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研究生:徐耀德
研究生(外文):Hsu, Yao-Te
論文名稱:多通道同步量測斑馬魚胚胎粒線體代謝之微流體感測裝置開發與應用
論文名稱(外文):Development of a multi-channel microfluidic sensing device for simultaneous monitoring of mitochondrial metabolism of a single developing zebrafish embryo
指導教授:黃士豪黃士豪引用關係
指導教授(外文):Huang, Shih-Hao
口試委員:吳志偉莊昀儒
口試委員(外文):Wu, Chih-WeiChuang, Yun-Ju
口試日期:2017-12-15
學位類別:碩士
校院名稱:國立臺灣海洋大學
系所名稱:機械與機電工程學系
學門:工程學門
學類:機械工程學類
論文種類:學術論文
論文出版年:2018
畢業學年度:106
語文別:中文
論文頁數:80
中文關鍵詞:光纖感測濃度梯度微流體裝置磷光相位斑馬魚胚胎耗氧率三氯沙粒線體
外文關鍵詞:Fiber optic sensingConcentration gradient generatorMicrofluidic devicePhosphorescence phaseZebrafish embryosOxygen consumption rateTriclosanMitochondrial
相關次數:
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  • 收藏至我的研究室書目清單書目收藏:0
本研究利用光纖系統結合磷光相位量測技術及濃度梯度微流體晶片,開發出可同時針對多顆斑馬魚胚胎之生物能量分析裝置,並透過氧氣消耗率(OCR, Oxygen Concentration Rate)的量測了解生物粒線體代謝之狀況。裝置包含了斑馬魚胚胎培養晶片、氣動封閉裝置、濃度梯度產生晶片及光纖磷光相位感測裝置四個部分。斑馬魚培養晶片亦為壓克力微孔晶片,藉由包含PtOEP氧氣感應層的微孔晶片及氣動封閉裝置可形成暫時封閉之量測區域,配合濃度梯度產生晶片可在量測時提供胚胎不同的培養環境。光纖磷光相位量測裝置由固定機構、光纖及光感測器構成,可對氧氣感應層發射激發光並利用光纖傳遞受激發後感應層發射之磷光至光感測器,最後由LabVIEW程式處理並分析光感測器之磷光訊號以計算斑馬魚胚胎之OCR。藉由同步量測12 hpf (hours post-fertilization)及36 hpf斑馬魚胚胎粒線體代謝曲線的實驗初步驗證了量測系統的可用性。
為證實裝置可確實應用於生物代謝量測中,本研究以不同缺氧及藥物濃度兩面向探討裝置的應用。三氯沙(Triclosan, TCS)為一種抗菌化合物,現今已廣泛應用於民生用品中,然而長年來其安全性卻飽受爭議,目前已有文獻針對斑馬魚胚胎進行表面形貌與代謝分析。本研究以濃度梯度產生晶片產生不同的TCS濃度之培養環境影響斑馬魚胚胎,並以光纖磷光相位感測系統量測分析呼吸量及代謝狀況,藉此探討設計之裝置於藥物試驗之應用。TCS已知是一種粒線體解偶聯劑,會直接造成胚胎的基礎呼吸量增加,但因質子洩漏率的增加降低了ATP生產效率及儲備能。經過粒線體功能指標的分析,發現隨TCS濃度增加會導致粒線體健康指標(Bioenergetic Health Index, BHI)會有明顯之下降,於30 M的TCS中24 hpf斑馬魚胚胎的BHI僅有正常培養環境的5.3%,反映了TCS會損害斑馬魚的粒線體功能。
此外,本研究亦有以不同氧氣濃度作用於斑馬魚胚胎並量測缺氧後之基礎呼吸量,以進一步瞭解不同缺氧程度對斑馬魚胚胎之影響,並探討裝置於缺氧相關研究之應用。48 hpf斑馬魚胚胎於氧濃度低於5%的低氧環境中,由於胚胎適應於環境氧濃度的改變,使得胚胎基礎呼吸量減少約47%。本研究提供了一套可針對多顆單一斑馬魚胚胎提供不同環境同步進行粒線體代謝分析的平台,且具有應用於其他模式生物及後續擴充之潛力。
In this study, we developed a bioenergetic sensing device that can simultaneous assess the biological metabolic status via oxygen consumption rate (OCR) of multiple zebrafish embryos by using fiber optic system based on phosphorescence phase measurement technique and concentration gradient generator. The microfluidic device combines four components: an array of PMMA microwells containing PtOEP as oxygen-sensitive layer, a microfluidic module with pneumatically actuated glass lids above the microwells to controllably seal the microwells of interest, a microfluidic concentration gradient generator, and a phase-based phosphorescence lifetime detection system which consists of optical fiber, and optical sensors. In addition, we calculated the OCR of zebrafish embryos by analyzing the phosphorescence signal of the photodetector using the LabVIEW program.
Triclosan (TCS) is an antibacterial agent used in many consumer products at millimolar concentrations, However, The safety of TCS has been the subject of controversy over the years. In order to investigate feasibility of our purposed device in the application of drug screening, we assess the TCS concentration effects on mitochondrial metabolism of zebrafish embryos using ours multi-channel microfluidic sensing device with concentration gradient generator. The result confirmed that TCS not only deceased ATP-Linked respiration and spare respiratory, but also increased proton leak, due to the mitochondrial uncoupler of TCS in a living organism. Calculated the bioenergetic health index (BHI) of 24 hpf (hours post-fertilization) zebrafish embryo in a solution of 30 M TCS, the BHI decreased to 5.3% of normal culturing environment of 24 hpf zebrafish embryo, suggesting that TCS can impair mitochondrial function.
Besides, we also assess the hypoxia effects on the basal respiration of zebrafish embryos at under a different oxygen (O2) concentrations to investigate the application of the device in hypoxia research. The results confirmed that the OCR decreased to 47% for 48 hpf embryos at below 5% pO2, due to the fact that embryos adapt the changes of ambient oxygen concentration. Our proposed platform provides the function of simultaneous analysis of mitochondrial metabolites in different environments for multiple single zebrafish embryos and the potential to be applied to other model organisms and expansion of measuring device.
摘要 I
Abstract II
目錄 III
圖目錄 V
表目錄 VII
第一章 緒論 1
1.1 前言 1
1.2研究背景 2
1.2-1 粒線體胞器 2
1.2-2 細胞代謝作用 3
1.2-3 三氯沙藥物特性介紹 5
1.2-4 生物微晶片 6
第二章 文獻回顧 8
2.1 濃度梯度晶片相關應用 8
2.2 光電感測裝置於生醫工程量測之應用 11
2.3 細胞與模式生物代謝量測相關研究 17
2.4 斑馬魚缺氧與藥物相關研究 19
2.5 研究動機與目的 21
第三章 實驗原理 24
3.1 磷光相位檢測原理 24
3.2 生物缺氧及代謝概論 25
3.2-1 粒線體電子傳遞概論 25
3.2-2 缺氧對粒線體造成之影響 29
3.2-3 生物能量健康指數(Bioenergetics Health Index, BHI) 30
3.3 濃度梯度晶片介紹與流路分析 31
第四章 實驗設計與架設 37
4.1 系統概述 37
4.2 光纖磷光相位系統設計與驗證 40
4.3 晶片及裝置製作 41
4.3-1 聚甲基丙烯酸甲酯(PMMA,壓克力) 44
4.3-2 磷光材料-PtOEP 44
4.4 氧氣感測系統程式計算 45
4.5 斑馬魚胚胎飼養裝置 48
4.6 斑馬魚與胚胎 48
4.7 藥物試驗評估用藥-三氯沙 50
4.8 生物粒線體抑制劑 50
第五章 結果與討論 52
5.1 氧氣及藥物濃度梯度產生與量測 52
5.2 同步量測系統之驗證 54
5.3 斑馬魚胚胎於環境氧氣濃度改變下之基礎呼吸量量測 57
5.4 斑馬魚胚胎於環境中添加藥物下之粒線體代謝狀況量測 59
第六章 結論與未來展望 65
6.1 結論 65
6.2 未來展望 65
參考文獻 67
附件 73
致謝 79
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[71] Thorlabs. Multimode Fiber Tutorial. Available: https://www.thorlabs.com/newgrouppage9.cfm?objectgroup_id=10417
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