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研究生: 黃博威
Huang,Po Wei
論文名稱: 改良微流道之設計與參數來加以提升精子篩選效率之成效
TO ENHANCE SPERM MOTILITY SORTING EFFICIENCY BY UTILIZING MICROFLUIDIC CHIP
指導教授: 饒達仁
Yao,Da Jeng
口試委員: 劉承賢
Liu,Cheng Hsien
范士岡
Fan,Shih Kang
學位類別: 碩士
Master
系所名稱: 工學院 - 動力機械工程學系
Department of Power Mechanical Engineering
論文出版年: 2015
畢業學年度: 104
語文別: 中文
論文頁數: 104
中文關鍵詞: 微流體晶片精子篩選層流特性螢光分析螢光分析
外文關鍵詞: Micro-fluidic chip, Sorting sperm, Laminar flow, Fluorescent analysis, In vitro fertilization
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  • 本論文利用流體在微尺度下呈現層流之特性,藉由此特性去篩選活動力精子,注射式幫浦控制系統有效地提供多個穩定流速使微流體晶片內的不同流體可以呈現出多個不同的層流現象,再利用精子本身的活動力條件,有效突破層流進入儲存槽內收集,以利日後的生醫的醫學應用。
    現今醫學上所使用的離心方式需要用快速旋轉的方式使精子分離出來,極大的重力離心條件可能對精蟲造成傷害。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。
    由於人工協助生殖技術(Assisted Reproductive Technology, ART)的出現,有助於解決許多不孕的問題存在,在醫學領域扮演相當重要的角色,不管是針對精子注射受精(Intracytoplasmic sperm injection, ICSI)還是體外受精技術(In Vitro Fertilization, IVF),精子的活動力、總數量、活率等因素將直接影響受精結果,本實驗透過生醫晶片在樣本前處理階段即可篩選出最適合進行人工協助生殖技術的精蟲。
    實驗晶片是由微機電製程與軟微影技術加工,並選用聚二甲基矽氧烷 (Polydimethylsiloxanes, PDMS) 作為實驗晶片,他具備幾項優點,如:有生物相容性、低成本、可拋棄式、製程簡易等特性質。
    本研究使用微尺度層流現象進而篩選精蟲的控制微流體系統,利用精子的活動力突破層流達到收集的成效。此實驗設計包含晶片設計、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。以緩慢流速提供精子在晶片裡游動的最佳環境,盡可能減少流體對精子游動方向的干擾,最終透過流式細胞儀的幫助,得知收集的精子隻數與比例都有明顯的提升,證實微流道晶片篩選之優勢。


    The thesis presents acontrolled microfluidic system which consists of laminar stream-based microchannels. This characteristic is useful for selecting motile sperm.There is some laminar flow phenomenon in chip because flow rate of sperm and buffer are different. Motile sperm has ability to pass through the laminar flow boundary, and reach the reservoir we want to collect.
    The bio-chip method is different from traditional methods and centrifugation. Centrifugation need to select sperm by rotating at fast speed, the great gravity maybe will cause the damage to sperm. Finally, the flow cytometric analysis is utilizedto confirm the sorting efficiency of this system. The result showed that our study is successfully for sorting sperm.
    Assisted Reproductive Technology (ART) has become an important role in the field of medical science. It provides an important solution to some couple which is suffer from infertility.No matter ICSI (Intracytoplasmic sperm injection) or IVF (in vitro fertilization), sperm motility, quantity, concentration are the main reason for fertilization. Therefore, the optimized microfluidic system provides a perfect opportunity to use an inexpensive requirement to select the most appropriate sperms before IVF process.
    The fabrication process of bio-chip include SU8 thick-film photolithography and soft-lithography which is used by PDMS (Polydimethylsiloxanes), which has the advantages of biocompatibility, low-cost, high-precision, disposable, and easy to produce etc., so that it is suitable and common used for biomedical chip.
    The study is to construct a microfluidic based controlled microfluidic system, which focus on the sorting and the classification based on sperm motility and viability. The experimental details including chip design, flow field simulation, pump parameter setup, flow cytometric analysis. All parts are a key point associated with the final result.Slow flow rate has favorable environment to increase the opportunity of motile sperm to pass through the laminar flow boundary. Moreover, this environment can reduce interference with direction of sperm swim forward. At the last, more progressive quantity and quality can be observed by the flow cytometric analysis. The result show that counted ratio of motile sperm can increase a lot. Therefore, using controlled microfluidic system is successful to enhance the sperm motility sorting efficiency. It confirms that the microchannel really has an advantage in Assisted Reproductive Technology.

    目錄 圖目錄 v 表目錄 xi 第一章 緒論 1 1.1 前言 1 1.2 研究背景 2 1.3 研究動機 2 第二章 文獻回顧 7 2.1. 生醫晶片之精子篩選種類 7 2.2. 微流體呈層流之特性 8 2.2.1. 分離具運動性的細胞 9 2.2.2. 以濃度梯度研究細胞趨性 10 2.3. 緩慢流速篩選精蟲 11 2.4. 應用在人工輔助生殖 15 2.5. 研究目的 18 第三章 晶片設計 20 3.1 微流道之篩選晶片 20 3.1.1 三入三出流道晶片設計 20 3.1.2 緩降晶片晶片設計 21 3.1.3 多入口流道設計 23 3.2 數值模擬 24 3.2.1 三入三出流道濃度分布 26 3.2.2 多入口流道濃度分布 28 3.3 微流體之主導參數 31 第四章 實驗製程與步驟 33 4.1 晶片製程 33 4.1.1. SU8-3050微流道結構製作 33 4.1.2. PDMS翻模 37 4.1.3. 氧電漿晶片接合 38 4.2 儀器與設備 39 4.3 檢體來源及前處理 40 4.3.1. 豬精 40 4.3.2. 人精 41 4.4 Syringe pump操作及應用 43 4.4.1. 流速計算 45 4.4.2. 幫浦設定 46 4.5 Flow cytometer分析 47 4.6 實驗操作流程 51 第五章 實驗結果 55 5.1 精蟲螢光染色 55 5.1.1 螢光染劑對精子的影響 56 5.1.2 螢光染劑的猝滅 58 5.2 精蟲層流篩選過程 59 5.2.1 呈現穩定的層流 59 5.2.2 活動力的精蟲穿越層流現象 60 5.2.3 無活動力的精蟲停留在原始層流 61 5.3 Flow cytometric分析結果 63 5.3.1 以活率和分離率來討論篩選效率 65 5.3.2 模擬入口濃度並比較出口濃度 66 5.3.3 三入三出流道 69 5.3.4 緩降流道 71 5.3.5 多入口流道 72 5.3.6 多入口流道進行緩慢流速篩選 74 第六章 問題與討論 79 6.1 表面改質與雜質堆積問題 79 6.1.1. BSA親水性改質 79 6.1.2. 出口端圓角設計 83 6.2 豬精之品質 85 6.3 人精之品質 86 6.3.1 品質較好人精 86 6.3.2 品質較差人精 88 6.4 插管方式比較 91 第七章 結論與未來展望 94 7.1 結論 94 7.2 未來展望 99 第八章 參考文獻 101

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