Rapid and high-throughput generation of agarose and gellan droplets by pump-free microfluidic step emulsification

Step emulsification generates droplets by a sharp change in confinement. It has emerged as a promising alternative to flow-focusing technology for high-throughput generation of droplets. Here we report a step emulsification technique consisting of two successive pipetting steps to generate more than 100,000 droplets from viscous hydrogel solutions in 5 min and using only 15 µL sample volume. We generated, on average, 1,17,556 $\pm$ 10,299 (mean $\pm$ SEM) and 1,75,704 $\pm$ 8771 droplets using agarose (0.1 % w/v) and gellan (0.7 % w/v) respectively. We also generated water droplets with 9 µm diameter which has rarely been reported using step emulsification. Further, we encapsulated red blood cells (RBC) or reaction mixtures (such as nucleic acids) inside droplets generated by our pump-free technique. Finally, we successfully demonstrated DNA amplification from the malarial parasite P. falciparum and a SARS-CoV-2 plasmid inside these droplets using loop-mediated isothermal amplification (LAMP). The ability to (a) generate droplets by step emulsification from a viscoelastic dispersed phase with a higher range of viscosities than what has been reported earlier, (b) generate > 100,000 hydrogel droplets from a very small sample volume within 5 min, (c) successfully encapsulate cells or a biological reaction mixture during droplet generation, and (d) perform DNA amplification within these hydrogel droplets, opens up possibilities of many point-of-care applications of this platform technology.