3f77cc1590f0f241a0fe19065295a500 biosensors-15-00694.pdf 41d3da99fa9c84f3e5c1340d86580dfdbf2833e4 biosensors-15-00694.pdf 3505a9378a230586f0222bd7a7aa62d689de35ebf6aa93ae20c80244130783d2 biosensors-15-00694.pdf Title: Exosome Biomarker Profiling Using a Paper-Based Vertical Flow Assay Subject: Exosomes are nanoscale extracellular vesicles that carry valuable biomolecular information. However, their characterization still depends on complex and costly techniques such as flow cytometry. In this study, a paper-based Vertical Flow Assay (VFA) specifically designed for the detection and profiling of exosomes derived from metastatic breast cancer cell lines is presented. The assay operates in an ELISA-like format, targeting exosomal surface proteins (CD9, CD63, CD81, and EGFR1) with specific antibodies and a secondary antibody conjugated to alkaline phosphatase. Upon reaction with the NBT/BCIP substrate, an insoluble indigo precipitate forms on the nitrocellulose membrane, generating a visual signal that can be further quantified by smartphone imaging. The VFA was optimized for membrane type, pore size, and blocking agents, reaching a detection limit of ~6 107 exosomes L-1 in less than 20 min. Comparative studies with bead-based flow cytometry confirmed consistent biomarker expression profiles, demonstrating the reliability of the method. By enabling exosome biomarker profiling in a simplified and low-cost format, this approach provides a promising alternative to flow cytometry and other applications required for exosome characterization. Keywords: exosome profiling; vertical flow assay; breast cancer; alkaline phosphatase; liquid biopsy; paper-based immunoassay; flow cytometry Author: Arnau Pallarès-Rusiñol, Jennifer Marfà, Rosanna Rossi, Mercè Martí and María Isabel Pividori Creator: LaTeX with hyperref Producer: pdfTeX-1.40.25; modified using OpenPDF 1.4.2 CreationDate: Tue Oct 14 07:49:38 2025 CEST ModDate: Tue Oct 14 08:06:48 2025 CEST Custom Metadata: yes Metadata Stream: no Tagged: no UserProperties: no Suspects: no Form: none JavaScript: no Pages: 15 Encrypted: no Page size: 595.276 x 841.89 pts (A4) Page rot: 0 File size: 2076069 bytes Optimized: no PDF version: 1.7 name type encoding emb sub uni object ID ------------------------------------ ----------------- ---------------- --- --- --- --------- RSXNQE+URWPalladioL-Ital Type 1 Custom yes yes yes 127 0 AIRHKL+VnURWPalladioL Type 1 Custom yes yes yes 128 0 VIWIOF+CMSY10 Type 1 Builtin yes yes yes 129 0 OVNEXW+EURM10 Type 1 Builtin yes yes yes 130 0 BGUGFY+URWPalladioL-Bold Type 1 Custom yes yes yes 131 0 QQTNTN+URWPalladioL-Roma Type 1 Custom yes yes yes 132 0 TXYQVB+VnURWPalladioL-Bold Type 1 Custom yes yes yes 133 0 GPDDMD+PalatinoLinotype TrueType WinAnsi yes yes no 184 0 GPDDMB+PalatinoLinotype,Bold TrueType WinAnsi yes yes no 185 0 GPDDMD+PalatinoLinotype TrueType WinAnsi yes yes no 244 0 GPDDMD+PalatinoLinotype TrueType WinAnsi yes yes no 269 0 GPDDMD+PalatinoLinotype TrueType WinAnsi yes yes no 284 0 GPDDMD+PalatinoLinotype TrueType WinAnsi yes yes no 304 0 Jhove (Rel. 1.28.0, 2023-05-18) Date: 2025-11-20 03:09:03 CET RepresentationInformation: biosensors-15-00694.pdf ReportingModule: PDF-hul, Rel. 1.12.4 (2023-03-16) LastModified: 2025-11-19 03:16:32 CET Size: 2076069 Format: PDF Version: 1.7 Status: Well-Formed and valid SignatureMatches: PDF-hul MIMEtype: application/pdf PDFMetadata: Objects: 448 FreeObjects: 1 IncrementalUpdates: 0 DocumentCatalog: ViewerPreferences: HideToolbar: false HideMenubar: false HideWindowUI: false FitWindow: true CenterWindow: false DisplayDocTitle: false NonFullScreenPageMode: UseNone Direction: L2R ViewArea: CropBox ViewClip: CropBox PrintArea: CropBox PageClip: CropBox PageLayout: SinglePage PageMode: UseNone Outlines: Item: Title: Introduction Item: Title: Materials and Methods Children: Item: Title: Instrumentation Item: Title: Chemicals and Biochemicals Item: Title: Cell Culturing, Exosome Isolation, and Purification Item: Title: Characterization of Exosomes by Nanoparticle Tracking Analysis, Cryogenic Transmission Electron Microscopy, and BCA Protein Assay Item: Title: Exosome Biomarker Profiling by Bead-Based Flow Cytometry Item: Title: Optimization of Vertical Flow Assay Design and Experimental Parameters Item: Title: Vertical Flow Assay for Exosome Biomarker Quantification and Profiling Item: Title: Safety Considerations Item: Title: Results Children: Item: Title: Characterization of Exosomes by Nanoparticle Tracking Analysis, Cryogenic Transmission Electron Microscopy, and BCA Protein Assay Item: Title: Exosome Biomarker Profiling by Bead-Based Flow Cytometry Item: Title: Optimization of Vertical Flow Assay Design and Experimental Parameters Item: Title: Vertical Flow Assay for Exosome Biomarker Quantification and Profiling Item: Title: Discussion Item: Title: Conclusions Item: Title: References Info: Title: Exosome Biomarker Profiling Using a Paper-Based Vertical Flow Assay Author: Arnau Pallarès-Rusiñol, Jennifer Marfà, Rosanna Rossi, Mercè Martí and María Isabel Pividori Subject: Exosomes are nanoscale extracellular vesicles that carry valuable biomolecular information. However, their characterization still depends on complex and costly techniques such as flow cytometry. In this study, a paper-based Vertical Flow Assay (VFA) specifically designed for the detection and profiling of exosomes derived from metastatic breast cancer cell lines is presented. The assay operates in an ELISA-like format, targeting exosomal surface proteins (CD9, CD63, CD81, and EGFR1) with specific antibodies and a secondary antibody conjugated to alkaline phosphatase. Upon reaction with the NBT/BCIP substrate, an insoluble indigo precipitate forms on the nitrocellulose membrane, generating a visual signal that can be further quantified by smartphone imaging. The VFA was optimized for membrane type, pore size, and blocking agents, reaching a detection limit of ~6 107 exosomes L-1 in less than 20 min. Comparative studies with bead-based flow cytometry confirmed consistent biomarker expression profiles, demonstrating the reliability of the method. By enabling exosome biomarker profiling in a simplified and low-cost format, this approach provides a promising alternative to flow cytometry and other applications required for exosome characterization. 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