Mass Activated Droplet Sorting (MADS) Enables High‐Throughput Screening of Enzymatic Reactions at Nanoliter Scale

Abstract

Microfluidic droplet sorting enables the high‐throughput screening and selection of water‐in‐oil microreactors at speeds and volumes unparalleled by traditional well‐plate approaches. Most such systems sort using fluorescent reporters on modified substrates or reactions that are rarely industrially relevant. We describe a microfluidic system for high‐throughput sorting of nanoliter droplets based on direct detection using electrospray ionization mass spectrometry (ESI‐MS). Droplets are split, one portion is analyzed by ESI‐MS, and the second portion is sorted based on the MS result. Throughput of 0.7 samples s−1 is achieved with 98 % accuracy using a self‐correcting and adaptive sorting algorithm. We use the system to screen ≈15 000 samples in 6 h and demonstrate its utility by sorting 25 nL droplets containing transaminase expressed in vitro. Label‐free ESI‐MS droplet screening expands the toolbox for droplet detection and recovery, improving the applicability of droplet sorting to protein engineering, drug discovery, and diagnostic workflows.

(R)evolution-on-a-chip

“…While the second part travels through the delay line, content analysis of the first part takes place and the sorting algorithm decides, above a given threshold, if the second droplet part corresponds to a positive hit. MADS has been tested over a range of enzyme-substrate concentrations and sorting accuracy was found at 98% [80]. Although promising, MADS performed at 0.7 Hz, which falls greatly behind FADS [80].…”

“…However, in these cases the droplet was destroyed in the process, thus the beneficial genotype could not be tracked down. Mass-activated droplet sorting (MADS) is based on an ingenious chip design and a programmable sorting algorithm [80]. Droplets injected for sorting are first split into two parts.…”

“…MADS has been tested over a range of enzyme-substrate concentrations and sorting accuracy was found at 98% [80]. Although promising, MADS performed at 0.7 Hz, which falls greatly behind FADS [80]. However, the universality that comes with its label-free nature leaves expectations for further development.…”

Production of High Value Amine Intermediates via Biocatalytic Cascades in Continuous Flow

“…Due to the relatively unexplored space of GOase-bioamination, these cascades represent the first of continuous biocatalyst substrate screening. This streamlined flow system will complement current high throughput biocatalytic screening methods, 36 and ultimately facilitate automation for biocatalytic cascade discovery in a highly efficient manner.…”

Can Bottom-Up Synthetic Biology Generate Advanced Drug-Delivery Systems?

“…As an alternative, Krafft and coworkers developed a simplified system relying solely on sucrose/salt solution as the inner and outer aqueous phases [58]. To completely dewet the Droplet-based microfluidics features various functional units: a droplet production unit to encapsulate a variety of chemical and biochemical contents [26]; a pico-injector for the sequential injection of picoliter volumes into individually produced droplets [94]; a fusion module to provoke the fusion of two distinct droplets, thus enabling the mixture of various chemical and biochemical reagents with high temporal control [95]; a mixing module to improve convection and thereby facilitate molecular reactions within the droplet [96]; a sorting module to precisely separate individual droplets based on various analytical signals such as fluorescence [62] or mass spectrometry [97]; a releasing unit to liberate the entrapped mixture from the droplet into an aqueous continuous phase by applying an electrical field [98] or by using a passive trapping structure [26]; and a detection or observation module that facilitates the assessment of a large droplet population [43,99]. Adding to the various possibilities of droplet-based microfluidics, capillary microfluidicswhich in contrast to PDMS-based microfluidics uses glass capillariesis even compatible with the use of organic solvent [52].…”