Fig. 2

Characterization and operation of the COC ^EVHB-Chip. a RT-PCR quantification of the RNA recovered from EVs captured on the microfluidic device coated with Cetuximab at different flow rates. RNA enrichment ratio in the microfluidic device was calculated between EGFRvIII and GAPDH and depicted by the dotted black line. The red line represents the total RNA quantity. b Comparison of RNA enrichment ratios between ultracentrifugation and different capture antibodies on the surface of the ^EVHB-Chip (*p < 0.05, one-way ANOVA). c Effect of linker size to captured tumor-specific EVs and cells at 1 mL h⁻¹. d Changes in Z_potential distribution for different complex configurations. EVs, EVs binded to antibodies, and EVs binded to linked antibodies. e Variation in Z_potential for different PEG linker size of the complex EV and antibodies. f Size and concentration of tumor EVs isolated with the microfluidic device. Different size distributions were identified within the range of 40–1000 nm. Insert shows oncosomes (top, scale bar 2 μm) and exosomes (bottom, scale bar 100 nm). g Performance comparison between the ^EVHB-Chip and standard EV isolation methods. Plasma from healthy donors with spiked-in fluorescent EVs isolated from Gli36-EGFRvIII cells was run through the microfluidic device coated with Cetuximab to capture tumor EVs. In parallel, tumor EVs were isolated using magnetic beads coated with Cetuximab or by ultracentrifugation. ddPCR was used to quantify the total number of EGFRvIII copies captured with the different techniques. h Fluorescent microscopy image of immunoaffinity stained tumor-specific EVs on the surface of a microfluidic chip (scale bar 50 μm). For a, b, c, e, and g n = 3 technical replicates; ±s.e.m. For d and f, a representative experiment is shown from n = 3 technical replicates