Ultrasensitive Single Extracellular Vesicle Detection Using High Throughput Droplet Digital Enzyme-Linked Immunosorbent Assay

Abstract

Extracellular vesicles (EVs) have attracted enormous attention for their diagnostic and therapeutic potential. However, it has proven challenging to achieve the sensitivity to detect individual nanoscale EVs, the specificity to distinguish EV subpopulations, and a sufficient throughput to study EVs among an enormous background. To address this fundamental challenge, we developed a droplet-based optofluidic platform to quantify specific individual EV subpopulations at high throughput. The key innovation of our platform is parallelization of droplet generation, processing, and analysis to achieve a throughput (∼20 million droplets/min) more than 100× greater than typical microfluidics. We demonstrate that the improvement in throughput enables EV quantification at a limit of detection = 9EVs/μL, a >100× improvement over gold standard methods. Additionally, we demonstrate the clinical potential of this system by detecting human EVs in complex media. Building on this work, we expect this technology will allow accurate quantification of rare EV subpopulations for broad biomedical applications.

Advancing microfluidic diagnostic chips into clinical use: a review of current challenges and opportunities

“…Digital assays have demonstrated utility as a platform for the ultrasensitive detection of proteins 10,37,48 and nucleic acids, 49,50 as well as the analysis of single cells 51,52 and single exosomes. [53][54][55][56]104 dELISA has been commercialized by Quanterix (Fig. 3c) and has found utility in several clinical applications, including traumatic brain injury and cancer diagnostics.…”

“…4d). In these assays, signals from scant quantities of protein on the surface of single EVs are amplified within each droplet using digital ELISA, 44,104 PCR, 80 or next-generation sequencing (NGS) 81 into a measurable signal at high throughput. While these techniques are still in their infancy, further work on these devices could produce useful research and clinical tools to improve our understanding of EVs and their role in disease.…”

Recent Advances in Digital Biosensing Technology

“…Improving droplet generation throughput is critical for refining assay sensitivity and achieving highly parallel reactions. A droplet microfluidic was designed to achieve a high throughput of 20 million droplets/min, which is more than 100 times greater than other microfluidic devices ( Figure 2 b) [ 33 ]. This device generated droplets in parallel using multiple droplet generation zones, which then converged into the reaction and detection zones on the chips.…”

“…The magnetic beads are then washed and added to the microfluidic device so they can be merged with droplets containing the enzyme substrate for detection. The droplets are then incubated and readout using either fluorescence or SERS methods under continuous flow [ 33 , 40 , 41 ] or within an incubation chamber ( Figure 2 c) [ 34 , 42 , 43 , 44 ].…”

“…(accessed on 22 July 2022). Copyright 2022 American Chemical Society [ 33 ]. ( c ) Flow focusing microfluidic device with reaction chamber.…”