Extracellular vesicles (EVs) are nanoscale, lipid bilayer-bound particles that are widely found in bodily fluids and carry a variety of biological cargo, including proteins, nucleic acids, and metabolites. EVs play critical roles in cellular communication, immune response regulation, and genetic material transfer. Over recent years, the potential of EVs in diagnostics and therapeutics has drawn increasing attention, particularly in the context of diseases such as cancer, neurodegenerative disorders, and immune diseases. However, despite their immense promise, efficiently isolating and analyzing EVs from complex biological fluids such as blood and urine remains a major technical challenge.

Traditional methods of EV isolation and analysis, such as ultracentrifugation, filtration, or affinity capture, are commonly used but suffer from several limitations, including low throughput, time consumption, and limited sensitivity. As a result, the need for a more efficient and sensitive EV capture and analysis platform has become a key focus in EV research. To address this challenge, our team has developed an innovative array-based approach utilizing an amphiphile–dendrimer supramolecular probe (ADSP) to significantly improve the efficiency of capturing and analyzing EVs from small sample volumes in a high-throughput manner.

Biological Functions of EVs and Their Potential Applications

EVs play an essential role in intercellular communication, carrying a wide range of biologically active molecules such as signaling proteins, non-coding RNAs, DNA fragments, lipids, and metabolites. These molecules can be transferred between cells of the same type and even across different tissues and organs, making EVs a crucial medium for cellular regulation and information exchange.

One of the most promising applications of EVs is as biomarkers for disease diagnosis. For example, tumor cells release EVs that carry specific tumor markers, reflecting the state and progression of the tumor. By analyzing EVs in patient biofluids, researchers can non-invasively monitor cancer development, metastasis, and treatment response. Additionally, EVs are being explored as drug delivery vehicles due to their biocompatibility and stability. They can deliver drugs, gene therapies, or other therapeutic agents directly to target cells or tissues.

However, a major barrier to the clinical application of EVs is the ability to efficiently and specifically isolate them from complex biological fluids (such as blood, urine, or saliva) and perform precise molecular analyses. Current isolation and analysis techniques, such as ultracentrifugation, can produce relatively pure EV samples but are labor-intensive, time-consuming, and have low throughput, making them unsuitable for clinical use. Therefore, improving the efficiency and sensitivity of EV capture and analysis has become a core issue in EV research.

Array-Based EV Capture and In Situ Protein Analysis

To overcome these challenges, we developed an array-based high-throughput EV capture and analysis method using ADSP. ADSP is an amphiphilic dendrimer supramolecular probe that has excellent self-assembly and membrane-binding properties. By coating ADSP onto a nitrocellulose membrane, we can construct an array capable of efficiently capturing EVs and facilitating high-throughput screening.

One of the core advantages of this array-based method is its high throughput and sensitivity. Traditional EV capture methods often require large sample volumes and long analysis times, but our array platform, combined with an automated workstation, allows for simultaneous processing of multiple samples, greatly enhancing experimental efficiency and reproducibility. During the experimental process, the ADSP-coated membrane preparation and array construction can be completed in a single day, and the EV capture process takes only 30 minutes, followed by immunoblotting analysis within 3 hours. This rapid workflow makes it suitable for high-throughput screening and the rapid analysis of clinical samples.

Detection of Glycosylation Modifications on EV Surfaces

We further demonstrated the potential of this array platform in detecting specific glycosylation modifications on EV surfaces. Glycosylation is a common post-translational modification of proteins and plays a critical role in many pathological processes, particularly in cancer, where changes in glycosylation are often associated with tumor progression and metastasis. By using metabolic labeling to introduce azide groups, we employed click chemistry to perform precise analyses of glycosylation on EV surfaces. This method efficiently detects glycosylation changes on EVs, further expanding the potential for EVs as biomarkers in disease diagnosis.

In our research, we successfully captured and analyzed EVs from various biological samples, including blood, urine, and cell culture media, and detected glycosylation patterns on the EV surface. This result provides strong evidence for the use of EVs as diagnostic tools, particularly in early cancer biomarker screening and dynamic disease monitoring.

 Advantages and Future Prospects of the Array-Based Approach

This ADSP-based array capture and analysis platform offers several key advantages. First, the array design enables high-throughput processing of multiple samples, significantly improving throughput. Second, the incorporation of an automated workstation ensures high reproducibility and accuracy, reducing human error. Additionally, the platform allows for in situ analysis while capturing EVs, providing a convenient tool for quickly obtaining EV molecular profiles. This innovative method can be applied in both basic EV research and clinical settings, with broad potential applications in diseases such as cancer, neurodegenerative disorders, and immune diseases.

Looking forward, we aim to further optimize this technology platform by enhancing its sensitivity and specificity to handle more complex biological fluid samples. Furthermore, by integrating this platform with other high-throughput omics technologies such as proteomics, metabolomics, and genomics, we hope to obtain a more comprehensive molecular profile of EVs, thus advancing the application of EVs in personalized medicine.

Overall, the ADSP-based array capture and analysis method provides a powerful tool for EV research, solving the challenges of low throughput, long analysis times, and limited sensitivity seen in traditional methods. The innovation and practicality of this technological platform bring new opportunities for both fundamental research and clinical translation of EVs, with the potential to play a critical role in future precision medicine and disease diagnostics.

 Conclusion

Extracellular vesicles hold vast potential in understanding cellular processes, diagnosing diseases, and developing targeted therapies. However, realizing this potential hinge on the development of effective and high-throughput methods for isolating and analyzing EVs from complex biological fluids. The ADSP-based array method presents a breakthrough in this regard, offering researchers a rapid, sensitive, and scalable platform for EV analysis. As this technology continues to evolve, it could unlock new pathways for early diagnosis, dynamic disease monitoring, and personalized therapeutic interventions, pushing the boundaries of EV-based research and clinical applications.