Abstract

Much of the functionality of multicellular systems arises from the spatial organization and dynamic behaviours within and between cells. Current single-cell genomic methods only provide a transcriptional ‘snapshot’ of individual cells. The real-time analysis and perturbation of living cells would generate a step change in single-cell analysis. Here we describe minimally invasive nanotweezers that can be spatially controlled to extract samples from living cells with single-molecule precision. They consist of two closely spaced electrodes with gaps as small as 10–20 nm, which can be used for the dielectrophoretic trapping of DNA and proteins. Aside from trapping single molecules, we also extract nucleic acids for gene expression analysis from living cells without affecting their viability. Finally, we report on the trapping and extraction of a single mitochondrion. This work bridges the gap between single-molecule/organelle manipulation and cell biology and can ultimately enable a better understanding of living cells.

Access optionsAccess options

Rent or Buy article

Get time limited or full article access on ReadCube.

from$8.99

All prices are NET prices.

Data availability

The data that support the plots within this paper and other findings of this study are available from the corresponding author upon reasonable request.

Additional information

Publisher’s note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

References

  1. 1.

    Tseng, F.-G. & Santra, T. S. Essentials of Single-cell Analysis: Concepts, Applications and Future Prospects (Springer, Berlin, 2016).

  2. 2.

    Borland, L. M., Kottegoda, S., Phillips, K. S. & Allbritton, N. L. Chemical analysis of single cells. Annu. Rev. Anal. Chem. 1, 191–227 (2008).

  3. 3.

    Trouillon, R., Passarelli, M. K., Wang, J., Kurczy, M. E. & Ewing, A. G. Chemical analysis of single cells. Anal. Chem. 85, 522–542 (2013).

  4. 4.

    Higgins, S. G. & Stevens, M. M. Extracting the contents of living cells. Science 356, 379–380 (2017).

  5. 5.

    Gawad, C., Koh, W. & Quake, S. R. Single-cell genome sequencing: current state of the science. Nat. Rev. Genet. 17, 175–188 (2016).

  6. 6.

    Blainey, P. C. & Quake, S. R. Dissecting genomic diversity, one cell at a time. Nat. Methods 11, 19–21 (2014).

  7. 7.

    Park, M. C., Hur, M. C., Cho, H. S., Park, S. H. & Suh, K. Y. High-throughput single-cell quantification using simple microwell-based cell docking and programmable time-course live-cell imaging. Lab Chip 11, 79–86 (2011).

  8. 8.

    Mu, X., Zheng, W. F., Sun, J. S., Zhang, W. & Jiang, X. Y. Microfluidics for manipulating cells. Small 9, 9–21 (2013).

  9. 9.

    Wang, J., Trouillon, R., Dunevall, J. & Ewing, A. G. Spatial resolution of single-cell exocytosis by microwell-based individually addressable thin film ultramicroelectrode arrays. Anal. Chem. 86, 4515–4520 (2014).

  10. 10.

    Kimmerling, R. J. et al. A microfluidic platform enabling single-cell RNA-seq of multigenerational lineages. Nat. Commun. 7, 10220 (2016).

  11. 11.

    Gao, J., Riahi, R., Sin, M. L. Y., Zhang, S. F. & Wong, P. K. Electrokinetic focusing and separation of mammalian cells in conductive biological fluids. Analyst 137, 5215–5221 (2012).

  12. 12.

    Fu, A. Y., Spence, C., Scherer, A., Arnold, F. H. & Quake, S. R. A microfabricated fluorescence-activated cell sorter. Nat. Biotechnol. 17, 1109–1111 (1999).

  13. 13.

    Liu, L., Cheung, T. H., Charville, G. W. & Rando, T. A. Isolation of skeletal muscle stem cells by fluorescence-activated cell sorting. Nat. Protoc. 10, 1612–1624 (2015).

  14. 14.

    Norregaard, K. et al. Manipulation and motion of organelles and single molecules in living cells. Chem. Rev. 117, 4342–4375 (2017).

  15. 15.

    Zhang, H. & Liu, K. K. Optical tweezers for single cells. J. R. Soc. Interface 5, 671–690 (2008).

  16. 16.

    Ashkin, A., Schütze, K., Dziedzic, J. M., Euteneuer, U. & Schliwa, M. Force generation of organelle transport measured in vivo by an infrared laser trap. Nature 348, 346–348 (1990).

  17. 17.

    Wang, Y. et al. Clonal evolution in breast cancer revealed by single nucleus genome sequencing. Nature 512, 155–160 (2015).

  18. 18.

    McConnell, M. J. et al. Mosaic copy number variation in human neurons. Science 342, 632–637 (2013).

  19. 19.

    Cai, X. et al. Single-cell, genome-wide sequencing identifies clonal somatic copy-number variation in the human brain. Cell Rep. 8, 1280–1289 (2014).

  20. 20.

    Villani, A. C. et al. Single-cell RNA-seq reveals new types of human blood dendritic cells, monocytes, and progenitors. Science 356, eaah4573 (2017).

  21. 21.

    Macaulay, I. C. et al. Single-cell RNA-sequencing reveals a continuous spectrum of differentiation in hematopoietic cells. Cell Rep. 14, 966–977 (2016).

  22. 22.

    Lan, F., Demaree, B., Ahmed, N. & Abate, A. R. Single-cell genome sequencing at ultra-high-throughput with microfluidic droplet barcoding. Nat. Biotechnol. 35, 640–646 (2017).

  23. 23.

    Regev, A. et al. The human cell atlas. eLife 6, 27041 (2017).

  24. 24.

    Guillaume-Gentil, O. et al. Tunable single-cell extraction for molecular analyses. Cell 166, 506–516 (2016).

  25. 25.

    Actis, P. Sampling from single cells. Small Methods 2, 1700300 (2018).

  26. 26.

    Guillaume-Gentil, O. et al. Force-controlled manipulation of single cells: from AFM to FluidFM. Trends Biotechnol. 32, 381–388 (2014).

  27. 27.

    Li, X., Tao, Y. L., Lee, D. H., Wickramasinghe, H. K. & Lee, A. P. In situ mRNA isolation from a microfluidic single-cell array using an external AFM nanoprobe. Lab Chip 17, 1635–1644 (2017).

  28. 28.

    Seger, R. A. et al. Voltage controlled nano-injection system for single-cell surgery. Nanoscale 4, 5843–5846 (2012).

  29. 29.

    Actis, P. et al. Compartmental genomics in living cells revealed by single-cell nanobiopsy. ACS Nano 8, 546–553 (2014).

  30. 30.

    Tóth, E. N. et al. Single-cell nanobiopsy reveals compartmentalization of mRNA in neuronal cells. J. Biol. Chem. 293, 4940–4951 (2018).

  31. 31.

    Kim, H., Okano, K., Osada, T., Ikai, A. & Yasuda, K. Development of mRNA analysis system in single living cells by atomic force microscopy. Biophys. J. 88, 154A–154A (2005).

  32. 32.

    Osada, T., Uehara, H., Kim, H. & Ikai, A. Advances in Clinical Chemistry, Vol. 38, Ch. 3 (Elsevier, London, 2004).

  33. 33.

    Osada, T., Uehara, H., Kim, H. & Ikai, A. mRNA analysis of single living cells. J. Nanobiotechnol. 1, 2 (2003).

  34. 34.

    Nawarathna, D., Turan, T. & Wickramasinghe, H. K. Selective probing of mRNA expression levels within a living cell. Appl. Phys. Lett. 95, 083117 (2009).

  35. 35.

    Cadinu, P. et al. Single molecule trapping and sensing using dual nanopores separated by a zeptoliter nanobridge. Nano Lett. 17, 6376–6384 (2017).

  36. 36.

    Cadinu, P. et al. Double barrel nanopores as a new tool for controlling single-molecule transport. Nano Lett. 18, 2738–2745 (2018).

  37. 37.

    Takahashi, Y. et al. Multifunctional nanoprobes for nanoscale chemical imaging and localized chemical delivery at surfaces and interfaces. Angew. Chem. Int. Ed. 50, 9638–9642 (2011).

  38. 38.

    Ren, R. et al. Nanopore extended field-effect transistor for selective single-molecule biosensing. Nat. Commun. 8, 586 (2017).

  39. 39.

    McKelvey, K. et al. Fabrication, characterization, and functionalization of dual carbon electrodes as probes for scanning electrochemical microscopy (SECM). Anal. Chem. 85, 7519–7526 (2013).

  40. 40.

    Freedman, K. J. et al. Nanopore sensing at ultra-low concentrations using single-molecule dielectrophoretic trapping. Nat. Commun. 7, 10217 (2016).

  41. 41.

    Ramos, A., Morgan, H., Green, N. G. & Castellanos, A. The role of electrohydrodynamic forces in the dielectrophoretic manipulation and separation of particles. J. Electrostat. 47, 71–81 (1999).

  42. 42.

    Barik, A. et al. Graphene-edge dielectrophoretic tweezers for trapping of biomolecules. Nat. Commun. 8, 1867 (2017).

  43. 43.

    Barik, A. et al. Ultralow-power electronic trapping of nanoparticles with sub-10 nm gold nanogap electrodes. Nano Lett. 16, 6317–6324 (2016).

  44. 44.

    Shivalingam, A. et al. The interactions between a small molecule and G-quadruplexes are visualized by fluorescence lifetime imaging microscopy. Nat. Commun. 6, 8178 (2015).

  45. 45.

    Gibbons, J. G., Branco, A. T., Yu, S. & Lemos, B. Ribosomal DNA copy number is coupled with gene expression variation and mitochondrial abundance in humans. Nat. Commun. 5, 4850 (2014).

Download references

Acknowledgements

J.B.E. has been funded in part by an ERC starting (NanoP) and consolidator (NanoPD) investigator grant. A.P.I. and J.B.E. acknowledge support from EPSRC grant EP/P011985/1 and BBSRC grant BB/R022429/1. A.P.I. acknowledges IC Research Fellowship funding. We thank B. Akpinar for helping with TEM and EDX spectroscopy and S. Rothery for helping with the cell viability studies. A.B. and S.-H.O. acknowledge support from the US National Science Foundation (NSF ECCS no. 1610333). M.J.D. is supported by a Wellcome Trust Clinical Postdoctoral Fellowship (106713/Z/14/Z) and J.T.K. received funding from an ERC starting grant (282430).

Author information

Author notes

  1. These authors contributed equally: Binoy Paulose Nadappuram, Paolo Cadinu.

Affiliations

  1. Department of Chemistry, Imperial College London, London, UK

    • Binoy Paulose Nadappuram
    • , Paolo Cadinu
    • , Alexander J. Ainscough
    • , Minkyung Kang
    • , Jorge Gonzalez-Garcia
    • , Keith R. Willison
    • , Ramon Vilar
    • , Aleksandar P. Ivanov
    •  & Joshua B. Edel
  2. Department of Electrical and Computer Engineering, University of Minnesota, Minneapolis, MN, USA

    • Avijit Barik
    •  & Sang-Hyun Oh
  3. Department of Experimental Medicine and Toxicology, Imperial College London, London, UK

    • Alexander J. Ainscough
    •  & Beata Wojciak-Stothard
  4. Department of Neuroscience, Physiology and Pharmacology, University College London, London, UK

    • Michael J. Devine
    •  & Josef T. Kittler
  5. School of Electronic and Electrical Engineering, Pollard Institute, University of Leeds, Leeds, UK

    • Paolo Actis

Authors

  1. Search for Binoy Paulose Nadappuram in:

  2. Search for Paolo Cadinu in:

  3. Search for Avijit Barik in:

  4. Search for Alexander J. Ainscough in:

  5. Search for Michael J. Devine in:

  6. Search for Minkyung Kang in:

  7. Search for Jorge Gonzalez-Garcia in:

  8. Search for Josef T. Kittler in:

  9. Search for Keith R. Willison in:

  10. Search for Ramon Vilar in:

  11. Search for Paolo Actis in:

  12. Search for Beata Wojciak-Stothard in:

  13. Search for Sang-Hyun Oh in:

  14. Search for Aleksandar P. Ivanov in:

  15. Search for Joshua B. Edel in:

Contributions

J.B.E. and A.P.I. designed and supervised the research. B.P.N. and P.C. performed the experiments and contributed equally to this work. B.P.N., P.C., J.B.E. and A.P.I. analysed the data and prepared the manuscript. A.B. and S.-H.O. developed the finite element model and performed the theoretical calculations. A.J.A., M.J.D., J.G.-G. and B.W.-S. prepared the cell samples and contributed to the cell biopsy experiments. M.K. recorded the electron micrographs. J.T.K., K.R.W., R.V. and P.A. helped with the experiments. All the authors discussed the results and commented on the manuscript.

Competing interests

The authors declare no competing interests.

Corresponding authors

Correspondence to Aleksandar P. Ivanov or Joshua B. Edel.

Supplementary information

  1. Supplementary Information

    Supplementary Results, Supplementary Figures 1–15, Supplementary Table 1, Supplementary References

  2. Reporting Summary

  3. Single-molecule trapping and writing (SI section 10b)

    Video showing the pick and release of single 10 kbp DNA molecule using the DEP nanotweezers. The DNA molecule was captured at the nanotweezer tip by turning on the a.c. field (DEP on). Transfer of the captured single molecule from one position to another was achieved by moving the nanotweezer using a micromanipulator while the DEP was on. Turning off the DEP resulted in the release of the captured molecule from the nanotweezers tip into the solution

  4. Protein trapping (SI section 7)

    Trapping of α-synuclein (SI section 7). Video showing the fluorescence profile at the nanotweezers tip during trapping and releasing of the α-Synuclein molecule. No fluoresce change was observed when DEP was off. Upon application of an a.c. field (DEP on) a sharp increase in fluorescence around the nanotweezer tip was observed. (n = 4.)

  5. Single Molecule trapping and writing (SI section 10a)

    Video showing the Pick and release of single 10 kbp DNA molecule using the DEP nanotweezers. The DNA molecule was captured at the nanotweezer tip by turning on the a.c. field (DEP on). Transfer of the captured single molecule from one position to another was achieved by moving the nanotweezer using a micromanipulator while the DEP was on. Turning off the DEP resulted in the release of the captured molecule from the nanotweezers tip into the solution

  6. Single organelle extraction (Figure 6)

    Video showing the trapping an extraction of a single mitochondrion inside a neuron. The nanotweezers (dark spot) was positioned close to a labelled mitochondrion (bright spot). Upon application of an a.c. field (DEP on), the mitochondrion gets trapped at the nanotweezer tip. Extraction of the mitochondrion from the neuron was achived by withdrawing the nanotweezer from the neuron while keeping the a.c. field turned on (DEP on). (n = 4.)

  7. Single Molecule trapping and writing (Figure 3)

    Video showing the Pick and release of single 10 kbp DNA molecule using the DEP nanotweezers. The DNA molecule was captured at the nanotweezer tip by turning on the a.c. field (DEP on). Transfer of the captured single molecule from one position to another was achieved by moving the nanotweezer using a micromanipulator while the DEP was on. Turning off the DEP resulted in the release of the captured molecule from the nanotweezers tip into the solution. (n = 5.)

  8. DNA trapping(Figure2)

    Video showing the trapping and release of 10 kbp DNA. When DEP is turned on, the force generated around the tip is sufficiently strong to capture freely diffusing DNA molecules in solution resulting in the accumulation of fluorescently tagged DNA molecule at the nanotweezers (bright spot). This operation is fully reversible; as soon as the DEP is turned off the trapped molecules are immediately released back into the solution. (n = 4.)

  9. DNA trapping in cells (Figure4)

    Video showing the trapping of DNA inside the cell nucleus. The tip was approached and then inserted into the cell nucleus. Application of an a.c. bias (DEP on) traps the DNA fragments at the nanotweezers tip as can be seen by an increase in fluorescence signal around the tip (bright spot) (n = 4).

About this article

Publication history

Received

Accepted

Published

DOI

https://doi.org/10.1038/s41565-018-0315-8