開發奈米材料-生物分子複合體及其在生物系統的應用

Abstract

近年來，奈米材料-生物分子的複合體應用在DNA偵測和RNA干擾是持續被發展的課題。在本研究中，我們發展了新的技術---利用二氧化矽螢光奈米管簡單並靈敏地偵測DNA。嵌進量子點的二氧化矽奈米管是以陽極氧化鋁為模板，藉著溶膠-凝膠法來製造。接著，將不同顏色的二氧化矽螢光奈米管固定上單股的DNA，把它當成奈米探針，在溶液中偵測具有螢光標示的目標DNA。我們利用光激螢光光譜，共軛焦顯微鏡和穿透式電子顯微鏡來檢驗二氧化矽螢光奈米探針的光學和結構特性。在普通顯微鏡下，經由肉眼可以很明顯地觀察到二氧化矽螢光奈米探針在成功偵測到目標DNA後的顏色改變。定量分析顯示，單根二氧化矽螢光奈米探針內只要有100 attomole (10-18摩爾) 的目標DNA，就可以產生可辨視，可觀察到的顏色變化。這些偵測的結果也證明我們的偵測試驗展現了高特異性，高選擇性和非常低的非特定吸附。我們以二氧化矽螢光奈米探針設計出的DNA偵測試驗被預期在快速的DNA掃描和偵測方面的應用是相當有用的。 更進一步地，我們藉著金奈米材料-短片段核糖核酸複合體的調控，來研究以短片段核糖核酸所造成的基因沉默現象的動力學。和動力學相關的因子---複合體的濃度，複合體的服藥次數和細胞繁殖雙倍的時間---分別被研究。這三個因子造成增強綠色螢光蛋白沉默50 %或更少百分比的持續時間也被研究。我們發現沉默持續時間和複合體的濃度呈現自然對數的關係。但是對於其他兩個因子---複合體的服藥次數和細胞繁殖雙倍的時間---則和沉默持續時間呈現線性的關係。根據這些關係，經由短片段核糖核酸調控的，預期的基因表現程度可以被設計出來。對於研究和治療的需求來說，延長的沉默持續時間可以透過改變複合體的濃度和(或)服藥次數來達成。除此之外，選擇模式細胞 (和細胞繁殖雙倍的時間有關)對於延長沉默持續時間是有幫助的。最重要的是，在核糖核酸干擾的應用方面，這些關係式可以大大地減少反覆嘗試錯誤的機會。我們的研究在核糖核酸干擾的治療應用方面具備很高的潛力。

The applications of nanomaterials-biomolecular conjugates in DNA detection and RNA interference (RNAi) have led to ever-growing developments in the past decades. In this work, we have developed a new technique used fluorescent silica nanotubes for simple and sensitive DNA detection. The quantum dots embedded silica nanotubes (QD-SNTs) were fabricated by a sol-gel reaction using anodic aluminum oxide (AAO) as a template. The fluorescent QD-SNTs of different colors were then immobilized with single stranded DNA and used as nanoprobes to detect dye-labeled target DNA in a solution phase. The optical and structural properties of QD-SNTs nanoprobes were examined using photoluminescence spectroscopy, confocal microscopy and transmission electron microscopy (TEM). The obvious color change of the QD-SNTs nanoprobes was observed by eyes under a simple microscope after the successful detection with target DNA. The quantitative analyses indicated that ~100 attomole of target DNA in one nanoprobe can generate the distinguishable and observable color change. The detection results also demonstrated that our assay exhibited high specificity, high selectivity and very low non-specific adsorption. Our simple DNA assay based on QD-SNTs nanoprobes is expected to be quite useful for the needs of fast DNA screening and detecting applications. Furthermore, we study the kinetics of siRNA-based gene-silencing by gold nanoparticles-siRNA conjugates mediation. The kinetics factors relative to the concentrations of conjugates, dosing times of conjugates and cell doubling time were studied separately. The resulting duration time of silence (TE, a period of time that EGFP expression level was regulated down to 50 % or less) by three variables mentioned above were also investigated. We found that TE showed natural logarithm relationship with the concentrations of conjugates. But for other two variables, dosing times of conjugates and cell doubling time, TE showed linear relationship with both of them. Based on these relationships, an expected siRNA-mediated gene expression level can be designed. For the need of researches and treatments, prolonged TE can be achieved by varying concentrations and/or dosing times of conjugates. Besides, choosing of model cell (relative to cell doubling time) may be helpful for the purpose of prolonged TE. Importantly, according to these relationships, the possibility of trial and error in RNAi based applications can be much reduced. Our study holds great potential for RNAi-based therapeutic applications.