(M1130-09-58) NTA Analysis: Linear Range Evaluation for Exosomes

Purpose: Multi laser nanoparticle tracking analysis is a new tool for evaluating particle concentration and size distribution of submicron particles. This technique, like many particle analysis techniques often requires dilution of the parent sample before measurement. Naturally, the degree of required dilution needs to be known and that is coupled to trade-off’s in measurement performance. One of the most critical questions is the linearity of the measurement results. That is how does one determine the range of concentrations for which results are sufficiently reliable. Here we review this determination for exosomes, which are biologically relevant. In addition, these materials have a more realistic, broader size distribution which requires the use of multiple lasers.

Methods: Lyophilized exosomes were obtained from Abcam (Lyophilized MCF7 Exosome Standards, lot GR3401014-1). These are derived from MCF7 a human cancer cell line and characterized by Abcam. Exosomes were reconstituted following manufacturer instructions, and diluted in Dulbeccos phosphate buffered saline (dPBS). NTA was performed with a HORIBA ViewSizer 3000 multi-laser NTA instrument. Multiple lasers were used to ensure analysis of all EV sizes in the sample. Laser power settings were 210 mW blue, 12 mW green, 8 mW red and camera gain was 30 dB.

Results: A single aliquot of reconstituted exosomes was diluted to varying degrees and the concentration of each resulting sample was determined by NTA. Dilutions ranged from 30,000 times to 1000 times and the resulting measured concentrations ranged from ~5e6 p/mL to 6e7 p/mL. A summary of concentration results is shown in Figure 1 below. The lowest measured concentrations showed distortion due to background particle concentration (particles that come from the PBS diluent) while the highest measured concentration showed an overly low concentration.

Conclusion: A linearity study is important to determine the acceptable range of measured concentrations. There is a trade-off between linearity (underreporting concentration) and uncertainty/measurement duration (measuring too few particles).

Figure 1: Measured concentration as a function of 1/dilution. Error bars (within points) are estimated from the number of analyzed particles using Poisson statistics.