Multi-step microfluidic droplet processing: kinetic analysis of an in vitro translated enzyme

Abstract

Microdroplets in water-in-oil emulsions can be used as microreactors with volumes 10(3) to 10(9) times smaller than the smallest working volumes in a microtitre plate well (1-2 microL). However, many reactions and assays require multiple steps where new reagents are added at defined times, to start, modify or terminate a reaction. The most flexible way to add new reagents to pre-formed droplets is by controlled, pairwise droplet fusion. We describe a droplet-based microfluidic system capable of performing multiple operations, including pairwise droplet fusion, to analyze complex and sequential multi-step reactions. It is exemplified by performing a series of six on-chip and two off-chip operations which enable the coupled in vitro transcription and translation of cotA laccase genes in droplets and, after performing a controlled fusion with droplets containing laccase assay reagents, the end-point and kinetic analysis of the catalytic activity of the translated protein. In vitro translation and the laccase assay must be performed sequentially as the conditions for the laccase assay are not compatible with in vitro translation. Droplet fusion was performed by electrocoalescence at a rate of approximately 3000 fusion events per second and nearly 90% of droplets were fused one-to-one (one droplet containing in vitro translated laccase fused to one droplet containing the reagents for the laccase assay). The ability to uncouple the enzymatic assay from in vitro translation greatly extends the range of activities of in vitro translated proteins that can potentially be screened in droplet-based microfluidic systems. Furthermore, the system also opens up the possibility of performing a wide range of other new (bio)chemical reactions in droplets.

Dynamics of microfluidic droplets

“…The two approaches have in common the need to first bring the drops into close proximity; this is often done passively, either by modulating the channel geometry in order to slow down the downstream drop until the upstream drop reaches it, [92][93][94] or by using drops of different sizes which flow at different velocities. 95 Active methods have also been developed for pushing drops into contact, for instance through electro-static attraction by using oppositely charged droplets 96 or by temporarily blocking the motion of a downstream drop with the opto-thermal blocking. 6 Finally, drops have also been captured in double wells in order to colocalise them and induce their fusion while they are stationary.…”

Basic Technologies for Droplet Microfluidics

“…To initiate a reaction in a droplet, two sets of droplets are separately generated and then brought into contact and merged through either active or passive processes. Under the occasions requiring multistep reactions or controlling the reaction kinetics, fusion steps are demanded to concentrate or dilute the reactants in droplets or to add new reagents into droplets [36][37][38][39].…”

Effect of Laser-Induced Heating on Raman Measurement within a Silicon Microfluidic Channel

“…The last decade has witnessed an explosive growth in applications of microfluidic channels to chemical and biological fields [1][2][3][4][5]. This has been driven by the trends of shrinking conventional benches to a small size to realize major advantages of efficiency, performance, integration, speed and cost.…”