KR20190034110A - 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 directionally clustered nanostructure of compartmentalized bimetal nanorods, a method for manufacturing the same, and a surface-enhanced Raman scattering (SERS)-based target substance detection method using the same. The nanostructure of the present invention can be applied as a metal nanoprobe for measuring a SERS image.

Description

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a clustered nanostructure having a directional cluster of double metal nanorods,

The present invention relates to clustered nanostructures having directionality of segmented double metal nanorods, a method for producing the same, and a surface-enhanced Raman scattering (SERS) -based target substance detection method using the same.

Raman spectroscopy has been studied for detection and identification of pathogens. In particular, surface-enhanced Raman scattering (SERS) has received great interest in spectroscopic detection and identification of small molecules, nucleic acids, proteins and cells, primarily due to its ultra-high sensitivity, narrow bandwidth and important multiplexing capabilities (Non-Patent Document 1).

Over the last decade, anisotropic nanoparticles with distinct compartments have been developed as state-of-the-art nanomaterials due to their exceptional electron, catalyst and photon properties. Isotropic nanostructures with single-compartments have inherent limitations due to their symmetrically distributed physical properties and chemical functionality. In contrast, anisotropic nanostructures have discrete functionality in each compartment as well as collective physicochemical properties due to asymmetric distribution of material and surface functionality. They have a number of biomedical applications, including drug delivery, high-efficiency immunoassays, remote actuators, catalysis, surfactants, coatings, and water repellents. In addition, although they are very difficult to produce, they can exhibit directionality within individual nanostructures, which allows directional covalent or noncovalent interactions between individual compartments of anisotropic nanoparticles. Partition-selective self-assembly generated by intergranular interaction can lead to very special nanostructures with special properties and functions.

Metal nanostructures can be used in photonic and electronic systems, contrast agents in cancer therapy, SERS-active platforms, photon-based imaging and tracking of tissues and cells, targeted release of drugs, antigens and DNA, detection and thermal removal of bacterial and cancer cells Has been widely applied in a number of biomedical fields. Depending on the morphology, size and surface functionality of the metal nanostructures, they have unique surface plasmon resonance properties, which are related to the excitation of the conduction band electrons and are included in a wide region from the visible to the infrared (IR) range . For example, the plasmonic response of silver and gold nanostructures is strongly shape-dependent because highly localized charge polarizations at their edges and boundaries lead to exceptional light scattering properties.

While the dimensions and morphology of single metal nanostructures are important factors in regulating their photon-and electron responses, the composition of anisotropic double- or triple-metal nanostructures is also an important factor controlling its response. For example, double metal anisotropic nanoparticles provide a variety of optical properties over single gold or silver nanostructures. The surface plasmon resonance (SPR) response of silver - gold nanoparticles is easily transformed from the visible to the near infrared region according to the gold or silver composition in each compartment. Compared to the two SPR peaks of Au nanoparticles with longitudinal and transverse modes, the gold core-silver shell nanoparticles have four plasmon modes contributing to the longitudinal and transverse modes and two 8-pole modes Respectively. Several methods for the synthesis of anisotropic metal nanoparticles have been reported, such as seed-mediated growth, sono-chemical production, co-reduction, biological templates, thermal degradation, and radiation degradation. Shells, alloys and nanocomposites having different shapes and compositions by optimizing critical parameters in the nucleation and growth stages, such as incubation time and temperature, solvent, ligand- and precursor-concentration, and order of experimental steps Reported. It has been reported that incorporation of different metal species into gold or silver nanoparticles affects morphology by adjusting the rate of reduction of the metal precursor. For example, gold-silver double metal nanostructures have been formed from a variety of metal seed nanoparticles. In addition, a growth mechanism for the production of double metal anisotropic nanoparticles has been recently studied. Although single metal nanoparticles are obtained by a variety of different parameters, such as reaction kinetics, surfactants, and by the addition of anion or cation molecules in the nucleation and growth steps, the introduction of another metal as a seed into single metal nanoparticles can lead to thermodynamic and / It complicates the growth process considering the dynamic factors. There have been considerable advances in making some double metal nanoparticles, including nanosized polyhedra, multi-dimers, core-shell nanoparticles, tadpole-nanostructures, and nanorods (NR). However, there have been few reports of specific assemblies of bi-metal anisotropic nanoparticles through covalent or non-covalent interactions that can provide improved optical properties for a variety of biomedical applications.

Surface-enhanced Raman scattering (SERS) as a highly sensitive, unlabeled optical detection technique is suitable for both in vitro and in vivo studies because it can detect both solid and liquid analytes non-destructively. It also has the exclusive ability to multiplex readings of different molecules using a single laser with engineered metal nanostructures. In a SERS-based biosensing study, one of the most basic challenges is to amplify the Raman intensity. This can be achieved by forming a hot spot between the metal nanostructures, since the hot spot creates a highly enhanced electromagnetic field in response to laser irradiation. Generally, the presence of a hotspot, a random aggregate of nanostructures, produces a variety of SERS signals because it is difficult to control the distance between metal agglomerates. In this regard, double metal anisotropic nanoparticles for directional clustering will be of great interest as an enhanced SERS nanoprobe with highly controlled hot-swath generation and improved SERS intensity.

In this study, we report the fabrication and characterization of dual metal Ag-Au-Ag nanorod clusters as highly sensitive functional nanoprobes in SERS-based biosensing applications. A double metal Ag-Au-Ag NR was prepared through directional overgrowth from a functionalized gold-ten-sided body as a seed. Directional clustering was achieved by non-covalent interactions through selective surface modification of the Au compartments of the double metal Ag-Au-Ag NR. As evidenced by zeta-dislocation, UV-Vis absorbance, dynamic light scattering, transmission electron microscopy and SERS analysis, they exhibited greatly improved physicochemical and optical properties compared to those of the individual double metal Ag-Au-Ag NR . As a proof of the concept that plasmonic nanostructures are useful as SERS nanoprobes, we have developed a sandwich immunoconjugate composed of magnetic beads (MB) as antibody-conjugated SERS nanoprobes and magnetic field-based separator in the presence of biological moieties . There was a linear correlation between Raman intensity and antigen concentration with minimum batch to batch change. In conclusion, these double-metal nanoclusters can be advantageous as highly sensitive, advanced functional nanoprobes (nanoprobes) in photon-based biosensing applications.

Accordingly, the present inventors have developed cluster nanostructures having directionality of segmented double metal 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 cluster nanostructures having directionality of segmented double metal nanorods.

Another object of the present invention is to provide a metal nanoprobe for measuring surface-enhanced Raman scattering (SERS) images using clustered nanostructures having directionality of segmented double metal nanorods according to the present invention.

It is still another object of the present invention to provide a method for producing cluster nanostructures having directionality of segmented double metal nanorods.

It is still another object of the present invention to provide a surface-enhanced Raman scattering (SERS) -based target substance detection method using the metal nanoprobe.

The present invention relates to a clustered nanostructure having directionality of segmented double metal nanorods comprising:

Cluster nanostructures in which the double-metal nanorods are self-assembled in a directionally bi-metallic nanoclust, with a segmented double-metal nanorods composed of a metal [A-B-A]

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

The segmented double metal nanorods refer to nanorodular double metal in which A and B, which are different metals, are in the form of A-B-A.

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

The B metal may be one whose surface has been modified with a carboxyl group.

The B metal is gold and the A metal is silver-gold-silver double metal nanorods composed of silver, and the B metal may be polyhedral gold nanoparticles or gold nanorods.

The polyhedral gold nanoparticles may be tetrahedral gold nanoparticles.

Raman reporters may be attached to the double metal nanorods.

The Raman reporter means a Raman active organic compound, and any of them can be used without limitation as long as it is widely used in the technical field. Specific examples thereof include malachite green isothiocyanate (MGITC), rhodamine B isothiocyanate (RITC), rhodamine 6G, adenine, 4-amino-pyrazole (3,4-d) pyrimidine, 2-fluoroadenine, Amino-adenine, zeatin, bromo-adenine, 8-aza-adenine, 8-azaguanine, 4-mercaptopyridine, 6-mercaptopurine, But are not necessarily limited to, 6-mercapto-pyrazolo (3,4-d) pyrimidine, 8-mercaptoadenine, 9-amino-acridine and mixtures thereof.

In the present invention, the term " metal nanorod cluster " is a term commonly used in this field to mean that metal nanorods are gathered.

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

In another aspect, the present invention provides metal nanoprobes for surface-enhanced Raman scattering (SERS) image measurement using segmented double metal nanorod cluster nanostructures according to the present invention.

In the present invention, the term " probe " means a substance capable of specifically binding to a target substance to be detected, and means a substance that can confirm the presence of a target substance through the binding.

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

The " nano " includes a size range to the extent understood by those of ordinary skill in the art. Specifically, the size range may be from 0.1 to 1000 nm, more specifically from 10 to 1000 nm, more preferably from 20 to 500 nm, even more preferably from 40 to 250 nm.

The present invention provides a method for preparing clustered nanostructures having directionality of a double metal nanorods partitioned into the following steps:

i) preparing a plurality of segmented Q double metal nanorodic seeds composed of [A-B-A] using a metal B surface-modified with a carboxyl group;

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

Iii) adding the EDC solution and then adding an amine compound and stirring;

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

v) In the B metal section, the amine compound is bonded to each other by the hydrophobic interaction between the alkyl groups of the amine compound, so that the B metal compartments face each other while the partitioned double metal nanorods form a directional cluster, felled;

≪ / RTI >

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

Activation of the carboxyl group of the B metal segment means activating the exposed carboxyl group of the B metal segment (carboxyl group of the portion not linked to A) with an amine group.

The B metal is gold and the A metal is silver-gold-silver double metal nanorods composed of silver, and the B metal may be polyhedral gold nanoparticles or gold nanorods.

The polyhedral gold nanoparticles may be tetrahedral gold nanoparticles.

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

The amine compound may be selected from the group consisting of hexadecylamine, heptadecylamine, octadecylamine, nonadecylamine, and dodecylamine. But is not limited thereto.

The present invention provides a surface-enhanced Raman scattering (SERS) -based target material detection method comprising the steps of:

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

b) immobilizing the magnetic nanoparticles with the first antibody against the target;

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

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

e) adding the second antibody-immobilized metal nanoprobe to a solution containing the immune complex to which the first antibody is conjugated to form a sandwich immunoconjugate of the first antibody of the metal nano-probe second antibody-target-magnetic nanoparticle Forming;

f) using magnetic fields to separate the magnetic nanoparticles and the metal nanoprobes that did not form the sandwich immunoconjugate; And

g) measuring the Raman signal of said sandwich immunoconjugate;

step.

The target material may be a protein or a pathogen.

The protein may be selected from the group consisting of an antigen, a biological aptamer, a receptor, an enzyme and a ligand.

In the present invention, the term "sandwich-type immunoconjugate" means an immunoconjugate bound through an antibody-antigen (target)-antibody reaction. Antigen was inserted in the middle of the antibody and was named as it showed a sandwich shape.

In one embodiment of the invention, clustered nanostructures with directionality of segmented double metal nanorods were made. We have investigated the fabrication and characterization of dual metal Ag-Au-Ag nanorods (NRs) and their orientation clusters as highly sensitive SERS nano probes in surface enhanced Raman scattering (SERS) -based biosensing applications. The double metal Ag-Au-Ag NRs were prepared through directional overgrowth from a functionalized gold tetrahedron as a seed. Directional clustering was achieved by non-covalent interactions through selective surface modification of the Au compartments of the double metal Ag-Au-Ag NRs. As evidenced by zeta-dislocation, UV-Vis absorbance, dynamic light scattering, transmission electron microscopy and SERS analysis, they exhibited greatly improved physicochemical and optical properties compared to those of the individual double metal Ag-Au-Ag NRs .

The clustered nanostructures having the directionality of the segmented double metal nanorods according to the present invention can be applied as metal nano probes for measuring surface-enhanced Raman scattering (SERS) images with improved Raman intensity.

BRIEF DESCRIPTION OF THE DRAWINGS Figure 1 is a schematic diagram of the synthesis of clusters formed by (A) a gold ten-sided and (B) gold nanorods, a double metal Ag-Au-Ag NRs and a directional self-assembly as seeds. (A) A double metal Ag-Au-Ag NRs (Type I) was prepared from the -COOH-functionalized Au decaploid prepared by the polyol method through the orientation and growth of Ag (B) Au rod seed-based Ag-Au-Ag bimetallic NRs (Type II) through and growth were also prepared. Ag-Au-Ag NRs with MUA-modified seeds activated by EDC and Sulfo-NHS and modified with ODA (octadecylamine) by means of hydrophobic interaction of ODA. Au-Ag NR clusters were formed.
Fig. 2 shows TEM images of (Au, Vis) absorbance spectra of (Au, Vis) absorbance spectra of Au ten-faces and their clustered nanostructures to investigate surface morphology, DLS profile.
3: (A) TEM image at high magnification of a double metal Ag-Au-Ag NR (Type I) having a single, thick Au ten-sided seed embedded in the body of a double metal NRs (Type I). (B) Energy-dispersive X-ray spectroscopy (EDS) maps of anisotropic properties of double metal NRs (Type I) consisting of Au and Ag. (CE) Mixed electronic image with its elemental map of Au of green and Ag of red.
Figure 4 shows TEM images of individual double metal NR (Type I) (A, B) and high angle annular dark field (HAADF) images of clusters of (C, D) Asymmetric growth of The Au compartments were selectively modified with both MGITC and ODA as Raman reporters to generate hotspots in the gaps between the individual double metal NRs (type I) through directional clustering to substantially amplify the SERS signal.
Figure 5 (A) UV-Vis absorbance spectrum of MGITC-labeled double metal NRs and clusters thereof, (B) DLS profile, and (C) SERS spectrum when MGITC concentration is 5.0 x ^10-6 M.
6, (AF) (A, B) Au rod seed, (C, D) Au rod HAADF image of a cluster of seeded-based double metal NRs (type II) and (E, F) (G) UV-Vis absorbance spectra of ODA-derived clusters of Au NRs seeds and (H) SERS intensity of clusters of Au rod seed-based double metal NRs (Type II) and (E, F).
Figure 7 (A) A schematic diagram of a SERS-based immunoassay method for the detection of IgG as a model protein using MGITC-labeled double metal NR (Type I). (B, C) Raman spectra as a function of IgG concentration for SERS-based quantitative analysis with 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 intended only to illustrate the present invention and do not limit or limit the scope of the present invention. It will be understood by those of ordinary skill in the art that various changes in form and details may be made therein without departing from the spirit and scope of the invention as defined by the appended claims. The references cited in the present invention are also incorporated herein by reference.

≪ Example 1 >

Diethylene glycol (DEG), gold (III) chloride hydrate (HAuCl ₄ .3H ₂ O), silver nitrate (AgNO _3, ≥ 99.0%) polyvinylpyrrolidone (PVP), octadecyl amine has a molecular weight of 55,000 ( ODA), ethanol (99.5%), aqueous ammonia (NH ₄ OH), 11- mercapto Featherstone deca acid (MUA, 95%), sulfo -N- hydroxysuccinimide ester (sulfo -NHS) and phosphate buffered saline (PBS) were purchased from Sigma-Aldrich (St. Louis, Missouri, USA). Malachite green isothiocyanate (MGITC) was obtained from Invitrogen (Grand Island, New York, USA). 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide (EDC) was purchased from Thermo Scientific (Rockford, Ill., USA). Aqua regia all glassware _{(aqua Regia, HCl: HNO 3} , 3: 1) was thoroughly rinsed with deionized water and then washed with. Deionized water purified by Milli-Q (Millipore Water Purification Systems; EMD Millipore, Bedford, Mass., USA) was used.

≪ Example 2 > Preparation of gold decahedron and gold nanorods as seeds

As a seed, a gold decahedron was prepared by slightly modifying the previously reported steps (Seo, D., Yoo, CI, Chung, IS, Park, SM, Ryu, S., & Song, H. (2008). The PVP solution (3.0 g, 27.0 mmol for monomers) was dissolved in 13 mL of DEG. ≪ tb >< TABLE > The PVP solution was stirred at room temperature for 2 hours and heated to reflux with vigorous stirring for 10 minutes. DEG (1.0 mL) of the HAuCl ₄ was quickly added (10.05 mg, 0.025 mmol) solution, and the solution immediately turned to a blue color of dark brown. The solution was left under stirring at room temperature for 15 minutes to obtain a homogeneous gold tetrahedron. The Au ten-sided solution was mixed with 35 mL ethanol and purified by centrifugation at 13,000 rpm for 15 minutes. This procedure yielded an Au 10 octahedron having an average edge length of 42 nm with a yield of approximately 92% of the shape. MUA was introduced to modify the surface of the gold ten-sided body with a carboxyl group. 1.5 mL of a 10 mM solution of MUA in ethanol was mixed with the Au 10-sphere in ethanol and the mixed solution was kept at room temperature. The Au decahedron was washed twice with ethanol and resuspended in DEG.

Au nanorods (NR) as seeds were prepared by MUA modification as reported previously (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 x 10 ^-4 M) dissolved in DEG solution was periodically added to the ten-sided seed dispersion in DEG for 30 minutes every 30 minutes, then the mixture was diluted with 245 ^Gt; C < / RTI > for 60 minutes. 5 ml of 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

1.5 mL of PVP (0.13 mM) in PEG and 1.5 mL of AgNO ₃ (3.4 x 10 ^-2 M) in DEG were added to 2.5 mL (0.15 mM) of PEG in order to produce double metal Ag-Au-Ag NRs using gold ten- RTI ID = 0.0 > 245 C < / RTI > over 3.7 minutes over 30 seconds. The reaction mixture was refluxed for 1 hour, cooled to room temperature and 10 mL ethanol was added for purification. The homogeneous double metal Ag-Au-Ag NRs were separated from the large agglomerates by centrifugation at 500 rpm for 5 minutes, washed with ethanol by precipitation and a dispersion period of 15 minutes at 12,000 rpm, and finally in 15 mL of ethanol Lt; / RTI >

Example 4 Clustering of Bimetallic Ag-Au-Ag NR

As a Raman reporter, MGITC was introduced into the double metal Ag-Au-Ag NRs and used for SERS characterization analysis, followed by directed self-assembly of the clustered double metal Ag-Au-Ag NRs through noncovalent interactions. 5 mL of double metal Ag-Au-Ag NRs was transferred to 5 mL deionized water by centrifugation at 12,000 rpm for 15 min. A colloidal solution of the double metal 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, 0.5 mM EDC was added to the double metal Ag-Au-Ag NRs solution to covalently attach ODA with long hydrophobic alkyl groups to the Au compartments of the double metal Ag-Au-Ag NRs through selective functionalization , And stirred for 1 hour to activate the remaining carboxyl groups on the Au compartments. The final ODA solution concentration in 10 ^-8 M THF was introduced dropwise into the EDC-activated double metal NRs solution and stirred for 1 hour to form an amide bond between the carboxyl functionality of the Au moiety of the double metal NRs and the amine functionality of the ODA . Through the hydrophobic interaction, the color of the reaction solution changed from deep yellow to reddish yellow due to ODA-mediated cluster formation of the double metal NR. Finally, the reaction solution was purified by centrifugation at 10,000 rpm for 15 minutes and resuspended in deionized water or PBS for further use.

<Example 5> Characterization of double metal Ag-Au-Ag NRs and clusters thereof

Zeta sizer Nano ZS instrument with a Ne-He laser with a wavelength of 633 nm and a scattering angle at 90 ° to determine the colloidal properties of the gold-10-sided, double-metal Ag-Au-Ag NRs and clusters thereof Dynamic light scattering (DLS) and zeta potential measurements were performed using a microfluidic device (Malvern Instruments, Malvern, UK). The sample was diluted to 10: 1 with deionized water and the temperature was adjusted to 25 ° C. The UV-Vis plot thereof was measured by a UV-visible spectroscope (UV-1800, Shimadzu, Japan) at 25 ° C using a fixed slit width of 1 nm in a single 10 scan mode with an intermediate scan rate of 300 nm to 800 nm Lt; / RTI &gt; The baseline was adjusted using two empty cells filled with deionized water and the average size was obtained from a minimum of 20 scan cycles. In addition, zeta-potential measurements were performed to determine surface charge in deionized water. Transmission electron microscopy (TEM) images were obtained using a JEM-2100F FE-STEM (JEOL, Germany) operating at an accelerating voltage of 80 to 200 kV and the nanoscale individual nanostructured Ag- Respectively. The samples were deposited on a 400 mesh copper lattice (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), a few drops of the sample were placed on silicon, dried and then coated by platinum sputtering using a turbo sputter coater (K575X) And imaged using SEM VEGA (TESCAN, USA) operating at an accelerating voltage of 0.5 kV to 30 kV. All Raman characterization analyzes were also performed using a Renishaw inVia Raman microscopy system (Renishaw, UK) equipped with a Reinshaw He-Ne laser operating at a wavelength (?) Of 632.8 nm for excitation sources with a laser output of 12.5 mW Respectively. The Rayleigh line was removed from the collected SERS profile using a hologram notch filter located in the acquisition filter. Raman scattering was obtained using a 1 cm ^-1 spectroscopic charge-coupled device (CCD) camera and all SERS spectra were corrected with a 520 cm ^-1 silicon line. The colloidal solution of MGITC labeled nanoparticles was transferred to a small glass capillary (general capillary, soda lime glass , inner diameter: 1.1 - 1.2 mm, Wall: 0.2 +/- 0.02 mm, length: 75 mm). The SERS spectra were collected over a 1 second exposure time using a 20x objective lens focusing the laser spot on a glass capillary in a wavelength range of 608 cm ^-1 to 738 cm ^-1 .

Figure 1 shows a schematic representation of the synthesis of clusters formed by gold ten-sided or gold nanorods, double metal Ag-Au-Ag NRs and directional self-assembly as seeds for SERS-based biosensing applications. Double metal Ag-Au-Ag NRs were prepared by directionally overgrowing from -COOH-functionalized Au 10-face or Au nanorods. Directional clustering was induced by non-covalent through selective surface modification of the Au compartments of the double metal Ag-Au-Ag NRs. The double metal NR was prepared using Ag salt and PVP as precursors and capping agents for MUA-functionalized Au 10-face or Au NR, and silver shells. Ag-Au-Ag NRs were formed by simultaneous growth of the Ag nanostructure on the surface of the Au ten-sided or Au NRs in both directions. The gold section of the double metal 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 a long hydrophobic alkyl group. The ODA on the surface Au compartments of the double metal NR were bonded to each other by hydrophobic interaction to allow the bi-metal NR to orient itself self-orientated into the double metal NR clusters. As shown in the Raman spectrum, the double metal NR clusters showed significantly improved Raman intensity compared to single bimetallic NR by increasing the hotspot contacts between them.

Figure 2 shows TEM images, UV / Vis absorbance spectra, and DLS profiles of Au ten-sided bodies and their clustered nanostructures to investigate surface morphology, plasmon absorbance change, and average size. As reported, Au 10-sided bodies were prepared by the technique employed in the polyol process (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 since DEG had a relatively high boiling point of 245 DEG C and had polarity sufficient to dissolve the gold salt and PVP. To clearly demonstrate the ODA-induced clustering of metal nanostructures, Au 10-sided bodies were functionalized with amide bonds between the residual carboxyl groups of the Au seed and the primary amine groups of the ODA using ODA with long hydrophobic alkyl groups. ODA on the Au seed surface was bonded to each other by hydrophobic interaction to form a gold tetrahedron cluster. TEM images of the Au ten-sided bodies and their clusters indicate that they are both uniform in shape and size. The average edge length of the Au ten-sided body was found to be 34 ± 8 nm by analysis of 50 randomly selected nanoparticles in the TEM image. The UV / Vis absorbance spectra of the Au 10 octahedron in Fig. 2 (C) show two characteristic peaks at 550 nm and 610 nm, representing polar-bipolar plasmon and azimuthal bipolar plasmon modes, respectively. When they were clustered, the two plasmon bands shifted towards red with a decrease in absorbance at longer wavelengths. Table 1 shows the zeta potential and the DLS value of the gold tetrahedron as a seed and a seed cluster. The thiol group of MUA was bound to the gold surface through covalent bonding while the carboxyl group of MUA showed the anionic character of the gold decahedron. The zeta potential of the gold decahydrate was-1.9 ± 0.5 mV and the zeta potential of the MUA-functionalized gold ten-sided body was -19.2 ± 0.5 mV due to the presence of -COOH on the gold seed surface. Because ODA is covalently conjugated to -COOH on the surface of the gold seed by EDC / NHS chemistry, the ODA-modified gold decahydrate was clustered through the hydrophobic interaction of the ODA moiety. The size of the gold octahedron clusters increased significantly from 33.0 nm to 50.3 nm in the gold ten - dodecahedron, and its zeta potential also greatly increased to - 9.4 ± 0.5 mV. In addition, the size distribution of both the gold ten-sided body and its clusters was uniform as shown in Fig. 2 (D).

The double metal Ag-Au-Ag NRs (Type I) was prepared via the directional overgrowth of Ag from a MUA-functionalized, twinned Au ten-sided as a seed in DEG as a reducing agent, 0.0 &gt; 245 C. &lt; / RTI &gt; Two equivalents of Ag salt and PVP (0.13 mM) were added periodically to the seed dispersion for gold concentration (1.7 x 10 ^-2 M) in DEG to form double metal NRs (Type I). As shown in Fig. 3 (A), its TEM image at high magnification showed a single, dense Au 10-face seed embedded in the body of a bimetallic NR (Type I). In addition, the energy-dispersive X-ray spectroscopy (EDS) map of FIG. 3 (B) shows anisotropic properties of the double metal NR (Type I) composed of Au and Ag. The mixed electronic image of Figure 3 (CE) clearly shows an elemental map of Au, which is green and Ag, which is red.

As shown in Figure 4, TEM images of (A, B) individual double metal NR (Type I) and HAADF images of clusters of (C, D) show the asymmetric growth of Ag on the -COOH- Lt; / RTI &gt; MGITC as a Rahman reporter was introduced into the colloidal state of the double metal NR solution to allow the isothiocyanate group (-NiCiS) of MGITC to be selectively bound to the surface of the double metal NR (type I). In addition, ODA was added to the MGITC-labeled double metal NR solution and the remaining carboxyl functionality from the Au 10 octahedral seed was chemically attached by amide coupling between the EDC-activated carboxyl functionality and the primary amine functionality of the ODA . The directional clustering of the double metal NR was made through the hydrophobic interactions between the hydrophobic long alkyl groups of ODA and the Au compartments of the double metal NR were faced with each other. As the ODA concentration varied, its degree of self-assembly was adjusted by its corresponding hydrophobicity.

Figure 5 shows the SERS spectra of MGITC-labeled bimetallic NR and its clusters when (A) UV-Vis absorbance spectrum, (B) DLS profile, and (C) MGITC concentration is 5.0 x ^10-6 M. The double metal NR (Type I) exhibits two different UV-Vis absorbance peaks at 420 nm and 510 nm, respectively, which represent the transverse mode of the silver and gold compartments. Due to the low concentration, there was no significant difference in the UV-Vis absorbance spectrum in the presence or absence of the introduced Raman reporter. The plasmon band shifted to red with increasing absorbance at longer wavelengths after cluster formation. Dynamic light scattering (DLS) measurements were also performed to characterize the size, size distribution, and colloid stability of MGITC-labeled bimetallic NR and its clusters under aqueous conditions. As shown in Fig. 5 (B), the average diameter of the double metal NR was 34.58 nm in diameter and the average length was 252.10 nm. On the other hand, its clusters exhibited only one size distribution at 279.80 nm, because its directional clustering created similar sizes of diameter and length with an aspect ratio of 1.0. Under the aqueous conditions (Table 1), the zeta potential values of MGITC-labeled double metal NR and its clusters were -18 mV-12 mV, respectively.

[Table 1]

Compared with the bimetallic NR, its clustered size and zeta potential values were greatly increased by the ODA junction. When orientated and self-assembled, the dual metal NR clusters were fabricated by selectively functionalizing the Au compartments with ODA. The SERS intensity at 1617 cm ^-1 was significantly improved by about 10-fold compared to individual ones, Because the electromagnetic field of the hot spot formed in the gap between the electrodes is amplified.

Au rod seed-based Ag-Au-Ag double metal NRs (Type II) were prepared to apply ODA-mediated directional clustering of the double metal NR to a new type of double metal NR. Au rods as seeds were prepared by the polyol method using PVP and HAuCl ₄ as capping agents and Au rod seed-based double metal NRs (Type II) was prepared as described above for the synthesis of bimetallic NRs (Type I) And orientation and growth of Ag from MUA-functionalized Au NR seeds. Figure 6 shows HAADF images of clusters of (AF) (A, B) Au bar seed, (C, D) Au bar seed-based double metal NR (type II) and (E, F). The Au rod was synthesized to have uniform size and shape. The average length of the Au rods was found to be 50 8 nm as determined by analyzing 50 nanoparticles randomly selected from the HAADF image. The Au rod seed-based double metal NRs (Type II) exhibited a single dark body embedded in the entire body, which was clearly observed at high magnification and whose average length was measured on 50 objects in the HAADF image Was 100 +/- 10 nm. As shown in the directional clusters of the double metal NR (Type I), directional clustering of the Au rod seed-based double metal NR (Type II) also results in hydrophobic interactions between the hydrophobic long alkyl groups of ODA on the surface of the Au NR seed . Similarly, ODA-induced clustering of Au NRs seeds was also demonstrated by the UV-Vis absorbance spectrum as shown in Figure 6 (G). Multiple hot spots in a double metal NR (type II) cluster generate a gap between them through directional clustering to substantially amplify the SERS intensity as compared to the individual as shown in Figure 6 (H). Thus, these clustered nanostructures of double metal NRs, including Type I and Type II, are a new class of SERS nanoprobes for biological detection.

Example 6 Bio-junction of a double metal NR cluster and a magnetic bead (MB) with an antibody

A double metal 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 bead (MB) to form an antibody-antigen-antibody interaction To form a sandwich immunoconjugate, where IgG as an antigen was present in solution. In a representative experiment, 10 ml of 0.2 mg / ml EDC was added to the bimetallic NR clusters in PBS at pH 7.4 and allowed to stir for 1.5 hours and then 10 ml of a 0.50 mg / ml solution of anti-human IgG- - activated double metal NR clusters were added and stirred for an additional 1.5 hours. Finally, double-metal NR clusters with anti-human IgG pAb-conjugated as SERS nanoprobes were purified with PBS using centrifugal force at 5,000 rpm. Similarly, magnetic nanoparticle-encapsulated polymer nanoparticles as magnetic beads (MB) were chemically conjugated to anti-human IgG mAbs using the residual carboxyl functional groups on MB after thermal 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 ultrasonic mill (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 resuspended in PBS for biosensing of IgG antigen.

Example 7 SERS-based biosensing of IgG antigens

Using MB with anti-human IgG mAb to bind IgG as antigen in PBS, a double metal NR cluster with anti-human IgG pAb was used as a SERS nanoprobe to probe IgG through the formation of sandwich-type immunoconjugates Respectively. First, anti-human IgG mAb-bound MB was introduced into the IgG solution in the concentration range of 410 ng / mL and 40 pg / mL. Immunoconjugates between anti-human mAb-bound MB and IgG were separated by magnetic force and then the solution was purified twice with PBS and resuspended in PBS. Second, the prepared anti-human mAb-conjugated MB and immunoglobulin complexes of IgG were mixed with anti-human IgG mAb-conjugated double metal NR clusters and stirred for 1 hour to obtain anti-human IgG pAb, IgG, and anti-human IgG mAb. Sandwich-type immune complexes in anti-human IgG pAb, IgG, and anti-human IgG mAbs were isolated using magnetic force and suspended for SERS recording. Non-specific binding was assessed by performing two control studies with no IgG and antibody conjugation of both MB and double metal NR clusters.

Figure 7 (A) shows a schematic diagram of a SERS-based immunoassay method for the detection of IgG as a model protein. First, anti-human IgG mAb conjugated magnetic bead solutions were added to solutions with different IgG concentrations. IgG assays were selectively captured with their corresponding magnetic beads through specific molecular interactions, purified by applying an external magnetic field, and resuspended in PBS. Second, an anti-human IgG pAb conjugated double metal NR cluster solution for SERS-based biosensing was added to a solution of complexed anti-human IgG mAb conjugated magnetic beads and IgG to form anti- A sandwich-type immunoconjugate consisting of human IgG mAb conjugated magnetic beads, IgG and anti-human IgG pAb conjugated double metal NR clusters was formed. Subsequently, the unattached SERS nanoprobe was removed by applying a magnetic field, and the resulting sandwich immunoconjugate was resuspended in PBS. Finally, Raman intensity was obtained as a function of IgG-concentration for SERS-based quantitation, and there was a linear correlation between relative Raman intensity and 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 double metal NR clusters linearly increased with the concentration of the sandwich immunoconjugate R ² = 0.937).

The present invention demonstrates that double metal NR clusters exhibit superior SERS activity relative to a single bimetallic NR. The platform method we have developed for the synthesis of double metal NRs (type I, II) and its clusters will be beneficial in synthesizing controlled double metal anisotropic nanostructures and clusters of different metals of various sizes and compositions.

Claims (13)

Clustered nanostructures in which the double-metal nanorods are self-assembled in a directionally bi-metallic nanocluster, with a segmented double-metal nanorods composed of a metal [ABA] as a seed,
Wherein A and B are each selected from the group consisting of silver, gold, copper and mixtures thereof, and wherein the A and B metals are not equal to each other.
Clustered nanostructures with directional orientation of segmented double metal nanorods.
The method according to claim 1,
Wherein the B metal has a surface modified with a carboxyl group.
The method according to claim 1,
The B metal is gold and the A metal is silver-gold-silver double metal nanorods composed of silver,
Wherein the B metal is a polyhedral gold nanoparticle or a gold nanorod.
The method of claim 3,
Wherein the polyhedral gold nanoparticle is a 10-sided gold nanoparticle, and the segmented double-metal nanorod has a directional cluster nanostructure.
The method according to claim 1,
Wherein the Raman reporter is attached to the double metal nanorods. &Lt; RTI ID = 0.0 &gt; 11. &lt; / RTI &gt;
A metal nano probe for surface-enhanced Raman scattering (SERS) image measurement using the cluster nanostructure of claim 5.
i) preparing a segmented Q plurality of double metal nanorodic seeds composed of [ABA] using a metal B surface-modified with a carboxyl group;
Ii) adding an EDC (1-ethyl-3- (3-dimethylaminopropyl) carbodiimide) solution to activate the carboxyl groups of the B metal section;
Iii) adding the EDC solution and then adding an amine compound and stirring;
Iv) forming an amide bond between the EDC-activated carboxyl group of the B metal section and the amine group of the amine-based compound;
v) In the B metal section, the amine compound is bonded to each other by the hydrophobic interaction between the alkyl groups of the amine compound, so that the B metal compartments face each other while the partitioned double metal nanorods form a directional cluster, felled;
&Lt; / RTI &gt;
Wherein A and B are each selected from the group consisting of silver, gold, copper and mixtures thereof, and wherein the A and B metals are not equal to each other.
A method for producing cluster nanostructures having directionality of segmented double metal nanorods.
8. The method of claim 7,
The B metal is gold and the A metal is silver-gold-silver double metal nanorods composed of silver,
Wherein the B metal is polyhedral gold nanoparticles or gold nanorods. &Lt; RTI ID = 0.0 &gt; 11. &lt; / RTI &gt;
8. The method of claim 7,
Wherein the polyhedral gold nanoparticle is a tetrahedral gold nanoparticle. The method of producing a cluster nanostructure having a directionality of a segmented double metal nanorod.
8. The method of claim 7,
Further comprising the step of attaching a Raman reporter to the surface of said double metal nanorods after said step i). &Lt; Desc / Clms Page number 20 &gt;
8. The method of claim 7,
Wherein the amine compound is selected from the group consisting of hexadecylamine, heptadecylamine, octadecylamine, nonadecylamine, and dodecylamine. Of producing directional clustered nanostructured nanostructured nanostructured.
a) preparing a sample solution containing the target substance to be detected;
b) immobilizing the magnetic nanoparticles with the first antibody against the target;
c) fixing the second antibody against the target to the metal nanoprobe of claim 6;
d) adding the magnetic nanoparticles immobilized with the first antibody to the sample liquid to form an immunocomplex in which the target and the first antibody of the magnetic nanoparticle are conjugated;
e) adding the second antibody-immobilized metal nanoprobe to a solution containing the immune complex to which the first antibody is conjugated to form a sandwich immunoconjugate of the first antibody of the metal nano-probe second antibody-target-magnetic nanoparticle Forming;
f) using magnetic fields to separate the magnetic nanoparticles and the metal nanoprobes that did not form the sandwich immunoconjugate; And
g) measuring the Raman signal of said sandwich immunoconjugate;
(SERS) based on the surface-enhanced Raman scattering (SERS).
13. The method of claim 12,
Wherein the target material is a protein or a pathogen, based on surface-enhanced Raman scattering (SERS).

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