本論文是製備出兩種化學感測分子並探討其性質與應用，內容可分為兩個主題，一是研究奈米碳粒子，可用來偵測鐵離子的存在，二是合成出有機小分子化合物，可用來偵測酪胺酸酶的存在，兩者皆可應用在細胞顯影實驗上。
第一個題目是N-doped carbon dots 可於水溶液中偵測鐵離子存在，將檸檬酸和離胺酸混合以高壓水熱法製備而成。鑑定其性質，得知合成出平均粒徑為4 nm之碳粒子，並在330 nm激發波長激發下，在420 nm會有藍色的螢光放光。選擇性實驗中，N-doped carbon dots可專一地偵測鐵三價離子，其偵測極限為127 nM，並以非常快速的時間( 20 sec ) 即可反應完全。而在pH酸鹼測試中，N-doped carbon dots不會因為環境酸鹼而影響其特性。在毒性測試中，細胞可維持80% 存活率， N-doped carbon dots可以成功應用在HeLa偵測鐵離子。
第二個題目為酪氨酸酶螢光化學感測分子F-TYR，以fluorescein為起始物，修飾上3-hydroxybenzyl作為辨識端，經由氧化羫基為酮基的機制，偵測酪胺酸酶的存在。感測器F-TYR沒有螢光放光，但當F-TYR加入酪胺酸脢的溶液中，用肉眼可明顯看到螢光，並以儀器測得，會有的綠色螢光放光(525 nm)。反應動力學數據為Vmax = 3.33μM/min，Km = 0.018μM，Kcat = 0.00398 min-1，偵測極限為0.0024 U/mL。在毒性測試中，細胞可維持90% 之存活率，故F-TYR可成功應用在B16f10細胞偵測酪氨酸酶活性。

In this thesis, there are two topics: N-doped carbon dots and fluorescent chemosensor F-TYR.
The first topic is the synthesis of N-doped carbon dots for Fe3+ detection. N-doped carbon dots were synthesized from critic acid and lysine by hydrothermal method. The average size of N-doped carbon dots was 4 nm and it has blue emission at 420 nm. In selectivity experiment, it shows specific detection toward Fe3+ with a detection limit 127 nM. The reaction time is less than 20 sec and it would not be affected in different pH environment. Besides, N-doped carbon dots show low cellular toxicity and can be used to detect Fe3+ in cells.
The second topic is the synthesis of fluorescent chemosensor F-TYR for tyrosinase detection. F-TYR consists of fluorescein and 3-hydroxybenzyl group which is used as an identification group for tyrosinase, F-TYR has no emission. In the presence of tyrosinase, tyrosinase oxidizes the phenol group into dopa and results in green emission at 525 nm. The reaction rate of F-TYR was calculated as Vmax = 3.33μM/min，Km = 0.018μM，Kcat = 0.00398 min-1. The detection limit is 0.0024 U/mL and the viability of cell is higher than 90 %. In addition, cell imaging experiments demonstrated that F-TYR is an effective fluorescent probe for tyrosinase detection in living cells.

摘要 I
Abstract II
Abstract Scheme III
謝誌 IV
Table of Contents V
List of Figures VII
List of Schemes XI
Chapter 1 Introduction 1
1.1 Fluorescent chemosensors 1
1.1.1 Chemosensor and Chemodosimeter 1
1.2 Carbon dots 2
1.3 Tyrosinase 4
1.4 Literature survey 6
1.4.1 Carbon dots for sensing metal ions 6
1.4.2 Chemodosimeter for Tyrosinase 11
1.5 Motivation 15
1.5.1 N-doped carbon dots for Fe3+ 15
1.5.2 Chemodosimeter F-TYR for Tyrosinase 15
Chapter 2 Results and discussion of N-doped CDs for Fe3+ 16
2.1 Synthesis of the probe N-doped CDs 16
2.2 Characterization of the N-doped CDs 16
2.3 Selectivity experiment of N-doped CDs for Fe3+ 19
2.4 Titration experiment of N-doped CDs for Fe3+ 22
2.5 Kinetic experiment of N-doped CDs for Fe3+ 24
2.6 pH experiment of N-doped CDs for Fe3+ 25
2.7 Quantum yield experiment of N-doped CDs for Fe3+ 26
2.8 The fluorescence quenching mechanism of N-doped CDs with Fe3+ 28
2.9 Cell imaging experiment of N-doped CDs with Fe3+ 30
2.10 Conclusion 32
Chapter 3 Results and discussion of F-TYR for Tyrosinase 33
3.1 Synthesis of the probe F-TYR 33
3.2 Selectivity experiment of F-TYR for Tyrosinase 34
3.3 Titration experiment of F-TYR with Tyrosinase 36
3.4 Detection limit experiment of F-TYR for Tyrosinase 37
3.5 Kinetic experiment of F-TYR for Tyrosinase 38
3.6 pH experiment of F-TYR for Tyrosinase 42
3.7 Quantum yield experiment of F-TYR for Tyrosinase 42
3.8 Proposed mechanism of r F-TYR for Tyrosinase 44
3.9 Cell imaging experiment of Chemodosimeter F-TYR for Tyrosinase 46
3.10 Conclusion 51
Chapter 4 Experimental 52
4.1 Materials 52
4.2 Instruments 52
4.3 Synthesis of N-doped CDs 53
4.4 Synthesis of the probe F-TYR 53
4.5 Method 56
4.5.1 Teflon-lined stainless-steel autoclave 56
4.5.2 Selectivity experiment 56
4.5.3 Titration experiment 56
4.5.4 Detection limit experiment 56
4.5.5 Kinetic experiment 56
4.5.6 pH experiment 57
4.5.7 Quantum yield experiment 57
4.5.8 Cytotoxicity assay 57
4.5.9 Cell experiment 57
Reference 59
Supplementary Information 61

 

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