Issue 20, 2009

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 103 to 109 times smaller than the smallest working volumes in a microtitre plate well (1–2 µL). 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 vitrotranscription 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 vitrotranslation and the laccase assay must be performed sequentially as the conditions for the laccase assay are not compatible with in vitrotranslation. Droplet fusion was performed by electrocoalescence at a rate of ∼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 vitrotranslation 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.

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

Supplementary files

Article information

Article type
Paper
Submitted
17 Apr 2009
Accepted
16 Jul 2009
First published
06 Aug 2009

Lab Chip, 2009,9, 2902-2908

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

L. Mazutis, J. Baret, P. Treacy, Y. Skhiri, A. F. Araghi, M. Ryckelynck, V. Taly and A. D. Griffiths, Lab Chip, 2009, 9, 2902 DOI: 10.1039/B907753G

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