Liquid crystal-enabled protease inhibition assays developed in a millifluidic device

Abstract

Protease inhibitors are essential drug molecules which can be used for treatments of protease-related diseases. Herein, we report an LC-based protease inhibition assay built inside a millifluidic device. This assay can be used to study the inhibition efficiency and reversibility of pefabloc (a serine protease inhibitor) against proteases. LC is employed for the detection of peptide fragments produced from protease activity inside the millifluidic device. LC gives a bright spot when the amount of peptide fragment exceeds a minimum value. By using this assay, we find that IC50 value of pefabloc for the immobilized protease is 0.45 mg/mL, which is lower than the IC50 value of pefabloc (0.90 mg/mL) obtained in a homogeneous assay. Moreover, proteases can be immobilized on the millifluidic device to build a heterogeneous protease assay. In this assay, pefabloc blocks the immobilized protease irreversibly after 60 min, which is longer than that in a homogenous protease assay. This reversibility study provides useful information about the transition period of pefabloc from reversible to irreversible inhibition. This protease inhibition assay is potentially useful for high throughput screening of unknown proteases and their inhibitor in a small sample volume. Moreover, this method can be used for dosage test of novel drug molecules which are protease inhibitors.