Article

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

Journal of the American Chemical Society (Impact Factor: 11.44). 11/2012; 135(5). DOI: 10.1021/ja307866z
Source: PubMed

ABSTRACT As principal degrading enzymes of the extracellular matrix, metalloproteinases contribute to various pathologies and represent a family of promising drug targets and biomarker candidates. However, multiple proteases and endogenous inhibitors interact to govern metalloproteinase 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 (<20µl) of complex biological fluids such as clinical samples which are only available in very limited amounts. We have developed a droplet-based microfluidic platform that injects the sample into thousands of picoliter-scale droplets from a barcoded droplet library 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 droplet library with three inhibitors and four FRET substrates, we apply the method to the peritoneal fluid of subjects with and without the invasive disease of endometriosis. Results show clear and physiologically relevant differences with disease; in particular, decreased MMP-2 and ADAM-9 activities.

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