Development of an ultrasensitive immunoassay for prostate specific antigen using DNA-gold nanoparticle assisted enzyme amplification method
- 발행기관 서강대학교 일반대학원
- 지도교수 오병근
- 발행년도 2009
- 학위수여년월 2009. 2
- 학위명 석사
- 실제URI http://www.dcollection.net/handler/sogang/000000045130
- 본문언어 한국어
초록/요약
암, 노인성 치매, 광우병 등과 같은 각종 질병에 대한 조기 진단을 위해서는 체내에 존재하는 질병에 대한 생물표지(biomarker)를 정량적으로 분석할 수 있는 고감도 바이오센서의 개발이 반드시 필요하다. 일례로 광우병의 경우, 그 질병의 원인체인 변형 프리온(prion)이 혈액 내에 극소량(ml 당 약 104개의 변형 프리온)이 존재할 것으로 예상되기 때문에 혈액을 이용한 조기 진단은 현재의 센서 기술로는 불가능하다고 알려져 있다. 따라서 민감도가 향상된 바이오센서의 개발을 위하여 전 세계적으로 활발한 연구가 진행되고 있다. 본 실험의 목적은 위의 내용을 토대로 enzyme을 증폭시켜 센서의 민감도를 향상시키는 것이다. 먼저 prostate specific antigen(PSA)의 항원을 측정하기 위하여 magnetic microparticle(MMP)와 Au nanoparticle(AuNP)에 항체를 결합시킨다. 또한 AuNP에는 DIG-labeled된 Oligouncleotide가 결합되어 있다. 여기에 다시 HRP가 포함된 anti-DIG을 넣어주게 되면 실험의 목적인 HRP의 수가 기존보다 더 많이 보유하게 된다. 항원과 위에서 만든 MMP와 AuNP를 반응시키고 magnetic field에서 분리시킨 후 여러번의 세척과정을 거쳐 기질인 tetramethylbenzidine(TMB)와 반응시킨 후 2M의 H2SO4로 반응을 중지시키면 UV-vis spectroscopy로 항원인 PSA를 측정할 수 있다.
more초록/요약
The enzyme amplification based bioassay for the detection of pathogen typically involves two types of particles, a magnetic microparticle (MMP) functionalized with an monoclonal antibody that has an affinity for a target of interest such as prostate specific antigen(PSA) and an Au nanoparticle functionalized with an polyclonal antibody that has an affinity for the same target of interest. The DIG-labeled DNA with thiol terminal group that is generated by DIG-High Prime according to the random primed labeling technique was covalently attached on the Au nanoparticle. And then HRP labeled anti-DIG was bound to DIG labeled DNA on Au nanoparticle. By this process, the number of HRP on Au nanoparticle could be enhanced and effectively act as reporter groups for the target of interest. The MMP probes are added to solution containing the target of interest. After the MMP probes have been given a chance to react with target, the Au nanoparticle probes are added to form a sandwich structure with the MMP probes that have captured target. A magnetic field is used to separate such sandwich complexes from the test solution, and the supernatant is discarded. After copious washing step, the immobilized enzyme on Au nanoparticle probes is reacted with tetramethylbenzidine (TMB) that is a substrate of HRP, followed by stopping the reaction with 2M H2SO4. The resulting end products were analyzed by UV-vis spectroscopy. The enzyme amplification based bioassay was applied to detect the pathogens such as PSA existing in contaminated environment and it could be successfully obtained colorimetric signal as a function of pathogen concentration. The proposed colorimetric detection schemes using enzyme are very attractive for bioassays because of their high sensitivity, simplicity, miniaturization, and low cost.
more목차
1. Introduction = 1
1.1 Definition of biotechnology = 1
1.2 Prostate Specific Antigen = 3
1.3 Enzyme Linked Immunosorbent Assay = 3
1.4 Scanning Electron Microscopy = 4
2. Materials and Methods = 7
2.1 Materials = 7
2.2 Method of fabrication magnetic microparticle probe = 9
2.3 Method of fabrication gold nanoparticle probe = 14
2.4 Protein detection and Enzyme Amplification = 20
3. Results and Discussion = 25
3.1 Comparison to Oligonucleotide tailing = 25
3.2 Comparison to amount of HRP = 27
3.3 Comparison to deferent gold nanoparticle size = 28
3.4 Scanning Electron Microscope (SEM) detection of MMP, AuNP and PSA = 30
3.5 Results of detection to Prostate Specific Antigen (PSA) = 32
4. Conclusion = 33
5. References = 34
List of Figure Captions
Figure 1.1. Biotechnology = 3
Figure 2.1. Principle of Dynabeads M-280 Tosylactivated = 38
Figure 2.2. SEM image of Dynabeads M-280 Tosylactivated = 39
Figure 2.3. Non-radioactive oligonucleotide tailing = 40
Figure 2.4. Oligonucleotide tailing with DIG-dUTP/dATP = 40
Figure 2.5. Kit contents = 40
Figure 2.6. A schematic of determination the AuNP and Pab = 40
Figure 2.7. A schematic of AuNP probes = 40
Figure 2.8. MMP probes and AuNP probes = 40
Figure 2.9. The concept of enzyme amplification = 40
Figure 2.10. A schematic of enzyme amplification assay = 40
Figure 3.1. Non-Oligonucleotide tailing = 60
Figure 3.2. DIG-Oligonucleotide tailing = 63
Figure 3.3. Comparison to amount of HRP = 63
Figure 3.4. The data of comparison to deferent gold nanoparticle size 1 = 63
Figure 3.5. The data of comparison to deferent gold nanoparticle size 2 = 63
Figure 3.6. SEM images of the prepared solution = 63
Figure 3.7. SEM images of comparison to deferent concentration = 63
Figure 3.8. Results of detection to PSA = 63
