近年來貴金屬奈米簇已經被廣泛研究，而銅奈米簇的研究相對稀少，銅元素與金、銀包含在同一族元素週期表中，且具備地球含量豐富、價格低廉和對人類相對較友善等特點。本研究中，直接以胃蛋白酶為模板，使具有高生物相容性及不須額外修飾的優點，以及使用低毒性的NaBH4及Ascorbic acid為還原劑，在鹼性環境下成功合成出以420 nm為激發波長，在496 nm處具有放射波長的銅奈米簇，推論以Cu13為主要合成的產物。之後利用血紅蛋白為吸收團，對銅奈米簇造成螢光內濾效應，利用此螢光強度降低的現象來偵測血紅蛋白，並可成功應用於真實尿液樣品中血紅蛋白的偵測，此方法方便快速，能對血紅蛋白進行專一性偵測，偵測極限為70 nM。此外在不同酸鹼環境中，做為模板的胃蛋白酶隨著官能基產生質子化與去質子化的情況，使其結構產生改變，導致其螢光強度有所變化，利用其螢光強度的變化，可以做為螢光探針偵測環境中的pH值改變，實驗結果可得到線性範圍為pH 2.0 ~ pH 6.5，可區分pH範圍為0.5的差異，且在酸鹼環境中重複調控後仍有高穩定性。後續將此偵測系統應用在尿素-尿素酶的偵測上，對尿素的偵測極限為0.1 mM，且可成功應用於人體尿液樣品的定量。最後，利用雷射共聚焦顯微鏡觀察攝入銅奈米簇後的細胞在不同pH值環境下，其螢光強度在pH 4.0 ~ pH 6.0之間具有增強的趨勢，表示該銅奈米簇具有高細胞穿透性及高生物相容性等特點。

In recent years, noble metal nanoclusters have been extensively studied, while the research on copper nanoclusters is relatively rare. Gold, silver, and copper are contained in the periodic table of the same group of elements. Copper is Earth-abundant, low prices and relatively friendly to humans. In this study, we direct use of pepsin as a template, so that with high biocompatibility and without additional modification. And we also use low toxicity reducing agents such as NaBH4 and ascorbic acid in the alkaline environment. Then, copper nanocluster is successfully synthesized with the excitation wavelength at 420 nm and the emission wavelength at 496 nm. It is deduced that Cu13 is the main product and uses its characteristics as a fluorescent probe. In the process, copper nanoclusters and hemoglobin were used as an IFE absorber/fluorophore pair and can be successfully used in real urine samples hemoglobin detection. This method is convenient, the detection limit of 70 nM. In addition, in different acid-base environments, the pepsin changes its structure due to the protonation and deprotonation of functional groups, resulting in a change in its fluorescence intensity. It can be used as a fluorescent probe to detect the change of pH value in the environment. The experimental results can be obtained in the linear range of pH 2.0 ~ pH 6.5, which can distinguish the difference of pH range of 0.5. After repeated regulation in the acid-base environment, they still keep high stability. After that, copper nanoclusters are used in the detection of urea, detection limit of 0.1mM, and can be successfully applied to the human urine sample. Finally, using laser scanning confocal microscopy to observe the copper nanoclusters, the fluorescence intensity of the copper nanoclusters showed an enhanced trend between pH 4.0 and pH 6.0, indicating that the copper nanoclusters have high cell penetration and high biocompatibility.

摘要 i
Abstract ii
目錄 iv
圖目錄 vi
表目錄 vii
1前言 1
1.1電化學合成法( Electrochemical synthesis method ) 1
1.2聲化學合成法( Sonochemical synthesis method) 2
1.3 微乳化法( Water-in-oil (w/o) microemulsion technique ) 2
1.4 模板合成法( Template-based synthesis ) 2
2實驗部分 4
2.1 實驗藥品 4
2.2儀器設備 7
2.3實驗溶液配置及樣品前處理步驟 9
3實驗結果與討論 17
3.1以胃蛋白酶為模板合成銅奈米簇之最適化條件探討 17
3.2 以胃蛋白酶為模板合成銅奈米簇之鑑定 23
3.2.1螢光光譜儀鑑定 23
3.2.1.1證明銅奈米簇的生成 23
3.2.1.2激發光源波長對銅奈米簇螢光放光現象之影響 24
3.2.2利用UV-Vis光譜圖證實銅奈米簇的生成 25
3.2.3 利用X射線光電子能譜鑑定銅奈米簇的生成 26
3.2.4 利用基質輔助雷射脫附游離串聯飛行時間質譜儀證實銅奈米簇的形成 28
3.2.5 銅奈米簇的螢光生物週期與量子產率 29
3.3以胃蛋白酶為模板合成銅奈米簇之穩定性測試 30
3.3.1溫度對銅奈米簇螢光放光的影響 30
3.3.2環境鹽類對銅奈米簇螢光放光現象之影響 32
3.3.3隨儲存時間增加對銅奈米簇螢光放光現象之影響 33
3.3.4銅奈米簇對金屬離子選擇性測試 34
3.4利用銅奈米簇偵測血紅蛋白 35
3.4.1偵測血紅蛋白之最適化實驗條件之探討 36
3.4.2偵測血紅蛋白之標準品 41
3.4.3偵測血紅蛋白之真實樣品 43
3.5利用銅奈米簇作為酸鹼感測器 44
3.5.1銅奈米簇偵測系統pH值及可調變化性測試 45
3.5.2將銅奈米簇應用在尿素-尿素酶系統 50
3.5.2.1偵測尿素之最適化實驗條件探討 52
3.5.2.2偵測尿素標準品 53
3.5.2.3偵測尿素之選擇性測試 55
3.5.2.4偵測尿液真實樣品中的尿素含量 56
3.5.3將銅奈米簇應用在不同pH環境下之細胞成像 57
4結論 58
5參考資料 59

1. Li, J.; Cheng, F.; Huang, H.; Li, L.; Zhu, J. J., Nanomaterial-based activatable imaging probes: from design to biological applications. Chem. Soc. Rev. 2015, 44, 7855-7880.
2. Huang, J.; Lin, L.; Sun, D.; Chen, H.; Yang, D.; Li, Q., Bio-inspired synthesis of metal nanomaterials and applications. Chem. Soc. Rev.2015, 44, 6330- 6374.
3. Hu, X.; Liu, T.T.; Zhuang, Y.; Wang, W.; Li, Y.; Fan, W.; Huang, Y., Recent advances in the analytical applications of copper nanoclusters. Trends Anal. Chem. 2016, 77, 66-75.
4. Noelia, V. V.; Blanco, C.; Arturo, L. Q.; Rivas, J.; Serra, C., Electrochemical synthesis of very stable photoluminescent copper clusters. J. Phys. Chem. C 2010, 114, 15924–15930.
5. Carlos, V. V; Manuel, B. L.; Atanu, M., Synthesis of small atomic copper clusters in microemulsions. Langmuir, 2009, 25, 8208–8216.
6. Gui, R.; Sun, J.; Cao, X.; Wang, Y.; Jin, H., Multidentate polymers stabilized water-dispersed copper nanoclusters: facile photoreduction synthesis and selective fluorescence turn-on response. RSC Adv. 2014, 4, 29083-29088.
7. Zhao, M. Q.; Sun, L.; Crooks, R. M., Preparation of Cu nanoclusters within dendrimer templates. J. Am. Chem. Soc. 1998, 120, 4877–4878.
8. Rama, G.; Amaresh, K. S.; Siddhartha, S. G.; Anumita, P.; Arun, C., Blue- emitting copper nanoclusters synthesized in the presence of lysozyme as candidates for cell labeling. ACS Appl. Mater. Interfaces 2014, 6, 3822-3828.
9. Huang, H.; Li, H.; Wang, A. J.; Zhong, S. X.; Fang, K. M.; Feng, J.J., Green synthesis of peptide-templated fluorescent copper nanoclusters for temperature sensing and cellular imaging. Analyst 2014, 139, 6536-6541.
10. Yang, X.; Feng, Y.; Zhu, S.; Luo, Y.; Zhuo, Y.; Dou, Y., One-step synthesis and applications of fluorescent Cu nanoclusters stabilized by L-cysteine in aqueous solution. Anal.Chim.Acta. 2014, 847, 49-54.
11. Jia, X.; Li, J.; Han, L.; Ren, J.; Yang, X.; Wang, E., DNA-hosted copper nanoclusters for fluorescent identification of single nucleotide polymorphisms. ACS Nano 2012, 6, 3311-3317.
12. Monica, F. U; Laura, T. A.; Jose, M. C. F.; Rosario, P.; Alfredo, S. M., One-step aqueous synthesis of fluorescent copper nanoclusters by direct metal reduction. Nanotechnology 2013, 24, 495601.
13. Cao, H.; Chen, A.; Zheng, H.; Hung, Y., Copper nanoclusters as a highly sensitive fluorescence sensor for ferric ions in serum and living cells by imaging. Biosens. Bioelectron. 2014, 62, 189-195.
14. Xie, J.; Zheng, Y.; Ying, J.Y., Protein-directed synthesis of highly fluorescent gold nanoclusters. J. Am. Chem. Soc. 2009, 131, 888–889.
15. Xavier, P. L.; Chaudhari, K.; Verma, P. K.; Pal, S.K.; Pradeep, T., Luminescent quantum clusters of gold in transferrin family protein, lactoferrin exhibiting FRET. Nanoscale 2010, 2, 2769-2776.
16. Kawasaki, H.; Hamaguchi, K.; Osaka, I.; Arakawa, R., pH-Dependent synthesis of pepsin-mediated gold nanoclusters with blue, green and red fluorescent emission. Adv. Funct. Mater. 2011, 21, 3508-3515.
17. Xu, Y.; Sherwood, J.; Qin, Y.; Crowley, D.; Bonizzoni, M.; Bao, Y., The role of protein characteristics in the formation and fluorescent of Au nanoclusters. Nanoscale 2014, 6, 1515-1524.

18. Xu, Y.; Palchoudhury, S.; Qin, Y.; Macher, T.; Bao, Y., Make conjugation simple: a facile approach to integrated nanostructures. Langmuir 2012, 28, 8767−8772.
19. Wang, Z.; Chen, B.; Rogach, A. L., Synthesis, optical properties and applications of light-emitting copper nanoclusters. Nanoscale horiz. 2017, 2, 135-146.
20. Jia, X.; Li, J.; Wang, E., Cu nanocluster with aggregation-induced emission enhancement. Small 2013, 9, 3873-3879.
21. Huang, H.; Li, H.; Wang, A. J.; Zhong, S. X.; Fang, K. M.; Feng, J. J., Green synthesis of peptide-templated fluorescent copper nanoclusters for temperature sensing and cellular imaging. Analyst 2014, 24, 6536-6541.
22. Wang, S.; Jiao, S.; Wang, J.; Chen, H. S.; Tian, D.; Lei, H.; Fang, D. N., High-performance aluminum-ion battery with CuS@C microsphere composite cathode. ACS Nano 2017, 11, 469−477.
23. Xie, J.; Zheng, Y.; Ying, J.Y., Protein-directed synthesis of highly fluorescent gold nanoclusters. J. Am. Chem. Soc. 2009, 131, 888–889.
24. Chib, R.; Butler, S.; Raut, S.; Borejdo, J.; Gryczynski, Z.; Gryczynski, L., Effect of quencher, denaturants, temperature and pH on the fluorescent properties of BSA protected gold nanoclusters. J. Lumin. 2015, 168, 62-68.
25. Chen, L. Y.; Huang, C. C.; Chen, W. Y.; Lin, H. J.; Chang, H.T., Using photoluminescent gold nanodots to detect hemoglobin in diluted blood samples. Biosens. Bioelectron. 2013, 43, 38-44.
26. Minoru, T.; Fuminobu, S.; Rumiko, H.; Yoshinori, S.; Yasufumi, M.; Takao, S.; Shigenori, Y.; Naoki, I., Low hemoglobin levels are associated with upper gastrointestinal bleeding. Biomedical Reports 2016, 5, 349-352.
27. Nirmal, K. D.; Subhadip, G.; Amulya, P.; Sunando, D.; Sunando, M., Luminescent copper nanoclusters as a specific cell-imaging probe and a selective metal ion sensor. J. Phys. Chem. C 2015, 119, 24657−24664.
28. Chang, H. C.; Ho, J.A., Gold nanocluster -assisted fluorescent detection for hydrogen peroxide and cholesterol based on the inner filter effect of gold nanoparticles. Anal. Chem. 2015, 87, 10362–10367.
29. Wang, C.; Wang, C.; Xu, L.; Cheng, H.; Lin, Q.; Zhang, C., Protein-directed synthesis of pH-responsive red fluorescent copper nanoclusters and their applications in cellular imaging and catalysis. Nanoscale 2014, 6, 1775-1781.
30. Chen, Y. N.; Chen, P. C.; Wang, C. W.; Lin, Y. S.; Wu, C. M.; Ho, L.C.; Chang, H. T., One-pot synthesis of fluorescent BSA–Ce/Au nanoclusters as ratiometric pH probes. Chem. Commun., 2014, 50, 8571-8574.
31. Qu, F.; Li, N. B.; Luo, H. Q., Highly sensitive fluorescent and colorimetric pH sensor based on polyethyleneimine-capped silver nanoclusters. Langmuir 2013, 29, 1199–1205.
32. Ding, W.; Liu, Y.; Li, Y.; Shi, Q.; Li, H.; Xia, H.; Wang, D.; Tao, X., Water-soluble gold nanoclusters with pH-dependent fluorescence and high colloidal stability over a wide pH range via co-reduction of glutathione and citrate. RSC Adv. 2014, 4, 22651–22659.
33. Deng, H. H.; Wu, G. W.; Zou, Z. Q.; Peng, H. P.; Liu, A. L.; Lin, X. H.; Xia, X. H.; Chen, W., pH-sensitive gold nanocluster: preparation and analytical application for urea, urease, and urease inhibitor detection. Chem. Comm. 2015, 54, 7847-7850.
34. Nair, L. V.; Philips, D. S.; Jayasree, R. S.; Ajayaghosh, A., A Near-infrared fluorescent nanosensor (AuC@Urease) for the selective detection of blood urea. Small 2013, 9, 2673-2677.