A Multiplexed Cell-Free Assay to Screen for Antimicrobial Peptides in Double Emulsion Droplets.

The global surge in bacterial resistance against traditional antibiotics triggered intensive research for novel compounds, with antimicrobial peptides (AMPs) identified as a promising candidate. Automated methods to systematically generate and screen AMPs according to their membrane preference, however, are still lacking. We introduce a novel microfluidic system for the simultaneous cell-free production and screening of AMPs for their membrane specificity. On our device, AMPs are cell-free produced within water-in-oil-in-water double emulsion droplets, generated at high frequency. Within each droplet, the peptides can interact with different classes of co-encapsulated liposomes, generating a membrane-specific fluorescent signal. The double emulsions can be incubated and observed in a hydrodynamic trapping array or analyzed via flow cytometry. Our approach provides a valuable tool for the discovery and development of membrane-active antimicrobials.

Compartmentalization - A Prerequisite for Maintaining and Changing an Identity.

The chemical manipulation of DNA is much more convenient than the manipulation of the bioproducts, such as enzymes, that it encodes. The optimization of bioproducts requires cycles of diversification of DNA followed by read-out of the information into the bioproduct. Maintaining the link between the information - the genotype - and the properties of the bioproduct - the phenotype - through some form of compartmentalization is therefore an essential aspect in directed evolution. While the ideal compartment is a biological cell, many projects involving more radical changes in the bioproduct, such as the introduction of novel cofactors, may not be suitable for expression of the information in cells, and alternative in vitro methods have to be applied. Consequently, the possibility to produce simple and advanced micro compartments at high rates and to combine them with the ability to translate the information into proteins represents a unique opportunity to explore demanding enzyme engineering projects that require the evaluation of at least hundreds of thousands of enzyme variants over multiple generations.

Silicon-Based 3D Microfluidics for Parallelization of Droplet Generation.

Both the diversity and complexity of microfluidic systems have experienced a tremendous progress over the last decades, enabled by new materials, novel device concepts and innovative fabrication routes. In particular the subfield of high-throughput screening, used for biochemical, genetic and pharmacological samples, has extensively emerged from developments in droplet microfluidics. More recently, new 3D device architectures enabled either by stacking layers of PDMS or by direct 3D-printing have gained enormous attention for applications in chemical synthesis or biomedical assays. While the first microfluidic devices were based on silicon and glass structures, those materials have not yet been significantly expanded towards 3D despite their high chemical compatibility, mechanical strength or mass-production potential. In our work, we present a generic fabrication route based on the implementation of vertical vias and a redistribution layer to create glass-silicon-glass 3D microfluidic structures. It is used to build different droplet-generating devices with several flow-focusing junctions in parallel, all fed from a single source. We study the effect of having several of these junctions in parallel by varying the flow conditions of both the continuous and the dispersed phases. We demonstrate that the generic concept enables an upscaling in the production rate by increasing the number of droplet generators per device without sacrificing the monodispersity of the droplets.

Direct electrification of silicon microfluidics for electric field applications.

Microfluidic systems are widely used in fundamental research and industrial applications due to their unique behavior, enhanced control, and manipulation opportunities of liquids in constrained geometries. In micrometer-sized channels, electric fields are efficient mechanisms for manipulating liquids, leading to deflection, injection, poration or electrochemical modification of cells and droplets. While PDMS-based microfluidic devices are used due to their inexpensive fabrication, they are limited in terms of electrode integration. Using silicon as the channel material, microfabrication techniques can be used to create nearby electrodes. Despite the advantages that silicon provides, its opacity has prevented its usage in most important microfluidic applications that need optical access. To overcome this barrier, silicon-on-insulator technology in microfluidics is introduced to create optical viewports and channel-interfacing electrodes. More specifically, the microfluidic channel walls are directly electrified via selective, nanoscale etching to introduce insulation segments inside the silicon device layer, thereby achieving the most homogeneous electric field distributions and lowest operation voltages feasible across microfluidic channels. These ideal electrostatic conditions enable a drastic energy reduction, as effectively shown via picoinjection and fluorescence-activated droplet sorting applications at voltages below 6 and 15 V, respectively, facilitating low-voltage electric field applications in next-generation microfluidics.