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.

Compartmentalization Approaches in Soft Matter Science: From Nanoreactor Development to Organelle Mimics

“…Besides GFP, also enzymes have been used as gene products to demonstrate the efficacy of the biological process in a microdroplet. For example, OpdA, a phosphotriesterase from Agrobacterium radiobacter P230, was expressed via IVTT, and its activity was demonstrated by the dephosphorylation of coumaphos (3‐chloro‐4‐methyl‐7‐coumarinyl diethylphosphate), leading to a fluorescent product . Although in principle one cognate gene is sufficient to lead to an enzyme with sufficient activity to show fluorescence, sensitivity however remains a critical point in the entire set up, and for a robust read‐out, it is preferred to have multiple copies of DNA.…”

Cytoskeletal Protein Expression and its Association within the Hydrophobic Membrane of Artificial Cell Models

“…4 Most recently, cell‐free expression of water‐soluble proteins such as green fluorescent protein (GFP), red‐shifted GFP (rsGFP) and an organophosphorus hydrolase enzyme, OpdA, has been performed within aqueous W/O microdroplets, mimicking, at a simple level, protein expression in a cell 5. 6, 7 W/O droplets, synthesised in microflow systems, are attractive for their simplicity and ease of production but provide only a limited cellular model. For example, they are surrounded by a continuous phase that is hydrophobic.…”

Fast fabrication of microfluidic devices using a low-cost prototyping method

“…Because of a capacity to mimic conditions similar to that of a single cell, micro-droplets can work as appropriate containers for in vitro biological reactions (Fredlake et al 2012;Kim et al 2011;Luo et al 2006;Minarik et al 2012). Moreover, through generating and dispersing solution within an immiscible continuous phase, they offer many advantages including an access to ultra-high throughput screen, reduced reagent consumption, improved efficiency through automation, and online analysis as a microreactor for combinatorial chemistry Osaki et al 2011;Pei et al 2010;Wegrzyn et al 2013;Wu et al 2011). Channel's performance can impose a great influence on the uniformity of micro-droplets' diameters which are directly related to their properties.…”