An anion exchange membrane sensor detects EGFR and its activity state in plasma CD63 extracellular vesicles from patients with glioblastoma

• Published in: Communications biology Vol. 7; no. 1; pp. 677 - 11
• Main Authors: Maniya, Nalin H., Kumar, Sonu, Franklin, Jeffrey L., Higginbotham, James N., Scott, Andrew M., Gan, Hui K., Coffey, Robert J., Senapati, Satyajyoti, Chang, Hsueh-Chia
• Format: Journal Article
• Language: English
• Published: London Nature Publishing Group UK 03.06.2024; Nature Publishing Group; Nature Portfolio
• Subjects: 631/67/2195; 692/53/2421; 82/62; 9/10; Biomarkers, Tumor - blood; Biomarkers, Tumor - metabolism; Biomedical and Life Sciences; Brain Neoplasms - blood; Brain Neoplasms - diagnosis; Brain Neoplasms - metabolism; CD63 antigen; Epidermal growth factor receptors; Epitopes; ErbB Receptors - metabolism; Extracellular vesicles; Extracellular Vesicles - metabolism; Glioblastoma; Glioblastoma - blood; Glioblastoma - diagnosis; Glioblastoma - metabolism; Glioma; Humans; Immunoassay - methods; Life Sciences; Monoclonal antibodies; Nanoparticles; Proteomics; Sensors; Tetraspanin 30 - metabolism
• ISSN: 2399-3642; 2399-3642
• DOI: 10.1038/s42003-024-06385-1

We present a quantitative sandwich immunoassay for CD63 Extracellular Vesicles (EVs) and a constituent surface cargo, EGFR and its activity state, that provides a sensitive, selective, fluorophore-free and rapid alternative to current EV-based diagnostic methods. Our sensing design utilizes a charge-gating strategy, with a hydrophilic anion exchange membrane functionalized with capture antibodies and a charged silica nanoparticle reporter functionalized with detection antibodies. With sensitivity and robustness enhancement by the ion-depletion action of the membrane, this hydrophilic design with charged reporters minimizes interference from dispersed proteins, thus enabling direct plasma analysis without the need for EV isolation or sensor blocking. With a LOD of 30 EVs/μL and a high relative sensitivity of 0.01% for targeted proteomic subfractions, our assay enables accurate quantification of the EV marker, CD63, with colocalized EGFR by an operator/sample insensitive universal normalized calibration. We analysed untreated clinical samples of Glioblastoma to demonstrate this new platform. Notably, we target both total and “active” EGFR on EVs; with a monoclonal antibody mAb806 that recognizes a normally hidden epitope on overexpressed or mutant variant III EGFR. Analysis of samples yielded an area-under-the-curve (AUC) value of 0.99 and a low p-value of 0.000033, surpassing the performance of existing assays and markers. Targeting conformally-active EGFR on small extracellular vesicles using a novel Charge-gated Ion Exchange Membrane sensor allows sensitive and non-invasive detection of Glioblastoma in human subjects from untreated plasma.