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

Abstract

IntroductionThe 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.MethodsExperiments 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.ResultsDaily 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/conclusionPore-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.

Challenges in Analysis of Circulating Extracellular Vesicles in Human Plasma Using Nanotracking and Tunable Resistive Pulse Sensing

“…Emerging techniques of NTA and TRPS have been increasingly used in detection and quantitation of EV of biological origin [14]. Although there is a significant body of literature looking at beads and liposomes potentially simulating body fluids there is a dearth of published literature estimating EVs in human plasma samples [13].…”

“…Therefore, based on our observations a 1:10 or 1:20 dilution appears optimal. Stretch and voltage was fixed for our TRPS experiments based on trial runs and published literature from urinary vesicles [14].…”

“…Dedicated flow cytometry is more accurate in determining the size of reference beads, but may have less accuracy when measuring biological vesicles, owing to heterogeneity of the refractive index of vesicles from body fluids or cells [18]. The main role of NTA and TRPS is to facilitate the measurement of EVs in the size range of 100 nm-300 nm which are usually not measured using standard techniques such as flow cytometry [1,14]. It may be critical to accurately measure vesicles in experiments where a correlation between particle count and function of EV is being ascertained [19].…”

“…Whilst nanotracking has potential for identifying specific EVs labelled with Qdots, TRPS has the potential of being able to mark EVs with aptamers. TRPS also has potential for particle by particle measurement of surface charge (known as zeta-potential) which may provide a discrimination between particles of varied surface charge and surrogate evidence for different cells of origin for the vesicles [11,14].…”

Tunable Resistive Pulse Sensing: Potential Applications in Nanomedicine

“…As a result, there have been a surge in the number of diagnostic modalities to study EVs. Over the last few years, a number of studies have employed TRPS as a diagnostic tool to characterise EVs [48][49][50][51][52].…”