中文摘要

蛋白質晶片時常都被用來偵測我們所感興趣的病原分子。但蛋白質晶片都會面臨到抗體方向性不固定以及抗體彼此空間分布不均導致空間障礙使得檢測效率降低。本篇論文研究想利用自組裝模方式結合C端帶有組胺酸的蛋白A的方式來解決以上蛋白質晶片所面臨到的問題。
我們選用了長短鍊混合硫醇類自組裝的方式希望達到空間分布均勻的效果。長鏈硫醇類選用11個碳化學官能基為COOH來結合蛋白質，而短鏈硫醇類選用5個碳化學官能基為CH3避免蛋白質結合。之前學長研究指出在50%混和比例當中分布比例最佳。我們在蛋白A 的尾端修飾組胺酸用來固定蛋白A方向性，且藉由蛋白A只接抗體Fc端的特性來固定抗體方向性。本實驗想同時藉由混和硫醇類以及蛋白A尾端修飾組胺酸來同時克服抗體方向性以及抗體空間障礙的問題以提高蛋白質晶片的檢測效率。
在本篇報導中，50%混合硫醇類那組蛋白A的結合抗體效率為7.9%而尾端有修飾組胺酸的蛋白A結合抗體效率為18.7%，在100%混合硫醇類蛋白A結合效率為29.9%而尾端有修飾組胺酸的蛋白A抗體結合效率為31.6%。可知有固定方向性的蛋白A的結合效率較好，但在表面修飾分布比例來取開蛋白兼具以提高抗體結合效率這部分，卻不如我們預期，此部分將是我們未來要改善的目標。

Angenendt, P., Nyarsik, L and Szaflarski, W. (2004). Cell-free protein expression and functional assay in nanowell chip format. Anal Chem 76, 1844-1849.

Chevalie, S., Cuestas-Ayllon, C., Valeria, G., Monica, L., Helene, F., and Jesus, M. (2010). Creating biomimetic surfaces through covalent and oriented binding of proteins. Langmuir 26(18), 14707-14715.

Delamarche, E., Bernard, A., Schmid, H., Michel, B., and Biebuyck, H. (1997), Patterned delivery of immunoglobulins to surfaces using microfluidic networks. Science 276, 779 -781.

Demirel A. M. O. C., ag˘layan, A. B., Garipcan A. M., and Duman A. E.(2007). Oriented immobilization of IgG on hydroxylated Si(001) surfaces via protein-A by a multiple-step process based on a self-assembly approach. J. Mater Sci 42,9402-9408.

Engvall, E., and Peter, P. (1971). Enzyme-linked immunosorbent assay (ELISA) quantitative assay of immunoglobulin G. Immunochemistry 8, 871-874.

Flink S., Van V. F., and Reinhoudt D. N. (2000). Sensor Functionalities in self-assembled monolayers. Adv Mater 12, 1315-1328.

Gershon, P. D., and Khilko, S. (1995). Stable chelating linkage for reversible of oligohistidine tagged proteins in the BIA core surface plasmon resonance detector. J Immunol Methods 183, 65-76.

George, B., Cynthia, B., Alcide, B., Jack, S., and George M., (1996). A self-assembled monolayer for the binding and study of histidine-tagged proteins by surface plasmon resonance. Anal Chem 68, 490-497.

Gamsjaeger, R., Wimmer, B., Kahr, H., Tinazli, A., Picuric, S., Lata, S., Tamp, R., Maulet, Y., Gruber, H. J., Hinterdorfer, P., and Romanin, C. (2004). Oriented binding of the His6-tagged carboxyl-tail of the L-type Ca2+ channel alpha1-subunit to a new NTA-functionalized self-assembled monolayer. Langmuir 20, 5885-5890.

Holloway A. J., Van Laar R. K., Tothill R. W., and Bowtell, D. D. (2002). Options available--from start to finish--for obtaining data from DNA microarrays II. Nat Genet 32, 481-489

Hua, W., Yanli L., Yunhui Y., Ting D., and Ruqin, Yu.(2004). A protein A-based orientation-controlled immobilization strategy for antibodies using nanometer-sized gold particles and plasma-polymerized film. Anal Biochem 324, 219-226.

Hyunhee, K., Da-Yeon K., Hyun-Jeong G., Byung-Keun O., Ravindra P., Soo-Min O., and Jeong-Woo, C. (2008). Analysis of direct immobilized recombinant protein G on a gold surface. Ultramicroscopy 108, 1152-1156.

Kersten, B., Possling, A., and Blaesing, F. (2004). Protein microarray technology and ultraviolet crosslinking combined with mass spectrometry for the analysis of protein-DNA interactions. Anal Biochem 331, 303-313.

Kukolka, F., Lovrinovic, M., Wacker, R., and Niemeyer, C. M. (2004). Bioconjugation protocols: Strategies and methods, Humana, New Jersey.

Khan, H., Chu F., Feng, Y., Zhang, Q. Qi, J. and Gao, G. F. (2008). Crystallization and preliminary crystallographic analysis of recombinant immunoglobulin G-binding protein from Streptococcus suis. Acta Cryst 64, 757-759.

Love, J. C., Estroff, L. A., Kriebel, J. K., Nuzzo, R. G., and Whitesides, G. M. (2005) Self-assembled monolayers of thiolates on metals as a form of nanotechnology. Chem Rev 105, 1103-1169.

Ludden, M. J. W., Mulder, A., Schulze, K., Subramaniam, V., Tamp, R., and Huskens, J. (2008). Anchoring of histidine-tagged proteins to molecular printboards: Selfassembly, thermodynamic modeling, and patterning. Chem Eur J 14, 2044-2051.

Liu, H. B., Venkataraman N. V., Spencer N. D., Textor, M., and Xiao, S. J. (2008). Structural evolution of self-assembled alkanephosphate monolayers on TiO2. Chemphyschem 9, 1979-1981.

Lisa-Jo, A.C., Davin, K., Ming, W., Joey, M., Caro, B., and Melisenda, J. M. (2009). Antibody orientation enhanced by selective polymer–protein noncovalent interactions. Anal Bioanal Chem 393,1531-1538

Marco, F., Franco, Mazzei ., and Tommaso, F. (2010). Protein immobilization at gold–thiol surfaces and potential for biosensing. Anal Bioanal Chem 398, 1545-1564.

Neves R. A., Salmon M. E., Russell P. E., and Troughton B. J. (2001). Spread coating of OPA on mica:  From multilayers to self-assembled monolayers. Langmuir 17, 8193-8198.

Peluso, P., Wilson, D. S., and Do, D. (2003). Optimizing antibody immobilization strategies for the construction of protein microarrays. Anal Biochem 312, 113-124.

Pascal, J., Dirk, W., Hendrik, S., Christof, M., and Herbert, W. (2008). Chemical strategies for generating protein biochips. Angew Chem. Int. Ed. 47, 9618-9647.

Pirrung, M. C. (2002). How to make a DNA chip. Angew Chem. 114, 1326-1341; Angew Chem. Int. Ed. 2002, 41, 1276-1289.

Sagiv, J. (1980). Organized monolayers by adsorption. 1. Formation and structure of oleophobic mixed monolayers on solid surfaces. J Am Chem Soc 102, 92-98.

Sven, L., Bengt, G., Mathias, U., Lennart, P., and Martin, L. (1983). Gene for staphylococcal protein A. Proc. Nat Acad Sci 80, 697-701.

Schmid, E. L., Keller , T. A., Dienes, Z., and Vogel, H. (1997). Reversible oriented surface immobilization of functional proteins on oxide surfaces. Anal Chem 69, 1979-1985.

Southern, E., Mir, M., and Shchepinov, M. (1999). Molecular interactions on microarrays. Nat Genet 21, 5-9.

Sin-Yi, H., and Pen-Cheng W. (2009). Effective enhancement of fluorescence detection efficiency in protein microarray assays: Application of a highly fluorinated organosilane as the blocking agent on the background surface by a facile vapor-phase deposition process. Anal Chem 81, 7908-7916.

Thompson, W. R., Pemberton, J. E. (1995). Thin sol-gel silica films on (3-Mercaptopropyl)trimethoxysilane-modified Ag and Au surfaces. Langmuir 11, 1720-1731.

Wen-Chi T., and Pi-Ju R. P. (2009). Surface plasmon resonance-based immunosensor with oriented immobilized antibody fragments on a mixed self-assembled monolayer for the determination of staphylococcal enterotoxin B. Microchim Acta 166,115-122.

Yeo, W. S., Min, D. H., Hsieh, R.W., Greene, G. L., and Mrksich, M. Label-Free Detection of protein–protein interactions on biochips. Angew Chem 2005, 117, 5616-5619; Angew Chem. Int. Ed.2005, 44, 5480 – 5483.

Zhu, H., and Snyder, M. (2001) Protein arrays and microarrays. Curr Opin Chem Biol 5, 40-45.