博碩士論文 91226013 詳細資訊




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姓名 李穎昌(Ying-Chang Li)  查詢紙本館藏   畢業系所 光電科學與工程學系
論文名稱 成對表面電漿波生物感知器應用在去氧核糖核酸及微型核糖核酸 雜交反應檢測
(DNA and miRNA analysis by Paired Surface Plasma-based biosensor )
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摘要(中) 表面電漿共振 (Surface Plasmon Resonance, SPR) 可以直接檢測偵測晶片上生物反應的特殊技術。SPR 因可監控偵測晶片上折射率的微量變化,在生化量測上排除了螢光標定過程,且能達到即時地分析生物分子之間交互作用的能力,縮短量測所需的時間。近年來,SPR技術已廣泛利用於生物分子診斷的許多相關領域,諸如抗原-抗體反應、蛋白質的非特異性吸附、去氧核糖核酸分子雜交等。
本次研究中,我們自行研發了相敏式表面電漿共振生物感知器(DP-SPRB)及成對表面電漿波生物感知器(PSPWB),它們具有相較於傳統SPR感知器,達到更高靈敏度且更好的雜訊抑制能力。其後我們利用成對表面電漿波生物感知器進行去氧核糖核酸分子雜交的量測,也成功區分單股核苷酸在互補配對及單點突變條件下雜交反應的差異,以及單股核苷酸序列長度不同時雜交反應的區別。我們也結合Langmuir isotherm equation 將高濃度去氧核糖核酸分子溶液的雜交結果的進行動力學分析。最後,我們利用表面電漿波生物感知器量測具有21個鹼基的微型核糖核酸 (miRNA) 雜交反應,並使用Langmuir isotherm方程式算出其结合速率常数。未來目標以金膜表面處理為優先,使系統能在血清或血漿作微量miRNA檢測。並將系統優化,使其維持高穩定性,以達到定量分析之能力,以期能讓系統在miRNA濃度分析上進行臨床檢驗,協助參與癌症預估及診斷分析。
摘要(英) Surface plasmon resonance spectroscopy is a surface characteristerization technique that can direct detect the biological interaction on the chip surface. The sensitivity to the refractive index of a substance is much higher than other detection methods and the feature of label-free detection can rule out some participations of the labeling process. It is particularly important to execute the biomolecular interaction analysis (BIA) with real-time monitoring, which is greatly shortened the detection period to avoid denaturation of surface-adsorbed antigens. In recent years, SPR spectroscopy has been widely applied in many areas of biomolecular diagnostics, such as receptor-ligand or antigen-antibody interactions, non-specific adsorption (NSA) of biomolecules, DNA hybridization, etc.
In this investigation, the development of differential phase surface plasmon resonance biosensor (DP-SPRB) and paired surface plasma waves biosensor (PSPWB) is to extend the detection limit and decrease the background fluctuation in biomaterial interactions. The discrimination between perfect-matched and single-base-pair-mismatched nucleic acid duplexes was performed by using PSPWB. An in vitro culture model was developed to make comparisons by using different sequence length of oligonucleotides for hybridization test on the platform of PSPWB, too. Finally, the kinetics information of short DNA oligonucleotides was obtained from PSPWB, and the hybridization detection of a 21-base-pair microRNA (miRNA) were proposed and discussed. In the future, we try to change the surface chemistry in blocking process to avoid the unwanted biomolecules and optimize the stability of our optical system to reach to quantitative detection of specific miRNA for the purpose of being used in cancer prognosis and diagnosis.
關鍵字(中) ★ 去氧核糖核酸
★ 表面電漿共振
★ 微型核糖核酸
★ 雜交
關鍵字(英) ★ microRNA
★ surface plasmon resonance
★ DNA
★ hybridization
論文目次 摘要 vi
ABSTRACT vii
ACKNOWLEDGEMENTS viii
LIST OF FIGURES xii
LIST OF TABLES xv
EXPLANATION OF SYMBOLS xvi
1 INTRODUCTION - 1 -
2 THEORY - 4 -
2.1 DNA and miRNA in general - 4 -
2.1.1 DNA - 4 -
2.1.2 MicroRNA - 7 -
2.2 SURFACE PLASMON RESONANCE - 9 -
2.2.1 Description of the SPR phenomenon - 10 -
2.2.2 The twe-interfaces system and its dispersion curves - 14 -
2.2.3 SPR profile including the resonance angle - 19 -
2.3 Langmuir Isotherm - 21 -
3 RESEARCH CONTENTS AND METHODS - 23 -
3.1 Interrogations of SPR-based sensor - 23 -
3.2 Experimental Methods - 33 -
3.2.1 Differential-phase surface plasmon resonance biosensor (DP-SPRB) setup ………………………………………………………………………...- 33 -
3.2.2 Paired Surface Plasma waves biosensor setup - 38 -
4 EXPERIMENTAL RESULTS IN DP-SPRB - 40 -
4-1 Detection Limit - 41 -
4-2 IgG/antimouse IgG interaction - 47 -
5 PSPWB OF DNA-RNA AND DNA-DNA HYBRIDIZATIONS - 51 -
5.1 Deteceion limit in short sequence length oligonucleotide hybridization - 52 -
5.1.1 Materials - 52 -
5.1.2 Immobilization of oligonucleotide probes - 54 -
5.1.3 The preparation of target oligonucleotide - 56 -
5.1.4 Measurement of affinity between oligonucleotides - 56 -
5.1.5 Construction of sensor chip - 57 -
5.1.6 Results and discussion - 60 -
5.2 Validation of the oligonucleotide binding specificity - 64 -
5.3 Length dependence of oligonucleotide target sequences - 66 -
5.4 Sequence dependence of oligonucleotide target - 68 -
5.5 Kinetics analysis - 70 -
5.6 DNA-RNA hybridization - 72 -
6 CONCLUSION - 75 -
6.1 DP-SPRB - 75 -
6.2 PSPWB detection and hybridizations of oligonucleotide or miRNA - 77 -
REFERENCE - 79 -
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指導教授 李正中、周晟
(Cheng-Chung Lee、Chien Chou)
審核日期 2012-7-26
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