{"paper_id":"c5160e2e-0a7b-4181-8073-594d818d9e03","body_text":"Multiplexed Protease Activity Assay for Low-Volume Clinical Samples Using Droplet-Based Microfluidics and Its Application to Endometriosis\nName\nGriffith_Multiplexed protease.pdf\nSize\n1.78 MB\nFormat\nAdobe PDF\nChecksum (MD5)\n4b1366d1dfac3988d1f5fbf01f66370b\nAuthor(s) • • • • • • •\nChen, Chia-Hung\nSarkar, Aniruddh\nBeste, Michael T.\nIsaacson, Keith B.\nLauffenburger, Douglas A.\nGriffith, Linda G.\nHan, Jongyoon\nMiller, Miles Aaron\nDate Issued\nNovember 2012\nJournal\nJournal of the American Chemical Society\nPublisher\nAmerican Chemical Society (ACS)\nCitation\nChen, 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.\nVersion\nAuthor's final manuscript\nAbstract\nAs 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.\nMIT Department\nMassachusetts Institute of Technology. Center for Gynepathology Research\nMassachusetts Institute of Technology. Department of Biological Engineering\nMassachusetts Institute of Technology. Department of Electrical Engineering and Computer Science\nMassachusetts Institute of Technology. Research Laboratory of Electronics\nTerms of Use\nArticle is made available in accordance with the publisher's policy and may be subject to US copyright law. Please refer to the publisher's site for terms of use.\nPersistent DSpace Link\nDOI of Published Version\nhttps://doi.org/10.1021/ja307866z","source_license":"CC0","license_restricted":false}