根據世界衛生組織的統計，光是在2018年，就有1810萬人罹患癌症並且有960萬人死於癌症當中，這也使得癌症成為全球致死率第二高的疾病。因此，為了提高癌症病人的存活率，發展一套有效率且精確的系統用於癌症診斷以利採用後續適當的治療方案，是非常重要的議題。在眾多癌症檢測方式當中，由於免疫組織化學染色法擁有快速且高度專一性這些特點，在臨床試驗上最為經常被使用來檢測惡性腫瘤細胞。然而，免疫組織化學染色法在臨床檢測當中還是面臨著一些挑戰。舉例來說，免疫組織化學染色法是相對來說較為耗時並且需要眾多繁複的步驟去完成的實驗。此外，也需要訓練有素的臨床檢驗人員去呈現穩定且可信的組織染色實驗。而儘管在微流體領域當中，已經有團隊也致力於開發晶片上的組織免疫染色，但是自動化的組織免疫染色平台目前仍未被實現。因此，根據目前臨床上的需求，本研究開發了一套整合型的微流體系統，能夠運用石蠟包埋組織並且以適體為基底來呈現螢光染色和免疫組織染色實驗用於診斷卵巢癌整體實驗部分，包含組織預處理以及染色步驟，只需要兩個小時，比傳統臨床染色方式快了一倍。此外，我們可以使用490 µL的試劑同時染十片組織，來達成高通量的染色結果。因此，此套新型微流體系統，可以作為強而有力的工具，用於診斷癌症，為臨床領域帶來重大的改變。

According to the World Health Organization (WHO), in 2018 about 18.1 million new cancer cases were lodged and about 9.6 million deaths were reported which made cancer the second preeminent cause of death worldwide. Therefore, there is immense importance in developing an efficient and precise method for diagnosing cancer and implement proper therapeutic strategies for increasing the chance of survival. In comparison with some other cancer detection methods, immunohistochemistry (IHC) is one of the most commonly and widely used in clinical tests for the diagnosis of abnormal tissues such as cancerous tumors due to its quick and high specificity features. However, there are still challenges as it is relatively labor-intensive, time-consuming process steps which require well-trained technicians to produce consistent and reliable staining results. Even though there were some previously reported articles which demonstrated IHC staining using microfluidic platforms but none of them had achieved full-automation yet. Therefore, considering the current demand we demonstrated an integrated microfluidic platform capable of performing the entire process for aptamer-based fluorescence and aptamer-based IHC staining of formalin-fixed paraffin embedded tissue samples for diagnosis of ovarian cancer. The entire pretreatment and staining procedure could be completed within 2 hours, which is 50% faster than conventional approach. Moreover, ten tissue sections could be simultaneously stained by only using 490 µL of reagents to achieve high throughput staining process. Thus, this newly designed microfluidic device brings significant promise to serve as a powerful tool for clinical diagnosis of cancer.

Table of contents
Acknowledgments I
Abstract III
中文摘要 V
Table of contents VI
List of Figures VIII
List of tables XII
Nomenclature and abbreviations XIII
Chapter 1 Introduction 1
1.1 Cancer 1
1.2 Cancer diagnosis methods 3
1.3 Comparison of nucleic acid aptamers and antibodies 5
1.4 Microfluidic chip 7
1.5 Microfluidic chip for clinical tissue staining 9
1.6 Motivation and novelty 11
Chapter 2 Materials and methods 13
2.1. Chip design and fabrication process 13
2.2. Custom-made air control system and temperature control module 18
2.3. Working principle and experimental procedures 20
2.3.1 Working principle 20
2.4 Tissue preparation 32
2.5 FFPET pretreatment process 32
2.6 On-chip aptamer-based IHC staining process 33
2.7 On-chip aptamer-based fluorescent staining process 34
2.8 H-score measurement for IHC staining image 36
2.9 Image acquisition and analysis for fluorescent staining 36
Chapter 3 Results and discussion…………………………...38
3.1 Characterization of the integrated microfluidic system 38
3.1.2 Mixing index of the micro-stirrer 42
3.2 Temperature performance of the temperature control module 45
3.3 Aptamer-based fluorescence staining 49
3.4 Optimization of operation conditions for on-chip IHC staining 55
3.5 On-chip Aptamer-based IHC staining 59
3.6 Assessment of on-chip aptamer-based assay at different incubation time 67
3.7 Uniformity testing of normal tissue sample by using integrated microfluidic system 71
3.8 Blind testing for on chip clinical tissue staining 73
Chapter 4 Conclusions and future prospective 75
4.1 Conclusions 75
4.2 Future prospective 77
Optimization of the operating conditions on the microfluidic system 77
Verification of clinical tissue samples 77
References 79

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