酵素固定化於中孔洞二氧化矽奈米材料:限制空間對於酵素之位向、結構與催化活性之影響

Enzyme Immobilized in Mesoporous Silica Nanoparticles: Effect of Confinement on Orientation, Conformation, and Catalytic Activity

高琨哲(Kun-Che Kao)

指導教授 ： 牟中原

國立臺灣大學/理學院/化學研究所/博士(2013年)

https://doi.org/10.6342/NTU.2013.11119

摘要

本論文主要發展了擴孔型中孔洞二氧化矽奈米材料，包含直徑在100 nm左右的中孔洞二氧化矽奈米粒子(mesoporous silica nanoparticles (MSNs))，和中孔洞二氧化矽薄膜(mesoporous silica thin films (MSTFs))。主要的概念是以陽離子界面活性劑(cationic surfactant: CTAB)當作孔道模板，加入有機矽烷(TEOS)，在鹼性的環境下水解縮合，自組裝(self-assembly)形成中孔洞二氧化矽材料。過去文獻中的合成方法，孔洞直徑往往侷限於2 ~ 3 奈米，我們透過添加正癸烷(n-decane)和乙醇當作助溶劑(co-solvent)形成油/水乳液系統(oil-in-water emulsion)，能夠順利將正癸烷引入微胞(micelle)中，透過微調正癸烷的添加量，能夠輕易合成孔洞直徑3 ~ 7奈米的中孔洞二氧化矽奈米材料，這些材料同時具有良好的週期性結構、孔道通透性、高表面積與高孔體積。主要實驗分為兩部份:
(一) 近年來奈米材料大量應用於生物醫學上，奈米材料與生物分子間的物理化學作用漸漸受到重視。我們進而想探討生物分子如酵素(enzyme)在限制空間中的位向選擇性、結構變化、和催化活性的影響。我們藉由中孔洞材料表面修飾不同的有機官能基，能將細胞色素c (cytochrome c)以不同的位向共價鍵結於孔道中，實驗的結果顯示，不同的位向決定了細胞色素c的活性中心位置，進而影響了催化活性與水熱穩定性。另外，我們將另一酵素溶菌酶(lysozyme)以靜電吸附力吸附到不同孔徑大小的材料中，目的是想探討酵素分子在孔道中的摺疊情況，由於中孔洞二氧化矽奈米粒子在水溶液中具有良好的懸浮性，我們能直接透過圓二色分光儀(circular dichroism spectroscopy)來觀察酵素分子的二級結構變化，實驗結果發現，酵素分子若是完全進入到孔道之中，水熱穩定性能夠提升至少20oC以上。這部份的研究也讓我們知道生物分子和中孔洞材料間的作用十分重要，透過良好的位向設計、吸附方法，對於將來應用於奈米醫學的酵素釋放治療(enzyme delivery therapy)，或是工業界上的催化反應有很好的幫助。
(二) 第二部份是中孔洞二氧化矽薄膜，其最大的應用價值在於它具有垂直於基板的奈米孔道，並且具有良好的週期性排列，此薄膜式的材料最被看好應用於成長光電半導體導線的模板。以往文獻中的合成方法是以旋轉塗佈法(spin coating)製程薄膜，孔道的排列容易平行於基板。我們的合成方法是直接將基材浸漬於合成溶液中，得到的垂直性中孔洞薄膜結構良好且大面積。因此，我們探討了此材料的生長機制，主要利用國家同步輻射的低掠X光小角度散射(grazing incidence small angle X-ray scattering)來分析此薄膜材料，我們發現能夠長出垂直性的孔道，主要原因是因為孔液系統中的正癸烷和乙醇助溶劑的影響，它們吸附於基材上，改變了微胞在基材上的排列，並且在有機矽烷縮合時，限制了孔道的成長方向，最終得到垂直性的孔道薄膜。我們後續也期望能將此垂直性的孔道薄膜當作模板，應用於成長垂直於基板的一維奈米材料。

關鍵字

中孔洞二氧化矽 ； 擴孔 ； 垂直孔道 ； 限制空間 ； 位向選擇 ； 酵素催化

並列摘要

In this dissertation, there are two topics mainly including the syntheses of pore-expanded mesoporous silica nanomaterials and the fundamental study of enzyme immobilized in mesoporous silica nanoparticles. In chapter 3, for the purposes of fast mass transport and high diffusion rate to immobilize large bio-molecules, mesoporous silica nanoparticles (MSNs) with particle size below 100 nm and pore size large than 6.0 nm were synthesized. We used alkanes as pore-expanded reagents to synthesize pore-expanded MSNs and several synthetic factors were systematically studied. Eventually, we can obtain pore-expanded MSNs with a pore size up to 6.0 nm and simultaneously maintained well-ordered mesostructures. In addition, in the same synthetic solution of pore expanded MSNs, mesoporous silica thin films (MSTFs) with vertical nanochannels could be synthesized on silicon wafers. MSTFs not only had well-ordered pore structures with pore size up to 5 – 6 nm, but the nanochannels were perpendicular to the silicon substrates. The synthetic mechanisms of vertical MSTF were investigated. Alkane molecules in the synthetic solution played an important role to construct the vertical nanochannels of MSTFs. Without the addition of alkane, the nanochannels of MSTFs were randomly oriented. Furthermore, concentration of ammonia solution could control the film thickness based on a nucleation and growth mechanism. Through a series of investigation, we finally could fabricate a MSTF with large scale and excellent mesostructure.
In chapter 4, pore-expanded MSNs were used to adsorb lysozyme for studying its structural stability in comparison to that on Stӧber silica nanoparticle (SSN). To really study the structure of lysozyme in a confined space, a well-designed desorption strategy were performed. We found that the secondary structures of lysozyme confined in the nanochannels of MSNs were unchanged and the thermal stability was significantly improved. The MSN-lysozyme nano-composites can be reused at least five times. In chapter 5, to study the orientation effect of enzyme inside the nanochannels of mesoporous silica, we modified silica surfaces with different functional groups to attach the model enzyme (cytochrome c) with different binding sites through covalent bonds. From molecular modeling, enzyme showed different orientations in the nanochannels and finally resulted in different enzyme stability and catalytic activity.

並列關鍵字

Mesoporous silica ； Pore-expansion ； Vertical channels ； Confined space ； Specific orientation ； Enzyme catalysis

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