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

Abstract

A nanoparticle heterodimer in which Raman-active molecules are located at a binding portion of the nanoparticle heterodimer is disclosed. A core-shell nanoparticle heterodimer includes a gold or silver core having a surface to which oligo nucleotides are bonded; and a gold or silver shell covering the core. In addition, a core-shell nanoparticle dimer, a method for preparing same, and uses thereof are disclosed.

Claims

1. A method for preparing a dimer comprising two Au/Ag core-shell composites labeled with a Raman active molecule at an interparticle junction between Au/Ag core-shell nanoparticle A and Au/Ag core-shell nanoparticle B, wherein the Au/Ag core-shell nanoparticle A comprises an Au nanoparticle as a core A; a target-capturing oligonucleotide (A) capable of complementary base pairing with one part of the target nucleic acid (T), of which one end is bonded to a surface of the Au nanoparticle; and an Ag layer as a shell surrounding the Au nanoparticle; the Au/Ag core-shell nanoparticle B comprises an Au nanoparticle as a core B; a target-capturing oligonucleotide (B) capable of complementary base pairing with the other part of the target nucleic acid (T), of which one end is bonded to a surface of the Au nanoparticle and the other end is modified with a Raman active molecule; and an Ag layer as a shell surrounding the Au nanoparticle; and the Au nanoparticle as core A and the Au nanoparticle as core B forms dimer via complementary base pairing through hydrogen bond between the target-capturing oligonucleotide (A) and the part of the target nucleic acid (T) and between the target-capturing oligonucleotide (B) and the other part of the target nucleic acid (T), the method comprising: forming a dimer of the Au nanoparticle as core A and the Au nanoparticle as core B via complementary base pairing through hydrogen bond between the target-capturing oligonucleotide (A) and the part of the target nucleic acid (T) and between the target-capturing oligonucleotide (B), of which the end is modified with a Raman active molecule, and the other part of the target nucleic acid (T), and then silver-staining on the Au nanoparticle as core A and the Au nanoparticle as core B in the dimer, while controlling the thickness of the Ag shell so that the distance between the Ag shells in the dimer of two Au/Ag core-shell composites is adjusted to be 1 nm or less, thereby placing a Raman active molecule at the interparticle junction adjusted to the distance of 1 nm or less, resulting in that the Raman signal amplification (SERS) effect is exerted by silver-staining, and false positives do not appear due to non-specific silver-staining.

2. The method for preparing the dimer of claim 1, wherein the Au nanoparticle as a core exhibits specific Surface Plasmon Resonance (SPR).

3. The method for preparing the dimer of claim 1, wherein one end of the target-capturing oligonucleotide is bonded to the Au nanoparticle as a core, and the target-capturing oligonucleotide is partially exposed to the outside of the Ag shell.

4. The method for preparing the dimer of claim 1, comprising: preparing the Au nanoparticle as core A functionalized with a protecting oligonucleotide and the target-capturing oligonucleotide A; and the Au nanoparticle as core B functionalized with a protecting oligonucleotide and the target-capturing oligonucleotide B, of which the end is modified with a Raman active molecule, respectively; forming, in the presence of the target nucleic acid (T), the dimer of the Au nanoparticle as core A and the Au nanoparticle as core B via complementary base paring between the target-capturing oligonucleotide (A) and the target nucleic acid (T) and complementary base paring between the target-capturing oligonucleotide B and the target nucleic acid (T); and forming Ag layers as a shell surrounding the respective Au nanoparticles in dimer.

5. The method for preparing the dimer of claim 1, wherein the Raman active molecule is selected from a group consisting of FAM, Dabcyl, TRIT (tetramethyl rhodamine isothiol), NBD (7-nitrobenz-2-1,3-diazole), Texas Red dye, phthalic acid, terephthalic acid, isophthalic acid, cresyl fast violet, cresyl blue violet, brilliant cresyl blue, para-aminobenzoic acid, erythrosine, biotin, digoxigenin, 5-carboxy-4,5-dichloro-2,7-dimethoxy, fluorescein, 5-carboxy-2,4,5,7-tetrachlorofluorescein, 5-carboxyfluorescein, 5-carboxyrhodamine, 6-carboxyrhodamine, 6-carboxytetramethyl aminophthalocyanine, azomethine, cyanine, xanthine, succinylfluorescein, aminoacridine, quantum dots, carbone nanotubes, carbon allotropes, cyanide, thiol, chlorine, bromine, methyl, phosphorus, sulfur, cyanine dyes (Cy3, Cy3.5, Cy5), and rhodamine.

6. The method for preparing the dimer of claim 1, wherein the target-capturing oligonucleotide is attached via a surface-bound functional group selected from the group consisting of thiol group, amine group and alcohol group to the surface of the Au nanoparticle as a core.

7. The method for preparing the dimer of claim 6, wherein the target-capturing oligonucleotide comprises a spacer sequence between the surface-bound functional group and the target-capturing oligonucleotide.

8. The method for preparing the dimer of claim 1, wherein the Au core diameter ranges in size from 10 nm to 40 nm, and the Ag shell thickness ranges in size from 1 nm to 20 nm.

9. The method for preparing the dimer of claim 4, further comprising: separating only the Au nanoparticle functionalized with the target-capturing oligonucleotide A and the Au nanoparticle functionalized with the target-capturing oligonucleotide B, by performing a hybridization reaction with magnetic microparticles having a sequence complementary to the target-capturing oligonucleotides A and B, respectively, after preparing the functionalized Au nanoparticles.

10. The method for preparing the dimer of claim 1, wherein the introduction of the Ag shell is achieved by reacting the dimer of the Au nanoparticle as core A and the Au nanoparticle as core B with a Ag shell precursor in the presence of a reducing agent and a stabilizer.

11. The method for preparing the dimer of claim 1, further comprising: after formation of the dimer of Au/Ag core-shell nanoparticle A and Au/Ag core-shell nanoparticle B on if any of the target nucleic acid, performing a Raman spectroscopy on the Raman active molecule located at the interparticle junction with a distance ranging 1 nm or less, thereby detecting whether the target nucleic acid is present or not.

12. The method for preparing the dimer of claim 11, wherein the detection of the target nucleic acid is for diagnosis of a disease.

13. The method for preparing the dimer of claim 11, wherein the Raman spectroscopy is Surface Enhanced Raman Scattering (SERS), Surface Enhanced Resonance Raman Spectroscopy (SERRS), hyper-Raman and/or Coherent Anti-Stokes Raman Spectroscopy (CARS).

14. The method for preparing the dimer of claim 11, wherein the target nucleic acids (T) are genes, viral RNAs and DNAs, bacterial DNAs, fungal DNAs, mammal DNAs, cDNAs, mRNAs, RNA and DNA fragments, oligonucleotides, synthetic oligonucleotides, modified oligonucleotides, single- and double-stranded nucleic acids, and natural or synthetic nucleic acids.

15. A method for preparing the dimer comprising a first Au/Ag core/shell nanoparticle, a second Au/Ag core/shell composite nanoparticle, and a target nucleotide (T), wherein the first Au/Ag core/shell nanoparticle comprises a first Au nanoparticle as a core, a second Ag layer as a shell surrounding the first Au nanoparticle, and a first nucleotide (A) of which one end is bound to the first Au nanoparticle via a functional group or a spacer molecule having the functional group and of which the other end has a Raman dye compound, wherein the second Au/Ag core/shell nanoparticle comprises a second Au nanoparticle as a core, a second Ag layer as a shell surrounding the second Au nanoparticle, and a second nucleotide (B) of which one end is bound to the second Au nanoparticle via a functional group or a spacer molecule having the functional group; wherein the first nucleotide (A) is capable of complementary base pairing with the target nucleotide (T), and the nucleotide (B) is capable of complementary base pairing with the oligonucleotide (T), and the first and the second Au/Ag core/shell composite nanoparticles form the dimer via the complementary base pairings between the target nucleotide (T), the first nucleotide (A) and the second nucleotide (B); wherein a closest distance between surface of the first Ag layer and surface of the second Ag layer of the dimer is 0.5 nm to 1 nm and the Raman dye compound is located at a gap of the closest distance between the first Ag layer and the second Ag layer thus the dimer has nano-junction exhibiting surface enhanced Raman Scattering effect at the gap of the dimer, the method comprising: preparing the first Au nanoparticle to which surface the first nucleotide (A) is bound via the functional group or the spacer molecule having the functional group and the second Au nanoparticle to which surface the nucleotide (B) is bound to the Au nanoparticle via the functional group or the spacer molecule having the functional group; forming, in the presence of the target nucleotide (T), a primary dimer of the first and the second Au nanoparticles via complementary base paring between the first nucleotide (A) and the target nucleotide (T) and complementary base paring between the second nucleotide (B) and the target nucleotide (T); and forming the first Ag layer surrounding the first Au nanoparticle of the primary dimer and the second Ag layer surrounding the second Au nanoparticle of the primary dimer until the gap of the closest distance between surface of the first Ag layer and surface of the second Ag layer reaches to 0.5 nm-1 nm to give the dimer.

Description

BRIEF DESCRIPTION OF THE DRAWINGS/FIGURES

[0119] The patent or application file contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawing(s) will be provided by the Office upon request and payment of the necessary fee.

[0120] FIGS. 1A and 1B are schematic diagrams showing the synthesis of Au nanoparticle dimers through magnetic purification, DNA hybridization and Ag-shell formation. The protecting sequence for probe A is 3-HS(CH.sub.2).sub.3-A.sub.10-PEG.sub.18-AAACTCTTTGCGCAC-5 (i.e., 3-HS(CH.sub.2).sub.3-SEQ ID NO: 1-PEG.sub.1-SEQ ID NO: 2-5), the target-capturing sequence for probe A is 3-HS(CH.sub.2)-A.sub.10-PEG.sub.18-CTCCCTAATAACAAT-5 (i.e., 3-HS(CH.sub.2).sub.3-SEQ ID NO: 1-PEG.sub.18-SEQ ID NO: 3-5), and the modified sequence for MMP-A is 3-NH.sub.2(CH.sub.2).sub.3-A.sub.10-PEG.sub.18-ATTGTTATTAGGGAG-5 (i.e., 3-NH.sub.2(CH.sub.2).sub.3-SEQ ID NO: 1-PEG.sub.18-SEQ ID NO: 4-5) (Tm=38 C.). The protecting sequence for probe B is 5-HS(CH2)6-A.sub.10-PEG.sub.18-AAACTCTTTGCGCAC-3 (i.e., 5-HS(CH2)6-SEQ ID NO: 1-PEG.sub.18-SEQ ID NO: 5-3), the target-capturing sequence for probe B is 5-HS(CH.sub.2).sub.3-A.sub.10-PEG.sub.18-ATCCTTATCAATATTAAA-Cy3-3 (i.e., 5-HS(CH.sub.2).sub.3-SEQ ID NO: 1-PEG.sub.18-SEQ ID NO: 6-Cy3-3) and the modified sequence for MMP-B is 5-NH.sub.2(CH.sub.2).sub.3-A.sub.10-PEG.sub.18-TTTAATATTGATAAGGAT-3(i.e., 5-NH.sub.2(CH.sub.2).sub.3-SEQ ID NO: 1-PEG.sub.18-SEQ ID NO: 7-3) (Tm=40 C.). The underlined parts represent spacer sequences designed to facilitate Ag-shell formation. The target-DNA sequence is 5-GAGGGATTATTGTTAAATATTGATAAGGAT-3 (i.e., SEQ ID NO: 8) (anthrax oligonucleotide). FIG. 1C shows an AFM-correlated nano-Raman spectroscopy set-up (laser focal diameter 250 nm) for the identification of SERS hot-spot from a single dimeric nanostructure. FIG. 1C shows a schematic of the Atomic Force Microscope (AFM) used to measure the Au nanoparticle dimers.

[0121] FIG. 2A shows UV-visible spectra corresponding to before and after Au nanoparticle dimer formation and shows the corresponding TEM and HR-TEM images. FIG. 2B shows UV-visible spectra before and after the introduction of an Ag-shell on the Au nanoparticle dimer with Plasmon resonance (peak at .sup.400 nm) of the nanostructure which varies depending on the silver-shell thickness. FIG. 2C shows HR-TEM images of AuAg core-shell monomers with a shell thickness of 5 nm and 10 nm, and shows AuAg core-shell heterodimers with a shell thickness of .sup.183 nm, .sup.185 nm, and .sup.1810 nm.

[0122] FIG. 3A shows an AFM (atomic force micrograph, 11 m) of the AuAg core-shell monomer and the heterodimer. FIG. 3B shows correlated SERS spectra taken from the monomeric or dimeric AuAg core-shell nanostructures. FIG. 3C shows all spectra taken with a 514.5 nm excitation laser, 1 s accumulation, 100 W sample, and a 250 nm laser focal diameter. Raman spectra were taken from Cy3-modified oligonucleotides (red line) and Cy3-free oligonucleotides (black line) in NaCl-aggregated silver colloids.

[0123] FIG. 4A shows the tapping-mode AFM images (5 m5 m) of the AuAg core-shell dimer (corresponding to the nanostructure with an Ag-shell thickness of .sup.5 nm and a gap of .sup.1 nm). FIG. 4B shows SERS spectra of Cy3 from the individual dimeric nanostructure with a laser wavelength of 514.5 nm, a laser power of .sup.80 W, a laser focal diameter of .sup.250 nm, and an integration time of 1 s.

[0124] FIGS. 5A and 5B show blinking SERS spectra taken from the nanostructure with an accumulation time of 1 s. FIG. 5C shows SERS spectra taken from AuAg core-shell heterodimers with different incident-laser polarizations. FIG. 5D shows polar plots of integrated SERS intensities of the 1470 and 1580 cm.sup.1 Raman bands with respect to . They were measured with a laser wavelength of 514.5 nm, a laser power of .sup.40 W, a laser focal diameter of .sup.250 nm, and an integration time of 20 s.

[0125] FIG. 6A shows Raman spectra from FAM-labeled oligonucleotides (1 nM) and Dabcyl-labeled oligonucleotides (1 nM) in solutions. FIG. 6B shows Raman spectra from dimeric AuAg core-shell nanostructures labeled with FAM (Ag-shell 5 nm) and Dabcyl (Ag shell, 5 nm).

[0126] FIG. 7 is a schematic view showing the method for preparing the Au/Ag core-shell composite in accordance with Example 7 of the present invention.

[0127] FIG. 8 shows UV spectrum of the Au/Ag core-shell composite in accordance with Example 7 of the present invention.

[0128] FIG. 9 is a transmission electron microscope (TEM) image of the Au/Ag core-shell composite in accordance with Example 7 of the present invention.

[0129] FIG. 10 is an enlarged image of FIG. 9.

[0130] FIG. 11 shows the EDX analysis result in accordance with Example 7 of the present invention.

[0131] FIG. 12 shows UV spectrum of an Au/Ag core-shell composite in accordance with Example 8 of the present invention.

[0132] FIG. 13 is a TEM image of the Au/Ag core-shell composite in accordance with Example 8 of the present invention.

[0133] FIG. 14 shows the variation of absorbance of an Au/Ag core-shell composite in accordance with Example 9 of the present invention, according to amounts of AgNO.sub.3 and hydroquinone.

[0134] FIG. 15 shows UV spectrum of a simple mixture of Au nanoparticle and Ag nanoparticle and an AuAg core-shell composite in Example 9.

[0135] FIG. 16 shows a base sequence of oligonucleotides A and B contained in the Au/Ag core-shell composite used in Example 10, and a target oligonucleotide having a complementary base sequence.

[0136] FIG. 17 shows the colorimetric assay result in Example 10.

[0137] FIG. 18 shows the variation of melting point with respect to time in Example 10.

[0138] FIGS. 19 and 20 are TEM images of Example 11.

DETAILED DESCRIPTION OF THE INVENTION

[0139] The above and other objects, features and other advantages of the present invention will be more clearly understood from the following examples, but it should be understood that the present invention is not limited to the following examples in any manner.

Example 1: Preparation of Dimeric AuAg Core-Shell Nanostructure Labeled with Raman Active Molecule (Cy3) Localized at Interparticle Junction

[0140] Based on a DNA-directed bridging method, the synthesis of a Raman active AuAg core-shell dimer was conducted using complementary target oligonucleotide-tethered Au nanoparticles, with an Ag-shell being formed from a controlled amount of Ag precursor, as shown in FIGS. 1A and 1B.

[0141] A gold nanoparticle (15 nm) for probe A was functionalized with two kinds of 3-thiol-modified oligonucleotides in such a manner that one target-capturing sequence was assigned to the surface of the gold nanoparticle. Also, a gold nanoparticle (30 nm) for probe B was functionalized with two kinds of 5-thiol-modified oligonucleotides. The molar ratios of the two kinds of sequence (protecting sequence/target-capture sequence) were 99:1 for core A and 199:1 for core B. These ratios were adopted to modify one target-capturing oligonucleotide per probe on the basis of nanoparticle size-dependent DNA loading capacity (FIGS. 1A and 1B). Importantly, the target-capturing sequence for probe B was labeled at the terminus with Cy3 which serves as a Raman tag. In order to remove the nanoparticle monomer to which no target capturing sequences are bonded, the oligonucleotide-modified probes A and B were purified by a magnetic-separation process. Tosyl-modified magnetic beads (diameter 1 m, Invitrogen) were functionalized by amine-modified target oligonucleotide sequences complementary to the target-capturing sequences for cores A and B, respectively. Only the core particles having target-capturing sequences bound thereto could be separated by the magnetic beads.

[0142] Next, purified probe A and B solutions were hybridized with a sufficient amount of the target sequence in 0.3 M PBS.

[0143] Highly purified Au nanoparticle heterodimers were produced by precisely controlling the molar ratio between the protecting oligonucleotide and the target-capturing oligonucleotide, followed by an effective purification. Because the maximum distance (gap distance) between the Au nanoparticle (AuNP) surface and the Cy3 molecule still remained 7.5 nm, it needed to be decreased so as to give an amplified electromagnetic enhancement. A silver nano-shell was introduced since silver enhances SERS signals several times more than gold.

[0144] In detail, the DNA tethered Au nanoparticle dimers were coated with silver by means of a well-known nanometer-scale silver-shell deposition process (Chem. Comm. 2008, J. Phys. Chem. B 2004, 108, 5882-5888) on the Au nanoparticle surface to shorten the distance between the nanoparticles, which leads to the amplification of SERS signals. In this regard, a 250 M Au nanoparticle dimer solution was reacted with various amounts of AgNO.sub.3 solution [10.sup.3 M] at room temperature for 3 h in the presence of 100 L of polyvinyl pyrrolidone as a stabilizer and 50 L of L-sodium ascorbate [10.sup.1 M] as a reductant in a 0.3 M PBS solution. The Ag shell thicknesses of the AuAg core-shell nanoparticles were .sup.3 nm, .sup.5 nm and .sup.10 nm when using 30 L, 40 L, and 70 L of an AgNO.sub.3 solution [10.sup.3 M], respectively. In this manner, target oligonucleotide-tethered AuAg core-shell heterodimeric nanostructures with an Ag shell thickness of .sup.3 nm, .sup.5 nm and .sup.10 nm were synthesized.

[0145] Likewise, oligonucleotide-modified AuAg core-shell nanoparticle dimers labeled with the Raman active molecule FAM or Dabcyl were prepared.

Example 2: UV-Visible Spectroscopy and HR-TEM Imaging Analysis

[0146] The formation of Au nanoparticle dimers (Cy3 used as a Raman Active molecule) was verified by UV-visible spectroscopy and high-resolution transmission electron microscope (HRTEM) images (FIGS. 2A, 2B and 2C). The UV-visible spectra show a very small red-shift after dimer formation, which is in agreement with the previously reported results by Oaul Alivisatos, et al. (Angew chem. 1999.38(12), 1808). FIG. 2A is of typical HR-TEM images of the Au nanoparticle dimers. By a statistical analysis of at least 200 particles, it was found that 25% of the particles existed as a monomer and 65% of the particles as a dimer, and less than 10% as a multimer (trimer, tetramer and so on). The interparticle distance between the gold particles was found to be ca. 2-3 nm as measured by HR-TEM. In a solution (0.3M PBS), the interparticle distance was expected to be .sup.15 nm which is far longer than that under dried conditions.

[0147] Also, silver nanoparticles for forming a Ag-shell were introduced at a nanometer scale to the surface of the Au nanoparticle dimer by a well-known method (Chem. Comm. 2008, J. Phys. Chem. B 2004, 108, 5882-5888) on the Au nanoparticle surface to shorten the distance between the nanoparticles, which leads to the amplification of SERS signals (see Example 1).

[0148] Au core-Ag shell monomers with an Ag-shell thickness of .sup.3 nm and .sup.10 nm (FIG. 2C) were also synthesized from a purified probe B solution (30 nm AuNP) under a similar condition. UV-visible spectra from individual solutions were separated at a plasmon resonance peak of .sup.400 nm according to shell thickness.

[0149] FIG. 2C is of HR-TEM images taken from individual AuAg core-shell heterodimers with a diameter of 26 nm-36 nm, 30 nm-40 nm and 40 nm-50 nm for a set of two core-shell nanoparticle spheres. FIG. 2C also shows HR-TEM images taken from individual AuAg core-shell monomers with a diameter of 40 nm (shell thickness=5 nm) and 50 nm (shell thickness=10 nm).

[0150] Next, the monomeric or dimeric core-shell nanostructures (using Cy3 as a Raman active molecule) were measured for SERS/AFM. In a typical experiment, aliquots (20 L) of the AuAg core-shell heterodimer solutions washed by repeated centrifugation (8,000 rpm, 20 min, three times) were applied to a poly-L-lysine-coated glass substrate by spin coating, washed many times with nanopure water, and dried in air. Immediately after being prepared, the samples were measured for AFM and SERS. SERS spectra were recorded using an AFM-correlated nano-Raman microscope equipped with an inverted optical microscope (Axovert 200, Zeiss) and a piezoelectic x-y sample scanner (Physik Instrument) was manipulated by an independent homemade scanning controller. The 514.5 nm line of an argon ion laser (Melles Griot) was used as the excitation source coupled with a single-mode optical fibre. A dichroic mirror (520DCLP, Chroma Technology Corp.) was set to direct the excitation laser beam from 50 nW to 1 mW into the oil-immersion microscope objective (100, 1.6 NA, Zeiss), which focused the beam to a diffraction-limited spot (250 nm) on the upper surface of the cover glass slip. The AFM (Bioscope, Digital Instruments, Veeco Metrology Group) with a Nanoscope IV controller was mounted on a micro-mechanical stage. The tapping-mode AFM module on top of the optical microscope stage was used to correlate the Raman signal with the AFM topographical image with an overlap precision of <100 nm. The laser focal spot was exactly matched with the center of the AFM tip for symmetrical scattering on the AFM tip end. The background Raman signals were collected on a liquid-nitrogen-cooled (125 C.) CCD (charge-coupled device). The scattering spectra of the sample were recorded in the range of 500-2,000 cm.sup.1, in one acquisition, 1 s accumulations and 400 W. All of the data were baseline-corrected by subtracting the background signals from Si.

Example 3: AFM (Atomic Force Micrograph) Analysis of AuAg Core-Shell Nanoparticles

[0151] FIG. 3A shows magnified AFM images (11 m) of the core-shell monomer and heterodimer nanostructures (using Cy3 as a Raman active molecule), which were coincident in shape and diameter with HR-TEM images. FIG. 3B shows the correlated SERS spectra from the corresponding single AFM-imaged particles in FIG. 3A. No Raman signals were detected for the monomeric AuAg core-shell nanoparticles with 5 nm and 10 nm silver shells, respectively because they had no hot spots and only one Cy3 molecule per particle. The Au nanoparticle heterodimers without Ag shells or with a gap distance less than 1 nm therebetween did not show any detectable SERS signal either. This is due to insufficient electromagnetic enhancement under 514.5 nm laser excitation conditions. When the Ag shell thickness was <3 nm, no Raman signals were detected even after using an elevated incident laser power (200 W). These results indicate that a thin silver shell (<3 nm) could not induce sufficient electromagnetic enhancements in SERS.

[0152] In contrast, when the Ag shell thickness was .sup.5 nm, a strong SERS signal from a Cy3 label, located in the junction of two core-shell particles, was observed. The characteristic Raman peaks for Cy3 dye, although low in intensity, were observed at 1,470 and 1,580 cm.sup.1, characteristic of fingerprint spectra, from a 514.5 nm laser excitation. The low intensity of these peaks is probably due to the presence of only one molecule within the hot junction region and relatively low laser power (100 W) compared with the power intensity used in other single-molecule SERS studies (Science 1997. 275(5203), 1102, Phys rev left 1997, 78(9), 1667, Nano left 2006, 6(1) 2173, Nano left DOI: 10.1021/ni803621x). Signals were taken from different species as in the oligonucleotide-modified Au nanoparticles. FIG. 3C shows the comparison of SERS spectra of Cy3-modified oligonucleotides (5-HS(CH.sub.2).sub.6-A.sub.10-PEG.sub.18-ATCCTTATCAATATTAAA (SEQ ID NO: 4)-Cy3-3, 1 nM, red line) with those of Cy3-free oligonucleotides (5-HS(CH.sub.2) 6-A.sub.10-PEG.sub.18-ATCCTTATCAATATTAAA (SEQ ID NO: 4)-3, 1 nM, black line) in aggregated Ag colloids. The SERS spectra of FIG. 3C (black line) features predominant adenine peaks (734 cm.sup.1, 1320 cm.sup.1) over other bases due to the enrichment of the adenine base (A10 used as a spacer sequence) (JACS 2008, 130(16), 5523). It is important that the lowest detection limit for non-adenine DNA bases, reported so far, is in the sub-micromolar range (JACS, 2006, 128, 15580). However, relatively strong signals were read at 1,470 cm.sup.1 and 1,580 cm.sup.1, characteristic of the Cy3 molecule, as shown in the SERS spectra of FIG. 3C (red line) (Anal chem. 2004, 76, 412-417). It is known that the Raman intensity and spectral positions of Cy3 molecules fluctuate with time, and the Raman spectra are different for each observed nanostructure (J. Phys. Chem. B 2002, 106, 8096). Therefore, spectral patterns are comparable, but not fully compatible with the reported ones. Irrespective of shell thickness, no observations of detectable SERS signals from the AuAg core-shell monomers under the experimental condition indicated that only Cy molecules localized at the interparticle junction could induce SERS peaks at 1,470 and 1,580 cm.sup.1. As shown in Raman spectra taken from the core-shell dimers with a shell thickness of .sup.10 nm (FIG. 3A), predominant adenine peaks were observed at 734 cm.sup.1 and 1320 cm.sup.1 along with a Cy3 peak at 1,480 cm.sup.1. Raman scattering intensities from other nanoparticles were not observed in a specific form. A thick Ag shell could cover Cy3 molecules which caused improper electromagnetic enhancement.

Example 4: Analysis of SERS Spectra from AuAg Core-Shell Nanoparticle Dimers According to Polarization of Incident Laser

[0153] Most of the core-shell nanoparticle dimers with a shell thickness of .sup.5 nm (using Cy3 as a Raman active molecule) showed detectable SERS signals from single molecules, as shown in FIGS. 4A and 4B. Considering that the incident laser light is not exactly polarized to the interparticle axes of the dimer (panels 1-5 in FIG. 4A), detectable SERS signals from each of the perpendicularly polarized nanoparticle dimers on the same surface were observed. However, FIG. 5C shows only small peaks at 1,470 cm.sup.1 because the dimer orientation is nearly perpendicular to the incident light. Herein, it was found that the core-shell nanoparticle dimers with the shell thickness optimized might be of hot spot structures highly applicable to the detection of a single DNA molecule.

[0154] It is experimentally known that on-off blinking behaviors are observed upon single-molecule detection (FIG. 5A) (J. Phys. Chem. B2002, 106, 8096). The absence of Raman intensity continues for 10 sec, after which Raman intensity is in an ON state. This On-Off cycling phenomenon may be repeated for several minutes during which signals ultimately disappear from an intense field. The SERS intensity fluctuation was observed on a second timescale owing to molecular movement around the hot spot. These blinking and fluctuation phenomena were in agreement with previous reports.

[0155] FIGS. 5C and 5D show the incident laser polarization dependence of the Raman signals for the AuAg core-shell dimer. All of the spectra were taken with a 514.5 nm excitation laser, 20 s accumulation time and 40 W laser power. Maximum Cy3 peaks were observed when the incident laser light was polarized parallel to the longitudinal axis of the heterodimer. When the laser light was rotated by 20-90 away from the longitudinal axis, the Cy3 signal was gradually reduced. The Raman peaks disappeared when the laser polarized perpendicular to the longitudinal axis (that is, 90 and 270). The enhancement factor (EF) at 1,580 cm.sup.1 of the hot spot in the dimeric nanostructure was calculated according to the following equation.

EF=(I.sub.sersN.sub.bulk)/(I.sub.bulkN.sub.molecule)

[0156] wherein

[0157] I.sub.sers and I.sub.bulk represent the same intensity of bands for SERS and bulk spectra, respectively,

[0158] N.sub.bulk is the number density of bulk molecules in a bulk sample, and

[0159] N.sub.molecule is the number density of Cy3 in SERS spectra (Nmolecule=1). The strongest spectrum band was read at 1,580 cm.sup.1 regions, so that it was used as the intensity for I.sub.sers and I.sub.bulk. In this manner, the EF of the hot spot was calculated to be 2.710.sup.12.

[0160] On the other hand, highly sensitive SERS spectra were detected from nanoparticle dimers modified with oligonucleotides labeled with FAM and Dabcyl, as described for Cy3 (FIG. 6). Thus, the dimeric nanostructures and preparation method in accordance with the present invention are applied to general Raman active molecules.

[0161] Materials

[0162] Au nanoparticle used herein was purchased from Ted pella (Redding, Calif., USA), and an AgNO.sub.3 solution as Ag ion (Ag.sup.+) source and a hydroquinone solution as a reducing agent was purchased from BBI international (Cardiff, UK). Oligonucleotide bonded with thiol group was purchased from IDT (Coralville, Iowa, USA) and thiol group was deprotected. Also, protein A and antibody was purchased from piercenet.com (USA). H2O used in the experiment was nanopure water.

Example 5: Preparation of Oligonucleotide

[0163] 3-alkylthiol modified oligonucleotide, 3-HO(CH.sub.2).sub.3SS(CH.sub.2).sub.3-A.sub.10-PEG.sub.18-CTCCCTAATAACAAT (SEQ ID NO: 2)-5, which was purchased from IDT, was added to 0.1 M dithiothreitol and a deprotection reaction was performed by leaving it at room temperature for 2 hours.

[0164] Oligonucleotide conglomerate A (3-HS(CH.sub.2).sub.3-A.sub.10-PEG.sub.10-CTCCCTAATAACAAT (SEQ ID NO: 2)-5) was prepared by purifying the deprotected solution while passing it through NAP-5 column (Sephadex G-25 medium, DNA grade).

[0165] AgNO.sub.3 (50 mM) dissolved in distilled water was added to 5-alkylthiol modified oligonucleotide (5-HO(CH.sub.2).sub.3SS(CH.sub.2).sub.6-PEG.sub.18-ACTCTTATCAATATT (SEQ ID NO: 7)-3) and left for 20 minutes, and the generated precipitation was removed by adding dithiothretol (10 mg/ml) for 5 minutes.

[0166] Oligonucleotide conglomerate B (5-HS(CH.sub.2).sub.6-A.sub.10-PEG.sub.18-ACTCTTATCAATATT (SEQ ID NO: 7)-3) was prepared by purifying supernatant while passing it through NAP-5 column (Sephadex G-25 medium, DNA grade).

[0167] By measuring extinction using a UV-visible spectrometer, an amount of oligonucleotide inside the solution was quantified.

Example 6: Bonding of Oligonucleotide (Receptor) on Surface of Au Nanoparticle

[0168] The oligonucleotide conglomerate deprotected through the procedure of Example 5 and bonded with thiol group and spacer site was added to 1 ml of the 3.8 nM solution of Au nanoparticle with a diameter of 15 nm and was mixed by shaking at room temperature for more than 12 hours.

[0169] The composition of the solution was adjusted so that the concentration of phosphate becomes 9 mM and the concentration of sodium dodecyl sulfonate becomes about 0.1%. After additional agitation for 30 minutes, the final salt concentration was adjusted to be 0.3 M NaCl.

[0170] After leaving for more than 12 hours, the solution is centrifuged and the supernatant was discharged. Then, 1 ml of 0.3 M phosphate solution (10 mM PB, 0.3 M NaCl) was added and diluted. Those steps were repeated two times.

[0171] In this way, oligonucleotide conglomerate was bonded on the surface of the Au nanoparticle.

Example 7: Preparation (1) of Au/Ag Core-Shell Composite Bonded with the Oligonucleotide Conglomerate

[0172] The concentration of the solution of the Au nanoparticle bonded with oligonucleotide conglomerate, which was synthesized in Example 6, was calculated using the extinction measured by the UV-visible spectrometer. The concentration of the solution was adjusted to 1 nM by concentrating or diluting the solution according to the result.

[0173] To 250 l of the solution, AgNO.sub.3 solution (12 l) diluted 10 times with distilled water, and hydroquinone solution (12 l) diluted 10 times with distilled water were sequentially added and then agitated for 30 minutes.

[0174] Thereafter, the extinction of the solution was measured by the UV-visible spectrometer. After leaving the solution at room temperature till there is no change in the extinction, it was centrifuged to remove the supernatant and was diluted with distilled water. Then, the solution was again centrifuged to remove the supernatant and was diluted with 250 l of distilled water.

[0175] In this way, the Au/Ag core-shell composite bonded with oligonucleotide in accordance with the embodiment of the present invention was prepared.

[0176] FIG. 7 is a schematic view showing the method for preparing the Au/Ag core-shell composite.

[0177] FIG. 8 shows variation in extinction of the solution measured by the UV-visible spectrometer with respect to time. As can be seen from FIG. 8, the absorbance was not substantially varied after reaction for about 30 minutes.

[0178] Furthermore, the shape and size of the prepared Au/Ag core-shell composite were confirmed using a transmission electron microscope (TEM) (see FIGS. 9 and 10). The Au/Ag core-shell composite of Example 7 was spherical in shape and was about 16 nm to about 17 nm in size, and the Ag nanoparticle layer was about 1.5 nm in thickness.

[0179] Moreover, by analyzing the solution using an energy dispersive X-ray microanalysis (EDX), the composition ratio of Au nanoparticle to Ag nanoparticle in the prepared Au/Ag core-shell composite was confirmed.

[0180] According to the EDX analysis of FIG. 11, silver (Ag) atoms and gold (Au) atoms in the Au/Ag core-shell composite were 25% and 75%, respectively. This result was identical to the TEM analysis result of FIGS. 9 and 10.

Example 8: Preparation (2) of Au/Ag Core-Shell Composite Bonded with Oligonucleotide Conglomerate

[0181] The concentration of the solution of the Au nanoparticle bonded with oligonucleotide, which was synthesized in Example 6, was calculated using the extinction measured by the UV-visible spectrometer. The concentration of the solution was adjusted 20 to 1 nM by concentrating or diluting the solution according to the result.

[0182] To the 250 l solution, AgNO.sub.3 solution (24 l) diluted 10 times with distilled water, and hydroquinone solution (24 l) diluted 10 times with distilled water were sequentially added and then agitated for 30 minutes.

[0183] Thereafter, the absorbance of the solution was measured by the UV-visible spectrometer. After leaving the solution at room temperature till there is no change in the extinction, it was centrifuged to remove the supernatant and was diluted with distilled water. Then, the solution was again centrifuged to remove the supernatant and was diluted with 250 l of distilled water.

[0184] In this way, the Au/Ag core-shell composite bonded with oligonucleotide conglomerate in accordance with the embodiment of the present invention was prepared.

[0185] FIG. 12 shows variation in absorbance of the solution measured by the UV-visible spectrometer with respect to time. As can be seen from FIG. 12, the absorbance was not substantially varied after reaction for about 30 minutes.

[0186] Furthermore, the shape and size of the prepared Au/Ag core-shell composite were confirmed using a TEM, and the result was shown in FIG. 13. An image shown on the left upper side of FIG. 13 is an enlarged image of the composite.

[0187] As a result of the TEM analysis, the Au/Ag core-shell composite of Example 8 was spherical in shape and was about 20 nm to about 22 nm in size, and the Ag nanoparticle layer was about 5 nm to about 7 nm in thickness.

Example 9: Comparison of Au/Ag Core-Shell Composite of the Above Examples and Mixture of Pure Au Nanoparticle and Au Nanoparticle

[0188] To confirm the structure of the Au/Ag core-shell composite of Example 7, the UV extinction of the Au/Ag core-shell composite of Example 7 was compared with the UV absorbance of the simple mixture of pure Au nanoparticle with a size of 15 nm and pure Ag nanoparticle with a size of 15 nm.

[0189] The UV absorbance of the Au/Ag core-shell composite, which was prepared according to Example 7 except that the thickness of the Ag nanoparticle layer was changed by adjusting amounts of AgNO.sub.3 and hydroquinone as shown in Table 1 below, was measured and shown in FIG. 14. Table 1 shows variation in UV absorbance of Au/Ag core-shell composite according to amounts of AgNO.sub.3 and hydroquinone.

TABLE-US-00001 TABLE 1 Amount of Amount of AgNO.sub.3 (l) Hydroquinone (l) Absorbance data 1.2 1.2 FIG. 14-a 2.0 2.0 FIG. 14-b 2.4 2.4 FIG. 14-c 3.2 3.2 FIG. 14-d 4.0 4.0 FIG. 14-3

[0190] In the case of the Au/Ag core-shell composite in accordance with the present invention, as shown in FIG. 14, a blue shift occurred at 520 nm, which is the maximum absorption peak of the Au nanoparticle, according to the thickness of the Ag nanoparticle layer, and the maximum absorption peak moved to 500 nm, 490 nm, and so on. The characteristic maximum absorption peak of the Ag nanoparticle occurred at 400 nm. Also, it was observed that the intensity of the absorbance was changed according to the thickness of the Ag nanoparticle layer.

[0191] Meanwhile, FIG. 15 shows the comparison of UV absorbance of the Au/Ag core-shell composite indicated by a of FIG. 14 and the simple mixture of the pure Au nanopartile with a size of 15 nm and the pure Ag nanopartile with a size of 15 nm.

[0192] The black curve of FIG. 15 is the UV absorbance of a simple mixture of pure 15 nm-sized gold nanoparticles and pure 15 nm-sized silver nanoparticles, and the red curve is the gold/silver core-shell composite according to the present invention shown by a in FIG. 14 is the UV absorbance.

[0193] As can be seen from FIG. 15, unlike the Au/Ag core-shell composite in accordance with the present invention, the maximum absorption peaks of the simple mixture occurred at the characteristic maximum absorption peaks of the Au nanoparticle and the Ag nanoparticle, that is, 400 nm corresponding to the Ag nanoparticle with a size of 15 nm and 520 nm corresponding to the Au nanoparticle with a size of 15 nm. In the Au/Ag core-shell composite, however, the blue shift occurred from about 520 nm to about 510 nm in the case of the maximum absorption peak of the Au nanoparticle, and the wide peak occurred at about 400 nm in the case of the Ag nanoparticle. Therefore, it was confirmed that the core-shell composite in accordance with the present invention does not exist in a form of the simple mixture of the Au nanoparticle and the Ag nanoparticle, but exists in a form of one core-shell nanoparticle.

Example 10: Colorimetric Assay Test

[0194] As described in Example 7, the Au/Ag core-shell composite where oligonucleotide A and oligonucleotide B were bonded was prepared.

[0195] FIG. 16 shows the base sequence of the oligonucleotide conglomerates A and B (i.e., SEQ ID No:4 and SEQ ID No:5) contained in the Au/Ag core-shell composite, and the target oligonucleotide having complementary base sequences thereto.

[0196] 300 l of Au/Ag core-shell composite A (1.5 pmol) bonded with oligonucleotide A dissolved in 0.3 M phosphate buffer solution was mixed with 375 l of Au/Ag core-shell composite B (1.5 pmol) bonded with oligonucleotide conglomerate B dissolved in phosphate buffer solution. 6.0 l (10 M) of target oligonucleotide was added to the mixed solution, the temperature of the mixed solution increased to 70 C., and then gradually decreased to room temperature. After about two hours, the solution changed from the initial orange color to the dark purple color.

[0197] The change of the color could be observed more clearly by dropping 2 l of the solution on a C18-coated glass plate. An image of FIG. 17-I shows the color (green) of the 15 nm Ag nanoparticle; an image of FIG. 17-II shows the color (purple) of the 15 nm Au nanoparticle; an image of FIG. 17-III shows the color (orange) of the 15 nm Au/Ag core-shell composites in accordance with the present invention; an image of FIG. 17-IV shows the color of the state where the Au/Ag core-shell composites were complementarily bonded with the target oligonucleotide base sequence and aggregated and an image of FIG. 17-V shows the color of the state where the temperature of the aggregated Au/Ag core-shell composite solution increased above the melting point (in this case, 53 C. of the oligonucleotide base sequence, so that the complementary hydrogen bond was broken to make the distance of the aggregated Au/Ag core-shell composites apart from each other, and thus, the color was restored to the original color. FIG. 18 shows the above experimental results as the variation of the melting point of the aggregated Au/Ag core-shell composite with respect to time. Specifically, FIG. 18C shows the absorbance measured at 260 nm of the aggregated Au/Ag core-shell composite while increasing the temperature from room temperature to 70 C. It can be seen from FIG. 18 that the bonded oligonucleotide was separated in a range of about 55 C. to about 65 C.

[0198] According to the result of the colorimetric assay test, the target material recognition site of the receptor was not embedded into the Ag nanoparticle layer, but was exposed to the outside of the Au nanoparticle layer. Thus, the normal target recognition function was carried out.

Example 11: Preparation of Au/Ag Core-Shell Composite when Au Nanoparticle is a Combination of Two or More Particles

[0199] The oligonucleotides A and B of Example 5 were bonded on the Au nanoparticle according to Example 6. 6.0 l of 10 M target oligonucleotide (see FIG. 16) was added to the mixed solution containing Au nanoparticle bonded with oligonucleotide conglomerate A and Au nanoparticle bonded with oligonucleotide conglomerate B, which were dissolved in 0.3 M phosphate buffer solution. The temperature of the mixed solution increased to 70 C. and then gradually decreased down to room temperature. It was observed through the TEM that the separate Au nanoparticles formed a dimer after about 2 hours.

[0200] To the 250 l of the solution, 50 l of AgNO.sub.3 (10-3 M) and 50 l of hydroquinone solution were added and then agitated for 3 hours. As a result of observing the progress of the reaction through the UV-visible spectroscopy, the extinction was increased at 400 nm as shown in FIG. 14. Moreover, as an observation result using the TEM, the Ag nanoparticle layer was formed even in the dimer and the combination of the dimer or more (see FIGS. 19 and 20).

[0201] In accordance with the embodiments of the present invention, even in the Au nanoparticle having the combination of the dimer or more, the Ag nanoparticle layer forming the shell can be formed while effectively adjusting its thickness. Although the method for preparing the dimer has been described as the method using oligonucleotide, it is merely exemplary and the present invention is not limited thereto.