臺灣博碩士論文加值系統

在此研究中，我們使用功能性奈米材料以雷射脫附游離質譜儀（Laser–induced desorption/ionization mass spectrometry, LDI-MS）簡單的同時檢測多種蛋白質。我們使用凝血酶（thrombin）、血管內皮生長因子-A165（vascular endothelial growth factor-A165, VEGF-A165）、血小板生長因子BB（platelet-derived growth factor-BB, PDGF-BB）作為目標蛋白來證實此檢測方法。以三種奈米粒子（金，銀和鉑奈米粒子）修飾上辨識目標蛋白質之適合體或是抗體以達到選擇性分析。修飾後之金屬奈米粒子會專一性地結合目標蛋白，之後將奈米粒子與目標蛋白混合液點樣於乙酸纖維薄膜（cellulose acetate membrane, CAM）以LDI-MS進行分析。多孔洞CAM在此時可以做為一種分離介質，隨著奈米粒子對應之目標蛋白濃度提高而導致奈米粒子質量增加亦或是降低奈米粒子對CAM的親和力，使得奈米粒子更容易滲入薄膜內部或是底部，在質譜中的結果顯示，經雷射照射後產生的金屬團簇離子的訊號（[Mn]¬¬+; M = Au和Ag, n = 1-3）以LDI-MS分析CAM表面金屬團簇離子訊號會因此降低。但是鉑團簇訊號卻是相反的，會因為增加目標蛋白質的濃度而提高鉑團簇訊號，這是因為功能化鉑奈米粒子在PDGF-BB的存在下會造成聚集的現象。在LDI-MS的分析過程中，奈米粒子搭配薄膜的系統具有將蛋白質濃度轉變為金屬團簇訊號，同時增幅訊號已達到更好的偵測極限。我們所開發的系統可以選擇性地檢測到不同蛋白質低至pM等級，並且能夠在樣品不需濃縮、分離或純化等預處理情況下，藉由不同金屬團簇的訊號而達到同時檢測的目的。我們所提出的使用奈米材料修飾上辨識分子搭配LDI-MS，應用於未來同時監測不同蛋白、腫瘤標記物、病原體或癌細胞具相當的發展潛力。

In this work we demonstrate a simple assay for the simultaneous detection of multiple proteins by functionalized nanomaterials assisted laser-induced desorption/ionization mass spectrometry (LDI-MS). We chose thrombin, vascular endothelial growth factor-A165 (VEGF-A165) and platelet-derived growth factor-BB (PDGF-BB) as model proteins to demonstrate our sensing method. Selective detection of these analytes was achieved by modifying nanoparticles of three different metals (Au, Ag and Pt) with their corresponding aptamer or antibody. Functionalized metal nanoparticles (NPs) bound to their specific analyte was subjected to LDI-MS on a cellulose acetate membrane (CAM). The microporous CAM served as a medium for an in situ separation of NPs based on the increase in particle weight and/or decreased affinity towards CAM with increasing analyte concentration. As a result, the intensities of the signals of the metal cluster ions ([Mn]¬¬+; M = Au or Ag n = 13) decreased in the mass spectra under pulsed laser irradiation. Platinum cluster ion signal being the only exception, signal increases with analyte concentration, due to aggregation of Pt NPs in the presence of PDGF-BB. The nanoparticles/CAM system can function as a signal transducer and amplifier through transformation of protein concentration to intense metal clusters ions signal during LDI-MS analysis. Our developed system can selectively detect proteins down to picomolar region. Most importantly, the system is able to detect multiple proteins simultaneously without any pre-concentration, separation or purification process since LDI-MS coupled with CAM effectively removes all signals except metal cluster ions. This sensing system shows great potential for the simultaneous detection of different protein, tumor markers, pathogens, and cancer cells by functionalizing with suitable ligands to nanomaterials.

中文摘要
Abstract
目錄
圖目錄
表目錄
第一章 緒論
1.1奈米材料於LDI-MS的應用
1.2脈衝雷射對金屬奈米粒子脫附游離的影響
1.3多重免疫法檢測的重要性
1.4雷射脫附游離法於免疫分析
1.5研究動機
第二章實驗材料與方法
2.1前言
2.2實驗藥品
2.3儀器設備
2.4適體修飾金奈米粒子合成
2.5抗體修飾銀奈米粒子合成
2.6抗體修飾鉑奈米粒子合成
2.7仿生理環境緩衝溶液配製
2.8乙酸纖維薄膜檢測平台製備
第三章 結果與討論
3.1以雷射脫附游離/薄膜檢測凝血酶
3.2薄膜優勢、選擇性與靈敏性
3.3多種蛋白質同時檢測
第四章 結論
全文圖表
參考文獻

1. Michalak, L.; Fisher, K. J.; Alderdice, D. S.; Jardine, D. R.; Willett, G. D. C60-assisted laser desorption-ionization mass spectrometry. Org. Mass Spectrom. 1994, 29, 512-515.
2. Rusling, J. F.; Kumar, C. V.; Gutkind, J. S.; Patel, V. Measurement of biomarker proteins for point-of-care early detection and monitoring of cancer. Analyst 2010, 135, 2496-2511.
3. Gedda, G.; Pandey, S.; Bhaisare, M. L.; Wu, H.-F. Carbon dots as nanoantennas for anti-inflammatory drug analysis using surface-assisted laser desorption/ionization time-of-flight mass spectrometry in serum. RSC Adv. 2014, 4, 38027-38033.
4. Chen, S.; Chen, L, Wang, J.; Hou, J.; He, Q.; Liu , J. Wang, J. Xiong, S. X.; Yang, G. Q.; Nie, Z. G. 2,3,4,5-Tetrakis(3’,4’-dihydroxylphenyl)thiophene: A New Matrix for the Selective Analysis of Low Molecular Weight Amines and Direct Determination of Creatinine in Urine by MALDI-TOF MS. Anal Chem. 2012, 84, 10291-10297.
5. Karas, M.; Hillenkamp, F. Laser desorption ionization of proteins with molecular masses exceeding 10,000 daltons. Anal. Chem. 1988, 60, 2299-2301.
6. Tanaka, K.; Waki, H.; Ido, Y.; Akita, S.; Yoshida, Y.; Yoshida, T. Protein and polymer analyses up to m/z 100,000 by laser ionization time-of-flight mass spectrometry. Rapid Commun. Mass Spectrom. 1988, 2, 151-153.
7. Mirkin, C. A. Programming the Assembly of Two- and Three-Dimensional Architectures with DNA and Nanoscale Inorganic Building Blocks. Inorg. Chem. 2000, 39, 2258-2272.
8. Chiang, C. -K.; Chen, W. -T.; Chang, H. -T. Nanoparticle-based mass spectrometry for the analysis of biomolecules. Chem. Soc. Rev. 2011, 40, 1269-1281.
9. Stolee, J. A.; Walker, B. N.; Zorba, V.; Russo, R. E.; Vertes, A. Laser–nanostructure interactions for ion production. Phys. Chem. Chem. Phys. 2012, 14, 8453-8752.
10. Liao, P.-C.; Allison, J. Ionization processes in matrix-assisted laser desorption/ionization mass spectrometry: Matrix-dependent formation of [M + H]+ vs [M + Na]+ ions of small peptides and some mechanistic comments. J. Mass Spectrom. 1995, 30, 408-423.
11. Zenobi, R.; Knochenmuss, R. Fourier transform ion cyclotron resonance mass spectrometry: a primer. Mass Spectrom. Rev. 1998, 17,337-366.
12. Hashimoto, S.; Werner, D.; Uwada, T. Studies on the interaction of pulsed lasers with plasmonic gold nanoparticles toward light manipulation, heat management, and nanofabrication J. Photochem. Photobiol. C-Photochem. Rev. 2012, 13, 28-54.
13. Werner, D.; Hashimoto, S. Improved Working Model for Interpreting the Excitation Wavelength- and Fluence-Dependent Response in Pulsed Laser-Induced Size Reduction of Aqueous Gold Nanoparticles. J. Phys. Chem. C. 2011, 115, 5063-5072.
14. Shoji, M.; Miyajima, K.; Mafuné, F. Ionization of gold nanoparticles in solution by pulse laser excitation as studied by mass spectrometric detection of gold cluster ions. J. Phys. Chem. C. 2008, 112, 1929-1932.
15. Kinumi, T.; Saisu, T.;Takayama, M.; Niwa, H. Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry using an inorganic particle matrix for small molecule analysis. J. Mass Spectrom. 2000, 35, 417-422.
16. Hua, Y.; Dagan, S.; Wickramasekara, S.; Boday, D. J.; Wysocki, V. H. Analysis of deprotonated acids with silicon nanoparticle-assisted laser desorption/ ionization mass spectrometry. J. Mass Spectrom. 2010, 45, 1394-1401.
17. Duffy, M. J.; Lamerz, R.; Haglund, C.; Nicolini, A.; Kalousová, M.; Holubec. L.; Sturgeon, C. Tumor markers in colorectal cancer, gastric cancer and gastrointestinal stromal cancers: European group on tumor markers 2014 guidelines update. Int. J. Cancer 2014, 134, 2513–2522.
18. Wu, G.G.; Datar, R. H.; Hansen, K. M.; Thundat, T.; Cote, R. J.; Majumdar.A. Bioassay of prostate-specific antigen (PSA) using microcantilevers. Nat. Biotechnol. 2001, 19, 856-860.
19. Keefe, A. D.; Pai, S.; Ellington, A. Aptamers as therapeutics. Nature Reviews Drug Discovery 2010, 9, 537-550.
20. Chen, A.; Yang, S. Replacing antibodies with aptamers in lateral flow immunoassay. Biosens. Bioelectron. 2015, 71, 230-242.
21. Gan, S. D.; Patel, K. R. Enzyme Immunoassay and Enzyme-Linked Immunosorbent Assay. J. Investig. Dermatol 2013, 133, 1-3.
22. Zhang, J., Bowers, J., Liu, L., Wei, S., Gowda, G. A. N., Hammoud, Z., Raftery D. Esophageal cancer metabolite biomarkers detected by LC-MS and NMR methods. PLos One 2012, 7, 1-10.
23. Duncan, M. W.; Nedelkov, D.; Walsh, R.; Hattan, S. J. Applications of MALDI Mass Spectrometry in Clinical Chemistry. Clin. Chem. 2016, 62, 134-143.
24. Kudina, O.; Eral, B.; Mugele, F. e‑MALDI: An Electrowetting-Enhanced Drop Drying Method for MALDI Mass Spectrometry. Anal. Chem. 2016, 88, 4669−4675.
25. Guinan, T.; Kirkbrid, P.; Ronci, M.; Kobus, H.; Voelcker, N. H. Surface-assisted laser desorption ionization mass spectrometry techniques for application in forensics. Mass Spectrom. Rev. 2015, 34, 627-640.
26. Liu, Q.; He L. Ionic matrix for matrix-enhanced surface-assisted laser desorption ionization mass spectrometry imaging (ME-SALDI-MSI). J. Am. Soc. Mass Spectrom. 2009, 20, 2229-2237.
27. Nierodzik, M. L.; Karpatkin, S. Thrombin induces tumor growth, metastasis, and angiogenesis: Evidence for a thrombin-regulated dormant tumor phenotype. Cancer cell 2006, 10, 355-362.
28. Bassus, S.; Herkert, O.; Kronemann, N.; Görlach, A.; Bremerich, D.; Kirchmaier, C. M.; Busse, R.; Schini-Kerth, V. B. Thrombin Causes Vascular Endothelial Growth Factor Expression in Vascular Smooth Muscle Cells: Role of Reactive Oxygen Species. Arteriosclerosis, Thrombosis, and Vascular Biology. 2001, 21, 1550-1555.
29. Jackson, A. L.; Davenport, S. M.; Herzog, T. J.; Coleman, R. L. Targeting angiogenesis: vascular endothelial growth factor and related signaling pathways. Transl. Cancer Res. 2015, 4, 70-83.
30. Ehnman, M.; Östman, A. Therapeutic targeting of platelet-derived growth factor receptors in solid tumors. Expert Opin. Invest. Drugs 2014, 23, 211-226.
31. Turkevich, J.; Stevenson, P. C.; Hillier, J. A study of the nucleation and growth processes in the synthesis of colloidal gold. Discuss. Faraday Soc. 1951, 11, 55-75.
32. So, Y.-H.; Chang, H.-T.; Chiu, W.-J.; Huang, C.-C. Graphene oxide modified with aptamer-conjugated gold nanoparticles and heparin: a potent targeted anticoagulant. Biomater. Sci. 2014, 2, 1332-1337.
33. Bigall, N. C.; Härtling, T.; Klose, M.; Simon, P.; Eng, L. M.; Eychmüller, A. Monodisperse Platinum Nanospheres with Adjustable Diameters from 10 to 100 nm: Synthesis and Distinct Optical Properties. Nano Lett. 2008, 8, 4588-4592.
34. Bock, L. C.;Griffin, L. C.; Latham, J. A.; Vermaas, E. H.; Toole, J. T. Selection of single-stranded DNA molecules that bind and inhibit human thrombin. Nature, 1992, 355, 564-566.
35. Tasset, D. M.; Kubik, M. F.; Steiner, W. Oligonucleotide inhibitors of human thrombin that bind distinct epitopes. J. Mol. Biol., 1997, 272, 688-698.
36. Huang, S.-S.; Wei S.-C., Chang, H.-T.; Lin, H.-J.; Huang, C.-C. Gold Nanoparticles Modified with Self-Assembled Hybrid Monolayer of Triblock Aptamers as a Photoreversible Anticoagulant. Journal of Controlled Release 2016, 221, 9-17.
37. Cordray, M. S.; Amdahl, M.; Richards-Kortum, R. R. Gold nanoparticle aggregation for quantification of oligonucleotides: Optimization and increased dynamic range. Anal. Biochem. 2012, 431, 99-105.
38. Li, Y.-J.; Tseng, Y.-T.; Unnikrishnan, B.; Huang, C.-C. Gold-nanoparticles-modified cellulose membrane coupled with laser desorption/ionization mass spectrometry for detection of iodide in urine. ACS Appl. Mater. Interfaces 2013, 5, 9161-9166.
39. https://www.applichem.com/fileadmin/Broschueren/Transfermembran_e_Internet1.pdf; (accessed June 2016).
40. Cans, A.-S.; Dean, S. L.; Reyes, F. E.; Keating, C. D. Synthesis and characterization of enzyme-Au bioconjugates: HRP and fluorescein-labeled HRP. NanoBiotechnol. 2007, 3, 12–22.