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研究生:劉彥珉
研究生(外文):Liou, Yan-Min
論文名稱:可同步量測酸化率及耗氧率微流體感測裝置應用於單一斑馬魚胚胎發育分析之研究
論文名稱(外文):Simultaneous monitoring of oxygen consumption and acidification rates of a single developing zebrafish embryo within a microfluidic device
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:莊昀儒吳志偉
口試委員(外文):Zhuang, Yun-RuWu, Chih-We
口試日期:2016-01-27
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:2016
畢業學年度:104
語文別:中文
論文頁數:56
中文關鍵詞:生物能量代謝微流體裝置斑馬魚胚胎磷光相位酸擠出率耗氧率
外文關鍵詞:Biological energy metabolismMicrofluidicLight intensityPhosphorescence phaseZebrafish embryosOxygen consumption rateAcid extrusion rate
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本研究利用微流體晶片裝置結合螢光強度及磷光相位系統,開發出檢測單一顆斑馬魚胚胎之發育分析機台。微流體晶片裝置配合Pt(Pt-porphyrin)的氧氣感應層,透過氧氣消耗率(OCR, oxygen consumption rate)的量測可以了解生物粒線體能量代謝之過程,以及配合5(6)-Carboxyfluorescein的酸鹼感應層,經由糖解、醱酵反應產生乳酸改變環境酸鹼值,我們稱為酸擠出率(AER, acid extrusion rate)可以了解生物非粒線體能量代謝之過程,透過兩者可以更深入的了解造成斑馬魚胚胎能量代謝之變化因數。其中生物的能量代謝分為粒線體產生的有氧呼吸和細胞基質中(不需粒線體)產生的無氧呼吸,藉由結合兩種感應層可以同時了解生物當下不同的呼吸作用,粒線體產生的能量大約佔生物體中能量來源的90%,如:ATP合成脢和質子滲漏,而配合了解非粒線體呼吸,如:糖解反應和發酵反應,本實驗可以分析絕大多數生物體的能量來源,並推斷出其健康和成長之狀況。微流體晶片裝置結合了二個組件:壓克力微孔陣列晶片、氣動封閉裝置。壓克力微孔陣列晶片: 又稱斑馬魚胚胎培養晶片,包合Pt(Pt-porphyrin)的氧氣感應層以及5(6)-Carboxyfluorescein的酸鹼感應層;氣動封閉裝置: 可以自由控制封閉晶片上感興趣且需量測之微孔槽。由於在低耗氧以及低酸化率的量測下防止量測區域的氣體擴散和潮解反應非常重要,因此利用氣動封閉裝置關閉微孔陣列晶片中的微孔槽(培養槽),形成只容得下單一顆斑馬魚胚胎的封閉的量測區域,並使用壓克力基材阻絕培養槽與外界液體接觸,防止氣體擴散及潮解反應,以利於斑馬魚胚胎發育初期低耗氧及低酸擠出率的量測。
有文獻表示生物早期成長階段受到缺氧會導致生物體變異,所以本實驗在胚胎缺氧狀態下量測呼吸量和酸化量的變化情形,得知斑馬魚胚胎在發育關鍵期間代謝能量的情況以及適應缺氧環境的代謝能力。經實驗了解斑馬魚胚胎逐漸發育長大形成幼魚,呼吸耗氧率及酸擠出率也隨得發育階段越來越大,斑馬魚胚胎代謝量與斑馬魚年紀成線性增加,經量測得知斑馬魚胚胎在發育關鍵期間代謝能量的情況。而缺氧實驗在斑馬魚胚胎成長到受精後48小時(48hpf),進行不同氧氣濃度程度對胚胎的影響做分析研究,經實驗證實將48hpf的胚胎處於氧氣20%、10%和3%的濃度下30分鐘,OCR以及AER是不變的,推測未達到胚胎缺氧狀態的條件,而氧氣濃度在3%的環境下30分鐘,量測出的耗氧率衰弱了30%而酸擠出率則上升90%,推測缺氧狀態下有氧呼吸功能下降,而無氧呼吸功能則大幅上升,斑馬魚藉由有氧呼吸和無氧呼吸的交替來適應不同環境的改變,為更加了解胚胎在缺氧下代謝變化,實驗從缺氧後量測2小時,發現代謝趨勢從缺氧後25分鐘OCR開使大幅下降而AER開始大幅上升,推測胚胎缺氧25分鐘這段時間,生物體首先偵測到ATP的供給量不足,所以使氧化磷酸化反應和糖解反應加劇,所以25分鐘前OCR大幅上升90%,但是在25分鐘後的時候胚胎因為環境缺氧的關係,可能此

環境已經無法靠氧化磷酸化反應了,但是糖解反應大量產生的丙酮酸依舊堆積在細胞中,此時胚胎為了排除這些過多的丙酮酸,開始加速發酵反應排除分解丙酮酸,造成發酵反應產生的乳酸大量生成並排出體外,所以導致AER的大幅增加,所以推測在25分鐘這個時間點是48hpf胚胎有氧呼吸和無氧呼吸的交替時間點。
綜合上述結論,此實驗方法提供了用於生物全方位能量代謝之檢測,不僅僅是市面上應用在細胞方面的生物能量測定儀,此平台適用生物樣本範圍更為廣泛,且具有生物體生理與疾病生物醫學應用之潛力。

A combination of a microfluidic device with a light modulation system was developed to detect the acid extrusion rate(AER) and the oxygen consumption rate (OCR) of a single developing zebrafish embryo via light intensity and phase-based phosphorescence lifetime detection. The microfluidic device combines two components: an array of PMMA microwells containing Pt(Pt-porphyrin) and 5(6)-Carboxyfluorescein as oxygen-sensitive and pH-sensitive luminescent layer and a microfluidic module with pneumatically actuated glass lids above the microwells to controllably seal the microwells of interest.
The acid extrusion rate(AER, in mpH/min/embryo) and oxygen consumption rate (OCR, in pmol O2/min/embryo) of a single developing zebrafish embryo inside a sealed microwell has been successfully measured from the blastula stage (3 h post-fertilization, 3 hpf) through the hatching stage (48 hpf). The AER and OCR increased in a linear and reproducible fashion with embryonic age. Continued adding 3% oxygen partial pressure of medium allows us to perform acid extrusion and respiratory measurements of a single zebrafish embryo at developmental stages and thus monitor changes in metabolism function in vivo that are coordinated with embryonic development. We have successfully measured the metabolic profiles of a single developing zebrafish embryo from 3 hpf to 48 hpf inside a microfluidic device.
The experiment confirmed that the oxygen 48hpf embryos in 20%, 10% and 6% partial pressure of oxygen for 30 minutes. The respiration and acidification amount is unchanged, suggesting that the embryo does not reach the hypoxic conditions on 3%partial pressure of oxygen environment of 30 minutes. Measured the oxygen consumption rate is decreased 30% and acid extrusion rate increased by 90%, suggesting that hypoxia aerobic respiratory function decline, while anaerobic respiration function rose sharply, thereby zebrafish adapt change to a different environment.
The Biological metabolism profiles is partitioned into Aerobic respiration and Anaerobic respiration. The changes in these respirations are correlated with zebrafish embryonic development stages. Our proposed platform provides the potential for studying bioenergetic metabolism in a developing organism and for a wide range of biomedical applications that relate physiology and disease

摘要............................................i
Abstract........................................iii
目錄............................................iv
圖目錄..........................................vii
表目錄..........................................viii
第一章 緒論......................................1
1.1前言.........................................1
1.2研究背景......................................3
1-2-1粒線體胞器..................................3
1-2-2細胞有氧呼吸 ................................4
1-2-3細胞發酵作用 ................................5
1-2-4生物微晶片..................................6
第二章 文獻回顧..................................8
2.1 微流道生物晶片裝置............................8
2.2量測系統......................................10
2.3缺氧再灌流....................................10
2.4實驗動機與目的 ................................14
第三章 實驗原理..................................18
3.1生物代謝概論..................................18
3.2磷光相位差檢測原理............................19
3.3胚胎酸擠出檢測原理............................21
第四章 實驗設計與架設.............................21
4.1實驗設計與機制 ................................22
4.2微流體晶片製作 ................................24
4.3 光強度及磷光相位感測系統......................27
4.4 pH值和氧氣感測系統程式計算....................32
4.5 斑馬魚胚胎飼養裝置............................34
4.6 斑馬魚與胚胎.................................35
第五章 實驗結果與討論.............................37
5.1斑馬魚胚胎存活測試.............................37
5.2斑馬魚胚胎基礎AER和OCR量測.....................38
5.3斑馬魚胚胎缺氧環境下AER和OCR檢測................41
第六章 斑馬魚熱休克及缺氧再灌流之粒線體活性研究......44
6.1熱休克及缺氧再灌流..............................44
6.2實驗結果與討論 .................................45
第七章 結論於未來展望..............................47
7.1結論...........................................47
7.2未來展望.......................................47
參考文獻..........................................48
附件..............................................51

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