Protein-polyelectrolyte interactions: Monitoring particle formation and growth by nanoparticle tracking analysis and flow imaging microscopy

The purpose of this study was to investigate the formation and growth kinetics of complexes of proteins and oppositely charged polyelectrolytes. Equal volumes of IgG and dextran sulfate (DS) solutions, 0.01 mg/ml each in 10 mM phosphate, pH 6.2, were mixed. At different time points, samples were taken and analyzed by nanoparticle tracking analysis (NTA), Micro-Flow Imaging (MFI) and size-exclusion chromatography (SEC). SEC showed a huge drop in monomer content (approximately 85%) already 2 min after mixing, while a very high nanoparticle (size up to 500 nm) concentration (ca. 9 × 10⁸/ml) was detected by NTA. The nanoparticle concentration gradually decreased over time, while the average particle size increased. After a lag time of about 1.5 h, a steady increase in microparticles was measured by MFI. The microparticle concentration kept increasing up to about 1.5 × 10⁶/ml until it started to slightly decrease after 10 h. The average size of the microparticles remained in the low-μm range (1-2 μm) with a slight increase and broadening of the size distribution in time. The experimental data could be fitted with Smoluchowski's perikinetic coagulation model, which was validated by studying particle growth kinetics in IgG:DS mixtures of different concentrations. In conclusion, the combination of NTA and MFI provided novel insight into the kinetics and mechanism of protein-polyelectrolyte complex formation.