Multiplexed Peptide-Based Fluorescent Probe Platform for Analysis of HER2-Positive Exosomal Membrane Proteins

Breast cancer (BC) exhibits substantial molecular heterogeneity, with HER2-Positive (HER2+) subtypes accounting for 15–20% of cases and associated with aggressive behavior. Current diagnostics rely on invasive biopsies and often fail to capture dynamic changes or enable longitudinal monitoring. Exosomes, nanoscale vesicles reflecting the molecular profiles of their parent cells, offer a promising liquid biopsy approach. Here, we present a peptide-based fluorescent probe platform for the sensitive detection of HER2-positive exosomes. HER2-binding peptides were rationally designed and optimized computationally, and their binding affinity was validated using molecular docking and isothermal titration calorimetry. Site-specific conjugation of C12 alkyl chains and fluorescent dyes enhanced interfacial localization and fluorescence output. The probes demonstrated preferential recognition of HER2-positive exosomes in cell-derived samples as well as in human plasma and urine. Multivariate analysis was applied to interpret the multiplexed fluorescence signals, illustrating the method’s potential for distinguishing HER2-enriched exosomes from diverse biological backgrounds. This work establishes a modular and noninvasive sensing strategy for profiling exosomal membrane proteins.