Year: 2019
Type: article
Abstract: 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.RBP4 and four Retinol binding aptamers (RBA's) were selected for this study.The aptamers shared conserved motifs that might play an ancillary role in binding.A small stem loop in the nucleuotide sequence -TAGTAAGTGCAA-at 62-73 nts was present for all predicted structures.Furthermore, RBA-1 and RBA-3 shared a conserved motif of -GTTG-at base 44-47, whereas, RBA-1 and RBA-2 shared a motif of -AGGGG-at base 26-32.Kinetic studies http://etd.uwc.ac.za/ iii revealed that all selected aptamers showed binding to RBP4, with dissociation constants in the nanomolar (nM) and picomolar range and that RBA-2 and RBA-1 bound to RBP4 at different sites.The AuNPs had a hydrodynamic size of 14 nm, a polydispersity index of 0.216 and a zeta potential of -30.31 mV.RBA-2 was chosen as a capture probe hence it was conjugated to AuNPs.Apt-AuNPs showed efficient conjugation at 50 µM.The development of the lateral flow test strip was unsuccessful and this could be attributed to desorption of the protein on the nitrocellulose membrane or insufficient protein concentration used.However, a colorimetric aptasensor for the detection of RBP4 was successful with a detection limit of 45.32 nM.This study demonstrated the feasibility of developing an aptamer based LFA for the detection of novel RBP4.Therefore, further optimizations on different types of membranes, buffers, pH and target concentrations are needed in order to obtain better resolutions and intensity on the test strips.Moreover, the study demonstrated the successful development of a simple yet effective colorimetric assay for RBP4 detection using one aptamer and AuNPs.
Author: Koena Leah Moabelo
Language: English
Subfield: Molecular Biology
Domain: Life Sciences
Open Access status: green
OpenAlex ID: W3010173556