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  • Brief Communication
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Digital quantification using amplified single-molecule detection

Abstract

We describe a scheme for biomolecule enumeration by converting nanometer-scale specific molecular recognition events mediated by rolling-circle amplification to fluorescent micrometer-sized DNA molecules amenable to discrete optical detection. Our amplified single-molecule detection (SMD) approach preserves the discrete nature of the molecular population, allowing multiplex detection and highly precise quantification of molecules over a dynamic range of seven orders of magnitude. We apply the method for sensitive detection and quantification of the bacterial pathogen Vibrio cholerae.

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Figure 1: Working mechanism for amplified SMD.
Figure 2: Characterization of different analysis parameters in the amplified SMD platform.
Figure 3: Sensitive and specific detection of bacteria.

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Acknowledgements

We thank O. Öhman at Åmic AB, L. Spångberg and S.E. Alm. This work was supported by the Wallenberg Foundation, the Swedish Defense Nanotechnology Program, Uppsala BioX, the EU Framework Programme 6 integrated project MolTools, the Swedish Research Council.

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Authors and Affiliations

Authors

Contributions

J.J., S.F. and M.N. conceived of the described method. J.J., J.M., J.G. and M.N. contributed to the technical development of the method. J.J., J.M. and J.G. performed laboratory work. C.G.-R. and S.B. contributed with the bacterial application. J.S. designed microbial specific padlock probes. J.M., J.J., J.G., S.B. and M.N. wrote the manuscript.

Corresponding author

Correspondence to Mats Nilsson.

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Competing interests

The authors declare no competing financial interests.

Supplementary information

Supplementary Fig. 1

Extended dynamic range using circle to circle amplification (C2CA). (PDF 104 kb)

Supplementary Fig. 2

Ligation and RCA in complex matrices. (PDF 47 kb)

Supplementary Fig. 3

Multiplex detection. (PDF 827 kb)

Supplementary Fig. 4

Protein detection using proximity ligation. (PDF 41 kb)

Supplementary Table 1

Oligonucleotide sequences. (PDF 35 kb)

Supplementary Methods (PDF 92 kb)

Supplementary Note (PDF 71 kb)

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Jarvius, J., Melin, J., Göransson, J. et al. Digital quantification using amplified single-molecule detection. Nat Methods 3, 725–727 (2006). https://doi.org/10.1038/nmeth916

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