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研究生： 陳毓宏
Chen, Yu-Hung
論文名稱： 新穎的奈米粒子合成方法與在生醫上的 應用：金銀，金鈀，金
A Novel Approach to synthesize nanoparticles and the applications on the Biomedical Aspect : AuAg，AuPd and Au
指導教授： 葉晨聖
Yeh, chen-sheng
學位類別： 博士
Doctor
系所名稱： 理學院 - 化學系
Department of Chemistry
論文出版年： 2004
畢業學年度： 92
語文別： 中文
論文頁數： 89
中文關鍵詞： 晶片 、基因轉殖 、合金 、奈米粒子 、核酸
外文關鍵詞： nanoparticles, genetransfection, chip, alloy, DNA
相關次數： 點閱：61  下載：8 
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    • 本論文中有三個研究主題，包括(1)合金奈米粒子新的合成方法，(2)利用DNA作用力形成一雙奈米粒子的網狀結構，(3)利用金奈米粒子當作載體，並結合微機電技術，發展一非病毒法的基因轉殖技術。
    首先，第一部份的研究是發展一合成金銀合金奈米粒子的方法。將金和銀奈米粒子溶液混合，並利用532 nm的雷射光照射。在UV圖譜中得到一介於金和銀特徵吸收峰之間的新吸收峰。將不同比例的金銀合金的最大吸收峰對金的莫耳成分比例作圖，可得到一良好的線性關係。不同比例的金銀合金(2/1，1/1，1/2)，我們也利用EDX對組成成分進行分析，結果非常吻合我們所預期的比例。XPS也得到相似的結果。由這些光譜的證據，我們認為所製備出的雙金屬奈米粒子是一個具有原子級均勻混合的合金結構，而非一核-殼結構。
    接著第二部分的研究是分別將金和金銀合金修飾上不同的DNA，並在0.3 M的磷酸緩衝溶液中進行雜交反應。由TEM可以很明顯的看出，較小的金銀合金(~4 nm)圍繞在金奈米粒子(~13 nm)周圍，形成一二維有規則的網狀結構，而在加熱後，這個結構消失了，此行為唯一可逆的過程。從溫度/時間的熱分析實驗，可以得知此結構的Tm為59.6 oC(半高寬為4.5 oC)。另一個雙股的系統(A21和T21)，由TEM可以很明顯的看出，其結構沒有之前三股系統具有的網狀結構，這可能是和DNA在金銀合金上的覆蓋率有關，其Tm為62.5 oC(半高寬為5.9 oC)。
    最後一部份的研究是利用金奈米粒子當作載體，並結合微機電技術，發展一非病毒法的基因轉殖技術。我們將單的DNA分子修飾在金奈米粒子表面，將此溶液加在已培養成骨細胞(MC3T3E)的電穿孔晶片上。利用外加的電壓分別先後執行電遷移和電穿孔。由實驗結果我們發現電遷移的施加會增加金奈米粒子進入細胞中的數量，這與理論計算相吻合。另一方面有修飾DNA的金奈米粒子比沒修飾的粒子更容易進入細胞中。

    In my thesis, there are three studies including (1) a new approach for the formation of alloy nanoparticles, (2) DNA-linked binary nanoparticle networks and (3) a nonviral transfection approach in vitro: the design of Au nanoparticle vector joint with MEMS.
    Initially, in the first part, a novel method is developed to synthesize the Au-Ag alloy nanoparticles. The alloy was prepared by irradiating a mixture of the gold and silver nanoparticle solutions using a 532 nm wavelength. The prepared alloy particles displayed only one plasmon band, rather than two bands for the case of a mixture of gold and silver nanoparticles. A linear relationship was attained from the plot of the surface plasmon bands vs. the gold mole fractions. The various compositions of the Au-Ag alloy, i.e. 2/1, 1/1 and 1/2, were confirmed by EDS analysis. The XPS analysis is agree with the results of EDS. These results indicate that the bimetallic particles consist of a mixture at the atomic level, instead of a core-shell structure. The most important is that this methodology is not unique, but can be universally applied to form other alloy nanoparticles, such as AuPd, AgPd and AuAgPd.
    Next, in the second part, this contribution is to perform noncovalent methods for assembling two kinds of nanoparticles into network materials. Both Au/Ag alloy and Au nanoparticles were modified with the complementary DNAs and hybridized as the assemblies for the first time. It was found that the modified Au/Ag alloy and Au nanoparticles could be stable on the alkaline solutions up to 0.3 M, which was required for the hybridization processes. TEM images revealed the network structures, showing the smaller Au/Ag (~ 4 nm) surrounding the Au particles (~ 13 nm) and form a ordered 2D network structure. After heating, the morphologies dramatically became to be dispersive . From the temperature / time dissociation experiment, the melting temperature was measured at 59.6 oC (FWHM = 4.5 oC). The DNAs used were two strands (12 bases) and another 24 bases strand which is complementary with them. Moreover, the results of the control experiment demonstrated that the noncomplementary DNAs would not build a high ordered structure as observed in the complementary case. Furthermore, the other system (thiolated A21 and T21) also was carried out and Tm was 62.5 oC (FWHM =5.9 oC).
    Finally,in the last part, Au and Au nanoparticles (13 nm) modified with 21-based thiolated-oligonucleotides have been examined as delivery vehicles for the development of the nonviral transfection platform. The electroporation was employed to perform on microchips. Electroporation introduces foreign materials into cells by applying impulses with an electric field to create multiple transient pores on the cell membrane through a dielectric breakdown of the cell membrane. Osteoblasts (MC3T3E cells) were cultured and studied for the transport of the nanoparticles. Based on the characteristic surface plasmon of the Au particles, UV-vis absorption was utilized to qualitatively judge whether the successful delivery occurred or not. TEM images provided the direct observation of the Au nanoparticles before and after taking electroporation function. Atomic absorption measurements gave rise to quantitative analysis. It was found that electroporation joint with electromigration largely enhanced the transportable efficiency as compared with electroporation alone. Most interestingly, Au capped with oligonucleotides led to optimal performance. Oligonucleotides seems to facilitate internalization process. In additions, theoretical analysis was conducted to investigate the electromigrating effect in increasing the local concentrations of the nanoparticles.

    目錄 誌謝……………………………………………………………………… 3 英文摘要………………………………………………………………… 4 中文摘要………………………………………………………………… 5 第一章 序論 9 1.1 論文規劃 9 1.2 奈米的時代 10 1.3 奈米粒子簡介 12 1.4 金屬奈米粒子簡介 15 1.5 影響奈米粒子穩定度的因素 18 1.6金屬奈米粒子的表面電漿共振(SURFACE PLASMON RESONANCE) 25 1.7雙金屬奈米粒子 30 1.8 胞膜電穿孔基因轉殖法簡介 31 1.9 本章相關文獻 33 第二章 一個新穎的合金奈米粒子合成方法 37 2.1 研究動機與目的 38 2.1實驗藥品與儀器 39 2.2.1 藥品 39 2.2.2 儀器 39 2.3 實驗步驟 40 2.3.1 金奈米粒子的製備 40 2.3.2 銀奈米粒子的製備 40 2.4 實驗結果與討論 41 紫外光-可見光譜分析 41 TEM圖分析 44 能量散佈光譜（EDX）分析 46 X光粉末繞射（XRD）和電子繞射光譜（ED）分析 46 X光光電子光譜（XPS）分析 47 金銀合金生成機制討論 48 金鈀及銀鈀合金奈米粒子的合成 51 2.5 結論 52 2.6 本章相關文獻 53 第三章 利用DNA形成一雙成分奈米粒子的網狀結構 54 3.1研究動機與目的 55 3.2實驗藥品與儀器 56 3.2.1 藥品 56 3.2.2 儀器 56 3.3實驗步驟 58 3.3.1 金奈米粒子的製備 58 3.3.2 銀奈米粒子的製備 58 3.3.3金銀合金奈米粒子製備 58 3.3.4製備5’端修飾硫醇的DNA（P1）與金奈米粒子形成的複合物（CONJUGATES） 58 3.3.5製備3’端修飾硫醇的DNA（P2）與金銀奈米粒子形成的複合物 59 3.3.6製備5’端修飾硫醇的DNA（T21）金奈米粒子形成的複合物 59 3.3.7製備3’端修飾硫醇的DNA（A21）與金銀奈米粒子形成的複合物 59 3.3.8雜交反應（HYBRIDIZATION） 59 3.4 實驗結果與討論 60 紫外光-可見光譜分析 60 TEM圖分析 61 金-金銀合金奈米粒子複合物的熱行為分析 64 雙股DNA系統 64 3.5 結論 69 3.6 本章相關文獻 70 第四章 以金奈米粒子當作載體，結合微機電技術在晶片上進行胞膜電穿孔和電遷移行為，進行DNA轉殖的研究 71 4.1研究動機與目的 72 4.2實驗藥品與儀器 74 4.2.1 藥品 74 4.2.2 儀器 74 4.3實驗步驟 75 4.3.1電穿孔微型晶片的設計和製作 75 4.3.2 金奈米粒子的製備 76 4.3.3製備5’端修飾硫醇的DNA(T21)與金奈米粒子形成的複合物 76 4.3.4細胞培養 76 4.3.5電遷移（ELECTROMIGRATION）和電穿孔（ELECTROPORATION） 操作模式 77 4.4 實驗結果與討論 79 紫外光-可見光譜分析 79 TEM圖分析 81 原子吸收光譜（AA）分析 82 理論分析 83 4.5 結論 87 4.6 本章相關文獻 88

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    22 我們要感謝Prof. Alivisatos 提供10 nm金奈米粒子及金-DNA的電泳移動率。我們的計算是利用10 nm金奈米粒子，其電泳移動率數值獲得自文獻，W. J. Parak, T. Pellegrino, C. M. Micheel, D. Gerion, S. C. Williams, and A. P. Alivisatos, Nano Lett. 2003, 3, 33.

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