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High-throughput capture and in situ protein analysis of extracellular vesicles by chemical probe-based array

Nature Protocols volume 20, pages 1057–1081 (2025)Cite this article

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Abstract

Extracellular vesicles (EVs) are small particles with phospholipid bilayers that carry a diverse range of cargoes including nucleic acids, proteins and metabolites. EVs have important roles in various cellular processes and are increasingly recognized for their ubiquitous role in cell–cell communications and potential applications in therapeutics and diagnostics. Although many methods have been developed for the characterization and measurement of EVs, analyzing them from biofluids remains a challenge with regard to throughput and sensitivity. Recently, we introduced an approach to facilitate high-throughput analysis of EVs from trace amounts of sample. In this method, an amphiphile–dendrimer supramolecular probe (ADSP) is coated onto a nitrocellulose membrane for array-based capture and to enable an in situ immunoblotting assay. Here, we describe the protocol for our array-based method of EV profiling. We describe an enhanced version of the method that incorporates an automated printing workstation, ensuring high throughput and reproducibility. We further demonstrate the use of our array to profile specific glycosylations on the EV surface using click chemistry of an azide group introduced by metabolic labeling. In this protocol, the synthesis of ADSP and the fabrication of ADSP nitrocellulose membrane array can be completed on the same day. EVs are efficiently captured from biological or clinical samples through a 30-min incubation, followed by an immunoblotting assay within a 3-h window, thus providing a high-throughput platform for EV isolation and in situ targeted analysis of EV proteins and their modifications.

Key points

  • This protocol uses an amphiphile–dendrimer supramolecular probe to capture extracellular vesicles from biofluids and cell culture medium to avoid time-consuming sample processing and the cocapture of nucleic acids and proteins associated with ultracentrifugation-based purification approaches.

  • The use of a chemical affinity probe coating onto nitrocellulose membrane enables high-throughput, array-based enrichment and in situ immunoblotting of extracellular vesicle proteins for relative concentration assay.

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Fig. 1: Scheme showing the interaction between ADSP and the phospholipid layer of EVs.
Fig. 2: Workflow for array-based high-throughput capture and analysis of EVs.
Fig. 3: Detection limit and linear range of ADSP array for plasma EVs.
Fig. 4: Detection limit and linear range of ADSP array for urine EVs.
Fig. 5: Analysis of clinical plasma samples by ADSP array.
Fig. 6: Analysis of clinical urine samples by ADSP array.
Fig. 7: Analysis of Ac4ManNAz-labeled EVs from cells by ADSP array.

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Data availability

The authors declare that the main data discussed in this protocol are available in the supporting primary research papers. Further parameters or details of the experiments are available from the corresponding author upon reasonable request.

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Acknowledgements

We sincerely thank X. Zhang at Jilin University/Tsinghua University for the help on concept proposing and experimental design of the project. This work was funded by National Science Foundation of China (22374056, 22174021), National Key R&D Program of China (2020YFE0202200, 2023YFA0915300) and Proteomic Navigator of The Human Body Project (π-HuB project).

Author information

Author notes

  1. Ao Shen

    Present address: School of Medicine, Shanghai Jiao Tong University, Shanghai, China

  2. These authors contributed equally: Xin Feng, Ao Shen.

Authors and Affiliations

  1. Center for Supramolecular Chemical Biology, State Key Laboratory of Supramolecular Structure and Materials, School of Life Sciences, Jilin University, Changchun, China

    Xin Feng, Ao Shen, Shengnan Jia, Wenke Zhang & Lianghai Hu

  2. School of Natural Sciences, Faculty of Science and Engineering, Macquarie University, Sydney, New South Wales, Australia

    Xin Feng, Wei Zhang & Yuling Wang

  3. Department of Biochemistry, Purdue University, West Lafayette, IN, USA

    Anton Iliuk & W. Andy Tao

  4. Department of Chemistry and NHC Key Laboratory of Glycoconjugates Research, Fudan University, Shanghai, China

    Ying Zhang

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Contributions

X.F., A.S. and L.H. designed the experimental protocol. X.F., A.S. and W.Z. performed all the experiments with help from S.J., A.I., Y.Z., Y.W. and W.Z. X.F. and A.S. wrote the manuscript with help from A.I., Y.Z., Y.W., W.Z. and W.A.T. X.F., A.S., W.A.T. and L.H. approved the final version. Y.Z., Y.W., W.Z., L.H. and W.A.T. provided advice and infrastructural support.

Corresponding authors

Correspondence to Ying Zhang, W. Andy Tao or Lianghai Hu.

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The authors declare no competing interests.

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Nature Protocols thanks Dennis Jeppesen and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.

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Key references

Feng, X. et al. Anal. Chem. 95, 2812–2821 (2023): https://doi.org/10.1021/acs.analchem.2c04190

Feng, X. et al. J. Proteome Res. 22, 2516–2524 (2023): https://doi.org/10.1021/acs.jproteome.3c00063

Shen, A. et al. Anal. Chim. Acta. 1309, 342699 (2024): https://doi.org/10.1016/j.aca.2024.342699

Extended data

Extended Data Fig. 1 Flowchart for metabolic glycan labeling of cells and array-based EV capture/detection.

a. Metabolic glycan labeling of cells. b. Collection of cell culture medium and click chemistry reaction of EVs. c. ADSP array-based capture of EVs and fluorescent detection of labeled glycans. Figure created using BioRender.

Extended Data Fig. 2 The synthesized ADSP solution and the use of ADSP to modify the membranes.

a. ADSP solution after dialysis. The A340 is ~0.62. b. The ADSP-modified NC membrane finished product. Left: blank membrane, right: ADSP-modified membrane. c. ADSP solution measured at 340 nm after each modification to show the content. The overall use of ADSP solution is 0.24 mL per cm2 membrane. The effectiveness of the array after each modification is shown, demonstrating that ADSP below an A340 of 0.4 might be insufficient for modification.

Extended Data Fig. 3 Distribution and flow cytometry charts of EVs captured by ADSP array, generated using nanoflow cytometry.

“Plasma”, “Urine” and “Cell culture medium” refer to particles from different samples captured by and eluted from the ADSP-coated membrane, respectively. “Original” refers to particles from plasma samples captured and eluted by the ADSP-coated membrane. “Filtrate” represents the sEVs obtained from the original sample after passing through a 0.22 μm membrane filter. “Residue” denotes the particles from the original sample retained on the membrane filter, which are collected by washing with PBS. a. Histograms depicting the distribution of particles obtained from the plasma, urine, and cell culture medium. b. A size reference chart provided by Apogee for standard microspheres, enabling the determination of the size distribution of the test sample based on comparison with the standard microspheres. The term 4-9 with “black gate” refers to different populations of particles with varying particle sizes, whereas the term 1-3 with “red gate” signifies particle populations with different sizes and the presence of fluorescence signals. c. The Original, Filtrate, and Residue samples are stained with a membrane dye DiO and subsequently subjected to detection through the 488-channel by Apogee. Particles exhibiting fluorescence signals above the red line are identified as particles having membrane structures, while those below are considered as lacking membrane structures. Among the particles above the red line, those with membrane structures and falling within the size range to the left of the black line are identified as sEVs, while those to the right are categorized as lEVs.

Extended Data Fig. 4 Comparison of EVs enrichment efficiency between ADSP-membrane and UC with characterization by NTA.

Comparison of EV enrichment efficiency between ADSP-membrane and ultracentrifugation using NTA. a–c show NTA characterization of plasma, urine and cell culture medium derived EVs captured by an ADSP membrane. The original volume of samples is: a. 10 µL of plasma diluted using PBS to a final volume of 2 mL; b. 2 mL of urine samples; c. 2 mL of cell culture medium samples. d–f show NTA characterization of plasma, urine and cell culture medium derived EVs enriched by ultracentrifugation. Original volumes of plasma, urine and cell culture medium were the same as a–c. DPBS was used to adjust the ultracentrifugation precipitate to 1 mL for NTA assay.

Supplementary information

Supplementary Video 1

The use of the automated pipetting workstation for array-based capture of EVs.

Supplementary Video 2

The use of Smart Blotter for array-based capture of EVs.

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Feng, X., Shen, A., Zhang, W. et al. High-throughput capture and in situ protein analysis of extracellular vesicles by chemical probe-based array. Nat Protoc 20, 1057–1081 (2025). https://doi.org/10.1038/s41596-024-01082-z

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