近年來由於生物感測技術的進步，藉由表面電漿共振技術可以即時的檢視分子間的交互作用，提高了生物分子交互作用分析(Biomolecular interaction analysis; BIA)的發展；而DNA與小分子物質間的交互作用，為篩選具有潛力之藥物的重要步驟之ㄧ。本研究的目的是以SPR 技術探討固定於SPR金片上之經Biotin 標記的DNA分子與小分子物質間之交互作用。本研究可以提供一個快速篩選具有生理活性小分子物質的平台。
本研究先將感測金片以食人魚溶液(Piranha solution)清洗金片上雜質。再使用16-mercaptohexadecanoic acid (16-MHDA)與6-mercapto-1
-hexanol(6-MCH)，兩種尾端分別帶有不同官能基之硫醇(Thiol)，以1：0、1：3、1：10與1：20四種不同比例之混合硫醇，於金片表面形成自組單層膜(self-assembled monolayer；SAM)，找出對金片表面具最佳修飾性的SAM。結果顯示，比例1：10之混合式自組單層膜，可得到最高的角度變化量。接著比較Avidin與Streptavidin於不同離子強度、pH值之環境下，對於Biotin-NF-κB固定量之影響。結果顯示，兩者在I=0.16、pH=5之環境下皆具有最好的Biotin-NF-κB固定量。隨後分別確定了Avidin、Biotin-NF-κB與Complementary NF-κB之最適濃度分別為250 μg/ml、10μg/ml與5μg/ml。在專一性測試中，使用Non-biotinylated DNA與Non-complementary DNA進行非專一性吸附測試。結果顯示，Biotin-NF-κB的部分所形成的角度變化量確為Avidin-Biotin之專一性吸附所造成；雜交反應時所造成之角度變化量確為Biotin-NF-κB與Complementary NF-κB之間的專一性雜交反應所造成。而再生性測試中，0.01 M HCl可達到不傷害金片表面其他的親和性分子，並且可以把Complementary NF-κB於雙股螺旋結構中分離，以0.01 M HCl做第一次再生可還原94.4 %的原偵測力，第二次再生可達到91.6 %。最後以Sanguinarine進行 DNA 與小分子間動力學分析，求得Sanguinarine與NF-κB DNA的親和常數 KA= 1.25 × 105 M-1。

The recent development of biosensor technologies for biomolecular interaction analysis enables monitoring of a variety of molecular reactions in real-time by surface Plasmon resonance (SPR). In this study, we aim to demonstrate the molecular interactions between small molecules and biotinylated target DNA probe immobilized on sensor chips is detectable by SPR technology. This work demonstrates the possibility of applying the biosensor approach in the search for bioactive constituents from small molecules.
The experiments start with cleansing the gold surface with Piranha solution. After the surface being cleaned, self-assembled monolayer (SAM) is adsorbed onto the surface. SAM with COOH functional group 16-mercaptohexadecanoic (16-MHDA) was mixed with SAM possessing OH functional group 6-mercapto-1-hexanol (6-MCH) in different ratios such as 1:0, 1:3, 1:10 and 1:20 to find the best ratio which can cause the maximum angle shift. The result show that the best mixed SAM for Avidin and double helix DNA immobilization is 16-MHDA/6-MCH in 1:10 ratio. Afterwards, compare with the angle shift caused by Biotin-NF-κB immobilization onto Avidin/Streptavidin modified surface in different pH and ion strength. The result show that both of Biotin-NF-κB immobilization on avidin/streptavidin cause the maximum angle shift in pH=5, 0.16 of the ion strength. As the follow experiments can decide that the best concentration of avidin, Biotin-NF-κB and Complementary NF-κB was 250μg/ml, 10μg/ml and 5μg/ml. In this study used Non-biotinylated DNA and Non-complementary DNA to decide the angle shift was caused by the specific interaction. The result prove that the angle shift was caused by the specific interaction between avidin-biotin and DNA hybridization. Using 0.01 M HCl as the regeneration solution for the first time leads to 94.4 % of the original detection ability; the second time result in 91.6 % of the original detection ability. The association constant (KA) of Sanguinarine binding to NF-κB DNA was 1.25 × 105 M-1.

目錄 i
表目錄 iv
圖目錄 v
縮寫表 vii
摘要 viii
Abstract x
第一章 緒論 - 1 -
1-1 研究動機與目的 - 1 -
1-2 小分子物質與DNA之作用機制 - 2 -
1-2-1 小分子物質與DNA 交互作用之前人研究 - 3 -
1-3 生物感測器 - 5 -
1-3-1 生物感測器之類型 - 7 -
1-3-2 表面電漿共振技術簡介 - 11 -
1-3-2-1 表面電漿共振技術原理 - 14 -
1-4 金片表面修飾 - 19 -
1-4-1 自組單層膜 - 21 -
1-4-2 生物親和性固定化 - 25 -
1-5 動力學參數分析 - 28 -
第二章 材料與方法 - 30 -
2-1 藥品試劑 - 30 -
2-2 儀器設備 - 32 -
2-3 實驗流程 - 35 -
2-4 SPR操作流程 - 36 -
2-5 金片前處理 - 37 -
2-6 金片表面修飾 - 37 -
2-6-1最佳SAMs比例探討 - 38 -
2-7 生物親和性分子固定 - 38 -
2-7-1 實驗用DNA序列 - 39 -
2-7-2 最佳固定化條件探討 - 40 -
2-8 DNA雜交反應 - 40 -
2-9 專一性試驗 - 40 -
2-10 再生性試驗 - 41 -
2-11 小分子動力學分析 - 41 -
第三章 結果與討論 - 43 -
3-1 最佳SAMs比例探討 - 43 -
3-2 生物親和性分子固定 - 47 -
3-2-1 Avidin/Streptavidin之比較 - 47 -
3-3 DNA雜交反應 - 55 -
3-4 專一性測試 - 58 -
3-5 再生性測試 - 61 -
3-6 DNA與小分子之動力學分析 - 63 -
第四章 結論與未來展望 - 66 -
4-1 結論 - 66 -
4-2 未來展望 - 68 -
參考文獻 - 69 -

白璧如 (2007) 利用表面電漿共振系統發展金黃色葡萄球菌腸毒素B
免疫感測器之研究。碩士論文 中國文化大學生物科技研究所。

林芷寧(2001) 利用TI-SPR 觀測生物分子間之交互作用。碩士論文 國立台灣科技大學化學工程研究所。

林哲瑩 (2005) 高敏度表面與高解析技術診斷具疾病表現之單一核苷酸多型性:運用SAMs 技術固定寡核苷酸。碩士論文 國立成功大學材料科學及工程研究所。

陳美滿 (2002) 自動組裝烷基二硫代羧酸薄膜結構與性質研究。碩士論文 東吳大學化學研究所。

徐怡琳 (2004) 利用矽烷類混合式自組裝單分子膜提高蛋白質晶片上抗體與檢體的接合效率。碩士論文 國立清華大學工程與系統科學研究所。

張怡南 (1997) 生物感測器-生物技術的發展與應用。九州出版社三版，303-320。
陳良宇 (2005) 表面電漿共振感測器觀測固定化蛋白質的構型變化
與島狀脂双層膜的建構。博士論文 國立中央大學化學工程與材料工程研究所。

蔡佩璇 (2005) 利用恆溫吸附曲線及恆溫滴定微卡計探討單股及雙股DNA與Hydroxyapatite間交互作用之機制與熱力學。碩士論文 國立中央大學化學工程與材料工程研究所。

賴怡潔 (2006) 表面電漿共振系統應用於金黃色葡萄球菌腸毒素之檢測。碩士論文 中國文化大學生物科技研究所。

賴銘智 (2003) 合成新的具有聚醯胺官能基之DNA結合試劑。博士論文 國立中山大學化學研究所。

謝振傑 (2006) 光纖生物感測器。物理雙月刊，第28卷第4期。

羅敏瑞 (2009) 親合性感測器應用於黃蓮生物活性物質的篩選及分離。碩士論文 中國文化大學生物科技研究所。

Berganza, J., Olabarria, G., Garc´ıa , R., Verdoy , D., Rebollo, A., and Arana, S. (2007) DNA microdevice for electrochemical detection of
Escherichia coli O157:H7 molecular markers. Biosensors and Bioelectronics 22, 2132-2137.


Cho, Y. K., Kim, S., Kim, Y. A, Lim, H. K., Lee, K., Yoon, D. S., Lim,G., Pak, Y. E. Ha , T. H. and Kim K. (2004) Characterization of DNA immobilization and subsequent hybridization using in situ quartz crystal microbalance, fluorescence spectroscopy, and surface plasmon resonance.
Journal of Colloid and Interface Science 278, 44-52.

Choi, S. H., Lee, J. W. and Sim, S. J. (2005) Enhanced performance of a surface plasmon resonanceimmunosensor for detecting Ab–GAD antibody based on the modified self-assembled monolayers. Biosensors and Bioelectronics 21, 378-383.

Ciolkowski, M. L., Fang M. M. and Lund, M. E. (2000) A surface
plasmon resonance method for detecting multiple modes of DNA–ligand
interactions. Pharmaceutical and Biomedical Analysis 22, 1037-1045.

Cooper, M. A. (2003) Biosensor profiling of molecular interaction in pharmacology. Current opinion in pharmacology 3, 557-562.

Cui, X., Pei, R., Wang, Z., Yang, F., Ma, Y., Dong, S.and Yang, X.(2003) Layer-by-layer assembly of multilayer films composed of avidin and
biotin-labeled antibody for immunosensing. Biosensors and Bioelectronics 18, 59 – 67.

Erdem, A. and Ozsoz, M. (2002) Electrochemical DNA biosensors based
on DNA-drug interactions. Electroanalysis 14, 965-974.

Finklea, H. O., Avery, S. and Lynch, M. (1987) Blocking oriented monolayers of alkylmercaptans on gold electrodes. Langmuir 3, 409-413.

Gambari, R. (2001) Biospecific interaction analysis (BIA) as a tool for
the design and development of gene transcription modifiers. Curr. Med.
Chem. Anti-cancer agents 1 , 277-291.

Gambari, R., Feriotto, G., Rutigliano, C., Blanchi, N. and Mischiati, C.
(2000) Biospecific Interaction Analysis (BIA) of low-molecular weight DNA-binding drugs. Pharmacology and Exprimental Therapeutics 294, 370-377.

Homola, J., Dosta´lek, J., Chen, S., Rasooly, A., Jiang, S.and Yee, S. S., (2002) Spectral surface plasmon resonance biosensor for detection of
staphylococcal enterotoxin B in milk. Food Microbiology 75, 61-69.

Ibrahim, M.S. (2001) Voltammetric studies of the interaction of nogalamycin antitumor drug with DNA. Analytica Chimica Acta 443 ,
63-72

Karadeniz, H., Gulmez, B., Sahinci, F., Erdem, A., Kaya, G. I., Unver N.,
Kivcak, B. and Ozsoz, M. (2003) Disposable electrochemical biosensor
for the detection of the interaction between DNA and lycorine based on
guanine and adenine signals. Pharmaceutical and Biomedical Analysis 33,
295-302.

Kett, W. C., Osmond, R. I. W., Moe, L., Skett, S. E., Kinnear, B. F. and Coombe, D. R. (2003) Avidin is a heparin-binding protein. Affinity, specificity and structural analysis. Biochimica et Biophysica Acta 1620, 225-234.

Lazcka, O., Del Campo, F. J.and Mu˜noz F. X. (2007) Pathogen detection: A perspective of traditional methods and biosensors. Biosensors and Bioelectronics 22, 1205-1217.

Lazerges, M., Perrot, H., Antoine, E., Defontaine, A.and Comperec, C. (Biosensors and Bioelectronics 21, 1355-1358.

Lee, J. W., Sim, S. J., Cho, S. M. and Lee, J. (2005) Characterization of a self-assembled monolayer of thiol on a gold surface and the fabrication of a biosensor chip based on surface plasmon resonance for detecting anti-GAD antibody. Biosensors and Bioelectronics 20, 1422-1427.

Lofas, S., Johnsson, B., Edstrom, A., Hansson, A., Lindquist, G., Hillgren, R. M. and Stigh, L. (1995) Methods for site controlled coupling to carboxymethyldextran surfaces in surface plasmon resonance sensors. Biosensors and Bioelectronics 10, 813-822.



Mannellia, I., Minunnia, M., Tombellia, S., Wanga, R., Spiritia, M. M. and Mascini, M. (2005) Direct immobilisation of DNA probes for the development of affinity biosensors. Bioelectrochemistry 66, 129-138.

Minunni,M., Tombelli, S., Mascini,M., Bili, A., Bergonzib, M. C. and
Vincierib, F.F. (2005) An optical DNA-based biosensor for the analysis
of bioactive constituents with application in drug and herbal drug
screening. Talanta 65, 578-585.

Mullett, W. M., Lai, E. P. C. and Yeung, J. M. (2000) Surface Plasmon Resonance-Based Immunoassays. Methods 22, 77-91.

Nguyen, B., Tanious, F. A. and Wilson, W. D. (2007) Biosensor-surface
plasmon resonance: Quantitative analysis of small molecule–nucleic
acid interactions. Methods 42 , 150-161.

Nuzzo, R. G., Fusco, F. A. and Allara, D. L., (1987) Spontaneously organized molecular assemblies. 3. preparation and properties of solution adsorbed monolayers of organic disulfides on gold surfaces. Journal of the American Chemical Society 109, 2358-2368.

Ostatná, V., Vaisocherová, H., Homola, J., and Hianik, T. (2008) Effect of the immobilisation of DNA aptamers on the detection of thrombin by means of surface plasmon resonance. Anal Bioanal Chem 391,1861-1869.

Rauf, S., Gooding, J.J., Akhtar, K., Ghauri, M.A., Rahman, M., Anwar,
M.A. and Khalid, A.M. (2005) Electrochemical approach of anticancer
drugs–DNA interaction. Pharmaceutical and Biomedical Analysis 37,
205-217.

Sen, A., Ray, A. and Maiti, M. (1996) Thermodynamics of the interactions of sanguinarine with DNA: influence of ionic strength and base composition. Biophysical Chemistry 59, 155-170.

Shankaran, D. R., Gobi, K. V. and Miura, N. (2006) Recent advancements in surface plasmon resonance immunosensors for detection of small molecules of biomedical, food and environmental interest. Sensors and Actuators. B, Chemical 121, 158-177.
Silin, V. and Plant, A., (1997) Biotechnological applications of surface
plasmon resonance. Tibtech September 15, 353-359.

Spiridonova, V., Rassokhin, T., Golovin, A., Petrova, E., Rohzdestvensky, T., Pakhomova, Y. and Kopylov, A. (2002) A comparative thermodynamic study for both natural and artificial RNA/DNA-protein binary complexes. Bioelectrochemistry 56, 95-97.

Steel, A. B., Levicky, R. L., Herne, T. M. and Tarlov M. J. (2000) Immobilization of Nucleic Acids at Solid Surfaces: Effect of Oligonucleotide Length on Layer Assembly. Biophysical Journal Volume 79, 975-981.

Watterson, J. H., Piunno, P. A. E. and Krull, U. J. (2002) Towards the optimization of an optical DNA sensor: control of selsctivity coefficients and relative surface affinities. Analytica chimica Acta 457, 29-38.

Wink, T., van Zuilen, S. J., Bult, A. and W. P. van Bennekom. (1997) Self-assembled Monolayers for Biosensors. Analyst 122, 43-50.

Yamasaki, R., Ito, M., Lee, B. K., Jung, H. S., Lee, H. Y. and Kawai, T. (2007) Single probe nucleic acid immobilization on chemically modified single protein by controlling ionic strength and pH. Analytica chimica acta 603, 76-81.

Yamasaki, R., Kim, J. M., Jung, H. S., Lee, H. Y. and Kawai, T. (2006) Dependence upon ionic strength in the immobilization of probing
oligonucleotides onto streptavidin-modified probe surfaces. Biochemical Engineering Journal 29, 125-128.

Zhu, X.-M., Lin, P.-H. Ao, P. and Sorensen, L. B. (2002) Surface treatments for surface Plasmon resonance biosensors. Senosrs and Actuators 84, 106-112.