Bustos-Quevedo, G. | bioRxiv Science

bioRxiv2026

PHoNUPS: Open-Source Software for Standardized Analysis and Visualization of Multi-Instrument Extracellular Vesicle Measurements

Accurate and transparent characterization of extracellular vesicle (EV) preparations is essential to ensure reproducibility, comparability, and adherence to MISEV reporting standards. However, data outputs from commonly used instruments for assessing EV size, concentration, and surface charge (zeta potential) vary widely in format and structure, complicating standardized analysis and integration across platforms. We present PHoNUPS (Plotting the Histogram of Non-Uniform Particles Sizes), free and open-source software (FOSS) developed in R, that enables unified processing, analysis, and visualization of EV characterization data. PHoNUPS computes statistics and generates standardized histograms and contour plots (for size against zeta potential) suitable for transparent reporting and cross-study comparison. The software produces high-quality, publication-ready figures. Third-party graphical editing tools allow users to refine and annotate visualizations for presentation or manuscript preparation. PHoNUPS supports multiple measurement file formats, thereby facilitating dataset integration from different instruments. PHoNUPS was developed with extensibility at its core, providing a basis for user-driven growth. We invite the EV community--researchers, analysts, and tool developers--to use PHoNUPS, share feedback on their experience and needs, and contribute to the platform by integrating additional input data formats, analytical routines, and visualization functionalities. Graphical abstractThe free software PHoNUPS processes the outputs of several different EV characterization instruments and it is extensible with further ones. It computes statistics of particle size and zeta potential distributions and it plots the corresponding histograms or contour plots. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=146 SRC="FIGDIR/small/702479v1_ufig1.gif" ALT="Figure 1"> View larger version (28K): org.highwire.dtl.DTLVardef@b2b3a1org.highwire.dtl.DTLVardef@2f2907org.highwire.dtl.DTLVardef@2ec521org.highwire.dtl.DTLVardef@903624_HPS_FORMAT_FIGEXP M_FIG C_FIG

bioinformatics↗

bioRxiv2025

VDisk: Microfluidic Cartridge for Multimodal High-Yield, High-Purity Isolation of Extracellular Vesicles from up to 1 mL of Plasma

Blood-derived extracellular vesicles (EVs) hold strong diagnostic potential, yet conventional isolation methods such as ultracentrifugation and size-exclusion chromatography (SEC) involve manual handling steps and show substantial run-to-run variability, hindering clinical translation. This study presents the Vesicle Disk (VDisk), a centrifugal microfluidics-based EV purification platform that combines cation-exchange chromatography, sequential filtration, and multimodal chromatography for automated, label-free EV isolation from up to 1 mL of plasma. VDisk configurations differing in filter membrane and plasma volume (0.1-1.0 mL) are benchmarked against SEC for yield, purity, reproducibility and robustness. VDisk matches SEC in EV yield and exceeds it in EV/contaminant ratios at reduced input volumes, while achieving greater reproducibility (intra-donor CV < 5% for CD9 and CD81, versus up to 16% for SEC) and maintaining consistent yield and contaminant removal under both fasting and postprandial sampling conditions. The platform is application-tunable: processing up to 1 mL of plasma maximizes EV yield and concentration, whereas processing 0.5 mL achieves approximately 2-3-fold higher EV/total protein ratios than SEC. These findings establish the VDisk as an automated, robust and adaptable alternative to existing EV isolation methods for both research and clinical translation.

bioengineering↗