本研究開發一種簡單的方法製備溶菌酶奈米粒子包覆金奈米團簇 (LysNP-AuNCs)，並將之作為雙放射螢光探針，以螢光比例法偵測自來水及生氰性醣苷植物中氰化物含量。首先以溶菌酶為模板，將金離子還原，合成金奈米團簇 (Lys-AuNCs)，據文獻報導，形成金奈米團簇時會自組裝而聚集，因此此時若加入戊二醛，可誘導溶菌酶交聯產生C=N，進而形成具藍色螢光的溶菌酶奈米粒子 (LysNPs)，使金奈米團簇均勻分布於溶菌酶奈米粒子中，並由穿透式電子顯微鏡及凝膠滲透層析中可得到證實。此LysNP-AuNCs具有雙螢光放射、較大的Stokes shift、光穩定度佳及鹽類穩定度高等優點。當溶液中存在氰化物時，會侵蝕LysNP-AuNCs中的金奈米團簇，進而抑制溶菌酶奈米粒子與金奈米團簇間的螢光共振能量轉移，可藉此定量氰化物，線性範圍為3 - 100 M，且此探針對氰化物的選擇性遠高於其它陰離子，最後也將此探針應用於自來水中氰化物定量及追蹤食物中氰化氫之釋放，證實其實用性。

This study developed a simple strategy to prepare lysozyme nanoparticle-encapsulated gold nanoclusters (LysNP-AuNCs) as a dual emission probe for ratiometric sensing of cyanide in tap water and cyanogenic glycoside-containing plants. The reduction of gold ion precursor with lysozyme generated lysozyme-stabilized AuNCs (Lys-AuNCs); they were demonstrated to be self-assembled into nano-aggregates during the formation of AuNCs. The aggregated Lys-AuNCs were treated with glutaraldehyde, triggering the conversion of aggregated lysozymes into blue-emitting lysozyme nanoparticles (LysNPs) via the formation of C=N bonds. As a result, the AuNCs were well-distributed inside the LysNPs, as demonstrated by transmission electron microscopy and size-exclusion chromatography. The as-prepared LysNP-AuNCs exhibited two separate emission bands, large Stokes shift, excellent photostability, and salt stability. The presence of cyanide triggered the etching of AuNCs in LysNP-AuNCs, leading to the suppression of fluorescence resonance energy transfer (FRET) from LysNPs to AuNCs. The LysNP-AuNCs probe was implemented for FRET detection of cyanide with linear range of 3100 M. Additionally, the selectivity of the LysNP-AuNCs probe for cyanide toward other anions was remarkably high. The practicality of the proposed probe was evaluated by quantifying cyanide in tap water and monitoring the liberation of hydrogen cyanide from ground cassava roots.

論文審定書 i
論文公開授權書 ii
中文摘要 iii
Abstract iv
目錄 vi
圖目錄 viii
表目錄 xiv
縮寫用語對照表 xv

合成溶菌酶奈米粒子包覆金奈米團簇作為比例法螢光探針應用於監控自來水及生氰性醣苷蔬果中之氰化物 1
1.1 前言 1
1.1.1 氰化物毒性簡介 1
1.1.2 檢測氰化物之方法回顧 3
1.1.3 雙放射螢光感測器之優點 13
1.2 實驗步驟 15
1.2.1 實驗藥品 15
1.2.2 儀器設備 18
1.2.3 實驗溶液配置 20
1.2.4 奈米材料之合成方法 22
1.2.5 真實樣品製備 24
1.3 結果與討論 26
1.3.1 合成及鑑定 LysNP-AuNCs 26
1.3.2 氰化物感測機制之建立 48
1.3.3 氰化物之定量分析 53
1.3.4 LysNP-AuNCs 對陰離子之選擇性探討 58
1.3.5 驗證氰化物感測機制 60
1.3.6 LysNP-AuNCs比例法螢光探針應用於追蹤生氰性醣苷植物之氰化物釋放 68
1.3.7 氰化物感測試紙之製備方法探討 72
1.4 結論 82
1.5 參考文獻 83

1. Suzuki, T.; Hioki, A.; Kurahashi, M., Development of a method for estimating an accurate equivalence point in nickel titration of cyanide ions. Anal. Chim. Acta 2003, 476, 159-165.
2. Blaedal, W.; Easty, D.; Anderson, L.; Farrell, T., Potentiometric determination of cyanide with an ion selective electrode. Application to cyanogenic glycosides in Sudan grasses. Anal. Chem. 1971, 43, 890-894.
3. Papežová, K.; Glatz, Z., Determination of cyanide in microliter samples by capillary electrophoresis and in-capillary enzymatic reaction with rhodanese. J. Chromatogr. A 2006, 1120, 268-272.
4. Martí, V.; Aguilar, M.; Yeung, E. S., Indirect fluorescence detection of free cyanide and related compounds by capillary electrophoresis. J. Chromatogr. A 1995, 709, 367-374.
5. Youso, S. L.; Rockwood, G. A.; Lee, J. P.; Logue, B. A., Determination of cyanide exposure by gas chromatography–mass spectrometry analysis of cyanide-exposed plasma proteins. Anal. Chim. Acta 2010, 677, 24-28.
6. Kage, S.; Nagata, T.; Kudo, K., Determination of cyanide and thiocyanate in blood by gas chromatography and gas chromatography-mass spectrometry. J. Chromatogr. B Biomed. Sci. Appl. 1996, 675, 27-32.
7. Calafat, A. M.; Stanfill, S. B., Rapid quantitation of cyanide in whole blood by automated headspace gas chromatography. J. Chromatogr. B 2002, 772, 131-137.
8. Haj-Hussein, A. T.; Christian, G. D.; Ruzicka, J., Determination of cyanide by atomic absorption using a flow injection conversion method. Anal. Chem. 1986, 58, 38-42.
9. Chattaraj, S.; Das, A. K., Indirect determination of free cyanide in industrial waste effluent by atomic absorption spectrometry. Analyst 1991, 116, 739-741.
10. Gong, W.-T.; Zhang, Q.-L.; Shang, L.; Gao, B.; Ning, G.-L., A new principle for selective sensing cyanide anions based on 2-hydroxy-naphthaldeazine compound. Sens. Actuators. B Chem. 2013, 177, 322-326.
11. Yang, Y.; Yin, C.; Huo, F.; Chao, J.; Zhang, Y.; Cheng, F., A new highly selective and turn-on fluorescence probe for detection of cyanide. Sens. Actuators. B Chem. 2014, 193, 220-224.
12. Peng, M.-J.; Guo, Y.; Yang, X.-F.; Wang, L.-Y.; An, J., A highly selective ratiometric and colorimetric chemosensor for cyanide detection. Dyes Pigm. 2013, 98, 327-332.
13. Huo, F.-J.; Su, J.; Sun, Y.-Q.; Yin, C.-X.; Chao, J.-B., A new ring-opening chromene molecule: colorimetric detection of cyanide anion. Chem. Lett. 2010, 39, 738-740.
14. Das, P.; Saha, S.; Baidya, M.; Ghosh, S. K.; Das, A., A CN− specific turn-on phosphorescent probe with probable application for enzymatic assay and as an imaging reagent. Chem. Commun. 2013, 49, 255-257.
15. Liu, C.-Y.; Tseng, W.-L., Colorimetric assay for cyanide and cyanogenic glycoside using polysorbate 40-stabilized gold nanoparticles. Chem. Commun. 2011, 47, 2550-2552.
16. Li, Y.; Wang, Q.; Zhou, X.; Wen, C.-y.; Yu, J.; Han, X.; Li, X.; Yan, Z.-f.; Zeng, J., A convenient colorimetric method for sensitive and specific detection of cyanide using Ag@ Au core–shell nanoparticles. Sens. Actuators. B Chem. 2016, 228, 366-372.
17. 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. Adv. Funct. Mater. 2010, 20, 951-956.
18. Zhang, G.; Qiao, Y.; Xu, T.; Zhang, C.; Zhang, Y.; Shi, L.; Shuang, S.; Dong, C., Highly selective and sensitive nanoprobes for cyanide based on gold nanoclusters with red fluorescence emission. Nanoscale 2015, 7, 12666-12672.
19. Dong, Z.-Z.; Yang, C.; Vellaisamy, K.; Li, G.; Leung, C.-H.; Ma, D.-L., Construction of a nano biosensor for cyanide anion detection and its application in environmental and biological systems. ACS sensors 2017, 2, 1517-1522.
20. Zhai, Y.; Jin, L.; Wang, P.; Dong, S., Dual-functional Au–Fe 3 O 4 dumbbell nanoparticles for sensitive and selective turn-on fluorescent detection of cyanide based on the inner filter effect. Chem. Commun. 2011, 47, 8268-8270.
21. Shojaeifard, Z.; Hemmateenejad, B.; Shamsipur, M., Efficient on–off ratiometric fluorescence probe for cyanide ion based on perturbation of the interaction between gold nanoclusters and a copper (II)-phthalocyanine complex. ACS Appl. Mater. Interfaces 2016, 8, 15177-15186.
22. Chen, Z.; Qian, S.; Chen, X.; Gao, W.; Lin, Y., Protein-templated gold nanoclusters as fluorescence probes for the detection of methotrexate. Analyst 2012, 137, 4356-4361.
23. Zhang, J.; Sajid, M.; Na, N.; Huang, L.; He, D.; Ouyang, J., The application of Au nanoclusters in the fluorescence imaging of human serum proteins after native PAGE: enhancing detection by low-temperature plasma treatment. Biosens. Bioelectron. 2012, 35, 313-318.
24. Wei, H.; Wang, Z.; Yang, L.; Tian, S.; Hou, C.; Lu, Y., Lysozyme-stabilized gold fluorescent cluster: synthesis and application as Hg2+ sensor. Analyst 2010, 135, 1406-1410.
25. Liu, C. L.; Wu, H. T.; Hsiao, Y. H.; Lai, C. W.; Shih, C. W.; Peng, Y. K.; Tang, K. C.; Chang, H. W.; Chien, Y. C.; Hsiao, J. K., Insulin‐Directed Synthesis of Fluorescent Gold Nanoclusters: Preservation of Insulin Bioactivity and Versatility in Cell Imaging. Angew. Chem.-Int. Edit. 2011, 50, 7056-7060.
26. 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.
27. Langer, K.; Balthasar, S.; Vogel, V.; Dinauer, N.; Von Briesen, H.; Schubert, D., Optimization of the preparation process for human serum albumin (HSA) nanoparticles. Int. J. Pharm. 2003, 257, 169-180.
28. Lee, S. H.; Heng, D.; Ng, W. K.; Chan, H.-K.; Tan, R. B., Nano spray drying: a novel method for preparing protein nanoparticles for protein therapy. Int. J. Pharm. 2011, 403, 192-200.
29. Patil, G. V., Biopolymer albumin for diagnosis and in drug delivery. Drug Dev. Res. 2003, 58, 219-247.
30. Fach, M.; Radi, L.; Wich, P. R., Nanoparticle Assembly of Surface-Modified Proteins. J. Am. Chem. Soc. 2016, 138, 14820-14823.
31. Yang, Q.; Ye, Z.; Zhong, M.; Chen, B.; Chen, J.; Zeng, R.; Wei, L.; Li, H.-w.; Xiao, L., Self-assembled fluorescent bovine serum albumin Nanoprobes for Ratiometric pH measurement inside living cells. ACS Appl. Mater. Interfaces 2016, 8, 9629-9634.
32. Ma, X.; Hargrove, D.; Dong, Q.; Song, D.; Chen, J.; Wang, S.; Lu, X.; Cho, Y. K.; Fan, T.-H.; Lei, Y., Novel green and red autofluorescent protein nanoparticles for cell imaging and in vivo biodegradation imaging and modeling. RSC Adv. 2016, 6, 50091-50099.
33. Migneault, I.; Dartiguenave, C.; Bertrand, M. J.; Waldron, K. C., Glutaraldehyde: behavior in aqueous solution, reaction with proteins, and application to enzyme crosslinking. Biotechniques 2004, 37, 790-802.
34. Lin, Y.-H.; Tseng, W.-L., Ultrasensitive sensing of Hg2+ and CH3Hg+ based on the fluorescence quenching of lysozyme type VI-stabilized gold nanoclusters. Anal. Chem. 2010, 82, 9194-9200.
35. Baksi, A.; Xavier, P. L.; Chaudhari, K.; Goswami, N.; Pal, S.; Pradeep, T., Protein-encapsulated gold cluster aggregates: the case of lysozyme. Nanoscale 2013, 5, 2009-2016.
36. Russell, B. A.; Jachimska, B.; Komorek, P.; Mulheran, P.; Chen, Y., Lysozyme encapsulated gold nanoclusters: effects of cluster synthesis on natural protein characteristics. Phys. Chem. Chem. Phys. 2017, 19, 7228-7235.
37. Le Guével, X.; Trouillet, V.; Spies, C.; Li, K.; Laaksonen, T.; Auerbach, D.; Jung, G.; Schneider, M., High photostability and enhanced fluorescence of gold nanoclusters by silver doping. Nanoscale 2012, 4, 7624-7631.
38. Le Guével, X.; Hötzer, B.; Jung, G.; Hollemeyer, K.; Trouillet, V.; Schneider, M., Formation of fluorescent metal (Au, Ag) nanoclusters capped in bovine serum albumin followed by fluorescence and spectroscopy. J. Phys. Chem. C 2011, 115, 10955-10963.
39. Le Guével, X.; Daum, N.; Schneider, M., Synthesis and characterization of human transferrin-stabilized gold nanoclusters. Nanotechnology 2011, 22, 275103.
40. Greenfield, N. J., Using circular dichroism spectra to estimate protein secondary structure. Nat. Protoc. 2006, 1, 2876.
41. Obliosca, J. M.; Babin, M. C.; Liu, C.; Liu, Y.-L.; Chen, Y.-A.; Batson, R. A.; Ganguly, M.; Petty, J. T.; Yeh, H.-C., A complementary palette of NanoCluster beacons. ACS Nano 2014, 8, 10150-10160.
42. Li, Z.-Y.; Wu, Y.-T.; Tseng, W.-L., UV-light-induced improvement of fluorescence quantum yield of DNA-templated gold nanoclusters: application to ratiometric fluorescent sensing of nucleic acids. ACS Appl. Mater. Interfaces 2015, 7, 23708-23716.
43. Wang, X.-B.; Wang, Y.-L.; Yang, J.; Xing, X.-P.; Li, J.; Wang, L.-S., Evidence of significant covalent bonding in Au (CN) 2−. J. Am. Chem. Soc. 2009, 131, 16368-16370.
44. Zhai, J.; Zhai, Y.; Dong, S., Direct dissolution of Au nanoparticles induced by potassium ferricyanide. Colloids Surf. A Physicochem. Eng. Asp. 2009, 335, 207-210.
45. Zong, C.; Zheng, L. R.; He, W.; Ren, X.; Jiang, C.; Lu, L., In situ formation of phosphorescent molecular gold (I) cluster in a macroporous polymer film to achieve colorimetric cyanide sensing. Anal. Chem. 2014, 86, 1687-1692.
46. Zhou, C.; Sun, M.; Yan, C.; Yang, Q.; Li, Y.; Song, Y., A new colorimetric and fluorescent chemodosimeter for fast detection of cyanide. Sens. Actuators. B Chem. 2014, 203, 382-387.
47. Wei, S.-C.; Hsu, P.-H.; Lee, Y.-F.; Lin, Y.-W.; Huang, C.-C., Selective detection of iodide and cyanide anions using gold-nanoparticle-based fluorescent probes. ACS Appl. Mater. Interfaces 2012, 4, 2652-2658.
48. Lu, D.; Liu, L.; Li, F.; Shuang, S.; Li, Y.; Choi, M. M.; Dong, C., Lysozyme-stabilized gold nanoclusters as a novel fluorescence probe for cyanide recognition. Spectrochim. Acta A Mol. Biomol. Spectrosc. 2014, 121, 77-80.
49. Lin, W.-C.; Fang, S.-K.; Hu, J.-W.; Tsai, H.-Y.; Chen, K.-Y., Ratiometric fluorescent/colorimetric cyanide-selective sensor based on excited-state intramolecular charge transfer− excited-state intramolecular proton transfer switching. Anal. Chem. 2014, 86, 4648-4652.
50. Lv, X.; Liu, J.; Liu, Y.; Zhao, Y.; Chen, M.; Wang, P.; Guo, W., A ratiometric fluorescent probe for cyanide based on FRET. Org. Biomol. Chem. 2011, 9, 4954-4958.
51. Chen, W.-Y.; Lan, G.-Y.; Chang, H.-T., Use of fluorescent DNA-templated gold/silver nanoclusters for the detection of sulfide ions. Anal. Chem. 2011, 83, 9450-9455.
52. Wang, C.-W.; Chen, Y.-N.; Wu, B.-Y.; Lee, C.-K.; Chen, Y.-C.; Huang, Y.-H.; Chang, H.-T., Sensitive detection of cyanide using bovine serum albumin-stabilized cerium/gold nanoclusters. Anal. Bioanal. Chem. 2016, 408, 287-294.
53. Zhang, D.; Chen, Z.; Omar, H.; Deng, L.; Khashab, N. M., Colorimetric peroxidase mimetic assay for uranyl detection in sea water. ACS Appl. Mater. Interfaces 2015, 7, 4589-4594.