吾人合成邊長為45 nm的奈米金方，並利用二氧化矽作為間隙物(spacer)，並以螢光素(Fluorescein, FITC)作為染料接在二氧化矽殼層的外圍，再藉由控制二氧化矽的厚度，定義染料與奈米金方之間的距離，吾人總共合成五種厚度之包覆二氧化矽的奈米金方，厚度(Ls/LA)分別為12/9、23/20、30/27、36/34、40/37，並利用自架式的靜態螢光光譜及時間解析之螢光光譜法研究中性及鹼性下奈米金方存在時染料之增強倍數(enhancement factor)及螢光生命期的改變。而由靜態螢光光譜可知，隨染料與奈米金方之間距離增加，其增強倍數也會隨之減少；由時間解析的螢光光譜可知，當染料與奈米金方之間距離下降，染料之螢光生命週期也會隨之減少。由實驗得到樣品之增強倍數與螢光生命期後，吾人利用本實驗組林等人所建立的動力學模型:在染料由基態被激發至激發態濃度設為([FiTC*, GNC]ex)之後，在激發態之染料可藉由以下三種路徑將能量釋出，在激發態之染料可直接藉由放出螢光(kr,m)或者以非放光(knr)的形式將能量釋出，此為第一種路徑，將此路徑之速率常數總和定義為k1，即k1 = kr,m + knr，而除了放光至基態之外，激發態之染料也可以將能量傳遞至奈米金方的明亮模式(dipole mode, 亦為bright mode , [FiTC , GNC*(bright)])，此過程之能量傳遞速率為k2，在將能量傳遞至明亮模式的奈米金方後，能量也可能回傳至激發態之染料，此過程之速率為k－2，除了回傳至激發態染料之外，能量也可藉由放光的形式將光子傳遞至遠場，此過程之能量傳遞速率為k3，但若是染料將能量傳遞至奈米金方的暗模式(dark mode, [FiTC , GNC*(dark)])，則能量不會以放光的形式釋出，而是以熱即聲子－聲子(phonon－phonon coupling)耦合等非放光的路徑將能量釋出，而能量由激發態之染料傳遞至暗模式的速率為k4，在藉由動力學模型解出鹼性下k1 = 4.5–0.77  109 s–1, k2 = 4.0–2.3  109 s–1, k-2 = 1.0–0.80  109 s–1,k4 = 20–2  109 s–1；中性下k1 = 5.1–0.84  109 s–1, k2 = 5.3–2.1  109 s–1, k-2 = 1.1–0.91  109 s–1,k4 = 29–2.3  109，比較各個速率常數可以看到k4相較於其他速率常數大，吾人認為這是因為奈米金方之表面電漿共振峰和染料分子放光峰之重疊程度高，造成奈米金方與染料之間能量傳遞速率快，且由於吾人合成之奈米金方邊長僅有45 nm，因此螢光增強效應較小，而在各速率常數中除k-2之大小，其他速率常數皆隨二氧化矽殼層厚度之上升而下降，造成此趨勢不符合預期的原因，吾人認為是因為當二氧化矽厚度上升時，染料與奈米金屬粒子間之能量傳遞速率下降，導致k3無法被忽略，且相較奈米金球，由於奈米金方之尖端也會與染料進行作用，因此即使在染料與奈米金方之間的距離到達40 nm仍可將能量傳遞至奈米金方之明亮模式。

We study the effects of metal-enhanced fluorescence (MEF) on fluorescein dye molecule by gold nanocubes. The silica shell was used as spacer to define the distance between fluorescein and gold nanocube (AuNC). Self-build steady state fluorescence spectroscopy and time-correlated single photon counting (TSCPC) technique were used to detect the weak intensities and interaction between gold nanocube and dye. AuNCs length 40-50 nm were synthesized with different thicknesses of silica shells. As the thickness of silica shell decreased, the enhancement factor increased due to the strong induced electric field by the nanoparticle. Besides, the fluorescence decay curves showed biexponential decay, indicating multiple pathways for relaxation of the excited-state fluorophore. We derived a kinetic model to explain the biexponential behavior to solve the rate constants and to interpret the processes of energy transfer. We obtained the rate constant k1 = 4.5–0.77  109 s–1, k2 = 4.0–2.3  109 s–1, k-2 = 1.0–0.80  109 s–1, and k4 = 20–2  109 s–1 in basic solution and k1 = 5.1–0.84  109 s–1, k2 = 5.3–2.1  109 s–1, k-2 = 1.1–0.91  109 s–1, and k4 = 29–2.3  109 s–1 in neutral solution. The values of k4 were the greatest among the other rate constants because the surface plasma resonance of the gold nanocubes and the emission of the dye molecule have a high degree of overlap resulting in rapid energy transfer rate between dye and AuNC. Since the side lengths of the nanocubes were only 45 nm, the fluorescence enhancement effect was small. All rate constants decreased with the increase in the thickness of the silica shell except for k-2. The energy transfer rate between the dye and nanoparticles decreased with distance, hence, the rate constant k-2 from nanocubes back to FITC became small. As a result, the radiation from nanocube k3 cannot be ignored at large distances. Compared with gold nanospheres, the tips of the AuNC have sharp edge electric field that interacts with the dye. Therefore, even if the distance between the dye and the nano-gold cube reaches 40 nm, the energy can be transferred to the bright modes of AuNC.

第一章 序論 17
1.1 螢光增強效應簡介 17
1.2 研究動機 18
1.3 研究方法 19
第二章 螢光增強效應之基本理論 20
2.1 金屬奈米粒子之表面電漿共振模式 20
2.1.1 德魯德模型(Drude model) 22
2.1.2 局域性表面電漿子共振模式 25
2.1.3 金屬奈米粒子之吸收及極化率 29
2-2 表面電漿共振效應與染料分子作用之局部電場增強效應 29
第三章 文獻回顧 31
3.1 影響金屬奈米粒子增強螢光效應之因素 31
3.1.1 距離因素 31
3.1.2 金屬奈米粒子共振吸收峰及染料之吸收及放光吸收峰耦合程度 36
3.1.3 金屬奈米粒子之尺寸 37
3.2 理論模型 39
3.3 金屬奈米粒子之螢光淬滅 47
第四章 樣品製備 51
4.1 合成奈米金方塊 51
4.2 合成二氧化矽金奈米粒子 52
4.3 修飾螢光素異硫氰酸酯(FiTC)於二氧化矽金奈米粒子 53
第五章 儀器架設 56
5.1 自架式靜態螢光光譜 56
5.2 時間相關單一光子計數系統 57
5.2.1 原理 57
5.2.2 架設及電子元件 61
5.3 樣品檢測 66
5.3.1 掃描式電子顯微鏡 66
5.3.2穿透式電子顯微鏡 69
5.4 靜態吸收光譜 69
5.5 靜態螢光光譜 71
第六章 實驗結果與討論 72
6.1 螢光素異硫氰酸酯之基本性質 72
6.2 螢光素修飾於包覆二氧化矽之奈米金方 78
6.2.1 樣品之SEM及TEM影像 78
6.2.2 樣品之靜態吸收光譜 83
6.2.3 螢光素修飾於二氧化矽奈米金方之時間解析螢光光譜 84
6.2.4 螢光素修飾於二氧化矽奈米金方之靜態營光光譜 91
6.3 GNC@SiO2@FiTC之動力學模型討論 95
第七章 結論 107
參考資料 113

1. Jayasmita Jana.; Mainak Gangulyb.;Tarasankar Pal*a. Enlightening surface plasmon resonance effect of metal nanoparticles for practical spectroscopic application. RSC Adv., 2016, 6, 86174.
2. Katherine A. Willets.; Richard P. Van Duyne. Localized Surface Plasmon Resonance Spectroscopy and Sensing. Annu. Rev. Phys. Chem. 2007. 58:267-97.
3. Metiu, H. Surface enhance spectroscopy. Prog. Surf.sci 1984,17,153-320
4. Van Dijk, M. A.; Tchebotareva, A. L.; Orrit, M.; Lipptz, M.;Berrciaud, S.; Lasne, D.;Cognet, L.; Lounis, B. Absorption and Scattering Microscopy of single metal Nanoparticle. PCCP. 2006, 8, 3486-3495
5. Tânia Ribeiro.; Carlos Baleizão.; José Paulo S. Farinha*. Artefact-free Evaluation of Metal Enhanced Fluorescence in Silica Coated Gold. Nanoparticles. Sci Rep 7, 2440 (2017).
6. Krishanu Ray; Ramachandram Badugu; Joseph R.; Lakowicz*. Distance-Dependent Metal-Enhanced Fluorescence from Langmuir-Blodgett Monolayers of Alkyl-NBD Derivatives on Silver Island Films. Langmuir 2006, 22, 8374-8378
7. Hirdyesh Mishra; Buddha L. Mali; Jan Karolin; Anatoliy I. Dragan ;Chris D. Geddes. Experimental and theoretical study of the distance dependence of metal-enhanced fluorescence, phosphorescence and delayed fluorescence in a single system. PCCP. 2013, 15, 19538
8. Daedu Lee; a Jaebeom Lee; b Junghyun Song; a Myungsam Jena; Yoonsoo Pang. Homogeneous silver colloidal substrates optimal for metal-enhanced fluorescence. Phys. Chem. Chem. Phys., 2019, 21, 11599
9. Fang Xie; Mark S. Baker; Ewa M. Goldys. Enhanced Fluorescence Detection on Homogeneous Gold Colloid Self-Assembled Monolayer Substrates. Chem. Mater. 2008, 20, 1788–1797
10. Geddes, C. D.; Lakowicz, J. R. Metal-Enhanced Fluorescence. J Fluoresc. 2002, 12, 121-129.
11. Yu-Zheng Su; Min-Wei Hung; Wen-Hong Wu; Kuo-Cheng Huang. Application of Metal-Enhanced Fluorescence Technology in Evanescent Wave Fluorescent Biosensor. IEEE Instrumentation & Measurement Technology Conference Proceedings, 2010, pp. 574-578.
12. Aslan, K.; Malyn, S. N.; Geddes, C. D., Metal-Enhanced Fluorescence from Gold Surfaces: Angular Dependent Emission. J Fluoresc. 2007, 17, 7-13.
13. Hsin-Lun Wu; Chun-Hong Kuo; Michael H. Huang. Seed-Mediated Synthesis of Gold Nanocrystals with Systematic Shape Evolution from Cubic to Trisoctahedral and Rhombic Dodecahedral Structures. Langmuir 2010, 26, 14, 12307–12313
14. Christopher J. Breshike; Ryan A. Riskowski; Geoffrey F. Strouse. Leaving Fö rster Resonance Energy Transfer Behind: Nanometal Surface Energy Transfer Predicts the Size-Enhanced Energy Coupling between a Metal Nanoparticle and an Emitting Dipole. J. Phys. Chem. C. 2013, 117, 23942−23949
15. Anatoliy I. Dragan; Eric S. Bishop; Jose R. Casas-Finet; Robert J. Strouse; James McGivney; Mark A. Schenerman; Chris D. Geddes. Distance Dependence of Metal-Enhanced Fluorescence. Plasmonics. 2012, 7(4), 739–744.
16. https://en.wikipedia.org/wiki/Quenching_(fluorescence)
17. Willets; Van Duyne. Localized Surface Plasmon Resonance Spectroscopy and Sensing. Annu. Rev. Phys. Chem. 2007. 58:267-97
18. Daniel Darvill; Anthony Centenoab; Fang Xie. Plasmonic fluorescence enhancement by metal nanostructures: shaping the future of bionanotechnology. Phys. Chem. Chem. Phys., 2013, 15, 15709-15726
19. Li, L.; Rao, G.; Lv, X.; Chen, R.; Cheng, X.; Wang, X.; Zeng, S.; Liu, X., Chemical Reactivation of Fluorescein Isothiocyanate Immunofluorescence-Labeled Resin-Embedded Samples. JBO 2018, 23, 020501.
20. Gerasimova, M. A.; Tomilin, F. N.; Malyar, E. Y.; Varganov, S. A.; Fedorov, D. G.; Ovchinnikov, S. G.; Slyusareva, E. A., Fluorescence and Photoinduced Proton Transfer in the Protolytic Forms of Fluorescein: Experimental and Computational Study. Dyes Pigm. 2020, 173, 107851.
21. Lin, H.-H.; Chen, I.-C., Study of the Interaction between Gold Nanoparticles and Rose Bengal Fluorophores with Silica Spacers by Time-Resolved Fluorescence Spectroscopy. J. Phys. Chem. C 2015, 119, 26663-26671.
22. Yi-Ting Liu; Xue-Feng Luo; Yin-Yu Lee; I-Chia Chen. Investigating the metal-enhanced fluorescence on fluorescein by silica core-shell gold nanoparticles using time-resolved fluorescence spectroscopy. Dyes Pigm. 2021, 190, 109263.
23. Jérémie Léonarda; Norbert Dumas; Jean-Pascal Caussé; Sacha Maillot; Naya Giannakopoulou; Sophie Barrea; Wilfried Uhring. High-throughput Time-Correlated Single Photon Counting. Lab Chip, 2014, 14, 4338
24. Kyung A Kang; Jianting Wang; Jacek B Jasinski; Samuel Achilefu. Fluorescence Manipulation by Gold Nanoparticles: From Complete Quenching to Extensive Enhancement. Journal of Nanobiotechnology, 2011, 9:16