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研究生:吳冠毅
研究生(外文):Kuan-yi Wu
論文名稱:金奈米粒子親水化及與DNA一對一鍵結之探討
論文名稱(外文):Study of One to One Linkage between Water Soluble Gold Nanoparticle and DNA
指導教授:阮若屈
學位類別:碩士
校院名稱:國立中央大學
系所名稱:化學工程與材料工程研究所
學門:工程學門
學類:化學工程學類
論文種類:學術論文
論文出版年:2007
畢業學年度:95
語文別:中文
論文頁數:96
中文關鍵詞:金奈米粒子親水化一對一鍵結
外文關鍵詞:DNAgold nanoparticle
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本研究主要的目的是合成一種能夠穩定的懸浮於高鹽高溫的水溶液中並且尺寸分布均一的金奈米粒子,然後利用這種水相金奈米粒子發展出一個可和DNA進行之ㄧ對一接合的方法。我們使用還原劑硼氫化鈉(NaBH4)將氯化金(HAuCl4)在甲苯中還原成粒徑7nm的金奈米粒子,再藉由兩性高分子Poly(maleic anhydride alt-1-tetradecene)修飾金奈米粒子表面使其親水化。結果顯示改質後的回收率可達到85.3%,另外在鹽穩定性方面在NaCl濃度低於200mM穩定度都可達到95%以上,即使在500mM的NaCl下也都有很好的穩定度。熱穩定性的部分,當溫度升高至80℃也不會產生聚集而沉澱。
在進行一對一接合反應時,先利用胺基修飾的DNA能經由活化劑(EDC)活化和羧基修飾的矽膠體(silica gel)產生鍵結,再加入互補DNA進行雜合反應(Hybridization),隨後將親水改質後的金奈米粒子鍵結至互補DNA末端,最後靠著去雜合反應(Dehybridization),即可獲得金奈米粒子與DNA之一對一接合。
透過毛細電泳(Capillary Electrophoresis)與穿透式電子顯微鏡(TEM)等儀器鑑定合成反應所得到的產物,藉此判斷此方法合成的一對一接合之成功性。
The main purposes of this study are to synthesize gold nanoparticles, to modify the surface of the gold nanoparticles from organic to aqueous solution by Poly(maleic anhydride alt-1-tetradecene)(PMATD), and to develop a method to conjugate one ssDNA onto this water soluble gold nanoparticles.
In the synthesis, HAuCl4 was reduced by NaBH4 to form 7nm Au nanoparticle. Then using PMATD to modify Au nanoparticle, we found that the recovery of modification is 85.3%. The salt endurability of water soluble Au nanoparticles can reach 95% when the NaCl concentration is below 200mM. Even if in the 500mM NaCl, the salt endurability can keep 87%. The Au nanoparticles won’t aggregate when the temperature increase to 80℃
In one-to-one synthesis, the COOH-silica gel can be activated by N-(3-Dimethylaminopropyl)-N-ethylcarbodiimide(EDC) and then conjugated with NH2-DNA (+). After conjugation, we added complementary NH2-DNA (-) to hybrid the ssDNA which onto the silica gel. Using EDC to activate the water soluble Au nanoparticles to conjugate with NH2-DNA (-). The last step, we adjusted the temperature to dehybride the dsDNA. We can get Au-DNA one-to-one conjugation.
Detail the production by capillary electrophoresis(CE) and transmission electron microscopy(TEM) to confirm the Au-DNA structure.
目錄
摘要 -----------------------------------------------------------------------------------------------------------I
Abstract -------------------------------------------------------------------------------------------------------II
致謝------------------------------------------------------------------------------------------------------------III
目錄 -----------------------------------------------------------------------------------------------------------IV
圖目錄 --------------------------------------------------------------------------------------------------------VII
表目錄 ---------------------------------------------------------------------------------------------------------IX
第一章 緒論 --------------------------------------------------------------------------------------------------------1
1.1研究動機 --------------------------------------------------------------------------------------------------------1
1.2研究目的 --------------------------------------------------------------------------------------------------------5
第二章 文獻回顧 --------------------------------------------------------------------------------------------------6
2.1奈米材料簡介 --------------------------------------------------------------------------------------------------6
2.2 金奈米粒子的特性 -------------------------------------------------------------------------------------------8
2.3 金奈米粒子的合成 -------------------------------------------------------------------------------------------10
2.3.1檸檬酸離子還原法(citrate method) ---------------------------------------------------------------------11
2.3.2二相合成法 ---------------------------------------------------------------------------------------------------11
2.4奈米粒子表面改質 --------------------------------------------------------------------------------------------14
2.4.1利用氫硫基化合物(Mercaptocarbonic acid)改質 -----------------------------------------------------14
2.4.2利用高分子披覆(Polymer-coating)方式改質 ----------------------------------------------------------16
2.4.3利用矽化處理(Silanization)方式改質 -------------------------------------------------------------------18
2.4.4利用磷脂質披覆(Phospholipid-coating)方式改質 ----------------------------------------------------19
2.5奈米粒子與生物分子的鍵結 --------------------------------------------------------------------------------21
2.5.1鍵結的方法 ----------------------------------------------------------------------------------------------------22
2.5.2奈米粒子與DNA之接合 ------------------------------------------------------------------------------------23
2.5.3奈米粒子與蛋白質之接合 ---------------------------------------------------------------------------------28
2.6奈米粒子和生物分子的一對一接合技術 ----------------------------------------------------------------29
2.6.1電泳分離一對一接合技術 ---------------------------------------------------------------------------------30
2.6.2官能基覆蓋/水解技術 ---------------------------------------------------------------------------------------33
2.7奈米粒子與生物分子的應用 --------------------------------------------------------------------------------35
第三章 實驗藥品、設備與方法 --------------------------------------------------------------------------------38
3.1實驗藥品 ----------------------------------------------------------------------------------------------------------38
3.2實驗設備 ----------------------------------------------------------------------------------------------------------42
3.3實驗方法 ----------------------------------------------------------------------------------------------------------44
3.3.1金奈米粒子濃度表示方式 ---------------------------------------------------------------------------------44
3.3.2金奈米粒子之合成 -------------------------------------------------------------------------------------------45
3.3.3金奈米粒子純化 ----------------------------------------------------------------------------------------------46
3.3.4金奈米粒子表面改質 ---------------------------------------------------------------------------------------46
3.3.4.1利用Poly(maleic anhydride alt-1-tetradecene)(PMATD)兩性高分子修飾金奈米粒子------46
3.3.4.2改變不同鹽濃度測試鹽穩定性 ------------------------------------------------------------------------48
3.3.4.3改變不同溫度測試熱穩定性 ---------------------------------------------------------------------------48
3.3.5單股寡核甘酸(ssDNA)鍵結固相基材 ------------------------------------------------------------------49
3.3.5.1單股寡核甘酸鹼量線 ------------------------------------------------------------------------------------49
3.3.5.2 EDC活化基材共價鍵結5’-單股寡核甘酸 --------------------------------------------------------50
3.3.6 DNA雜合反應 -----------------------------------------------------------------------------------------------50
3.3.7一對一接合之金奈米粒子 --------------------------------------------------------------------------------51
3.3.7.1去雜合脫附金奈米粒子-DNA一對一接合 ---------------------------------------------------------51
3.3.8金奈米粒子-DNA一對一接合之雜合反應 ------------------------------------------------------------52
第四章 結果與討論 -----------------------------------------------------------------------------------------------53
4.1金奈米粒子的合成---------------------------------------------------------------------------------------------53
4.1.1金奈米粒子的粒徑分布 -----------------------------------------------------------------------------------54
4.1.2金奈米粒子的吸收光光譜 --------------------------------------------------------------------------------55
4.2金奈米粒子的表面親水化改質 ----------------------------------------------------------------------------57
4.2.1改質前後光譜的差異 --------------------------------------------------------------------------------------58
4.2.2改質前後粒徑的差異 --------------------------------------------------------------------------------------60
4.2.3金奈米粒子的鹽穩定性測試 ----------------------------------------------------------------------------61
4.2.4金奈米粒子的熱穩定性測試 ----------------------------------------------------------------------------63
4.3金奈米粒子與DNA一對一鍵結 ----------------------------------------------------------------------------66
4.3.1矽膠體與DNA之鍵結 --------------------------------------------------------------------------------------68
4.3.2活化劑濃度之影響 -----------------------------------------------------------------------------------------68
4.3.3改變不同DNA序列長度之影響 -------------------------------------------------------------------------70
4.3.4不同pH值的緩衝液中反應對鍵結效率之影響 -----------------------------------------------------71
4.4固相雜合DNA --------------------------------------------------------------------------------------------------73
4.4.1溫度對雜合反應之影響 -----------------------------------------------------------------------------------73
4.5奈米粒子的接合 -----------------------------------------------------------------------------------------------75
4.5.1活化劑對奈米粒子的影響 --------------------------------------------------------------------------------76
4.5.2利用金奈米粒子鍵結至矽膠體表面 --------------------------------------------------------------------77
4.5.3 pH值對脫附之影響 -----------------------------------------------------------------------------------------78
4.6奈米金-DNA一對一接合的鑑定 ---------------------------------------------------------------------------80
4.6.1利用光譜的變化鑑定一對一接合 -----------------------------------------------------------------------80
4.6.2利用粒徑關係鑑定一對一接合 --------------------------------------------------------------------------85
4.6.3利用zeta potential鑑定一對一接合 ----------------------------------------------------------------------85
4.6.4利用毛細電泳與穿透式電子顯微鏡鑑定一對一接合-------86
第五章 結論與建議 ------------------------------------------------------------------------------------------------91
第六章 參考文獻 ---------------------------------------------------------------------------------------------------93
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