Fluorescence-activated droplet sorting (FADS): efficient microfluidic cell sorting based on enzymatic activity

Abstract

We describe a highly efficient microfluidic fluorescence-activated droplet sorter (FADS) combining many of the advantages of microtitre-plate screening and traditional fluorescence-activated cell sorting (FACS). Single cells are compartmentalized in emulsion droplets, which can be sorted using dielectrophoresis in a fluorescence-activated manner (as in FACS) at rates up to 2000 droplets s(-1). To validate the system, mixtures of E. coli cells, expressing either the reporter enzyme beta-galactosidase or an inactive variant, were compartmentalized with a fluorogenic substrate and sorted at rates of approximately 300 droplets s(-1). The false positive error rate of the sorter at this throughput was <1 in 10(4) droplets. Analysis of the sorted cells revealed that the primary limit to enrichment was the co-encapsulation of E. coli cells, not sorting errors: a theoretical model based on the Poisson distribution accurately predicted the observed enrichment values using the starting cell density (cells per droplet) and the ratio of active to inactive cells. When the cells were encapsulated at low density ( approximately 1 cell for every 50 droplets), sorting was very efficient and all of the recovered cells were the active strain. In addition, single active droplets were sorted and cells were successfully recovered.

High-throughput screening for industrial enzyme production hosts by droplet microfluidics

“…4). This is in good agreement with the theoretically maximal enrichment, 28 η max , of 13 (see ESI † for calculation). This result indicates that the enzyme screening and the sorting work very well, as η max is derived assuming that the only limitation to the enrichment is the Poisson governed encapsulation of cells.…”

“…This is also in line with the sorter error rate being on the order of 10 −4 indicating that it should not significantly affect the enrichment, which is more than ten times lower. A good agreement between η max and the actual enrichment was also reported by Fallah-Araghi et al 28 In contrast, Kintses et al (2012) 30 found an enrichment of ca. 1/20 of η max , likely due to the fact that their sorting experiment involved a library rather than a binary mixture of two strains, the library being a more difficult model system.…”

“…27 This principle can be used to sort droplets based on their fluorescence using fluorescently activated droplet sorting (FADS). 28 Droplet microfluidics has recently been demonstrated for directed evolution of enzymes. [28][29][30][31] The throughputs of these prototype systems are at least a 100 times higher than a microtiter plate robot and the reagent consumption is lowered by about a million fold, vastly reducing the total cost associated with enzyme evolution.…”

Encapsulation of single cells into monodisperse droplets by fluorescence-activated droplet formation on a microfluidic chip

“…In addition to compartmentalizing reactions, droplet-based microfluidics can also be used to encapsulate prokaryotic [14][15][16][17][18] and eukaryotic cells [19][20][21][22][23], and even the embryos of multicellular organisms [24,25], which opens up a new avenue for cell analysis. Recently, Brouzes et al [19] developed a droplet-based viability assay that permitted quantitative analysis of cell viability and growth within compartmentalized aqueous droplets.…”

An Amplex Red-based fluorometric and spectrophotometric assay for l-asparaginase using its natural substrate

“…Moreover, the limited sensitivity of absorption-based spectrophotometric assays [5][6][7]9,10] is a major handicap to significantly reducing reaction volumes. A noteworthy example of such a case is droplet-based microfluidics, which has emerged as a powerful tool for high-throughput screening in directed protein evolution [13,14]. In these experimental setups, the assay volume is minimized to 1 nl, to 1 fl, scaling down light path lengths to 1 lm.…”