Title

利用螢光奈米鑽石做生物標記的開發與應用

Translated Titles

Development and Use of Albumin-Conjugated Fluorescent Nanodiamond for Biolabeling

Authors

張璧名

Key Words

螢光奈米鑽石 ; 白蛋白 ; 抗體 ; fluorescencet nanodiamond ; Albumin protein ; antibody

PublicationName

臺灣師範大學化學系學位論文

Volume or Term/Year and Month of Publication

2011年

Academic Degree Category

碩士

Advisor

張煥正

Content Language

繁體中文

Chinese Abstract

生物研究上,螢光標記是常用的研究方法之一,但常用的螢光標記物 質,如有機螢光染劑、量子點等有光漂白的問題或是具生物毒性而影響生物活性, 這些都會降低研究結果的準確性。相較而言,螢光奈米鑽石能永久維持螢光性質 並具有非常好的生物相容性,沒有量子點會光閃爍而不利於觀察的缺點,在生物 標記上也能做長期及連續的追蹤,因此做為生物標記的工具極具潛力。然而螢光 奈米鑽石在生物研究常用的緩衝溶液中容易聚集 (agglomeration) 的問題,讓 生物標記無法成功達到專一性及良好的影像解析。因此本篇利用白蛋白(albumin protein) 以簡易吸附的方式修飾到螢光奈米鑽石表面上,避免螢光奈米鑽石聚 集的問題,且證明能長時間在生物緩衝溶液 (Phosphate buffered saline, PBS) 中維持良好的懸浮性。 再者,本實驗利用懸浮性良好的螢光奈米鑽石 (albumin-FND)表面的白 蛋白之胺基 (amine group) 位置,將生物素 (biotin) 分子共價鍵結在白蛋白 上成為我們的標記探針 (biotin-albumin conjugated FND)。而我們將具有生物 素分子的 CD44 抗體或霍亂毒素次單元 B 在辨認細胞表面專一性的位置後,利用 卵白素 (streptavidin protein) 與生物素之間極高的親和性來與標記探針 (biotin-albumin conjugated FND) 做連接,並成功得到專一性的螢光標記及良 好的螢光解析。

English Abstract

Immunofluorescence labeling is a common method offering visualization and detection of key cellular processes. Fluorescent probes, such as, organic dye and quantum dots suffer from some limitations, like photo bleaching and toxicity. Recently, fluorescent nanodiamond (FND) has emerged as a promising tool for fluorescence analysis owing to its non-photobleaching and high biocompatibility characteristics. Here we show that albumin-conjugated FNDs disperse well in high ionic strength bio-medium without agglomeration and can keep good stability for at least 9 days just using simple non-covalent conjugation methods. Also, we demonstrate that this albumin-conjugated FNDs coupled with biotins, through covalent cross-linkage with the albumin amine groups, can bind selectively with streptavidin and subsequently with biotinylated CD44 antibodies or biotinylated Cholera toxin subunit B. With this technique, we achieve high-specificity targeting of membrane proteins or glycolipid, and obtain high-quality fluorescence images of fixed and live cells.

Topic Category 基礎與應用科學 > 化學
理學院 > 化學系
Reference
  1. 18. Gruber, A.; Dräbenstedt, A.; Tietz, C.; Fleury, L.; Wrachtrup, J.; Borczyskowski, C. v., Scanning Confocal Optical Microscopy and Magnetic Resonance on Single Defect Centers. Science 1997, 276 (5321), 2012-2014. 19. Yu, S.-J.; Kang, M.-W.; Chang, H.-C.; Chen, K.-M.; Yu, Y.-C., Bright Fluorescent Nanodiamonds: No Photobleaching and Low Cytotoxicity. Journal of the American Chemical Society 2005, 127 (50), 17604-17605.
    連結:
  2. 20. Vairakkannu, V.; et al., The biocompatibility of fluorescent nanodiamonds and their mechanism of cellular uptake. Nanotechnology 2009, 20 (42), 425103. 21. Huang, L. C. L.; Chang, H.-C., Adsorption and Immobilization of Cytochrome c on Nanodiamonds. Langmuir 2004, 20 (14), 5879-5884.
    連結:
  3. 22. CHAFFEY, N., Alberts, B., Johnson, A., Lewis, J., Raff, M., Roberts, K. and Walter, P. Molecular biology of the cell. 4th edn. Annals of Botany 2003, 91 (3), 401. 23. Ormö, M.; Cubitt, A. B.; Kallio, K.; Gross, L. A.; Tsien, R. Y.; Remington, S. J., Crystal Structure of the Aequorea victoria Green Fluorescent Protein. Science 1996, 273 (5280), 1392-1395.
    連結:
  4. 38. Wee, T.-L.; Tzeng, Y.-K.; Han, C.-C.; Chang, H.-C.; Fann, W.; Hsu, J.-H.; Chen, K.-M.; Yu, Y.-C., Two-photon Excited Fluorescence of Nitrogen-Vacancy Centers in Proton-Irradiated Type Ib Diamond†. The Journal of Physical Chemistry A 2007, 111 (38), 9379-9386.
    連結:
  5. 1. Schröder, U.; Sabel, B. A., Nanoparticles, a drug carrier system to pass the blood-brain barrier, permit central analgesic effects of i.v. dalargin injections. Brain Research 1996, 710 (1-2), 121-124. 2. Neugart, F.; Zappe, A.; Jelezko, F.; Tietz, C.; Boudou, J. P.; Krueger, A.; Wrachtrup, J., Dynamics of Diamond Nanoparticles in Solution and Cells. Nano Letters 2007, 7 (12), 3588-3591.
  6. 45
  7. 46
  8. 36. Jaiswal, J. K.; Goldman, E. R.; Mattoussi, H.; Simon, S. M., Use of quantum dots for live cell imaging. Nat Meth 2004, 1 (1), 73-78. 37. Wang, Y.; Iqbal, Z.; Mitra, S., Rapidly Functionalized, Water-Dispersed Carbon Nanotubes at High Concentration. Journal of the American Chemical Society 2005, 128 (1), 95-99.
  9. 39. Chang, Y.-R.; Lee, H.-Y.; Chen, K.; Chang, C.-C.; Tsai, D.-S.; Fu, C.-C.; Lim, T.-S.; Tzeng, Y.-K.; Fang, C.-Y.; Han, C.-C.; Chang, H.-C.; Fann, W., Mass production and dynamic imaging of fluorescent nanodiamonds. Nat Nano 2008, 3 (5), 284-288.
  10. 43. Quantum-Dot Leap. Science News Online. Retrieved on 2005-06-17 44. Electric Field Assisted Assembly of Functionalized Quantum Dots into Multiple Layer Thin Films D.A. Dehlinger, B.D. Sullivan, S. Esener and M.J. Heller 45. contributors, w. Diamond. (accessed 29 May) 46. "Basic Properties of Diamond". DiamondBladeSelect.com 47. Davies, G., Properties and Growth of Diamond. The Institution of electric engineers: London, 1994. 48. Smith BR, Niebert M, Plakhotnik T, Zvyagin AV (2007) J Lumin 127:260-263 49. Vul’ A Ya (2006) In: Shenderova O, Gruen D (eds), Ultra-nanocrystalline diamond: syntheses, properties and applications. William Andrew Publisher, Norwich, NY, USA, pp 379–404 50. H. Tracy Hall, "The Synthesis of Diamond," J. Chem. Educ., 38, 484-489 (1961). 51. Davies G (ed) (1994) Properties and growth of diamond, emis datareviews series No. 9, INSPEC. The Institute of Electrical Engineers, London, Chap. 3 52. Jelezko F, Tietz C, Gruber A, Popa I, Nizovtsev A, Kilin S, Wrachtrup J (2001) Single Mol 2:255–260 53. Collins AT, Thomaz MF, Jorge MIB (1983) J Phys C 16:2177–2181 54. Rand SC (1994) In: Davies G (ed.), Properties and growth of diamond, emis datareviews series no. 9, INSPEC. The Institute of Electrical Engineers, London, Chap. 7.4 55. 10.1517/17460440903186118,2009 Informa UK Ltd ISSN 1746-0441 56. C. A. Wurm, D. Neumann, R. Schmidt, A. Egner, S. Jakobs, Methods in Molecular Biology 591, 185-199 (2010) 57. J.M. Zazula (1997). "On Graphite Transformations at High Temperature and Pressure Induced by Absorption of the LHC Beam