Natural polymers represent promising materials for sustainable applications such as food packaging. However, their use remains limited due to poor mechanical performance, high water sensitivity, and insufficient barrier properties. Multilayer structures can overcome these drawbacks by integrating complementary materials, for which interfacial compatibility is critical. Herein, colloidal-probe atomic force microscopy (AFM) was used to quantify nanoscale adhesion forces and elucidate interfacial interactions in biopolymer-based multilayer films. The films were constructed from polysaccharides, specifically carboxymethylcellulose (CMC), and proteins, casein and zein, assembled with and without tannic acid (TA) as a cross-linker. AFM adhesion measurements obtained using a casein-modified probe were correlated with macroscale delamination, surface wettability, water vapor permeability, and water uptake. Casein-CMC interfaces exhibited a low normalized pull-off force (0.005 mN·m-1), which increased to 0.02 and 0.04 mN·m-1 upon incorporating 5% and 30% of TA, respectively. Enhanced adhesion with longer probe-substrate contact time supported interfacial cross-linking between CMC and proteins mediated by TA. This cross-linking also improved moisture resistance, evidenced by an 80% reduction in equilibrium moisture content in TA-containing casein-CMC bilayer films. The adhesion between casein and zein (both in terms of normalized pull-off forces and normalized adhesion energies) was significantly higher than between casein and CMC, indicating strong protein-protein affinity. However, this protein-protein adhesion was insensitive to TA, likely due to intralayer cross-linking as opposed to interlayer cross-linking. Colloidal-probe AFM was an effective approach for resolving interfacial interactions to expand the versatility of biopolymer-based multilayer packaging, and for highlighting the role of nanoscale adhesion in guiding materials selection and interface engineering.
