High-throughput quantification of interfacial protein load in monodisperse oil-in-water emulsions

Food proteins play a critical role as emulsifiers, where adsorption at the oil and water interface governs emulsion formation and stability. However, quantitative characterization of interfacial protein load remains challenging due to polydispersity in conventional emulsification methods. We combine microfluidic generation of monodisperse emulsions with colorimetric protein quantification to measure the post-collection initial interfacial load ($Γ_0$) and subsequently monitored over 48 h to obtain the saturation load ($Γ_\infty$). Interfacial load exhibited a strong bulk concentration-dependent trend and was well described by a pseudo-first-order model. Both $Γ_0$ and $Γ_\infty$ followed sublinear scaling with concentration ($Γ_0$ ~ C^{0.21}$, $Γ_\infty$ ~ C^{0.12}$). Interfacial loads were largely independent of droplet diameter, with modest reductions in initial loading for the smallest droplets at low protein concentration. Measured loads exceeded idealized monolayer predictions and fell between bilayer and trilayer bounds, consistent with accumulation beyond a simplified monolayer model. Additionally, high-throughput mapping was performed across three proteins, seven concentrations, three pH values, and two ionic strengths, revealing that $α$-Casein exhibited higher interfacial loads than $β$-Casein and Whey protein Isolate, with maximum adsorption near the isoelectric point and at elevated ionic strength. In summary, this study establishes a quantitative experimental framework for linking protein properties, formulation conditions, and interfacial behavior using monodisperse microfluidic emulsions, while requiring only milligram-scale protein quantities.