Please use this identifier to cite or link to this item: http://hdl.handle.net/11455/36106
標題: 利用未修飾的奈米金顆粒之靜電力作用以呈色分析偵測病毒核酸之研究
Colorimetric detection of viral RNA based on electrostatic interactions with unmodified gold nanoparticles
作者: 賴奕丞
Lai, Yi-Chen
關鍵字: gold nanoparticles;奈米金粒子;Bamboo mosaic virus;竹嵌紋病毒
出版社: 生物科技學研究所
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摘要: 
由前人研究指出,奈米金溶液對於單股和雙股核酸的吸附程度有所不同;單股核酸會因為本身較具彈性而容易利用其鹼基(base)端吸附到金粒子上,雙股核酸則因其周圍帶負電而產生斥力無法吸附於金粒子。本研究是利用檸檬酸鈉當作還原劑,將四氯金酸還原成奈米金粒子,使其粒徑達到12至15奈米而形成紅色膠狀金溶液。藉由金粒子聚集會使顏色改變之原理,建立一套快速分析單股病毒RNA的雜配系統。本實驗是以竹嵌紋病毒衛星核酸(Bamboo mosaic virus satellite RNA)當作檢測之目標物,首先分別利用構築於載體pUC119以及pGEM-4上的質體pBSF4及pBSHE,經由限制酶酵素切割和RNA聚合酶之作用合成竹嵌紋衛星核酸病毒RNA的負股及正股,當作探針以及標的之RNA序列。將純化之RNA經由混合後加入製備之紅色金奈米溶液中,利用鹽類作為呈色劑,使溶液中之金奈米顆粒產生聚集,顆粒粒徑變大,顏色產生改變而能被肉眼所辨識。若混合後的RNA皆以單股方式呈現,由於單股核酸吸附在金顆粒表面形成保護作用,使得在鹽類作用下不會聚集,則溶液會維持紅色;若形成互補型態之雙股RNA,由於雙股無法吸附金顆粒表面,使得在鹽類之作用下造成聚集,則溶液會由紅色轉為灰藍色。其結果可來作為樣品是否含有互補核酸序列之依據。由實驗結果發現,鹽類濃度對於此套系統之呈色結果會有所影響,但此系統並不會受到溫度改變之影響。另外,雜配後所形成之雙股核酸若有部分為裸露之單股核酸,會對於呈色結果造成影響。經由測試結果發現,此系統之靈敏度可以達picomoles。此系統最大之優勢,在於不需透過儀器就能得到實驗結果,實驗分析所需時間在10分鐘內,因而可以避免RNA在室溫下被分解的問題。

The fact that single- and double- stranded oligonucleotides with different propensities to adsorb on gold nanoparticles in colloidal solution has been reported previously. Repulsion between the charged phosphate backbone of dsDNA and the adsorbed citrate ions dominates the electrostatic interaction between the gold and dsDNA so that dsDNA will not adsorb. Because the ssDNA is sufficiently flexible to partially uncoil its bases, they can be exposed to the gold nanoparticles. This study used a colloidal solution of gold nanoparticles around 12~15 nm in diameter synthesized by citrate reduction of HAuCl4. We have set up a quick hybridization assay based on color changes associated with gold aggregation. The satellite RNA of Bamboo mosaic virus(satBaMV)was need in this experiment. The plasmid pBSF4 and pBSHE were digested by restriction enzymes to synthesize positive- and negative-strand satBaMV RNA using RNA polymerase. Unlabeled target RNA and probes sequence were mixed prior to exposure to the gold nanoparticles to enable efficient hybridization. After the trial hybridization, the trial solution was mixed with gold colloid and immediately followed by addition of salt solution. With enough ssRNA, the colloid retains a pink color, whereas hybridization of the trial solution to form dsRNA leads to a bluish colloid. This assay system was used complementary single stranded RNA based on a color change of colloid gold with sensitivity up to a few picomoles scale even without instruments. This assay system can be effected by salt concentration and by the probe length. The time for whole assay requires less than 10 min.
URI: http://hdl.handle.net/11455/36106
其他識別: U0005-1107200616063300
Appears in Collections:生物科技學研究所

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