- 學位論文
功能化磁性奈米探針應用於目標分子純化與偵測
Functionalized Magnetic Nanoparticle as Affinity Probe for Target Molecule Purification and Detection
摘要
磁性奈米粒子為目前廣泛的運用於生物學研究熱門材料之一，因其高粒子總表面積對體積比的優勢，可以在粒子表面產生較高密度的親和官能基修飾，而能提升分子間的交互作用力。此外利用磁性易分離的優勢，將氧化鐵磁性奈米粒子進行表面修飾，並結合生物分子特有的專一辨識性，此功能化奈米粒子可以有效的應用在生物分離方面。在本論文中，將發展表面功能化修飾的氧化鐵奈米粒子，並運用於目標分子的純化與偵測。 利用磁性奈米粒子表面覆蓋聚乙二醇，並同時修飾良好的金屬離子螯和試劑nitrilotriacetic acid為親和性探針，經由調控反應最佳化的條件，提供磷酸蛋白質萃取與純化另一種更快速且互補於現今的方法。除了對於低濃度的胜肽樣品具有良好的純化效率之外，在較複雜的生物細胞的純化，亦能同時針對單磷酸化胜肽與多磷酸化胜肽進行萃取純化。實驗結果證實我們所發展的磁性奈米探針不僅能和現今常用的方法互補，亦能提供較高比例的多磷酸化純化效率。 另外，亦採用氟化物進行奈米粒子表面覆蓋，以及利用不同的表面修飾方法如CuAAC和疏水性長碳鏈鑲嵌而將nitrilotriacetic acid固化於表面形成親和探針，並探討其在磷酸化胜肽純化時的不同效率。最後更將亦能做為良好的金屬離子螯合試劑的di-pyridine化合物，固化於磁性奈米粒子表面，藉由與不同的金屬的親和作用力，表面功能化修飾的氧化鐵奈米粒子能同時達到磷酸化胜肽的純化，並幫助微量金屬的偵測提升其偵測效率。
並列摘要
Magnetic nanoparticle (MNP) has emerged as a promising new material in biological applications. Due to the high surface area to volume ratio, the high surface ligand density of MNP provides significant enhancement of interaction affinity. Furthermore, the unique magnetic property of MNP facilitates the rapid separation by simple magnetic separation without the need of centrifugation. In this thesis, diverse functionalized MNP have been developed and applied on phosphopeptide enrichment and metal ion detection. We developed a surface-blocked nanoprobe-based immobilized metal ion affinity chromatography (NB-IMAC) method for enhanced purification specificity and enrichment of multiply phosphorylated peptides. Titanium (IV) ion charged nitrilotriacetic acid-conjugated MNP (Ti4+NTA@MNP) showed unbiased extraction of phosphorylated peptides from diluted β-casein (2×10-10 M). By blocking MNP surface with low molecular weight polyethylene glycol and controlling pH and concentration of acetic acid of loading buffers, the developed NB-IMAC allowed rapid and specific one-step enrichment. Compared to the magnetic micro-sized particle (MMP, 2-10 m), the NB-IMAC identified more phosphopeptides as well as higher percentage of multiply phosphorylated peptides (31%) on proteome scale. Furthermore, NB-IMAC complements chromatography-based IMAC and TiO2 method. The MNP surface was also functionalized with fluorous compounds (by amide bond formation), trivalent-NTA (by CuAAC reaction) and lipid-NTA (by hydrophobic interaction), respectively. Furthermore, di-pyridine compound, a good metal chelator, was also immobilized on the MNP surface. The phosphopeptide enrichment of the resulting MNPs was investigated. All of them, the NTA-C18@MNP (by hydrophobic interaction) has shown the best efficiency of low abundant phosphopeptide enrichment (2×10-10 M, α- and β-casein). Besides, the tri-NTA@MNP (by CuAAC reaction) and NTA-C18@MNP provide the similar potential for phosphopeptide enrichment form more complex mixture (α- and β-casein mixed with BSA, the molar ratio is 1:1:50). As a good metal chelator, the NTA@MNP and di-Py@MNP, immobized with different metal ions, would show their potential in phosphopeptide enrichment with complementary result. Besides, both of them would improve the metal ion detection efficiency through the strong interaction between the chelator with metal ions. Owing to the high sensitivity and specificity of MNPs, functionalized MNPs provide a rapid, effective, and specific platform for target molecule, such as phosphopeptides and trace metal ions purification. In this dissertation, through appropriate surface modification, MNPs present excellent performance in various target molecule extractions and detection.