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Single-molecule detection of protein efflux from microorganisms using fluorescent single-walled carbon nanotube sensor arrays

Nature Nanotechnology volume 12, pages 368–377 (2017)Cite this article

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Abstract

A distinct advantage of nanosensor arrays is their ability to achieve ultralow detection limits in solution by proximity placement to an analyte. Here, we demonstrate label-free detection of individual proteins from Escherichia coli (bacteria) and Pichia pastoris (yeast) immobilized in a microfluidic chamber, measuring protein efflux from single organisms in real time. The array is fabricated using non-covalent conjugation of an aptamer-anchor polynucleotide sequence to near-infrared emissive single-walled carbon nanotubes, using a variable chemical spacer shown to optimize sensor response. Unlabelled RAP1 GTPase and HIV integrase proteins were selectively detected from various cell lines, via large near-infrared fluorescent turn-on responses. We show that the process of E. coli induction, protein synthesis and protein export is highly stochastic, yielding variability in protein secretion, with E. coli cells undergoing division under starved conditions producing 66% fewer secreted protein products than their non-dividing counterparts. We further demonstrate the detection of a unique protein product resulting from T7 bacteriophage infection of E. coli, illustrating that nanosensor arrays can enable real-time, single-cell analysis of a broad range of protein products from various cell types.

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Figure 1: Characterization of the aptamer-anchor structure on nanotube.
Figure 2: Calibration of nanosensor response to recombinant protein.
Figure 3: Detection of protein from crude cell lysate and from E. coli engineered to secrete target protein.
Figure 4: Imaging the secretion of single proteins from individual microorganisms.
Figure 5: Real-time monitoring of bacteriophage T7RAP1 infection of E. coli cells and resulting cell lysis.

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Acknowledgements

This work was supported by a Burroughs Wellcome Fund Career Award at the Scientific Interface (CASI), the Simons Foundation, a BBRF young investigator award and a Beckman Foundation Young Investigator Award (to M.P.L.). M.S.S. acknowledges a grant from the National Science Foundation (NSF) to support this work. H.A. is supported by fellowships from the Japan Society for the Promotion of Science and the Naito Foundation. D.Y. acknowledges support from an NSF GRFP fellowship and L.C. acknowledges support from a LAM research fellowship. A.Y.C. acknowledges graduate research support from the Hertz Foundation, the Department of Defense and NIH Medical Scientist Training Program grant T32GM007753. This work was also supported by the National Institutes of Health (DP2 OD008435 and P50 GM098792), the Office of Naval Research (N00014-13-1-0424) and the NSF (MCB-1350625). The authors thank P. Perez-Pinera (University of Illinois Urbana-Champaign) for providing the parental Pichia cells.

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Authors and Affiliations

  1. Department of Chemical and Biomolecular Engineering, University of California Berkeley, Berkeley, 94720, California, USA

    Markita Patricia Landry, Linda Chio & Darwin Yang

  2. California Institute for Quantitative Biosciences (qb3), University of California-Berkeley, Berkeley, 94720, California, USA

    Markita Patricia Landry

  3. Department of Electrical Engineering & Computer Science and Department of Biological Engineering, Massachusetts Institute of Technology, Cambridge, 02139, Massachusetts, USA

    Hiroki Ando, Allen Y. Chen, Jicong Cao & Timothy K. Lu

  4. MIT Synthetic Biology Center, Massachusetts Institute of Technology, Cambridge, 02139, Massachusetts, USA

    Hiroki Ando, Allen Y. Chen, Jicong Cao & Timothy K. Lu

  5. Biophysics Program, Harvard University, Cambridge, 02138, Massachusetts, USA

    Allen Y. Chen

  6. The Rowland Institute at Harvard University, Cambridge, 02142, Massachusetts, USA

    Vishal Isaac Kottadiel

  7. Department of Biology, The Catholic University of America, Washington, 20064, District of Columbia, USA

    Vishal Isaac Kottadiel

  8. Department of Chemical Engineering, Massachusetts Institute of Technology, Cambridge, 02138, Massachusetts, USA

    Juyao Dong & Michael S. Strano

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Contributions

M.P.L. and M.S.S. conceived of the aptamer-anchor nanosensor platform and designed experiments. M.P.L. synthesized aptamer–nanotube conjugates, performed protein selectivity screens and carried out in vitro and cell-based experiments with J.D. and analysed data. H.A., A.C., J.C. and V.I.K. constructed recombinant E. coli strains, H.A. constructed T7RAP1 bacteriophage. M.P.L., H.A., A.C., V.I.K., L.C., D.Y., T.K.L. and M.S.S. discussed the experimental results and wrote the manuscript. All authors discussed the results and commented on the manuscript.

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Correspondence to Michael S. Strano.

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The authors declare no competing financial interests.

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Landry, M., Ando, H., Chen, A. et al. Single-molecule detection of protein efflux from microorganisms using fluorescent single-walled carbon nanotube sensor arrays. Nature Nanotech 12, 368–377 (2017). https://doi.org/10.1038/nnano.2016.284

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