Label-free direct visual analysis of hydrolytic enzyme activity using aqueous two-phase system droplet phase transitions

Dextran hydrolysis-mediated conversion of polyethylene glycol (PEG)-dextran (DEX) aqueous two-phase system droplets to a single phase was used to directly visualize Dextranase activity. DEX droplets were formed either by manual micropipetting or within a continuous PEG phase by computer controlled actuation of an orifice connecting rounded channels formed by backside diffused light lithography. The time required for the two-phase to one-phase transition was dependent on the Dextranase concentration, pH of the medium, and temperature. The apparent Michaelis constants for Dextranase were estimated based on previously reported catalytic constants, the binodal polymer concentration curves for PEG-DEX phase transition for each temperature, and pH condition. The combination of a microfluidic droplet system and phase transition observation provides a new method for label-free direct measurement of enzyme activity.