Reliable microfluidic on-chip incubation of droplets in delay-lines

Together with droplet creation, fusion and sorting, the incubation of droplets is one of the most important and essential operations for droplet-based microfluidic assays. This manuscript concerns the development of delay-lines, which are necessary to allow incubation of reactions for precise time periods. We analyze the problems associated with creating delay-lines for incubation in the minute to hour time range, which arise from back-pressure and from the dispersion in the incubation time due to the unequal speeds with which droplets pass through the delay-line. We describe delay-line systems which resolve these problems and demonstrate their use to measure reaction kinetics over several minutes in droplets.