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研究生： 李健瑋
Lee, Chien-Wei
論文名稱： 化學界面結合電磁場增強光學感應技術於生物感測應用
Electromagnetic enhancement combined with binding selectivity technology for novel optical detection of biological sensing applications
指導教授： 黃志嘉
Huang, Chih-Chia
學位類別： 博士
Doctor
系所名稱： 理學院 - 光電科學與工程學系
Department of Photonics
論文出版年： 2021
畢業學年度： 109
語文別： 英文
論文頁數： 206
中文關鍵詞： 柔軟性 、表面增強拉曼散射 、隱形墨水 、氫化 、無標籤檢測 、抗生素 、對苯二酚 、濕化學種子成長反應 、奧斯瓦爾德熟化 、非線性光學 、三倍頻光子 、介孔結構 、奈米棒
外文關鍵詞： Flexible, SERS, invisible ink, hydrogenation, label-free sensing, antibiotic, Au nanoparticles, seed-meditated growth reaction, Ostwald ripening, nonlinear optical, third-harmonic generation, mesostructured, nanorod
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    • 近年來，將高靈敏的表面增強拉曼散射（Surface Enhanced Raman Scattering; SERS）技術與高柔軟性基材結合在一起的研究已經引起越來越多科學家的關注。傳統硬式基材需要藉由高耗能蒸濺鍍系統或是複雜的蝕刻步驟來進行SERS基板的製作，不僅原料成本高、能源浪費多，更會有廢棄物不易處理的問題。因此尋找一快速、環保、大批量製作且符合綠色合成概念的軟性SERS基板製備方法變得相當重要。
    在本篇論文中，第一部分是關於我們利用浸泡法將天然抗氧化物－單寧酸搭載在具備吸水特性的可撓式軟性纖維濾紙上，並透過自發性的氧化還原反應直接在微米纖維的表面合成出板狀奈米金顆粒，成功開發出一款簡單且符合綠色化學的環境友善軟性Au-SERS基板，這種無保護劑的合成方法可以減少廢棄產物且使用完畢後可以燃燒銷毀減少環境汙染。我們展現此軟性基板可以成功檢測到多種毒化物以及抗生素分子，其中包含了不同有機溶液與水溶液這顯示了在檢測應用上的廣泛性，且定量範圍在微米到奈米莫爾濃度可以產生高SERS強度、訊號再現性與良好均勻度。此外，我們發現此軟性Au-SERS基板具有良好氫化反應的催化特性（K = 2.14×10-2 s-1），並利用SERS光譜變化來監控催化反應過程。同時我們也展現了這種製備方法可以被應用在各種吸水基材上以達到不同的使用需求，也可以進一步地開發出具有奈米金銀顆粒的軟性SERS基板。
    接著第二部份我們將膠體奈米溶液常見的種子成長法引入軟性Au-SERS基板中，而這種透過濕化學成長法優化軟性SERS基板是過去很少被討論的研究。我們探討了常用的四種還原劑後發現對苯二酚較溫和的還原力可以使得金原子緩慢成長而使得纖維濾紙表面的奈米金密度變高並且形成類似海膽的奈米金結構，同時對苯二酚會在奈米金表面形成一層膜提供帶正電的分子緊密吸附作用力，讓SERS活性位點與拉曼橫截面都能增加使得SERS訊號順利提升6-7倍。此外搭配選用更接近奈米金共振吸收的雷射波長，可以有效地提升訊號強度並且使偵測極限達到ppb等級以及分析物增強因子為1.4-7.4×105。同時我們也展現了軟性基板可以應用在曲面量測、高度吸水性、高重複性等等特質。
    最後第三部分，我們開發了一套種子/生長啟發性的合成方法，其中結合了礦化作用和配體輔助的生長策略來製作介孔結構的α-FeOOH奈米棒。這種材料本身的高折射率使得我們使用近紅外光（1230 nm）進行激發下觀察到產生強烈的三倍頻光子訊號，其中長棒狀的α-FeOOH的三倍頻光子訊號強度比球型的α-FeOOHs強11倍。我們展現了這種介孔結構的α-FeOOH奈米棒在非線性光學顯微鏡下，由於其光學訊號不會隨時間降低而可以作為一種非漂白的造影劑併用於長期標記細胞，以及修飾凝集素讓α-FeOOH奈米棒可以停留在血管壁上，並以此來實現小鼠耳內的血管造影功能。我們的結果為以鐵奈米顆粒作為非線性光學顯微鏡的探針提供了新的見識，並相信未來能為磁成像系統建立互補的顯微成像方法。

    In recent years, many scientists had worked hard for creating flexible substrates that owning an ultrasensitive Surface Enhanced Raman Scattering (SERS). Traditional solid substrates required high-energy sputtering systems or complex etching steps to produce SERS substrates, which not only high material costs and energy waste but also the problem of difficulty dealing with waste. Therefore, it is very important to find a method for preparing a flexible SERS substrate that fasts, environmentally friendly, mass-produced, and the concept of green synthesis.
    In the first work of my dissertation, we through the soaking process of an invisible-ink-inspired to mount a common green reagent - Tannic acid (TNA), and then we designed an on-site redox strategy to fabricate plasmonic Au plate nanoparticles in the surface of micro-fiber. It had successfully fabricated simple and environmentally friendly soft Au@TNA-SERS substrates that conformed to green chemistry. This non-protective synthesis method could reduce waste environmental pollution and be burned to destroy after use. Through amphiphilic inorganic-organic surface structure to absorb various analyte for excellent SERS enhancement, good reproducibility, wettability, and signal uniformity. In addition, we found a good catalytic property (K = 2.14×10-2 s-1) of soft Au@TNA-SERS substrate and monitoring the hydrogenation process. Moreover, this strategy also could be used to fabricate an AuAg SERS substrate that could improve the SERS intensity and detect the lower concentration of pollution.
    Then second work, we introduced the common seed growth method of colloidal nano solution into the soft Au@TNA-SERS substrate, and optimized the soft SERS substrate through the wet chemical growth method was rarely discussed in the past. We discussed the four common reducing agents, the milder reducing power of hydroquinone could slowly grow Au atoms, increased the density of Au nanoparticles, and formed sea urchins structure. Hydroquinone would form a film on the surface of the Au NPs that could provide adsorb force for positive-charge molecules, increased the SERS active site and Raman cross-section, so that increased 6-7 times the SERS signal, detection limit down to the ppb level, and the analyte enhancement factor was 1.4-7.4 × 105. For higher SERS performance, the 632.8 nm laser that more matches with plasmonic resonance peak would be used. We demonstrate the different properties of the soft substrate about detecting in curve surface, wettability, and batch-to-batch reproducibility.
    The final part, mesostructured α-FeOOH nanorods (NRs) prepared by a seed/growth- synthesis strategy that combined ligand-assisted secondary growth and primary mineralization. Under NIR-II (1230 nm) femtosecond laser excitation, mesostructured α-FeOOH NRs would enhance third-harmonic generation (THG) signals because of the high refractive index and exhibited an 11-times stronger THG intensity than bare mesostructured α-FeOOH. We demonstrated that the α-FeOOH NRs could be used as nonbleaching contrast agents for long-term tracking in vitro and angiography in vivo based on the unique nonlinear optical properties. Because mesostructured α-FeOOH NRs are all iron-based composites, they exhibit low cytotoxicity in the test of cell viability. Our results provided an opportunity about Fe-based material maybe could establish an additional bioimaging method for the magnetic imaging system, because it could emit coherent light and employ as a probe in optical microscopy.

    口試合格證明 I 中文摘要 II Abstract IV 致謝 VI Contents VII Figure contents X Table contents XXI 1. Introduction 1 1.1 Surface-enhanced Raman scattering 1 1.1.1 Raman scattering effect 1 1.1.2 Introducing surface-enhanced Raman scattering 2 1.1.3 Electromagnetic Effect 3 1.1.4 Chemical Effect 4 1.1.5 Enhancement Factors calculation 5 1.1.6 Application of SERS 6 1.2 Introduction of SERS substrates 7 1.2.1 Solid SERS substrates 7 1.2.2 Flexible SERS substrates 7 1.2.3 Flexible cellulose paper-nanoparticle hybrid substrates 9 1.2.4 Tannic acid 10 1.2.5 In situ seed-mediated growth soft SERS substrates 10 1.2.6 Improved SERS effect from roughness surface 11 1.3 Third-harmonic generation 12 1.3.1 Advantage of NIR-II wavelength selectivity and Bio-window 12 1.3.2 Introducing Third-harmonic generation 13 1.3.3 Ostwald ripening 14 1.3.4 Introducing of α-FeOOH nanoparticles 14 2. Motivation 26 2.1 Invisible-ink-assisted strategic soft cellulose SERS substrates for various sensing platforms 26 2.2 To engineer and improve SERS efficiency of Au-based SERS substrate through a facile wet-chemistry strategy 27 2.3 Mesostructured FeOOH for third-harmonic generation bioimaging from Ostwald ripening process 28 3. Method and Materials 29 3.1 Materials 29 3.2 Equipment 32 3.3 Synthesis methods of soft SERS substrates 34 3.3.1 Synthesis of Au@TNA-SERS substrate 34 3.3.2 Fabrication of SERS substrates with in-situ reduction strategy 34 3.3.3 Fabrication of the sputtered Au substrate, 13 nm Au-coated substrate, and 80 nm Ag@PVP-coated substrate, and 36 3.3.4 Quantified the concentration of Au atom within different soft substrates 37 3.4 SERS experiment and parameter 37 3.4.1 SERS measurement 37 3.4.2 Confocal microscope for Raman spectrum 38 3.4.3 The analytical enhancement calculating 38 3.4.4 Finite-Difference Time-Domain simulation 38 3.4.5 Mimic experiments of melamine 39 3.4.6 Monitoring hydrogenation with Au@TNA-SERS substrate 39 3.4.7 Electrochemical measurement 39 3.4.8 Characterization 39 3.5 Synthesis methods of α-FeOOH nanorod 40 3.5.1 Synthesis of α-FeOOH nanorods 40 3.5.2 Synthesis of FeOOH@Dopa-Lectin nanorods 40 3.5.3 Synthesis of FeOOH@Dopa-QD-Lectin nanorods 41 3.5.4 Calculation of Fe concentration 41 3.6 Preparation methods of cell study and nonlinear microscopy system 42 3.6.1 In-vitro cell study of 42 3.6.2 Cellular toxicity test of MTT assay 42 3.6.3 Cellular toxicity test of LDH assay 43 3.6.4 Cellular toxicity test of Hemolysis assay 43 3.6.5 Evaluation of laser peak intensity 44 3.6.6 In vitro nonlinear microscopy system 45 3.6.7 In vivo nonlinear microscopy system 45 3.6.8 Animal model and anesthesia 46 4. Results and discussion 47 4.1 Invisible-ink-assisted strategic soft cellulose SERS substrates for various sensing platforms 47 4.1.1 Synthesis and analysis of the Au@TNA-SERS substrate 47 4.1.2 SERS measurements of various SERS substrates 50 4.1.3 Label-free SERS detection for practical application 53 4.1.4 Used SERS to monitor the hydrogenation of an Au@TNA-SERS substrate 55 4.1.5 Fabricated the bimetallic AuAg-SERS substrate 56 4.2 To engineer and improve SERS efficiency of Au-based SERS substrate through a facile wet-chemistry strategy 83 4.2.1 Synthesis and analysis of the Hydroquinone-Au SERS substrate 83 4.2.2 SERS measurements of the Hydroquinone-Au SERS substrate 85 4.2.3 Compared different reductants and reduction condition 88 4.2.4 Advantages of paper-based SERS substrate 92 4.3 Mesostructured FeOOH for third-harmonic generation bioimaging from Ostwald ripening process 124 4.3.1 Synthesis and characterization of mesostructured FeOOH 124 4.3.2 In vitro cell viability study 129 4.3.3 THG test of mesostructured FeOOH 130 4.3.4 In situ THG image 131 4.3.5 In vivo THG image 132 5. Conclusion 171 Reference 176 Publication list 202 Appendix 205  

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