中文摘要 I Abstract II 目錄 III 表目錄 V 圖目錄 VI 第一章 緒論 1 第二章 文獻回顧 2 2-1 H. salinarum、紫色細胞膜(PM)和細菌視紫質(BR) 2 2-1-1 H. salinarum 2 2-1-2 PM和BR 3 2-2 BR光循環及質子傳遞 6 2-3 生物親和固定法 8 2-4 BR對連續光源與脈衝雷射之光電響應 9 2-4-1 脈衝雷射光電響應 9 2-4-2 連續光源光電響應 12 2-4-3 脈衝響應與微分響應的比較與關係 16 2-4-4 PM之I-V 曲線 18 2-5 BR與奈米金 19 第三章 實驗 22 3-1 實驗目的 22 3-2 實驗流程 23 3-3 實驗藥品與分析儀器 24 3-3-1 實驗藥品 24 3-3-2 實驗設備 25 3-4 藥品配製 27 3-5 實驗步驟 29 3-5-1 製備 biotin-PM 29 3-5-2 清洗ITO玻璃 29 3-5-3 自排性單分子膜(SAM)修飾ITO玻璃 30 3-5-4 奈米金之修飾 30 3-5-5 水滴接觸角量測 33 3-5-6 循環伏安法分析 33 3-5-7 D1、D2微分光電訊號量測 34 3-5-8 B1、B2脈衝光電訊號量測 35 第四章 結果與討論 36 4-1 PM晶片的NHS與EDC活化 36 4-1-1 PM經EDC與NHS活化後表面性質變化 37 4-1-2 質子泵機制探討 47 4-1-3 EDC與NHS最適化 51 4-2 PM晶片上之NH2-C6-DNA1共價鍵結 57 4-2-1 PM共價鍵結NH2-C6-DNA1後之表面性質變化 57 4-2-2 PM晶片共價鍵結NH2-C6-DNA1後之光電響應變化 65 4-2-3 DNA1濃度不同之AFM分析 73 4-2-4 NH2-C6-DNA1之最適化鍵結濃度 75 4-2-5 PM/DNA1晶片以ethanolamine 之處理 77 4-3 以PM/DNA1晶片結合帶有DNA2之奈米金(Au-DNA2) 82 4-3-1 PM/DNA1晶片接上Au-DNA2後AFM分析 82 4-3-2 不同濃度DNA1接上相同濃度DNA2 85 4-3-3 用相同濃度DNA1接上不同濃度DNA2 88 4-4 利用共價鍵結與雜交作用製程與生物親和性吸附製程比較 94 4-4-1 循環伏安法 94 4-4-2 AFM分析比較 95 4-4-3 D1與D2微分光電響應探討 102 第五章 結論 105 第六章 參考文獻 106   表目錄 Table 2-1 Dependence of the decay lifetime of the intermediate I460 on the gold nanoparticle concentration and size. (Biesso, et al., 2008) 19   圖目錄 Fig. 2-1The four archaeal rhodopsins in H. salinarum (Spudich, 1998). 3 Fig. 2-2 High-resolution AFM topographs of membranes：BR molecules assembled into (a) trimers, (b) dimers, and (c) monomers. Top row, surveys show the membranes being adsorbed flat on mica. (Spara et al., 2006). 4 Fig. 2-3 Scheme of bR amino acid sequence in the membrane . (Voïtchovsky et al., 2007). 5 Fig. 2-4 Photoisomerization of all-trans to 13-cis retinal in bR. (Jin et al., 2008). 5 Fig. 2-5 A schematic representation of (a) the bacteriorhodopsin tertiary structure and (b) the main photocycle and the branching reactions studied here. (Gillespie et al., 2002). 7 Fig. 2-6 X-ray crystallographic structures of the Schiff base region in BR. (Kandori, 2004). 7 Fig. 2-7 (A) The photoelectric response signal of normal PM in (bilayer lipid membrane) BLM; (B) biotinylated PM. ( Su et al., 2002) . 8 Fig. 2-8 Photocurrent observed on the micro-millisecond time scales from an oriented bR film in 10 mM KCl, 2 mM K2HPO4 at room temperature. (A) pH = 8.0 and (B) pH = 3.0 (Wang et al., 1997a). 10 Fig. 2-9 Effect of ionic strength on the rise and decay of B3 and D1. (A) Effect of buffer concentration at pH 8.0; (B) KCl concentration effect on B3, in 1 mM K2HPO4; and (C) KCl concentration effect on D1/D2, in 1 mM K2HPO4 at pH 8.5 (Wang et al., 1997a). 10 Fig. 2-10 Photocurrent signal under pulsed-laser excitation, in the time domain of 500 ms. Various film orientation on ITO glass are shown. (Wang, 2000). 11 Fig. 2-11 The typical differential response of PM. (Hong, 1997). 12 Fig. 2-12 Waveforms of the BR-LB photocell response to the interval light, measured by the digital oscilloscope DC and AC modes at 1 MΩ input impedance. A: Waveforms obtained at DC. B: Waveforms obtained at AC. (Yao et al., 2002)。 13 Fig. 2-13 Comparison of the typical photoelectric response from a sandwich-type photocell. (Koyama, et al., 1995)。 14 Fig. 2-14 Typical response profile from a wild-type bR thin film immobilized at the SnO2 relectrolyte (0.1 M KCl, pH 8.0 ) interface in an electrochemicall cell. The cell was irradiated with green light supplied by a 150-W xenon arc lamp. Light intensity pattern of irradiation is given in the lower half(Koyama and Miyasaka., 2001). 15 Fig. 2-15 Response profiles at electrochemical cells with various mutant proteins. (Koyama and Miyasaka., 2001). 15 Fig. 2-16 Photocurrent components from an oriented bR film at various time scales at two typical pHs. (Wang et al., 1997b ) 17 Fig. 2-17 Membrane orientation effect on the photocurrent components. (Wang et al., 1997b ) 17 Fig. 2-18 Current–voltage (I–V) curves of Au/bR/APTMS–AlOx–Al junction, with an oriented bR monolayer, prepared by bR–OTG vesicle fusion, measured under ambient conditions, in the dark, upon λ < 550 nm illumination and upon illumination with (380 nm < λ < 440 nm) light. The arrows show that the I–V responses can be cycled by switching between the two types of illumination. Au pad area: 2 ╳ 10-3 cm2. Inset: absorption difference spectra of two bR monolayers, adsorbed on each side of an APTMS-modified quartz slide, as a result of green light irradiation and dark adaptation. Green curve: irradiated–dark; black curve: (dark after illumination)–irradiated (Jin et al., 2008 ). 18 Fig. 2-19 Effect of increasing the concentration of 40 nm Au-NPs on the dynamics of the primary step (the decay kinetics of I460) of the photosynthetic system of bacteriorhodopsin. (Biesso, et al., 2009). 20 Fig. 2-20 The effect of Au-NPs size (plasmonic field) on the dynamic of the primary step (the decay kinetics of I460) of bacteriorhodopsin (Biesso, et al., 2009). 20 Fig. 2-21 Dependence of the decay kinetics of M412 on Au NRs concentration in the presence of NIR laser irradiation with an intensity of 0.256 W/cm2. Each decay lifetime value was obtained by fitting the decay kinetic with a single exponential function (Biesso, et al., 2009). 21 Fig. 3-1 奈米金於PM晶片上的不同固定化之方法：(A)利用共價鍵結與DNA雜交為機制；(B)利用生物親和性吸附為機制. 22 Fig. 3-2 Experimental procedures. 23 Fig. 3-3 Scheme of PM chip. 32 Fig. 3-4 Equipment setup for the measurements of D1 and D2 photocurrent signals. 34 Fig. 3-5 Equipment setup for the measurements of B1 and B2 photocurrent signals. 35 Fig. 4-1 The Mechanism of EDC/NHS reaction. 36 Fig. 4-2 Contact angles of various ITO coating layers. 37 Fig. 4-3 The cyclic voltammetry analysis of a PM chip 39 Fig. 4-4 The cyclic voltammetry analysis of a PM chip activated with 0.15M EDC/NHS. 40 Fig. 4-5 Oxidation and reduction diffusion coefficients of ferricyanide on PM chips before and after EDC/NHS activated. 41 Fig. 4-6 AFM image of a PM chip (Data scale : 5 μm), Ra=5.79 nm. (以化工系 AFM儀器分析 林聖為操作) 42 Fig. 4-7 AFM image of a PM chip activated by 0.15M EDC/NHS (Data scale : 5 μm), Ra=5.73 nm. (以化工系 AFM儀器分析 林聖為操作) 42 Fig. 4-8 AFM image of a PM chip (Data scale : 2 μm), Ra=5.06 nm. (以化工系 AFM儀器分析 林聖為操作) 43 Fig. 4-9 AFM image of a PM chip activated by 0.15M EDC/NHS (Data scale : 2 μm), Ra=4.24 nm. (以化工系 AFM儀器分析 林聖為操作) 43 Fig. 4-10 3D AFM image of a PM chip ( Data scale : 5 μm). (以化工系 AFM儀器分析 林聖為操作) 44 Fig. 4-11 3D AFM image of a PM chip activated by 0.15M EDC/NHS (Data scale : 5 μm). (以化工系 AFM儀器分析 林聖為操作) 44 Fig. 4-12 3D AFM image of a PM chip ( Data scale : 2 μm). (以化工系 AFM儀器分析 林聖為操作) 45 Fig. 4-13 3D AFM image of a PM chip activated by 0.15M EDC/NHS (Data scale : 2 μm). (以化工系 AFM儀器分析 林聖為操作) 45 Fig. 4-14 The section profile AFM image of PM chips before and after EDC/NHS activation. (以化工系張家耀實驗室 AFM儀器分析 吳佩容操作) 46 Fig. 4-15 Photocurrent of PM chips before and after EDC/NHS activation. 48 Fig. 4-16 Ratio of D1 peak-on over D2 peak-off photocurrent of PM chips before and after EDC/NHS activation. 48 Fig. 4-17 The D1 peak-on photocurrent of PM chips before and after EDC/NHS activation. 49 Fig. 4-18 The D2 peak-off photocurrent of PM chips before and after EDC/NHS activation. 49 Fig. 4-19 Overlapped responses of D1 peak-on photocurrent response of PM chips with and without EDC/NHS activation. 50 Fig. 4-20 Overlapped responses of D2 peak-off photocurrent response of PM chips with and without EDC/NHS activation. 50 Fig. 4-21 Photocurrent density of PM chips activated with different EDC+NHS concentrations. 52 Fig. 4-22 The D1 peak-on photocurrent density of PM chips activated with different EDC+NHS concentrations. 53 Fig. 4-23 The D2 peak-off photocurrent density of PM chips activated with different EDC+NHS concentrations. 53 Fig. 4-24 Overlapped responses of D1 peak-on photocurrent signals of PM chips with different EDC+NHS concentrations. 54 Fig. 4-25 Overlapped responses of D2 peak-off photocurrent signals of PM chips with different EDC+NHS concentrations. 54 Fig. 4-26 Overlapped curves of normalized D1 peak-on signals of PM chips with different EDC+NHS concentrations. 55 Fig. 4-27 Overlapped curves of normalized D2 peak-off signals of PM chips with different EDC+NHS concentrations. 55 Fig. 4-28 Ratio of D1 peak-on over D2 peak-off photocurrents of PM chips activated with different concentrations of EDC+NHS. 56 Fig. 4-29 Contact angles of PM chips activated with EDC/NHS and conjugated different concentrations of DNA1. 58 Fig. 4-30 Oxidation diffusion coefficients of ferricyanide on PM chips activated with EDC/NHS and conjugated with different concentrations of DNA1. 59 Fig. 4-31 Reduction diffusion coefficients of ferricyanide on PM chips activated with EDC/NHS and conjugated with different concentrations of DNA1. 59 Fig. 4-32 The scheme diagram of DNA1 molecules covalently bonded to PM. 60 Fig. 4-33 C.V. curves of PM chips, activated with EDC/NHS, aminated with ethylenediamine, and blocked with ethanolamine. Scan rate :0.1V/s. 60 Fig. 4-34 C.V. curves of PM chips, activated with EDC/NHS, conjugated with DNA1, and blocked with ethanolamine. Scan rate :0.1V/s. 61 Fig. 4-35 AFM image of a DNA1-coated PM chip (Data scale : 5 μm), Ra=3.89 nm. (以化工系 AFM儀器分析 林聖為操作) 62 Fig. 4-36 AFM image of a DNA1-coated PM chip (Data scale : 2 μm), Ra=3.05 nm. 62 Fig. 4-37 3D AFM image of a DNA1-coated PM chip (Data scale : 5 μm). (以化工系 AFM儀器分析 林聖為操作) 63 Fig. 4-38 3D AFM image of a DNA1-coated PM chip (Data scale : 2 μm ). (以化工系 AFM儀器分析 林聖為操作) 63 Fig. 4-39 Section profile of the AFM image of DNA 1 coated PM chip (Data scale : 5 μm ). (以化工系 AFM儀器分析 林聖為操作) 64 Fig. 4-40 Photocurrent density of PM chips, activated with EDC/NHS, and conjugated with 1 μM, 5 μM, 10 μM DNA1. 66 Fig. 4-41 Ratio of D1 peak-on over D2 peak-off photocurrents of PM chips, activated with EDC/NHS, and conjugated with 1 μM, 5 μM, 10 μM DNA1. 66 Fig. 4-42 Overlapped responses of D1 peak-on photocurrent signals of PM chips, activated with EDC/NHS, and conjugated with 1 μM, 5 μM, 10 μM DNA1. 67 Fig. 4-43 Overlapped responses of D2 peak-off photocurrent signals of PM chips, activated with EDC/NHS, and conjugated with 1 μM, 5 μM, 10 μM DNA1. 67 Fig. 4-44 D1 peak-on photocurrents of PM chips, activated with EDC/NHS, and conjugated with 1 μM, 5 μM, 10 μM DNA1. 68 Fig. 4-45 D2 peak-off photocurrents of PM chips, activated with EDC/NHS, and conjugated with 1 μM, 5 μM, 10 μM DNA1. 68 Fig. 4-46 Decay time of D1 peak-on signals of PM chips ,activated with EDC/NHS, and conjugated with 1 μM,5 μM,10 μM DNA1. 69 Fig. 4-47 Decay time of D2 peak-off signals of PM chips ,activated with EDC/NHS, and conjugated with 1 μM,5 μM,10 μM DNA1. 69 Fig. 4-48 The B1 and B2 pulse signals of a PM chip. 71 Fig. 4-49 The B1 and B2 pulse signals of a PM chip activated with 0.15 M EDC+NHS . 72 Fig. 4-50 The B1 and B2 pulse signals of a 10 μM DNA1-coated PM chips. 72 Fig. 4-51 AFM images of PM chips coated with (a) 10μM and (b) 1 μM DNA1, (Data scale : 10 μm ). (以化工系張家耀實驗室 AFM儀器分析 吳佩容操作) 73 Fig. 4-52 AFM images of PM chips coated with (a) 10μM and (b) 1 μM DNA1,(Data scale : 5 μm ). (以化工系張家耀實驗室 AFM儀器分析 吳佩容操作) 74 Fig. 4-53 AFM images of PM chips coated with (a) 10μM and (b) 1 μM DNA1, (Data scale : 2 μm ). (以化工系張家耀實驗室 AFM儀器分析 吳佩容操作) 74 Fig. 4-54 C.V. curves of PM chips ,activated with EDC/NHS, conjugated with 0.2 μM and 2 μM DNA1, Scan rate :0.1V/s. 76 Fig. 4-55 C.V. curves of PM chips, activated with EDC/NHS, conjugated with 1 μM, 5 μM and 10 μM DNA1, Scan rate :0.1V/s. 76 Fig. 4-56 Photocurrent density of PM chips, activated with EDC/NHS, and conjugated with 1 μM, 5 μM, 10 μM DNA1 with and without ethanolamine blocking. 78 Fig. 4-57 Ratio of D1 peak-on over D2 peak-off photocurrents of PM chips, activated with EDC/NHS, and conjugated with 1 μM, 5 μM, 10 μM DNA1 with and without ethanolamine blocking. 78 Fig. 4-58 D1 peak-on photocurrents of PM chips, activated with EDC/NHS, and conjugated with 1 μM, 5 μM, 10 μM DNA1 with and without ethanolamine blocking. 79 Fig. 4-59 D2 peak-off photocurrents of PM chips, activated with EDC/NHS, and conjugated with 1 μM, 5 μM, 10 μM DNA1 with and without ethanolamine blocking. 79 Fig. 4-60 Decay time of D1 peak-on signals of PM chips, activated with EDC/NHS, and conjugated with 1 μM, 5 μM, 10 μM DNA1 with and without ethanolamine blocking. 80 Fig. 4-61 Decay time of D2 peak-off signals of PM chips, activated with EDC/NHS, and conjugated with 1 μM, 5 μM, 10 μM DNA1 with and without ethanolamine blocking. 80 Fig. 4-62 Oxidation diffusion coefficients of ferricyanide on PM chips, activated with EDC/NHS, and conjugated with 1 μM, 5 μM, 10 μM DNA1 with and without ethanolamine blocking. 81 Fig. 4-63 Reduction diffusion coefficients of ferricyanide on PM chips, activated with EDC/NHS, and conjugated with 1 μM, 5 μM, 10 μM DNA1 with and without ethanolamine blocking. 81 Fig. 4-64 AFM images of a Au/DNA2-coated PM chips (Data scale : 5 μm), Ra=6.48 nm. (以化工系 AFM儀器分析 林聖為操作) 82 Fig. 4-65 AFM images of a Au/DNA2-coated PM chips (Data scale : 2 μm ) ,Ra=3.3 nm. (以化工系 AFM儀器分析 林聖為操作) 83 Fig. 4-66 3D AFM images of a Au/DNA2-coated PM chips ( Data scale : 5 μm ). (以化工系 AFM儀器分析 林聖為操作) 83 Fig. 4-67 3D AFM images of a Au/DNA2-coated PM chips ( Data scale : 2 μm ). (以化工系 AFM儀器分析 林聖為操作) 84 Fig. 4-68 Section profile of the AFM images of a Au/DNA2-coated PM chips (Data scale : 5 μm ). (以化工系 AFM儀器分析 林聖為操作) 84 Fig. 4-69 Effect of DNA1 coating conditions (1μM , 5 μM , 10 μM ) on the photocurrents of DNA1-coated PM chips, subsequently coated with or without 1 nM (50 ppm) 80 nm Au-16MA-DNA2. 85 Fig. 4-70 Effect of DNA1 coating conditions (1μM , 5 μM , 10 μM ) on the D1 peak-on photocurrents of DNA1-coated PM chips, subsequently coated with or without 1 nM (50ppm) 80 nm Au-16MA-DNA2. 86 Fig. 4-71 Effect of DNA1 coating condition (1μM , 5 μM , 10 μM ) on the D2 peak-off photocurrents of DNA1-coated PM chips, subsequently coated with or without 1 nM (50ppm) 80 nm Au-16MA-DNA2. 86 Fig. 4-72 Effect of DNA1 coating condition (1μM , 5 μM , 10 μM ) on the D1 peak-on decay time of DNA1-coated PM chips, subsequently coated with or without 1 nM (50ppm) 80 nm Au-16MA-DNA2. 87 Fig. 4-73 Effect of DNA1 coating condition (1μM , 5 μM , 10 μM ) on the D2 peak-off decay time of DNA1-coated PM chips, subsequently coated with or without 1 nM (50ppm) 80 nm Au-16MA-DNA2. 87 Fig. 4-74 Effect of DNA1 coating condition (1 μM , 5 μM , 10 μM ) on the ratio of peak-on over peak-off photocurrents of DNA1-coated PM chips, subsequently coated with or without 1 nM (50ppm) 80 nm Au-16MA-DNA2. 88 Fig. 4-75 Effect of different 80 nm Au-16MA-DNA2 coating concentrations (0.001~1 nM) on the photocurrents PM chips, and activated by EDC/NHS, conjugated with 10 μM DNA1). 89 Fig. 4-76 Effect of different 80 nm Au-16MA-DNA2 coating concentrations (0.001~1 nM) on the D1 peak-on photocurrents PM chips, and activated by EDC/NHS, conjugated with 10 μM DNA1). 89 Fig. 4-77 Effect of different 80 nm Au-16MA-DNA2 coating concentrations (0.001~1 nM) on the D2 peak-off photocurrents PM chips, and activated by EDC/NHS, conjugated with 10 μM DNA1). 90 Fig. 4-78 Effect of different 80 nm Au-16MA-DNA2 coating concentrations (0.001~1nM) on the photocurrents of 10μM DNA1-coated PM chips. 90 Fig. 4-79 Effect of different 80 nm Au-16MA-DNA2 coating concentrations (0.001~1 nM) on Decay time of D1 peak-on PM chips, and activated by EDC/NHS, conjugated with 10 μM DNA1). 91 Fig. 4-80 Effect of different 80 nm Au-16MA-DNA2 coating concentrations (0.001~1 nM) on Decay time of D2 peak-off PM chips, and activated by EDC/NHS, conjugated with 10 μM DNA1). 91 Fig. 4-81 Overlapped D1 peak-on responses of 10μM DNA1-coated chips binding with different concentration 80 Au-16MA-DNA2. 92 Fig. 4-82 Overlapping D2 peak-off responses of 10μM DNA1-coated chips binding with different concentration 80 Au-16MA-DNA2. 92 Fig. 4-83 Overlapped curves of normalized D1 peak-on signals of 10μM DNA1-coated chips binding with different concentration 80 Au-16MA-DNA2. 93 Fig. 4-84 Overlapped curves of normalized D2 peak-off signals of 10μM DNA1-coated chips binding with different concentration 80 Au-16MA-DNA2. 93 Fig. 4-85 C.V. curves of PM chips ,activated with EDC/NHS, conjugated with 2μM DNA1, binding with different concentrations (0.002~2 nM) 20nm Au-16MA-DNA2. Scan rate :0.1V/s. 94 Fig. 4-86 C.V. curves of PM chips ,modified with biotin, adsorbed with 2mg/mL Avidin, binding with different concentrations (0.2~2 nM) 20nm Au-16MA-DNA2. Scan rate :0.1V/s. 95 Fig. 4-87 AFM image of a PM chip modified by biotin (Data scale : 5 μm), Ra=4.885 nm. (以化工系 AFM儀器分析 林聖為操作) 96 Fig. 4-88 AFM image of a PM chip coated with the second avidin layer (Data scale : 5 μm), Ra=6.043 nm. (以化工系 AFM儀器分析 林聖為操作) 96 Fig. 4-89 AFM image of a 2 nM 20 nm biotin-Au coated-PM chip. (Data scale : 5 μm), Ra=6.545 nm. (以化工系 AFM儀器分析 林聖為操作) 97 Fig. 4-90 AFM image of a PM chip modified by biotin (Data scale : 2 μm), Ra=3.621nm. (以化工系 AFM儀器分析 林聖為操作) 97 Fig. 4-91 AFM image of a PM chip coated with the second avidin layer. (Data scale : 2 μm), Ra=4.309 nm. (以化工系 AFM儀器分析 林聖為操作) 98 Fig. 4-92 AFM image of a 2 nM 20 nm biotin-Au coated-PM chip. (Data scale : 2 μm), Ra=3.721 nm. (以化工系 AFM儀器分析 林聖為操作) 98 Fig. 4-93 3D AFM image of a PM chip modified by biotin ( Data scale : 5 μm ) (以化工系 AFM儀器分析 林聖為操作) 99 Fig. 4-94 3D AFM image of a PM chip coated with the second avidin layer ( Data scale : 5 μm ) (以化工系 AFM儀器分析 林聖為操作) 99 Fig. 4-95 3D AFM image of a 2 nM 20 nm biotin-Au coated-PM chip. ( Data scale : 5 μm ) (以化工系 AFM儀器分析 林聖為操作) 100 Fig. 4-96 3D AFM image of a PM chip modified by biotin ( Data scale : 2 μm ) (以化工系 AFM儀器分析 林聖為操作) 100 Fig. 4-97 3D AFM image of a PM chip coated with the second avidin layer ( Data scale : 2 μm ) (以化工系 AFM儀器分析 林聖為操作) 101 Fig. 4-98 3D AFM image of a 2 nM 20 nm biotin-Au coated-PM chip. ( Data scale : 2 μm ) (以化工系 AFM儀器分析 林聖為操作) 101 Fig. 4-99 Photocurrents for decay profile of PM chips, subsequently modified by either bioaffinity linkage or covalent –DNA hybridization methods. 102 Fig. 4-100 Overlapped responses of D1 peak-on photocurrent signals of different layer for Bioaffinity linkage method. 103 Fig. 4-101 Overlapped responses of D2 peak-off photocurrent signals of different layer for Bioaffinity linkage method. 103 Fig. 4-102 Overlapped curves of normalized D1 peak-on photocurrent signals of different layer for Bioaffinity linkage method. 104 Fig. 4-103 Overlapped curves of normalized D2 peak-off photocurrent signals of different layer for Bioaffinity linkage method. 104