12. DIRECT DETECTION OF DEGRADATIVE ENZYME ACTIVITY IN WHOLE BLOOD: ELIMINATING SAMPLE PREPARATION
Department: Bioengineering
Faculty Advisor(s):
Michael Heller
Primary Student
Name: Roy Brian Lefkowitz
Email: rlefkowi@ucsd.edu
Phone: 858-822-1276
Grad Year: 2010
Abstract
Degradative enzymes (DE) (proteases, lipases, amylases, nucleases, matrix metalloproteases (MMPs)) have a significant role in many major diseases and medical conditions. In the early stages of 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/screening, 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. Unfortunately, current 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 (reducing measurement 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 specific DE, a uniquely charged fluorescent cleavage product that can rapidly be removed from whole blood by electrophoresis and detected. 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. To further improve the detection sensitivity and reduce the assay time, we are using high voltage gradient, high gel density microelectrophoresis to concentrate CIFS cleavage products into micron-scale bands within seconds. Current results have demonstrated a 20x improvement in signal concentration. Our goal is to optimize this technique to bring the detection sensitivity to at least 1 nM for an assay time of several minutes.
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