Molecular tools for the rapid and cost-effective detection of small molecules

Identifier:  TDX:3262

Handle: https://hdl.handle.net/20.500.11797/TDX3262

Authors:  Shkembi, Xhensila

Abstract:
This thesis was focused on the identification of novel aptamers binding to small molecules and their application to the development of highly sensitive bioanalytical assays and biosensors. The target molecules were the marine toxin Tetrodotoxin and the anabolic steroid Nandrolone. Small molecules are biologically active and monitoring their levels in different types of samples is of high interest in the biomedical field, environmental monitoring, food safety and forensic analysis. The first objective was the selection of aptamers required for the development of the assays and biosensors. Aptamers are biorecognition molecules alternative to antibodies. They are artificial synthetic nucleic acids (RNA/DNA) that bind specifically to their target, and they are selected through an in vitro iterative process called Systematic Evolution of Ligands by Exponential enrichment (SELEX). Two different types of selection methodologies were employed for the identification of aptamers in combination with Next Generation Sequencing. Different binding assays were also developed to enable the characterization of the binding properties of the selected aptamer candidates and the chosen aptamers were finally applied in different assays for target detection. A hybrid antibody-aptamer assay was developed for Tetrodotoxin detection in contaminated puffer fish extracts in a microtiter plate format as well as in the portable lateral flow dipstick format. For Nandrolone, a label-free colorimetric assay was developed using gold nanoparticles (AuNPs) with a red-to-blue color change to indicate the presence of nandrolone.