Extracellular vesicles nanoarray technology: Immobilization of individual extracellular vesicles on nanopatterned polyethylene glycol-lipid conjugate brushes

Arraying individual extracellular vesicles (EVs) on a chip is expected one of the promising approaches for investigating their inherent properties. In this study, we immobilized individual EVs on a surface using a nanopatterned tethering chip-based versatile platform. A microfluidic device was used to ensure soft, reproducible exposure of the EVs over the whole chip surface. The device is incorporated with a high-density nanoarray chip patterned with 200-nm diameter nanospots composed of polyethylene glycol (PEG)-lipid conjugate brushes. We present a procedure adopted for fabricating high-density PEG-lipid modified nanospots (200 nmϕ, 5.0 × 10(5) spots/mm(2) in 2 × 2 mm(2) area). This procedure involves nanopatterning using electron beam lithography, followed by multistep selective chemical modification. Aqueous treatment of a silane coupling agent, used as a linker between PEG-lipid molecules and the silicon surface, was the key step that enabled surface modification using a nanopatterned resist film as a mask. The nanoarray chip was removed from the device for subsequent measurements such as atomic force microscopy (AFM). We developed a prototype device and individually immobilized EVs derived from different cell lines (Sk-Br-3 and HEK293) on tethering nanospots. We characterized EV's morphology using AFM and showed the possibility of evaluating the deformability of EVs using the aspect ratio as an indicator.