論文第一部分是以自行合成的碲化鎘量子點為輔助基質，對小分子及胜肽標準品進行分析。實驗利用α、β及γ-cyclodextrin的混合物，對碲化鎘量子點濃度做最適化探討；並比較LDI、加入CHCA傳統基質及碲化鎘量子點輔助基質三種方式，發現以碲化鎘量子點作為輔助基質能有效提升訊號絕對強度，證明碲化鎘量子點可作為基質分析小分子胜肽樣品。此部分實驗也在偵測極限上做了探討，所能偵測的樣品最低濃度為12.5 nM。另外，實驗也以碲化鎘量子點作為微波輔助酵素消化蛋白質的親和探針(affinity probe)，將BSA微波消化後，對其胜肽片段進行分析，結果觀察到加入量子點後，能有效增益BSA胜肽片段的訊號強度。
論文第二部分以碲化鎘量子點為輔助基質對蛋白質樣品進行分析，實驗在雷射能量、pH值影響、incubation time及修飾官能基的各種效應上做了探討。結果發現以碲化鎘量子點為輔助基質不僅能成功偵測到蛋白質單一訊號，和傳統基質所觀察到質子化的[M+H]+或[M+2H]2+訊號並不相同，且解析度相較於傳統有機基質可提升6~95倍，推測可能的游離機制為電荷轉移反應，意即碲化鎘量子點和分析物混合形成共結晶，經由雷射轟擊時，碲化鎘量子點內部電子會躍遷至傳導帶中，此時容易進行電子的轉移，產生氧化還原反應，使分析物可形成正電荷自由基(M+．)或負電荷自由基(M-．)。而實驗能測得的質量上限可達78 kDa，為了和SALDI作區分，將此方法命為電荷轉移-量子點直接脫附游離法(charge transfer-quantum dots laser desorption ionization, CT-QDLDI)。實驗也嘗試以高溫燒結的方式，將量子點塗佈於鋁箔基板表面，在高溫下烘烤，冷卻後再點上樣品，此結果有效地提升訊號靈敏度及偵測到的機率。此外，將蛋白質混合檸檬酸銨溶液進行偵測，證明檸檬酸銨為主要質子提供來源，和本實驗所觀察到的高解析現象屬於不同結果，反應機制也不相同。
論文第三部分是以電荷轉移-量子點直接脫附游離法(CT-QDLDI)對小分子結構樣品進行機制探討。文獻報導中，由於選用的基質游離能比分析物低，經由雷射轟擊會產生光游離形成帶正電的自由基(R+•) ，於氣相中再與分析物進行電荷轉移反應，使分析物失去電子形成M+•，因此會觀察到分子離子的訊號。而實驗以半導體材料-碲化鎘量子點為輔助基質，選用perylene及pyrene作為主要分析物，成功的偵測到分析物分子離子峰訊號 (M+•)，而加入氧化劑glucose及1,4-benzoquinone，分析物分子離子訊號靈敏度有增加的趨勢，證明氧化劑有助於電荷轉移的進行。實驗中也意外地發現以鋁箔作為基板時，能有效提升訊號的靈敏度。

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中文摘要 ………………………………………………………………………I
謝誌 …………………………………………………………………………...III
目錄 …………………………………………………………………………...IV
圖表目錄 ………………………………………………………………..…….IX
第一章 緒論 …………………………………………………………………...1
1-1 前言 ………………………………………………………………………..1
1-2 SALDI-MS的發展歷史 …………………………………………………..1
1-2-1 奈米碳管 …………………………………………………………..2
1-2-2鑽石奈米粒子 ………………..……………………………………3
1-2-3金奈米粒子 ………………..………………………………………3
1-2-4 氧化鐵奈米粒子 …………………………………………………..4
1-2-5 硫化鋅奈米粒子………………….………………………………...5
1-3 量子點 (Quantum dots) ……………………………………………………5
1-3-1 量子點特性 ………………………………………………………..5
1-3-2 量子點的合成發展 ………………………………………………..6
1-3-3 量子點的應用 ……………………………………………………..8
1-3-4 量子點在質譜中的應用……………………………………..……10
1-4 研究目標 …………………………………………………………………11
第二章 碲化鎘量子點輔助基質偵測小分子生化樣品及作為蛋白質消化親
和探針.……………………………………………………...………...12
2-1 目的………………………………………………………………..………12
2-2 實驗部分…………………………………………………………………..12
2-2-1 藥品材料 ……………………………………………………….12
2-2-2 實驗儀器 …………………………………………………………14
2-2-3 實驗步驟與流程 …………………………………………………16
2-2-3-1碲化鎘量子點表面修飾之官能基 ……………………..16
2-2-3-2碲化鎘量子點的合成 ……………………………….…..17
2-2-3-3小分子胜肽及蛋白質溶液的配製.………………….…...19
2-2-3-4微波輔助酵素消化蛋白質溶液配製……………..…..….19
2-2-3-5基質溶液的配製 ………………………………….……..19
2-3 結果與討論 ……………………………………………………………....20
2-3-1 碲化鎘量子點材料性質之鑑定 …………………………………20
2-3-1-1以TEM觀察量子點結果 ……………………………...…20
2-3-1-2紫外-可見光吸收光譜與光激發光譜分析 …….………..23
2-3-1-3 FT-IR光譜分析 ……………………………………….…27
2-3-1-4 碲化鎘量子點濃度計算……………………………….....28
2-3-1-5估算溶液中1μL 量子點修飾官能基分子總數 ..............28
2-3-2 以碲化鎘量子點為基質偵測小分子胜肽樣品………………..…29
2-3-2-1 碲化鎘量子點濃度最適化探討 ………………………...30
2-3-2-2 偵測小分子比較LDI、CHCA及加入量子點為基質結果.32
2-3-2-3 α, β, γ-cyclodextrin混合物偵測極限探討 ….………..….34
2-3-3 碲化鎘量子點應用於蛋白質微波輔助酵素消化 …………...….35
2-3-3-1 比較使用CHCA及2,5-DHB基質差異 ……………....….36
2-3-3-2 探討碲化鎘量子點吸收微波輻射效應……………..…...38
2-3-3-3碲化鎘量子點在BSA微波輔助酵素消化反應……….….39
2-3-3-4 cytochrome c加入碲化鎘量子點的微波輔助酵素消化....43
2-3-3-5 Lysozyme加入碲化鎘量子點的微波輔助酵素消化 …...46
2-4 結論 ……………………………………………………………………....50
第三章 碲化鎘量子點為輔助基質偵測蛋白質高解析的應用……………...51
3-1 目的………………………………………………………………………..51
3-2 實驗部分……………………………………………………………..51
3-2-1 藥品及材料………………………………………………………..51
3-2-2 實驗儀器…………………………………………………………..52
3-2-3 實驗樣品製備……………………………………………………..53
3-2-3-1 蛋白質溶液的配製……………………………..………...53
3-2-3-2 傳統基質溶液的配..…………………………..………….53
3-2-3-3 檸檬酸銨溶液的配製………………………..…………...54
3-3 結果與討論……………………………………………………………..…54
3-3-1偵測蛋白質碲化鎘量子點濃度最適化探討 …………..………54
3-3-2 Incubation 效應的探討 ……………………………………...…55
3-3-3 TFA效應探討 ……………………….……………….…………..56
3-3-4雷射能量對蛋白質在質譜中訊號的影響 ……….……...............57
3-3-5 pH值對蛋白質質譜訊號的影響 ………………….………….…59
3-3-6 以表面修飾不同官能基之碲化鎘量子點偵測蛋白質………...61
3-3-7 電荷轉移機制探討………………………………………………..68
3-3-8 以檸檬酸銨證明電荷轉移機制……………………………..……69
3-3-8-1 檸檬酸銨濃度最適化探討………………………….....…69
3-3-8-2 比較檸檬酸銨及檸檬酸結果…………………...……..…70
3-3-8-3 比較蛋白質混合檸檬酸銨溶液中加入量子點之結果.…71
3-3-9 利用高溫燒結對蛋白質高解析進行偵測…….………….…...….73
3-4 結論……………………………………………………………………….79
第四章 利用小分子對碲化鎘量子點電荷轉移機制的探討 ………….……81
4-1 目的……………………………………………………………………..…81
4-2 實驗部份………………………………………………………………..…82
4-2-1 藥品材料………………………………………………………..…82
4-2-2 實驗儀器………………………………………………………..…82
4-2-3 實驗流程與製備………………………………………………..…83
4-2-3-1 碲化鎘量子點溶液………………………………..…...…83
4-2-3-2 基質溶液與小分子溶液的配製…………………….……83
4-3 結果與討論……………………………………………………………..…84
4-3-1 選用特定小分子進行電荷轉移機制探討……………..……84
4-3-2 小分子電荷轉移機制………………………………………..85
4-3-3 小分子於質譜中的電荷轉移……………………………………86
4-3-4 將小分子點樣於鋁箔基板進行電荷轉移探討...……..……..90
4-4 結論………………………………………………………………………98
第五章 總結………………………………………………………………….100
第六章 參考文獻………………………………………………………….…102

1. Sunner, J.; Dratz, E.; Chen, Y.-C.“Graphite surface-assisted laser desorption/ionization time-of-flight mass spectrometry of peptides and proteins from liquid solutions” Anal. Chem. 1995, 67,4335-4342.
2. 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.
3. McLean, J. A.; Stumpo, K. A.; Russell, D. H. “Size-selected (2-10 nm) gold nanoparticles for matrix assisted laser desorption ionization of peptides” J. Am .Chem. Soc. 2005, 127, 5304-5305.
4. Schurenberg, M.; Dreisewerd, K.; Hillenkamp, F. “Laser desorption/ ionization mass spectrometry of peptides and proteins with particle suspension matrixes” Anal. Chem.1999, 71, 221-229.
5. Shrivas, K.; Kailasa, S. K.; Wu, H. F. “Quantum dots laser desorption/ ionization MS:multifunctional CdSe quantum dots as the matrix,
concentrating probes and acceleration for microwave enzymatic digestion for peptide analysis and high resolution detection of proteins in a linear MALDI-TOF MS” Proteomics, 2009, 9, 2656–2667.
6. Huang, J. P.; Yuan, C. H.; Shiea, J.; Chen, Y. C. “Rapid screening for diuretic doping agents in urine by C-60-assisted laser-desorption- ionization-time-of-flight mass spectrometry” J. Anal. Toxicol. 1999, 23, 337-342.
7. Chen, W. Y.; Wang, L. S.; Chiu, H. T.; Chen, Y. C.; Lee, C. Y. “Carbon nanotubes as affinity probes for peptides and proteins in MALDI MS analysis” J. Am. Soc. Mass. Spectrom. 2004, 15, 1629-1635.
8. Xu, S. Y.; Li, Y. F.; Zou, H. F.; Qiu, J. S.; Guo, Z.; Guo, B. C. “Carbon nanotubes as assisted matrix for laser desorption/ionization time-of-flight mass spectrometry” Anal. Chem. 2003, 75, 6191-6195.
9. Ren, S.-F.; Guo, Y.-L. “Oxidized carbon nanotubes as matrix for matrix- assisted laser desorption/ionization time-of-flight mass spectrometric analysis of biomolecules” Rapid Commun. Mass Spectrom. 2005, 19, 255-260.
10. Kong, X. L.; Huang, L. C.; Hsu, C. M.; Chen, W. H.; Han, C. C.; Chang , H. C.“ High-affinity capture of proteins by diamond nano- particles for mass spectrometric analysis” Anal. Chem. 2005, 77, 259-265.
11. Kong, X.; Huang, L. C.; Liau, S. C.; Han,C. C.; Chang, H. C. “Polylysine -Coated Diamond Nanocrystals for MALDI-TOF Mass Analysis of DNA Oligonucleotides” Anal. Chem. 2005, 77, 4273-4277.
12. Huang, Y.-F.; Chang, H.-T. “Nile red-adsorbed gold nanoparticle matrixes for determining aminothiols through surface-assisted laser desorption/ ionization mass spectrometry” Anal. Chem. 2006, 78, 1485-1493.
13. Chen, W.-Y.; Chen, Y.-C. “Affinity-based mass spectrometry using magnetic iron oxide particles as the matrix and concentrating probes for SALDI MS analysis of peptides and proteins” Anal. Bioanal. Chem. 2006, 386, 699-704.
14. Kailasa, S. K.; Wu, H. F.“Interference free detection for small molecules: Probing the Mn2+-doped effect and cysteine capped effect on the ZnS nanoparticles for coccidiostats and peptide analysis in SALDI-TOF MS” Analyst, 2010, 135, 1115 – 1123.
15. Quinlan, F.T.; Kuther, J.; Tremel, W.; Knoll, W.; Risbud, S.; Stroeve, P. “Reverse micelle synthesis and characterization of ZnSe nano- particles” Langmuir, 2000, 16, 4049-4051.
16. Medintz, I. L.; Uyeda, H. T.; Goldman, E. R.; Mattoussi, H. “Quantum dot bioconjugates for imaging, labelling and sensing” Nature Materials, 2005, 4, 435-446.
17. Murray, C. B.; Norris, D. J.; Bawendi, M. G. “Synthesis and characterization of nearly monodisperse CdE (E = S, Se, Te) semiconductor nanocrystallites”J. Am. Chem. Soc. 1993, 115, 8706-8715.
18. Mews, A.; Eychmiiller, A.; Giersig, M.; Schooss, D.; Weller, H. “Preparation, characterization, and photophysics of the quantum dot quantum well system CdS/HgS/CdS” J. Phys. Chem. 1994, 98, 934-941.
19. Hines, M. A.; Philippe, G.-S. “Synthesis and characterization of strongly luminescing ZnS-capped CdSe nanocrystals” J. Phys. Chem. 1996, 100, 468-471.
20. Peng, Z. A.; Peng, X. “Formation of high-quality CdTe, CdSe, and CdS nanocrystals using CdO as precursor” J. Am. Chem. Soc. 2001, 123, 183-184.
21. Willard, D. M.; Carillo, L. L.; Jung, J.; Van Orden, A. “CdSe-ZnS quantum dots as resonance energy transfer donors in a model protein-protein binding assay.” Nano Lett.2001, 1, 469–474.
22. Gerion, D.; Pinaud, F.; Williams, S. C.; Parak, W. J.; Zanchet, D.; Weiss, S.; Alivisatos, A. P. “Synthesis and properties of biocompatible water-soluble silicacoated CdSe/ZnS semiconductor quantum dots” J. Phys. Chem. B. 2001, 105, 8861–8871.
23. Pellegrino, T.; Parak, W. J.; Boudreau, R.; Legros, M. A.; Gerion, D.; Alivisatos, A. P.; Larabell, C. A. “Quantum dot-based cell motility assay” Differentiation. 2003, 71,542–548.
24. Osaki, F.; Kanamori, T.; Sando, S.; Sera, T.; Aoyama, Y. “A quantum dot conjugated sugar ball and its cellular uptake on the size effects of endocytosis in the subviral region” J. Am. Chem. Soc. 2004, 126, 6520–6521.
25. Gao, X.; Cui, Y.; Levenson, R. M.; Chung, L. W. K.; Nie, S. “In vivo cancer targeting and imaging with semiconductor quantum dots” Nature Biotechnol.2004, 22, 969–976.
26. Pinaud, F.; King, D.; Moore, H.-P.; Weiss, S. “Bioactivation and cell targeting of semiconductor CdSe/ZnS nanocrystals with phytochelatin -related peptides” J. Am. Chem. Soc.2004, 126, 6115–6123.
27. 揚智慧, 黃耿祥, 王英基, 林裕城, 量子點-奈米彩虹標籤, 科學發展, 2008, 422期, 46.
28. Zhang, H.; Zhou, Z.; Yang, B. “The influence of carboxyl Groups on the photoluminescence of mercaptocarboxylic acid-stabilized CdTe
nanoparticles” J. Phys. Chem. B, 2003, 107, 8-13.
29. Shastri, L. A.; Kailasa, S. K.; Wu, H. F. “Cysteine-capped ZnSe quantum dots as affinity and accelerating probes for microwave enzymatic digestion of proteins via direct matrix-assisted laser desorption/ionization time-of-flight mass spectrometric analysis” Rapid Commun. Mass Spectrom. 2009, 23, 2247–2252.
30. Giz, M. J.; Duong, B.; Tao, N. J. “In situ STM study of self-assembled mercaptopropionic acid monolayers for electrochemical detection of dopamine” J. Electro. Chem.1999, 465,72.
31. Lo, C. Y.; Chen, W. Y.; Chen, C. T.; Chen, Y. C. “Rapid enrichment of phosphopeptides from tryptic digests of proteins using iron oxide nanocomposites of magnetic particles coated with zirconia as the concentrating probes” J. Proteome Res., 2007, 6 (2), 887–893.
32. Idowu, M.; Lamprecht, E.; Nyokong, T. “Interaction of water-soluble thiol capped CdTe quantum dots and bovine serum albumin” Journal of Photochemistry and Photobiology A: Chemistry, 2008, 198, 7-12.
33. Huanga, C. C.; Chang, H. T. “Parameters for selective colorimetric sensing of mercury(II) in aqueous solutions using mercaptopropionic acid-modified gold nanoparticles” Chem. Commun., 2007, 1215–1217.
34. Hagerman, A. E.; Butler, L. G. “Protein precipitation method for the quantitative determination of tannins” J. Agric. Food Chem., 1978, 26, 809-812.
35. Hao, E.; Zhang, H.; Yang, B.; Ren, H.; Shen, J. “Preparation of luminescent polyelectrolyte/Cu-doped ZnSe nanoparticle multilayer composite films” J.Colloid Interface Sci. 2001, 238, 285–290.
36. Qiao, L.; Bi, H.; Busnel, J.-M.; Liu, B.; Girault, H. H.“In-source photocatalytic reduction of disulfide bonds during laser desorption ionizationw” Chem. Commun. 2008, 6357–6359.
37. Qiao, L.; Bi, H.; Busnel, J.-M.; Waser, J.; Yang, P.; Girault, H. H.; Liu, B. “Photocatalytic redox reactions for in-source peptide fragmentation” Chem. Eur. J. 2009, 15, 6711 – 6717.
38. Masumoto, Y.; Sonobe, K. “Size-dependent energy levels of CdTe quantum dots” Physical Review B, 1997, 56, 9734-9737.
39. Chiang, C.-K.; Chiang, N.-C.; Lin, Z.-H.; Lan, G.-Y.; Lin, Y.-W.; Chang, H.-T. “Nanomaterial-based surface-assisted laser desorption /ionization mass spectrometry of peptides and proteins” J Am Soc Mass Spectrom, 2010, 21, 1204-1207.
40. Chiang, C.-K.; Yang, Z.; Lin, Y.-W.; Chen, W.-T.; Lin, H.-J.; Chang, H.-T. “Detection of proteins and protein-ligand complexes using HgTe nanostructure matrixes in surface-assisted laser desorption /ionization mass spectrometry” Anal. Chem., 2010, 82, 4543–4550.
41. Macha, S. F.; McCarley, T. D.; Limbach, P. A. “Influence of ionization energy on charge-transfer ionization in matrix-assisted laser desorption/ionization mass spectrometry” Analytica Chimica Acta, 1999, 397, 235-245.
42. McCarley, T. D.; McCarley, R. L.; Limbach, P. A. “Electron-transfer ionization in matrix-assisted laser desorption/ionization mass spectrometry” Anal. Chem., 1998, 70, 4376-4379.
43. http://www.engineeringtoolbox.com/thermal-conductivity-d_429.html