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Digitally encoded DNA nanostructures for multiplexed, single-molecule protein sensing with nanopores

Nature Nanotechnology volume 11, pages 645–651 (2016)Cite this article

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Abstract

The simultaneous detection of a large number of different analytes is important in bionanotechnology research and in diagnostic applications. Nanopore sensing is an attractive method in this regard as the approach can be integrated into small, portable device architectures, and there is significant potential for detecting multiple sub-populations in a sample. Here, we show that highly multiplexed sensing of single molecules can be achieved with solid-state nanopores by using digitally encoded DNA nanostructures. Based on the principles of DNA origami, we designed a library of DNA nanostructures in which each member contains a unique barcode; each bit in the barcode is signalled by the presence or absence of multiple DNA dumbbell hairpins. We show that a 3-bit barcode can be assigned with 94% accuracy by electrophoretically driving the DNA structures through a solid-state nanopore. Select members of the library were then functionalized to detect a single, specific antibody through antigen presentation at designed positions on the DNA. This allows us to simultaneously detect four different antibodies of the same isotype at nanomolar concentration levels.

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Figure 1: Signal for a single bit formed of dumbbell hairpins.
Figure 2: Design and nanopore measurement of DNA structures with multiple zones of protruding secondary structures.
Figure 3: Multiplexed barcode design and readout efficiency.
Figure 4: Binding-site presentation and analysis for the bound antibody in translocations.
Figure 5: Selective detection of multiple antibodies.

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Acknowledgements

The authors thank J. Kong and V. Thacker for useful discussions. N.A.W.B. and U.F.K. acknowledge funding from an ERC starting grant (Passmembrane 261101) and an ERC consolidator grant (Designerpores 647144). N.A.W.B. also acknowledges funding from an EPSRC doctoral prize award.

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  1. Cavendish Laboratory, University of Cambridge, JJ Thomson Avenue, Cambridge, CB3 0HE, UK

    Nicholas A. W. Bell & Ulrich F. Keyser

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  1. Nicholas A. W. Bell
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Contributions

N.A.W.B. conceived the idea, N.A.W.B. and U.F.K. designed the experiments. N.A.W.B. performed the experiments and analysed the data, and N.A.W.B. and U.F.K. wrote the manuscript.

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Correspondence to Nicholas A. W. Bell or Ulrich F. Keyser.

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Bell, N., Keyser, U. Digitally encoded DNA nanostructures for multiplexed, single-molecule protein sensing with nanopores. Nature Nanotech 11, 645–651 (2016). https://doi.org/10.1038/nnano.2016.50

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