KR20230104003A - Sensor for detecting house dust allergen and preparing method thereof - Google Patents
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- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
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Abstract
일 양상은 집먼지 알러젠 검출용 센서에 관한 것이다. 일 양상에 따라 제작된 집먼지 알러젠 검출용 센서는 제작 과정이 간단하고, 신호 증폭 효과가 우수함을 확인하였다. 또한, 상기 센서는 기존의 알러젠 검출 방법에 비해 우수한 감도를 나타내고, 현장에서 소량의 먼지 시료에서도 신속/정확하게 알러젠의 검출이 가능함을 확인하였는 바, 집먼지 진드기로 인해 유발되는 여러 질병들의 예방이 가능하다.One aspect relates to a sensor for detecting house dust allergens. It was confirmed that the sensor for detecting house dust allergen manufactured according to one aspect had a simple manufacturing process and excellent signal amplification effect. In addition, it was confirmed that the sensor exhibits superior sensitivity compared to conventional allergen detection methods and can detect allergens quickly and accurately even in a small amount of dust samples in the field, so it is possible to prevent various diseases caused by house dust mites. .
Description
집먼지 알러젠 검출용 센서에 관한 것이다.It relates to a sensor for detecting house dust allergens.
집먼지 진드기는 살코프티포르메스(Sarcoptiformes)목 파이로글리프(Pyroglyphidae)과에 속하는 진드기의 한 종류이다. 집먼지 진드기는 알레르기 증상의 원인 중 하나이며, 집먼지 진드기에 의해 야기되는 천식, 아토피성피부염, 비염 등의 각 종 알레르기 질환은 전세계적으로 6천 5백만 명 내지 1억 2천만 명에게 영향을 미치고 있다. 집먼지 진드기는 각질이나 비듬 등이 흔히 사람들이 많이 활동하거나 생활하는 곳에 쌓이게 되는 매트리스, 가구, 카펫 등에서 많이 발생하고 있으며, 집먼지 진드기에 의해 유발되는 알레르기 질환 중에서, 천식은 높은 사망률로 인하여 가장 중요한 질환이다. House dust mite is a type of mite belonging to the family Pyroglyphidae in the order Sarcoptiformes . House dust mites are one of the causes of allergic symptoms, and various allergic diseases such as asthma, atopic dermatitis, and rhinitis caused by house dust mites affect 65 to 120 million people worldwide. . House dust mites occur frequently in mattresses, furniture, carpets, etc., where keratin and dandruff are often accumulated in places where people are active or live. Among allergic diseases caused by house dust mites, asthma is the most important disease due to its high mortality rate. .
진드기 유래 알러젠 중 Der P1이 대표적인 물질로서, 알레르기 반응의 조절에 관여하는 것으로 보고되었다. 그러나, Der P1 물질이 집먼지 내에 존재하는지 여부를 검출할 수 있는 방법은 매우 제한적으로, ELISA와 같은 실험실 내에서 분석이 가능한 방법이 개발되었다.Among mite-derived allergens, Der P1 is a representative substance and has been reported to be involved in the control of allergic reactions. However, a method capable of detecting whether or not Der P1 material is present in house dust is very limited, and a method enabling analysis in a laboratory such as ELISA has been developed.
따라서, 진드기가 번식할 수 있는 환경 현장에서 빠른 시간내에 진드기 유래 알러젠을 검출할 수 없으므로 새로운 센서 기술이 필요한 실정이다.Therefore, since mite-derived allergen cannot be detected in a short time in an environment where mites can breed, a new sensor technology is required.
일 양상은 금 단분자층(Monolayer);One aspect is a gold monolayer (Monolayer);
상기 금 단분자층의 표면에 부착된 링커; a linker attached to the surface of the gold monolayer;
상기 링커에 연결된 고정화제; 및an immobilizing agent connected to the linker; and
상기 고정화제에 고정된 항체;를 포함하는 집먼지 알러젠 검출용 센서를 제공하는 것이다.It is to provide a sensor for detecting a house dust allergen comprising; an antibody immobilized on the immobilizing agent.
다른 양상은 금 단분자층(Monolayer)에 링커를 부착하는 단계; Another aspect is attaching a linker to a gold monolayer;
상기 부착된 링커에 고정화제를 연결하는 단계; 및linking an immobilizing agent to the attached linker; and
상기 연결된 고정화제에 항체를 고정시키는 단계;를 포함하는 집먼지 알러젠 검출용 센서의 제조방법을 제공하는 것이다.It is to provide a method for manufacturing a sensor for detecting house dust allergens, including immobilizing the antibody to the immobilized agent.
또 다른 양상은 상기 센서에 먼지 시료를 처리하는 단계; 및Another aspect includes processing a dust sample to the sensor; and
상기 센서에서 알러젠 검출 여부를 확인하는 단계;를 포함하는 알러젠의 검출 방법을 제공하는 것이다.It is to provide an allergen detection method comprising the step of checking whether the allergen is detected by the sensor.
일 양상은 금 단분자층(Monolayer);One aspect is a gold monolayer (Monolayer);
상기 금 단분자층의 표면에 부착된 링커; a linker attached to the surface of the gold monolayer;
상기 링커에 연결된 고정화제; 및an immobilizing agent connected to the linker; and
상기 고정화제에 고정된 항체;를 포함하는 집먼지 알러젠 검출용 센서를 제공한다.It provides a sensor for detecting a house dust allergen comprising; an antibody immobilized on the immobilizing agent.
상기 용어 "단분자층(Monolayer)'은 분자 하나의 두께를 지닌 다른 분리된(isolate) 분자막을 의미한다.The term "monolayer" refers to an isolate molecular film having a thickness of one molecule.
일 실시예에 있어서, 상기 링커는 11-메르캅토운데카산(11-mercaptoundecanoic acid), 3-메르캅토프로피온(3-mercaptopropionic acid), 11-메르캅토운데카놀(11-mercaptoundecanol), 6-메캅토-1-헥산올 (6-mecapto-1-hexanol), 메르캅토폴리(에틸렌 글리콜) 카르복실산 (mercaptopoly(ethylene glycol) carboxylic acid) 및 메르캅토폴리(에틸렌 글리콜) 아민(mercaptopoly(ethylene glycol) amine)으로 이루어진 군에서 선택되는 하나 이상일 수 있다.In one embodiment, the linker is 11-mercaptoundecanoic acid, 3-mercaptopropionic acid, 11-mercaptoundecanol, 6- mercapto-1-hexanol (6-mecapto-1-hexanol), mercaptopoly(ethylene glycol) carboxylic acid and mercaptopoly(ethylene glycol) amine ) amine) may be one or more selected from the group consisting of.
일 구체예에서, 상기 링커는 11-메르캅토운데카산(11-mercaptoundecanoic acid)일 수 있다.In one embodiment, the linker may be 11-mercaptoundecanoic acid.
일 실시예에 있어서, 상기 고정화제는 EDC/NHS (N-ethyl-N'-(3-(dimethylamino)propyl)carbodiimide)/(N-hydroxysuccinimide) 또는 DCC(dicyclohexylcarbodiimide)일 수 있다.In one embodiment, the immobilizing agent may be EDC/NHS (N-ethyl-N'-(3-(dimethylamino)propyl)carbodiimide)/(N-hydroxysuccinimide) or DCC (dicyclohexylcarbodiimide).
일 실시예에 있어서, 상기 항체는 상기 집먼지 알러젠 (Allergen)과 특이적으로 결합하는 것일 수 있다.In one embodiment, the antibody may specifically bind to the house dust allergen.
상기 용어 "알러젠 (Allergen)"은 알레르기를 유발하는 물질을 의미한다. 예를 들어, 상기 알러젠은 꽃가루, 생선, 금속, 먼지일 수 있다.The term "allergen" means a substance that causes allergy. For example, the allergen may be pollen, fish, metal, or dust.
일 실시예에 있어서, 상기 집먼지 알러젠은 Der P1, Der P2, Der f1, Der f2 및 Der m1으로 이루어진 군에서 선택되는 하나 이상일 수 있다.In one embodiment, the house dust allergen may be one or more selected from the group consisting of Der P1, Der P2, Der f1, Der f2 and Der m1.
일 구체예에서, 상기 집먼지 알러젠은 Der P1일 수 있다.In one embodiment, the house dust allergen may be Der P1.
일 실시예에 있어서, 상기 센서의 알러젠 검출 민감도(Kd 값)는 1 내지 10 fg/ml일 수 있다.In one embodiment, the allergen detection sensitivity (Kd value) of the sensor may be 1 to 10 fg/ml.
일 구체예에서, 상기 센서의 알러젠 검출 민감도(Kd 값)는 1 fg/ml 내지 10 fg/ml, 1 fg/ml 내지 8 fg/ml, 1 fg/ml 내지 6 fg/ml, 2 fg/ml 내지 10 fg/ml, 2 fg/ml 내지 8 fg/ml, 2 fg/ml 내지 6 fg/ml, 4 fg/ml 내지 10 fg/ml, 4 fg/ml 내지 8 fg/ml 또는 4 fg/ml 내지 6 fg/ml일 수 있다.In one embodiment, the allergen detection sensitivity (Kd value) of the sensor is 1 fg/ml to 10 fg/ml, 1 fg/ml to 8 fg/ml, 1 fg/ml to 6 fg/ml, 2 fg/ml to 10 fg/ml, 2 fg/ml to 8 fg/ml, 2 fg/ml to 6 fg/ml, 4 fg/ml to 10 fg/ml, 4 fg/ml to 8 fg/ml or 4 fg/ml to 6 fg/ml.
상기 용어 "검출"은 측정된 대상의 농도를 정량하는 것을 의미한다.The term “detection” means quantifying the concentration of a measured object.
일 실시예에 있어서, 상기 상기 센서의 알러젠 검출 농도는 0.05 pg/ml 내지 5,000 pg/ml일 수 있다.In one embodiment, the allergen detection concentration of the sensor may be 0.05 pg/ml to 5,000 pg/ml.
일 구체예에서, 상기 센서의 알러젠 검출 농도는 0.05 pg/ml 내지 5,000 pg/ml, 0.05 pg/ml 내지 4,500 pg/ml, 0.05 pg/ml 내지 4,000 pg/ml, 0.08 pg/ml 내지 5,000 pg/ml, 0.08 pg/ml 내지 4,500 pg/ml, 0.08 pg/ml 내지 4,000 pg/ml, 0.1 pg/ml 내지 5,000 pg/ml, 0.1 pg/ml 내지 4,500 pg/ml 또는 0.1 pg/ml 내지 4,000 pg/ml일 수 있다.In one embodiment, the allergen detection concentration of the sensor is 0.05 pg/ml to 5,000 pg/ml, 0.05 pg/ml to 4,500 pg/ml, 0.05 pg/ml to 4,000 pg/ml, 0.08 pg/ml to 5,000 pg/ml. ml, 0.08 pg/ml to 4,500 pg/ml, 0.08 pg/ml to 4,000 pg/ml, 0.1 pg/ml to 5,000 pg/ml, 0.1 pg/ml to 4,500 pg/ml or 0.1 pg/ml to 4,000 pg/ml can be ml.
다른 양상은 금 단분자층(Monolayer)에 링커를 부착하는 단계; Another aspect is attaching a linker to a gold monolayer;
상기 부착된 링커에 고정화제를 연결하는 단계; 및linking an immobilizing agent to the attached linker; and
상기 연결된 고정화제에 항체를 고정시키는 단계;를 포함하는 집먼지 알러젠 검출용 센서의 제조방법을 제공한다.It provides a method for manufacturing a sensor for detecting house dust allergens, including the step of immobilizing the antibody on the linked immobilizing agent.
상기 "금 단분자층(Monolayer)", "링커", "고정화제", "항체", "집먼지 얼러젠", "검출", "센서" 등은 전술한 범위 내일 수 있다.The "gold monolayer", "linker", "immobilizing agent", "antibody", "house dust allergen", "detection", "sensor" and the like may be within the above-described range.
일 실시예에 있어서, 상기 금 단분자층은 자가조립(self-assembled)된 것일 수 있다.In one embodiment, the gold monolayer may be self-assembled.
일 실시예에 있어서, 상기 링커를 부착하는 단계는 5 mM 내지 15 mM 농도의 링커 용액을 처리하여 수행되는 것일 수 있다.In one embodiment, the step of attaching the linker may be performed by treating the linker solution at a concentration of 5 mM to 15 mM.
일 구체예에서, 상기 링커를 부착하는 단계는 5 mM 내지 15 mM, 5 mM 내지 13 mM, 5 mM 내지 11 mM, 7 mM 내지 15 mM, 7 mM 내지 13 mM, 7 mM 내지 11 mM, 9 mM 내지 15 mM, 9 mM 내지 13 mM 또는 9 mM 내지 11 mM 농도의 링커 용액을 처리하여 수행되는 것일 수 있다.In one embodiment, the step of attaching the linker is 5 mM to 15 mM, 5 mM to 13 mM, 5 mM to 11 mM, 7 mM to 15 mM, 7 mM to 13 mM, 7 mM to 11 mM, 9 mM to 15 mM, 9 mM to 13 mM, or 9 mM to 11 mM concentration of the linker solution.
일 실시예에 있어서, 상기 고정화제를 연결하는 단계는 10 mg/mL 내지 30 mg/mL 농도의 고정화제 용액을 처리하여 수행되는 것일 수 있다.In one embodiment, the step of connecting the immobilizing agent may be performed by treating a solution of the immobilizing agent having a concentration of 10 mg/mL to 30 mg/mL.
일 구체예에서, 상기 고정화제를 연결하는 단계는 10 mg/mL 내지 30 mg/mL, 10 mg/mL 내지 26 mg/mL, 10 mg/mL 내지 23 mg/mL, 14 mg/mL 내지 30 mg/mL, 14 mg/mL 내지 26 mg/mL, 14 mg/mL 내지 23 mg/mL, 17 mg/mL 내지 30 mg/mL, 17 mg/mL 내지 26 mg/mL 또는 17 mg/mL 내지 23 mg/mL 농도의 고정화제 용액을 처리하여 수행되는 것일 수 있다.In one embodiment, the step of linking the immobilizing agent is 10 mg / mL to 30 mg / mL, 10 mg / mL to 26 mg / mL, 10 mg / mL to 23 mg / mL, 14 mg / mL to 30 mg /mL, 14 mg/mL to 26 mg/mL, 14 mg/mL to 23 mg/mL, 17 mg/mL to 30 mg/mL, 17 mg/mL to 26 mg/mL or 17 mg/mL to 23 mg /mL concentration of the immobilizing agent solution may be carried out.
일 실시예에 있어서, 상기 항체를 고정시키는 단계는 항체를 집먼지 알러젠 (Allergen)과 특이적으로 결합시켜 고정하여 수행되는 것일 수 있다.In one embodiment, the step of immobilizing the antibody may be performed by specifically binding the antibody to a house dust allergen and fixing the antibody.
일 실시예에 있어서, 상기 집먼지 알러젠은 Der P1, Der P2, Der f1, Der f2 및 Der m1으로 이루어진 군에서 선택되는 하나 이상일 수 있다.In one embodiment, the house dust allergen may be one or more selected from the group consisting of Der P1, Der P2, Der f1, Der f2 and Der m1.
일 구체예에서, 상기 집먼지 알러젠은 Der P1일 수 있다.In one embodiment, the house dust allergen may be Der P1.
또 다른 양상은 상기 센서에 먼지 시료를 처리하는 단계; 및Another aspect includes processing a dust sample to the sensor; and
상기 센서에서 알러젠 검출 여부를 확인하는 단계;를 포함하는 알러젠의 검출 방법을 제공한다.It provides an allergen detection method comprising the step of checking whether allergen is detected by the sensor.
상기 "센서", "알러젠", "검출" 등은 전술한 범위 내일 수 있다.The "sensor", "allergen", "detection", etc. may be within the aforementioned range.
일 양상에 따라 제작된 집먼지 알러젠 검출용 센서는 제작 과정이 간단하고, 신호 증폭 효과가 우수함을 확인하였다. 또한, 상기 센서는 기존의 알러젠 검출 방법에 비해 우수한 감도를 나타내고, 현장에서 소량의 먼지 시료에서도 신속/정확하게 알러젠의 검출이 가능함을 확인하였는 바, 집먼지 진드기로 인해 유발되는 여러 질병들의 예방이 가능하다.It was confirmed that the sensor for detecting house dust allergen manufactured according to one aspect had a simple manufacturing process and excellent signal amplification effect. In addition, it was confirmed that the sensor exhibits superior sensitivity compared to conventional allergen detection methods and can detect allergens quickly and accurately even in a small amount of dust samples in the field, so it is possible to prevent various diseases caused by house dust mites. .
도 1은 진드기 알러젠 검출용 센서의 계략도이다.
도 2는 상기 센서의 알러젠 검출의 민감도를 확인한 결과를 나타내는 도이다.
도 3은 상기 센서의 바이러스 표면 단백질인 헤마글루티닌(HA: hemagglutinin)에 대한 반응성을 비교한 결과를 나타내는 도이다.
도 4는 ELISA 방법과 상기 센서를 이용하여 실제 6종의 집먼지를 채취한 후 집먼지 시료에서 검출되는 진드기 알러젠을 검출한 결과를 나타내는 도이다.1 is a schematic diagram of a sensor for detecting mite allergens.
2 is a diagram showing the result of confirming the sensitivity of allergen detection of the sensor.
3 is a diagram showing the result of comparing the reactivity of the sensor to hemagglutinin (HA), a viral surface protein.
4 is a diagram showing the results of detecting mite allergens detected in house dust samples after actually collecting 6 types of house dust using the ELISA method and the sensor.
이하 본 발명을 실시예를 통하여 보다 상세하게 설명한다. 그러나, 이들 실시예는 본 발명을 예시적으로 설명하기 위한 것으로 본 발명의 범위가 이들 실시예에 한정되는 것은 아니다. Hereinafter, the present invention will be described in more detail through examples. However, these examples are intended to illustrate the present invention by way of example, and the scope of the present invention is not limited to these examples.
실시예Example
1. 알러젠 검출용 센서의 제작1. Fabrication of sensors for detecting allergens
알러젠 농도에 따른 전기화학신호의 변화를 감지하기 위한 센서 제작을 위하여 금 평면 인쇄 전극 (SPE: gold screen printed electrode)을 사용하였다. 11-메르캅토운데카산(11-MUA: 11-mercaptoundecanoic acid)을 자기조립한 금 센서 표면을 제작하기 위하여 10 mM 11-MUA 20 μL를 SPE 샘플 전극에 점적한 후 2시간 동안 상온 방치하였다. 고순도 에탄올로 전극 표면을 세척한 후, 질소 가스를 가하여 표면을 건조하였다. Der P1 항체를 11-MUA 자기조립 표면에 고정하기 위하여 EDC/NHS를 20 mg/mL 농도로 제조하여 20 μL를 전극 표면에 점적한 후, 상온에서 1시간 동안 방치하였다. 반응 후, 식염수로 표면을 세척하고, 질소 가스를 가하여 건조하였다. Der P1 항체를 10 μg/mL 농도로 희석하여 10 μL를 센서 표면에 점적하고, 상온에서 1시간 방치한 후, 식염수로 세척하고 질소 가스로 건조하였다. 마지막으로, 0.1% BSA 40 μL를 점적한 후 상온에서 1시간 동안 방치하고, 식염수 세척 및 질소가스 건조를 진행하였다. A gold screen printed electrode (SPE) was used to fabricate a sensor for detecting changes in electrochemical signals according to allergen concentration. To fabricate a gold sensor surface self-assembled with 11-mercaptoundecanoic acid (11-MUA), 20 μL of 10 mM 11-MUA was spotted onto the SPE sample electrode and left at room temperature for 2 hours. After washing the surface of the electrode with high-purity ethanol, the surface was dried by applying nitrogen gas. In order to immobilize the Der P1 antibody on the 11-MUA self-assembled surface, EDC/NHS was prepared at a concentration of 20 mg/mL, and 20 μL was applied onto the electrode surface, and then left at room temperature for 1 hour. After the reaction, the surface was washed with saline and dried by applying nitrogen gas. Der P1 antibody was diluted to a concentration of 10 μg/mL, 10 μL was applied onto the sensor surface, left at room temperature for 1 hour, washed with saline, and dried with nitrogen gas. Finally, after dripping 40 μL of 0.1% BSA, the sample was left at room temperature for 1 hour, washed with saline and dried with nitrogen gas.
2. 알러젠 검출용 센서를 이용한 알러젠 검출 효과 확인2. Confirmation of allergen detection effect using sensor for allergen detection
상기 제작된 Der P1 항체가 고정화된 센서에 Der P1 표준 물질을 처리할 경우, 알러젠 검출의 민감도를 확인하였다. Der P1 항원 표준 물질을 0 pg/mL, 0.001 pg/mL, 0.01 pg/mL, 0.1 pg/mL, 1 pg/mL, 10 pg/mL, 100 pg/mL, 1,000 pg/mL 및 10,000 pg/mL 농도로 식염수로 희석하여 준비하였다 (총 9개 농도). 항체가 고정화된 Der P1 센서 9개를 준비하고, 센서 1개당 1개의 표준 물질 용액과 결합시키고, 결합 전후에 센서 칩에 흐르는 전기 신호 변화 (전류 변화, uA)를 DY2000 potentiostat 기기 (Digi-Ivy 사, 미국)를 사용하여 측정하였다. 이를 위하여 1 mL 항원 표준 물질 용액에 항체가 고정화된 센서칩의 전극 부분을 담지하고, 37℃에서 30분간 방치하여 항원-항체 반응을 유도하였다. 항원 결합 전후의 전극으로 흐르는 전류 세기 변화 측정을 위하여 DY2000 potentiostat에 - 0.2 V 내지 0.6 V, 50 Hz 세기의 전위를 흘리면서 SWV(square wave voltammetry) 방식으로 전기화학 신호를 수집하였다. 가장 높은 전류가 흐르는 전위 (potential, V)에서 항원-항체 결합 전후의 전류 세기 차이를 각 항원 농도에서 나타나는 전기화학 신호 값으로 산정하여 항원 농도별 전기 신호 그래프를 작성하였다. 이와 같이 농도-신호 관계를 도시한 그래프에서 최고 전류 신호의 50% 세기의 전류 흐름을 나타내는 항원 농도를 센서 민감도를 판정하는 기준인 결합상수 KD 값으로 산정하였다. Sensitivity of allergen detection was confirmed when the prepared Der P1 antibody was immobilized with the Der P1 standard material. Der P1 antigen standards were prepared at 0 pg/mL, 0.001 pg/mL, 0.01 pg/mL, 0.1 pg/mL, 1 pg/mL, 10 pg/mL, 100 pg/mL, 1,000 pg/mL, and 10,000 pg/mL. It was prepared by diluting with saline to the concentration (9 concentrations in total). Nine antibody-immobilized Der P1 sensors were prepared, and each sensor was combined with one standard solution, and the change in electrical signal (current change, uA) flowing through the sensor chip before and after binding was measured using a DY2000 potentiostat (manufactured by Digi-Ivy). , USA) was used. To this end, the electrode part of the sensor chip on which the antibody was immobilized was supported in a 1 mL antigen standard solution, and left at 37° C. for 30 minutes to induce an antigen-antibody reaction. To measure the change in current intensity flowing through the electrode before and after antigen binding, electrochemical signals were collected by SWV (square wave voltammetry) method while passing a potential of -0.2 V to 0.6 V, 50 Hz to a DY2000 potentiostat. The difference in current strength before and after antigen-antibody binding at the potential (V) where the highest current flows was calculated as the electrochemical signal value at each antigen concentration, and an electrical signal graph was prepared for each antigen concentration. In this graph showing the concentration-signal relationship, the antigen concentration representing the current flow of 50% of the highest current signal was calculated as the coupling constant K D value, which is a criterion for determining sensor sensitivity.
그 결과, Kd 값이 5 fg/ml 수준으로 매우 높은 민감도를 나타냄을 확인하였다(기존 ELISA kit의 검출 한계 0.78 ng/ml)(도 2).As a result, it was confirmed that the Kd value showed very high sensitivity at the level of 5 fg/ml (detection limit of the existing ELISA kit was 0.78 ng/ml) (FIG. 2).
또한, 상기 제작된 센서의 바이러스 표면 단백질인 헤마글루티닌(HA: hemagglutinin)에 대한 반응성 비교하였다. HA에 대한 반응성을 측정하기 위하여 HA 항원 표준물질을 0 pg/mL, 0.001 pg/mL, 0.01 pg/mL, 0.1 pg/mL, 1 pg/mL, 10 pg/mL, 100 pg/mL, 1,000 pg/mL 및 10,000 pg/mL 농도로 식염수로 희석하여 준비하였다(총 9개 농도). Der P1 항체가 고정화된 Der P1 센서 9개를 준비하고, 센서 1개당 1개의 HA 표준물질 용액과 결합시키고, 결합 전후에 센서 칩에 흐르는 전기 신호 변화 (전류 변화, uA)를 DY2000 potentiostat 기기 (Digi-Ivy 사, 미국)를 사용하여 측정하였다. HA 표준 물질 용액에 대한 Der P1 센서의 교차 반응도 산정은 Der P1 표준 용액에 대한 Der P1 센서의 검출 능력 시험과 동일한 조건에서 진행하였다. In addition, the reactivity of the fabricated sensor to hemagglutinin (HA), a viral surface protein, was compared. To measure reactivity to HA, HA antigen standards were 0 pg/mL, 0.001 pg/mL, 0.01 pg/mL, 0.1 pg/mL, 1 pg/mL, 10 pg/mL, 100 pg/mL, 1,000 pg /mL and 10,000 pg/mL concentrations were prepared by dilution with saline (9 concentrations in total). Prepare 9 Der P1 sensors immobilized with Der P1 antibody, bind one HA standard solution per sensor, and measure the electrical signal change (current change, uA) flowing through the sensor chip before and after binding with a DY2000 potentiostat instrument (Digi -Ivy Co., USA) was used for measurement. The cross reactivity of the Der P1 sensor for the HA standard solution was calculated under the same conditions as the Der P1 sensor's detection ability test for the Der P1 standard solution.
그 결과, Der P1 센서에 바이러스 단백질인 HA를 0.01 pg/ml에서 10 ng/ml까지 처리하였을 때, 전기 신호의 변화가 거의 나타나지 않아, Der P1 단백질에 대한 특이적 반응성이 있음을 확인하였다(도 3). 또한, Der P1 1 pg/ml 이하 농도 검출이 가능할 경우 집먼지 내 알러젠이 fM 수준의 낮은 농도로 존재할 경우에도 검출 가능할 것으로 예상하였다.As a result, when the Der P1 sensor was treated with HA, a viral protein, from 0.01 pg/ml to 10 ng/ml, almost no change in electrical signal was observed, confirming that there was specific reactivity to the Der P1 protein (Fig. 3). In addition, if the concentration of Der P1 1 pg/ml or less can be detected, it is expected that the allergen in house dust can be detected even when the concentration is low at the fM level.
3. 집먼지 시료에서 검출되는 진드기 알러젠의 분석 방법의 비교3. Comparison of analysis methods for mite allergens detected in house dust samples
집먼지 시료에서 검출되는 진드기 알러젠을 ELISA 방법과 상기 제작된 센서를 이용하여 분석하고자 하였다. 집먼지 시료는 임의로 선택된 가정집 6곳에서 채집한 먼지에서 수용성 분획을 추출하여 준비하였다. 집먼지로부터 진드기 알러젠 분획을 추출하여 집먼지 시료를 준비하는 과정은 다음과 같다: 1) 먼지를 1 mm 혹은 더 작은 크기의 메쉬로 걸러 큰 사이즈의 파티클들을 제거한다. 2) 메쉬를 통과한 먼지 10 mg 내지 100 mg을 5 mL 튜브에 넣는다. 3) 먼지 100 mg 기준 2 mL PBS-T (0.05% Tween 20 in phosphate buffered saline, pH 7.4)를 넣는다. 20 μL/mg의 PBS-T 용량으로 추출을 진행한다. 먼지 분산액은 볼텍스(vortex)를 사용해 섞는다. 4) 2시간 동안 라커(rocker)를 이용해 상온에서 회전하면서 수용성 물질들이 용출되도록 섞는다. 5) 2,500 x g, 4℃에서 20분간 원심분리 한다. 6) 상등액을 취해 -20℃에 보관하고 침전물은 폐기한다.Mite allergens detected in house dust samples were analyzed using the ELISA method and the fabricated sensor. House dust samples were prepared by extracting water-soluble fractions from dust collected from six randomly selected households. The process of preparing a house dust sample by extracting the mite allergen fraction from house dust is as follows: 1) Filter the dust through a 1 mm or smaller mesh to remove large particles. 2) Put 10 mg to 100 mg of dust that has passed through the mesh into a 5 mL tube. 3) Based on 100 mg of dust, add 2 mL PBS-T (0.05% Tween 20 in phosphate buffered saline, pH 7.4). Proceed with extraction with a PBS-T volume of 20 μL/mg. The dust dispersion is mixed using a vortex. 4) Rotate at room temperature using a rocker for 2 hours and mix so that water-soluble substances are eluted. 5) Centrifuge at 2,500 x g and 4℃ for 20 minutes. 6) Take the supernatant and store it at -20℃, and discard the precipitate.
Der P1 분석은 상용 시판되고 있는 ELISA 키트 (Indoor Biotechnologies, 영국)를 구입하여, 제조사의 지침에 따라 분석하였다. ELISA 표준물질은 0.2 ng/mL 내지 100 ng/mL 범위로 준비하였다.For Der P1 analysis, a commercially available ELISA kit (Indoor Biotechnologies, UK) was purchased and analyzed according to the manufacturer's instructions. ELISA standards were prepared in the range of 0.2 ng/mL to 100 ng/mL.
그 결과, 상기 제작된 센서를 이용하여 분석한 경우에는 6개 시료 모두에서 Der p1 농도가 검출되었으나, ELISA 방법으로 분석한 경우에는 2개의 시료에서 검출되지 않았으며, 5개의 시료에서 검출된 Der P1은 ELISA 키트의 검출 한계에 미치지 않는 저농도임을 확인하였다(도 4). 구체적으로, ELISA로 분석한 6종 집먼지 시료내 진드기 알러젠 농도는 각각 0.31 ng/mL, 0 ng/mL, 0.21 ng/mL, 0.155 ng/mL, 0 ng/mL 및 2.339 ng/mL로 나타났고, 상기 제작된 센서를 이용한 경우, 진드기 알러젠 농도는 각각 0.12 ng/mL, 0.38 ng/mL, 0.37 ng/mL, 0.11 ng/mL, 1.37 ng/mL 및 11.44 ng/mL로 나타났다. As a result, Der p1 concentration was detected in all 6 samples when analyzed using the prepared sensor, but not detected in 2 samples and Der P1 detected in 5 samples when analyzed by ELISA method. It was confirmed that the low concentration did not reach the detection limit of the ELISA kit (FIG. 4). Specifically, the mite allergen concentrations in the six house dust samples analyzed by ELISA were 0.31 ng/mL, 0 ng/mL, 0.21 ng/mL, 0.155 ng/mL, 0 ng/mL, and 2.339 ng/mL, respectively, In the case of using the prepared sensor, the mite allergen concentrations were 0.12 ng/mL, 0.38 ng/mL, 0.37 ng/mL, 0.11 ng/mL, 1.37 ng/mL, and 11.44 ng/mL, respectively.
Claims (14)
상기 금 단분자층의 표면에 부착된 링커;
상기 링커에 연결된 고정화제; 및
상기 고정화제에 고정된 항체;를 포함하는 집먼지 알러젠 검출용 센서.
gold monolayer;
a linker attached to the surface of the gold monolayer;
an immobilizing agent connected to the linker; and
A sensor for detecting a house dust allergen comprising; an antibody immobilized on the immobilizing agent.
The method according to claim 1, wherein the linker is 11-mercaptoundecanoic acid (11-mercaptoundecanoic acid), 3-mercaptopropion (3-mercaptopropionic acid), 11-mercaptoundecanol (11-mercaptoundecanol), 6-methyl Capto-1-hexanol (6-mecapto-1-hexanol), mercaptopoly(ethylene glycol) carboxylic acid and mercaptopoly(ethylene glycol) amine (mercaptopoly(ethylene glycol) At least one sensor selected from the group consisting of amine).
The sensor according to claim 1, wherein the immobilizing agent is EDC/NHS (N-ethyl-N'-(3-(dimethylamino)propyl)carbodiimide)/(N-hydroxysuccinimide) or DCC (dicyclohexylcarbodiimide).
The sensor according to claim 1, wherein the antibody specifically binds to the house dust allergen.
The sensor according to claim 4, wherein the house dust allergen is at least one selected from the group consisting of Der P1, Der P2, Der f1, Der f2, and Der m1.
The method according to claim 1, Allergen detection sensitivity (Kd value) of the sensor is 1 to 10 fg / ml, the sensor.
The sensor according to claim 1, wherein the sensor has an allergen detection concentration of 0.05 ng/ml to 5,000 pg/ml.
상기 부착된 링커에 고정화제를 연결하는 단계; 및
상기 연결된 고정화제에 항체를 고정시키는 단계;를 포함하는 집먼지 알러젠 검출용 센서의 제조방법.
attaching a linker to a gold monolayer;
linking an immobilizing agent to the attached linker; and
A method of manufacturing a sensor for detecting house dust allergens comprising the step of immobilizing the antibody to the immobilizing agent connected thereto.
The method according to claim 8, wherein the gold monomolecular layer is self-assembled.
The method according to claim 8, wherein the step of attaching the linker is performed by treating the linker solution at a concentration of 5 mM to 15 mM.
The method according to claim 8, wherein the linking of the immobilizing agent is performed by treating a solution of the immobilizing agent at a concentration of 10 mg/mL to 30 mg/mL, the sensor.
The method according to claim 8, wherein the step of immobilizing the antibody is performed by specifically binding the antibody to a house dust allergen and fixing the antibody.
The method according to claim 12, wherein the house dust allergen is at least one selected from the group consisting of Der P1, Der P2, Der f1, Der f2 and Der m1.
상기 센서에서 알러젠 검출 여부를 확인하는 단계;를 포함하는 알러젠의 검출 방법.Processing a dust sample to the sensor of claim 1; and
An allergen detection method comprising the step of checking whether the allergen is detected by the sensor.
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