Multiplexed Protease Activity\nAssay for Low-Volume\nClinical Samples Using Droplet-Based Microfluidics and Its Application\nto Endometriosis

As principal degrading enzymes of the extracellular matrix,\nmetalloproteinases\n(MPs) contribute to various pathologies and represent a family of\npromising drug targets and biomarker candidates. However, multiple\nproteases and endogenous inhibitors interact to govern MP activity,\noften leading to highly context-dependent protease function that unfortunately\nhas impeded associated clinical utility. We present a method for rapidly\nassessing the activity of multiple specific proteases in small volumes\n(<20 μL) of complex biological fluids such as clinical samples\nthat are available only in very limited amounts. It uses a droplet-based\nmicrofluidic platform that injects the sample into thousands of picoliter-scale\ndroplets from a barcoded droplet library (DL) containing mixtures\nof unique, moderately selective FRET-based protease substrates and\nspecific inhibitors and monitors hundreds of the reactions thus initiated\nsimultaneously by tracking these droplets. Specific protease activities\nin the sample are then inferred from the reaction rates using a deconvolution\ntechnique, proteolytic activity matrix analysis (PrAMA). Using a nine-member\nDL with three inhibitors and four FRET substrates, we applied the\nmethod to the peritoneal fluid of subjects with and without the invasive\ndisease endometriosis. The results showed clear and physiologically\nrelevant differences with disease, in particular, decreased MMP-2\nand ADAM-9 activities.