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Development of a bienzyme system for the electrochemical determination of nitrate in ambient air

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Abstract

This work reports the development of a bienzyme system consisting of salicylate hydroxylase (SHL) and nitrate reductase (NaR) for the electrochemical determination of nitrate. This method measures the concentration of nitrate directly under ambient air without suffering from oxygen interferences. The determination is based on the detection of NADH consumption, and the principle is as follows: NADH initiates the irreversible decarboxylation and hydroxylation of salicylate by SHL in the presence of oxygen to produce catechol, which results in a detectable signal due to its oxidation at the working electrode; the second enzyme, NaR, in the presence of nitrate, reduced the availability of NADH, and consequently, the current difference after the injection of nitrate is proportional to its concentration. This method shows high performance characteristics for nitrate determination with a broad detection range between 10 μM and 1,000 μM, a short measuring time of around 5 min, and a simple operation without sample pretreatment by inert gas purge or oxygen scavenger.

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Acknowledgement

The authors gratefully acknowledge Hong Kong Research Grant Council (HKUST6253-02P and 510-0091-02-44000-0) for the financial support.

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Authors and Affiliations

  1. Department of Chemical Engineering and Department of Chemistry, Hong Kong University of Science and Technology, Clear Water Bay, Kowloon, Hong Kong, People’s Republic of China

    Yue Cui, John P. Barford & Reinhard Renneberg

Authors
  1. Yue Cui
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  2. John P. Barford
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  3. Reinhard Renneberg
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Corresponding author

Correspondence to Yue Cui.

Electronic supplementary material

Development of a bienzyme system for the electrochemical determination of nitrate under ambient air

Fig. S1

Investigation of SHL and glutaraldehyde (GA) loadings (n=3) (Buffer: 100 mM Tris-HCl buffer containing 10 mM salicylate at pH 8.0. Substrate: 1 mM NADH). The relative response (%) was calculated by normalizing the signal to the maximum signal (DOC 30 kb)

Fig. S2

Investigation of buffer pH (n=3) (Enzyme electrode: 0.25 U SHL and 1% glutaraldehyde in 0.5 μL enzyme matrix. Buffer: 100 mM Tris-HCl buffer containing 10 mM salicylate at variou pH values. Substrate: 1 mM NADH). The relative response (%) was calculated by normalizing the signal to the maximum signal (DOC 31 kb)

Fig. S3

Investigation of buffer pH (n=3) (Enzyme electrode: 0.25 U SHL and 1% glutaraldehyde in 0.5 μL enzyme matrix. Buffer: 100 mM Tris-HCl buffer containing 10 mM salicylate at variou pH values. Substrate: 1 mM NADH). The relative response (%) was calculated by normalizing the signal to the maximum signal (DOC 30 kb)

Fig. S4

Investigation of NADH loading (n=3) (Enzyme electrode: 0.25 U SHL and 1% glutaraldehyde in 0.5 μL enzyme matrix. Buffer: 100 mM Tris-HCl buffer containing 10 mM salicylate at pH 8.0. Substrate: various amounts of NADH) (DOC 30 kb)

Fig. S5

Investigation of NaR loading (n=3) (Enzyme electrode: 0.25 U SHL and 1% glutaraldehyde in 0.5 μL enzyme matrix. Buffer: 100 mM Tris-HCl buffer containing 10 mM salicylate, 1 mM NADH and various loadings of NaR at pH 8.0. Substrate: 1 mM Nitrate) (DOC 30 kb)

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Cui, Y., Barford, J.P. & Renneberg, R. Development of a bienzyme system for the electrochemical determination of nitrate in ambient air. Anal Bioanal Chem 386, 1567–1570 (2006). https://doi.org/10.1007/s00216-006-0673-1

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  • DOI: https://doi.org/10.1007/s00216-006-0673-1

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