目錄 中文摘要 I ABSTRACT II 目錄 IV 表目錄 VI 圖目錄 VII 第一章 緒論 1 第二章 文獻回顧 2 2-1 Halobacterium salinarum、PM及BR 2 2-1-1 簡介 2 2-1-2 BR的光循環 4 2-1-3 BR光電流響應 6 2-2 自排性單分子膜（self-assembly monolayer，SAM） 8 2-2-1 SAM與基板 8 2-2-2 SAM長度與尾端官能基之比較 14 2-3 原子力顯微鏡 （Atomic force microscopy，AFM） 15 2-3-1 AFM原理 15 2-3-2 以AFM觀察PM在雲母上的物理吸附( Muller et al., 1997 ) 19 2-3-3 以TM-AFM判別PM的EC與CP側 21 2-3-4 Time-lapse AFM 22 第三章 實驗 24 3-1 實驗目的 24 3-2 實驗藥品 25 3-3 實驗設備 26 3-4 藥品配製與實驗流程 28 3-4-1 藥品配製 28 3-4-2 實驗流程 31 3-5 實驗步驟 32 3-5-1 PM修飾 32 3-5-2 ITO玻璃清洗 32 3-5-3 SAM修飾ITO 33 3-5-4 PM固定化 34 3-5-5 循環伏安法分析 35 3-5-6 水滴接觸角量測 36 3-5-7 AFM分析與操作 36 3-5-8 D1、D2光電訊號量測 37 3-5-9 B1、B2脈衝光電訊號量測 38 第四章 結果與討論 39 4-1 SAM對以生物親和吸附法製備PM晶片之影響 39 4-1-1 羧酸的SAM製備條件的探討 40 4-1-2 羧亞磷酸SAM條件對於PM晶片的影響 45 4-1-3 ITO來源對於後續塗佈層的影響 63 4-1-4 AFM分析 73 4-1-5 B1與B2訊號分析 91 4-2 以共價鍵結法製備PM晶片 113 4-2-1 懸浮液pH值對共價鍵結固定化的PM與digested PM之晶片的影響 113 4-2-2 以EDC與NHS活化後之PM塗佈於ITO/APPA之PM晶片製作 122 4-3 生物親和吸附與共價鍵結固定化PM晶片之總結比較 122 第五章 結論 126 第六章 參考文獻 128   表目錄 Table 2-1不同基材上不同尾端官能基SAM的比較 15 Table 2-2 PM之EC和CP側之TM-AFM分析比較 ( Dong et al., 2009 ) 22 Table 4-1 Measured solubility of various SAMs in butanol ( average of 3 runs) 44 Table 4-2 SAM coating condition chosen to prepare PM chips. 45 Table 4-3 Polarity indexes of different solvents 57 Table 4- 4 pKa indexes of different function group. 58 Table 4- 5 The parameter of the B1B2 fitting. 112   圖目錄 Fig. 2-1 Cytoplasmic surface of the native purple membrane from Halobacterium salinarum. Individual bacteriorhodopsin molecules form trimers（outline）that assemble into a two-dimensional hexagonal lattice（Muller, 2008）. 3 Fig. 2-2 A secondary structure model of bacteriorhodopsin. All-trans-retinal is attached as a Schiff base to Lys-216 in helix G（Khorana, 1988）. 3 Fig. 2-3 A current model interpreting the intermediates detected in the photochemical cycolfe purple membrane by U-Vvisible absorption spectroscopy at low temperature （ Khorana, 1988 ）. 5 Fig. 2-4 Scheme showing steps in light-driven proton transport by bacteriorhodopsin from the inside the cell to the outside（Balashov, 2000）. 5 Fig. 2-5 D1 and D2, upon turning on and turning off the CW light source ( Wang et al., 1997 ). 6 Fig. 2-6 The three major photocurrent components of purple membrane, BI, B2, and B3, as seen with different time axes（ Liu, 1990 ）. 7 Fig. 2-7 Surface-active organosulfur compounds that form monolayers on gold （ Ulman, 1996 ）. 9 Fig. 2-8 XP spectra after chemical vapor deposition of octadecanethiol: （a） sputter-clean ITO, （b） 4.5 A, （c） 16 A, （d） 18 A, and（e） 21 A（ Yan et al., 2000 ）. 11 Fig. 2-9 A schematic description of fatty acid monolayers on AgO and on Al2O3（Ulman, 1996）. 12 Fig. 2-10 O 1s XP spectra of （a） bare ITO and （b） stearic acid on ITO（ Yan et al., 2000 ）. 13 Fig. 2-11 Organic phosphonic acid binding modes on metal oxide surfaces（Zhang et al., 2010）. 14 Fig. 2-12 AFM-based nanotools provide a laboratory on a tip for imaging and probing biological membranes at the molecular level（Muller, 2008）. 16 Fig. 2-13 The tip-sample system. D is the actual tip-sample distance, whereas Z is the distance between the sample and the cantilever rest position. ( Cappella and Dietler, 1999 ) 17 Fig. 2-14 Graphical construction of an AFM force-displacement curve. In panel ( a ) the curve F(D) represents the tip-sample interaction and the lines 1, 2, and 3 represent the elastic force of the cantilever. At each distance the cantilever deflects until the elastic force equals the tip-sample force and the system is in equilibrium. The force values fa, fb, and fc. at equilibrium are given by the intersections a, b, and c between lines 1, 2, and 3 and the curve F(D). These force values must be assigned to the distances Z between the sample and the cantilever rest positions, i.e., the distancesα, β, andγ given by the intersections between lines 1, 2, and 3 and the horizontal axis. This graphical construction has to be made going both from right to left and from left to right. The result is shown in panel ( b ). The points A, B, B', C, and C' correspond to the points a, b, b', c, and c' respectively. BB' and CC' are two discontinuities. The origin O of axis in panel ( b ) is usually put at the intersection between the prolongation of the zero line and the contact line of the approach curve. The force fc', eventually coincides with the zero force. ( Cappella et al., 1999 ) 18 Fig. 2-15 Total force per unit area ( 1 nm2 ) between purple membrane and the mica surface. FDLVO was calculated for different concentrations of the monovalent electrolyte. While the attractive van der Waals force is mainly unaffected by the electrolyte, the double-layer repulsion decreases with increasing salt concentration（Muller et al., 1997）. 20 Fig. 2-16 Attachment of purple membrane to mica dependent on the electrolyte oncentration（Muller et al., 1997）. 21 Fig. 2-17 High-resolution mapping of protein flexibility. a–c, Height （a）, phase （b） and quantitative flexibility （c） maps of two adjacent patches of purple membranes. The scan size is 3.2_ 3.2 mm2. d,e, High-resolution flexibility maps of the cytoplasmic （d） and extracellular （e） sides obtained from the regions indicated by CP and EC in c. f–h, Numerical values plotted from left to right across the dashed lines in a–c, respectively. （Dong et al., 2009） 22 Fig. 2-18 Time-lapse AFM depicts the remodeling of a DPPC bilayer by hospholipase A2. （A） DPPC bilayer imaged in buffer solution before addition of PLA2. Dark holes are defects in the supported bilayer. （B） Addition of PLA2 induces remodeling of the lipid bilayer. Arrows point out small channels that are indicative of phospholipid hydrolysis by PLA2. （C） With increasing time （4 min）, the phospholipid bilayer becomes increasingly hydrolyzed. （D） Scanning the DPPC bilayer while applying a strong force （2 nN） to the AFM probe induces small perturbations that provide PLA2 a possibility for hydrolysis along the scanned line （topography and illustration）( Grandbois et al., 1998 ). 23 Fig. 3-1實驗流程圖 31 Fig. 3-2 Setup to measure differential photocurrents. 37 Fig. 3-3 Setup to measure pulse photocurrents. 38 Fig. 4-1 Fabrication scheme of PM chip prepared via bioaffinity linkage. 39 Fig. 4-2 Contact angle of ITO coated with 16PHDA SAM at different SAM concentrations for 72 h ( Vertical coating position，v.p. ). 40 Fig. 4-3 AFM image of ITO coated with 16PHDA at different concentrations. ( a ) 5 μM, ( b ) 1 μM, ( c ) 0.5 μM, ( d ) 0.1 μM and ( e ) 0 μM (Scan size : 5μm) 41 Fig. 4-4 AFM image of ITO coated with 11PUA at different concentration. ( a ) 1 μM, ( b ) 0.5 μM, ( c ) 0.1 μM and ( d ) 0 μM (Scan size : 5μm) 42 Fig. 4-5 Contact angle of ITO coated with 5 μM 11PUA at the vertical position for various time. 43 Fig. 4-6 Photocurrent of ITO coated with 5 μM 11PUA at the vertical position (v.p.) for various time. 43 Fig. 4-7 Contact angle of ITO coated with 1 μM SAM dissolved in butanol for various time. 44 Fig. 4-8 Photocurrent of ITO coated with 1 μM SAM dissolved in butanol for various time. 45 Fig. 4-9 Contact angle of ITO coated with different SAMs. 46 Fig. 4-10 Contact angle of ITO coated with activated avidin on different SAMs. 47 Fig. 4-11 (a) CV curves of ITO coated with different SAMs. (b) Zoomed-in reduction peaks, (c) Zoomed-in oxidation peaks. ( Reference electrode : AgCl / Ag ) 49 Fig. 4-12 (a) Cyclic voltammograms of ITO at different scan rate. Data Fitting with Randles-Sevcik equation for (b) oxidation reactions and (c) reduction reactions. ( Reference electrode : AgCl / Ag ) 51 Fig. 4-13 (a) Cyclic voltammograms of 16PHDA coated on ITO at different scan rate. Data Fitting with Randles-Sevcik equation for (b) oxidation reactions and (c) reduction reactions. ( Reference electrode : AgCl / Ag ) 52 Fig. 4-14 (a) Cyclic voltammograms of 11PUA coated on ITO at different scan rate. Data Fitting with Randles-Sevcik equation for (b) oxidation reactions and (c) reduction reactions. ( Reference electrode : AgCl / Ag ) 53 Fig. 4-15 Cyclic voltammograms of 6PHA coated on ITO at different scan rate. Data Fitting with Randles-Sevcik equation for (b) oxidation reactions and (c) reduction reactions. ( Reference electrode : AgCl / Ag ) 54 Fig. 4-16 Cyclic voltammograms of 3PPA coated on ITO at different scan rate. Data Fitting with Randles-Sevcik equation for (b) oxidation reactions and (c) reduction reactions. ( Reference electrode : AgCl / Ag ) 55 Fig. 4-17 Ferricyanide diffusion coefficients of ITO coated with different lengths of SAM. ( Reference electrode : AgCl / Ag ) 56 Fig. 4-18 Photocurrents of b-PM chips coated with different SAMs. 57 Fig. 4-19 Hypothesis of SAM arrangement on ITO coated in different solvents. 59 Fig. 4-20 Contact angles of ITO coated with 6PHA in different solvents. 59 Fig. 4-21 Contact angles of activated avidin coated on ITO with 6PHA as the SAM in different solvents. 60 Fig. 4-22 Photocurrent of b-PM chip prepared by using 6PHA as the SAM in different solvents. 60 Fig. 4-23 Contact angle of ITO coated with 3PPA and APPA in DI water. 62 Fig. 4-24 Photocurrent of b-PM prepared by 3PPA and APPA in DI water as the SAM. 62 Fig. 4-25 Contact angle of APPA and 16PHDA coated different thickness of ITO perchased from different suppliers. 63 Fig. 4-26 Photocurrents of b-PM chips prepared with ITO purchased from different supplier than coating on b-PM. 64 Fig. 4-27 AFM analysis of ITO purchased from different suppliers. (a) Merk 0.5 mm (b) Merk 0.7 mm (c) CN 0.7 mm 67 Fig. 4-28 Image of b-PM chips prepared from ITO purchased from different suppliers. (a) Merk 0.7 mm (b) CN 0.7 mm 69 Fig. 4-29 D1 and D2 photocurrents of b-PM (pH 4.5) chips prepared by using 16PHDA as the SAM layer coated on ITO purchased from different suppliers. ( a ) Peak on signal ( b ) Peak off signal 70 Fig. 4-30 D1 and D2 photocurrents of b-PM (pH 4.5) chips prepared by using APPA as the SAM layer coated on ITO purchased from different suppliers. ( a ) Peak on signal ( b ) Peak off signal 71 Fig. 4-31 D1 and D2 photocurrents of b-PM (pH 4.5) chips prepared by using different SAM coated on ITO purchased from different suppliers. ( a ) Peak on signal ( b ) Peak off signal 72 Fig. 4-32 AFM images of various coated ITO, (b)-(d) 5 μM 16PHDA coated at vertical position and (e)-(g) 100 μM 3PPA coated at Horizontal position. 77 Fig. 4-33 Arithmetic mean roughness (Ra) of different coating layers ( Data from the AFM analysis of Fig. 4-32 ). 77 Fig.4-34 AFM images of (a) ITO and b-PM coated ITO with 5 μM as the (b) 16PHDA, (c) 11PUA, (d) 6PHA, (e) 3PPA SAM. All SAMs were coated at Horizontal position. Scan size : 10μm 79 Fig. 4-35 3D images of (a) ITO and b-PM coated ITO with 5 μM as the (b) 16PHDA, (c) 11PUA, (d) 6PHA, (e) 3PPA SAM. All SAMs were coated at Horizontal position. Scan size : 10μm 80 Fig.4-36 Section profile of (a) ITO and b-PM coated ITO with 5 μM as the (b) 16PHDA, (c) 11PUA, (d) 6PHA, (e) 3PPA SAM. All SAMs were coated at Horizontal position. Scan size : 10μm 82 Fig. 4-37 AFM images of (a) ITO and b-PM coated ITO with 5 μM as the (b) 16PHDA, (c) 11PUA, (d) 6PHA, (e) 3PPA SAM. All SAMs were coated at Horizontal position. Scan size : 5μm 83 Fig. 4-38 3D images of (a) ITO and b-PM coated ITO with 5 μM as the (b) 16PHDA, (c) 11PUA, (d) 6PHA, (e) 3PPA SAM. All SAMs were coated at Horizontal position. Scan size : 5μm 84 Fig.4-39 Section profile of (a) ITO and b-PM coated ITO with 5 μM as the (b) 16PHDA, (c) 11PUA, (d) 6PHA, (e) 3PPA SAM. All SAMs were coated at Horizontal position. Scan size : 5μm 86 Fig. 4-40 AFM images of (a) ITO and b-PM coated ITO with 5 μM as the (b) 16PHDA, (c) 11PUA, (d) 6PHA, (e) 3PPA SAM. All SAMs were coated at Horizontal position. Scan size : 2μm 87 Fig. 4-41 AFM images of (a) ITO and b-PM coated ITO with 5 μM as the (b) 16PHDA, (c) 11PUA, (d) 6PHA, (e) 3PPA SAM. All SAMs were coated at Horizontal position. Scan size : 2μm 88 Fig.4-42 Section profile of (a) ITO and b-PM coated ITO with 5 μM as the (b) 16PHDA, (c) 11PUA, (d) 6PHA, (e) 3PPA SAM. All SAMs were coated at Horizontal position. Scan size : 2μm 90 Fig. 4-43 Roughness of PM layer which coated on different SAM. Data scale : (a) 10μm, (b) 5μm, (c) 2μm 91 Fig. 4-44 B1 and B2 signals, Laser energy 7.9 mJ . (a) ITO (b) 0.1 mM APPA modified ITO, (c) b-PM chip. (a.1) (b.1) (c.1) present the beat signals within the first three laser exicitations. (a.2) (b.2) (c.2) present the breakdown after over-excitation. 92 Fig.4-45 Photocurrent signal under pulsed-laser excitation, in the time domain of 500 μs. Various film orientation on ITO/glass are shown, ITO/EC-CP (a), ITO/Random (b), and ITO/CP-EC (c). B1/B2 are useful in determining membrane orientation ( J. Wang, 2000). 93 Fig. 4-46 B1 and B2 signals of b-PM chips prepared by ITO coated by (a) 16PHDA, (b) 11PUA, (c) 6PHA, and (d) 3PPA SAM, followed by amination, activated avidin and b-PM coating. 95 Fig.4-47 The equivalent circuit of the photoelectric effect genetrated from a BLM film constituted of PM ( Hong, 1997 ). 96 Fig. 4-48 B1 and B2 signal of b-PM chip prepared by ITO coated by 11PUA followed by amination, activated avidin and b-PM coating. 98 Fig. 4-49 Fabrication scheme PM chip prepared by covalent linkage. 113 Fig.4-50 PM bending at different pH (Mostafa et al., 1996). 114 Fig.4-51 CV analyses of (a) ITO, (b) 5 μM 16PHDA modified ITO, (c) ITO/16PHDA/PM pH = 4.5, (d) ITO/16PHDA/PM pH = 5.2, (e) ITO/16PHDA/PM pH = 7 (2 chips for each analysis ; Reference electrode : AgCl / Ag ) 115 Fig. 4-52 B1 and B2 signals of Native PM chip prepared by covalent linkage, with PM suspended at different pHs ( a ) ITO, ( b ) pH 4.5, ( c ) pH 5.2, ( d ) pH 7. 117 Fig. 4-53 (a) L-aspartic acid (b) L-glutamic acid 118 Fig. 4-54 SDS-PAGE Electrophoresis of Native PM and Native PM treated with papain 119 Fig. 4-55 B1 and B2 signals of Native papain-digested PM chip prepared by covalent linkage with PM suspended in different pH suspension buffer ( a ) pH 4.5, ( b ) pH 5.2, ( c ) pH 7. 121 Fig. 4-56 Photocurrent of Native PM chips prepared by covalent linkage. PM were treated with and without papain digestion and suspended in suspension buffers at different pHs. 121 Fig. 4-57 Cyclic Voltammetry of various coated ITO. b-PM suspended at pH 4.5 was immobilized by bioaffinity linkage. ( Reference electrode : AgCl / Ag ) 122 Fig. 4-58 Cyclic Voltammetry of various coated ITO. Unmodified PM suspended at pH 4.5 was immobilized by covalent linkage. ( Reference electrode : AgCl / Ag ) 123 Fig. 4-59 Photocurrent responses of PM chips prepared by ( a ) bioaffinity linkage ( b ) covalent linkage 124 Fig. 4-60 AFM image and section profile of PM chips coated by ( a ) bioaffinity linkage and ( b ) covalent linkage at pH 4.5. Scan size : 5μm 124 Fig. 4-61 B1 and B2 signals of PM chips prepared by ( a ) bioaffinity linkage at pH 4.5 and ( b ) covalent linkage at pH 4.5. 125