細胞膜在人體當中扮演著重要的角色，除了作為細胞的屏障以外，調控細胞內的 pH 值、免疫的調節以及細胞與細胞間的溝通都是非常的重要，因此有必要對細胞膜進行觀測。在本論文研究中，旨在開發一個能夠同時擁有螢光與拉曼訊號之細胞質膜探針，首先利用實驗室已經開發的螢光分子2,3-二氫喹啉-4-亞胺 (2,3-dihydroquinolin-4-imines, DQI)，並在 DQI 分子上修飾上炔丙基作為拉曼標籤，進而合成出模型分子 DQI-CDI，接著利用 DQI-CDI 對癌細胞進行標記，並利用螢光以及拉曼訊號觀測癌細胞之細胞膜。經過 DQI-CDI 分子的測試後的結果證實了螢光-拉曼探針的可行性，因此同樣是利用 DQI 分子作為基本的骨架，在 DQI 分子上修飾炔丙基以及長碳鏈的脂質，成功合成出對於細胞膜具有專一性的螢光-拉曼探針 DQI-FR，然而 DQI-FR 在共軛焦雷射掃描顯微鏡中可以發現對細胞膜有良好的專一性，但是在利用拉曼光譜儀測量 DQI-FR 的拉曼訊號時發現其拉曼訊號強度非常微弱，無法對癌細胞之細胞膜進行成像，因此後續要如何提升 DQI-FR 的拉曼訊號強度仍有待調整。

Cell membrane plays a crucial role in human body. Besides serving as a barrier for the cell, it is essential for regulating intracellular pH, immune modulation, and cell-cell communication. Therefore, it is necessary to monitor cell membrane. To this purpose, this study aims to develop a probe for the plasma membrane that exhibits both fluorescence and Raman signals at the same time. The fluorescence molecule 2,3-dihydroquinolin-4-imine (DQI), which has been developed in our laboratory, was used as the basic skeleton, and a propargyl group was introduced as a Raman tag on the DQI molecule which is regarded as a model molecule DQI-CDI. The DQI-CDI probe was then used to label cancer cells, and both fluorescence and Raman signals were observed on cancer cell membranes. These results confirmed the feasibility of the fluorescence-Raman dual-mode probe after cell test. Therefore, DQI molecule is also used as the basic skeleton, and a propargyl group and long-chain lipid were further modified on the DQI molecule, which exhibits membrane-specific fluorescence and Raman signaling. In confocal laser scanning microscopy (CLSM), the images show that DQI-FR has good specificity of cell membrane. However, the Raman signal of DQI-FR was measured by Raman spectroscopy, it was found that the intensity of Raman signal was very weak and failed in imaging the cell membrane of cancer cells. Therefore, further optimization is required to enhance the intensity of Raman signal of DQI-FR.

論文審定書 i
謝誌 ii
中文摘要 iii
Abstract iv
目錄 v
圖目錄 vii
流程目錄 xi
光譜附圖 xii
縮寫表 xiv
第一章、緒論 1
第一節、 研究背景 1
1.1.1 細胞膜的組成 1
1.1.2 細胞膜形變與生物調節之關係 5
1.1.3 觀測細胞膜之方法 8
1.1.3.1 非光學方法 (Non-Optical Methods) 8
1.1.3.1.1 穿透式電子顯微鏡 (Transmission Electron Microscope) 8
1.1.3.1.2 原子力顯微鏡 (Atomic Force Microscope) 11
1.1.3.2 光學方法 (Optical Methods) 17
1.1.3.2.1 螢光光譜 (Fluorescence Spectroscopy) 17
1.1.3.2.1.1 螢光探針的放光種類 17
1.1.3.2.1.1.1 分子內電荷轉移 17
1.1.3.2.1.1.2 激發態分子內質子轉移 20
1.1.3.2.1.1.3 聚集誘導放光 23
1.1.3.2.1.1.4 光誘導電子轉移 25
1.1.3.2.2 拉曼光譜 28
1.1.4 螢光-拉曼探針發展現況 36
第二節、 研究動機與實驗設計 38
第二章、實驗結果與討論 42
2.1 模型分子 DQI-CDI 之合成 42
2.2 模型分子 DQI-CDI 光物理性質數據及標記測試 44
2.3 螢光-拉曼分子 DQI-N3之合成 49
2.4 長碳鏈脂質之合成 51
2.5 環張力炔類分子之合成 52
2.6 細胞膜螢光-拉曼探針 DQI-FR 之合成 52
2.7 細胞膜螢光-拉曼探針 DQI-FR 光物理數據及癌細胞標記 56
第三章、結果與未來展望 60
第四章、參考文獻 62
第五章、實驗材料與方法 66
第一節、 儀器設備與藥品材料 66
第二節、 合成步驟與數據 68
第三節、 實驗步驟 88
5.3.1. HeLa cell culture 88
5.3.2. Confocal laser scanning microscope (CLSM) in cells 88
5.3.3. Raman microscope in cells 89
第六章、光譜數據 90

1. Casey, J. R.; Grinstein, S.; Orlowski, J. Nat. Rev. Mol. Cell Biol. 2010, 11, 50–61.
2. Lin, J. C.; Duell, K.; Konopka, J. B. Mol. Cell Biol. 2004, 24, 2041–2051.
3. Jiang, Y.; Lee, A.; Chen, J.; Ruta, V.; Cadene, M.; Chait, B. T.; MacKinnon, R. Nature. 2003, 423, 33–41.
4. Sezgin, E.; Levental, I.; Mayor, S.; Eggeling, C. Nat. Rev. Mol. Cell Biol. 2017, 18, 361–374.
5. Harayama, T.; Riezman, H. Nat. Rev. Mol. Cell Biol. 2018, 19, 281–296.
6. Griffiths, W. J.; Abdel-Khalik, J.; Yutuc, E.; Morgan, A. H.; Gilmore, I.; Hearn, T.; Wang, Y. Anal. Biochem. 2017, 524, 56–67.
7. Nagahashi, M.; Ramachandran, S.; Kim, E. Y.; Allegood, J. C.; Rashid, O. M.; Yamada, A.; Zhao, R.; Milstien, S.; Zhou, H.; Spiegel, S. Cancer Res. 2012, 72, 726–735.
8. Guo, Y. X.; Ma, Y. J.; Han, L.; Wang Y. J.; Han, J. A.; Zhu, Y. Int. J. Clin. Exp. Med. 2015, 8, 20349–20354.
9. Tsuchida, J.; Nagahashi, M.; Takabe, K.; Wakai, T. Mediators Inflamm. 2017, 9, 2076239.
10. Ingolfsson, H. I.; Melo, M. N.; van Eerden, F. J.; Arnarez, C.; Lopez, C. A.; Wassenaar, T. A.; Periole, X.; de Vries, A. H.; Tieleman, D. P.; Marrink, S. J. J. Am. Chem. Soc. 2014, 136, 14554–14559.
11. Preta, G. Front. Cell Dev. Biol. 2020, 8, 571237.
12. Szlasa, W.; Zendran, I.; Zalesinska, A.; Tarek, M.; Kulbacka, J. J. Bioenerg. Biomembr. 2020, 52, 321–342.
13. Tan, L. T.; Chan, K. G.; Pusparajah, P.; Lee, W. L.; Chuah, L. H.; Khan, T. M.; Lee, L. H.; Goh, B. H. Front. Pharmacol. 2017, 8, 12.
14. Rizzo, W. B.; S'Aulis, D.; Dorwart, E.; Bailey, Z. Mol. Genet. Metab. Rep. 2022, 30, 100839.
15. Liang, H.; Mu, H.; Jean-Francois, F.; Lakshman, B.; Sarkar-Banerjee, S.; Zhuang, Y.; Zeng, Y.; Gao, W.; Zaske, A. M.; Nissley, D. V. Life Sci. Alliance. 2019, 2, e201900343.
16. Bozelli, J. C., Jr.; Epand, R. M. J. Mol. Biol. 2020, 432, 5124–5136.
17. Muller, D. A. Nat. Mater. 2009, 8, 263–270.
18. Graham, L.; Orenstein, J. M. Nat. Protoc. 2007, 2, 2439–2450.
19. Palade, G. E. J. Histochem. Cytochem. 1953, 1, 188–211.
20. Briggs, J. A.; Riches, J. D.; Glass, B.; Bartonova, V.; Zanetti, G.; Krausslich, H. G. Proc. Natl. Acad. Sci. U.S.A. 2009, 106, 11090–11095.
21. Binnig, G.; Quate, C. F.; Gerber, C. Phys. Rev. Lett. 1986, 56, 930–933.
22. Shan, Y.; Wang, H. Chem. Soc. Rev. 2015, 44, 3617–3638.
23. Butt, H. J.; Wolff, E. K.; Gould, S. A.; Dixon Northern, B.; Peterson, C. M.; Hansma, P. K. J. Struct. Biol. 1990, 105, 54–61.
24. Radmacher, M. IEEE Eng. Med. Biol. Mag. 1997, 16, 47–57.
25. Frederix, P. L. T. M.; Bosshart, P. D.; Engel, A. Biophys. J. 2009, 96, 329–338.
26. Dumitru, A. C.; Conrard, L.; Lo Giudice, C.; Henriet, P.; Veiga-da-Cunha, M.; Derclaye, S.; Tyteca, D.; Alsteens, D. Chem. Commun. 2018, 54, 6903–6906.
27. Georgiev, N. I.; Bakov, V. V.; Anichina, K. K.; Bojinov, V. B. Pharmaceuticals. 2023, 16, 381.
28. Ma, Y.; Chen, Q.; Pan, X.; Zhang, J. Top. Curr. Chem. 2021, 379, 10.
29. Liu, Y.; Zhang, T.; Huo, F.; Yin, C. Dyes Pigm. 2022, 205, 110540.
30. Weller, A. Naturwissenschaften. 1955, 42, 175–176.
31. Padalkar, V. S.; Seki, S. Chem. Soc. Rev. 2016, 45, 169–202.
32. Sedgwick, A. C.; Wu, L.; Han, H. H.; Bull, S. D.; He, X. P.; James, T. D.; Sessler, J. L.; Tang, B. Z.; Tian, H.; Yoon, J. Chem. Soc. Rev. 2018, 47, 8842–8880.
33. Li, K.; Lyu, Y.; Huang, Y.; Xu, S.; Liu, H. W.; Chen, L.; Ren, T. B.; Xiong, M.; Huan, S.; Yuan, L. Proc. Natl. Acad. Sci. U. S. A. 2021, 118, e2018033118.
34. Luo, J.; Xie, Z.; Lam, J. W.; Cheng, L.; Chen, H.; Qiu, C.; Kwok, H. S.; Zhan, X.; Liu, Y.; Zhu, D. Chem. Commun. 2001, 1740–1741.
35. Hong, Y.; Lam, J. W.; Tang, B. Z. Chem. Commun. 2009, 4332–4353.
36. Wang, D.; Su, H.; Kwok, R. T. K.; Hu, X.; Zou, H.; Luo, Q.; Lee, M. M. S.; Xu, W.; Lam, J. W. Y.; Tang, B. Z. Chem. Sci. 2018, 9, 3685–3693.
37. de Silva, A. P.; Gunaratne, H. Q. N.; Habib‐Jiwan, J. L.; McCoy, C. P.; Rice, T. E.; Soumillion, J. P. Angew. Chem. Int. Ed. 1995, 34, 1728–1731.
38. de Silva, A. P. J. Phys. Chem. Lett. 2011, 2, 28658–2871.
39. Podder, A.; Joseph, M. M.; Biswas, S.; Samanta, S.; Maiti, K. K.; Bhuniya, S. Chem. Commun. 2021, 57, 6078–610.
40. Epstein, T.; Xu, L.; Gillies, R. J.; Gatenby, R. A. Cancer Metab. 2014, 2, 7.
41. Raman, C. V.; Krishnan, K. S. Nature. 1928, 121, 501–502.
42. Mosca, S.; Conti, C.; Stone, N.; Matousek, P. Nat. Rev. Methods Primers. 2021, 1, 21.
43. Smith, R.; Wright, K. L.; Ashton, L. Analyst. 2016, 141, 3590–3600.
44. Fujita, K.; Smith, N. I. Mol. Cells. 2008, 26, 530–535.
45. Dodo, K.; Fujita, K.; Sodeoka, M. J. Am. Chem. Soc. 2022, 144, 19651–19667.
46. Yamakoshi, H.; Dodo, K.; Palonpon, A.; Ando, J.; Fujita, K.; Kawata, S.; Sodeoka, M. J. Am. Chem. Soc. 2012, 134, 20681–20689.
47. Hamada, K.; Fujita, K.; Smith, N. I.; Kobayashi, M.; Inouye, Y.; Kawata, S. J Biomed. Opt. 2008, 13, 044027.
48. Bentinger, M.; Tekle, M.; Dallner, G. Biochem. Biophys. Res. Commum. 2010, 396, 74–79.
49. Hu, F.; Zeng, C.; Long, R.; Miao, Y.; Wei, L.; Xu, Q.; Min, W. Nat. Methods. 2018, 15, 194–200.
50. Lin, J.; Graziotto, M. E.; Lay, P. A.; New, E. J. Cells. 2021, 10, 1699.
51. Collot, M.; Ashokkumar, P.; Anton, H.; Boutant, E.; Faklaris, O.; Galli, T.; Mely, Y.; Danglot, L.; Klymchenko, A. S. Cell Chem. Biol. 2019, 26, 600–614.
52. Collot, M.; Kreder, R.; Tatarets, A. L.; Patsenker, L. D.; Mely, Y.; Klymchenko, A. S. Chem. Commun 2015, 51, 17136–17139.
53. Kucherak, O. A.; Oncul, S.; Darwich, Z.; Yushchenko, D. A.; Arntz, Y.; Didier, P.; Mely, Y.; Klymchenko, A. S. J. Am. Chem. Soc. 2010, 132, 4907–4916.
54. Yang, L.; Chen, Q.; Wang, Z.; Zhang, H.; Sun, H. Coord. Chem. Rev. 2023, 474.
55. Chou, C. H.; Rajagopal, B.; Liang, C. F.; Chen, K. L.; Jin, D. Y.; Chen, H. Y.; Tu, H. C.; Shen, Y. Y.; Lin, P. C. Chem. Eur. J. 2018, 24, 1112–1120.
56. Edwards, H. G. M. Handbook of vibrational spectroscopy, 2006.
57. Wiergowska, G.; Stasilowicz, A.; Miklaszewski, A.; Lewandowska, K.; Cielecka-Piontek, J. Pharmaceutics. 2021, 13, 384
58. Czamara, K.; Majzner, K.; Pacia, M. Z.; Kochan, K.; Kaczor, A.; Baranska, M. J. Raman Spectrosc. 2015, 46, 4–20.
59. 靳惇淵 2022 利用金屬催化建構具有可調式放光之環境敏感性螢光分子衍生物並應用於生醫影像顯影劑之開發與應用 博士論文 國立中山大學化學研究所.
60. 楊淨雯 2022 開發新型環境敏感螢光分子應用於癌細胞質膜之 pH 動態觀測 碩士論文 國立中山大學化學研究所.
61. Tu, H. C.; Lee, Y. P.; Liu, X. Y.; Chang, C. F.; Lin, P. C. ACS Appl. Bio. Mater. 2019, 2, 1286–1297.