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研究生:曾新堯
研究生(外文):Tseng, Hsin-Yao
論文名稱:利用控制微流體系統以提升精蟲篩選之效能
論文名稱(外文):Using Controlled Microfluidic System to Enhance the Sperm Motility Sorting Efficiency
指導教授:饒達仁
指導教授(外文):Yao, Da-Jeng
學位類別:碩士
校院名稱:國立清華大學
系所名稱:奈米工程與微系統研究所
學門:工程學門
學類:材料工程學類
論文種類:學術論文
論文出版年:2013
畢業學年度:101
語文別:中文
論文頁數:100
中文關鍵詞:注射式幫浦層流精蟲篩選生醫晶片體外授精
外文關鍵詞:Syringe pumpBio-chipSorting spermLaminar flowIn vitro fertilization
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本論文提出一注射式幫浦(Syringe pump)控制系統佐以微流體晶片,利用流體在微尺度下呈現層流之特性,針對原始濃度及活動力不同的精蟲樣本進行一次性的篩選。運用此技術直接取代傳統篩選方式與人工挑選之過程,同時符合不需離心即可篩選原則以避免可能對精蟲造成的傷害。為證實該系統的精蟲篩選能力,透過流式細胞儀進行實驗分析,結果顯示本研究成功發展出具有篩選精蟲的生醫檢體處理系統。
本研究晶片是經由微機電製程與軟微影技術加工,並選用具備生物相容、低成本、可拋棄式、高精密度及製程簡易等特性之聚二甲基矽氧烷 (Polydimethylsiloxanes, PDMS) 作為實驗基材,相當符合微流體生醫晶片的需求。
人工協助生殖技術(Assisted Reproductive Technology, ART)在醫學領域扮演相當重要的角色,其中,體外受精技術(In Vitro Fertilization, IVF)是將樣本進行前處理,於體外讓一群精蟲與卵細胞自然受精,故精蟲的活動力、總數量等因素將直接影響受精結果。因此可透過生醫晶片於樣本前處理階段篩選出最適合進行IVF的精蟲。
本研究共分為二階段,第一階段完成一微尺度層流篩選精蟲之控制微流體系統,主要針對幫浦控制系統與晶片間的連結匹配,其中包含晶片設計、流場模擬、幫浦參數設定以及流式細胞檢測計數的分析結果等。第二階段則延續第一階段的研究,加強篩選效率性與流道設計。從篩選結果可知,活精蟲比例皆可提升至90%以上,濃度與數量上亦有明顯的提升。因此,利用控制微流體系統提供長時間穩定推進之能力與多次設計高效率高穩定度之微流道晶片,成功提升精蟲篩選之效能。最後本論文於第六章提出部分未來可望加以思考的研究方向。

The thesis presents a controlled microfluidic system which consists of laminar stream-based microchannels. The control microfluidic system is consists of syringe pump and connective tube module. The working concept lies in the microfluidic systems that successfully replace traditional methods, which could select motile sperms without centrifugation steps. Simultaneously, the damage to the sperms could be avoided. The flow cytometric analysis is utilized to confirm the sorting efficiency of this system. The result showed that our study is successfully for sorting sperm.
The fabrication process of our bio-chip include SU8 thick-film photolithography and soft-lithography, which has the advantages of biocompatibility, low-cost, high-precision, disposable, and easy to produce etc., so that it is suitable for biomedical chip.
Reproductive medicine has become an important role in the field of medical science. The main reasons of infertility from male are the abnormality of sperms and the lack of sperms. IVF (in vitro fertilization) technology is a natural in vitro fertilization process that comes after a series of sample pre-treatment. The semen property, including motility, total number, and the concentration of sperms will directly affect the outcome. Therefore, the optimized microfluidic system provides a good opportunity to use and an inexpensive requirement to select the most appropriate sperms before IVF process.
The study is divided into two parts. In the first part, the goal is to construct a microfluidic based controlled microfluidic system, which focus on the sorting and the classification based on sperm motility and viability. The details including chip design, flow field simulation, pump parameter setup, flow cytometric analysis, and the experiments of semen samples. In the second part, the sorting efficiency and chip redesign were focused to enhance the sorting efficiency in the quality and quantity of sperm. The result show that counted ratio of motile sperm can increase to 90%. Therefore, using controlled microfluidic system is successful to enhance the sperm motility sorting efficiency. At the end, some ideas of future works were proposed.

致謝 i
中文摘要 ii
Abstract iv
目錄 v
圖目錄 x
表目錄 xvi
第一章 緒論 1
1.1 前言 1
1.2 研究背景 1
1.3 研究動機 2
第二章 文獻回顧 6
2.1 微流體呈層流之特性 6
2.2 以緩慢的流速篩選並排列精蟲 10
2.3 微技術應用於人工輔助生殖 12
2.4 精液狀況評估研究 17
2.5 研究目的 22
第三章 實驗架構與方法 23
3.1 微流道之篩選晶片 23
3.1.1 設計概念 23
3.1.2 微流體之特性分析 24
3.2 數值模擬 25
3.3 晶片製程 28
3.3.1 SU8-3050微流道結構製作 28
3.3.2 PDMS翻模 30
3.3.3 氧電漿晶片接合 31
3.4 實驗材料 33
3.4.1 儀器與設備 33
3.4.2 檢體來源及前處理 34
3.5 Syringe pump的應用 35
3.5.1 流速計算 36
3.5.2 幫浦設定 37
3.5.2.1針筒的架設 37
3.5.2.2管徑、體積以及注射流率的設定 38
3.5.2.3開始、停止以及體積流率的變換 39
3.6 Flow cytometer分析 40
3.7 實驗流程 43
3.8 實驗結果 44
3.8.1 精蟲螢光染色 44
3.8.2 精蟲層流篩選過程 46
3.8.2.1呈現穩定的層流 46
3.8.2.2具活動力的精蟲穿越層流 47
3.8.2.3無活動力的精蟲停留在原始層流 48
3.8.3 Flow cytometric分析結果 49
3.9 問題與討論 53
3.9.1 微流道尺寸結構 53
3.9.1.1流量問題 53
3.9.1.2雜質堆積問題 55
3.9.2 Syringe pump改變體積流率過程 56
第四章 多出口式微流道精蟲篩選研究 58
4.1 設計概念 58
4.2 數值模擬 59
4.3 實驗樣本 61
4.4 實驗流程 63
4.5 實驗結果 65
4.5.1 豬精篩選結果 65
4.5.2 人精篩選結果 67
4.5.3 以濃度來討論篩選效率 70
4.6 問題與討論 71
4.6.1 二入四出晶片內流場變化 71
4.6.2 樣本篩選後濃度過低問題 72
第五章 多進多出式微流道精蟲篩選研究 73
5.1 設計概念 73
5.2 數值模擬 74
5.3 實驗流程 77
5.4 實驗結果 80
5.4.1 豬精篩選結果 80
5.4.2 人精篩選結果 82
5.4.2.1品質較好人精 82
5.4.2.2品質較差人精 85
5.4.2.3特殊狀況人精 88
5.4.3 以濃度來討論篩選效率 89
5.5 問題與討論 90
5.5.1 豬精團聚雜質的影響 90
5.5.2 實驗樣本不稀釋問題 91
第六章 結論與未來展望 93
6.1 結論 93
6.2 未來展望 94
6.2.1 精蟲篩選效率與濃度再提升 94
6.2.2 整合微流道篩選機制以實現IVF 95
第七章 參考文獻 97

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