The interference of HEPES buffer during amperometric detection of ATP in clinical applications.
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, (31)P NMR, and (1)H NMR studies indicate that complexation of ATP and HEPES induced by the presence of Ca(2+) 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.
Masson
Jean-Francois
JF
School of Chemistry and Biochemistry, Georgia Institute of Technology, 901 Atlantic Drive, Atlanta, GA 30332-0400, USA.
Gauda
Estelle
E
Mizaikoff
Boris
B
Kranz
Christine
C
D000255:Adenosine Triphosphate / Q000032:analysis*; D015374:Biosensing Techniques / Q000379:methods*; D002021:Buffers; D004563:Electrochemistry; D004566:Electrodes; D004800:Enzymes, Immobilized / Q000737:chemistry; D006531:HEPES / Q000737:chemistry*; D006593:Hexokinase / Q000737:chemistry; D006861:Hydrogen Peroxide / Q000737:chemistry; D009682:Magnetic Resonance Spectroscopy / Q000379:methods; D013058:Mass Spectrometry / Q000379:methods; D010084:Oxidation-Reduction; D010088:Oxidoreductases / Q000737:chemistry; D010777:Photochemistry; D015203:Reproducibility of Results; D013499:Surface Properties; D013997:Time Factors
D002021:Buffers; D004800:Enzymes, Immobilized; D000255:Adenosine Triphosphate; D006861:Hydrogen Peroxide; D010088:Oxidoreductases; D006593:Hexokinase; D006531:HEPES