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研究生： 普麗雅
Priya, Gopinathan
論文名稱： 利用整合型微流體平台篩選和選擇親和試劑及其在生物學上的應用
Screening and selection of affinity reagents using integrated microfluidic platforms: applications in biology
指導教授： 李國賓
Lee, Gwo-Bin
口試委員: 洪上程
Hung, Shang-Cheng
陳致真
Chen, Chih-Chen
謝淑珠
Shiesh, Shu-Chu
沈延盛
Shan, Yan-Shan
學位類別： 博士
Doctor
系所名稱： 工學院 - 奈米工程與微系統研究所
Institute of NanoEngineering and MicroSystems
論文出版年： 2019
畢業學年度： 107
語文別： 英文
論文頁數： 119
中文關鍵詞： 微流體 、親和試劑 、適體 、膽管癌 、心肌梗塞 、配體指數增強進化技術 、循環腫瘤細胞 、糖胺聚糖
外文關鍵詞： SELEX, glycosaminoglycan, microfluidics, affinity reagent, aptamer, cholangiocarcinoma, acute myocardial infarction, circulating tumor cells
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    • 近年來，微流體在生醫領域蓬勃發展，用於快速和精準診斷的微流體系統日益增多，已儼然自成一家。由於抗體具有高成本，以及批次間差異導致的再現性問題，一些人工抗體的體外篩選方式越來越受到歡迎。在本篇研究中，我們介紹了適體的篩選方式及其在世界上兩種主要疾病(癌症和心血管疾病中)的應用。首先，我們先行開發了一微流體系統，該系統可針對膽管癌細胞（cholangiocarcinoma ，CCA），進行適體篩選。利用配體指數增強系統進化技術 (Systematic Evolution of Ligands by EXponential enrichment, SELEX)能在連續6次的篩選過程中找到2種具有高親和力和特異性的適體，比傳統方式需15次連續篩選來的更有優勢。在後續實驗中，也證明該篩選適體能成功的分辨目標和非目標CCA細胞。接著，我們設計了另一款微流體裝置，結合上述篩選出的適體，能成功地從30位以上的CCA患者血液中分離出循環腫瘤細胞（circulating tumor cells ，CTC），其分離率高達100%。此外，我們也嘗試使用另一種親和試劑-醣胺聚醣 (glycosaminoglycans)，測試其抓取CCA細胞的潛力，最後更使用該醣成功的從 CCA患者周邊血液中分離出CTC，該部分研究也針對CTC在CCA中的預後意義作探討。最後，我們也針對急性心肌梗塞（acute myocardial infarction，AMI）的顯著生物標誌 (肌鈣蛋白I (cardiac troponin I) )進行適體篩選。更進一步在另一開發之微流體平台上，成功的利用酶聯DNA適體方法測定該蛋白。本研究中使用的所有微流體平台都已經實驗驗證其功效性。相信這些微量全分析系統（μTAS）具有能用於臨床診斷或即時護理系統的潛力。

    Microfluidics has emerged in recent years as a distinct new area of biomedical fields and an increasing number of assays have been demonstrated on microfluidic systems for rapid and accurate diagnosis. Due to the high cost, batch-to-batch variation and reproducibility issues in antibodies, in-vitro screening methods have gained popularity recently. In this work, we presented the screening of aptamers and their applications for two of the major diseases in the world i.e., cancers and cardiovascular diseases. First, a new integrated microfluidic system for screening of aptamers against cholangiocarcinoma (CCA) cells was reported. Two aptamers with high affinity and specificity were screened in just six rounds of the process called Systematic evolution of ligands by exponential enrichment (SELEX) in comparison to the fifteen or more rounds required in a traditional process. This system could successfully distinguish between target and non-target CCA cells. Furthermore, the screened aptamers were then used to isolate circulating tumor cells (CTCs) from the blood of more than 30 CCA patients with 100% response on a newly designed integrated microfluidic platform. In the quest for new alternative affinity agents, glycosaminoglycans were also analyzed for their potential to recognize CCA cells. It was also used for the isolation of CTCs from peripheral blood of CCA patients. The prognostic significance of CTCs in CCA was also explored. Finally, aptamers were screened against a prominent acute myocardial infarction (AMI) biomarker i.e., troponin I. It was further used to develop an enzyme-linked DNA aptamer assay on an integrated microfluidic platform. All microfluidic platforms used in this study were thoroughly validated. Experimental results indicated that these micro-total-analysis-systems (μTAS) could serve as promising tools for clinical diagnosis or point-of-care systems in the near future.

    Table of Contents Abstract III 摘要 IV List of Tables VIII List of figures IX Abbreviations and Nomenclature XIII Chapter 1 : Introduction 1 1.1 Microfluidic technologies and Lab-on-a-chip 1 1.2 Affinity reagents and types 3 1.3 Systematic Evolution of ligands by exponential enrichment (SELEX) using conventional platforms and limitations 4 1.4 Improvements in SELEX 5 1.5 Glycosaminoglycans 7 1.6 Cholangiocarcinoma 8 1.7 Cholangiocarcinoma and circulating tumor cells 9 1.8 Acute myocardial infarction and cardiac troponins 9 1.9 Motivation and objectives 10 1.9.1 Selection of aptamers against intrahepatic cholangiocarcinoma cells using an integrated microfluidic platform 10 1.9.2 Isolation of circulating tumor cells from blood of patients with cholangiocarcinoma using aptamers on an integrated microfluidic platform 11 1.9.3 Glycosaminoglycan as a potential affinity reagent for the diagnosis of CCA metastasis: Exploring the capabilities using an integrated microfluidic platform. 13 1.9.4 Aptamer for human cardiac Troponin I (TnI) detection and optimization of an Enzyme-linked DNA aptamer assay on an integrated microfluidic platform. 13 1.10 Scope and structure of the dissertation 14 Chapter 2 : Selection of aptamers against intrahepatic cholangiocarcinoma using an integrated microfluidic platform 16 2.1 Introduction 16 2.2 Materials and methods 17 2.2.1 Cell lines and cell culture 17 2.2.2 Immuno-magnetic beads and ssDNA library 18 2.2.3 Fabrication process of the microfluidic chip 18 2.2.4 Characterization of mixing and pumping devices 19 2.2.5 Working conditions and PCR 20 2.2.6 Negative selection and WBC isolation 22 2.2.7 Monitoring the enrichment of the selected DNA pool 22 2.2.8 Cloning, sequencing and synthesis of selected aptamers 23 2.2.9 Determination of dissociation constants by flow cytometry 24 2.2.10 Fluorescence imaging of FAM labelled aptamers bound to CCA cells and negative control cells 24 2.2.11 Testing the binding selectivity of CCA aptamers to different cell lines 25 2.3 Results and discussion 26 2.3.1 Microfluidic chip, its operation and characterization of microdevices 26 2.3.2 Screening aptamers against CCA through cell-SELEX 32 2.3.3 Monitoring the enrichment of the selected DNA pool 35 2.3.4 Selection of aptamer 35 2.3.5 Cell staining and fluorescence imaging 39 2.3.6 Binding selectivity of CCA specific aptamers 40 2.4 Summary 41 Chapter 3 : Isolation of circulating tumor cells from blood of patients with cholangiocarcinoma using aptamers on an integrated microfluidic platform 42 3.1 Introduction 42 3.2 Materials and methods 43 3.2.1 Microfabrication and design of the microfluidic chip 43 3.2.2 Characterization of micromixer and micropump 45 3.2.3 Conjugation of HN16 to magnetic beads 46 3.2.4 Variation in aptamer binding as a factor of pH and various ion concentrations in the buffer 47 3.2.5 Cell culture and spiking experiments 47 3.2.6 Blood specimen collection 48 3.2.7 Experimental set-up 50 3.2.8 Assessment of CTCs in metastatic CCA patients before and after chemotherapy 56 3.3 Results and discussion 57 3.3.1 Microfluidic device 57 3.3.2 Characterization of the micromixers and micropump 59 3.3.3 Variation in aptamer binding as a factor of pH and various ion concentrations in the buffer 61 3.3.4 Spiking experiments and CTCs isolated using aptamer on the integrated microfluidic platform 62 3.3.5 Role of CTCs in assessing disease status 64 3.4 Summary 67 Chapter 4 : Glycosaminoglycan as a potential affinity reagent for the diagnosis of CCA metastasis: Exploring the capabilities using an integrated microfluidic platform 69 4.1 Introduction 69 4.2 Materials and methods 70 4.2.1 Glycosaminoglycans 70 4.2.2 Conjugation of GAGs to magnetic beads 72 4.2.3 Capture rate percentages of GAGs with different cell lines 72 4.2.4 Conjugation of SCH45-biotin with Strepavidin-FAM and staining experiments 73 4.2.5 Blood samples, experimental setup and integrated microfluidic platform. 74 4.2.6 Spiking experiments and isolation of CTCs 74 4.3 Results and discussion 75 4.3.1 Results of Capture rate tests 75 4.3.2 Results of spiking experiments 76 4.3.3 Staining with SCH45-Alexa Fluor 488 78 4.3.4 CTC detection using SCH45 on the integrated microfluidic chip 79 4.3.5 Role of CTCs in assessing disease status 82 4.4 Summary 85 Chapter 5 : Aptamer against human cardiac Troponin I and optimization of an Enzyme-linked DNA aptamer assay on an integrated microfluidic platform. 88 5.1 Introduction 88 5.2 Materials and methods 89 5.2.1 Aptamer screening against Troponin I on an integrated microfluidic platform. 89 5.2.2 Fabrication and operational principle of the newly designed microfluidic chip 91 5.2.3 Samples and reagents 91 5.2.4 Magnetic beads coated with aptamers 92 5.2.5 Optimization of operational parameters on the microfluidic chip 93 5.2.6 Optimization of analytical conditions 94 5.2.7 Aptamer-magnetic bead-based sandwich assay 94 5.3 Results and discussion 96 5.3.1 Microfluidic chip 96 5.3.2 Characterization of the microfluidic platform 98 5.3.3 Optimization of conditions on the microfluidic platform 99 5.3.4 Results of the as developed assay and specificity test 100 5.4 Summary 104 Chapter 6 : Conclusions and future prospective 106 6.1 Conclusions 106 6.1.1 Pros and cons of the three affinity reagents used 107 6.2 Future prospective 109 References 111

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