SELEX Of Functional Nucleic Acids And Their Applications To The Analytical Detection

Functional nucleic acids are single-stranded oligonucleotides selected from a huge combinatorial library by a process known as "systematic evolution of ligand by exponential enrichment"(SELEX). Numerous high-affinity and highly specific oligonucleotides have been selected against a wide variety of targets including organic/inorganic molecules, large proteins and even cells. Functional nucleic acids have been proved to have advantages over antibodies with regard to simple modifiability, small size, chemical stability, high affinity, high specificity, remarkable flexibility and so on. Recently, a variety of functional nucleic acids-based analytical assays have been developed for molecular recognition and detection. However, most of these techniques suffer from the disadvantages of high costs, sophisticated instruments and sample pretreatment, therefore, the development of a simple and sensitive sensor system is still a focus of research. Inspired by this observation, we focus on the design of novel, simple, rapid, sensitive, and selective technology, based on the traditional optical methods (SERS, UV-vis and fluorescence spectroscopy). The main work of this thesis is summarized as follows:(1) SELEX of L-miR122(Chapter2). miR122is one of liver-specific miRNA, which regulates the metabolism of fatty acid and cholesterol, and is closely related to the occurrence and development of the hepatitis C virus. Herein, we took L-miR122as the model molecule, and obtained aptamer of L-miR122via SELEX. The similarity and homology of the sequences had been investigated. All these pave the way for application in clinic diagnosis.(2) Design of SERS assay (Chapter3). We had synthesized a stable, sensitive and specific surface-enhanced Raman tag, and demonstrated its application in human thrombin detection. The tag consisted of aptamer-modified core-shell nanoparticles with hydrophobic Au@Ag as core and silica as shell encapsulating Raman active molecules. The method described in this work had been proved effective, selective and reproducible, and could be applied to analytes in complex matrices. The method also showed the following benefits:firstly, avoiding the competition between the Raman dyes and the biomolecules; secondly, avoiding the background signal from the biological system; thirdly, avoiding the problems of surface adsorption, substrate variations, and poor data reproducibility.(3) Design of colorimetric assay (Chapter4). We prepared Pt-DNA complexes and demonstrated their use in catalyzing the oxidation of a peroxidase substrate3,3’,5,5’-tetramethylbenzidine (TMB) to the oxidized product which provided a colorimetric detection of H2O2and glucose. We also identified the concentration of glucose in sprite with naked eyes without referring to any sophisticated apparatus. More importantly, Pt-DNA complexes could rival natural enzymes due to their easy preparation, robustness, and stability. The detection limit of glucose was0.1μM after denaturalization of as-prepared Pt-DNA complexes.(4) Design of label-free fluorescent assay (Chapter5). Pb2+is one of the most toxic metallic pollutants, and it can cause gastrointestinal disturbance, liver damages and neurological damage at high concentrations. In this work, we developed a label-free protocol for Pb2+, combining the specific DNAzyme with polythiophene. Upon addition of Pb2+, the DNAzyme catalyzed the hydrolytic cleavage of dsDNA into two pieces, resulting in electrostatic interactions between ssDNA and PT, and leading to a planar conformation with a decreased fluorescence signal. As low as10nM Pb2+could be detected with a detection range from10nM to100μM via this method. Furthermore, this method was highly selective and only minimally perturbed by nonspecific metal ions.(5) Design of fluorescent assay (Chapter6and Chapter7). A selective and sensitive fluorescence biosensing strategy for Ag+, Hg2+, thrombin, DNA and cysteine was constructed, based on the conformational change of FAM-ssDNA and the electrostatic affinity of graphene oxide (GO). In the absence of target molecules, FAM-ssDNA adsorbs onto the surface of GO through π-π stacking interaction between the ring structure in the nucleobases and the hexagonal cells of GO, and the fluorescence of the dye is effectively quenched. Upon adding target molecules, the random coil structure changes into a hairpin-like structure (or other conformation). As a result, the interaction between FAM-ssDNA signal probe and GO becomes weaker, which significantly disrupts the energy transfer from FAM-ssDNA to GO and, thus, recovers the fluorescence emission of FAM-ssDNA. The change in fluorescent intensity could be used for detection of target molecules.