Determination of particle number concentration for biological particles using AF4-MALS: Dependencies on light scattering model and refractive index

Determining accurate counts and size distributions for biological particles (bioparticles) is crucial in wide-ranging fields, but current ensemble methods to this end are susceptible to bias from polydispersity in size. This bias can be mitigated by incorporating a separation step prior to characterization. For this reason, asymmetrical flow field-flow fractionation (AF4) with on-line multiangle light scattering (MALS) has become an important platform for determining particle size. AF4-MALS has been used to report particle concentration, particularly for complex biological particles, yet the impact of light scattering models and particle refractive indices (RI) have not been quantitatively assessed. Here, we develop an analysis workflow using AF4-MALS to simultaneously separate and determine particles sizes and concentrations. The impacts of the MALS particle counting model used to process data and the chosen RI value(s) on particle counts are systematically assessed for polystyrene latex (PSL) particles and bacterial outer membrane vesicles (OMVs) in the 20-500 nm size range. Across spherical models, PSL and OMV particle counts varied up to 13% or 200%, respectively. For the coated-sphere model used in the analysis of OMV samples, the sphere RI value greatly impacts particle counts. As the sphere RI value approaches the RI of the suspending medium, the model becomes increasingly sensitive to the light scattering signal-to-noise ultimately causing erroneous particle counts. Overall, this work establishes the importance of selecting appropriate MALS models and RI values for bioparticles to obtain accurate counts and provides an AF4-MALS method to separate, enumerate, and size polydisperse bioparticles

Frit inlet field-flow fractionation techniques for the characterization of polyion complex self-assemblies

International audiencePolymer self-assemblies joining oppositely charged chains, known as polyion complexes (PICs), have been formed using poly(ethyleneoxide – b – acrylic acid)/poly(l-lysine), poly(ethyleneoxide-b-acrylic acid)/dendrigraft poly(l-lysine) and poly[(3-acrylamidopropyl) trimethylammonium chloride – b – N – isopropyl acrylamide]/poly(acrylic acid). The self-assemblies have been first characterized in batch by Dynamic Light Scattering. In a second step, their analysis by Flow Field-Flow Fractionation techniques (FlFFF) was examined. They were shown to be very sensitive to shearing, especially during the focus step of the fractionation, and this led to an incompatibility with asymmetrical FlFFF. On the other hand, Frit Inlet FlFFF proved to be very efficient to observe them, either in its symmetrical (FI-FlFFF) or asymmetrical version (FI-AsFlFFF). Conditions of elution were found to optimize the sample recovery in pure water. Spherical self-assemblies were detected, with a size range between 70–400nm depending on the polymers. Compared to batch DLS, FI-AsFlFFF clearly showed the presence of several populations in some cases. The influence of salt on poly(ethyleneoxide-b-acrylic acid) (PEO-PAA) 6000-3000/dendrigraft poly(l-lysine) (DGL 3) was also assessed in parallel in batch DLS and FI-AsFlFFF. Batch DLS revealed a first process of swelling of the self-assembly for low concentrations up to 0.8M followed by the dissociation. FI-AsFlFFF furthermore indicated a possible ejection of DGL3 from the PIC assembly for concentrations as low as 0.2M, which could not be observed in batch DLS