現今科學與醫療的發展日新月異，有許多新型態之檢測生物分子的方法被提出並備受矚目。其中，微陣列技術（Microarray）已廣泛被應用於醫學診斷和生物學研究，包括單核苷酸多態性（SNP）檢測、基因表達譜、比較基因組雜交和表位作圖等。與其他現有的分析方法相比，微陣列主要有小型化、高通量、並行性、快速、多功能和低成本等優點。儘管微陣列技術可迅速且同時篩選和分析多種分析物，但大多數分析物僅限於大分子，例如蛋白質、抗體以及核酸等。因此在這篇論文中，我們開發出一種基於微陣列平台上的半合成蛋白質開關，以調控親和力的方式來偵測小分子。其原理是利用小分子與配體競爭結合位點，進而調控生物素與鏈黴親和素的結合能力。
在尚未添加小分子分析物時，由於生物素衍生物周圍空間的立障較大，無法與修飾有螢光基團的鏈黴親和素結合，訊號呈現關閉狀態；當添加小分子分析物後，即消除生物素衍生物周圍的空間立障，而修飾有螢光基團的鏈黴親和素則可與其結合，此時訊號呈現開啟狀態。藉此，我們利用該訊號表達的機制、探針模塊化的特性和微陣列技術的優點，來達到偵測不同小分子的目的。基於這項設計，我們相信半合成蛋白質開關與微陣列技術的結合可以成為未來檢測各種重要生物小分子的實用分析平台。

Nowadays, with the rapid development of science and medical treatments, numerous novel techniques for detecting biomolecules have been developed and attracted wide attention. Among them, microarray technology has been widely used in medical diagnosis and biology research, including single nucleotide polymorphism (SNP) genotyping, gene expression profiling, comparative genomic hybridization, and epitope mapping. Compared with other traditional analysis methods, microarray have the advantages of miniaturization, high throughput, parallelism, rapidity, versatility, and inexpensive.
Although microarray technique enables screening and analyzing multiple analytes rapidly and simultaneously, most analytes are limited to macromolecules, for example, proteins, antibodies, and nucleic acids. Therefore, we developed an affinity-tunable semisynthetic protein switches based on microarray platform to detect small molecules, utilizing the competition between small molecules and ligands to regulate the binding affinity of biotin and streptavidin.
In the absence of a small molecule target, biotin cannot bind to the streptavidin due to the large steric hindrance around the biotin derivative. Therefore, the signal is turned off. If an analyte is present, the steric hindrance around the biotin derivative will be removed. Thus, the streptavidin can bind to the biotin, and the signal will be turned on. In this way, we use this new signal transduction mechanism and modular design on microarray to detect various small molecules. Based on this design, we believe that the combination of semisynthetic protein switches and microarray detection system can become the practical analysis platform for detecting various important small biomolecules in the future.

摘要 I
Abstract II
謝誌 III
目錄 VI
第一章、緒論 1
1-1 小分子介紹 1
1-1.1 常見生物小分子 2
1-1.2 神經傳遞物質 3
1-1.3 小分子藥物 5
1-2 檢測小分子之方法 6
1-2.1 核磁共振光譜分析法 7
1-2.2 液相層析質譜分析法 9
1-2.3 酵素結合免疫吸附分析法 11
第二章、文獻回顧 13
2-1 微陣列 14
2-1.1 DNA微陣列 15
2-1.2 抗體微陣列 18
2-1.3 蛋白質-配體微陣列 20
2-2 蛋白質開關 22
2-2.1 G蛋白偶聯受體 22
2-2.2 核受體 24
2-3 合成蛋白質開關 25
2-3.1 螢光蛋白質開關 25
2-3.2 酶蛋白質開關 28
第三章、探針設計 32
3-1 設計構想 32
3-2 合成設計與策略 37
3-2.1 固定端 38
3-2.2 配體端 38
3-2.3 反應端 40
3-3 探針結構與運作機制 41
第四章、實驗結果及討論 44
4-1 探針1之合成與實驗結果 44
4-1.1 探針1之合成 44
4-1.2 探針1之實驗結果 46
4-2 探針2之合成與實驗結果 50
4-2.1 探針2之合成 50
4-2.2 探針2之實驗結果 52
4-3 探針3之合成與實驗結果 58
4-3.1 探針3之合成 58
4-3.2 探針3之實驗結果 60
第五章、實驗結論 63
第六章、實驗部分 64
6-1 實驗器材與藥品 64
6-2 微陣列實驗 66
6-3 有機合成及光譜資料 67
第七章、參考資料 93
附錄 101

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