臺灣博碩士論文加值系統

bR (bacteriorhodopsin) 為一個具有光驅動質子唧筒功能的蛋白質，存在於 Halobacterium halobium 的紫色細胞膜 (purple membrane, PM)內。本文主要的研究目的在於建立一個電化學光電系統，以量測PM溶液受光激發後所引起的電流變化。實驗中先由基本的比色管光電化學系統進行探討，以尋求系統的最適化操作條件與參數；而後再將此條件應用到 PDMS 陣列系統，並比較此二系統之差異。由初步的比色管實驗，發現工作電極以ITO玻璃、逆電極以白金棒、導電介質為銅片時最為適當，其光電流訊號值在微安培 (μA) 的範圍內；而將相同的操作條件應用到PDMS陣列系統也同樣可行。文中並就光電流訊號產生的機制進行探討。對比色管系統進一步探討，發現對系統最適合的PM 酸鹼值為 6；而電解液之最適值則是 7；且在不同酸鹼值下 PM 細胞膜的外觀有所變化。同時在固定 PM 溶液量下， PM 的濃度與燈源瓦數不會影響光電流訊號的大小；照光頻率越高，光電流訊號值會越小；而藍色與黃色濾片所測得之光電流訊號會較綠色及紅色濾片為大。以比色管與 PDMS 陣列系統做比較可以發現，PM體積的大小以及電極與銅片間的距離皆會影響光電流訊號的大小；且在相近的 PM 用量下由於系統尺寸的縮小，PDMS 陣列系統可較比色管系統測得較大的光電流訊號值。此光電化學系統的建立對於 bR 感光材料的應用上，樹立相當程度的研發基礎。

Bacteriorhodopsin (bR) is a protein of light-driven proton pump located in the purple membrane (PM) of Halobacterium halobium. In this study, two photo-electrochemical systems were set up to measure the photocurrent produced from illuminated PM. A cuvette system was first used to study the optimal operation condition including the intermediate, the pH values of PM and electrolyte solution, PM concentration, and the incident light intensity, which was subsequently applied to a PDMS array system. The cuvette study showed the highest photocurrent, which was in a range of microamperes, was achieved when ITO glass as the working electrode, a Pt wire as the counter electrode, and a Cu film as the intermediate were used. The production mechanism of the photocurrent was proposed according to its signal profile. The photocurrent of the PDMS array system was also obtained when the same electrodes/intermediate setup as that of the cuvette system was built. The studies on the cuvette system also revealed an optimal operation pH value of PM as 6, while the light intensity and the PM concentration did not have any effect on photocurrents if the same PM amount was used. The comparison between the cuvette and PDMS systems suggested that the distances between the electrodes and the Cu intermediate film determined the photocurrent values, yielding a higher signal in the latter system than in the former one if the same PM amount was used. This study provides valuable informations for the future photoelectric applications of the photosensitive bR.

中文摘要 Ⅰ
英文摘要 Ⅱ
目錄 Ⅲ
表目錄 Ⅶ
圖目錄 Ⅷ

第一章 緒論 1

第二章 文獻回顧 2

2-1 Halobacterium halobium 2
2-1-1 菌體介紹 2
2-1-2紫色細胞膜 ( PM ) 2
2-2 Bacteriorhodopsin (bR) 6
2-2-1 bR 之結構 6
2-2-2 bR的光循環機制 10
2-2-3 氫離子之傳輸路徑 18
2-2-4 bR 在感光材料上的應用 28
2-3 PM 的光電流訊號 30
2-3-1 PM光電訊號流 30
2-3-2 影響光電流訊號的因子 31
2-3-2-1 電極的選擇 31
2-3-2-2固定化方式 31
2-3-2-3酸鹼值與溫度效應 35
2-3-2-4光源強度之效應 37
2-3-2-5照光頻率的影響 38
2-3-2-6 添加劑 39
2-4 PDMS (poly-dimethylsiloxane) 41
2-4-1 PDMS材料簡介 41
2-4-2 PDMS之應用 43

第三章 實驗目的 44

第四章 實驗 45

4-1實驗藥品 45
4-2藥品配製 46
4-3實驗設備 47
4-4實驗流程 49
4-5實驗步驟 52
4-5-1 H. halobium-S9菌株培養 52
1.固態培養 52
2.液態搖瓶培養 52
3.發酵培養 52
4-5-2 PM細胞膜純化 53
4-5-3 光電化學系統的建立 54
1.比色管系統 (cuvette system) 54
2.清洗ITO玻璃 55
3.蝕刻 ITO 玻璃 55
4.PDMS 陣列系統 (PDMS array system) 56
4-5-4 偵測光電流訊號 58
1.光電系統架設 58
2.光電流訊號影響因子 59
(a)中間介質對光電流訊號之影響 59
(b) PM 與 KCl 酸鹼值對光電流訊號之影響 59
(c) PM 濃度對光電流訊號之影響 60
(d)燈源瓦數對光電流訊號之影響 61
(e)照光頻率對光電流訊號之影響 61
(f)光源顏色對光電流訊號之影響 61
(g)PDMS 陣列系統之光電流訊號 61

第五章 結果與討論 62

5-1菌種培養及觀察 62
5-2 PM 純化程序及其特性 64
5-3 光電化學系統 66
5-3-1 中間介質對光電流訊號之影響 66
5-3-2 比色管光電流訊號之機制 66
5-3-3 酸鹼值對光電流訊號之影響 73
5-3-4 PM 細胞膜濃度對光電流訊號之影響 98
5-3-5 燈源瓦數對光電訊號之影響 103
5-3-6 照光頻率對光電訊號之影響 105
5-3-7 光源顏色對光電流訊號值的影響 110
5-3-8 PDMS 陣列系統之光電流訊號 113
5-3-9 比色管與 PDMS 陣列系統之比較 113
第六章 結論 117

參考文獻 119

附錄I. 發酵槽操作流程 126

Armani, D., Liu, C., and Aluru, N., “Re-configurable fluid circuits by PDMS elastomer micromaching” IEEE, 1999, 222-227 (1999)

Balashov, S. P., “Protonation reactions and their coupling in bacteriorhodopsin” Biochim. Biophy. Acta, 1460, 75-94 (2000)

Betancourt, F. M. H. and Glaeser, R. M., “Chemical and physical evidence for multiple functionl steps comprising the M state of the bacteriorhodopsin photocycle” Biochim. Biophy. Acta, 1460, 106-118 (2000)

Birge, R. R., Govender, D. S. K., Gross, R. B., Lawrence, A. F., Stuart, J. A., Tallent, J. R., Tan, E., and Vought, B. W., “Bioelectronics, three-dimensional memories and hybrid computers” IEDM, 4, 3-6 (1994)

Bondar, A. N., Elstner, M., Suhai, S., Smith, J. C., and Fischer, S., “Mechanism of primary proton transfer in bcteriorhodopsin” Structure, 12, 1281-1288 (2004)

Boyer, A., Dery, M., Selles, P., Arbour, C., and Boucher, F., “Colour discrimination by forward and reverse photocurrents in bacteriorhodopsin-based photosensor” Biosen. Bioelectron., 10, 415-422 (1995)

Chan, V. S. S., Koek, W. D., Barnhart, D. H., Bhattacharya, N., Braat J. J. M., and Westerweel, J., “Application of holography to fluid flow measurements using bacteriorhodopsin (bR)” Meas. Sci. Technol.,15, 647-655 (2004)

Chen, Z., Govender, D., Gross, R., and Birge, R., “Advances in protein-based three-dimensional optical memories” BioSystems, 35, 145-151 (1995)

Choi, J. W., Ding, Y., Ahn, C. H., Halsall, H. B., and Heineman, W. R., “A microchip electrochemical immunosensor fabricated using micromaching techniques” Proc. 19th Intern. Conf. –IEEE/EMBS, Oct. 30–Nov. 2, 2264-2266 (1997)

Choi, H. G., Jung, W. C., Lee, W. H., and Choi, J. W., “Color image detection by biomolecular photoreceptor using bacteriorhodopsin-based complex LB films” Biosen. Bioelectron., 16, 925-935 (2001)

Choi, H. G., Min, J., Lee, W. H., and Choi, J. W., “Adsorption behavior and photoelectric response characteristics of bacteriorhodopsin thin films fabricated by self-assembly technique” Colloids and Surfaces B: Biointerfaces, 23, 327-337 (2002)

Chu, J., Li, X., Zhang, J., and Tang, J., “Fabrication and photoelectric response of poly(allylamine hydrochloride)/PM thin films by layer-by-layer deposition technique” Biochim. Biophy. Res. Commu., 305, 116-121 (2003)

Crittenden, S., Howell, S., Reifenberger, R., Hillebrecht, J., and Birge, R. R., “Humidity-dependent open-circuit photovoltage from a bacteriorhodopsin/indium tin oxide bioelectronic heterostructure” Nanotechnol., 14, 562-565 (2003)

Drachev, L. A., Frolov, V. N., Kaulen, A. D., Liberman, E. A., Ostroumov, S. A., Plakunova, V. G., Semenov, A. Y., and Skulaachev, V. P., “Reconstitution of biological molecular generators of electric currrent” J. Biol. Chem., 251, 7059-7065 (1976)

Dyukova, T., Robertson, B., and Weetall, H., “Optical and electrical characterization of bacteriorhodopsin films” BioSystems, 41, 91-98 (1997)

Frydrych, M., Silfsten, P., Parkkinen, S., Parkkinen, J., and Jaaskelainen, T., “Color sensitive retina based on bacteriorhodopsin” BioSystems, 54, 131-140 (2000)

Fukuzawa, K., Yanagisawa, K. I., and Kuwano, H., “Photoelectrical cell utilizing bacteriorhodopsin on a hole array fabricated by micromachining technique” Sens. Actuators, B 30, 121-126 (1996)

Hampp, N., Brauchle, C., and Oesterhelt, D., “Bacteriorhodopsin as a reversible holographic medium in optical processing” Ann. Inter. Confer. IEEE Engin. In Med. Bio. Soci., 12, 1719-1729 (1990)

Haronian, D. and Lewis, A., “Microfabricating bacteriorhodopsin film for imaging and computing ” Appl. Phys. Lett., 61, 2237-2239 (1992)

Heberle, J., “Proton transfer reactions across bacteriorhodopsin and along the membrane” Biochim. Biophy. Acta, 1458, 135-147 (2000)

Heberle, J., Fitter, J., Sass, H. J., and Buldt, G., “Bacteriorhodopsin: the functional details of a molecular machine are being resolved” Biophy. Chem., 85, 229-248 (2000)

Henderson, R., “The purple membrane from Halobacterium halobium” Ann. Rev. Biophys. Bioeng., 6, 87-109 (1977)

Hendler, R. W. and Dracheva, S., “Importance of lipids for bacteriorhodopsin structure, photocycle, and function” Biochem., 66, 1311-1314 (2001)

Hirai, T. and Subramaniam, S., “Structural insights into the mechanism of proton pumping by bacteriorhodopsin” FEBS Lett., 545, 2-8 (2003)

Hong, F. T., “Molecular sensors based on the photoelectric effect of bacteriorhodopsin: origin of differential responsivity” Mater. Sci. Eng., C4, 267-285 (1997)

Hong, J. W., Hosokawa, K., Fujii, T., Seki, M., and Endo, I., “Capillary gel electrophoresis on a disposable PDMS (polydimethylsiloxane) microchip” Proc. 1st Joint BMES/EMBS Conf. Serving Humanity, Advancing Technol., Oct. 13-16, 728 (1999)

Hong, J. W., Fujii, T., Seki, M., Yamamoto, T., and Endo, I., “PDMS-glass hybrid microchip for gene amplification” 1st Ann. Intern. IEEE-EMBS Spec. Top. Conf. Microtechnol. Med. Biol., 80, 407-410 (2000)

Jo, B. H., Lerberghe, L. M. V., Motsegood, K. M., and Beebe, D. J., “Three-dimensional micro-channel fabrication in polydimethylsiloxane (PDMS) elastomer” J. Microelectromech. Sys., 9, 76-81 (2000)

Juchem, T. and Hampp, N., “Interferometric system for non-destructive testing based on large diameter bacteriorhodopsin films” Opt. Lasers Eng., 34, 87-100 (2000)

Kalaidzidis, I. V., Kaulen, A. D., Radionov, A. N., and Khitrina, L. V., “Photoelectrochemical cycle of bacteriorhodopsin” Biochem., 66, 1220-1233 (2001)

Kandori, H., “Hydration switch model for the proton transfer in the Schiff base region of bacteriorhodopsin” Biochim. Biophys. Acta, 1658, 72-79 (2004)

Lanyi, J. K. and Luecke, H., “Bacteriorhodopsin” Curr. Opin. Struct. Biol., 11, 415-419 (2001)

Lanyi, J. K. and Schobert. B., “Mechanism of proton transport in bacteriorhodopsin from crystallographic structures of the K, L, M1, M2, and M2` intermediates of the photocycle” J. Mol. Biol., 328, 439-450 (2003)

Lanyi, J. K., “Bacteriorhodopsin” Annu. Rev. Physiol., 66, 665-88 (2004)

Lensu, L., Frydrych, M., Parkkinen, J., Parkkinen, S., and Jaaskelainen, T., “Photoelectric properties of bacteriorhodopsin analogs for color-sensitive optoelectronic device” Opt. Mater., 27, 57-62 (2004)

Lewis, A., Albeck, Y., Lange, Z., Benchowski, J., and Weizman, G., “Optical computation with negative light intensity with a plastic bacteriorhodopsin film” Science, 275, 1462-1464 (1997)

Libertino, S., Fichera, M., D’Arrigo, G., La Mantia, A., and Ricceri, D., “Characterization and pattering of bacteriorhodopsin films on Si-based materials” Synth. Metal, 138, 71-74 (2003)

Li, B. F., Li, J. R., Song, Y. C., and Jiang, L., “A study of the improvement of photoelectric responses on a Langmuir-Blodgett film containing bacteriorhodopsin” Mater. Sci. Eng., C3, 219-222 (1995)

Manoj, A. G. and Narayan, K. S., “Opto-electrical processes in a conducting polyer-bacteriorhodopsin system” Biosen. Bioelectron., 19, 1067-1074 (2004)

Marcy, D., Tetley, W., Stuart, J., and Birge, R., “Three-dimensional data storage using the photochromic protein bacteriorhodopsin” Proc. 22nd Annu. EMBS Intern. Conf., Chicago IL, July 23-28, 1003-1006 (2000)

Min, J., Choi, H. G., Choi, J. W., Lee, W. H., and Kim, U. R., “Photocurrent of bacteriorhodopsin films deposited by electrophoretic method” Thin Solid Films, 327-329, 698-702 (1998)

Min, J., Choi, H. G., Oh, B. K., Lee, W. H., Paek, S. H., and Choi, J. W., “Visual information processing using bacteriorhodopsin-based complex LB film” Biosen. Bioelectron., 16, 917-923 (2001)

Miyasaka, T., Koyama, K., and Itoh, I., “Quantum conversion and image detection by a bacteriorhodopsin-based artificial photoreceptor” Science, 255, 342-344 (1992)

Muneyuki, E., Okuno, D., Yoshida, M., Ikai, A., and Arakawa, H., “A new system for the measurement of electrogenicity produced by ion pumps using a thin polymer film: examination of wild type bacteriorhodopsin and the D96N mutant over a wide pH range” FEBS Lett., 427, 109-114 (1998)

Nishimura S., Mashimo T., Hiraki K., Hamanaka T., Kito Y., and Yoshiya I., “Volatile anesthetics cause conformational changes of bacteriorhodopsin in purple membrane” Biochim. Biophys. Acta., 818, 421-424 (1985)

Nollert, P., “Lipidic cubic phases as matrices for membrane protein crystallization” Methods, 34, 348-353 (2004)

Persike, N., Pfeiffer, M., Guckenberger, R., and Fritz, M., “Changes in the surface structure of purple membrane upon illumination measured by atomic force microscopy” Colloids and Surfaces B: Biointerfaces, 19, 325-332 (2000)

Qutub, Y., Reviakine, I., Maxwell, C., Navarro J., Landau, E. M., and Vekilov, P. G., “Crystallization of transmembrane proteins in cubo: mechanisms of crystal growth and defect formation” J. Mol. Biol., 343, 1243-1254 (2004)

Saga, Y., Watanabe, T., Koyama, K., and Miyasaka, T., “pH-dependent photocurrent response from bacteriorhodopsin at electrode-electrolyte interfaces” Chem. Lett., 1998, 961-962 (1998)

Saga, Y., Watanabe, T., Koyama, K., and Miyasaka, T., “Mechanism of photocurrent generation from bacteriorhodopsin on gold electrodes” J. Phys. Chem. B, 103, 234-238 (1999)

Saga, Y., Ishikawa, T., and Watanabe, T., “Effect of lanthanum ions on the photoelectrochemical response of bacterorhodopsin” Chem. Lett., 2001, 106-107 (2001)

Saga, Y., Ishikawa, T., and Watanabe, T., “Effect of metal ion exchange on the photocurrent response from bacteriorhodopsin on tin oxide electrodes” Bioelectrochem., 57, 17-22 (2002)

Sharma, M. K., Jattani, H., and Gilchrist, M. L., Jr., “Bacteriorhodopsin conjugates as anchors for supported membranes” Bioconj. Chem., 15, 942-947 (2004)

Shibata, A., Yorimitsu, A., Ikema, H., Minami, K., Ueno, S., Muneyuki, E., and Higuti, T., “Photocurrent of purple membrane adsorbed onto a thin polymer film: action characteristics of the local anesthetics” Collod Surface B: Biointerf., 23, 375-382 (2002)

Singh, C. P. and Roy, S., “All-optical logic gates with bacteriorhodopsin” Curr. Appl. Phys., 3, 163-169 (2003)

Stern, S., Brooks, C., Strachan, M., Kopf-Sill, A., and Parce, J. W., “Microfluidic thermocyclers for genetic analysis” Intern. Soc. Conf. Therm. Phen., 2002, 1033-1038 (2002)

Stuart, J. A., Tallent, J. R., Tan, E. H. L., and Birge, R. R., “Protein-based volumetric memories” Int’l NonVolatile Memory Technol. Conf., 1996, 45-51 (1996)

Tan, E. H. L., Govender, D. S. K., and Birge, R. R., “Large organic cations can replace Mg2+ and Ca2+ ions in bacteriorhodopsin and maintain proton pumping ability” J. Am. Chem. Soc., 118, 2752-2753 (1996)

Tokes, S., Orzo, L., Varo, G., and Roska, T., “Bacteriorhodopsin as an analog holographic memory for joint fourier implementation of CNN computers” Research Report, DNS-3-2000 (2000)

Wang, G., Lu, T., Hu, K. S., and Jiang, L., “Photoresponse discrimination of bacteriorhodopsin films to light stimuli of different frequencies” J. Photochem. Photobiol. B: Biol., 52, 86-91 (1999)

Weetall, H. H. and Druzhko A. B., “Optical and electrical characteristics of bacteriorhodopsin gelatin films and tin-oxide coated electrodes” Curr. Appl. Phys., 3, 281-291 (2003)

Wise, K. J., Gillespie, N. B., Stuart, J. A., Krebs, M. P., and Birge, R. R., “Optimization of bacteriorhodopsin for bioelectronic devices” Trends Biotechnol., 20, 387-394 (2002)

Xu, J. Bhattacharya, P., and Varo, G., “Photo-induced anisotropic photoelectric response in oriented bacteriorhodopsin films” Opt. Mater., 22, 321-326 (2003)

Xu, J., Bhattacharya, P., and Varo, G., “Monolithically integrated bacteriorhodopsin/semiconductor opto-electronic integrated circuit for a bio-photoreceiver” Biosens. Bioelectron., 19, 885-892 (2004)

Yang, J. and Wang, G., “Image edge detecting by using the bacteriorhodopsin-based artificial ganglion cell receptive field” Thin Solid Films, 324, 281-284 (1998)

Yao, B., Wang, Y., Lei, M., and Zheng, Y., “Characteristics and mechanisms of the two types of photoelectric differential response of bacteriorhodopsin-based photocell” Biosens. Bioelectron., 19, 283-287 (2003)

Zeringue, H. C., Glasgow, I. K., Beebe, D. J., Lyaman, J. T., and Wheeler, M. B., “Micro fluidic single embryo culture systems in PDMS” Proc. 1st Joint BMES/EMBS Conf. Serving Humanity, Advancing Technol., Oct. 13-16, 851 (1999)

Zhang, G., Li, B., and Zhang, J., “A study on the fabrication of chemically modified bacteriorhodopsin film and its photochemical properties” Biochem. Biophys. Res. Commun., 313, 733-737 (2004)