"表面增強拉曼光譜細菌藥敏測試法” 所利用的生物標記之分子來源探討

簡易檢索 / 詳目顯示

回結果列表

研究生：	邱文瑜 Chiu, Shirley Wen-Yu
論文名稱：	"表面增強拉曼光譜細菌藥敏測試法” 所利用的生物標記之分子來源探討 Molecular Origin of the Surface-Enhanced-Raman-Scattering Biomarkers Exploited for Bacterial Antibiotic Susceptibility Test
指導教授：	王玉麟 Wang, Yuh-Lin 楊家銘 Yang, Chia-Min
口試委員:	倪其焜 Ni, Chi-Kung 劉定宇 Liu, Ting-Yu 王俊凱 Wang, Juen-Kai 黃念祖 Huang, Nien-Tsu
學位類別：	博士 Doctor
系所名稱：	理學院 - 化學系 Department of Chemistry
論文出版年：	2020
畢業學年度：	108
語文別：	英文
論文頁數：	90
中文關鍵詞：	表面增強式拉曼光譜、抗生素藥物敏感測試、超高效液相層析串聯電噴灑式質譜、嘌呤、核糖體核糖核酸降解
外文關鍵詞：	Antibiotic susceptibility test (AST), UPLC/ESI-MS, purine metabolites, ribosomal RNA degradation
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

表面增強式拉曼光譜由於其快速偵測及非侵入性的特性，已經被廣泛地應用在微生物領域的研究上。近年來，細菌表面增強式拉曼光譜中的生物標記也被成功地運用在細菌的抗生素藥物敏感測試。本論文的目標為探討細菌在表面增強拉曼光譜結合藥物敏感測試中的生物標記之分子來源及其所代表的生理意義。我們使用了表面增強拉曼光譜及超高效液相層析串聯電噴灑式質譜儀，鑑定了格蘭氏陽性菌金黃色葡萄球菌與格蘭氏陰性菌大腸桿菌之生物標記的分子來源為來自細菌代謝釋放之數個嘌呤衍生物，更利用質譜將細菌在飢餓壓力之下釋放的分子隨時間作定量分析
。除此之外，為了瞭解細菌釋放此嘌呤衍生物之緣由，我們分析了數個核糖核酸降解基因缺失的大腸桿菌細菌株的拉曼光譜及質譜，證實細菌釋放之嘌呤衍生物與飢餓誘發之核醣體核糖核酸降解之關聯性。

Surface-enhanced Raman scattering (SERS), due to its rapid and non-invasive characteristics, has be widely used in microbiological fields. Recently, the SERS biomarkers from bacteria were also applied to bacterial antibiotic susceptibility testing (AST) successfully. The aim of this work is to explore the origin of the biomolecules responsible for the bacterial biomarkers in the SERS-AST method and the underlying physiological implication. We used both SERS and UPLC/ESI-MS to identify the molecules released from Gram-positive bacteria Staphylococcus aureus and Gram-negative bacteria Escherichia coli as several purine derivatives, as well as to quantify the time-dependence concentration of the purine derivatives from bacteria in starvation stress. Furthermore, changes in both the SERS and MS spectral features in Escherichia coli mutants without specific RNA degrading enzymes were observed, verifying that the origin of the purine derivatives from bacteria is attributed to the ribosomal RNA degradation upon starvation.

TABLE OF CONTENTS

ABSTRACT                   I
TABLE OF CONTENTS         III
LIST OF FIGURES                   V

CHAPTER I  Introduction           1
1  Motivation                   1
2  Raman scattering           2
3  Surface-enhanced Raman Scattering            3
4  Applications of SERS on microbiology    6
5  SERS-based antibiotic susceptibility test (SERS-AST)    9
6  Hypotheses of the molecules responsible for bacterial SERS spectra         13
6.1  Signal contribution from bacterial outer structures    13
6.2  Signal contribution from bacterial purine derivative molecules    14
CHAPTER II  Research methods    18
1    Bacterial sample preparation    18
1.1  Bacterial strains    18
1.2  Bacterial growth media    18
1.3  Bacterial sample pretreatment    19
2    Reagents and Materials    20
3  Fabrication of SERS-active substrates    21
4    Raman spectra acquisition and processing    23
5    UPLC/ESI-MS analysis    24
CHAPTER III  Identification and quantification of the responsible biomolecules    26
1    SERS spectra of bacterial suspension and supernatant    26
2    SERS spectra of bacterial supernatants after water incubation    28
3  Mass spectra of bacterial supernatants    30
4    Isotope substitution of bacteria    37
5    Quantification of excreted purine derivatives from bacteria    38
5.1  Calibration curves of internal and external standards    39
5.2  Time-dependence of released purine nucleobases from bacteria    43
6  Viability of bacteria incubated in water    49
7  Simulation of bacterial SERS spectra    50
8  Discussion    53
8.1  The challenges in the identification and quantification of molecules in SERS    53
8.2  Purine-releasing behaviors in bacteria    57
CHAPTER IV Purine derivatives from RNA degradation    60
1  Motivation    60
2  RNA degradation    61
3  Starvation-induced RNA degradation             65
4  Results and discussion                         68
4.1  SERS spectra of E.coli wild-type and RNase deletion strains
68
4.2  Mass analysis of E.coli wild-type and RNase deletion strains         74
4.3  Regulation of RNase PH by RNase II upon starvation    78
CONCLUSIONS    83
REFERENCES    84


                                

1. C. Y. Liu, Y. Y. Han, P. H. Shih, W. N. Lian, H. H. Wang, C. H. Lin, P. R. Hsueh, J. K. Wang and Y. L. Wang, Sci Rep, 2016, 6, 15.
2. Y.-Y. Han, Y.-C. Lin, W.-C. Cheng, Y.-T. Lin, L.-J. Teng, J.-K. Wang and Y.-L. Wang, Sci Rep, 2020, 10, 12538.
3. C. V. Raman and K. S. Krishnan, Nature, 1928, 121, 501-502.
4. G. Keresztury, in Handbook of Vibrational Spectroscopy2006.
5. P. R. G. Derek J. Gariner, 1989.
6. G. D. Ewen Smith, Modern Raman Spectroscopy – A Practical Approach, Wiley 2004.
7. C. Handapangoda, S. Nahavandi and M. Premaratne, 2013, pp. 439-472.
8. M. Fleischmann, P. Hendra and A. J. C. p. l. McQuillan, 1974, 26.
9. D. L. Jeanmaire and R. P. Van Duyne, Journal of Electroanalytical Chemistry and Interfacial Electrochemistry, 1977, 84, 1-20.
10. M. G. Albrecht and J. A. Creighton, J. Am. Chem. Soc., 1977, 99, 5215-5217.
11. P. L. Stiles, J. A. Dieringer, N. C. Shah and R. R. Van Duyne, Annu. Rev. Anal. Chem., 2008, 1, 601-626.
12. J. Langer, D. Jimenez de Aberasturi, J. Aizpurua, R. A. Alvarez-Puebla, B. Auguié, J. J. Baumberg, G. C. Bazan, S. E. J. Bell, A. Boisen, A. G. Brolo, J. Choo, D. Cialla-May, V. Deckert, L. Fabris, K. Faulds, F. J. García de Abajo, R. Goodacre, D. Graham, A. J. Haes, C. L. Haynes, C. Huck, T. Itoh, M. Käll, J. Kneipp, N. A. Kotov, H. Kuang, E. C. Le Ru, H. K. Lee, J.-F. Li, X. Y. Ling, S. A. Maier, T. Mayerhöfer, M. Moskovits, K. Murakoshi, J.-M. Nam, S. Nie, Y. Ozaki, I. Pastoriza-Santos, J. Perez-Juste, J. Popp, A. Pucci, S. Reich, B. Ren, G. C. Schatz, T. Shegai, S. Schlücker, L.-L. Tay, K. G. Thomas, Z.-Q. Tian, R. P. Van Duyne, T. Vo-Dinh, Y. Wang, K. A. Willets, C. Xu, H. Xu, Y. Xu, Y. S. Yamamoto, B. Zhao and L. M. Liz-Marzán, ACS Nano, 2020, 14, 28-117.
13. E. C. Le Ru and P. G. Etchegoin, in Principles of Surface-Enhanced Raman Spectroscopy, eds. E. C. Le Ru and P. G. Etchegoin, Elsevier, Amsterdam2009, pp. 1-27.
14. J. P. Camden, J. A. Dieringer, Y. Wang, D. J. Masiello, L. D. Marks, G. C. Schatz and R. P. Van Duyne, J. Am. Chem. Soc., 2008, 130, 12616-12617.
15. A. Otto, 2005, 36, 497-509.
16. B. Sharma, R. R. Frontiera, A.-I. Henry, E. Ringe and R. P. Van Duyne, Materials Today, 2012, 15, 16-25.
17. P. Mosier-Boss, Nanomaterials, 2017, 7, 142.
18. H. H. Wang, C. Y. Liu, S. B. Wu, N. W. Liu, C. Y. Peng, T. H. Chan, C. F. Hsu, J. K. Wang and Y. L. Wang, Adv. Mater., 2006, 18, 491-+.
19. H. H. Wang, T. Y. Cheng, P. Sharma, F. Y. Chiang, S. W. Y. Chiu, J. K. Wang and Y. L. Wang, Nanotechnology, 2011, 22, 5.
20. W. E. Huang, R. I. Griffiths, I. P. Thompson, M. J. Bailey and A. S. Whiteley, Anal. Chem., 2004, 76, 4452-4458.
21. C. G. Xie, C. Goodman, M. A. Dinno and Y. Q. Li, Opt. Express, 2004, 12, 6208-6214.
22. R. M. Jarvis and R. Goodacre, Anal. Chem., 2004, 76, 40-47.
23. C. Fan, Z. Q. Hu, L. K. Riley, G. A. Purdy, A. Mustapha and M. S. Lin, J. Food Sci., 2010, 75, M302-M307.
24. W. R. Premasiri, D. T. Moir, M. S. Klempner, N. Krieger, G. Jones and L. D. Ziegler, J. Phys. Chem. B, 2005, 109, 312-320.
25. T.-T. Liu, Y.-H. Lin, C.-S. Hung, T.-J. Liu, Y. Chen, Y.-C. Huang, T.-H. Tsai, H.-H. Wang, D.-W. Wang, J.-K. Wang, Y.-L. Wang and C.-H. Lin, Plos One, 2009, 4.
26. T. Y. Liu, K. T. Tsai, H. H. Wang, Y. Chen, Y. H. Chen, Y. C. Chao, H. H. Chang, C. H. Lin, J. K. Wang and Y. L. Wang, Nat. Commun., 2011, 2, 8.
27. J.-Y. Chien, Y.-C. Gu, H.-M. Tsai, C.-H. Liu, C.-Y. Yen, Y.-L. Wang, J.-K. Wang, C.-H. J. J. o. F. Lin and D. Analysis, 2020, 28.
28. T. T. Liu, Y. H. Lin, C. S. Hung, T. J. Liu, Y. Chen, Y. C. Huang, T. H. Tsai, H. H. Wang, D. W. Wang, J. K. Wang, Y. L. Wang and C. H. Lin, PLoS One, 2009, 4, 10.
29. R. M. Jarvis, A. Brooker and R. Goodacre, Faraday Discuss., 2006, 132, 281-292.
30. H. Y. Chu, Y. W. Huang and Y. P. Zhao, Appl. Spectrosc., 2008, 62, 922-931.
31. J. Guicheteau, L. Argue, D. Emge, A. Hyre, M. Jacobson and S. Christesen, Appl. Spectrosc., 2008, 62, 267-272.
32. W. R. Premasiri, A. F. Sauer-Budge, J. C. Lee, C. M. Klapperich and L. D. Ziegler, Spectroscopy (Springf), 2012, 27, s8-31.
33. L. Cui, P. Y. Chen, S. D. Chen, Z. H. Yuan, C. P. Yu, B. Ren and K. S. Zhang, Anal. Chem., 2013, 85, 5436-5443.
34. L. Brown, J. M. Wolf, R. Prados-Rosales and A. Casadevall, Nat. Rev. Microbiol., 2015, 13, 620-630.
35. W. R. Premasiri, A. F. Sauer-Budge, J. C. Lee, C. M. Klapperich and L. D. Ziegler, Spectroscopy (Springfield, Or.), 2012, 27, s8-31.
36. S. E. J. Bell and N. M. S. Sirimuthu, Journal of the American Chemical Society, 2006, 128, 15580-15581.
37. K. Kneipp and J. Flemming, J. Mol. Struct., 1986, 145, 173-179.
38. W. R. Premasiri, J. C. Lee, A. Sauer-Budge, R. Theberge, C. E. Costello and L. D. Ziegler, Anal. Bioanal. Chem., 2016, 408, 4631-4647.
39. S. W.-Y. Chiu, H.-W. Cheng, Z.-X. Chen, H.-H. Wang, M.-Y. Lai, J.-K. Wang and Y.-L. Wang, Phys. Chem. Chem. Phys., 2018.
40. M. Morhac and V. Matousek, Appl. Spectrosc., 2008, 62, 91-106.
41. B. D. Bennett, J. Yuan, E. H. Kimball and J. D. Rabinowitz, Nat. Protoc., 2008, 3, 1299-1311.
42. A. Otto and M. Futamata, in Surface-Enhanced Raman Scattering: Physics and Applications, eds. Katrin Kneipp, Martin Moskovits and H. Kneipp, Springer-Verlag Berlin Heidelberg2006, pp. 147-182.
43. N. Colthup, L. Daly and S. Wiberley, in Introduction to Infrared and Raman Spectroscopy, 3rd Edition, Academic Press, New York1990, ch. 13.
44. G. Limousin, J. P. Gaudet, L. Charlet, S. Szenknect, V. Barthes and M. Krimissa, Appl. Geochem., 2007, 22, 249-275.
45. K. Y. Foo and B. H. Hameed, Chem. Eng. J., 2010, 156, 2-10.
46. A. Otto, Phys. Status Solidi A-Appl. Mat., 2001, 188, 1455-1470.
47. U. Rinas, K. Hellmuth, R. J. Kang, A. Seeger and H. Schlieker, Appl. Environ. Microbiol., 1995, 61, 4147-4151.
48. M. J. Brauer, J. Yuan, B. D. Bennett, W. Y. Lu, E. Kimball, D. Botstein and J. D. Rabinowitz, Proc. Natl. Acad. Sci. U. S. A., 2006, 103, 19302-19307.
49. H. Link, T. Fuhrer, L. Gerosa, N. Zamboni and U. Sauer, Nat. Methods, 2015, 12, 1091-1097.
50. K. Kneipp, Y. Wang, H. Kneipp, L. T. Perelman, I. Itzkan, R. Dasari and M. S. Feld, Phys. Rev. Lett., 1997, 78, 1667-1670.
51. J. D. Watson and F. H. C. Crick, Nature, 1953, 171, 737-738.
52. M. Bonora, S. Patergnani, A. Rimessi, E. De Marchi, J. M. Suski, A. Bononi, C. Giorgi, S. Marchi, S. Missiroli, F. Poletti, M. R. Wieckowski and P. Pinton, Purinergic Signal., 2012, 8, 343-357.
53. G. Burnstock, Bioessays, 2012, 34, 218-225.
54. A. Verkhratsky and G. Burnstock, Bioessays, 2014, 36, 697-705.
55. G. Burnstock and A. Verkhratsky, Acta Physiol., 2009, 195, 415-447.
56. B. McCarthy, Biochimica et Biophysica Acta (BBA)-Specialized Section on Nucleic Acids and Related Subjects, 1962, 55, 880-889.
57. H. Maruyama and D. i. Mizuno, Biochimica et Biophysica Acta (BBA) - Nucleic Acids and Protein Synthesis, 1970, 199, 159-165.
58. R. Kaplan and D. Apirion, Journal of Biological Chemistry, 1975, 250, 1854-1863.
59. F. Ben-Hamida and D. Schlessinger, Biochimica et Biophysica Acta (BBA) - Nucleic Acids and Protein Synthesis, 1966, 119, 183-191.
60. R. KAPLAN and D. APIRION, 1975.
61. M. P. Deutscher, Nucleic acids research, 2006, 34, 659-666.
62. Z. Li and M. P. J. M. i. e. Deutscher, 2008, 447, 31-45.
63. K. Piir, A. Paier, A. Liiv, T. Tenson and U. Maiväli, EMBO Rep., 2011, 12, 458-462.
64. M. P. Deutscher, Progress in molecular biology and translational science, 2009, 85, 369-391.
65. A. W. Nicholson, Fems Microbiol. Rev., 1999, 23, 371-390.
66. Z. Li and M. Deutscher, 2004.
67. J. M. Andrade, V. Pobre, I. J. Silva, S. Domingues and C. M. Arraiano, in Progress in Molecular Biology and Translational Science, Academic Press2009, vol. 85, pp. 187-229.
68. M. P. Deutscher, Journal of Biological Chemistry, 2003, 278, 45041-45044.
69. M. A. Zundel, G. N. Basturea and M. P. Deutscher, Rna, 2009, 15, 977-983.
70. S. Sulthana, G. N. Basturea and M. P. J. R. Deutscher, 2016, 22, 1163-1171.
71. R. F. dos Santos, A. P. Quendera, S. Boavida, A. F. Seixas, C. M. Arraiano and J. M. Andrade, in Progress in Molecular Biology and Translational Science, ed. D. B. Teplow, Academic Press2018, vol. 159, pp. 101-155.
72. S. Sulthana, E. Quesada and M. P. J. R. Deutscher, 2017, 23, 1456-1464.
73. Z.-F. Cheng and M. P. Deutscher, Proc. Natl. Acad. Sci. U. S. A., 2003, 100, 6388-6393.

簡易檢索 / 詳目顯示

相關論文