本研究是利用電解剝離法製備石墨烯，使石墨烯表面接有聚苯乙烯磺酸鈉，減少石墨烯之間的凡得瓦爾力作用，以提升其在水中的分散性。此方法所使用的聚苯乙烯磺酸鈉毒性低，可增加生物相容性，適合應用在生物領域。相較於其他製程，電解剝離法可以低溫、低成本、高產率的方式快速製備石墨烯。
為了評估石墨烯的刺激性與在體內的毒性，以瞭解石墨烯作為體內功能性材料的可能性。本實驗分為兩部分，第一部分於兔子皮膚上塗抹石墨烯，在經過24小時接觸後，連續追蹤達三天都未發現皮膚出現紅斑或浮腫。眼睛滴入石墨烯溶液後在後續的72小時內皆沒有發現結膜的充血刺激，角膜染色的結果也未出現潰瘍。這些結果顯示石墨烯並不會對皮膚及眼睛等黏膜產生刺激性。第二部分是在小鼠尾靜脈注射不同濃度的石墨烯，發現在注射後第一天高濃度組別的小鼠，血清肝功能出現異常，且在第七天以後回到正常範圍。組織切片的結果顯示，注射高濃度石墨烯的小鼠可在肺、肝、脾等器官中發現石墨烯的團簇，並且累積達90天都無法排除體外。體內即時追蹤的結果則顯試，帶有螢光的石墨烯在短時間就分布到全身，並且在24小時有部分經由尿液排泄，另一部分逐漸累積到肝臟。

In order to acquire well dispersed graphene solution, we chose electrochemical exfoliation method to synthesize graphene. In this method, poly sodium 4-styrenesulfonate was bound on graphene during the process, therefore improve the hydrophilicity of graphene and prevent it from re-stacking by strong Van der Waals interaction. This method offers advantages such as low process temperature, low cost, and rapid and efficient graphene fabrication. Also, the polymer is biocompatible and thus suitable for biomedical application.
To evaluate possible applications of graphene in vivo, this study conducted irritation and toxicity tests of graphene synthesized by the exfoliation method. First, skin irritation test on rabbits reveals that graphene did not cause any skin irritation and corrosion even in a long exposure time up to 24 h. Eye irritation test indicates that graphene could neither cause eye irritation nor induce corneal ulcer.
On the other hand, mouse after tail vein injection of graphene at a high concentration showed acute abnormality of liver function on day 1, although the liver function result dropped to normal range on day 7. Histopathology analysis of tissue section uncovered that after injection of high dose graphene, there were obvious accumulates of graphene found in lung, liver, and spleen, and could be retained by these organs till 90 days post injection. In order to trace the distribution of graphene in mice after intravenous injection, in vivo and in situ image system was applied to acquire fluorescence images at different time points post injection. It shows that graphene is widely distributed in the entire mouse within one minute, and tends to retain in liver and bladder after one day.

摘要 I
Abstract II
致謝 III
總目錄 IV
表目錄 VII
圖目錄 VIII
縮寫表 XIV
第1章 緒論 1
1-1 石墨烯的簡介 1
1-1-1 石墨烯的起源與結構 1
1-1-2 石墨烯的製備方法 3
1-2 石墨烯與生物科學領域 9
1-2-1 石墨烯在生醫工程上的應用 9
1-3 奈米材料的潛在危害 9
1-3-1 暴露途徑 10
1-3-2 石墨烯對動物的影響 10
第2章 研究目的與石墨烯溶液製備 20
2-1-1 研究目的 20
2-1-2 實驗設計 21
2-2 電解剝離之石墨烯溶液製備 22
2-3 石墨烯形貌與性質分析 24
2-4 標記螢光之石墨烯溶液製備 25
第3章 石墨烯局部刺激性之實驗方法 36
3-1 實驗動物-兔子 36
3-2 皮膚刺激性試驗 36
3-3 眼睛刺激性試驗 39
第4章 小鼠28天亞急毒性試驗 47
4-1 實驗動物-小鼠 47
4-2 尾靜脈注射方法 47
4-3 毒理表現分析 50
4-3-1 臨床檢察 50
4-3-2 血液採集 50
4-3-3 組織病理檢查 52
4-4 標記螢光之石墨烯在小鼠體內的分布情形 52
第5章 結果與討論 58
5-1 石墨烯溶液製備之結果 58
5-2 石墨烯形貌與性質分析結果 58
5-2-1 場發射掃描式電子顯微鏡觀察材料之結果 58
5-2-2 拉曼光譜儀之分析結果 59
5-2-3 傅立葉轉換紅外線光譜儀觀察材料之結果 61
5-2-4 原子力顯微鏡之分析結果 62
5-2-5 雷射光散射儀之分析結果 62
5-3 石墨烯皮膚刺激性試驗結果 63
5-4 石墨烯眼睛刺激性試驗結果 63
5-5 石墨烯之毒性試驗結果 64
5-5-1 臨床檢察結果 64
5-5-2 血清生化分析結果 65
5-5-3 組織病理檢查結果 66
5-6 石墨烯在組織內的鑑定結果 68
5-7 標記螢光之石墨烯在小鼠體內分布之結果 69
第6章 結論 95
參考文獻 97

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