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Protein microarrays with carbon nanotubes as multicolor Raman labels

Nature Biotechnology volume 26, pages 1285–1292 (2008)Cite this article

Abstract

The current sensitivity of standard fluorescence-based protein detection limits the use of protein arrays in research and clinical diagnosis. Here, we use functionalized, macromolecular single-walled carbon nanotubes (SWNTs) as multicolor Raman labels for highly sensitive, multiplexed protein detection in an arrayed format. Unlike fluorescence methods, Raman detection benefits from the sharp scattering peaks of SWNTs with minimal background interference, affording a high signal-to-noise ratio needed for ultra-sensitive detection. When combined with surface-enhanced Raman scattering substrates, the strong Raman intensity of SWNT tags affords protein detection sensitivity in sandwich assays down to 1 fM—a three-order-of-magnitude improvement over most reports of fluorescence-based detection. We use SWNT Raman tags to detect human autoantibodies against proteinase 3, a biomarker for the autoimmune disease Wegener's granulomatosis, diluted up to 107-fold in 1% human serum. SWNT Raman tags are not subject to photobleaching or quenching. By conjugating different antibodies to pure 12C and 13C SWNT isotopes, we demonstrate multiplexed two-color SWNT Raman-based protein detection.

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Figure 1: Carbon nanotubes as Raman labels for protein microarray detection.
Figure 2: Highly selective recognition of surface-bound proteins by SWNT-antibody conjugates.
Figure 3: Femtomolar protein detection using SWNT Raman labels, compared with fluorescence-based protein microarray detection.
Figure 4: Calibration curve of mouse anti-human serum albumin measured in microarray format from nine duplicate protein spots at each analyte concentration by SWNT Raman tags.
Figure 5: Raman versus fluorescence-based protein microarray detection of aPR3, a biomarker for Wegener's granulomatosis, in human serum.
Figure 6: Multi-color SWNT Raman labels for multiplexed protein detection.

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References

  1. Bailey, R.C., Kwong, G.A., Radu, C.G., Witte, W.N. & Heath, J.R. DNA-encoded antibody libraries: a unified platform for multiplexed cell sorting and detection of genes and proteins. J. Am. Chem. Soc. 129, 1959–1967 (2007).

    Article  CAS  Google Scholar 

  2. Hayes, F.J., Halsall, H.B. & Heineman, W.R. Simultaneous immunoassay using electrochemical detection of metal ion labels. Anal. Chem. 66, 1860–1865 (1994).

    Article  CAS  Google Scholar 

  3. Landry, J.P., Zhu, X.D. & Gregg, J.P. Label-free detection of microarrays of biomolecules by oblique-incidence reflectivity difference microscopy. Opt. Lett. 29, 581–583 (2004).

    Article  CAS  Google Scholar 

  4. Wegner, G.J., Lee, H.J. & Corn, R.M. Characterization and optimization of peptide arrays for the study of epitope-antibody interactions using surface plasmon resonance imaging. Anal. Chem. 74, 5161–5168 (2002).

    Article  CAS  Google Scholar 

  5. Cao, Y.C., Jin, R., Nam, J.M., Thaxton, C.S. & Mirkin, C.A. Raman dye-labeled nanoparticle probes for proteins. J. Am. Chem. Soc. 125, 14676–14677 (2003).

    Article  CAS  Google Scholar 

  6. Li, T., Guo, L. & Wang, Z. Microarray based Raman spectroscopic detection with gold nanoparticle probes. Biosens. Bioelectron. 23, 1125–1130 (2008).

    Article  CAS  Google Scholar 

  7. Grubisha, D.S., Lipert, R.J., Park, H.Y., Driskell, J. & Porter, M.D. Femtomolar detection of prostate-specific antigen: an immunoassay based on surface-enhanced Raman scattering and immunogold labels. Anal. Chem. 75, 5936–5943 (2003).

    Article  CAS  Google Scholar 

  8. Wu, G. et al. Bioassay of prostate-specific antigen (PSA) using microcantilevers. Nat. Biotechnol. 19, 856–860 (2001).

    Article  CAS  Google Scholar 

  9. Goldman, E.R. et al. Conjugation of luminescent quantum dots with antibodies using an engineered adaptor protein to provide new reagents for fluoroimmunoassays. Anal. Chem. 74, 841–847 (2002).

    Article  CAS  Google Scholar 

  10. 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).

    Article  CAS  Google Scholar 

  11. Chen, R.J. et al. An investigation of the mechanisms of electronic sensing of protein adsorption on carbon nanotube devices. J. Am. Chem. Soc. 126, 1563–1568 (2004).

    Article  CAS  Google Scholar 

  12. Kong, J. et al. Nanotube molecular wires as chemical sensors. Science 287, 622–625 (2000).

    Article  CAS  Google Scholar 

  13. Zheng, G., Patolsky, F., Cui, Y., Wang, W.U. & Lieber, C.M. Multiplexed electrical detection of cancer markers with nanowire sensor arrays. Nat. Biotechnol. 23, 1294–1301 (2005).

    Article  CAS  Google Scholar 

  14. MacBeath, G. & Schreiber, S.L. Printing proteins as microarrays for high-throughput function determination. Science 289, 1760–1763 (2000).

    CAS  PubMed  Google Scholar 

  15. Hudson, M.E., Pozdnyakova, I., Haines, K., Mor, G. & Snyder, M. Identification of differentially expressed proteins in ovarian cancer using high-density protein microarrays. Proc. Natl. Acad. Sci. USA 104, 17494–17499 (2007).

    Article  CAS  Google Scholar 

  16. Robinson, W.H. et al. Autoantigen microarrays for multiplex characterization of autoantibody responses. Nat. Med. 8, 295–301 (2002).

    Article  CAS  Google Scholar 

  17. Song, S. et al. A cancer protein microarray platform using antibody fragments and its clinical applications. Mol. Biosyst. 3, 151–158 (2007).

    Article  CAS  Google Scholar 

  18. Espina, V. et al. Protein microarray detection strategies: focus on direct detection technologies. J. Immunol. Methods 290, 121–133 (2004).

    Article  CAS  Google Scholar 

  19. Hartwell, L., Mankoff, D., Paulovich, A., Ramsey, S. & Swisher, E. Cancer biomarkers: a systems approach. Nat. Biotechnol. 24, 905–908 (2006).

    Article  CAS  Google Scholar 

  20. Jensen, L. & Schatz, G.C. Resonance Raman scattering of rhodamine 6G as calculated using time-dependent density functional theory. J. Phys. Chem. A 110, 5973–5977 (2006).

    Article  CAS  Google Scholar 

  21. Baughman, R.H., Zakhidov, A.A. & de Heer, W.A. Carbon nanotubes–the route toward applications. Science 297, 787–792 (2002).

    Article  CAS  Google Scholar 

  22. Heller, D.A., Baik, S., Eurell, T.E. & Strano, M.S. Single-walled carbon nanotube spectroscopy in live cells: towards long-term labels and optical sensors. Adv. Mater. 17, 2793–2799 (2005).

    Article  CAS  Google Scholar 

  23. Sun, Y.P., Fu, K.F., Lin, Y. & Huang, W.J. Functionalized carbon nanotubes: properties and applications. Acc. Chem. Res. 35, 1096–1104 (2002).

    Article  CAS  Google Scholar 

  24. Bahr, J.L. & Tour, J.M. Covalent chemistry of single-wall carbon nanotubes. J. Mater. Chem. 12, 1952–1958 (2002).

    Article  CAS  Google Scholar 

  25. Kam, N.W.S., Liu, Z. & Dai, H.J. Functionalization of carbon nanotubes via cleavable disulfide bonds for efficient intracellular delivery of siRNA and potent gene silencing. J. Am. Chem. Soc. 127, 12492–12493 (2005).

    Article  CAS  Google Scholar 

  26. Liu, Z. et al. In vivo biodistribution and highly efficient tumor targeting of carbon nanotubes in mice. Nat. Nanotechnol. 2, 47–52 (2007).

    Article  CAS  Google Scholar 

  27. Chen, R.J. et al. Noncovalent functionalization of carbon nanotubes for highly specific electronic biosensors. Proc. Natl. Acad. Sci. USA 100, 4984–4989 (2003).

    Article  CAS  Google Scholar 

  28. Schipper, M.L. et al. A pilot toxicology study of single-walled carbon nanotubes in a small sample of mice. Nat. Nanotechnol. 3, 216–221 (2008).

    Article  CAS  Google Scholar 

  29. Welsher, K. & Liu, Z. Daranciang & Dai, H.J. Selective probing and imaging of cells with single walled carbon nanotubes as near-infrared fluorescence molecules. Nano Lett. 8, 586–590 (2008).

    Article  CAS  Google Scholar 

  30. Nie, S. & Emory, S.R. Probing single molecules and single nanoparticles by surface-enhanced Raman scattering. Science 275, 1102–1106 (1997).

    Article  CAS  Google Scholar 

  31. Kneipp, K., Kneipp, H. & Kneipp, J. Surface-enhanced Raman scattering in local optical fields of silver and gold nanoaggregates - from single-molecule Raman spectroscopy to ultrasensitive probing in live cells. Acc. Chem. Res. 39, 443–450 (2006).

    Article  CAS  Google Scholar 

  32. Shim, M., Kam, N.W.S., Chen, R.J., Li, Y. & Dai, H.J. Functionalization of carbon nanotubes for biocompatibility and biomolecular recognition. Nano Lett. 2, 285–288 (2002).

    Article  CAS  Google Scholar 

  33. Diamandis, E.P. & Christopoulos, T.K. Immunoassay (Academic Press, San Diego, 1996).

  34. Fang, Y., Seong, N.H. & Dlott, D.D. Measurement of the distribution of site enhancement in surface-enhanced Raman scattering. Science 321, 388–392 (2008).

    Article  CAS  Google Scholar 

  35. Jenne, D.E., Tschopp, J., Lüdemann, J., Utecht, B. & Gross, W.L. Wegener's autoantigen decoded. Nature 346, 520 (1990).

    Article  CAS  Google Scholar 

  36. Seo, P. & Stone, J.H. The antineutrphil cytoplasmic antibody-associated vasculitides. Am. J. Med. 117, 39–50 (2004).

    Article  CAS  Google Scholar 

  37. Li, X. et al. Selective synthesis combined with chemical separation of single-walled carbon nanotubes for chirality selection. J. Am. Chem. Soc. 129, 15770–15771 (2007).

    Article  CAS  Google Scholar 

  38. Liu, L. & Fan, S.S. Isotope labeling of carbon nanotubes and formation of 12C-13C nanotube junctions. J. Am. Chem. Soc. 123, 11502–11503 (2001).

    Article  CAS  Google Scholar 

  39. Ferraro, J.R., Nakamoto, K. & Brown, C.W . Introductory Raman Spectroscopy edn. 2 (Academic Press, San Diego, 2003).

  40. Arnold, M.S., Green, A.A. & Hulvat, J.F. Stupp. S.I. & Hersam, M.C. Sorting carbon nanotubes by electronic structure using density differentiation. Nat. Nanotechnol. 1, 60–65 (2006).

    Article  CAS  Google Scholar 

  41. Tu, X. & Zheng, M. A DNA-based approach to the carbon nanotube sorting problem. Nano. Res. 1, 185–194 (2008).

    Article  CAS  Google Scholar 

  42. Liu, Z. et al. Multiplexed mutli-color Raman imaging of live cells with isotopically modified single walled carbon nanotubes. J. Am. Chem. Soc. doi:10.1021/ja806242t (20 September 2008).

  43. Liu, Z. et al. Circulation and long-term fate of functionalized, biocompatible single-walled carbon nanotubes in mice probed by Raman spectroscopy. Proc. Natl. Acad. Sci. USA 105, 1410–1415 (2008).

    Article  CAS  Google Scholar 

  44. Kam, N.W.S., Connell, M.O., Wisdom, J.A. & Dai, H.J. Carbon nanotubes as multifunctional biological transporters and near-infrared agents for selective cancer cell destruction. Proc. Natl. Acad. Sci. USA 102, 11600–11605 (2005).

    Article  CAS  Google Scholar 

Download references

Acknowledgements

This work was supported by the National Institutes of Health/National Cancer Institute–funded Center for Cancer Nanotechnology Excellence Focused on Therapeutic Response U54 CA119367 at Stanford University, and NIH-NCI R01 CA135109-01. The authors would like to thank Nozomi Nakayama-Ratchford and Sarunya Bangsaruntip for their assistance in developing carbon nanotube–protein conjugates.

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Author notes

  1. Zhuo Chen and Scott M Tabakman: These authors contributed equally to the work.

Authors and Affiliations

  1. Department of Chemistry and Laboratory for Advanced Materials, Stanford University, 333 Campus Drive, Mudd Building, Room 121, Stanford, 94305, California, USA

    Zhuo Chen, Scott M Tabakman, Andrew P Goodwin, Dan Daranciang, Xinran Wang, Guangyu Zhang, Xiaolin Li, Zhuang Liu & Hongjie Dai

  2. School of Medicine, Stanford University, 269 Campus Drive, Stanford, 94305, California, USA

    Michael G Kattah & Paul J Utz

  3. Department of Physics, Tsinghua-Foxconn Nanotechnology Research Center, Tsinghua University, Beijing, 100084, China

    Kaili Jiang & Shoushan Fan

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  1. Zhuo Chen
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Contributions

Z.C. and S.M.T. contributed equally to the work, developing the SWNT passivation and conjugation schemes presented, as well as the procedure for obtaining SERS-active surfaces and the related protein immobilization methodology. A.P.G. contributed synthesis of 6-arm branched carboxy-PEG. M.G.K. and P.J.U. contributed to fluorescence-based detection of anti-proteinase 3. D.D. and Z.L. contributed by assisting in preparing SWNT-antibody and SWNT-peptide conjugates. X.W. and G.Z. contributed by assisting in the preparation of SERS-active substrates. X.L., K.J. and S.F. contributed by providing isotopomer SWNTs. H.D. designed the research and contributed much direction and assistance to this project.

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Correspondence to Hongjie Dai.

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Supplementary Figures 1–15, Supplementary Table 1, Supplementary Methods (PDF 724 kb)

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Chen, Z., Tabakman, S., Goodwin, A. et al. Protein microarrays with carbon nanotubes as multicolor Raman labels. Nat Biotechnol 26, 1285–1292 (2008). https://doi.org/10.1038/nbt.1501

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