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

Research output: Journal Publications and Reviews (RGC: 21, 22, 62)21_Publication in refereed journalpeer-review

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Author(s)

  • Miles A. Miller
  • Aniruddh Sarkar
  • Michael T. Beste
  • Keith B. Isaacson
  • Douglas A. Lauffenburger
  • Linda G. Griffith
  • Jongyoon Han

Detail(s)

Original languageEnglish
Pages (from-to)1645-1648
Journal / PublicationJournal of the American Chemical Society
Volume135
Issue number5
Online published18 Nov 2012
Publication statusPublished - 6 Feb 2013
Externally publishedYes

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. © 2012 American Chemical Society.

Citation Format(s)

Multiplexed Protease Activity Assay for Low-Volume Clinical Samples Using Droplet-Based Microfluidics and Its Application to Endometriosis. / Chen, Chia-Hung; Miller, Miles A.; Sarkar, Aniruddh et al.

In: Journal of the American Chemical Society, Vol. 135, No. 5, 06.02.2013, p. 1645-1648.

Research output: Journal Publications and Reviews (RGC: 21, 22, 62)21_Publication in refereed journalpeer-review