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Preparation of electrode-immobilized, redox-modified oligonucleotides for electrochemical DNA and aptamer-based sensing

Nature Protocols volume 2, pages 2875–2880 (2007)Cite this article

Abstract

Recent years have seen the development of a number of reagentless, electrochemical sensors based on the target-induced folding or unfolding of electrode-bound oligonucleotides, with examples reported to date, including sensors for the detection of specific nucleic acids, proteins, small molecules and inorganic ions. These devices, which are often termed electrochemical DNA (E-DNA) and E-AB (electrochemical, aptamer-based) sensors, are comprised of an oligonucleotide probe modified with a redox reporter (in this protocol methylene blue) at one terminus and attached to a gold electrode via a thiol-gold bond at the other. Binding of an analyte to the oligonucleotide probe changes its structure and dynamics, which, in turn, influences the efficiency of electron transfer to the interrogating electrode. This class of sensors perform well even when challenged directly with blood serum, soil and other complex, multicomponent sample matrices. This protocol describes the fabrication of E-DNA and E-AB sensors. The protocol can be completed in 12 h.

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Figure 1: The original E-DNA sensor is comprised of a stem-loop DNA modified on one terminus with a redox reporter and chemi-absorbed at the other to an interrogating electrode7.
Figure 2
Figure 3: The E-AB sensor is based on the target binding-induced folding of electrode-bound DNA aptamer.
Figure 4: The signal gain of E-AB sensors depends on the relative collision dynamics in the folded (bound) and unfolded (target-free) states and thus can be either signal-on or signal-off depending on the structure of the final folded form of the aptamer.
Figure 5
Figure 6: We employ oxidation and reduction scanning under basic conditions (0.5 M NaOH) to clean our electrodes before sensor fabrication.

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Acknowledgements

This work was supported by Lawrence Livermore National Laboratory (URP-06-019), by the Institute for Collaborative Biotechnologies through grant DAAD19-03-D-0004 from the US Army Research Office. We also thank Arica Lubin and Francesco Ricci for critically reading this manuscript.

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Authors and Affiliations

  1. Departments of Chemistry and Biochemistry, University of California, Santa Barbara, 93106, California, USA

    Yi Xiao, Rebecca Y Lai & Kevin W Plaxco

  2. Department of Physics, University of California, Santa Barbara, 93106, California, USA

    Yi Xiao & Rebecca Y Lai

  3. Interdepartmental Program in BioMolecular Science and Engineering, University of California, Santa Barbara, 93106, California, USA

    Kevin W Plaxco

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Correspondence to Kevin W Plaxco.

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Xiao, Y., Lai, R. & Plaxco, K. Preparation of electrode-immobilized, redox-modified oligonucleotides for electrochemical DNA and aptamer-based sensing. Nat Protoc 2, 2875–2880 (2007). https://doi.org/10.1038/nprot.2007.413

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