This report summarises the presentations and activities of the ISEV Workshop on extracellular vesicle biomarkers held in Birmingham, UK during December 2017. Among the key messages was broad agreement about the importance of biospecimen science. Much greater attention needs to be paid towards the provenance of collected samples. The workshop also highlighted clear gaps in our knowledge about pre-analytical factors that alter extracellular vesicles (EVs). The future utility of certified standards for credentialing of instruments and software, to analyse EV and for tracking the influence of isolation steps on the structure and content of EVs were also discussed. Several example studies were presented, demonstrating the potential utility for EVs in disease diagnosis, prognosis, longitudinal serial testing and stratification of patients. The conclusion of the workshop was that more effort focused on pre-analytical issues and benchmarking of isolation methods is needed to strengthen collaborations and advance more effective biomarkers.

Research in the field of extracellular vesicles (EVs) is challenged by the small size of the nano-sized particles. Apart from the use of transmission and scanning electron microscopy, established technical platforms to visualize, quantify, and characterize nano-sized EVs were lacking. Recently, methodologies to characterize nano-sized EVs have been developed. This chapter aims to summarize physical principles of novel and conventional technologies to be used in the EV field and to discuss advantages and limitations.

Liposomes loaded with water-soluble and water-insoluble quantum dots (QD) were for the first time applied as labels in different heterogeneous immunoassays for the determination of food contaminants, using mycotoxin zearalenone (ZEN) as a model. A great deal of work was devoted to the optimal choice of phospholipids for the liposomes preparation and to the factors which are important for the stability and size of obtained liposomes. Thin-film hydration and reverse-phase evaporation techniques were evaluated in terms of stability of the obtained liposomes and their efficiency for QD loading. Conjugation of liposomes with proteins and the influence of cross-linkers to the nonspecific interaction of the obtained liposomes with the surface of microtiter plates and cartridges were investigated and 3-(2-pyridyldithio)propionic acid N-hydroxysuccinimide ester was found as the optimal cross-linker. The limits of detection (LOD) for ZEN of fluorescence-labeled immunosorbent assays were 0.6 μg kg(-1), 0.08 μg kg(-1), and 0.02 μg kg(-1), using QD, liposomes loaded with water-soluble QD, and water-insoluble QD, respectively. Similarly, the developed qualitative on-site tests using the different QD labels and taking into account the EU maximum residues level for ZEN in unprocessed cereals showed cutoff levels of 100, 50, and 20 μg kg(-1).

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We introduce photophoretic velocimetry as a new technique for characterization of particulate matter on the basis of optical particle properties. Complementary to well-established techniques, we could show that, by measuring the photophoretic velocity of the single particles, it is possible to distinguish particles of different sizes as well as particles of one size but different refractive indices. The difference in photophoretic migration of particles can be applied to the separation of particles. Polystyrene, melamine, and SiO2 microparticles (0.3-10 mum) suspended in purified water were used as test samples for validation of a cross-flow setup. The particles were pushed perpendicular to a uniform, pulsation-free fluid flow by a focused He-Ne laser (lambda = 633 nm, P = 47 mW, I(max) = 14.0 kW cm(-2)) providing a well-defined Gaussian-shaped flux distribution. The migration behavior was observed by means of a video camera system, and the velocities and displacements were calculated by using an adapted particle imaging velocimetry code as an approach to automatic characterization. The photophoretic displacement depends on both flow conditions and particle properties and can be applied for separation means.