초록 ▼

본 연구는 “생체 기능 모니터링용 바이오 센서의 구성요소인 생물 분자 설계, 생산 기술 및 바이오 검출시스템 확립” 을 최종 목표로 급성 또는 민성염증, 동맥경화, 고혈압, 협심등, 고지혈증, 뇌경색, 심근경색등과 같은 환자들의 모니터링을 위한 생물소재 (항원, 항체) 및 진단기술을 개발하고, 이를 나노바이오센서에 응용하여 질병의 예방/예후 목적으로 진단기술의 산업화를 목표로 함.
이를 위하여 구성요소인 생물분자(CRP, troponin 또는 CK-MB)들의 분리, 정제 및 단백질 발현시 스템을 이용한 단백질 발현 및 이들에

본 연구는 “생체 기능 모니터링용 바이오 센서의 구성요소인 생물 분자 설계, 생산 기술 및 바이오 검출시스템 확립” 을 최종 목표로 급성 또는 민성염증, 동맥경화, 고혈압, 협심등, 고지혈증, 뇌경색, 심근경색등과 같은 환자들의 모니터링을 위한 생물소재 (항원, 항체) 및 진단기술을 개발하고, 이를 나노바이오센서에 응용하여 질병의 예방/예후 목적으로 진단기술의 산업화를 목표로 함.
이를 위하여 구성요소인 생물분자(CRP, troponin 또는 CK-MB)들의 분리, 정제 및 단백질 발현시 스템을 이용한 단백질 발현 및 이들에 대한 항체들을 생산하여 혈액에서 이들의 농도 측정이 가능한 지 또는 향후 센서에 적용될 수 있도록 sandwich ELISA 법을 확립하였으며, 100 case 이상의 임상시 료를 대상으로 이를 측정하여 임상적 유의성을 검증하였음. 또한 개발된 생물소재 및 진단법을 공동연구팀에 제공하여, 바이오센서에 적용함.
1 cm 크기의 유리 기질 위에 500 nm 이하의 금나노패턴을 만들어내어 생체분자가 효율적으로 결합하도록 제작하였고, 항체(결합 단백질) 혹은 항원(타겟 단백질)의 최적 어레이 조건을 단일분자 수준에서 확립, 심혈관질환 시료인 트로포닌을 이용하여 심혈관질환 진단을 효과적으로 이뤄낼 수 있음을 확인하였고, 고감도 바이오센서 기술을 심혈관질환 시료에 이용하여 실시간, 조기, 신속, 현장 질병진단 및 치료에 응용할 수 있음을 확인했고, 이를 통해 다양한 바이오마커에도 응용이 가능함을 확인함.

Abstract ▼

Ⅳ. Results
1. Separation and purification of CRP protein
- For antibody production we separated and purified the CRP antigen from CRP-rich fluid (synovial, ascitie fluids) which we obtained from patients suffering fever or inflammation. CRP was separated and purified by manufacturing p-Diazoni

Ⅳ. Results
1. Separation and purification of CRP protein
- For antibody production we separated and purified the CRP antigen from CRP-rich fluid (synovial, ascitie fluids) which we obtained from patients suffering fever or inflammation. CRP was separated and purified by manufacturing p-Diazonium phenylphosphoryl choline (DPPC) affinity column in the first step and fractions obtained from DPPC column used Hydroxylapatite (HA) column chromatography for CRP purification. As a result we were able to obtain purely separated CRP protein.
2. Production of CRP protein and antibody
- Mass production of CRP antigen is required for development of future diagnostic technology and diagnostic reagents. So we produced and expressed the CRP antigen for stable supply of antigen by using not only separation and purification but also E. coli expression system. We produced the specific antibodies by hybridoma technology by using these antigens for mice immunization.
3. Production of Troponin and CK-MB protein and antibody
- Antigens were produced in large scale by protein expression in Troponin and CK-MB, separated and purified antigens were checked by western blot using prototype antibody. As a result antigens manufactured by expression systems are suitable for manufacturing antibodies and were verified to react specifically on existing antibodies. These Troponin and CK-MB proteins were immunized on mice to produce antibodies and we checked the subtype and titer of the antibody.
4. Constructing analysis technique using antibody
- To analyze the usefulness of the marker, we applied it on clinical samples (blood or serum) to determine whether it is possible to check the disease or not using manufactured biomarker proteins and antibodies
- Construction of diagnostic technology and production of antigen and antibody for practicalization of nano-biosensor
- Establishment of measurement condition by the existing measurement techniques and the developed nano-biosensor
- Establishment of measurement condition using clinical examples by collaboration with KAIST
5. Analysis of sensor performance and quality control
- Measurement of CRP concentration using nano-biosensor Before applying to the nano-biosensor of KAIST using developed antignes and antibodies, we did an experiment fixing antibodies on the glass surface. A succeed on fixing antibody we obtained a result 2nd antibody-FITC conjugate successfully reacting. Also after fixing the antibody, we tried to quantify CRP concentration by loading different CRP standard samples on the surface of sensors.
- Fabrication of gold nanoarray chip Fabrication of gold nanoarray chip with 100-500 nm spot for high sensitive detection
- Development of single-biomolecule detection technology Detection of single cTnI using fluorescence and non-fluorescence optical detection.
- Quantification of bio-marker at < aM concentration on nanoarray chip and research application to real sample High sensitivity detection of cTnI protein and application of various sample (bio-marker) on immuno-protein chip. The quantity analysis and detection of cTnI and CRP at single molecule level.
6. Application on clinical samples of CRP, Troponin I, Troponin T, and CK-MB
- Using manufactured antigens antibodies, to establish immune analysis technology used ELISA technique to measure the CRP concentration in the blood of patients suffering inflammation and fever. Also we used the same method to obtain the Troponin I,Troponin T, and CK-MB concentration in the blood of patients suffering cardiovascular diseases. These antigens, antibodies, and bloods of patients were applied on the nano-biosensor and established the sensor detection technique with the joint research with Kyunghee Univ. and KAIST.
7. Establishment of the mass production system of bio-substance and sensor
- Quantification of these biomarkers was tried by application of the nano-biosensor manufactured by KAIST on clinical specimens. It shows similarity with the concentration measured by existing techniques.