Abstract
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.