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

Abstract

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 2.4·109±3.8·109 vesicles/mL (range 1.4·108-1.8·1010). 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 3.2-8.2·108 vesicles/mL with fixed stretch and 6.4-7.8·108 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.

Keywords: exosomes; extracellular vesicles; microparticles; nanoparticles; resistive pulse sensing; size determination.

Fig. 1

Resistive pulse sensing operating principle. When a non-conductive vesicle in a conductive medium passes through a pore, a brief increase in electrical resistance of the pore results. This is measured by monitoring the electrical current through the pore. Panel A shows a schematic representation of a pore with a vesicle passing from position a to d. Panel B shows the current from a particle moving through the pore, the letters a–d correspond to vesicle positions a–d in panel A. Panel C shows the qNano system on the right with the air-based variable pressure module (VPM) on the left. The pore and fluid cells are contained within the green square. A detail of the bottom fluid cell and the crucifix containing the pore mounted on the stretching mechanism is shown in the insert.

Fig. 2

(A) Particle size distribution obtained from 3 measurement of the standard sample. The size distribution is shown as a histogram with bin width 10 nm. The minimum detected size for these 3 distributions is indicated with vertical arrows below the x-axis. (B) Histogram of the minimum detected size of the standard sample for 102 consecutive measurements measured on 102 days. The dashed line shows a fit of a normal distribution with a mean size of 127 nm and standard deviation of 19 nm.

Fig. 3

Effect of set voltage and stretch on baseline current and blockade height. Each point represents the average of 3 pores. Lines are linear fits to the data. Panel A shows the relationship between baseline current, stretch and voltage. Panel B shows the relationship between blockade height and baseline current. The combination of parameters selected for further optimization is indicated with a bold circle.

Fig. 4

Impact of pressure on measurement. The data from 3 pores are shown for beads (black markers) and urine (blue markers). The particle rate versus pressure is shown in panel A. The blockade height versus pressure is shown in panel B.

Fig. 5

Flow chart for fixed blockade height method.

Fig. 6

Inter- and intra-pore reproducibility (both n=3) of particle rate (panel A), minimum detected diameter (panel B) and concentration (panel C) for 2 configuration methods. Labels below panel C refer to the methods fixed stretch and voltage and fixed blockade height.