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Live-cell synthesis of biocompatible quantum dots

Nature Protocols volume 20, pages 1884–1914 (2025)Cite this article

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Abstract

Quantum dots (QDs) exhibit fluorescence properties with promising prospects for biomedical applications; however, the QDs synthesized in organic solvents shows poor biocompatibility, limiting their use in biological systems. We developed an approach for synthesizing QDs in live cells by coupling a series of intracellular metabolic pathways in a precise spatial and temporal sequence. We have validated this approach in yeast (Saccharomyces cerevisiae), Staphylococcus aureus, Michigan Cancer Foundation-7 (MCF-7) and Madin-Darby canine kidney (MDCK) cells. The intracellularly synthesized QDs are inherently stable and biocompatible, making them suitable for the direct in situ labeling of cells and cell-derived vesicles. Here, we provide an optimized workflow for the live-cell synthesis of QDs by using S. cerevisiae, S. aureus or MCF-7 cells. In addition, we detail a cell-free aqueous synthetic system (quasi-biosynthesis) containing enzymes, electrolytes, peptides and coenzymes, which closely mimics the intracellular synthetic conditions used in our cell culture system. In this solution, we synthesize biocompatible ultrasmall QDs that are easier to purify and characterize than those synthesized in cells. The live-cell-synthesized QDs can be used for bioimaging and microvesicle detection, whereas the quasi-biosynthesized QDs are suitable for applications such as biodetection, biolabeling and real-time imaging. The procedure can be completed in 3–4 d for live-cell QD synthesis and 2 h for the quasi-biosynthesis of QDs. The procedure is suitable for users with expertise in chemistry, biology, materials science and synthetic biology. This approach encourages interested researchers to engage in the field of QDs and develop further biomedical applications.

Key points

  • In yeast, selenium and cadmium are supplied to the culture, where they are taken up by cells, to intracellularly generate CdSe QDs by the converging of glutathione/NADPH-involved selenite reduction and Cd2+ detoxication pathways.

  • The live-cell synthesis of QDs can be replicated in an aqueous solution that closely mimics the intracellular environment containing enzymes, electrolytes, peptides, coenzymes and metal ions.

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Fig. 1: Metabolic pathways in ARLCS of fluorescent QDs in live cells.
Fig. 2: Schematic overview of the workflow for live-cell synthesis of QDs and their applications.
Fig. 3: Schematic overview of the workflow for quasi-biological synthesis of QDs.
Fig. 4: Apparatus setup for the quasi-biological synthesis of Ag2Se QDs.
Fig. 5: Synthesis and purification of QDs in live yeast cells.
Fig. 6: Fluorescent nanoprobes for detection of virus.
Fig. 7: Live-cell synthesis of QDs for in situ labeling of MVs.
Fig. 8: Characterization of Ag2Se QDs.
Fig. 9: ECL of Ag2Se QDs for detection of dopamine.
Fig. 10: Characterization of Ag2Se@Mn QDs.
Fig. 11: Ag2Se@Mn QDs for labeling and real-time tracking of MVs.

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Data availability

Source data for the figures in this protocol are available in the listed supporting primary research articles: references3,5,6,23,24,28,34,45. Further parameters or details of the experiments are available from the corresponding author upon reasonable request.

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Acknowledgements

We thank Z.-L. Zhang, Z. Xie, H.-H. Liu, Y. Li, Y.-P. Gu, L.-H. Xiong, C.-Q. Wu and X. Li for their work and contributions to the project. This work was supported by the National Natural Science Foundation of China (Nos. 22293030, 22293032, 21535005, 20921062, 20621502 and 20025311) and the National Basic Research Program of China (973 Program, 2006CB933100 and 2011CB933600).

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Author notes

  1. These authors contributed equally: An-An Liu, Ran Cui.

Authors and Affiliations

  1. State Key Laboratory of Medicinal Chemical Biology, Frontiers Science Centre for New Organic Matter, Tianjin Key Laboratory of Biosensing and Molecular Recognition, Research Centre for Analytical Sciences, College of Chemistry, Frontiers Science Center for Cell Responses, Nankai University, Tianjin, People’s Republic of China

    An-An Liu, Xia Zong, Jianhong Jia, Yusi Hu & Dai-Wen Pang

  2. College of Chemistry and Molecular Sciences, Wuhan University, Wuhan, People’s Republic of China

    Ran Cui & Jing-Ya Zhao

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  1. An-An Liu
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  3. Xia Zong
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Contributions

D.-W.P. is the group leader who conceived and initiated the project and supervised the study and the entire manuscript preparation. R.C., J.-Y.Z. and Y.H. developed the procedures for live-cell synthesis and quasi-biosynthesis of QDs and data processing. A.-A.L., X.Z. and J.J. organized and wrote the manuscript. All authors reviewed and edited the manuscript and approved the final draft.

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Correspondence to Dai-Wen Pang.

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

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Key references

Hu, Y. S. et al. Natl Sci. Rev. 11, nwae021 (2024): https://doi.org/10.1093/nsr/nwae021

Xiong, L. H. et al. ACS Nano 8, 5116–5124 (2014): https://doi.org/10.1021/nn501174g

Zhao, J. Y. et al. J. Am. Chem. Soc. 138, 1893–1903 (2016): https://doi.org/10.1021/jacs.5b10340

Cui, R. et al. Adv. Funct. Mater. 19, 2359–2364 (2009): https://doi.org/10.1002/adfm.200801492

Gu, Y. P. et al. J. Am. Chem. Soc. 134, 79–82 (2012): https://doi.org/10.1021/ja2089553

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Liu, AA., Cui, R., Zong, X. et al. Live-cell synthesis of biocompatible quantum dots. Nat Protoc 20, 1884–1914 (2025). https://doi.org/10.1038/s41596-024-01133-5

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