Enhancement of Fluorescence in Protease Assays for Biowarfare Toxin

In this complex world of ever-increasing chemical and biological threats, rapid and simple assays for detecting the release of hazardous compounds or microorganisms are of major interest. Because of the sensitivity and availability of novel fluorogenic formats, fluorescence assays have come of age. Bacterial toxins expected to be involved in the pathogenicity of biowarfare agents include the lethal toxin (LT) of Bacillus anthracis and the neurotoxins (BoNT) of Clostridium botulinum which also have medical applications that warrant sensitive assays for inhibitor studies. The anthrax LT actually consists of two components, the protective antigen (PA) and the lethal factor (LF), a zinc metalloprotease. The LF is responsible for the devastating effects of all forms of anthrax. This Phase I effort will focus on, i) optimizing the fluorescent substrate assays currently being developed at Luna and elsewhere, ii) preparing and characterizing fluorescence enhancing silver colloids and silver complexes, and iii) integrating the silver nanoparticles with the fluorescent substrates by adsorption and covalent coupling for fluorescent signal enhancement. Upon successful completion of the proof-of-complex feasibility studies, Phase I Option and Phase II work will include further studies and collaborations in which the enhancement of fluorescent assays for BoNT and other toxin proteases will be examined.