NADH amperometric sensor based on poly(3-methylthiophene)-coated cylindrical carbon fiber microelectrodes: application to the enzymatic determination of L-lactate

Abstract

Abstract A NADH amperometric microsensor based on cylindrical carbon fiber microelectrodes (CFMEs) coated with poly(3-methylthiophene) (P3MT) is reported. Cyclic voltammograms for NADH showed an oxidation response at the P3MT-coated CFME 320 mV lower than that obtained at a conventional glassy carbon electrode. A higher resistance to the electrode fouling was observed when P3MT-coated CFMEs were compared with P3MT-coated GC electrodes of conventional size. A potential of +0.45 V was selected for the detection of NADH by amperometry in stirred solutions. The steady-state current was reached very rapidly (5–7 s), and the same modified micro-electrode could be used during a whole working day without any cleaning treatment. A linear calibration graph for NADH was obtained in the range 1.0×10 −6 –10×10 −4 mol l −1 with a detection limit of 7.6×10 −7 mol l −1 . The influence of the presence of high concentrations of NAD + or ascorbic acid on the NADH amperometric response was also tested. An amperometric flow-cell adapted for the use of microelectrodes was designed. Improved signal-to-background ratios were obtained in this case, which allowed NADH amperometric measurements to be carried out at potentials lower than +0.45 V. Taking into account the influence of an excess of NAD + on the NADH response, a potential value of +0.30 V was selected. The stability of the polymer coating on the CFME was shown to be good in the carrier stream, and the analytical characteristics of the calibration graph were slightly better than those obtained by amperometry under batch conditions. A flow-injection method for the amperometric-enzymatic determination of L-lactate has been developed by using a reticulated vitreous carbon-based enzyme (lactate dehydrogenase) reactor inserted into the flow cell. Practically constant lactate responses were obtained over the first 8 d. A good selectivity for lactate was achieved against citric, malic, tartaric, and acetic acids, and ethanol.