Small extracellular vesicles (sEVs) present fairly distinctive lipid membrane features in the extracellular environment. These include high curvature, lipid-packing defects and a relative abundance in lipids such as phosphatidylserine and ceramide. sEV membrane could be then considered as a "universal" marker, alternative or complementary to traditional, characteristic, surface-associated proteins. Here, we introduce the use of membrane-sensing peptides as new, highly efficient ligands to directly integrate sEV capturing and analysis on a microarray platform. Samples were analysed by label-free, singleparticle counting and sizing, and by fluorescence co-localisation immune staining with fluorescent anti-CD9/anti-CD63/anti-CD81 antibodies. Peptides performed as selective yet general sEV baits and showed a binding capacity higher than anti-tetraspanins antibodies. Insights into surface chemistry for optimal peptide performances are also discussed, as capturing efficiency is strictly bound to probes surface orientation effects. We anticipate that this new class of ligands, also due to the versatility and limited costs of synthetic peptides, may greatly enrich the molecular toolbox for EV analysis.

Veronesi, Marina et al. (2020) Chemical perturbation of oncogenic protein folding: from the prediction of locally unstable structures to the design of disruptors of Hsp90-Client interactions. Chemistry -A European Journal, 26 (43). pp. 9459-9465.

Despite their clinical potential, Extracellular Vesicles (EVs) struggle to take the scene as a preeminent source of biomarkers in liquid biopsy. Limitations in the use of EVs origin from their inherent complexity and heterogeneity and from the sensitivity demand in detecting low to very low abundant disease-specific sub-populations. Such need can be met by digital detection, namely capable to reach the single-molecule sensitivity. Here we set to compare, side by side, two digital detection platforms that have recently gained increasing importance in the field of EVs. The platforms, both commercially available, are based on the principles of the Single Particle Interferometric Reflectance Imaging Sensing (SP-IRIS) and the Single Molecule Array technology (SiMoA) respectively. Sensitivity in immune-phenotyping of a well characterized EV sample is reported, discussing possible applicative implications and rationales for alternative or complementary use of the two platforms in biomarker discovery or validation.

Canonical immunoassays rely on highly sensitive and specific capturing of circulating biomarkers by interacting biomolecular baits. In this frame, bioprobe immobilization in spatially discrete three-dimensional (3D) spots onto analytical surfaces by hydrogel encapsulation was shown to provide relevant advantages over conventional two-dimensional (2D) platforms. Yet, the broad application of 3D systems is still hampered by hurdles in matching their straightforward fabrication with optimal functional properties. Herein, we report on a composite hydrogel obtained by combining a self-assembling peptide (namely, Q3 peptide) with low-temperature gelling agarose that is proved to have simple and robust application in the fabrication of microdroplet arrays, overcoming hurdles and limitations commonly associated with 3D hydrogel assays. We demonstrate the real-case scenario feasibility of our 3D system in the profiling of Covid-19 patients' serum IgG immunoreactivity, which showed remarkably improved signal-to-noise ratio over canonical assays in the 2D format and exquisite specificity. Overall, the new two-component hydrogel widens the perspectives of hydrogel-based arrays and represents a step forward towards their routine use in analytical practices.