Abstract
ABSTRACT
Understanding how biomolecules organize at metal interfaces is of central importance for a wide range of sensing and biointerfacial technologies, yet it is still challenging to decipher their microscopic organization because conventional macroscopic measurements cannot reveal the hidden assembly states of flexible adsorbates. Here, a multiscale analytical strategy integrates electrochemical active surface area (ECSA), gap‐mode surface‐enhanced Raman spectroscopy (SERS), and ensemble‐based all‐atom molecular dynamics simulations to connect macroscopic adsorption behavior with microscopic assembly states on gold surfaces. Tyr‐Tyr‐Tyr (YYY), an extraordinarily adhesive peptide with an unresolved mechanism for its strong interfacial affinity, is used as a model system. The results show that fundamentally distinct microscopic organizations can exhibit nearly identical macroscopic adsorption states. Systematic sequence mutants further reveal that consecutive Tyr residues sustain the hydrogen‐bond‐driven lateral aggregation, from which Tyr‐Phe‐Tyr (YFY) emerges as a compact anchoring motif that retains strong gold affinity while suppressing aggregation. Beyond resolving this peptide‐gold assembly problem, our work advances an ensemble‐based perspective for interpreting interfacial Raman spectra and provides a foundation for programmable bio‐interface engineering, with potential applications in biosensing, peptide/protein arrays, and other functional bio‐metal platforms.