A lactate dehydrogenase amperometric pyruvate electrode exploiting direct detection of NAD+ at a poly(3-methylthiophene):poly(phenol red) modified platinum surface

An amperometric L(+) lactic dehydrogenase, pyruvate sensing electrode has been developed in which NADH is regenerated at a poly (3-methylthiophene)/poly (phenol red) electrode. A key part of the sensor is the use of a dual poly (3-methylthiophene)/poly (phenol red) film which permits the reduction of enzymically active NAD+ at an over potential of only −0.2 V (vs. Ag/AgCl) in comparison to − 1 V at a bare electrode. NAD+ reduction is proposed to take place via the quinone group of poly (phenol red) to poly (3-methylthiophene) facilitating electron transfer from the base Pt electrode. A pyruvate sensor was constructed using gel entrapped L(+) lactic dehydrogenase. The pH, cofactor concentration, buffer type and concentration all had effects on the pyruvate response. In phosphate saline buffer, pyruvate gave a response independent of L(+) lactic dehydrogenase activity, and were attributed to the acid doping of the poly(3-methylthiophene) film. With 2-[N-morpholino(ethanesulfonic acid)] (MES) saline buffer the pyruvate electrode gave a decrease in cathodic currents. The nonspecific doping effect by pyruvate was negligible and this behaviour was therefore attributed to an increase in poly(3-methylthiophene) conductivity during interaction with enzymically produced NAD+. Under optimised assay conditions of (40 mM MES pH 6 containing 50 mM KCl, 1 mM NADH) the drop in cathodic response could be related to solution pyruvate concentration. The KM for pyruvate reduction by L(+) lactic dehydrogenase was determined to be 3.7 mM. The different properties of poly(3-methylthiophene)/poly(phenol red) modified electrodes in phosphate and MES buffers were studied using impedance spectroscopy. It is proposed that phosphate entry contributes significantly to the charge transfer resistance of the dual polymer film despite the presence of mobile K+ and Cl− ions. However, in MES buffer the K+ and Cl− ions appeared to be the dominant charge carriers. The reasons for the different sensitivities of poly (3-methylthiophene)/poly(phenol red) modified electrodes to pyruvate in phosphate and MES buffers are discussed in terms of the ion exchange properties of the dual film.