| Summary: | 博士 === 國立臺灣大學 === 化學研究所 === 98 === This thesis focuses on developments of highly sensitivity and selectivity oligonucleotides-based sensor for mercury detection, monitoring the binding stoichiometries of oligonucleotides/ligand noncovalent complexes by electrospray ionization mass spectrometry (ESI-MS), developing a practical method for the determination aminothiols through surface-assisted laser desorption/ionization mass spectrometry (SALDI-MS) in conjunction with an internal standard, and using HgTe nanomaterials as matrices in the SALDI-MS analyses of peptides, protein-drug complexes, and proteins. The thesis is divided into five parts. Chapter one introduces the framework and background of nucleic acid-based sensors and basic principle and two most commonly used soft ionization techniques of mass spectrometry. In chapter two, fluorescence detection of Hg2+ in aqueous solution was demonstrated using a sensor composed of TOTO-3 and a polythymine oligonucleotide T33. The formation of folded structure of T33 induced by specific Hg2+-mediated thymine–Hg–thymine pairs between T-residues in the T33 sequence, allowing strong interaction of TOTO-3 with folded T33, leading to increased fluorescence. Under the optimum conditions, this TOTO-3/T33 sensor provided high sensitivity and specificity for Hg2+ over other metal ions in aqueous solutions with a limit of detection of 0.6 ppb. The third chapter describes the performance of ESI-MS, fluorescence and circular dichroism (CD) spectroscopies to explore the binding of ethidium bromide (EthBr) to non-self-complementary polyT strands in the absence and presence of Hg2+ ions. ESI-MS results revealed that Hg2+ ions have greater affinity, through T–Hg2+–T coordination, toward polyT strands than do other metal ions. These findings are consistent with fluorescence and CD results obtained in solution; they revealed that the T33–EthBr–Hg2+ complexes become more stable upon increasing the concentrations of Hg2+ ions. Chapter four describes an internal standard approach using d N-2-mercaptopropionylglycine (MPG) bound 4-nm-diameter gold nanoparticles (Au NPs) as an internal to improve the precision of SALDI-MS. This approach provided good quantitative linearity of the three aminothiols (R2 = ca. 0.99), with good reproducibility (relative standard deviations: <10%). The practicality of the SALDI-MS approach has been validated by the analyses of glutathione (GSH) in the lysates of human red blood cells and MCF-7 cancer breast cells in the presence and absence of the anti-inflammatory drug sulfasalazine. In the last chapter, mercury-tellurium (HgTe) nanomaterials were chosen as SALDI-MS matrices in the analyses of peptides, proteins, and protein-drug complexes. Relative to other commonly used nanomaterials like gold nanoparticles, HgTe nanostructures provide low background from metal clusters, fewer fragment ions, less interference from alkali adducted analyte ions, and higher mass range (150 000 Da). The present approach provides limits of detection (LODs) of 200 pM and 22 nM for angiotensin I and bovine serum albumin, respectively, with great reproducibility (RSD <25%). The applicability of the present method has been validated by the identification of recombinant proteins transformed in E. coli and detection of weak protein-drug complexes. My studies clearly show that DNA and nanomaterials are useful materials for developing sensitive and/or selective analytical techniques.
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