Nanoscience and Microsystems ETDs


Jingsh Zhu

Publication Date



In this dissertation, we have designed a series of recombinant fluorescent full-length protease substrates containing the cleavage sites separated by a biotinylation tag and a GFP domain, which can be associated with streptavidin-coated microspheres/nanospheres. We have developed microsphere-based platforms to detect the cleavage activities of several proteases simultaneously. In addition, we have performed high-throughput screening (HTS) to identify potential inhibitors for the Bacillus anthracis lethal factor protease (LF) and the Botulinum neurotoxin type A & F light chain proteases (BoNT/A & F LC). In order to select the efficient inhibitors, a chemical library of 350,000 compounds was screened with the HyperCyt® high-throughput flow cytometry system, which can screen thousands of compounds per day for drug discovery. In order to understand the kinetic mechanisms of these proteases as well as the inhibitory mechanisms of some inhibitor candidates, we have carried out surface-based analysis to characterize the enzymatic reaction in terms of the key kinetic parameters of BoNT/A LC (Km, kcat etc.) for surface-bound substrates by flow cytometry. In my work, we have also created a FRET full-length substrate solution assay to determine the kinetic constants of the proteases and inhibitory mechanisms of some known inhibitors. However, despite the advantages of the microsphere-based assay, the surface area to volume ratio of microspheres limits us to sub-saturating concentrations of substrate (in the low nM range). Therefore, we developed a nanosphere-based protease assay platform to increase substrate concentrations. To demonstrate this approach, we have implemented our nanosphere-based protease assay using the toxin protease models to prove the concept. The surface-based system is a promising method for the use of multiple protease assays to evaluate the kinetic mechanisms and to select inhibitors for several different proteases simultaneously. This provides the advantages of robustness, sensitivity, low substrate cost and reproducibility.


Protease, Microsphere, Multiplex, High-throughput screening, Drug Discovery, Inhibitors, Kinetic mechanism, Nanosphere


Center for Biomedical Engineering, Center for Molecular Discovery, Cancer Nanotechnology Training Center

Document Type




Degree Name

Nanoscience and Microsystems

Level of Degree


Department Name

Nanoscience and Microsystems

First Advisor

Graves, Steven

Second Advisor

Edwards, Bruce

First Committee Member (Chair)

Sklar, Larry

Second Committee Member

Shreve, Andrew

Third Committee Member

Whitten, David

Fourth Committee Member

Graves, Steven

Fifth Committee Member

Edwards, Bruce