Aptamer-Facilitated Biomarker Discovery of Leukemia Cells with Mass Spectrometry and Their Detection with Luminescent Nanoparticles

Abstract

Aptamers have shown a great potential due to their cheaper synthesis and easy chemical modification compared to antibodies, and have been employed in various biological assays and applications throughout the last two decades. Despite of their limitations, such as non-specific binding and low nuclease resistance, aptamers could be successfully used in the biomarker discovery and for the development of the aptamer-based imaging probes for in vitro assays. In this thesis, luminescent aptamer-conjugated nanoparticles were developed and utilized for leukemia cell detection with fluorescent microscopy. It was shown that for the bioconjugation of an aptamer with luminescent nanoparticles it is more beneficial to use carboxyl-modified nanoparticles, which results in a stable luminescence after the conjugation and the absence of unsaturated and unstable conjugates, unlike with amino-modified nanoparticles. Moreover, a cell viability assay was performed and it was revealed that aptamer-conjugated nanoparticles did not induce spontaneous apoptosis and necrosis of leukemia cells, which can be further explored with additional cytotoxicity tests, whether the aptamer-conjugated nanoparticles are biocompatible, or not. Aptamer-based biomarker discovery implies disease biomarker identification, and most commonly used methods are tedious and require a relatively high concentration of captured aptamer-target complexes. For that, AptaBiD was used in order to optimize aptamer-target identification method. Using Sgc8-aptamer, it was first shown with flow cytometry that it binds to both, healthy and malignant T lymphocytes, which requires further improvements for this aptamer to be used for leukemia detection. Among three tested detergents for the aptamer-target purification, DDM happened to be the most suitable one, due to its gentle cell lysis and solubilization properties. However, the cross-linking with formaldehyde has not positively affected the results obtained and could be replaced with photocross-linking in future experiments, which would allow to selectively cross-link an aptamer with a photomodified nucleobase with its target. Lastly, a high number of intracellular proteins identified within samples could be associated with the aptamer non-specific binding and internalization, which could be improved in future with an alternative cell fractionation with a membrane isolation approach used for the identification of transmembrane target protein.