Single-Fluorophore Detection in Femtoliter Droplets Generated by Flow Focusing

Aqueous microdroplets with a volume of a few femtoliters are an ideal sample size for single-molecule fluorescence experiments. In particular, they enable prolonged measurements to be made on individual molecules that can diffuse freely in the surrounding medium. However, the rapid production of monodisperse droplets in a hydrodynamic flow, such as microfluidic flow focusing, will often involve volumes that are typically too large (>0.5 pL) for single-molecule studies. Desired volumes of a few femtoliters, or smaller, can be produced by either tip streaming or step emulsification in a flow-focusing device; however, in both of these methods, the aqueous droplets are dispersed in a large volume of the continuous phase, where individual droplets can diffuse perpendicular to the flow direction, and the monodispersity of droplet size produced by tip streaming is difficult to sustain for more than transient time scales. We show here that the optimized design and fabrication of microfluidic devices with shallow channel depths can result in the reliable production of stable droplets of a few femtoliters at a high rate in the dripping regime of flow focusing. Furthermore, the generated microdroplets are localized in a two-dimensional plane to enable immediate analysis. We have demonstrated the fluorescence monitoring of single molecules of encapsulated green fluorescent protein. The apparatus is straightfoward, inexpensive, and readily assembled within an ordinary laboratory environment.