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博碩士論文 etd-0723107-113008 詳細資訊
Title page for etd-0723107-113008
論文名稱
Title
原子力顯微鏡之奈米圖案顯影與核酸雜化反應探討
AFM-Based Nanolithography and Detection of DNA Hybridization Reactions at the Nanoscale
系所名稱
Department
畢業學年期
Year, semester
語文別
Language
學位類別
Degree
頁數
Number of pages
104
研究生
Author
指導教授
Advisor
召集委員
Convenor
口試委員
Advisory Committee
口試日期
Date of Exam
2007-07-11
繳交日期
Date of Submission
2007-07-23
關鍵字
Keywords
原子力顯微鏡、奈米顯影術、自組裝單層膜、核酸雜化反應
atomic force microscopy, DNA hybridization, self-assembled monolayers, nanolithography
統計
Statistics
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中文摘要
本研究以原子力顯微術於大氣下掃瞄雲母、石墨、二硫化鉬和鍍金玻璃片等無機材料表面,取得高解析度晶格週期成像,掌握了顯微系統之穩定度和極佳成像條件。並將原子力顯微鏡應用於奈米顯影,結合自組裝薄膜,與可置換液體的流動液相設備,在烷硫醇及DNA自組裝薄膜上製作出奈米圖案。針對掃描速度、加工次數及作用力等參數進行優化,取得高品質且具再現性的奈米圖案。當選用不同頭基的烷硫醇分子,還可實現原位薄膜表面親疏水性之改質。比較不同鹼基數目探針核酸所製作的奈米圖案,發現核酸長度會影響奈米圖案品質。鹼基雜化反應研究顯示,過長的探針核酸鏈可能與基板有非特異性吸附,形成立體障礙而影響標靶核酸的配對,導致未能見到雜化反應發生的證據。未來應朝縮短探針核酸與標靶核酸的長度,改變核酸序列,採用變溫裝置提高反應溫度,調配溶液組成,及延長反應時間進行探討。
Abstract
High-resolution lattice periodicity images of a variety of well-defined surfaces, including graphite, mica, and Au(111), validated the good stability of our atomic force microscope (AFM) system. Combining self-assembled monolayer (SAM) and AFM technology, we demonstrated the capabilities of pattern fabrication as well as modification of surface functionality. AFM-based nanolithography operating conditions, such as scan rate, deflection setpoint, and number of scan were studied to obtain the optimized quality of the fabricated patterns. Thiolated-DNA probe molecules could be patterned at a nanometer scale on a gold substrate. However, we found that the surface coverage began to drop notably with the probe length (number of DNA bases). Therefore, the displaced DNA molecules during nanoshaving were reversibly adsorbed, and patterning became unreliable. We were unsuccessful in detecting the subsequent hybridization reactions at these nanopatterns from AFM measurements. To realize the DNA hybridization, further studies on the incubation temperature, probe length and even DNA sequences are required to demonstrate that this AFM-based gene diagnostic method is truly operational.
目次 Table of Contents
中文摘要 i
英文摘要 ii
目錄 iii
圖表目錄 vii
第壹章 緒論 1
1-1 前言 1
1-2 研究背景 1
1-2.1 核酸雜化反應 2
1-2.2 核酸鹼基雜化的相關研究 3
1-3 研究動機 5
第貳章 儀器與實驗 7
2-1 實驗儀器 7
2-2 原子力顯微鏡原理 7
2-3 Contact mode AFM氣、液相之掃描 8
氣相操作步驟 10
液相操作步驟 11
2-4 實驗材料 12
基板 12
樣品 12
緩衝液 12
成像環境 12
2-5 實驗操作方法 14
2-5.1 清洗容器 14
2-5.2 製備gold substrates
火焰鍛燒 14
2-5.3 生物樣品配置 16
凍乾oligonucleotides之處理 16
Oligonucleotides原液稀釋 17
Reducing thiol-modified oligonucleotides 17
萃取oligonucleotides 18
DNA annealing 19
2-5.4 製備自組裝薄膜 20
DNA自組裝薄膜 20
烷基硫醇自組裝薄膜 20
2-6 原子力顯微鏡奈米顯影術操作步驟 20
2-6.1 Nanoshaving 21
2-6.2 Nanografting 21
2-7 實驗分析量測 23
第參章 校正AFM scanner 26
3-1 Calibration reference 26
3-2 Linearity Correction Procedure 27
3-2.1 Set up SPM for contact AFM 27
3-2.2 Align calibration reference 27
3-2.3 Adjust sample orthogonality 28
3-2.4 Adjust mag0 and arg0 29
Adjusting Fast mag0 30
Adjusting Fast arg 31
Adjusting Slow mag0 31
Adjusting Slow arg 32
Adjusting Fast mag1 33
Fine Adjustment 34
Adjusting Slow mag1 35
3-3 X-Y Calibration 36
3-3.1 Capture Calibration 36
3-3.2 Autocalibration 36
3-4 Fine-tuning for X-Y Measuring Accuracy 37
3-4.1 Measure horizontally at 440 V scan size 37
3-4.2 Measure vertically at 440 V scan size 38
3-4.3 Measure horizontally at 150 V scan size 39
3-4.4 Measure vertically at 150 V scan size 40
3-4.5 Measure horizontally at 440 V scan size 41
3-4.6 Measure vertically at 440 V scan size 42
3-4.7 Measure horizontally at 150 V scan size 43
3-4.8 Measure vertically at 150 V scan size 44
3-5 Calibrating Z 45
3-6 Calculate Retracted and Extended offset deratings 47
3-6.1 Set Extended offset der 47
3-6.2 Set Retracted offset der 48
第肆章 成像與奈米圖案製作 50
4-1 AFM原子成像 50
4-1.1 Atomic resolution images 50
4-1.2 Lattice periodicity 51
4-1.3 晶格成像原理:stick-slip behavior 52
4-1.4 幾種無機材料表面的AFM晶格成像研究 53
雲母 53
石墨 56
二硫化鉬 58
Gold substrate (Arrandee) 59
4-2 烷基硫醇奈米圖案製作 60
4-2.1 烷基硫醇自組裝薄膜 60
4-2.2 AFM奈米顯影術. 61
4-2.3 C10S matrix的奈米圖案 62
Negative patterns 62
Positive patterns 64
4-2.4 Spatially constrained self-assembly reaction 66
4-2.5 區域調控自組裝薄膜表面的親疏水性質 67
4-2.6 MCH SAM matrix上的單股DNA nanoisland 68
4-3 DNA奈米圖案製作 70
4-3.1 DNA自組裝薄膜 70
4-3.2 DNA matrix的奈米圖案 71
ssDNA (Oligo1) matrix 71
dsDNA (Oligo1-Oligo2) matrix 72
4-3.3 核酸長度對自組裝行為的影響 73
4-3.4 核酸雜化反應研究與雜化效率分析-文獻報導 75
4-3.5 探針核酸固定與雜化反應研究 79
4-3.6 影響雜化反應的因素 82
第伍章 結論 84
參考文獻 86
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