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Currently, one of the most prominent methods used to impart biocompatibility to aqueous-in-oil droplets is to synthesize a triblock copolymer surfactant composed of perfluoropolyether and polyether blocks. The resulting surfactants (EA surfactant, KryJeffa, etc.) allow generation of highly biocompatible droplet surfaces while maintaining the heat stability of the starting material. However, production of these surfactants requires expertise in synthetic organic chemistry, creating a barrier to widespread adoption in the field. Herein, we describe a simple alternative to synthetic modification of surfactants to impart biocompatibility. We have observed that aqueous-in-oil droplet surfaces can be made biocompatible and heat stable by merely exploiting binding interactions between polyetherdiamine additives in the aqueous phase and carboxylated perfluorocarbon surfactants in the oil phase. Droplets formed under these conditions are shown to possess biocompatible surfaces capable of supporting picoliter-scale protein assays, droplet polymerase chain reaction (PCR), and droplet DNA amplification with isothermal recombinase polymerase amplification (RPA). Droplets formed with polyetherdiamine aqueous additives are stable enough to withstand temperature cycling during PCR (30-40 cycles at 60-94 °C) while maintaining biocompatibility, and the reaction efficiency of RPA is shown to be similar to that with a covalently modified surfactant (KryJeffa). The binding interaction was confirmed with various methods, including FT-IR spectroscopy, NMR spectroscopy, electrospray ionization mass spectrometry (ESI-MS), and fluorescence microscopy. Overall, our results suggest that, by simply introducing a commercially-available, polyetherdiamine additive (Jeffamine ED-900) to the aqueous phase, researchers can avoid synthetic methods in generating biocompatible droplet surfaces capable of supporting DNA and protein analysis at the subnanoliter scale.

Creating Biocompatible Oil–Water Interfaces without Synthesis: Direct Interactions between Primary Amines and Carboxylated Perfluorocarbon Surfactants