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.


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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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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

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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