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Single-molecule enzyme-linked immunosorbent assay detects serum proteins at subfemtomolar concentrations

Abstract

The ability to detect single protein molecules1,2 in blood could accelerate the discovery and use of more sensitive diagnostic biomarkers. To detect low-abundance proteins in blood, we captured them on microscopic beads decorated with specific antibodies and then labeled the immunocomplexes (one or zero labeled target protein molecules per bead) with an enzymatic reporter capable of generating a fluorescent product. After isolating the beads in 50-fl reaction chambers designed to hold only a single bead, we used fluorescence imaging to detect single protein molecules. Our single-molecule enzyme-linked immunosorbent assay (digital ELISA) approach detected as few as 10–20 enzyme-labeled complexes in 100 μl of sample (10−19 M) and routinely allowed detection of clinically relevant proteins in serum at concentrations (<10−15 M) much lower than conventional ELISA3,4,5. Digital ELISA detected prostate-specific antigen (PSA) in sera from patients who had undergone radical prostatectomy at concentrations as low as 14 fg/ml (0.4 fM).

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Figure 1: Digital ELISA based on arrays of femtoliter-sized wells.
Figure 2: Digitization of enzyme-linked complexes greatly increases sensitivity compared with bulk, ensemble measurements.
Figure 3: Subfemtomolar detection of proteins in serum using digital ELISA.
Figure 4: Digital detection of prostate-specific antigen (PSA) in serum samples of patients who had undergone radical prostatectomy.

References

  1. 1

    Tessler, L.A., Reifenberger, J.G. & Mitra, R.D. Protein quantification in complex mixtures by solid phase single-molecule counting. Anal. Chem. 81, 7141–7148 (2009).

    CAS  Article  Google Scholar 

  2. 2

    Todd, J. et al. Ultrasensitive flow-based immunoassays using single-molecule counting. Clin. Chem. 53, 1990–1995 (2007).

    CAS  Article  Google Scholar 

  3. 3

    Gosling, J.P.A. Decade of development in immunoassay methodology. Clin. Chem. 36, 1408–1427 (1990).

    CAS  PubMed  Google Scholar 

  4. 4

    Wild, D. The Immunoassay Handbook 3rd Edn. (Elsevier, 2005).

  5. 5

    Zhang, H.Q., Zhao, Q., Li, X.F. & Le, X.C. Ultrasensitive assays for proteins. Analyst (Lond.) 132, 724–737 (2007).

    CAS  Article  Google Scholar 

  6. 6

    Giljohann, D.A. & Mirkin, C.A. Drivers of biodiagnostic development. Nature 462, 461–464 (2009).

    CAS  Article  Google Scholar 

  7. 7

    Srinivas, P.R., Kramer, B.S. & Srivastava, S. Trends in biomarker research for cancer detection. Lancet Oncol. 2, 698–704 (2001).

    CAS  Article  Google Scholar 

  8. 8

    Galasko, D. Biomarkers for Alzheimer's disease—clinical needs and application. J. Alzheimers Dis. 8, 339–346 (2005).

    CAS  Article  Google Scholar 

  9. 9

    de Jong, D., Kremer, B.P.H., Olde Rikkert, M.G.M. & Verbeek, M.M. Current state and future directions of neurochemical biomarkers for Alzheimer's disease. Clin. Chem. Lab. Med. 45, 1421–1434 (2007).

    CAS  Article  Google Scholar 

  10. 10

    Barletta, J.M., Edelman, D.C. & Constantine, N.T. Lowering the detection limits of HIV-1 viral load using real-time immuno-PCR for HIV-1 p24 antigen. Am. J. Clin. Pathol. 122, 20–27 (2004).

    CAS  Article  Google Scholar 

  11. 11

    Adler, M., Wacker, R. & Niemeyer, C.M. Sensitivity by combination: immuno-PCR and related technologies. Analyst (Lond.) 133, 702–718 (2008).

    CAS  Article  Google Scholar 

  12. 12

    Nam, J.M., Thaxton, C.S. & Mirkin, C.A. Nanoparticle-based bio-bar codes for the ultrasensitive detection of proteins. Science 301, 1884–1886 (2003).

    CAS  Article  Google Scholar 

  13. 13

    Armani, A.M., Kulkarni, R.P., Fraser, S.E., Flagan, R.C. & Vahala, K.J. Label-free, single-molecule detection with optical microcavities. Science 317, 783–787 (2007).

    CAS  Article  Google Scholar 

  14. 14

    Cui, Y., Wei, Q.Q., Park, H.K. & Lieber, C.M. Nanowire nanosensors for highly sensitive and selective detection of biological and chemical species. Science 293, 1289–1292 (2001).

    CAS  Article  Google Scholar 

  15. 15

    Gaster, R.S. et al. Matrix-insensitive protein assays push the limits of biosensors in medicine. Nat. Med. 15, 1327–1332 (2009).

    CAS  Article  Google Scholar 

  16. 16

    Fan, R. et al. Integrated barcode chips for rapid, multiplexed analysis of proteins in microliter quantities of blood. Nat. Biotechnol. 26, 1373–1378 (2008).

    CAS  Article  Google Scholar 

  17. 17

    Thaxton, C.S. et al. Nanoparticle-based bio-barcode assay redefines “undetectable” PSA and biochemical recurrence after radical prostatectomy. Proc. Natl. Acad. Sci. USA 106, 18437–18442 (2009).

    CAS  Article  Google Scholar 

  18. 18

    Bao, Y.P. et al. Detection of protein analytes via nanoparticle-based bio bar code technology. Anal. Chem. 78, 2055–2059 (2006).

    CAS  Article  Google Scholar 

  19. 19

    Anderson, N.L. & Anderson, N.G. The human plasma proteome: history, character, and diagnostic prospects. Mol. Cell. Proteomics 1, 845–867 (2002).

    CAS  Article  Google Scholar 

  20. 20

    Gorris, H.H., Rissin, D.M. & Walt, D.R. Stochastic inhibitor release and binding from single-enzyme molecules. Proc. Natl. Acad. Sci. USA 104, 17680–17685 (2007).

    CAS  Article  Google Scholar 

  21. 21

    Rissin, D.M., Gorris, H.H. & Walt, D.R. Distinct and long-lived activity states of single enzyme molecules. J. Am. Chem. Soc. 130, 5349–5353 (2008).

    CAS  Article  Google Scholar 

  22. 22

    Rissin, D.M. & Walt, D.R. Digital readout of target binding with attomole detection limits via enzyme amplification in femtoliter arrays. J. Am. Chem. Soc. 128, 6286–6287 (2006).

    CAS  Article  Google Scholar 

  23. 23

    Rissin, D.M. & Walt, D.R. Digital concentration readout of single enzyme molecules using femtoliter arrays and Poisson statistics. Nano Lett. 6, 520–523 (2006).

    CAS  Article  Google Scholar 

  24. 24

    Tan, W. & Yeung, E.S. Monitoring the reactions of single enzyme molecules and single metal ions. Anal. Chem. 69, 4242–4248 (1997).

    CAS  Article  Google Scholar 

  25. 25

    Rissin, D.M. & Walt, D.R. Duplexed sandwich immunoassays on a fiber-optic microarray. Anal. Chim. Acta 564, 34–39 (2006).

    CAS  Article  Google Scholar 

  26. 26

    Ferguson, R., Yu, H., Kalyvas, M., Zammit, S. & Diamandis, E. Ultrasensitive detection of prostate-specific antigen by a time-resolved immunofluorometric assay and the Immulite immunochemiluminescent third-generation assay: potential applications in prostate and breast cancers. Clin. Chem. 42, 675–684 (1996).

    CAS  PubMed  Google Scholar 

  27. 27

    Jackson, T.M. & Ekins, R.P. Theoretical limitations on immunoassay sensitivity. Current practice and potential advantages of fluorescent Eu3+ chelates as non-radioisotopic tracers. J. Immunol. Methods 87, 13–20 (1986).

    CAS  Article  Google Scholar 

  28. 28

    Bock, J.L. & Klee, G.G. How sensitive is a prostate-specific antigen measurement? How sensitive does it need to be? Arch. Pathol. Lab. Med. 128, 341–343 (2004).

    PubMed  Google Scholar 

  29. 29

    Trock, B.J. et al. Prostate cancer-specific survival following salvage radiotherapy vs observation in men with biochemical recurrence after radical prostatectomy. Jama-Journal of the American Medical Association 299, 2760–2769 (2008).

    CAS  Article  Google Scholar 

  30. 30

    Pantano, P. & Walt, D.R. Ordered nanowell arrays. Chem. Mater. 8, 2832–2835 (1996).

    CAS  Article  Google Scholar 

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Acknowledgements

The project described was supported by Award Number R43CA133987 from the National Cancer Institute.

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Authors

Contributions

D.M.R., C.W.K., D.R.F., D.R.W. and D.C.D. conceived the approach. D.R.F. built the imaging system. D.M.R., C.W.K., T.G.C., S.C.H., L.S., P.P.P., A.J.R., E.P.F., J.D.R. and G.K.P. conducted the experiments. T.P. wrote the image analysis software. L.C. prepared reagents. D.M.R. and D.C.D. wrote the manuscript. All authors were involved in designing experiments, reviewing and discussing data, and commenting on the manuscript.

Corresponding author

Correspondence to David C Duffy.

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Competing interests

All authors are employees or advisors of Quanterix Corporation who have a minority ownership or ownership option position in the company.

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Supplementary Text and Figures

Supplementary Tables 1–3, Supplementary Figs. 1–3 and Supplementary Methods (PDF 250 kb)

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Rissin, D., Kan, C., Campbell, T. et al. Single-molecule enzyme-linked immunosorbent assay detects serum proteins at subfemtomolar concentrations. Nat Biotechnol 28, 595–599 (2010). https://doi.org/10.1038/nbt.1641

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