Kinetic measurement of an alkaline phosphatase enzyme label was performed with amperometric detection using conversion of substrate 4-aminophenyl phosphate to the electroactive phenolic, measured at +125 to +200 mV (vs. Ag/AgCl). Calibration plots for thyroxine-binding globulin (TBG) and cortisol were obtained by excess reagent and limited reagent immunoassays, respectively, which were comparable with those obtained using colorimetric detection. These provided a measurement range of 31-1000 pg/L for TBG and 100-2000 nM for cortisol. An antibody-bound label was monitored in this system, and covering the electrode with a simple dialysis or polycarbonate membrane avoided electrode fouling. The kinetic mode of detection reduced assay time and helped to simplie the assay protocol.
Reduced nicotinamide adenine dinucleotide (NADH), produced from glucose-6-phosphate dehydrogenase (G6PDH) was catalytically oxidized using diaphorase and 1,4-benzoquinone to yield 1,4-hydroquinone. This benzoquinone redox mediator was readily detected at an electrode polarized at +0.5 V (vs. Ag/AgCI) so avoiding the high overpotentials required for a direct electrochemical measurement of NADH. Also, the lipophilic nature of the hydroquinone enabled a novel high selectivity plasticized PVC membrane mounted over the electrode to be exploited. The mediator/PVC combination achieved NADH calibration linear up to at least 1 0 0~~. When used to assay G6PDH activity, a linear calibration from 0.1-1.0 U/mL was produced. The detection system offers the possibility of highly selective measurement of dehydrogenase enzyme activity in biological samples without sample preparation and could provide the basis for simplified homogeneous immunoassays as well as dehydrogenase amperometric enzyme electrodes.
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