Extracellular vesicles (EVs), including exosomes, hold great promise as biomarkers, as novel therapeutics and generally as a source for new understanding of biological processes. Their physical characterization however has until now presented two significant challenges: Both the size and concentration of EVs span multiple orders of magnitude, and they scatter light weakly, making accurate characterization by optical techniques difficult. Spectradyne's nCS1 instrument, based on microfluidic resistive pulse sensing (MRPS), delivers practical solutions to both challenges in a fast and robust analysis platform.
Fraction Analysis. Urinary exosomes were separated using sucrose gradient ultracentrifugation, and three fractions were analyzed with the nCS1. Results are shown in the figure at right. The nCS1 measurements revealed subtle differences in the size distribution of EVs in the fractions, and importantly, show that each fraction comprises EVs spanning more than 400 nm in diameter, and four orders of magnitude in concentration. Characterization of these samples in such fine detail would not be possible using optical techniques due to the low refractive index contrast of the samples.
Robustness & Repeatability. The reliability of EV measurements obtained with the nCS1 was demonstrated using cell culture media. Triplicate measurements were performed of the EV size distribution in cell culture supernatant. Importantly, no sample preparation of any kind was required before measurement. The results show very high repeatability: Variance in the measured concentration on the size range of 250-2000 nm was less than 3% (see table below).
The high resolution of the nCS1 enables quantitative differentiation of EVs from different sources. The figure on the right demonstrates this application for five different EV samples, with the concentration values shown below over the range from 140-400 nm in diameter.
The Spectradyne nCS1 is a practical tool for high-fidelity EV characterization: Typical measurement time is about 3 min/sample, and the system delivers resolution and accuracy that are superior to other metrologies. Disposable microfluidic cartridges require only 3 μL of sample per measurement, eliminate cleaning between samples, and provide engineered features to reduce or eliminate blockages of the sensing constriction.
The table below enumerates the integrated concentration levels for each EV sample plotted at right.