Fluorescent detection of COVID-19 via Aptamer-initiated EXPAR.

Abstract

The rapid and accurate detection of severe acute respiratory syndrome-related coronavirus (SARS-CoV-2) remains a critical aspect of managing infectious disease outbreaks. This thesis introduces a novel diagnostic platform that integrates trimeric aptamers and exponential amplification reaction (EXPAR) with real-time fluorescent detection to enable the detection of SARS-CoV-2. Building on previous advances in nucleic acid biosensors, trimeric aptamers were designed to leverage multivalent interactions, notably enhancing their binding affinity and specificity for the SARS-CoV-2 Omicron BA.5 spike protein. These aptamers were integrated with EXPAR, an isothermal amplification technique, to achieve rapid signal amplification while minimizing non-specific interactions. The novel trimeric aptamers demonstrated high binding affinity and specificity. They exhibited up to a 17.8-fold improvement in affinity compared to their monomeric counterparts. The EXPAR system employed DNA templates and primers to achieve efficient amplification, with a detection threshold of 1 pM of initial primer. Following integration of the trimeric aptamer and EXPAR, the new approach detected SARS-CoV-2 with a detection threshold of 1×10^6 viral particles/ml. Fluorescence monitoring enabled real-time analysis, providing a quantitative and scalable diagnostic readout. Despite promising results, the diagnostic sensitivity fell short of clinical requirements for low viral load detection. Challenges included background amplification and the production of shorter-than-expected DNA amplicons, likely caused by enzymatic star activity. However, the compatibility of trimeric aptamers with EXPAR highlights a promising avenue for future diagnostic innovations. Optimization strategies, including refined buffer conditions and enhanced sequence design, are proposed to improve diagnostic performance. This research underscores the potential of combining trimeric aptamers with EXPAR for nucleic acid-based diagnostics, offering a platform that bridges high specificity, rapid amplification, and adaptability for emerging pathogens. These innovations hold significant promise for the early detection and containment of future pandemics.