由於近年來的奈米科技快速發展，奈米材料已應用在相當多的範圍。奈米材料對環境與生物的衝擊與影響必須要審慎地被討論，尤其是常見的奈米材料對環境生物的生長與行為的影響更值得我們研究。氧化鐵是常見補充人體內鐵質的元素，也被應用在許多廣泛的地方，例如：磁紀錄原件、區域熱治療和應用在藥物傳送的相關研究。本實驗將針對氧化鐵奈米粒子對斑馬魚的生物相容性及運動行為進行研究。利用奈米氧化鐵的磁性物理特質進一步觀察攝取氧化鐵奈米粒子後的斑馬魚對外加磁場的環境下其行為上之變化。

Magnetic nanomaterial has extensive potential for many applications, such as magnetic recording media, localize heating treatment, and drug delivery systems for pharmaceutical cure in vivo. However, the toxicology and biocompatibility of magnetic nanoparticles(NPs) should be studied. In this work, the security and impact of magnetic NPs, iron oxide, were investigated by injecting and feeding iron oxide NPs into zebrafish embryos and adult zebrafish to check the ethology, respectively. From death, mortality, hatching, deformity rate and morphology during embyro-larval stages, and ethology analysis for adult zebrafish, we infer that the toxicity of iron oxide nanoparticles is a dose-dependent tendency in zebrafish. When the feeding magnetic NPs concentration was increasing, the magnetic field will strong affect the moving trace of the zebrafish, like an invisiable wall to limit the movement.

目錄

第一章	介紹.……………………………………………………………1
簡介.………………………………………………………………………1
1.1科技的發展……………………………………………………………1
1.2環境毒理研究…………………………………………………………2
1.3磁性奈米粒子…………………………………………………………3
i.  介紹.…………………………………………………………………3
ii. 奈米粒子的磁性介紹.………………………………………………4
iii.四氧化三鐵奈米粒子備製.…………………………………………6
1.4人體對鐵元素的需求…………………………………………………7
1.5生物磁感應……………………………………………………………9
1.6實驗動物發展史簡介.………………………………………………10
1.7斑馬魚介紹.…………………………………………………………12
i.  物種介紹……………………………………………………………12
ii. 優勢與研究方向……………………………………………………12
第二章  研究動機………………………………………………………16
第三章  實驗裝置與流程………………………………………………18
3.1斑馬魚胚胎注射實驗.………………………………………………18
3.1.1  藥品配製.………………………………………………………18
i.  奈米粒子乾燥過程…………………………………………………18
ii. 胚胎重量量測………………………………………………………18
iii.注射劑量……………………………………………………………19
iv. 藥品秤重……………………………………………………………19
3.1.2  放魚概述.………………………………………………………20
3.1.3  藥品注射.………………………………………………………20
i.  注射針頭……………………………………………………………20
ii. 固定胚胎……………………………………………………………21
iii.濃度調控……………………………………………………………21
iv. 胚胎的顯微注射……………………………………………………23
3.1.4  記錄統計.………………………………………………………24
3.1.5  行為分析.………………………………………………………24
i.  實驗平台……………………………………………………………24
ii. 實驗流程……………………………………………………………24
3.2  斑馬魚成魚行為實驗.……………………………………………25
3.2.1  分辨公母.………………………………………………………25
3.2.2  藥品配製.………………………………………………………25
i.  奈米粒子乾燥過程..………………………………………………25
ii. 斑馬魚秤重…………………………………………………………25
iii.餵食劑量……………………………………………………………26
iv. 藥品秤重……………………………………………………………26
3.2.3  實驗裝置.………………………………………………………27
i.  實驗平台……………………………………………………………27
ii. 磁場設計……………………………………………………………27
3.2.4  實驗流程.………………………………………………………28
i.  釹鐵硼磁鐵磁場強度測量…………………………………………28
ii. 斑馬魚行為分析實驗………………………………………………29
3.2.5  影片分析.………………………………………………………30
3.3    成魚組織切片.…………………………………………………30
3.3.1  福馬林配製.……………………………………………………30
3.3.2  切片相關與特殊染色.…………………………………………31
第四章  數據分析與討論………………………………………………43
4.1   材料分析…………………………………………………………43
4.1.1  FE-HRTEM低倍率分析.…………………………………………43
4.1.2  元素分析.………………………………………………………44
i.   EELS分析.…………………………………………………………44
ii.  EDX分析……………………………………………………………44
4.1.3 高解析FE-HRTEM分析……………………………………………45
4.2   胚胎注射實驗……………………………………………………45
4.2.1  存活率.…………………………………………………………45
4.2.2  孵化率.…………………………………………………………46
4.2.3  畸形率.…………………………………………………………47
4.2.4  泳速量化.………………………………………………………47
i.  靜置期………………………………………………………………48
ii. 驚嚇期………………………………………………………………49
4.3 成魚行為分析………………………………………………………50
4.3.1  泳速量化與軌跡分析.…………………………………………50
i.  未加磁場……………………………………………………………50
ii. 外加磁場……………………………………………………………51
4.3.2  移動時間比較.…………………………………………………53
4.3.3  速度差值比較.…………………………………………………54
4.4   切片數據分析……………………………………………………55
4.4.1  普魯士藍染色.…………………………………………………55
4.4.2  成魚切片成果.…………………………………………………55
第五章  結論……………………………………………………………76
附錄………………………………………………………………………78
參考文獻…………………………………………………………………83




圖目錄

【圖1】積體電路發展趨勢與摩爾定律.………………………………15
【圖3.1】釹鐵硼磁鐵吸附儲存於酒精中的四氧化三鐵奈米粒子.…32
【圖3.2】傾斜式真空減壓濃縮機.……………………………………32
【圖3.3】微量天平.……………………………………………………33
【圖3.4】公母魚比較.…………………………………………………33
【圖3.5】拉針機.………………………………………………………34
【圖3.6】磨針機.………………………………………………………34
【圖3.7】胚胎固定盤示意圖.…………………………………………35
【圖3.8】超音波洗淨器.………………………………………………35
【圖3.9】顯微注射機.…………………………………………………36
【圖3.10】顯微注射量對應注射位置關係圖…………………………36
【圖3.11】此為2-cell(約0.5hpf)胚胎注射實體圖…………………37
【圖3.12】幼魚行為分析平台…………………………………………37
【圖3.13】斑馬魚餵食法實照…………………………………………38
【圖3.14】成魚行為實驗裝置示意圖…………………………………39
【圖3.15】磁場設計圖形及意義………………………………………40
【圖3.16】磁場感應器…………………………………………………41
【圖3.17】EthoVision XT軟體設定.…………………………………41
【圖3.18】EthoVision XT軟體設定.…………………………………42
【圖3.19】EthoVision XT軟體設定.…………………………………42
【圖4.1】四氧化三鐵奈米粒子在FE-HRTEM的低倍率分析圖.………59
【圖4.2】四氧化三鐵奈米粒子的EELS分析結果.……………………59
【圖4.3】四氧化三鐵奈米粒子的EELS mapping材料分析.…………60
【圖4.4】四氧化三鐵奈米粒子的EDX line scan分析………………60
【圖4.5】四氧化三鐵奈米粒子的EDX mapping材料分析……………61
【圖4.6】四氧化三鐵奈米粒子在FE-HRTEM的高倍率分析.…………61
【圖4.7】胚胎注射的存活率統計.……………………………………62
【圖4.8】胚胎注射的孵化率統計.……………………………………62
【圖4.9】注射不同濃度所發現的畸形魚實照.………………………63
【圖4.10】有發現畸形魚的濃度之畸形率統計………………………63
【圖4.11】96hpf靜置期的軌跡圖.……………………………………64
【圖4.12】 96hpf驚嚇期的軌跡圖……………………………………65
【圖4.13】成魚未加磁場………………………………………………66
【圖4.14】在外加大圓形磁場排列的情況……………………………66
【圖4.15】在外加小圓形磁場排列的情況……………………………67
【圖4.16】在外加磁場排列的情況……………………………………67
【圖4.17】斑馬魚的移動時間與不同劑量做圖比較…………………68
【圖4.18】靜置期與驚嚇期的速度表現差值比較……………………69
【圖4.19】一般魚類器官位置分佈解剖圖……………………………70
【圖4.20】斑馬魚組織切片(資料庫)…………………………………70
【圖4.21】斑馬魚組織切片……………………………………………71
【圖4.22】斑馬魚組織切片……………………………………………72
【圖4.23】斑馬魚組織切片……………………………………………73
【圖4.24】斑馬魚組織切片……………………………………………74
【圖4.25】斑馬魚組織切片……………………………………………75
【附錄圖1】玻璃針與注射針頭尖端大小的比較.……………………82

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