title: A simple method to evaluate the biochemical compatibility of oil/surfactant mixtures for experiments in microdroplets.
abstract: The enormous reduction of assay volume afforded by compartmentalization into picolitre water-in-oil droplets is an exciting prospect for high-throughput biology. Maintaining the activity of encapsulated proteins is critical for experimental success, for example in in vitro directed evolution, where protein variants are expressed in droplets to identify mutants with improved properties. Here, we present a simple and rapid method to quantitatively compare concentrations of fluorescent molecules in microdroplets. This approach allows an assessment of different emulsification procedures and several oil/surfactant mixtures for biochemical compatibility, in particular in vitro protein expression. Based on determining droplet fluorescence vs. droplet diameter, the method uses the gradient of such curves as a 'concentration correlation coefficient' (CCC) that is directly proportional to fluorophore concentration. Our findings suggest that generation of droplets using a microfluidic flow-focusing device gave no more protein expression than droplet production by the bulk methods of vortexing and homogenizing. The choice of oil/surfactant, however, was found to be critical for protein expression and even encapsulation of purified protein, highlighting the importance of careful selection of these components when carrying out biochemical experiments in droplets. This methodology will serve as a quantitative test for the rapid optimization of droplet-based experiments such as in vitro protein expression or enzymatic assays.
authors.affiliation: Department of Biochemistry, University of Cambridge, 80 Tennis Court Road, CB2 1GA, Cambridge, UK.
authors.forename: Miriam
authors.initials: M
authors.lastname: Kaltenbach
authors.forename: Sean R A
authors.initials: SR
authors.lastname: Devenish
authors.forename: Florian
authors.initials: F
authors.lastname: Hollfelder
mesh_terms: D019020:Directed Molecular Evolution / Q000295:instrumentation / Q000379:methods; D004655:Emulsions; D005453:Fluorescence; D055767:Immobilized Proteins; D046210:Microfluidic Analytical Techniques / Q000295:instrumentation* / Q000379:methods*; D008899:Mineral Oil / Q000737:chemistry*; D014176:Protein Biosynthesis; D011993:Recombinant Fusion Proteins / Q000737:chemistry; D013501:Surface-Active Agents / Q000737:chemistry*
chemical_list: D004655:Emulsions; D055767:Immobilized Proteins; D011993:Recombinant Fusion Proteins; D013501:Surface-Active Agents; D008899:Mineral Oil