Freeze-Float Selection of Ice Nucleators.

In this manuscript, we developed a screening system that employs the difference in density between liquid water and ice (0.9998 g/cm3 vs 0.9168 g/cm3 at 0 °C) to identify ice-nucleating agents (INAs) that are encapsulated into droplets of water suspended in silicone oil of intermediate density (0.939 g/cm3). Droplets of liquid water stably reside at the interface of the silicone oil and perfluoro oil (1.6658 g/cm3); freezing causes the aqueous droplets to float to the top of the silicone oil layer. We demonstrated the feasibility of this screening system by using droplets that contained well-defined ice-nucleator Snomax. The droplets with and without Snomax froze at different temperatures and separated into two groups in our system. We employed the screening system to test samples that have different ice-nucleating activities. Starting from known ice-nucleating active bacteria Pseudomonas syringae, we confirmed that droplets that contain an increasing amount of ice-nucleating bacteria per droplet exhibit a dose-dependent increase in ice nucleation. When droplets containing different amounts of P. syringae were separated using a freeze-float setup, we observed that the droplets that floated at higher temperature contained more ice-nucleating active bacteria. The outlined system, thus, permits simple power-free separation of droplets that contain effective INA from those that contain weak or no INA. Such a setup can be used as a starting point for the development of high-throughput approaches for the discovery of new INAs.