Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

  • Article
  • Published:

Nanoscale tweezers for single-cell biopsies

Nature Nanotechnology volume 14, pages 80–88 (2019)Cite this article

Subjects

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.

This is a preview of subscription content, access via your institution

Access options

Buy this article

  • Purchase on Springer Link
  • Instant access to full article PDF
Buy now

Prices may be subject to local taxes which are calculated during checkout

Fig. 1: Schematic and characterization of the DEP nanotweezer.
Fig. 2: Trapping and extraction of 10 kbp DNA.
Fig. 3: Nanotweezer aided single-molecule trapping and extraction.
Fig. 4: DNA extraction from the cell nucleus.
Fig. 5: Extraction of mRNA from the cytoplasm.
Fig. 6: Single organelle extraction.

Similar content being viewed by others

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.

References

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

  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).

    Article  CAS  Google Scholar 

  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).

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

  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).

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

  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).

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

  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).

    Article  CAS  Google Scholar 

  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).

    Article  CAS  Google Scholar 

  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).

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

  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).

    Article  CAS  Google Scholar 

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

    Article  Google Scholar 

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

    Article  CAS  Google Scholar 

  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).

    Article  CAS  Google Scholar 

  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).

    Article  Google Scholar 

  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).

    Article  CAS  Google Scholar 

  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).

    Article  CAS  Google Scholar 

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

    Article  Google Scholar 

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

    Article  CAS  Google Scholar 

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

    Article  Google Scholar 

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

    Article  CAS  Google Scholar 

  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).

    Article  CAS  Google Scholar 

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

    Article  Google Scholar 

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

    Article  CAS  Google Scholar 

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

    Article  Google Scholar 

  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).

    Article  Google Scholar 

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

    Google Scholar 

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

    Article  Google Scholar 

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

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

  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).

    Article  CAS  Google Scholar 

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

    Article  Google Scholar 

  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).

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

  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).

    Article  CAS  Google Scholar 

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

    Article  Google Scholar 

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

    Article  CAS  Google Scholar 

  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).

    Article  CAS  Google Scholar 

  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).

    Article  CAS  Google Scholar 

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.

Authors and 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. Binoy Paulose Nadappuram
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Paolo Cadinu
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Avijit Barik
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Alexander J. Ainscough
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Michael J. Devine
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Minkyung Kang
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Jorge Gonzalez-Garcia
    View author publications

    You can also search for this author in PubMed Google Scholar

  8. Josef T. Kittler
    View author publications

    You can also search for this author in PubMed Google Scholar

  9. Keith R. Willison
    View author publications

    You can also search for this author in PubMed Google Scholar

  10. Ramon Vilar
    View author publications

    You can also search for this author in PubMed Google Scholar

  11. Paolo Actis
    View author publications

    You can also search for this author in PubMed Google Scholar

  12. Beata Wojciak-Stothard
    View author publications

    You can also search for this author in PubMed Google Scholar

  13. Sang-Hyun Oh
    View author publications

    You can also search for this author in PubMed Google Scholar

  14. Aleksandar P. Ivanov
    View author publications

    You can also search for this author in PubMed Google Scholar

  15. Joshua B. Edel
    View author publications

    You can also search for this author in PubMed Google Scholar

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.

Corresponding authors

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

Ethics declarations

Competing interests

The authors declare no competing interests.

Additional information

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

Supplementary information

Supplementary Information

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

Reporting Summary

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

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.)

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

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.)

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.)

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.)

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).

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Nadappuram, B.P., Cadinu, P., Barik, A. et al. Nanoscale tweezers for single-cell biopsies. Nature Nanotech 14, 80–88 (2019). https://doi.org/10.1038/s41565-018-0315-8

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

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

This article is cited by

Search

Advanced search

Quick links

Nature Briefing: Translational Research

Sign up for the Nature Briefing: Translational Research newsletter — top stories in biotechnology, drug discovery and pharma.

Get what matters in translational research, free to your inbox weekly. Sign up for Nature Briefing: Translational Research