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標題: | 以金奈米粒子修飾3-(三氟甲基)苯基硫醇結合白鳳豆凝集素運用於偵測蛋白質間作用力與乳腺癌细胞的顯像 Detection of Protein-Protein Interactions and Imaging MCF-7 Cells with (3-(2,2,2-Trifluoroethyl)phenyl)me- thanethiol-ConA Capped Gold Nanoparticles |
作者: | Cheng-Han Yang 楊承翰 |
指導教授: | 陳昭岑(Chao-Tsen Chen) |
關鍵字: | 金奈米粒子,白鳳豆凝集素,蛋白質間作用力, Gold Nanoparticles,Concanavalin A,Protein-Protein Interactions, |
出版年 : | 2008 |
學位: | 碩士 |
摘要: | 本論文的研究重點是利用化學修飾的方法將醣結合蛋白修飾於不同粒徑的金奈米粒子,探討及評估其應用於檢測蛋白質交互作用及乳癌細胞造影的可能性。利用具有光親和性配基 8 的甘露糖分子與白鳳豆凝集素有專一性結合能力,將光親和性配基 8 帶到白鳳豆集素表面。再透過光照反應,使光親和性配基 8 的光反應基團以共價鍵結固定在白鳳豆凝集素的表面,接著將甘露糖分子移除,即可得到表面具有硫醇官能基的白鳳豆凝集素(SH-ConA)。SH-ConA 和作為辨識訊號輸出單元的 32 nm金奈米粒間可藉由連接分子(linker)結合。在此,我們也設計了幾種功能性及非功能性的連接端分子,功能性連接端(Linker MMUA 和 Linker MTA),帶有馬來醯亞胺順丁烯二醯亞胺(Maleimide),經過硫醇的加成之後會形成穩定的硫醚鍵,用來與 SH-ConA 作用;非功能性連接端(Linker DO、Linker DOEG 和 Linker DA)則是用來稀釋功能性連接端在金奈米粒子表面的濃度。將兩者以不同比例修飾在金奈米粒子上。我們發現非功能性連接端能夠藉由親水性和帶電荷的多寡幫助金奈米粒子均勻分佈在水溶液中。當修飾 Linker DA 時,金奈米粒子排斥的效果最好。在蛋白質交互作用實驗中,利用金奈米粒子的電漿共振光譜吸收的變化,我們成功地找出適當氯化鈉水溶液的濃度,使 ConA-GNP-MTA 表面電荷中和而能與 BS-I 作用,但是對其它蛋白質選擇性不如預期。應用相同的化學修飾策略,我們也成功地將 SH-ConA 修飾在1.2 nm的螢光奈米金上(ConA-Audot-MTA),希冀 ConA-Audot-MTA 與乳癌細胞(MCF-7)表面過度表現的甘露糖分子作用,而達到細胞顯影的目的。但是 ConA-Audot-MTA 在細胞實驗的條件下,有聚集的現象,使得細胞顯影結果不盡理想。所以未來希望可藉由改變連接端分子或輸出端分子的特性來達到偵測的目的。 The objective of this thesis is to develop a chemical method to immobilize sugar binding proteins onto gold nanoparticles with different sizes as well as to investigate the possibilities of using the resultant proteins-capped gold nanoparticles in screening protein/protein interactions and imaging surface tomography of breast cancer cells. The cognate substrate of ConA appended with 3-(trifluoroethyl)-3- phenyldiazirine was synthesized and the resultant photoaffinity ligand 8 was introduced to the surface of ConA based on the characteristics of ConA’s specific substrate binding and photo-activated labeling. By removing 8, ConA not only displaying the surface thiol groups (denoted as SH-ConA) but also possessing the free binding site for the mannose are thus generated. The structural features of linkers play some roles in the dispersion of gold nanoparticles. Thus, we design and synthesize some functional (namely Linker MMUA and Linker MTA) linkers with maleimide functionality and nonfunctional (Linker DO, Linker DOEG and Linker DA) linkers. SH-ConA is chemically modified to 32-nm gold nanoparticles by reacting with maleimide of functional linkers via 1,4-addition yielding ConA-GNP-MTA. Nonfunctional linkers not only dilute the concentration of functional linkers on the gold nanoparticle surface, but also increase the repulsion among gold nanoparticle by hydrophilicity and charge density. Based on experimental observation, the best linker to serve our purposes is Linker DA. By monitoring the changes of plasmon resonance absorption bands of gold nanoparticles, we successfully find the optimal concentration of sodium chloride solution, which can neutralize surface charge of ConA-GNP-MTA, for ConA-GNP-MTA to interact with BS-I in the protein-protein interaction experiment. However, the selectivity of ConA-GNP-MTA to other proteins is not as good as what we expected. Applying the same immobilization strategy, SH-ConA successfully encapsulates 1.2-nm gold nanodots, which fluoresce strongly in aqueous solution. The resultant ConA-Audot-MTA is thus evaluated in the application of MCF-7 breast cancer cells imaging. However, ConA-Audot-MTA aggregates under the cell imaging experiment condition. The experiment results could be possibly improved by changing the signal transduction unit or characteristic features of linkers. |
URI: | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/26302 |
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顯示於系所單位: | 化學系 |
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