Ultrahigh-throughput screening in drop-based microfluidics for directed evolution

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Original Entry: Tom Dimiduk APPY 225 Fall 2010

Ultrahigh-throughput screening in drop-based microfluidics for directed evolution


Microfluidics Droplet Microfludics Fluorocarbon Surfactant Plug Flow


Figure 1: "Photographs of device in operation. (A and B) Droplet formation (C) droplets with cells (D) Fluorescent Product detection (E) Sorting".

Figure 1: "Cartoon of SELEX process. They generate a gene library which expresses the protein on the surface of cells. They use a enzyme substrate which becomes florescent after processing, allowing detection and sorting based on enzyme activity. ".

The authors have developed a droplet microfluidics based system for directed evolution (SELEX) that is ~1000 fold faster and ~1,000,000 cheaper than current state of the art robotic techniques. They are able to sort through ~10^8 mutant enzymes in less than a day to develop an improved peroxidase enzyme that is ten fold faster than its already highly efficient wild type precursor.

They use a 2 stage mutation process, the first with broadly dispersed mutations across the entire protein and selecting any proteins that still have appreciative activity, and then a second stage of targeted mutations at the active site of the enzyme and selection for the highest activity. Their rational here is that some structural mutations might initially decrease activity but might stabilize mutant active sites with higher affinity.

They conclude with some of the usual microfluidics selling points that this form of drastic density increase and cost reduction will hopefully enable a biotech revolution in the way computer semiconductor technology and moore's law has revolutionized the world.

Soft Matter Discussion

Their system is water droplets containing cells in fluorocarbon oil. They do not say why they use the fluorocarbon oil, but I would presume it is for density matching. They use a flourosurfactant to stabilize the droplets by reducing their surface energy to the point where they are not prone to coalescence or oswald ripening.

They use an incubation tube which they configure such that the droplets proceed through it in a plug flow regime. This allows approximately equal incubation times for all droplets as opposed to a laminar flow system where the droplets near the tube wall would flow slower and thus have longer incubation times.

Their flow rate is limited by shear on the droplets in the curved arms which would tend to break up the droplets. Thus keeping this shear low turns out to be the limiting design factor.