蛋白質包覆金奈米團簇(Protein-gold nanoclusters)由於具有良好的光學穩定性及生物相容性，並能夠保留原本蛋白的生物活性，近年來在許多領域中已是一種非常熱門的螢光材料，被廣泛應用於生物傳感、檢測和生物顯影上。過去對於其合成方法已報導過非常多，但是對於蛋白質包覆金奈米團簇的分子內部結構尚未被明確的研究出來。在本研究中我們嘗試以離子交換層析、膠體電泳以及質譜來分離牛血清蛋白包覆金奈米團簇及α-乳清蛋白包覆金奈米團簇，以了解Protein-AuNCs形成時的結構變化。

首先利用先前實驗室優化出的條件合成藍光和紅光的牛血清蛋白包覆金奈米團簇，並另外合成出α-乳清蛋白包覆金奈米團簇和溶菌酶包覆金奈米團簇，以UV/Vis分光光譜儀、螢光光譜儀來鑑定光學性質，藍光BSA-AuNCs在約425 nm放出螢光；而其他的紅光Protein-AuNCs在約635 nm放出螢光。

利用離子交換層析法分別分離紅光BSA-AuNCs和ALAB-AuNCs，在BSA-AuNCs中確認出可能為BSA-AuNCs的吸收峰，分離出BSA和BSA-AuNCs，而ALAB-AuNCs層析圖顯示合成出的ALAB-AuNCs並未有反應不完全的ALAB且成分相對單純。我再用不連續膠體電泳(Native-PAGE)和SDS膠體電泳(SDS-PAGE)將BSA、BSA_pH 12、BSA_pH 8、BSA-AuNCs_red、BSA-AuNCs_blue及ALAB、ALAB_pH 12、ALAB-AuNCs_red分離，發現形成AuNCs過程主要受到NaOH的影響造成蛋白質的構型改變再形成AuNCs。

BSA、BSA_pH 12、BSA_pH 8、BSA-AuNCs_red、BSA-AuNCs_blue使用奈升級四極棒飛行式串聯質譜 (nanoESI-Q-TOF)分析並結合理論計算的方式找到Au的指紋訊號以及在BSA-AuNCs_blue中fitting出7-9顆Au的質量，符合文獻中MALDI實驗的平均8顆Au結果。

第二部分利用由下而上的蛋白質體學方法分析甲狀腺癌組織，發現在腫瘤組織中可能藉由蛋白中的磷酸修飾形成nanocluster的磷酸鈣，類似於蛋白質為基板的奈米團簇，並以鈣比色測定試劑盒及感應電漿耦合質譜儀測得組織中的鈣離子濃度並找出之間的相關性。

Protein-gold nanoclusters have good optical stability, biocompatibility, and retain the biological activity of the original protein. In recent years, it has become a popular material in many fields. In the past many reports on its synthesis methods, but the molecular internal structure of the protein-gold nanoclusters has not been clearly studied. In this study, we tried to separate bovine serum albumin protected gold nanoclusters and α-lactalbumin protected gold nanoclusters by ion exchange chromatography, gel electrophoresis, and mass spectrometry to understand the formation of protein-AuNCs structure.
First, I used the optimized method in the previous laboratory to synthesize the blue and red bovine serum albumin protected gold nanoclusters, and additionally synthesize α-lactalbumin protected gold nanoclusters and lysozyme protected gold nanoclusters. The properties of the clusters were identified by UV/Vis spectrometer and fluorescence spectrometer. The blue BSA-AuNCs emitted fluorescence at 425 nm; the other red protein-AuNCs emitted fluorescence at 635 nm.
The BSA-AuNCs_red and ALAB-AuNCs_red were separated by ion exchange chromatography. We can identify the peak of BSA-AuNCs, and ALAB-AuNCs product looks more purity. I used Native-PAGE and SDS-PAGE to separate standard, controller, AuNCs, then it was found that the process of forming AuNCs was mainly affected by adding NaOH, which caused the protein conformation change and then form AuNCs.
Using nanoESI-Q-TOF analysis and combine theoretical calculations to find the fingerprint of Au and the mass of 7-9 Au fittings in BSA-AuNCs_blue, the same results of the MALDI experiment in the literature.
The second part used the bottom-up proteomics method to analyze the thyroid cancer tissues. It was found that the calcium phosphate of nanocluster may be formed by the phosphor modification protein in the tumor tissue, which is similar to the protein-based nanocluster. Calcium colorimetric kit and ICP measure the calcium concentration in the tissue and find the correlation between them.

1. Dixon, J. M.; Egusa, S., Conformational Change-Induced Fluorescence of Bovine Serum Albumin-Gold Complexes. J Am Chem Soc 2018, 140 (6), 2265-2271.
2. Majorek, K. A.; Porebski, P. J.; Dayal, A.; Zimmerman, M. D.; Jablonska, K.; Stewart, A. J.; Chruszcz, M.; Minor, W., Structural and immunologic characterization of bovine, horse, and rabbit serum albumins. Mol Immunol 2012, 52 (3-4), 174-82.
3. Dietler, G.; Anselmetti, D.; Haeberli, A.; Meister, J.-J., Quasistatic and dynamic force microscopy of single antigen-antibody complexes and fibrin-fibrinogen systems. 2004.
4. Permyakov, E. A.; Berliner, L. J., α-Lactalbumin: structure and function. FEBS Letters 2000, 473 (3), 269-274.
5. Wu, L.-Z.; Sheng, Y.-B.; Xie, J.-B.; Wang, W., Photoexcitation of tryptophan groups induced reduction of disulfide bonds in hen egg white lysozyme. Journal of Molecular Structure 2008, 882 (1-3), 101-106.
6. Qu, X.; Li, Y.; Li, L.; Wang, Y.; Liang, J.; Liang, J., Fluorescent Gold Nanoclusters: Synthesis and Recent Biological Application. Journal of Nanomaterials 2015, 2015, 1-23.
7. Zheng, J.; Nicovich, P. R.; Dickson, R. M., Highly Fluorescent Noble-Metal Quantum Dots. Annual Review of Physical Chemistry 2007, 58 (1), 409-431.
8. Zhu, M.; Aikens, C. M.; Hollander, F. J.; Schatz, G. C.; Jin, R., Correlating the Crystal Structure of A Thiol-Protected Au25. J Am Chem Soc 2008, (130), 5883–5885.
9. Yu, P.; Wen, X.; Toh, Y.-R.; Ma, X.; Tang, J., Fluorescent Metallic Nanoclusters: Electron Dynamics, Structure, and Applications. Particle & Particle Systems Characterization 2015, 32 (2), 142-163.
10. Chen, L. Y.; Wang, C. W.; Yuan, Z.; Chang, H. T., Fluorescent gold nanoclusters: recent advances in sensing and imaging. Anal Chem 2015, 87 (1), 216-29.
11. Zheng, J.; Dickson, R. M., Individual Water-Soluble Dendrimer-Encapsulated Silver Nanodot. J Am Chem Soc 2002, (124), 13982-13983.
12. Maya, L.; Muralidharan, G.; Thundat, T. G.; Kenik, E. A., Polymer-Mediated Assembly of Gold Nanoclusters. Langmuir : the ACS journal of surfaces and colloids 2000.
13. Chevrier, D. M.; Chatt, A.; Zhang, P., Properties and applications of proteinstabilized. Journal of Nanophotonics 2012, 6 (1), 1-17, 17.
14. Yu, Y.; Yao, Q.; Luo, Z.; Yuan, X.; Lee, J. Y.; Xie, J., Precursor engineering and controlled conversion for the synthesis of monodisperse thiolate-protected metal nanoclusters. Nanoscale 2013, 5 (11), 4606-20.
15. Halawa, M. I.; Lai, J.; Xu, G., Gold nanoclusters: synthetic strategies and recent advances in fluorescent sensing. Materials Today Nano 2018, 3, 9-27.
16. Luo, Z.; Zheng, K.; Xie, J., Engineering ultrasmall water-soluble gold and silver nanoclusters for biomedical applications. Chem Commun (Camb) 2014, 50 (40), 5143-55.
17. Xie, J.; Zheng, Y.; Ying, J. Y., Protein-Directed Synthesis of Highly Fluorescent Gold Nanoclusters. J. AM. CHEM. SOC. 2009, 131, 888–889.
18. 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.; Cheng, J. T.; Chou, P. T., Insulin-directed synthesis of fluorescent gold nanoclusters: preservation of insulin bioactivity and versatility in cell imaging. Angew Chem Int Ed Engl 2011, 50 (31), 7056-60.
19. Kawasaki, H.; Hamaguchi, K.; Osaka, I.; Arakawa, R., ph-Dependent Synthesis of Pepsin-Mediated Gold Nanoclusters with Blue Green and Red Fluorescent Emission. Advanced Functional Materials 2011, 21 (18), 3508-3515.
20. Chandirasekar, S.; You, J.-G.; Xue, J.-H.; Tseng, W.-L., Synthesis of gold nanocluster-loaded lysozyme nanoparticles for label-free ratiometric fluorescent pH sensing: applications to enzyme–substrate systems and cellular imaging. Journal of Materials Chemistry B 2019, 7 (24), 3876-3883.
21. 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 (12), 2769-76.
22. Yan, L.; Cai, Y.; Zheng, B.; Yuan, H.; Guo, Y.; Xiao, D.; Choi, M. M. F., Microwave-assisted synthesis of BSA-stabilized and HSA-protected gold nanoclusters with red emission. J. Mater. Chem. 2012, 22 (3), 1000-1005.
23. Xie, J.; Zheng, Y.; Ying, J. Y., Highly selective and ultrasensitive detection of Hg(2+) based on fluorescence quenching of Au nanoclusters by Hg(2+)-Au(+) interactions. Chem Commun (Camb) 2010, 46 (6), 961-3.
24. 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, 9194–9200.
25. Hu, D.; Sheng, Z.; Fang, S.; Wang, Y.; Gao, D.; Zhang, P.; Gong, P.; Ma, Y.; Cai, L., Folate receptor-targeting gold nanoclusters as fluorescence enzyme mimetic nanoprobes for tumor molecular colocalization diagnosis. Theranostics 2014, 4 (2), 142-53.
26. Li, H.; Zhu, W.; Wan, A.; Liu, L., The mechanism and application of the protein-stabilized gold nanocluster sensing system. Analyst 2017, 142 (4), 567-581.
27. Lin, C.-A. J.; Yang, T.-Y.; Lee, C.-H.; Zanella, M.; Li, J. K.; Shen, J.-L.; Chang, W. H.; Huang, S. H.; Sperling, R. A.; Wang, H.-H.; Yeh, H.-I.; Parak, W. J., Synthesis, Characterization, andBioconjugation of Fluorescent GoldNanoclusters toward Biological LabelingApplications. ACSnano 2009, 3 (2), 395–401
28. Yu, Y.; Luo, Z.; Teo, C. S.; Tan, Y. N.; Xie, J., Tailoring the protein conformation to synthesize different-sized gold nanoclusters. Chem Commun (Camb) 2013, 49 (84), 9740-2.
29. Chaudhari, K.; Xavier, P. L.; Pradeep, T., Understanding the evolution of luminescent gold quantum clusters in protein templates. ACS Nano 2011, 5 (11), 8816-27.
30. Xu, Y.; Sherwood, J.; Qin, Y.; Crowley, D.; Bonizzoni, M.; Bao, Y., The role of protein characteristics in the formation and fluorescence of Au nanoclusters. Nanoscale 2014, 6 (3), 1515-24.
31. Khan, S.; Gupta, A.; Verma, N. C.; Nandi, C. K., Kinetics of protein adsorption on gold nanoparticle with variable protein structure and nanoparticle size. J Chem Phys 2015, 143 (16), 164709.
32. Hsu, Y. C.; Hung, M. J.; Chen, Y. A.; Wang, T. F.; Ou, Y. R.; Chen, S. H., Identifying Reducing and Capping Sites of Protein-Encapsulated Gold Nanoclusters. Molecules 2019, 24 (8).
33. Mudedla, S. K.; Singam, E. R. A.; Vijay Sundar, J.; Pedersen, M. N.; Murugan, N. A.; Kongsted, J.; Ågren, H.; Subramanian, V., Enhancement of Internal Motions of Lysozyme through Interaction with Gold Nanoclusters and its Optical Imaging. The Journal of Physical Chemistry C 2014, 119 (1), 653-664.
34. Christodoulou, J.; Sadler, P. J.; Tucker, A., A new structural transition of serum albumin dependent on the state of Cys34. European Journal of Biochemistry 1994, 225 (1), 363-368.
35. Navas-Carrillo, D.; Rios, A.; Rodriguez, J. M.; Parrilla, P.; Orenes-Pinero, E., Familial nonmedullary thyroid cancer: screening, clinical, molecular and genetic findings. Biochim Biophys Acta 2014, 1846 (2), 468-76.
36. Hemminki, K.; Li, X., Familial risk of cancer by site and histopathology. Int J Cancer 2003, 103 (1), 105-9.
37. Woodruff, S. L.; Arowolo, O. A.; Akute, O. O.; Afolabi, A. O.; Nwariaku, F., Global variation in the pattern of differentiated thyroid cancer. Am J Surg 2010, 200 (4), 462-6.
38. Bonora, E.; Tallini, G.; Romeo, G., Genetic Predisposition to Familial Nonmedullary Thyroid Cancer: An Update of Molecular Findings and State-of-the-Art Studies. J Oncol 2010, 2010, 385206.
39. Molina, F. B., M.; Pau, B.; Granier, C., Characterization of the type-1 repeat from thyroglobulin, a cysteine-rich module found in proteins from different families. Eur. J. Biochem. 1996, 240, 125–133
40. Coscia, F.; Taler-Vercic, A.; Chang, V. T.; Sinn, L.; O'Reilly, F. J.; Izore, T.; Renko, M.; Berger, I.; Rappsilber, J.; Turk, D.; Lowe, J., The structure of human thyroglobulin. Nature 2020, 578 (7796), 627-630.
41. Lacout, A.; Chevenet, C.; Thariat, J.; Marcy, P. Y., Thyroid calcifications: a pictorial essay. J Clin Ultrasound 2016, 44 (4), 245-51.
42. Ferreira, L. B.; Gimba, E.; Vinagre, J.; Sobrinho-Simoes, M.; Soares, P., Molecular Aspects of Thyroid Calcification. Int J Mol Sci 2020, 21 (20).
43. Holt, C.; Lenton, S.; Nylander, T.; Sørensen, E. S.; Teixeira, S. C. M., Mineralisation of soft and hard tissues and the stability of biofluids. Journal of Structural Biology 2014, 185 (3), 383-396.
44. Holt, C., Inorganic constituents of milk III. The colloidal calcium phosphate of cow's milk. Journal of Dairy Research 1982, 49 (1), 29-38.
45. Wei, H.; Wang, Z.; Yang, L.; Tian, S.; Hou, C.; Lu, Y., Lysozyme-stabilized gold fluorescent cluster: Synthesis and application as Hg(2+) sensor. Analyst 2010, 135 (6), 1406-10.
46. Yarramala, D. S.; Baksi, A.; Pradeep, T.; Rao, C. P., Green Synthesis of Protein-Protected Fluorescent Gold Nanoclusters (AuNCs): Reducing the Size of AuNCs by Partially Occupying the Ca2+ Site by La3+ in Apo-α-Lactalbumin. ACS Sustainable Chemistry & Engineering 2017, 5 (7), 6064-6069.
47. Holt, C.; Sorensen, E. S.; Clegg, R. A., Role of calcium phosphate nanoclusters in the control of calcification. FEBS J 2009, 276 (8), 2308-23.
48. Lenton, S.; Wang, Q.; Nylander, T.; Teixeira, S.; Holt, C., Structural Biology of Calcium Phosphate Nanoclusters Sequestered by Phosphoproteins. Crystals 2020, 10 (9).