Towards Point-Of-Care Diagnostics: Eliminating Sample Preparation in the Detection of Degradative Enzyme Activity in Whole Blood

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Towards Point-Of-Care Diagnostics: Eliminating Sample Preparation in the Detection of Degradative Enzyme Activity in Whole Blood
Roy Lefkowitz1, Jennifer Y. Marciniak1, Che-Ming Hu1, Geert W. Schmid-Sch├Ânbein1,2, and Michael J. Heller1,3
From 1UCSD, Department of Bioengineering, 2UCSD, Department of Medicine, & 3UCSD, Department of Nanoengineering

Degradative enzymes (e.g. proteases) are elevated in the blood in several types of cancer (including pancreatic), physiological shock, cardiovascular disease, and in many other diseases. Therefore, the measurement of their activity in whole blood is important for the development of novel diagnostics. Unfortunately, current techniques for measuring this activity require considerable sample preparation, which makes these assays more time-consuming and costly and lowers their accuracy. In general, sample preparation also makes these assays more complex, which precludes them from point-of-care (POC) applications because complex tests must be performed in CLIA-approved laboratories. Therefore, in order to develop viable, cost-effective POC tests that measure degradative enzyme activity, it is important to avoid sample preparation and measure this activity directly in unprocessed whole blood.

Toward these goals, we have developed a simple electrophoretic device that allows rapid detection of degradative enzyme activity directly in whole blood using charge-changing fluorescent peptide substrates. Using only microliters of sample in a quick 1-hour reaction, these negatively charged substrates produce positively charged fluorescent cleavage products upon cleavage by the target enzyme. After several minutes of simple electrophoresis, the cleavage products are readily separated from the components of whole blood (blood cells, proteins, etc.), which are predominantly negatively charged, and then quantified with a fluorescent detector.

Thus far, we have developed a library of charge-changing substrates for the specific detection of activity of ╬▒-chymotrypsin, trypsin, elastase, and matrix metalloproteinases-2 and -9. We have demonstrated detection of these enzymes in 1X PBS, human plasma, and in whole rat blood. We have also developed various agarose and polyacrylamide gel-based detection formats that allow different speeds and sensitivities for detection with these substrates, with detection as low as several picograms in microliters of sample, which is approaching clinically relevant levels of these enzymes. This simple assay overcomes a major limitation in developing viable, cost effective POC diagnostics because it can measure disease biomarkers directly in clinical samples, without sample preparation.