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An electrochemical clamp assay for direct, rapid analysis of circulating nucleic acids in serum

Nature Chemistry volume 7, pages 569–575 (2015)Cite this article

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Abstract

The analysis of cell-free nucleic acids (cfNAs), which are present at significant levels in the blood of cancer patients, can reveal the mutational spectrum of a tumour without the need for invasive sampling of the tissue. However, this requires differentiation between the nucleic acids that originate from healthy cells and the mutated sequences shed by tumour cells. Here we report an electrochemical clamp assay that directly detects mutated sequences in patient serum. This is the first successful detection of cfNAs without the need for enzymatic amplification, a step that normally requires extensive sample processing and is prone to interference. The new chip-based assay reads out the presence of mutations within 15 minutes using a collection of oligonucleotides that sequester closely related sequences in solution, and thus allow only the mutated sequence to bind to a chip-based sensor. We demonstrate excellent levels of sensitivity and specificity and show that the clamp assay accurately detects mutated sequences in a collection of samples taken from lung cancer and melanoma patients.

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Figure 1: The clamp chip for the electrochemical analysis of mutated cfNAs.
Figure 2: Proof of principle and validation of the probes.
Figure 3: Limit-of-detection studies and time dependence for KRAS cfNAs.
Figure 4: Limit-of-detection studies and time dependence for BRAF cfNAs.
Figure 5: Determination of target analyte.

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Acknowledgements

This research was sponsored by the Ontario Research Fund (Research Excellence Award to S.O.K.), the Canadian Institutes for Health Research (Emerging Team Grant to S.O.K. and E.H.S.), the Canadian Cancer Society Research Institute (Innovation Grant No. 702414 to S.O.K. and J.R.) and the Natural Science and Engineering Research Council (Discovery Grant to S.O.K.).

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Authors and Affiliations

  1. Department of Pharmaceutical Sciences, Leslie Dan Faculty of Pharmacy, University of Toronto, Toronto, M5S 3M2, Canada

    Jagotamoy Das, Ivaylo Ivanov & Shana O. Kelley

  2. The Research Institute of the McGill University Health Centre, Montreal Children's Hospital, 1001 Decarie Blvd, Montreal, Quebec, H4A 3J1, Canada

    Laura Montermini & Janusz Rak

  3. Department of Electrical and Computer Engineering, Faculty of Engineering, University of Toronto, Toronto, Canada

    Edward H. Sargent

  4. Department of Biochemistry, Faculty of Medicine, University of Toronto, Toronto, M5S 3M2, Ontario, Canada

    Shana O. Kelley

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  1. Jagotamoy Das
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Contributions

J.D., I.I., J.R., E.H.S. and S.O.K. conceived the experiments, J.D. and I.I. designed the experiments, L.M. contributed critical materials and J.D., I.I., E.H.S. and S.O.K. co-wrote the paper. All the authors reviewed and improved the paper.

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Correspondence to Shana O. Kelley.

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The authors declare no competing financial interests.

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Das, J., Ivanov, I., Montermini, L. et al. An electrochemical clamp assay for direct, rapid analysis of circulating nucleic acids in serum. Nature Chem 7, 569–575 (2015). https://doi.org/10.1038/nchem.2270

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