Biology subjects

Sanchez-Martin, P.

Publications and source records attributed to Sanchez-Martin, P..

5 recordsLinked to original sources

bioRxiv2026

Reference-free single-vesicle profiling of small extracellular vesicles from liquid biopsies with the PICO assay

Small extracellular vesicles (sEV) are membrane-enclosed nanoparticles found in body fluids that carry molecular cargo from their cells of origin. Their stability and disease-associated molecular signatures make them promising targets for the development of non-, or minimally invasive liquid biopsies, yet scalable approaches enabling single-vesicle quantification of sEV while resolving their heterogeneity remain limited. Here, we present PICO (Protein Interaction Coupling), a reference-free quantitative assay adapted for sensitive multiplex profiling of individual intact vesicles. PICO detects vesicle markers by requiring colocalization of two or more copies of the same protein or of distinct proteins (e.g., CD9 or CD9/CD63) on individual vesicles, using DNA-barcoded antibodies and digital PCR (dPCR) for quantitative readout. We demonstrate that this unique architecture of the assay provides high specificity by distinguishing EV-bound proteins from soluble counterparts, and can be adapted to target either surface-exposed or intravesicular biomarkers. PICO requires minimal sample input (1 {micro}l) and no specialized instrumentation beyond standard digital PCR. In a head-to-head comparison with nano-flow cytometry, PICO achieved a comparable limit of detection for sEV subpopulations. Profiling sEV isolates from blood for canonical markers (CD9, CD63 and CD81) and HER2 demonstrates precise, high-resolution quantification of sEV subpopulations in complex clinical samples and supports integration of scalable single-EV analysis into research and diagnostic workflows.

molecular biology↗

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↗

bioRxiv2024

Lossless Single-Molecule Counting To Absolute QuantifyProteoforms

A novel immunoassay, termed Protein Interaction Coupling (PICO), is introduced to deliver unequivocal, reference-free quantification of proteoforms - absolute quantification. PICO employs a compartmentalized, homogeneous single-molecule assay with a lossless and highly sensitive signal generation, capable of detecting down to a few molecules per reaction. Additionally, it utilises a background-free, digital enumeration principle, known as the decouplexing. PICO is presented as exact mathematical theories, providing a theoretical comprehension of its chemistry. Consequently, PICO demonstrates absolute quantification, as exemplified with recombinant and non-recombinant ErbB2 and multi-tagged peptide rTRX targets, validating absolute quantification against internal and external references in both analytical and cellular matrices. Furthermore, PICO enables combinatorial multiplexing (cplex), a readout between any two antibodies, demonstrated by an 8-plex antibody, 12-cplex PICO, measuring functional changes of ErbB pathway upon mock and dactolisib treatment delivering absolute quantitative cellular stoichiometry. PICO possesses immense potential for versatile, standardized, and accurate protein measurements, offering insights into physiological and perturbed cellular processes.

molecular biology↗

bioRxiv2022

LRBA balances antigen presentation and T-cell responses by facilitating autophagy through the binding to PIK3R4 and FYCO1

Reduced autophagy is associated with the aberrant humoral response observed in lipopolysaccharide-responsive beige-like anchor protein (LRBA) deficiency; however, the exact molecular mechanism and its impact on T-cell responses remain unknown. We identified two novel LRBA interactors, phosphoinositide 3-kinase regulatory subunit 4 (PIK3R4) and FYVE And Coiled-Coil Domain Autophagy Adaptor 1 (FYCO1). Both proteins play essential roles in different stages of autophagy. PIK3R4 facilitates the production of phosphatidylinositol-3 phosphate (PI(3)P) required for autophagosome formation and autophagosome-lysosome fusion, whereas FYCO1 allows autophagosome movement. LRBA-KO cells showed an impaired PI(3)P production, a delayed autophagosome-lysosome fusion, an accumulation of enlarged autophagosomes, and an atypical lysosomal positioning. These abnormalities led to decreased cargo material degradation and prolonged antigen presentation to T-cells via autophagy, resulting in increased production of proinflammatory cytokines, as autophagy is a major intracellular degradation system for major histocompatibility class II complex (MHCII) loading. Aberrant autophagosome formation, cargo degradation and antigen presentation were rescued by ectopic expression of WT-LRBA. In summary, we identified a novel function of LRBA that is crucial for T-cell-driven response through the interaction with two proteins of the autophagy machinery. These observations may contribute to the exacerbated T-cell dysregulation observed in LRBA-deficient patients.

immunology↗

bioRxiv2019

NBR1-mediated p62-liquid droplets enhance the Keap1-Nrf2 system

p62/SQSTM1 is a multivalent protein that has an ability to cause a liquid-liquid phase separation and serves as a receptor protein that participates in cargo isolation during selective autophagy. This protein is also involved in the non-canonical activation of the Keap1-Nrf2 system, a major oxidative stress response pathway. Here we show a role of Neighbor of BRCA1 gene 1 (NBR1), an autophagy receptor structurally similar to p62/SQSTM1, in the p62-liquid droplet formation and the Keap1-Nrf2 pathway. The overexpression of NBR1 blocked selective degradation of p62/SQSTM1 through autophagy and promoted the accumulation and phosphorylation of p62/SQSTM1 in liquid-like bodies, which is required for the activation of Nrf2. NBR1 was induced in response to oxidative stress, and then the p62-mediated Nrf2 activation was up-regulated. Conversely, loss of Nbr1 suppresses not only the formation of p62/SQSTM1-liquid droplets but also p62-dependent Nrf2 activation during oxidative stress. Taken together, our results show that NBR1 mediates p62/SQSTM1-liquid droplet formation to activate the Keap1-Nrf2 pathway.

cell biology↗