KR102561465B1 - Water-soluble Carbon-coated magnetic nanoparticle by pyrene derivatives, its preparation method and its applications for purification of biological samples - Google Patents

Water-soluble Carbon-coated magnetic nanoparticle by pyrene derivatives, its preparation method and its applications for purification of biological samples Download PDF

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KR102561465B1
KR102561465B1 KR1020160164204A KR20160164204A KR102561465B1 KR 102561465 B1 KR102561465 B1 KR 102561465B1 KR 1020160164204 A KR1020160164204 A KR 1020160164204A KR 20160164204 A KR20160164204 A KR 20160164204A KR 102561465 B1 KR102561465 B1 KR 102561465B1
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carbon
hydrophilic
coated magnetic
pyrene
coated
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KR20180064584A (en
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김영규
이승재
최지원
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주식회사 루미맥
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    • H—ELECTRICITY
    • H01—ELECTRIC ELEMENTS
    • H01F—MAGNETS; INDUCTANCES; TRANSFORMERS; SELECTION OF MATERIALS FOR THEIR MAGNETIC PROPERTIES
    • H01F1/00—Magnets or magnetic bodies characterised by the magnetic materials therefor; Selection of materials for their magnetic properties
    • H01F1/0036—Magnets or magnetic bodies characterised by the magnetic materials therefor; Selection of materials for their magnetic properties showing low dimensional magnetism, i.e. spin rearrangements due to a restriction of dimensions, e.g. showing giant magnetoresistivity
    • H01F1/0045—Zero dimensional, e.g. nanoparticles, soft nanoparticles for medical/biological use
    • H01F1/0054—Coated nanoparticles, e.g. nanoparticles coated with organic surfactant
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B82—NANOTECHNOLOGY
    • B82B—NANOSTRUCTURES FORMED BY MANIPULATION OF INDIVIDUAL ATOMS, MOLECULES, OR LIMITED COLLECTIONS OF ATOMS OR MOLECULES AS DISCRETE UNITS; MANUFACTURE OR TREATMENT THEREOF
    • B82B1/00—Nanostructures formed by manipulation of individual atoms or molecules, or limited collections of atoms or molecules as discrete units
    • B82B1/008—Nanostructures not provided for in groups B82B1/001 - B82B1/007

Abstract

본 발명은 생물학적 응용을 위한 탄소가 코팅된 자성나노입자의 표면처리방법, 더욱 구체적으로 친수성 파이렌(pyrene)유도체를 소수성 탄소코팅 자성나노입자 표면에 흡착시킴으로서 우수한 물분산력과 화학적 안정성을 특징으로 하는 수용성 자성나노입자 복합체 및 그 제조방법에 관한 것이다.
본 발명에 따르면, 코팅된 탄소에 파이렌 유도체를 π-π stacking이 이루어지게 한 후 pyrene 유도체에 친수성 기능기 (카르복실기, 아민기 등)가 존재함으로 표면 외곽은 물에 분산이 용이하게 된다. 이 기능기의 존재는 단순히 물 친화력 뿐 아니라 다음 단계인 생화학물질과의 접합에 이용되기에 생명과학, 의학 및 진단학과 같은 분야에서 효과적으로 이용할 수 있다. 구체적인 예로서 Protein A 단백질을 부착시켜서 항체정제용 자성입자를 제조하거나 Ni-NTA(Nickel-Nitrilo acetic acid) 물질을 부착시켜서 His-tag단백질 정제용 자성입를 제조할 수 있다. The present invention is a surface treatment method of carbon-coated magnetic nanoparticles for biological applications, more specifically, by adsorbing a hydrophilic pyrene derivative on the surface of hydrophobic carbon-coated magnetic nanoparticles, characterized by excellent water dispersibility and chemical stability. It relates to a water-soluble magnetic nanoparticle composite and a manufacturing method thereof.
According to the present invention, after the π-π stacking of the pyrene derivative on the coated carbon, the presence of a hydrophilic functional group (carboxyl group, amine group, etc.) in the pyrene derivative makes it easy to disperse in water on the outside of the surface. The existence of this functional group can be used effectively in fields such as life sciences, medicine, and diagnostics because it is used not only for water affinity but also for conjugation with biochemical substances, which is the next step. As a specific example, magnetic particles for antibody purification may be prepared by attaching Protein A protein, or magnetic particles for His-tag protein purification may be prepared by attaching Ni-NTA (Nickel-Nitrilo acetic acid) material.

Description

파이렌 유도체에 의한 수용성 탄소코팅 자성나노입자 복합체, 그 제조방법 및 생물질 분리 정제를 위한 그의 이용 {Water-soluble Carbon-coated magnetic nanoparticle by pyrene derivatives, its preparation method and its applications for purification of biological samples}Water-soluble carbon-coated magnetic nanoparticle by pyrene derivatives, its preparation method and its applications for purification of biological samples}

본 발명은 생물학적 응용을 위한 탄소코팅 자성나노입자의 표면처리를 통한 친수성화, 더욱 구체적으로 친수성 기능기(카르복실기, 아민기 등)가 포함된 파이렌(pyrene)유도체가 탄소표면과 π-π stacking이 이루어지게 한 후 친수성 기능기 (카르복실기, 아민기 등)가 존재함으로 표면 외곽은 물에 분산이 용이하게 되는 것을 특징으로 하는 제조방법이며 이러한 복합체는 단백질 그리고 각종 생화학 물질과 공유결합을 통한 최종 산물화로 이어진다. 구체적인 예로서 Protein A 단백질을 부착시켜서 항체정제용 자성입자를 제조하거나 Ni-NTA(Nickel-Nitrilo acetic acid) 물질을 부착시켜서 His-tag단백질 정제용 자성입를 제조할 수 있다. The present invention is hydrophilicization through surface treatment of carbon-coated magnetic nanoparticles for biological applications, more specifically, pyrene derivatives containing hydrophilic functional groups (carboxyl groups, amine groups, etc.) are stacked with carbon surfaces by π-π After this is made, the presence of a hydrophilic functional group (carboxyl group, amine group, etc.) makes the surface outline easy to disperse in water, and this complex is the final product through covalent bonding with proteins and various biochemical substances leads to anger As a specific example, magnetic particles for antibody purification may be prepared by attaching Protein A protein, or magnetic particles for His-tag protein purification may be prepared by attaching Ni-NTA (Nickel-Nitrilo acetic acid) material.

최초의 자성입자의 생명과학적 응용은 1970년대 John Ugelstad (노르웨이)가 도입하였고, 샘플내 관련물질 분리가 주목적이었다. 주 소재는 거의 대부분 산화철을 이용한 것이고 물분산력을 갖추기 위해 친수성기를 포함하는 폴리머 코팅재가 주 제품이다. 기능기가 부착된 폴리머를 이용하여 각종 생화학물질이 부착된 형태로 상업적으로 이용된다. 최초로 도입했던 John Ugelstad의 DynaBead라는 제품군이 대표적이다. 이러한 제품은 주로 분리 정제 관련 영역뿐 아니라 면역진단기법에 많이 이용되고 있다.The first life science application of magnetic particles was introduced by John Ugelstad (Norway) in the 1970s, and the main purpose was to separate related substances in samples. The main material is almost always iron oxide, and the main product is a polymer coating material containing a hydrophilic group to have water dispersibility. It is commercially used in the form of attaching various biochemical substances by using a polymer attached with a functional group. John Ugelstad's DynaBead, which was introduced for the first time, is a representative product. These products are mainly used in areas related to separation and purification, as well as in immunodiagnostic techniques.

차세대 제품으로서 등장한 것은 카본코팅된 자성소재이다. Turbo Bead라 불리는 이 제품은 탄소코팅에 의하여 강한 자성을 보일뿐 아니라 좋은 화학적 안정도의 특성을 가지고 있다. 중금속제거와 같은 환경적 측면에서의 이용의 예는 있으나 아직 DynaBead와 같은 광범위한 쓰임새는 보고되지 않고 있다. 빛을 이용한 측정방법이 추가된 형태의 다기능성 소재로서 CardioGenics라는 자성소재가 있다. 이는 자성입자 표면에 은을 입힘으로서 빛에 대한 민감도가 현저히 증가된 형태이다.What appeared as a next-generation product was a carbon-coated magnetic material. This product called Turbo Bead not only shows strong magnetism by carbon coating but also has good chemical stability. There are examples of use in environmental aspects such as heavy metal removal, but a wide range of uses such as DynaBead have not been reported yet. There is a magnetic material called CardioGenics as a multifunctional material in which a measurement method using light is added. This is a form in which the sensitivity to light is remarkably increased by coating the surface of the magnetic particle with silver.

산화철 자성나노입자의 경우 한국화학연구원에서 개발한 초음파 조사를 이용한 제조기법(한국특허등록: 10-1350400)을 이용하여 고품질의 산화철 나노소재를 저가에 대량으로 제조할 수 있는 기술이 중요 장점이다. 더 나아가 기존제품의 코팅재인 실리카보다 상대적으로 저가이며 용이한 탄소코팅 기법(국내특허등록:10-1335520)의 추가는 성능과 생산비 절감차원에서 월등히 앞선 기술이다.In the case of magnetic iron oxide nanoparticles, a technology that can mass-produce high-quality iron oxide nanomaterials at low cost using a manufacturing technique using ultrasonic irradiation developed by the Korea Research Institute of Chemical Technology (Korean Patent Registration: 10-1350400) is an important advantage. Furthermore, the addition of carbon coating technique (domestic patent registration: 10-1335520), which is relatively inexpensive and easy to use compared to silica, which is a coating material of existing products, is a far more advanced technology in terms of performance and production cost reduction.

철-코발트 자성입자의 경우 그 소재의 특성인 우수한 자기력 때문에 기존 산화철 제품군보다 성능 면에서 우위에 있다. 위에서 언급한 바와 같은 초음파 조사와 탄소코팅 기법(국내특허등록:10-1335520)을 이용하여 제조가 가능하다.In the case of iron-cobalt magnetic particles, they are superior to existing iron oxide products in terms of performance because of the excellent magnetic force, which is a characteristic of the material. As mentioned above, it can be manufactured using ultrasonic irradiation and carbon coating technique (domestic patent registration: 10-1335520).

그럼에도 불구하고, 이러한 탄소코팅 자성나노입자의 생물학적 응용을 위한 표면처리방법 개발이 미흡하며, 탄소코팅 자체의 소수성적 측면 때문에 수용액상의 바이오시료의 직접적 이용이 이루어지지 않고 있다.Nevertheless, the development of surface treatment methods for biological applications of these carbon-coated magnetic nanoparticles is insufficient, and the direct use of biosamples in an aqueous solution is not achieved due to the hydrophobic aspect of the carbon coating itself.

이에, 본 발명자들은 상기 문제점들을 극복하기 위하여 예의 연구노력한 결과, 탄소코팅 자성나노소재에 파이렌 유도체를 반응시킴으로서 파이렌과 코팅된 탄소의 강한 결합력에 파이렌에서 연결된 친수성 기능기에 의한 수용액상의 안정성을 부여함으로 강한 자성과 화학적 안정성 그리고 바이오 응용능력을 갖춘 복합체를 형성시키는 것을 확인하고, 본 발명을 완성하게 되었다. Accordingly, as a result of intensive research efforts to overcome the above problems, the inventors of the present invention reacted the pyrene derivative with the carbon-coated magnetic nanomaterial, thereby improving the stability of the aqueous phase by the hydrophilic functional group connected from the pyrene to the strong bonding force between the pyrene and the coated carbon. By imparting, it was confirmed that a complex having strong magnetism, chemical stability, and bioapplication ability was formed, and the present invention was completed.

USUS 2012020802620120208026 A1A1 KRKR 10-133552010-1335520 BB

탄소가 코팅된 자성나노입자의 제조방법은 금속전구체와 탄소가 포함된 용매를 사용하여 화학적으로 불안정한 자성나노입자의 표면에 탄소 코팅층을 형성시켜, 개별 자성나노입자의 자기적 특성을 감소시키지 않으면서 화학적, 열적 안정성이 향상된 나노영역의 균일한 크기를 가지는 자성나노입자를 대량으로 생산하는 것이 최종결과이다. 이 경우 금속 전구체 용액 제조시 탄소를 포함하는 용매를 이용함으로서 금속전구체 용액 제조를 위한 역할 뿐 아니라 탄소를 코팅하기위한 원료물질로서의 역할도 수행한다. 따라서 탄소코팅을 위한 별도의 재료를 분산 내지 첨가할 필요가 없다는 장점을 가진다. 이후 초음파조사와 석출, 열처리의 과정을 거쳐 최종 산물을 얻게 된다.A method for producing carbon-coated magnetic nanoparticles uses a metal precursor and a carbon-containing solvent to form a carbon coating layer on the surface of chemically unstable magnetic nanoparticles, without reducing the magnetic properties of individual magnetic nanoparticles. The final result is the mass production of magnetic nanoparticles having a uniform size in the nanodomain with improved chemical and thermal stability. In this case, by using a solvent containing carbon when preparing the metal precursor solution, it serves not only for preparing the metal precursor solution but also as a raw material for coating carbon. Therefore, it has the advantage that there is no need to disperse or add a separate material for carbon coating. After that, the final product is obtained through the process of ultrasonic irradiation, precipitation, and heat treatment.

준비된 탄소코팅 나노소재가 바이오분야에 이용되기 위하여, 탄소표면위에 친수성 표면개질이 필요하고 기능기의 부착으로 단백질이나 각종 생화학 물질이 부착되도록 하는 것이 해결해야 되는 과제이다. 추가적으로 독성문제가 없고, 제조가 간편하며 대량생산이 가능해야 되는 점 역시 주요 목적이다.In order for the prepared carbon-coated nanomaterial to be used in the bio field, hydrophilic surface modification is required on the carbon surface, and proteins or various biochemical substances are attached by attaching functional groups. In addition, it is also a major purpose that there is no toxicity problem, manufacturing is simple, and mass production is possible.

본 발명의 한 양태에 따르면, 본 발명은 친수성 기능기를 포함하는 파이렌 유도체를 이용하여 파이렌과 자성나노입자의 표면의 탄소층과의 강한 결합을 형성시켜 파이렌 유도체-탄소코팅 자성나노입자의 복합체를 제공한다.According to one aspect of the present invention, the present invention uses a pyrene derivative containing a hydrophilic functional group to form a strong bond between pyrene and the carbon layer on the surface of the magnetic nanoparticles, thereby forming the pyrene derivative-carbon-coated magnetic nanoparticles. provides a complex.

본 발명에 있어서, 상기 친수성 기능기를 포함하는 파이렌 유도체는 친수성을 나타내는 어떤 화합물도 가능하나 바람직하게는 카르복실기(carboxyl group)나 아민기(amine group)를 기능기로 가지고 있는 pyrene butylic acid나 aminopyrene인 것을 특징으로 하는 파이렌 유도체-탄소코팅 자성나노입자의 복합체를 제공한다. 이러한 친수성 기능기들은 수용성 (생체적합성)형태의 나노입자들로 전환된다. In the present invention, the pyrene derivative containing a hydrophilic functional group can be any compound exhibiting hydrophilicity, but is preferably pyrene butylic acid or aminopyrene having a carboxyl group or an amine group as a functional group. A pyrene derivative-carbon-coated magnetic nanoparticle complex is provided. These hydrophilic functional groups are converted into water-soluble (biocompatible) nanoparticles.

본 발명에 있어서, 탄소코팅 나노입자에 직접 항체나 단백질과 같은 생화학 물질을 부착하는 것이 아니라, 탄소코팅 위에 수용성을 부여하며 이러한 물질들과 공유결합 반응성이 유도되는 파이렌 유도체를 이용한 중간매개복합체를 만든 후 생화학 물질을 크로스링커(crosslinker)를 이용하여 부착시키는 것을 특징으로 하는 생화학 물질-파이렌 유도체-탄소코팅 자성나노입자 복합체를 형성한다.In the present invention, rather than directly attaching biochemical substances such as antibodies or proteins to carbon-coated nanoparticles, an intermediate complex using a pyrene derivative that imparts water solubility on the carbon coating and induces covalent bond reactivity with these substances After making, a biochemical material-pyrene derivative-carbon-coated magnetic nanoparticle complex is formed, characterized in that the biochemical material is attached using a crosslinker.

본 발명에 있어서, 바람직하게는 상기 친수성 파이렌 유도체로 코팅된 탄소코팅 자성나노입자의 표면 친수성 화학적 기능기에 단백질A가 공유결합으로 연결되어, 항체를 분리 정제하는데 사용된는 것을 특징으로 하는 파이렌-탄소코팅 자성나노입자 복합체를 제공한다.In the present invention, preferably, protein A is covalently linked to a hydrophilic chemical functional group on the surface of the carbon-coated magnetic nanoparticles coated with the hydrophilic pyrene derivative, and is used to separate and purify antibodies. Provided is a carbon-coated magnetic nanoparticle composite.

구체적으로, 항체의 분리 정제를 위하여 Protein A가 부착된 자성입자의 제조는 1-Pyrenebutylic Acid로 표면처리된 자성입자에 1-Ethyl-3-[3-dimethylaminopropyl] carbodiimide (EDC) Crosslinker를 이용하여 Protein A의 일차 아민기와 자성입자 표면의 카르복실기와의 공유결합을 유도하여 제조한다.Specifically, for the separation and purification of antibodies, the production of magnetic particles to which Protein A is attached is performed by using 1-Ethyl-3-[3-dimethylaminopropyl] carbodiimide (EDC) crosslinker on magnetic particles surface-treated with 1-pyrenebutylic acid. It is prepared by inducing a covalent bond between the primary amine group of A and the carboxyl group on the surface of magnetic particles.

본 발명에 있어서, 상기 친수성 파이렌 유도체로 코팅된 탄소코팅 자성나노입자의 표면 친수성 화학적 기능기에 NTA(Nickel-Nitrilo acetic acid)가 공유결합으로 연결되어, Ni 이온을 매개로 His-tag 단백질을 분리 정제하는데 사용된는 것을 특징으로 하는 파이렌-탄소코팅 자성나노입자 복합체를 제공한다.In the present invention, NTA (Nickel-Nitrilo acetic acid) is covalently linked to the hydrophilic chemical functional group on the surface of the carbon-coated magnetic nanoparticles coated with the hydrophilic pyrene derivative to separate His-tag proteins through Ni ions. It provides a pyrene-carbon coated magnetic nanoparticle complex, characterized in that it is used for purification.

구체적으로, His-tag 단백질의 분리 정제를 위한 Ni-NTA(nitriloacetic acid)의 부착은 EDC를 이용하여 amino-NTA의 일차 아민가와 자성입자 표면의 카르복실기간의 공유결합을 유도하여 제조하고 후에 니켈이온을 충진하여 이용한다.Specifically, the attachment of Ni-NTA (nitriloacetic acid) for separation and purification of His-tag protein is prepared by inducing covalent bonding between the primary amine value of amino-NTA and the carboxyl group on the surface of magnetic particles using EDC, and then nickel ions are added. Fill up and use.

본 발명의 다른 양태에 따르면, 다음 단계를 포함하는 제1항의 친수성 파이렌-탄소코팅 자성나노입자 복합체의 제조방법을 제공한다:According to another aspect of the present invention, there is provided a method for preparing the hydrophilic pyrene-carbon-coated magnetic nanoparticle composite of claim 1, comprising the following steps:

(a) 탄소코팅 자성나노입자에 친수성 파이렌을 코팅하는 단계;(a) coating hydrophilic pyrene on carbon-coated magnetic nanoparticles;

(b) 상기 친수성 파이렌으로 코팅된 탄소코팅 자성나노입자에 단백질 A 또는 NTA(Nickel-Nitrilo acetic acid)를 결합시키는 단계. (b) binding protein A or nickel-nitrilo acetic acid (NTA) to the carbon-coated magnetic nanoparticles coated with hydrophilic pyrene.

본 발명에 따르면, 파이렌 유도체 코팅된 탄소코팅 자성나노입자는 수용성이며 상기 나노입자들을 바이오 용도에 사용될 수 있도록, 그들을 생리학적 조건(인산-버퍼 염수, PBS, pH 7.2-7.4)에서 수개월간 4℃ 냉장고에서 어떤 물리화학적 특성도 잃지 않고 매우 안정하다. 또한, 상기 나노입자들은 동결건조될 수 있고 어떤 특성들을 유의하게 잃지 않고 생리학적 버퍼의 첨가시 재수화될 수 있다.According to the present invention, carbon-coated magnetic nanoparticles coated with pyrene derivatives are water-soluble, and in order to use the nanoparticles for bio-use, they are stored in physiological conditions (phosphate-buffered saline, PBS, pH 7.2-7.4) for several months. It is very stable in the refrigerator without losing any physicochemical properties. Additionally, the nanoparticles can be lyophilized and rehydrated upon addition of a physiological buffer without significant loss of any properties.

본 발명에 따른 파이렌 유도체-탄소코팅 자성나노입자의 제조방법은 무독성의 금속전구체와 탄소용매의 이용을 통하여 불안정한 자성나노입자의 표면에 안정한 탄소 코팅층이 형성된 탄소코팅 자성나노입자 제조후 생물학적인 응용을 위하여 탄소 코팅층 위에 새로운 친수성 코팅을 형성시키는 것이다.The method for producing pyrene derivative-carbon-coated magnetic nanoparticles according to the present invention is applied to biological applications after preparing carbon-coated magnetic nanoparticles having a stable carbon coating layer formed on the surface of unstable magnetic nanoparticles through the use of a non-toxic metal precursor and a carbon solvent. To this end, a new hydrophilic coating is formed on the carbon coating layer.

이러한 친수성 파이렌 유도체-탄소코팅 자성나노입자는 개별 자성나노입자의 자기적 특성을 유지시키면서 화학적, 물리적 안정성, 그리고 수용액상 적용적합성을 가진다. 또한 이 친수성 기능기는 탄소 코팅층에 생리활성물질과 같은 생화학 물질들과 결합이 가능한 화학적 기능기로서, 여러 다른 형태의 표면개질이 가능하여 생화학, 온열치료 및 진단등의 많은 영역에 응용하여 사용할 수 있다.These hydrophilic pyrene derivative-carbon coated magnetic nanoparticles maintain the magnetic properties of individual magnetic nanoparticles while maintaining chemical and physical stability and suitability for use in aqueous solution. In addition, this hydrophilic functional group is a chemical functional group capable of bonding with biochemical substances such as bioactive substances in the carbon coating layer, and various types of surface modification are possible, so it can be applied and used in many areas such as biochemistry, thermotherapy and diagnosis. .

도 1은 본 발명의 실시형태에 따른 친수성 파이렌 유도체-탄소코팅 자성나노입자 복합체의 형성에서 결합 유도체인 EDC를 통한 NTA 결합과 니켈 이온의 충진을 통한 his-tag 단백질 분리, 정제용 최종산물 제조방법을 나타내는 개략도이다.
도2 는 실시예 1에서 친수성 파이렌 유도체-탄소코팅 자성나노입자 복합체 제조에 사용된 탄소코팅 자성나노입자의 투과전자현미경 사진이다.
도3 는 실시예 1에서 친수성 파이렌 유도체-탄소코팅 자성나노입자 복합체 제조에 사용된 탄소코팅 자성나노입자의 X-선 회절분석 결과이다.
도4 는 실시예 1에서 친수성 파이렌 유도체-탄소코팅 자성나노입자 복합체 제조에 사용된 탄소코팅 자성나노입자와 일반적인 자성입자인 Fe2O3dp 대한 자기 이력 곡선 (magnetic hystersis curves)결과이다.
도5 는 실시예 1에서 친수성 파이렌 유도체-탄소코팅 자성나노입자 복합체 제조 과정에 대한 결과이다.
도6는 클로로포름 용매에 있는 탄소코팅된 자성나노입자(왼쪽)사진과 이를 수용액에 용해되어있는 pyrene butylic acid과 코팅반응을 진행한 친수성 파이렌 유도체-탄소코팅 자성나노입자 복합체(오른쪽) 사진이다.
도7은 친수성 파이렌 유도체-탄소코팅 자성나노입자 복합체와 생리활성물질 중 Horse Radish Peroxidase (HRP) 효소를 반응시켜 HRP-친수성 파이렌 유도체-탄소코팅 자성나노입자 최종복합체의 효소활성테스트(TMB Test)의 결과이다. TMB를 첨가후 효소활성에 따른 색변화테스트로서 친수성 파이렌 유도체-탄소코팅 자성나노입자 복합체(왼쪽)반응사진과 HRP-친수성 파이렌 유도체-탄소코팅 자성나노입자 최종복합체(오른쪽)반응 사진이다.
도8 는 실시예 2에서 니트릴로트리아세트산 (NTA)이 결합된 탄소코팅 자성나노입자 복합체에 Ni을 배위시켜 E. Coli RNAP alpha subunit 분리 실험 후 상층액에 남은 E. Coli RNAP alpha subunit의 280nm에서의 흡수 측정표와 그래프이다. 재현성 실험을 위하여 7번 반복 실험을 진행하였다.
도9 는 Ni이 배위된 탄소코팅 자성나노입자에 분리된 E. Coli RNAP alpha subunit을 250mM Imidazole 용액으로 용출하여 E. Coli RNAP alpha subunit를 확인하기 위하여 사용한SDS-PAGE 사진이다.
도10는 항체의 분리정제를 위하여 ProteinA-친수성 파이렌 유도체-탄소코팅 자성나노입자 복합체와 human Immunoglobulin G (항체)를 반응시켜 human Immunogloculin G-ProteinA-친수성 파이렌 유도체-탄소코팅 자성나노입자 최종복합체의 생성양에 관련된 도표이다. ProteinA-친수성 파이렌 유도체-탄소코팅 나노입자 복합체에 1mg/ml 농도의 human Immunoglobulin G 200μl를 첨가하고 반응이 끝난 후 첨가했던 용액을 다시 회수하여 280nm 에서의 흡광도를 측정한 결과이다. 좌측에 측정된 280nm 의 흡광도는 처음 첨가한 용액과 회수한 용액의 흡광도이고 우측에 측정된 280nm 의 흡광도는 이를 기반으로 human Immunoglobulin G-ProteinA-친수성 파이렌 유도체-탄소코팅 자성나노입자 최종복합체를 형성한 human Immunoglobulin G 의 예상 흡광도이다. 우측의 농도, 무게의 값은 비어-람버트 법칙을 사용하여 도출하였다. 1 is a graph showing the formation of a hydrophilic pyrene derivative-carbon-coated magnetic nanoparticle complex according to an embodiment of the present invention, NTA binding through EDC, a binding derivative, and separation of his-tag protein through filling of nickel ions, and preparation of final products for purification. It is a schematic diagram showing the method.
2 is a transmission electron micrograph of carbon-coated magnetic nanoparticles used in preparing the hydrophilic pyrene derivative-carbon-coated magnetic nanoparticle composite in Example 1.
3 is a result of X-ray diffraction analysis of carbon-coated magnetic nanoparticles used in preparing the hydrophilic pyrene derivative-carbon-coated magnetic nanoparticle complex in Example 1.
4 is a result of magnetic hysteresis curves for the carbon-coated magnetic nanoparticles used in the preparation of the hydrophilic pyrene derivative-carbon-coated magnetic nanoparticle composite in Example 1 and Fe2O3dp, which is a general magnetic particle.
5 is a result of the manufacturing process of the hydrophilic pyrene derivative-carbon-coated magnetic nanoparticle composite in Example 1.
6 is a photograph of carbon-coated magnetic nanoparticles (left) in a chloroform solvent and a photograph of a hydrophilic pyrene derivative-carbon-coated magnetic nanoparticle complex (right) obtained through a coating reaction with pyrene butylic acid dissolved in an aqueous solution.
7 is an enzyme activity test (TMB Test) of the final HRP-hydrophilic pyrene derivative-carbon coated magnetic nanoparticle complex by reacting the hydrophilic pyrene derivative-carbon coated magnetic nanoparticle complex with the Horse Radish Peroxidase (HRP) enzyme among physiologically active substances. ) is the result of As a color change test according to enzyme activity after adding TMB, these are the reaction pictures of the hydrophilic pyrene derivative-carbon-coated magnetic nanoparticle complex (left) and the final HRP-hydrophilic pyrene derivative-carbon-coated magnetic nanoparticle complex (right) reaction picture.
Figure 8 shows the E. Coli RNAP alpha subunit remaining in the supernatant after the E. Coli RNAP alpha subunit separation experiment at 280 nm by coordinating Ni to the nitrilotriacetic acid (NTA)-coupled carbon-coated magnetic nanoparticle complex in Example 2. It is an absorption measurement table and graph. For the reproducibility test, the experiment was repeated 7 times.
9 is an SDS-PAGE image used to confirm the E. Coli RNAP alpha subunit by eluting the E. Coli RNAP alpha subunit separated from the Ni-coordinated carbon-coated magnetic nanoparticles with a 250 mM Imidazole solution.
Figure 10 shows the final complex of human Immunoglobulin G-ProteinA-hydrophilic pyrene derivative-carbon-coated magnetic nanoparticles by reacting ProteinA-hydrophilic pyrene derivative-carbon-coated magnetic nanoparticle complex with human Immunoglobulin G (antibody) for separation and purification of antibodies It is a diagram related to the amount of production of This is the result of measuring the absorbance at 280 nm by adding 200 μl of human Immunoglobulin G at a concentration of 1 mg/ml to the ProteinA-hydrophilic pyrene derivative-carbon-coated nanoparticle complex, and recovering the added solution after the reaction was finished. The absorbance at 280nm measured on the left is the absorbance of the first added solution and the recovered solution, and the absorbance at 280nm measured on the right is based on this to form the final complex of human Immunoglobulin G-ProteinA-hydrophilic pyrene derivative-carbon-coated magnetic nanoparticles. It is the expected absorbance of one human Immunoglobulin G. The concentration and weight values on the right side were derived using the Beer-Lambert law.

이하, 실시예를 통하여 본 발명을 더욱 상세히 설명하기로 한다. 이들 실시예는 단지 본 발명을 예시하기 위한 것이므로, 본 발명의 범위가 이들 실시예에 의해 제한되는 것으로 해석되지는 않는다.Hereinafter, the present invention will be described in more detail through examples. Since these examples are intended to illustrate the present invention only, the scope of the present invention is not to be construed as being limited by these examples.

<실시예 1> 친수성 파이렌 유도체-탄소코팅 자성나노입자 복합체의 제조<Example 1> Preparation of a hydrophilic pyrene derivative-carbon-coated magnetic nanoparticle composite

단계 1: 탄소코팅 자성나노입자 20mg을 1ml 클로로포름 유기용매에 섞는다. Step 1: Mix 20mg of carbon-coated magnetic nanoparticles with 1ml of chloroform organic solvent.

단계 2: 같은 부피의 1M NaOH에 녹인 50mM 파이렌 부틸릭산을 3ml 첨가한 후 탄소코팅 자성나노입자와 반응을 상온에서 1시간 혼합기(Shaker)를 이용하여 섞어준다.Step 2: After adding 3ml of 50mM pyrene butyric acid dissolved in 1M NaOH in the same volume, the carbon-coated magnetic nanoparticles and the reaction were mixed at room temperature using a shaker for 1 hour.

단계 3: 반응이 완료 한 후 자석을 이용하여 파이렌 유도체-탄소코팅 자성나노입자 복합체를 분리하고 증류수로 세척과정을 거친다.Step 3: After the reaction is completed, the pyrene derivative-carbon-coated magnetic nanoparticle complex is separated using a magnet and washed with distilled water.

단계 4: 세척과정을 거친 파이렌 유도체-탄소코팅 자성나노입자 복합체는 증류수를 첨가하여 냉장 보관한다.Step 4: After washing, the pyrene derivative-carbon-coated magnetic nanoparticle complex is refrigerated after adding distilled water.

상기 단계 2에 의하여 유기용매층에 존재하는 탄소코팅 자성나노입자가 수용액층으로 분리되며 이는 파이렌 유도체-탄소코팅 자성나노입자 복합체가 친수성을 가짐을 의미한다. In step 2, the carbon-coated magnetic nanoparticles present in the organic solvent layer are separated into the aqueous solution layer, which means that the pyrene derivative-carbon-coated magnetic nanoparticle composite has hydrophilicity.

<실시예 2> HRP(Horse Radish Peroxidase)-친수성 파이렌 유도체-탄소코팅 자성나노입자 복합체의 제조<Example 2> Preparation of HRP (Horse Radish Peroxidase)-hydrophilic pyrene derivative-carbon-coated magnetic nanoparticle complex

단계 1: 인산 버퍼 염수에 보관된 친수성 파이렌 유도체-탄소코팅 자성나노입자 복합체 (2mg 자성입자/ml 인산버퍼 염수)에서 0.5 ml의 슬러리를 준비한다.Step 1: Prepare a slurry of 0.5 ml from the hydrophilic pyrene derivative-carbon coated magnetic nanoparticle complex (2mg magnetic particles/ml phosphate buffered saline) stored in phosphate buffered saline.

단계 2: 140 ug의 1-Ethyl-3-[3-dimethylaminopropyl] carbodiimide와 300 ug의 Sulfo NHS를 100 ul의 인산버퍼염수에 용해 시킨 후 상기 단계 1의 슬러리에서 상층액을 제거한 친수성 파이렌 유도체-탄소코팅 자성나노입자 복합체와 보텍스 믹서로 10분간 반응시킨 다.Step 2: Hydrophilic pyrene derivative obtained by dissolving 140 ug of 1-Ethyl-3-[3-dimethylaminopropyl] carbodiimide and 300 ug of Sulfo NHS in 100 ul of phosphate buffered saline and removing the supernatant from the slurry of step 1- The carbon-coated magnetic nanoparticle composite was reacted with a vortex mixer for 10 minutes.

단계 3: 상층액을 제거한 후 인산버퍼 염수로 두 차례 세척한다.Step 3: After removing the supernatant, wash twice with phosphate buffered saline.

단계 4: 100 ul의 인산 버퍼염수에 넣어서 냉장고에 보관한다.Step 4: Put in 100 ul of phosphate buffered saline and store in the refrigerator.

단계 5: HRP 접합을 테스트하기 위하여 100 ul의 Tetramethylbenzidine (TMB) 용액을 HRP 접합 전과 후의 샘플 1 ul 슬러리에 반응시킨다. Step 5: To test HRP bonding, 100 ul of Tetramethylbenzidine (TMB) solution was reacted with 1 ul slurry of samples before and after HRP bonding.

<실시예 3> 니트릴로트리아세트산 (NTA) 결합된 자성나노입자 복합체의 제조와 단백질 분리, 정제 실험<Example 3> Preparation of Nitrilotriacetic Acid (NTA) Coupled Magnetic Nanoparticle Complex and Protein Separation and Purification Test

실시예 1에서 제조 한 파이렌 유도체-탄소코팅 자성나노입자 복합체를 사용한다.The pyrene derivative-carbon-coated magnetic nanoparticle composite prepared in Example 1 was used.

단계 1: 파이렌 유도체-탄소코팅 자성나노입자 복합체 2mg을 튜브에 넣고 자석을 이용하여 200μl의 0.1M MES buffer (pH 4.6)를 사용하여 3회 세척과정을 거친다.Step 1: Put 2 mg of the pyrene derivative-carbon-coated magnetic nanoparticle complex into a tube and wash it three times using a magnet and 200 μl of 0.1M MES buffer (pH 4.6).

단계 2: 튜브에 15.3mg의 1-Ethyl-3-[3-dimethylaminopropyl] carbodiimide (EDC)를 200μl 0.1M MES buffer (pH 4.6)에 녹여서 준비한다.Step 2: Prepare by dissolving 15.3mg of 1-Ethyl-3-[3-dimethylaminopropyl] carbodiimide (EDC) in 200μl 0.1M MES buffer (pH 4.6) in a tube.

단계 3: 0.1M MES buffer (pH 4.6)를 사용하여 50mM Na,Na-Bis(carboxymethyl)-L- lysine hydrate (NTA) 용액을 제조하여 준비한다.Step 3: Prepare a 50 mM Na,Na-Bis(carboxymethyl)-L-lysine hydrate (NTA) solution using 0.1M MES buffer (pH 4.6).

단계 4: 세척이 완료된 파이렌 유도체-탄소코팅 자성나노입자 복합체의 용액을 자석을 이용하여 제거한 후 200μl의 1-Ethyl-3-[3-dimethylaminopropyl] carbodiimide (EDC)를 첨가하여 10분 동안 상온에서 혼합기(Shaker)를 이용하여 섞어준다. Step 4: After removing the solution of the pyrene derivative-carbon-coated magnetic nanoparticle complex that has been washed using a magnet, 200 μl of 1-Ethyl-3-[3-dimethylaminopropyl] carbodiimide (EDC) is added and left at room temperature for 10 minutes. Mix using a shaker.

단계 5: 반응이 완료 된 후 200μl 0.1M MES buffer (pH 4.6)를 사용하여 3회 세척 후 용액을 제거한다(잔여 EDC 제거).Step 5: After the reaction is complete, wash 3 times with 200μl 0.1M MES buffer (pH 4.6) and remove the solution (remove residual EDC).

단계 6: 세척 완료된 EDC-파이렌 유도체-탄소코팅 자성나노입자 복합체에 100μl 50mM a,Na-Bis(carboxymethyl)-L- lysine hydrate (NTA) 용액을 첨가하여 1시간 동안 상온에서 혼합기(Shaker)를 이용하여 섞어준다.Step 6: Add 100μl 50mM a,Na-Bis(carboxymethyl)-L-lysine hydrate (NTA) solution to the washed EDC-pyrene derivative-carbon-coated magnetic nanoparticle complex and shaker at room temperature for 1 hour. Mix using

단계 7: 반응 완료 후 증류수로 3회 세척하여 준비한다.Step 7: After completion of the reaction, prepare by washing with distilled water three times.

단계 8: 세척 한 자성나노입자 복합체에 500μl 0.1M NiSO4 용액을 첨가한 후 10분 동안 상온에서 혼합기(Shaker)를 이용하여 섞어준다. Step 8: After adding 500 μl 0.1M NiSO 4 solution to the washed magnetic nanoparticle composite, it was mixed for 10 minutes using a shaker at room temperature.

단계 9: 혼합 후 500μl Ni binding buffer로 3회 세척하여 준비한다.Step 9: After mixing, prepare by washing three times with 500 μl Ni binding buffer.

단계 10: 100μl E. Coli RNAP alpha subunit 용액을 첨가 한 후 30 ~ 60분 동안 상온에서 혼합기(Shaker)를 이용하여 섞어준다. Step 10: After adding 100 μl E. Coli RNAP alpha subunit solution, mix using a shaker at room temperature for 30 to 60 minutes.

단계 11: 반응 완료 후 자석을 이용하여 상층액을 제거하고 Ni binding buffer를 사용하여 3회 세척한다. 상층액은 280nm의 흡수를 측정하여 남은 단백질의 양을 확인한다. Step 11: After completion of the reaction, the supernatant is removed using a magnet and washed three times using Ni binding buffer. The supernatant is measured for absorption at 280 nm to determine the amount of remaining protein.

단계 12: 100μl 250mM imidazole이 첨가 된 Ni binding buffer를 사용하여 Ni에 붙어 있는 E. Coli RNAP alpha subunit을 분리한다.Step 12: Use Ni binding buffer with 100μl 250mM imidazole to separate the E. coli RNAP alpha subunit attached to Ni.

단계 13: 분리 된 E. Coli RNAP alpha subunit을 SDS-PAGE를 이용하여 확인한다.Step 13: Confirm the isolated E. Coli RNAP alpha subunit by SDS-PAGE.

<실시예 4> 항체정제를 위한 ProteinA-친수성 파이렌 유도체-탄소코팅 자성나노입자 복합체의 제조<Example 4> Preparation of ProteinA-hydrophilic pyrene derivative-carbon-coated magnetic nanoparticle complex for antibody purification

단계 1: 인산-버퍼 염수(PBS, pH 7.2-7.4)에 보관된 친수성 파이렌 유도체-탄소코팅 자성나노입자 복합체 (10mg 자성입자/200μl 인산-버퍼 염수)에서 40μl의 슬러리를 준비한다.Step 1: Prepare a slurry of 40 μl from the hydrophilic pyrene derivative-carbon-coated magnetic nanoparticle complex (10 mg magnetic particles/200 μl phosphoric acid-buffered saline) stored in phosphoric acid-buffered saline (PBS, pH 7.2-7.4).

단계 2: 자석이 있는 튜브거치대를 이용하여 친수성 파이렌 유도체-탄소코팅 자성나노입자 복합체에서 인산-버퍼 염수를 제거 하고, 0.1M 2-(N-morpholino)ethanesulfonic acid (pH :4.6), 0.5M NaCl 200μl 를 이용하여 두 차례 세척한다.Step 2: Remove the phosphoric acid-buffer brine from the hydrophilic pyrene derivative-carbon coated magnetic nanoparticle complex using a tube holder with a magnet, and add 0.1M 2-(N-morpholino)ethanesulfonic acid (pH:4.6), 0.5M Wash twice with 200 μl of NaCl.

단계 3: 15.3 ug의 1-Ethyl-3-[3-dimethylaminopropyl] carbodiimide 를 200 ul의 0.1M 2-(N-morpholino)ethanesulfonic acid (pH :4.6), 0.5M NaCl 에 용해 시킨 후 상기 단계 2의 세척을 마친 슬러리에서 상층액을 제거한 친수성 파이렌 유도체-탄소코팅 자성나노입자 복합체와 보텍스 믹서로 상온에서 10분간 반응시킨다.Step 3: After dissolving 15.3 ug of 1-Ethyl-3-[3-dimethylaminopropyl] carbodiimide in 200 ul of 0.1M 2-(N-morpholino)ethanesulfonic acid (pH:4.6), 0.5M NaCl, The supernatant from the washed slurry is reacted with the hydrophilic pyrene derivative-carbon-coated magnetic nanoparticle complex at room temperature for 10 minutes with a vortex mixer.

단계 4: 상층액을 제거한 후 인산-버퍼 염수로 두 차례 세척한다.Step 4: After removing the supernatant, wash twice with phosphoric acid-buffered saline.

단계 5: 200μl 인산 버퍼 염수를 넣어서 ProteinA-친수성 파이렌 유도체-탄소코팅 자성나노입자 복합체 (10mg 자성입자/200μl 인산버퍼 염수) 슬러리를 준비한다.Step 5: Prepare ProteinA-hydrophilic pyrene derivative-carbon-coated magnetic nanoparticle complex (10mg magnetic particles/200μl phosphate buffered saline) slurry by adding 200μl phosphate buffer saline.

단계 6: 상기 단계 5의 슬러리의 상층액을 제거한다.Step 6: The supernatant of the slurry of step 5 is removed.

단계 7: 200 ul의 인산 버퍼 염수 (pH : 7.4), 0.5M NaCl 에 용해되어 있는 1mg/ml 농도의 human Immunoglobulin G 를 상기 단계 6의 상층액을 제거한 슬러리 ProteinA-친수성 파이렌 유도체-탄소코팅 자성나노입자 복합체와 보텍스 믹서로 10분간 반응시킨다.Step 7: 200 ul of phosphate buffered saline (pH: 7.4), 1mg/ml human Immunoglobulin G dissolved in 0.5M NaCl slurry from which the supernatant of step 6 was removed ProteinA-hydrophilic pyrene derivative-carbon coating magnetic React the nanoparticle complex with a vortex mixer for 10 minutes.

단계 8: 반응이 끝난 상층액을 제거하여 따로 보관 후 인산-버퍼 염수로 두 차례 세척한다.Step 8: After the reaction is over, the supernatant is removed, stored separately, and washed twice with phosphate-buffered saline.

단계 9: 상기 단계 8에서 제거한 상층액과 상기 단계 3에서 넣어준 200 ul의 인산 버퍼 염수 (pH : 7.4), 0.5M NaCl 에 용해되어 있는 1mg/ml 농도의 human Immunoglobulin G 의 280nm 파장에서의 흡광도를 비교하여 human Immunoglobulin G-ProteinA-친수성 파이렌 유도체-탄소코팅 자성 나노입자 복합체의 제조에 들어간 human Immunoglobulin G 의 양을 Beer-Lambert 법 (280nm에서 human Immunoglobuling G 의 extinction coefficient : 210,000 M-1cm-1, human Immunoglobulin G 의 분자량 : 150,000) 을 이용하여 산정한다.Step 9: Absorbance at 280 nm wavelength of human Immunoglobulin G at a concentration of 1 mg/ml dissolved in the supernatant removed in step 8, 200 μl of phosphate buffered saline (pH: 7.4), and 0.5 M NaCl added in step 3 By comparing the amount of human Immunoglobulin G-ProteinA-hydrophilic pyrene derivative-carbon-coated magnetic nanoparticle composite, the amount of human Immunoglobulin G entered into the preparation was calculated by Beer-Lambert method (extinction coefficient of human Immunoglobulin G at 280 nm: 210,000 M -1 cm - 1 , molecular weight of human Immunoglobulin G: 150,000).

이상 설명한 바와 같이, 본 발명에 따르면, 친수성 기능기를 포함하는 파이렌 유도체를 이용하여 파이렌과 자성나노입자의 표면의 탄소층과의 강한 결합을 형성시켜 파이렌 유도체-탄소코팅 자성나노입자의 복합체를 만듬으로써 자성나노입자의 수용상상의 용해도, 안정성 그리고 분리, 정제, 농축 및 검출용 각종 생물질에 공유결합에 의한 부착이 가능하다. 따라서 이 자성나노입자 복합체는 생물학, 의학 및 진단학등과 같은 햄염과학 분야에 효과적으로 이용할 수 있다.As described above, according to the present invention, a pyrene derivative-carbon-coated magnetic nanoparticle complex is formed by forming a strong bond between pyrene and the carbon layer on the surface of magnetic nanoparticles using a pyrene derivative containing a hydrophilic functional group. By making the solubility and stability of the aqueous phase of magnetic nanoparticles, it is possible to attach them to various biological substances for separation, purification, concentration and detection by covalent bonds. Therefore, this magnetic nanoparticle complex can be effectively used in the fields of hematology such as biology, medicine and diagnostics.

Claims (5)

탄소코팅된 자성나노입자 및 그 외부에 코팅된 친수성 파이렌(pyrene) 유도체로 이루어지고, 상기 친수성 파이렌 유도체는 파이렌 분자에 카르복실기가 결합된 파이렌 부틸릭산이고,　상기 파이렌 부틸릭산은　1-에틸-3-[3-디메틸아미노프로필]　카르보디이미드　(EDC)와 커플링되어 있는 것을 특징으로 하는 친수성 파이렌-탄소코팅 자성나노입자 복합체.
It consists of carbon-coated magnetic nanoparticles and a hydrophilic pyrene derivative coated on the outside, and the hydrophilic pyrene derivative is pyrene butyric acid in which a carboxyl group is bonded to a pyrene molecule, -Ethyl-3-[3-dimethylaminopropyl] carbodiimide (EDC) and hydrophilic pyrene-carbon coated magnetic nanoparticle complex.
 삭제delete 제1항에 있어서, 상기 친수성 파이렌 유도체로 코팅된 탄소코팅 자성나노입자의 표면 친수성 화학적 기능기에 단백질A가 공유결합으로 연결되어, 항체를 분리 정제하는데 사용되는 것을 특징으로 하는 친수성 파이렌-탄소코팅 자성나노입자 복합체.
The hydrophilic pyrene-carbon according to claim 1, wherein protein A is covalently linked to a hydrophilic chemical functional group on the surface of the carbon-coated magnetic nanoparticles coated with the hydrophilic pyrene derivative, and is used to separate and purify the antibody. Coated magnetic nanoparticle composite.
 제1항에 있어서, 상기 친수성 파이렌 유도체로 코팅된 탄소코팅 자성나노입자의 표면 친수성 화학적 기능기에 NTA(Nickel-Nitrilo acetic acid)가 공유결합으로 연결되어, Ni 이온을 매개로 His-tag 단백질을 분리 정제하는데 사용되는 것을 특징으로 하는 친수성 파이렌-탄소코팅 자성나노입자 복합체. The method of claim 1, wherein NTA (Nickel-Nitrilo acetic acid) is covalently linked to the hydrophilic chemical functional group on the surface of the carbon-coated magnetic nanoparticles coated with the hydrophilic pyrene derivative, thereby forming His-tag proteins through Ni ions. A hydrophilic pyrene-carbon coated magnetic nanoparticle complex, characterized in that it is used for separation and purification. 삭제delete
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