Executive Summary : | The increasing number of novel or profound virulent pathogens has led to the need for fast, efficient, and sensitive detection systems. This proposal proposes novel methods to develop fluorescence-based probes to monitor specific pathogenic targets or events associated with disease progression. The goal is to develop point-of-care diagnostic devices that are time-effective and require minimal sample quantities. The model system used is dengue. The first method involves identifying and designing fluorescent probes for efficient sensing of pathogens or monitoring disease progression using a fluorescence turn-off mechanism. Unique surface targets on pathogens are identified using bioinformatics tools, and high affinity aptamer probes are generated through phage-display screening. These probes are tagged with FRET pairs, and upon sensing specific surface targets, a conformational modification is induced to block FRET. The second method uses a fluorescent turn-ON/OFF mechanism to monitor the activity of enzymes that serve as marker elements while sensing disease progression. The target is dengue causing virus (DENV), which exhibits RNA dependent RNA polymerase activity and plays a major role in viral replication. Monitoring these events will aid in diagnosing DENV infection and screen therapeutic targets. This method does not require the use of radiolabeled 32P, unlike current diagnostic methods. |