Rapid Separation and Analysis of Exosomes in Milk Sample by on-Line Nano-Liquid Chromatography

Exosomes are very small vesicles of 30-150 nm average particle size and hold great potential in new therapeutic applications. The aim of this study is to develop a new method for the isolation and analysis of exosomes in milk via hydrophobic interaction chromatography (HIC), including salting-out process in nano-liquid chromatography (nano-LC). On the basis of this approach, a trap column combined with graphene oxide (GO)-based monolithic nano-column was used for on-line analysis of exosomes in nano-LC. The monolith was prepared by an in situ polymerization of butyl methacrylate (BMA), ethylene glycol dimethacrylate (EDMA), and methacryloyl graphene oxide nanoparticles (MGONPs). The final solution was introduced into a fused silica capillary with a 50 µm i.d. for polymerization. After preparation, the column was further modified with dimethyloctadecylchlorosilane (DODCS) to increase its hydrophobicity. The characterization of monolith was performed using scanning electron microscopy (SEM) and chromatographic examination. The final monolith was applied for the isolation and analysis of exosomes in milk via HIC-nano-LC. Nanoparticle tracking analysis (NTA), SEM, and Fourier transform-infrared (FT-IR) for the tandem characterization of milk exosomes were used, whereas step gradient elution was employed for HIC. The results demonstrated good ability to isolate exosomes from milk with three dilution factors, and a loading capacity of 7.3 ± 02 × 10¹¹ exosomes could be obtained using the on-line nano-LC system. The developed method holds many advantages and may be adapted for the isolation of exosomes from a diverse range of media.