FTIR Imaging as a Tool for Studying Protein Dynamics and Interactions at the Oil/Water Interface with Spatial Resolution: A Single Droplet Analysis Approach

Emulsions play a pivotal role in the food, pharmaceutical, and cosmetic industries due to their unique structural properties and versatility in delivering active ingredients. Developing emulsion systems with optimized structural and functional properties is a key focus, requiring a comprehensive understanding of their physicochemical attributes. A major challenge in emulsion research is accurately characterizing the molecular-level interactions at the interface, which are critical for stability, performance, and functionality. In this study, Fourier-transform infrared (FTIR) imaging was employed as a nondestructive technique to chemically map single emulsion droplet interfaces <i>in situ</i> with spatial resolution. A microfluidic platform was utilized to generate single emulsion droplets stabilized by bovine serum albumin (BSA), enabling the comparison of three systems: droplets stabilized by native BSA, droplets stabilized by native BSA followed by heat treatment, and droplets stabilized by preheat-treated BSA. Conformational changes induced by processing and protein adsorption at the oil/water interface were evaluated through characterization of the amide I band. FTIR imaging effectively distinguished protein-rich and oil phases, revealing detailed structural variations at the interface. Spectral analysis of the water phase identified distinct protein zones with varying structural motifs and concentrations surrounding the droplets. Both the secondary structure and spatial distribution of proteins at the interface were significantly influenced by the processing conditions. This study demonstrates FTIR imaging as a powerful tool for investigating emulsion interfaces and tracking structural changes associated with protein processing history. These findings provide critical insights into the design of protein-stabilized emulsions with tailored functional properties.