Functional nanomaterials and aptamers for the analysis of biological and environmental samples
Date Issued
2009
Date
2009
Author(s)
Liu, Chi-Wei
Abstract
This thesis describes highly selective and sensitive optical sensors for enzyme, mercury(II), lead(II), and single nucleotide polymorphisms (SNPs) using functional nanomaterials, including proteins modified quantum dots (QDs) and DNA modified gold nanoparticles (Au NPs), and aptamers. The thesis is divided in six chapters. In the first chapter, the framework and background of sensors were discussed. In chapter two, protein-conjugated QDs were used for detecting trypsin (Try) and trypsin inhibitor (TI) through fluorescence resonance energy transfer (FRET). Green-fluorescent CdTe QDs served as the energy donors and rhodamine isothiocyanate (RITC) conjugated to bovine serum albumin (BSA-RITC) was the acceptor. By simply mixing the two fluorophores, FRET occurred when BSA-RITC bound to the CdTe QDs. When Try was used to digest BSA, the FRET efficiency decreased and the fluorescence intensity ratio (IF574/IF520) decreased, allowing the detection of Try and TI. The LOD for TI was down to 250 pM. The third chapter describes control of the surface DNA density on Au NPs for selective and sensitive detection of mercury(II). When Hg2+ ions interacted with the thymine (T) units of the DNA molecules bound to the Au NPs through Au-S bonds, the conformations of these DNA derivatives changed from linear to hairpin structures, causing the release of some of the DNA molecules from the surface of the Au NPs into the bulk solution to react with OliGreen (high density, > 60 DNA/Au NP). The fluorescence of OliGreen-DNA complexes increased with increasing concentration of Hg2+, and Hg2+ could be detected at concentrations over the range 0.05-2.5 μM (R2= 0.98). In chapter four, the detection of mercury(II) was created by Hg2+-DNA complexes inducing the aggregation of Au NPs, and the sensor was detectable by naked eye as a result of the shift of surface plasmon resonance (SPR) for the aggregated Au NPs. The probe for sensing Hg2+ using the formation of DNA-Hg2+ complexes through T-Hg2+-T coordination to control the negative charge density of the DNA strands—thereby varying their structures—adsorbed onto Au NPs. Therefore, the ability of protection of them in the presence and absence of Hg2+ was different and we can easily distinguish between them in the high concentration of salt. Chapter five describes the fluorescence detection of SNPs using a thymine-based molecule beacon (MB). A T7-MB-T7, which contains a 19-mer loop and a stem comprising a pair of seven T bases, forms double-stranded structures with target DNA molecules, leading to increases in the fluorescence of ethidium bromide (EthBr) as a result of intercalation. The interactions of the beacon with perfectly matched (DNApm) and single-base mismatched (DNAmm) DNA strands were stronger and weaker, respectively, than those with Hg2+ ions. As a result, the fluorescence of a solution containing DNApm was higher than that of a corresponding solution containing DNAmm, because the former had a greater number of intercalation sites for EthBr. In the last chapter, the sensor of two metal ions was created using the thrombin-binding aptamer (TBA), a random coil structure that changed into a G-quartet structure and a hairpin-like structure upon binding Pb2+ and Hg2+ ions, respectively. As a result, the fluorescence decreased through FRET between the fluorophore and quencher labeled on the termini of TBA. These changes in fluorescence intensity allowed the selective detection of Pb2+ and Hg2+ ions at concentrations as low as 300 pM and 5.0 nM using this TBA probe in the presence of phytic acid and a random DNA/NaCN mixture, respectively.
Subjects
quantum dots (QDs)
gold nanoparticles (Au NPs)
aptamer
enzyme
mercury
lead
single nucleotide polymorphisms (SNPs)
Type
thesis
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