Development of Surface Based Platforms for Biomolecular Recognition and Protease Assays

The discovery of analytical tools for biomolecular recognition in the area of diagnostics is a research trend rich in innovative methods. Some of the main drivers found in this area are the constant need for low cost devices and biosensors, simplicity in design and operation, and time efficiency. In this thesis we present novel approaches for the development of protease activity assays. Our focus of study is the protease assay development process on two types of characterized platforms: microspheres and selfassembled monolayers (SAMs) on flat surfaces. The surface preparation and conjugation process with biological components of interest is presented along with surface plasmon resonance (SPR) and flow cytometry results. These platforms were engineered in order to develop assays for bacterial toxin activity. Bacterial toxins are comprised in part of proteases, which selectively cleave peptides bonds in proteins. In this thesis we present, protease assays from our main focus of studies, the Clostridium botulinum Neurotoxin type A Light Chain (BoNTA/LC) metalloprotease. Supplementary studies include assays performed involving the Bacillus anthracis Lethal factor metalloprotease. In the work described we have prepared active