and amplifying the nucleic acid in the sample; 4) performing hybridization by reacting the core-shell nanoparticle dimer with a specific sequence of the amplified nucleic acid; And 5) performing a Raman spectroscopy on the nucleic acid to which the nanoparticle dimer is bound.
  • This same type of method can also be used to detect other information about the nucleic acid, such as one or more single base polymorphisms (SNPs) or other genetic variations in the sample, and furthermore, applications in DNA sequencing. It is possible.
  • SNPs single base polymorphisms
  • the Raman active substrate may be operatively coupled with one or more Raman detection units.
  • Raman detection units 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).
  • 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.
  • the excitation beam is generated by a frequency superimposed Nd: YAG laser at 532 nm wavelength or a frequency superimposed Ti: sapphire laser at 365 nm wavelength.
  • Pulsed laser beams or continuous laser beams may 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
  • U.S. Patent No. 5,306,403 which is a Spex Model equipped with a gallium-arsenide photomultiplier tube (RCA Model C31034 or Burle Industries Model C3103402) operating in a single photon counting manner. 1403 dual grating spectrometer. Excitation sources include SpectraPhysics, 514.5 nm line argon-ion laser from Model 166, and 647.1 nm line of krypton-ion laser (Innova 70, non-coherent).
  • excitation beam is spectrally purified by a bandpass filter on a Raman active substrate using a 6X objective lens (Newport, Model L6X).
  • the objective lens uses a holographic beam splitter (Kaiser Optical Systems, Inc., Model HB 647-26N18) to excite the analyte and collect the Raman signal to form a right angle shape for the excitation beam and the emitted Raman signal.
  • Holographic notch filters can be used to reduce Rayleigh scattered radiation.
  • the furnace includes an ISA HR-320 spectrometer equipped with a red-enhanced high-sensitivity charge-coupled device (RE-ICCD) detection system (Princeton Instruments): a Fourier transform spectrometer (based on a Michaelson interferometer), a charged implant device, a photodiode Other types of detectors may be used, such as arrays, InCaAs detectors, electron multiplication CCDs, high sensitivity CCDs and / or phototransistor arrays.
  • RE-ICCD red-enhanced high-sensitivity charge-coupled device
  • 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 , Non-coherent antistock Raman spectroscopy (CARS), stimulus 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 microspectroscopy, three-dimensional or scanning Raman, Raman saturation spectroscopy, time resolved resonance Raman, Raman dissociation spectroscopy or UV-Raman microscopy.
  • CARS Non-coherent antistock Raman spectroscopy
  • MOLE molecular optical laser examiner
  • Raman microprobe or Raman Microscopy or confocal Raman microspectroscopy three-dimensional or scanning Raman, Raman saturation spectroscopy, time resolved resonance Raman, Raman
  • the Raman detection device may comprise a computer.
  • the example 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).
  • the Raman detection device may be operatively coupled with a computer.
  • Data from the detection device may be processed by the processor and the data may be stored in the main memory. Data on release profiles for standard analytes may also be stored in main memory or ROM.
  • 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. Thus, the structure of the system may be different in different embodiments of the present invention.
  • the data After the data collection task, the data will typically be sent to a data analysis task. 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.
  • the present invention relates to an analyte detection kit comprising the nanoparticle dimer of the present invention.
  • the analyte to be detected when it is a nucleic acid, it may be a kit including an element necessary for performing RT-PCR to amplify the nucleic acid contained in the sample.
  • RT-PCR kits include test tubes or other suitable containers, reaction buffers (pH and magnesium concentrations vary), deoxynucleotides (dNTPs), Taq-polymerases and reverse transcription, in addition to each primer pair specific for the nucleic acid to be detected.
  • Enzymes such as enzymes, DNase, RNAse inhibitors, DEPC-water (DEPC-water), sterile water and the like may be further included. It may also further comprise a primer pair specific for the gene used as a quantitative control.
  • the kit of the present invention may be a detection kit containing essential elements necessary for carrying out a DNA chip.
  • the DNA chip kit may further include a substrate to which a cDNA corresponding to a gene or a fragment thereof is attached as a probe, and a reagent, an agent, an enzyme, and the like for preparing a fluorescent probe.
  • the substrate may further comprise a cDNA corresponding to the quantitative control gene or fragment thereof.
  • the analyte to be detected when it is a protein, it may further include a substrate, an appropriate buffer solution, a secondary antibody labeled with a nanoparticle dimer of the present invention, a chromogenic substrate, etc. for immunological detection of the antibody.
  • the substrate may be a nitrocellulose film, a 96 well plate synthesized with a polyvinyl resin, a 96 well plate synthesized with a polystyrene resin, a slide glass made of glass, or the like.
  • Such detection kits include tools, reagents, and the like commonly used in the art.
  • tools / reagents include, but are not limited to, suitable carriers, labeling materials capable of producing detectable signals, solubilizers, detergents, buffers, stabilizers, and the like.
  • labeling substance is an enzyme, it may include a substrate and a reaction terminator capable of measuring enzyme activity.
  • Suitable carriers include, but are not limited to, soluble carriers such as physiologically acceptable buffers known in the art, such as PBS, insoluble carriers such as polystyrene, polyethylene, polypropylene, polyesters, Polyacrylonitrile, fluorine resin, crosslinked dextran, polysaccharides, polymers such as magnetic fine particles plated with latex metal, other papers, glass, metals, agarose and combinations thereof.
  • physiologically acceptable buffers known in the art, such as PBS, insoluble carriers such as polystyrene, polyethylene, polypropylene, polyesters, Polyacrylonitrile, fluorine resin, crosslinked dextran, polysaccharides, polymers such as magnetic fine particles plated with latex metal, other papers, glass, metals, agarose and combinations thereof.
  • Antigen-antibody complex formation may include tissue immunostaining, radioimmunoassay (RIA), enzyme immunoassay (ELISA), Western blotting, immunoprecipitation assay, immunodiffusion assay, and complement fixation assay. (Complement Fixation Assay), FACS, protein chip, etc., but are not limited thereto.
  • the Raman active molecule is located at the junction of two nanoparticles
  • the Raman active molecule is precisely located at the junction of two nanoparticles
  • the distance between the surface of the Raman active molecule and the nanoparticle is silver or gold nanoparticles. Because of the precise thickness control with the particles, a very enhanced surface enhanced Raman scattering (SERS) signal can be obtained. Moreover, despite the presence of only one Raman active molecule, a strong Raman signal can be obtained.
  • SERS surface enhanced Raman scattering
  • the method of synthesizing the core-shell nanoparticle dimer is a useful method for synthesizing the dimer with a very high purity, in particular, the equivalent control of oligonucleotides during the synthesis of cores A and B, core A using magnetic nanoparticles Through the purification of B, etc., the desired nanostructure could be synthesized with high purity.
  • the gap between the two nanoparticles can be adjusted to the distance between the gap in a nano-level adjustable way. Therefore, this core-shell nanostructure is a nanostructure in which the signal is amplified to a very high degree, and thus it is highly applicable to the detection of analytes such as DNA and proteins (biomarkers) related to the development and progression of specific diseases. It can be used for sequence analysis, detection of single nucleotide polymorphism (SNP), sequence comparison, genotyping, disease correlation, and drug development.
  • SNP single nucleotide polymorphism
  • FIGS. 1 (A) and (B) are schematic diagrams illustrating the synthesis of gold nanoparticle dimers by magnetic purification, DNA hybridization, and silver-shell formation.
  • a probe sequences protected 3'-HS- (CH 2) 3 - A 10 -PEG 18 -AAACTCTTTGCGCAC-5 '
  • probe A target binding sequence is 3'-HS- (CH 2) 3 - A 10 -PEG 18 -
  • Probe B protection sequence 5'-HS- (CH 2) 6 - A 10 -PEG 18 -AAACTCTTTGCGCAC-3 '
  • probe B target binding sequence is 5'-HS- (CH 2) 3 - A 10 -PEG 18 -
  • Underlined sequences are spacer sequences designed to promote silver-shell formation.
  • the target DNA sequence is 5'-GAGGGATTATTGTTAAATATTGATAAGGAT-3 '(anthrax sequence).
  • FIG. 1 (C) shows an experimental device using a calibrated AFM, confocal Raman spectrometer (laser focal diameter 250 nm) to determine the SERS hot spot structure from each dimer nanoparticle.
  • FIG. 2 shows ultraviolet-visible spectra and HR-TEM images before and after forming gold nanoparticle dimers.
  • B shows ultraviolet-visible spectra and HR-TEM images before and after the introduction of silver-shells into gold nanoparticle dimers.
  • C shows the plasmon resonance of silver nanoparticles of ⁇ 400 nm depending on the silver-shell thickness. Corresponds to the HR-TEM image of the gold-silver core-shell dimer structure.
  • the HR-TEM images C.1a and C.1b of the core-shell nanoparticles shown in the drawings represent gold-silver core-shell monomers having silver shell thicknesses of 5 nm and 10 nm, respectively.
  • C.2, C.3 and C.4 represent gold-silver core-shell heterodimeric particles having silver shell thicknesses of ⁇ 3 nm, ⁇ 5 nm and ⁇ 10 nm, respectively.
  • A shows AFM (atomic force micrograph, 1 ⁇ m ⁇ 1 ⁇ m) of gold-silver core-shell monomer and heterodimeric nanoparticles.
  • B shows the calibrated SERS spectra obtained from monomers or dimers of gold-silver core-shell nanoparticles.
  • C shows all spectra obtained at 514.5 nm excitation laser, 1 s accumulation, 100 ⁇ W of sample, 250 nm laser focal diameter.
  • Raman spectra red line
  • Raman spectra black line
  • Raman spectra black line
  • FIG. 4 shows that each analyzed gold-silver core-shell dimer nanoparticle exhibits a SERS signal corresponding to Cy3 with single molecule activity.
  • A shows a tapping-mode AFM image (5 ⁇ m ⁇ 5 ⁇ m) of a gold-silver core-shell dimer (corresponding to a silver-shell thickness of 5 nm and a distance of 1 nm or less in FIG. 2 (B) -2).
  • B) shows the surface-enhanced Raman spectra of Cy3 obtained in each dimer structure, measured at a laser wavelength of 514.5 nm, a laser power of 80 ⁇ W, a laser focal diameter of 250 nm, and an integration time of 1 second.
  • (A) and (B) show the Blinking SERS spectra obtained on the same particles at 1 second accumulation time, and (C) shows the SERS spectra obtained on the gold-silver core-shell heterodimers at different incident laser polarizations.
  • (D) represents the polar spot of the integrated SERS intensity of the 1470 and 1580 cm ⁇ 1 Raman bands for ⁇ . The measurement was performed at a laser wavelength of 514.5 nm, a laser power of 40 ⁇ W, a laser focal diameter of 250 nm, and an integration time of 20 seconds.
  • Figure 6 shows SERS spectra obtained from nanoparticle dimers modified with oligonucleotides labeled with FAM and Dabcyl.
  • (a) shows Raman spectra of FAM labeled oligonucleotides (1 nM) and Dabcyl labeled oligonucleotides (1 nM) in solution.
  • (b) shows Raman spectra of FAM labeled gold-silver core-shell nanoparticle dimers (silver shell, 5 nm), Dabcyl labeled gold-silver core-shell nanoparticle dimers (silver shell, 5 nm).
  • silver nanoshells can be modified (Chem. Comm. 2008, J. Phys. Chem. B 2004, 108, 5882-5888).
  • gold nanoparticle dimer 250 ⁇ M gold nanoparticle dimer solution 250 ⁇ M in the presence of 100 ⁇ L of poly (vinyl) pyrrolidone as stabilizer and 50 ⁇ L of L-sodium ascorbic acid [10 ⁇ 1 M] as reducing agent in a 0.3 M PBS solution for 3 hours at room temperature was reacted with various amounts of AgNO 3 [10 ⁇ 3 M].
  • Gold-silver core-shell heterodimeric nanoparticles having silver-shell thicknesses of ⁇ 3 nm, ⁇ 5 nm, and ⁇ 10 nm were synthesized using 30 ⁇ L, 40 ⁇ L, and 70 ⁇ L of AgNO 3 [10 ⁇ 3 M] solution, respectively.
  • the silver shell thickness was successfully adjusted in nano units such as ⁇ 3 nm, ⁇ 5 nm, and ⁇ 10 nm, thereby obtaining a gold-silver core-shell heterodimer structure coupled with the target oligonucleotide.
  • the gold nanoparticles (15 nm) for probe A are functionalized with two kinds of 3 'terminal thiol-modified oligonucleotide sequences to control one target oligonucleotide sequence to be modified on the surface of the gold nanoparticles. (functionalize).
  • Gold nanoparticles (30 nm) for probe B were also functionalized by two 5 'end-thiol-modified oligonucleotide sequences. The molar ratio of the two kinds of sequences was adjusted to be 199: 1 for probe A and 99: 1 for probe B, based on the nanoparticle-size dependent loading ability of oligonucleotides on the gold nanoparticle surface (FIG. One).
  • the targeting oligonucleotide sequence bound Cy3 to the end, which functions as a Raman tag.
  • Oligonucleotide modified probes A and B are also purified by magnetic separation techniques to remove monomer particles to which the target capturing sequence is not bound.
  • the tosyl group of magnetic beads (1 ⁇ m in diameter, Invitrogen) was substituted by the amine-modified oligonucleotide complementary sequence to the target capture sequence of probe A or B, respectively. Only gold nanoparticles bound to the target capture sequence can be separated by magnetic beads.
  • the purified probe A and B solutions hybridized with a sufficient amount of target oligonucleotide sequence in 0.3M PBS.
  • the spacing distance between nanoparticles in solution is expected to be -15 nm which is much longer than the dry state.
  • silver nanoparticles have been applied to known modification methods (Chem. Comm. 2008, J. Phys. Chem. B 2004, 108, 5882-5888). By introducing it into the surface of the gold nanoparticle dimer while adjusting to nanometer (see Example 1). Gold-silver core-shell monomers (1a, 1b in FIG. 2C) having a silver-shell thickness of ⁇ 3 nm to 10 nm were also synthesized from probe B solution (30 nm AuNP) purified under similar conditions.
  • the ultraviolet-visible spectrum (FIG. 2B) of each solution was distinguished at plasmon resonance peaks of ⁇ 400 nm depending on the silver shell thickness.
  • 2C shows HR-TEM images of individual gold-silver core-shell heterodimers, 26 nm-36 nm in diameter (FIG. 2C-2), 30 nm-40 nm (FIG. 2C-3), 40 nm-50 nm (FIG. 2C-3) Are the two core-shell nanoparticle spheres and silver shell thickness. There is a narrow gap of less than 1 nm between the two nanoparticles.
  • SERS / AFM of the monomer or heterodimer of the core-shell nanoparticle was measured.
  • an aliquot (20 ⁇ L) of gold-silver core-shell heterodimer solution washed by repeated centrifugation (8000 rpm, 20 minutes, 3 times) was spin coated onto a poly-L-lysine coated glass surface. coated), washed several times with ultrapure water and then dried in air. After sample preparation, it was immediately used for AFM and SERS measurements.
  • SERS spectra were recorded by an AFM-associated NanoRaman microscope equipped with an inverted optical microscope (Axovert 200, Zeiss) and a piezoelectric xy sample scanner (Physik Instrument) by a separate homemade scanning controller. Adjusted.
  • the 514.5 nm line of the argon ion laser (Melles Griot, USA) was used as the excitation source coupled with the single-mode optical fiber.
  • the dichroic mirror (520DCLP, Chroma Technology Corp.) is an immersion objective microscope with an excitation laser beam focused at a diffraction-limit spot (250 nm) on the upper surface of the glass cover slip from 250 nW to 1 mW.
  • FIG. 3A shows an enlarged atomic force micrograph (AMF) image (1 ⁇ m ⁇ 1 ⁇ m) of representative core-shell monomer and heterodimeric nanostructures (using Cy3 as Raman active molecule). Shape and diameter were consistent with the results of HR-TEM analysis.
  • FIG. 3B shows the SERS spectrum corrected by AFM image for the single particles indicated in FIG. 3A. Since there is no hot spot in the core-shell monomer structure and there is only one Cy3 molecule, gold-silver core-shell monomers with silver shell thicknesses of 5 nm (FIG. 3A-1) or 10 nm (FIG. 3A-2) show SERS signals. Was not detected.
  • AFM atomic force micrograph
  • Gold dimers without silver shells or gold dimers with gap distances of less than 1 nm also did not detect SERS signals. This is due to the lack of electromagnetic enhancement under 514.5 nm laser excitation conditions. In the case of the thin shell thickness (less than 3 nm) in Figs. 3A-4, no signal was detected even when the incident laser power ( ⁇ 200 ⁇ M) was increased. These results indicate that very thin silver layers cannot produce sufficient electromagnetic enhancement. On the other hand, in the case of FIG. 3A-5 with a shell thickness of ⁇ 5 nm, a relatively high SERS signal was detected from a single gold-silver core-shell dimer where one Cy3 was located at the junction between the nanoparticles.
  • 3C shows Cy3-free SERS spectra of Cy3 modified oligonucleotides (5-HS- (CH 2 ) 6 -A 10 -PEG 18 -ATCCTTATCAATATTAAA-Cy3-3 ', 1 nM, red line) in aggregated silver colloids oligonucleotide shows a nucleotide compared to SERS spectra of (5-HS- (CH 2) 6 -A 10 -PEG 18 -ATCCTTATCAATATTAAA-3 ', 1nM, black line).
  • 3C shows that the adenine mode (734 cm -1 , 1320 cm -1 ) is predominant due to the abundance of adenine bases (A 10 was used as the spacer sequence) and is more augmented than other bases. (JACS 2008, 130 (16), 5523). It is also important that the lowest detection limit reported so far for other DNA bases is in the sub-micromolar range (JACS, 2006, 128, 15580). However, SERS spectra (red line) in FIG. 3C shows a relatively high signal in the signal of 1470cm -1, 1580cm -1 generated in the Cy3 molecules (Ananl chem. 2004, 76, 412-417).
  • FIGS. 4A and B Most core-shell dimer nanoparticles (using Cy3 as Raman active molecule) with a shell thickness of ⁇ 5 nm exhibited detectable SERS from single particles, as shown in FIGS. 4A and B.
  • the incident laser light is not exactly polarized on the interparticle axis of the dimer (Figs. 4A 1, 2, 3, 4, 5)
  • each of the vertically polarized dimer nanoparticles is detectable SERS. Indicates a signal.
  • FIG. 5C shows only a small peak at 1470 cm ⁇ 1 because dimer formation is nearly perpendicular to the incident tube.
  • these core-shell dimer nanostructures with optimized shell thickness may be hot spot structures that are highly applicable to single DNA detection.
  • 5C and 5D show the incident laser polarized dependence of the SERS signal on gold-silver core-shell dimers. All spectra are detected at 514.5 nm excitation laser, 20 s accumulation, and 40 ⁇ W of sample. The Cy3 peak was maximized when the incident laser light was polarized parallel to the longitudinal axis of the dimer. As the laser rotated 20 and 40 degrees from the longitudinal axis, the Cy3 signal gradually decreased. Finally, the Cy3 peak disappeared (eg, 90 and 270 degrees) when light polarized perpendicular to the vertical axis. Enhancement factor (EF) of the hot spot in the dimer structure was measured by the following formula at 1580 cm -1 .
  • EF Enhancement factor
  • I ser s and I bulk represent the same band intensities for SERS and bulk spectra
  • N bulk represents the number of bulk molecular labels for bulk samples
  • the strongest spectra bands were found in the 1580 cm -1 band region, which was used for I sers and I bulk intensities. Following this approach, the EF of the hot spot was calculated to be 2.7 ⁇ 10 12 .

Abstract

L'invention concerne une nanoparticule hétérodimère dans laquelle des molécules actives à effet Raman sont situées au niveau d'une partie de liaison de la nanoparticule hétérodimère, et plus particulièrement, une nanoparticule hétérodimère coeur-écorce comprenant: un noyau d'or ou d'argent possédant une surface à laquelle des oligonucléotides sont liés; et une écorce d'or ou d'argent recouvrant le noyau. En outre, cette invention concerne une nanoparticule hétérodimère coeur-écorce, un procédé destiné à préparer celle-ci, et son utilisation.
PCT/KR2010/008862 2008-05-07 2010-12-10 Nanoparticle coeur-écorce hétérodimère dans laquelle des molécules actives à effet raman sont situées au niveau d'une partie de liaison d'une nanoparticule hétérodimère, utilisation de celle-ci, et procédé de préparation correspondant WO2011071343A2 (fr)

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US13/514,920 US20130029360A1 (en) 2009-12-11 2010-12-10 Dimeric core-shell nanostructure labeled with raman active molecule localized at interparticle junction, use thereof, and method for preparing the same
BR112012013999-1A BR112012013999B1 (pt) 2009-12-11 2010-12-10 nanoestrutura dimérica de casca-núcleo marcada com molécula raman ativa localizada na junção de interpartículas e método para preparação do mesmo e para a detecção de um analito
JP2012543027A JP5701896B2 (ja) 2009-12-11 2010-12-10 ラマン活性分子がナノ粒子二量体の接合部に位置する二量体コア−シェルナノ粒子、その用途およびその製造方法
CA2783788A CA2783788C (fr) 2009-12-11 2010-12-10 Nanoparticle coeur-ecorce heterodimere dans laquelle des molecules actives a effet raman sont situees au niveau d'une partie de liaison d'une nanoparticule heterodimere, utilisation de celle-ci, et procede de preparation correspondant
AU2010328768A AU2010328768B2 (en) 2009-12-11 2010-12-10 A dimeric core-shell nanostructure labeled with a raman active molecule localized at an interparticle junction, use thereof, and method for preparing the same
CN201080060485.3A CN102811943B (zh) 2009-12-11 2010-12-10 以位于粒子间的结合部的拉曼活性分子标记的二聚体核壳纳米结构、其用途及制备方法
EP10836238.5A EP2511231B1 (fr) 2009-12-11 2010-12-10 Nanoparticle coeur-écorce hétérodimère dans laquelle des molécules actives à effet raman sont situées au niveau d'une partie de liaison d'une nanoparticule hétérodimère, utilisation de celle-ci, et procédé de préparation correspondant
RU2012129158/10A RU2542386C2 (ru) 2009-12-11 2010-12-10 Димерная окклюдантная наноструктура, меченная молекулой, активной в отношении рамановского рассеяния, локализованной в межчастичном соединении, ее использование и способ ее получения
IL220294A IL220294A (en) 2009-12-11 2012-06-10 Nano structure with a dimeric nucleus shell labeled with a raman active molecule is located at the intersection of particles, its use and method for making it
US15/069,063 US20160266104A1 (en) 2008-05-07 2016-03-14 Heterodimeric core-shell nanoparticle in which raman-active molecules are located at a binding portion of a nanoparticle heterodimer, use thereof, and method for preparing same
US16/906,093 US20200385790A1 (en) 2008-05-07 2020-06-19 Heterodimeric core-shell nanoparticle in which raman-active molecules are located at a binding portion of a nanoparticle heterodimer, use thereof, and method for preparing same

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PCT/KR2009/002399 Continuation-In-Part WO2009136741A1 (fr) 2008-05-07 2009-05-07 Nouveau composite cœur-coque or-argent convenant comme biocapteur
US13/514,920 A-371-Of-International US20130029360A1 (en) 2009-12-11 2010-12-10 Dimeric core-shell nanostructure labeled with raman active molecule localized at interparticle junction, use thereof, and method for preparing the same
US15/069,063 Continuation-In-Part US20160266104A1 (en) 2008-05-07 2016-03-14 Heterodimeric core-shell nanoparticle in which raman-active molecules are located at a binding portion of a nanoparticle heterodimer, use thereof, and method for preparing same

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CN102590174A (zh) * 2012-02-14 2012-07-18 厦门大学 用Fe3O4@Au核壳纳米探针检测生物分子的方法
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WO2014022330A2 (fr) * 2012-07-31 2014-02-06 Northwestern University Nanofeuilles dispersibles pour spectrométrie laser de l'effet raman exalté de surface
JP5835584B2 (ja) * 2010-06-15 2015-12-24 日産化学工業株式会社 表面増強ラマン散乱用金属粒子及び分子センシング
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