Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

The bio-barcode assay for the detection of protein and nucleic acid targets using DTT-induced ligand exchange

Nature Protocols volume 1, pages 324–336 (2006)Cite this article

This article has been updated

Abstract

The recently developed bio-barcode assay for the detection of nucleic acid and protein targets without PCR has been shown to be extraordinarily sensitive, showing high sensitivity for both nucleic acid and protein targets. Two types of particles are used in the assay: (i) a magnetic microparticle with recognition elements for the target of interest; and (ii) a gold nanoparticle (Au-NP) with a second recognition agent (which can form a sandwich around the target in conjunction with the magnetic particle) and hundreds of thiolated single-strand oligonucleotide barcodes. After reaction with the analyte, a magnetic field is used to localize and collect the sandwich structures, and a DTT solution at elevated temperature is used to release the barcode strands. The barcode strands can be identified on a microarray via scanometric detection or in situ if the barcodes carry with them a detectable marker. The recent modification to the original bio-barcode assay method, utilizing DTT, has streamlined and simplified probe preparation and greatly enhanced the quantitative capabilities of the assay. Here we report the detailed methods for performing the ligand exchange bio-barcode assay for both nucleic acid and protein detection. In total, reagent synthesis, probe preparation and detection require 4 d.

*Note: In the version of the article initially published online, incorrect (non-final) versions of the article were posted in both the PDF and HTML formats. The errors have been corrected in all versions of the article.

This is a preview of subscription content, access via your institution

Relevant articles

Open Access articles citing this article.

Access options

Rent or buy this article

Get just this article for as long as you need it

$39.95

Learn more

Prices may be subject to local taxes which are calculated during checkout

Figure 1: Bio-barcode assay for DNA and protein detection.
Figure 2: Testing antibody loading.
Figure 3: Scanometric image.
Figure 4: DNA detection image.

Change history

  • 20 July 2006

    In the version of the article initially published online, incorrect (non-final) versions of the article were posted in both the PDF and HTML formats. The errors have been corrected in all versions of the article.

References

  1. 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  PubMed  Google Scholar 

  2. Nam, J.M., Stoeva, S.I. & Mirkin, C.A. Bio-bar code–based DNA detection with PCR-like sensitivity, J. Am. Chem. Soc. 126, 5932–5933 (2004).

    Article  CAS  PubMed  Google Scholar 

  3. Mirkin, C.A., Letsinger, R.L., Mucic, R.C. & Storhoff, J.J. A DNA-based method for rationally assembling nanoparticles into macroscopic materials, Nature 382, 607–609 (1996).

    Article  CAS  PubMed  Google Scholar 

  4. Storhoff, J.J. et al. What controls the optical properties of DNA-linked gold nanoparticle assemblies? J. Am. Chem. Soc. 122, 4640–4650 (2000).

    Article  CAS  Google Scholar 

  5. Storhofff, J.J., Elghanian, R., Mirkin, C.A. & Letsinger, R.L. Sequence-dependent stability of DNA-modified gold nanoparticles. Langmuir 18, 6666–6670 (2002).

    Article  Google Scholar 

  6. Jin, R.C. et al. What controls the melting properties of DNA-linked gold nanoparticle assemblies? J. Am. Chem. Soc. 125, 1643–1654 (2003).

    Article  CAS  PubMed  Google Scholar 

  7. Lytton-Jean, A.K.R. & Mirkin, C.A. A thermodynamic investigation into the binding properties of DNA functionalized gold nanoparticle probes and molecular fluorophore probes. J. Am. Chem. Soc. 127, 12754–12755 (2005).

    Article  CAS  PubMed  Google Scholar 

  8. Demers, L.M. et al. Thermal desorption behavior and binding properties of DNA bases and nucleosides on gold. J. Am. Chem. Soc. 124, 11248–11249 (2002).

    Article  CAS  PubMed  Google Scholar 

  9. Thaxton, C.S. et al. A bio-bar-code assay based upon dithiothreitol-induced oligonucleotide release. Anal. Chem. 77, 8174–8178 (2005).

    Article  CAS  PubMed  Google Scholar 

  10. Demers, L.M. et al. A fluorescence-based method for determining the surface coverage and hybridization efficiency of thiol-capped oligonucleotides bound to gold thin films and nanoparticles. Anal. Chem. 72, 5535–5541 (2000).

    Article  CAS  PubMed  Google Scholar 

  11. Taton, T.A., Mirkin, C.A. & Letsinger, R.L. Scanometric DNA array detection with nanoparticle probes, Science 289, 1757–1760 (2000).

    Article  CAS  PubMed  Google Scholar 

  12. Cao, Y.W.C., Jin, R.C. & Mirkin, C.A. Nanoparticles with Raman spectroscopic fingerprints for DNA and RNA detection. Science 297, 1536–1540 (2002).

    Article  CAS  PubMed  Google Scholar 

  13. Oh, B.K., Nam, J.M., Lee, S.W. & Mirkin, C.A. A fluorophore-based bio-barcode amplification assay for proteins, Small 2, 103–108 (2006).

    Article  CAS  PubMed  Google Scholar 

  14. Stoeva, S.I. et al. Multiplexed detection of protein cancer markers with bio-barcoded nanoparticle probes, J. Am. Chem. Soc. (2006) DI: 10.1020/ja0613106.

  15. Stoeva, S.I., Lee, J.-S., Thaxton, C.S. & Mirkin, C.A. Multiplexed DNA detection with biobarcoded nanoparticle probes. Angew. Chem. Int. Ed. Engl. 45, 3303–3306 (2006).

    Article  CAS  PubMed  Google Scholar 

  16. Frens, G. Controlled nucleation for regulation of particle-size in monodisperse gold suspensions. Nat. Phys. Sci. 241, 20–22 (1973).>

    Article  CAS  Google Scholar 

Download references

Acknowledgements

C.A.M. acknowledges HSARPA, NIH Pioneer Award, NIH NIAID, AFOSR, Doris Duke Charitable Foundation, NSF/NSEC and NCI. H.D.H: This work was performed while on appointment as a U.S. Department of Homeland Security (DHS) Fellow under the DHS Scholarship and Fellowship Program, a program administered by the Oak Ridge Institute for Science and Education (ORISE) for DHS through an interagency agreement with the U.S. Department of Energy (DOE). ORISE is managed by Oak Ridge Associated Universities under DOE contract number DE-AC05-06OR23100. All opinions expressed in this paper are the author's and do not necessarily reflect the policies and views of DHS, DOE or ORISE.

Author information

Authors and Affiliations

  1. Northwestern University Department of Chemistry and The International Institute for Nanotechnology, 2145 Sheridan Road, Evanston, 60208, Illinois, USA

    Haley D Hill & Chad A Mirkin

Authors
  1. Haley D Hill
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Chad A Mirkin
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Chad A Mirkin.

Ethics declarations

Competing interests

The authors declare no competing financial interests.

Rights and permissions

Reprints and Permissions

About this article

Cite this article

Hill, H., Mirkin, C. The bio-barcode assay for the detection of protein and nucleic acid targets using DTT-induced ligand exchange. Nat Protoc 1, 324–336 (2006). https://doi.org/10.1038/nprot.2006.51

Download citation

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/nprot.2006.51

This article is cited by

Comments

By submitting a comment you agree to abide by our Terms and Community Guidelines. If you find something abusive or that does not comply with our terms or guidelines please flag it as inappropriate.

Search

Advanced search

Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing