Biological nanoparticles include lipoproteins, protein aggregates, viruses, extracellular vesicles, and liposomes. Ranging in size from 1-1000 nm, these particles have important roles in diverse fields like health and sustainability. OMV separation and enumeration are essential to understanding the impact of their biochemical heterogeneity in processes such as biopolymer degradation. Counting techniques, e.g., flow cytometry, Coulter counter, and nanoparticle tracking analysis currently lack the sensitivity to count particles with diameter below ~200 nm or encounter difficulties when addressing polydisperse populations.<br/>Asymmetrical flow field-flow fractionation (AF4) is a sized-based separation technique for particles that span 1 - 1000 nm in diameter when utilizing the normal mode separation mechanism. AF4 coupled to multi-angle light scattering (MALS) provides the separation needed to produce the more monodisperse size subpopulations required for accurate size and count distributions by MALS. Recent work with AF4-MALS has shown successful enumeration of virus particles and colloids in solution by measuring the amount of scattered light from the particles and known information on particle shape and refractive index (RI). However the extent of how RI and particle shape impact particle counts have not been studied in detail. The work presented here utilizes liposome standards to better understand how light scattering intensity for particles of known size distribution and concentration translates into particle counts and the impact of RI and particle shape. Increases in liposome concentration led to a proportional increase in counts, matching theoretical calculations. Increasing RI values used during data treatment by 10% decreased particle counts up to 95%. Particle shape (hard sphere versus coated sphere) also affected particle counts (up to 98%). This work highlights the need for accurate RI values when using light scattering as a particle counting method and the particularly challenging nature of particles with a core-shell type structure.

Impacts of refractive indices on counting biological particles with AF4-MALS

ACS Spring Meeting 2021

The official archive and repository for all ACS Meetings & Expositions abstracts and select posters or presentations.