Surface Plasmon Resonance Unveils Diffusion Fingerprints of Biomolecular Mixtures in Ocular Fluid Models

Analyzing diffusion processes within complex biomolecular mixtures is essential but technically challenging, especially for understanding molecular interactions in physiological environments. Here, we apply the diffusion-based surface plasmon resonance ( D -SPR), a recent, label-free methodology that combines accurate SPR-based diffusion measurements with stochastic computational simulations, to address biomolecular complexity in ocular fluid models. By combining discrete Fréchet distance analysis with Gillespie algorithm simulations, D -SPR effectively detects unique diffusion patterns in binary (BSA/glycine, BSA/glucose) and ternary (HSA/ubiquitin/glycine) mixtures, enabling composition characterization without the need for external fluorophores or chromatographic techniques. Demonstrating the method sensitivity, as a proof-of-concept, we applied our workflow to probe the oligomeric transitions of bovine lens α-crystallin induced by mild acidification (pH 6.5). Our findings revealed subtle oligomer dissociation events and identified smaller, rapidly diffusing subunits, undetectable by conventional dynamic light scattering, thus providing insights into protein structural changes potentially pertinent to cataractogenesis. Considering the critical role of diffusion-based processes in eye health, the proposed D -SPR approach provides an advanced and versatile analytical tool with significant potential applications in medical research, especially ophthalmology.