Development of a commercial amperometric biosensor electrode for the ketone D-3-hydroxybutyrate.

Representatives of the common classes of quinoid NADH redox mediator, including Meldola Blue (MB) 3, 4-methyl-1,2-benzoquinone (4-MBQ) 4, 1-methoxy phenazine methosulphate (1-MeO-PMS) 5 and 2,6-dichloroindophenol (DCIP) 6, are shown to inhibit the NAD-dependent enzyme D-3-hydroxybutyrate dehydrogenase (HBDH), severely limiting their utility in the construction of a stable biosensor electrode for the ketone body D-3-hydroxybutyrate (3-OHB). It is proposed that these mediators bind covalently to important thiol groups in the enzyme. This mode of inhibition is overcome through the use of mediators such as 1,10-phenanthroline quinone (1,10-PQ) 7, which avoid 1,4-nucleophilic addition with enzyme amino acid residues such as Cys. As a result, 1,10-PQ 7 was selected for incorporation in a biosensor electrode for 3-OHB. The resulting MediSense Optiumtrade mark beta-Ketone electrode is stable (<or=10% loss in response at 30 degrees C versus 4 degrees C) with a long shelf life of 18 months. Diabetics can determine their D-3-hydroxybutyrate level with good precision (0.43 mM 3-OHB, 10.5% CV; 1.08 mM, 5.9%; 3.55 mM, 3.2%; n=20 per level) and accuracy (versus reference assay: slope=0.98; intercept=0.02 mM, r=0.97, n=120) over the range 0.0-6.0 mM in 30 s using a small volume of blood (5 microl). The electrode has a low operating potential (+200 mV versus Ag/AgCl) such that the effect of electroactive agents in blood is minimised.