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研究生:林廷恩
研究生(外文):Ting-En Lin
論文名稱:單官能化卵白素奈米探針技術之開發
論文名稱(外文):Development and Characterization of Nanoprobes Conjugated with Monovalent Streptavidin
指導教授:林政鞍張恒雄
指導教授(外文):Cheng-An J. LinHomg-Shong Chang
學位類別:碩士
校院名稱:中原大學
系所名稱:生物醫學工程研究所
學門:工程學門
學類:生醫工程學類
論文種類:學術論文
論文出版年:2012
畢業學年度:100
語文別:中文
論文頁數:93
中文關鍵詞:單一官能化卵白素奈米探針卵白素
外文關鍵詞:StreptavidinMonovalent StreptavidinNanoprobes
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近年來控制奈米粒子表面官能基和精準接枝生物分子數目之技術開發已成為生物奈米科技重要之研究課題,要精確地調控奈米粒子表面官能基或生物分子接枝之數目仍然是一大瓶頸。其中以單一官能化技術為最難克服。奈米生醫探針設計強調其精準性,過多之接位造成探針結構複雜性,亦使在奈米組裝上難以控制,若是應用在追蹤活體細胞上無法精準追蹤細胞表面上的受質,使得細胞過度交聯造成複雜影像,而無法有效診斷結果。本研究用卵白素-生物素(Streptavidin – Biotin)來建立起快速組裝單一官能化探針平台,因為卵白素具能與四個生物素產生鍵結之特性。卵白素-生物素這系統早就廣泛用於生醫探針檢測以及生物檢測上。本研究設計兩種胜肽皆帶有生物素,其一帶有電性、其二不具電性,調控胜肽與卵白素比例,成不對稱修飾,讓卵白素嫁接上不同數目的胜肽,利用胜肽末端His-tag與Ni離子之高親和性以Ni-NTA 管柱分離與切膠純化這兩種方法,分離出單一官能化之卵白素。探討哪種性質(帶電性、分子量大小)胜肽能夠有效利用垂直電泳檢測出不同接枝數目卵白素以及兩種快速純化之方式,最後以垂直電泳(PAGE)以及奈米粒子組裝已證明本研究所製備出單官能卵白素之結構。
To control the Surface functional groups of nano particle and the technique accurate grafting on the molecules. These kinds of technologies has playing an important role in this area. However , to control the Surface functional groups of nano particle or to grafted on how much number of molecules are still big tough questions. Among these technologies , single functional technology is the most difficult issue.The design of bio nanotechnology probes emphasize the great precision. Too many positions may make the structures of the probes becoming more complicated and becoming more difficult to compos the nano particle.If we can’t chasing the substrate of the living cells’ surface accurately may make the cells over cross-linked and bring about some complex images. Therefore, this kind of result is helpless. In this research is going to use Streptavidin – Biotin to build a platform which can construct a Single functionalized probes quickly. Streptavidin can bond with four Biotin . So this system is widely used in biomedical probes for the detection and biological detection already. In this experiment is going to design two peptides which have Biotin. One of them with electrical and the other does not. To adjust the rate of peptides and the Streptaviding becoming asymmetric in order to make the Streptavidin can grafted different numbers of peptides. There are two ways to Isolate from a single function’s avidin. One of them is using the high affinity of the peptide terminal His-tag and Ni ions to Ni-of NTA column separation. And the other is purified gel. In this case, is going to study which kind of peptide(the size of molecular or the Electrical ) will able to use the vertical pages to check out different numbers of Streptavidin and two ways to purify quickly. In the end, we take vertical pages and nano particle to prove the structure of the monofunctional Streptavidin prepared in this experiment.
摘要 I
ABSTRACT II
致謝 III
目錄 IV
圖索引 VI
第一章 前言 1
1-1. 研究計畫之背景 1
第二章 文獻回顧 4
2-1 膠體奈米粒子(colloidal nanoparticle) 4
2-2 金屬奈米粒子 5
2-3 奈米粒子表面改質官能化 6
2-4 奈米粒子單官能化修飾 8
2-5 Streptavidin 21
2-6 Streptavidin的應用 23
2-7 單一官能Streptavidin 24
2-8 單一官能化Streptavidin接枝奈米粒子應用 26
2-9 研究目的 30
第三章 材料與方法 32
3-1 實驗設計 32
3-2 實驗架構 33
3-3 實驗藥品 34
3-4 實驗儀器設備 36
3-5 實驗步驟 37
3-5.1 基因工程製備單一官能化卵白素(streptavidin) 37
3-5.2 Native PAGE垂直電泳分析 42
3-5.3 Streptavidin與D-Biotin、胜肽一(His×6-G×4-Biotin)、胜肽二(Biotin-G-G-His×6)接枝測試及反應參數 43
3-5.4 多官能化Streptavidin放大製程 43
3-5.5 製備單一官能化Streptavidin運用垂直電泳分離純化 44
3-5.6 製備單一官能化Streptavidin 運用Ni-NTA-Column分離純化 44
3-5.7 六奈米金粒子合成製備 46
3-5.8 六奈米金粒子的兩性高分子披覆(Polymercoating) 47
3-5.9 兩性高分子披覆的純化 48
3-5.10 親水性六奈米金粒子修飾NH2-PEG(5000)-Biotin 49
3-5.11 六奈米金-NH2-PEG(5000)-Biotin接枝不同官能化Streptavidin鑑定其官能化數目 50
3-5.12 1.8nm Ni-NTA-金奈米粒子接枝不同官能化Streptavidin鑑定其官能化數目 50
第四章 結果與討論 51
4-1. 基因工程製備單一官能化卵白素(streptavidin) 52
4-2. Streptavidin與D-Biotin、胜肽一(His×6-G×4-Biotin)、胜肽二(Biotin-G-G-His×6)接枝測試及反應參數 53
4-3. 多官能化Streptavidin放大製程 56
4-4. 製備單一官能化Streptavidin運用垂直電泳分離純化 57
4-5. 製備單一官能化Streptavidin 運用Ni-NTA-Column分離純化 59
4-6. 製備六奈米金粒子及兩性高分子披覆純化 61
4-7. 親水性六奈米金粒子修飾NH2-PEG(5000)-Biotin 63
4-8. 六奈米金-NH2-PEG(5000)-Biotin接枝不同官能化Streptavidin測試其與Biotin鍵結活性 67
4-1. 1.8nm Ni-NTA-金奈米粒子接枝不同官能化Streptavidin測試其與Biotin鍵結活性 68
第五章 結論 69
參考文獻 70
附錄一、以大分子量Biotin-PEG(3000)-NH2鑑定不同官能數目Streptavidin結構 79

圖索引
圖2- 1 不同粒徑大小奈米金溶液顏色:12, 18, 30, 40 及 72 nm(由左至右)。------------------------------------------------------------------------------------------------------5
圖2- 2 Poly(maleic anhydride alt-1-tetradecene)兩性高分子披覆有機疏水性奈米粒子示意圖。----------------------------------------------------------------------------------------7
圖2- 3 兩性高分子披覆奈米粒子示意圖。(上:聚馬來酸酐未開環前(有機疏水性);中:聚馬來酸酐開環後(水相);下:修飾嫁接其他生物分子,如:醣類、PEG 等)。
------------------------------------------------------------------------------------------------------7
圖2-4 奈米粒子嫁接不同官能基團示意圖。---------------------------------------------------8
圖2-5 固相合成單官能法的一般原理。-----------------------------------------------------9
圖2-6 固相樹脂交換反應合成單一羧基配體金奈米粒子示意圖。------------------10
圖2-7 矽烷化玻璃基底單官能化修飾法示意圖。---------------------------------------11
圖2-8 單晶聚合物基底單官能化修飾法示意圖。---------------------------------------12
圖2-9 大尺寸微粒子基底單官能化修飾法示意圖。------------------------------------13
圖2-10 利用PS樹脂修飾上帶有胺基配體單官能金奈米粒子示意圖。--------------14
圖2-11 運用帶有車輪烷磁性奈米粒子純化單官能金奈米粒子示意圖。-----------15
圖2-12 單官能奈米金粒子利用車輪烷自組裝能二聚體與三聚體示意圖。--------15
圖2-13 螢光金奈米團簇嫁接PEG-Biotin電泳跑膠圖。---------------------------------16
圖2-14奈米粒子自組裝樹枝狀的概念示意圖。------------------------------------------17
圖2-15 DNA結構編碼。-----------------------------------------------------------------------18
圖2-16 金奈米粒子接枝純化以及自組裝洋菜膠電泳膠圖。--------------------------18
圖2-17 Streptavidin-Biotin組裝電化學免疫催乳激素感測器示意圖。---------------19
圖2-18 量子點奈米粒子透過Strepavidin-Biotin系統嫁接上一個抗體示意圖。---20
圖2-19 利用混合凝膠純化量子點奈米粒子。--------------------------------------------20
圖2-20 單官能化Streptavidin組裝純化示意圖。-----------------------------------------22
圖2-21 Streptavidin-Alexa Fluor568 標定海馬神經細胞,所示單官能化Streptavidin標定細胞解析力高於一般Streptavidi。-----------------------------------------------------25
圖2-22 Streptavidin-Alexa Fluor568 標定神經細胞,加入突觸標記囊泡谷氨酸轉運體(VGLUT1), -----------------------------------------------------------------------------25
圖2-23 單官能化Streptavidin製備示意圖。----------------------------------------------26
圖2-24 利用洋菜膠(agarose gel)將不同接枝單官能化Streptavidin進行切膠純化分離。------------------------------------------------------------------------------------------------26
圖2-25 在活細胞標定中,很明顯呈現出單官能化Streptavidin其細胞成像較佳。YFP是被用來作為轉染標記,並說明,Streptavidin-QD標籤是有專一性。DIC為明視野。------------------------------------------------------------------------------------------27
圖2-26 量子點(QD)批覆胜肽接枝Streptavidin 示意圖。------------------------------27
圖2-27 利用洋菜膠(agarose gel)將不同接枝數目Streptavidin純化分離,並且加入Biotin-PEG測試其探針是否還具有與Biotin高親和性及穩定性。--------------------28
圖2-28 利用Biotin-A647(紅色) 與Streptavidin-QD(綠色)鍵結後所產生Total internal reflection fluorescence (TIRF)來計算Streptavidin與單官能化Streptavidin兩者與Biotin鍵結位子的數量。----------------------------------------------------------------28
圖2-29 Membrane-anchored cyan fluorescent protein fusion (AP-CFP-TM)表面帶有Biotin利用單官能化Streptavidin進行追蹤,並且記錄下其移動軌跡,計算出Streptavidin螢光探針的移動率。------------------------------------------------------------29
圖3 1 Dead和 Alive以3:1之形式組裝,並以Ni-NTA純化。-------------------------37
圖3-2 奈米金粒子合成示意圖。------------------------------------------------------------46
圖3-3 兩性高分子披覆奈米粒子示意圖。------------------------------------------------47
圖3- 4 兩性高分子合成示意圖。------------------------------------------------------------48
圖4-1 基因工程利用異源細胞表現重組蛋白的方法製備出單官能化卵白素未經純化。PAGE-12%膠。--------------------------------------------------------------------------52
圖4-2 基因工程利用異源細胞表現重組蛋白的方法製備出單官能化卵白素經Ni-NTA親和性管柱純化只有一個Band,。PAGE-12%膠。-----------------------------52
圖4-3 D-Biotin序列濃度增加。PAGE-8%膠。---------------------------------------------53
圖4-4 His×6-G×4-Biotin濃度座序列增加。PAGE-8%膠。------------------------------54
圖4-5 Biotin-G-G-His×6濃度座序列增加。PAGE-8%膠。------------------------------55
圖4-6 一步法與漸進式垂直電泳分析。PAGE-8%膠。----------------------------------56
圖4-7 (A)為最佳反應參數放大製備以垂直電泳進行分離。(0):未接枝Streptavidin。(1):一個胜肽接枝Streptavidin。(2): 二個胜肽接枝Streptavidin。(3): 三個胜肽接枝Streptavidin。(4): 四個胜肽接枝Streptavidin。----------------------------------------57
圖4-8 (A)一次切膠純化。PAGE-8%膠。(B) 加入過量胜肽測試純化後Streptavidin活性,其Band皆有往下移動。---------------------------------------------------------------58
圖4-9二次切膠純化,Band變得很不清楚。PAGE-8%膠。------------------------------58
圖4-10 Ni-NTA-Column分離純化垂直電泳(膠體8%、150V,150min)檢測。-------60
圖4-11 (A)兩性高分子披覆六奈米金粒子示意圖。(B) a)未批覆兩性高分子六奈米金溶於Toiuene中。b) 成功批覆兩性高分子六奈米金溶於水相中。(C)批覆兩性高分子六奈米金以2%洋菜膠膠體電泳檢測(75V,90 分鐘)。------------------------61
圖4-12 六奈米金與批覆兩性高分子六奈米金UV吸收圖。--------------------------62
圖4-13 NH2-PEG(5000)-Biotin @AuNP 的 EDC 反應濃序列測試。----------------63
圖4-14 NH2-PEG(5000)-Biotin反應濃度序列測試(1mM~0.625mM)。----------------64
圖4-15 NH2-PEG(5000)-Biotin @AuNP放大製程。-----------------------------------------65
圖4-16切膠純化1-NH2-PEG(5000)-Biotin@AuNP切膠純化。----------------------------------------------------------------------------------------------------65
圖4-17 黑線為Au-6nm特徵吸收峰為520,接枝NH2-PEG(5000)-Biotin其特徵吸收峰並無位移。---------------------------------------------------------------------------------------66
圖4-18 NH2-1-PEG(5000)-Biotin與濃度序列Streptavidin反應以及不同官能Streptavidin反應。------------------------------------------------------------------------------67
圖4-19 1.8nm AuNPs-Ni-NTA與濃度序列Streptavidin反應以及不同官能Streptavidin反應。------------------------------------------------------------------------------68
圖5-1 SA-0與Biotin-PEG(3000)-NH2濃度序列反應。-----------------------------------79
圖5-2 SA-1與Biotin-PEG(3000)-NH2濃度序列反應。-----------------------------------80
圖5-3 SA-2與Biotin-PEG(3000)-NH2濃度序列反應。-----------------------------------81
圖5-4 SA-3與Biotin-PEG(3000)-NH2濃度序列反應。-----------------------------------82
圖5-5 SA-4與Biotin-PEG(3000)-NH2濃度序列反應。-----------------------------------83
圖5-6 不同接枝數目Streptavidin與Biotin-PEG(3000)-NH2濃度10μM反應鑑定其結構。---------------------------------------------------------------------------------------------85
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