Static microdroplet array generated by spraying and analyzed with automated microscopy and image processing.

Microdroplets have received increasing interest as practical platforms for high-throughput biochemical analysis. Typically, numerous discrete aqueous microdroplets containing biochemical targets are generated in a continuous oil phase and characterized using a flow-through configuration. Although this approach is capable of extremely high throughput, it is challenging to provide dynamic characterization of time-dependent reaction kinetics. In this paper, we present a practical and affordable method to create and analyze a massive array of static aqueous microdroplets immersed in oil for biochemical analysis. The discrete microdroplets were produced by an air-spray gun, imaged by automated microscopy, and then characterized by image processing. The location, area, and fluorescence intensity of randomly generated individual microdroplets were automatically registered for high-throughput characterization. With this approach, we rapidly produced and characterized a static microdroplet array of over 0.7 million microdroplets with an average volume of 300 fL and a mean population density of 1.5*10 microdroplets/cm. Using the developed setup, we demonstrated the dependency of the microdroplets' fluorescence intensity on their volume, as well as characterized the time-dependent enzyme reaction kinetics of β-galactosidase-mediated cleavage of the substrate fluorescein di-β-d-galactopyranoside (FDG). The new approach described herein provides an inexpensive alternative solution for high-throughput analysis of dynamic biochemical processes.