THE DEVELOPMENT OF APTAMER 5’UTR-GOLD NANOPARTICLE BASED DIAGNOSTIC TEST FOR COVID-19 DETECTION

• Main Author: Syafira, Dinda
• Format: Theses
• Language: Indonesia
• Online Access: https://digilib.itb.ac.id/gdl/view/71438
• Institution: Institut Teknologi Bandung

A fast and accurate diagnostic method for detecting COVID-19 is needed to monitor and control the spread of the virus. Aptamer-gold nanoparticle (AuNP) based detection has gained much attention in the diagnostic field due to its simplicity, sensitive and cost effectiveness. This research aims to develop a COVID-19 detection method using computationally designed RNA aptamers from the conserved regions of the SARS-CoV-2 genome. The research method was conducted using an in silico approach that includes aptamer design by performing multiple sequence alignment, prediction of aptamer secondary structure, and molecular docking to predict the interaction between the aptamer and the RNA target. Then, the aptamer design was synthesized, and the AuNP material was obtained from Sigma Aldrich. Moreover, tests were conducted to evaluate the detection potency of the biosensor components, including characterization of AuNP quality, aggregation testing, and immobilization testing of the aptamer. The optimization of detection conditions was carried out, including optimization of NaCl concentration (0-7 M), aptamer concentration (0-2 ?M), sample incubation time (0-75 minutes), and AuNP size (5 nm and 15 nm) (n=3). The detection limit was determined based on a standard curve created through testing of positive control samples with a concentration range of 0-20 ?M (n=3). The method was validated through testing of 47 positive and 19 negative COVID-19 samples that have been confirmed by qPCR. The results of the research indicate that the 5’UTR of SARS-CoV-2 can be used in aptamer design. The aptamer has a secondary structure that meets reference standards with a ?G value of -14.83 kcal/mol, GC content of 41%, and 19 unpaired bases. The results of molecular docking indicate that the aptamer has the potential to bind to the RNA target with a binding energy value of -74.1 kcal/mol and RMSD of 1.8 Å. Based on the results of the detection potential test, the AuNP and aptamer have good quality and have potential to be used in detection. The testing reached optimal conditions with the use of a 5M NaCl concentration, 1 ?M aptamer concentration, 60 minutes sample incubation time, and 5 nm AuNP size. The developed method has a detection limit of RNA target concentration of 0,819 ?M. Of the total 66 samples tested, 60 samples showed results that were consistent with the gold standard. Based on the obtained results, it can be concluded that the aptamer-AuNP-based diagnostic method developed can be used in COVID-19 detection.