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論文名稱 Title |
開發以單股寡核苷酸和金屬奈米簇為材料之感測器 Development of oligonucleotides- and metal nanoclusters-based sensors |
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系所名稱 Department |
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畢業學年期 Year, semester |
語文別 Language |
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學位類別 Degree |
頁數 Number of pages |
140 |
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研究生 Author |
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指導教授 Advisor |
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召集委員 Convenor |
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口試委員 Advisory Committee |
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口試日期 Date of Exam |
2012-07-18 |
繳交日期 Date of Submission |
2012-08-03 |
關鍵字 Keywords |
單股寡核苷酸 nanoclusters, oligonucleotide |
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統計 Statistics |
本論文已被瀏覽 5700 次,被下載 758 次 The thesis/dissertation has been browsed 5700 times, has been downloaded 758 times. |
中文摘要 |
本篇論文是利用單股寡核苷酸(Oligonucleotide)和金屬奈米簇(Nanoclusters)為材料,開發快速、簡便、高靈敏度以及高專一性的分析技術,應用於環境汙染物或是生物小分子的檢測。首先在單股寡核苷酸部分,我們分別設計重複有20個胞嘧啶之單股寡核苷酸—Polycytosine (Poly C20),以及重複40個腺嘌呤之寡核苷酸(Polyadenosine,Poly A40),同時再搭配一種形成雙股螺旋時螢光會增強11倍的螢光試劑—SYBR Green Ι (SG)的使用,做為重金屬物種銀離子和生物小分子Coralyne的感測器,實驗方法是當溶液中僅有Poly C20或Poly A40的單股寡核苷酸存在時,依據SG螢光試劑特性,此時螢光強度相對低;但若是這兩條單股寡核苷酸與目標分析物鍵結,分別形成C-Ag+-C和A-coralyne-A錯合物,則會誘導原先為隨意線狀(Coil)之單股寡核苷酸,摺疊成雙股螺旋結構,使SG螢光試劑可以嵌入雙股螺旋結構中,而增強其螢光染劑訊號,達到偵測目標分析物目的,之後,也分別將此兩技術應用於銀奈米粒子(AgNPs)的間接偵測,以及核糖核酸多聚腺苷酸化反應(Polyadenylation reaction)的監控,觀察核糖核酸尾端長出腺嘌呤鹼基的個數,與聚腺嘌呤寡核苷酸聚合酶的濃度,對反應速率的影響。此技術的成功開發,提供我們一套簡單(One-step)、免標記(Label-free)、便宜、快速、高選擇性且高靈敏度的檢測方法。隨後,我們也將Poly A40單股寡核苷酸可以與Coralyne形成A-coralyne-A錯合物之概念,於室溫下,應用於分子信籤(Molecular beacons,MBs)的開發,直接區分目標寡核苷酸之單一鹼基點突變序列。方法是將16個腺嘌呤重複之Poly A16設計於分子信籤的兩邊莖(Stem)端,中間環(Loop)端則設計15個能辨識目標寡核苷酸的鹼基,並在分子信籤的5’與 3’尾端標示上一螢光試劑和一消光劑。過程中,當分子信籤與Coralyne反應,形成A-coralyne-A錯合物時,則分子信籤的5’與3’莖端會相互靠近,形成髮夾彎(Hairpin)形狀,縮短螢光試劑和消光劑間的距離,發生碰撞消光現象,降低螢光試劑的螢光;但若分子信籤遇見與其環端序列完全互補之DNA序列時,分子信籤會與此互補DNA形成雙股螺旋DNA,取代原先Coralyne與分子信籤形成的錯合物,使螢光試劑與消光試劑間的距離拉開,而恢復螢光,達到偵測目的。此方法的開發,除了改善傳統須於高溫下,進行單一鹼基點突變序列反應之缺點外,此分子信籤相較於其他C-Ag+-C和T-Hg2+-T的分子信籤,A-coralyne-A分子信籤反應速度快、無毒,且不受胺基酸硫醇類(Aminothiols)、單股寡核苷酸鍵結蛋白質(Single-stranded DNA-binding protein)和核酸內切酶(Endogenous nuclease)的影響,可以進一步應用於生物樣品血清中偵測。論文另一部分,我們也以生物相容之蛋白質物種—溶菌酶(Lysozyme type VI,Lys VI),於37oC環境下,一步(One-step)直接合成出具發光性質之金奈米簇,實驗中發現奈米簇的合成,與溶菌酶蛋白之濃度有極大相關,當蛋白質濃度增加,合成出來之金奈米簇顆粒逐漸降低,放光波長會有藍位移現象,且量子產率會增加,顯示溶菌酶蛋白質不僅扮演穩定試劑角色,亦扮演還原劑的角色,此外,此合成之金奈米簇相當穩定,即使在高濃度的穀胱甘肽或氯化鈉鹽類環境下,螢光強度仍不受影響,適於廣範圍應用;隨後,我們也透過奈米簇表面之Au+與Hg2+或CH3Hg+反應,開發做為Hg2+和CH3Hg+的檢測器,偵測極限分別為3 pM 和4 nM ,最後,也成功地應用於基質複雜之海水中進行Hg2+和CH3Hg+偵測,可測得之最低濃度分別為1 nM和20 nM。 |
Abstract |
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目次 Table of Contents |
摘要……………………………………….……………………………………...………i 目錄………………………………………………………………………………..……iii 圖目錄…………………………………………………………………………...….…viii 表目錄…………………………………………………………………………………xiv 壹、 緒論........................................................................................................................1 1.1 去氧核醣核酸(DNA)簡介....................................................................................1 1.1.1 去氧核醣核酸的種類與作用方式................................................................1 1.1.1.1 Watson-Crick鹼基配對法則..............................................................2 1.1.1.2 非Watson-Crick鹼基配對法則.........................................................2 1.1.1.2.1 G-quadruplex和i-motif四級結構...........................................2 1.1.1.2.2 單股核酸適合體(Aptamers)......................................................4 1.1.1.2.3 去氧核醣核酸酶(DNAzymes,deoxyribozyme,DNA enzymes or catalytic DNA).......................................................................5 1.1.1.2.4 金屬-鹼基鍵結(Metal-base binding)........................................6 1.1.1.2.4.1 Thymine - Hg2+- Thymine鍵結........................................6 1.1.1.2.4.2 Cytosine - Ag+ - Cytosine鍵結..........................................7 1.1.1.2.4.3 Adenosine 2-Coralyne - Adenosine 2鍵結.........................7 1.2 奈米簇(Nanoclusters)簡介..................................................................................9 1.2.1 奈米簇原理介紹—The jellium model.....................................................10 1.2.2 奈米簇發光原理...…………...…………...……………....…...…………13 1.2.3 奈米簇的合成...…………...…………...…………...……………...….…15 1.3 研究動機...…………...…………...…………...…………...…………………..17 1.4 參考文獻...…………...…………...…………...…………...………………..…18 貳、 水溶液中以寡核苷酸做為螢光探針進行高靈敏度與高選擇性之銀離子與銀奈米粒子的偵測……...…………...……………...…...……24 2.1 摘要……...…………...…………………………………………...……...……24 2.2 前言………………………………………………….………………………...24 2.3 實驗部分…………………….……………………….………………….…….25 2.3.1 實驗藥品…………………….…….…….…….……………………...…25 2.3.2 儀器設備…………………….……………………….…………….……28 2.3.3 實驗過程與樣品製備方法…………………….………………….….…28 2.4 結果與討論…………………….……………………….………………..……30 2.4.1 反應系統的建立以及最佳化條件的探討………………..……...…..…30 2.4.2 專一性以及靈敏度的探討………………….………………...…..….…35 2.4.3 銀奈米粒子的應用…………………….…………………………......…39 2.5 結論…………………….……………………….……………………...…...…40 2.6 參考文獻…………………….………………………………………...………41 參、 以寡核苷酸做為螢光探針進行生物鹼分子的偵測以及核糖核酸多聚腺苷酸化反應的監控………………………………………………43 3.1 摘要……………………………………………………………………………43 3.2 前言……………………………………………………………………………43 3.3 實驗部分………………………………………………………………………45 3.3.1 實驗藥品………………………………………………………...………45 3.3.2 儀器設備………………………………………………………...………47 3.3.3 實驗過程與樣品製備方法………………………………...……………47 3.4 結果與討論………………………………………………………...……….…49 3.4.1 反應系統的建立以及最佳化條件的探討………………………...……49 3.4.2 反應系統的偵測靈敏度……………………………………...…………54 3.4.3 核糖核酸多聚腺苷酸化的應用………………………………...………55 3.5 結論………………………………………………………………………....…57 3.6 參考文獻…………………………………………………………….…...……58 肆、 開發於室溫下以腺嘌呤為基礎之分子信籤進行高靈敏度的核苷酸偵測………………………………………………………………….….…61 4.1 摘要……………………………………………………………………………61 4.2 前言……………………………………………………………………………61 4.3 實驗部分………………………………………………………...…….………63 4.3.1 實驗藥品……………………………………………….………...…….…63 4.3.2 儀器設備……………………………………….…………………………65 4.3.3 實驗過程與樣品製備方法………………………….…………….………65 4.4 結果與討論.…………………….……...……………………………….……...68 4.4.1 反應系統的建立以及最佳化條件的探討……………………….……...68 4.4.2 Coralyne-A16-MB-A16分子信籤偵測目標DNA之靈敏度……......…..72 4.4.3 評估單股寡核苷酸鍵結蛋白質和核酸內切酶對系統的影響….….........76 4.4.4 比較Coralyne-A16-MB-A16、Hg2+-T16-MB-T16和Ag+-C16-MB-C16分子信籤…….........……………….……………………………………………79 4.4.5 評估A16-MB-A16、T16-MB-T16和C16-MB-C16三條分子信籤受胺基酸硫醇類分子影響………………………........................……..........…………87 4.4.6 真實樣品血清中的應用…………….………………………………...…87 4.5 結論…….…………………………….…………………………….………..…90 4.6 參考文獻…….…………………………….…………………………….…..…91 伍、 以溶菌酶穩定之金奈米簇做為螢光消光探針進行汞離子和甲基汞的超靈敏偵測……………………..…….…………………………….…93 5.1 摘要……………….………………………….………………..…………….…93 5.2 前言……………….…………………………………………………...…….…93 5.3 實驗部分……………….………………………………….…………..…….…95 5.3.1 實驗藥品……………….……………………..………………..……….…95 5.3.2 儀器設備……………….……………………………………………….…97 5.3.3 實驗過程與樣品製備方法……………….…………………………….…98 5.4 結果與討論……………….……………………………………………………99 5.4.1 Lys VI溶菌酶蛋白濃度的影響…….……………………………………99 5.4.2 Lys VI-AuNCs的光學性質和穩定性…….………………….…...……103 5.4.3 Hg2+和CH3Hg+的超靈敏檢測…….…………………….……….….…107 5.4.4 Hg2+和CH3Hg+於真實樣品中的定量…….………………………..….112 5.5 結論…….…………………………….………………………….…….………116 5.6 參考文獻…….…………………………….………………………………..…117 陸、 總結論…….…………………………….……………………...……………121 附錄…….…………………………….……………………...………………………..123 |
參考文獻 References |
[1]. Taton, T. A.; Mirkin, C. A.; Letsinger, R. Scanometric DNA array detection with nanoparticle probes. Science 2000, 289, 1757-1760. [2]. Zhang, C.-Y.; Hu, J. Single quantum dot-based nanosensor for multiple DNA detection. Anal. Chem. 2010, 82, 1921-1927. [3]. Singh, A. K.; Senapati, D.; Wang, S.; Griffin, J.; Neely, A.; Candice, P.; Naylor, K. M.; Varisli, B.; Kalluri, J. R.; Ray, P. C. Gold nanorod based selective identification of Escherichia coli bacteria using two-photon Rayleigh scattering spectroscopy. ACS Nano 2009, 3, 1906-1912. [4]. Chen, C. - T.; Chen, W. -J.; Liu, C. -Z.; Chang, L. -Y.; Chen, Y. -C. Glutathione-bound gold nanoclusters for selective-binding and detection of glutathione S-transferase-fusion proteins from cell lysates. Chem. Commun. 2009, 7515-7517. [5]. Li, T.; Shi, L.; Wang, E.; Dong, S. Silver-ion-mediated DNAzyme switch for the ultarsensitive and selective colorimetric detection of aqueous Ag+ and cystein. Chem. Eur. J. 2009, 15, 3347-3350. [6]. Xia, F.; Zuo, X.; Yang, R.; Xiao, Y.; Kang, D. Vallee-Belisle, A.; Gong, X.; Yuen, J. D.; Hsu, B. B. Y.; Heeger, A. J.; Plaxco, K. W. Colorimetric detection of DNA, small molecules, proteins, and ions using unmodified gold nanoparticles and conjugated polyelectrolytes. Proc. Natl. Acad. Sci. 2010, 107, 10837-10841. [7]. Lyon, L. A.; Keating, C. D.; Fox, A. P.; Baker, B. E.; He, L.; Nicewarner, S. R.; Mulvaney, S. P.; Natan, M. J. Raman Spectroscopy. Anal. Chem. 1998, 70, 341-362. [8]. Zhang, J.; Lao, R.; Song, S.; Yan, Z.; Fan, C. Design of an oligonucleotide-incorporated nonfouling surface and its application in electrochemical DNA sensors for highly sensitive and sequence-specific detection of target DNA. Anal. Chem. 2008, 80, 9029-9033. [9]. Wang, S.; Gaylord, B. S.; Bazan, G. C. Fluorescein provides a resonance gate for FRET from conjugated polymers to DNA intercalated dyes. J. Am. Chem. Soc. 2004, 126, 5446-5451. [10]. Liu, X.; Freeman, R.; Golub, E.; Willner, I. Chemiluminescence and chemiluminescence resonance energy transfer (CRET) aptamer sensors using catalytic hemin/G-quadruplexes. ACS nano 2011, 5, 7648-7655. [11]. Navin,J. K.; Grass, M. E.; Somorjai, G. A.; Marsh, A. L. Characterization of colloidal platinum nanoparticles by MALDI-TOF mass spectrometry. Anal. Chem. 2009, 81, 6295-6299. [12]. Carrillo-Carrión, C.; Moliner-Martínez, Y.; Simonet, B. M.; Valcárcel, M. Capillary electrophoresis method for the characterization and separation of CdSe quantum dots. Anal. Chem. 2011, 83, 2807-2813. [13]. Phan, A.T.; Mergny, J.-L. Human telomeric DNA: G-quadruplex, i-motif and Watson-Crick double helix. Nucleic Acids Res. 2002, 30, 4618-4625. [14]. Gehring, K.; Leroy, J.-L.; Gueron. M. A tetrameric DNA structure with protonated cytosine•cytosine base pairs. Nature 1993, 363, 561-565. [15]. Liedl, T.; Simmel, F. C. Switching the conformation of a DNA molecule with a chemical oscillator. Nano Lett. 2005, 5, 1894-1898. [16]. http://altair.sci.hokudai.ac.jp/g6/Projects/images/selex.gif [17]. Breaker, R. R.; Joyce, G. F. A DNA enzyme that cleaves RNA. Chem. Biol. 1994, 1, 223-229. [18]. Joyce, G. F. Nucleic acid enzymes: playing with a fuller deck. Proc. Natl. Acad. Sci. USA 1998, 95, 5845-5847. [19]. Miyake, Y.; Togash, H.; Tashiro, M.; Yammaguchi, H.; Oda, S.; Kudo, M.; Tanaka, Y.; Kondo, Y.; Sawa, R.; Fujimoto, T.; Machinami, T.; Ono, A. MercuryII-mediated formation of thymine−HgII−thymine base pairs in DNA duplexes. J. Am. Chem. Soc. 2006, 128, 2172-2173. [20]. Ono, A.; Cao, S.; Togashi, H.; Tashiro, M.; Fujimoto, T.; Machinami, T.; Oda, S.; Miyake, Y.; Okamoto, I.; Tanaka, Y. Specific interactions between silver(I) ions and cytosine–cytosine pairs in DNA duplexes. Chem. Commun. 2008, 4825-4827. [21]. Ren, J.; Chaires, J. B. Sequence and structural selectivity of nucleic acid binding ligands. Biochemistry 1999, 38, 16067-16075. [22]. Song, G.; Chen, C.; Qu, X.; Miyoshi, D.; Ren, J.; Sugimoto, N. Small-molecule- directed assembly: a gold nanoparticle-based strategy for screening of homo-adenine DNA duplex binders. Adv. Mater. 2008, 20, 706-710. [23]. Link, S.; El-Sayed, M. A. Optical properties and ultrafast dynamics of metallic nanocrystals. Annu. Rev. Phys. Chem. 2003, 54, 331-366. [24]. Sherry, L. J.; Chang, S.-H.; Schatz, G. C.; Duyne, R. P. V. Localized surface plasmon resonance spectroscopy of single silver nanocubes. Nano Lett. 2005, 5, 2034-2038. [25]. Kreibig, U.; Vollmer, M. Optical Properties of Metal Clusters; Springer: New York, 1995. [26]. Zheng, J.; Nicovich, P. R.; Dickson, R. M. Highly fluorescent noble metal quantum dots. Annu. Rev. Phys. Chem. 2007, 58, 409-431. [27]. Bigioni, T. P.; Whetten, R. L.; Dag, O. Near-unfrared luminescence from small gold nanocrystals. J. Phys. Chem. B 2000, 104, 6983-6986. [28]. Schaaff, T. G.; Whetten, R. L. Giant gold−glutathione cluster compounds:  intense optical activity in metal-based transitions. J. Phys. Chem. B 2000, 104, 2630-2641. [29]. Gautier, C.; Burgi, T. Chiral inversion of gold nanoparticles. J. Am. Chem. Soc. 2008, 130, 7077-7084. [30]. Crespo, P.; Litran, R.; Rojas, T. C.; Multigner, M.; de laFuente, J. M.; Sanchez-Lopez, J. C.; Garcia, M. A.; Hernando, A.; Penades, S.; Fernandez, A. Permanent magnetism, magnetic anisotropy, and hysteresis of thiol-capped gold nanoparticles. Phys. Rev. Lett. 2004, 93, 087204-1-087204-4. [31]. Chen, S.; Ingram, R. S.; Hostetler, M. J.; Pietron, J. J.; Murray, R. W.; Schaaff, T. G.; Khoury, J. T.; Alvarez, M. M.; Whetten, R. L. Gold nanoelectrodes of varied size: transition to molecule-like charging. Science 1998, 280, 2098-2101. [32]. Huang, C. C.; Yang, Z.; Lee, K. H.; Chang, H. T. Synthesis of highly fluorescent gold nanoparticles for sensing mercury(II). Angew. Chem.-Int. Ed. 2007, 46, 6824-6828. [33]. Triulzi, R. C.; Micic, M.; Giordani, S.; Serry, M.; Chiou, W. A.; Leblanc, R. M. Immunoasssay based on the antibody-conjugated PAMAM-dendrimer-gold quantum dot complex. Chem. Commun. 2006, 5068-5070. [34]. Lee, T. H.; Gonzalez, J. I.; Zheng, J.; Dickson, R. M. Single-molecule optoelectronics. Acc. Chem. Res. 2005, 38, 534-541. [35]. Touboul, D.; Halgand, F.; Brunelle, A.; Kersting, R.; Tallarek, E.; Hagenhoff, B.; Laprevote, O. Tissue molecular ion imaging by gold cluster ion bombardment. Anal. Chem. 2004, 76, 1550-1559. [36]. Knight , W. D.; Clemenger, K.; Heer, W. A. D.; Saunders, W. A.; Chou, M. Y.; Cohen, M. L. Electronic shell structure and abundances of sodium clusters. Phys. Rev. Lett. 1984, 52, 2141-2143. [37]. Heer, W. A. D. The physics of simple metal clusters: experimental aspects and simple models. Rev. Mod. Phys.1993, 65, 611-659. [38]. Girifalco, L. A. Statistical mechanics of solids; Oxford: U. S. A.,2000. [39]. Grypeos, M. E.; Kotsos, B. A. Determination of the harmonic oscillator energy level spacing for atomic clusters. J. Phys. B: At. Mol. Opt. Phys. 1996, 29, L473-L481. [40]. Hodes, G. When small is different: some recent advances in concepts and applications of nanoscale phenomena. Adv. Mater. 2007, 19, 639-655. [41]. Zheng, J.; Petty, J. T.; Dickson, R. M. High quantum yield blue emission from water-soluble Au8 nanodots. J. Am. Chem. Soc. 2003, 125, 7780-7781. [42]. Zheng, J.; Zhang, C. ; Dickson, R. M. Highly fluorescent, water-soluble, size-tunable gold quantum dots. Phys. Rev. Lett. 2004, 93, 077402-077404. [43]. Link, S.; Beeby, A.; FitzGerald, S.; El-Sayed, M. A.; Schaaff, T. G.; Whetten, R. L. Visible to infrared luminescence from a 28-atom gold cluster. J. Phys. Chem. B 2002, 106, 3410-3415. [44]. Brust, M.; Walker, M.; Bethell, D.; Schiffrin, D. J.; Whyman, R. Synthesis of thiol-derivatised gold nanoparticles in a two-phase liquid-liquid system. J. Chem. Soc., Chem. Commun. 1994, 801-802. [45]. Templeton, A. C.; Wuelfing, W. P.; Murray, R. W. Monolayer-protected cluster molecules. Acc. Chem. Res. 2000, 33, 27-36. [46]. Hostetler, M. J.; Wingate, J. E.; Zhong, C.-Z.; Harris, J. E.; Vachet, R. W.; Clark, M. R.; Londono, J. D.; Green, S. J.; Stokes, J. J.; Wignall, G. D.; Glish, G. L.; Porter, M. D.; Evans, N. D.; Murray, R. W. Alkanethiolate gold cluster molecules with core diameters from 1.5 to 5.2 nm: core and monolayer properties as a function of core size. Langmuir 1998, 14, 17-30. [47]. Fabris, L.; Antonello, S.; Armelao, L.; Donkers, R. L.; Polo, F.; Toniolo, C.; Maran, F. Gold nanoclusters protected by conformationally constrained peptides. J. Am. Chem. Soc. 2006, 128, 326-336. [48]. Xie, J. ; Zheng, Y. ; Ying, J. Y. Protein-directed synthesis of highly fluorescent gold nanoclusters. J. Am. Chem. Soc. 2009, 131, 888-889. |
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