本篇論文以金奈米粒子（Gold nanoparticles﹐AuNPs）偵測生物小分子及酵素活性，分為以下兩部分：
一、利用修飾氟界面活性劑的金奈米粒子（FSN-capped gold nanoparticles﹐FSN-AuNPs）與鄰苯二醛（o-Phthaldialdehyde﹐OPA）偵測尿液中同半胱胺酸（Homocysteine﹐HCys）：本篇研究中，使用修飾氟界面活性劑的金奈米粒子為萃取試劑、鄰苯二醛為衍生試劑，結合兩者可選擇性偵測樣品溶液中同半胱胺酸。以FSN-AuNPs萃取樣品內HCys及半胱胺酸（Cysteine﹐Cys），接著離心將上清液移除，隨後加入2-硫基乙醇（2-Mercaptoethanol﹐2-ME）取代鍵結於奈米粒子表面之HCys與Cys，將其釋放至溶液中，並以OPA衍生，激發波長370 nm，HCys與OPA衍生後放射波長於485 nm有最大螢光值，但是Cys與OPA反應在波長457 nm僅有非常微弱的螢光，因此可選擇性偵測樣品溶液中HCys。與其他種類的胺基硫醇（Aminothiols）相較下，此系統偵測HCys之選擇性可達一百倍以上，HCys偵測極限為180 nM，最後也成功地運用這項技術於尿液樣品中HCys含量的檢測。
二、以修飾氟界面活性劑的金奈米粒子比色試驗偵測S-腺苷高半胱胺酸水解酶（S-adenosylhomocysteine Hydrolase﹐SAHH）活性及抑制效應：此篇研究我們利用FSN-AuNPs做為感測器，偵測S-腺苷高半胱胺酸水解酶酵素活性及其抑制劑對酵素活性的影響，設計一套免標記（Label free）、方便、靈敏、選擇性佳且肉眼（Naked eyes）即可辨識的分析方法。SAHH催化S-腺苷高半胱胺酸（S-adenosylhomocysteine﹐SAH）水解產生HCys。由於SAH結構中沒有裸露的硫醇基，很難吸附於FSN-AuNPs表面，一旦溶液中含有SAHH催化SAH水解而產生HCys，HCys於FSN-AuNPs表面形成Au-S鍵結，並且透過粒子間表面電荷降低或氫鍵作用力，促使金奈米粒子聚集，因此利用溶液顏色的變化可偵測SAHH酵素活性。另一方面，腺苷類似物（Adenosine analogs）可以有效地抑制SAHH活性，因此實驗中討論腺苷、單磷酸腺苷（Adenosine monophosphate﹐AMP）、雙磷酸腺苷（Adenosine diphosphate﹐ADP）及三磷酸腺苷（Adenosine triphosphate﹐ATP）等四種腺苷類似物對SAHH活性的影響，利用FSN-AuNPs為感測器分析抑制劑抑制酵素活性的效率，Adenosine與AMP、ADP及ATP相較，低濃度劑量即可抑制SAHH酵素活性。

none

摘要I
目錄III
圖表目錄V
縮寫表VIII
第一章、利用修飾氟界面活性劑的金奈米粒子與鄰苯二醛偵測尿液中同半胱胺酸
壹、前言1
貳、實驗部分9
　一、藥品與樣品製備9
　二、儀器設備13
　三、實驗過程17
參、結果與討論19
　一、利用FSN-AuNPs萃取溶液中之HCys19
　二、FSN-AuNPs濃度的影響26
　三、選擇性、靈敏度及應用30
肆、結論38
伍、參考文獻39

第二章、以修飾氟界面活性劑的金奈米粒子之比色試驗偵測S-腺苷高半胱胺酸水解酶活性及抑制效應
壹、前言48
貳、實驗部分55
　一、藥品與樣品製備55
　二、儀器設備58
　三、實驗過程61
參、結果與討論63
　一、利用FSN-AuNPs為感測器偵測酵素水解產物63
　二、SAHH酵素活性試驗69
　三、抑制劑效率與比色試驗73
肆、結論79
伍、參考文獻80

第一章、利用修飾氟界面活性劑的金奈米粒子與鄰苯二醛偵測尿液中同半胱胺酸
1. Refsum, H.; Ueland, P. M.; Nygard, O.; Vollset, S. E. “Homocysteine and cardiovascular disease” Annu. Rev. Med. 1998, 49, 31-62.
2. Seshadri, S.; Beiser, A.; Selhub, J.; Jacques, P. F.; Rosenberg, I. H.; D'Agostino, R. B.; Wilson, P. W.; Wolf, P. A. “Homocysteine and dementia” N. Engl. J. Med. 2002, 346, 476-483.
3. Lochman, P.; Adam, T.; Friedecký, D.; Hlídková, E.; Skopková, Z. “High-throughput capillary electrophoretic method for determination of total aminothiols in plasma and urine” Electrophoresis 2003, 24, 1200-1207.
4. Refsum, H.; Smith, A. D.; Ueland, P. M.; Nexo, E.; Clarke, R.; McPartlin, J.; Johnston, C.; Engbaek, F.; Schneede, J.; McPartlin, C.; Scott, J. M. “Facts and recommendations about total homocysteine determinations: an expert opinion” Clin. Chem. 2004, 50, 3-32.
5. Nekrassova, O.; Lawrence, N. S.; Compton, R. G. “Analytical determination of homocysteine: a review” Talanta 2003, 60, 1085-1095.
6. Steegers-Theunissen, R. P.; Boers, G. H.; Trijbels, F. J.; Eskes, T. K. “Neural-tube defects and derangement of homocysteine metabolism” N. Engl. J. Med. 1991, 324, 199-200.
7. van Meurs, J. B.; Dhonukshe-Rutten, R. A.; Pluijm, S. M.; van der Klift, M.; de Jonge, R.; Lindemans, J.; de Groot, L. C.; Hofman, A.; Witteman, J. C.; van Leeuwen, J. P.; Breteler, M. M.; Lips, P.; Pols, H. A.; Uitterlinden, A. G. “Homocysteine levels and the risk of osteoporotic fracture” N. Engl. J. Med. 2004, 350, 2033-2041.
8. Wu, L. L.; Wu, J. T. “Hyperhomocysteinemia is a risk factor for cancer and a new potential tumor marker” Clin. Chim. Acta 2002, 322, 21-28.
9. Satterfield, M. B.; Sniegoski, L. T.; Welch, M. J.; Nelson, B. C.; Pfeiffer, C. M. “Comparison of isotope dilution mass spectrometry methods for the determination of total homocysteine in plasma and serum” Anal. Chem. 2003, 75, 4631-4638.
10. Nelson, B. C.; Satterfield, M. B.; Sniegoski, L.T.; Welch, M. J. “Simultaneous quantification of homocysteine and folate in human serum or plasma using liquid chromatography/tandem mass spectrometry” Anal. Chem. 2005, 77, 3586-3593.
11. Nelson, B. C.; Pfeiffer, C. M.; Sniegoski, L. T.; Satterfield, M. B. “Development and evaluation of an isotope dilution LC/MS method for the determination of total homocysteine in human plasma” Anal Chem. 2003, 75,775-784.
12. Bayle, C.; Caussé, E.; Couderc, F. “Determination of aminothiols in body fluids, cells, and tissues by capillary electrophoresis” Electrophoresis 2004, 25, 1457-1472.
13. Zhang, D.; Zhang, M.; Liu, Z., Yu, M.; Li, F.; Yi, T.; Huang, C. “Highly selective colorimetric sensor for cysteine and homocysteine based on azo derivatives” Tetrahedron Lett. 2006, 47, 7093-7096.
14. Rusin, O.; St Luce, N. N.; Agbaria, R. A.; Escobedo, J. O.; Jiang, S.; Warner, I. M.; Dawan, F. B.; Lian, K.; Strongin, R. M. “Visual detection of cysteine and homocysteine” J. Am. Chem. Soc. 2004, 126, 438-439.
15. Wang, W.; Rusin, O.; Xu, X.; Kim, K. K.; Escobedo, J. O.; Fakayode, S. O.; Fletcher, K. A.; Lowry, M.; Schowalter, C. M.; Lawrence, C. M.; Fronczek, F. R.; Warner, I. M.; Strongin, R. M. “Detection of Homocysteine and Cysteine” J. Am. Chem. Soc. 2005, 127, 15949-15958.
16. Kawatsura, M.; Ata, F.; Hirakawa, T.; Hayase, S.; Itoh, T. “Ruthenium-catalyzed linear selective allylic aminations of monosubstituted allyl acetates” Tetrahedron Lett. 2008, 49, 4873-4875.
17. Chen, H.; Zhao, Q.; Wu, Y.; Li, F.; Yang, H.; Yi, T.; Huang, C. “Selective Phosphorescence Chemosensor for Homocysteine Based on an Iridium(III) Complex” Inorg. Chem. 2007, 46, 11075-11081.
18. Wang, W.; Escobedo, J. O.; Lawrence, C. M.; Strongin, R. M. “Direct detection of homocysteine” J. Am. Chem. Soc. 2004, 126, 3400-3401.
19. Zhang, M.; Yu, M.; Li, F.; Zhu, M.; Li, M.; Gao, Y.; Li, L.; Liu, Z.; Zhang, J.; Zhang, D.; Yi, T.; Huang, C. “A Highly Selective Fluorescence Turn-on Sensor for Cysteine/Homocysteine and Its Application in Bioimaging” J. Am. Chem. Soc. 2007, 129, 10322-10323.
20. Zhang, F. X.; Han, L.; Israel, L. B.; Daras, J. G.; Maye, M. M.; Ly, N. K.; Zhong, C. J. “Colorimetric detection of thiol-containing amino acids using gold nanoparticles” Analyst 2002, 127, 462-465.
21. Chen, S.-J.; Chang, H.-T. “Nile red-adsorbed gold nanoparticles for selective determination of thiols based on energy transfer and aggregation” Anal. Chem. 2004, 76, 3727-3734.
22. Hong, R.; Fernández, J. M.; Nakade, H.; Arvizo, R.; Emrick, T.; Rotello, V. M. “In situ observation of place exchange reactions of gold nanoparticles. Correlation of monolayer structure and stability” Chem. Commun. 2006, 2347-2349.
23. Lim, I. I.; Ip, W.; Crew, E.; Njoki, P. N.; Mott, D.; Zhong, C. J.; Pan, Y.; Zhou, S. “Homocysteine-mediated reactivity and assembly of gold nanoparticles” Langmuir 2007, 23, 826-833.
24. Lim, I. I.; Mott, D.; Engelhard, M. H.; Pan, Y.; Kamodia, S.; Luo, J.; Njoki, P. N.; Zhou, S.; Wang, L.; Zhong, C. J. “Interparticle chiral recognition of enantiomers: a nanoparticle-based regulation strategy” Anal. Chem. 2009, 81, 689-698.
25. Daniel, M. C.; Astruc, D. “Gold nanoparticles: assembly, supramolecular chemistry, quantum-size-related properties, and applications toward biology, catalysis, and nanotechnology” Chem. Rev. 2004, 104, 293-346.
26. Stewart, M. E.; Anderton, C. R.; Thompson, L. B.; Maria, J.; Gray, S. K.; Rogers, J. A.; Nuzzo, R. G. “Nanostructured plasmonic sensors” Chem. Rev. 2008, 108, 494-521.
27. Rance, G. A.; Marsh, D. H.; Khlobystov, A. N. “Extinction coefficient analysis of small alkanethiolate-stabilised gold nanoparticles” Chem. Phys. Lett. 2008, 460, 230-236.
28. Thomas, K. G.; Kamat, P. V. “Chromophore-functionalized gold nanoparticles” Acc. Chem. Res. 2003, 36, 888-898.
29. Lu, C.; Zu, Y.; Yam, V. W. “Specific postcolumn detection method for HPLC assay of homocysteine based on aggregation of fluorosurfactant-capped gold nanoparticles” Anal. Chem. 2007, 79, 666-672.
30. Tang, Y.; Yan, J.; Zhou, X.; Fu, Y.; Mao, B. “An STM study on nonionic fluorosurfactant zonyl FSN self-assembly on Au(111): large domains, few defects, and good stability” Langmuir 2008, 24, 13245-13249.
31. Huang, C.-C.; Tseng, W.-L. “ Role of fluorosurfactant-modified gold nanoparticles in selective detection of homocysteine thiolactone: remover and sensor” Anal. Chem. 2008, 80, 6345-6350.
32. Wu, H.-P.; Huang, C.-C.; Tseng, W.-L. “Sodium hydroxide as pretreatment and fluorosurfactant-capped gold nanoparticles as sensor for the highly selective detection of cysteine” Talanta 2008, 76, 347-352.
33. Lu, C.; Zu, Y. “ Specific detection of cysteine and homocysteine: recognizing onemethylene difference using fluorosurfactant-capped gold nanoparticles” Chem. Commun. 2007, 3871-3873.
34. Thaxton, C. S.; Hill, H. D.; Georganopoulou, D. G.; Stoeva, S. I.; Mirkin, C. A. “A bio-bar-code assay based upon dithiothreitol-induced oligonucleotide release” Anal. Chem. 2005, 77, 8174-8178.
35. Hurst, S. J.; Lytton-Jean, A. K.; Mirkin, C. A. “Maximizing DNA loading on a range of gold nanoparticle sizes” Anal. Chem. 2006, 78, 8313-8318.
36. Li, M.-D.; Cheng, T,-L.; Tseng, W.-L. “Nonionic surfactant-capped gold nanoparticles for selective enrichment of aminothiols prior to CE with UV absorption detection” Electrophoresis 2009, 30, 388-395.
37. Zuman, P. “Reactions of orthophthalaldehyde with nucleophiles” Chem. Rev. 2004, 104, 3217-3238.
38. Benson, J. R.; Hare, P. E. “o-phthalaldehyde: fluorogenic detection of primary amines in the picomole range. Comparison with fluorescamine and ninhydrin” Proc. Natl. Acad. Sci. U. S. A. 1975, 72, 619-622.
39. Tcherkas, Y. V.; Denisenko, A. D. “Simultaneous determination of several amino acids, including homocysteine, cysteine and glutamic acid, in human plasma by isocratic reversed-phase high-performance liquid chromatography with fluorimetric detection” J. Chromatogr. A 2001, 913, 309-313.
40. Lee, P. C.; Meisel, D. “Adsorption and surface-enhanced Raman of dyes on silver and gold sols” J. Phys. Chem. 1982, 86, 3391-3395.
41. Huang, C.-C.; Chen, C.-T.; Shiang, Y.-C.; Lin, Z.-H.; Chang, H.-T. “Synthesis of fluorescent carbohydrate-protected Au nanodots for detection of Concanavalin A and Escherichia coli” Anal. Chem. 2009, 81, 875-882.
42. Yu, C.-J.; Tseng, W.-L. “Online concentration and separation of basic proteins using a cationic polyelectrolyte in the presence of reversed electroosmotic flow” Electrophoresis 2006, 27, 3569-3577.
43. Chang, C.-W.; Tseng, W.-L. “Gold nanoparticle extraction followed by capillary electrophoresis to determine the total, free, and protein-bound aminothiols in plasma” Anal. Chem. 2010, 82, 2696-2702.
44. Mukai, Y.; Togawa, T.; Suzuki, T.; Ohata, K.; Tanabe, S. “Determination of homocysteine thiolactone and homocysteine in cell cultures using high-performance liquid chromatography with fluorescence detection” J. Chromatogr. B 2002, 767, 263-268.
45. Chwatko, G.; Jakubowski, H. “Urinary excretion of homocysteine-thiolactone in humans” Clin. Chem. 2005, 51, 408-415.
46. Seiwert, B.; Karst, U. “Simultaneous LC/MS/MS determination of thiols and disulfides in urine samples based on differential labeling with ferrocene-based maleimides” Anal. Chem. 2007, 79, 7131-7138.
47. Fiskerstrand, T.; Refsum, H.; Kvalheim, G.; Ueland, P. M. “Homocysteine and other thiols in plasma and urine: automated determination and sample stability” Clin. Chem. 1993, 39, 263-271.
第二章、以修飾氟界面活性劑的金奈米粒子之比色試驗偵測S-腺苷高半胱胺酸水解酶活性及抑制效應
1. Nelson, D. L.; Cox, M, M. Cox Lehninger Principles of Biochemistry, 4th ed.; W. H. Freeman & company: New York, 2004.
2. Ghadiali, J. E.; Stevens, M. M. “Enzyme-Responsive Nanoparticle Systems” Adv. Mater. 2008, 20, 4359-4363.
3. Xu, X.; Han, M. S.; Mirkin, C. A. “A gold-nanoparticle-based real-time colorimetric screening method for endonuclease activity and inhibition” Angew. Chem. Int. Ed. 2007, 46, 3468-3470.
4. Song, G.; Chen, C.; Ren, J.; Qu, X. “A simple, universal colorimetric assay for endonuclease/methyltransferase activity and inhibition based on an enzyme-responsive nanoparticle system” ACS Nano 2009, 3, 1183-1189.
5. Laromaine, A.; Koh, L.; Murugesan, M.; Ulijn, R. V.; Stevens, M. M. “Protease-triggered dispersion of nanoparticle assemblies” J. Am. Chem. Soc. 2007, 129, 4156-4157.
6. Guarise, C.; Pasquato, L.; De Filippis, V.; Scrimin, P. “Gold nanoparticles-based protease assay” Proc. Natl. Acad. Sci. U. S. A. 2006, 103, 3978-3982.
7. Liu, R.; Liew, R.; Zhou, J.; Xing, B. “A simple and specific assay for real-time colorimetric visualization of beta-lactamase activity by using gold nanoparticles” Angew. Chem. Int. Ed. 2007, 46, 8799-8803.
8. Jiang, T.; Liu, R.; Huang, X.; Feng, H.; Teo, W.; Xing, B. “Colorimetric screening of bacterial enzyme activity and inhibition based on the aggregation of gold nanoparticles” Chem. Commun. 2009, 1972-1974.
9. Wang, M.; Gu, X.; Zhang, G.; Zhang, D.; Zhu, D. “Continuous colorimetric assay for acetylcholinesterase and inhibitor screening with gold nanoparticles” Langmuir 2009, 25, 2504-2507.
10. Shen, Q.; Nie, Z.; Guo, M.; Zhong, C. J.; Lin, B.; Li, W.; Yao, S. “Simple and rapid colorimetric sensing of enzymatic cleavage and oxidative damage of single-stranded DNA with unmodified gold nanoparticles as indicator” Chem. Commun. 2009, 929-931.
11. Lou, X.; Xiao, Y.; Wang, Y.; Mao, H.; Zhao, J. “Label-free colorimetric screening of nuclease activity and substrates by using unmodified gold nanoparticles” Chembiochem 2009, 10, 1973-1977.
12. Choi, Y.; Ho, N. H.; Tung, C. H. “Sensing phosphatase activity by using gold nanoparticles” Angew. Chem. Int. Ed. 2007, 46, 707-709.
13. Zhao, W.; Chiuman, W.; Lam, J. C. F.; Brook, M. A.; Li, Y. “Simple and rapid colorimetric enzyme sensing assays using non-crosslinking gold nanoparticle aggregation” Chem. Commun. 2007, 3729-3731.
14. Nakanishi, M. “S-adenosyl-L-homocysteine hydrolase as an attractive target for antimicrobial drugs” Yakugaku Zasshi 2007, 127, 977-982.
15. Carlucci, F.; Tabucchi, A.; Aiuti, A.; Rosi, F.; Floccari, F.; Pagani, R.; Marinello, E. “Capillary electrophoresis in diagnosis and monitoring of adenosine deaminase deficiency” Clin. Chem. 2003, 49, 1830-1838.
16. Yuan, C. S.; Ault-Riche, D. B.; Borchardt, R. T. “Chemical modification and site-directed mutagenesis of cysteine residues in human placental S-adenosylhomocysteine hydrolase” J. Biol. Chem. 1996, 271, 28009-28016.
17. Baric, I.; Fumic, K.; Glenn, B.; Cuk, M.; Schulze, A.; Finkelstein, J. D.; James, S. J.; Mejaski-Bosnjak, V.; Pazanin, L.; Pogribny, I. P.; Rados, M.; Sarnavka, V.; Scukanec-Spoljar, M.; Allen, R. H.; Stabler, S.; Uzelac, L.; Vugrek, O.; Wagner, C.; Zeisel, S.; Mudd, S. H. “S-adenosylhomocysteine hydrolase deficiency in a human: a genetic disorder of methionine metabolism” Proc. Natl. Acad. Sci. U. S. A. 2004, 101, 4234-4239.
18. Struys, E. A.; Jansen, E. E. W.; de Meer, K.; Jakobs, C. “Determination of S-adenosylmethionine and S-adenosylhomocysteine in plasma and cerebrospinal fluid by stable-isotope dilution tandem mass spectrometry” Clin. Chem. 2000, 46, 1650-1656.
19. Sbrana, E.; Bramanti, E.; Spinetti, M. C.; Raspi, G. “S-Adenosyl methionine/S-adenosyl-L-homocysteine ratio determination by capillary electrophoresis employed as a monitoring tool for the antiviral effectiveness of adenosine analogs” Electrophoresis 2004, 25, 1518-1521.
20. Huang, C.-C.; Tseng, W.-L. “Role of fluorosurfactant-modified gold nanoparticles in selective detection of homocysteine thiolactone: remover and sensor” Anal. Chem. 2008, 80, 6345-6350.
21. Lin, J.-H.; Chang, C.-W.; Tseng, W.-L. “Fluorescent sensing of homocysteine in urine: using fluorosurfactant-capped gold nanoparticles and o-Phthaldialdehyde” Analyst 2010, 135, 104-110.
22. Lu, C.; Zu, Y.; Yam, V. W. “Specific postcolumn detection method for HPLC assay of homocysteine based on aggregation of fluorosurfactant-capped gold nanoparticles” Anal. Chem. 2007, 79, 666-672.
23. De Clercq, E. “Carbocyclic adenosine analogues as S-adenosylhomocysteine hydrolase inhibitors and antiviral agents: recent advances” Nucleosides Nucleotides 1998, 17, 625-634.
24. Kloor, D.; Yao, K.; Delabar, U.; Osswald, H. “Simple and sensitive binding assay for measurement of adenosine using reduced S-adenosylhomocysteine hydrolase” Clin. Chem. 2000, 46, 537-542.
25. Huang, C.-C.; Chen, C.-T.; Shiang, Y.-C.; Lin, Z.-H.; Chang, H.-T. Anal. Chem. 2009, 81, 875-882.
26. Thaxton, C. S.; Hill, H. D.; Georganopoulou, D. G.; Stoeva, S. I.; Mirkin, C. A. “A bio-bar-code assay based upon dithiothreitol-induced oligonucleotide release” Anal. Chem. 2005, 77, 8174-8178.
27. Ueland, P. M. “Pharmacological and biochemical aspects of S-adenosylhomocysteine and S-adenosylhomocysteine hydrolase” Pharmacol. Rev. 1982, 34, 223-253.
28. Wu, C.-S.; Wu, C.-T.; Yang, Y.-S.; Ko, F.-H. “An enzymatic kinetics investigation into the significantly enhanced activity of functionalized gold nanoparticles” Chem. Commun. 2008, 14, 5327-5329.
29. Zhang, S.; Xia, J.; Li, X. “Electrochemical biosensor for detection of adenosine based on structure-switching aptamer and amplification with reporter probe DNA modified Au nanoparticles” Anal. Chem. 2008, 80, 8382-8388.
30. Lu, N.; Shao, C.; Deng, Z. “Rational design of an optical adenosine sensor by conjugating a DNA aptamer with split DNAzyme halves” Chem. Commun. 2008, 6161-6163.
31. Xu, W.; Lu, Y. “Label-free fluorescent aptamer sensor based on regulation of malachite green fluorescence” Anal. Chem. 2010, 82, 574-578.