此篇論文的目的在於探討如何結合氧化石墨烯(graphene oxide，GO)與紫膜(purple membrane，PM)以整合開發一種新穎的生物感測系統。GO是氧化態的石墨烯，其價位低廉也易於生產。而來自嗜鹽菌Halobacterium salinarum的PM則含有單一種蛋白質，細菌視紫質 (bacteriorhodopsin ，BR)，為一種光驅動質子泵，具有獨特的光電性質。本研究開發一種簡易的方法將GO應用於PM生物晶片技術上，且並以原子力顯微鏡（AFM）、接觸角（contact angle）與光電流等分析來探討添加GO的成效。先製作結合GO與avidin的複合物（GO-avidin complex），然後當作架橋以將經biotin修飾的PM（b-PM）固定於以自排性單分子膜胺基化之 ITO表面。AFM分析的結果顯示，GO-avidin複合物的添加可增加PM晶片表面的平坦度，且GO用量的多寡對於平坦度影響十分重要。然而，GO添加對PM的光電流響應並沒有明顯的抑制效果。此外，也發現利用biotin與avidin作用的生物親和性固定化對於晶片上PM的塗覆率是很重要的，因為GO與PM間的非特異性吸附作用僅能產生較不穩定且微弱的光電流訊號。另一方面，僅單純將GO應用於PM晶片之層層塗覆製程：先以不同的塗覆方法將GO塗覆於晶片上，接著依序固定化avidin 與b-PM。接觸角分析証實每一種方法都可將GO塗覆上去，然而光電流也會因GO的結合而下降。在未來，此種整合GO的PM生物晶片可能開創一種獨特且功能較佳的生物分析技術。

The objective of this research was to explore the integration of graphene oxide (GO) with purple membrane (PM) in the approach for a novel bio-sensing system. As the oxidized form of graphene, GO possesses unique and stable structure derived from graphene. In addition, it is relatively cheap and easy to produce. PM from Halobacterium salinarum contains only one protein constituent, bacteriorhodopsin (BR) that acts as a light-driven proton pump providing novel photonic properties.
A facile way to introduce GO into PM chip technology was developed with evaluation of the GO addition effect by AFM, contact angle, and photocurrent analyses. GO-avidin complex was prepared and subsequently used as a linker to immobilize biotinylated PM onto ITO fabricated with an amine-terminated self-assembled monolayer. Morphological studies conducted with AFM revealed that GO addition through GO-avidin complex enhanced surface flatness of the PM chips and the amount of GO employed was critical to yield a distinguishable effect on the surface. Nevertheless, no significant inhibition of photoelectric responses generated from PM was observed on GO addition. The bioaffinity immobilization through biotin-avidin interaction was found to be important for high PM coverage, because the nonspecific binding directly between GO and PM yielded less stable and lower photoelectric responses. In addition, GO alone was integrated into the PM chip fabrication during the layer-by-layer assembly. GO was added with different coating methods prior to avidin and subsequently b-PM immobilization. The contact angle analysis confirmed the presence of coated GO in each method, while the chip photocurrents were reduced by GO integration. In the future, GO-integrated PM biochips might lead to a bioassay with unique and enhanced properties.

Shen J.F., Yan B., Shi M., Ma H.W., Li N., and Ye M.X, “Synthesis of graphene oxide-based biocomposites through diimide-activated amidation”, Journal of Colloid and Interface Science 356, p. 543–549 (2011)

Vengadesh P, Abdul Majid W.H., Shanmugam S.A., and Low K.S., “Fabrication and photoresponse of novel carboxymethylcellulose (CMC) based bacteriorhodopsin (bR) sensor”, Organic electronics 7, p. 300–304 (2006)

Wang Y., Li Z.H., Wang J., Li J.H., and Lin Y.H., “Graphene and graphene oxide:biofunctionalization and applicationsin biotechnology”, Trends in Biotechnology 29, p. 205-212 (2011)

Geim A.K. and K.S. Novoselov, “The rise of graphene”, Nature Materials 6, p.183-191 (2007)

Mouras, S., Hamm, A., Djurado, D., and Cousseins J.C., “Synthesis of first stage graphite intercalation compounds with fluorides”, Revue de Chimie Minerale 24, p. 572-582 (1987)

Novoselov K.S., Geim A.K., Morozov S. V., Jiang D., Zhang Y., Dubonos S.V., Grigorieva I.V., and Firsov A.A., “Electric field efffect in automically thin carbon films”, Science 306, p. 666–669 (2004)

Zhu Y.W., Murali S. , Cai W.W., Li X.S., Suk J.W., Potts J.R., and Ruoff R.S., “Graphene and Graphene oxide: synthesis, properties, and applications”, Advanced Materials 22, p. 3906–3924 (2010)

Zborˇil R., Karlicky’ F., Bourlinos A.B., Steriotis T.A., Stubos A.K., Georgakilas V., Sˇafarˇova K., Jancˇik D., Trapalis C., and Otyepka M., “Graphene fluoride: a stable stoichiometric graphene derivative and its chemical conversion to graphene”, Small 6, p. 2885–2891 (2010)

Dreyer D.R., Park S.J., Bielawski C.W., and Ruoff R.S., “The chemistry of graphene oxide”, Chemical Society Reviews 39, p. 228–240 (2010)

Kim H.W., Abdala A.A., and Macosko C.W., “Graphene/polymer nanocomposites”, Macromolecules 43, p. 6515–6530 (2010)

Wan C.Y. and Chen B.Q., “Reinforcement and interphase of polymer/graphene oxide nanocomposites”, Journals of Materials Chemistry 22, p. 3637-3646 (2012)

Hummers Jr. W.S. and Offeman R.E., “Preparation of graphitic oxide”, Journals of American Chemical Society 80, p. 1339–1339 (1958)

Su C.Y., Xu Y.P., Zhang W.J., Zhao J.W., Liu A.P., Tang X.H., Tsai C.H., Huang Y.Z., and Li L.J., “Highly efficient restoration of graphitic structure in graphene oxide using alcohol vapors”, ACS Nano 4, p. 5285-5292 (2010)

Pandey D., Reifenberger R., and Piner R., “Scanning probe microscopy study of exfoliated oxidized graphene sheets”, Surface Science 602, p.1607–1613 (2008)

Schniepp H.C., Li J.L., McAllister M.J., Sai H., Herrera-Alonso M., Adamson D.H., Prud’homme R.K., Car R., Saville D.A., and Aksay I.A., “Functionalized single graphene sheets derived from splitting graphite oxide”, The Journal of Physical Chemistry B, 110, p. 8535-8539 (2006)

Stankovich S., Piner R.D., Chen X.Q., Wu N.Q., Nguyen S.B.T., and Ruoff R.S., “Stable aqueous dispersions of graphitic nanoplatelets via the reduction of exfoliated graphite oxide in the presence of poly(sodium 4-styrenesulfonate)”, Journal of Materials Chemistry 16, p. 155–158 (2006)

Pan S. and Aksay I.A., “Factors controlling the size of graphene oxide sheets produced via the graphite oxide route”, ACS Nano 5, p. 4073–4083 (2011)

Hamilton C.E., PhD Thesis, Rice University, 2009 (http://cnx.org/content/m29187/latest/)

Kang D.W. and Shin H.S., “Control of size and physical properties of graphene oxide by changing the oxidation temperature”, Carbon Letters 13, p. 39-43 (2012)

Zhou X.F. and Liu Z.P., “A scalable,solution-phase processing route to graphene oxide and graphene ultralarge sheets”, Chemical Communications 46, p. 2611–2613 (2010)

Graphos (http://www.graphene.it/), accessed June 2012

Cheap Tubes Inc. (http://www.cheaptubes.com/graphene-oxide.htm), accessed June 2012

Liu Z.F., Jiang L.H., Galli F., Nederlof I., Olsthoorn R.C.L., Lamers G.E.M., Oosterkamp T.H., and Abrahams J.P., “A graphene oxide˙streptavidin complex for biorecognition – towards affinity purification”, Advanced Functional Materials 20, p. 2857–2865 (2010)

Paredes J.I., Villar-Rodil S., Martı’nez-Alonso A., and Tasco’n J. M. D., “Graphene oxide dispersions in organic solvents”, Langmuir 24, p. 10560-10564 (2008)

Gomez-Navarro C., Weitz R.T., Bittner A.M., Scolari M., Mews A., Burghard M., and Kern K., “Electronic transport properties of individual chemically reduced graphene oxide sheets”, Nano Letters 7, p. 3499-3503 (2007)

Wang G.M., Qian F., Saltikov C.W., Jiao Y.Q., and Li Y., “Microbial reduction of graphene oxide by Shewanella”, Nano Research 4, p.563–570 (2011)

Lei Z.B., Lu L., and Zhao X.S., “The electrocapacitive properties of graphene oxide reduced by urea”, Energy and Environmental Science 5, p. 6391–6399 (2012)

Zhang J.L., Yang H.J., Shen G.X., Cheng P., Zhang J.Y., and Guo S.W., “Reduction of graphene oxide via L-ascorbic acid”, Chemical Communications 46, p. 1112–1114 (2010)

Xu B., Yue S.F., Sui Z.Y., Zhang X.T., Hou S.S., Cao G.P., and Yang Y.S., “What is the choice for supercapacitors: graphene or graphene oxide?”, Energy and Environmental Science 4, p. 2826–2830 (2011)

McCarren C. and Polyakova E. “Graphene oxide: hands guide practical applications” http://www.graphene-info.com/graphene-oxide-hands-guide-practical-applications, accessed June 2012

Wang S.J., Geng Y., Zheng Q.B., and Kim J.K., “Fabrication of highly conducting and transparent graphene films”, Carbon 48, p.1815 –1823 (2010)

Nair R.R., Wu H.A., Jayaram P.N., Grigorieva I.V., and Geim A.K., “Unimpeded permeation of water through helium-leak–tight graphene-based membranes” Science 335, p. 442-444 (2012)

Wang L., Lee K.H., Sun Y.Y., Lucking M., Chen Z.F., Zhao J.J., and Zhang S.B., “Graphene oxide as an ideal substrate for hydrogen storage”, ACS Nano 3, p. 2995–3000 ( 2009)

Yang X.Y., Wang Y.S., Huang X., Ma Y.F., Huang Y., Yang R.C., Duan H.Q., and Chen Y.S., “Multi-functionalized graphene oxide based anticancer drug-carrier with dual-targeting function and pH-sensitivity”, Journal of Materials Chemistry 21, p. 3448-3454 (2011)

Hu W.B., Peng C., Luo W.J., Lu M., Li X.M., Li D., Huang Q., and Fan C.H., “Graphene-based antibacterial paper”, ACS Nano 4, p. 4317–4323 (2010)

Cote L.J., Kim J.M., Tung V.C., Luo J.Y., Kim F., and Huang J.X., “Graphene oxide as surfactant sheets”, Pure and Applied Chemistry 83(1), p. 95–110 (2011)

Kim J.M., Cote L.J., Kim F., Yuan W., Shull K.R., and Huan J.X., “Graphene oxide sheets at interfaces”, Journal of the American Chemical Society 132, p. 8180-8186 (2010)

Hampp N., “Bacteriorhodopsin as a photochromic retinal protein for optical memories”, Chemical Reviews 100, p. 1755-1776 (2000)

Nanonic Imaging Ltd., (http://www.nanonics.co.il/index.php?page_id=765), copyright 2007, accessed June 2012

Theoritical Biophysiscs Group, Beckman Institute, University of Illinois at Urbana-Champaign( http://www.ks.uiuc.edu/Research/newbr/heptamer_fig.html), accessed June 2012

Theoritical Biophysiscs Group, Beckman Institute, University of Illinois at Urbana-Champaign (http://www.ks.uiuc.edu/Research/Method/quant_sim/), accessed June 2012

Kuhlbrandt W., “Bacteriorhodopsin — the movie”, Nature 406, p. 569-570 (2000)

Cartailler J.P. and Luecke H., “X- Ray crystallopraphic analysis of lipid-protein interactions in the bacteriorhodopsin purple membrane”, Annual Review of Biophysics and Biomolecular Structure 32, p. 285-310 (2003)

Wang J.P., Yoo S.K., Song L., and El-Sayed M.A., “Molecular mechanism of the differential photoelectric response of bacteriorhodopsin”, The Journal of Physical Chemistry B 1101, p. 3420-3423(1997)

Chu L.K., Yen C.W., and El-Sayed M.A., “Bacteriorhodopsin-based photo-electrochemical cell”, Biosensors and Bioelectronics 26, p. 620–626(2010)

Trivedi S, Choudhary O.P., and Gharu J., “Different proposed applications of bacteriorhodopsin”, Recent Patents on DNA & Gene Sequences 5, p. 35-40 (2011)

Webb K., Hlady V., and Tresco P.A., “Relative importance of surface wettability and charged functional groups on NIH 3T3 fibroblast attachment, spreading, and cytoskeletal organization”, Journal of Biomedical Material Research 41, p. 422–430 (1998)

Ophardt C.E., “Polarity of Organic Compounds” Virtual E-book Elmhurst College (c 2003) (http://www.elmhurst.edu/~chm/vchembook/213organicfcgp.html), accesed July 2012

Guo L.H. and Yang X.Q., “A new chemically amplified electrochemical system for the detection of biological affinity reactions: direct and competitive biotin assay”, Analyst 130, p. 1027–1031 (2005)

WangS., Zhang Y., Abidi N. and Cabrales L., “Wettability and Surface Free Energy of Graphene Films” Langmuir 25, pp 11078–11081 (2009)