Nano‐Optical Biomolecular Interaction Sensing Within Complex Media via Peptide Hybrid Ligand Conjugation

Protein-protein interactions (PPIs) govern most biological processes and are frequently altered in disease. However, in vitro characterization methods are often limited to binding affinities, specialized instrumentation, or are susceptible to extended-surface artifacts. Moreover, many remain incompatible with physiological media. Nano-optical biosensing based on nanoparticle (NP) localized surface plasmon resonances (LSPRs) offers an attractive in-solution alternative, but their performance has been limited in complex environments. Here, we present a solution-phase optical platform based on plasmonic nanoparticles functionalized with programmable peptide-hybrid ligands. We use sequence-defined ligands that combine nanoparticle anchoring, suppression of non-specific interactions, and site-specific bioconjugation. Unlike conventional polymers, these ligands are molecularly precise via solid-phase peptide synthesis. We show how this architecture effectively suppresses nonspecific adsorption while preserving colloidal stability and optical performance, and can be adapted to diverse protein targets through click chemistry and Sortase-mediated ligation. This platform enables high-sensitivity, label-free PPI detection via standard UV-vis spectrophotometry and minimal sample amounts. We quantify binding affinities of low molecular-weight proteins directly in undiluted serum, along with kinetic profiling in buffer. Furthermore, we demonstrate applicability over multiple protein pairs in buffer and serum, as well as proof-of-concept detection and quantification in human serum, highlighting its potential for future diagnostic applications.