University of California
San Diego

Departments of Nanoengineering
and Bioengineering

Roy Lefkowitz

Current Research

Degradative enzymes (DE) (proteases, lipases, amylases, nucleases, matrix metalloproteases (MMPs)) have a significant role in many major diseases and medical conditions. In physiological shock, pancreatic enzymes escape into the bloodstream from the lumen of the intestine and are involved in the progression of shock into multi organ dysfunction syndrome (MODS), and ultimately death. Further evidence has shown that there are elevated levels of pancreatic enzymes in the blood in acute/chronic pancreatitis, pancreatic cancer, and irritable bowel syndrome as well as elevated levels of MMPs in hypertension, acute coronary syndrome, diabetes, and several types of cancer. Thus, the measurement of DE activity in whole blood is useful for the elucidation of disease mechanisms, for biomarker and drug target discovery, and, ultimately, for the development of novel drugs and early stage diagnostic tests. This is especially important for physiological shock, which has a high hospital mortality rate (52%) and no early diagnostic test.

Earlier methods for detecting DE activity, such as fluorogenic/chromogenic substrates, FRET-based substrates, fluorescent polarization assays, and zymography, require considerable sample preparation, which is time-consuming and can significantly alter the sample and reduces accuracy. This limits the usefulness of these assays for disease diagnostics, particularly for fast-progressing diseases and conditions such as physiological shock. Therefore, our long-term goal is to eliminate the need for sample preparation by performing rapid and sensitive detection of DE activity directly in whole blood.

Toward this end, we have developed charge inverting fluorescent substrates (CIFS) that produce, upon cleavage by a target DE, a uniquely charged fluorescent cleavage product that can be rapidly resolved from blood and uncleaved substrate by electrophoresis. Using CIFS, we have demonstrated detection limits of 10 nM and 20 nM for the pancreatic proteases α-chymotrypsin and trypsin, respectively, in 1X phosphate buffered saline (PBS) and in human plasma for a 1.5 hour assay time utilizing 2-3 μl of sample. In whole rat blood, these detection limits were 20 nM and 50 nM, respectively.

We are also developing a technique, microelectrophoretic focusing (MEF), that improves the detection sensitivity by concentrating CIFS cleavage products using high electric field strength electrophoresis in high-density gels. This technique can use as little as nL of sample and concentrate CIFS removed from blood into micron-wide bands in seconds. Since similar techniques have achieved 10- to 10,000-fold sample concentration, incorporation of MEF into the CIFS detection method will result in dramatic improvement in detection time and sensitivity.