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A Bienzyme Carbon Paste Electrode for the Sensitive Detection of NADPH and the Measurement of Glucose‐6‐phosphate Dehydrogenase
Author(s) -
Huang Tina,
Warsinke Axel,
KoroljovaSkorobogatko Olga V.,
Makower Alexander,
Kuwana Theodore,
Scheller Frieder W.
Publication year - 1999
Publication title -
electroanalysis
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.574
H-Index - 128
eISSN - 1521-4109
pISSN - 1040-0397
DOI - 10.1002/(sici)1521-4109(199905)11:5<295::aid-elan295>3.0.co;2-0
Subject(s) - biosensor , chemistry , electrode , detection limit , dehydrogenase , laccase , chromatography , carbon paste electrode , enzyme , nuclear chemistry , biochemistry , electrochemistry , cyclic voltammetry
A novel bienzyme‐based biosensor was constructed for the sensitive detection of NADPH. The sensing system consists of two enzymes, p ‐hydroxybenzoate hydroxylase (HBH) and laccase, both immobilized inside a carbon paste electrode. The detection scheme for NADPH is as followed: first, the HBH converts p ‐hydroxybenzoate (pHB) to 3,4‐dihydroxybenzoate (3,4‐DHB red ) in the presence of NADPH. Then the 3,4‐DHB red is further oxidized by laccase to its corresponding o ‐quinone form (3,4‐DHB ox ) and reduced back to 3,4 DHB red at the surface of the electrode (at E appl =–50 mV vs. Ag/AgCl). The recycling of the 3,4‐DHB red between the laccase and the electrode results in an amplified current signal that is proportional to NADPH concentration. The low applied potential is a desirable feature for minimizing interferants in biological applications. This bienzyme system showed an increased sensitivity for NADPH by a factor of ca. 18 when compared with the single enzyme electrode (HBH only, 3,4‐DHB oxidation at E appl = +350 mV vs. Ag/AgCl). A linear NADPH calibration plot was obtained from 5–30 μM, with a detection limit of 1 μM. The pH optimum of this sensor was around 6. The bienzyme sensor showed consistent response over 7 hours in pH 6.2 phosphate buffer at room temperature. Both enzymes remained stable inside the carbon paste for 2 months when stored dry at 4°C. The utility of this NADPH‐detecting biosensor was demonstrated by measuring the activity of glucose‐6‐phosphate dehydrogenase in test blood samples. The results obtained with the sensor setup were compared with those obtained with the spectrophotometric assay. Good correlation was found between the two methods with a correlation coefficient of 0.98.