Development of an aptamer based lateral flow test strip for a diabetes biomarker
Moabelo, Koena Leah
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Diabetes mellitus is one of the major chronic diseases that poses a significant risk to human health globally. Current diagnostics for diabetes are based on blood glucose measurements, which can go unnoticed for years. As such, there is an urgent need for diagnosis of diabetes at the point-of-care (PoC), and in low-income settings. Recent developments in the field of nanotechnology, and the use of aptamers show great promise in developing sensitive and disease-specific assays. Aptamers have high specificity, selectivity, low molecular weight, nontoxicity, non-immunogenicity, and are easy to produce. The aim of the study was to characterize and identify dual aptamers for the development of an aptamer-based PoC diagnostic kit for diabetes. Retinol binding protein 4 (RBP4) and its binding aptamers were identified through in silico approach. The secondary structures of the selected retinol binding aptamers (RBA) were predicted by M-fold and Quadruplex forming G-Rich Sequences mapper. Binding affinity of aptamers to the biomarker was characterized by Surface Plasmon Resonance(SPR), Electrophoretic mobility shift assay (EMSA) and Enzyme-Linked Aptamer Assay (ELAA) and the aptamer pairs were evaluated by sandwich-based SPR and ELAA. Gold nanoparticles (AuNPs) were synthesized using the Turkevich method, and characterized by UV-visible (UV-vis) spectrophotometry, Zetasizer and Transmission Electron Microscopy. One aptamer was conjugated to AuNPs and the conjugate was characterized by UV-Vis, Zetasizer, Qubit assay and gel electrophoresis. The apt-AuNPs were used in the preparation of the lateral flow test strips and a colorimetric aptasensor for the detection of RBP4.