Multiplexed protease activity assay for low-volume clinical samples using droplet-based microfluidics and its application to endometriosis.

TitleMultiplexed protease activity assay for low-volume clinical samples using droplet-based microfluidics and its application to endometriosis.
Publication TypeJournal Article
Year of Publication2013
AuthorsChen, C-H, Miller, MA, Sarkar, A, Beste, MT, Isaacson, KB, Lauffenburger, DA, Griffith, LG, Han, J
JournalJ Am Chem Soc
Volume135
Issue5
Pagination1645-8
Date Published2013 Feb 6
ISSN1520-5126
KeywordsEndometriosis, Enzyme Assays, Female, Fluorescence Resonance Energy Transfer, Humans, Metalloproteases, Microfluidic Analytical Techniques, Particle Size, Protease Inhibitors, Substrate Specificity, Surface Properties
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.

DOI10.1021/ja307866z
Alternate JournalJ. Am. Chem. Soc.
PubMed ID23157326
PubMed Central IDPMC3566300
Grant ListP50-GM68762 / GM / NIGMS NIH HHS / United States
R01 EB010246 / EB / NIBIB NIH HHS / United States
U54 CA112967 / CA / NCI NIH HHS / United States
U54-CA112967 / CA / NCI NIH HHS / United States