Multiplexed Protease Activity Assay for Low-Volume Clinical Samples Using Droplet-Based Microfluidics and Its Application to Endometriosis
Author(s)Chen, Chia-Hung; Sarkar, Aniruddh; Beste, Michael T.; Isaacson, Keith B.; Lauffenburger, Douglas A.; Griffith, Linda G.; Han, Jongyoon; Miller, Miles Aaron; ... Show more Show less
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As principal degrading enzymes of the extracellular matrix, metalloproteinases (MPs) contribute to various pathologies and represent a family of promising drug targets and biomarker candidates. However, multiple proteases and endogenous inhibitors interact to govern MP activity, often leading to highly context-dependent protease function that unfortunately has impeded associated clinical utility. We present a method for rapidly assessing the activity of multiple specific proteases in small volumes (<20 μL) of complex biological fluids such as clinical samples that are available only in very limited amounts. It uses a droplet-based microfluidic platform that injects the sample into thousands of picoliter-scale droplets from a barcoded droplet library (DL) containing mixtures of unique, moderately selective FRET-based protease substrates and specific inhibitors and monitors hundreds of the reactions thus initiated simultaneously by tracking these droplets. Specific protease activities in the sample are then inferred from the reaction rates using a deconvolution technique, proteolytic activity matrix analysis (PrAMA). Using a nine-member DL with three inhibitors and four FRET substrates, we applied the method to the peritoneal fluid of subjects with and without the invasive disease endometriosis. The results showed clear and physiologically relevant differences with disease, in particular, decreased MMP-2 and ADAM-9 activities.
DepartmentMassachusetts Institute of Technology. Center for Gynepathology Research; Massachusetts Institute of Technology. Department of Biological Engineering; Massachusetts Institute of Technology. Department of Electrical Engineering and Computer Science; Massachusetts Institute of Technology. Research Laboratory of Electronics
Journal of the American Chemical Society
American Chemical Society (ACS)
Chen, Chia-Hung, Miles A. Miller, Aniruddh Sarkar, Michael T. Beste, Keith B. Isaacson, Douglas A. Lauffenburger, Linda G. Griffith, and Jongyoon Han. “Multiplexed Protease Activity Assay for Low-Volume Clinical Samples Using Droplet-Based Microfluidics and Its Application to Endometriosis.” Journal of the American Chemical Society 135, no. 5 (February 6, 2013): 1645–1648.
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