敗血症是一種可能危及生命的疾病，定義為感染所引發的全身性嚴重發炎反應，細菌感染是血液感染的最常見原因，然而也可能由病毒和真菌感染引起。其所引起的發炎反應可能導致組織損傷，器官衰竭，休克甚至死亡，因此敗血症在臨床上是一種可能會危及性命的嚴重併發症，致死率可能高達七成以上。最標準的細菌檢測方式為利用培養基進行細菌增殖，再利用一系列生化反應，例如微生物發酵、特定受質的利用、還原與降解等特性來鑑定微生物種類，或是利用聚合酵素連鎖反應 (Polymerase Chain Reaction，PCR)，質譜鑑定(Mass Spectrometry，MS)等大型機台進行判讀，這些方法精準度高，但至少需耗費一天以上作增殖反應，若遇上培養困難、生長緩慢或是無適當培養基的菌種再更為耗時。
為了克服上述問題，本研究提出了兩種微流體介電泳晶片，基於介電泳力(Dielecteophoresis, DEP)與電滲流(Electroosmotic flow)分離與聚集大腸桿菌(Escherichia coli, E. coli)，並利用SERS指紋識別技術檢測微流道中的細菌。第一種為分離晶片，藉由指叉型交錯之金屬電極結構，產生不均勻電場之介電泳力分離出大腸桿菌，並利用自摻雜磺化聚苯胺(self-doped sulfonated polyaniline, SPANI)作為塗層材料整合於Au電極上，改善表面非特異性吸附造成的沾黏問題；第二種為聚集晶片，藉由上與下之銦錫氧化物玻璃(ITO-coated glass)作為電極，產生之介電泳力與電滲流聚集大腸桿菌。最後，於待測溶液中加入能夠增加拉曼訊號之碘化奈米銀粒子(AgINMs)，並應用聚集晶片濃縮待測物，目前已可辨識出大腸桿菌之SERS訊號。

Sepsis is a potentially life-threatening condition and defined as the systemic inflammatory response syndrome (SIRS) caused by infections. Bacterial infections are the most common cause of bloodstream infection; however, they can also be caused by viral and fungal infections. The inflammatory reaction caused by sepsis may lead to tissue damage, organ failure, shock, or even death. Therefore, sepsis is a serious complications that can endanger lives, and the mortality rate might reach up to 70%. The most standard method of bacterial detection is to use the culture medium for bacterial proliferation, and then use a series of biochemical reactions, such as microbial fermentation, specific substrate utilization, reduction and degradation, to identify microbial species, or to use large-scale machines such as polymerase chain reaction (PCR) or mass spectrometry (MS) for interpretation. These methods are highly accurate, but required at least one day for proliferative reactions. It is more time consuming to encounter strains that are difficult to grow, slow to grow, or have no suitable medium.
To overcome aforementioned problems, this study proposed two different microfluidic dielectrophoresis devices to separate and concentrate Escherichia coli (E. coli), and utilize SERS fingerprint identification to detect bacteria in microfluidic chips. In terms of separation, E. coli are separated by DEP force generated from the gold interdigitated electrodes. And self-doped sulfonated polyaniline (SPANI) is used as a coating material on the Au electrodes to improve the fouling of electrodes caused by non-specific adsorption; In terms of concentration, E. coli are collected by DEP and electroosmotic forces generated from the top and bottom indium tin oxide (ITO)-coated glasses. To identify the bacteria, the AgINMs which can increase the Raman signal are mixed with the solution to be detected and injected into concentration chips, and the SERS signal of E. coli could be recognized after concentration.

摘要 i
Abstract ii
致謝 iv
目錄 v
圖目錄 viii
第一章 緒論 1
1.1 研究背景 1
1.2 研究動機 2
1.3 研究目標 4
第二章 文獻回顧 5
2.1 毛細管電泳(Capillary Electrophoresis, CE) 5
2.1.1 電泳發展歷史 5
2.1.2 電泳原理(帶電微粒的電泳遷移運動) 7
2.1.3 電滲流(Electro-osmosis flow, EOF) 10
2.2 介電泳(Dielectrophoresis, DEP) 14
2.2.1 微粒與介質之極化效應 14
2.2.2 介電泳原理 16
2.2.3 介電泳種類與應用 17
2.3 介電濕潤(Electrowetting-on-dielectric, EWOD) 19
2.3.1 介電濕潤原理 19
2.3.2 電濕潤、介電濕潤應用 21
2.4 表面修飾 23
第三章 實驗設計與方法 24
3.1 分離晶片設計與實驗原理 24
3.1.1 實驗方法與步驟 25
3.2 聚集晶片設計與實驗原理 33
3.2.1 實驗方法與步驟 34
3.3 實驗設備介紹 38
3.4 實驗藥品/化學品 40
3.5 藥品製備處理 43
3.5.1 配置血液之緩衝溶液 43
3.5.2 檢體分離及染色流程 44
第四章 實驗結果與討論 46
4.1 表面改質測試 46
4.2 介電泳力理論計算 50
4.3 分離晶片測試結果 52
4.3.1 大腸桿菌與PS球之分離(去離子水) 53
4.3.2 大腸桿菌、PS球、白血球與血小板之分離 55
4.4 聚集晶片測試結果 58
4.5 E. coli Raman spectrum 63
第五章 結論與未來工作 65
5.1 未來工作 65
參考文獻 67

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