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Inside Dolomite Labs: Double Emulsions for FACS

 

 

Novel biopharmaceutical development forms an integral part of the fast-growing medicine and biologics market. Whilst targeted biologic therapies such as enzymes, monoclonal antibodies and many more show specificity and promise in the treatment of cancer and other diseases, they lack the functional validation necessary for their use as therapeutics.

 

Library screening in combination with fluorescence-activated cell sorting (FACs) offers a way of analysing the expression of cell surface and intracellular molecules within a heterogenous population one cell at a time. Predominantly, the fluorescent signal intensity emitted as a result of antibody binding to proteins, cell surface markers and ligands is measured during sorting. Traditional subcloning methods can be performed on the displayed surface markers which demand further optimisation, but this technique lacks the ability and throughput necessary for early phenotypic screening.

 

FACS sorting gene library using double emulsions with droplets containing all components required for protein expression. a) a gene in single droplets with reaction mix b) gene variants expressed in bacteria which are then encapsulated one cell per droplet. The active translated enzyme in both scenarios interacts with a fluorogenic substrate to produce and fluorescent product. This is then sorted using FACS.

 

Combining fluorescence detection in droplets can also allow for quantitative measurement of reaction progress and outcome. Microfluidic technology offers a platform for producing monodispersed droplets capable of encapsulating biological cells and molecules in nano or pico-litre volumes. These mini bioreactors can help drive high-throughput screening by performing up to 107 reactions at a time with dramatically lower costs per reaction 1. The droplets compartmentalise individual cells entrapping all secreted molecules and analytes, preventing interference with other cells and products. These droplets can be sorted by one or more signals and their nucleic acid content analysed to identify variants responsible for the activity. In other words, this technology can provide genotype to phenotype linkage 2.

 

A prime example where compartmentalising cells in droplets can be beneficial is directed evolution. A series of random mutations are introduced into a target gene, upon encapsulation in droplets and FACS sorting, desired characteristics can be selected for. The size of the gene libraries that can be obtained from these experiments easily exceeds the throughput of any screening system, implying that screening is the bottleneck in the exploration of sequence space 3.

 

Droplet microfluidics exceeds the current screening system at providing high-throughput capabilities. In order to FACS sort droplets (water-in-oil) the surrounding sheath fluid needs to be compatible with the FACs platform. It, therefore, necessitates the droplets to be re-encapsulated in an aqueous buffer, creating double emulsion (water-in-oil-in-water) 4. Dolomite’s µEncapsulator system offers precisely the solution by allowing the user to create droplet sizes varying from 1-70 µm, compatible with various FACS nozzles. The two-step encapsulation technique allows the processing of small sample volumes (100 µl) at a time accommodating the use of rare or precious samples.

 

 

To find out more please refer to our application notes, protocols and webinars. Alternatively, get in touch with our Experts via our contact form or at info@dolomite-microfluidics.com and see how you can use double emulsions to increase the quality and throughput of your assays!

 

  1. Sukovich, D. J., Lance, S. T., & Abate, A. R. (2017) Sequence Specific Sorting of DNA Molecules with FACS Using 3dPCR, Scientific Reports, 7, 1–9
  2. Zinchenko, A., Devenish, S. R. A., Kintses, B., Colin, P., Fischlechner, M. & Hollfelder F. (2014) One in a Million: Flow Cytometric Sorting of Single Cell-Lysate Assays in Monodisperse Picolitre Double Emulsion Droplets for Directed Evolution, Analytical Chemistry, 86.5, 2526–33
  3. van Tatenhove-Pel, R. J., Hernandez-Valdes, J. A., Teusink, B., Kuipers, O. P., Fischlechner, M., & amp; Bachmann, H. (2020) Microdroplet Screening and Selection for Improved Microbial Production of Extracellular Compounds, Current Opinion in Biotechnology, 61, 72–81
  4. Brower, K.K., Carswell-Crumpton, C., Klemm, S., Cruz, B., Kim, G., Calhoun, S.G., Nichols, L. & Fordyce, P.M., (2020) Double emulsion flow cytometry with high-throughput single droplet isolation and nucleic acid recovery Lab on a Chip 20, 2062-2074