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

Jean Francois Masson, Estelle Gauda, Boris Mizaikoff, Christine Kranz

Research output: Contribution to journalArticlepeer-review

8 Scopus citations


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.

Original languageEnglish (US)
Pages (from-to)2067-2071
Number of pages5
JournalAnalytical and Bioanalytical Chemistry
Issue number8
StatePublished - Apr 2008
Externally publishedYes


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

ASJC Scopus subject areas

  • Analytical Chemistry
  • Biochemistry


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