Enhancement of Fluorescence in Protease Assays for Biowarfare Toxins

Recently, there have been a growing number of rapid fluorescence-based assays for detection and study of biothreat agents, particularly bacterial pathogens and toxins, and inhibitors thereof. Of the bacterial toxins, foremost are 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. Both classes of the toxins are comprised of a zinc metalloprotease which is responsible for the devastating effects of all forms of anthrax and botulism. This Phase II effort will focus on expanding upon Phase I work in which Luna, i) optimized the fluorescent substrate assays for bacterial proteases, ii) prepared and characterized fluorescence enhancing silver colloids and silver nanoparticles, and iii) began integrating the silver nanoparticles with the fluorescent assays using adsorptive and covalent chemistries. During Phase II Luna will, i) refine the synthesis and characterization of fluorescence enhancing silver and silver/gold nanoparticles, ii) further develop stabilizing and sequestering dendrimer, polymer and silica colloid “spacer” coatings, iii) expand the protease assays to include both solution-based and surface-based microassay formats. Additional fluorescence enhancing applications will be examined including FRET-based assays for the apoptosis-related caspases and HIV proteases.