Nomadics will adapt amplifying fluorescent polymer (AFP) technology to nucleic acid detection. AFPs are a class of polymers that amplify optical signatures resulting from FRET or fluorescence quenching events. Combining this inherent signal amplification with the specificity exhibited by nucleic acid recognition events will enable a new generation of nucleic acid sensors that do not rely on target amplification or catalyzed reporter deposition. This AFP-based technology should make a significant contribution to the adaptation of nucleic acid diagnostics towards the demands of field operations in reducing size, weight, and power consumption. This will make the technology suitable for use in countering bioterrorism. The research will focus on nanoparticle AFP detection systems, as the particle format offers a number of advantages in the adaptation of AFP technology to PCR-less molecular analyses-principally, the ability to harness 3-dimensional signal amplification in the solid particle while retaining an optically clear fluid matrix. Particle systems can also minimize background and increase sensitivity through single-particle analyses. Further, variations in the AFP composition allow us to tune the emission color of the bead; this, in turn, will enable multiplex analyses to screen for multiple agents. In Phase I, Nomadics will demonstrate APP nanoparticle technology for the detection of nucleic acids. This will require fabrication and characterization of AFP nanoparticles and the development of efficient coupling chemistries for the attachment of nucleic acid probes. This will enable hybridization studies to quantify the sensitivity and selectivity of APP nanoparticles. Experience in the handling and performance of AFP systems will enable design and fabrication of laboratory prototype systems. Studies will focus on anthrax. Synthesis and testing of a molecular beacon for a specific anthrax targets will only be performed in Phase II. Phase I will include selection of an appropriate anthrax target based on a complete literature search and design of a molecular beacon in silico. In order to test the concept using APP nanoparticles in Phase I, a probe that has already been designed, synthesized, and tested by Nomadics for another target will be used. The goal of Phase I will be to demonstrate highly specific nucleic acid detection in the low (<10) femtomole range.
