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Towards a Real-Time, Label-Free, Diamond-Based DNA Sensor

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Hasselt University and Transnationale Universiteit Limburg, School for Life Sciences, Biomedical Research Institute, Agoralaan, Building A, B-3590 Diepenbeek, Belgium, Hasselt University and Transnationale Universiteit Limburg, School for Life Sciences, Institute for Materials Research, Wetenschapspark 1, B-3590 Diepenbeek, Belgium, and IMEC vzw, Division IMOMEC, Wetenschapspark 1, B-3590 Diepenbeek, Belgium
Cite this: Langmuir 2007, 23, 26, 13193–13202
Publication Date (Web):November 16, 2007
https://doi.org/10.1021/la702143d
Copyright © 2007 American Chemical Society

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    Abstract

    Abstract Image

    Most challenging in the development of DNA sensors is the ability to distinguish between fully complementary target ssDNA (single-strand DNA) and 1-mismatch ssDNA. To deal with this problem, we performed impedance spectroscopy on DNA-functionalized nanocrystalline diamond (NCD) layers during hybridization and denaturation. In both reactions, a difference in behavior was observed for 1-mismatch target DNA and complementary target DNA in real-time. During real-time hybridization, a decrease of the impedance was observed at lower frequencies when the complementary target DNA was added, while the addition of 1-mismatch target ssDNA caused no significant change. Fitting these results to an electrical circuit demonstrates that this is correlated with a decrease of the depletion zone in the space charge region of the diamond. During real-time denaturation, differentiation between 1-mismatch and complementary target DNA was possible at higher frequencies. Denaturation of complementary DNA showed the longest exponential decay time of the impedance, while the decay time during 1-mismatch denaturation was the shortest. The real-time hybridization and denaturation experiments were carried out on different NCD samples in various buffer solutions at temperatures between 20 and 80 °C. It was revealed that the best results were obtained using a Microhyb hybridization buffer at 80 °C and 10× PCR buffer at 30 °C for hybridization and 0.1 M NaOH at temperatures above 40 °C for denaturation. We demonstrate that the combination of real-time hybridization spectra and real-time denaturation spectra yield important information on the type of target. This approach may allow a reliable identification of the mismatch sequence, which is the most biologically relevant.

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     Biomedical Research Institute, Hasselt University and Transnationale Universiteit Limburg.

     These authors contributed equally to this publication.

    §

     Institute for Materials Research, Hasselt University and Transnationale Universiteit Limburg.

     IMEC vzw, Division IMOMEC.

    *

     To whom correspondence should be addressed. E-mail:  luc.michiels@ uhasselt.be; tel.:  (+32)-11-26-92-31; fax:  (+32)-11-26-92-35.

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