蛋白質於金奈米粒子表面吸附之探討

Investigation of Proteins Adsorption on Colloidal Gold Nanoparticles

林宏聲(Hong-Sheng Lin)

指導教授 ： 阮若屈 ； 劉英麟

中原大學/工學院/化學工程研究所/碩士(2004年)

https://doi.org/10.6840/cycu200400397

摘要

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。

關鍵字

表面改質 ； 蛋白質吸附 ； 金奈米粒子

並列摘要

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.

並列關鍵字

gold nanoparticles ； bioconjugation ； surface modification ； proteins adsorption

參考文獻

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