title: Single-Fluorophore Detection in Femtoliter Droplets Generated by Flow Focusing.
abstract: 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.
authors.affiliation: Department of Biochemistry, University of Leicester , Leicester, LE1 9HN, United Kingdom.|Department of Chemistry, University of Leicester , Leicester, LE1 7RH, United Kingdom.
authors.forename: Robert
authors.initials: R
authors.lastname: Weinmeister
authors.affiliation: Department of Chemistry, University of Leicester , Leicester, LE1 7RH, United Kingdom.
authors.forename: Emma
authors.initials: E
authors.lastname: Freeman
authors.affiliation: Department of Biochemistry, University of Leicester , Leicester, LE1 9HN, United Kingdom.
authors.forename: Ian C
authors.initials: IC
authors.lastname: Eperon
authors.affiliation: Department of Chemistry, University of Leicester , Leicester, LE1 7RH, United Kingdom.
authors.forename: Alison M
authors.initials: AM
authors.lastname: Stuart
authors.affiliation: Department of Chemistry, University of Leicester , Leicester, LE1 7RH, United Kingdom.
authors.forename: Andrew J
authors.initials: AJ
authors.lastname: Hudson
mesh_terms: D004867:Equipment Design; D005453:Fluorescence; D005456:Fluorescent Dyes / Q000032:analysis; D049452:Green Fluorescent Proteins / Q000032:analysis; D057446:Hydrodynamics; D049408:Luminescent Agents / Q000032:analysis; D046210:Microfluidic Analytical Techniques / Q000295:instrumentation*; D061848:Optical Imaging / Q000295:instrumentation*; D038761:Photobleaching; D018401:Sample Size
chemical_list: D005456:Fluorescent Dyes; D049408:Luminescent Agents; D049452:Green Fluorescent Proteins
keywords: digital microfluidics; droplet encapsulation; flow focusing; in vitro compartmentalization; lab-on-a-chip; single-molecule fluorescence; soft lithography