本文提出利用離心式濾膜結構以離心過濾的方式來分離微小粒子及偵測稀少循環腫瘤細胞(Circulating Tumor Cells, CTCs)。近年，國內外許多研究團隊將研究著重於利用微流道實現微小粒子的分離，而目前微流體分離技術中，最簡易及常見的方法即為過濾法，但由於過濾分離技術最大的缺點為容易因欲分離樣本濃度過高或數量太多而導致流道發生阻塞的現象，降低其分離效率，故本文提出利用離心式濾膜設計，並結合劇烈改變轉速而產生切向流的方式來改善此阻塞現象，並以PS beads (聚苯乙烯微球)分離實驗來加以佐證。
本研究以離心式濾膜結構應用於不同直徑大小的PS beads分離，再將此分離概念應用於稀少CTCs分離。利用抗體對於在細胞表面上的抗原具專一性結合的特性，使接有特定抗體的PS beads能和癌細胞結合，使癌細胞體積變大以體積差異和其他血球細胞區分。
本研究證實可以濾膜式結構搭配切向流的分離方式減少阻塞現象。在檢測稀少腫瘤細胞方面，實驗證實本濾膜可在CTCs 與血球細胞濃度比為1:1,000,000的情況下成功將CTCs檢測分離。離心濾膜晶片是由微機電製程和軟微影技術製作，並以聚二甲基矽氧烷作為基材，因具生物相容、低成本、可拋棄、材質透明易於觀察及可大量製造等特性，非常符合微流體生醫晶片的需求；而離心設備及晶片體積小巧，易於攜帶。

關鍵字 : 循環腫瘤細胞(Circulating Tumor Cells, CTCs)、切向流、離心式濾膜結構。

In this study, a centrifugal multilayer filter device is applied for particles and Circulating Tumor Cells (CTCs) separation. In recent years, many researches emphasize separating microparticles by using microfluid chips. Size-based separation is the most straightforward and common approach for microfluid based separation. However, the main disadvantage of size-based filtration encountered is clogging. The reason is that the concentration of particle sample is too high and the clogging will lead to reducing separation efficiency. In this study, a multilayer concentric filter device with different pore sizes combined with changing Revolution(s) Per Minute(RPM) instantly to generate cross-flow and reduce the phenomenon of particles clogging.

We use the centrifugal filter device to separate microparticles and further apply this concept to CTCs detection. Tumor cells can be bounded by specific antibody coated with PS beads. The antibody has the ability to bind specific antigen on tumor cells .The conjugated size of CTCs and PS bead will become larger depending on how many tumor cells and PS beads bounded. The results show a significant clogging reducing with particle separation and the rare targeted tumor cells mixed with blood cells in the ratio of 1:1,000,000 are successful demonstrated.

The centrifugal filter device was fabricated using photolithography and soft-lithography techniques. It is fabricated by regular PDMS modeling process. The advantages of biocompatible, low cost, disposable, and transparent of PDMS make , it fulfill with the requirements of useful microfluidic biochips. The centrifugal filter device and centrifuge used in this research are portable.

目錄
第一章 緒論…………………………….. 1
1.1 前言與研究背景 1
1.2 研究動機與目的 1
第二章 文獻回顧 5
2.1 微流體晶片應用 5
2.1.1 流體力學特性分離(Hydrodynamic Based Separation) 5
2.1.2 免疫磁球分離(Immune-magnetic Enrichment) 11
2.1.3 流式細胞儀分離(Flow Cytometry Sorting) 16
2.1.4 密度梯度分離(Density radient ?Centrifugation) 18
2.1.5 過濾方式分離(Size-based Separation) 20
第三章 實驗架構與方法 29
3.1 離心式濾膜結構 29
3.1.1 切向流(Cross-flow)降低流道阻塞之設計概念 31
3.1.2 晶片分離PS beads之設計概念 32
3.1.3 晶片分離CTCs之設計概念 33
3.1.3.1. 以結合PS beads的Jurkat cell為目標細胞 33
3.1.3.2. 以結合PS beads的MCF-7 為目標細胞 34
3.2 晶片製程 36
3.2.1 SU8-3025微流道結構製作 36
3.2.2 PDMS翻模 39
3.2.3 氧電漿晶片接合 40
3.3 影像軟體與實驗儀器 42
3.3.1 影像量測軟體 42
3.3.2 儀器設備 43
3.4 實驗材料與方法 49
3.4.1 PS beads (聚苯乙烯微球) 49
3.4.2 細胞培養 49
3.4.2.1 細胞解凍 50
3.4.2.2 細胞繼代 51
3.4.3 細胞和抗體結合 52
3.4.3.1 PS beads和抗體結合步驟 52
3.4.3.2 細胞和有接枝抗體的PS beads 結合 53
3.5 實驗步驟 55
3.5.1 切向流對阻塞影響之步驟 55
3.5.2 PS beads分離之實驗步驟 56
3.5.2.1 有切向流之步驟 56
3.5.2.2 無切向流之步驟 57
3.5.3 CTCs檢測及分離之實驗步驟 57
3.5.3.1. 以結合PS beads的Jurkat cell 為目標細胞 57
3.5.3.2. 以結合PS beads的MCF-7為目標細胞 58
3.5.3.3. 非特異性結合之實驗步驟 61
第四章 實驗結果與討論 62
4.1 切向流對阻塞降低之影響 62
4.2 PS beads分離效率 64
4.3 CTCs 檢測及分離結果 68
4.3.1. 以結合PS beads的Jurkat cell 為目標細胞 68
4.3.2. 以結合PS beads的MCF-7為目標細胞 72
4.3.3. 分離篩選後於晶片培養 80
4.3.4 非特異性結合(Non-specific binding) 84
第五章 未來工作 86
5.1 CTCs檢測及分離 86
5.1.1 改善晶片內可容樣本體積 86
5.1.2 人類血液中分離循環腫瘤細胞 86
5.1.3 MCF-7 螢光標記 87
5.1.4 未來展望 87
第六章 參考文獻 88

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