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.

  • Protocol
  • Published:

Electrochemical detection of nucleic acids, proteins, small molecules and cells using a DNA-nanostructure-based universal biosensing platform

Nature Protocols volume 11, pages 1244–1263 (2016)Cite this article

Subjects

Abstract

The occurrence and prognosis of many complex diseases, such as cancers, is associated with the variation of various molecules, including DNA at the genetic level, RNA at the regulatory level, proteins at the functional level and small molecules at the metabolic level (defined collectively as multilevel molecules). Thus it is highly desirable to develop a single platform for detecting multilevel biomarkers for early-stage diagnosis. Here we report a protocol on DNA-nanostructure-based programmable engineering of the biomolecular recognition interface, which provides a universal electrochemical biosensing platform for the ultrasensitive detection of nucleic acids (DNA/RNA), proteins, small molecules and whole cells. The protocol starts with the synthesis of a series of differentially sized, self-assembled tetrahedral DNA nanostructures (TDNs) with site-specifically modified thiol groups that can be readily anchored on the surface of a gold electrode with high reproducibility. By exploiting the rigid structure, nanoscale addressability and versatile functionality of TDNs, one can tailor the type of biomolecular probes appended on individual TDNs for the detection of specific molecules of interest. Target binding occurring on the gold surface patterned with TDNs is quantitatively translated into electrochemical signals via a coupled enzyme-based catalytic process. This uses a sandwich assay strategy in which biotinylated reporter probes recognize TDN-bound target biomolecules, which then allow binding of horseradish-peroxidase-conjugated avidin (avidin–HRP). Hydrogen peroxide (H2O2) is then reduced by avidin–HRP in the presence of TMB (3,3′,5,5′-tetramethylbenzidine) to generate a quantitative electrochemical signal. The time range for the entire protocol is ∼1 d, whereas the detection process takes ∼30 min to 3 h.

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

Access options

Buy this article

  • Purchase on SpringerLink
  • Instant access to full article PDF
Buy now

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

Figure 1: A tetrahedral DNA-nanostructure-based universal biosensing platform for electrochemical detection of nucleic acids, proteins, small molecules and cells.
Figure 2: The process for TDN assembly and PAGE characterization.
Figure 3: Using FRET analysis to confirm the integrity of TDNs.
Figure 4: Characterization of the size and morphology of TDNs by AFM.
Figure 5: TDN capture probes.
Figure 6: Biosensing platform for the detection of DNA targets.
Figure 7: Detection of microRNAs, small molecules and proteins.

Similar content being viewed by others

References

  1. Bonventre, J.V., Vaidya, V.S., Schmouder, R., Feig, P. & Dieterle, F. Next-generation biomarkers for detecting kidney toxicity. Nat. Biotechnol. 28, 436–440 (2010).

    Article  CAS  Google Scholar 

  2. Etzioni, R. et al. The case for early detection. Nat. Rev. Cancer 3, 243–252 (2003).

    Article  CAS  Google Scholar 

  3. Ferrari, M. Cancer nanotechnology: opportunities and challenges. Nat. Rev. Cancer 5, 161–171 (2005).

    Article  CAS  Google Scholar 

  4. Sawyers, C.L. The cancer biomarker problem. Nature 452, 548–552 (2008).

    Article  CAS  Google Scholar 

  5. Das, J. et al. An electrochemical clamp assay for direct, rapid analysis of circulating nucleic acids in serum. Nat. Chem. 7, 569–575 (2015).

    Article  CAS  Google Scholar 

  6. Fredriksson, S. et al. Multiplexed protein detection by proximity ligation for cancer biomarker validation. Nat. Methods 4, 327–329 (2007).

    Article  CAS  Google Scholar 

  7. Kelley, S.O. et al. Advancing the speed, sensitivity and accuracy of biomolecular detection using multi-length-scale engineering. Nat. Nanotechnol. 9, 969–980 (2014).

    Article  CAS  Google Scholar 

  8. Kohannim, O. et al. Boosting power for clinical trials using classifiers based on multiple biomarkers. Neurobiol. Aging 31, 1429–1442 (2010).

    Article  Google Scholar 

  9. Mitchell, P.S. et al. Circulating microRNAs as stable blood-based markers for cancer detection. Proc. Natl. Acad. Sci. USA 105, 10513–10518 (2008).

    Article  CAS  Google Scholar 

  10. Osterfeld, S.J. et al. Multiplex protein assays based on real-time magnetic nanotag sensing. Proc. Natl. Acad. Sci. USA 105, 20637–20640 (2008).

    Article  CAS  Google Scholar 

  11. Sreekumar, A. et al. Metabolomic profiles delineate potential role for sarcosine in prostate cancer progression. Nature 457, 910–914 (2009).

    Article  CAS  Google Scholar 

  12. Urdea, M. et al. Requirements for high impact diagnostics in the developing world. Nature 444, 73–79 (2006).

    Article  Google Scholar 

  13. Wang, T.J. et al. Multiple biomarkers for the prediction of first major cardiovascular events and death. New Engl. J. Med. 355, 2631–2639 (2006).

    Article  CAS  Google Scholar 

  14. Zethelius, B. et al. Use of multiple biomarkers to improve the prediction of death from cardiovascular causes. New Engl. J. Med. 358, 2107–2116 (2008).

    Article  CAS  Google Scholar 

  15. Zheng, G.F., 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 

  16. Das, J. et al. An ultrasensitive universal detector based on neutralizer displacement. Nat. Chem. 4, 642–648 (2012).

    Article  CAS  Google Scholar 

  17. Xia, F. et al. Colorimetric detection of DNA, small molecules, proteins, and ions using unmodified gold nanoparticles and conjugated polyelectrolytes. Proc. Natl. Acad. Sci. USA 107, 10837–10841 (2010).

    Article  CAS  Google Scholar 

  18. Xiang, Y. & Lu, Y. Using personal glucose meters and functional DNA sensors to quantify a variety of analytical targets. Nat. Chem. 3, 697–703 (2011).

    Article  CAS  Google Scholar 

  19. Pei, H. et al. A DNA nanostructure-based biomolecular probe carrier platform for electrochemical biosensing. Adv. Mater. 22, 4754–4758 (2010).

    Article  CAS  Google Scholar 

  20. Lin, M.H. et al. Programmable engineering of a biosensing interface with tetrahedral DNA nanostructures for ultrasensitive DNA detection. Angew. Chem. Int. Edit. 54, 2151–2155 (2015).

    Article  CAS  Google Scholar 

  21. Chen, X.Q. et al. Ultrasensitive electrochemical detection of prostate-specific antigen by using antibodies anchored on a DNA nanostructural scaffold. Anal. Chem. 86, 7337–7342 (2014).

    Article  CAS  Google Scholar 

  22. Ge, Z.L. et al. Hybridization chain reaction amplification of microRNA detection with a tetrahedral DNA nanostructure-based electrochemical biosensor. Anal. Chem. 86, 2124–2130 (2014).

    Article  CAS  Google Scholar 

  23. Lin, M.H. et al. Target-responsive, DNA nanostructure-based E-DNA sensor for microRNA analysis. Anal. Chem. 86, 2285–2288 (2014).

    Article  CAS  Google Scholar 

  24. Wen, Y.L. et al. DNA nanostructure-based interfacial engineering for PCR-free ultrasensitive electrochemical analysis of microRNA. Sci. Rep. 2 (2012).

  25. Wen, Y.L. et al. DNA nanostructure-decorated surfaces for enhanced aptamer-target binding and electrochemical cocaine sensors. Anal. Chem. 83, 7418–7423 (2011).

    Article  CAS  Google Scholar 

  26. Zhou, G.B. et al. Multivalent capture and detection of cancer cells with DNA nanostructured biosensors and multibranched hybridization chain reaction amplification. Anal. Chem. 86, 7843–7848 (2014).

    Article  CAS  Google Scholar 

  27. Ge, Z.L., Pei, H., Wang, L.H., Song, S.P. & Fan, C.H. Electrochemical single nucleotide polymorphisms genotyping on surface immobilized three-dimensional branched DNA nanostructure. Sci. China Chem. 54, 1273–1276 (2011).

    Article  CAS  Google Scholar 

  28. Wu, J., Campuzano, S., Halford, C., Haake, D.A. & Wang, J. Ternary surface monolayers for ultrasensitive (zeptomole) amperometric detection of nucleic acid hybridization without signal amplification. Anal. Chem. 82, 8830–8837 (2010).

    Article  CAS  Google Scholar 

  29. Soleymani, L., Fang, Z.C., Sargent, E.H. & Kelley, S.O. Programming the detection limits of biosensors through controlled nanostructuring. Nat. Nanotechnol. 4, 844–848 (2009).

    Article  CAS  Google Scholar 

  30. Yang, F. et al. A bubble-mediated intelligent microscale electrochemical device for single-step quantitative bioassays. Adv. Mater. 26, 4671–4676 (2014).

    Article  CAS  Google Scholar 

  31. Deng, W.P. et al. Diagnosis of schistosomiasis japonica with interfacial co-assembly-based multi-channel electrochemical immunosensor arrays. Sci. Rep. 3 1789 (2013).

  32. Zuo, X.L., Xiao, Y. & Plaxco, K.W. High specificity, electrochemical sandwich assays based on single aptamer sequences and suitable for the direct detection of small-molecule targets in blood and other complex matrices. J. Am. Chem. Soc. 131, 6944–6945 (2009).

    Article  CAS  Google Scholar 

  33. Zhang, J., Song, S.P., Wang, L.H., Pan, D. & Fan, C.H. A gold nanoparticle-basedchronocoulometric DNA sensor for amplified detection of DNA. Nat. Protoc. 2, 2888–2895 (2007).

    Article  CAS  Google Scholar 

  34. Xiao, Y., Lai, R.Y. & Plaxco, K.W. Preparation of electrode-immobilized, redox-modified oligonucleotides for electrochemical DNA and aptamer-based sensing. Nat. Protoc. 2, 2875–2880 (2007).

    Article  CAS  Google Scholar 

  35. Steel, A.B., Herne, T.M. & Tarlov, M.J. Electrochemical quantitation of DNA immobilized on gold. Anal. Chem. 70, 4670–4677 (1998).

    Article  CAS  Google Scholar 

Download references

Acknowledgements

This work was supported by the National Basic Research Program (973 Program grants 2012CB932600, 2013CB933802 and 2013CB932803), NSFC (21422508, 31470960, 21390414, 21227804, 91123037 and 21329501) and the Chinese Academy of Sciences. A.A. extends his sincere appreciation to the Deanship of Scientific Research at King Saud University and the Distinguished Scientist Fellowship Program of King Saud University (RG-1436-005).

Author information

Authors and Affiliations

  1. Division of Physical Biology and Bioimaging Center, Shanghai Synchrotron Radiation Facility, CAS Key Laboratory of Interfacial Physics and Technology, Shanghai Institute of Applied Physics, Chinese Academy of Sciences, Shanghai, China

    Meihua Lin, Ping Song, Guobao Zhou, Xiaolei Zuo, Jiye Shi & Chunhai Fan

  2. Department of Chemistry, King Saud University, Riyadh, Saudi Arabia

    Ali Aldalbahi

  3. Key Laboratory for Large-Format Battery Materials and System, Ministry of Education, School of Chemistry and Chemical Engineering, Huazhong University of Science and Technology, Wuhan, China

    Xiaoding Lou

  4. Kellogg College,

    Jiye Shi

  5. , University of Oxford, Oxford, UK

    Jiye Shi

  6. UCB Pharma, Slough, UK

    Jiye Shi

  7. School of Life Science and Technology, ShanghaiTech University, Shanghai, China

    Chunhai Fan

Authors
  1. Meihua Lin
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Ping Song
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Guobao Zhou
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Xiaolei Zuo
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Ali Aldalbahi
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Xiaoding Lou
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Jiye Shi
    View author publications

    You can also search for this author in PubMed Google Scholar

  8. Chunhai Fan
    View author publications

    You can also search for this author in PubMed Google Scholar

Contributions

X.Z. and C.F. conceived the projects; M.L., P.S., G.Z., X.L., X.Z. and C.F. designed and conducted the experiments; M.L., A.A., X.Z., J.S., X.L. and C.F. analyzed the data; and M.L., X.Z. and C.F. wrote the manuscript.

Corresponding author

Correspondence to Xiaolei Zuo.

Ethics declarations

Competing interests

The authors declare no competing financial interests.

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Lin, M., Song, P., Zhou, G. et al. Electrochemical detection of nucleic acids, proteins, small molecules and cells using a DNA-nanostructure-based universal biosensing platform. Nat Protoc 11, 1244–1263 (2016). https://doi.org/10.1038/nprot.2016.071

Download citation

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/nprot.2016.071

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