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研究生： 盧鈺心
Lu, Yu-Hsin
論文名稱： 應用於寡精症之活動力精蟲篩選微流道
Utilization of Motility-Driven Sperm Sorting Microfluidic Chip for Oligozoospermia patients
指導教授： 饒達仁
Yao, Da-Jeng
口試委員: 劉承賢
Liu, Cheng-Hsien
鍾添淦
Chung, Tien-Kan
學位類別： 碩士
Master
系所名稱： 工學院 - 奈米工程與微系統研究所
Institute of NanoEngineering and MicroSystems
論文出版年： 2019
畢業學年度： 108
語文別： 中文
論文頁數： 110
中文關鍵詞： 微流體晶片 、精子篩選 、層流特性 、人工生殖 、寡精症
外文關鍵詞： microfluidic chip, sperm sorting, laminar flow, artificial reproduction, oligozoospermia
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    • 　　隨著醫學與科技進展，不少輔助生殖技術，如：體外受精（in vitro fertilization, IVF）、卵胞漿內單精子顯微注射（Intracytoplasmic Sperm Injection, ICSI），為不孕症夫婦帶來一大福音，然而上述方法往往需要採集高質量、活動力佳的精子進行妊娠。本研究以機械式幫浦控制系統穩定緩慢的流場提供精蟲合適的游動環境，利用微流道晶片內層流特性區隔活動力較差的精蟲，並藉由精蟲本身之泳動性篩選出高品質的精蟲。
    　　本論文使用微機電製程與軟微影技術製成的聚二甲基矽氧烷 （Polydimethylsiloxanes, PDMS）晶片以及經由射出成型加工而成的聚碳酸酯（Polycarbonate, PC）晶片，此二種材料具有生物相容性、可拋棄式、低成本、製程簡易等優點。實驗包含晶片微流道設計、流場模擬、幫浦參數設定、流式細胞檢測計數以及各精子濃度、活動力篩選的分析結果。
    　　首先以PDMS晶片針對不同黏滯度的原液樣本採不同流速比進行分選，雖能提升活蟲率，然而當稀釋液流速提高時，篩選效率降低。故而後先將檢體固定稀釋成2mPa×s黏滯度後，使用相同8:1 （稀釋液:精液）之流速比進行篩選，可提高篩選效率且無論是正常、寡精或弱精檢體之活蟲率均有約15%左右的提升，證實本晶片確實兼具提升檢體的活蟲率和收集活精蟲之能力。
    　　接著使用PC晶片進行固定黏滯度搭配8:1流速比之篩選實驗，發現精液會匯集到儲存槽，需提高流速比做篩選，推論可能原因為在流道與熱熔線間縫隙的回流現象導致，未來會改善PC晶片的接合方式，量產晶片以提供予人工生殖技術使用。

    　　With advances in medicine, many assisted reproductive technologies, such as in vitro fertilization (IVF) and intracytoplasmic Sperm Injection (ICSI), bring benefits to a large number of infertility couples. However, the above methods often require the high-quality, highly active sperms for pregnancy. In this study, the stable flow field of the syringe pump provides a suitable swimming environment for sperms. We can separate poor sperms by laminar flow and collect high quality sperms by motility.
    　　In this paper, polydimethylsiloxanes (PDMS) chips fabricated by SU-8 thick film photolithography and soft lithography are used, as well as polycarbonate (Polycarbonate, PC) chips processed by injection molding. These two materials are biocompatible, disposable, low cost, and easy to manufacture. The experiment included microchannel design, flow field simulation, pump settings, sperm counting with flow cytometry, and also the sorting results analysis.
    　　From the results of stock sample sorting with PDMS chip, the viability was obviously improved by the modified flow rate, and the separation with respect to 8:1 has been greatly improved. However, sorting efficiency decreases as the flow rate ratio increases. Therefore, the semen was fixedly diluted to 2mPa×s viscosity, and sorted by the flow rate 8: 1 (buffer: semen). This method can enhance the viability for about 15% on whether normal, oligozoospermia, or asthenospermia samples. It was confirmed that the chip can indeed collect motile sperms and increase the viability.
    　　Then in PC chip sorting experiment with fixed viscosity and 8:1 flow rate ratio, we find that the semen will concentrate into the storage tank; hence the flow rate ratio needs to be increased. It may because of the reflux phenomenon in the gap between the channel and the bonding line. The bonding way of PC chip will be improved, and it is expected that the PC sperm sorting chips will be mass-produced in the future to provide for artificial reproduction technology.

    目錄 5 圖目錄 9 表目錄 15 第一章 緒論 16 1.1 前言 16 1.2 研究背景 17 1.3 研究動機 18 1.4 本文架構 21 第二章 文獻回顧 22 2.1 傳統精蟲收集法 22 2.1.1 離心洗精法 22 2.1.2 直接上泳法 23 2.1.2 非連續密度梯度離心法 24 2.2 現有之微流體精蟲篩選方式 24 2.2.2 螺旋微流道分選精蟲與白血球 25 2.2.3 平行微通道篩選高DNA完整性精子 26 2.2.4 結合磁珠篩選活精蟲 27 2.3 本研究之相關參考文獻 28 2.3.2 微流道晶片精子篩選與其游速的關係 28 2.3.3 緩慢流速篩選精蟲 29 2.4 文獻比較 31 2.5 前代實驗結果回顧與研究目標 31 第三章 晶片設計 33 3.1 精子篩選之微流道晶片 33 3.2 微流體之主導參數 34 3.3 數值模擬 35 3.3.2 流道精液濃度分布模擬 37 第四章 實驗製程與步驟 41 4.1 PDMS晶片製程 41 4.1.2 SU8-3050微流道結構製作 41 4.1.3 PDMS翻模 44 4.1.4 氧電漿晶片接合 45 4.2 PC晶片製程 46 4.2.2 PC材質 48 4.2.3 射出成型 49 4.2.4 超音波熔接 51 4.2.5 晶片改良 54 4.3 儀器與設備 57 4.4 Syringe pump操作及應用 58 4.4.2 流速計算 59 4.4.3 幫浦設定 60 4.5 檢體來源及前處理 61 4.5.2 豬精 61 4.5.3 人精 62 4.6 PDMS晶片實驗操作流程 64 4.6.2 原液檢體篩選 64 4.6.3 固定黏滯度之稀釋精液篩選 67 4.7 PC晶片實驗操作流程 70 4.7.2 晶片量測 70 4.7.3 固定黏滯度之稀釋精液篩選 71 4.8 精蟲螢光染色 72 4.9 Flow cytometer分析 73 4.10 流道組與離心組精蟲細胞膜完整性檢測 76 第五章 實驗結果 78 5.1 精蟲層流篩選過程 78 5.1.2 呈現穩定的層流 78 5.1.3 活動力的精蟲穿越層流現象 78 5.2 流式細胞儀分析結果 79 5.2.2 以活蟲率和篩選效率討論分離效果 81 5.2.3 模擬注入隻數並比較收集隻數 82 5.3 PDMS晶片 85 5.3.2 原液檢體篩選 85 5.3.3 固定黏滯度之稀釋精液篩選 87 5.4 PC晶片 91 5.4.2 親水性測試 91 5.4.3 層流現象測試 92 5.4.4 固定黏滯度之稀釋精液篩選 93 第六章 問題與討論 96 6.1 精蟲濃度與精液黏滯度之相關性 96 6.2 PC晶片滲漏問題 97 6.3 PC晶片流場探討 98 6.3.1 回流現象 98 6.3.2 流場模擬 99 第七章 結論與未來展望 101 7.3 結論 101 7.4 未來展望 102 7.4.1 PC晶片量產與臨床應用 102 7.4.2 溶劑型黏合法 103 7.4.3 廢液槽流道加寬 105 7.4.4 整合型IVF晶片 106 參考文獻 107

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