Microfluidics Immunoaffinity Selection and Profiling of Disease-associated Extracellular Vesicles for Early Detection of Ovarian Cancer

Extracellular vesicles (EVs) are small membrane-bound vesicles secreted by cells that serve as promising biomarkers for early cancer detection. Recent studies have identified integrin beta-3 (ITGB3) and integrin alpha-5 (ITGA5) as EV-associated proteins overexpressed in ovarian cancer patients, particularly those in early stages. These markers demonstrated high diagnostic performance, with area under the curve (AUC) values of 0.98 for ITGB3 and 0.95 for ITGA5. Given the heterogeneity of EV populations and the variability in marker expression, we employed a mixed monolayer of antibodies targeting both ITGB3 and ITGA5 to enhance the capture efficiency of ovarian cancer-associated EVs. An automated, microfluidic device with two million micropillars was used for EV isolation and controlled release via a photocleavable linker. Protein quantification using the BCA assay confirmed high antibody capture efficiency. Significantly elevated levels of ITGB3 and ITGA5 EVs were observed in ovarian cancer plasma samples compared to healthy controls (p < 0.05). Nanoparticle tracking analysis (NTA) and transmission electron microscopy (TEM) confirmed the size distribution and morphology of isolated EVs. Early-stage ovarian cancer patients showed a notably higher EV expression than healthy donors. Further analysis will be performed using the Spectradyne ARC particle analyzer, which combines microfluidic resistive pulse sensing (MRPS) with flow cytometry to provide comprehensive profiling of isolated EVs. This approach can support the use of expanded biomarker panels to improve diagnostic accuracy in ovarian cancer.

Key words: extracellular vesicles, microfluidics, integrins, ovarian cancer, early detection