14. A NOVEL DISEASE DIAGNOSTIC USING NET CHARGE CHANGING FLUORESCENT PROTEASE SUBSTRATES

Department: Bioengineering
Faculty Advisor(s): Michael Heller

Primary Student
Name: Roy Brian Lefkowitz
Email: rlefkowi@ucsd.edu
Phone: 858-822-1276
Grad Year: 2011

Abstract
Increasing evidence suggests that physiological shock, diabetes, cardiovascular diseases, tumors, and other diseases are associated with an inflammatory cascade. This cascade is accompanied by elevated permeability of the endothelium and release of degradative enzymes that are targeted towards a variety of autologous proteins and lipids. Such evidence now provides a great opportunity to develop a variety of therapeutic interventions to ameliorate shock and treat inflammatory diseases. Unfortunately, such interventions will be highly dependent on the ability to diagnose and monitor a highly complex series of events which occurs rapidly in shock scenarios and more subtly in chronic inflammatory diseases. The goal of this proposal is to develop a novel monitoring and diagnostic system to meet this important clinical need.

We are developing a novel protease activity detection system for the monitoring of clinically relevant inflammatory responses and for disease diagnosis. The basic premise for our approach is to use electric fields to actively concentrate fluorescent labeled peptide substrates when cleaved by a particular enzyme. Cleavage of the fluorescent peptide substrates will result in a change in the net charge on the complex and the cleaved products can then be separated from the intact peptide substrate by application of a directed electrophoretic field. Subsequent detection is performed with a high sensitivity fluorescent detection device. These unique substrates will allow highly sensitive and selective detection in clinical samples of the key enzymes (chymotrypsin, trypsin, elastase, matrix metallo-proteases, lipases, amylases) associated with the inflammatory cascade.

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