Dual recognition of beta-sheet proteinopathy and glycan moieties enables noninvasive detection of pancreatic ductal adenocarcinoma

Early detection of pancreatic ductal adenocarcinoma (PDAC) remains a formidable challenge due to the absence of reliable and accessible screening tools. In contrast to other cancers with established diagnostic methods, PDAC is often detected only after metastasis, resulting in poor survival outcomes. Current biomarkers such as CA19-9 offer limited diagnostic accuracy and frequently yield false positives, emphasizing the urgent need for a more specific and cost-effective approach. Here, we introduce EV-FRET, a biosensing assay that detects PDAC through fluorescence resonance energy transfer (FRET) signaling from tumor-derived extracellular vesicles (EVs). EV-FRET focuses on the molecular cargo of EVs secreted by cancer cells, by simultaneously targeting beta-sheet-rich misfolded proteins, indicative of tumor proteinopathy, and N-acetyl-D-galactosamine (GalNAc), a glycan enriched in pancreatic epithelial cells. The assay uses thioflavin T (ThT) and fluorescein-labeled Dolichos biflorus agglutinin (FITC-DBA) as the donor-acceptor pair, generating a quantifiable FRET signal only when both tumor and organ markers coexist on EV surfaces. Without requiring EV isolation, the assay provides a single-step, reproducible (coefficient of variation < 4%) and low-cost method. The diagnostic performance revealed an AUC of 0.95 compared with 0.72 for CA 19-9, demonstrating improved specificity. EV-FRET demonstrates the potential for noninvasive PDAC detection and presents a flexible framework for adapting protein structure based diagnostics to other malignancies.