Single-Exosome-Counting Immunoassays for Cancer Diagnostics

Abstract

Exosomes shed by tumor cells have been recognized as promising biomarkers for cancer diagnostics due to their unique composition and functions. Quantification of low concentrations of specific exosomes present in very small volumes of clinical samples may be used for noninvasive cancer diagnosis and prognosis. We developed an immunosorbent assay for digital qualification of target exosomes using droplet microfluidics. The exosomes were immobilized on magnetic microbeads through sandwich ELISA complexes tagged with an enzymatic reporter that produces a fluorescent signal. The constructed beads were further isolated and encapsulated into a sufficient number of droplets to ensure only a single bead was encapsulated in a droplet. Our droplet-based single-exosome-counting enzyme-linked immunoassay (droplet digital ExoELISA) approach enables absolute counting of cancer-specific exosomes to achieve unprecedented accuracy. We were able to achieve a limit of detection (LOD) down to 10 enzyme-labeled exosome complexes per microliter (∼10 M). We demonstrated the application of the droplet digital ExoELISA platform in quantitative detection of exosomes in plasma samples directly from breast cancer patients. We believe our approach may have the potential for early diagnosis of cancer and accelerate the discovery of cancer exosomal biomarkers for clinical diagnosis.

Potential roles of extracellular vesicles in the pathophysiology, diagnosis, and treatment of autoimmune diseases

“…At present, microfluidic filtering techniques are considered the most promising strategies for EVs separation in clinic based on their expandable flux, high degree of automation, and solid reproducibility [34,37]. Liu C et al introduced a method for EV purification by microfluidic filtering combined with droplet enzyme-linked immunosorbent assay (ELISA), which fills the gap in accurate quantification of EVs [38]. Wu et al introduced a method based on the acoustic principle combined with microfluidic technology whereby it would become relatively simple to isolate EVs from whole blood, in a label-free and contact-free manner that would protect the peculiarities, structures and functions of isolated EVs [39].…”

Progress in Microfluidics‐Based Exosome Separation and Detection Technologies for Diagnostic Applications

“…The emergence of a digital ELISA platform is expected to improve the sensitivity by confining exosomes to a small volume . Researchers have developed an immunosorbent assay that uses an ELISA to count cancer exosomes in absolute numbers to achieve unprecedented accuracy ( Figure ) . First, an exosome suspension is mixed with immunomagnetic beads.…”

“…D) The positive droplets with the target exosomes are counted. Reproduced with permission . Copyright 2018, American Chemical Society.…”

“…Simultaneously, detection techniques for analyzing and quantifying exosomes have been developed for both research and clinical applications. These widely used detection techniques include scanning electron microscopy (SEM), transmission electron microscopy (TEM), atomic force microscopy (AFM), enzyme‐linked immunosorbent assay (ELISA), dynamic light scattering (DLS), and nanoparticle tracking analysis (NTA) . Despite the great advances in exosome detection techniques, further development is needed to meet the requirements of rapid, sensitive, and low‐cost detections.…”

Lab-on-a-Chip Technologies for the Single Cell Level: Separation, Analysis, and Diagnostics

“…This is generally done with an x-intersection on a microfluidic chip, where the aqueous phase is pinched off into droplets by two streams of oil (see Figure 9a) Using any kind of forces (shear, drag, coulomb, inertial, etc. ), droplets can be merged to combine their contents and to start reactions towards a diagnostic signal (see Figure 9c), like for example an exosome immunoassay for cancer diagnosis [166].…”