A bienzyme carbon paste electrode for the sensitive detection of NADPH andthe measurement of glucose-6-phosphate dehydrogenase

A novel bienzyme-based biosensor was constructed for the sensitive detectio n of NADPH. The sensing system consists of two enzymes, p-hydroxybenzoate h ydroxylase (HBH) and laccase, both immobilized inside a carbon paste electr ode. The detection scheme for NADPH is as followed: first, the HBH converts p-hydroxybenzoate (pHB) to 3,4-dihydroxybenzoate (3,4-DHBred) in the prese nce of NADPH. Then the 3,4-DHBred is further oxidized by laccase to its cor responding o-quinone form (3,4-DHBox) and reduced back to 3,4 DHBred at the surface of the electrode (at E-appl = -50 mV vs. Ag/AgCl). The recycling o f the 3,4-DHBred between the laccase and the electrode results in an amplif ied current signal that is proportional to NADPH concentration. The low app lied potential is a desirable feature for minimizing interferants in biolog ical 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 N ADPH calibration plot was obtained from 5-30 mu M, with a detection limit o f 1 mu M. The pH optimum of this sensor was around 6. The bienzyme sensor s howed consistent response over 7 hours in pH 6.2 phosphate buffer at room t emperature. Both enzymes remained stable inside the carbon paste for 2 mont hs when stored dry at 4 degrees C. The utility of this NADPH-detecting bios ensor was demonstrated by measuring the activity of glucose-6-phosphate deh ydrogenase in test blood samples. The results obtained with the sensor setu p were compared with those obtained with the spectrophotometric assay. Good correlation was found between the two methods with a correlation coefficie nt of 0.98.