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

Zinchenko, A.; Devenish, S. R.; Hollfelder, F.

doi:10.1101/2023.04.23.538007

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bioRxiv · 10.1101/2023.04.23.538007

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

Zinchenko, A.·Devenish, S. R.·Hollfelder, F.

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Abstract

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.

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Zinchenko, A., Devenish, S. R., Hollfelder, F.. 2023-04-23. Rapid quantitative assessment of small molecule leakage from microdroplets by flow cytometry and improvement of fluorophore retention in biochemical assays. https://doi.org/10.1101/2023.04.23.538007

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