Highly sensitive conductometric biosensors for total lactate, d- and l-lactate determination in dairy products

An original method combining two conductometric biosensors is proposed for the determination of total lactate, l- and d-lactate in dairy products. The biosensors were prepared through cross-linking of l-lactate oxidase from Pediococcus sp. (LODP) or a combination of LODP and horseradish peroxidase (HRP) at the surface of gold interdigitated microelectrodes using glutaraldehyde (GA) vapors. LODP is reported to catalyze specifically l-lactate oxidation into pyruvate and hydrogen peroxide in solution. In this work, we showed that LODP chiral selectivity was lost following enzyme cross-linking, rendering LODP sensor suitable for total lactate determination. Biosensor sensitivity towards both stereoisomers was 1.16 ± 0.04 μS μM−1. The addition of HRP significantly improved the linear range and stability of LODP biosensor, but also increased its sensitivity, the effect being more pronounced for d-lactate (+381%) than for l-lactate (+260%). This result was attributed to a modification in LODP chiral selectivity combined with the production of additional ions (acetate, H+ and HCO3−) via the HRP-catalyzed oxidation of pyruvate. A method, taking advantage of the difference in d- and l-lactate behaviours and combining the monoenzymatic and bienzymatic biosensors calibration data, was proposed for the calculation of their concentrations in unknown samples. Different parameters (HRP/LODP ratio, exposure time to GA vapors, pH and concentration of measurement medium) were optimized in order to achieve the best compromise between sensitivity and stability of the biosensors. The best limit of detection for l- and d-lactate, calculated as three times the signal to noise ratio was achieved with the bi-enzymatic sensor and was equal to 0.05 μM. LODP/HRP biosensor response towards l-lactate was not significantly affected by glucose, fructose and lactose. The proposed biosensors were finally applied to the determination of total lactate, d- and l-lactate concentrations in yogurt samples. Results were in good agreement with those obtained using a reference colorimetric enzymatic method.