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In-Droplet Electrophoretic Separation and Enrichment of Biomolecules.

We demonstrate the in-droplet separation and enrichment of biomolecules, from small organic molecules to long nucleic acids (lambda DNA). Electric potentials are applied via two parallel three-dimensional liquid electrodes, which interface the nanodroplets through polydimethylsiloxane (PDMS)-carbon composite membranes. These membranes enable the generation of uniform electric fields inside the droplets, while simultaneously preventing the formation of electrolytic byproducts. Bi-omolecules of different sizes migrate towards one side of the droplets, according to their net charge, when exposed to the electric field. Directly afterwards, a Y-junction promotes droplet splitting, resulting in the generation of two biomolecules-enriched daughter droplets. The biomolecules were fluorescently labelled, and fluorescence microscopy was employed to as-sess their electrophoretic separation and enrichment. Experimental results demonstrate how the enrichment of biomolecules is influenced by their size, charge and concentration, by the ionic strength, viscosity and pH of the suspending medium, and by the in-droplet flow profile. Enrichments above 95% were observed for small molecules and highly-charged nucleic acids at velocities over 10 mm/s (13 droplets per second). Moreover, the enrichment performance asymptotically approached a value of 38% for velocities as high as 50 mm/s, demonstrating the potential of this technique for the high-throughput separation of charged species. We finally demonstrate the applicability of our system by cleaving a peptide and selectively separating the cleaved fragments and enzyme in different daughter droplets. This method adds a versatile module to the large toolbox of droplet analysis and manipulation.