Multiplexed Protease Activity Assay for Low-Volume Clinical Samples Using Droplet-Based Microfluidics and Its Application to Endometriosis

Chia‐Hung Chen, Chia-Hung Chen, Miles A. Miller, Aniruddh Sarkar, Michael T. Beste, Beste MT, Keith B Isaacson, Keith Isaacson, Isaacson KB, Douglas A. Lauffenburger, Lauffenburger DA, Linda G. Griffith, Jongyoon Han, Han J
Journal of the American Chemical Society · 2012 · vol. 135(5) , pp. 1645–1648 · doi:10.1021/ja307866z · PMID:23157326 · PMC3566300 · W2070168562
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This paper details a droplet-based microfluidic method to measure multiple protease activities in small clinical samples, demonstrating decreased MMP-2 and ADAM-9 activity in endometriosis patients.

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Researchers developed a droplet-based microfluidic platform to measure the activity of multiple specific proteases in low-volume clinical samples using a barcoded library of FRET substrates and inhibitors. The study applied this multiplexed assay to peritoneal fluid from subjects with and without endometriosis, utilizing proteolytic activity matrix analysis to deconvolve complex reaction rates. Results indicated physiologically relevant differences in disease states, specifically showing decreased activities of MMP-2 and ADAM-9 in patients with endometriosis. This paper is centrally about endometriosis — specifically analyzing protease activity profiles in peritoneal fluid as potential biomarkers for the invasive disease.

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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.
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Multiplexed Protease Activity Assay for Low-Volume Clinical Samples Using Droplet-Based Microfluidics and Its Application to Endometriosis Name Griffith_Multiplexed protease.pdf Size 1.78 MB Format Adobe PDF Checksum (MD5) 4b1366d1dfac3988d1f5fbf01f66370b 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 Terms of Use Article 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. Persistent DSpace Link DOI of Published Version https://doi.org/10.1021/ja307866z

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Condition tags

endometriosis

MeSH descriptors

Endometriosis Endometriosis Enzyme Assays Metalloproteases Microfluidic Analytical Techniques Endometriosis Endometriosis Female Fluorescence Resonance Energy Transfer Humans Metalloproteases Metalloproteases Metalloproteases Particle Size Protease Inhibitors Protease Inhibitors Substrate Specificity Surface Properties

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