WO2018030785A1

WO2018030785A1 - Bimetal-conductive polymer janus composite nanostructure having electrical stimulus response, colloid self-assembled structure thereof, preparing method, and bio-sensing, bio-imaging, drug delivery, and industrial application

Info

Publication number: WO2018030785A1
Application number: PCT/KR2017/008620
Authority: WO
Inventors: 임동우; 황은영
Original assignee: 한양대학교 에리카산학협력단
Priority date: 2016-08-09
Filing date: 2017-08-09
Publication date: 2018-02-15

Abstract

The present invention relates to a bimetal-conductive polymer Janus composite nanostructure having electrical stimulus response, a colloid self-assembled structure thereof, a preparing method therefor, and bio-sensing, bio-imaging, drug delivery, and industrial application using the same.

Description

Double metal-conductive polymer Janus composite nanostructures with electrical stimulation responsiveness, colloidal self-assembled structures thereof, methods of preparation and biosensing, bioimaging, drug delivery and industrial applications

The present invention relates to double metal-conductive polymer Janus composite nanostructures having electrical stimulation reactivity, colloidal self-assembled structures thereof, methods for their preparation and biosensing, bioimaging, drug delivery and industrial applications using the same.

Many optical biosensors based on fluorescence (FL) and surface plasmon resonance (SPR) have been developed for applications for detection because of their high sensitivity.

Raman spectroscopy has been studied for the detection and identification of pathogens. Specifically, Surface-enhanced Raman scattering (SERS) is of great interest for spectroscopic detection and identification of small molecules, nucleic acids, proteins and cells, mainly due to its ultra-high sensitivity, narrow bandwidth and significant multiplexing capabilities. . Raman spectroscopy has been used as a versatile tool for obtaining structural information on materials based on vibrational transitions, but traditional Raman scattering is limited by low sensitivity. In this regard, SERS spectroscopy is a powerful analytical technique that provides a remarkable signal up to 10 ¹⁴ near the surface of metal nanoparticles compared to normal Raman scattering. This improvement is due to hot spots present in non-uniformly distributed electromagnetic fields across the particle surface, ie, nanoscale bonds, which are sharp protrusions or gaps between nanoparticles. On the other hand, much effort has been made to develop plasmon metal nanoparticles and their high optical efficiency as well as their useful optical properties. These plasmon properties are highly dependent on size, morphology and cohesion.

Metal nanoparticles, on the other hand, have been extensively studied in a variety of applications, including electronic, catalytic, biological imaging, and surface enhanced Raman spectroscopy, due to their electrical, chemical, and optical properties, depending on their structure and size. In addition, multicomponent metal nanoparticles have new or improved optical properties as well as the aggregate physicochemical properties from each component due to synergistic effects compared to single component metal nanoparticles.

An object of the present invention is to self-assembled double metal-polymer Janus nanostructures, self-assembled nanostructures thereof, methods for their preparation and metal nanoprobes, drug carriers, surface-enhanced Raman scattering (SERS) using the same It is to provide a method for detecting a target material based.

Another object of the present invention is a Janus nanostructure consisting of a double-metal nanoparticle compartment and a polymer compartment of a core-satellite structure, a method for manufacturing the same, and a metal nanoprobe for detecting a target substance based on surface-enhanced Raman scattering (SERS), SERS It is to provide a method for detecting a target material based.

Another object of the present invention is to provide an asymmetric Janus nanoprobe for surface-enhanced Raman scattering (SERS) based target material detection, a method for preparing the same, and a method for detecting the target material using the same.

Another object of the present invention is an asymmetric Janus nanostructure consisting of a double metal nanoparticle compartment and a polymer compartment comprising a directional metal nanorod cluster, a manufacturing method thereof and a surface-enhanced Raman scattering (SERS) -based target using the same It is to provide a method for detecting a substance.

제 1First 발명: invent:

The present invention

Double metal nanocluster cores; And a conductive polymer shell radially positioned around the core. The self-assembled double metal-polymer Janus nanostructure is provided.

The double metal nanocluster core is composed of a first metal and a second metal surrounding the first metal surface.

Each of the first metal and the second metal may be selected from the group consisting of gold, silver, copper, and mixtures thereof, but is not necessarily limited thereto, and any metal widely used in the art is not limited thereto. Can be used.

The first metal and the second metal may not be the same.

The conductive polymer may be at least one selected from the group consisting of polypyrrole, polythiophene, poly (3,4-ethylene dioxythiophene) (PEDOT), and polyaniline, but is not limited thereto. But preferably may be polyaniline.

The double metal nano cluster core may further include a Raman dye.

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

In the present invention, the double metal means two kinds of metals forming a metal core-metal shell structure.

In the present invention, the metal nanocluster is a term generally used in the art as a term meaning aggregates (aggregates) in which metal nanoparticles are collected and aggregated.

In the present invention, the double metal nano cluster refers to agglomerated aggregates of double metal nanoparticles forming a core-shell structure.

In the present invention, the "Janus nanostructure" or "hybrid nanostructure" refers to a nanostructure composed of two different compartments (double metal nanocluster compartment (core) and conductive polymer compartment (shell)) that are physically and chemically separated. .

The double metal nano cluster is composed of a first metal and a second metal surrounding the first metal surface. Conductive polymer compartments are attached to only one side of the double metal nanocluster compartment to grow, that is, eccentrically deposited to form asymmetric Janus nanoparticles, and the double metal nanoparticles in the Janus nanoparticles undergo hydrophobic interactions. Self-assembled through to form a double metal nanocluster core and a polymer compartment radially positioned around the core to exhibit a shell shape. Hydrophobic interaction of the double metal nanoparticles in the Janus nanoparticles is achieved by covalently binding ODA (octadecylamine) to the Janus nanoparticles to induce selective functionalization.

As used herein, "Janus nanostructures" are also referred to as "double metal-polymer Janus nanoparticles" or "Janus nanoparticles" or "Janus nanoprobes", as their structure includes a double metal nano cluster core-conductive polymer shell. . In addition, in the present specification, "a double metal nanocluster core and a polymer compartment radially positioned around the core to form a shell shape" are called "superparticular structures".

In another aspect, the present invention provides metal nanoprobes for surface-enhanced Raman scattering (SERS) based biosensing and / or bioimaging measurement using Janus nanostructures according to the present invention.

The self-assembled double metal-polymer Janus nanostructures according to the present invention comprise a Raman dye, thereby providing a metal nanoprobe for surface-enhanced Raman scattering based biosensing and / or bioimaging. have.

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

In the present invention, nanoprobe means a probe of a nano size.

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

The present invention provides metal nanoprobes for fluorescence based biosensing and / or bioimaging measurement using Janus nanostructures according to the present invention.

Janus nanostructures of self-assembled double metal-polymers according to the present invention are cyanine-based fluorescent molecules, rodamine-based fluorescent molecules, oxazine-based fluorescent molecules, Alexa-based fluorescent molecules, By including a fluorescein isothiocyanate (FITC) fluorescent molecule, a 5-carboxy fluorescein (FAM) fluorescent molecule, and a Texas red (Texas Red) fluorescent molecule, it can be provided as a metal nanoprobe for measuring fluorescence-based images. Specifically, the fluorescent dye (R ₂ ) is cy 3 (Cy 3), cy 5 (Cy 5), fluorescein isothiocyanate (FITC), tetramethyltamine isothiocyanate (RITC), Alexa (Alexa) , 4,4, -difluoro-4-boro-3a, 4a-diaza-s-indacene (BODIPY), Texas Red, biotin rhodamine, coumarin, Cy, EvoBlue, Fluorescent dyes having derivatives of oxazine, carbopyronine, naphthalene, biphenyl, anthracene, phenanthrene, pyrene, carbazole and the like as basic skeletons and derivatives of the fluorescent dyes thereof can be exemplified. Specifically, CR110: carboxyrodamine 110: Rhodamine Green (trade name), TAMRA: carboxytetramethylhodamine: TMR, carboxyrodamine 6G: CR6G, ATTO655 (trade name), BODIPY FL (trade name): 4,4-difluoro-5,7-dimethyl -4-bora-3a, 4a-diaza-s-indacene-3-propionic acid, BODIPY 493/503 (trade name): 4,4-difluoro-1,3,5,7-tetramethyl-4- Bora-3a, 4a-diaza-s-indacene-8-propionic acid, BOD IPY R6G (trade name): 4,4-difluoro-5- (4-phenyl-1,3-butadienyl) -4-bora-3a, 4a-diaza-s-indacene-3-propionic acid, BODIPY 558/568 (trade name): 4,4-difluoro-5- (2-thienyl) -4-bora-3a, 4a-diaza-s-indacene-3-propionic acid, BODIPY 564/570 Trademark): 4,4-Difluoro-5-styryl-4-bora-3a, 4a-diaza-s-indacene-3-propionic acid, BODIPY 576/589 (trade name): 4,4-difluoro Rho-5- (2-pyrrolyl) -4-bora-3a, 4a-diaza-s-indacene-3-propionic acid, BODIPY 581/591 (trade name): 4,4-difluoro-5- ( 4-phenyl-1,3-butadienyl) -4-bora-3a, 4a-diaza-s-indacene-3-propionic acid, Cy3 (trade name), Cy3B (trade name), Cy3.5 (trade name), Cy5 (trade name), Cy5.5 (trade name), EvoBlue10 (trade name), EvoBlue30 (trade name), MR121, ATTO 390 (trade name), ATTO 425 (trade name), ATTO 465 (trade name), ATTO 488 (trade name), ATTO 495 (Trade name), ATTO 520 (trade name), ATTO 532 (trade name), ATTO Rho6G (trade name), ATTO 550 (trade name), ATTO 565 (trade name), ATTO Rho3B (trade name), ATTO R ho11 (trade name), ATTO Rho12 (trade name), ATTO Thio12 (trade name), ATTO 610 (trade name), ATTO 611X (trade name), ATTO 620 (trade name), ATTO Rho14 (trade name), ATTO 633 (trade name), ATTO 647 ( Trademark name), ATTO 647N (trade name), ATTO 655 (trade name), ATTO Oxa12 (trade name), ATTO 700 (trade name), ATTO 725 (trade name), ATTO 740 (trade name), Alexa Fluor 350 (trade name), Alexa Fluor 405 ( Trademark), Alexa Fluor 430 (trade name), Alexa Fluor 488 (trade name), Alexa Fluor 532 (trade name), Alexa Fluor 546 (trade name), Alexa Fluor 555 (trade name), Alexa Fluor 568 (trade name), Alexa Fluor 594 (trade name) ), Alexa Fluor 633 (trade name), Alexa Fluor 647 (trade name), Alexa Fluor 680 (trade name), Alexa Fluor 700 (trade name), Alexa Fluor 750 (trade name), Alexa Fluor 790 (trade name), Rhodamine Red-X (trade name) ), Texas Red-X (trade name), 5 (6) -TAMRA-X (trade name), 5TAMRA (trade name), and SFX (trade name).

The present invention provides a drug carrier using Janus nanostructures according to the present invention.

The drug carrier may be one that is electric field stimulant reactive.

Specifically, the polymer compartment of the self-assembled double metal-polymer Janus nanostructure according to the present invention was composed of a conductive polymer and exhibited electric field reactivity. The present invention supported the drug into the polymer compartment through the electrostatic interaction of the negatively charged drug and the positively charged conductive polymer monomer, and the concentrated drug-supported nanoparticles were added to the PEG solution and irradiated with UV to PEG- Nanoparticle hydrogels were formed. De-protonation of the conducting polymer monomer (Repeating unit) when a voltage of -1.5V was applied, the drug release was made while the electrostatic interaction is reduced (Fig. 6).

In another aspect, the present invention provides a method for producing a self-assembled double metal-polymer Janus nanostructure comprising the following steps.

i) preparing metal nanoparticles to form a seed;

ii) adding the seed metal nanoparticles to an aqueous solution in which a conductive polymer monomer and a surfactant are dissolved,

iii) adding a metal ion solution to the solution to which the seed metal nanoparticles of ii) are added to perform a redox reaction between the metal ion and the conductive polymer monomer;

iv) the metal ions are deposited on the surface of the seed metal nanoparticles while being reduced by receiving electrons provided by the conductive polymer to form a double metal nanoparticle compartment, and the conductive polymer monomer is oxidized to one side of the double metal nanoparticle compartment. Is deposited only to form a conductive polymer compartment asymmetrically while growing into a conductive polymer, thereby making Janus nanoparticles composed of double metal-polymers;

v) octadecylamine (ODA) is added to the solution containing Janus nanoparticles; And

vi) the double metal nanoparticles in the Janus nanoparticles self-assemble covalently with the ODA to form a double metal nano cluster core and a radially located polymer shell around the core; step.

After step iv), the method may further include attaching a Raman dye to the surface of the double metal nanoparticles of the nanoparticles composed of the double metal-polymer.

In one embodiment of the invention, two kinds of Raman dyes, RBITC and MGITC, are added to a colloidal solution of double metal-polymer Janus nanoparticles, followed by the isothiocyanate group of the Raman dye (-N = C〓). Through S), RBITC and MGITC were fixed on the surface of the double metal nanoparticle consisting of Au core-Ag shell, and selectively adsorbed to the double metal nanocluster compartment.

The seed metal of step i) may be selected from the group consisting of gold, silver, copper and mixtures thereof, but is not necessarily limited thereto, and any metal widely used in the art may be used without limitation.

The metal ion of step iii) may be selected from the group consisting of gold ions, silver ions, copper ions and mixtures thereof.

Specifically, the gold ions are gold (III) chloride hydrate, chlorocarbonylgold, hydrogen tetrachloroaurate, tetrachloroaurate hydrate, chlorotriethylphosphine gold compound. (Chlorotriethylphosphinegold), Chlorotrimethylphosphinegold, Dimethyl (acetylacetonate) gold compound, Gold (I) chloride, Gold cyanide, Gold sulfide sulfide) and mixtures thereof, and may be, but is not necessarily limited to, chlorinated chloride.

The silver ions include silver nitrate (AgNO3), silver tetrafluoroborate (AgBF4), trifluoromethanesulfonate silver (AgCF3SO3), silver perchlorate (AgClO4), silver acetate (Ag (CH3COO)), hexafluorophosphate silver (AgPF6), It may be selected from the group consisting of Ag (CF 3 COO) and mixtures thereof, but is not necessarily limited thereto, and may preferably be silver nitrate.

The copper ions may be copper (II) acetylacetonate (Cu (acac) 2), copper chloride (CuCl), copper chloride (II) (CuCl 2), copper (II) hexafluoroacetylacetonate (Cu (hfac) 2 ), Copper (II) trifluoroacetylchloride (Cu (tfac) 2), copper (II) dipiballomethacrylate (Cu (dpm) 2), copper (II) pentafluorodimethylheptanedione (Cu (ppm) 2), copper (II) heptafluorodimethyloctane (Cu (fod) 2), copper (II) iminopentanone (Cu (acim) 2), copper (II) hexafluoro-[(trifluoro Ethyl) imino] -pentanone (Cu (nona-F) 2), copper (II) acetylacetoethylenediamine (Cu (acen) 2), copper nitrate (Cu (NO3) 2), copper sulfate (CuSO4) and its It may be selected from the group consisting of the mixture, but is not necessarily limited thereto.

In one embodiment of the present invention, the double metal nanocluster core may be composed of a seed metal nanoparticle (first metal) and a second metal surrounding the seed metal surface.

The conductive polymer of step ii) may be at least one selected from the group consisting of polypyrrole, polythiophene, poly (3,4-ethylene dioxythiophene) (PEDOT), and polyaniline, It is not necessarily limited thereto, but may preferably be polyaniline.

The growth of the conductive polymer of step iv) may be by surface-templated polymerization.

The "surface template polymerization method" refers to a polymerization method based on an oxidation-reduction reaction, and in this specification, polyaniline, which is a conductive polymer, is deposited on a double metal nanocluster through a spontaneous redox reaction between silver nitrate and an aniline monomer. Specifically, aniline monomers having a primary amine group donate electrons to silver nitrate and silver ions receive corresponding electrons to balance the oxidation-reduction reaction so that the conductive polymer monomers are oxidatively polymerized on the double metal nanoclusters. Polyaniline was deposited.

The surfactant of step ii) may be at least one selected from the group consisting of sodium dodecyl sulfate (SDS), sodium deoxycholate, and Triton X-200, It is not necessarily limited to this, but may preferably be SDS.

A schematic diagram of an experimental method for preparing self-assembled double metal-polymer Janus nanostructures and their specific structures and an experimental method for SERS-based biosensing and / or application is shown in FIG. 1.

Figure 1 (a) shows the synthesis of double metal-polymer Janus nanoparticles and their specific structure. First, double metal-polymer Janus nanoparticles were prepared through oxidative polymerization of aniline monomers and further growth of Ag nanoparticles. Specifically, the concentrated Au nanoparticles were added to a solution containing aniline and a surfactant, SDS, and then silver nitrate was added to initiate an oxidation-reduction reaction. The resulting solution was further incubated overnight in a 3.6 mM SDS solution to prepare double metal-polymer Janus nanoparticles consisting of a double metal nanocluster compartment consisting of Au core-Ag shell and a polymer compartment consisting of poly (aniline). The partitioning of the double metal-polymer Janus nanoparticles is due to the balanced interfacial tension between three phase systems of Ag, poly (aniline) and water. The addition of SDS affects the interfacial tension between two adjacent phases, poly (aniline) -Ag and poly (aniline) -water, followed by the formation of separate poly (aniline) polymer compartments on one side of the Au nanoparticles Energy was minimized. In addition, when the double metal cluster compartments of the double metal-polymer Janus nanoparticles are selectively functionalized with ODA containing long hydrophobic alkyl chains, the directional self-assembles to form a specific structure, where the double metal cluster compartments exhibit hydrophobic interactions. Facing through. This is due to the covalent bonding of ODA to the surface of the double metal cluster compartment by the amide bonding reaction of the residual carboxyl groups of the Au nanoparticles and the first amine groups of the ODA.

Figure 1 (b) shows the spontaneous redox reaction between the aniline monomer and silver nitrate for the synthesis of double metal-polymer Janus nanoparticles. The redox reaction was associated with the transfer of electrons between the two precursors, where aniline was oxidized to poly (aniline) and silver nitrate was reduced to silver particles. Aniline monomers, which are electron donor molecules due to the first amine group, donated electrons to silver nitrate and received corresponding electrons to balance the silver nitrate redox reaction. As can be seen in Figure 1 (c), the specific structure of the double metal-polymer Janus nanoparticles can be applied to a new kind of SERS nanoprobe for biosensing. Specifically, the bimetallic compartments are selectively functionalized with Raman dyes and ODA to create hot spots in the gaps between the bimetallic nanoparticles through directional clustering, thereby improving significant Raman signals. Specific antibodies that were induced and were part of the target were introduced onto the polymer compartment via molecular binding. In the presence of the target, a sandwiched immune complex consisting of specific structure, target and magnetic beads was formed and washed in the magnetic field. Finally, Raman scattering-based detection of targets was confirmed via Raman shift.

In another aspect, the present invention provides a surface-enhanced Raman scattering (SERS) based target substance detection method comprising the following steps:

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

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

c) preparing by immobilizing a second antibody against the target material on a metal nanoprobe containing the Raman dye;

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

e) adding the metal nanoprobe to which the second antibody of c) is immobilized to a solution containing the immunocomplex conjugated with the first antibody of d) to prepare a second antibody-target material-magnetic nanoparticle of the metal nanoprobe. Forming a sandwich immunocomplex of the first antibody;

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

g) measuring the Raman signal of said sandwich immunocomplex; step.

As used herein, the term "sandwich immune complex" refers to an immunocomplex bound through an antibody-antigen (target) -antibody reaction. The antigen was named as it is inserted in the middle of the antibody to form a sandwich.

A schematic of SERS-based immunoassay for detection of target protein is shown in FIG. 7. In step 1, anti-human IgG mAb binding or anti-human CEA mAb bound magnetic beads were added to a solution containing different concentrations of IgG or CEA. The target was selectively captured by the corresponding magnetic beads and resuspended in PBS by applying an external magnetic field. In step 2, anti-human IgG pAb binding or anti-human CEA pAb bound SERS nanoprobes were added to the solution to form a sandwich immune complex consisting of magnetic beads, target protein and SERS based nanoprobes. The unbound SERS nanoprobe was then removed by applying a magnetic field, and the resulting sandwiched immune complex was resuspended in PBS. In the final step, Raman spectra were identified following Raman shift for SERS-based quantitative analysis of the target with a linear correlation between relative Raman intensity and concentration of target protein.

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.

제 22nd 발명: invent:

The present invention provides Janus nanostructures consisting of:

A double-metal nanoparticle compartment having a core-satellite structure comprising a metal nanoparticle core to which a ligand is adsorbed, and a metal satellite reduced to a ligand adsorption portion of the core; And

Conductive polymer block.

The ligand may be a charged ligand or a ligand having two reactors, and the metal nanoparticle core may be a positively charged metal nanoparticle core or a negatively charged metal nanoparticle core.

The chargeable ligand may be a polymeric ligand including a charging unit, and the ligand having two reactors may be a small molecule ligand.

Polymeric ligands including the charging unit (repeating unit) are PS (poly (sodium-4-styrenesulfonate)), PVP (poly (N-vinyl pyrrolidone)), PDADMAC (poly (diallyldimethylammonium chloride)), PAA (polyacrylic acid ) Or PAH (poly (allylamine) hydrochloride) may be at least one selected from the group consisting of, but is not necessarily limited thereto, preferably PSS.

In the ligand having two reactors, the two reactors may be, but are not necessarily limited to, thiol group (-SH) and amine group (-NH 2).

The small molecule ligand having the two reactors is selected from the group consisting of ATP (4-aminothiophenol), BDT (1,4-benzenedithiol), MBA (4-mercaptobenzoic acid) and MBIA (2-mercaptobenzoimidazole-5-carboxylic acid) At least one may be, but is not necessarily limited thereto, and may be preferably ATP.

The positively charged metal nanoparticle core may be a metal nanoparticle capped with a positively charged material, but is not necessarily limited thereto. Specifically, the positively charged material may be cetyltrimethylammonium bromide (CTAB).

The negatively charged metal nanoparticle core may be a metal nanoparticle capped with a negatively charged material, but is not necessarily limited thereto. Specifically, the negatively charged material may be citric acid.

In the dual metal nanoparticle compartment of the core-satellite structure, the core metal nanoparticle and the metal of the satellite may each be selected from the group consisting of silver, gold, copper and mixtures thereof, but are not limited thereto. Any metal that is widely used in the art may be used without limitation.

The core metal and the satellite metal may not be identical to each other.

The metal nanoparticles of the metal nanoparticle core may be metal nanorods or metal nanospheres, but are not necessarily limited thereto, and any metal nanoparticles having a form widely used in the art may be used. It can be used without limitation.

The double metal nanoparticle compartment of the core-satellite structure may further include a Raman dye.

In the present invention, the "Janus nanostructure" refers to a nanostructure composed of two different compartments (core-satellite double metal nanoparticle compartment and conductive polymer compartment).

In the present invention, the double metal nanoparticle means a nanoparticle having a metal core-metal satellite structure. The metal core-metal satellite structure is named after the shape of a planet and its orbiting satellites. The core-satellite form of the present invention is a shape in which a satellite metal is embedded in a metal core.

The double metal nanoparticle compartment consists of a metal nanoparticle core onto which ligand is adsorbed, and a metal satellite reduced to the ligand adsorption portion of the core. In addition, the conductive polymer compartment is attached to only one side of the double metal nanoparticle compartment and grows, that is, eccentrically deposited to form an asymmetric Janus nanostructure.

As used herein, "Janus nanostructures" refers to "Janus nanoparticles" or Janus nanoprobes or asymmetric Janus nanostructures or asymmetrical, as the structure comprises a double metal nanoparticle compartment-conductive polymer compartment of a core-satellite structure. It is also named nanostructure.

In another aspect, the present invention provides a metal nanoprobe for surface-enhanced Raman scattering (SERS) based target material detection using Janus nanostructures according to the present invention.

Janus nanostructures according to the invention can be provided as metal nanoprobes for surface-enhanced Raman scattering based target material detection by including Raman dyes.

In the present invention, nanoprobe means a probe of a nano size.

The present invention provides a method for producing Janus nanostructures comprising the following steps:

i) preparing positively or negatively charged core metal nanoparticles;

ii) adsorbing a negatively charged ligand or a ligand having two reactors to the core metal nanoparticle;

iii) adding the ligand-adsorbed core metal nanoparticles to an aqueous solution in which the conductive polymer monomer and the surfactant are dissolved,

iv) adding a metal ion solution to the solution to which the core metal nanoparticles of iii) are added to perform a redox reaction between the metal ion and the conductive polymer monomer; And

v) the metal ions in the portion adsorbed by the ligand of the core metal nanoparticles to receive the electrons provided by the conductive polymer is reduced to form a satellite metal, thereby forming a double metal nanoparticle compartment of the core-satellite structure The conductive polymer monomer is oxidized and deposited on only one side of the double metal nanoparticle compartment to grow into a conductive polymer to form asymmetrically conductive polymer polymer compartment to form Janus nanoparticles; step.

In the positively or negatively charged core metal nanoparticles of step i),

The positively charged core metal nanoparticle may be a metal nanoparticle capped with a positively charged material, but is not necessarily limited thereto. Specifically, the positively charged material may be cetyltrimethylammonium bromide (CTAB).

The negatively charged core metal nanoparticle may be a metal nanoparticle capped with a negatively charged material, but is not limited thereto. Specifically, the negatively charged material may be citric acid.

In the negatively charged ligand of step ii) or a ligand having two reactors,

The negatively charged ligand may be a polymeric ligand including a charging unit, and a ligand having two reactors may be a small molecule ligand.

Specifically, the polymeric ligand including the charging unit may include PSS (poly (sodium-4-styrenesulfonate)), PVP (poly (N-vinyl pyrrolidone)), PDADMAC (poly (diallyldimethylammonium chloride)), PAA (polyacrylic acid) or PAH (poly (allylamine) hydrochloride) may be at least one selected from the group consisting of, but is not necessarily limited thereto, preferably may be PSS.

In the double metal nanoparticle compartment of the core-satellite structure of step v), the core metal nanoparticle and the metal of the satellite may be selected from the group consisting of silver, gold, copper and mixtures thereof, respectively. It is not necessarily limited thereto, and any metal widely used in the art may be used without limitation.

The metal ions of step iv) may be selected from the group consisting of gold ions, silver ions, copper ions, and mixtures thereof.

The core metal and the satellite metal may not be identical to each other.

The conductive polymer of step iii) may be at least one selected from the group consisting of polypyrrole, polythiophene, poly (3,4-ethylene dioxythiophene) (PEDOT), and polyaniline, It is not necessarily limited thereto, but may preferably be polyaniline.

The metal nanoparticles of the core metal nanoparticles of step i) may be metal nanorods or metal nanospheres, but are not necessarily limited thereto, and are widely used in the art. Any particle can be used without limitation.

The growth of the conductive polymer of step v) may be by surface-templated polymerization.

The "surface template polymerization method" refers to a polymerization method based on an oxidation-reduction reaction, and in this specification, polyaniline, which is a conductive polymer, is deposited on a double metal nanoparticle through a spontaneous redox reaction between silver nitrate and an aniline monomer. Specifically, aniline monomers having a primary amine group donate electrons to silver nitrate and silver ions receive corresponding electrons to balance the oxidation-reduction reaction so that the conductive polymer monomer is oxidatively polymerized on the double metal nanoparticles. Polyaniline was deposited.

After the step v), it may further comprise the step of attaching a Raman dye on the surface of the double metal nanoparticles of the Janus nanoparticles.

The surfactant of step iii) may be at least one selected from the group consisting of sodium dodecyl sulfate (SDS), sodium deoxycholate, and Triton X-200, It is not necessarily limited to this, but may preferably be SDS.

The production of Janus nanostructures consisting of a double-metal nanoparticle compartment and a polymer compartment of a core-satellite structure according to the present invention and a method of applying SERS-based biosensing are shown in FIG. 1. Figure 1 (a, b) shows a method for producing Janus nanostructure consisting of a double metal Au core-Ag satellite nanoparticle compartment and a polymer compartment through ligand-mediated interface control and spontaneous redox reaction. As can be seen in Figure 1 (a), for the synthesis of double metal AuNR core-Ag satellite nanoparticle compartments, the AuNR surface was functionalized by coating CTAB-capped AuNR with PSS, a negatively charged polymer electrolyte. PSS, a polymeric ligand and negatively charged polyelectrolyte, was adsorbed onto AuNR capped with positively charged CTAB through electrostatic interaction. A specific concentration of PSS with a molecular weight of 70,000 g / mol was added to the AuNR solution in the presence of NaCl. Flexible polymers with long chain lengths compared to the size of metal nanoparticles allow electrically charged polymers to be coated on oppositely charged MNPs. In addition, because the salt concentration affects the electrostatic interaction between the electrically charged polymer and the metal nanoparticles, NaCl was used to have an extended form of high charge polymer, which is the oppositely charged metal nano It is sufficient to coat the particles efficiently. High salt concentrations can lead to uncontrolled aggregation of metal nanoparticles due to increased ionic strength. In addition, in order to produce stable polymer-metal nanoparticle composites, polymers in a suitable concentration range are needed to prevent precipitation of metal nanoparticles. In addition, a Janus structure having a double metal nanoparticle compartment and a polymer compartment was prepared through an oxidation-reduction reaction between the aniline monomer and the silver nitrate in the presence of the surfactant SDS. Specifically, the polymer-coated AuNR was added to the aniline solution and SDS and silver nitrate were added to initiate oxidative polymerization of the aniline monomer and further reduction of silver ions. Interestingly, silver ions were reduced on the surface modified by ligands of gold nanorod particles (AgNPs), so that many silver nanoparticles (AgNPs) were used as satellite particles on AuNR coated with polymers.

We preferentially adsorb Ag ⁺ ions onto the polymer-coated AuNR through electrostatic interaction between positively charged silver ions and negatively charged sulfonic acid functional groups on the core surface, and AgNPs to satellite particles. It was judged that it is heterogeneous deposition. In addition, the co-oxidative polymerization of aniline was initiated by silver ions to form poly (aniline) compartments deposited on only one side of the double metal core-satellite nanoparticles in the presence of SDS. The partitioning of Janus nanostructures with double metal core-satellite nanoparticle compartments and polymer compartments was achieved by the balance of total surface energy, which means that SDS has two adjacent phases, poly (aniline) -water and poly (aniline) -metal. This may affect the interfacial tension between nanoparticles. In a control experiment, a negatively charged molecule, sodium citrate, was introduced as a small ligand to further confirm ligand mediated surface functionalization, but no core-satellite nanostructures were observed under these conditions. As CTAB was densely packed along the transverse axis of AuNR, the citrate ions were placed between AuNRs so that the AuNRs were arranged side by side through electrostatic interactions between the CTAB and the bilayer of citrate ions. This is because it is difficult for small ligands to replace the CTAB coating to functionalize the entire AuNR surface.

Meanwhile, Janus nanostructures having double metal nanoparticle compartments and polymer compartments of AuNP (AuNS) core-Ag satellites were synthesized by small ligand mediated surface control and redox reactions as shown in FIG. Specifically, to prepare double metal AuNP core-Ag satellite nanoparticle compartments, citric acid-capped AuNPs were functionalized with small molecule ligands. ATP, a small molecule ligand containing thiol groups and amine groups distributed in the radial direction, was introduced onto citric acid-capped AuNPs (AuNS). After simultaneous oxidation of the aniline monomer and reduction of the silver ions, Janus nanostructures having anisotropic polymer compartments were prepared. The ligand density on AuNP (AuNS) determines the degree of Ag ion coordination and the reduction rate of Ag. Small molecule ligands have been used to control the interfacial tension between two metal nanoparticle layers in metal nanoparticle growth pathway control. Ligands embedded at the interface between Au and Ag are the main elements that bind the second metal. In a control experiment, a positively charged polyelectrolyte, poly (dimethylaminoethyl methacrylate), was introduced into the AuNP solution as a polymer ligand, but the electrostatic between citrate-capped AuNP and positively charged polymer ligand Due to the miraculous interaction, stable ligand-coated metal nanoparticle structures were not formed and large metal nanoparticle aggregates were formed. Since AuNPs are weakly capped with citrate ions, AuNPs tend to aggregate easily upon addition of heterogeneous materials. In this regard, the small ligand ATP was chosen at a concentration sufficient to maintain the colloidal stability of AuNPs. Figure 1 (c) shows a spontaneous redox reaction between the aniline monomer and silver nitrate. Aniline monomers were oxidized to poly (aniline) by providing electrons to silver nitrate, while silver ions received electrons and were reduced.

As can be seen in Figure 1 (d), Janus nanostructure consisting of a core-satellite double metal nanoparticle compartment and a polymer compartment can be applied as SERS nano probe for bio sensing. The polymer compartment provided the antibody conjugation site for target detection, while the double metal particle compartment was functionalized with a Raman dye for SERS. When the target was present, a sandwich immune complex consisting of SERS nanoprobe, target and magnetic beads was formed, and both quantitative and qualitative SERS based biosensing was achieved as a function of Raman migration.

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

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

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

c) preparing by immobilizing a second antibody against the target on the metal nanoprobe;

d) adding the magnetic nanoparticle 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 nanoparticle are conjugated;

e) a sandwich immunocomplex of the first antibody of the second antibody-target-magnetic nanoparticle of the metal nanoprobe by adding a metal nanoprobe to which the second antibody is immobilized to a solution containing the immunocomplex to which the first antibody is conjugated 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 said sandwich immunocomplex; step.

By "sandwich immune complex" is meant herein an immunocomplex bound through an antibody-antigen-antibody reaction. The antigen was named as it is inserted in the middle of the antibody to form a sandwich.

The target substance may be a protein or a pathogen.

Janus nanostructures comprising a double metal Au core-Ag satellite nanoparticle compartment and a polymer compartment according to the present invention are functionalized by coating a CTAB-capped AuNR with PSS, a negatively charged polymer electrolyte, to functionalize the AuNR surface, Capping AuNPs were functionalized with small molecule ligands to form double metal nanoparticle compartments modified with charged polymers or ligands. In this manner, the silver ions are reduced only in the surface-modified portion to form a nano gap, thereby greatly improving Raman strength. Accordingly, the Janus nanostructures of the present invention can be used as metal nanoprobes for surface-enhanced Raman scattering (SERS) based target material detection for target material detection.

제 33rd 발명: invent:

The present invention provides an asymmetric Janus nanoprobe for surface-enhanced Raman scattering (SERS) based target material detection comprising:

A double metal nanocluster compartment having a Raman dye and consisting of a core-shell structure;

And conductive polymer compartments;

Nanoprobe consisting of,

The nanoprobe exhibits an asymmetrical structure in which the conductive polymer compartment is oxidized on only one side of the double metal nanocluster compartment.

In the present invention, the probe refers to a substance that can specifically bind to a target (target) substance to be detected, and means a substance that can confirm the presence of the target substance through the binding.

In the present invention, nanoprobe means a probe of a nano size.

In the present invention, the Raman dye means a Raman active organic compound, and any one widely used in the art may be used without limitation. Specific examples include Malachite green isothiocyanate (MGITC), rhodamine B isothiocyanate (RBITC), rhodamine 6G, adenine, 4-amino-pyrazole (3,4-d) pyrimidine, 2-ruoroadenine, N6-benzoyl Adenine, kinetin, dimethyl-allyl-amino-adenine, zeatin, bromo-adenine, 8-aza-adenine, 8-azaguanine, 4-mercaptopyridine, 6-mercaptopurine, 4-amino- 6-mercaptopyrazolo (3,4-d) pyrimindine, 8-mercaptoadenin, 9-amino-acridine and mixtures thereof, but is not necessarily limited thereto.

The double metal nanocluster compartment may comprise a core selected from the group consisting of gold, silver, copper and mixtures thereof; It may be a double metal nanocluster consisting of; a shell selected from the group consisting of gold, silver, copper and mixtures thereof.

The core metal and the shell metal may be the same metal or may not be the same metal.

In the present invention, the "asymmetric Janus nanoprobe" is a nanoprobe composed of two different compartments (a double metal nanocluster compartment and a conductive polymer compartment having a Raman dye and having a core-shell structure), and a conductive polymer compartment. It exhibits an asymmetric structure that is oxidized and grown, i.e., eccentrically deposited, on only one side of the double metal nanocluster compartment. As used herein, “asymmetric Janus nanoprobe” refers to “asymmetric Janus nanocluster-polymer nanoparticles” or asymmetric nanoprobes or asymmetric Janus nanostructures, as the structure includes a double metal nanocluster compartment-conductive polymer compartment. Or asymmetric nanostructures.

In the present invention, the double metal nanocluster refers to aggregates in which a different kind of metal ion is reduced on the core metal nanocluster to form a core-shell structure.

A double metal nanocluster compartment consisting of a core-shell structure according to the present invention; And asymmetric Janus nanostructures consisting of conductive polymer compartments can highly enhance SERS properties from interparticle coupling between core-shell nanoparticles in the double metal nanoclusters. It is also possible to improve the reactivity between the target and the antibody by attaching the antibody to the target to be detected in the polymer compartment. Therefore, the asymmetric Janus nanoprobe according to the present invention can be utilized as a nanoprobe for detecting SERS-based target material.

In another aspect, the present invention provides a method of making an asymmetric Janus nanoprobe for detecting surface-enhanced Raman scattering (SERS) based target material, comprising the following steps:

i) mixing and heating or agglomerating the metal nanoparticles and the Raman dye forming the core to form a core metal nanoparticle cluster having a Raman dye;

ii) adding the core metal nanoparticle cluster to an aqueous solution in which a conductive polymer monomer and a surfactant are dissolved;

iii) performing a redox reaction between the metal ions and the conductive polymer monomer by adding a metal ion solution to the solution to which the core metal nanoclusters of ii) are added; And

iv) the metal ions are deposited on the surface of the core metal nanoparticles while receiving and reducing electrons provided by the conductive polymer to form a double-metal nanoparticle cluster section having a core-shell structure; The conductive polymer monomer is oxidized and deposited on only one side of the double metal nanocluster compartment to grow into a conductive polymer to form an asymmetrically conductive polymer compartment; step.

After step i), the method may further include stabilizing the core metal nanocluster with a protein.

The protein may be selected from the group consisting of avidin (avidin), streptavidin (streptavidin), bovine serum albumin (BSA), insulin (insulin), soy protein, casein, gelatin and mixtures thereof, but are not limited thereto. But may preferably be BSA.

The core metal of step i) may be selected from the group consisting of gold, silver, copper and mixtures thereof, but is not necessarily limited thereto, and any metal widely used in the art may be used without limitation.

Specifically, the core metal may be selected from the group consisting of gold, silver, copper and mixtures thereof.

The metal ion of step iii) may be selected from the group consisting of gold ions, silver ions, copper ions and mixtures thereof, but is not necessarily limited thereto.

The silver ions include silver nitrate (AgNO ₃ ), tetrafluoroborate silver (AgBF ₄ ), trifluoromethanesulfonate silver (AgCF ₃ SO ₃ ), silver perchlorate (AgClO ₄ ), silver acetate (Ag (CH ₃ COO)), Hexafluorophosphate may be selected from the group consisting of silver (AgPF ₆ ), Ag (CF ₃ COO) and mixtures thereof, but is not necessarily limited thereto, and preferably silver nitrate.

The copper ions include copper (II) acetylacetonate (Cu (acac) ₂ ), copper chloride (CuCl), copper chloride (II) (CuCl ₂ ), copper (II) hexafluoroacetylacetonate (Cu (hfac) ₂ ), copper (II) trifluoroacetylchloride (Cu (tfac) ₂ ), copper (II) dipiballomethacrylate (Cu (dpm) ₂ ), copper (II) pentafluorodimethylheptanedione (Cu ( ppm) ₂ ), copper (II) heptafluorodimethyloctane (Cu (fod) ₂ ), copper (II) iminopentanone (Cu (acim) ₂ ), copper (II) hexafluoro-[(trifluoro Roethyl) imino] -pentanone (Cu (nona-F) ₂ ), copper (II) acetylacetoethylenediamine (Cu (acen) ₂ ), copper nitrate (Cu (NO ₃ ) ₂ ), copper sulfate (CuSO ₄ ) And mixtures thereof, but is not necessarily limited thereto.

The conductive polymer of step ii) may be at least one selected from the group consisting of polypyrrole, polythiophene, poly (3,4-ethylene dioxythiophene) (PEDOT), and polyaniline. Although not limited thereto, it may be preferably polyaniline.

After the oxidation-reduction reaction of step iii), the reaction solution may further comprise the step of incubating the surfactant solution.

After the redox reaction of step ii) and step iii), the surfactant is sodium dodecyl sulfate (SDS), sodium deoxycholate, and Triton X-200 (Triton X-200) It may be at least one selected from the group consisting of, but is not necessarily limited thereto, and preferably may be SDS.

The "surface template polymerization method" refers to a polymerization method based on an oxidation-reduction reaction. In this specification, polyaniline, which is a conductive polymer, is deposited on a double metal nanocluster through a spontaneous redox reaction between silver nitrate and an aniline monomer. Specifically, aniline monomers having a primary amine group donate electrons to silver nitrate and silver ions receive corresponding electrons to balance the redox reaction so that the conductive polymer monomer is oxidatively polymerized on the double metal nanoclusters. Polyaniline was deposited.

A schematic diagram of the synthesis and SERS-based application of the asymmetric Janus nanocluster-polymer nanoparticles according to the present invention is shown in FIG. 1. Figure 1 (a) shows a method for producing SERS nanoprobe based on the reduction oxidation reaction. First, gold nanoparticle (AuNP) clusters were formed by agglomeration of AuNPs in the presence of Raman dye and stabilized by BSA coating. Specifically, citric acid capped AuNP was mixed with Raman dye MGITC or RBITC at a final concentration of 1.5 μM or 3.8 μM, respectively. In addition, silver nitrate and sodium citrate were added to the AuNP colloidal solution and incubated at 95 ° C. for 10-60 minutes. When silver ions were reduced to Au, Au nanoparticles grew and agglomerated at the same time by adsorbing Raman dye on the surface. When the isothiocyanate group (-N = C = S) of MGITC and RBITC is strongly bound to the AuNP surface, the electrostatic interaction between negatively charged particles and particles labeled with Raman dye with cationic surface charge This occurred to form AuNP clusters. Au nanoclusters containing Raman dyes were stabilized by BSA to prevent further aggregation. Second, asymmetric double metal nanocluster-polymer nanostructures were prepared by redox reactions between silver nitrate and aniline monomers. Concentrated AuNP clusters were added to the solution containing the aniline monomer and the surfactant SDS. After mixing, silver nitrate was added to the solution to initiate the oxidative polymerization to poly (aniline) by reducing silver ions in the AuNP cluster to form a double metal Au (core) -Ag (shell) nanocluster. In the presence of SDS, anisotropic (asymmetric) partitioning of double metal nanoclusters and poly (aniline) was formed to balance the interfacial tension between poly (aniline) -double metal nanoclusters and-water by minimizing the surface area. Figure 1 (b) shows a spontaneous redox reaction between two precursors. Aniline monomers with primary amine groups donated electrons to silver nitrate and silver ions received corresponding electrons to balance the redox reaction. Figure 1 (c) shows a SERS-based biosensing (biosensing) method using an asymmetric Janus nanocluster-polymer nanoprobe. Specifically, antibodies were introduced by selectively functionalizing the polymer compartments. When the target molecule is present, the SERS nanoprobe to which the target specific antibody is bound and the magnetic beads capture the corresponding target to form a sandwich immune complex consisting of the SERS nanoprobe, the target and the magnetic beads. These complexes were washed by applying a magnetic field and Raman shift appeared according to the target concentration.

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

g) measuring the Raman signal of said sandwich immunocomplex; step.

The target material may be a protein or a pathogen.

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

제 44th 발명: invent:

The present invention provides an asymmetric Janus nanostructure consisting of:

A dual metal nanorod cluster compartment comprising a directional metal nanorod cluster seed and a metal shell structure; And

Conductive polymer block.

In the seed-shell structured double metal nanorod cluster compartment,

The metal nanorod cluster seed and the shell metal, respectively, may be selected from the group consisting of silver, gold, copper, and mixtures thereof, but are not necessarily limited thereto, and any metal widely used in the art is limited. Can be used without.

The directional metal nanorod cluster seed is formed in a side-by-side assmebly form in which side surfaces of the individual metal nanorod particles are aligned, or in which ends of the individual metal nanorod particles are connected to each other. -to-end assembly).

In the present invention, the "side of the metal nanorod particles" refers to two side portions having a long length in a narrow and long form of the metal nanorods.

In the present invention, the “side-by-side assmebly form” in which the side surfaces of the metal nanorod particles are arranged side by side is formed in a narrow and long form of the metal nanorod, in which two long surface portions contact each other, continuously It means the form that is arranged and assembled. In the present specification, the "side-by-side assmebly form" of the metal nanorod particles is also referred to as "side-to-side assembly", as the form is made by the side-to-side assembling. do.

In the present invention, the "end of the metal nanorod particles" refers to two surface portions having a short length in a narrow and long form of the metal nanorods.

In the present invention, the "end-to-end assembly form of the ends of the metal nanorod particles" in the narrow and long form of the metal nanorods, the two surface portions of short length abut each other in a diagonal direction, It means the form that is continuously arranged and assembled. In the present specification, "end-to-end assembly form of the metal nanorod particles" is also referred to as "end-to-end assembly", as the form is formed by the end-to-end assembly. .

The double metal nanorod cluster compartment may further comprise a Raman dye.

In the present invention, the "asymmetric Janus nanostructure" refers to a nanostructure composed of two different partitions (seed-shell structure double metal nanorod cluster compartment and conductive polymer compartment) which are not symmetrical with each other. In the present invention, the double metal nanorod cluster compartment is composed of a directional metal nanorod cluster seed and a metal shell, and the conductive polymer compartment is attached to only one side of the double metal nanorod cluster compartment and grows, that is, eccentrically. deposited to form an asymmetric Janus nanostructure.

Asymmetric Janus nanostructures herein are also termed "double metal-polymer Janus nanoparticles" or "Janus nanoparticles" or Janus nanoprobes.

In the present invention, the double metal means two kinds of metals forming a metal seed-metal shell structure.

In the present invention, the metal nanorod cluster is a term used to mean that the metal nanorods are collected, 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 forming a seed-shell structure are collected.

The present invention provides a metal nanoprobe for surface-enhanced Raman scattering (SERS) signal measurement using an asymmetric Janus nanostructure according to the present invention.

Janus nanostructures according to the present invention can be provided as a metal nanoprobe for measuring surface-enhanced Raman scattering signal by including a Raman dye.

In the present invention, nanoprobe means a probe of a nano size.

The present invention provides a method for preparing an asymmetric Janus nanostructure comprising the following steps:

i) mixing the metal nanorod particles forming the seed and the negatively charged stimulatory copolymer having a thiol group at the organic anion or the terminal to form a metal nanorod cluster seed having an orientation;

ii) adding the metal nanorod cluster seed to an aqueous solution in which the conductive polymer monomer and the surfactant were dissolved,

iii) adding a metal ion solution to the solution to which the metal nanorod cluster seed of ii) is added to perform a redox reaction between the metal ion and the conductive polymer monomer;

iv) the metal ions are deposited on the surface of the seed metal nanorod particles while receiving and reducing electrons provided by the conductive polymer to form a seed metal shell double metal nanorod cluster section; The conductive polymer monomer is oxidized and deposited only on one side of the double metal nanorod cluster compartment to grow into a conductive polymer to form an asymmetrically conductive polymer compartment; step.

After step i), the method may further include attaching a Raman dye on the surface of the metal nanorod cluster seed.

The metal nanorod cluster seed having the directionality of step i) may be formed in a side-by-side assmebly side by side of the individual metal nanorod particles, or the ends of the individual metal nanorod particles are connected to each other. It may be in an end-to-end assembly form.

Sides of the individual metal nanorod particles are arranged side by side (by side-by-side assmebly) form of metal nanorod cluster seed, it may be prepared by including the following steps:

Preparing metal nanorod particles in which a positively charged surfactant is present on the surface using the metal seeds;

Adding a Raman dye to the solution containing the metal nanorods; And

Adding an organic anion to the metal nanorod solution containing the Raman dye;

Electrostatic attraction between the positively charged surfactant attached to the side of the metal nanorods and the organic anion causes the side surfaces of the individual metal nanorods to be arranged side by side with other side metal nanorods to form a metal nanorod cluster seed. Steps.

By the electrostatic attraction between the positively charged surfactant attached to the side of the metal nanorods and the organic anion, the sides of the individual metal nanorods are arranged side by side with the other side of the other metal nanorods to assemble the metal nanorod cluster seeds. Forming " specifically refers to the positively charged surfactant-organic anion-second metal nanorod side attached to the first metal nanorod side by electrostatic attraction (interaction) between the positively charged surfactant and the organic anion. A bond consisting of a positively charged surfactant attached to the substrate is continuously generated, and means that the side surfaces of the individual metal nanorods are assembled by being aligned with the side surfaces of other individual metal nanorods. Since the assembly occurs spontaneously by electrostatic attraction, it is referred to herein as "self assembly".

In one embodiment of the present invention, the electrostatic attraction between the CTAB bilayer and citrate induced side-to-side self-assembled gold nanorod (AuNR) clusters.

The positively charged surfactant may be selected from the group consisting of hexadecyltrimethylammonium bromide (CTAB), dodecyltrimethylammonium bromide (DTAB), and trimethyltetradecylammonium bromide (TTAB), but is not necessarily limited thereto, and preferably may be CTAB.

The organic anion may be selected from the group consisting of citrate, maleate, fumarate, tartrate, succinate, oxalate, and gluconate, but is not necessarily limited thereto, preferably citrate, that is, citric acid It may be a salt.

Ends of the individual metal nanorod particles are connected to the end-to-end assembly of the metal nanorod cluster seed, which may be prepared by the following steps.

Adding a negatively charged stimulatory copolymer having a thiol group at the end to the solution containing the metal nanorods; And

Stirring the metal nanorod solution to which the negatively charged stimulatory copolymer is added;

Adding Raman dye to the stirred solution;

The positively charged surfactant at the end of the metal nanorod and the thiol group of the negatively charged stimuli-reactive copolymer combine with each other to form a metal-thiol group bond, and the positively charged surfactant at the side of the individual metal nanorod and the other individual metal nanorod Bonding the terminal negatively charged stimuli-responsive copolymers with electrostatic attraction so that the ends of the individual metal nanorod particles are joined together to form a metal nanorod cluster seed.

"The positively charged surfactant at the end of the metal nanorod and the thiol group of the negatively charged stimulatory copolymer combine with each other to form a metal-thiol group bond", whereby the positively charged surfactant at the end of the metal nanorod is Exchanged for coalescence.

"The positively charged surfactant on the side of the individual metal nanorods and the negatively charged stimulatory copolymer at the ends of the other individual metal nanorods are bonded together by electrostatic attraction so that the ends of the individual metal nanorod particles are assembled and assembled to form a metal nanorod cluster. Seed formation "is specifically defined as" positive charge-surfactant-second metal nanorods on the side of the first metal nanorods by electrostatic attraction (interaction) between the positively charged surfactant and the negatively charged stimulatory copolymer. The bond consisting of the negatively charged stimuli-copolymer on the end face of is continuously generated, meaning that the ends of the individual metal nanorods (more specifically, the side-ends) are connected in a diagonal direction and assembled. Since the assembly occurs spontaneously by electrostatic attraction, it is referred to herein as "self assembly".

In one embodiment of the present invention, the CTAB ligand attached to the terminal of the surface-modified AuNR was exchanged with a negatively charged poly (AAc-b-NIPAM) -SH through metal-thiol group bonds. Accordingly, the electrostatic attraction between positively charged CTAB on the side of AuNR and poly (AAc-b-NIPAM) on the end face of the other AuNR induced an end-to-end self-assembled AuNR cluster.

As used herein, the term "stimulatory responsiveness" refers to a property that changes behavior in response to a stimulus (eg, heat).

In the present invention, the "negative stimulus-reactive copolymer" refers to a copolymer having a negative charge, in particular a copolymer having a negative charge whose behavior changes in response to a stimulus.

The negatively charged stimuli copolymer may be a copolymer consisting of a negatively charged moiety and a stimulatory polymer. The copolymerization method of the negatively charged moiety and the stimuli polymer may be any copolymerization method known in the art. In one embodiment of the present invention it was synthesized by a sequential reversible addition-fragmentation chain transfer (RAFT) polymerization, followed by additional amino decomposition and hydrolysis processes.

In the present invention, the term "moiety" means a substance, and means a part that indicates the properties of the substance.

In the present invention, the "negative moiety" means a material having negative charge properties.

The negatively charged moiety is selected from the group consisting of acrylic acid, methacrylic acid, itaconic acid, maleic acid and mixtures thereof. It may be, but is not necessarily limited to this, it may be preferably an acrylic acid.

In the present invention, the "stimulatory polymer" means a polymer whose behavior changes in response to a stimulus.

The stimulatory polymer is poly (N-isopropylacrylamide) [poly (N-isopropylacrylamide): polyNIPAM], poly (N-diethyl acrylamide) [poly (N, N'-diethyl acrylamide): polyDEAAm], poly (Dimethylamino ethyl methacrylate) [poly (dimethylamino ethyl methacrylate): polyDMAEMA], poly (N-hydroxymethyl propyl methacrylamide) [poly (N- (L)-(1-hydroxymethyl) propyl methacrylamide)], Poly [oligo (ethylene glycol) methyl ether methacrylate]: POEGMA], poly (2-vinyl pyridine): P2VP], poly ( 4-vinyl pyridine) may be selected from the group consisting of [poly (4-vinyl pyridine): P4VP] and mixtures thereof, but is not necessarily limited thereto, and may be preferably poly (N-isopropylacrylamide). have.

The negatively charged stimuli-reactive copolymer is not necessarily limited thereto, but may be preferably poly (acc-b-NIPAM) (poly (acrylic acid-block-N-isopropylacrylamide)). Poly (AAc-b-NIPAM) according to the present invention exhibits negative charge properties due to acrylic acid. In addition, since it has a thiol group (-SH) at the terminal, it is also named "poly (AAc-b-NIPAM) -SH" in this specification.

According to the present invention, the synthesis step of poly (AAc-b-NIPAM), a negatively charged irritant copolymer synthesized by a subsequent amino reversible addition-fragmentation chain transfer (RAFT) polymerization, followed by further amino decomposition and hydrolysis processes, is shown in FIG. 3. Shown in First, poly (tBA) (poly (tBA)) was prepared by RAFT polymerization using tBA (tert-butyl acrylate) as monomer, CDTPA as chain transfer agent (CTA) and AIBN (azobisisobutyronitrile) as initiator, 2,2'-azobis (2-methylpropionitrile). (tBA) -macro CDTPA) was synthesized. Poly (tBA-b-NIPAM) was prepared by RAFT polymerization using NIPAM (N-isopropylacrylamide 97%) as a monomer, poly (tBA) -macro CDTPA previously synthesized with CTA, and AIBN as an initiator. The thiol-terminated poly (tBA-b-NIPAM) was formed by amino decomposition of the thiocarbonylthio group in the prepared poly (tBA-b-NIPAM) to thiol (Fig. 3 (a)), and the thiol-terminated formed Thiol-terminated poly (AAc-b-NIPAM) was prepared by converting large hydrophobic end groups of poly (tBA) blocks in poly (tBA-b-NIPAM) through hydrolysis to carboxyl groups (FIG. 3 (b)).

Poly (AAc-b-NIPAM), a negatively charged stimuli copolymer prepared as described above, is used to selectively modify the end face of AuNR.

After the oxidation-reduction reaction of step iii), the method may further include incubating the reaction solution with a surfactant solution.

The surfactant after the redox reaction of step iii) or step iii) comprises sodium dodecyl sulfate (SDS), sodium deoxycholate, and Triton X-200. It may be at least one selected from the group, but is not necessarily limited thereto, preferably may be SDS.

A schematic diagram of the synthesis of side-by-side or end-to-end directional self-assembled AuNR and its Janus nanostructures is shown in FIG. 1. Figure 1 (a) shows the controlled assembly process of AuNR through electrostatic interaction. Side-to-side assembly of AuNR was achieved by the addition of citrate anions. During the synthesis of AuNR, CTAB surfactants were used to induce asymmetric forms and to maintain the colloidal stability of AuNR. As the citrate anion and the Raman dye MGITC were added to the concentrated AuNR solution, the electrostatic attraction between the CTAB bilayer and the citrate induced side-to-side assembled AuNR clusters. MGITC was effectively embedded in the CTAB bilayer and located at the interparticle junctions between adjacent AuNRs in side-to-side assembled clusters. The assembled side-to-side AuNR clusters were stabilized by coating with PSS. On the other hand, end-to-end assembly of AuNR exchanges the CTAB ligand attached to the end of AuNR with poly (AAc-b-NIPAM) through thiol group-metal bonds, and the end face exchanged with positively charged CTAB on the side of AuNR. Electrostatic attraction between poly (AAc-b-NIPAM) on the phase led to end-to-end self-assembly of AuNR. Figure 1 (b) shows the synthesis of an asymmetric double metal nanorod cluster-polymer Janus nanostructures using directional self-assembled AuNR clusters as seeds. Janus nanostructures were prepared through redox-reduction between silver nitrate and aniline monomers. Poly (aniline) compartments were formed through surface template polymerization of the aniline monomers disclosed by reducing silver nitrate in the presence of the surfactant SDS, so that poly (aniline) was eccentrically deposited on the aromatically assembled AuNR. The reaction proceeded without mechanical agitation for 24 hours and further incubated in SDS solution overnight to prepare asymmetric Janus nanostructured double metal Au core-Ag shell nanorod cluster sections and poly (aniline) sections. Upon addition of SDS, the double metal nanorod cluster compartment was partially captured by the poly (aniline) compartment. SDS, a surfactant, influenced the balance of interfacial tension (σ) in three phases including double metal, poly (aniline) and water to determine the equilibrium arrangement. That is, σ _poly _(aniline) via SDS _{- water} by reducing the interfacial tension (σ _poly _{(aniline) -water)} may balance the three-phase interaction; σ _Ag _- _poly _(aniline) > σ _Ag _-water + σ _poly _{(aniline) -water} . Figure 1 (c) shows the equation for the oxidation bond reaction between the aniline monomer and silver nitrate. Redox reactions include electron transfer in which the aniline monomer provides silver ions to poly (aniline) by oxidation and the silver ions accept the electrons. As can be seen in Figure 1 (d), Janus nanostructures according to the present invention can be applied as a promising SERS nanoprobe for biosensing. Double metal nanorod cluster compartments can exhibit high SERS efficiency, due to hot spot junctions between the interparticle gaps of the directionally assembled AuNR nanoclusters, with the poly (aniline) compartment being the target. Provide an antibody binding site for detection. When the target was present, a sandwich immune complex consisting of SERS nanoprobe, target and magnetic beads was formed, and both quantitative and qualitative SERS based biosensing was achieved as a function of Raman migration.

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

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

g) measuring the Raman signal of said sandwich immunocomplex; step.

The target substance may be a protein or a pathogen.

The asymmetric double metal nanorod cluster-polymer Janus nanostructure according to an embodiment of the present invention is characterized by electrostatic attraction between CTAB-capped AuNR and organic anions or electrostatic attraction between CTAB-capped AuNR and negatively charged stimulatory copolymers. By inducing directional self-assembly using, a double metal nanorod cluster compartment comprising a directional AuNR cluster seed and a metal shell structure is formed, and the gaps between particles of the directionally assembled AuNR nanoclusters are interspersed. Raman strength is greatly improved by hot spot junctions. In addition, since the poly (aniline) compartments provide antibody binding sites for target detection, the asymmetric Janus nanostructures consisting of polymer compartments and double metal nanoparticle compartments comprising directional metal nanorod clusters are used for surface-detection of target substances. It can be used as metal nanoprobe for measuring enhanced Raman scattering (SERS) signal.

제 1First 발명: invent:

The double metal-polymer Janus nanostructure according to the present invention is composed of a double metal nanocluster compartment and a conductive polymer compartment covalently bonded to ODA to induce selective functionalization, and the SERS strength is greatly improved by forming a controlled self-assembly thereof. It became. Therefore, the Janus nanostructures of the present invention can be used as metal nanoprobes for surface-enhanced Raman scattering (SERS) -based or fluorescence-based biosensing and / or bioimaging measurement for target material detection. In addition, the double metal-polymer Janus nanostructures and their self-assembled Janus nanostructure clusters, according to the present invention, contain a positively charged conductive polymer compartment, thereby providing a drug through an electrostatic interaction with a negatively charged drug. It can be used as a drug carrier that can control drug release because it can be supported into the polymer compartment, and can induce drug release by forming a hydrogel containing concentrated drug-supported nanoparticles and changing voltage or pH conditions have.

제 22nd 발명: invent:

Janus nanostructures comprising a double metal Au core-Ag satellite nanoparticle compartment and a polymer compartment according to the present invention have a surface with significantly improved SERS strength as it comprises a double metal nanoparticle compartment modified with a charged polymer or ligand. -Can be used as metal nanoprobes for detection of enhanced Raman scattering (SERS) based target materials.

제 33rd 발명: invent:

The asymmetric Janus nanoprobe according to the present invention highly enhances SERS properties from interparticle coupling between core-shell nanoparticles in metal nanoclusters. Target detection in the polymer compartment and SERS in the metal nanocluster compartment can be measured to improve target detection and optical properties simultaneously. Therefore, the asymmetric Janus nanoprobe of the present invention can be utilized as a functional nanoprobe for SERS-based biosensing.

제 44th 발명: invent:

The asymmetric Janus nanostructure consisting of a double metal nanoparticle compartment and a polymer compartment comprising a directional metal nanorod cluster according to the present invention has a surface-enhanced Raman scattering with a significant improvement in Raman strength as it comprises a directional assembled double metal nanorod cluster. It can be applied as metal nano probe for (SERS) signal measurement.

제 1First 발명: invent:

1 is a schematic diagram of an experimental method for preparing a double metal-polymer Janus nanostructure, its self-assembled Janus nanostructure cluster and its specific structure, and an experimental method for SERS-based biosensing applications; (a) a method of synthesizing a double metal-polymer Janus nanostructure, its self-assembled Janus nanostructure cluster and its specific structure, (b) a spontaneous redox reaction between aniline monomer and silver nitrate for the synthesis of double metal-polymer Janus nanostructure , (c) SERS-based biosensing method using the specific structure of self-assembled Janus nanostructure cluster of double metal-polymer Janus nanostructure.

2 is the UV-Vis absorbance and hydrodynamic diameters of Au nanoparticles, double metal-polymer Janus nanostructures, their self-assembled Janus nanostructure clusters and their specific structures; (a) UV-Vis absorption peaks of AuNP, double metal-polymer Janus nanoparticles and their specific structures (Nanoclusters I, II, III), (b) ^10-6 M Raman dye and 2.968 μM ODA When added respectively, the RBITC- or MGITC-labeled double metal-polymer Janus nanostructures (RB / with MG) and their self-assembled Janus nanostructure cluster-specific structures (Nanoclusters with RB / with MG) UV-Vis absorption spectrum, (c) hydrodynamic diameters of AuNP, double metal-polymer hybrid nanostructures and their specific structures (Nanoclusters I, II, III). The average diameters of Au nanoparticles and double metal-polymer Janus nanoparticles were 30.1 ± 0.5 nm and 62.8 ± 2.3 nm, respectively, and the average diameters of specific structures according to different clustering levels were 168.3 ± 1.3 nm, 192.4 ± 2.4 nm and 266.3. ± 6.0 nm. The z-potential values of Au nanoparticles, double metal-polymer Janus nanoparticles and their self-assembled Janus nanostructure cluster specific structures were -29.5 ± 0.7 mV, -28.0 ± 0.6 mV and -11.2 ± 0.9 mV, respectively (d ) Hydrodynamic diameters of RBITC-labeled, MGITC-labeled double metal-polymer Janus nanoparticles and their self-assembled Janus nanostructure cluster specific structures, respectively. The average diameters of RBITC-labeled and MGITC-labeled double metal-polymer Janus nanoparticles (Hybrid nanostructure with RB / with MG) were 122.1 ± 2.4 nm and 112.0 ± 15.9 nm, respectively, and RBITC-labeled and MGITC-labeled specific structures The hydrodynamic diameters of (Nanoclusters with RB / with MG) were 450.1 ± 3.1 nm and 401.1 ± 10.0 nm, respectively.

3 is a relative Raman spectrum and Raman intensity for optimizing the proper concentration of Raman dye in the concentration range 10 ^-5 -10 ^-8 M. (a) Relative Raman spectra of specific structures labeled with RBITC. R ² = 0.9826, (b) RBITC Raman intensity at 1646 cm ⁻¹ , (c) relative Raman spectrum of specific structure labeled with MGITC, (d) MGITC Raman intensity at 1617 cm ⁻¹ . R ² = 0.9162, (e) Raman spectra of double metal-polymer Janus nanoparticles and their specific structures at 2.968 μM concentration of ODA and 10 ^-6 M concentration of RBITC, (f) double metal at 10 ^-6 M concentration of RBITC Raman intensity of polymer Janus nanoparticles and their specific structures, (g) Raman spectra of double metal-polymer Janus nanoparticles and their specific structures, at 2.968 μM concentration of ODA and 10 ^-6 M concentration of MGITC, (h) of MGITC Raman strength of double metal-polymer Janus nanoparticles and their specific structure at 10 ^-6 M concentration.

4 shows colloidal stability and inter-batch variability over time of specific structures. (a) Colloidal stability over time of a specific structure labeled with MGITC, (b) Colloidal stability over time with a specific structure labeled with RBITC, (c) Variability between batches (S1-S5) of a specific structure labeled with MGITC (d) Variability between batches (S1-S5) of specific structures labeled with RBITC.

5 is a TEM and SEM image to confirm size and shape; (a) TEM image of Au nanoparticles, (b) TEM image of double metal-polymer Janus nanostructures before further incubation overnight in 3.6 mM SDS solution, (c) double incubation overnight in 3.6 mM SDS solution TEM image of metal-polymer Janus nanoparticles. The black part is the double metal cluster compartment and the bright part is the polymer compartment. (D, e, f) TEM image of the specific structure according to the degree of clustering. As the level of clustering increased, self-assembled double metal-polymer Janus nanostructure clusters in the form of dimers, trimers or tetramers were formed. (G) SEM images of double metal-polymer Janus nanostructures. , (h) SEM image of self-assembled double metal-polymer Janus nanostructure cluster specific structure. The scale bar of the TEM image is (a) 100 nm and (b-f) 200 nm, and the scale bar of the SEM image is (g-h) 1.0 μm.

FIG. 6 shows the cumulative release rate of fluorescein from double metal-polymer Janus nanostructures embedded in PEG hydrogels measured with an electric field of + 1.5 v or − 1.5 v every 30 minutes; (a) cumulative release rate upon electrical stimulation of + 1.5 v, (b) cumulative release rate upon electrical stimulation of -1.5 v, (c) fluorescein-filled double metal-polymer Janus nanoparticles at

pH

4, 7 and 11 UV-vis absorbance, (d) Raman spectra of MGITC originating from fluorescein-supported double metal-polymer Janus nanostructures. Raman peaks of MGITC and fluorescein were 1617 cm ⁻¹ and 1176 cm ^−1, respectively.

7 is a schematic of SERS-based immunoassay for the detection of target proteins.

8 is Raman spectrum and Raman intensity of different target concentrations according to IgG concentration. SERS peak intensities of RBITC or MGITC-labeled specific structures at 1646 cm ⁻¹ and 1617 cm ⁻¹ increased linearly with IgG concentration. R ² = 0.9435-0.9806 and R ² = 0.9572-0.9953. control 1 and control 2 are controls without anti-human IgG pAb and IgG, respectively.

9 is Raman spectrum and Raman intensity of different target concentrations according to CEA concentrations. SERS peak intensities of RBITC or MGITC-labeled specific structures increased linearly with CEA concentrations at 1646 cm ⁻¹ and 1617 cm ⁻¹ . R ² = 0.9801-0.9856 and R ² = 0.9257-0.9838. control 1 and control 2 are controls without anti-human CEA pAb and CEA, respectively.

제 22nd 발명: invent:

FIG. 10 is a schematic diagram of the fabrication of Janus nanostructures consisting of a double-metal nanoparticle compartment and a polymer compartment of a core-satellite structure and its SERS-based biosensing application method; FIG. (a) a method of making a Janus nanostructure comprising a double metal nanoparticle compartment and a polymer compartment of an AuNR core-Ag satellite, (b) a Janus comprising a double metal nanoparticle compartment and a polymer compartment of an AuNP core-Ag satellite. Method for preparing nanostructures, (c) spontaneous redox reaction between aniline monomer and silver nitrate, (d) surface-enhanced Raman scattering using Janus nanostructures consisting of core-satellite double metal nanoparticle compartments and polymer compartments (SERS) based target substance detection method.

11 shows Janus nanostructures comprising Au nanoparticles, PSS coated AuNR (AuNR-PSS) or ATP coated AuNP (AuNP-ATP) and their Au core-Ag satellite double metal compartments and polymer compartments (AuNR- UV-Vis absorbance and hydrodynamic diameter of PSS Ag PANI or AuNP-ATP Ag PANI); (a) Absorbance of Janus nanostructures (AuNR-PSS Ag PANI) comprising AuNR, PSS coated AuNR (AuNR-PSS) and Au core-Ag satellite double metal compartments and polymer compartments thereof. After synthesis of Janus nanostructures comprising Au core-Ag satellite double metal compartments and polymer compartments, new Ag absorption peaks appeared in the range of 380 nm to 480 nm, and the longitudinal and transverse axes of AuNR at 525 nm and 664 nm. LSPR peaks were blue-shifted to 508 nm and 595 nm, respectively. (B) Janus nanostructures comprising AuNR, PSS coated AuNR (AuNR-PSS) and Au core-Ag satellite double metal compartments and polymer compartments thereof ( Hydrodynamic diameter of AuNR-PSS Ag PANI). The mean diameters of the longitudinal and transverse axes of AuNR were 1.5 ± 0.1 nm and 49.9 ± 0.9 nm, respectively, and the average diameter of PSS coated AuNR was 2.8 ± 0.1 nm, 63.4 ± 0.5 nm, and the average of double metal core-satellite nanostructures. The diameter was 3.1 ± 0.1 nm, 73.0 ± 0.8 nm due to the non-spherical morphology, (c) Janus comprising AuNP, ATP coated AuNP (AuNP-ATP) and its Au core-Ag satellite double metal compartment and polymer compartment Absorbance of Nanostructures (AuNP-ATP Ag PANI). The UV-vis absorption spectrum of citric acid-capped AuNP at 524 nm was red-shifted to 526 nm, so that ATP was adsorbed on the AuNP surface, (d) AuNP, ATP coated AuNP (AuNP-ATP) and its Au core -Hydrodynamic diameter of Janus nanostructures (AuNP-ATP Ag PANI) including Ag satellite double metal compartments and polymer compartments. The average diameters of AuNPs, ATP coated AuNPs and their double metal core-satellite Janus nanostructures were 19.0 ± 0.8 nm, 33.3 ± 0.4 nm and 72.6 ± 0.6 nm, respectively.

Figure 12 shows (a) relative Raman shift with Raman dye concentration of AuNR core-Ag satellites labeled with MGITC in the concentration range of 10 ^-7 -10 ^-5.5 M, (b) double metal at MGITC at 10 ^-5.5 M Relative Raman shift between AuNR core-Ag satellites (Au-PSS Ag PANI, Example 1) and -Ag shell nanoparticles (Au-Ag PANI, Comparative Example 1), (c) 10 ^-6 -5.0 x 10 ^-6 Relative Raman shift with Raman dye concentration of AuNP core-Ag satellites labeled with ATP in the M concentration range, (d) AuNP core-Ag satellites labeled with ATP at 2.5 × 10 ^-6 M concentration (Au-ATP Ag Relative Raman shift of PANI, Example 2) and -Ag shell nanoparticles (Au-Ag PANI, Comparative Example 1).

FIG. 13 is transmission electron microscopy (TEM) and elevation annular dark field scanning TEM (HAADF-STEM) images for each nanoparticle; FIG. (a) TEM image of AuNRs, (b) TEM image of double metal AuNR core-Ag nanoparticles, (cd) TEM image of double metal AuNR core-Ag satellite nanoparticles at various magnifications, (gh) at various magnifications HAADF-STEM image of double metal AuNR core-Ag satellite nanoparticles. Scale bars are (a, c, e) 100 nm, (b, g) 200 nm, (d, f, h) 20 nm.

FIG. 14 is transmission electron microscopy (TEM) and elevation annular dark field scanning TEM (HAADF-STEM) images for each nanoparticle; FIG. (a) TEM image of AuNPs, (b) TEM image of double metal AuNP core-Ag nanoparticles, (cd) TEM image of double metal AuNP core-Ag satellite nanoparticles at various magnifications, (gh) at various magnifications HAADF-STEM image of double metal AuNP core-Ag satellite nanoparticles of. The scale bar is (a) 50 nm, (b, c, e, g) 200 nm, (d, f, h) 20 nm.

15 shows Raman spectra and Raman intensities of different target concentrations according to CEA concentrations. The SERS peak intensity of Janus nanostructures comprising MGITC-labeled double metal Au core-Ag satellite compartments and polymer compartments at 1618 cm ⁻¹ increased linearly with CEA concentration. R2 = 0.9954. control is a control without CEA.

제 33rd 발명: invent:

16 is a schematic diagram of the synthesis of asymmetric Janus nanocluster-polymer nanostructures and SERS-based biosensing applications; (a) a method for preparing an asymmetric Janus nanocluster-polymer nanostructure based on a reduction oxidation reaction, (b) a spontaneous redox reaction between silver nitrate and aniline monomers, (c) a SERS using an asymmetric Janus nanocluster-polymer nanostructure Based Biosensing Method.

17 is a UV-Vis absorbance spectrum of AuNPs, Raman dye derived Au nanoclusters and asymmetric Janus nanoclusters-polymer nanostructures; (a) UV-Vis absorption peaks of AuNP (Au) and MGITC induced Au nanoclusters (Au_MG), (b) UV-Vis absorption peaks of AuNP (Au) and RBITC derived Au nanoclusters (Au_RB), (c ) UV-Vis absorption peaks of asymmetric Janus nanocluster-polymer nanostructures (Au-MG @ Ag PANI or Au-RB @ Ag PANI), (d) AuNP, Au nanoclusters and asymmetric Janus nanocluster-polymer nanostructures Hydrodynamic diameter and size distribution of (Au-MG @ Ag PANI or Au-RB @ Ag PANI). The average diameters of AuNP, MGITC- or RBITC-derived Au nanoclusters were 18.9 ± 0.4 nm, 152.9 ± 2.8 nm and 115.7 ± 1.8 nm, and the MGITC- or RBITC-induced asymmetric Janus nanocluster-polymer nanostructures were 205 ± 4.5 nm and 186.3 ± 2.1 nm.

18 is a relative Raman spectrum of MGITC or RBITC derived Au nanoclusters during cluster formation at various incubation times; (a) the relative Raman spectrum of the MGITC derived Au nanoclusters, (b) the Raman intensity of the MGITC derived Au nanoclusters, (c) the relative Raman spectrum of the RBITC derived Au nanoclusters, and (d) the RBITC derived Au nanoclusters Raman strength of (e) MGITC-labeled AuNPs (Au), MGITC-derived Au nanoclusters (AuNC) and asymmetric Janus nanocluster-polymer nanostructures comprising them (Au-Ag cluster PANI, Au-MG @ Ag PANI) Relative Raman spectra of (a), (f) are RBITC-labeled AuNPs (Au), RBITC-derived Au nanoclusters (AuNC) and asymmetric Janus nanocluster-polymer nanostructures (Au-Ag cluster PANI, Au-RB) Relative Raman spectrum of @Ag PANI).

19 is a TEM image of asymmetric Janus nanocluster-polymer nanoparticles induced with MGITC at a final concentration of 1.5 μM; (a, b) Asymmetric Janus nanocluster-polymer nanoparticles without BSA stabilization. No asymmetric Janus nanocluster-polymer nanoparticles were formed, (c, d) BSA stabilized asymmetric Janus nanocluster-polymer nanoparticles. The scale bar is (a) 100 nm, (b) 10 nm, (c) 200 nm, (d) 20 nm.

20 is a TEM image of asymmetric Janus nanocluster-polymer nanoparticles induced with RBITC at a final concentration of 3.75 μM; (a, b) Asymmetric Janus nanocluster-polymer nanoparticles without BSA stabilization. No asymmetric Janus nanocluster-polymer nanoparticles were formed, (c, d) BSA stabilized asymmetric Janus nanocluster-polymer nanoparticles. Scale bars are (a, c) 100 nm, (b, d) 20 nm.

21 is a TEM image of asymmetric Janus nanocluster-polymer nanoparticles induced with MGITC at a final concentration of 0.75 μM; (a, b) Asymmetric Janus nanocluster-polymer nanoparticles without BSA stabilization. Low levels of clustering resulted in the formation of asymmetric Janus nanocluster-polymer nanoparticles without BSA coating. (C, d) BSA stabilized asymmetric Janus nanocluster-polymer nanoparticles. There was no significant difference between the two nanostructures with or without BSA coating. Scale bars are (a, c) 200 nm, (b) 50 nm, (d) 20 nm.

22 is a TEM image of asymmetric Janus nanocluster-polymer nanoparticles induced with RBITC at a final concentration of 1.9 μM; (a, b) Asymmetric Janus nanocluster-polymer nanoparticles without BSA stabilization. Low levels of clustering resulted in the formation of asymmetric Janus nanocluster-polymer nanoparticles without BSA coating. (C, d) BSA stabilized asymmetric Janus nanocluster-polymer nanoparticles. There was no significant difference between the two nanostructures with or without BSA coating. Scale bars are (a, c) 200 nm, (b, d) 20 nm.

FIG. 23 shows Raman spectra and Raman intensities of different target concentrations according to CEA concentrations. The SERS peak intensity of MGITC-induced asymmetric Janus nanocluster-polymer nanoparticles at 1618 cm ⁻¹ increased linearly with CEA concentration. R ² = 0.9047. control is a control without CEA.

제 44th 발명: invent:

FIG. 24 is a schematic diagram of the synthesis and application of side-by-side or end-to-end directional self-assembled AuNR and its Janus nanostructures; (a) controlled assembly of AuNR through electrostatic interaction, (b) synthesis of asymmetric double metal nanorod cluster-polymer Janus nanostructures using directional self-assembled AuNR clusters as seeds, (c) aniline monomers Spontaneous redox reaction between and silver nitrate, (d) Surface-enhanced Raman scattering (SERS) based target material detection using Janus nanostructures.

FIG. 25 shows UV-Vis absorbance spectra and hydrodynamic diameters of side-to-side assembled AuNR clusters and asymmetric Janus nanostructures comprising the same; (a) UV-Vis absorbance spectra of side-to-side assembled AuNR clusters with increasing incubation time from 1 to 5 minutes, (b) of side-to-side assembled AuNR clusters with increasing incubation time of 1 to 5 minutes. Hydrodynamic diameter, (c) UV-Vis absorbance spectrum of asymmetric Janus nanostructures with lateral Audirectional clusters, and (d) hydrodynamic diameters of asymmetric Janus nanostructures with lateral AuNR clusters. Control was a double metal nanorod-polymer Janus nanostructure without AuNR cluster.

Fig. 26 is a schematic diagram showing the synthesis steps of (a) thiol-terminated poly (tBA-b-NIPAM) and (b) poly (AAc-b-NIPAM).

(A) 1H NMR spectra of poly (tBA) and poly (tBA-b-NIPAM) measured in CDCl ₃ , poly (AAc-b-NIPAM) measured in DMSO-d6 at 400 MHz, (b) retention GPC traces of poly (tBA) and poly (tBA-b-NIPAM) over time (reaction), (c) are UV-Vis absorption graphs of poly (tBA-b-NIPAM) before and after amino degradation, ( d) inherent thermal deformation properties of poly (AAc-b-NIPAM) at 0.05 w / v% PBS 10 mM over temperature.

FIG. 28 shows UV-Vis absorbance spectra and hydrodynamic diameters of end-to-end assembled AuNR clusters and asymmetric Janus nanostructures comprising the same; (a) UV-Vis absorbance spectra of deionized water at different temperatures or end-to-end assembled AuNR clusters in PBS, (b) hydrodynamic diameters of deionized water at different temperatures or end-to-end assembled AuNR clusters in PBS, (c A) UV-Vis absorbance spectra of asymmetric Janus nanostructures containing end-to-end AuNR clusters at different temperatures or deionized water at different temperatures, (d) deionized water at different temperatures or end-to-end AuNR clusters at PBS Hydrodynamic Diameter of Asymmetric Janus Nanostructures. Control was a double metal nanorod-polymer Janus nanostructure without AuNR cluster.

29 (a) Relative Raman migration of side-to-side assembled AuNR clusters labeled with 10 ⁻⁶ M MGITC in deionized water and asymmetric Janus nanostructures comprising the same, (b) end-to-end assembled in deionized water at room temperature and PBS Relative Raman shift of Janus nanostructures containing AuNR nanoclusters. Control was individual AuNR.

30 is transmission electron microscopy (TEM) for each nanoparticle; Asymmetric including (a) individual AuNR, (b) double metal nanorod-polymer Janus nanoparticles without AuNR clusters, (c, d) side-to-side assembled AuNR clusters, and (e, f) side-to-side assembled AuNR clusters Janus nanostructures. Scale bars are (a) 100 nm, (b, c, e) 200 nm, (d) 10 nm, (f) 20 nm.

FIG. 31 is transmission electron microscopy (TEM) for each nanoparticle; FIG. An asymmetric Janus nanostructure comprising (a, b) end-to-end assembled AuNR clusters, and (c, d) end-to-end assembled AuNR clusters. Scale bars are (a, c) 100 nm, (b, d) 50 nm.

32 shows Raman spectra and Raman intensities of different target concentrations according to CEA concentrations. SERS peak intensities of asymmetric Janus nanostructures containing MGITC-derived side-to-side assembled AuNR clusters at 1618 cm ⁻¹ increased linearly with CEA concentrations. R ² = 0.9412. control is a control without CEA.

Hereinafter, the present invention will be described in more detail in the following examples. The following examples of the present invention are intended to embody the present invention, but do not limit or limit the scope of the present invention. From the detailed description and examples of the present invention, those skilled in the art to which the present invention pertains can be easily inferred to be within the scope of the present invention. References cited in the present invention are also incorporated into the specification of the present invention.

제 1First 발명: invent:

Example 1 Synthesis of Double Metal-Polymer Janus Structure

Citrate-capped gold nanoparticles (AuNPs) were synthesized according to the citrate reduction process. Specifically, gold chloride (III) hydrate (Gold (III) chloride hydrate) (HAuCl 4 .3H 2 O) stock solution (stock solution) of the stirring the mixture, and the solution so that the total concentration of 0.01% in deionized water 100 mL After heating with heating, stirring was continued while adding 1.5 mL of 1% sodium citrate solution to the boiling solution. The solution turned red within 5 minutes, indicating a reduction of gold ions, and the reaction proceeded further for 20 minutes. The solution was then cooled to room temperature. Double metal-polymer Janus nanostructures consisting of a double metal Au core-Ag shell compartment and a poly (aniline) counter compartment were prepared via surface template polymerization based on reduction-oxidation. Specifically, 15 mL of citrate-attached AuNP solution was concentrated by centrifugation at 10,000 rpm for 15 minutes, and then the supernatant was removed. Aniline and SDS were dissolved in 7.5 mL of deionized water to a final concentration of 5 mM and 0.9 mM, respectively. Concentrated AuNP was added to the solution, followed by brief voltexing, and 2.5 ml of silver nitrate solution was added and mixed to a final concentration of 2.5 mM. The reaction proceeded without stirring for 24 hours at room temperature under dark conditions to synthesize a double metal consisting of Au seed (first metal) and Ag (second metal) surrounding the seed metal. The reaction solution was incubated overnight in a 3.6 mM SDS solution to eccentrically deposit poly (aniline) on only one side of the double metal to form polymer compartments. The resulting solution was purified by centrifugation at 8,000 rpm for 10 minutes and resuspended in 3.6 mM SDS solution to prevent aggregation to prepare a double metal-polymer Janus nanostructure comprising a double metal nano cluster compartment and a polymer compartment. It was.

Example 2 Self-assembled double metal-polymer Janus nanostructure cluster specific structure synthesis consisting of a double metal nanocluster core and a polymer shell radially located around the core

Both types of Raman reporters, RBITC (rhodamine B isothiocyanate) and MGITC (Malachite green isothiocyanate), were selectively introduced into double metal-polymer Janus nanocluster compartments, and directional self-assembly into specific structures was achieved through noncovalent interactions. The double metals in the double metal-polymer Janus nanostructures of Example 1 self-assemble through hydrophobic interaction to form a double metal nanocluster core, and the polymer compartment extends radially in the opposite direction of the double metal nanoclusters that abut each other. While forming a specific structure while forming a polymer shell (Fig. 1 (a)). Specifically, 1 mL of the double metal-polymer Janus nanoparticle solution was centrifuged at 10,000 rpm for 10 minutes and transferred to 1 mL of deionized water. The colloidal solution was mixed with freshly prepared RBITC or MGITC at a concentration range of 10 ⁻⁵ to 10 ⁻⁸ M and incubated for 2 hours each. In addition, 0.46 mM EDC (1-ethyl-3- (3-dimethylaminopropyl) carbodiimide) was first used to covalently bond ODA (octadecylamine) containing a long hydrophobic alkyl chain to the double metal cluster compartment through selective functionalization. It was added to the particle solution to activate the residual carboxyl groups on the double metal cluster compartment and stirred for 1 hour. ODA dissolved in THF (tetrahydrofuran) was slowly added to the reaction solution to be 0.742 μM, 1.484 μM and 2.968 μM and stirred for 1 hour to amide bond reaction between the carboxyl group and the amine group of ODA. Finally, the reaction solution was centrifuged at 10,000 rpm for 10 minutes and resuspended in deionized water or phosphate buffered saline (PBS) to carry out the experiment.

Example 3 Synthesis of Magnetic Nanoparticles (MNPs) and Magnetic Beads by Electrohydrodynamic (EHD) Injection

Magnetic nanoparticles (MNPs) were synthesized using iron chloride precursors. Iron oxide nanoparticles (Fe ₃ O ₄ ) was prepared by chemical coprecipitation using a mixture of Fe ² ⁺ and Fe ³ ⁺ in a molar ratio of 1: 2 in an aqueous ammonia solution as a precipitant. Specifically, 0.86 g of ferrous chloride tetrahydrate (FeCl ₂ ) and 2.35 g of ferric chloride (FeCl ₃ ) were stirred and mixed in 40 mL of deionized water, and degassed with nitrogen gas for 30 minutes. The temperature was raised to 80 ° C. while stirring and 5 mL of ammonia hydroxide (NH ₄ OH) was added by syringe and then heated for 30 minutes. 1 g citric acid was added to the reaction flask and the reaction solution was heated to 90 ° C. and then stirred for an additional 90 minutes. Finally, Fe ₃ O ₄ magnetic nanoparticles (MNPs) were washed twice with deionized water under a static magnetic field of several hundred Gauss. In addition, a small aliquot of the MNP solution was concentrated using a magnetic field, added to the polymer solution, and electrophoretic (EHD) injection to prepare magnetic beads. 4.5 w / v% of poly (acrylamide-co-acrylic acid, poly (AAm-co-AA)) is prepared in a 3: 1 volume mixture of deionized water and ethylene glycol, Concentrated MNP was uniformly suspended in the polymer solution. Suspensions of dispersed MNPs were placed in a 1.0 mL syringe (BD, Franklin Lakes, USA) with a 23 gage stainless steel capillary for the electrohydrodynamic (EHD) dispersion process. In order to achieve a stable Taylor cone and con-jet mode, optimized viscosity was obtained by dissolving the polymer in a viscous solvent such as ethylene glycol without increasing the polymer concentration. The syringe was equipped with a micro syringe pump KDS-100 (KD Scientific, Inc, USA) which flows the MNPs suspension at a constant rate. An aluminum foil of 0.018 mm thickness (Fisherbrand; Thermo Fisher Scientific, USA) was used as the capture substrate. High voltage was applied between the capillary connected to the positive electrode and the aluminum foil connected to the negative electrode using a high voltage power supply NNC HV 30 (Nano NC, Korea). The distance between the two electrodes was 20-25 cm. The high voltage was maintained in the range of 15-20 kV and the flow rates of the two solutions were maintained at 0.08-0.15 ml / hour. High resolution digital cameras (D-90, Nikon Corporation, Japan) were used to visualize and capture single-phase Taylor cones, jet streams, and jet break-ups during EHD injection. After EHD injection, the resulting magnetic beads were thermally crosslinked overnight at 175 ° C. Finally, magnetic beads in powder form were collected by scraping off the foil and used for later experiments.

Example 4 Properties of Double Metal-Polymer Janus Nanoparticles and Specific Structures

The UV-Vis spectrum of double metal-polymer Janus nanoparticles is a UV-visible spectrometer (UV-1800, which changes the wavelength from 300 to 900 nm to a fixed slit width of 1 nm in a one-time 10 scan mode at medium scan speed at room temperature. Shimadzu, Japan). Baseline was calibrated using two empty cells filled with deionized water. Colloidal solution characteristics are dynamic light scattering (DLS) (Zeta-sizer Nano ZS90, Malvern Instruments, UK) with a maximum power of 5 mW as a light source at a scattering angle of 90 ° and supplied with a Ne-He laser at 633 nm. Was used to characterize the hydrodynamic diameter and its size distribution and the temperature was controlled to 25 ° C. Samples were diluted 2-fold in deionized water at a volume ratio of 1: 1, and their average size was measured at least 20 scan cycles. Zeta potential measurements were also performed to characterize the surface charge in deionized water. Individual AuNPs and double metal-polymer Janus nanoparticles and their specific structures were analyzed using a JEM-2100F FE-STEM (JEOL, Germany) operating at an acceleration voltage of 80 to 200 kV via transmission electron microcopy. . Samples were deposited on a 400 mesh copper grating coated with an ultra thin layer of carbon (Ted Pella, Inc., USA). Average diameter, size distribution and surface morphology were measured by scanning electron microscope (SEM) (VEGA-SB3, TESCAN, Czech Republic) with a focused beam of 0.5-30 kV. A few nanoparticle solutions were placed on silicon wafers and dried at room temperature. Samples were coated with a thin layer of conductive platinum using a coater (K575X Turbo Sputter Coater, Emitech Ltd, UK). The average size of the particles was analyzed by measuring approximately 50-100 particles randomly selected from TEM and SEM images using ImageJ software developed by the National Institutes of Health. In addition, all SERS measurements were performed using a Renishaw inVia Raman microscope system equipped with a Renishaw He-Ne laser operating at a wavelength (λ) of 632.8 nm for a stimulus source with a laser power of 12.5 mW. Rayleigh lines were removed from the collected SERS spectrum using a holographic notch filter located in the acquisition 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 a 520 cm ⁻¹ silicon line. Colloidal solutions of nanostructures labeled with RBITC or MGITC were prepared using small glass capillary tubes (Kimble Chase, plain capillary tubes, soda-lime glass, inner diameter: 1.1-1.2 mm, wall: 0.2 ± 0.02 mm, length: 75 mm). ) SERS spectra were collected for 1 second of exposure time used to focus the laser spot on the glass capillary in the wavelength range of 608-1738 cm ⁻¹ using a 20 × objective lens.

As a result, the hydrodynamic diameter and zeta potential for each nanoparticle were as shown in Table 1 below.

	AuNPsAuNPs	ABP NPs^* ABP NPs ^*	superparticular nanostructuresuperparticular nanostructure	ABP NPs with RBABP NPs with RB	ABP NPs with MGABP NPs with MG	superparticular nanostructure with RBsuperparticular nanostructure with RB	superparticular nanostructure with MGsuperparticular nanostructure with MG
hydrodynamic diameter(nm)hydrodynamic diameter (nm)	30.1±0.530.1 ± 0.5	62.8±2.362.8 ± 2.3	266.3±6.0266.3 ± 6.0	122.1±2.4122.1 ± 2.4	112.0±15.9112.0 ± 15.9	450.1±3.1450.1 ± 3.1	401.1±10.0401.1 ± 10.0
ξ-potential(mV)ξ-potential (mV)	-29.5±0.7-29.5 ± 0.7	-28.0±0.6-28.0 ± 0.6	-11.2±0.9-11.2 ± 0.9	-32.2±0.2-32.2 ± 0.2	-37±1.1-37 ± 1.1	-5.9±0.2-5.9 ± 0.2	-9.8±0.2-9.8 ± 0.2

^* ABP NPs (anisotropic bimetal-polymer nanoparticles): double metal-polymer Janus nanostructures.

FIG. 2 shows the UV-Vis absorbance and hydrodynamic diameter of AuNP, double metal-polymer Janus nanoparticles and self-assembled double metal-polymer Janus nanostructure cluster specific structures. As shown in Figure 2 (a), the UV-Vis absorption peak of AuNP appeared at 520 nm. After synthesis of the double metal-polymer Janus nanostructures, a new absorption peak appears in the range from 410 nm to 490 nm and Au absorption is blue-shifted to 480 nm, indicating the presence of double metal nano clusters consisting of Au core-Ag shells. Indicated. This change in plasmon absorption was consistent with the color change from red to brown of the colloidal solution. In addition, due to the plasmon absorption band of the aggregated metal nanostructures, additional peaks appeared at 650 nm after directional self-assembly into specific structures. 2 (b) shows RBITC-labeled or MGITC-labeled double metal-polymer Janus nanoparticles and their self-assembled double metal-polymer Janus when 10 ⁻⁶ M Raman dye and 2.968 μM ODA were added, respectively. The UV-Vis absorption spectrum of the nanocluster specific structure is shown. There was no significant difference in the UV-Vis absorption spectrum in the presence or absence of the Raman reporter due to the low concentration of the introduced Raman reporter. However, after absorbing Raman dyes on the double-metal nanocluster compartments correlated with the UV-vis absorption wavelengths of RBITC and MGITC, a broad peak appeared in the range from 410 nm to 500 nm. In addition, as shown in Figure 2 (c) and Table 1, the average diameter of Au nanoparticles and double metal-polymer Janus nanostructures were 30.1 ± 0.5 nm and 62.8 ± 2.3 nm, respectively, according to different clustering levels The average diameters of the specific structures were 168.3 ± 1.3 nm, 192.4 ± 2.4 nm and 266.3 ± 6.0 nm. Zeta potential values for Au nanoparticles in deionized water, double metal-polymer Janus nanostructures, and their self-assembled double metal-polymer Janus nanostructure cluster-specific structures are -29.5 ± 0.7 mV, -28.0 ± 0.6 mV, and -11.2, respectively. ± 0.9 mV. The surface charge of the double metal-polymer Janus nanostructure cluster specific structure was greatly reduced, indicating that ODA was well bound to the double metal cluster compartment through an amide bond reaction. 2 (d) shows the hydrodynamic diameters of the RBITC-labeled and MGITC-labeled double metal-polymer Janus nanostructures and their double metal-polymer Janus nanostructure cluster specific structures. The average diameters of the RBITC-labeled and MGITC-labeled double metal-polymer Janus nanostructures were 122.1 ± 2.4 nm and 112.0 ± 15.9 nm, respectively. This result indicated that the uptake of positively charged Raman dyes induced small aggregates despite no significant change in surface charge as shown in Table 1. This was analyzed as a result of the contrary zeta potential values of the particles, Cl ⁻ and ClO ₄ ⁻ , which are low concentrations of Raman dye and the opposite anions associated with the surface, which are inappropriate to cover the entire surface of the double metal cluster compartment. In addition, positively charged Raman dyes may connect the double metal nanocluster compartments with each other to increase net charge. After directional clustering, the hydrodynamic diameters of the RBITC-labeled and MGITC-labeled specific structures were 450.1 ± 3.1 nm and 401.1 ± 10.0 nm, respectively. Because of the more hydrophobic nature of RBITC, there was a significant difference in the degree of self-assembly of specific structures labeled with Raman dyes at the same concentration of Raman dyes.

FIG. 3 shows the relative Raman spectra of specific structures (a) labeled with RBITC and (c) MGITC to optimize the proper concentration of Raman dye for highly sensitive SERS based biosensing. Raman intensity of (b) RBITC and (d) MGITC was linearly proportional to the concentration of Raman dye in the range of 10 ⁻⁵ to 10 ⁻⁸ M of 1646 cm ⁻¹ and 1617 cm ⁻¹ . That is, as the Raman dye concentration increased, the Raman scattering intensity increased linearly (R ² = 0.9826 for RBITC, R ² = 0.9162 for MGITC). In particular, the strength of RBITC reached its maximum at 10 ^-5 M, but decreased as colloidal stability of large aggregates was lowered. This is in great agreement with the DLS profile of FIG. As can be seen in FIG. 3 (eh), Raman spectra of (e) RBITC and (g) dual metal-polymer Janus nanostructures and their double metal-polymer Janus nanostructure cluster specific structures at ^10-6 M concentration of MGITC are clustered It was measured to compare the Raman intensity according to the degree. The double metal-polymer Janus nanostructures exhibited relatively low Raman strength, and insufficient absorption of the Raman dye resulted in the polymer compartment (polymer), although the double metal Au core-Ag shell has a high capacity for local surface plasmon resonance. Thickness of the shell) impedes the generation of high Raman signals. On the other hand, when directional self-assembly is formed through selective deformation of the double metal nanocluster compartment, Raman strength is significantly enhanced due to the increased electromagnetic field in the gaps between the nanoparticles. Raman intensity of the RBITC and MGITC-labeled specific structures at 1646 cm ⁻¹ and 1617 cm ⁻¹ were about 15.92 and 15.59 times higher than the specific structures at ODA concentrations of 2.968 μM, respectively.

4 shows colloidal stability over time (a, b) and (c, d) batch variability of (a, c) MGITC or (b, d) specific structures labeled with RBITC. Raman spectra were measured in aqueous solution using five different batches (S1-S5). Directional self-assembly of the double metal nanocluster compartments exposed the polymer compartments under aqueous conditions, leading to reproducibility of the SERS signal through several days of good colloidal stability and stabilization of specific structures.

FIG. 5 shows (a) Au nanoparticles, (b, c, g) double metal-polymer Janus nanoparticles and (d, e, f, h) self-assembled double metal-polymer Janus to characterize size and shape. TEM and SEM images of three-dimensional morphologies of nanostructure cluster-specific structures at different magnifications. The average size of Au nanoparticles was 21.82 ± 2.59 nm. The resulting nanoparticles consisted of two separate compartments containing a double metal Au core-Ag shell and poly (aniline) as shown in FIG. 5 (b). After overnight incubation of the nanoparticles in a 3.6 mM SDS solution, a more eccentric polymer compartment was formed on the Au nanoparticles, as shown in Figure 5 (c). When different concentrations of ODA containing hydrophobic long alkyl chains were selectively introduced onto the double metal nanocluster compartments, directional self-assembly was formed as shown in FIG. 5 (d-f). The degree of self-assembly was controlled by the concentration of ODA. 5 (g) and 5 (h) are SEM images and clearly show the three-dimensional surface morphology of the double metal-polymer Janus nanostructure and its self-assembled double metal-polymer Janus nanostructure cluster specific structure. The average diameters of the double metal-polymer Janus nanostructures and their cluster structures were 69 ± 15 nm and 148 ± 24 nm, respectively.

FIG. 6 shows the accumulation of fluorescein from double metal-polymer Janus nanoparticles embedded in PEG hydrogels with electrical stimulation of (a) + 1.5 v and (b) -1.5 v every 30 minutes. Shows the release. Fluorescein was supported into the polymer compartment through the electrostatic interaction of negatively charged fluorescein and positively charged aniline monomers. Concentrated fluorescein-supported nanoparticles were added to the PEG solution and irradiated with UV to form PEG-nanoparticle hydrogels. When stimulated at 480 nm, the emission intensity of the fluorescent material was calculated by measuring the fluorescence intensity at the maximum emission wavelength of 512 nm. The polymer compartment of the double metal-polymer Janus nanostructure was composed of poly (aniline), which represents a conductive polymer, and exhibited electric field reactivity. When a voltage of -1.5 v was applied between the two electrodes, the amount of fluorescein emitted was higher than when a voltage of + 1.5 v was applied. When a voltage of -1.5 v was applied, the positive charge in the polymer compartment reduced the electrostatic forces of the negatively charged fluorescein and poly (aniline) compartments, resulting in good drug release. Figure 6 (c) shows the UV-vis absorption of fluorescein-filled double metal-polymer Janus nanoparticles at various pHs of 4, 7 and 11. At high pH, the deprotonation of the apeline unit reduced the electrostatic interaction of fluorescein. As a result, the electrostatic force of fluorescein, a negatively charged drug, was weakened, resulting in good drug release. Figure 6 (d) shows the Raman spectrum of MGITC generated from fluorescein-supported double metal-polymer Janus nanostructures. Raman peaks of MGITC and fluorescein were 1617 cm ⁻¹ and 1176 cm ⁻¹ , respectively, indicating that the dual metal-polymer Janus nanostructures are fluorescent and SERS based nanoprobes.

Example 5 Antibody Binding to Self-assembled Double Metal-Polymer Janus Nanostructure Cluster Specific Structures of Magnetic Beads and Double Metal-Polymer Janus Nanostructures

Self-assembled double metal-polymer Janus nanostructure cluster Specific structures and magnetic beads were combined with two different sets of monoclonal antibodies (mAb) and polyclonal antibodies (pAbs) against two types of target proteins, IgG (Immunogloblin G) and CEA (carcinoembryonic antigen). First, a polymer structure (polymer shell) having a specific structure and an anti-human IgG pAb or an anti-human antibody are obtained by using an amide bond reaction between an amine group remaining in the poly (aniline) compartment and a carboxyl group present in the antibody. Bioconjugation of the human CEA polyclonal antibody (anti-human CEA pAb) was performed. This binding reaction was based on the chemistry of sulfo-N-hydroxysuccinimide ester (EDC) and sulfo-NHS. Specifically, 1.0 mg / ml or 2.0 mg / ml of anti-human IgG pAb in a dispersion solution of double metal-polymer Janus nanoparticles containing 10 mM PBS at pH 7.4 containing 60 mM EDC and 9.2 mM sulfo-NHS. Or 5 μl of anti-human CEA pAb was added and stirred for 3 hours to bring the total pAb concentration to 5 μg / ml or 10 μg / ml, respectively. Anti-human IgG pAb- or anti-human CEA pAb- bound specific structures were washed by centrifugation at 3,000 rpm and resuspended in PBS. In addition, magnetic beads were chemically reacted with an anti-human IgG mAb or an anti-human CEA mAb by activating the residual carboxyl groups on the polymer nanoparticles heat stabilized overnight at 175 ° C. Combined. Specifically, 1.25 mg of magnetic beads were suspended in 0.9 ml of PBS and sonicated for 2 minutes using a tip-sonicator using a 3/3 second on / off cycle at 20.0% amplitude. The uniformly suspended magnetic beads were mixed with 5.0 mM EDC and 5.0 mM sulfo-NHS and stirred for 1 hour. Dilute 2.96 mg / ml or 3.56 mg / ml anti-human IgG mAb or anti-human CEA mAb with 100 μl of PBS buffer, and then add a final concentration of 7.4 μg / ml or 8.9 μg / ml to the magnetic bead solution. Add slowly and stir for 1 hour. Unbound anti-human IgG mAb or anti-human CEA mAb was isolated using a magnetic field and antibody-bound magnetic beads were resuspended in PBS for SERS-based biosensing of IgG and CEA.

Example 6 SERS-Based Biosensing of Target Proteins IgG and CEA Using Specific Structures and Magnetic Beads

A specific structure labeled with a Raman reporter was used as a SERS nanoprobe for quantitative analysis of target proteins IgG and CEA, and sandwiched immune complexes were prepared using anti-human IgG mAbs or magnetic beads combined with anti-human CEA mAbs. The immune complexes were selectively isolated by forming tools by forming them. First, magnetic beads bound with anti-human IgG mAb or anti-human CEA mAb were added to a buffer containing 6 different concentrations of IgG or CEA ranging from 2 μg / ml to 1 ng / ml and reacted for 1 hour. I was. The target protein was washed with an external magnetic field and resuspended in fresh PBS buffer. Subsequently, anti-human IgG pAb or anti-human CEA pAb bound SERS nanoprobe was added to each immune complex in which the target protein and magnetic beads were formed and reacted for 1 hour to constitute the magnetic beads, target protein and SERS nanoprobe. Sandwich immune complexes were prepared (FIG. 1 (c)). Unbound SERS nanoprobes were removed using a magnetic field and the product sandwiched immune complex was resuspended in PBS for SERS measurements (FIG. 7). Experiments with two controls were also performed together to assess the selective binding capacity of SERS nanoprobes without each of the bound antibodies or target proteins.

8 and 9 show plots of Raman spectra and Raman intensity at different target concentrations according to IgG and CEA concentrations. As IgG concentration increased in the range from 1.0 ng / ml to 2.0 μg / ml, Raman intensity of RBITC-labeled or MGITC-labeled specific structures increased due to increased formation of sandwich immune complexes. Control 1 and Control 2 represent controls without anti-human IgG pAb and IgG, respectively. In addition, the representative SERS peak intensities of RBITC and MGITC at 1646 cm ⁻¹ and 1617 cm ⁻¹ increased linearly with IgG concentration as shown in FIG. 8 (R ² = 0.9435-0.9806 and R ² = 0.9572-0.9953). Likewise, as shown in FIG. 9, as the CEA concentration increased, the Raman intensity of the RBITC-labeled or MGITC-labeled specific structure increased linearly (R ² = 0.9801-0.9856 and R ² = 0.9257-0.9838). . Control 1 and Control 2 represent controls without anti-human CEA pAb and CEA, respectively. Detection limits for IgG and CEA were less than 1 ng / ml.

제 22nd 발명: invent:

Example 1 Synthesis of Janus Nanostructures Containing AuNR Core-Ag Satellite Double Metal Compartments and Polymer Compartments

Gold nanorods (AuNRs) were synthesized through a seed-mediated growth method. Specifically, 5 mL of 0.20 M hexadecyltrimethylammonium bromide (Sigma-Aldrich, USA) was dissolved at 29-30 ° C. and 0.0005 M of Gold (III) chloride hydrate (HAuCl ₄ .3H ₂ O) ( Sigma-Aldrich, USA) and 5 mL of cold 0.010 M NaBH ₄ were then added. The yellow color of the reaction solution was changed to a brownish yellow solution, and the resultant seed solution was maintained at 29-30 ° C. and used within 2 to 2.5 hours. To grow the nanorods on the seed particles, 0.25 mL of 0.004 M AgNO ₃ and 5 ml of 0.20 M CTAB were mixed at 29-30 ° C. Then, 5.0 mL of 0.001 M HAuCl ₄ was added to the solution, followed by stirring. After mixing for 30-40 minutes, ascorbic acid, a reducing agent, was added to induce a color change of the growth solution from dark yellow to colorless. In the final step 12 μL of the seed solution was added to the colorless solution and the solution color was slowly changed in 10-20 minutes. The solution was stirred and stored at 29-30 ° C overnight. In order to change the surface properties of the seed metal nanoparticles, metal-polymer composite formation was prepared through electrostatic interaction. CTAB capped AuNR was centrifuged at 10,000 rpm for 10 minutes and resuspended in 1 mM NaCl solution. Poly (styrene sulfonate) (PSS) (Sigma-Aldrich, USA), a negatively charged polymeric ligand dissolved in 1 mM NaCl solution, was prepared to a final concentration of 0.06-0.2 w / v% and added to AuNR solution -Polymer (AuNR-PSS) complexes were formed. The metal-polymer (AuNR-PSS) complex was purified by centrifugation at 8,000 rpm for 10 minutes and concentrated, followed by oxidation of double metal-polymer Janus nanoparticles consisting of AuNR core-Ag satellite double metal compartments and poly (aniline) compartments. Prepared via surface mold polymerization based on reduction. Specifically, aniline and SDS were dissolved in 7.5 mL of deionized water at a final concentration of 5 mM and 0.9 mM, respectively. The concentrated metal-polymer (AuNR-PSS) complex was added to the solution and stirred, followed by 2.5 mL of silver nitrate solution and mixed to a final concentration of 2.5 mM. The reaction proceeded without stirring for 24 hours at room temperature under dark conditions. The reaction was further incubated overnight in a 3.6 mM SDS solution so that poly (aniline) was eccentrically deposited on only one side of the double metal nanostructure of AuNR core-Ag satellites. The resulting solution was purified by centrifugation at 8,000 rpm for 10 minutes and resuspended in 3.6 mM SDS solution to prevent aggregation. 1 mL of Janus nanoparticle solution consisting of AuNR core-Ag satellite double metal compartment and poly (aniline) compartment was centrifuged at 10,000 rpm for 10 minutes and transferred to 1 mL of deionized water. The colloidal solution was mixed with freshly prepared MGITC at a concentration range of 10 ⁻⁵ to 10 ^−5.5 M, and incubated for 2 hours each. Selective adsorption on Ag satellite surfaces on AuNR by immobilization of MGITC on the surface of double metal nanoparticles consisting of AuNR core-Ag satellites via isothiocyanate groups (-N = C〓S) of the Raman dye I was.

Example 2 Synthesis of Janus Nanostructures Comprising AuNP (AuNS) Core-Ag Satellite Double Metal Nanoparticle Compartments and Polymer Compartments

Citrate-capped gold nanoparticles (AuNPs) or gold nanospheres (AuNSs) were synthesized according to the citrate reduction procedure. Specifically, a stock solution of gold (III) chloride hydrate was added to 100 mL of deionized water so that the total concentration was 0.01%, and rapidly adding 1.5 ml of 1% sodium citrate solution while stirring and boiling the solution. . The solution turned red within 5 minutes, indicating a reduction of gold ions, and the reaction proceeded further for 20 minutes. The resulting solution was cooled to room temperature. In addition, double metal AuNP core-Ag satellite nanostructures having polymer compartments were synthesized by ligand mediated surface control of AuNP seed particles and redox reaction between silver nitrate and aniline. The surface properties of the metal nanoparticles were modified by adjusting the interfacial energy using ligands containing -SH and -NH2 groups to control the deposition of Ag onto AuNP seed particles. Specifically, citrate-capped AuNPs were centrifuged at 10,000 rpm for 10 minutes and resuspended in deionized water. The small molecule ligand 4-aminothiophenol (ATP) containing -SH and -NH2 groups on the AuNP seed was bound to a final concentration of 10 ^-5 M. After purification by centrifugation, double metal-polymer Janus nanoparticles consisting of AuNP core-Ag satellite double metal compartments and poly (aniline) compartments were prepared via surface template polymerization based on redox. Specifically, aniline and SDS were dissolved in 7.5 mM deionized water at a final concentration of 5 mM and 0.9 mM, respectively. These concentrated metal-ligand (AuNP-ATP) complexes were added to the solution and stirred, followed by 2.5 mL of silver nitrate solution and mixed to a final concentration of 2.5 mM. The reaction proceeded without stirring for 24 hours at room temperature under dark conditions. The reaction was further incubated overnight in a 3.6 mM SDS solution so that poly (aniline) was eccentrically deposited on only one side of the double metal AuNP core-Ag satellite nanostructure. The resulting solution was purified by centrifugation at 8,000 rpm for 10 minutes and resuspended in 3.6 mM SDS solution to prevent aggregation. 1 mL of Janus nanoparticle solution consisting of AuNP core-Ag satellite double metal compartment and poly (aniline) compartment was centrifuged at 10,000 rpm for 10 minutes and transferred to 1 mL of deionized water.

Comparative Example 1 Synthesis of Janus Nanostructure Containing AuNR Core-Ag Shell Nanoparticles and Polymer Compartments

Oxidation-reduction based double metal-polymer Janus nanoparticles consisting of a double metal AuNR core-Ag shell compartment and a poly (aniline) compartment, prepared in the same manner as in Example 1 but without modifying the surface properties of the seed AuNR with PSS Prepared via surface mold polymerization. Specifically, aniline and SDS were dissolved in 7.5 mM deionized water at a final concentration of 5 mM and 0.9 mM, respectively. Concentrated AuNR was added to the solution and stirred, followed by 2.5 mL of silver nitrate solution and mixed to a final concentration of 2.5 mM. The reaction proceeded without stirring for 24 hours at room temperature under dark conditions. The reaction was further incubated overnight in a 3.6 mM SDS solution so that poly (aniline) was eccentrically deposited on only one side of the AuNR core-Ag shell double metal nanostructures. The resulting solution was purified by centrifugation at 8,000 rpm for 10 minutes and resuspended in 3.6 mM SDS solution to prevent aggregation. 1 mL of Janus nanoparticle solution consisting of AuNR core-Ag shell double metal compartment and poly (aniline) compartment was centrifuged at 10,000 rpm for 10 minutes and transferred to 1 mL of deionized water. The colloidal solution was mixed with freshly prepared MGITC at a concentration range of 10 ⁻⁵ to 10 ^−5.5 M, and incubated for 2 hours each. MGITC was immobilized on the surface of the double metal nanoparticles consisting of AuNR core-Ag shells through isothiocyanate groups (-N = C〓S) of the Raman dye and selectively adsorbed onto the Ag shell surface on AuNR.

Iron oxide nanoparticles (Fe ₃ O ₄ ) was prepared by chemical coprecipitation using a mixture of Fe ^{2 +} and Fe ³ ⁺ mixed in a molar ratio of 1: 2 in an aqueous ammonia solution as a precipitant. Specifically, 0.86 g of ferrous chloride tetrahydrate (FeCl ₂ ) and 2.35 g of ferric chloride (FeCl ₃ ) were stirred and mixed in 40 mL of deionized water, and degassed with nitrogen gas for 30 minutes. The temperature was raised to 80 ° C. while stirring and 5 mL of ammonia hydroxide (NH ₄ OH) was added by syringe and then heated for 30 minutes. 1 g citric acid was added to the reaction flask and the reaction solution was heated to 90 ° C. and then stirred for an additional 90 minutes. Finally, Fe ₃ O ₄ magnetic nanoparticles (MNPs) were washed twice with deionized water under a static magnetic field of several hundred Gauss. In addition, a small aliquot of the MNP solution was concentrated using a magnetic field, added to the polymer solution, and electrophoretic (EHD) injection to prepare magnetic beads. 4.5 w / v% of poly (acrylamide-co-acrylic acid, poly (AAm-co-AA)) was prepared in a 3: 1 volume mixture of deionized water and ethylene glycol, Concentrated MNP was uniformly suspended in the polymer solution. A suspension of dispersed MNPs for an electrohydrodynamic (EHD) spraying process was placed in a 1.0 mL syringe (BD, Franklin Lakes, USA) with a 23 gage stainless steel capillary. In order to achieve a stable Taylor cone and con-jet mode, optimized viscosity was obtained by dissolving the polymer in a viscous solvent such as ethylene glycol without increasing the polymer concentration. The syringe was equipped with a micro syringe pump KDS-100 (KD Scientific, Inc, USA) which flows the MNPs suspension at a constant rate. An aluminum foil (Fisherbrand; Thermo Fisher Scientific, USA) with a thickness of 0.018 mm was used as the collecting substrate. High voltage was applied between the capillary connected to the positive electrode and the aluminum foil connected to the negative electrode using a high voltage power supply NNC HV 30 (Nano NC, Korea). The distance between the two electrodes was 20-25 cm. The high voltage was maintained in the range of 10-15 kV and the flow rates of the two solutions were maintained at 0.08-0.15 ml / hour. High resolution digital cameras (D-90, Nikon Corporation, Japan) were used to visualize and capture single-phase Taylor cones, jet streams, and jet break-ups during EHD injection. After EHD injection, the resulting magnetic beads were thermally crosslinked overnight at 175 ° C. Finally, magnetic beads in powder form were collected by scraping off the foil and used for later experiments.

Example 4 Properties of Janus Nanostructure Including Au Core-Ag Satellite Double Metal Compartment and Polymer Compartment

UV-visible spectrometer (Cary-100 Bio, Varian Biotech, USA) was used to measure the optical properties of single Au-Ag core-shell nanoparticles with eccentrically deposited poly (aniline) compartments and cluster nanostructures. When the wavelength was changed in the range of 300 ~ 900 nm, the UV-vis absorption spectrum of the single nanoparticles and their clusters were confirmed. The hydrodynamic diameter and size distribution of nanoparticles was determined using dynamic light scattering (DLS) (Zeta-sizer Nano ZS90, Malvern Instruments, Malvern, UK) using Ne-He laser at 633 nm and 90 ° scattering angles. Analyzed using. Zeta potential measurements were also performed to characterize the surface charge of deionized water. Transmission electron microscopy analysis was performed using JEM-2100F FE-STEM (JEOL, Germany) operating at an acceleration voltage of 80-200 kV. The average diameter, size distribution and surface morphology of the nanoparticles were characterized by scanning electron microscopy (SEM) (VEGA-SB3, TESCAN, Czech Republic) with a focused beam of 0.5-30 kV. The nanoparticles were coated with a thin layer of conductive platinum using a K575X Turbo Sputter Coater (Emitech Ltd, Ashford, UK). All SERS measurements were performed using a Renishaw inVia Raman microscope system equipped with a Renishaw He-Ne laser operating at a wavelength (λ) of 632.8 nm for a stimulus source with a laser power of 12.5 mW. Rayleigh lines were removed from the collected SERS spectrum using a holographic notch filter located in the acquisition 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 a 520 cm ⁻¹ silicon line. SERS spectra were collected for 1 second of exposure time used to focus the laser spot on the glass capillary in the wavelength range of 608-1738 cm ⁻¹ using a 20 × objective lens.

As a result, the ζ potential and the hydrodynamic diameter of each nanoparticle were as shown in Table 2 below.

	ξ-potential(mV)ξ-potential (mV)	hydrodynamic diameter(nm)hydrodynamic diameter (nm)
AuNRAuNR	36.6±0.736.6 ± 0.7	49.9±0.9, 1.5±0.149.9 ± 0.9, 1.5 ± 0.1
AuNR-PSSAuNR-PSS	-36.7±1.1-36.7 ± 1.1	63.37±0.5, 2.8±0.163.37 ± 0.5, 2.8 ± 0.1
AuNR-PSS Ag PANI(polyaniline)AuNR-PSS Ag PANI (polyaniline)	-26.5±0.3-26.5 ± 0.3	73.0±0.8, 3.1±0.173.0 ± 0.8, 3.1 ± 0.1
AuNPAuNP	-26.8±1.1-26.8 ± 1.1	19.0±0.819.0 ± 0.8
AuNP-ATPAuNP-ATP	-28.2±1.2 -28.2 ± 1.2	33.3±0.433.3 ± 0.4
AuNP-ATP Ag PANI(polyaniline)AuNP-ATP Ag PANI (polyaniline)	-25.2±0.7-25.2 ± 0.7	72.6±0.672.6 ± 0.6

FIG. 11 shows UV-Vis absorption spectra and hydrodynamic diameters of Janus nanostructures comprising Au nanoparticles, PSS or ATP coated Au nanoparticles and Au core-Ag satellite double metal compartments and polymer compartments. As shown in FIG. 11 (a), the absorption spectrum of AuNR changed according to PSS binding. The longitudinal local surface plasmon resonance (LSPR) peaks of AuNRs were blue-shifted from 666 nm to 664 nm after being coated with PSS, indicating that AuNR was partially wrapped with PSS to provide a silver reduction site. In addition, AuNR aggregation or clustering did not occur since no additional peak was observed during the process. When metal nanoparticles were completely coated with PSS, red-shifts of LSPR peaks were observed due to an increase in local dielectric function. However, AuNR completely wrapped with PSS prevented silver ions from being reduced to core metal nanoparticles. On the other hand, insufficient PSS coating triggered the precipitation of AuNR due to electrostatic interaction. In this regard, the proper concentration of PSS at optimal conditions has been carefully considered. In addition, after the synthesis of the Janus nanostructures comprising Au core-Ag satellite double metal compartments and polymer compartments through redox reactions of silver ions and aniline monomers, new Ag absorption peaks appear in the range of 380 nm to 480 nm. The longitudinal and transverse LSPR peaks of AuNR at 525 nm and 664 nm were blue-shifted to 508 nm and 595 nm, respectively. This UV-Vis absorption change is consistent with the blue to brown color change of the MNP solution, indicating the presence of a double metal Janus nanostructure of Au-Ag. Dynamic light scattering (DLS) and ζ potential measurements were also performed to analyze the hydrodynamic diameter, size distribution and surface charge of AuNR, PSS coated AuNR and its double metal core-satellite Janus nanostructures. As can be seen in Figure 11 (b) and Table 2, the average diameter of the longitudinal axis and the transverse axis of AuNR is 1.5 ± 0.1 nm, 49.9 ± 0.9 nm, respectively, the average diameter of PSS coated AuNR is 2.8 ± 0.1 nm, 63.4 ± 0.5 nm. In addition, the average diameter of the double metal core-satellite Janus nanostructures was 3.1 ± 0.1 nm, 73.0 ± 0.8 nm due to the aspherical morphology. The ζ potential values of AuNRs, PSS-coated AuNRs and their double metal core-satellite Janus nanostructures were 36.6 ± 0.7 nm, -36.7 ± 1.1 nm and -26.5 ± 0.3 nm, respectively. The presence of negatively charged PSS on the AuNR surface drastically reduced the ζ potential of the AuNR coated with PSS. As the silver ions were trapped in the polymer ligand-bound AuNR and reduced by the redox reaction, the negative charge present in the AuNR was reduced, which reduced the ζ potential value of the double metal AuNR core-Ag satellite Janus nanostructure with polymer compartment. . Similarly, citric acid-capped AuNPs for core-satellite Janus nanostructures were functionalized with a small ligand, ATP. As shown in FIG. 11 (c), the UV-vis absorption spectrum of citric acid-capped AuNP at 524 nm red-shifted to 526 nm, supporting that ATP was adsorbed on the AuNP surface. After synthesis of double metal core-satellite Janus nanostructures with polymer compartments, Ag plasmon peaks and additional peaks appeared in the 400-550 nm range due to the Au-Ag interface. In addition, as shown in Fig. 11 (d) and Table 2, the average diameter of AuNPs, ATP coated AuNPs and their double metal core-satellite Janus nanostructures were 19.0 ± 0.8 nm, 33.3 ± 0.4 nm and 72.6 ± 0.6, respectively. nm, and ζ potential values were −26.8 ± 1.1 mV, −28.2 ± 1.2 mV, and −25.2 ± 0.7 mV, respectively.

FIG. 12 shows relative Raman shifts according to Raman dye concentrations of (a) AuNR core-Ag satellites labeled with MGITC and (c) AuNP core-Ag satellites labeled with ATP to optimize high sensitivity SERS based biosensing conditions. Indicates. As MGITC concentrations increased, the SERS intensity increased in the concentration range from 10 ⁻⁷ M to 10 ^−5.5 M. However, it labeled with the colloidal stability of the core MGITC AuNR -Ag satellites was reduced due to MGITC induced aggregation at a concentration of 10 ^-5 M MGITC. Figure 12 (b) shows the relative Raman shift of double metal AuNR core-Ag satellites and -Ag shell nanoparticles (Comparative Example 1) at MGITC with a concentration of 10 ^-5.5 M. The interparticle bonding between Ag satellites provides electromagnetic field enhancement, resulting in a five-fold increase in SERS strength due to hot spots. Similarly, the relative Raman shift of the double metal AuNP core-Ag satellites increased in the concentration range from 10 ⁻⁶ M to 2.5 × 10 ⁻⁶ M as shown in FIG. 12 (c). The SERS strength of these nanostructures was greatly reduced due to deposition at ATP concentrations of 5.0 × 10 ⁻⁶ M. During the synthesis, silver satellites formed on the surface of the ATP-coated AuNP so that ATP was buried in the inner gap between the AuNP core and Ag satellite. Therefore, a large improvement in the electromagnetic field can be observed at this position where the Raman active ATP is located as shown in FIG. 12 (d).

FIG. 13 shows transmission electron microscopy (TEM) images of (a) AuNRs, (b) double metal AuNR core-Ag nanoparticles, and (c-h) -Ag satellites at various magnifications. The double metal compartments of the AuNR core and Ag satellites were clearly visible and the distinct polymer compartments were grayed out against the background. In addition, high-angle annular dark-field scanning TEM (HAADF-STEM) was performed to confirm the composition of the double metal compartment. Due to the high atomic number, the AuNR cores appeared brighter than Ag satellites.

14 shows transmission electron microscope (TEM) images of (a) AuNPs, (b) double metal AuNP core -Ag nanoparticles and (c-h) -Ag satellites at various magnifications. The double metal compartments of the AuNP core and Ag satellites were clearly visible and the distinct polymer compartments were grayed out against the background. In addition, a high angle annular dark scanning TEM (HAADF-STEM) was performed to confirm the composition of the double metal compartment. The high atomic number made AuNP cores look brighter than Ag satellites.

Example 5 Antibody Binding of Janus Nanostructures Containing Magnetic Beads and Au Core-Ag Satellite Double Metal Compartments and Polymer Compartments

Janus nanostructures and magnetic beads, including Au core-Ag satellite double metal compartments and polymer compartments, bind to monoclonal antibodies (mAb) and polyclonal antibodies (pAbs) against two types of target proteins, carcinoembryonic antigens (CEA), respectively It became. First, the polymer compartment and the anti-human CEA of the Janus nanostructure including Au core-Ag satellite double metal compartment and polymer compartment are utilized by the amide bond reaction between the amine group remaining in the poly (aniline) compartment and the carboxyl group present in the antibody. A bioconjugation reaction between polyclonal antibodies (anti-human CEA pAbs) was performed. This binding reaction was based on the chemistry of EDC (1-ethyl-3- (3-dimethylaminopropyl) carbodiimide) and sulfo-N-hydroxysuccinimide ester (sulfo-NHS). Specifically, 5 μl of 2.0 mg / ml anti-human CEA pAb was added to a dispersion solution of double metal-polymer Janus nanoparticles containing 10 mM PBS at pH 7.4 containing 60 mM EDC and 9.2 mM sulfo-NHS. After stirring for 3 hours, the total pAb concentration was 10 μg / ml. Anti-human CEA pAb-coupled Janus nanostructures were washed by centrifugation at 3,000 rpm and resuspended in PBS. In addition, magnetic beads were chemically bound to anti-human CEA mAbs by activating residual carboxyl groups on polymer nanoparticles that were heat stabilized at 175 ° C. overnight. Specifically, 1.25 mg of magnetic beads were suspended in 0.9 ml of PBS and sonicated for 2 minutes using a tip-sonicator using a 3/3 second on / off cycle at 20.0% amplitude. The uniformly suspended magnetic beads were mixed with 5.0 mM EDC and 5.0 mM sulfo-NHS and stirred for 1 hour. 3.56 mg / ml anti-human CEA mAb was diluted with 100 μl of PBS buffer, then slowly added to the magnetic bead solution to a final concentration of 8.9 μg / ml and stirred for 1 hour. Unbound anti-human CEA mAbs were isolated using a magnetic field and antibody-bound magnetic beads were resuspended in PBS for SERS-based biosensing of CEA.

Example 6 SERS-Based Biosensing of Target Protein CEA Using Janus Nanostructures and Magnetic Beads Containing Au Core-Ag Satellite Double Metal Compartments and Polymer Compartments

Janus nanostructures comprising Raman reporter labeled double metal Au core-Ag satellite compartments and polymer compartments were used as SERS nanoprobes for quantitative analysis of the target protein CEA and magnetic beads bound to anti-human CEA mAb. Immune complexes were selectively isolated with a separation tool by forming sandwiched immune complexes. First, magnetic beads bound with anti-human CEA mAb antibody were added to a buffer containing three different concentrations of CEA ranging from 22.5-67.5 ng / ml and allowed to react for 1 hour. The target protein was washed with an external magnetic field and resuspended in fresh PBS buffer. Subsequently, SERS nanoprobe with anti-human CEA pAb bound was added to each immune complex in which the target protein and the magnetic beads were formed and reacted for 1 hour to prepare a sandwich immune complex consisting of the magnetic beads, the target protein and the SERS nanoprobe. (FIG. 10 (d)). Unbound SERS nanoprobes were removed using a magnetic field and the product sandwiched immune complex was resuspended in PBS for SERS measurements. Experiments with controls were also performed to assess the selective binding capacity of SERS nanoprobes without the target protein.

15 shows a plot of Raman spectra and Raman intensity of different target concentrations according to CEA concentrations. As the CEA concentration increased in the range of 22.5-67.5 ng / ml, Raman strength of Janus nanostructures containing MGITC-labeled Au core-Ag satellite double metal compartments and polymer compartments increased due to increased formation of sandwich immune complexes. It was. Experiments on the control were performed in the absence of CEA. In addition, the representative SERS peak intensity of MGITC at 1618 cm ⁻¹ increased linearly with CEA concentration (R ² = 0.9954).

제 33rd 발명: invent:

Example 1 Preparation of gold nanoparticle clusters (AuNCs)

Gold nanoparticles (AuNPs) were synthesized according to the citric acid reduction procedure. Specifically, a stock solution of gold (III) chloride hydrate (HAuCl4.3H2O, Sigma-Aldrich, USA) is added to 100 mL of deionized water to a total concentration of 0.01%, and the solution is added to 1.5 ml of 1% sodium citrate (sodium citrate) solution was added rapidly and boiled with vigorous stirring. The solution turned red within 5 minutes, indicating a reduction of gold ions, and the reaction proceeded further for 20 minutes. The resulting solution was cooled to room temperature. AuNP clusters were also prepared by agglomeration of AuNPs in the presence of Raman dyes. Seed AuNPs in glass vials were mixed with the mother liquor, silver nitrate, and sodium citrate of the Raman active molecule Malachite green isothiocyanate (Invitrogen, USA), resulting in a final concentration of 1.5 μM or 0.75 μM MGITC, 0.5 mM silver nitrate and 1.0 mM citric acid. Sodium. The mixture was stirred for 5 minutes and then the vial was heated to 95 ° C. for 10-60 minutes. Small aliquots were collected at known intervals during heating to specify the UV-Vis absorption band and Raman intensity of the gold nanoclusters with MGITC. In the same way, AuNP clusters with RODC (rhodamine B isothiocyanate, Sigma-Aldrich, USA) were prepared with a final concentration of 3.8 μM or 1.9 μM RBITC. After sufficient clustering, the reaction solution was rapidly cooled to room temperature and 0.5% BSA was added to stabilize the cluster to prevent subsequent aggregation.

Example 2 Preparation of Asymmetric Janus Nanoprobe of Double Metal Nanocluster-Polymer

Asymmetric Janus nanocluster-polymer nanoparticles consisting of a double metal nanocluster compartment and a poly (aniline) compartment of an Au core-Ag shell were prepared via surface template polymerization based on redox reactions. To prepare an Au core-Ag shell, 15 mL of the BSA stabilized AuNP cluster solution of Example 1 was concentrated by centrifugation at 7,000 rpm for 5 minutes and then the supernatant was removed. Aniline and SDS were dissolved in 7.5 mL of deionized water to a final concentration of 5 mM and 0.9 mM, respectively. Concentrated AuNP clusters were added to the prepared solution, then briefly voltexed, and 2.5 ml of silver nitrate solution was added and mixed to a final concentration of 2.5 mM. The reaction proceeded without stirring for 24 hours at room temperature under dark conditions. At this time, an Ag shell was formed in the Au core. The reaction was incubated overnight in a 3.6 mM SDS solution to eccentrically deposit poly (aniline) on only one side of the Au seed. SDS, a surfactant, affects the interfacial tension between two adjacent phases, poly (aniline) -Ag and poly (aniline) -water, and separates poly (aniline) on one side of the Au seed to minimize total surface energy. A compartment was formed. The resulting solution was purified by centrifugation at 8,000 rpm for 10 minutes and resuspended in 3.6 mM SDS solution to prevent aggregation.

TEM images of the asymmetric Janus nanostructures including the double metal nanoclusters derived with MGITC at a final concentration of 1.5 μM are shown in FIG. 19. As shown in FIGS. 19A and 19B, when the Au nanoclusters were not BSA coated, the double metal nanoclusters having the Au core-Ag shells were not well formed. However, coating Au nanoclusters with BSA resulted in the formation of an asymmetric Janus nanostructure consisting of a double metal nanocluster compartment-polymer compartment, as shown in FIGS. 19 (c) and (d). Similarly, asymmetric Janus nanostructures were formed via redox reactions from the RBITC-derived Au nanoclusters at a final concentration of 3.8 μM RBITC.

20 (a) and (b) show that in the absence of the BSA coating, there is no asymmetric Janus nanostructure. However, as shown in FIGS. 20 (c) and (d), when stabilizing the MNP cluster with BSA, Janus nanostructures having an asymmetric polymer compartment were formed.

21 shows a TEM image of an asymmetric Janus nanostructure comprising Janus nanoclusters derived with MGITC at a final concentration of 0.75 μM. Raman dye concentrations were optimized to control cluster size. When low concentrations of MGITC were used to induce aggregation of AuNPs, several nanoclusters were observed. 21 (a) and (b) show asymmetric Janus nanostructures without BSA stabilization. Compared to nanostructures prepared using MGITC at 1.5 μM concentration, Janus nanostructures with Janus nanoclusters can be formed due to low levels of metal clustering without BSA coating. As can be seen in Figure 21 (c) and (d) there was no significant difference between the two nanostructures, without or without BSA coating.

FIG. 22 shows a TEM image of an asymmetric Janus nanostructure comprising Janus nanoclusters derived with RBITC at a final concentration of 1.9 μM. Likewise, the low concentration of RBITC did not affect the formation of asymmetric Janus nanostructures.

Iron oxide nanoparticles (Fe3O4) were prepared using a chemical coprecipitation method using ammonia water mixed with Fe ²⁺ and Fe ³⁺ in a molar ratio of 1: 2 as a precipitant. 0.86 g of ferrous chloride (FeCl ₂ ) and 2.35 g of ferric chloride (FeCl ₃ ) were mixed in 40 mL of deionized water under vigorous stirring and degassed with nitrogen gas for 30 minutes. The reaction solution was heated to 80 ° C. and 5 mL of ammonium hydroxide (NH ₄ OH) was added under mechanical stirring for 30 minutes. 1 g citric acid was added to the reaction flask and the temperature was increased to 90 ° C. and then vigorously stirred for an additional 90 minutes. Finally, Fe ₃ O ₄ magnetic nanoparticles (MNPs) were washed twice with deionized water under a static magnetic field of several hundred Gauss. In addition, small aliquots of the MNP solution were concentrated using a magnetic field and added to the polymer solution to prepare magnetic beads. 4.5 w / v% of poly (acrylamide-co-acrylic acid), poly (AAm-co-AA) in a mixture of deionized water and ethylene glycol in a volume ratio of 3: 1 Was prepared and uniformly dispersed MNP in this polymer solution to prepare a suspension of MNPs. A suspension of dispersed MNPs for an electrohydrodynamic (EHD) spraying process was placed in a 1.0 mL syringe (BD, Franklin Lakes, USA) with a 23 gage stainless steel capillary. To achieve a stable Taylor cone and con-jet mode, optimized viscosity was obtained by dissolving the polymer in a viscous solvent such as ethylene glycol without increasing the polymer concentration. The syringe was equipped with a micro syringe pump KDS-100 (KD Scientific, Inc, USA) which flows the MNPs suspension at a constant rate. An aluminum foil of 0.018 mm thickness (Fisherbrand; Thermo Fisher Scientific, USA) was used as the capture substrate. High voltage was applied between the capillary connected to the positive electrode and the aluminum foil connected to the negative electrode using a high voltage power supply NNC HV 30 (Nano NC, Korea). The distance between the two electrodes was 20-25 cm. The high voltage was maintained in the range of 10-15 kV and the flow rates of the two solutions were maintained at 0.08-0.15 ml / hour. High resolution digital cameras (D-90, Nikon Corporation, Japan) were used to visualize and capture single-phase Taylor cones, jet streams, and jet break-ups during EHD injection. After EHD injection, the resulting magnetic beads were thermally crosslinked overnight at 175 ° C. Finally, magnetic beads in powder form were collected and used for later experiments.

Example 4 Properties of Asymmetric Janus Nanostructures Having Double Metal Nanocluster Compartments and Polymer Compartments

The UV-Vis spectrum of the bimetallic nanocluster-polymer asymmetric Janus nanostructures is a UV-vis spectrometer with a wavelength of 300-900 nm changed to a fixed slit width of 1 nm in one scan mode of 10 scans at medium scan rate at room temperature. Obtained using (UV-1800, Shimadzu, Japan). Baseline was calibrated using two empty cells filled with deionized water. Colloidal solution characteristics are dynamic light scattering (DLS) (Zeta-sizer Nano ZS90, Malvern Instruments, UK) with a maximum power of 5 mW as a light source at a scattering angle of 90 ° and supplied with a Ne-He laser at 633 nm. ) Was used to characterize the hydrodynamic diameter and its size distribution, and the temperature was controlled to 25 ° C. Samples were diluted twice in a volume ratio of 1: 1 in deionized water and their average size was measured at a minimum of 20 scan cycles. Zeta-potential measurements were also performed to characterize the surface charge in deionized water. Individual AuNPs and asymmetric Janus nanocluster-polymer nanoparticles were analyzed using JEM-2100F FE-STEM (JEOL, Germany) operating at 80-200 kV acceleration voltage using Transmission Electron Microcopy. Samples were deposited on a 400 mesh copper grating coated with an ultra thin layer of carbon (Ted Pella, Inc., USA). Average diameter, size distribution and surface morphology were measured by scanning electron microscope (SEM) (VEGA-SB3, TESCAN, Czech Republic) with a focused beam of 0.5-30 kV. A few nanoparticle solutions were placed on silicon wafers and dried at room temperature. Samples were coated with a thin layer of conductive platinum using a coater (K575X Turbo Sputter Coater, Emitech Ltd, UK). The average size of the particles was analyzed by measuring approximately 50-100 particles randomly selected from TEM and SEM images using ImageJ software developed by the National Institutes of Health. In addition, all SERS measurements were performed using a Renishaw inVia Raman microscope system equipped with a Renishaw He-Ne laser operating at a wavelength (λ) of 632.8 nm for a stimulus source with a laser power of 12.5 mW. Rayleigh lines were removed from the collected SERS spectrum using a holographic notch filter located in the acquisition 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 a 520 cm ⁻¹ silicon line. Colloidal solutions of nanoparticles labeled with RBITC or MGITC were prepared in small glass capillary tubes (Kimble Chase, plain capillary tubes, soda-lime glass, inner diameter: 1.1-1.2 mm, wall: 0.2 ± 0.02 mm, length: 75 mm). ) SERS spectra were collected for 1 second of exposure time used to focus the laser spot on the glass capillary in the wavelength range of 608-1738 cm ⁻¹ using a 20 × objective lens.

17 shows the UV-Vis absorbance spectra of the asymmetric Janus nanostructures with AuNP, Raman dye induced Au nanoclusters and double metal nanoclusters. UV-Vis absorption peak of AuNP was shown at 510 nm (Fig. 17 (a)). When MGITC was added to AuNP solution and incubated for 10-90 minutes, the original absorption peak was red-shifted and new absorption peak appeared in the range of 650-850 nm. As the incubation time increased both peaks showed red shift due to the aggregation of AuNPs. Figure 17 (b) shows the UV-Vis absorbance of RBITC induced Au nanoclusters at different incubation times from 10 minutes to 90 minutes. Similar to the MGITC derived nanoclusters, the peak shifted red as the incubation time increased. Figure 17 (c) shows the UV-Vis absorbance of the asymmetric Janus nanostructure having a double metal nanocluster. After synthesis of the double metal nanoclusters, new absorption peaks appeared in the range of 410-550 nm, indicating the presence of Ag in the Au nanoclusters. In addition, wide peaks appeared in the range of 600-700 nm due to the aggregation of AuNPs. Dynamic light scattering (DLS) was performed to characterize the hydrodynamic diameter and size distribution of AuNP, Au nanoclusters and asymmetric Janus nanostructures as shown in FIG. 17 (d). The average diameters of AuNP, MGITC- or RBITC-induced Au nanoclusters were 18.9 ± 0.4 nm, 152.9 ± 2.8 nm and 115.7 ± 1.8 nm, and asymmetric Janus nanostructures with MGITC- or RBITC-induced double metal nanoclusters were 205 ± 4.5 nm and 186.3 ± 2.1 nm.

FIG. 18 shows the relative Raman spectra of (a) MGITC or (c) RBITC derived Au nanoclusters during clustering at various incubation times from 10 minutes to 90 minutes to optimize the degree of clustering for high SERS efficiency. In addition, Raman dye concentration and BSA coating of Au nanoclusters were adjusted to control cluster size and prevent further aggregation. It was found that as the incubation time increased, the Raman strength gradually decreased, resulting in the formation and precipitation of larger aggregates. As can be seen in Figure 18 (b) and (d), the Raman intensity of the MGITC- and RBITC- induced Au nanoclusters were 1618 cm ^-1 and 1648 cm ^-1 . 18 (e) and (f) show the relative Raman spectra of MGITC- or RBITC-labeled AuNPs, MGITC- or RBITC-induced Au nanoclusters and their asymmetric Janus nanostructures at the same Raman dye and particle concentrations. The Raman intensity of the MGITC- and RBITC-derived Au nanoclusters was 10.42 and 2.32 times that of AuNPs, which means that interparticle coupling between AuNP clusters significantly improves SERS efficiency due to hot spots. In addition, the Raman intensity of asymmetric Janus nanostructures with MGITC- or RBITC-induced double nanoclusters was 8.87 and 1.82 times higher than Raman dye-labeled AuNPs. Compared to the Raman intensity of the MGITC- and RBITC-induced Au nanoclusters, the Raman intensity of the asymmetric Janus nanostructures was somewhat reduced. Asymmetric Janus nanostructures with MGITC- or RBITC-induced double nanoclusters have better optical signals than MGITC- or RBITC-labeled AuNPs at the same Raman dye and particle concentration.

Example 5 Antibody-Binding with Asymmetric Janus Nanostructures Having Double Metal Nanocluster Compartments and Polymer Compartments

The bimetallic nanocluster-polymer asymmetric Janus nanostructures were combined with monoclonal antibodies (mAb) and polyclonal antibodies (pAbs) against the target protein carcinoembryonic antigen (CEA), respectively. First, the bioconjugation reaction of the polymer compartment and the anti-human CEA polyclonal antibody (anti-human CEA pAb) is performed by using an amide bond reaction between the amine group remaining in the poly (aniline) compartment and the carboxyl group present in the antibody. It was. This binding reaction was based on the chemistry of EDC (1-ethyl-3- (3-dimethylaminopropyl) carbodiimide) and sulfo-N-hydroxysuccinimide ester (sulfo-NHS). Specifically, 5 μl of 2.0 mg / ml anti-human CEA pAb was added to a dispersion solution of asymmetric Janus nanostructures containing 10 mM PBS at pH 7.4 containing 60 mM EDC and 9.2 mM sulfo-NHS, 3 Stirring for hours allowed the total pAb concentration to be 10 μg / ml. Anti-human CEA pAb-linked polymer compartments were washed by centrifugation at 3,000 rpm and resuspended in PBS. In addition, magnetic beads were chemically bound with anti-human CEA monoclonal antibodies (anti-human CEA mAbs) by activating residual carboxyl groups on polymer nanoparticles heat stabilized overnight at 175 ° C. Specifically, 1.25 mg of magnetic beads were suspended in 0.9 ml of PBS and sonicated for 2 minutes using a tip-sonicator using a 3/3 second on / off cycle at 20.0% amplitude. The uniformly suspended magnetic beads were mixed with 5.0 mM EDC and 5.0 mM sulfo-NHS and stirred for 1 hour. 3.56 mg / ml anti-human CEA mAb was diluted with 100 μl of PBS buffer, then slowly added to the magnetic bead solution to a final concentration of 8.9 μg / ml and stirred for 1 hour. Unbound anti-human CEA mAbs were separated using a magnetic field, and antibody-bound magnetic beads were resuspended in PBS for SERS-based biosensing of CEA (FIG. 16 (c)).

Example 6 SERS-Based Biosensing Using Asymmetric Janus Nanostructures

Quantitative analysis of the target protein CEA was carried out using asymmetric Janus nanostructures labeled with a Raman reporter as SERS nanoprobes. Immune complexes were selectively isolated with a separation tool (magnet) by forming sandwiched immune complexes using magnetic beads combined with anti-human CEA mAb. First, magnetic beads bound with anti-human CEA monoclonal antibody were added to a buffer containing three different concentrations of CEA ranging from 22.5 to 67.5 ng / ml and reacted for 1 hour. The target protein was washed with an external magnetic field and resuspended in fresh PBS buffer. Subsequently, SERS nanoprobe with anti-human CEA pAb bound was added to each immune complex in which the target protein and the magnetic beads were formed and reacted for 1 hour to prepare a sandwich immune complex consisting of the magnetic beads, the target protein and the SERS nanoprobe. It was. Unbound SERS nanoprobes were removed using a magnetic field and the product sandwiched immune complex was resuspended in PBS for SERS measurements. Experiments with controls were also performed to assess the selective binding capacity of SERS nanoprobes without the target protein.

제 44th 발명: invent:

Example 1 Synthesis of Double Metal Nanorod Clusters by Side-by-Side

Side-to-side assembled gold nanorods (AuNRs) clusters were prepared by the addition of citrate anions for electrostatic interaction with positively charged hexadecyltrimethylammonium bromide (CTAB) in AuNR. AuNRs were synthesized via a seed-mediated growth method. Specifically, the 0.20 M CTAB dissolved in 5 mL of 29-30 ℃ was mixed with 0.0005 M of gold chloride (III) hydrate (Gold (III) chloride hydrate, HAuCl ₄ .3H ₂ O), and then 5 mL of 0.010 M 0.010 mL of cold NaBH ₄ was added. The yellow color of the reaction solution turned into a brownish yellow solution, and the resulting seed solution was maintained at 29-30 ° C. and used within 2 to 2.5 hours. 0.25 mL of 0.004 M AgNO ₃ was mixed with 0.20 M CTAB at 29-30 ° C. to grow nanorods on seed particles. Subsequently, 5.0 mL of 0.001 M HAuCl ₄ was added to the solution and stirred. After mixing for 30-40 minutes, ascorbic acid, a reducing agent, was added to induce a color change of the growth solution from dark yellow to colorless. In the final step, 12 μL of the seed solution was added to the colorless solution and the solution color was slowly changed within 10-20 minutes. The solution was stirred and stored at 29-30 ° C overnight. The resulting solution was centrifuged at 10,000 rpm for 10 minutes and resuspended in 1 mM CTAB to prevent aggregation of AuNR, finally producing AuNR capped CTAB. MGITC was introduced into the AuNR solution at a final concentration of ^10-6 M and fixed to the AuNR surface via the isothiocyanate group (-N〓C〓S) of MGITC. For side-to-side assembly of AuNR, 30 uL of 0.175 mM sodium citrate solution was added to 1 mL of AuNR solution and incubated for a short period of 1 to 5 minutes. Through electrostatic interactions between citrate ions, which are anions, and CTAB, which are cations, the side-to-side self-assembled AuNR clusters were stabilized by 200 μL of 1 w / v% PSS coating (FIG. 24 (a)).

Example 2 Synthesis of Diblock Poly (AAc-b-NIPAM)

Poly (acrylic acid-block-N-isopropylacrylamide), a negatively charged stimulatory copolymer and diblock polymer, is a sequential reversible addition-fragmentation chain transfer (RAFT) polymerization and poly (tBA-b-NIPAM) was synthesized by an additional hydrolysis process of the tBA group of poly (tert-butyl acrylate-block-N-isopropylacrylamide). NIPAM monomer was dissolved in n-hexane at 40 ° C. Impurities and inhibitors were removed by recrystallization below 4 ° C. 10 g of NIPAM was dissolved in 200 ml of n-hexane in a beaker to a concentration of 5 w / v% When crystalline NIPAM was formed at low temperature, filter paper (Whatman The solution was filtered through an aspirator device (EYELA 1000S, US) using qualitative filter paper grade 1) and the product was dried under vacuum to remove n-hexane tBA was distilled at 40 ° C. and 26 mmHg to remove the polymerization inhibitor. Purified.

Poly (tBA), i.e. poly (tBA) -macro CDTPA (4-cyano-4-[(dodecylsulfanylthiocarbonyl) sulfanyl] pentanoic acid) is a monomer, tB (tert-butyl acrylate), CTA (chain transfer agent) [A Monomer]: [CTA]: [Initiator] = 1000: 10 with zero AIBN (azobisisobutyronitrile, 2,2'-azobis (2-methylpropionitrile)) under reaction solvent 1,4-dioxane (1,4-dioxane) Synthesis was carried out by RAFT polymerization of tBA added in a: 1 molar ratio. Specifically, tBA (5 mL, 34 mmol), CDTPA (0.137 g, 0.34 mmol) and 1,4-dioxane (5 mL) were placed in a Schlenk flask. The solution was degassed with nitrogen gas for 20 minutes before polymerization. AIBN (0.0055 g, 0.034 mmol) was added to the reaction mixture to initiate the polymerization reaction. The polymerization was carried out with mechanical stirring at 60 ° C. for 5 or 6 hours and quenched in ice water. The resulting poly (tBA) -macro CDTPA was obtained by precipitation in a methanol: H 2 O = 50: 50 (v / v) solution and dried overnight in a vacuum oven.

Poly (tBA-b-NIPAM) was prepared in a similar manner. For the synthesis of poly (tBA-b-NIPAM), NIPAM (N-isopropylacrylamide 97%) as monomer, poly (tBA) -macro CDTPA as CTA and AIBN as initiator [monomer]: [CTA]: [initiator] = Add at 1500: 5: 1 molar ratio. Specifically, 0.411 g of poly (tBA ₁₄₅ ) -macro CDTPA (Mn ^NMR = 18,584 g / mol) and 0.75 g of NIPAM were dissolved in 2.5 ml of 1,4-dioxane and degassed with nitrogen gas for 20 minutes. To this solution was added 0.00075 g of AIBN and polymerized at 60 ° C. for 6 hours. The flask was immersed in ice water to stop the reaction and the solution was exposed to air. The poly (tBA ₁₄₅ -b-NIPAM ₃₀₀ ) solution was purified by precipitation in ether to remove unreacted monomers and initiators and dried overnight in a vacuum oven.

Since the thiocarbonylthio- group exists as a protected thiol group (masked -SH), the trithiocarbonate group of poly (tBA-b-NIPAM) is bound to the thiol group coupling. It was cleaved by aminolysis using a nucleophilic reagent. Specifically, 0.5 g of poly (tBA-b-NIPAM) (9.5 μmol), MTS (18 μL, 190 μmol), hexylamine (252 μL, 1.9 mmol) and triethylamine (266 μL, 1.9 mmol) was dissolved in 5 mL of THF (Tetrahydrofuran) and the mixture was stirred at rt for 24 h. The prepared thiol-terminated poly (tBA ₁₄₅ -b-NIPAM ₃₀₀ ) (poly (tBA ₁₄₅ -b-NIPAM ₃₀₀ ) -SH) was precipitated three times in hexane and dried in a vacuum oven overnight (FIG. 26 (a)).

Thiol-terminated poly (AAc ₁₄₅ -b-NIPAM ₃₀₀ ) (poly (AAc ₁₄₅ -b-NIPAM ₃₀₀ ) -SH) is a thiol-terminated poly (tBA ₁₄₅ -b-NIPAM ₃₀₀ ) (TBA) using trifluoroacetic acid (TFA) TBA in poly (tBA ₁₄₅ -b-NIPAM ₃₀₀ ) -SH) was prepared by hydrolysis with AAc groups. Specifically, 0.2 g of thiol-terminated poly (tBA ₁₄₅ -b-NIPAM ₃₀₀ ) (poly (tBA ₁₄₅ -b-NIPAM ₃₀₀ ) -SH) (3.8 μmol) and 583 μL of TFA (7.6 mmol) were added to 5 mL of It was dissolved in dichloromethane (DCM). After 24 hours a light brown gelatin mass formed and precipitated out of solution. The resulting material was dissolved in DCM and precipitated in n-hexane. Finally, the product was dissolved in THF and dialyzed against deionized water for 2 days. The obtained thiol-terminated poly (AAc ₁₄₅ -b-NIPAM ₃₀₀ ) (poly (AAc ₁₄₅ -b-NIPAM ₃₀₀ ) -SH) was lyophilized with freeze dryer MCFD8508 (Ilshin Lab, Korea) under vacuum (FIG. 26 (b)). ).

Example 3 Synthesis of Double Metal Nanorod Clusters through End-to-End Assembly

An end-to-end assembled AuNR cluster was prepared by selectively attaching the poly (AAc ₁₄₅ -b-NIPAM ₃₀₀ ) -SH of Example 2 to the ends of the AuNR capped CTAB of Example 1. Specifically, 4 mL of CTAB capped AuNR solution was centrifuged at 10,000 rpm for 10 minutes and reproduced in 0.5 mL of deionized water. Concentrated CTAB capped AuNR was injected rapidly under sonication into a 10 mL DMSO solution containing 5 mg of poly (AAc ₁₄₅ -b-NIPAM ₃₀₀ ) -SH to a final concentration of 0.05 w / v%. The mixture solution was sonicated in an sonicator for 30 minutes and incubated for 1 hour at room temperature. The CTAB ligand attached to the terminal end of AuNR is converted to poly (AAc ₁₄₅ -b-NIPAM ₃₀₀ ) -SH through a metal-thiol group bond with a thiol group (-SH) in poly (AAc ₁₄₅ -b-NIPAM ₃₀₀ ) -SH. Exchanged. The AuNR side is then through an electrostatic interaction between positively charged CTAB on the side of AuNR in 20 ° C. or 50 ° C. or PBS and poly (AAc ₁₄₅ -b-NIPAM ₃₀₀ ) -SH on the end face of AuNR. And AuNR ends are bonded to form self-assembly between ends. End-to-end self-assembled AuNR clusters were purified by centrifugation at 6,000 rpm for 6 minutes and resuspended in 1 mL of deionized water. MGITC was introduced into the AuNR solution at a final concentration of 10 ^-5 M and fixed to the AuNR surface via the isothiocyanate group (-N〓C〓S) of MGITC. End-to-end assembled AuNR was stabilized by 0.5% BSA (bovine serum albumin) coating (FIG. 24 (b)).

Example 4 Synthesis of Asymmetric Double Metal Nanorod Cluster-Polymer Janus Nanostructures

Asymmetric Janus nanostructures consisting of double metal nanorod clusters and polymer (poly (aniline)) compartments were synthesized through reduction of silver based on surface template polymerization and reduction oxidation of aniline. A mother-side or end-to-end assembled AuNR cluster was used as the mother liquor of the seed particles. Specifically, aniline and SDS were dissolved in 0.5 mL of deionized water to a final concentration of 5 mM and 0.9 mM, respectively. Seed particle solution was added to the mixture and stirred, after which 0.5 mL of silver nitrate solution was added and mixed with the solution to a final concentration of 2.5 mM. The reaction proceeded without stirring for 24 hours at room temperature under dark conditions. The reaction was further incubated overnight in a 3.6 mM SDS solution so that the poly (aniline) compartments were eccentrically deposited on only one side of the Au seed. The resulting solution was purified by centrifugation at 8,000 rpm for 10 minutes and resuspended in deionized water or 10 mM PBS (phosphate buffer saline).

Comparative Example 1 Preparation of Double Metal Nanorod-Polymer Janus Nanoparticles Without AuNR Cluster

Double metal nanorod-polymer Janus nanoparticles without AuNR clusters were prepared in the same manner as in Example 4, but a general AuNR solution was used as a mother liquor of the seed particles.

The prepared double metal nanorod-polymer Janus nanoparticles without AuNR cluster were used as a control.

Example 5 Synthesis of Magnetic Nanoparticles (MNPs) and Magnetic Beads by Electrohydrodynamic (EHD) Injection

Iron oxide nanoparticles (Fe ₃ O ₄ ) was prepared using a chemical coprecipitation method using ammonia water mixed with Fe ²⁺ and Fe ³⁺ in a molar ratio of 1: 2 as a precipitant. 0.86 g of ferrous chloride (FeCl ₂ ) and 2.35 g of ferric chloride (FeCl ₃ ) were mixed in 40 mL of deionized water under vigorous stirring and degassed with nitrogen gas for 30 minutes. The reaction solution was heated to 80 ° C. and 5 mL of ammonium hydroxide (NH ₄ OH) was added under mechanical stirring for 30 minutes. 1 g citric acid was added to the reaction flask and the temperature was increased to 90 ° C. and then vigorously stirred for an additional 90 minutes. Fe ₃ O ₄ magnetic nanoparticles (MNPs) were washed twice with deionized water under a static magnetic field of several hundred Gauss. In addition, small aliquots of the MNP solution were prepared as magnetic beads via electrohydrodynamic (EHD) spraying which was concentrated using a magnetic field and added to the polymer solution. 4.5 w / v% of poly (acrylamide-co-acrylic acid), poly (AAm-co-AA) in a mixture of deionized water and ethylene glycol in a volume ratio of 3: 1 Was prepared and uniformly dispersed MNP in this polymer solution to prepare a suspension of MNPs. A suspension of dispersed MNPs for the EHD injection process was placed in a 1.0 mL syringe (BD, Franklin Lakes, USA) with a 23 gage stainless steel capillary. To achieve a stable Taylor cone and con-jet mode, optimized viscosity was obtained by dissolving the polymer in a viscous solvent such as ethylene glycol without increasing the polymer concentration. The syringe was equipped with a micro syringe pump KDS-100 (KD Scientific, Inc, USA) which flows the MNPs suspension at a constant rate. An aluminum foil (Fisherbrand; Thermo Fisher Scientific, USA) with a thickness of 0.018 mm was used as the collecting substrate. High voltage was applied between the capillary connected to the positive electrode and the aluminum foil connected to the negative electrode using a high voltage power supply NNC HV 30 (Nano NC, Korea). The distance between the two electrodes was 20-25 cm. The high voltage was maintained in the range of 15-20 kV and the flow rates of the two solutions were maintained at 0.08-0.15 ml / hour. High resolution digital cameras (D-90, Nikon Corporation, Japan) were used to visualize and capture single-phase Taylor cones, jet streams, and jet break-ups during EHD injection. After EHD injection, the resulting magnetic beads were thermally crosslinked overnight at 175 ° C. Magnetic beads in powder form were collected from the foil and used for later experiments.

Example 6 Antibody Conjugation of Asymmetric Double Metal Nanorod Cluster-Polymer Janus Nanostructures and Magnetic Beads

Asymmetric Janus nanostructures and magnetic beads were conjugated separately with two different sets of monoclonal antibodies (mAb) and polyclonal antibodies (pAb) against the target protein carcinoembryonic antigen (CEA). First, an anti-human CEA polyclonal antibody (anti-human CEA pAb) was introduced into a Janus nanostructured poly (aniline) compartment through an amide coupling reaction of an amine group and a carboxyl group present in the poly (aniline) compartment. This binding reaction was based on the chemistry of EDC (1-ethyl-3- (3-dimethylaminopropyl) carbodiimide) and sulfo-NHS (sulfo-N-hydroxysuccinimide ester). To a nanostructure solution of 10 mM PBS, pH 7.4, containing 60 mM EDC and 9.2 mM sulfo-NHS, 5 μl of anti-human CEA pAb 2.0 mg / ml was added and stirred for 3 hours to give a total pAb concentration of 5 μg / to ml. Anti-human CEA pAb-binding Janus nanostructures were washed by centrifugation at 3,000 rpm and resuspended in PBS. In addition, magnetic beads were chemically bound to anti-human CEA monoclonal antibodies (anti-human CEA mAbs) by activating residual carboxyl groups on magnetic beads that were thermally stabilized at 175 ° C. overnight. Specifically, 1.25 mg of magnetic beads were suspended in 0.9 ml of PBS and sonicated for 2 minutes using a tip-sonicator using a 3/3 second on / off cycle at 20.0% amplitude. The uniformly suspended magnetic beads were mixed with 5.0 mM EDC and 5.0 mM sulfo-NHS and stirred for 1 hour. 3.56 mg / ml of anti-human CEA mAb was diluted with 100 μl of PBS buffer, then slowly added to the magnetic bead solution until the final concentration was 8.9 μg / ml and stirred for 1 hour. Unconjugated anti-human CEA mAbs were separated using a magnetic field and antibody-bound magnetic beads were resuspended in PBS for SERS-based biosensing of CEA.

Example 7 SERS-Based Biosensing for Target Protein CEA Using Asymmetric Double Metal Nanorod Cluster-Polymer Janus Nanostructures and Magnetic Beads

Asymmetric Janus nanostructures labeled with Raman reporters were used as SERS nanoprobes for quantitative analysis of the target protein CEA and sandwiched immune complexes using anti-human IgG mAbs or magnetic beads coupled with anti-human CEA mAbs. Immune complexes were selectively isolated by means of a separation tool by forming. First, magnetic beads bound with anti-human IgG monoclonal antibody or anti-human CEA monoclonal antibody were added to a buffer containing IgG or CEA at three different concentrations ranging from 22.5 to 67.5 ng / ml and reacted for 1 hour. . The target protein was washed with an external magnetic field and resuspended in fresh PBS buffer. Subsequently, SERS nanoprobe with anti-human CEA pAb bound was added to each immune complex in which the target protein and the magnetic beads were formed and reacted for 1 hour to prepare a sandwich immune complex consisting of the magnetic beads, the target protein and the SERS nanoprobe. It was. Unbound SERS nanoprobes were removed using a magnetic field and the product sandwiched immune complex was resuspended in PBS for SERS measurements. Experiments with controls were performed together to assess the selective binding capacity of SERS nanoprobes without the target protein.

Example 8 Characterization of Diblock Poly (AAc-b-NIPAM)

1H nuclear magnetic resonance (1H NMR) instrument operating at 400 MHz frequency using dimethyl sulfoxide (d-6) and chloroform-d (CDCl3) as a solvent, AVANCE III 400, Bruker BioSpin AG, Fallennden, Switzerland) to analyze the chemical composition of the poly (AAc-b-NIPAM) of Example 2. The apparent molar ratio of poly (AAc-b-NIPAM) was obtained by comparing the relative peak signals of the corresponding protons from each monomer. To determine its number-average molecular weight, weight-average molecular weight and polydispersity index, gel-permeation chromatography (GPC) measurements were performed on Shodex. High-performance liquid chromatography (HPLC) 1260 series apparatus (Agilent Technologies, Palo Alto, CA, USA) using GPC column KF-803 (Shodex GPC system-21; Showa Denko Co., Tokyo, Japan) It was performed using. THF and polystyrene of 1,270 to 139,000 g / mol were used as the mobile and stationary phases with a flow rate of 1.0 ml / min, respectively.

The thermal properties of the poly (AAc-b-NIPAM) were confirmed by measuring the UV absorbance of the poly (AAc-b-NIPAM) solution and the hydrodynamic diameter of the micelle structures in phosphate buffer was specified with temperature. Samples were prepared by dissolving poly (AAc-b-NIPAM) in PBS at a concentration of 0.05 w / v%. Low critical solution temperature (LCST) of poly (AAc-b-NIPAM) uses UV-Vis spectrometer Cary-100 Bio (Varian Biotech, US) with temperature control of peltier thermostat Was determined by monitoring the absorbance of the poly (AAc-b-NIPAM) solution at 350 nm. The measurement was carried out in a temperature range of 20 to 70 ° C. at a heating rate of 1 ° C./min. The hydrodynamic radius of poly (AAc-b-NIPAM) was measured over temperature using dynamic light scattering (DLS) (Zeta-sizer Nano ZS90, Malvern Instruments, Malvern, UK).

A UV-Vis spectrometer Shimadzu model UV-1800 series (Shimadzu, Japan) was used to examine the UV absorbance of the sample to analyze the cleavage of trithiocarbonate groups. Poly (tBA-b-NIPAM) -macro CDTPA and poly (AAc-b-NIPAM) -SH were dissolved in CHCl ₃ at a concentration of 0.5 w / v%. Each sample was scanned at a wavelength of 200-700 nm.

As a result, poly (tBA ₁₄₅₎ and poly (tBA _145- b-NIPAM ₃₀₀₎ a number average molecular weight (number-average molecular weight, Mn ^GPC), the weight average molecular weight (weight-average molecular weight, ^GPC Mw) and polydispersity of The index (polydispersity index, PDI) is shown in Table 3.

	[Monomer]:[CTA]:[Initiater][Monomer]: [CTA]: [Initiater]	Retention (Reaction) time(h)Retention (Reaction) time (h)	Mn^NMR(g/mol)Mn ^NMR (g / mol)	Mn^GPC(g/mol)Mn ^GPC (g / mol)	Mw^GPC(g/mol)Mw ^GPC (g / mol)	PDIPDI
poly(tBA₁₄₅) poly (tBA ₁₄₅ )	1000:10:11000: 10: 1	55	18,58418,584	21,15221,152	25,59325,593	1.211.21
poly(tBA_145-b-NIPAM₃₀₀)poly (tBA _145- b-NIPAM ₃₀₀ )	1500:5:11500: 5: 1	66	52,53252,532	55,86355,863	70,94670,946	1.271.27

Figure 27 (a) shows the 1H NMR spectra of poly (tBA) and poly (tBA-b-NIPAM) measured in CDCl ₃ , poly (AAc-b-NIPAM) measured in DMSO-d6 at 400 MHz. The peak at 1.45 ppm showed the terminal methyl proton of the tBA block and the single peak at 3.9 ppm showed the C-2 proton of the isopropyl group on the NIPAM block. After the tBA group was hydrolyzed to the AAc group, the peak of the methyl ester proton at 1.45 ppm disappeared, indicating that all ester groups of poly (tBA-b-NIPAM) were converted to acrylic acid. FIG. 27 (b) and Table 3 show GPC of poly (tBA) and poly (tBA-b-NIPAM) using THF as a mobile phase to measure molecular weight, polydispersity index and copolymer distribution according to holding time (reaction) time. Indicates a trace. Poly (tBA ₁₄₅ ) has a weight average molecular weight of 25,593 g / mol, PDI 1.21 and poly (tBA ₁₄₅ -b-NIPAM ₃₀₀ ) has a weight average molecular weight of 70,946 g / mol and PDI of 1.27. (C) is a UV-Vis absorption graph of poly (tBA-b-NIPAM) before and after amino degradation. After removing the thiocarbonylthio group via amino decomposition, the absorption peak disappeared at 307 nm. In addition, the heat deformation characteristic of poly (AAc-b-NIPAM) was measured according to the temperature through UV absorbance and dynamic light scattering, and as shown in FIG. 27 (d). Poly (NIPAM) -based copolymers or block copolymers exhibited a water soluble state under a lower critical solution temperature (LCST), whereas above a LCST it showed an insoluble state due to hydrophobic interactions. The LCST of the copolymer was determined as the temperature at which the maximum value of the derivative was reached in temperature dependent UV absorbance. Poly (AAc-b-NIPAM) showed an LCST of 39.5 ° C. at a final concentration of 0.05 w / v% in 10 mM PBS. The hydrodynamic radius of poly (AAc-b-NIPAM) was 12.05 nm below LCST and the diameter was 39.2 nm above LCST.

Example 9 Characterization of Asymmetric Nanorod Cluster-Polymer Janus Nanostructures

The UV-Vis spectra of the asymmetric Janus nanostructures are UV-visible spectrometers (UV-1800, Shimadzu, Ltd.) that vary the wavelength from 300 to 900 nm to a fixed slit width of 1 nm in a one-time 10 scan mode at medium scan rate at room temperature. Japan). Baseline was calibrated using two empty cells filled with deionized water. Colloidal solution characteristics are dynamic light scattering (DLS) (Zeta-sizer Nano ZS90, Malvern Instruments, UK) with a maximum power of 5 mW as a light source at a scattering angle of 90 ° and supplied with a Ne-He laser at 633 nm. Was used to characterize the hydrodynamic diameter and its size distribution, and the temperature was controlled to 25 ° C. Samples were diluted 10-fold in deionized water or PBS at a volume ratio of 1: 1, and their average size was measured at a minimum of 20 scan cycles. Zeta potential measurements were also performed to characterize the surface charge in deionized water. Individual AuNRs and asymmetric Janus nanostructures were analyzed using JEM-2100F FE-STEM (JEOL, Germany) operating at 80-200 kV acceleration voltage through Transmission Electron Microcopy. Samples were deposited on a 400 mesh copper lattice coated with an ultra thin layer of carbon (Ted Pella, Inc., USA). All SERS measurements were performed using a Renishaw inVia Raman microscope system (Renishaw, UK) equipped with a Renishaw He-Ne laser operating at a wavelength (λ) of 632.8 nm for a stimulus source with a laser power of 12.5 mW. Rayleigh lines were removed from the collected SERS spectrum using a holographic notch filter located in the acquisition 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 a 520 cm ⁻¹ silicon line. Colloidal solutions of nanoparticles labeled with MGITC were placed in small glass capillaries (Kimble Chase, plain capillary tubes, soda-lime glass, inner diameter: 1.1-1.2 mm, wall diameter: 0.2 ± 0.02 mm, length: 75 mm). Put in. SERS spectra were collected for 1 second of exposure time used to focus the laser spot on the glass capillary in the wavelength range of 608-1738 cm ⁻¹ using a 20 × objective lens.

As a result, side-by-side assembled AuNR nanoclusters prepared by adding the citrate anion to the AuNR solution at different incubation times, and an asymmetric hybrid nanoparticles comprising the same Hydrodynamic diameter and zeta potential of (Citrate anion incubation time: 1-5 minutes) were as Table 4 below.

	side-by-side assembled AuNR nanoclustersside-by-side assembled AuNR nanoclusters					Anisotropic hybrid nanoparticlesAnisotropic hybrid nanoparticles
	incubation time(min)incubation time (min)
	00	1One	22	33	55
hydrodynamic diameter(nm)hydrodynamic diameter (nm)	transverse:46.8±0.1, longitudinal: 1.1±0.1transverse: 46.8 ± 0.1, longitudinal: 1.1 ± 0.1	transverse: 68.2±2.5, longitudinal: 3.7±0.5transverse: 68.2 ± 2.5, longitudinal: 3.7 ± 0.5	transverse: 99.8±3.0, longitudinal: 8.9±0.7transverse: 99.8 ± 3.0, longitudinal: 8.9 ± 0.7	transverse: 87.6±1.1, longitudinal: 6.4±0.2transverse: 87.6 ± 1.1, longitudinal: 6.4 ± 0.2	transverse: 92.1±2.5, longitudinal: 7.4±0.6transverse: 92.1 ± 2.5, longitudinal: 7.4 ± 0.6	78.6±0.278.6 ± 0.2
ζ-potential(mV) ζ-potential (mV)	31.1±1.3331.1 ± 1.33	N/A^* N / A ^*	N/A^* N / A ^*	N/A^* N / A ^*	-41.4±0.4-41.4 ± 0.4	-7.8±0.3-7.8 ± 0.3

^* N / A: not available.

In addition, the hydrodynamic diameters of AuNRs (original AuNPs), end-to-end assembled AuNR nanoclusters and asymmetric Janus nanostructures including the same are shown in Table 5 below. AuNR clusters and asymmetric Janus nanostructures were self-assembled in deionized water (DW) at room temperature or PBS at different temperatures.

	original AuNPsoriginal AuNPs	DW(RT)DW (RT)		PBS(20 ℃)PBS (20 degreeC)		PBS(50 ℃)PBS (50 degreeC)
	original AuNPsoriginal AuNPs	end-to-end assembled AuNR nanoclustersend-to-end assembled AuNR nanoclusters	Anisotropic hybrid nanoparticlesAnisotropic hybrid nanoparticles	end-to-end assembled AuNR nanoclustersend-to-end assembled AuNR nanoclusters	Anisotropic hybrid nanoparticlesAnisotropic hybrid nanoparticles	end-to-end assembled AuNR nanoclustersend-to-end assembled AuNR nanoclusters	Anisotropic hybrid nanoparticlesAnisotropic hybrid nanoparticles
hydrodynamic diameter(nm)hydrodynamic diameter (nm)	transverse: 60.9±0.5, longitudinal:2.0±0.1transverse: 60.9 ± 0.5, longitudinal: 2.0 ± 0.1	transverse: 499.5, 101.8, longitudinal: 5.1transverse: 499.5, 101.8, longitudinal: 5.1	388388	transverse: 455.3, longitudinal: 57.9transverse: 455.3, longitudinal: 57.9	735735	transverse: 329.7, longitudinal: 46.0transverse: 329.7, longitudinal: 46.0	590590

FIG. 25 shows UV-Vis absorbance spectra and hydrodynamic diameters of the side-to-side assembled AuNR and asymmetric Janus nanostructures comprising the same. In the side-to-side assembly of AuNR, the terminal plasmon peak shifted blue from 700 nm to 610 nm with reduced intensity as the incubation time increased from 1 minute to 5 minutes as shown in FIG. 25 (a). In contrast, the transverse peak shifted slightly red from 510 nm to 525 nm with increased intensity. The degree of shift of the longitudinal and transverse peaks of AuNRs correlated with the level of clustering in terms of incubation time. This change in plasmon absorption peak is due to plasmon coupling in the lateral cross-sectional assembly of AuNRs. Dynamic light scattering (DLS) measurements were also performed to characterize the hydrodynamic diameter, size distribution, and colloidal stability of the side-to-side self-assembled AuNR and its Janus nanostructures. Figure 25 (b) and Table 4 shows the hydrodynamic diameter of the assembled nano-cluster between the side and the existing AuNR with increasing incubation time of 1 to 5 minutes. The mean diameters of the longitudinal and transverse axes of AuNR were 1.1 ± 0.1 nm and 46.8 ± 0.1 nm, respectively. When the citrate anion was added to the AuNR solution, the diameter of both the longitudinal and transverse axes of AuNR increased as the incubation time was extended from 0 to 5 minutes. After PSS coating of side-to-side assembled AuNRs, the longitudinal and transverse axis mean diameters of the nanoclusters were 7.4 ± 0.6 nm and 92.1 ± 2.5 nm, respectively. The ζ-potential values of the conventional AuNR and the assembled side-to-side AuNR were 31.1 ± 1.3 mV and-41.4 ± 0.4 mV, respectively. The dramatic change in the surface charge value of the AuNR cluster was due to the presence of PSS, a negatively charged polymer electrolyte. As shown in FIG. 25 (c), after oxidation polymerization and silver deposition onto poly (aniline) on the lateral AuNR clusters, a new Ag absorption peak appears in the range of 330-450 nm and the termination plasmon peak is Red-shifts were shown as compared to the peaks of Janus nanoparticles seeded with AuNR, indicating the presence of double metal Au core-Ag shell nanorod cluster sections. Figure 25 (d) shows the hydrodynamic diameter of the asymmetric double metal nanorod cluster-polymer Janus nanostructures through side-to-side assembly of AuNR. The mean diameter of Janus nanostructures was 78.6 ± 0.2 nm, and the cluster of nanoparticles containing the existing AuNRs as seeds was 93.2 ± 2.3 nm.

FIG. 28 shows UV-Vis absorbance spectra and hydrodynamic diameters of end-to-end assembled AuNR and asymmetric Janus nanostructures comprising the same. In the end-to-end assembly of AuNR, the longitudinal plasmon absorption peak was red-shifted from 645 nm to 660 nm as shown in Fig. 28 (a), and the transverse peak was little changed. This change was caused by alternating dipoles along the AuNR chain. When AuNR was assembled in the end-to-end direction in PBS at 20 ° C., the shoulder peak appeared at about 800 nm, indicating that these nanoclusters were partially attributable to the decrease in electrostatic repulsion between CTAB bilayers at high ionic strength of PBS. Showed aggregation. At 50 ° C. above LCST of poly (AAc-b-NIPAM) the longitudinal plasmon absorption peak was slightly blue-shifted. 28 (b) and Table 5 show the hydrodynamic diameters of the assembled end-to-end AuNR clusters in deionized water or PBS at different temperatures. The mean diameters of the vertical and horizontal axes of the conventional AuNR were 2.0 ± 0.1 nm and 60.9 ± 0.5 nm, respectively. The diameters of AuNR nanoclusters in deionized water were 5.1 nm, 101.8 nm and 499.5 nm, indicating both the presence of individual AuNR and the end-to-end assembled AuNR. When the nanoclusters were present in PBS at 20 ° C, the diameters were 57.9 nm and 455.3 nm due to the increase in ionic strength. At 50 ° C., higher than the transition temperature of poly (AAc-b-NIPAM), the collapse of the NIPAM block greatly reduces the diameter, leading to a reduction in the nanogap between AuNRs. As shown in FIG. 28 (c), after oxidation polymerization to poly (aniline) and silver reduction on the end-to-end directional AuNR cluster, a new Ag absorption peak appears in the range of 350-450 nm, and the longitudinal plasmon peak is terminal Due to the inter-assembled AuNR, it was slightly blue-shifted compared to Janus nanoparticles seeded with conventional AuNR. Figure 28 (d) shows the hydrodynamic diameter of asymmetric double metal nanorod cluster-polymer Janus nanostructures via end-to-end assembly of AuNR. The mean diameter of Janus nanostructures was 388 nm in deionized water and the cluster of nanoparticles consisting of AuNR was 246.4 ± 30.7 nm. When these Janus nanostructures were suspended in PBS at 20 ° C., the average diameter was 735 nm, indicating partial cohesion between the double metal nanorod cluster compartments of Janus nanostructures. However, at 50 ° C above LCST, the particle gap between adjacent nanorods was reduced due to the collapse of the NIPAM block, resulting in a decrease in hydrodynamic diameter to 590 nm.

FIG. 29 (a) shows the relative Raman shift of the AuNR labeled MGITC at 10 ⁻⁶ M MGITC and the assembled side-to-side AuNR cluster. As MGITC is anchored to the AuNR surface via the isothiocyanate group of MGITC (-N = C = S), the Raman strength of MGITC embedded in the interparticle junction between adjacent AuNRs of the lateral oriented nanoclusters is AuNR (original AuNPs) were approximately 11.0 times higher than the Raman strength of MGITC immobilized on the surface. The SERS intensity of MGITC from this Janus nanostructure was about 6.53 times higher at 1617 cm −1 than the MGITC SERS intensity immobilized on the AuNR (original AuNPs) surface. In addition, relative Raman spectra of the end-assembled AuNR cluster and Janus nanostructure including the same as shown in FIG. 29 (b) were measured in deionized water and PBS at room temperature. MGITC was introduced to investigate SERS efficiency after self-assembly. Compared to the individual AuNRs, the Raman intensity of the end-to-end assembled AuNR nanoclusters increased significantly by 38.2 times in deionized water. The higher the curved surface of the AuNRs, the greater the electromagnetic field, and thus the higher SERS strength of the end-to-end assembly compared to the individual AuNR. The SERS strength of Janus nanostructures increased by 17.3 times compared to the individual AuNR.

30 shows an asymmetric double metal nanorod-polymer Janus nanostructure using each AuNR, AuNR as a seed to specify size and shape, directional self-assembled AuNR nanoclusters through side-to-side assembly, and an asymmetric double including the same TEM images of metal nanorod cluster-polymer Janus nanostructures at various magnifications.

FIG. 31 is a TEM image at various magnifications of directional self-assembled AuNR nanoclusters and asymmetric bimetallic nanorod cluster-polymer Janus nanostructures comprising the same by end-to-end assembly to specify size and shape.

Claims

Double metal nanocluster cores; And

A conductive polymer shell radially located around the core;

Self-assembled double metal-polymer Janus nanostructure consisting of.
The method of claim 1,

The double metal nano cluster core is composed of a first metal and a second metal surrounding the first metal surface,

The first metal and the second metal are each selected from the group consisting of silver, gold, copper and mixtures thereof,

The first metal and the second metal are not the same, self-assembled double metal-polymer Janus nanostructures.
The method of claim 1,

The conductive polymer is at least one selected from the group consisting of polypyrrole, polythiophene, poly (3,4-ethylene dioxythiophene) (PEDOT) and polyaniline, self-assembled double metal-polymer Janus nanostructures.
The method of claim 1,

The double metal nano cluster core further comprises a Raman dye, self-assembled double metal-polymer Janus nanostructure.
Metal nanoprobe for surface-enhanced Raman scattering (SERS) based biosensing and / or bioimaging measurement using Janus nanostructure of claim 4.
Metal nanoprobe for fluorescence based biosensing and / or bioimaging measurement using the Janus nanostructures according to claim 1.
Drug delivery using the Janus nanostructures according to any one of claims 1 to 4.
i) preparing metal nanoparticles to form a seed;

ii) adding the seed metal nanoparticles to an aqueous solution in which a conductive polymer monomer and a surfactant are dissolved,

iii) adding a metal ion solution to the solution to which the seed metal nanoparticles of ii) are added to perform a redox reaction between the metal ion and the conductive polymer monomer;

iv) the metal ions are deposited on the surface of the seed metal nanoparticles while being reduced by receiving electrons provided by the conductive polymer to form a double metal nanoparticle compartment, and the conductive polymer monomer is oxidized to one side of the double metal nanoparticle compartment. Is deposited only to form a conductive polymer compartment asymmetrically while growing into a conductive polymer, thereby making Janus nanoparticles composed of double metal-polymers;

v) octadecylamine (ODA) is added to the solution containing Janus nanoparticles; And

vi) the double metal nanoparticles in the Janus nanoparticles self-assemble covalently with the ODA to form a double metal nano cluster core and a radially located polymer shell around the core;

Comprising the steps,

Method for producing self-assembled double metal-polymer Janus nanostructures.
The method of claim 8,

After step iv),

A method of producing a self-assembled double metal-polymer Janus nanostructure, further comprising attaching a Raman dye to the surface of the double metal nanoparticle of the double metal-polymer nanoparticles.
The method of claim 8,

The seed metal of step i) is selected from the group consisting of gold, silver, copper and mixtures thereof;

Wherein the metal ions of step iii) is selected from the group consisting of gold ions, silver ions, copper ions and mixtures thereof, a method for producing self-assembled double metal-polymer Janus nanostructures.
The method of claim 8,

The conductive polymer of step ii) is at least one selected from the group consisting of polypyrrole, polythiophene, poly (3,4-ethylene dioxythiophene) (PEDOT) and polyaniline, self-assembled Method for producing double metal-polymer Janus nanostructures.
The method of claim 8,

The growth of the conductive polymer of step iv) is by surface-templated polymerization, a method for producing self-assembled double metal-polymer Janus nanostructures.
a) preparing a sample solution containing the target substance to be detected;

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

c) preparing by immobilizing a second antibody against the target material on the metal nanoprobe of claim 5;

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

e) adding the metal nanoprobe to which the second antibody of c) is immobilized to a solution containing the immunocomplex conjugated with the first antibody of d) to prepare a second antibody-target material-magnetic nanoparticle of the metal nanoprobe. Forming a sandwich immunocomplex of the first antibody;

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

g) measuring the Raman signal of said sandwich immunocomplex;

A surface-enhanced Raman scattering (SERS) based target substance detection method comprising the step.
The method of claim 13,

The target material is a protein or pathogen, surface-enhanced Raman scattering (SERS) based target material detection method.
A double-metal nanoparticle compartment having a core-satellite structure comprising a metal nanoparticle core to which a ligand is adsorbed, and a metal satellite reduced to a ligand adsorption portion of the core; And

Conductive polymer compartments;

Janus nanostructure consisting of.
The method of claim 15,

The ligand is a negatively charged ligand or a ligand having two reactors,

The metal nanoparticle core is a positively charged metal nanoparticle core or a negatively charged metal nanoparticle core, Janus nanostructure.
The method of claim 16,

The negatively charged ligand is a Janus nanostructure, which is a polymeric ligand comprising a charging unit (Repeating unit) or a small molecule ligand having two reactors.
The method of claim 17,

The polymeric ligand is PS (poly (sodium-4-styrenesulfonate)), PVP (poly (N-vinyl pyrrolidone)), PDADMAC (poly (diallyldimethylammonium chloride)), PAA (polyacrylic acid) or PAH (poly (allylamine) hydrochloride At least one selected from the group consisting of

The small molecule ligand is at least one selected from the group consisting of ATP (4-aminothiophenol), BDT (1,4-benzenedithiol), MBA (4-mercaptobenzoic acid), and MBIA (2-mercaptobenzoimidazole-5-carboxylic acid). Nanostructures.
The method of claim 15,

In the double metal nanoparticle compartment of the core-satellite structure,

Janus nanostructures, wherein the core metal nanoparticles and the metal of the satellite are each selected from the group consisting of silver, gold, copper and mixtures thereof, and the core metal and the satellite metal are not identical to each other.
The method of claim 15,

The conductive polymer is at least one selected from the group consisting of polypyrrole, polythiophene, polythiophene, poly (3,4-ethylene dioxythiophene) (PEDOT), and polyaniline, Janus nanostructure.
The method of claim 15,

Janus nanostructures of the metal nanoparticle core of the metal nanoparticles are metal nanorods (nanorods) or metal nanospheres (nanospheres).
The method of claim 15,

Janus nanostructure, wherein the core-satellite structure of the double metal nanoparticle compartment further comprises a Raman dye.
A metal nanoprobe for detecting surface-enhanced Raman scattering (SERS) based target substance using Janus nanostructure of claim 22.
i) preparing positively or negatively charged core metal nanoparticles;

ii) adsorbing a negatively charged ligand or a ligand having two reactors to the core metal nanoparticle;

iii) adding the ligand-adsorbed core metal nanoparticles to an aqueous solution in which the conductive polymer monomer and the surfactant are dissolved,

iv) adding a metal ion solution to the solution to which the core metal nanoparticles of iii) are added to perform a redox reaction between the metal ion and the conductive polymer monomer; And

v) the metal ions in the portion adsorbed by the ligand of the core metal nanoparticles to receive the electrons provided by the conductive polymer is reduced to form a satellite metal, thereby forming a double metal nanoparticle compartment of the core-satellite structure The conductive polymer monomer is oxidized and deposited on only one side of the double metal nanoparticle compartment to grow into a conductive polymer to form asymmetrically conductive polymer polymer compartment to form Janus nanoparticles;

Method of producing a Janus nanostructure, comprising the step.
The method of claim 24,

The negatively charged ligand of step ii) is a polymeric ligand comprising a charging unit (Repeating unit) or a small molecule ligand having two reactors, the manufacturing method of Janus nanostructures.
The method of claim 25,

The polymeric ligand is PS (poly (sodium-4-styrenesulfonate)), PVP (poly (N-vinyl pyrrolidone)), PDADMAC (poly (diallyldimethylammonium chloride)), PAA (polyacrylic acid) or PAH (poly (allylamine) hydrochloride At least one selected from the group consisting of

The small molecule ligand is at least one selected from the group consisting of ATP (4-aminothiophenol), BDT (1,4-benzenedithiol), MBA (4-mercaptobenzoic acid), and MBIA (2-mercaptobenzoimidazole-5-carboxylic acid). Method of producing nanostructures.
The method of claim 24,

In the double metal nanoparticle compartment of the core-satellite structure of step v),

The core metal nanoparticle and the metal of the satellite are each selected from the group consisting of silver, gold, copper and mixtures thereof, wherein the core metal and the satellite metal are not identical to each other. Way.
The method of claim 24,

The conductive polymer of step iii) is at least one selected from the group consisting of polypyrrole, polythiophene, poly (3,4-ethylene dioxythiophene) (PEDOT) and polyaniline, Janus nanostructure Method of preparation.
The method of claim 24,

The metal nanoparticles of the core metal nanoparticles of step i) are metal nanorods or metal nanospheres.
The method of claim 24,

The growth of the conductive polymer of step v) is by the surface-templated polymerization (surface-templated polymerization), the manufacturing method of Janus nanostructures.
The method of claim 24,

After step v),

Attaching a Raman dye on the surface of the double metal nanoparticles of the Janus nanoparticles, manufacturing method of Janus nanostructures.
The method of claim 24,

The surfactant of step iii) is at least one selected from the group consisting of sodium dodecyl sulfate (SDS), sodium deoxycholate, and Triton X-200, Janus nanostructure Method of preparation.
a) preparing a sample solution containing the target substance to be detected;

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

c) preparing by immobilizing a second antibody against the target on the metal nanoprobe of claim 23;

d) adding the magnetic nanoparticle 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 nanoparticle are conjugated;

e) a sandwich immunocomplex of the first antibody of the second antibody-target-magnetic nanoparticle of the metal nanoprobe by adding a metal nanoprobe to which the second antibody is immobilized to a solution containing the immunocomplex to which the first antibody is conjugated 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 said sandwich immunocomplex;

A surface-enhanced Raman scattering (SERS) based target substance detection method comprising the step.
The method of claim 33, wherein

The target material is a protein or pathogen, surface-enhanced Raman scattering (SERS) based target material detection method.
A double metal nanocluster compartment having a Raman dye and consisting of a core-shell structure; And

Conductive polymer compartments;

Nanoprobe consisting of,

The nanoprobe is that the conductive polymer compartment is oxidized only on one side of the double metal nanocluster compartment to exhibit an asymmetric structure,

Asymmetric Janus nanoprobes for surface-enhanced Raman scattering (SERS) based target material detection.
36. The method of claim 35 wherein

The double metal nanocluster compartment,

A core selected from the group consisting of gold, silver, copper and mixtures thereof;

A shell selected from the group consisting of gold, silver, copper and mixtures thereof;

Asymmetric Janus nanoprobe for surface-enhanced Raman scattering (SERS) based target material detection, which is a bimetallic nanocluster composed of:
36. The method of claim 35 wherein

The conductive polymer is at least one selected from the group consisting of polypyrrole, polythiophene, poly (3,4-ethylene dioxythiophene) (PEDOT) and polyaniline, surface-enhanced Raman scattering (SERS) ) Asymmetric Janus nanoprobes for detection of target material.
i) mixing and heating or agglomerating the metal nanoparticles and the Raman dye forming the core to form a core metal nanoparticle cluster having a Raman dye;

ii) adding the core metal nanoparticle cluster to an aqueous solution in which a conductive polymer monomer and a surfactant are dissolved;

iii) performing a redox reaction between the metal ions and the conductive polymer monomer by adding a metal ion solution to the solution to which the core metal nanoclusters of ii) are added; And

iv) the metal ions are deposited on the surface of the core metal nanoparticles while receiving and reducing electrons provided by the conductive polymer to form a double-metal nanoparticle cluster section having a core-shell structure; The conductive polymer monomer is oxidized and deposited on only one side of the double metal nanocluster compartment to grow into a conductive polymer to form an asymmetrically conductive polymer compartment;

Comprising the steps,

Method for preparing asymmetric Janus nanoprobes for surface-enhanced Raman scattering (SERS) based target material detection.
The method of claim 38,

After step i), further comprising stabilizing the core metal nanocluster with a protein, wherein the asymmetric Janus nanoprobe for surface-enhanced Raman scattering (SERS) based target material detection.
The method of claim 39,

The protein is surface-enhanced Raman scattering (avidin) selected from the group consisting of avidin (streptavidin), bovine serum albumin (BSA), insulin (sulin), soy protein, casein, gelatin and mixtures thereof SERS) based asymmetric Janus nanoprobe method for the detection of target substances.
The method of claim 38,

The core metal of step i) is selected from the group consisting of gold, silver, copper and mixtures thereof;

The metal ion of step iii) is selected from the group consisting of gold ions, silver ions, copper ions and mixtures thereof. The method for preparing asymmetric Janus nanoprobes for surface-enhanced Raman scattering (SERS) based target material detection.
The method of claim 38,

The conductive polymer is at least one selected from the group consisting of polypyrrole, polythiophene, poly (3,4-ethylene dioxythiophene) (PEDOT) and polyaniline, surface-enhanced Raman scattering (SERS) ) A method for producing an asymmetric Janus nanoprobe for detecting a target material.
The method of claim 38,

After the redox reaction of step iii), further comprising incubating the reaction solution with a surfactant solution, wherein the asymmetric Janus nanoprobe for surface-enhanced Raman scattering (SERS) based target material detection is prepared. .
The method of claim 38 or 43, wherein

The surfactant is at least one selected from the group consisting of sodium dodecyl sulfate (SDS), sodium deoxycholate, and Triton X-200, surface-enhanced Raman scattering (SERS) ) A method for producing an asymmetric Janus nanoprobe for detecting a target material.
The method of claim 38,

Wherein the growth of the conductive polymer of step iv) is by surface-templated polymerization (surface-templated polymerization), manufacturing method of asymmetric Janus nanoprobe for surface-enhanced Raman scattering (SERS) -based target material detection.
a) preparing a sample solution containing the target substance to be detected;

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

c) preparing by immobilizing a second antibody against the target on the metal nanoprobe of any one of claims 35-37;

d) adding the magnetic nanoparticle 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 nanoparticle are conjugated;

e) a sandwich immunocomplex of the first antibody of the second antibody-target-magnetic nanoparticle of the metal nanoprobe by adding a metal nanoprobe to which the second antibody is immobilized to a solution containing the immunocomplex to which the first antibody is conjugated 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 said sandwich immunocomplex;

Comprising the steps,

Surface-enhanced Raman scattering (SERS) based target material detection method.
47. The method of claim 46 wherein

The target material is a protein or pathogen, surface-enhanced Raman scattering (SERS) based target material detection method.
A dual metal nanorod cluster compartment comprising a directional metal nanorod cluster seed and a metal shell structure; And

Conductive polymer block:

Asymmetric Janus nanostructure consisting of.
The method of claim 48,

In the seed-shell structured double metal nanorod cluster compartment,

The metal nanorod cluster seed and the shell metal are each selected from the group consisting of silver, gold, copper and mixtures thereof, wherein the core metal and the shell metal are not identical to each other.
The method of claim 48,

The oriented metal nanorod cluster seed,

Sides of the individual metal nanorod particles are arranged side by side (by side-by-side assmebly) form, or end-to-end assembly of the ends of the individual metal nanorod particles are connected (assembled form) , Asymmetric Janus nanostructures.
The method of claim 48,

The conductive polymer is at least one selected from the group consisting of polypyrrole, polythiophene, polythiophene, poly (3,4-ethylene dioxythiophene) (PEDOT), and polyaniline, asymmetric Janus nanostructure.
The method of claim 48,

Wherein the double metal nanorod cluster compartment is asymmetric Janus nanostructure further comprising a Raman dye.
A metal nanoprobe for measuring surface-enhanced Raman scattering (SERS) signals using the asymmetric Janus nanostructure of claim 52.
i) mixing the metal nanorod particles to form a seed and the negatively charged stimuli responsive copolymer having a thiol group at the organic anion or terminal to form a metal nanorod cluster seed having an orientation;

ii) adding the metal nanorod cluster seed to an aqueous solution in which the conductive polymer monomer and the surfactant were dissolved,

iii) adding a metal ion solution to the solution to which the metal nanorod cluster seed of ii) is added to perform a redox reaction between the metal ion and the conductive polymer monomer;

iv) the metal ions are deposited on the surface of the seed metal nanorod particles while receiving and reducing electrons provided by the conductive polymer to form a seed metal shell double metal nanorod cluster section; The conductive polymer monomer is oxidized and deposited only on one side of the double metal nanorod cluster compartment to grow into a conductive polymer to form an asymmetrically conductive polymer compartment;

Method of producing an asymmetric Janus nanostructure, comprising the step.
The method of claim 54,

And after step i), further comprising attaching a Raman dye to the surface of the metal nanorod cluster seed.
The method of claim 54,

The metal nanorod cluster seed having the directionality of step i) may be formed in a side-by-side assmebly side by side of the individual metal nanorod particles, or the ends of the individual metal nanorod particles are connected to each other. Method of producing an asymmetric Janus nanostructure, which will be in the form of end-to-end assembly.
The method of claim 56, wherein

Method for producing an asymmetric Janus nanostructure, wherein the metal nanorod cluster seed of the side-by-side assmebly form of the side of the individual metal nanorod particles are prepared, comprising the following steps:

Preparing metal nanorod particles in which a positively charged surfactant is present on the surface using the metal seeds;

Adding a Raman dye to the solution containing the metal nanorods; And

Adding an organic anion to the metal nanorod solution containing the Raman dye;

Electrostatic attraction between the positively charged surfactant attached to the side of the metal nanorods and the organic anion causes the side surfaces of the individual metal nanorods to be arranged side by side with other side metal nanorods to form a metal nanorod cluster seed. Steps.
The method of claim 57,

The positively charged surfactant is selected from the group consisting of hexadecyltrimethylammonium bromide (CTAB), dodecyltrimethylammonium bromide (DTAB), and trimethyltetradecylammonium bromide (TTAB),

The organic anion is selected from the group consisting of citrate, malate, fumarate, tartrate, succinate, oxalate, and gluconate.
The method of claim 56, wherein

Method for producing an asymmetric Janus nanostructure, wherein the metal nanorod cluster seed in the form of the end-to-end assembly of the ends of the individual metal nanorod particles are prepared, comprising the following steps:

Preparing metal nanorod particles in which a positively charged surfactant is present on the surface using the metal seeds;

Adding a negatively charged stimulatory copolymer having a thiol group at the end to the solution containing the metal nanorods; And

Stirring the metal nanorod solution to which the negatively charged stimulatory copolymer is added;

Adding Raman dye to the stirred solution;

The positively charged surfactant at the end of the metal nanorod and the thiol group of the negatively charged stimuli-reactive copolymer combine with each other to form a metal-thiol group bond, and the positively charged surfactant at the side of the individual metal nanorod and the other individual metal nanorod Bonding the terminal negatively charged stimuli-responsive copolymers with electrostatic attraction so that the ends of the individual metal nanorod particles are joined together to form a metal nanorod cluster seed.
The method of claim 59,

The positively charged surfactant is selected from the group consisting of hexadecyltrimethylammonium bromide (CTAB), dodecyltrimethylammonium bromide (DTAB), and trimethyltetradecylammonium bromide (TTAB),

The negatively charged stimuli-responsive copolymer is a copolymer consisting of a negatively charged moiety and a stimulation-responsive polymer, a method for producing an asymmetric Janus nanostructure.
The method of claim 60,

The negatively charged moiety is selected from the group consisting of acrylic acid, methacrylic acid, itaconic acid, maleic acid and mixtures thereof. ;

The stimulatory polymer is poly (N-isopropylacrylamide) [poly (N-isopropylacrylamide): polyNIPAM], poly (N-diethyl acrylamide) [poly (N, N'-diethyl acrylamide): polyDEAAm], poly (Dimethylamino ethyl methacrylate) [poly (dimethylamino ethyl methacrylate): polyDMAEMA], poly (N-hydroxymethyl propyl methacrylamide) [poly (N- (L)-(1-hydroxymethyl) propyl methacrylamide)], Poly [oligo (ethylene glycol) methyl ether methacrylate]: POEGMA], poly (2-vinyl pyridine): P2VP], poly ( 4-vinyl pyridine) [poly (4-vinyl pyridine): P4VP] and a mixture thereof, the method for producing an asymmetric Janus nanostructure.
The method of claim 59,

The negatively charged stimuli-responsive copolymer is poly (AAc-b-NIPAM) (poly (acrylic acid-block-N-isopropylacrylamide)) a method for producing an asymmetric Janus nanostructure.
The method of claim 54,

The conductive polymer of step ii) is at least one selected from the group consisting of polypyrrole, polythiophene, poly (3,4-ethylene dioxythiophene) (PEDOT), and polyaniline, asymmetrical Janus Method of producing nanostructures.
The method of claim 54,

After the redox reaction of step iii), further comprising incubating the reaction solution with a surfactant solution.
The method of claim 54 or 64, wherein

The surfactant is at least one selected from the group consisting of sodium dodecyl sulfate (SDS), sodium deoxycholate, and Triton X-200, the production of asymmetric Janus nanostructures Way.
The method of claim 54,

The growth of the conductive polymer of step iv) is by surface-templated polymerization (surface-templated polymerization), a method for producing an asymmetric Janus nanostructure.
a) preparing a sample solution containing the target substance to be detected;

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

c) preparing by immobilizing a second antibody against the target on the metal nanoprobe of claim 53;

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

e) a sandwich immunocomplex of the first antibody of the second antibody-target-magnetic nanoparticle of the metal nanoprobe by adding a metal nanoprobe to which the second antibody is immobilized to a solution containing the immunocomplex to which the first antibody is conjugated 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 said sandwich immunocomplex;

A surface-enhanced Raman scattering (SERS) based target substance detection method comprising the step.
The method of claim 67,

The target material is a protein or pathogen, surface-enhanced Raman scattering (SERS) based target material detection method.