Analytical and Bioanalytical Chemistry

, Volume 390, Issue 8, pp 2067–2071 | Cite as

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

  • Jean-Francois Masson
  • Estelle Gauda
  • Boris Mizaikoff
  • Christine KranzEmail author
Short Communication


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.


4-(2-Hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) Phototoxicity ATP microbiosensor Hexokinase Glucose oxidase Ringer’s solution 



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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Copyright information

© Springer-Verlag 2008

Authors and Affiliations

  • Jean-Francois Masson
    • 1
  • Estelle Gauda
    • 2
  • Boris Mizaikoff
    • 1
  • Christine Kranz
    • 1
    Email author
  1. 1.School of Chemistry and BiochemistryGeorgia Institute of TechnologyAtlantaUSA
  2. 2.Division of Neonatology Research LaboratoriesJohns Hopkins HospitalBaltimoreUSA

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