KR20130106044A - Biosensor using the graphene oxide by the polymer of enzyme substrate and method of detecting using it - Google Patents
Biosensor using the graphene oxide by the polymer of enzyme substrate and method of detecting using it Download PDFInfo
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Abstract
Description
본 발명은 효소 기질의 고분자화에 의한 그라핀옥사이드의 형광저하를 이용한 바이오센서 및 이를 이용한 검출방법에 관한 것으로, 보다 상세하게는, 형광 염료의 사용없이 그라핀옥사이드 자체의 고유한 형광 특성을 이용하여, 검출 물질의 존재 여부를 광학적으로 식별할 수 있는 효소 기질의 고분자화에 의한 그라핀옥사이드의 형광저하를 이용한 바이오센서 및 이를 이용한 검출방법에 관한 것이다.
The present invention relates to a biosensor using fluorescence reduction of graphene oxide by polymerizing an enzyme substrate and a detection method using the same, and more particularly, using the inherent fluorescence characteristics of graphene oxide itself without the use of fluorescent dyes. The present invention relates to a biosensor using fluorescence reduction of graphene oxide by polymerizing an enzyme substrate that can optically identify the presence of a detection substance, and a detection method using the same.
최근에 의료비 절감 및 생명 연장을 위해서 질병의 조기 진단에 대한 필요성이 점점 증가하고 있다. 특정 질병 여부를 알려주는 생체지표물질은 대부분의 경우, 매우 낮은 농도로 시료 속에 존재하기 때문에, 생체지표물질의 존재 및 농도를 고감도로 측정할 수 있는 바이오 센서가 필요하다.In recent years, the need for early diagnosis of diseases is increasing to reduce medical expenses and prolong life. Biomarkers that indicate whether a particular disease is present in most cases are present in the sample at very low concentrations, and thus, a biosensor capable of measuring the presence and concentration of the biomarker with high sensitivity is needed.
바이오센서란 생체감지물질(bio receptor)과 신호 변환기(signal transducer)로 구성되어 인식 가능한 신호로 변환하여 분석하고자하는 물질을 선택적으로 감지하는 장치이다. 생체감지물질로는 특정 물질과 선택적으로 반응 및 결합할 수 있는 효소, 항체, 항원, 호르몬 수요체 등이 있으며, 신호 변환 방법으로는 전기화학, 형광, 발색, SPR(surface plasmon resonance), FET(fieldeffecttransistor), QCM(quartz crystal microbalance), 열센서 등 다양한 물리화학적 방법을 사용한다. A biosensor is a device that selectively detects a substance to be analyzed by converting it into a recognizable signal composed of a bioreceptor and a signal transducer. Biosensors include enzymes, antibodies, antigens, and hormone demanders that can selectively react with and bind to specific substances. Signal transduction methods include electrochemistry, fluorescence, coloration, surface plasmon resonance (SPR), and FET ( Various physicochemical methods such as field effect transistors, quartz crystal microbalance (QCM), and thermal sensors are used.
상기의 바이오센서의 신호 변환 방법 중에서 형광 방식은 물질간의 결합에 따른 형광 특성 변화를 유도하기 위하여 염료를 사용하고, 표적 물질(센싱 대상 물질)과 프로브 물질(특정 물질과 선택적으로 반응 및 결합하는 물질)을 염료로 표지화(labeling)시켜야하므로 제조 공정이 복잡하고, 염료의 높은 가격으로 인해 경제성이 떨어지는 문제점이 있다. Among the signal conversion methods of the biosensor, the fluorescence method uses a dye to induce a change in fluorescence characteristics according to the binding between materials, and a material that selectively reacts with and binds to a target material (a sensing target material) and a probe material (a specific material). ) Has to be labeled with a dye (complicated) manufacturing process is complicated, due to the high price of the dye, there is a problem that the economy is poor.
이에, 한국 출원특허 제10-2009-0084855호는 그라핀옥사이드를 이용하여, 표적 물질(센싱 대상 물질)을 금 나노입자로 표지화한 다음, 금 나노입자의 성장으로 인하여 그라핀옥사이드의 형광 특성을 감소시키는 바이오센서를 제조하였으나, 표적 물질에 금 나노입자를 표지화하는 공정이 복잡하고, 표적물질인 단일 스트랜드 DNA의 길이가 짧거나 긴 경우에는 금 나노입자에 의한 형광 저하가 발생하지 않아 표적 물질을 검출하지 못하는 문제점이 있다.Accordingly, Korean Patent Application No. 10-2009-0084855 uses a graphene oxide to label a target material (sensing material) with gold nanoparticles, and then display the fluorescence characteristics of the graphene oxide due to the growth of the gold nanoparticles. Although biosensors have been prepared for reducing, the process of labeling gold nanoparticles on a target material is complicated, and when the length of a single stranded DNA, which is a target material, is short or long, fluorescence deterioration by the gold nanoparticles does not occur. There is a problem that cannot be detected.
이에, 본 발명자들은 상기 문제점을 해결하기 위하여 예의 노력한 결과, 그라핀옥사이드층에 고정화된 포획 항체와 표적 항원을 결합시키고, 효소를 함유하는 검출 항체를 결합시켜 효소 기질을 첨가한 다음, 효소 기질의 고분자화를 유도하여 표적 항원을 검출할 경우, 형광 염료의 사용없이 우수한 검출 한도를 확인하고, 본 발명을 완성하게 되었다. Accordingly, the present inventors have made diligent efforts to solve the above problems. As a result, the present inventors have combined the capture antibody immobilized on the graphene oxide layer with the target antigen, add the enzyme substrate by binding the detection antibody containing the enzyme, and then add the enzyme substrate. When the target antigen is detected by inducing polymerization, excellent detection limits are confirmed without the use of fluorescent dyes, thereby completing the present invention.
본 발명의 목적은 형광 염료의 사용없이 그라핀옥사이드 자체의 고유한 형광 특성을 이용하여, 검출 물질의 존재 여부를 광학적으로 식별할 수 있는 효소 기질의 고분자화에 의한 그라핀옥사이드의 형광저하를 이용한 바이오센서 및 이를 이용한 검출방법을 제공하는데 있다.
An object of the present invention is to use the intrinsic fluorescence properties of graphene oxide itself without the use of fluorescent dyes, and to utilize the fluorescence degradation of graphene oxide by polymerizing an enzyme substrate that can optically identify the presence of a detection substance. It provides a biosensor and a detection method using the same.
상기 목적을 달성하기 위하여, 본 발명은 (a) 기판 상에 그라핀옥사이드층을 형성하는 단계; (b) 상기 형성된 그라핀옥사이드층에 포획 항체(capture antibody)를 고정화시키는 단계; (c) 상기 고정화된 포획 항체에 표적 항원을 결합시키는 단계; (d) 상기 결합된 표적 항원에 효소를 함유하는 검출 항체(detecrion antibody)를 결합시키는 단계; (e) 상기 결합된 효소를 함유하는 검출 항체에 효소 기질을 첨가하여 효소 기질의 고분자화를 유도하는 단계; 및 (f) 상기 효소 기질의 고분자화가 유도된 그라핀옥사이드의 형광 강도를 측정하여 표적 항원을 검출하는 단계를 포함하는 효소 기질의 고분자화에 의한 그라핀옥사이드의 형광저하를 이용한 표적 항원의 검출방법을 제공한다.In order to achieve the above object, the present invention (a) forming a graphene oxide layer on a substrate; (b) immobilizing a capture antibody on the formed graphene oxide layer; (c) binding a target antigen to the immobilized capture antibody; (d) binding a detection antibody containing an enzyme to the bound target antigen; (e) adding an enzyme substrate to the detection antibody containing the bound enzyme to induce polymerization of the enzyme substrate; And (f) detecting the target antigen by measuring the fluorescence intensity of the graphene oxide induced by the polymerization of the enzyme substrate, thereby detecting the target antigen. To provide.
본 발명은 또한, 그라핀옥사이드층; 상기 그라핀옥사이드층에 고정화된 포획 항체; 상기 포획 항체에 결합된 표적 항원; 및 상기 표적 항원에 결합된 효소를 함유하는 검출 항체를 포함하며, 여기서 상기 효소를 함유하는 검출 항체의 효소와 효소 기질이 결합하는 경우, 효소 기질의 고분자화에 의해 그라핀옥사이드의 형광강도를 감소시키는 것을 특징으로 하는 효소 기질의 고분자화에 의한 그라핀옥사이드의 형광저하를 이용한 바이오센서를 제공한다.
The present invention also provides a graphene oxide layer; Capture antibody immobilized on the graphene oxide layer; A target antigen bound to the capture antibody; And a detection antibody containing an enzyme bound to the target antigen, wherein when the enzyme and the enzyme substrate of the detection antibody containing the enzyme bind, the fluorescence intensity of graphene oxide is reduced by polymerizing the enzyme substrate. The present invention provides a biosensor using fluorescence reduction of graphene oxide by polymerizing an enzyme substrate.
본 발명에 따른 효소 기질의 고분자화에 의한 그라핀옥사이드의 형광저하를 이용한 바이오센서는 형광 염료의 사용없이 그라핀옥사이드 자체의 형광 특성을 이용한 바이오 물질 검출센서로, 합성 공정이 복잡하고 비싼 염료의 사용이 제한되므로 경제성이 우수하다. 또한, 직접적으로 다양한 표적 물질을 검출할 수 있고, 간단한 공정으로 바이오센서의 검출 한도를 향상시킬 수 있다.
Biosensor using fluorescence reduction of graphene oxide by polymerizing enzyme substrate according to the present invention is a biomaterial detection sensor using fluorescence properties of graphene oxide itself without the use of fluorescent dyes. It is economical because its use is limited. In addition, various target substances can be detected directly, and the detection limit of the biosensor can be improved by a simple process.
도 1은 본 발명에 따른 효소 기질의 고분자화에 의한 그라핀옥사이드의 형광저하를 이용한 바이오센서의 개략도로, (a)는 효소와 효소 기질의 반응 전이고, (b)는 효소와 효소 기질의 반응 후이다.
도 2는 본 발명에 따른 그라핀옥사이드의 UV-Vis 스펙트럼 결과 그래프로, (a)는 그라핀옥사이드 수용액 내의 그라핀옥사이드이고, (b)는 그라핀옥사이드층이 형성된 아민-개질된 유리 슬라이드의 그라핀옥사이드이다.
도 3은 본 발명에 따른 그라핀옥사이드의 형광도 그래프로, (a)는 DAB 중합 전 그라핀옥사이드이고, (b)는 DAB 중합 후 그라핀옥사이드이다.
도 4는 본 발명에 따른 아민-개질된 유리 및 DAB 중합 전·후 그라핀옥사이드 증착 유리의 라만 스펙트럼 결과 그래프이다.
도 5는 본 발명에 따른 DAB 중합 전·후 그라핀옥사이드 증착 유리 표면의 UV-Vis 스펙트럼 결과 그래프이다.
도 6은 본 발명에 따른 그라핀옥사이드의 AFM 분석으로, (a)는 그라핀옥사이드 증착-아민-개질된 유리 표면이고, (b)는 항IL-5 항체를 고정시킨 그라핀옥사이드의 표면이다.
도 7은 본 발명에 따른 IL-5의 농도 변화에 따른 그라핀옥사이드의 상대 형광도 그래프이다.
도 8은 본 발명에 따른 효소 기질의 고분자화에 의한 그라핀옥사이드의 형광저하를 이용한 바이오센서의 사이토카인의 종류에 대한 상대 형광도 그래프이다.
도 9는 본 발명에 따른 효소 기질의 고분자화에 의한 그라핀옥사이드의 형광저하를 이용한 바이오센서의 인간 혈청 내의 IL-5에 대한 상대형광도 그래프이다.1 is a schematic diagram of a biosensor using fluorescence reduction of graphene oxide by polymerizing an enzyme substrate according to the present invention, (a) before the reaction between the enzyme and the enzyme substrate, and (b) the reaction between the enzyme and the enzyme substrate. After.
Figure 2 is a graph of the UV-Vis spectrum results of the graphene oxide according to the present invention, (a) is a graphene oxide in the aqueous solution of graphene oxide, (b) is an amine-modified glass slide of the graphene oxide layer is formed Graphene oxide.
Figure 3 is a graph of the fluorescence graphene oxide according to the present invention, (a) is a graphene oxide before DAB polymerization, (b) is a graphene oxide after DAB polymerization.
4 is a Raman spectral result graph of amine-modified glass and graphene oxide deposited glass before and after DAB polymerization according to the present invention.
5 is a graph of UV-Vis spectra of the graphene oxide deposited glass surface before and after DAB polymerization according to the present invention.
6 is an AFM analysis of graphene oxide according to the present invention, (a) is a graphene oxide deposited-amine-modified glass surface, and (b) is a surface of graphene oxide immobilized with an anti-IL-5 antibody. .
7 is a graph of the relative fluorescence of graphene oxide according to the concentration change of IL-5 according to the present invention.
FIG. 8 is a graph of relative fluorescence of cytokines of biosensors using fluorescence reduction of graphene oxide by polymerizing an enzyme substrate according to the present invention.
9 is a relative fluorescence graph of IL-5 in human serum of a biosensor using fluorescence reduction of graphene oxide by polymerizing an enzyme substrate according to the present invention.
본 명세서에서 사용되는 '포획 항체(capture antibody)'는 원하는 항원과 특이적, 선택적으로 결합 가능한 항체를 의미하고, '표적 항원'은 센싱 대상 항원을 의미한다. 또한, 본 명세서에 사용되는 '검출 항체(detecrion antibody)'는 센싱 대상 항원과 특이적, 선택적으로 결합 가능한 항체를 의미한다.As used herein, 'capture antibody' refers to an antibody that can be selectively and selectively bound to a desired antigen, and 'target antigen' refers to an antigen to be sensed. In addition, as used herein, 'detecrion antibody' refers to an antibody that can be specifically and selectively bound to a antigen to be sensed.
본 발명은 일 관점에서, (a) 기판 상에 그라핀옥사이드층을 형성하는 단계; (b) 상기 형성된 그라핀옥사이드층에 포획 항체(capture antibody)를 고정화시키는 단계; (c) 상기 고정화된 포획 항체에 표적 항원을 결합시키는 단계; (d) 상기 결합된 표적 항원에 효소를 함유하는 검출 항체(detecrion antibody)를 결합시키는 단계; (e) 상기 결합된 효소를 함유하는 검출 항체에 효소 기질을 첨가하여 효소 기질의 고분자화를 유도하는 단계; 및 (f) 상기 효소 기질의 고분자화가 유도된 그라핀옥사이드의 형광 강도를 측정하여 표적 항원을 검출하는 단계를 포함하는 효소 기질의 고분자화에 의한 그라핀옥사이드의 형광저하를 이용한 표적 항원의 검출방법에 관한 것이다.The present invention in one aspect, (a) forming a graphene oxide layer on a substrate; (b) immobilizing a capture antibody on the formed graphene oxide layer; (c) binding a target antigen to the immobilized capture antibody; (d) binding a detection antibody containing an enzyme to the bound target antigen; (e) adding an enzyme substrate to the detection antibody containing the bound enzyme to induce polymerization of the enzyme substrate; And (f) detecting the target antigen by measuring the fluorescence intensity of the graphene oxide induced by the polymerization of the enzyme substrate, thereby detecting the target antigen. It is about.
본 발명에 따른, 바이오센서는 다양한 표적 항원을 검출하기 위하여 고유한 형광 특성을 가진 그라핀옥사이 상에 포획 항체, 표적 항원 및 효소를 함유하는 검출 항체가 연결되어 결합하고, 여기서 검출 항체의 효소가 효소 기질과 결합하여 생성된 고분자에 의하여 그라핀옥사이드의 형광 강도를 감소시켜 표적 항원을 검출한다.According to the present invention, a biosensor is linked to a detection antibody containing a capture antibody, a target antigen and an enzyme on a graphene oxime having inherent fluorescent properties to detect various target antigens, wherein the enzyme of the detection antibody is The target antigen is detected by reducing the fluorescence intensity of graphene oxide by the polymer produced by binding to the enzyme substrate.
상기 그라핀옥사이드(graphene oxide)는 그래파이트(graphite)를 강산과 강산화제 하에서 산화시켜 제조되며, 판상의 면 부분에 다수의 에폭시기, 알콜기 또는 카보닐기를 포함하고 판상의 끝부분에는 카르복실기를 포함하는 형태로 이루어져 있다. 또한, 그라핀옥사이드는 그라핀(graphene)에 비하여 불규칙적이고 다양한 모양의 판상 구조를 형성하며, 상기 에폭시기, 알콜기 또는 카보닐기에 의하여 판상과 판상 사이의 거리를 그라핀에 비하여 넓게 유지하고 있어 용매 및 다른 유기물의 침투를 용이하게 하기 때문에 용매 및 고분자 물질과의 상용성을 향상시킬 수 있다. 특히, 상기 알콜기 및 카르복실기의 경우 친수성을 가지기 때문에 그라핀에 비하여 물이나 극성용매에 대하여 우수한 분산성을 보인다. 또한, 그라핀옥사이드는 들뜬 에너지를 광자로 방출하는 형광 특성을 자기고 있다. 이에, 본 발명은 그라핀옥사이드의 고유한 형광 특성을 바이오센서에 이용하여 검출 물질의 존재 여부를 광학적으로 식별하는 것을 핵심 요지로 한다.The graphene oxide is prepared by oxidizing graphite under a strong acid and a strong oxidizing agent, and includes a plurality of epoxy groups, alcohol groups or carbonyl groups on a plate surface, and a carboxyl group on the plate. It consists of forms. In addition, the graphene oxide forms a plate-like structure of irregular and various shapes compared to graphene (graphene), and the distance between the plate and the plate by the epoxy group, alcohol group or carbonyl group is kept wider than the graphene solvent And because it facilitates the penetration of other organics can improve the compatibility with the solvent and the polymeric material. In particular, since the alcohol group and the carboxyl group have hydrophilicity, they show excellent dispersibility in water or polar solvents compared to graphene. In addition, graphene oxide has a fluorescence property that emits excited energy as photons. Thus, the present invention is to use the intrinsic fluorescence characteristics of the graphene oxide in the biosensor to optically identify the presence of a detection material.
본 발명에 따른 그라핀옥사이드 기반 항체-항원-항체 바이오센서는 도 1에 나타낸 바와 같이, 기판상에 그라핀옥사이드(10), 포획 항체(11), 표적 항원(12), HRP-검출 항체(13)를 포함한다. 일 실시예에서는 포획 항체, 표적 항원 및 검출 항체에 IL-5를 사용하였으나, 본 발명의 범위는 이에 제한되지 않는다.Graphene oxide-based antibody-antigen-antibody biosensor according to the present invention is a graphene oxide (10), capture antibody (11), target antigen (12), HRP-detecting antibody ( 13). In one embodiment, IL-5 was used in the capture antibody, target antigen and detection antibody, but the scope of the present invention is not limited thereto.
상기 기판은 실리콘(silicon), 석영(quartz), 알루미나, 타티타니아, 세라믹(ceramic) 및 유리(glass)로 구성된 군에서 선택되고, 바람직하게는 유리를 사용할 수 있다. 또한, 상기 (a)단계에서 그라핀옥사이드층의 형성은 아민, 암모니움 및 아실 양이온으로 구성된 군에서 선택하여 사용할 수 있으며, 바람직하게는 아민을 사용하여 기능기로 기판을 개질시킨 다음, 그라핀옥사이드와 반응시켜 수행할 수 있다. The substrate is selected from the group consisting of silicon, quartz, alumina, tatitania, ceramic and glass, preferably glass. In addition, the formation of the graphene oxide layer in the step (a) may be selected from the group consisting of amine, ammonium and acyl cation, preferably modifying the substrate with a functional group using an amine, then graphene oxide It can be carried out by reacting with.
일 실시예로, 상기 개질된 기판 상에 그라핀옥사이드의 함량이 10-6 ~ 10-3 중량부인 그라핀옥사이드 수용액을 도핑한 다음, 항온항습기에서 12시간동안 배양을 수행하여 증착시킬 수 있다.In one embodiment, the content of the graphene oxide is doped with a graphene oxide aqueous solution of 10 -6 to 10 -3 parts by weight on the modified substrate, it can be deposited by incubation for 12 hours in a thermo-hygrostat.
상기 포획 항체는 모든 질병에 관한 항체가 가능하고, 바람직하게는 인터류킨-2(IL-2) 항체, 인터류킨-5(IL-5) 항체, 인터류킨-6(IL-6) 항체, 인터류킨-7(IL-7) 항체, 인터페론-감마(INF-γ) 항체, aFGF/FGF-1 항체, CDKN2D 항체, Prostatic Acid Phosphatase 등의 암생체표식체(cancer biomarker) 및 hCG 항체, LH 항체, HIV 항체, HVC 항체, HBV 항체, HepA 항체, HepB 항체 등의 병원성 단백질 항체로 구성된 군에서 선택하여 사용할 수 있으며, 이에 한정하는 것은 아니다. 상기 (b)단계에서 포획 항체의 고정화는정전기 결합, 안히드라이드, 불화페틸에스터 및 카르보디이미드-간접 아미드화 반응으로 구성된 군에서 선택되고, 바람직하게는 카르보이미드-간접 아미드화 반응 및 정전기 결합 반응을 사용하여 그라핀옥사이층에 고정시킨다. 일 실시예로, 포획 항체의 고정화는 3시간 동안 온도 10℃ ~ 20℃에서 수행되고, 바람직하게는 10℃에서 수행할 수 있다. 30℃를 벗어날 경우, 항체 및 항체의 특이적 활성 저하의 문제점이 있다.The capture antibody may be an antibody to any disease, preferably, interleukin-2 (IL-2) antibody, interleukin-5 (IL-5) antibody, interleukin-6 (IL-6) antibody, interleukin-7 ( IL-7) cancer biomarkers such as antibodies, interferon-gamma (INF-γ) antibodies, aFGF / FGF-1 antibodies, CDKN2D antibodies, Prostatic Acid Phosphatase and hCG antibodies, LH antibodies, HIV antibodies, HVC It can select from the group which consists of pathogenic protein antibodies, such as an antibody, an HBV antibody, a HepA antibody, and a HepB antibody, It is not limited to this. Immobilization of the capture antibody in step (b) is selected from the group consisting of electrostatic bonds, anhydrides, fluorinated peteyl esters and carbodiimide-indirect amidation reactions, preferably carbodiimide-indirect amidation reactions and electrostatic The binding reaction is used to fix the graphene oxime layer. In one embodiment, the immobilization of the capture antibody is carried out at a temperature of 10 ° C to 20 ° C for 3 hours, preferably at 10 ° C. If it is outside the 30 ℃, there is a problem of lowering the specific activity of the antibody and antibody.
또한, 상기 포획 항체와 결합하는 표적 항원은 모든 질병에 관한 항체가 가능하고, 바람직하게는 인터류킨-2(IL-2) 항원, 인터류킨-5(IL-5) 항원, 인터류킨-6(IL-6) 항원, 인터류킨-7(IL-7) 항원, 인터페론-감마(INF-γ) 항원, aFGF/FGF-1 항원, CDKN2D 항원, Prostatic Acid Phosphatase 등의 암생체표식체(cancer biomarker) 및 hCG 항원, LH 항원, HIV 항원, HVC 항원, HBV 항원, HepA 항원, HepB 항원 등의 병원성 단백질 항원으로 구성된 군에서 선택하여 사용할 수 있으며, 이에 한정하는 것은 아니다. 일 실시예로, 상기 (c)단계에서 표적 항원의 결합은 1시간 동안 온도 2℃ ~ 25℃에서 수행되고, 바람직하게는 10 ℃에서 수행할 수 있다. 30℃를 벗어날 경우, 항원 및 항원의 특이적 활성 저하의 문제점이 있다.In addition, the target antigen binding to the capture antibody may be an antibody of any disease, preferably, interleukin-2 (IL-2) antigen, interleukin-5 (IL-5) antigen, interleukin-6 (IL-6) ) Cancer biomarkers and hCG antigens such as antigen, interleukin-7 (IL-7) antigen, interferon-gamma (INF-γ) antigen, aFGF / FGF-1 antigen, CDKN2D antigen, Prostatic Acid Phosphatase, It can be selected from the group consisting of pathogenic protein antigens, such as LH antigen, HIV antigen, HVC antigen, HBV antigen, HepA antigen, HepB antigen, but is not limited thereto. In one embodiment, the binding of the target antigen in step (c) is carried out at a temperature of 2 ℃ to 25 ℃ for 1 hour, preferably at 10 ℃. If it is outside the 30 ℃, there is a problem of lowering the specific activity of the antigen and antigen.
상기 효소를 함유하는 검출 항체는 모든 질병에 관한 항체가 가능하고, 바람직하게는 인터류킨-2(IL-2) 항체, 인터류킨-5(IL-5) 항체, 인터류킨-6(IL-6) 항체, 인터류킨-7(IL-7) 항체, 인터페론-감마(INF-γ) 항체, aFGF/FGF-1 항체, CDKN2D 항체, Prostatic Acid Phosphatase 등의 암생체표식체(cancer biomarker) 및 hCG 항체, LH 항체, HIV 항체, HVC 항체, HBV 항체, HepA 항체, HepB 항체 등의 병원성 단백질 항체로 구성된 군에서 선택하여 사용할 수 있으며, 이에 한정하는 것은 아니다. 상기 (d)단계에서 효소를 함휴하는 검출 항체는 표적 항원에 대응하여 결합할 수 있다.The detection antibody containing the enzyme can be an antibody to any disease, preferably, interleukin-2 (IL-2) antibody, interleukin-5 (IL-5) antibody, interleukin-6 (IL-6) antibody, Cancer biomarkers such as interleukin-7 (IL-7) antibody, interferon-gamma (INF-γ) antibody, aFGF / FGF-1 antibody, CDKN2D antibody, Prostatic Acid Phosphatase and hCG antibody, LH antibody, It can select from the group which consists of pathogenic protein antibodies, such as HIV antibody, HVC antibody, HBV antibody, HepA antibody, HepB antibody, It is not limited to this. In step (d), the detection antibody containing the enzyme may bind to the target antigen.
상기 효소는 시토크롬c 과산화효소(cytochrome c peroxidase), 글루타티온 과산화효소(glutathione peroxidase), 미엘로퍼옥시다아제(myeloperoxidase), vanadium bromoperoxidase(바나듐 브로모과산화효소), 타이로이드 과산화효소(thyroid peroxidase), 락토페르옥시다아제(lactoperoxidase) 및 호스래디시 과산화효소(HRP ; horseradish peroxidase)로 구성된 군에서 선택되고, 바람직하게는 호스래디시 과산화효소(HRP ; horseradish peroxidase)를 사용할 수 있다. 또한, 상기 효소 기질은 N,N-bis(4-sulfobutyl)-3,5-dimethylaniline, N-ethyl-N-(2-hydroxy-3-sulfopropyl)-3-methoxyaniline, N-ethyl-N-(3-sulfopropyl)-3-methoxyaniline, N-ethyl-N-(3-sulfopropyl)aniline, N-ethyl-N-(2-hydroxy-3-sulfopropyl)-3,5-dimethoxyaniline, N-(2-hydroxy-3-sulfopropyl)-3,5-dimethoxyaniline, N-ethyl-N-(2-hydroxy-3-sulfopropyl)-3,5-dimethylaniline, N,N-bis(4-sulfobutyl)-3-methylaniline, N-ethyl-N-(2-hydroxy-3-sulfopropyl)-3-methylaniline, N-ethyl-N- (3-sulfopropyl)-3-methylaniline로 구성된 군에서 선택되는 화합물과 4-aminoantipyrine의 혼합물, N-(4-aminobutyl)-N-ethylisoluminol, 3-Amino-9-ethylcarbazole, N-(6- aminohexyl)-N-ethylisoluminol, 4-aminophthalhydrazide, 5- aminosalicylic acid, 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid), 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) diammonium, 4-chloro-1-naphthol, 4-chloro-7-nitrobenzofurazan, o-dianisidine, dicarboxidine dihydrochloride, guaiacol, iodonitrotetrazolium chloride, MTT formazan, nitrotetrazolium blue chloride, o-phenylenediamine, 3,3′,5,5′-tetramethylbenzidine (TMB), tetrazolium violet, 2,3,5-triphenyltetrazolium chloride, 3,3’-diaminobenzidine tetrahydrochloride 및 디아미노벤지딘(DAB;3,3' -diaminobenzidine)으로 구성된 군에서 선택되고, 바람직하게는 디아미노벤지딘(DAB;3,3' -diaminobenzidine)을 사용할 수 있다.The enzymes include cytochrome c peroxidase, glutathione peroxidase, myeloperoxidase, vanadium bromoperoxidase, vanadium bromoperoxidase, thyroid peroxidase, lactoperoxidase (lactoperoxidase) and horseradish peroxidase (HRP; horseradish peroxidase) selected from the group consisting of, preferably horseradish peroxidase (HRP; horseradish peroxidase) can be used. In addition, the enzyme substrate is N, N-bis (4-sulfobutyl) -3,5-dimethylaniline, N-ethyl-N- (2-hydroxy-3-sulfopropyl) -3-methoxyaniline, N-ethyl-N- ( 3-sulfopropyl) -3-methoxyaniline, N-ethyl-N- (3-sulfopropyl) aniline, N-ethyl-N- (2-hydroxy-3-sulfopropyl) -3,5-dimethoxyaniline, N- (2-hydroxy -3-sulfopropyl) -3,5-dimethoxyaniline, N-ethyl-N- (2-hydroxy-3-sulfopropyl) -3,5-dimethylaniline, N, N-bis (4-sulfobutyl) -3-methylaniline, N A mixture of 4-aminoantipyrine and a compound selected from the group consisting of -ethyl-N- (2-hydroxy-3-sulfopropyl) -3-methylaniline and N-ethyl-N- (3-sulfopropyl) -3-methylaniline, N- (4-aminobutyl) -N-ethylisoluminol, 3-Amino-9-ethylcarbazole, N- (6-aminohexyl) -N-ethylisoluminol, 4-aminophthalhydrazide, 5- aminosalicylic acid, 2,2′-azino-bis (3- ethylbenzothiazoline-6-sulfonic acid), 2,2′-azino-bis (3-ethylbenzothiazoline-6-sulfonic acid) diammonium, 4-chloro-1-naphthol, 4-chloro-7-nitrobenzofurazan, o-dianisidine, dicarboxidine dihydrochloride , guaiacol, iodonitrotetrazolium chloride, MTT formazan, nitrotetrazolium blue chloride, o-phenylenediamine, 3,3 ′, 5,5′-tetramethylbenzidine (TMB), tetrazolium violet, 2,3,5-triphenyltetrazolium chloride, 3,3'-diaminobenzidine tetrahydrochloride and diaminobenzidine DAB; 3,3'-diaminobenzidine), preferably diaminobenzidine (DAB; 3,3'-diaminobenzidine) can be used.
상기 (e)단계에서 효소 기질을 첨가한 다음, 검출 항체의 효소와 결합하여 진한 갈색의 oxidized DAB 또는 polymerized DAB이 그라핀옥사이층 위에 박막을 생성하여 그라핀옥사이드의 형광 특성을 감소시킨다. 즉, 본 발명은 검출 물질의 유무를 확인하기 위하여, 그라핀옥사이드의 형광 특성 및 그라핀옥사이드의 형광 강도를 저하시킬 수 있는 효소와 효소 기질의 고분자화에 의한 oxidized DAB 또는 polymerized DAB의 박막 형성을 이용한다.
After the addition of the enzyme substrate in step (e), the dark brown oxidized DAB or polymerized DAB is combined with the enzyme of the detection antibody to form a thin film on the graphene oxime layer to reduce the fluorescence characteristics of the graphene oxide. In other words, in order to confirm the presence or absence of a detection substance, the present invention is directed to the formation of a thin film of oxidized DAB or polymerized DAB by polymerizing an enzyme and an enzyme substrate which may lower the fluorescence property of graphene oxide and the fluorescence intensity of graphene oxide. I use it.
본 발명은 다른 관점에서, 그라핀옥사이드층; 상기 그라핀옥사이드층에 고정화된 포획 항체; 상기 포획 항체에 결합된 표적 항원; 및 상기 표적 항원에 결합된 효소를 함유하는 검출 항체를 포함하며, 여기서 상기 효소를 함유하는 검출 항체의 효소와 효소 기질이 결합하는 경우, 효소 기질의 고분자화에 의해 그라핀옥사이드의 형광강도를 감소시키는 것을 특징으로 하는 바이오센서에 관한 것이다.In another aspect, the present invention, the graphene oxide layer; Capture antibody immobilized on the graphene oxide layer; A target antigen bound to the capture antibody; And a detection antibody containing an enzyme bound to the target antigen, wherein when the enzyme and the enzyme substrate of the detection antibody containing the enzyme bind, the fluorescence intensity of graphene oxide is reduced by polymerizing the enzyme substrate. It relates to a biosensor characterized in that.
구체적으로, 효소 기질의 고분자화에 의한 그라핀옥사이드의 형광저하를 이용한 바이오센서는 형광 염료의 사용없이 그라핀옥사이드 자체의 형광 특성과 효소와 효소 기질의 고분자화에 의한 oxidized DAB 또는 polymerized DAB의 박막 형성을 이용하여 검출 물질의 존재 여부 및 농도를 광학적으로 식별할 수 있다. 또한, 직접적으로 다양한 표적 물질을 검출할 수 있고, 간단한 공정으로 바이오센서의 검출 한도를 향상시킬 수 있다.
Specifically, a biosensor using fluorescence reduction of graphene oxide by polymerizing an enzyme substrate is characterized in that the fluorescent properties of graphene oxide itself and the thin film of oxidized DAB or polymerized DAB by polymerization of enzyme and enzyme substrate without the use of fluorescent dyes. Formation can be used to optically identify the presence and concentration of a detection substance. In addition, various target substances can be detected directly, and the detection limit of the biosensor can be improved by a simple process.
이하, 실시예를 통하여 본 발명을 더욱 상세히 설명하고자 한다. 이들 실시예는 오로지 본 발명을 예시하기 위한 것으로, 본 발명의 범위가 이들 실시예에 의해 제한되는 것으로 해석되지 않는 것은 당업계에서 통상의 지식을 가진 자에게 있어서 자명할 것이다.
Hereinafter, the present invention will be described in more detail with reference to Examples. It is to be understood by those skilled in the art that these embodiments are only for illustrating the present invention and that the scope of the present invention is not construed as being limited by these embodiments.
실시예Example : : 그라핀옥사이드를Graphene oxide 이용한 바이오센서 제조 Bio sensor manufacturing using
1-1 : 그라핀옥사이드 제조 1-1: Graphene Oxide Preparation
천연 그래파이트(Graphit Kropfmuhl AG, Germany)를 변형된 Hummer법에 의해 제조하였다. 흑연 플래이크를 NaNO3 및 KMnO4 와 함께 진한 황산에 넣고 산화시킨 후 5 중량%의 질산으로 세척한 후 과산화수소용액으로 산화시킨 후 염산 및 증류수로 세척하고 투석한다.
Natural graphite (Graphit Kropfmuhl AG, Germany) was prepared by the modified Hummer method. The graphite flakes are placed in concentrated sulfuric acid with
1-2 : 그래핀옥사이드층이 형성된 유리 슬라이드 제조1-2: graphene oxide layer formed glass slide
유리 슬라이드를 피라니아 용액(황산:H2O2 = 1:3)에 침지시켜 70℃에서 15분간 처리했다. 피라니아 용액으로 처리한 유리 슬라이드는 2차 증류수와 에탄올로 세척한 다음, 실온에서 4시간 동안 1% 3-아미노프로필 트리메틸실록산(APTMS, 3-aminopropyltrimethoxysilane; Sigma Aldich, USA) 에탄올 용액에 침지시켰다. 침지 후 유리 슬라이드를 120℃에서 30분간 건조하여 아민-개질된 유리 슬라이드를 생성하였다. The glass slide was immersed in a piranha solution (sulfuric acid: H 2 O 2 = 1: 3) and treated at 70 ° C. for 15 minutes. The glass slides treated with the piranha solution were washed with secondary distilled water and ethanol and then immersed in 1% 3-aminopropyl trimethoxysiloxane (APTMS, 3-aminopropyltrimethoxysilane; Sigma Aldich, USA) ethanol solution for 4 hours at room temperature. After immersion the glass slides were dried at 120 ° C. for 30 minutes to produce amine-modified glass slides.
아민-개질된 유리 슬라이드 상에 그라핀옥사이드 수용액(~1mg/ml)을 떨어뜨린 다음, 24시간 동안 항온항습기에서 배양하였다. 배양 후, 수득된 그라핀옥사이드층이 형성된 유리 슬라이드는 H2O2로 세척하고 건조하여 어두운 곳에 보관하였다.
An aqueous solution of graphene oxide (˜1 mg / ml) was dropped onto an amine-modified glass slide and then incubated in a thermohygrostat for 24 hours. After incubation, the obtained glass slide with the graphene oxide layer was washed with H 2 O 2 , dried and stored in a dark place.
1-3 : 효소 기질의 고분자화에 의한 그라핀옥사이드의 형광저하를 이용한 바이오센서 제조1-3: Preparation of biosensor using fluorescence reduction of graphene oxide by polymerizing enzyme substrate
상기 실시예 1-1에서 수득된 그라핀옥사이드층이 형성된 유리 슬라이드 상에 40mM 1-에틸-3-(디메틸아미노프로필)카르복디이미드 (EDC,1-ethyl-3-[3-dimethylaminopropyl]carbodiimide; Sigma Aldich, USA)와 10mM N-하이드록시석신이미드(NHS, N-hydroxysuccinimide; Sigma Aldich, USA)가 함유된 pH 6.0인 10mM 인산 완충용액(phosphate buffer, PB)을 15분간 첨가한 다음, IL-5 포획 항체 2㎕/㎖ 용액이 함유된 pH 7.0인 10mM 인산 완충용액을 실온에서 3시간 동안 배양시킨 후, 인산 완충 식염수(PBS, phosphate buffer saline)로 세척하여 항체가 고정화된 그라핀옥사이드층이 형성된 유리 슬라이드를 수득하였다. 상기 항체가 고정화된 그라핀옥사이드층이 형성된 유리 슬라이드의 특정 스폿에 소 혈청 알부민(BSA, borvine serum albumin) 30mg/㎖를 첨가하고, 실온에서 1시간 30분 동안 배양시켰다. 이후, 0.001% tween 20과 PBS를 함유한 IL-5 용액을 항체가 고정화된 스폿에 첨가하여 1시간 동안 실온에서 배양시킨 다음, PBS로 세척하였다. 비오틴-항 IL-5 항체(BD Pharmingen, USA) 10㎍/㎖에 스트렙타아비딘-홀스래디쉬 퍼옥시다아제 칸저게이트(STA-HRP, streptavidin-horseradish peroxidase conjugate; BD Pharmingen, USA)를 첨가하여 1시간 동안 혼합한 다음, 비오틴-BSA 5.2㎍/㎖를 첨가하여 1시간 동안 혼합하여 제조한 항 IL-5 항체-HRP 용액 2.5㎍/㎖를 IL-5를 첨가한 스폿에 떨어뜨렸다. 항체-HRP 용액으로 세척하고 건조한 다음, 2mM H2O2를 함유한 1mM 3,3'-디아미노벤지딘(DAB, 3,3'-diaminobenzidine; Sigma Aldich, USA)을 첨가하고 8분 동안 반응시켰다.
40 mM 1-ethyl-3- (dimethylaminopropyl) carboximide (EDC, 1-ethyl-3- [3-dimethylaminopropyl] carbodiimide on a glass slide having a graphene oxide layer obtained in Example 1-1 10 mM phosphate buffer (PB) at pH 6.0 containing Sigma Aldich, USA) and 10 mM N-hydroxysuccinimide (NHS, N-hydroxysuccinimide; Sigma Aldich, USA) 10 mM phosphate buffer, pH 7.0, containing 2 μl / ml solution of IL-5 capture antibody was incubated at room temperature for 3 hours, and then washed with phosphate buffer saline (PBS) to fix the graphene oxide. A layered glass slide was obtained. Bovine serum albumin (BSA) 30 mg / ml was added to a specific spot of a glass slide on which the graphene oxide layer on which the antibody was immobilized was incubated for 1 hour and 30 minutes at room temperature. Thereafter, an IL-5 solution containing 0.001
실험예Experimental Example 1 : One : UVUV 특성 분석 Character analysis
실시예 1-2에 의해 수득된 그라핀옥사드층이 형성된 유리 슬라이드의 표면에 그라핀옥사이드층의 형성을 확인하기 위해 UV/Vis 스펙트럼(Jasco, Japan)으로 측정하였다.The graphene oxide layer was measured by UV / Vis spectra (Jasco, Japan) to confirm the formation of the graphene oxide layer on the surface of the glass slide on which the graphene oxade layer obtained in Example 1-2 was formed.
그 결과, 도 2에 나타난 바와 같이, (a)와 (b)의 그라핀옥사이드는 각각 230nm와 220nm에서 전형적인 강한 흡수 밴드를 나타냈고, 그라핀옥사이드 수용액의 310nm로에서 그라핀옥사이드층이 형성된 아민-개질된 유리 슬라이드의 320nm로 피크가 이동한 것은 그라핀옥사이드의 음전하와 유리 표면의 양전하사이의 결합에 의한 미세 환경 변화 때문이다. As a result, as shown in Figure 2, the graphene oxide of (a) and (b) showed a typical strong absorption band at 230nm and 220nm, respectively, and the amine with the graphene oxide layer formed at 310nm of the aqueous solution of graphene oxide The peak shift to 320 nm of the modified glass slide is due to the microenvironmental change caused by the coupling between the negative charge of graphene oxide and the positive charge on the glass surface.
또한, 그라핀옥사이드의 DAB 중합 전 후 형광도를 확인하기 위해 UV/Vis 스펙트럼(Jasco, Japan)으로 측정하였다. 그 결과, 도 3에 나타낸 바와 같이, DAB 중합 전 그라핀옥사이드는 480nm에서 최대 545nm까지 광범위한 형광 방출 밴드를 보여주지만, DAB 중합 후 그라핀옥사이드의 형광도는 95%이상 감소한다. 이와 같은 현상은 그라핀옥사이드층이 형성된 아민-개질된 유리 슬라이드에서도 확인하였다(도 3의 Inset graph). DAB는 HRP와 H2O2에 의해 산화 중합 반응을 통해 진한 갈색의 고분자화합물을 생성하고 이에 의해 그라핀옥사이드의 형광 특성을 감소시킨다. 또한, 도 4에 나타낸 바와 같이, DAB 중합 전·후로 그라핀옥사이드의 고유한 특성 피크인 1338㎝- 1(D)와 1597㎝-1(G)의 피크가 감소함을 확인하였고, 도 5의 UV/Vis 스펙트럼 측정 결과에서도 DAB 중합 전 그라핀옥사이드의 흡수 밴드가 450nm에서 DAB 중합 후 300nm ~ 700nm로 넓은 흡수 밴드로 변화를 보여주었다.
In addition, it was measured by UV / Vis spectrum (Jasco, Japan) to confirm the fluorescence before and after DAB polymerization of graphene oxide. As a result, as shown in Figure 3, the graphene oxide before DAB polymerization shows a wide range of fluorescence emission band from 480nm up to 545nm, the fluorescence of graphene oxide after the DAB polymerization decreases by more than 95%. This phenomenon was also confirmed in the amine-modified glass slide in which the graphene oxide layer was formed (Inset graph of FIG. 3). DAB produces a dark brown high molecular compound through oxidative polymerization with HRP and H 2 O 2 , thereby reducing the fluorescence properties of graphene oxide. In addition, as shown in Fig. 4, DAB polymerization before and graphene unique characteristic peaks of the
실험예Experimental Example 2 : 2 : AFMAFM 특성 분석 Character analysis
실시예 1-2에 의해 수득된 그라핀옥사이드층이 형성된 아민-개질된 유리 슬라이드와 실시예 1-3에 의해 수득된 항IL-5 항체를 고정시킨 그라핀옥사이드의 단면을 확인하기 위해 Nanoscope IIID Multimode AFM(Digital Instrument Inc., USA)으로 측정하였다. 그 결과, 도 6에 나타난 바와 같이, 단면 분석에 의해 그라핀옥사이드 상에 고정화된 항IL-5 항체의 높이는 그라핀옥사이드에 부착된 항체의 방향에 따라 0.8 ~ 1.7nm로 측정되었다.
Nanoscope IIID to confirm the cross section of the graphene oxide immobilized with the amine-modified glass slide with the graphene oxide layer obtained in Examples 1-2 and the anti-IL-5 antibody obtained in Examples 1-3. It was measured by Multimode AFM (Digital Instrument Inc., USA). As a result, as shown in Figure 6, the height of the anti-IL-5 antibody immobilized on the graphene oxide by cross-sectional analysis was measured to 0.8 ~ 1.7nm depending on the direction of the antibody attached to the graphene oxide.
실험예Experimental Example 3 : 상대 형광도 특성 분석 3: Relative Fluorescence Characterization
실시예 1-3에 의해 수득된 효소 기질의 고분자화에 의한 그라핀옥사이드의 형광저하를 이용한 바이오센서의 상대 형광도를 확인하기 위해 GenePix Professional 4200A(Axon Instrument, USA)로 측정하였다.GenePix Professional 4200A (Axon Instrument, USA) was used to determine the relative fluorescence of the biosensor using fluorescence reduction of graphene oxide by polymerizing the enzyme substrate obtained in Examples 1-3.
그 결과, 도 7에 나타난 바와 같이, 그라핀옥사이드의 형광 강도를 감소시키는 DAB와 비례 관계인 IL-5의 농도가 증가할수록 그라핀옥사이드의 상대 형광도가 감소함을 보여준다. 그라핀옥사이드를 이용한 바이오센서의 형광도는 IL-5의 농도가 4.0ng/㎖일 때, 약 20%정도 감소하였다. 본 발명에 따른 그라핀옥사이드를 이용한 바이오센서의 IL-5 검출 한도는 5pg/㎖로 다른 IL-5 센서보다 우수하였다. As a result, as shown in Figure 7, the relative fluorescence of graphene oxide decreases as the concentration of IL-5, which is proportional to DAB which decreases the fluorescence intensity of graphene oxide, increases. The fluorescence of the biosensor using graphene oxide decreased about 20% when IL-5 concentration was 4.0ng / ml. IL-5 detection limit of the biosensor using graphene oxide according to the present invention was 5pg / ㎖ was superior to other IL-5 sensor.
또한, 도 8에 나타난 바와 같이, 실시예 1-3에서 수득된 효소 기질의 고분자화에 의한 그라핀옥사이드의 형광저하를 이용한 바이오센서에 각각 IL-2, IL-5, IL-6, IL-7 및 INF-γ와 같은 사이토카인의 종류에 따른 그라핀옥사이드의 상대 형광도의 경우, IL-5가 50%의 상대 형광도 감소를 나타내어 효소 기질의 고분자화에 의한 그라핀옥사이드의 형광저하를 이용한 바이오센서가 IL-5 검출에 가장 우수함을 확인하였다.In addition, as shown in Figure 8, IL-2, IL-5, IL-6, IL- in the biosensor using fluorescence reduction of graphene oxide by polymerizing the enzyme substrate obtained in Example 1-3, respectively In the case of the relative fluorescence of graphene oxides according to the types of cytokines such as 7 and INF-γ, IL-5 showed a 50% relative fluorescence decrease, thereby reducing the fluorescence degradation of graphene oxide by polymerizing the enzyme substrate. It was confirmed that the biosensor used was the best for IL-5 detection.
또한, 도 9에 나타난 바와 같이, 항IL-5 항체와 BSA에 대한 그라핀옥사이드를 이용한 바이오센서의 상대 형광도는 IL-5의 농도 변화에 따른 BSA와 결합한 그라핀옥사이드의 상대 형광도 변화는 거의 없었다. 반면에, 항IL-5 항체가 고정화된 그라핀옥사이드의 상대 형광도는 IL-5의 농도가 0 ~ 100ng/㎖ 까지 증가할 때, 50% 정도 감소하였다. 인간 혈청 용액을 이용한 검출 한도는 PBS 완충용액을 이용한 검출 한도 10pg/㎖와 같았다. In addition, as shown in Figure 9, the relative fluorescence of the biosensor using the graphene oxide for the anti-IL-5 antibody and BSA, the relative fluorescence change of the graphene oxide combined with BSA according to the change in the concentration of IL-5 There was little. On the other hand, the relative fluorescence of the graphene oxide immobilized with the anti-IL-5 antibody decreased by 50% when the concentration of IL-5 increased from 0 to 100 ng / ml. The detection limit using human serum solution was the same as the detection limit of 10 pg / ml using PBS buffer.
그라핀옥사이드를 이용한 바이오센서는 포획 항체를 변경하여 다른 화학물질의 검출과 분자생물학에서 다양한 타겟 단백질의 고속 대량 분석에 적용할 수 있다.
Biosensors using graphene oxide can be adapted to the detection of other chemicals and the rapid mass analysis of a variety of target proteins in molecular biology.
이상으로 본 발명 내용의 특정한 부분을 상세히 기술하였는바, 당업계의 통상의 지식을 가진 자에게 있어서 이러한 구체적 기술은 단지 바람직한 실시 양태일 뿐이며, 이에 의해 본 발명의 범위가 제한되는 것이 아닌 점은 명백할 것이다. 따라서, 본 발명의 실질적인 범위는 첨부된 청구항들과 그것들의 등가물에 의하여 정의된다고 할 것이다.
While the present invention has been particularly shown and described with reference to specific embodiments thereof, those skilled in the art will appreciate that such specific embodiments are merely preferred embodiments and that the scope of the present invention is not limited thereto will be. Accordingly, the actual scope of the present invention will be defined by the appended claims and their equivalents.
10 : 그라핀옥사이드
11 : 포획 항체
12 : 표적 항원
13 : HRP-검출 항체10: graphene oxide
11: capture antibody
12: target antigen
13: HRP-detecting antibody
Claims (18)
(a) 기판 상에 그라핀옥사이드층을 형성하는 단계;
(b) 상기 형성된 그라핀옥사이드층에 포획 항체를 고정화시키는 단계;
(c) 상기 고정화된 포획 항체에 표적 항원을 결합시키는 단계;
(d) 상기 결합된 표적 항원에 효소를 함유하는 검출 항체를 결합시키는 단계;
(e) 상기 결합된 효소를 함유하는 검출 항체에 효소 기질을 첨가하여 효소 기질의 고분자화를 유도하는 단계; 및
(f) 상기 효소 기질의 고분자화가 유도된 그라핀옥사이드의 형광 강도를 측정하여 표적 항원을 검출하는 단계.
A method for detecting a target antigen using fluorescence reduction of graphene oxide by polymerizing an enzyme substrate, comprising the following steps:
(a) forming a graphene oxide layer on the substrate;
(b) immobilizing the capture antibody on the formed graphene oxide layer;
(c) binding a target antigen to the immobilized capture antibody;
(d) binding a detection antibody containing an enzyme to the bound target antigen;
(e) adding an enzyme substrate to the detection antibody containing the bound enzyme to induce polymerization of the enzyme substrate; And
(f) detecting the target antigen by measuring the fluorescence intensity of the graphene oxide induced polymerization of the enzyme substrate.
The method of claim 1, wherein the substrate is selected from the group consisting of silicon, quartz, ceramic, alumina, titania, and glass. Method for detecting target antigen using fluorescence reduction of pin oxide.
The method of claim 1, wherein the forming of the graphene oxide layer in the step (a) is performed by modifying the substrate with a functional group selected from the group consisting of amine, ammonium and acyl theory, and then reacting with graphene oxide. A method for detecting a target antigen using fluorescence reduction of graphene oxide by polymerizing an enzyme substrate.
The method of claim 1, wherein the capture antibody is an interleukin-2 (IL-2) antibody, an interleukin-5 (IL-5) antibody, an interleukin-6 (IL-6) antibody, an interleukin-7 (IL-7) antibody, Interferon-gamma (INF-γ) antibody, aFGF / FGF-1 antibody, CDKN2D antibody, Prostatic Acid Phosphatase, hCG antibody, LH antibody, HIV antibody, HVC antibody, HBV antibody, HepA antibody and HepB antibody A method for detecting a target antigen using fluorescence reduction of graphene oxide by polymerizing an enzyme substrate.
The method of claim 1, wherein the immobilization of the capture antibody in step (b) is immobilized on the graphene oxide layer by a method selected from the group consisting of electrostatic bonds, anhydrides, phenyl fluoride and carbodiimide-indirect amidation reactions. A method for detecting a target antigen using fluorescence reduction of graphene oxide by polymerizing an enzyme substrate, characterized in that
The method of claim 1, wherein the target antigen is an interleukin-2 (IL-2) antigen, an interleukin-5 (IL-5) antigen, an interleukin-6 (IL-6) antigen, an interleukin-7 (IL-7) antigen, Interferon-gamma (INF-γ) antigen, aFGF / FGF-1 antigen, CDKN2D antigen, Prostatic Acid Phosphatase, hCG antigen, LH antigen, HIV antigen, HVC antigen, HBV antigen, HepA antigen and HepB antigen A method for detecting a target antigen using fluorescence reduction of graphene oxide by polymerizing an enzyme substrate.
The method of claim 1, wherein the detection antibody is an interleukin-2 (IL-2) antibody, an interleukin-5 (IL-5) antibody, an interleukin-6 (IL-6) antibody, an interleukin-7 (IL-7) antibody, Interferon-gamma (INF-γ) antibody, aFGF / FGF-1 antibody, CDKN2D antibody, Prostatic Acid Phosphatase, hCG antibody, LH antibody, HIV antibody, HVC antibody, HBV antibody, HepA antibody and HepB antibody A method for detecting a target antigen using fluorescence reduction of graphene oxide by polymerizing an enzyme substrate.
The enzyme of claim 1, wherein the enzyme is cytochrome c peroxidase, glutathione peroxidase, myeloperoxidase, vanadium bromoperoxidase, vanadium bromoperoxidase, or thyroid peroxidase. peroxidase), lactoperoxidase (lactoperoxidase) and horseradish peroxidase (HRP; horseradish peroxidase) characterized in that the target antigen detection method using the fluorescent degradation of the graphene oxide by polymerizing the enzyme substrate, characterized in that selected from the group .
The method of claim 1, wherein the enzyme substrate is N, N-bis (4-sulfobutyl) -3,5-dimethylaniline, N-ethyl-N- (2-hydroxy-3-sulfopropyl) -3-methoxyaniline, N-ethyl -N- (3-sulfopropyl) -3-methoxyaniline, N-ethyl-N- (3-sulfopropyl) aniline, N-ethyl-N- (2-hydroxy-3-sulfopropyl) -3,5-dimethoxyaniline, N- (2-hydroxy-3-sulfopropyl) -3,5-dimethoxyaniline, N-ethyl-N- (2-hydroxy-3-sulfopropyl) -3,5-dimethylaniline, N, N-bis (4-sulfobutyl) -3 4-aminoantipyrine and compounds selected from the group consisting of -methylaniline, N-ethyl-N- (2-hydroxy-3-sulfopropyl) -3-methylaniline, and N-ethyl-N- (3-sulfopropyl) -3-methylaniline Mixture, N- (4-aminobutyl) -N-ethylisoluminol, 3-Amino-9-ethylcarbazole, N- (6-aminohexyl) -N-ethylisoluminol, 4-aminophthalhydrazide, 5- aminosalicylic acid, 2,2′-azino- bis (3-ethylbenzothiazoline-6-sulfonic acid), 2,2′-azino-bis (3-ethylbenzothiazoline-6-sulfonic acid) diammonium, 4-chloro-1-naphthol, 4-chloro-7-nitrobenzofurazan, o- dianisidine, dicarboxidine dihydrochloride, guaiacol, iodonitrotetrazol ium chloride, MTT formazan, nitrotetrazolium blue chloride, o-phenylenediamine, 3,3 ′, 5,5′-tetramethylbenzidine (TMB), tetrazolium violet, 2,3,5-triphenyltetrazolium chloride, 3,3'-diaminobenzidine tetrahydrochloride and dia A method for detecting a target antigen using fluorescence reduction of graphene oxide by polymerizing an enzyme substrate, characterized in that it is selected from the group consisting of minobenzidine (DAB; 3,3 '-diaminobenzidine).
The method of claim 1, wherein detecting the target antigen of (f) comprises polymerizing the enzyme substrate to reduce the fluorescence characteristics of graphene oxide when the target antigen and the detection antibody containing the enzyme bind in the presence of the enzyme substrate. A method for detecting a target antigen using fluorescence reduction of graphene oxide by polymerizing an enzyme substrate, characterized in that
Graphene oxide layer; Capture antibody immobilized on the graphene oxide layer; A target antigen bound to the capture antibody; And a detection antibody containing an enzyme bound to the target antigen, wherein when the enzyme and the enzyme substrate of the detection antibody containing the enzyme bind, the fluorescence intensity of graphene oxide is reduced by polymerizing the enzyme substrate. Biosensor characterized in that.
The biosensor of claim 11, wherein the substrate is selected from a group consisting of silicon, quartz, ceramic, alumina, titania, and glass.
The method of claim 11, wherein the capture antibody is an interleukin-2 (IL-2) antibody, an interleukin-5 (IL-5) antibody, an interleukin-6 (IL-6) antibody, an interleukin-7 (IL-7) antibody, Interferon-gamma (INF-γ) antibody, aFGF / FGF-1 antibody, CDKN2D antibody, Prostatic Acid Phosphatase, hCG antibody, LH antibody, HIV antibody, HVC antibody, HBV antibody, HepA antibody and HepB antibody Biosensor, characterized in that.
The method of claim 13, wherein the target antigen is an interleukin-2 (IL-2) antigen, an interleukin-5 (IL-5) antigen, an interleukin-6 (IL-6) antigen, an interleukin-7 (IL-7) antigen, Interferon-gamma (INF-γ) antigen, aFGF / FGF-1 antigen, CDKN2D antigen, Prostatic Acid Phosphatase, hCG antigen, LH antigen, HIV antigen, HVC antigen, HBV antigen, HepA antigen and HepB antigen Biosensor, characterized in that.
The method of claim 13, wherein the detection antibody containing the enzyme is interleukin-2 (IL-2) antibody, interleukin-5 (IL-5) antibody, interleukin-6 (IL-6) antibody, interleukin-7 (IL- 7) consisting of antibody, interferon-gamma (INF-γ) antibody, aFGF / FGF-1 antibody, CDKN2D antibody, Prostatic Acid Phosphatase, hCG antibody, LH antibody, HIV antibody, HVC antibody, HBV antibody, HepA antibody and HepB antibody Biosensor, characterized in that selected from the group.
The method of claim 11, wherein the enzyme is cytochrome c peroxidase, glutathione peroxidase, myeloperoxidase, vanadium bromoperoxidase, vanadium bromoperoxidase, thyroid peroxidase peroxidase), lactoperoxidase (lactoperoxidase) and horseradish peroxidase (HRP; horseradish peroxidase), characterized in that the biosensor selected from the group consisting of.
The method of claim 11, wherein the enzyme substrate is N, N-bis (4-sulfobutyl) -3,5-dimethylaniline, N-ethyl-N- (2-hydroxy-3-sulfopropyl) -3-methoxyaniline, N-ethyl -N- (3-sulfopropyl) -3-methoxyaniline, N-ethyl-N- (3-sulfopropyl) aniline, N-ethyl-N- (2-hydroxy-3-sulfopropyl) -3,5-dimethoxyaniline, N- (2-hydroxy-3-sulfopropyl) -3,5-dimethoxyaniline, N-ethyl-N- (2-hydroxy-3-sulfopropyl) -3,5-dimethylaniline, N, N-bis (4-sulfobutyl) -3 4-aminoantipyrine and compounds selected from the group consisting of -methylaniline, N-ethyl-N- (2-hydroxy-3-sulfopropyl) -3-methylaniline, and N-ethyl-N- (3-sulfopropyl) -3-methylaniline Mixture, N- (4-aminobutyl) -N-ethylisoluminol, 3-Amino-9-ethylcarbazole, N- (6-aminohexyl) -N-ethylisoluminol, 4-aminophthalhydrazide, 5- aminosalicylic acid, 2,2′-azino- bis (3-ethylbenzothiazoline-6-sulfonic acid), 2,2′-azino-bis (3-ethylbenzothiazoline-6-sulfonic acid) diammonium, 4-chloro-1-naphthol, 4-chloro-7-nitrobenzofurazan, o- dianisidine, dicarboxidine dihydrochloride, guaiacol, iodonitrotetrazo lium chloride, MTT formazan, nitrotetrazolium blue chloride, o-phenylenediamine, 3,3 ′, 5,5′-tetramethylbenzidine (TMB), tetrazolium violet, 2,3,5-triphenyltetrazolium chloride, 3,3'-diaminobenzidine tetrahydrochloride and diadia Biosensor, characterized in that selected from the group consisting of minobenzidine (DAB; 3,3 '-diaminobenzidine).
A method for detecting a target antigen by the biosensor of claim 11.
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WO2015147594A1 (en) * | 2014-03-28 | 2015-10-01 | 에스케이이노베이션 주식회사 | Electrochemical biosensor using dual electrode pair |
CN105424776A (en) * | 2015-11-03 | 2016-03-23 | 东南大学 | Biosensor based on carbon nano composite material and preparation method thereof |
KR20220001897A (en) * | 2020-06-30 | 2022-01-06 | 광주과학기술원 | One-pot biosensor and method of immunoassay using the same |
CN115165991A (en) * | 2022-07-06 | 2022-10-11 | 岭南师范学院 | Preparation method of reduced glutathione photoelectrochemical sensor |
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WO2015147594A1 (en) * | 2014-03-28 | 2015-10-01 | 에스케이이노베이션 주식회사 | Electrochemical biosensor using dual electrode pair |
CN105424776A (en) * | 2015-11-03 | 2016-03-23 | 东南大学 | Biosensor based on carbon nano composite material and preparation method thereof |
KR20220001897A (en) * | 2020-06-30 | 2022-01-06 | 광주과학기술원 | One-pot biosensor and method of immunoassay using the same |
US11650203B2 (en) | 2020-06-30 | 2023-05-16 | GIST (Gwangju Institute of Science and Technology) | One-pot biosensor and immunoassay method using the same |
CN115165991A (en) * | 2022-07-06 | 2022-10-11 | 岭南师范学院 | Preparation method of reduced glutathione photoelectrochemical sensor |
CN115165991B (en) * | 2022-07-06 | 2023-11-07 | 岭南师范学院 | Preparation method of reduced glutathione photoelectrochemical sensor |
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