KR20240037851A - Nanostructure comprising polymersomes and quantum dots, and method for detecting virus using the same - Google Patents
Nanostructure comprising polymersomes and quantum dots, and method for detecting virus using the same Download PDFInfo
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- 239000002086 nanomaterial Substances 0.000 title claims abstract description 56
- 239000002096 quantum dot Substances 0.000 title claims abstract description 38
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- 238000000034 method Methods 0.000 title claims description 5
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- UHYPYGJEEGLRJD-UHFFFAOYSA-N cadmium(2+);selenium(2-) Chemical compound [Se-2].[Cd+2] UHYPYGJEEGLRJD-UHFFFAOYSA-N 0.000 claims description 21
- 239000005083 Zinc sulfide Substances 0.000 claims description 17
- 241001135549 Porcine epidemic diarrhea virus Species 0.000 claims description 11
- DRDVZXDWVBGGMH-UHFFFAOYSA-N zinc;sulfide Chemical compound [S-2].[Zn+2] DRDVZXDWVBGGMH-UHFFFAOYSA-N 0.000 claims description 11
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- 238000006243 chemical reaction Methods 0.000 claims description 10
- CCCMONHAUSKTEQ-UHFFFAOYSA-N octadec-1-ene Chemical compound CCCCCCCCCCCCCCCCC=C CCCMONHAUSKTEQ-UHFFFAOYSA-N 0.000 claims description 10
- 229910052717 sulfur Inorganic materials 0.000 claims description 10
- 239000011593 sulfur Substances 0.000 claims description 10
- RMZAYIKUYWXQPB-UHFFFAOYSA-N trioctylphosphane Chemical compound CCCCCCCCP(CCCCCCCC)CCCCCCCC RMZAYIKUYWXQPB-UHFFFAOYSA-N 0.000 claims description 9
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- LQJBNNIYVWPHFW-UHFFFAOYSA-N 20:1omega9c fatty acid Natural products CCCCCCCCCCC=CCCCCCCCC(O)=O LQJBNNIYVWPHFW-UHFFFAOYSA-N 0.000 claims description 4
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- 239000005642 Oleic acid Substances 0.000 claims description 4
- BUGBHKTXTAQXES-UHFFFAOYSA-N Selenium Chemical compound [Se] BUGBHKTXTAQXES-UHFFFAOYSA-N 0.000 claims description 4
- 239000004480 active ingredient Substances 0.000 claims description 4
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- QXJSBBXBKPUZAA-UHFFFAOYSA-N isooleic acid Natural products CCCCCCCC=CCCCCCCCCC(O)=O QXJSBBXBKPUZAA-UHFFFAOYSA-N 0.000 claims description 4
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- ZQPPMHVWECSIRJ-KTKRTIGZSA-N oleic acid Chemical compound CCCCCCCC\C=C/CCCCCCCC(O)=O ZQPPMHVWECSIRJ-KTKRTIGZSA-N 0.000 claims description 4
- 238000010926 purge Methods 0.000 claims description 4
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- WGPCGCOKHWGKJJ-UHFFFAOYSA-N sulfanylidenezinc Chemical compound [Zn]=S WGPCGCOKHWGKJJ-UHFFFAOYSA-N 0.000 claims description 3
- JBQYATWDVHIOAR-UHFFFAOYSA-N tellanylidenegermanium Chemical compound [Te]=[Ge] JBQYATWDVHIOAR-UHFFFAOYSA-N 0.000 claims description 3
- 241000701386 African swine fever virus Species 0.000 claims description 2
- 241001397616 Influenza A virus (H1N1) Species 0.000 claims description 2
- AQCDIIAORKRFCD-UHFFFAOYSA-N cadmium selenide Chemical compound [Cd]=[Se] AQCDIIAORKRFCD-UHFFFAOYSA-N 0.000 claims description 2
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- 238000001338 self-assembly Methods 0.000 claims description 2
- 238000001514 detection method Methods 0.000 abstract description 10
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- AFCARXCZXQIEQB-UHFFFAOYSA-N N-[3-oxo-3-(2,4,6,7-tetrahydrotriazolo[4,5-c]pyridin-5-yl)propyl]-2-[[3-(trifluoromethoxy)phenyl]methylamino]pyrimidine-5-carboxamide Chemical compound O=C(CCNC(=O)C=1C=NC(=NC=1)NCC1=CC(=CC=C1)OC(F)(F)F)N1CC2=C(CC1)NN=N2 AFCARXCZXQIEQB-UHFFFAOYSA-N 0.000 description 2
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- 241000127282 Middle East respiratory syndrome-related coronavirus Species 0.000 description 1
- NIPNSKYNPDTRPC-UHFFFAOYSA-N N-[2-oxo-2-(2,4,6,7-tetrahydrotriazolo[4,5-c]pyridin-5-yl)ethyl]-2-[[3-(trifluoromethoxy)phenyl]methylamino]pyrimidine-5-carboxamide Chemical compound O=C(CNC(=O)C=1C=NC(=NC=1)NCC1=CC(=CC=C1)OC(F)(F)F)N1CC2=C(CC1)NN=N2 NIPNSKYNPDTRPC-UHFFFAOYSA-N 0.000 description 1
- 241000315672 SARS coronavirus Species 0.000 description 1
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- 229910007339 Zn(OAc)2 Inorganic materials 0.000 description 1
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- KGBXLFKZBHKPEV-UHFFFAOYSA-N boric acid Chemical compound OB(O)O KGBXLFKZBHKPEV-UHFFFAOYSA-N 0.000 description 1
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- 229940098773 bovine serum albumin Drugs 0.000 description 1
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- 241000712461 unidentified influenza virus Species 0.000 description 1
- DJWUNCQRNNEAKC-UHFFFAOYSA-L zinc acetate Chemical compound [Zn+2].CC([O-])=O.CC([O-])=O DJWUNCQRNNEAKC-UHFFFAOYSA-L 0.000 description 1
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Abstract
본 발명은 폴리머좀(polymersome) 및 양자점(quantum dot)을 포함하는 나노구조체 및 이를 이용한 바이러스 검출 방법에 관한 것으로, 상기 나노구조체를 통한 바이러스 검출 방법이 기존의 바이러스 검출 방법보다 고민감도 및 고특이도를 나타내어, 낮은 민감도를 나타내는 환자에서도 바이러스를 정확하게 검출할 수 있음을 확인함으로써, 바이러스 검출 방법으로써 유용하게 활용될 수 있다.The present invention relates to a nanostructure containing polymersomes and quantum dots and a virus detection method using the same. The virus detection method using the nanostructure has higher sensitivity and specificity than existing virus detection methods. By confirming that the virus can be accurately detected even in patients with low sensitivity, it can be usefully used as a virus detection method.
Description
본 발명은 폴리머좀(polymersome) 및 양자점(quantum dot)을 포함하는 나노구조체 및 이를 이용한 바이러스 검출 방법에 관한 것이다.The present invention relates to a nanostructure containing polymersomes and quantum dots and a virus detection method using the same.
2018년 세계보건기구(WHO)는 "추후 세계 대유행을 일으킬 바이러스 8가지"를 발표하였고, 상기 8가지의 바이러스를 "질병 X(disease X)"라 명명하였다. 상기 바이러스는 에볼라 바이러스, 말버그 바이러스, 라싸열, 지카바이러스, 중증급성호흡기증후군 코로나바이러스, 중동호흡기증후군 코로나바이러스 등을 포함하고, 이 중 최초로 "질병 X"로 명명된 바이러스는 최근 확산된 코로나19 바이러스이다.In 2018, the World Health Organization (WHO) announced “8 viruses that will cause global pandemics in the future” and named these 8 viruses “disease X.” The above viruses include Ebola virus, Malberg virus, Lassa fever, Zika virus, Severe Acute Respiratory Syndrome coronavirus, Middle East Respiratory Syndrome coronavirus, etc. Among these, the first virus to be named "Disease X" is the recently spread COVID-19. It's a virus.
대유행인 감염병을 빠른 시간 내 진단; 전파 차단; 열악한 지역에서의 검체 진단 목적 등을 위해서는 1시간 이내로 결과가 나오는 현장진단의 필요성은 매우 절대적이다. 신속진단은 표준검사와 달리 현장에서 신속하게 감염여부를 판단할 수 있는 장점이 있지만, 타 진단 방법에 비해 민감도가 낮아 무증상 또는 감염 초기 환자를 놓칠 수 있다는 단점이 있다. 따라서, 낮은 민감도를 가지는 기존 현장진단의 단점을 극복한 고민감도 및 고특이도를 가지는 새로운 현장진단 플랫폼 개발이 필요한 상황이다.Rapid diagnosis of pandemic infectious diseases; blocking radio waves; For purposes such as sample diagnosis in poor areas, the need for on-site diagnosis that produces results within one hour is absolutely necessary. Unlike standard tests, rapid diagnosis has the advantage of being able to quickly determine infection on site, but it has the disadvantage of being less sensitive than other diagnostic methods and potentially missing asymptomatic or early-stage infected patients. Therefore, there is a need to develop a new on-site diagnostic platform with high sensitivity and specificity that overcomes the shortcomings of existing on-site diagnostics with low sensitivity.
본 발명의 목적은 폴리머좀(polymersome) 및 양자점(quantum dot)을 포함하는 나노구조체를 제공하는 것이다.The purpose of the present invention is to provide a nanostructure containing polymersomes and quantum dots.
본 발명의 다른 목적은 상기 나노구조체 제조방법을 제공하는 것이다.Another object of the present invention is to provide a method for manufacturing the nanostructure.
본 발명의 또 다른 목적은 항체를 결합한 상기 나노구조체를 유효성분으로 포함하는 바이러스 검출용 조성물을 제공하는 것이다.Another object of the present invention is to provide a composition for detecting viruses containing the nanostructure bound to an antibody as an active ingredient.
본 발명의 또 다른 목적은 상기 항체를 결합한 상기 나노구조체를 이용한 바이러스 검출 방법을 제공하는 것이다.Another object of the present invention is to provide a virus detection method using the nanostructure bound to the antibody.
상기 목적을 달성하기 위해, 본 발명은 폴리머좀(polymersome) 및 양자점(quantum dot)을 포함하는 나노구조체를 제공한다.To achieve the above object, the present invention provides a nanostructure containing polymersomes and quantum dots.
또한, 본 발명은 양친매성 고분자를 자기조립하여 폴리머좀(polymersome)을 제조하는 단계(제1단계); Cd(OAc)2, Zn(OAc)2 및 올레익산(oleic acid)을 혼합하고, 가스를 제거한 후, 1-ODE(1-octadecene)를 혼합하고, 탈기하는 단계(제2단계); 상기 제2단계의 탈기한 혼합물을 질소 퍼징(purging) 및 온도 조절을 통해 Cd(OAc)2 및 Zn(OAc)2 용액을 제조하는 단계(제3단계); TOP(trioctylphosphine)에 셀레늄(Se) 및 황(S)을 용해시키고, 상기 제3단계에서 제조한 용액과 혼합하여 셀렌화 카드뮴 및 황화아연 결합체(CdSe@ZnS)를 제조하는 단계(제4단계); 상기 제4단계에서 제조한 셀렌화 카드뮴 및 황화아연 결합체(CdSe@ZnS)에 황(S)을 혼합하여 아연황 쉘(ZnS shell)로 오버코팅하는 단계(제5단계); 상기 제5단계에서 오버코팅한 셀렌화 카드뮴 및 황화아연 결합체(CdSe@ZnS)에 아연(Zn) 및 황(S)을 혼합하고 반응시켜 양자점(quantum dot)을 제조하는 단계(제6단계); 및 상기 제1단계에서 제조한 폴리머좀(polymersome); 및 상기 제6단계에서 제조한 양자점(quantum dot)을 혼합하는 단계(제7단계)를 포함하는, 폴리머좀-양자점 나노구조체 제조방법을 제공한다.In addition, the present invention includes the steps of self-assembling amphiphilic polymers to produce polymersomes (first step); Mixing Cd(OAc) 2 , Zn(OAc) 2 and oleic acid, removing gas, mixing 1-ODE (1-octadecene), and degassing (second step); Preparing Cd(OAc) 2 and Zn(OAc) 2 solutions from the degassed mixture of the second step through nitrogen purging and temperature control (third step); A step of dissolving selenium (Se) and sulfur (S) in TOP (trioctylphosphine) and mixing with the solution prepared in the third step to prepare a cadmium selenide and zinc sulfide conjugate (CdSe@ZnS) (step 4) ; Mixing sulfur (S) with the cadmium selenide and zinc sulfide composite (CdSe@ZnS) prepared in the fourth step and overcoating it with a zinc sulfur shell (ZnS shell) (step 5); Mixing and reacting zinc (Zn) and sulfur (S) with the cadmium selenide and zinc sulfide conjugate (CdSe@ZnS) overcoated in the fifth step to produce quantum dots (step 6); And polymersome prepared in the first step; and a step (seventh step) of mixing the quantum dots prepared in the sixth step.
또한, 본 발명은 항체를 결합한 상기 나노구조체를 유효성분으로 포함하는 바이러스 검출용 조성물을 제공한다.In addition, the present invention provides a composition for detecting a virus comprising the nanostructure bound to an antibody as an active ingredient.
또한, 본 발명은 상기 나노구조체에 항체를 결합하는 단계(제1단계); 및 상기 제1단계의 항체를 결합한 나노구조체; 및 항원의 항원-항체 반응을 측정하는 단계(제2단계)를 포함하는, 바이러스 검출 방법을 제공한다.In addition, the present invention includes the step of binding an antibody to the nanostructure (first step); and a nanostructure combining the antibodies of the first step; and measuring the antigen-antibody reaction of the antigen (second step).
본 발명에 따르면, 폴리머좀(polymersome) 및 양자점(quantum dot)을 포함하는 나노구조체를 통한 바이러스 검출 방법이 기존의 바이러스 검출 방법보다 고민감도 및 고특이도를 나타내어, 낮은 민감도를 나타내는 환자에서도 바이러스를 정확하게 검출할 수 있음을 확인함으로써, 바이러스 검출 방법으로써 유용하게 활용될 수 있다.According to the present invention, a virus detection method using a nanostructure containing polymersomes and quantum dots exhibits higher sensitivity and specificity than existing virus detection methods, so that viruses can be detected even in patients with low sensitivity. By confirming that it can be detected accurately, it can be usefully used as a virus detection method.
도 1은 실시예 1-2에서 제조한 양자점의 특성을 분석한 결과이다. Q.D; quantum dot.
도 2는 실시예 1-3에서 제조한 폴리머좀-양자점 하이브리드 나노구조체의 특성(크기)을 분석한 결과이다.
도 3은 실시예 1-4에서 제조한 항체 부착 폴리머좀-양자점 하이브리드 나노구조체의 특성을 분석한 결과이다. QP@Psome; 폴리머좀-양자점 나노구조체, QP@Psome+Ab; 항체 부착 폴리머좀-양자점 나노구조체.
도 4는 상기 폴리머좀-양자점 하이브리드 나노구조체에 부착하는 바이러스 항체 종류에 따라 항원-항체 반응에 차이가 나타나는지 분석한 결과이다. QP@Psome; 폴리머좀-양자점 나노구조체, QP@Psome+H1N1; H1N1 항체 부착 폴리머좀-양자점 나노구조체; QP@Psome+PEDV; PEDV(Porcine epidemic diarrhea virus) 항체 부착 폴리머좀-양자점 나노구조체.Figure 1 shows the results of analyzing the characteristics of quantum dots prepared in Example 1-2. QD; quantum dot.
Figure 2 shows the results of analyzing the characteristics (size) of the polymersome-quantum dot hybrid nanostructure prepared in Examples 1-3.
Figure 3 shows the results of analyzing the characteristics of the antibody-attached polymersome-quantum dot hybrid nanostructure prepared in Examples 1-4. QP@Psome; Polymersome-quantum dot nanostructure, QP@Psome+Ab; Antibody-attached polymersome-quantum dot nanostructure.
Figure 4 shows the results of analyzing whether there is a difference in the antigen-antibody reaction depending on the type of viral antibody attached to the polymersome-quantum dot hybrid nanostructure. QP@Psome; polymersome-quantum dot nanostructure, QP@Psome+H1N1; H1N1 antibody-attached polymersome-quantum dot nanostructure; QP@Psome+PEDV; Polymersome-quantum dot nanostructure with PEDV (Porcine epidemic diarrhea virus) antibody attached.
이하, 본 발명을 보다 상세하게 설명한다.Hereinafter, the present invention will be described in more detail.
본 발명은 폴리머좀(polymersome) 및 양자점(quantum dot)을 포함하는 나노구조체를 제공한다.The present invention provides a nanostructure containing polymersomes and quantum dots.
상기 폴리머좀은 양친매성 고분자로 제조한 것일 수 있고, 상기 양친매성 고분자는 mPEG-b-PLA, NHS-PEG-PLA, COOH-PEG-PLA 및 NH2-PEG-PLA로 이루어진 군에서 선택된 하나 이상을 포함할 수 있으나, 이에 한정되는 것은 아니다. The polymersome may be prepared from an amphipathic polymer, and the amphiphilic polymer is one or more selected from the group consisting of mPEG-b-PLA, NHS-PEG-PLA, COOH-PEG-PLA, and NH 2 -PEG-PLA. It may include, but is not limited to this.
또한, 상기 폴리머좀은 자기조립(self-assembly) 특성을 나타내는 것일 수 있다.Additionally, the polymersome may exhibit self-assembly characteristics.
상기 양자점은 셀렌화 카드뮴(CdSe) 또는 황화아연(ZnS)을 포함할 수 있다.The quantum dots may include cadmium selenide (CdSe) or zinc sulfide (ZnS).
상기 나노구조체의 입경은 150 내지 250nm일 수 있다.The particle diameter of the nanostructure may be 150 to 250 nm.
또한, 본 발명은 양친매성 고분자를 자기조립하여 폴리머좀(polymersome)을 제조하는 단계(제1단계); Cd(OAc)2, Zn(OAc)2 및 올레익산(oleic acid)을 혼합하고, 가스를 제거한 후, 1-ODE(1-octadecene)를 혼합하고, 탈기하는 단계(제2단계); 상기 제2단계의 탈기한 혼합물을 질소 퍼징(purging) 및 온도 조절을 통해 Cd(OAc)2 및 Zn(OAc)2 용액을 제조하는 단계(제3단계); TOP(trioctylphosphine)에 셀레늄(Se) 및 황(S)을 용해시키고, 상기 제3단계에서 제조한 용액과 혼합하여 셀렌화 카드뮴 및 황화아연 결합체(CdSe@ZnS)를 제조하는 단계(제4단계); 상기 제4단계에서 제조한 셀렌화 카드뮴 및 황화아연 결합체(CdSe@ZnS)에 황(S)을 혼합하여 아연황 쉘(ZnS shell)로 오버코팅하는 단계(제5단계); 상기 제5단계에서 오버코팅한 셀렌화 카드뮴 및 황화아연 결합체(CdSe@ZnS)에 아연(Zn) 및 황(S)을 혼합하고 반응시켜 양자점(quantum dot)을 제조하는 단계(제6단계); 및 상기 제1단계에서 제조한 폴리머좀(polymersome); 및 상기 제6단계에서 제조한 양자점(quantum dot)을 혼합하는 단계(제7단계)를 포함하는, 폴리머좀-양자점 나노구조체 제조방법을 제공한다.In addition, the present invention includes the steps of self-assembling amphiphilic polymers to produce polymersomes (first step); Mixing Cd(OAc) 2 , Zn(OAc) 2 and oleic acid, removing gas, mixing 1-ODE (1-octadecene), and degassing (second step); Preparing Cd(OAc) 2 and Zn(OAc) 2 solutions from the degassed mixture of the second step through nitrogen purging and temperature control (third step); A step of dissolving selenium (Se) and sulfur (S) in TOP (trioctylphosphine) and mixing with the solution prepared in the third step to prepare a cadmium selenide and zinc sulfide conjugate (CdSe@ZnS) (step 4) ; Mixing sulfur (S) with the cadmium selenide and zinc sulfide composite (CdSe@ZnS) prepared in the fourth step and overcoating it with a zinc sulfur shell (ZnS shell) (step 5); Mixing and reacting zinc (Zn) and sulfur (S) with the cadmium selenide and zinc sulfide conjugate (CdSe@ZnS) overcoated in the fifth step to produce quantum dots (step 6); And the polymersome prepared in the first step; and a step (seventh step) of mixing the quantum dots prepared in the sixth step.
또한, 본 발명은 항체를 결합한 상기 나노구조체를 유효성분으로 포함하는 바이러스 검출용 조성물을 제공한다.In addition, the present invention provides a composition for detecting a virus comprising the nanostructure bound to an antibody as an active ingredient.
상기 항체는 A형 인플루엔자 바이러스(H1N1) 항체, 아프리카 돼지 열병 바이러스(African swine fever virus) 항체 및 돼지 유행성 설사병 바이러스(Porcine epidemic diarrhea virus; PEDV) 항체로 이루어진 군에서 선택된 하나 이상일 수 있으나, 이에 한정되는 것은 아니다.The antibody may be one or more selected from the group consisting of influenza A virus (H1N1) antibodies, African swine fever virus antibodies, and porcine epidemic diarrhea virus (PEDV) antibodies, but is limited thereto. That is not the case.
또한, 본 발명은 상기 나노구조체에 항체를 결합하는 단계(제1단계); 및 상기 제1단계의 항체를 결합한 나노구조체; 및 항원의 항원-항체 반응을 측정하는 단계(제2단계)를 포함하는, 바이러스 검출 방법을 제공한다.In addition, the present invention includes the steps of binding an antibody to the nanostructure (first step); and a nanostructure combining the antibodies of the first step; and measuring the antigen-antibody reaction of the antigen (second step).
이하, 본 발명의 이해를 돕기 위하여 실시예를 들어 상세하게 설명하기로 한다. 다만 하기의 실시예는 본 발명의 내용을 예시하는 것일 뿐 본 발명의 범위가 하기 실시예에 한정되는 것은 아니다. 본 발명의 실시예는 당업계에서 평균적인 지식을 가진 자에게 본 발명을 보다 완전하게 설명하기 위해 제공되는 것이다.Hereinafter, the present invention will be described in detail through examples to aid understanding. However, the following examples only illustrate the content of the present invention and the scope of the present invention is not limited to the following examples. Examples of the present invention are provided to more completely explain the present invention to those skilled in the art.
[실시예 1] 폴리머좀-양자점 나노구조체 제조[Example 1] Preparation of polymersome-quantum dot nanostructure
1-1. 양자점 제조1-1. Quantum dot manufacturing
양자점(quantum dot)을 제조하기 위해, 50mL 3구 플라스크에 0.14mmol(32.27mg)의 Cd(OAc)2, 3.41mmol(625.67mg)의 Zn(OAc)2 및 7mL의 올레익산(oleic acid; 이하 OA라 함)을 첨가하고 진공 하에서 150℃에서 가스를 제거한 후, 온도를 100℃로 낮추고 15mL의 1-octadecene(이하 1-ODE라 함)를 플라스크에 주입한 후, 30분 동안 탈기하였다. 그 후, 질소 퍼징(purging)을 하면서 Cd(OAc)2 및 Zn(OAc)2의 투명한 용액을 얻기 위해 온도를 310℃로 상승시켰다. 5mL의 trioctylphosphine(이하 TOP라 함)에 5mmol(394.8mg)의 Se(셀레늄) 및 5mmol(160.33mg)의 S(황)를 용해하여 제조한 2mL의 TOPSeS를 310℃에서 플라스크에 빠르게 주입한 후, CdSe@ZnS의 성장을 위해 10분 동안 반응시켰다. 그 후, 2.4mL의 S 공급원[4.8mL의 1-ODE에서 3.2mmol의 S(102.61mg)]을 CdSe@ZnS 코어의 현탁액에 주입하여 CdSe@ZnS 코어를 ZnS 쉘(shell)로 오버코팅하고, 12분 동안 반응시켰다. 그 후, 5mL의 Zn 공급원[4.92mmol(902.72mg)의 Zn(OAc)2, 2mL의 OA 또는 8mL의 ODE(octadecene)]을 현탁액에 주입하고, 270℃에서 쿨링(cooling)하였다. 그 후, 5mL S의 전구체(10mL TOP의 내 19.3mmol(618.85mg)의 S)를 반응조에 0.5mL/분의 속도로 적가하고 20분 동안 반응시켰다. 반응을 완료하기 위해, 반응기를 실온으로 빠르게 냉각하고, 50mL 에탄올 및 10mL 헥산을 첨가하여 침전시킨 후, 8000rpm에서 10분 동안 원심분리하고 헥산(hexane)에 재분산시켰다. 정제 단계(헥산:에탄올 = 1:4)를 3회 반복하고, 양자점 추가 사용을 위해 클로로포름(30mL)에 분산시켰다.To prepare quantum dots, 0.14 mmol (32.27 mg) of Cd(OAc) 2 , 3.41 mmol (625.67 mg) of Zn(OAc) 2 and 7 mL of oleic acid (hereinafter) were added to a 50 mL three-necked flask. After adding OA) and removing the gas at 150°C under vacuum, the temperature was lowered to 100°C and 15 mL of 1-octadecene (hereinafter referred to as 1-ODE) was injected into the flask and degassed for 30 minutes. Afterwards, the temperature was raised to 310°C to obtain a transparent solution of Cd(OAc) 2 and Zn(OAc) 2 while purging with nitrogen. 2 mL of TOPSeS prepared by dissolving 5 mmol (394.8 mg) of Se (selenium) and 5 mmol (160.33 mg) of S (sulfur) in 5 mL of trioctylphosphine (hereinafter referred to as TOP) was quickly injected into the flask at 310°C. For the growth of CdSe@ZnS, the reaction was performed for 10 minutes. Afterwards, 2.4 mL of S source [3.2 mmol of S (102.61 mg) in 4.8 mL of 1-ODE] was injected into the suspension of CdSe@ZnS core to overcoat the CdSe@ZnS core with ZnS shell; It was reacted for 12 minutes. Afterwards, 5 mL of Zn source [4.92 mmol (902.72 mg) of Zn(OAc)2, 2 mL of OA or 8 mL of ODE (octadecene)] was injected into the suspension and cooled at 270°C. Afterwards, 5 mL of S precursor (19.3 mmol (618.85 mg) of S in 10 mL TOP) was added dropwise to the reaction tank at a rate of 0.5 mL/min and reacted for 20 minutes. To complete the reaction, the reactor was quickly cooled to room temperature, precipitated by adding 50 mL ethanol and 10 mL hexane, centrifuged at 8000 rpm for 10 minutes, and redispersed in hexane. The purification step (hexane:ethanol = 1:4) was repeated three times, and the quantum dots were dispersed in chloroform (30 mL) for further use.
상기 제조한 양자점의 특성을 분석한 결과, 도 1과 같이, 530nm 파장을 가지는 녹색 형광이 나타나는 것을 PL(Photoluminescence) 강도 분석을 통해 확인하였고, 양자점 크기는 12nm로 나타나, ZnS 쉘 오버코팅을 통해 기존 양자점 대비 크기가 증가했음을 투과전자현미경(Transmission Electron Microscope; TEM) 관찰을 통해 확인하였다. 또한, 양자점 응집에 의한 형광 파장 변화를 분석한 결과, 도 1과 같이, 양자점 응집 시, 기존 방출 파장과 다른 방출 파장으로 변화하는 것을 확인하였다.As a result of analyzing the characteristics of the manufactured quantum dots, it was confirmed through PL (Photoluminescence) intensity analysis that green fluorescence with a wavelength of 530 nm appeared, as shown in Figure 1, and the size of the quantum dots was found to be 12 nm. It was confirmed through transmission electron microscope (TEM) observation that the size increased compared to the quantum dots. Additionally, as a result of analyzing the change in fluorescence wavelength due to quantum dot aggregation, as shown in Figure 1, it was confirmed that when quantum dots agglomerate, the emission wavelength changes to a different emission wavelength from the existing emission wavelength.
1-2. 폴리머좀-양자점 나노구조체 제조1-2. Manufacturing of polymersome-quantum dot nanostructures
폴리머좀-양자점 나노구조체(이하 하이브리드 나노구조체라 함)를 제조하기 위해, 양친매성 고분자[=폴리머좀, mPEG(2K)-b-PLA(8K)] 5mg을 0.5ml의 CHCl3에 용해하고, 상기 용해한 용액에 상기 실시예 1-2에서 제조한 양자점의 용액을 첨가한 후, 60℃에서 10분 동안 초음파 처리(Ultrasonication)하였다.To prepare polymersome-quantum dot nanostructures (hereinafter referred to as hybrid nanostructures), 5 mg of amphiphilic polymer [=polymersome, mPEG(2K)-b-PLA(8K)] was dissolved in 0.5 ml of CHCl 3 , The quantum dot solution prepared in Example 1-2 was added to the dissolved solution, and then ultrasonication was performed at 60°C for 10 minutes.
상기 제조한 하이브리드 나노구조체의 크기를 분석한 결과, 도 2와 같이, 상기 하이브리드 나노구조체의 평균 크기는 224.67nm로, 약 200nm의 크기로 형성된 것을 확인하였다.As a result of analyzing the size of the hybrid nanostructure prepared above, it was confirmed that the average size of the hybrid nanostructure was 224.67nm, which was formed at a size of about 200nm, as shown in FIG. 2.
1-3. 항체 부착 하이브리드 나노구조체 제조1-3. Manufacture of antibody-attached hybrid nanostructure
상기 실시예 1-3에서 제조한 하이브리드 나노구조체에 항체를 부착하기 위해, 하이브리드 나노구조체 10mL에 K2C03 200μL 및 항체(H1N1 항체, invitrogen, 5mg/mL) 100μL를 첨가하고, 30분 동안 상온에서 교반하였다. 그 후, 10% 소혈청알부민(Bovine Serum Albumin; 이하 BSA라 함) 1mL를 첨가하고, 15분 동안 추가 교반한 후, 8000rpm, 20분 및 상온 조건에서 원심 분리하였다. 그 후, 상등액을 제거하고, 1% BSA(in 붕산 20mM) 10mL를 첨가하였다.In order to attach an antibody to the hybrid nanostructure prepared in Example 1-3, 200 μL of K 2 CO 3 and 100 μL of antibody (H1N1 antibody, invitrogen, 5 mg/mL) were added to 10 mL of the hybrid nanostructure, and incubated at room temperature for 30 minutes. It was stirred. Afterwards, 1 mL of 10% bovine serum albumin (hereinafter referred to as BSA) was added, stirred for additional 15 minutes, and then centrifuged at 8000 rpm, 20 minutes, and room temperature. Afterwards, the supernatant was removed, and 10 mL of 1% BSA (in boric acid 20mM) was added.
상기 제조한 항체 부착 하이드리드 나노구조체의 특성을 분석한 결과, 도 3과 같이, 평균 크기는 319.17nm로, 항체를 부착하지 않은 하이브리드 나노구조체 대비 약 80nm 유체학적 크기가 증가하였다. 또한, 항체 부착 전후로 하이브리드 나노구조체의 형광 방출 파장에는 유의한 변화가 나타나지 않았다.As a result of analyzing the characteristics of the antibody-attached hybrid nanostructure prepared above, as shown in Figure 3, the average size was 319.17nm, which increased the fluidic size by about 80nm compared to the hybrid nanostructure without an antibody attached. Additionally, there was no significant change in the fluorescence emission wavelength of the hybrid nanostructure before and after antibody attachment.
[실시예 2] 바이러스 검출 효과 분석[Example 2] Virus detection effect analysis
상기 하이브리드 나노구조체에 부착하는 바이러스 항체 종류에 따라 항원-항체 반응에 차이가 나타나는지 확인하기 위해, 상기 하이브리드 나노구조체에 인플루엔자 바이러스인 H1N1 항체(invitrogen); 및 돼지 유행성 설사병 바이러스(Porcine epidemic diarrhea virus; 이하 PEDV라 함, invitrogen) 항체를 각각 부착하여 항체 부착 하이브리드 나노구조체를 제조하고, 각각의 항원과의 반응성을 확인하기 위해, 상기 항체 부착 하이브리드 나노구조체; 및 항원 50μl를 상온에서 15분 동안 반응시킨 후, 형광 강도를 분석한 결과, 도 4와 같이, 항체 미부착 하이브리드 나노구조체(대조군) 대비 H1N1 항체 부착 하이브리드 나노구조체의 형광 강도가 감소한 반면, PEDV 항체 부착 하이브리드 나노구조체의 형광 강도는 유의한 변화가 나타나지 않았고, 이를 통해 H1N1가 PEDV보다 높은 항원-항체 반응을 나타내는 것을 확인하였다. 상기 결과로부터, 상기 하이브리드 나노구조체는 부착하는 바이러스 항체 종류에 따라 항원-항체 반응에 차이가 나타남을 확인하였다.In order to determine whether there is a difference in the antigen-antibody reaction depending on the type of viral antibody attached to the hybrid nanostructure, an H1N1 antibody (invitrogen), which is an influenza virus, is added to the hybrid nanostructure; and Porcine epidemic diarrhea virus (hereinafter referred to as PEDV, invitrogen) antibodies were attached to prepare an antibody-attached hybrid nanostructure, and to confirm the reactivity with each antigen, the antibody-attached hybrid nanostructure; And after reacting 50 μl of the antigen for 15 minutes at room temperature, the fluorescence intensity was analyzed. As shown in Figure 4, the fluorescence intensity of the H1N1 antibody-attached hybrid nanostructure decreased compared to the antibody-unattached hybrid nanostructure (control group), whereas the PEDV antibody-attached hybrid nanostructure decreased. There was no significant change in the fluorescence intensity of the hybrid nanostructure, confirming that H1N1 exhibits a higher antigen-antibody reaction than PEDV. From the above results, it was confirmed that the hybrid nanostructure showed differences in antigen-antibody reactions depending on the type of viral antibody attached.
이상으로 본 발명 내용의 특정한 부분을 상세히 기술하였는 바, 당업계의 통상의 지식을 가진 자에게 있어서, 이러한 구체적 기술은 단지 바람직한 실시양태일 뿐이며, 이에 의해 본 발명의 범위가 제한되는 것이 아닌 점은 명백하다. 즉, 본 발명의 실질적인 범위는 첨부된 청구항들과 그것들의 등가물에 의하여 정의된다.As the specific parts of the present invention have been described in detail above, it is clear to those skilled in the art that these specific techniques are merely preferred embodiments and do not limit the scope of the present invention. do. That is, the practical scope of the present invention is defined by the appended claims and their equivalents.
Claims (10)
Cd(OAc)2, Zn(OAc)2 및 올레익산(oleic acid)을 혼합하고, 가스를 제거한 후, 1-ODE(1-octadecene)를 혼합하고, 탈기하는 단계(제2단계);
상기 제2단계의 탈기한 혼합물을 질소 퍼징(purging) 및 온도 조절을 통해 Cd(OAc)2 및 Zn(OAc)2 용액을 제조하는 단계(제3단계);
TOP(trioctylphosphine)에 셀레늄(Se) 및 황(S)을 용해시키고, 상기 제3단계에서 제조한 용액과 혼합하여 셀렌화 카드뮴 및 황화아연 결합체(CdSe@ZnS)를 제조하는 단계(제4단계);
상기 제4단계에서 제조한 셀렌화 카드뮴 및 황화아연 결합체(CdSe@ZnS)에 황(S)을 혼합하여 아연황 쉘(ZnS shell)로 오버코팅하는 단계(제5단계);
상기 제5단계에서 오버코팅한 셀렌화 카드뮴 및 황화아연 결합체(CdSe@ZnS)에 아연(Zn) 및 황(S)을 혼합하고 반응시켜 양자점(quantum dot)을 제조하는 단계(제6단계); 및
상기 제1단계에서 제조한 폴리머좀(polymersome); 및 상기 제6단계에서 제조한 양자점(quantum dot)을 혼합하는 단계(제7단계)를 포함하는, 폴리머좀-양자점 나노구조체 제조방법.Self-assembling amphiphilic polymers to produce polymersomes (first step);
Mixing Cd(OAc) 2 , Zn(OAc) 2 and oleic acid, removing gas, mixing 1-ODE (1-octadecene), and degassing (second step);
Preparing Cd(OAc) 2 and Zn(OAc) 2 solutions from the degassed mixture of the second step through nitrogen purging and temperature control (third step);
A step of dissolving selenium (Se) and sulfur (S) in TOP (trioctylphosphine) and mixing with the solution prepared in the third step to prepare a cadmium selenide and zinc sulfide conjugate (CdSe@ZnS) (step 4) ;
Mixing sulfur (S) with the cadmium selenide and zinc sulfide composite (CdSe@ZnS) prepared in the fourth step and overcoating it with a zinc sulfur shell (ZnS shell) (step 5);
Mixing and reacting zinc (Zn) and sulfur (S) with the cadmium selenide and zinc sulfide conjugate (CdSe@ZnS) overcoated in the fifth step to produce quantum dots (step 6); and
Polymersome prepared in the first step; and a step (seventh step) of mixing the quantum dots prepared in the sixth step.
상기 제1단계의 항체를 결합한 나노구조체; 및 항원의 항원-항체 반응을 측정하는 단계(제2단계)를 포함하는, 바이러스 검출 방법.Binding an antibody to the nanostructure of claim 1 (first step); and
A nanostructure combining the antibodies of the first step; and measuring the antigen-antibody reaction of the antigen (second step).
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