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

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.