An anion exchange membrane sensor detects EGFR and its activity state in plasma CD63 extracellular vesicles from patients with glioblastoma
Abstract 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...
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Nature Portfolio
2024-06-01
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| Series: | Communications Biology |
| Online Access: | https://doi.org/10.1038/s42003-024-06385-1 |
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| author | Nalin H. Maniya Sonu Kumar Jeffrey L. Franklin James N. Higginbotham Andrew M. Scott Hui K. Gan Robert J. Coffey Satyajyoti Senapati Hsueh-Chia Chang |
| author_facet | Nalin H. Maniya Sonu Kumar Jeffrey L. Franklin James N. Higginbotham Andrew M. Scott Hui K. Gan Robert J. Coffey Satyajyoti Senapati Hsueh-Chia Chang |
| author_sort | Nalin H. Maniya |
| collection | DOAJ |
| description | Abstract 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. |
| format | Article |
| id | doaj-art-73f922b965a546b2a477dcf2d44e21d8 |
| institution | Kabale University |
| issn | 2399-3642 |
| language | English |
| publishDate | 2024-06-01 |
| publisher | Nature Portfolio |
| record_format | Article |
| series | Communications Biology |
| spelling | doaj-art-73f922b965a546b2a477dcf2d44e21d82025-08-20T03:46:11ZengNature PortfolioCommunications Biology2399-36422024-06-017111110.1038/s42003-024-06385-1An anion exchange membrane sensor detects EGFR and its activity state in plasma CD63 extracellular vesicles from patients with glioblastomaNalin H. Maniya0Sonu Kumar1Jeffrey L. Franklin2James N. Higginbotham3Andrew M. Scott4Hui K. Gan5Robert J. Coffey6Satyajyoti Senapati7Hsueh-Chia Chang8Department of Chemical and Biomolecular Engineering, University of Notre DameDepartment of Chemical and Biomolecular Engineering, University of Notre DameDepartment of Medicine, Vanderbilt University Medical CenterDepartment of Medicine, Vanderbilt University Medical CenterTumour Targeting Laboratory, Olivia Newton-John Cancer Research InstituteTumour Targeting Laboratory, Olivia Newton-John Cancer Research InstituteDepartment of Medicine, Vanderbilt University Medical CenterDepartment of Chemical and Biomolecular Engineering, University of Notre DameDepartment of Chemical and Biomolecular Engineering, University of Notre DameAbstract 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.https://doi.org/10.1038/s42003-024-06385-1 |
| spellingShingle | Nalin H. Maniya Sonu Kumar Jeffrey L. Franklin James N. Higginbotham Andrew M. Scott Hui K. Gan Robert J. Coffey Satyajyoti Senapati Hsueh-Chia Chang An anion exchange membrane sensor detects EGFR and its activity state in plasma CD63 extracellular vesicles from patients with glioblastoma Communications Biology |
| title | An anion exchange membrane sensor detects EGFR and its activity state in plasma CD63 extracellular vesicles from patients with glioblastoma |
| title_full | An anion exchange membrane sensor detects EGFR and its activity state in plasma CD63 extracellular vesicles from patients with glioblastoma |
| title_fullStr | An anion exchange membrane sensor detects EGFR and its activity state in plasma CD63 extracellular vesicles from patients with glioblastoma |
| title_full_unstemmed | An anion exchange membrane sensor detects EGFR and its activity state in plasma CD63 extracellular vesicles from patients with glioblastoma |
| title_short | An anion exchange membrane sensor detects EGFR and its activity state in plasma CD63 extracellular vesicles from patients with glioblastoma |
| title_sort | anion exchange membrane sensor detects egfr and its activity state in plasma cd63 extracellular vesicles from patients with glioblastoma |
| url | https://doi.org/10.1038/s42003-024-06385-1 |
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