在細胞代謝過程中，每一個細胞器都有著特定和不可或缺的作用。例如，核調節基因表達，線粒體負責有氧代謝。因此，監測生物物種內特定細胞器的微環境提供了至關重要的準確信息，以了解其在細胞器的病理與生理學中的作用。
然而，由於大多數近紅外染料擁有傾向於積聚在線粒體中的陽離子電荷，這使得它們不適用於特定細胞器的細胞影像。在本論文中，我們提出了一種新型的無電荷、可切換近紅外光的部份花青螢光染料。與傳統的部份花青相比，此新染料對擁擠的環境非常敏感，並在近紅外區域內展示出顯著的吸收和放射。將能夠與蛋白質專一性結合的配體整合到螢光團，探針在與靶標蛋白結合時表現出顯著的螢光增加（高達300倍）。大差距的螢光差異能夠擁有最小的背景螢光、可免去洗滌步驟並且針對不同細胞器來標記。
除此之外，我們也開發出新型籠閉生物素探針，搭配醯胺耦合的設計，提出一種細胞膜成像的方法。所設計的籠閉生物素探針與鏈親和素蛋白的結合，可以偵測細胞表面上的過氧亞硝酸分泌，此方法可達到極小背景訊號、螢光增益，且可應用不同的螢光分子。在目標分析物存在下引發去籠閉情況，將含有多個螢光團連接的鏈親和素蛋白來進行信號放大。

Cellular organelles play a specific and indispensable role in cellular processes. Consequently, monitoring of biological species and the microenvironment within specific organelles are crucial to provide accurate information to understand their roles in the physiopathology of organelles. Because of permanent cationic charge, most near-infrared dyes tend to accumulate preferentially in the mitochondria which make them not suitable for organelle-specific imaging. In this thesis, we present a novel charge-free fluorescence switchable near-IR dye based on merocyanine. As compared to the classical merocyanine, the new dye is highly sensitive to crowded surroundings and exhibits substantial red-shifted absorption and emission within the near-IR region. By incorporating a protein specific ligand to the dye, the probe P-Mero4BG exhibit dramatic fluorescence increases (up to 300-fold) upon binding to its target protein. The large fluorescence enhancement enabled no-wash labeling and imaging of the SNAP-tag protein in different subcellular compartments with minimum background fluorescence.
In the second part of my thesis, I demonstrated a novel approach for specific imaging of plasma membrane by using caged-biotin chemical probes by regulating biotin-streptavidin binding. The caged-biotin chemical probe design takes advantage of the specific and rapid biotin-streptavidin binding. One application is to anchor caged-biotin on the cell surface to accomplish background free and signal amplified detection of the secreted peroxynitrite upon PMA stimulation.

Abstract i
摘要 iii
謝誌 iv
Publication vi
Contents vii
Table of Abbreviation xi
Chapter 1 Introduction 1
1-1 Preface 1
1-2 Biosensor 1
1-2.1 Bioreceptor 2
1-2.2 Transducer 3
1-2.2.1 Optical Fibres 4
1-2.2.2 Surface Plasmon Resonance (SPR) 4
1-2.2.3 Fluorescence Transducer 6
1-3 Mechanism of Fluorescence Enhancement 7
1-3.1 Self-Assembly/Disassembly 7
1-3.2 Aggregation-Induced Emission (AIE) 8
1-3.3 Environment-Sensitive 9
Part 1. 15
Chapter 2 Literature Review 16
2-1 Near-Infrared Fluorophore 16
2-2 Organelles-Specific Imaging 17
2-3 Protein Labeling 19
2-3.1 Fluorescence Protein Labeling 21
2-3.2 Fluorescence Probe Labeling 23
Chapter 3 Probe Design and Concept 25
Chapter 4 Results and Discussion 27
4-1 Spectroscopic Property of Mero4 and P-Mero4 27
4-2 Organelle-Specific Imaging 29
4-3 Permeability and Labeling Rates in Living Cells 32
4-4 The Specific Labeling of Pre-Mature Lamin A 34
4-5 Experimental Conclusion and Future Research 35
Part 2. 37
Chapter 5 Literature Review 38
5-1 Reactive Caged Probe 38
5-1.1 Caged-Luciferin 38
5-1.2 Caged-Fluorophore 39
5-1.3 Caged-Biotin 41
Chapter 6 Probe Design and Concept 43
6-1 Design Concept 43
6-2 The Motivation of Detection Peroxynitrite 46
6-3 Strategy for Cell Membrane Sensor 48
Chapter 7 Results and Discussion 50
7-1 Selectivity Studies and Imaging of Exogenous ONOO- 50
7-2 Secreted Peroxynitrite under PMA Stimulation 53
7-3 Initiation and Duration of Peroxynitrite Burst in RAW264.7 Cells under PMA Stimulation 56
7-4 Determine Amount of Secreted Endogeneous Peroxynitrite on Extracellular Surface 59
7-5 Experimental Conclusion 67
Chapter 8 Conclusion of the Thesis 68
Chapter 9 Experimental Section 70
9-1 Materials and Apparatus 70
9-2 Fluorescence Spectroscopic Evaluation 71
9-3 Protein Expression and Purification 71
9-3.1 Protein Expression 71
9-3.2 Purification of Protein 71
9-3.3 SDS-PAGE 72
9-4 Cell Culture and Image 74
9-4.1 Culture Medium and Reagent 74
9-4.2 Subculture of Cells 75
9-4.3 Cell Transfection and Fluorescence Imaging 76
9-5 Study of Peroxynitrite Released from SIN-1 in DMEM Medium 78
9-6 Preparation of Various ROS and RNS Solutions for Selectivity Studies 78
9-7 Flow Cytometry Analysis 79
References 80

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