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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

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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.

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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.

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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).

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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

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  1. Meihua Lin
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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.

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Correspondence to Xiaolei Zuo.

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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

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