Development of Protease Reactivity Assays for Packed Microcolumns

Proteases play critical roles in biological pathways.  A significant number of them are involved in disease. In this work we focus our attention to proteases from the botulinum neurotoxin serotypes A and F due to their high toxicity. Therefore, it is of great importance to understand their proteolytic kinetic mechanisms in order to contribute to the discovery of effective in vivo inhibitors. We describe a simple method of protease detection via fluorescent-based techniques inside polydimethylsiloxane (PDMS)-based microfluidic channels. This approach addresses simple molecular diffusion constrains inherited by solution-based assays. Streptavidin-bearing polystyrene microspheres have been utilized in order to immobilize biotinylated substrate proteins tagged by green fluorescence protein (GFP). Microfluidic system designs capable of performing multiple parallel bioassays simultaneously in order to analyze the proteases from different types of botulinum neurotoxins will be presented. We will present the study of receptor-ligand kinetics as particles are monitored in laminar flow in microchannels. Qualitative analysis via fluorescence microscopy will indicate loss of fluorescence from the substrates upon interactions with their specific proteases. Qualitative analysis will be presented via an acquisition setup that transforms fluorescence intensity into voltage signals, thus allowing one to calculate the amount of GFP that is cleaved by the protease. We have presented an inexpensive, portable, and efficient diagnostic system capable of detecting biological components linked to disease, and as an example we have demonstrated those capabilities by analyzing components present in botulism intoxication.