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Piezoelectric Plate Sensor (PEPS) for Analysis of Specific KRAS Point Mutations at Low Copy Number in Urine Without DNA Isolation or Amplification

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Biosensors and Biodetection

Part of the book series: Methods in Molecular Biology ((MIMB,volume 1572))

Abstract

We have examined in situ detection of single-nucleotide KRAS mutations in urine using a (Pb(Mg1/3Nb2/3)O3)0.65(PbTiO3)0.35 (PMN-PT) piezoelectric plate sensor (PEPS) coated with a 17-nucleotide (nt) locked nucleic acid (LNA) probe DNA complementary to the KRAS mutation without DNA isolation and amplification. In situ mutant (MT) DNA in urine in a wild type (WT) background was carried out at a flow rate of 4 mL/min and at 63 °C with the PEPS vertically situated at the center of the flow. Both the temperature and the impingement flow force discriminated the wild type. Under these conditions PEPS was shown to specifically detect KRAS MT in situ within 30 min with an analytical sensitivity of 60 copies/mL in a clinically relevant background of WT with concentrations 1000-fold greater than that of MT without DNA isolation, amplification, or labeling. For validation, detection was performed in a mixture of blue MT fluorescent reporter microspheres (FRMs) (MT FRMs) that bound to only the captured MT, and orange WT FRMs that bound to only the captured WT. The captured blue MT FRMs still outnumbered the orange WT FRMs by a factor of 4–1 even though WT was 1000-fold of MT in urine, illustrating the specificity of the point mutation detection.

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Acknowledgment

This work was supported in part by the Coulter-Drexel Translational Research Partnership grant, the Nanotechnology Institute of Benjamin Franklin Partnership of Southeastern Pennsylvania, and National Institute of Health Grants No. R41AI112224 and R41AI120445.

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Authors and Affiliations

  1. School of Biomedical Engineering, Science, and Health Systems, Drexel University, Philadelphia, PA, 10104, USA

    Ceyhun E. Kirimli & Wan Y. Shih

  2. Department of Materials Science and Engineering, Drexel University, Philadelphia, PA, 10104, USA

    Wei-Heng Shih

Authors
  1. Ceyhun E. Kirimli
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  2. Wei-Heng Shih
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  3. Wan Y. Shih
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Corresponding author

Correspondence to Wan Y. Shih .

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Editors and Affiliations

  1. National Cancer Institute, National Institutes of Health, Rockville, Maryland, USA

    Ben Prickril

  2. National Cancer Institute, National Institutes of Health, Rockville, Maryland, USA

    Avraham Rasooly

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Kirimli, C.E., Shih, WH., Shih, W.Y. (2017). Piezoelectric Plate Sensor (PEPS) for Analysis of Specific KRAS Point Mutations at Low Copy Number in Urine Without DNA Isolation or Amplification. In: Prickril, B., Rasooly, A. (eds) Biosensors and Biodetection. Methods in Molecular Biology, vol 1572. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-6911-1_22

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  • DOI: https://doi.org/10.1007/978-1-4939-6911-1_22

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  • Publisher Name: Humana Press, New York, NY

  • Print ISBN: 978-1-4939-6910-4

  • Online ISBN: 978-1-4939-6911-1

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