Enzyme synthesis and activity assay in microfluidic droplets on a chip

Abstract

There is growing demand for high-throughput measurement of biochemical reactions in drug discovery and directed evolution programs. To meet this need, a powerful platform based on droplet-based bioreactors manipulated by microfluidic systems is being developed, which can overcome the limitations of scale and power encountered in conventional screening methods. This paper reports our progress in the synthesis of enzymes and assay of their activity within a microfluidic droplet system. The model system we use involves the organophosphorus hydrolase enzyme OpdA from Agrobacterium radiobacter and a robust microchip made from polymethyl methacrylate (PMMA). Synthesis of OpdA from cognate DNA within water-in-oil droplets was tested using both in-house and commercial in vitro transcription and translation (IVTT) kits. OpdA activity was measured using coumaphos as substrate and by monitoring the fluorescence released by its product, chlorferone. OpdA was demonstrated to be synthesized and assayed within the droplets using the commercial in vitro transcription and translation kit, although the activity measured within the droplets diminished over time, apparently due to leakage of chlorferone out of the droplets.