KR101917439B1 - A metallic nanostructures having a cube-in-cube shape, a method for prepaing the same, and use thereof - Google Patents

Abstract

The present invention relates to a nanocomposite nanocomposite core comprising: A nanogap formed at a predetermined thickness on the outer periphery of the core; And a first metal and a second metal alloy shell spaced apart from the core at regular intervals through the nanogap to a predetermined thickness, a method for producing the metal nano structure, and a use thereof .

Description

[0001] The present invention relates to a cube-in-cube type metal nanostructure, a method for manufacturing the same, and a method for preparing the same,

The present invention relates to a nanocomposite nanocomposite core comprising: A nanogap formed at a predetermined thickness on the outer periphery of the core; And a second metal and a third metal alloy shell spaced apart from the core at regular intervals through the nanogap to have a constant thickness, a method for producing the metal nano structure, and a use thereof .

The direct PL of metals was first reported by Mooradian in 1969, but has not been studied for many years due to the low QY of metals. After Boyd reported local enhancement of PL from smooth and rough metal surfaces, metal nanoparticles, and more specifically gold nanorods (AuNRs), showed markedly increased PL. Over the decades, PL signals from various nanostructures, including gold nanospheres and gold nanorods, have been studied. It has been reported that the gold nano-bipyramid exhibits a quantum yield (QY) of ~ 10-5 higher than gold nanorods. However, most of these studies have focused on the relationship between PL and localized surface plasmon resonance (LSPR) for different polarization angles or excitation wavelengths. It has been found that PL spectra can be tuned by nanostructure geometry changes, as for LSPs. Recently, PLs from plasmonically coupled nanostructures have been extensively studied. It has been reported that the QY of the gold dimer, consisting of two spherical gold nanoparticles, is similar to the value for the monomers constituting it, despite the higher localization between the intersections of the dimers. On the other hand, increased PL in the case of nanostructures located on the gold thin film has been reported, which is attributed to gap plasmon. Thus, the effect of plasmon coupling on the PL intensity is clearly illustrated. To date, several mechanisms have been proposed to explain the observed phenomena of PL in nanostructures: (1) eh recombination after interband transition, (2) LSPs excited by interband transition , (3) interband transitions leading to (eh) recombination, (4) inelastic scattering of sp-band electrons, and (5) direct emission of LSPR leading to radioactive emission. However, the exact mechanism is controversial.

To date, plasmatically coupled systems have been largely limited to lithographically fabricated structures or simple nanostructures located on metal films. PL from nanoparticles that can be plasmatically coupled and precisely regulated at the nanometer scale has not yet been achieved. In addition, nanoparticles exhibiting both strong PL signals and high QY were not devised or synthesized.

The inventors of the present invention have made intensive efforts to discover a metal nanostructure exhibiting strong PL and a method for producing the same. As a result, the present inventors have found that, through galvanic substitution / reduction reaction from cubic metal nanoparticles, The present invention has been accomplished by confirming that these particles exhibit a strong photo-thermal effect as well as a strong PL.

One object of the present invention is to provide a nanocomposite nanocomposite comprising: a cubic first metal nanoparticle core; A nanogap comprising a void space formed by removing a second metal from a second metal coating layer formed to a constant thickness on an outer periphery of the core and a second metal containing nanograid; And a second metal and a third metal alloy shell spaced apart from the core at regular intervals through the nanogap to have a constant thickness, to provide a cube-in-cube type metal nanostructure.

Another object of the present invention is to provide a method of manufacturing a nanoparticle comprising: a first step of preparing nanoparticles comprising a cubic first metal nanoparticle core and a second metal coating layer of a constant thickness on the core; And a second step of performing a galvanic substitution / reduction reaction on the nanoparticles of the first step. The present invention also provides a method of manufacturing the cube-in-cube type metal nanostructure.

Another object of the present invention is to provide a biosensing composition comprising the metal nanostructure of the cube-in-cube type.

Another object of the present invention is to provide a photothermal therapy (PTT) composition for a cancer disease comprising the cube-in-cube type metal nanostructure.

It is still another object of the present invention to provide a photothermal therapy composition comprising: a first step of administering the photothermal therapy composition to a subject in need thereof; And a second step of irradiating the subject with light.

According to one aspect of the present invention, there is provided a method of manufacturing a metal nanocomposite material, A nanogap comprising a void space formed by removing a second metal from a second metal coating layer formed to a constant thickness on an outer periphery of the core and a second metal containing nanograid; And a second metal and a third metal alloy shell spaced apart from the core at regular intervals through the nanogap to a predetermined thickness. The metal nano structure of the cube-in-cube type is provided.

The term " nano bridge " of the present invention is a structure formed of a low residual second metal to form an empty space while the second metal is removed from the second metal coating layer, Metal and the third metal alloy shell to maintain a constant thickness of the nanogap and to maintain a constant gap between the core and the shell. As can be inferred from the above-described process of forming a nano bridge, the nano bridge of the present invention has been described as such in that it plays a role of maintaining a space by connecting and supporting the core and the shell. The shape and / It does not. For example, the content of the second metal in the nano bridge may be determined depending on the kind of the second metal, the thickness of the second metal coating layer, the condition of the second metal removal reaction, and / or the rate of formation of the shell, .

For example, the metal nanostructure may be prepared by performing a galvanic substitution / reduction reaction on a nanoparticle including a cubic first metal nanoparticle core and a second metal coating layer having a predetermined thickness on the core.

For example, in the metal nanostructure of the present invention, the nanogap may have a controlled thickness at a sub-nanometer level, and the thickness of the nanogap may be adjusted by adjusting the thickness of the second metal coating layer formed on the cubic first metal nanoparticle core Can be determined by the thickness.

At this time, the thickness of the nano gap may be 0.5 to 20 nm, but is not limited thereto.

The cube-in-cube type metal nanostructure of the present invention may have an improved photoluminescence intensity at a level of 10 to 100 times higher than that of the cubic first metal nanoparticle having the same volume as the core or the entire metal nanostructure. The term " identical " does not mean that it is literally an exact match, but allows variations of the order of +/- 15%.

Specifically, in the metal nanostructure of the present invention, the first metal forming the core and the third metal serving as one component of the alloy forming the shell may be the same metal.

For example, in the metal nanostructure of the present invention, the first metal and the third metal may be all gold and the second metal may be silver. Since the gold and silver disclosed as examples of the first metal and the third metal and the second metal respectively exhibit a low lattice mismatch, that is, the lattice constant difference between these metals is small, epitaxial growth. Thus, the metal nanostructure in the form of a cube-in-cube, which is the final product, can be grown while maintaining the cubic shape of the core, which is the first reaction material.

According to another aspect of the present invention, there is provided a nanoparticle comprising: a first step of preparing nanoparticles comprising a cubic first metal nanoparticle core and a second metal coating layer of a constant thickness on the core; And a second step of performing a galvanic substitution / reduction reaction on the nanoparticles of the first step. The present invention also provides a method of manufacturing the cube-in-cube type metal nanostructure.

In the manufacturing method of the present invention, the first step includes a first step of preparing cubic first metal nanoparticles and a second step of forming a second metal coating layer on the first metal nanoparticles, Step < / RTI > The reaction of each of the above steps can be carried out by using methods known in the art.

For example, the step 1-1 may be carried out by reacting a first metal precursor, such as a gold precursor, with a reducing agent in an aqueous solution. The step 1-1 may be performed in the presence of CTAB (cetyltrimethylammonium bromide), but is not limited thereto. At this time, HAuCl ₄ may be used as the gold precursor and ascorbic acid may be used as the reducing agent, but the present invention is not limited thereto.

Meanwhile, the step 1-2 may be performed by reacting a second metal precursor such as silver precursor with a reducing agent. The step 1-2 may be performed in the presence of CTAC (cetyltrimethylammonium chloride), but is not limited thereto. At this time, AgNO ₃ may be used as the silver precursor and ascorbic acid may be used as the reducing agent, but the present invention is not limited thereto.

Specifically, in the manufacturing method of the present invention, the second step is to remove the second metal from the second metal coating layer included in the nanoparticles obtained from the first step formed to a constant thickness on the outer surface of the core to form a void space Thereby forming an alloy shell including both a second metal and a third metal at an outer angle, wherein the oxidation of the second metal is performed in the second metal coating layer, and the oxidation of the second metal and the third metal Is reduced at the same time.

For example, the second step may be performed in the presence of a cationic third metal precursor. Specifically, the cationic third metal precursor can provide a third metal cation, which can act as an oxidant for the second metal in the second metal coating layer to oxidize the second metal atom to ions to form an empty space have. Specifically, in the case of a nanostructure containing silver as a second metal, HAuCl ₄ , which can provide a gold cation having a lower ionization tendency than silver, can be used as the third metal precursor, The type of the third metal precursor is not limited thereto.

In addition, the second step may be carried out in the presence of a reducing agent. As described above, in the second step, the second metal is oxidized, and at the outer angle, the second metal ion and the third metal ion are reduced together to form an alloy shell of the second metal and the third metal. Therefore, a reducing agent may be further added to assist the reduction of the second metal ion and the third metal ion. At this time, as the reducing agent, ascorbic acid may be used, but is not limited thereto.

In addition, the second step may be performed at a relatively high temperature of 40 to 100 < 0 > C. Specifically, it may be carried out at 60 to 80 占 폚, but is not limited thereto.

In the case where the second step is carried out under the presence of a reducing agent and / or at a high temperature, it is possible to provide a cube-in-cube metal nanostructure which has little joints and is firm and robust. On the other hand, if these conditions are not met, a shell with a rough or porous surface may be formed, which may hinder uniform and adjustable plasmon coupling.

As described above, through the second step, the pores are formed by the oxidation of the second metal by the cationic third metal precursor, and at the same time, the coexistence of the cationic third metal precursor and the second metal ion formed by the oxidation The second metal-third metal alloy shell can be formed by reduction. The remaining second metal, which is removed by oxidation of the second metal, may act as a nanobridge connecting the first metal core therein to the second metal and third metal alloy shell formed on the outer periphery. Due to the presence of such a nano bridge, it is possible to maintain the nanogap of the hollow space formed between the core and the shell at a constant thickness.

At this time, the first metal, the second metal and the third metal may be selected so that the first metal and the second metal, and the second metal and the third metal exhibit a low lattice mismatch. Thus, when forming the second metal and third metal alloy shells from the surface of the second metal coating layer and the second metal coating on the first metal core by selecting metals in combination with a low lattice mismatch between them, Because it is capable of epitaxial growth, it can be grown along the crystal form of the cubic first metal nanoparticle core, which is the first reaction material acting as a seed, so that the final product can also be made to have a cubic shape.

For example, the lattice constant difference between the first metal and the second metal and between the second metal and the third metal may be selected to be 10 pm or less. Specifically, the first to third metals may be selected to have a lattice constant difference of 5 pm or less, more specifically, 2 pm or less, but the present invention is not limited thereto.

In a specific embodiment of the present invention, gold and silver having lattice constants of 407.82 pm and 408.53 pm, respectively, were used as the first and third metals and the second metal, but the present invention is not limited thereto. Since the difference between the values of the lattice constants of gold and silver is very low to 1 pm or less and has a crystal structure of FCC (face centered cubic) in common, it can be advantageous to maintain the shape of the core by laminating while maintaining the crystal structure.

In another aspect, the present invention provides biosensing compositions comprising metal nanostructures in the cube-in-cube form.

For example, the metal nanostructure may further include, but is not limited to, a substance that specifically binds to a substance to be detected, which is directly or indirectly connected to the metal nanostructure.

Specifically, the cube-in-cube type metal nanostructure of the present invention has a remarkably increased light emission intensity as compared with a single metal nanoparticle of a similar volume, and exhibits a photobleach or a light flicker, which is a disadvantage in organic phosphors and quantum dots Therefore, even if the light is irradiated for a long time, the luminescence intensity is not reduced, and therefore, it can be usefully used as a marker for detecting living organisms.

In another aspect, the present invention provides a photothermal therapy (PTT) composition comprising a metal nanostructure in the form of the cube-in-cube.

Specifically, the composition may be useful for the treatment of cancer, but the present invention is not limited thereto. The composition can be used without limitation in the treatment of diseases in which lesion cells or tissues become more heat-sensitive than normal cells or tissues.

For example, the metal nanostructure may have an absorption spectrum in the near infrared region. Since the light in the near infrared region has a lower energy than that of ultraviolet rays or visible rays but has a long wavelength, it has excellent permeability and can reach deep inside the body even when irradiated from the outside of the body, Non-invasive treatment may be possible.

The present invention also provides a method for preparing a photothermal therapy composition, comprising the steps of: And a second step of irradiating the subject with light.

The term " administering " of the present invention means introducing a predetermined substance into an individual by an appropriate method, and the administration route of the composition can be administered through any conventional route so long as it can reach the target tissue. But are not limited to, intravenous, intraperitoneal, intravenous, intramuscular, subcutaneous, intradermal, oral, topical, intranasal, intrapulmonary, rectal.

The term " individual " refers to all animals including mice, mice, livestock, and the like, including humans suffering from cancer. Preferably, it may be a mammal, including a human. The phototherapy composition of the present invention may be effectively administered to an individual and irradiated with light of an appropriate wavelength to treat the cancer disease. The photothermal therapy using the photothermal therapy composition of the present invention may be used alone or in combination Surgery, hormonal therapy, drug therapy, and methods using biological response modifiers.

In the present invention, the term " treatment " means all the actions of improving or alleviating symptoms of a cancerous subject by administration of the composition and light irradiation. The treatment of cancer diseases by the photothermal therapeutic composition of the present invention can be achieved by inducing the death of cancer cells by the heat energy absorbed and emitted from the cube-in-cube type metal nanostructure .

The term " photothermal therapy " may refer to the act of using electromagnetic radiation to treat a variety of medical conditions, including cancer. Specifically, it refers to a method for treating cancer, wherein photon energies are converted into thermal energy to induce apoptosis. At this time, the wavelengths in the near infrared or infrared region are most widely used. This is an extension of photodynamic therapy that is performed by exciting a photosensitizer to light of a certain wavelength. This can cause the sensitizer to be excited and excite the target cell, for example, cancer cells, using the vibrational energy, i.e., heat, emitted from the excitatory agent.

Unlike photodynamic therapy, photothermal therapy does not require oxygen to interact with target cells or tissues. It can be performed using light of a long wavelength with less harm to other cells and tissues since it has lower energy. On the other hand, since cancer cells are generally more heat-sensitive than normal cells, the therapeutic effect can be maximized when the temperature is selectively increased in cancer cells.

The cube-in-cube type metal nanostructure having an internal nanogap of the present invention is formed by a galvanic substitution / reduction reaction from a structure including a second metal coating layer coated with a uniform thickness on cubic first metal nanoparticles, And a shell made of a first metal and a second metal alloy connected to each other by a predetermined gap and a second metal nano bridge. The nanostructure can exhibit a strong PL as well as a photothermal effect Therefore, it can be used not only for detection of living organism but also for photothermal treatment such as cancer.

1 is a schematic representation of a method for the preparation of CiC nanoparticles having an adjustable internal nanogap (interior nanogap-engineerable CiC NPs) according to the present invention and a method for synthesizing CiC NPs via a controlled two-step GVF process.
FIG. 2 is a TEM image corresponding to each step of the process of synthesizing CiC nanoparticles having an adjustable internal nanogap according to the present invention. FIG. The degree of insertion represents a representative single-particle image and the scale bar is 100 nm.
FIG. 3 is a view showing the morphology of the precisely regulated Ag shell-formed CiC nanoparticles according to the present invention. Specifically, (a) TEM and (b) SEM images of particles in which Ag shells having different thicknesses are formed on AuNCs are shown. Insertion degrees represent representative nanostructures, and the scale bars in (a) and (b) are 200 nm and 1 μm, respectively.
4 is a TEM image showing a structural change observed during the synthesis of CiC nanoparticles according to the present invention. TEM images were taken at 4 minutes (left) and 8 minutes (right) after the start of the first galvanic permutation reaction, and the scale bars were all 50 nm.
FIG. 5 is a graph showing the influence of various conditions on a controlled galvanic substitution reaction in the process of synthesizing CiC nanoparticles according to the present invention. FIG. (a) shows an SEM image of the product formed after the first galvanic substitution reaction under different conditions, and the scale bars are all 1 μm. (b) show the corresponding UV-vis spectra.
FIG. 6 is a diagram showing a large-area TEM image of the final product of high yield according to the method of synthesizing CiC nanoparticles of the present invention. The degree of insertion represents a representative SEM image of CiC NPs, with a scale bar of 1 μm.
7 is a SEM image showing a structural morphology of CiCs having a 9-nm gap prepared by the method of synthesizing CiC nanoparticles according to the present invention. Some truncation was observed at the corners of the CiCs. The scale bar is 200 nm.
8 is a diagram showing (a) HAADF-STEM image (lower right) and corresponding EDS map and (b) electron diffraction pattern of CiC nanoparticles according to the present invention. (a), red and green represent the elements Au and Ag, respectively, and the bottom left image is their superimposed mapping image and the scale bar is 50 nm. The electron diffraction pattern of (b) represents the single-crystalline nature of CiC NP. The electron diffraction pattern was recorded with an electron beam vertically aligned on one side of CiC NPs.
FIG. 9 is a diagram showing (a) TEM observation and (b) optical characteristics of CiCs having an inner gap controlled according to the present invention. FIG. Specifically, CiCs with a gap size of 1 nm (left side of a), 3 nm (middle of a), and 15 nm (right side of a) were synthesized and observed. . The scale bar is 100 nm. (b) show the calculated (upper) and experimental (lower) extinction spectra of these CiCs. For computed spectra for CiCs with a 3 nm gap, it is worth noting that the increase at about 1,000 nm, associated with the bonding plasmon peak.
FIG. 10 is a diagram showing a plasmon hybridization diagram of a plasmon coupling model for CiC NPs according to the present invention. FIG. The basic coupling between the core and the shell creates two plasmon modes. The antiparallel coupling that occurs at the lower energy level represents quasi-radioactive, whereas the parallel coupling represents super-radioactivity with an increased total dipole moment. The embedded image represents the surface charge distribution for the corresponding plasmon mode.
11 is a diagram showing the optical measurement results of CiC NPs according to the present invention. (a) is the calculated optical spectrum of the core AuNC, shell, and CiC obtained through FEM simulation, the solid line corresponds to extinction, and the dotted and dashed lines show absorption and scattering, respectively. (b) shows an experimental UV-Vis spectrum for a 10 pM CiC dispersion.
12 is a diagram showing the surface charge distribution of the separated shell corresponding to the shoulder peak at the LSPR wavelength of ~ 560 nm of CiC NPs according to the present invention.
13 shows (a) TEM images of the 87-nm AuNCs and (b) calculated optical spectra according to the present invention. The scale bar is 100 nm.
14 is a diagram showing optical spectra of CiC NPs and two other AuNCs according to the present invention. (a) shows the extinction spectrum obtained by UV-Vis measurement, and the edge lengths of 47-nm and 87-nm of AuNCs correspond to those of the core cube and CiCs of CiCs, respectively. The concentration of the sample was fixed at 5 pM. (b) shows the photoluminescence (PL) spectra of 47- and 87-nm AuNCs measured with a 532-nm excitation laser and CiCs dispersions with various internal gap sizes. All spectra were measured by exposing samples at 10 pM concentration to 5 mW laser power for 10 seconds.
Figure 15 is cross-sectional images of the CiC internal electric field corresponding to (a) visible light peak and (b) near infrared peak for each LSPR wavelength. The section is perpendicular to the direction of light propagation. All drawings use the same scale for field strength.
16 is a graph showing photoluminescence intensity according to the concentration of CiC NPs according to the present invention.
17 is a graph showing photoluminescence intensity according to laser power of CiC NPs according to the present invention.
18 is a diagram showing photoluminescence characteristics from CiC NPs according to the present invention. (a) shows the comparison of PL characteristics from CiCs, 47- and 87-nm AuNCs, and AuNR, and shows 13 × 46 nm AuNRs (Au nanorods) and Rhodamine 6G (R6G) As shown in FIG. Quantum yield (QY) and total PL intensity were obtained by integration from 535 nm to 1,000 nm. The conditions were the same for all samples (concentration 10 pM). First, the spectra of AuNRs and R6G were measured using 300 and 100 pM concentrations, respectively, and then re-calculated at the same concentrations. The error bars are the standard deviations for three independent measurements. (b) shows the PL intensity for CiC measured using a single TIRF microscope under continuous illumination from a 532-nm laser for 1 hour. Images were acquired every 30 seconds and the signal and error bars were averaged over 20 nanoparticles.
19 is a diagram showing a photoluminescence spectrum of R6G used as a reference. The measurement conditions are similar for CiCs except for the concentration (100 pM).
20 shows (a) TEM images and (b) optical characteristics of AuNRs. The scale of AuNRs determined from the TEM image of (a) is 13 x 46 nm and the scale bar is 50 nm. Specifically, (b) represents the extinction (top) and the photo-emission (bottom) spectra of AuNRs, and the photoluminescence measurement conditions are similar for CiCs except for the concentration (300 pM).
21 is a microscope image of CiC NPs according to the present invention. Specifically, images of CiCs were obtained from the same area using bright-field microscopy (left) and total internal reflection fluorescence microscopy (right), and in the two images, CiCs Respectively. The scale bars are all 2 μm.
22 is a diagram showing the photothermal effect of CiC NPs according to the present invention. 1 mL of CiC NPs dispersion was irradiated with a laser for a certain period of time, and the temperature of the solution was measured with time.
23 is a diagram showing the results of cell imaging using CiC NPs according to the present invention.

Hereinafter, the present invention will be described in more detail with reference to Examples. These examples are for further illustrating the present invention, and the scope of the present invention is not limited by these examples.

<Material>

Cetyltrimethylammonium bromide (CTAB) and cetyltrimethylammonium chloride (CTAC) were purchased from TCI. Ascorbic acid, gold chloride trihydrate, and silver nitrate were purchased from Sigma-Aldrich. Sodium borohydride was purchased from Daejung Chemical & Metals. All reagents were used on the market without further purification. For all experiments, deionized water (DIW) obtained using Millipore system (> 18.0 MΩ, Milli-Q) was used.

Manufacturing example  One: Of gold nanocubes (AuNCs)  synthesis

Dovgolevsky, E .; Gold nanocubes (AuNCs) were synthesized using the seed-growth method disclosed in Haick, H., Small, 2008, 4 (11): 2059-2066. All solutions used below were prepared using DIW.

Specifically, in order to synthesize a gold seed solution, 7.5 mL of a 100 mM CTAB solution was prepared in a round bottom flask in a water bath heated to 30 ° C. To the flask was added 250 μL of 10 mM HAuCl ₄ solution and the mixture was stirred at 500 rpm. Then, 600 μL of ice-cold 10 mM NaBH ₄ solution was rapidly mixed, at which time the solution turned brown. The solution was stirred for 2 minutes, incubated at 30 &lt; 0 &gt; C for a minimum of 1 hour, and then used as a seed solution in the next step. Thereafter, growth was carried out in 50 mL conical tubes.

1.6 mL of 100 mM CTAB solution was mixed with 8 mL of DIW and 200 μL of 10 mM HAuCl ₄ solution and 950 μL of 100 mM ascorbic acid solution were added to the mixture in turn. After adding ascorbic acid, the solution turned transparent. Then, 5 [mu] L of a 10-fold diluted seed solution was added dropwise to the tube and mixed gently with the prepared mixture using gentle inversion mixing. After overnight incubation, AuNCs were recovered by centrifugation (5500 rpm, 10 min) twice. After removing the supernatant, the recovered particles were dispersed again in DIW for later use.

Manufacturing example  2: AuNCs  Silver shell coating on

All solutions used in the present preparation examples were prepared using DIW. Ma, Y .; Li, W .; Cho, EC; Li, Z .; Yu, T .; Zeng, J .; Xie, Z .; and Xia, Y., ACS Nano, 2010, 4 (11): 6725-6734, silver shells were introduced on AuNCs prepared according to Preparation Example 1, by optimizing and performing the protocol for forming Ag shells. Specifically, 400 μL of a 10 pM AuNC dispersion was mixed with 400 μL of a 10 mM CTAC solution and 200 μL of a 50 mM ascorbic acid solution. After mixing, 40 μL of AgNO ₃ solution at various concentrations (0.5 to 5.0 mM) was added and mixed with a vortex mixer. After incubation at 60 DEG C for 3 hours, the coated nanoparticles were recovered by centrifugation twice (5000 to 6000 rpm, 10 minutes) and redispersed in DIW for subsequent use.

Example  1: cube-in-cube nanostructures ; CiCs )

All solutions used were prepared using DIW. First, 400 μL of 10 μM shell-coated nanocube dispersion was mixed with 400 μL of 100 mM CTAB and 87 μL of 100 mM ascorbic acid. The mixture was incubated at 70 &lt; 0 &gt; C for 3 minutes with stirring at 300 rpm. Then 400 μL of HAuCl ₄ solution (0.06-0.15 mM) was injected into the mixture at a rate of 20 μL / min using a syringe pump. After the infusion procedure was completed, the solution was incubated for 5 minutes or more and centrifuged twice at 4500 rpm for 5 minutes. Finally, the solution was mixed with 400 [mu] L of DIW. Next, it was mixed with 400 μL of 10 mM CTAB. Then, 400 μL of HAuCl ₄ solution (0.008 to 0.13 mM) was immediately injected into the mixture. The mixture was incubated at 30 DEG C for 20 minutes. After the injection procedure, the solution was centrifuged twice (4500 rpm, 5 minutes) and the CiCs were dispersed in DIW.

Experimental Example  1: Characterization of nanostructures

Transmission electron microscopy (TEM) and scanning electron microscopy (TEM) were carried out using JEM-2100 (JEOL) and Helios NanoLab 650 (FEI) systems at the National Center for Inter-University Research Facilities (NCIRF, Microscope (SEM) images were obtained. HAADF-STEM data (and corresponding EDS map) and SAED pattern were obtained by using JEM-2100F (JEOL) system at Research Institute of Advanced Materials (RIAM, Korea) at Seoul National University.

Experimental Example  2: electromagnetic calculations

For the theoretical analysis, FEM simulation was performed using COMSOL Multiphysics which is commercialized software. The model used was based on the analysis of the CiC structure determined from TEM and SEM images. Electric fields and extinction cross-sections were calculated in a scattered-field mode under plane-wave excitation. The core was modeled as gold (Au) and the shell was modeled as a gold-silver alloy. The surrounding medium and the gap region between the core and the shell were modeled with water. An empirical dielectric function reported by Hale and Querry (Hale, G. M .; and Querry, M. R., Appl. Opt., 1973, 12 (3): 555-563) was used for water. For the gold and gold-silver alloys, an analytical model (Rioux, D .; Vallieres, S .; Besner, S.; Munoz, P.; Mazur, E .; and Meunier, M., Advanced Optical Materials, 2): 176-182).

Experimental Example  3: Optical measurement

Ultraviolet-visible (UV-vis) spectra of the nanostructures were measured using a UV-vis spectrophotometer (Agilent 8453 spectrophotometer, USA). PL measurements were performed using a HORIBA LabRAM ARAMIS Raman microscope. The excitation laser was a ND: Yag laser with a 600 gr / mm grating and a multi-channel 1024x256 air-cooled charge-coupled device detector (pixel size: 26 x 26 μm) 532 nm) was used. All spectra were acquired using a 5x objective for a laser power of 5 mW and an exposure time of 10 seconds. Specifically, 12 μL of the nanostructure dispersion to be tested was placed on a silicon isolator on a glass substrate for measurement. Notch filters were used to block direct scattering of the laser causing a significant reduction in the number of photocounts near the excitation laser wavelength of the spectrum. The spectrum from the empty glass cover slip and DIW used as references from the measured PL spectra was subtracted. In addition, all PL spectra were corrected by subtracting the background signal for glass and DIW to eliminate noise, and further smoothed using software origin 8.0. For TIRF measurement, 1 μL of CiC solution was spin-coated on a glass slide, washed with ethanol and dried.

Experimental Example  4: Photoluminescence  century( I _PL ) And Quantum yield (QY)  Calculation

The second time-derivative square of the total dipole moment at the LSPR wavelength (47-nm AuCs) and the absorption cross-sections at the excitation wavelength (2-fold increase from 47-nm AuNCs to CiCs) nm AuNCs to CiCs 12.3 times) were calculated by FEM simulation.

The quantum yield (QYs) of the nanostructure was obtained based on the above equation. Where?,?, And c are the QY values, the molar absorption coefficient at the excitation wavelength, and the solution concentration, respectively. The suffixes s and r refer to the sample and R6G, respectively. And σ and c of R6G were 1.160 × 10 ⁵ cm ^-1 · M ^-1 and 0.95, respectively. The molecular absorption coefficient of AuNRs has been previously reported (Fang, Y., Chang, W.-S., Willingham, B .; Swanglap, P.; Dominguez-Medina, S .; , 2012, 6 (8): 7177-7184).

Experimental Example  5: Photothermal effect ( photothermal  effect)

In order to confirm the photothermal effect of CiC NPs according to the present invention, an 808 nm laser was continuously irradiated to 1 mL of the above-prepared dispersion of CiC NPs at a concentration of 10 pM for 5 minutes at a power of 0.35 W / cm ² , The temperature change was measured while keeping it for 10 minutes. The laser irradiation for 5 minutes and the process for blocking and maintaining the laser for 10 minutes were performed in a single cycle, and the same process was repeated several times to measure the temperature. As a typical CiC NP, a cubic nanostructure having a side dimension of 85 nm with a nanometer gap of 9 nm and a shell thickness of 10 nm formed on cubic gold nanoparticles with a lateral size of 47 nm was used.

Experimental Example  6: Cell Imaging

The glioblastoma U87MG cells in 96-well plates 1 × 10 ⁴ DMEM (Gibco) containing the cells / divided by the density of the wells, 10% FBS and 1% PS (100 U / mL penicillin and 100 μg / mL streptomycin) After culturing for 3 days at 5% carbon dioxide and 37 ° C using a culture medium, a 100 pM concentration of the CiC NPs dispersion according to the present invention was added at a volume ratio of 1: 1 for 24 hours, and a 488 nm laser was excited Were used as a light source and observed with a confocal fluorescence microscope.

<Result>

In the present invention, the nanogap includes a nanogap, which is an empty space between the core and the shell in a cube-in-cube form, and the nanogap is adjustable at the nm level, and is based on a metal capable of providing a strong PL signal with high QY The present invention relates to nanoparticles and a method of manufacturing the nanoparticles and a method of forming the cube-in-cube nanoparticles (CiC NPs or CiCs) and a specific synthesis method. Furthermore, we tried to clarify the mechanism of strong PL in these metallic nanoparticles. The interior gap-based plasmonic coupling was strong and tunable, with signal fluctuations between individual particles being minimized. As the core material, gold nanocubes (AuNCs) exhibiting higher QY than nanospheres and nanorods (NRs) were selected. Its well-defined faces, edges, and vertices also lead to well-defined nanogaps with uniform electromagnetic fields between the core and the shell. The present invention has developed a GVF process that includes replacement / void and void formation steps to form an outer shell from the core in the presence of an internal gap (FIG. 1). This resulted in the synthesis of the desired nanostructures with high structural accuracy and yield (~ 88%). In the present invention, a super-radiant PL mechanism for PL enhancement based on plasmon coupling from metal nanostructures was first proposed, and the optical properties of CiC NPs were analyzed based on the mechanism and plasmon hybridization theory. In conclusion, CiC NPs exhibited highly enhanced PL intensity and higher QY compared to AuNCs, and the PL intensity and QY from the CiC NPs were the highest reported values for metallic nanoparticles.

First, Preparation Examples 1 and 2 were sequentially performed to synthesize ~ 47-nm AuNCs, which were coated with a ~13-nm Ag shell and confirmed by TEM image measurement (i and ii in FIG. 2). Specifically, the thickness and the vertex sharpness of the Ag shell were controlled in a large number of particles by precisely controlling the concentration of cetyltrimethylammonium chloride (CTAC) surfactant and Ag precursor ( Ii and Fig. 3). Next, a galvanic replacement / reduction step was performed with ascorbic acid at 70 &lt; 0 &gt; C. The time-dependent TEM image was used to confirm the synthesis mechanism, that is, the oxidation of Ag atoms by Au precursor and the simultaneous co-reduction of Au and Ag ions by ascorbic acid Iii and Fig. 4). In particular, the synergistic effect of the presence of high temperature and reducing agent resulted in a nearly seamless, solid, robust cube Au-Ag alloy shell (FIG. 5). Conversely, when the galvanic substitution reaction was carried out at 25 &lt; 0 &gt; C and / or in the absence of a reducing agent, a rough or porous surface was formed which inhibited uniform and adjustable plasmon coupling. The subsequent void formation step, carried out in the absence of a reducing agent at reduced temperature, pushed out the remaining internal Ag atoms and formed a clean pore between the core and the shell (iv in FIG. 2). Scanning electron microscopy (SEM) was used to confirm vertex truncation, such as ~ 25% of edge length. The cleavage was induced to minimize the total surface energy and produces a {111} plane instead of less stable {100} planes (FIG. 6, inset and FIG. 7). In addition, it was observed that connecting junctions were formed between the core and the shell (the inset of iv in Fig. 2 and the lower right image of Fig. 8a). The resulting CiC NPs were homogeneous and had a corner length of 85.11 ± 1.07 nm (n = 50), a gap size of ~9 nm, and a shell thickness of ~10 nm. 200 nano structures were analyzed and the synthesis yield was found to be ~ 88% (FIG. 6). The present inventors synthesized CiC NPs having various gap sizes while varying the Ag shell thickness (FIG. 9). The gap distance was varied to ~ 1, 3 or 15 nm while the outer shell thickness was kept constant at ~ 10 nm. In order to determine the elemental composition of the nanostructures as well as the spatial distribution of each constituent element, a high-angle annular dark-field scanning transmission electron microscopy (HAADF-STEM) and an energy dispersive X- Energy-dispersive X-ray spectroscopy (EDS) mapping was performed. The obtained HAADF-STEM image provided information on the formation of an Au-Ag alloy shell with the presence of an internal nanogap (FIG. 8A). Most of the Ag atoms are mainly in the inner part of the shell, while the gold atoms are distributed evenly in the core and shell. These results support the proposed synthetic mechanism described above. The electron diffraction pattern of CiC NPs was symmetrical and showed a single - crystal structure. More specifically, the pattern suggested that the preferred growth direction of the surface follows the fcc {100} facet. This is indicative of epitaxial growth from NCs to CiCs due to low lattice mismatch between Au and Ag (Fig. 8B).

To illustrate the coupling between the core and the shell of CiCs, a plasmon hybridization model was used. When two dipolar parent modes of a cube and a truncated cubic shell are hybridized, they form two dominant plasmon modes: bonding dipolar and antibonding dipolar modes (Figure 10). Asymmetric hybridization is due to the phase-retardation effect. The half-coupled mode exhibits supra-radioactivity with an increased total dipole moment while the coupled mode is sub-radiant with a reduced total dipole moment. The concept of super-radioactivity was first disclosed in the field of fluorescence. In contrast to the case of isolated scatterers, they exhibited cooperative emission when the emitters interacted and coherently interfere with each other. When the collective oscillation of the free electrons of a metal is considered to be similar to an oscillating dipole, the radiative damping rate of this plasmon mode is proportional to the square of the second derivative of the dipole moment. As the plasmon mode became super-radioactive, the degree of radiative damping increased significantly.

The plasmon coupling results were further described by the extinction spectrum. The spectra obtained using the finite element method (FEM) simulation showed the energy levels of each mode (FIG. 11A). The AuNCs exhibited quenching peaks in the visible light region, while the nanoshells showed strong quenching peaks in the near-infrared region with small shoulders associated with the quadrupole mode resulting from larger sizes (Figs. 11A and 12). The calculated extinction spectrum of the coupled CiCs showed two dominant plasmon modes, as expected. The semi-coupling mode increased the line-width reflecting the ultra-radioactivity, and the quasi-radioactivity of the coupling mode was reflected in the sharpness of the line width. It should be noted that the peak in the visible light region is mainly due to scattering, and the peak in the near infrared region exhibits strong absorption. Further, the smaller the gap distance, the stronger the coupling between the core and the shell was, thereby lowering the energy of the bond plasmon mode (FIG. 9). The experimentally determined quenching spectra of CiC NPs agree well with the calculated spectra, although the Ag remaining in the gap is not accounted for in the FEM model, since the remaining Ag atoms are in far-field optical properties (Fig. 11 (b)). The broadening in the experimental spectrum was due to the intrinsic heterogeneity of the nanostructures. CiCs showed two distinct peaks at ~ 1.45 eV (855 nm) and ~ 2.15 eV (577 nm). The spectrum of CiCs with a 15-nm gap did not show any definite peak due to the large amount of residual Ag remaining from the thick Ag shell after the galvanic substitution reaction (Figure 9). These residues fill the cavity between the core and the shell, thus inhibiting plasmon coupling.

To clarify the effect of internal nanogap on PL, two different sized AuNCs were synthesized. The size of the smaller AuNCs (47 nm) was equal to the size of the core of CiCs, and larger AuNCs (87 nm) were similar in size to CiCs (Fig. 13a). The extinction spectra of 47- and 87-nm AuNCs contained a single LSPR peak (Fig. 14A). The maximum LSPR wavelength of the 87-nm AuNCs was red-shifted compared to the smaller AuNCs, and the extinction intensity of the 87-nm AuNCs was the highest of the three nanostructures due to the large size of the AuNCs. Further, the calculated extinction spectrum of 87-nm AuNCs was similar to the observed spectrum (Figure 13b). Based on the cross-sectional image of the electric field in the metal, the present inventors have confirmed that the intensities in the core of CiCs are stronger than those in the shell in the visible light region (Fig. 15). In the case of peaks in the near infrared region, strong electric fields of CiCs were mainly distributed in the shell region. This indicates that the peak in the visible light region is core-driven whereas the peak in the near-infrared region is shell-dominated.

The PL intensity is maximized when the excitation wavelength is superimposed on the LSPR wavelength of the nanostructure of interest, so it is important to select an appropriate excitation laser source when studying the PL of nanostructures. In the present invention, PL measurement was performed using a 532-nm laser as an excitation source, and PL intensity was measured in an ensemble mode for a wavelength of 535 to 1000 nm. During the ensemble measurement, the inner-filter effect, which needs to be accounted for by re-absorption by neighboring particles, proved negligible (Fig. 16). The linear relationship between laser power and PL intensity suggests that PL is a one-photon process (Fig. 17). The PL intensity of CiCs with a 9-nm gap was significantly different from that of two AuNCs as well as those of CiCs with different gap sizes (Fig. 14B). The larger AuNCs showed a significantly lower PL compared to CiCs, indicating that the internal nanogap of CiCs affects PL signal enhancement. Since the nanostructures synthesized in the present invention were obtained at a high yield, the difference in PL intensity in various NPs can be mainly due to their structural differences. It is believed that the enhancement of supersensitivity of the plasmonic mode is due to the improved absorption capacity. With respect to the super-radioactive PL from the plasmonic nanostructure, the PL intensity, i.e., IPL, can be expressed by the following equation.

Considering the absorption and emission steps associated with the PL process, the IPL is proportional to the product of σ _abs and (P ") ² , where σ _abs is the absorption cross section at the excitation wavelength, (P") ² is the total dipole moment, P is the radial decay rate, expressed as the square of the second derivative of P. The radial decay rate can be regarded as QY in experimental values, in that both represent emission efficiencies. Compared to 47-nm AuNCs, absorption cross-sections and radial decay rates of 1.9-fold and 12.3-fold higher, respectively, were obtained for CiCs. Based on this, we determined the total PL intensity ratio of CiCs to 47-nm AuNCs to be 24.7, which is comparable to the experimentally obtained value of 31.3 (Fig. 18A). Taking into account other factors such as the relaxation parameter based on the PL mechanism, the enhancement factor can be calculated with higher accuracy. One possible explanation for the low PL intensity of 87-nm AuNCs is that relatively large energy differences between excitation and maximum LSPR wavelengths and AuNCs exhibit ordinary and super-radiative dipole oscillations Can be true.

Interestingly, the PL spectrum of CiC NPs contained a single peak, although its scattering spectrum had two LSPR peaks. This does not follow the existing studies suggesting that PL spectra of nanostructures are similar to individual scattering spectra. Furthermore, many researchers considered that local field enhancement is the reason for improved PL intensity of nanostructures. However, the present inventors failed to observe the PL peak in the near-infrared region, even though the calculated electric field corresponding to the LSPR peak in the near-infrared region is stronger than that for the peak of the visible light region, It does not always guarantee a high PL intensity. Considering the excitation energy and the quasi-radioactivity of near-infrared peaks can help explain this phenomenon. The energy from the excitation laser can be redistributed by electron-electron and electron-phonon coupling and mitigated exponentially. This reduces the excitation probability of the LSPR mode away from the excitation wavelength. In addition, the quasi-radioactive LSPR mode has less radiation opportunity.

Finally, the present inventors have discovered that the total PL intensity integrated with organic dye and various nanostructures (including cubic gold nanoparticles of 47 nm and 87 nm in size, and gold nanorods) and QC of CiCs, (Fig. 18A, Fig. 19 and Table 1). The QY of each sample was determined in consideration of the molar absorption coefficient and the total PL intensity calculated at 532 nm, and R6G was used as a reference. The QY of CiC NPs was 4.38 × 10 ^-4 , which was 16.2-fold and 12.3-fold higher than that of 47-nm and 87-nm AuNCs, respectively. The QY of 47-nm AuNCs was determined to be 2.80 × 10 ^-5 . The superlattivity of the plasmon mode resulting from the plasmon coupling has an increased radiated attenuation allowing an increase in QY. , The AuNRs was used as an internal standard, the measured QY is 7.53 × 10 thereof to conform to the ^{measured-value} of ^6, which was consistent with the recently reported data (Fig. 18a and 20). The inclusion of increased QY and higher absorption cross-sections provides 31.3-fold higher PL intensity for CiC NPs compared to 47-nm AuNCs. In addition, although CiCs share similar absorption cross-sectional values, they have a PL that is 13.5 times stronger than 87-nm AuNCs, suggesting that super-radioactive plasmon mode plays an important role in improving PL from plasmonic nanostructures . The total PL intensity of CiCs is higher than that of the conventional organic phosphor, R6G, by about 10 ³ for the same concentration. The PL signal of CiC NPs stably remained without photobleaching or optical flickering even when measured using total internal reflection fluorescence (TIRF) microscopy under continuous laser irradiation for at least 1 hour (Figs. 18B and 21). This indicates that the observed PL originates from the nanostructures rather than from the surrounding molecules, considering that the fluorescence from organic molecules is bleached over time.

竜 (M ^-1揃 cm ^-1 ) I _total (au) QY 47-nm AuNCs 3.06 × 10 ¹⁰ 4.55 × 10 ⁵ ± 1.51 × 10 ⁴ 2.73 × 10 ^-5 ± 9.07 × 10 ^-7 87-nm AuNCs 5.38 × 10 ¹⁰ 1.02 10 ⁶ 1.89 10 ⁴ 3.25 × 10 ^-5 ± 6.02 × 10 ^-7 AuNRs 1.27 × 10 ¹⁰ 5.08 × 10 ⁴ ± 6.96 × 10 ² 7.53 × 10 ^-6 ± 1.03 × 10 ^-7 CiCs with 9-nm gap 5.91 × 10 ¹⁰ 1.37 × 10 ⁷ ± 9.04 × 10 ⁴ 4.78 × 10 ^-4 ± 3.14 × 10 ^-6 R6G 1.16 x 10 ⁵ 5.85 × 10 ⁵ 9.5 x 10 ^-1

Overall, in this study, we have devised and synthesized CiCs capable of regulating the internal nanogap that exhibit a highly enhanced PL through superlattice plasmon coupling in CiCs. The controlled two - step GVF reaction, consisting of substitution / reduction and pore - forming steps, produced CiCs with uniform and controllable internal nanogaps in high yield. Above all, the plasmon coupling between the core and the shell has been found to induce a super-radioactive LSPR mode, which significantly increases the radiative attenuation of the corresponding LSPs to provide a highly enhanced PL from CiCs. CiC NPs exhibited ~ 2-fold and ~ 16-fold higher absorption cross-sections and QY, respectively, leading to ~ 31-fold higher PL intensity than 47-nm AuNCs, indicating the highest PL intensity reported for metallic nanostructures and QY to be. Furthermore, the total PL intensity of CiC NPs was ~ 10 ³ higher than the organic phosphor (R6G in this case). Based on the above results, it is possible to suggest a strategy based on the strategy for improving PL from nanostructures and a mechanism based on plasmon hybridization. Further, PL wavelengths from these plasmonic nanostructures can be easily controlled by tuning the LSPR of the nanostructures based on their structure and composition. The PL represented by the metal is optically stable and is distinguished from the PL from the organic phosphor and the quantum dot.

Further, photothermal effects of CiC NPs were confirmed through experiments, and the results are shown in FIG. As shown in FIG. 22, when the 808 nm laser was continuously irradiated with 0.35 W / cm ² power for 5 minutes, the temperature of the control (PBS solution) was less than 30 ° C, However, when the nanostructure according to the present invention was irradiated, it was confirmed that the solution temperature was increased by about 30 ° C to 51 ° C or more. This indicates that the effect of CiC NPs as a photothermal therapeutic agent is superior to that required for apoptosis (42 ° C). As a result of repetitive laser irradiation and interception, it is confirmed that similar temperature increase / decrease width is observed every time, and it is possible to show repetitive light heat effect without abnormality such as collapse of nanoparticles or loss of ability even in the laser power intensity to be irradiated Respectively.

U87MG cells were treated with CiC NPs and observed with a confocal fluorescence microscope. The results are shown in Fig. As shown in FIG. 23, the fluorescence signal of CiC NPs appeared at 488 nm laser irradiation, and the signal position was in good agreement with the cell position. This indicates that fluorescence signals can be successfully observed from intracellular injected CiC NPs, suggesting the possibility of being introduced into the intracellular imaging field.

The strategy and results show that the full understanding, coordination, and enhancement of PL signals from plasmonic nanostructures with many advantages over SERS and MEF, and optical, biosensing, bioimaging, and energy applications And the possibility of using PL nanoprobe based on plasmonic nanomaterials for various applications including.

Claims (18)

A cubic first metal nanoparticle core;
A nanogap comprising a void space formed by removing a second metal from a second metal coating layer formed to a constant thickness on an outer periphery of the core and a second metal containing nanograid; And
And a second metal and a third metal alloy shell spaced apart from the core at regular intervals through the nanogaps to a constant thickness.
As a metal nano structure in the form of a cube-in-cube,
Wherein the metal nanostructure has an absorption spectrum including a superadiant peak in a visible light region and a near infrared region and exhibits photoluminescence.
The method according to claim 1,
Wherein the metal nanostructure is formed by performing a galvanic substitution / reduction reaction using a third metal on a nanoparticle including a cubic first metal nanoparticle core and a second metal coating layer having a predetermined thickness on the core, structure.
The method according to claim 1,
Wherein the nanogap is controlled in thickness at subnano-meter levels.
The method according to claim 1,
Wherein the nanogap has a thickness of 0.5 to 20 nm.
5. The method of claim 4,
Wherein the metal nanostructure has a photoluminescence intensity of about 10 to 100 times higher than that of the first metal nanoparticle having the same volume as the core or the entire metal nanostructure.
The method according to claim 1,
Wherein the first metal and the third metal are the same metal.
The method according to claim 1,
Wherein the first metal and the third metal are all gold and the second metal is silver.
A first step of preparing nanoparticles comprising a cubic first metal nanoparticle core and a second metal coating layer of a constant thickness on the core; And
And a second step of performing a galvanic substitution / reduction reaction on the nanoparticles of the first step,
A method for manufacturing a cube-in-cube type metal nanostructure according to any one of claims 1 to 7.
9. The method of claim 8,
Wherein the second step is performed in the presence of a cationic third metal precursor.
9. The method of claim 8,
Wherein the second step is carried out in the presence of a reducing agent.
9. The method of claim 8,
And the second step is carried out at 40 to 100 &lt; 0 &gt; C.
9. The method of claim 8,
Wherein the second step comprises forming the pores by oxidation of the second metal by the cationic third metal precursor and simultaneously reducing the second metal ions formed by the cationic third metal precursor and the oxidation, - a third metal alloy shell is formed.
9. The method of claim 8,
Wherein the first metal and the second metal, and the second metal and the third metal are selected so that the lattice constant difference is less than or equal to 10 pm.
A biosensing composition comprising a metal nanostructure in the form of a cube-in-cube of any one of claims 1 to 7.
15. The method of claim 14,
Wherein the metal nanostructure comprises a substance that specifically binds to a substance to be detected.
A photothermal therapy (PTT) composition comprising the metal nanostructure of the cube-in-cube type of any one of claims 1 to 7.
17. The method of claim 16,
Wherein said composition is for the treatment of cancer.
17. The method of claim 16,
Wherein the metal nanostructure has an absorption spectrum in the near infrared region.

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