開發三向功能的奈米探針, 使 SERS (表面增強拉曼光譜) 於無背景干擾下偵測細胞表面的唾液酸

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研究生：	芮娜妲 Renata, Septila
論文名稱：	開發三向功能的奈米探針, 使 SERS (表面增強拉曼光譜) 於無背景干擾下偵測細胞表面的唾液酸 Development of a Tri-Functional Nanoprobe for Background-Free SERS (Surface-enhanced Raman Spectroscopy) Detection of Sialic Acid on Cell Surface
指導教授：	林俊宏 Lin, Chun-Hung 潘榮隆 Pan, Rong-Long
口試委員:	郭俊宏 Kuo, Chun-Hong 謝馬利歐 Hofmann, Mario 凃智傑 Tu, Zhijay
學位類別：	博士 Doctor
系所名稱：	生命科學暨醫學院 - 生物資訊與結構生物研究所 Institute of Bioinformatics and Structural Biology
論文出版年：	2021
畢業學年度：	109
語文別：	英文
論文頁數：	83
中文關鍵詞：	表面增強拉曼光譜
相關次數：	點閱：2 下載：0
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細胞表面的唾液酸在許多生理和病理學過程中都是不可缺少的，唾液酸的靈敏且可重複的檢測對於許多疾病的診斷和治療至關重要。在本論文中，我們開發了一種三重功能的奈米探針，作為靈敏、簡單的表面增強拉曼光譜（SERS）奈米探針，用於細胞表面上的唾液酸檢測。此報導分子的設計旨在提供三個關鍵功能，使其成為唾液酸檢測的理想選擇。首先，我們採用了兩個識別基團，即苯基硼酸和一個銨基，它們可增強唾液酸的識別和捕獲效率。其次，使用氰基作為拉曼光譜報告分子，因為其分子振動光譜會落在細胞拉曼無聲區中。最後，硫醇充當錨定劑以使報告分子能與銀奈米立方體結合，從而達成SERS並保護銀奈米立方體免於氧化。我們的分子奈米探針設計證明了能夠以高靈敏度和選擇性檢測細胞表面的唾液酸，為細胞診斷開闢了新途徑。

Sialic acids, especially those on cell surface, are indispensable in numerous physiological and pathological processes. Sensitive and reproducible detection of sialic acid is crucial for diagnosis and therapy in many diseases. In this thesis, we developed a tri-functional nanoprobe as a sensitive and straightforward surface-enhanced Raman spectroscopy (SERS) nanoprobe for detecting sialic acid-containing glycan on cell surfaces. The reporter was designed to provide three key functionalities that make it ideal for sialic acid detection. First, we employed two recognition groups, phenylboronic acid and an ammonium group, to enhance sialic acid recognition and capture efficiency. Second, we used cyano as the Raman reporter because its vibrational energy is shown in the cellular Raman silent region. Finally, thiol acted as an anchoring agent that facilitate the conjugation of reporter onto silver nanocubes to provide SERS enhancement and protect silver nanocubes from oxidation. Our molecular nanoprobe design demonstrated the ability to detect sialic acid on the cell surface with high sensitivity and selectivity, opening up new routes to cellular diagnostics.

Table of Contents
Abstract    i
摘要    ii
Acknowledgements    iii
List of Abbreviations    v
Chapter 1. Introduction    1
1.1. Biological Importance of Sialic Acid    1
1.2. Raman and SERS Spectroscopy    3
1.2.1. Electromagnetic Enhancement (EM)    5
1.2.2. Chemical Enhancement (CE)    6
1.3. SERS Nanoprobe    7
1.4. Synthesis of Gold and Silver Nanocubes    9
1.4.1. Seed-Growth Method    10
1.4.2. Polyol Method    10
1.5. Raman Reporter    11
1.5.1. Selection of Raman Reporter    11
1.5.2. Bioorthogonal Raman Reporter    12
1.6. Boronic Acid and Its Binding Towards Monosaccharides    13
1.7. SERS-Based Detection of Sialic Acid and Challenges    17
1.8. Research Goal of Thesis    26
Chapter 2. Results and Discussion    28
2.1. Synthesis and Characterization of AgNCs    28
2.2. Synthesis and Characterization of AuNCs    31
2.3. Design and Synthesis of Reporter    32
2.4. Characterization of Tri-functional Nanoprobe    36
2.5. Sialic Acid Expression on HeLa cells    38
2.6. Examining Sialic Acid Expression on Normal (Non-Cancerous) and Cancer Cells    43
Chapter 3. Conclusions    46
Chapter 4. Materials and Methods    48
4.1. Instruments and Characterization    48
4.2. Synthesis of AuNCs    49
4.3. Synthesis of AgNCs    50
4.4. Synthetic Procedures    51
4.5. Synthesis of Tri-Functional Nanoprobe (Reporter 6 Modified AgNCs)    56
4.6. Sensitivity of AgNCs Towards Reporter 6    57
4.7. Cell Culture and Treatment with Nanoprobe    57
4.8. Nanoprobe Specificity Toward Sialic Acid    58
4.9. SERS Measurement    59
References    60
List of Publications    66
Appendix: NMR Spectra    67
1H NMR spectrum of compound 1 (500 MHz, CD3OD)    68
13C NMR spectrum of compound 1 (125 MHz, CD3OD)    69
1H NMR spectrum of compound 2 (500 MHz, CD3OD)    70
13C NMR spectrum of compound 2 (125 MHz, CD3OD)    71
1H NMR spectrum of compound 3 (500 MHz, CD3OD)    72
13C NMR spectrum of compound 3 (125 MHz, CD3OD)    73
1H NMR spectrum of compound 4 (500 MHz, CD3OD)    74
13C NMR spectrum of compound 4 (125 MHz, CD3OD)    75
1H NMR spectrum of compound 5 (500 MHz, CD3OD)    76
13C NMR spectrum of compound 5 (125 MHz, CD3OD)    77
1H NMR spectrum of compound 6 (500 MHz, CD3OD)    78
13C NMR spectrum of compound 6 (125 MHz, CD3OD)    79
1H-13C 2D HMQC NMR spectrum of 6 (500/125 MHz, CD3OD)    80
1H-13C 2D HMBC NMR spectrum of 6 (500/125 MHz, CD3OD)    81
1H-1H 2D COSY NMR spectrum of 6 (500 MHz, CD3OD)    82
High Resolution Mass Spectra of compound 6    83


                                

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