臺灣博碩士論文加值系統

中文摘要


本研究最主要的目的是利用不同鏈長的硫氫化合物(mercapto-aliphatic acids)來修飾奈米金粒子表面，探討人血清蛋白(Human serum albumin)及澱粉分解脢(Bacillus amyloliquefaciens α-amylase)於不同表面之吸附行為，以及蛋白吸附後構形的變化。
在金奈米粒子表面改質方面，我們發現長鏈的MUA (mercaptoundecanoic acid)於奈米金表面有最高的鍵結密度，且經MUA修飾後的奈米金粒子之鹽穩定性也最高。這是因為當硫氫化合物碳鏈較長時，其彼此間之靜電排斥力較低，導致MUA分子於金奈米粒子表面之鍵結密度較其他鏈長較短的硫化物高之緣故。此外，我們也發現，金奈米粒子經硫氫化合物表面修飾後，其鹽穩定度會隨粒子表面鍵結密度增加及硫化物鏈長增加而提高。
在蛋白質吸附的部分，由HSA及BAA之吸附曲線得知，HSA於金奈米粒子表面之吸附量皆遠大於BAA，此外我們也發現，HSA於不同表面改質奈米金粒子之吸附量大致上與奈米金表面硫化物之鍵結密度成正比，而BAA之吸附量與其成反比。我們推測這是因為HSA分子鏈中之雙硫鍵(disulfide bond)取代了奈米金粒子表面與硫化物間的Au-S鍵結所導致；而BAA之組成氨基酸中因不含cysteine，使得BAA無法以雙硫鍵取代粒子表面之硫化物，以致於其吸附量遠低於HSA。由此我們認為，HSA與金奈米粒子間之作用力主要是以Au-S鍵結為主，而BAA於粒子表面之吸附主要是依賴Au-N。
從本實驗中發現，奈米金粒子經HSA吸附後其鹽穩定度均大幅度地提高，特別是MAA (mercaptoacetic acid)改質的奈米粒子。原本穩定度最低的Au-MAA透過HSA的吸附，其穩定度僅次於最高的Au-MHDA (mercaptohexaundecanoic acid)。經比較後我們發現，金奈米粒子之穩定度會隨其表面蛋白吸附量或修飾硫化物鏈長增加而提升。
我們也利用螢光光譜分析HSA與BAA吸附前後之光譜，並以一比值I350/I305代表蛋白中tryptophan及tyrosine之相對位置。由實驗結果發現，HSA吸附後之I350/I305均比吸附前低，由此透露出少許變形的跡象。而在比較BAA之I350/I305後發現，BAA吸附在短鏈硫化物修飾之奈米金粒子表面其構形變化程度不大，而在Au-MUA及Au-MHDA表面吸附後其變形程度較大。此外，從HSA及BAA吸附在金奈米粒子後之螢光光譜最大散射波長發現，HSA吸附後產生藍位移(blue shift)，而BAA並無此現象，由此推測HSA於金奈米粒子表面吸附時歷經較大程度之構形變化。

Abstract

The major purposes of this study are to modify the surfaces of the colloidal gold nanoparticles by mercapto-aliphatic acids of various lengths (C2 to C16), to study the effects of aliphatic chain length on the adsorption of Human serum albumin (HSA) and Bacillus amyloliquefaciens α-amylase (BAA), and to elucidate conformational changes of these two proteins upon adsorption to the modified surfaces.

Thiolated aliphatic acids of different concentrations were added into solutions containing Au nanoparticle, it is found that the binding capacity of mercaptoundecanoic acid (MUA) on Au nanoparticle is the highest and the resulting modified Au particles has the highest stability in salt. It is suggested that the salt endurability of these modified Au particles is affected by both the coverage of thiolated aliphatic acids as well as the chain length.

We also found that the amount of HSA adsorption was much higher than that of BAA. And it was found that the amounts of HSA adsorption was roughly followed the order of the degree of surface thiolation, but that of BAA basically increases as the degree of thiolation decreases. After HSA adsorption, the stability in salt of these particles was dramatically increased, especially for the MAA-modified one. The MAA-modified nanogold was the least stable one among all the modified particles under study, and became the the most stable one after HSA adsorption. This phenomenon might be attributed to its high protein adsorption.

The structural alteration of proteins after adsorption was defined by the fluorescent emission intensities ratio of 350 nm to 305 nm (I350/I305). By comparing the ratios prior to and after adsorption, conformational changes of HSA and BAA on different Au surfaces were analyzed. It was found that the value of I350/I305 after HSA adsorption was smaller than that before adsorption. We also found that the adsorbed HSA exhibited blue shift in its fluorescent emission spectra. It indicated that HSA adsorbed on various Au nanoparticles undergoes a certain degree of structural changes. On the contrary, we did not find any indication of conformational changes of adsorbed BAA.

總目錄

中文摘要-------------------------------------------------------------- Ⅰ
英文摘要-------------------------------------------------------------- Ⅲ
總目錄----------------------------------------------------------------- Ⅴ
圖目錄---------------------------------------------------------------- Ⅷ
表目錄----------------------------------------------------------------- Ⅹ

第一章 簡介--------------------------------------------------------- 1
第二章 文獻回顧--------------------------------------------------- 3
2.1奈米材料的世界------------------------------------------------- 3
2.2金屬奈米材料的發展------------------------------------------ 3
2.3金屬奈米粒子之製備方式------------------------------------ 4
2.3.1 雷射消熔法-------------------------------------------- 5
2.3.2 金屬氣相合成法-------------------------------------- 6
2.3.3 化學還原法-------------------------------------------- 7
2.3.3.1 鹽類還原法---------------------------------------- 8
2.3.3.2 電化學法------------------------------------------- 10
2.3.3.3 聲化學製備法------------------------------------- 11
2.3.3.4 晶種促進成長法---------------------------------- 12
2.3.3.5 利用逆微胞、微乳液來製備奈米粒子------- 13
2.3.3.6 利用Dendrimer製備奈米粒子------------------ 15
2.4 奈米粒子在生物上的應用--------------------------------- 16
2.4.1 高分子奈米粒子-------------------------------------- 16
2.4.2 磁性奈米粒子----------------------------------------- 18
2.4.3 金屬與半導體奈米粒子----------------------------- 19
2.4.3.1 金奈米粒子與DNA之結合---------------------- 21
2.3.1 半導體奈米粒子與生物分子之結合----------- 26
2.5 蛋白吸附後結構探討--------------------------------------- 33
第三章 實驗藥品、設備與方法--------------------------------- 38
3.1 實驗藥品-------------------------------------------------------- 38
3.2 實驗設備-------------------------------------------------------- 40
3.3 實驗方法-------------------------------------------------------- 42
3.3.1 奈米金顆粒之合成----------------------------------- 42
3.3.2 金奈米顆粒之表面改質----------------------------- 43
3.3.2.1 利用MAA修飾奈米金表面--------------------- 43
3.3.2.2 Ellman’s method------------------------------------- 44
3.3.2.4 利用MPA修飾奈米金表面--------------------- 45
3.3.2.5 利用Ellman’s method分析MPA鍵結量------- 46
3.3.2.5利用MUA修飾奈米金表面--------------------- 47
3.3.2.6利用Ellman’s method分析MUA鍵結量------- 48
3.3.2.7利用MHDA修飾奈米金表面------------------- 49
3.3.2.8利用Ellman’s method分析MHDA鍵結量----- 50
3.3.3 不同表面改質奈米金穩定性分析------------------ 51
3.3.4 HSA及BAA在不同表面之吸附曲線---------------- 52
3.3.4.1 奈米金表面改質---------------------------------- 52
3.3.4.2 吸附蛋白濃度測定------------------------------- 53
3.3.5 吸附後構形變化分析--------------------------------- 55
第四章 結果與討論------------------------------------------------ 56
4.1 奈米金顆粒之合成--------------------------------------- 56
4.1.1 粒徑分佈-------------------------------------------------- 57
4.1.2 莫耳消光係數------------------------------------------ 60
4.2 金奈米粒子之表面改質-------------------------------------- 63
4.2.1 短鏈硫化物mercaptoacetic acid (MAA)
mercaptopropionic acid (MPA)於奈米金表面之鍵結------------------------------------------------------- 64
4.2.2 長鏈硫氫化合物mercaptoundecanoic acid (MUA)與mercaptohexaundecanoic acid (MHDA)於奈米金表面之鍵結-------------------------------------------------------- 67
4.2.3 探討mercaptoaliphatic acids在奈米金表面之鍵結行為--------------------------------------------------- 69
4.2.4 探討奈米金粒子經不同硫化物改質後之穩定性---------------------------------------------------------- 72
4.3探討Human serum albumin (HSA)與Bacillus
amyloliquefaciens -amylase (BAA)於不同表面改質奈米金之吸附----------------------------------------------------- 76
4.3.1人血清蛋白(HSA)吸附曲線--------------------------- 76
4.3.2澱粉分解脢(BAA)吸附曲線--------------------------- 78
4.3.3探討HSA、BAA吸附後奈米粒子鹽穩定性-------- 80
4.3.4蛋白質與奈米金表面之作用力---------------------- 82
4.3.5蛋白質(HSA、BAA)與奈米金粒子間可能之吸附模式-------------------------------------------------------- 83
4.4探討蛋白質吸附前後之結構變化--------------------------- 87
第五章 結論--------------------------------------------------------- 99
參考文獻-------------------------------------------------------------- 102

圖目錄

圖2.1 雷射消熔法實驗裝置簡圖---------------------------------- 5
圖2.2 利用界面活性劑NR4Br保護金奈米顆粒--------------------------------- 10
圖2.3 逆微胞三維球型結構簡圖---------------------------------- 14
圖2.4 利用dendrimer製造奈米金屬顆粒流程簡圖------------------- 14
圖2.5 (A)藥物分子自由擴散至細胞膜並通過
(B)藥物分子藉由奈米粒子輸送至細胞膜，經生物分解作用釋放出
藥物分子-------------------------------------------------------------------- 17
圖2.6 利用DNA修飾奈米金顆粒並使其進行自我組裝，接著進行雜交反應---------------------------------------------------------------------------------- 22
圖2.7 DNA雜交與金奈米顆粒吸收值隨溫度之變化--------------------------- 24
圖2.8 利用TEM電子顯微鏡觀察金奈米粒子聚集----------------------------- 25
圖2.9 不同大小奈米晶體之螢光光譜------------------------------ 27
圖2.10 以363nm雷射激發CdSe/ZnS core shell奈米晶體標記
3T3老鼠纖維細胞之共軛焦雷射顯微鏡影像------------------- 29
圖2.11 比較Fluorescein與半導體奈米粒子標記之光穩定性----------------- 29
圖2.12 (A)以MAA修飾量子點CdSe/ZnS鍵結蛋白質示意圖
(B)鍵結輸鐵蛋白量子點之TEM影像------------------------------------- 30
圖2.13 (A)未修飾之奈米晶體
(B)MAA修飾後之奈米晶體-------------------------------------------------
(C)IgG鍵結後之奈米晶體 31
圖2.14 (A)含有BSA蛋白之奈米晶體螢光影像。
(B)IgG修飾奈米晶體後，經抗體抗原反應使奈米晶體產生聚集之螢光影像 32
圖2.15 血紅素吸附於silica奈米粒子後(a)二級結構(b)三級結構 隨時間之變化------------------------------------------------------------------- 34
圖2.16 BSA吸附前後之螢光光譜J代表BSA吸附前I代表BSA吸附後 35
圖2.17 Aspartic protease於奈米金粒子表面吸附前後之螢光光譜------------ 36
圖3.1 金奈米粒子合成裝置圖------------------------------------------------------- 42
圖3.2 DTNB與氫硫化合物反應示意圖------------------------------------------- 44
圖3.3 DTNB與不同濃度MAA反應之吸收值變化---------------------------- 45
圖3.4 DTNB與不同濃度MPA反應之吸收值變化------------------------------ 47
圖3.5 DTNB與不同濃度MUA反應之吸收值變化---------------------------- 49
圖3.6 DTNB與不同濃度MHDA反應之吸收值變化-------------------------- 51
圖3.7 不同濃度HSA之螢光強度-------------------------------------------------- 53
圖3.8 不同濃度BAA之螢光強度-------------------------------------------------- 54
圖4.1 利用粒徑分析儀分析奈米金之粒徑分佈圖------------------------------- 58
圖4.2 利用AFM分析奈米金粒子之表面型態-2D------------------------------ 59
圖4.3 利用AFM分析奈米金粒子之表面型態-3D------------------------------ 59
圖4.4 利用AFM分析奈米金粒子之粒徑大小----------------------------------- 60
圖4.5 金奈米粒子之吸收光譜------------------------------------------------------- 61
圖4.6 金奈米粒子組成示意圖------------------------------------------------------- 63
圖4.7 MAA分子鍵結曲線----------------------------------------------------------- 65
圖4.8 MPA分子鍵結曲線------------------------------------------------------------ 65
圖4.9 MUA分子鍵結曲線----------------------------------------------------------- 68
圖4.10 MHDA分子鍵結曲線--------------------------------------------------------- 68
圖4.11 不同鏈長之硫氫化合物於金奈米粒子表面之鍵結曲線---------------- 70
圖4.12 金奈米粒子因鹽濃度增加而導致聚集、沈澱示意圖------------------- 72
圖4.13 經不同碳鏈長短硫氫化合物修飾後之金奈米粒子於不同NaCl濃度下之穩定性---------------------------------------------------------------------- 74
圖4.14 經ME後修飾之金奈米粒子於不同NaCl濃度下之穩定性----------- 75
圖4.15 HSA於不同表面改質金奈米粒子之吸附曲線--------------------------- 77
圖4.16 BAA於不同表面改質金奈米粒子之吸附曲線--------------------------- 79
圖4.17 經HSA吸附之不同表面改質奈米粒子之鹽穩定度-------------------- 81
圖4.18 經BAA吸附之不同表面改質奈米粒子之鹽穩定度-------------------- 81
圖4.19 蛋白質與奈米金粒子交互作用力示意圖---------------------------------- 82
圖4.20 BAA於ME後改質奈米金粒子之吸附曲線------------------------------ 85
圖4.21 HSA於ME後改質奈米金粒子之吸附曲線------------------------------- 85
圖4.22 Tryptophan與Tyrosine在280 nm激發波長下之螢光光譜--------- 87
圖4.23 不同濃度HSA吸附於Au之I350/305------------------------------------- 90
圖4.24 不同濃度HSA吸附於Au-MAA之I350/305---------------------------- 90
圖4.25 不同濃度HSA吸附於Au-MPA之I350/305----------------------------- 91
圖4.26 不同濃度HSA吸附於Au-MAA-ME之I350/305----------------------- 91
圖4.27 不同濃度HSA吸附於Au-MPA-ME之I350/305------------------------- 92
圖4.28 不同濃度HSA吸附於Au-MUA之I350/305----------------------------- 92
圖4.29 不同濃度HSA吸附於Au-MHDA之I350/305--------------------------- 93
圖4.30 不同濃度BAA吸附於Au之I350/305------------------------------------- 93
圖4.31 不同濃度BAA吸附於Au-MAA之I350/305---------------------------- 94
圖4.32 不同濃度BAA吸附於Au-MPA之I350/305----------------------------- 94
圖4.33 不同濃度BAA吸附於Au-MAA-ME之I350/305----------------------- 95
圖4.34 不同濃度BAA吸附於Au-MPA-ME之I350/305------------------------ 95
圖4.35 不同濃度BAA吸附於Au-MPA-ME之I350/305------------------------ 96
圖4.36 不同濃度BAA吸附於Au-MPA-ME之I350/305------------------------ 96
圖4.37 HSA吸附後之螢光光譜------------------------------------------------------ 97
圖4.38 BAA吸附後之螢光光譜------------------------------------------------------ 97
圖4.39 BAA吸附後之比活性--------------------------------------------------------- 98

表目錄

表4.1 利用粒徑分析儀分析自行合成奈米金粒子之粒徑分佈----------------- 57
表4.2 金奈米微粒顆粒大小與莫耳消光係數比較-------------------------------- 62

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