KR20170142601A - Matter for detecting virus using ZnO-carbon based quantom dot and method for fabricating the same - Google Patents
Matter for detecting virus using ZnO-carbon based quantom dot and method for fabricating the same Download PDFInfo
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Abstract
Description
본 발명은 산화아연-탄소기반 양자점을 이용한 바이러스 검출물질 및 그 제조방법에 관한 것으로서, 보다 상세하게는 FRET 현상을 이용하는 바이러스 검출물질을 구성함에 있어서 산화아연-탄소기반 양자점을 적용함으로써 인체 유해성을 회피함과 함께 바이러스 존재 유무를 용이하게 확인할 수 있는 산화아연-탄소기반 양자점을 이용한 바이러스 검출물질 및 그 제조방법에 관한 것이다.The present invention relates to a virus detecting substance using zinc oxide-carbon based quantum dots and a method for preparing the same, and more particularly, to a method for preparing a virus detecting substance using a FRET phenomenon by applying a zinc oxide- The present invention relates to a virus detection material using zinc oxide-carbon based quantum dots capable of easily confirming the presence or absence of a virus and a manufacturing method thereof.
형광공명에너지전이(FRET, fluorescence resonance energy transfer) 현상을 이용하여 바이오물질을 검출하는 바이오센서가 제시된 바 있다.A biosensor for detecting a biomaterial using fluorescence resonance energy transfer (FRET) phenomenon has been proposed.
형광공명에너지전이 현상 즉, FRET 현상은 2가지 형광물질의 상호작용을 이용한 것으로, 장거리 쌍극자-쌍극자 상호작용에 의하여 여기된(excited) 형광분자에서 다른 형광분자로 비복사 과정을 통해 에너지가 전이되는 물리적 현상이다. 이 때, 에너지를 주는 분자를 도너(doner), 에너지를 받는 분자를 억셉터(acceptor)라 한다.Fluorescence resonance energy transfer phenomenon, that is, FRET phenomenon, is based on the interaction of two fluorescent materials. Energy is transferred from fluorescent molecules excited by long-dipole-dipole interaction to other fluorescent molecules through non-radiation processes It is a physical phenomenon. At this time, the molecule that gives energy is called a donor, and the molecule that receives energy is called an acceptor.
특정 파장대의 빛을 방출하는 형광물질인 도너와, 도너로부터 방출되는 에너지를 흡수할 수 있는 형광물질인 억셉터가 소정 거리 이내로 근접하면, 외부에서 도너를 여기시키기 위해 조사된 빛 에너지가 억셉터로 비복사 전이되어 도너의 고유파장대의 발광이 감소하고, 도너로부터 에너지를 전달받은 억셉터의 고유파장대의 빛이 방출된다. 따라서, 억셉터가 Fㆆrester 반경 이내에 존재하는 경우, 도너의 방출강도는 감소한다.When the donor, which is a fluorescent material emitting light of a specific wavelength band, and the acceptor, which is a fluorescent material capable of absorbing energy emitted from the donor, are within a predetermined distance, light energy irradiated to excite the donor from the outside is transmitted to the acceptor The non-radiation transition causes the emission of the intrinsic wavelength band of the donor to decrease, and the light of the intrinsic wavelength band of the acceptor which has transferred the energy from the donor is emitted. Thus, if the acceptor is within the F ㆆ rester radius, the emission intensity of the donor decreases.
FRET 현상은 생물 내 매크로 분자의 치수(dimension)에 상당하는 약 10∼100Å 범위에서 일어나기 때문에 생물 분자 연구로의 응용과 관련하여 많은 관심을 받고 있으며, 앞서 언급한 바와 같이 바이오물질의 검출에 응용될 수 있다.Since the FRET phenomenon takes place in the range of about 10 to 100 Å, which corresponds to the dimension of macromolecules in living organisms, the FRET phenomenon has attracted much interest in application to biomolecular research. As mentioned above, .
한편, 양자점(quantom dot)은 나노크기의 결정으로서, 독특한 광학특성을 갖고 있어 최근 바이오분야에서 많은 주목을 받아왔다. 통상의 유기발색단, 형광단백질과 비교하면, 크기에 따른 스펙트럼 변화, 개선된 휘도, 광표백에 대한 우수한 안정성 및 동시 다중 형광 여기 등과 같은 특유의 유용한 광학특성을 갖고 있다. 또한, 양자점의 넓은 흡수 스펙트럼 및 좁은 방출 스펙트럼으로 안하여 FRET 현상을 기반으로 하는 바이오센서 또는 진단 시스템 내에서 도너(donor)로서 적합한 특성을 갖고 있다.On the other hand, quantum dots are nano-sized crystals and have unique optical properties and have received much attention in recent years in the field of biotechnology. Compared to conventional organic chromophores and fluorescent proteins, they have unique useful optical properties such as spectral changes according to size, improved brightness, excellent stability against photobleaching, and simultaneous multi-fluorescence excitation. It also has suitable characteristics as a donor in a biosensor or diagnostic system based on the FRET phenomenon, not on the broad absorption and narrow emission spectra of the quantum dots.
양자점과 FRET 현상을 이용한 바이오센서에 대한 연구로, Michele D. Kattke et al. 연구팀은 CdSe/ZnS 양자점과 항체 결합으로 말단에 흡수체(quencher) 붙여서 항원에 의해서 치환 및 소실에 의한 상태로 균(바이러스) 유무를 광 검출하는 방법을 개발하였고, 누룩곰팡이 세균으로 오염된 시료에 CdSe 양자점 결합을 반응시키면 항원-항체의 특이적 반응(흡수체 치환)에 의해 CdSe 양자점 결합이 균과 반응하며, 이 결합물에 일정한 파장 범위에서 자외선 광을 조사하면 CdSe 양자점 결합의 양자점이 형광을 일으키게 되어 검출하는 연구를 보고하였다 (Sensors 2011, 11, 6396-6410). 또한, 김기영 연구팀은 식중독균인 살모넬라 세균의 검출감도를 높이기 위해 CdSe 양자점 결합을 개발하여 연구 보고를 한 바 있다(Journal of Biosystems Eng. 35 (2010) 458).A study of biosensors using quantum dots and FRET phenomena, Michele D. Kattke et al. The team developed a method to detect the presence of a virus (virus) by substitution and disappearance by attaching an absorber (quencher) to the end by binding to CdSe / ZnS quantum dots and antibodies. In the samples contaminated with the yeast fungus bacteria, CdSe When the quantum dot bond is reacted, the CdSe quantum dot bond reacts with the bacterium by the specific reaction of the antigen-antibody (absorber substitution). When the ultraviolet light is irradiated to the conjugate in a certain wavelength range, the quantum dots of the CdSe quantum dot bond cause fluorescence (Sensors 2011, 11, 6396-6410). In addition, Kim, Ki-young and colleagues developed a CdSe quantum dot binding assay to increase detection sensitivity of Salmonella bacteria, a food poisoning bacterium (Journal of Biosystems Eng. 35 (2010) 458).
한편, 상술한 바이오센서는 모두 CdSe 양자점을 기반으로 하고 있어, 카드뮴(Cd)에 의한 인체 유해성이 문제가 되며, FRET 현상을 확인하기 위해 고가의 형광분석장비가 요구되는 단점이 있다.On the other hand, all of the above-described biosensors are based on CdSe quantum dots, which is a problem of human hazards due to cadmium (Cd), and there is a disadvantage that expensive fluorescence analysis equipment is required to confirm FRET phenomenon.
본 발명은 상기와 같은 문제점을 해결하기 위해 안출한 것으로서, FRET 현상을 이용하는 바이러스 검출물질을 구성함에 있어서 산화아연-탄소기반 양자점을 적용함으로써 인체 유해성을 회피함과 함께 바이러스 존재 유무를 용이하게 확인할 수 있는 산화아연-탄소기반 양자점을 이용한 바이러스 검출물질 및 그 제조방법을 제공하는데 그 목적이 있다.DISCLOSURE Technical Problem The present invention has been devised in order to solve the above problems, and it is an object of the present invention to provide a virus detection material using a FRET phenomenon by applying a zinc oxide-carbon based quantum dot to avoid human harmfulness, The present invention also provides a method for detecting a virus using the zinc oxide-carbon based quantum dot.
상기의 목적을 달성하기 위한 본 발명에 따른 산화아연-탄소기반 양자점을 이용한 바이러스 검출물질은 산화아연-탄소기반 양자점; 상기 산화아연-탄소기반 양자점의 일측에 연결된 항체; 및 상기 항체의 일단에 연결된 흡수체를 포함하여 이루어지는 것을 특징으로 한다.In order to accomplish the above object, a virus detecting material using zinc oxide-based carbon nanotubes according to the present invention comprises a zinc oxide-carbon based quantum dot; An antibody linked to one side of the zinc oxide-carbon based quantum dot; And an absorber connected to one end of the antibody.
상기 산화아연-탄소기반 양자점은 산화아연-그래핀 양자점 또는 산화아연-플러렌 양자점이다. 또한, 상기 산화아연-그래핀 양자점 및 산화아연-플러렌 양자점은, 산화아연이 중심부에 위치하고, 그래핀 또는 플러렌이 산화아연과 화학적으로 결합함과 함께 산화아연의 둘레에 위치하는 코어-쉘(core-shell) 구조를 이룬다.The zinc oxide-carbon based quantum dot is a zinc oxide-graphene quantum dot or a zinc oxide-fullerene quantum dot. The zinc oxide-graphene quantum dot and the zinc oxide-fullerene quantum dot may be formed in such a manner that zinc oxide is located at the center and graphene or fullerene is chemically bonded to the zinc oxide, -shell) structure.
상기 산화아연-양자점과 흡수체는 산화아연-탄소기반 양자점과 흡수체는 FRET(fluorescence resonance energy transfer) 현상을 유발하는 형광물질이며, 산화아연-탄소기반 양자점과 흡수체는 FRET 현상이 발생될 수 있는 Fㆆrester 반경 내에 위치한다.The zinc oxide-quantum dot and absorber are fluorescent materials that cause a fluorescence resonance energy transfer (FRET) phenomenon, and zinc oxide-carbon based quantum dots and absorbers are F It is located within the rester radius.
상기 흡수체는 산화아연-탄소기반 양자점에 의해 발광된 빛을 흡수하는 특성을 가지며, 상기 흡수체는 300∼600nm 파장대의 빛을 흡수하는 물질이다. 또한, 상기 흡수체는 BHQ-0로 구성할 수 있으며, BHQ-0는 아래와 같은 화학구조를 갖는 다.The absorber has a property of absorbing light emitted by a zinc oxide-carbon based quantum dot, and the absorber is a material that absorbs light in a wavelength range of 300 to 600 nm. Further, the absorber may be composed of BHQ-0, and BHQ-0 has the following chemical structure.
상기 항체는 검출대상 바이러스와 항원-항체 반응이 가능한 물질이며, 상기 흡수체는 상기 항체와 검출대상 바이러스의 항원-항체 반응에 따라, 검출대상 바이러스가 존재하는 환경에서 검출대상 바이러스에 의해 치환 가능하다.The antibody is a substance capable of reacting with a detection target virus by an antigen-antibody reaction, and the absorber can be replaced by a virus to be detected in an environment in which the detection subject virus is present according to an antigen-antibody reaction between the antibody and the detection subject virus.
자외선이 조사됨과 함께 검출대상 바이러스가 존재하는 환경 하에서, 상기 항체와 검출대상 바이러스의 항원-항체 반응에 의해 상기 흡수체가 검출대상 바이러스에 의해 치환되면, 자외선 조사에 의해 산화아연-탄소기반 양자점으로부터 발광된 빛은 흡수체에 흡수되지 않는다.When the absorber is replaced with the virus to be detected by an antigen-antibody reaction between the antibody and the detection subject virus in an environment in which ultraviolet rays are irradiated and the virus to be detected exists, ultraviolet light is irradiated from the zinc oxide- Is not absorbed by the absorber.
본 발명에 따른 산화아연-탄소기반 양자점을 이용한 바이러스 검출물질 제조방법은 산화아연-탄소기반 양자점을 준비하는 단계; 산화아연-탄소기반 양자점과 항체를 결합시키는 단계; 및 상기 항체의 일단에 흡수체를 결합시키는 단계를 포함하여 이루어지는 것을 특징으로 한다.The method for preparing a virus detection material using zinc oxide-carbon based quantum dots according to the present invention comprises: preparing a zinc oxide-carbon based quantum dot; Binding the zinc oxide-carbon based Qdot to the antibody; And binding the absorber to one end of the antibody.
상기 산화아연-그래핀 양자점은, 산화된 흑연(graphite oxide)이 분산된 용액과 Zinc acetate dehydrate[Zn(COO)2-H2O]용액을 혼합하여 제조할 수 있다. 또한, 상기 산화아연-플러렌 양자점은, 산화된 플러렌(fullerene oxide)이 분산된 용액과 Zinc acetate dehydrate[Zn(COO)2-H2O]용액을 혼합하여 제조할 수 있다.The zinc oxide-graphene quantum dot can be prepared by mixing a solution of oxidized graphite oxide and a solution of zinc acetate dehydrate [Zn (COO) 2 -H 2 O]. The zinc oxide-fullerene quantum dot can be prepared by mixing a solution of oxidized fullerene oxide and a solution of zinc acetic acid dehydrate [Zn (COO) 2 -H 2 O].
본 발명에 따른 산화아연-탄소기반 양자점을 이용한 바이러스 검출물질 및 그 제조방법은 다음과 같은 효과가 있다.The virus detection material using zinc oxide-based carbon nanotubes according to the present invention and the manufacturing method thereof have the following effects.
산화아연-탄소기반의 양자점으로 바이러스 검출물질을 구성함에 따라 인체 유해성을 회피할 수 있으며, 바이러스 검출물질을 대기 중에 분사하고 자외선과의 반응 여부에 따라 바이러스의 존재 유무를 용이하게 파악할 수 있다.By constituting the virus detection material with the zinc oxide-carbon based quantum dots, it is possible to avoid human harm, and it is possible to easily detect the presence or absence of the virus according to whether the virus detection material is sprayed into the air and reacted with ultraviolet rays.
도 1은 본 발명의 일 실시예에 따른 산화아연-탄소기반 양자점을 이용한 바이러스 검출물질의 FRET 현상 유무에 따른 여기된 빛의 흡수 및 방출을 나타낸 참고도.
도 2a는 실시예 1 및 실시예 2에 따라 제조된 기능화된(functionalized) 산화아연-탄소기반 양자점의 모식도이고, 도 2b는 기능화된 항체의 모식도.
도 3은 실시예 3에 따른 산화아연-탄소기반 양자점과 항체의 결합 과정을 나타낸 모식도.
도 4는 실시예 4에 따른 항체와 흡수체의 결합 과정을 나타낸 모식도.FIG. 1 is a reference view showing excitation and emission of excited light according to presence or absence of FRET phenomenon of a virus detecting material using zinc oxide-carbon based quantum dots according to an embodiment of the present invention.
Figure 2a is a schematic diagram of functionalized zinc oxide-based carbon-based quantum dots prepared according to Example 1 and Example 2, and Figure 2b is a schematic diagram of a functionalized antibody.
FIG. 3 is a schematic view showing a binding process of an antibody with zinc oxide-based QDs according to Example 3. FIG.
4 is a schematic view showing a binding process of an antibody and an absorber according to Example 4. Fig.
본 발명은 산화아연-탄소기반 양자점 및 FRET 현상을 이용하여 바이러스를 검출하는 바이러스 검출물질에 대한 기술을 제시한다.The present invention provides a technique for detecting a virus detecting substance using zinc oxide-carbon based quantum dots and FRET phenomenon.
'발명의 배경이 되는 기술'에서 설명한 바와 같이, FRET(fluorescence resonance energy transfer) 현상은 두 형광물질의 상호작용에 의해 여기된(excited) 형광물질에서 다른 형광물질로 에너지가 전이되는 현상을 일컫는다.As described in the Background of the Invention, a fluorescence resonance energy transfer (FRET) phenomenon refers to a phenomenon in which energy is transferred from a fluorescent material excited by the interaction of two fluorescent materials to another fluorescent material.
본 발명에 따른 산화아연-탄소기반 양자점을 이용한 바이러스 검출물질은 산화아연-탄소기반 양자점에 항체(anti-body) 및 흡수체(quencher)가 구비된 구조를 이루며, 산화아연-탄소기반 양자점과 흡수체가 FRET 현상을 일으키는 두 형광물질에 해당된다. 또한, 산화아연-탄소기반 양자점과 흡수체는 FRET 현상이 발생될 수 있는 거리 즉, Fㆆrester 반경 내에 위치한다.The virus detection material using zinc oxide-based carbon nanotubes according to the present invention has a structure in which an anti-body and an absorber are provided on a zinc oxide-carbon based quantum dot, and a zinc oxide-carbon based quantum dot and an absorber It corresponds to two fluorescent substances causing FRET phenomenon. In addition, the zinc oxide-carbon based quantum dots and absorbers are located within a distance that the FRET phenomenon can occur, that is, a radius of F ㆆ rester.
본 발명에 있어서, 산화아연-탄소기반 양자점이라 함은 산화아연(ZnO)과 탄소물질이 코어-쉘(core-shell) 구조를 이루는 양자점을 일컫는 것으로서, 산화아연이 중심부(core)에 위치하고 탄소물질이 산화아연의 둘레(shell)에 위치하는 구조이다. 또한, 상기 탄소물질은 그래핀(graphene) 또는 플러렌(fullerene)이며, 이에 본 발명의 산화아연-탄소기반 양자점이라 함은 산화아연-그래핀 양자점 또는 산화아연-플러렌 양자점을 의미한다.In the present invention, the zinc oxide-carbon based quantum dot refers to a quantum dot in which zinc oxide (ZnO) and a carbon material form a core-shell structure, in which zinc oxide is located in the core, Is located in the shell of zinc oxide. Also, the carbon material is graphene or fullerene, and the zinc oxide-carbon based quantum dot of the present invention means zinc oxide-graphene quantum dot or zinc oxide-fullerene quantum dot.
산화아연(ZnO)는 광 밴드갭(band gap)이 3.0 eV 이상으로 자외선을 흡수할 수 있으며, 산화아연(ZnO)에 산소(O)를 매개로 화학적으로 결합되는 그래핀 또는 플러렌은 전자이동도, 열전도도 및 유연성이 뛰어나다. 이와 같은 산화아연과 그래핀(또는 플러렌)이 코어-쉘 구조를 이루게 되면 전계발광이 가시광선 영역으로 확대되는 특성을 갖는다.Zinc oxide (ZnO) can absorb ultraviolet rays with a band gap of 3.0 eV or more. Graphene or fullerene, which is chemically bonded to zinc oxide (ZnO) through oxygen (O), has electron mobility , Excellent thermal conductivity and flexibility. When the zinc oxide and the graphene (or fullerene) have a core-shell structure, the electroluminescence expands to the visible light region.
본 발명에 따른 산화아연-탄소기반 양자점을 이용한 바이러스 검출물질은 바이러스의 존재 유무를 확인함에 이용되며, 그 원리는 다음과 같다.The virus detection material using zinc oxide-based carbon nanotubes according to the present invention is used for confirming the presence or absence of a virus, and the principle thereof is as follows.
본 발명에 따른 산화아연-탄소기반 양자점을 이용한 바이러스 검출물질은 앞서 언급한 바와 같이, 산화아연-탄소기반 양자점에 항체(anti-body) 및 흡수체(quencher)가 구비된 구조를 이룬다. 세부적으로, 산화아연-탄소기반 양자점의 일지점에 항체가 연결되고, 항체의 일단에 흡수체가 구비된다. 또한, 산화아연-탄소기반 양자점과 흡수체는 FRET(fluorescence resonance energy transfer) 현상을 유발하는 형광물질이며, 산화아연-탄소기반 양자점과 흡수체는 FRET 현상이 발생될 수 있는 거리 즉, Fㆆrester 반경 내에 위치한다.As described above, the virus detecting material using the zinc oxide-carbon based quantum dot according to the present invention has a structure in which an anti-body and an absorber are provided in a zinc oxide-carbon based quantum dot. Specifically, the antibody is linked to one point of the zinc oxide-carbon based quantum dot, and an absorber is provided at one end of the antibody. The zinc oxide-carbon based quantum dots and absorbers are fluorescence materials that cause FRET (fluorescence resonance energy transfer) phenomenon. The zinc oxide-carbon based quantum dots and absorbers are located at a distance where FRET phenomenon can occur, that is, Located.
상기 산화아연-탄소기반 양자점은 자외선 조사에 의해 여기(excited)되어 발광하는 특성을 갖고 있으며, 상기 흡수체는 산화아연-탄소기반 양자점에 의해 발광된 빛을 흡수하는 특성을 갖고 있다(도 1 참조). 산화아연-탄소기반 양자점의 여기된 빛이 흡수체에 흡수되는 현상은 FRET(fluorescence resonance energy transfer) 현상에 기인한다.The zinc oxide-carbon based quantum dots have a characteristic of being excited by ultraviolet irradiation to emit light, and the absorber has a characteristic of absorbing light emitted by zinc oxide-carbon based quantum dots (see FIG. 1) . The phenomenon that the excited light of the zinc oxide-carbon based quantum dots is absorbed by the absorber is due to the fluorescence resonance energy transfer (FRET) phenomenon.
한편, 상기 흡수체는 항원인 바이러스가 존재하는 환경에서 바이러스에 의해 치환될 수 있다. 즉, 검출대상 바이러스가 존재하는 환경 하에 본 발명에 따른 바이러스 검출물질이 노출되면, 항원-항체 반응에 의해 항체 일단에 구비된 흡수체는 검출대상 바이러스에 의해 치환된다. 여기서, 상기 항원-항체 반응은 항원과 항체의 결합 또는 그에 따른 반응결과를 의미한다.On the other hand, the absorber can be replaced by a virus in an environment where a virus as an antigen is present. That is, when the virus detecting substance according to the present invention is exposed under the environment in which the virus to be detected exists, the absorber provided at one end of the antibody by the antigen-antibody reaction is replaced by the virus to be detected. Here, the antigen-antibody reaction refers to the binding of the antigen to the antibody or the result of the reaction.
형광물질인 흡수체가 검출대상 바이러스에 의해 치환되면, FRET 현상은 발생되지 않는다(도 1 참조). 즉, 항체의 일단에 연결된 흡수체가 검출대상 바이러스에 의해 치환된 상태에서, 자외선 조사에 의해 산화아연-탄소기반 양자점이 발광되더라도 산화아연-탄소기반 양자점의 여기된 빛을 흡수할 수 있는 흡수체가 존재하지 않음에 따라 FRET 현상은 발생되지 않는다. 산화아연-탄소기반 양자점의 여기된 빛이 흡수체에 의해 흡수되지 않음은 산화아연-탄소기반 양자점의 여기된 빛에 대한 감지가 가능함을 의미한다.When the absorber, which is a fluorescent substance, is replaced by the virus to be detected, the FRET phenomenon does not occur (see Fig. 1). That is, even if the zinc oxide-based QDs are emitted by ultraviolet irradiation in a state where the absorber connected to one end of the antibody is substituted by the target virus, an absorber capable of absorbing the excited light of the zinc oxide- The FRET phenomenon does not occur. The fact that the excited light of the zinc oxide-carbon based Qdot is not absorbed by the absorber means that it is possible to detect the excited light of the zinc oxide-carbon based Qdot.
정리하면, 검출대상 바이러스가 존재하지 않는 환경 하에서는 산화아연-탄소기반 양자점의 여기된 빛이 흡수체에 의해 흡수되어 감지가 불가능하나, 검출대상 바이러스가 존재하는 환경 하에서는 흡수체가 검출대상 바이러스에 의해 치환됨으로 인해 산화아연-탄소기반 양자점의 여기된 빛이 흡수체에 흡수되지 않아 여기된 빛에 대한 감지가 가능하다. 이러한 여기된 빛에 대한 감지를 통해 검출대상 바이러스의 존재를 확인할 수 있다.In summary, under an environment where no target virus is present, the excited light of the zinc oxide-carbon based quantum dots is absorbed by the absorber and can not be detected. However, under the environment where the target virus is present, Due to the fact that the excited light of the zinc oxide-carbon based quantum dots is not absorbed by the absorber, it is possible to detect the excited light. The presence of the virus to be detected can be confirmed by detecting the excited light.
산화아연-탄소기반 양자점의 일측에 구비되는 항체는 검출대상 바이러스와 항원-항체 반응을 일으키는 물질로 이루어진다. 예를 들어, 검출대상 바이러스가 조류인플레엔자(AI) 바이러스인 경우, 항체로는 AI 바이러스와 항원-항체 반응을 일으키는 H9N2 단백질이 적용될 수 있다.The antibody provided on one side of the zinc oxide-carbon based quantum dots consists of a substance that causes an antigen-antibody reaction with a detection target virus. For example, when the virus to be detected is an avian infectious agent (AI) virus, H9N2 protein that causes an antigen-antibody reaction with an AI virus may be applied as an antibody.
한편, 항체의 일단에 구비되는 흡수체는 산화아연-탄소기반 양자점에서 여기되는 빛을 흡수할 수 있는 형광물질로 이루어진다. 산화아연-탄소기반 양자점의 여기된 빛의 파장은 385 nm, 415 nm and 435 nm이며, 상기 흡수체는 300∼600nm 파장대의 빛을 흡수할 수 있는 물질로 구성될 수 있다. 일 실시예로, 상기 흡수체는 BHQ(black hole quencher)-0를 이용할 수 있다. BHQ-0는 Biosearch Technologies社에서 제조 판매하는 상표명으로서, 아래의 화학구조를 갖고 있으며, 300∼600nm 파장대의 빛을 흡수할 수 있고 430-521 nm 파장대에서 흡수율이 최대치를 나타내는 특성을 갖고 있다.On the other hand, the absorber provided at one end of the antibody is composed of a fluorescent material capable of absorbing light excited at the zinc oxide-carbon based quantum dots. The wavelengths of the excited light of the zinc oxide-carbon based quantum dots are 385 nm, 415 nm and 435 nm, and the absorber can be composed of a material capable of absorbing light in a wavelength range of 300 to 600 nm. In one embodiment, the absorber may utilize BHQ (black hole quencher) -0. BHQ-0 is a brand name manufactured and sold by Biosearch Technologies, Inc. It has the following chemical structure and is capable of absorbing light in the wavelength range of 300 to 600 nm and exhibiting the maximum absorption in the wavelength range of 430 to 521 nm.
<BHQ-0의 화학구조><Chemical structure of BHQ-0>
이하에서는, 실시예를 통해 본 발명에 대해 보다 구체적으로 설명하기로 한다.Hereinafter, the present invention will be described in more detail by way of examples.
<실시예 1 : 산화아연-그래핀 양자점 제조>≪ Example 1: Preparation of zinc oxide-graphene quantum dot >
40ml의 디메틸포름아마이드(N,N-dimethylforamide)에 산화된 흑연(graphite oxide) 40mg을 넣고 분산기에서 10분간 넣고 분산시켜 준다. 그리고 200ml 디메틸포름아마이드(N,N-dimethylforamide)에 0.93g의 Zinc acetate dehydrate [Zn(COO)2-H2O]를 넣고 저어준다. 10분 후 분산시킨 산화된 흑연 용액과 Zinc acetate dehydrate[Zn(COO)2-H2O]용액을 섞고 온도는 95℃로 맞춰주고 5시간 150rpm으로 유지시킨다. 처음 용액의 색은 검은색이지만 30분 후 용액은 투명하게 변하게 되고 1시간이 지나면 용액은 뿌옇게 변하면서 점차 흰색을 띄는 용액이 된다. 5시간 후 투명한 용액에 검은 갈색 빛이 있는 파우더가 생기는데, 이 파우더를 에탄올로 세척하고 다시 증류수로 세척한 다음 55℃ 오븐에서 천천히 건조시켜 준다. 이렇게 하여 생성된 산화아연-그래핀 양자점(ZnO@graphene) 파우더를 제조하였다.Add 40 mg of oxidized graphite oxide to 40 ml of N, N-dimethylforamide and allow to disperse in the disperser for 10 minutes. Add 0.93 g of Zinc acetate dehydrate [Zn (COO) 2 -H 2 O] to 200 ml of N, N-dimethylforamide and stir. After 10 minutes, the oxidized graphite solution and Zinc acetate dehydrate [Zn (COO) 2 -H 2 O] solution are mixed and the temperature is kept at 95 ° C and maintained at 150 rpm for 5 hours. The color of the first solution is black, but after 30 minutes, the solution turns transparent and after 1 hour, the solution becomes cloudy and gradually becomes a white solution. After 5 hours, a clear brownish-colored powder is formed in the clear solution. The powder is washed with ethanol, again with distilled water, and slowly dried in an oven at 55 ° C. The thus-produced zinc oxide-graphene quantum dot (ZnO @ graphene) powder was prepared.
<실시예 2 : 산화아연-플러렌 양자점 제조>≪ Example 2: Preparation of zinc oxide-fullerene quantum dot &
40ml의 디메틸포름아마이드(N,N-dimethylforamide)에 산화된 플러렌(fullerene oxide) 40mg을 넣고 분산기에서 10분간 분산시켜 준다. 그리고 200ml 디메틸포름아마이드(N,N-dimethylforamide)에 0.93g의 Zinc acetate dehydrate [Zn(COO)2-H2O]를 넣고 저어준다. 분산시킨 산화된 플러렌 용액과 Zinc acetate dehydrate[Zn(COO)2-H2O]용액을 섞고 온도는 140℃로 맞춰주고 3시간 정도로 하고 150rpm으로 유지시킨다. 처음 용액의 색은 검은색이지만 30분 후 용액은 투명하게 변하게 되고 1시간이 지나면 용액은 뿌옇게 변하면서 점차 흰색을 띄는 용액이 된다. 5시간 후 투명한 용액에 검은 갈색 빛이 있는 파우더가 생기는데, 이 파우더를 에탄올로 세척하고 다시 증류수로 세척한 다음 55℃ 오븐에서 천천히 건조시켜 준다. 이렇게 하여 생성된 산화아연-플러렌 양자점(ZnO@fullerene) 파우더를 제조하였다.Add 40 mg of oxidized fullerene oxide to 40 ml of N, N-dimethylformamide and disperse in a disperser for 10 minutes. Add 0.93 g of Zinc acetate dehydrate [Zn (COO) 2 -H 2 O] to 200 ml of N, N-dimethylforamide and stir. Mix the dispersed oxidized fullerene solution with Zinc acetate dehydrate [Zn (COO) 2 -H 2 O] solution. Set the temperature at 140 ° C and maintain the temperature at 150 rpm for about 3 hours. The color of the first solution is black, but after 30 minutes, the solution turns transparent and after 1 hour, the solution becomes cloudy and gradually becomes a white solution. After 5 hours, a clear brownish-colored powder is formed in the clear solution. The powder is washed with ethanol, again with distilled water, and slowly dried in an oven at 55 ° C. The zinc oxide-fullerene quantum dot (ZnO @ fullerene) powder thus produced was prepared.
<실시예 3 : 산화아연-탄소기반 양자점과 항체의 결합>≪ Example 3: Binding of zinc oxide-carbon based Qdots to antibodies >
10 mM의 succinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate crosslinker (SMCC) 를 15 분 동안 37 ℃로 평형화 하였다. 이어, 4 μM 아민유도체(amine-derivatized) 125 μL에 14 μL를 첨가하여 아민유도체 용액을 준비하였다. 실시예 1과 실시예 2를 통해 제조된 산화아연-탄소기반 양자점을 폴리에틸렌글리콜(PEG)로 코팅한 양자점 용액을 준비하였다. 양자점 용액에 아민유도체 용액을 혼합하였다. H9N2 단백질이 용해된 항체 용액을 준비하고, 아민유도체가 포함된 양자점 용액에 항체 용액을 혼합하여 항체 결합 반응을 유도하였다. H9N2에 존재하는 티올(thiols)은 SMCC 활성화 나노결정에 존재하는 반응성 maleimide groups에 결합하였다. 반응물을 30 분 동안 10 mM의 2-mercaptoethanol 10 μL로 켄칭시켰다. 이어, 50kDa의 MWCO 필터를 15 분 동안 7000 RPM에서 원심 분리하여 40 μL의 최종 부피로 농축시켰다. 농축된 접합체(산화아연-탄소기반 양자점과 항체가 결합된 물질)는 분리 칼럼을 이용하여 정제하였다. 도 2a는 실시예 1 및 실시예 2에 따라 제조된 기능화된(functionalized) 산화아연-탄소기반 양자점의 모식도이고, 도 2b는 기능화된 항체의 모식도이며, 도 3은 실시예 3에 따른 산화아연-탄소기반 양자점과 항체의 결합 과정을 나타낸 모식도이다.10 mM succinimidyl-4- (N-maleimidomethyl) cyclohexane-1-carboxylate crosslinker (SMCC) was equilibrated at 37 ° C for 15 minutes. Then, 14 μL was added to 125 μL of 4 μM amine-derivatized to prepare an amine derivative solution. A quantum dot solution prepared by coating zinc oxide-carbon based quantum dots prepared in Example 1 and Example 2 with polyethylene glycol (PEG) was prepared. The amine derivative solution was mixed with the quantum dot solution. The antibody solution in which the H9N2 protein was dissolved was prepared, and the antibody solution was mixed with the quantum dot solution containing the amine derivative to induce the antibody binding reaction. The thiols present in H9N2 bound to reactive maleimide groups present in SMCC activated nanocrystals. The reaction was quenched with 10 [mu] L of 10 mM 2-mercaptoethanol for 30 minutes. The 50 kDa MWCO filter was then centrifuged at 7000 RPM for 15 minutes to concentrate to a final volume of 40 [mu] L. Concentrated conjugates (zinc oxide-carbon based quantum dots and antibody bound material) were purified using a separation column. FIG. 2A is a schematic diagram of functionalized zinc oxide-based carbon nanotubes prepared according to Example 1 and Example 2, FIG. 2B is a schematic diagram of a functionalized antibody, and FIG. 3 is a schematic view of a zinc oxide- Fig. 2 is a schematic diagram showing the binding process of a carbon-based Qdot and an antibody.
<실시예 4 : 흡수체의 결합>≪ Example 4: bonding of absorber >
5mg BHQ-0를 50 uL의 DMSO에 용해시키고, 200mg의 1,5-diaminopentane dihydrochloride(DAP, Sigma-Aldrich, St. Louis, MO)를 0.136 M 붕산나트륨 완충액 1ml(pH 8.5)를 용해시킨 다음, 두 용액을 혼합, 교반시켰다. 이어, 10 % 글리세롤이 첨가된 100 uL HE 완충액을 준비하고 이를 상기 혼합용액에 첨가하였다. 실시예 3을 통해 제조된 산화아연-탄소기반 양자점과 항체가 결합된 물질을 상기 최종 혼합용액에 혼합한 다음, 암실에서 써모믹서를 이용하여 90도 에서 5분 동안 교반한 후 상온에서 2시간 동안 교반시켜 항체와 흡수체의 결합을 유도하였다. 항체 및 흡수체가 결합된 양자점을 탄산수소나트륨을 이용하여 세척하였다. 도 4는 실시예 4에 따른 항체와 흡수체의 결합 과정을 나타낸 모식도이다.5 mg BHQ-0 was dissolved in 50 uL of DMSO and 1 ml of 0.136 M sodium borate buffer (pH 8.5) was dissolved in 200 mg of 1,5-diaminopentane dihydrochloride (DAP, Sigma-Aldrich, St. Louis, MO) The two solutions were mixed and stirred. Then, 100 uL HE buffer solution to which 10% glycerol was added was prepared and added to the mixed solution. The zinc oxide-carbon based quantum dots and the antibody-bound material prepared in Example 3 were mixed in the final mixed solution, stirred in a dark room at 90 degrees for 5 minutes using a thermomixer, and then incubated at room temperature for 2 hours Followed by stirring to induce binding between the antibody and the absorber. The quantum dots to which the antibody and the absorber were bound were washed with sodium bicarbonate. 4 is a schematic view showing a binding process of an antibody and an absorber according to Example 4. Fig.
Claims (15)
상기 산화아연-탄소기반 양자점의 일측에 연결된 항체; 및
상기 항체의 일단에 연결된 흡수체를 포함하여 이루어지는 것을 특징으로 하는 산화아연-탄소기반 양자점을 이용한 바이러스 검출물질.
Zinc oxide-carbon based quantum dots;
An antibody linked to one side of the zinc oxide-carbon based quantum dot; And
And an absorber connected to one end of the antibody.
The virus detection material according to claim 1, wherein the zinc oxide-carbon based quantum dots are zinc oxide-graphene quantum dots or zinc oxide-fullerene quantum dots.
산화아연이 중심부에 위치하고, 그래핀 또는 플러렌이 산화아연과 화학적으로 결합함과 함께 산화아연의 둘레에 위치하는 코어-쉘(core-shell) 구조를 이루는 것을 특징으로 하는 산화아연-탄소기반 양자점을 이용한 바이러스 검출물질.
The method of claim 2, wherein the zinc oxide-graphene quantum dot and the zinc oxide-
Characterized in that zinc oxide is located in the center and graphene or fullerene is chemically bonded to the zinc oxide and forms a core-shell structure located around the zinc oxide. Used virus detection substance.
The method of claim 1, wherein the zinc oxide-based carbon nanotubes and the absorber are zinc oxide-carbon based quantum dots and the absorber is a fluorescent material that causes FRET (fluorescence resonance energy transfer) Wherein the zinc oxide-based QDs are located within a F ㆆ rester radius at which development can occur.
The method of claim 1, wherein the absorber has a property of absorbing light emitted by zinc oxide-based carbon-based quantum dots, and the absorber is a material that absorbs light in a wavelength range of 300 to 600 nm. Virus detection substance using quantum dots.
The virus detection material of claim 1, wherein the absorber is BHQ-0 and the BHQ-0 has the following chemical structure.
상기 흡수체는 상기 항체와 검출대상 바이러스의 항원-항체 반응에 따라, 검출대상 바이러스가 존재하는 환경에서 검출대상 바이러스에 의해 치환 가능한 것을 특징으로 하는 산화아연-탄소기반 양자점을 이용한 바이러스 검출물질.
The method according to claim 1, wherein the antibody is a substance capable of reacting with a detection target virus with an antigen-
Wherein the absorber is replaceable with a virus to be detected in an environment in which a detection subject virus is present according to an antigen-antibody reaction between the antibody and a detection subject virus.
상기 항체와 검출대상 바이러스의 항원-항체 반응에 의해 상기 흡수체가 검출대상 바이러스에 의해 치환되면, 자외선 조사에 의해 산화아연-탄소기반 양자점으로부터 발광된 빛은 흡수체에 흡수되지 않는 것을 특징으로 하는 산화아연-탄소기반 양자점을 이용한 바이러스 검출물질.
8. The method according to claim 7, wherein, in an environment in which ultraviolet light is irradiated and a detection target virus is present,
Wherein the light emitted from the zinc oxide-carbon based quantum dots by ultraviolet irradiation is not absorbed by the absorber when the absorber is replaced by the virus to be detected by the antigen-antibody reaction between the antibody and the detection target virus. - Detection of viruses using carbon-based QDs.
The virus detection substance according to claim 1, wherein the antibody is a H9N2 protein.
산화아연-탄소기반 양자점과 항체를 결합시키는 단계; 및
상기 항체의 일단에 흡수체를 결합시키는 단계를 포함하여 이루어지는 것을 특징으로 하는 산화아연-탄소기반 양자점을 이용한 바이러스 검출물질 제조방법.
Preparing a zinc oxide-carbon based quantum dot;
Binding the zinc oxide-carbon based Qdot to the antibody; And
And attaching an absorber to one end of the antibody. The method for producing a virus-detecting substance using the zinc oxide-carbon based quantum dot according to claim 1,
11. The method of claim 10, wherein the zinc oxide-carbon based quantum dots are zinc oxide-graphene quantum dots or zinc oxide-fullerene quantum dots.
산화아연이 중심부에 위치하고, 그래핀 또는 플러렌이 산화아연과 화학적으로 결합함과 함께 산화아연의 둘레에 위치하는 코어-쉘(core-shell) 구조를 이루는 것을 특징으로 하는 산화아연-탄소기반 양자점을 이용한 바이러스 검출물질 제조방법.
The method of claim 11, wherein the zinc oxide-graphene quantum dot and the zinc oxide-
Characterized in that zinc oxide is located in the center and graphene or fullerene is chemically bonded to the zinc oxide and forms a core-shell structure located around the zinc oxide. A method for producing a virus detection substance using the same.
상기 흡수체는 산화아연-탄소기반 양자점에 의해 발광된 빛을 흡수하는 특성을 갖는 것을 특징으로 하는 산화아연-탄소기반 양자점을 이용한 바이러스 검출물질 제조방법.
11. The method of claim 10, wherein the zinc oxide-based carbon nanotubes and the absorber are fluorescent materials that cause a fluorescence resonance energy transfer (FRET) phenomenon, and the zinc oxide- Lt; / RTI >
Wherein the absorber has a property of absorbing light emitted by zinc oxide-carbon based quantum dots.
12. The method of claim 11, wherein the zinc oxide-graphene quantum dot is prepared by mixing a solution of oxidized graphite oxide and a solution of zinc acetic acid dehydrate [Zn (COO) 2 -H 2 O] A method for producing a virus detection substance using zinc oxide-carbon based quantum dots.
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