應用功能性金奈米簇為細菌之親和探針

Abstract

螢光金奈米簇由於具高光穩度性且合成步驟簡單等優點，因此已廣泛應用為細菌、細胞或特定分子的螢光奈米探針。本論文即以開發細菌螢光探針為研究目標，合成的方法則以快速簡便的微波輔助加熱合成方式進行製作具功能性的金奈米簇探針。本論文第一部份利用具硫醇基之甘露醣做為還原劑，合成出具放光波長在~650 nm的紅色螢光金奈米簇，且表面上還保有甘露醣官能基，粒徑直徑大小約為~2 nm，量子產率為~0.4%，可應用於感測具甘露醣受體之大腸桿菌。感測的方法為讓表面具甘露醣的金奈米簇和目標細菌辨識後，利用低轉速離心的方法，將表面被紅色螢光金奈米簇標定的細菌沉降於離心管底部，即可於紫外光燈照射下利用肉眼觀察目標細菌存在與否，感測極限可達到~106 cells/mL左右。本論文第二部份為利用人類血清蛋白為前驅物，合成表面具有人類血清蛋白包覆的紅色螢光金奈米簇為篩選細菌之探針，此金奈米簇之最大放光波長在~655 nm左右，而量子產率為~3.50 %。而實驗中以此金奈米簇當作細菌感測探針時，成功篩選出金黃色葡萄球菌和抗甲氧苯青黴素金黃色葡萄球菌可和此蛋白質金奈米簇有相當的親和辨識能力。論文中也確認在金奈米簇表面的人類血清蛋白和此兩株菌辨識的胜肽片段，並進一步直接以此胜肽片段合成金奈米簇，實驗結果也證實此胜肽合成之金奈米簇具有和金黃色葡萄球菌及抗甲氧苯青黴素金黃色葡萄球菌辨識的能力。本論文所製作的兩種功能性金奈米簇均適用於辨識在如處於尿液及蘋果汁等複雜基質的目標細菌，肉眼感測極限均可低至約~106 cells/mL左右。

Fluorescent gold nanoclusters (NCs) are nanomaterials with the features of high light stability and ease of synthesis. They have been widely used as fluorescent nanoproobes for bacteria, cells or specific target molecules. The objective of this thesis is to develop bacterial fluorescent probes via a quick and easy microwave-assisted heating synthesis method for the production of functional gold NCs. The first part of this thesis discussed the use of a mannose derivative with a thiol group as the reducing agent, for the synthesis fluorescent gold NCs with red-emission (λem= ~650 nm; λex=~ 375 nm) that retain mannose groups on the surface (AuNCs@Mann). The diameter of the NCs was about ~ 2 nm and the quantum yield was 0.4%. The AuNCs@Mann can be applied as a sensor for Escherichia coli with mannose receptor. When the AuNCs@Mann recognizing their target bacteria, the target bacteria-AuNCs@Mann conjugates with red emission can be readily spun down at the bottom of the centrifuge tube at low-speed centrifugation. The presence or absence of target bacteria can be observed if red fluorescence conjugates appear at the bottom of the centrifuge tube under illumination of UV light (λmax= 365 nm) with the naked eye. The detection limit of this approach is in the level of ~106 cells/mL. The second part of the thesis explored the feasibility of employing human serum albumin (HSA) encapsulated gold NCs (AuNCs@HSA), which was generated by reacting HSA with tetrachloroacuric under microwave-heating, as bacterial sensing-probes. The generated gold NCs have the maximum emission at ~655 nm (λex= 368 nm) with a quantum yield of ~ 3.50%. In this study, the AuNCs@HSA was used to successfully screen S. aureus and methicillin-resistant Staphylococcus aureus (MRSA) as their target bacteria. This study also identified two HSA peptide fragments, which were the binding sites when using AuNCs@HSA to interact with S. aureus and MRSA. These two identified peptides were further used as the reagents to generate peptide direct synthesis of gold NCs. The experimental results confirmed that the peptide synthesis of gold NCs enables identification of S. aureus and MRSA. The feasibility of using these two newly generated gold NCs, namely AuNCs@Mann and AuNCs@HSA, were successfully used to detect target bacteria from complex samples such as urine and apple juice with the naked eye. The detection limit was as low as ~106 cells/mL.