Fluorescence Resonance Energy Transfer Reveals β-Sheet–Dominant Structural Features in Extracellular Vesicles from Pancreatic Cancer Detection

Protein misfolding and β-sheet aggregation, long recognized as hallmarks of neurodegenerative proteinopathies, are increasingly implicated in cancer biology. In pancreatic ductal adenocarcinoma (PDAC), tumor cells release extracellular vesicles (EVs) containing β-sheet–rich protein assemblies that may alter intercellular communication and promote malignancy. We report a fluorescence resonance energy transfer (FRET) assay that directly detects these β-sheet–dominated structures in circulating EVs as a physical biomarker for PDAC. The assay couples Thioflavin T (ThT), an amyloid-binding fluorophore, with fluorescein-labeled Dolichos biflorus agglutinin (DBA–FITC), which recognizes N-acetyl-D-galactosamine residues abundant on pancreatic epithelial membranes. When both probes engage the same EV surface within a Förster distance (<10 nm), energy transfer occurs, producing a distinct FRET emission signature at 520 nm. EVs from PDAC samples exhibited significantly stronger FRET signals than those from healthy or benign controls, confirming that pancreatic tumor–derived vesicles are enriched in β-sheet structural motifs. Quantitative analysis yielded an area under the ROC curve of 0.95, surpassing the clinical standard CA19-9 (0.72), with high reproducibility and minimal background noise. The single-step measurement requires no vesicle isolation and has a low cost per test. These results reveal a measurable link between protein misfolding and cancer-derived extracellular vesicles, demonstrating that structural biophysics can provide new routes for disease detection. Beyond diagnostics, this approach offers a physical framework for studying protein aggregation phenomena across cancer and other misfolding-driven pathologies.