Multiplexed Protease Activity Assay for Low-Volume Clinical Samples Using Droplet-Based Microfluidics and Its Application to Endometriosis
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Griffith_Multiplexed protease.pdf
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Author(s)
Chen, Chia-Hung
Sarkar, Aniruddh
Beste, Michael T.
Isaacson, Keith B.
Lauffenburger, Douglas A.
Griffith, Linda G.
Han, Jongyoon
Miller, Miles Aaron
Date Issued
November 2012
Journal
Journal of the American Chemical Society
Publisher
American Chemical Society (ACS)
Citation
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.
Version
Author's final manuscript
Abstract
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.
MIT Department
Massachusetts 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
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DOI of Published Version
https://doi.org/10.1021/ja307866z
