KR100989289B1 - Magnetic active surface enhanced raman scattering nano particle, method for production thereof, and biosensor using the same - Google Patents

Abstract

The present invention relates to magnetic surface-enhanced Raman scattering particles (M-SERS dots) and a method of manufacturing the same, and a biosensor using the same. Specifically, a magnetic-surface enhanced Raman scattering particle comprising a particle aggregate and a silica shell surrounding the particle aggregate, wherein the particle aggregate comprises: a magnetic core particle comprising a magnetic material; Silver nano particles introduced on the surface of the magnetic central particle; And magnetic-surface enhanced Raman scattering particles, and a method of manufacturing the same, wherein the particles including the labeling substance fixed around the silver nanoparticles are formed by aggregation of one or more particles. Functional groups introduced to the surface of the magnetic-surface enhanced Raman scattering particles; And it relates to a biosensor comprising a receptor attached to the functional group, and a manufacturing method thereof.

The biosensor according to the present invention has the advantage that the induction of hot spots is made efficiently by using magnetic-surface-enhanced Raman scattering particles including magnetic material and silver nanoparticles, and the detection of the substance bound to the biosensor is easy. In addition, the biosensor according to the present invention is free of labels or has no particular limitation on the number of labels and has non-toxic properties, and thus may be variously used in fields such as medicine and pharmacy, in which detection of biomaterials is important.

Magnetic-Surface Enhanced Raman Scattering Particles, Biosensors, Silver Nanoparticles, Magnetic Particles, Hot Spots

Description

MAGNETIC ACTIVE SURFACE ENHANCED RAMAN SCATTERING NANO PARTICLE, METHOD FOR PRODUCTION THEREOF, AND BIOSENSOR USING THE SAME}

The present invention relates to magnetic surface-enhanced Raman scattering particles, a preparation method thereof, and a biosensor using the same. In particular, the present invention includes magnetic materials and silver nanoparticles, has the advantage that the induction of hot spots are made efficiently, and that the detection of the material bound to the biosensor is easy, and it is label free or specific to the number of labels. A magnetic-surface enhanced Raman scattering particle having unlimited and non-toxic properties.

With advances in bioanalytical science and bioengineering, recent studies in genomics and proteomics produce more sequence data. Thus, there is a need for new technologies that can quickly screen large numbers of biomolecules and their ligands simultaneously.

At first, a method for analyzing substances using radioisotopes was developed. However, this method has a problem that it emits harmful radiation to the human body and has a long half-life and stored for a long time.

Subsequently, fluorescent nanomaterials have been developed and most widely used in biological applications. However, the method of using the fluorescent nanomaterials has problems such as photobleaching, a narrow excitation region having a wide emission profile, overlap of peaks in multiple tests, and a limitation in the number of labeling materials.

Recently, a method using quantum dots has been developed and used. However, this also has problems such as the use of cadmium during the manufacturing process is toxic, the problem of losing the intrinsic color of the quantum dots when modifying the surface and the number of labeling substances is limited.

 There is no photobleaching, there is no label-free or many kinds of labeling material, there is no limit to the number of labels, low or no toxicity, the development of particles that enable efficient detection of the material is required.

In order to solve the above problems, an object of the present invention is to provide a magnetic-surface-enhanced Raman scattering particle, a preparation method thereof, and a biosensor using the same.

In order to solve the above problems, the present invention is a magnetic-surface enhanced Raman scattering particles comprising a particle aggregate and a silica shell surrounding the particle aggregate, wherein the particle aggregate is a magnetic core particle comprising a magnetic material; Silver nano particles introduced on the surface of the magnetic central particle; And it provides a magnetic-surface-enhanced Raman scattering particles that are formed by agglomerated one or more particles comprising a labeling material fixed around the silver nanoparticles.

In addition, the present invention comprises the steps of introducing the silver nanoparticles on the surface of the magnetic core particles containing the magnetic material; Fixing a labeling material around the silver nanoparticles; Agglomerating at least one particle including the magnetic central particle, silver nanoparticles introduced into the magnetic central particle, and a labeling substance fixed around the silver nanoparticles to provide a particle aggregate; And it provides a method for producing a magnetic-surface enhanced Raman scattering particles comprising the step of forming a silica shell around the particle aggregate.

The present invention also provides a magnetic-surface enhanced Raman scattering particle according to the present invention; Functional groups introduced to the surface of the magnetic-surface enhanced Raman scattering particles; And it provides a biosensor comprising a receptor attached to the functional group.

In addition, the present invention is characterized in that the use of the biosensor according to the present invention, the detection method of the target biomaterial that binds to or reacts with the receptor of the biosensor, the detection is a Raman scattering, magnetic field, or Raman scattering and magnetic field It provides a detection method characterized in that the use.

Magnetic-surface-enhanced Raman scattering particles and silver nano-magnetic particles according to the present invention can be easily formed Raman analysis because of the efficient formation of hot spots, there is no label-free or many kinds of labeling material, there is no limitation in the labeling material, the detection of the material It can be widely used in this important field of medicine and pharmacy. In addition, the magnetic surface-enhanced Raman scattering particles and silver nano-magnetic particles have no photobleaching, low toxicity or no effect, has the effect of enabling the efficient detection of the material.

The above objects, features, and advantages will become more apparent from the following description taken in conjunction with the accompanying drawings. Hereinafter, the present invention will be described in detail.

Justice

Definitions of terms used in the present invention are as follows.

In the present invention, "magnetic core particle" means a particle containing a magnetic material.

In the present invention, "magnetic-surface-enhanced Raman scattering particles (M-SERS dots)" means particles comprising particle aggregates and silica shells surrounding the particle aggregates. Here, the particle aggregates are magnetic central particles comprising a magnetic material; Silver nano particles introduced on the surface of the magnetic central particle; And one or more particles including a labeling substance fixed around the silver nanoparticles.

In the present invention, "silver nano magnetic particles (Ag-M dots)" is a magnetic core particle containing a magnetic material; And one or more particles including silver nanoparticles introduced on the surface of the magnetic central particle.

Detailed description of the invention

The present invention provides a magnetic-surface-enhanced Raman scattering particle comprising a particle aggregate and a silica shell surrounding the particle aggregate, wherein the particle aggregate comprises: a magnetic core particle comprising a magnetic material; Silver nano particles introduced on the surface of the magnetic central particle; And it provides a magnetic-surface-enhanced Raman scattering particles that are formed by agglomerated one or more particles comprising a labeling material fixed around the silver nanoparticles.

The magnetic central particle is a particle including a magnetic material, and the magnetic material is a material having magnetic properties. Magnetic core particles impart magnetic properties to the biosensor according to the present invention to facilitate the induction of hot spots and the detection of target biomaterials. The magnetic material may be Co, Mn, Fe, Ni, Gd, MM ' ₂ O ₄ , M _x O _y (M or M' = Co, Fe, Ni, Mn, Zn, Gd, Cr, 0 <x≤3, 0 <y≤5) and the like are preferred, but are not limited thereto.

In addition, the magnetic core particles may be particles in which a magnetic material is selectively silica coated, and may include particles including a magnetic material and a silica coating surrounding the magnetic material. In the case of the magnetic core particle formed of silica coated magnetic material, the size is preferably 20 nm to 300 nm, more preferably 40 nm to 80 nm, but is not limited thereto. If the size of the central particle is less than 20 nm Raman surface enhancement effect may be small, if it exceeds 300 nm may be limited in biological applications.

The silica coating fixes a magnetic material inside the magnetic central particle, and then protects the magnetic material in the process of forming magnetic-surface enhanced Raman scattering particles, and facilitates surface modification of the magnetic central particle, and magnetic central particle. It plays a role to control the size of. In order to perform the above role and to sufficiently exhibit the magnetic properties of the magnetic material in the magnetic-surface enhanced Raman scattering particles, the thickness of the silica coating is preferably 100 nm or less, but is not limited thereto. .

Silver nanoparticles introduced into the magnetic central particle surface facilitate the use of Raman scattering spectroscopy. Between the dense silver nanoparticles, there is a junction called a hot spot or a nano junction, and at this position, a surface enhanced Raman scattering phenomenon is more strongly generated. In other words, the dense silver nanoparticles have an advantage of facilitating the application of the nano-labeled particles of the present invention based on Raman spectroscopy. The size of the silver nanoparticles to be introduced is preferably 100 nm or less, preferably 8 nm to 20 nm, but is not limited thereto. When the size of the silver nanoparticles exceeds 100 nm, the signal enhancement for Raman scattering may be small. In addition, even when the size of the silver nanoparticles is 10 nm or less, the signal augmentation for Raman scattering may be reduced, but may be applied by inducing hot spots by a magnetic field.

Before introducing the labeling material into the magnetic central particles into which the silver nanoparticles are introduced, the polar polymer may be introduced. The polar polymer thus introduced may serve to facilitate the formation of the silica shell.

The labeling substance is preferably a chemical substance having a binding force with the silver nanoparticles, and may show a Raman spectrum in a distinct fingerprint region while having a simple structure. For this reason, the labeling material facilitates detection of a target biomaterial using a biosensor comprising the magnetic-surface enhanced Raman scattering particles according to the present invention.

Labeling materials that can be used in the present invention are 4-merctotoluene (4-MT), 3,5-dimethyl benzenethiol (3,5-DMT), thiophenol (TP), 4-amino thiophenol (4- ATP), benzenethiol (BT), 4-bromo benzenethiol (4-BBT), 2-bromo benzenethiol (2-BBT), 4-isopropyl benzenethiol (4-IBT), 2-naphthalene thiol ( 2-NT), 3,4-dichloro benzenethiol (3,4-DCT), 3,5-dichloro benzenethiol (3,5-DCT), 4-chloro benzenethiol (4-CBT), 2-chloro benzene Thiol (2-CBT), 2-fluoro benzenethiol (2-FBT), 4-fluoro benzenethiol (4-FBT), 4-methoxy benzenethiol (4-MOBT), 3,4-dimethoxy benzene Thiols (3,4-DMOBT), 2-mercetopyrimidine (2-MPY), 2-merceto-1-methyl imidazole (2-MMI), 2-merceto-5-methyl benzimidazole (2 -MBI), 2-amino-4- (trifluoromethyl) benzenethiol (2-ATFT), benzyl merethane (BZMT), benzyl disulfide (BZDSF), 2-amino-4-chloro benzenethiol (2-ACBT ), 3-mercenzo benzoic acid (3-MBA), 1-phenyltetrazol-5-thiol (1-PTET), 5-phenyl-1,2,3-tria 3-thiol (5-PTRT), 2-iodoaniline (2-IAN), phenyl isothiocyanate (PITC), 4-nitrophenyl disulfide (4-NPDSF) and 4-azido-2-bromoa It is preferred that any one or more selected from the group consisting of cetophenone (ABAPN), but can be used without limitation as long as it is a chemical substance capable of binding to silver nanoparticles. Since the present invention has a large number of Raman labeling substances and any combination thereof, the present invention has the same effect as the number of labeling substances is not limited. In the above, "a chemical substance having a bonding force with silver nanoparticles" is a thiol group (-SH), an amine group (-NH ₂ ), cyanide group (-CN), isocyanide group (-CNO), isothiosi An amide group (-CNS), a disulfide group (-SS-) or an azide group (-N ₃ ) means a chemical present, such as thiol group, amine group, cyanide group, isocyanide Chemicals having groups, isothiocyanide groups, disulfide groups, or azide groups have a strong affinity with silver nanoparticles, and thus can be preferably used in the present invention.

The agglomeration during the formation of the magnetic-surface enhanced Raman scattering particles or the silver nanomagnetic particles is a phenomenon in which the electrostatic repulsive force of the silica coating surface of the magnetic core particles is reduced by the introduction of the silver nanoparticles, thereby weakening the repulsive force between the particles.

The magnetic-surface enhanced Raman scattering particles according to the present invention comprise a silica shell surrounding the particle aggregates. The silica shell facilitates the surface modification of the magnetic-surface enhanced Raman scattering particles, and serves to control the size of the magnetic-surface enhanced Raman scattering particles. The silica shell preferably has a thickness of 5 nm to 10 nm, but is not limited thereto. If the thickness of the silica shell is too thin to less than 5 nm, it is difficult to protect the silver nanoparticles coated with the labeling material from the external environment, and if the thickness of the silica shell exceeds 10 nm, it may be a limitation in biological application to cells or tissues.

The size of the magnetic-surface-enhanced Raman scattering particles according to the present invention can be varied without limitation depending on the conditions applied. The magnetic-surface enhanced Raman scattering particles according to the present invention preferably have a size of 36 nm to 8 μm, more preferably 50 nm to 800 nm, but are not limited thereto. If the size of the magnetic-surface-enhanced Raman scattering particles is less than 36 nm, surface modification is difficult, signal enhancement for Raman scattering may be lowered, and when the size of the magnetic-surface enhanced Raman scattering particles is greater than 8 µm, binding efficiency with cells may be lowered.

In addition, the present invention comprises the steps of introducing the silver nanoparticles on the surface of the magnetic core particles containing the magnetic material; Fixing a labeling material around the silver nanoparticles; Agglomerating at least one particle including the magnetic central particle, silver nanoparticles introduced into the magnetic central particle, and a labeling substance fixed around the silver nanoparticles to provide a particle aggregate; And it provides a method for producing a magnetic-surface enhanced Raman scattering particles comprising the step of forming a silica shell around the particle aggregate. Magnetic materials, magnetic core particles, silver nanoparticles, labeling substances, particle aggregates and silica shells are described above.

In the case of a magnetic core particle formed by silica coating of a magnetic material, the silica coating on the magnetic material may be performed according to a known method such as using a silica precursor or a microemulsion method.

The step of introducing the silver nanoparticles to the magnetic central particle surface may be made by a known method. For example, a known method such as a method of adding silver nanoparticles after introducing a thiol group (-SH) on the surface of the magnetic central particle by adding 3-mercetopropyltriethoxysilane to the magnetic central particle may be used. Can be.

Fixing the labeling material around the silver nanoparticles may be performed according to any known method. The type of labeling substance to be immobilized and the functional group for facilitating the fixing of the labeling substance are as described above.

Aggregating one or more particles including the magnetic core particles, silver nanoparticles introduced to the magnetic core particles, and a labeling material fixed around the silver nanoparticles to provide a particle aggregate may include silica coating of the magnetic core particles. The negative charge repulsion of the surface is largely offset by the introduction of silver nanoparticles, which may result in weak repulsion between the particles.

Forming a silica shell around the particle aggregate may be accomplished according to a known method using a silica precursor such as tetraethyl orthosilicate or tetramethyl orthosilicate.

On the other hand, the present invention is magnetic-surface enhanced Raman scattering particles; Functional groups introduced to the surface of the magnetic-surface enhanced Raman scattering particles; And it provides a biosensor comprising a receptor attached to the functional group. The magnetic-surface enhanced Raman scattering particles are as described above.

The functional group that can be introduced to the surface of the magnetic-surface enhanced Raman scattering particles are functional groups that can be generally used in biosensors, but are not limited thereto, such as amine groups, thiol groups, carboxyl groups. The functional group serves to connect the receptor to which the target material is bound and the magnetic-surface enhanced Raman scattering particles according to the present invention. The presence of such a functional group allows the attachment of various receptors to the functional group, whereby the biosensor according to the present invention can detect various target substances.

In addition, the receptor attached to the functional group is also a receptor generally used in biosensors, enzyme substrates, ligands, amino acids, peptides, proteins, nucleic acids, lipids, cofactors, carbohydrates, antibodies, etc., but is not limited thereto. no. Each of the receptors can be combined or reacted with a specific target material corresponding thereto. Then, the biosensor that is bound or reacted with a specific target material through the receptor can be identified using magnetic field, Raman scattering, or magnetic field and Raman scattering. . This enables the biosensor according to the invention to selectively detect various target substances.

In addition, the present invention comprises the steps of introducing a functional group on the surface of the magnetic-enhanced Raman scattering particles; And attaching a receptor to the functional group.

The step of introducing a functional group on the surface of the magnetic-surface enhanced Raman scattering particle and the attaching method of the receptor corresponding to the functional group may be performed according to a known method. For example, Korean Patent No. 10-0525764 discloses a method of introducing an amine group into a functional group using aminoalkyloxysilane, and the like. Patent Nos. 20-0525764 and 10-0549051 describe a thiol group. The introduction method and the method of attaching a receptor corresponding to the thiol group, Patent No. 10-0580642 and the like disclose a method of introducing a carboxyl group and a method of attaching a receptor corresponding to the carboxyl group.

In another aspect, the present invention provides a method for detecting a target biomaterial that binds to or reacts with a receptor of the biosensor, using a biosensor according to the invention, wherein the detection is performed using Raman scattering, magnetic field, or Raman scattering and magnetic field. It provides a detection method characterized in that.

In the present invention, the target biomaterial is a substance capable of serving as a target to be detected by reacting with or binding to the receptor of the biosensor, preferably an enzyme, a protein, a nucleic acid, an oligosaccharide, a peptide, an amino acid, a carbohydrate, a lipid, a cell, Cancer cells, cancer stem cells, antigens, aptamers or other bio-derived biomolecules, more preferably proteins related to disease, but are not limited thereto. Preferably, the cancer cells are any one of breast cancer cells, lung cancer cells, and leukemia cells, but are not limited thereto.

The detection is carried out using the Raman scattering effect by the hot spot which is the characteristic of the magnetic-surface enhanced Raman scattering particles according to the present invention and the magnetic properties due to the magnetic material. The Raman scattering effect enables the detection of the target biomaterial by detecting the Raman scattering of the label. In this case, the target biomaterial may be detected by performing Raman analysis with a confocal Raman system (Laboam 300, JY-Horiba, Edison, NJ, USA). In addition, the magnetic properties facilitate the detection by the magnetic field of the target material bound or reacted with the biosensor using the magnetic-surface enhanced Raman scattering particles. In order to facilitate the detection of the target material, the strength of the magnetic field is preferably 100 to 7000 gauss, but is not limited thereto.

According to the present invention, since the magnetic-central particles include a magnetic material, the magnetic-surface enhanced Raman scattering particles can be aggregated by a magnetic field, so that the magnetic-surface enhanced Raman scattering particles are conventional surface-enhanced Raman scattering nanoparticles (SERS). Better signal amplification effects (dots of hot spots and aggregation) than dots. In addition, magnetic-surface-enhanced Raman scattering particles agglomerated by a magnetic field according to such effects are highly efficient target materials because they can amplify 1000 times or more Raman signals compared to non-agglomerated dispersed magnetic-surface-enhanced Raman scattering particles. A detection method can be provided. Therefore, there is an advantage that can facilitate the application of the nano-labeled particles of the present invention based on Raman spectroscopy.

On the other hand, the present invention is a magnetic core particle containing a magnetic material; And silver nano magnetic particles formed by agglomeration of one or more particles including silver nano particles introduced on the magnetic central particle surface. Magnetic core particles, silver nanoparticles, and the description of the aggregation is as described above.

In addition, the present invention comprises the steps of introducing the silver nanoparticles on the surface of the magnetic core particles containing the magnetic material; And aggregating one or more magnetic central particles into which the silver nanoparticles are introduced. A description of the step of introducing the silver nanoparticles to the magnetic central particle surface is as described above.

The aggregation step of the magnetic nanoparticles into which the silver nanoparticles are introduced may be performed because the negative charge repulsion force on the surface of the silica coating of the magnetic nanoparticles is reduced by introduction of the silver nanoparticles, thereby weakening the repulsive force between the particles. .

In addition, the present invention is a method for detecting a substance capable of binding to silver nanoparticles in the silver nano-magnetic particles, characterized in that using the silver nano-magnetic particles according to the present invention, the detection is Raman scattering, magnetic field, or Raman scattering and magnetic field It provides a detection method characterized in that made using. The description of the silver nano magnetic particles is as described above.

Since the repulsive force of the silica coating surface of the magnetic core particles is canceled in the introduction step of the silver nanoparticles, the silver nanomagnetic particles according to the present invention may be agglomerated by an external magnetic field, and thus the label-free material of the silver nanoparticles may bind to the silver nanoparticles. It can be used in the detection method. In order to facilitate the detection of a substance capable of binding to the silver nanoparticles, the strength of the external magnetic field is preferably 100 to 7000 gauss, but is not limited thereto.

The material capable of binding to the silver nanoparticles detected by the detection method using the silver nanomagnetic particles according to the present invention is preferably a compound having a bonding force with the silver nanoparticles described above, but is not limited thereto. In addition, the combination of the silver nanoparticles and the material capable of bonding with the silver nanoparticles is preferably a chemical bond, more preferably a series of covalent to coordination bonds, but is not limited thereto. According to a preferred embodiment of the present invention, the material capable of binding to the silver nanoparticles may be methyl parathion, phenol or adenine. The methyl parathion has a non-covalent electron pair at the sulfur atom of the thion group, phenol has a non-covalent electron pair at the oxygen atom of the hydroxy group, adenine has a non-covalent electron pair at the nitrogen atom of the azide group (azide, N ₃ ), metal particles For example, silver nanoparticles can form chemical bonds with the lone pairs of electrons, which are weaker than CC bonds but chemically strong. By such bonding through the silver nanoparticles, methyl parathion, phenol or adenine is combined with the silver nanomagnetic particles according to the present invention to form a self-assembled monolayer. Thus, using the silver nano magnetic particles according to the present invention can be detected by label-free analysis even when the methyl parathion, phenol or adenine is present in a low concentration. This also uses the Raman scattering effect by the hot spot and the magnetic properties due to the magnetic material.

Example

Hereinafter, exemplary embodiments of the present invention will be described in detail with reference to the accompanying drawings. However, the following examples are merely to illustrate the invention, but the content of the present invention is not limited to the following examples.

[ Example  1] Magnetic-Surface Enhancement Raman Scattering  Preparation of Particles

1-1. Preparation of Magnetic Core Particles

First, 10.8 g of iron chloride (FeCl ₃ · 6H ₂ O) and 36.5 g of sodium oleate were dissolved in a mixture of 80 ml of ethanol, 60 ml of distilled water, and 140 ml of hexane, and then heated to 70 ° C. for 4 hours to react. -Olate complex was obtained. After the reaction, the iron-oleate complex was dissolved in the organic layer, which was the upper layer, which was washed three times with distilled water, then washed with hexane, and dried. Thus prepared 36 g of iron-oleate complex and 5.7 g of oleic acid were dissolved in 200 g of 1-eicosene, and the temperature was gradually raised to 320 ° C. After cooling to room temperature to precipitate the reaction product into ethanol 500 ㎖ (Nature materials , 2004, 3 , 891). This material was again silica coated by microemulsion method. The diameter of the iron-oleate composite was 18 nm and the thickness of the silica coating was 16 nm. As a result, magnetic central particles were obtained.

1-2. On the magnetic core particle surface Silver nano  Introduction of particles

1 ml of the magnetic core particles prepared in Example 1-1 (the content of the magnetic substance in the magnetic core particles determined by Inductively Coupled Plasma (ICP) analysis was 2.2 mg / ml) were added to 1 ml of ethanol. After dispersion, 10 μl of 1% (v / v) 3-mercaptopropyltrimethoxysilane (MPTS) and 10 μl of 25% aqueous ammonia (NH ₄ OH _{( aq )} ) were added thereto. Stirring at 25 ° C. for 12 hours introduces a thiol group to the magnetic central particle surface. In order to introduce silver nanoparticles that can cause surface enhanced Raman scattering around the thiol-group-introduced magnetic central particles, the magnetic central particles are washed with ethanol, and then the magnetic central particles are AgNO _3. 0.92 mg was dispersed in 1 ml of dissolved ethylene glycol and stirred at 50 ° C. for 12 hours. Thereafter, the mixture was washed five times with ethanol, and purified. In the final washing, the magnetic core particles into which silver nanoparticles were introduced were precipitated, and the filtrate was discarded with only 100 μl of ethanol. After stirring, the magnetic central particles into which silver nanoparticles were introduced were dispersed in ethanol well. As a result, silver nanoparticles were introduced to the magnetic central particle surface. The method is a kind of polyol method.

1-3-1. Introduced to the magnetic core particle surface Silver nano  Fixation of labeling substance around particles

In order to give the Raman scattering effect to the magnetic nanoparticles into which the silver nanoparticles prepared in Example 1-2 were introduced, benzenethiol, a Raman labeling substance, was fixed. 1 mg of 2 mM benzenethiol was added to 50 mg of the magnetic nanoparticles containing silver nanoparticles as a labeling material and stirred at 25 ° C. for 30 minutes, and benzenethiol was used as a Raman labeling substance around the silver nanoparticles introduced on the magnetic core particle surface. Fixed. After washing three times with ethanol to obtain the magnetic nanoparticles containing silver nanoparticles and benzenethiol immobilized around the silver nanoparticles.

1-3-2. Introduced to the magnetic core particle surface Silver nano  Introduction of polar polymer around particles and fixation of labeling substance

In order to introduce polyvinylpyrrolidone into the polar polymer to facilitate the formation of the silica shell later on the magnetic core particles into which the silver nanoparticles prepared in Example 1-2 were introduced, and to give a Raman scattering effect, Raman marker benzenethiol was fixed. First, 30 ml of 0.5% polyvinylpyrrolidone aqueous solution was added to 50 mg of the magnetic central particles into which silver nanoparticles were introduced, and reacted at 25 ° C. for 25 hours. After washing once with distilled water and ethanol, 1 ml of 2 mM benzenethiol was added as a label, stirred at 25 ° C. for 30 minutes, and washed three times with ethanol. As a result, polar polymers were introduced around the silver nanoparticles introduced on the magnetic central particle surface, and the labeling material was fixed.

1-4. Preparation of Particle Aggregates

The hot spots-derived particle aggregates were prepared by allowing the magnetic core particles, silver nanoparticles, and benzenethiol-containing particles prepared in Example 1-3-1 or Example 1-3-2 to stand and aggregate. The aggregation principle is as described above.

1-5. Formation of Silica Shell around Particle Aggregates

In order to form a silica shell around the particle aggregates prepared in Examples 1-4, the particle aggregates were dispersed in 15 ml of aqueous solution, and then 1.83 ml of 0.025% (v / v) sodium silicate aqueous solution was added thereto, and at 25 ° C. Stir for 12 hours. The obtained particle aggregate was washed three times with water and ethanol, respectively. This resulted in magnetic-surface enhanced Raman scattering particles.

[ Example  2] Biosensor Manufacturing

2-1. Magnetic-Surface Enhancement Raman Scattering  Which is a functional group on the particle surface Amine  Introduction

In order to introduce a functional group having biocompatibility and functionality to the surface of the magnetic-surface enhanced Raman scattering particles prepared in Example 1, 0.1 ml of the magnetic-surface enhanced Raman scattering particles (the magnetic-surface enhanced Raman quantified by ICP analysis) The amount of magnetic material in the scattering particles was 22 mg / ml) to 1 ml of 3-aminopropyltriethoxysilane (APTS) solution (5% by volume in ethanol), followed by 10 µl of ammonium hydroxide. . The resulting dispersion was stirred at 25 ° C. for 12 hours. After washing with ethanol and redispersed in 200 μl of N, N-dimethylformamide (DMF) to obtain a dispersion of magnetic-surface enhanced Raman scattering particles surface-modified with an amine group.

2-2-1. As a receptor  Antibodies HER2 Attachment of

1 ml of the dispersion of the magnetically-surface-enhanced Raman scattering particles surface-modified with the amine group prepared in Example 2-1 (content of the magnetic substance in the dispersion of the magnetic-surface-enhanced Raman scattering particles surface-modified by the amine group as determined by ICP analysis) Silver is 2.2 mg / ml), and 10 μmol of Fmoc-NH-TEG-COOH is added to (benzotriazol-1-yloxy) tris (dimethylamino) phosphonium hexafluorophosphate (BOP), 1-hydroxybenzo 12 mol of triazole (HOBt) and N, N-diisopropyl ethylamine (DIEA) were added respectively. The resulting mixture was stirred at 25 ° C. for 6 hours. After washing with DMF, Fmoc protecting groups were removed by treatment with 500 μl of piperidine (20% by volume in DMF) for 40 minutes. Succinic acid in 1 ml of the dispersion of the magnetic-surface-enhanced Raman scattering particles dispersed in the obtained DMF (the content of the magnetic substance in the magnetic-surface-enhanced Raman scattering particle dispersion dispersed in the DMF as determined by ICP analysis was 2.2 mg / ml). Anhydrous (succinic anhydride) and DIEA were each added 20 μmol, and stirred at room temperature for 6 hours to introduce carboxylic acid functional groups. After washing the surface-modified magnetic surface-enhanced Raman scattering particles with carboxylic acid three times with DMF, and then dispersed in 1 ml DMF, 20 μmol of N-hydroxysuccinimide (NHS), 30 μmol each of DIC and DIEA Stir at room temperature for 3 hours. The carboxyl groups attached to the magnetic-surface enhanced Raman scattering particles were activated, and then dispersed in 1 ml of PBS buffer solution. To this dispersion was added 10 [mu] g of antibody HER2 (SantaCruz Biotechnology, USA) and the resulting mixture (the content of magnetic material in the obtained mixture quantified by ICP analysis was 0.58 mg / ml) at 4 ° C. overnight or at 25 ° C. After stirring for 2 hours, the obtained magnetic-surface enhanced Raman scattering particles to which the antibody HER2 was attached were centrifuged and washed with a PBS solution containing Tween 20 (0.1% by weight). After treatment with bovine serum albumin (bovine serum albumin) (1% by weight in PBS solution) for 30 minutes at 25 ℃, washed several times with PBS solution containing Tween 20 (0.1% by weight), magnetic-surface enhanced Raman A biosensor was prepared in which the antibody HER2 was attached to the scattering particles as a receptor.

2-2-2. The amine functional group As a receptor  Antibodies CD10 Attachment of

In a similar manner to Example 2-2-1, a biosensor was prepared by attaching the antibody CD10 to the magnetic-surface enhanced Raman scattering particles with a receptor.

[ Example  3] Cancer Cell Detection Experiment of Biosensor

Cancer cell detection experiment was performed using the biosensors prepared in Example 2.

3-1. Breast cancer cells ( SKBR3 ) Detection

First, magnetic-surface enhanced Raman scattering particles having 2-naphthalenethiol introduced as a labeling material were prepared in the same manner as in Example 1. Subsequently, a biosensor was prepared by introducing antibody HER2 as a receptor specific for breast cancer cell antigen to magnetic-surface enhanced Raman scattering particles in the same manner as in Example 2-2-1, and the biosensor was combined with breast cancer cells (SKBR3). Incubated at 4 ° C. for 12 h. Then, when the cell was attracted by a bar magnet of 4000 gauss or a round magnet of 3000 gauss, it was confirmed that the cells were attracted in a few seconds, and then the confocal Raman system (Laboam 300, JY-Horiba, Edison, NJ). , USA) and the results of the Raman analysis are shown in FIG. 2.

As shown in Figure 2, the cells combined with the biosensor was confirmed that the Raman signal of 2-naphthalene thiol was generated at the same time as attracted to the magnet.

3-2. Leukemia cells SP2 / O ( floating leukemia cells ) Detection

First, magnetic-surface-enhanced Raman scattering particles into which 4-merctotoluene was introduced as a labeling material were prepared in the same manner as in Example 1. Subsequently, a biosensor was prepared by introducing antibody HER2 or antibody CD10 as a receptor specific for leukemia cells to magnetic-surface enhanced Raman scattering particles in the same manner as in Example 2-2-1 or Example 2-2-2. The biosensor was incubated with leukemia cells at 4 ° C. for 12 hours. After 4,000 gauss of bar magnets, the cells were attracted in seconds, followed by Raman analysis using the confocal Raman system (Laboam 300, JY-Horiba, Edison, NJ, USA). The results are shown in FIG. 3.

As shown in FIG. 3, the cells combined with the biosensors were attracted to the magnet, and the Raman signal of 4-merctotoluene was generated at the same time.

[ Example  4] Non-toxic test of biosensor

Biosensors of 0.144 mg / ml, 0.288 mg / ml, 0.575 mg / ml, 1.15 mg / ml and 2.3 mg / ml were prepared in the same manner as in Examples 1 and 2. Thereafter, the biosensor was incubated with lung cancer cells (A549) to perform an experiment on cell viability. The results are shown in FIG.

As shown in Figure 4, the biosensor according to the present invention was confirmed that there is little toxicity and biocompatibility.

[ Example  5] Silver nano  Magnetic particles ( Ag -M dots Manufacturing

In the same manner as in Examples 1-1 and 1-2, magnetic central particles were prepared, and silver nanoparticles were introduced to the magnetic central particle surfaces. Silver nano magnetic particles were prepared by leaving the magnetic central particles into which silver nano particles were introduced on the surface and agglomerating. The aggregation principle is as described above. The silver nano-magnetic particles prepared as described above are agglomerated by an external magnetic field because the repulsive force of the silica coating surface of the magnetic core particles is canceled in the introduction of the silver nano particles, so that the label-free material of the silver nano particles can be combined with the silver nano particles. It can be used in the detection method.

[ Example  6] by magnetic field Silver nano  Magnetic particles ( Ag -M dots ) And Hotspot  Assay

In order to confirm the controllability by the magnet of the silver nano-magnetic particle movement prepared in Example 5, an experiment using a capillary (capillary) was performed. First, magnetic core particles having silver nanoparticles introduced therein were prepared by the same method as in Examples 1-1 and 1-2. Silver nano magnetic particles were prepared by leaving the magnetic central particles into which silver nano particles were introduced on the surface and agglomerating. The silver nano magnetic particles were placed in a 10 cm long and 0.5 mm diameter capillary tube. Then, the silver nano magnetic particle group was formed using a magnet of 4000 gauss, the formed silver nano magnetic particle group was moved, and the experiment was performed to disperse it by sonication again. Was performed. The results are shown in FIG.

As shown in FIG. 5, it was confirmed that the hot spots can be induced by inducing the formation of the silver nano magnetic particle group by the magnetic field, and at the same time, the movement of the silver nano magnetic particles can be controlled by the magnetic field.

[ Example  7] Silver nano  Magnetic particles Hotspot  Assay

7-1. methyl Parathion  analysis

The silver nanomagnetic particles prepared in Example 5 were used to detect the pesticide methyl parathion, thereby demonstrating that the present invention enables label-free analysis of specific substances.

The silver nano magnetic particles were dispersed in 1 ml of an aqueous solution, and 100 µl of the aqueous solution was aliquoted and placed in a 1 ml aqueous solution containing 1 ppm, 0.1 ppm, and 0.01 ppm of methyl parathion. It was stirred at 25 ° C. for 1 hour and then washed five times with water. Subsequently, a drop of silver nano-magnetic particles forming a self-assembled monolayer by chemical bonding of methyl parathion on a slide glass was dropped, and then agglomerated silver nano-magnetic particles were obtained using a rod magnet of 4000 gauss. Thus prepared hot spot-induced agglomerated silver nano magnetic particles were measured by Raman, and the results are shown in FIG.

As shown in FIG. 6, the Raman signal was well seen when 1 ppm methyl parathion was used. In addition, the silver nano-magnetic particles of the present invention were sensitive to the Raman scattering analysis so that the Raman signal in the case of using 0.001 ppm methyl parathion could also be measured. In other words, the silver nano-magnetic particles according to the present invention showed that even a very low concentration of the material can be analyzed.

7-2. Phenolic Analysis

The silver nano magnetic particles prepared in Example 5 were used to analyze phenols that are known to be harmful and not easy to analyze with Raman.

The silver nano magnetic particles were dispersed in 1 ml of aqueous solution, 100 µl of the aqueous solution was collected, 1 ml of 1 mM phenol was added thereto, and the mixture was stirred at 25 ° C. for 1 hour. Thereafter, a drop of silver nanomagnetic particles containing 1 mM phenol was dropped on the slide glass, and then agglomerated silver nanomagnetic particles were obtained using a 4000 gauss magnet. The hot spot-induced agglomerated silver nanomagnetic particles were measured by Raman. The results are shown in FIG.

As shown in FIG. 7, by analyzing the phenol having no functional group such as a thiol group or an amine group capable of binding to silver using the silver nanomagnetic particles of the present invention, the present invention can be used to analyze a material without a thiol group or an amine group. Showed that there is. That is, the applicability and label-free analysis of the surface-enhanced Raman scattering magnetic nanoparticles according to the present invention were confirmed.

7-3. Adenine analysis

The silver nano magnetic particles prepared in Example 5 were used to detect adenine, a biomaterial, thereby demonstrating that the present invention enables label-free analysis of specific materials.

The silver nano magnetic particles were dispersed in 1 ml of an aqueous solution, and then 100 µl of the aqueous solution was collected and placed in a 1 ml aqueous solution containing 1 pM to 1 µM of adenine. It was stirred at 25 ° C. for 1 hour and then washed five times with water. Thereafter, a drop of silver nanomagnetic particles having adenine self-assembled monolayer on the surface of the slide glass was dropped by one drop, and then agglomerated silver nanomagnetic particles were obtained using a rod magnet of 4000 gauss. Thus prepared hot spot-induced agglomerated silver nano magnetic particles were measured by Raman, and the results are shown in FIG.

As shown in FIG. 8, the Raman signal was well seen when 1 pM adenine was used. In other words, the silver nano-magnetic particles according to the present invention showed that even a very low concentration of the material can be analyzed.

1 is a schematic and TEM image of the structure and manufacturing process of the silver nano-magnetic particles, magnetic-surface enhanced Raman scattering particles and biosensor according to an embodiment of the present invention:

1A is a schematic diagram of a synthesis process of silver nano magnetic particles, magnetic-surface enhanced Raman scattering particles, and a biosensor according to an embodiment of the present invention;

1B is a TEM image of 18 nm magnetite in a magnetic material according to one embodiment of the present invention;

1C is a TEM image of a magnetic central particle according to an embodiment of the present invention;

1D is a TEM image of silver nano magnetic particles according to an embodiment of the present invention;

FIG. 1E is an FE-SEM image of silver nano magnetic particles when the particles of FIG. 1D are dropped onto a thiol group-modified silicon wafer; FIG.

1F is an HR-TEM image of magnetic-surface enhanced Raman scattering particles in accordance with an embodiment of the present invention.

2 is a schematic diagram of a detection process of a target biomaterial using a biosensor according to an embodiment of the present invention:

Figure 2a is a schematic diagram of the reaction of the biosensor with the antigen of the cancer cell according to an embodiment of the present invention;

Figure 2b is an optical microscope image of a biosensor (2-naphthalene thiol as a label, antibody HER2 as a receptor), confocal laser scanning microscope (Confocal) according to an embodiment of the present invention bound to the antigen of human breast cancer cells (SKBR3) Laser Scanning Microscopy (CLSM) image, and Raman data of a biosensor according to an embodiment of the present invention bound to the antigen of the cancer cells;

Figure 2c is an optical microscope image of a breast cancer cell (SKBR3) detection process coupled to the biosensor according to an embodiment of the present invention by an external magnetic field.

3 is a light microscopy image of a biosensor (4-mersutotoluene as a label, antibody CD10 as a receptor) and confocal laser scanning microscope according to an embodiment of the present invention bound to leukemia cells (SP2 / O) antigen Raman data of the biosensor according to an embodiment of the present invention bound to the image, and the antigen of the cancer cells.

4 is a graph showing cell viability for potential cytotoxicity of biosensors according to one embodiment of the present invention in various cell lines.

5 is a view of the control by the magnetic field of the silver nano-magnetic particles experimented in the capillary tube:

FIG. 5A is a diagram showing the formation of the silver nano magnetic particle group by the magnetic field and the movement by the magnetic field of the formed silver nano magnetic particle group; FIG.

Fig. 5B shows (a) silver nano magnetic particles dispersed from the top as a result of experiments in capillaries, (ii) a group of silver nano magnetic particles formed by a magnetic field, (iii) silver nano magnetic particles redispersed by ultrasonic treatment, A diagram showing a silver nano magnetic particle group reformed by a magnetic field;

FIG. 5C is a graph of Raman data of silver nano magnetic particles, and (i) and (ii) surface enhanced Raman scattering peaks of silver nano magnetic particle groups formed by magnetic fields (b. (Ii) and (iii)) and (iii) Peaks (b. (I) and (iii)) of dispersed silver nano magnetic particles.

6 is a surface-enhanced Raman scattering image showing the results of the analysis of the pesticide methyl parathion using silver nano-magnetic particles according to the present invention.

7 is a surface-enhanced Raman scattering image showing the results of analyzing the phenol using silver nano-magnetic particles according to the present invention.

8 is a surface-enhanced Raman scattering image showing the results of analyzing adenine using silver nano-magnetic particles according to the present invention:

8A is a surface enhanced Raman scattering image when adenine 10 pM concentration;

Figure 8b is a surface enhanced Raman scattering image when adenine 1 pM concentration.

9 is a graph showing the Raman peak of the magnetic-surface enhanced Raman scattering particles introduced with various Raman labeling material according to the present invention.

Claims (20)

A magnetic-surface enhanced Raman scattering particle comprising a particle aggregate and a silica shell surrounding the particle aggregate, The particle aggregate is Magnetic central particles comprising a magnetic material; Silver nano particles introduced on the surface of the magnetic central particle; And One or more particles comprising a labeling substance fixed around the silver nanoparticles are formed by aggregation Magnetic-Surface Enhanced Raman Scattering Particles. The method of claim 1, The magnetic core particle is a particle including a magnetic material and a silica coating surrounding the magnetic material. Magnetic-Surface Enhanced Raman Scattering Particles. The method of claim 1, The magnetic material is Co, Mn, Fe, Ni, Gd, MM ' ₂ O ₄ And M _x O _y (M or M ′ = Co, Fe, Ni, Mn, Zn, Gd, Cr, 0 <x ≦ 3, 0 <y ≦ 5). Magnetic-Surface Enhanced Raman Scattering Particles. The method of claim 1, The labeling substance is a chemical substance having a binding force with silver nano particles. Magnetic-Surface Enhanced Raman Scattering Particles. The method of claim 4, wherein The labeling substance is 4-mercotto toluene (4-MT), 3,5-dimethyl benzenethiol (3,5-DMT), thiophenol (TP), 4-amino thiophenol (4-ATP), benzenethiol ( BT), 4-bromo benzenethiol (4-BBT), 2-bromo benzenethiol (2-BBT), 4-isopropyl benzenethiol (4-IBT), 2-naphthalene thiol (2-NT), 3 , 4-dichloro benzenethiol (3,4-DCT), 3,5-dichloro benzenethiol (3,5-DCT), 4-chloro benzenethiol (4-CBT), 2-chloro benzenethiol (2-CBT) , 2-fluoro benzenethiol (2-FBT), 4-fluoro benzenethiol (4-FBT), 4-methoxy benzenethiol (4-MOBT), 3,4-dimethoxy benzenethiol (3,4- DMOBT), 2-mercetopyrimidine (2-MPY), 2-merceto-1-methyl imidazole (2-MMI), 2-merceto-5-methyl benzimidazole (2-MBI), 2- Amino-4- (trifluoromethyl) benzenethiol (2-ATFT), benzyl mertantan (BZMT), benzyl disulfide (BZDSF), 2-amino-4-chloro benzenethiol (2-ACBT), 3-mersanto Benzoic acid (3-MBA), 1-phenyltetrazol-5-thiol (1-PTET), 5-phenyl-1,2,3-triazole-3-thiol (5-PTRT), 2-eye Any one selected from the group consisting of doaniline (2-IAN), phenyl isothiocyanate (PITC), 4-nitrophenyl disulfide (4-NPDSF) and 4-azido-2-bromoacetophenone (ABAPN) Ideal Magnetic-Surface Enhanced Raman Scattering Particles. Introducing silver nanoparticles into the surface of the magnetic central particle including the magnetic material; Fixing a labeling material around the silver nanoparticles; Agglomerating at least one particle including the magnetic central particle, silver nanoparticles introduced into the magnetic central particle, and a labeling substance fixed around the silver nanoparticles to provide a particle aggregate; And A method of manufacturing a magnetic-surface enhanced Raman scattering particle comprising forming a silica shell around the particle aggregate. The method of claim 6, The magnetic core particle is a particle including a magnetic material and a silica coating surrounding the magnetic material. Method for producing magnetic surface-enhanced Raman scattering particles. Magnetic-surface enhanced Raman scattering particles according to claim 1 or 2; Functional groups introduced to the surface of the magnetic-surface enhanced Raman scattering particles; And Biosensor comprising a receptor attached to the functional group. The method of claim 8, The functional group is any one or more selected from the group consisting of an amine group, a carboxyl group and a thiol group Biosensor. The method of claim 8, The receptor is any one or more selected from the group consisting of enzyme substrates, ligands, amino acids, peptides, proteins, nucleic acids, lipids, cofactors, carbohydrates and antibodies. Biosensor. Introducing a functional group to the surface of the magnetic-surface enhanced Raman scattering particles according to claim 1; And A method of manufacturing a biosensor comprising attaching a receptor to the functional group. A method for detecting a target biomaterial that binds to or reacts with a receptor of the biosensor, comprising using the biosensor according to claim 8. The detection method is characterized by using Raman scattering, magnetic field, or Raman scattering and magnetic field. The method of claim 12, The target biomaterial is any one or more selected from the group consisting of enzymes, proteins, nucleic acids, oligosaccharides, peptides, amino acids, cells, cancer cells, cancer stem cells, antigens and aptamers. Detection method. The method of claim 13, The cancer cell is any one of breast cancer cells, lung cancer cells and leukemia cells Detection method. Magnetic central particles comprising a magnetic material; And One or more particles including silver nanoparticles introduced on the magnetic central particle surface are aggregated. Silver nano magnetic particles. The method of claim 15, The magnetic core particle is a particle including a magnetic material and a silica coating surrounding the magnetic material. Silver nano magnetic particles. Introducing silver nanoparticles into the surface of the magnetic central particle including the magnetic material; And A method for producing silver nano-magnetic particles comprising the step of agglomerating one or more magnetic central particles into which the silver nano particles are introduced. The method of claim 17, The magnetic core particle is a particle including a magnetic material and a silica coating surrounding the magnetic material. Method for producing silver nano magnetic particles. A method for detecting a substance capable of binding to silver nanoparticles in the silver nanomagnetic particles, wherein the silver nanomagnetic particles according to claim 15 or 16 are used. The detection method is characterized by using Raman scattering, magnetic field, or Raman scattering and magnetic field. The method of claim 19, The material capable of bonding with the silver nanoparticles is any one of methyl parathion, phenol and adenine. Detection method.

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