Capture Beads: To a microtube containing the washed Tim-4 conjugated magnetic beads, we added 40 µL PBS buffer containing 0.125 w/w% BSA and 0.0025 w/w% Tween-20 to prevent aggregation of magnetic beads. After adding 30 µL of the EV solution, a 10000-fold dilution of the binding enhancer solution containing Ca 2+ ions was added to the mixture. This concentration of binding enhancer was chosen such that it promotes the binding of the EVs and Tim-4, but does not generate non-specific aggregation of magnetic beads. This mixture was then gently pipetted 10 times to uniformly disperse in the microtube. To prevent nonspecific adsorption of EVs and magnetic beads, the surfaces of the PDMS substrate and cover glass were treated with 0.5 wt% ethanol solution of a phospholipid-mimic polymer (Lipidure-CM5206, NOF Corp., Japan) for 3 min and dried on a hotplate at 40 °C. Five microliters of the mixture were dropped onto the center of the pillar substrate. Finally, the mixture on the substrate was covered with the cover glass.