Encapsulation and release of living tumor cells using hydrogels with the hybridization chain reaction


Circulating tumor cells (CTCs) enable noninvasive liquid biopsy and identification of cancer. Various approaches exist for the capture and release of CTCs, including microfluidic methods and those involving magnetic beads or nanostructured solid interfaces. However, the concomitant cell damage and fragmentation that often occur during capture make it difficult to extensively characterize and analyze living CTCs. Here, we describe an aptamer-trigger-clamped hybridization chain reaction (atcHCR) method for the capture of CTCs by porous 3D DNA hydrogels. The 3D environment of the DNA networks minimizes cell damage, and the CTCs can subsequently be released for live-cell analysis. In this protocol, initiator DNAs with aptamer-toehold biblocks specifically bind to the epithelial cell adhesion molecule (EpCAM) on the surface of CTCs, which triggers the atcHCR and the formation of a DNA hydrogel. The DNA hydrogel cloaks the CTCs, facilitating quantification with minimal cell damage. This method can be used to quantitively identify as few as 10 MCF-7 cells in a 2-µL blood sample. Decloaking of tumor cells via gentle chemical stimulus (ATP) is used to release living tumor cells for subsequent cell culture and live-cell analysis. We also describe how to use the protocol to encapsulate and release cells of cancer cell lines, which can be used in preliminary experiments to model CTCs. The whole protocol takes ~2.5 d to complete, including downstream cell culture and analysis.

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Fig. 1: Multiscale characterization of DNA hydrogels.
Fig. 2: DNA hydrogel–based cloaking and decloaking of living tumor cells.
Fig. 3: The release and live-cell analysis of MCF-7 cells.
Fig. 4: Comparison of clamped HCR and traditional HCR.
Fig. 5: Characterization of cloaked tumor cells and DNA hydrogels.
Fig. 6: The kinetics of DNA hydrogel formation monitored using a bacterial indicator.
Fig. 7: The detection of MCF-7 cells in DNA hydrogel.

Data availability

The main data supporting the examples of this protocol are available within the article and its Supplementary Information files. Extra data are available from the corresponding author upon reasonable request. The source data underlying Figs. 3e,f, 6d,e and 7c–f and Supplementary Figs. 2, 3, 5, 6b, 8a–c and 9 are provided as source data files.


  1. 1.

    Nagrath, S. et al. Isolation of rare circulating tumour cells in cancer patients by microchip technology. Nature 450, 1235–1239 (2007).

    CAS  Article  Google Scholar 

  2. 2.

    Hou, S. et al. Capture and stimulated release of circulating tumor cells on polymer-grafted silicon nanostructures. Adv. Mater. 25, 1547–1551 (2013).

    CAS  Article  Google Scholar 

  3. 3.

    Ke, Z. F. et al. Programming thermoresponsiveness of nano velcro substrates enables effective purification of circulating tumor cells in lung cancer patients. ACS Nano 9, 62–70 (2015).

    CAS  Article  Google Scholar 

  4. 4.

    Ozkumur, E. et al. Inertial focusing for tumor antigen-dependent and -independent sorting of rare circulating tumor cells. Sci. Transl. Med. 5, 179ra147 (2013).

    Article  Google Scholar 

  5. 5.

    Stott, S. L. et al. Isolation of circulating tumor cells using a microvortex-generating herringbone-chip. Proc. Natl Acad. Sci. USA 107, 18392–18397 (2010).

    CAS  Article  Google Scholar 

  6. 6.

    Zhou, G. B. et al. Multivalent capture and detection of cancer cells with DNA nanostructured biosensors and multibranched hybridization chain reaction amplification. Anal. Chem. 86, 7843–7848 (2014).

    CAS  Article  Google Scholar 

  7. 7.

    Yoon, H. J. et al. Sensitive capture of circulating tumour cells by functionalized graphene oxide nanosheets. Nat. Nanotechnol. 8, 735–741 (2013).

    CAS  Article  Google Scholar 

  8. 8.

    Zhang, P. C. et al. Programmable fractal nanostructured interfaces for specific recognition and electrochemical release of cancer cells. Adv. Mater. 25, 3566–3570 (2013).

    CAS  Article  Google Scholar 

  9. 9.

    Zhao, W. A. et al. Bioinspired multivalent DNA network for capture and release of cells. Proc. Natl Acad. Sci. USA 109, 19626–19631 (2012).

    CAS  Article  Google Scholar 

  10. 10.

    Cushing, M. C. & Anseth, K. S. Materials science. Hydrogel cell cultures. Science 316, 1133–1134 (2007).

    CAS  Article  Google Scholar 

  11. 11.

    Li, J. & Mooney, D. J. Designing hydrogels for controlled drug delivery. Nat. Rev. Mater. 1, 16071 (2016).

    CAS  Article  Google Scholar 

  12. 12.

    Li, J. et al. Self-assembly of DNA nanohydrogels with controllable size and stimuli-responsive property for targeted gene regulation therapy. J. Am. Chem. Soc. 137, 1412–1415 (2015).

    CAS  Article  Google Scholar 

  13. 13.

    Seliktar, D. Designing cell-compatible hydrogels for biomedical applications. Science 336, 1124–1128 (2012).

    CAS  Article  Google Scholar 

  14. 14.

    Shen, Q. L. et al. Specific capture and release of circulating tumor cells using aptamer-modified nanosubstrates. Adv. Mater. 25, 2368–2373 (2013).

    CAS  Article  Google Scholar 

  15. 15.

    Liu, Q. et al. Valency-controlled framework nucleic acid signal amplifiers. Angew. Chem. Int. Ed. 57, 7131–7135 (2018).

    CAS  Article  Google Scholar 

  16. 16.

    Guo, W. W. et al. pH-stimulated DNA hydrogels exhibiting shape-memory properties. Adv. Mater. 27, 73–78 (2015).

    CAS  Article  Google Scholar 

  17. 17.

    Guo, W. W. et al. Switchable bifunctional stimuli-triggered poly-N-isopropylacrylamide/DNA hydrogels. Angew. Chem. Inter. Ed. 53, 10134–10138 (2014).

    CAS  Article  Google Scholar 

  18. 18.

    Jin, J. et al. A triggered DNA hydrogel cover to envelop and release single cells. Adv. Mater. 25, 4714–4717 (2013).

    CAS  Article  Google Scholar 

  19. 19.

    Xing, Y. Z. et al. Self-assembled DNA hydrogels with designable thermal and enzymatic responsiveness. Adv. Mater. 23, 1117–1121 (2011).

    CAS  Article  Google Scholar 

  20. 20.

    Zhu, Z. et al. Au@Pt nanoparticle encapsulated target-responsive hydrogel with volumetric bar-chart chip readout for quantitative point-of-care testing. Angew. Chem. Int. Ed. 53, 12503–12507 (2014).

    CAS  Google Scholar 

  21. 21.

    Song, Y. L. et al. Selection of DNA aptamers against epithelial cell adhesion molecule for cancer cell imaging and circulating tumor cell capture. Anal. Chem. 85, 4141–4149 (2013).

    CAS  Article  Google Scholar 

  22. 22.

    Song, P. et al. DNA hydrogel with aptamer-toehold-based recognition, cloaking, and decloaking of circulating tumor cells for live cell analysis. Nano Lett. 17, 5193–5198 (2017).

    CAS  Article  Google Scholar 

  23. 23.

    Chen, X. Q. et al. Ultrasensitive electrochemical detection of prostate-specific antigen by using antibodies anchored on a DNA nanostructural scaffold. Anal. Chem. 86, 7337–7342 (2014).

    CAS  Article  Google Scholar 

  24. 24.

    Ge, Z. L. et al. Hybridization chain reaction amplification of microRNA detection with a tetrahedral DNA nanostructure-based electrochemical biosensor. Anal. Chem. 86, 2124–2130 (2014).

    CAS  Article  Google Scholar 

  25. 25.

    Ge, Z. L., Pei, H., Wang, L. H., Song, S. P. & Fan, C. H. Electrochemical single nucleotide polymorphisms genotyping on surface immobilized three-dimensional branched DNA nanostructure. Sci. China Chem. 54, 1273–1276 (2011).

    CAS  Article  Google Scholar 

  26. 26.

    Lin, M. H. et al. Programmable engineering of a biosensing interface with tetrahedral DNA nanostructures for ultrasensitive DNA detection. Angew. Chem. Int. Ed. 54, 2151–2155 (2015).

    CAS  Article  Google Scholar 

  27. 27.

    Lin, M. H. et al. Target-responsive, DNA nanostructure-based e-DNA sensor for microRNA analysis. Anal. Chem. 86, 2285–2288 (2014).

    CAS  Article  Google Scholar 

  28. 28.

    Pei, H. et al. A DNA Nanostructure-based biomolecular probe carrier platform for electrochemical biosensing. Adv. Mater. 22, 4754–4758 (2010).

    CAS  Article  Google Scholar 

  29. 29.

    Shen, J. et al. Valence-engineering of quantum dots using programmable DNA scaffolds. Angew. Chem. Int. Ed. 56, 16077–16081 (2017).

    CAS  Article  Google Scholar 

  30. 30.

    Ye, D. K., Zuo, X. L. & Fan, C. H. DNA nanostructure-based engineering of the biosensing interface for biomolecular detection. Prog. Chem. 29, 36–46 (2017).

    Google Scholar 

  31. 31.

    Zhu, D. et al. A surface-confined proton-driven DNA pump using a dynamic 3D DNA scaffold. Adv. Mater. 28, 6860–6865 (2016).

    CAS  Article  Google Scholar 

  32. 32.

    Dirks, R. M. & Pierce, N. A. Triggered amplification by hybridization chain reaction. Proc. Natl Acad. Sci. USA 101, 15275–15278 (2004).

    CAS  Article  Google Scholar 

  33. 33.

    Wang, J. et al. Clamped hybridization chain reactions for the self-assembly of patterned DNA hydrogels. Angew. Chem. Int. Ed. 56, 2171–2175 (2017).

    CAS  Article  Google Scholar 

  34. 34.

    Li, C. et al. Rapid formation of a supramolecular polypeptide-DNA hydrogel for in situ three-dimensional multilayer bioprinting. Angew. Chem. Int. Ed. 54, 3957–3961 (2015).

    CAS  Article  Google Scholar 

  35. 35.

    Zhu, Z. et al. An aptamer cross-linked hydrogel as a colorimetric platform for visual detection. Angew. Chem. Int. Ed. 49, 1052–1056 (2010).

    CAS  Article  Google Scholar 

  36. 36.

    Kosuri, S. & Church, G. M. Large-scale de novo DNA synthesis: technologies and applications. Nat. Methods 11, 499–507 (2014).

    CAS  Article  Google Scholar 

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This work was financially supported by the Ministry of Science and Technology of China (2016YFA0201200), the National Natural Science Foundation of China (21904086, 21804088, 21804091), the Shanghai Municipal Education Commission-Gaofeng Clinical Medicine Grant Support (20171913), the “Shuguang Program” supported by the Shanghai Education Development Foundation, and the Shanghai Municipal Education Commission (18SG16).

Author information




X. Zuo, Q.L. and C.F. supervised the projects; D.Y., M.L., T.Z., P.S., L.S., H.W., X.M., X. Zuo and C.F. designed and conducted the experiments; F.W., X. Zhang, J.S., Z.G., L.W. and Q.L. analyzed the data; and Q.L., X. Zuo and C.F. wrote the manuscript.

Corresponding authors

Correspondence to Qian Li or Xiaolei Zuo.

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The authors declare no competing interests.

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Related links

Key reference using this protocol

Song, P. et al. Nano Lett. 17, 5193–5198 (2017): https://doi.org/10.1021/acs.nanolett.7b01006

Key data used in this protocol

Song, P. et al. Nano Lett. 17, 5193–5198 (2017): https://doi.org/10.1021/acs.nanolett.7b01006

Supplementary information


E. coli movement in PBS


E. coli movement in DNA hydrogel

Supplementary Information

Supplementary Figures 1–9.

Reporting Summary

Supplementary Video 1

E. coli movement in PBS

Supplementary Video 2

E. coli movement in DNA hydrogel

Supplementary Data 1

Statistical source data for Supplementary Figure 2

Supplementary Data 2

Statistical source data for Supplementary Figure 3

Supplementary Data 3

Statistical source data for Supplementary Figure 5

Supplementary Data 4

Statistical source data for Supplementary Figure 6

Supplementary Data 5

Statistical source data for Supplementary Figure 8

Supplementary Data 6

Statistical source data for Supplementary Figure 9

Source data

Source Data Fig. 3

Statistical source data

Source Data Fig. 4

Unprocessed gels

Source Data Fig. 6

Statistical source data

Source Data Fig. 7

Statistical source data

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Ye, D., Li, M., Zhai, T. et al. Encapsulation and release of living tumor cells using hydrogels with the hybridization chain reaction. Nat Protoc 15, 2163–2185 (2020). https://doi.org/10.1038/s41596-020-0326-4

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