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Ultra-High-Throughput Absorbance-Activated Droplet Sorting for Enzyme Screening at Kilohertz Frequencies.

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Medcalf, Elliot J 
Gantz, Maximilian 
Hollfelder, Florian  ORCID logo


Droplet microfluidics is a valuable method to "beat the odds" in high throughput screening campaigns such as directed evolution, where valuable hits are infrequent and large library sizes are required. Absorbance-based sorting expands the range of enzyme families that can be subjected to droplet screening by expanding possible assays beyond fluorescence detection. However, absorbance-activated droplet sorting (AADS) is currently ∼10-fold slower than typical fluorescence-activated droplet sorting (FADS), meaning that, in comparison, a larger portion of sequence space is inaccessible due to throughput constraints. Here we improve AADS to reach kHz sorting speeds in an order of magnitude increase over previous designs, with close-to-ideal sorting accuracy. This is achieved by a combination of (i) the use of refractive index matching oil that improves signal quality by removal of side scattering (increasing the sensitivity of absorbance measurements); (ii) a sorting algorithm capable of sorting at this increased frequency with an Arduino Due; and (iii) a chip design that transmits product detection better into sorting decisions without false positives, namely a single-layered inlet to space droplets further apart and injections of "bias oil" providing a fluidic barrier preventing droplets from entering the incorrect sorting channel. The updated ultra-high-throughput absorbance-activated droplet sorter increases the effective sensitivity of absorbance measurements through better signal quality at a speed that matches the more established fluorescence-activated sorting devices.



Biotechnology, Bioengineering

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Anal Chem

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American Chemical Society (ACS)
European Commission Horizon 2020 (H2020) ERC (695664)
Biotechnology and Biological Sciences Research Council (2273624)
Biotechnology and Biological Sciences Research Council (BB/M011194/1)
European Commission Horizon 2020 (H2020) Marie Sk?odowska-Curie actions (750772)
European Commission Horizon 2020 (H2020) Marie Sk?odowska-Curie actions (813786)
BBSRC Doctoral Training Account, (E.J.M., BB/M011194/1) Trinity College / Benn W Levy SBS DTP PhD Studentship (M.G.), H2020 ERC Advanced Investigator Award (695669).