An effective strategy based on electrostatic interaction for the simultaneous sequential purification and isolation of exosomes.

A material-based strategy was developed to achieve the simultaneous purification and isolation of exosomes from serum and urine. Based on the combination of electrostatic interaction and hydrophobic interaction, polyacrylic acid (PAA)-coated anionic nanoparticles (Fe3O4@PAA) were used to remove positively charged contaminant proteins and neutral proteins, and polyethyleneimine (PEI)-functionalized cationic nanoparticles (Fe3O4@PEI) were applied to remove negatively charged contaminant proteins as well as capture and mild release of negatively charged exosome. By employing this strategy (denoted as PAA-PEI), a high recovery (> 95%) of serum exosomes was achieved with a high removal efficiency of protein contaminants (87%). The strategy was further applied to purify and isolate urinary exosomes, followed by downstream proteomics analysis. Compared with the standard isolation method of ultracentrifugation (UC), the PAA-PEI strategy shows high contaminant protein removal efficiency (98.6%) and obtains a higher concentration of exosomes. Using the PAA-PEI strategy, more urinary exosomal proteins (124) than UC (92) were identified. These results indicate that the PAA-PEI strategy is not only excellent in exosome capture but also effectively mitigates the interference of protein contamination. Given that the PAA-PEI strategy demonstrates a high protein contaminant removal efficiency and requires less cost and time (1 h) than UC (3 h), it would be a promising candidate method for efficiently purifying and isolating exosomes from complex biological samples for early discovery and diagnosis diseases. Furthermore, this study provides a new direction for emphasizing the issue of protein interference in the development of biological sample isolation methods.