Decoding Biofilm–Surface Interactions: A Hyperspectral Infrared Platform Links Interfacial Chemistry To Stress Adaptation and Assembly Mechanism

The formation of resilient biofilms at material interfaces remains a persistent challenge across biomedical, industrial, and environmental systems. Progress has been hindered by the lack of tools that can correlate the spatial chemical heterogeneity of biofilms with the properties of the underlying surface. In this work, we introduce a transformative hyperspectral infrared imaging platform that uniquely integrates three previously disparate capabilities: (i) biomimetic surface engineering to mimic tissue interfaces, (ii) synchrotron infrared microspectroscopy for label-free chemical mapping, and (iii) integration with transcriptomic sequencing to link chemical phenotypes with genetic mechanisms. Using collagen I-functionalized surfaces to mimic biotic interfaces and bare CaF2 as an abiotic control, we demonstrate that surface biochemistry actively guides biofilm architecture and composition. Quantitative analysis revealed clear differences in biofilm thickness and biochemical makeup between biotic and abiotic surfaces. Biofilms formed on collagen exhibited an unrecognized matrix-enriched phenotype, characterized by surface polysaccharide enrichment under copper stress, a response correlated with the upregulation of exopolysaccharide biosynthesis genes. In contrast, planktonic cells underwent acute metabolic disruption. By bridging spatial chemometrics with molecular biology, this study provides a mechanistic framework for understanding and controlling biofilm formation through rational interface design. The approach holds direct implications for antibiofilm surface engineering and infection control strategies.