ja307866z_si_003.avi (4.01 MB)
Multiplexed Protease Activity Assay for Low-Volume Clinical Samples Using Droplet-Based Microfluidics and Its Application to Endometriosis
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posted on 2013-02-06, 00:00 authored by Chia-Hung Chen, Miles
A. Miller, Aniruddh Sarkar, Michael
T. Beste, Keith B. Isaacson, Douglas A. Lauffenburger, Linda
G. Griffith, Jongyoon HanAs 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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drug targetsreaction ratesfluidSpecific protease activitiesMMPbiomarker candidatesactivity matrix analysisADAMmethoddeconvolution techniquebarcoded droplet librarydisease endometriosisdropletsinhibitorDLsampleextracellular matrixMP activityMultiplexed Protease Activity AssayFRET substratesmonitors hundreds
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