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Title: | 研究基材表面粗糙度對DNA在脂雙層上擴散行為之影響 Research on the influence of the surface roughness induced potential well to DNA diffusion on supported lipid bilayers |
Authors: | Chieh-Jung Lee 李杰容 |
Advisor: | 謝之真(Chih-Chen Hsieh) |
Keyword: | 脂雙層,DNA,擴散行為,表面曲率,位能井, Lipid bilayers,DNA,Diffusion behavior,Surface curvature,Potential well, |
Publication Year : | 2020 |
Degree: | 碩士 |
Abstract: | 先前的研究發現當DNA吸附於於脂雙層上時,會在短延遲時間下出現次擴散現象,且會隨著脂雙層正電荷濃度增加或溶液離子強度下降而更為顯著,雖然研究結果指向DNA在脂雙層上出現次擴散現象與靜電作用力有關,然而目前尚未有相對應的研究以更進一步了解它的成因。而在另一個研究中,我們發現當帶負電的DNA吸附在帶正電的脂雙層上時,DNA會自發性聚集於表面具有正曲率之處,這是因為DNA在正曲率之處能感應到的正電荷脂質分子較多,導致靜電位能較低的關係,也可將該處視為DNA之靜電位能井。我們推測DNA的次擴散現象也是由於靜電位能井的存在所造成,也就是和基材表面曲率分布有關,具有高度正曲率之處對DNA有強大的吸引力,造成DNA不容易脫離該處,形成次擴散現象。 為了驗證上述假設,我們改變基材的表面曲率,並觀察DNA在脂雙層上的型態及擴散行為之變化。在第一階段的研究中,我們先以具有不同天然粗糙度的雲母和硼玻璃做為基材,發現雖然DNA在粗糙度較低的雲母上仍然出現了次擴散現象,但已經比在較粗糙的玻璃表面減輕許多。而當脂雙層正電荷濃度高時,DNA在粗糙度較大的玻璃上無法展開並蜷曲成一團,然而在粗糙度較小的雲母上卻可以自由地展開。因為表面粗糙度與曲率為高度正相關,我們可以初步確認基材表面曲率會影響吸附於脂雙層上DNA之擴散行為及型態。 為了更進一步了解基材表面曲率對於DNA的影響,並排除基材本身材質之變因,在第二階段的研究中,我們利用乾蝕刻改變玻璃表面粗糙度及曲率,並觀察DNA的型態及行為變化。我們發現當乾蝕刻秒數增加時,玻璃表面粗糙度及平均正曲率皆上升。當脂雙層正電荷濃度低時,DNA在粗糙度較小的玻璃上可以自由地展開。然而當粗糙度開始增加時,DNA會開始蜷縮,使纏繞半徑下降,最終蜷縮成一團,且與其在三維溶液中的纏繞半徑相同,表示DNA吸附在脂雙層後仍維持在溶液中的大小,推測是因為表面具有很深的靜電位能井,使DNA吸附後被位能井所侷限,而無法展開。在短延遲時間下,DNA在粗糙度越大的玻璃上也具有更顯著的次擴散效應。此外,我們也比較脂質分子與DNA擴散速率隨基材表面粗糙度的變化。我們發現脂質分子及DNA的擴散速率皆隨基材表面粗糙度增加而下降,但DNA受到的影響遠比脂質分子顯著。我們認為這是因為DNA的帶電量遠高於脂質分子,所以受靜電位能井影響較小。 此外,我們也在高粗糙度的玻璃上觀察溶液離子強度對DNA行為的影響。當離子強度增加時,DNA會由原本蜷縮成一團的型態逐漸展開,且次擴散現象也隨之減弱,這是因為離子強度增高時,削弱了DNA與帶正電脂雙層之間的吸引力,所以由表面正曲率造成的位能井也因此減弱,DNA之間的鏈段排斥力便可以克服位能井的侷限,回復到正常展開的型態。而次擴散現象的減弱,也支持是靜電位能井而不是表面粗糙度所形成的物理性障礙造成DNA的次擴散。 最後,我們透過濕蝕刻改變玻璃的粗糙度及曲率。濕蝕刻及乾蝕刻兩種蝕刻原理不同,前者為等向性蝕刻後者則為非等向性蝕刻,故於玻璃上會產生兩種情況供我們探討:粗糙度相似但曲率不同,以及粗糙度不同但曲率相似的情形。我們比較粗糙度相似的玻璃,DNA在曲率較大的玻璃上,其次擴散效應較明顯,且擴散速率也較小;而在粗糙度不同但曲率相似的玻璃上,DNA則具有相近的擴散行為及擴散速率,此結果似乎也說明表面曲率是更直接影響DNA在脂雙層上的擴散行為。 Previous studies have reported that DNA adsorbed on a lipid bilayer shows a sub-diffusion behavior at short delay time. The phenomenon becomes more significant with increasing cationic lipid concentration and decreasing solution ionic strength. Although the previous results suggest the origin of DNA sub-diffusion must relate to the electrostatic interaction between DNA and lipids, the exact mechanism is still not clear. On the other hand, a related phenomenon had been studied in our lab is that DNA adsorbed on cationic lipid bilayer spontaneously accumulate at the place with positive curvature. It was found that DNA at the place with positive curvature can interact with more cationic lipids and therefore has lower electrostatic energy, resulting in an electrostatic potential well for DNA. We speculate that DNA sub-diffusion is also caused by these electrostatic potential wells due to the curvature of the substrate. In order to confirm our hypothesis, we examined how the local curvature of the substrate affects the DNA conformation as well as its diffusive behavior. In the first stage of the study, mica with less rough surface and borosilicate glass with rougher surface were used as the substrates. We found the sub-diffusion phenomenon is less significant on mica than on glass. When the concentration of the cationic lipid is high, DNA was found collapsed on the glass surface, but it can unfold freely on the mica. Because the surface roughness is highly correlated with the local curvature of the substrate, we preliminarily confirmed our hypothesis about the origin of DNA sub-diffusion. In the second stage of the study, we prepared glass substrates with various roughness and observed the diffusive behavior of DNA on the lipid bilayer set on the glass. The goal of this stage is to exclude the influence of the material properties of substrate and conduct a quantitative investigation on the influence of the substrate roughness to the adsorbed DNA. The experimental results were in agreement with those obtained in the first stage. DNA sub-diffusion becomes more server and DNA diffusivity also reduces significantly with the increasing surface roughness. DNA adsorbed on low roughness surface can unfold freely while DNA on high roughness surface eventually collapses to the size the same as that in solution. The collapse of DNA is presumably because the high surface roughness results in very deep electrostatic potential wells which trap DNA segments upon the moment of adsorption. Besides, we also measured the diffusivity of lipids and found that it also decreases with increasing surface roughness, although much less affected than DNA. The motion of lipid molecules is less affected by the surface roughness is because they carry many fewer charges than DNA. In addition, we examined the influence of the ionic strength to DNA adsorbed on high roughness glass substrates. At low ionic strength, DNA collapsed on the bilayer. As the ionic strength increases, DNA gradually unfolds from the collapsed conformation, and the sub-diffusion phenomenon is also weakened. This is because when the ionic strength increases, the attraction between the DNA and the positively charged lipid bilayer is weakened, so the potential well caused by the positive surface curvature is also weakened, and the repulsive force between the DNA segments can overcome the potential. The weakening of the sub-diffusion phenomenon also supports that the sub-diffusion of DNA is not caused by the physical barriers but by the electrostatic potential well. Finally, we changed the curvature and roughness of the glass through two etching methods. Wet etching is isotropic etching and dry etching is anisotropic etching. Due to different etching principles, two situations will occur on the glass: similar roughness but different curvature, and different roughness but similar curvature. We compare glasses with similar roughness but different curvature. The sub-diffusion phenomenon of DNA on glass with larger curvature is more obvious. While on glasses with different roughness but similar curvature, DNA has similar diffusion behavior. This result seems to indicate that the surface curvature more directly affects the diffusion behavior of DNA on the lipid bilayer. |
URI: | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/49665 |
DOI: | 10.6342/NTU202003048 |
Fulltext Rights: | 有償授權 |
Appears in Collections: | 化學工程學系 |
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