指導教授：張煥宗臺灣大學：化學研究所李季霖Li, Chi-LinChi-LinLi2014-11-252018-07-102014-11-252018-07-102014http://ntur.lib.ntu.edu.tw//handle/246246/261341本論文主要著重於功能性核苷酸適合體、金與碳奈米材料之製備，將其應用於重金屬分析、細胞影像與癌症治療。第一章主要簡介核酸奈米感測器和螢光碳奈米點之背景，及其在生物和環境分析上之應用。在第二章中，主要介紹一個螢光DNA感測器於重金屬鉛離子偵測，結合Amplex UltraRed (AUR)試劑和寡核苷酸AGRO100/鐵血紅素(hemin)可定量溶液中鉛離子的含量。鉛離子能誘導AGRO100/heminr具酵素(DNAzyme)的催化活性，在H2O2媒介作用下可與AUR反應，最後得到Rusorufin的螢光產物。於最佳化條件下，相較其他金屬離子，AGRO100/hemin-AUR感測器對於鉛有極佳之靈敏度與專一性，偵測極限為0.4 nM。第三章主要描述寡核苷酸(T30695)鍵結金奈米粒子(Au NPs)並結合AUR試劑(T30695–Au NPs/AUR)來偵測鉛離子。Au NPs表面之核苷酸(T30695)密度本身扮演很重要的角色，不僅能調控金–鉛合金(Au-Pb alloys)以及核苷酸–鉛複合體 (T30695–Pb2+ complexes)之形成，亦會影響T30695–Au NPs的催化活性。在Au NPs表面T30695密度最佳化條件下(~40 T30695 per Au NP)，對於鉛離子的偵測有極高的靈敏度(偵測極限為0.05 nM)與專一性。第四章主要是描述利用新鮮的嫩薑汁經由水熱法(hydrothermal process)來合成具有螢光的碳奈米點(C-dots)材料，這是一個相較於傳統技術更為便利且環保的方法。所合成的碳奈米點不僅具有良好的光子激發放光特性(量子產率約13.4%)，對於人類肝癌細胞株(HepG2)亦提供極高的選擇性與抑制效果；另外，對於正常細胞有較少的生物毒性。螢光C-dots在HepG2細胞中產生大量的活性氧分子（ROS增加18.2倍），誘導p53蛋白的表現。從表面輔助雷射脫附游離飛行時間質譜儀結果得知：螢光碳奈米點表面存有高度抗癌活性的薑黃素(curcumin)，這可能有助於促凋亡蛋白的表現，進而使HepG2細胞產生凋亡。首次，我們成功將C-dots材料應用於裸鼠中被HepG2細胞所誘導的腫瘤，有效地抑制腫瘤的生長（約為96.4%）。第五章主要是利用半胱氨酸(cysteine)以水熱法製備螢光碳奈米點用來偵測維他命B12及環境水中的鈷離子。鈷離子與溶液中或碳奈米點表面所殘留之半胱氨酸與硫分子反應後即可形成大顆粒碳奈米點/硫化鈷(C-dots/CoxSy)之聚集，導致碳奈米點螢光有明顯的猝滅作用，表明其間發生了電荷轉移。於最佳化條件下，螢光碳奈米點對於鈷離子有極佳之靈敏度(偵測極限約為5 nM)與線性(10 nM至100 μM, R2 = 0.992)。This thesis focuses on preparation and application of functional nanomaterials (e.g., apamer, gold, and carbon) for bioassay (e.g., heavy metal ions and bioimaging) in addition to cancer therapy. Chapter one introduces the framework and background of DNA-based nano-sensors and photoluminescent carbon nanodots (C-dots) in biomedical and environmental applications. In chapter two, fluorescence detection of Pb2+ in aqueous solution was demonstrated using a sensor composed of Amplex UltraRed (AUR) and a G-quadruplex oligonucleotide AGRO100. The sensing strategy is based on Pb2+ ions inducing increased DNAzyme activity of AGRO100 in the presence of hemin, which acts as a cofactor to catalyze H2O2-mediated oxidation of AUR. Under optimized conditions, this AGRO100-AUR sensor provided high sensitivity and specificity for Pb2+ over other metal ions in aqueous solutions with a limit of detection of 0.4 nM. The third chapter describes a simple assay employing a G-quadruplex oligonucleotide T30695 modified gold nanoparticles, and AUR (T30695–Au NPs/AUR) for the detection of Pb2+ ions. The surface density of T30695 units on Au NP surface played an important role in controlling the formation of the Au-Pb alloys and T30695–Pb2+ complexes and, therefore, the catalytic activity of the T30695–Au NPs. Under optimized conditions, the 40T30695–Au NP/AUR probe was highly sensitive (LOD = 0.05 nM) and selective toward Pb2+ ions. Chapter four describes a facile and green method to synthesize fluorescent C-dots from fresh tender ginger juice via a hydrothermal process. The as-prepared C-dots not only exhibited favorable photoluminescent (PL) properties (quantum yield~13.4%), but also provided extremely high selectivity and suppression efficiency on the growth of human hepatocellular carcinoma cells (HepG2), with low toxicity to normal cells. The C-dots generated greater amounts of reactive oxygen species (ROS, 18.2-fold increased) in the HepG2 cells, resulting in enhanced expression of p53 protein. Surface-assisted laser desorption/ionization time-of-flight mass spectrometry (SALDI-TOF MS) results revealed the existence of curcumin molecules (a highly active anti-cancer agent) on the surface of C-dots, which likely assisted in triggering the pro-apoptotic factor to promote HepG2 cell apoptosis. For the first time, C-dots have been used for significantly reducing the weight of HepG2 cells induced tumor in nude mice by about 96.4%. The final chapter describes a label-free probe based on C-dots that were prepared from cysteine through a hydrothermal process for real-time monitoring of Co2+ ions in vitamin B12 and nature water samples. The proposed strategy utilizes Co2+ ions to react with cysteine/residual sulfur-related molecules in solution and/or on the surfaces of C-dots to form cobalt sulfide (CoxSy) nanoparticles that further undergo aggregation to form large granular C-dots/CoxSy nanomaterials, leading to the PL quenching via a charge transfer-dependent dynamic quenching process. Under optimum conditions, this C-dots based PL assay allows detection of Co2+ ions with great sensitivity (down to 5 nM) and linearity (10 nM to 100 μM, R2 = 0.992).中文摘要 I 關鍵詞 II Abstract III Keywords IV Contents V Table Contents IX Figure Contents IX Conclusions and Prospect 162 Publications 164 Chapter 1 Introduction 1 1.1 Nanomaterials 2 1.2 Aptamer 2 1.3 Biosensors 3 1.3.1 Colorimetry 3 1.3.2 Fluorescence 5 1.4 DNA functional Au NPs (DNA–Au NPs) 6 1.4.1 Preparation and optical properties of DNA functional Au NPs 6 1.4.1.1 Preparation 7 1.4.1.2 Optical properties 8 1.4.2 Environmental application of DNA–Au NPs 8 1.4.2.1 Mercury assay 8 1.4.2.2 Lead assay 10 1.4.2.3 Copper assay 11 1.4.2.4 Silver assay 11 1.4.3 Biomedical application of DNA–Au NPs 12 1.4.3.1 Proteins assay 12 1.4.3.2 Single nucleotide polymorphisms (SNPs) assay 13 1.4.3.3 Other biomedical applications 13 1.5 Photoluminescent carbon nanodots 14 1.5.1 Synthetic Methods 14 1.5.2 Optical properties 15 1.5.2.1 Absorbance 15 1.5.2.2 PL 15 1.5.3 Application 16 1.5.3.1 Bioimaging 16 1.5.3.2 Inhibition of cancer cells 17 1.5.3.3 Sensor 17 1.5.3.4 Optoelectronic 18 1.6 Motive of Research 18 1.7 References 19 Chapter 2 Fluorescence Detection of Lead(II) Ions Through Their Induced Catalytic Activity of DNAzymes 42 2.1 Abstract 43 2.2 Introduction 43 2.3 Experimental Section 45 2.3.1 Chemicals and Oligonucleotides 45 2.3.2 Hemin/G-Quadruplex Complexes Modulated by Pb2+ Ions 45 2.3.3 Sample Pretreatment 46 2.3.4 Circular Dichroism 46 2.4 Results and Discussion 47 2.4.1 Sensing Strategy 47 2.4.2 Catalytic Activities of Eight Aptamers 48 2.4.3 Effect of Buffer Concentration on Activity and Selectivity of AGRO100-AUR 49 2.4.4 Sensitivity and Selectivity of AGRO100-AUR Probe Toward Pb2+ Ions 50 2.4.5 Detection of Pb2+ Ions in Soil 51 2.5 Conclusions 51 2.6 References 52 Chapter 3 Peroxidase mimicking DNA–gold nanoparticles for fluorescence detection of the lead ions in blood 65 3.1 Abstract 66 3.2 Introduction 67 3.3 Experimental Section 69 3.3.1 Chemicals 69 3.3.2 Synthesis of Au NPs 69 3.3.3 Preparation of DNA-Au NPs 70 3.3.4 Preparation of 40T30695–Au NPs 70 3.3.5 T30695–Au NP/AUR probe for Pb2+ ions 71 3.3.6 Blood sample pretreatment 72 3.4 Results and Discussion 73 3.4.1 Sensing strategy 73 3.4.2 Characterization of Au-Pb alloys and Pb2+–oligonucleotide on Au NPs 74 3.4.3 Effects of the surface oligonucleotide 75 3.4.4 Effect of pH 78 3.4.5 Sensitivity and selectivity of 40T30695–Au NP/AUR probe toward Pb2+ ions 79 3.4.6 Detection of Pb2+ ions in blood 80 3.5 Conclusions 81 3.6 References 81 Chapter 4 Carbon dots prepared from ginger exhibiting efficient inhibition of human hepatocellular carcinoma cells 101 4.1 Abstract 102 4.2 Introduction 102 4.3 Experimental Section 104 4.3.1 Chemicals 104 4.3.2 Preparation of Ginger Juice 105 4.3.3 Synthesis of C-dots 105 4.3.4 Characterization 106 4.3.5 Cell Culture 107 4.3.6 Cell Imaging 108 4.3.7 Cytotoxicity Assays 108 4.3.8 Imaging Cytometry and Intracellular Reactive Oxygen Species (ROS) Assay 109 4.3.9 Western Blot Assay 109 4.3.10 In Vivo Tumorigenicity Tests 110 4.4 Results and Discussion 110 4.4.1 Characterization and Properties of C-dots 110 4.4.2 Cell Imaging and Cytotoxicity of C-dots 112 4.4.3 Surface Components of C-Dots 115 4.4.4 In Vivo Tumor Growth Inhibition 116 4.5 Conclusions 116 4.6 References 117 Chapter 5 Synthesis of photoluminescent carbon dots for the detection of cobalt ions 135 5.1 Abstract 136 5.2 Introduction 136 5.3 Experimental Section 137 5.3.1 Chemicals 138 5.3.2 Synthesis of C-dots 138 5.3.3 Characterization of C-dots 139 5.3.4 Detection of Co2+ Ions 140 5.3.5 Analysis of Co2+ Ions in Real Samples 140 5.4 Results and Discussion 141 5.4.1 Characterization of C-dots 141 5.4.2 Sensing of Co2+ 142 5.4.3 Sensitivity and Selectivity 144 5.4.4 Detection of Co2+ ions in water and vitamin B12 samples 145 5.5 Conclusions 146 5.6 References 1468060448 bytesapplication/pdf論文公開時間：2015/08/01論文使用權限：同意有償授權(權利金給回饋學校)奈米粒子螢光鉛離子碳奈米點薑鈷離子[SDGs]SDG3thesishttp://ntur.lib.ntu.edu.tw/bitstream/246246/261341/1/ntu-103-D96423002-1.pdf