1. 金奈米粒子應用於偵測蛋白質間作用力與純化特定蛋白質 2. 螢光二氧化矽奈米粒子應用於鼠傷寒沙門氏桿菌的顯像

Abstract

摘要 本論文主要分成兩部分，第一部份是在金奈米粒子表面分別修飾醣配基、活性標示分子及進行Staudinger Ligation與1,3-Dipolar Cycloaddition所需的Phosphine與Propargyl ether。 我們所合成的一系列醣修飾金奈米粒子，不但可以用來測試醣受體蛋白與何種醣配基作用，也可以用來偵測蛋白質間的作用力。我們結合醣修飾金奈米粒子及其相對應的凝集素來偵測凝集素與其他蛋白質間之作用力。此方法具有很高的偵測極限 (5 nM)，且實驗時間短無需修飾蛋白質等優點，再者肉眼即可輕易判斷實驗結果。此外，此方法不只可用來定性分析，也可用來定量測量蛋白質間的的結合常數。由於此方法非常簡單、快速，所以適合用來做為大量篩選 (High-Throughput Screening) 蛋白質間作用力的工具，用於蛋白質體學上的分析應該有相當的濳力，甚至可用來篩選影響醣受體蛋白與細胞之間作用力的藥物。 醣修飾金奈米粒子也可用於純化醣受體蛋白。藉由表面電漿帶是否有紅位移的現象，來判斷細胞萃取液中是否含有可與醣修飾金奈米粒子作用的醣受體蛋白，若表面電漿帶有紅位移的現象，可進一步利用離心的方式純化醣受體蛋白，並藉由SDS-PAGE及MALDI-TOF來鑑定醣受體蛋白。與傳統的方式比較，我們可縮減偵測及分離所需的時間，也可藉由結合MALDI-TOF，提高偵測醣受體蛋白的靈敏度，其偵測極限約為0.05-0.10

Abstract This thesis is divided into two parts. The first part used substrate-capped gold nanoparticles to detect protein-protein interactions and to purify specific protein. We designed and synthesized a series of sugar-capped gold nanoparticles (Sugar-GNPs) used in competition colorimetric assay for studying protein-protein interactions, particularly lectins. The competition assay uses the ensemble of lectin and Sugar-GNPs, which display blue color due to multivalent interactions between lectin and Sugar-GNPs, to identify the binding partners for lectin. Protein-Protein interaction can be evaluated qualitatively as well as quantitatively by using this competition assay. By using this methodology, a broad range of proteins can be rapidly evaluated for their abilities to interact with the protein of interest in real time, rendering high throughput screening possible. Sugar-GNPs can be used not only in detection of protein-protein interaction but also in purification of carbohydrate-binding protein. If the surface plasmon resonance band is red-shifted, it indicates that the binding occurs between Sugar-GNPs and the protein in the solution. By centrifuging the colloid, the complex can be isolated and purified. The identity of the isolated carbohydrate-binding protein can be further confirmed by SDS-PAGE and MALDI-TOF-MS. The feasibility of Sugar-GNPs for the simultaneous enrichment and isolation of ConA from protein mixture is demonstrated. Furthermore, very few quantity of agglutinin can be purified from a small amount of dry weight of algae and the biological activity still remains after recovering the protein from the complex. In addition, activity-based probe was designed and synthesized to form a covalent bond between the specific protein and substrate-capped gold nanoparticles through “click” reaction. Our experimental results indicate that labeled enzymes are easily separated from the unlabeled ones by centrifuge. SDS-PAGE and MALDI-TOF-MS are applied to analyze the purified samples. In second part, two methods were employed to synthesize fluorescent silica nanoparticles, which were then modified by mannose derivatives on the surface, namely Man-F-SNP and Man-R-SNP. Both types of particles were used to stain Salmonella typhimurium for bacteria imaging and the results showed that photostability of Man-R-SNP is better than Man-F-SNP.