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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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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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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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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DOI: https://doi.org/10.1038/nnano.2016.284
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