Abstract
The rapid growth of nanotechnology, especially the development of magnetic nanoparticles (MNPs) has promised a new platform in biological applications due to their high surface area-to-volume ratio and unique magnetic properties. In this thesis, the surface functionalized MNPs, including matrix-conjugated MNPs (Matrix@MNPs) and boronic acid-decorated lectin functionalized MNPs (BAD-lectin@MNPs), have been fabricated and optimized for two applications, small molecule detection and glycoprotein enrichment, respectively. The size and morphology of the functionalized nanoprobes were characterized to be about 5–30 nm by transmission electron mircroscopy (TEM). In addition, Fourier transform infrared spectroscopy (FT-IR) and superconducting quantum interference device (SQUID) are used to validate the characteristic functionality on the surface of MNPs and the superparamagnetic property. To develop the effective method for small molecule identification by matrix-assisted laser desorption/ionization mass spectrometry (MALDI MS), the hybrid of immobilized silica and 2,5-dihydrobenzoic acid (DHB) on MNP (DHB@MNP) was fabricated as a general matrix for background-free MALDI detection of low molecular weight compounds and various metal ions. The combination of Matrix@MNPs and MALDI MS was developed as a general method for direct and rapid screening for low molecular-weight compounds. Herein, we also demonstrated that Matrix@MNP can provide as a soft ionization element for analyte detection with MALDI MS analysis. Besides, the ratio between silane and MNP during the synthesis of Matrix@MNP was examined and was found to affect the surface immobilization of matrix on the nanoparticle, critically influence the ionization efficiency of analyte and the interference background in low molecular weight range of mass spectrum. Compared with commercial DHB, the Matrix@MNP-assisted MALDI MS provided superior soft ionization on the detection of new types of synthetic materials used for solar cells, light emitting devices, and glycolipids, including the analytes with either thermally labile structures or poor protonation tendencies. Moreover, the enhancements of the molecular ion signal also provided high quality product-ion spectra allowing structural characterization and unambiguous small molecule identification. Furthermore, this Matrix@MNP-assisted approach was established as a novel technique for simple and rapid analysis of metal ions. Matrix@MNPs significantly enhance the detection sensitivity of metal ions through characteristic isotope patterns and accurate mass. With the advantages stable ability and simple sample preparation, the use of DHB-functionalized nanoparticles combined with high-resolution MALDI MS provides a generic platform for rapid and unambiguous structure determination of small molecules and metal ion. Detection of aberrant glycoproteins in diseases can provide an opportunity to develop glycoprotein biomarkers for diagnosis and prognosis. However, comprehensive identification of carrier proteins and site of glycan-specific alternation still remain intangible. Lectin-based affinity probe can provide glycan-specific recognition, but is restricted by their weak noncovalent affinity with glycoprotein. The weak and variable binding affinities exhibited by lectin–carbohydrate interactions have often compromised the practical utility of lectin in capturing glycoproteins for glycoproteomic applications. We report here the development and applications of a new type of hybrid biomaterial, namely a boronic acid-decorated lectin (BAD-lectin), for efficient bifunctional glycoprotein labeling and enrichment. Our binding studies showed an enhanced affinity by BAD-lectin, likely to be mediated via the formation of boronate ester linkages between the lectin and glycan subsequent to the initial recognition process and thus preserving its glycan-specificity. Moreover, when attached to magnetic nanoparticles (BAD-lectin@MNPs), 2 to 30-fold improvement on detection sensitivity and enrichment efficiency for specific glycoproteins was observed over the use of either lectin or BA. Tested at the level of whole cell lysates for glycoproteomic applications, three different types of BAD-lectin@MNPs exhibited excellent specificities with only 6% overlapping among the 295 N-linked glycopeptides identified. As many as 236 N-linked glycopeptides (80%) were uniquely identified by one of the BAD-lectin@MNPs. These results indicated that the enhanced glycan-selective recognition and binding affinity of BAD-lectin@MNPs will facilitate a complementary identification of the under-explored glycoproteome. Thus, taken together, we demonstrated several advantages, including that iron oxide (Fe3O4) nanoparticles can serve as the general building unit with flexible surface chemistry in biological applications; the unique magnetic separation avoids tedious de-salting and purification process and reduces the handling time to prevent target biomolecules from degradation; the high sensitivity and specificity performance of functionalized MNPs deal with the low affinity interaction and low abundant biomolecule detection. In future perspective, we anticipate that MNPs can serve as a promising platform to tackle the interesting but challenging issues in various biological applications. For small molecule and metal ion detection, DHB@MNP potentially can be developed as an efficient and directive alternative tool for the fast, sensitive, and accurate determination of trace small molecule and metal ions in real samples. In glycoproteomic study, we expect that BAD-lectin@MNPs provide facile and comprehensive glycoprotein analysis for biomarker discovery in clinical specimens.