Single-Fluorophore Detection in Femtolitre Droplets Generated by Flow Focussing

Aqueous microdroplets with a volume of a few femtolitres 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 focussing, will often involve volumes that are typically too large (>0.5 pl) for single molecule studies. Desired volumes of a few femtolitres, or smaller, can be produced by either tip streaming or step emulsification in a flow-focussing device; however, in both of these methods, the aqueous droplets are dispersed in a large volume of the continuous phase, where individual dropets 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 timescales. We show here that the optimised design and fabrication of microfluidic devices with shallow channel depths can result in the reliable production of stable droplets of a few femtolitres at a high rate in the dripping regime of flow focussing. Furthermore, the generated microdroplets are localised 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.