Automated generation of libraries of nL droplets.

We demonstrate an integrated system for rapid and automated generation of multiple, chemically distinct populations of 10(3)-10(4) sub-nanoliter droplets. Generation of these 'libraries of droplets' proceeds in the following automated steps: i) generation of a sequence of micro-liter droplets of individually predetermined composition, ii) injection of these 'parental' droplets onto a chip, iii) transition from a mm- to a μm-scale of the channels and splitting each of the parental drops with a flow-focusing module into thousands of tightly monodisperse daughter drops and iv) separation of such formed homogeneous populations with plugs of a third immiscible fluid. This method is compatible both with aspiration of microliter portions of liquid from a 96-well plate with a robotic station and with automated microfluidic systems that generate (μL) droplets of preprogrammed compositions. The system that we present bridges the techniques that provide elasticity of protocols executed on microliter droplets with the techniques for high-throughput screening of small (~pL, ~nL) droplet libraries. The method that we describe can be useful in exploiting the synergy between the ability to rapidly screen distinct chemical environments and to perform high-throughput studies of single cells or molecules and in digital droplet PCR systems.