Microfluidic chip device for fluorescence-activated droplet sorting (FADS) of ≈ 3 pL-sized droplets (offline-incubation). Blue: silanised area for emulsion handling, depth: 21.5–23 μm. Red: signal electrode (-). Black: ground electrode (+).
D. Schnettler Fernández, O. J. Klein, T. S. Kaminski, P.-Y. Colin, F. Hollfelder.
Ultrahigh-throughput directed evolution of a metal-free α/β-hydrolase with a Cys-His-Asp triad into an efficient phosphotriesterase.
Submitted by: David Schnettler Fernández
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Optics and electronics of the microfluidic on-chip sorting device were set-up as previously described (, ). After incubation at room temperature, 3 pL droplets were reinjected from the collection tubing into the sorting device at a rate of 10–25 μL/h. To enable precise sorting of single droplets, the distance between the droplets was increased by injection of spacing oil (Novec HFE-7500, 3M, USA) into the device at a flow rate of 100–300 μL/h. The asymmetric Y- shaped junction in the device ensures that all droplets automatically flow into the waste channel, unless deviated by an electrical pulse into the sorting channel. A 488-nm laser was focused 100 μm upstream of the sorting junction through a 40× microscope objective (UPlanFLN, Olympus, Japan) for fluorophore excitation and the emitted fluorescent light was collected and amplified using photomultiplier tubes (PMM02, Thorlabs, USA). Whenever the fluorescence peak reached a user-defined threshold, an electric field was applied by the two electrodes on the sorting device, attracting the highly fluorescent droplet towards the narrower sorting channel. Droplets were sorted into a collection tube pre-filled with 100 μL nuclease-free water. DSF