Multiplexed Protease Activity Assay for Low-Volume Clinical Samples Using Droplet-Based Microfluidics and Its Application to Endometriosis
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 hig...
| Main Authors: | , , , , , , , |
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| Other Authors: | |
| Format: | Article |
| Language: | en_US |
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American Chemical Society (ACS)
2014
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| Online Access: | http://hdl.handle.net/1721.1/88925 https://orcid.org/0000-0002-1801-5548 https://orcid.org/0000-0001-7215-1439 |
| _version_ | 1826217782242443264 |
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| author | Chen, Chia-Hung Sarkar, Aniruddh Beste, Michael T. Isaacson, Keith B. Lauffenburger, Douglas A. Griffith, Linda G. Han, Jongyoon Miller, Miles Aaron |
| author2 | Massachusetts Institute of Technology. Center for Gynepathology Research |
| author_facet | Massachusetts Institute of Technology. Center for Gynepathology Research Chen, Chia-Hung Sarkar, Aniruddh Beste, Michael T. Isaacson, Keith B. Lauffenburger, Douglas A. Griffith, Linda G. Han, Jongyoon Miller, Miles Aaron |
| author_sort | Chen, Chia-Hung |
| collection | MIT |
| description | 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. |
| first_indexed | 2024-09-23T17:09:04Z |
| format | Article |
| id | mit-1721.1/88925 |
| institution | Massachusetts Institute of Technology |
| language | en_US |
| last_indexed | 2024-09-23T17:09:04Z |
| publishDate | 2014 |
| publisher | American Chemical Society (ACS) |
| record_format | dspace |
| spelling | mit-1721.1/889252022-10-03T10:45:10Z Multiplexed Protease Activity Assay for Low-Volume Clinical Samples Using Droplet-Based Microfluidics and Its Application to Endometriosis Chen, Chia-Hung Sarkar, Aniruddh Beste, Michael T. Isaacson, Keith B. Lauffenburger, Douglas A. Griffith, Linda G. Han, Jongyoon Miller, Miles Aaron 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 Chen, Chia-Hung Miller, Miles Aaron Sarkar, Aniruddh Beste, Michael T. Isaacson, Keith B. Lauffenburger, Douglas A. Griffith, Linda G. Han, Jongyoon 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. National Institutes of Health (U.S.) (P50-GM68762) National Institutes of Health (U.S.) (U54-CA112967) United States. Defense Advanced Research Projects Agency. Centers in Integrated Photonics Engineering Research Program 2014-08-19T20:35:22Z 2014-08-19T20:35:22Z 2012-11 2012-08 Article http://purl.org/eprint/type/JournalArticle 0002-7863 1520-5126 http://hdl.handle.net/1721.1/88925 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. https://orcid.org/0000-0002-1801-5548 https://orcid.org/0000-0001-7215-1439 en_US http://dx.doi.org/10.1021/ja307866z Journal of the American Chemical Society 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. application/pdf American Chemical Society (ACS) ACS |
| spellingShingle | Chen, Chia-Hung Sarkar, Aniruddh Beste, Michael T. Isaacson, Keith B. Lauffenburger, Douglas A. Griffith, Linda G. Han, Jongyoon Miller, Miles Aaron Multiplexed Protease Activity Assay for Low-Volume Clinical Samples Using Droplet-Based Microfluidics and Its Application to Endometriosis |
| title | Multiplexed Protease Activity Assay for Low-Volume Clinical Samples Using Droplet-Based Microfluidics and Its Application to Endometriosis |
| title_full | Multiplexed Protease Activity Assay for Low-Volume Clinical Samples Using Droplet-Based Microfluidics and Its Application to Endometriosis |
| title_fullStr | Multiplexed Protease Activity Assay for Low-Volume Clinical Samples Using Droplet-Based Microfluidics and Its Application to Endometriosis |
| title_full_unstemmed | Multiplexed Protease Activity Assay for Low-Volume Clinical Samples Using Droplet-Based Microfluidics and Its Application to Endometriosis |
| title_short | Multiplexed Protease Activity Assay for Low-Volume Clinical Samples Using Droplet-Based Microfluidics and Its Application to Endometriosis |
| title_sort | multiplexed protease activity assay for low volume clinical samples using droplet based microfluidics and its application to endometriosis |
| url | http://hdl.handle.net/1721.1/88925 https://orcid.org/0000-0002-1801-5548 https://orcid.org/0000-0001-7215-1439 |
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