Droplet based microfluidics are promising new tools for biological and chemical assays. In this paper, a high throughput and high sensitivity microfluidic droplet platform is described for in vitro protein expression using crude Escherichia coli S30 extract. A flow-focusing polymethylmethacrylate (PMMA) microchip was designed and integrated with different functions involving droplet generation, storage, separation and detection. The material used for the chip is superior to the previously tested polydimethylsiloxane (PDMS) due to its mechanical and chemical properties. Droplet formation characteristics such as size and generation rate are investigated systematically. The effect of surfactants Abil EM90 and Span80 in the oil phase on droplet formation and optical detection is also studied. The performance of the system is demonstrated by the high throughput and stable droplet generation and ultralow detection limit. The robustness of the system is also demonstrated by the successful synthesis of a green fluorescent protein (GFP) using E. coli S30 extract as a source of RNA translation reagents.

Increasingly over the past two decades, biotechnologists have been exploiting various molecular technologies for high-throughput screening of genes and their protein products to isolate novel functionalities with a wide range of industrial applications. One particular technology now widely used for these purposes involves directed evolution, an artificial form of evolution in which genes and proteins are evolved towards new or improved functions by imposing intense selection pressures on libraries of mutant genes generated by molecular biology techniques and expressed in heterologous systems such as Escherichia coli. Most recently, the rapid development of droplet-based microfluidics has created the potential to dramatically increase the power of directed evolution by increasing the size of the libraries and the throughput of the screening by several orders of magnitude. Here, we review the methods for generating and controlling droplets in microfluidic systems, and their applications in directed evolution. We focus on the methodologies for cell-based assays, in vitro protein expression and DNA amplification, and the prospects for using such platforms for directed evolution in next-generation biotechnologies.

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