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作者(中文):林育男
論文名稱(中文):修飾負電微米粒子之紙晶片應用於豬X-Y精子分選
論文名稱(外文):Isolation of Farrow X-Y Spermatozoa on A Paper Chip Modified with Negative Micro Particles
指導教授(中文):曾繁根
口試委員(中文):潘力誠
王本誠
學位類別:碩士
校院名稱:國立清華大學
系所名稱:工程與系統科學系
學號:101011525
出版年(民國):103
畢業學年度:102
語文別:中文
論文頁數:64
中文關鍵詞:紙晶片精子XY分選
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隨著畜牧業的高度發展和乳製品的高度需求,牲畜經濟儼然變成一個炙手可熱的方向,但是現今的研究方向和已存在的方式卻不十分著墨,因此本篇論文想要發展一個方式用來分選豬的XY精子,讓牲畜的下一代可以決定是生公的或母的,如此一來便有非常龐大的經濟價值。
生醫晶片已經是非常成熟的技術,舉凡蛋白質、細菌和病毒,有無數的文獻利用晶片來達成不管是篩選或是檢測的目的,因此本篇研究想要利用晶片來達成分選精子的目標,但同時又要開創一個利用新的基材-紙來製作。傳統用來分選的方法如pure sperm和白蛋白液注所得出的分選效率都不十分顯著,但像是效果很好的電泳分離法和流式細胞儀,儘管效果明顯,由於實驗過程中會傷害精子,導致回收出來的精子存活率低,因此要如何在提高分選效率的同時又能維持精子的存活率也是本篇研究的重點。
利用製成簡易的優勢來製作紙晶片,過程中只需使用印表機和烘箱在紙上製造出流道,再利用表面修飾帶負電的微米例子,最後再使用靜電產生器對表面做改質,就能完成整個晶片。檢測時只需把原液直接滴入晶片,靜待一分鐘就能完成實驗,最後再把跑完晶片的液體拿去流式細胞儀做分析。
本研究能夠達到將死活精子分離開來,而且能夠確保活精子裡面都是品質比較好的,最後X精子和Y精子的分選比例結果可以達到61:39。
As long as the blooming of livestock and high demand for dairy, livestock economy seems to turn into a hot direction. But the research and methods nowadays are not very value, so this paper wants to develop a method to differentiate the X and Y sperm of farrow. In that we can choose the offspring of livestock, which will become a huge property.
Biochip is already become a mature way to select or detect not only protein but also germ and virus. So this paper wants to use the theory of biochip to differentiate the sperm in X and Y, but also wants to open up a new base-paper to make the chip. Traditional methods for differentiation efficiency like puresperm and albumin are not very powerful. Although the high efficiency ways like electrophoresis and flow cytometric will harm to the sperm. Because electrophoresis way will use electricity direct among the sperm so it will harm to them and flow cytometerichave to use the fluorescent dye. So these ways will make the survival rate of sperm be very low. In conclusion, this paper will focus on how to raise not only the differentiation efficiency but also the survival rate.
This paper has the advantage of easy fabrication, just need printer and oven to make the chip on the filter paper. After the fabrication, spin the polystyrenebeads that modified with negative functional groups on the surface of the chip. At last, use plasma treatment on the chip. When run the experiment, just drop the sperm of farrow on the chip, and then wait for one minute to collect the liquid in the specific position. At last, use the flow cytometric to analysis.
This research can achieve to separate the live sperm from dead and live sperm is in good quality. The differentiation efficiency can reach to 61:39 between X and Y sperm.
總目錄
摘要 i
Abstract ii
總目錄 iii
圖目錄 v
表目錄 vii
第一章 緒論 1
1.1 研究背景 1
1.1.1 機電系統 1
1.1.2 微流體生醫晶片 2
1.2 研究動機 3
1.2.1 牲畜經濟 3
1.2.2 性連遺傳疾病 4
1.2.3 現今使用的X,Y精子分離法 6
第二章 文獻回顧 10
2.1 各種方法用來分離X,Y精子 10
2.1.1 電泳分離法 10
2.1.2 PureSperm / 牛血清蛋白離心分離法 12
2.1.3 流式細胞分選方法 17
2.2 判別XY精子的辦法 21
2.2.1 螢光染色 21
2.2.2 流式細胞儀 23
2.3 精子的表面蛋白 24
2.3.1 唾液酸 24
2.3.2 唾液酸 26
2.4 研究目的 29
第三章 紙晶片分析 31
3.1 流道設計 31
3.2 靜電排斥力計算 34
3.3 精子掉落距離計算 36
3.4 晶片製程 37
3.5 實驗方法 40
3.4.1 檢體來源其前處理 40
3.4.2 定量 42
3.4.3 晶片分析 44
3.4.4 精子取樣 46
3.4.5 精子染色 47
第四章 實驗結果 49
4.1 聚苯乙烯球排列之情形 49
4.2 晶片分選功效 50
4.2.1 分選精子之死活 50
4.2.2 精子死活螢光圖 52
4.2.3 速度分析 54
4.2.4 X-Y精子比例 56
第五章 結論與展望 60
5.1 結論 60
5.2 未來工作 61
5.2.1 提高辨別X和Y精子的準確性 61
5.2.2 解決檢體濃度因為染色而降低 61
5.3 展望 62
參考文獻 63
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