Doctor of Philosophy (PhD)
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Aptamer, biosensor, SELEX, functional nucleic acids, small molecule
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Aptamers have recently gained considerable attention for small-molecule detection in diverse applications such as drug identification, medical diagnostics, and environmental monitoring. However, the performance of aptamer-based sensors has been greatly limited by the low target affinity and responsiveness of small-molecule binding aptamers. This dissertation describes several novel aptamer engineering and isolation strategies to remedy this problem. Specifically, we first develop a generally applicable strategy to engineer split aptamers containing two binding domains termed cooperative-binding split aptamers (CBSAs). CBSAs exhibit higher target binding affinity and are far more responsive in terms of target-induced split aptamer assembly compared to single-domain parent split aptamers from which they are derived. Using a cocaine-binding CBSA, we achieve specific fluorescence detection of as low as 50 nM cocaine in 10% saliva within 15 minutes. We then develop a general approach for creating rapid and sensitive CBSA-based enzyme-assisted target recycling (EATR)-amplified small-molecule sensors for sensitive target detection. Using this strategy, we develop a fluorescence assay for dehydroisoandrosterone-3-sulfate which achieves 100-fold enhanced target sensitivity relative to a non-EATR-based assay, and a colorimetric assay for visual detection of low-micromolar concentrations of cocaine. To simplify the sensor development process, , we establish a novel and simple SELEX strategy for directly isolating aptamers with intrinsic dye-displacement functionality which transduce target-binding events into a change of dye absorbance. As a demonstration, we isolate an aptamer against the synthetic cathinone 3,4-methylenedioxypyrovalerone (MDPV) that can detect MDPV at concentrations as low as 300 nM in a label-free, rapid, and simple dye-displacement assay. To further control the target-binding spectra of aptamers, we employ a novel parallel-and-serial SELEX strategy to isolate an aptamer binding 12 synthetic cathinones with nanomolar affinity but not 11 non-target compounds that are closely related in structure. Using this aptamer, instantaneous visual detection of synthetic cathinones at nanomolar concentrations in biological samples is achieved. In summary, this work demonstrates the great potential of novel aptamer engineering and isolation strategies in generating functional signal-reporting aptamers for sensitive small molecule detection. Importantly, the strategies described here are generalizable and can be used to develop aptamer-based assays for virtually any small-molecule targets.
Yu, Haixiang, "Functionalized Aptamers for Detection of Small-Molecule Targets" (2019). FIU Electronic Theses and Dissertations. 4249.
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