A Nanofluidic Device for Multiplexed Analysis of Single Exosomes

Abstract

Detailed characterization of exosomes on the single particle level is challenging because of their small size and heterogeneous nature. We have developed a nanofluidic device that enables single particle fluorescence-based tracking and characterization of nano-sized biological particles, which overcomes this problem. This device allows simultaneous three-color fluorescence detection and Brownian-motion based measurement of each particle's size, opening new possibilities for the analysis of population heterogeneity and the detection of scarce events. Our nanofluidic device is fabricated in fused silica and consists of an array of 300nm × 300nm nanochannels connecting to two microchannels (for respectively sample loading and post-analysis collection). The device is fitted onto a conventional fluorescence microscope equipped with a LED light source and a multi-bandpass filter. We have developed software to determine nanoparticle size based on their Brownian motion when passed through the nanochannel at low speed, reaching better accuracy in size determination than with commercial nanoparticle tracking instruments. The device operates with very small sample volumes (2-5 µL) and a data collection time of 2-20 minutes. We demonstrate that it is possible to accurately detect, count, and determine the size of exosomes collected directly from conditioned cell media and without need of potentially sample-altering purification steps, enabling “real-time” exosome profiling from cell culture and other live biological specimen. Additionally, we have been able to visualize the co-localization of Alzheimer peptide amyloid-β peptide with exosomes and demonstrate possibilities to detect size changes associated with protein binding to the exosome surface. Altogether, our device constitutes a new tool to analyze biological nanoparticles, allowing firm establishment of correlations between their biophysical attributes (size), contents and interactions, which is important to understand their biofunctionality.