Multi-step microfluidic droplet processing: kinetic analysis of an in vitro translated enzyme (original) (raw)
* Corresponding authors
a Institut de Science et d'Ingénierie Supramoléculaire (ISIS), Université de Strasbourg, CNRS UMR 7006, 8 allée Gaspard Monge, Strasbourg Cedex, FR
E-mail: griffiths@unistra.fr, vtaly@isis.u-strasbg.fr
b Medical Research Council, Laboratory of Molecular Biology, Hills Road, Cambridge, UK
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 vitro_transcription and translation of _cot_A 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 ∼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.
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Supplementary files
- Details of optical set-up, emulsion storage off-chip and description of the electro-coalescence device. PDF (5369K)
- Movie M1 shows 2 pL IVT droplet production. The device contains a Ψ-shaped connection, in which two aqueous streams are merged and emulsified using 10 μm wide flow focusing junction. The fluorinated HFE-7500 oil used to produce droplets contained 3% (w/w) EA-surfactant. The droplets were collected at the exhaust. MPG (1533K)
- Movie M2 shows IVT emulsion re-injection after 6 hours of incubation off-chip. The re-injection rate was 20 ?L/hr for the IVT emulsion and 180 μL/hr for the carrier oil used to space droplets. The carrier oil was FC40 with 3% (w/w) EA-surfactant. MPG (2878K)
- Movie M3 shows droplet re-injection, pairing and electro-coalescence. 2 pL IVT droplets were reinjected at 20 μL/hr and spaced by carrier oil at 180 μL/hr. 10 pL Assay droplets were generated by the on-chip droplet generation module using 100 μL/hr for the aqueous phase and 200 μL/hr for the carrier oil. Carrier oil was FC40 with 3% (w/w) EA-surfactant. The fused droplets were collected atthe exhaust. MPG (2252K)
- Movie M4 shows the fusion module during electro-coalescence of 2 pL IVT droplets with 10 pLAssay droplets. MPG (2158K)
- Movie M5 shows the fused droplets after electro-coalescence, collection off-chip and re-injection. Re-injection rate was 20 μL/hr for the emulsion and 120 μL/hr for the carrier oil used to spacedroplets. MPG (1844K)
Article information
DOI
https://doi.org/10.1039/B907753G
Article type
Paper
Submitted
17 Apr 2009
Accepted
16 Jul 2009
First published
06 Aug 2009
Download Citation
Lab Chip, 2009,9, 2902-2908
Permissions
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, 2902DOI: 10.1039/B907753G
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