Chemical and Biological Engineering ETDs

Jose Cornejo

Publication Date

7-11-2013

Abstract

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 self-assembled 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 recombinant protease substrates in our laboratory, capable of binding with the surfaces of study. In addition, we present methods to on how to address non-specific binding issues inherited by the nature of these assays. Commercially available recombinant proteases have been utilized throughout these studies. These studies present novel methods for BoNT protease detection based on previous microsphere-based assays. Our biomimetic detection platforms show promise for further understanding BoNTs toxicity, the biological pathways of BoNTs substrates, and possible contributions to the discovery of protease inhibitors.

Keywords

Surface Plasmon Resonance, Supported Lipid Bilayers, Biosensing, Diagnostics, Protease Assays, Botulinum Neurotoxins, Flow Cytometry

Document Type

Thesis

Language

English

Degree Name

Chemical Engineering

Level of Degree

Masters

Department Name

Chemical and Biological Engineering

First Committee Member (Chair)

Freyer, James

Second Committee Member

Shreve, Andrew

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