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:

A multiplexed, electrochemical interface for gene-circuit-based sensors

Nature Chemistry volume 12, pages 48–55 (2020)Cite this article

Subjects

Abstract

The field of synthetic biology has used the engineered assembly of synthetic gene networks to create a wide range of functions in biological systems. To date, gene-circuit-based sensors have primarily used optical proteins (for example, fluorescent, colorimetric) as reporter outputs, which has limited the potential to measure multiple distinct signals. Here we present an electrochemical interface that permits expanded multiplexed reporting for cell-free gene-circuit-based sensors. We have engineered a scalable system of reporter enzymes that cleave specific DNA sequences in solution, which results in an electrochemical signal when these newly liberated strands are captured at the surface of a nanostructured microelectrode. We describe the development of this interface and show its utility using a ligand-inducible gene circuit and toehold switch-based sensors by demonstrating the detection of multiple antibiotic resistance genes in parallel. This technology has the potential to expand the field of synthetic biology by providing an interface for materials, hardware and software.

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

Access options

Buy this article

  • Purchase on SpringerLink
  • Instant access to full article PDF
Buy now

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

Fig. 1: A gene-circuit/electrode interface for cell-free synthetic gene networks.
Fig. 2: Development of orthogonal, restriction-enzyme-based reporters.
Fig. 3: Electrochemical detection of restriction-enzyme reporters.
Fig. 4: Application of the gene-circuit/electrochemical interface for small-molecule- and RNA-actuated electrochemical signalling.
Fig. 5: Detection of mcr genes.

Similar content being viewed by others

Data availability

All raw data presented in the manuscript are available upon request from the corresponding authors.

Code availability

All custom computer code used in the manuscript is available upon request from the corresponding authors.

References

  1. Cameron, D. E., Bashor, C. J. & Collins, J. J. A brief history of synthetic biology. Nat. Rev. Microbiol. 12, 381–390 (2014).

    CAS  PubMed  Google Scholar 

  2. Cheng, A. A. & Lu, T. K. Synthetic biology: an emerging engineering discipline. Annu. Rev. Biomed. Eng. 14, 155–178 (2012).

    CAS  PubMed  Google Scholar 

  3. Fossati, E. et al. Reconstitution of a 10-gene pathway for synthesis of the plant alkaloid dihydrosanguinarine in Saccharomyces cerevisiae. Nat. Commun. 5, 3283 (2014).

    PubMed  Google Scholar 

  4. Smanski, M. J. et al. Synthetic biology to access and expand nature’s chemical diversity. Nat. Rev. Microbiol. 14, 135–149 (2016).

    CAS  PubMed  PubMed Central  Google Scholar 

  5. Green, A. A. et al. Complex cellular logic computation using ribocomputing devices. Nature 548, 117–121 (2017).

    CAS  PubMed  PubMed Central  Google Scholar 

  6. Yehl, K. & Lu, T. Scaling computation and memory in living cells. Curr. Opin. Biomed. Eng 4, 143–151 (2017).

    PubMed  PubMed Central  Google Scholar 

  7. Kitada, T., DiAndreth, B., Teague, B. & Weiss, R. Programming gene and engineered-cell therapies with synthetic biology. Science 359, eaad1067 (2018).

    PubMed  Google Scholar 

  8. Mao, N., Cubillos-Ruiz, A., Cameron, D. E. & Collins, J. J. Probiotic strains detect and suppress cholera in mice. Sci. Transl. Med. 10, eaao2586 (2018).

    PubMed  Google Scholar 

  9. Kotula, J. W. et al. Programmable bacteria detect and record an environmental signal in the mammalian gut. Proc. Natl Acad. Sci. USA 111, 4838–4843 (2014).

    CAS  PubMed  Google Scholar 

  10. Keasling, J. D. Synthetic biology and the development of tools for metabolic engineering. Metab. Eng. 14, 189–195 (2012).

    CAS  PubMed  Google Scholar 

  11. Shimizu, Y. et al. Cell-free translation reconstituted with purified components. Nat. Biotechnol. 19, 751–755 (2001).

    CAS  PubMed  Google Scholar 

  12. Jewett, M. C., Calhoun, K. A., Voloshin, A., Wuu, J. J. & Swartz, J. R. An integrated cell-free metabolic platform for protein production and synthetic biology. Mol. Syst. Biol. 4, 220–230 (2008).

    PubMed  PubMed Central  Google Scholar 

  13. Shin, J., Jardine, P. & Noireaux, V. Genome replication, synthesis, and assembly of the bacteriophage T7 in a single cell-free reaction. ACS Synth. Biol 1, 408–413 (2012).

    CAS  PubMed  Google Scholar 

  14. Pardee, K. et al. Paper-based synthetic gene networks. Cell 159, 940–954 (2014).

    CAS  PubMed  PubMed Central  Google Scholar 

  15. Pardee, K. et al. Rapid, low-cost detection of Zika virus using programmable biomolecular components. Cell 165, 1255–1266 (2016).

    CAS  PubMed  Google Scholar 

  16. Pardee, K. et al. Portable, on-demand biomolecular manufacturing. Cell 167, 248–259.e12 (2016).

    CAS  PubMed  Google Scholar 

  17. Huang, A. et al. BioBitsTM explorer: a modular synthetic biology education kit. Sci. Adv. 4, eaat5105 (2018).

    CAS  PubMed  PubMed Central  Google Scholar 

  18. Stark, J. C. et al. BioBitsTM bright: a fluorescent synthetic biology education kit. Sci. Adv. 4, eaat5107 (2018).

    CAS  PubMed  PubMed Central  Google Scholar 

  19. Wen, K. Y. et al. A cell-free biosensor for detecting quorum sensing molecules in P. aeruginosa-infected respiratory samples. ACS Synth. Biol. 6, 2293–2301 (2017).

    CAS  PubMed  Google Scholar 

  20. Takahashi, M. K. et al. A low-cost paper-based synthetic biology platform for analyzing gut microbiota and host biomarkers. Nat. Commun. 9, 3347 (2018).

    PubMed  PubMed Central  Google Scholar 

  21. Alligrant, T. M., Nettleton, E. G. & Crooks, R. M. Lab on a chip electrochemical detection of individual DNA. Lab Chip 13, 349–354 (2013).

    CAS  PubMed  Google Scholar 

  22. Fan, C., Plaxco, K. W. & Heeger, A. J. Electrochemical interrogation of conformational changes as a reagentless method for the sequence-specific detection of DNA. Proc. Natl Acad. Sci. USA 100, 9134–9137 (2003).

    CAS  PubMed  Google Scholar 

  23. Khan, H. U. et al. In situ, label-free DNA detection using organic transistor sensors. Adv. Mater. 22, 4452–4456 (2010).

    CAS  PubMed  Google Scholar 

  24. Patolsky, F., Lichtenstein, A. & Willner, I. Detection of single-base DNA mutations by enzyme-amplified electronic transduction. Nat. Biotechnol. 19, 253–257 (2001).

    CAS  PubMed  Google Scholar 

  25. Slinker, J. D., Muren, N. B., Gorodetsky, A. A. & Barton, J. K. Multiplexed DNA-modified electrodes. J. Am. Chem. Soc. 132, 2769–2774 (2010).

    CAS  PubMed  PubMed Central  Google Scholar 

  26. Das, J. et al. An ultrasensitive universal detector based on neutralizer displacement. Nat. Chem. 4, 642–648 (2012).

    CAS  PubMed  Google Scholar 

  27. Zuo, X., Xiao, Y. & Plaxco, K. W. High specificity, electrochemical sandwich assays based on single aptamer sequences and suitable for the direct detection of small-molecule targets in blood and other complex matrices. J. Am. Chem. Soc. 131, 6944–6945 (2009).

    CAS  PubMed  PubMed Central  Google Scholar 

  28. Kuang, Z., Kim, S. N., Crookes-Goodson, W. J., Farmer, B. L. & Naik, R. R. Biomimetic chemosensor: designing peptide recognition elements for surface functionalization of carbon nanotube field effect transistors. ACS Nano 4, 452–458 (2010).

    CAS  PubMed  Google Scholar 

  29. Liu, H., Xiang, Y., Lu, Y. & Crooks, R. M. Aptamer-based origami paper analytical device for electrochemical detection of adenosine. Angew. Chem. Int. Ed. 51, 6925–6928 (2012).

    CAS  Google Scholar 

  30. Das, J. & Kelley, S. O. Protein detection using arrayed microsensor chips: tuning sensor footprint to achieve ultrasensitive readout of CA-125 in serum and whole blood. Anal. Chem. 83, 1167–1172 (2011).

    CAS  PubMed  Google Scholar 

  31. Tang, D., Yuan, R. & Chai, Y. Ultrasensitive electrochemical immunosensor for clinical immunoassay using thionine-doped magnetic gold nanospheres as labels and horseradish peroxidase as enhancer. Anal. Chem. 80, 1582–1588 (2008).

    CAS  PubMed  Google Scholar 

  32. Sage, A. T., Besant, J. D., Lam, B., Sargent, E. H. & Kelley, S. O. Ultrasensitive electrochemical biomolecular detection using nanostructured microelectrodes. Acc. Chem. Res. 47, 2417–2425 (2014).

    CAS  PubMed  Google Scholar 

  33. Li, J. J., Geyer, R. & Tan, W. Using molecular beacons as a sensitive fluorescence assay for enzymatic cleavage of single-stranded DNA. Nucleic Acids Res 28, e52 (2000).

    CAS  PubMed  PubMed Central  Google Scholar 

  34. Soleymani, L., Fang, Z., Sargent, E. H. & Kelley, S. O. Programming the detection limits of biosensors through controlled nanostructuring. Nat. Nanotechnol. 4, 844–848 (2009).

    CAS  PubMed  Google Scholar 

  35. Karig, D. K., Iyer, S., Simpson, M. L. & Doktycz, M. J. Expression optimization and synthetic gene networks in cell-free systems. Nucleic Acids Res. 40, 3763–3774 (2012).

    CAS  PubMed  Google Scholar 

  36. Green, A. A., Silver, P. A., Collins, J. J. & Yin, P. Toehold switches: de-novo-designed regulators of gene expression. Cell 159, 925–939 (2014).

    CAS  PubMed  PubMed Central  Google Scholar 

  37. Zhang, J. et al. Molecular detection of colistin resistance genes (mcr-1, mcr-2 and mcr-3) in nasal/oropharyngeal and anal/cloacal swabs from pigs and poultry. Sci. Rep. 8, 3705 (2018).

    PubMed  PubMed Central  Google Scholar 

  38. García, V. et al. Co-occurrence of mcr-1, mcr-4 and mcr-5 genes in multidrug-resistant ST10 enterotoxigenic and Shiga toxin-producing Escherichia coli in Spain (2006–2017). Int. J. Antimicrob. Agents 52, 104–108 (2018).

    PubMed  Google Scholar 

  39. Zadeh, J. N. et al. NUPACK: analysis and design of nucleic acid systems. J. Comput. Chem. 32, 170–173 (2011).

    CAS  PubMed  Google Scholar 

  40. Gootenberg, J. S. et al. Nucleic acid detection with CRISPR-Cas13a/C2c2. Science 356, 438–442 (2017).

    CAS  PubMed  PubMed Central  Google Scholar 

  41. Davis, J. H., Rubin, A. J. & Sauer, R. T. Design, construction and characterization of a set of insulated bacterial promoters. Nucleic Acids Res. 39, 1131–1141 (2011).

    CAS  PubMed  Google Scholar 

Download references

Acknowledgements

The molecular components of the ligand-inducible gene circuit were kindly provided by the Doktycz lab. The plasmid pSB3C5-proD-B0032-E0051 was a gift from J. Davis and R. Sauer (Addgene plasmid no. 107241). J.B.C. was funded by an Ontario Graduate Scholarship. This work was supported by the NSERC Discovery Grants Program (RGPIN-2016-06352), the CIHR Foundation Grant Program (201610FDN-375469), The University of Toronto’s Connaught New Research Award and the CIHR Canada Research Chair Program (950-231075) to K.P.; the University of Toronto’s Medicine by Design initiative, which receives funding from the Canada First Research Excellence Fund (C1TPA-2016-06), and the National Institute of Allergy and Infectious Diseases of the National Institutes of Health under award number R21AI136571 (the content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health) to K.P. and S.O.K.; an NIH Director’s New Innovator Award (1DP2GM126892), an Arizona Biomedical Research Commission New Investigator Award (ADHS16-162400), an Alfred P. Sloan Research Fellowship (FG-2017-9108), Gates Foundation funds (OPP1160667), and Gordon and Betty Moore Foundation funds (no. 6984) to A.A.G. We thank S. Cicek for her help enhancing the high-throughput data analysis. We thank M. Labib and C. Nemr for their advice and support of the project.

Author information

Author notes

  1. These authors contributed equally: Peivand Sadat Mousavi, Sarah J. Smith, Jenise B. Chen.

Authors and Affiliations

  1. Leslie Dan Faculty of Pharmacy, University of Toronto, Toronto, Ontario, Canada

    Peivand Sadat Mousavi, Sarah J. Smith, Margot Karlikow, Aidan Tinafar, Clare Robinson, Shana O. Kelley & Keith Pardee

  2. Department of Chemistry, Bucknell University, Lewisburg, PA, USA

    Sarah J. Smith

  3. Department of Chemistry, University of Toronto, Toronto, Ontario, Canada

    Jenise B. Chen & Shana O. Kelley

  4. Institute of Biomaterials and Biomedical Engineering, University of Toronto, Toronto, Ontario, Canada

    Wenhan Liu & Shana O. Kelley

  5. Biodesign Center for Molecular Design and Biomimetics, The Biodesign Institute and the School of Molecular Sciences, Arizona State University, Tempe, AZ, USA

    Duo Ma & Alexander A. Green

Authors
  1. Peivand Sadat Mousavi
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Sarah J. Smith
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Jenise B. Chen
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Margot Karlikow
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Aidan Tinafar
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Clare Robinson
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Wenhan Liu
    View author publications

    You can also search for this author in PubMed Google Scholar

  8. Duo Ma
    View author publications

    You can also search for this author in PubMed Google Scholar

  9. Alexander A. Green
    View author publications

    You can also search for this author in PubMed Google Scholar

  10. Shana O. Kelley
    View author publications

    You can also search for this author in PubMed Google Scholar

  11. Keith Pardee
    View author publications

    You can also search for this author in PubMed Google Scholar

Contributions

P.S.M. designed and performed molecular experiments and co-wrote the manuscript; S.J.S. designed and performed electrochemical experiments and co-wrote the manuscript; J.B.C. designed and performed electrochemical experiments and co-wrote the manuscript; M.K. designed and performed molecular experiments and edited the manuscript; A.T. designed and performed molecular experiments and edited the manuscript; C.R. designed and performed molecular experiments; W.L. contributed to designing the DNA duplex reporter and electrochemical chip; D.M. designed the toehold switches; A.A.G. designed the toehold switches and edited the manuscript; S.O.K. was responsible for project supervision, designed experiments and edited the manuscript; K.P. was responsible for project design and supervision, designed experiments and co-wrote the manuscript. All authors discussed the results and commented on the manuscript.

Corresponding authors

Correspondence to Shana O. Kelley or Keith Pardee.

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 Materials

Supplementary materials and methods, Figs. 1–18 and Tables 1–10.

Supplementary Sequence Information

DNA sequences used for this project.

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Sadat Mousavi, P., Smith, S.J., Chen, J.B. et al. A multiplexed, electrochemical interface for gene-circuit-based sensors. Nat. Chem. 12, 48–55 (2020). https://doi.org/10.1038/s41557-019-0366-y

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/s41557-019-0366-y

This article is cited by

Search

Advanced search

Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing