KR101792123B1 - Nanoflower consisting of magnetic nanoparticles and oxidative enzymes and method for manufacturing the same - Google Patents
Nanoflower consisting of magnetic nanoparticles and oxidative enzymes and method for manufacturing the same Download PDFInfo
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Abstract
본 발명은 자성 나노입자와 산화효소가 결합된 나노플라워 및 그 제조방법에 관한 것으로, 더욱 상세하게는 과산화효소 활성을 가지는 자성 나노입자와 산화효소가 정전기적 인력에 의해 결합된 나노플라워 및 그 제조방법에 관한 것이다.
본 발명에 따른 자성 나노입자와 산화효소가 결합된 나노플라워는 과산화효소 활성을 가지고 있고, 정전기적 인력을 이용하여 합성된 것으로 기존의 효소 나노플라워에 비해 회수 및 재사용이 매우 용이하다. 또한, 각 물질의 특성을 잃지 않고 나노플라워를 제조함으로써 효소를 효과적으로 고정시킬 수 있으며, 산화효소와 연계시켜 사용함으로써 당뇨병의 마커가 되는 포도당의 검출이 가능한 것을 확인하였으며, 따라서 다양한 질병의 정보를 제공할 수 있는 마커 물질(갈락토스, 콜레스테롤 등의 소분자 물질)의 손쉬운 발색 검출에 유용하게 사용될 수 있다.The present invention relates to a nanoflower in which magnetic nanoparticles and an oxidizing enzyme are combined and a method for producing the same, and more particularly, to a nanoflower in which magnetic nanoparticles having a peroxidase activity are combined with electroactive grafts ≪ / RTI >
The magnetic nanoparticles and oxidase-bound nanoflowers according to the present invention have a peroxidase activity and are synthesized using an electrostatic attraction. Therefore, they are easier to recover and reuse than conventional enzyme nanoflowers. In addition, it has been confirmed that the enzymes can be effectively fixed by producing the nanoflower without losing the properties of each substance, and it is possible to detect glucose as a marker of diabetes by using it in association with oxidizing enzymes. Accordingly, And can be usefully used for easy color detection of a marker substance (such as a small molecule substance such as galactose or cholesterol).
Description
본 발명은 자성 나노입자와 산화효소가 결합된 나노플라워 및 그 제조방법에 관한 것으로, 더욱 상세하게는 과산화효소 활성을 가지는 자성 나노입자와 산화효소가 정전기적 인력에 의해 결합된 나노플라워 및 그 제조방법에 관한 것이다.The present invention relates to a nanoflower in which magnetic nanoparticles and an oxidizing enzyme are combined and a method for producing the same, and more particularly, to a nanoflower in which magnetic nanoparticles having a peroxidase activity are combined with electroactive grafts ≪ / RTI >
사회가 고도화·현대화될수록 보다 오래, 건강하게 살고 싶은 인간의 욕구는 증가하고 있다. 특히 최근 의학기술의 비약적인 발전은 어떤 질병이라도 조기에만 발견하면 완치 가능한 사회를 만들어가고 있기 때문에 질병 진단 기술이 더욱 중요해지고 있다. 또한, 비싼 검출 기계 혹은 숙련된 기술자가 필요한 질병 진단 기술보다, 일반인도 쉽고 간편하게 이용할 수 있는 진단방식의 기술 개발 역시 경제적 실현성 면에서 매우 중요하다. 이와 같은 필요성에 따라 질병원인이 되는 포도당, 갈락토스, 콜레스테롤 등의 소분자 물질, 단백질, DNA, 병원균 등을 보다 쉽게 고감도로 검출 및 정량하는 기술은 경제적·기술적으로 매우 중요하고, 산업적으로도 큰 파급효과를 가지며, 세계적으로 활발히 연구되고 있는 분야이다. 또한, 질병진단의 또 하나의 중요한 흐름은 POCT(Point of care testing)라고 하는 현장진단기술 분야로서, 이 기술은 숙련된 분석 수행자 없이 현장에서 즉각적이고 손쉽게 진단의 결과를 낼 수 있는 기술과 장치를 포함한다. 최근 새롭게 형성되고 있는 세계 POCT 시장은 보다 빠르고 손쉽게 질병을 진단할 수 있는 새로운 기술을 요구하고 있다.As society becomes more sophisticated and modernized, human desire to live longer and healthier is increasing. Especially, since the breakthrough of medical technology is making a society that can be cured if any disease is found early, disease diagnosis technology becomes more important. In addition, the development of diagnostic techniques that can be easily and easily used by the general public is more important than the disease diagnosis techniques that require expensive detection machines or skilled technicians. Techniques for detecting and quantifying small molecular substances such as glucose, galactose, and cholesterol, proteins, DNA, pathogens and the like, which are causative of diseases, with high sensitivity are very important economically and technically according to the necessity, It is a field that is being actively studied all over the world. Another important flow of disease diagnosis is the field of diagnostic technology called Point of care testing (POCT), which is a technique and device that can produce an immediate and easy diagnostic result in the field without a skilled analyst. . Recently, the newly formed world POCT market is demanding new technology that can diagnose diseases more quickly and easily.
진단 기술 개발을 위해 지난 수십 년간 단백질 기반 유기효소의 활성에 기반한 바이오센서 기술이 다양하게 연구되었고 많은 분야에서 이미 상품화가 진행되었다. 이들 유기효소에 기반한 기술은 선택적이고 민감하게 대상 물질을 진단할 수 있다는 장점이 있지만, 반응 환경과 보관 시간에 따라 효소의 활성이 변하여 궁극적으로 진단결과가 변할 수 있다는 단점이 있다. For the development of diagnostic technology, biosensor technology based on the activity of protein-based organic enzymes has been studied for many decades and commercialization has already been done in many fields. These organic enzyme-based technologies have the advantage of being capable of selectively and sensitively diagnosing the target substance, but they have disadvantages in that the activity of the enzyme changes depending on the reaction environment and storage time, ultimately resulting in a change in the diagnosis result.
이를 보완하기 위해 효소를 고정화시켜 그 활성을 오래 유지하거나 증폭시키는 방법에 대한 연구가 수십 년간 활발히 진행되었다. 특히 최근, 효소를 CuSO4 와 반응시킴으로써 만들어지는 효소 나노플라워가 각광을 받고 있다. 효소 나노플라워는 기존의 고정화 방법에 비해 활성 보존(Activity retention)이 우수하고, 동시에 획기적으로 높은 안정성을 보인다고 알려져 있다(Nature Nanotechnology, 2012, 7(7), 428-432; Journal of Nanobiotechnology, 2015, 13:54). 그러나, 기존의 효소 나노플라워는 원심분리를 이용하여 재사용시 구조가 손상되는 문제 때문에, 원할한 재사용이 어려운 단점을 가지고 있다. In order to compensate for this, studies have been actively conducted for several decades to immobilize the enzyme and to maintain or amplify its activity for a long time. In recent years, enzyme nanoflowers, which are produced by reacting enzymes with CuSO 4 , are in the spotlight. The enzyme nanoflower is known to exhibit excellent activity retention and remarkably high stability at the same time as compared with the existing immobilization method (Nature Nanotechnology, 2012, 7 (7), 428-432; Journal of Nanobiotechnology, 2015, 13:54). However, the conventional enzyme nanoflower has disadvantages in that it is difficult to reuse the enzyme nanoflower because of the problem that the structure is damaged when it is reused by centrifugation.
이에, 본 발명자들은 상기 문제점을 해결하기 위하여 예의 노력한 결과 자성 나노입자와 산화효소를 정전기적 인력으로 결합시켜 나노플라워를 제조함으로써, 포도당, 갈락토스, 콜레스테롤, 단백질 등의 소분자 물질들을 보다 효과적으로 발색 진단할 수 있는 것을 확인하였으며, 자성을 이용하여 간편히 회수 및 재사용이 가능한 것을 확인하고 본 발명을 완성하게 되었다.Accordingly, the present inventors have made intensive efforts to solve the above-mentioned problems. As a result, the inventors of the present invention have found that by producing nanoflower by combining magnetic nanoparticles and oxidizing enzyme with electrostatic attraction, it is possible to more effectively diagnose small molecules such as glucose, galactose, cholesterol, And it was confirmed that recovery and reuse were easy by using magnetism, and the present invention was completed.
본 발명의 목적은 자성 나노입자와 산화효소가 정전기적 인력으로 결합된 나노플라워를 제공하는데 있다.It is an object of the present invention to provide a nanoflower in which magnetic nanoparticles and an oxidizing enzyme are bound by electrostatic attraction.
본 발명의 다른 목적은 상기 나노플라워를 포함하는 바이오 센서 및 이를 이용하여 소분자 물질을 진단하는 방법을 제공하는데 있다.It is another object of the present invention to provide a biosensor including the nanoflower and a method of diagnosing a small molecule using the biosensor.
본 발명의 다른 목적은 상기 나노플라워의 제조방법을 제공하는데 있다.It is another object of the present invention to provide a method for producing the nanoflower.
상기 목적을 달성하기 위하여, 본 발명은 자성 나노입자와 산화효소가 정전기적 인력으로 결합된 나노플라워를 제공한다.In order to achieve the above object, the present invention provides a nanoflower in which magnetic nanoparticles and an oxidizing enzyme are combined with electrostatic attraction.
본 발명은 또한, 상기 나노플라워를 포함하는 바이오 센서 및 이를 이용하여 소부자 물질을 진단하는 방법을 제공한다.The present invention also provides a biosensor including the nanoflower and a method of diagnosing a small-matter substance using the biosensor.
본 발명은 또한, a) 자성 나노입자에 아민기를 코팅시키는 단계; b) 상기 아민이 코팅된 자성 나노입자와 산화효소를 정전기적 인력으로 결합시키는 단계; 및 c) 상기 결합된 자성 나노입자 및 산화효소를 염 용액과 반응시키는 단계를 포함하는 나노플라워 제조방법을 제공한다.The present invention also provides a method for preparing a magnetic nanoparticle comprising: a) coating an amine group on a magnetic nanoparticle; b) electrostatically attracting the amine-coated magnetic nanoparticles to the oxidizing enzyme; And c) reacting the coupled magnetic nanoparticles and the oxidizing enzyme with a salt solution.
본 발명에 따른 자성 나노입자와 산화효소가 결합된 나노플라워는 과산화효소 활성을 가지고 있고, 정전기적 인력을 이용하여 합성된 것으로 기존의 효소 나노플라워에 비해 회수 및 재사용이 매우 용이하다. 또한, 각 물질의 특성을 잃지 않고 나노플라워를 제조함으로써 효소를 효과적으로 고정시킬 수 있으며, 산화효소와 연계시켜 사용함으로써 당뇨병의 마커가 되는 포도당의 검출이 가능한 것을 확인하였으며, 따라서 다양한 질병의 정보를 제공할 수 있는 마커 물질(갈락토스, 콜레스테롤 등의 소분자 물질)의 손쉬운 발색 검출에 유용하게 사용될 수 있다.The magnetic nanoparticles and oxidase-bound nanoflowers according to the present invention have a peroxidase activity and are synthesized using an electrostatic attraction. Therefore, they are easier to recover and reuse than conventional enzyme nanoflowers. In addition, it has been confirmed that the enzymes can be effectively fixed by producing the nanoflower without losing the properties of each substance, and it is possible to detect glucose as a marker of diabetes by using it in association with oxidizing enzymes. Accordingly, And can be usefully used for easy color detection of a marker substance (such as a small molecule substance such as galactose or cholesterol).
도 1은 본 발명의 일 실시예에 따른 자성 나노입자와 산화효소가 결합된 나노플라워의 제조방법을 도식화한 것이다.
도 2는 본 발명의 일 실시예에 따른 자성 나노입자의 TEM 이미지를 나타낸 것이다.
도 3은 본 발명의 일 실시예에 따른 자성 나노입자 및 아민이 코팅된 자성 나노입자의 FT-IR 분석 결과를 나타낸 것이다.
도 4는 본 발명의 일 실시예에 따른 산화효소 나노플라워의 SEM 이미지를 나타낸 것이다.
도 5는 본 발명의 일 실시예에 따른 아민이 코팅된 자성 나노입자 나노플라워 및 자성 나노입자와 산화효소가 결합된 나노플라워의 SEM 이미지를 나타낸 것이다.
도 6은 본 발명의 일 실시예에 따른 산화효소 및 아민이 코팅된 자성 나노입자의 제타전위를 측정한 결과를 나타낸 것이다.
도 7은 본 발명의 일 실시예에 따른 자성 나노입자-산화효소 나노플라워, 자성 나노입자 나노플라워 및 산화효소 나노플라워가 외부 자력에 대해 끌리는 성질을 나타낸 것이다.
도 8은 본 발명의 일 실시예에 따른 자성 나노입자-포도당 산화효소 나노플라워를 이용하여 포도당 진단 결과를 나타낸 것이다. FIG. 1 is a diagram illustrating a method of manufacturing a nanoflower in which a magnetic nanoparticle and an oxidizing enzyme are combined according to an embodiment of the present invention.
2 is a TEM image of a magnetic nanoparticle according to an embodiment of the present invention.
FIG. 3 shows FT-IR analysis results of magnetic nanoparticles and amine-coated magnetic nanoparticles according to an embodiment of the present invention.
4 is an SEM image of an oxidized enzyme nanoflower according to an embodiment of the present invention.
FIG. 5 is a SEM image of an amine-coated magnetic nanoparticle nanoflower and a nanoflower in which magnetic nanoparticles and oxidizing enzyme are combined according to an embodiment of the present invention.
FIG. 6 shows the results of measurement of zeta potential of magnetic nanoparticles coated with oxidase and amine according to an embodiment of the present invention.
FIG. 7 shows the magnetic nanoparticle-oxidizing enzyme nanoflower, the magnetic nanoparticle nanoflower, and the oxidized enzyme nanoflower according to an embodiment of the present invention, which are attracted to external magnetic force.
8 is a graph showing the results of glucose diagnosis using a magnetic nanoparticle-glucose oxidase nanoflower according to an embodiment of the present invention.
본 발명은 하기의 설명에 의하여 모두 달성될 수 있다. 하기의 설명은 본 발명의 바람직한 구체적인 예를 기술하는 것으로 이해되어야 하며, 본 발명이 반드시 이에 한정되는 것은 아니다. 또한, 첨부된 도면은 이해를 돕기 위한 것으로, 본 발명이 이에 한정되는 것은 아니며, 개별 구성에 관한 세부 사항은 후술하는 관련 기재의 구체적 취지에 의하여 적절히 이해될 수 있다.The present invention can be all accomplished by the following description. It is to be understood that the following description is only illustrative of preferred embodiments of the invention, but the invention is not necessarily limited thereto. It is to be understood that the accompanying drawings are included to provide a further understanding of the invention and are not to be construed as limiting the present invention. The details of the individual components may be properly understood by reference to the following detailed description of the related description.
다른 식으로 정의되지 않는 한, 본 명세서에서 사용된 모든 기술적 및 과학적 용어들은 본 발명이 속하는 기술 분야에서 숙련된 전문가에 의해서 통상적으로 이해되는 것과 동일한 의미를 가진다. 일반적으로, 본 명세서에서 사용된 명명법은 본 기술 분야에서 잘 알려져 있고 통상적으로 사용되는 것이다.Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. In general, the nomenclature used herein is well known and commonly used in the art.
본 발명에서는 과산화효소 활성을 가지는 자성 나노입자와 산화효소가 정전기적 인력에 의해 결합된 나노플라워를 제조하였으며, 기존의 효소 나노플라워에 비해 회수 및 재사용이 매우 용이하다. 또한, 각 물질의 특성을 잃지 않고 나노플라워를 제조함으로써 효소를 효과적으로 고정시킬 수 있고, 산화효소와 연계시켜 사용함으로써 당뇨병의 마커가 되는 포도당의 검출이 가능한 것을 확인하였으며, 따라서 다양한 질병의 정보를 제공할 수 있는 마커 물질(갈락토스, 콜레스테롤, 단백질 등의 소분자 물질)의 손쉬운 발색 검출에 유용하게 사용될 수 있다.In the present invention, a magnetic nanoparticle having a peroxidase activity and a nanoflower in which an oxidizing enzyme is bound by an electrostatic attraction are prepared, and it is very easy to recover and reuse it compared to a conventional enzyme nanoflower. Further, it has been confirmed that the enzyme can be effectively fixed by producing the nanoflower without losing the properties of each substance, and it is possible to detect glucose, which is a marker of diabetes, by using it in association with an oxidase, (Small molecule materials such as galactose, cholesterol, and proteins) that can be easily detected.
본 발명은 일 관점에서 자성 나노입자와 산화효소가 정전기적 인력으로 결합된 나노플라워에 관한 것이다.In one aspect, the present invention relates to a nanoflower in which magnetic nanoparticles and an oxidizing enzyme are electrostatically attracted.
본 발명의 자성 나노입자는 과산화효소 활성을 가지는 것을 특징으로 하고, 자성 나노입자는 입자의 크기가 10 내지 20 nm인 것이 바람직하다.The magnetic nanoparticles of the present invention are characterized by having a peroxidase activity, and the magnetic nanoparticles preferably have a particle size of 10 to 20 nm.
또한, 본 발명의 자성 나노입자는 APTES((3-Aminopropyl) triethoxysilane), poly-L-lysine 및 APTMS((3-Aminopropyl) trimethoxysilane)으로 구성된 군에서 선택된 1종 이상의 아민으로 코팅된 것이 바람직하고, APTES를 이용하여 자성 나노입자를 코팅하는 것이 가장 바람직하다.The magnetic nanoparticles of the present invention are preferably coated with at least one amine selected from the group consisting of APTES ((3-Aminopropyl) triethoxysilane), poly-L-lysine and APTMS ((3-Aminopropyl) trimethoxysilane) It is most preferred to coat the magnetic nanoparticles using APTES.
본 발명의 산화효소는 포도당 산화효소, 글루코스 산화효소, 콜레스테롤 산화효소 또는 단백질 산화효소인 것이 바람직하나, 이에 한정되는 것은 아니다.The oxidizing enzyme of the present invention is preferably a glucose oxidase, a glucose oxidase, a cholesterol oxidase or a protein oxidase, but is not limited thereto.
또한, 본 발명의 자성 나노입자와 산화효소가 정전기적 인력으로 결합된 다음, 염 용액을 추가로 혼합하는 것이 바람직하다.In addition, it is preferable that the magnetic nanoparticles of the present invention and the oxidase are bound by electrostatic attraction, and then the salt solution is further mixed.
또한, 본 발명의 자성 나노입자와 산화효소가 정전기적 인력으로 결합된 나노플라워는 pH 4 내지 pH 7에서 혼합하는 것이 바람직하고, pH 5.5이 가장 바람직하다.In addition, the nanoflower in which the magnetic nanoparticles of the present invention and the oxidase are bound by an electrostatic attraction is preferably mixed at pH 4 to pH 7, and most preferably at pH 5.5.
본 발명은 다른 관점에서 자성 나노입자와 산화효소가 정전기적 인력으로 결합된 나노플라워를 포함하는 바이오 센서에 관한 것이다.The present invention relates to a biosensor comprising a nanoflower in which magnetic nanoparticles and an oxidizing enzyme are electrostatically coupled in a different aspect.
본 발명은 다른 관점에서 상기 바이오 센서를 이용하여 소분자 물질을 진단하는 방법에 관한 것이다. The present invention relates to a method for diagnosing a small molecule material using the biosensor from another viewpoint.
본 발명에 있어서, 상기 소분자 물질은 포도당, 갈락토스 또는 콜레스테롤이 바람직하나, 이에 한정되는 것은 아니다.In the present invention, the small molecule material is preferably glucose, galactose or cholesterol, but is not limited thereto.
본 발명은 다른 관점에서, a) 자성 나노입자에 아민기를 코팅시키는 단계; b) 상기 아민이 코팅된 자성 나노입자와 산화효소를 정전기적 인력으로 결합시키는 단계; 및 c) 상기 결합된 자성 나노입자 및 산화효소를 염 용액과 반응시키는 단계를 포함하는 나노플라워 제조방법에 관한 것이다.In another aspect, the present invention provides a magnetic nanoparticle comprising: a) coating an amine group on a magnetic nanoparticle; b) electrostatically attracting the amine-coated magnetic nanoparticles to the oxidizing enzyme; And c) reacting the bound magnetic nanoparticles and the oxidase with a salt solution.
본 발명에 있어서, 상기 염 용액은 Cu, Zn, Ti, Sn 등의 금속 이온을 포함하는 것이 바람직하고, Cu 금속 함유 염 용액의 제조가 용이하다. CuSO4 염 용액이 가장 바람직하다. In the present invention, it is preferable that the salt solution contains metal ions such as Cu, Zn, Ti, and Sn, and it is easy to produce a Cu metal-containing salt solution. The CuSO 4 salt solutions are most preferred.
본 발명의 자성 나노입자와 산화효소가 결합된 나노플라워는 합성 및 회수가 매우 용이하다. 유기효소의 경우 필연적인 단백질 구조의 변성에 의한 활성의 감소가 있지만, 본 발명의 나노플라워는 꽃 모양의 구조체를 형성하여 효소를 고정시켜 안정성과 효소활성을 극대화할 수 있으며, APTES를 통해 아민 작용기를 자성나노입자 표면에 코팅하여도 자성을 잃지 않아 자석으로 회수가 용이하다는 장점이 있고 자성나노입자 자체가 가지고 있는 과산화효소 성질을 이용하여, 목적 기질의 분석시 추가적인 시약의 필요를 최소로 하여 경제적인 측면에서도 매우 우수한 장점을 가지고 있다.The nanoflower in which the magnetic nanoparticles of the present invention and the oxidase are combined can be easily synthesized and recovered. In the case of the organic enzyme, there is inevitably a decrease in the activity due to denaturation of the protein structure. However, the nanoflower of the present invention forms a flower-like structure to immobilize the enzyme, thereby maximizing the stability and enzyme activity. Is coated on the surface of magnetic nanoparticles, it is advantageous in that it can be recovered as a magnet because it does not lose its magnetism. Also, by using the peroxidase properties possessed by the magnetic nanoparticles themselves, the need for additional reagents is minimized And it has very good advantages.
[[ 실시예Example ]]
이하 본 발명을 실시예에 의하여 더욱 상세히 설명한다. 이들 실시예는 단지 본 발명을 보다 구체적으로 설명하기 위한 것으로, 이에 의해 본 발명의 기술적 범위가 이들 실시예에 국한되지 않는다는 것은 당업계에서 통상의 지식을 가진 자에게 있어서 자명할 것이다.Hereinafter, the present invention will be described in more detail by way of examples. It will be apparent to those skilled in the art that these embodiments are merely illustrative of the present invention and that the technical scope of the present invention is not limited to these embodiments.
실시예Example 1: One: 아민이Amine 코팅된 자성 나노입자 제조 Manufacture of coated magnetic nanoparticles
1-1. 자성 나노입자의 합성1-1. Synthesis of magnetic nanoparticles
자성 나노입자의 합성은 FeCl3와 FeCl2의 침전을 통해 제조하였다(Mehta et al., Biotechnology Techniques, 11(7), 493-496, 1997). 균일하게 침전된 자성 나노입자는 진공 오븐에서 충분히 건조 시킨 다음, TEM 이미지를 통하여 나노입자의 크기 및 형태를 확인하였다.Synthesis of magnetic nanoparticles was prepared by precipitation of FeCl 3 and FeCl 2 (Mehta et al., Biotechnology Techniques, 11 (7), 493-496, 1997). The uniformly precipitated magnetic nanoparticles were sufficiently dried in a vacuum oven, and the size and shape of the nanoparticles were confirmed by TEM image.
1-2. 1-2. 아민이Amine 코팅된 자성 나노입자의 합성 Synthesis of Coated Magnetic Nanoparticles
아민이 코팅된 자성 나노입자는 에탄올과 톨루엔이 1:1(v/v)로 혼합된 용액 100 mL에 1-1에서 제조된 자성 나노입자 0.5 g을 넣어 분산시킨 다음, 3mM 농도의 APTES((3-Aminopropyl)triethoxysilane) 용액 100 ㎕을 넣어 코팅하였다.Amine coated magnetic nanoparticles were prepared by dispersing 0.5 g of the magnetic nanoparticles prepared in 1-1 in 100 mL of a 1: 1 (v / v) mixed solution of ethanol and toluene, and then adding 3 mM of APTES ( 3-Aminopropyl) triethoxysilane (100 μL) was added and coated.
도 2는 본 실시예에서 제조된 자성 나노입자(MNP)와 아민이 코팅 된 자성 나노입자(APTES-MNP)의 TEM 이미지를 나타낸 것으로, 지름이 약 10nm인 것을 확인 할 수 있었다.FIG. 2 shows a TEM image of the magnetic nanoparticles (MNP) and amine-coated magnetic nanoparticles (APTES-MNP) prepared in this example, and it was confirmed that the diameter was about 10 nm.
또한, 도 3은 본 실시예에서 제조된 자성 나노입자(MNP) 및 아민이 코팅된 자성 나노입자(APTES-MNP)의 FT-IR 분석 결과를 나타낸 것으로, 일반 자성나노입자에 아민이 코팅된 것은 아민의 특정 피크파장이 나타난 것을 통해 알 수 있었다.FIG. 3 shows FT-IR analysis results of the magnetic nanoparticles (MNP) and amine-coated magnetic nanoparticles (APTES-MNP) prepared in this example, and the amine-coated magnetic nanoparticles The specific peak wavelength of the amine was revealed.
실시예Example 2: 자성 나노입자와 산화효소가 2: Magnetic nanoparticles and oxidase 결합된Combined 나노플라워Nano flower 제조 Produce
2-1. 포도당 산화효소 2-1. Glucose oxidase 나노플라워의Nano-flower 합성 synthesis
본 실시예에서는 포도당 산화효소만을 이용하여 나노플라워를 합성하였다. PBS 4 mL, pH 7.4에서 0.02 mg/ml 농도의 포도당 산화효소와 120 mM의 CuSO4 20 μL를 혼합한 다음, 상온에서 3일간 배양하였다. 합성된 나노플라워는 DI water로 세 차례 washing하여 포도당 산화효소 나노플라워를 수득하였다. In this Example, a nanoflower was synthesized using only glucose oxidase. 4 mL of PBS and 0.02 mg / mL of glucose oxidase and 20 mM of CuSO 4 were mixed at pH 7.4 and incubated at room temperature for 3 days. The synthesized nanoflower was washed three times with DI water to obtain glucose oxidase nanoflower.
도 4는 본 실시예에서 제조된 포도당 산화효소 나노플라워의 SEM 이미지를 나타낸 것으로, Cu와 포도당 산화효소의 복합체로 꽃 모양의 구조체가 생성된 것을 확인 할 수 있었다.FIG. 4 is a SEM image of the glucose oxidase nanoflower prepared in this Example. It was confirmed that a flower-like structure was formed by a complex of Cu and glucose oxidase.
2-2. 자성 나노입자 2-2. Magnetic nanoparticle 나노플라워의Nano-flower 합성 synthesis
본 실시예에서는 자성 나노입자만을 이용하여 나노플라워를 합성하였다. PBS 4 mL, pH 5.5의 조건에서 0.4 mg/mL 농도의 MNP-APTES(실시예 1-2에서 제조)와 120mM의 CuSO4 20 μL를 혼합한 다음, 상온에서 3일간 배양하였다. 합성된 나노플라워는 DI water로 세 차례 washing하여 아민이 코팅된 자성 나노입자(MNP-APTES) 나노플라워를 수득하였다. In this embodiment, a nanoflower was synthesized using only magnetic nanoparticles. MNP-APTES (manufactured in Example 1-2) and 20 μL of 120 mM CuSO 4 were mixed at a concentration of 0.4 mg / mL under the conditions of 4 mL of PBS and pH 5.5, and then cultured at room temperature for 3 days. The synthesized nanoflower was washed with DI water three times to obtain amine-coated magnetic nanoparticles (MNP-APTES) nanoflower.
도 5는 본 실시예에서 제조된 아민이 코팅된 자성 나노입자(MNP-APTES) 나노플라워의 SEM 이미지(a) 및 상기 자성 나노입자(MNP-APTES)와 산화효소가 결합된 나노플라워의 SEM 이미지(b)를 나타낸 것으로, 포도당 산화효소로 만든 나노플라워와 모양이 다른 것을 확인 할 수 있었다.FIG. 5 is a SEM image (a) of the amine-coated magnetic nanoparticles (MNP-APTES) nanoflower prepared in this example and a SEM image of the nanoparticles combined with the magnetic nanoparticles (MNP-APTES) (b), which was different from that of nanoflower made of glucose oxidase.
2-3. 자성 나노입자와 산화효소가 2-3. Magnetic nanoparticles and oxidizing enzymes 결합된Combined 나노플라워의Nano-flower 합성 synthesis
본 실시예에서는 자성 나노입자와 산화효소가 결합된 나노플라워를 합성하였다. In this embodiment, a nanoflower having a magnetic nanoparticle coupled with an oxidase was synthesized.
나노플라워의 합성에 앞서 1-2에서 제조된 아민이 코팅된 자성 나노입자(APTES-MNP)와 포도당 산화효소(GOx)의 제타전위를 측정하였다. 도 5에서 나타난 바와 같이, APTES-MNP은 20.05 mV, GOx은 -25 mV의 제타전위를 갖는 것을 확인하였으며, 그 결과 두 물질이 정전기적 인력으로 결합되는 것을 확인할 수 있었다.The zeta potential of amine-coated magnetic nanoparticles (APTES-MNP) and glucose oxidase (GOx) prepared in 1-2 was measured prior to the synthesis of nanoflower. As shown in FIG. 5, it was confirmed that APTES-MNP had a zeta potential of 20.05 mV and GOx had a zeta potential of -25 mV. As a result, it was confirmed that the two materials were combined with an electrostatic attractive force.
PBS 4 mL, pH 5.5의 조건에서 0.02 mg/ml 농도의 포도당 산화효소 및 0.4 mg/mL 농도의 MNP-APTES(실시예 1-2에서 제조)와 120mM의 CuSO4 20 μL를 혼합한 다음, 상온에서 3일간 배양하였다. 합성된 나노플라워는 DI water로 세 차례 washing하여 자성 나노입자와 산화효소가 결합된 나노플라워를 수득하였다. 또한 합성된 산화효소-자성 나노입자 나노플라워는 자석을 이용해 간단히 회수가 가능한 것을 확인하여, 자성 성질을 유지하고 있음을 확인하였다. 도 7-(a)는 자성 나노입자-산화효소 나노플라워, (b)는 자성 나노입자 나노플라워 (c)는 포도당 산화효소 나노플라워이다.After mixing 0.02 mg / ml of glucose oxidase and 0.4 mg / ml of MNP-APTES (prepared in Example 1-2) and 20 μl of 120 mM CuSO 4 in 4 ml of PBS and pH 5.5, And cultured for 3 days. The synthesized nanoflower was washed three times with DI water to obtain a nanoflower in which magnetic nanoparticles and oxidizing enzyme were combined. In addition, it was confirmed that the synthesized oxidase - magnetic nanoparticle nanoflower can be recovered easily by using a magnet, and maintained magnetic properties. 7 (a) is a magnetic nanoparticle-oxidizing enzyme nanoflower, (b) is a magnetic nanoparticle nanoflower (c) is a glucose oxidase nanoflower.
2-4. 자성 나노입자-포도당 산화효소가 동시 2-4. Magnetic nanoparticles - glucose oxidase simultaneously 결합된Combined 나노플라워를Nano flower 이용한 포도당 진단 Diagnosis of glucose used
본 실시예에서는 자성 나노입자-포도당 산화효소가 동시에 결합된 나노플라워를 이용하여 포도당을 발색 진단하였다. In this example, glucose nanoparticle coupled with magnetic nanoparticle-glucose oxidase was used to diagnose glucose color development.
실험에 사용한 시료는 포도당(500 mM, pH 7.4), Sodium acetate buffer(100 mM, pH 4), 포도당 산화효소(0.1 mg/ml), 자성 나노입자와 포도당 산화효소가 결합된 나노플라워(각 0.1mg/ml), 자성 나노입자(0.1 mg/ml)와 색의 변화를 시각적으로 볼 수 있게 하는 TMB(3,3’,5,5’-tetramethylbenzidine)가 있다.The samples used were glucose (500 mM, pH 7.4), sodium acetate buffer (100 mM, pH 4), glucose oxidase (0.1 mg / ml), nanoparticles mg / ml), magnetic nanoparticles (0.1 mg / ml) and TMB (3,3 ', 5,5'-tetramethylbenzidine), which allows visual changes in color.
도 8에서 A는 포도당 : 나노플라워 : buffer : TMB = 5:1:3:1 의 비율로 총 부피가 1ml가 되도록 하였다.In FIG. 8, the total volume was 1 ml in the ratio of glucose: nanoflow: buffer: TMB = 5: 1: 3: 1.
B는 포도당 : 포도당 산화효소 : buffer : TMB 가 5:1:3:1B is glucose: glucose oxidase: buffer: TMB is 5: 1: 3: 1
C는 포도당 : 자성 나노입자 : buffer : TMB 가 5:1:3:1C is glucose: magnetic nanoparticles: buffer: TMB is 5: 1: 3: 1
D는 buffer : 나노플라워 : buffer : TMB 가 5:1:3:1 비율로 들어 있는 실험 조건을 만든 후 37도에서 50분 동안 반응시킨 결과이다.D is the result of reaction at 37 ° C for 50 minutes after making the experimental conditions containing buffer: nanoflow: buffer: TMB in the ratio of 5: 1: 3: 1.
MNP(magnetic nanoparticles) : 자성 나노입자
APTES-MNP : 아민(APTES) 코팅된 자성 나노입자
GOx(Glucose Oxygenase) : 포도당 산화효소MNP (magnetic nanoparticles): Magnetic nanoparticles
APTES-MNP: Amine (APTES) coated magnetic nanoparticles
GOx (Glucose Oxygenase): glucose oxidase
Claims (8)
Nanoflowers in which amine-coated magnetic nanoparticles and oxidizing enzymes are combined with electrostatic attraction.
The magnetic nanoparticle according to claim 1, wherein the magnetic nanoparticles are coated with at least one amine selected from the group consisting of APTES ((3-Aminopropyl) triethoxysilane), poly-L-lysine and APTMS ((3-Aminopropyl) trimethoxysilane) Nano flower which we do with.
The nano-flower according to claim 1, wherein the oxidizing enzyme is glucose oxidase, glucose oxidase, cholesterol oxidase or protein oxidase.
A biosensor comprising the nanoflower according to any one of claims 1 to 3.
b) 상기 아민이 코팅된 자성 나노입자와 산화효소를 정전기적 인력으로 결합시키는 단계; 및
c) 상기 산화효소가 결합된 자성 나노입자를 염 용액과 반응시키는 단계;
를 포함하는 나노플라워 제조방법.
a) coating the magnetic nanoparticles with an amine group;
b) electrostatically attracting the amine-coated magnetic nanoparticles to the oxidizing enzyme; And
c) reacting the oxidized enzyme-bound magnetic nanoparticles with a salt solution;
≪ / RTI >
8. The method of claim 7, wherein the salt solution comprises at least one metal ion selected from the group consisting of Cu, Zn, Ti, and Sn.
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| Ge et al., Nature nanotechnology, Vol.7, pp.428-432 (2012. 07.)* |
| Misson et al., J.R.Soc.Interface 12:20140891.(2015.01.)* |
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