UC San Diego

The measurement of protease activity in blood is important for the development of novel diagnostics and for biomedical research. Presently, most protease assays require a considerable amount of sample preparation, making them time-consuming, costly and less accurate. Sample preparation also increases the complexity of these assays, precluding them from point-of-care applications. The current dissertation explores the development of a simple and rapid electrophoretic assay for measurement of protease activity directly in microliters of whole blood. This assay utilizes charge-changing fluorescent peptide substrates that produce a positively charged fluorescent product fragment upon cleavage by the target enzyme. This fragment is then rapidly separated from whole blood by electrophoresis and quantified with a fluorescent detector. Charge-changing substrates were developed for detection of [alpha]-chymotrypsin, trypsin, elastase, matrix metalloproteinase-2 and matrix metalloproteinase-9. Kinetics and specificity studies were then performed in order to make comparisons with substrates from previous assays. Electrophoretic conditions were optimized in order to achieve lower detection limits. During this optimization, various electrophoretic gel formats were explored, such as agarose and polyacrylamide slab gels. A novel technique, called polyanion focusing gel electrophoresis, was developed in order to achieve even lower detection limits. In this method, we used to polyanionic poly-L-glutamic acid-doped polyacrylamide gels to focus the fluorescent cleavage product and markedly improve the detection limits of the assay toward clinically relevant levels of protease activity. For all of this study's five target proteases, this technique was able to demonstrate detection limits that were below the normal reference levels established in the literature. Using this assay, protease activity was measured in blood, plasma, and serum samples from rats, mice, or humans that have type II diabetes, pancreatic cancer, or that are undergoing physiological shock. In these small proof-of- concept studies, the assay has been able to consistently measure both normal baseline and abnormal levels, showing that this technology may be useful for the development of novel diagnostics. This straightforward technique now allows for the rapid measurement of clinically relevant levels of protease activity in microliter volumes of whole blood, providing a useful tool for the development of novel point-of-care diagnostics