開發三向功能的奈米探針, 使 SERS (表面增強拉曼光譜) 於無背景干擾下偵測細胞表面的唾液酸

簡易檢索 / 詳目顯示

回結果列表

研究生：	芮娜妲 Renata, Septila
論文名稱：	開發三向功能的奈米探針, 使 SERS (表面增強拉曼光譜) 於無背景干擾下偵測細胞表面的唾液酸 Development of a Tri-Functional Nanoprobe for Background-Free SERS (Surface-enhanced Raman Spectroscopy) Detection of Sialic Acid on Cell Surface
指導教授：	林俊宏 Lin, Chun-Hung 潘榮隆 Pan, Rong-Long
口試委員:	郭俊宏 Kuo, Chun-Hong 謝馬利歐 Hofmann, Mario 凃智傑 Tu, Zhijay
學位類別：	博士 Doctor
系所名稱：	生命科學暨醫學院 - 生物資訊與結構生物研究所 Institute of Bioinformatics and Structural Biology
論文出版年：	2021
畢業學年度：	109
語文別：	英文
論文頁數：	83
中文關鍵詞：	表面增強拉曼光譜
相關次數：	點閱：3 下載：0
分享至:	分享至facebook 分享至twitter

查詢本校圖書館目錄查詢臺灣博碩士論文知識加值系統勘誤回報

細胞表面的唾液酸在許多生理和病理學過程中都是不可缺少的，唾液酸的靈敏且可重複的檢測對於許多疾病的診斷和治療至關重要。在本論文中，我們開發了一種三重功能的奈米探針，作為靈敏、簡單的表面增強拉曼光譜（SERS）奈米探針，用於細胞表面上的唾液酸檢測。此報導分子的設計旨在提供三個關鍵功能，使其成為唾液酸檢測的理想選擇。首先，我們採用了兩個識別基團，即苯基硼酸和一個銨基，它們可增強唾液酸的識別和捕獲效率。其次，使用氰基作為拉曼光譜報告分子，因為其分子振動光譜會落在細胞拉曼無聲區中。最後，硫醇充當錨定劑以使報告分子能與銀奈米立方體結合，從而達成SERS並保護銀奈米立方體免於氧化。我們的分子奈米探針設計證明了能夠以高靈敏度和選擇性檢測細胞表面的唾液酸，為細胞診斷開闢了新途徑。

Sialic acids, especially those on cell surface, are indispensable in numerous physiological and pathological processes. Sensitive and reproducible detection of sialic acid is crucial for diagnosis and therapy in many diseases. In this thesis, we developed a tri-functional nanoprobe as a sensitive and straightforward surface-enhanced Raman spectroscopy (SERS) nanoprobe for detecting sialic acid-containing glycan on cell surfaces. The reporter was designed to provide three key functionalities that make it ideal for sialic acid detection. First, we employed two recognition groups, phenylboronic acid and an ammonium group, to enhance sialic acid recognition and capture efficiency. Second, we used cyano as the Raman reporter because its vibrational energy is shown in the cellular Raman silent region. Finally, thiol acted as an anchoring agent that facilitate the conjugation of reporter onto silver nanocubes to provide SERS enhancement and protect silver nanocubes from oxidation. Our molecular nanoprobe design demonstrated the ability to detect sialic acid on the cell surface with high sensitivity and selectivity, opening up new routes to cellular diagnostics.

Table of Contents
Abstract    i
摘要    ii
Acknowledgements    iii
List of Abbreviations    v
Chapter 1. Introduction    1
1.1. Biological Importance of Sialic Acid    1
1.2. Raman and SERS Spectroscopy    3
1.2.1. Electromagnetic Enhancement (EM)    5
1.2.2. Chemical Enhancement (CE)    6
1.3. SERS Nanoprobe    7
1.4. Synthesis of Gold and Silver Nanocubes    9
1.4.1. Seed-Growth Method    10
1.4.2. Polyol Method    10
1.5. Raman Reporter    11
1.5.1. Selection of Raman Reporter    11
1.5.2. Bioorthogonal Raman Reporter    12
1.6. Boronic Acid and Its Binding Towards Monosaccharides    13
1.7. SERS-Based Detection of Sialic Acid and Challenges    17
1.8. Research Goal of Thesis    26
Chapter 2. Results and Discussion    28
2.1. Synthesis and Characterization of AgNCs    28
2.2. Synthesis and Characterization of AuNCs    31
2.3. Design and Synthesis of Reporter    32
2.4. Characterization of Tri-functional Nanoprobe    36
2.5. Sialic Acid Expression on HeLa cells    38
2.6. Examining Sialic Acid Expression on Normal (Non-Cancerous) and Cancer Cells    43
Chapter 3. Conclusions    46
Chapter 4. Materials and Methods    48
4.1. Instruments and Characterization    48
4.2. Synthesis of AuNCs    49
4.3. Synthesis of AgNCs    50
4.4. Synthetic Procedures    51
4.5. Synthesis of Tri-Functional Nanoprobe (Reporter 6 Modified AgNCs)    56
4.6. Sensitivity of AgNCs Towards Reporter 6    57
4.7. Cell Culture and Treatment with Nanoprobe    57
4.8. Nanoprobe Specificity Toward Sialic Acid    58
4.9. SERS Measurement    59
References    60
List of Publications    66
Appendix: NMR Spectra    67
1H NMR spectrum of compound 1 (500 MHz, CD3OD)    68
13C NMR spectrum of compound 1 (125 MHz, CD3OD)    69
1H NMR spectrum of compound 2 (500 MHz, CD3OD)    70
13C NMR spectrum of compound 2 (125 MHz, CD3OD)    71
1H NMR spectrum of compound 3 (500 MHz, CD3OD)    72
13C NMR spectrum of compound 3 (125 MHz, CD3OD)    73
1H NMR spectrum of compound 4 (500 MHz, CD3OD)    74
13C NMR spectrum of compound 4 (125 MHz, CD3OD)    75
1H NMR spectrum of compound 5 (500 MHz, CD3OD)    76
13C NMR spectrum of compound 5 (125 MHz, CD3OD)    77
1H NMR spectrum of compound 6 (500 MHz, CD3OD)    78
13C NMR spectrum of compound 6 (125 MHz, CD3OD)    79
1H-13C 2D HMQC NMR spectrum of 6 (500/125 MHz, CD3OD)    80
1H-13C 2D HMBC NMR spectrum of 6 (500/125 MHz, CD3OD)    81
1H-1H 2D COSY NMR spectrum of 6 (500 MHz, CD3OD)    82
High Resolution Mass Spectra of compound 6    83


                                

References
1. Schauer, R.; Kamerling, J. P., Chapter one - exploration of the sialic acid world. In Advances in carbohydrate chemistry and biochemistry, Baker, D. C., Ed. Academic Press: Massachusetts, US, 2018, Vol. 75, 1-213.
2. Varki, A., Sialic acids in human health and disease. Trends Mol. Med. 2008, 14, 351-360.
3. Zhou, X.; Yang, G.; Guan, F., Biological functions and analytical strategies of sialic acids in tumor. Cells 2020, 9, 273.
4. Smith, E.; Dent, G., Introduction, basic theory and principles. In Modern Raman spectroscopy – a practical approach, John Wiley and Sons Ltd: West Sussex, England, 2005, 1-21.
5. Aroca, R., Theory of molecular vibrations. The origin of infrared and Raman spectra. In Surface‐enhanced vibrational spectroscopy, John Wiley and Sons Ltd: West Sussex, England, 2006, 1-33.
6. Fleischmann, M.; Hendra, P. J.; McQuillan, A. J., Raman spectra of pyridine adsorbed at a silver electrode. Chem. Phys. Lett. 1974, 26, 163-166.
7. Le Ru, E. C.; Blackie, E.; Meyer, M.; Etchegoin, P. G., Surface enhanced Raman scattering enhancement factors: A comprehensive study. J. Phys. Chem. C 2007, 111, 13794-13803.
8. Radziuk, D.; Moehwald, H., Prospects for plasmonic hot spots in single molecule SERS towards the chemical imaging of live cells. Phys. Chem. Chem. Phys. 2015, 17, 21072-21093.
9. Kumari, G.; Kandula, J.; Narayana, C., How far can we probe by SERS? J. Phys. Chem. C 2015, 119, 20057-20064.
10. Su, J.-P.; Lee, Y.-T.; Lu, S.-Y.; Lin, J. S., Chemical mechanism of surface-enhanced Raman scattering spectrum of pyridine adsorbed on Ag cluster: Ab initio molecular dynamics approach. J. Comput. Chem. 2013, 34, 2806-2815.
11. Campion, A.; Kambhampati, P., Surface-enhanced Raman scattering. Chem. Soc. Rev. 1998, 27, 241-250.
12. Wang, Y.; Yan, B.; Chen, L., SERS tags: Novel optical nanoprobes for bioanalysis. Chem. Rev. 2013, 113, 1391-1428.
13. Sajanlal, P. R.; Sreeprasad, T. S.; Samal, A. K.; Pradeep, T., Anisotropic nanomaterials: Structure, growth, assembly, and functions. Nano Rev. 2011, 2.
14. Siekkinen, A. R.; McLellan, J. M.; Chen, J.; Xia, Y., Rapid synthesis of small silver nanocubes by mediating polyol reduction with a trace amount of sodium sulfide or sodium hydrosulfide. Chem. Phys. Lett. 2006, 432, 491-496.
15. Koczkur, K. M.; Mourdikoudis, S.; Polavarapu, L.; Skrabalak, S. E., Polyvinylpyrrolidone (PVP) in nanoparticle synthesis. Dalton Trans. 2015, 44, 17883-17905.
16. Im, S. H.; Lee, Y. T.; Wiley, B.; Xia, Y., Large-scale synthesis of silver nanocubes: The role of HCl in promoting cube perfection and monodispersity. Angew. Chem. Int. Ed. 2005, 44, 2154-2157.
17. Zhang, Q.; Li, W.; Wen, L.-P.; Chen, J.; Xia, Y., Facile synthesis of Ag nanocubes of 30 to 70 nm in edge length with CF3COOAg as a precursor. Chem. Eur. J. 2010, 16, 10234-10239.
18. Hong, S.; Lin, L.; Xiao, M.; Chen, X., Live-cell bioorthogonal Raman imaging. Curr. Opin. Chem. Biol. 2015, 24, 91-96.
19. Brooks, W. L. A.; Sumerlin, B. S., Synthesis and applications of boronic acid-containing polymers: From materials to medicine. Chem. Rev. 2016, 116, 1375-1397.
20. Springsteen, G.; Wang, B., A detailed examination of boronic acid–diol complexation. Tetrahedron 2002, 58, 5291-5300.
21. Peters, J. A., Interactions between boric acid derivatives and saccharides in aqueous media: Structures and stabilities of resulting esters. Coord. Chem. Rev. 2014, 268, 1-22.
22. Djanashvili, K.; Koning, G. A.; van der Meer, A. J. G. M.; Wolterbeek, H. T.; Peters, J. A., Phenylboronate 160Tb complexes for molecular recognition of glycoproteins expressed on tumor cells. Contrast Media Mol. Imaging 2007, 2, 35-41.
23. Lin, L.; Tian, X.; Hong, S.; Dai, P.; You, Q.; Wang, R.; Feng, L.; Xie, C.; Tian, Z.-Q.; Chen, X., A bioorthogonal Raman reporter strategy for SERS detection of glycans on live cells. Angew. Chem. Int. Ed. 2013, 52, 7266-7271.
24. Liang, L.; Qu, H.; Zhang, B.; Zhang, J.; Deng, R.; Shen, Y.; Xu, S.; Liang, C.; Xu, W., Tracing sialoglycans on cell membrane via surface-enhanced Raman scattering spectroscopy with a phenylboronic acid-based nanosensor in molecular recognition. Biosens. Bioelectron. 2017, 94, 148-154.
25. Lizzi, A. R.; D’Alessandro, A. M.; Bozzi, A.; Cinque, B.; Oratore, A.; D’Andrea, G., Pattern expression of glycan residues in AZT-treated K562 cells analyzed by lectin cytochemistry. Mol. Cell. Biochem. 2007, 300, 29-37.
26. Di, H.; Liu, H.; Li, M.; Li, J.; Liu, D., High-precision profiling of sialic acid expression in cancer cells and tissues using background-free surface-enhanced Raman scattering tags. Anal. Chem. 2017, 89, 5874-5881.
27. Gong, T.; Cui, Y.; Goh, D.; Voon, K. K.; Shum, P. P.; Humbert, G.; Auguste, J.-L.; Dinh, X.-Q.; Yong, K.-T.; Olivo, M., Highly sensitive SERS detection and quantification of sialic acid on single cell using photonic-crystal fiber with gold nanoparticles. Biosens. Bioelectron. 2015, 64, 227-233.
28. Stiles, P. L.; Dieringer, J. A.; Shah, N. C.; Duyne, R. P. V., Surface-enhanced Raman spectroscopy. Annu. Rev. Anal. Chem. 2008, 1, 601-626.
29. He, X.-N.; Wang, Y.-N.; Wang, Y.; Xu, Z.-R., Accurate quantitative detection of cell surface sialic acids with a background-free SERS probe. Talanta 2020, 209, 120579.
30. Ying, T.-H.; Yang, S.-F.; Tsai, S.-J.; Hsieh, S.-C.; Huang, Y.-C.; Bau, D.-T.; Hsieh, Y.-H., Fisetin induces apoptosis in human cervical cancer HeLa cells through ERK1/2-mediated activation of caspase-8-/caspase-3-dependent pathway. Arch. Toxicol 2012, 86, 263-273.
31. Evanoff Jr., D. D.; Chumanov, G., Synthesis and optical properties of silver nanoparticles and arrays. ChemPhysChem 2005, 6, 1221-1231.
32. Evanoff, D. D.; Chumanov, G., Size-controlled synthesis of nanoparticles. 2. Measurement of extinction, scattering, and absorption cross sections. J. Phys. Chem. B 2004, 108, 13957-13962.
33. Rycenga, M.; Kim, M. H.; Camargo, P. H. C.; Cobley, C.; Li, Z.-Y.; Xia, Y., Surface-enhanced Raman scattering: Comparison of three different molecules on single-crystal nanocubes and nanospheres of silver. J. Phys. Chem. A 2009, 113, 3932-3939.
34. Stamplecoskie, K. G.; Scaiano, J. C.; Tiwari, V. S.; Anis, H., Optimal size of silver nanoparticles for surface-enhanced Raman spectroscopy. J. Phys. Chem. C 2011, 115, 1403-1409.
35. Fievet, F.; Lagier, J. P.; Blin, B.; Beaudoin, B.; Figlarz, M., Homogeneous and heterogeneous nucleations in the polyol process for the preparation of micron and submicron size metal particles. Solid State Ionics 1989, 32-33, 198-205.
36. Kha, N. M.; Chen, C.-H.; Su, W.-N.; Rick, J.; Hwang, B.-J., Improved Raman and photoluminescence sensitivity achieved using bifunctional Ag@SiO2 nanocubes. Phys. Chem. Chem. Phys. 2015, 17, 21226-21235.
37. Bhattacharjee, S., DLS and zeta potential – what they are and what they are not? J. Controlled Release 2016, 235, 337-351.
38. Wu, H.-L.; Kuo, C.-H.; Huang, M. H., Seed-mediated synthesis of gold nanocrystals with systematic shape evolution from cubic to trisoctahedral and rhombic dodecahedral structures. Langmuir 2010, 26, 12307-12313.
39. Kuo, B.-H.; Hsia, C.-F.; Chen, T.-N.; Huang, M. H., Systematic shape evolution of gold nanocrystals achieved through adjustment in the amount of HAuCl4 solution used. J. Phys. Chem. C 2018, 122, 25118-25126.
40. Matsumoto, A.; Stephenson-Brown, A. J.; Khan, T.; Miyazawa, T.; Cabral, H.; Kataoka, K.; Miyahara, Y., Heterocyclic boronic acids display sialic acid selective binding in a hypoxic tumor relevant acidic environment. Chem. Sci. 2017, 8, 6165-6170.
41. Moran, C. H.; Rycenga, M.; Zhang, Q.; Xia, Y., Replacement of poly(vinyl pyrrolidone) by thiols: A systematic study of Ag nanocube functionalization by surface-enhanced Raman scattering. J. Phys. Chem. C 2011, 115, 21852-21857.
42. Madeira, A.; Plissonneau, M.; Servant, L.; Goldthorpe, I. A.; Tréguer-Delapierre, M., Increasing silver nanowire network stability through small molecule passivation. Nanomaterials 2019, 9, 899.
43. Loza, K.; Diendorf, J.; Sengstock, C.; Ruiz-Gonzalez, L.; Gonzalez-Calbet, J. M.; Vallet-Regi, M.; Köller, M.; Epple, M., The dissolution and biological effects of silver nanoparticles in biological media. J. Mater. Chem. B 2014, 2, 1634-1643.
44. Michieli, N.; Pilot, R.; Russo, V.; Scian, C.; Todescato, F.; Signorini, R.; Agnoli, S.; Cesca, T.; Bozio, R.; Mattei, G., Oxidation effects on the SERS response of silver nanoprism arrays. RSC Adv. 2017, 7, 369-378.
45. Pearson, D. A.; Blanchette, M.; Baker, M. L.; Guindon, C. A., Trialkylsilanes as scavengers for the trifluoroacetic acid deblocking of protecting groups in peptide synthesis. Tetrahedron Lett. 1989, 30, 2739-2742.
46. Wehner, J. W.; Lindhorst, T. K., Glycocluster synthesis by native chemical ligation. Synthesis 2010, 2010, 3070-3082.
47. Li, S.; Zhou, Q.; Chu, W.; Zhao, W.; Zheng, J., Surface-enhanced Raman scattering behaviour of 4-mercaptophenyl boronic acid on assembled silver nanoparticles. Phys. Chem. Chem. Phys. 2015, 17, 17638-17645.
48. Song, W.; Ding, L.; Chen, Y.; Ju, H., Plasmonic coupling of dual gold nanoprobes for SERS imaging of sialic acids on living cells. Chem. Comm. 2016, 52, 10640-10643.
49. Wang, D.-E.; Yan, J.; Jiang, J.; Liu, X.; Tian, C.; Xu, J.; Yuan, M.-S.; Han, X.; Wang, J., Polydiacetylene liposomes with phenylboronic acid tags: A fluorescence turn-on sensor for sialic acid detection and cell-surface glycan imaging. Nanoscale 2018, 10, 4570-4578.

簡易檢索 / 詳目顯示

相關論文