在人類文明中，癌症是最致命、最無法醫治的疾病之一。目前雖然已經發展出許多先進的治療技術，但現今還是難以找到一個普及而且有效的治療方法，尤其是在後期階段。要提升治療的效果，癌症專一的標記技術是不可或缺得，若能將癌症專一標記技術與現有的治療方法結合，便有機會得到更好的治療效果。因此我們極需一個個人化並且有效的癌腫瘤辨別技術。現今，適體已被證實是個可以有效辨別生物表現的一種分子，它在藥物傳輸應用方面也是一項非常具有潛力的工具。找出一種針對癌症具有專一性與高親和性的適體絕對是在腫瘤學領域中最重要的目標之一。這些具有癌症專一性與高親和性的適體通常可以利用配體指數增強進化技術進行挑選，然而配體指數增強進化技術本身是一項步驟繁複、耗時且勞力密集的技術。粗略計算，在大型系統上利用配體指數增強進化技術操作10至20次的配體篩選需要好幾個禮拜的時間。本研究所開發的自動化微流體系統將使用臨床的癌組織檢體連續操作6次配體篩選以分離出針對癌症具有專一性與高親和性的適體。其中所有繁複的操作流程例如:試劑傳輸、混合、沖洗、適體放大以及熱處裡，全部都由12個電熱製冷晶片與36個電磁閥整合而成的系統完成。此系統只需花費15小時即可完成整個6次自動化的配體篩選程序。實驗數據顯示此系統已成功篩選出對癌組織具有專一性的適體。其中三個適體已被證實對目標檢體具有專一性，透過親合性分析測試得到Kd值約為129 nM。此系統在檢測應用上將會是一個具有潛力的一項工具。

Cancer is one of the deadliest incurable diseases known to man. Although several sophisticated treatments have already been developed, it is still a challenge to find an effective universal methodology to treat cancers, especially in advanced stages. To establish an effective treatment regimen, cancer specific targeting technique is required. With the combination of cancer labeling techniques and current cancer treatment, it is possible to obtain better results. Therefore, a personalized and effective tumor targeting technique is of great demand. Aptamer is considered to be a useful affinity reagent for biomarker recognition and has great potential in drug delivery. Thus finding a potential aptamer with high affinity and specificity towards specific target cancer cells or tissues is definitely crucial in oncology. By repeating rounds of systematic evolution of ligands by exponential enrichment (SELEX) with cancer biopsies, aptamers with high specificity and affinity could be screened. However, SELEX is a complicated, time-consuming and labor-intensive process and takes several weeks to complete 10-20 cycles of screening on a bench-top system. In this work, an automatic microfluidic system was therefore developed to screen specific aptamers by performing on-chip SELEX with clinical cancer tissue samples in a consecutive format. The integrated microfluidic system consisted of 12 thermoelectric chips and 36 electromagnetic valves to carry out the complicated SELEX steps of reagent transportation, mixing, washing, amplification, and thermal processes. It only took 15 hours to operate 6 rounds of SELEX automatically. Experimental results showed that cancer-specific aptamers could be successfully screened on the integrated system. Three screened aptamers were confirmed with high specificity toward the target tissue. Affinity analysis showed that dissociation constant was around 129 nM. The developed system may be a powerful tool for screening affinity reagents for diagnostic applications.

Table of contents
Abstract I
摘要 III
Table of contents V
List of figures VII
List of tables X
Abbreviations and nomenclature XI
Chapter 1 Introduction 1
1-1 Cancer 1
1-2 Microfluidics 3
1-3 Aptamer and antibody 5
1-4 Systematic evolution of ligands by exponential enrichment (SELEX) 7
1-5 Literature survey 9
1-5-1 Biomarker applications and screening on bench-top methods 9
1-5-2 SELEX applications on microfluidic systems 11
1-6 Novelty and motivation 13
Chapter 2 Materials and methods 15
2-1 Experimental setup 15
2-2 Tissues, ssDNA library, primers, reagents and operating conditions 24
2-3 Chip design and fabrication 27
2-4 Tissue slide pretreatment 32
2-5 Experiment procedure 33
2-6 Fluorescent imaging of aptamer and determination of dissociation constants 37
Chapter 3 Results and discussion 39
3-1 Performance of microfluidic devices 39
3-1-1 Pumping volume and mixing index 39
3-1-2 Thermal cycling of thermoelectric chips 44
3-2 Results of tissue-SELEX 53
3-2-1 Consecutive tissue-SELEX 53
3-2-2 Aptamer candidates 56
3-2-3 Identification of OvCa specificity and affinity 59
Chapter 4 Conclusions and Future perspective 64
4-1 Conclusions 64
4-2 Future perspective 66
References 68

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