Extracellular vesicles INFOCUS : a novel technique for biological nanoparticle size analysis

Abstract

Small extracellular vesicles (EVs) such as exosomes are physiologically and pathophysiologically relevant biological nanoparticles derived from cells. Their diverse roles in homeostasis and pathology could allow for the sensitive diagnosis and monitoring of various conditions such as cancers, infections, and genetic diseases. However, due to their complex structures, lack of specific markers, broad population heterogeneity, similarity to other functional particles, and very small size, the tools and methods used to study EVs are often compromised in their utility or reliability. Further advancement in EV diagnostic medicine will require the development of new techniques that can quickly and reliably produce accurate, multiplexed information about different types of EVs. The author proposes the Inferred Focal Centers of Uniform Spheres (INFOCUS) method as a solution to address the many demands required by the EV field. This method takes advantage of the properties of small EVs: sub-diffraction limit diameters, lipid membranes, and high sphericality due to membrane tension, to measure particle sizes with high accuracy and throughput, while also allowing for parallel functional analyses of EV markers. Using a set of polystyrene nanoparticles, the INFOCUS method was demonstrated in its entirety from sample preparation to analysis and output. Various considerations were examined and corrected as necessary. Experimental parameters, sample preparation, and analysis were optimized, while a set of optical and spectral variables were controlled through analytical corrections. INFOCUS was then compared to commercial tunable resistive-pulse sensing (TRPS), a competitive EV size analysis solution. The INFOCUS method was found to have superior accuracy to the reported sizes than TRPS, with measured means of particles ranging between 40 and 170 nm in diameter being within 10 nm of the manufacturer reported specification. Finally, initial EV testing illuminated the next set of challenges for INFOCUS to overcome in order to address the demands of the EV diagnostic field.