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研究生:蘇祈彰
研究生(外文):Chi-Chang Su
論文名稱:金與銀奈米粒子於增進聚合酶連鎖反應效能之研究
論文名稱(外文):The Study of Enhancing the Efficiency of Polymerase Chain Reaction with Gold and Silver Nanoparticles
指導教授:林裕城林裕城引用關係
指導教授(外文):Yu-Cheng Lin
學位類別:碩士
校院名稱:國立成功大學
系所名稱:工程科學系碩博士班
學門:工程學門
學類:綜合工程學類
論文種類:學術論文
論文出版年:2006
畢業學年度:95
語文別:中文
論文頁數:98
中文關鍵詞:酵素活性比表面積奈米粒子聚合酶連鎖反應
外文關鍵詞:NanoparticlesSurface Volume rationPCREnzyme activativity
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本論文主要研究重點在於將水相的金奈米粒子與銀奈米粒子添加於聚合酶連鎖反應,藉以探討奈米粒子增加聚合酶連鎖反應中效率的原因與添加方式,實驗發現金奈米粒子能有效縮短聚合酶連鎖反應的時間與提升DNA產物複製效率,而奈米粒子的粒徑、濃度與保護劑將會影響聚合酶連鎖反應的進行。實驗中所採用之10-30 nm金奈米粒子與15 nm銀奈米粒子分別添加於聚合酶配方中,藉由173 bp之核苷酸片段於即時聚合酶連鎖反應系統中加以分析,再將實驗結果利用熔解溫度(melting temperature curve)分析方式與瓊膠電泳(gel electrophoresis)交相驗證金奈米粒子與銀奈米粒子增進聚合酶連鎖反應的效率,並進行定性與定量分析。
實驗結果發現以檸檬酸鈉做為保護劑的金奈米粒子能有效增進聚合酶連鎖反應之效率高達1000倍,並能縮短時間為參考反應時間為1/6倍,以相同濃度做為標準進行不同粒徑的比較,在實驗中發現當金奈米粒子粒徑愈小時,增進聚合酶連鎖反應的效率更佳,奈米粒子的其中一種特性便是具有極高的反應表面積,因此將金原子總體積相同做為基準,調配成不同粒徑的金奈米粒子,此時實驗結果証實當表面積愈高,增進聚合酶連鎖反應的效率更佳;反觀以硼氫化鈉(NaBH4)調配的商用的水相銀奈米粒子其水溶液呈中性(pH7.0-7.5),對聚合酶連鎖反應的效率並無顯著的提升效益,但是卻能有效維持Taq酵素的活性,聚合酶連鎖反應的酵素會因溫度的增加而降低活性,故實驗設計上首先將酵素事先增溫至97℃、加熱5 分鐘,使得原有酵素活性降約為1/2倍,再將銀奈米粒子加入後卻能保持一定的酵素活性,這項實驗結果能應用於提升酵素反應與延長酵素活性上,因此本論文將分別討論金奈米粒子與銀奈米粒子對聚合酶連鎖反應的增進效率的機制與環境控制做一系列的探討,相關成果十分有助於分子生物工程領域的提升。
In recent decade, many researches of nanoparticle applications are published for molecular biology, material engineer, medical diagnostics, etc. The metal nanoparticle, for example, gold and silver, is excellent thermo-transmitting and high surface-volume ratio. The polymerase chain reaction, PCR, is used with Taq DNA polymerase to amplify a specific DNA fragment in 30-50 heating-cooling cycles. Therefore, the efficiency of thermo-transmitting is important condition of PCR efficiency. In this thesis, gold and silver nanoparticles are used to enhance the efficiency of PCR with excellent thermo-transmitting. The real-time PCR is a quickly method to quantify the amount of DNA. This method could analyze easily and quickly the efficiency of PCR by melting temperature curve analysis. Therefore, the real-time PCR is used for estimating efficiency of PCR. In real-time PCR, the DNA template is used with the 173 bp specific green fluorescent protein which is designed from pEGFP-N1 plasmid. After PCR, DNA products are confirmed with agarose gel electrophoresis.


In this study, 13, 20 and 30 nm gold nanoparticles are used with enhancing efficiency of PCR. The efficiency of PCR could be enhancing 1000 fold with gold nanoparticle than without it. Besides enhancing efficiency, the PCR reaction time could be shortened to 1/6 standard reaction time. Comparison of the same concentration of 13-30 nm gold nanoparticles, the 30 nm gold nanoparticle has the best efficiency for enhancing PCR efficiency. The high surface-volume ratio of nanoparticle is very important condition for efficiency of thermo-transmitting. The large surface-volume ratio has excellent thermo-transmitting in nanoparticles. Therefore, comparing with the same volume of 13-30 nm gold nanoparticle, 13 nm gold nanopraticle is the largest surface-volume ratio and is the best effect of enhancing PCR.

The 15 nm silver nanoparticle is similar condition with 13 nm gold nanoparticle. Unfortunately, the silver nanoparticle is very poor effect of enhancing PCR. When Taq DNA polymerase is heating 97.5℃ for 5-6 minutes to destroy activation of enzyme, the PCR efficiency is down to 1/2 original efficiency. In this condition, the efficiency of PCR is maintained at original efficiency by adding 15 nm silver nanoparticle. Summary, the gold nanoparticles could enhance efficiency of PCR and silver nanoparticle could maintain activation of enzyme in PCR. These metal nanopartilces applications would promote researches in biomedical diagnostics and molecular biology.
摘要 I
ABSTRACT III
致謝 VI
圖目錄 X
表目錄 XIV
第一章 緒論 1
1.1. 前言 1
1.2. 文獻回顧 2
1.3. 研究架構 6
第二章 金屬奈米粒子物理性質、合成及應用 7
2.1. 奈米粒子定義及物理特性 7
2.1.1. 奈米粒子之尺寸效應 9
2.1.2. 奈米粒子之表面積效應 10
2.1.3. 奈米粒子之量子效應 12
2.2. 金屬奈米粒子製備方法 13
2.3. 金與銀奈米粒子合成技術 15
2.3.1. 金奈米粒子合成 15
2.3.2. 銀奈米粒子合成 17
2.4. 金屬奈米粒子合成分析 18
2.4.1. 奈米粒子之紫外-可見光吸收光譜分析 19
2.4.2. 穿透式電子顯微鏡影像分析 22
2.5. 聚合酶連鎖反應發展與原理 24
2.5.1. 聚合酶連鎖反應簡介 24
2.5.2. 聚合酶連鎖反應原理 25
2.5.3. 即時聚合酶連鎖反應系統簡介 30
第三章 奈米粒子應用於聚合酶連鎖反應介紹與分析方法 33
3.1. 聚合酶連鎖反應與核苷酸片段準備 33
3. 2. 聚合酶即時偵測系統分析模式 34
3.3. 瓊膠電泳與熔解曲線分析聚合酶連鎖反應 36
3.4. 奈米粒子對聚合酶連鎖反應的影響 39
3.4.1. 金奈米粒子對聚合酶連鎖反應的影響 40
3.4.2. 銀奈米粒子對聚合酶連鎖反應的影響 42
第四章 奈米粒子增加聚合酶連鎖反應效率研究 44
4.1. 金奈米粒子對聚合酶連鎖反應效率影響 44
4.1.1. 檸檬酸鈉對聚合酶連鎖反應的影響 44
4.1.4. 金奈米粒子體積表面積比對聚合酶連鎖反應的影響 53
4.1.5. 金奈米粒子粒徑對聚合酶連鎖反應的影響 58
4.1.6. 金奈米粒子對縮短聚合酶連鎖反應時間之影響 62
4.2. 銀奈米粒子對聚合酶連鎖反應效率影響 67
4.2.1. 銀奈米粒子濃度對聚合酶連鎖反應之影響 67
4.2.2. 銀奈米粒子溶劑成份對聚合酶連鎖反應之影響 69
4.2.3. 銀奈米粒子於聚合酶連鎖反應之變化 72
4.2.4. 銀奈米粒子對聚合酶連鎖反應效率之效果 74
4.2.5. 銀奈米粒子對DNA聚合酶的影響 77
第五章 結論與建議 87
5.1. 結論 87
5.2. 建議 89
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