Rapid quantitative assessment of small molecule leakage from microdroplets by flow cytometry and improvement of fluorophore retention in biochemical assays

Microdroplets are compartments made in the laboratory that allow the miniaturisation of chemical and biological experiments to the femto- to picolitre scale, replacing the classical test tube with a droplet. Ideally containment of the contents of individual droplets would be perfect, but in reality this situation rarely occurs. Instead the leaking of molecules even from intact droplets presents a challenge to the success of miniaturisation and must be assessed on a case-by-case basis. We now present a new method for quantitative determination of leakage: a sheath fluid-free flow cytometer (Guava EasyCyte) is used to directly determine the fluorescence of water-in-oil droplets as a function of time. We validate this method by demonstrating that this assessment of leakage provides a framework for experimental improvements that reduce the leakage of two widely used fluorophores. A 40-fold better retention compared to current protocols is achieved for resorufin with an optimized mix (oil: FC-70, surfactant: 0.1% w/w AZ900C, additive: 1% BSA) to maintain useful retention for up to 130 hours. Likewise leakage of the fluorophore methylumbelliferone is reduced by 75-fold. The availability of a method to quantitate leakage quickly for a variety of experimental conditions will facilitate future applications of droplet-based experiments (e.g. in directed evolution or diagnostics), aid miniaturisation of lab-scale assays into this format, and improve the degrees of freedom in setting up such ultrahigh-throughput experiments.