KR102153950B1 - Core-satellite Nanostructures with Stimuli-responsiveness, method thereof and its uses - Google Patents

Abstract

The present invention relates to a stimulus-responsive core-satellite nanostructure, a method for manufacturing the same, and a method for detecting a target substance based on surface-enhanced Raman scattering (SERS) using the same. The stimulus-responsive core-satellite nanostructure according to the present invention can be applied as a metal nanoprobe for measuring a surface-enhanced Raman scattering (SERS) image with improved Raman strength.

Description

Stimulus-responsive core-satellite nanostructures, manufacturing method, and application thereof {Core-satellite Nanostructures with Stimuli-responsiveness, method thereof and its uses}

The present invention relates to a stimulus-responsive core-satellite nanostructure, a method for manufacturing the same, and a method for detecting a target substance based on surface-enhanced Raman scattering (SERS) using the same.

Raman spectroscopy has been studied for detection and identification of pathogens. Specifically, surface-enhanced Raman scattering (SERS) has attracted great interest in spectroscopic detection and identification of small molecules, nucleic acids, proteins and cells mainly due to its ultra-high sensitivity, narrow bandwidth, and important multiplexing capabilities. (Non-patent document 1). SERS has been extensively studied on a variety of metallic nanoparticles (MNP) substrates, and provides unique plasmon properties for spectroscopic detection of target molecules. In the development of remarkably advanced new MNPs, plasmon assembly has attracted great interest in SERS-based biosensing as it exhibits remarkable SERS enhancement due to plasmon interparticle coupling.

Due to their structure- and size-dependent electrical, chemical and optical properties, the novel metallic nanoparticles (MNPs) are extensively studied in a variety of applications including electronics, catalysis, biological imaging and surface-enhanced Raman spectroscopy. It has been (Non-Patent Document 2).

Stimulus-responsive nanostructures respond to physical or chemical triggers through phase transitions of structural states. The reversible phase transition of these nanostructures is useful for bio-actuators, biochemical sensors, tissue engineering, and drug delivery. When stimulus-reactive polymers are bonded to the surface of plasmonic nanostructures, plasmonic coupling and electromagnetic field enhancement are controlled by changes in the three-dimensional structure of the polymer in response to environmental factors including temperature, pH and ionic strength. (Non-Patent Document 3). The structure of the stimulus-responsive polymer on the plasmonic nanostructure can reversibly alter the steric hindrance, solubility, or light scattering properties of the colloidal nanoparticles.

Accordingly, the present inventors have developed a stimulus-responsive core-satellite nanostructure by continuing research on the stimulus-responsive nanostructure.

K. Kneipp, Y. Wang, H. Kneipp, L. T. Perelman, I. Itzkan, R. R. Dasari, et al., "Single Molecule Detection Using Surface-Enhanced Raman Scattering (SERS)," Physical Review Letters, vol. 78, pp. 1667-1670, 03/03/1997. Daniel, M.-C.; Astruc, D., Gold nanoparticles: assembly, supramolecular chemistry, quantum-size-related properties, and applications toward biology, catalysis, and nanotechnology. Chemical reviews 2004, 104 (1), 293-346. Tokarev, I.; Minko, S., Tunable plasmonic nanostructures from noble metal nanoparticles and stimuli-responsive polymers. Soft Matter 2012, 8 (22), 5980-5987.

An object of the present invention is to provide a stimulus-responsive core-satellite nanostructure.

Another object of the present invention is to provide a metal nanoprobe for measuring a surface-enhanced Raman scattering (SERS) image using a stimulus-responsive core-satellite nanostructure according to the present invention.

Another object of the present invention is to provide a method of manufacturing a stimulus-responsive core-satellite nanostructure.

Another object of the present invention is to provide a method for detecting a target material based on surface-enhanced Raman scattering (SERS) using the stimulus-responsive core-satellite nanostructure.

The present invention provides a stimulus-responsive core-satellite nanostructure consisting of:

A core structure of a metal nanorod in which a diblock copolymer composed of a positively charged polymer block having a thiol end group and a stimulus-reactive polymer block are bonded to each other; And

Satellite structure of metal nanoparticles in which a negatively charged polymer block having a thiol end group and a diblock copolymer composed of a stimulation-reactive polymer block are conjugated.

The positively charged polymer block is allyamine, 2-(dimethyl amino)ethylmethacrylate, ethyleneimine, vinyl amine, 2-vinylpyridine (2-vinyl pyridine), and 4-vinyl pyridine (4-vinyl pyridine) may include one or more monomers, but is not necessarily limited to this, 　 If a compound that can impart a positive charge in the block, it may be included without limitation have.

The negatively charged polymer block contains one or more monomers of acrylic acid, methacrylic acid, itaconic acid, and maleic acid. It may be included, but is not limited thereto, and any compound capable of imparting a negative charge in a block may be included without limitation.

The stimulation-reactive polymer block 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] [Poly[oligo(ethylene glycol) methyl ether methacrylate]: POEGMA], poly(2-vinyl pyridine) [poly(2-vinyl pyridine): P2VP], And poly(4-vinyl pyridine) [poly(4-vinyl pyridine): P4VP] may include one or more monomers, but is not limited thereto.

The stimulus-responsive core-satellite nanostructure prevents an electrostatic interaction between a diblock copolymer of a positively charged polymer bonded to the metal rod and a diblock copolymer of a negatively charged polymer bonded to the metal nanoparticles. It can be formed through.

A gap between the metal nanorods and the metal nanoparticles may decrease above the lower limit critical solution temperature (LCST) of the core-satellite nanostructure.

The stimulation may be heat.

The core structure of the metal rod may further include a Raman dye.

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

In the present invention, the term "irritation responsiveness" refers to a property in which behavior changes in response to a stimulus (eg, heat).

The term "stimulation-reactive polymer block" used in the present invention is a compartment whose properties are changed in response to a stimulus, and the stimulus is preferably a temperature (heat) condition, and the property changed by the stimulus is gelation due to temperature ( gelatioin) or mechanical strength. In other words, it shrinks under conditions equal to or higher than the lower limit critical solution temperature (LCST) to change the structure of the copolymer.

In the present invention, the "core-satellite nanostructure" refers to a nanostructure composed of a core structure and a satellite structure around it. In the present invention, the core-satellite nanostructure represents that metal nanoparticles form a satellite structure in a metal rod core structure.

Specifically, a stimulation-responsive core-satellite nanostructure through an electrostatic interaction between a diblock copolymer of a positively charged polymer bonded to a metal rod and a diblock copolymer of a negatively charged polymer bonded to a metal nanoparticle. Is formed.

In this specification, the electrostatic interaction is also referred to as "polyelectrolyte complexation".

In another aspect, the present invention provides a metal nanoprobe for measuring a surface-enhanced Raman scattering (SERS) image using a stimulus-responsive core-satellite nanostructure according to the present invention.

The stimulation-responsive core-satellite nanostructure according to the present invention may be provided as a metal nanoprobe for measuring a surface-enhanced Raman scattering image by including a Raman dye.

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

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

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

In another aspect, the present invention provides a method for preparing a stimulus-responsive core-satellite nanostructure comprising the following steps:

i) preparing a diblock copolymer (A) composed of a negatively charged polymer block and a stimulus reactive polymer block, and a diblock copolymer (B) composed of a positively charged polymer block and a stimulus reactive polymer block, respectively;

ii) Diblock copolymer (A-SH) and diblock copolymer (B) in which thiol groups are introduced by introducing thiol groups at the ends of each of the diblock copolymers (A) and diblock copolymers (B) through amino decomposition -SH) respectively;

iii) Metal nanoparticles in which the diblock copolymer (A-SH) into which the thiol group is introduced is bonded to the metal nanoparticles, and the diblock copolymer (A) consisting of a negatively charged polymer block and a stimulation-reactive polymer block is bonded To prepare;

iv) A metal nanorod in which the diblock copolymer (B-SH) into which the thiol group is introduced is bonded to a metal nanorod, and a diblock copolymer (B) composed of a positively charged polymer block and a stimulus-reactive polymer block is bonded. To prepare; And

v) mixing and stirring the metal nanoparticle to which (A) is bonded and the metal nanorod to which (B) is bonded;

step.

Detailed descriptions of the positively charged polymer block, negatively charged polymer block, stimulus-responsive polymer block, and stimulus are the same as above.

In step v), a core-satellite nanostructure may be formed through an electrostatic interaction between (B) bonded to the metal rod and (A) bonded to the metal nanoparticles.

A gap between the metal nanorods and the metal nanoparticles may decrease above the lower limit critical solution temperature (LCST) of the core-satellite nanostructure.

The step iv) may further include attaching a Raman dye to the metal nanorod.

The description of the Raman dye is the same as before.

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

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

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

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

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

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

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

g) measuring the Raman signal of the sandwich immunocomplex;

step.

The target substance may be a protein or a pathogen.

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

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

The block copolymer-mediated assembly approach shown in the present invention is useful for the production of plasmonic core-satellite nanostructures with controlled spatial gaps. Reversible Addition-Fragmentation Chain Transfer (RAFT) polymerization is a controlled molar mass and low polydispersity using different types of monomers and chain transfer agents (CTA) under various polymerization conditions. It facilitates the synthesis of a block copolymer having. The major advantage of RAFT polymerization in the preparation of well-designed block copolymers is that the thiocarbonylthio group acts as a masked thiol against thiol-based coupling chemistry, which is a nucleophilic reagent as a thiocarbonylthiol- It can be easily accessed by treating the end groups of the functionalized polymer. In addition, RAFT polymerization allows the preparation of stimulus-responsive copolymers with each block length controlled.

In one embodiment of the present invention, negatively charged poly(acrylic acid) [poly(acrylic acid), poly(AAc)] or positively charged poly(N,N-dimethylaminoethyl methacrylate)[poly(N ,N-dimethylaminoethyl methacrylate, poly(DMAEMA)) and heat-reactive poly(N-isopropylacrylamide) [thermo-responsive poly(N-isopropylacrylamide), poly(NIPAM)] two different diblock copolymers After preparation by RAFT polymerization, aminolysis was performed to have a thiol group at each end of the diblock copolymer. This diblock copolymer containing a thiol-terminus was conjugated to metal nanorods (AuNRs) and metal nanoparticles (AuNPs) through thiol bonds to negatively charged p(AAc-b-NIPAM)-coated AuNPs (AuNPs). -p(AAc-b-NIPAM)) and positively charged p(DMAEMA-b-NIPAM)-coated AuNR (AuNRs-p(DMAEMA-b-NIPAM)) were formed. Plasmonic core-satellite by polyelectrolyte complexation of positively charged AuNRs-p (DMAEMA-b-NIPAM) and negatively charged AuNPs-p (AAc-b-NIPAM) A nanostructure was formed.

These complexed nanostructures of AuNPs-p(AAc-b-NIPAM)-AuNRs-p(DMAEMA-b-NIPAM) as a core-satellite form are individual AuNPs-p(AAc-b-NIPAM) and AuNRs-p( DMAEMA-b-NIPAM) showed significantly improved physicochemical and optical performance, potentially due to the enhancement of the electromagnetic field in the gap between AuNP and AuNR.

In addition, in the present invention, surface-enhanced Raman scattering (SERS)-based biosensing of immunoglobulin G (IgG) was performed using such a core-satellite nanostructure as a nanotag. Polyclonal antibodies to IgG (pAb) were chemically conjugated using SERS nanotags. On the other hand, a monoclonal antibody (mAb) against IgG was chemically attached using magnetic beads (MB). We demonstrated that a sandwich-type immunocomplex consisting of SERS nanotags, IgG and magnetic beads was formed, and that there was a linear relationship between antigen concentration and Raman intensity. These complexed nanostructures can be used as SERS nanoprobes in highly sensitive biosensing fields.

The stimulus-responsive core-satellite nanostructure according to the present invention can be applied as a metal nanoprobe for measuring a surface-enhanced Raman scattering (SERS) image with improved Raman strength.

1 is a diagram for preparing a core-satellite nanostructure of heat-reactive AuNPs-p (AAc-b-NIPAM)-AuNRs-p (DMAEMA-b-NIPAM). As a complexed nanostructure, (a) AuNPs-p (AAc-b-NIPAM), AuNRs-p (DMAEMA-b-NIPAM) and (c) AuNPs-p (AAc-b-NIPAM)-AuNRs-p (DMAEMA -b-NIPAM) schematic diagram for the synthesis. As shown in the Raman spectrum, the nanostructure complexed as a core-satellite nanostructure exhibited a very strong SERS signal through the reversible LCST phase transition of the heat-reactive diblock copolymer. The scale bar in the TEM image of the composite nanostructure is 20 nm.
Figure 2 shows (A) negatively charged poly(AAc- b- NIPAM) diblock copolymer and (B) positively charged poly(DMAEMA- b ) through RAFT polymerization using CDTPA as CTA and AIBN as initiator. -NIPAM) Chemical reaction formula for the synthesis of diblock copolymer.
Figure 3 (A) ¹ H NMR spectra of poly(DMAEMA ₁₁₁ ) and poly(DMAEMA ₁₁₁ - b- NIPAM ₂₇₃ ) in CDCl ₃ . (B) ¹ H NMR spectra of poly(AAc ₁₃₁ ) and poly(AAc ₁₃₁ -b -NIPAM ₂₇₂ ) in D ₂ O and DMSO-d ₆ , respectively. (C) GPC trace of poly(DMAEMA- b- NIPAM) and (D) poly(AAc- b- NIPAM).
Figure 4 (A, B) Thermal profiles of poly(DMAEMA- b- NIPAM) and poly(AAc- b- NIPAM) in PBS by turbidity measurement and dynamic light scattering. UV profiles of (C) poly(DMAEMA- b- NIPAM) and (D) poly(AA- b- NIPAM) before and after aminolysis. After aminolysis, the absorption peak of the trithiocarbonate group decreased at about 307 nm.
5 is AuNR, AuNR-p (DMAEMA-b-NIPAM), AuNP, AuNP-p (AAc-b-NIPAM), and (I) 0.057:1, (II) 0.114:1, (III) 0.284:1, (IV ) AuNP-p (AAc-b) with five different molar concentration ratios of AuNP-p (AAc-b-NIPAM) to AuNR-p (DMAEMA-b-NIPAM) ranging from 0.568:1 and (V) 2.273:1 UV-Vis absorption spectrum and hydrodynamic diameter of -NIPAM)-AuNR-p (DMAEMA-b-NIPAM).
6 shows AuNP-p (AAc-b-NIPAM) in the range of (I) 0.057:1, (II) 0.114:1, (III) 0.284:1, (IV) 0.568:1 and (V) 2.273:1. (A) Raman of the core-satellite nanostructure, AuNP-p(AAc-b-NIPAM)-AuNR-p(DMAEMA-b-NIPAM) with different molar concentration ratios of AuNR-p(DMAEMA-b-NIPAM) Spectrum, (b) Core-satellite nanoparticles in the range of (I)-(V) by varying AuNP-p (AAc-b-NIPAM) of different molar concentrations to a constant AuNR-p (DMAEMA-b-NIPAM) molar concentration. Raman intensity when the structure was formed, with different molar concentration ratios of AuNP-p (AAc-b-NIPAM) to AuNR-p (DMAEMA-b-NIPAM) in the range of (cg) (I)-(V) Raman spectrum as a function of temperature of core-satellite nanostructures. (h) AuNR, AuNRs-p (DMAEMA-b-NIPAM), AuNP, AuNPs-p (AAc-b-NIPAM), and AuNPs-p (AAc-b-NIPAM) with different molar ratios of AuNP to AuNR -AuNRs-p (DMAEMA-b-NIPAM) ξ-potential graph.
7 is a TEM image of (a, b) citrate-capped AuNP and (c, d) CTAB-capped AuNR at different magnification ratios. Scale bars are 50 nm in (a), 10 nm in (b), 200 nm in (c), and 100 nm in (d).
8 is TEM image of a series of complexed nanostructures of AuNPs-p(AAc-b-NIPAM)-AuNRs-p(DMAEMA-b-NIPAM) having different densities of AuNPs for AuNR, the density is AuNP/AuNR molar concentration It increases as the ratio increases from (a, b) 0.057:1 to (c, d) 0.114:1 and then (e, f) 0.284:1. Scale bars are 100 nm in (a), 20 nm in (b), 200 nm in (c, e), and 50 nm in (d, f).
9 is (a) Schematic of preparation of anti-human IgG pAb-conjugated core-satellite nanostructure and anti-human IgG mAb-conjugated MB. (b) Schematic diagram of a SERS-based immunoassay using complexed nanostructures as SERS nanoprobes for biosensing. (C) Relative Raman spectrum and (d) Raman intensity of the tertiary immunocomplex consisting of AuNPs-p(AAc-b-NIPAM)-AuNRs-p(DMAEMA-b-NIPAM), IgG, and MB as a function of IgG concentration .

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

<Example 1> Materials

Gold (III) chloride trihydrate (HAuCl ₄ .3H ₂ O), sodium borohydride (purity 98.0%), silver nitrate, hexadecyltrimethylammonium bromide (CTAB, 98%), hydroquinone, sodium hydroxide, 4- Cyano-4-[(dodecylsulfanylthiocarbonyl)sulfanyl]pentanoic acid (CDTPA), phosphate buffered saline (PBS), hexylamine, dimethylphenylphosphine (DMPP), tris(2-carboxyethyl) Phosphine hydrochloride (TCEP), anti-human IgG (γ-chain specific) pAb produced in goat, anti-human IgG (Fc-specific) mAb produced in mice, IgG from human serum and sulfo-N- Hydroxysuccinimide ester (Sulfo-NHS) was purchased from Sigma-Aldrich (St. Louis, Missouri, USA). 2,2'-azobis(2-methylpropionitrile (AIBN) provided by Thermo Fisher Scientific (Walsom, Massachusetts, USA) was purified by recrystallization from methanol. N-isopropylacrylamide (NIPAM) was obtained. Purchased from Tokyo Chemical Industry Co., Ltd. (Tokyo, Japan) and recrystallized from hexane prior to use N,N -dimethylaminoethyl methacrylate (DMAEMA) and acrylic acid (AAc) were prepared by Sigma Aldrich (St. Louis, Mo. (Trimethylsilyl) diazomethane 2.0 M in hexane (Acros Organics), 1,4-dioxane (Kanto Chemical Co., Inc), diethyl ether ( Samchun), tetrahydrofuran (Daejung), chloroform (Daejung), N-hexane (Daejung), triethylamine (Junsei Chemical Co., Ltd.) and dimethyl sulfoxide (DMSO) (Junsei Chemical Co., Ltd) Malachite Green Isothiocyanate (MGITC) was obtained from Invitrogen Corporation (Grand Island, NY) 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC) was used. Purchased from Thermo Scientific (Rockford, Illinois, USA) Magnetic beads with carboxyl surface functional groups were purchased from Bioneer Corporation (Daejon, Korea) Millipore Milli-Q deionized water was used in all experiments.

<Example 2> Preparation of AuNPs and AuNRs

Citrate-stabilized gold nanoparticles (AuNPs) were converted to Turkevich (Turkevich, J.; Stevenson, PC; Hillier, J. (1951). "A study of the nucleation and growth processes in the synthesis of colloidal gold". Discuss.Faraday.Soc. 11: 55-75.), prepared by HAuCl ₄ reduction using sodium citrate according to the method described by Frens ( Frens, G., Controlled nucleation for the regulation of the particle size in). monodisperse gold suspensions. Nature 1973, 241 (105), 20-22.) and purified by a slight modification. in a typical experiment, 0.5 mM of gold (III) chloride tree boiled by heating an aqueous solution of 100.0 ml of a hydrate under rapid stirring Then 1.5 mL of 5% sodium citrate tribasic dehydration was added The solution was boiled for 15 minutes, cooled to room temperature, and stored for further experiments.

Seeds for gold nanorods (AuNRs) with high aspect ratio were synthesized through the seed-mediated growth method as described in other literatures (Vigderman, L.; Zubarev, ER, High-yield synthesis of gold nanorods). with longitudinal SPR peak greater than 1200 nm using hydroquinone as a reducing agent.Chemistry of Materials 2013, 25 (8), 1450-1457.). Briefly, 0.50 mL of 0.010 M gold (III) chloride trihydrate solution was added to 9.5 mL of 0.10 M CTAB. An ice-cooled, freshly prepared 0.46 mL of 0.010 M sodium borohydride solution in 0.010 M sodium hydroxide was rapidly added to the gold solution with vigorous stirring. After stirring for 10 minutes, the solution was left overnight. To synthesize AuNR with an aspect ratio ranging from 6 to 8, a solution containing 0.50 mL of 0.010 M gold (III) chloride trihydrate and 8.0 mL of 0.10 M CTAB was prepared. Next, 40 μL of 0.10 M silver nitrate was added to the growth solution, and the solution was stirred. Then 0.50 mL of 0.10 M hydroquinone was added, and the solution was gently inverted to mix (inverted). When the solution became clear, 2.0 mL of the seed solution was added, and the solution was left overnight without stirring.

<Example 3> Poly(AAc- b -NIPAM) and poly (DMAEMA- b -NIPAM) synthesis

Poly(AAc- b- NIPAM) was prepared using AIBN as initiator, NIPAM as monomer, and poly(AAc)-macro CDTPA as CTA. First, poly(AAc)-macro CDTPA was prepared by slightly modifying the previously reported method (Pelet, JM; Putnam, D., Poly(acrylic acid) Undergoes Partial Esterification During RAFT Synthesis in Methanol and Interchain Disulfide Bridging Upon NaOH. Treatment.Macromolecular Chemistry and Physics 2012, 213 (23), 2536-2540.). In a representative experiment, AAc (0.9 g, 12.5 mmol) and CDTPA (0.0202 g, 0.05 mmol) were added to 4 ml 1,4-dioxane in a 100 ml 2-neck round bottom flask equipped with a magnetic stir bar, and N ₂ purged with gas. After 15 minutes of purging, 1 ml of a solution of AIBN (0.0021 g, 0.0125 mmol) in 1,4-dioxane was added to the mixture and nitrogen purging was continued for an additional 15 minutes before heating at 70° C. for 15 hours. The polymerization was terminated by quenching with cold water. The resulting poly(AAc ₁₆₆ )-macro CDTPA was precipitated in ethyl acetate and redissolved in deionized water. The free monomer was discarded by dialysis against deionized water for 2 days, and the polymer was freeze-dried. Second, the synthesis of poly(AAc- b- NIPAM) was carried out in a similar manner. In short, 0.445 g poly(AAc ₁₆₆ )-macro CDTPA and 0.81 g NIPAM monomer were dissolved in 11 ml of ethanol:water (3:1) mixture, and the mixture was placed in a 100 ml two-neck round bottom flask equipped with a magnetic stir bar. Put on. During N ₂ gas purging, 0.0015 g AIBN in 1 ml ethanol:water mixture (3:1) was added and the flask was sealed with a rubber stopper. The polymerization was carried out in a water bath at 70 °C for 15 hours. The flask was opened and immersed in cold water to terminate the reaction. Poly(AAc ₁₆₆ - b -NIPAM ₁₆₂ ) was precipitated in ethyl acetate and redissolved in phosphate buffer at pH 10. The polymer solution was then dialyzed against deionized water for 2 days to remove salt and freeze-dried for further use.

Poly(DMAEMA)-macro CDTPA was synthesized with some modifications of the previously reported method (Sahnoun, M.; Charreyre, M.-T.; Veron, L.; Delair, T.; D'Agosto, F., Synthetic and characterization aspects of dimethylaminoethyl methacrylate reversible addition fragmentation chain transfer (RAFT) polymerization.Journal of Polymer Science Part A: Polymer Chemistry 2005, 43 (16), 3551-3565.). In short, DMAEMA (3.77 g, 24 mmol) and CDTPA (0.0484 g, 0.12 mmol) were dissolved in 11 ml 1,4-dioxane in a 100 ml two-neck round bottom flask equipped with a magnetic stir bar, and N ₂ gas Purged with. After 15 minutes of purging, 1 ml of a solution of AIBN (0.0066 g, 0.04 mmol) in 1,4-dioxane was added to the mixture and nitrogen purging was continued for an additional 15 minutes before heating at 90° C. for 5 hours. The polymerization was stopped by quenching with cold water and exposed to outside air. Poly(DMAEMA ₁₁₁ )-macro CDTPA was precipitated in hexane and redissolved in deionized water before freeze drying. Next, 0.5 g poly(DMAEMA ₁₁₁ )-macro CDTPA, 0.98 g NIPAM monomer and 8.3 ml 1,4-dioxane were placed in a 100 ml two-neck round bottom flask equipped with a magnetic stir bar to obtain poly(DMAEMA ₁₁₁ - b). -NIPAM ₂₇₃ ) was synthesized. The mixture was then purged with N ₂ gas for 30 minutes to remove any oxygen in the solution. During the purge, 0.0012 g AIBN in 1 ml 1,4-dioxane was added to the mixture and the flask was sealed with a rubber stopper. Polymerization was carried out in a water bath at 70° C. for 10 hours, and then the flask was exposed to outside air to terminate the reaction. Poly(DMAEMA ₁₁₁ - b- NIPAM ₂₇₃ ) was precipitated in hexane and purified using the concentrated salt before redissolving in deionized water. After dialysis to remove the salt, the polymer was dried for further use.

<Example 4> Poly(AAc- b -NIPAM) and poly (DMAEMA- b -NIPAM) Aminolysis of CTA

Aminolysis of the trithiocarbonate group of poly(AAc- b- NIPAM) was performed with a procedure similar to that previously reported (Boyer, C.; Granville, A.; Davis, TP; Bulmus, V., Modification of RAFT -polymers via thiol-ene reactions: A general route to functional polymers and new architectures.Journal of Polymer Science Part A: Polymer Chemistry 2009, 47 (15), 3773-3794.). 300 mg poly(AAc ₁₃₇ - b- NIPAM ₂₇₂ ) (M _nNMR = 41,055 g/mol) was dissolved in 7 ml deionized water, triethylamine (4.5 mg; 0.044 mmol) was added and stirred for 15 minutes. Next, hexylamine (2.2 mg; 0.022 mmol) was added dropwise to the mixture, and the resulting mixture was purged with N ₂ gas for 30 minutes. The reaction was maintained at room temperature for 24 hours under N ₂ gas. The resulting poly(AAc ₁₃₇ - b- NIPAM ₂₇₂ )-SH was purified by precipitation in a concentrated salt solution, and redissolved in a phosphate buffer solution of pH 10. The polymer was dialyzed against deionized water before freeze drying. After aminolysis, the UV absorbance of the polymer was measured. Cleavage of the trithiocarbonate group reduced the absorbance at about 307 nm.

Aminolysis of the trithiocarbonate group of poly(DMAEMA- b- NIPAM) was performed with a slight modification of the previously reported procedure (Ho, TH Synthesis of thermoresponsive copolymers by RAFT polymerization: characterization and study of their interaction with proteins. du Maine, 2012.). For example, 0.35 g poly(DMAEMA _{111 -NIPAM 273} ) (M _nNMR = 48,746 g/mol) and DMPP (1.4 mg, 0.01 mmol) are dissolved in 5 ml THF, and the mixture is 100 ml 2-neck equipped with a magnetic bar. Stir in a round bottom flask for 15 minutes. Hexylamine (2.5 mg, 0.025 mmol) was added to the mixture and the flask was purged with N ₂ gas for 30 minutes. The solution was stirred for 4 hours at room temperature under N ₂ gas. The reaction was stopped by pouring the solution into hexane, and the precipitate was washed three times with hexane to remove excess hexylamine and DMPP. Colorless poly(DMAEMA _111- NIPAM ₂₇₃ )-SH was redissolved in deionized water and then dried in a freeze dryer for further characterization.

<Example 5> Preparation of core-satellite nanostructure

As the thiol end group of p(AAc- b- NIPAM)-SH showed strong affinity for the surface of MNPs, p(AAc- b- NIPAM)-SH was attached to the AuNPs. In short, a p(AAc- b- NIPAM)-SH solution dissolved in deionized water was added to the AuNPs solution so that the final concentration became 0.858 μM, and stirred for 1 hour. Next, unconjugated p(AAc-b-NIPAM)-SH and other impurities were removed by centrifugation at 10,000 rpm for 10 minutes.

To prepare AuNRs-p (DMAEMA-b-NIPAM), CTAB-capped AuNRs were centrifuged at 10,000 rpm for 10 minutes, repeated twice, and then resuspended in deionized water. A stock solution of MGITC dissolved in DMSO was added to the AuNRs solution with a final concentration of 10 ^-7 M, and a 3.225 mM p(DMAEMA-b-NIPAM)-SH solution was added to the solution so that the final concentration became 8.58 μM. After that, it was incubated for 2 hours. Unconjugated MGITC and p(DMAEMA-b-NIPAM)-SH were removed by centrifugation at 10,000 rpm for 10 minutes, and the remaining pellet was resuspended in deionized water.

In order to prepare a series of AuNPs-p(AAc-b-NIPAM)-AuNRs-p(DMAEMA-b-NIPAM) in the form of a core-satellite nanostructure, AuNPs/AuNRs of different molar concentration ratios were used. Five different concentrations of AuNPs of 10 pM, 20 pM, 50 pM, 100 pM and 400 pM were used under the same AuNRs concentration of 176 pM. In short, 5 different concentrations of AuNPs-p (AAc-b-NIPAM) ranging from 10 pM to 400 pM were added to 176 pM AuNRs-p (DMAEMA-b-NIPAM) solution, and the mixture solution was gently stirred. It was kept for 1 hour without stirring.

<Example 6> Characterization of charged block polymers and nanostructures

The number average molecular weight (M _n ), weight average molecular weight (M _w ) and polydispersity (PDI) of poly(DMAEMA) and p(DMAEMA- b- NIPAM) were determined using a refractive index (RI) detector and a Shodex GPC column SB- Gel Permeation Chromatography (HPLC) 1260 series equipment (Agilent Technologies, Palo Alto, CA, USA) equipped with 804HQ (Shodex GPC system-21; Showa Denko Co., Tokyo, Japan) GPC). An aqueous solution of 0.1 M NaNO ₃ and 0.5 M CH ₃ COOH at 20° C. was used as an eluent at a flow rate of 0.5 ml/min. The GPC column was calibrated with pullulan with a low dispersion in the range of 5,900 to 200,000 g/mol and the column temperature was maintained at 20°C. Poly(AAc) and poly(AAc- b- NIPAM) were modified with (trimethylsilyl)diazomethane prior to GPC measurement (McHale, R.; Aldabbagh, F.; Carroll, WM; Yamada, B., Polymerization) of N-isopropylacrylamide in the presence of poly(acrylic acid) and poly(methacrylic acid) containing ω-unsaturated end-groups.Journal of Polymer Science Part A: Polymer Chemistry 2007, 45 (18), 4394-4400.). Before adding 2.0 M of (trimethylsilyl) diazomethane in hexane dropwise, 5 mg poly(AAc- b- NIPAM) was dissolved in a 1 ml mixture of THF:methanol (3:2). Upon addition, air bubbles were formed and the solution naturally turned colorless. (Trimethylsilyl)diazomethane was added in excess until the yellow color remained. The solution was continued to stir at room temperature for an additional 6 hours. THF, methanol and excess methylating agent were evaporated at ambient temperature and the resulting polymeric membrane was dissolved in THF. Shodex GPC column KF-804 (Shodex GPC system-21; Showa Denko Co., Tokyo, Japan) maintained at 20°C as a stationary phase with its M _n , M _w and PDI and THF at a flow rate of 1 ml/min as a mobile phase It was analyzed with the same installed GPC system. The GPC column was calibrated with low-dispersity polystyrene in the range of 3,180 to 333,000 g/mol and the column temperature was maintained at 20°C. The block length of each polymer was measured using a ¹ H nuclear magnetic resonance ( ¹ H NMR) instrument (AVANCE III 400, Bruker BioSpin AG, Falanden, Switzerland) operating at a frequency of 400 MHz. Poly(AAc) monoblock was dissolved in D ₂ O while poly(AAc- b- NIPAM) was dissolved in DMSO-D _{6 at} a concentration of 5 mg/ml. Poly(DMAEMA) monoblock and poly(DMAEMA- b- NIPAM) were dissolved in CDCl ₃ at the same concentration. Low critical solution temperature (LCST) was analyzed for turbidity at 350 nm using a UV-Vis spectrometer Cary-100 Bio (Varian Biotech, USA) with a Peltier thermostatted temperature control and a heating rate of 0.5 °C/min. The characteristics were analyzed. The LCST point was calculated using the MATLAB program (SI), which is determined by the highest value of the first derivative of absorbance as a function of temperature. Samples were prepared by dissolving the block copolymer in PBS or phosphate buffer having different pH values at a concentration of 0.05 w/v%. Cleavage of the trithiocarbonate group was analyzed by scanning the UV absorbance of the sample through a UV-Vis spectrometer (Shimadzu UV-1800 series, Japan). Poly(AAc- b- NIPAM)-SH was dissolved in 10 mM Tris buffer at a pH of 8.8 at a concentration of 0.5 w/v%, and poly(DMAEMA- b- NIPAM)-SH was dissolved in CHCl ₃ at a concentration of 0.5 w/v%. I did. Each sample was scanned at a wavelength of 200-700 nm.

Colloids of AuNPs, AuNRs, AuNPs-p (AAc-b-NIPAM), AuNRs-p (DMAEMA-b-NIPAM), and AuNPs-p (AAc-b-NIPAM)-AuNRs-p (DMAEMA-b-NIPAM) To understand the properties of the solution, dynamic light scattering (DLS) and zeta potential measurements were taken on a Zeta sizer Nano ZS instrument equipped with a Ne-He laser with a wavelength of 633 nm and a scattering angle of 90° (Malvern Instruments, Melbourne). , UK). The sample was diluted 10:1 with deionized water. Transmission electron microscopy (TEM) imaging was performed on a transmission electron microscope JEM-2100F FE-STEM (JEOL, Germany) operating at an acceleration voltage of 200 kV using a copper grating coated with an ultra-thin layer of carbon (Ted Pella Inc. USA). Were used to investigate individual and complexed nanostructures. The optical properties of the nanostructures were characterized using UV-Vis absorbance spectroscopy. UV-Vis absorbance measurements were measured at 300 to 1200 nm wavelength using a UV-1800 (Shimadzu, Japan) with a fixed slit width of 1 nm in a single 10 scan mode with an intermediate scan rate at room temperature. In addition, Raman measurements were performed using a Raman spectroscopy with a microscope system (Renishaw 2000, Renishaw, UK) and Rayleigh lines were removed from the collected spectra using a holographic notch filter. The MGITC was excited as a Raman reporter with an exposure time of 1 second at maximum laser power using a He/Ne laser beam excited at 633 nm. The laser spot was focused on the capillary using visible light from a 20X objective.

A schematic diagram for the preparation of a new class of stimulation-responsive plasmonic core-satellite nanostructures AuNPs-p (AAc-b-NIPAM)-AuNRs-p (DMAEMA-b-NIPAM) as SERS nanoprobes is shown in FIG. 1. . Figure 1 (a) shows the synthesis of negatively charged AuNPs-p (AAc-b-NIPAM) and positively charged AuNRs-p (DMAEMA-b-NIPAM) through thiol bonding. Two different diblock copolymers consisting of negatively charged poly(AAc) or positively charged poly(DMAEMA) and heat-reactive poly(NIPAM) blocks were prepared by RAFT polymerization, followed by aminolysis to diblock It was made to have a thiol group at each end of the copolymer. Negatively charged diblock copolymer of p (AAc-b-NIPAM) containing thiol-terminus was conjugated to AuNPs, while positively charged p (DMAEMA-b-NIPAM) was conjugated to MGITC-labeled AuNR I did. The MGITC molecule was introduced as a Raman reporter. Figure 1 (b) is a polymer electrolyte complex between the oppositely charged diblock copolymer of p (AAc-b-NIPAM) and p (DMAEMA-b-NIPAM) as shown in the TEM image of Figure 1 (c) The stimulation of AuNPs-p(AAc-b-NIPAM)-AuNRs-p(DMAEMA-b-NIPAM) through the formation of a plasmonic core-satellite is shown. We found that the non-covalent electrostatic interactions were individual AuNPs-p(AAc-b-NIPAM) and AuNRs-p(DMAEMA-b-NIPAM) and AuNPs-p(AAc-b-NIPAM)-AuNRs-p(DMAEMA- b-NIPAM) was found to be assembled in a controlled manner to form a core-satellite nanostructure. In addition, when the p(NIPAM) block of the polymer electrolyte complex was thermally triggered and contracted in the core-satellite nanostructure, the gap between AuNRs as cores and AuNPs as shells was dramatically reduced. This significantly improves the SERS intensity of the plasmonic core-satellite compared to that of the individual AuNPs-p (AAc-b-NIPAM) and AuNRs-p (DMAEMA-b-NIPAM) due to the increased electric field in the gap between AuNPs and AuNRs. I could have it. These core-satellite nanostructures are smart SERS nanoprobes that can induce temperature-dependent induction of electromagnetic coupling through the reversible phase transition from the expanded state in the hydrated form of the polymer electrolyte complex to the contracted state in the dehydrated form. Could Therefore, the gap between the particles of the core and the satellite was reduced to exceed the LCST, which greatly increased the SERS intensity as seen in the Raman spectrum of FIG. 1 (d).

Figure 2 shows a chemical reaction formula for the preparation of (A) negatively charged poly (AAc-b-NIPAM) diblock copolymer and (B) positively charged poly (DMAEMA-b-NIPAM) diblock copolymer. . Synthesis of two different types of thermally reactive diblock p(AAc-b-NIPAM) and poly(DMAEMA-b-NIPAM) diblock copolymers by continuous RAFT polymerization using CDTPA as CTA and AIBN as initiator. I did. CDTPA is a trithiocarbonate-type CTA, which provides a stable tertiary carbon during the addition-fragmentation step, allowing skillful control of polymerization. Next, a diblock copolymer was prepared using poly(DMAEMA) and poly(AAc) homopolymers as macro-CTA. Experimental conditions for the synthesis of monoblock and diblock copolymers and their polymer characterization are shown in Table 1.

[Table 1]

3 shows (A, B) 1 H NMR and (C, D) GPC plots of (A, C) poly(DMAEMA-b-NIPAM) and (B, D) poly(AAc-b-NIPAM). Peaks of δ-6-7 ppm (in CDCl3) and δ-7-8 ppm (in DMSO-d6) due to the amide proton of poly(NIPAM) are poly(AAc) or as macro-CTA via RAFT polymerization It shows the continuous synthesis of poly(NIPAM) blocks from poly(DMAEMA). In addition, GPC tracking indicates that the poly(NIPAM) block was synthesized in a controlled manner.

4 shows (A) poly(DMAEMA-b-NIPAM) and (B) poly(AAc-b-NIPAM) as a function of temperature for characterizing their stimulus-responsiveness such as LCST behavior and hydrodynamic radius of self-assembled micelles. ) (A, B) UV absorption- and DLS profiles are shown. Both poly(AAc-b-NIPAAM) and poly(DMAEMA-b-NIPAAM) were heat-reactive due to the poly(NIPAM) block. The onset temperature of the phase transition behavior obtained by UV spectroscopy and DLS shows the difference due to the difference in heating rate. As shown in Table 1, the LCST of the poly(DMAEMA111) monoblock was 67.3°C, because poly(DMAEMA) is a heat-reactive polymer having a transition temperature of more than 50°C. In addition, the LCST of poly(DMAEMA111-b-NIPAM273) was 35.4°C and 71°C for the first and second LCST, showing two different phase transition behaviors of both poly(NIPAM) and poly(DMAEMA), respectively. The incorporation of poly(NIPAM) into poly(DMAEMA) in the form of a diblock structure caused the transition temperature of poly(DMAEMA) to have a higher value, indicating that poly(DMAEMA) became more hydrophilic. The transition temperature of poly(AAc166-b-NIPAM162) was 38.1°C. As the temperature increased above LCST, poly(AAc166-b-NIPAM162) and poly(DMAEMA111-b-NIPAM273) self-assembled into micelle structures because poly(NIPAM) blocks agglomerated. The hydrodynamic radii of poly(DMAEMA111-b-NIPAM273) and poly(AAc166-b-NIPAM162) micelles in PBS at 37° C. were 91±0.4 nm and 103±3 nm, respectively. 4(C) and (D) show the UV profiles of poly(DMAEMA111-b-NIPAM273) and poly(AAc166-b-NIPAM162) before and after aminolysis. The absorption peak of the trithiocarbonate group at about 307 nm decreased significantly after aminolysis.

5 is Different of AuNR, AuNR-p (DMAEMA-b-NIPAM), AuNP, AuNP-p (AAc-b-NIPAM), and AuNP-p (AAc-b-NIPAM) versus AuNR-p (DMAEMA-b-NIPAM) It shows the UV-Vis absorption spectrum and hydrodynamic diameter of AuNP-p (AAc-b-NIPAM)-AuNR-p (DMAEMA-b-NIPAM) of the core-satellite nanostructure having a molar concentration. Five different concentrations of AuNPs of 10 pM, 20 pM, 50 pM, 100 pM and 400 pM were used, while maintaining the AuNR concentration at 176 pM. As shown in Figure 5 (a), the excitation spectrum of AuNP was generated when p (AAc-b-NIPAM) binding. The UV-vis absorption spectrum of the citrate-capped AuNPs at 520 nm shifted to red to 524 nm, which supports the successful attachment to AuNPs through thiol bonding. 5(b) shows the blue-shift of the LSPR peak in the longitudinal direction of AuNR from 911 nm to 908 nm. The average diameters of AuNP and AuNP-p (AAc-b-NIPAM) were 29.5 ± 0.2 nm and 35.9 ± 1.0 nm, respectively, as shown in FIG. 5(c). In addition, the average diameter of the horizontal axis of AuNRs shifted from 98.3 ± 20.6 nm to 295.5 ± 23.2 nm, and the average diameter of the vertical axis was at 9.1 ± 11.1 nm by the introduction of positively charged AuNRs of p (DMAEMA-b-NIPAM). Increased to 59.4 ± 1.8 nm. 5 (e) and (f) show UV-Vis absorption spectra and hydrodynamic diameters with various molar ratios of AuNP to AuNR. Polymer electrolyte complexation of AuNPs-p (AAc-b-NIPAM) as a satellite with AuNR-p (DMAEMA-b-NIPAM) as a core (I) 0.057:1, (II) 0.114:1, (III) 0.284: 1, (IV) 0.568:1 and (V) 2.273:1 occurred when incubating with different molar ratios of AuNPs to AuNRs. These complexed nanostructures through electrostatic interactions were termed “plasmonic core-satellite nanostructures”. As the AuNPs concentration increased, the longitudinal LSPR peak gradually shifted toward red, and the average size increased. Table 2 shows AuNR, AuNR-p (DMAEMA-b-NIPAM), AuNP, AuNP-p (AAc-b-NIPAM), and AuNP-p (AAc-b-NIPAM) with different molar ratios of AuNP to AuNR- Shows the hydrodynamic diameter of AuNR-p (DMAEMA-b-NIPAM).

[Table 2]

Figure 6(a) shows the SERS intensity of MGITC as a Raman reporter when the concentration of AuNP-p (AAc-b-NIPAM) is increased from 0 pM to 400 pM under the same conditions of 176 pM AuNR-p (DMAEMA-b-NIPAM). Higher, suggesting that the AuNP concentration is directly related to the specific degree of assembly of AuNPs around AuNR through electrostatic interactions. As core-satellite nanostructures, these complexed nanostructures exhibited significantly improved SERS strength compared to individual AuNRs, potentially due to the enhancement of the electric field in the gap between AuNPs and AuNRs. However, a reduced SERS intensity was observed at 400 pM AuNPs, potentially due to partial precipitation of the composite nanostructures. 6(b) shows the temperature-induced enhancement of the SERS signal of the complexed nanostructure. 6(c) to (g), the temperature-triggered shrinkage of the poly(NIPAM) block of poly(AAc-b-NIPAM) and poly(DMAEMA-b-NIPAM) in water exceeding LCST is LCST It produced a higher SERS spectrum than was observed at <below, because the gap between AuNP and AuNR was potentially reduced. In particular, when the molar ratio of AuNP to AuNR was (IV) 0.568:1 and (V) 2.273:1, there was no significant improvement in SERS strength. This was due to the low colloidal stability of the composite nanostructure above the LCST, and because irreversible aggregation occurred. This observation is in good agreement with the ξ-potential value as shown in Fig. 6(h) and Table 2.

7 shows TEM images of AuNPs and AuNRs at different magnifications. A series of complexed nanostructures of AuNPs-p(AAc-b-NIPAM)-AuNRs-p(DMAEMA-b-NIPAM) as core-satellite nanostructures are shown in FIG. 8. The density of AuNPs on AuNRs increased to 0.284:1 after the AuNP/AuNR molar ratio increased from 0.057:1 to 0.114:1.

<Example 7> Bioconjugation of antibody and core-satellite nanostructure and magnetic beads

AuNPs-p(AAc-b-NIPAM)-AuNRs-p(DMAEMA-b-NIPAM) And core-satellite nanostructures of magnetic beads (MBs) were individually conjugated with two different sets of monoclonal antibodies (mAb) and polyclonal antibodies (pAb) against immunoglobulin G (IgG), respectively. First, bioconjugation of the carboxyl group of anti-human IgG polyclonal antibody (anti-human IgG pAb) and AuNPs-p (AAc-b-NIPAM)-AuNRs-p (DMAEMA-b-NIPAM) was amide coupling. It was carried out through the reaction. This conjugation reaction was based on EDC and sulfo-NHS chemistry. In short, 5 μl of 1.0 mg/ml anti-human IgG pAb was added to AuNPs-p(AAc-b-NIPAM)-AuNRs-p with 1 mM EDC and 1 mM Sulfo-NHS in 10 mM PBS, pH 7.4. (DMAEMA-b-NIPAM) was added and stirred for 1 hour. Anti-human IgG pAb-conjugated nanostructures were washed by centrifugation at 3,000 rpm and resuspended in PBS. In addition, MB was chemically conjugated with an anti-human IgG monoclonal antibody (anti-human IgG mAb). In a typical experiment, homogeneously suspended MB was mixed with 5.0 mM EDC and 5.0 mM Sulfo-NHS and stirred for 1 hour. 2.96 mg/ml of anti-human IgG mAb solution was diluted in 100 μL of PBS buffer and then slowly added dropwise to the MB solution to a final concentration of 7.4 μg/ml, followed by stirring for 1 hour. Unconjugated anti-human IgG was separated using a magnetic field, and anti-human IgG mAb-conjugated MB was resuspended in PBS for SERS-based biosensing of IgG.

<Example 8> SERS-based biosensing of IgG

Anti-human IgG pAb-conjugated core-satellite nanostructure, AuNPs-p (AAc-b-NIPAM)-AuNRs-p (DMAEMA-b-NIPAM), as a SERS nanotag for quantitative analysis of target protein IgG I did. On the other hand, immunocomplexes were specifically isolated as a separation tool through the formation of sandwich-type immunocomplexes using MB conjugated with anti-human IgG mAb. First, anti-human IgG mAb-conjugated MB was added to a buffer solution containing four different concentrations of IgG ranging from 0.083 μg/ml to 83 ng/ml and incubated for 1 hour. Subsequently, the target protein was washed with an external magnetic field and resuspended in fresh PBS? buffer. Subsequently, as a SERS nanoprobe, an anti-human IgG pAb-conjugated core-satellite nanostructure was added to the immunocomplex, and incubated for 1 hour to induce the formation of a sandwich-type immunocomplex consisting of magnetic beads, IgG, and SERS nanoprobes. . The unattached SERS nanoprobe was removed using a magnetic field, and the resulting sandwich-type immunocomplex was resuspended in PBS for SERS measurement. Control experiments were also performed without IgG as target.

As a SERS nanoprobe, the complexed nanostructures of AuNPs-p (AAc-b-NIPAM) and AuNRs-p (DMAEMA-b-NIPAM), as shown in Fig. 9 (a), were AuNPs-p (AAc-b- NIPAM)-AuNRs-p(DMAEMA-b-NIPAM) It was chemically conjugated to anti-human IgG pAb by activating the -COOH group present on the surface. Similarly, magnetic beads (MB) as a refining tool were reported as (Ali, A.; Hwang, EY; Choo, J.; Lim, DW, Nanoscale graphene oxide-induced metallic nanoparticle clustering for surface-enhanced Raman scattering- based IgG detection.Sensors and Actuators B: Chemical 2018, 255 (Part 1), 183-192.) Anti-human IgG using -COOH groups remaining on the polymer chain for the formation of sandwich-type immunocomplexes in the presence of IgG Conjugated to mAb. 9(b) shows a schematic diagram of a method in which an immunocomplex is produced through a two-step process. First, the anti-human IgG mAb-conjugated MB was mixed with a human IgG solution to specifically capture IgG as a model, and the complex between the anti-human IgG mAb-conjugated MB and the IgG was separated using a magnetic field. The solution was washed and resuspended. Second, the prepared complex was further mixed with an anti-human IgG pAb-conjugated core-satellite nanostructure as a SERS nanoprobe. Anti-human IgG pAb-conjugated AuNPs-p(AAc-b-NIPAM)-AuNRs-p (DMAEMA-b-NIPAM), IgG, and anti-human IgG mAb-conjugated MBs are anti-conjugated through specific molecular interactions. A sandwich type immunocomplex was formed between -IgG pAb, IgG, and anti-IgG mAb. The sandwich-type immunocomplex was isolated using a magnetic field, and Raman intensity was generated which is directly proportional to the IgG concentration. 9(c) shows the relative Raman spectrum and Raman intensity of the tertiary immunocomplex as a function of the IgG concentration when the immunocomplex is separated by a magnetic field. As the IgG concentration increased, the Raman intensity of the immunocomplex increased. On the other hand, as a control experiment, a negligible Raman intensity was observed in the absence of IgG, because the immune complex was not formed without IgG. The Raman intensity of the tertiary immunocomplex was linearly correlated with IgG, indicating that its SERS signal is directly proportional to the IgG concentration. One of the advantages of AuNPs-p(AAc-b-NIPAM)-AuNRs-p(DMAEMA-b-NIPAM) as a SERS nanoprobe is that it enables rapid molecular interactions between SERS nanoprobes, biomarkers and MBs in solution. , Which shows high sensitivity without any photobleaching and long assay times for SERS-based biosensing.

In the present invention, a shape consisting of positively charged, heat-reactive AuNRs-p (DMAEMA-b-NIPAM) and negatively charged, heat-reactive AuNPs-p (AAc-b-NIPAM) through electrostatic interaction- Custom plasmonic core-satellite nanostructures were shown. Two different diblock copolymers of negatively charged poly (AAc) or positively charged poly (DMAEMA) and heat-reactive poly (NIPAM) were prepared by RAFT polymerization, followed by aminolysis to obtain each diblock copolymer. It was made to have the thiol end group of the coalescence. By attaching this diblock copolymer containing a thiol-terminus to AuNRs or AuNPs through thiol bonds, negatively charged AuNPs-p (AAc-b-NIPAM) and positively charged AuNRs-p (DMAEMA-b-NIPAM) ) Was formed. As core-satellite nanostructures, the complexed nanostructures of AuNPs-p(AAc-b-NIPAM)-AuNRs-p(DMAEMA-b-NIPAM) are individual AuNPs-p(AAc-b-NIPAM) and AuNRs-p( DMAEMA-b-NIPAM) showed significantly improved physicochemical and optical performance, potentially due to the enhancement of the electric field in the gap between AuNP and AuNR. In conclusion, these core-satellite nanostructures have biomedical applications such as single-molecule recognition, plasmonics, and biosensing.

Claims (19)

A core structure of a metal nanorod in which a diblock copolymer composed of a positively charged polymer block having a thiol end group and a stimulus-reactive polymer block are bonded to each other; And
A satellite structure of metal nanoparticles in which a negatively charged polymer block having a thiol end group and a diblock copolymer composed of a stimulation-reactive polymer block are bonded;
As a stimulus-responsive core-satellite nanostructure composed of,
The stimulation-responsive core-satellite nanostructure,
A stimulation-responsive core formed through an electrostatic interaction between a diblock copolymer of a positively charged polymer bonded to the metal nanorod and a diblock copolymer of a negatively charged polymer bonded to a metal nanoparticle -Satellite nanostructures.
The method of claim 1,
The positively charged polymer block is allyamine, 2-(dimethyl amino)ethylmethacrylate, ethyleneimine, vinyl amine, 2-vinylpyridine (2-vinyl pyridine), and 4-vinyl pyridine (4-vinyl pyridine) that contains one or more monomers, stimulation-responsive core-satellite nanostructure.
The method of claim 1,
The negatively charged polymer block contains one or more monomers of acrylic acid, methacrylic acid, itaconic acid, and maleic acid. The stimulus-responsive core-satellite nanostructure comprising.
The method of claim 1,
The stimulation-reactive polymer block 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] [Poly[oligo(ethylene glycol) methyl ether methacrylate]: POEGMA], poly(2-vinyl pyridine) [poly(2-vinyl pyridine): P2VP], And poly(4-vinyl pyridine) [poly(4-vinyl pyridine): P4VP] that contains one or more monomers, stimulation-responsive core-satellite nanostructures.
delete The method of claim 1,
Wherein the gap between the metal nanorod and the metal nanoparticles decreases above the lower limit critical solution temperature (LCST) of the core-satellite nanostructure, the stimulation-responsive core-satellite nanostructure.
The method of claim 1,
The stimulation is heat, stimulation responsive core-satellite nanostructure.
The method of claim 1,
The core structure of the metal nanorod further includes a Raman dye, stimulation-responsive core-satellite nanostructure.
The stimulus-responsive core of claim 8-a metal nanoprobe for measuring a surface-enhanced Raman scattering (SERS) image using a satellite nanostructure.
i) preparing a diblock copolymer (A) composed of a negatively charged polymer block and a stimulus reactive polymer block and a diblock copolymer (B) composed of a positively charged polymer block and a stimulus reactive polymer block, respectively;
ii) Diblock copolymer (A-SH) and diblock copolymer (B) in which thiol groups are introduced by introducing thiol groups at the ends of each of the diblock copolymers (A) and diblock copolymers (B) through amino decomposition -SH) respectively;
iii) Metal nanoparticles in which the diblock copolymer (A-SH) into which the thiol group is introduced is bonded to the metal nanoparticles, and the diblock copolymer (A) consisting of a negatively charged polymer block and a stimulation-reactive polymer block is bonded To prepare;
iv) A metal nanorod in which the diblock copolymer (B-SH) into which the thiol group is introduced is bonded to a metal nanorod, and a diblock copolymer (B) composed of a positively charged polymer block and a stimulus-reactive polymer block is bonded. To prepare; And
v) mixing and stirring the metal nanoparticles to which (A) is bonded and the metal nanorods to which (B) is bonded;
A method for producing a stimulus-responsive core-satellite nanostructure comprising the step.
The method of claim 10,
Positively charged polymer blocks include allyamine, 2-(dimethyl amino)ethylmethacrylate, ethyleneimine, vinyl amine, and 2-vinylpyridine ( 2-vinyl pyridine), and 4-vinyl pyridine (4-vinyl pyridine) that contains one or more monomers, stimulation-reactive core-a method for producing a satellite nanostructure.
The method of claim 10,
The negatively charged polymer block contains one or more monomers of acrylic acid, methacrylic acid, itaconic acid, and maleic acid. The method for producing a stimulus-responsive core-satellite nanostructure containing.
The method of claim 10,
The stimulation-reactive polymer block 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] [Poly[oligo(ethylene glycol) methyl ether methacrylate]: POEGMA], poly(2-vinyl pyridine) [poly(2-vinyl pyridine): P2VP], And poly(4-vinyl pyridine) [poly(4-vinyl pyridine): P4VP] that contains one or more monomers, stimulation-responsive core-satellite nanostructure manufacturing method.
The method of claim 10,
In step iv),
The method of manufacturing a stimulus-responsive core-satellite nanostructure, further comprising attaching a Raman dye to the metal nanorod.
The method of claim 10,
In step v),
Through the electrostatic interaction between (B) bonded to the metal nanorod and (A) bonded to the metal nanoparticles, a core-satellite nanostructure is formed, a stimulation-responsive core-satellite nanostructure manufacturing method.
The method of claim 10,
A method of manufacturing a stimulus-responsive core-satellite nanostructure, wherein the gap between the metal nanorod and the metal nanoparticle decreases above the lower limit critical solution temperature (LCST) of the core-satellite nanostructure.
The method of claim 10,
The stimulation is heat, the stimulation-responsive core-satellite nanostructure manufacturing method.
a) preparing a sample solution containing a target substance to be detected;
b) preparing the magnetic nanoparticles by immobilizing the first antibody against the target;
c) preparing by immobilizing a second antibody against the target to the metal nanoprobe of claim 9;
d) adding the magnetic nanoparticles to which the first antibody is immobilized to the sample solution to form an immunocomplex in which the target and the first antibody of the magnetic nanoparticles are conjugated;
e) The metal nano-probe to which the second antibody is immobilized is added to the solution containing the immunocomplex to which the first antibody is conjugated to obtain a sandwich immunocomplex of the first antibody of the second antibody-target-magnetic nanoparticle. To form;
f) separating magnetic nanoparticles and metal nanoprobes that do not form the sandwich immunocomplex using a magnetic field; And
g) measuring the Raman signal of the sandwich immunocomplex;
Including the step of, surface-enhanced Raman scattering (SERS)-based target material detection method.
The method of claim 18,
The target material is a protein or a pathogen, surface-enhanced Raman scattering (SERS)-based target material detection method.

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