Controlling the retention of small molecules in emulsion microdroplets for use in cell-based assays.
Water-in-oil microdroplets in microfluidics are well-defined individual picoliter reaction compartments and, as such, have great potential for quantitative high-throughput biological screening. This, however, depends upon contents of the droplets not leaking out into the oil phase. To assess the mechanism of possible leaking, the retention of various fluorescein derivatives from droplets formed in mineral oil and stored for hours in a reservoir on chip was studied. Leaking into the oil phase was observed and was shown to be dependent on the nature of the compounds and on the concentration of the silicone-based polymeric surfactant Abil EM 90 used. In experiments in which droplets filled with fluorescein were mixed with droplets filled with only buffer, the rate of efflux from filled droplets to empty droplets was dependent on the number of neighboring droplets of different composition. Buffer droplets with five fluorescein-containing neighbors took up the fluorophore 4.5 times faster than buffer droplets without fluorescein neighbors. The addition of bovine serum albumin (BSA) substantially reduced leaking. A formulation with 5% BSA reduces leaking of the fluorophore from 45% to 3%. Inclusion of BSA enabled experiments to be carried out over periods up to 18 h, without substantial leaking (<5%). We demonstrate the utility of this additive by following the enzymatic activity of alkaline phosphatase expressed by Escherichia coli cells. The ability to reliably compartmentalize genotype (cell) and phenotype (reaction product) is the basis for using microdroplets in directed evolution studies, and the approaches described herein provide a test system for assessing emulsion formulations for such purposes.
Courtois
Fabienne
F
Department of Biochemistry, University of Cambridge, 80 Tennis Court Road, Cambridge, CB2 1GA, UK.
Olguin
Luis F
LF
Whyte
Graeme
G
Theberge
Ashleigh B
AB
Huck
Wilhelm T S
WT
Hollfelder
Florian
F
Abell
Chris
C
D000818:Animals; D002417:Cattle; D004655:Emulsions / Q000737:chemistry*; D004926:Escherichia coli / Q000166:cytology*; D019793:Fluorescein / Q000737:chemistry; D046210:Microfluidic Analytical Techniques; D008856:Microscopy, Fluorescence; D008863:Microspheres; D008899:Mineral Oil / Q000737:chemistry; D014867:Water / Q000737:chemistry
D004655:Emulsions; D014867:Water; D008899:Mineral Oil; D019793:Fluorescein