An amperometric lactate sensor based on a NAD(+)-analog and lactate dehydrogenase coimmobilized on reticulated vitreous carbon

A NAD(+)-analog was coimmobilized with lactate dehydrogenase (LDH) on reticulated vitreous carbon (RVC) to give an amperometric lactate biosensor. Both LDH and the NAD(+)-analog were bound covalently with carbodiimide to the surface of the porous RVC-material. The electrode was operated in a FIA-arrangement in the presence or absence of a soluble mediator, Meldola Blue. The stability was poor when the electrode was operated at +400 mV (vs. Ag/AgCl) in the absence of mediator but improved most significantly in the presence of 5 mu M mediator, so that 65% of the original activity remained after 16 days. The amperometric currents were smaller with regeneration by mediator at -100mV than with direct electrochemical oxidation at +400 mV, indicating that the additional steps slow down the reaction rate. Linear calibration plots were obtained from the detection limit, 1 mu M, to 500 lambda M lactate with 5 mu M mediator, reoxidized at -100mV. The sample throughput was about 60 h(-1).