KR102153949B1 - Directionally Clustered Nanostructures of Compartmentalized Bimetal Nanorods as Surface Enhanced Raman Scattering Nanoprobes for Biosensing and the Methods Thereof - Google Patents

Abstract

The present invention relates to a cluster nanostructure having a directionality of a partitioned bimetallic nanorod, a method for manufacturing the same, and a method for detecting a target material based on surface-enhanced Raman scattering (SERS) using the same. In the present invention, the nanostructure can be applied as a metal nanoprobe for measuring a surface-enhanced Raman scattering (SERS) image.

Description

Directionally Clustered Nanostructures of Compartmentalized Bimetal Nanorods as Surface Enhanced Raman Scattering Nanoprobes for Biosensing and the Methods Thereof}

The present invention relates to a cluster nanostructure having a directionality of a partitioned bimetallic nanorod, a method for manufacturing the same, and a method for detecting a target material based on surface-enhanced Raman scattering (SERS) using the same.

Raman spectroscopy has been studied for detection and identification of pathogens. Specifically, surface-enhanced Raman scattering (SERS) has attracted great interest in spectroscopic detection and identification of small molecules, nucleic acids, proteins and cells mainly due to its ultra-high sensitivity, narrow bandwidth, and important multiplexing capabilities. (Non-patent document 1).

Over the past decade, anisotropic nanoparticles with distinct compartments have been developed as advanced nanomaterials due to their exceptional electronic, catalytic and photonic properties. Isotropic nanostructures with single-compartments have inherent limitations due to their symmetrically distributed physical properties and chemical functionality. Conversely, anisotropic nanostructures have discrete functionalities in each compartment as well as collective physicochemical properties due to the asymmetric distribution of substances and surface functionalities. They have a number of biomedical applications including drug delivery, high efficiency immunoassays, remote actuation devices, catalysis, surfactants, coatings, and water repellent materials. In addition, although it is very difficult to prepare them, they can exhibit directionality within individual nanostructures, which allows directional covalent- or non-covalent interactions between individual compartments of anisotropic nanoparticles. Compartment-selective self-assembly created by interparticle interactions can lead to very specific nanostructures with special properties and functions.

Metallic nanostructures include photonic and electronic systems, contrast agents in cancer treatment, SERS-activated platforms, photon-based imaging and tracking of tissues and cells, targeted release of drugs, antigens and DNA, detection and thermal removal of bacteria and cancer cells. It has been widely applied in a number of biomedical fields such as. Depending on the shape, size and surface functionality of the metal nanostructures, they have unique surface plasmon resonance properties, which are related to the collective excitation of electrons in the conduction band and are covered in a wide range from the visible to infrared (IR) range. . For example, the plasmon reaction of silver and gold nanostructures is strongly form-dependent because the highly localized charge polarization at its edges and boundaries leads to exceptional light scattering properties.

While the size and shape of a single metal nanostructure is an important factor controlling its photon- and electron reactions, the composition of an anisotropic double- or triple-metal nanostructure is also an important factor controlling its reaction. For example, bimetallic anisotropic nanoparticles provide a variety of optical properties compared to a single gold or silver nanostructure. The surface plasmon resonance (SPR) reaction of silver-gold nanoparticles is easily converted from the visible region to the near-infrared region depending on the gold or silver composition in each compartment. Compared to the two SPR peaks of Au nanoparticles with longitudinal mode and transverse mode, the gold core-silver shell nanoparticles have a longitudinal mode and a transverse mode and four plasmon modes that contribute to the two eight-pole modes. Indicated. Several methods have been reported for the synthesis of anisotropic metal nanoparticles, such as seed-mediated growth, sono-chemical preparation, co-reduction, biological template, thermal decomposition, and radiolysis. Various bimetallic core-shells, alloys and nanocomposites of different morphology and composition are produced by optimizing important parameters in the nucleation and growth stages, such as incubation time and temperature, solvent, ligand- and precursor-concentration, and sequence of experimental steps. Is reported. It has been reported that the introduction of different metal species into gold or silver nanoparticles affects morphology by adjusting the reduction rate of the metal precursor. For example, gold-silver bimetallic nanostructures have been formed from various metal seed nanoparticles. In addition, a growth mechanism for the production of bimetallic anisotropic nanoparticles has been recently studied. Single metal nanoparticles are obtained by a variety of different parameters such as reaction kinetics, surfactants, and by the addition of anionic or cationic molecules in the nucleation and growth steps, but the introduction of another metal as a seed to a single metal nanoparticle results in thermodynamics and It complicates the growth process by taking into account the kinetic factor. Significant advances have been made in the manufacture of several bimetallic nanoparticles, including nanosized polyhedra, multi-dimer, core-shell nanoparticles, tadpole-nanostructures, and nanorods (NR). However, there have been few reports of specific assemblies of bimetallic anisotropic nanoparticles via covalent- or non-covalent interactions, which can provide improved optical properties for a variety of biomedical applications.

As a highly sensitive, label-free optical detection technique, surface-enhanced Raman scattering (SERS) is suitable for in vitro and in vivo studies as it can non-destructively detect both solid and liquid analytes. In addition, it has the exclusive ability to multiplex the readings of different molecules using a single laser with engineered metal nanostructures. In SERS-based biosensing studies, one of the most basic challenges is to amplify Raman intensity. This can be achieved by forming hotspots between the metal nanostructures, because the hotspots respond to laser irradiation and generate a very enhanced electromagnetic field. In general, the presence of hot spots, which are random aggregates of nanostructures, produces various SERS signals because it is difficult to control the distance between the metal aggregates. In this regard, bimetallic anisotropic nanoparticles for directional clustering will be of great interest as improved SERS nanoprobes with highly controlled hot-shot generation and improved SERS strength.

In this study, we report the fabrication and characterization of a bimetallic Ag-Au-Ag nanorod cluster as a highly sensitive functional nanoprobe in SERS-based biosensing applications. The bimetallic Ag-Au-Ag NR was prepared via directional overgrowth from a gold octahedron functionalized as a seed. Directional clustering was achieved by non-covalent interactions through selective surface modification of the Au compartment of the bimetallic Ag-Au-Ag NR. As verified by zeta-potential, UV-Vis absorbance, dynamic light scattering, transmission electron microscopy and SERS analysis, they showed very improved physicochemical and optical properties compared to those of the individual bimetallic Ag-Au-Ag NR. . As proof of the concept that plasmon composition nanostructures are useful as SERS nanoprobes, we have shown that we have a sandwich-type immunocomplex consisting of antibody-conjugated SERS nanoprobes in the presence of biological moieties and magnetic beads (MB) as magnetic field-based separating agents. Showed the formation of. There was a linear correlation between Raman intensity and antigen concentration with minimum batch versus batch change. In conclusion, these bimetallic nanoclusters can be advantageous as highly sensitive advanced functional nanoprobes (nanoprobes) in photon-based biosensing applications.

Accordingly, the present inventors have developed a cluster nanostructure having the direction of the partitioned bimetallic nanorods.

K. Kneipp, Y. Wang, H. Kneipp, L. T. Perelman, I. Itzkan, R. R. Dasari, et al., "Single Molecule Detection Using Surface-Enhanced Raman Scattering (SERS)," Physical Review Letters, vol. 78, pp. 1667-1670, 03/03/1997.

It is an object of the present invention to provide a clustered nanostructure having the orientation of partitioned bimetallic nanorods.

Another object of the present invention is to provide a metal nanoprobe for measuring a surface-enhanced Raman scattering (SERS) image using a cluster nanostructure having a directionality of a partitioned bimetallic nanorod according to the present invention.

Another object of the present invention is to provide a method for producing a clustered nanostructure having a direction of partitioned bimetallic nanorods.

Another object of the present invention is to provide a method for detecting a target material based on surface-enhanced Raman scattering (SERS) using the metal nano probe.

The present invention is a cluster nanostructure with the orientation of the partitioned bimetallic nanorods consisting of:

A cluster nanostructure in which the bimetallic nanorods are oriented self-assembled into bimetallic nanoclusters by using a partitioned bimetallic nanorod composed of metal [A-B-A] as a seed,

Each of A and B is selected from the group consisting of silver, gold, copper, and mixtures thereof, and the A and B metals are not identical to each other.

The partitioned double metal nanorod refers to a nanorod-shaped double metal in which different metals A and B are in the form of A-B-A.

The directional cluster nanostructure refers to a clustered nanostructure of A-B-A type partitioned bimetallic nanorods in a specific direction through B metal-specific surface modification.

The B metal may have a surface modified with a carboxyl group.

The B metal is gold, and the A metal is a silver-gold-silver double metal nanorod composed of silver, and the B metal may be a polyhedral gold nanoparticle or a gold nanorod.

The polyhedral gold nanoparticles may be octahedral gold nanoparticles.

A Raman reporter may be attached to the double metal nanorod.

The Raman reporter means a Raman active organic compound, and any one that is widely used in the art may be used without limitation. Specific examples include MGITC (Malachite green isothiocyanate), RBITC (rhodamine B isothiocyanate), rhodamine 6G, adenine, 4-amino-pyrazole (3,4-d) pyrimidine, 2-luoroadenin, N6-benzoyl Adenine, kinetine, dimethyl-allyl-amino-adenine, zeatin, bromo-adenine, 8-aza-adenine, 8-azaguanine, 4-mercaptopyridine, 6-mercaptopurine, 4-amino- 6-mercaptopyrazolo(3,4-d)pyrimidine, 8-mercaptoadenin, 9-amino-acridine, and mixtures thereof may be selected from the group consisting of, but are not limited thereto.

In the present invention, the term "metal nanorod cluster" refers to a group of metal nanorods, and is a term generally used in this field.

In the present invention, the "double metal nanorod cluster" means that the double metal nanorod particles are gathered.

In another aspect, the present invention provides a metal nanoprobe for measuring a surface-enhanced Raman scattering (SERS) image using the partitioned bimetallic nanorod cluster nanostructure according to the present invention.

In the present invention, "probe" means a substance capable of specifically binding to a target (target) substance to be detected, and means a substance capable of confirming the presence of a target substance through the binding.

In the present invention, "nanoprobe" means a nano-sized probe.

The term "nano" includes a size range that is understood by those skilled in the art. Specifically, the size range may be 0.1 to 1000 nm, more specifically 10 to 1000 nm, more preferably 20 to 500 nm, and even more preferably 40 to 250 nm.

The present invention provides a method for producing a clustered nanostructure having the orientation of a bimetallic nanorod partitioned by the following steps:

i) preparing a plurality of partitioned bimetallic nanorod seeds composed of [A-B-A] by using the metal B whose surface is modified with a carboxyl group;

Ii) adding EDC (1-ethyl-3-(3-dimethylaminopropyl) carbodiimide) solution to activate the carboxyl group of the B metal compartment;

Iii) adding and stirring an amine-based compound after adding the EDC solution;

Iv) forming an amide bond between the EDC-activated carboxyl group of the B metal compartment and the amine group of the amine-based compound;

v) The amine compounds are bonded to each other by hydrophobic interactions between the alkyl groups of the amine compounds in the B metal compartment, so that the B metal compartments face each other, while the compartmentalized bimetallic nanorods form a cluster with directionality and self-assembly. felled;

In the manufacturing method comprising the step,

Each of A and B is selected from the group consisting of silver, gold, copper, and mixtures thereof, and the A and B metals are not identical to each other.

The activation of the carboxyl group in the B metal compartment means activating the exposed carboxyl group (remaining carboxyl group, a carboxyl group in a portion not connected to A) of the B metal compartment to bind with an amine group.

The B metal is gold, and the A metal is a silver-gold-silver double metal nanorod composed of silver, and the B metal may be a polyhedral gold nanoparticle or a gold nanorod.

The polyhedral gold nanoparticles may be octahedral gold nanoparticles.

After the step i), a step of attaching a Raman reporter to the surface of the double metal nanorod may be further included.

The amine compound may be selected from the group consisting of hexadecylamine, heptadecylamine, octadecylamine, nonadecylamine, and dodecylamine, but must be It is not limited to this.

The present invention provides a method for detecting a target substance based on surface-enhanced Raman scattering (SERS) comprising the following steps:

a) preparing a sample solution containing a target substance to be detected;

b) preparing the magnetic nanoparticles by immobilizing the first antibody against the target;

c) preparing by immobilizing a second antibody against the target on the metal nanoprobe according to the present invention;

d) adding the magnetic nanoparticles to which the first antibody is immobilized to the sample solution to form an immunocomplex in which the target and the first antibody of the magnetic nanoparticles are conjugated;

e) The metal nano-probe to which the second antibody is immobilized is added to the solution containing the immunocomplex to which the first antibody is conjugated to obtain a sandwich immunocomplex of the first antibody of the second antibody-target-magnetic nanoparticle. To form;

f) separating magnetic nanoparticles and metal nanoprobes that do not form the sandwich immunocomplex using a magnetic field; And

g) measuring the Raman signal of the sandwich immunocomplex;

step.

The target substance may be a protein or a pathogen.

The protein may be selected from the group consisting of antigens, biological aptamers, receptors, enzymes and ligands.

In the present invention, "sandwich-type immune complex" refers to an immunocomplex bound through an antibody-antigen (target)-antibody reaction. It was named as the antigen was inserted in the middle of the antibody to give it a sandwich shape.

In an embodiment of the present invention, a cluster nanostructure having the orientation of the partitioned bimetallic nanorods was made. Fabrication and characterization of bimetallic Ag-Au-Ag nanorods (NRs) and directional clusters thereof as a highly sensitive SERS nanoprobe in surface-enhanced Raman scattering (SERS)-based biosensing applications were analyzed. Bimetallic Ag-Au-Ag NRs were prepared via directional overgrowth from gold octahedrons functionalized as seeds. Directional clustering was achieved by non-covalent interactions through selective surface modification of the Au compartment of the bimetallic Ag-Au-Ag NRs. As verified by zeta-potential, UV-Vis absorbance, dynamic light scattering, transmission electron microscopy and SERS analysis, they showed very improved physicochemical and optical properties compared to those of individual bimetallic Ag-Au-Ag NRs. .

The cluster nanostructure having the directionality of the partitioned bimetallic nanorods according to the present invention can be applied as a metal nanoprobe for measuring a surface-enhanced Raman scattering (SERS) image with improved Raman strength.

1 is a schematic diagram of the synthesis of (A) gold octahedron and (B) gold nanorods, bimetallic Ag-Au-Ag NRs, and clusters thereof formed by directional self-assembly as seeds. (A) Bimetallic Ag-Au-Ag NRs (Type I) were prepared from the -COOH functionalized Au octahedron prepared by the polyol method through directional overgrowth of Ag and (B) -COOH functionalized Au NR Au rod seed-based Ag-Au-Ag bimetallic NRs (Type II) via overgrowth were also prepared. Bimetallic Ag- by directional self-assembly via hydrophobic interaction of ODA from MUA-modified seeds, activated by EDC and sulfo-NHS and modified with octadecylamine (ODA), with MUA-modified seeds. Au-Ag NR clusters were formed.
FIG. 2 is a (A, B) TEM image, (C) UV/Vis absorbance spectrum, and (D) of Au octahedron and clustered nanostructures thereof to investigate surface morphology, plasmon absorbance change, and average size. DLS profile.
3 is a TEM image at high magnification of (A) a bimetallic Ag-Au-Ag NR (Type I) having a single dark Au octahedral seed embedded in the body of the bimetallic NRs (Type I). (B) Energy-dispersive X-ray spectroscopy (EDS) map of the anisotropic properties of bimetallic NRs (type I) composed of Au and Ag. (CE) Mixed electron image with its elemental maps of green Au and red Ag.
Figure 4 shows (A, B) TEM images of individual bimetallic NR (Type I) and (C, D) HAADF (high angle annular dark field) images of clusters thereof -COOH functionalized Ag on the surface of Au octahedron Clearly indicates the asymmetric growth of The Au segment was selectively modified with both MGITC and ODA as Raman reporters to create hot spots in the gap between individual bimetallic NRs (Type I) through directional clustering, thereby substantially amplifying the SERS signal.
5 is (A) UV-Vis absorbance spectra, (B) DLS profiles, and (C) SERS spectra of MGITC-labeled bimetallic NRs and clusters thereof when MGITC concentration is 5.0×10 ⁻⁶ M.
Figure 6 (AF) HAADF images of (A, B) Au rod seed, (C, D) Au rod seed-based bimetallic NRs (Type II) and (E, F) clusters thereof. (G) UV-Vis absorbance spectra of ODA-induced clusters of Au NRs seeds and (H) SERS intensity of Au rod seed-based bimetallic NRs (Type II) and (E, F) clusters thereof.
Figure 7 (A) Schematic schematic diagram of a SERS-based immunoassay method for detection of IgG as a model protein using MGITC-labeled bimetallic NR (Type I). (B, C) Raman spectra as a function of IgG concentration for SERS-based quantitative analysis with a linear correlation between relative Raman intensity and IgG concentration.

Hereinafter, the present invention will be described in more detail in the following examples. The following examples of the present invention are for embodiing the present invention and are not intended to limit or limit the scope of the present invention. What can be easily inferred by those skilled in the art from the detailed description and examples of the present invention is construed as belonging to the scope of the present invention. In addition, references cited in the present invention are incorporated as part of the specification of the present invention.

<Example 1> Materials

Diethylene glycol (DEG), gold (III) chloride hydrate (HAuCl ₄ .3H ₂ O), silver nitrate (AgNO ₃ , ≥ 99.0%), polyvinylpyrrolidone (PVP) with a molecular weight of 55,000, octadecylamine ( ODA), ethanol (99.5%), aqueous ammonia (NH ₄ OH), 11-mercaptoundecanoic acid (MUA, 95%), sulfo-N-hydroxysuccinimide ester (sulfo-NHS) and phosphate buffered saline (PBS) was purchased from Sigma-Aldrich (St. Louis, Missouri, USA). Malachite green isothiocyanate (MGITC) was obtained from Invitrogen (Grand Island, NY, USA). 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC) was purchased from Thermo Scientific (Rockford, Illinois, USA). All glassware was washed with aqua Regia (HCl: HNO ₃ , 3:1) and then thoroughly rinsed with deionized water. Deionized water purified by Milli-Q (Millipore Water Purification Systems; EMD Millipore, Bedford, Massachusetts, USA) was used.

<Example 2> Preparation of gold octahedron and gold nanorods as seeds

Gold octahedron as a seed was prepared by slightly modifying the previously reported steps (Seo, D., Yoo, CI, Chung, IS, Park, SM, Ryu, S., & Song, H. (2008). The Journal of Physical Chemistry C, 112(7), 2469-2475.) PVP solution (3.0 g, 27.0 mmol for monomer) was dissolved in 13 mL DEG. The PVP solution was stirred at room temperature for 2 hours and heated for 10 minutes with vigorous stirring to reflux. A solution of HAuCl ₄ (10.05 mg, 0.025 mmol) in DEG (1.0 mL) was added rapidly, and the solution immediately turned dark brown blue. The solution was allowed to stand under stirring at room temperature for 15 minutes to obtain a homogeneous octahedron. The Au octahedral solution was mixed with 35 mL ethanol and purified by centrifugation at 13,000 rpm for 15 minutes. This process produced an Au octahedron having an average edge length of 42 nm with a yield of about 92% shape. In order to modify the surface of the gold octahedron with a carboxyl group, MUA was introduced. 1.5 mL of a 10 mM solution of MUA in ethanol was mixed with the Au octahedron in ethanol and the mixed solution was kept at room temperature. Au octahedron was washed twice with ethanol and resuspended in DEG.

Au nanorods (NR) as seeds were prepared by MUA modification as previously reported (Seo, D., Yoo, CI, Jung, J., & Song, H. (2008). Journal of the American Chemical Society , 130(10), 2940-2941.). 1.5 mL of PVP (0.13 mM) and 1.5 mL of HAuCl ₄ (1.0 × 10 ^-4 M) dissolved in the DEG solution were periodically added every 30 seconds for 3.8 minutes to the octahedral seed dispersion in DEG, then the mixture was added to 245. ^Refluxed at ^° C for 60 minutes. 5 ml ethanol was added to the reaction mixture and purified by centrifugation at 13,000 rpm for 15 minutes. Au NRs were incubated with 10 ^-5 M MUA, sonicated for 10 minutes, and purified by centrifugation at 13,000 rpm for 15 minutes.

<Example 3> Preparation of double metal Ag-Au-Ag NRs

To prepare bimetallic Ag-Au-Ag NRs using gold octahedron and NRs as seeds, 1.5 mL PVP in PEG (0.13 mM) and 1.5 mL AgNO _{3 in} DEG (3.4 × 10 ^-2 M) 2.5 mL Gold octahedron or NRs seeds in DEG of were added dropwise every 30 seconds over 3.7 minutes at a boiling temperature of 245°C. The reaction mixture was refluxed for 1 hour, cooled to room temperature, and then 10 mL ethanol was added for purification. Homogeneous bimetallic Ag-Au-Ag NRs were separated from large aggregates by centrifugation at 500 rpm for 5 minutes, washed with ethanol by precipitation and dispersion cycles for 15 minutes at 12,000 rpm, and finally in 15 mL ethanol. Dispersed.

<Example 4> Clustering of double metal Ag-Au-Ag NR

As a Raman reporter, MGITC was introduced into bimetallic Ag-Au-Ag NRs and used for SERS characterization, and then directional self-assembly into clustered bimetallic Ag-Au-Ag NRs was achieved through non-covalent interactions. By centrifugation at 12,000 rpm for 15 minutes, 5 mL of bimetallic Ag-Au-Ag NRs were transferred to 5 mL of deionized water. A colloidal solution of bimetallic Ag-Au-Ag NRs was added to the freshly prepared MGITC at a concentration ranging from 10 ^-5 M to 10 ^-8 M, and left under stirring for 1 hour. In addition, to covalently attach ODA with a long hydrophobic alkyl group to the Au compartment of the bimetallic Ag-Au-Ag NRs through selective functionalization, 0.5 mM EDC was added to the bimetallic Ag-Au-Ag NRs solution. , Stirred for 1 hour to activate the remaining carboxyl groups on the Au compartment. Concentration of the final ODA solution in 10 ^-8 M THF was introduced dropwise into the EDC-activated bimetallic NRs solution and stirred for 1 hour to amide bonds between the carboxyl functional groups of the Au compartment of the bimetallic NRs and the amine functional groups of the ODA Formed. The color of the reaction solution changed from dark yellow to reddish yellow due to the formation of ODA-mediated clusters of the bimetallic NR through hydrophobic interactions. Finally, the reaction solution was purified by centrifugation at 10,000 rpm for 15 minutes and resuspended in deionized water or PBS for later use.

<Example 5> Double metal Ag-Au-Ag NRs and characterization of clusters thereof

To determine the colloidal properties of gold octahedron, bimetallic Ag-Au-Ag NRs and clusters thereof, a Zeta sizer Nano ZS instrument equipped with a Ne-He laser with a wavelength of 633 nm and a scattering angle at 90 ° ( Dynamic light scattering (DLS) and zeta potential measurements were performed using Malvern Instruments, Malvern, UK). The sample was diluted 10:1 with deionized water and the temperature was adjusted to 25°C. Its UV-Vis plot is from 300 nm to 800 nm using a fixed slit width of 1 nm in a single 10 scan mode with an intermediate scan rate at 25°C by a UV-visible spectrometer (UV-1800, Shimatsu, Japan). Obtained at wavelength. The baseline was adjusted using two empty cells filled with deionized water and their average size was obtained from at least 20 scan cycles. In addition, zeta-potential measurements were performed to determine the surface charge in deionized water. Transmission electron microscopy (TEM) images were obtained using JEM-2100F FE-STEM (JEOL, Germany) operating at an acceleration voltage of 80 to 200 kV, and nano-sized individual bimetallic Ag-Au-Ag NR and clusters thereof Indicated. Samples were deposited on a 400 mesh copper grid (Ted Pella, Inc. USA) coated with an ultra-thin layer of carbon. To characterize the diameter, diameter distribution, and surface morphology by scanning electron microscopy (SEM), several drops of sample were placed on silicon, dried, and coated with platinum sputtering using a turbo sputter coater (K575X). Imaged using SEM VEGA (TESCAN, USA) operating at an acceleration voltage of 0.5 kV to 30 kV. In addition, all Raman characterization was performed using a Renishaw inVia Raman microscope system (Renishaw, UK) equipped with a Reinshaw He-Ne laser, operating at a wavelength (λ) of 632.8 nm for an excitation source with a laser power of 12.5 mW. Performed. Rayleigh lines were removed from the collected SERS profiles using a holographic notch filter placed on the collection filter. Raman scattering was obtained using a charge-coupled device (CCD) camera with a spectral resolution of 1 cm ^-1 and all SERS spectra were corrected with 520 cm ^-1 silicon lines. The colloidal solution of the nanoparticles labeled with MGITC was mixed with a small glass capillary tube (Kimble Chase, ordinary capillary tube, soda lime glass , inner diameter: 1.1-1.2 mm, Wall: 0.2 ± 0.02 mm, length: 75 mm). SERS spectra were collected during the exposure time of 1 second used, at which time the laser spot was focused on the glass capillary in the wavelength range of 608 cm ^-1 to 738 cm ^-1 using a 20x objective lens.

1 shows a schematic diagram of the synthesis of gold octahedron or gold nanorods as seeds for SERS-based biosensing applications, bimetallic Ag-Au-Ag NRs, and clusters thereof formed by directional self-assembly. Bimetallic Ag-Au-Ag NRs were prepared by directional overgrowth from -COOH functionalized Au octahedron or Au nanorods. The directional clustering of the bimetallic Ag-Au-Ag NRs was induced by non-covalent non-covalent via selective surface modification of the Au compartment. The bimetallic NR was prepared using MUA-functionalized Au octahedron or Au NR, and Ag salt and PVP as precursor and capping agent of silver shell. Double-metal Ag-Au-Ag NRs were formed by simultaneously growing Ag nanostructures in both directions on the surface of Au octahedron or Au NRs. The gold segment of the bimetallic NR was selectively functionalized through the formation of an amide bond between the residual carboxyl group of the Au seed and the primary amine group of the ODA using ODA with long hydrophobic alkyl groups. ODAs on the surface Au compartment of the bimetallic NR were bonded to each other by hydrophobic interactions, so that the bimetallic NRs were oriented and self-assembled into bimetallic NR clusters. As shown in the Raman spectrum, the bimetallic NR cluster showed a very improved Raman intensity compared to the single bimetallic NR due to an increase in hotspot junctions between them.

2 shows TEM images, UV/Vis absorbance spectra, and DLS profiles of Au octahedron and clustered nanostructures thereof to investigate surface morphology, plasmon absorbance changes, and average size. As a seed, an Au octahedron was prepared, as reported, by the technique adopted in the polyol method (Seo, D., Yoo, CI, Chung, IS, Park, SM, Ryu, S., & Song, H. (2008).. The Journal of Physical Chemistry C, 112(7), 2469-2475.). The reaction was carried out in DEG because DEG has a relatively high boiling point of 245° C. and has sufficient polarity to dissolve gold salts and PVP. To clearly demonstrate the ODA-induced clustering of metal nanostructures, the Au octahedron was functionalized through an amide bond between the residual carboxyl group of the Au seed and the primary amine group of the ODA using ODA with long hydrophobic alkyl groups. ODAs on the Au seed surface were bonded to each other by hydrophobic interaction to form gold octahedral clusters. TEM images of the Au octahedron and its clusters show that they are uniform in both shape and size. The average edge length of the Au octahedron was found to be 34 ± 8 nm by analysis of 50 randomly selected nanoparticles in the TEM image. The UV/Vis absorbance spectrum of the Au octahedron of FIG. 2(C) shows two characteristic peaks at 550 nm and 610 nm, which represent polar-positive plasmon mode and oriented bipolar plasmon mode, respectively. When they were clustered, the two plasmon bands shifted toward red with a decrease in absorbance at longer wavelengths. Table 1 shows the zeta potential and DLS values of the gold octahedron as seeds and seed clusters. The thiol group of MUA was bonded to the gold surface through a covalent bond, whereas the carboxyl group of MUA showed the anionic properties of the gold octahedron. The zeta potential value of the gold octahedron was -1.9 ± 0.5 mV, and the zeta potential value of the MUA-functionalized gold octahedron was-19.2 ± 0.5 mV due to the presence of -COOH on the surface of the gold seed. Because ODA was covalently conjugated with -COOH on the surface of the gold seed by EDC/NHS chemistry, the ODA-modified gold octahedron was clustered through the hydrophobic interaction of the ODA moiety. The size of the gold octahedron cluster increased significantly from 33.0 nm to 50.3 nm of the gold octahedron, and its zeta potential also increased significantly to -9.4 ± 0.5 mV. In addition, the size distribution of both the gold octahedron and its clusters was uniform as shown in Fig. 2(D).

The bimetallic Ag-Au-Ag NRs (Type I) were prepared through directional overgrowth of Ag from MUA-functionalized, multi-twinned Au octahedron as a seed in DEG as a reducing agent. It was heated to a boiling point of 245 °C. Bimetallic NRs (Type I) were formed by periodically adding 2 equivalents of Ag salt and PVP (0.13mM) to the seed dispersion relative to the gold concentration in DEG (1.7×10 ⁻² M). As shown in Fig. 3(A), its TEM image at high magnification showed a single dark Au octahedral seed embedded in the body of the double metal NR (Type I). In addition, the energy-dispersive X-ray spectroscopy (EDS) map of FIG. 3(B) shows the anisotropic properties of the bimetallic NR (Type I) composed of Au and Ag. The mixed electron image of FIG. 3(CE) clearly shows its elemental map, which is green Au and red Ag.

As shown in Figure 4, (A, B) TEM images of individual bimetallic NR (Type I) and (C, D) HAADF images of clusters thereof are asymmetric growth of Ag on the surface of -COOH functionalized Au octahedron Clearly indicate. As a Raman reporter, MGITC was introduced into the colloidal bimetallic NR solution so that the isothiocyanate groups (-N〓C〓S) of MGITC were selectively bound to the surface of the bimetallic NR (type I). In addition, ODA was added to the MGITC-labeled bimetallic NR solution, and residual carboxyl functional groups from the Au octahedral seed were chemically attached by amide coupling between the EDC-activated carboxyl functional group and the primary amine functional group of the ODA. . The directional clustering of the bimetallic NR was made through hydrophobic interactions between the hydrophobic long alkyl groups of the ODA and the Au compartments of the bimetallic NR were made to face each other. As the ODA concentration varied, its degree of self-assembly was adjusted by its corresponding hydrophobicity.

5 shows (A) UV-Vis absorbance spectrum, (B) DLS profile, and (C) SERS spectrum of MGITC-labeled bimetallic NR and clusters thereof when the MGITC concentration is 5.0 × 10 ^-6 M. The bimetallic NR (Type I) exhibits two different UV-Vis absorbance peaks at 420 nm and 510 nm, respectively, indicating the transverse mode of the silver and gold compartments. Due to the low concentration, there was no significant difference in UV-Vis absorbance spectra in the presence or absence of the introduced Raman reporter. The plasmon band shifted to red with an increase in absorbance at a longer wavelength after cluster formation. In addition, to characterize the size, size distribution, and colloidal stability of MGITC-labeled bimetallic NR and its clusters under aqueous conditions, dynamic light scattering (DLS) measurements were performed. As shown in FIG. 5(B), the average diameter of the double metal NR was 34.58 nm in diameter and 252.10 nm in average length. On the other hand, its clusters showed only one size distribution at 279.80 nm, because their directional clustering produced similar diameters and lengths with an aspect ratio of 1.0. The zeta potential values of the MGITC-labeled bimetallic NR and its clusters under aqueous conditions (Table 1) were-18 mV-12 mV, respectively.

[Table 1]

Compared to the bimetallic NR, its clustered size and zeta potential value increased significantly due to ODA conjugation. When self-assembled with orientation, the bimetallic NR cluster was prepared by selectively functionalizing the Au compartment with ODA, and the SERS strength at 1617 cm ^-1 was greatly improved by about 10 times compared to that of the individual, and the reason is that the bimetallic NRs This is because the electromagnetic field of the hot spot formed in the gap between them is amplified.

To apply ODA-mediated directional clustering of bimetallic NR to a new type of bimetallic NR, Au rod seed-based Ag-Au-Ag bimetallic NRs (type II) were prepared. As mentioned above for the synthesis of double metal NRs (type I), Au rods as seeds were prepared by the polyol method using PVP and HAuCl ₄ as capping agents, and Au rods seed-based bimetallic NRs (type II) were Prepared via directional overgrowth of Ag from MUA-functionalized Au NR seeds. 6 shows HAADF images of (AF) (A, B) Au rod seeds, (C, D) Au rod seeds-based bimetallic NR (Type II) and (E, F) clusters thereof. The Au rod was synthesized to have a uniform size and shape. The average length of the Au bars was found to be 50 ± 8 nm as determined by analyzing 50 randomly selected nanoparticles in the HAADF image. Au rod seed-based bimetallic NRs (Type II) represented a single dark object buried within the entire body, which was clearly observed at high magnification, and its average length was determined by the measurement of 50 objects in the HAADF image. By 100 ± 10 nm. As shown in the directional cluster of the bimetallic NR (Type I), the directional clustering of the Au rod seed-based bimetallic NR (Type II) is also a hydrophobic interaction between the hydrophobic long alkyl groups of ODA on the surface of the Au NR seed. Was done through. Likewise, ODA-induced clustering of Au NRs seeds was also demonstrated by UV-Vis absorbance spectra as shown in Figure 6(G). Multiple hotspots of bimetallic NR (Type II) clusters created gaps between them through directional clustering, substantially amplifying the SERS intensity compared to the individual ones as shown in Fig. 6(H). Thus, these clustered nanostructures of bimetallic NRs, including type I and type II, are a new class of SERS nanoprobes for biodetection.

<Example 6> Bimetallic NR cluster and bioconjugation of magnetic beads (MB) and antibody

The bimetallic NR cluster is chemically bound to the anti-human IgG pAb using the carboxyl group of the MUA molecule present on the cluster surface, and the anti-human IgG mAb is conjugated to the magnetic beads (MB), thereby interacting with the antibody-antigen-antibody. A sandwich-type immunocomplex was formed through, and at this time, IgG as an antigen was present in the solution. In a representative experiment, 10 ml of 0.2 mg/mL EDC was added to the bimetallic NR cluster in PBS at pH 7.4, left under stirring for 1.5 hours, and then 10 ml of a 0.50 mg/ml solution of anti-human IgG-pAb was added to EDC. -Added activated bimetallic NR cluster and stirred for an additional 1.5 hours. Finally, anti-human IgG pAb-conjugated, bimetallic NR clusters as SERS nanoprobes were purified with PBS using centrifugal force at 5,000 rpm. Similarly, magnetic nanoparticle-encapsulated polymeric nanoparticles as magnetic beads (MB) were chemically conjugated to anti-human IgG mAb using residual carboxyl functional groups on MB after heat stabilization as reported. In a representative experiment, 1.25 mg MB was introduced into 2.0 mL PBS and sonicated for 1 minute at 30% amplitude using a tip sonicator (VC 505 Sonics and Materials Inc.). MB solution was incubated with 100 ml of 3.5 mg/ml EDC and 100 μl of 3.5 mg/ml sulfo-NHS in PBS at a final concentration of 1.766 M EDC and sulfo-NHS. Unbound anti-human IgG mAb was removed using magnetic force and the solution was resuspended in PBS for biosensing of the IgG antigen.

<Example 7> SERS-based biosensing of IgG antigen

MB with anti-human IgG mAb was used to bind IgG as antigen in PBS, while double metal NR cluster with anti-human IgG pAb was used as SERS nanoprobe to search for IgG through formation of sandwich-type immunocomplex. I did. First, anti-human IgG mAb-bound MB was introduced into the IgG solution at concentration ranges of 410 ng/mL and 40 pg/mL. The immunocomplex between anti-human mAb-conjugated MB and IgG was separated by applying a magnetic force, and the solution was purified twice with PBS and then resuspended in PBS. Second, the prepared anti-human mAb-conjugated MB and the immunocomplex of IgG was mixed with the anti-human IgG mAb-conjugated bimetal NR cluster and stirred for 1 hour to obtain anti-human IgG through specific molecular interactions. Sandwich-type immunocomplexes were generated in pAb, IgG, and anti-human IgG mAb. Sandwich-type immunocomplexes in anti-human IgG pAb, IgG, and anti-human IgG mAb were isolated using magnetic force and suspended for SERS recording. Nonspecific binding was assessed by conducting two control studies without antibody conjugation of both the IgG and the MB and bimetallic NR clusters.

7(A) shows a schematic diagram of a SERS-based immunoassay method for detection of IgG as a model protein. First, anti-human IgG mAb conjugated magnetic bead solutions were added to solutions with different IgG concentrations. The IgG analyte was selectively captured with its corresponding magnetic beads through specific molecular interactions, purified by applying an external magnetic field, and resuspended in PBS. Second, anti-human IgG pAb conjugated bimetal NR cluster solution for SERS-based biosensing was added to a solution of complexed anti-human IgG mAb conjugated magnetic beads and IgG, and anti- A sandwich type immunocomplex consisting of human IgG mAb conjugated magnetic beads, IgG and anti-human IgG pAb conjugated bimetallic NR clusters was formed. Subsequently, the unattached SERS nanoprobe was removed by applying a magnetic field, and the resulting sandwich-type immunocomplex was resuspended in PBS. Finally, Raman intensity was obtained as a function of IgG-concentration for SERS-based quantitative analysis, and there was a linear correlation between the relative Raman intensity and the IgG concentration as shown in Figures (B, C). As the IgG concentration increased from 410 ng/mL to 0.041 ng/mL, the Raman intensity of the MGITC-labeled bimetallic NR cluster increased linearly with the concentration of IgG due to the increase in the concentration of the sandwich-type immunocomplex ( R ² = 0.937) increased.

The present invention has demonstrated that the double metal NR cluster exhibits superior SERS activity compared to the single double metal NR. The platform method we developed for the synthesis of bimetallic NRs (types I, II) and clusters thereof would be beneficial for synthesizing controlled bimetallic anisotropic nanostructures and clusters thereof composed of several metals of various sizes and compositions.

Claims (13)

A cluster nanostructure in which the bimetallic nanorods are directionally self-assembled into a bimetallic nanocluster, using a partitioned bimetallic nanorod composed of metal [ABA] as a seed,
The A and B are each selected from the group consisting of silver, gold, copper, and mixtures thereof, and the A and B metals are not identical to each other,
The bimetallic nanorods are self-assembled in a direction while facing each other in the B metal compartments,
Cluster nanostructures with the orientation of partitioned bimetallic nanorods.
The method of claim 1,
The B metal is a cluster nanostructure having a directionality of a partitioned bimetallic nanorod, whose surface is modified with a carboxyl group.
The method of claim 1,
The B metal is gold and the A metal is a silver-gold-silver double metal nanorod composed of silver,
The B metal is a polyhedral gold nanoparticle or a gold nanorod, a cluster nanostructure having the orientation of a partitioned bimetallic nanorod.
The method of claim 3,
The polyhedral gold nanoparticles are octahedral gold nanoparticles, clustered nanostructures having the orientation of partitioned bimetallic nanorods.
The method of claim 1,
Cluster nanostructures having the direction of the partitioned bimetallic nanorods to which a Raman reporter is attached to the bimetallic nanorods.
Surface-enhanced Raman scattering (SERS) using the cluster nanostructures of claim 5, metal nano-probe for image measurement.
i) preparing a plurality of partitioned bimetallic nanorod seeds composed of [ABA] by using the metal B whose surface is modified with a carboxyl group;
Ii) adding EDC (1-ethyl-3-(3-dimethylaminopropyl) carbodiimide) solution to activate the carboxyl group of the B metal compartment;
Iii) adding and stirring an amine-based compound after adding the EDC solution;
Iv) forming an amide bond between the EDC-activated carboxyl group of the B metal compartment and the amine group of the amine-based compound;
v) The amine compounds are bonded to each other by hydrophobic interactions between the alkyl groups of the amine compounds in the B metal compartment, so that the B metal compartments face each other, while the compartmentalized bimetallic nanorods form a cluster with directionality and self-assembly. felled;
In the manufacturing method comprising the step,
The A and B are each selected from the group consisting of silver, gold, copper, and mixtures thereof, and the A and B metals are not the same as each other,
Method for producing a clustered nanostructure with the direction of the partitioned bimetallic nanorods.
The method of claim 7,
The B metal is gold and the A metal is a silver-gold-silver double metal nanorod composed of silver,
The B metal is a polyhedral gold nanoparticle or a gold nanorod, a method of manufacturing a cluster nanostructure having a direction of a partitioned bimetallic nanorod.
The method of claim 7,
The polyhedral gold nanoparticles are octahedral gold nanoparticles, a method of manufacturing a cluster nanostructure having a direction of a partitioned bimetallic nanorod.
The method of claim 7,
After the step i), further comprising the step of attaching a Raman reporter to the surface of the double metal nanorods, the method of producing a cluster nanostructure having the direction of the partitioned double metal nanorods.
The method of claim 7,
The amine-based compound is selected from the group consisting of hexadecylamine, heptadecylamine, octadecylamine, nonadecylamine, and dodecylamine. Method for producing a clustered nanostructure with the orientation of the double metal nanorods.
a) preparing a sample solution containing a target substance to be detected;
b) preparing the magnetic nanoparticles by immobilizing the first antibody against the target;
c) preparing by immobilizing a second antibody against the target to the metal nanoprobe of claim 6;
d) adding the magnetic nanoparticles to which the first antibody is immobilized to the sample solution to form an immunocomplex in which the target and the first antibody of the magnetic nanoparticles are conjugated;
e) The metal nano-probe to which the second antibody is immobilized is added to the solution containing the immunocomplex to which the first antibody is conjugated to obtain a sandwich immunocomplex of the first antibody of the second antibody-target-magnetic nanoparticle. To form;
f) separating magnetic nanoparticles and metal nanoprobes that do not form the sandwich immunocomplex using a magnetic field; And
g) measuring the Raman signal of the sandwich immunocomplex;
Including the step of, surface-enhanced Raman scattering (SERS)-based target material detection method.
The method of claim 12,
The target material is a protein or a pathogen, surface-enhanced Raman scattering (SERS)-based target material detection method.

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