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

• A liquid crystal-based protease inhibition assay is developed. • This assay is used to assess inhibition efficiency of a protease inhibitor pefabloc. • IC 50 value of pefabloc can be detected by using the color of liquid crystal. • The assay provides detailed information about the transition period from reversible to irreversible inhibition. • Pefabloc inhibits protease activity irreversibly if the incubation time is more than 60 min. 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 IC 50 value of pefabloc for the immobilized protease is 0.45 mg/mL, which is lower than the IC 50 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. [ABSTRACT FROM AUTHOR]