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The interference of HEPES buffer during amperometric detection of ATP in clinical applications

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Abstract

HEPES-based biological buffer is subject to photooxidation upon exposure to fluorescent illumination. Thereby hydrogen peroxide is generated, which interferes with amperometric oxidoreductase-based biosensors for glucose or adenosine triphosphate (ATP). These biosensors operate at an oxidation potential above 500 mV vs. the standard calomel electrode (SCE) and involve hydrogen peroxide as the electroactive molecule detected at the electrode surface. False-positive detection of ATP was observed in HEPES buffer utilizing an amperometric microbiosensor based on the co-immobilization of glucose oxidase and hexokinase for detection of ATP in biological specimens. Electrochemical, mass spectrometric, 31P NMR, and 1H NMR studies indicate that complexation of ATP and HEPES induced by the presence of Ca2+ in HEPES buffer decreases the photooxidation of HEPES. Consequently, the hydrogen peroxide background concentration is reduced, thereby leading to erroneous ATP detection at the dual-enzyme microbiosensor, which determines an increase in ATP via a reduced hydrogen peroxide signal.

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Acknowledgments

The authors would like to thank Dr Leslie Gelbaum of the NMR Center, School of Chemistry and Biochemistry, Georgia Tech, for valuable help with acquisition and interpretation of NMR spectra. The authors acknowledge financial support by NIH grant R01 HL080725 and R21 HL082860.

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Author notes

  1. Jean-Francois Masson

    Present address: Département de chimie, Université de Montréal, C.P. 6128, Succ. Centre-Ville, Montréal, QC, Canada, H3C 3J7

Authors and Affiliations

  1. School of Chemistry and Biochemistry, Georgia Institute of Technology, 901 Atlantic Drive, Atlanta, GA, 30332-0400, USA

    Jean-Francois Masson, Boris Mizaikoff & Christine Kranz

  2. Division of Neonatology Research Laboratories, Johns Hopkins Hospital, 600 N. Wolfe St., Baltimore, MD, 21287-3200, USA

    Estelle Gauda

Authors
  1. Jean-Francois Masson
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  2. Estelle Gauda
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  3. Boris Mizaikoff
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  4. Christine Kranz
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Correspondence to Christine Kranz.

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Masson, JF., Gauda, E., Mizaikoff, B. et al. The interference of HEPES buffer during amperometric detection of ATP in clinical applications. Anal Bioanal Chem 390, 2067–2071 (2008). https://doi.org/10.1007/s00216-008-2015-y

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  • DOI: https://doi.org/10.1007/s00216-008-2015-y

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