新型態中孔洞材料之合成與應用：磷酸化蛋白質純化與去氧核醣核酸藥物載體之設計

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姓名

劉依欣(Yi-hsin Liu) 查詢紙本館藏

畢業系所

化學學系

論文名稱

新型態中孔洞材料之合成與應用：磷酸化蛋白質純化與去氧核醣核酸藥物載體之設計
(Synthesis and application of a new type mesoporous silica: phosphopeptide purification and DNA drug carrier)

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摘要(中)

本篇論文分為兩個部分:
第一部分，本實驗室成功在具有羧酸官能基的中孔洞材料表面，進一步修飾可調節環狀大小之雙胺官能基，合成新型環狀中孔洞材料，命名為XC-CAR-10，X代表雙端胺基化合物中間的碳數。此環狀中孔洞材料可利用大環分子效應，提升對金屬的吸附效果，以鑭系元素中的鉺金屬為例，其吸附效果可達174 μg/mg，並藉由鉺金屬和磷酸化蛋白質間的螯合作用，溶液中的磷酸化蛋白質會與中孔洞材料中的鉺金屬產生鍵結，利用固定化金屬離子層析法(IMAC)，達到使磷酸化蛋白質具有增量純化的成果，回收率可高達百分之30以上。
第二部分，以表面修飾胺基的中孔洞材料AM-SBA-15發展出新型藥物載體，將孔洞材料內部吸附螢光物質，且因為胺基在中性環境下帶正電荷因此可以吸附負電荷的 DNA雙股螺旋，因此我們期望將DNA作為藥物載體之蓋子，保護孔洞內部的螢光物質不被釋放，但若加入可以使DNA斷裂之切割酶，則可以控制藥物載體使螢光物質不受DNA的保護進而被釋放出來。

摘要(英)

The first part of my research is about annular rings with controllable sizes are successfully created on the surface of COOH-functionalized mesoporous silica (namely, XC-CAR-10, where X represents the number of carbons in the diamines used) for the adsorption of metal ions. Particularly, excellent adsorption ability of 3C-CAR-10 towards Er3+ (ca. 174 μg/mg) was achieved.
Reversible phosphorylation of proteins is a common theme in the regulation of important cellular functions such as growth, metabolism, and differentiation. Although, the number of cellular phosphoproteins is relatively high, the phosphorylated residues themselves are generally of low abundance due to the sub-stoichiometric nature. Additionally, phosphopeptides with phosphate groups are able to form stable complexes with the lanthanide ions by the formation of precipitation. In this study, thus, we reported an annular mesoporous adapted with trivalent lanthanide ions Er3+ on for the applications of phosphopeptide enrichment and cleaning-up.
The second part of my research is about a new style drug carrier with designed DNA as a cap to control drug release. We synthesized mesoporous silicas with an amine group on the surface, named AM-SBA15. Due to owning positive charge of AM-SBA15, it can be formation of positive-negative interaction between AM-SBA15 and DNA phosphate groups at neutral condition. Thus, we designed a short length DNA with 17 EcoRV cutting cites as caps on the surface of AM-SBA15 mesoporous silica. According to investigation results, DNA shows a good capability for covering pores on the surface of AM-SBA15 so as to not allow fluorescein released. Remarkably, the fluorescein will be released upon EcoRV added because restriction enzyme of EcoRV digests DNA into smaller fragments. Finally, we develop a novel platform as a drug carrier and release system. Drugs will be released when composites are triggered by specific restriction enzyme.

關鍵字(中)

★ 中孔洞材料
★ 藥物載體
★ 磷酸化蛋白
★ 純化

關鍵字(英)

論文目次

中文摘要 i
Abstract ii
致謝辭 iii
目錄 iv
圖目錄 viii
表目錄 x
Part I 1
第一章緒論 1
1-1 中孔洞材料的簡介 1
1-2 中孔洞材料的發展 2
1-3 中孔洞材料的應用 3
1-4 吸附理論 6
1-4-1 簡介 6
1-4-2 等溫吸附模式 7
1-4-2-1 Freundlich等溫吸附模式 7
1-4-2-2 Langmuir等溫吸附模式 8
1-5蛋白質純化方法的簡介 9
1-6 固定化金屬親和層析法發展與應用 12
1-6-1 載體 13
1-6-2 螯合基 13
1-6-3 金屬離子 14
1-7磷酸化蛋白質與其純化方法 15
1-7-1 磷酸化蛋白質 15
1-7-2 磷酸化蛋白質之純化方法 16
1-8研究動機與目標 19
第二章實驗部分 20
2-1 實驗藥品與設備 20
2-1-1 實驗藥品 20
2-1-2 實驗設備 21
2-2 中孔洞材料合成與鑑定 21
2-2-1 SBA-15合成步驟 22
2-2-2 合成具羧酸官能基的SBA-15 22
2-2-3 移除中孔洞材料中的模板 23
2-2-4 合成具環狀結構的YC-CAR-X 23
2-3 鑑定設備與原理 25
2-3-1 實驗儀器 25
2-3-2 X射線粉末繞射儀 26
2-3-3 氮氣等溫吸/脫附儀 27
2-3-4傅立葉轉換紅外線吸收光譜儀 30
2-3-5固態核磁共振儀 32
2-3-6場發掃描式電子顯微鏡附加能量分散光譜儀 32
2-4 磷酸化蛋白質純化實驗 34
2-4-1利用中孔洞材料吸附鑭系金屬實驗─鉺(Er, Erbium) 34
2-4-2以重力管柱進行純化實驗 34
2-4-3以微量離心管進行純化實驗 35
2-5蛋白質純化分析方法 37
2-5-1 十二烷基磺酸鈉－聚丙烯醯胺膠體電泳（Sodium Dodecyl Sulfate－Polyacrylamide Gel Electrophoresis, SDS－PAGE） 37
2-5-2 蛋白質濃度定量─Bradford Protein Assay 41
第三章結果與討論 43
3-1內環狀孔洞材料3C-CAR-10之鑑定 43
3-1-1 13C CP/MAS NMR 鑑定3C-CAR-10 43
3-1-2 FT-IR 光譜鑑定3C-CAR-10 44
3-1-3 XRD 鑑定3C-CAR-10 45
3-1-4 TEM 影像鑑定3C-CAR-10 45
3-1-5 氮氣等溫吸脫附鑑定3C-CAR-10 46
3-1-6 SEM影像及EDS 鑑定3C-CAR-10 47
3-2 蛋白質純化結果 50
3-2-1 重力管柱實驗結果 50
3-2-2 微量離心管實驗結果 52
第四章結論 55
Part Ⅱ 56
第五章緒論 56
5-1聚合酶鏈式反應(Polymerase chain reaction，PCR)之簡介 56
5-2 限制酶(Restriction enzyme)之簡介 59
5-3 研究動機與目標 60
第六章實驗部分 61
6-1 實驗藥品與儀器 61
6-2合成具胺基之中孔洞材料AM-SBA-15 61
6-3利用中孔洞材料吸附螢光物質(Fluorescein) 62
6-4利用中孔洞材料AM-SBA-15吸附DNA 62
6-5 以PCR法擴增DNA片段最佳化實驗 63
6-6 設計藥物載體實驗 65
6-6-1 A:中孔洞材料+螢光物質 65
6-6-2 B:中孔洞材料+螢光物質+包覆DNA 65
6-6-3 C:中孔洞材料+螢光物質+包覆DNA+限制酶切割DNA 66
6-7 DNA膠體電泳 67
第七章結果與討論 68
7-1 中孔洞材料AM-SBA-15 鑑定 68
7-1-1 13C CP/MAS NMR 鑑定AM-SBA-15 68
7-1-2 FT-IR 光譜鑑定AM-SBA-15 68
7-1-3 XRD 鑑定AM-SBA-15 69
7-2 AM-SBA-15對DNA的吸附 70
7-3 AM-SBA-15 對螢光物質的吸附 70
7-4 PCR條件最佳化 71
7-5 藥物載體設計之結果 72
第八章結論 75
參考文獻 76
附錄 80

參考文獻

1. McBain, J. W., The Sorption of Gases and Vapors by Solid 1932.
2. Lee, C.-H.; Lin, T.-S.; Mou, C.-Y., Mesoporous materials for encapsulating enzymes. Nano Today 2009, 4 (2), 165.
3. Hudson, S. P.; Padera, R. F.; Langer, R.; Kohane, D. S., The biocompatibility of mesoporous silicates. Biomaterials 2008, 29 (30), 4045.
4. Zhao, X. S.; Bao, X. Y.; Guo, W.; Lee, F. Y., Immobilizing catalysts on porous materials. Materials Today 2006, 9 (3), 32.
5. Zhang, Y.; Yuan, Q.; Chen, T.; Zhang, X.; Chen, Y.; Tan, W., DNA-Capped Mesoporous Silica Nanoparticles as an Ion-Responsive Release System to Determine the Presence of Mercury in Aqueous Solutions. Analytical Chemistry 2012, 84 (4), 1956.
6. Shieh, F.-K.; Hsiao, C.-T.; Wu, J.-W.; Sue, Y.-C.; Bao, Y.-L.; Liu, Y.-H.; Wan, L.; Hsu, M.-H.; Deka, J. R.; Kao, H.-M., A bioconjugated design for amino acid-modified mesoporous silicas as effective adsorbents for toxic chemicals. Journal of Hazardous Materials 2013, 260 (0), 1083.
7. Thu, P.; Thanh, T.; Phi, H.; Kim, S.; Vo, V., Adsorption of lead from water by thiol-functionalized SBA-15 silicas. Journal of Materials Science 2010, 45 (11), 2952.
8. Peters, The sorption of gases and vapours by solids. Angewandte Chemie 1932, 45 (21), 365.
9. Kresge, C. T.; Leonowicz, M. E.; Roth, W. J.; Vartuli, J. C.; Beck, J. S., Ordered mesoporous molecular sieves synthesized by a liquid-crystal template mechanism. Nature 1992, 359 (6397), 710.
10. Heilman, B. J.; St. John, J.; Oliver, S. R. J.; Mascharak, P. K., Light-Triggered Eradication of Acinetobacter baumannii by Means of NO Delivery from a Porous Material with an Entrapped Metal Nitrosyl. Journal of the American Chemical Society 2012, 134 (28), 11573.
11. Sayari, A., Catalysis by Crystalline Mesoporous Molecular Sieves. Chemistry of Materials 1996, 8 (8), 1840.
12. Hoffmann, F.; Cornelius, M.; Morell, J.; Froba, M., Silica-based mesoporous organic-inorganic hybrid materials. Angew Chem Int Ed Engl 2006, 45 (20), 3216.
13. (a) Beck, J. S.; Vartuli, J. C.; Roth, W. J.; Leonowicz, M. E.; Kresge, C. T.; Schmitt, K. D.; Chu, C. T. W.; Olson, D. H.; Sheppard, E. W., A new family of mesoporous molecular sieves prepared with liquid crystal templates. Journal of the American Chemical Society 1992, 114 (27), 10834; (b) Vartuli, J. C.; Schmitt, K. D.; Kresge, C. T.; Roth, W. J.; Leonowicz, M. E.; McCullen, S. B.; Hellring, S. D.; Beck, J. S.; Schlenker, J. L., Effect of Surfactant/Silica Molar Ratios on the Formation of Mesoporous Molecular Sieves: Inorganic Mimicry of Surfactant Liquid-Crystal Phases and Mechanistic Implications. Chemistry of Materials 1994, 6 (12), 2317.
14. Lee, Y.-C.; Chen, C.-T.; Chiu, Y.-T.; Wu, K. C. W., An Effective Cellulose-to-Glucose-to-Fructose Conversion Sequence by Using Enzyme Immobilized Fe3O4-Loaded Mesoporous Silica Nanoparticles as Recyclable Biocatalysts. ChemCatChem 2013, 5 (8), 2153.
15. Shieh, F.-K.; Hsiao, C.-T.; Kao, H.-M.; Sue, Y.-C.; Lin, K.-W.; Wu, C.-C.; Chen, X.-H.; Wan, L.; Hsu, M.-H.; Hwu, J. R.; Tsung, C.-K.; Wu, K. C. W., Size-adjustable annular ring-functionalized mesoporous silica as effective and selective adsorbents for heavy metal ions. RSC Advances 2013, 3 (48), 25686.
16. (a) Lin, Q. N.; Huang, Q.; Li, C. Y.; Bao, C. Y.; Liu, Z. Z.; Li, F. Y.; Zhu, L. Y., Anticancer Drug Release from a Mesoporous Silica Based Nanophotocage Regulated by Either a One- or Two-Photon Process. J. Am. Chem. Soc. 2010, 132 (31), 10645; (b) Zhu, C. L.; Lu, C. H.; Song, X. Y.; Yang, H. H.; Wang, X. R., Bioresponsive Controlled Release Using Mesoporous Silica Nanoparticles Capped with Aptamer-Based Molecular Gate. J. Am. Chem. Soc. 2011, 133 (5), 1278; (c) Halamova, D.; Badanicova, M.; Zelenak, V.; Gondova, T.; Vainio, U., Naproxen drug delivery using periodic mesoporous silica SBA-15. Appl. Surf. Sci. 2010, 256 (22), 6489; (d) Yang, Q.; Wang, S.; Fan, P.; Wang, L.; Di, Y.; Lin, K.; Xiao, F.-S., pH-Responsive Carrier System Based on Carboxylic Acid Modified Mesoporous Silica and Polyelectrolyte for Drug Delivery. Chemistry of Materials 2005, 17 (24), 5999; (e) Yu, H.; Zhai, Q.-Z., Mesoporous SBA-15 molecular sieve as a carrier for controlled release of nimodipine. Microporous and Mesoporous Materials 2009, 123 (1–3), 298.
17. Setoguchi, T.; Takao, M., Current status of self rectifying air turbines for wave energy conversion. Energy Conversion and Management 2006, 47 (15–16), 2382.
18. Shieh, F.-K.; Hsiao, C.-T.; Wu, J.-W.; Sue, Y.-C.; Bao, Y.-L.; Liu, Y.-H.; Wan, L.; Hsu, M.-H.; Deka, J. R.; Kao, H.-M., A bioconjugated design for amino acid-modified mesoporous silicas as effective adsorbents for toxic chemicals. Journal of Hazardous Materials 2013, 260 (0), 1083.
19. Haxel, O.; Volz, H., über die Absorption von Neutronen in wässerigen Lösungen. Zeitschrift für Physik 1943, 120 (7-10), 493.
20. Langmuir, I., THE ADSORPTION OF GASES ON PLANE SURFACES OF GLASS, MICA AND PLATINUM. Journal of the American Chemical Society 1918, 40 (9), 1361.
21. Hearon, J. Z., The configuration of cobaltodihistidine and oxy-bis (cobaltodihistidine). Journal of the National Cancer Institute 1948, 9 (1), 1.
22. Porath, J.; Carlsson, J.; Olsson, I.; Belfrage, G., Metal chelate affinity chromatography, a new approach to protein fractionation. Nature 1975, 258 (5536), 598.
23. Hochuli, E.; Dobeli, H.; Schacher, A., New metal chelate adsorbent selective for proteins and peptides containing neighbouring histidine residues. Journal of chromatography 1987, 411, 177.
24. Chaga, G. S., Twenty-five years of immobilized metal ion affinity chromatography: past, present and future. Journal of Biochemical and Biophysical Methods 2001, 49 (1–3), 313.
25. Pearson, R. G., Hard and Soft Acids and Bases. Journal of the American Chemical Society 1963, 85 (22), 3533.
26. Ueda, E. K. M.; Gout, P. W.; Morganti, L., Current and prospective applications of metal ion–protein binding. Journal of Chromatography A 2003, 988 (1), 1.
27. Arnold, F. H., Metal-Affinity Separations: A New Dimension in Protein Processing. Nat Biotech 1991, 9 (2), 151.
28. Cheng, H.-C.; Qi, R. Z.; Paudel, H.; Zhu, H.-J., Regulation and Function of Protein Kinases and Phosphatases. Enzyme Research 2011, 2011, 3.
29. Baker, A. F.; Dragovich, T.; Ihle, N. T.; Williams, R.; Fenoglio-Preiser, C.; Powis, G., Stability of phosphoprotein as a biological marker of tumor signaling. Clinical cancer research : an official journal of the American Association for Cancer Research 2005, 11 (12), 4338.
30. Ptacek, J.; Devgan, G.; Michaud, G.; Zhu, H.; Zhu, X.; Fasolo, J.; Guo, H.; Jona, G.; Breitkreutz, A.; Sopko, R.; McCartney, R. R.; Schmidt, M. C.; Rachidi, N.; Lee, S. J.; Mah, A. S.; Meng, L.; Stark, M. J.; Stern, D. F.; De Virgilio, C.; Tyers, M.; Andrews, B.; Gerstein, M.; Schweitzer, B.; Predki, P. F.; Snyder, M., Global analysis of protein phosphorylation in yeast. Nature 2005, 438 (7068), 679.
31. Hubbard, M. J.; Cohen, P., On target with a new mechanism for the regulation of protein phosphorylation. Trends in biochemical sciences 1993, 18 (5), 172.
32. Braich, N.; Codd, R., Immobilised metal affinity chromatography for the capture of hydroxamate-containing siderophores and other Fe(III)-binding metabolites directly from bacterial culture supernatants. The Analyst 2008, 133 (7), 877.
33. (a) Mirza, M. R.; Rainer, M.; Guzel, Y.; Choudhary, I. M.; Bonn, G. K., A novel strategy for phosphopeptide enrichment using lanthanide phosphate co-precipitation. Analytical and bioanalytical chemistry 2012, 404 (3), 853; (b) Mirza, M. R.; Rainer, M.; Messner, C. B.; Guzel, Y.; Schemeth, D.; Stasyk, T.; Choudhary, M. I.; Huber, L. A.; Rode, B. M.; Bonn, G. K., A new type of metal chelate affinity chromatography using trivalent lanthanide ions for phosphopeptide enrichment. The Analyst 2013, 138 (10), 2995; (c) Cheng, G.; Liu, Y. L.; Zhang, J. L.; Sun, D. H.; Ni, J. Z., Lanthanum silicate coated magnetic microspheres as a promising affinity material for phosphopeptide enrichment and identification. Analytical and bioanalytical chemistry 2012, 404 (3), 763; (d) Mirza, M.; Rainer, M.; Güzel, Y.; Choudhary, I.; Bonn, G., A novel strategy for phosphopeptide enrichment using lanthanide phosphate co-precipitation. Analytical and bioanalytical chemistry 2012, 404 (3), 853; (e) Pink, M.; Verma, N.; Polato, F.; Bonn, G. K.; Baba, H. A.; Rettenmeier, A. W.; Schmitz-Spanke, S., Precipitation by lanthanum ions: a straightforward approach to isolating phosphoproteins. Journal of proteomics 2011, 75 (2), 375.
34. Zhao, D.; Huo, Q.; Feng, J.; Chmelka, B. F.; Stucky, G. D., Nonionic Triblock and Star Diblock Copolymer and Oligomeric Surfactant Syntheses of Highly Ordered, Hydrothermally Stable, Mesoporous Silica Structures. Journal of the American Chemical Society 1998, 120 (24), 6024.
35. Tsai, C.-T.; Pan, Y.-C.; Ting, C.-C.; Vetrivel, S.; Chiang, A. S. T.; Fey, G. T. K.; Kao, H.-M., A simple one-pot route to mesoporous silicas SBA-15 functionalized with exceptionally high loadings of pendant carboxylic acid groups. Chemical Communications 2009, (33), 5018.
36. Maria Chong, A. S.; Zhao, X. S., Functionalization of SBA-15 with APTES and Characterization of Functionalized Materials. The Journal of Physical Chemistry B 2003, 107 (46), 12650.
37. Brunauer, S.; Emmett, P. H.; Teller, E., Adsorption of Gases in Multimolecular Layers. Journal of the American Chemical Society 1938, 60 (2), 309.
38. Brunauer, S.; Deming, L. S.; Deming, W. E.; Teller, E., On a Theory of the van der Waals Adsorption of Gases. Journal of the American Chemical Society 1940, 62 (7), 1723.
39. Barrett, E. P.; Joyner, L. G.; Halenda, P. P., The Determination of Pore Volume and Area Distributions in Porous Substances. I. Computations from Nitrogen Isotherms. Journal of the American Chemical Society 1951, 73 (1), 373.
40. 國立台灣大學生物技術研究中心蛋白質分析-膠體電泳法. http://juang.bst.ntu.edu.tw/Protein/Analysis/A3.htm.
41. Bradford, M. M., A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Analytical biochemistry 1976, 72, 248.
42. Kleppe, K.; Ohtsuka, E.; Kleppe, R.; Molineux, I.; Khorana, H. G., Studies on polynucleotides: XCVI. Repair replication of short synthetic DNA′s as catalyzed by DNA polymerases. Journal of Molecular Biology 1971, 56 (2), 341.
43. Saiki, R. K.; Scharf, S.; Faloona, F.; Mullis, K. B.; Horn, G. T.; Erlich, H. A.; Arnheim, N., Enzymatic amplification of beta-globin genomic sequences and restriction site analysis for diagnosis of sickle cell anemia. Science (New York, N.Y.) 1985, 230 (4732), 1350.

指導教授

謝發坤

審核日期

2014-8-14

推文