Substrate Engineering Enabling Fluorescence Droplet Entrapment for IVC-FACS-Based Ultrahigh-Throughput Screening

Abstract

In vitro compartmentalization-based fluorescence-activated cell sorting (IVC-FACS) is a powerful screening tool for directed evolution of enzymes. However, the efficiency of IVC-FACS is limited by the tendency of the fluorescent reporter to diffuse out of the droplets, which decouples the genotype and phenotype of the target gene. Herein we present a new strategy called fluorescence droplet entrapment (FDE) to solve this problem. The substrate is designed with a polarity that enables it to pass through the oil phase, react with the enzyme and generate an oil-impermeable and fluorescent product that remains entrapped inside the droplet. Several FDE substrates were designed, using two distinct substrate engineering strategies, for the detection of phosphotriesterases, carboxylesterases, and glycosidases activities. Model screening assays in which rare phosphotriesterase-active cells were enriched from large excesses of inactive cells showed that the enrichment efficiency achievable using an FDE substrate was as high as 900-fold: the highest yet reported in such an IVC-FACS system. Thus, FDE provides a means to tightly control the onset of the enzymatic reaction, minimize droplet cross-talk, and lower the background fluorescence. It therefore may serve as a useful strategy for the IVC-FACS screening of enzymes, antibodies, and other proteins.

Droplet flow cytometry for single-cell analysis

“…The usage of uorinated oil (e.g., HFE-7500 and FC-40) acting as a selective barrier can reduce hydrophilic mass transfer and communication between DEs. 49,55,56 The aqueous external phase allows the suspension of DEs in nutrient medium for long-term cell culture and the compatibility with commercial cytometers for high-throughput assays 18,49,57,58 (with the sorting speed >10 7 events per hour 31,32,56 ).…”

“…123 Another approach that may circumvent this limitation is the chemical or enzymatic modication of the hydrophobic products into hydrophilic, charged molecules that cannot diffuse into the oil shell. 55 There is still a need for further development of new uorescence dyes and metabolic biomarkers that can be conned within the droplets and new biochemical technologies that can prevent the leakage of small molecules from droplets.…”

Ultrahigh-Throughput Improvement and Discovery of Enzymes Using Droplet-Based Microfluidic Screening

“…However, the possibility of re-emulsifying w/o droplets into water-in-oil-in-water (w/o/w) double emulsions made it possible to screen droplets using fluorogenic assays and FACS [ 26 ]. By combining the advantages of MTP and FACS, IVC allowed improving the activity of many enzymes [ 27 , 28 , 29 , 30 , 31 , 32 , 33 , 34 , 35 , 36 , 37 , 38 ], a list that may lengthen in the future with the development of new strategies [ 39 ]. Finally, the small droplet volume (femtoliters to picoliters) permits screening directly at the level of single cells, and even single genes.…”

Scalable production of double emulsion drops with thin shells

“…Emulsion drops are often used as picoliter--sized vessels to conduct chemical 1,2 and biochemical reactions, 3,4 and for high throughput screening assays. [5][6][7] These drops can be produced at high frequencies allowing compartmentalization of large quantities of reagents in a short amount of time. 8--10 To take advantage of the possibility to produce large quantities of samples in a minimum amount of time, they must be analyzed at high rates.…”