Key Factors for Stable Retention of Fluorophores and Labeled Biomolecules in Droplet-Based Microfluidics

Abstract

Water-in-oil emulsion droplets created in droplet-based microfluidic devices have been tested and used recently as well-defined picoliter-sized 3D compartments for various biochemical and biomedical applications. In many of these applications, fluorescence measurements are applied to reveal the protein content, spatial distribution, and dynamics in the droplets. However, emulsion droplets do not always provide entirely sealed compartments, and partitioning of dyes or labeled molecules to the oil phase is frequently observed. Therefore, stable molecular retention in the droplets represents a challenge, and many physical and chemical key factors of microfluidic system components have to be considered. In this study, we investigated the retention of 12 commonly used water-soluble dyes in droplets having six different aqueous phase conditions. We demonstrate that the physicochemical properties of the dyes have a major influence on the retention level. In particular, hydrophilicity has a strong influence on retention, with highly hydrophilic dyes (LogD < -7) showing stable, buffer/medium independent retention. In the case of less hydrophilic dyes, we showed that retention can be improved by adjusting the surfactants physical properties, such as geometry, length, and concentration. Furthermore, we analyzed the retention stability of labeled biomolecules such as antibodies, streptavidin, and tubulin proteins and showed that stable retention can be strongly dependent on dye and surfactants selection.

Surfactant adsorption kinetics in microfluidics

“…The practical consequence for emulsification in microfluidics is that although channels can be designed to give enough time for the surfactant to adsorb at the interface, the chemical binding to the interface is the key parameter determining the emulsion stabilization timescale. An important consequence of our analysis is that we also model the kinetics of equilibration of the chemical potential across the interface (33)(34)(35). The transfer of protons toward the aqueous droplet is balanced by the extraction of a counter ion from the droplet to the oil.…”

Stabilisers for water-in-fluorinated-oil dispersions: Key properties for microfluidic applications

“…A direct link between half-life of retention of the fluorophores in the emulsion droplets and the predicted partition coefficient of the dye was found. Recently the modulation of exchange rates with various buffers and additives was also demonstrated [49,92].…”

Selectively Permeable Double Emulsions

“…Indeed, double emulsions are frequently employed to store and deliver reagents, as picoliter‐sized reaction vessels to conduct chemical and biochemical reactions, or as templates to produce microcapsules . Certain surfactant‐stabilized double emulsions are permeable toward hydrophilic encapsulants, even though their solubility in the oil phase is very low . These double emulsions allow passive exchange of reagents.…”

“…These double emulsions allow passive exchange of reagents. However, such exchanges are difficult to control and often hamper the use of emulsions as tight containers . To improve the control over the reagent transport, surfactants that respond to stimuli, such as changes in pH and the presence of certain salts, have been developed .…”