有效增進表面核酸探針對於短鏈基因序列的辨識能力，無論從光學或電化學的方式，一直是基因晶片改良的目標。本研究著眼於探針分子的設計，利用改變探針分子的結構（線型或髮夾型）及核酸組成（DNA或LNA）達到有效辨識的目的。我們結合自組裝薄膜（SAM）與AFM奈米顯影術（AFM-based nanolithography），透過無標記技術（label-free）在探針分子所形成的自組裝薄膜刻劃奈米尺度凹形圖案，依序導入完全互補、含有單一錯配及不互補的標靶分子進行辨識的雜化反應，同時使用AFM的高度訊號於原位（in situ）量測表面單股探針分子因雜化反應形成雙股鏈所引起的剛性變化，藉此驗證具備髮夾構型、配合修飾鎖核酸的探針分子的設計對於單一錯配的標靶分子能夠展現最高的分辨能力。其次並針對雜化反應的鹽類濃度、反應溫度、雜化區域大小進行雜化反應效率的探討。

Improving the identification ability of surfaced-immobilized nucleic acid probes for small size DNA or RNA targets, utilizing optical or electrochemical methods, has been the goal for the gene chip technology. This study focuses on new probe design for introducing hairpin structural features and locked nucleic acid modification. We use three kinds of probes (DNA-LN, DNA-HP and LNA-HP) to prepare recognition layers via self-assembly processes on a gold substrate, and utilize AFM-based nanolithography technique to produce nanofeatures to observe the stiffness changes of oligonucleotide chains resulting from the formation of rigid double stranded duplexes when target sequence hybridizes to the probe. We also monitor the topographic changes upon exposure to the single mismatched and non-complementary targets as a function of time. The results reveal LNA-HP probes exhibit the highest response to discriminating single-point mutation in the base sequence. In addition, we study the effects of salt concentration, reaction temperature and the small size on the hybridization efficiency.

目 錄
中文摘要………….………………………………………………………i
英文摘要…………………………………………………………………ii
目錄……………………………………………………………………...iii
圖表目錄………………………………………………………………....v

第壹章 緒論…………………………………..……………………..…1
1-1 前言………….…………………………………………..…1
1-2 探針分子的設計……….…………………………………..2
1-3 使用AFM對雜化反應進行觀測…………………………6
1-4 研究動機…………………………………………………...8
第貳章 儀器與實驗……………………………………………………9
2-1 實驗儀器…………………………………………………...9
2-2 原子力顯微鏡原理………………………………………...9
2-3 Contact Mode AFM之液相掃描………………………..10
2-4 實驗材料………………………………………………….11
2-5 實驗操作方法…………………………………………….13
2-5.1 清洗容器……………….………..………………...13
2-5.2 製備gold substrate/Au(111)…...…….…………….14
2-5.3 生物樣品配製………………....…………………..15
2-5.4 製備基質薄膜(matrix)………..……..…………….17
2-5.5 製備核酸核酸自組裝單層膜………...…………...18
2-6 原子力顯微鏡奈米顯影術操作步驟……………..……...19
2-7 核酸雜化反應………………………………………….....19
2-8 實驗量測分析…………………………………………….20
第參章 實驗結果與討論……………………………………………..24
3-1 液相機板掃描…………………………………….............24
3-2 LNA-HP……………………………….………………….25
3-3 DNA-HP………………………………..………………...33
3-4 DNA-LN.............................................................................37
3-5 鹽類濃度對雜化反應之影響……..……….……………..43
3-6 溫度對雜化反應之影響………………………………….46
3-7 雜化區域微小化………………………………………….50
第肆章 結論…………………………………………..………………55
參考文獻………………………………………………………………..57

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