Advances in subpopulation separation and detection of extracellular vesicles: for liquid biopsy and downstream research

Abstract

Extracellular vesicles (EVs) are carriers of a diverse array of bioactive molecules, making them valuable clinical tools for liquid biopsy in disease diagnosis and prognosis evaluation. These molecules play critical roles in various physiological and pathological conditions, and effective separation of EVs is essential to achieve these objectives. Due to the high heterogeneity of EVs, particularly with regard to their cargo molecules, merely isolating the general EV population is inadequate for liquid biopsy and biological function studies. Therefore, separating EV subpopulations becomes crucial. Traditional separation methods, such as differential ultracentrifugation and size exclusion chromatography, along with burgeoning techniques like classical microfluidic chips and covalent chemistry, often prove time-consuming, yield low purity, and have limited ability to address cargo heterogeneity. Thus, precise separation of EV subpopulations is of utmost importance. Additionally, detecting subpopulation-specific cargo is vital for validating the effectiveness of separation methods and supporting clinical biopsy applications. However, reviews that focus specifically on detection methods for EV subpopulations are limited. This paper provides a comprehensive overview of the methods for separating and detecting EV subpopulations with surface marker heterogeneity, comparing the advantages and limitations of each technique. Furthermore, it discusses challenges and future prospects for these methods in the context of liquid biopsy and downstream research. Collectively, this review aims to offer innovative insights into the separation and detection of EV subpopulations, guiding researchers to avoid common pitfalls and refine their investigative approaches.

Keywords: detection; extracellular vesicle; liquid biopsy; separation; subpopulation.

Figure 1

(A) Classical and some potential cargo molecules carried by EVs, along with separation methods for EV subpopulations, including approaches based on microfluidic chip, click/covalent chemistry, DNA nanoflowers, robust double-positive strategy, receptor-ligand interaction and negative strategy. (B) Detection methods for EV subpopulations, including approaches based on NanoFCM, Simoa, HRP, electrochemical sensor and Raman beads.

Figure 2

Microfluidic chip-based subpopulation separation methods. (A) The on-chip capture microfluidic device. (B) Separation of single-positive and double-positive EV subpopulations using AND and NOT logic algorithm. (C) The Sub-ExoProfile chip-based EV subpopulations separation and proteins profiling.

Figure 3

Click chemistry/covalent chemistry-based subpopulation separation methods. (A) Click-bubble-based EV subpopulation separation and fluorescent aptamer binding, facilitated by flipping and buoyancy-induced bubble self-aggregation. (B) LINGO-1⁺ EV separation mediated by click chemistry between Tz and TCO.

Figure 4

Aptamer-based subpopulation separation methods. (A) Traceless separation of PD-L1⁺ EV for accurate dissection of its subpopulation signature and function. Adapted with permission from , copyright 2023 American Chemical Society. (B) EpCAM⁺ EV separation and subsequent applications based on DFs. (C) Robust separation and release of EV with double-positive membrane protein. Adapted with permission from , copyright 2024 John Wiley and Sons Ltd.

Figure 5

Subpopulation separation methods not based on antibodies or aptamers and based on negative strategy. (A) ACE2⁺ EV separation based on receptor-ligand interaction between ACE2 receptor and RBD spike viral protein. (B) Natural tumor-cell derived EV separation by removing non-tumor-cell derived EVs. Adapted with permission from , copyright 2022 Taylor and Francis Ltd.

Figure 6

Color/Fluorescence reaction-based optical subpopulation detection methods. (A) Simoa immunoassay for detecting EpCAM and PD-L1 double-positive EV subpopulation by the increasing fluorescent signal from the catalytic reaction of SBG with RGP, confined within the micro-well. (B) The ExoAptaSensor with HRP accelerated dopamine polymerization and deposition for EV subpopulation detection. Adapted with permission from , copyright 2020 Elsevier Ltd.

Figure 7

Electrochemical sensor-based and Raman bead-based subpopulation detection methods. (A) The assay for electrochemical detecting FAM134B⁺ EV using cadmium-containing quantum dots. (B) Detection of EpCAM⁺ EV using Raman beads.

Figure 8

EV subpopulation separation and detection prospects for liquid biopsy and downstream research, including SERS, new microfluidic chips for fine separation, strategies based on biological detection technologies with ultra-high accuracy, for liquid biopsy; and DNA nanostructures, well-designed aptamer for fine separation, strategies based on acoustic, electricity, fluid dynamics, for downstream research.