Format

Send to

Choose Destination
See comment in PubMed Commons below
J Am Chem Soc. 2013 Feb 6;135(5):1645-8. doi: 10.1021/ja307866z. Epub 2012 Nov 29.

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

Author information

1
Department of Electrical Engineering and Computer Science, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139, United States.

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.

PMID:
23157326
PMCID:
PMC3566300
DOI:
10.1021/ja307866z
[Indexed for MEDLINE]
Free PMC Article
PubMed Commons home

PubMed Commons

0 comments
How to join PubMed Commons

    Supplemental Content

    Full text links

    Icon for American Chemical Society Icon for PubMed Central
    Loading ...
    Support Center