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A sensitive and stable amperometric nitrate biosensor employing Arabidopsis thaliana nitrate reductase

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Abstract

Nitrate reductase (NR) from the plant Arabidopsis thaliana has been employed in the development of an amperometric nitrate biosensor that functions at physiological pH. The anion anthraquinone-2-sulfonate (AQ) is used as an effective artificial electron transfer partner for NR at a glassy carbon (GC) electrode. Nitrate is enzymatically reduced to nitrite and the oxidized form of NR is electrochemically reduced by the hydroquinone form of the mediator (AQH2). The GC/NR electrode shows a pronounced cathodic wave for nitrate reduction and the catalytic current increases linearly in the nitrate concentration range of 10–400 µM with a correlation coefficient of 0.989. Using an amperometric method, a low detection limit of 0.76 nM (S/N = 3) was achieved. The practical application of the present electrochemical biosensor was demonstrated by the determination of nitrate concentration in natural water samples and the results agreed well with a standard spectroscopic method.

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Notes

  1. In all cases we assume that the heme cofactor of NR relays electrons rapidly from AQH2 to the Mo active site.

Abbreviations

AQ:

Anthraquinone-2-sulfonate

CV:

Cyclic voltammetry

FAD:

Flavin adenine dinucleotide

GC:

Glassy carbon

MV:

Methyl viologen

NADH:

Nicotinamide adenine dinucleotide

NHE:

Normal hydrogen electrode

NR:

Nitrate reductase

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Acknowledgments

We gratefully acknowledge support from the Australian Research Council (DP120101465).

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Authors and Affiliations

  1. School of Chemistry and Molecular Biosciences, University of Queensland, Brisbane, 4072, Australia

    Palraj Kalimuthu & Paul V. Bernhardt

  2. Department of Chemistry and Center for Molecular Medicine, Institute of Biochemistry, Cologne University, Zülicher Str. 47, 50674, Cologne, Germany

    Katrin Fischer-Schrader & Günter Schwarz

Authors
  1. Palraj Kalimuthu
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  2. Katrin Fischer-Schrader
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  3. Günter Schwarz
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  4. Paul V. Bernhardt
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Corresponding author

Correspondence to Paul V. Bernhardt.

Additional information

Responsible Editors: José Moura and Paul Bernhardt.

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Kalimuthu, P., Fischer-Schrader, K., Schwarz, G. et al. A sensitive and stable amperometric nitrate biosensor employing Arabidopsis thaliana nitrate reductase. J Biol Inorg Chem 20, 385–393 (2015). https://doi.org/10.1007/s00775-014-1171-0

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  • DOI: https://doi.org/10.1007/s00775-014-1171-0

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