Reproducible extracellular vesicle size and concentration determination with tunable resistive pulse sensing

INTRODUCTION: The size of extracellular vesicles (EVs) can be determined with a tunable resistive pulse sensor (TRPS). Because the sensing pore diameter varies from pore to pore, the minimum detectable diameter also varies. The aim of this study is to determine and improve the reproducibility of TRPS measurements. METHODS: Experiments were performed with the qNano system (Izon) using beads and a standard urine vesicle sample. With a combination of voltage and stretch that yields a high blockade height, we investigate whether the minimum detected diameter is more reproducible when we configure the instrument targeting (a) fixed stretch and voltage, or (b) fixed blockade height. RESULTS: Daily measurements with a fixed stretch and voltage (n=102) on a standard urine sample show a minimum detected vesicle diameter of 128±19 nm [mean±standard deviation; coefficient of variation (CV) 14.8%]. The vesicle concentration was 2.4·10(9)±3.8·10(9) vesicles/mL (range 1.4·10(8)–1.8·10(10)). When we compared setting a fixed stretch and voltage to setting a fixed blockade height on 3 different pores, we found a minimum detected vesicle diameter of 118 nm (CV 15.5%, stretch), and 123 nm (CV 4.5%, blockade height). The detected vesicle concentration was 3.2–8.2·10(8) vesicles/mL with fixed stretch and 6.4–7.8·10(8) vesicles/mL with fixed blockade height. SUMMARY/CONCLUSION: Pore-to-pore variability is the cause of the variation in minimum detected size when setting a fixed stretch and voltage. The reproducibility of the minimum detectable diameter is much improved by setting a fixed blockade height.