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.