螢光奈米貴金屬團簇之合成與應用

Synthesis and Applications of Fluorescent Noble Metal Nanoclusters

陳威宇(Wei-Yu Chen)

指導教授 ： 張煥宗

國立臺灣大學/理學院/化學研究所/博士(2014年)

https://doi.org/10.6342/NTU.2014.02651

摘要

螢光貴金屬奈米團簇具獨特光學和催化的特性，如尺寸量化的放光、順磁性及高光穩定性等，故已被廣泛應用於生物感測與生物顯影上。本論文主要著重於水相螢光貴金屬(金和銀)奈米團簇製備、鑑定及應用。首先以特定序列單股去氧核醣核酸(DNA)當模板，以硼氫化鈉(NaBH4)還原銀和金離子成DNA保護的金/銀奈米團簇 (Au/Ag nanoclusters)，此團簇具高螢光亮度與光穩定性。利用電噴灑質譜儀與感應耦合電漿質譜儀鑑定得知此奈米團簇由兩金原子與一個銀原子所組成。相對於銀奈米團簇，金/銀奈米團簇在高離子強度的環境中有較好的穩定性。藉由硫離子(S2−)造成團簇放光淬息(quenching)現象，此金/銀奈米團簇感測器對硫離子偵測極限可達0.83 nM，對其它陰離子選擇性高達55倍以上。我們也進一步結合微脂體(Liposome)與螢光11-巰基十一酸–金微奈米粒子(11-MUA‒Au NDs)製備出11-巰基十一酸–金微奈米粒子/微脂體複合體。此奈米複合體可應用於偵測磷脂酶C (phospholipase C)。磷脂酶C水解微脂體後，產生的疏水二醯基甘油(diacylglycerol)，可與長碳鏈之11-巰基十一酸作用，降低11-MUA‒Au NDs的螢光放光淬息現象。此複合體探針對磷脂酶C偵測極限為0.21 nM，對其他蛋白質和酵素選擇性大於95倍以上。此探針亦應用在乳癌細胞(MCF-7 與 MDA-MB-231細胞株)以及正常細胞(MCF-10A細胞株)中之磷脂酶C活性的分析。為提升金微奈米粒子放光效率與穩定度，我們以不同帶電性之兩親配體分子(如癸酸、十二烷酸、十四烷酸、溴化十二烷基三甲銨、溴化十四烷基三甲銨和溴化十六烷基三甲銨等)與11-巰基十一醇共同修飾於金微奈米粒子。其中以溴化十六烷基三甲銨/11-巰基十一醇–金量子點展現優異的量子產率高達40%。十二烷酸/11-巰基十一醇–金量子點量子產率僅有2.57%，而優異的分散性以及高螢光亮度之特性，可利用此探針作為偵測亞硝酸離子(NO2−)之分析，此探針對亞硝酸離子偵測極限為40 nM，對其他陰離子選擇性高達70倍以上，並應用在自然水體(湖水與海水)中之亞硝酸離子的分析。透過自組裝抗菌表面活性肽(surfactin)與十二烷基硫醇‒金微奈米粒子(DT‒Au NDs)，製備出具有抗菌表面活性肽/十二烷基硫醇‒金微奈米粒子。此抗菌表面活性肽本身已具有抗細菌、抗病毒、抗真菌、抗黴菌以及溶血之作用與性質。而相對於表面活性肽，此金微奈米粒子有著更優異抑制金黃色葡萄球菌(Staphylococcus aureus)、枯草桿菌(Bacillus subtilis)、大腸桿菌(Escherichia coli)、變形桿菌(Proteus vulgaris)與多重抗藥性金黃色葡萄球菌(Methicillin-resistant S. aureus)。我們也探討十二烷基硫醇與抗菌表面活性肽之合成比例，有效提升金微奈米粒子之水溶性、螢光強度以及抗菌效果。金微奈米粒子亦進行哺乳類動物細胞與溶血實驗測試，顯現低毒性與無顯著性的溶血的結果。

關鍵字

奈米團簇 ； 微奈米粒子 ； 微脂體 ； 硫離子 ； 亞硝酸離子 ； 磷脂酶C ； 兩親配體 ； 表面活性肽 ； 抗菌胜肽 ； 多重抗藥性細菌

並列摘要

Fluorescent noble metal nanoclusters (NCs) and nanodots (NDs) are interesting materials and widely employed in the biosensing and bioimaging, mainly because of their unique optical and catalytic properties, including strong fluorescence, size-dependent emission wavelengths, magnetism, and high photostability. This thesis focuses on the preparation, characterization, and application of water-soluble fluorescent noble metallic nanoclusters/nanodots (NCs/NDs). First, we have employed cytosine-rich oligonucleotides to prepare strongly fluorescent and highly photostable DNA-templated gold/silver nanoclusters (DNA–Au/Ag NCs) through the NaBH4-mediated reduction method. Electrospray ionization-mass spectrometry (ESI-MS) and inductively coupled plasma mass spectrometry (ICP-MS) were employed to characterize the DNA–Au/Ag NCs, revealing that each DNA–Au/Ag NCs contain two Au atoms and one Ag atom. Based on that fact that sulfide (S2−) ion-induced fluorescence quenching of DNA–Au/Ag NCs, we further developed a fluorescence turn-off assay for the high sensitive detection of S2− ions at concentrations as low as 0.83 nM. For preparation the functional Au NDs, we combined the biocompatible liposomes (Lip) and fluorescent 11-mercaptoundecanoic acid–gold nanodots (11-MUA–Au NDs) to prepare the 11-MUA–Au ND/Lip hybrids by incorporation of gold nanoparticles (∼3 nm) and 11-MUA molecules in hydrophobic phospholipid membranes that self-assemble to form small unilamellar vesicles. A simple and homogeneous fluorescence assay for phospholipase C (PLC) was developed on the basis of the fluorescence quenching of 11-MUA–Au ND/Lip hybrids in aqueous solution. The fluorescence of the 11-MUA–Au ND/Lip hybrids is quenched by oxygen (O2) molecules in solution, and quenching is reduced in the presence of PLC. PLC catalyzes the hydrolysis of phosphatidylcholine units from Lip to yield diacylglycerol (DAG) and phosphocholine (PC) products, leading to the decomposition of Lip. The diacylglycerol further interacts with 11-MUA–Au NDs via hydrophobic interactions, leading to inhibition of O2 quenching. The 11-MUA–Au ND/Lip probe provides a limit of detection of 0.21 nM for PLC, with high selectivity over other proteins, enzymes, and phospholipases. For preparation of self-assembly Au NDs, hybridized ligands were used to etching and stabilization of gold nanoparticles (~3 nm). These NDs were employed to detect nitrite based on analyte-induced photoluminescence (PL) quenching. 11-Mercaptoundecanol (11-MU) and its complexes with amphiphilic ligands (ALs) etch Au nanoparticles through hydrophobic interactions and form a densely packed ligand shell on the surface of each core in the as-formed Au NDs. We tested such ALs as three fatty acids and three quaternary ammonium surfactants with alkyl chain lengths of 10–16 carbons. The results show that chain length, ligand density, and functional group (charge) of ALs play important roles in determining the optical properties of Au NDs. Tetradecanoic acid (TA)/11-MU–Au NDs are highly dispersible in aqueous solution and allow detection of nitrite down to 40 nM with selectivities (>100-fold) greater than that for common ions present in natural (lake and sea) water samples. We further prepared antimicrobial Au NDs which surfaces were co-immobilized with antibacterial peptide (surfactin; SFT) and 1-dodecanethiol (DT). SFT, a cyclic lipopeptide, has been credited with antibacterial, antiviral, antifungal, anti-mycoplasma and hemolytic activities. The hybrid SFT/DT-capped Au NDs (SFT/DT-Au NDs) were prepared through the self-assembly of antimicrobial peptides (SFT) on DT-anchored Au NDs by the nonspecific hydrophobic interactions between the alkyl chains of the SFT and the DT molecules. Relative to SFT and DT-Au NDs, SFT/DT-Au NDs possessed superior antimicrobial activity toward non-multi-drug resistant (non-MDR) Escherichia coli (E. coli), Proteus vulgaris (P. vulgaris), Proteus vulgaris (P. vulgaris), Salmonella enterica serovar Enteritidis (S. enteritidis), and Staphylococcus aureus (S. aureus) bacteria as well as the multi-drug resistant (MDR) bacteria, methicillin-resistant S. aureus (MRSA). We demonstrated the water solubility, PL as well as antibacterial activity of Au NDs were highly dependent on the ligand ratio of SFT/DT on Au NDs. In vitro haemolysis and cytotoxicity analyses of SFT/DT-Au NDs have revealed their insignificant haemolysis in red blood cells (RBCs) and low toxicity in selected cell lines.

並列關鍵字

nanoclusters ； nanodots ； liposomes ； sulfide ions ； phospholipase C ； amphiphilic ligands ； nitrite ions ； surfactin ； antibacterial peptides ； multidrug-resistant bacteria

參考文獻

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