在本篇研究中提出了一個簡單且只需一步的方式來獲得鉑奈米簇。本實驗利用溶菌酶作為模板，並控制在鹼性的條件中來合成極小的鉑奈米簇。利用X光光電子光譜、基質輔助雷射脫附游離飛行時間質譜儀及傅立葉轉換紅外光譜儀來證實鉑奈米簇的形成。以370 nm激發光激發可得到鉑奈米簇最大放射光位置在434 nm。此外，鉑奈米簇展現出良好的量子產率 (8 %)、短的螢光生命週期 (2.7 μs)以及類氧化酶的活性。相較於尺寸較大的鉑奈米粒子，鉑奈米簇利用氧氣作為介質來催化2,2-聯氮-二(3-乙基-苯並噻唑-6-磺酸)二銨鹽 (2,2'-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid, ABTS)、3,3’,5,5’-四甲基聯苯胺 (3,3',5,5'-Tetramethylbenzidine, TMB)和多巴胺 (dopamine)，展現出較佳的催化能力。接著利用TMB作為受質來計算Km及Vmax，其值分別為0.63 mM和2.7 mM‧s-1，此結果意味著此奈米簇對於過氧化物酶受質具有高親和力及高效率的催化能力，並藉由此催化特性來對湖水中之有機汙染物進行降解，如亞甲基藍 (Methylene blue)。
當榖胱甘肽存在時，會對鉑奈米簇造成核蝕刻 (Core-etching)的現象，進而造成螢光焠熄。然而，在沒有任何氧化劑存在的情況下，鉑奈米簇會誘導ABTS產生陽離子自由基，而此陽離子自由基與抗氧化劑具有高反應性。藉此可測量抗氧化劑的活性及偵測穀胱甘肽；其穀胱甘肽的偵測極限為1 μM。此外，利用Ellman’s試劑 (5,5'-dithiobis-(2-nitrobenzoic acid), DTNB)來作為定量穀胱甘肽的標準方法，由t-test (95% confidence level, 4 degrees of freedom) 與 F-test (95% confidence level)中顯示兩者具有類似的定量結果。因此證實鉑奈米簇可用來測量一滴血中穀胱甘肽的濃度。

We present a simple, one-pot approach for synthesizing ultrafine platinum (Pt) nanocluster (NCs) under alkaline conditions using lysozyme (Lys) as a template. Pt NC formation was confirmed using X-ray photoelectron spectrometry, matrix-assisted laser desorption/ionization time-of-flight mass spectrometry, and Fourier-transformed infrared spectroscopy. The maximal fluorescence of Pt NCs appears at 434 nm. Pt NCs exhibit a satisfactory quantum yield, a short fluorescence lifetime, excitation-dependent emission wavelength behavior, and intrinsic oxidase-like activity. Compared with larger Pt nanoparticles (NPs), the Pt NCs produce substantially greater catalytic activity in the O2-mediated oxidation of 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid) (ABTS), 3,3',5,5'-Tetramethylbenzidine (TMB) and dopamine. When incubating the Pt NCs and ABTS, the produced ABTS radical cation was highly reactive toward antioxidants. Thus, without any oxidizing agent, Pt NC-induced formation of ABTS radical cation was used for evaluating antioxidant activity and sensing glutathione (GSH); the limit of detection (LOD) for GSH at a signal-to-noise ratio of 3 was 1 μM. We demonstrated the practicality of using Pt NCs by determining the concentration of GSH in a single drop of blood.

摘要 ............................................................................................................................. I
ABSTRACT ........................................................................................................................................ II
目錄 ...................................................................................................................................... III
圖目錄 .................................................................................................................................... V
縮寫表 ................................................................................................................................. VII
第一章、緒論 ........................................................................................................................ 1
一、前言 ............................................................................................................................ 1
1.1 奈米簇 (Nanocluster) ............................................................................................ 1
1.2 金屬奈米簇的合成方法 ....................................................................................... 2
1.3 金屬奈米簇的應用 ................................................................................................ 3
1.3.1 金屬奈米簇應用於感測器 ............................................................................ 3
1.3.1.1 偵測金屬離子 .......................................................................................... 3
1.3.1.2 偵測硫醇分子 .......................................................................................... 4
1.3.1.3 偵測氰化物 ............................................................................................... 5
1.3.2 金屬奈米簇作為催化劑 ................................................................................ 5
1.3.3 金屬奈米簇應用於生物影像 ....................................................................... 6
1.4 研究動機 ................................................................................................................. 7
第二章、合成溶菌酶修飾鉑奈米簇來作為仿生酵素催化氧化反應以及偵測穀胱
甘肽 ......................................................................................................................................... 8
一、 前言 .......................................................................................................................... 8
二、實驗部分 .................................................................................................................. 11
2.1 實驗藥品 ............................................................................................................... 11
2.2 儀器設備 ............................................................................................................... 15
2.3 實驗過程與樣品配置方法 ................................................................................. 18
三、結果與討論 ............................................................................................................. 23
3.1 探討溶菌酶修飾鉑奈米簇之特性 .................................................................... 23
3.2 鉑奈米簇之光學特性及穩定度 ........................................................................ 29
3.3 溶菌酶修飾鉑奈米簇作為仿生酵素催化受質 .............................................. 35
3.4 鉑奈米簇的動力學探討 ..................................................................................... 42
3.5 不同物質間於氧氣下的催化能力 .................................................................... 44
3.6 鉑奈米簇應用於湖水中有機染料的降解 ....................................................... 49
3.7 穀胱甘肽造成溶菌酶修飾鉑奈米簇的螢光淬熄之路徑探討 .................... 51
3.8 利用鉑奈米簇探討不同抗氧化劑的活性 ....................................................... 55
3.9 利用鉑奈米簇定量穀胱甘肽標準品 ............................................................... 57
3.10 鉑奈米簇選擇性探討 ....................................................................................... 60
3.11 紅血球中穀胱甘肽的定量 ............................................................................... 62
四、結論 .......................................................................................................................... 65
五、參考資料 .................................................................................................................. 66

1. Sherry, L. J.; Chang, S.-H.; Schatz, G. C.; Van Duyne, R. P.; Wiley, B. J.; Xia, Y., Localized Surface Plasmon Resonance Spectroscopy of Single Silver Nanocubes. Nano Letters 2005, 5 (10), 2034-2038.

2. Crespo P Fau - Litran, R.; Litran R Fau - Rojas, T. C.; Rojas Tc Fau - Multigner, M.; Multigner M Fau - de la Fuente, J. M.; de la Fuente Jm Fau - Sanchez-Lopez, J. C.; Sanchez-Lopez Jc Fau - Garcia, M. A.; Garcia Ma Fau - Hernando, A.; Hernando A Fau - Penades, S.; Penades S Fau - Fernandez, A.; Fernandez, A., Permanent magnetism, magnetic anisotropy, and hysteresis of thiol-capped gold nanoparticles.

3. Bigioni, T. P.; Whetten, R. L.; Dag, Ö., Near-Infrared Luminescence from Small Gold Nanocrystals. The Journal of Physical Chemistry B 2000, 104 (30), 6983-6986.

4. Gautier, C.; Bürgi, T., Chiral Inversion of Gold Nanoparticles. Journal of the American
Chemical Society 2008, 130 (22), 7077-7084.

5. (a) Shang, L.; Dong, S.; Nienhaus, G. U., Ultra-small fluorescent metal nanoclusters: Synthesis and biological applications. Nano Today 2011, 6 (4), 401-418; (b) Lu, Y.; Chen, W., Sub-nanometre sized metal clusters: from synthetic challenges to the unique property discoveries. Chemical Society reviews 2012, 41 (9), 3594-623; (c) Xavier, P. L.; Chaudhari, K.; Baksi, A.; Pradeep, T., Protein-protected luminescent noble metal quantum clusters: an emerging trend in atomic cluster nanoscience. Nano reviews 2012, 3.


6. Qian, H.; Jin, R., Ambient Synthesis of Au144(SR)60 Nanoclusters in Methanol. Chemistry of Materials 2011, 23 (8), 2209-2217.

7. Habeeb Muhammed, M.; Ramesh, S.; Sinha, S.; Pal, S.; Pradeep, T., Two distinct fluorescent quantum clusters of gold starting from metallic nanoparticles by pH-dependent ligand etching. Nano Res. 2008, 1 (4), 333-340.

8. Duan, H.; Nie, S., Etching Colloidal Gold Nanocrystals with Hyperbranched and Multivalent Polymers:  A New Route to Fluorescent and Water-Soluble Atomic Clusters. Journal of the American Chemical Society 2007, 129 (9), 2412-2413.

9. Xie, J.; Zheng, Y.; Ying, J. Y., Protein-Directed Synthesis of Highly Fluorescent Gold Nanoclusters. Journal of the American Chemical Society 2009, 131 (3), 888-889.

10. Guo, C.; Irudayaraj, J., Fluorescent Ag Clusters via a Protein-Directed Approach as a Hg(II) Ion Sensor. Analytical Chemistry 2011, 83 (8), 2883-2889.

11. Valden, M.; Lai, X.; Goodman, D. W., Onset of Catalytic Activity of Gold Clusters on Titania with the Appearance of Nonmetallic Properties. Science 1998, 281 (5383), 1647-1650.

12. Belloni, J., Photography: enhancing sensitivity by silver-halide crystal doping. Radiation Physics and Chemistry 2003, 67 (3–4), 291-296.

13. Wang, H.-H.; Lin, C.-A. J.; Lee, C.-H.; Lin, Y.-C.; Tseng, Y.-M.; Hsieh, C.-L.; Chen, C.-H.; Tsai, C.-H.; Hsieh, C.-T.; Shen, J.-L.; Chan, W.-H.; Chang, W. H.; Yeh, H.-I., Fluorescent Gold Nanoclusters as a Biocompatible Marker for In Vitro and In Vivo Tracking of Endothelial Cells. ACS Nano 2011, 5 (6), 4337-4344.

14. Jang, K.; Eom, K.; Lee, G.; Han, J.-H.; Haam, S.; Yang, J.; Kim, E.; Kim, W.-J.; Kwon, T., Water-stable single-walled carbon nanotubes coated by pyrenyl polyethylene glycol for fluorescence imaging and photothermal therapy. BioChip J 2012, 6 (4), 396-403.

15. Holmes, P.; James, K. A. F.; Levy, L. S., Is low-level environmental mercury exposure of concern to human health? Science of The Total Environment 2009, 408 (2), 171-182.

16. Xie, J.; Zheng, Y.; Ying, J. Y., Highly selective and ultrasensitive detection of Hg2+ based on fluorescence quenching of Au nanoclusters by Hg2+-Au+ interactions. Chemical communications 2010, 46 (6), 961-963.

17. Lin, Y.-H.; Tseng, W.-L., Ultrasensitive Sensing of Hg2+ and CH3Hg+ Based on the Fluorescence Quenching of Lysozyme Type VI-Stabilized Gold Nanoclusters. Analytical Chemistry 2010, 82 (22), 9194-9200.

18. Chen, W.; Tu, X.; Guo, X., Fluorescent gold nanoparticles-based fluorescence sensor for Cu2+ ions. Chemical communications 2009, 0 (13), 1736-1738.

19. Shang, L.; Dong, S., Silver nanocluster-based fluorescent sensors for sensitive detection of Cu(II). Journal of Materials Chemistry 2008, 18 (39), 4636-4640.

20. Goswami, N.; Giri, A.; Bootharaju, M. S.; Xavier, P. L.; Pradeep, T.; Pal, S. K., Copper Quantum Clusters in Protein Matrix: Potential Sensor of Pb2+ Ion. Analytical Chemistry 2011, 83 (24), 9676-9680.

21. Chen, X.; Zhou, Y.; Peng, X.; Yoon, J., Fluorescent and colorimetric probes for detection of thiols. Chemical Society reviews 2010, 39 (6), 2120-2135.

22. Han, B.; Wang, E., Oligonucleotide-stabilized fluorescent silver nanoclusters for sensitive detection of biothiols in biological fluids. Biosensors and Bioelectronics 2011, 26 (5), 2585-2589.

23. Shang, L.; Dong, S., Sensitive detection of cysteine based on fluorescent silver clusters. Biosensors and Bioelectronics 2009, 24 (6), 1569-1573.

24. Liu, Y.; Ai, K.; Cheng, X.; Huo, L.; Lu, L., Gold-Nanocluster-Based Fluorescent Sensors for Highly Sensitive and Selective Detection of Cyanide in Water. Advanced Functional Materials 2010, 20 (6), 951-956.

25. Chen, W.; Chen, S., Oxygen Electroreduction Catalyzed by Gold Nanoclusters: Strong Core Size Effects. Angewandte Chemie International Edition 2009, 48 (24), 4386-4389.

26. Wang, X.-X.; Wu, Q.; Shan, Z.; Huang, Q.-M., BSA-stabilized Au clusters as peroxidase mimetics for use in xanthine detection. Biosensors and Bioelectronics 2011, 26 (8), 3614-3619.


27. Judai, K.; Abbet, S.; Wörz, A. S.; Heiz, U.; Henry, C. R., Low-Temperature Cluster Catalysis. Journal of the American Chemical Society 2004, 126 (9), 2732-2737.

28. Vajda, S.; Pellin, M. J.; Greeley, J. P.; Marshall, C. L.; Curtiss, L. A.; Ballentine, G. A.; Elam, J. W.; Catillon-Mucherie, S.; Redfern, P. C.; Mehmood, F.; Zapol, P., Subnanometre platinum clusters as highly active and selective catalysts for the oxidative dehydrogenation of propane. Nature materials 2009, 8 (3), 213-6.

29. Yamamoto, K.; Imaoka, T.; Chun, W. J.; Enoki, O.; Katoh, H.; Takenaga, M.; Sonoi, A., Size-specific catalytic activity of platinum clusters enhances oxygen reduction reactions. Nature chemistry 2009, 1 (5), 397-402.

30. Makarava, N.; Parfenov, A.; Baskakov, I. V., Water-soluble hybrid nanoclusters with extra bright and photostable emissions: a new tool for biological imaging. Biophysical journal 2005, 89 (1), 572-80.

31. Wu, X.; He, X.; Wang, K.; Xie, C.; Zhou, B.; Qing, Z., Ultrasmall near-infrared gold nanoclusters for tumor fluorescence imaging in vivo. Nanoscale 2010, 2 (10), 2244-2249.

32. Herzing, A. A.; Kiely, C. J.; Carley, A. F.; Landon, P.; Hutchings, G. J., Identification
of Active Gold Nanoclusters on Iron Oxide Supports for CO Oxidation. Science 2008, 321 (5894), 1331-1335.

33. Yamamoto, H.; Yano, H.; Kouchi, H.; Obora, Y.; Arakawa, R.; Kawasaki, H., N,N-Dimethylformamide-stabilized gold nanoclusters as a catalyst for the reduction of 4-nitrophenol. Nanoscale 2012, 4 (14), 4148-4154.
34. Dobrin, S., CO oxidation on Pt nanoclusters, size and coverage effects: a density functional theory study. Physical Chemistry Chemical Physics 2012, 14 (35), 12122-12129.

35. (a) Luo, W.; Zhu, C.; Su, S.; Li, D.; He, Y.; Huang, Q.; Fan, C., Self-Catalyzed, Self-Limiting Growth of Glucose Oxidase-Mimicking Gold Nanoparticles. ACS Nano 2010, 4 (12), 7451-7458; (b) Ma, M.; Zhang, Y.; Gu, N., Peroxidase-like catalytic activity of cubic Pt nanocrystals. Colloids and Surfaces A: Physicochemical and Engineering Aspects 2011, 373 (1–3), 6-10; (c) Jv, Y.; Li, B.; Cao, R., Positively-charged gold nanoparticles as peroxidiase mimic and their application in hydrogen peroxide and glucose detection. Chemical communications 2010, 46 (42), 8017-8019; (d) Wang, S.; Chen, W.; Liu, A.-L.; Hong, L.; Deng, H.-H.; Lin, X.-H., Comparison of the Peroxidase-Like Activity of Unmodified, Amino-Modified, and Citrate-Capped Gold Nanoparticles. ChemPhysChem 2012, 13 (5), 1199-1204; (e) Jiang, H.; Chen, Z.; Cao, H.; Huang, Y., Peroxidase-like activity of chitosan stabilized silver nanoparticles for visual and colorimetric detection of glucose. Analyst 2012, 137 (23), 5560-5564.

36. Zheng, J.; Zhang, C.; Dickson, R. M., Highly fluorescent, water-soluble, size-tunable gold quantum dots. Physical review letters 2004, 93 (7), 077402.

37. Petty, J. T.; Zheng, J.; Hud, N. V.; Dickson, R. M., DNA-Templated Ag Nanocluster Formation. Journal of the American Chemical Society 2004, 126 (16), 5207-5212.

38. Kawasaki, H.; Yamamoto, H.; Fujimori, H.; Arakawa, R.; Inada, M.; Iwasaki, Y., Surfactant-free solution synthesis of fluorescent platinum subnanoclusters. Chemical communications 2010, 46 (21), 3759-3761.

39. Tanaka, S.-I.; Miyazaki, J.; Tiwari, D. K.; Jin, T.; Inouye, Y., Fluorescent Platinum Nanoclusters: Synthesis, Purification, Characterization, and Application to Bioimaging. Angewandte Chemie 2011, 123 (2), 451-455.

40. Le Guével, X.; Trouillet, V.; Spies, C.; Jung, G.; Schneider, M., Synthesis of Yellow-Emitting Platinum Nanoclusters by Ligand Etching. The Journal of Physical Chemistry C 2012, 116 (10), 6047-6051.

41. Yang, T.; Li, Z.; Wang, L.; Guo, C.; Sun, Y., Synthesis, Characterization, and Self-Assembly of Protein Lysozyme Monolayer-Stabilized Gold Nanoparticles. Langmuir 2007, 23 (21), 10533-10538.

42. Eby, D. M.; Schaeublin, N. M.; Farrington, K. E.; Hussain, S. M.; Johnson, G. R., Lysozyme Catalyzes the Formation of Antimicrobial Silver Nanoparticles. ACS Nano 2009, 3 (4), 984-994.

43. Luckarift, H. R.; Dickerson, M. B.; Sandhage, K. H.; Spain, J. C., Rapid, Room-Temperature Synthesis of Antibacterial Bionanocomposites of Lysozyme with Amorphous Silica or Titania. Small 2006, 2 (5), 640-643.

44. Chen, W.-Y.; Lin, J.-Y.; Chen, W.-J.; Luo, L.; Wei-Guang Diau, E.; Chen, Y.-C., Functional gold nanoclusters as antimicrobial agents for antibiotic-resistant bacteria. Nanomedicine 2010, 5 (5), 755-764.



45. Chen, T.-H.; Tseng, W.-L., (Lysozyme Type VI)-Stabilized Au8 Clusters: Synthesis Mechanism and Application for Sensing of Glutathione in a Single Drop of Blood. Small 2012, 8 (12), 1912-1919.

46. Tao, Y.; Lin, Y.; Huang, Z.; Ren, J.; Qu, X., Incorporating Graphene Oxide and Gold Nanoclusters: A Synergistic Catalyst with Surprisingly High Peroxidase-Like Activity Over a Broad pH Range and its Application for Cancer Cell Detection. Advanced Materials 2013,
25 (18), 2594-2599.

47. Doerrer, L. H., Steric and electronic effects in metallophilic double salts. Dalton Transactions 2010, 39 (15), 3543-3553.

48. Sennuga, A.; van Marwijk, J.; Whiteley, C. G., Ferroxidase activity of apoferritin is increased in the presence of platinum nanoparticles. Nanotechnology 2012, 23 (3), 035102.

49. Teranishi, T.; Hosoe, M.; Tanaka, T.; Miyake, M., Size Control of Monodispersed Pt
Nanoparticles and Their 2D Organization by Electrophoretic Deposition. The Journal of Physical Chemistry B 1999, 103 (19), 3818-3827.

50. (a) Gao, L.; Zhuang, J.; Nie, L.; Zhang, J.; Zhang, Y.; Gu, N.; Wang, T.; Feng, J.; Yang, D.; Perrett, S.; Yan, X., Intrinsic peroxidase-like activity of ferromagnetic nanoparticles. Nature nanotechnology 2007, 2 (9), 577-83; (b) Liu, C.-H.; Tseng, W.-L., Oxidase-functionalized Fe3O4 nanoparticles for fluorescence sensing of specific substrate. Analytica Chimica Acta 2011, 703 (1), 87-93.


51. (a) Asati, A.; Santra, S.; Kaittanis, C.; Nath, S.; Perez, J. M., Oxidase-Like Activity of Polymer-Coated Cerium Oxide Nanoparticles. Angewandte Chemie International Edition 2009, 48 (13), 2308-2312; (b) He, W.; Liu, Y.; Yuan, J.; Yin, J.-J.; Wu, X.; Hu, X.; Zhang, K.; Liu, J.; Chen, C.; Ji, Y.; Guo, Y., Au@Pt nanostructures as oxidase and peroxidase mimetics for use in immunoassays. Biomaterials 2011, 32 (4), 1139-1147.

52. Bisaglia, M.; Mammi, S.; Bubacco, L., Kinetic and structural analysis of the early oxidation products of dopamine: analysis of the interactions with alpha-synuclein. The Journal of biological chemistry 2007, 282 (21), 15597-605.

53. Dhakshinamoorthy, A.; Navalon, S.; Alvaro, M.; Garcia, H., Metal nanoparticles as heterogeneous Fenton catalysts. ChemSusChem 2012, 5 (1), 46-64.

54. Yuan, X.; Tay, Y.; Dou, X.; Luo, Z.; Leong, D. T.; Xie, J., Glutathione-Protected Silver Nanoclusters as Cysteine-Selective Fluorometric and Colorimetric Probe. Analytical Chemistry 2012, 85 (3), 1913-1919.

55. Chen, W.; Zhao, Y.; Seefeldt, T.; Guan, X., Determination of thiols and disulfides via HPLC quantification of 5-thio-2-nitrobenzoic acid. Journal of pharmaceutical and biomedical analysis 2008, 48 (5), 1375-80.

56. Rahman, I.; Kode, A.; Biswas, S. K., Assay for quantitative determination of glutathione and glutathione disulfide levels using enzymatic recycling method. Nature protocols 2006, 1 (6), 3159-65.