Direct Visualization and Quantitative Modeling of Mass Transport Limitation in Biomolecular Kinetics via Imaging-Based Meta-SPR

Directly observing how mass transport limitation (MTL) distorts biomolecular kinetics has long remained out of reach. Here, we harness an imaging-based metasurface plasmon resonance (Meta-SPR) platform to both visualize and chemically quantify MTL effects in real time, revealing a pronounced flow-path signal decay beyond bulk depletion. This spatially resolved capability uniquely links optical contrast to the local analyte concentration, enabling quantitative mapping of MTL severity. Inspired by neural network loss-landscape theory, we show that the traditional mass transport coefficient (<i>k</i>_m) is fundamentally unidentifiable, exhibiting flat loss valleys that prevent robust fitting. To address this, we introduce two practical alternatives: a pseudoactivity model replacing <i>k</i>_m with a single factor (α_p) for intuitive MTL quantification and a free-<i>R</i>_max model that extracts robust kinetic parameters without known ligand density. Both strategies are compatible with standard fitting software and validated across Meta-SPR, SPR, and BLI platforms, offering broadly applicable tools for accurate affinity and kinetics determination.