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研究生: 賴怡穎
I-ying Lai
論文名稱: 金奈米粒子與DNA一對一鍵結及其在檢測單一核苷酸變異的應用
One to One Linkage between Gold Nanoparticle and DNA and Its Application in Detecting Single Nucleotide Mutation
指導教授: 阮若屈
Ruoh-chyu Ruaan
口試委員:
學位類別: 碩士
Master
系所名稱: 工學院 - 化學工程與材料工程學系
Department of Chemical & Materials Engineering
畢業學年度: 96
語文別: 中文
論文頁數: 78
中文關鍵詞: 金奈米粒子DNA單一核苷酸變異
外文關鍵詞: Single Nucleotide Mutation, Gold nanoparticle, DNA
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  • 本研究利用金奈米粒子ㄧ對ㄧ鍵結單一條聚核苷酸,來檢測36個鹼基之DNA片段中的單一核苷酸變異(single nucleotide mutation)。首先,我們使用氫硫基化合物Mercaptoundecanoic acid包覆金奈米粒子表面使其親水化;改質後金奈米粒子於高溫80℃與200 mM鹽溶液中有很好的穩定性,並可穩定保存約十個月。此外,利用固相合成方式使金奈米粒子鍵結單一條聚核苷酸,兩條互補且與奈米金形成一對一鍵結的探針進行雜合反應,會形成二聚體結構,可經由穿透式顯微鏡與動態粒徑分析儀證實此結構的存在。我們以一對一鍵結的金奈米探針來偵測單一核苷酸變異的ssDNA,並與一對多鍵結的金奈米探針進行比較。實驗結果發現,一對一與一對多鍵結的金奈米探針均可清楚辨別單一核苷酸變異,但經多次操作後,也發現一對一鍵結的金奈米探針造成的背景干擾遠比一對多鍵結的金奈米探針來得低。


    Water-soluble gold nanoparticles were stabily dispersed in 200mM NaCl solution for ten months by surface modification with mercaptoundecanoic acid. We developed an efficient method to link nanogold with single strain DNA (ssDNA). One to one conjugates of the Au/ssDNA(+) were dimerized with the complimentary Au/ssDNA(-) and imaged by TEM and nanoparticle tracking analysis. Both one to one conjugates of Au/ssDNA and randomly linked Au/ssDNA were able to detect single nucleotide mutation by absorb intensity. The lower background absorb intensity of one to one conjugates can be estimated by the detection results.

    中文摘要 I 英文摘要 II 總目錄 III 圖目錄 V 表目錄 VII 第一章 緒論 1 1.1 研究動機 1 1.2 研究目的與進行策略 2 第二章 文獻回顧 4 2.1 奈米材料簡介 4 2.2 金奈米粒子的特性 5 2.3 金奈米粒子的製備方法 7 2.4 金奈米粒子親水化改質 9 2.4.1 氫硫基化合物(Mercaptocarbonic acid)改質 9 2.4.2 高分子披覆(Polymer-coating)改質 11 2.5 金奈米粒子與生物分子鍵結 12 2.5.1 奈米粒子與蛋白質接合 14 2.5.2 奈米粒子與聚核苷酸 (DNA)接合 16 2.6 奈米粒子鍵結DNA數量 18 2.7 奈米粒子在生物學上應用 22 第三章 實驗藥品、設備與方法 25 3.1實驗藥品 25 3.2實驗設備 28 3.3 實驗方法 30 3.3.1 金奈米粒子的製備 30 3.3.2 金奈米粒子表面親水化改質 32 3.3.3 金奈米粒子穩定性試驗 33 3.3.4金奈米粒子鍵結單根ssDNA 34 3.3.5 吸附基材表面處理 36 第四章 結果與討論 39 4.1 金奈米粒子合成 39 4.1.1 金奈米粒子於甲苯溶液中的粒徑分布 41 4.1.2 金奈米粒子於甲苯溶液的吸收光光譜 42 4.2 金奈米粒子表面親水化改質 43 4.2.1 利用氫硫基化合物MUA修飾金奈米粒子表面 45 4.2.2 室溫下鹽穩定性試驗 49 4.2.3 鹽、熱穩定性試驗 52 4.2.4 長時間觀察改質後金奈米粒子粒徑變化 54 4.3 金奈米粒子與ssDNA的一對一鍵結 55 4.3.1 脫附一對一鍵結的金奈米粒子 56 4.3.2 鑑定與ssDNA一對一鍵結的金奈米粒子 57 4.3.3 金奈米粒子二聚體結構 58 4.4 檢測單一核苷酸變異之DNA 62 4.4.1 金奈米粒子表面ssDNA與石英片表面ssDNA固相雜合 63 4.4.2 金奈米粒子表面ssDNA與石英片表面single mismatch ssDNA進行固相雜合 66 4.4.3 脫附夾具內雜合反應的金奈米粒子 69 第五章 結論與結論與未來研究方向 71 5.1 結論 71 5.2 未來研究方向 73 參考文獻 74

    Alivisatos A.P., Zanchet D., Micheel C. M., Parak W. J., Gerion D., “Electrophoretic Isolation of Discrete Au Nanocrystal/DNA Conjugates” Nano Lett., 1, 32-35, 2001.
    Alivisatos A.P., Zanchet D., Micheel C. M., Parak W. J., Gerion D., Williams S.C., “Electrophoretic and Structural Studies of DNA-Directed Au Nanoparticle Groupings” J. Phys. Chem. B, 106, 11758-11763, 2002.
    Astruc D., Daniel M. C.,“Gold Nanoparticles: Assembly, Supramolecular Chemistry, Quantum-Size-Related Properties, and Applications toward Biology, Catalysis, and Nanotechnology” Chem. Rev., 104, 293-346, 2004.
    Bohren C.F., Huffman D. R., “Absorption and Scattering of Light by Small Particles” Wiley, New York, 1983.
    Brust M., Walker M., Bethell D., “Synthesis of Thiol-derivatised Gold Nanopartilces in a Two-phase Liquid-Liquid”, J. Chem. Soc., Chem. Comm., 801-802, 1994.
    Bradley, J. S.; “The Chemistry of Transition Metal Colloids. In Clusters and Colloids”, VCH Publishers: New York, 459-530, 1994.
    Blum L. J., Audrey Sassolas, Be´atrice D. Leca-Bouvier., “DNA Biosensors and Microarrays .” Chem. Rev, 108, 109-139, 2008
    Crooks R. M., Sun L., Chechik V., “Preparation of Polycyclodextrin Hollow Spheres by Templating Gold Nanoparticles” Chem. Comm., 359-360, 2001.
    Caruso F., Gittins D. I., “ Biological and Physical Applications of Water-Based Metal Nanoparticles Synthesised in Ordanic Solution.” CHEMPHYSCHEM, 1, 110-113, 2002
    Dubertret B., Calame M., Libchaber A. J., “Single-Mismatch Detection Using Gold-Quenched Fluorescent Oligonucleotides” Nature Biotech., 19, 365-370, 2001.
    Dubertret B., Skourides P., Norris D. J., Noireaux V., Brivanlou A. H., Libchaber A.,“In Vivo Imaging of Quantum Dots Encapsulated in Phospholipid Micells” Science, 298, 1759-1762, 2002.
    Frens G., “Controlled Nucleation for the Regulation of the Particle Size in Monodisperse Gold Suspensions”, Nature Phys. Sci., 241, 20-22, 1973.
    Gerron D., Pinaud F., Williams S. C., Parak W. J., Zanchet D., Weiss S., Alivisates A. P., “Synthesis and Properties of Biocompatible Water-Soluble Silica-Coated CdSe / ZnS Semiconductor Quantum Dots” J. Phys. Chem. B, 105, 8861-8871, 2001.
    Hermanson G. T., Mallia A. K., Smith P. K., “Immobilized Affinity Ligand Techniques” Academic, San Diego, 1992.
    Hupp T. J., Kim Y., Johnson R. C., “Gold Nanoparticle-Based Sensing of “Spectroscopically Slient”Heavy Metal Ions” Nano Lett., 13, 165-167, 2001.
    Jacobson J. M., Sung K. M., Mosley D. W., Peelle B. R., Zhang S., “Synthesis of Monofunctionalized Gold Nanoparticles by Fmoc Solid-Phase Reactions” J. Am. Chem. Soc., 126, 5064-5065, 2004.
    Kerker M., “The Scattering of Light and Other Electromagnetic Radiation”Academic, New York, 1969.
    Lu Y., Lin J., “Adenosine-Dependent Assembly of Aptazyme-Functionalized Gold Nanoparticles and Its Application as a Colorimetric Biosensor” Anal. Chem., 76, 1627-1632, 2004.
    Medintz I. L., Uyeda H. T., Goldman E. R., Mattoussi H., “Quantum Dot Bioconjugates for Imaging, Labeling and Sensing” Nature Mater., 4, 435-446, 2005.
    Mirkin C. A., Letsinger R. L., Mucic R. C., Storhoff J. J., “A DNA-based Method for Rationally Assembling Nanoparticles into Macroscopic Materials” Nature, 382, 607-609, 1996.
    Mirkin C. A., Elghanian R., Storhoff J. J., Mucic R. C., Letsinger R. L., “Selective Colorimetric Detection of Polynucleotides Based on the Distance-Dependent Optical Properties of Gold Nanoparticles ” Science, 277, 1078-1081, 1997.
    Mirkin C. A, Storhoff J. J., Elghanian R., Mucic R. C., Letsinger R. L., “One-Pot Colorimetric Differentiation of Polynucleotides with Single Base Imperfections Using Gold Nanoparticle Probes” J. Am. Chem. Soc., 120, 1959-1964, 1998.
    Mirkin C. A., T. Andrew Taton, Robert L. L., “Scanometric DNA Array Detection with Nanoparticle Probes.” Science, 289, 1757-1760, 2000
    Mirkin C. A, Storhoff J. J., Lazarides A. A., Mucic R. C., Letsinger R. L., Schatz G. C., “What Controls the Optical Properties of DNA-Linked Gold Nanoparticle Assemble?” J. Am. Chem. Soc., 122, 4640-4650, 2000.
    Mirkin C. A., Demers L. M., Mucic R. C., Reynolds R. A., Letsinger R. L., Elghanian R., Viswanadham G., “A Fluorescence-Based Method for Determining the Surface Coverage and Hybridization Efficiency of Thiol-Capped Oligonucleotides Bound to Gold Thin Films and Nanoparticles” Anal. Chem., 72, 5535-5541, 2000.
    Mirkin C. A., Storhoff J. J., Elghanian R., Letsinger R. T., “Squence-Dependent Stability of DNA-Modified Gold Nanoparticles” Langmuir, 18, 6666-6670, 2002.
    Mulvaney P., Underwood S., “Effect of the Solution Refractive Index on the Color of Gold Colloids”, Langmuir, 10 , 3427-3430, 1994.
    Mo Zhihong., Yang X., Qian J., Wan Q., “Functional Gold Clusters and Their Applications in Biomedicine. ” PROGRESS IN CHEMISTRY, 19, 5, 2007
    Nie S., Warren C. W., “Quantum Dot Bioconjugates for Ultrasensitive Nonisotopic Detection.” Science, 281, 2016-2018, 1998.
    Nie S., Maxwell D. J., Taylor J.R., “Self-Assembled Nanoparticle Probes for Recognition and Detection of Biomolecules” J. Am. Chem. Soc., 5, 9606-9612, 2002.
    Peng X., Aldana J., Wang Y. A., “Photochemical Instability of CdSe Nanocrystals Coated by Hydrophilic Thiols” J. Am. Chem. Soc., 123, 8844-8850, 2001.
    Pellegrino T., Manna L., Kudera S., Liedl T., Koktysh D., Rogach A. L., Keller S., Radler J., Natile G., Parak W. J., “Hydrophobic Nanocrystals Coated with an Amphiphilic Polymer Shell: A General Route to Water Soluble Nanocrystals.” Nano Lett., 4, 703-707, 2004.
    Rotello V. M., Joseph Simard., Cheryl Briggs., Andrew K. Boal., “Formation and pH-controlled assembly of amphiphilic gold nanoparticles.” ChemComm., 1943-1944, 2000
    Rotello V. M., Catherine M.M., Edward A. E., Andrew K. B.,Joseph M.S., Craig T. M., “Inhibition of DNA Transcription Using Cationic Mixed Monolayer Protected Gold Clusters.” J. Am. Chem. Soc., 123, 7626-7629, 2001
    Rotello V. M., Nicholas O. F., Ayush V., Catherine M. G., Joseph M. S., “Reversible“Irreversible” Inhibition of Chymotrypsin Using Nanoparticle Receptors.” J. Am. Chem. Soc., 125, 13387-13391, 2003
    Rotello V. M., Ayush Verma., “Surface recognition of biomacromolecules using nanoparticle receptors.” ChemComm., 303-312, 2004
    Rotello V. M., Chang-Cheng You., Ayush Verma., “Engineering the nanoparticle–biomacromolecule interface.” Soft Matter, 2, 190-204, 2006
    Schatz G. C., Mirkin C. A., Kelly K. L., “Photoinduced Conversion of Silver Nanospheres to Nanoprisms”, Science, 294, 1901-1903, 2001.
    Schiffrin D.J., Fink J., Kiely C.J., “Self-Organization of Nanosized Gold Particles”, Chem. Mater., 10, 922-926, 1998.
    Storhoff J. J., Mucic R.C., Mirkin C. A., “Strategies for Organizing Nanoparticles into Aggregation Structures and Functional Materials” J. Cluster Sci., 8, 179-216, 1997.
    Turkevitch J., Stevenson P.C., Hillier J., “Nucleation and Growth Process in the Synthesis of Colloidal Gold”, Discuss. Faraday Soc., 11, 55-57, 1951.
    Wang S., Mamedova N., Kotov N.A., Chen W., Studer J., “Antigen/Antibody Immuncomplex from CdTe Nanoparticle Bioconguates” Nano Lett., 2, 817-822, 2002.
    Weimer B. C., Walsh M. K., Wang X., “Optimizing the Immobilization of Single-Stranded DNA onto Glass Beads” J. Biochem. Biophys. Methods, 47, 221-231, 2001.
    Willard D.M., Carillo L.L., Jung J., Orden A. V., “CdSe-ZnS Quantum Dots as Resonance Energy Transfer Donors in a Model Protein-Protein Binding Assay” Nano Lett., 1, 469-474, 2001.
    Yung L. I. L, Qinw. J., “ Well-Defined Nanoassemblies Using Gold Nanoparticle Bearing Specific Number of DNA Strands” Bioconjugate Chem., 19, 385-390, 2008

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