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    Title: 利用毛細管電泳偵測單點突變DNA和分析不同方式合成的金奈米粒子
    Other Titles: Using capillary electrophoresis for single point mutation DNA analysis and characterization of gold nanopaticles synthesized by different methods.
    Authors: 楊承熹;Yang, Cheng-his
    Contributors: 淡江大學化學學系博士班
    吳俊弘;Wu, Chunhung
    Keywords: 突變DNA;微胞膠體;金奈米粒子;表面修飾劑;mutation DNA;micellar gel;gold nanoparticle;modification reagent
    Date: 2007
    Issue Date: 2010-01-11 02:50:12 (UTC+8)
    Abstract: 本論文主要在利用毛細管電泳技術開發便捷的分析方法,應用於突變DNA的偵測,以及合成金奈米粒子的的分析。

    首先我們發現三嵌段共聚合物F127,即EO99PO69EO99(其中EO和PO分別為環氧乙烷和環氧丙烷),所形成的微胞膠狀結構,可經由添加直鏈高分聚葡萄糖(Dextran),來調節其孔洞大小及分布情形,作為毛細管電泳分離介質,以達到高解析度之DNA分離。此外,在尋找最佳分離條件時,我們研究分別添加不同正二價離子於分離介質以及變性DNA的重新黏合(reannealing)反應中對於DNA分離解析的效應,並探討電泳分離溫度,毛細管偵測長度,以及偵測方式對於分離正常型和突變DNA的影響。根據實驗結果,在分離介質組成為30% F127 + 0.1% Dextran2M + 50µM Co2+離子,分離溫度為20℃,分離有效距離為20cm,電場強度為600V/cm時,可將人類乳突狀病毒(Human papilloma virus)第十六型( HPV-16)中表現E6蛋白質的一段DNA序列,以及其單點突變(single point mutation,亦即正常型DNA的第12號位置由GC改變成AT)樣品分離。若是基因DNA所發生的單點突變使得E6蛋白質的第14個胺基酸由原本的Glutamine變異成Histidine,則將會引發較為嚴重的p53蛋白質降解,在臨床的表現則會使得致癌的機率升高。利用前述的分離條件也可將這兩個DNA樣品分離,若以雷射激發螢光偵測方法(LIF),並加長偵測長度為30cm,提高施加電場強度為700V/cm,則可達到更佳的解析效果。

    另外,我們也發現,利用毛細管電泳技術分離經中性界面活性劑修飾的金奈米粒子,可簡便、快速地分析具不同表面性質的金奈米粒子之顆粒大小以及分布情形。由不同方式所合成的奈米粒子表面通常會吸附或鍵結不同性質的穩定劑或氧化還原產物分子,所以在進行奈米粒子的電泳實驗時,除了粒子的顆粒大小之外其表面性質也會影響奈米粒子的電泳行為。因此,我們以一系列界面活性劑修飾具不同表面性質的金奈米粒子希望使其表面性質均一化後,再進行電泳分析,以期從電泳數據,直接獲得奈米粒子的大小和分布情形。根據電泳最佳條件的實驗結果,我們採用10mM Sodium tetraborate與10mM Sodium phosphate並添加200mM Sodium dodecyl sulfate(SDS)作為緩衝溶液(pH 8.8),以經實驗室合成的界面活性劑(hexyl-oligo (p-phenyleneethyny-lene)-poly(ethyleneoxide),Hex-OPE-PEO)修飾過後的金奈米粒子標準品作為樣品進行電泳實驗,將所測得的電泳遷移率與標準品顆粒大小做一校正曲線,可以得到最佳的電泳遷移率-粒徑大小之線性關係(R2>0.99)。以不同方法所合成的金奈米粒子,經此電泳方法分析所得的結果,與經穿透式電子顯微鏡(TEM)測量的結果互相吻合。我們也利用此分析方法分別探討在中性和陰離子界面活性劑的環境中所合成的金奈米粒子的性質。




    在不添加還原劑的情況下,三嵌段共聚物F127(一種中性界面活性劑)可同時扮演還原劑和表面穩定劑的角色,成功合成金奈米粒子。我們在F127的環境中,分段逐次進行金奈米粒子的合成步驟,如此可得到顆粒較小,分布較均勻,且合成再現性較好的金奈米粒子。由於F127的微胞行為會受溫度影響,我們分別在4℃、25℃和95℃進行合成反應,發現溫度改變並不會影響到所合成金奈米粒子的顆粒大小以及分布。當F127的濃度介於5%到13%之間,所合成的金奈米粒子的顆粒大小會隨著F127的濃度增加從大約26nm減小到12nm,而當F127的濃度繼續由13%增加到40%,金奈米粒子的顆粒大小就不會再改變。利用我們的合成方法,只要改變F127的濃度就可以得到不同顆粒大小且分布均勻的金奈米粒子。

    以相同的分段逐次合成法,再添加還原劑,以及具不同碳鏈長度的陰離子界面活性劑 ( CnH2n+1SO4Na, n=8, 10, 12, 14,分別是SOS、SDeS、SDS、STS)的環境中所合成的金奈米粒子的性質,也可經由電泳方法加以分析。在25℃合成的金奈米粒子之電泳吸收峰相當寬,表示此金奈米粒子顆粒大小分布相當廣,而當我們將合成溫度降低到4℃後發現,在5mM和20mM的 SDS環境中所合成的金奈米粒子之顆粒大小都明顯變小,而且分布變得相當均勻。此外我們也在陰離子界面活性劑中添加了飽和的芘(pyrene),由於pyrene 本身會與金奈米粒子作用,而且其本身的疏水性質,可將剛成核的金奈米粒子帶入微胞中進行成長反應,所以在我們的實驗結果中都可以明顯觀察到在含有飽和的pyrene的系統中,即使是在25℃的反應溫度下,也可以非常有效的減小所合成金奈米粒子的顆粒大小,並使得顆粒分布相當均勻,而且此時顆粒大小會隨著界面活性劑的濃度增加而減小。此外所合成的金奈米粒子的顆粒大小也會隨著界面活性劑的碳鏈長度增加而減小,約從6.7nm減小到3.6nm。從實驗結果可以證實,我們所提出的毛細管電泳結合奈米粒子表面修飾方法,確實是一種方便而且有效率的奈米粒子分析工具,可以快速地分析經由不同方法所合成的奈米粒子,使我們可以較有效率地找出許多不同合成奈米粒子方法的最佳條件。
    In this report we demonstrated to find appropriate and convenient separation conditions for fast mutation DNA detection and analysis the nanoparticles synthesis by different ways.
    First, we find the micellar gel structure of triblock copolymer F127, EO99PO69EO99 (EO and PO being ethylene oxide and propylene oxide, respectively.), could be adjusted to modify the pore size and its distribution with the additions of Dextran which could be the separation mediums for high separation resolution of DNA. Besides, we investigated the effect of cation of the separation medium additions and for reannealing of denature DNA. Then we also demonstrated to the separation effect on the separation temperature, detection length and detection mode of wild type and mutation type DNA. In our result we were able to resolve the single point mutation of the DNA sequencing result in variants human papillomavirus type 16 (HPV-16) E6 proteins, when gel composition of 30% F127 + 0.1% Dextran2M containing 50µM Co2+ at 20℃, detection length was 20cm and electric field strength of 600V/cm. This variant made the Glutamin variant to Histidine was induced the degradation of p53 protein in vitro. If we used the LIF detection mode and increase the detection length to 30cm, electric field strength adjust to 700V/cm, we can obtain the optimums separation conditions.
    Besides, we also find the capillary electrophoresis (CE) was used to separate gold nanoparticles (AuNPs) modified with neutral surfactants. This method provided us a simple and fast approach to the analysis of particle size and size distribution of AuNPs having different surface properties. The surface of AuNPs synthesized by different ways usually adsorbed or bonded different stabilizers or product molecules of redox reactions. Therefore, besides the particle size, the surface properties of AuNPs would also influence their electrophoretic behaviors in CE experiments. In order to homogenize the surface properties of differently synthesized AuNPs before proceeding to electrophoretic analysis, we used a series of surfactants to modify the surface properties of these AuNPs. In so doing, we expected to obtain the information regarding particle size and size distribution of AuNPs directly from the electrophoresis results. In this experiment we used 10mM Sodium tetraborate and 10mM Sodium phosphate containing 200mM Sodium dodecyl sulfate (SDS) as electrophoretic run buffers (pH 8.8), and AuNP Standards were treated with the synthesized compound hexyl-oligo(p-phenyleneethyny-lene)-poly (ethyleneoxide) as the sample and separated by CE. The optimum linear relationship (R2 > 0.995) between electrophoretic mobilities and sizes for AuNP standards could be achieved. The results were all in agreements with those obtained from TEM measurements. In this report we demonstrated that CE combined with surface modifying reagent could provide a convenient and efficient method for nanoparticle analysis.
    The Au nanoparticles can be obtained in air-saturated aqueous solutions that contain triblock copolymers F127 but not any other reducing agent, these block copolymers act as both reductants and colloidal stabilizers and prove very efficient in both functions. In F127 aqueous solutions the process of AuNPs synthesis was step by step, then we can obtain the AuNPs of small particle size, narrow distribution and good reproducible. Because the temperature was influence to the micelle behavior of F127, we synthesized the AuNPs at 4℃,25℃and 95℃, respectively, and observed the particle size and distribution of AuNP was not influence by temperature. When the concentration of F127 increase from 5% to 13%, the particle size was decreased from 26nm to 12nm, as the F127 concentration up to 40% the particle size of AuNP was not changed. Use our synthesis method, we can obtain the AuNP of different particle sizes and narrow distribution to different F127 concentration.
    With the same step by step synthesis method, Au nanoparticles (NPs) were synthesized by added reductant in different alkyl length of anionic surfactants (CnH2n+1SO4Na, n=8, 10, 12 and 14, SOS, SDeS, SDS and STS) solutions could also be characterization by capillary electrophoresis. When synthesis temperature at 25℃, a very broad peak of AuNP was observed, it means the particle distribution of AuNP was very widespread but when the synthesis temperature adjust to 4℃, we could find out the particle size decreasing and narrowing in 5mM or 20mM SDS solution. Then the pyrene was added in all anionic surfactant solutions, the chemical reaction between pyrene and gold complexes was attributed to the formation of AuNPs inside the micelles core at the embryonic stage. Further, pyrene has the extraordinary effect in decreasing the size and narrowing the dispersity of AuNPs, besides, the particle size was also decreasing with the increasing concentration of anionic surfactants. Compared with the experimental results in particle size and distribution it seems that particle size smaller and narrower in the more hydrophobic surfactant. In this report we demonstrated that CE combined with surface modifying reagent could provide a convenient and efficient method for nanoparticle analysis.
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