Halobacterial salinarum紫色細胞膜(purple membrane，PM) 含有具光趨動單一方向質子泵功能的細菌視紫質(bacteriorhodopsin) BR蛋白。本研究先以biotin衍生物選擇性地鍵結於PM膜兩側，而後藉由biotin/avidin親和性吸附將PM分別定向固定化於ITO與玻璃基材上，並探討其光電與二倍頻響應。經由接觸角監控PM之塗覆可發現，其會改變ITO基材表面由高度親水性轉變為中度親水性，而接觸角會由<10°增加至32-55°。以XPS分析不同塗覆層的元素成分，可發現In、Sn、C、N、O、P等元素的強度隨著塗覆而變化之趨勢合乎預期，且藉由AFM分析可觀察到片狀PM膜貼附於基材表面，顯示塗覆成功。進一步分析各PM晶片之光電響應，發現PM若被蛋白酶降解過或在酸性電解液酸鹼中會造成響應的極性發生反轉，但PM的定向性對晶片的響應極性沒有影響。

其次，對PM玻璃晶片進行二倍頻效應探討，發現當PM膜以胞外側朝向玻璃的方式堆疊，且層數達三至五層時始可測得晶片於入射雷射光為P偏振時所產生之二倍頻效應；然而於S偏振下，其二倍頻效應極為微弱而偵測不到，顯示PM膜確實被定向固定於玻璃上。此外，對摻雜有PM之β-alanine單晶體進行量測，發現其所產生之二倍頻效應強度會隨著光入射面與入射雷射光偏振方向有所變化。再者，當雷射光入射角度為-8°~ 0°時可得到最佳之S偏振二倍頻效應，隨著此入射角偏轉其強度也逐漸減弱， 因此可推測摻雜在以單晶體內之PM的視黃醛分子呈現均一方向性的排列。

Bacteriorhodopsin (BR) is the unidirectional light-driven proton pump residing in the purple membrane (PM) of Halobacterial salinarum. In this study, both sides of PM were each selectively conjugated with biotin for directional immobilization on ITO and plan glass, respectively, via biotin-avidin affinity interactions, and the photoelectric responses and second harmonic generation from the prepared chips were investigated. The coating of PM changed the ITO substrate from highly to moderately hydrophilic as revealed by the increase of chip contact angles from <10° to 32-55°. The XPS analyses showed that the element contents of C, N, O, P, In, and Sn in each fabrication layer varied accordingly with the coating scheme. In addition, the PM patches pasted on the glass substrate were observed by AFM analyses, confirming the successful fabrication of PM on substrates. The photocurrent polarity of the prepared PM-coated ITO glass was reversed both upon prolonged papain-digestion of BR and in acidic electrolytes, but remained the same for both PMs with either immobilization orientation. The second harmonic generation from both three-layer and five-layer PM-coated glass with the PM extracellular side facing the substrate was successfully detected upon the illumination with a P-polarized Nd:YAG laser pulse, while no signal was observed with an S-polarized pulse, implying that the PM patches immobilized on glass were in a high degree of uniform orientation. Finally, the second harmonic generation from a PM-doped β-alanine single crystal depended on not only the incident faces and polarizations of the laser pulse but also the incident angles. The strongest signals were obtained when the incident angles were -8°to 0° for the S-polarization, confirming the orientational arrangement of retinal molecules of BR within the single crystal.

安棣，“製備具高度方向性Bacteriorhodopsin生物光電晶片”，國立台灣科技大學化學工程研究所碩士論文 (2009)
Aktsipetrov, O. A., Akhmediev, N. N., Vsevolodov, N. N., Esikov, D. A., Shutov, D. A. “Photochromism in nonlinear optics: photocontrolled second harmonic generation by bacteriorhodopsin molecules,” (in Russian) Sov. Phys. Dokl., 32, 219-220 (1987)
Aktsipetrov, O. A., Fedyanin, A. A., Murzina, T. V., Borisevich, G. P., Kononenko, A. A., “Electroinduced and photoinduced effects in optical second-harmonic generation and hyper-rayleigh scattering from thin films of bacteriorhodopsin,” J. Opt. Soc. Am. B, 14, 771-776 (1997)
Aktsipetrov, O. A., Fedyanin, A. A., Melnikov, A. V., Mishina, E. D., Murzina, T. V., “Second harmonic generation as a nondestructive readout of optical (photo(electro)chromic and magnetic) memories,” Jpn. J. Appl. Phys., 37, 122-127 (1998)
Balashov, S. P., “Protonation reactions and their coupling in bacteriorhodopsin,” Biochim. Biophys. Acta., 1460, 75-94 (2000)
Chen, D. L., Lu, Y. J., Sui, S. F., Xu, B., Hu, K. S., “Oriented assembly of purple membrane on solid support, mediated by molecular recognition,” J. Phys. Chem. B, 107, 3598-3605 (2003)
Clays, K., Elshocht, S. V., Persoons, A., “Bacteriorhodopsin: a natural (nonlinear) photonic bandgap material,” Opt. Lett., 25, 1391-1393 (2000)
Clays, K., Elshocht, S. V., Chi, M., Lepoudre, E., Persoons, A., “Bacteriorhodopsin: a natural efficient (nonlinear) photonic crystal,” J. Opt. Soc. Am. B, 18, 1474-1482 (2001)
Dementjev, A. P., de Graaf, A., van de Sanden, M. C. M., Maslakov, K. I., Naumkin, A. V., Serov, A. A., “X-Ray photoelectron spectroscopy reference data for identification of the C3N4 phase in carbon-nitrogen films,” Diamond Relat. Mater., 9, 1904-1907 (2000)
Grezesiek, S., Dencher, N. A., “Time-course and stoichiometry of fight-induced proton release and uptake during the photocycle of bacteriorhodopsin,” FEBS Lett., 208, 337-342 (1986)
Groma, G. I., Raksi, F., Szabo, G., Varo, G., “Picosecond and nanosecond components in bacteriorhodopsin light-induced electric response signal,” Biopys. J., 54, 77-80 (1988)
Hampp, N., “Bacteriorhodopsin as a photochromic retinal protein for optical memories,” Chem. Rev., 100, 1755-1776 (2000)
Hampp, N., Oesterhelt, D., “Bacteriorhodopsin and its potential in technical applications,” in Niemeyer, C. M., Mirkin, C. A., ed., Nanobiotechnology, Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim, 160, (2004)
Heberle, J., Dencher, N.A., “Surface-bound optical probes monitor proton translocation and surface potential changes during the bacteriorhodopsin photocycle,” Proc. Natl. Acad. Sci. USA, 89, 5996-6000 (1992)
Henderson, R., Jubb, J. S., Whytock, S. “Specific labeling of the protein and lipid on the extracellular surface of purple membrane,” J. Mol. Biol., 123, 259-274 (1978)
Ho, D., Chu, B., Lee, H., Montemagno, C. D., “Directed protein orientation by site-specific labeling,” IEEE Proc. Nanotechnol., 12-14 (2003)
Hong, F. T., Montal, M., “Bacteriorhodopsin in model membranes a new component of the displacement photocurrent in the microsecond time scale,” Biophys. J., 25, 465-472 (1979)
Huang, J., Lewis, A., Rasing, T., “Second harmonic generation from Langmuir-Blodgett films of retinal and retinal Schiff bases” J. Phys. Chem., 92, 1756-1759 (1988)
Huang, J. Y., Chen, Z., Lewis, A., “Second-harmonic generation in purple membrane-poly(vinyl alcohol) films: probing the dipolar characteristics of the bacteriorhodopsin chromophore in BR570 and M412,” J. Phys. Chem., 93, 3314-3320 (1989)
Khorana, H. G., “Bacteriorhodopsin, a membrane protein that uses light to translocate proton,” J. Biol. Chem., 263, 7439-7442 (1988)
Kim, Y. S., Cho, J. H., Ansari, S. G., Kim, H. I., Dar, M. A., Seo, H. K., Kim, G. S., Lee, D. S., Khang, G., Shin, H. S., “Immobilization of avidin on the functionalized carbon nanotubes,” Synth. Met., 156, 938-943 (2006)
Kimura, Y., Vassylyev, D. G., Miyazawa, A., Kidera, A., Matsushima, M., Mitsuoka, K., Murata, K., Hirai, T., Fujuyoshi, Y., “Surface of bacteriorhodopsin revealed by high-resolution electron crystallography,” Nature, 289, 206-211 (1997)
Lewis, A., Khatchatouriants, A., Treinin, M., Chen, Z., Peleg, G., Friedman, N., Bouevitch, O., Rothman, Z., Loew, L., Sheres, M., “Second-harmonic generation of biological interfaces: probing the membrane protein bacteriorhodopsin and imaging membrane potential around GFP molecules at specific sites in neuronal cells of C. elegans,” Chem. Phys., 245, 133-144 (1999)
Liu, S. Y., “Light induced currents from oriented purple membrane, I. Correlation of the microsecond component (B2) with the L-M photocycle transition,” Biophys. J., 57, 943-950 (1990)
Lo, Y. S., Huefner, N. D., Chan, W. S., Stevens, F., Harris, J. M., Beebe, T. P. Jr., “Specific interactions between biotin and avidin studied by atomic force microscopy using the poisson statistical analysis method,” Langmuir, 15, 1373-1382 (1999)
Lukashev, E. P., Vozary, E., Kononenko, A. A., Rubin A. B., “Electric field promotion of the bacteriorhodopsin BR570 to BR412 photoconversion in films of Halobacterium halobium,” Biochim. Biophys. Acta, 592, 258-266 (1980)
Maker, P. D., Terhune, R. W., Nisenoff, M., Savage, C. M., “Effects of dispersion focusing on the production of optical harmonic,” Phys. Rev. Lett., 8, 21-22 (1962)
Oesterhelt, D, “The structure and mechanism of the family of retinal proteins from halophilic archaea,” Curr. Opin. Struc. Biol., 8, 489-500 (1998)
Ovchinnikov, Y. A., Abdulaev, N. G., Feigina, M. Y., Kiselev, A. V., Lobanov, N. A., “The structural basis of the functioning of bacteriorhodopsin: an overview,” FEBS Lett., 100, 219-224 (1979)
Ramanathan, T., Fisher, F. T., Ruoff, R. S., Brinson, L. C., “Amino-functionalized carbon nanotubes for binding to polymers and biological systems,” Chem. Mater., 17, 1290-1295 (2005)
Sharma, M. K., Jattani, H., Gilchrist, M. L., “Bacteriorhodopsin conjugate as anchors for supported membrans,” Bioconjug. Chem., 15, 942-947 (2004)
Shibata, Y., Miyazaki, T., “Anode Glow Discharge Plasma Treatment Enhances Calcium Phosphate Adsorption onto Titanium Plates,” J. Den.t Res., 81, 841-844 (2002)
Sirokman, G., Fasman, G.D., “Refolding and proton pumping activity of a polyethylene glycol-bacteriorhodopsin water-soluble conjugate,” Protein Sci., 2, 1161-1170 (1993)
Spudich, J. L., “Variations on a molecular switch: transport and sensory signalling by archaeal rhodopsins,” Mol. Microbiol., 28, 1051-1058 (1998)
Stoeckenius W. “Bacterial rhodopsins: evolution of a mechanistic model for the ion pumps,” Protein Sci., 8, 447-459 (1999)
Su, T., Zhong, S., Zhang, Y., Hu, K. S., “Asymmetric distribution of biotin labeling on the purple membrane,” J. Photochem. Photobiol. B, Biology 92, 123-127 (2008)
Wang, J. P., Yoo, S. K., Song, L., El-sayed, M. A., “Molecular mechanism of the differential photoelectric response of bacteriorhodopsin,” J. Phys. Chem. B, 101, 3420-3423 (1997)