Development of Aptamer-Based Biolayer Interferometry Biosensors for the Detection of Cancer and Viral Biomarkers

Abstract

This thesis used Biolayer Interferometry (BLI) and DNA aptamers to develop simple and reliable biosensors for detecting important biological targets, including proteins, cancer-derived exosomes, and viral-like particles (VLPs), ranked from simple to complex biomolecules. Aptamers are short single-stranded DNA molecules that can be considered as a bio recognition element that are able to specifically recognize proteins or particles, similar to antibodies but with better stability and easier preparation. This study sought to develop a rapid, sensitive, and label-free technology for quantitative detection by integrating aptamers with the BLI method, which enables real-time monitoring of molecular interactions without the need for labels. In the first project, an aptamer designed for the Ki-67 protein, a known marker of cell proliferation, was studied using a BLI platform. The results showed a strong and preferable specific interaction between the aptamer and Ki-67 protein, with a dissociation constant (KD) of 10.9 ± 0.6 nM and a clear linear relationship across the working range of 50-400 nM. This confirmed that the BLI-based aptasensor can provide accurate and reproducible data for protein detection. The second project focused on the detection of cancer-derived exosomes from MDA-MB-231 cells using three known aptamers: CD63, MUC1, and 5TR1. Exosomes were isolated through a gold standard isolation method which is ultracentrifugation and characterized by Dynamic Light Scattering (DLS) and Transmission Electron Microscopy (TEM), which confirmed their typical size and structure. The binding tests showed that all three aptamers interacted strongly with exosomes, with KD values in the picomolar range and good linearity in the calibration plots. These results demonstrated the high sensitivity and specificity of the aptamer-based BLI system for exosome analysis. In the third project, newly selected aptamers were tested for recognizing SARS-CoV-2 pseudoviruses (VLPs). Among them, the S1-SP10 aptamer showed the best performance with a KD of 112 ± 5 nM and a limit of detection (LOD) of 33 nM. The assay showed clear dose-dependent signals and high specificity compared to the scrambled control sequence, confirming that the aptamer could effectively bind the viral spike protein in its native form.

Overall, this research shows that BLI combined with aptamers is a powerful and flexible approach for studying molecular interactions and detecting disease-related biomarkers. The developed aptasensors worked well for different biological systems, proteins, exosomes, and viral particles, proving their potential for future use in medical diagnostics and real-sample testing.